Simultaneous liquid chromatography/mass spectrometry determination of both polar and "multiresidue" pesticides in food using parallel hydrophilic interaction/reversed-phase liquid chromatography and a hybrid sample preparation approach.

PubMed

Robles-Molina, José; Gilbert-López, Bienvenida; García-Reyes, Juan F; Molina-Díaz, Antonio

2017-09-29

Pesticide testing of foodstuffs is usually accomplished with generic wide-scope multi-residue methods based on liquid chromatography tandem mass spectrometry (LC-MS/MS). However, this approach does not cover some special pesticides, the so called "single-residue method" compounds, that are hardly compatible with standard reversed-phase (RP) separations due to their specific properties. In this article, we propose a comprehensive strategy for the integration of single residue method compounds and standard multiresidue pesticides within a single run. It is based on the use of a parallel LC column assembly with two different LC gradients performing orthogonal hydrophilic interaction chromatography (HILIC) and reversed-phase (RPLC) chromatography within one analytical run. Two sample aliquots were simultaneously injected on each column, using different gradients, being the eluents merged post-column prior to mass spectrometry detection. The approach was tested with 41 multiclass pesticides covering a wide range of physicochemical properties across several orders of log K ow (from -4 to +5.5). With this assembly, distinct separation from the void was attained for all the pesticides studied, keeping similar performance in terms of sensitivity, peak area reproducibility (<6 RSD% in most cases) and retention time stability of standard single column approaches (better than±0.1min). The application of the proposed approach using parallel HILIC/RPLC and RPLC/aqueous normal phase (Obelisc) were assessed in leek using LC-MS/MS. For this purpose, a hybrid QuEChERS (Quick, easy, cheap, effective, rugged and safe)/QuPPe (quick method for polar pesticides) method was evaluated based on solvent extraction with MeOH and acetonitrile followed by dispersive solid-phase extraction, delivering appropriate recoveries for most of the pesticides included in the study within the log K ow in the range from -4 to +5.5. The proposed strategy may be extended to other fields such as sport drug testing or environmental analysis, where the same type of variety of analytes featuring poor retention within a single chromatographic separation occurs. Copyright © 2017 Elsevier B.V. All rights reserved.

Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD)

PubMed Central

Rhoades, Seth D.

2017-01-01

Introduction Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Objective Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). Methods We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. Results LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. Conclusions The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method. PMID:28348510

Determination of linuron and related compounds in soil by microwave-assisted solvent extraction and reversed-phase liquid chromatography with UV detection.

PubMed

Molins, C; Hogendoorn, E A; Dijkman, E; Heusinkveld, H A; Baumann, R A

2000-02-11

The combination of microwave-assisted solvent extraction (MASE) and reversed-phase liquid chromatography (RPLC) with UV detection has been investigated for the efficient determination of phenylurea herbicides in soils involving the single-residue method (SRM) approach (linuron) and the multi-residue method (MRM) approach (monuron, monolinuron, isoproturon, metobromuron, diuron and linuron). Critical parameters of MASE, viz, extraction temperature, water content and extraction solvent were varied in order to optimise recoveries of the analytes while simultaneously minimising co-extraction of soil interferences. The optimised extraction procedure was applied to different types of soil with an organic carbon content of 0.4-16.7%. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. A comparative study between the applicability of RPLC-UV without and with the use of column switching for the processing of uncleaned extracts, was carried out. For some of the tested analyte/matrix combinations the one-column approach (LC mode) is feasible. In comparison to LC, coupled-column LC (LC-LC mode) provides high selectivity in single-residue analysis (linuron) and, although less pronounced in multi-residue analysis (all six phenylurea herbicides), the clean-up performance of LC-LC improves both time of analysis and sample throughput. In the MRM approach the developed procedure involving MASE and LC-LC-UV provided acceptable recoveries (range, 80-120%) and RSDs (<12%) at levels of 10 microg/kg (n=9) and 50 microg/kg (n=7), respectively, for most analyte/matrix combinations. Recoveries from aged residue samples spiked at a level of 100 microg/kg (n=7) ranged, depending of the analyte/soil type combination, from 41-113% with RSDs ranging from 1-35%. In the SRM approach the developed LC-LC procedure was applied for the determination of linuron in 28 sandy soil samples collected in a field study. Linuron could be determined in soil with a limit of quantitation of 10 microg/kg.

Electrochemistry coupled online to liquid chromatography-mass spectrometry for fast simulation of biotransformation reactions of the insecticide chlorpyrifos.

PubMed

Mekonnen, Tessema F; Panne, Ulrich; Koch, Matthias

2017-05-01

An automated method is presented for fast simulation of (bio)transformation products (TPs) of the organophosphate insecticide chlorpyrifos (CPF) based on electrochemistry coupled online to liquid chromatography-mass spectrometry (EC-LC-MS). Oxidative TPs were produced by a boron doped diamond (BDD) electrode, separated by reversed phase HPLC and online detected by electrospray ionization-mass spectrometry (ESI-MS). Furthermore, EC oxidative TPs were investigated by HPLC-tandem mass spectrometry (LC-MS/MS) and FT-ICR high resolution mass spectrometry (HRMS) and compared to in vitro assay metabolites (rat and human liver microsomes). Main phase I metabolites of CPF: chlorpyrifos oxon (CPF oxon), trichloropyridinol (TCP), diethylthiophosphate (DETP), diethylphosphate (DEP), desethyl chlorpyrifos (De-CPF), and desethyl chlorpyrifos oxon (De-CPF oxon), were successfully identified by the developed EC-LC-MS method. The EC-LC-MS method showed similar metabolites compared to the in vitro assay with possibilities of determining reactive species. Our results reveal that online EC-(LC)-MS brings an advantage on time of analysis by eliminating sample preparation steps and matrix complexity compared to conventional in vivo or in vitro methods.

Comprehensive Optimization of LC-MS Metabolomics Methods Using Design of Experiments (COLMeD).

PubMed

Rhoades, Seth D; Weljie, Aalim M

2016-12-01

Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.

Separation and characterization of chemical constituents in Ginkgo biloba extract by off-line hydrophilic interaction×reversed-phase two-dimensional liquid chromatography coupled with quadrupole-time of flight mass spectrometry.

PubMed

Ji, Shuai; He, Dan-Dan; Wang, Tian-Yun; Han, Jie; Li, Zheng; Du, Yan; Zou, Jia-Hui; Guo, Meng-Zhe; Tang, Dao-Quan

2017-11-30

Ginkgo biloba extract (GBE), derived from the leaves of Ginkgo biloba L., is one of the most widely used traditional Chinese medicines worldwide. Due to high structural diversity and low abundance of chemical constituents in GBE, conventional reversed-phase liquid chromatography has limited power to meet the needs of its quality control. In this study, an off-line hydrophilic interaction×reversed-phase two-dimensional liquid chromatography (HILIC×RP 2D-LC) system coupled with diode array detection (DAD) and quadrupole time-of-flight mass spectrometry (qTOF-MS) was established to comprehensively analyze the chemical constituents of GBE. After optimizing the chromatographic columns and mobile phase of 2D-LC, a Waters XBridge Amide column using acetonitrile/water/formic acid as the mobile phase was selected as the first dimension to fractionate GBE, and the obtained fractions were further separated on an Agilent Zorbax XDB-C18 column with methanol/water/formic acid as the mobile phase. As a result, a total of 125 compounds were detected in GBE. The orthogonality of the 2D-LC system was 69.5%, and the practical peak capacity was 3864 and 2994, respectively, calculated by two different methods. The structures of 104 compounds were tentatively characterized by qTOF-MS analysis, and 21 of them were further confirmed by comparing with reference standards. This established HILIC×RP 2D-LC-qTOF/MS system can greatly improve the separation and characterization of natural products in GBE or other complicated herbal extracts. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous determination of bifonazole and tinctures of calendula flower in pharmaceutical creams by reversed-phase liquid chromatography.

PubMed

Ferreyra, Carola F; Ortiz, Cristina S

2005-01-01

The aim of this research was to develop and validate a sensitive, rapid, easy, and precise reversed-phase liquid chromatography (LC) method for stability studies of bifonazole (I) formulated with tinctures of calendula flower (II). The method was especially developed for the analysis and quantitative determination of I and II in pure and combined forms in cream pharmaceutical formulations without using gradient elution and at room temperature. The influence on the stability of compound I of temperature, artificial radiation, and drug II used for the new pharmaceutical design was evaluated. The LC separation was carried out using a Supelcosil LC-18 column (25 cm x 4.6 mm id, 5 microm particle size); the mobile phase was composed of methanol-0.1 M ammonium acetate buffer (85 + 15, v/v) pumped isocratically at a flow rate of 1 mL/min; and ultraviolet detection was at 254 nm. The analysis time was less than 10 min. Calibration graphs were found to be linear in the 0.125-0.375 mg/mL (rI = 0.9991) and 0.639-1.916 mg/mL (rII = 0.9995) ranges for I and II, respectively. The linearity, precision, recovery, and limits of detection and quantification were satisfactory for I and II. The results obtained suggested that the developed LC method is selective and specific for the analysis of I and II in pharmaceutical products, and that it can be applied to stability studies.

Simple method for the extraction and reversed-phase high-performance liquid chromatographic analysis of carotenoid pigments from red yeasts (Basidiomycota, Fungi).

PubMed

Weber, Roland W S; Anke, Heidrun; Davoli, Paolo

2007-03-23

A simple method for the extraction of carotenoid pigments from frozen wet cells of red yeasts (Basidiomycota) and their analysis by reversed-phase HPLC using a C(18) column and a water/acetone solvent system is described. Typical red yeast carotenoids belonging to an oxidative series from the monocyclic gamma-carotene to 2-hydroxytorularhodin and from the bicyclic beta-carotene to astaxanthin were separated. Pigment identity was confirmed by LC-atmospheric pressure chemical ionisation (APCI) mass spectrometry using similar chromatographic conditions.

Porous Graphitic Carbon Liquid Chromatography-Mass Spectrometry Analysis of Drought Stress-Responsive Raffinose Family Oligosaccharides in Plant Tissues.

PubMed

Jorge, Tiago F; Florêncio, Maria H; António, Carla

2017-01-01

Drought is a major limiting factor in agriculture and responsible for dramatic crop yield losses worldwide. The adjustment of the metabolic status via accumulation of drought stress-responsive osmolytes is one of the many strategies that some plants have developed to cope with water deficit conditions. Osmolytes are highly polar compounds, analysis of whcih is difficult with typical reversed-phase chromatography. Porous graphitic carbon (PGC) has shown to be a suitable alternative to reversed-phase stationary phases for the analysis of highly polar compounds typically found in the plant metabolome. In this chapter, we describe the development and validation of a PGC-based liquid chromatography tandem mass spectrometry (LC-MS n ) method suitable for the target analysis of water-soluble carbohydrates, such as raffinose family oligosaccharides (RFOs). We present detailed information regarding PGC column equilibration, LC-MS n system operation, data analysis, and important notes to be considered during the steps of method development and validation.

Fully automated method for the liquid chromatographic determination of cyproterone acetate in plasma using restricted access material for sample pre-treatment.

PubMed

Christiaens, B; Chiap, P; Rbeida, O; Cello, D; Crommen, J; Hubert, Ph

2003-09-25

A new fully automated method for the quantitative analysis of an antiandrogenic substance, cyproterone acetate (CPA), in plasma samples has been developed using on-line solid-phase extraction (SPE) prior to the determination by reversed-phase liquid chromatography (LC). The automated method was based on the use of a precolumn packed with an internal-surface reversed-phase packing material (LiChrospher RP-4 ADS) for sample clean-up coupled to LC analysis on an octadecyl stationary phase using a column-switching system. A 200-microL volume of plasma sample was injected directly on the precolumn packed with restricted access material using a mixture of water-acetonitrile (90:10, v/v) as washing liquid. The analyte was then eluted in the back-flush mode with the LC mobile phase which consisted of a mixture of phosphate buffer, pH 7.0-acetonitrile (54:46, v/v). The elution profiles of CPA and blank plasma samples on the precolumn and the time needed for analyte transfer from the precolumn to the analytical column were determined. Different compositions of washing liquid and mobile phase were tested to reduce the interference of plasma endogenous components. UV detection was achieved at 280 nm. Finally, the developed method was validated using a new approach, namely the application of the accuracy profile based on the interval confidence at 90% of the total measurement error (bias+standard deviation). The limit of quantification of cyproterone acetate in plasma was determined at 15 ng mL(-1). The validated method should be applicable to the determination of CPA in patients treated by at least 50 mg day(-1).

Limitation of predictive 2-D liquid chromatography in reducing the database search space in shotgun proteomics: in silico studies.

PubMed

Moskovets, Eugene; Goloborodko, Anton A; Gorshkov, Alexander V; Gorshkov, Mikhail V

2012-07-01

A two-dimensional (2-D) liquid chromatography (LC) separation of complex peptide mixtures that combines a normal phase utilizing hydrophilic interactions and a reversed phase offers reportedly the highest level of 2-D LC orthogonality by providing an even spread of peptides across multiple LC fractions. Matching experimental peptide retention times to those predicted by empirical models describing chromatographic separation in each LC dimension leads to a significant reduction in a database search space. In this work, we calculated the retention times of tryptic peptides separated in the C18 reversed phase at different separation conditions (pH 2 and pH 10) and in TSK gel Amide-80 normal phase. We show that retention times calculated for different 2-D LC separation schemes utilizing these phases start to correlate once the mass range of peptides under analysis becomes progressively narrow. This effect is explained by high degree of correlation between retention coefficients in the considered phases. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction.

PubMed

Behrens, Beate; Engelen, Jeannine; Tiso, Till; Blank, Lars Mathias; Hayen, Heiko

2016-04-01

Rhamnolipids are surface-active agents with a broad application potential that are produced in complex mixtures by bacteria of the genus Pseudomonas. Analysis from fermentation broth is often characterized by laborious sample preparation and requires hyphenated analytical techniques like liquid chromatography coupled to mass spectrometry (LC-MS) to obtain detailed information about sample composition. In this study, an analytical procedure based on chromatographic method development and characterization of rhamnolipid sample material by LC-MS as well as a comparison of two sample preparation methods, i.e., liquid-liquid extraction and solid-phase extraction, is presented. Efficient separation was achieved under reversed-phase conditions using a mixed propylphenyl and octadecylsilyl-modified silica gel stationary phase. LC-MS/MS analysis of a supernatant from Pseudomonas putida strain KT2440 pVLT33_rhlABC grown on glucose as sole carbon source and purified by solid-phase extraction revealed a total of 20 congeners of di-rhamnolipids, mono-rhamnolipids, and their biosynthetic precursors 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs) with different carbon chain lengths from C8 to C14, including three rhamnolipids with uncommon C9 and C11 fatty acid residues. LC-MS and the orcinol assay were used to evaluate the developed solid-phase extraction method in comparison with the established liquid-liquid extraction. Solid-phase extraction exhibited higher yields and reproducibility as well as lower experimental effort.

Investigating the Retention Mechanisms of Liquid Chromatography Using Solid-Phase Extraction Cartridges

ERIC Educational Resources Information Center

O'Donnell, Mary E.; Musial, Beata A.; Bretz, Stacey Lowery; Danielson, Neil D.; Ca, Diep

2009-01-01

Liquid chromatography (LC) experiments for the undergraduate analytical laboratory course often illustrate the application of reversed-phase LC to solve a separation problem, but rarely compare LC retention mechanisms. In addition, a high-performance liquid chromatography instrument may be beyond what some small colleges can purchase. Solid-phase…

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography.

PubMed

Vanhoenacker, Gerd; Dos Santos Pereira, Alberto; Kotsuka, Takashi; Cabooter, Deirdre; Desmet, Gert; Sandra, Pat

2010-05-07

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height (h) decreased from 3.4 at 25 degrees C (optimal flow 0.5 mL/min) to 2.4 at 150 degrees C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 degrees C within the pH range 1-9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC-MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol. Copyright (c) 2009 Elsevier B.V. All rights reserved.

CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

EPA Science Inventory

A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

A RP-LC method with evaporative light scattering detection for the assay of simethicone in pharmaceutical formulations.

PubMed

Moore, Douglas E; Liu, Tina X; Miao, William G; Edwards, Alison; Elliss, Russell

2002-09-05

A reversed-phase liquid chromatographic method has been developed and validated for the determination of the polydimethylsiloxane (PDMS) component of Simethicone, which is used as an anti-foaming agent in pharmaceutical formulations. The method involves acidification to neutralise antacid components of the formulation, then a single extraction of the PDMS with dichloromethane. This is followed by separation with a reversed-phase column using an acetonitrile-chloroform solvent gradient, and quantification by an evaporative light scattering detector. An assay precision of 3% was achieved in intraday and interday determinations. No interference was found from the aluminium and magnesium hydroxide components of antacid formulations.

Analysis of triacylglycerols on porous graphitic carbon by high temperature liquid chromatography.

PubMed

Merelli, Bérangère; De Person, Marine; Favetta, Patrick; Lafosse, Michel

2007-07-20

The retention behaviour of several triacylglycerols (TAGs) and fats on Hypercarb, a porous graphitic carbon column (PGC), was investigated in liquid chromatography (LC) under isocratic elution mode with an evaporative light scattering detector (ELSD). Mixtures of chloroform/isopropanol were selected as mobile phase for a suitable retention time to study the influence of temperature. The retention was different between PGC and non-aqueous reversed phase liquid chromatography (NARP-LC) on octadecyl phase. The retention of TAGs was investigated in the interval 30-70 degrees C. Retention was greatly affected by temperature: it decreases as the column temperature increases. Selectivity of TAGs was also slightly influenced by the temperature. Moreover, this chromatographic method is compatible with a mass spectrometer (MS) detector by using atmospheric pressure chemical ionisation (APCI): same fingerprints of cocoa butter and shea butter were obtained with LC-ELSD and LC-APCI-MS. These preliminary results showed that the PGC column could be suitable to separate quickly triacylglycerols in high temperature conditions coupled with ELSD or MS detector.

Development and validation of LC methods for the separation of misoprostol related substances and diastereoisomers.

PubMed

Kahsay, Getu; Song, Huiying; Eerdekens, Fran; Tie, Yaxin; Hendriks, Danny; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

2015-01-01

Misoprostol is a synthetic prostaglandin E1 analogue which is mainly used for prevention and treatment of gastric ulcers, but also for abortion due to its labour inducing effect. Misoprostol exists as a mixture of diastereoisomers (1:1) and has several related impurities owing to its instability at higher temperatures and moisture. A simple and robust reversed phase liquid chromatographic (RPLC) method is described for the separation of the related substances and a normal phase (NP) LC method for the separation of misoprostol diastereoisomers. The RPLC method was performed using an Ascentis Express C18 (150 mm × 4.6 mm, 5 μm) column kept at 35 °C. The mobile phase was a gradient mixture of mobile phase A (ACN-H2O-MeOH, 28:69:3 v/v/v) and mobile phase B (ACN-H2O-MeOH, 47:50:3 v/v/v) eluted at a flow rate of 1.5 mL/min. UV detection was performed at 200 nm. The NPLC method was undertaken by using an XBridge bare silica (150 mm × 2.1 mm, 3.5 μm) column at 35 °C. The mobile phase contained 1-propanol-heptane-TFA (4:96:0.1%, v/v/v), pumped at a flow rate of 0.5 mL/min. UV detection was performed at 205 nm. This LC method can properly separate the two diastereoisomers (Rs > 2) within an analysis time of less than 20 min. Both methods were validated according to the ICH guidelines. Furthermore, these new LC methods have been successfully applied for purity control and diastereoisomers ratio determination of misoprostol bulk drug, tablets and dispersion. Copyright © 2015 Elsevier B.V. All rights reserved.

Nanomaterials as stationary phases and supports in liquid chromatography.

PubMed

Beeram, Sandya R; Rodriguez, Elliott; Doddavenkatanna, Suresh; Li, Zhao; Pekarek, Allegra; Peev, Darin; Goerl, Kathryn; Trovato, Gianfranco; Hofmann, Tino; Hage, David S

2017-10-01

The development of various nanomaterials over the last few decades has led to many applications for these materials in liquid chromatography (LC). This review will look at the types of nanomaterials that have been incorporated into LC systems and the applications that have been explored for such systems. A number of carbon-based nanomaterials and inorganic nanomaterials have been considered for use in LC, ranging from carbon nanotubes, fullerenes and nanodiamonds to metal nanoparticles and nanostructures based on silica, alumina, zirconia and titanium dioxide. Many ways have been described for incorporating these nanomaterials into LC systems. These methods have included covalent immobilization, adsorption, entrapment, and the synthesis or direct development of nanomaterials as part of a chromatographic support. Nanomaterials have been used in many types of LC. These applications have included the reversed-phase, normal-phase, ion-exchange, and affinity modes of LC, as well as related methods such as chiral separations, ion-pair chromatography and hydrophilic interaction liquid chromatography. Both small and large analytes (e.g., dyes, drugs, amino acids, peptides and proteins) have been used to evaluate possible applications for these nanomaterial-based methods. The use of nanomaterials in columns, capillaries and planar chromatography has been considered as part of these efforts. Potential advantages of nanomaterials in these applications have included their good chemical and physical stabilities, the variety of interactions many nanomaterials can have with analytes, and their unique retention properties in some separation formats. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS.

PubMed

Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J

2013-01-01

In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV < 13%). Sixteen dietary supplements were tested with the developed methods. Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

Method for the Determination of Aconitum Alkaloids in Dietary Supplements and Raw Materials by Reversed-Phase Liquid Chromatography with Ultraviolet Detection and Confirmation by Tandem Mass Spectrometry: Single-Laboratory Validation

PubMed Central

Tang, Wai-Tong; Wong, Sui-Kay; Law, Tin-Yau; Pang, Kwok-Chu; Sin, Della; Tam, Yin-King

2008-01-01

A study of single-laboratory validation (SLV) of a reversed-phase liquid Chromatography (RP-LC) method was conducted for the determination of diester-diterpene Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in a variety of dietary supplements, including single-arid multiple-ingredient dry powder extracts, pills, capsules, and raw materials. The Aconitum alkaloids in the samples were extracted by diethyl ether in the presence of ammonia. After cleanup with solid-phase extraction to remove the matrix interferences, the alkaloids were determined by RP-LC with UV detection at 235 nm, and the results were confirmed by tandem mass Spectrometry. The linear responses for aconitine, mesaconitine, and hypaconitine based on the present LC system ranged from 0.5 to 200 μg/mL. Relative standard deviations of 2.0 to 6.9% were obtained from duplicate analysis of 6 test materials of different matrixes for the 3 Aconitum alkaloids performed by 2 analysts on 5 different days. The recoveries determined for supplements and raw materials spiked with 3 Aconitum alkaloids at levels of 2.5–10 μg/g were in the range of 86–99%. In view of the attainment of satisfactory results for accuracy, precision, and recovery in the SLV study, it is recommended that the method validation process proceed to a collaborative study. PMID:17225594

Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping.

PubMed

Eckard, Anahita D; Dupont, David R; Young, Johnie K

2018-01-01

N -lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N -glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography-mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N -glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N -glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of <5% for peaks with >0.5% relative area.

Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatography-mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides.

PubMed

Eeltink, Sebastiaan; Wouters, Sam; Dores-Sousa, José Luís; Svec, Frantisek

2017-05-19

This review focuses on the preparation of organic polymer-based monolithic stationary phases and their application in the separation of biomolecules, including antibodies, intact proteins and protein isoforms, oligonucleotides, and protein digests. Column and material properties, and the optimization of the macropore structure towards kinetic performance are also discussed. State-of-the-art liquid chromatography-mass spectrometry biomolecule separations are reviewed and practical aspects such as ion-pairing agent selection and carryover are presented. Finally, advances in comprehensive two-dimensional LC separations using monolithic columns, in particular ion-exchange×reversed-phase and reversed-phase×reversed-phase LC separations conducted at high and low pH, are shown. Copyright © 2017 Elsevier B.V. All rights reserved.

Semi-permeable surface analytical reversed-phase column for the improved trace analysis of acidic pesticides in water with coupled-column reversed-phase liquid chromatography with UV detection. Determination of bromoxynil and bentazone in surface water.

PubMed

Hogendoorn, E A; Westhuis, K; Dijkman, E; Heusinkveld, H A; den Boer, A C; Evers, E A; Baumann, R A

1999-10-08

The coupled-column (LC-LC) configuration consisting of a 3 microm C18 column (50 x 4.6 mm I.D.) as the first column and a 5 microm C18 semi-permeable-surface (SPS) column (150 x 4.6 mm I.D.) as the second column appeared to be successful for the screening of acidic pesticides in surface water samples. In comparison to LC-LC employing two C18 columns, the combination of C18/SPS-C18 significantly decreased the baseline deviation caused by the hump of the co-extracted humic substances when using UV detection (217 nm). The developed LC-LC procedure allowed the simultaneous determination of the target analytes bentazone and bromoxynil in uncleaned extracts of surface water samples to a level of 0.05 microg/l in less than 15 min. In combination with a simple solid-phase extraction step (200 ml of water on a 500 mg C18-bonded silica) the analytical procedure provides a high sample throughput. During a period of about five months more than 200 ditch-water samples originating from agricultural locations were analyzed with the developed procedure. Validation of the method was performed by randomly analyzing recoveries of water samples spiked at levels of 0.1 microg/l (n=10), 0.5 microg/l (n=7) and 2.5 microg/l (n=4). Weighted regression of the recovery data showed that the method provides overall recoveries of 95 and 100% for bentazone and bromoxynil, respectively, with corresponding intra-laboratory reproducibilities of 10 and 11%, respectively. Confirmation of the analytes in part of the samples extracts was carried out with GC-negative ion chemical ionization MS involving a derivatization step with bis(trifluoromethyl)benzyl bromide. No false negatives or positives were observed.

Identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized LC-DAD-ESI/MS profiling method.

PubMed

Lin, Long-Ze; Harnly, James M

2008-11-12

A screening method using LC-DAD-ESI/MS was developed for the identification of common hydroxycinnamoylquinic acids based on direct comparison with standards. A complete standard set for mono-, di-, and tricaffeoylquinic isomers was assembled from commercially available standards, positively identified compounds in common plants (artichokes, asparagus, coffee bean, honeysuckle flowers, sweet potato, and Vernonia amygdalina leaves) and chemically modified standards. Four C18 reversed phase columns were tested using the standardized profiling method (based on LC-DAD-ESI/MS) for 30 phenolic compounds, and their elution order and retention times were evaluated. Using only two columns under standardized LC condition and the collected phenolic compound database, it was possible to separate all of the hydroxycinnamoylquinic acid conjugates and to identify 28 and 18 hydroxycinnamoylquinic acids in arnica flowers (Arnica montana L.) and burdock roots (Arctium lappa L.), respectively. Of these, 22 are reported for the first time.

Identification and measurement of beta-lactam antibiotic residues in milk: integration of screening kits with liquid chromatography.

PubMed

Harik-Khan, R; Moats, W A

1995-01-01

A procedure for identifying and quantitating violative beta-lactams in milk is described. This procedure integrates beta-lactam residue detection kits with the multiresidue automated liquid chromatographic (LC) cleanup method developed in our laboratory. Spiked milk was deproteinized, extracted, and subjected to reversed-phase LC using a gradient program that concentrated the beta-lactams. Amoxicillin, ampicillin, cephapirin, ceftiofur, cloxacillin, and penicillin G were, thus, separated into 5 fractions that were subsequently tested for activity by using 4 kits. beta-lactams in the positive fractions were quantitated by analytical LC methods developed in our laboratory. The LC cleanup method separated beta-lactam antibiotics from each other and from interferences in the matrix and also concentrated the antibiotics, thus increasing the sensitivity of the kits to the beta-lactam antibiotics. The procedure facilitated the task of identifying and measuring the beta-lactam antibiotics that may be present in milk samples.

Microwave assisted solvent extraction and coupled-column reversed-phase liquid chromatography with UV detection use of an analytical restricted-access-medium column for the efficient multi-residue analysis of acidic pesticides in soils.

PubMed

Hogendoom, E A; Huls, R; Dijkman, E; Hoogerbrugge, R

2001-12-14

A screening method has been developed for the determination of acidic pesticides in various types of soils. Methodology is based on the use of microwave assisted solvent extraction (MASE) for fast and efficient extraction of the analytes from the soils and coupled-column reversed-phase liquid chromatography (LC-LC) with UV detection at 228 nm for the instrumental analysis of uncleaned extracts. Four types of soils, including sand, clay and peat, with a range in organic matter content of 0.3-13% and ten acidic pesticides of different chemical families (bentazone, bromoxynil, metsulfuron-methyl, 2,4-D, MCPA, MCPP, 2,4-DP, 2,4,5-T, 2,4-DB and MCPB) were selected as matrices and analytes, respectively. The method developed included the selection of suitable MASE and LC-LC conditions. The latter consisted of the selection of a 5-microm GFF-II internal surface reversed-phase (ISRP, Pinkerton) analytical column (50 x 4.6 mm, I.D.) as the first column in the RAM-C18 configuration in combination with an optimised linear gradient elution including on-line cleanup of sample extracts and reconditioning of the columns. The method was validated with the analysis of freshly spiked samples and samples with aged residues (120 days). The four types of soils were spiked with the ten acidic pesticides at levels between 20 and 200 microg/kg. Weighted regression of the recovery data showed for most analyte-matrix combinations, including freshly spiked samples and aged residues, that the method provides overall recoveries between 60 and 90% with relative standard deviations of the intra-laboratory reproducibility's between 5 and 25%; LODs were obtained between 5 and 50 microg/kg. Evaluation of the data set with principal component analysis revealed that the parameters (i) increase of organic matter content of the soil samples and (ii) aged residues negatively effect the recovery of the analytes.

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF

PubMed Central

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D.; Yanjanin, Nicole M.; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S.; Jiang, Xuntian

2014-01-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and “dilute and shoot” procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. PMID:24868096

Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF.

PubMed

Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian

2014-07-01

2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Measurement of bromate in bread by liquid chromatography with post-column flow reactor detection.

PubMed

Himata, K; Noda, M; Ando, S; Yamada, Y

2000-01-01

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30,000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10-52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.

Development of high-throughput multi-residue method for non-steroidal anti-inflammatory drugs monitoring in swine muscle by LC-MS/MS.

PubMed

Castilhos, Tamara S; Barreto, Fabiano; Meneghini, Leonardo; Bergold, Ana Maria

2016-07-01

A reliable and simple method for the detection and quantification of residues of 14 non-steroidal anti-inflammatory drugs and a metamizole metabolite in swine muscle was developed using liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). The samples were extracted with acetonitrile (ACN) in solid-liquid extraction followed by a low-temperature partitioning (LLE-LTP) process at -20 ± 2°C. After evaporation to dryness, the residue was reconstituted with hexane and a mixture of water:acetonitrile (1:1). LC separation was achieved on a reversed-phase (RP18) column with gradient elution using water (phase A) and ACN (phase B) both containing 1 mmol l(-)(1) ammonium acetate (NH4COO) with 0.025% acetic acid. Analysis was carried out on a triple-quadrupole tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode using an electrospray interface in negative and positive mode in a single run. Method validation was performed according to the criteria of Commission Decision No. 2002/657/EC. The matrix effect and linearity were evaluated. Decision limit (CCα), detection capability (CCβ), accuracy and repeatability of the method are also reported. The proposed method proved to be simple, easy and adequate for high-throughput analysis and was applied to routine analysis by the Brazilian Ministry of Agriculture, Livestock and Food Supply.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

NASA Astrophysics Data System (ADS)

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-12-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS

PubMed Central

Sitnikov, Dmitri G.; Monnin, Cian S.; Vuckovic, Dajana

2016-01-01

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34–80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics. PMID:28000704

Photo-switchable membrane and method

DOEpatents

Marshall, Kenneth L; Glowacki, Eric

2013-05-07

Switchable gas permeation membranes in which a photo-switchable low-molecular-weight liquid crystalline (LC) material acts as the active element, and a method of making such membranes. Different LC eutectic mixtures were doped with mesogenic azo dyes and infused into track-etched porous membranes with regular cylindrical pores. Photo-induced isothermal phase changes in the imbibed mesogenic material afforded large, reversible changes in the permeability of the photo-switchable membrane to nitrogen. For example, membranes imbibed with a photo-switchable cyanobiphenyl LC material demonstrated low permeability in the nematic state, while the photo-generated isotropic state demonstrated a 16.times.-greater sorption coefficient. Both states obey a high linear sorption behavior in accordance with Henry's Law. In contrast, membranes imbibed with a photo-switchable phenyl benzoate LC material showed the opposite permeability behavior to the biphenyl-imbibed membrane, along with nonlinear sorption behavior.

Development of a solid-phase extraction method for determination of pheophorbide a and pyropheophorbide a in health foods by liquid chromatography.

PubMed

Oshima, Harumi; Ueno, Eiji; Saito, Isao; Matsumoto, Hiroshi

2004-01-01

A simple solid-phase extraction (SPE) method was developed for the liquid chromatography (LC) determination of pheophorbide (Phor) a and pyropheophorbide (Pyro) a in health foods such as chlorella, spirulina, etc. The food sample was extracted with 85% (v/v) acetone. The extract was acidified with hydrochloric acid and loaded on a C18 cartridge. After washing with water, Phor a and Pyro a were eluted with the LC mobile phase. Phor a and Pyro a were separated by isocratic reversed-phase LC and quantitated by fluorescence detection. The recoveries for spiked samples of chlorella and the extract were 87.1-102.0%. Commercial health foods (chlorella, spirulina, aloe, kale, Jews mallow, and green tea leaves) were analyzed using the SPE method. The values found for Phor a and Pyro a ranged from 2 to 788 microg/g and from <1 to 24 microg/g, respectively. There was no significant difference between the SPE method and the official method in Japan (spectrophotometry after liquid-liquid extraction). The advantages of the SPE method are the short extraction times, lack of emulsions, and reduced consumption of organic solvents compared with the official method in Japan. The SPE method is considered to be useful for the screening of Phor a and Pyro a in health foods.

An Improved LC-MS/MS Method for Simultaneous Determination of the Eleven Bioactive Constituents for Quality Control of Radix Angelicae Pubescentis and Its Related Preparations

PubMed Central

Li, Jin; Zhang, Qiu-Hong; He, Jun; Liu, Er-wei; Gao, Xiu-mei; Chang, Yan-xu

2015-01-01

An improved LC-MS/MS method was developed for simultaneous determination of eleven bioactive constituents of Radix Angelicae Pubescentis and its related preparations. It was the first report on the quantification of bioactive constituents in different preparations of Radix Angelicae Pubescentis by LC-MS/MS analytical method. These samples were separated with an Agilent Zorbax Extend reversed-phase C18 column (1.8 μm, 4.6 × 100 mm) by linear gradient elution using aqueous ammonium acetate and acetonitrile as mobile phase. The flow rate was 0.3 mL min−1. The eleven bioactive constituents showed good regression (R > 0.990) within test ranges and the recoveries were in the range of 87.1–110%. The limit of detections and quantifications for most of the major constituents were less than 0.5 and 1.0 ng mL−1, respectively. All results indicated that the developed method could be readily utilized as a suitable quality control method for Radix Angelicae Pubescentis and related preparations. PMID:26078992

CHARACTERIZATION OF DANSYLATED CYSTEINE, CYSTINE, GLUTATHIONE, AND GLUTATHIONE DISULFIDE BY NARROW BORE LIQUID CHROMATOGRAPHY - ELECTROSPRAY IONIZATION MASS SPECTROMETRY

EPA Science Inventory

A method using reversed phase high performance liquid chromtography/electrospray ionization-mass spectrometry (RP-LC/ESI-MS) has been developed to confirm the dientity of dansylated derivatives of cysteine (C) and glutathione (GSH), and their respective dimers, cystine (CSSC) and...

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip.

PubMed

Yin, Hongfeng; Killeen, Kevin; Brennen, Reid; Sobek, Dan; Werlich, Mark; van de Goor, Tom

2005-01-15

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.

A LC/UV/Vis method for determination of cyanocobalamin in multivitamin dietary supplements with on-line sample clean-up

USDA-ARS?s Scientific Manuscript database

A HPLC-UV method using a two-column strategy with a switching valve for on-line sample clean-up was developed for the determination of cyanocobalamin (CN-CBL-vitamin B12, in dietary supplements. The method uses two columns, an Agilent Zorbax C8 (150 mm x 4.6 mm, 5 um particle) reversed-phase column...

Reduction of Solvent Effect in Reverse Phase Gradient Elution LC-ICP-MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sullivan, Patrick Allen

2005-12-17

Quantification in liquid chromatography (LC) is becoming very important as more researchers are using LC, not as an analytical tool itself, but as a sample introduction system for other analytical instruments. The ability of LC instrumentation to quickly separate a wide variety of compounds makes it ideal for analysis of complex mixtures. For elemental speciation, LC is joined with inductively coupled plasma mass spectrometry (ICP-MS) to separate and detect metal-containing, organic compounds in complex mixtures, such as biological samples. Often, the solvent gradients required to perform complex separations will cause matrix effects within the plasma. This limits the sensitivity ofmore » the ICP-MS and the quantification methods available for use in such analyses. Traditionally, isotope dilution has been the method of choice for LC-ICP-MS quantification. The use of naturally abundant isotopes of a single element in quantification corrects for most of the effects that LC solvent gradients produce within the plasma. However, not all elements of interest in speciation studies have multiple naturally occurring isotopes; and polyatomic interferences for a given isotope can develop within the plasma, depending on the solvent matrix. This is the case for reverse phase LC separations, where increasing amounts of organic solvent are required. For such separations, an alternative to isotope dilution for quantification would be is needed. To this end, a new method was developed using the Apex-Q desolvation system (ESI, Omaha, NE) to couple LC instrumentation with an ICP-MS device. The desolvation power of the system allowed greater concentrations of methanol to be introduced to the plasma prior to destabilization than with direct methanol injection into the plasma. Studies were performed, using simulated and actual linear methanol gradients, to find analyte-internal standard (AIS) pairs whose ratio remains consistent (deviations {+-} 10%) over methanol concentration ranges of 5%-35% (simulated) and 8%-32% (actual). Quadrupole (low resolution) and sector field (high resolution) ICP-MS instrumentation were utilized in these studies. Once an AIS pair is determined, quantification studies can be performed. First, an analysis is performed by adding both elements of the AIS pair post-column while performing the gradient elution without sample injection. A comparison of the ratio of the measured intensities to the atomic ratio of the two standards is used to determine a correction factor that can be used to account for the matrix effects caused by the mobile phase. Then, organic and/or biological molecules containing one of the two elements in the AIS pair are injected into the LC column. A gradient method is used to vary the methanol-water mixture in the mobile phase and to separate out the compounds in a given sample. A standard solution of the second ion in the AIS pair is added continuously post-column. By comparing the ratio of the measured intensities to the atomic ratio of the eluting compound and internal standard, the concentration of the injected compound can be determined.« less

Determination of D-malic acid in apple juice by liquid chromatography: collaborative study.

PubMed

Eisele, T A

1996-01-01

Eleven laboratories collaboratively studied a liquid chromatographic (LC) method for determination of D-malic acid in apple juice. The mobile phase consisted of mM L-valine and 8 mM copper acetate adjusted to pH 5.5 with NaOH. The UV detector was set at 330 nm, and a single reversed-phase LC column was used. Seven paired samples containing various amounts of D-malic acid ranging from 0 to 188 mg/100 mL of 12 Brix pasteurized apple juice were tested by each collaborator. Repeatability and reproducibility coefficients of variation ranged from 1.0 to 3.5% and 7.7 to 11.7%, respectively, within the range of 26 to 188 mg D-malic acid/100 mL of 12 Brix apple juice. The collaborative study results demonstrated that the method could quantitate the economic adulteration of apple juice with DL-malic acid at lower levels than those reported with previous methods. The LC method for determination of D-malic acid in apple juice has been adopted first action by AOAC INTERNATIONAL.

Determination of capsaicinoids in topical cream by liquid-liquid extraction and liquid chromatography.

PubMed

Kaale, Eliangiringa; Van Schepdael, Ann; Roets, Eugène; Hoogmartens, Jos

2002-11-07

A reversed-phase liquid chromatography (LC) method has been developed, optimised and validated for the separation and quantitation of capsaicin (CP) and dihydrocapsaicin (DHCP) in a topical cream formulation. Sample preparation involves liquid-liquid extraction prior to LC analysis. The method uses a Hypersil C(18) BDS, 5 micrometer, 250x4.6 mm I.D. column maintained at 35 degrees C. The mobile phase comprises methanol, water, acetonitrile (ACN) and acetic acid (47:42:10:1, v/v/v/v) at a flow rate of 1.0 ml/min. Robustness was evaluated by performing a central composite face-centred design (CCF) experiment. The method shows good selectivity, linearity, sensitivity and repeatability. The conditions allow the separation and quantitation of CP and DHCP without interference from the other substances contained in the cream.

Size-Sorting Combined with Improved Nanocapillary-LC-MS for Identification of Intact Proteins up to 80 kDa

PubMed Central

Vellaichamy, Adaikkalam; Tran, John C.; Catherman, Adam D.; Lee, Ji Eun; Kellie, John F.; Sweet, Steve M.M.; Zamdborg, Leonid; Thomas, Paul M.; Ahlf, Dorothy R.; Durbin, Kenneth R.; Valaskovic, Gary A.; Kelleher, Neil L.

2010-01-01

Despite the availability of ultra-high resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for on-line LC-MS to drive high-throughput top-down proteomics in a fashion similar to bottom-up. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary-LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier-Transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation (NSD) and detection of fragment ions with <5 ppm mass accuracy for highly-specific database searching using custom software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines pre-fractionated by their molecular weight using a gel-based sieving system. PMID:20073486

Development and Validation of Eco-Friendly Liquid Chromatographic and Spectrophotometric Methods for Simultaneous Determination of Coformulated Drugs: Phenylephrine Hydrochloride and Prednisolone Acetate.

PubMed

Mostafa, Nadia M; Elsayed, Ghada M; Hassan, Nagiba Y; El Mously, Dina A

2017-11-01

Five simple, sensitive, and eco-friendly LC and UV spectrophotometric methods have been developed for the simultaneous determination of phenylephrine hydrochloride (PHE) and prednisolone acetate (PRD) in their combined dosage form. The first method was reversed-phase (RP) LC using methanol-water-heptane-1-sulfonic acid sodium salt (75 + 25 + 0.1, v/v/w) as a mobile phase. Separation was achieved using an XSelect HSS reversed-phase C18 analytical column (250 × 4.6 mm, 5µm). The flow rate was 1.0 mL/min and UV detection was done at 230 nm. Quantification was achieved over the concentration ranges of 5-50 µg/mL for PHE and 2-90 µg/mL for PRD. Four spectrophotometric methods were proposed, namely dual wavelength, first derivative of ratio spectra, ratio difference, and mean-centering of ratio spectra. Linearity was observed in the concentration ranges of 10-120 and 5-35 µg/mL for PHE and PRD, respectively, for the spectrophotometric methods. Green solvents were used in the proposed methods because they play a vital role in the analytical methods' influence on the environment. The suggested methods were validated regarding linearity, accuracy, and precision according to the International Conference on Harmonization guidelines, with satisfactory results. These methods could be used as harmless substitutes for routine analysis of the mentioned drugs, with no interference from excipients.

Comparison of various liquid chromatographic methods involving UV and atmospheric pressure chemical ionization mass spectrometric detection for the efficient trace analysis of phenylurea herbicides in various types of water samples.

PubMed

van der Heeft, E; Dijkman, E; Baumann, R A; Hogendoorn, E A

2000-05-19

The performance of mass spectrometric (MS) detection and UV detection in combination with reversed-phase liquid chromatography without and with the use of coupled column RPLC (LC-LC) has been compared for the trace analysis of phenylurea herbicides in environmental waters. The selected samples of this comparative study originated from an inter-laboratory study. For both detection modes, a 50 mm x 4.6 mm I.D. column and a 100 mm x 4.6 mm I.D. column packed with 3 microm C18 were used as the first (C-1) and second (C-2) column, respectively. Atmospheric pressure chemical ionization mass spectrometry was performed on a magnetic sector instrument. The LC-LC-MS analysis was carried out on-line by means of direct large volume (11.7 ml) injection (LVI). The performance of both on-line (LVI, 4 ml of sample) and off-line LC-LC-UV (244 nm) analysis was investigated. The latter procedure consisted of a solid-phase extraction (SPE) of 250 ml of water sample on a 500 mg C18 cartridge. The comparative study showed that LC-LC-MS is more selective then LC-LC-UV and, in most cases, more sensitive. The LVI-LC-LC-MS approach combines direct quantification and confirmation of most of the analytes down to a level of 0.01 microg/l in water samples in less then 30 min. As regards LC-LC-UV, the off-line method appeared to be a more viable approach in comparison with the on-line procedure. This method allows the screening of phenylurea's in various types of water samples down to a level of at least 0.05 microg/l. On-line analysis with LVI provided marginal sensitivity (limits of detection of about 0.1 microg/l) and selectivity was sometimes less in case of surface water samples. Both the on-line LVI-LC-LC-MS method and the LC-LC-UV method using off-line SPE were validated by analysing a series of real-life reference samples. These samples were part of an inter-laboratory test and contained residues of herbicides ranging from 0.02 to 0.8 microg/l. Beside good correlation between the methods the data agreed very well with the true values of the samples.

Determination of acetone in saliva by reversed-phase liquid chromatography with fluorescence detection and the monitoring of diabetes mellitus patients with ketoacidosis.

PubMed

Fujii, Shinya; Maeda, Toshio; Noge, Ichiro; Kitagawa, Yutaka; Todoroki, Kenichiro; Inoue, Koichi; Min, Jun Zhe; Toyo'oka, Toshimasa

2014-03-20

In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and β-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis. 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of phenoxyacid herbicides and their phenolic metabolites in surface and drinking water.

PubMed

Marchese, Stefano; Perret, Daniela; Gentili, Alessandra; D'Ascenzo, Guiseppe; Faberi, Angelo

2002-01-01

An evaluation was made of the feasibility of using reversed-phase liquid chromatography/tandem mass spectrometry with an electrospray interface (LC/ESI-MS/MS) to measure traces of phenoxyacid herbicides and their metabolites in surface and drinking water samples. The procedure involved passing 0.5 L of river and drinking water samples through a 0.5 g graphitized carbon black (GCB) extraction cartridge. Recovery was higher than 85% irrespective of the aqueous matrix in which the analytes were dissolved. A conventional 4.6-mm i.d. reversed-phase LC C-18 column operating with a mobile phase flow rate of 1 mL/min was used to chromatograph the analytes. A flow of 200 microL/min of the column effluent was diverted to the ESI source. The limits of detection (signal-to-noise ratio = 3) of the method for the pesticides considered in drinking and surface water samples are less than 0.1 ng/L for phenoxyacid herbicides, and about 5-10 ng/L for their metabolites (2,4-dichlorophenol and 4-chloro-2-methylphenol). Copyright 2001 John Wiley & Sons, Ltd.

Determination of artificial sweeteners in water samples by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Ordóñez, Edgar Y; Quintana, José Benito; Rodil, Rosario; Cela, Rafael

2012-09-21

The development and performance evaluation of an analytical method for the determination of six artificial sweeteners in environmental waters using solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry are presented. To this end, different SPE alternatives have been evaluated: polymeric reversed-phase (Oasis HLB, Env+, Plexa and Strata X), and mixed-mode with either weak (Oasis WAX) or strong anionic-exchange (Oasis MAX and Plexa PAX) sorbents. Among them, reversed-phase sorbents, particularly Oasis HLB and Strata X, showed the best performance. Oasis HLB provided good trueness (recoveries: 73-112%), precision (RSD<10%) and limits of quantification (LOQ: 0.01-0.5 μg/L). Moreover, two LC separation mechanisms were evaluated: reversed-phase (RPLC) and hydrophilic interaction (HILIC), with RPLC providing better performance than HILIC. The final application of the method showed the presence of acesulfame, cyclamate, saccharin and sucralose in the wastewater and surface water samples analyzed at concentrations up to 54 μg/L. Copyright © 2012 Elsevier B.V. All rights reserved.

Methylammonium formate as a mobile phase modifier for reversed-phase liquid chromatography

PubMed Central

Grossman, Shau; Danielson, Neil D.

2009-01-01

Although alkylammonium ionic liquids such as ethylammonium nitrate and ethylammonium formate have been used as mobile phase “solvents” for liquid chromatography (LC), we have shown that methylammonium formate (MAF), in part because of its lower viscosity, can be an effective replacement for methanol (MeOH) in reversed-phase LC. Plots of log retention factor versus the fraction of MeOH and MAF in the mobile phase indicate quite comparable solvent strength slope values of 2.50 and 2.05, respectively. Using a polar endcapped C18 column, furazolidone and nitrofurantoin using 20% MAF-80% water could be separated in 22 min but no baseline separation was possible using MeOH as the modifier, even down to 10%. Suppression of silanol peak broadening effects by MAF is important permitting a baseline separation of pyridoxine, thiamine, and nicotinamide using 5% MAF-95% water at 0.7 mL/min. Using 5% MeOH-95% water, severe peak broadening for thiamine is evident. The compatibility of MAF as a mobile phase modifer for LC with mass spectrometry detection of water soluble vitamins is also shown. PMID:18849044

LC-MS n Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

NASA Astrophysics Data System (ADS)

Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

2011-09-01

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS n . The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS n fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS n experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS n methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

LC-MSn Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

PubMed Central

Huang, Rongrong; Pomin, Vitor H.; Sharp, Joshua S.

2011-01-01

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MSn. The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MSn fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MSn experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MSn methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications. PMID:21953261

Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desai, Meera Jay

The purpose of this research was to develop sensitive LC-MS methods for enantiomeric separation and detection, and then apply these methods for determination of enantiomeric composition and for the study of pharmacokinetic and pharmacodynamic properties of a chiral nutraceutical. Our first study, evaluated the use of reverse phase and polar organic mode for chiral LC-API/MS method development. Reverse phase methods containing high water were found to decrease ionization efficiency in electrospray, while polar organic methods offered good compatibility and low limits of detection with ESI. The use of lower flow rates dramatically increased the sensitivity by an order of magnitude.more » Additionally, for rapid chiral screening, the coupled Chirobiotic column afforded great applicability for LC-MS method development. Our second study, continued with chiral LC-MS method development in this case for the normal phase mode. Ethoxynonafluorobutane, a fluorocarbon with low flammability and no flashpoint, was used as a substitute solvent for hexane/heptane mobile phases for LC-APCI/MS. Comparable chromatographic resolutions and selectivities were found using ENFB substituted mobile phase systems, although, peak efficiencies were significantly diminished. Limits of detection were either comparable or better for ENFB-MS over heptane-PDA detection. The miscibility of ENFB with a variety of commonly used organic modifiers provided for flexibility in method development. For APCI, lower flow rates did not increase sensitivity as significantly as was previously found for ESI-MS detection. The chiral analysis of native amino acids was evaluated using both APCI and ESI sources. For free amino acids and small peptides, APCI was found to have better sensitivities over ESI at high flow rates. For larger peptides, however, sensitivity was greatly improved with the use of electrospray. Additionally, sensitivity was enhanced with the use of non-volatile additives, This optimized method was then used to simultaneously separate all 19 native amino acids enantiomerically in less than 20 minutes, making it suitable for complex biological analysis. The previously developed amino acid method was then used to enantiomerically separate theanine, a free amino acid found in tea leaves. Native theanine was found to have lower limits of detection and better sensitivity over derivatized theanine samples. The native theanine method was then used to determine the enantiomeric composition of six commercially available L-theanine products. Five out of the six samples were found to be a racemic mixture of both D- and L-theanine. Concern over the efficacy of these theanine products led to our final study evaluating the pharmacokinetics and pharmacodynamics of theanine in rats using LC-ESI/MS. Rats were administered D-, L, and QL-theanine both orally and intra-peritoneally. Oral administration data demonstrated that intestinal absorption of L-theanine was greater than that of D-theanine, while i.p. data showed equal plasma uptake of both isomers. This suggested a possible competitive binding effect with respect to gut absorption. Additionally, it was found that regardless of administration method, the presence of the other enantiomer always decreased overall theanine plasma concentration. This indicated that D- and L- theanine exhibit competitive binding with respect to urinary reabsorption as well. The large quantities of D-theanine detected in the urine suggested that D-themine was eliminated with minimal metabolism, while L-theanine was preferentially reabsorbed and metabolized to ethylamine. Clearly, the metabolic fate of racemic theanine and its individual enantiomers was quite different, placing into doubt the utility of the commercial theanine products.« less

Multiple, simultaneous, independent gradients for a versatile multidimensional liquid chromatography. Part II: Application 1 - Large increases in isoform resolution of human transferrin by use of dual simultaneous independent gradients of pH & acetonitrile on a mixed bed (anion exchange plus reversed phase) stationary phase.

PubMed

Tsonev, Latchezar I; Hirsh, Allen G

2016-10-14

We have previously described a liquid chromatographic (LC) method for uncoupling controlled, wide range pH gradients and simultaneous controlled gradients of a non-buffering solute on ion exchange resins (Hirsh and Tsonev, 2012) [1]. Here we report the application of this two dimensional LC technique to the problem of resolving Human Transferrin (HT) isoforms. This important iron transporting protein should theoretically occur in several thousand glycoforms, but only about a dozen have been reported. Using dual simultaneous independent gradients (DSIGs) of acetonitrile (ACN) and pH on a mixed bed stationary phase (SP) consisting of a mixture of an anion exchange resin and a reversed phase (RP) resin we partially resolve about 60 isoforms. These are likely to be partially refolded glycoforms generated by interaction of HT with the highly hydrophobic RP SP, as well as distinct folded glycoforms. Thus this study should have interesting implications for both glycoform separation and the study of protein folding. Copyright © 2016 Elsevier B.V. All rights reserved.

Post-column mobile phase adjustment: a strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma.

PubMed

Zheng, Shirui; Ma, Zhiyuan; Han, Haixia; Ye, Jianfeng; Wang, Ruwei; Cai, Sheng; Zhou, Hui; Yu, Lushan; Zeng, Su; Jiang, Huidi

2014-07-01

Flavonoids are a group of important naturally occurring polyphenolic compounds with a wide range of biological effects. In this study, a sensitive liquid chromatography tandem mass spectrometry method was developed to simultaneously determine multiple active flavonoids, including quercetin (Que), kaempferol (Kae), apigenin (Api), isorhamnetin (Iso), luteolin (Lut), and naringenin (Nar), in rat plasma. To achieve a satisfied peak shape and LC separation, formic acid with the concentration between 0.05 and 0.2%, or in some case 5%, was generally used to acidify the LC mobile phase in reported studies. Here we found that even 0.05% formic acid could lead to strong mass signal suppression, and the absence of formic acid could reverse the signal suppression but cause serious peak tailing. There is an irreconcilable contradiction between liquid chromatography (LC) and mass spectrometry (MS). In order to simultaneously satisfy LC and MS, LC mobile phase with 0.00075% formic acid and post column mobile phase adjustment with 0.0677% ammonium solution in isopropanol were applied. Compared with the conventional method with mobile phase containing 0.05% formic acid, the mass signal response of Que, Kae, Api, Iso, Lut, Nar, and Oka increased 26.2, 18.6, 13.6, 23.5, 17.5, 15.6 and 15.4 fold, respectively. In addition, the post column mobile phase addition exhibited the better peak shape for the reduction of analytes longitudinal diffusion. The method has been fully validated according to FDA guidelines within the linear range between 0.328 ng mL⁻¹ and 168 ng mL⁻¹, and successfully applied to a pilot pharmacokinetic study of rats after administering 5.43 g kg⁻¹ Pollen of Brassica campestris. Copyright © 2014 Elsevier B.V. All rights reserved.

Validated reversed phase LC method for quantitative analysis of polymethoxyflavones in citrus peel extracts.

PubMed

Wang, Zhenyu; Li, Shiming; Ferguson, Stephen; Goodnow, Robert; Ho, Chi-Tang

2008-01-01

Polymethoxyflavones (PMFs), which exist exclusively in the citrus genus, have biological activities including anti-inflammatory, anticarcinogenic, and antiatherogenic properties. A validated RPLC method was developed for quantitative analysis of six major PMFs, namely nobiletin, tangeretin, sinensetin, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone. The polar embedded LC stationary phase was able to fully resolve the six analogues. The developed method was fully validated in terms of linearity, accuracy, precision, sensitivity, and system suitability. The LOD of the method was calculated as 0.15 microg/mL and the recovery rate was between 97.0 and 105.1%. This analytical method was successfully applied to quantify the individual PMFs in four commercially available citrus peel extracts (CPEs). Each extract shows significant difference in the PMF composition and concentration. This method may provide a simple, rapid, and reliable tool to help reveal the correlation between the bioactivity of the PMF extracts and the individual PMF content.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yuzhan; Zhang, Yuehong; Rios, Orlando

In this study, a liquid crystalline epoxy network (LCEN) with exchangeable disulfide bonds is synthesized by polymerizing a biphenyl-based epoxy monomer with an aliphatic dicarboxylic acid curing agent containing a disulfide bond. The effect of disulfide bonds on curing behavior and liquid crystalline (LC) phase formation of the LCEN is investigated. The presence of the disulfide bonds results in an increase in the reaction rate, leading to a reduction in liquid crystallinity of the LCEN. In order to promote LC phase formation and stabilize the self-assembled LC domains, a similar aliphatic dicarboxylic acid without the disulfide bond is used asmore » a co-curing agent to reduce the amount of exchangeable disulfide bonds in the system. After optimizing the molar ratio of the two curing agents, the resulting LCEN exhibits improved reprocessability and recyclability because of the disulfide exchange reactions, while preserving LC properties, such as the reversible LC phase transition and macroscopic LC orientation, for shape memory applications.« less

Recent development in liquid chromatography stationary phases for separation of Traditional Chinese Medicine components.

PubMed

Jin, Hongli; Liu, Yanfang; Guo, Zhimou; Wang, Jixia; Zhang, Xiuli; Wang, Chaoran; Liang, Xinmiao

2016-10-25

Traditional Chinese Medicine (TCM) is an ancient medical practice which has been used to prevent and cure diseases for thousands of years. TCMs are frequently multi-component systems with mainly unidentified constituents. The study of the chemical compositions of TCMs remains a hotspot of research. Different strategies have been developed to manage the significant complexity of TCMs, in an attempt to determine their constituents. Reversed-phase liquid chromatography (RPLC) is still the method of choice for the separation of TCMs, but has many problems related to limited selectivity. Recently, enormous efforts have been concentrated on the development of efficient liquid chromatography (LC) methods for TCMs, based on selective stationary phases. This can improve the resolution and peak capacity considerably. In addition, high-efficiency stationary phases have been applied in the analysis of TCMs since the invention of ultra high-performance liquid chromatography (UHPLC). This review describes the advances in LC methods in TCM research from 2010 to date, and focuses on novel stationary phases. Their potential in the separation of TCMs using relevant applications is also demonstrated. Copyright © 2016 Elsevier B.V. All rights reserved.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins.

PubMed

Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

2014-01-01

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins

PubMed Central

Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

2014-01-01

N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented. PMID:24848368

Development and Validation of RP-LC Method for the Determination of Cinnarizine/Piracetam and Cinnarizine/Heptaminol Acefyllinate in Presence of Cinnarizine Reported Degradation Products

PubMed Central

EL-Houssini, Ola M.; Zawilla, Nagwan H.; Mohammad, Mohammad A.

2013-01-01

Specific stability indicating reverse-phase liquid chromatography (RP-LC) assay method (SIAM) was developed for the determination of cinnarizine (Cinn)/piracetam (Pira) and cinnarizine (Cinn)/heptaminol acefyllinate (Hept) in the presence of the reported degradation products of Cinn. A C18 column and gradient mobile phase was applied for good resolution of all peaks. The detection was achieved at 210 nm and 254 nm for Cinn/Pira and Cinn/Hept, respectively. The responses were linear over concentration ranges of 20–200, 20–1000 and 25–1000 μgmL−1 for Cinn, Pira, and Hept respectively. The proposed method was validated for linearity, accuracy, repeatability, intermediate precision, and robustness via statistical analysis of the data. The method was shown to be precise, accurate, reproducible, sensitive, and selective for the analysis of Cinn/Pira and Cinn/Hept in laboratory prepared mixtures and in pharmaceutical formulations. PMID:24137049

Microwave assisted synthesis for A2E and development of LC-ESI-MS method for quantification of ocular bisretinoids in human retina.

PubMed

Kotnala, A; Senthilkumari, S; Halder, N; Kumar, A; Velpandian, T

2018-01-15

To develop a microwave assisted method for the rapid synthesis of A2E and also to develop a method to quantify N-retinylidene-N-retinylethanolamine(A2E), all-trans retinal dimer (ATRD), A2-glycerophospho ethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE) and monofuran A2E (MFA2E) in age matched retina. The development of microwave assisted synthesis of A2E, its purification and characterization for its utility in quantification in human retina. The semi-quantitative method development using LC-ESI-MS, LC-ESI-MS/MS and LC-APCI-MS/MS from pooled macula and peripheral retina for the bisretinoid analysis has been done. Maximum A2E conversion using microwave assisted process took place at 80°C for 45min with a yield of 55.01%. Highly sensitive and specific mass spectrometric method was developed using reverse phase C-18 separation with positive electrospray ionization and positive atmospheric phase chemical ionization of tandom mass spectrometry. A gradient mobile phase separation was achieved using water and methanol with 0.1% TFA. Multiple reaction monitoring acquisition for ESI and APCI was performed at ATRD m/z 551.2/522.2, A2GPE m/z 746.4/729.5, A2DHPEm/z 594.4/576.5, MFA2E m/z 608.2/591.2, A2E m/z 592.4/418.2. Method was validated using LC-ESI-SIM mode to determine selectivity, linearity, sensitivity, precision and accuracy. An attempt towards optimization of the synthetic procedure of A2E was made so as to reduce the lengthy reaction time without compromising the yield. Developed method was capable enough for the detection of low level of bisretinids in retina. Copyright © 2017 Elsevier B.V. All rights reserved.

Spectroscopic characterization and quantitative determination of atorvastatin calcium impurities by novel HPLC method

NASA Astrophysics Data System (ADS)

Gupta, Lokesh Kumar

2012-11-01

Seven process related impurities were identified by LC-MS in the atorvastatin calcium drug substance. These impurities were identified by LC-MS. The structure of impurities was confirmed by modern spectroscopic techniques like 1H NMR and IR and physicochemical studies conducted by using synthesized authentic reference compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. These impurities were detected by newly developed gradient, reverse phase high performance liquid chromatographic (HPLC) method. The system suitability of HPLC analysis established the validity of the separation. The analytical method was validated according to International Conference of Harmonization (ICH) with respect to specificity, precision, accuracy, linearity, robustness and stability of analytical solutions to demonstrate the power of newly developed HPLC method.

A serially coupled stationary phase method for the determination of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine by liquid chromatography ion trap tandem mass spectrometry☆

PubMed Central

Rota, Cristina; Cristoni, Simone; Trenti, Tommaso; Cariani, Elisabetta

2013-01-01

Oxidative attack to DNA is of particular interest since DNA modifications can lead to heritable mutations. The most studied product of DNA oxidation is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG). While 8-oxodG determination in blood and tissue cells is prone to artifacts, its measurement in urine employing liquid chromatography tandem mass spectrometry (LC-MS/MS) has gained more and more interest for increased reliability. LC-MS/MS can be affected by matrix effects and this is particularly true when ion trap is used as MS analyzer, due to ion accumulation in the trap and related space charge effect. In the present work, we have developed a LC-MS/MS method where the combination of cation exchange and reverse phase solid phases resulted in LC separation optimization. This together with the employment of an isotopically labeled internal standard, allowed the usage of ion trap LC-MS/MS, typically not employed for quantitative measurement in biological samples, for the measurement of 8-oxodG in urine samples from control populations. Four different urine matrices were employed for method validation. Limit of quantitation was set at least at 0.5 ng/ml. While analyzing urine samples from healthy volunteers, 8-oxodG levels reported as ng/ml were statistically different comparing males with females (p<0.05, Mann Whitney test); while comparing results normalized for creatinine no statistical significant difference was found. Mean urinary 8-oxodG level found in healthy volunteers was 1.16±0.46 nmol/mmol creatinine. The present method by enhancing at best the chromatographic performances allows the usage of ion trap LC-MS/MS for the measurement of 8-oxodG in urine samples from control populations. PMID:24251117

Development and validation of a rapid reverse-phase HPLC method for the determination of methotrexate from nanostructured liquid crystalline systems.

PubMed

Zuben, E S Von; Oliveira, A G; Chorilli, M; Scarpa, M V

2018-03-05

A reversed-phase liquid chromatography (RP-LC) method was successfully developed and validated for the determination of methotrexate in nanostructured liquid crystalline systems composed by polyether functional siloxane and silicone polyether copolymer. The LC method was performed on RP C18-ODS column, Agilent Zorbax® (4.6 x 250 mm, 5 μm), maintained at room temperature, with a mobile phase constituted by a mixture of 50 mM ammonium acetate buffer (pH 6.0) and methanol (77:23,v/v) with a flow rate of 1.0 mL/min, using ultraviolet detection at 313 nm. The parameters used in the validation process were linearity, specificity, intra and inter-day precision, accuracy, robustness. The quantitation and detection limits yielded good results. The calibration plot assumed linear behavior from 5.0-150.0 μg. mL-1 (r2 = 0.9999). The methotrexate was subjected to oxidation, acid, base and neutral degradation, photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of methotrexate. The nanostructured liquid crystalline systems did not interfere with the analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.20 %. The method developed proved to be simple, sensitive, accurate, precise, reproducible and therefore adequate for routine analysis of methotrexate in nanostructured liquid crystalline systems.

Determination of nicotianamine in soy sauce and other plant-based foods by LC-MS/MS.

PubMed

Yamaguchi, Hitomi; Uchida, Riichiro

2012-10-10

Nicotianamine is a nonproteinogenic amino acid, known to be an important metal chelator in plants. Recently, the antihypertensive effect of nicotianamine was discovered. In this study, a simple method to determine nicotianamine was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a multimode ODS column. This method does not need derivatizing or ion-pairing reagents to retain nicotianamine, which is known for its poor retention on reversed-phase columns because of its high polarity. Moreover, this method showed a sufficient limit of detection (0.5 ng/mL), so it was found to be suitable for the analysis of nicotianamine in soy sauce and other foods, without cleanup. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of nicotianamine in soy sauce ranged from <0.25 to 71 μg/g. Nicotianamine was also determined in other foods, including soy milk, vegetable juice, fruit juice, and bottled tea.

Determination of β-Carotene in Supplements and Raw Materials by Reversed-Phase High Pressure Liquid Chromatography

PubMed Central

Szpylka, John; DeVries, Jonathan W.; Bhandari, S.; Bui, M.H.; Ji, D.; Konings, E.; Lewis, R.; Maas, P.; Parish, H.; Post, B.; Schierle, J.; Sullivan, D.; Taylor, A.; Wang, J.; Ware, G.; Woollard, D.; Wu, T.

2008-01-01

Twelve laboratories representing 4 countries participated in an interlaboratory study conducted to determine all-trans-β-carotene and total β-carotene in dietary supplements and raw materials. Thirteen samples were sent as blind duplicates to the collaborators. Results obtained from 11 laboratories are reported. For products composed as softgels and tablets that were analyzed for total β-carotene, the reproducibility relative standard deviation (RSDR) ranged from 3.35 to 23.09% and the HorRat values ranged from 1.06 to 3.72. For these products analyzed for trans β-carotene, the reproducibility relative standard deviation (RSDR) ranged from 4.28 to 22.76% and the HorRat values ranged from 0.92 to 3.37. The RSDr and HorRat values in the analysis of a beadlet raw material were substantial and it is believed that the variability within the material itself introduced significant variation in subsampling. The method uses high pressure liquid chromatography (LC) in the reversed-phase mode with visible light absorbance for detection and quantitation. If high levels of α-carotenes are present, a second LC system is used for additional separation and quantitation of the carotene species. It is recommended that the method be adopted as an AOAC Official Method. PMID:16385976

Sensitive and simultaneous determination of HIV protease inhibitors in rat biological samples by liquid chromatography-mass spectrometry.

PubMed

Gao, Weihua; Kishida, Tomoyuki; Kimura, Keisuke; Kageyama, Michiharu; Sumi, Masaki; Yoshikawa, Yukako; Shibata, Nobuhito; Takada, Kanji

2002-06-01

A sensitive and simultaneous liquid chromatographic-mass spectrometric (LC/MS) method for the determination of current four HIV protease inhibitors (PIs), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV) and amprenavir (APV) in rat plasma and liver dialysate by a microdialysis method was described. An isocratic LC/MS method in combination with atmospheric pressure chemical ionization was developed for the determination of these four PIs in biological samples in the same run. The analytes including an internal standard were extracted from 100 microL of plasma or 150 microL of liver dialysate samples by salting-out with 100 microL of ice-cold 2 M K(3)PO(4) followed by ether extraction. The separation of analytes was carried out on a reversed-phase semi-micro column using 50% of acetonitrile containing 1% acetic acid as mobile phase at a flow rate of 0.2mL/min(-1). The separation was completed within 5 min. Precision, recovery and limits of detection indicated that the method was suitable for the quantitative determination of these PIs in rat plasma or liver dialysate. This simple, sensitive and highly specific LC/MS method is suitable for pharmacokinetic studies and therapeutic drug monitoring in AIDS patients who receive double protease therapy. Copyright 2002 John Wiley & Sons, Ltd.

Simple Sodium Dodecyl Sulfate-Assisted Sample Preparation Method for LC-MS-based Proteomic Applications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Jianying; Dann, Geoffrey P.; Shi, Tujin

2012-03-10

Sodium dodecyl sulfate (SDS) is one of the most popular laboratory reagents used for highly efficient biological sample extraction; however, SDS presents a significant challenge to LC-MS-based proteomic analyses due to its severe interference with reversed-phase LC separations and electrospray ionization interfaces. This study reports a simple SDS-assisted proteomic sample preparation method facilitated by a novel peptide-level SDS removal protocol. After SDS-assisted protein extraction and digestion, SDS was effectively (>99.9%) removed from peptides through ion substitution-mediated DS- precipitation with potassium chloride (KCl) followed by {approx}10 min centrifugation. Excellent peptide recovery (>95%) was observed for less than 20 {mu}g of peptides.more » Further experiments demonstrated the compatibility of this protocol with LC-MS/MS analyses. The resulting proteome coverage from this SDS-assisted protocol was comparable to or better than those obtained from other standard proteomic preparation methods in both mammalian tissues and bacterial samples. These results suggest that this SDS-assisted protocol is a practical, simple, and broadly applicable proteomic sample processing method, which can be particularly useful when dealing with samples difficult to solubilize by other methods.« less

Isolation of n-decyl-alpha(1-->6) isomaltoside from a technical APG mixture and its identification by the parallel use of LC-MS and NMR spectroscopy

PubMed

Billian; Hock; Doetzer; Stan; Dreher

2000-10-15

The identification of n-decyl alpha(1-->6)isomaltoside as a main component of technical alkyl polyglucoside (APG) mixtures by the parallel use of liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy is described. Following enrichment on a styrene-divinylbenzene-based solid-phase extraction material, unknown components were separated by reversed-phase liquid chromatography (LC). Chemical characterization was achieved by both mass spectrometry and NMR spectroscopy. It is demonstrated that the combination of LC-MS with various NMR techniques is very suitable for stereochemical assignment of unknown components in technical APG mixtures.

Liquid crystalline epoxy networks with exchangeable disulfide bonds

DOE PAGES

Li, Yuzhan; Zhang, Yuehong; Rios, Orlando; ...

2017-06-09

In this study, a liquid crystalline epoxy network (LCEN) with exchangeable disulfide bonds is synthesized by polymerizing a biphenyl-based epoxy monomer with an aliphatic dicarboxylic acid curing agent containing a disulfide bond. The effect of disulfide bonds on curing behavior and liquid crystalline (LC) phase formation of the LCEN is investigated. The presence of the disulfide bonds results in an increase in the reaction rate, leading to a reduction in liquid crystallinity of the LCEN. In order to promote LC phase formation and stabilize the self-assembled LC domains, a similar aliphatic dicarboxylic acid without the disulfide bond is used asmore » a co-curing agent to reduce the amount of exchangeable disulfide bonds in the system. After optimizing the molar ratio of the two curing agents, the resulting LCEN exhibits improved reprocessability and recyclability because of the disulfide exchange reactions, while preserving LC properties, such as the reversible LC phase transition and macroscopic LC orientation, for shape memory applications.« less

Determination of patulin in apple juice by liquid chromatography: collaborative study.

PubMed

Brause, A R; Trucksess, M W; Thomas, F S; Page, S W

1996-01-01

An AOAC International-International Union of Pure and Applied Chemistry-International Fruit Juice Union (AOAC-IUPAC-IFJU) collaborative study was conducted to evaluate a liquid chromatographic (LC) procedure for determination of patulin in apple juice. Patulin is a mold metabolite found naturally in rotting apples. Patulin is extracted with ethyl acetate, treated with sodium carbonate solution, and determined by reversed-phase LC with UV detection at 254 or 276 nm. Water, water-tetrahydrofuran, or water-acetonitrile was used as mobile phase. Levels determined in spiked test samples were 20, 50, 100, and 200 micrograms/L. A test sample naturally contaminated at 31 micrograms/L was also included. Twenty-two collaborators in 10 countries analyzed 12 test samples of apple juice. Recoveries averaged 96%, with a range of 91-108%. Repeatability relative standard deviations (RSDr) ranged from 10.9 to 53.8%. The reproducibility relative standard deviation (RSDR) ranged from 15.1 to 68.8%. The LC method for determination of patulin in apple juice has been adopted first action by AOAC INTERNATIONAL.

LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

NASA Astrophysics Data System (ADS)

Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi

2014-06-01

Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.

N-linked glycoprotein analysis using dual-extraction ultrahigh-performance liquid chromatography and electrospray tandem mass spectrometry.

PubMed

Siu, S O; Lam, Maggie P Y; Lau, Edward; Yeung, William S B; Cox, David M; Chu, Ivan K

2010-01-01

Although reverse-phase liquid chromatography (RP-LC) is a common technique for peptide separation in shotgun proteomics and glycoproteomics, it often provides unsatisfactory results for the analysis of glycopeptides and glycans. This bias against glycopeptides makes it difficult to study glycoproteins. By coupling mass spectrometry (MS) with a combination of RP-LC and normal-phase (NP)-LC as an integrated front-end separation system, we demonstrate that effective identification and characterization of both peptides and glycopeptides mixtures, and their constituent glycan structures, can be achieved from a single sample injection event.

Separation of Cis-Trans Phospholipid Isomers using Reversed Phase LC with High Resolution MS Detection

PubMed Central

Bird, Susan S.; Marur, Vasant R.; Stavrovskaya, Irina G.; Kristal, Bruce S.

2012-01-01

The increased presence of synthetic trans fatty acids into western diets has been shown to have deleterious effects on physiology and raising an individual’s risk of developing metabolic disease, cardiovascular disease, and stroke. The importance of these fatty acids for health and the diversity of their (patho) physiological effects suggest that not only should the free trans fatty acids be studied, but that monitoring the presence of these fats into the side-chains of biological lipids, such as glycerophospholipids, is also essential. We developed a high resolution LC-MS method that quantitatively monitors the major lipid classes found in biospecimens in an efficient, sensitive and robust manner while also characterizing individual lipid side-chains through the use of HCD fragmentation and chromatographic alignment. We herein show how this previously described reversed phase method can baseline separate the cis-trans isomers of phosphatidylglycerol and phosphatidylcholine (PC) with two 18:1 side chains, in both positive and negative mode, as neat solutions and when spiked into a biological matrix. Endogenous PC (18:1/18:1) cis and PC (18:1/18:1) trans isomers were examined in mitochondrial and serum profiling studies, where rats were fed diets enriched in either trans 18:1 fatty acids, or cis 18:1 fatty acids. In this study, we determined the cis:trans isomer ratios of PC (18:1/18:1) and related this ratio to dietary composition. This generalized LC-MS method enables the monitoring of trans fats in biological lipids in the context of a non-targeted method, allowing for relative quantitation and enhanced identification of unknown lipids in complex matrices. PMID:22656324

Determination of malachite green and its leuco form in water

USGS Publications Warehouse

Allen, J.L.; Meinertz, J.R.; Gofus, J.E.

1992-01-01

Liquid chromatographic (lc) analysis can detect malachite green residues in water at less than 10 mu-g/l. Water samples were concentrated on disposable diol columns, eluted with 0.05m P-toluene-sulfonic acid in methanol, and determined by reversed-phase lc. When combined with a lead oxide postcolumn reactor, the lc method can simultaneously determine both leuco and chromatic forms of malachite green. Recoveries averaged 95.4% For the chromatic form and 57.3% For the leuco form of malachite green oxalate and leuco malachite green in spiked pond water samples. Recoveries of the carbinol form of malachite green (an equilibrium product of the dye in water) from spiked tap water samples averaged 98.6%. Recoveries of leuco malachite green were low and ph-dependent.

Sensitive and selective liquid chromatography-tandem mass spectrometry method for the quantification of aniracetam in human plasma.

PubMed

Zhang, Jingjing; Liang, Jiabi; Tian, Yuan; Zhang, Zunjian; Chen, Yun

2007-10-15

A rapid, sensitive and selective LC-MS/MS method was developed and validated for the quantification of aniracetam in human plasma using estazolam as internal standard (IS). Following liquid-liquid extraction, the analytes were separated using a mobile phase of methanol-water (60:40, v/v) on a reverse phase C18 column and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode using the respective [M+H]+ ions, m/z 220-->135 for aniracetam and m/z 295-->205 for the IS. The assay exhibited a linear dynamic range of 0.2-100 ng/mL for aniracetam in human plasma. The lower limit of quantification (LLOQ) was 0.2 ng/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The validated LC-MS/MS method has been successfully applied to study the pharmacokinetics of aniracetam in healthy male Chinese volunteers.

High-performance liquid chromatography with nuclear magnetic resonance detection applied to organosilicon polymers. Part 2. Comparison with other methods.

PubMed

Blechta, Vratislav; Kurfürst, Milan; Sýkora, Jan; Schraml, Jan

2007-03-23

LC-NMR utilizing (1)H and (29)Si NMR spectroscopy is ideally suited for the analysis of silicones. It is shown that reversed phase gradient LC-NMR surpasses standard gel permeation chromatography (GPC) and diffusion ordered spectroscopy (DOSY) in the analysis of model hydride terminated polydimethylsiloxane. (1)H and (29)Si NMR in the stopped-flow arrangement leads to full identification of the components. Concentration gradient introduces a dependence of the (29)Si shifts on solvent composition, this dependence can be substantially reduced by a proposed method of referencing. It is shown that the ADEQUATE version of powerful but insensitive 2D INADEQUATE experiment can be used for complete line assignment.

A method for the direct injection and analysis of small volume human blood spots and plasma extracts containing high concentrations of organic solvents using revered-phase 2D UPLC/MS.

PubMed

Rainville, Paul D; Simeone, Jennifer L; Root, Dan S; Mallet, Claude R; Wilson, Ian D; Plumb, Robert S

2015-03-21

The emergence of micro sampling techniques holds great potential to improve pharmacokinetic data quality, reduce animal usage, and save costs in safety assessment studies. The analysis of these samples presents new challenges for bioanalytical scientists, both in terms of sample processing and analytical sensitivity. The use of two dimensional LC/MS with, at-column-dilution for the direct analysis of highly organic extracts prepared from biological fluids such as dried blood spots and plasma is demonstrated. This technique negated the need to dry down and reconstitute, or dilute samples with water/aqueous buffer solutions, prior to injection onto a reversed-phase LC system. A mixture of model drugs, including bromhexine, triprolidine, enrofloxacin, and procaine were used to test the feasibility of the method. Finally an LC/MS assay for the probe pharmaceutical rosuvastatin was developed from dried blood spots and protein-precipitated plasma. The assays showed acceptable recovery, accuracy and precision according to US FDA guidelines. The resulting analytical method showed an increase in assay sensitivity of up to forty fold as compared to conventional methods by maximizing the amount loaded onto the system and the MS response for the probe pharmaceutical rosuvastatin from small volume samples.

Stability-indicating RP-HPLC method for the simultaneous determination of escitalopram oxalate and clonazepam.

PubMed

Kakde, Rajendra B; Satone, Dinesh D; Gadapayale, Kamalesh K; Kakde, Megha G

2013-07-01

The objective of the current study was to develop a validated, specific stability-indicating reversed-phase liquid chromatographic (LC) method for the quantitative determination of escitalopram oxalate and clonazepam and their related substances in bulk drugs and pharmaceutical dosage forms in the presence of degradation products. Forced degradation studies were performed on the pure drugs of escitalopram oxalate and clonazepam, as per the stress conditions prescribed by the International Conference on Harmonization (ICH) using acid, base, oxidation, thermal stress and photolytic degradation to show the stability-indicating power of the method. Significant degradation was observed during acid and alkaline hydrolysis and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies. Good resolution between the peaks corresponded to the active pharmaceutical ingredients, escitalopram oxalate and clonazepam, and degradation products from the analyte were achieved on an ODS Hypersil C18 column (250 × 4.6 mm) using a mobile phase consisting of a mixture of acetonitrile-50 mM phosphate buffer + 10 mM triethylamine (70:30, v/v). The detection was conducted at 268 nm. The limit of detection and the limit of quantitation for escitalopram oxalate and clonazepam were established. The stress test solutions were assayed against the qualified working standards of escitalopram oxalate and clonazepam, which indicated that the developed LC method was stability-indicating. Validation of the developed LC method was conducted as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of escitalopram oxalate and clonazepam.

Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation

PubMed Central

Liu, Liyao; Zhou, Cuiping; Xia, Xuejun; Liu, Yuling

2016-01-01

Purpose Here, we investigated the formation and functional properties of self-assembled lecithin/chitosan nanoparticles (L/C NPs) loaded with insulin following insulin–phospholipid complex preparation, with the aim of developing a method for oral insulin delivery. Methods Using a modified solvent-injection method, insulin-loaded L/C NPs were obtained by combining insulin–phospholipid complexes with L/C NPs. The nanoparticle size distribution was determined by dynamic light scattering, and morphologies were analyzed by cryogenic transmission electron microscopy. Fourier transform infrared spectroscopy analysis was used to disclose the molecular mechanism of prepared insulin-loaded L/C NPs. Fast ultrafiltration and a reversed-phase high-performance liquid chromatography assay were used to separate free insulin from insulin entrapped in the L/C NPs, as well as to measure the insulin-entrapment and drug-loading efficiencies. The in vitro release profile was obtained, and in vivo hypoglycemic effects were evaluated in streptozotocin-induced diabetic rats. Results Our results indicated that insulin-containing L/C NPs had a mean size of 180 nm, an insulin-entrapment efficiency of 94%, and an insulin-loading efficiency of 4.5%. Cryogenic transmission electron microscopy observations of insulin-loaded L/C NPs revealed multilamellar structures with a hollow core, encircled by several bilayers. In vitro analysis revealed that insulin release from L/C NPs depended on the L/C ratio. Insulin-loaded L/C NPs orally administered to streptozotocin-induced diabetic rats exerted a significant hypoglycemic effect. The relative pharmacological bioavailability following oral administration of L/C NPs was 6.01%. Conclusion With the aid of phospholipid-complexation techniques, some hydrophilic peptides, such as insulin, can be successfully entrapped into L/C NPs, which could improve oral bioavailability, time-dependent release, and therapeutic activity. PMID:26966360

The differences in matrix effect between supercritical fluid chromatography and reversed phase liquid chromatography coupled to ESI/MS.

PubMed

Svan, Alfred; Hedeland, Mikael; Arvidsson, Torbjörn; Pettersson, Curt E

2018-02-13

For many sample matrices, matrix effects are a troublesome phenomenon using the electrospray ionization source. The increasing use of supercritical fluid chromatography with CO 2 in combination with the electrospray ionization source for MS detection is therefore raising questions: is the matrix effect behaving differently using SFC in comparison with reversed phase LC? This was investigated using urine, plasma, influent- and effluent-wastewater as sample matrices. The matrix effect was evaluated using the post-extraction addition method and through post-column infusions. Matrix effect profiles generated from the post-column infusions in combination with time of flight-MS detection provided the most valuable information for the study. The combination of both qualitative and semi-quantitative information with the ability to use HRMS-data for identifying interfering compounds from the same experiment was very useful, and has to the authors' knowledge not been used this way before. The results showed that both LC and SFC are affected by matrix effects, however differently depending on sample matrix. Generally, both suppressions and enhancements were seen, with a higher amount of enhancements for LC, where 65% of all compounds and all sample matrices were enhanced, compared to only 7% for SFC. Several interferences were tentatively identified, with phospholipids, creatinine, and metal ion clusters as examples of important interferences, with different impact depending on chromatographic technique. SFC needs a different strategy for limiting matrix interferences, owing to its almost reverse retention order compared to RPLC. Copyright © 2017 Elsevier B.V. All rights reserved.

A New LC-MS-based Strategy to integrate chemistry, morphology, and evolution of eggplant (Solanum) species

USDA-ARS?s Scientific Manuscript database

The economically valuable giant genus Solanum, containing dozens of functional food species such as eggplant and tomato, affords an excellent system to compare and correlate metabolic chemistry with species morphology and evolution. Here, we devised a strategy based on repeatable reversed-phase LC-T...

ICH guidance in practice: validated reversed-phase HPLC method for the determination of active mangiferin from extracts of Mangifera indica Linn.

PubMed

Gowda, Nagaraj; Kumar, Pradeep; Panghal, Surender; Rajshree, Mashru

2010-02-01

This study presents the development and validation of a reversed-phase liquid chromatographic method for the determination of mangiferin (MGN) in alcoholic extracts of mangifera indica. A Lichrospher 100 C(18)-ODS (250 x 4.6 mm, 5 microm size) (Merck, Whitehouse Station, NJ) prepacked column and a mobile phase of potassium dihydrogen orthophosphate (0.01M) pH 2.7 +/- 0.2-acetonitrile (15:85, v/v) with the flow rate of 1 mL/min was used. MGN detection was achieved at a wavelength monitored at 254 nm with SPD-M 10A vp PDA detector or SPD 10AD vp UV detector in combination with class LC 10A software. The proposed method was validated as prescribed by International Conference on Harmonization (ICH) with respect to linearity, specificity, accuracy, precision, stability, and quantification. The method validation was realized using alcoholic extracts and raw materials of leaves and barks. All the validation parameters were within the acceptable limits, and the developed analytical method can successfully be applied for MGN determination.

Simultaneous determination of emamectin and ivermectin residues in Atlantic salmon muscle by liquid chromatography with fluorescence detection.

PubMed

van de Riet, J M; Brothers, N N; Pearce, J N; Burns, B G

2001-01-01

A liquid chromatographic (LC) method for determining residues of the antiparasitic drugs emamectin (EMA) and ivermectin (IVR) in fish tissues has been developed. EMA and IVR residues are extracted with acetonitrile and cleaned up on a C18 solid-phase extraction column. Extracts are derivatized with 1-methylimidazole and trifluoroacetic anhydride and the components are determined by LC on a C18 reversed-phase column with fluorescence detection (excitation: 365 nm, emission: 470 nm). The mobile phase is 94% acetonitrile-water run isocratically. Calibration curves were linear between 1 and 32 ng injected for both EMA and IVR. The limit of detection for both analytes was 0.5 ng/g, with a limit of quantitation of 1.5 ng/g. Recoveries of EMA and IVR added to salmon muscle averaged 96 +/- 9% and 86 +/- 6%, respectively, at levels between 5 and 80 ng/g. The percent relative standard deviation for the described method was less than 7% over the range of concentrations studied. The operational errors, interferences, and recoveries for fortified samples compare favorably with an established IVR method. The recommended method is simple, rapid, and specific for monitoring residues of EMA and IVR in Atlantic salmon muscle.

Systematical Optimization of Reverse-phase Chromatography for Shotgun Proteomics

PubMed Central

Xu, Ping; Duong, Duc M.; Peng, Junmin

2009-01-01

Summary We report the optimization of a common LC/MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 μm internal diameter × 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC). The peptide mix was analyzed on a highly reproducible, automated nanoLC/MS/MS system with systematic adjustment of loading amount, flow rate, elution gradient range and length. Interestingly, the column was found to be almost saturated by loading ~1 μg of the sample. Whereas the optimal flow rate (~0.2 μl/min) and elution buffer range (13–32% of acetonitrile) appeared to be independent of the loading amount, the best gradient length varied according to the amount of samples: 160 min for 1 μg of the peptide mix, but 40 min for 10 ng of the same sample. The effect of these parameters on elution peptide peak width is evaluated. After full optimization, 1,012 proteins (clustered in 806 groups) with an estimated protein false discovery rate of ~3% were identified in 1 μg of yeast lysate in a single 160-min LC/MS/MS run. PMID:19566079

Optimized liquid chromatography tandem mass spectrometry approach for the determination of diquat and paraquat herbicides.

PubMed

Hao, Chunyan; Zhao, Xiaoming; Morse, David; Yang, Paul; Taguchi, Vince; Morra, Franca

2013-08-23

Liquid chromatography tandem mass spectrometry (LC-MS/MS) determination of quaternary ammonium herbicides diquat (DQ) and paraquat (PQ) can be very challenging due to their complicated chromatographic and mass spectrometric behaviors. Various multiple reaction monitoring (MRM) transitions from radical cations M(+) and singly charged cations [M-H](+), have been reported for LC-MS/MS quantitation under different chromatographic and mass spectrometric conditions. However, interference peaks were observed for certain previously reported MRM transitions in our study. Using a Dionex Acclaim(®) reversed-phase and HILIC mixed-mode LC column, we evaluated the most sensitive MRM transitions from three types of quasi-molecular ions of DQ and PQ, elucidated the cross-interference phenomena, and demonstrated that the rarely mentioned MRM transitions from dications M(2+) offered the best selectivity for LC-MS/MS analysis. Experimental parameters, such as IonSpray (IS) voltage, source temperature, declustering potential (DP), column oven temperature, collision energy (CE), acid and salt concentrations in the mobile phases were also optimized and an uncommon electrospray ionization (ESI) capillary voltage of 1000V achieved the highest sensitivity. Employing the proposed dication transitions 92/84.5 for DQ and 93/171 for PQ, the direct aqueous injection LC-MS/MS method developed was able to provide a method detection limit (MDL) of 0.1μg/L for the determination of these two herbicides in drinking water. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

A not-stop-flow online normal-/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry method for comprehensive lipid profiling of human plasma from atherosclerosis patients.

PubMed

Li, Min; Tong, Xunliang; Lv, Pu; Feng, Baosheng; Yang, Li; Wu, Zheng; Cui, Xinge; Bai, Yu; Huang, Yining; Liu, Huwei

2014-11-03

A not-stop-flow online two-dimensional (2D) liquid chromatography (LC) method was developed for comprehensive lipid profiling by coupling normal- and reversed-phase LC with quadrupole time-of-flight mass spectrometry (QToF-MS), which was then applied to separate and identify the lipid species in plasma, making its merits in quality and quantity of the detection of lipids. Total 540 endogenous lipid species from 17 classes were determined in human plasma, and the differences in lipid metabolism products in human plasma between atherosclerosis patients and control subjects were explored in detail. The limit of detections (LODs) of 19 validation standards could all reach ng/mL magnitude, and the RSDs of peak area and retention time ranged 0.4-8.0% and 0.010-0.47%, respectively. In addition, a pair of isomers, galactosylceramides (GalC) and glucosylceramides (GluC), was successfully separated, showing that only the levels of GalC in atherosclerosis patients were significantly increasing, rather than GluC, compared with the controls (controls vs. patients: the ratio was 1.5-2.8-fold increasing). It would be helpful to the further research of the atherosclerosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS.

PubMed

Kakitani, Ayano; Inoue, Tomonori; Matsumoto, Keiko; Watanabe, Jun; Nagatomi, Yasushi; Mochizuki, Naoki

2014-01-01

An LC-MS/MS method was developed for the simultaneous determination of 15 water-soluble vitamins that are widely used as additives in beverages and dietary supplements. This combined method involves the following simple pre-treatment procedures: dietary supplement samples were prepared by centrifugation and filtration after an extraction step, whereas beverage samples were diluted prior to injection. Chromatographic analysis in this method utilised a multi-mode ODS column, which provided reverse-phase, anion- and cation-exchange capacities, and therefore improved the retention of highly polar analytes such as water-soluble vitamins. Additionally, the multi-mode ODS column did not require adding ion pair reagents to the mobile phase. We optimised the chromatographic separation of 15 water-soluble vitamins by adjusting the mobile phase pH and the organic solvent. We also conducted an analysis of a NIST Standard Reference Material (SRM 3280 Multi-vitamin/Multi-element tablets) using this method to verify its accuracy. In addition, the method was applied to identify the vitamins in commercial beverages and dietary supplements. By comparing results with the label values and results obtained by official methods, it was concluded that the method could be used for quality control and to compose nutrition labels for vitamin-enriched products.

Structure-Activity Relationships of Agents Modifying Cholinergic Transmissions

DTIC Science & Technology

1983-09-01

t .Li ,.L,: "."c’S .!Cetylchoiine vithin a choliner .,-ic synapse. 3o*e poss~lle .n~r.Ic:,. .- .ire I lecrease the content of acecylcholine wiithia the... choliner .,iLc tu,.,I’, by •nterfering aith synthesis, (2) desensitizing cholinergic receptors I.t 7o-’--n..tic qites, (3) decreasing the release of...method of Potter et al (1983). This method uses reverse phase HPLC to .;eparate acetylcholine and choline . The effluent S emerging from the column is

Enhancing Sensitivity of Liquid Chromatography-Mass Spectrometry of Peptides and Proteins Using Supercharging Agents.

PubMed

Nshanian, Michael; Lakshmanan, Rajeswari; Chen, Hao; Ogorzalek Loo, Rachel R; Loo, Joseph A

2018-04-01

Trifluoroacetic acid (TFA) is often used as a mobile phase modifier to enhance reversed phase chromatographic performance. TFA adjusts solution pH and is an ion-pairing agent, but it is not typically suitable for electrospray ionization-mass spectrometry (ESI-MS) and liquid chromatography/MS (LC/MS) because of its significant signal suppression. Supercharging agents elevate peptide and protein charge states in ESI, increasing tandem MS (MS/MS) efficiency. Here, LC/MS protein supercharging was effected by adding agents to LC mobile phase solvents. Significantly, the ionization suppression generally observed with TFA was, for the most part, rescued by supercharging agents, with improved separation efficiency (higher number of theoretical plates) and lowered detection limits.

LC-MS/MS-based quantification of kynurenine metabolites, tryptophan, monoamines and neopterin in plasma, cerebrospinal fluid and brain.

PubMed

Fuertig, René; Ceci, Angelo; Camus, Sandrine M; Bezard, Erwan; Luippold, Andreas H; Hengerer, Bastian

2016-09-01

The kynurenine (KYN) pathway is implicated in diseases such as cancer, psychiatric, neurodegenerative and autoimmune disorders. Measurement of KYN metabolite levels will help elucidating the involvement of the KYN pathway in the disease pathology and inform drug development. Samples of plasma, cerebrospinal fluid or brain tissue were spiked with deuterated internal standards, processed and analyzed by LC-MS/MS; analytes were chromatographically separated by gradient elution on a C18 reversed phase analytical column without derivatization. We established an LC-MS/MS method to measure 11 molecules, namely tryptophan, KYN, 3-OH-KYN, 3-OH-anthranilic acid, quinolinic acid, picolinic acid, kynurenic acid, xanthurenic acid, serotonin, dopamine and neopterin within 5.5 min, with sufficient sensitivity to quantify these molecules in small sample volumes of plasma, cerebrospinal fluid and brain tissue.

Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry.

PubMed

Penney, Lisa; Smith, Anderson; Coates, Brent; Wijewickreme, Arosha

2005-01-01

A new liquid chromatography/mass spectrometry (LC/MS) method is presented for the determination of chloramphenicol (CAP) residues in milk, eggs, chicken muscle and liver, and beef muscle and kidney. CAP is extracted from the samples with acetonitrile and defatted with hexane. The acetonitrile extracts are then evaporated, and residues are reconstituted in 10mM ammonium acetate--acetonitrile mobile phase and injected into the LC system. CAP is determined by reversed-phase chromatography using an Inertsil ODS-2 column and MS detection with negative ion electrospray ionization. Calibration curves were linear between 0.5-5.0 ng/g for all matrixes studied. The relative standard deviations for measurements by this method were generally <12%, and average recoveries ranged from 80 to 120%, depending on the matrix involved. The method detection limits of CAP ranged from 0.2 to 0.6 ng/g, which are comparable to previously reported results. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 40 samples in a regular working day.

Development and validation of an LC-UV method for the quantification and purity determination of the novel anticancer agent C1311 and its pharmaceutical dosage form.

PubMed

den Brok, Monique W J; Nuijen, Bastiaan; Hillebrand, Michel J X; Grieshaber, Charles K; Harvey, Michael D; Beijnen, Jos H

2005-09-01

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. The pharmaceutical development of C1311 necessitated the availability of an assay for the quantification and purity determination of C1311 active pharmaceutical ingredient (API) and its pharmaceutical dosage form. A reversed-phase liquid chromatographic method (RP-LC) with ultraviolet (UV) detection was developed, consisting of separation on a C18 column with phosphate buffer (60 mM; pH 3 with 1 M citric acid)-acetonitrile-triethylamine (83:17:0.05, v/v/v) as the mobile phase and UV-detection at 280 nm. The method was found to be linear over a concentration range of 2.50-100 microg/mL, precise and accurate. Accelerated stress testing showed degradation products, which were well separated from the parent compound, confirming its stability-indicating capacity. Moreover, the use of LC-MS and on-line photo diode array detection enabled us to propose structures for four degradation products. Two of these products were also found as impurities in the API and more abundantly in an impure lot of API.

Extraction and LC determination of lysine clonixinate salt in water/oil microemulsions.

PubMed

Pineros, I; Ballesteros, P; Lastres, J L

2002-02-01

A new reversed-phase high performance liquid chromatography method has been developed and validated for the quantitative determination of lysine clonixinate salt in water/oil microemulsions. The mobile phase was acetonitrile-buffer phosphate pH 3.3. Detection was UV absorbance at 252 nm. The precision and accurately of the method were excellent. The established linearity range was 5-60 microg ml(-1) (r(2)=0.999). Microemulsions samples were dispersed with chloroform and extracted lysine clonixinate salt with water. This easy method employing chloroformic extraction has been done three times. The recovery of lysine clonixinate salt from spiked placebo and microemulsion were >90% over the linear range.

Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry.

PubMed

Ho, Emmie N M; Kwok, W H; Wong, April S Y; Wan, Terence S M

2012-01-13

Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction (SPE) using an ISOLUTE(®) CBA SPE column and analysed by LC/MS/MS in the positive electrospray ionisation mode. Separation of the 38 QADs was achieved on a polar group embedded C18 LC column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the 38 QADs at sub-ppb to low-ppb levels in equine urine could be achieved within 16 min using selected reaction monitoring (SRM). Matrix interference of the target transitions at the expected retention times was not observed. Other method validation data, including precision and recovery, were acceptable. The method was successfully applied to the analyses of drug-administration samples. Copyright © 2011 Elsevier B.V. All rights reserved.

ALS/FTD Mutation-Induced Phase Transition of FUS Liquid Droplets and Reversible Hydrogels into Irreversible Hydrogels Impairs RNP Granule Function

PubMed Central

Murakami, Tetsuro; Qamar, Seema; Lin, Julie Qiaojin; Schierle, Gabriele S. Kaminski; Rees, Eric; Miyashita, Akinori; Costa, Ana R.; Dodd, Roger B.; Chan, Fiona T.S.; Michel, Claire H.; Kronenberg-Versteeg, Deborah; Li, Yi; Yang, Seung-Pil; Wakutani, Yosuke; Meadows, William; Ferry, Rodylyn Rose; Dong, Liang; Tartaglia, Gian Gaetano; Favrin, Giorgio; Lin, Wen-Lang; Dickson, Dennis W.; Zhen, Mei; Ron, David; Schmitt-Ulms, Gerold; Fraser, Paul E.; Shneider, Neil A.; Holt, Christine; Vendruscolo, Michele; Kaminski, Clemens F.; St George-Hyslop, Peter

2015-01-01

Summary The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins. PMID:26526393

[Simultaneous determination of ochratoxin A, B and citrinin in foods by HPLC-FL and LC/MS/MS].

PubMed

Tabata, Setsuko; Iida, Kenji; Kimura, Keisuke; Iwasaki, Yumiko; Nakazato, Mitsuo; Kamata, Kunihiro; Hirokado, Masako

2008-04-01

Methods using high-performance liquid chromatography with fluorescence detection (HPLC-FL) and using liquid chromatography with tandem mass spectrometry (LC/MS/MS) were developed for simultaneous determination of ochratoxin A (OTA), ochratoxin B (OTB) and citrinin (CIT) in cereal, fruit, and coffee products. The samples were extracted with ethyl acetate under an acidic condition, and then cleaned up with liquid-liquid separation. The test solutions were analyzed by reverse-phase HPLC-FL and LC/MS/MS. Mass spectral acquisition was performed in positive ion mode by applying multiple reaction monitoring. The performances of both detectors were almost equivalent. The recoveries of OTA and OTB were 87-111%, and that of CIT were 70-88%. The limits of quantification (S/N> or =10) of OTA, OTB and CIT was 0.1 mug/kg or less. These methods were considered to be useful for the determination of the three mycotoxins at low levels (0.1 microg/kg).

Development and validation of LC-MS/MS method for the determination of Ochratoxin A and its metabolite Ochratoxin α in poultry tissues and eggs.

PubMed

Paoloni, Angela; Solfrizzo, Michele; Bibi, Rita; Pecorelli, Ivan

2018-05-04

The objective of this study was to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of Ochratoxin A (OTA) and Ochratoxin α (OTα) in poultry tissues and eggs. The two toxins were extracted by a mixture of acetonitrile/water, purified with a reversed phase C18 solid phase extraction column (SPE) and determined by LC-MS/MS. The LC-MS/MS method performances were evaluated in terms of linearity in solvent and in matrix (ranged from 0.5 to 15.10 µg L -1 for OTA and from 0.60 to 17.85 µg L -1 for OTα), limit of detection (LOD), limit of quantitation (LOQ), specificity, accuracy and precision in repeatability conditions. Recovery experiments were performed by spiking poultry liver, kidney, muscle and eggs around 1 µg kg -1 and 10 µg kg -1 . LODs were 0.27 and 0.26 µg kg -1 while LOQs were fixed at 1.0 and 1.2 µg kg -1 for OTA and OTα, respectively. Main recoveries for OTA ranged from 82 to 109% and for OTα ranged from 55 to 89%. The values of within-laboratory relative standard deviation (RSD r ) were equal to or below 20%. Considering the results obtained and that all analytical performance criteria were fulfilled, the new extraction and purification method developed for OTA and OTα determination in animal tissues and eggs was found appropriate for control laboratories and research activities designed to ensure food safety.

Determination of Aconitum Alkaloids in Dietary Supplements and Raw Botanical Materials by Liquid Chromatography/UV Detection with Confirmation by Liquid Chromatography/Tandem Mass Spectrometry: Collaborative Study

PubMed Central

Wong, Siu-Kay

2010-01-01

An interlaboratory study was conducted to evaluate a method for the determination of 3 Aconitum alkaloids, viz., aconitine, mesaconitine, and hypaconitine, in raw botanical material and dietary supplements. The alkaloids were extracted with diethyl ether in the presence of ammonia. After cleanup by solid-phase extraction to remove matrix interferences, the alkaloids were determined by reversed-phase liquid chromatography (LC)/UV detection at 235 nm with confirmation by LC/tandem mass spectrometry (MS/MS). A total of 14 blind duplicates were successfully analyzed by 12 collaborators. For repeatability, the relative standard deviation (RSDr) values ranged from 1.9 to 16.7%, and for reproducibility, the RSDR values ranged from 6.5 to 33%. The HorRat values were all <2 with only one exception at 2.3. All collaborating laboratories had calibration curves with correlation coefficients of >0.998. In addition, 6 collaborators performed the confirmation and were able to verify the identities of the alkaloids by using LC/MS/MS. PMID:19382567

One-pot preparation of a mixed-mode organic-silica hybrid monolithic capillary column and its application in determination of endogenous gibberellins in plant tissues.

PubMed

Zhang, Zheng; Hao, Yan-Hong; Ding, Jun; Xu, Sheng-Nan; Yuan, Bi-Feng; Feng, Yu-Qi

2015-10-16

A newly improved one-pot method, based on "thiol-ene" click chemistry and sol-gel approach in microemulsion system, was developed for the preparation of C8/PO(OH)2-silica hybrid monolithic capillary column. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The monolithic column was demonstrated to have cation exchange/reversed-phase (CX/RP) mixed-mode retention for analytes on nano-liquid chromatography (nano-LC). On the basis of the developed nano-LC system with MS detector coupled to pipette tip solid phase extraction (PT-SPE) and derivatization process, we then realized simultaneous determination of 10 gibberellins (GAs) with low limits of detection (LODs, 0.003-0.025 ng/mL). Furthermore, 6 endogenous GAs in only 5mg rice leaves (fresh weight) were successfully detected and quantified. The developed PT-SPE-nano-LC-MS strategy may offer promising applications in the determination of low abundant bioactive molecules from complex matrix. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of aliskiren in tablet dosage forms by a validated stability-indicating RP-LC method.

PubMed

Wrasse-Sangoi, M; Sangoi, M S; Oliveira, P R; Secretti, L T; Rolim, C M B

2011-02-01

A reversed-phase liquid chromatography (RP-LC) method is validated for the determination of aliskiren in tablet dosage form. The LC method is carried out on a Waters XBridge C(18) column (150 × 4.6 mm i.d.), maintained at 25°C. The mobile phase consisted of acetonitrile:water (95:5, v/v)/phosphoric acid (25 mM, pH 3.0) (40:60, v/v), run at a flow rate of 1.0 mL/min, with photodiode array detector set at 229 nm. The chromatographic separation is obtained with aliskiren retention time of 3.68 min, and it is linear in the range of 10-300 μg/mL (r = 0.9999). The limits of detection and quantitation are 2.38 and 7.93 μg/mL, respectively. The specificity and stability-indicating capability of the method are proven through degradation studies, which also showed that there is no interference of the formulation excipients, showing that peak is free from any coeluting peak. The method showed adequate precision, with a relative standard deviation (RSD) values lower than 0.92%. Good values of accuracy were also obtained, with a mean value of 99.55%. Experimental design is used during validation to calculate method robustness. The proposed method is applied for the analysis of the tablet dosage forms, contributing to improve the quality control and to assure the therapeutic efficacy.

NOVEL CONTINUOUS PH/SALT GRADIENT AND PEPTIDE SCORE FOR STRONG CATION EXCHANGE CHROMATOGRAPHY IN 2D-NANO-LC/MSMS PEPTIDE IDENTIFICATION FOR PROTEOMICS

EPA Science Inventory

Tryptic digests of human serum albumin (HSA) and human lung epithelial cell lysates were used as test samples in a novel proteomics study. Peptides were separated and analyzed using 2D-nano-LC/MSMS with strong cation exchange (SCX) and reverse phase (RP) chromatography and contin...

Novel angiotensin I-converting enzyme inhibitory peptides produced in fermented milk by specific wild Lactococcus lactis strains.

PubMed

Rodríguez-Figueroa, J C; González-Córdova, A F; Torres-Llanez, M J; Garcia, H S; Vallejo-Cordoba, B

2012-10-01

The ability of specific wild Lactococcus lactis strains to hydrolyze milk proteins to release angiotensin I-converting enzyme (ACE) inhibitory peptides was evaluated. The peptide profiles were obtained from the <3 kDa water-soluble extract and subsequently fractionated by reversed-phase HPLC. The fractions with the lowest half-maximal inhibitory concentration estimated values (peptide concentration necessary to inhibit ACE activity by 50%) were Lc. lactis NRRL B-50571 fraction (F)1 (0.034 ± 0.002 μg/mL; mean ± SD) and Lc. lactis NRRL B-50572B F 0005 (0.041 ± 0.003 μg/mL; mean ± SD). All peptide fractions were analyzed by reversed-phase HPLC tandem mass spectrometry. Twenty-one novel peptide sequences associated with ACE inhibitory (ACEI) activity were identified. Several novel ACEI peptides presented peptides encrypted with proven hypotensive activity. In conclusion, specific wild Lc. lactis strains were able to hydrolyze milk proteins to generate potent ACEI peptides. However, further studies are necessary to find out the relationship between Lc. lactis strain proteolytic systems and their ability to biogenerate hypotensive peptides. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Self-assembled lecithin/chitosan nanoparticles for oral insulin delivery: preparation and functional evaluation.

PubMed

Liu, Liyao; Zhou, Cuiping; Xia, Xuejun; Liu, Yuling

2016-01-01

Here, we investigated the formation and functional properties of self-assembled lecithin/chitosan nanoparticles (L/C NPs) loaded with insulin following insulin-phospholipid complex preparation, with the aim of developing a method for oral insulin delivery. Using a modified solvent-injection method, insulin-loaded L/C NPs were obtained by combining insulin-phospholipid complexes with L/C NPs. The nanoparticle size distribution was determined by dynamic light scattering, and morphologies were analyzed by cryogenic transmission electron microscopy. Fourier transform infrared spectroscopy analysis was used to disclose the molecular mechanism of prepared insulin-loaded L/C NPs. Fast ultrafiltration and a reversed-phase high-performance liquid chromatography assay were used to separate free insulin from insulin entrapped in the L/C NPs, as well as to measure the insulin-entrapment and drug-loading efficiencies. The in vitro release profile was obtained, and in vivo hypoglycemic effects were evaluated in streptozotocin-induced diabetic rats. Our results indicated that insulin-containing L/C NPs had a mean size of 180 nm, an insulin-entrapment efficiency of 94%, and an insulin-loading efficiency of 4.5%. Cryogenic transmission electron microscopy observations of insulin-loaded L/C NPs revealed multilamellar structures with a hollow core, encircled by several bilayers. In vitro analysis revealed that insulin release from L/C NPs depended on the L/C ratio. Insulin-loaded L/C NPs orally administered to streptozotocin-induced diabetic rats exerted a significant hypoglycemic effect. The relative pharmacological bioavailability following oral administration of L/C NPs was 6.01%. With the aid of phospholipid-complexation techniques, some hydrophilic peptides, such as insulin, can be successfully entrapped into L/C NPs, which could improve oral bioavailability, time-dependent release, and therapeutic activity.

Determination of imidacloprid, metalaxyl, myclobutanil, propham, and thiabendazole in fruits and vegetables by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry.

PubMed

Pous, X; Ruíz, M J; Picó, Y; Font, G

2001-09-01

Imidacloprid, metalaxyl, myclobutanil, propham, and thiabendazole have been simultaneously determined in strawberries, oranges, potatoes, pears, and melons by matrix solid-phase dispersion (MSPD) followed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) in positive-ion mode. The samples were homogenized with C8 bonded silica as MSPD sorbent, placed in a glass column, and eluted with dichloromethane. Chromatographic separation of the compounds was achieved on a reversed-phase LC column using a methanol-ammonium formate (50 mmol L(-1)) gradient as a mobile phase. Samples were screened by monitoring the protonated molecular ion at m/z 256 for imidacloprid, 280 for metalaxyl, 289 for myclobutanil, and 202 for thiabendazole, and the main fragment at m/z 138 for propham. Positive samples were confirmed by multiple-ion monitoring. The repeatability (<20%) and recovery (>57%) of the method were good, and limits of detection (<0.05 mg kg(-1)) were adequate.

A validated LC method for determination of 2,3-dichlorobenzoic acid and its associated regio isomers.

PubMed

Krishnaiah, Ch; Sri, Khagga Bhavya

2012-05-01

A simple, selective and sensitive gradient reversed-phase liquid chromatography method has been developed for the separation and determination of 2,3-dichlorobenzoic acid, which is an intermediate of the lamotrizine drug substance, and its regio isomers. The separation was achieved on a reversed-phase United States Pharmacopeia L1 (C-18) column using 0.01 M ammonium acetate buffer at pH 2.5 and methanol (50:50 v/v) mixture as mobile phase A and a methanol and water mixture (80:20 v/v) as mobile phase B in a gradient elution at flow rate 1.2 mL/min with ultraviolet detection at 210 nm. The method is found to be selective, precise, linear, accurate and robust. It was used for quality assurance and monitoring the synthetic reactions involved in the process development of lamotrizine. The method is found to be simple, rapid, specific and reliable for the determination of unreacted levels of raw materials and isomers in reaction mixtures and finished product lamotrizine. The method was fully validated as per International Conference of Harmonization guidelines and results from validation confirm that the method is highly suitable for its intended purpose. © The Author [2012]. Published by Oxford University Press. All rights reserved.

Analysis of Soft Drinks: UV Spectrophotometry, Liquid Chromatography, and Capillary Electrophoresis

NASA Astrophysics Data System (ADS)

McDevitt, Valerie L.; Rodriguez, Alejandra; Williams, Kathryn R.

1998-05-01

Instrumental analysis students analyze commercial soft drinks in three successive laboratory experiments. First, UV multicomponent analysis is used to determine caffeine and benzoic acid in Mello YelloTM using the spectrophotometer's software and manually by the simultaneous equations method. The following week, caffeine, benzoic acid and aspartame are determined in a variety of soft drinks by reversed-phase liquid chromatography using 45% methanol/55% aqueous phosphate, pH 3.0, as the mobile phase. In the third experiment, the same samples are analyzed by capillary electrophoresis using a pH 9.4 borate buffer. Students also determine the minimum detection limits for all three compounds by both LC and CE. The experiments demonstrate the analytical use and limitations of the three instruments. The reports and prelab quizzes also stress the importance of the chemistry of the three compounds, especially the relationships of acid/base behavior and polarity to the LC and CE separations.

Liquid chromatographic determination of benzo(a)pyrene in total particulate matter of cigarette smoke

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomkins, B.A.; Jenkins, R.A.; Griest, W.H.

The benzo(a)pyrene (BaP) delivery of reference and commercially available tobacco cigarettes, as well as reference and placebo marijuana cigarettes, is determined using a sequential liquid chromatographic/liquid chromatographic procedure. The total particulate matter of sample cigarette smoke is collected using a Cambridge filter pad, which is ultrasonically extracted with acetone. The resulting extract is filtered, then fractionated using semipreparative-scale normal phase liquid chromatography (LC). Quantitative determination is achieved using analytical-scale reverse phase LC equipped with a fluorescence detector. The method is precise (+/- 10-15% relative standard deviation) and yields 85% or better BaP recovery at the ng/cig. level. A single padmore » may be analyzed in 8 person-hours, while a more typical lot of 12 pads (6 pads each for 2 cigarette brands) may be analyzed in 10 person-days.« less

Annotation of the human serum metabolome by coupling three liquid chromatography methods to high-resolution mass spectrometry.

PubMed

Boudah, Samia; Olivier, Marie-Françoise; Aros-Calt, Sandrine; Oliveira, Lydie; Fenaille, François; Tabet, Jean-Claude; Junot, Christophe

2014-09-01

This work aims at evaluating the relevance and versatility of liquid chromatography coupled to high resolution mass spectrometry (LC/HRMS) for performing a qualitative and comprehensive study of the human serum metabolome. To this end, three different chromatographic systems based on a reversed phase (RP), hydrophilic interaction chromatography (HILIC) and a pentafluorophenylpropyl (PFPP) stationary phase were used, with detection in both positive and negative electrospray modes. LC/HRMS platforms were first assessed for their ability to detect, retain and separate 657 metabolite standards representative of the chemical families occurring in biological fluids. More than 75% were efficiently retained in either one LC-condition and less than 5% were exclusively retained by the RP column. These three LC/HRMS systems were then evaluated for their coverage of serum metabolome. The combination of RP, HILIC and PFPP based LC/HRMS methods resulted in the annotation of about 1328 features in the negative ionization mode, and 1358 in the positive ionization mode on the basis of their accurate mass and precise retention time in at least one chromatographic condition. Less than 12% of these annotations were shared by the three LC systems, which highlights their complementarity. HILIC column ensured the greatest metabolome coverage in the negative ionization mode, whereas PFPP column was the most effective in the positive ionization mode. Altogether, 192 annotations were confirmed using our spectral database and 74 others by performing MS/MS experiments. This resulted in the formal or putative identification of 266 metabolites, among which 59 are reported for the first time in human serum. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of large volume injection of aliphatic alcohols as sample diluents on the retention of low hydrophobic solutes in reversed-phase liquid chromatography.

PubMed

David, Victor; Galaon, Toma; Aboul-Enein, Hassan Y

2014-01-03

Recent studies showed that injection of large volume of hydrophobic solvents used as sample diluents could be applied in reversed-phase liquid chromatography (RP-LC). This study reports a systematic research focused on the influence of a series of aliphatic alcohols (from methanol to 1-octanol) on the retention process in RP-LC, when large volumes of sample are injected on the column. Several model analytes with low hydrophobic character were studied by RP-LC process, for mobile phases containing methanol or acetonitrile as organic modifiers in different proportions with aqueous component. It was found that starting with 1-butanol, the aliphatic alcohols can be used as sample solvents and they can be injected in high volumes, but they may influence the retention factor and peak shape of the dissolved solutes. The dependence of the retention factor of the studied analytes on the injection volume of these alcohols is linear, with a decrease of its value as the sample volume is increased. The retention process in case of injecting up to 200μL of upper alcohols is dependent also on the content of the organic modifier (methanol or acetonitrile) in mobile phase. Copyright © 2013 Elsevier B.V. All rights reserved.

[Simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using solid phase extraction-reversed-phase high performance liquid chromatography].

PubMed

Zhang, Hua; Yang, Xin; Ma, Ying; Dong, Aijun; Zhang, Yingchun

2008-05-01

A method was developed for the simultaneous determination of canthaxanthin and astaxanthin in feedstuffs using reversed-phase high performance liquid chromatography (RP-HPLC). The sample was extracted by acetonitrile, and cleaned up by an LC-NH2 column. An Agilent ZORBAX Eclipse XDB-C18 analytical column (150 mm x 4.6 mm, 5 microm) was used and kept at 25 degrees C. Acetonitrile-methanol (95 : 5, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min. The detection was performed by a diode array detector at 474 nm. The quantitive analysis of external standard calibration curves was used. The linear ranges of the method for canthaxanthin and astaxanthin were 1.0 - 30.0 mg/L (r = 0.999 0) and 1.0 - 20.0 mg/L (r = 0.999 1), respectively. The average recoveries were 90% - 101% with the relative standard deviations of 0.62% - 3.68%. The detection limits were 0.84 and 0.60 mg/L for canthaxanthin and astaxanthin, respectively. The method is simple, precise, sensitive and reproductive. It can be used to determine the contents of canthaxanthin and astaxanthin in feedstuffs.

Liquid chromatographic method for determining the concentration of bisazir in water

USGS Publications Warehouse

Scholefield, Ronald J.; Slaght, Karen S.; Allen, John L.

1997-01-01

Barrier dams, traps, and lampricides are the techniques currently used by the Great Lakes Fishery Commission to control sea lampreys (Petromyzon marinus) in the Great Lakes. To augment these control techniques, a sterile-male-release research program was initiated at the Lake Huron Biological Station. Male sea lampreys were sterilized by intraperitoneal injection of the chemical sterilant P,P-bis(1-aziridinyl)-N-methylphosphinothioic amide (bisazir). An analytical method was needed to quantitate the concentration of bisazir in water and to routinely verify that bisazir (>25 μg/L) does not persist in the treated effluent discharged from the sterilization facility to Lake Huron. A rapid, accurate, and sensitive liquid chromatographic (LC) method was developed for determining bisazir in water. Bisazir was dissolved in Lake Huron water; extracted and concentrated on a C18 solid-phase extraction column; eluted with methanol; and quantitated by reversed-phase LC using a C18 column, a mobile phase of 70% water and 30% methanol (v/v), and UV detection (205 nm). Bisazir retention time was 7-8 min; total run time was about 20 min. Method detection limit for bisazir dissolved in Lake Huron water was about 15 μg/L. Recovery from Lake Huron water fortified with bisazir at 100 μg/L was 94% (95% confidence interval, 90.2-98.2%).

Reverse-phase liquid chromatography with electrospray ionization/mass spectrometry for the quantification of pseudoephedrine in human plasma and application to a bioequivalence study.

PubMed

Kim, Jin-Ki; Jee, Jun-Pil; Park, Jeong-Sook; Kim, Hyung Tae; Kim, Chong-Kook

2011-01-01

A sensitive and selective reverse-phase liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) method was developed and validated to quantify pseudoephedrine (CAS 90-82-4) in human plasma. Phenacetin was used as the internal standard (I.S.). Sample preparation was performed with a deproteinization step using acetonitrile. Pseudoephedrine and I.S. were successfully separated using gradient elution with 0.5% trifluoroacetic acid (TFA) in water and 0.5% TFA in methanol at a flow-rate of 0.2 mL/min. Detection was performed on a single quadrupole mass spectrometer by a selected ion monitoring (SIM) mode via electrospray ionization (ESI) source. The ESI source was set at positive ionization mode. The ion signals of m/z 166.3 and 180.2 were measured for the protonated molecular ions of pseudoephedrine and I.S., respectively. The lower limit of quantification (LLOQ) of pseudoephedrine in human plasma was 10 ng/mL and good linearity was observed in the range of concentrations 10-500 ng/mL (R2 = 1). The intra-day accuracy of the drug containing plasma samples was more than 97.60% with a precision of 3.99-11.82%. The inter-day accuracy was 99.36% or more, with a precision of 7.65-18.42%. By using this analytical method, the bioequivalence study of the pseudoephedrine preparation was performed and evaluated by statistical analysis of the log transformed mean ratios of pharmacokinetic parameters. All the results fulfilled the standard criteria of bioequivalence, being within the 80-125% range which is required by the Korea FDA, US FDA, and EMEA to conclude bioequivalence. Consequently, the developed reverse-phase LC-ESI-MS method was successfully applied to bioequivalence studies of pseudoephedrine in healthy male volunteers.

Determination of target fat-soluble micronutrients in rainbow trout's muscle and liver tissues by liquid chromatography with diode array-tandem mass spectrometry detection.

PubMed

Pérez Fernández, Virginia; Ventura, Salvatore; Tomai, Pierpaolo; Curini, Roberta; Gentili, Alessandra

2017-03-01

This paper describes an analytical approach, based on LC-diode array detector-MS/MS (LC-DAD-MS/MS), for characterizing the fat-soluble micronutrient fraction in rainbow trout (Oncorhynchus mykiss). Two different procedures were applied to isolate the analytes from liver and muscle tissue: overnight cold saponification to hydrolyze bound forms and to simplify the analysis; matrix solid-phase dispersion to avoid artifacts and to maintain unaltered the naturally occurring forms. Analytes were separated on a C 30 analytical column by using a nonaqueous reversed mobile phase compatible with the atmospheric pressure chemical ionization. Compared to other works, the most relevant advantage of the here illustrated method is the large amount of information obtained with few analytical steps: nine fat-soluble vitamins (3,4-dehydroretinol, retinol, cholecalciferol, ergocalciferol, α-tocopherol, γ-tocopherol, δ-tocopherol, phylloquinone, and menaquinone-4) and eight carotenoids (all-trans-lutein, all-trans-astaxanthin, all-trans-zeaxanthin, all-trans-β-cryptoxanthin, all-trans-canthaxanthin, all-trans-ζ-carotene, all-trans-β-carotene, and all-trans-γ-carotene) were quantified after the method validation, while other untargeted carotenoids were tentatively identified by exploiting the identification power of the LC-DAD-MS/MS hyphenation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Light-melt adhesive based on dynamic carbon frameworks in a columnar liquid-crystal phase

NASA Astrophysics Data System (ADS)

Saito, Shohei; Nobusue, Shunpei; Tsuzaka, Eri; Yuan, Chunxue; Mori, Chigusa; Hara, Mitsuo; Seki, Takahiro; Camacho, Cristopher; Irle, Stephan; Yamaguchi, Shigehiro

2016-07-01

Liquid crystal (LC) provides a suitable platform to exploit structural motions of molecules in a condensed phase. Amplification of the structural changes enables a variety of technologies not only in LC displays but also in other applications. Until very recently, however, a practical use of LCs for removable adhesives has not been explored, although a spontaneous disorganization of LC materials can be easily triggered by light-induced isomerization of photoactive components. The difficulty of such application derives from the requirements for simultaneous implementation of sufficient bonding strength and its rapid disappearance by photoirradiation. Here we report a dynamic molecular LC material that meets these requirements. Columnar-stacked V-shaped carbon frameworks display sufficient bonding strength even during heating conditions, while its bonding ability is immediately lost by a light-induced self-melting function. The light-melt adhesive is reusable and its fluorescence colour reversibly changes during the cycle, visualizing the bonding/nonbonding phases of the adhesive.

Lipidomic analysis of plasma in patients with lacunar infarction using normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry.

PubMed

Yang, Li; Lv, Pu; Ai, Wanpeng; Li, Linnan; Shen, Sensen; Nie, Honggang; Shan, Yabing; Bai, Yu; Huang, Yining; Liu, Huwei

2017-05-01

Stroke is a major cause of mortality and long-term disability worldwide. The study of biomarkers and pathogenesis is vital for early diagnosis and treatment of stroke. In the present study, a continuous-flow normal-phase/reversed-phase two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (NP/RP 2D LC-QToF/MS) method was employed to measure lipid species in human plasma, including healthy controls and lacunar infarction (LI) patients. As a result, 13 lipid species were demonstrated with significant difference between the two groups, and a "plasma biomarker model" including glucosylceramide (38:2), phosphatidylethanolamine (35:2), free fatty acid (16:1), and triacylglycerol (56:5) was finally established. This model was evaluated as an effective tool in that area under the receiver operating characteristic curve reached 1.000 in the discovery set and 0.947 in the validation set for diagnosing LI patients from healthy controls. Besides, the sensitivity and specificity of disease diagnosis in validation set were 93.3% and 96.6% at the best cutoff value, respectively. This study demonstrates the promising potential of NP/RP 2D LC-QToF/MS-based lipidomics approach in finding bio-markers for disease diagnosis and providing special insights into the metabolism of stroke induced by small vessel disease. Graphical abstract Flow-chart of the plasma biomarker model establishment through biomarker screening and validation.

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase.

PubMed

Lv, Haitao; Palacios, Gustavo; Hartil, Kirsten; Kurland, Irwin J

2011-04-01

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Identification of polar, ionic, and highly water soluble organic pollutants in untreated industrial wastewaters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castillo, M.; Alonso, M.C.; Riu, J.

1999-04-15

This paper presents a generic protocol for the determination of polar, ionic, and highly water soluble organic pollutants on untreated industrial wastewaters involving the use of two different solid-phase extraction (SPE) methodologies followed by liquid chromatography-mass spectrometry (LC-MS). Untreated industrial wastewaters might contain natural and synthetic dissolved organic compounds with total organic carbon (TOC) values varying between 100 and 3000 mg/L. All polar, ionic and highly water soluble compounds comprising more than 95% of the organic content and with major contribution to the total toxicity of the sample cannot be analyzed by conventional gas chromatography-mass spectrometry (GC-MS), and LC-MS ismore » a good alternative. In this work two extraction procedures were used to obtain fractionated extracts of the nonionic polar compounds: a polymeric Isolute ENV + SPE cartridge for the preconcentration of anionic analytes and a sequential solid-phase extraction (SSPE) method percolating the samples first in octadecylsilica cartridge in series with the polymeric Lichrolut EN cartridge. Average recoveries ranging from 72% to 103% were obtained for a variety of 23 different analytes. Determination of nonionic pollutants was accomplished by reverse-phase liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS), while anionic compounds were analyzed by ion pair chromatography-electrospray-mass spectrometry (IP-ESI-MS) and LC-ESI-MS. This protocol was applied to a pilot survey of textile and tannery wastewaters leading to the identification and quantification of 33 organic pollutants.« less

Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos.

PubMed

Lebold, Katie M; Kirkwood, Jay S; Taylor, Alan W; Choi, Jaewoo; Barton, Carrie L; Miller, Galen W; La Du, Jane; Jump, Donald B; Stevens, Jan Frederik; Tanguay, Robert L; Traber, Maret G

2013-01-01

To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π-π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography-mass spectrometry (LC-MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E- or E+ diets for 4 months, and then were spawned to obtain E- and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E- (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.

Analysis of imazaquin in soybeans by solid-phase extraction and high-performance liquid chromatography.

PubMed

Guo, C; Hu, J-Y; Chen, X-Y; Li, J-Z

2008-02-01

An analytical method for the determination imazaquin residues in soybeans was developed. The developed liquid/liquid partition and strong anion exchange solid-phase extraction procedures provide the effective cleanup, removing the greatest number of sample matrix interferences. By optimizing mobile-phase pH water/acetonitrile conditions with phosphoric acid, using a C-18 reverse-phase chromatographic column and employing ultraviolet detection, excellent peak resolution was achieved. The combined cleanup and chromatographic method steps reported herein were sensitive and reliable for determining the imazaquin residues in soybean samples. This method is characterized by recovery >88.4%, precision <6.7% CV, and sensitivity of 0.005 ppm, in agreement with directives for method validation in residue analysis. Imazaquin residues in soybeans were further confirmed by high performance liquid chromatography-mass spectrometry (LC-MS). The proposed method was successfully applied to the analysis of imazaquin residues in soybean samples grown in an experimental field after treatments of imazaquin formulation.

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC-MS/MS and its application to a pharmacokinetic study.

PubMed

Bae, Jung-Woo; Choi, Chang-Ik; Jang, Choon-Gon; Lee, Seok-Yong

2011-11-01

A simple and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed and validated for the determination of sibutramine and its N-desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t-butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse-phase Luna C18 column with a mobile phase of acetonitrile-10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI-MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05-20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra- and inter-day validation for sibutramine, M1 and M2 were acceptable. This LC-MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.

Is nothing sacred?

USGS Publications Warehouse

Hoffman, G.L.

1980-01-01

N-sodium-N-chloro-rho-toluenesulfonamide (chloramine-T) effectively controls bacterial gill disease (BGD) in cultured fishes, BGD, a common disease of hatchery-reared salmonids, causes more fish losses than any other disease among these species, This study describes a liquid chromatographic (LC) method that is capable of direct, simultaneous analysis of chloramine-T and its primary degradation product, rho-toluenesulfonamide (rho-TSA), in water. The procedure involves reversed-phase (C-18) LC analysis with ion suppression, using 0.01 M phosphate buffer at pH 3. The mobile phase is phosphate buffer-acetonitrile (60 + 40) at 1 mL/min. Both chemicals can be detected with a UV spectrophotometer at 229 nm; the method is linear up to 40 mg, chloramine-T or rho-TSA/L. Mean recoveries were 96.4 +/- 6.1% for water samples fortified with 0.03 mg chloramine-T/L and 95.3 +/- 4.6% for water samples fortified with 0.005 mg rho-TSA/L. Limits of detection without sample enrichment for chloramine-T and rho-TSA are 0.01 mg/L and 0.001 mg/L, respectively.

Liquid chromatographic determination of chloramine-T and its primary degradation product, p-toluenesulfonamide, in water

USGS Publications Warehouse

Dawson, Verdel K.; Davis, Ruth A.

1997-01-01

N-sodium-N-chloro-rho-toluenesulfonamide (chloramine-T) effectively controls bacterial gill disease (BGD) in cultured fishes, BGD, a common disease of hatchery-reared salmonids, causes more fish losses than any other disease among these species. This study describes a liquid chromatographic (LC) method that is capable of direct, simultaneous analysis of chloramine-T and its primary degradation product, rho-toluenesulfonamide (rho-TSA), in water. The procedure involves reversed-phase (C-18) LC analysis with ion suppression, using 0.01 M phosphate buffer at pH 3. The mobile phase is phosphate buffer-acetonitrile (60 + 40) at 1 mL/min. Both chemicals can be detected with a UV spectrophotometer at 229 nm; the method is linear up to 40 mg, chloramine-T or rho-TSA/L. Mean recoveries were 96.4 +/- 6.1% for water samples fortified with 0.03 mg chloramine-T/L and 95.3 +/- 4.6% for water samples fortified with 0.005 mg rho-TSA/L. Limits of detection without sample enrichment for chloramine-T and rho-TSA are 0.01 mg/L and 0.001 mg/L, respectively.

Liquid chromatographic determination of chloramine-T and its primary degradation product, p-toluenesulfonamide, in water

USGS Publications Warehouse

Dawson, V.K.; Davis, R.A.

1997-01-01

N-sodium-N-chloro-rho-toluenesulfonamide (chloramine-T) effectively controls bacterial gill disease (BGD) in cultured fishes, BGD, a common disease of hatchery-reared salmonids, causes more fish losses than any other disease among these species, This study describes a liquid chromatographic (LC) method that is capable of direct, simultaneous analysis of chloramine-T and its primary degradation product, rho-toluenesulfonamide (rho-TSA), in water. The procedure involves reversed-phase (C-18) LC analysis with ion suppression, using 0.01 M phosphate buffer at pH 3. The mobile phase is phosphate buffer-acetonitrile (60 + 40) at 1 mL/min. Both chemicals can be detected with a UV spectrophotometer at 229 nm; the method is linear up to 40 mg, chloramine-T or rho-TSA/L. Mean recoveries were 96.4 +/- 6.1% for water samples fortified with 0.03 mg chloramine-T/L and 95.3 +/- 4.6% for water samples fortified with 0.005 mg rho-TSA/L. Limits of detection without sample enrichment for chloramine-T and rho-TSA are 0.01 mg/L and 0.001 mg/L, respectively.

Comprehensive Two-Dimensional Hydrophilic Interaction Chromatography (HILIC) × Reversed-Phase Liquid Chromatography Coupled to High-Resolution Mass Spectrometry (RP-LC-UV-MS) Analysis of Anthocyanins and Derived Pigments in Red Wine.

PubMed

Willemse, Chandré M; Stander, Maria A; Vestner, Jochen; Tredoux, Andreas G J; de Villiers, André

2015-12-15

Changes in anthocyanin chemistry represent some of the most important transformations involved in red wine aging. However, accurate analysis of the derived pigments, as required to study the evolution of anthocyanins and tannins during aging, is hampered by their extreme structural diversity, low levels, and the fact that many of these compounds have identical mass spectral characteristics. In this context, chromatographic separation is critical. In this contribution, the application of online hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography (RP-LC) separation coupled to high-resolution mass spectrometry (MS) is described for the detailed characterization of anthocyanins and their derived pigments in aged red wine. A systematic approach was followed for the optimization of HILIC × RP-LC separation parameters using a capillary liquid chromatography (LC) system in the first dimension and an ultrahigh-pressure LC system in the second dimension to ensure maximum sensitivity and performance. Ninety four (94) anthocyanin-derived pigments were tentatively identified in one- and six-year-old Pinotage wines using accurate mass and fragmentation information obtained using quadrupole-time-of-flight mass spectrometry (Q-TOF-MS). Online HILIC × RP-LC-MS was found to offer high-resolution separation, because of the combination of two different separation modes, while the structured elution order observed improved the certainty in compound identification. Therefore, this approach shows promise for the detailed elucidation of the chemical alteration of anthocyanins during wine aging.

Simultaneous determination of diclofenac potassium and methocarbamol in ternary mixture with guaifenesin by reversed phase liquid chromatography.

PubMed

Elkady, Ehab F

2010-09-15

New, simple, rapid and precise reversed phase liquid chromatographic (RP-LC) method has been developed for the simultaneous determination of diclofenac potassium (DP) and methocarbamol (MT) in ternary mixture with guaifenesin (GF), degradation product of methocarbamol. Chromatographic separation was achieved on a Symmetry Waters C18 column (150 mm x 4. 6mm, 5 microm). Gradient elution based on phosphate buffer pH (8)-acetonitrile at a flow rate of 1 mL min(-1) was applied. The UV detector was operated at 282 nm for DP and 274 nm for MT and GF. Linearity, accuracy and precision were found to be acceptable over the concentration ranges of 0.05-16, 0.5-160 and 0.5-160 microg mL(-1) for DP, MT and GF, respectively. The optimized method proved to be specific, robust and accurate for the quality control of the cited drugs in pharmaceutical preparation. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Rapid determination of ampicillin in bovine milk by liquid chromatography with fluorescence detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ang, C.Y.W.; Luo, Wenhong

1997-01-01

A rapid and sensitive liquid chromatographic (LC) method was developed for the determination of ampicillin residues in raw bovine milk, processed skim milk, and pasteurized, homogenized whole milk with vitamin D. Milk samples were deproteinized with trichloroacetic acid (TCA) and acetonictrile. After centrifugation, the clear supernatant was reacted with formaldehyde and TCA under heat. The major fluorescent derivative of ampicillin was then determined by reversed-phase LC with fluorescence detection. Average recoveries of ampicillin fortified at 5, 10, and 20 ppb (ng/mL) were all >85% with coefficients of variation <10%. Limits of detection ranged from 0.31 to 0.51 ppb and limitsmore » of quantitation, from 0.66 to 1.2 ppb. After appropriate validation, this method should be suitable for rapid analysis of milk for ampicillin residues at the tolerance level of 10 ppb. 16 refs., 4 figs., 3 tabs.« less

[Separation and identification of bovine lactoferricin by high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/ time of flight mass spectrometry].

PubMed

An, Meichen; Liu, Ning

2010-02-01

A high performance liquid chromatography-matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (HPLC-MALDI-TOF/TOF MS) method was developed for the separation and identification of bovine lactoferricin (LfcinB). Bovine lactoferrin was hydrolyzed by pepsin and then separated by ion exchange chromatography and reversed-phase liquid chromatography (RP-LC). The antibacterial activities of the fractions from RP-LC separation were determined and the protein concentration of the fraction with the highest activity was measured, whose sequence was indentified by MALDI-TOF/TOF MS. The relative molecular mass of LfcinB was 3 124.89 and the protein concentration was 18.20 microg/mL. The method of producing LfcinB proposed in this study has fast speed, high accuracy and high resolution.

Simultaneous flame ionization and absorbance detection of volatile and nonvolatile compounds by reversed-phase liquid chromatography with a water mobile phase.

PubMed

Bruckner, C A; Ecker, S T; Synovec, R E

1997-09-01

A flame ionization detector (FID) is used to detect volatile organic compounds that have been separated by water-only reversed-phase liquid chromatography (WRP-LC). The mobile phase is 100% water at room temperature, without use of organic solvent modifiers. An interface between the LC and detector is presented, whereby a helium stream samples the vapor of volatile components from individual drops of the LC eluent, and the vapor-enriched gas stream is sent to the FID. The design of the drop headspace cell is simple because the water-only nature of the LC separation obviates the need to do any organic solvent removal prior to gas phase detection. Despite the absence of organic modifier, hydrophobic compounds can be separated in a reasonable time due to the low phase volume ratio of the WRP-LC columns. The drop headspace interface easily handles LC flows of 1 mL/min, and, in fact, compound detection limits are improved at faster liquid flow rates. The transfer efficiency of the headspace interface was estimated at 10% for toluene in water at 1 mL/min but varies depending on the volatility of each analyte. The detection system is linear over more than 5 orders of 1-butanol concentration in water and is able to detect sub-ppb amounts of o-xylene and other aromatic compounds in water. In order to analyze volatile and nonvolatile analytes simultaneously, the FID is coupled in series to a WRP-LC system with UV absorbance detection. WRP-LC improves UV absorbance detection limits because the absence of organic modifier allows the detector to be operated in the short-wavelength UV region, where analytes generally have significantly larger molar absorptivities. The selectivity the headspace interface provides for flame ionization detection of volatiles is demonstrated with a separation of 1-butanol, 1,1,2-trichloroethane (TCE), and chlorobenzene in a mixture of benzoic acid in water. Despite coelution of butanol and TCE with the benzoate anion, the nonvolatile benzoate anion does not appear in the FID signal, allowing the analytes of interest to be readily detected. The complementary selectivity of UV-visible absorbance detection and this implementation of flame ionization detection allows for the analysis of volatile and nonvolatile components of complex samples using WRP-LC without the requirement that all the components of interest be fully resolved, thus simplifying the sample preparation and chromatographic requirements. This instrument should be applicable to routine automated water monitoring, in which repetitive injection of water samples onto a gas chromatograph is not recommended.

Separation and analysis of phenolic acids from Salvia miltiorrhiza and its related preparations by off-line two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography coupled with ion trap time-of-flight mass spectrometry.

PubMed

Sun, Wanyang; Tong, Ling; Miao, Jingzhuo; Huang, Jingyi; Li, Dongxiang; Li, Yunfei; Xiao, Hongting; Sun, Henry; Bi, Kaishun

2016-01-29

Salvia miltiorrhiza (SM) is one of the most widely used Traditional Chinese Medicine. Active constituents of SM mainly contain hydrophilic phenolic acids (PAs) and lipophilic tanshinones. However, due to the existing of multiple ester bonds and unsaturated bonds in the structures, PAs have numerous chemical conversion products. Many of them are so low-abundant that hard to be separated using conventional methods. In this study, an off-line two-dimensional liquid chromatography (2D-LC) method was developed to separate PAs in SM and its related preparations. In the first dimension, samples were fractionated by hydrophilic interaction chromatography (HILIC) (Acchrom×Amide, 4.6×250mm, 5μm) mainly based on the hydrogen bonding effects. The fractions were then separated on reversed-phase liquid chromatography (RP-LC) (Acquity HSS T3, 2.1×50mm, 1.7μm) according to hydrophobicity. For the selective identification of PAs, diode array detector (DAD) and electrospray ionization tandem ion trap time-of-flight mass spectrometry (ESI-IT-TOF-MS) were employed. Practical and effective peak capacities of all the samples were greater than 2046 and 1130, respectively, with the orthogonalities ranged from 69.7% to 92.8%, which indicated the high efficiency and versatility of this method. By utilizing the data post-processing techniques, including mass defect filter, neutral loss filter and product ion filter, a total of 265 compounds comprising 196 potentially new PAs were tentatively characterized. Twelve kinds of derivatives, mainly including glycosylated compounds, O-alkylated compounds, condensed compounds and hydrolyzed compounds, constituted the novelty of the newly identified PAs. The HILIC×RP-LC/TOF-MS system expanded our understanding on PAs of S. miltiorrhiza and its related preparations, which could also benefit the separation and characterization of polar constituents in complicated herbal extracts. Copyright © 2016. Published by Elsevier B.V.

The Use of Ammonium Formate as a Mobile-Phase Modifier for LC-MS/MS Analysis of Tryptic Digests

PubMed Central

Johnson, Darryl; Boyes, Barry; Orlando, Ron

2013-01-01

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage. PMID:24294112

The use of ammonium formate as a mobile-phase modifier for LC-MS/MS analysis of tryptic digests.

PubMed

Johnson, Darryl; Boyes, Barry; Orlando, Ron

2013-12-01

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance. Trifluoroacetic acid (TFA) is a commonly used mobile-phase modifier, as it produces peptide separations that are far superior to other additives. However, TFA leads to signal suppression when incorporated with electrospray ionization (ESI), and thus, other modifiers, such as formic acid (FA), are used for LC-MS applications. FA exhibits significantly less signal suppression, but is not as effective of a modifier as TFA. An alternative mobile-phase modifier is the combination of FA and ammonium formate (AF), which has been shown to improve peptide separations. The ESI-MS compatibility of this modifier has not been investigated, particularly for proteomic applications. This work compares the separation metrics of mobile phases modified with FA and FA/AF and explores the use of FA/AF for the LC-MS analysis of tryptic digests. Standard tryptic-digest peptides were used for comparative analysis of peak capacity and sample load tolerance. The compatibility of FA/AF in proteomic applications was examined with the analysis of soluble proteins from canine prostate carcinoma tissue. Overall, the use of FA/AF improved online RP-LC separations and led to significant increases in peptide identifications with improved protein sequence coverage.

Estimation of thermodynamic acidity constants of some penicillinase-resistant penicillins.

PubMed

Demiralay, Ebru Çubuk; Üstün, Zehra; Daldal, Y Doğan

2014-03-01

In this work, thermodynamic acidity constants (pssKa) of methicillin, oxacillin, nafcillin, cloxacilin, dicloxacillin were determined with reverse phase liquid chromatographic method (RPLC) by taking into account the effect of the activity coefficients in hydro-organic water-acetonitrile binary mixtures. From these values, thermodynamic aqueous acidity constants of these drugs were calculated by different approaches. The linear relationships established between retention factors of the species and the polarity parameter of the mobile phase (ET(N)) was proved to predict accurately retention in LC as a function of the acetonitrile content (38%, 40% and 42%, v/v). Copyright © 2013 Elsevier B.V. All rights reserved.

Determination of tafenoquine in dried blood spots and plasma using LC and fluorescence detection.

PubMed

Römsing, Susanne; Lindegardh, Niklas; Bergqvist, Yngve

2011-08-01

The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment. A bioanalytical method for the determination of tafenoquine in 100 µl of capillary blood applied onto sampling paper and in 100 µl of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l. The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.

Rapid and precise measurement of serum branched-chain and aromatic amino acids by isotope dilution liquid chromatography tandem mass spectrometry.

PubMed

Yang, Ruiyue; Dong, Jun; Guo, Hanbang; Li, Hongxia; Wang, Shu; Zhao, Haijian; Zhou, Weiyan; Yu, Songlin; Wang, Mo; Chen, Wenxiang

2013-01-01

Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C18 column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode. Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%. A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.

Determination of osthol and its metabolites in a phase I reaction system and the Caco-2 cell model by HPLC-UV and LC-MS/MS

PubMed Central

Yuan, Zhenting; Xu, Haiyan; Wang, Ke; Zhao, Zhonghua; Hu, Ming

2012-01-01

A straightforward and sensitive reversed-phase high-performance liquid chromatography (HPLC) assay was developed and validated for the analysis of osthol and its phase I metabolites (internal standard: umbelliferone). The method was validated for the determination of osthol with respect to selectivity, precision, linearity, limit of detection, recovery, and stability. The linear response range was 0.47 ~ 60 μM, and the average recoveries ranged from 98 to 101%. The inter-day and intra-day relative standard deviations were both less than 5%. Using this method, we showed that more than 80% of osthol was metabolized in 20 min in a phase I metabolic reaction system. Transport experiments in the Caco-2 cell culture model indicated that osthol was easily absorbed with high absorptive permeability (>10×10-6 cm/sec). The permeability did not display concentration-dependence or vectorial-dependence and is mildly temperature sensitive (activation energy less than 10 Kcal/mole), indicating passive mechanism of transport. When analyzed by LC-MS/MS, five metabolites were detected in a phase I reaction system and in the receiver side of a modified Caco-2 cell model, which was supplemented with the phase I reaction system. The major metabolites appeared to be desmethyl-osthol and multiple isomers of dehydro-osthol. In conclusion, a likely cause of poor osthol bioavailability is rapid phase I metabolism via the cytochrome P-450 pathways. PMID:19304430

METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

EPA Science Inventory

Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensi...

Light-Driven Reversible Transformation between Self-Organized Simple Cubic Lattice and Helical Superstructure Enabled by a Molecular Switch Functionalized Nanocage.

PubMed

Zhou, Kang; Bisoyi, Hari Krishna; Jin, Jian-Qiu; Yuan, Cong-Long; Liu, Zhen; Shen, Dong; Lu, Yan-Qing; Zheng, Zhi-Gang; Zhang, Weian; Li, Quan

2018-04-23

Self-organized stimuli-responsive smart materials with adjustable attributes are highly desirable for a plethora of device applications. Simple cubic lattice is quite uncommon in soft condensed matter due to its lower packing factor. Achieving a stable simple cubic soft lattice and endowing such a lattice with dynamic reconstruction capability solely by a facile light irradiation are of paramount significance for both fundamental studies and engineering explorations. Herein, an elegant stable self-organized simple cubic soft lattice, i.e., blue phase II, in a chiral liquid crystal (LC) system is disclosed, which is stable down to room temperature and exhibits both reversible lattice deformation and transformation to a helical superstructure, i.e., cholesteric LC, by light stimulation. Such an amazing trait is attained by doping a judiciously designed achiral photoresponsive molecular switch functionalized polyhedral oligomeric silsesquioxane nanocage into a chiral LC host. An unprecedented reversible collapse and reconstruction of such a high symmetric simple cubic blue phase II driven by light has been achieved. Furthermore, a well-defined conglomerate micropattern composed of simple cubic soft lattice and helical superstructure, which is challenging to fabricate in organic and inorganic crystalline materials, is produced using photomasking technology. Moreover, the promising photonic application based on such a micropattern is demonstrated. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of free formaldehyde in cosmetics containing formaldehyde-releasing preservatives by reversed-phase dispersive liquid-liquid microextraction and liquid chromatography with post-column derivatization.

PubMed

Miralles, Pablo; Chisvert, Alberto; Alonso, M José; Hernandorena, Sandra; Salvador, Amparo

2018-03-30

An analytical method for the determination of traces of formaldehyde in cosmetic products containing formaldehyde-releasing preservatives has been developed. The method is based on reversed-phase dispersive liquid-liquid microextraction (RP-DLLME), that allows the extraction of highly polar compounds, followed by liquid chromatography-ultraviolet/visible (LC-UV/vis) determination with post-column derivatization. The variables involved in the RP-DLLME process were studied to provide the best enrichment factors. Under the selected conditions, a mixture of 500 μL of acetonitrile (disperser solvent) and 50 μL of water (extraction solvent) was rapidly injected into 5 mL of toluene sample solution. The extracts were injected into the LC-UV/vis system using phosphate buffer 6 mmol L -1 at pH 2 as mobile phase. After chromatographic separation, the eluate merged with a flow stream of pentane-2,4-dione in ammonium acetate solution as derivatizing reagent and passed throughout a post-column reactor at 85 °C in order to derivatize formaldehyde into 3,5-diacetyl-1,4-dihydrolutidine, according to Hantzsch reaction, which was finally measured spectrophotometrically at 407 nm. The method was successfully validated showing good linearity, an enrichment factor of 86 ± 2, limits of detection and quantification of 0.7 and 2.3 ng mL -1 , respectively, and good repeatability (RSD < 9.2%). Finally, the proposed analytical method was applied to the determination of formaldehyde in different commercial cosmetic samples containing formaldehyde-releasing preservatives, such as bronopol, diazolidinyl urea, imidazolidinyl urea, and DMDM hydantoin, with good relative recovery values (91-113%) thus showing that matrix effects were negligible. The good analytical features of the proposed method besides of its simplicity and affordability, make it useful to carry out the quality control of cosmetic products containing formaldehyde-releasing preservatives. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of the mycotoxin moniliformin in cultures of Fusarium subglutinans and in naturally contaminated maize by high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

PubMed

Sewram, V; Nieuwoudt, T W; Marasas, W F; Shephard, G S; Ritieni, A

1999-07-02

A LC-MS method employing triethylamine as ion-pairing reagent for the determination of moniliformin in culture material and naturally contaminated maize samples is described. Mass spectrometric detection of moniliformin was accomplished following atmospheric pressure chemical ionization to yield the deprotonated molecular ion [M-H]- at m/z 97. The moniliformin response was found to be linear over the injected range 10 ng to 700 ng and a detection limit of 10 ng was attainable at a signal-to-noise (S/N) ratio of 4. Five South African strains of Fusarium subglutinans were grown on maize kernels and moniliformin extracted with an acetonitrile-water (95:5) mixture. Following sample clean up with reversed-phase (C18) solid-phase extraction cartridges, the extracts were subjected to LC-MS analysis. Triethylamine was used as an ion-pair reagent and found to improve the retention characteristics of moniliformin without any detrimental effects to the instrument. Moniliformin concentrations ranged between 130 mg/kg and 1460 mg/kg culture. Application of this method to naturally contaminated maize samples from Transkei showed that it was capable of measuring moniliformin levels down to 10 micrograms/kg in selected moldy maize cobs. This is the first report on the application of LC-MS to the analysis of moniliformin in cultures of F. subglutinans and in naturally contaminated maize.

Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography/tandem mass spectrometry.

PubMed

Pruvost, A; Becher, F; Bardouille, P; Guerrero, C; Creminon, C; Delfraissy, J F; Goujard, C; Grassi, J; Benech, H

2001-01-01

The objective was to develop and validate a routine assay for active intracellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase inhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitivity, specificity and high sample throughput. After cellular lysis in a Tris/methanol buffer, the extract spiked with 2[H(8)]-ATP (internal standard) is directly injected into the LC/MS/MS system. Phosphorylated metabolites of d4T as well as deoxythymidine-triphosphate, the competitor on the reverse transcriptase, are separated from d4T on a reverse-phase microbore column with ion pairing. The detection is performed in the multiple reaction monitoring (MRM) mode after drug ionisation in negative mode electrospray. The limit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 10(6) cells) and CV% for repeatability and intermediate precision were lower than 15%. Stability of compounds was checked before and during the process of isolation of PBMC. Cellular samples from several d4T-treated patients were successfully analysed using this method and d4T-triphosphate and deoxythymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determination of mono-, di- and triphosphorylated anabolites of d4T in PBMC as well as the natural corresponding triphosphate in one analysis. For the first time, the chain terminator ratio (d4T-TP/dT-TP) could be directly measured. This method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development. Copyright 2001 John Wiley & Sons, Ltd.

Improving LC-MS sensitivity through increases in chromatographic performance: comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS.

PubMed

Churchwell, Mona I; Twaddle, Nathan C; Meeker, Larry R; Doerge, Daniel R

2005-10-25

Recent technological advances have made available reverse phase chromatographic media with a 1.7 microm particle size along with a liquid handling system that can operate such columns at much higher pressures. This technology, termed ultra performance liquid chromatography (UPLC), offers significant theoretical advantages in resolution, speed, and sensitivity for analytical determinations, particularly when coupled with mass spectrometers capable of high-speed acquisitions. This paper explores the differences in LC-MS performance by conducting a side-by-side comparison of UPLC for several methods previously optimized for HPLC-based separation and quantification of multiple analytes with maximum throughput. In general, UPLC produced significant improvements in method sensitivity, speed, and resolution. Sensitivity increases with UPLC, which were found to be analyte-dependent, were as large as 10-fold and improvements in method speed were as large as 5-fold under conditions of comparable peak separations. Improvements in chromatographic resolution with UPLC were apparent from generally narrower peak widths and from a separation of diastereomers not possible using HPLC. Overall, the improvements in LC-MS method sensitivity, speed, and resolution provided by UPLC show that further advances can be made in analytical methodology to add significant value to hypothesis-driven research.

Chromatographic methods for determination of macrolide antibiotic residues in tissues and milk of food-producing animals.

PubMed

Moats, W A

1985-01-01

Tylosin, an antibiotic developed specifically for agricultural use, and erythromycin are the main macrolide antibiotics used in animal production. Two-dimensional thin layer chromatography has been used for detection of tylosin in poultry meat, eggs, and milk and for erythromycin in poultry meat. Detection limits reported are, for tylosin, 0.1 ppm in poultry meat, 0.05 ppm in egg, and 0.01 ppm in milk, and for erythromycin, 0.25 ppm in poultry meat. Liquid chromatography (LC) has also been used for determination of tylosin in milk, blood, and tissues of animals. Samples (milk, blood serum, or tissue homogenates in water or pH 2.2 buffer) were deproteinized with acetonitrile, tylosin was partitioned into methylene chloride, and the extracts were concentrated and dissolved in acetonitrile. Chromatography was done on a reverse phase end-capped C18 column using 0.002-0.005 M ammonium dihydrogen phosphate-acetonitrile-methanol (10 + 60 + 30-5 + 80 + 15). Solvent composition was varied with the type of sample analyzed. The method will detect 0.1 ppm tylosin in tissues and less in milk and blood serum. The LC method was more sensitive than microbiological assays for detection of tylosin in tissues of treated swine; recoveries of tylosin by the LC method were frequently several-fold higher.

Progress in a selective method for the determination of the acetaldehyde-derived DNA adducts by using HILIC-ESI-MS/MS.

PubMed

Murakami, Hiroya; Horiba, Ruri; Iwata, Tomoko; Miki, Yuta; Uno, Bunji; Sakai, Tadao; Kaneko, Kazuhiro; Ishihama, Yasushi; Teshima, Norio; Esaka, Yukihiro

2018-01-15

Acetaldehyde (AA), which is present in tobacco smoke, automobile exhaust gases and alcohol beverage, is a mutagen and carcinogen. AA reacts with 2'-deoxyguanosine (dG) in DNA to form N 2 -ethyl-dG (EtdG) and cyclic, 1, N 2 -propano-dG (CPrdG), which are considered to have a critical role in carcinogenesis induced by AA. In this study, we have developed a highly sensitive method for the quantitation of the two AA-derived DNA adducts by using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in which hydrophilic interaction chromatography (HILIC) employing mobile phases of high organic solvent concentration was selected to improve the ionization efficiency in the ESI process. Fourteen times and 11 times larger peak areas for EtdG and CPrdG, respectively, in HILIC-ESI-MS/MS were obtained compared with those in reversed phase (RP)-LC-ESI-MS/MS. Furthermore, 6.9 times (for EtdG) and 2.4 times (for CPrdG) larger peak areas were also obtained as additional enhancement by varying additive compounds in the HILIC mobile phases from ammonium acetate to ammonium bicarbonate. In total, the enhancements in detected MS signal intensities by exchanging from the RP-LC system to the HILIC system are 97 times for EtdG and 26 times for CPrdG, respectively. Three commercially available HILIC columns with different polar functional groups were examined and sufficient separation between normal 2'-deoxynucleosides and the AA-derived DNA adducts was achieved by a carbamoyl-bonded HILIC column. Finally, we applied the established method to quantify EtdG and CPrdG in the damaged calf thymus DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous determination of bioactive constituents in Danggui Buxue Tang for quality control by HPLC coupled with a diode array detector, an evaporative light scattering detector and mass spectrometry.

PubMed

Yi, Ling; Qi, Lian-Wen; Li, Ping; Ma, Yi-Han; Luo, Yong-Jing; Li, Hai-Yun

2007-09-01

Danggui Buxue Tang (DBT), a classical traditional Chinese formula comprising Radix Angelicae Sinensis (RAS) and Radix Astragali (RA), has been widely used to treat menopausal irregularity in Chinese women for nearly 800 years. In this study, a comprehensive analytical method of simultaneously determining the main types of bioactive constituents, eighteen in all from the formula, involving flavonoids, saponins, organic acid and some volatile compounds, was developed. This method was based on HPLC coupled to a diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) on a common reverse-phase C(18) column. Liquid chromatography coupled with on-line electrospray ionization mass spectrometry (LC-ESI-MS) was also used to further validate and analyze the constituents. It was found that 0.3% aqueous formic acid and acetonitrile was the optimum mobile phase for gradient elution. This method, which showed good precision and accuracy, was successfully used to quantify the bioactive constituents in six products. As a result, the validated HPLC method, together with the LC-ESI-MS analysis, provided a new basis for assessing the quality of traditional Chinese medicinal compound preparations (TCMCPs) consisting of many bioactive components.

Qualitative analysis of MDR-reversing Anastasia Black (Russian black sweet pepper, Capsicum annuum, Solanaceae) extracts and fractions by HPLC and LC-MS-MS methods.

PubMed

Schelz, Zsuzsanna; Molnár, Joseph; Fogliano, Vincenzo; Ferracane, Rosalia; Pernice, Rita; Shirataki, Yoshiaki; Motohashi, Noboru

2006-01-01

In earlier experiments, the MDR (multidrug resistance)-reversal activities of Anastasia Black (Russian black sweet pepper) extracts had been analysed. Recently, the most effective MDR reversing extracts and fractions have been separated by HPLC (high-performance liquid chromatography, for carotenoids) and LC-MS-MS (HPLC combined with mass spectrometry, for phenolic compounds) methods. As a result of the analytical studies, the following flavonoids had been identified: feruloyl glucopyranoside, quercetin rhamnopyranoside glucopyranoside, luteolin glucopyranoside arabinopyranoside, apigenin glucopyranoside arabinopyranoside, quercetin rhamnopyranoside, luteolin arabinopyranoside diglucopy-ranoside, hesperidine and luteolin glucuronide. According to the literature, the aglycones of these phenolic compounds exhibit MDR-reversal activity in vitro, and the connection between the phenolic content of Anastasia Black and MDR-reversal action was therefore studied by different analytical methods. The results of this study revealed that the identified flavonoids of Anastasia Black may be only partially responsible for the modulation of the MDR of mouse lymphoma cells. Other lipophilic compounds, most probably carotenoids, present in Russian black sweet pepper may act as inhibitors of MDR reversal.

On-line solid phase extraction coupled to capillary LC-ESI-MS for determination of fluoxetine in human blood plasma.

PubMed

Saber, Amr L

2009-04-15

An instrumental setup including on-line solid phased extraction coupled to capillary liquid chromatography-electrospray ionization-mass spectrometry (SPE-capLC-ESI-MS) has been constructed to improve the sensitivity for quantification of fluoxetine hydrochloride in human plasma. Prior to injection, 0.5 mL of plasma spiked with metronidazole (internal standard) was mixed with ammonium formate buffer for effective chloroform liquid-liquid extraction. The method was validated in the range 5-60 ng mL(-1) fluoxetine, yielding a correlation coefficient of 0.999 (r(2)). The within-assay and between-assay precisions were between (8.5 and 11%) and (6.6 and 7.5%), respectively. The method was used to determine the amount of fluoxetine in a healthy male 14 h after an intake of one capsule of the antidepressant and anorectic Flutin, which contains 20mg fluoxetine per each capsule. Fluoxetine was detected, and the concentration was calculated to 9.0 ng mL(-1) plasma. In the preliminary experiments, conventional LC-UV instrumentation was employed. However, it was found that employing a capillary column with an inner diameter of (0.3mm I.D. x 50 mm, Zorbax C(18)) increased the sensitivity by a factor of approximately 100, when injecting the same mass of analyte. Incorporating an easily automated C(18) reversed phase column switching system with SPE (1.0mm I.D. x 5.0mm, 5 microm) made it possible to inject up to 100 microL of solution, and the total analysis time was 5.5 min.

High-temperature LC-MS/MS of permethylated glycans derived from glycoproteins.

PubMed

Zhou, Shiyue; Hu, Yunli; Mechref, Yehia

2016-06-01

Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Simple and Sensitive LC-MS/MS Method for the Determination of Free 8-Hydroxy-2'-Deoxyguanosine in Human Urine

NASA Technical Reports Server (NTRS)

Wang, Zuwei; Smith, Scott M.

2016-01-01

Urinary free 8-hydroxy-2'-deoxyguanosine (8OHdG), an oxidized product of DNA, and is frequently chosen as a biomarker of oxidative stress in humans, including studies of oxidative DNA damage during space flight. It is challenging to accurately and efficiently quantify urinary free 8OHdG in large scale human studies. LC-MS/MS is emerging as a preferable analytical technique owing its high sensitivity, selectivity and efficiency, compared to some traditional methods such as ELISA and HPLC. A simple and sensitive LC-MS/MS method has been developed for the determination of free 8OHdG in human urine. Sample preparation was done by solid phase extraction with a Waters Oasis HLB 96 well plate. A Waters Alliance 2795 HT Separation Module combined with a Quattro Micro tandem mass spectrometer was used as the LC-MS/MS system. The runtime of one injection can be less than 5 minutes using a reversed phase C18 column and an isocratic flow of methanol/water. ESI positive ions were quantified in the multiple reaction modes (MRM) using m/z 284 yields 168 for 8OHdG and m/z 289 yields173 for stable isotope labeled internal standard [(15)N5] 8OHdG. With this method for 8OHdG, a lower limit of quantitation of 1.0 nM (0.28 ng/mL) has been achieved using 100 microliter urine sample. The analytical range is between 1.0 and 100 nM with a correlation coefficient greater than or equal to 0.99. Good reproducibility can be obtained with intra-assay and inter-assay CVs less than or equal to 10% for 8OHdG spiked urine QC samples. This method can be used in high-throughput routine analysis of free 8OHdG in human urine.

Comprehensive two-dimensional liquid chromatographic analysis of poloxamers.

PubMed

Malik, Muhammad Imran; Lee, Sanghoon; Chang, Taihyun

2016-04-15

Poloxamers are low molar mass triblock copolymers of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), having number of applications as non-ionic surfactants. Comprehensive one and two-dimensional liquid chromatographic (LC) analysis of these materials is proposed in this study. The separation of oligomers of both types (PEO and PPO) is demonstrated for several commercial poloxamers. This is accomplished at the critical conditions for one of the block while interaction for the other block. Reversed phase LC at CAP of PEO allowed for oligomeric separation of triblock copolymers with regard to PPO block whereas normal phase LC at CAP of PPO renders oligomeric separation with respect to PEO block. The oligomeric separation with regard to PEO and PPO are coupled online (comprehensive 2D-LC) to reveal two-dimensional contour plots by unconventional 2D IC×IC (interaction chromatography) coupling. The study provides chemical composition mapping of both PEO and PPO, equivalent to combined molar mass and chemical composition mapping for several commercial poloxamers. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

Evaluation of reversed-phase nano liquid chromatography conditions by using reversed-phase thin layer chromatography based on Hansen solubility parameters for the analysis of amphiphilic glycosylsphingolipid transformations.

PubMed

Kanie, Yoshimi; Taniuchi, Mizuki; Kanie, Osamu

2018-01-26

Pulse chase analysis is often used in investigating dynamics of cellular substances. Fluorescently labeled lactosyl sphingosine molecule is useful in chasing its transformation, however the analysis of such metabolites in attomole level is of extreme difficult due to the presence of large amount of endogenous amphiphilic molecules such as glycosphingolipids, sphingomyerin, and glycerophospholipids. Nano LC suites for analyzing the attomole scale metabolites, therefore removal of endogenous substances prior to nano LC and finding appropriate nano LC conditions are necessary. Thus, we focused on the solubility of fluorescent BODIPY-labeled lactosylsphingosine (Lac-Sph-BODIPY) to identify suitable solvents to remove endogenous compounds. In this study, we evaluated solvents by using C18 thin layer chromatography (RP TLC). The mobility (R f ) of Lac-Sph-BODIPY against several solvent mixtures on RP TLC were plotted against polarity and hydrogen bonding capability followed by Hansen solubility parameters (HSPs). The optimum solvent mixture with R f  = 0.3 ± 0.1 was chosen for elimination of endogenous phospholipids on a ZrO 2 -SiO 2 cartridge column and subsequent separation by nano LC. Efficient removal of endogenous phospholipids was demonstrated, and good resolution in nano LC analysis of Lac-Sph-BODIPY extracted from Chinese hamster ovary (CHO)-K1 cells was achieved. It was also shown that the amount of exogenously added compound was important in the investigation of metabolites using cultured cells. Copyright © 2017 Elsevier B.V. All rights reserved.

[Rapid identification of two new isomers in bear bile powder by LC-Q-TOF-MS combined with PCC oxidation].

PubMed

Jian, Long-Hai; Hu, Chun; Yu, Hong; Wang, Ke; Ji, Shen

2013-07-01

A rapid method of Liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) combined with pyridinium chlorochromate (PCC) oxidation has been developed to determine chemical structures of two novel isomers in bear bile powder. Derivatives of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) were semi-synthesized by PCC oxidation, then were analyzed by LC-Q-TOF-MS. Separation was carried out on a reverse column with the mobile phase of acetonitrile-0.1% formic acid (45:55). The data of Q-TOF-MS was acquired by MS, MS/MS, positive and negative modes. Since UDCA and CDCA were stereochemical isomeric at an alcohol position, two oxidation products were same and have been confirmed by LC-Q-TOF-MS. Other two products were also determined based on the PCC oxidation theory. Samples of bear bile powder were dissolved by methanol and measured by LC-Q-TOF-MS. Two unknown peaks were found and identified by matching their retention times and accurate mass spectra ions with PCC oxidation productS. Finally, the structures of two new bile acids in bear bile powder were confirmed as 3alpha-hydroxy-7-oxo-5beta-cholanic acid, 7alpha-hydroxy-3-oxo-5beta-cholanic acid, respectively.

Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

NASA Astrophysics Data System (ADS)

Catron, Brittany; Caruso, Joseph A.; Limbach, Patrick A.

2012-06-01

A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.

Analysis of iodinated quorum sensing peptides by LC-UV/ESI ion trap mass spectrometry.

PubMed

Janssens, Yorick; Verbeke, Frederick; Debunne, Nathan; Wynendaele, Evelien; Peremans, Kathelijne; De Spiegeleer, Bart

2018-02-01

Five different quorum sensing peptides (QSP) were iodinated using different iodination techniques. These iodinated peptides were analyzed using a C 18 reversed phase HPLC system, applying a linear gradient of water and acetonitrile containing 0.1% (m/v) formic acid as mobile phase. Electrospray ionization (ESI) ion trap mass spectrometry was used for the identification of the modified peptides, while semi-quantification was performed using total ion current (TIC) spectra. Non-iodinated peptides and mono- and di-iodinated peptides (NIP, MIP and DIP respectively) were well separated and eluted in that order. Depending on the used iodination method, iodination yields varied from low (2%) to high (57%).

ANALYSIS OF ALKYLPHENOLS AND ALKYLPHENOL ETHOXYLATES WITH SEPARATION OF ETHOXYMERS USING REVERSED PHASE LC/MS

EPA Science Inventory

Alkylphenol polyethoxylates are non-ionic surfactants, widely used in detergents, paints, personal care products, etc., which enter the environment primarily through wastewater treatment systems. Their biodegradation during wastewater treatment leads to the formation of persisten...

Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma.

PubMed

Wang, Jixia; Kong, Song; Yan, Jingyu; Jin, Gaowa; Guo, Zhimou; Shen, Aijin; Xu, Junyan; Zhang, Xiuli; Zou, Lijuan; Liang, Xinmiao

2014-06-01

Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC-MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5,000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Validation of an electrospray ionisation LC-MS/MS method for quantitative analysis of telaprevir and its R-diastereomer.

PubMed

Penchala, Sujan Dilly; Tjia, John; El Sherif, Omar; Back, David J; Khoo, Saye H; Else, Laura J

2013-08-01

A sensitive high-performance reverse phase liquid chromatography-positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of telaprevir and its inactive R-diastereomer (VRT-127394) in human plasma. The analytes and the internal standard (telaprevir-d11) were extracted from plasma by liquid-liquid extraction using tert-Butyl methyl ether (TBME). Chromatographic separation was achieved on a reversed-phase Accucore C18 column with a gradient programme consisting of water:ammonia (25%), 100:0.01 (v/v) (mobile phase A) and ACN:MeOH:ammonia (25%), 15:85:0.01 (v/v/v) (mobile phase B). The MS acquisition was performed with selective reaction monitoring mode using the respective [M+H](+) ions, m/z 680.59→322.42 for telaprevir and VRT-127394, and 691.15→110.13 for telaprevir-d11. The assay exhibited a linear dynamic range of 5-5000ng/mL for telaprevir and VRT-127394. Acceptable precision (%RSD<6.5%) and accuracy (94-108%) were obtained for concentrations over the range of the standard curve. A procedure was established to stabilise the plasma to prevent ex vivo interconversion of the isomers. Copyright © 2013 Elsevier B.V. All rights reserved.

Simultaneous LC-MS/MS determination of phenylbutyrate, phenylacetate benzoate and their corresponding metabolites phenylacetylglutamine and hippurate in blood and urine.

PubMed

Laryea, Maurice D; Herebian, Diran; Meissner, Thomas; Mayatepek, Ertan

2010-12-01

Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 μmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.

Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography–tandem mass spectrometry.

PubMed

Liu, Wei; Kong, Yu; Zu, Yuangang; Fu, Yujie; Luo, Meng; Zhang, Lin; Li, Ji

2010-07-09

A novel method using liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) has been optimized and established for the qualitative and quantitative analysis of ten active phenolic compounds originating from the pigeon pea leaves and a medicinal product thereof (Tongluo Shenggu capsules). In the present study, the chromatographic separation was achieved by means of a HiQ Sil C18V reversed-phase column with a mobile phase consisting of methanol and 0.1% formic acid aqueous solution. Low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) using the selected reaction monitoring (SRM) analysis was employed for the detection of ten analytes which included six flavonoids, two isoflavonoids and two stilbenes. All calibration curves showed excellent coefficients of determination (r(2) ≥ 0.9937) within the range of tested concentrations. The intra- and inter-day variations were below 5.36% in terms of relative standard deviation (RSD). The recoveries were 95.08-104.98% with RSDs of 2.06-4.26% for spiked samples of pigeon pea leaves. The method developed was a rapid, efficient and accurate LC-MS/MS method for the detection of phenolic compounds, which can be applied for quality control of pigeon pea leaves and related medicinal products.

Tailored liquid chromatography-mass spectrometry analysis improves the coverage of the intracellular metabolome of HepaRG cells.

PubMed

Cuykx, Matthias; Negreira, Noelia; Beirnaert, Charlie; Van den Eede, Nele; Rodrigues, Robim; Vanhaecke, Tamara; Laukens, Kris; Covaci, Adrian

2017-03-03

Metabolomics protocols are often combined with Liquid Chromatography-Mass Spectrometry (LC-MS) using mostly reversed phase chromatography coupled to accurate mass spectrometry, e.g. quadrupole time-of-flight (QTOF) mass spectrometers to measure as many metabolites as possible. In this study, we optimised the LC-MS separation of cell extracts after fractionation in polar and non-polar fractions. Both phases were analysed separately in a tailored approach in four different runs (two for the non-polar and two for the polar-fraction), each of them specifically adapted to improve the separation of the metabolites present in the extract. This approach improves the coverage of a broad range of the metabolome of the HepaRG cells and the separation of intra-class metabolites. The non-polar fraction was analysed using a C18-column with end-capping, mobile phase compositions were specifically adapted for each ionisation mode using different co-solvents and buffers. The polar extracts were analysed with a mixed mode Hydrophilic Interaction Liquid Chromatography (HILIC) system. Acidic metabolites from glycolysis and the Krebs cycle, together with phosphorylated compounds, were best detected with a method using ion pairing (IP) with tributylamine and separation on a phenyl-hexyl column. Accurate mass detection was performed with the QTOF in MS-mode only using an extended dynamic range to improve the quality of the dataset. Parameters with the greatest impact on the detection were the balance between mass accuracy and linear range, the fragmentor voltage, the capillary voltage, the nozzle voltage, and the nebuliser pressure. By using a tailored approach for the intracellular HepaRG metabolome, consisting of three different LC techniques, over 2200 metabolites can be measured with a high precision and acceptable linear range. The developed method is suited for qualitative untargeted LC-MS metabolomics studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of n-hexane metabolites by liquid chromatography/mass spectrometry. 2. Glucuronide-conjugated metabolites in untreated urine samples by electrospray ionization.

PubMed

Manini, P; Andreoli, R; Mutti, A; Bergamaschi, E; Niessen, W M

1998-01-01

A liquid chromatography atmospheric pressure electrospray mass spectrometry (ESI-LC/MS) system was evaluated for the identification and characterization of n-hexane conjugated metabolites (glucuronides) in untreated urine samples. Chromatography of glucuronides was obtained under ion-suppressed reversed-phase conditions, by using high-speed (3 cm, 3 microns) columns and formic acid (2 mM) as modifier in the mobile phase. The mass spectrometer was operated in negative ion (NI) mode. For the first time, four glucuronides were identified by ESI-LC/MS in untreated urine samples of rats exposed to n-hexane: 2-hexanol-glucuronide, 5-hydroxy-2-hexanone-glucuronide, 2,5-hexanediol-glucuronide and 4,5-dihydroxy-2-hexanone-glucuronide. Confirmation of the conjugated metabolites was obtained by LC/MS/MS experiments. Gas chromatography/mass spectrometry (GC/MS) and atmospheric pressure chemical ionization (APCI) LC/MS analyses were performed on the same samples. An integrated approach GC/MS-LC/MS for the semi-quantitative analysis of n-hexane glucuronides, whose standards are not commercially available, is discussed and proposed here. In order to understand the fate of the metabolites during sample pre-treatment, a study about the effects of enzymatic and acid hydrolysis on urine samples was conducted on glucuronides isolated by solid-phase extraction. Combined analyses by GC/MS and LC/MS enabled us to distinguish 'true' n-hexane metabolites from compounds resulting from sample treatment and handling (i.e. enzymatic and acid hydrolysis, extraction and GC injection).

The versatility of heart-cutting and comprehensive two-dimensional liquid chromatography in monoclonal antibody clone selection.

PubMed

Sandra, Koen; Steenbeke, Mieke; Vandenheede, Isabel; Vanhoenacker, Gerd; Sandra, Pat

2017-11-10

In recent years, two-dimensional liquid chromatography (2D-LC) has seen an enormous evolution and one of the fields where it is being widely adopted is in the analysis of therapeutic monoclonal antibodies (mAbs). We here further add to the many flavours of this powerful technology. Workflows based on heart-cutting (LC-LC) and comprehensive (LC×LC) 2D-LC are described that allow to guide the clone selection process in mAb and biosimilar development. Combining Protein A affinity chromatography in the first dimension with size exclusion (SEC), cation exchange (CEX) or reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) in the second dimension simultaneously allows to assess mAb titer and critical structural aspects such as aggregation, fragmentation, charge heterogeneity, molecular weight (MW), amino acid sequence and glycosylation. Complementing the LC-LC measurements at intact protein level with LC×LC based peptide mapping provides the necessary information to make clear decisions on which clones to take further into development. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of Aristolochic Acid in Botanicals and Dietary Supplements by Liquid Chromatography with Ultraviolet Detection and by Liquid Chromatography/Mass Spectrometry: Single Laboratory Validation Confirmation

PubMed Central

Trujillo, William A.; Sorenson, Wendy R.; La Luzerne, Paul; Austad, John W.; Sullivan, Darryl

2008-01-01

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 μg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid Chromatography with ultraviolet detection (LC-UV) and LC/mass Spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 μg/g level, between 102 and 103% at the 10 μg/g level, and 104% at the 30 μg/g level. PMID:16915829

Determination of aristolochic acid in botanicals and dietary supplements by liquid chromatography with ultraviolet detection and by liquid chromatography/mass spectrometry: single laboratory validation confirmation.

PubMed

Trujillo, William A; Sorenson, Wendy R; La Luzerne, Paul; Austad, John W; Sullivan, Darryl

2006-01-01

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 microg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid chromatography with ultraviolet detection (LC-UV) and LC/mass spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 microg/g level, between 102 and 103% at the 10 microg/g level, and 104% at the 30 microg/g level.

Capillary liquid chromatography combined with pressurized liquid extraction and dispersive liquid-liquid microextraction for the determination of vitamin E in cosmetic products.

PubMed

Viñas, Pilar; Pastor-Belda, Marta; Campillo, Natalia; Bravo-Bravo, María; Hernández-Córdoba, Manuel

2014-06-01

Capillary liquid chromatography (LC) is used for the determination of tocopherols and tocotrienols in cosmetic products. Dispersive liquid-liquid microextraction (DLLME) allows the analytes to be preconcentrated into a very small volume of organic solvent which is then injected into the chromatograph running at a very low flow rate. Pressurized liquid extraction (PLE) at a high temperature and pressure was used to isolate vitamin E forms from cosmetics. The Taguchi experimental method was used to optimize the factors affecting DLLME. The parameters selected were 2mL of acetonitrile (disperser solvent), 100μL carbon tetrachloride (extraction solvent) and 10mL aqueous solution. A volume of 5μL of the organic phase was injected into the reversed-phase capillary LC system equipped with a diode array detector and using an isocratic mobile phase composed of an 95:5 (v/v) methanol:water mixture at a flow-rate of 20μLmin(-1). Quantification was carried out using aqueous standards and detection limits were in the range 0.1-0.5ngmL(-1), corresponding to 3-15ngg(-1) in the cosmetic sample. The recoveries were in the 87-105% range, with RSDs lower than 7.8%. The method was validated according to international guidelines and using a certified reference material. Copyright © 2014 Elsevier B.V. All rights reserved.

Lacticin LC14, a new bacteriocin produced by Lactococcus lactis BMG6.14: isolation, purification and partial characterization.

PubMed

Lasta, Samar; Ouzari, Hadda; Andreotti, Nicolas; Fajloun, Ziad; Mansuelle, Pascal; Boudabous, Abdellatif; Sampieri, Francois; Sabatier, Jean Marc

2012-08-01

A new bacteriocin, lacticin LC14, produced by Lactococcus lactis BMG6.14, was isolated and characterized. It was purified to homogeneity from overnight broth culture by ammonium sulfate precipitation, Sep-Pak chromatography, and two steps of reversed-phase HPLC. Lacticin LC14 showed bactericidal-type antimicrobial activity against several lactic acid bacteria and pathogenic strains including Listeria monocytogenes. It was inactivated by proteinase K and pronase E, but was resistant to papain, lysozyme, lipase and catalase. Lacticin LC14 was heat resistant, stable over a wide range of pH (2-10) and after treatment by solvents and detergents. Its N-terminal end was found unreactive towards Edman sequencing. Based on MALDI-TOF mass spectrometry, its molecular mass was 3333.7 Da. LC14 amino acid composition revealed a high proportion of hydrophobic residues, but no modified ones. LC14 may be able to challenge other well known other bacteriocins in probiotic and therapeutic applications.

Supramolecular separation mechanism of pentafluorophenyl column using ibuprofen and omeprazole as markers: LC-MS and simulation study.

PubMed

Hussain, Afzal; AlAjmi, Mohamed F; Ali, Imran

2018-06-01

The pentafluorophenyl (PFP) column is emerging as a new advancement in separation science to analyze a wide range of analytes and, thus, its separation mechanism at supramolecular level is significant. We developed a mechanism for the separation of ibuprofen and omeprazole using different combinations (ranging from 50:50 to 60:40) of water-acetonitrile containing 0.1% formic acid as the mobile phase. The column used was Waters Acquity UPLC HSS PFP (75 × 2.1 mm, 1.8 μm). The reverse order of elution was observed in different combinations of the mobile phases. The docking study indicated hydrogen bonding between ibuprofen and PFP stationary phase (binding energy was -11.30 kJ/mol). Separation at PFP stationary phase is controlled by hydrogen bonding along with π-π interactions. This stationary phase may be used to analyze both aromatic and aliphatic analytes. The developed mechanism will be useful to separate various analytes by considering the possible interactions, leading to saving of energy, time and money. In addition, this work will be highly useful in preparative chromatography where separation is the major problem at a large scale. Moreover, the developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample owing to the low value of detection limits. Copyright © 2018 John Wiley & Sons, Ltd.

Characterization and screening of pyrrolizidine alkaloids and N-oxides from various parts of many botanicals and dietary supplements using liquid chromatography high resolution mass spectrometry

USDA-ARS?s Scientific Manuscript database

The UHPLC-QToF-MS analysis of pyrrolizidine alkaloids from various parts of 37 botanicals and 7 dietary supplements was performed. A separation by LC was achieved using a reversed-phase column and a gradient of water/acetonitrile each containing formic acid as the mobile phase. MS-MS detection was u...

Facile preparation of a stable and functionalizable hybrid monolith via ring-opening polymerization for capillary liquid chromatography.

PubMed

Lin, Hui; Ou, Junjie; Tang, Shouwan; Zhang, Zhenbin; Dong, Jing; Liu, Zhongshan; Zou, Hanfa

2013-08-02

An organic-inorganic hybrid monolith was prepared by a single-step ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS) with poly(ethylenimine) (PEI). The obtained hybrid monoliths possessed high ordered 3D skeletal microstructure with dual retention mechanism that exhibits reversed-phase (RP) mechanism under polar mobile phase and hydrophilic-interaction liquid chromatography (HILIC) retention mechanism under less polar mobile phase. The high column efficiencies of 110,000N/m can be achieved for separation of alkylbenzenes in capillary reversed-phase liquid chromatography (cLC). Due to the robust property of hybrid monolith and the rich primary and secondary amino groups on its surface, the resulting hybrid monolith was easily modified with γ-gluconolactone and physically coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), respectively. The former was successfully applied for HILIC separation of neutral, basic and acidic polar compounds as well as small peptides, and the latter for enantioseparation of racemates in cLC. The high column efficiencies were achieved in all of those separations. These results demonstrated that the hybrid monolith (POSS-PEI) possessed high stability and good surface tailorbility, potentially being applied for other research fields. Copyright © 2013 Elsevier B.V. All rights reserved.

Liquid chromatography-tandem mass spectrometry method for the analysis of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine, a biocidal disinfectant, in dairy products.

PubMed

Slimani, Kahina; Pirotais, Yvette; Maris, Pierre; Abjean, Jean-Pierre; Hurtaud-Pessel, Dominique

2018-10-01

A novel and reliable method to quantify residual levels of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in dairy products using ion-pairing reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and fully validated. Sample extraction was done with salting-out technique using acetonitrile and sodium chloride. For LC-MS/MS, the analyte was detected using positive electrospray ionization (ESI+) and two multiple reaction monitoring (MRM) transitions were monitored. The method was validated in the 5-150 µg kg -1 range using total error approach. Thus, performance criteria of the method were evaluated. Relative standard deviations for trueness and precision were lower than 10%; with the exception of hard pressed cheese at 5 µg kg -1 for precision. The limit of quantification (LOQ) was around 5-7 µg kg -1 depending on the matrix of interest. The method was successfully applied to accurately quantify N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in 146 various dairy products with a maximum contamination level of 225 µg kg -1 in cheese. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fully automated analysis of four tobacco-specific N-nitrosamines in mainstream cigarette smoke using two-dimensional online solid phase extraction combined with liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Jie; Bai, Ruoshi; Yi, Xiaoli; Yang, Zhendong; Liu, Xingyu; Zhou, Jun; Liang, Wei

2016-01-01

A fully automated method for the detection of four tobacco-specific nitrosamines (TSNAs) in mainstream cigarette smoke (MSS) has been developed. The new developed method is based on two-dimensional online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE/LC-MS/MS). The two dimensional SPE was performed in the method utilizing two cartridges with different extraction mechanisms to cleanup disturbances of different polarity to minimize sample matrix effects on each analyte. Chromatographic separation was achieved using a UPLC C18 reversed phase analytical column. Under the optimum online SPE/LC-MS/MS conditions, N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were baseline separated with good peak shapes. This method appears to be the most sensitive method yet reported for determination of TSNAs in mainstream cigarette smoke. The limits of quantification for NNN, NNK, NAT and NAB reached the levels of 6.0, 1.0, 3.0 and 0.6 pg/cig, respectively, which were well below the lowest levels of TSNAs in MSS of current commercial cigarettes. The accuracy of the measurement of four TSNAs was from 92.8 to 107.3%. The relative standard deviations of intra-and inter-day analysis were less than 5.4% and 7.5%, respectively. The main advantages of the method developed are fairly high sensitivity, selectivity and accuracy of results, minimum sample pre-treatment, full automation, and high throughput. As a part of the validation procedure, the developed method was applied to evaluate TSNAs yields for 27 top-selling commercial cigarettes in China. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple-Reaction–Monitoring Mass Spectrometry

PubMed Central

Agger, Sean A.; Marney, Luke C.; Hoofnagle, Andrew N.

2011-01-01

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs <6% and interassay CVs <12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay – 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability. PMID:20923952

Development and validation of stability indicating the RP-HPLC method for the estimation of related compounds of guaifenesin in pharmaceutical dosage forms

PubMed Central

Reddy, Sunil Pingili; Babu, K. Sudhakar; Kumar, Navneet; Sekhar, Y. V. V. Sasi

2011-01-01

Aim and background: A stability-indicating gradient reverse phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of related substances of guaifenesin in pharmaceutical formulations. Materials and methods: The baseline separation for guaifenesin and all impurities was achieved by utilizing a Water Symmetry C18 (150 mm × 4.6 mm) 5 μm column particle size and a gradient elution method. The mobile phase A contains a mixture of 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 90:10 v/v, while the mobile phase B contains 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 10:90 v/v, respectively. The flow rate of the mobile phase was 0.8 ml/min with a column temperature of 25°C and detection wavelength at 273 nm. Results: Guaifenesin was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Conclusion: The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness. PMID:23781462

Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.

PubMed

t'Kindt, Ruben; Jorge, Lucie; Dumont, Emmie; Couturon, Pauline; David, Frank; Sandra, Pat; Sandra, Koen

2012-01-03

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%). © 2011 American Chemical Society

Online extraction LC-MS/MS method for the simultaneous quantitative confirmation of urine drugs of abuse and metabolites: amphetamines, opiates, cocaine, cannabis, benzodiazepines and methadone.

PubMed

de Jager, Andrew D; Bailey, Neville L

2011-09-01

A rapid LC-MS/MS method for confirmatory testing of five major categories of drugs of abuse (amphetamine-type substances, opiates, cocaine, cannabis metabolites and benzodiazepines) in urine has been developed. All drugs of abuse mandated by the Australian/New Zealand Standard AS/NZS 4308:2008 are quantified in a single chromatographic run. Urine samples are diluted with a mixture of isotope labelled internal standards. An on-line trap-and-flush approach, followed by LC-ESI-MS/MS has been successfully used to process samples in a functioning drugs of abuse laboratory. Following injection of diluted urine samples, compounds retained on the trap cartridge are flushed onto a reverse-phase C18 HPLC column (5-μm particle size) with embedded hydrophylic functionality. A total chromatographic run-time of 15 min is required for adequate resolution. Automated quantitation software algorithms have been developed in-house using XML scripting to partially automate the identification of positive samples, taking into account ion ratio (IR) and retention times (Rt). The sensitivity of the assay was found to be adequate for the quantitation of drugs in urine at and below the confirmation cut-off concentrations prescribed by AS/NZS 4308:2008. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitation of tamsulosin in human plasma by liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Din, Li; Li, Limin; Tao, Ping; Yang, Jin; Zhang, Zhengxing

2002-02-05

A highly sensitive method for quantitation of tamsulosin in human plasma using 1-(2,6-dimethyl-3-hydroxylphenoxy)-2-(3,4-methoxyphenylethylamino)-propane hydrochloride as the internal standard (I.S.) was established using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). After alkalization with saturated sodium bicarbonate, plasma were extracted by ethyl acetate and separated by HPLC on a C18 reversed-phase column using a mobile phase of methanol-water-acetic acid-triethylamine (620:380:1.5:1.5, v/v). Analytes were quantitated using positive electrospray ionization in a quadrupole spectrometer. LC-ESI-MS was performed in the selected ion monitoring (SIM) mode using target ions at m/z 228 for tamsulosin and m/z 222 for the I.S. Calibration curves, which were linear over the range 0.2-30 ng/ml, were analyzed contemporaneously with each batch of samples, along with low (0.5 ng/ml), medium (3 ng/ml) and high (30 ng/ml) quality control samples. The intra- and inter-assay variability ranged from 2.14 to 8.87% for the low, medium and high quality control samples. The extraction recovery of tamsulosin from plasma was in the range of 84.2-94.5%. The method has been used successfully to study tamsulosin pharmacokinetics in adult humans.

A validated specific stability-indicating RP-HPLC assay method for Ambrisentan and its related substances.

PubMed

Narayana, M B V; Chandrasekhar, K B; Rao, B M

2014-09-01

A validated specific stability-indicating reverse-phase liquid chromatographic method was developed for the quantitative determination of Ambrisentan as well as its related substances in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its related impurities. Forced degradation studies were performed on bulk samples of Ambrisentan as per the ICH-prescribed stress conditions using acid, base, oxidative, thermal stress and photolytic degradation to show the stability-indicating power of the LC method. Significant degradation in acidic, basic stress conditions was observed and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from the forced degradation studies and the impurity-spiked solution. Good resolution between the peaks corresponds to Ambrisentan-related impurities and degradation products from the analyte were achieved on a SunFire C18 column using a mobile phase consisting of a mixture of potassium dihydrogen orthophosphate at a pH adjusted to 2.5 with ortho-phosphoric acid in water and a mixture of acetonitrile:methanol using a simple linear gradient. The detection was carried out at 225 nm. The limit of detection and the limit of quantification for the Ambrisentan and its related impurities were established. The stressed test solutions were assayed against the qualified working standard of Ambrisentan and the mass balance in each case was between 98.9 and 100.3%, indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per the ICH requirements. The developed method was found to be suitable to check the quality of bulk samples of Ambrisentan at the time of batch release and also during its storage (long-term and accelerated stability). © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A validated stability-indicating RP-HPLC method for levofloxacin in the presence of degradation products, its process related impurities and identification of oxidative degradant.

PubMed

Lalitha Devi, M; Chandrasekhar, K B

2009-12-05

The objective of current study was to develop a validated specific stability indicating reversed-phase liquid chromatographic method for the quantitative determination of levofloxacin as well as its related substances determination in bulk samples, pharmaceutical dosage forms in the presence of degradation products and its process related impurities. Forced degradation studies were performed on bulk sample of levofloxacin as per ICH prescribed stress conditions using acid, base, oxidative, water hydrolysis, thermal stress and photolytic degradation to show the stability indicating power of the method. Significant degradation was observed during oxidative stress and the degradation product formed was identified by LCMS/MS, slight degradation in acidic stress and no degradation was observed in other stress conditions. The chromatographic method was optimized using the samples generated from forced degradation studies and the impurity spiked solution. Good resolution between the peaks corresponds to process related impurities and degradation products from the analyte were achieved on ACE C18 column using the mobile phase consists a mixture of 0.5% (v/v) triethyl amine in sodium dihydrogen orthophosphate dihydrate (25 mM; pH 6.0) and methanol using a simple linear gradient. The detection was carried out at 294 nm. The limit of detection and the limit of quantitation for the levofloxacin and its process related impurities were established. The stressed test solutions were assayed against the qualified working standard of levofloxacin and the mass balance in each case was in between 99.4 and 99.8% indicating that the developed LC method was stability indicating. Validation of the developed LC method was carried out as per ICH requirements. The developed LC method was found to be suitable to check the quality of bulk samples of levofloxacin at the time of batch release and also during its stability studies (long term and accelerated stability).

N-nitrosodimethylamine (NDMA) removal by reverse osmosis and UV treatment and analysis via LC-MS/MS.

PubMed

Plumlee, Megan H; López-Mesas, Montserrat; Heidlberger, Andy; Ishida, Kenneth P; Reinhard, Martin

2008-01-01

N-nitrosodimethylamine (NDMA) is a probable human carcinogen found in ng/l concentrations in chlorinated and chloraminated water. A method was developed for the determination of ng/l levels of NDMA using liquid chromatography-tandem mass spectrometry (LC-MS/MS) preceded by sample concentration via solid-phase extraction with activated charcoal. Recoveries were greater than 90% and allowed a method reporting limit as low as 2ng/l. Using this method, the removal of NDMA was determined for the Interim Water Purification Facility (IWPF), an advanced wastewater treatment facility operated by the Orange County Water District (OCWD) in Southern California. The facility treats effluent from an activated sludge treatment plant with microfiltration (MF), reverse osmosis (RO), and an ultraviolet-hydrogen peroxide advanced oxidation process (UV-AOP). Six nitrosamines were surveyed: NDMA, N-nitrosomethylethylamine (NMEA), N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopiperidine (NPip), and N-nitrosopyrrolidine (NPyr). Only NDMA was detected and at all treatment steps in the IWPF, with influent concentrations ranging from 20 to 59 ng/l. Removals for RO and UV ranged from 24% to 56% and 43% to 66%, respectively. Overall, 69+/-7% of the original NDMA concentration was removed from the product water across the advanced treatment process and, in combination with blending, the final concentration did not exceed the California drinking water notification level of 10 ng/l. NDMA removal data are consistent with findings reviewed for other advanced treatment facilities and laboratory studies.

A novel two-dimensional liquid-chromatography method for online prediction of the toxicity of transformation products of benzophenones after water chlorination.

PubMed

Li, Jian; Ma, Li-Yun; Xu, Li; Shi, Zhi-Guo

2015-08-01

Benzophenone-type UV filters (BPs) are ubiquitous in the environment. Transformation products (TPs) of BPs with suspected toxicity are likely to be produced during disinfection of water by chlorination. To quickly predict the toxicity of TPs, in this study, a novel two-dimensional liquid-chromatography (2D-LC) method was established in which the objective of the first dimension was to separate the multiple components of the BPs sample after chlorination, using a reversed-phase liquid-chromatography mode. A biochromatographic system, i.e. bio-partitioning micellar chromatography with the polyoxyethylene (23) lauryl ether aqueous solution as the mobile phase, served as the second dimension to predict the toxicity of the fraction from the first dimension on the basis of the quantitative retention-activity relationships (QRARs) model. Six BPs, namely 2,4-dihydroxybenzophenone, oxybenzone, 4-hydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone, were the target analytes subjected to chlorination. The products of these BPs after chlorination were directly injected to the 2D-LC system for analysis. The results indicated that most TPs may be less toxic than their parent chemicals, but some may be more toxic, and that intestinal toxicity of TPs may be more obvious than blood toxicity. The proposed method is time-saving, high-throughput, and reliable, and has great potential for predicting toxicity or bioactivity of unknown and/or known components in a complex sample. Graphical Abstract The scheme for the 2D-LC online prediction of toxicity of the transformation products of benzophenone-type UV filters after chlorination.

Resolving the chemical heterogeneity of natural organic matter: new insights from comprehensive two-dimensional liquid chromatography.

PubMed

Duarte, Regina M B O; Barros, Ana C; Duarte, Armando C

2012-08-03

For the purpose of resolving the chemical heterogeneity of natural organic matter (NOM), comprehensive two-dimensional liquid chromatography (LC×LC) was employed for the first time to map the hydrophobicity versus molecular weight (MW) distribution of two well-known complex organic mixtures: Suwannee River Fulvic Acids (SR-FA) and Pony Lake Fulvic Acids (PL-FA). Two methods have been developed using either a conventional reversed-phase (RP) silica column or a mixed-mode hydrophilic interaction column operating under aqueous RP mode in the first dimension, and a size-exclusion column in the second dimension. The LC×LC fractions were screened on-line by UV at 254 nm, molecular fluorescence at excitation/emission wavelengths (λ(Exc)/λ(Em)) of 240/450 nm, and by evaporative light scattering. The MW distributions of these two NOM samples were further characterized by number (Mn) and weight (Mw) average MW, and by polydispersity (Mw/Mn). Findings suggest that the combination of two independent separation mechanisms is promising in extend the range of NOM separation. For the cases where NOM separation was accomplished, smaller Mw group fractions seem to be related to a more hydrophobic nature. Regardless of the detection method, the complete range of MW distribution provided by both comprehensive LC×LC methods was found to be lower than those reported in the literature. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantitative determination of triterpenes from Amphiptherygium adstringens by liquid chromatography and thin-layer chromatography and morphological analysis of cuachalalate preparations.

PubMed

Navarrete, Andres; Avula, Bharathi; Joshi, Vaishali C; Ji, Xiuhong; Hersh, Paul; Khan, Ikhlas A

2006-01-01

Amphiptherygium adstringens (Anacardiaceae/Julianaceae), local name "cuachalalate," is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolemia, gastritis, gastric ulcers, and cancer of the gastrointestinal tract. The development of column high-performance liquid chromatography-photodiode array detector (LC-PDA) and high-performance thin-layer chromatography (HPTLC)-densitometry methods for the determination of masticadienonic acid and 3-hydroxymasticadienonic acid in cuachalalate preparations is described in this paper. Good separation of the compounds could be achieved by both methods. Either might be preparable depending on the requirements. The LC separation was performed on a Phenomenex Synergi MAX-RP 80A reversed-phase column operated at 40 degrees C with detection at 215 nm. The plant materials were extracted with methanol by sonication. The triterpenes present in the plant material and commercial extracts were separated with an acetonitrile-water reagent alcohol isocratic system. The limit of detection was 0.1-0.2 microg/mL. The relative standard deviation values for the determination of triterpenes in plant extracts were less than 1.00%. This is the first report of an analytical method developed for the quantitative analysis of triterpenes from Amphiptherygium adstringens by LC-PDA and HPTLC. The stem bark showed higher amounts of triterpenes, and low amounts in root and stem root. The microscopic description of the crude drug of cuachalalate was also provided.

Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

PubMed

Mladic, Marija; de Waal, Tessa; Burggraaff, Lindsey; Slagboom, Julien; Somsen, Govert W; Niessen, Wilfried M A; Manjunatha Kini, R; Kool, Jeroen

2017-10-01

This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

Bioanalytical LC-MS/MS method validation for plasma determination of topiramate in healthy Indian volunteers.

PubMed

Goswami, Dipanjan; Kumar, Ajay; Khuroo, Arshad H; Monif, Tausif; Rab, Shamsur

2009-11-01

A LC-MS/MS method for plasma topiramate analysis is delineated involving least number of healthy volunteers. Topiramate and amlodipine internal standard (IS) were extracted by simple centrifuge-coupled solid-phase extraction and reverse-phase chromatographic separation was performed on an Ascentis C(18) column. Turbo-spray negative-ion mode multiple-reaction monitoring was selected for mass pair detection at m/z 338.3 --> 78.0 and m/z 407.3 --> 295.5 for analyte and IS respectively. The method showed a dynamic linearity range from 10.4 to 2045.0 ng/mL, lower limit of quantitation achieved at 10.4 ng/mL and finally a mass spectrometric total run time of within 2.5 min for human sample analysis. Bioequivalence was assessed successfully using this fully validated method on 16 fasted Indian male subjects with 25 mg topiramate tablet administration. An appropriate study design describes plasma samples collection up to 216 h post dose in two periods, separated by a 28 day washout period. The challenge of half-life matching for test and reference drug was achieved with 73.43 +/- 9.68 and 73.06 +/- 14.03 h, respectively, and intra-subject coefficient of variation achieved within 11% for AUCs and C(max) evaluated by non-compartmental pharmacokinetic analysis. The results of LCMS topiramate complete method validation supported by pharmacokinetic study have not been published before, and are presented and discussed for the first time in this article. Copyright (c) 2009 John Wiley & Sons, Ltd.

Morphological and electro optic studies of polymer dispersed liquid crystal in reverse mode

NASA Astrophysics Data System (ADS)

Sharma, Vandna; Kumar, Pankaj; Chinky, Malik, Praveen; Raina, K. K.

2018-05-01

Present work deals with reverse mode polymer dispersed liquid crystals (PDLCs) sensitive to electric field. Contrary to the conventional PDLCs operate from opaque (OFF state) to transparent state (ON state) with the application of field, reverse mode PDLCs work in transparent to opaque state. Reverse mode PDLC composed of nematic LC and UV curable optical adhesive polymer were prepared by the polymerization induced phase separation. The polarizing optical microscope study shows the vertical alignment of LCs within droplets with initial dark state under cross polarizers and confirms preliminary natural transparent state. The electro optic (EO) results show that the reverse mode PDLC lowered the threshold and operating voltages significantly compared with reported values. The contrast ratio of the film was also studied.

Evaluation of a method based on liquid chromatography-diode array detector-tandem mass spectrometry for a rapid and comprehensive characterization of the fat-soluble vitamin and carotenoid profile of selected plant foods.

PubMed

Gentili, Alessandra; Caretti, Fulvia

2011-02-04

The feasibility of using reversed-phase liquid chromatography/diode array/tandem mass spectrometry (LC-DAD-MS/MS) for a rapid and comprehensive profiling of fat soluble vitamins and pigments in some foods of plant origin (maize flour, green and golden kiwi) was evaluated. The instrumental approach was planned for obtaining two main outcomes within the same chromatographic run: (i) the quantitative analysis of ten target analytes, whose standards are commercially available; (ii) the screening of pigments occurring in the selected matrices. The quantitative analysis was performed simultaneously for four carotenoids (lutein, zeaxanthin, β-cryptoxanthin, and β-carotene) and six compounds with fat-soluble activity (α-tocopherol, δ-tocopherol, γ-tocopherol, ergocalciferol, phylloquinone and menaquinone-4), separated on a C30 reversed-phase column and detected by atmospheric pressure chemical ionization (APCI) tandem mass spectrometry, operating in Selected Reaction Monitoring (SRM) mode. Extraction procedure was based on matrix solid-phase dispersion with recoveries of all compounds under study exceeding 78 and 60% from maize flour and kiwi, respectively. The method intra-day precision ranged between 3 and 7%, while the inter-day one was below 12%. The mild isolation conditions precluded artefacts creation, such as cis-isomerization phenomena for carotenoids. During the quantitative LC-SRM determination of the ten target analytes, the identification power of the diode array detector joined to that of the triple quadrupole (QqQ) allowed the tentatively identification of several pigments (chlorophylls and carotenoids), without the aid of standards, on the basis of: (i) the UV-vis spectra recorded in the range of 200-700nm; (ii) the expected retention time; (iii) the two SRM transitions, chosen for the target carotenoids but also common to many of isomeric carotenoids occurring in the selected foods. Copyright © 2010 Elsevier B.V. All rights reserved.

Development and validation of stability indicating the RP-HPLC method for the estimation of related compounds of guaifenesin in pharmaceutical dosage forms.

PubMed

Reddy, Sunil Pingili; Babu, K Sudhakar; Kumar, Navneet; Sekhar, Y V V Sasi

2011-10-01

A stability-indicating gradient reverse phase liquid chromatographic (RP-LC) method was developed for the quantitative determination of related substances of guaifenesin in pharmaceutical formulations. The baseline separation for guaifenesin and all impurities was achieved by utilizing a Water Symmetry C18 (150 mm × 4.6 mm) 5 μm column particle size and a gradient elution method. The mobile phase A contains a mixture of 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 90:10 v/v, while the mobile phase B contains 0.02 M KH2PO4 (pH 3.2) and methanol in the ratio of 10:90 v/v, respectively. The flow rate of the mobile phase was 0.8 ml/min with a column temperature of 25°C and detection wavelength at 273 nm. Guaifenesin was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection and quantification, accuracy, precision, and robustness.

Real-time liquid-crystal atmosphere turbulence simulator with graphic processing unit.

PubMed

Hu, Lifa; Xuan, Li; Li, Dayu; Cao, Zhaoliang; Mu, Quanquan; Liu, Yonggang; Peng, Zenghui; Lu, Xinghai

2009-04-27

To generate time-evolving atmosphere turbulence in real time, a phase-generating method for our liquid-crystal (LC) atmosphere turbulence simulator (ATS) is derived based on the Fourier series (FS) method. A real matrix expression for generating turbulence phases is given and calculated with a graphic processing unit (GPU), the GeForce 8800 Ultra. A liquid crystal on silicon (LCOS) with 256x256 pixels is used as the turbulence simulator. The total time to generate a turbulence phase is about 7.8 ms for calculation and readout with the GPU. A parallel processing method of calculating and sending a picture to the LCOS is used to improve the simulating speed of our LC ATS. Therefore, the real-time turbulence phase-generation frequency of our LC ATS is up to 128 Hz. To our knowledge, it is the highest speed used to generate a turbulence phase in real time.

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines.

PubMed Central

Lewis, R N; McElhaney, R N

2000-01-01

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains. PMID:11023908

Cellulose-lanthanum hydroxide nanocomposite as a selective marker for detection of toxic copper

PubMed Central

2014-01-01

In this current report, a simple, reliable, and rapid method based on modifying the cellulose surface by doping it with different percentages of lanthanum hydroxide (i.e., 1% La(OH)3-cellulose (LC), 5% La(OH)3-cellulose (LC2), and 10% La(OH)3-cellulose (LC3)) was proposed as a selective marker for detection of copper (Cu(II)) in aqueous medium. Surface properties of the newly modified cellulose phases were confirmed by Fourier transform infrared spectroscopy, field emission scanning electron microscope, energy dispersive X-ray spectroscopy, X-ray diffraction, and X-ray photoelectron spectroscopic analysis. The effect of pH on the adsorption of modified cellulose phases for Cu(II) was evaluated, and LC3 was found to be the most selective for Cu(II) at pH 6.0. Other parameters, influencing the maximum uptake of Cu(II) on LC3, were also investigated for a deeper mechanistic understanding of the adsorption phenomena. Results showed that the adsorption capacity for Cu(II) was improved by 211% on the LC3 phase as compared to diethylaminoethyl cellulose phase after only 2 h contact time. Adsorption isotherm data established that the adsorption process nature was monolayer with a homogeneous adsorbent surface. Results displayed that the adsorption of Cu(II) onto the LC3 phase obeyed a pseudo-second-order kinetic model. Selectivity studies toward eight metal ions, i.e., Cd(II), Co(II), Cr(III), Cr(VI), Cu(II), Fe(III), Ni(II), and Zn(II), were further performed at the optimized pH value. Based on the selectivity study, it was found that Cu(II) is highly selective toward the LC3 phase. Moreover, the efficiency of the proposed method was supported by implementing it to real environmental water samples with adequate results. PMID:25258599

Reversed phase liquid chromatography with UV absorbance and flame ionization detection using a water mobile phase and a cyano propyl stationary phase Analysis of alcohols and chlorinated hydrocarbons.

PubMed

Quigley, W W; Ecker, S T; Vahey, P G; Synovec, R E

1999-10-01

The development of liquid chromatography with a commercially available cyano propyl stationary phase and a 100% water mobile phase is reported. Separations were performed at ambient temperature, simplifying instrumental requirements. Excellent separation efficiency using a water mobile phase was achieved, for example N=18 800, or 75 200 m(-1), was obtained for resorcinol, at a retention factor of k'=4.88 (retention time of 9.55 min at 1 ml min(-1) for a 25 cmx4.6 mm i.d. column, packed with 5 mum diameter particles with the cyano propyl stationary phase). A separation via reversed phase liquid chromatography (RP-LC) with a 100% water mobile phase of six phenols and related compounds was compared to a separation of the same compounds by traditional RP-LC, using octadecylsilane (ODS), i.e. C18, bound to silica and an aqueous mobile phase modified with acetonitrile. Nearly identical analysis time was achieved for the separation of six phenols and related compounds using the cyano propyl stationary phase with a 100% water mobile phase, as compared to traditional RP-LC requiring a relatively large fraction of organic solvent modifier in the mobile phase (25% acetonitrile:75% water). Additional understanding of the retention mechanism with the 100% water mobile phase was obtained by relating measured retention factors of aliphatic alcohols, phenols and related compounds, and chlorinated hydrocarbons to their octanol:water partition coefficients. The retention mechanism is found to be consistent with a RP-LC mechanism coupled with an additional retention effect due to residual hydroxyl groups on the cyano propyl stationary phase. Advantages due to a 100% water mobile phase for the chemical analysis of alcohol mixtures and chlorinated hydrocarbons are reported. By placing an absorbance detector in-series and preceding a novel drop interface to a flame ionization detector (FID), selective detection of a separated mixture of phenols and related compounds and aliphatic alcohols is achieved. The compound class of aliphatic alcohols is selectively and sensitively detected by the drop interface/FID, and the phenols and related compounds are selectively and sensitively detected by absorbance detection at 200 nm. The separation and detection of chlorinated hydrocarbons in a water sample matrix further illustrated the advantages of this methodology. The sensitivity and selectivity of the FID signal for the chlorinated hydrocarbons are significantly better than absorbance detection, even at 200 nm. This methodology is well suited to continuous and automated monitoring of water samples. The applicability of samples initially in an organic solvent matrix is explored, since an organic sample matrix may effect retention and efficiency. Separations in acetonitrile and isopropyl alcohol sample matrices compared well to separations with a water sample matrix.

Easy and Fast Method for the Determination of Biogenic Amines in Fish and Fish Products with Liquid Chromatography Coupled to Orbitrap Tandem Mass Spectrometry.

PubMed

Kaufmann, Anton; Maden, Kathryn

2018-03-01

A quantitative method for the determination of biogenic amines was developed. The method is characterized by the virtual absence of sample cleanup and does not require a derivatization reaction. Diluted extracts are centrifuged, filtrated, and directly injected into an ultra-HPLC column, which is coupled to a single-stage high-resolution mass spectrometer (Orbitrap). The chromatography is based on a reversed-phase column and an eluent containing an ion-pairing agent (heptafluorobutyric acid). The high sensitivity of the instrument permits the injection of very diluted extracts, which ensures stable retention times and the virtual absence of signal suppression effects. In addition, the quantification of histamine (a regulated compound) is further aided by the use of an isotopically labeled internal standard. The method was validated for three fish-based matrixes. Both the sample processing and the analytical measurement are very fast; hence, the methodology is ideal for high-throughput work. In addition, the method is significantly more selective than conventional methods (i.e., derivatization followed by LC with UV/fluorescence (FL) detection) for biogenic amines. A comparison showed that LC-UV/FL methods can produce false-positive findings due to coeluting matrix compounds.

Absolute quantification of histone PTM marks by MRM-based LC-MS/MS.

PubMed

Gao, Jun; Liao, Rijing; Yu, Yanyan; Zhai, Huili; Wang, Yingqi; Sack, Ragna; Peters, Antoine H F M; Chen, Jiajia; Wu, Haiping; Huang, Zheng; Hu, Min; Qi, Wei; Lu, Chris; Atadja, Peter; Oyang, Counde; Li, En; Yi, Wei; Zhou, Shaolian

2014-10-07

The N-terminal tails of core histones harbor the sites of numerous post-translational modifications (PTMs) with important roles in the regulation of chromatin structure and function. Profiling histone PTM marks provides data that help understand the epigenetics events in cells and their connections with cancer and other diseases. Our previous study demonstrated that specific derivatization of histone peptides by NHS propionate significantly improved their chromatographic performance on reversed phase columns for LC/MS analysis. As a step forward, we recently developed a multiple reaction monitoring (MRM) based LC-MS/MS method to analyze 42 targeted histone peptides. By using stable isotopic labeled peptides as internal standards that are spiked into the reconstituted solutions, this method allows to measure absolute concentration of the tryptic peptides of H3 histone proteins extracted from cancer cell lines. The method was thoroughly validated for the accuracy and reproducibility through analyzing recombinant histone proteins and cellular samples. The linear dynamic range of the MRM assays was achieved in 3 orders of magnitude from 1 nM to 1 μM for all targeted peptides. Excellent intrabatch and interbatch reproducibility (<15% CV) was obtained. This method has been used to study translocated NSD2 (a histone lysine methyltransferase that catalyzes the histone lysine 36 methylation) function with its overexpression in KMS11 multiple myeloma cells. From the results we have successfully quantitated both individual and combinatorial histone marks in parental and NSD2 selective knockout KMS11 cells.

Development and validation of sensitive LC/MS/MS method for quantitative bioanalysis of levonorgestrel in rat plasma and application to pharmacokinetics study.

PubMed

Ananthula, Suryatheja; Janagam, Dileep R; Jamalapuram, Seshulatha; Johnson, James R; Mandrell, Timothy D; Lowe, Tao L

2015-10-15

Rapid, sensitive, selective and accurate LC/MS/MS method was developed for quantitative determination of levonorgestrel (LNG) in rat plasma and further validated for specificity, linearity, accuracy, precision, sensitivity, matrix effect, recovery efficiency and stability. Liquid-liquid extraction procedure using hexane:ethyl acetate mixture at 80:20 v:v ratio was employed to efficiently extract LNG from rat plasma. Reversed phase Luna column C18(2) (50×2.0mm i.d., 3μM) installed on a AB SCIEX Triple Quad™ 4500 LC/MS/MS system was used to perform chromatographic separation. LNG was identified within 2min with high specificity. Linear calibration curve was drawn within 0.5-50ng·mL(-1) concentration range. The developed method was validated for intra-day and inter-day accuracy and precision whose values fell in the acceptable limits. Matrix effect was found to be minimal. Recovery efficiency at three quality control (QC) concentrations 0.5 (low), 5 (medium) and 50 (high) ng·mL(-1) was found to be >90%. Stability of LNG at various stages of experiment including storage, extraction and analysis was evaluated using QC samples, and the results showed that LNG was stable at all the conditions. This validated method was successfully used to study the pharmacokinetics of LNG in rats after SubQ injection, providing its applicability in relevant preclinical studies. Copyright © 2015 Elsevier B.V. All rights reserved.

High throughput screening and antioxidant assay of dibenzo[a,c]cyclooctadiene lignans in modified-ultrasonic and supercritical fluid extracts of Schisandra chinensis Baill by liquid chromatography--mass spectrometry and a free radical-scavenging method.

PubMed

Wang, Ming-Chih; Lai, Yih-Cherng; Chang, Chia-Lin

2008-05-01

Dibenzo[a,c]cyclooctadiene lignans of Schisandra chinensis Baill are well known because of their hepatoprotective activity, antioxidant activity, and anticancer effect. For the isolation of the dibenzo[a,c]cyclooctadiene lignans of Schisandra chinensis Baill two extraction methods were used: modified-ultrasonic extraction and supercritical fluid extraction. A specific and fast analytical method for structure identification is established for quality control because structure elucidation could be accomplished by means of liquid chromatography-mass spectrometry (LC-MS) technologies. The separation and identification of the compounds were completed by: (i) a water-acetonitrile gradient system using a C18 reversed-phase column; (ii) UV detection at 225 nm; (iii) MS/MS experiments with electrospray ionization interface (ESI) ion trap mass spectrometry in the positive mode. Normalized collision energy was used to obtain fragment ions of structural relevance in the LC-MS/MS. These results provided a reliable LC-MS/MS method for the determination of the dibenzo[a,c]cyclooctadiene lignans from Schisandra chinensis Baill. Finally, we also detected 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging effects (%) of the modified-ultrasonic and supercritical fluid extracts of Schisandra chinensis Baill compared with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). The antioxidant activities of the modified-ultrasonic and supercritical fluid extracts were lower than that of trolox.

Improved quality-by-design compliant methodology for method development in reversed-phase liquid chromatography.

PubMed

Debrus, Benjamin; Guillarme, Davy; Rudaz, Serge

2013-10-01

A complete strategy dedicated to quality-by-design (QbD) compliant method development using design of experiments (DOE), multiple linear regressions responses modelling and Monte Carlo simulations for error propagation was evaluated for liquid chromatography (LC). The proposed approach includes four main steps: (i) the initial screening of column chemistry, mobile phase pH and organic modifier, (ii) the selectivity optimization through changes in gradient time and mobile phase temperature, (iii) the adaptation of column geometry to reach sufficient resolution, and (iv) the robust resolution optimization and identification of the method design space. This procedure was employed to obtain a complex chromatographic separation of 15 antipsychotic basic drugs, widely prescribed. To fully automate and expedite the QbD method development procedure, short columns packed with sub-2 μm particles were employed, together with a UHPLC system possessing columns and solvents selection valves. Through this example, the possibilities of the proposed QbD method development workflow were exposed and the different steps of the automated strategy were critically discussed. A baseline separation of the mixture of antipsychotic drugs was achieved with an analysis time of less than 15 min and the robustness of the method was demonstrated simultaneously with the method development phase. Copyright © 2013 Elsevier B.V. All rights reserved.

Electrospray and MALDI mass spectrometry in the identification of spermicides in criminal investigations.

PubMed

Hollenbeck, T P; Siuzdak, G; Blackledge, R D

1999-07-01

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry have been used to examine evidence in a sexual assault investigation. Because condoms are being used increasingly by sexual assailants and some condom brands include the spermicide nonoxynol-9 (nonylphenoxy polyethoxyethanol) in the lubricant formulation, the recovery, and identification of nonoxynol-9 from evidence items may assist in proving corpus delicti. A method was developed for the recovery of nonoxynol-9 from internal vaginal swabs and for its identification by reverse phase liquid chromatography/electrospray ionization mass spectrometry (LC ESI-MS), nanoelectrospray ionization (nanoESI) mass spectrometry, and high resolution MALDI Fourier transform mass spectrometry (MALDI-FTMS). The method was tested on extracts from precoitus, immediate postcoitus, and four-hours postcoitus vaginal swabs provided by a volunteer whose partner does not normally use condoms, but for this trial used a condom having a water-soluble gel-type lubricant that includes 5% nonoxynol-9 in its formulation. Subsequently, LC ESI-MS was used to identify traces of nonoxynol-9 from the internal vaginal swab of a victim of a sexual assault.

Liquid chromatographic determination of urinary 5-methyl-2'-deoxycytidine and pseudouridine as potential biological markers for leukaemia.

PubMed

Zambonin, C G; Aresta, A; Palmisano, F; Specchia, G; Liso, V

1999-12-01

A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2'-deoxycytidine (m5dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 microg/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m5dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (psi), a modified nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary psi/Cre ratio was found significantly increased for leukaemia patients, the urinary m5dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease.

Development and validation of a highly sensitive LC-MS/MS method for the determination of dexamethasone in nude mice plasma and its application to a pharmacokinetic study.

PubMed

Yuan, Yin; Zhou, Xuan; Li, Jian; Ye, Suofu; Ji, Xiwei; Li, Liang; Zhou, Tianyan; Lu, Wei

2015-04-01

In the current study, a simple, sensitive and rapid analytical method for the determination of dexamethasone was developed and applied to a pharmacokinetic study in nude mice. Using testosterone as an internal standard, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach after one-step precipitation with acetonitrile was validated and used to determine the concentrations of dexamethasone in nude mice plasma. The method utilized a simple isocratic reverse phase separation over a Dionex C18 column with a mobile phase composed of acetonitrile-water (40:60, v/v). The analyte was detected by a triple quadrupole tandem mass spectrometer via electrospray and multiple reaction monitoring was employed to select both dexamethasone at m/z 393.0/147.1 and testosterone at m/z 289.5/97.3 in the positive ion mode. The calibration curves were linear (r >0.99) ranging from 2.5 to 500 ng/mL with a lower limit of quantitation of 2.5 ng/mL. The relative standard deviation ranged from 1.69 to 9.22% while the relative error ranged from -1.92 to -8.46%. This method was successfully applied to a preclinical pharmacokinetic study of dexamethasone and its pharmacokinetics was characterized by a two-compartment model with first-order absorption in female nude mice. Copyright © 2014 John Wiley & Sons, Ltd.

Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

PubMed

Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

2017-06-02

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

Analysis of selected sugars and sugar phosphates in mouse heart tissue by reductive amination and liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Han, Jun; Tschernutter, Vera; Yang, Juncong; Eckle, Tobias; Borchers, Christoph H

2013-06-18

Sensitive and reliable analysis of sugars and sugar phosphates in tissues and cells is essential for many biological and cell engineering studies. However, the successful analysis of these endogenous compounds in biological samples by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is often difficult because of their poor chromatographic retention properties in reversed-phase LC, the complex biological matrices, and the ionization suppression in ESI. This situation is further complicated by the existence of their multiple structural isomers in vivo. This work describes the combination of reductive amination using 3-amino-9-ethylcarbazole, with a new LC approach using a pentafluorophenyl core-shell ultrahigh performance (UP) LC column and methylphosphonic acid as an efficient tail-sweeping reagent for improved chromatographic separation. This new method was used for selected detection and accurate quantitation of the major free and phosphorylated reducing sugars in mouse heart tissue. Among the detected compounds, accurate quantitation of glyceraldehyde, ribose, glucose, glycerylaldehyde-3-phosphate, ribose-5-phosphate, glucose-6-phosphate, and mannose-6-phosphate was achieved by UPLC/multiple-reaction monitoring (MRM)-MS, with analytical accuracies ranging from 87.4% to 109.4% and CVs of ≤8.5% (n = 6). To demonstrate isotope-resolved metabolic profiling, we used UPLC/quadrupole time-of-flight (QTOF)-MS to analyze the isotope distribution patterns of C3 to C6 free and phosphorylated reducing sugars in heart tissues from (13)C-labeled wild type and knockout mice. In conclusion, the preanalytical derivatization-LC/ESI-MS method has resulted in selective determination of free and phosphorylated reducing sugars without the interferences from their nonreducing structural isomers in mouse heart tissue, with analytical sensitivities in the femtomole to low picomole range.

Validated LC-MS-MS Method for Multiresidual Analysis of 13 Illicit Phenethylamines in Amniotic Fluid.

PubMed

Burrai, Lucia; Nieddu, Maria; Carta, Antonio; Trignano, Claudia; Sanna, Raimonda; Boatto, Gianpiero

2016-04-01

A multi-residue analytical method was developed for the determination in amniotic fluid (AF) of 13 illicit phenethylamines, including 12 compounds never investigated in this matrix before. Samples were subject to solid-phase extraction using; hydrophilic-lipophilic balance cartridges which gave good recoveries and low matrix effects on analysis of the extracts. The quantification was performed by liquid chromatography electrospray tandem mass spectrometry. The water-acetonitrile mobile phase containing 0.1% formic acid, used with a C18 reversed phase column, provided adequate separation, resolution and signal-to-noise ratio for the analytes and the internal standard. The final optimized method was validated according to international guidelines. A monitoring campaign to assess fetal exposure to these 13 substances of abuse has been performed on AF test samples obtained from pregnant women. All mothers (n = 194) reported no use of drugs of abuse during pregnancy, and this was confirmed by the analytical data. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry.

PubMed

Uutela, Päivi; Ketola, Raimo A; Piepponen, Petteri; Kostiainen, Risto

2009-02-09

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, beta-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r(2)=0.996-0.999, range=100-8500nmolL(-1)). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.

Unifying expression scale for peptide hydrophobicity in proteomic reversed phase high-pressure liquid chromatography experiments.

PubMed

Grigoryan, Marine; Shamshurin, Dmitry; Spicer, Victor; Krokhin, Oleg V

2013-11-19

As an initial step in our efforts to unify the expression of peptide retention times in proteomic liquid chromatography-mass spectrometry (LC-MS) experiments, we aligned the chromatographic properties of a number of peptide retention standards against a collection of peptides commonly observed in proteomic experiments. The standard peptide mixtures and tryptic digests of samples of different origins were separated under the identical chromatographic condition most commonly employed in proteomics: 100 Å C18 sorbent with 0.1% formic acid as an ion-pairing modifier. Following our original approach (Krokhin, O. V.; Spicer, V. Anal. Chem. 2009, 81, 9522-9530) the retention characteristics of these standards and collection of tryptic peptides were mapped into hydrophobicity index (HI) or acetonitrile percentage units. This scale allows for direct visualization of the chromatographic outcome of LC-MS acquisitions, monitors the performance of the gradient LC system, and simplifies method development and interlaboratory data alignment. Wide adoption of this approach would significantly aid understanding the basic principles of gradient peptide RP-HPLC and solidify our collective efforts in acquiring confident peptide retention libraries, a key component in the development of targeted proteomic approaches.

Quantitation of Tenofovir and Emtricitabine in Dried Blood Spots (DBS) with LC-MS/MS

PubMed Central

Zheng, Jia-Hua; Guida, Louis A; Rower, Caitlin; Castillo-Mancilla, Jose; Meditz, Amie; Klein, Brandon; Kerr, Becky Jo; Langness, Jacob; Bushman, Lane; Kiser, Jennifer; Anderson, Peter L.

2013-01-01

A reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of tenofovir (TFV) and emtricitabine (FTC) in dried blood spots (DBS) from human whole blood was developed and validated. Whole blood samples were spotted, dried, and a 3mm punch was extracted with methanol for analysis by LC-MS/MS utilizing stable isotope labeled internal standards. The assay was validated over the range of 2.5ng/mL to 1,000ng/mL for TFV and 2.5ng/mL to 5,000ng/mL for FTC. The method was accurate (within ± 15% of control) and precise (coefficient of variation ≤ 15%) for hematocrit concentrations ranging from 25% to 76%; using edge punches versus center punches; and spot volumes of 10µL to 50µL. Analytes were stable for five freeze/thaw cycles and up to 6 days at room temperature, whereas long-term storage required −20°C or −80°C. Comparison of TFV and FTC in DBS versus plasma yielded r2 ≥ 0.96, indicating that DBS can be used as a plasma alternative for pharmacokinetic analyses in vivo. PMID:24055850

Fast-HPLC Fingerprinting to Discriminate Olive Oil from Other Edible Vegetable Oils by Multivariate Classification Methods.

PubMed

Jiménez-Carvelo, Ana M; González-Casado, Antonio; Pérez-Castaño, Estefanía; Cuadros-Rodríguez, Luis

2017-03-01

A new analytical method for the differentiation of olive oil from other vegetable oils using reversed-phase LC and applying chemometric techniques was developed. A 3 cm short column was used to obtain the chromatographic fingerprint of the methyl-transesterified fraction of each vegetable oil. The chromatographic analysis took only 4 min. The multivariate classification methods used were k-nearest neighbors, partial least-squares (PLS) discriminant analysis, one-class PLS, support vector machine classification, and soft independent modeling of class analogies. The discrimination of olive oil from other vegetable edible oils was evaluated by several classification quality metrics. Several strategies for the classification of the olive oil were used: one input-class, two input-class, and pseudo two input-class.

Evaluation of an International Pharmacopoeia method for the analysis of nelfinavir mesilate by liquid chromatography.

PubMed

Yekkala, Raja Satyanarayana; Vandenwayenberg, Stephanie; Hoogmartens, Jos; Adams, Erwin

2006-11-17

A gradient LC method for the determination of related substances in nelfinavir mesilate (NFVM) has been recently published in the International Pharmacopoeia. The method uses a base deactivated reversed phase C18 column (25 cm x 4.6 mm I.D.), 5 microm kept at a temperature of 35 degrees C. The mobile phases consist of acetonitrile, methanol, phosphate buffer pH 3.4 and water. The flow rate is 1.0 ml/min. UV detection is performed at 225 nm. A system suitability test (SST) is described to govern the quality of the separation. The separation towards NFVM components was investigated on 18 C18 columns and correlation was made with the column classification system developed in our laboratory. The method was evaluated using a Hypersil BDS C18 column (25 cm x 4.6 mm I.D.), 5 microm. A two level fractional factorial design was applied to examine the robustness of the method. The method shows good selectivity, precision, linearity and sensitivity. Seven commercial samples were examined using this method.

Prepolymer Characterization

DTIC Science & Technology

1988-10-01

and ZIP Code) 10 SOURCE OF FUNDING NUMBERS PROGRAM PROJECT TASK WORK UNIT ELEMENT NO. NO. NO ACCESSION NO. 62302f ---5730 - o 00 AN 11. TITLE (Include...32 Preparative-Scale Reverse-Phase LC Fractionation of Polystyrene Homologs ..................................... 35 Work -Up of...were also employed. In addition, much of the work was based upon R-45M. However, the fundamental analytical developments and resultant practical

Simultaneous estimation of lisofylline and pentoxifylline in rat plasma by high performance liquid chromatography-photodiode array detector and its application to pharmacokinetics in rat.

PubMed

Italiya, Kishan S; Sharma, Saurabh; Kothari, Ishit; Chitkara, Deepak; Mittal, Anupama

2017-09-01

Lisofylline (LSF) is an anti-inflammatory and immunomodulatory agent with proven activity in serious infections associated with cancer chemotherapy, hyperoxia-induced acute lung injury, autoimmune disorders including type-1 diabetes (T1DM) and islet rejection after islet transplantation. It is also an active metabolite of another anti-inflammatory agent, Pentoxifylline (PTX). LSF bears immense therapeutic potential in multiple pharmacological activities and hence appropriate and accurate quantification of LSF is very important. Although a number of analytical methods for quantification of LSF and PTX have been reported for pharmacokinetics and metabolic studies, each of these have certain limitations in terms of large sample volume required, complex extraction procedure and/or use of highly sophisticated instruments like LC-MS/MS. The aim of current study is to develop a simple reversed-phase HPLC method in rat plasma for simultaneous determination of LSF and PTX with the major objective of ensuring minimum sample volume, ease of extraction, economy of analysis, selectivity and avoiding use of instruments like LC-MS/MS to ensure a widespread application of the method. A simple liquid-liquid extraction method using methylene chloride as extracting solvent was used for extracting LSF and PTX from rat plasma (200μL). Samples were then evaporated, reconstituted with mobile phase and injected into HPLC coupled with photo-diode detector (PDA). LSF, PTX and 3-isobutyl 1-methyl xanthine (IBMX, internal standard) were separated on Inertsil® ODS (C18) column (250×4.6mm, 5μm) with mobile phase consisting of A-methanol B-water (50:50v/v) run in isocratic mode at flow rate of 1mL/min for 15min and detection at 273nm. The method showed linearity in the concentration range of 50-5000ng/mL with LOD of 10ng/mL and LLOQ of 50ng/mL for both LSF and PTX. Weighted linear regression analysis was also performed on the calibration data. The mean absolute recoveries were found to be 80.47±3.44 and 80.89±3.73% for LSF and PTX respectively. The method was successfully applied for studying the pharmacokinetics of LSF and PTX after IV bolus administration at dose of 25mg/kg in Wistar rat. In conclusion, a simple, sensitive, accurate and precise reversed-phase HPLC-UV method was established for simultaneous determination of LSF and PTX in rat plasma. Copyright © 2017 Elsevier B.V. All rights reserved.

Feasibility and application of an HPLC/UVD to determine dinotefuran and its shorter wavelength metabolites residues in melon with tandem mass confirmation.

PubMed

Rahman, Md Musfiqur; Park, Jong-Hyouk; Abd El-Aty, A M; Choi, Jeong-Heui; Yang, Angel; Park, Ki Hun; Nashir Uddin Al Mahmud, Md; Im, Geon-Jae; Shim, Jae-Han

2013-01-15

A new analytical method was developed for dinotefuran and its metabolites, MNG, UF, and DN, in melon using high-performance liquid chromatography (HPLC) coupled with an ultraviolet detector (UVD). Due to shorter wavelength, lower sensitivity to UV detection, and high water miscibility of some metabolites, QuEChERs acetate-buffered version was modified for extraction and purification. Mobile phases with different ion pairing or ionisation agents were tested in different reverse phase columns, and ammonium bicarbonate buffer was found as the best choice to increase the sensitivity of target analytes to the UV detector. After failure of dispersive SPE clean-up with primary secondary amine, different solid phase extraction cartridges (SPE) were used to check the protecting capability of analytes against matrix interference. Finally, samples were extracted with a simple and rapid method using acetonitrile and salts, and purified through C(18)SPE. The method was validated at two spiking levels (three replicates for each) in the matrix. Good recoveries were observed for all of the analytes and ranged between 70.6% and 93.5%, with relative standard deviations of less than 10%. Calibration curves were linear over the calibration ranges for all the analytes with r(2)≥ 0.998. Limits of detection ranged from 0.02 to 0.05 mg kg(-1), whereas limits of quantitation ranged from 0.06 to 0.16 mg kg(-1) for dinotefuran and its metabolites. The method was successfully applied to real samples, where dinotefuran and UF residues were found in the field-incurred melon samples. Residues were confirmed via LC-tandem mass spectrometry (LC-MS/MS) in positive-ion electrospray ionisation (ESI(+)) mode. Copyright © 2012 Elsevier Ltd. All rights reserved.

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography-ultraviolet detection.

PubMed

Kim, Dalho; Han, Jungho; Choi, Yongwook

2013-01-01

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol-water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar-polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng L(-1), except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples.

Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

PubMed

Spreafico, Filippo; Bongarzone, Italia; Pizzamiglio, Sara; Magni, Ruben; Taverna, Elena; De Bortoli, Maida; Ciniselli, Chiara M; Barzanò, Elena; Biassoni, Veronica; Luchini, Alessandra; Liotta, Lance A; Zhou, Weidong; Signore, Michele; Verderio, Paolo; Massimino, Maura

2017-07-11

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column.

PubMed

Magda, Balázs; Márta, Zoltán; Imre, Tímea; Kalapos-Kovács, Bernadett; Klebovich, Imre; Fekete, Jenő; Szabó, Pál T

2015-01-01

The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation ("inverse gradient elution" on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250 mm × 4.6 mm, i.d.; 5 μm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r = 0.9987) over the concentration range from 1 to 1000 nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0-10.2% and -6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1 nM (0.446 ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides. Copyright © 2015. Published by Elsevier B.V.

Supercritical fluid chromatography-photodiode array detection-electrospray ionization mass spectrometry as a framework for impurity fate mapping in the development and manufacture of drug substances.

PubMed

Pirrone, Gregory F; Mathew, Rose M; Makarov, Alexey A; Bernardoni, Frank; Klapars, Artis; Hartman, Robert; Limanto, John; Regalado, Erik L

2018-03-30

Impurity fate and purge studies are critical in order to establish an effective impurity control strategy for approval of the commercial filing application of new medicines. Reversed phase liquid chromatography-diode array-mass spectrometry (RPLC-DAD-MS) has traditionally been the preferred tool for impurity fate mapping. However, separation of some reaction mixtures by LC can be very problematic requiring combination LC-UV for area % analysis and a different LC-MS method for peak identification. In addition, some synthetic intermediates might be chemically susceptible to the aqueous conditions used in RPLC separations. In this study, the use of supercritical fluid chromatography-photodiode array-electrospray ionization mass spectrometry (SFC-PDA-ESIMS) for fate and purge of two specified impurities in the 1-uridine starting material from the synthesis of a bis-piv 2'keto-uridine, an intermediate in the synthesis of uprifosbuvir, a treatment under investigation for chronic hepatitis C infection. Readily available SFC instrumentation with a Chiralpak IC column (4.6 × 150 mm, 3 μm) and ethanol: carbon dioxide based mobile phase eluent enabled the separation of closely related components from complex reaction mixtures where RLPC failed to deliver optimal chromatographic performance. These results illustrate how SFC combined with PDA and ESI-MS detection can become a powerful tool for direct impurity fate mapping across multiple reaction steps. Copyright © 2018 Elsevier B.V. All rights reserved.

A compensation method for the full phase retardance nonuniformity in phase-only liquid crystal on silicon spatial light modulators.

PubMed

Teng, Long; Pivnenko, Mike; Robertson, Brian; Zhang, Rong; Chu, Daping

2014-10-20

A simple and efficient compensation method for the full correction of both the anisotropic and isotropic nonuniformity of the light phase retardance in a liquid crystal (LC) layer is presented. This is achieved by accurate measurement of the spatial variation of the LC layer's thickness with the help of a calibrated liquid crystal wedge, rather than solely relying on the light intensity profile recorded using two crossed polarizers. Local phase retardance as a function of the applied voltage is calculated with its LC thickness and a set of reference data measured from the intensity of the reflected light using two crossed polarizers. Compensation of the corresponding phase nonuniformity is realized by applying adjusted local voltage signals for different grey levels. To demonstrate its effectiveness, the proposed method is applied to improve the performance of a phase-only liquid crystal on silicon (LCOS) spatial light modulator (SLM). The power of the first diffraction order measured with the binary phase gratings compensated by this method is compared with that compensated by the conventional crossed-polarizer method. The results show that the phase compensation method proposed here can increase the dynamic range of the first order diffraction power significantly from 15~21 dB to over 38 dB, while the crossed-polarizer method can only increase it to 23 dB.

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

PubMed

Qamar, Seema; Wang, GuoZhen; Randle, Suzanne J; Ruggeri, Francesco Simone; Varela, Juan A; Lin, Julie Qiaojin; Phillips, Emma C; Miyashita, Akinori; Williams, Declan; Ströhl, Florian; Meadows, William; Ferry, Rodylyn; Dardov, Victoria J; Tartaglia, Gian G; Farrer, Lindsay A; Kaminski Schierle, Gabriele S; Kaminski, Clemens F; Holt, Christine E; Fraser, Paul E; Schmitt-Ulms, Gerold; Klenerman, David; Knowles, Tuomas; Vendruscolo, Michele; St George-Hyslop, Peter

2018-04-19

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Determination of tetraalkyllead compounds in gasoline by liquid chromatography-atomic absorption spectrometry

USGS Publications Warehouse

Messman, J.D.; Rains, T.C.

1981-01-01

A liquid chromatography-atomic absorption spectrometry (LC-AAS) hybrid analytical technique is presented for metal speciation measurements on complex liquid samples. The versatility and inherent metal selectivity of the technique are Illustrated by the rapid determination of five tetraalkyllead compounds in commercial gasoline. Separation of the individual tetraalkyllead species is achieved by reversed-phase liquid chromatography using an acetonitrile/water mobile phase. The effluent from the liquid Chromatograph Is introduced directly into the aspiration uptake capillary of the nebulizer of an air/acetylene flame atomic absorption spectrometer. Spectral interferences due to coeluting hydrocarbon matrix constituents were not observed at the 283.3-nm resonance line of lead used for analysis. Detection limits of this LC-AAS hydrid analytical technique, based on a 20-??L injection, are approximately 10 ng Pb for each tetraalkyllead compound.

Method development and application of offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis for comprehensive characterization of the saponins from Xueshuantong Injection.

PubMed

Yang, Wenzhi; Zhang, Jingxian; Yao, Changliang; Qiu, Shi; Chen, Ming; Pan, Huiqin; Shi, Xiaojian; Wu, Wanying; Guo, Dean

2016-09-05

Xueshuantong Injection (XSTI), derived from Notoginseng total saponins, is a popular traditional Chinese medicine injection for the treatment of thrombus-resultant diseases. Current knowledge on its therapeutic basis is limited to five major saponins, whereas those minor ones are rarely investigated. We herein develop an offline two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry-fast data directed analysis (offline 2D LC/QTOF-Fast DDA) approach to systematically characterize the saponins contained in XSTI. Key parameters affecting chromatographic separation in 2D LC (including stationary phase, mobile phase, column temperature, and gradient elution program) and the detection by QTOF MS (involving spray voltage, cone voltage, and ramp collision energy) were optimized in sequence. The configured offline 2D LC system showed an orthogonality of 0.84 and a theoretical peak capacity of 8976. Total saponins in XSTI were fractionated into eleven samples by the first-dimensional hydrophilic interaction chromatography, which were further analyzed by reversed-phase UHPLC/QTOF-Fast DDA in negative ion mode. The fragmentation features evidenced from 36 saponin reference standards, high-accuracy MS and Fast-DDA-MS(2) data, elemental composition (C<80, H<120, O<50), double-bond equivalent (DBE 5-15), and searching an in-house library of Panax notoginseng, were simultaneously utilized for structural elucidation. Ultimately, 148 saponins were separated and characterized, and 80 have not been isolated from P. notoginseng. An in-depth depiction of the chemical composition of XSTI was achieved. The results obtained would benefit better understanding of the therapeutic basis and significant promotion on the quality standard of XSTI as well as other homologous products. Copyright © 2016. Published by Elsevier B.V.

Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice.

PubMed

Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia

2016-08-11

Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

ANALYSIS OF PROTEIN DIGESTS BY nano- SCX/RP/MSMS WITH pH SALT GRADIENT SCX ELUTION

EPA Science Inventory

The objective of this study was to optimize chromatographic parameters for complex peptide mixture analyses using two dimensional nano-LC/MSMS system. It used a strong cation exchange (SCX) and reversed phase chromatography (RP). The SCX solvent system was designed to promote pep...

Validation of an HPLC method for the determination of fleroxacin and its photo-degradation products in pharmaceutical forms.

PubMed

Djurdjevic, Predrag; Laban, Aleksandra; Jelikic-Stankov, Milena

2004-01-01

HPLC determination of fleroxacin in dosage forms was carried out using either reversed-phase column YMC pack ODS-AQ or Supelco LC Hisep shielded hydrophobic phase column, with UV detection at 280 nm. The mobile phase for ODS column consisted of 50:50:0.5 v/v/v and for Hisep column 15:85:0.5 v/v/v acetonitrile-water-triethylamine. The pH of the mobile phase was adjusted to 6.30 for ODS column and to 6.85 for Hisep column, with H3PO4. Linear response was obtained in the concentration range of fleroxacin between 0.01 and 1.30 micrograms/mL. Detection limit was 4.8 ng/mL. Recovery test in the determination of fleroxacin in "Quinodis" tablets (Hoffmann La Roche, nominal mass 400 or 200 mg) was 98-101% for both columns. The effect of the composition and pH of the mobile phase on spectra, retention time and dissociation constants of fleroxacin was discussed. The proposed method could be also used for separation of the photo-degradation products of fleroxacin. Ten degradation products were separated on the ODS-AQ column, thus confirming the suitability of the proposed method for stability study of fleroxacin in pharmaceuticals.

Method development for the analysis of ionophore antimicrobials in dairy manure to assess removal within a membrane-based treatment system.

PubMed

Hurst, Jerod J; Wallace, Josh S; Aga, Diana S

2018-04-01

Ionophore antimicrobials are heavily used in the livestock industries, both for preventing animal infection by coccidia protozoa and for increasing feed efficiency. Ionophores are excreted mostly unmetabolized and are released into the environment when manure is land-applied to fertilize croplands. Here, an analytical method was optimized to study the occurrences of five ionophore residues (monensin, lasalocid, maduramycin, salinomycin, and narasin) in dairy manure after solid-liquid separation and further treatment of the liquid manure by a membrane-based treatment system. Ionophore residues from the separated solid manure (dewatered manure) and suspended solids of manure slurry samples were extracted using ultrasonication with methanol, followed by sample clean-up using solid phase extraction (SPE) and subsequent analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of an ethyl acetate and methanol (1:1 v:v) mixture as an SPE eluent resulted in higher recoveries and lower method quantitation limits (MQL), when compared to using methanol. Overall recoveries from separated solid manure ranged from 73 to 134%. Liquid manure fractions were diluted with Nanopure™ water and cleaned up using SPE, where recoveries ranged from 51 to 100%. The developed extraction and LC-MS/MS methods were applied to analyze dairy manure samples subjected to an advanced manure treatment process involving a membrane-based filtration step (reverse osmosis). Monensin and lasalocid were detected at higher concentrations in the suspended solid fractions (4.40-420 ng/g for lasalocid and 85-1950 ng/g for monensin) compared to the liquid fractions (

Considerations on comprehensive and off-line supercritical fluid chromatography x reversed-phase liquid chromatography for the analysis of triacylglycerols in fish oil.

PubMed

François, Isabelle; Pereira, Alberto dos Santos; Sandra, Pat

2010-06-01

The separation of the triacylglycerols in fish oil was performed by comprehensive and off-line supercritical fluid chromatography combined with RP-LC. The first dimension consisted of two serially coupled silver-ion (SI)-loaded columns operated with a supercritical mobile phase (supercritical fluid chromatography, SFC) in both the cases, whereas the second dimension was performed in non-aqueous RP mode (NARP-LC) on a 10-cm monolithic octadecyl silica (ODS) or a 45-cm long ODS column packed with 1.8 microm particles for the comprehensive and off-line separations, respectively. Despite the outstanding performance of the SI-SFC x NARP-LC interface, the high complexity of the sample rendered the online separation far from complete. The off-line approach gave much better separation mainly because of the higher peak capacity of the second-dimension column, but even in this case, the use of MS was mandatory to elucidate the different triacylglycerols in fish oil. The disadvantage of the off-line procedure was the long analysis time.

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma.

PubMed

Jiang, Hongliang; Wang, Yurong; Shet, Manjunath S; Zhang, Yang; Zenke, Duane; Fast, Douglas M

2011-09-01

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples. Copyright © 2011 Elsevier B.V. All rights reserved.

A new acetonitrile-free mobile phase method for LC-ELSD quantification of fructooligosaccharides in onion (Allium cepa L.).

PubMed

Downes, Katherine; Terry, Leon A

2010-06-30

Onion soluble non-structural carbohydrates consist of fructose, glucose and sucrose plus fructooligosaccharides (FOS) with degrees of polymerisation (DP) in the range of 3-19. In onion, sugars and FOS are typically separated using liquid chromatography (LC) with acetonitrile (ACN) as a mobile phase. In recent times, however, the production of ACN has diminished due, in part, to the current worldwide economic recession. A study was therefore undertaken, to find an alternative LC method to quantify sugars and FOS from onion without the need for ACN. Two mobile phases were compared; the first taken from a paper by Vågen and Slimestad (2008) using ACN mobile phase, the second, a newly reported method using ethanol (EtOH). The EtOH mobile phase eluted similar concentrations of all FOS compared to the ACN mobile phase. In addition, limit of detection, limit of quantification and relative standard deviation values were sufficiently and consistently lower for all FOS using the EtOH mobile phase. The drawback of the EtOH mobile phase was mainly the inability to separate all individual sugar peaks, yet FOS could be successfully separated. However, using the same onion extract, a previously established LC method based on an isocratic water mobile phase could be used in a second run to separate sugars. Although the ACN mobile phase method is more convenient, in the current economic climate a method based on inexpensive and plentiful ethanol is a valid alternative and could potentially be applied to other fresh produce types. In addition to the mobile phase solvent, the effect of extraction solvents on sugar and FOS concentration was also investigated. EtOH is still widely used to extract sugars from onion although previous literature has concluded that MeOH is a superior solvent. For this reason, an EtOH-based extraction method was compared with a MeOH-based method to extract both sugars and FOS. The MeOH-based extraction method was more efficacious at extracting sugars and FOS from onion flesh, eluting significantly higher concentrations of glucose, kestose, nystose and DP5-DP8. Copyright 2010 Elsevier B.V. All rights reserved.

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gritsenko, Marina A.; Xu, Zhe; Liu, Tao

Comprehensive, quantitative information on abundances of proteins and their post-translational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labelling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification andmore » quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples, and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.« less

Large-Scale and Deep Quantitative Proteome Profiling Using Isobaric Labeling Coupled with Two-Dimensional LC-MS/MS.

PubMed

Gritsenko, Marina A; Xu, Zhe; Liu, Tao; Smith, Richard D

2016-01-01

Comprehensive, quantitative information on abundances of proteins and their posttranslational modifications (PTMs) can potentially provide novel biological insights into diseases pathogenesis and therapeutic intervention. Herein, we introduce a quantitative strategy utilizing isobaric stable isotope-labeling techniques combined with two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) for large-scale, deep quantitative proteome profiling of biological samples or clinical specimens such as tumor tissues. The workflow includes isobaric labeling of tryptic peptides for multiplexed and accurate quantitative analysis, basic reversed-phase LC fractionation and concatenation for reduced sample complexity, and nano-LC coupled to high resolution and high mass accuracy MS analysis for high confidence identification and quantification of proteins. This proteomic analysis strategy has been successfully applied for in-depth quantitative proteomic analysis of tumor samples and can also be used for integrated proteome and PTM characterization, as well as comprehensive quantitative proteomic analysis across samples from large clinical cohorts.

Simultaneous determination of esculin and its metabolite esculetin in rat plasma by LC-ESI-MS/MS and its application in pharmacokinetic study.

PubMed

Li, Ying-yi; Song, Ye-ying; Liu, Chang-hui; Huang, Xiao-tao; Zheng, Xia; Li, Neng; Xu, Mei-li; Mi, Sui-qing; Wang, Ning-sheng

2012-10-15

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method operated in the negative electrospray ionization (ESI) switching mode has been developed and validated for the simultaneous determination of esculin and its metabolite esculetin in rat plasma. After addition of internal standards scopoletin, the plasma sample was pretreated by solid-phase extraction (SPE), and separated on a reversed phase C(18) column with a mobile phase of 0.01% formic acid in water (solvent A) and methanol (solvent B) using isocratic elution (A:B=20:80, v/v). The detection of target compounds was done in multiple reaction monitoring (MRM) mode. The MRM detection was operated in the negative ESI mode using the transitions of m/z 339.1 ([M-H](-))→176.7 for esculetin, m/z 176.9 ([M-H](-))→133.0 and m/z 191.0 ([M-H](-))→175.9 for scopoletin. The standard curves, which ranged from 25 to 3200 ng/mL for esculin with the lowest limit of quantification (LLOQ) of 0.25 ng/mL and from 1.25 to 160 ng/mL for esculetin with the LLOQ of 1.25 ng/mL, were fitted to a 1/x weighted quadratic regression model. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and inter-day, RSD<8.73%), accuracy, recovery as well as the stability of the analyte under various conditions. The method was successfully applied to study the pharmacokinetics of esculin and its metabolite esculetin in rat plasma after oral administration of esculin at a dose of 100mg/kg. Copyright © 2012 Elsevier B.V. All rights reserved.

Development, validation and application of the liquid chromatography tandem mass spectrometry method for simultaneous quantification of azilsartan medoxomil (TAK-491), azilsartan (TAK-536), and its 2 metabolites in human plasma.

PubMed

Kuze, Yoji; Kogame, Akifumi; Jinno, Fumihiro; Kondo, Takahiro; Asahi, Satoru

2015-09-15

Azilsartan medoxomil potassium salt (TAK-491) is an orally administered angiotensin II type 1 receptor blocker for the treatment of hypertension and is an ester-based prodrug that is rapidly hydrolyzed to the pharmacologically active moiety, azilsartan (TAK-536), during absorption. TAK-536 is biotransformed to the 2 metabolites M-I by decarboxylation and M-II by dealkylation. In this study, we developed and validated a LC/MS/MS method which can simultaneously determine 4 analytes, TAK-491, TAK-536, M-I and M-II. The bioanalytical method can be outlined as follows: two structural analogues are used as the internal standards. The analytes and the IS are extracted from human plasma using solid phase extraction. After evaporating, the residue is reconstituted and injected into a LC/MS/MS system with an ESI probe and analyzed in the positive ion mode. Separation is performed through a conventional reversed-phase column with a mobile phase of water/acetonitrile/acetic acid (40:60:0.05, v/v/v) mixture at a flow rate of 0.2mL/min. The total run time is 8.5min. The calibration range is 1-2500ng/mL in human plasma for all the analytes. Instability issues of the prodrug, TAK-491, were overcome and all the validation results met the acceptance criteria in accordance with the regulatory guideline/guidance. As a result of the clinical study, the human PK profiles of TAK-536, M-I and M-II were successfully obtained and also it was confirmed that TAK-491 was below the LLOQ (1ng/mL) in the human plasma samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and validation of an HPLC method to quantify camptothecin in polymeric nanocapsule suspensions.

PubMed

Granada, Andréa; Murakami, Fabio S; Sartori, Tatiane; Lemos-Senna, Elenara; Silva, Marcos A S

2008-01-01

A simple, rapid, and sensitive reversed-phase column high-performance liquid chromatographic method was developed and validated to quantify camptothecin (CPT) in polymeric nanocapsule suspensions. The chromatographic separation was performed on a Supelcosil LC-18 column (15 cm x 4.6 mm id, 5 microm) using a mobile phase consisting of methanol-10 mM KH2PO4 (60 + 40, v/v; pH 2.8) at a flow rate of 1.0 mL/min and ultraviolet detection at 254 nm. The calibration graph was linear from 0.5 to 3.0 microg/mL with a correlation coefficient of 0.9979, and the limit of quantitation was 0.35 microg/mL. The assay recovery ranged from 97.3 to 105.0%. The intraday and interday relative standard deviation values were < 5.0%. The validation results confirmed that the developed method is specific, linear, accurate, and precise for its intended use. The current method was successfully applied to the evaluation of CPT entrapment efficiency and drug content in polymeric nanocapsule suspensions during the early stage of formulation development.

Top-down approach for the direct characterization of low molecular weight heparins using LC-FT-MS.

PubMed

Li, Lingyun; Zhang, Fuming; Zaia, Joseph; Linhardt, Robert J

2012-10-16

Low molecular heparins (LMWHs) are structurally complex, heterogeneous, polydisperse, and highly negatively charged mixtures of polysaccharides. The direct characterization of LMWH is a major challenge for currently available analytical technologies. Electrospray ionization (ESI) liquid chromatography-mass spectrometry (LC-MS) is a powerful tool for the characterization complex biological samples in the fields of proteomics, metabolomics, and glycomics. LC-MS has been applied to the analysis of heparin oligosaccharides, separated by size exclusion, reversed phase ion-pairing chromatography, and chip-based amide hydrophilic interaction chromatography (HILIC). However, there have been limited applications of ESI-LC-MS for the direct characterization of intact LMWHs (top-down analysis) due to their structural complexity, low ionization efficiency, and sulfate loss. Here we present a simple and reliable HILIC-Fourier transform (FT)-ESI-MS platform to characterize and compare two currently marketed LMWH products using the top-down approach requiring no special sample preparation steps. This HILIC system relies on cross-linked diol rather than amide chemistry, affording highly resolved chromatographic separations using a relatively high percentage of acetonitrile in the mobile phase, resulting in stable and high efficiency ionization. Bioinformatics software (GlycReSoft 1.0) was used to automatically assign structures within 5-ppm mass accuracy.

Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies.

PubMed

Chhonker, Yashpal S; Sleightholm, Richard L; Li, Jing; Oupický, David; Murry, Daryl J

2018-01-01

Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50×4.6mm, 3μ) column, with gradient elution using 0.2% formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1 to 2000ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of an LC-MS/MS method for simultaneous quantification of levodopa and MD01 in rat plasma and its application to a pharmacokinetic study of mucuna pruriens extract.

PubMed

Yang, Guangjie; Zhang, Fangrong; Deng, Linfang; Chen, Chang; Cheng, Zhongzhe; Huang, Jiangeng; Liu, Jiangyun; Jiang, Hongliang

2016-09-01

Mucuna pruriens, an ancient Indian herbal medicine containing levodopa, is widely used for Parkinson's disease. In order to simultaneously determine levodopa and 1,1-dimethyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (MD01) in rat plasma, an improved LC-MS/MS method was developed and validated for a pharmacokinetic study in rats orally administered levodopa or Mucuna pruriens extract (MPE). Elimination of matrix effect and improvement of extraction recovery were achieved through systematic optimization of reversed-phase and hydrophilic interaction chromatographic conditions together with sample clean-up procedures. A satisfactory chromatographic performance was obtained with a Thermo Aquasil C18 column (50 × 2.1 mm, 3 µm) using acetonitrile and water containing 0.2% formic acid as mobile phases. Futhermore, sodium metabisulfite and formic acid were used as stabilizers in neat solutions as well as rat plasma. The method was validated in a dynamic range of 20.0-10,000 ng/mL for levodopa and MD01; the intra- and inter-day precision and accuracy were acceptable. The method was successfully utilized to determine the levodopa level in plasma samples of rats administered levodopa or MPE. Pharmacokinetic results showed that an increase in the AUC of levodopa was observed in rats following oral administration of multiple doses of MPE. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Polar Aprotic Modifiers for Chromatographic Separation and Back-Exchange Reduction for Protein Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

NASA Astrophysics Data System (ADS)

Valeja, Santosh G.; Emmett, Mark R.; Marshall, Alan G.

2012-04-01

Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein-protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.

Pattern-Directed Ordering of Spin-Dewetted Liquid Crystal Micro- or Nanodroplets as Pixelated Light Reflectors and Locomotives.

PubMed

Ravi, Bolleddu; Chakraborty, Snigdha; Bhattacharjee, Mitradip; Mitra, Shirsendu; Ghosh, Abir; Gooh Pattader, Partho Sarathi; Bandyopadhyay, Dipankar

2017-01-11

Chemical pattern directed spin-dewetting of a macroscopic droplet composed of a dilute organic solution of liquid crystal (LC) formed an ordered array of micro- and nanoscale LC droplets. Controlled evaporation of the spin-dewetted droplets through vacuum drying could further miniaturize the size to the level of ∼90 nm. The size, periodicity, and spacing of these mesoscale droplets could be tuned with the variations in the initial loading of LC in the organic solution, the strength of the centripetal force on the droplet, and the duration of the evaporation. A simple theoretical model was developed to predict the spacing between the spin-dewetted droplets. The patterned LC droplets showed a reversible phase transition from nematic to isotropic and vice versa with the periodic exposure of a solvent vapor and its removal. A similar phase transition behavior was also observed with the periodic increase or reduction of temperature, suggesting their usefulness as vapor or temperature sensors. Interestingly, when the spin-dewetted droplets were confined between a pair of electrodes and an external electric field was applied, the droplets situated at the hydrophobic patches showed light-reflecting properties under the polarization microscopy highlighting their importance in the development of micro- or nanoscale LC displays. The digitized LC droplets, which were stationary otherwise, showed dielectrophoretic locomotion under the guidance of the external electric field beyond a threshold intensity of the field. Remarkably, the motion of these droplets could be restricted to the hydrophilic zones, which were confined between the hydrophobic patches of the chemically patterned surface. The findings could significantly contribute in the development of futuristic vapor or temperature sensors, light reflectors, and self-propellers using the micro- or nanoscale digitized LC droplets.

Determination of phenolic xenoestrogens in water by liquid chromatography with coulometric-array detection.

PubMed

Inoue, Koichi; Yoshie, Yuriko; Kondo, Sachiko; Yoshimura, Yoshihiro; Nakazawa, Hiroyuki

2002-02-08

A sensitive method for the simultaneous determination of phenolic xenoestrogens such as bisphenol A, 2,4-dichlorophenol, 4-tert,-butylphenol, 4-n2-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol, 4-octylphenol, 4-nonylphenol was developed using reversed-phase LC and coulometric-array detection. Stepwise gradient elution with phosphoric acid in water-acetonitrile was used. The calibration curves were linear in the range of 5.0 (or 10.0)-1000 ng ml(-1) with correlation coefficients of 0.9978-0.9999, the limits of detection were 0.01-0.02 ng ml(-1). Sample clean-up was performed by solid-phase extraction (SPE) using 3M Empore extraction disks. Three commercial sorbents, C18, SDB-XD (styrene-divinylbenzene polymer) and SDB-RPS (sulfonated styrene-divinylbenzene polymer) were compared. The highest recoveries were obtained with SDB-RPS. They were above 70% with a relative standard deviation of less than 15%. The proposed method was applied to the determination of phenolic xenoestrogens in various water samples.

Liquid chromatographic determination of L-ascorbic acid in candies and soft drinks.

PubMed

Maeda, Y; Ochi, S; Masui, T; Matubara, S

1988-01-01

The L-ascorbic acid (AsA) contents of candies and soft drinks available in the market were determined by liquid chromatography (LC). Samples are cleaned up on a disposable Sep-Pak C18 cartridge followed by reverse phase separation on an ODS column using a mobile phase of 0.1% phosphoric acid (pH 2.2). The AsA peak is detected on the basis of the UV absorption at 254 nm. The detection limit was 1 microgram/mL final concentration. Recoveries of AsA added at levels of 1-10 mg/g candy and 1-10 mg/10 mL soft drink were 99.2-101.7% with a coefficient of variation of 0.52-1.20% (n = 5). The present method allows rapid and accurate assays because it is a simple procedure compared with the official dye-titration method, and it is suitable for the routine analysis of AsA in selected candies and soft drinks.

A simple, rapid atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry method for the determination of 25-hydroxyvitamin D2 and D3.

PubMed

Garg, Uttam; Munar, Ada; Frazee, Clinton; Scott, David

2012-09-01

Vitamin D plays a vital role not only in bone health but also in pathophysiology of many other body functions. In recent years, there has been significant increase in testing of 25-hydroxyvitamin D (25-OH vitamin D), a marker of vitamin D deficiency. The most commonly used methods for the measurement of 25-OH vitamin D are immunoassays and liquid chromatography tandem mass spectrometry (LC-MS-MS). Since immunoassays suffer from inaccuracies and interferences, LC-MS-MS is a preferred method. In LC-MS-MS methods, 25-OH vitamin D is extracted from serum or plasma by solid-phase or liquid-phase extraction. Because these extraction methods are time consuming, we developed an easy method that uses simple protein precipitation followed by injection of the supernatant to LC-MS-MS. Several mass-to-charge (m/z) ratio transitions, including commonly used transitions based on water loss, were evaluated and several tube types were tested. The optimal transitions for 25-OH vitamin D2 and D3 were 395.5 > 269.5 and 383.4 > 257.3, respectively. The reportable range of the method was 1-100 ng/mL, and repeatability (within-run) and within-laboratory imprecision were <4% and <6%, respectively. The method agreed well with the solid-phase extraction methods. © 2012 Wiley Periodicals, Inc.

Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography-tandem mass spectrometry

PubMed Central

Li, Guoyun; Li, Lingyun; Xue, Changhu; Middleton, Dustin; Linhardt, Robert J.; Avci, Fikri Y.

2015-01-01

Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumonia infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides. PMID:25913329

Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography-tandem mass spectrometry.

PubMed

Li, Guoyun; Li, Lingyun; Xue, Changhu; Middleton, Dustin; Linhardt, Robert J; Avci, Fikri Y

2015-06-05

Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumoniae infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides. Copyright © 2015 Elsevier B.V. All rights reserved.

Suspect screening of maternal serum to identify new environmental chemical biomonitoring targets using liquid chromatography-quadrupole time-of-flight mass spectrometry.

PubMed

Gerona, Roy R; Schwartz, Jackie M; Pan, Janet; Friesen, Matthew M; Lin, Thomas; Woodruff, Tracey J

2018-03-01

The use and advantages of high-resolution mass spectrometry (MS) as a discovery tool for environmental chemical monitoring has been demonstrated for environmental samples but not for biological samples. We developed a method using liquid chromatography-quadrupole time-of-flight MS (LC-QTOF/MS) for discovery of previously unmeasured environmental chemicals in human serum. Using non-targeted data acquisition (full scan MS analysis) we were able to screen for environmental organic acids (EOAs) in 20 serum samples from second trimester pregnant women. We define EOAs as environmental organic compounds with at least one dissociable proton which are utilized in commerce. EOAs include environmental phenols, phthalate metabolites, perfluorinated compounds, phenolic metabolites of polybrominated diphenyl ethers and polychlorinated biphenyls, and acidic pesticides and/or predicted acidic pesticide metabolites. Our validated method used solid phase extraction, reversed-phase chromatography in a C18 column with gradient elution, electrospray ionization in negative polarity and automated tandem MS (MS/MS) data acquisition to maximize true positive rates. We identified "suspect EOAs" using Agilent MassHunter Qualitative Analysis software, to match chemical formulas generated from each sample run with molecular formulas in our unique database of 693 EOAs assembled from multiple environmental literature sources. We found potential matches for 282 (41%) of the EOAs in our database. Sixty-five of these suspect EOAs were detected in at least 75% of the samples; only 19 of these compounds are currently biomonitored in National Health and Nutrition Examination Survey. We confirmed two of three suspect EOAs by LC-QTOF/MS using a targeted method developed through LC-MS/MS, reporting the first confirmation of benzophenone-1 and bisphenol S in pregnant women's sera. Our suspect screening workflow provides an approach to comprehensively scan environmental chemical exposures in humans. This can provide a better source of exposure information to help improve exposure and risk evaluation of industrial chemicals.

Liquid chromatography-electrospray mass spectrometry of beta-carotene and xanthophylls. Validation of the analytical method.

PubMed

Careri, M; Elviri, L; Mangia, A

1999-08-27

The investigation of beta-carotene and the xanthophylls beta-cryptoxanthin, lutein, zeaxanthin, canthaxanthin and astaxanthin using reversed-phase liquid chromatography-electrospray mass spectrometry interfaced with TurboIonspray (LC-TurboISP-MS) is described. Two narrow-bore C18 columns connected in series and an isocratic solvent system containing acetonitrile-methanol (0.1 M ammonium acetate)-dichloromethane at a flow-rate of 300 microl/min (without splitting) were used. Operating in the positive-ion mode over m/z 500-650, the effects on the formation of the molecular ion species or adduct ions and the MS detector response were investigated for carotenoids, varying the orifice plate voltage, the ring voltage and the ISP voltage. Both conventional ISP and TurboISP were performed; using the TurboISP-MS system, ionization efficiency increased with respect to ISP-MS, particularly at the highest temperature (500 degrees C). Good results were particularly obtained for beta-carotene, which was detectable at the low ng level, without the use of solution-phase oxidants. Using LC columns and acquiring in single-ion monitoring mode, detection limits were estimated to be in the 0.1-1 ng range; dynamic range was established between one- and two-orders of magnitude. Better sensitivity under positive-ion than negative-ion conditions was demonstrated.

Separation and quantification of 15 carotenoids by reversed phase high performance liquid chromatography coupled to diode array detection with isosbestic wavelength approach.

PubMed

Mitrowska, Kamila; Vincent, Ursula; von Holst, Christoph

2012-04-13

The manuscript presents the development of a new reverse phase high performance liquid chromatography (RP-HPLC) photo diode array detection method allowing the separation and quantification of 15 carotenoids (adonirubin, adonixanthin, astaxanthin, astaxanthin dimethyl disuccinate, asteroidenone, beta-apo-8'-carotenal, beta-apo-8'-carotenoic acid ethyl ester, beta-carotene, canthaxanthin, capsanthin, citranaxanthin, echinenone, lutein, lycopene, and zeaxanthin), 10 of which are feed additives authorised within the European Union. The developed method allows for the reliable determination of the total carotenoid content in one run using the corresponding E-isomer as calibration standard while taking into account the E/Z-isomers composition. This is a key criterion for the application of the method, since for most of the analytes included in this study analytical standards are only available for the E-isomers. This goal was achieved by applying the isosbestic concept, in order to identify specific wavelengths, at which the absorption coefficients are identical for all stereoisomers concerned. The second target referred to the optimisation of the LC conditions. By means of an experimental design, an optimised RP-HPLC method was developed allowing for a sufficient chromatographic separation of all carotenoids. The selected method uses a Suplex pKb-100 HPLC column and applying a gradient with a mixture of acetonitrile, tert-butyl-methyl ether and water as mobile phases. The limits of detection and limits of quantification ranged from 0.06 mg L(-1) to 0.14 mg L(-1) and from 0.20 mg L(-1) to 0.48 mg L(-1), respectively. Copyright © 2012 Elsevier B.V. All rights reserved.

Diagnosis and therapeutic monitoring of inborn errors of creatine metabolism and transport using liquid chromatography-tandem mass spectrometry in urine, plasma and CSF.

PubMed

Haas, Dorothea; Gan-Schreier, Hongying; Langhans, Claus-Dieter; Anninos, Alexandros; Haege, Gisela; Burgard, Peter; Schulze, Andreas; Hoffmann, Georg F; Okun, Jürgen G

2014-03-15

Biochemical detection of inborn errors of creatine metabolism or transport relies on the analysis of three main metabolites in biological fluids: guanidinoacetate (GAA), creatine (CT) and creatinine (CTN). Unspecific clinical presentation of the diseases might be the cause that only few patients have been diagnosed so far. We describe a LC-MS/MS method allowing fast and reliable diagnosis by simultaneous quantification of GAA, CT and CTN in urine, plasma and cerebrospinal fluid (CSF) and established reference values for each material. For quantification deuterated stable isotopes of each analyte were used as internal standards. GAA, CT and CTN were separated by reversed-phase HPLC. The characterization was carried out by scanning the ions of each compound by negative ion tandem mass spectrometry. Butylation is needed to achieve sufficient signal intensity for GAA and CT but it is not useful for analyzing CTN. The assay is linear in a broad range of analyte concentrations usually found in urine, plasma and CSF. Comparison of the "traditional" cation-exchange chromatography and LC-MS/MS showed proportional differences but linear relationships between the two methods. The described method is characterized by high speed and linearity over large concentration ranges comparable to other published LC-MS methods but with higher sensitivity for GAA and CT. In addition, we present the largest reference group ever published for guanidino compounds in all relevant body fluids. Therefore this method is applicable for high-throughput approaches for diagnosis and follow-up of inborn errors of creatine metabolism and transport. Copyright © 2014 Elsevier B.V. All rights reserved.

Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography.

PubMed

Stoll, Dwight R; Sajulga, Ray W; Voigt, Bryan N; Larson, Eli J; Jeong, Lena N; Rutan, Sarah C

2017-11-10

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension ( 1 D) column into the second dimension ( 2 D) column leads to severe 2 D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1 D mobile phase overwhelms the 2 D column with each injection of 1 D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2 D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2 D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4-80μL), loop filling level (25-100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (-20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of -1.0 and -4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real injection profiles obtained from 2D-LC interface valves are used to simulate the introduction of samples into the 2 D column. These profiles are highly asymmetric - simulation using simple rectangular pulses leads to peak widths that are far too narrow under many conditions. We believe the simulation approach developed here will be useful for addressing practical questions in the development of 2D-LC methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization.

PubMed

Welch, Leslie; Dong, Xiao; Hewitt, Daniel; Irwin, Michelle; McCarty, Luke; Tsai, Christina; Baginski, Tomasz

2018-06-02

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative Determination of Fluorinated Alkyl Substances by Large-Volume-Injection LC/MS/MS—Characterization of Municipal Wastewaters

PubMed Central

Schultz, Melissa M.; Barofsky, Douglas F.; Field, Jennifer A.

2008-01-01

A quantitative method was developed for the determination of fluorinated alkyl substances in municipal wastewater influents and effluents. The method consisted of centrifugation followed by large-volume injection (500 μL) of the supernatant onto a liquid chromatograph with a reverse-phase column and detection by electrospray ionization, and tandem mass spectrometry (LC/MS/MS). The fluorinated analytes studied include perfluoroalkyl sulfonates, fluorotelomer sulfonates, perfluorocarboxylates, and select fluorinated alkyl sulfonamides. Recoveries of the fluorinated analytes from wastewater treatment plant (WWTP) raw influents and final effluent ranged from 77% – 96% and 80% – 99%, respectively. The lower limit of quantitation ranged from 0.5 to 3.0 ng/L depending on the analyte. The method was applied to flow-proportional composites of raw influent and final effluent collected over a 24 hr period from ten WWTPs nationwide. Fluorinated alkyl substances were observed in wastewater at all treatment plants and each plant exhibited unique distributions of fluorinated alkyl substances despite similarities in treatment processes. In nine out of the ten plants sampled, at least one class of fluorinated alkyl substances exhibited increased concentrations in the effluent as compared to the influent concentrations. In some instances, decreases in certain fluorinated analyte concentrations were observed and attributed to sorption to sludge. PMID:16433363

Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction.

PubMed

Ma, Zhen; Ge, Liya; Lee, Anna S Y; Yong, Jean Wan Hong; Tan, Swee Ngin; Ong, Eng Shi

2008-03-10

Coconut (Cocos nucifera L.) water, which contains many uncharacterized phytohormones is extensively used as a growth promoting supplement in plant tissue culture. In this paper, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of various classes phytohormones, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N(6)-benzyladenine (BA), alpha-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid, pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detection made at 265 nm wavelength. The method was validated for specificity, quantification, accuracy and precision. After preconcentration of putative endogenous phytohormones in CW using C(18) solid-phase extraction (SPE) cartridges, the HPLC method was able to screen for putative endogenous phytohormones present in CW. Finally, the identities of the putative phytohormones present in CW were further confirmed using independent liquid chromatography-tandem mass spectrometry (LC-MS/MS) equipped with an electrospray ionization (ESI) interface.

Expanded separation technique for chlorophyll metabolites in Oriental tobacco leaf using non aqueous reversed phase chromatography.

PubMed

Ishida, Naoyuki

2011-08-26

An improved separation method for chlorophyll metabolites in Oriental tobacco leaf was developed. While Oriental leaf still gives the green color even after the curing process, little attention has been paid to the detailed composition of the remaining green pigments. This study aimed to identify the green pigments using non aqueous reversed phase chromatography (NARPC). To this end, liquid chromatograph (LC) equipped with a photo diode array detector (DAD) and an atmospheric pressure chemical ionization/mass spectrometer (APCI/MSD) was selected, because it is useful for detecting low polar non-volatile compounds giving green color such as pheophytin a. Identification was based on the wavelength spectrum, mass spectrum and retention time, comparing the analytes in Oriental leaf with the commercially available and synthesized components. Consequently, several chlorophyll metabolites such as hydroxypheophytin a, solanesyl pheophorbide a and solanesyl hydroxypheophorbide a were newly identified, in addition to typical green pigments such as chlorophyll a and pheophytin a. Chlorophyll metabolites bound to solanesol were considered the tobacco specific components. NARPC expanded the number of detectable low polar chlorophyll metabolites in Oriental tobacco leaf. Copyright © 2011 Elsevier B.V. All rights reserved.

Concentration determination of urinary metabolites of N,N-dimethylacetamide by high-performance liquid chromatography-tandem mass spectrometry

PubMed Central

Yamamoto, Shinobu; Matsumoto, Akiko; Yui, Yuko; Miyazaki, Shota; Kumagai, Shinji; Hori, Hajime; Ichiba, Masayoshi

2017-01-01

Objectives: N,N-Dimethylacetamide (DMAC) is widely used in industry as a solvent. It can be absorbed through human skin. Therefore, it is necessary to determine exposure to DMAC via biological monitoring. However, the precision of traditional gas chromatography (GC) is low due to the thermal decomposition of metabolites in the high-temperature GC injection port. To overcome this problem, we have developed a new method for the simultaneous separation and quantification of urinary DMAC metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods: Urine samples were diluted 10-fold in formic acid, and 1-μl aliquots were injected into the LC-MS/MS equipment. A C18 reverse-phase Octa Decyl Silyl (ODS) column was used as the analytical column, and the mobile phase consisted of a mixture of methanol and aqueous formic acid solution. Results: Urinary concentrations of DMAC and its known metabolites (N-hydroxymethyl-N-methylacetamide (DMAC-OH), N-methylacetamide (NMAC), and S- (acetamidomethyl) mercapturic acid (AMMA) ) were determined in a single run. The dynamic ranges of the calibration curves were 0.05-5 mg/l (r≥0.999) for all four compounds. The limits of detection for DMAC, DMAC-OH, NMAC, and AMMA in urine were 0.04, 0.02, 0.05, and 0.02 mg/l, respectively. Within-run accuracies were 96.5%-109.6% with relative standard deviations of precision being 3.43%-10.31%. Conclusions: The results demonstrated that the proposed method could successfully quantify low concentrations of DMAC and its metabolites with high precision. Hence, this method is useful for evaluating DMAC exposure. In addition, this method can be used to examine metabolite behaviors in human bodies after exposure and to select appropriate biomarkers. PMID:29213009

Liquid chromatography-mass spectrometry (LC-MS): a powerful combination for selenium speciation in garlic (Allium sativum).

PubMed

Dumont, Emmie; Ogra, Yasumitsu; Vanhaecke, Frank; Suzuki, Kazuo T; Cornelis, Rita

2006-03-01

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se-78 Se, 80Se: and 82Se. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.

Single Laboratory Validated Method for Determination of Microcystins and Nodularin in Ambient Freshwaters by Solid Phase Extraction and Liquid Chromatography/ Tandem Mass Spectrometry (LC/MS/MS)

EPA Pesticide Factsheets

This document is a standardized, single laboratory validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the detection of cyanotoxins—microsystins and nodularin (combined intracellular and extracellular)—in ambient freshwaters.

Ultrapressure liquid chromatography-tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for quantification of 4-methoxydiphenylmethane in pharmacokinetic evaluation.

PubMed

Farhan, Nashid; Fitzpatrick, Sean; Shim, Yun M; Paige, Mikell; Chow, Diana Shu-Lian

2016-09-05

4-Methoxydiphenylmethane (4-MDM), a selective augmenter of Leukotriene A4 Hydrolase (LTA4H), is a new anti-inflammatory compound for potential treatment of chronic obstructive pulmonary disease (COPD). Currently, there is no liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the quantification of 4-MDM. A major barrier for developing the LC-MS/MS method is the inability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) to ionize 4-MDM due to its hydrophobicity and lack of any functional group for ionization. With the advent of atmospheric pressure photoionization (APPI) technique, many hydrophobic compounds have been demonstrated to ionize by charge transfer reactions. In this study, a highly sensitive ultrapressure liquid chromatography tandem mass spectrometry assay using atmospheric pressure photoionization (UPLC-APPI-MS/MS) for the quantifications of 4-MDM in rat plasma has been developed and validated. 4-MDM was extracted from the plasma by solid phase extraction (SPE) and separated chromatographically using a reverse phase C8 column. The photoionization (PI) was achieved by introducing anisole as a dopant to promote the reaction of charge transfer. The assay with a linear range of 5 (LLOQ)-400ngmL(-1) met the regulatory requirements for accuracy, precision and stability. The validated assay was employed to quantify the plasma concentrations of 4-MDM after an oral dosing in Sprague Dawley (SD) rats. Copyright © 2016 Elsevier B.V. All rights reserved.

Therapeutic Drug Monitoring of Fluoxetine, Norfluoxetine and Paroxetine: A New Tool Based on Microextraction by Packed Sorbent Coupled to Liquid Chromatography.

PubMed

Magalhães, Paulo; Alves, Gilberto; Llerena, Adrián; Falcão, Amílcar

2017-09-01

The present article reports the first liquid chromatography (LC) assay for the simultaneous quantification of fluoxetine (FLU), its metabolite norfluoxetine (NFLU) and paroxetine (PAR) in human plasma, applying the microextraction by packed sorbent (MEPS) technology in sample preparation. Chromatographic analysis of FLU, NFLU and PAR was achieved in <13 min on a reverse-phase C18 column using isocratic elution and fluorescence detection (FLD). The mobile phase was composed by an aqueous solution of 25 mM sodium phosphate monobasic anhydrous and 7.5 mM di-potassium hydrogen phosphate anhydrous (pH 3.0)/acetonitrile/methanol (70:23:7, v/v/v). The detector was set at 240/312 nm for FLU, NFLU and IS and at 295/350 nm for PAR. The method showed linearity in the ranges of 20-750 ng mL-1 for FLU and NFLU, and 5-750 ng mL-1 for PAR (r2 ≥ 0.9919). The overall intra- and interday precision did not exceed 13.6% and the corresponding accuracy (bias) ranged from 0.02 to 16.7%. The method was successfully applied in the analysis of authentic plasma samples. Hence, this new MEPS/LC-FLD assay ensures robust and low-cost analyses representing, therefore, a good alternative to support therapeutic drug monitoring and clinical studies involving these antidepressant drugs. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development, validation and utilization of a highly sensitive LC-MS/MS method for quantification of levonorgestrel released from a subdermal implant in human plasma.

PubMed

Cirrincione, Lauren R; Penchala, Sujan Dilly; Scarsi, Kimberly K; Podany, Anthony T; Winchester, Lee C; Back, David J; Khoo, Saye H; Fletcher, Courtney V; Siccardi, Marco; Else, Laura J

2018-05-01

Levonorgestrel (LNG) is a synthetic progestin that is available in oral contraceptive tablets, a subdermal implant, and an intrauterine system for contraception. LNG pharmacokinetics are a pivotal determinant of contraceptive efficacy and essential in assessing drug-drug interactions influencing LNG exposure following different routes of LNG administration. A highly sensitive LC-MS/MS method was developed and validated to quantify levonorgestrel in human plasma. Liquid-liquid extraction was utilized with a sample volume of 500 μL to extract levonorgestrel from plasma. Chromatographic separation of LNG was achieved with a Fortis™ C18 (3 μm: 100 mm × 2.1 mm) reverse phase analytical column. The mobile phases consisted of de-ionized water plus 0.1% NH 4 OH (100:0.1%, v/v) (A), and methanol plus 0.1% NH 4 OH (100:0.1%, v/v) (B) delivered as a gradient at a flow rate of 400 μL/min. Detection of LNG and internal standard (D-(-)-norgestrel-d7) was achieved using positive polarity mode monitoring at 313.2-245.2 amu and 320.1-251.2 amu, respectively. The assay was linear over the calibration range of 49.6 to 1500 pg/mL. This method was used to quantify plasma LNG released by subdermal implant in support of a drug interaction study among women with HIV receiving efavirenz- or nevirapine-based antiretroviral therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of oligonucleotide photoproducts produced by UV-A light and a riboflavin photosensitizer

NASA Astrophysics Data System (ADS)

Gelhaus, Stacy L.; LaCourse, William R.

2004-12-01

DNA damage is caused by a variety of foreign and endogenous compounds. There are endogenous photosensitizers in cells, such as porphyrins and flavins, which may create damage in the presence of UV-A light. Typically, samples are analyzed by 32P-postlabelling and electrophoretic separation or by LC-MS separation and detection. Separation by HPLC is common; however, in all instances, the DNA sample is hydrolyzed down to nucleosides prior to analysis. It will be shown here that ion-pairing reversed phase high performance liquid chromatography (IP-RPLC) has the ability to provide biophysical information concerning the sites of UV-A induced photosensitizer damage on an intact oligonucleotide concurrent with the separation. IP-RPLC is less labor intensive and faster than electrophoretic methods and it is less costly than LC-MS. IP-RPLC can also be used to purify modified oligonucleotides for further use and analysis. This technique is sensitive to the charge, conformation, and sequence characteristics of the nucleic acid sample and may be used to determine the damage or modifications made to DNA by a variety of compounds.

Comparison of various second-dimension gradient types in comprehensive two-dimensional liquid chromatography.

PubMed

Jandera, Pavel; Hájek, Tomás; Cesla, Petr

2010-06-01

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions. In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC x LC are compared: (i) "full in fraction", (ii) "segment in fraction" and (iii) "continuously shifting" gradients, applied in orthogonal LC x LC separations of phenolic acids and flavones on a polyethylene glycol column in the first dimension and two types of porous shell fused-core C18 columns in the second dimension (Ascentis Express and Kinetex). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated. The type of gradient program controls the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. This range can be calibrated using alkylbenzene standards, to design the separation conditions for complete sample separation, avoiding harmful wrap around of non-eluted compounds to the subsequent second-dimension fractions.

Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hong; Yang, Yanling; Li, Yuxin

2015-02-06

Development of high resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase column (100 μm x 150 cm) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phasemore » fractionation of peptide ions further increased the number of peptide identification by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.« less

A novel mass spectrometry-based method for simultaneous determination of asymmetric and symmetric dimethylarginine, l-arginine and l-citrulline optimized for LC-MS-TOF and LC-MS/MS.

PubMed

Wiśniewski, Jerzy; Fleszar, Mariusz G; Piechowicz, Joanna; Krzystek-Korpacka, Małgorzata; Chachaj, Angelika; Szuba, Andrzej; Lorenc-Kukula, Katarzyna; Masłowski, Leszek; Witkiewicz, Wojciech; Gamian, Andrzej

2017-11-01

Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l-arginine to l-citrulline. l-Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l-arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l-arginine, l-citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6-pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse-phase column. Serum aliquots (100 μL) were spiked with isotope-labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R 2  = 0.9966-0.9986) for all derivatized compounds. Good accuracy (94.67-99.91%) and precision (1.92-11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l-arginine and l-citrulline in angiologic patients. This robust and fast LC-ESI-MS method may be a useful tool in quantitative analysis of l-arginine, ADMA, SDMA and l-citrulline. Copyright © 2017 John Wiley & Sons, Ltd.

Development of liquid chromatography high resolution mass spectrometry strategies for the screening of complex organic matter: Application to astrophysical simulated materials.

PubMed

Eddhif, Balkis; Allavena, Audrey; Liu, Sylvie; Ribette, Thomas; Abou Mrad, Ninette; Chiavassa, Thierry; d'Hendecourt, Louis Le Sergeant; Sternberg, Robert; Danger, Gregoire; Geffroy-Rodier, Claude; Poinot, Pauline

2018-03-01

The present work aims at developing two LC-HRMS setups for the screening of organic matter in astrophysical samples. Their analytical development has been demonstrated on a 100-µg residue coming from the photo-thermo chemical processing of a cometary ice analog produced in laboratory. The first 1D-LC-HRMS setup combines a serially coupled columns configuration with HRMS detection. It has allowed to discriminate among different chemical families (amino acids, sugars, nucleobases and oligopeptides) in only one chromatographic run without neither a priori acid hydrolysis nor chemical derivatisation. The second setup is a dual-LC configuration which connects a series of trapping columns with analytical reverse-phase columns. By coupling on-line these two distinct LC units with a HRMS detection, high mass compounds (350

Identification and Quantification of Dimethylamylamine in Geranium by Liquid Chromatography Tandem Mass Spectrometry

PubMed Central

Li, J.S.; Chen, M.; Li, Z.C.

2012-01-01

A sensitive and reliable method of liquid chromatography–electrospray ionization/tandem mass spectrometry (LC-ESI/MS/ MS) was developed and validated for determining 1,3-dimethylamylamine (1,3-DMAA) and 1,4-dimethylamylamine (1,4-DMAA) in geranium plants (Pelargonium graveolens). The sample was extracted with 0.5 M HCl and purified by liquid-liquid partition with hexane. The parameters for reverse-phase (C18) LC and positive ESI/MS/MS were optimized. The matrix effect, specificity, linearity, precision, accuracy and reproducibility of the method were determined and evaluated. The method was linear over a range of 0.10–10.00 ng/mL examined, with R2 of 0.99 for both 1,3-DMAA and 1,4-DMAA. The recoveries from spiked concentrations between 5.00–40.00 ng/g were 85.1%–104.9% for 1,3-DMAA, with relative standard deviation (RSD) of 2.9%–11.0%, and 82.9%–101.8% for 1,4-DMAA, with RSD of 3.2%–11.7%. The instrument detection limit was 1–2 pg for both DMAAs. The quantification limit was estimated to be 1–2 ng/g for the plant sample. This method was successfully applied to the quantitative determination of 1,3- and 1,4-DMAA in both geranium plant and geranium oil. PMID:22915838

Simultaneous determination of UV-filters and estrogens in aquatic invertebrates by modified quick, easy, cheap, effective, rugged, and safe extraction and liquid chromatography tandem mass spectrometry.

PubMed

He, Ke; Timm, Anne; Blaney, Lee

2017-08-04

Ultraviolet-filters (UV-filters) and estrogens have attracted increased attention as contaminants of emerging concern (CECs) due to their widespread occurrence in the environment. Most of these CECs are hydrophobic and have the potential to accumulate in aquatic organisms. To date, co-analysis of UV-filters and estrogens has not been reported due, in part, to the complex environmental matrices. Here, a multi-residue method has been developed for simultaneous determination of five UV-filters and three estrogens in tissue from aquatic and marine organisms. The procedure involved a modified Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction with a novel reverse-solid-phase extraction (reverse-SPE) cleanup in place of dispersive-SPE, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The tissue mass, acetonitrile content, and salt conditions for QuEChERS extraction, along with the reverse-SPE cartridge material and elution conditions, were thoroughly investigated and optimized. Five UV-filters (i.e., 3-(4-methylbenzylidene) camphor, benzophenone-3, ethylhexylmethoxycinnamate, homosalate, and octocrylene) and three estrogens (i.e., estrone, 17β-estradiol, and 17α-ethinylestradiol) were simultaneously analyzed by taking advantage of wrong-way-round ionization in LC-MS/MS. The optimized analytical protocol exhibited good recoveries (>80%) for target compounds and enabled their detection at concentrations as low as 0.2ng/g in 50mg tissue samples. The method was applied to determine concentrations of target analytes in four invertebrates (i.e., Orconectes virilis, Procambarus clarkii, Crassostrea virginica, and Ischadium recurvum). All eight target analytes were detected at least once in the tissue samples, with the highest concentration being 399ng/g of homosalate in O. virilis. These results highlight the ubiquitous bioaccumulation of CECs in aquatic and marine invertebrates. Copyright © 2017 Elsevier B.V. All rights reserved.

Liquid chromatography/electrospray ionization/isotopic dilution mass spectrometry analysis of n-(phosphonomethyl) glycine and mass spectrometry analysis of aminomethyl phosphonic acid in environmental water and vegetation matrixes.

PubMed

Grey, L; Nguyen, B; Yang, P

2001-01-01

A liquid chromatography/electrospray/mass spectrometry (LC/ES/MS) method was developed for the analysis of glyphosate (n-phosphonomethyl glycine) and its metabolite, aminomethyl phosphonic acid (AMPA) using isotope-labelled glyphosate as a method surrogate. Optimized parameters were achieved to derivatize glyphosate and AMPA using 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer prior to a reversed-phase LC analysis. Method spike recovery data obtained using laboratory and real world sample matrixes indicated an excellent correlation between the recovery of the native and isotope-labelled glyphosate. Hence, the first performance-based, isotope dilution MS method with superior precision, accuracy, and data quality was developed for the analysis of glyphosate. There was, however, no observable correlation between the isotope-labelled glyphosate and AMPA. Thus, the use of this procedure for the accurate analysis of AMPA was not supported. Method detection limits established using standard U.S. Environmental Protection Agency protocol were 0.06 and 0.30 microg/L, respectively, for glyphosate and AMPA in water matrixes and 0.11 and 0.53 microg/g, respectively, in vegetation matrixes. Problems, solutions, and the method performance data related to the analysis of chlorine-treated drinking water samples are discussed. Applying this method to other environmental matrixes, e.g., soil, with minimum modifications is possible, assuring accurate, multimedia studies of glyphosate concentration in the environment and the delivery of useful multimedia information for regulatory applications.

Concentration determination of urinary metabolites of N,N-dimethylacetamide by high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Yamamoto, Shinobu; Matsumoto, Akiko; Yui, Yuko; Miyazaki, Shota; Kumagai, Shinji; Hori, Hajime; Ichiba, Masayoshi

2018-03-27

N,N-Dimethylacetamide (DMAC) is widely used in industry as a solvent. It can be absorbed through human skin. Therefore, it is necessary to determine exposure to DMAC via biological monitoring. However, the precision of traditional gas chromatography (GC) is low due to the thermal decomposition of metabolites in the high-temperature GC injection port. To overcome this problem, we have developed a new method for the simultaneous separation and quantification of urinary DMAC metabolites using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples were diluted 10-fold in formic acid, and 1-μl aliquots were injected into the LC-MS/MS equipment. A C18 reverse-phase Octa Decyl Silyl (ODS) column was used as the analytical column, and the mobile phase consisted of a mixture of methanol and aqueous formic acid solution. Urinary concentrations of DMAC and its known metabolites (N-hydroxymethyl-N-methylacetamide (DMAC-OH), N-methylacetamide (NMAC), and S- (acetamidomethyl) mercapturic acid (AMMA) ) were determined in a single run. The dynamic ranges of the calibration curves were 0.05-5 mg/l (r≥0.999) for all four compounds. The limits of detection for DMAC, DMAC-OH, NMAC, and AMMA in urine were 0.04, 0.02, 0.05, and 0.02 mg/l, respectively. Within-run accuracies were 96.5%-109.6% with relative standard deviations of precision being 3.43%-10.31%. The results demonstrated that the proposed method could successfully quantify low concentrations of DMAC and its metabolites with high precision. Hence, this method is useful for evaluating DMAC exposure. In addition, this method can be used to examine metabolite behaviors in human bodies after exposure and to select appropriate biomarkers.

Reverse phase high performance liquid chromatographic method development based on ultravioletvisible detector for the analysis of 1-hydroxypyrene (PAH biomarker) in human urine.

PubMed

Kamal, Atif; Gulfraz, Mohammad; Anwar, Mohammad Asad; Malik, Riffat Naseem

2015-01-01

1-hydroxypyrene is an important biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs), which appears in the urine of exposed human subjects. In developing countries, where advanced instruments are not available, the importance of this biomarker demands convenient and sensitive methods for determination purposes. This study aimed at developing a methodology to quantify 1-hydroxypyrene (a biomarker of PAHs exposure) based on the UV-visible detector in the reverse phase high pressure liquid chromatography (HPLC). A 20 μl injection of sample was used for manual injection into the HPLC Shimadzu, equipped with the SPD-20 A UV-visible detector, the LC-20AT pump and the DGU-20A5 degasser. The C-18 column was used for the purpose of the analysis. The method showed a good linearity (the range: R² = 0.979-0.989), and high detectability up to the nmol level. The average retention was 6.37, with the accuracy of 2%, and the percentage of recovery remained 108%. The overall performance of this method was comparable (in terms of detection sensitivity) and relatively better than previously reported studies using the HPLC system equipped with the UV-detector. This method is suitable and reliable for the detection/quantification of the 1-OHP in human urine samples, using the UV-detector, however, it is less sensitive as compared to the results of a florescence detector. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Determination of phosphatidylethanolamine molecular species in various food matrices by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS2).

PubMed

Zhou, Li; Zhao, Minjie; Ennahar, Saïd; Bindler, Françoise; Marchioni, Eric

2012-04-01

A liquid chromatographic-electrospray ionization-tandem mass spectrometric (LC-ESI-MS(2)) method has been developed for determination of the molecular species of phosphatidylethanolamine (PE) in four food matrices (soy, egg yolk, ox liver, and krill oil). The extraction and purification method consisted of a pressurized liquid extraction procedure for total lipid (TL) extraction, purification of phospholipids (PLs) by adsorption on a silica gel column, and separation of PL classes by semi-preparative normal-phase HPLC. Separation and identification of PE molecular species were performed by reversed-phase HPLC coupled with electrospray ionization tandem mass spectrometry (ESI-MS(2)). Methanol containing 5 mmol L(-1) ammonium formate was used as the mobile phase. A variety of PE molecular species were detected in the four food matrices. (C16:0-C18:2)PE, (C18:2-C18:2)PE, and (C16:0-C18:1)PE were the major PE molecular species in soy. Egg yolk PE contained (C16:0-C18:1)PE, (C18:0-C18:1)PE, (C18:0-C18:2)PE, and (C16:0-C18:2)PE as the major molecular species. Ox liver PE was rich in the species (C18:0-C18:1)PE, (C18:0-C20:4)PE, and (C18:0-C18:2)PE. Finally, krill oil which was particularly rich in (C16:0(alkyl)-C22:6(acyl))plasmanylethanolamine (PakE), (C16:0-C22:6)PE, and (C16:0-C20:5)PE, seemed to be an interesting potential source for supplementation of food with eicosapentaenoic acid and docosahexaenoic acid.

Mixed-mode chromatography/isotope ratio mass spectrometry.

PubMed

McCullagh, James S O

2010-03-15

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high-precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment delta(13)C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies. There is strong potential to extend these to new compounds and complex matrices but several challenges face the development of LC/IRMS methods. To achieve accurate isotopic measurements, HPLC separations must provide baseline-resolution between analyte peaks; however, the design of current liquid interfaces places severe restrictions on compatible flow rates and in particular mobile phase compositions. These create a significant challenge on which reports associated with LC/IRMS have not previously focused. Accordingly, this paper will address aspects of chromatography in the context of LC/IRMS, in particular focusing on mixed-mode separations and their benefits in light of these restrictions. It aims to provide an overview of mixed-mode stationary phases and of ways to improve high aqueous separations through manipulation of parameters such as column length, temperature and mobile phase pH. The results of several practical experiments are given using proteogenic amino acids and nucleosides both of which are of noted importance in the LC/IRMS literature. This communication aims to demonstrate that mixed-mode stationary phases provide a flexible approach given the constraints of LC/IRMS interface design and acts as a practical guide for the development of new chromatographic methods compatible with LC/IRMS applications. Copyright 2010 John Wiley & Sons, Ltd.

Dispersive liquid-liquid microextraction for the determination of vitamins D and K in foods by liquid chromatography with diode-array and atmospheric pressure chemical ionization-mass spectrometry detection.

PubMed

Viñas, Pilar; Bravo-Bravo, María; López-García, Ignacio; Hernández-Córdoba, Manuel

2013-10-15

A simple and rapid method was developed using reversed-phase liquid chromatography (LC) with both diode array (DAD) and atmospheric pressure chemical ionization mass spectrometric (APCI-MS) detection, for the simultaneous analysis of the vitamins ergocalciferol (D2), cholecalciferol (D3), phylloquinone (K1), menaquinone-4 (K2) and a synthetic form of vitamin K, menadione (K3). The Taguchi experimental method, an orthogonal array design (OAD), was used to optimize an efficient and clean preconcentration step based on dispersive liquid-liquid microextraction (DLLME). A factorial design was applied with six factors and three levels for each factor, namely, carbon tetrachloride volume, methanol volume, aqueous sample volume, pH of sample, sodium chloride concentration and time of the centrifugation step. The DLLME optimized procedure consisted of rapidly injecting 3 mL of acetonitrile (disperser solvent) containing 150 µL carbon tetrachloride (extraction solvent) into the aqueous sample, thereby forming a cloudy solution. Phase separation was performed by centrifugation, and the sedimented phase was evaporated with nitrogen, reconstituted with 50 µL of acetonitrile, and injected. The LC analyses were carried out using a mobile phase composed of acetonitrile, 2-propanol and water, under gradient elution. Quantification was carried out by the standard additions method. The APCI-MS spectra, in combination with UV spectra, permitted the correct identification of compounds in the food samples. The method was validated according to international guidelines and using a certified reference material. The validated method was applied for the analysis of vitamins D and K in infant foods and several green vegetables. There was little variability in the forms of vitamin K present in vegetables, with the most abundant vitamer in all the samples being phylloquinone, while menadione could not be detected. Conversely, cholecalciferol, which is present in food of animal origin, was the main form in infant foods, while ergocalciferol was not detected. Copyright © 2013 Elsevier B.V. All rights reserved.

In tube-solid phase microextraction-nano liquid chromatography: Application to the determination of intact and degraded polar triazines in waters and recovered struvite.

PubMed

Serra-Mora, P; Jornet-Martinez, N; Moliner-Martinez, Y; Campíns-Falcó, P

2017-09-01

In-tube solid-phase microextraction (IT-SPME) coupled to miniaturized liquid chromatography (LC) techniques are attractive mainly due to the column efficiency improvement, sensitivity enhancement and reduction of solvent consumption. In addition, the nanomaterials based sorbents can play a key role in the improvement of the extraction efficiency taking into account their interesting physical and chemical properties. Thus, in this work the performance of IT-SPME coupled to nano LC (NanoLC) has been compared with the performance of IT-SPME coupled to capillary LC (CapLC) with similar configurations for the determination of polar triazines including their degradation products. In both cases, a DAD detector was used. Different extractive phases such as TRB-5, TRB-5/c-SWNTs, TRB-5/c-MWNTs capillary columns have been tested. The dimensions of the capillary columns were 0.32mm id×40cm length and 0.1 or 0.075mm i.d.×15cm length for the couplings with CapLC and NanoLC, respectively. The processed volume was 4mL for CapLC and 0.5mL for NanoLC. The elution was carried out with ACN:H 2 O (30:70, v/v). IT-SPME-NanoLC has shown a higher performance than IT-SPME-CapLC for the target analytes demonstrating the enhancement of the extraction efficiency with the former configuration. A new phase TEOS-MTEOS-SiO 2 NPs has been also proposed for IT-SPME-NanoLC, which improves the retention of polar compounds. Compared with previously published works, improved LODs were achieved (0.025-0.5μgL -1 ). The practical application of the proposed procedure has been demonstrated for the analysis of water samples and recovered struvite samples from wastewater treatment plants. Therefore, the proposed procedure can be an alternative method for regulatory purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

A Rapid Novel HPLC Method for Estimation of Eight Related Compounds in Azilsartan Kamedoxomil and Identification of Degradation Compounds by Using LC-MS.

PubMed

Sreenivasulu, J; Venkata Ramana, P; Sampath Kumar Reddy, G; Nagaraju, Ch V S; Thirumalai Rajan, S; Eswaraiah, S

2015-10-01

A novel, rapid, specific and stability-indicating reverse-phase high-performance liquid chromatography method was developed for the quantitative determination of related compounds, obtained from two different synthetic routes and degradation products of Azilsartan kamedoxomil (AZL). The method was developed by using a YMC-Pack pro C18 (150 × 4.6 mm, 3 µm) column with a mobile phase containing a gradient mobile phase combination. The eluted compounds were measured at wavelength 220 nm. The developed method run time was 25 min, within which AZL and its eight impurities were well separated with minimum 3.0 resolution. The drug substance was subjected to stress conditions of hydrolysis (acid, base and water), oxidation, photolysis, sunlight, 75% relative humidity and thermal degradation as per International Conference on Harmonization (ICH) prescribed stress conditions to ascertain the stability-indicating power of the method. Significant degradation was observed during acid, base, peroxide, water hydrolysis and 75% relative humidity studies. The mass balance of AZL was close to 100% in all the stress condition. The developed method was validated as per the ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

PubMed Central

Lv, Haitao; Palacios, Gustavo; Hartil, Kirsten; Kurland, Irwin J.

2014-01-01

In this study a UPLC-tandem (Waters Xevo TQ) MRM based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice; including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a “fingerprint” characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples. PMID:21322650

Current progress and technical challenges of flexible liquid crystal displays

NASA Astrophysics Data System (ADS)

Fujikake, Hideo; Sato, Hiroto

2009-02-01

We focused on several technical approaches to flexible liquid crystal (LC) display in this report. We have been developing flexible displays using plastic film substrates based on polymer-dispersed LC technology with molecular alignment control. In our representative devices, molecular-aligned polymer walls keep plastic-substrate gap constant without LC alignment disorder, and aligned polymer networks create monostable switching of fast-response ferroelectric LC (FLC) for grayscale capability. In the fabrication process, a high-viscosity FLC/monomer solution was printed, sandwiched and pressed between plastic substrates. Then the polymer walls and networks were sequentially formed based on photo-polymerization-induced phase separation in the nematic phase by two exposure processes of patterned and uniform ultraviolet light. The two flexible backlight films of direct illumination and light-guide methods using small three-primary-color light-emitting diodes were fabricated to obtain high-visibility display images. The fabricated flexible FLC panels were driven by external transistor arrays, internal organic thin film transistor (TFT) arrays, and poly-Si TFT arrays. We achieved full-color moving-image displays using the flexible FLC panel and the flexible backlight film based on field-sequential-color driving technique. Otherwise, for backlight-free flexible LC displays, flexible reflective devices of twisted guest-host nematic LC and cholesteric LC were discussed with molecular-aligned polymer walls. Singlesubstrate device structure and fabrication method using self-standing polymer-stabilized nematic LC film and polymer ceiling layer were also proposed for obtaining LC devices with excellent flexibility.

Analysis of Human Plasma Metabolites across Different Liquid Chromatography - Mass Spectrometry Platforms: Cross-platform Transferable Chemical Signatures

PubMed Central

Telu, Kelly H.; Yan, Xinjian; Wallace, William E.; Stein, Stephen E.; Simón-Manso, Yamil

2016-01-01

RATIONALE The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different LC-MS platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. METHODS Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC-MS platforms using reversed phase chromatography and different chromatographic scales (nano, conventional and UHPLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). RESULTS Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (RSD < 2%); however, substantial differences were found in the LC-MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. CONLUSIONS Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable. PMID:26842580

Phytonadione Content in Branded Intravenous Fat Emulsions.

PubMed

Forchielli, Maria Luisa; Conti, Matteo; Motta, Roberto; Puggioli, Cristina; Bersani, Germana

2017-03-01

Intravenous fat emulsions (IVFE) with different fatty acid compositions contain vitamin E as a by-product of vegetable and animal oil during the refining processes. Likewise, other lipid-soluble vitamins may be present in IVFE. No data, however, exist about phytonadione (vitamin K1) concentration in IVFE information leaflets. Therefore, our aim was to evaluate the phytonadione content in different IVFE. Analyses were carried out in triplicate on 6 branded IVFE as follows: 30% soybean oil (100%), 20% olive-soybean oil (80%-20%), 20% soybean-medium-chain triglycerides (MCT) coconut oil (50%-50%), 20% soybean-olive-MCT-fish oil (30%-25%-30%-15%), 20% soybean-MCT-fish oil (40%-50%-10%), and 10% pure fish oil (100%). Phytonadione was analyzed and quantified by a quali-quantitative liquid chromatography-mass spectrometry (LC-MS) method after its extraction from the IVFE by an isopropyl alcohol-hexane mixture, reverse phase-liquid chromatography, and specific multiple-reaction monitoring for phytonadione and vitamin d3 (as internal standard). This method was validated through specificity, linearity, and accuracy. Average vitamin K1 content was 500, 100, 90, 100, 95, and 70 µg/L in soybean oil, olive-soybean oil, soybean-MCT coconut oil, soybean-olive-MCT-fish oil, soybean-MCT-fish oil, and pure fish oil intravenous lipid emulsions (ILEs), respectively. The analytical LC-MS method was extremely effective in terms of specificity, linearity ( r = 0.99), and accuracy (coefficient of variation <5%). Phytonadione is present in IVFE, and its intake varies according to IVFE type and the volume administered. It can contribute to daily requirements and become clinically relevant when simultaneously infused with multivitamins during long-term parenteral nutrition. LC-MS seems adequate in assessing vitamin K1 intake in IVFE.

Dispersive solid-phase extraction followed by high-performance liquid chromatography/tandem mass spectrometry for the determination of ricinine in cooking oil.

PubMed

Cai, Meiqiang; Chen, Xiaohong; Wei, Xiaoqing; Pan, Shengdong; Zhao, Yonggang; Jin, Micong

2014-09-01

A rapid and accurate method by liquid chromatography/tandem mass spectrometry (LC-MS/MS) using positive electrospray was established for the determination of ricinine in cooking oils. The homogenized samples, spiked with (13)C6-labelled ricinine as an internal standard, were extracted using ethanol/water (20:80, v/v) and purified by dispersive solid-phase extraction (dSPE) using primary-secondary amine (PSA) and C18 as adsorbents. The extract was separated in a short C18 reversed-phase column using methanol/water (25:75, v/v) as the mobile phase and detected in multiple reaction monitoring (MRM) mode with the absolute matrix effect of 93.2-102.2%. The alkali-metal adduct ions were discussed and the mass/mass fragmentation pathway was explained. Ricinine showed good linearity in the range of 0.5-50.0 μg/kg with the limit of quantitation 0.5 μg/kg. The recoveries were between 86.0% and 98.3% with the intra- and inter-day RSDs of 2.6-7.0%, 5.5-10.8%, respectively. This method could be applied to the rapid quantification of ricinine in cooking oils. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Study on the generation of a vortex laser beam by using phase-only liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Ma, Haotong; Hu, Haojun; Xie, Wenke; Xu, Xiaojun

2013-09-01

The generation of vortex laser beam by using phase-only liquid crystal spatial light modulator (LC-SLM) combined with the spiral phase screen is experimentally and theoretically studied. Results show that Gaussian and dark hollow vortex laser beams can be generated by using this method successfully. Differing with the Gaussian and dark hollow beams, far field intensities of the generated vortex laser beams still exhibit dark hollow distributions. The comparisons between the ideal generation and experimental generation of vortex laser beams with different optical topological charges by using phase only LC-SLM is investigated in detail. Compared with the ideal generated vortex laser beam, phase distribution of the experimental generated vortex laser beam contains many phase singularities, the number of which is the same as that of the optical topological charges. The corresponding near field and far field dark hollow intensity distributions of the generated vortex laser beams exhibit discontinuous in rotational direction. Detailed theoretical analysis show that the main reason for the physical phenomenon mentioned above is the response error of phase only LC-SLM. These studies can provide effective guide for the generation of vortex laser beam by using phase only LC-SLM for optical tweezers and free space optical communication.

Accelerated wavefront determination technique for optical imaging through scattering medium

NASA Astrophysics Data System (ADS)

He, Hexiang; Wong, Kam Sing

2016-03-01

Wavefront shaping applied on scattering light is a promising optical imaging method in biological systems. Normally, optimized modulation can be obtained by a Liquid-Crystal Spatial Light Modulator (LC-SLM) and CCD hardware iteration. Here we introduce an improved method for this optimization process. The core of the proposed method is to firstly detect the disturbed wavefront, and then to calculate the modulation phase pattern by computer simulation. In particular, phase retrieval method together with phase conjugation is most effective. In this way, the LC-SLM based system can complete the wavefront optimization and imaging restoration within several seconds which is two orders of magnitude faster than the conventional technique. The experimental results show good imaging quality and may contribute to real time imaging recovery in scattering medium.

Direct chiral determination of free amino acid enantiomers by two-dimensional liquid chromatography: application to control transformations in E-beam irradiated foodstuffs.

PubMed

Guillén-Casla, Vanesa; León-González, María Eugenia; Pérez-Arribas, Luis Vicente; Polo-Díez, Luis María

2010-05-01

Changes in free amino acids content and its potential racemization in ready-to-eat foods treated with E-beam irradiation between 1 and 8 kGy for sanitation purposes were studied. A simple heart cut two-dimensional high performance liquid chromatographic method (LC-LC) for the simultaneous enantiomeric determination of three pairs of amino acids used as markers (tyrosine, phenylalanine, and tryptophan) is presented. The proposed method involves the use of two chromatographs in an LC-LC achiral-chiral coupling. Amino acids and their decomposition products were firstly separated in a primary column (C(18)) using a mixture of ammonium acetate buffer (20 mM, pH 6) (94%) and methanol (6%) as the mobile phase. Then, a portion of each peak was transferred by heart cutting through a switching valve to a teicoplanin-chiral column. Methanol (90%)/water (10%) was used as the mobile phase. Ultraviolet detection was at 260 nm. Detection limits were between 0.16 and 3 mg L(-1) for each enantiomer. Recoveries were in the range 79-98%. The LC-LC method combined with the proposed sample extraction procedure is suitable for complex samples; it involves an online cleanup, and it prevents degradation of protein, racemization of L-enantiomers, and degradation of tryptophan. Under these conditions, D-amino acids were not found in any of the analyzed samples at detection levels of the proposed method.

Anchoring Energy Measurements at the Aqueous Phase/Liquid Crystal Interface with Cationic Surfactants Using Magnetic Fréedericksz Transition.

PubMed

Yesil, Fatma; Suwa, Masayori; Tsukahara, Satoshi

2018-01-09

We constructed the apparatus to observe the Fréedericksz transition of liquid crystal in contact with water. The Fréedericksz transition is a distortion of nematic liquid crystals (LCs) induced by external fields. In the present system, sweeping homogeneous magnetic field was applied to the sample, and the distortion of the LC was visualized with a polarized light microscope with the crossed Nichols configuration. The anchoring energy (W AQ/LC ) at the aqueous phase/LC interface was measured in the presence of surfactant from the threshold magnetic field of the Fréedericksz transition. We studied two cationic surfactants: dodecyltrimethylammonium bromide and tetradecyltrimethylammonium bromide. A nematic LC, 4-cyano-4'-pentylbiphenyl (5CB), was examined, which was confined in a copper grid on an octadecyltrichlorosilane-treated microscope glass plate. Measured W AQ/LC were reproducible and showed consistence with the reported region for the water/LC interface. Interfacial excess of surfactants was also measured by the pendant drop method, and the relationship between the obtained W AQ/LC and the interfacial excess was investigated. Experiments showed that an increase in the anchoring energy depends on the surfactant and its interfacial excess. The region of the interfacial coverage, at which W AQ/LC increases, varied with the chain length of the surfactant. The measurement of the anchoring energy will provide new fundamental information on aqueous phase/LC interface.

Novel liquid chromatography–mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos☆

PubMed Central

Lebold, Katie M.; Kirkwood, Jay S.; Taylor, Alan W.; Choi, Jaewoo; Barton, Carrie L.; Miller, Galen W.; Du, Jane La; Jump, Donald B.; Stevens, Jan Frederik; Tanguay, Robert L.; Traber, Maret G.

2013-01-01

To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π–π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography–mass spectrometry (LC–MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E− or E+ diets for 4 months, and then were spawned to obtain E− and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E− (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E− embryos. At 36 hpf, E− embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids. PMID:24416717

Therapeutic drug monitoring of seven psychotropic drugs and four metabolites in human plasma by HPLC-MS.

PubMed

Choong, Eva; Rudaz, Serge; Kottelat, Astrid; Guillarme, Davy; Veuthey, Jean-Luc; Eap, Chin B

2009-12-05

A simple and sensitive LC-MS method was developed and validated for the simultaneous quantification of aripiprazole (ARI), atomoxetine (ATO), duloxetine (DUL), clozapine (CLO), olanzapine (OLA), sertindole (STN), venlafaxine (VEN) and their active metabolites dehydroaripiprazole (DARI), norclozapine (NCLO), dehydrosertindole (DSTN) and O-desmethylvenlafaxine (OVEN) in human plasma. The above mentioned compounds and the internal standard (remoxipride) were extracted from 0.5 mL plasma by solid-phase extraction (mix mode support). The analytical separation was carried out on a reverse phase liquid chromatography at basic pH (pH 8.1) in gradient mode. All analytes were monitored by MS detection in the single ion monitoring mode and the method was validated covering the corresponding therapeutic range: 2-200 ng/mL for DUL, OLA, and STN, 4-200 ng/mL for DSTN, 5-1000 ng/mL for ARI, DARI and finally 2-1000 ng/mL for ATO, CLO, NCLO, VEN, OVEN. For all investigated compounds, good performance in terms of recoveries, selectivity, stability, repeatability, intermediate precision, trueness and accuracy, was obtained. Real patient plasma samples were then successfully analysed.

Development of a LC-MS/MS method for the simultaneous screening of seven water-soluble vitamins in processing semi-coarse wheat flour products.

PubMed

Nurit, Eric; Lyan, Bernard; Piquet, Agnès; Branlard, Gérard; Pujos-Guillot, Estelle

2015-05-01

Wheat is the second largest crop cultivated around the world and constitutes a major part of the daily diet in Europe. It is therefore important to determine the content of micronutrient in wheat and wheat-based food products to define the contribution of wheat-based foods to the nutrition of the consumers. The aim of the present work was to develop a simple and rapid method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of seven water-soluble vitamins in various wheat-based food materials. The vitamins present in the test material were separated in less than 15 min by using a reverse-phase C18 column, and analyzed by positive ion electrospray selected reaction monitoring MS/MS. The MS response for all the vitamins was linear over the working range (0.05 to 9 μg/mL) with correlation coefficients ranging between 0.991 and 1. Limits of quantification in the different food materials ranged from 0.09 to 3.5 μg/g. Intra-day and inter-day precision were found satisfactory. The developed method was applied for the simultaneous analysis of the water-soluble vitamin natural content of different semi-coarse wheat flours and in their corresponding baking products.

Liquid chromatographic determination of histamine in fish, sauerkraut, and wine: interlaboratory study.

PubMed

Beljaars, P R; Van Dijk, R; Jonker, K M; Schout, L J

1998-01-01

An interlaboratory study of the liquid chromatographic (LC) determination of histamine in fish, sauerkraut, and wine was conducted. Diminuted and homogenized samples were suspended in water followed by clarification of extracts with perchloric acid, filtration, and dilution with water. After LC separation on a reversed-phase C18 column with phosphate buffer (pH 3.0)--acetonitrile (875 + 125, v/v) as mobile phase, histamine was measured fluorometrically (excitation, 340 nm; emission, 455 nm) in samples and standards after postcolumn derivatization with o-phthaldialdehyde (OPA). Fourteen samples (including 6 blind duplicates and 1 split level) containing histamine at about 10-400 mg/kg or mg/L were analyzed singly according to the proposed procedure by 11 laboratories. Results from one participant were excluded from statistical analysis. For all samples analyzed, repeatability relative standard deviations varied from 2.1 to 5.6%, and reproducibility relative standard deviations ranged from 2.2 to 7.1%. Averaged recoveries of histamine for this concentration range varied from 94 to 100%.

Orientational fluctuations and phase transitions in 8CB confined by cylindrical pores of the PET film

NASA Astrophysics Data System (ADS)

Maksimochkin, G. I.; Shmeliova, D. V.; Pasechnik, S. V.; Dubtsov, A. V.; Semina, O. A.; Kralj, S.

2016-08-01

Results of optical investigations of the isotropic-nematic and nematic-smectic A phase transitions in porous polyethyleneterephthalate (PET) films filled with octyl-cyanobihenyl (8CB) liquid crystal (LC) are reported. Samples of porous films of thickness 23 µm with normally oriented cylindrical pores of a radius R ranging from 10 nm to 1000 nm were prepared using the track-etched membrane technology. The dynamic light scattering method was used to probe the nematic orientational fluctuations of confined LC samples. The corresponding relaxation time τ was measured as a function of R and temperature T at slow enough cooling rates (0.3-0.6 K/h) to locate the phase transition temperatures. Changes in τ(T) dependencies relatively sensitivity fingerprint the LC phase transformations. Experimental results are analysed using the Landau-de Gennes-Ginzburg phenomenological approach.

Protein mass analysis of histones.

PubMed

Galasinski, Scott C; Resing, Katheryn A; Ahn, Natalie G

2003-09-01

Posttranslational modification of chromatin-associated proteins, including histones and high-mobility-group (HMG) proteins, provides an important mechanism to control gene expression, genome integrity, and epigenetic inheritance. Protein mass analysis provides a rapid and unbiased approach to monitor multiple chemical modifications on individual molecules. This review describes methods for acid extraction of histones and HMG proteins, followed by separation by reverse-phase chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS). Posttranslational modifications are detected by analysis of full-length protein masses. Confirmation of protein identity and modification state is obtained through enzymatic digestion and peptide sequencing by MS/MS. For differentially modified forms of each protein, the measured intensities are semiquantitative and allow determination of relative abundance and stoichiometry. The method simultaneously detects covalent modifications on multiple proteins and provides a facile assay for comparing chromatin modification states between different cell types and/or cellular responses.

Development of multiclass methods for drug residues in eggs: hydrophilic solid-phase extraction cleanup and liquid chromatography/tandem mass spectrometry analysis of tetracycline, fluoroquinolone, sulfonamide, and beta-lactam residues.

PubMed

Heller, David N; Nochetto, Cristina B; Rummel, Nathan G; Thomas, Michael H

2006-07-26

A method was developed for detection of a variety of polar drug residues in eggs via liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI). A total of twenty-nine target analytes from four drug classes-sulfonamides, tetracyclines, fluoroquinolones, and beta-lactams-were extracted from eggs using a hydrophilic-lipophilic balance polymer solid-phase extraction (SPE) cartridge. The extraction technique was developed for use at a target concentration of 100 ng/mL (ppb), and it was applied to eggs containing incurred residues from dosed laying hens. The ESI source was tuned using a single, generic set of tuning parameters, and analytes were separated with a phenyl-bonded silica cartridge column using an LC gradient. In a related study, residues of beta-lactam drugs were not found by LC/MS/MS in eggs from hens dosed orally with beta-lactam drugs. LC/MS/MS performance was evaluated on two generations of ion trap mass spectrometers, and key operational parameters were identified for each instrument. The ion trap acquisition methods could be set up for screening (a single product ion) or confirmation (multiple product ions). The lower limit of detection for screening purposes was 10-50 ppb (sulfonamides), 10-20 ppb (fluoroquinolones), and 10-50 ppb (tetracyclines), depending on the drug, instrument, and acquisition method. Development of this method demonstrates the feasibility of generic SPE, LC, and MS conditions for multiclass LC/MS residue screening.

Automated solid-phase extraction workstations combined with quantitative bioanalytical LC/MS.

PubMed

Huang, N H; Kagel, J R; Rossi, D T

1999-03-01

An automated solid-phase extraction workstation was used to develop, characterize and validate an LC/MS/MS method for quantifying a novel lipid-regulating drug in dog plasma. Method development was facilitated by workstation functions that allowed wash solvents of varying organic composition to be mixed and tested automatically. Precision estimates for this approach were within 9.8% relative standard deviation (RSD) across the calibration range. Accuracy for replicate determinations of quality controls was between -7.2 and +6.2% relative error (RE) over 5-1,000 ng/ml(-1). Recoveries were evaluated for a wide variety of wash solvents, elution solvents and sorbents. Optimized recoveries were generally > 95%. A sample throughput benchmark for the method was approximately equal 8 min per sample. Because of parallel sample processing, 100 samples were extracted in less than 120 min. The approach has proven useful for use with LC/MS/MS, using a multiple reaction monitoring (MRM) approach.

Magnetic field induced quantum dot brightening in liquid crystal synergized magnetic and semiconducting nanoparticle composite assemblies

DOE PAGES

Amaral, Jose Jussi; Wan, Jacky; Rodarte, Andrea L.; ...

2014-10-22

The design and development of multifunctional composite materials from artificial nano-constituents is one of the most compelling current research areas. This drive to improve over nature and produce ‘meta-materials’ has met with some success, but results have proven limited with regards to both the demonstration of synergistic functionalities and in the ability to manipulate the material properties post-fabrication and in situ. Here, magnetic nanoparticles (MNPs) and semiconducting quantum dots (QDs) are co-assembled in a nematic liquid crystalline (LC) matrix, forming composite structures in which the emission intensity of the quantum dots is systematically and reversibly controlled with a small appliedmore » magnetic field (<100 mT). This magnetic field-driven brightening, ranging between a two- to three-fold peak intensity increase, is a truly cooperative effect: the LC phase transition creates the co-assemblies, the clustering of the MNPs produces LC re-orientation at atypical low external field, and this re-arrangement produces compaction of the clusters, resulting in the detection of increased QD emission. These results demonstrate a synergistic, reversible, and an all-optical process to detect magnetic fields and additionally, as the clusters are self-assembled in a fluid medium, they offer the possibility for these sensors to be used in broad ranging fluid-based applications.« less

Comparison of gamma-oryzanol contents in crude rice bran oils from different sources by various determination methods.

PubMed

Yoshie, Ayano; Kanda, Ayato; Nakamura, Takahiro; Igusa, Hisao; Hara, Setsuko

2009-01-01

Although there are various determination methods for gamma -oryzanol contained in rice bran oil by absorptiometry, normal-phase HPLC, and reversed-phase HPLC, their accuracies and the correlations among them have not been revealed yet. Chloroform-containing mixed solvents are widely used as mobile phases in some HPLC methods, but researchers have been apprehensive about its use in terms of safety for the human body and the environment.In the present study, a simple and accurate determination method was developed by improving the reversed-phase HPLC method. This novel HPLC method uses methanol/acetonitrile/acetic acid (52/45/3 v/v/v), a non-chlorinated solvent, as the mobile phase, and shows an excellent linearity (y = 0.9527x + 0.1241, R(2) = 0.9974) with absorptiometry. The mean relative errors among the existing 3 methods and the novel method, determined by adding fixed amounts of gamma-oryzanol into refined rice salad oil, were -4.7% for the absorptiometry, -6.8% for the existing normal-phase HPLC, +4.6% for the existing reversed-phase HPLC, and -1.6% for the novel reversed-phase HPLC method. gamma -Oryzanol content in 12 kinds of crude rice bran oils obtained from different sources were determined by the four methods. The mean content of those oils were 1.75+/-0.18% for the absorptiometry, 1.29+/-0.11% for the existing normal-phase HPLC, 1.51+/-0.10% for the existing reversed-phase HPLC, and 1.54+/-0.19% for the novel reversed-phase HPLC method.

Two fatal intoxication cases with imidacloprid: LC/MS analysis.

PubMed

Proença, Paula; Teixeira, Helena; Castanheira, Fernando; Pinheiro, João; Monsanto, Paula V; Marques, Estela P; Vieira, Duarte Nuno

2005-10-04

Imidacloprid [1-(6-chloro-3pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] is a new and potent nitromethylene insecticide with high insecticidal activity at very low application rates. It is the first highly effective insecticide that, like nicotine, acts on the nervous system, causing blockage of postsynaptic nicotinergic acetylcholine receptors. Two fatal cases with this insecticide in two male individuals, of 33 and 66 years old, are presented. An LC/MS with electrospray method for measuring imidacloprid and its metabolites in post-mortem samples is described. In the chromatographic separation, a reverse-phase column XTerra MS C18 (2.1mm i.d.x 150 mm, 5 microm) was used and the mobile phase composed with acetonitrile and 0.1% formic acid (15:85), at a 0.25 mL/min flow rate. Samples were prepared with a liquid-liquid extraction procedure with dichloromethane. Calibration curves for imidacloprid in blood and urine samples were linear from 0.2 to 15 microg/mL. The mean recovery was 86% with a coefficient of variation of +/-5.9%. The detection limit was 0.002 microg/mL. Quantitative results were obtained for all post-mortem matrices available of the two fatal cases: blood, urine, stomach contents, lung, liver and kidney. The imidacloprid blood concentrations found in two-cases were 12.5 and 2.05 microg/mL. The authors validated a method to detect and quantify imidacloprid in post-mortem samples, and to our knowledge for the first time a post-mortem tissue distribution was performed on various samples for this insecticide.

Stable isotope labeling-solid phase extraction-mass spectrometry analysis for profiling of thiols and aldehydes in beer.

PubMed

Zheng, Shu-Jian; Wang, Ya-Lan; Liu, Ping; Zhang, Zheng; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

2017-12-15

In this study, we developed a strategy for profiling of thiols and aldehydes in beer samples by stable isotope labeling-solid phase extraction-liquid chromatography-double precursor ion scan/double neutral loss scan-mass spectrometry analysis (SIL-SPE-LC-DPIS/DNLS-MS). A pair of isotope reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d 7 bromide, BQB-d 7 ) were used to label thiols; while for the aldehydes, a pair of isotope reagents (4-(2-(trimethylammonio) ethoxy) benzenaminium halide, 4-APC; 4-(2-(trimethylammonio) ethoxy) benzenaminium halide-d 4 , 4-APC-d 4 ) were used. The labeled thiols and aldehydes were extracted and purified with solid-phase extraction, respectively, followed by LC-MS analysis. Using the proposed SIL-SPE-LC-DPIS/DNLS-MS methods, 76 thiol and 25 aldehyde candidates were found in beer. Furthermore, we established SIL-SPE-LC-MRM-MS methods for the relative quantitation of thiols and aldehydes in different beer samples. The results showed that the contents of thiols and aldehydes are closely related to the brands and origins of beers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Multi-residue analysis method for analysis of pharmaceuticals using liquid chromatography-time of flight/mass spectrometry (LC-TOF/MS) in water sample

NASA Astrophysics Data System (ADS)

Al-Qaim, Fouad Fadhil; Abdullah, Md Pauzi; Othman, Mohamed Rozali

2013-11-01

In this work, a developed method using solid - phase extraction (SPE) followed by liquid chromatography - time of flight mass spectrometry (LC-ESI-TOF/MS) was developed and validated for quantification and confirmation of eleven pharmaceuticals with different therapeutic classes in water samples, Malaysia. These compounds are caffeine (CAF), prazosin (PRZ), enalapril (ENL), carbamazepine (CBZ), nifedipine (NFD), levonorgestrel (LNG), simvastatin (SMV), hydrochlorothiazide (HYD), gliclazide (GLIC), diclofenac-Na (DIC-Na) and mefenamic acid (MEF). LC was performed on a Dionex Ultimate 3000/LC 09115047 (USA) system. Chromatography was performed on a Thermo Scientific C18 (250 mm × 2.1 mm, i.d.: 5μm) column. Several parameters were optimised such as; mobile phase, gradient elution, collision energy and solvent elution for extraction of compounds from water. The recoveries obtained ranged from 30-148 % in river water. Five pharmaceutical compounds were detected in the surface water samples: caffeine, prazosin, enalpril, diclofenac-Na and mefenamic acid. The developed method is precise and accepted recoveries were got. In addition, this method is suitable to identify and quantify trace concentrations of pharmaceuticals in surface water.

Separation and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS.

PubMed

Xu, Yu; Wang, DanDan; Tang, Lan; Wang, Jian

2017-10-25

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5μm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MS n data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability. Copyright © 2017. Published by Elsevier B.V.

An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Erin Shammel; Livesay, Eric A.; Orton, Daniel J.

2010-02-05

A high-throughput approach and platform using 15 minute reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking twenty reference peptides at varying concentrations from 1 ng/mL to 10 µg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected thirteen out of the twenty spiked peptides that had concentrations ≥100 ng/mL.more » In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for nineteen of the twenty peptides with all spiking level present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects, but in turn limits the achievable dynamic range compared to the TOF detector.« less

Unusually high levels of bisphenol A (BPA) in thermal paper cash register receipts (CRs): development and application of a robust LC-UV method to quantify BPA in CRs.

PubMed

Babu, Sainath; Uppu, Sannihith N; Martin, Brittany; Agu, Ogad A; Uppu, Rao M

2015-01-01

We have developed a simple, reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of bisphenol A (BPA) in thermal paper cash register receipts (CRs). The method is suitable for analysis of other types of bisphenols and it involves an overnight extraction of CRs with acetonitrile (AN) at 50 °C followed by the HPLC analysis on a Supelcosil LC18 column (150 × 4.6 mm, particle size: 5 μ) using 50% AN in water as the mobile phase (5 min, isocratic). The composition of AN in the mobile phase changed to 100% over a 10 min period (linear gradient) and then held at 100% AN for 10 min (isocratic). The flow rate was set at 1 mL/min (injection volume: 20 μL) and the eluent was monitored at 234 nm. The authentic BPA eluted with a retention time of 5.9 min and gave a linear detector response in the concentration range of 0.23-50 mg/L. BPA in the CR extracts also eluted with the same retention and had identical absorbance properties as the standard. When CR extracts were co-injected with authentic BPA, they were resolved as a single peak. Further, GC/MS/EI analysis of authentic BPA and the HPLC-purified CR extracts have identical ion chromatograms and fragmentation of the molecular ion (m/z = 228). We have analyzed 170 CRs collected from 62 different vendors including supermarkets, fast food restaurants, gas stations and banking outlets. Almost all cash receipts (n = 168) showed the presence of BPA in the concentration range of 0.45-4.26% (M ± SD, 1.54 ± 0.73%).

Determination of cyclic guanosine- and cyclic adenosine monophosphate (cGMP and cAMP) in human plasma and animal tissues by solid phase extraction on silica and liquid chromatography-triple quadrupole mass spectrometry.

PubMed

Van Damme, Thomas; Zhang, Yanhua; Lynen, Frédéric; Sandra, Pat

2012-11-15

3',5'-Cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP) are essential second messenger molecules. They are involved in signal transduction within cells, in physiological functions such as neurotransmission and in the modulation of cell growth and differentiation of organisms, respectively. A quantitative solid phase extraction method (SPE) based on hydrophilic interaction on silica was developed and applied to both plasma and tissue samples. The stable isotope-labeled internal standards ²D₁, ¹⁵N₃-3',5'-cGMP and ¹³C₁₀, ¹⁵N₅-3',5'-cAMP were added prior to the sample preparation to ensure high precision and accuracy. The samples were analyzed by reversed-phase liquid chromatography (RP-LC). Negative electrospray (ESI)-MS/MS was used to selectively monitor several transitions of each metabolite. The method for the analysis of 3',5'-cAMP and 3',5'-cGMP in plasma was validated in the range of 0.15-20 ng/mL (R²=0.9996 and 0.9994 for 3',5'-cAMP and 3',5'-cGMP, respectively). Basal plasma concentrations for fifteen healthy human patients determined with this method varied between 4.66-9.20 ng/mL for 3',5'-cAMP and between 0.30-1.20 ng/mL for 3',5'-cGMP, with precisions better than 9.1%. 3',5'-cGMP and 3',5'-cAMP together with their 2',3'-isomers were also determined in a semi quantitative way in animal tissues. The structures of the isomers were confirmed by analysis with LC-high resolution time-of-flight MS and subsequently by comparison of retention times with standards. Copyright © 2012 Elsevier B.V. All rights reserved.

A sensitive and accurate method for the determination of perfluoroalkyl and polyfluoroalkyl substances in human serum using a high performance liquid chromatography-online solid phase extraction-tandem mass spectrometry.

PubMed

Yu, Chang Ho; Patel, Bhupendra; Palencia, Marilou; Fan, Zhihua Tina

2017-01-13

A selective, sensitive, and accurate analytical method for the measurement of perfluoroalkyl and polyfluoroalkyl substances (PFASs) in human serum, utilizing LC-MS/MS (liquid chromatography-tandem mass spectrometry), was developed and validated according to the Centers for Disease Control and Prevention (CDC) guidelines for biological sample analysis. Tests were conducted to determine the optimal analytical column, mobile phase composition and pH, gradient program, and cleaning procedure. The final analytical column selected for analysis was an extra densely bonded silica-packed reverse-phase column (Agilent XDB-C 8 , 3.0×100mm, 3.5μm). Mobile phase A was an aqueous buffer solution containing 10mM ammonium acetate (pH=4.3). Mobile phase B was a mixture of methanol and acetonitrile (1:1, v/v). The gradient program was programmed by initiating a fast elution (%B, from 40 to 65%) between 1.0 and 1.5min, followed by a slow elution (%B: 65-80%) in the period of 1.5-7.5min. The cleanup procedures were augmented by cleaning with (1) various solvents (isopropyl alcohol, methanol, acetonitrile, and reverse osmosis-purified water); (2) extensive washing steps for the autosampler and solid phase extraction (SPE) cartridge; and (3) a post-analysis cleaning step for the whole system. Under the above conditions, the resolution and sensitivity were significantly improved. Twelve target PFASs were baseline-separated (2.5-7.0min) within a 10-min of acquisition time. The limits of detection (LODs) were 0.01ng/mL or lower for all of the target compounds, making this method 5 times more sensitive than previously published methods. The newly developed method was validated in the linear range of 0.01-50ng/mL, and the accuracy (recovery between 80 and 120%) and precision (RSD<20%) were acceptable at three spiked levels (0.25, 2.5, and 25ng/mL). The method development and validation results demonstrated that this method was precise, accurate, and robust, with high-throughput (∼10min per sample); thus suitable for large-scale epidemiological studies. Published by Elsevier B.V.

Simultaneous determination of Vitamin A, 25-hydroxyl vitamin D3 α-tocopherol in small biological fluids by liquid chromatography-tandem mass spectrometry.

PubMed

Zhang, Haifeng; Quan, Li; Pei, Pei; Lin, Ye; Feng, Chao; Guan, Hongyan; Wang, Fang; Zhang, Ting; Wu, Jianxin; Huo, Junsheng

2018-03-15

A sensitive method for the simultaneous analysis of vitamin A (VA), 25-hydroxyl vitamin D 3 (25-OH VD 3 ) and α-tocopherol (VE) in children plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Sample preparation chose the solid phase extraction. 100 μL of plasma was mixed with 300 μL ethanol contained 4 μL isotope-labelled analytes. After a series operation, the supernatant was applied to the solid phase extraction (SPE) plate (HLB μElution plate). The eluate was evaporated, and reconstituted in 100 μL methanol. And then, 6 μL reconstituted sample was injected into LC-MS/MS. Quantitative analysis was carried out by multiple reaction monitoring (MRM) with a positive mode electrospray (ESi + ). Separations of VA, 25-OH VD 3 and VE were performed on an Acquity UPLC reversed-phase Phenyl-Hexyl analytical column (CSH, 2.1 × 50 mm, 1.7 μm). Gradient elution was used at a flow rate of 0.4 mL/min with a mobile phase consisting of 0.1% formic acid solution and 0.1% formic acid, 5 mM ammonium formate in acetonitrile. The total time of analysis was 10 min. The method had a limit of quantification (LOQ) of 10.03, 1.20, and 0.04 ng/mL for VA, 25-OH VD 3 and VE in methanol, respectively. The linear calibration curves were fitted over the range of 0.14-14.32 μg/mL, 1.80-180.29 ng/mL, and 6.03-602.99 ng/mL for VA, 25-OH VD 3 and VE in methanol. The correlation coefficients were greater than 0.998 for all analytes. The recoveries for all analytes were between 80 and 120% with the inter- and intra-day precisions (presented as relative standard deviation, RSD%) less than 10.0%. Analysis of VA, 25-OH VD 3 and VE in recurrent respiratory tract infection children plasma and anemic infants' fingertip blood was then carried out using this method and statistical analysis of the data with statistic package for social science 20.0 (SPSS 20.0). Using this method, multiple fat-soluble vitamins could be detected at the same time. Solid phase extraction was used to simplify sample pretreatment. μElution plate used here could reduce the sample volume, only 100 μL sample was used in this method, and 6 μL reconstituted sample was injected into LC-MS/MS. This makes the method appropriate for larger sample pretreatment, and suitable for children, especially infants and newborns' sample detection, in whom the circulation blood was low. Copyright © 2017. Published by Elsevier B.V.

Simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: application to a bioequivalence study.

PubMed

Hu, Ziyan; Zou, Qiaogen; Tian, Jixin; Sun, Lili; Zhang, Zunjian

2011-12-15

A rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma has been developed and validated. Following liquid-liquid extraction, the analytes were separated on a reversed-phase C(18) column (150 mm × 2.0 mm, 3 μm) using formic acid:10 mM ammonium acetate:methanol (0.2:62:38, v/v/v) as mobile phase at a flow rate of 0.2 mL/min and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode. The method was linear for all analytes over the following concentration (ng/mL) ranges: codeine 0.08-16; ephedrine 0.8-160; guaiphenesin 80-16,000; chlorpheniramine 0.2-40. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC-MS/MS method was successfully applied to a bioequivalence study in 6 healthy beagle dogs. Copyright © 2011 Elsevier B.V. All rights reserved.

Establishment of Protocols for Global Metabolomics by LC-MS for Biomarker Discovery.

PubMed

Saigusa, Daisuke; Okamura, Yasunobu; Motoike, Ikuko N; Katoh, Yasutake; Kurosawa, Yasuhiro; Saijyo, Reina; Koshiba, Seizo; Yasuda, Jun; Motohashi, Hozumi; Sugawara, Junichi; Tanabe, Osamu; Kinoshita, Kengo; Yamamoto, Masayuki

2016-01-01

Metabolomics is a promising avenue for biomarker discovery. Although the quality of metabolomic analyses, especially global metabolomics (G-Met) using mass spectrometry (MS), largely depends on the instrumentation, potential bottlenecks still exist at several basic levels in the metabolomics workflow. Therefore, we established a precise protocol initially for the G-Met analyses of human blood plasma to overcome some these difficulties. In our protocol, samples are deproteinized in a 96-well plate using an automated liquid-handling system, and conducted either using a UHPLC-QTOF/MS system equipped with a reverse phase column or a LC-FTMS system equipped with a normal phase column. A normalization protocol of G-Met data was also developed to compensate for intra- and inter-batch differences, and the variations were significantly reduced along with our normalization, especially for the UHPLC-QTOF/MS data with a C18 reverse-phase column for positive ions. Secondly, we examined the changes in metabolomic profiles caused by the storage of EDTA-blood specimens to identify quality markers for the evaluation of the specimens' pre-analytical conditions. Forty quality markers, including lysophospholipids, dipeptides, fatty acids, succinic acid, amino acids, glucose, and uric acid were identified by G-Met for the evaluation of plasma sample quality and established the equation of calculating the quality score. We applied our quality markers to a small-scale study to evaluate the quality of clinical samples. The G-Met protocols and quality markers established here should prove useful for the discovery and development of biomarkers for a wider range of diseases.

Determination of a flame retardant hydrolysis product in human urine by SPE and LC-MS. Comparison of molecularly imprinted solid-phase extraction with a mixed-mode anion exchanger.

PubMed

Möller, Kristina; Crescenzi, Carlo; Nilsson, Ulrika

2004-01-01

Diphenyl phosphate is a hydrolysis product and possible metabolite of the flame retardant and plasticiser additive triphenyl phosphate. A molecularly imprinted polymer solid-phase extraction (MISPE) method for extracting diphenyl phosphate from aqueous solutions has been developed and compared with SPE using a commercially available mixed-mode anion exchanger. The imprinted polymer was prepared using 2-vinylpyridine (2-Vpy) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, and a structural analogue of the analyte as the template molecule. The imprinted polymer was evaluated for use as a SPE sorbent, in tests with both aqueous standards and spiked urine samples, by comparing recovery and breakthrough data obtained using the imprinted form of the polymer and a non-imprinted form (NIP). Extraction from aqueous solutions resulted in more than 80% recovery. Adsorption by the molecularly imprinted polymer (MIP) was non-selective, but selectivity was achieved by selective desorption in the wash steps. Diphenyl phosphate could also be selectively extracted from urine samples, although the urine matrix reduced the capacity of the MISPE cartridges. Recoveries from urine extraction were higher than 70%. It was important to control pH during sample loading. The MISPE method was found to yield a less complex LC-ESI-MS chromatogram of the urine extracts compared with the mixed-mode anion-exchanger method. An LC-ESI-MS method using a Hypercarb LC column with a graphitised carbon stationary phase was also evaluated for organophosphate diesters. LC-ESI-MS using negative-ion detection in selected ion monitoring (SIM) mode was shown to be linear for diphenyl phosphate in the range 0.08-20 ng microL(-1).

Mesenchymal Stem Cells Reverse Bone Marrow Dysfunction Following Injury and Stress

PubMed Central

Gore, Amy V.; Bible, Letitia E.; Livingston, David H.; Mohr, Alicia M.; Sifri, Ziad C.

2015-01-01

Background Bone marrow (BM) dysfunction following experimental lung contusion (LC) resolves in 7 days, however, if followed by chronic stress (CS) following, BM dysfunction is persistent. Mesenchymal stem cells (MSC) have protective immunomodulatory effects. We hypothesize that MSC can protect the BM against the deleterious effect of CS following LC. Methods Male Sprague-Dawley rats (n=6–7/group) underwent LC or LC/CS ± MSC injection. CS consisted of a daily 2-hour period of restraint with repositioning and alarming every 30 minutes to prevent habituation. A single intravenous dose of 5 × 106 MSC was given within ten minutes following LC. Animals were sacrificed at day seven and peripheral blood (PB) and BM were collected. Flow cytometry was used to assess hematopoietic progenitor cells (HPCs) mobilized to PB. Plasma G-CSF levels were measured by ELISA. BM cellularity and growth of BM HPC colonies (CFU-E, BFU-E, CFU-GEMM) were also evaluated. Results As previously reported, the addition of CS to LC resulted in a 32% decrease in BM cellularity, significant decreases in CFU-GEMM, BFU-E, and CFU-E and marked increase in HPC in the PB as compared naïve animals. The addition of MSC to LC/CS resulted in a 22% increase in BM cellularity and significant increases in CFU-GEMM, BFU-E, and CFU-E cultured from the BM. MSCs additionally reduced plasma G-CSF, prevented prolonged mobilization of HPC to PB, and restored colony growth to naïve levels. Conclusion Chronic stress following LC results in persistent BM dysfunction manifested by a significant decrease in cellularity, HPC colony growth, and increased G-CSF levels and HPC mobilization to the PB at seven days following injury. The addition of a single dose of MSCs following acute traumatic injury reverses the deleterious effects of CS on BM function. Further study is warranted to better understand the mechanisms behind MSC-mediated protection of BM function in the setting of CS. PMID:26402534

Small Angle Neutron Scattering (sans) Studies on "SIDE-END FIXED" and "SIDE-ON FIXED" Liquid Crystal Polymers

NASA Astrophysics Data System (ADS)

Hardouin, F.; Noirez, L.; Keller, P.; Leroux, N.; Cotton, J. P.

The following sections are included: * Introduction * Experimental * Results and discussion * Determination of the backbone conformation in the nematic and smectic A phases of "side-end fixed" L.C. polymethacrylates (PMA) or polyacrylates (PA) * Determination of the global and backbone conformation in the nematic and smectic A phases of "side-end fixed" L.C. polysiloxanes (PMS) * Determination of the backbone conformation in the unique nematic phase (without smectic A phase) or in the reentrant nematic phase (below smectic A phase) of "side-end fixed" L.C. polyacrylates (PA) * Determination of the global conformation in the nematic phase of "side-on fixed" L.C. polysiloxanes (PMS) * Determination of the global conformation in the nematic phase of "diluted side-on fixed" L.C. copolysiloxanes * Determination of the backbone conformation in the nematic phase of "side-on fixed" L.C. polyacrylates * Conclusions * References

Reversible switching of liquid crystalline order permits synthesis of homogeneous populations of dipolar patchy microparticles

DOE PAGES

Wang, Xiaoguang; Miller, Daniel S.; de Pablo, Juan J.; ...

2014-08-15

The spontaneous positioning of colloids on the surfaces of micrometer-sized liquid crystal (LC) droplets and their subsequent polymerization offers the basis of a general and facile method for the synthesis of patchy microparticles. The existence of multiple local energetic minima, however, can generate kinetic traps for colloids on the surfaces of the LC droplets and result in heterogeneous populations of patchy microparticles. To address this issue, in this paper it is demonstrated that adsorbate-driven switching of the internal configurations of LC droplets can be used to sweep colloids to a single location on the LC droplet surfaces, thus resulting inmore » the synthesis of homogeneous populations of patchy microparticles. The surface-driven switching of the LC can be triggered by addition of surfactant or salts, and permits the synthesis of dipolar microparticles as well as “Janus-like” microparticles. Finally, by using magnetic colloids, the utility of the approach is illustrated by synthesizing magnetically responsive patchy microdroplets of LC with either dipolar or quadrupolar symmetry that exhibit distinct optical responses upon application of an external magnetic field.« less

Phase retardation vs. pretilt angle in liquid crystal cells with homogeneous and inhomogeneous LC director configuration.

PubMed

Belyaev, Victor; Solomatin, Alexey; Chausov, Denis

2013-02-25

Phase retardation of both extraordinary and ordinary polarized rays passing through a liquid crystal (LC) cell with homogeneous and inhomogeneous LC director distribution is calculated as a function of the LC pretilt angle θ₀ on the cell substrates in the range 0 ≤ θ₀ ≤ 90°. The LC pretilt on both substrates can have the same or opposite direction, thereby forming homogeneous, splay, or bend director configurations. At the same pretilt angle value, the largest phase retardation ΔΦ is observed in splay LC cells, whereas the smallest phase retardation is observed in bend cells. For the θ₀ values close to 0, 45°, and 90°, analytical approximations are derived, showing that phase retardation depends on LC birefringence variation.

Ultra trace determination of 31 pesticides in water samples by direct injection-rapid resolution liquid chromatography-electrospray tandem mass spectrometry.

PubMed

Díaz, Laura; Llorca-Pórcel, Julio; Valor, Ignacio

2008-08-22

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the detection of pesticides in tap and treated wastewater was developed and validated according to the ISO/IEC 17025:1999. Key features of this method include direct injection of 100 microL of sample, an 11 min separation by means of a rapid resolution liquid chromatography system with a 4.6 mm x 50 mm, 1.8 microm particle size reverse phase column and detection by electrospray ionization (ESI) MS-MS. The limits of detection were below 15 ng L(-1) and correlation coefficients for the calibration curves in the range of 30-2000 ng L(-1) were higher than 0.99. Precision was always below 20% and accuracy was confirmed by external evaluation. The main advantages of this method are direct injection of sample without preparative procedures and low limits of detection that fulfill the requirements established by the current European regulations governing pesticide detection.

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

PubMed

Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to have impact on the binding affinity to the antigen. In this work, we utilized a mass spectrometry-based peptide mapping approach to detect and quantitate the Asp isomerization in the CDRs of mAb-A. To routinely monitor the CDR isomerization of mAb-A, a focused peptide mapping method utilizing reversed phase chromatographic separation and UV detection has been developed and qualified. This approach is generally applicable to monitor isomerization and other post-translational modifications of proteins in a specific and high-throughput mode to ensure product quality. © PDA, Inc. 2016.

Determination of flumazenil in serum by liquid chromatography-mass spectrometry: Application to kinetics study in acute diazepam overdose.

PubMed

Djordjević, Snezana; Jović-Stosić, Jasmina; Kilibarda, Vesna; Segrt, Zoran; Perković-Vukcević, Natasa

2016-02-01

Flumazenil is benzodiazepine receptor antagonist. It has been studied for a various indications, including reversal of sedation after surgery or diagnostic procedures, awakening of comatose patients in benzodiazepine overdose, or for symptomatic treatment of hepatic encephalopathy. Some drugs, like theophylline, may prolong its elimination half-life. Considering the long half-life of diazepam and its metabolites, concomitant use of theophylline may reduce the need for repeated dosing of flumazenil in patients with acute diazepam poisoning. The aim of this study was to introduce a reliable and accurate method for determining the concentration of flumazenil after therapeutic application in patients with acute poisoning, and using that method to assess whether the kinetics of flumazenil change in the presence of aminophylline (combination of theophylline and ethylenediamine in a 2:1 ratio) applied as concomitant therapy. Blood samples from patients with acute diazepam poisoning that received flumazenil at the dose of 0.5 mg, or the same dose with 3 mg/kg of body weight of aminophylline, were collected 1, 3, 10, 30, 60, 120 and 240 min after its intravenous administration. Samples were prepared by solid-phase extraction on Oasis HLB cartridges with ethylacetate as extracting agens. Flumazenil was determined by liquid chromatography with mass spectrometry (LC-MS) in single ionmonitoring mode at m/z 304. Separation of flumazenil from matrix compound was performed on Lichrospher RP-8 column usingthe mixture of acidic acetonitrile and 20 mM of ammonium acetatein water (55 : 45) as a mobile phase. The applied analitycal method showed excellent recovery (94.65%). The obtained extracts were much cleaner than the extracts obtained by the sameextractant in the process of liquid-liquid extraction. The limit ofdetection of the LC-MS method described in this paper was 0.5 ng/mL and the limit of quantitation was 1 ng/mL. In the patientstreated with both flumazenil and aminophylline, the eliminationconstant for flumazenil was significantly lower and the elimination half-life was longer (p < 0.05) in comparison with the same parameters in.the patients who received flumazenil alone. The applied LC-MS method for the determination of flumazenil in serum samples of patients with acute diazepam poisoning is rapid, sensitive, precise and specific. Concomitant use with theophylline significantly prolonged elimination of flumazenil during the treatment of acute poisonings with diazepam.

Fast response liquid crystal devices

NASA Astrophysics Data System (ADS)

Wu, Yung-Hsun

Liquid crystal (LC) has been widely used for displays, spatial light modulators, variable optical attenuators (VOAs) and other tunable photonic devices. The response time of these devices is mainly determined by the employed liquid crystal material. The response time of a LC device depends on the visco-elastic coefficient (gamma1/K11), LC cell gap (d), and applied voltage. Hence, low visco-elastic coefficient LC materials and thinner cell gap are favorable for reducing the response time. However, low visco-elastic coefficient LCs are usually associated with a low birefringence because of shorter molecular conjugation. For display applications, such as LCD TVs, low birefringence (Deltan<0.1) LCs are commonly used. However, for optical communications at 1550 nm, low birefringence requires to a thick cell gap which, in turn, increases the response time. How to obtain fast response for the LC devices is a fundamentally important and technically challenging task. In this dissertation, we investigate several methods to improve liquid crystal response time, for examples, using dual-frequency liquid crystals, polymer stabilized liquid crystals, and sheared polymer network liquid crystals. We discover a new class of material, denoted as sheared polymer network liquid crystal (SPNLC) which exhibits a submillisecond response time. Moreover, this response time is insensitive to the LC cell gap. This is the first LC device exhibiting such an interesting property. Chapters 1 and 2 describe the motivation and background of this dissertation. From chapter 3 to chapter 6, dual-frequency liquid crystals and polymer network methods are demonstrated as examples for the variable optical attenuators. Variable optical attenuator (VOA) is a key component in optical communications. Especially, the sheared PNLC VOA shows the best result; its dynamic range reaches 43 dB while the response time is in the submillisecond range at 1550 nm wavelength, which is 50 times faster than the commercial LC-based VOA. In Chapter 7, we report a new device called axially-symmetric sheared polymer network liquid crystals (AS-SPNLC) and use it as LC devices. Through analyzing the structure of this axially-symmetric SPNLC, we construct a 3-D model to explain the observed phenomena. An axially-symmetric sheared polymer network liquid crystal has several attractive features: (1) it is polarization independent, (2) it has gradient phase change, and (3) its response time is fast. It can be used for polarization converter and divergent LC lens. In addition, a new method for simultaneously measuring the phase retardation and optic axis of a compensation film is demonstrated using an axially-symmetric sheared polymer network liquid crystal. By overlaying a tested compensation film with a calibrated SPNLC cell between crossed polarizers, the optic axis and phase retardation value of the compensation film can be determined. This simple technique can be used for simultaneously measuring the optic axis and phase retardations of both A- and C-plates. These compensation films have been used extensively in wide-view LCD industry. Therefore, this method will make an important impact to the LCD industry.

Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics.

PubMed

Kahsay, Getu; Song, Huiying; Van Schepdael, Ann; Cabooter, Deirdre; Adams, Erwin

2014-01-01

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Profiling of phytohormones and their major metabolites in rice using binary solid-phase extraction and liquid chromatography-triple quadrupole mass spectrometry.

PubMed

Cao, Zhao-Yun; Sun, Li-Hua; Mou, Ren-Xiang; Zhang, Lin-Ping; Lin, Xiao-Yan; Zhu, Zhi-Wei; Chen, Ming-Xue

2016-06-17

A high-throughput method was developed using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) for the profiling and quantification of 43 phytohormones and their major metabolites, including auxins, abscisic acid, jasmonic acid, salicylic acid, cytokinins and gibberellins in a single sample extract. Considerable matrix effects (MEs) were observed (with most ME values in the range of 29%-84%, but maximum MEs of more than 115%, even up to 206%, existed) in sample extracts for most of the compounds studied. The application of the proposed binary solid-phase extraction using polymer anion and polymer cation exchange resins, was performed to purify 25 acidic and 18 alkaline phytohormones and their major metabolites prior to the LC-MS/MS analysis, which markedly reduced the MEs to acceptable levels, with ME values in the range of ±15%. Moreover, all of the isomers of cytokinins and their metabolites were fully separated on a sub-2μm particle C18 reverse-phase column with the optimized mobile phase consisting of methanol and 5mM ammonium formate. The method showed good linearity for all 43 analytes with regression coefficients (R(2))>0.991. Limits of detection ranged from 0.19 to 7.57 fmol for auxin, gibberellins, abscisic acid and their metabolites, 29.7 fmol for jasmonic acid, 18.1 fmol for salicylic acid, and from 0.03 to 0.31 fmol for cytokinins and their metabolites. The mean recoveries for all of the analytes were from 70.7 to 118.5%, and the inter-day precisions (n=6) were less than 18.7%, with intra-day precisions (n=6) within 25.4%. Finally, 20 compounds were successfully quantified in rice sample profiles using the proposed method, which will greatly facilitate the understanding of hormone-related regulatory networks that influence rice growth and development. To our knowledge, there are limited reports that measure this level of phytohormone species in rice samples using a single analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of in utero cannabis exposure by umbilical cord analysis.

PubMed

Kim, Jiyoung; de Castro, Ana; Lendoiro, Elena; Cruz-Landeira, Angelines; López-Rivadulla, Manuel; Concheiro, Marta

2018-04-01

According to the 2014 National Survey on Drug Use and Health, 5.3% of pregnant women smoked marijuana in the past month. This prevalence is expected to increase as a growing number of states and countries are now considering legalization. Although the umbilical cord is becoming a useful objective tool to detect in utero drug exposure, currently data about analytical methods and its utility to detect cannabis exposure are scarce. The objective of this work was to develop a method for the determination of Δ 9 -tetrahydrocannabinol (THC), 11-hydroxyTHC (THC-OH), 11-nor-9-carboxy-THC (THCCOOH), 8-β-11-dihydroxyTHC (THC-diOH), THC and THCCOOH glucuronides, and cannabidiol (CBD) in the umbilical cord by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with dual ionization source. Umbilical cord samples (0.5 g) were homogenized in methanol and extracted by solid-phase extraction. Reversed-phase chromatographic separation was performed in 14 minutes, and 2 transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1-10 to 20-200 ng/g), precision (4.1%-23.4%), accuracy (87.5%-111.4%), matrix effect (-54.8% to -5.8%), extraction efficiency (25%-45.6%), limits of detection and quantification (1-10 ng/g), and endogenous (n = 5) or exogenous interferences (not detected). The method was applied to 13 authentic samples from cannabis-exposed newborns, which meconium samples had tested positive for cannabis. Twelve cord specimens tested positive for THCCOOH-glucuronide (1.6-19.1 ng/g). We developed and validated a specific and sensitive method for the simultaneous determination of THC, its metabolites, including THC and THCCOOH glucuronides, and CBD in umbilical cord samples by LC-MS/MS. The analysis of authentic samples showed the usefulness of umbilical cord to detect cannabis in utero exposure. Copyright © 2017 John Wiley & Sons, Ltd.

Analytical methods in environmental effects-directed investigations of effluents.

PubMed

Hewitt, L Mark; Marvin, Chris H

2005-05-01

Effluent discharges are released into aquatic environments as complex mixtures for which there is commonly either no knowledge of the toxic components or a lack of understanding of how known toxicants interact with other effluent components. Effects-directed investigations consist of chemical extraction and iterative fractionation steps directed by a biological endpoint that is designed to permit the identification or characterization of the chemical classes or compounds in a complex mixture responsible for the observed biological activity. Our review of the literature on effects-directed analyses of effluents for non-mutagenic as well as mutagenic endpoints showed that common extraction and concentration methods have been used. Since the mid-1980s, the methods have evolved from the use of XAD resins to C18 solid-phase extraction (SPE). Blue cotton, blue rayon, and blue chitin have been used specifically for investigations of mutagenic activity where polycyclic compounds were involved or suspected. After isolation, subsequent fractionations have been accomplished using SPE or a high-pressure liquid chromatography (HPLC) system commonly fitted with a C18 reverse-phase column. Substances in active fractions are characterized by gas chromatography/mass spectrometry (GC-MS) and/or other spectrometric techniques for identification. LC-MS methods have been developed for difficult-to-analyze polar substances identified from effects-directed studies, but the potential for LC-MS to identify unknown polar compounds has yet to be fully realized. Salmonella-based assays (some miniaturized) have been coupled with fractionation methods for most studies aimed at identifying mutagenic fractions and chemical classes in mixtures. Effects-directed investigations of mutagens have focused mostly on drinking water and sewage, whereas extensive investigations of non-mutagenic effects have also included runoff, pesticides, and pulp mill effluents. The success of effects-directed investigations should be based on a realistic initial objective of each project. Identification of chemical classes associated with the measured biological endpoint is frequently achievable; however, confirmation of individual compounds is much more difficult and not always a necessary goal of effects-directed chemical analysis.

LC-MS/MS analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen.

PubMed

Trettin, Arne; Jordan, Jens; Tsikas, Dimitrios

2014-09-01

Paracetamol (acetaminophen, APAP) is a commonly used analgesic drug. Known paracetamol metabolites include the glucuronide, sulfate and mercapturate. N-Acetyl-benzoquinonimine (NAPQI) is considered the toxic intermediate metabolite of paracetamol. In vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites. For example, metabolomic studies in urine samples of APAP-treated mice revealed metabolites such as APAP-sulfate-APAP and APAP-S-S-APAP in addition to the classical phase II metabolites. Here, we report on the development and application of LC-MS and LC-MS/MS approaches to study reactions of unlabelled and (2)H-labelled APAP with unlabelled and (15)N-labelled nitrite in aqueous phosphate buffers (pH 7.4) upon their immersion into liquid nitrogen (-196°C). In mechanistic studies, these reactions were also studied in aqueous buffer prepared in (18)O-labelled water. LC-MS and LC-MS/MS analyses were performed on a reverse-phase material (C18) using gradient elution (2mM ammonium acetate/acetonitrile), in positive and negative electrospray mode. We identified a series of APAP metabolites including di-, tri- and tetra-APAP, mono- and di-nitro-APAP and nitric ester of di-APAP. Our study indicates that nitrite induces oxidation, i.e., polymerization and nitration of APAP, when buffered APAP/nitrite solutions are immersed into liquid nitrogen. These reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of NAPQI. Potassium ions and physiological saline but not thiols inhibit nitrite- and shock-freeze-induced reactions of paracetamol. The underlying mechanism likely involves in situ formation of NO2 radicals from nitrite secondary to profound pH reduction (down to pH 1) and disproportionation. Polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by LC-MS but not by GC-MS. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of metallic silver nanoparticles on the alignment and relaxation behaviour of liquid crystalline material in smectic C* phase

NASA Astrophysics Data System (ADS)

Vimal, Tripti; Kumar Gupta, Swadesh; Katiyar, Rohit; Srivastava, Atul; Czerwinski, Michal; Krup, Katarzyna; Kumar, Sandeep; Manohar, Rajiv

2017-09-01

The influence of silver nanoparticles dispersed in a Ferroelectric Liquid Crystal (FLC) on the properties of the resultant composite system has been investigated by thermal, electro-optical, and dielectric methods. We show that the concentration of thiol capped silver nanoparticles is a critical factor in governing the alignment of nanoparticles (NPs) in the host FLC. The orientation of NPs in composite samples affects the ordering of the LC (Liquid Crystal) phase and consequently changes the various phase transition temperatures of the host LC. Formation of self-assembled 2D (two dimensional) arrays of nanoparticles is observed for high concentration of dopant in the LC, oriented perpendicular to the direction of rubbing. We propose that the molecular interaction between the thiol capped NPs and LC molecules is the key factor behind such an arrangement of NPs. Orientation of NPs has affected the relaxation behaviour and various other material parameters, significantly. A noteworthy change in DC conductivity articulates our proposed idea of the formation of 2D array of NPs perpendicular to the direction of rubbing. This comprehensive study endorses the importance of dopant concentration in modifying the properties of the host LC material.

Analytical determination of virginiamycin drug residues in edible porcine tissues by LC-MS with confirmation by LC-MS/MS.

PubMed

Boison, Joe; Lee, Stephen; Gedir, Ron

2009-01-01

A liquid chromatographic-mass spectrometric (LC-MS) method was developed and validated for the determination and confirmation of virginiamycin (VMY) M1 residues in porcine liver, kidney, and muscle tissues at concentrations > or =2 ng/g. Porcine liver, kidney, or muscle tissue is homogenized with methanol-acetonitrile. After centrifugation, the supernatant is diluted with phosphate buffer and cleaned up on a C18 solid-phase extraction cartridge. VMY in the eluate is partitioned into chloroform and the aqueous upper layer is removed by aspiration. After evaporating the chloroform in the residual mixture to dryness, the dried extract is reconstituted in mobile phase and VMY is quantified by LC-MS. Any samples eliciting quantifiable levels of VMY M1 (i.e., at concentrations > or =2 ng/g) are subjected to confirmatory analysis by LC-MSIMS. VMY S1, a minor component of the VMY complex, is monitored but not quantified or confirmed.

Direct high-performance liquid chromatographic determination of the enantiomeric purity of levodopa and methyldopa: comparison with pharmacopoeial polarimetric methods.

PubMed

Dolezalová, M; Tkaczyková, M

1999-03-01

Chiral high-performance liquid chromatography was employed for determination of the enantiomeric purity of levodopa and methyldopa. The determination of D-DOPA in levodopa was accomplished using a chiral ligand-exchange chromatograpy with an ordinary C18 column and a chiral mobile phase containing N,N-dimethyl-L-phenylalanine and Cu(II) acetate or by means of LC on a teicoplanin column in conjunction with ethanol-water (65:35, v/v). Both methods gave good performance, however, the latter was faster and more convenient and suitable for routine analyses. For the determination of D-methyldopa a LC method based on the use of a teicoplanin column in polar organic mode with methanol-acetic acid-triethylamine (1,000:0.05:0.05, v/v/v) mobile phase was developed. The precision, accuracy, linearity and selectivity were satisfactory. In comparison with pharmacopoeial polarimetric methods (according to the European Pharmacopoeia and the Pharmacopoea Bohemoslovaca), the LC methods proved to be much more sensitive giving detection limits 0.04% of D-DOPA and 0.3% of D-methyldopa.

Investigation of Soman Adducts of Human Hemoglobin by Liquid Chromatography

DTIC Science & Technology

2004-04-01

acid standard, with fifteen primary amino acids , was used to evaluate and refine the chromatographic methods . An LC/MS/MS was used to analyze the non...several chromatographic conditions and stationary phases were used to create an LC/MS/MS method to directly analyze the amino acids , these studies...terminated because of a lack of resolution of the amino acid peaks. Also, initial attempts to develop an HPLC method to separate individual amino acids

Preparation and characterization of two new forced degradation products of letrozole and development of a stability-indicating RP-LC method for its determination.

PubMed

Elkady, Ehab Farouk; Fouad, Marwa Ahmed

2015-11-01

Two new hydrolytic products of letrozole were identified and proved to be true degradation products obtained by alkaline and acidic degradation of the drug. The acid and amide forms of the nitrile groups of letrozole were prepared and identified by IR and mass spectroscopic techniques. Subsequently, a simple, precise and selective stability-indicating RPLC method was developed and validated for the determination of letrozole in the presence of its degradation products. Letrozole was subjected to alkali and acid hydrolysis, oxidation, thermal degradation and photo-degradation. The degradation products were well isolated from letrozole. The chromatographic method was achieved using gradient elution of the drug and its degradation products on a reversed phase Zorbax Eclipse C18 column (100mm x 4.6mm, 3.5 μm) using a mobile phase consisting of 0.01M KH₂PO₄and methanol at a flow rate of 1 mL min⁻¹. Quantitation was achieved with UV detection at 230 nm. Linearity, accuracy and precision were found to be acceptable over the concentration range of 0.01-80 μgmL⁻¹. The proposed method was successfully applied to the determination of letrozole in bulk, plasma and in its pharmaceutical preparation.

Determination of 7alpha-OH cholesterol by LC-MS/MS: Application in assessing the activity of CYP7A1 in cholestatic minipigs

USDA-ARS?s Scientific Manuscript database

An LC-MS/MS method was developed and validated to determine 7alpha-OH cholesterol in liver microsome. This method was convenient and fast with high specificity and sensitivity. Briefly, a gradient elution was performed on a Synergi polar-C18 column (50 x 4.6mm i.d., 3microm). The mobile phase (cons...

Isolation and characterization of gluten protein types from wheat, rye, barley and oats for use as reference materials.

PubMed

Schalk, Kathrin; Lexhaller, Barbara; Koehler, Peter; Scherf, Katharina Anne

2017-01-01

Gluten proteins from wheat, rye, barley and, in rare cases, oats, are responsible for triggering hypersensitivity reactions such as celiac disease, non-celiac gluten sensitivity and wheat allergy. Well-defined reference materials (RM) are essential for clinical studies, diagnostics, elucidation of disease mechanisms and food analyses to ensure the safety of gluten-free foods. Various RM are currently used, but a thorough characterization of the gluten source, content and composition is often missing. However, this characterization is essential due to the complexity and heterogeneity of gluten to avoid ambiguous results caused by differences in the RM used. A comprehensive strategy to isolate gluten protein fractions and gluten protein types (GPT) from wheat, rye, barley and oat flours was developed to obtain well-defined RM for clinical assays and gluten-free compliance testing. All isolated GPT (ω5-gliadins, ω1,2-gliadins, α-gliadins, γ-gliadins and high- and low-molecular-weight glutenin subunits from wheat, ω-secalins, γ-75k-secalins, γ-40k-secalins and high-molecular-weight secalins from rye, C-hordeins, γ-hordeins, B-hordeins and D-hordeins from barley and avenins from oats) were fully characterized using analytical reversed-phase high-performance liquid chromatography (RP-HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequencing, electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS) and untargeted LC-MS/MS of chymotryptic hydrolyzates of the single GPT. Taken together, the analytical methods confirmed that all GPT were reproducibly isolated in high purity from the flours and were suitable to be used as RM, e.g., for calibration of LC-MS/MS methods or enzyme-linked immunosorbent assays (ELISAs).

Quantitative and comparative liquid chromatography-electrospray ionization-mass spectrometry analyses of hydrogen sulfide and thiol metabolites derivaitized with 2-iodoacetanilide isotopologues.

PubMed

Lee, Der-Yen; Huang, Wei-Chieh; Gu, Ting-Jia; Chang, Geen-Dong

2018-06-01

Hydrogen sulfide (H 2 S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H 2 S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H 2 S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). 13 C 6 -Labeled and label-free 2-IAN efficiently react with H 2 S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of H 2 S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13 C 6 -labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 μM linear ranges for DL-homocysteine and glutathione and 1 nM-1 μM linear ranges for l-cysteine and H 2 S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13 C 6 -2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide.

PubMed

Wang, Jian; Xu, Yu; Wen, Chunmei; Wang, Zhijian

2017-11-01

High-resolution mass spectrometry had been routinely used for structure identification of impurity. However, all LC-MS methods were based on a volatile mobile phase, and a non-volatile system is used in the official analytical method of United States Pharmacopoeia for cefpiramide which limited the use of mass spectrometry for structure characterization of the impurities. Here we presented the utilization of a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) with positive and negative modes of electrospray ionization for characterization of eight impurities in cefpiramide. Trap-free two-dimensional liquid chromatography and online desalting technique made it possible to characterize the impurity in cefpiramide in the condition of official standard, and the TIC chromatogram of LC-MS was in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities, which could further improve the method of official monographs in pharmacopoeias. Each peak separated by the non-volatile mobile phase was trapped by a 20 μL quantitative loop then transferred into a system with a volatile mobile phase connected to a MS detector. In the first dimension, the column was Kromasil C 8 analytical column (250 mm × 4.6 mm, 5 μm) with a non-volatile salt mobile phase at the flow rate of 0.8 mL min -1 . In the second dimension, the column was Shimadzu Shim-pack GISS C 18 (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase at the flow rate of 0.3 mL min -1 . Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely. The fragmentation behavior of cefpiramide and its eight impurities were studied. The structures of eight impurities in cefpiramide drug substance were deduced based on the HPLC-MS n data, in which seven impurities were novel impurities. The forming mechanisms of degradation products in cefpiramide were also studied. Copyright © 2017 Elsevier B.V. All rights reserved.

A Method for LC-MS/MS Profiling of Coumarins in Zanthoxylum zanthoxyloides (Lam.) B. Zepernich and Timler Extracts and Essential Oils.

PubMed

Tine, Yoro; Renucci, Franck; Costa, Jean; Wélé, Alassane; Paolini, Julien

2017-01-22

The metabolites from the coumarin class, present in tissues of plants belonging mainly to the Rutaceae and Apiaceae families, included compounds with high chemical diversity such as simple coumarins and furocoumarins. These health-promoting components are recognized for their valuable biological activities in herbal preparations but also for their phototoxic effects. In this work, a targeted liquid chromatography (LC) coupled with tandem mass spectrometry (MS²) was developed for the screening of 39 reference standards of coumarins and furocoumarins in essential oils and plant extracts. Chromatographic separation was accomplished on reversed phase column using water/acetonitrile as the mobile phase and detection was performed on a hybrid QqQ/linear ion trap spectrometer fitted with an atmospheric pressure chemical ionization (APCI) source operating in positive ion mode. This analytical approach was applied to investigate the coumarin compositions of fruit essential oils and methanolic extracts obtained from separated parts (fruit, leaf, stem, trunk, and root) of Zanthoxylum zanthoxyloides . Ten coumarins and six furanocoumarins were reported in this species and data analyses were used to assess the suitability of these compounds to the metabolomics-based differentiation of plant organs. The quantification criteria of the metabolites in extract samples included linearity, limit of quantification, limit of detection, and matrix effect were validated. As reported for other species of the Rutaceae family, the concentration of coumarins was drastically higher in Z. zanthoxyloides fruits than in other plant organs.

Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography-electrospray ionization-mass spectrometry.

PubMed

Ho, Jenny T C; White, Jim F; Grisshammer, Reinhard; Hess, Sonja

2008-05-01

The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)-MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI-MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography-ESI-MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC-ESI-tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.

Dioctyl sulfosuccinate analysis in near-shore Gulf of Mexico water by direct-injection liquid chromatography-tandem mass spectrometry.

PubMed

Mathew, Johnson; Schroeder, David L; Zintek, Lawrence B; Schupp, Caitlin R; Kosempa, Michael G; Zachary, Adam M; Schupp, George C; Wesolowski, Dennis J

2012-03-30

Dioctyl sulfosuccinate (DOSS) was a major component of the dispersants most used in the 2010 Deepwater Horizon Oil Spill incident response. This analytical method quantifies salt water DOSS concentrations to a reporting limit of 20 μg/L, which was below the United States Environmental Protection Agency's (U.S. EPA) 40 μg/L DOSS Aquatic Life Benchmark. DOSS in Gulf of Mexico water samples were analyzed by direct-injection reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample preparation with 50% acetonitrile (ACN) enabled quantitative transfer of DOSS and increased DOSS response 20-fold by reducing aggregation. This increased sensitivity enabled the detection of a confirmatory transition over the calibration range of 10-200 μg/L. U.S. EPA Region 5 and Region 6 laboratories analyzed hundreds of near-shore surface Gulf of Mexico water samples, none contained more than the 20 ppb reporting limit. The matrix spike DOSS/deuterated surrogate (DOSS-D34) correlation of determination varied with mobile phase modifier (ammonium formate R(2)=0.95 and formic acid R(2)=0.27). Using ammonium formate, DOSS-D34 accurately measured DOSS matrix effect. The near-shore sodium concentrations varied more than 10,000-fold, but were not strongly correlated with DOSS recovery. DOSS detection by LC-MS/MS enabled rapid analysis which was valuable in guiding incident response. Published by Elsevier B.V.

The Prognostic Value of Epithelial Membrane Protein 1 (EMP-1) in Patients with Laryngeal Carcinoma

PubMed Central

Liu, Chang; Wei, Xiaojun; Li, Feng; Wang, Li; Ruan, Xinjian; Jia, Jia; Zhang, Xia

2017-01-01

Background In the present study, we aimed to investigate the prognostic value of epithelial membrane protein 1 (EMP-1) gene in patients diagnosed with laryngeal carcinoma (LC). Material/Methods Patients who were pathologically diagnosed with LC were enrolled in the present study. The expression levels of EMP-1 in tumor tissues and corresponding normal tissues collected from the LC patients were detected by semi-reverse transcriptase polymerase chain reaction (semi-RT-PCR). Chi-square analysis was used to evaluate the relationship between EMP-1 expression level and clinical characteristics. Survival analysis for the study population was analyzed by Kaplan-Meier method with log rank test. Additionally, Cox regression model was applied to evaluate the prognostic value of EMP-1 in LC patients. Results 106 LC patients, including 55 men and 51 women, were enrolled in the present study. Semi-RT-PCR demonstrated that the expression level of EMP-1 was decreased in tumor tissues, compared with adjacent normal tissues (p<0.001). Moreover, the level was significantly associated with lymph node metastasis, histological grade, and clinical stage (p<0.05 for all). In addition, low levels of EMP-1 was significantly correlated with poor survival rate (log rank test, p=0.020). Cox regression analysis indicated that EMP-1 was an independent marker for LC prognosis (HR=2.755, 95% CI=1.123–6.760, p=0.027). Conclusions The abnormal expression of EMP-1 may be associated with progression of LC and the gene may act as a prognostic marker for LC. PMID:28779068

The Prognostic Value of Epithelial Membrane Protein 1 (EMP-1) in Patients with Laryngeal Carcinoma.

PubMed

Liu, Chang; Wei, Xiaojun; Li, Feng; Wang, Li; Ruan, Xinjian; Jia, Jia; Zhang, Xia

2017-08-05

BACKGROUND In the present study, we aimed to investigate the prognostic value of epithelial membrane protein 1 (EMP-1) gene in patients diagnosed with laryngeal carcinoma (LC). MATERIAL AND METHODS Patients who were pathologically diagnosed with LC were enrolled in the present study. The expression levels of EMP-1 in tumor tissues and corresponding normal tissues collected from the LC patients were detected by semi-reverse transcriptase polymerase chain reaction (semi-RT-PCR). Chi-square analysis was used to evaluate the relationship between EMP-1 expression level and clinical characteristics. Survival analysis for the study population was analyzed by Kaplan-Meier method with log rank test. Additionally, Cox regression model was applied to evaluate the prognostic value of EMP-1 in LC patients. RESULTS 106 LC patients, including 55 men and 51 women, were enrolled in the present study. Semi-RT-PCR demonstrated that the expression level of EMP-1 was decreased in tumor tissues, compared with adjacent normal tissues (p<0.001). Moreover, the level was significantly associated with lymph node metastasis, histological grade, and clinical stage (p<0.05 for all). In addition, low levels of EMP-1 was significantly correlated with poor survival rate (log rank test, p=0.020). Cox regression analysis indicated that EMP-1 was an independent marker for LC prognosis (HR=2.755, 95% CI=1.123-6.760, p=0.027). CONCLUSIONS The abnormal expression of EMP-1 may be associated with progression of LC and the gene may act as a prognostic marker for LC.

High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry

PubMed Central

Li, Xingnan; Franke, Adrian A.

2015-01-01

An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2′-deoxycytidine (5MedC) to 2′-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC–ESI-MS/MS. The cost-effective internal standards 15N3-dC and 15N5-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4 femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring. PMID:21843675

Determination of Glyphosate, Maleic Hydrazide, Fosetyl Aluminum, and Ethephon in Grapes by Liquid Chromatography/Tandem Mass Spectrometry.

PubMed

Chamkasem, Narong

2017-08-30

A simple high-throughput liquid chromatography/tandem mass spectrometry (LC-MS-MS) method was developed for the determination of maleic hydrazide, glyphosate, fosetyl aluminum, and ethephon in grapes using a reversed-phase column with weak anion-exchange and cation-exchange mixed mode. A 5 g test portion was shaken with 50 mM HOAc and 10 mM Na 2 EDTA in 1/3 (v/v) MeOH/H 2 O for 10 min. After centrifugation, the extract was passed through an Oasis HLB cartridge to retain suspended particulates and nonpolar interferences. The final solution was injected and directly analyzed in 17 min by LC-MS-MS. Two MS-MS transitions were monitored in the method for each target compound to achieve true positive identification. Four isotopically labeled internal standards corresponding to each analyte were used to correct for matrix suppression effects and/or instrument signal drift. The linearity of the detector response was demonstrated in the range from 10 to 1000 ng/mL for each analyte with a coefficient of determination (R 2 ) of ≥0.995. The average recovery for all analytes at 100, 500, and 2000 ng/g (n = 5) ranged from 87 to 111%, with a relative standard deviation of less than 17%. The estimated LOQs for maleic hydrazide, glyphosate, fosetyl-Al, and ethephon were 38, 19, 29, and 34 ng/g, respectively.

A simple and inexpensive on-column frit fabrication method for fused-silica capillaries for increased capacity and versatility in LC-MS/MS applications.

PubMed

Wang, Ling-Chi; Okitsu, Cindy Yen; Kochounian, Harold; Rodriguez, Anthony; Hsieh, Chih-Lin; Zandi, Ebrahim

2008-05-01

A modified sol-gel method for a one-step on-column frit preparation for fused-silica capillaries and its utility for peptide separation in LC-MS/MS is described. This method is inexpensive, reproducible, and does not require specialized equipments. Because the frit fabrication process does not damage polyimide coating, the frit-fabricated column can be tightly connected on-line for high pressure LC. These columns can replace any capillary liquid transfer tubing without any specialized connections up-stream of a spray tip column. Therefore multiple columns with different phases can be connected in series for one- or multiple-dimensional chromatography.

A LC-MS method allowing the analysis of HMX and RDX present at the picogram level in natural aqueous samples without a concentration step.

PubMed

Vigneau, Olivier; Machuron-Mandard, Xavier

2009-03-15

The introduction of chloroform into the nebulising gas of a LC/MS electrospray interface (ESI), in a perfectly controlled way, leads to the formation of intense adducts ([M+Cl](-)) when a mobile phase containing HMX (1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane or octogen) and RDX (1,3,5-trintro-1,3,5-triazacyclohexane or hexogen) is eluted. This LC/MS method allows the direct analysis of aqueous samples containing HMX and RDX at the pictogram level without a concentration step. The method is used to determine HMX and RDX concentrations in ground water samples from a military site.

Lattice-patterned LC-polymer composites containing various nanoparticles as additives

PubMed Central

2012-01-01

In this study, we show the effect of various nanoparticle additives on phase separation behavior of a lattice-patterned liquid crystal [LC]-polymer composite system and on interfacial properties between the LC and polymer. Lattice-patterned LC-polymer composites were fabricated by exposing to UV light a mixture of a prepolymer, an LC, and SiO2 nanoparticles positioned under a patterned photomask. This resulted in the formation of an LC and prepolymer region through phase separation. We found that the incorporation of SiO2 nanoparticles significantly affected the electro-optical properties of the lattice-patterned LC-polymer composites. This effect is a fundamental characteristic of flexible displays. The electro-optical properties depend on the size and surface functional groups of the SiO2 nanoparticles. Compared with untreated pristine SiO2 nanoparticles, which adversely affect the performance of LC molecules surrounded by polymer walls, SiO2 nanoparticles with surface functional groups were found to improve the electro-optical properties of the lattice-patterned LC-polymer composites by increasing the quantity of SiO2 nanoparticles. The surface functional groups of the SiO2 nanoparticles were closely related to the distribution of SiO2 nanoparticles in the LC-polymer composites, and they influenced the electro-optical properties of the LC molecules. It is clear from our work that the introduction of nanoparticles into a lattice-patterned LC-polymer composite provides a method for controlling and improving the composite's electro-optical properties. This technique can be used to produce flexible substrates for various flexible electronic devices. PMID:22222011

LC-QTOF-MS-based targeted metabolomics of arginine-creatine metabolic pathway-related compounds in plasma: application to identify potential biomarkers in pediatric chronic kidney disease.

PubMed

Benito, Sandra; Sánchez, Alicia; Unceta, Nora; Andrade, Fernando; Aldámiz-Echevarria, Luis; Goicolea, M Aránzazu; Barrio, Ramón J

2016-01-01

Chronic kidney disease (CKD) is a major epidemiologic problem which causes several disturbances in adults and in pediatrics. Despite being a worldwide public health problem, information available for CKD in the pediatric population is scarce. For that reason, an ion-pair reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) method has been developed and validated in order to analyze 16 amino acids, amino acid derivatives, and analogous compounds related to the arginine-creatine metabolic pathway that are suspicious of being increased or decreased in plasma from patients with CKD. The analytical method involved the addition of dithiothreitol, a reducing agent which reduces disulfide and thus giving total aminothiol concentration, as well as a simple precipitation of plasma proteins. Moreover, despite amino acids being usually derivatized to improve their retention time and to enhance their signal, for this method, an ion-pairing reagent was used, thus avoiding the need for derivatization. Subsequently, analysis of plasma from pediatric patients suffering from CKD and control pediatrics was carried out. As a result, glycine, citrulline, creatinine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were significantly increased in patients with CKD, regardless of their creatinine level, whereas in addition to these compounds dimethylglycine was also increased when CKD patients had plasma creatinine concentrations above 12 μg mL(-1), thus all are suggested as potential biomarkers for renal impairment.

A new, direct analytical method using LC-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils.

PubMed

Yamazaki, K; Ogiso, M; Isagawa, S; Urushiyama, T; Ukena, T; Kibune, N

2013-01-01

A new, direct analytical method for the determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) was developed. The targeted 3-MCPD esters included five types of monoester and 25 [corrected] types of diester. Samples (oils and fats) were dissolved in a mixture of tert-butyl methyl ether and ethyl acetate (4:1), purified using two solid-phase extraction (SPE) cartridges (C(18) and silica), then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Five monoesters and five diesters with the same fatty acid group could be separated and quantified. Pairs of 3-MCPD diesters carrying the same two different fatty acid groups, but at reversed positions (sn-1 and sn-2), could not be separated and so were expressed as a sum of both compounds. The limits of quantification (LOQs) were estimated to be between 0.02 to 0.08 mg kg(-1), depending on the types of 3-MCPD ester. Repeatability expressed as relative standard deviation (RSD(r)%) varied from 5.5% to 25.5%. The new method was shown to be applicable to various commercial edible oils and showed levels of 3-MCPD esters varying from 0.58 to 25.35 mg kg(-1). The levels of mono- and diesters ranged from 0.10 to 0.69 mg kg(-1) and from 0.06 to 16 mg kg(-1), respectively.

Analytical methodologies based on LC-MS/MS for monitoring selected emerging compounds in liquid and solid phases of the sewage sludge.

PubMed

Boix, C; Ibáñez, M; Fabregat-Safont, D; Morales, E; Pastor, L; Sancho, J V; Sánchez-Ramírez, J E; Hernández, F

2016-01-01

In this work, two analytical methodologies based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed for quantification of emerging pollutants identified in sewage sludge after a previous wide-scope screening. The target list included 13 emerging contaminants (EC): thiabendazole, acesulfame, fenofibric acid, valsartan, irbesartan, salicylic acid, diclofenac, carbamazepine, 4-aminoantipyrine (4-AA), 4-acetyl aminoantipyrine (4-AAA), 4-formyl aminoantipyrine (4-FAA), venlafaxine and benzoylecgonine. The aqueous and solid phases of the sewage sludge were analyzed making use of Solid-Phase Extraction (SPE) and UltraSonic Extraction (USE) for sample treatment, respectively. The methods were validated at three concentration levels: 0.2, 2 and 20 μg L(-1) for the aqueous phase, and 50, 500 and 2000 μg kg(-1) for the solid phase of the sludge. In general, the method was satisfactorily validated, showing good recoveries (70-120%) and precision (RSD < 20%). Regarding the limit of quantification (LOQ), it was below 0.1 μg L(-1) in the aqueous phase and below 50 μg kg(-1) in the solid phase for the majority of the analytes. The method applicability was tested by analysis of samples from a wider study on degradation of emerging pollutants in sewage sludge under anaerobic digestion. The key benefits of these methodologies are: • SPE and USE are appropriate sample procedures to extract selected emerging contaminants from the aqueous phase of the sewage sludge and the solid residue. • LC-MS/MS is highly suitable for determining emerging contaminants in both sludge phases. • Up to our knowledge, the main metabolites of dipyrone had not been studied before in sewage sludge.

Nano-liquid chromatography applied to enantiomers separation.

PubMed

Fanali, Salvatore

2017-02-24

This paper presents the state of the art concerning the separation of chiral compounds by means of nano-liquid chromatography (nano-LC). The enantiomers' separation and determination are a subject of fundamental importance in various application fields such as pharmaceutical industry, biomedicine, food, agrochemical etc. Nano-LC is a miniaturized chromatographic technique offering some advantages over conventional ones such as low consumption of mobile phase, sample volume and amount of chiral stationary phase, reduced costs etc. This is reported in the first part of the paper illustrating the features of the nano-LC. In addition, chiral resolution methods are briefly illustrated. Some chiral selectors, used in high-performance liquid chromatography have also been applied in nano-LC including cyclodextrins, glycopeptide antibiotics, modified polysaccharides etc. This is discussed in the second part of the review. Finally some examples of the applications available in literature are reported. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitation of the enantiomers of tramadol and its three main metabolites in human whole blood using LC-MS/MS.

PubMed

Haage, Pernilla; Kronstrand, Robert; Carlsson, Björn; Kugelberg, Fredrik C; Josefsson, Martin

2016-02-05

The analgesic drug tramadol and its metabolites are chiral compounds, with the (+)- and (-)-enantiomers showing different pharmacological and toxicological effects. This novel enantioselective method, based on LC-MS/MS in reversed phase mode, enabled measurement of the parent compound and its three main metabolites O-desmethyltramadol, N-desmethyltramadol and N,O-didesmethyltramadol simultaneously. Whole blood samples of 0.5g were fortified with internal standards (tramadol-(13)C-D3 and O-desmethyl-cis-tramadol-D6) and extracted under basic conditions (pH 11) by liquid-liquid extraction. Chromatography was performed on a chiral alpha-1-acid glycoprotein (AGP) column preceded by an AGP guard column. The mobile phase consisted of 0.8% acetonitrile and 99.2% ammonium acetate (20mM, pH 7.2). A post-column infusion with 0.05% formic acid in acetonitrile was used to enhance sensitivity. Quantitation as well as enantiomeric ratio measurements were covered by quality controls. Validation parameters for all eight enantiomers included selectivity (high), matrix effects (no ion suppression/enhancement), calibration model (linear, weight 1/X(2), in the range of 0.25-250ng/g), limit of quantitation (0.125-0.50ng/g), repeatability (2-6%) and intermediate precision (2-7%), accuracy (83-114%), dilution integrity (98-115%), carry over (not exceeding 0.07%) and stability (stable in blood and extract). The method was applied to blood samples from a healthy volunteer administrated a single 100mg dose and to a case sample concerning an impaired driver, which confirmed its applicability in human pharmacokinetic studies as well as in toxicological and forensic investigations. Copyright © 2015 Elsevier B.V. All rights reserved.

Increasing reconstruction quality of diffractive optical elements displayed with LC SLM

NASA Astrophysics Data System (ADS)

Cheremkhin, Pavel A.; Evtikhiev, Nikolay N.; Krasnov, Vitaly V.; Rodin, Vladislav G.; Starikov, Sergey N.

2015-03-01

Phase liquid crystal (LC) spatial light modulators (SLM) are actively used in various applications. However, majority of scientific applications require stable phase modulation which might be hard to achieve with commercially available SLM due to its consumer origin. The use of digital voltage addressing scheme leads to phase temporal fluctuations, which results in lower diffraction efficiency and reconstruction quality of displayed diffractive optical elements (DOE). Due to high periodicity of fluctuations it should be possible to use knowledge of these fluctuations during DOE synthesis to minimize negative effect. We synthesized DOE using accurately measured phase fluctuations of phase LC SLM "HoloEye PLUTO VIS" to minimize its negative impact on displayed DOE reconstruction. Synthesis was conducted with versatile direct search with random trajectory (DSRT) method in the following way. Before DOE synthesis begun, two-dimensional dependency of SLM phase shift on addressed signal level and time from frame start was obtained. Then synthesis begins. First, initial phase distribution is created. Second, random trajectory of consecutive processing of all DOE elements is generated. Then iterative process begins. Each DOE element sequentially has its value changed to one that provides better value of objective criterion, e.g. lower deviation of reconstructed image from original one. If current element value provides best objective criterion value then it left unchanged. After all elements are processed, iteration repeats until stagnation is reached. It is demonstrated that application of SLM phase fluctuations knowledge in DOE synthesis with DSRT method leads to noticeable increase of DOE reconstruction quality.

Lung autophagic response following exposure of mice to whole body irradiation, with and without amifostine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zois, Christos E.; Giatromanolaki, Alexandra; Kainulainen, Heikki

2011-01-07

Research highlights: {yields} We investigated the effect 6 Gy of WBI on the autophagic machinery of normal mouse lung. {yields} Irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. {yields} The membrane bound LC3A-II protein levels increased in the cytosolic fraction (not in the pellet), contrasting the patterns noted after starvation-induced autophagy. {yields} Administration of amifostine, reversed all the LC3A and p62 findings, suggesting protection of the normal autophagic function. -- Abstract: Purpose: The effect of ionizing irradiation on the autophagic response ofmore » normal tissues is largely unexplored. Abnormal autophagic function may interfere the protein quality control leading to cell degeneration and dysfunction. This study investigates its effect on the autophagic machinery of normal mouse lung. Methods and materials: Mice were exposed to 6 Gy of whole body {gamma}-radiation and sacrificed at various time points. The expression of MAP1LC3A/LC3A/Atg8, beclin-1, p62/sequestosome-1 and of the Bnip3 proteins was analyzed. Results: Following irradiation, the LC3A-I and LC3A-II protein levels increased significantly at 72 h and 7 days. Strikingly, LC3A-II protein was increased (5.6-fold at 7 days; p < 0.001) only in the cytosolic fraction, but remained unchanged in the membrane fraction. The p62 protein, was significantly increased in both supernatant and pellet fraction (p < 0.001), suggesting an autophagosome turnover deregulation. These findings contrast the patterns of starvation-induced autophagy up-regulation. Beclin-1 levels remained unchanged. The Bnip3 protein was significantly increased at 8 h, but it sharply decreased at 72 h (p < 0.05). Administration of amifostine (200 mg/kg), 30 min before irradiation, reversed all the LC3A and p62 findings on blots, suggesting restoration of the normal autophagic function. The LC3A and Beclin1 mRNA levels significantly declined following irradiation (p < 0.01), whereas Bnip3 levels increased. Conclusions: It is suggested that irradiation induces dysfunction of the autophagic machinery in normal lung, characterized by decreased transcription of the LC3A/Beclin-1 mRNA and accumulation of the LC3A, and p62 proteins. Whether this is due to defective maturation or to aberrant degradation of the autophagosomes requires further investigation.« less

Comprehensive solid-phase extraction of multitudinous bioactive peptides from equine plasma and urine for doping detection.

PubMed

Guan, Fuyu; Robinson, Mary A

2017-09-08

The ability to analyze biological samples for multitudinous exogenous peptides with a single analytical method is desired for doping control in horse racing. The key to achieving this goal is the capability of extracting all target peptides from the sample matrix. In the present study, theory of mixed-mode solid-phase extraction (SPE) of peptides from plasma is described, and a generic mixed-mode SPE procedure has been developed for recovering multitudinous exogenous peptides with remarkable sequence diversity, from equine plasma and urine in a single procedure. Both the theory and the developed SPE procedure have led to the development of a novel analytical method for comprehensive detection of multitudinous bioactive peptides in equine plasma and urine using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). Thirty nine bioactive peptides were extracted with strong anion-exchange mixed-mode SPE sorbent, separated on a reversed-phase C 18 column and detected by HRMS and data-dependent tandem mass spectrometry. The limit of detection (LOD) was 10-50 pg mL -1 in plasma for most of the peptides and 100 pg mL -1 for the remaining. For urine, LOD was 20-400 pg mL -1 for most of the peptides and 1-4 ng mL -1 for the others. In vitro degradation of the peptides in equine plasma and urine was examined at ambient temperature; the peptides except those with a D-amino acid at position 2 were unstable not only in plasma but also in urine. The developed method was successful in analysis of plasma and urine samples from horses administered dermorphin. Additionally, dermorphin metabolites were identified in the absence of reference standards. The developed SPE procedure and LC-HRMS method can theoretically detect virtually all peptides present at a sufficient concentration in a sample. New peptides can be readily included in the method to be detected without method re-development. The developed method also generates such data that can be retrospectively analyzed for peptides unknown at the time of sample analysis. It is the first generic analytical method for comprehensive detection of multitudinous exogenous peptides in biological samples, to the authors' knowledge. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel IMSL tunable phase shifter for HMSIW-LWA-fed rectangular patches based on nematic liquid crystal

NASA Astrophysics Data System (ADS)

Fu, JiaHui; Raheem, Odai H.

2017-07-01

A novel IMSL tunable phase shifter for HMSIW-LWA-fed rectangular patches based on liquid crystal technology is proposed. Rectangular patches are used as radiators for the opening sidewall of the waveguide and matched section part for a unit cell. The transition structure is added for enhancing the efficiency of HMSIW-LWA due to converting most input power to the leaky mode. The novel IMSL phase shifter is used for investigating the tunable dielectric characteristics of N-LC by applying an electric field to the LC cell, which is controlled by the orientation angle of the LC molecules. Theoretically, the orientation angle is derived and solved numerically with the accurate method. As a result, the HMSIW-LWA can be tuned up to ± 25° for a fixed frequency by tuning the nematic LC with applied voltage from 0 to 20 V. In addition, the realized gain changed from 6 to 9.4 dB for a fixed tuned frequency, and 46° steerable for rest main beams range of the HMSIW-LWA in both forward and backward directions.

Study of different HILIC, mixed-mode, and other aqueous normal-phase approaches for the liquid chromatography/mass spectrometry-based determination of challenging polar pesticides.

PubMed

Vass, Andrea; Robles-Molina, José; Pérez-Ortega, Patricia; Gilbert-López, Bienvenida; Dernovics, Mihaly; Molina-Díaz, Antonio; García-Reyes, Juan F

2016-07-01

The aim of the study was to evaluate the performance of different chromatographic approaches for the liquid chromatography/mass spectrometry (LC-MS(/MS)) determination of 24 highly polar pesticides. The studied compounds, which are in most cases unsuitable for conventional LC-MS(/MS) multiresidue methods were tested with nine different chromatographic conditions, including two different hydrophilic interaction liquid chromatography (HILIC) columns, two zwitterionic-type mixed-mode columns, three normal-phase columns operated in HILIC-mode (bare silica and two silica-based chemically bonded columns (cyano and amino)), and two standard reversed-phase C18 columns. Different sets of chromatographic parameters in positive (for 17 analytes) and negative ionization modes (for nine analytes) were examined. In order to compare the different approaches, a semi-quantitative classification was proposed, calculated as the percentage of an empirical performance value, which consisted of three main features: (i) capacity factor (k) to characterize analyte separation from the void, (ii) relative response factor, and (iii) peak shape based on analytes' peak width. While no single method was able to provide appropriate detection of all the 24 studied species in a single run, the best suited approach for the compounds ionized in positive mode was based on a UHPLC HILIC column with 1.8 μm particle size, providing appropriate results for 22 out of the 24 species tested. In contrast, the detection of glyphosate and aminomethylphosphonic acid could only be achieved with a zwitterionic-type mixed-mode column, which proved to be suitable only for the pesticides detected in negative ion mode. Finally, the selected approach (UHPLC HILIC) was found to be useful for the determination of multiple pesticides in oranges using HILIC-ESI-MS/MS, with limits of quantitation in the low microgram per kilogram in most cases. Graphical Abstract HILIC improves separation of multiclass polar pesticides.

Robust trace analysis of polar (C2-C8) perfluorinated carboxylic acids by liquid chromatography-tandem mass spectrometry: method development and application to surface water, groundwater and drinking water.

PubMed

Janda, Joachim; Nödler, Karsten; Brauch, Heinz-Jürgen; Zwiener, Christian; Lange, Frank T

2018-03-19

A simple and robust analytical method for the determination of perfluorinated carboxylic acids (PFCAs) with C 2 to C 8 chains, based on solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), was developed, validated and applied to tap water, groundwater and surface water. Two stationary phases for LC (Obelisc N and Kinetex C 18 ) and two materials with weak anion-exchange properties for SPE (Strata X-AW and Oasis WAX) were evaluated. Robust separation and retention was achieved with the reversed phase column and an acidic eluent. Quantitative extraction recoveries were generally achieved for PFCAs with C > 3, but extraction efficiencies were different for the two shortest chained analytes: 36 to 114% of perfluoropropanoate (PFPrA) and 14 to 99% of trifluoroacetate (TFA) were recovered with Strata X-AW, while 93 to 103% of PFPrA and 40 to 103% of TFA were recovered with Oasis WAX. The sample pH was identified as a key parameter in the extraction process. One-step elution-filtration was introduced in the workflow, in order to remove sorbent particles and minimise sample preparation steps. Validation resulted in limits of quantification for all PFCAs between 0.6 and 26 ng/L. Precision was between 0.7 and 15% and mean recoveries ranged from 83 to 107%. In groundwater samples from sites impacted by per- and polyfluoroalkyl substances (PFASs), PFCA concentrations ranged from 0.056 to 2.2 μg/L. TFA and perfluorooctanoate were the predominant analytes. TFA, however, revealed a more ubiquitous occurrence and was found in concentrations between 0.045 and 17 μg/L in drinking water, groundwater and surface water, which were not impacted by PFASs.

Automated Online Solid-Phase Derivatization for Sensitive Quantification of Endogenous S-Nitrosoglutathione and Rapid Capture of Other Low-Molecular-Mass S-Nitrosothiols.

PubMed

Wang, Xin; Garcia, Carlos T; Gong, Guanyu; Wishnok, John S; Tannenbaum, Steven R

2018-02-06

S-Nitrosothiols (RSNOs) constitute a circulating endogenous reservoir of nitric oxide and have important biological activities. In this study, an online coupling of solid-phase derivatization (SPD) with liquid chromatography-mass spectrometry (LC-MS) was developed and applied in the analysis of low-molecular-mass RSNOs. A derivatizing-reagent-modified polymer monolithic column was prepared and adapted for online SPD-LC-MS. Analytes from the LC autosampler flowed through the monolithic column for derivatization and then directly into the LC-MS for analysis. This integration of the online derivatization, LC separation, and MS detection facilitated system automation, allowing rapid, laborsaving, and sensitive detection of RSNOs. S-Nitrosoglutathione (GSNO) was quantified using this automated online method with good linearity (R 2 = 0.9994); the limit of detection was 0.015 nM. The online SPD-LC-MS method has been used to determine GSNO levels in mouse samples, 138 ± 13.2 nM of endogenous GSNO was detected in mouse plasma. Besides, the GSNO concentrations in liver (64.8 ± 11.3 pmol/mg protein), kidney (47.2 ± 6.1 pmol/mg protein), heart (8.9 ± 1.8 pmol/mg protein), muscle (1.9 ± 0.3 pmol/mg protein), hippocampus (5.3 ± 0.9 pmol/mg protein), striatum (6.7 ± 0.6 pmol/mg protein), cerebellum (31.4 ± 6.5 pmol/mg protein), and cortex (47.9 ± 4.6 pmol/mg protein) were also successfully quantified. When the derivatization was performed within 8 min, followed by LC-MS detection, samples could be rapidly analyzed compared with the offline manual method. Other low-molecular-mass RSNOs, such as S-nitrosocysteine and S-nitrosocysteinylglycine, were captured by rapid precursor-ion scanning, showing that the proposed method is a potentially powerful tool for capture, identification, and quantification of RSNOs in biological samples.

PEGylated-nanoliposomal clusterin for amyloidogenic light chain-induced endothelial dysfunction.

PubMed

Guzman-Villanueva, Diana; Migrino, Raymond Q; Truran, Seth; Karamanova, Nina; Franco, Daniel A; Burciu, Camelia; Senapati, Subhadip; Nedelkov, Dobrin; Hari, Parameswaran; Weissig, Volkmar

2018-06-01

Light chain (AL) amyloidosis is a disease associated with significant morbidity and mortality arising from multi-organ injury induced by amyloidogenic light chain proteins (LC). There is no available treatment to reverse the toxicity of LC. We previously showed that chaperone glycoprotein clusterin (CLU) and nanoliposomes (NL), separately, restore human microvascular endothelial function impaired by LC. In this work, we aim to prepare PEGylated-nanoliposomal clusterin (NL-CLU) formulations that could allow combined benefit against LC while potentially enabling efficient delivery to microvascular tissue, and test efficacy on human arteriole endothelial function. NL-CLU was prepared by a conjugation reaction between the carboxylated surface of NL and the primary amines of the CLU protein. NL were made of phosphatidylcholine (PC), cholesterol (Chol) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG 2000 carboxylic acid) at 70:25:5 mol%. The protective effect of NL-CLU was tested by measuring the dilation response to acetylcholine and papaverine in human adipose arterioles exposed to LC. LC treatment significantly reduced the dilation response to acetylcholine and papaverine; co-treatment of LC with PEGylated-nanoliposomal CLU or free CLU restored the dilator response. NL-CLU is a feasible and promising approach to reverse LC-induced endothelial damage.

Improved LC-MS/MS method for the quantification of hepcidin-25 in clinical samples.

PubMed

Abbas, Ioana M; Hoffmann, Holger; Montes-Bayón, María; Weller, Michael G

2018-06-01

Mass spectrometry-based methods play a crucial role in the quantification of the main iron metabolism regulator hepcidin by singling out the bioactive 25-residue peptide from the other naturally occurring N-truncated isoforms (hepcidin-20, -22, -24), which seem to be inactive in iron homeostasis. However, several difficulties arise in the MS analysis of hepcidin due to the "sticky" character of the peptide and the lack of suitable standards. Here, we propose the use of amino- and fluoro-silanized autosampler vials to reduce hepcidin interaction to laboratory glassware surfaces after testing several types of vials for the preparation of stock solutions and serum samples for isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS). Furthermore, we have investigated two sample preparation strategies and two chromatographic separation conditions with the aim of developing a LC-MS/MS method for the sensitive and reliable quantification of hepcidin-25 in serum samples. A chromatographic separation based on usual acidic mobile phases was compared with a novel approach involving the separation of hepcidin-25 with solvents at high pH containing 0.1% of ammonia. Both methods were applied to clinical samples in an intra-laboratory comparison of two LC-MS/MS methods using the same hepcidin-25 calibrators with good correlation of the results. Finally, we recommend a LC-MS/MS-based quantification method with a dynamic range of 0.5-40 μg/L for the assessment of hepcidin-25 in human serum that uses TFA-based mobile phases and silanized glass vials. Graphical abstract Structure of hepcidin-25 (Protein Data Bank, PDB ID 2KEF).

Optogenetic silencing of locus coeruleus activity in mice impairs cognitive flexibility in an attentional set-shifting task

PubMed Central

Janitzky, Kathrin; Lippert, Michael T.; Engelhorn, Achim; Tegtmeier, Jennifer; Goldschmidt, Jürgen; Heinze, Hans-Jochen; Ohl, Frank W.

2015-01-01

The locus coeruleus (LC) is the sole source of noradrenergic projections to the cortex and essential for attention-dependent cognitive processes. In this study we used unilateral optogenetic silencing of the LC in an attentional set-shifting task (ASST) to evaluate the influence of the LC on prefrontal cortex-dependent functions in mice. We expressed the halorhodopsin eNpHR 3.0 to reversibly silence LC activity during task performance, and found that silencing selectively impaired learning of those parts of the ASST that most strongly rely on cognitive flexibility. In particular, extra-dimensional set-shifting (EDS) and reversal learning was impaired, suggesting an involvement of the medial prefrontal cortex (mPFC) and the orbitofrontal cortex. In contrast, those parts of the task that are less dependent on cognitive flexibility, i.e., compound discrimination (CD) and the intra-dimensional shifts (IDS) were not affected. Furthermore, attentional set formation was unaffected by LC silencing. Our results therefore suggest a modulatory influence of the LC on cognitive flexibility, mediated by different frontal networks. PMID:26582980

Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

PubMed Central

Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

2012-01-01

Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti-tumor mechanism of ORI as treatment for prostate cancer. PMID:22745580

Phase retrieval with the reverse projection method in the presence of object's scattering

NASA Astrophysics Data System (ADS)

Wang, Zhili; Gao, Kun; Wang, Dajiang

2017-08-01

X-ray grating interferometry can provide substantially increased contrast over traditional attenuation-based techniques in biomedical applications, and therefore novel and complementary information. Recently, special attention has been paid to quantitative phase retrieval in X-ray grating interferometry, which is mandatory to perform phase tomography, to achieve material identification, etc. An innovative approach, dubbed ;Reverse Projection; (RP), has been developed for quantitative phase retrieval. The RP method abandons grating scanning completely, and is thus advantageous in terms of higher efficiency and reduced radiation damage. Therefore, it is expected that this novel method would find its potential in preclinical and clinical implementations. Strictly speaking, the reverse projection method is applicable for objects exhibiting only absorption and refraction. In this contribution, we discuss the phase retrieval with the reverse projection method for general objects with absorption, refraction and scattering simultaneously. Especially, we investigate the influence of the object's scattering on the retrieved refraction signal. Both theoretical analysis and numerical experiments are performed. The results show that the retrieved refraction signal is the product of object's refraction and scattering signals for small values. In the case of a strong scattering, the reverse projection method cannot provide reliable phase retrieval. Those presented results will guide the use of the reverse projection method for future practical applications, and help to explain some possible artifacts in the retrieved images and/or reconstructed slices.

Development and Multi-laboratory Verification of U.S. EPA Method 540 for the Analysis of Drinking Water Contaminants by Solid Phase Extraction-LC/MS/MS

EPA Science Inventory

A drinking water method for 12 chemicals, predominately pesticides, is presented that addresses the occurrence monitoring needs of the U.S. Environmental Protection Agency (EPA) for a future Unregulated Contaminant Monitoring Regulation (UCMR). The method employs solid phase ext...

Classification of LC columns based on the QSRR method and selectivity toward moclobemide and its metabolites.

PubMed

Plenis, Alina; Olędzka, Ilona; Bączek, Tomasz

2013-05-05

This paper focuses on a comparative study of the column classification system based on the quantitative structure-retention relationships (QSRR method) and column performance in real biomedical analysis. The assay was carried out for the LC separation of moclobemide and its metabolites in human plasma, using a set of 24 stationary phases. The QSRR models established for the studied stationary phases were compared with the column test performance results under two chemometric techniques - the principal component analysis (PCA) and the hierarchical clustering analysis (HCA). The study confirmed that the stationary phase classes found closely related by the QSRR approach yielded comparable separation for moclobemide and its metabolites. Therefore, the QSRR method could be considered supportive in the selection of a suitable column for the biomedical analysis offering the selection of similar or dissimilar columns with a relatively higher certainty. Copyright © 2013 Elsevier B.V. All rights reserved.

Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC-MS/MS.

PubMed

Dettmer, Katja; Stevens, Axel P; Fagerer, Stephan R; Kaspar, Hannelore; Oefner, Peter J

2012-01-01

Two mass spectrometry-based methods for the quantitative analysis of free amino acids are described. The first method uses propyl chloroformate/propanol derivatization and gas chromatography-quadrupole mass spectrometry (GC-qMS) analysis in single-ion monitoring mode. Derivatization is carried out directly in aqueous samples, thereby allowing automation of the entire procedure, including addition of reagents, extraction, and injection into the GC-MS. The method delivers the quantification of 26 amino acids. The isobaric tagging for relative and absolute quantification (iTRAQ) method employs the labeling of amino acids with isobaric iTRAQ tags. The tags contain two different cleavable reporter ions, one for the sample and one for the standard, which are detected by fragmentation in a tandem mass spectrometer. Reversed-phase liquid chromatography of the labeled amino acids is performed prior to mass spectrometric analysis to separate isobaric amino acids. The commercial iTRAQ kit allows for the analysis of 42 physiological amino acids with a respective isotope-labeled standard for each of these 42 amino acids.

Monosialoganglioside-Containing Nanoliposomes Restore Endothelial Function Impaired by AL Amyloidosis Light Chain Proteins.

PubMed

Franco, Daniel A; Truran, Seth; Weissig, Volkmar; Guzman-Villanueva, Diana; Karamanova, Nina; Senapati, Subhadip; Burciu, Camelia; Ramirez-Alvarado, Marina; Blancas-Mejia, Luis M; Lindsay, Stuart; Hari, Parameswaran; Migrino, Raymond Q

2016-06-13

Light chain amyloidosis (AL) is associated with high mortality, especially in patients with advanced cardiovascular involvement. It is caused by toxicity of misfolded light chain proteins (LC) in vascular, cardiac, and other tissues. There is no treatment to reverse LC tissue toxicity. We tested the hypothesis that nanoliposomes composed of monosialoganglioside, phosphatidylcholine, and cholesterol (GM1 ganglioside-containing nanoliposomes [NLGM1]) can protect against LC-induced human microvascular dysfunction and assess mechanisms behind the protective effect. The dilator responses of ex vivo abdominal adipose arterioles from human participants without AL to acetylcholine and papaverine were measured before and after exposure to LC (20 μg/mL) with or without NLGM1 (1:10 ratio for LC:NLGM1 mass). Human umbilical vein endothelial cells were exposed for 18 to 20 hours to vehicle, LC with or without NLGM1, or NLGM1 and compared for oxidative and nitrative stress response and cellular viability. LC impaired arteriole dilator response to acetylcholine, which was restored by co-treatment with NLGM1. LC decreased endothelial cell nitric oxide production and cell viability while increasing superoxide and peroxynitrite; these adverse effects were reversed by NLGM1. NLGM1 increased endothelial cell protein expression of antioxidant enzymes heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 and increased nuclear factor, erythroid 2 like 2 (Nrf-2) protein. Nrf-2 gene knockdown reduced antioxidant stress response and reversed the protective effects of NLGM1. NLGM1 protects against LC-induced human microvascular endothelial dysfunction through increased nitric oxide bioavailability and reduced oxidative and nitrative stress mediated by Nrf-2-dependent antioxidant stress response. These findings point to a potential novel therapeutic approach for light chain amyloidosis. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Quantification of phenolic acids and their methylates, glucuronides, sulfates and lactones metabolites in human plasma by LC-MS/MS after oral ingestion of soluble coffee.

PubMed

Marmet, Cynthia; Actis-Goretta, Lucas; Renouf, Mathieu; Giuffrida, Francesca

2014-01-01

Chlorogenic acids and derivatives like phenolic acids are potentially bioactive phenolics, which are commonly found in many foods. Once absorbed, chlorogenic and phenolic acids are highly metabolized by the intestine and the liver, producing glucuronidated and/or sulphated compounds. These metabolites were analyzed in human plasma using a validated liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method. After protein precipitation, phenolic acids and their metabolites were extracted by using ethanol and chromatographic separation was achieved by reversed-phase using an Acquity UPLC BEH C18 column combined with a gradient elution system using 1% acetic acid aqueous solution and 1% acetic acid with 100% acetonitrile. The method was able to quantify 56 different compounds including 24 phenolic acids, 4 lactones, 15 sulfates and 13 glucuronides metabolites between 5 and 1000nM in plasma for most of them, except for m-dihydrocoumaric acid, 5-ferulloylquinic-glucuronide, 4-methoxycinnamic acid, 3-phenylpropionic acid, 3-(4-methoxyphenyl)propionic acid (25 to 1000nM) and p-dihydrocoumaric acid (50-1000nM). Values of repeatability and intermediate reproducibility were below 15% of deviation in general, and maximum 20% for the lowest concentrations. The validated method was successfully applied to quantify phenolic acids and their metabolites in plasma obtained after oral ingestion of soluble coffee. In conclusion, the developed and validated method is proved to be very sensitive, accurate and precise for the quantification of these possible dietary phenols. Copyright © 2013 Elsevier B.V. All rights reserved.

Improvement of Nicotinic Acid and Nicotinamide Analysis in Meats and Meat Products by HPLC and LC-MS/MS with Solid-Phase Extraction.

PubMed

Hiki, Asako; Yamajima, Yukiko; Uematsu, Yoko

2016-01-01

A method for nicotinic acid (NA) and nicotinamide (NAA) analysis in meats was developed. NA and NAA were extracted from meats or meat products with metaphosphate aqueous solution. The extract was cleaned up with an Oasis MCX cartridge. The cartridge was washed with 2% acetic acid (v/v) and acetic acid-methanol solution. NA and NAA were eluted with ammonia-methanol solution. NA and NAA in the eluate were chromatographed on a Scherzo SM-C18 (3.0×150 mm, 3.0 μm) column with 20 mmol/L ammonium acetate containing 0.1% acetic acid-acetonitrile (97 : 3) as a mobile phase and were monitored at 261 nm. Quantification was performed by LC and LC-MS/MS. Calibration curves showed high linearity (correlation coefficient>0.998) between 1-25 μg/mL for LC and LC-MS/MS. Recoveries were 84-108% (CV≦5.8%) by HPLC and 79-105% (CV≦9.0%) by LC-MS/MS. The limit of quantitation for NA was 0.005-0.01 g/kg and that for NAA was 0.01-0.02 g/kg.

Synthesis, Self-Assembly, and Drug-Release Properties of New Amphipathic Liquid Crystal Polycarbonates

PubMed Central

Xie, Yujiao; Liu, Xiaofeng; Hu, Zhuang; Hou, Zhipeng; Chen, Zhangpei; Hu, Jianshe; Yang, Liqun

2018-01-01

New amphiphilic liquid crystal (LC) polycarbonate block copolymers containing side-chain cholesteryl units were synthesized. Their structure, thermal stability, and LC phase behavior were characterized with Fourier transform infrared (FT-IR) spectrum, 1H NMR, gel permeation chromatographic (GPC), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), polarizing optical microscope (POM), and XRD methods. The results demonstrated that the LC copolymers showed a double molecular arrangement of a smectic A phase at room temperature. With the elevating of LC unit content in such LC copolymers, the corresponding properties including decomposition temperature (Td), glass temperature (Tg), and isotropic temperature (Ti) increased. The LC copolymers showed pH-responsive self-assembly behavior under the weakly acidic condition, and with more side-chain LC units, the self-assembly process was faster, and the formed particle size was smaller. It indicated that the self-assembly driving force was derived from the orientational ability of LC. The particle size and morphologies of self-assembled microspheres loaded with doxorubicin (DOX), together with drug release tracking, were evaluated by dynamic light scattering (DLS), SEM, and UV–vis spectroscopy. The results showed that DOX could be quickly released in a weakly acidic environment due to the pH response of the self-assembled microspheres. This would offer a new strategy for drug delivery in clinic applications. PMID:29584691

Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

PubMed

Anderson, M A; Wachs, T; Henion, J D

1997-02-01

A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and that using solid-phase extraction from 100 to 5000 pg ml-1. The lower level of quantitation (LLQ) using liquid-liquid and solid-phase extraction was 50 and 200 pg ml-1, respectively. The lower level of detection for reserpine by LC/MS/MS was 10 pg ml-1. The intra-assay accuracy did not exceed 13% for liquid-liquid and 12% for solid-phase extraction. The recoveries for the LLQ were 68% for liquid-liquid and 58% for solid-phase extraction.

A Liquid Chromatography – Tandem Mass Spectrometry Approach for the Identification of Mebendazole Residue in Pork, Chicken, and Horse

PubMed Central

Lee, Ji Sun; Cho, Soo Hee; Lim, Chae Mi; Chang, Moon Ik; Joo, Hyun Jin; Park, Hyun Jin

2017-01-01

A confirmatory and quantitative method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of mebendazole and its hydrolyzed and reduced metabolites in pork, chicken, and horse muscles was developed and validated in this study. Anthelmintic compounds were extracted with ethyl acetate after sample mixture was made alkaline followed by liquid chromatographic separation using a reversed phase C18 column. Gradient elution was performed with a mobile phase consisting of water containing 10 mM ammonium formate and methanol. This confirmatory method was validated according to EU requirements. Evaluated validation parameters included specificity, accuracy, precision (repeatability and within-laboratory reproducibility), analytical limits (decision limit and detection limit), and applicability. Most parameters were proved to be conforming to the EU requirements. The decision limit (CCα) and detection capability (CCβ) for all analytes ranged from 15.84 to 17.96 μgkg-1. The limit of detection (LOD) and the limit of quantification (LOQ) for all analytes were 0.07 μgkg-1 and 0.2 μgkg-1, respectively. The developed method was successfully applied to monitoring samples collected from the markets in major cities and proven great potential to be used as a regulatory tool to determine mebendazole residues in animal based foods. PMID:28085912

Validation of LC-TOF-MS screening for drugs, metabolites, and collateral compounds in forensic toxicology specimens.

PubMed

Guale, Fessessework; Shahreza, Shahriar; Walterscheid, Jeffrey P; Chen, Hsin-Hung; Arndt, Crystal; Kelly, Anna T; Mozayani, Ashraf

2013-01-01

Liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis provides an expansive technique for identifying many known and unknown analytes. This study developed a screening method that utilizes automated solid-phase extraction to purify a wide array of analytes involving stimulants, benzodiazepines, opiates, muscle relaxants, hypnotics, antihistamines, antidepressants and newer synthetic "Spice/K2" cannabinoids and cathinone "bath salt" designer drugs. The extract was applied to LC-TOF-MS analysis, implementing a 13 min chromatography gradient with mobile phases of ammonium formate and methanol using positive mode electrospray. Several common drugs and metabolites can share the same mass and chemical formula among unrelated compounds, but they are structurally different. In this method, the LC-TOF-MS was able to resolve many isobaric compounds by accurate mass correlation within 15 ppm mass units and a narrow retention time interval of less than 10 s of separation. Drug recovery yields varied among spiked compounds, but resulted in overall robust area counts to deliver an average match score of 86 when compared to the retention time and mass of authentic standards. In summary, this method represents a rapid, enhanced screen for blood and urine specimens in postmortem, driving under the influence, and drug facilitated sexual assault forensic toxicology casework.

Liquid chromatographic and ultraviolet spectrophotometric determination of bevantolol and hydrochlorothiazide in feeds.

PubMed

Spurlock, C H; Schneider, H G

1984-01-01

Separate assay methods have been developed for the 2 components of an 80 + 20 drug blend of bevantolol and hydrochlorothiazide (HCT) in admixtures with animal feed. Drug/diet admixtures are extracted with methanol for reverse phase ion-pair liquid chromatographic (LC) assay of bevantolol, and with acetonitrile for ultraviolet spectrophotometric assay of HCT. Bevantolol, a cardioselective beta blocker, is separated from soluble feed components with an RP-18 column, using methanol-water-acetic acid (60 + 40 + 1) containing 0. 005M octane-sulfonic acid, sodium salt, as ion-pairing reagent. HCT is determined spectrophotometrically in acetonitrile extracts, using a suitable blank extract as reference. Average recovery of HCT from an admixture of 0.5 mg blend/g diet is 94.5% +/- 4.3 RSD and at 2.0 mg/g, 101.5% +/- 3.5 RSD. Bevantolol recovery from the same admixtures is 101.8% +/- 2.7 RSD and 99.0% +/- 3.5 RSD, respectively, using the method as described.

Microfludic Sensing Devices Employing In Situ-Formed Liquid Crystal Thin Film for Detection of Biochemical Interactions1†

PubMed Central

Liu, Ye; Cheng, Daming; Lin, I-Hsin; Abbott, Nicholas L.; Jiang, Hongrui

2012-01-01

Although biochemical sensing using liquid crystals (LC) has been demonstrated, relatively little attention has been paid towards the fabrication of in situ-formed LC sensing devices. Herein, we demonstrate a highly reproducible method to create uniform LC thin film on treated substrates, as needed, for LC sensing. We use shear forces generated by the laminar flow of aqueous liquid within a microfluidic channel to create LC thin films stabilized within microfabricated structures. The orientational response of the LC thin films to targeted analytes in aqueous phases was transduced and amplified by the optical birefringence of the LC thin films. The biochemical sensing capability of our sensing devices was demonstrated through experiments employing two chemical systems: dodecyl trimethylammonium bromide (DTAB) dissolved in an aqueous solution, and the hydrolysis of phospholipids by the enzyme phospholipase A2 (PLA2). PMID:22842797

Quasi-targeted analysis of hydroxylation-related metabolites of polycyclic aromatic hydrocarbons in human urine by liquid chromatography-mass spectrometry.

PubMed

Tang, Caiming; Tan, Jianhua; Fan, Ruifang; Zhao, Bo; Tang, Caixing; Ou, Weihui; Jin, Jiabin; Peng, Xianzhi

2016-08-26

Metabolite identification is crucial for revealing metabolic pathways and comprehensive potential toxicities of polycyclic aromatic hydrocarbons (PAHs) in human body. In this work, a quasi-targeted analysis strategy was proposed for metabolite identification of monohydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) in human urine using liquid chromatography triple quadruple mass spectrometry (LC-QqQ-MS/MS) combined with liquid chromatography high resolution mass spectrometry (LC-HRMS). Potential metabolites of OH-PAHs were preliminarily screened out by LC-QqQ-MS/MS in association with filtering in a self-constructed information list of possible metabolites, followed by further identification and confirmation with LC-HRMS. The developed method can provide more reliable and systematic results compared with traditional untargeted analysis using LC-HRMS. In addition, data processing for LC-HRMS analysis were greatly simplified. This quasi-targeted analysis method was successfully applied to identifying phase I and phase II metabolites of OH-PAHs in human urine. Five metabolites of hydroxynaphthalene, seven of hydroxyfluorene, four of hydroxyphenanthrene, and three of hydroxypyrene were tentatively identified. Metabolic pathways of PAHs in human body were putatively revealed based on the identified metabolites. The experimental results will be valuable for investigating the metabolic processes of PAHs in human body, and the quasi-targeted analysis strategy can be expanded to the metabolite identification and profiling of other compounds in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Off-Line Multidimensional Liquid Chromatography and Auto Sampling Result in Sample Loss in LC/LC–MS/MS

PubMed Central

2015-01-01

Large-scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures. Fractionation can involve ion exchange separation coupled to reversed-phase separation or, more recently, two reversed-phase separations performed at different pH values. When multidimensional separations are combined with tandem mass spectrometry for protein identification, the strategy is often referred to as multidimensional protein identification technology (MudPIT). MudPIT has been used in either an automated (online) or manual (offline) format. In this study, we evaluated the performance of different MudPIT strategies by both label-free and tandem mass tag (TMT) isobaric tagging. Our findings revealed that online MudPIT provided more peptide/protein identifications and higher sequence coverage than offline platforms. When employing an off-line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high-pressure device, a 5.3% loss in protein identifications is observed. When off-line fractionated samples are loaded via an autosampler, a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column. Moreover, peptide recovery was significantly lower after offline fractionation than in online fractionation. Signal-to-noise (S/N) ratio, however, was not significantly altered between experimental groups. It is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces. Therefore, the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples. PMID:25040086

Ion-pair in-tube solid-phase microextraction and capillary liquid chromatography using a titania-based column: application to the specific lauralkonium chloride determination in water.

PubMed

Prieto-Blanco, M C; Moliner-Martínez, Y; López-Mahía, P; Campíns-Falcó, P

2012-07-27

A quick, miniaturized and on-line method has been developed for the determination in water of the predominant homologue of benzalkonium chloride, dodecyl dimethyl benzyl ammonium chloride or lauralkonium chloride (C(12)-BAK). The method is based on the formation of an ion-pair in both in-tube solid-phase microextraction (IT-SPME) and capillary liquid chromatography. The IT-SPME optimization required the study of the length and nature of the stationary phase of capillary and the processed sample volume. Because to the surfactant character of the analyte both, the extracting and replacing solvents, have played a decisive role in the IT-SPME optimized procedure. Conditioning the capillary with the mobile phase which contains the counter ion (acetate), using an organic additive (tetrabutylammonium chloride) added to the sample and a mixture water/methanol as replacing solvent (processed just before the valve is switched to the inject position), allowed to obtain good precision of the retention time and a narrow peak for C(12)-BAK. A reversed-phase capillary based TiO(2) column and a mobile phase containing ammonium acetate at pH 5.0 for controlling the interactions of cationic surfactant with titania surface were proposed. The optimized procedure provided adequate linearity, accuracy and precision at the concentrations interval of 1.5-300 μg L(-1) .The limit of detection (LOD) was 0.5 μg L(-1) using diode array detection (DAD). The applicability of proposed IT-SPME-capillary LC method has been assessed in several water samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Analytical method for the accurate determination of tricothecenes in grains using LC-MS/MS: a comparison between MRM transition and MS3 quantitation.

PubMed

Lim, Chee Wei; Tai, Siew Hoon; Lee, Lin Min; Chan, Sheot Harn

2012-07-01

The current food crisis demands unambiguous determination of mycotoxin contamination in staple foods to achieve safer food for consumption. This paper describes the first accurate LC-MS/MS method developed to analyze tricothecenes in grains by applying multiple reaction monitoring (MRM) transition and MS(3) quantitation strategies in tandem. The tricothecenes are nivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, fusarenon X, 3-acetyl-deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, and HT-2 and T-2 toxins. Acetic acid and ammonium acetate were used to convert the analytes into their respective acetate adducts and ammonium adducts under negative and positive MS polarity conditions, respectively. The mycotoxins were separated by reversed-phase LC in a 13.5-min run, ionized using electrospray ionization, and detected by tandem mass spectrometry. Analyte-specific mass-to-charge (m/z) ratios were used to perform quantitation under MRM transition and MS(3) (linear ion trap) modes. Three experiments were made for each quantitation mode and matrix in batches over 6 days for recovery studies. The matrix effect was investigated at concentration levels of 20, 40, 80, 120, 160, and 200 μg kg(-1) (n = 3) in 5 g corn flour and rice flour. Extraction with acetonitrile provided a good overall recovery range of 90-108% (n = 3) at three levels of spiking concentration of 40, 80, and 120 μg kg(-1). A quantitation limit of 2-6 μg kg(-1) was achieved by applying an MRM transition quantitation strategy. Under MS(3) mode, a quantitation limit of 4-10 μg kg(-1) was achieved. Relative standard deviations of 2-10% and 2-11% were reported for MRM transition and MS(3) quantitation, respectively. The successful utilization of MS(3) enabled accurate analyte fragmentation pattern matching and its quantitation, leading to the development of analytical methods in fields that demand both analyte specificity and fragmentation fingerprint-matching capabilities that are unavailable under MRM transition.

Fast method for simultaneous quantification of tamoxifen and metabolites in dried blood spots using an entry level LC-MS/MS system.

PubMed

Tré-Hardy, Marie; Capron, Arnaud; Antunes, Marina Venzon; Linden, Rafael; Wallemacq, Pierre

2016-11-01

The purpose of this study was to develop and validate a new liquid chromatography-tandem mass spectrometric (LC-MSMS) assay for the simultaneous quantification of tamoxifen (TAM) and its main therapeutically active metabolites, N-desmethyltamoxifen (NDT), 4-hydroxytamoxifen (4HT) and endoxifen (END) in dried blood spots. Ultrasound assisted methanolic extraction was used for TAM and metabolites extraction from dried blood spot. After evaporation and methanol reconstitution, the extract was injected into a LC-MSMS system. Reversed phase chromatography was performed on a C18 grafted column in gradient mode. TAM, metabolites, and internal standard (diazepam-d 5 ; IS) were identified in positive electrospray ionization mode using m/z transition of 372.5>72.1 (TAM); 374.23>58.10 (END); 358.27>58.10 (NDT); 388.23>44.80 (4HT) and 290.00>198.00 (IS). Total analytical run time was 6.5min. Assay was linear from 1 to 500ng/mL for all substances and presented intra and inter-assay precision and accuracy <15%. TAM, NDT, 4HT and END limits of quantification and detection were of 1 and 0.5ng/mL; 1 and 3ng/mL; 1.7 and 3ng/mL; 0.6 and 2ng/mL, respectively. Recovery ranged from 83.8 to 96.3% with matrix effect ranged from 4.3 to 29.8% for TAM and its metabolites. Hematocrit value ≤40% appeared to negatively influence accuracy of the method. In conclusion, the method described here is somewhat accessible, relatively fast, sensitive and selective with no interference. This assay might be used to investigate the level of TAM and its metabolites in DBS for therapeutic drug monitoring purposes. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Determination of vitamin K1 in powdered infant formulas, using supercritical fluid extraction and liquid chromatography with electrochemical detection.

PubMed

Schneiderman, M A; Sharma, A K; Mahanama, K R; Locke, D C

1988-01-01

Vitamin K1 (phylloquinone) is extracted from commercial soy protein-based and milk-based powdered infant formulas by using supercritical fluid extraction with CO2 at 8000 psi and 60 degrees C. Quantitative extraction requires only 15 min, and does not suffer from the problems associated with conventional solvent extraction of lipophilic materials from media such as formulas. Vitamin K1 is determined in the extracts by using reverse-phase liquid chromatography (LC) with reductive mode electrochemical detection at a silver electrode polarized at -1.1 V vs SCE. LC run time is 9 min. The minimum detectable quantity is 80 pg, and response is linear over at least 5 orders of magnitude. Recovery of vitamin K1 from a milk-based powdered formula was 95.6% with RSD of 7.4%, and from a soy protein-based product, 94.4% recovery with RSD of 6.5%.

Identification of selenium-containing proteins in HEK 293 kidney cells using multiple chromatographies, LC-ICPMS and nano-LC-ESIMS.

PubMed

Chitta, Karnakar R; Landero-Figueroa, Julio A; Kodali, Phanichand; Caruso, Joseph A; Merino, Edward J

2013-09-30

Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS). Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of synthetic dyes by comprehensive two-dimensional liquid chromatography combining ion-exchange chromatography and fast ion-pair reversed-phase chromatography.

PubMed

Pirok, Bob W J; Knip, Jitske; van Bommel, Maarten R; Schoenmakers, Peter J

2016-03-04

In the late 19th century, newly invented synthetic dyes rapidly replaced the natural dyes on the market. The characterization of mixtures of these so-called early synthetic dyes is complicated through the occurrence of many impurities and degradation products. Conventional one-dimensional liquid chromatography does not suffice to obtain fingerprints with sufficient resolution and baseline integrity. Comprehensive two-dimensional liquid chromatography (LC×LC) is employed in this study, with ion-exchange chromatography in the first dimension and fast ion-pair liquid chromatography in the second. Retention in the first dimension is largely determined by the number of charges, while the selection of a small ion-pair reagent (tetramethylammonium hydroxide) in the second dimension causes retention to be largely determined by the molecular structure of the dye. As a result, there is a high degree of orthogonality of the two dimensions, similar to the values typically encountered in GC×GC. The proposed LC×LC method shows a theroretical peak capacity of about 2000 in an analysis time of about three hours. Clear, informative fingerprints are obtained that open a way to a more efficient characterization of dyes used in objects of cultural heritage. Copyright © 2016 Elsevier B.V. All rights reserved.

Hydrophilic Strong Anion Exchange (hSAX) Chromatography for Highly Orthogonal Peptide Separation of Complex Proteomes

PubMed Central

2013-01-01

Due to its compatibility and orthogonality to reversed phase (RP) liquid chromatography (LC) separation, ion exchange chromatography, and mainly strong cation exchange (SCX), has often been the first choice in multidimensional LC experiments in proteomics. Here, we have tested the ability of three strong anion exchanger (SAX) columns differing in their hydrophobicity to fractionate RAW264.7 macrophage cell lysate. IonPac AS24, a strong anion exchange material with ultralow hydrophobicity, demonstrated to be superior to other materials by fractionation and separation of tryptic peptides from both a mixture of 6 proteins as well as mouse cell lysate. The chromatography displayed very high orthogonality and high robustness depending on the hydrophilicity of column chemistry, which we termed hydrophilic strong anion exchange (hSAX). Mass spectrometry analysis of 34 SAX fractions from RAW264.7 macrophage cell lysate digest resulted in an identification of 9469 unique proteins and 126318 distinct peptides in one week of instrument time. Moreover, when compared to an optimized high pH/low pH RP separation approach, the method presented here raised the identification of proteins and peptides by 10 and 28%, respectively. This novel hSAX approach provides robust, reproducible, and highly orthogonal separation of complex protein digest samples for deep coverage proteome analysis. PMID:23294059

A Comprehensive Workflow of Mass Spectrometry-Based Untargeted Metabolomics in Cancer Metabolic Biomarker Discovery Using Human Plasma and Urine

PubMed Central

Zou, Wei; She, Jianwen; Tolstikov, Vladimir V.

2013-01-01

Current available biomarkers lack sensitivity and/or specificity for early detection of cancer. To address this challenge, a robust and complete workflow for metabolic profiling and data mining is described in details. Three independent and complementary analytical techniques for metabolic profiling are applied: hydrophilic interaction liquid chromatography (HILIC–LC), reversed-phase liquid chromatography (RP–LC), and gas chromatography (GC). All three techniques are coupled to a mass spectrometer (MS) in the full scan acquisition mode, and both unsupervised and supervised methods are used for data mining. The univariate and multivariate feature selection are used to determine subsets of potentially discriminative predictors. These predictors are further identified by obtaining accurate masses and isotopic ratios using selected ion monitoring (SIM) and data-dependent MS/MS and/or accurate mass MSn ion tree scans utilizing high resolution MS. A list combining all of the identified potential biomarkers generated from different platforms and algorithms is used for pathway analysis. Such a workflow combining comprehensive metabolic profiling and advanced data mining techniques may provide a powerful approach for metabolic pathway analysis and biomarker discovery in cancer research. Two case studies with previous published data are adapted and included in the context to elucidate the application of the workflow. PMID:24958150

Proteomic Analysis of the Extracellular Matrix Produced by Mesenchymal Stromal Cells: Implications for Cell Therapy Mechanism

PubMed Central

Harvey, Adam; Yen, Ten-Yang; Aizman, Irina; Tate, Ciara; Case, Casey

2013-01-01

Mesenchymal stromal cells (MSCs) transiently transfected with notch1 intracellular domain (NICD) are beneficial for neurological disorders as observed in several preclinical studies. Extracellular matrix (ECM) derived from NICD-transfected MSCs has been previously shown to support in vitro neural cell growth and survival better than that of un-transfected MSCs. To understand the underlying mechanism(s) by which NICD-transfected MSC-derived ECM supports neural cell growth and survival, we investigated the differences in NICD-transfected MSC- and MSC-derived ECM protein quantity and composition. To compare the ECM derived from MSCs and NICD-transfected MSCs, the proteins were sequentially solubilized using sodium dodecyl sulfate (SDS) and urea, quantified, and compared across four human donors. We then analyzed ECM proteins using either in-gel digests or in-solution surfactant-assisted trypsin digests (SAISD) coupled with reverse phase nano-liquid chromatography and tandem mass spectrometry (nLC-MS/MS). Analyses using nLC-MS/MS identified key components of ECM from NICD-transfected MSCs and un-transfected MSCs and revealed significant differences in their respective compositions. This work provides a reproducible method for identifying and comparing in vitro cell-derived ECM proteins, which is crucial for exploring the mechanisms underlying cellular therapy. PMID:24244468

Medial Prefrontal Cortex and HPA Axis Roles in Generation of PTSD-Like Symptoms in SPS Model

DTIC Science & Technology

2010-09-01

phenytoin , 5) SPS alters locus coeruleus (LC) activity. The research conducted to date has compelled us to change some of our hypotheses and/or the...antikindling drug Phenytoin can reverse these effects. This experiment was proposed in specific aim #4 (hypothesis #4b). SPS enhanced fear renewal without...affecting fear conditioning or extinction. Systemic administration of phenytoin reversed this effect. This is shown in Figure 7. 5) SPS sensitizes LC

Confinement effects on lyotropic nematic liquid crystal phases of graphene oxide dispersions

NASA Astrophysics Data System (ADS)

Al-Zangana, Shakhawan; Iliut, Maria; Turner, Michael; Vijayaraghavan, Aravind; Dierking, Ingo

2017-12-01

Graphene oxide (GO) forms well ordered liquid crystal (LC) phases in polar solvents. Here, we map the lyotropic phase diagram of GO as a function of the lateral dimensions of the GO flakes, their concentration, geometrical confinement configuration and solvent polarity. GO flakes were prepared in water and transferred into other polar solvents. Polarising optical microscopy (POM) was used to determine the phase evolution through the isotropic-biphasic-nematic transitions of the GO LC. We report that the confinement volume and geometry relative to the particle size is critical for the observation of the lyotropic phase, specifically, this determines the low-end concentration limit for the detection of the GO LC. Additionally, a solvent with higher polarisability stabilises the LC phases at lower concentrations and smaller flake sizes. GO LCs have been proposed for a range of applications from display technologies to conductive fibres, and the behaviour of LC phase formation under confinement imposes a limit on miniaturisation of the dimensions of such GO LC systems which could significantly impact on their potential applications.

Determination of Grayanotoxins from Rhododendron brachycarpum in Dietary Supplements and Homemade Wine by Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry.

PubMed

Hwang, Taeik; Noh, Eunyoung; Jeong, Ji Hye; Park, Sung-Kwan; Shin, Dongwoo; Kang, Hoil

2018-02-28

A sensitive and specific high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS) method combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the determination of grayanotoxins I and III in dietary supplements and homemade wine. Grayanotoxins I and III were successfully extracted using solid-phase extraction cartridges, characterized by LC-QTOF-MS, and quantitated by LC-MS/MS. The LC-MS/MS calibration curves were linear over concentrations of 10-100 ng/mL (grayanotoxin I) and 20-400 ng/mL (grayanotoxin III). Grayanotoxins I and III were found in 51 foodstuffs, with quantitative determinations revealing total toxin concentrations of 18.4-101 000 ng/mL (grayanotoxin I) and 15.3-56 000 ng/mL (grayanotoxin III). The potential of the validated method was demonstrated by successful quantitative analysis of grayanotoxins I and III in dietary supplements and homemade wine; the method appears suitable for the routine detection of grayanotoxins I and III from Rhododendron brachycarpum.

Rapid Isocratic Liquid Chromatographic Separation and Quantification of Tryptophan and Six kynurenine Metabolites in Biological Samples with Ultraviolet and Fluorimetric Detection

PubMed Central

Badawy, Abdulla A-B; Morgan, Christopher J

2010-01-01

A simple, rapid isocratic liquid chromatographic procedure with ultraviolet and fluorimetric detection is described for the separation and quantification of L-tryptophan (Trp) and six of its kynurenine metabolites (kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic, kynurenic, xanthurenic and anthranilic acids). Using the Perkin Elmer LC 200 system, a reverse phase Synergi 4 μ fusion-RP80 A column (250 × 4.6 mm) (Phenomenex), and a mobile phase of 10 mM sodium dihydrogen phosphate: methanol (73:27, by vol) at pH 2.8 and a flow rate of 1.0–1.2 ml/min at 37 °C, a run took ∼13 min. The run took <7 min at 40 °C and a 1.4 ml/min flow rate. Limits of detection of all 7 analytes were 5–72 nM and their recoveries from human plasma and rat serum and liver varied between 62% and 111%. This simple method is suitable for high throughput work and can be further developed to include quinolinic acid and other Trp metabolites. PMID:22084598

Analysis and stability study of retinoids in pharmaceuticals by LC with fluorescence detection.

PubMed

Gatti, R; Gioia, M G; Cavrini, V

2000-08-01

Liquid chromatographic (HPLC) methods with fluorescence detection at different wavelengths were developed for measurements of retinoic acids (13-cis and all-trans) in pharmaceutical dosage forms and components of 'retinoid solution' (all-trans retinoic acid, vitamin A palmitate and beta-carotene), a galenical of 'Di Bella therapy', using reversed phase columns under isocratic conditions. The stability of all-trans retinoic acid in cream and all-trans retinoic acid and vitamin A palmitate in 'retinoid solution' was investigated. Solid-phase extraction (SPE), using C18 sorbent was applied to the analysis of retinoic acids (9-cis, 13-cis and all-trans) in the 'retinoid solution' to obtain a practical and reliable sample clean-up. The results showed that these preparations (cream and solution) can be conveniently stored in the dark (t.a. or 2-8 degrees C): under these conditions about 86-87% of the all-trans retinoic acid initial concentration in both formulations and about 73-78% of vitamin A palmitate in the 'retinoid solution' remained after 90 days, while under sunlight exposure rapid degradation of the drugs was observed.

Isolation and characterization of antimicrobial food components.

PubMed

Papetti, Adele

2012-04-01

Nowadays there is an evident growing interest in natural antimicrobial compounds isolated from food matrices. According to the type of matrix, different isolation and purification steps are needed and as these active compounds belong to different chemical classes, also different chromatographic and electrophoretic methods coupled with various detectors (the most used diode array detector and mass spectrometer) have to be performed. This review covers recent steps made in the fundamental understanding of sample preparation methods as well as of analytical tools useful for the complete characterization of bioactive food compounds. The most commonly used methods for extraction of natural antimicrobial compounds are the conventional liquid-liquid or solid-liquid extraction and the modern techniques such as pressurized liquid extraction, microwave-assisted extraction, ultrasound-assisted extraction, solid-phase micro-extraction, supercritical fluid extraction, and matrix solid phase dispersion. The complete characterization of the compounds is achieved using both monodimensional chromatographic processes (LC, nano-LC, GC, and CE coupled with different type of detectors) and, recently, using comprehensive two-dimensional systems (LC×LC and GC×GC). Copyright © 2011 Elsevier Ltd. All rights reserved.

In-line carbon nanofiber reinforced hollow fiber-mediated liquid phase microextraction using a 3D printed extraction platform as a front end to liquid chromatography for automatic sample preparation and analysis: A proof of concept study.

PubMed

Worawit, Chanatda; Cocovi-Solberg, David J; Varanusupakul, Pakorn; Miró, Manuel

2018-08-01

A novel concept for automation of nanostructured hollow-fiber supported microextraction, combining the principles of liquid-phase microextraction (LPME) and sorbent microextraction synergically, using mesofluidic platforms is proposed herein for the first time, and demonstrated with the determination of acidic drugs (namely, ketoprofen, ibuprofen, diclofenac and naproxen) in urine as a proof-of-concept applicability. Dispersed carbon nanofibers (CNF) are immobilized in the pores of a single-stranded polypropylene hollow fiber (CNF@HF) membrane, which is thereafter accommodated in a stereolithographic 3D-printed extraction chamber without glued components for ease of assembly. The analytical method involves continuous-flow extraction of the acidic drugs from a flowing stream donor (pH 1.7) into an alkaline stagnant acceptor (20 mmol L -1 NaOH) containing 10% MeOH (v/v) across a dihexyl ether impregnated CNF@HF membrane. The flow setup features entire automation of the microextraction process including regeneration of the organic film and on-line injection of the analyte-laden acceptor phase after downstream neutralization into a liquid chromatograph (LC) for reversed-phase core-shell column-based separation. Using a 12-cm long CNF@HF and a sample volume of 6.4 mL, linear dynamic ranges of ketoprofen, naproxen, diclofenac and ibuprofen, taken as models of non-steroidal anti-inflammatory drugs, spanned from ca. 5-15 µg L -1 to 500 µg L -1 with enhancement factors of 43-97 (against a direct injection of 10 µL standards into LC), and limits of detection from 1.6 to 4.3 µg L -1 . Relative recoveries in real urine samples ranged from 97% to 105%, thus demonstrating the reliability of the automatic CNF@HF-LPME method for in-line matrix clean-up and determination of drugs in urine at therapeutically relevant concentrations. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of hydroxylated polychlorinated biphenyls (HO-PCBs) in blood plasma by high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry.

PubMed

Letcher, R J; Li, H X; Chu, S G

2005-01-01

Hydroxylated metabolites of polychlorinated biphenyls (HO-PCBs) and pentachlorophenol (PCP) are halogenated phenolic compounds, and they are increasingly common as environmental contaminants mainly in the blood of wildlife and humans. A methodology based on high-performance liquid chromatography (reversed-phase)-electrospray (negative) ionization-tandem quadrupole mass spectrometry (LC-ESI(-)-MS-MS) in the select ion monitoring or multiple reaction monitoring modes was developed for HO-PCB and PCP determination in blood plasma and serum. Among 11 environmentally relevant HO-PCB congeners and PCP spiked to fetal calf serum, quantitative assessments, including matrix effects on ESI(-) suppression/ enhancement, showed process (recovery) efficiencies of 73% to 89% without internal standard (IS) correction, and 88% to 103% with IS correction, and method limits of quantification ranging from 1 to 50 pg/g (wet weight). Using the developed LC-ESI(-)-MS methodology in comparison with GC-MS and GC-ECD based approaches, similar results were found for HO-PCB identification and quantification in the plasma of polar bear (Ursus maritimus) from the Canadian arctic. LC-ESI(-)-MS identified four HO-PCB congeners [4'-HO-2,2',4,6,6'-pentachlorobiphenyl (4'-HO-CB104), 4-HO-2,3,3',4',5-pentachlorobiphenyl (4-HO-CB107), 4-HO-2,3,3',5,5',6-hexachlorobiphenyl (4-HO-CB165) and 3'-HO-2,2',3,4,4',5,5'-heptachlorobiphenyl (3'-HO-CB180)], and 14 additional tetra- to hepta-chlorinated HO-PCBs isomers in the polar bear plasma.

Analysis of β-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry

PubMed Central

2014-01-01

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications. PMID:25120905

Analysis of β-N-methylamino-L-alanine (BMAA) in spirulina-containing supplements by liquid chromatography-tandem mass spectrometry.

PubMed

McCarron, Pearse; Logan, Alan C; Giddings, Sabrina D; Quilliam, Michael A

2014-01-01

Over the last decade the amino acid beta-N-methylamino-L-alanine (BMAA) has come under intense scrutiny. International laboratory and epidemiological research continues to support the hypothesis that environmental exposure to BMAA (e.g., through dietary practices, water supply) can promote the risk of various neurodegenerative diseases. A wide variety of cyanobacteria spp. have previously been reported to produce BMAA, with production levels dependent upon species, strain and environmental conditions. Since spirulina (Arthrospira spp.) is a member of the cyanobacteria phylum frequently consumed via dietary supplements, the presence of BMAA in such products may have public health implications. In the current work, we have analyzed ten spirulina-containing samples for the presence of BMAA; six pure spirulina samples from two separate raw materials suppliers, and four commercially-available multi-ingredient products containing 1.45 g of spirulina per 8.5 g serving. Because of controversy surrounding the measurement of BMAA, we have used two complementary liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods: one based on reversed phase LC (RPLC) with derivatization and the other based on hydrophilic interaction LC (HILIC). Potential matrix effects were corrected for by internal standardization using a stable isotope labeled BMAA standard. BMAA was not detected at low limits of detection (80 ng/g dry weight) in any of these product samples. Although these results are reassuring, BMAA analyses should be conducted on a wider sample selection and, perhaps, as part of ongoing spirulina production quality control testing and specifications.

Distribution of the hallucinogens N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in rat brain following intraperitoneal injection: application of a new solid-phase extraction LC-APcI-MS-MS-isotope dilution method.

PubMed

Barker, S A; Littlefield-Chabaud, M A; David, C

2001-02-10

A method for the solid-phase extraction (SPE) and liquid chromatographic-atmospheric pressure chemical ionization-mass spectrometric-mass spectrometric-isotope dilution (LC-APcI-MS-MS-ID) analysis of the indole hallucinogens N,N-dimethyltryptamine (DMT) and 5-methoxy DMT (or O-methyl bufotenin, OMB) from rat brain tissue is reported. Rats were administered DMT or OMB by the intraperitoneal route at a dose of 5 mg/kg and sacrificed 15 min post treatment. Brains were dissected into discrete areas and analyzed by the methods described as a demonstration of the procedure's applicability. The synthesis and use of two new deuterated internal standards for these purposes are also reported.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS.

PubMed

Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom

2009-10-01

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.

Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry.

PubMed

Liu, Ai-Hua; Guo, Hui; Ye, Min; Lin, Yan-Hua; Sun, Jiang-Hao; Xu, Man; Guo, De-An

2007-08-17

By using HPLC-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)) in negative ion mode, we have analyzed the fragmentation pathways of 11 phenolic acids which were isolated from Danshen. Then the extract of Danshen was analyzed, and a total of 42 phenolic acids, including sixteen new minor constituents, were identified or tentatively identified for the first time. A new solid-phase extraction (SPE) method, new HPLC separation method, new liquid chromatography (LC)-MS and LC-MS(n) (n=3-5) data and proposed fragmentation pathways, LC retention time for phenolic acids are reported.

Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

PubMed

Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

2011-11-01

In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

On-line high-performance liquid chromatography-ultraviolet-nuclear magnetic resonance method of the markers of nerve agents for verification of the Chemical Weapons Convention.

PubMed

Mazumder, Avik; Gupta, Hemendra K; Garg, Prabhat; Jain, Rajeev; Dubey, Devendra K

2009-07-03

This paper details an on-flow liquid chromatography-ultraviolet-nuclear magnetic resonance (LC-UV-NMR) method for the retrospective detection and identification of alkyl alkylphosphonic acids (AAPAs) and alkylphosphonic acids (APAs), the markers of the toxic nerve agents for verification of the Chemical Weapons Convention (CWC). Initially, the LC-UV-NMR parameters were optimized for benzyl derivatives of the APAs and AAPAs. The optimized parameters include stationary phase C(18), mobile phase methanol:water 78:22 (v/v), UV detection at 268nm and (1)H NMR acquisition conditions. The protocol described herein allowed the detection of analytes through acquisition of high quality NMR spectra from the aqueous solution of the APAs and AAPAs with high concentrations of interfering background chemicals which have been removed by preceding sample preparation. The reported standard deviation for the quantification is related to the UV detector which showed relative standard deviations (RSDs) for quantification within +/-1.1%, while lower limit of detection upto 16mug (in mug absolute) for the NMR detector. Finally the developed LC-UV-NMR method was applied to identify the APAs and AAPAs in real water samples, consequent to solid phase extraction and derivatization. The method is fast (total experiment time approximately 2h), sensitive, rugged and efficient.

Effect of menstrual cycle phase on corticolimbic brain activation by visual food cues.

PubMed

Frank, Tamar C; Kim, Ginah L; Krzemien, Alicja; Van Vugt, Dean A

2010-12-02

Food intake is decreased during the late follicular phase and increased in the luteal phase of the menstrual cycle. While a changing ovarian steroid milieu is believed to be responsible for this behavior, the specific mechanisms involved are poorly understood. Brain activity in response to visual food stimuli was compared during the estrogen dominant peri-ovulatory phase and the progesterone dominant luteal phase of the menstrual cycle. Twelve women underwent functional magnetic resonance imaging during the peri-ovulatory and luteal phases of the menstrual cycle in a counterbalanced fashion. Whole brain T2* images were collected while subjects viewed pictures of high calorie (HC) foods, low calorie (LC) foods, and control (C) pictures presented in a block design. Blood oxygen level dependent (BOLD) signal in the late follicular phase and luteal phase was determined for the contrasts HC-C, LC-C, HC-LC, and LC-HC. Both HC and LC stimuli activated numerous corticolimbic brain regions in the follicular phase, whereas only HC stimuli were effective in the luteal phase. Activation of the nucleus accumbens (NAc), amygdala, and hippocampus in response to the HC-C contrast and the hippocampus in response to the LC-C contrast was significantly increased in the late follicular phase compared to the luteal phase. Activation of the orbitofrontal cortex and mid cingulum in response to the HC-LC contrast was greater during the luteal phase. These results demonstrate for the first time that brain responses to visual food cues are influenced by menstrual cycle phase. We postulate that ovarian steroid modulation of the corticolimbic brain contributes to changes in ingestive behavior during the menstrual cycle. Copyright © 2010 Elsevier B.V. All rights reserved.

Oral Administration of (S)-Allyl-l-Cysteine and Aged Garlic Extract to Rats: Determination of Metabolites and Their Pharmacokinetics.

PubMed

Park, Taehoon; Oh, Ju-Hee; Lee, Joo Hyun; Park, Sang Cheol; Jang, Young Pyo; Lee, Young-Joo

2017-11-01

( S )-Allyl-l-cysteine is the major bioactive compound in garlic. ( S )-Allyl-l-cysteine is metabolized to ( S )-allyl-l-cysteine sulfoxide, N -acetyl-( S )-allyl-l-cysteine, and N -acetyl-( S )-allyl-l-cysteine sulfoxide after oral administration. An accurate LC-MS/MS method was developed and validated for the simultaneous quantification of ( S )-allyl-l-cysteine and its metabolites in rat plasma, and the feasibility of using it in pharmacokinetic studies was tested. The analytes were quantified by multiple reaction monitoring using an atmospheric pressure ionization mass spectrometer. Because significant quantitative interference was observed between ( S )-allyl-l-cysteine and N -acetyl-( S )-allyl-l-cysteine as a result of the decomposition of N -acetyl-( S )-allyl-l-cysteine at the detector source, chromatographic separation was required to discriminate ( S )-allyl-l-cysteine and its metabolites on a reversed-phase C 18 analytical column with a gradient mobile phase consisting of 0.1% formic acid and acetonitrile. The calibration curves of ( S )-allyl-l-cysteine, ( S )-allyl-l-cysteine sulfoxide, N -acetyl-( S )-allyl-l-cysteine, and N -acetyl-( S )-allyl-l-cysteine sulfoxide were linear over each concentration range, and the lower limits of quantification were 0.1 µg/mL [( S )-allyl-l-cysteine and N -acetyl-( S )-allyl-l-cysteine] and 0.25 µg/mL [( S )-allyl-l-cysteine sulfoxide and N -acetyl-( S )-allyl-l-cysteine sulfoxide]. Acceptable intraday and inter-day precisions and accuracies were obtained at three concentration levels. The method satisfied the regulatory requirements for matrix effects, recovery, and stability. The validated LC-MS/MS method was successfully used to determine the concentration of ( S )-allyl-l-cysteine and its metabolites in rat plasma samples after the administration of ( S )-allyl-l-cysteine or aged garlic extract. Georg Thieme Verlag KG Stuttgart · New York.

Liquid Chromatography-Tandem Mass Spectrometric Analysis of Octaethylene Glycol Monodecyl Ether in Rat Plasma and its Application to Pharmacokinetic Studies.

PubMed

Kim, Hyeon; Kim, Hyeong Jun; Choi, Min Sun; Kim, In Sook; Gye, Myung Chan; Yoo, Hye Hyun

2017-05-01

Alcohol ethoxylates (AEs) are a major class of non-ionic surfactants, which are widely used in household, institutional and industrial cleaners, and they are considered as an alternative of nonylphenol. In this study, a rapid, sensitive and reliable bioanalytical method was developed for the determination of octaethylene glycol monodecyl ether (C10E8, an AE) in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Chromatographic separation was performed on a reversed-phase C18 column (2.1 mm × 50 mm, 2.1 μm). The mobile phase consisted of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile (40:60% v/v). The flow rate was 0.3 mL/min. For mass spectrometric detection, the multiple reaction monitoring (MRM) mode was used; the MRM transitions were m/z 511.5 → m/z 133.1 for C10E8 and m/z 423.3 → m/z 133.1 for hexaethylene glycol monodecyl ether (internal standard) in the positive ion mode. A calibration curve was constructed within the range of 2-2,000 ng/mL; the intra- (n = 5) and inter-day (n = 3) precision and accuracy were within 10%. The LC-MS-MS method was specific, accurate and reproducible, and this method was successfully applied in a pharmacokinetic study of C10E8 in rats. C10E8 was intravenously (1 mg/kg, n = 6) and orally (10 mg/kg, n = 7) administered to rats. The kinetic parameters were analyzed based on a noncompartmental statistical model using the pharmacokinetic modeling software (WinNonlin). The oral bioavailability of C10E8 was 34.4%. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Determination of Aflatoxins and Ochratoxin A in Traditional Turkish Concentrated Fruit Juice Products by Multi-Immunoaffinity Column Cleanup and LC Fluorescence Detection: Single-Laboratory Validation.

PubMed

Kaymak, Tugrul; Türker, Levent; Tulay, Hüseyin; Stroka, Joerg

2018-04-27

Background : Pekmez and pestil are traditional Turkish foods made from concentrated grapejuice, which can be contaminated with mycotoxins such as aflatoxins and ochratoxin A (OTA). Objective : To carry out a single-laboratory validation of a method to simultaneously determine aflatoxins B 1 , B₂, G 1 , and G₂ and ochratoxin A in pekmez and pestil. Methods : The homogenized sample is extracted with methanol-water (80 + 20) using a high-speed blender. The (sample) extract is filtered, diluted with phosphate-buffered saline solution, and applied to a multi-immunoaffinity column (AFLAOCHRA PREP®). Aflatoxins and ochratoxin A are removed with (neat) methanol and then directly analyzed by reversed-phase LC with fluorescence detection using post-column bromination (Kobra cell®). Results : Test portions of blank pekmez and pestil were spiked with a mixture of aflatoxins and ochratoxin A to give levels ranging from 2.6 to 10.4 μg/kg and 1.0-4.0 μg/kg, respectively. Recoveries for total aflatoxins and ochratoxin A ranged from 84 to 106% and 80-97%, respectively, for spiked samples. Based on results for spiked pekmez and pestil (30 replicates each at three levels), the repeatability RSD ranged from 1.6 to 12% and 2.7-11% for total aflatoxins and ochratoxin A, respectively. Conclusions : The method performance in terms of recovery, repeatability, and detection limits has been demonstrated to be suitable for use as an Official Method. Highlights : First immunoaffinity column method validated for simultaneous analysis of aflatoxins and ochratoxin A in pekmez and pestil. Suitability for use for official purposes in Turkey, demonstrated by single-laboratory validation. Co-occurrence of aflatoxins and OTA in mulberry and carob pekmez reported for the first time.

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography.

PubMed

Chiap, P; Rbeida, O; Christiaens, B; Hubert, Ph; Lubda, D; Boos, K S; Crommen, J

2002-10-25

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.

Mouse pharmacokinetics and metabolism of the curcumin analog, 4-Piperidione,3,5-bis[(2-fluorophenyl)methylene]-acetate(3E,5E) (EF-24; NSC 716993)

PubMed Central

Reid, Joel M.; Buhrow, Sarah A.; Gilbert, Judith A.; Jia, Lee; Shoji, Mamoru; Snyder, James P.; Ames, Matthew M.

2015-01-01

Purpose Curcumin, a keto-enol constituent of turmeric, has in vitro and in vivo antitumor activity. However, in vivo potency is low due to poor oral absorption. The mono-carbonyl analog, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone acetate (EF-24, NSC 716993), exhibited broad spectrum activity in the NCI anti-cancer cell line screen and potent anti-angiogenesis activity in a HUVEC cell migration assay. The purpose of this study was to characterize the preclinical pharmacology of EF-24 in mice. Methods EF-24 plasma stability, protein binding, pharmacokinetics and metabolism were characterized utilizing an LC/MS/MS assay. Results An LC/MS/MS assay that incorporated protein precipitation with methanol, reverse-phase HPLC separation under gradient elution using an aqueous methanol mobile phase containing 0.1% formic acid and positive electrospray ionization detection of the m/z 312 > 149 transition for EF-24. The assay was linear over the range 7.8-1000 nM. Plasma protein binding was > 98% with preferential binding to albumin. EF-24 plasma disposition in mice after i.v. administration of a 10 mg/kg dose was best fit to a 3-compartment open model. The terminal elimination half-life and plasma clearance values were 73.6 min and 0.482 L/min/kg, respectively. EF-24 bioavailability was 60% and 35% after oral and i.p. administration, respectively. NADPH-dependent metabolism of EF-24 loss in liver microsomal preparations yielded several metabolites consistent with EF-24 hydroxylation and reduction. PMID:24760417

Economic Burden for Informal Caregivers of Lung and Colorectal Cancer Patients

PubMed Central

Ramsey, Scott D.; Hornbrook, Mark C.; Atienza, Audie A.; van Ryn, Michelle

2010-01-01

Background. Informal care provides many benefits to cancer patients, but can be costly to caregivers. This study quantified the economic burden for informal caregivers of lung cancer (LC) and colorectal cancer (CRC) patients, examining differences by cancer type, phase of disease, stage at diagnosis, patient age, and relationship. Methods. A cross-sectional survey of caregivers of LC and CRC patients participating in the Share Thoughts on Care survey was conducted. Economic burden was calculated using the opportunity cost of caregiver time, the value of work hours lost, and out-of-pocket expenditures. Factors associated with economic burden to caregivers were modeled using fixed-effects generalized least squares estimation. Results. Informal caregivers (1,629) completed mailed surveys. Of these, 663, 822, and 144 were surveyed during the patient's initial phase (first year after diagnosis, not within 6 months of death), continuing phase (after 1 year, not within 6 months of death), and terminal phase (within 6 months of death) of disease, respectively. The accumulated economic burdens for caregivers were $7,028, $19,701, and $14,234 for those evaluated during the patient's initial phase, continuing phase, and terminal phase of disease, respectively. Economic burden was higher for caregivers of LC patients than CRC patients (p = .044) and for caregivers of patients diagnosed at stage 4 versus stage 1 (p = .001). Spouses faced higher economic burden than other relatives (p = .000) or friends (p = .000). Conclusions. Economic burden for informal caregivers of LC and CRC patients is substantial and should be included in estimates of the societal cost of cancer care. PMID:20667966

Liquid chromatography-tandem MS/MS method for simultaneous quantification of paracetamol, chlorzoxazone and aceclofenac in human plasma: An application to a clinical pharmacokinetic study.

PubMed

Mohamed, Dalia; Hegazy, Maha A; Elshahed, Mona S; Toubar, Safaa S; Helmy, Marwa I

2018-07-01

A facile, fast and specific method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of paracetamol, chlorzoxazone and aceclofenac in human plasma was developed and validated. Sample preparation was achieved by liquid-liquid extraction. The analysis was performed on a reversed-phase C 18 HPLC column (5 μm, 4.6 × 50 mm) using acetonitrile-10 mM ammonium formate pH 3.0 (65:35, v/v) as the mobile phase where atrovastatin was used as an internal standard. A very small injection volume (3 μL) was applied and the run time was 2.0 min. The detection was carried out by electrospray positive and negative ionization mass spectrometry in the multiple-reaction monitoring mode. The developed method was capable of determining the analytes over the concentration ranges of 0.03-30.0, 0.015-15.00 and 0.15-15.00 μg/mL for paracetamol, chlorzoxazone and aceclofenac, respectively. Intraday and interday precisions (as coefficient of variation) were found to be ≤12.3% with an accuracy (as relative error) of ±5.0%. The method was successfully applied to a pharmacokinetic study of the three analytes after being orally administered to six healthy volunteers. Copyright © 2018 John Wiley & Sons, Ltd.

Stability-Indicating Reversed-Phase UHPLC Method Development and Characterization of Degradation Products of Almotriptan Maleate by LC-QTOF-MS/MS.

PubMed

Saibaba, B; Vishnuvardhan, Ch; Johnsi Rani, P; Satheesh Kumar, N

2018-01-01

Almotriptan maleate (ALMT), a highly selective 5-hydroxy tryptamine 1B/1D (5-HT1B/1D) receptor agonist used in the treatment of migraine headache was subjected to various ICH (Q1A (R2)) specified guidelines. The drug underwent significant degradation under hydrolytic (acid, base and neutral), oxidative and photolytic stress conditions, while it was stable under thermal stress condition. A total of seven significant degradation products (DPs) were obtained. A simple, selective and reliable UPLC method has been developed for the separation of ALMT and its DPs using Acquity UPLC HSS Cyano (100 × 2.1 mm, 1.8 μm) column with mobile phase consisting of ammonium acetate (10 mM, pH 4.4) buffer and acetonitrile in gradient elution mode. Chromatographic analysis was performed at a flow rate of 0.3 mL/min using a PDA detector at a wavelength of 230 nm. All the DPs (DP-1 to DP-7) were characterized using UHPLC-ESI-QTOF based on mass fragmentation pattern and accurate m/z values. The developed UPLC method was validated in terms of specificity, linearity, precision and accuracy. The developed stability-indicating method helps in quantification of drug in the presence of DPs. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative LC-MS/MS analysis of seven ginsenosides and three aconitum alkaloids in Shen-Fu decoction

PubMed Central

2013-01-01

Background Shen-Fu decoction is a traditional Chinese medicine prescription with a 3:2 ratio of Radix Ginseng and Fuzi (Radix Aconiti lateralis praeparata). Ginsenosides and alkaloids are considered to be the main active components of Shen-Fu decoction. However, no analytical methods have been used to quantitatively analyse both components in Shen-Fu decoction simultaneously. Results We successfully developed a rapid resolution liquid chromatography coupled with tandem mass spectrometry (RRLC-MS/MS) method for the simultaneous analysis of seven ginsenosides and three aconitum alkaloids in Shen-Fu decoction, the decoction of Radix ginseng and Fuzi (Radix Aconiti lateralis praeparata). Chromatogrpahic separation by RPLC was achieved using a reversed-phase column and a water/acetonitrile mobile phase, containing 0.05% formic acid and using a gradient system. The method was optimized to allow for simultaneous analysis of all analytes in 11minutes without the need for baseline resolution of the components. Furthermore, the separation demonstrated good linearity (r > 0.9882), repeatability (RSD < 7.01%), intra- and inter-day precisions (RSD < 5.06%) and high yields of recovery (91.13-111.97%) for ten major constituents, namely ginsenoside-Re, Rg1, Rb1, Rc, Rb2, Rd, Rf, aconitine, hypacoitine and mesaconitine. Conclusions The developed method could be used as a rapid and reliable approach for assessment of the quantity of the major constituents in Shen-Fu decoction. PMID:24107599

Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of tigecycline in rat brain tissues.

PubMed

Munyeza, Chiedza F; Shobo, Adeola; Baijnath, Sooraj; Bratkowska, Dominika; Naiker, Suhashni; Bester, Linda A; Singh, Sanil D; Maguire, Glenn E M; Kruger, Hendrik G; Naicker, Tricia; Govender, Thavendran

2016-06-01

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra-abdominal infections. A selective, accurate and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel-Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C18 column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150-1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time-points. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Analysis of street drugs in seized material without primary reference standards.

PubMed

Laks, Suvi; Pelander, Anna; Vuori, Erkki; Ali-Tolppa, Elisa; Sippola, Erkki; Ojanperä, Ilkka

2004-12-15

A novel approach was used to analyze street drugs in seized material without primary reference standards. Identification was performed by liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS), essentially based on accurate mass determination using a target library of 735 exact monoisotopic masses. Quantification was carried out by liquid chromatography/chemiluminescence nitrogen detection (LC/CLND) with a single secondary standard (caffeine), utilizing the detector's equimolar response to nitrogen. Sample preparation comprised dilution, first with methanol and further with the LC mobile phase. Altogether 21 seized drug samples were analyzed blind by the present method, and results were compared to accredited reference methods utilizing identification by gas chromatography/mass spectrometry and quantification by gas chromatography or liquid chromatography. The 31 drug findings by LC/TOFMS comprised 19 different drugs-of-abuse, byproducts, and adulterants, including amphetamine and tryptamine designer drugs, with one unresolved pair of compounds having an identical mass. By the reference methods, 27 findings could be confirmed, and among the four unconfirmed findings, only 1 apparent false positive was found. In the quantitative analysis of 11 amphetamine, heroin, and cocaine findings, mean relative difference between the results of LC/CLND and the reference methods was 11% (range 4.2-21%), without any observable bias. Mean relative standard deviation for three parallel LC/CLND results was 6%. Results suggest that the present combination of LC/TOFMS and LC/CLND offers a simple solution for the analysis of scheduled and designer drugs in seized material, independent of the availability of primary reference standards.

Rapid determination of underivatized pyroglutamic acid, glutamic acid, glutamine and other relevant amino acids in fermentation media by LC-MS-MS.

PubMed

Qu, Jun; Chen, Wei; Luo, Guoan; Wang, Yiming; Xiao, Shengyuan; Ling, Zhihua; Chen, Guoqiang

2002-01-01

Determination of amino acids in a complex matrix without derivatization is advantageous, however, difficulties are found in both the detection and the separation of those compounds. In this study, a rapid and reliable LC-MS-MS method for the quantitation of underivatized amino acids in exocellular media was established. Injections were made directly after centrifugation of the samples, without further preparation. The separation of seven underivatized amino acids was achieved on a reversed-phase C18 column with pentadecafluorooctanoic acid as a volatile ion-pair reagent, and the specific detection of most amino acids was achieved by MS-MS of the specific transitions [M + H]+-->[M + H - 46]+. The calibration curves of all analytes were linear over the range of 1.0-1000 microg ml(-1) and the detection limits ranged from 0.1 to 5 ng ml(-1), with an injection volume of 20 microl. The inter-day and intra-day precisions ranged from 2.6 to 5.7% and 4.8 to 8.2%, respectively; the mean recoveries of the seven analytes were 81-104%, 91-107% and 93-101% respectively at the spiked level of 10, 40 and 200 microg ml(-1). A large number of fermentation samples were analysed using this method. The technique is simple, rapid, selective and sensitive, and shows potential for the high-throughput quantitation of amino acids from other biological matrices.

Liquid chromatographic determination of para-toluenesulfonamide in edible fillet tissues from three species of fish

USGS Publications Warehouse

Meinertz, J.R.; Schmidt, L.J.; Stehly, G.R.; Gingerich, W.H.

1999-01-01

Chloramine-T (N-sodium-N-chloro-p-toluene-sulfonamide) is a candidate therapeutic drug for treating bacterial gill disease, a predominant disease of a variety of fish species. Research has been initiated to obtain the U.S. Food and Drug Administration's (FDA) approval for the use of chloramine-T on a variety of fish species. An attribute of a therapeutic aquaculture drug that must be characterized before the FDA approves its use is depletion of the drug's marker residue (the drug's parent compound or metabolite of highest concentration in an edible tissue). Para-Toluenesulfonamide (p-TSA) is the primary degradation product and marker residue for chloramine-T in rainbow trout. To conduct residue depletion studies for chloramine-T in fish, a robust analytical method sensitive and specific for p-TSA residues in edible fillet tissue from a variety of fish was required. Homogenized fillet tissues from rainbow trout (Oncorhynchus mykiss), walleye (Stizostedion vitreum), and channel catfish (Ictalurus punctatus) were fortified at nominal p-TSA concentrations of 17, 67, 200, 333, and 1000 ng/g. Samples were analyzed by isocratic reversed-phase liquid chromatography (LC) with absorbance detection at 226 nm. Mean recoveries of p-TSA ranged from 77 to 93.17%; relative standard deviations ranged from 1.5 to 14%; method quantitation limits ranged from 13 to 18 ng/g; and method detection limits ranged from 3.8 to 5.2 ng/g. The LC parameters produced p-TSA peaks without coelution of endogenous compounds and excluded chromatographic interference from at least 20 chemicals and drugs of potential use in aquaculture.

Quantitative determination of cyclic phosphatidic acid and its carba analog in mouse organs and plasma using LC-MS/MS.

PubMed

Shimizu, Yoshibumi; Ishikawa, Masaki; Gotoh, Mari; Fukasawa, Keiko; Yamamoto, Shinji; Iwasa, Kensuke; Yoshikawa, Keisuke; Murakami-Murofushi, Kimiko

2018-02-15

Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid, is involved in the regulation of many cellular processes. A sensitive and specific method to quantify the molecular species of cPA is important for studying the physiological and pathophysiological roles of cPA. Here, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based quantification method for the simultaneous detection of cPA species having various fatty acids (16:0, 18:0, 18:1, and 18:2) as well as 2-carba-cPA, a chemically synthesized analog of cPA. Chromatography was performed using a reversed-phase C18 column. cPA species were detected using a triple quadrupole mass spectrometer. cPA 17:0 was used as an internal standard. Intra- and interday precision values (CV%) were within 10%. The linear range of detection for each cPA species was 0.01 μg/mL to 5 μg/mL, with correlation coefficients of 0.998 or higher. The developed method was applied to the quantification of cPA species in mouse plasma and organs. The concentrations of cPA 16:0, 18:0, and 18:1 were revealed to be significantly reduced in the brains of cuprizone-treated mice, a model of multiple sclerosis, compared with control mice. These findings could be important for understanding the roles of cPA in the neurodegenerative processes associated with multiple sclerosis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

PubMed

Fanali, Chiara; Dugo, Laura; D'Orazio, Giovanni; Lirangi, Melania; Dachà, Marina; Dugo, Paola; Mondello, Luigi

2011-01-01

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and validation of an LC-ESI-MS/MS method for the quantification of D-84, reboxetine and citalopram for their use in MS Binding Assays addressing the monoamine transporters hDAT, hSERT and hNET.

PubMed

Neiens, Patrick; De Simone, Angela; Ramershoven, Anna; Höfner, Georg; Allmendinger, Lars; Wanner, Klaus T

2018-03-03

MS Binding Assays represent a label-free alternative to radioligand binding assays. In this study, we present an LC-ESI-MS/MS method for the quantification of (R,R)-4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol [(R,R)-D-84, (R,R)-1], (S,S)-reboxetine [(S,S)-2], and (S)-citalopram [(S)-3] employed as highly selective nonlabeled reporter ligands in MS Binding Assays addressing the dopamine [DAT, (R,R)-D-84], norepinephrine [NET, (S,S)-reboxetine] and serotonin transporter [SERT, (S)-citalopram], respectively. The developed LC-ESI-MS/MS method uses a pentafluorphenyl stationary phase in combination with a mobile phase composed of acetonitrile and ammonium formate buffer for chromatography and a triple quadrupole mass spectrometer in the multiple reaction monitoring mode for mass spectrometric detection. Quantification is based on deuterated derivatives of all three analytes serving as internal standards. The established LC-ESI-MS/MS method enables fast, robust, selective and highly sensitive quantification of all three reporter ligands in a single chromatographic run. The method was validated according to the Center for Drug Evaluation and Research (CDER) guideline for bioanalytical method validation regarding selectivity, accuracy, precision, calibration curve and sensitivity. Finally, filtration-based MS Binding Assays were performed for all three monoamine transporters based on this LC-ESI-MS/MS quantification method as read out. The affinities determined in saturation experiments for (R,R)-D-84 toward hDAT, for (S,S)-reboxetine toward hNET, and for (S)-citalopram toward hSERT, respectively, were in good accordance with results from literature, clearly demonstrating that the established MS Binding Assays have the potential to be an efficient alternative to radioligand binding assays widely used for this purpose so far. Copyright © 2018 John Wiley & Sons, Ltd.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis of endogenous steroids in the luteal phase and early pregnancy in dogs: a pilot study.

PubMed

Holst, Bodil S; Kushnir, Mark M; Bergquist, Jonas

2015-12-01

Blood samples from dogs are often limited in volume, only allowing few steroids to be quantified with immunoassays. In addition, immunoassays may be compromised by interferences such as anti-reagent antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used for the simultaneous quantitation of several steroids. This has not been described in dogs before. The aims were to use LC-MS/MS to study steroid profiles in early pregnancy and luteal phase in dogs, and to determine if differences exist between pregnant (P) and nonpregnant (NP) dogs. Nine female dogs were included, 4 during a NP luteal phase, 4 during a P luteal phase, and one during one NP and one P luteal phase. Blood samples were collected around the time of the LH surge (Day 0) and on Day 26. Serum was analyzed for 5 classes of steroids, including glucocorticoids, androgens, estrogens, pregnanes, and progestins, using LC-MS/MS methods. The concentration of progesterone was significantly higher on Day 26 in P than in NP bitches. Distribution of concentrations of glucocorticoids, androgens, estrogens, or pregnanes in P and NP dogs were not statistically different. The predominating glucocorticoid was cortisol, and dihydroepiandrosterone (DHEA) was the predominating androgen. Concentration of estrone was comparable to oestradiol, whereas concentrations of pregnenolone were higher than those of 17-OH pregnenolone. Only concentration of progesterone differed between P and NP bitches, being significantly higher on Day 26 in P than in NP bitches. LC-MS/MS offers interesting possibilities for studies of canine reproductive endocrinology. © 2015 American Society for Veterinary Clinical Pathology.

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

LC-UV assay method and UPLC/Q-TOF-MS characterisation of saponins from Ilex paraguariensis A. St. Hil. (mate) unripe fruits.

PubMed

Peixoto, Maria Paula Garofo; Kaiser, Samuel; Verza, Simone Gasparin; de Resende, Pedro Ernesto; Treter, Janine; Pavei, Cabral; Borré, Gustavo Luís; Ortega, George González

2012-01-01

Ilex paraguariensis A. St. Hil. (mate) is known in several South American countries because of the use of its leaves in stimulant herbal beverages. High saponin contents were reported in mate leaves and unripe fruits that possess a dissimilar composition. Two LC-UV methods previously reported for mate saponins assay focused on mate leaves and the quantification of the less polar saponin fraction in mate fruits. To develop and validate a LC-UV method to assay the total content of saponins in unripe mate fruits and characterise the chemical structure of triterpenic saponins by UPLC/Q-TOF-MS. From unripe fruits of mate a crude ethanolic extract was prepared (EX40) and the mate saponin fraction (MSF) purified by solid phase extraction. The LC-UV method was validated using ilexoside II as external standard. UPLC/Q-TOF-MS was adjusted from the LC-UV method to obtain the fragmentation patterns of the main saponins present in unripe fruits. Both LC-UV and UPLC/Q-TOF-MS methods indicate a wide range of Ilex saponins polarity. The ilexoside II and total saponin content of EX40 were 8.20% (w/w) and 47.60% (w/w), respectively. The total saponin content in unripe fruits was 7.28% (w/w). The saponins present in MSF characterised by UPLC/Q-TOF-MS are derived mainly from ursolic/oleanolic, acetyl ursolic or pomolic acid. The validated LC-UV method was shown to be linear, precise, accurate and to cover several saponins previously isolated from Ilex species and could be applied for the quality control of unripe fruit saponins. Copyright © 2011 John Wiley & Sons, Ltd.

Fatty Acids Suppress Autophagic Turnover in β-Cells*

PubMed Central

Las, Guy; Serada, Sam B.; Wikstrom, Jakob D.; Twig, Gilad; Shirihai, Orian S.

2011-01-01

Recent studies have shown that autophagy is essential for proper β-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in β-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in β-cells, contributing to their toxic effect. INS1 cells expressing GFP-LC3 (an autophagosome marker) were treated with 0.4 mm palmitate, 0.4 mm oleate, and various concentrations of glucose for 22 h. Kinetics of the effect of fatty acids on autophagy showed a biphasic response. During the second phase of autophagy, the size of autophagosomes and the content of autophagosome substrates (GFP-LC3, p62) and endogenous LC3 was increased. During the same phase, fatty acids suppressed autophagic degradation of long lived protein in both INS1 cells and islets. In INS1 cells, palmitate induced a 3-fold decrease in the number and the acidity of Acidic Vesicular Organelles. This decrease was associated with a suppression of hydrolase activity, suppression of endocytosis, and suppression of oxidative phosphorylation. The combination of fatty acids with glucose synergistically suppressed autophagic turnover, concomitantly suppressing insulin secretion. Rapamycin treatment resulted in partial reversal of the inhibition of autophagic flux, the inhibition of insulin secretion, and the increase in cell death. Our results indicate that excess nutrient could impair autophagy in the long term, hence contributing to nutrient-induced β-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes. PMID:21859708

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

PubMed

Nguyen, Duc Doan; Busetti, Francesco; Johnson, Stuart Keith; Solah, Vicky Ann

2018-03-01

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

New ordered metastable phases between the gel and subgel phases in hydrated phospholipids.

PubMed Central

Tenchov, B; Koynova, R; Rapp, G

2001-01-01

Formation of low-temperature ordered gel phases in several fully hydrated phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) with saturated chains as well as in dipalmitoylphosphatidylglycerol (DPPG) was observed by synchrotron x-ray diffraction, microcalorimetry, and densitometry. The diffraction patterns recorded during slow cooling show that the gel-phase chain reflection cooperatively splits into two reflections, signaling a transformation of the usual gel phase into a more ordered phase, with an orthorhombic chain packing (the Y-transition). This transition is associated with a small decrease (2-4 microl/g) or inflection of the partial specific volume. It is fully reversible with the temperature and displays in heating direction as a small (0.1-0.7 kcal/mol) endothermic event. We recorded a Y-transition in distearoyl PE, dipalmitoyl PE (DPPE), mono and dimethylated DPPE, distearoyl PC, dipalmitoyl PC, diC(15)PC, and DPPG. No such transition exists in dimyristoyl PE and dilauroyl PE where the gel L(beta) phase transforms directly into subgel L(c) phase, as well as in the unsaturated dielaidoyl PE. The PE and PC low-temperature phases denoted L(R1) and SGII, respectively, have different hydrocarbon chain packing. The SGII phase is with tilted chains, arranged in an orthorhombic lattice of two-nearest-neighbor type. Except for the PCs, it was also registered in ionized DPPG. In the L(R1) phase, the chains are perpendicular to the bilayer plane and arranged in an orthorhombic lattice of four-nearest-neighbor type. It was observed in PEs and in protonated DPPG. The L(R1) and SGII phases are metastable phases, which may only be formed by cooling the respective gel L(beta) and L(beta') phases, and not by heating the subgel L(c) phase. Whenever present, they appear to represent an indispensable intermediate step in the formation of the latter phase. PMID:11259300

A novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) based bioanalytical method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) and its application in pharmacokinetic study.

PubMed

Viswanathan, Sekarbabu; Verma, P R P; Ganesan, Muniyandithevar; Manivannan, Jeganathan

2017-07-15

Omega-3 fatty acids are clinically useful and the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are prevalent in fish and fish oils. Omega-3 fatty acid formulations should undergo a rigorous regulatory step in order to obtain United States Food and Drug Administration (USFDA) approval as prescription drug. In connection with that, despite quantifying EPA and DHA fatty acids, there is a need for quantifying the level of ethyl esters of them in biological samples. In this study, we make use of reverse phase high performance liquid chromatography coupled with mass spectrometry (RP-HPLC-MS)technique for the method development. Here, we have developed a novel multiple reaction monitoring method along with optimized parameters for quantification of EPA and DHA as ethyl esters. Additionally, we attempted to validate the bio-analytical method by conducting the sensitivity, selectivity, precision accuracy batch, carryover test and matrix stability experiments. Furthermore, we also implemented our validated method for evaluation of pharmacokinetics of omega fatty acid ethyl ester formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

Confirmatory and quantitative analysis of beta-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry.

PubMed

Fagerquist, Clifton K; Lightfield, Alan R; Lehotay, Steven J

2005-03-01

A simple, rapid, rugged, sensitive, and specific method for the confirmation and quantitation of 10 beta-lactam antibiotics in fortified and incurred bovine kidney tissue has been developed. The method uses a simple solvent extraction, dispersive solid-phase extraction (dispersive-SPE) cleanup, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) for confirmation and quantitation. Dispersive-SPE greatly simplifies and accelerates sample cleanup and improves overall recoveries compared with conventional SPE cleanup. The beta-lactam antibiotics tested were as follows: deacetylcephapirin (an antimicrobial metabolite of cephapirin), amoxicillin, desfuroylceftiofur cysteine disulfide (DCCD, an antimicrobial metabolite of ceftiofur), ampicillin, cefazolin, penicillin G, oxacillin, cloxacillin, naficillin, and dicloxacillin. Average recoveries of fortified samples were 70% or better for all beta-lactams except DCCD, which had an average recovery of 58%. The LC/MS/MS method was able to demonstrate quantitative recoveries at established tolerance levels and provide confirmatory data for unambiguous analyte identification. The method was also tested on 30 incurred bovine kidney samples obtained from the USDA Food Safety and Inspection Service, which had previously tested the samples using the approved semiquantitative microbial assay. The results from the quantitative LC/MS/MS analysis were in general agreement with the microbial assay for 23 samples although the LC/MS/MS method was superior in that it could specifically identify which beta-lactam was present and quantitate its concentration, whereas the microbial assay could only identify the type of beta-lactam present and report a concentration with respect to the microbial inhibition of a penicillin G standard. In addition, for 6 of the 23 samples, LC/MS/MS analysis detected a penicillin and a cephalosporin beta-lactam, whereas the microbial assay detected only a penicillin beta-lactam. For samples that do not fall into the "general agreement" category, the most serious discrepancy involves two samples where the LC/MS/MS method detected a violative level of a cephalosporin beta-lactam (deacetylcephapirin) in the first sample and a possibly violative level of desfuroylceftiofur in the second, whereas the microbial assay identified the two samples as having only violative levels of a penicillin beta-lactam.

Combination of Bottom-up 2D-LC-MS and Semi-top-down GelFree-LC-MS Enhances Coverage of Proteome and Low Molecular Weight Short Open Reading Frame Encoded Peptides of the Archaeon Methanosarcina mazei.

PubMed

Cassidy, Liam; Prasse, Daniela; Linke, Dennis; Schmitz, Ruth A; Tholey, Andreas

2016-10-07

The recent discovery of an increasing number of small open reading frames (sORF) creates the need for suitable analytical technologies for the comprehensive identification of the corresponding gene products. For biological and functional studies the knowledge of the entire set of proteins and sORF gene products is essential. Consequently in the present study we evaluated analytical approaches that will allow for simultaneous analysis of widest parts of the proteome together with the predicted sORF. We performed a full proteome analysis of the methane producing archaeon Methanosarcina mazei strain Gö1 cytosolic proteome using a high/low pH reversed phase LC-MS bottom-up approach. The second analytical approach was based on semi-top-down strategy, encompassing a separation at intact protein level using a GelFree system, followed by digestion and LC-MS analysis. A high overlap in identified proteins was found for both approaches yielding the most comprehensive coverage of the cytosolic proteome of this organism achieved so far. The application of the second approach in combination with an adjustment of the search criteria for database searches further led to a significant increase of sORF peptide identifications, finally allowing to detect and identify 28 sORF gene products.

Chiral HPLC for a study of the optical purity of new liquid crystalline materials derived from lactic acid

NASA Astrophysics Data System (ADS)

Vojtylová, T.; Kašpar, M.; Hamplová, V.; Novotná, V.; Sýkora, D.

2014-08-01

New liquid crystalline (LC) materials were prepared by derivatization of lactic acid. First compound possesses the lactic acid unit as the only chiral center and the second group of LC materials contains two chiral centers. Mesomorphic properties of both the newly synthesized LC materials were studied and the presence of the SmA*-SmC* or exhibit the twist grain boundary (TGB) phases, namely TGBA and TGBC, in a wide range of temperatures down to the room temperature was established. The potential of high-performance liquid chromatography (HPLC) applying chiral stationary phases to separate enantiomers or diastereoisomers of the synthesized LC compounds was evaluated. Two different brands of commercial chiral sorbents, Lux Amylose-2 and Chiralpak AD-3, both based on modified silica with derivatized polysaccharide, were employed in the development of separation procedures. The optimized chiral HPLC method provided a baseline separation of the individual enantiomers for the LC material containing one chiral center. In the case of the more complex compound with two asymmetric carbon atoms, where four isomers exist, partial separation was reached only using the current chiral HPLC.

Supercritical fluid reverse micelle separation

DOEpatents

Fulton, John L.; Smith, Richard D.

1993-01-01

A method of separating solute material from a polar fluid in a first polar fluid phase is provided. The method comprises combining a polar fluid, a second fluid that is a gas at standard temperature and pressure and has a critical density, and a surfactant. The solute material is dissolved in the polar fluid to define the first polar fluid phase. The combined polar and second fluids, surfactant, and solute material dissolved in the polar fluid is maintained under near critical or supercritical temperature and pressure conditions such that the density of the second fluid exceeds the critical density thereof. In this way, a reverse micelle system defining a reverse micelle solvent is formed which comprises a continuous phase in the second fluid and a plurality of reverse micelles dispersed in the continuous phase. The solute material is dissolved in the polar fluid and is in chemical equilibrium with the reverse micelles. The first polar fluid phase and the continuous phase are immiscible. The reverse micelles each comprise a dynamic aggregate of surfactant molecules surrounding a core of the polar fluid. The reverse micelle solvent has a polar fluid-to-surfactant molar ratio W, which can vary over a range having a maximum ratio W.sub.o that determines the maximum size of the reverse micelles. The maximum ratio W.sub.o of the reverse micelle solvent is then varied, and the solute material from the first polar fluid phase is transported into the reverse micelles in the continuous phase at an extraction efficiency determined by the critical or supercritical conditions.

Supercritical fluid reverse micelle separation

DOEpatents

Fulton, J.L.; Smith, R.D.

1993-11-30

A method of separating solute material from a polar fluid in a first polar fluid phase is provided. The method comprises combining a polar fluid, a second fluid that is a gas at standard temperature and pressure and has a critical density, and a surfactant. The solute material is dissolved in the polar fluid to define the first polar fluid phase. The combined polar and second fluids, surfactant, and solute material dissolved in the polar fluid is maintained under near critical or supercritical temperature and pressure conditions such that the density of the second fluid exceeds the critical density thereof. In this way, a reverse micelle system defining a reverse micelle solvent is formed which comprises a continuous phase in the second fluid and a plurality of reverse micelles dispersed in the continuous phase. The solute material is dissolved in the polar fluid and is in chemical equilibrium with the reverse micelles. The first polar fluid phase and the continuous phase are immiscible. The reverse micelles each comprise a dynamic aggregate of surfactant molecules surrounding a core of the polar fluid. The reverse micelle solvent has a polar fluid-to-surfactant molar ratio W, which can vary over a range having a maximum ratio W[sub o] that determines the maximum size of the reverse micelles. The maximum ratio W[sub o] of the reverse micelle solvent is then varied, and the solute material from the first polar fluid phase is transported into the reverse micelles in the continuous phase at an extraction efficiency determined by the critical or supercritical conditions. 27 figures.

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

PubMed

Periat, Aurélie; Kohler, Isabelle; Thomas, Aurélien; Nicoli, Raul; Boccard, Julien; Veuthey, Jean-Luc; Schappler, Julie; Guillarme, Davy

2016-03-25

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode. Copyright © 2015 Elsevier B.V. All rights reserved.

Optically addressed and submillisecond response phase only liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Zhao, Xiangjie; Duan, Jiazhu; Zhang, Dayong; Luo, Yongquan

2014-10-01

Liquid crystal based phase only spatial light modulator has attracted many research interests since last decades because of its superior advantage. Until now the liquid crystal spatial light modulator has been applied in many fields, but the response speed of nematic LC limited its further application. In this paper, an optically addressed phase only LC spatial light modulator was proposed based on polymer network liquid crystal. Morphology effect on the light scattering of PNLC was studied, which was mainly consisted of fiber and fiber bundles. The morphology nearly determined the light scattering and electro-optical property. Due to the high threshold voltage, to address the PNLC phase modulator was also concerned. Optical addressing method was proposed, in which BSO crystal was selected to replace one of the glass substrate. The response speed of PNLC was so fast that the reorientation of liquid crystal director will follow the change of effective voltage applied on LC layer, which was related with the voltage signal and especially with electron transport of photo-induced carriers due to diffusion and drift. The on state dynamic response of phase change was investigated. Based on this device, beam steering was also achieved by loading 488nm laser strip on the optical addressed phase only spatial light modulator.

Determination of alkylphenols and alkylphenol mono- and diethoxylates in environmental samples by high-performance liquid chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahel, M.; Giger, W.

1985-07-01

A routine method is described for the quantitative determination of 4-nonylphenol (NP) and 4-nonylphenol mono-(NP1EO) and diethoxylate (NP2EO) in samples from wastewater and sludge treatment and from the aquatic environment. An exhaustive steam-distillation/solvent-extraction procedure was employed to enrich the analytes from aqueous and solid samples. Quantitative determinations were performed by normal-phase high-performance liquid chromatography (HP-LC) using aminosilica columns. Relative standard deviations were 3.0-4.4% in a river water containing 3.9 ..mu..g/L NP, 23.4 ..mu..g/L NP1EO, and 9.4 ..mu..g/L NP2EO. A digested sewage sludge with 1.6 g of NP/kg of dry matter was analyzed with a relative standard deviation of 3.7%. Recoveriesmore » were higher than 80%, and the estimated detection limit in water samples was 0.5 ..mu..g/L. Reversed-phase HPLC on octylsilica provided complementary qualitative data, particularly on homologous alkylphenolic compounds. Good agreement was found between quantitative determinations by HPLC and by high-resolution gas chromatography with flame ionization detection and directly coupled mass spectrometry. Municipal wastewater effluents, sewage sludges, and natural waters were analyzed to demonstrate the method's broad applicability. 19 references, 4 tables, 4 figures.« less

Analytical Stability-Indicating Methods for Alogliptin in Tablets by LC-CAD and LC-UV.

PubMed

Bertol, Charise Dallazem; Friedrich, Maria Tereza; Carlos, Graciela; Froehlich, Pedro Eduardo

2017-03-01

Stability-indicating LC methods using a UV detector and a charged aerosol detector (CAD) simultaneously were validated for the assessment of alogliptin (ALG) in tablets. The analysis was performed on a C8 column (250 × 4.6 mm, 5 μm) at a flow of 0.8 mL/min, using acetonitrile-10 mM ammonium acetate buffer (pH 3.5; 90 + 10, v/v) as mobile phase and UV detection at 275 nm. Validation followed the International Conference on Harmonization guidelines. The method was linear over the range of 25-200 μg/mL. Normality of the residuals showed a normal distribution, no autocorrelation, and homoscedasticity. LODs were 6.25 and 2.65 µg/mL and LOQs were 20.85 and 8.84 µg/mL for the CAD and the UV detector, respectively. The methods were precise and accurate. Excipients and degradation products did not interfere in the methods in studies of specificity. None of the factors studied in the analysis of robustness had a significant effect on the quantification of the ALG by the Pareto chart. The results of the assay obtained with LC-CAD and LC-UV were similar. The methods could be considered interchangeable and stability-indicating, and can be applied as an appropriate QC tool for analysis of ALG in tablets.

Determination of mercury compounds in fish by microwave-assisted extraction and liquid chromatography-vapor generation-inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Chiou, Chwei-Sheng; Jiang, Shiuh-Jen; Kumar Danadurai, K. Suresh

2001-07-01

A method employing a vapor generation system and LC combined with inductively coupled plasma mass spectrometry (LC-ICP-MS) is presented for the determination of mercury in biological tissues. An open vessel microwave digestion system was used to extract the mercury compounds from the sample matrix. The efficiency of the mobile phase, a mixture of L-cysteine and 2-mercaptoethanol, was evaluated for LC separation of inorganic mercury [Hg(II)], methylmercury (methyl-Hg) and ethylmercury (ethyl-Hg). The sensitivity, detection limits and repeatability of the liquid chromatography (LC) ICP-MS system with a vapor generator were comparable to, or better than, that of an LC-ICP-MS system with conventional pneumatic nebulization, or other sample introduction techniques. The experimental detection limits for various mercury species were in the range of 0.05-0.09 ng ml -1 Hg, based on peak height. The proposed method was successfully applied to the determination of mercury compounds in a swordfish sample purchased from the local market. The accuracy of the method was evaluated by analyzing a marine biological certified reference material (DORM-2, NRCC).

[Identification of impurity peaks in the HPLC chromatogram by LC-MS and two-dimensional chromatographic correlation spectroscopy].

PubMed

Chen, Zhen-Zhen; Zhang, Dou-Sheng; Wang, Nan; Feng, Fang; Hu, Chang-Qin

2012-04-01

A novel qualitative analytical method by using two-dimensional chromatographic correlation spectroscopy techniques for recognizing impurity peaks of HPLC methods of quality control and LC-MS chromatographic system was established. The structures of major degradation products of ceftizoxime and cefdinir were identified by LC-MS and MassWorks application; the standard chromatographic and spectral data of the degradation impurities were obtained by high-performance liquid chromatography with diode array detection. The impurity peaks of two-dimensional chromatography were matched by comparison of spectra and calculating correlation coefficients. Peaks in chromatography can be identified accurately and rapidly in different chromatographic systems such as column and mobile phase changed. The method provides a new way and thought to identify the peaks in quality control of impurities without reference impurity substances.

Multiplicative effects model with internal standard in mobile phase for quantitative liquid chromatography-mass spectrometry.

PubMed

Song, Mi; Chen, Zeng-Ping; Chen, Yao; Jin, Jing-Wen

2014-07-01

Liquid chromatography-mass spectrometry assays suffer from signal instability caused by the gradual fouling of the ion source, vacuum instability, aging of the ion multiplier, etc. To address this issue, in this contribution, an internal standard was added into the mobile phase. The internal standard was therefore ionized and detected together with the analytes of interest by the mass spectrometer to ensure that variations in measurement conditions and/or instrument have similar effects on the signal contributions of both the analytes of interest and the internal standard. Subsequently, based on the unique strategy of adding internal standard in mobile phase, a multiplicative effects model was developed for quantitative LC-MS assays and tested on a proof of concept model system: the determination of amino acids in water by LC-MS. The experimental results demonstrated that the proposed method could efficiently mitigate the detrimental effects of continuous signal variation, and achieved quantitative results with average relative predictive error values in the range of 8.0-15.0%, which were much more accurate than the corresponding results of conventional internal standard method based on the peak height ratio and partial least squares method (their average relative predictive error values were as high as 66.3% and 64.8%, respectively). Therefore, it is expected that the proposed method can be developed and extended in quantitative LC-MS analysis of more complex systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Study of matrix effects on the direct trace analysis of acidic pesticides in water using various liquid chromatographic modes coupled to tandem mass spectrometric detection.

PubMed

Dijkman, E; Mooibroek, D; Hoogerbrugge, R; Hogendoorn, E; Sancho, J V; Pozo, O; Hernández, F

2001-08-10

This study investigated the effects of matrix interferences on the analytical performance of a triple quadrupole mass spectrometric (MS-MS) detector coupled to various reversed-phase liquid chromatographic (LC) modes for the on-line determination of various types of acidic herbicides in water using external calibration for quantification of the analytes tested at a level of 0.4 microg/l. The LC modes included (i) a single-column configuration (LC), (ii) precolumn switching (PC-LC) and (iii) coupled-column LC (LC-LC). As regards detection, electrospray (ESI) and atmospheric pressure chemical ionization (APCI) in both positive (PI) and negative (NI) ionization modes were examined. Salinity and dissolved organic carbon (DOC) were selected as interferences to study matrix effects in this type of analysis. Therefore, Milli-Q and tap water samples both fortified with 12 mg/l DOC and spiked with sulfometuron-methyl, bentazone, bromoxynil, 2-methyl-4-chlorophenoxyacetic acid, and 2-methyl-4-chlorophenoxypropionic acid at a level of about 0.4 microg/l were analyzed with the various LC-MS approaches. Direct sample injection was performed with volumes of 0.25 ml or 2.0 ml on a column of 2.1 mm I.D. or 4.6 mm I.D. for the ESI and APCI modes, respectively. The recovery data were used to compare and evaluate the analytical performance of the various LC approaches. As regards matrix effects, the salinity provided a dramatic decrease in response for early eluting analytes (k value of about 1) when using the LC mode. Both PC-LC and LC-LC efficiently eliminated this problem. The high DOC content hardly effected the responses of analytes in the ESI mode, while in most cases the responses increased when using APCI-MS-MS detection. Of all the tested configurations, LC-LC-ESI-MS-MS with the column combination Discovery C18/ABZ+ was the most favorable as regards elimination of matrix effects and provided reliable quantification of all compounds using external calibration at the tested low level. The major observed effects were verified with statistical evaluation of the data employing backwards ordinary least-square regression. All tested column-switching modes hyphenated to ESI- or APCI-MS-MS allowed the on-line multi-residue analysis of acidic pesticides in the reference water down to a level of 0.1 microg/l in less than 10 min, emphasizing the feasibility of such an approach in this field of analysis.

Fine golden rings: Tunable surface plasmon resonance from assembled nanorods in topological defects of liquid crystals

DOE PAGES

Lee, Elaine; Xia, Yu; Ferrier, Jr., Robert C.; ...

2016-02-08

Unprecedented, reversible, and dynamic control over an assembly of gold nanorods dispersed in liquid crystals (LC) is demonstrated. The LC director field is dynamically tuned at the nanoscale using microscale ring confinement through the interplay of elastic energy at different temperatures, thus fine-tuning its core replacement energy to reversibly sequester nanoscale inclusions at the microscale. As a result, this leads to shifts of 100 nm or more in the surface plasmon resonance peak, an order of magnitude greater than any previous work with AuNR composites.

Advantages of a validated UPLC-MS/MS standard addition method for the quantification of A-type dimeric and trimeric proanthocyanidins in cranberry extracts in comparison with well-known quantification methods.

PubMed

van Dooren, Ines; Foubert, Kenn; Theunis, Mart; Naessens, Tania; Pieters, Luc; Apers, Sandra

2018-01-30

The berries of Vaccinium macrocarpon, cranberry, are widely used for the prevention of urinary tract infections. This species contains A-type proanthocyanidins (PACs), which intervene in the initial phase of the development of urinary tract infections by preventing the adherence of Escherichia coli by their P-type fimbriae to uroepithelial cells. Unfortunately, the existing clinical studies used different cranberry preparations, which were poorly standardized. Because of this, the results were hard to compare, which led sometimes to conflicting results. Currently, PACs are quantified using the rather non-specific spectrophotometric 4-dimethylaminocinnamaldehyde (DMAC) method. In addition, a normal phase HPTLC-densitometric method, a HPLC-UV method and three LC-MS/MS methods for quantification of procyanidin A2 were recently published. All these methods contain some shortcomings and errors. Hence, the development and validation of a fast and sensitive standard addition LC-MS/MS method for the simultaneous quantification of A-type dimers and trimers in a cranberry dry extract was carried out. A linear calibration model could be adopted for dimers and, after logaritmic transformation, for trimers. The maximal interday and interconcentration precision was found to be 4.86% and 4.28% for procyanidin A2, and 5.61% and 7.65% for trimeric PACs, which are all acceptable values for an analytical method using LC-MS/MS. In addition, twelve different cranberry extracts were analyzed by means of the newly validated method and other widely used methods. There appeared to be an enormous variation in dimeric and trimeric PAC content. Comparison of these results with LC-MS/MS analysis without standard addition showed the presence of matrix effects for some of the extracts and proved the necessity of standard addition. A comparison of the well-known and widely used DMAC method, the butanol-HCl assay and this newly developed LC-MS/MS method clearly indicated the need for a reliable method able to quantify A-type PACs, which are considered to be the pharmacologically active constituents of cranberry, since neither the DMAC or butanol-HCl assays are capable of distinguishing between A and B-type PACs and therefore cannot detect adulterations with, for example, extracts with a high B-type PAC content. Hence, the combination of the DMAC method or butanol-HCl assay with this more specific LC-MS/MS assay could overcome these shortcomings. Copyright © 2017 Elsevier B.V. All rights reserved.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

PubMed

Venkatramani, C J; Al-Sayah, Mohammad; Li, Guannan; Goel, Meenakshi; Girotti, James; Zang, Lisa; Wigman, Larry; Yehl, Peter; Chetwyn, Nik

2016-02-01

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented. Copyright © 2015 Elsevier B.V. All rights reserved.

Multiresidue method for N-methyl carbamates and metabolite pesticide residues at the parts-per-billion level in selected representative commodities of fruit and vegetable crop groups.

PubMed

Podhorniak, Lynda V; Schenck, Frank J; Krynitsky, Alexander; Griffith, Francis

2004-01-01

A reversed-phase liquid chromatographic method with both fluorescence and mass spectrometric detection is presented for the determination of 13 parent N-methyl carbamate pesticides and their metabolites, as well as piperonyl butoxide, for a total of 24 compounds in selected fruits and vegetables. The commodities chosen were of special concern to the U.S. Environmental Protection Agency (EPA) because they had the least amount of monitoring data for dietary exposure estimates used in risk assessment. The method is based on a judicious selection of procedures from U.S. Food and Drug Administration sources such as the Pesticide Analytical Manual (Volume I), and Laboratory Information Bulletins, plus additional material from the chemical literature combined in a manner to recover the N-methyl carbamates and their metabolites at the 1 microg/kg or 1 part-per-billion level. The method uses an acetone extraction, followed by an aminopropyl solid-phase extraction cleanup. Determination of residues is by RP-LC, in which the liquid chromatograph is interfaced with either a fluorescence or a mass spectrometric detector. The method is designed so that a set of 6 samples can be prepared in 1 working day for overnight instrumental analysis. Recovery data are presented from analyses of selected commodities in some of EPA's fruit and vegetable crop groupings. A table listing relative retention times is presented for the N-methyl carbamates and their metabolites.

Quantitative determination of flavonoids by column high-performance liquid chromatography with mass spectrometry and ultraviolet absorption detection in Artemisia afra and comparative studies with various species of Artemisia plants.

PubMed

Avula, Bharathi; Wang, Yan-Hong; Smillie, Troy J; Mabusela, Wilfred; Vincent, Leszek; Weitz, Frans; Khan, Ikhlas A

2009-01-01

A simple and specific analytical method for the quantitative determination of flavonoids from the aerial parts of the Artemisia afra plant samples was developed. By column high-performance liquid chromatography (HPLC) with UV absorption and mass spectrometry (MS) detection, separation was achieved on a reversed-phase octadecylsilyl (C18) column with water, methanol, and acetonitrile, all containing 0.1% acetic acid, as the mobile phase. These methods were used to analyze various species of Artemisia plant samples. The wavelength used for quantification of flavonoids with the diode array detector was 335 nm. The limits of detection (LOD) by HPLC/MS were found to be 7.5, 7.5, 10, 2.0, and 2.0 ng/mL; and by LC-UV the LODs were 500, 500, 500, 300, and 300 ng/mL for apigenin, chrysoeriol, tamarixetin, acacetin, and genkwanin, respectively. The HPLC/MS method was found to be 50-150 times more sensitive than the HPLC-UV method. HPLC/MS coupled with an electrospray ionization interface is described for the identification and quantification of flavonoids in various plant samples. This method involved the use of the [M+H]+ ions of the compounds at mass-to-charge ratio of 1.0606, 301.0712, 317.0661, 285.0763, and 285.0763 (calculated mass), respectively, in the positive ion mode with extractive ion monitoring.

A validated ultra-high-performance liquid chromatography-tandem mass spectrometry method for the selective analysis of free and total folate in plasma and red blood cells.

PubMed

Kiekens, Filip; Van Daele, Jeroen; Blancquaert, Dieter; Van Der Straeten, Dominique; Lambert, Willy E; Stove, Christophe P

2015-06-12

A stable isotope dilution LC-MS/MS method is the method of choice for the selective quantitative determination of several folate species in clinical samples. By implementing an integrated approach to determine both the plasma and red blood cell (RBC) folate status, the use of consumables and time remains limited. Starting from a single 300μl whole blood sample, the folate status in plasma and RBCs can be determined after separating plasma and RBCs and sequential washing of the latter with isotonic buffer, followed by reproducible lysis using an ammonium-based buffer. Acidification combines both liberation of protein bound folates and protein precipitation. Sample cleanup is performed using a 96-well reversed-phase solid-phase extraction procedure, similar for both plasma and RBC samples. Analyses are performed by UHPLC-MS/MS. Method validation was successfully performed based on EMA-guidelines and encompassed selectivity, carry-over, linearity, accuracy, precision, recovery, matrix effect and stability. Plasma and RBC folates could be quantified in the range of 1-150nmol/l and 5-1500nmol/l, respectively. This method allows for the determination of 6 folate monoglutamates in both plasma and RBCs. It can be used to determine short and long term folate status in both normal and severely deficient subjects in a single analytical sequence. Copyright © 2015 Elsevier B.V. All rights reserved.

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256

PubMed Central

Sroka, Jolanta; Krecioch, Izabela; Zimolag, Eliza; Lasota, Slawomir; Rak, Monika; Kedracka-Krok, Sylwia; Borowicz, Pawel; Gajek, Marta; Madeja, Zbigniew

2016-01-01

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement—specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1–3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways. PMID:26863616

Lamellipodia and Membrane Blebs Drive Efficient Electrotactic Migration of Rat Walker Carcinosarcoma Cells WC 256.

PubMed

Sroka, Jolanta; Krecioch, Izabela; Zimolag, Eliza; Lasota, Slawomir; Rak, Monika; Kedracka-Krok, Sylwia; Borowicz, Pawel; Gajek, Marta; Madeja, Zbigniew

2016-01-01

The endogenous electric field (EF) may provide an important signal for directional cell migration during wound healing, embryonic development and cancer metastasis but the mechanism of cell electrotaxis is poorly understood. Additionally, there is no research addressing the question on the difference in electrotactic motility of cells representing various strategies of cell movement-specifically blebbing vs. lamellipodial migration. In the current study we constructed a unique experimental model which allowed for the investigation of electrotactic movement of cells of the same origin but representing different modes of cell migration: weakly adherent, spontaneously blebbing (BC) and lamellipodia forming (LC) WC256 cells. We report that both BC and LC sublines show robust cathodal migration in a physiological EF (1-3 V/cm). The directionality of cell movement was completely reversible upon reversing the field polarity. However, the full reversal of cell direction after the change of EF polarity was much faster in the case of BC (10 minutes) than LC cells (30 minutes). We also investigated the distinct requirements for Rac, Cdc42 and Rho pathways and intracellular Ca2+ in electrotaxis of WC256 sublines forming different types of cell protrusions. It was found that Rac1 is required for directional movement of LC to a much greater extent than for BC, but Cdc42 and RhoA are more crucial for BC than for LC cells. The inhibition of ROCK did not affect electrotaxis of LC in contrast to BC cells. The results also showed that intracellular Ca2+ is essential only for the electrotactic reaction of BC cells. Moreover, inhibition of MLCK and myosin II did not affect the electrotaxis of LC in contrast to BC cells. In conclusion, our results revealed that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of WC 256 carcinosarcoma cells, however directional migration is mediated by different signalling pathways.

Quantitative determination of a synthetic amide derivative of gallic acid, SG-HQ2, using liquid chromatography tandem mass spectrometry, and its pharmacokinetics in rats.

PubMed

Seo, Seung-Yong; Kang, Wonku

2016-11-30

An amide derivative of gallic acid (GA), 3,4,5-trihydroxy-N-(8-hydroxyquinolin-2-yl)benzamide) (SG-HQ2) was recently synthesized, and its inhibitory actions were previously shown on histamine release and pro-inflammatory cytokine expression. In this study, a simultaneous quantification method was developed for the determination of SG-HQ2 and its possible metabolite, GA, in rat plasma using liquid chromatography with a tandem mass spectrometry (LC-MS/MS). After simple protein precipitation with acetonitrile including diclofenac (internal standard, IS), the analytes were chromatographed on a reversed phased column with a mobile phase of acetonitrile and water (60:40, v/v, including 0.1% formic acid). The ion transitions of the precursor to the product ion were principally protonated ion [M+H] + at m/z 313.2→160.6 for SG-HQ2, and deprotonated ions [M-H] - at m/z 168.7→124.9 for GA and 296.0→251.6 for the IS. The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was successfully applied to a pharmacokinetic study of SG-HQ2 after intravenous administration in rats. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization.

PubMed

Ziegler, Jörg; Abel, Steffen

2014-12-01

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC-ESI-MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using L-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using L-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).

Blue phase liquid crystal phase transition for cyano compound chiral nematic liquid crystal mixtures with three two-ring core structures and chiral dopant concentrations

NASA Astrophysics Data System (ADS)

Shin, Jaesun; Kim, Beomjong; Jung, Wansu; Fahad, Mateen; Park, SangJin; Hong, Sung-Kyu

2017-05-01

Blue phase (BP) temperature range of a chiral nematic liquid crystal (LC) mixture is dependent upon the host nematic LC chemical structure and chiral dopant concentration. In this study, we investigated BP phase transition behaviour and helical twisting power (HTP) using three chiral dopant concentrations of cyano compound chiral nematic LC mixtures incorporating three two-ring core structures in the host nematic LCs. The effect of the host nematic LC core structure, HTP and chiral dopant concentrations were considered on BP temperature ranges, for two types of complete BPI and BPII without isotropic phase (Iso) and two types of coexistence state of BPI+Iso and BPII+Iso.

Sensitive determination of nitrophenol isomers by reverse-phase high-performance liquid chromatography in conjunction with liquid-liquid extraction

USDA-ARS?s Scientific Manuscript database

A method for the highly sensitive determination of 2-, 3- and 4- nitrophenols was developed using reverse-phase high-performance liquid chromatography (RP-HPLC) with a UV photodiode array detector. Using a reverse-phase column and 40% aqueous acetonitrile as an eluent (i.e. isocratic elution), the i...

Development and validation of a high throughput assay for the quantification of multiple green tea-derived catechins in human plasma.

PubMed

Mawson, Deborah H; Jeffrey, Keon L; Teale, Philip; Grace, Philip B

2018-06-19

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilises protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed-phase liquid chromatography - tandem mass spectrometry (LC-MS/MS). Traditional issues such as lengthy chromatographic run times, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high throughput sample analysis studies. This article is protected by copyright. All rights reserved.

Direct aqueous determination of glyphosate and related compounds by liquid chromatography/tandem mass spectrometry using reversed-phase and weak anion-exchange mixed-mode column.

PubMed

Hao, Chunyan; Morse, David; Morra, Franca; Zhao, Xiaoming; Yang, Paul; Nunn, Brian

2011-08-19

Analysis of the broad-spectrum herbicide glyphosate and its related compounds is quite challenging. Tedious and time-consuming derivatization is often required for these substances due to their high polarity, high water solubility, low volatility and molecular structure which lacks either a chromophore or fluorophore. A novel liquid chromatography/tandem mass spectrometry (LC/MS-MS) method has been developed for the determination of glyphosate, aminomethylphosphonic acid (AMPA) and glufosinate using a reversed-phase and weak anion-exchange mixed-mode Acclaim® WAX-1 column. Aqueous environmental samples are directly injected and analyzed in 12 min with no sample concentration or derivatization steps. Two multiple reaction monitoring (MRM) channels are monitored in the method for each target compound to achieve true positive identification, and ¹³C, ¹⁵N-glyphosate is used as an internal standard to carry out isotope dilution mass spectrometric (IDMS) measurement for glyphosate. The instrument detection limits (IDLs) for glyphosate, AMPA and glufosinate are 1, 2 and 0.9 μg/L, respectively. Linearity of the detector response with a minimum coefficient of determination (R² value (R² > 0.995) was demonstrated in the range of ∼10 to 10³ μg/L for each analytes. Spiked drinking water, surface water and groundwater samples were analyzed using this method and the average recoveries of analytes in three matrices ranged from 77.0 to 102%, 62.1 to 101%, 66.1 to 93.7% while relative standard deviation ranged from 6.3 to 10.2%, 2.7 to 14.8%, 2.9 to 10.7%, respectively. Factors that may affect method performance, such as metal ions, sample preservation, and storage time, are also discussed. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Spatial resolution limitation of liquid crystal spatial light modulator

NASA Astrophysics Data System (ADS)

Wang, Xinghua; Wang, Bin; McManamon, Paul F., III; Pouch, John J.; Miranda, Felix A.; Anderson, James E.; Bos, Philip J.

2004-10-01

The effect of fringing electric fields in a liquid crystal (LC) Optical Phased Array (OPA), also referred to as a spatial light modulator (SLM), is a governing factor that determines the diffraction efficiency (DE) of the LC OPA for high resolution spatial phase modulation. In this article, the fringing field effect in a high resolution LC OPA is studied by accurate modeling the DE of the LC blazed gratings by LC director simulation and Finite Difference Time Domain (FDTD) simulation. Influence factors that contribute significantly to the DE are discussed. Such results provide fundamental understanding for high resolution LC devices.

Surrogate analyte approach for quantitation of endogenous NAD(+) in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

PubMed

Liu, Liling; Cui, Zhiyi; Deng, Yuzhong; Dean, Brian; Hop, Cornelis E C A; Liang, Xiaorong

2016-02-01

A high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for the quantitative determination of NAD(+) in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. (13)C5-NAD(+) was used as the surrogate analyte for authentic analyte, NAD(+). The standard curve ranging from 0.250 to 25.0μg/mL in acidified human blood for (13)C5-NAD(+) was fitted to a 1/x(2) weighted linear regression model. The LC-MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD(+) concentration from the (13)C5-NAD(+) standard curve since the percent difference was less than 5%. The precision and accuracy of the LC-MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of (13)C5-NAD(+) was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD(+) in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD(+) in 10 human subjects. Copyright © 2015 Elsevier B.V. All rights reserved.

Microstrip technology and its application to phased array compensation

NASA Technical Reports Server (NTRS)

Dudgeon, J. E.; Daniels, W. D.

1972-01-01

A systematic analysis of mutual coupling compensation using microstrip techniques is presented. A method for behind-the-array coupling of a phased antenna array is investigated as to its feasibility. The matching scheme is tried on a rectangular array of one half lambda 2 dipoles, but it is not limited to this array element or geometry. In the example cited the values of discrete components necessary were so small an L-C network is needed for realization. Such L-C tanks might limit an otherwise broadband array match, however, this is not significant for this dipole array. Other areas investigated were balun feeding and power limits of spiral antenna elements.

Morphology and kinetics of the formation of polyamide benzimidazole in N-methylpyrrolidone

NASA Astrophysics Data System (ADS)

Khanchich, O. A.; Larionov, V. B.; Biryukov, A. L.

2018-02-01

Polymerization-optical methods were used to study the supermolecular structure in the gelation of systems based on polyamidobenzimidazole (PABI) in N-methylpyrrolidone (N-MP) and the kinetics of phase transformations during polymer coagulation from a the solution. It was found that the use of N-MP leads to the appearance in the PABI-N-MP-H2O system of spherulites having a structure characteristic of LC structures. It is shown that the alternation of metastable structures upon gelation of the PABY - N-MP-H2O system is consistent with the appearance of the LC phase of the cholesteric type.

LC separation of calcipotriol from its photodegradation products and protection possibilities using adjuvants.

PubMed

Cirunay, J J; Vander Heyden, Y; Plaizier-Vercammen, J

2001-08-01

Mobile phase optimization and reversed-phase column characteristics were used to separate photodegradation products from the parent compound, 24-cyclopropyl-9-,10-secochola-5,7,10(19),22-tetraene-1alpha,3beta,24-triol (calcipotriol). Separation between calcipotriol and its degradation products was obtained with an acetonitrile/water (53:47, v/v) mobile phase on a C(18) Hypersil ODS column (250 mm length, 4.6 mm id, 5 microm particle size) and a flow rate of 1 ml/min. Using this system, the influence of commonly used solvents in dermatology on degradation was studied. The addition of a UV filter in two concentrations was also evaluated for its possible protective effect to light exposure. Propylene glycol and polyethylene glycol 400 decreased the speed of degradation. The sunscreen 2-hydroxy-4-methoxybenzophenone affords a protection proportional to the filter concentration used in the study.

Measurement of Intracellular Ribavirin Mono-, Di- and Triphosphate Using Solid Phase Extraction and LC-MS/MS Quantification

PubMed Central

Jimmerson, Leah C.; Ray, Michelle L.; Bushman, Lane R.; Anderson, Peter L.; Klein, Brandon; Rower, Joseph E.; Zheng, Jia-Hua; Kiser, Jennifer J.

2014-01-01

Ribavirin (RBV) is a nucleoside analog used to treat a variety of DNA and RNA viruses. RBV undergoes intracellular phosphorylation to a mono- (MP), di- (DP), and triphosphate (TP). The phosphorylated forms have been associated with the mechanisms of antiviral effect observed in vitro, but the intracellular pharmacology of the drug has not been well characterized in vivo. A highly sensitive LC-MS/MS method was developed and validated for the determination of intracellular RBV MP, DP, and TP in multiple cell matrix types. For this method, the individual MP, DP, and TP fractions were isolated from lysed intracellular matrix using strong anion exchange solid phase extraction, dephosphorylated to parent RBV, desalted and concentrated and quantified using LC-MS/MS. The method utilized a stable labeled internal standard (RBV-13C5) which facilitated accuracy (% deviation within ±15%) and precision (coefficient of variation of ≤15%). The quantifiable linear range for the assay was 0.50 to 200 pmol/sample. The method was applied to the measurement of RBV MP, DP, and TP in human peripheral blood mononuclear cells (PBMC), red blood cells (RBC), and dried blood spot (DBS) samples obtained from patients taking RBV for the treatment of chronic Hepatitis C virus infection. PMID:25555148

Improvement of a stability-indicating method by Quality-by-Design versus Quality-by-Testing: a case of a learning process.

PubMed

Hubert, C; Lebrun, P; Houari, S; Ziemons, E; Rozet, E; Hubert, Ph

2014-01-01

The understanding of the method is a major concern when developing a stability-indicating method and even more so when dealing with impurity assays from complex matrices. In the presented case study, a Quality-by-Design approach was applied in order to optimize a routinely used method. An analytical issue occurring at the last stage of a long-term stability study involving unexpected impurities perturbing the monitoring of characterized impurities needed to be resolved. A compliant Quality-by-Design (QbD) methodology based on a Design of Experiments (DoE) approach was evaluated within the framework of a Liquid Chromatography (LC) method. This approach allows the investigation of Critical Process Parameters (CPPs), which have an impact on Critical Quality Attributes (CQAs) and, consequently, on LC selectivity. Using polynomial regression response modeling as well as Monte Carlo simulations for error propagation, Design Space (DS) was computed in order to determine robust working conditions for the developed stability-indicating method. This QbD compliant development was conducted in two phases allowing the use of the Design Space knowledge acquired during the first phase to define the experimental domain of the second phase, which constitutes a learning process. The selected working condition was then fully validated using accuracy profiles based on statistical tolerance intervals in order to evaluate the reliability of the results generated by this LC/ESI-MS stability-indicating method. A comparison was made between the traditional Quality-by-Testing (QbT) approach and the QbD strategy, highlighting the benefit of this QbD strategy in the case of an unexpected impurities issue. On this basis, the advantages of a systematic use of the QbD methodology were discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Performance of plasma free metanephrines measured by liquid chromatography-tandem mass spectrometry in the diagnosis of pheochromocytoma.

PubMed

Peaston, Robert T; Graham, Kendon S; Chambers, Erin; van der Molen, Jan C; Ball, Stephen

2010-04-02

Plasma free metanephrines have proved a highly sensitive biochemical test for the diagnosis of pheochromocytoma. We have developed and validated a simple, LC-MS/MS method to determine plasma metanephrines and compared the diagnostic efficacy of the method with an enzyme immunoassay procedure in 151 patients, 38 with histologically confirmed pheochromocytoma. Off-line solid phase extraction in a 96-well plate format was used to isolate metanephrines from 100-microL of plasma, followed by rapid separation with hydrophilic interaction chromatography. Mass spectrometry detection was performed in multiple-reaction monitoring mode using a tandem quadrupole mass spectrometer with positive electrospray ionization. Detection limits were <0.1nmol/l with method linearity up to 23.0nmol/L for normetanephrine (NMN), metanephrine (MN) and 3-methoxytyramine (3-MT). Method comparison with an automated LC-MS/MS yielded Deming regression slopes of r=0.94 for NMN, r=0.98 for MN and r=0.94 for 3-MT. Method comparison with enzyme immunoassay revealed regression slope of r=1.28 (NMN) and 1.25 (MN) with values approximately 25% lower than LC-MS/MS. Plasma metanephrines by LC-MS/MS identified all 38 patients with phaeochromocytoma compared with 36 cases by immunoassay. Plasma metanephrines measured by LC-MS/MS are a reliable and sensitive test for the biochemical detection of pheochromocytoma. 2010 Elsevier B.V. All rights reserved.

LC-MS/MS signal suppression effects in the analysis of pesticides in complex environmental matrices.

PubMed

Choi, B K; Hercules, D M; Gusev, A I

2001-02-01

The application of LC separation and mobile phase additives in addressing LC-MS/MS matrix signal suppression effects for the analysis of pesticides in a complex environmental matrix was investigated. It was shown that signal suppression is most significant for analytes eluting early in the LC-MS analysis. Introduction of different buffers (e.g. ammonium formate, ammonium hydroxide, formic acid) into the LC mobile phase was effective in improving signal correlation between the matrix and standard samples. The signal improvement is dependent on buffer concentration as well as LC separation of the matrix components. The application of LC separation alone was not effective in addressing suppression effects when characterizing complex matrix samples. Overloading of the LC column by matrix components was found to significantly contribute to analyte-matrix co-elution and suppression of signal. This signal suppression effect can be efficiently compensated by 2D LC (LC-LC) separation techniques. The effectiveness of buffers and LC separation in improving signal correlation between standard and matrix samples is discussed.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

PubMed

Parr, Maria Kristina; Wuest, Bernhard; Naegele, Edgar; Joseph, Jan F; Wenzel, Maxi; Schmidt, Alexander H; Stanic, Mijo; de la Torre, Xavier; Botrè, Francesco

2016-09-01

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases.

PubMed

Borges, Endler M

2014-01-07

Three RP-LC column characterization protocols [Tanaka et al. (1989), Snyder et al. (PQRI, 2002), and NIST SRM 870 (2000)] were evaluated using both Euclidian distance and Principal Components Analysis to evaluate effectiveness at identifying equivalent columns. These databases utilize specific chromatographic properties such as hydrophobicity, hydrogen bonding, shape/steric selectivity, and ion exchange capacity of stationary phases. The chromatographic parameters of each test were shown to be uncorrelated. Despite this, the three protocols were equally successful in identifying similar and/or dissimilar stationary phases. The veracity of the results has been supported by some real life pharmaceutical separations. The use of Principal Component Analysis to identify similar/dissimilar phases appears to have some limitations in terms of loss of information. In contrast, the use of Euclidian distances is a much more convenient and reliable approach. The use of auto scaled data is favoured over the use of weighted factors as the former data transformation is less affected by the addition or removal of columns from the database. The use of these free databases and their corresponding software tools shown to be valid for identifying similar columns with equivalent chromatographic selectivity and retention as a "backup column". In addition, dissimilar columns with complimentary chromatographic selectivity can be identified for method development screening strategies. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry

PubMed Central

Jimmerson, Leah C.; Zheng, Jia-Hua; Bushman, Lane R.; MacBrayne, Christine E.; Anderson, Peter L.; Kiser, Jennifer J.

2014-01-01

Efficient, inexpensive and sensitive assays for the measurement of drugs are of interest for pharmacokinetic and pharmacodynamics (PK-PD) analysis. Dried blood spots (DBS) are a unique bioanaltyical matrix with the potential to fulfill this interest for the measurement of numerous analytes. Here we describe the development and validation of a reversed-phase high performance liquid chromatographic (LC), tandem mass spectrometry (MS/MS) assay for the determination of ribavirin (RBV) in DBS. A 3mm punch from spotted and dried whole blood was extracted in methanol utilizing isotopically labeled internal standard for LC-MS/MS analysis. Validation was performed over a range of 0.05 μg/mL to 10.0 μg/mL and the method was shown to be precise (coefficient of variation ≤ 15%) and accurate (within ±15% of control). These acceptance criteria were met for hematocrit ranges of 20-54%, for center versus edge punches and for spot volumes from 10-60 μL. RBV was stable for up to 140 days at room temperature and −20°C as well as for three freeze/thaw cycles. Correlation of RBV in DBS versus in plasma yielded r2 ≥ 0.98 demonstrating that DBS can be used as an alternative to plasma for PK-PD studies in human subjects. PMID:24291608

Simultaneous determination of 17 sulfonamides and the potentiators ormetoprim and trimethoprim in salmon muscle by liquid chromatography with tandem mass spectrometry detection.

PubMed

Potter, Ross A; Burns, B Garth; van de Riet, Jeffrey M; North, David H; Darvesh, Rozina

2007-01-01

A simple, robust method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of 17 sulfonamides [sulfanilamide (SNL), sulfacetamide (SAA), sulfaguanidine (SGD), sulfapyridine (SPY), sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethoxazole (SOZ), sulfamoxole (SXL), sulfisoxazole (SXZ), sulfamethizole (SML), sulfamethazine (SMZ), sulfamethoxypyridazine (SMP), sulfamonomethoxine (SMM), sulfachloropyridazine (SCP), sulfaquinoxaline (SQX), and sulfadimethoxine (SDM)] and 2 potentiators [ormetoprim (OMP) and trimethoprim (TMP)] in fish tissue has been developed. The analytes were extracted from homogenized fish tissue with water-acetonitrile (50 + 50). The extract was clarified by centrifugation and a portion defatted with hexane. The analytes were partitioned into chloroform and evaporated to dryness. The redissolved residue was applied to a C18 reversed-phase column with a water-acetonitrile (0.1% acetic acid) gradient. All of the compounds were completely separated and detected in <10 min at 30 degrees C using LC/MS/MS. Standard curves were linear over the range of 0.02 to 5 ng injected. The limit of detection varied from 0.1 ng/g for SMZ and OMP to 0.9 ng/g for SXL and SOZ. Recoveries varied from 100% for SDM, SOZ, and SQX and 85% for SMR, OMP, and TMP to approximately 30% for SAA. Relative standard deviations for repeat analysis varied from 4% for SMZ and SCP to 23% for SAA.

Development and Validation of an LC-MS-MS Method for Determination of Simvastatin and Simvastatin Acid in Human Plasma: Application to a Pharmacokinetic Study.

PubMed

Partani, Pankaj; Verma, Saurabh Manaswita; Monif, Tausif

2016-09-01

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of simvastatin (SV) and simvastatin acid (SVA) in human plasma. To improve assay sensitivity and achieve simultaneous analysis, SVA monitored in (-)ESI (electrospray ionization) mode within the first 4.5 min and SV thereafter in (+)ESI mode. The separation of all compounds was achieved in about 6.2 min using a C18 reverse-phase fused-core(®) column (Ascentis(®) Express C18) and a mobile phase, which was composed of 2.00 ± 0.05 mM ammonium acetate buffer titrated to pH 3.8 with glacial acetic acid-acetonitrile (25:75, v/v), in isocratic mode at a flow rate of 0.500 mL/min. Additionally, a solid-phase extraction step was performed to reduce any ion-suppression and/or enhancement effects. The developed method was linear in the concentration range of 0.100-74.626 ng/mL for SV, and 0.100-48.971 ng/mL for SVA, with correlation coefficient greater than 0.99 for both analytes. The method has shown tremendous reproducibility, with intra- and inter-day precision <7.6%, and intra- and interday accuracy within ±10.9% of nominal values, for the both analytes. The method was successfully applied to characterize the pharmacokinetic profiles of SV and SVA following an oral administration of 40 mg SV tablet to healthy human volunteers. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

UPLC and LC-MS studies on degradation behavior of irinotecan hydrochloride and development of a validated stability-indicating ultra-performance liquid chromatographic method for determination of irinotecan hydrochloride and its impurities in pharmaceutical dosage forms.

PubMed

Kumar, Navneet; Sangeetha, Dhanaraj; Reddy, Sunil P

2012-10-01

The objective of the current investigation was to study the degradation behavior of irinotecan hydrochloride under different International Conference on Harmonization (ICH) recommended stress conditions using ultra-performance liquid chromatography and liquid chromatography-mass spectrometry and to establish a validated stability-indicating reverse-phase ultra-performance liquid chromatographic method for the quantitative determination of irinotecan hydrochloride and its seven impurities and degradation products in pharmaceutical dosage forms. Irinotecan hydrochloride was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal and photolytic degradation. Irinotecan hydrochloride was found to degrade significantly in oxidative and base hydrolysis and photolytic degradation conditions. The degradation products were well resolved from the main peak and its impurities, thus proving the stability-indicating power of the method. Chromatographic separation was achieved on a Waters Acquity BEH C8 (100 × 2.1 mm) 1.7-µm column with a mobile phase containing a gradient mixture of solvent A (0.02M KH(2)PO(4) buffer, pH 3.4) and solvent B (a mixture of acetonitrile and methanol in the ratio of 62:38 v/v). The mobile phase was delivered at a flow rate of 0.3 mL/min with ultraviolet detection at 220 nm. The run time was 8 min, within which irinotecan and its seven impurities and degradation products were satisfactorily separated. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also suitable for the assay determination of irinotecan hydrochloride in pharmaceutical dosage forms.

Automated solid-phase extraction-liquid chromatography-tandem mass spectrometry analysis of 6-acetylmorphine in human urine specimens: application for a high-throughput urine analysis laboratory.

PubMed

Robandt, P V; Bui, H M; Scancella, J M; Klette, K L

2010-10-01

An automated solid-phase extraction-liquid chromatography- tandem mass spectrometry (SPE-LC-MS-MS) method using the Spark Holland Symbiosis Pharma SPE-LC coupled to a Waters Quattro Micro MS-MS was developed for the analysis of 6-acetylmorphine (6-AM) in human urine specimens. The method was linear (R² = 0.9983) to 100 ng/mL, with no carryover at 200 ng/mL. Limits of quantification and detection were found to be 2 ng/mL. Interrun precision calculated as percent coefficient of variation (%CV) and evaluated by analyzing five specimens at 10 ng/mL over nine batches (n = 45) was 3.6%. Intrarun precision evaluated from 0 to 100 ng/mL ranged from 1.0 to 4.4%CV. Other opioids (codeine, morphine, oxycodone, oxymorphone, hydromorphone, hydrocodone, and norcodeine) did not interfere in the detection, quantification, or chromatography of 6-AM or the deuterated internal standard. The quantified values for 41 authentic human urine specimens previously found to contain 6-AM by a validated gas chromatography (GC)-MS method were compared to those obtained by the SPE-LC-MS-MS method. The SPE-LC-MS-MS procedure eliminates the human factors of specimen handling, extraction, and derivatization, thereby reducing labor costs and rework resulting from human error or technique issues. The time required for extraction and analysis was reduced by approximately 50% when compared to a validated 6-AM procedure using manual SPE and GC-MS analysis.

Dynamic evolution of light-induced orientation of dye-doped liquid crystals in liquid phase studied by time-resolved optically heterodyned optical Kerr effect technique.

PubMed

Yang, Pei; Liu, Liying; Xu, Lei

2008-02-28

Transient evolution of light-induced molecular reorientation both in 1-amino-anthraquinone (1AAQ) dye and azobenzene doped isotropic liquid crystals (LCs) were studied by time-resolved optically heterodyned optical Kerr effect method. The results give clear direct experimental proof that under short pulse (30 ps) excitation, LC molecules orientate toward the excitation light polarization direction in the 1AAQ/LC system. However, LC molecular orientation becomes orthogonal to the light polarization in azobenzene/LC system. Time-resolved excited-state absorption of 1AAQ and wavelength dependent excited-state absorption of azobenzene were also observed and their contributions to the early dynamics of the third order optical responses of the two systems were confirmed. A simplified two-level mean-field theory was derived to reveal the intensity dependence of orientation enhancement factor in azobenzene/LC system considering the photoisomerization process.

Characterization of the components of meleumycin by liquid chromatography with photo-diode array detection and electrospray ionization tandem mass spectrometry.

PubMed

Wang, Ming-Juan; Li, Ya-Ping; Wang, Yan; Li, Jin; Hu, Chang-Qin; Hoogmartens, Jos; Van Schepdael, Ann; Adams, Erwin

2013-10-01

Reversed-phase liquid chromatography coupled with photo-diode array (PDA) detection and electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to characterize the components of meleumycin, a 16-membered macrolide antibiotic produced by fermentation. In total 31 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 others that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known components. Their ultraviolet spectra and chromatographic behavior were used to confirm the proposed structures: e.g. λmax shift from 232 nm to 282 nm would indicate the presence of an α-, β-, γ-, δ-unsaturated ketone instead of a normal α-, β-, γ-, δ-unsaturated alcohol in the 16-membered ring of the examined components. Compared to other methods, this LC/MS(n) method is particularly advantageous to characterize minor components at trace levels in multi-components antibiotics, in terms of sensitivity and efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study.

PubMed

Lee, Heon-Woo; Seo, Ji-Hyung; Choi, Seung-Ki; Lee, Kyung-Tae

2007-01-30

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5>166.1 for itopride and m/z 342.3>111.6 for IS, respectively. Analytes were chromatographed on an YMC C18 reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r2=0.9999) over the studied range (0.5-1000 ng mL(-1)) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

Analysis of phenolic choline esters from seeds of Arabidopsis thaliana and Brassica napus by capillary liquid chromatography/electrospray- tandem mass spectrometry.

PubMed

Böttcher, Christoph; von Roepenack-Lahaye, Edda; Schmidt, Jürgen; Clemens, Stephan; Scheel, Dierk

2009-04-01

Total phenolic choline ester fractions prepared from seeds of Arabidopsis thaliana and Brassica napus were analyzed by capillary LC/ESI-QTOF-MS and direct infusion ESI-FTICR-MS. In addition to the dominating sinapoylcholine, 30 phenolic choline esters could be identified based on accurate mass measurements, interpretation of collision-induced dissociation (CID) mass spectra, and synthesis of selected representatives. The compounds identified so far include substituted hydroxycinnamoyl- and hydroxybenzoylcholines, respective monohexosides as well as oxidative coupling products of phenolic choline esters and monolignols. Phenolic choline esters are well separable by reversed-phase liquid chromatography and sensitively detectable using electrospray ionization mass spectrometry in positive ion mode. CID mass spectra obtained from molecular ions facilitate the characterization of both the type and substitution pattern of such compounds. Therefore, LC/ESI-MS/MS represents a valuable tool for comprehensive qualitative and quantitative analysis of this compound class. Copyright (c) 2009 John Wiley & Sons, Ltd.

Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.

2008-03-07

Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® andmore » X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.« less

Transient phases during crystallization of solution-processed organic thin films

NASA Astrophysics Data System (ADS)

Wan, Jing; Li, Yang; Ulbrandt, Jeffery; Smilgies, Detlef-M.; Hollin, Jonathan; Whalley, Adam; Headrick, Randall

We report an in-situ study of 2,7-dioctyl[1]benzothieno[3,2-b][1]benzothiophene (C8-BTBT) organic semiconductor thin film deposition from solution via hollow pen writing, which exhibits multiple transient phases during crystallization. Under high writing speed (25 mm/s) the films have an isotropic morphology, although the mobilities range up to 3.0 cm2/V.s. To understand the crystallization in this highly non-equilibrium regime, we employ in-situ microbeam grazing incidence wide-angle X-ray scattering combined with optical video microscopy at different deposition temperatures. A sequence of crystallization was observed in which a layered liquid-crystalline (LC) phase of C8-BTBT precedes inter-layer ordering. For films deposited above 80ºC, a transition from LC phase to a transient crystalline state that we denote as Cr1 occurs after a temperature-dependent incubation time, which is consistent with classical nucleation theory. After an additional ~ 0.5s, Cr1 transforms to the final stable structure Cr2. Based on these results, we demonstrate a method to produce large crystalline grain size and high carrier mobility during high-speed processing by controlling the nucleation rate during the transformation from the LC phase. Nsf DMR-1307017, NSF DMR-1332208.

Can Mesenchymal Stem Cells Reverse Chronic Stress-Induced Impairment of Wound Healing Following Traumatic Injury?

PubMed Central

Gore, Amy V.; Bible, Letitia E.; Livingston, David H.; Mohr, Alicia M.; Sifri, Ziad C.

2015-01-01

Intro One week following unilateral lung contusion (LC), rat lungs demonstrate full histologic recovery. When animals undergo LC plus the addition of chronic restraint stress (CS), wound healing is significantly delayed. Mesenchymal stem cells (MSC) are pluripotent cells capable of immunomodulation that have been the focus of much research in wound healing and tissue regeneration. We hypothesize that the addition of MSCs will improve wound healing in the setting of CS. Methods Male Sprague-Dawley rats (n=6-7/group) were subjected to LC/CS with or without the injection of MSCs. MSCs were given as a single IV dose of 5 × 106 cells in 1mL IMDM media at the time of LC. Rats were subjected to two hours of restraint stress on days 1-6 following LC. Seven days following injury, rats were sacrificed and lungs examined for histologic evidence of wound healing using a well-established histologic lung injury score (LIS) to grade injury. LIS examines inflammatory cells/high power field (hpf) averaged over 30 fields, interstitial edema, pulmonary edema, and alveolar integrity with scores ranging from 0 (normal) to 11 (highly damaged). Peripheral blood was analyzed by flow cytometry for the presence of T-regulatory (C4+CD25+FoxP3+) cells. Data analyzed by ANOVA followed by Tukey’s multiple comparison test, expressed as mean ± SD. Results As previously shown, seven days following isolated LC, LIS has returned to 0.83 ± 0.41, with a subscore of zero for inflammatory cells/hpf. The addition of CS results in a LIS score of 4.4 ± 2.2, with a subscore of 1.9 ± 0.7 for inflammatory cells/hpf. Addition of MSC to LC/CS decreased LIS score to 1.7 ± 0.8 with a subscore of zero for inflammatory cells/hpf. Furthermore, treatment of animals undergoing LC/CS with MSCs increased the %T-regulatory cells by 70% in animals undergoing LC/CS alone (12.9 ± 2.4% vs 6.2 ± 1.3%) Conclusion Stress-induced impairment of wound healing is reversed by addition of MSCs given at the time of injury in this rat lung contusion model. This improvement in lung healing is associated with a decrease in the number of inflammatory cells and an increase in the number of T regulatory cells. Further study into the mechanisms by which MSCs hasten wound healing is warranted. PMID:25807405

Reversible, on-demand generation of aqueous two-phase microdroplets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collier, Charles Patrick; Retterer, Scott Thomas; Boreyko, Jonathan Barton

The present invention provides methods of on-demand, reversible generation of aqueous two-phase microdroplets core-shell microbeads, microparticle preparations comprising the core-shell microbeads, and drug delivery formulation comprising the microparticle preparations. Because these aqueous microdroplets have volumes comparable to those of cells, they provide an approach to mimicking the dynamic microcompartmentation of biomaterial that naturally occurs within the cytoplasm of cells. Hence, the present methods generate femtoliter aqueous two-phase droplets within a microfluidic oil channel using gated pressure pulses to generate individual, stationary two-phase microdroplets with a well-defined time zero for carrying out controlled and sequential phase transformations over time. Reversible phasemore » transitions between single-phase, two-phase, and core-shell microbead states are obtained via evaporation-induced dehydration and water rehydration.« less

Androgen profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in healthy normal-weight ovulatory and anovulatory late adolescent and young women.

PubMed

Fanelli, Flaminia; Gambineri, Alessandra; Belluomo, Ilaria; Repaci, Andrea; Di Lallo, Valentina Diana; Di Dalmazi, Guido; Mezzullo, Marco; Prontera, Olga; Cuomo, Gaia; Zanotti, Laura; Paccapelo, Alexandro; Morselli-Labate, Antonio Maria; Pagotto, Uberto; Pasquali, Renato

2013-07-01

Physiological transient imbalance typical of adolescence needs to be distinguished from hyperandrogenism-related dysfunction. The accurate determination of circulating androgens is the best indicator of hyperandrogenism. However, reliable reference intervals for adolescent and young women are not available. The aim of the study was to define androgen reference intervals in young women and to analyze the impact of the menstrual phase and ovulation efficiency over the androgen profile as assessed by reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. Female high school students aged 16-19 years were included in the study. The study was performed on reference subjects properly selected among an unbiased population. Normal-weight, drug and disease free, eumenorrheic females with no signs of hyperandrogenism were included. The steroid hormone profile was determined by a validated in-house LC-MS/MS method. A statistical estimation of overall and menstrual phase-specific reference intervals was performed. A subgroup of anovulatory females was identified based on progesterone circulating levels. The impact of ovulation efficiency over hormonal profile was analyzed. A total of 159 females satisfied healthy criteria. Androgen levels did not vary according to menstrual phase, but a significantly higher upper reference limit was found for T in the luteal phase compared to the follicular phase. Higher T and androstenedione levels were observed in anovulatory compared to ovulatory females, paralleled by higher LH and FSH and lower 17-hydroxyprogesterone and 17β-estradiol levels. This is the first study providing LC-MS/MS-based, menstrual phase-specific reference intervals for the circulating androgen profile in young females. We identified a subgroup of anovulatory healthy females characterized by androgen imbalance.

A new approach based on off-line coupling of high-performance liquid chromatography with gas chromatography-mass spectrometry to determine acrylamide in coffee brew.

PubMed

Blanch, Gracia Patricia; Morales, Francisco José; Moreno, Fernando de la Peña; del Castillo, María Luisa Ruiz

2013-01-01

A new method based on off-line coupling of LC with GC in replacement of conventional sample preparation techniques is proposed to analyze acrylamide in coffee brews. The method involves the preseparation of the sample by LC, the collection of the selected fraction, its concentration under nitrogen, and subsequent analysis by GC coupled with MS. The composition of the LC mobile phase and the flow rate were studied to select those conditions that allowed separation of acrylamide without coeluting compounds. Under the conditions selected recoveries close to 100% were achieved while LODs and LOQs equal to 5 and 10 μg/L for acrylamide in brewed coffee were obtained. The method developed enabled the reliable detection of acrylamide in spiked coffee beverage samples without further clean-up steps or sample manipulation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated on-line SPE LC-MS/MS method to quantitate 6beta-hydroxycortisol and cortisol in human urine: use of the 6beta-hydroxycortisol to cortisol ratio as an indicator of CYP3A4 activity.

PubMed

Barrett, Yu Chen; Akinsanya, Billy; Chang, Shu-Ying; Vesterqvist, Ole

2005-07-25

A sensitive method for quantitation of urinary 6beta-hydroxycortisol (6beta-HC) and cortisol using on-line SPE and LC-MS/MS was developed and validated. Human urine samples were injected directly onto an on-line solid phase extraction apparatus, Prospekt-2, followed by HPLC separation and electrospray triple quadrupole LC-MS/MS detection. The inter-day precision for the 6beta-HC:cortisol ratio was 7-9%. The lower limit of quantitation was 1 and 0.2 ng/mL for 6beta-HC and cortisol, respectively. Using the method we observed a diurnal variation on the 6beta-HC:cortisol ratio in healthy volunteers with the maximal ratio observed in the 2-10 pm urine collection period.

Parallel achiral-chiral determination of oxybutynin, N-desethyl oxybutynin and their enantiomers in human plasma by LC-MS/MS to support a bioequivalence trial.

PubMed

Sharma, Primal; Patel, Daxesh P; Sanyal, Mallika; Guttikar, Swati; Shrivastav, Pranav S

2014-01-01

A parallel achiral and chiral determination of oxybutynin, its pharmacologically active metabolite N-desethyl oxybutynin and their enantiomers in human plasma is described using LC-MS/MS. Both the methods were developed and validated using deuterated analogues as internal standards. Achiral analysis of racemic oxybutynin and N-desethyl oxybutynin was carried out on Phenomenex Gemini C18 (150mm×4.6mm, 5μm) column under isocratic conditions using acetonitrile-5.0mM ammonium acetate, pH 4.0 (90:10, v/v) as the mobile phase. Separation of (S)- and (R)-enantiomers of the analytes was performed on Phenomenex Lux Amylose-2 (150mm×4.6mm, 3μm) chiral column using a mixture of solvent A [acetonitrile:10mM ammonium bicarbonate, 80:20 (v/v)] and solvent B [2-propanol:methanol, 50:50 (v/v)] in 20:80 (v/v) ratio as the mobile phase. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate-diethyl ether-n-hexane solvent mixture. A linear range was established from 0.025 to 10.0ng/mL and 0.25 to 100ng/mL for the enantiomers of oxybutynin and N-desethyl oxybutynin respectively. The extraction recovery varied from 96.0 to 105.1%, while the IS-normalized matrix factors ranged from 0.96 to 1.07 for all the enantiomers. The validated method was applied for a pilot bioequivalence study with 5mg oxybutynin tablet formulation in 8 healthy subjects. The pharmacokinetic profiles showed that the plasma concentration of (R)-oxybutynin was lower than that of (S)-oxybutynin, while a reverse trend was observed for the enantiomers of N-desethyl oxybutynin. The reproducibility in the measurement of study data was demonstrated by reanalysis of 20 incurred samples. Copyright © 2013 Elsevier B.V. All rights reserved.

In-situ phase transition from microemulsion to liquid crystal with the potential of prolonged parenteral drug delivery.

PubMed

Ren, Xiazhong; Svirskis, Darren; Alany, Raid G; Zargar-Shoshtari, Sara; Wu, Zimei

2012-07-15

This study is the first to investigate and demonstrate the potential of microemulsions (MEs) for sustained release parenteral drug delivery, due to phase transition behavior in aqueous environments. Phase diagrams were constructed with Miglyol 812N oil and a blend of (co)surfactants Solutol HS 15 and Span 80 with ethanol. Liquid crystal (LC) and coarse emulsion (CE) regions were found adjacent to the ME region in the water-rich corner of the phase diagram. Two formulations were selected, a LC-forming ME and a CE-forming ME and each were investigated with respect to their rheology, particle size, drug release profiles and particularly, the phase transition behavior. The spreadability in an aqueous environment was determined and release profiles from MEs were generated with gamma-scintigraphy. The CE-forming ME dispersed readily in an aqueous environment, whereas the LC-forming ME remained in a contracted region possibly due to the transition of ME to LC at the water/ME interface. Gamma-scintigraphy showed that the LC-forming ME had minimal spreadability and a slow release of (99m)Tc in the first-order manner, suggesting phase conversion at the interface. In conclusion, owing to the potential of phase transition, LC-forming MEs could be used as extravascular injectable drug delivery vehicles for prolonged drug release. Copyright © 2012 Elsevier B.V. All rights reserved.

Quality assessment of Herba Leonuri based on the analysis of multiple components using normal- and reversed-phase chromatographic methods.

PubMed

Dong, Shuya; He, Jiao; Hou, Huiping; Shuai, Yaping; Wang, Qi; Yang, Wenling; Sun, Zheng; Li, Qing; Bi, Kaishun; Liu, Ran

2017-12-01

A novel, improved, and comprehensive method for quality evaluation and discrimination of Herba Leonuri has been developed and validated based on normal- and reversed-phase chromatographic methods. To identify Herba Leonuri, normal- and reversed-phase high-performance thin-layer chromatography fingerprints were obtained by comparing the colors and R f values of the bands, and reversed-phase high-performance liquid chromatography fingerprints were obtained by using an Agilent Poroshell 120 SB-C18 within 28 min. By similarity analysis and hierarchical clustering analysis, we show that there are similar chromatographic patterns in Herba Leonuri samples, but significant differences in counterfeits and variants. To quantify the bio-active components of Herba Leonuri, reversed-phase high-performance liquid chromatography was performed to analyze syringate, leonurine, quercetin-3-O-robiniaglycoside, hyperoside, rutin, isoquercitrin, wogonin, and genkwanin simultaneously by single standard to determine multi-components method with rutin as internal standard. Meanwhile, normal-phase high-performance liquid chromatography was performed by using an Agilent ZORBAX HILIC Plus within 6 min to determine trigonelline and stachydrine using trigonelline as internal standard. Innovatively, among these compounds, bio-active components of quercetin-3-O-robiniaglycoside and trigonelline were first determined in Herba Leonuri. In general, the method integrating multi-chromatographic analyses offered an efficient way for the standardization and identification of Herba Leonuri. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two-dimensional liquid chromatography (LC) of phenolic compounds from the shoots of Rubus idaeus 'Glen Ample' cultivar variety.

PubMed

Kula, Marta; Głód, Daniel; Krauze-Baranowska, Mirosława

2016-03-20

In this study the application of two-dimensional LC (2D LC) for qualitative analysis of polyphenols and simple phenols in the shoots of Rubus idaeus 'Glen Ample' variety is presented. In the preliminary analysis, the methanol extract of the shoots was analyzed by one-dimensional LC. One-dimensional LC separation profiles of phenolics from R. idaeus 'Glen Ample' shoots were dependent on column type, mobile phase composition and gradient program used. Two-dimensional LC system was built from connecting an octadecyl C-18 silica column in the first dimension and pentafluorophenyl column in the second dimension, coupled with DAD and MS (ESI, APCI, DUIS ionization) detectors. A total of 34 phenolic compounds belonging to the groups of phenolic acids, ellagitannins, flavan-3-ols, flavonols and ellagic acid conjugates were identified in the shoots of R. idaeus 'Glen Ample'. The established 2D LC method offers an effective tool for analysis of phenolics present in Rubus species. Copyright © 2015 Elsevier B.V. All rights reserved.

ANALYSIS OF SELECTED PYRETHROID PESTICIDES USING REVERSE PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY/UV

EPA Science Inventory

This research was conducted in cooperation with EPA Region 4 in Athens, GA to develop a method to analyze selected pyrethroid pesticides using Reverse Phase-High Pressure Liquid Chromatography (HPLC). This HPLC method will aid researchers in separating and identifying these py...

Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

PubMed

Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

2015-05-01

Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.

Simultaneous determination and characterization of tannins and triterpene saponins from the fruits of various species of Terminalia and Phyllantus emblica using a UHPLC-UV-MS method: application to triphala.

PubMed

Avula, Bharathi; Wang, Yan-Hong; Wang, Mei; Shen, Yun-Heng; Khan, Ikhlas A

2013-01-01

Terminalia species are a rich source of tannins. Many preparations of these species are used in traditional medicine and have many different ethnobotanical applications. A simple UHPLC method was developed for the simultaneous analysis of such hydrolysable tannins and triterpene saponins from the fruit rinds of different species of Terminalia (T. chebula, T. arjuna, T. bellirica) and Phyllantus emblica. A separation by LC was achieved using a reversed-phase column and a water/acetonitrile mobile phase, both containing formic acid, using a gradient system and a temperature of 40°C. Eight hydrolysable tannins (gallic acid, gallic acid methyl ester, corilagin, chebulagic acid, 1,2,3,6-tetra-O-galloyl-β-D-glucose, ellagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-β-D-glucose) and six triterpene saponins (arjunglucoside-I, arjunglucoside-III, chebuloside II, bellericoside, arjunetin, and arjunglucoside-II) could be separated within 20 minutes. The wavelength used for detection with the diode array detector was 254 and 275 nm for tannins and 205 nm for triterpene saponins. The method was validated for linearity, repeatability, limits of detection, and limits of quantification. The developed method is economical, fast, and especially suitable for quality control analysis of tannins and triterpene saponins in various plant samples and commercial products of Terminalia. Georg Thieme Verlag KG Stuttgart · New York.

[Multi-residue method for screening of pesticides in crops by liquid chromatography with tandem mass spectrometry].

PubMed

Tanizawa, Haruna; Shima, Mikie; Ikehara, Chieko; Kobata, Masakazu; Sato, Motoaki

2005-10-01

A simple and rapid method was developed for the screening of 82 pesticides/metabolites in a wide variety of crops, using solid-phase extraction and liquid chromatography with tandem mass spectrometry (LC/MS/MS). After extraction with methanol, the filtered extracts were made up to 100 mL and a 2 mL aliquot was subjected to solid-phase extraction. Co-extractives were removed with a C18 mini-column, while pesticides were retained on 3 kinds of mini-columns (HLB, SAX, activated carbon), and then eluted with acetonitrile. Analysis was performed by LC/MS/MS, and MS acquisition parameters were established in positive and negative ESI modes. The utility of the method was demonstrated by the analysis of 6 crops (carrot, cabbage, onion, spinach, lemon, brown rice) and one mixed vegetable juice. Of 82 compounds tested, 75 in carrot and 62 in lemon were obtained with recoveries ranging from 70-120%. For all samples tested, 75 compounds could be obtained with recoveries of over 50%, and the detection limits of most compounds were lower than 0.01 microg/g. This method provides acceptable performance for analysis of these 75 compounds. Further, by using aliquots of the extracts with small-scale mini-columns, purified samples could be obtained. This proposed method with small matrix effects, is effective and suitable for screening of multiple residual pesticides by using LC/MS/MS.

Ethanol Reversal of Cellular Tolerance to Morphine in Rat Locus Coeruleus Neurons

PubMed Central

Llorente, Javier; Withey, Sarah; Rivero, Guadalupe; Cunningham, Margaret; Cooke, Alex; Saxena, Kunal; McPherson, Jamie; Oldfield, Sue; Dewey, William L.; Bailey, Chris P.; Kelly, Eamonn; Henderson, Graeme

2013-01-01

Consumption of ethanol is a considerable risk factor for death in heroin overdose. We sought to determine whether a mildly intoxicating concentration of ethanol could alter morphine tolerance at the cellular level. In rat locus coeruleus (LC) neurons, tolerance to morphine was reversed by acute exposure of the brain slice to ethanol (20 mM). Tolerance to the opioid peptide [d-Ala2,N-MePhe4,Gly-ol]-enkephalin was not reversed by ethanol. Previous studies in LC neurons have revealed a role for protein kinase C (PKC)α in μ-opioid receptor (MOPr) desensitization by morphine and in the induction and maintenance of morphine tolerance, but we have been unable to demonstrate that 20 mM ethanol produces significant inhibition of PKCα. The ability of ethanol to reverse cellular tolerance to morphine in LC neurons was absent in the presence of the phosphatase inhibitor okadaic acid, indicating that dephosphorylation is involved. In human embryonic kidney 293 cells expressing the MOPr, ethanol reduced the level of MOPr phosphorylation induced by morphine. Ethanol reversal of tolerance did not appear to result from a direct effect on MOPr since acute exposure to ethanol (20 mM) did not modify the affinity of binding of morphine to the MOPr or the efficacy of morphine for G-protein activation as measured by guanosine 5′-O-(3-[35S]thio)triphosphate binding. Similarly, ethanol did not affect MOPr trafficking. We conclude that acute exposure to ethanol enhances the effects of morphine by reversing the processes underlying morphine cellular tolerance. PMID:23716621

Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

PubMed

Lin, Chia-Wei; Haeuptle, Micha A; Aebi, Markus

2016-09-06

Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.

Determination of 17alpha-ethynylestradiol, carbamazepine, diazepam, simvastatin, and oxybenzone in fish livers.

PubMed

Kwon, Jeong-Wook; Armbrust, Kevin L; Vidal-Dorsch, Doris; Bay, Steven M

2009-01-01

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of 17alpha-ethynylestradiol in fish liver; a second method using LC/MS was developed for the determination of carbamazepine, diazepam, simvastatin, and oxybenzone in fish liver. The fish liver samples were extracted and cleaned up by using liquid-liquid extraction and solid-phase extraction before the extracts were analyzed by LC/MS or LC/MS/MS with electrospray negative and positive ionization. Recoveries of the 5 target compounds from spiked catfish liver ranged from 72 +/- 2 to 100 +/- 3%. Limits of quantification for the 5 compounds were between 4.2 and 12.3 ng/g (wet weight). Ten turbot (Pleuronichthys verticalis) liver samples were analyzed; levels of 17alpha-ethynylestradiol, carbamazepine, simvastatin, and oxybenzone were below the detection limits. Diazepam was detected in all 10 fish liver samples at concentrations ranging from 23 to 110 ng/g (wet weight).

LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs.

PubMed

Spanjer, Martien C; Rensen, Peter M; Scholten, Jos M

2008-04-01

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC-MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B1, B2, G1 and G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, alpha-zearalenol, alpha-zearalanol, beta-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B1, B2 and B3, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, alpha-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 microg kg(-1) and for deoxynivalenol 50 microg kg(-1). The quantification limits for the other mycotoxins were in the range 10-200 microg kg(-1). The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC-MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B1 and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.

High throughput screening of active pharmaceutical ingredients by UPLC.

PubMed

Al-Sayah, Mohammad A; Rizos, Panagiota; Antonucci, Vincent; Wu, Naijun

2008-07-01

Ultra performance LC (UPLC) was evaluated as an efficient screening approach to facilitate method development for drug candidates. Three stationary phases were screened: C-18, phenyl, and Shield RP 18 with column dimensions of 150 mm x 2.1 mm, 1.7 microm, which should theoretically generate 35,000 plates or 175% of the typical column plate count of a conventional 250 mm x 4.6 mm, 5 microm particle column. Thirteen different active pharmaceutical ingredients (APIs) were screened using this column set with a standardized mobile-phase gradient. The UPLC method selectivity results were compared to those obtained for these compounds via methods developed through laborious trial and error screening experiments using numerous conventional HPLC mobile and stationary phases. Peak capacity was compared for columns packed with 5 microm particles and columns packed with 1.7 microm particles. The impurities screened by UPLC were confirmed by LC/MS. The results demonstrate that simple, high efficiency UPLC gradients are a feasible and productive alternative to more conventional multiparametric chromatographic screening approaches for many compounds in the early stages of drug development.