Development of a novel amide-silica stationary phase for the reversed-phase HPLC separation of different classes of phytohormones.

PubMed

Aral, Hayriye; Aral, Tarık; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2013-11-15

A novel amide-bonded silica stationary phase was prepared starting from N-Boc-phenylalanine, cyclohexylamine and spherical silica gel (4 µm, 60 Å). The amide ligand was synthesised with high yield. The resulting amide bonded stationary phase was characterised by SEM, IR and elemental analysis. The resulting selector bearing a polar amide group is used for the reversed-phase chromatography separation of different classes of thirteen phytohormones (plant hormones). The chromatographic behaviours of these analytes on the amide-silica stationary phase were compared with those of RP-C18 column under same conditions. The effects of different separation conditions, such as mobile phase, pH value, flow rate and temperature, on the separation and retention behaviours of the 13 phytohormones in this system were studied. The optimum separation was achieved using reversed-phase HPLC gradient elution with an aqueous mobile phase containing pH=6.85 potassium phosphate buffer (20 mM) and acetonitrile with a 22 °C column temperature. Under these experimental conditions, the 12 phytohormones could be separated and detected at 230 or 270 nm within 26 min. Copyright © 2013 Elsevier B.V. All rights reserved.

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

PubMed

Nakamura, A; Watanabe, T

2001-04-01

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.

Development of a perfusion reversed-phase high performance liquid chromatography method for the characterisation of maize products using multivariate analysis.

PubMed

Rodriguez-Nogales, J M; Garcia, M C; Marina, M L

2006-02-03

A perfusion reversed-phase high performance liquid chromatography (RP-HPLC) method has been designed to allow rapid (3.4 min) separations of maize proteins with high resolution. Several factors, such as extraction conditions, temperature, detection wavelength and type and concentration of ion-pairing agent were optimised. A fine optimisation of the gradient elution was also performed by applying experimental design. Commercial maize products for human consumption (flours, precocked flours, fried snacks and extruded snacks) were characterised for the first time by perfusion RP-HPLC and their chromatographic profiles allowed a differentiation among products relating the different technological process used for their preparation. Furthermore, applying discriminant analysis makes it possible to group the samples according with the technological process suffered by maize products, obtaining a good prediction in 92% of the samples.

Simultaneous separation and analysis of water- and fat-soluble vitamins on multi-modal reversed-phase weak anion exchange material by HPLC-UV.

PubMed

Dabre, Romain; Azad, Nazanin; Schwämmle, Achim; Lämmerhofer, Michael; Lindner, Wolfgang

2011-04-01

Several methods for the separation of vitamins on HPLC columns were already validated in the last 20 years. However, most of the techniques focus on separating either fat- or water-soluble vitamins and only few methods are intended to separate lipophilic and hydrophilic vitamins simultaneously. A mixed-mode reversed-phase weak anion exchange (RP-WAX) stationary phase was developed in our laboratory in order to address such mixture of analytes with different chemical characteristics, which are difficult to separate on standard columns. The high versatility in usage of the RP-WAX chromatographic material allowed a baseline separation of ten vitamins within a single run, seven water-soluble and three fat-soluble, using three different chromatographic modes: some positively charged vitamins are eluted in ion exclusion and ion repulsion modes whereas the negatively charged molecules are eluted in the ion exchange mechanism. The non-charged molecules are eluted in a classical reversed-phase mode, regarding their polarities. The method was validated for the vitamin analysis in tablets, evaluating selectivity, robustness, linearity, accuracy, and precision. The validated method was finally employed for the analysis of the vitamin content of some commercially available supplement tablets. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimization of the high-performance liquid chromatographic separation of a complex mixture containing urinary steroids, boldenone and bolasterone: application to urine samples.

PubMed

Gonzalo-Lumbreras, R; Izquierdo-Hornillos, R

2000-05-26

An HPLC separation of a complex mixture containing 13 urinary anabolics and corticoids, and boldenone and bolasterone (synthetic anabolics) has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation was studied. The optimum separation was achieved by using a water-acetonitrile (60:40, v/v) mobile phase in reversed-phase HPLC at 30 degrees C, allowing the separation of all the analytes in about 24 min. Calibration graphs were obtained using bolasterone or methyltestosterone as internal standards. Detection limits were in the range 0.012-0.107 microg ml(-1). The optimized separation was applied to the analysis, after liquid-liquid extraction, of human urine samples spiked with steroids.

Gradient Scouting in Reversed-Phase HPLC Revisited

ERIC Educational Resources Information Center

Alcazar, A.; Jurado, J. M.; Gonzalez, A. G.

2011-01-01

Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio [delta]t/t[subscript G] (where [delta]t is the difference in the retention time between the last and the first peak and t[subscript G] is…

Effect of /sup 60/Co-irradiation on penicillin G procaine in veterinary mastitis products

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

Tsuji, K.; Goetz, J.F.; Vanmeter, W.

The effect of /sup 60/Co-irradation on penicillin G procaine in a peanut oil-based veterinary mastitis product was examined by reversed-phase high-performance liquid chromatography (HPLC). The HPLC method is capable of separating and quantifiying procaine, penicillin G, and various degradation compounds. Values obtained by the HPLC method on the product irradiated and stored at various temperatures correlated well with those of the microbiological assay. No significant decrease in the procaine was detected even after 4.0-Mrad irradiation. The HPLC method is applicable for analysis of other beta-lactam antibiotics.

Reverse-phase HPLC analysis of human alpha crystallin.

PubMed

Swamy, M S; Abraham, E C

1991-03-01

A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

The Quorum-Sensing Regulon of Vibriofischeri: Novel Components of the Autoinducer/LuxR Regulatory Circuit

DTIC Science & Technology

1999-06-01

cpdP, from the marine symbiotic bacterium Vibrio fische ri 160 Table of abbreviations 30C6-HSL AI-1 AI-2 C8-HSL CHAPS CNP EDTA FMN GFP HPLC ...using a Zorbax C18 1.0 mm by 150 mm reverse-phase column on a Hewlett-Packard 1090 HPLC /1040 diode array detector at the Harvard Microchemistry...separated by reversed-phase HPLC , and sequenced (Table 2; 10-PK12, 10-PK39, and 10-PK51). From two of the three peptide sequences (Materials and

A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC.

PubMed

Akhlaghi, Yousef; Ghaffari, Solmaz; Attar, Hossein; Alamir Hoor, Amir

2015-11-01

Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

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.

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves.

PubMed

Katekhaye, S; Kale, M S; Laddha, K S

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C(18) column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r(2)>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves.

Development and Validation of an HPLC Method for Karanjin in Pongamia pinnata linn. Leaves

PubMed Central

Katekhaye, S; Kale, M. S.; Laddha, K. S.

2012-01-01

A rapid, simple and specific reversed-phase HPLC method has been developed for analysis of karanjin in Pongamia pinnata Linn. leaves. HPLC analysis was performed on a C18 column using an 85:13.5:1.5 (v/v) mixtures of methanol, water and acetic acid as isocratic mobile phase at a flow rate of 1 ml/min. UV detection was at 300 nm. The method was validated for accuracy, precision, linearity, specificity. Validation revealed the method is specific, accurate, precise, reliable and reproducible. Good linear correlation coefficients (r2>0.997) were obtained for calibration plots in the ranges tested. Limit of detection was 4.35 μg and limit of quantification was 16.56 μg. Intra and inter-day RSD of retention times and peak areas was less than 1.24% and recovery was between 95.05 and 101.05%. The established HPLC method is appropriate enabling efficient quantitative analysis of karanjin in Pongamia pinnata leaves. PMID:23204626

Authentication and Quantitation of Fraud in Extra Virgin Olive Oils Based on HPLC-UV Fingerprinting and Multivariate Calibration

PubMed Central

Carranco, Núria; Farrés-Cebrián, Mireia; Saurina, Javier

2018-01-01

High performance liquid chromatography method with ultra-violet detection (HPLC-UV) fingerprinting was applied for the analysis and characterization of olive oils, and was performed using a Zorbax Eclipse XDB-C8 reversed-phase column under gradient elution, employing 0.1% formic acid aqueous solution and methanol as mobile phase. More than 130 edible oils, including monovarietal extra-virgin olive oils (EVOOs) and other vegetable oils, were analyzed. Principal component analysis results showed a noticeable discrimination between olive oils and other vegetable oils using raw HPLC-UV chromatographic profiles as data descriptors. However, selected HPLC-UV chromatographic time-window segments were necessary to achieve discrimination among monovarietal EVOOs. Partial least square (PLS) regression was employed to tackle olive oil authentication of Arbequina EVOO adulterated with Picual EVOO, a refined olive oil, and sunflower oil. Highly satisfactory results were obtained after PLS analysis, with overall errors in the quantitation of adulteration in the Arbequina EVOO (minimum 2.5% adulterant) below 2.9%. PMID:29561820

A simple method for plasma total vitamin C analysis suitable for routine clinical laboratory use.

PubMed

Robitaille, Line; Hoffer, L John

2016-04-21

In-hospital hypovitaminosis C is highly prevalent but almost completely unrecognized. Medical awareness of this potentially important disorder is hindered by the inability of most hospital laboratories to determine plasma vitamin C concentrations. The availability of a simple, reliable method for analyzing plasma vitamin C could increase opportunities for routine plasma vitamin C analysis in clinical medicine. Plasma vitamin C can be analyzed by high performance liquid chromatography (HPLC) with electrochemical (EC) or ultraviolet (UV) light detection. We modified existing UV-HPLC methods for plasma total vitamin C analysis (the sum of ascorbic and dehydroascorbic acid) to develop a simple, constant-low-pH sample reduction procedure followed by isocratic reverse-phase HPLC separation using a purely aqueous low-pH non-buffered mobile phase. Although EC-HPLC is widely recommended over UV-HPLC for plasma total vitamin C analysis, the two methods have never been directly compared. We formally compared the simplified UV-HPLC method with EC-HPLC in 80 consecutive clinical samples. The simplified UV-HPLC method was less expensive, easier to set up, required fewer reagents and no pH adjustments, and demonstrated greater sample stability than many existing methods for plasma vitamin C analysis. When compared with the gold-standard EC-HPLC method in 80 consecutive clinical samples exhibiting a wide range of plasma vitamin C concentrations, it performed equivalently. The easy set up, simplicity and sensitivity of the plasma vitamin C analysis method described here could make it practical in a normally equipped hospital laboratory. Unlike any prior UV-HPLC method for plasma total vitamin C analysis, it was rigorously compared with the gold-standard EC-HPLC method and performed equivalently. Adoption of this method could increase the availability of plasma vitamin C analysis in clinical medicine.

Highly efficient peptide separations in proteomics. Part 2: bi- and multidimensional liquid-based separation techniques.

PubMed

Sandra, Koen; Moshir, Mahan; D'hondt, Filip; Tuytten, Robin; Verleysen, Katleen; Kas, Koen; François, Isabelle; Sandra, Pat

2009-04-15

Multidimensional liquid-based separation techniques are described for maximizing the resolution of the enormous number of peptides generated upon tryptic digestion of proteomes, and hence, reduce the spatial and temporal complexity of the sample to a level that allows successful mass spectrometric analysis. This review complements the previous contribution on unidimensional high performance liquid chromatography (HPLC). Both chromatography and electrophoresis will be discussed albeit with reversed-phase HPLC (RPLC) as the final separation dimension prior to MS analysis.

Molecular-level characterization of crude oil compounds combining reversed-phase high-performance liquid chromatography with off-line high-resolution mass spectrometry

USGS Publications Warehouse

Sim, Arum; Cho, Yunju; Kim, Daae; Witt, Matthias; Birdwell, Justin E.; Kim, Byung Ju; Kim, Sunghwan

2014-01-01

A reversed-phase separation technique was developed in a previous study (Loegel et al., 2012) and successfully applied to the de-asphalted fraction of crude oil. However, to the best of our knowledge, the molecular-level characterization of oil fractions obtained by reversed-phase high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (MS) has not yet been reported. A detailed characterization of the oil fractions prepared by reversed-phase HPLC was performed in this study. HPLC fractionation was carried out on conventional crude oil and an oil shale pyrolysate. The analyses of the fractions showed that the carbon number of alkyl chains and the double bond equivalent (DBE) value were the major factors determining elution order. The compounds with larger DBE (presumably more condensed aromatic structures) and smaller carbon number (presumably compounds with short side chains) were eluted earlier but those compounds with lower DBE values (presumably less aromatic structures) and higher carbon number (presumably compounds with longer alkyl chains) eluted later in the chromatograms. This separation behavior is in good agreement with that expected from the principles of reversed-phase separation. The data presented in this study show that reversed-phase chromatography is effective in separating crude oil compounds and can be combined with ultrahigh-resolution MS data to better understand natural oils and oil shale pyrolysates.

Reversed phase HPLC for strontium ranelate: Method development and validation applying experimental design.

PubMed

Kovács, Béla; Kántor, Lajos Kristóf; Croitoru, Mircea Dumitru; Kelemen, Éva Katalin; Obreja, Mona; Nagy, Előd Ernő; Székely-Szentmiklósi, Blanka; Gyéresi, Árpád

2018-06-01

A reverse-phase HPLC (RP-HPLC) method was developed for strontium ranelate using a full factorial, screening experimental design. The analytical procedure was validated according to international guidelines for linearity, selectivity, sensitivity, accuracy and precision. A separate experimental design was used to demonstrate the robustness of the method. Strontium ranelate was eluted at 4.4 minutes and showed no interference with the excipients used in the formulation, at 321 nm. The method is linear in the range of 20-320 μg mL-1 (R2 = 0.99998). Recovery, tested in the range of 40-120 μg mL-1, was found to be 96.1-102.1 %. Intra-day and intermediate precision RSDs ranged from 1.0-1.4 and 1.2-1.4 %, resp. The limit of detection and limit of quantitation were 0.06 and 0.20 μg mL-1, resp. The proposed technique is fast, cost-effective, reliable and reproducible, and is proposed for the routine analysis of strontium ranelate.

High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP.

PubMed

Petrova, Olga E; Sauer, Karin

2017-01-01

The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

Regio- and stereospecific analysis of glycerolipids.

PubMed

Kuksis, Arnis; Itabashi, Yutaka

2005-06-01

In recent years researchers have recognized the potential value of comprehensive lipid profiling (lipidomics), which was invented and promoted by lipidologists who recognized the many valuable applications that grew out of the fields of DNA profiling (genomics) and protein profiling (proteonomics). Through lipid class-selective intrasource ionization and subsequent analysis of two-dimensional cross-peak intensities, the chemical identity and mass composition of individual molecular species of most lipid classes can now be determined in a chloroform extract. There remains, however, the necessity to distinguish the enantiomers and isobaric regioisomers resulting from enzymatic and chemical reactions, which conventional high performance liquid chromatography/mass spectrometry (HPLC/MS) has been slow to accommodate, and tandem MS unable to provide. While reversed-phase HPLC can separate regioisomers, normal-phase HPLC can resolve diastereomers, and chiral-phase HPLC can effect dramatic resolution of enantiomers, the full potential of the combined systems has seldom been exploited. The present chapter calls attention to both recent and earlier combinations of these methodologies with mass spectrometry, which allows the HPLC/ESI (electrospray ionization)-MS/MS separation and identification of enantiomeric diacylglycerols, triacylglycerols, and glycerophospholipids as well as their isobaric regioisomers. These developments permit further expansion of lipid profiling (lipidomics) and better understanding of lipid metabolism.

Preparation of a polybutadiene stationary phase immobilized by gamma radiation for reversed-phase high-performance liquid chromatography.

PubMed

Lopes, Nilva P; Collins, Kenneth E; Jardim, Isabel C S F

2003-02-14

Polybutadiene (PBD) has been immobilized on HPLC silica by gamma radiation doses in the range from 5 to 180 kGy. Columns prepared from these reversed-phase materials, as well as from similar non-irradiated materials, were tested with standard sample mixtures and characterized by elemental analysis (% C) and infrared spectroscopy. A low dose of 5 kGy is sufficient to produce a layer of immobilized PBD which functions as an efficient and stable stationary phase. Higher doses give thicker immobilized layers having less favorable chromatographic properties.

Analysis of sesquiterpene lactones, lignans, and flavonoids in wormwood (Artemisia absinthium L.) using high-performance liquid chromatography (HPLC)-mass spectrometry, reversed phase HPLC, and HPLC-solid phase extraction-nuclear magnetic resonance.

PubMed

Aberham, Anita; Cicek, Serhat Sezai; Schneider, Peter; Stuppner, Hermann

2010-10-27

Today, the medicinal use of wormwood (Artemisia absinthium) is enjoying a resurgence of popularity. This study presents a specific and validated high-performance liquid chromatography (HPLC)-diode array detection method for the simultaneous determination and quantification of bioactive compounds in wormwood and commercial preparations thereof. Five sesquiterpene lactones, two lignans, and a polymethoxylated flavonoid were baseline separated on RP-18 material, using a solvent gradient consisting of 0.085% (v/v) o-phosphoric acid and acetonitrile. The flow rate was 1.0 mL/min, and chromatograms were recorded at 205 nm. The stability of absinthin was tested exposing samples to light, moisture, and different temperatures. Methanolic and aqueous solutions of absinthin were found to be stable for up to 6 months. This was also the case when the solid compound was kept in the refrigerator at -35 °C. In contrast, the colorless needles, when stored at room temperature, turned yellow. Three degradation compounds (anabsin, anabsinthin, and the new dimer 3'-hydroxyanabsinthin) were identified by HPLC-mass spectrometry and HPLC-solid-phase extraction-nuclear magnetic resonance and quantified by the established HPLC method.

Development and validation of reverse phase high performance liquid chromatography for citral analysis from essential oils.

PubMed

Gaonkar, Roopa; Yallappa, S; Dhananjaya, B L; Hegde, Gurumurthy

2016-11-15

Citral is a widely used monoterpene aldehyde in aromatherapy, food and pesticide industries. A new validated reverse phase high performance liquid chromatography (RP - HPLC) procedure for the detection and quantification of cis-trans isomers of citral was developed. The RP-HPLC analysis was carried out using Enable C - 18G column (250×4.6mm, 5μ), with acetonitrile and water (70: 30) mobile phase in isocratic mode at 1mL/min flow. A photodiode array (PDA) detector was set at 233nm for the detection of citral. The method showed linearity, selectivity and accuracy for citral in the range of 3-100μg/mL. In order to compare the new RP-HPLC method with the available methods, one of the commercially available essential oil from Cymbopogon flexuosus was analyzed using new RP-HPLC method and the same was analyzed using GC-MS for the comparison of the method for the detection of citral. The GC-MS analysis was done using mass selective detector (MSD) showed citral content to be of 72.76%; wherein the new method showed to contain that same at 74.98%. To prove the application of the new method, essential oils were extracted from lemongrass, lemon leaves and mosambi peels by steam distillation. The citral content present in the essential and also in the condensate was analyzed. The method was found to be suitable for the analysis of citral in essential oils and water based citral formulations with a very good resolution of its components geranial and neral. Copyright © 2016 Elsevier B.V. All rights reserved.

'Click Chemistry' in the preparation of porous polymer-basedparticulate stationary phases for mu-HPLC separation of peptides andproteins

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

Slater, Michael; Snauko, Marian; Svec, Frantisek

With the use of the copper(I)-catalyzed (3 + 2) azide-alkynecycloaddition, an element of "click chemistry," stationary phasescarrying long alkyl chains or soybean trypsin inhibitor have beenprepared for use in HPLC separations in the reversed-phase and affinitymodes, respectively. The ligands were attached via a triazole ring tosize monodisperse porous beads containing either alkyne or azide pendantfunctionalities. Alkyne-containing beads prepared by directcopolymerization of propargyl acrylate with ethylene dimethacrylate wereallowed to react with azidooctadecane to give a reversed-phase sorbent.Azide-functionalized beads were prepared by chemical modification ofglycidyl methacrylate particles. Subsequent reaction with a terminalaliphatic alkyne produced a reversed-phase sorbent similar to thatobtained from themore » alkyne beads. Soybean trypsin inhibitor wasfunctionalized with N-(4-pentynoyloxy) succinimide to carry alkyne groupsand then allowed to react with the azide-containing beads to produce anaffinity sorbent for trypsin. The performance of these stationary phaseswas demonstrated with the HPLC separations of a variety of peptides andproteins.« less

Separation and structural analysis of saponins in a bark extract from Quillaja saponaria Molina.

PubMed

Nord, L I; Kenne, L

1999-07-20

Six major saponins were isolated from a bark extract from Quillaja saponaria Molina. Solid-phase extraction, followed by a two-step reversed-phase HPLC separation procedure with phosphate and ammonium acetate buffers of different pH values, was used. The compounds were characterised using NMR spectroscopy, mass spectrometry and chemical methods.

Composition and Molecular Weight Distribution of Carob Germ Proteins Fractions

USDA-ARS?s Scientific Manuscript database

Biochemical properties of carob germ proteins were analyzed using a combination of selective extraction, reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALS) and electrophoretic analysis. Using a mo...

Analyses of polyphenols in cacao liquor, cocoa, and chocolate by normal-phase and reversed-phase HPLC.

PubMed

Natsume, M; Osakabe, N; Yamagishi, M; Takizawa, T; Nakamura, T; Miyatake, H; Hatano, T; Yoshida, T

2000-12-01

The antioxidant polyphenols in cacao liquor, a major ingredient of chocolate and cocoa, have been characterized as flavan-3-ols and proanthocyanidin oligomers. In this study, various cacao products were analyzed by normal-phase HPLC, and the profiles and quantities of the polyphenols present, grouped by molecular size (monomers to approximately oligomers), were compared. Individual cacao polyphenols, flavan-3-ols (catechin and epicatechin), and dimeric (procyanidin B2), trimeric (procyanidin C1), and tetrameric (cinnamtannin A2) proanthocyanidins, and galactopyranosyl-ent-(-)-epicatechin (2alpha-->7, 4alpha-->8)-(-)-epicatechin (Gal-EC-EC), were analyzed by reversed-phase HPLC and/or HPLC/MS. The profile of monomers (catechins) and proanthocyanidin in dark chocolate was similar to that of cacao liquor, while the ratio of flavan-3-ols to the total amount of monomeric and oligomeric polyphenols in the case of pure cocoa powder was higher than that in the case of cacao liquor or chocolate.

Analysis of Cocoa Proanthocyanidins Using Reversed Phase High-Performance Liquid Chromatography and Electrochemical Detection: Application to Studies on the Effect of Alkaline Processing.

PubMed

Stanley, Todd H; Smithson, Andrew T; Neilson, Andrew P; Anantheswaran, Ramaswamy C; Lambert, Joshua D

2015-07-01

Flavan-3-ols and proanthocyanidins play a key role in the health beneficial effects of cocoa. Here, we developed a new reversed phased high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method for the analysis of flavan-3-ols and proanthocyanidins of degree of polymerization (DP) 2-7. We used this method to examine the effect of alkalization on polyphenol composition of cocoa powder. Treatment of cocoa powder with NaOH (final pH 8.0) at 92 °C for up to 1 h increased catechin content by 40%, but reduced epicatechin and proanthocyanidins by 23-66%. Proanthocyanidin loss could be modeled using a two-phase exponential decay model (R(2) > 0.7 for epicatchin and proanthocyanidins of odd DP). Alkalization resulted in a significant color change and 20% loss of total polyphenols. The present work demonstrates the first use of HPLC-ECD for the detection of proanthocyanidins up to DP 7 and provides an initial predictive model for the effect of alkali treatment on cocoa polyphenols.

Integrating Elemental Analysis and Chromatography Techniques by Analyzing Metal Oxide and Organic UV Absorbers in Commercial Sunscreens

ERIC Educational Resources Information Center

Quin~ones, Rosalynn; Bayline, Jennifer Logan; Polvani, Deborah A.; Neff, David; Westfall, Tamara D.; Hijazi, Abdullah

2016-01-01

A series of undergraduate laboratory experiments that utilize reversed-phase HPLC separation, inductively coupled plasma spectroscopy (ICP), and scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS) are described for the analysis of commercial sunscreens. The active ingredients of many sunscreen brands include zinc or titanium…

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...

Rapid Development and Validation of Improved Reversed-Phase High-performance Liquid Chromatography Method for the Quantification of Mangiferin, a Polyphenol Xanthone Glycoside in Mangifera indica

PubMed Central

Naveen, P.; Lingaraju, H. B.; Prasad, K. Shyam

2017-01-01

Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica, is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica. RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography–mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica. SUMMARY The present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica. The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica. Abbreviations Used: M. indica: Mangifera indica, RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification. PMID:28539748

Rapid Development and Validation of Improved Reversed-Phase High-performance Liquid Chromatography Method for the Quantification of Mangiferin, a Polyphenol Xanthone Glycoside in Mangifera indica.

PubMed

Naveen, P; Lingaraju, H B; Prasad, K Shyam

2017-01-01

Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica , is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica . RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography-mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica . The present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica . The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica . Abbreviations Used: M. indica : Mangifera indica , RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification.

Reversed Phase Column HPLC-ICP-MS Conditions for Arsenic Speciation Analysis of Rice Flour.

PubMed

Narukawa, Tomohiro; Matsumoto, Eri; Nishimura, Tsutomu; Hioki, Akiharu

2015-01-01

New measurement conditions for arsenic speciation analysis of rice flour were developed using HPLC-ICP-MS equipped with a reversed phase ODS column. Eight arsenic species, namely, arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), dimethylarsinic acid (DMAA), trimethylarsine oxide (TMAO), tetramethylarsonium (TeMA), arsenobetaine (AsB) and arsenocholine (AsC), were separated and determined under the proposed conditions. In particular, As(III) and MMAA and DMAA and AsB were completely separated using a newly proposed eluent containing ammonium dihydrogen phosphate. Importantly, the sensitivity changes, in particular those of As(V) and As(III) caused by coexisting elements and by complex matrix composition, which had been problematical in previously reported methods, were eliminated. The new eluent can be applied to C8, C18 and C30 ODS columns with the same effectiveness and with excellent repeatability. The proposed analytical method was successfully applied to extracts of rice flour certified reference materials.

Comprehensive hydrophilic interaction and ion-pair reversed-phase liquid chromatography for analysis of di- to deca-oligonucleotides.

PubMed

Li, Qin; Lynen, Frédéric; Wang, Jian; Li, Hanlin; Xu, Guowang; Sandra, Pat

2012-09-14

A comprehensive two-dimensional HPLC approach with a high degree of orthogonality was developed for analysis of di- to deca-oligonucleotides (ONs). Hydrophilic interaction liquid chromatography (HILIC) was used in the first dimension, and ion-pair reversed-phase liquid chromatography (IP-RPLC) was employed in the second dimension. The two dimensions were connected via a ten-port valve interface equipped with octadecyl silica (ODS) traps to immobilize and focus the ONs eluting from the first dimension prior to IP-RPLC separation. An aqueous make-up flow was used for effective trapping. The comprehensive two-dimensional HPLC system was optimized with a mixture consisting of 27 oligonucleotide standards. An overall chromatographic peak capacity of 500 was obtained. The use of the volatile buffer triethylamine acetate in the second dimension allowed straightforward coupling to electrospray ionization mass spectrometry (ESI-MS) and detection of each ON in the negative ionization mode. Copyright © 2011 Elsevier B.V. All rights reserved.

Reversed-phase thin-layer chromatography of homologs of Antimycin-A and related derivatives

USGS Publications Warehouse

Abidi, Sharon L.

1989-01-01

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

Quality Analysis of Chlorogenic Acid and Hyperoside in Crataegi fructus

PubMed Central

Weon, Jin Bae; Jung, Youn Sik; Ma, Choong Je

2016-01-01

Background: Crataegi fructus is a herbal medicine for strong stomach, sterilization, and alcohol detoxification. Chlorogenic acid and hyperoside are the major compounds in Crataegi fructus. Objective: In this study, we established novel high-performance liquid chromatography (HPLC)-diode array detection analysis method of chlorogenic acid and hyperoside for quality control of Crataegi fructus. Materials and Methods: HPLC analysis was achieved on a reverse-phase C18 column (5 μm, 4.6 mm × 250 mm) using water and acetonitrile as mobile phase with gradient system. The method was validated for linearity, precision, and accuracy. About 31 batches of Crataegi fructus samples collected from Korea and China were analyzed by using HPLC fingerprint of developed HPLC method. Then, the contents of chlorogenic acid and hyperoside were compared for quality evaluation of Crataegi fructus. Results: The results have shown that the average contents (w/w %) of chlorogenic acid and hyperoside in Crataegi fructus collected from Korea were 0.0438% and 0.0416%, respectively, and the average contents (w/w %) of 0.0399% and 0.0325%, respectively. Conclusion: In conclusion, established HPLC analysis method was stable and could provide efficient quality evaluation for monitoring of commercial Crataegi fructus. SUMMARY Quantitative analysis method of chlorogenic acid and hyperoside in Crataegi fructus is developed by high.performance liquid chromatography.(HPLC).diode array detectionEstablished HPLC analysis method is validated with linearity, precision, and accuracyThe developed method was successfully applied for quantitative analysis of Crataegi fructus sample collected from Korea and China. Abbreviations used: HPLC: High-performance liquid chromatography, GC: Gas chromatography, MS: Mass spectrometer, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation, RRT: Relative retention time, RPA: Relation peak area. PMID:27076744

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 general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin.

PubMed

Germann, M W; Pon, R T; van de Sande, J H

1987-09-01

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.

Hydrogen sulfide measurement using sulfide dibimane: critical evaluation with electrospray ion trap mass spectrometry

PubMed Central

Shen, Xinggui; Chakraborty, Sourav; Dugas, Tammy R; Kevil, Christopher G

2015-01-01

Accurate measurement of hydrogen sulfide bioavailability remains a technical challenge due to numerous issues involving sample processing, detection methods used, and actual biochemical products measured. Our group and others have reported that reverse phase HPLC detection of sulfide dibimane (SDB) product from the reaction of H2S/HS− with monobromobimane allows for analytical detection of hydrogen sulfide bioavailability in free and other biochemical forms. However, it remains unclear whether possible interfering contaminants may contribute to HPLC SDB peak readings that may result in inaccurate measurements of bioavailable sulfide. In this study, we critically compared hydrogen sulfide dependent SDB detection using reverse phase HPLC (RP-HPLC) versus quantitative SRM electrospray ionization mass spectrometry (ESI/MS) to obtain greater clarity into the validity of the reverse phase HPLC method for analytical measurement of hydrogen sulfide. Using an LCQ-deca ion-trap mass spectrometer, SDB was identified by ESI/MS positive ion mode, and quantified by selected reaction monitoring (SRM) using hydrocortisone as an internal standard. Collision induced dissociation (CID) parameters were optimized at MS2 level for SDB and hydrocortisone. ESI/MS detection of SDB standard was found to be a log order more sensitive than RP-HPLC with a lower limit of 0.25 nM. Direct comparison of tissue and plasma SDB levels using RP-HPLC and ESI/MS methods revealed comparable sulfide levels in plasma, aorta, heart, lung and brain. Together, these data confirm the use of SDB as valid indicator of H2S bioavailability and highlights differences between analytical detection methods. PMID:24932544

Aqueous Reversed-Phase HPLC/FT-IR Using Diffuse Reflectance Detections

NASA Astrophysics Data System (ADS)

Kalasinsky, Victor F.; Pai, T. H.; Kenton, R. C.; Kalasinsky, Kathryn S.

1989-12-01

Solvent-elimination HPLC/FT-IR has become a viable combination of two important techniques, and we have been developing a system which is adaptable to both normal and reversed-phase liquid chromatography. The interface involves the deposition of HPLC eluites onto a KCI-laden train with subsequent analysis via diffuse reflectance spectroscopy, and with minor modifications, the system can be used with microbore and analytical columns. With aqueous solvents, the water is converted to methanol and acetone in a post-column reaction with 2,2-dimethoxypropane before the eluites are deposited. A number of different samples have been used to demonstrate the interface and its flexibility. Steroids, analgesics, and other pharmaceutical preparations have been separated with reverse-phase solvents and identified by their infrared spectra. For some of the compounds studied, different infrared spectra of a given compound have been found to exhibit intensity variations, which arise from different crystalline states. The differences can be concentration dependent and may be useful in obtaining semi-quantitative information from the infrared spectra. Applications involving both gradient elution and isocratic separations have been successful. The former provides the same advantages for HPLC/FT-IR as one finds in conventional HPLC. More recent work has been applied to the use of buffers such as those frequently used in bioanalytical separations. In trying to simplify the post-column reaction with water, we have immobilized dehydration reagents onto silica particles and packed these materials into a column which is inserted in-line after the analytical column. Of the reagents utilized to date, 3,3-dimethoxypropyltrimethoxysilane has been found to perform most efficiently. It has advantages over the simpler reagents because it can be regenerated in the reaction column. Results and the efficiency of the dehydration process and its relation to the type of reagent and its coverage will be discussed.

Alterations in the sarcoplasmic protein fraction of beef muscle with postmortem aging and hydrodynamic pressure processing

USDA-ARS?s Scientific Manuscript database

Capillary electrophoresis (CE) and reversed-phase high performance liquid chromatography (RP-HPLC) analysis were utilized to detect differences in the sarcoplasmic protein profiles of beef strip loins subjected to aging and hydrodynamic pressure processing (HDP) treatments. At 48 h postmortem, stri...

Development and Validation of a Simultaneous RP-HPLCUV/DAD Method for Determination of Polyphenols in Gels Containing S. terebinthifolius Raddi (Anacardiaceae)

PubMed Central

Carvalho, Melina G.; Aragão, Cícero F. S; Raffin, Fernanda N.; de L. Moura, Túlio F. A.

2017-01-01

Topical gels containing extracts of Schinus terebinthifolius have been used to treat bacterial vaginosis. It has been reported that this species has antimicrobial, anti-inflammatory and anti-ulcerogenic properties, which can be attributed to the presence of phenolic compounds. In this work, a sensitive and selective reversed-phase HPLC-UV/DAD method for the simultaneous assay of six polyphenols that could be present in S. terebinthifolius was developed. The method was shown to be accurate and precise. Peak purity and similarity index both exceeded 0.99. Calibration curves were linear over the concentration range studied, with correlation coefficients between 0.9931 and 0.9974. This method was used to determine the polyphenol content of a hydroalcoholic extract and pharmacy-compounded vaginal gel. Although the method is useful to assess the 6 phenolic compounds, some compounds could not be detected in the products. SUMMARY A sensitive, selective, accurate and precise reversed-phase HPLC-UV/DAD method for the simultaneous assay of six polyphenols in S. terebinthifolius Raddi Abbreviations used: RP-HPLC-UV/DAD: Reverse Phase High Performance Liquid Chromatograph with Ultraviolet and Diode Array Detector, HPLC: High Performance Liquid Chromatograph, HPLC-UV: High Performance Liquid Chromatograph with Ultraviolet Detector, ANVISA: Brazilian National Health Surveillance Agency, LOD: Limit of detection, LOQ: Limit of quantitation PMID:28539726

Utilization of Photochemically Induced Fluorescence Detection for HPLC Determination of Genotoxic Impurities in the Vortioxetine Manufacturing Process.

PubMed

Douša, Michal; Doubský, Jan; Srbek, Jan

2016-07-01

An analytical reversed-phase high-performance liquid chromatography (HPLC) method for the detection and quantitative determination of two genotoxic impurities at ppm level present in the vortioxetine manufacturing process is described. Applying the concept of threshold of toxicological concern, a limit of 75 ppm each for both genotoxic impurities was calculated based on the maximum daily dose of active pharmaceutical ingredients. The novel reversed-phase HPLC method with photochemically induced fluorescence detection was developed on XSELECT Charged Surface Hybrid Phenyl-Hexyl column using the mobile phase consisted a mixture of 10 mM ammonium formate pH 3.0 and acetonitrile. The elution was performed using an isocratic composition of 48:52 (v/v) at a flow rate of 1.0 mL/min. The photochemically induced fluorescence detection is based on the use of UV irradiation at 254 nm through measuring the fluorescence intensity at 300 nm and an excitation wavelength of 272 nm to produce fluorescent derivatives of both genotoxic impurities. The online photochemical conversion and detection is easily accomplished for two expected genotoxic impurities and provides a sufficiently low limit detection and quantification for the target analysis. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Liquid chromatography of hydrocarbonaeous quaternary amines on cyclodextrin bonded silica

USGS Publications Warehouse

Abidi, S.L.

1986-01-01

Mixtures of n-alkylbenzyldimethylammonium chloride (ABDAC) were resolved into homologous components by high-performance liquid chromatography (HPLC) with a cyclodextrin-bonded silica stationary phase. With a few exceptions, results from this study are similar to those obtained from traditional reversed-phase HPLC. It was found that the presence of electrolytes in aqueous mobile phases is not a critical factor in determining the success of HPLC separation. Under normal HPLC conditions, a mobile phase consisting of either methanol–water (50:50) or acetonitrile–water (30:70) was employed for obtaining adequate resolution of the quaternary ammonium mixtures. Although the percent organic modifier–water profiles were similar to those in previous studies with these compounds, resolution (R) and selectivity (α) parameters were found to be quite susceptible to changes in the mobile phase solvent composition. The retention behavior of the cationic analytes in the homologous series is consistent with the hydrophobic-interaction concept proposed for the retention mechanism via dominant inclusion complex formation. Several electrolytes were chosen for a study of the counter ion effect on the chromatographic characteristics of ABDAC components. Among the electrolytes examined, the perchlorate ion was found most likely to act as an ion-pairing counter ion for ammonium cations in the HPLC system studied. A correlation study established linear relationships between the chain length of ABDAC and the logarithmic capacity factor (k2). The analytical utility of the HPLC method was demonstrated by the analysis of various unknown mixtures.

Using reversed phase high performance liquid chromatography to study the complexation of anthocyanins with β-cyclodextrin

NASA Astrophysics Data System (ADS)

Deineka, V. I.; Lapshova, M. S.; Deineka, L. A.

2014-06-01

It is shown by means of reversed phase high performance liquid chromatography (RP HPLC) with mobile phases containing additions of β-cyclodextrin that 5-glucosides of cyanidin and pelargonidin form stronger inclusion complexes than 3-glucosides; this is explained by the steric interference of the glucoside radical.

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

PubMed Central

Kamal, Abid; Khan, Washim; Ahmad, Sayeed; Ahmad, F. J.; Saleem, Kishwar

2015-01-01

Objective: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga. Materials and Methods: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase. Results: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin. Conclusions: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient. PMID:26681890

An innovative approach to the analysis of 3-[4-(2-methylpropyl)phenyl]propanoic acid as an impurity of ibuprofen on a carbon-coated zirconia stationary phase.

PubMed

Kalafut, P; Kucera, R; Klimes, J; Sochor, J

2009-07-12

3-[4-(2-Methylpropyl)phenyl]propanoic acid has been introduced as impurity F to the European Pharmacopoeia in its Supplement 4.2. In contrast to other impurities, which are evaluated by HPLC, the content of impurity F is determined by gas chromatography after previous derivatization. Thus a novel reversed-phase HPLC method was developed to simplify the evaluation of pharmacopoeial impurity F of ibuprofen. Favourable properties of zirconia stationary phases were employed for this purpose. The HPLC separation was achieved on a Zr-CARB column (150 mm x 4.6mm i.d., 5 microm) using the mobile phase acetonitrile-phosphate buffer (pH 3.5, 25 mM) (38:62, v/v), temperature 80 degrees C and the flow rate 1.2 ml min(-1). The fluorescence detection was employed to enhance the sensitivity of the method. Optimal detection parameters were chosen on the basis of fluorescence spectra of the analytes. The excitation and emission wavelengths were 220 nm and 285 nm, respectively. The analysis was completed within 25 min. The subsequent validation of the method confirmed the applicability of method for the analytical assay of impurity F.

Analysis of metallic nanoparticles and their ionic counterparts in complex matrix by reversed-phase liquid chromatography coupled to ICP-MS.

PubMed

Yang, Yuan; Luo, Li; Li, Hai-Pu; Wang, Qiang; Yang, Zhao-Guang; Qu, Zhi-Peng; Ding, Ru

2018-05-15

Developing quantification and characterization methodology for metallic nanoparticles (MNPs) and their ionic component in complex matrix are crucial for the evaluation of their environmental behavior and health risks to humans. In this study, reversed phase high performance liquid chromatography combined ICP-MS was established for the characterization of MNPs in complex matrix. The ionic component could be separated from NPs with the optimized parameters of aqueous mobile phase. Good linear relationship between average diameter and retention time of NPs was obtained using HPLC-ICP-MS and the size smaller than 40 nm could be determined with this method, the detected results were in accordance with TEM results. The low detection limit of AuNPs and Au(Ⅲ) (both in sub-μg/L level) showed that this method was promising for the characterization of AuNPs and Au(Ⅲ) in environmental water. The mass concentration of ionic Au(Ⅲ) in environmental water could be detected using the proposed HPLC-ICP-MS and the concentration of AuNPs was obtained by subtracting the Au(Ⅲ) concentration from the total Au (The concentration of total Au was detected by ICP-MS after microwave digestion). Furthermore this proposed HPLC-ICP-MS method and single particle-ICPMS (SP-ICP-MS) was used for the analysis of the Ag speciation in commercial antibacterial products. Copyright © 2018 Elsevier B.V. All rights reserved.

Determination of tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations by HPLC.

PubMed

Almeida, M M; Alves, J M P; Patto, D C S; Lima, C R R C; Quenca-Guillen, J S; Santoro, M I R M; Kedor-Hackmann, E R M

2009-12-01

A rapid HPLC method was developed for the assay of tocopheryl acetate and ascorbyl tetraisopalmitate in cosmetic formulations. The validated method was applied for quantitative determination of these vitamins in simulated emulsion formulation. Samples were analysed directly on a RP-18 reverse phase column with UV detection at 222 nm. A mixture of methanol and isopropanol (25 : 75 v/v) was used as mobile phase. The retention time of tocopheryl acetate and ascorbyl tetraisopalmitate were 3.0 min and 5.9 min, respectively. Recovery was between 95% and 104%. In addition, the excipients did not interfere in the analysis. The method is simple, reproducible, selective and is suitable for routine analyses of commercial products.

Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

PubMed

Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

2015-07-07

The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

High Performance Liquid Chromatographic Analysis of Phytoplankton Pigments Using a C16-Amide Column

EPA Science Inventory

A reverse-phase high performance liquid chromatographic (RP-HPLC) method was developed to analyze in a single run, most polar and non-polar chlorophylls and carotenoids from marine phytoplankton. The method is based on a RP-C16-Amide column and a ternary gradient system consistin...

Comparison of the phenolic composition of fruit juices by single step gradient HPLC analysis of multiple components versus multiple chromatographic runs optimised for individual families.

PubMed

Bremner, P D; Blacklock, C J; Paganga, G; Mullen, W; Rice-Evans, C A; Crozier, A

2000-06-01

After minimal sample preparation, two different HPLC methodologies, one based on a single gradient reversed-phase HPLC step, the other on multiple HPLC runs each optimised for specific components, were used to investigate the composition of flavonoids and phenolic acids in apple and tomato juices. The principal components in apple juice were identified as chlorogenic acid, phloridzin, caffeic acid and p-coumaric acid. Tomato juice was found to contain chlorogenic acid, caffeic acid, p-coumaric acid, naringenin and rutin. The quantitative estimates of the levels of these compounds, obtained with the two HPLC procedures, were very similar, demonstrating that either method can be used to analyse accurately the phenolic components of apple and tomato juices. Chlorogenic acid in tomato juice was the only component not fully resolved in the single run study and the multiple run analysis prior to enzyme treatment. The single run system of analysis is recommended for the initial investigation of plant phenolics and the multiple run approach for analyses where chromatographic resolution requires improvement.

Major and Modified Nucleosides, RNA, and DNA

NASA Astrophysics Data System (ADS)

Gehrke, Charles W.; Kuo, Kenneth C.

Most analytical chemists are well aware of the rapid rate of development of high-performance liquid chromatography (HPLC) over the past 5 years. A number of articles have been published in Analytical Chemistry on different topics in HPLC and many papers appear in the chromatographic journals. Some books also have been published covering this subject. HPLC has proved to be a very effective, broadly applicable chromatographic method for the separation and analysis of complex molecules in fields as diverse as biochemistry and environmental, pharmaceutical, medical, and polymer chemistry. HPLC is now having a major impact on the clinical and research aspects of medical biochemistry. Although the contributions of HPLC to other disciplines generally complements gas-liquid chromatography, this method is destined to play a much greater role in medical and biochemical research. This is because many of the biomolecules, owing to their molecular complexity and size, are thermally unstable or nonvolatile, preventing or complicating an analysis by GC. A major factor contributing to the powerful advances in biomedical liquid chromatography is the development of reversed-phase high-performance liquid chromatography (RP-HPLC) using n-alkyl and phenyl chemically bonded substrates.

Analysis of condensed and hydrolysable tannins from commercial plant extracts.

PubMed

Romani, A; Ieri, F; Turchetti, B; Mulinacci, N; Vincieri, F F; Buzzini, P

2006-05-03

High performance liquid chromatography (HPLC)/DAD and MS qualitative and quantitative analyses of polyphenols, hydrolysable and condensed tannins from Pinus maritima L. and tannic acid (TA) extracts were performed using normal and reverse phase. Normal-phase HPLC was more suitable for pine bark (PBE) and tannic acid extracts analysis. The chromatographic profile revealed that P. maritima L. extract was mainly composed by polymeric flavanols (containing from two to seven units) and tannic acid (characterized by a mixture of glucose gallates containing from three to seven units of gallic acid). Concerning their antimycotic properties, P. maritima L. extract exhibited a broad activity towards yeast strains of the genera Candida, Cryptococcus, Filobasidiella, Issatchenkia, Saccharomyces: MICs from 200 to 4000 microg/ml (corresponding to 140-2800 microg/ml of active polyphenols) were determined. Conversely, no activity of tannic acid was observed over the same target microorganisms. Taken into consideration the above results of HPLC analysis and on the basis of the current literature, we may conclude that only 70.2% of polyphenols (recognized as condensed tannins) occurring in P. maritima L. extract can be apparently considered responsible for its antimycotic activity.

[Determination of glycyrrhizinic acid in biotransformation system by reversed-phase high performance liquid chromatography].

PubMed

Li, Hui; Lu, Dingqiang; Liu, Weimin

2004-05-01

A method for determining glycyrrhizinic acid in the biotransformation system by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The HPLC conditions were as follows: Hypersil C18 column (4.6 mm i.d. x 250 mm, 5 microm) with a mixture of methanol-water-acetic acid (70:30:1, v/v) as the mobile phase; flow rate at 1.0 mL/min; and UV detection at 254 nm. The linear range of glycyrrhizinic acid was 0.2-20 microg. The recoveries were 98%-103% with relative standard deviations between 0.16% and 1.58% (n = 3). The method is simple, rapid and accurate for determining glycyrrhizinic acid.

Quantitative high-performance liquid chromatography of nucleosides in biological materials.

PubMed

Gehrke, C W; Kuo, K C; Davis, G E; Suits, R D; Waalkes, T P; Borek, E

1978-03-21

A rigorous, comprehensive, and reliable reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the analysis of ribonucleosides in urine (psi, m1A, m1I, m2G, A, m2(2)G). An initial isolation of ribonucleosides with an affinity gel containing an immobilized phenylboronic acid was used to improve selectivity and sensitivity. Response for all nucleosides was linear from 0.1 to 50 nmoles injected and good quantitation was obtained for 25 microliter or less of sample placed on the HPLC column. Excellent precision of analysis for urinary nucleosides was achieved on matrix dependent and independent samples, and the high resolution of the reversed-phase column allowed the complete separation of 9 nucleosides from other unidentified UV absorbing components at the 1-ng level. Supporting experimental data are presented on precision, recovery, chromatographic methods, minimum detection limit, retention time, relative molar response, sample clean-up, stability of nucleosides, boronate gel capacity, and application to analysis of urine from patients with leukemia and breast cancer. This method is now being used routinely for the determination of the concentration and ratios of nucleosides in urine from patients with different types of cancer and in chemotherapy response studies.

Simultaneous analysis of insect repellent DEET, sunscreen oxybenzone and five relevant metabolites by reversed-phase HPLC with UV detection: application to an in vivo study in a piglet model.

PubMed

Kasichayanula, Sreeneeranj; House, James D; Wang, Tao; Gu, Xiaochen

2005-08-05

N,N-Diethyl-m-toluamide (DEET) and oxybenzone are two essential active ingredients in insect repellent and sunscreen preparations. We developed and validated a simple, sensitive, and selective HPLC assay to simultaneously measure DEET, oxybenzone and five primary metabolites of DEET and oxybenzone in biological samples including plasma, urine and skin strips. The compounds were separated on a reversed-phase C18 column using three-stage gradient steps with methanol and water. DEET and two relevant metabolites were detected at 254 nm, while oxybenzone and three relevant metabolites were detected at 289 nm. The limit of detection was 0.6 ng for DEET and 0.5 ng for oxybenzone, respectively. The developed method was further applied to analyze various biological samples from an in vivo animal study that evaluated concurrent use of commercially available insect repellent and sunscreen preparations.

Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins.

PubMed

Zhu, Zaifang; Chen, Huang; Ren, Jiangtao; Lu, Juan J; Gu, Congying; Lynch, Kyle B; Wu, Si; Wang, Zhe; Cao, Chengxi; Liu, Shaorong

2018-03-01

We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

High-performance liquid-chromatographic separation of subcomponents of antimycin-A

USGS Publications Warehouse

Abidi, S.L.

1988-01-01

Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

Reverse phase HPLC method for detection and quantification of lupin seed γ-conglutin.

PubMed

Mane, Sharmilee; Bringans, Scott; Johnson, Stuart; Pareek, Vishnu; Utikar, Ranjeet

2017-09-15

A simple, selective and accurate reverse phase HPLC method was developed for detection and quantitation of γ-conglutin from lupin seed extract. A linear gradient of water and acetonitrile containing trifluoroacetic acid (TFA) on a reverse phase column (Agilent Zorbax 300SB C-18), with a flow rate of 0.8ml/min was able to produce a sharp and symmetric peak of γ-conglutin with a retention time at 29.16min. The identity of γ-conglutin in the peak was confirmed by mass spectrometry (MS/MS identification) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The data obtained from MS/MS analysis was matched against the specified database to obtain the exact match for the protein of interest. The proposed method was validated in terms of specificity, linearity, sensitivity, precision, recovery and accuracy. The analytical parameters revealed that the validated method was capable of selectively performing a good chromatographic separation of γ-conglutin from the lupin seed extract with no interference of the matrix. The detection and quantitation limit of γ-conglutin were found to be 2.68μg/ml and 8.12μg/ml respectively. The accuracy (precision and recovery) analysis of the method was conducted under repeatable conditions on different days. Intra-day and inter-day precision values less than 0.5% and recovery greater than 97% indicated high precision and accuracy of the method for analysis of γ-conglutin. The method validation findings were reproducible and can be successfully applied for routine analysis of γ-conglutin from lupin seed extract. Copyright © 2017 Elsevier B.V. All rights reserved.

ON-COLUMN ENRICHMENT OF HYDROPHOBIC CYP450 PROTEINS IN HPLC FRACTIONATION OF MOUSE MICROSOMES PRIOR TO PROTEIN DIGESTION AND NANOSPRAY-LC/MSMS ANALYSIS

EPA Science Inventory

Introduction

Membrane proteins play crucial role in many cellular processes and are promising candidates for biomarker discovery but are under-represented in the field of proteomics due to their hydrophobic nature. Although standard reversed-phase LC methods often exhibit ...

Reverse Phase HPLC Method for Analysis of TNT, RDX, HMX and 2,4-DNT in Munitions Wastewater,

DTIC Science & Technology

1984-12-01

Materials Agency (USATHAMA), Capt. P. Rissell (USATHAMA) and Dr. R. Westerdahl (LCWSL). project monitors. The authors ac- knowledge the support given by...Capt. Rissell and Dr. Westerdahl throughout this effort, and their technical review of the report. The contents of this report are not to be used for

Analytical Method Development and Validation for the Simultaneous Estimation of Abacavir and Lamivudine by Reversed-phase High-performance Liquid Chromatography in Bulk and Tablet Dosage Forms.

PubMed

Raees Ahmad, Sufiyan Ahmad; Patil, Lalit; Mohammed Usman, Mohammed Rageeb; Imran, Mohammad; Akhtar, Rashid

2018-01-01

A simple rapid, accurate, precise, and reproducible validated reverse phase high performance liquid chromatography (HPLC) method was developed for the determination of Abacavir (ABAC) and Lamivudine (LAMI) in bulk and tablet dosage forms. The quantification was carried out using Symmetry Premsil C18 (250 mm × 4.6 mm, 5 μm) column run in isocratic way using mobile phase comprising methanol: water (0.05% orthophosphoric acid with pH 3) 83:17 v/v and a detection wavelength of 245 nm and injection volume of 20 μl, with a flow rate of 1 ml/min. In the developed method, the retention times of ABAC and LAMI were found to be 3.5 min and 7.4 min, respectively. The method was validated in terms of linearity, precision, accuracy, limits of detection, limits of quantitation, and robustness in accordance with the International Conference on Harmonization guidelines. The assay of the proposed method was found to be 99% - 101%. The recovery studies were also carried out and mean % recovery was found to be 99% - 101%. The % relative standard deviation from reproducibility was found to be <2%. The proposed method was statistically evaluated and can be applied for routine quality control analysis of ABAC and LAMI in bulk and in tablet dosage form. Attempts were made to develop RP-HPLC method for simultaneous estimation of Abacavir and Lamivudine for the RP-HPLC method. The developed method was validated according to the ICH guidelines. The linearity, precision, range, robustness were within the limits as specified by the ICH guidelines. Hence the method was found to be simple, accurate, precise, economic and reproducible. So the proposed methods can be used for the routine quality control analysis of Abacavir and Lamivudine in bulk drug as well as in formulations. Abbreviations Used: HPLC: High-performance liquid chromatography, UV: Ultraviolet, ICH: International Conference on Harmonization, ABAC: Abacavir, LAMI: Lamivudine, HIV: Human immunodeficiency virus, AIDS: Acquired immunodeficiency syndrome, NRTI: Nucleoside reverse transcriptase inhibitors, ARV: Antiretroviral, RSD: Relative standard deviation, RT: Retention time, SD: Standard deviation.

A rapid solid-phase extraction method for measurement of non-metabolised peripheral benzodiazepine receptor ligands, [(18)F]PBR102 and [(18)F]PBR111, in rat and primate plasma.

PubMed

Katsifis, Andrew; Loc'h, Christian; Henderson, David; Bourdier, Thomas; Pham, Tien; Greguric, Ivan; Lam, Peter; Callaghan, Paul; Mattner, Filomena; Eberl, Stefan; Fulham, Michael

2011-01-01

To develop a rapid and reliable method for estimating non-metabolised PBR ligands fluoroethoxy ([(18)F]PBR102)- and fluoropropoxy ([(18)F]PBR111)-substituted 2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridine-3-yl)-N,N-diethylacetamides in plasma. Rats and baboons were imaged with PET up to 2 h postinjection of [(18)F]PBR102 and [(18)F]PBR111 under baseline conditions, after pre-blocking or displacement with PK11195. Arterial plasma samples were directly analysed by reverse-phase solid-phase extraction (RP-SPE) and RP-HPLC and by normal-phase TLC. SPE cartridges were successively washed with acetonitrile/water mixtures. SPE eluant radioactivity was measured in a γ-counter to determine the parent compound fraction and then analysed by HPLC and TLC for validation. In SPE, hydrophilic and lipophilic radiolabelled metabolites were eluted in water and 20% acetonitrile/water. All non-metabolised [(18)F]PBR102 and [(18)F]PBR111 were in SPE acetonitrile fraction as confirmed by HPLC and TLC analysis. Unchanged (%) [(18)F]PBR102 and [(18)F]PBR111 from SPE analysis in rat and baboon plasma agreed with those from HPLC and TLC analysis. In rats and baboons, the fraction of unchanged tracer followed a bi-exponential decrease, with half-lives of 7 to 10 min for the fast component and >80 min for the slow component for both tracers. Direct plasma SPE analysis of [(18)F]PBR102 and [(18)F]PBR111 can reliably estimate parent compound fraction. SPE was superior to HPLC for samples with low activity; it allows rapid and accurate metabolite analysis of a large number of plasma samples for improved estimation of metabolite-corrected input function during quantitative PET imaging studies. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

Speciation of As(III) and As(V) in water and sediment using reverse-phase ion-pair high-performance liquid chromatography-neutron activation analysis (HPLC-NAA).

PubMed

Tulasi, Delali; Adotey, Dennis; Affum, Andrews; Carboo, Derick; Serfor-Armah, Yaw

2013-10-01

Total As content and the As species distribution in water and sediments from the Kwabrafo stream, a major water body draining the Obuasi gold mining community in southwestern Ghana, have been investigated. Total As content was determined by instrumental neutron activation analysis (INAA). Ion-pair reverse phase high-performance liquid chromatography-neutron activation analysis (HPLC-NAA) was used for speciation of As species. Solid phase extraction with phosphate buffer was used to extract soluble As species from lyophilized sediment. The mass balance after phosphate extraction of soluble As species in sediment varied from 89 to 96 %. Compositionally appropriate reference material International Atomic Energy Agency (IAEA)-Lake Sediment (SL)-1 was used to check the validity of INAA method for total As determination. The measured values are in good agreement with the IAEA recommended value and also within the 95 % confidence interval. The accuracy of the measurement in terms of relative deviation from the IAEA recommended value was ±0.83 %. "In-house" prepared As(III) and As(V) standards were used to validate the HPLC-INAA method used for the As species determination. Total As concentration in the water samples ranged from 1.15 to 9.20 mg/L. As(III) species in water varied from 0.13 to 0.7 mg/L, while As(V) species varied from 0.79 to 3.85 mg/L. Total As content in sediment ranged from 2,134 to 3,596 mg/kg dry mass. The levels of As(III) and As(V) species in the sediment ranges from 138 to 506 mg/kg dry mass and 156 to 385 mg/kg dry mass, respectively.

A Simple, Fast, Low Cost, HPLC/UV Validated Method for Determination of Flutamide: Application to Protein Binding Studies.

PubMed

Esmaeilzadeh, Sara; Valizadeh, Hadi; Zakeri-Milani, Parvin

2016-06-01

The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r(2) > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide.

A Simple, Fast, Low Cost, HPLC/UV Validated Method for Determination of Flutamide: Application to Protein Binding Studies

PubMed Central

Esmaeilzadeh, Sara; Valizadeh, Hadi; Zakeri-Milani, Parvin

2016-01-01

Purpose: The main goal of this study was development of a reverse phase high performance liquid chromatography (RP-HPLC) method for flutamide quantitation which is applicable to protein binding studies. Methods: Ultrafilteration method was used for protein binding study of flutamide. For sample analysis, flutamide was extracted by a simple and low cost extraction method using diethyl ether and then was determined by HPLC/UV. Acetanilide was used as an internal standard. The chromatographic system consisted of a reversed-phase C8 column with C8 pre-column, and the mobile phase of a mixture of 29% (v/v) methanol, 38% (v/v) acetonitrile and 33% (v/v) potassium dihydrogen phosphate buffer (50 mM) with pH adjusted to 3.2. Results: Acetanilide and flutamide were eluted at 1.8 and 2.9 min, respectively. The linearity of method was confirmed in the range of 62.5-16000 ng/ml (r2 > 0.99). The limit of quantification was shown to be 62.5 ng/ml. Precision and accuracy ranges found to be (0.2-1.4%, 90-105%) and (0.2-5.3 %, 86.7-98.5 %) respectively. Acetanilide and flutamide capacity factor values of 1.35 and 2.87, tailing factor values of 1.24 and 1.07 and resolution values of 1.8 and 3.22 were obtained in accordance with ICH guidelines. Conclusion: Based on the obtained results a rapid, precise, accurate, sensitive and cost-effective analysis procedure was proposed for quantitative determination of flutamide. PMID:27478788

Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

PubMed

Wan, Jun-Hui; Tian, Pei-Ling; Luo, Wei-Hao; Wu, Bing-Yi; Xiong, Fu; Zhou, Wan-Jun; Wei, Xiang-Cai; Xu, Xiang-Min

2012-07-15

Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-β, β, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and β thalassemia) were found in the area ratio of α/pre-β+β applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/β and α/pre-β+β area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating β thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/β for β thalassemia carriers and 0.626 of α/pre-β+β for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Human and porcine immunoreactive gastric inhibitory polypeptides (IR-GIP) are not identical.

PubMed

Bacarese-Hamilton, A J; Adrian, T E; Bloom, S R

1984-03-12

Immunoreactive gastric inhibitory polypeptide (IR-GIP) from human and porcine intestine was quantified by radioimmunoassay and the molecular forms characterised by gel permeation and reverse-phase high pressure liquid chromatography (HPLC). Gel filtration revealed two major immunoreactive peaks corresponding to the previously described 5-kDa and 8-kDa molecular forms, which appeared similar in both species. Isocratic reverse-phase HPLC revealed that the major immunoreactive GIP peak (5-kDa) in the human tissue eluted earlier than the corresponding porcine molecular form, indicating the latter to be less hydrophobic. These findings suggest significant species differences between human and porcine GIP.

In vivo biosynthesis of L-(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin: rapid estimation using reversed phase high pressure liquid chromatography. [Rats

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

Franco-Bourland, R.E.; Fernstrom, J.D.

1981-01-01

L(/sup 35/S)Cys-arginine vasopressin, -oxytocin, and -somatostatin were purified from hypothalami and neurohypophyses 4 h after rats received L(/sup 35/S)Cys via the third ventricle. After acetic acid extraction, Sephadex G-25 filtration, and chemoadsorption to C18-silica (Sep-Pak cartridges), the labeled peptides were rapidly separated by gradient elution, reversed phase, high pressure liquid chromatography (HPLC). The identity and isotopic purity of the labeled peptides were determined by several reversed phase HPLC procedures in conjunction with chemical modification. The labeled peptide fractions were at least 50% radiochemically pure. Using this HPLC isolation procedure, incorporation of L-(/sup 35/S)Cys into each peptide was determined in hydratedmore » and dehydrated rats. Label incorporation into arginine vasopressin and oxytocin in the hypothalamus and the neurohypophysis of dehydrated rats was 2-3 times greater than that in hydrated rats. Incorporation of label into hypothalamic and neurohypophyseal somatostatin was unaffected by the hydration state of the animal. This procedure thus provides a very rapid, but sensitive, set of techniques for studying the control of small peptide biosynthesis in the brain.« less

Comparison of analytical methods for the determination of histamine in reference canned fish samples

NASA Astrophysics Data System (ADS)

Jakšić, S.; Baloš, M. Ž.; Mihaljev, Ž.; Prodanov Radulović, J.; Nešić, K.

2017-09-01

Two screening methods for histamine in canned fish, an enzymatic test and a competitive direct enzyme-linked immunosorbent assay (CD-ELISA), were compared with the reversed-phase liquid chromatography (RP-HPLC) standard method. For enzymatic and CD-ELISA methods, determination was conducted according to producers’ manuals. For RP-HPLC, histamine was derivatized with dansyl-chloride, followed by RP-HPLC and diode array detection. Results of analysis of canned fish, supplied as reference samples for proficiency testing, showed good agreement when histamine was present at higher concentrations (above 100 mg kg-1). At a lower level (16.95 mg kg-1), the enzymatic test produced some higher results. Generally, analysis of four reference samples according to CD-ELISA and RP-HPLC showed good agreement for histamine determination (r=0.977 in concentration range 16.95-216 mg kg-1) The results show that the applied enzymatic test and CD-ELISA appeared to be suitable screening methods for the determination of histamine in canned fish.

Virtual Cross-Linking of the Active Nemorubicin Metabolite PNU-159682 to Double-Stranded DNA.

PubMed

Scalabrin, Matteo; Quintieri, Luigi; Palumbo, Manlio; Riccardi Sirtori, Federico; Gatto, Barbara

2017-02-20

The DNA alkylating mechanism of PNU-159682 (PNU), a highly potent metabolite of the anthracycline nemorubicin, was investigated by gel-electrophoretic, HPLC-UV, and micro-HPLC/mass spectrometry (MS) measurements. PNU quickly reacted with double-stranded oligonucleotides, but not with single-stranded sequences, to form covalent adducts which were detectable by denaturing polyacrylamide gel electrophoresis (DPAGE). Ion-pair reverse-phase HPLC-UV analysis on CG rich duplex sequences having a 5'-CCCGGG-3' central core showed the formation of two types of adducts with PNU, which were stable and could be characterized by micro-HPLC/MS. The first type contained one alkylated species (and possibly one reversibly bound species), and the second contained two alkylated species per duplex DNA. The covalent adducts were found to produce effective bridging of DNA complementary strands through the formation of virtual cross-links reminiscent of those produced by classical anthracyclines in the presence of formaldehyde. Furthermore, the absence of reactivity of PNU with CG-rich sequence containing a TA core (CGTACG), and the minor reactivity between PNU and CGC sequences (TACGCG·CGCGTA) pointed out the importance of guanine sequence context in modulating DNA alkylation.

Synthesis, liquid chromatographic fractionation and partial characterization of polybrominated dibenzofuran congeners.

PubMed

Gallistl, Christoph; Vetter, Walter

2016-04-15

Polybrominated dibenzofurans (PBDFs) are a class of highly toxic environmental contaminants which comprises 135 structurally different congeners. While the gas chromatographic separation and analysis of the most polychlorinated dibenzofurans (PCDFs) are well-documented, comparably little data is currently available in the case of PBDFs. In this study dibenzofuran was brominated to give a mixture of ∼40 PBDFs with one to seven bromine atoms. This synthesis mixture was fractionated by both countercurrent chromatography (CCC) with the solvent system n-hexane/toluene/acetonitrile and non-aqueous reversed-phase high performance liquid chromatography (RP-HPLC) with acetonitrile as the mobile phase. All together 80 consecutive CCC fractions and 40 HPLC fractions were taken and analyzed for PBDFs by gas chromatography coupled to mass spectrometry (GC/MS). CCC and RP-HPLC offered orthogonal separation of the PBDF mixture. As a consequence, selected CCC fractions were further fractionated by RP-HPLC. In this way, eight PBDFs could be isolated and the structures of twelve PBDFs were elucidated by proton magnetic resonance spectroscopy ((1)H NMR). Copyright © 2016 Elsevier B.V. All rights reserved.

Separation and quantitation of debrisoquine and 4-hydroxydebrisoquine in human urine by capillary electrophoresis and high-performance liquid chromatography.

PubMed

Cifuentes, A; Valencia, J; Sanz, E; Sánchez, M J; Rodríguez-Delgado, M A

1997-08-22

A comparative study on the use of reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE) for the determination of debrisoquine (D) and its metabolite, 4-hydroxydebrisoquine (4-HD), in human urine is presented. Four different urine pre-treatments are compared for purification of samples prior to their injection in HPLC and CE. The use of a solid-phase extraction with a C18 cartridge provides the best results for the urine sample treatment, with good recoveries, i.e., 94.5% for D and 93.4% for 4-HD, and high reproducibility, i.e., R.S.D. N = 10 values of 1.7% and 1.2%, respectively. Under our separation conditions it is shown that CE is twice as fast and provides slightly better analysis time reproducibility than HPLC for this type of sample. Both the sensitivity and peak area reproducibility are better when HPLC is used. The two techniques show good agreement when employed for determination of phenotypes for hydroxylation, which seems to corroborate the usefulness of CE for this type of study.

Solid cation exchange phase to remove interfering anthocyanins in the analysis of other bioactive phenols in red wine.

PubMed

da Silva, Letícia Flores; Guerra, Celito Crivellaro; Klein, Diandra; Bergold, Ana Maria

2017-07-15

Bioactive phenols (BPs) are often targets in red wine analysis. However, other compounds interfere in the liquid chromatography methods used for this analysis. Here, purification procedures were tested to eliminate anthocyanin interference during the determination of 19 red-wine BPs. Liquid chromatography, coupled to a diode array detector (HPLC-DAD) and a mass spectrometer (UPLC-MS), was used to compare the direct injection of the samples with solid-phase extractions: reversed-phase (C18) and strong cation-exchange (SCX). The HPLC-DAD method revealed that, out of 13BPs, only six are selectively analyzed with or without C18 treatment, whereas SCX enabled the detection of all BPs. The recovery with SCX was above 86.6% for eight BPs. Moreover, UPLC-MS demonstrated the potential of SCX sample preparation for the determination of 19BPs. The developed procedure may be extended to the analysis of other red wine molecules or to other analytical methods where anthocyanins may interfere. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

PubMed

Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

2015-01-01

A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation conditions for seven phenolic compounds was also achieved using reversed-phase HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and seven phenolic compounds could be separated and detected at 230 nm within 16 min. Copyright © 2014 Elsevier B.V. All rights reserved.

HPLC-DAD-MS identification of bioactive secondary metabolites from Ferula communis roots.

PubMed

Arnoldi, Lolita; Ballero, Mauro; Fuzzati, Nicola; Maxia, Andrea; Mercalli, Enrico; Pagni, Luca

2004-06-01

A simple HPLC method was developed to distinguish between 'poisonous' and 'non-poisonous' chemotypes of Ferula communis. The method was performed on a C8 reverse phase analytical column using a binary eluent (aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition. The two chemotypes showed different fingerprints. The identification of five coumarins and eleven daucane derivatives by HPLC-diode array detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, was detected. Copyright 2004 Elsevier B.V.

Quantitative determination of ambroxol in tablets by derivative UV spectrophotometric method and HPLC.

PubMed

Dinçer, Zafer; Basan, Hasan; Göger, Nilgün Günden

2003-04-01

A derivative UV spectrophotometric method for the determination of ambroxol in tablets was developed. Determination of ambroxol in tablets was conducted by using first-order derivative UV spectrophotometric method at 255 nm (n = 5). Standards for the calibration graph ranging from 5.0 to 35.0 microg/ml were prepared from stock solution. The proposed method was accurate with 98.6+/-0.4% recovery value and precise with coefficient of variation (CV) of 1.22. These results were compared with those obtained by reference methods, zero-order UV spectrophotometric method and reversed-phase high-performance liquid chromatography (HPLC) method. A reversed-phase C(18) column with aqueous phosphate (0.01 M)-acetonitrile-glacial acetic acid (59:40:1, v/v/v) (pH 3.12) mobile phase was used and UV detector was set to 252 nm. Calibration solutions used in HPLC were ranging from 5.0 to 20.0 microg/ml. Results obtained by derivative UV spectrophotometric method was comparable to those obtained by reference methods, zero-order UV spectrophotometric method and HPLC, as far as ANOVA test, F(calculated) = 0.762 and F(theoretical) = 3.89, was concerned. Copyright 2003 Elsevier Science B.V.

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.

Novel HPLC-UV Method for Simultaneous Determination of Fat-soluble Vitamins and Coenzyme Q10 in Medicines and Supplements.

PubMed

Temova-Rakuša, Žane; Srečnik, Eva; Roškar, Robert

2017-09-01

A precise, accurate and rapid HPLC-UV method for simultaneous determination of fat-soluble vitamins (vitamin D3, E-acetate, K1, β-carotene, A-palmitate) and coenzyme Q10 was developed and validated according to ICH guidelines. Optimal chromatographic separation of the analytes in minimal analysis time (8 min) was achieved on a Luna C18 150 × 4.6 mm column using a mixture of acetonitrile, tetrahydrofuran and water (50:45:5, v/v/v). The described reversed phase HPLC method is the first published for quantification of these five fat-soluble vitamins and coenzyme Q10 within a single chromatographic run. The method was further applied for quantification of the analytes in selected liquid and solid dosage forms, registered as nutritional supplements and prescription medicines, which confirmed its suitability for routine analysis.

Stability Indicating Reverse Phase HPLC Method for Estimation of Rifampicin and Piperine in Pharmaceutical Dosage Form.

PubMed

Shah, Umang; Patel, Shraddha; Raval, Manan

2018-01-01

High performance liquid chromatography is an integral analytical tool in assessing drug product stability. HPLC methods should be able to separate, detect, and quantify the various drug-related degradants that can form on storage or manufacturing, plus detect any drug-related impurities that may be introduced during synthesis. A simple, economic, selective, precise, and stability-indicating HPLC method has been developed and validated for analysis of Rifampicin (RIFA) and Piperine (PIPE) in bulk drug and in the formulation. Reversed-phase chromatography was performed on a C18 column with Buffer (Potassium Dihydrogen Orthophosphate) pH 6.5 and Acetonitrile, 30:70), (%, v/v), as mobile phase at a flow rate of 1 mL min-1. The detection was performed at 341 nm and sharp peaks were obtained for RIFA and PIPE at retention time of 3.3 ± 0.01 min and 5.9 ± 0.01 min, respectively. The detection limits were found to be 2.385 ng/ml and 0.107 ng/ml and quantification limits were found to be 7.228ng/ml and 0.325ng/ml for RIFA and PIPE, respectively. The method was validated for accuracy, precision, reproducibility, specificity, robustness, and detection and quantification limits, in accordance with ICH guidelines. Stress study was performed on RIFA and PIPE and it was found that these degraded sufficiently in all applied chemical and physical conditions. Thus, the developed RP-HPLC method was found to be suitable for the determination of both the drugs in bulk as well as stability samples of capsule containing various excipients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Rapid method for measuring rotenone in water at piscicidal concentrations

USGS Publications Warehouse

Dawson, V.K.; Harman, P.D.; Schultz, D.P.; Allen, J.L.

1983-01-01

A high-performance liquid chromatography (HPLC) procedure that is rapid, specific, and sensitive (limit of detection <0.005 mg/liter) was developed for monitoring application and degradation rates of rotenone. For analysis, a water sample is buffered to pH 5 and injected through a Sep Pak(R) C18 disposable cartridge. The cartridge adsorbs and retains the rotenone which then can be eluted quantitatively from the cartridge with a small volume of methanol. This step effectively concentrates the sample and provides sample cleanup. The methanol extract is analyzed directly by HPLC on an MCH 10 reverse-phase column; methanol: water (75:25, volume : volume) is the mobile phase and flow rate is 1.5 ml/minute. The rotenone is detected by ultraviolet spectrophotometry at a wavelength of 295 nm.

Identification of ionic chloroacetanilide-herbicide metabolites in surface water and groundwater by HPLC/MS using negative ion spray

USGS Publications Warehouse

Ferrer, I.; Thurman, E.M.; Barcelo, D.

1997-01-01

Solid-phase extraction (SPE) was combined with high-performance liquid chromatography/high-flow pneumatically assisted electrospray mass spectrometry (HPLC/ESP/MS) for the trace analysis of oxanilic and sulfonic acids of acetochlor, alachlor, and metolachlor. The isolation procedure separated the chloroacetanilide metabolites from the parent herbicides during the elution from C18 cartridges using ethyl acetate for parent compounds, followed by methanol for the anionic metabolites. The metabolites were separated chromatographically using reversed-phase HPLC and analyzed by negative-ion MS using electrospray ionization in selected ion mode. Quantitation limits were 0.01 ??g/L for both the oxanilic and sulfonic acids based on a 100-mL water sample. This combination of methods represents an important advance in environmental analysis of chloroacetanilide-herbicide metabolites in surface water and groundwater for two reasons. First, anionic chloroacetanilide metabolites are a major class of degradation products that are readily leached to groundwater in agricultural areas. Second, anionic metabolites, which are not able to be analyzed by conventional methods such as liquid extraction and gas chromatography/mass spectrometry, are effectively analyzed by SPE and high-flow pneumatically assisted electrospray mass spectrometry. This paper reports the first HPLC/MS identification of these metabolites in surface water and groundwater.

Separating DDTs in edible animal fats using matrix solid-phase dispersion extraction with activated carbon filter, Toyobo-KF.

PubMed

Furusawa, Naoto

2006-09-01

A technique is presented for the economical, routine, and quantitative analysis of contamination by dichloro-diphenyl-trichloroethanes (DDTs) [pp'-DDT, pp'-dichlorodiphenyl dichloroethylene, and pp'-dichlorodiphenyl dichloreothane in beef tallow and chicken fat samples, based on their separation using matrix solid-phase dispersion (MSPD) extraction with Toyobo-KF, an activated carbon fiber. Toyobo-KF is a newly applied MSPD sorbent, and it is followed by reversed-phase high-performance liquid chromatography (HPLC) with a photodiode array detector. The resulting analytical performance parameters [recoveries of spiked DDTs (0.1, 0.2, and 0.4 microg/g) > or = 81%, with relative standard deviations of < or = 8% (n = 5), and quantitation limits < or = 0.03 microg/g], with minimal handling and cost-efficiency, indicate that the present MSPD-HPLC method may be a useful tool for routine monitoring of DDT contamination in meat.

Investigation of folic acid stability in fortified instant noodles by use of capillary electrophoresis and reversed-phase high performance liquid chromatography.

PubMed

Hau Fung Cheung, Rodney; Morrison, Paul D; Small, Darryl M; Marriott, Philip J

2008-12-05

A single enzyme treatment with alpha-amylase, prior to the quantification of added folic acid (FA) in fortified instant fried Asian noodles with analysis performed by capillary zone electrophoresis (CZE) and reversed-phase high performance liquid chromatography (RP-HPLC) with UV detection, is described. The method was validated and optimized for capillary electrophoresis (CE) with separation achieved using a 8 mM phosphate-12 mM borate run buffer with 5% MeOH at pH 9.5. FA was well separated from matrix components with nicotinic acid (NA) employed as an internal standard. In a comparative study, separation of FA was performed using HPLC with a mobile phase consisting of 27% MeOH (v/v) in aqueous potassium phosphate buffer (3.5 mM KH(2)PO(4) and 3.2 mM K(2)HPO(4)), pH 8.5, and containing 5 mM tetrabutylammonium dihydrogen phosphate as an ion-pairing agent. For both methods, excellent results were obtained for various analytical parameters including linearity, accuracy and precision. The limit of detection was calculated to be 2.2 mg/L for CE without sample stacking and 0.10 mg/L with high performance liquid chromatography (HPLC). Sample extraction involved homogenization and enzymatic extraction with alpha-amylase. Results indicated that FA was stable during four main stages of instant fried noodle manufacturing (dough crumbs, cut sheets, steaming and frying).

Development and validation of an HPLC method for simultaneous determination of trimethoprim and sulfamethoxazole in human plasma.

PubMed

Sayar, Esin; Sahin, Selma; Cevheroglu, Semsettin; Hincal, A Atilla

2010-09-01

The combination of trimethoprim (TMP) and sulfamethoxazole (SMX) is used in the treatment of many common infections such as urinary, respiratory and gastrointestinal tract infections. The aim of this study was to determine TMP and SMX simultaneously in human plasma samples by high performance liquid chromatography (HPLC) using antipyrine as the internal standard. Separation of the compounds was achieved on a reverse-phase C8 column packed with 5 microm dimethyl octadecylsilyl bonded amorphous silica (4.6 mm x 250 mm) column using a mobile phase consisted of potassium hydrogen phosphate, acetonitrile, methanol and water adjusted to pH 6.2. The mobile phase was delivered at a flow rate of 1 mL min- and the effluent was monitored using Max plot technique at 25 derees C. Retention times were 5 min for TMP, 7 min for antipyrine and 9 min for SMX. Quantitation limits were 10 ng mL(-1) for TMP and 50 ng mL(-1) for SMX. Our findings indicated that the developed HPLC method was precise, accurate, specific and sensitive for simultaneous determination of TMP and SMX. Proposed HPLC method was successfully applied for the analysis of TMP and SMX in human plasma after oral administration of a co-trimoxazole tablet to human volunteers.

The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

NASA Astrophysics Data System (ADS)

Karsten, Ulf; Escoubeyrou, Karine; Charles, François

2009-09-01

Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

Optimisation of ultrasound-assisted reverse micelles dispersive liquid-liquid micro-extraction by Box-Behnken design for determination of acetoin in butter followed by high performance liquid chromatography.

PubMed

Roosta, Mostafa; Ghaedi, Mehrorang; Daneshfar, Ali

2014-10-15

A novel approach, ultrasound-assisted reverse micelles dispersive liquid-liquid microextraction (USA-RM-DLLME) followed by high performance liquid chromatography (HPLC) was developed for selective determination of acetoin in butter. The melted butter sample was diluted and homogenised by n-hexane and Triton X-100, respectively. Subsequently, 400μL of distilled water was added and the microextraction was accelerated by 4min sonication. After 8.5min of centrifugation, sedimented phase (surfactant-rich phase) was withdrawn by microsyringe and injected into the HPLC system for analysis. The influence of effective variables was optimised using Box-Behnken design (BBD) combined with desirability function (DF). Under optimised experimental conditions, the calibration graph was linear over the range of 0.6-200mgL(-1). The detection limit of method was 0.2mgL(-1) and coefficient of determination was 0.9992. The relative standard deviations (RSDs) were less than 5% (n=5) while the recoveries were in the range of 93.9-107.8%. Copyright © 2014. Published by Elsevier Ltd.

Preparation and Characterization of a Polymeric Monolithic Column for Use in High-Performance Liquid Chromatography (HPLC)

ERIC Educational Resources Information Center

Bindis, Michael P.; Bretz, Stacey Lowery; Danielson, Neil D.

2011-01-01

The high-performance liquid chromatography (HPLC) experiment, most often done in the undergraduate analytical instrumentation laboratory course, generally illustrates reversed-phase chromatography using a commercial C[subscript]18 silica column. To avoid the expense of periodic column replacement and introduce a choice of columns with different…

A NEW HPLC METHOD FOR SEPARATION OF PHYTOPLANKTON PIGMENTS IN NATURAL SAMPLES

EPA Science Inventory

A new high-performance liquid chromatographic (HPLC) method was developed to analyze, in a single run, most polar and non-polar chlorophylls and carotenoids from marine phytoplankton. The method is based on a reverse-phase amide C16 (RP-amide C16) column and an elution gradient o...

Simplified RP-HPLC method for multi-residue analysis of abamectin, emamectin benzoate and ivermectin in rice.

PubMed

Xie, Xianchuan; Gong, Shu; Wang, Xiaorong; Wu, Yinxing; Zhao, Li

2011-01-01

A rapid, reliable and sensitive reverse-phase high-performance liquid chromatography method with fluorescence detection (RP-FLD-HPLC) was developed and validated for simultaneous analysis of the abamectin (ABA), emamectin (EMA) benzoate and ivermectin (IVM) residues in rice. After extraction with acetonitrile/water (2 : 1) with sonication, the avermectin (AVMs) residues were directly derivatised by N-methylimidazole (N-NMIM) and trifluoroacetic anhydride (TFAA) and then analysed on RP-FLD-HPLC. A good linear relationship (r(2 )> 0.99) was obtained for three AVMs ranging from 0.01 to 5 microg ml(-1), i.e. 0.01-5.0 microg g(-1) in rice matrix. The limit of detection (LOD) and the limit of quantification (LOQ) were between 0.001 and 0.002 microg g(-1) and between 0.004 and 0.006 microg g(-1), respectively. Recoveries were from 81.9% to 105.4% and precision less than 12.4%. The proposed method was successfully applied to routine analysis of the AVMs residues in rice.

High-performance liquid chromatography-mass spectrometry for mapping and sequencing glycosaminoglycan-derived oligosaccharides

PubMed Central

Volpi, Nicola; Linhardt, Robert J

2012-01-01

Glycosaminoglycans (GAGs) have proven to be very difficult to analyze and characterize because of their high negative charge density, polydispersity and sequence heterogeneity. As the specificity of the interactions between GAGs and proteins results from the structure of these polysaccharides, an understanding of GAG structure is essential for developing a structure–activity relationship. Electrospray ionization (ESI) mass spectrometry (MS) is particularly promising for the analysis of oligosaccharides chemically or enzymatically generated by GAGs because of its relatively soft ionization capacity. Furthermore, on-line high-performance liquid chromatography (HPLC)-MS greatly enhances the characterization of complex mixtures of GAG-derived oligosaccharides, providing important structural information and affording their disaccharide composition. A detailed protocol for producing oligosaccharides from various GAGs, using controlled, specific enzymatic or chemical depolymerization, is presented, together with their HPLC separation, using volatile reversed-phase ion-pairing reagents and on-line ESI-MS structural identification. This analysis provides an oligosaccharide map together with sequence information from a reading frame beginning at the nonreducing end of the GAG chains. The preparation of oligosaccharides can be carried out in 10 h, with subsequent HPLC analysis in 1–2 h and HPLC-MS analysis taking another 2 h. PMID:20448545

Exploring the fatty acids of vernix caseosa in form of their methyl esters by off-line coupling of non-aqueous reversed phase high performance liquid chromatography and gas chromatography coupled to mass spectrometry.

PubMed

Hauff, Simone; Vetter, Walter

2010-12-24

Vernix caseosa is a greasy biofilm formed on the skin of the human fetus in the last period of pregnancy. This matrix is known to contain a range of uncommon branched chain fatty acids. In this study, we studied the fatty acid composition of vernix caseosa by non-aqueous reversed phase high performance liquid chromatography (RP-HPLC) fractionation followed by gas chromatography-electron ionization mass spectrometry (GC/EI-MS) of the fractions. For this purpose the fatty acids from vernix caseosa were converted into the corresponding methyl esters. These were fractionated by non-aqueous RP-HPLC using three serially connected C(18)-columns and pure methanol as the eluent. Aliquots of the HPLC fractions were directly analyzed by GC/EI-MS in the selected ion monitoring mode. Data analysis and visualization were performed by the creation of a two dimensional (2D) contour plot, in which GC retention times were plotted against the HPLC fractions. Inspection of the plot resulted in the detection of 133 different fatty acids but only 16 of them contributed more than 1% to the total fatty acids detected. Identification was based on HPLC and GC retention data, GC/MS-SIM and full scan data, as well as plotting the logarithmic retention times against the longest straight carbon chain. In selected cases, aliquots of the HPLC fractions were hydrogenated or studied by means of the picolinyl esters. Using these techniques, the number of double bonds could be unequivocally assigned to all fatty acids. Moreover, the number of methyl branches, and in many cases the positions of methyl branches could be determined. The enantioselective analysis of chiral anteiso-fatty acids resulted in the dominance of the S-enantiomers. However, high proportions of R-a13:0, R-a15:0, and R-a17:1 were also detected while a17:0 was virtually S-enantiopure. Copyright © 2010 Elsevier B.V. All rights reserved.

HPLC analysis of functionalized poly(amidoamine) dendrimers and the interaction between a folate-dendrimer conjugate and folate binding protein.

PubMed

Shi, Xiangyang; Bi, Xiangdong; Ganser, T Rose; Hong, Seungpyo; Myc, Lukasz A; Desai, Ankur; Holl, Mark M Banaszak; Baker, James R

2006-07-01

Poly(amidoamine) (PAMAM) dendrimers of different generations with carboxyl, acetyl, and hydroxyl terminal groups and a folic acid (FA)-dendrimer conjugate were separated and analyzed using reverse-phase high performance liquid chromatography (HPLC). Analysis of both the individual PAMAM derivatives and the separation of mixed generations can be achieved using a linear gradient 0-50% acetonitrile (ACN) (balance water) within 40 min. We also show that PAMAMs with defined acetylation and carboxylation degrees can be analyzed using HPLC. Furthermore, a generation 5 dendrimer-FA conjugate (G5.75Ac-FA4; Ac denotes acetyl) was analyzed and its specific binding with a bovine folic acid binding protein (FBP) was monitored. The HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results indicate the formation of three complexes after the binding of G5.75Ac-FA4 with FBP. Dendrimers with FA moieties show much higher specific binding capability with FBP than those without FA moieties. Findings from this study indicate that HPLC is an effective technique not only for characterization and separation of functionalized PAMAM dendrimers and conjugates but also for investigation of the interaction between dendrimers and biomolecules.

Offline solid-phase extraction for preconcentration of pharmaceuticals and personal care products in environmental water and their simultaneous determination using the reversed phase high-performance liquid chromatography method.

PubMed

G Archana; Dhodapkar, Rita; Kumar, Anupama

2016-09-01

The present study reports a precise and simple offline solid-phase extraction (SPE) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of five representative and commonly present pharmaceuticals and personal care products (PPCPs), a new class of emerging pollutants in the aquatic environment. The target list of analytes including ciprofloxacin, acetaminophen, caffeine benzophenone and irgasan were separated by a simple HPLC method. The column used was a reversed-phase C18 column, and the mobile phase was 1 % acetic acid and methanol (20:80 v/v) under isocratic conditions, at a flow rate of 1 mL min(-1). The analytes were separated and detected within 15 min using the photodiode array detector (PDA). The linearity of the calibration curves were obtained with correlation coefficients 0.98-0.99.The limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and ruggedness demonstrated the reproducibility, specificity and sensitivity of the developed method. Prior to the analysis, the SPE was performed using a C18 cartridge to preconcentrate the targeted analytes from the environmental water samples. The developed method was applied to evaluate and fingerprint PPCPs in sewage collected from a residential engineering college campus, polluted water bodies such as Nag river and Pili river and the influent and effluent samples from a sewage treatment plant (STP) situated at Nagpur city, in the peak summer season. This method is useful for estimation of pollutants present in microquantities in the surface water bodies and treated sewage as compared to nanolevel pollutants detected by mass spectrometry (MS) detectors.

Simple and simultaneous determination of the hiv-protease inhibitors amprenavir, atazanavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir plus M8 nelfinavir metabolite and the nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine in human plasma by reversed-phase liquid chromatography.

PubMed

Poirier, Jean-Marie; Robidou, Pascal; Jaillon, Patrice

2005-04-01

Several studies suggest that therapeutic drug monitoring of protease inhibitors and nonnucleoside reverse transcriptase inhibitors may contribute to the clinical outcome of HIV-infected patients. Because of the growing number of antiretroviral drugs and of drug combinations than can be administered to these patients, an accurate high-performance liquid chromatographic (HPLC) method allowing the simultaneous determination of these drugs may be useful. To date, the authors present the first simultaneous HPLC determination of the new protease inhibitor atazanavir with all the others currently in use (M8 nelfinavir metabolite included) and the 2 widely used nonnucleoside reverse transcriptase inhibitors efavirenz and nevirapine. This simple HPLC method allows the analysis all these drugs at a single ultraviolet wavelength following a 1-step liquid-liquid extraction procedure. A 500-muL plasma sample was spiked with internal standard and subjected to liquid-liquid extraction using by diethyl ether at pH 10. HPLC was performed using a Symmetry Shield RP18 and gradient elution. All the drugs of interest and internal standard were detected with ultraviolet detection at 210 nm. Calibration curves were linear in the range 50-10,000 ng/mL. The observed concentrations of the quality controls at plasma concentrations ranging from 50 to 5000 ng/mL for these drugs showed that the overall accuracy varied from 92% to 104% and 92% to 106% for intraday and day-to-day analysis, respectively. No metabolites of the assayed compounds or other drugs commonly coadministered to HIV-positive patients were found to coelute with the drugs of interest or with the internal standard. This assay was developed for the purpose of therapeutic monitoring (TDM) in HIV-infected patients.

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC.

PubMed

López-Cervantes, J; Sánchez-Machado, D I; Gutiérrez-Coronado, M A; Ríos-Vázquez, N J

2006-10-01

In the present study, a simple and rapid reversed-phase HPLC method for the determination of astaxanthin in shrimp waste hydrolysate has been developed and validated. The analytical procedure involves the direct extraction of astaxanthin from the lipid fraction with methanol. The analytical column, SS Exil ODS, was operated at 25C. The mobile phase consisted of a mixture of water:methanol:dichloromethane:acetonitrile (4.5:28:22:45.5 v/v/v/v) at a flow rate of 1.0 mL/min. Detection and identification were performed using a photodiode array detector (lambda(detection) = 476 nm). The proposed HPLC method showed adequate linearity, repeatability and accuracy.

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.

HPLC purification and re-evaluation of chemical identity of two circular bacteriocins, gassericin A and reutericin 6.

PubMed

Arakawa, K; Kawai, Y; Ito, Y; Nakamura, K; Chujo, T; Nishimura, J; Kitazawa, H; Saito, T

2010-04-01

The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6. Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results. Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues. The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Separation and identification of various carotenoids by C30 reversed-phase high-performance liquid chromatography coupled to UV and atmospheric pressure chemical ionization mass spectrometric detection.

PubMed

Lacker, T; Strohschein, S; Albert, K

1999-08-27

In this paper the application of on-line HPLC-UV-APCI (atmospheric pressure chemical ionization) mass spectrometry (MS) coupling for the separation and determination of different carotenoids as well as cis/trans isomers of beta-carotene is reported. All HPLC separations were carried out under RP conditions on self-synthesized polymeric C30 phases. The analysis of a carotenoid mixture containing astaxanthin, canthaxanthin, zeaxanthin, echinenone and beta-carotene by HPLC-APCI-MS was achieved by scanning the mass range from m/z 200 to 700. For the characterization of a sample containing cis/trans isomers of beta-carotene as well as their oxidation products, a photodiode-array UV-visible absorbance detector was used in addition between the column and the mass spectrometer for structural elucidation of the geometrical isomers. The detection limit for beta-carotene in positive-ion APCI-MS was determined to be 1 pmol. In addition, an extract of non-polar substances in vegetable juice has been analyzed by HPLC-APCI-MS. The included carotenoids could be identified by their masses and their retention times.

Distribution or adsorption: the major dilemma in reversed-phase HPLC

NASA Astrophysics Data System (ADS)

Deineka, V. I.

2008-06-01

A method is suggested for analyzing the dependences obtained for different compositions of mobile eluent system phases, their slopes and intercepts, log k( i, B) = a + b log k ( i, A), where a is the intercept for the A and B stationary phases and b is the proportionality factor. An analysis requires parallel investigation of sorbate retention on at least three stationary phases with different lengths of grafted hydrocarbon radicals. The dependence of correlation parameters on the sorbate retention mechanism is discussed. It is shown that the hypothetical dependences coincide with the experimental dependences for surface sorption of resveratrol and volume distribution of triglycerides.

A cytocidal tissue kallikrein isolated from mouse submandibular glands.

PubMed

Murakami, K; Ikigai, H; Nagumo, N; Tomita, M; Shimamura, T

1989-11-06

A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.

Purification and Pore Forming Activity of Two Hydrophobic Polypeptides from the Secretion of the Red Sea Moses Sole (Pardachirus marmoratus)

DTIC Science & Technology

1986-11-01

Uppsala, Sweden. Ovalbumin, bovine albumin fractio n V, soybean lecithin , sodium cnolate, gramicidin D and Dowex 50 x 8 (50-100 mesh) were obtained from...A-,.ino acid analysis 50 ug duplIcate samples of PXI and PXII, from reverse phase HPLC, were dissolved in 0.4 ml of 6 N HCI and hydrolyzed ocr 24 hr

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.

Simultaneous determination of 11 antibiotics and their main metabolites from four different groups by reversed-phase high-performance liquid chromatography-diode array-fluorescence (HPLC-DAD-FLD) in human urine samples.

PubMed

Fernandez-Torres, R; Consentino, M Olías; Lopez, M A Bello; Mochon, M Callejon

2010-05-15

A new, accurate and sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) as analytical method for the quantitative determination of 11 antibiotics (drugs) and the main metabolites of five of them present in human urine has been worked out, optimized and validated. The analytes belong to four different groups of antibiotics (sulfonamides, tetracyclines, penicillins and anphenicols). The analyzed compounds were sulfadiazine (SDI) and its N(4)-acetylsulfadiazine (NDI) metabolite, sulfamethazine (SMZ) and its N(4)-acetylsulfamethazine (NMZ), sulfamerazine (SMR) and its N(4)-acetylsulfamerazine (NMR), sulfamethoxazole (SMX), trimetroprim (TMP), amoxicillin (AMX) and its main metabolite amoxicilloic acid (AMA), ampicillin (AMP) and its main metabolite ampicilloic acid (APA), chloramphenicol (CLF), thiamphenicol (TIF), oxytetracycline (OXT) and chlortetracycline (CLT). For HPLC analysis, diode array (DAD) and fluorescence (FLD) detectors were used. The separation of the analyzed compounds was conducted by means of a Phenomenex Gemini C(18) (150mm x 4.6mm I.D., particle size 5microm) analytical column with LiChroCART LiChrospher C(18) (4mm x 4mm, particle size 5microm) guard column. Analyzed drugs were determined within 34min using formic acid 0.1% in water and acetonitrile in gradient elution mode as mobile phase. A linear response was observed for all compounds in the range of concentration studied. Two procedures were optimized for sample preparation: a direct treatment with methanol and acetonitrile and a solid phase extraction procedure using Bond Elut Plexa columns. The method was applied to the determination of the analytes in human urine from volunteers under treatment with different pharmaceutical formulations. This method can be successfully applied to routine determination of all these drugs in human urine samples.

Quantitative study of flavonoids in leaves of citrus plants.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M; Koizumi, M; Ito, C; Furukawa, H

2000-09-01

Leaf flavonoids were quantitatively determined in 68 representative or economically important Citrus species, cultivars, and near-Citrus relatives. Contents of 23 flavonoids including 6 polymethoxylated flavones were analyzed by means of reversed phase HPLC analysis. Principal component analysis revealed that the 7 associations according to Tanaka's classification were observed, but some do overlap each other. Group VII species could be divided into two different subgroups, namely, the first-10-species class and the last-19-species class according to Tanaka's classification numbers.

Quantitative analysis of PMR-15 polyimide resin by HPLC

NASA Technical Reports Server (NTRS)

Roberts, Gary D.; Lauver, Richard W.

1987-01-01

The concentration of individual components and of total solids of 50 wt pct PMR-15 resin solutions was determined using reverse-phase HPLC to within + or - 8 percent accuracy. Acid impurities, the major source of impurities in 3,3', 4,4'-benzophenonetetracarboxylic acid (BTDE), were eliminated by recrystallizing the BTDE prior to esterification. Triester formation was not a problem because of the high rate of esterification of the anhydride relative to that of the carboxylic acid. Aging of PMR-15 resin solutions resulted in gradual formation of the mononadimide and bisnadimide of 4,4'-methylenedianiline, with the BTDE concentration remaining constant. Similar chemical reactions occurred at a reduced rate in dried films of PMR-15 resin.

Retention properties of novel beta-CD bonded stationary phases in reversed-phase HPLC mode.

PubMed

Zhao, Yanyan; Guo, Zhimou; Zhang, Yongping; Xue, Xingya; Xu, Qing; Li, Xiuling; Liang, Xinmiao; Zhang, Yukui

2009-05-15

With the given special structures, the CD bonded stationary phases are expected to have complementary retention properties with conventional C18 stationary phase, which will be helpful to enhance the polar selectivity in RP mode separation. In this work, two beta-cyclodextrin (beta-CD) bonded stationary phases for reversed-phase HPLC, including 1, 12-dodecyldiol linked beta-CD stationary phase (CD1) and olio (ethylene glycol) (OEG) linked beta-CD stationary phase (CD2), have been synthesized via click chemistry. The resulting materials were characterized with FT-IR and elemental analysis, which proved the successful immobilization of ligands. The similarities and differences in retention characteristics between the CD and C18 stationary phases have been elucidated by using comparative linear solvation energy relationships (LSERs). The force related to solute McGowan volume has no significant difference, while the hydrogen bonding and dipolar interactions between solutes and CD stationary phases are stronger than between solutes and C18, which is attributed to the special structures (CD and triazole groups) of CD stationary phases. Chemical origins are interpreted by comparison between CD1 and CD2. Similar dispersive interactions of CD1 and CD2 are attributed to their similar length of spacer arms. CD2 which contains OEG spacer arm has relative weaker HBD acidity but stronger HBA basicity. CD stationary phases display no serious different methylene selectivity and higher polar selectivity than in the case of C18. Higher acid selectivity and lower basic selectivity are observed on CD2 than on CD1. Distinctive retention properties and good complementary separation selectivity to C18 make the novel CD bonded stationary phases available for more application in RPLC.

A unified classification of stationary phases for packed column supercritical fluid chromatography.

PubMed

West, C; Lesellier, E

2008-05-16

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.

A novel reversed-phase HPLC method for the determination of urinary creatinine by pre-column derivatization with ethyl chloroformate: comparative studies with the standard Jaffé and isotope-dilution mass spectrometric assays.

PubMed

Leung, Elvis M K; Chan, Wan

2014-02-01

Creatinine is an important biomarker for renal function diagnosis and normalizing variations in urinary drug/metabolites concentration. Quantification of creatinine in biological fluids such as urine and plasma is important for clinical diagnosis as well as in biomonitoring programs and urinary metabolomics/metabonomics research. Current methods for creatinine determination either are nonselective or involve the use of expensive mass spectrometers. In this paper, a novel reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of creatinine of high hydrophilicity by pre-column derivatization with ethyl chloroformate is presented. N-Ethyloxycarbonylation of creatinine significantly enhanced the hydrophobicity of creatinine, facilitating its chromatographic retention as well as quantification by HPLC. Factors governing the derivatization reaction were studied and optimized. The developed method was validated and applied for the determination of creatinine in rat urine samples. Comparative studies with isotope-dilution mass spectrometric method revealed that the two methods do not yield systematic differences in creatinine concentrations, indicating the HPLC method is suitable for the determination of creatinine in urine samples.

First derivative ratio spectrophotometric, HPTLC-densitometric, and HPLC determination of nicergoline in presence of its hydrolysis-induced degradation product.

PubMed

Ahmad, Abdel Kader S; Kawy, M Abdel; Nebsen, M

2002-10-15

Three methods are presented for the determination of Nicergoline in presence of its hydrolysis-induced degradation product. The first method was based on measurement of the first derivative of ratio spectra amplitude of Nicergoline at 291 nm. The second method was based on separation of Nicergoline from its degradation product followed by densitometric measurement of the spots at 287 nm. The separation was carried out on HPTLC silica gel F(254) plates, using methanol-ethyl acetate-glacial acetic acid (5:7:3, v/v/v) as mobile phase. The third method was based on high performance liquid chromatographic (HPLC) separation and determination of Nicergoline from its degradation product on a reversed phase, nucloesil C(18) column using a mobile phase of methanol-water-glacial acetic acid (80:20:0.1, v/v/v) with UV detection at 280 nm. Chlorpromazine hydrochloride was used as internal standard. Laboratory prepared mixtures containing different percentages of the degradation product were analysed by the proposed methods and satisfactory results were obtained. These methods have been successfully applied to the analysis of Nicergoline in Sermion tablets. The validities of these methods were ascertained by applying standard addition technique, the mean percentage recovery +/- R.S.D.% was found to be 99.47 +/- 0.752, 100.01 +/- 0.940, 99.75 +/- 0.740 for the first derivative of ratio spectra method, the HPTLC method and the HPLC method, respectively. The proposed methods were statistically compared with the manufacturer's HPLC method of analysis of Nicergoline and no significant difference was found with respect to both precision and accuracy. They have the advantage of being stability indicating. Therefore, they can be used for routine analysis of the drug in quality control laboratories. Copyright 2002 Elsevier Science B.V.

An analysis of dissolved organic matter from freshwater Karelian Lakes using reversed-phase high-performance liquid chromatography with online absorbance and fluorescence analysis

NASA Astrophysics Data System (ADS)

Khundzhua, D. A.; Patsaeva, S. V.; Trubetskoj, O. A.; Trubetskaya, O. E.

2017-01-01

The spectral and optical properties of the fractionated components of dissolved organic matter (DOM) of three freshwater lakes in Karelia were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) with online detection of fluorescence and absorption spectra. It is shown that the DOM fractions are qualitatively similar, but differ quantitatively in the ratio of components and consist of at least three types of fluorophores: (1) hydrophilic "humic-like" fluorophore(s) with the emission maximum in the region of 420 nm and an absorption band at 260-270 nm; (2) hydrophobic "humic-like" fluorophore(s) with the emission maximum at approximately 450 nm that has no characteristic absorption maxima in the region from 220 to 400 nm; and (3) a "protein-like" fluorophore with the emission maximum in the region of 340-350 nm, which is typical of proteins and peptides containing tryptophan.

Identification and Quantitation of Asparagine and Citrulline Using High-Performance Liquid Chromatography (HPLC)

PubMed Central

Bai, Cheng; Reilly, Charles C.; Wood, Bruce W.

2007-01-01

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (μMol ml−1/μMol ml−1)], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides. PMID:19662174

Identification and quantitation of asparagine and citrulline using high-performance liquid chromatography (HPLC).

PubMed

Bai, Cheng; Reilly, Charles C; Wood, Bruce W

2007-03-28

High-performance liquid chromatography (HPLC) analysis was used for identification of two problematic ureides, asparagine and citrulline. We report here a technique that takes advantage of the predictable delay in retention time of the co-asparagine/citrulline peak to enable both qualitative and quantitative analysis of asparagine and citrulline using the Platinum EPS reverse-phase C18 column (Alltech Associates). Asparagine alone is eluted earlier than citrulline alone, but when both of them are present in biological samples they may co-elute. HPLC retention times for asparagine and citrulline were influenced by other ureides in the mixture. We found that at various asparagines and citrulline ratios [= 3:1, 1:1, and 1:3; corresponding to 75:25, 50:50, and 25:75 (microMol ml(-1)/microMol ml(-1))], the resulting peak exhibited different retention times. Adjustment of ureide ratios as internal standards enables peak identification and quantification. Both chemicals were quantified in xylem sap samples of pecan [Carya illinoinensis (Wangenh.) K. Koch] trees. Analysis revealed that tree nickel nutrition status affects relative concentrations of Urea Cycle intermediates, asparagine and citrulline, present in sap. Consequently, we concluded that the HPLC methods are presented to enable qualitative and quantitative analysis of these metabolically important ureides.

Separation and quantitation of colour pigments of chili powder (Capsicum frutescens) by high-performance liquid chromatography-diode array detection.

PubMed

Cserháti, T; Forgács, E; Morais, M H; Mota, T; Ramos, A

2000-10-27

The performance of reversed-phase thin-layer (RP-TLC) and reversed-phase high-performance liquid chromatography (RP-HPLC) was compared for the separation and determination of the colour pigments of chili (Capsicum frutescens) powder using a wide variety of eluent systems. No separation of pigments was achieved in RP-TLC, however, it was established that tetrahydrofuran shows an unusually high solvent strength. RP-HPLC using water-methanol-acetonitrile gradient elution separated the chili pigments in many fractions. Diode array detection (DAD) indicated that yellow pigments are eluted earlier than the red ones and chili powder contains more yellow pigments than common paprika powders. It was established that the very different absorption spectra of pigments make the use of DAD necessary.

Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles.

PubMed

das Neves, José; Sarmento, Bruno; Amiji, Mansoor M; Bahia, Maria Fernanda

2010-06-05

The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile-0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1-50 microg/ml (R(2)=0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17+/-0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 microg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Analysis of nine food additives in red wine by ion-suppression reversed-phase high-performance liquid chromatography using trifluoroacetic acid and ammonium acetate as ion-suppressors.

PubMed

Zhao, Yong-Gang; Chen, Xiao-Hong; Yao, Shan-Shan; Pan, Sheng-Dong; Li, Xiao-Ping; Jin, Mi-Cong

2012-01-01

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous determination of nine food additives, i.e., acesulfame, saccharin, caffeine, aspartame, benzoic acid, sorbic acid, stevioside, dehydroacetic acid and neotame in red wine. The effects of ion-suppressors, i.e., trifluoroacetic acid (TFA) and ammonium acetate (AmAc) on retention behavior of nine food additives in RP-HPLC separation were discussed in detail. The relationships between retention factors of solutes and volume percent of ion-suppressors in the mobile-phase systems of acetonitrile-TFA aqueous solution and acetonitrile-TFA-AmAc aqueous solution were quantitatively established, respectively. The results showed that the ion suppressors had not only an ion suppression effect, but also an organic modification effect on the acidic analytes. The baseline separation of nine food additives was completed by a gradient elution with acetonitrile-TFA(0.01%, v/v)-AmAc(2.5 mmol L(-1)) aqueous solution as the mobile phase. The recoveries were between 80.2 - 99.5% for all analytes with RSDs in the range of 1.5 - 8.9%. The linearities were in the range of 0.2 - 100.0 mg L(-1) with determination coefficients (r(2)) higher than 0.9991 for all analytes. The limits of quantification (LOQs) were between 0.53 - 0.99 mg L(-1). The applicability of the proposed method to detect and quantify food additives has been demonstrated in the analysis of 30 real samples.

Determination of 13-cis-retinoic acid and its major metabolite, 4-oxo-13-cis-retinoic acid, in human blood by reversed-phase high-performance liquid chromatography.

PubMed

Vane, F M; Stoltenborg, J K; Buggé, C J

1982-02-12

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10--2000 and 50--2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.

Simultaneous determination of cations, zwitterions and neutral compounds using mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography.

PubMed

Li, Jingyi; Shao, Shan; Jaworsky, Markian S; Kurtulik, Paul T

2008-03-28

A novel mixed-mode reversed-phase and cation-exchange high-performance liquid chromatography (HPLC) method is described to simultaneously determine four related impurities of cations, zwitterions and neutral compounds in developmental Drug A. The commercial column is Primesep 200 containing hydrophobic alkyl chains with embedded acidic groups in H(+) form on a silica support. The mobile phase variables of acid additives, contents of acetonitrile and concentrations of potassium chloride have been thoroughly investigated to optimize the separation. The retention factors as a function of the concentrations of potassium chloride and the percentages of acetonitrile in the mobile phases are investigated to get an insight into the retention and separation mechanisms of each related impurity and Drug A. Furthermore, the elution orders of the related impurities and Drug A in an ion-pair chromatography (IPC) are compared to those in the mixed-mode HPLC to further understand the chromatographic retention behaviors of each related impurity and Drug A. The study found that the positively charged Degradant 1, Degradant 2 and Drug A were retained by both ion-exchange and reversed-phase partitioning mechanisms. RI2, a small ionic compound, was primarily retained by ion-exchange. RI4, a neutral compound, was retained through reversed-phase partitioning without ion-exchange. Moreover, the method performance characteristics of selectivity, sensitivity and accuracy have been demonstrated to be suitable to determine the related impurities in the capsules of Drug A.

Structural Elucidation and Antioxidant Activities of Proanthocyanidins from Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Leaves

PubMed Central

Fu, Yu; Qiao, Liping; Cao, Yuming; Zhou, Xiaozhou; Liu, Yu; Ye, Xingqian

2014-01-01

Proanthocyanidins in Chinese bayberry leaves (PCBLs) were qualitatively analyzed. NMR data suggest that PCBLs are mostly composed of (epi)gallocatechin gallate units. Matrix-assisted laser desorption time-of-flight MS data indicate 95 possible prodelphinidin structures, ranging from dimers to tridecamers. Preparative normal-phase HPLC and further analysis by reverse-phase HPLC together with electrospray ionization MS enabled detection of 20 compounds, including seven newly identified compounds in Chinese bayberry leaves. The antioxidant capacity of PCBLs was evaluated by (1,1-diphenyl-2-picryl-hydrazyl), ferric-reducing antioxidant power, and oxygen radical absorption capacity assays. The EC50 of DPPH radical scavenging activities (as 50% decrease in the initial DPPH concentration) were 7.60 µg. The FRAP and ORAC values were 8859.33±978.39 and 12991.61±1553.34 µmol Trolox equivalents per gram, respectively. The results indicate the high antioxidant potency of PCBLs. PMID:24805126

Quantitative estimation of itopride hydrochloride and rabeprazole sodium from capsule formulation.

PubMed

Pillai, S; Singhvi, I

2008-09-01

Two simple, accurate, economical and reproducible UV spectrophotometric methods and one HPLC method for simultaneous estimation of two component drug mixture of itopride hydrochloride and rabeprazole sodium from combined capsule dosage form have been developed. First developed method involves formation and solving of simultaneous equations using 265.2 nm and 290.8 nm as two wavelengths. Second method is based on two wavelength calculation, wavelengths selected for estimation of itopride hydrochloride was 278.0 nm and 298.8 nm and for rabeprazole sodium 253.6 nm and 275.2 nm. Developed HPLC method is a reverse phase chromatographic method using phenomenex C(18) column and acetonitrile: phosphate buffer (35:65 v/v) pH 7.0 as mobile phase. All developed methods obey Beer's law in concentration range employed for respective methods. Results of analysis were validated statistically and by recovery studies.

Quantitative Estimation of Itopride Hydrochloride and Rabeprazole Sodium from Capsule Formulation

PubMed Central

Pillai, S.; Singhvi, I.

2008-01-01

Two simple, accurate, economical and reproducible UV spectrophotometric methods and one HPLC method for simultaneous estimation of two component drug mixture of itopride hydrochloride and rabeprazole sodium from combined capsule dosage form have been developed. First developed method involves formation and solving of simultaneous equations using 265.2 nm and 290.8 nm as two wavelengths. Second method is based on two wavelength calculation, wavelengths selected for estimation of itopride hydrochloride was 278.0 nm and 298.8 nm and for rabeprazole sodium 253.6 nm and 275.2 nm. Developed HPLC method is a reverse phase chromatographic method using phenomenex C18 column and acetonitrile: phosphate buffer (35:65 v/v) pH 7.0 as mobile phase. All developed methods obey Beer's law in concentration range employed for respective methods. Results of analysis were validated statistically and by recovery studies. PMID:21394269

A Novel HPLC Method for the Concurrent Analysis and Quantitation of Seven Water-Soluble Vitamins in Biological Fluids (Plasma and Urine): A Validation Study and Application

PubMed Central

Grotzkyj Giorgi, Margherita; Howland, Kevin; Martin, Colin; Bonner, Adrian B.

2012-01-01

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B1, B2, B5, B6, B9, B12) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%. PMID:22536136

A novel HPLC method for the concurrent analysis and quantitation of seven water-soluble vitamins in biological fluids (plasma and urine): a validation study and application.

PubMed

Giorgi, Margherita Grotzkyj; Howland, Kevin; Martin, Colin; Bonner, Adrian B

2012-01-01

An HPLC method was developed and validated for the concurrent detection and quantitation of seven water-soluble vitamins (C, B(1), B(2), B(5), B(6), B(9), B(12)) in biological matrices (plasma and urine). Separation was achieved at 30°C on a reversed-phase C18-A column using combined isocratic and linear gradient elution with a mobile phase consisting of 0.01% TFA aqueous and 100% methanol. Total run time was 35 minutes. Detection was performed with diode array set at 280 nm. Each vitamin was quantitatively determined at its maximum wavelength. Spectral comparison was used for peak identification in real samples (24 plasma and urine samples from abstinent alcohol-dependent males). Interday and intraday precision were <4% and <7%, respectively, for all vitamins. Recovery percentages ranged from 93% to 100%.

Quantitative and Qualitative Determination of Polysulfide Species in the Electrolyte of a Lithium-Sulfur Battery using HPLC ESI/MS with One-Step Derivatization

DOE PAGES

Zheng, Dong; Qu, Deyu; Yang, Xiao-Qing; ...

2015-01-29

The polysulfide species dissolved in aprotic solvents can be separated and analyzed by reverse phase (RP) high performance liquid chromatography (HPLC) in tandem with electrospray-mass spectroscopy. The relative distribution of polysulfide species in the electrolyte recovered from Li-S batteries is quantitatively and reliably determined for the first time.

Quantitative and Qualitative Determination of Polysulfide Species in the Electrolyte of a Lithium-Sulfur Battery using HPLC ESI/MS with One-Step Derivatization

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

Zheng, Dong; Qu, Deyu; Yang, Xiao-Qing

The polysulfide species dissolved in aprotic solvents can be separated and analyzed by reverse phase (RP) high performance liquid chromatography (HPLC) in tandem with electrospray-mass spectroscopy. The relative distribution of polysulfide species in the electrolyte recovered from Li-S batteries is quantitatively and reliably determined for the first time.

Quantitative thin-layer chromatography/mass spectrometry analysis of caffeine using a surface sampling probe electrospray ionization tandem mass spectrometry system.

PubMed

Ford, Michael J; Deibel, Michael A; Tomkins, Bruce A; Van Berkel, Gary J

2005-07-15

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 mum/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methods determined from the calibration curve statistics were 0.20 ng injected (0.50 muL) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by approximately 8% or more) than the literature values.

Quantitative Caffeine Analysis Using a Surface Sampling Probe Electrospray Ionization Tandem Mass Spectrometry System

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

Ford, Michael J; Deibel, Michael A.; Tomkins, Bruce A

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 {mu}m/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methodsmore » determined from the calibration curve statistics were 0.20 ng injected (0.50 {mu}L) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by 8% or more) than the literature values.« less

Analysis of Organic Acids, Deacetyl Asperulosidic Acid and Polyphenolic Compounds as a Potential Tool for Characterization of Noni (Morinda citrifolia) Products.

PubMed

Bittová, Miroslava; Hladůkova, Dita; Roblová, Vendula; Krácmar, Stanislav; Kubán, Petr; Kubán, Vlastimil

2015-11-01

Organic acids, deacetyl asperulosidic acid (DAA) and polyphenolic compounds in various noni (Morinda citrifolia L.) products (4 juices, 4 dry fruit powders and 2 capsules with dry fruit powder) were analyzed. Reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a variable wavelength detector (VWD) and electrospray ionization time-of-flight mass spectrometer (ESI-TOF MS) was applied for simultaneous analysis of organic acids (malic, lactic, citric and succinic acid) and DAA. An RP-HPLC method with diode-array detector (DAD) was developed for the analysis of polyphenolic compound content (rutin, catechin, quercitrin, kaempferol, gallic acid, caffeic acid and p-coumaric acid). The developed methods can contribute to better characterization of available noni products that is required from the consumers. In our study, we discovered significant dissimilarities in the content of DAA, citric acid and several phenolic compounds in some samples.

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.

Conventional sample enrichment strategies combined with high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance analysis allows analyte identification from a single minuscule Corydalis solida plant tuber.

PubMed

Sturm, Sonja; Seger, Christoph; Godejohann, Markus; Spraul, Manfred; Stuppner, Hermann

2007-09-07

Identification of putative biomarker molecules within the genus Corydalis (Papaveraceae) was pursued by combining conventional off-line sample enrichment with high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR) based structure elucidation. Off-line reversed phase solid phase extraction (SPE) was used to enrich the desired analytes from a methanolic extract (93 mg dry weight) of a miniscule single tuber (233 mg dry weight) of C. solida. An aliquot of the SPE fraction (2.1 mg) was subjected to separation in the HPLC-SPE-NMR hyphenation. Chromatographic peaks bearing the metabolites under investigation were trapped in the SPE device in a single experiment and transferred to a 600 MHz NMR spectrometer equipped with a 30 microl cryofit insert fed into a 3 mm cryoprobe. Recorded homo- and heteronuclear 1D and 2D NMR data allowed the identification of the three analytes under investigation as protopine, allocryptopine, and N-methyl-laudanidinium acetate. The latter is a rare alkaloid, which has been isolated only once before.

Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics.

PubMed

Grassetti, Andrew V; Hards, Rufus; Gerber, Scott A

2017-07-01

Technological advances in liquid chromatography and tandem mass spectrometry (LC-MS/MS) have enabled comprehensive analyses of proteins and their post-translational modifications from cell culture and tissue samples. However, sample complexity necessitates offline prefractionation via a chromatographic method that is orthogonal to online reversed-phase high-performance liquid chromatography (RP-HPLC). This additional fractionation step improves target identification rates by reducing the complexity of the sample as it is introduced to the instrument. A commonly employed offline prefractionation method is high pH reversed-phase (Hi-pH RP) chromatography. Though highly orthogonal to online RP-HPLC, Hi-pH RP relies on buffers that interfere with electrospray ionization. Thus, samples that are prefractionated using Hi-pH RP are typically desalted prior to LC-MS/MS. In the present work, we evaluate an alternative offline prefractionation method, pentafluorophenyl (PFP)-based reversed-phase chromatography. Importantly, PFP prefractionation results in samples that are dried prior to analysis by LC-MS/MS. This reduction in sample handling relative to Hi-pH RP results in time savings and could facilitate higher target identification rates. Here, we have compared the performances of PFP and Hi-pH RP in offline prefractionation of peptides and phosphopeptides that have been isolated from human cervical carcinoma (HeLa) cells. Given the prevalence of isobaric mass tags for peptide quantification, we evaluated PFP chromatography of peptides labeled with tandem mass tags. Our results suggest that PFP is a viable alternative to Hi-pH RP for both peptide and phosphopeptide offline prefractionation.

Characterization of nutraceuticals and functional foods by innovative HPLC methods.

PubMed

Corradini, Claudio; Galanti, Roberta; Nicoletti, Isabella

2002-04-01

In recent years there is a growing interest in food and food ingredient which may provide health benefits. Food as well as food ingredients containing health-preserving components, are not considered conventional food, but can be defined as functional food. To characterise such foods, as well as nutraceuticals specific, high sensitive and reproducible analytical methodologies are needed. In light of this importance we set out to develop innovative HPLC methods employing reversed phase narrow bore column and high-performance anion-exchange chromatographic methods coupled with pulsed amperometric detection (HPAEC-PAD), which are specific for carbohydrate analysis. The developed methods were applied for the separation and quantification of citrus flavonoids and to characterize fructooligosaccharide (FOS) and fructans added to functional foods and nutraceuticals.

[Determination of cyclamate in complex matrix using HPLC after column derivatization with 4-fluoro-7-nitrobenzofurazan].

PubMed

Hauck, M; Köbler, H

1990-01-01

A method for the analysis of cyclamate in complex foodstuffs has been developed. This method is applicable in strongly coloured and protein-rich foodstuffs. The quantitative determination depends on oxidation of cyclamate to cyclohexylamine and derivatisation with 4-fluoro-7-nitrobenzofuran (NBD-F). The derivatives are analysed by HPLC on a C18: reversed-phase column, their minimal stability being 12 h. There are two possible methods of detection: (a) absorbance at 485 nm and (b) fluorescence with excitation at 485 nm and emission at 530 nm. The detection limit of cyclamate is 5 mg/kg foodstuff, with fluorescence detection 0.4 mg/kg. The recoveries are in the range of 88% to 104%.

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

Chenna, Ahmed; Gupta, Ramesh C.; Bonala, Radha R.

N2-(4-Hydroxyphenyl)-2'-deoxyguanosine-5'-O-DMT-3'-phosphoramidite has been synthesized and used to incorporate the N2-(4-hydroxyphenyl)-2'-dG (N2-4-HOPh-dG) into DNA, using solid-state synthesis technology. The key step to obtaining the xenonucleoside is a palladium (Xantphos-chelated) catalyzed N2-arylation (Buchwald-Hartwig reaction) of a fully protected 2'-deoxyguanosine derivative by 4-isobutyryloxybromobenzene. The reaction proceeded in good yield and the adduct was converted to the required 5'-O-DMT-3'-O-phosphoramidite by standard methods. The latter was used to synthesize oligodeoxynucleotides in which the N2-4-HOPh-dG adduct was incorporated site-specifically. The oligomers were purified by reverse-phase HPLC. Enzymatic hydrolysis and HPLC analysis confirmed the presence of this adduct in the oligomers.

Analysis of some selected catechins and caffeine in green tea by high performance liquid chromatography.

PubMed

El-Shahawi, M S; Hamza, A; Bahaffi, S O; Al-Sibaai, A A; Abduljabbar, T N

2012-10-15

Green tea seems to have a positive impact on health due to the catechins-found as flavanols. Thus, the present study was aimed to develop a low cost reversed phase high performance liquid chromatographic (HPLC) method for simultaneous determination of flavanol contents, namely catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin 3-gallate (ECG) and epigallocatechin 3-gallate (EGCG) and caffeine in 29 commercial green tea samples available in a Saudi Arabian local market. A C-18 reversed-phase column, acetonitrile-trifluoroacetic acid as a mobile phase, coupled with UV detector at 205 nm, was successfully used for precise analysis of the tested analytes in boiled water of digested tea leaves. The average values of N (No. of theoretical plates), HETP (height equivalent of theoretical plates) and R(s) (separation factor) (at 10 μg ml(-1) of the catechins EC, EGC, EGCG and ECG) were 2.6×10(3)±1.2×10(3), 1.7×10(-3)±4.7×10(-4) cm and 1.7±5.53×10(-2), respectively. The developed HPLC method demonstrated excellent performance, with low limits of detection (LOD) and quantification (LOQ) of the tested catechins of 0.004-0.05 μg ml(-1) and 0.01-0.17 μg ml(-1), respectively, and recovery percentages of 96-101%. The influence of infusion time (5-30 min) and temperature on the content of the flavanols was investigated by HPLC. After a 5 min infusion of the tea leaves, the average concentrations of caffeine, catechin, EC, EGC, ECG and EGCG were found to be in the ranges 0.086-2.23, 0.113-2.94, 0.58-10.22, 0.19-24.9, 0.22-13.9 and 1.01-43.3 mg g(-1), respectively. The contents of caffeine and catechins followed the sequence: EGCG>EGC>ECG>EC>C>caffeine. The method was applied satisfactorily for the analysis of (+)-catechin, even at trace and ultra trace concentrations of catechins. The method was rapid, accurate, reproducible and ideal for routine analysis. Copyright © 2012 Elsevier Ltd. All rights reserved.

Quantitative study of fruit flavonoids in citrus hybrids of King (C. nobilis) and Mukaku Kishu (C. kinokuni).

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Nonomura-Nakano, M; Nesumi, H; Yoshida, T; Sugiura, M; Yano, M

2001-08-01

Twenty-four Citrus hybrids of King (C. nobilis) and Mukaku Kishu (C. kinokuni) were examined for their flavonoid profiles of the edible part by reversed-phase HPLC analysis. Two hybrids (G-155 and G-156) contained higher amounts of natsudaidain than their parents, whereas the remainder of the hybrids had a character intermediate between those of King and Mukaku Kishu on the basis of polymethoxylated flavone composition. Principal component analysis revealed the distribution of the hybrids by quantifying 23 flavonoid contents.

Liquid chromatographic separation of terpenoid pigments in foods and food products.

PubMed

Cserháti, T; Forgács, E

2001-11-30

The newest achievements in the use of various liquid chromatographic techniques such as adsorption and reversed-phase thin-layer chromatography and HPLC employed for the separation and quantitative determination of terpenoid-based color substances in foods and food products are reviewed. The techniques applied for the analysis of individual pigments and pigments classes are surveyed and critically evaluated. Future trends in the separation and identification of pigments in foods and food products are delineated.

Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high performance liquid chromatography.

PubMed

Pistos, C; Tsantili-Kakoulidou, A; Koupparis, M

2005-09-15

The retention/pH profiles of three fluoroquinolones, ofloxacin, norfloxacin and ciprofloxacin, was investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC) and reversed-phase ion-interaction chromatography (RP-IIC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexanesulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion-interaction chromatography. The retention/pH profiles under in RP-HPLC were compared to the corresponding lipophilicity/pH profiles. Despite the rather hydrophilic nature of the three fluoroquinolones positive retention factors were obtained while there was a shift of the retention maximum towards more acidic pH values. This behavior was attributed mainly to non-hydrophobic silanophilic interactions with the silanized silica gel material of the stationary phase. In ion-interaction chromatography the effect of counter ions over a broad pH range was found to be ruled rather by the ion pair formation in the mobile phase which led to a drastic decrease in retention as a consequence of the disruption of the zwitterionic structure and thereupon the deliberation of a net charge in the molecules. At pH values at which zwitterionic structure was not favored both the ion-exchange and ion pair formation mechanisms were assumed to contribute to the retention.

Spectrophotometric and HPLC Methods for Simultaneous Estimation of Amlodipine Besilate, Losartan Potassium and Hydrochlorothiazide in Tablets

PubMed Central

Wankhede, S. B.; Raka, K. C.; Wadkar, S. B.; Chitlange, S. S.

2010-01-01

Two UV-spectrophotometric and one reverse phase high performance liquid chromatography methods have been developed for the simultaneous estimation of amlodipine besilate, losartan potassium and hydrochlorothiazide in tablet dosage form. The first UV spectrophotometric method was a determination using the simultaneous equation method at 236.5, 254 and 271 nm over the concentration range 5-25, 10-50 and 5-25 μg/ml for amlodipine besilate, losartan potassium and hydrochlorothiazide, respectively. The second UV method was a determination using the area under curve method at 231.5-241.5, 249-259 and 266-276 nm over the concentration range of 5-25, 5-25 and 10-50 μg/ml for amlodipine besilate, hydrochlorothiazide and losartan potassium, respectively. In reverse phase high performance liquid chromatography analysis is carried out using 0.025 M phosphate buffer (pH 3.7):acetonitrile (57:43 v/v) as the mobile phase and Kromasil C18 (4.6 mm i.d×250 mm) column as stationery phase with detection wavelength of 232 nm linearity was obtained in the concentration range of 2-14, 20-140 and 5-40 μg/ml for amlodipine besilate, losartan potassium and hydrochlorothiazide, respectively. Both UV-spectrophotometric and reverse phase high performance liquid chromatography methods were statistically validated and can be used for analysis of combined dose tablet formulation containing amlodipine besilate, losartan potassium and hydrochlorothiazide. PMID:20582208

Reversed-phase high-performance liquid chromatography of unsubstituted aminobenzoic acids

USGS Publications Warehouse

Abidi, S.L.

1989-01-01

High-performance liquid chromatographic (HPLC) characteristics of three position isomers of aminobenzoic acids (potential metabolites of important anesthetic drugs), were delineated with respect to their interactions with various mobile phases and stationary phases. HPLC with five hydrocarbonaceous phase, I?-cyclodextrin silica (CDS), macrophase MP-1 polymer (MP), macroporous polystyrene/divinylbenzene (MPD), octadecylsilica (ODS), and propylphenylsilica (PPS), yielded results explicable in terms of substituent effects derived from the bifunctional amino- and carboxy groups. For cases where mobile phases contained sulfonates or quaternary ammonium salts both having longer chain alkyls, retention of analytes on all but CDS appeared to proceed predominantly via an ion-pairing mechanism. The extent of the corresponding counter-ion effects decreased in the order: MPD > ODS > PPS > MP, while the analyte retention order paralleled thier pH2 values. On the other hand, an inverse relationship between the magnitude of capacity factors (k') and pK1 values of the title compounds was observed in experiments that produced retention data incompatible with ion-pair interaction rationales. The unique HPLC results obtained with the CDS phase are compared with those obtained with other phases.

[A reversed-phase HPLC method for determining tretinoin].

PubMed

Jiang, X G; Xi, N Z

1994-09-01

Tretinoin (Tre) and its active stereo isomer isotretinoin (Iso) were simultaneously determined by reversed-phase high pressure liquid chromatographic method with a uv detector adjusted to 348 nm. Separation was accomplished on YWG-C18 column by using a MeOH:NH4Ac buffer (pH 6.0) 85:15 (vol:vol), chlorpromazine (Chl) being chosen as internal standard. Minimal detectable amount of Tre was 0.5 ng. Calibration curve was linear (r = 0.9999) in the concentration range of 25-2500 ng.ml-1. This method was used to determinate the transdermal amounts of Tre from three different preparations in Franz diffusion cell in vitro. The results showed that the proposed method could distinguish the transdermal differences from various formulations or different skin samples. In addition, it is able to be used in quantitative analysis of Tre and Iso.

Validation of AN Hplc-Dad Method for the Classification of Green Teas

NASA Astrophysics Data System (ADS)

Yu, Jingbo; Ye, Nengsheng; Gu, Xuexin; Liu, Ni

A reversed phase high performance liquid chromatography (RP-HPLC) separation coupled with diode array detection (DAD) and electrospray ionization mass spectrometer (ESI/MS) was developed and optimized for the classification of green teas. Five catechins [epigallocatechin (EGC), epigallocatechin gallate (EGCG), epicatechin (EC), gallocatechin gallate (GCG), epicatechin gallate (ECG)] had been identified and quantified by the HPLC-DAD-ESI/MS/MS method. The limit of detection (LOD) of five catechins was within the range of 1.25-15 ng. All the analytes exhibited good linearity up to 2500 ng. These compounds were considered as chemical descriptors to define groups of green teas. Chemometric methods including principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied for the purpose. Twelve green tea samples originating from different regions were subjected to reveal the natural groups. The results showed that the analyzed green teas were differentiated mainly by provenance; HCA afforded an excellent performance in terms of recognition and prediction abilities. This method was accurate and reproducible, providing a potential approach for authentication of green teas.

Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection.

PubMed

Novak, Ivana; Janeiro, Patricia; Seruga, Marijan; Oliveira-Brett, Ana Maria

2008-12-23

Several flavonoids present in red grape skins from four varieties of Portuguese grapes were determined by reverse-phase high-performance liquid chromatography (RP-HPLC) with electrochemical detection (ECD). Extraction of flavonoids from red grape skins was performed by ultrasonication, and hydrochloric acid in methanol was used as extraction solvent. The developed RP-HPLC method used combined isocratic and gradient elution with amperometric detection with a glassy carbon-working electrode. Good peak resolution was obtained following direct injection of a sample of red grape extract in a pH 2.20 mobile phase. Eleven different flavonoids: cyanidin-3-O-glucoside (kuromanin), delphinidin-3-O-glucoside (myrtillin), petunidin-3-O-glucoside, peonidin-3-O-glucoside, malvidin-3-O-glucoside (oenin), (+)-catechin, rutin, fisetin, myricetin, morin and quercetin, can be separated in a single run by direct injection of sample solution. The limit of detection obtained for these compounds by ECD was 20-90 pg/L, 1000 times lower when compared with photodiode array (PDA) limit of detection of 12-55 ng/L. RP-HPLC-ECD was characterized by an excellent sensitivity and selectivity, and appropriate for the simultaneous determination of these electroactive phenolic compounds present in red grape skins.

Quantitative Determination of α-Arbutin, β-Arbutin, Kojic Acid, Nicotinamide, Hydroquinone, Resorcinol, 4-Methoxyphenol, 4-Ethoxyphenol, and Ascorbic Acid from Skin Whitening Products by HPLC-UV.

PubMed

Wang, Yan-Hong; Avonto, Cristina; Avula, Bharathi; Wang, Mei; Rua, Diego; Khan, Ikhlas A

2015-01-01

An HPLC-UV method was developed for the quantitative analysis of nine skin whitening agents in a single injection. These compounds are α-arbutin, β-arbutin, kojic acid, nicotinamide, resorcinol, ascorbic acid, hydroquinone, 4-methoxyphenol, and 4-ethoxyphenol. The separation was achieved on a reversed-phase C18 column within 30 min. The mobile phase was composed of water and methanol, both containing 0.1% acetic acid (v/v). The stability of the analytes was evaluated at different pH values between 2.3 and 7.6, and the extraction procedure was validated for different types of skin whitening product matrixes, which included two creams, a soap bar, and a capsule. The best solvent system for sample preparation was 20 mM NaH2PO4 containing 10% methanol at pH 2.3. The analytical method was validated for accuracy, precision, LOD, and LOQ. The developed HPLC-UV method was applied for the quantitation of the nine analytes in 59 skin whitening products including creams, lotions, sera, foams, gels, mask sheets, soap bars, tablets, and capsules.

On-line comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of Peucedanum praeruptorum.

PubMed

Wang, Xin-Yuan; Li, Jia-Fu; Jian, Ya-Mei; Wu, Zhen; Fang, Mei-Juan; Qiu, Ying-Kun

2015-03-27

A new on-line comprehensive preparative two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was developed for the separation of complicated natural products. It was based on the use of a silica gel packed medium-pressure column as the first dimension and an ODS preparative HPLC column as the second dimension. The two dimensions were connected with normal-phase (NP) and reversed-phase (RP) enrichment units, involving a newly developed airflow assisted adsorption (AAA) technique. The instrument operation and the performance of this NPLC × RPLC separation method were illustrated by gram-scale isolation of ethanol extract from the roots of Peucedanum praeruptorum. In total, 19 compounds with high purity were obtained via automated multi-step preparative separation in a short period of time using this system, and their structures were comprehensively characterized by ESI-MS, (1)H NMR, and (13)C NMR. Including two new compounds, five isomers in two groups with identical HPLC and TLC retention values were also obtained and identified by 1D NMR and 2D NMR. This is the first report of an NPLC × RPLC system successfully applied in an on-line preparative process. This system not only solved the interfacing problem of mobile-phase immiscibility caused by NP and RP separation, it also exhibited apparent advantages in separation efficiency and sample treatment capacity compared with conventional methods. Copyright © 2015 Elsevier B.V. All rights reserved.

Calcium-binding protein from mouse Ehrlich ascites-tumour cells is homologous to human calcyclin.

PubMed Central

Kuźnicki, J; Filipek, A; Hunziker, P E; Huber, S; Heizmann, C W

1989-01-01

A Ca2+-binding protein was purified from mouse Ehrlich ascites-tumour cells. The protein forms monomers and disulphide-linked dimers, which can be separated by reverse-phase h.p.l.c. A partial amino acid sequence analysis demonstrated that the protein has an EF-hand structure. A striking homology was found to rat and human calcyclin (a member of the S-100 protein family), which is possibly involved in cell-cycle regulation. Images Fig. 1. Fig. 2. PMID:2597136

Isocratic RP-HPLC method for rutin determination in solid oral dosage forms.

PubMed

Kuntić, Vesna; Pejić, Natasa; Ivković, Branka; Vujić, Zorica; Ilić, Katarina; Mićić, Svetlana; Vukojević, Vladana

2007-01-17

A rapid and sensitive assay for quantitative determination of rutin in oral dosage forms based on isocratic reversed phase high performance liquid chromatography (RP-HPLC) was developed and validated. Using a C(18) reverse-phase analytical column, the following conditions were chosen as optimal: mobile phase methanol-water 1:1 (v/v), pH 2.8 (adjusted with phosphoric acid), flow rate=1 mL min(-1) and temperature T=40.0 degrees C. Linearity was observed in the concentration range 8-120 microg mL(-1) with a correlation coefficient of 0.99982 and the limit of detection (LOD)=2.6 microg mL(-1), and limit of quantification (LOQ)=8.0 microg mL(-1). Intra- and inter-day precision were within acceptable limits. Robustness test indicated that the mobile phase composition and pH influence mainly the separation. The proposed method allowed direct determination of rutin in pharmaceutical dosage forms in the presence of excipients, but is not suitable for preparations where compounds structurally/chemically related to rutin may be present.

Quantification of appetite suppressing steroid glycosides from Hoodia gordonii in dried plant material, purified extracts and food products using HPLC-UV and HPLC-MS methods.

PubMed

Janssen, Hans-Gerd; Swindells, Chris; Gunning, Philip; Wang, Weijun; Grün, Christian; Mahabir, Krishna; Maharaj, Vinesh J; Apps, Peter J

2008-06-09

High-performance liquid chromatography (HPLC)-UV and HPLC-Mass Spectrometry (MS) methods were developed for the quantitative analysis of the family of Hoodia gordonii steroid glycosides with appetite suppressing properties in dried plant material, in purified and enriched extracts and in various prototype food-products fortified with H. gordonii extracts. For solid materials, e.g. dried plants or for non-fatty foods, extraction of the steroid glycosides is performed using methanol. For products where the steroid glycosides are present in an oil matrix, direct injection of the oil after dilution in tetrahydrofuran is applied. The HPLC separation is performed on an octyl-modified reversed-phase column in the gradient mode with UV detection at lambda = 220 nm. Quantification is performed against an external calibration line prepared using either one of the pure steroid glycosides or geranyl-tiglate. Short- and long-term repeatabilities of the methods are better than 3 and 6%, respectively. Recoveries are better than 85%, even in the analysis of the least abundant steroid glycosides in a complex yoghurt drink. Linearity is better than 3-4 orders of magnitude and the detection limits are below approximately 2 microg g(-1) for the individual steroid glycosides in dried plant material and food products. HPLC-MS is used to confirm that the steroid glycosides contain the characteristic steroid core, the carbohydrate chain and the tigloyl group.

Influence of the Structure of Molecules of Derivatives of 1,2,4-Triazole and 1,2,4-Triazine on Chromatographic Retention Under Conditions of Reversed Phase HPLC

NASA Astrophysics Data System (ADS)

Karaseva, I. N.; Karasev, M. O.; Nechaeva, O. N.; Kurbatova, S. V.

2018-07-01

The dependence of the chromatographic retention of 1,2,4-triazine and 1,2,4-triazole derivatives from water-acetonitrile solutions over octadecyl silica on the structure of sorbate molecules is studied. The effect the physicochemical parameters and topology of heterocycle molecules have on the retention characteristics under RP HPLC conditions is analyzed.

Microwave-assisted extraction with water for fast extraction and simultaneous RP-HPLC determination of phenolic acids in radix Salviae Miltiorrhizae.

PubMed

Fang, Xinsheng; Wang, Jianhua; Zhou, Hongying; Jiang, Xingkai; Zhu, Lixiang; Gao, Xin

2009-07-01

An optimized microwave-assisted extraction method using water (MAE-W) as the extractant and an efficient HPLC analysis method were first developed for the fast extraction and simultaneous determination of D(+)-(3,4-dihydroxyphenyl) lactic acid (Dla), salvianolic acid B (SaB), and lithospermic acid (La) in radix Salviae Miltiorrhizae. The key parameters of MAE-W were optimized. It was found that the degradation of SaB was inhibited when using the optimized MAE-W and the stable content of Dla, La, and SaB in danshen was obtained. Furthermore, compared to the conventional extraction methods, the proposed MAE-W is a more rapid method with higher yield and lower solvent consumption with a reproducibility (RSD <6%). In addition, using water as extractant is safe and helpful for environment protection, which could be referred to as green extraction. The separation and quantitative determination of the three compounds was carried out by a developed reverse-phase high-performance liquid chromatographic (RP-HPLC) method with UV detection. Highly efficient separation was obtained using gradient solvent system. The optimized HPLC analysis method was validated to have specificity, linearity, precision, and accuracy. The results indicated that MAE-W followed by HPLC-UV determination is an appropriate alternative to previously proposed method for quality control of radix Salviae Miltiorrhizae.

Determination of salicylic acid by HPLC in plasma and saliva from children with juvenile chronic arthritis.

PubMed

Legaz, M E; Acitores, E; Valverde, F

1992-12-01

A high performance liquid chromatography (HPLC) method has been developed for measuring salicylic acid in the plasma and saliva of children with juvenile chronic arthritis (JCA). Samples were extracted with diethyl ether and, after drying, redissolved in methanol to be chromatographed. Quantitation of salicylic acid was performed by reverse phase HPLC on a spherisorb ODS-2 column, using methanol: water: acetic acid as mobile phase. Phenolic was monitored by absorbance at 237 nm. Linearity between the amount of mass injected and the response in the detector was determined. This method was applied to compare concentrations of salivary and plasma salicylic acid. The method also permitted the quantitation of salivary salicylate as a non-invasive, indirect method for monitoring the concentration of plasma salicylate in patients with JCA.

HPLC, NMR and MALDI-TOF MS analysis of condensed tannins from Lithocarpus glaber leaves with potent free radical scavenging activity.

PubMed

Zhang, Liang Liang; Lin, Yi Ming

2008-12-04

Using acid-catalyzed degradation in the presence of cysteamine, the condensed tannins from Lithocarpus glaber leaves were characterized, following thiolysis, by means of reversed-phase HPLC, 13C-NMR and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. The thiolysis reaction products showed the presence of the procyanidin (PC) and prodelphinidin (PD) structures. The 13C-NMR spectrum revealed that the condensed tannins were comprised of PD (72.4%) and PC (27.6%), and with a greater content of cis configuration rather than the trans configuration of C2-C3. The MALDI-TOF MS analysis proved the presence of PD units, and the maximum degree of polymerization (DP) was an undecamer. The antioxidant activity of condensed tannins from L. glaber leaves was evaluated by using a free radical scavenging activity assay.

Journal of Chemical Education: Software.

ERIC Educational Resources Information Center

Journal of Chemical Education, 1988

1988-01-01

Describes a chemistry software program that emulates a modern binary gradient HPLC system with reversed phase column behavior. Allows for solvent selection, adjustment of gradient program, column selection, detectory selection, handling of computer sample data, and sample preparation. (MVL)

Ion-pairing reversed-phase chromatography coupled to inductively coupled plasma mass spectrometry as a tool to determine mercurial species in freshwater fish.

PubMed

Cheng, Heyong; Chen, Xiaopan; Shen, Lihuan; Wang, Yuanchao; Xu, Zigang; Liu, Jinhua

2018-01-05

Most of analytical community is focused on reversed phase high performance liquid chromatography (RP-HPLC) for mercury speciation by employing mobile phases comprising of high salts and moderate amounts of organic solvents. This study aims at rapid mercury speciation analysis by ion-pairing RP-HPLC with inductively coupled plasma mass spectrometry (ICP-MS) detection only using low salts for the sake of green analytical chemistry. Two ion-pairing HPLC methods were developed on individual usage of positively and negatively charged ion-pairing reagents (tetrabutylammonium hydroxide -TBAH and sodium dodecylbenzene sulfonate -SDBS), where sodium 3-mercapto-1-propysulfonate (MPS) and l-cysteine (Cys) were individually added in mobile phases to transform mercury species into negative and positive Hg-complexes for good resolution. Addition of phenylalanine was also utilized for rapid baseline separation in combination of short C 18 guard columns. Optimum mobile phases of 2.0mM SDBS+2.0mM Cys+1.0mM Phe (pH 3.0) and 4.0mM TBAH+2.0mM MPS+2.0mM Phe (pH 6.0) both achieved baseline separation of inorganic mercury (Hg 2+ ), methylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg) on two consecutive 12.5-mm C 18 columns. The former mobile phase was selected for mercury speciation in freshwater fish because of short separation time (3.0min). Detection limits of 0.015 for Hg 2+ , 0.014 for MeHg, 0.028 for EtHg and 0.042μgL -1 for PhHg were obtained along with satisfactory precisions of peak height and area (1.0-2.8% for 5.0μgL -1 Hg-mixture standard). Good accordance of determined values of MeHg and total mercury in certified reference materials of fish tissue (GBW 10029) and tuna fish (BCR-463) with certified values as well as good recoveries (91-106%) proved good accuracy of the proposed method. An example application to freshwater fish indicated its potential in routine analysis, where MeHg was presented at 3.7-20.3μgkg -1 as the dominate species. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative Thin-Layer Chromatography/Mass Spectrometry Analysis of Caffeine Using a Surface Sampling Probe Electrospray Ionization Tandem Mass Spectrometry System

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

Ford, Michael J; Deibel, Michael A.; Tomkins, Bruce A

Quantitative determination of caffeine on reversed-phase C8 thin-layer chromatography plates using a surface sampling electrospray ionization system with tandem mass spectrometry detection is reported. The thin-layer chromatography/electrospray tandem mass spectrometry method employed a deuterium-labeled caffeine internal standard and selected reaction monitoring detection. Up to nine parallel caffeine bands on a single plate were sampled in a single surface scanning experiment requiring 35 min at a surface scan rate of 44 {mu}m/s. A reversed-phase HPLC/UV caffeine assay was developed in parallel to assess the mass spectrometry method performance. Limits of detection for the HPLC/UV and thin-layer chromatography/electrospray tandem mass spectrometry methodsmore » determined from the calibration curve statistics were 0.20 ng injected (0.50 {mu}L) and 1.0 ng spotted on the plate, respectively. Spike recoveries with standards and real samples ranged between 97 and 106% for both methods. The caffeine content of three diet soft drinks (Diet Coke, Diet Cherry Coke, Diet Pepsi) and three diet sport drinks (Diet Turbo Tea, Speed Stack Grape, Speed Stack Fruit Punch) was measured. The HPLC/UV and mass spectrometry determinations were in general agreement, and these values were consistent with the quoted values for two of the three diet colas. In the case of Diet Cherry Coke and the diet sports drinks, the determined caffeine amounts using both methods were consistently higher (by 8% or more) than the literature values.« less

Arsenic-containing fatty acids and hydrocarbons in marine oils - determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS.

PubMed

Sele, Veronika; Sloth, Jens J; Holmelid, Bjarte; Valdersnes, Stig; Skov, Kasper; Amlund, Heidi

2014-04-01

Arsenolipids are the major arsenic species present in marine oils. Several structures of arsenolipids have been elucidated the last 5 years, demonstrating the chemical complexity of this trace element in the marine environment. Several commercial fish oils and marine oils, ranging in total arsenic concentrations from 1.6 to 12.5 mg kg(-1) oil, were analyzed for arsenolipids using reversed-phase high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The arsenolipids were quantified using three different arsenic-containing calibration standards; dimethylarsinate (DMA), triphenylarsinoxide (Ph₃AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C₂₁H₄₃AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time-resolved response factors for the arsenolipids. Isotopes of germanium ((74)Ge) and indium ((115)In) were suited as internal standards for arsenic, and were used for verification of the arsenic signal response factors during the gradient elution. Dimethylarsinate was the most suitable calibration standard for the quantification of arsenolipids, with recoveries between 91% and 104% compared to total arsenic measurements in the same extracts. A range of marine oils was investigated, including oils of several fish species, cod liver and seal, as well as three commercial fish oils. The AsHCs - C₁₇H₃₈AsO, C₁₉H₄₂AsO and C₂₃H₃₈AsO - were identified as the major arsenolipids in the extracts of all oils by HPLC coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). Minor amounts of two arsenic-containing fatty acids (AsFAs) (C₂₃H₃₈AsO₃ and C₂₄H₃₈AsO₃) were also detected in the oils. The sum of the AsHCs and the AsFAs determined in the present study accounted for 17-42% of the total arsenic in the oils. Copyright © 2014 Elsevier B.V. All rights reserved.

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

NASA Astrophysics Data System (ADS)

Zeb, Alam; Ullah, Fareed

2017-04-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins and two chlorophylls. Lutein (806.0 µg/g), chlorophyll b' (410.0 µg/g), chlorophyll a (162.4 µg/g), 9'-Z-neoxanthin (142.8 µg/g) and all-E-violaxanthin (82.2 µg/g)) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of thirteen compounds, namely p-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5-O-caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g) and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for possible medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a potential source of nutraceuticals or as a functional food ingredient.

Chromatographic behaviour predicts the ability of potential nootropics to permeate the blood-brain barrier.

PubMed

Farsa, Oldřich

2013-01-01

The log BB parameter is the logarithm of the ratio of a compound's equilibrium concentrations in the brain tissue versus the blood plasma. This parameter is a useful descriptor in assessing the ability of a compound to permeate the blood-brain barrier. The aim of this study was to develop a Hansch-type linear regression QSAR model that correlates the parameter log BB and the retention time of drugs and other organic compounds on a reversed-phase HPLC containing an embedded amide moiety. The retention time was expressed by the capacity factor log k'. The second aim was to estimate the brain's absorption of 2-(azacycloalkyl)acetamidophenoxyacetic acids, which are analogues of piracetam, nefiracetam, and meclofenoxate. Notably, these acids may be novel nootropics. Two simple regression models that relate log BB and log k' were developed from an assay performed using a reversed-phase HPLC that contained an embedded amide moiety. Both the quadratic and linear models yielded statistical parameters comparable to previously published models of log BB dependence on various structural characteristics. The models predict that four members of the substituted phenoxyacetic acid series have a strong chance of permeating the barrier and being absorbed in the brain. The results of this study show that a reversed-phase HPLC system containing an embedded amide moiety is a functional in vitro surrogate of the blood-brain barrier. These results suggest that racetam-type nootropic drugs containing a carboxylic moiety could be more poorly absorbed than analogues devoid of the carboxyl group, especially if the compounds penetrate the barrier by a simple diffusion mechanism.

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

PubMed Central

Zeb, Alam; Ullah, Fareed

2017-01-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins, and two chlorophylls. Lutein (806.0 μg/g), chlorophyll b′ (410.0 μg/g), chlorophyll a (162.4 μg/g), 9′-Z-neoxanthin (142.8 μg/g) and all-E-violaxanthin (82.2 μg/g) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of 13 compounds, namely 4-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5-O-caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g), and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of Adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for the potential medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a possible source of nutraceuticals or as a functional food ingredient. PMID:28497036

Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves.

PubMed

Zeb, Alam; Ullah, Fareed

2017-01-01

Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins, and two chlorophylls. Lutein (806.0 μg/g), chlorophyll b ' (410.0 μg/g), chlorophyll a (162.4 μg/g), 9'- Z -neoxanthin (142.8 μg/g) and all- E -violaxanthin (82.2 μg/g) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of 13 compounds, namely 4-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5- O -caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g), and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of Adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for the potential medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a possible source of nutraceuticals or as a functional food ingredient.

An industry consensus study on an HPLC fluorescence method for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products.

PubMed

Shumow, Laura; Bodor, Alison

2011-07-05

This manuscript describes the results of an HPLC study for the determination of the flavan-3-ol monomers, (±)-catechin and (±)-epicatechin, in cocoa and plain dark and milk chocolate products. The study was performed under the auspices of the National Confectioners Association (NCA) and involved the analysis of a series of samples by laboratories of five member companies using a common method. The method reported in this paper uses reversed phase HPLC with fluorescence detection to analyze (±)-epicatechin and (±)-catechin extracted with an acidic solvent from defatted cocoa and chocolate. In addition to a variety of cocoa and chocolate products, the sample set included a blind duplicate used to assess method reproducibility. All data were subjected to statistical analysis with outliers eliminated from the data set. The percent coefficient of variation (%CV) of the sample set ranged from approximately 7 to 15%. Further experimental details are described in the body of the manuscript and the results indicate the method is suitable for the determination of (±)-catechin and (±)-epicatechin in cocoa and chocolate products and represents the first collaborative study of this HPLC method for these compounds in these matrices.

Chromatographic and Spectrophotometric Analysis of Phenolic Compounds from Fruits of Libidibia ferrea Martius

PubMed Central

Ferreira, Magda R. A.; Fernandes, Mônica T. M.; da Silva, Wliana A. V.; Bezerra, Isabelle C. F.; de Souza, Tatiane P.; Pimentel, Maria F.; Soares, Luiz A. L.

2016-01-01

Background: Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz (Fabaceae) is a tree which is native to Brazil, widely known as “Jucá,” where its herbal derivatives are used in folk medicine with several therapeutic properties. The constituents, which have already been described in the fruit, are mainly hydrolysable tannins (gallic acid [GA] and ellagic acid [EA]). Objective: The aim of this study was to investigate the phenolic variability in the fruit of L. ferrea by ultraviolet/visible (UV/VIS) and chromatographic methods (high-performance liquid chromatography [HPLC]/high-performance thin layer chromatography [HPTLC]). Materials and Methods: Several samples were collected from different regions of Brazil and the qualitative (fingerprints by HPTLC and HPLC) and quantitative analysis (UV/VIS and HPLC) of polyphenols were performed. Results: The HPTLC and HPLC profiles allowed separation and identification of both major analytical markers: EA and GA. The chemical profiles were similar in a number of spots or peaks for the samples, but some differences could be observed in the intensity or area of the analytical markers for HPTLC or HPLC, respectively. Regarding the quantitative analysis, the polyphenolic content by UV/VIS ranged from 13.99 to 37.86 g% expressed as GA or from 10.75 to 29.09 g% expressed as EA. The contents of EA and GA by liquid chromatography-reversed phase (LC-RP) method ranged from 0.57 to 2.68 g% and from 0.54 to 3.23 g%, respectively. Conclusion: The chemical profiles obtained by HPTLC or HPLC, as well as the quantitative analysis by spectrophotometry or LC-RP method, were suitable for discrimination of each herbal sample and can be used as tools for the comparative analysis of the fruits from L. ferrea. SUMMARY The polyphenols of fruits of Libidibia ferrea can be quantified by UV/VIS and HPLCThe HPLC method was able to detect the gallic and ellagic acids in several samples of fruits of Libidibia ferreaThe phenolic profiles of fruits from Libidibia ferrea by HPTLC and HPLC were reproductible. Abbreviations used: HPTLC: high performance thin layer chromatography, HPLC: high performance liquid chromatography, UV-Vis: spectrophotometry PMID:27279721

Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp).

PubMed

Brighenti, Virginia; Pellati, Federica; Steinbach, Marleen; Maran, Davide; Benvenuti, Stefania

2017-09-05

The present work was aimed at the development and validation of a new, efficient and reliable technique for the analysis of the main non-psychoactive cannabinoids in fibre-type Cannabis sativa L. (hemp) inflorescences belonging to different varieties. This study was designed to identify samples with a high content of bioactive compounds, with a view to underscoring the importance of quality control in derived products as well. Different extraction methods, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and supercritical-fluid extraction (SFE) were applied and compared in order to obtain a high yield of the target analytes from hemp. Dynamic maceration for 45min with ethanol (EtOH) at room temperature proved to be the most suitable technique for the extraction of cannabinoids in hemp samples. The analysis of the target analytes in hemp extracts was carried out by developing a new reversed-phase high-performance liquid chromatography (HPLC) method coupled with diode array (UV/DAD) and electrospray ionization-mass spectrometry (ESI-MS) detection, by using an ion trap mass analyser. An Ascentis Express C 18 column (150mm×3.0mm I.D., 2.7μm) was selected for the HPLC analysis, with a mobile phase composed of 0.1% formic acid in both water and acetonitrile, under gradient elution. The application of the fused-core technology allowed us to obtain a significant improvement of the HPLC performance compared with that of conventional particulate stationary phases, with a shorter analysis time and a remarkable reduction of solvent usage. The analytical method optimized in this study was fully validated to show compliance with international requirements. Furthermore, it was applied to the characterization of nine hemp samples and six hemp-based pharmaceutical products. As such, it was demonstrated to be a very useful tool for the analysis of cannabinoids in both the plant material and its derivatives for pharmaceutical and nutraceutical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Cyclic interconversion of methionine containing peptide between oxidized and reduced phases monitored by reversed-phase HPLC and ESI-MS/MS.

PubMed

Jin, Yulong; Huang, Yanyan; Xie, Yunfeng; Hu, Wenbing; Wang, Fuyi; Liu, Guoquan; Zhao, Rui

2012-01-30

The cyclic oxidation and reduction of methionine (Met) containing peptides and proteins play important roles in biological system. This work was contributed to analysis the cyclic oxidation and reduction processes of a methionine containing peptide which is very likely to relate in the cell signal transduction pathways. To mimic the biological oxidation condition, hydrogen peroxide was used as the reactive oxygen species to oxidize the peptide. Reversed-phase high-performance liquid chromatography and mass spectrometry were employed to monitor the reactions and characterize the structural changes of the products. A rapid reduction procedure was developed by simply using KI as the reductant, which is green and highly efficient. By investigation of the cyclic oxidation and reduction process, our work provides a new perspective to study the function and mechanism of Met containing peptides and proteins during cell signaling processes as well as diseases. Copyright © 2011 Elsevier B.V. All rights reserved.

Synthesis and evaluation of porous polymethylsilsesquioxane microspheres as low silanol activity chromatographic stationary phase for basic compound separation.

PubMed

Huo, Zhixia; Wan, Qianhong; Chen, Lei

2018-06-08

Polymethylsilsesquioxanes (PMSQ) are potentially useful materials for liquid chromatography owing to their unique chemical, electrical and mechanical properties. Surprisingly however, no systematic studies on the use of spherical PMSQ particles as chromatographic packing have been reported. Accordingly, we present a comprehensive study aimed to characterize the chromatographic properties of this material in high performance liquid chromatography (HPLC) and to compare them with those observed on methyl (C 1 ) bonded silica phase under comparable conditions. Porous spherical particles were synthesized by a two-step hydrolysis and condensation procedure from methyltrimethoxysilane (MTMS) as a sole precursor. The as-synthesized microspheres possess spherical shape, narrow size distribution, mesoporous structure, high surface area (817 m 2  g -1 ) and reasonable carbon load (16.6%). They can be used directly as the HPLC stationary phase without the need for size classification. The PMSQ phase exhibits typical reversed-phase chromatographic properties with higher methylene selectivity and low silanol activity compared with the C 1 column. The retention mechanism for basic compounds was systematically evaluated by studying the effect of pH, ionic and solvent strength of the mobile phase. Basic compounds displayed lower retention factor and symmetric peak shape on the PMSQ column whereas longer retention and strong tailing peaks were observed on the C 1 column. The difference in retention behavior between the two columns is explained in terms of different principal retention mechanisms. Because of the low silanol activity, retention of basic compounds on the PMSQ column is governed solely by a reversed-phase mechanism. By contrast, multiple interactions including reversed-phase, cation exchange and simultaneous reversed-phase/cationic exchange interaction contribute to the retention on the C 1 column, as previously observed on other silica based reversed-phases. Furthermore, the PMSQ phase exhibited significantly enhanced stability under alkaline conditions compared with its silica-based counterpart. Taken together, the favorable morphology and pore structure combined with the benefits of low silanol activity, high pH stability and prolonged column lifetime make the newly developed PMSQ phase a promising and viable alternative to silica based reversed-phase packings for separation of basic compounds. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative structure-retention relationship models for the prediction of the reversed-phase HPLC gradient retention based on the heuristic method and support vector machine.

PubMed

Du, Hongying; Wang, Jie; Yao, Xiaojun; Hu, Zhide

2009-01-01

The heuristic method (HM) and support vector machine (SVM) were used to construct quantitative structure-retention relationship models by a series of compounds to predict the gradient retention times of reversed-phase high-performance liquid chromatography (HPLC) in three different columns. The aims of this investigation were to predict the retention times of multifarious compounds, to find the main properties of the three columns, and to indicate the theory of separation procedures. In our method, we correlated the retention times of many diverse structural analytes in three columns (Symmetry C18, Chromolith, and SG-MIX) with their representative molecular descriptors, calculated from the molecular structures alone. HM was used to select the most important molecular descriptors and build linear regression models. Furthermore, non-linear regression models were built using the SVM method; the performance of the SVM models were better than that of the HM models, and the prediction results were in good agreement with the experimental values. This paper could give some insights into the factors that were likely to govern the gradient retention process of the three investigated HPLC columns, which could theoretically supervise the practical experiment.

Gas release-based prescreening combined with reversed-phase HPLC quantitation for efficient selection of high-γ-aminobutyric acid (GABA)-producing lactic acid bacteria.

PubMed

Wu, Qinglong; Shah, Nagendra P

2015-02-01

High γ-aminobutyric acid (GABA)-producing lactobacilli are promising for the manufacture of GABA-rich foods and to synthesize GRAS (generally recognized as safe)-grade GABA. However, common chromatography-based screening is time-consuming and inefficient. In the present study, Korean kimchi was used as a model of lactic acid-based fermented foods, and a gas release-based prescreening of potential GABA producers was developed. The ability to produce GABA by potential GABA producers in de Man, Rogosa, and Sharpe medium supplemented with or without monosodium glutamate was further determined by HPLC. Based on the results, 9 isolates were regarded as high GABA producers, and were further genetically identified as Lactobacillus brevis based on the sequences of 16S rRNA gene. Gas release-based prescreening combined with reversed-phase HPLC confirmation was an efficient and cost-effective method to identify high-GABA-producing LAB, which could be good candidates for probiotics. The GABA that is naturally produced by these high-GABA-producing LAB could be used as a food additive. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Direct determination of purine bases in tea by reversed-phase high performance liquid chromatography].

PubMed

Ding, M; Yang, H; Xiao, S; Chen, P

1999-09-01

A reversed-phase high performance liquid chromatographic(RP-HPLC) method for the direct determination of three purine bases(theobromin, theophyllin and caffeine) in tea was developed. An ODS column with Zorbax SB-C18(4.6 mm i.d. x 250 mm, 5 microns) was employed. The aqueous solution of methanol containing 0.05% of acetic acid and 0.25% of N,N-dimethylformamide(DMF) was used as eluent with a flow rate of 0.8 mL/min. In this method, the aqueous extract of tea can be injected into HPLC directly, but in current HPLC methods for purine bases the coexisted tea polyphenols must be pre-separated. The three purine bases in tea were separated without any interference from the coexisted tea polyphenols. This method is simple (without any special sample pretreatment) and sensitive with detection limits (S/N = 3) of 0.7, 0.9 and 1.8 mg/L for theobromin, theophyllin and caffeine respectively. The linear range of the calibration curve of peak area for the three purine bases were from 6 mg/L to 1,000 mg/L with a correlation coefficient (r) of 0.998-0.999.

H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

PubMed

Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

2016-04-01

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

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.

Preparation of clenbuterol imprinted monolithic polymer with hydrophilic outer layers by reversible addition-fragmentation chain transfer radical polymerization and its application in the clenbuterol determination from human serum by on-line solid-phase extraction/HPLC analysis.

PubMed

Li, Xiaobing; Zhou, Man; Turson, Mamat; Lin, Shen; Jiang, Ping; Dong, Xiangchao

2013-05-21

A novel imprinted monolithic material with the ability of protein exclusion was developed for the selective extraction of clenbuterol (CLE) from biological samples by direct injection in the HPLC analysis. The material has an imprinted inner structure and hydrophilic outer layer. The reversible addition-fragmentation chain transfer (RAFT) polymerization was employed in the material preparation by a two-step procedure. In the first step, clenbuterol imprinted monolithic polymer was synthesized by combining the molecular imprinting and the RAFT polymerization techniques. The resulting monolithic polymer has a RAFT chain transfer agent (trithioester groups) in its structure, which was used to graft poly(glycerol mono-methacrylate) [pGMMA] in the second step by post-RAFT polymerization. The hydrophilic pGMMA layers grafted on the surface of the imprinted monolith created barriers for protein diffusion. More than 90% of bovine serum albumin can be excluded from the pGMMA coated monolithic column. Meanwhile the clenbuterol was retained selectively with a large retention factor. The result indicated that the column, denoted as RA-MIM, has both the merits of a molecularly imprinted polymer and restricted access material. By using RA-MIM as the solid-phase extraction pre-column, an on-line column-switching HPLC method for the determination of clenbuterol in human serum has been established and validated. The recoveries of clenbuterol from the serum were 87.3-96.9% in the spiked level 2-1000 ng mL(-1). Both good linearity (R = 0.999) and acceptable reproducibility (RSD < 7.0%) were obtained. The limit of detection and the limit of quantitation were 0.7 ng mL(-1) and 2.0 ng mL(-1) respectively, which is sensitive in terms of UV detection. The results have demonstrated that the RAFT polymerization can be used to synthesize bi-functional monolithic columns by using its living reaction property. The resulting RA-MIM in this research can be used for efficient clenbuterol determination by HPLC from biological samples.

Speciation of Selenium in Selenium-Enriched Sunflower Oil by High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry/Electrospray-Orbitrap Tandem Mass Spectrometry.

PubMed

Bierla, Katarzyna; Flis-Borsuk, Anna; Suchocki, Piotr; Szpunar, Joanna; Lobinski, Ryszard

2016-06-22

The reaction of sunflower oil with selenite produces a complex mixture of selenitriglycerides with antioxidant and anticancer properties. To obtain insight into the identity and characteristics of the species formed, an analytical approach based on the combination of high-performance liquid chromatography (HPLC) with (78)Se-specific selenium detection by inductively coupled plasma mass spectrometry (ICP MS) and high-resolution (100 000), high mass accuracy (<1 ppm) molecule-specific detection by electrospray-Orbitrap MS(3) was developed. For the first time, a non-aqueous mobile phase gradient was used in reversed-phase HPLC-ICP MS for the separation of a complex mixture of selenospecies and a mathematical correction of the background signal was developed. The identical chromatographic conditions served for the sample introduction into electrospray MS. Two types of samples were analyzed: sunflower oil dissolved in isopropanol and methanol extract of the oil containing 65% selenium. HPLC-ICP MS showed 14 peaks, 11 of which could also be detected in the methanol extract. Isotopic patterns corresponding to molecules with one or two selenium atoms could be attributed by Orbitrap MS at the retention times corresponding to the HPLC-ICP MS peak apexes. Structural data for these species were acquired by MS(2) and MS(3) fragmentation of protonated or sodiated ions using high-energy collisional dissociation (HCD). A total of 11 selenium-containing triglycerol derivatives resulting from the oxidation of one or two double bonds of linoleic acid and analogous derivatives of glycerol-mixed linoleate(s)/oleinate(s) have been identified for the first time. The presence of these species was confirmed by the targeted analysis in the total oil isopropanol solution. Their identification corroborated the predicted elution order in reversed-phase chromatography: LLL (glycerol trilinoleate), LLO (glycerol dilinoleate-oleinate), LOO (glycerol linoleate-dioleinate), OOO (glycerol trioleinate), of which the extrapolation allowed for the prediction of the identity [glycerol dioleinate-stearate (OOS) and glycerol oleinate-distearate (OSS)] of the nonpolar species detected by ICP MS in the oil but not detected by electrospray MS.

High-performance liquid chromatography analysis methods developed for quantifying enzymatic esterification of flavonoids in ionic liquids.

PubMed

Lue, Bena-Marie; Guo, Zheng; Xu, Xuebing

2008-07-11

Methods using reversed-phase high-performance liquid chromatography (RP-HPLC) with ELSD were investigated to quantify enzymatic reactions of flavonoids with fatty acids in the presence of diverse room temperature ionic liquids (RTILs). A buffered salt (preferably triethylamine-acetate) was found essential for separation of flavonoids from strongly polar RTILs, whereby RTILs were generally visible as two major peaks identified based on an ion-pairing/exchanging hypothesis. C8 and C12 stationary phases were optimal while mobile phase pH (3-7) had only a minor influence on separation. The method developed was successfully applied for primary screening of RTILs (>20), with in depth evaluation of substrates in 10 RTILs, for their evaluation as reaction media.

Quantitative analysis of the major constituents of St John's wort with HPLC-ESI-MS.

PubMed

Chandrasekera, Dhammitha H; Welham, Kevin J; Ashton, David; Middleton, Richard; Heinrich, Michael

2005-12-01

A method was developed to profile the major constituents of St John's wort extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The objective was to simultaneously separate, identify and quantify hyperforin, hypericin, pseudohypericin, rutin, hyperoside, isoquercetrin, quercitrin and chlorogenic acid using HPLC-MS. Quantification was performed using an external standardisation method with reference standards. The method consisted of two protocols: one for the analysis of flavonoids and glycosides and the other for the analysis of the more lipophilic hypericins and hyperforin. Both protocols used a reverse phase Luna phenyl hexyl column. The separation of the flavonoids and glycosides was achieved within 35 min and that of the hypericins and hyperforin within 9 min. The linear response range in ESI-MS was established for each compound and all had linear regression coefficient values greater than 0.97. Both protocols proved to be very specific for the constituents analysed. MS analysis showed no other signals within the analyte peaks. The method was robust and applicable to alcoholic tinctures, tablet/capsule extracts in various solvents and herb extracts. The method was applied to evaluate the phytopharmaceutical quality of St John's wort preparations available in the UK in order to test the method and investigate if they contain at least the main constituents and at what concentrations.

Automated on-line renewable solid-phase extraction-liquid chromatography exploiting multisyringe flow injection-bead injection lab-on-valve analysis.

PubMed

Quintana, José Benito; Miró, Manuel; Estela, José Manuel; Cerdà, Víctor

2006-04-15

In this paper, the third generation of flow injection analysis, also named the lab-on-valve (LOV) approach, is proposed for the first time as a front end to high-performance liquid chromatography (HPLC) for on-line solid-phase extraction (SPE) sample processing by exploiting the bead injection (BI) concept. The proposed microanalytical system based on discontinuous programmable flow features automated packing (and withdrawal after single use) of a small amount of sorbent (<5 mg) into the microconduits of the flow network and quantitative elution of sorbed species into a narrow band (150 microL of 95% MeOH). The hyphenation of multisyringe flow injection analysis (MSFIA) with BI-LOV prior to HPLC analysis is utilized for on-line postextraction treatment to ensure chemical compatibility between the eluate medium and the initial HPLC gradient conditions. This circumvents the band-broadening effect commonly observed in conventional on-line SPE-based sample processors due to the low eluting strength of the mobile phase. The potential of the novel MSFI-BI-LOV hyphenation for on-line handling of complex environmental and biological samples prior to reversed-phase chromatographic separations was assessed for the expeditious determination of five acidic pharmaceutical residues (viz., ketoprofen, naproxen, bezafibrate, diclofenac, and ibuprofen) and one metabolite (viz., salicylic acid) in surface water, urban wastewater, and urine. To this end, the copolymeric divinylbenzene-co-n-vinylpyrrolidone beads (Oasis HLB) were utilized as renewable sorptive entities in the micromachined unit. The automated analytical method features relative recovery percentages of >88%, limits of detection within the range 0.02-0.67 ng mL(-1), and coefficients of variation <11% for the column renewable mode and gives rise to a drastic reduction in operation costs ( approximately 25-fold) as compared to on-line column switching systems.

Chemically bonded stationary phases that use synthetic hosts containing aromatic binding clefts: HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons.

PubMed Central

Zimmerman, S C; Saionz, K W; Zeng, Z

1993-01-01

The synthesis of hosts with improved binding affinities for nitroaromatic guests is described. Association constants for several host-guest complexes were measured in chloroform solution and ranged over three orders of magnitude. Two hosts were covalently linked to silica gel to produce chemically bonded stationary phases for HPLC. The use of these phases for HPLC analysis of nitro-substituted polycyclic aromatic hydrocarbons is discussed. PMID:8433981

Retention behavior of long chain quaternary ammonium homologues and related nitroso-alkymethylamines

USGS Publications Warehouse

Abidi, S.L.

1985-01-01

Several chromatographic methods have been utilized to study the retentionbehavior of a homologous series of n-alkylbenzyldimethylammonium chlorides (ABDAC) and the corresponding nitroso-n-alkylmethylamines (NAMA). Linear correlation of the logarithmic capacity factor (k') with the number of carbons in the alkyl chain provides useful information on both gas chromatographic (GC) and high-performance liquid chromatographich (HPLC) retention parameters of unknown components. Under all conditions empolyed, GC methodology has proved effective in achieving complete resolution of the homologous mixture of NMA despite its obvious inadequacy in the separation of E-Z configurational isomers. Conversely, normal-phase HPLC on silica demonstrates that the selectivity (a) value for an E-Z pair is much higher than that for an adjacent homologous pair. In the reversed-phase HPLC study, three different silica-based column systems were examined under various mobile phase conditions. The extent of variation in k' was found to be a function of the organic modifier, counter-ion concentration, eluent pH, nature of counter-ion, and the polarity and type of stationary phase. The k'—[NaClO4] profiles showed similar trends between the ABDAC and the NAMA series, supporting the dipolar electronic structures of the latter compounds. Mobile phase and stationary phase effects on component separation are described. The methodology presented establishes the utility of HPLC separation techniques as versatile analytical tools for practical application.

Expanding the term "Design Space" in high performance liquid chromatography (I).

PubMed

Monks, K E; Rieger, H-J; Molnár, I

2011-12-15

The current article presents a novel approach to applying Quality by Design (QbD) principles to the development of high pressure reversed phase liquid chromatography (HPLC) methods. Four common critical parameters in HPLC--gradient time, temperature, pH of the aqueous eluent, and stationary phase--are evaluated within the Quality by Design framework by the means of computer modeling software and a column database, to a satisfactory degree. This work proposes the establishment of two mutually complimentary Design Spaces to fully depict a chromatographic method; one Column Design Space (CDS) and one Eluent Design Space (EDS) to describe the influence of the stationary phase and of the mobile phase on the separation selectivity, respectively. The merge of both Design Spaces into one is founded on the continuous nature of the mobile phase influence on retention and the great variety of the stationary phases available. Copyright © 2011 Elsevier B.V. All rights reserved.

N,N-(2,4-dinitrophenyl)octylamine derivatives for the isolation, purification, and mass spectrometric characterization of oligosaccharides.

PubMed

Zhang, Y; Cedergren, R A; Nieuwenhuis, T J; Hollingsworth, R I

1993-02-01

A simple, sensitive method for the structural characterization of oligosaccharides by fast atom bombardment-mass spectrometry (FAB-MS) has been designed. Oligosaccharides are labeled with a uv chromophore (which also serves as a charge stabilizing group) and with a hydrophobic alkyl tail. The chromophore, a 2,4-dinitrophenyl group, aids uv detection during HPLC and stabilizes negative ion species formed during analysis by FAB-MS. The hydrophobic tail, provided by an octyl group, enhances the surface activity of the analytes and makes them amenable to separation by reverse-phase chromatography using a C18 bonded phase. This method was applied to the structural analysis of the components of a mixture of starch maltodextrins with a degree of polymerization 1-16, to the analysis of the structure of pure maltohexaose, and to a previously characterized oligosaccharide from a Rhizobium capsular polysaccharide. The method gave a good yield of [M-H]- anions for the derivatized compounds, which in most cases were detectable at a level of about 1 pmol. In the case of maltohexaose, four series of sequence anions corresponding to sequential loss of glycosyl residues from the reducing and nonreducing end by different mechanisms were observed. The mixture of derivatized malto-oligosaccharides could easily be separated by HPLC. Based on the relative proportions of the individual oligomers in the mixture calculated from HPLC analysis, even though the higher oligomers were present in amounts of about 0.1%, they could still be easily detected in mass spectra of the entire mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Chiral-phase high-performance liquid chromatography of rotenoid racemates

USGS Publications Warehouse

Abidi, S.L.

1987-01-01

The high-performance liquid chromatograhic (HPLC) behavior of parent rotenoids (type I) and the hydroxyl-analogues (type II) on three different chiral stationary phases (CSPs) was studied. Separations of optical isomers were achieved in various degrees depending largely upon the rotenoidal structures and the CSP types employed. Enantiomers of all but elliptone compounds were separable on β-cyclodextrin-bonded silica (CDS). Without exception, the 12a-hydroxyrotenoid antipodes were resolved on Pirkle's phenylglycine-bonded silica (PGS) despite unsuccessful attenmpts to resolve the type I rotenoidal racemates. Conversely, optical resolution of the latter rotenoids was accomplished by using a helical polytriphenylmethylacrylate-coated silica (TPS) column and the observed separation factors (α values) ranged from 1.14 to 1.90. The results from HPLC of type II rotenoids on TPS (α = 1.00–1.63) suggested that variations in E-ring structures had profound influence on the resolution outcome. Conjugated double bonds on the E-ring and the desisopropylation of the five-membered E-ring ot type II rotenoids appeared to be important structural features for chiral recognition involving the TPS substrate. In both reversed-phase (CDS) and normal-pahse (PGS and TPS) HPLC modes, the less polar enantiomers were the 6aβ,12aβ-rotenoids as observed in most cases, though this relationship was reversed in the cases of deguelin and hydroxyelliptone probably due to conformational effects of rotenoidal ring systems.

[Determination of canthaxanthin and astaxanthin in egg yolks by reversed phase high performance liquid chromatography with diode array detection].

PubMed

He, Kang-Hao; Zou, Xiao-Li; Liu, Xiang; Zeng, Hong-Yan

2012-01-01

A method using reversed phase high performance liquid chromatography (RP-HPLC) coupled with diode array detector (DAD) was developed for the simultaneous determination of canthaxanthin and astaxanthin in egg yolks. Samples were extracted with acetonitrile in ultrasonic bath for 20 minutes and then purified by freezing-lipid filtration and solid phase extraction (SPE). After being vaporized to dryness by nitrogen blowing and made up to volume with methanol, the extract solution was chromatographically separated in C18 column with a unitary mobile phase consisting of acetonitrile. The proposed method was validated in terms of linearity, precision, accuracy, and limit of detection (LOD). Regression analysis revealed a good linearity between peak area of each analyte and its concentration (r > or = 0.998). The intra- and inter-day relative standard deviations (RSDs) were less than 3.6% and 5.2%, respectively. LODs of canthaxanthin and astaxanthin were 0.035 and 0.027 microg/mL (S/N = 3). The average recoveries of canthaxanthin and astaxanthin were 91.5% and 88.7%. The proposed method is simple, fast and easy to apply.

Simultaneous determination of gatifloxacin and ambroxol hydrochloride from tablet dosage form using reversed-phase high performance liquid chromatography.

PubMed

Shahed, Mirza; Nanda, Rabindra; Dehghan, Muhammad Hassan; Nasreen, Huda; Feroz, Shaikh

2008-05-01

A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm x 4.6 mm, 5 microm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70 : 30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10 -60 microg/mL and that for AM was found to be 5 - 30 microg/mL. The correlation coefficients were 0.999 6 and 0.999 3 for GA and AM respectively. The results of the tablet analysis (n = 5) were found to be 99.94% with +/- 0.25% standard deviation (SD) and 99.98% with +/- 0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92% - 100.02% and that of AM was 99.86% - 100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.

High-performance liquid chromatography of human glycoprotein hormones.

PubMed

Chlenov, M A; Kandyba, E I; Nagornaya, L V; Orlova, I L; Volgin, Y V

1993-02-12

The chromatographic behavior of the glycoprotein hormones from human pituitary glands and of placental origin [thyroid-stimulating hormone, luteinizing hormone and chorionic gonadotropin (CG)] was studied. It was shown that hydrophobic interaction chromatography on a microparticulate packing and anion-exchange HPLC can be applied for the purification of these hormones. Reversed-phase HPLC on wide-pore C4-bonded silica at neutral pH can be applied for the determination of the above hormones and for the isolation of pure CG and its subunits.

The Effect of Acid Neutralization on Analytical Results Produced from SW846 Method 8330 after the Alkaline Hydrolysis of Explosives in Soil

DTIC Science & Technology

2012-09-01

basic form of phosphoric acid or sodium phosphate NO2- Nitrite OH- Hydroxide ion ERDC/EL TR-12-14 1 1 Introduction Alkaline hydrolysis has...into amber sample vials and refrigerated until analyzed. TNT analyses were conducted by high performance liquid chromatography (HPLC) with a C-18...The explosives concentrations of the different soils were quantified using a DIONEX HPLC system equipped with a C-18 reverse phase column and a

Carboxylate modified porous graphitic carbon: a new class of hydrophilic interaction liquid chromatography phases.

PubMed

Wahab, M Farooq; Ibrahim, Mohammed E A; Lucy, Charles A

2013-06-18

Stationary phases for hydrophilic interaction liquid chromatography (HILIC) are predominantly based on silica and polymer supports. We present porous graphitic carbon particles with covalently attached carboxylic acid groups (carboxylate-PGC) as a new HILIC stationary phase. PGC particles were modified by adsorbing the diazonium salt of 4-aminobenzoic acid onto the PGC, followed by reduction of the adsorbed salt with sodium borohydride. The newly developed carboxylate-PGC phase exhibits different selectivity than that of 35 HPLC columns, including bare silica, zwitterionic, amine, reversed, and unmodified PGC phases. Carboxylate-PGC is stable from pH 2.0 to 12.6, yielding reproducible retention even at pH 12.6. Characterization of the new phase is presented by X-ray photoelectron spectroscopy, thermogravimetry, zeta potentials, and elemental analysis. The chromatographic performance of carboxylate-PGC as a HILIC phase is illustrated by separations of carboxylic acids, nucleotides, phenols, and amino acids.

Method development for speciation analysis of nanoparticle and ionic forms of gold in biological samples by high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Malejko, Julita; Świerżewska, Natalia; Bajguz, Andrzej; Godlewska-Żyłkiewicz, Beata

2018-04-01

A new method based on coupling high performance liquid chromatography (HPLC) to inductively coupled plasma mass spectrometry (ICP MS) has been developed for the speciation analysis of gold nanoparticles (AuNPs) and dissolved gold species (Au(III)) in biological samples. The column type, the composition and the flow rate of the mobile phase were carefully investigated in order to optimize the separation conditions. The usefulness of two polymeric reversed phase columns (PLRP-S with 100 nm and 400 nm pore size) to separate gold species were investigated for the first time. Under the optimal conditions (PLRP-S400 column, 10 mmol L-1 SDS and 5% methanol as the mobile phase, 0.5 mL min-1 flow rate), detection limits of 2.2 ng L-1 for Au(III), 2.8 ng L-1 for 10 nm AuNPs and 3.7 ng L-1 for 40 nm AuNPs were achieved. The accuracy of the method was proved by analysis of reference material RM 8011 (NIST) of gold nanoparticles of nominal diameter of 10 nm. The HPLC-ICP MS method has been successfully applied to the detection and size characterization of gold species in lysates of green algae Acutodesmus obliquus, typical representative of phytoplankton flora, incubated with 10 nm AuNPs or Au(III).

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography-searching for alternatives to organic solvents.

PubMed

Sutton, Adam T; Fraige, Karina; Leme, Gabriel Mazzi; da Silva Bolzani, Vanderlan; Hilder, Emily F; Cavalheiro, Alberto J; Arrua, R Dario; Funari, Cristiano Soleo

2018-06-01

Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of "green analytical chemistry," new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a "greener" alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50 °C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses. Graphical abstract Natural deep eutectic solvents versus traditional solvents in HPLC.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin

PubMed Central

Natal, Fabio Luis Nogueira; Ribela, Maria Teresa Carvalho Pinto; de Almeida, Beatriz Elane; de Oliveira, João Ezequiel; Bartolini, Paolo

2016-01-01

Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical–chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (tR = 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone. PMID:27297410

Identification of phenolic compounds from the leaf part of Teucrium pseudo-Scorodonia Desf. collected from Algeria.

PubMed

Belarbi, Karima; Atik-Bekkara, Fawzia; El Haci, Imad Abdelhamid; Bensaid, Ilhem; Bekhechi, Chahrazed

2018-02-01

In the present paper,we reported for the first time, the identification of the phenolic compounds in butanolic fraction obtained from the leaf part of Teucrium pseudo-Scorodonia Desf. collected from Algeria using RP-HPLC-PDA (Reversed Phase High Performance Liquid Chromatography/Photo Diode Array) technique. Several standards were used for this purpose. The analysis led to the identification of six phenolic acids (ferulic, sinapic, rosmarinic, syringique, caffeic, p-coumaric acids) and one flavonoid (rutin), the last one, has interesting pharmacological properties.

Is the C-terminal flanking peptide of rat cholecystokinin double sulphated?

PubMed

Adrian, T E; Domin, J; Bacarese-Hamilton, A J; Bloom, S R

1986-02-03

A specific radioimmunoassay was developed to the predicted nine amino acid C-terminal flanking peptide of cholecystokinin (peptide serine serine, PSS). In aqueous extracts of rat brain, PSS was undetectable unless the extracts were first treated with arylsulphatase, which also resulted in desulphation of cholecystokinin. The reverse-phase HPLC analysis of partially desulphated extracts showed the presence of two peaks intermediate to the naturally occurring and the completely desulphated forms. It is therefore proposed that the CCK-flanking peptide PSS has both tyrosine residues sulphated.

Report: An ex vivo up-take of levamisole molecules by cestode (Monezia expensa) of goat (Capra hirsa) and its detection through RP-HPLC.

PubMed

Ayaz, Muhammad Mazhar; Sajid, Muhammad; Das, Sanjota Nirmal; Hanif, Muhammad

2018-05-01

Detection of various molecules of drugs remained a prime issue especially in tissues of animals, humans and in their target parasites. The cestode/tapeworms pose a dilemma because of their weird body composition and uptake pattern of nutrients and medicines especially through absorption by tegument. We selected levamisole; thought to be potent antiparasitic/ani-cestodal drug. The uptake of levamisole (LEV) through cestodeal tissues is studied through HPCL in this paper. High performance liquid chromatography technique has been utilized to know the uptake of levamisole in tissues of cestodes of Goat (Monezia expensa) in small ruminants. The drug was exposed to M. expensa by in vitro till its death or a parasite ceases its movement. The tissue/ part of proglattids of the M. expensa were homogenized with some modifications and levamisole extraction was performed with liquid phase extraction method. The evaporation of solvent was done and the residual cestodal tissues were cleaned by solid phase. After the solid phase extraction method, the recovery of drug, detection and quantification of levamisole from cestodal tissues was determined through Reverse Phase Column High Performance Liquid Chromatography (RP-HPLC). Levamisole (LEV) molecules assay was obtained on a C18 reverse-phase (20um, 6mm x 150mm) column at flow rate of 1ml/min using acetonitrile and ammonium acetate as mobile phase and UV detection was done at 254nm. The development of method of Levamisole (LEV) detection from cestodal tissues by HPLC in vitro samples has been demonstrated first time in Pakistan, which can provide the solution of parasitic control and provide in sight in to the uptake of anti cestodal drugs either against human or livestock parasites.

Chromatographic Behaviour Predicts the Ability of Potential Nootropics to Permeate the Blood-Brain Barrier

PubMed Central

Farsa, Oldřich

2013-01-01

The log BB parameter is the logarithm of the ratio of a compound’s equilibrium concentrations in the brain tissue versus the blood plasma. This parameter is a useful descriptor in assessing the ability of a compound to permeate the blood-brain barrier. The aim of this study was to develop a Hansch-type linear regression QSAR model that correlates the parameter log BB and the retention time of drugs and other organic compounds on a reversed-phase HPLC containing an embedded amide moiety. The retention time was expressed by the capacity factor log k′. The second aim was to estimate the brain’s absorption of 2-(azacycloalkyl)acetamidophenoxyacetic acids, which are analogues of piracetam, nefiracetam, and meclofenoxate. Notably, these acids may be novel nootropics. Two simple regression models that relate log BB and log k′ were developed from an assay performed using a reversed-phase HPLC that contained an embedded amide moiety. Both the quadratic and linear models yielded statistical parameters comparable to previously published models of log BB dependence on various structural characteristics. The models predict that four members of the substituted phenoxyacetic acid series have a strong chance of permeating the barrier and being absorbed in the brain. The results of this study show that a reversed-phase HPLC system containing an embedded amide moiety is a functional in vitro surrogate of the blood-brain barrier. These results suggest that racetam-type nootropic drugs containing a carboxylic moiety could be more poorly absorbed than analogues devoid of the carboxyl group, especially if the compounds penetrate the barrier by a simple diffusion mechanism. PMID:23641330

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I.

PubMed

Zolla, Lello; Rinalducci, Sara; Timperio, Anna Maria; Huber, Christian G

2002-12-01

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.

Validated RP-HPLC/DAD Method for the Quantification of Insect Repellent Ethyl 2-Aminobenzoate in Membrane-Moderated Matrix Type Monolithic Polymeric Device.

PubMed

Islam, Johirul; Zaman, Kamaruz; Chakrabarti, Srijita; Sharma Bora, Nilutpal; Mandal, Santa; Pratim Pathak, Manash; Srinivas Raju, Pakalapati; Chattopadhyay, Pronobesh

2017-07-01

A simple, accurate and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the estimation of ethyl 2-aminobenzoate (EAB) in a matrix type monolithic polymeric device and validated as per the International Conference on Harmonization guidelines. The analysis was performed isocratically on a ZORBAX Eclipse plus C18 analytical column (250 × 4.4 mm, 5 μm) and a diode array detector (DAD) using acetonitrile and water (75:25 v/v) as the mobile phase by keeping the flow-rate constant at 1.0 mL/min. Determination of EAB was not interfered in the presence of excipients. Inter- and intra-day relative standard deviations were not higher than 2%. Mean recovery was between 98.7 and 101.3%. Calibration curve was linear in the concentration range of 0.5-10 µg/mL. Limits of detection and quantification were 0.19 and 0.60 µg/mL, respectively. Thus, the present report put forward a novel method for the estimation of EAB, an emerging insect repellent, by using RP-HPLC technique. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Isolation and characterization of (15Z)-lycopene thermally generated from a natural source

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

Takehara, Munenori, E-mail: takehara@mat.usp.ac.jp; Kuwa, Takahiro; Inoue, Yoshinori

(15Z)-Lycopene was prepared by thermal isomerization of (all-E)-lycopene derived from tomatoes, and isolated by using a series of chromatographies. The fine red crystalline powder of (15Z)-lycopene was obtained from 556 mg of (all-E)-lycopene with a yield of 0.6 mg (purity: reversed-phase HPLC, 97.2%; normal-phase HPLC, ≥99.9%), and {sup 1}H and {sup 13}C NMR spectra of the isomer were fully assigned. More refined computational analyses that considered differences in the energy levels of the conformers involved in isomerization have also determined the stabilities of (15Z)-lycopene and other geometric isomers, along with the activation energies during isomerization from the all-E form. The fine controlmore » of conditions for HPLC separation and an advanced theoretical insight into geometric isomerization have led to the discovery of the 15Z-isomer generated from a natural source. - Highlights: • (15Z)-lycopene, isomerized from the all-E form of a natural source, was purified. • The obtained (15Z)-lycopene was structurally identified by an NMR analysis. • A modified theoretical study accounted for the generation of the 15Z-isomer. • This study demonstrated the occurrence of the isomer from a natural origin.« less

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%).

[The determination of molecular sulphur in Matsesta mineral water and its analog Novonukutskaya mineral water].

PubMed

Khutorianskiĭ, V A; Smirnov, A I; Matveev, D A

2014-01-01

The method of microcolumn reversed phase high performance liquid chromatography (rp-HPLC) was employed to determine the content of elemental sulphur in mineral waters. The study envisaged the analysis of the samples of sulphide-containing mineral waters Novonukutskaya and Matsesta obtained by the solid phase extraction technique. Based on these data, the authors discuss the origin and the circulation of sulphur in the hydrogen sulphide sources. The elution conditions selected in this study ensured the high-resolution separation of the octasulphur peak from the peaks of allotropic components of the extract whereas the two-wave detection technique allowed to identify the peaks of molecular sulphur.

Online reverse phase-high-performance liquid chromatography-fluorescence detection-electrospray ionization-mass spectrometry separation and characterization of heparan sulfate, heparin, and low-molecular weight-heparin disaccharides derivatized with 2-aminoacridone.

PubMed

Galeotti, Fabio; Volpi, Nicola

2011-09-01

A high-resolution online reverse-phase-high-performance liquid chromatography (RP-HPLC)-fluorescence detector (Fd)-electrospray ionization-mass spectrometry (ESI-MS) separation and structural characterization of disaccharides prepared from heparin (Hep), heparan sulfate (HS), and various low-molecular-weight (LMW)-Hep using heparin lyases and derivatization with 2-aminoacridone (AMAC) are described. A total of 12 commercially available Hep/HS-derived unsaturated disaccharides were separated and unambiguously identified on the basis of their retention times and mass spectra. The constituent disaccharides of various samples, including unfractionated Hep/HS, fast-moving and slow-moving Hep components, and several marketed products, were characterized. Furthermore, for the first time, the saturated trisulfated disaccharide belonging to the nonreducing end of Heps was detected as being approximately 2% in unfractionated samples and ~15-21% in LMW-Heps prepared by nitrous acid depolymerization. No desalting of the commercial products prior to enzymatic digestion or prepurification steps to eliminate any excess of AMAC reagent or interference from proteins, peptides, and other sample impurities before RP-HPLC-Fd-ESI-MS injection were necessary. This method has applicability for the rapid differentiation of pharmaceutical Heps and LMW-Heps prepared by means of different depolymerization processes and for compositional analysis of small amounts of samples derived from biological sources by using the highly sensitive fluorescence detector.

Reversed phase HPLC analysis of stability and microstructural effects on degradation kinetics of β-carotene encapsulated in freeze-dried maltodextrin-emulsion systems.

PubMed

Harnkarnsujarit, Nathdanai; Charoenrein, Sanguansri; Roos, Yrjö H

2012-09-26

Degradation of dispersed lipophilic compounds in hydrophilic solids depends upon matrix stability and lipid physicochemical properties. This study investigated effects of solid microstructure and size of lipid droplets on the stability of dispersed β-carotene in freeze-dried systems. Emulsions of β-carotene in sunflower oil were dispersed in maltodextrin systems (M040/DE6, M100/DE11, and M250/DE25.5) (8% w/w oil) and prefrozen at various freezing conditions prior to freeze-drying to control nucleation and subsequent pore size and structural collapse of freeze-dried solids. The particle size, physical state, and β-carotene contents of freeze-dried emulsions were measured during storage at various water activity (a(w)) using a laser particle size analyzer, differential scanning calorimeter, and high performance liquid chromatography (HPLC), respectively. The results showed that M040 stabilized emulsions in low temperature freezing exhibited lipid crystallization. Collapse of solids in storage at a(w) which plasticized systems to the rubbery state led to flow and increased the size of oil droplets. Degradation of β-carotene analyzed using a reversed-phase C(30) column followed first-order kinetics. Porosity of solids had a major effect on β-carotene stability; however, the highest stability was found in fully plasticized and collapsed solids.

Development and Optimization of HPLC Analysis of Metronidazole, Diloxanide, Spiramycin and Cliquinol in Pharmaceutical Dosage Forms Using Experimental Design.

PubMed

Elkhoudary, Mahmoud M; Abdel Salam, Randa A; Hadad, Ghada M

2016-11-01

A new simple, sensitive, rapid and accurate gradient reversed-phase high-performance liquid chromatography with photodiode array detector (RP-HPLC-DAD) was developed and validated for simultaneous analysis of Metronidazole (MNZ), Spiramycin (SPY), Diloxanidefuroate (DIX) and Cliquinol (CLQ) using statistical experimental design. Initially, a resolution V fractional factorial design was used in order to screen five independent factors: the column temperature (°C), pH, phosphate buffer concentration (mM), flow rate (ml/min) and the initial fraction of mobile phase B (%). pH, flow rate and initial fraction of mobile phase B were identified as significant, using analysis of variance. The optimum conditions of separation determined with the aid of central composite design were: (1) initial mobile phase concentration: phosphate buffer/methanol (50/50, v/v), (2) phosphate buffer concentration (50 mM), (3) pH (4.72), (4) column temperature 30°C and (5) mobile phase flow rate (0.8 ml min -1 ). Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9999. Limits of detection for all of the analyzed compounds ranged between 0.02 and 0.11 μg ml -1 ; limits of quantitation ranged between 0.06 and 0.33 μg ml -1 The proposed method showed good prediction ability. The optimized method was validated according to ICH guidelines. Three commercially available tablets were analyzed showing good % recovery and %RSD. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Impurity profiling of liothyronine sodium by means of reversed phase HPLC, high resolution mass spectrometry, on-line H/D exchange and UV/Vis absorption.

PubMed

Ruggenthaler, M; Grass, J; Schuh, W; Huber, C G; Reischl, R J

2017-09-05

For the first time, a comprehensive investigation of the impurity profile of the synthetic thyroid API (active pharmaceutical ingredient) liothyronine sodium (LT 3 Na) was performed by using reversed phase HPLC and advanced structural elucidation techniques including high resolution tandem mass spectrometry (HRMS/MS) and on-line hydrogen-deuterium (H/D) exchange. Overall, 39 compounds were characterized and 25 of these related substances were previously unknown to literature. The impurity classification system recently developed for the closely related API levothyroxine sodium (LT 4 Na) could be applied to the newly characterized liothyronine sodium impurities resulting in a wholistic thyroid API impurity classification system. Furthermore, the mass-spectrometric CID-fragmentation of specific related substances was discussed and rationalized by detailed fragmentation pathways. Moreover, the UV/Vis absorption characteristics of the API and selected impurities were investigated to corroborate chemical structure assignments derived from MS data. Copyright © 2017 Elsevier B.V. All rights reserved.

A study of elastase peptides from bovine white matter proteolipid.

PubMed

Lees, M B; Macklin, W B; Chao, B H

1981-10-01

Bovine white matter proteolipid has been digested with elastase in the presence of deoxycholate. After acidification, the digest was separated into an acid-soluble and an acid-insoluble fraction. The acid-insoluble fraction was enriched in nonpolar amino acids and, by a combination of solvent fractionation and chromatography, a fraction was obtained which consisted of a mixture of two peptides with a molecular weight of approximately 4000 daltons. The acid-soluble peptides were separated by molecular sieve, ion exchange and high performance liquid chromatography (HPLC) in the reverse phase mode. The purified peptides were smaller than expected on the basis of their elution position from a molecular sieve column, suggesting they were in an aggregated state during the initial chromatography. Reverse phase HPLC was shown to be useful for fingerprinting these peptide mixtures. The data demonstrate the difficulties associated with the study of this proteolipid and emphasize the tendency of both the protein and the peptides derived from it to aggregate.

Structural characterization of osmoregulator peptides from the brain of the leech Theromyzon tessulatum: IPEPYVWD and IPEPYVWD-amide.

PubMed

Salzet, M; Vandenbulcke, F; Verger-Bocquet, M

1996-12-31

Neurons immunoreactive to an antiserum (a-OT) directed specifically against the C-terminal part (prolyl-leucyl-glycinamide) of vertebrate oxytocin (OT) were detected in the brain of the leech Theromyzon tessulatum. With high pressure gel permeation chromatography followed by reversed-phase HPLC on brain extracts, evidence was given of the presence of three peptides (P1, P2, P3) immunoreactive to a-OT. Results of injection experiments in T. tessulatum and of titrations of each peptide at the different physiological stages of the animals which showed a peak in peptide P1 amount at stage 3B, indicated that P1 is the active OT-like peptide. Using three steps of reversed-phase HPLC, Edman degradation and electrospray mass spectrometry, two sequences for P1 (IPEPYVWD and IPEPYVWD-amide) were found. These peptides differ from peptides to the oxytocin/vasopressin family and are unique in the animal kingdom. Confirmation of their action on the hydric balance and their distribution in the CNS were presented.

Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC. 1. Separation behavior of aromatic carboxylic acids

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

Kawamura, K.; Okuwaki, A.; Verheyen, T.

In order to develop separation processes and analytical methods for aromatic carboxylic acids for the coal oxidation products, the separation behavior of aromatic carboxylic acids on a reversed-phase HPLC using eluent containing quaternary ammonium salt has been investigated. The retention mechanism of aromatic carboxylic acids was discussed on the basis of both ion-pair partition model and ion-exchange model. The retention behavior of aromatic carboxylic acids possessing one (or two) carboxylic acid group(s) followed the ion-pair partition model, where linear free energy relationship was observed between the capacity factor and the extraction equilibrium constants of benzoic acid and naphthalene carboxylic acid.more » Besides, the retention behavior followed ion-exchange model with increasing the number of carboxylic acids, where the capacity factor of benzene polycarboxylic acids is proportional to the association constants between aromatic acids and quaternary ammonium ions calculated on the basis of an electrostatic interaction model.« less

Simultaneous determination of secondary metabolites from Vinca rosea plant extractives by reverse phase high performance liquid chromatography

PubMed Central

Siddiqui, Mohammad Jamshed Ahmad; Ismail, Zhari; Saidan, Noor Hafizoh

2011-01-01

Background: Vinca rosea (Apocynaceae) is one of the most important and high value medicinal plants known for its anticancer alkaloids. It is the iota of the isolated secondary metabolites used in chemotherapy to treat diverse cancers. Several high performance liquid chromatography (HPLC) methods have been developed to quantify the active alkaloids in the plant. However, this method may serve the purpose in quantification of V. rosea plant extracts in totality. Objective: To develop and validate the reverse phase (RP)-HPLC method for simultaneous determination of secondary metabolites, namely alkaloids from V. rosea plant extracts. Materials and Methods: The quantitative determination was conducted by RP-HPLC equipped with ultraviolet detector. Optimal separation was achieved by isocratic elution with mobile phase consisting of methanol:acetonitrile:ammonium acetate buffer (25 mM) with 0.1% triethylamine (15:45:40 v/v) on a column (Zorbax Eclipse plus C18, 250 mm % 4.6 mm; 5 μm). The standard markers (vindoline, vincristine, catharanthine, and vinblastine) were identified by retention time and co-injected with reference standard and quantified by external standard method at 297 nm. Results: The precision of the method was confirmed by the relative standard deviation (R.S.D.), which was lower than 2.68%. The recoveries were in the range of 98.09%-108%. The limits of detection (LOD) for each marker alkaloids were lower than 0.20 μg. Different parts of the V. rosea extracts shows different concentrations of markers, flower samples were high in vinblastine content, while methanol extract from the leaves contains all the four alkaloids in good yield, and there is no significant presence of markers in water extracts. Conclusion: HPLC method established is appropriate for the standardization and quality assurance of V. rosea plant extracts. PMID:21716929

Characterization of the isomeric configuration and impurities of (Z)-endoxifen by 2D NMR, high resolution LC⬜MS, and quantitative HPLC analysis.

PubMed

Elkins, Phyllis; Coleman, Donna; Burgess, Jason; Gardner, Michael; Hines, John; Scott, Brendan; Kroenke, Michelle; Larson, Jami; Lightner, Melissa; Turner, Gregory; White, Jonathan; Liu, Paul

2014-01-01

(Z)-Endoxifen (4-hydroxy-N-desmethyltamoxifen), an active metabolite generated via actions of CYP3A4/5 and CYP2D6, is a more potent selective estrogen receptor modulator (SERM) than tamoxifen. In the MCF-7 human mammary tumor xenograft model with female athymic mice, (Z)-endoxifen, at an oral dose of 4⬜8 mg/kg, significantly inhibits tumor growth. (Z)-Endoxifen's potential as an alternative therapeutic agent independent of CYP2D6 activities, which can vary widely in ER+ breast cancer patients, is being actively evaluated. This paper describes confirmation of the configuration of the active (Z)-isomer through 2D NMR experiments, including NOE (ROESY) to establish spatial proton⬜proton correlations, and identification of the major impurity as the (E)-isomer in endoxifen drug substance by HPLC/HRMS (HPLC/MS-TOF). Stability of NMR solutions was confirmed by HPLC/UV analysis. For pre-clinical studies, a reverse-phase HPLC⬜UV method, with methanol/water mobile phases containing 10 mM ammonium formate at pH 4.3, was developed and validated for the accurate quantitation and impurity profiling of drug substance and drug product. Validation included demonstration of linearity, method precision, accuracy, and specificity in the presence of impurities, excipients (for the drug product), and degradation products. Ruggedness and reproducibility of the method were confirmed by collaborative studies between two independent laboratories. The method is being applied for quality control of the API and oral drug product. Kinetic parameters of Z- to E-isomerization were also delineated in drug substance and in aqueous formulation, showing conversion at temperatures above 25 °C. Copyright © 2014 Elsevier B.V. All rights reserved.

Pharmacokinetics and tissue distribution study of Praeruptorin D from Radix peucedani in rats by high-performance liquid chromatography (HPLC).

PubMed

Liang, Taigang; Yue, Wenyan; Du, Xue; Ren, Luhui; Li, Qingshan

2012-01-01

Praeruptorin D (PD), a major pyranocoumarin isolated from Radix Peucedani, exhibited antitumor and anti-inflammatory activities. The aim of this study was to investigate the pharmacokinetics and tissue distribution of PD in rats following intravenous (i.v.) administration. The levels of PD in plasma and tissues were measured by a simple and sensitive reversed-phase high-performance liquid chromatography (HPLC) method. The biosamples were treated by liquid-liquid extraction (LLE) with methyl tert-butyl ether (MTBE) and osthole was used as the internal standard (IS). The chromatographic separation was accomplished on a reversed-phase C(18) column using methanol-water (75:25, v/v) as mobile phase at a flow rate of 0.8 mL/min and ultraviolet detection wave length was set at 323 nm. The results demonstrate that this method has excellent specificity, linearity, precision, accuracy and recovery. The pharmacokinetic study found that PD fitted well into a two-compartment model with a fast distribution phase and a relative slow elimination phase. Tissue distribution showed that the highest concentration was observed in the lung, followed by heart, liver and kidney. Furthermore, PD can also be detected in the brain, which indicated that PD could cross the blood-brain barrier after i.v. administration.

Reverse isotope dilution method for determining benzene and metabolites in tissues

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

Bechtold, W.E.; Sabourin, P.J.; Henderson, R.F.

1988-07-01

A method utilizing reverse isotope dilution for the analysis of benzene and its organic soluble metabolites in tissues of rats and mice is presented. Tissues from rats and mice that had been exposed to radiolabeled benzene were extracted with ethyl acetate containing known, excess quantities of unlabeled benzene and metabolites. Butylated hydroxytoluene was added as an antioxidant. The ethyl acetate extracts were analyzed with semipreparative reversed-phase HPLC. Isolated peaks were collected and analyzed for radioactivity (by liquid scintillation spectrometry) and for mass (by UV absorption). The total amount of each compound present was calculated from the mass dilution of themore » radiolabeled isotope. This method has the advantages of high sensitivity, because of the high specific activity of benzene, and relative stability of the analyses, because of the addition of large amounts of unlabeled carrier analogue.« less

Determination of fenoterol in human plasma by HPLC with fluorescence detection after derivatization.

PubMed

Meineke, Ingolf; Steinmetz, Hannelore; Kramer, Skaidrit; Gleiter, Christoph H

2002-06-20

A new method for the determination of fenoterol is described, which uses HPLC separation with fluorescence detection. Dobutamine is employed as an internal standard. The separation was achieved on a short reversed phase column with a mobile phase consisting of water, acetonitrile and methanol. Prior to chromatography both analytes are derivatized with 9-chloroformyl-carbazole. Isolation of the analytes from plasma is carried out by liquid-liquid extraction into 2-butanol after protein precipitation with acetonitrile. The method is capable of estimating fenoterol concentrations in the sub-nanogram per ml range with sufficient accuracy and precision. The determination of fenoterol can now be carried out in the average laboratory without radiolabelled material.

Analysis of Dithiocarbamate Fungicides in Vegetable Matrices Using HPLC-UV Followed by Atomic Absorption Spectrometry.

PubMed

Al-Alam, Josephine; Bom, Laura; Chbani, Asma; Fajloun, Ziad; Millet, Maurice

2017-04-01

A simple method combining ion-pair methylation, high-performance liquid chromatography (HPLC) analysis with detection at 272 nm and atomic absorption spectrometry was developed in order to determine 10 dithiocarbamate fungicides (Dazomet, Metam-sodium, Ferbam, Ziram, Zineb, Maneb, Mancozeb, Metiram, Nabam and Propineb) and distinguish ethylenbisdithiocarbamates (EBDTCs) Zineb, Maneb and Mancozeb in diverse matrices. This method associates reverse phase analysis by HPLC analysis with detection at 272 nm, with atomic absorption spectrometry in order to distinguish, with the same extraction protocol, Maneb, Mancozeb and Zineb. The limits of detection (0.4, 0.8, 0.5, 1.25 and 1.97) and quantification (1.18, 2.5, 1.52, 4.2 and 6.52) calculated in injected nanogram, respectively, for Dazomet, Metam-Na, dimethyldithiocarbamates (DMDTCs), EBDTCs and propylenebisdithiocarbamates (PBDTCs) justify the sensitivity of the method used. The coefficients of determination R2 were 0.9985, 0.9978, 0.9949, 0.988 and 0.9794, respectively, for Dazomet, Metam-Na, DMDTCs, EBDTCs and PBDTCs, and the recovery from fortified apple and leek samples was above 90%. Results obtained with the atomic absorption method in comparison with spectrophotometric analysis focus on the importance of the atomic absorption as a complementary specific method for the distinction between different EBDTCs fungicides. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative determination for cytotoxic Friedo-nor-oleanane derivatives from five morphological types of Maytenus ilicifolia (Celastraceae) by reverse-phase high-performance liquid chromatography.

PubMed

Buffa Filho, Waldemar; Corsino, Joaquim; Bolzani, da Silva Vanderlan; Furlan, Maysa; Pereira, Ana Maria S; França, Suzelei Castro

2002-01-01

Five different morphological types of Maytenus ilicifolia of the same age and harvested under the same conditions showed distinct accumulations of some friedo-nor-oleananes. A rapid, sensitive and reliable reverse-phase HPLC method (employing an external standard) was used for the determination of the cytotoxic triterpenoids, 20 alpha-hydroxymaytenin, 22 beta-hydroxymaytenin, maytenin, celastrol and pristimerin in each of the five types. Well resolved peaks with good detection response and linearity in the range 1.0-100 micrograms/mL were obtained.

validated microbiological and HPLC methods for the determination of moxifloxacin in pharmaceutical preparations and human plasma.

PubMed

Abdelaziz, Ahmed A; Elbanna, Tarek E; Gamaleldeen, Noha M

2012-10-01

The article presents a comparison between microbiological and high performance liquid chromatographic (HPLC) assays for quantification of moxifloxacin in tablets, ophthalmic solutions and human plasma. The microbiological method employed a cylinder-plate agar diffusion assay using a strain of Esherichia coli ATCC 25922 as the test organism and phosphate buffer (pH8) as the diluent. The calibration curves were linear (R(2) > 0.98) over a concentration range of 0.125 to 16 µgml(-1). The within day and between days precisions were ≤ 4.47% and ≤ 6.39% respectively. Recovery values were between 89.4 and 110.2%. The HPLC assay used Hypersil(®) BDS C18 reversed phase column (250×4.6 mm, 5µm) with a mobile phase comprising 20 mM ammonium dihydrogen orthophosphate (pH3) and acetonitrile (75:25) and flowing at 1.5 ml/min. The detection was at 295nm. The calibration curves were linear (R(2) > 0.999) over the range of 0.125 to 16 µg ml(-1). The within day and between days precisions were ≤ 4.07% and ≤ 5.09% respectively. Recovery values were between 97.7 and 107.6%. Similar potencies were obtained after the analysis of moxifloxacin tablets and ophthalmic solutions by both methods. Also pharmacokinetic parameters were calculated after the analysis of plasma samples of six male healthhy volunteers by both validated methods.

Analysis of glycerophosphocholine molecular species as derivatives of 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin.

PubMed

Wheelan, P; Zirrolli, J A; Clay, K L

1992-01-01

A method has been developed for the analysis of derivatized diradylglycerols obtained from glycerophosphocholine (GPC) of transformed murine bone marrow-derived mast cells that provided high performance liquid chromatography (HPLC) separation of GPC subclasses and molecular species separation with on-line quantitation using UV detection. In addition, the derivatized diradylglycerol species were unequivocably identified by continuous flow fast-atom bombardment mass spectrometry. GPC was initially isolated by thin-layer chromatography (TLC), the phosphocholine group was hydrolyzed, and the resultant diradylglycerol was derivatized with 7-[(chlorocarbonyl)-methoxy]-4-methylcoumarin (CMMC). After separation of the derivatized subclasses by normal phase HPLC, the individual molecular species of the alkylacyl and diacyl subclasses were quantitated and collected during a subsequent reverse phase HPLC step. With an extinction coefficient of 14,700 l mol-1 cm-1 at a wavelength detection of 320 nm, the CMMC derivatives afforded sensitive UV detection (100 pmol) and quantitation of the molecular species. Continuous flow fast-atom bombardment mass spectrometry of the alkylacyl CMMC derivatives yielded abundant [MH]+ ions and a single fragment ion formed by loss of alkylketene from the sn-2 acyl group, [MH-(R = C = O)]+. No fragmentation of the sn-1 alkyl chain was observed. Diacyl derivatives also produced abundant [MH]+ ions plus two fragment ions arising from loss of RCOOH from each of the acyl substituents and two fragment ions from the loss of alkyketene from each acyl group. Individual molecular species substituents were assigned from these ions.

[Synthesis of porous spherical silicon oxynitride material and evaluation of its properties in reversed-phase chromatographic separation].

PubMed

Zhong, Hongmin; Zhang, Hua; Wan, Huihui

2013-04-01

Silica has been widely used as HPLC column packing material. However, the fact that base can attack the silanol and dissolve the silica embarrasses the utilization of silica stationary phase in high pH mobile phases (pH >8). In our previous research, the use of porous spherical silicon oxynitride (sph-SiON) material from high temperature nitridation of silica microspheres as stationary phase for HPLC has been explored, and the sph-SiON is stable to alkaline mobile phases and demonstrates excellent separation of a variety of polar compounds in hydrophilic interaction liquid chromatography (HILIC) mode. Herein, the degree of nitridation was studied as a function of temperature of nitridation at 750-1 050 degrees C, yielding the silicon oxynitride with 0.40%-12.0% (mass fraction) nitrogen from elemental analysis. At the temperature of 1 050 degrees C, the nitrogen content increased from 12.0% to 24.5% with the nitridation time increasing from 20 h to 120 h. The sph-SiON is stable when disposed in different pH aqueous solutions for one week. The sph-SiON material can be modified to give hydrophobic surface through the reaction of surface Si-NHx with dimethyloctadecylchlorosilane. Elemental analysis and 13C cross-polarization magic-angle spinning (CP/MAS) NMR spectrum of C18-sph-SiON prove the integration of C18 alkyl groups attached onto the sph-SiON surface. The chromatographic evaluation of C18-sph-SiON in reversed-phase separation mode was performed with alkylbenzenes as hydrophobic probes. Three alkylbenzene compounds can be separated and retained well on C18-sph-SiON even in the mobile phase of methanol/H2O (70/30, v/v) with 78 507 plates/m, and an excellent tailing factor (0.95) can be obtained for ethylbenzene. In comparison with C18-SiO2, C18-sph-SiON shows distinct differences with respect to different classes of analytes, i. e. neutral analyte naphthalene, acidic analyte ibuprofen, and basic analyte amitriptyline.

Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data.

PubMed

Mortera, Pablo; Zuljan, Federico A; Magni, Christian; Bortolato, Santiago A; Alarcón, Sergio H

2018-02-01

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20mmolL -1 phosphate buffer at pH 2.20) and a flow-rate of 1.0mLmin -1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0mmolL -1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes. Copyright © 2017 Elsevier B.V. All rights reserved.

Evidence for tyrosine-linked glycosaminoglycan in a bacterial surface protein.

PubMed

Peters, J; Rudolf, S; Oschkinat, H; Mengele, R; Sumper, M; Kellermann, J; Lottspeich, F; Baumeister, W

1992-04-01

The S-layer protein of Acetogenium kivui was subjected to proteolysis with different proteases and several high molecular mass glycosaminoglycan peptides containing glucose, galactosamine and an unidentified sugar-related component were separated by molecular sieve chromatography and reversed-phase HPLC and subjected to N-terminal sequence analysis. By methylation analysis glucose was found to be uniformly 1,6-linked, whereas galactosamine was exclusively 1,4-linked. Hydrazinolysis and subsequent amino-acid analysis as well as two-dimensional NMR spectroscopy were used to demonstrate that in these peptides carbohydrate was covalently linked to tyrosine. As all of the four Tyr-glycosylation sites were found to be preceded by valine, a new recognition sequence for glycosylation is suggested.

A robust high resolution reversed-phase HPLC strategy to investigate various metabolic species in different biological models.

PubMed

D'Alessandro, Angelo; Gevi, Federica; Zolla, Lello

2011-04-01

Recent advancements in the field of omics sciences have paved the way for further expansion of metabolomics. Originally tied to NMR spectroscopy, metabolomic disciplines are constantly and growingly involving HPLC and mass spectrometry (MS)-based analytical strategies and, in this context, we hereby propose a robust and efficient extraction protocol for metabolites from four different biological sources which are subsequently analysed, identified and quantified through high resolution reversed-phase fast HPLC and mass spectrometry. To this end, we demonstrate the elevated intra- and inter-day technical reproducibility, ease of an MRM-based MS method, allowing simultaneous detection of up to 10 distinct features, and robustness of multiple metabolite detection and quantification in four different biological samples. This strategy might become routinely applicable to various samples/biological matrices, especially for low-availability ones. In parallel, we compare the present strategy for targeted detection of a representative metabolite, L-glutamic acid, with our previously-proposed chemical-derivatization through dansyl chloride. A direct comparison of the present method against spectrophotometric assays is proposed as well. An application of the proposed method is also introduced, using the SAOS-2 cell line, either induced or non-induced to express the TAp63 isoform of the p63 gene, as a model for determination of variations of glutamate concentrations.

[Sensitive and selective HPLC methods with prechromatographic derivatization for the determination of cyclamate in foods].

PubMed

Rüter, J; Raczek, D I

1992-06-01

A sensitive and selective high pressure liquid chromatography (HPLC) procedure for the determination of sodium cyclamate in juices and preserves is presented. The method depends on the oxidation of cyclamate to cyclohexylamine, which then is converted prechromatographically into a fluorescent derivative. It is analyzed by HPLC on a C18:reversed-phase column and determined with fluorescence detection (excitation at 350 nm, emission at 440-650 nm). The detection limit of sodium cyclamate was 0.5-5 mg/kg, depending on the nature and dilution of the samples. The relative standard deviations thus obtained were +/- 1.0 to +/- 2.6%. The average recovery was 90%.

[Influences of ion-suppressors on retention behaviors of nine food additives in reversed-phase high performance liquid chromatographic separation].

PubMed

Zhao, Yonggang; Chen, Xiaohong; Li, Xiaoping; Yao, Shanshan; Jin, Micong

2011-10-01

The influences of ion-suppressors on retention behaviors of nine food additives, i.e., acesulfame, saccharin, caffeine, aspartame, benzoic acid, sorbic acid, stevioside, dehydroacetic acid and neotame in reversed-phase high performance liquid chromatographic (RP-HPLC) separation were investigated. The organic modification effects of acids, i. e. , trifluoroacetic acid (TFA) and buffer salts, i. e. , TFA-ammonium acetate (AmAc) were studied emphatically. The relationships between retention factors of solutes and volume percentages of ion-suppressors in the mobile phase systems of acetonitrile-TFA aqueous solution and acetonitrile-TFA-AmAc aqueous solution were quantitatively established, separately. The separation of nine food additives was completed by a gradient elution with acetonitrile-TFA (0.01%, v/v)-AmAc (2. 5 mmol/L) aqueous solution as the mobile phases. An RP-HPLC method was established for the simultaneous determination of nine food additives in red wine. In the range of 10. 0 - 100. 0 mg/L, nine food additives showed good linearity with the correlation coefficients ( r2 ) larger than 0. 999 1. The limits of detection (LODs) were in the range of 0. 33 - 2. 36 mg/L and the limits of quantification (LOQs) were in the range of 1. 11 - 7. 80 mg/L. The spiked recoveries were between 87. 61% and 108. 4% with the relative standard deviations (RSDs) of 2. 2% -9. 4%. These results are of referential significance for the rapid establishment and accu- rate optimization of RP-HPLC separation for the simultaneous determination of food additives in other foods.

Methacrylate-bonded covalent-organic framework monolithic columns for high performance liquid chromatography.

PubMed

Liu, Li-Hua; Yang, Cheng-Xiong; Yan, Xiu-Ping

2017-01-06

Covalent-organic frameworks (COFs) are a newfangled class of intriguing microporous materials. Considering their unique properties, COFs should be promising as packing materials for high performance liquid chromatography (HPLC). However, the irregular shape and sub-micrometer size of COFs synthesized via the traditional methods render the main obstacles for the application of COFs in HPLC. Herein, we report the preparation of methacrylate-bonded COF monolithic columns for HPLC to overcome the above obstacles. The prepared COF bonded monolithic columns not only show good homogeneity and permeability, but also give high column efficiency, good resolution and precision for HPLC separation of small molecules including polycyclic aromatic hydrocarbons, phenols, anilines, nonsteroidal anti-inflammatory drugs and benzothiophenes. Compared with the bare polymer monolithic column, the COF bonded monolithic columns show enhanced hydrophobic, π-π and hydrogen bond interactions in reverse phase HPLC. The results reveal the great potential of COF bonded monoliths for HPLC and COFs in separation sciences. Copyright © 2016 Elsevier B.V. All rights reserved.

Versatile ligands for high-performance liquid chromatography: An overview of ionic liquid-functionalized stationary phases.

PubMed

Zhang, Mingliang; Mallik, Abul K; Takafuji, Makoto; Ihara, Hirotaka; Qiu, Hongdeng

2015-08-05

Ionic liquids (ILs), a class of unique substances composed purely by cation and anions, are renowned for their fascinating physical and chemical properties, such as negligible volatility, high dissolution power, high thermal stability, tunable structure and miscibility. They are enjoying ever-growing applications in a great diversity of disciplines. IL-modified silica, transforming the merits of ILs into chromatographic advantages, has endowed the development of high-performance liquid chromatography (HPLC) stationary phase with considerable vitality. In the last decade, IL-functionalized silica stationary phases have evolved into a series of branches to accommodate to different HPLC modes. An up-to-date overview of IL-immobilized stationary phases is presented in this review, and divided into five parts according to application mode, i.e., ion-exchange, normal-phase, reversed-phase, hydrophilic interaction and chiral recognition. Specific attention is channeled to synthetic strategies, chromatographic behavior and separation performance of IL-functionalized silica stationary phases. Copyright © 2015 Elsevier B.V. All rights reserved.

Chromatographic determination of itopride hydrochloride in the presence of its degradation products.

PubMed

Kaul, Neeraj; Agrawal, Himani; Maske, Pravin; Rao, Janhavi Ramchandra; Mahadik, Kakasaheb Ramoo; Kadam, Shivajirao S

2005-08-01

Two sensitive and reproducible methods are described for the quantitative determination of itopride hydrochloride (IH) in the presence of its degradation products. The first method is based on HPLC separation on a reversed phase Kromasil column [C18 (5-microm, 25 cm x 4.6 mm, ID)] at ambient temperature using a mobile phase consisting of methanol and water (70:30, v/v) adjusted to pH 4.0 with orthophosphoric acid with UV detection at 258 nm. The flow rate was 1.0 mL per min with an average operating pressure of 180 kg/cm2. The second method is based on HPTLC separation on silica gel 60 F254 using toluene:methanol:chloroform:10% ammonia (5.0:3.0:6.0:0.1, v/v/v/v) as mobile phase at 270 nm. The analysis of variance (ANOVA) and Student's t-test were applied to correlate the results of IH determination in dosage form by means of HPLC and HPTLC methods. The drug was subjected to acid and alkali hydrolysis, oxidation, dry heat, wet heat treatment, UV, and photodegradation. The proposed HPLC method was utilized to investigate the kinetics of the acidic, alkaline, and oxidative degradation processes at different temperatures and the apparent pseudo-first-order rate constant, half-life, and activation energy were calculated. In addition the pH-rate profile of degradation of IH in constant ionic strength buffer solutions in the pH range 2-11 was studied.

Steroids in porcine follicular fluid: analysis by HPLC, capillary CG and capillary CG/MS after purification on SEP-PAK C18 and ion exchange chromatography.

PubMed

Khalil, M W; Lawson, V

1983-04-01

Steroids in porcine follicular fluid have been concentrated by reverse phase chromatography in SEP-PAK C18 and purified further on the cation exchanger SP-Sephadex C-25. Fractionation into unconjugated neutral and phenolic steroids, glucuronides and sulfates was carried out on triethylaminohydroxypropyl Sephadex LH-20 (TEAP-LH-20). The unconjugated neutral fraction was analysed by high pressure liquid chromatography (HPLC) on a C18 radial cartridge 5 mm I.D.; 10 mu, or on a C18 5 mu RESOLVE column, and by capillary gas chromatography (GC) on a 12 M OV-1 cross linked fused silica column. Testosterone, progesterone and androstenedione were the major steroids detected by HPLC monitored at 254 nm, although 17- hydroxy-, 20 alpha-dihydro- and 20 beta-dihydroprogesterone were also present. Pregnenolone, pregnanediol, dehydroepiandrosterone, 17-hydroxypregnenolone and androsterone were detected by capillary CG as their 0-methyloxime trimethylsilyether derivatives. Further confirmation of structure was provided by complete mass spectral data or by selective ion monitoring (SIM).

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

PubMed Central

Strickler, M P; Neill, R J; Stone, M J; Hunt, R E; Brinkley, W; Gemski, P

1989-01-01

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity. Images PMID:2745678

HPLC and chemometrics-assisted UV-spectroscopy methods for the simultaneous determination of ambroxol and doxycycline in capsule

NASA Astrophysics Data System (ADS)

Hadad, Ghada M.; El-Gindy, Alaa; Mahmoud, Waleed M. M.

2008-08-01

High-performance liquid chromatography (HPLC) and multivariate spectrophotometric methods are described for the simultaneous determination of ambroxol hydrochloride (AM) and doxycycline (DX) in combined pharmaceutical capsules. The chromatographic separation was achieved on reversed-phase C 18 analytical column with a mobile phase consisting of a mixture of 20 mM potassium dihydrogen phosphate, pH 6-acetonitrile in ratio of (1:1, v/v) and UV detection at 245 nm. Also, the resolution has been accomplished by using numerical spectrophotometric methods as classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS-1) applied to the UV spectra of the mixture and graphical spectrophotometric method as first derivative of the ratio spectra ( 1DD) method. Analytical figures of merit (FOM), such as sensitivity, selectivity, analytical sensitivity, limit of quantitation and limit of detection were determined for CLS, PLS-1 and PCR methods. The proposed methods were validated and successfully applied for the analysis of pharmaceutical formulation and laboratory-prepared mixtures containing the two component combination.

Simultaneous determination of eight major bioactive compounds in Dachengqi Tang (DT) by high-performance liquid chromatography.

PubMed

Tang, Wenfu; Wan, Meihua; Zhu, Zhengyan; Chen, Guanyuan; Huang, Xi

2008-04-29

Dachengqi Tang (DT) is a common traditional Chinese medicine formula for expelling neire ('internal heat') in the stomach and intestines. There was no reliable analytical method available for the quality control of DT. A high-performance liquid chromatography (HPLC) method with a reverse phase C18 column (150 x 4.6 mm) was developed. The mobile phase was methanol with 0.2% acetic acid. Eight markers including naringin, hesperidin, aloe emodin, rhein, honokiol, magnolol, emodin and chrysophanol were determined. Regression analysis revealed a linear relationship between the concentrations of the markers and the peak area ratio of the standards and internal standard. The limit of detection (S/N = 3) and the limit of qualification (RSD < 20%) ranged from 0.21 to 0.43 ng/microl and 0.76 to 1.74 ng/microl respectively. The recovery was between 95.6% and 103.4%. The tests on the samples from three batches of DT showed that the profiles of the markers did not vary significantly among batches. A reliable HPLC method for simultaneous determination of the eight markers in DT was developed.

Application of ion-trap mass spectrometry for identification and structural determination of an unknown impurity in simvastatin.

PubMed

Reddy, G V Ram; Kumar, A Praveen; Reddy, B Venkateswara; Sreeramulu, J

2009-10-01

Anhydro-simvastatin and simvastatin dimer are the two main impurities in the fermentation broth as well as in the final product of simvastatin, which is a hypolipidemic drug. An unknown impurity with m/z 451 for [(M + H)(+)] was detected in the analysis of final simvastatin drug sample. By using reverse phase high performance liquid chromatography (HPLC)-mass spectrometry (MS) and MS/MS spectra, the unknown impurity was detected and identified. Separation was achieved on ACE-5 C18 (150 x 4.6 mm, 3 microm column) at the flow rate of 1.2 ml min(-1) applying gradient elution of mobile phase A consisting of Milli-Q water of pH 3.0 with formic acid and B consisting of acetonitrile. MS/MS spectrum of the unknown impurity was obtained using HPLC-MS equipped with positive electrosoray ionization (ESI). The unknown impurity is named as 7-[7-(2,2-dimethyl-butyryloxy)-2,6-dimethyl-1,2,6,7,8,8a-hexahydro-naphthalen-1 -yl]-3-hydroxy-5-hydroxymethyl-heptanoic acid.

HPLC and chemometrics-assisted UV-spectroscopy methods for the simultaneous determination of ambroxol and doxycycline in capsule.

PubMed

Hadad, Ghada M; El-Gindy, Alaa; Mahmoud, Waleed M M

2008-08-01

High-performance liquid chromatography (HPLC) and multivariate spectrophotometric methods are described for the simultaneous determination of ambroxol hydrochloride (AM) and doxycycline (DX) in combined pharmaceutical capsules. The chromatographic separation was achieved on reversed-phase C(18) analytical column with a mobile phase consisting of a mixture of 20mM potassium dihydrogen phosphate, pH 6-acetonitrile in ratio of (1:1, v/v) and UV detection at 245 nm. Also, the resolution has been accomplished by using numerical spectrophotometric methods as classical least squares (CLS), principal component regression (PCR) and partial least squares (PLS-1) applied to the UV spectra of the mixture and graphical spectrophotometric method as first derivative of the ratio spectra ((1)DD) method. Analytical figures of merit (FOM), such as sensitivity, selectivity, analytical sensitivity, limit of quantitation and limit of detection were determined for CLS, PLS-1 and PCR methods. The proposed methods were validated and successfully applied for the analysis of pharmaceutical formulation and laboratory-prepared mixtures containing the two component combination.

Development and Validation of High Performance Liquid Chromatography Method for Determination Atorvastatin in Tablet

NASA Astrophysics Data System (ADS)

Yugatama, A.; Rohmani, S.; Dewangga, A.

2018-03-01

Atorvastatin is the primary choice for dyslipidemia treatment. Due to patent expiration of atorvastatin, the pharmaceutical industry makes copy of the drug. Therefore, the development methods for tablet quality tests involving atorvastatin concentration on tablets needs to be performed. The purpose of this research was to develop and validate the simple atorvastatin tablet analytical method by HPLC. HPLC system used in this experiment consisted of column Cosmosil C18 (150 x 4,6 mm, 5 µm) as the stationary reverse phase chomatography, a mixture of methanol-water at pH 3 (80:20 v/v) as the mobile phase, flow rate of 1 mL/min, and UV detector at wavelength of 245 nm. Validation methods were including: selectivity, linearity, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ). The results of this study indicate that the developed method had good validation including selectivity, linearity, accuracy, precision, LOD, and LOQ for analysis of atorvastatin tablet content. LOD and LOQ were 0.2 and 0.7 ng/mL, and the linearity range were 20 - 120 ng/mL.

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.

Simultaneous analysis and retention behavior of major isoflavonoids in Radix Puerariae lobatae and Radix Puerariae thomsonii by high performance liquid chromatography with cyclodextrins as a mobile phase modifier.

PubMed

Zeng, Aiguo; Xing, Jianfeng; Wang, Changhe; Song, Jie; Li, Cong; Yang, Xin; Yang, Guangde

2012-01-27

In order to differentiate two species of Radix Puerariae (Radix Puerariae lobatae and Radix Puerariae thomsonii) and to determine major isoflavonoids (puerarin, daidzin, daidzein and genistein) in the samples, a simple high performance liquid chromatography (HPLC) method with isocratic elution employing cyclodextrins (CDs) as mobile phase additives was developed. Various factors affecting the retention of isoflavonoids in the C(18) reversed-phase column, such as the nature of CDs, the concentration of hydroxypropyl-β-cyclodextrin (HP-β-CD) and the methanol percentage in the mobile phase, were studied. Experimental results confirmed that HP-β-CD, as a very effective mobile phase additive, could markedly reduce the retention of isoflavonoids, especially daidzein and genistein. The elution of four isoflavonoids could be achieved on a Kromasil(®) C(18) column within 56 min by using the methanol-water contained 5 mM HP-β-CD (25/75, v/v) mixture as the mobile phase. The formation of the inclusion complexes between isoflavonoids and HP-β-CD explained the modification of the retention of analytes. The apparent formation constants determined by HPLC confirmed that the stoichiometry of HP-β-CD-isoflavonoid complexes was 1:1, and the stability of the complexes depended on the size and property of isoflavonoids. The optimized method was successfully applied for the simultaneous determination of major isoflavonoids in P. lobatae and P. thomsonii samples. This work provides a useful method for the analysis of traditional Chinese herbs. Copyright © 2011 Elsevier B.V. All rights reserved.

Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

PubMed

Gupta, Shweta; Kesarla, Rajesh; Chotai, Narendra; Omri, Abdelwahab

2017-01-01

Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed-phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1-300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard) were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

International Symposium on Neurotoxins in Neurobiology (4th) Held in Bath, United Kingdom on 19th-23rd September 1993. Speakers’ Abstracts 1-25

DTIC Science & Technology

1993-01-01

experiments with plant materials following, in part, ethnopharmacological clues, has led to the discovery that several flavonoids have medium to high...1990), FEBS Letters, 270, 45-48. 27 0 AN HPLC ASSAY FOR THE NORDITERPENOID ALKALOID, METHYLLYCACONITINE P A Co , I S Blagbrough, B V L Potter, M G Rowan...Accordingly, we have developed a reverse phase HPLC assay for MLA for routine monitoring of alkaloid samples isolated from MLA-containing plant sources

HPLC-DAD-ESI/MS identification of light harvesting and light screening pigments in the lake sediments at Edmonson Point.

PubMed

Giovannetti, Rita; Alibabaei, Leila; Zannotti, Marco; Ferraro, Stefano; Petetta, Laura

2013-01-01

The composition of sedimentary pigments in the Antarctic lake at Edmonson Point has been investigated and compared with the aim to provide a useful analytical method for pigments separation and identification, providing reference data for future assessment of possible changes in environmental conditions. Reversed phase high performance liquid chromatography (HPLC) with electrospray-mass spectrometry (ESI-MS) detection and diode array detection (DAD) has been used to identify light screening and light harvesting pigments. The results are discussed in terms of local environmental conditions.

Separation of flavonol-2-O-glycosides from Calendula officinalis and Sambucus nigra by high-performance liquid and micellar electrokinetic capillary chromatography.

PubMed

Pietta, P; Bruno, A; Mauri, P; Rava, A

1992-02-28

Calendula officinalis and Sambucus nigra flowers were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) and micellar electrokinetic capillary chromatography (MECC). RP-HPLC was performed on C8 Aquapore RP 300 columns with eluents containing 2-propanol and tetrahydrofuran. MECC was carried out on a 72-cm fused-silica capillary using sodium dodecyl sulphate and sodium borate (pH 8.3) as the running buffer. The results obtained by these techniques are compared.

Speciation of chromium using reversed phase-high performance liquid chromatography coupled to different spectrometric detection methods

NASA Astrophysics Data System (ADS)

Andrle, C. M.; Jakubowski, N.; Broekaert, J. A. C.

1997-02-01

Speciation of Cr(III) and Cr(VI) based on the formation of different complexes with ammonium-pyrrolidinedithioate (APDC) in a continuous flow technique and their preconcentration using solid phase extraction (SPE) have been elaborated and applied to the analysis of waste waters from the galvanic industry. The Cr complexes were separated and determined using reversed phase-high performance liquid chromatography (RP-HPLC) coupled to different detection methods, namely UV-detection, graphite furnace-atomic absorption spectrometry (GF-AAS) and inductively coupled plasma mass spectrometry with hydraulic high pressure nebulization (HHPN/ICP-MS). After optimization the detection limits for Cr(III) and Cr(VI) of all methods are at the μg 1 -1 level and the precision in terms of RSD is 5% ( cCr = 100 μg 1 -1, N = 10). The procedure was applied to the determination of Cr(III) and Cr(VI) at the μg 1 -1 level in galvanic waste waters, and its accuracy was approved by comparing the results with those of independent methods.

Kinetics on cocondensation between phenol and urea through formaldehyde I

Treesearch

Yasunori Yoshida; Bunchiro Tomita; Chung-Yun Hse

1995-01-01

The kinetics of the reactions of methylolphenols and urea were investigated using 2- and 4-hydroxybenzyl alcohols. The high-performance liquid chromatography (HPLC) using a reverse-phase column gave a clear separation between methylolphenols and hydroxybenzylureas. The molar ratios of hydroxybenzylureas to the corresponding methylolphenols in reaction mixtures were...

Kinetics on cocondensation between phenol and urea through formaldehyde I.

Treesearch

Yasunori Yoshida; Bunichiro Tomita; Chung-Yun Hse

1995-01-01

The kinetics of the ractions of methylolphenols and urea were investigated using 2- and 4- hydroxybenzyl alcohols. The high-performance liquid chromatogrpahy (HPLC) using a reverse-phase column gave a clear separation between methylolphenols and hydroxybenzylureas. The molar ratios of hydroxybenzylureas to be corresponding methylolphenols in reaction mixtures were...

Hot Chili Peppers: Extraction, Cleanup, and Measurement of Capsaicin

NASA Astrophysics Data System (ADS)

Huang, Jiping; Mabury, Scott A.; Sagebiel, John C.

2000-12-01

Capsaicin, the pungent ingredient of the red pepper or Capsicum annuum, is widely used in food preparation. The purpose of this experiment was to acquaint students with the active ingredients of hot chili pepper (capsaicin and dihydrocapsaicin), the extraction, cleanup, and analysis of these chemicals, as a fun and informative analytical exercise. Fresh peppers were prepared and extracted with acetonitrile, removing plant co-extractives by addition to a C-18 solid-phase extraction cartridge. Elution of the capsaicinoids was accomplished with a methanol-acetic acid solution. Analysis was completed by reverse-phase HPLC with diode-array or variable wavelength detection and calibration with external standards. Levels of capsaicin and dihydrocapsaicin were typically found to correlate with literature values for a specific hot pepper variety. Students particularly enjoyed relating concentrations of capsaicinoids to their perceived valuation of "hotness".

Micellar liquid chromatography

NASA Astrophysics Data System (ADS)

Basova, Elena M.; Ivanov, Vadim M.; Shpigun, Oleg A.

1999-12-01

Background and possibilities of practical applications of micellar liquid chromatography (MLC) are considered. Various retention models in MLC, the effects of the nature and concentration of surfactants and organic modifiers, pH, temperature and ionic strength on the MLC efficiency and selectivity are discussed. The advantages and limitations of MLC are demonstrated. The performance of MLC is critically evaluated in relationship to the reversed-phase HPLC and ion-pair chromatography. The potential of application of MLC for the analysis of pharmaceuticals including that in biological fluids and separation of inorganic anions, transition metal cations, metal chelates and heteropoly compounds is described. The bibliography includes 146 references.

Determination of tylosin residues in pig tissues using high-performance liquid chromatography.

PubMed

De Liguoro, M; Anfossi, P; Angeletti, R; Montesissa, C

1998-06-01

In accordance with the maximum residue limit of 100 micrograms kg-1 established by EU legislation, a simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for the measurement of tylosin residues in pig tissues (fat, kidney, liver and muscle). Tylosin, a macrolide antibiotic, is extracted with water-methanol and cleaned-up by solid-phase extraction (SPE) on cation-exchange cartridges using methanol elution. Tylosin was determined by reversed-phase HPLC with UV detection at 280 nm and the mean recovery from pig tissues fortified in the range 50-200 micrograms kg-1 was 70-85%, with intra- and inter-day RSDs in the ranges 3.4-9.1 and 3.9-10.1% respectively.

[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.

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b

PubMed Central

2011-01-01

Background The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. Results The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Conclusion Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin. PMID:21299876

Isolation and Purification of Cu-free Methanobactin from Methylosinus trichosporium OB3b

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

M Pesch; I Christl; K Barmettler

The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/Nmore » ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin.« less

Preparation and characterization of a new microwave immobilized poly(2-phenylpropyl)methylsiloxane stationary phase for reversed phase high-performance liquid chromatography.

PubMed

Begnini, Fernanda R; Jardim, Isabel C S F

2013-07-05

A new reversed phase high-performance liquid chromatography (RP-HPLC) stationary phase was prepared and its chromatographic and physical-chemical properties were evaluated. The new stationary phase was prepared with a silica support and poly(2-phenylpropyl)methylsiloxane (PPPMS), a phenyl type polysiloxane copolymer. Since this is a new copolymer and there is little information in the literature, it was submitted to physical-chemical characterization by infrared spectroscopy and thermogravimetry. The chromatographic phase was prepared through sorption and microwave immobilization of the copolymer onto a silica support. The chromatographic performance was evaluated by employing test procedures suggested by Engelhardt and Jungheim, Tanaka and co-workers, Neue, and Szabó and Csató. These test mixtures provide information about the hydrophobic selectivity, silanophilic activity, ion-exchange capacity, shape selectivity and interaction with polar analytes of the new Si-PPPMS reversed phase. Stability tests were developed using accelerated aging tests under both basic and acidic conditions to provide information about the lifetime of the packed columns. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Antioxidant capacities of ten edible North American plants.

PubMed

Acuña, Ulyana Muñoz; Atha, Daniel E; Ma, Jun; Nee, Michael H; Kennelly, Edward J

2002-02-01

The EtOAc extract obtained from ten edible North American plants, Acorus calamus, Clintonia borealis, Gaultheria shallon, Juniperus osteosperma, Opuntia polyacantha, Prunus americana, Prunus virginiana, Sambucus cerulea, Sorbus americana and Vaccinium parvifolium, were tested in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay. High antioxidant activity was obtained from the extracts of three fruits, Gaultheria shallon, Sambucus cerulea and Prunus americana and one extracted rhizome, Acorus calamus. Catechin and epicatechin, potent polyphenolic antioxidants, were identified in the EtOAc extracts of Gaultheria shallon and Sambucus cerulea by reversed-phase thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (HPLC). Copyright 2002 John Wiley & Sons, Ltd.

HPLC Characterization of Phenol-Formaldehyde Resole Resin Used in Fabrication of Shuttle Booster Nozzles

NASA Technical Reports Server (NTRS)

Young, Philip R.

1999-01-01

A reverse phase High Performance Liquid Chromatographic method was developed to rapidly fingerprint a phenol-formaldehyde resole resin similar to Durite(R) SC-1008. This resin is used in the fabrication of carbon-carbon composite materials from which Space Shuttle Solid Rocket Booster nozzles are manufactured. A knowledge of resin chemistry is essential to successful composite processing and performance. The results indicate that a high quality separation of over 35 peaks in 25 minutes were obtained using a 15 cm Phenomenex LUNA C8 bonded reverse phase column, a three-way water-acetonitrile-methanol nonlinear gradient, and LTV detection at 280 nm.

Comparison of UV spectrophotometry and high performance liquid chromatography methods for the determination of repaglinide in tablets.

PubMed

Dhole, Seema M; Khedekar, Pramod B; Amnerkar, Nikhil D

2012-07-01

Repaglinide is a miglitinide class of antidiabetic drug used for the treatment of type 2 diabetes mellitus. A fast and reliable method for the determination of repaglinide was highly desirable to support formulation screening and quality control. UV spectrophotometric and reversed-phase high performance liquid chromatography (RP-HPLC) methods were developed for determination of repaglinide in the tablet dosage form. The UV spectrum recorded between 200 400 nm using methanol as solvent and the wavelength 241 nm was selected for the determination of repaglinide. RP-HPLC analysis was carried out using Agilent TC-C18 (2) column and mobile phase composed of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the International Conference on Harmonization (ICH) guidelines. The developed methods illustrated excellent linearity (r(2) > 0.999) in the concentration range of 5-30 μg/ml and 5-50 μg/ml for UV spectrophotometric and HPLC methods, respectively. Precision (%R.S.D < 1.50) and mean recoveries were found in the range of 99.63-100.45% for UV spectrophotometric method and 99.71-100.25% for HPLC method which shows accuracy of the methods. The developed methods were found to be reliable, simple, fast, accurate and successfully used for the quality control of repaglinide as a bulk drug and in pharmaceutical formulations.

Comparison of UV spectrophotometry and high performance liquid chromatography methods for the determination of repaglinide in tablets

PubMed Central

Dhole, Seema M.; Khedekar, Pramod B.; Amnerkar, Nikhil D.

2012-01-01

Background: Repaglinide is a miglitinide class of antidiabetic drug used for the treatment of type 2 diabetes mellitus. A fast and reliable method for the determination of repaglinide was highly desirable to support formulation screening and quality control. Objective: UV spectrophotometric and reversed-phase high performance liquid chromatography (RP-HPLC) methods were developed for determination of repaglinide in the tablet dosage form. Materials and Methods: The UV spectrum recorded between 200 400 nm using methanol as solvent and the wavelength 241 nm was selected for the determination of repaglinide. RP-HPLC analysis was carried out using Agilent TC-C18 (2) column and mobile phase composed of methanol and water (80:20 v/v, pH adjusted to 3.5 with orthophosphoric acid) at a flow rate of 1.0 ml/min. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the International Conference on Harmonization (ICH) guidelines. Results: The developed methods illustrated excellent linearity (r2 > 0.999) in the concentration range of 5-30 μg/ml and 5-50 μg/ml for UV spectrophotometric and HPLC methods, respectively. Precision (%R.S.D < 1.50) and mean recoveries were found in the range of 99.63-100.45% for UV spectrophotometric method and 99.71-100.25% for HPLC method which shows accuracy of the methods. Conclusion: The developed methods were found to be reliable, simple, fast, accurate and successfully used for the quality control of repaglinide as a bulk drug and in pharmaceutical formulations. PMID:23781481

Correlation of sensory bitterness in dairy protein hydrolysates: Comparison of prediction models built using sensory, chromatographic and electronic tongue data.

PubMed

Newman, J; Egan, T; Harbourne, N; O'Riordan, D; Jacquier, J C; O'Sullivan, M

2014-08-01

Sensory evaluation can be problematic for ingredients with a bitter taste during research and development phase of new food products. In this study, 19 dairy protein hydrolysates (DPH) were analysed by an electronic tongue and their physicochemical characteristics, the data obtained from these methods were correlated with their bitterness intensity as scored by a trained sensory panel and each model was also assessed by its predictive capabilities. The physiochemical characteristics of the DPHs investigated were degree of hydrolysis (DH%), and data relating to peptide size and relative hydrophobicity from size exclusion chromatography (SEC) and reverse phase (RP) HPLC. Partial least square regression (PLS) was used to construct the prediction models. All PLS regressions had good correlations (0.78 to 0.93) with the strongest being the combination of data obtained from SEC and RP HPLC. However, the PLS with the strongest predictive power was based on the e-tongue which had the PLS regression with the lowest root mean predicted residual error sum of squares (PRESS) in the study. The results show that the PLS models constructed with the e-tongue and the combination of SEC and RP-HPLC has potential to be used for prediction of bitterness and thus reducing the reliance on sensory analysis in DPHs for future food research. Copyright © 2014 Elsevier B.V. All rights reserved.

Determination of pterins in urine by HPLC with UV and fluorescent detection using different types of chromatographic stationary phases (HILIC, RP C8, RP C18).

PubMed

Kośliński, Piotr; Jarzemski, Piotr; Markuszewski, Michał J; Kaliszan, Roman

2014-03-01

Pterins are a class of potential cancer biomarkers. New methods involving hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) high-performance liquid chromatography have been developed for analysis of eight pterin compounds: 6,7-dimethylpterin, pterin, 6-OH-methylpterin, biopterin, isoxanthopterin, neopterin, xanthopterin, and pterin-6-carboxylic acid. The effect of mobile phase composition, buffer type, pH and concentration on retention using HILIC, C8 and C18 RP stationary phases were examined. Separation of pterins on RP and HILIC stationary phase was performed and optimized. Eight pterins were successfully separated on HILIC Luna diol-bonded phases, Aquasil C18 RP column and LiChrospher C8 RP column. Determination and separation of the pterins from urine samples were performed on HILIC Luna and LiChrospher C8 RP columns which were chosen as the most appropriate ones. Finally, LiChrospher C8 RP column with fluorescence detection was selected for further validation of the method. The optimum chromatographic condition was mobile phase methanol (A)/phosphoric buffer pH 7, 10mM (B), isocratic elution 0-15min 5% A flow=0.5ml/min 15-17min. 5% A, flow=0.5-1ml/min the linearity (R(2)>0.997) and retention time repeatability (RSD%<1) were at satisfactory level. The precision of peak areas expressed as RSD in % was between 0.55 and 14. Pterins detection limits varied from 0.041ng/ml to 2.9ng/ml. Finally, HPLC method was used for the analysis of pterins in urine samples with two different oxidation procedures. Concentration levels of pterin compounds in bladder cancer patients and healthy subjects were compared. Copyright © 2013 Elsevier B.V. All rights reserved.

Green chiral HPLC study of the stability of Chiralcel OD under high temperature liquid chromatography and subcritical water conditions.

PubMed

Droux, S; Roy, M; Félix, G

2014-10-01

We report here the study of the stability under subcritical water conditions of one of the most popular polysaccharide chiral stationary phase (CSP): Chiralcel OD. This CSP was used under high temperature and reversed phase conditions with acetonitrile and 2-propanol as modifier, respectively. The evolution of selectivity and resolution was investigated both in normal and reversed mode conditions with five racemates after packing, heating at 150 °C and separations of some racemic compounds under different high temperatures and mobile phase conditions. The results show that after using at high temperature and subcritical water conditions the selectivity was only moderately affected while the resolution fell dramatically especially in reversed mode due to the creation of a void at the head of the columns which reflects the dissolution of the silica matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of celecoxib, hydroxycelecoxib, and carboxycelecoxib in human plasma using gradient reversed-phase liquid chromatography with ultraviolet absorbance detection.

PubMed

Störmer, Elke; Bauer, Steffen; Kirchheiner, Julia; Brockmöller, Jürgen; Roots, Ivar

2003-01-05

A new HPLC method for the simultaneous determination of celecoxib, carboxycelecoxib and hydroxycelecoxib in human plasma samples has been developed. Following a solid-phase extraction procedure, the samples were separated by gradient reversed-phase HLPC (C(18)) and quantified using UV detection at 254 nm. The method was linear over the concentration range 10-500 ng/ml. The intra-assay variability for the three analytes ranged from 4.0 to 12.6% and the inter-assay variability from 4.9 to 14.2%. The achieved limits of quantitation (LOQ) of 10 ng/ml for each analyte allowed the determination of the pharmacokinetic parameters of the analytes after administration of 100 mg celecoxib.

Analytical and semi-preparative enantioresolution of (RS)-ketorolac from pharmaceutical formulation and in human plasma by HPLC.

PubMed

Lal, Manohar; Bhushan, Ravi

2016-10-01

An efficient, simple, validated, analytical and semi-preparative HPLC method has been developed for direct enantioresolution of (RS)-Ketorolac (Ket) using monochloro-methylated derivatives of cellulose and amylose, i.e. cellulose (tris-3-chloro-4-methylphenylcarbamate) and amylose (tris-5-chloro-2-methylphenylcarbamate) as chiral stationary phases (CSPs) with photo diode array detection at 320 nm. Enantioresolution was carried out in samples of human plasma spiked with (RS)-Ket under normal and reversed-phase elution modes with suitable mobile phase compositions. The effect of nature of alcohols (MeOH, EtOH, PrOH and n-BuOH) and other solvents (MeCN and MeOH) as organic modifiers in the mobile phase was investigated on the separation performance of two CSPs in terms of retention and separation of enantiomers. The best resolution was observed on cellulose-based CSP using EtOH, while using 2-PrOH (15%) and amylose-based CSP obtained the highest retention. Under reversed-phase elution mode the best enantioseparation was observed using 30% MeCN with ammonium formate buffer. The elution order of enantiomers was ascertained by determining specific rotations. The limit of detection and quantitation values were 5 and 15.5 ng/mL for each enantiomer of (RS)-Ket, respectively. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Toxicity and metabolism of 2,4-dichlorophenol by the aquatic angiosperm Lemna gibba

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

Ensley, H.E.; Barber, J.T.; Polito, M.A.

1994-02-01

The toxicity and metabolism of 2,4-dichlorophenol with regard to the aquatic macrophyte Lemna gibba (duckweed), have been studied. Toxicity is described in terms of the effect of 2,4-dichlorophenol (2,4-DCP) on the vegetative reproduction of duckweed over a 10-d growth period; the EC10 and EC50 were 2.5 and 9.2 [mu]M, respectively. Metabolism of 2,4-dichlorophenol was monitored by incubation of the plants with radiolabeled substrate, and periodic sampling and analysis by reversed-phase HPLC of the plant growth medium. Depending on the growth conditions, up to 95% of the 2,4-DCP was metabolized over a 6-d growth period. To analyze the metabolites, the plantsmore » were grown in the presence of sublethal concentrations of [U-[sup 14]C]-2,4-DCP. The growth medium was lyophilized and then mixed with the plants, extracted, and analyzed using reversed-phase HPLC, followed by scintillation counting of the fractions. The major metabolite was isolated and identified as 2,4-dichlorophenol-[beta]-D-glucopyranoside by high-field NMR and MS. The structure of the metabolite was confirmed by synthesis and by enzymatic cleavage of the [beta]-glucosidic linkage to afford 2,4-DCP. An important consequence of conjugate formation is the masking of the presence of 2,4-DCP to the usual analytical techniques used for its detection and quantitation. This finding is probably applicable to other contaminants and organisms.« less

Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: A potential local regulator of IGF action

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

Mohan, S.; Bautista, C.M.; Wergedal, J.

1989-11-01

Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C{sub 4} reverse-phase, HPLC CN reverse-phase and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known PBs. (iii) In-IGF-BP exhibited a single band with molecular mass of 25 kDa under reducing conditions on sodiummore » dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of {sup 125}I-labeled IGF-I or {sup 125}I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increases synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, the authors conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.« less

Ionic liquids improved reversed-phase HPLC on-line coupled with ICP-MS for selenium speciation.

PubMed

Chen, Beibei; He, Man; Mao, Xiangju; Cui, Ran; Pang, Daiwen; Hu, Bin

2011-01-15

Room-temperature ionic liquids (RTILs) improved reversed-phase high performance liquid chromatography (RP-HPLC) on-line combined with inductively coupled plasma mass spectrometry (ICP-MS) was developed for selenium speciation. The different parameters affecting the retention behaviors of six target selenium species especially the effect of RTILs as mobile phase additives have been studied, it was found that the mobile phase consisting of 0.4% (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 0.4% (v/v) 1-butyl-2,3-dimethylimidazolium tetrafluroborate ([BMMIM]BF(4)) and 99.2% (v/v) water has effectively improved the peak profile and six target selenium species including Na(2)SeO(3) (Se(IV)), Na(2)SeO(4) (Se(VI)), L-selenocystine (SeCys(2)), D,L-selenomethionine (SeMet), Se-methylseleno-l-cysteine (MeSeCys), seleno-D,L-ethionine (SeEt) were separated in 8 min. In order to validate the accuracy of the method, a Certified Reference Material of SELM-1 yeast sample was analyzed and the results obtained were in good agreement with the certified values. The developed method was also successfully applied to the speciation of selenium in Se-enriched yeasts and clover. For fresh Se-enriched yeast cells, it was found that the spiked SeCys(2) in living yeast cells could be transformed into SeMet. Compared with other ion-pair RP-HPLC-ICP-MS approaches for selenium speciation, the proposed method possessed the advantages including ability to regulate the retention time of the target selenium species by selecting the suitable RTILs and their concentration, simplicity, rapidness and low injection volume, thus providing wide potential applications for elemental speciation in biological systems. Copyright © 2010 Elsevier B.V. All rights reserved.

Establishment and reliability evaluation of the design space for HPLC analysis of six alkaloids in Coptis chinensis (Huanglian) using Bayesian approach.

PubMed

Dai, Sheng-Yun; Xu, Bing; Zhang, Yi; Li, Jian-Yu; Sun, Fei; Shi, Xin-Yuan; Qiao, Yan-Jiang

2016-09-01

Coptis chinensis (Huanglian) is a commonly used traditional Chinese medicine (TCM) herb and alkaloids are the most important chemical constituents in it. In the present study, an isocratic reverse phase high performance liquid chromatography (RP-HPLC) method allowing the separation of six alkaloids in Huanglian was for the first time developed under the quality by design (QbD) principles. First, five chromatographic parameters were identified to construct a Plackett-Burman experimental design. The critical resolution, analysis time, and peak width were responses modeled by multivariate linear regression. The results showed that the percentage of acetonitrile, concentration of sodium dodecyl sulfate, and concentration of potassium phosphate monobasic were statistically significant parameters (P < 0.05). Then, the Box-Behnken experimental design was applied to further evaluate the interactions between the three parameters on selected responses. Full quadratic models were built and used to establish the analytical design space. Moreover, the reliability of design space was estimated by the Bayesian posterior predictive distribution. The optimal separation was predicted at 40% acetonitrile, 1.7 g·mL(-1) of sodium dodecyl sulfate and 0.03 mol·mL(-1) of potassium phosphate monobasic. Finally, the accuracy profile methodology was used to validate the established HPLC method. The results demonstrated that the QbD concept could be efficiently used to develop a robust RP-HPLC analytical method for Huanglian. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Determination of thiopurine S-methyltransferase activity by hydrophilic interaction liquid chromatography hyphenated with mass spectrometry.

PubMed

Pecher, Daniel; Dokupilová, Svetlana; Zelinková, Zuzana; Peppelenbosch, Maikel; Mikušová, Veronika; Mikuš, Peter

2017-08-05

Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry (MS). In comparison with reversed-phase high-performance liquid chromatography (RP-HPLC), that has been typically applied in determination of TPMT activity, the HILIC significantly improved the analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution. The HILIC-HPLC-MS method was optimized and validated, providing favorable parameters of detection and quantitation limits (5.5 and 16.5pmol/mL, respectively), linearity (coefficient of determination 0.9999 in the range of 0.01-1.0nmol/mL), recovery and precision (93.25-100.37% with RSD 1.06-1.32% in the whole concentration range of QC samples). Moreover, in contrast to the conventional RP-HPLC-UV approach, the complex phenotype TPMT profiles can be reliably and without interferences monitored using the HILIC-HPLC-MS method. Such advanced monitoring can provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non-methylated 6-mercaptopurine) and, by that, TPMT action in biological systems before and during the therapy of IBD. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of phenol, m-, o- and p-cresol, p-aminophenol and p-nitrophenol in urine by high-performance liquid chromatography.

PubMed

Brega, A; Prandini, P; Amaglio, C; Pafumi, E

1990-12-28

A method for the biological monitoring of human exposure to aromatic hydrocarbons, nitrocompounds, amines and phenols has been developed. Phenol, cresols, p-aminophenol, p-nitrophenol and their glucorono- or sulpho-conjugates, were quantified by HPLC; 4-chlorphenol was added as internal standard. After enzymatic hydrolysis, the free compounds were extracted with an organic solvent and analyzed by an isocratic HPLC Perkin Elmer system at ambient temperature and at a flow-rate of 1 ml/min. The column was a reversed-phase Pecosphere 3 x 3 C18 Perkin Elmer; the mobile phase was a 30:70:0.1 (v/v/v) methanol-water-orthophosphoric acid mixture and the chromatogram was monitored at 215 nm. Identification was based on retention time and quantification was performed by automatic peak height determination, corrected for the internal standard. The recovery was ca. 95% for phenol and cresols; 90% for p-nitrophenol; 85% for p-aminophenol; the coefficients of variance were less than 6% within analysis (n = 20) and less than 10% between analysis (n = 20). The detection limits, at a signal/noise ratio of 2, were 0.5 mg/l for phenol and cresols and 1 mg/l for p-aminophenol and p-nitrophenol.

Comprehensive two-dimensional chromatography with coupling of reversed phase high performance liquid chromatography and supercritical fluid chromatography.

PubMed

Stevenson, Paul G; Tarafder, Abhijit; Guiochon, Georges

2012-01-13

A 2D comprehensive chromatographic separation of blackberry sage fragrant oil was performed by using HPLC in the first dimension and SFC in the second. A C(18)-bonded silica column eluted with an ACN gradient was used in the HPLC dimension and an amino-bonded silica column eluted with ACN as a modifier in the SFC dimension. This 2D separation was completed in the off-line mode, the fractions from the HPLC column being collected and injected in the SFC column. The retention factors on the two columns have a -0.757 correlation coefficient. The method provides a practical peak capacity of 2400 in 280 min. The first eluted peaks in HPLC are the last ones eluted in SFC and vice versa. The results demonstrate that the coupling of an HPLC and an SFC separation have a great potential for 2D chromatographic separations. Copyright © 2011 Elsevier B.V. All rights reserved.

SEPARATION AND CHARACTERIZATION OF TETROL METABOLITES OF BENZO[A]PYRENE-DNA ADDUCTS USING HPLC AND SOLID-MATRIX ROOM TEMPERATURE LUMINESCENCE. (R824100)

EPA Science Inventory

Abstract

Four tetrols of benzo[a]pyrene-DNA adducts were separated using reversed-phase high performance liquid chromatography. Chromatographic fractions containing a given tetrol were readily characterized with solid-matrix room temperature luminescence techniques. So...

DETERMINATION OF CARBENDAZIM IN WATER BY HIGH-PERFORMANCE IMMUNOAFFINITY CHROMATOGRAPHY ON-LINE WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH DIODE-ARRAY OR MASS SPECTROMETRIC DETECTION

EPA Science Inventory

An automated method for the determination of carbendazim in water that combines high-performance immunoaffinity chromatography (HPIAC), high-performance liquid chromatography (HPLC) in the reversed-phase mode, and detection by either UV-Vis diode array detector (DAD) spectroscopy...

Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.

PubMed

Nakajima, Kazuki; Kitazume, Shinobu; Angata, Takashi; Fujinawa, Reiko; Ohtsubo, Kazuaki; Miyoshi, Eiji; Taniguchi, Naoyuki

2010-07-01

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

Antimicrobial activity of Marcetia DC species (Melastomataceae) and analysis of its flavonoids by reverse phase-high performance liquid chromatography coupled-diode array detector.

PubMed

Leite, Tonny Cley Campos; de Sena, Amanda Reges; Dos Santos Silva, Tânia Regina; Dos Santos, Andrea Karla Almeida; Uetanabaro, Ana Paula Trovatti; Branco, Alexsandro

2012-07-01

Marcetia genera currently comprises 29 species, with approximately 90% inhabiting Bahia (Brazil), and most are endemic to the highlands of the Chapada Diamantina (Bahia). Among the species, only M. taxifolia (A.St.-Hil.) DC. populates Brazil (state of Roraima to Paraná) and also Venezuela, Colombia, and Guyana. This work evaluated the antimicrobial activity of hexane, ethyl acetate, and methanol extracts of three species of Marcetia (Marcetia canescens Naud., M. macrophylla Wurdack, and M. taxifolia A.StHil) against several microorganism. In addition, the flavonoids were analyzed in extracts by HPLC-DAD. The tests were made using Gram-positive (three strains of Staphylococcus aureus) and Gram-negative (two strains of Escherichia coli, a strain of Pseudomonas aeruginosa and another of Salmonella choleraesius) bacteria resistant and nonresistant to antibiotics and yeasts (two strains of Candida albicans and one of C. parapsilosis) by the disk diffusion method. Solid-phase extraction (SPE) was performed on the above extracts to isolate flavonoids, which were subsequently analyzed by high performance liquid chromatography coupled diode array detector (HPLC-DAD). Results showed that extracts inhibited the Gram-positive bacteria and yeast. The hexane extracts possessed the lowest activity, while the ethyl acetate and methanolic extracts were more active. Marcetia taxifolia was more effective (active against 10 microorganisms studied), and only its methanol extract inhibited Gram-negative bacteria (P. aeruginosa and S. choleraesius). SPE and HPLC-DAD analysis showed that M. canescens and M. macrophylla contain glycosylated flavonoids, while the majority of extracts from M. taxifolia were aglycone flavonoids.

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%).

High-performance liquid chromatographic analysis of as-synthesised N,N'-dimethylformamide-stabilised gold nanoclusters product

NASA Astrophysics Data System (ADS)

Xie, Shunping; Paau, Man Chin; Zhang, Yan; Shuang, Shaomin; Chan, Wan; Choi, Martin M. F.

2012-08-01

Reverse-phase high-performance liquid chromatographic (RP-HPLC) separation and analysis of polydisperse water-soluble gold nanoclusters (AuNCs) stabilised with N,N'-dimethylformamide (DMF) were investigated. Under optimal elution gradient conditions, the separation of DMF-AuNCs was monitored by absorption and fluorescence spectroscopy. The UV-vis spectral characteristics of the separated DMF-AuNCs have been captured and they do not possess distinct surface plasmon resonance bands, indicating that all DMF-AuNCs are small AuNCs. The photoluminescence emission spectra of the separated DMF-AuNCs are in the blue-light region. Moreover, cationic DMF-AuNCs are for the first time identified by ion chromatography. Our proposed RP-HPLC methodology has been successfully applied to separate AuNCs of various Au atoms as well as DMF-stabilised ligands. Finally, the composition of the separated DMF-AuNCs was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and electrospray ionisation mass spectrometry, proving that the as-synthesised DMF-AuNCs product consists of Au10+, Au10, Au11, Au12, Au13, and Au14 NCs stabilised with various numbers of DMF ligands.Reverse-phase high-performance liquid chromatographic (RP-HPLC) separation and analysis of polydisperse water-soluble gold nanoclusters (AuNCs) stabilised with N,N'-dimethylformamide (DMF) were investigated. Under optimal elution gradient conditions, the separation of DMF-AuNCs was monitored by absorption and fluorescence spectroscopy. The UV-vis spectral characteristics of the separated DMF-AuNCs have been captured and they do not possess distinct surface plasmon resonance bands, indicating that all DMF-AuNCs are small AuNCs. The photoluminescence emission spectra of the separated DMF-AuNCs are in the blue-light region. Moreover, cationic DMF-AuNCs are for the first time identified by ion chromatography. Our proposed RP-HPLC methodology has been successfully applied to separate AuNCs of various Au atoms as well as DMF-stabilised ligands. Finally, the composition of the separated DMF-AuNCs was confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and electrospray ionisation mass spectrometry, proving that the as-synthesised DMF-AuNCs product consists of Au10+, Au10, Au11, Au12, Au13, and Au14 NCs stabilised with various numbers of DMF ligands. This article was submitted as part of a Themed Issue on metallic clusters. Other papers on this topic can be found in issue 14 of vol. 4 (2012). This issue can be found from the Nanoscale homepage [http://www.rsc.org/nanoscale].

Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma.

PubMed

Vielhauer, S; Rudolphi, A; Boos, K S; Seidel, D

1995-04-21

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C18-, C8- or C4-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C8-Alkyl-Diol Silica, 25 microns, 25 x 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5-101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2-3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproducibility. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).

Simultaneous quantification of flavonoids and triterpenoids in licorice using HPLC.

PubMed

Wang, Yuan-Chuen; Yang, Yi-Shan

2007-05-01

Numerous bioactive compounds are present in licorice (Glycyrrhizae Radix), including flavonoids and triterpenoids. In this study, a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous quantification of three flavonoids (liquiritin, liquiritigenin and isoliquiritigenin) and four triterpenoids (glycyrrhizin, 18alpha-glycyrrhetinic acid, 18beta-glycyrrhetinic acid and 18beta-glycyrrhetinic acid methyl ester) from licorice was developed, and further, to quantify these 7 compounds from 20 different licorice samples. Specifically, the reverse-phase HPLC was performed with a gradient mobile phase composed of 25 mM phosphate buffer (pH 2.5)-acetonitrile featuring gradient elution steps as follows: 0 min, 100:0; 10 min, 80:20; 50 min, 70:30; 73 min, 50:50; 110 min, 50:50; 125 min, 20:80; 140 min, 20:80, and peaks were detected at 254 nm. By using our technique, a rather good specificity was obtained regarding to the separation of these seven compounds. The regression coefficient for the linear equations for the seven compounds lay between 0.9978 and 0.9992. The limits of detection and quantification lay in the range of 0.044-0.084 and 0.13-0.25 microg/ml, respectively. The relative recovery rates for the seven compounds lay between 96.63+/-2.43 and 103.55+/-2.77%. Coefficient variation for intra-day and inter-day precisions lay in the range of 0.20-1.84 and 0.28-1.86%, respectively. Based upon our validation results, this analytical technique is a convenient method to simultaneous quantify numerous bioactive compounds derived from licorice, featuring good quantification parameters, accuracy and precision.

Reversed-phase HPLC analysis of levetiracetam in tablets using monolithic and conventional C18 silica columns.

PubMed

Can, Nafiz O; Arli, Goksel

2010-01-01

Development and validation of an RP-HPLC method for determination of levetiracetam in pharmaceutical tablets is described. The separation and quantification of levetiracetam and caffeine (internal standard) were performed using a single analytical procedure with two different types of stationary phases, conventional Phenomenex Gemini C18 (100 x 4.6 mm, 5 microm) and Merck Chromolith Performance RP18e (100 x 4.6 mm, macropore size 2 mm, micropore size 13 nm) monolithic silica. Five-microliter aliquots of samples were injected into the system and eluted using water-acetonitrile (90 + 10, v/v) mobile phase pumped at the rate of 1 mL/min. The analyte peaks were detected at 200 nm using a diode array detector with adequate resolution. Validation studies were performed using the method recommended by the International Conference on Harmonization, the U.S. Pharmacopeia, and AOAC INTERNATIONAL, which includes accuracy, precision, range, limits, robustness, and system suitability parameters. Levetiracetam and caffeine were detected in about 7 min using the conventional column, whereas less than 5 min was required when the monolithic column was used. Calibration plots had r values close to unity in the range of 0.8-8.0 microg/mL. Assay of levetiracetam in a tablet formulation was demonstrated as an application to real samples.

Integration of magnetic solid phase fishing and off-line two-dimensional high-performance liquid chromatography-diode array detector-mass spectrometry for screening and identification of human serum albumin binders from Radix Astragali.

PubMed

Zhang, Yuping; Nie, Mingkun; Shi, Shuyun; You, Qingping; Guo, Junfang; Liu, Liangliang

2014-03-01

Radix Astragali is one of the most popular traditional medicinal herb and healthy dietary supplement. Isoflavonoids and astragalosides are the main bioactive ingredients. However, the systematic bioactive component analysis is inadequate so far. Then a facile method based on Fe3O4@SiO2-human serum albumin (Fe3O4@SiO2-HSA) magnetic solid phase fishing integrated with two-dimensional high-performance liquid chromatography-diode array detector-mass spectrometry (2D HPLC-DAD-MS(n)) was developed to fish out and identify HSA binders from Radix Astragali. The immobilized HSA displayed a high stability with 96.2% retained after ten consecutive cycles. 2D HPLC system (size exclusion chromatography×reversed phase chromatography, SEC×RP) were developed and optimised. Forty-seven bioactive compounds including thirty-four isoflavonoids and thirteen astragalosides were screened and identified or tentatively deduced based on their retention time, ultraviolet (UV), accurate molecular weight and diagnostic fragment ions. The results indicated that the integrated method could be widely applied for systematical fishing and identification of bioactive compounds, especially for low-abundance and overlapped compounds, from complex mixtures. Copyright © 2013 Elsevier Ltd. All rights reserved.

A simple, sensitive determination of ganciclovir in infant plasma by high-performance liquid chromatography with fluorescence detection.

PubMed

Yoshida, Terumitsu; Takahashi, Ryohei; Imai, Koichi; Uchida, Hiroshi; Arai, Yasutoshi; Oh-ishi, Tsutomu

2010-03-01

This study developed a simple and sensitive method using reversed-phase high-performance liquid chromatography (HPLC) for ganciclovir (GCV) plasma concentrations in cytomegalovirus infectious infants with hearing loss. The method involves a simple protein precipitation procedure that uses no solid-phase or liquid-liquid extraction. The HPLC separation was carried out on a Cadenza CD-C(18) column (3 microm, 4.6 mm x 150 mm) with phosphate buffer (pH 2.5, 25 mM) containing 1% methanol-acetonitrile mixture (4:3, v/v) as a mobile phase at a 0.7 mL/min flow rate. GCV was detected using a fluorescence detection (lambdaex/em: 265/380 nm). The quantification limit was 0.025 microg/mL for 100 microL of plasma sample at which good intra- and inter-assay coefficient of variation values (< 4.96%) and recoveries (94.9-96.5%) were established.

Simultaneous determination of related substances of telmisartan and hydrochlorothiazide in tablet dosage form by using reversed phase high performance liquid chromatographic method

PubMed Central

Mukhopadhyay, Sutirtho; Kadam, Kiran; Sawant, Laxman; Nachane, Dhanashree; Pandita, Nancy

2011-01-01

Objective: Telmisartan is a potent, long-lasting, nonpeptide antagonist of the angiotensin II type-1 (AT1) receptor that is indicated for the treatment of essential hypertension. Hydrochlorothiazide is a widely prescribed diuretic and it is indicated for the treatment of edema, control of essential hypertension and management of diabetes insipidus. In the current article a new, accurate, sensitive, precise, rapid, reversed phase high performance liquid chromatography (RP-HPLC) method was developed for determination of related substances of Telmisartan and Hydrochlorthiazide in tablet dosage form. Materials and Methods: Simultaneous determination of related substances was performed on Kromasil C18 analytical column (250 × 4.6 mm; 5μm pertical size) column at 40°C employing a gradient elution. Mobile phase consisting of solvent A (solution containing 2.0 g of potassium dihydrogen phosphate anhydrous and 1.04 g of Sodium 1- Hexane sulphonic acid monohydrate per liter of water, adjusted to pH 3.0 with orthophosphoric acid) and solvent B (mixture of Acetonitrile: Methanol in the ratio 80:20 v/v) was used at a flow rate of 1.0 ml min–1. UV detection was performed at 270 nm. Results: During method validation parameter such as precision, linearity, accuracy, specificity, limit of detection and quantification were evaluated, which remained within acceptable limits. Conclusions: HPLC analytical method is linear, accurate, precise, robust and specific, being able to separate the main drug from its degradation products. It may find application for the routine analysis of the related substances of both Telmisartan and Hydrochlorthiazide in this combination tablets. PMID:21966158

HPLC Separation of Sulforaphane Enantiomers in Broccoli and Its Sprouts by Transformation into Diastereoisomers Using Derivatization with (S)-Leucine.

PubMed

Okada, Makiko; Yamamoto, Atsushi; Aizawa, Sen-Ichi; Taga, Atsushi; Terashima, Hiroyuki; Kodama, Shuji

2017-01-11

Racemic sulforaphane, which was derivatized with (S)-leucine (l-leucine), was resolved by reversed phase HPLC with UV detection. The optimum mobile phase conditions were found to be 10 mM citric acid (pH 2.8) containing 22% methanol at 35 °C using detection at 254 nm. Sulforaphane enantiomers in florets and stems of five brands of broccoli and leaves and stems of three brands of broccoli sprouts were analyzed by the proposed HPLC method. Both sulforaphane enantiomers were detected in all of the samples. The S/R ratios of sulforaphane in broccoli samples were 1.5-2.6/97.4-98.5% for florets and 5.0-12.1/87.9-95.0% for stems. The S/R ratios in broccoli sprout samples were higher than those in broccoli samples and were found to be 8.3-19.7/80.3-91.7% for leaves and 37.0-41.8/58.2-63.0% for stems. (S)-Sulforaphane detected in the broccoli and its sprout samples was positively identified by separately using an HPLC with a chiral column (Chiralpak AD-RH) and mass spectrometry.

Monitoring gradient profile on-line in micro- and nano-high performance liquid chromatography using conductivity detection.

PubMed

Zhang, Min; Chen, Apeng; Lu, Joann J; Cao, Chengxi; Liu, Shaorong

2016-08-19

In micro- or nano-flow high performance liquid chromatography (HPLC), flow-splitters and gradient elutions are commonly used for reverse phase HPLC separations. When a flow splitter was used at a high split-ratio (e.g., 1000:1 or higher), the actual gradient may deviate away from the programmed gradient. Sometimes, mobile phase concentrations can deviate by as much as 5%. In this work, we noticed that the conductivity (σ) of a gradient decreased with the increasing organic-solvent fraction (φ). Based on the relationship between σ and φ, a method was developed for monitoring gradient profile on-line to record any deviations in these HPLC systems. The conductivity could be measured by a traditional conductivity detector or a capacitively coupled contactless conductivity detector (C(4)D). The method was applied for assessing the performance of an electroosmotic pump (EOP) based nano-HPLC. We also observed that σ value of the gradient changed with system pressure; a=0.0175ΔP (R(2)=0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated. Copyright © 2016. Published by Elsevier B.V.

Simultaneous preparation of naturally abundant and rare catechins by tannase-mediated biotransformation combining high speed counter current chromatography.

PubMed

Xia, Guobin; Hong, Shan; Liu, Songbai

2014-05-15

Simultaneous preparation of naturally rare catechins, EGC and EC, has been realized by tannase-mediated biotransformation combining high speed counter current chromatography. In addition, simultaneous preparation of the four catechins, EGCG, ECG, EGC, and EC in green tea extract has also been achieved by HSCCC under the normal phase and the reversed phase modes. The identity of the catechins was determined by HPLC-DAD-ESI-MS and quantification of the catechins was performed by HPLC-DAD. In a typical HSCCC separation, 27.2 mg 98.8% EGCG, 14.1 mg 94.7% EGC, and 9.3 mg 97.5% EC were obtained. This new method is efficient, time-saving and valuable for biological studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Strategy for Simultaneous Isolation of Less Polar Ginsenosides, Including a Pair of New 20-Methoxyl Isomers, from Flower Buds of Panax ginseng.

PubMed

Li, Sha-Sha; Li, Ke-Ke; Xu, Fei; Tao, Li; Yang, Li; Chen, Shu-Xiao; Gong, Xiao-Jie

2017-03-10

The present study was designed to simultaneously isolate the less polar ginsenosides from the flower buds of Panax ginseng (FBPG). Five ginsenosides, including a pair of new 20-methoxyl isomers, were extracted from FBPG and purified through a five-step integrated strategy, by combining ultrasonic extraction, Diaion Hp-20 macroporous resin column enrichment, solid phase extraction (SPE), reversed-phase high-performance liquid chromatography (RP-HPLC) analysis and preparation, and nuclear magnetic resonance (NMR) analysis. The quantification of the five ginsenosides was also discussed by a developed method with validations within acceptable limits. Ginsenoside Rg5 showed content of about 1% in FBPG. The results indicated that FBPG might have many different ginsenosides with diverse chemical structures, and the less polar ginsenosides were also important to the quality control and standardization of FBPG.

Measurement of the alpha4beta2* nicotinic acetylcholine receptor ligand 2-[(18)F]Fluoro-A-85380 and its metabolites in human blood during PET investigation: a methodological study.

PubMed

Sorger, Dietlind; Becker, Georg A; Patt, Marianne; Schildan, Andreas; Grossmann, Udo; Schliebs, Reinhard; Seese, Anita; Kendziorra, Kai; Kluge, Magnus; Brust, Peter; Mukhin, Alexey G; Sabri, Osama

2007-04-01

2-[(18)F]fluoro-A-85380 (2-[(18)F]FA) is a new radioligand for noninvasive imaging of alpha4beta2* nicotinic acetylcholine receptors (nAChRs) by positron emission tomography (PET) in human brain. In most cases, quantification of 2-[(18)F]FA receptor binding involves measurement of free nonmetabolized radioligand concentration in blood. This requires an efficient and reliable method to separate radioactive metabolites from the parent compound. In the present study, three analytical methods, thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and solid phase extraction (SPE) have been tested. Reversed-phase TLC of deproteinized aqueous samples of plasma provides good estimates of 2-[(18)F]FA and its metabolites. However, because of the decreased radioactivity in plasma samples, this method can be used in humans over the first 2 h after radioligand injection only. Reliable quantification of the parent radioligand and its main metabolites was obtained using reversed-phase HPLC, followed by counting of eluted fractions in a well gamma counter. Three main and five minor metabolites of 2-[(18)F]FA were detected in human blood using this method. On average, the unchanged 2-[(18)F]FA fraction in plasma of healthy volunteers measured at 14, 60, 120, 240 and 420 min after radioligand injection was 87.3+/-2.2%, 74.4+/-3%, 68.8+/-5%, 62.3+/-8% and 61.0+/-8%, respectively. In patients with neurodegenerative disorders, the values corresponding to the three last time points were significantly lower. The fraction of nonmetabolized 2-[(18)F]FA in plasma determined using SPE did not differ significantly from that obtained by HPLC (+gamma counting) (n=73, r=.95). Since SPE is less time-consuming than HPLC and provides comparable results, we conclude that SPE appears to be the most suitable method for measurement of 2-[(18)F]FA parent fraction during PET investigations.

Intracellular nucleotide and nucleotide sugar contents of cultured CHO cells determined by a fast, sensitive, and high-resolution ion-pair RP-HPLC.

PubMed

Kochanowski, N; Blanchard, F; Cacan, R; Chirat, F; Guedon, E; Marc, A; Goergen, J-L

2006-01-15

Analysis of intracellular nucleotide and nucleotide sugar contents is essential in studying protein glycosylation of mammalian cells. Nucleotides and nucleotide sugars are the donor substrates of glycosyltransferases, and nucleotides are involved in cellular energy metabolism and its regulation. A sensitive and reproducible ion-pair reverse-phase high-performance liquid chromatography (RP-HPLC) method has been developed, allowing the direct and simultaneous detection and quantification of some essential nucleotides and nucleotide sugars. After a perchloric acid extraction, 13 molecules (8 nucleotides and 5 nucleotide sugars) were separated, including activated sugars such as UDP-glucose, UDP-galactose, GDP-mannose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine. To validate the analytical parameters, the reproducibility, linearity of calibration curves, detection limits, and recovery were evaluated for standard mixtures and cell extracts. The developed method is capable of resolving picomolar quantities of nucleotides and nucleotide sugars in a single chromatographic run. The HPLC method was then applied to quantify intracellular levels of nucleotides and nucleotide sugars of Chinese hamster ovary (CHO) cells cultivated in a bioreactor batch process. Evolutions of the titers of nucleotides and nucleotide sugars during the batch process are discussed.

Conjugated linoleic acid-rich soy oil triacylglycerol identification.

PubMed

Lall, Rahul K; Proctor, Andrew; Jain, Vishal P; Lay, Jackson O

2009-03-11

Conjugated linoleic acid (CLA)-rich soy oil has been produced by soy oil linoleic acid (LA) photoisomerization, but CLA-rich oil triacylglycerol (TAG) characterization was not described. Therefore, the objectives were to identify and quantify new TAG fractions in CLA-rich oil by nonaqueous reversed-phase high-performance liquid chromatography (NARP-HPLC). Analytical NARP-HPLC with an acetonitrile/dichloromethane (ACN/DCM) gradient and an evaporating light scattering detector/ultraviolet (ELSD/UV) detector was used. New TAG peaks from LA-containing TAGs were observed. The LnLL, LLL, LLO, and LLP (Ln, linolenic; L, linoleic; O, oleic; and P, palmitic) peaks reduced after isomerization with an increase in adjacent peaks that coeluted with LnLnO, LnLO, LnOO, and LnPP. The newly formed peaks were wider than those of the original oil and absorbed at 233 nm, suggesting the possibility of various CLA containing TAGs. The HPLC profile showed five fractions of mixed TAGs, and fatty acid analysis showed that CLA isomers were found predominately in fractions 2 and 3, which originally contained most LA. The CLA isomers were 70-80% trans,trans and 20-30% cis,trans and trans,cis.

Demonstration and Evaluation of Solid Phase Microextraction for the Assessment of Bioavailability and Contaminant Mobility

DTIC Science & Technology

2012-05-01

methods demonstrated that desorption into solvents suitable for subsequent chemical analysis (into acetonitrile for HPLC analysis or hexane for GC...SPME. Analysis by HPLC with EPA 8310 with fluorescent detection. a) surface water quality criteria (NRWQC) are given for comparison to detection... analysis ) or hexane (for PCB analysis ) was added to the inserts. The vials were then analyzed directly by HPLC (PAHs) or GC-ECD (PCBs). Fiber achieved

Chemometrics-based Approach in Analysis of Arnicae flos

PubMed Central

Zheleva-Dimitrova, Dimitrina Zh.; Balabanova, Vessela; Gevrenova, Reneta; Doichinova, Irini; Vitkova, Antonina

2015-01-01

Introduction: Arnica montana flowers have a long history as herbal medicines for external use on injuries and rheumatic complaints. Objective: To investigate Arnicae flos of cultivated accessions from Bulgaria, Poland, Germany, Finland, and Pharmacy store for phenolic derivatives and sesquiterpene lactones (STLs). Materials and Methods: Samples of Arnica from nine origins were prepared by ultrasound-assisted extraction with 80% methanol for phenolic compounds analysis. Subsequent reverse-phase high-performance liquid chromatography (HPLC) separation of the analytes was performed using gradient elution and ultraviolet detection at 280 and 310 nm (phenolic acids), and 360 nm (flavonoids). Total STLs were determined in chloroform extracts by solid-phase extraction-HPLC at 225 nm. The HPLC generated chromatographic data were analyzed using principal component analysis (PCA) and hierarchical clustering (HC). Results: The highest total amount of phenolic acids was found in the sample from Botanical Garden at Joensuu University, Finland (2.36 mg/g dw). Astragalin, isoquercitrin, and isorhamnetin 3-glucoside were the main flavonol glycosides being present up to 3.37 mg/g (astragalin). Three well-defined clusters were distinguished by PCA and HC. Cluster C1 comprised of the German and Finnish accessions characterized by the highest content of flavonols. Cluster C2 included the Bulgarian and Polish samples presenting a low content of flavonoids. Cluster C3 consisted only of one sample from a pharmacy store. Conclusion: A validated HPLC method for simultaneous determination of phenolic acids, flavonoid glycosides, and aglycones in A. montana flowers was developed. The PCA loading plot showed that quercetin, kaempferol, and isorhamnetin can be used to distinguish different Arnica accessions. SUMMARY A principal component analysis (PCA) on 13 phenolic compounds and total amount of sesquiterpene lactones in Arnicae flos collection tended to cluster the studied 9 accessions into three main groups. The profiles obtained demonstrated that the samples from Germany and Finland are characterized by greater amounts of phenolic derivatives than the Bulgarian and Polish ones. The PCA loading plot showed that quercetin, kaemferol and isorhamnetin can be used to distinguish different arnica accessions. PMID:27013791

Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

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

King, L.C.; Gallagher, J.E.; Lewtas, J.

The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. Amore » second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.« less

Quantum Yields of Soluble Particulate Material in the Ocean

DTIC Science & Technology

1998-01-01

Mycosporine - like amino acids (MAAs) were separated by reverse-phase isocratic HPLC on a... Mycosporine - like Amino Acids in Phaeocystis antarctica Karsten. Marine Biology (1999b) (Submitted) Moisan, T.A., Olaizola, M. & Mitchell, B.G. Xanthophyll...monochromator and detector. We have carried out work to evaluate the role of photoprotective pigments including mycosporine amino acids and the

Simultaneous determination of eight major bioactive compounds in Dachengqi Tang (DT) by high-performance liquid chromatography

PubMed Central

Tang, Wenfu; Wan, Meihua; Zhu, Zhengyan; Chen, Guanyuan; Huang, Xi

2008-01-01

Background Dachengqi Tang (DT) is a common traditional Chinese medicine formula for expelling neire ('internal heat') in the stomach and intestines. There was no reliable analytical method available for the quality control of DT. Methods A high-performance liquid chromatography (HPLC) method with a reverse phase C18 column (150 × 4.6 mm) was developed. The mobile phase was methanol with 0.2% acetic acid. Eight markers including naringin, hesperidin, aloe emodin, rhein, honokiol, magnolol, emodin and chrysophanol were determined. Results Regression analysis revealed a linear relationship between the concentrations of the markers and the peak area ratio of the standards and internal standard. The limit of detection (S/N = 3) and the limit of qualification (RSD < 20%) ranged from 0.21 to 0.43 ng/μl and 0.76 to 1.74 ng/μl respectively. The recovery was between 95.6% and 103.4%. The tests on the samples from three batches of DT showed that the profiles of the markers did not vary significantly among batches. Conclusion A reliable HPLC method for simultaneous determination of the eight markers in DT was developed. PMID:18445276

Quantitation of fumonisin B1 and B2 in feed using FMOC pre-column derivatization with HPLC and fluorescence detection.

PubMed

Smith, Lori L; Francis, Kyle A; Johnson, Joseph T; Gaskill, Cynthia L

2017-11-01

Pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was determined to be effective for quantitation of fumonisins B 1 and B 2 in feed. Liquid-solid extraction, clean-up using immunoaffinity solid phase extraction chromatography, and FMOC-derivatization preceded analysis by reverse phase HPLC with fluorescence. Instrument response was unchanged in the presence of matrix, indicating no need to use matrix-matched calibrants. Furthermore, high method recoveries indicated calibrants do not need to undergo clean-up to account for analyte loss. Established method features include linear instrument response from 0.04-2.5µg/mL and stable derivatized calibrants over 7days. Fortified cornmeal method recoveries from 0.1-30.0μg/g were determined for FB 1 (75.1%-109%) and FB 2 (96.0%-115.2%). Inter-assay precision ranged from 1.0%-16.7%. Method accuracy was further confirmed using certified reference material. Inter-laboratory comparison with naturally-contaminated field corn demonstrated equivalent results with conventional derivatization. These results indicate FMOC derivatization is a suitable alternative for fumonisins B 1 and B 2 quantitation in corn-based feeds. Copyright © 2017 Elsevier Ltd. All rights reserved.

Use of itaconic acid-based polymers for solid-phase extraction of deoxynivalenol and application to pasta analysis.

PubMed

Pascale, Michelangelo; De Girolamo, Annalisa; Visconti, Angelo; Magan, Naresh; Chianella, Iva; Piletska, Elena V; Piletsky, Sergey A

2008-02-25

Molecular modelling and computational design were used to identify itaconic acid (IA) as a functional monomer with high affinity towards deoxynivalenol (DON), a Fusarium-toxin frequently occurring in cereals. IA-based polymers were photochemically synthesised in dimethyl formamide (porogen) using ethylenglycol dimethacrylate as cross-linker and 1,1'-azo-bis(cyclohexane carbonitrile) as initiator, and the relevant binding interactions with DON in solvents with different polarity were investigated. The performances of the non-imprinted IA-based polymer (blank polymer, BP) and the corresponding molecularly imprinted polymer (MIP) were compared using DON as a template. Both BP and MIP were able to bind about 90% DON either in toluene, water or water containing 5% polyethylene glycol. Non-imprinted polymers with different molar ratios of IA to cross-linker were evaluated as adsorbents for solid-phase extraction (SPE) clean-up and pre-concentration of DON from wheat and pasta samples prior to HPLC analysis. Samples were extracted with PBS/0.1M EDTA solution and cleaned up through a cartridge containing blank IA-based polymer. The column was washed with PBS (pH 9.2) and the toxin was eluted with methanol and quantified by reversed-phase HPLC with UV detector (lambda=220nm), using methanol:water:acetic acid (15:85:0.1, v/v/v) as the mobile phase. Effective removal of matrix interferences was observed only for pasta with DON recoveries higher than 70% (RSD<7%, n=3) at levels close to or higher than EU regulatory limit.

Development and Validation of a Reversed-Phase Chiral HPLC Method to Determine the Chiral Purity of Bulk Batches of (S)-Enantiomer in Afoxolaner.

PubMed

Padivitage, Nilusha; Kumar, Satish; Rustum, Abu

2017-01-01

Afoxolaner is a new antiparasitic molecule from the isoxazoline family that acts on insect acarine g-aminobutyric acid and glutamate receptors. Afoxolaner is a racemic mixture, which has a chiral center at the isoxazoline ring. A reversed-phase chiral HPLC method has been developed to determine the chiral purity of bulk batches of (S)-enantiomer in afoxolaner for the first time. This method can also be used to verify that afoxolaner is a racemic mixture, which was demonstrated by specific rotation. ChromSword, an artificial intelligence method development tool, was used for initial method development. The column selected for the final method was CHIRALPAK AD-RH (150 × 4.6 mm, 5 μm particle size), maintained at 45°C, and isocratic elution using water-isopropanol-acetonitrile (40 + 50 + 10, v/v/v) as the mobile phase with a detection wavelength of 312 nm. The run time for the method was 11 min. The resolution and selectivity factors of the two enantiomers were 2.3 and 1.24, respectively. LOQ and LOD of the method were 1.6 and 0.8 μg/mL, respectively. This method was appropriately validated according to International Conference on Harmonization guidelines for its intended use.

[Simultaneous Determination of Three Kinds of Effective Constituents in Cannabis Plants by Reversed-phase HPLC].

PubMed

Fu, Q; Shu, Z; Deng, K; Luo, X; Zeng, C G

2016-08-01

To establish a high performance liquid chromatographic （HPLC） method for simultaneous determination of three effective constituents, including tetrahydrocannabinol （THC）, cannabidiol （CBD） and cannabinol （CBN） in Cannabis plants. A C₁₈ column was used in this study, and acetonitrile-phosphate buffer （0.015 mol/L KH₂PO₄） was used as mobile phase at a flow rate of 1.0 mL/min. At a detection wavelength of 220 mm, UV absorption spectra were collected at the wavelength range of 190-400 nm, and the spectra and retention time were counted as qualitative evidence. THC, CBD and CBN could be well separated by this method. Three components had good linear relationship in the range of 0.4-40 μg/mL （ R ²≥0.999 3）. The recoveries were over 87%. The limits of detection were 1.8 ng, 2.0 ng and 1.3 ng, respectively. The relative standard deviation （RSD） were less than 5% for both inter-day and intra-day precisions. Reversed-phase HPLC method is simple, rapid and accurate, and it is suitable for the qualitative and quantitative detection of THC, CBD and CBN in Cannabis plants. Copyright© by the Editorial Department of Journal of Forensic Medicine

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.

High-performance liquid chromatography of quinoidal imminium compounds derived from triphenylmethanes

USGS Publications Warehouse

Abidi, S.L.

1983-01-01

A series of eleven p-aminotriphenylmethane dyes have been studied by high-performance liquid chromatography (HPLC). The combined use of HPLC and spectrophotometry permits specific detection of these compounds in the visible range around 600 nm. As the high affinity of the imminium cations for the active sites of the hydrocarbonaceous stationary phase has presented difficulties for reversed-phase HPLC with pure solvents, organic electrolytes were added to the mobile phase to facilitate the elution of the components with improved selectivity, sensitivity (minimum detection limit, 0.1 μg/ml), and peak symmetry. The effects of chromatographic variables on the component retentivity were investigated. Retention times of the dye analytes decreased with increasing concentration of the added ionic reagent and with decreasing number of the hydrophobic alkyl substituents on the nitrogen atom. The influence of pH on the retention parameters appears to parallel that observed previously for cationic quaternary ammonium compounds. Among the acidic reagents employed, naphthalenesulfonic acid yielded the most satisfactory results. The use of binary electrolyte systems invariably improved the chromatographic behavior of the imminium solutes analyzed. Results obtained with two different octadecylsilica columns have been compared.

Simultaneous analysis of hydrochlorothiazide, triamterene and reserpine in rat plasma by high performance liquid chromatography and tandem solid-phase extraction.

PubMed

Li, Hang; He, Junting; Liu, Qin; Huo, Zhaohui; Liang, Si; Liang, Yong

2011-03-01

A tandem solid-phase extraction method (SPE) of connecting two different cartridges (C(18) and MCX) in series was developed as the extraction procedure in this article, which provided better extraction yields (>86%) for all analytes and more appropriate sample purification from endogenous interference materials compared with a single cartridge. Analyte separation was achieved on a C(18) reversed-phase column at the wavelength of 265 nm by high-performance liquid chromatography (HPLC). The method was validated in terms of extraction yield, precision and accuracy. These assays gave mean accuracy values higher than 89% with RSD values that were always less than 3.8%. The method has been successfully applied to plasma samples from rats after oral administration of target compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparing the selectivity and chiral separation of d- and l- fluorenylmethyloxycarbonyl chloride protected amino acids in analytical high performance liquid chromatography and supercritical fluid chromatography; evaluating throughput, economic and environmental impact.

PubMed

Vera, C M; Shock, D; Dennis, G R; Farrell, W; Shalliker, R A

2017-04-14

The chiral separation of d- and l- FMOC amino acids was undertaken using the Lux Cellulose-1 polysaccharide based chiral column in HPLC (normal phase and reverse phase) and SFC conditions. This was done to compare the relative selectivity and separation between the three separation modes and to evaluate the potential benefits of SFC separations with regards to resolution, throughput, economic and environmental impact. It was established that the separation of d- and l- FMOC amino acids in SFC displayed behaviours that were similar to both normal phase and reversed phase, rather than distinctly one or the other. Additionally, although reversed phase conditions yielded significantly higher resolution values between enantiomers across the range of amino acids studied, improvements in selectivity in SFC via the introduction of higher concentrations of formic acid in the mobile phase allowed for better resolution per unit of time. Moreover since the SFC mobile phase is composed mostly of recyclable CO 2 , there is a reduction in organic solvent consumption, which minimises the economic and environmental costs. Copyright © 2017. Published by Elsevier B.V.

Characterization of biliary conjugates of 4,4'-methylenedianiline in male versus female rats

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

Chen, Kan; Cole, Richard B.; Santa Cruz, Vicente

2008-10-15

4,4'-Methylenedianiline (4,4'-diaminodiphenylmethane; DAPM) is an aromatic diamine used in the production of numerous polyurethane foams and epoxy resins. Previous studies in rats revealed that DAPM initially injures biliary epithelial cells of the liver, that the toxicity is greater in female than in male rats, and that the toxic metabolites of DAPM are excreted into bile. Since male and female rats exhibit differences in the expression of both phase I and phase II enzymes, our hypothesis was that female rats either metabolize DAPM to more toxic metabolites or have a decreased capacity to conjugate metabolites to less toxic intermediates. Our objectivemore » was thus to isolate, characterize, and quantify DAPM metabolites excreted into bile in both male and female bile duct-cannulated Sprague Dawley rats. The rats were gavaged with [{sup 14}C]-DAPM, and the collected bile was subjected to reversed-phase HPLC with radioisotope detection. Peaks eluting from HPLC were collected and analyzed using electrospray MS and NMR spectroscopy. HPLC analysis indicated numerous metabolites in both sexes, but male rats excreted greater amounts of glutathione and glucuronide conjugates than females. Electrospray MS and NMR spectra of HPLC fractions revealed that the most prominent metabolite found in bile of both sexes was a glutathione conjugate of an imine metabolite of a 4'-nitroso-DAPM. Seven other metabolites were identified, including acetylated, cysteinyl-glycine, glutamyl-cysteine, glycine, and glucuronide conjugates. While our prior studies demonstrated increased covalent binding of DAPM in the liver and bile of female compared to male rats, in these studies, SDS-PAGE with autoradiography revealed 4-5 radiolabeled protein bands in the bile of rats treated with [{sup 14}C]-DAPM. In addition, these bands were much more prominent in female than in male rats. These studies thus suggest that a plausible mechanism for the increased sensitivity of female rats to DAPM toxicity may be decreased conjugation of reactive DAPM metabolites, leading to greater levels of protein adduct formation.« less

Quantitative analysis of ginger components in commercial products using liquid chromatography with electrochemical array detection

PubMed Central

Shao, Xi; Lv, Lishuang; Parks, Tiffany; Wu, Hou; Ho, Chi-Tang; Sang, Shengmin

2010-01-01

For the first time, a sensitive reversed-phase HPLC electrochemical array method has been developed for the quantitative analysis of eight major ginger components ([6]-, [8]-, and [10]-gingerol, [6]-, [8]-, and [10]-shogaol, [6]-paradol, and [1]-dehydrogingerdione) in eleven ginger-containing commercial products. This method was valid with unrivaled sensitivity as low as 7.3 – 20.2 pg of limit of detection and a range of 14.5 to 40.4 pg of limit of quantification. Using this method, we quantified the levels of eight ginger components in eleven different commercial products. Our results found that both levels and ratios among the eight compounds vary greatly in commercial products. PMID:21090746

Chiral ligand exchange high-speed countercurrent chromatography: mechanism, application and comparison with conventional liquid chromatography in enantioseparation of aromatic α-hydroxyl acids

PubMed Central

Tong, Shengqiang; Shen, Mangmang; Cheng, Dongping; Ito, Yoichiro; Yan, Jizhong

2014-01-01

This work concentrates on the separation mechanism and application of chiral ligand exchange high-speed countercurrent chromatography (HSCCC) in enantioseparations, and comparison with traditional chiral ligand exchange high performance liquid chromatography (HPLC). The enantioseparation of ten aromatic α-hydroxyl acids were performed by these two chromatographic methods. Results showed that five of the racemates were successfully enantioseparated by HSCCC while only three of the racemates could be enantioseparated by HPLC using a suitable chiral ligand mobile phase additive. For HSCCC, the two-phase solvent system was composed of butanol-water (1:1, v/v), to which N-n-dodecyl-L-proline was added in the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transition metal ion. Various operation parameters in HSCCC were optimized by enantioselective liquid-liquid extraction. Based on the results of the present studies the separation mechanism for HSCCC was proposed. For HPLC, the optimized mobile phase composed of aqueous solution containing 6 mmol L−1 L-phenylalanine and 3 mmol L−1 cupric sulfate and methanol was used for enantioseparation. Among three ligands tested on a conventional reverse stationary phase column, only one was found to be effective. In the present studies HSCCC presented unique advantages due to its high versatility of two-phase solvent systems and it could be used as an alternative method for enantioseparations. PMID:25087742

Urea, the most abundant component in urine, cross-reacts with a commercial 8-OH-dG ELISA kit and contributes to overestimation of urinary 8-OH-dG.

PubMed

Song, Ming-Fen; Li, Yun-Shan; Ootsuyama, Yuko; Kasai, Hiroshi; Kawai, Kazuaki; Ohta, Masanori; Eguchi, Yasumasa; Yamato, Hiroshi; Matsumoto, Yuki; Yoshida, Rie; Ogawa, Yasutaka

2009-07-01

Urinary 8-OH-dG is commonly analyzed as a marker of oxidative stress. For its analysis, ELISA and HPLC methods are generally used, although discrepancies in the data obtained by these methods have often been discussed. To clarify this problem, we fractionated human urine by reverse-phase HPLC and assayed each fraction by the ELISA method. In addition to the 8-OH-dG fraction, a positive reaction was observed in the first eluted fraction. The components in this fraction were examined by the ELISA. Urea was found to be the responsible component in this fraction. Urea is present in high concentrations in the urine of mice, rats, and humans, and its level is influenced by many factors. Therefore, certain improvements, such as a correction based on urea content or urease treatment, are required for the accurate analysis of urinary 8-OH-dG by the ELISA method. In addition, performance of the ELISA at 4 degrees C reduced the recognition of urea considerably and improved the 8-OH-dG analysis.

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples.

PubMed

Fulcrand, Hélène; Mané, Carine; Preys, Sébastien; Mazerolles, Gérard; Bouchut, Claire; Mazauric, Jean-Paul; Souquet, Jean-Marc; Meudec, Emmanuelle; Li, Yan; Cole, Richard B; Cheynier, Véronique

2008-12-01

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis. Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection.

PubMed

Kand'ár, Roman; Záková, Pavla; Jirosová, Jana; Sladká, Michaela

2009-01-01

The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], alpha-keto acids derived from BCAA [BCKA; alpha-ketoisovaleric acid (KIV), alpha-ketoisocaproic acid (KIC), alpha-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples. Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27-41 years of age and 35 males, 28-43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3-5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 microm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5+/-0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol. Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%-110%. We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Isolation and purification of series bioactive components from Hypericum perforatum L. by counter-current chromatography.

PubMed

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Xu, Chunming; Ito, Yoichiro

2011-03-01

Counter-current chromatography (CCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate-water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, ¹HNMR and ¹³CNMR. Copyright © 2011 Elsevier B.V. All rights reserved.

Isolation and purification of series bioactive components from Hypericum Perforatum L. by high-speed counter-current chromatography

PubMed Central

Cao, Xueli; Wang, Qiaoe; Li, Yan; Bai, Ge; Ren, Hong; Ito, Yiochiro

2011-01-01

High-speed counter-current chromatography (HSCCC) combined with pre-separation by ultrasonic solvent extraction was successively used for the separation of series bioactive compounds from the crude extract of Hypericum perforatum L. The petroleum ether extract was separated by the solvent system of n-heptane-methanol-acetonitrile (1.5:0.5:0.5, v/v) and n-heptane-methanol (1.5:1, v/v) in gradient elution, yielding a phloroglucinol compound, hyperforin with HPLC purity over 98%. The ethyl acetate extract was separated by using the solvent system composed of hexane-ethyl acetate-methanol-water (1:1:1:1 and 1:3:1:3, v/v) in gradient through both reverse phase and normal phase elution mode, yielding a naphthodianthrone compound, hypericin with HPLC purity about 95%. The n-butanol extract was separated with the solvent system composed of n-butanol-ethyl acetate–water (1:4:5 and 1.5:3.5:5, v/v) in elution and back-extrusion mode, yielding two of flavones, rutin and hyperoside, with HPLC purity over 95%. HPLC-MS, reference sample and UV spectrum were selectively used in separation to search for target compounds from HPLC-DAD profiles of different sub-extracts. The structures of isolated compounds were further identified by ESI-MS, 1HNMR and 13CNMR. PMID:21306961

Purification and partial characterization of analogous 26-kDa rat submandibular and parotid gland integral membrane phosphoproteins that may have a role in exocytosis.

PubMed

Quissell, D O; Deisher, L M

1992-04-01

Rat submandibular and parotid gland exocytosis is primarily controlled by beta-adrenergic receptor stimulation. Although its precise role in the regulation of salivary gland exocytosis is not fully understood, protein phosphorylation, mediated by the activation of cAMP-dependent protein kinase, may be directly involved. Previous studies suggest that analogous 26-kDa integral membrane phosphoproteins may play a direct role in regulating exocytosis. Studies were here undertaken to purify and partially characterize both phosphoproteins. After endogenous phosphorylation with 32P, subcellular fraction and solubilization of the microsomal fraction in n-octyl beta-glucopyranoside, the 26-kDa integral membrane phosphoproteins were purified by high performance liquid chromatography (HPLC), followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroelution of the proteins. Amino acid analysis indicated a significant number of serine amino acids: N-terminal sequence data demonstrated a high level of homology; and trypsin digestion followed by reversed-phase HPLC indicated the possibility of multiple phosphorylation sites.

A convenient method for the quantitative determination of elemental sulfur in coal by HPLC analysis of perchloroethylene extracts

USGS Publications Warehouse

Buchanan, D.H.; Coombs, K.J.; Murphy, P.M.; Chaven, C.

1993-01-01

A convenient method for the quantitative determination of elemental sulfur in coal is described. Elemental sulfur is extracted from the coal with hot perchloroethylene (PCE) (tetrachloroethene, C2Cl4) and quantitatively determined by HPLC analysis on a C18 reverse-phase column using UV detection. Calibration solutions were prepared from sublimed sulfur. Results of quantitative HPLC analyses agreed with those of a chemical/spectroscopic analysis. The HPLC method was found to be linear over the concentration range of 6 ?? 10-4 to 2 ?? 10-2 g/L. The lower detection limit was 4 ?? 10-4 g/L, which for a coal sample of 20 g is equivalent to 0.0006% by weight of coal. Since elemental sulfur is known to react slowly with hydrocarbons at the temperature of boiling PCE, standard solutions of sulfur in PCE were heated with coals from the Argonne Premium Coal Sample program. Pseudo-first-order uptake of sulfur by the coals was observed over several weeks of heating. For the Illinois No. 6 premium coal, the rate constant for sulfur uptake was 9.7 ?? 10-7 s-1, too small for retrograde reactions between solubilized sulfur and coal to cause a significant loss in elemental sulfur isolated during the analytical extraction. No elemental sulfur was produced when the following pure compounds were heated to reflux in PCE for up to 1 week: benzyl sulfide, octyl sulfide, thiane, thiophene, benzothiophene, dibenzothiophene, sulfuric acid, or ferrous sulfate. A sluury of mineral pyrite in PCE contained elemental sulfur which increased in concentration with heating time. ?? 1993 American Chemical Society.

Industrial application of green chromatography--I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase.

PubMed

Yang, Yu; Strickland, Zackary; Kapalavavi, Brahmam; Marple, Ronita; Gamsky, Chris

2011-03-15

In this work, chromatographic separation of niacin and niacinamide using pure water as the sole component in the mobile phase has been investigated. The separation and analysis of niacinamide have been optimized using three columns at different temperatures and various flow rates. Our results clearly demonstrate that separation and analysis of niacinamide from skincare products can be achieved using pure water as the eluent at 60°C on a Waters XTerra MS C18 column, a Waters XBridge C18 column, or at 80°C on a Hamilton PRP-1 column. The separation efficiency, quantification quality, and analysis time of this new method are at least comparable with those of the traditional HPLC methods. Compared with traditional HPLC, the major advantage of this newly developed green chromatography technique is the elimination of organic solvents required in the HPLC mobile phase. In addition, the pure water chromatography separations described in this work can be directly applied in industrial plant settings without further modification of the existing HPLC equipment. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Application of cyanuric chloride-based six new chiral derivatizing reagents having amino acids and amino acid amides as chiral auxiliaries for enantioresolution of proteinogenic amino acids by reversed-phase high-performance liquid chromatography.

PubMed

Bhushan, Ravi; Dixit, Shuchi

2012-04-01

Six dichloro-s-triazine (DCT) reagents having L-Leu, D-Phg, L-Val, L-Met, L-Ala and L-Met-NH(2) as chiral auxiliaries in cyanuric chloride were introduced for enantioseparation of 13 proteinogenic amino acids. Four other DCTs and six monochloro-s-triazine (MCT) reagents having amino acid amides as chiral auxiliaries were also synthesized. These 16 chiral derivatizing reagents (CDRs) were used for synthesis of diastereomers of all the 13 analytes using microwave irradiation, which were resolved by reversed-phase high-performance liquid chromatography (RP-HPLC) using C18 column and gradient eluting mixture of aqueous TFA and acetonitrile with UV detection at 230 nm. It required only 60-90 s for derivatization using microwave irradiation. Better resolution and lower retention times were observed for the diastereomers prepared with CDRs having amino acids as chiral auxiliaries as compared to counterparts prepared with reagents having amino acid amides as chiral auxiliaries. As the best resolution of all the 13 analytes was observed for their diastereomers prepared using the DCT reagent having L-Leu as chiral auxiliary, this CDR was further employed for derivatization of Lys, Tyr, His and Arg followed by RP-HPLC analysis of resulting diastereomers. The results are discussed in light of acid and amide groups of chiral auxiliaries constituting CDRs, electronegativities of the atoms of achiral moieties constituting CDRs and hydrophobicities of side chains of amino acids constituting CDRs and analytes.

Influence of variation in mobile phase pH and solute pK(a) with the change of organic modifier fraction on QSRRs of hydrophobicity and RP-HPLC retention of weakly acidic compounds.

PubMed

Han, Shu-ying; Liang, Chao; Zou, Kuan; Qiao, Jun-qin; Lian, Hong-zhen; Ge, Xin

2012-11-15

The variation in mobile phase pH and ionizable solute dissociation constant (pK(a)) with the change of organic modifier fraction in hydroorganic mobile phase has seemingly been a troublesome problem in studies and applications of reversed phase high performance liquid chromatography (RP-HPLC). Most of the early studies regarding the RP-HPLC of acid-base compounds have to measure the actual pH of the mixed mobile phase rigorously, sometimes bringing difficulties in the practices of liquid chromatographic separation. In this paper, the effect of this variation on the apparent n-octanol/water partition coefficient (K(ow)″) and the related quantitative structure-retention relationship (QSRR) of logK(ow)″ vs. logk(w), the logarithm of retention factor of analytes in neat aqueous mobile phases, was investigated for weakly acidic compounds. This QSRR is commonly used as a classical method for K(ow) measurement by RP-HPLC. The theoretical and experimental derivation revealed that the variation in mobile phase pH and solute pK(a) will not affect the QSRRs of acidic compounds. This conclusion is proved to be suitable for various types of ion-suppressors, i.e., strong acid (perchloric acid), weak acid (acetic acid) and buffer salt (potassium dihydrogen phosphate/phosphoric acid, PBS). The QSRRs of logK(ow)″ vs. logk(w) were modeled by 11 substituted benzoic acids using different types of ion-suppressors in a binary methanol-water mobile phase to confirm our deduction. Although different types of ion-suppressor all can be used as mobile phase pH modifiers, the QSRR model obtained by using perchloric acid as the ion-suppressor was found to have the best result, and the slightly inferior QSRRs were obtained by using acetic acid or PBS as the ion-suppressor. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation and identification of three potential impurities of pholcodine bulk drug substance.

PubMed

Denk, O M; Gray, A I; Skellern, G G; Watson, D G

2000-07-01

Three previously unreported manufacturing impurities were isolated from a pholcodine mother liquor using preparative reversed-phase HPLC. The liquor was the residue remaining after recrystallisation of a production batch of pholcodine. The impurities, which are structurally related to pholcodine, were initially detected by thin-layer chromatography (TLC). Their structures were determined after separation by preparative HPLC (Econo-Prep 5 microm C18 column, 30 cm x 21.2 mm i.d.). Structure elucidation was carried out using nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and ultra violet (UV) spectroscopy. The impurities were identified as alkylated derivatives of pholcodine possessing second 2-morpholinoethyl substituents at various positions.

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.

Antimicrobial activity of Marcetia DC species (Melastomataceae) and analysis of its flavonoids by reverse phase-high performance liquid chromatography coupled-diode array detector

PubMed Central

Leite, Tonny Cley Campos; de Sena, Amanda Reges; dos Santos Silva, Tânia Regina; dos Santos, Andrea Karla Almeida; Uetanabaro, Ana Paula Trovatti; Branco, Alexsandro

2012-01-01

Background: Marcetia genera currently comprises 29 species, with approximately 90% inhabiting Bahia (Brazil), and most are endemic to the highlands of the Chapada Diamantina (Bahia). Among the species, only M. taxifolia (A.St.-Hil.) DC. populates Brazil (state of Roraima to Paraná) and also Venezuela, Colombia, and Guyana. Objective: This work evaluated the antimicrobial activity of hexane, ethyl acetate, and methanol extracts of three species of Marcetia (Marcetia canescens Naud., M. macrophylla Wurdack, and M. taxifolia A.StHil) against several microorganism. In addition, the flavonoids were analyzed in extracts by HPLC-DAD. Materials and methods: The tests were made using Gram-positive (three strains of Staphylococcus aureus) and Gram-negative (two strains of Escherichia coli, a strain of Pseudomonas aeruginosa and another of Salmonella choleraesius) bacteria resistant and nonresistant to antibiotics and yeasts (two strains of Candida albicans and one of C. parapsilosis) by the disk diffusion method. Solid-phase extraction (SPE) was performed on the above extracts to isolate flavonoids, which were subsequently analyzed by high performance liquid chromatography coupled diode array detector (HPLC-DAD). Results: Results showed that extracts inhibited the Gram-positive bacteria and yeast. The hexane extracts possessed the lowest activity, while the ethyl acetate and methanolic extracts were more active. Conclusion: Marcetia taxifolia was more effective (active against 10 microorganisms studied), and only its methanol extract inhibited Gram-negative bacteria (P. aeruginosa and S. choleraesius). SPE and HPLC-DAD analysis showed that M. canescens and M. macrophylla contain glycosylated flavonoids, while the majority of extracts from M. taxifolia were aglycone flavonoids. PMID:23060695

Determination of some phenolic compounds in red wine by RP-HPLC: method development and validation.

PubMed

Burin, Vívian Maria; Arcari, Stefany Grützmann; Costa, Léa Luzia Freitas; Bordignon-Luiz, Marilde T

2011-09-01

A methodology employing reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for simultaneous determination of five phenolic compounds in red wine. The chromatographic separation was carried out in a C(18) column with water acidify with acetic acid (pH 2.6) (solvent A) and 20% solvent A and 80% acetonitrile (solvent B) as the mobile phase. The validation parameters included: selectivity, linearity, range, limits of detection and quantitation, precision and accuracy, using an internal standard. All calibration curves were linear (R(2) > 0.999) within the range, and good precision (RSD < 2.6%) and recovery (80-120%) was obtained for all compounds. This method was applied to quantify phenolics in red wine samples from Santa Catarina State, Brazil, and good separation peaks for phenolic compounds in these wines were observed.

Paired-ion chromatography and high performance liquid chromatography of labetalol in feeds.

PubMed

Townley, E R; Ross, B

1980-11-01

A high performance liquid chromatographic (HPLC) method using reverse phase paired-ion chromatography and ultraviolet detection at 280 nm has been developed to determine labetalol, an alpha and beta adrenoceptor blocking agent, in Purina No. 5001 rodent chow. The method is simple and rapid, and demonstrates a separation technique applicable to other acidic and basic drugs. It requires only extraction of the drug with methanol--water--acetic acid (66 + 33 + 1) and separation of insoluble material by filtration before HPLC. Labetalol, is chromatographically separated from soluble feed components by means of a microBondapak C18 column and methanol--water--acetic acid (66 + 33 + 1) mobile phase, 0.005M with respect to sodium dioctylsulfosuccinate paired-ion reagent. Average recovery is 98.7% with a relative standard deviation of +/- 2.3% for the equipment described.

Demonstration and Evaluation of Solid Phase Microextraction for the Assessment of Bioavailability and Contaminant Mobility. ESTCP Cost and Performance Report

DTIC Science & Technology

2012-08-01

subsequent chemical analysis (into acetonitrile for high-performance liquid chromatography [ HPLC ] analysis or hexane for gas chromatography [GC... analysis ) is rapid and complete. In this work, PAHs were analyzed by Waters 2795 HPLC with fluorescent detection (USEPA Method 8310) and PCBs were...detection limits by direct water injection versus SPME with PDMS and coefficient of variation and correlation coefficient for SPME. Analysis by HPLC

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

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

Smith, L.I.; Bodwell, J.E.; Mendel, D.B.

1988-05-17

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. The authors have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with (/sup 3/H)dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single (/sup 3/H)dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the (/sup 3/H)dexamethasone 21-mesylate was located at position 5 frommore » the amino terminus. Dual-isotope labeling studies with (/sup 3/H)dexamethasone 21-mesylate and (/sup 35/S)methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of (/sup 3/H)dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, their data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate. They have confirmed this finding by demonstrating that a synthetic peptide representing the amino acid sequence 640-650 of the murine glucocorticoid receptor behaves in an identical manner on reversed-phase HPLC as the trypsin-generated peptide from intact cells.« less

[Pharmacokinetics of phenolic antioxidant 4-methyl-2,6-diisobornylphenol upon intravenous injection].

PubMed

Chernyshova, G A; Smol'iakova, V I; Plotnikov, M B; Ianovskaia, E A; Gurto, R V; Udut, V V; Kuchin, A V; Chukicheva, I Iu

2011-01-01

The pharmacokinetics of 4-methyl-2,6-diisobornylphenol (MDIBP) in rat blood plasma has been studied after intravenous injection. The drug concentration in the plasma was determined using a reverse-phase HPLC procedure. It is shown that MDIBP rapidly penetrates into intensively perfused organs, but is slowly eliminated from the organism (MRT value amounting to 9 h).

18, 19-Dihydroxycorticosterone: a new metabolite in human urine.

PubMed

Godzsa, J; Vecsei, P; Iwuanyanwu, T; Harnik, M

1989-01-01

Urines of patients with primary aldosteronism were extracted, the extract repeatedly chromatographed with reversed phase HPLC. The fractions immunoactive against 18-hydroxycorticosterone antiserum and being more polar than the 18-hydroxycorticosterone were further purified, derivatized and investigated by GC/MS. In this manner the natural occurrence of the 18, 19-dihydroxycorticosterone, which was lately synthetized, in human urine was confirmed.

Quantitative determination of triterpene saponins and alkenated-phenolics from Labisia pumila using LC-UV/ELSD method and confirmation by LC-ESI-TOF

USDA-ARS?s Scientific Manuscript database

This study describes the first analytical method for the determination of saponins and alkenated-phenolics from the leaves, leaves/stems and roots of Labisia pumila using a HPLC-UV-ELSD method. The separation was achieved using a reversed phase column, PDA and ELS detection, and a water/acetonitrile...

Quantitative determination of triperpene saponins and alkenated-phenolics from Labisia pumila using LC-UV/ELSD method and confirmation by LC-ESI-TOF

USDA-ARS?s Scientific Manuscript database

This study describes the first analytical method for the determination of saponins and alkenated-phenolics from the leaves, leaves/stems and roots of Labisia pumila using a HPLC-UV-ELSD method. The separation was achieved using a reversed phase column, PDA and ELS detection, and a water/acetonitrile...

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.

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

PubMed

Krstanović, Marina; Frkanec, Ruza; Vranesić, Branka; Ljevaković, Durdica; Sporec, Vesna; Tomasić, Jelka

2002-06-25

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

Stress Degradation Studies on Varenicline Tartrate and Development of a Validated Stability-Indicating HPLC Method

PubMed Central

Pujeri, Sudhakar S.; Khader, Addagadde M. A.; Seetharamappa, Jaldappagari

2012-01-01

A simple, rapid and stability-indicating reversed-phase liquid chromatographic method was developed for the assay of varenicline tartrate (VRT) in the presence of its degradation products generated from forced decomposition studies. The HPLC separation was achieved on a C18 Inertsil column (250 mm × 4.6 mm i.d. particle size is 5 μm) employing a mobile phase consisting of ammonium acetate buffer containing trifluoroacetic acid (0.02M; pH 4) and acetonitrile in gradient program mode with a flow rate of 1.0 mL min−1. The UV detector was operated at 237 nm while column temperature was maintained at 40 °C. The developed method was validated as per ICH guidelines with respect to specificity, linearity, precision, accuracy, robustness and limit of quantification. The method was found to be simple, specific, precise and accurate. Selectivity of the proposed method was validated by subjecting the stock solution of VRT to acidic, basic, photolysis, oxidative and thermal degradation. The calibration curve was found to be linear in the concentration range of 0.1–192 μg mL−1 (R2 = 0.9994). The peaks of degradation products did not interfere with that of pure VRT. The utility of the developed method was examined by analyzing the tablets containing VRT. The results of analysis were subjected to statistical analysis. PMID:22396908

New Stability Indicating RP-HPLC Method for the Estimation of Cefpirome Sulphate in Bulk and Pharmaceutical Dosage Forms.

PubMed

Rao, Kareti Srinivasa; Kumar, Keshar Nargesh; Joydeep, Datta

2011-01-01

A simple stability indicating reversed-phase HPLC method was developed and subsequently validated for estimation of Cefpirome sulphate (CPS) present in pharmaceutical dosage forms. The proposed RP-HPLC method utilizes a LiChroCART-Lichrosphere100, C18 RP column (250 mm × 4mm × 5 μm) in an isocratic separation mode with mobile phase consisting of methanol and water in the proportion of 50:50 % (v/v), at a flow rate 1ml/min, and the effluent was monitored at 270 nm. The retention time of CPS was 2.733 min and its formulation was exposed to acidic, alkaline, photolytic, thermal and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The described method was linear over a range of 0.5-200μg/ml. The percentage recovery was 99.46. F-test and t-test at 95% confidence level were used to check the intermediate precision data obtained under different experimental setups; the calculated value was found to be less than the critical value.

Precolumn Derivatization with Bromine to Improve Separation and Detection Sensitivity of Triacylglycerols in Edible Oil by Reversed-Phase High Performance Liquid Chromatography.

PubMed

Shan, Xiao-Lin; Liu, Xiao-Ting; Gong, Can; Xu, Xu

2018-01-01

The complexity of triacylglycerols (TAGs) in edible oils is largely due to the many similar unsaturated TAG compounds, which makes profiling TAGs difficult. In this study, precolumn derivatization with bromine (Br 2 ) was used to improve the separation and detection sensitivity of TAGs in edible oils by RP-HPLC. Oil samples dissolved in n-hexane and TAGs were derived by reaction with a Br2-CCl 4 (1:1, v/v) solution for 3 h at room temperature. The derivate product solution was stable and was best separated and detected by RP-HPLC using a C18 column, with a mobile phase of methanol-n-hexane (91.5:8.5, v/v) at 25°C. A detection wavelength of 230 nm was used. The results showed that the approach enabled the separation and detection of more similar TAGs by RP-HPLC. The method was applied to profile 20 types of edible oil, and the results presented the differences in the TAG profiles of various edible oils, which may be useful in the identification of edible oils.

Simultaneous Determination of Ursodeoxycholic Acid and Chenodeoxycholic Acid in Pharmaceutical Dosage Form by HPLC-UV Detection.

PubMed

Khairy, Mostafa A; Mansour, Fotouh R

2017-01-01

A reversed-phase HPLC method was developed for the simultaneous determination of ursodeoxycholic acid (UDCA) and the epimeric isomer, chenodeoxycholic acid (CDCA), in their synthetic mixtures and in tablet dosage form. The proposed HPLC method uses a C18 column and mobile phase consisting of an acetonitrile-phosphate buffer mixture (pH 2.3, 100 mM; 50 + 50, v/v) at a flow rate of 2.0 mL/min with UV detection at 210 nm. The method was validated according to the International Conference on Harmonization guidelines; and linearity, range, accuracy, precision, robustness, and system suitability were studied. The LOD and LOQ were also calculated and found to be 1.23 and 3.73 μg/mL for UDCA and 0.83 and 2.52 μg/mL for CDCA, respectively. The method was adapted for UHPLC, in which baseline separation was achieved in <2.5 min. The assay results of Ursomix tablets by the developed method were statistically compared with those obtained by the reference method using t- and F-tests, and no significant differences were observed.

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

PubMed Central

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC50 value of 2.27 μg mL−1 and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation. PMID:22489129

Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

PubMed

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.

Quantitative determination of reserpine, ajmaline, and ajmalicine in Rauvolfia serpentina by reversed-phase high-performance liquid chromatography.

PubMed

Srivastava, A; Tripathi, A K; Pandey, R; Verma, R K; Gupta, M M

2006-10-01

A sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) method using photodiode array detection is established for the simultaneous quantitation of important root alkaloids of Rauvolfia serpentina, namely, reserpine, ajmaline, and ajmalicine. A Chromolith Performance RP-18e column (100 x 4.6-mm i.d.) and a binary gradient mobile phase composed of 0.01 M (pH 3.5) phosphate buffer (NaH(2)PO(4)) containing 0.5% glacial acetic acid and acetonitrile are used. Analysis is run at a flow rate of 1.0 mL/min with the detector operated at a wavelength of 254 nm. The calibration curves are linear over a concentration range of 1-20 microg/mL (r = 1.000) for all the alkaloids. The various other aspects of analysis (i.e., peak purity, similarity, recovery, and repeatability) are also validated. For the three components, the recoveries are found to be 98.27%, 97.03%, and 98.38%, respectively. The limits of detection are 6, 4, and 8 microg/mL for ajmaline, ajmalicine, and reserpine, respectively, and the limits of quantitation are 19, 12, and 23 microg/mL for ajmaline, ajmalicine, and reserpine, respectively. The developed method is simple, reproducible, and easy to operate. It is useful for the evaluation of R. serpentina.

Determination of modafinil in plasma and urine by reversed phase high-performance liquid-chromatography.

PubMed

Schwertner, Harvey A; Kong, Suk Bin

2005-03-09

Modafinil (Provigil) is a new wake-promoting drug that is being used for the management of excessive sleepiness in patients with narcolepsy. It has pharmacological properties similar to that of amphetamine, but without some of the side effects associated with amphetamine-like stimulants. Since modafinil has the potential to be abused, accurate drug-screening methods are needed for its analysis. In this study, we developed a high-performance liquid-chromatographic procedure (HPLC) for the quantitative analysis of modafinil in plasma and urine. (Phenylthio)acetic acid was used as an internal standard for the analysis of both plasma and urine. Modafinil was extracted from urine and plasma with ethyl acetate and ethyl acetate-acetic acid (100:1, v/v), respectively, and analyzed on a C18 reverse phase column with methanol-water-acetic acid (500:500:1, v/v) as the mobile phase. Recoveries from urine and plasma were 80.0 and 98.9%, respectively and the limit of quantitation was 0.1 microg/mL at 233 nm. Forty-eight 2-h post-dose urine samples from sham controls and from individuals taking 200 or 400 mg of modafinil were analyzed without knowledge of drug administration. All 16-placebo urine samples and all 32 2-h post-dose urine samples were correctly classified. The analytical procedure is accurate and reproducible and can be used for therapeutic drug monitoring, pharmacokinetic studies, and drug abuse screening.

Analysis of quaternary ammonium and phosphonium ionic liquids by reversed-phase high-performance liquid chromatography with charged aerosol detection and unified calibration.

PubMed

Stojanovic, Anja; Lämmerhofer, Michael; Kogelnig, Daniel; Schiesel, Simone; Sturm, Martin; Galanski, Markus; Krachler, Regina; Keppler, Bernhard K; Lindner, Wolfgang

2008-10-31

Several hydrophobic ionic liquids (ILs) based on long-chain aliphatic ammonium- and phosphonium cations and selected aromatic anions were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) employing trifluoroacetic acid as ion-pairing additive to the acetonitrile-containing mobile phase and adopting a step-gradient elution mode. The coupling of charged aerosol detection (CAD) for the non-chromophoric aliphatic cations with diode array detection (DAD) for the aromatic anions allowed their simultaneous analysis in a set of new ILs derived from either tricaprylmethylammonium chloride (Aliquat 336) and trihexyltetradecylphosphonium chloride as precursors. Aliquat 336 is a mix of ammonium cations with distinct aliphatic chain lengths. In the course of the studies it turned out that CAD generates an identical detection response for all the distinct aliphatic cations. Due to lack of single component standards of the individual Aliquat 336 cation species, a unified calibration function was established for the quantitative analysis of the quaternary ammonium cations of the ILs. The developed method was validated according to ICH guidelines, which confirmed the validity of the unified calibration. The application of the method revealed molar ratios of cation to anion close to 1 indicating a quantitative exchange of the chloride ions of the precursors by the various aromatic anions in the course of the synthesis of new ILs. Anomalies of CAD observed for the detection of some aromatic anions (thiosalicylate and benzoate) are discussed.

Sensitive method for the quantitation of droloxifene in plasma and serum by high-performance liquid chromatography employing fluorimetric detection.

PubMed

Tess, D A; Cole, R O; Toler, S M

1995-12-15

A simple and highly sensitive reversed-phase fluorimetric HPLC method for the quantitation of droloxifene from rat, monkey, and human plasma as well as human serum is described. This assay employs solid-phase extraction and has a dynamic range of 25 to 10,000 pg/ml. Sample extraction (efficiencies > 86%) was accomplished using a benzenesulfonic acid (SCX) column with water and methanol rinses. Droloxifene and internal standard were eluted with 1 ml of 3.5% (v/v) ammonium hydroxide (30%) in methanol. Samples were quantitated using post-column UV-photochemical cyclization coupled with fluorimetric detection with excitation and emission wavelengths of 260 nm and 375 nm, respectively. Relative ease of sample extraction and short run times allow for the analysis of approximately 100 samples per day.

Quantitative analysis of matrine in liquid crystalline nanoparticles by HPLC.

PubMed

Peng, Xinsheng; Li, Baohong; Hu, Min; Ling, Yahao; Tian, Yuan; Zhou, Yanxing; Zhou, Yanfang

2014-01-01

A reversed-phase high-performance liquid chromatographic method has been developed to quantitatively determine matrine in liquid crystal nanoparticles. The chromatographic method is carried out using an isocratic system. The mobile phase was composed of methanol-PBS(pH6.8)-triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min with SPD-20A UV/vis detector and the detection wavelength was at 220 nm. The linearity of matrine is in the range of 1.6 to 200.0  μ g/mL. The regression equation is y = 10706x - 2959 (R (2) = 1.0). The average recovery is 101.7%; RSD = 2.22%  (n = 9). This method provides a simple and accurate strategy to determine matrine in liquid crystalline nanoparticle.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system.

PubMed

Yasuda, Makoto; Ota, Tatsuhiro; Morikawa, Atsushi; Mawatari, Ken-ichi; Fukuuchi, Tomoko; Yamaoka, Noriko; Kaneko, Kiyoko; Nakagomi, Kazuya

2013-09-01

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

A simple, sensitive and rapid isocratic reversed-phase high-performance liquid chromatography method for determination and stability study of curcumin in pharmaceutical samples

PubMed Central

Amanolahi, Farjad; Mohammadi, Ali; Kazemi Oskuee, Reza; Nassirli, Hooriyeh; Malaekeh-Nikouei, Bizhan

2017-01-01

Objective: This study was designed to develop and validate a new reversed-phase high-performance liquid chromatography (RP-HPLC) method based on Q2 (R1) International Conference on Harmonization (ICH) guideline for determination of curcumin in pharmaceutical samples. Materials and Methods: The HPLC instrument method was optimized with isocratic elution with acetonitrile: ammonium acetate (45:55, v/v, pH 3.5), C18 column (150 mm×4.6 mm×5 µm particle size) and a flow rate of 1 ml/min in ambient condition and total retention time of 17 min. The volume of injection was set at 20 µl and detection was recorded at 425 nm. The robustness of the method was examined by changing the mobile phase composition, mobile phase pH, and flow rate. Results: The method was validated with respect to precision, accuracy and linearity in a concentration range of 2-100 µg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.5 µg/ml, respectively. The percentage of recovery was 98.9 to 100.5 with relative standard deviation (RSD) < 0.638%. Conclusion: The method was found to be simple, sensitive and rapid for determination of curcumin in pharmaceutical samples and had enough sensitivity to detect degradation product of curcumin produced under photolysis and hydrolysis stress condition. PMID:29062806

Chromatographic evaluation of self-immobilized stationary phases for reversed-phase liquid chromatography.

PubMed

Bottoli, Carla B G; Collins, Kenneth E; Collins, Carol H

2003-02-14

The preparation of stationary phases for HPLC using polymers deposited on silica usually includes an immobilization step involving cross-linking by free radicals induced by ionizing radiation or by other radical initiators. The present paper reports changes which occur at ambient temperature in the character of poly(methyloctylsiloxane) deposited on porous silica particles as a function of the time interval between particle loading and column packing. Column performance and retention factors increase with time and these changes are attributed to rearrangement (self-assembly) which result in "self-immobilization" of the polymer molecules on the silica surface.

Chromatographic analysis of salicylic compounds in different species of the genus Salix.

PubMed

Pobłocka-Olech, Loretta; van Nederkassel, Anne-Marie; Vander Heyden, Yvan; Krauze-Baranowska, Mirosława; Glód, Daniel; Baczek, Tomasz

2007-11-01

The separation of nine phenol glycosides--salicin, salicortin, 2'-acetylsalicortin, populin, tremulacin, salidroside, triandrin, picein and helicin--by normal phase (NP), reversed phase (RP) HPLC techniques and a coupling of NP and RP monolithic silica columns was studied. Among the above nine compounds only five--salicin, populin, tremulacin, salidroside and triandrin--were resolved in an NP system with a mobile phase comprising hexane/isopropanol/methanol (87:12:1, v/v/v). Optimized separation was performed with two coupled monolithic silica columns of different polarity (bare silica and RP-18). The method was applied to verify the presence of salicylic compounds and other phenolic derivatives in the bark of six species from the genus Salix, namely S. purpurea, S. daphnoides clone 1095, S. alba clone 1100, S. triandra, S. viminalis, and S. herbacea. Gradient elution with a mobile phase composed of acetonitrile and water containing 0.05% of trifluoroacetic acid, with increasing acetonitrile concentration from 3% to 48%, was chosen as optimal. For the selective detection of the salicylic compounds, an evaporative light scattering detector was employed along with a UV detector. The differences in the composition of phenols in the different plant materials were confirmed. Additionally, it must be emphasized that for the first time the presence of 2'-acetylsalicortin was revealed in S. alba clone 1100. Furthermore, an SPE-HPLC method was developed for the rapid analysis of the salicin content, analyzed as free and total fraction, in willow barks. The determined concentrations of total salicin varied from 25.4 mg/g in S. alba clone 1100 to 96.47 mg/g in S. daphnoides clone 1095.

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.

Spectrophotometric and Reversed-Phase High-Performance Liquid Chromatographic Method for the Determination of Doxophylline in Pharmaceutical Formulations

PubMed Central

Joshi, HR; Patel, AH; Captain, AD

2010-01-01

Two methods are described for determination of Doxophylline in a solid dosage form. The first method was based on ultraviolet (UV)-spectrophotometric determination of the drug. It involves absorbance measurement at 274 nm (λmax of Doxophylline) in 0.1 N hydrochloric acid. The calibration curve was linear, with the correlation coefficient between 0.99 and 1.0 over a concentration range of 0.20–30 mg/ml for the drug. The second method was based on high-performance liquid chromatography (HPLC) separation of the drug in reverse-phase mode using the Hypersil ODS C18 column (250 × 4.6 mm, 5 mm). The mobile phase constituted of buffer acetonitrile (80:20) and pH adjusted to 3.0, with dilute orthophosphoric acid delivered at a flow rate 1.0 ml/min. Detection was performed at 210 nm. Separation was completed within 7 min. The calibration curve was linear, with the correlation coefficient between 0.99 and 1.0 over a concentration range of 0.165–30 mg/ml for the drug. The relative standard deviation was found to be <2.0% for the UV-spectrophotometry and HPLC methods. Both these methods have been successively applied to the solid dosage pharmaceutical formulation, and were fully validated according to ICH guidelines. PMID:21042488

Increased concentrations of endogenous 13-cis- and all-trans-retinoic acids in diffuse idiopathic skeletal hyperostosis, as demonstrated by HPLC.

PubMed

Periquet, B; Lambert, W; Garcia, J; Lecomte, G; De Leenheer, A P; Mazieres, B; Thouvenot, J P; Arlet, J

1991-11-09

Endogenous 13-cis- and all-trans-retinoic acids have been quantitated in human serum using a solvent extraction procedure followed by isocratic reversed phase high performance liquid chromatography and UV detection. In healthy adults, after an overnight fasting period, the concentrations of 13-cis- and all-trans-retinoic acids yielded 5.3 +/- 2.43 nmol/l and 11.8 +/- 3.3 nmol/l, respectively (mean +/- SD). The method has been successfully applied to the analysis of both isomers in serum from patients with idiopathic skeletal hyperostosis in whom, the 13-cis- as well as all-trans-retinoic acid levels were raised as compared to the control group.

Rapid and sensitive determination of beta-phenylethylamine in animal brains by high performance liquid chromatography with fluorometric detection.

PubMed

Taga, C; Tsuji, M; Nakajima, T

1989-05-01

A reversed phase HPLC method with fluorometric detection for the analysis of beta-phenylethylamine has been developed using p-methoxyphenylethylamine as an internal standard. Two columns, containing 200 microL of Dowex 50-X8 and Amberlite CG-50 respectively, were used to prepare a fraction containing beta-phenylethylamine. The recoveries of beta-phenylethylamine and p-methoxyphenylethylamine were 53.9 +/- 9.4% and 68.1 +/- 12.4%, respectively, and elution profile of p-methoxyphenylethylamine was sufficiently well correlated with that of beta-phenylethylamine. Regional distributions of beta-phenylethylamine in rat and mouse brains were determined. The highest concentrations were found in hypothalamus and hippocampus in both animals.

Profiling of tryptophan-related plasma indoles in patients with carcinoid tumors by automated, on-line, solid-phase extraction and HPLC with fluorescence detection.

PubMed

Kema, I P; Meijer, W G; Meiborg, G; Ooms, B; Willemse, P H; de Vries, E G

2001-10-01

Profiling of the plasma indoles tryptophan, 5-hydroxytryptophan (5-HTP), serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) is useful in the diagnosis and follow-up of patients with carcinoid tumors. We describe an automated method for the profiling of these indoles in protein-containing matrices as well as the plasma indole concentrations in healthy controls and patients with carcinoid tumors. Plasma, cerebrospinal fluid, and tissue homogenates were prepurified by automated on-line solid-phase extraction (SPE) in Hysphere Resin SH SPE cartridges containing strong hydrophobic polystyrene resin. Analytes were eluted from the SPE cartridge by column switching. Subsequent separation and detection were performed by reversed-phase HPLC combined with fluorometric detection in a total cycle time of 20 min. We obtained samples from 14 healthy controls and 17 patients with metastasized midgut carcinoid tumors for plasma indole analysis. In the patient group, urinary excretion of 5-HIAA and serotonin was compared with concentrations of plasma indoles. Within- and between-series CVs for indoles in platelet-rich plasma were 0.6-6.2% and 3.7-12%, respectively. Results for platelet-rich plasma serotonin compared favorably with those obtained by single-component analysis. Plasma 5-HIAA, but not 5-HTP was detectable in 8 of 17 patients with carcinoid tumors. In the patient group, platelet-rich plasma total tryptophan correlated negatively with platelet-rich plasma serotonin (P = 0.021; r = -0.56), urinary 5-HIAA (P = 0.003; r = -0.68), and urinary serotonin (P <0.0001; r = -0.80). The present chromatographic approach reduces analytical variation and time needed for analysis and gives more detailed information about metabolic deviations in indole metabolism than do manual, single-component analyses.

Analysis of anti-neoplastic drug in bacterial ghost matrix, w/o/w double nanoemulsion and w/o nanoemulsion by a validated 'green' liquid chromatographic method.

PubMed

Youssof, Abdullah M E; Salem-Bekhit, Mounir M; Shakeel, Faiyaz; Alanazi, Fars K; Haq, Nazrul

2016-07-01

The objective of the present investigation was to develop and validate a 'green' reversed phase high-performance liquid chromatography (RP-HPLC) method for rapid analysis of a cytotoxic drug 5-fluorouracil (5-FU) in bulk drug, marketed injection, water-in-oil (w/o) nanoemulsion, double water-in-oil-in-water (w/o/w) nanoemulsion and bacterial ghost (BG) matrix. The chromatography study was carried out at room temperature (25±1°C) using an HPLC system with the help of ultraviolet (UV)-visible detector. The chromatographic performance was achieved with a Nucleodur 150mm×4.6mm RP C8 column filled with 5µm filler as a static phase. The mobile phase consisted of ethyl acetate: methanol (7:3% v/v) which was delivered at a flow rate of 1.0mLmin(-1) and the drug was detected in UV mode at 254nm. The developed method was validated in terms of linearity (r(2)=0.998), accuracy (98.19-102.09%), precision (% RSD=0.58-1.17), robustness (% RSD=0.12-0.53) and sensitivity with satisfactory results. The efficiency of the method was demonstrated by the assay of the drug in marketed injection, w/o nanoemulsion, w/o/w nanoemulsion and BG with satisfactory results. The successful resolution of the drug along with its degradation products clearly established the stability-indicating nature of the proposed method. Overall, these results suggested that the proposed analytical method could be effectively applied to the routine analysis of 5-FU in bulk drug, various pharmaceutical dosage forms and BG. Copyright © 2016 Elsevier B.V. All rights reserved.

HPLC Determination of Esculin and Esculetin in Rat Plasma for Pharmacokinetic Studies.

PubMed

Rehman, Shaheed Ur; Kim, In Sook; Kang, Ki Sung; Yoo, Hye Hyun

2015-09-01

An optimized, sensitive and validated reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection is described for simultaneous determination of esculin and its aglycone, esculetin, in rat plasma. After addition of internal standard (chrysin), plasma samples were pretreated by solid-phase extraction and introduced into the HPLC system. Analytes were separated on a RP C18 column with a mobile phase of 0.075% acetic acid in water (solvent A) and 90% acetonitrile in solvent A (solvent B) using gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 338 nm. Calibration curves for esculin and esculetin were constructed over a range of 10-1,000 ng/mL. The developed method was found to be specific, precise and accurate. The method was successfully applied to study the pharmacokinetics of esculin and esculetin in rats. After oral administration of 120 mg/kg, the mean Cmax values were 340.3 and 316.5 ng/mL and the AUClast values were 377.3 and 1276.5 h ng/mL for esculin and esculetin, respectively. The bioavailability of esculin was calculated to be 0.62%. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Method 447.0 - Determination of Chlorophylls a and b and Identification of Other Pigments of Interest in Marine and Freshwater Algae Using High Performance Liquid Chromatography with Visible Wavelength Detection

EPA Science Inventory

This method provides a procedure for determination of chlorophylls a (chl a) and b (chl b) found in marine and freshwater phytoplankton. Reversed phase high performance liquid chromatography (HPLC) with detection at 440 nm is used to separate the pigments from a complex pigment ...

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...

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 rapid high-performance liquid-chromatographic method for simultaneously determining the concentrations of TFM and Bayer 73 in water during lampricide treatments

USGS Publications Warehouse

Dawson, V.K.

1982-01-01

The high-performance liquid-chromatography (HPLC) procedure requires only minutes per sample, is specific, and is relatively sensitive (limit of detection 18 disposable cartridge. The cartridge adsorbs and retains both the lampricides and the internal standard. The quantitative elution of the three chemicals from the cartridge with a small volume of methanol effectively concentrates the sample and provides sample cleanup. The methanol extract is then analyzed directly by HPLC on an MCH 10 reverse phase column by using a methanol:0.01 mol/L acetate buffer (87:13, v:v) as the mobile phase at 2 mL/min and detected by ultraviolet spectrophotometry at 330 (or 254) nm. A microprocessor data system further facilitates the procedure by quantifying off-scale peaks and yielding results directly in units of concentration (mg/L).

HPLC separation of acetaminophen and its impurities using a mixed-mode reversed-phase/cation exchange stationary phase.

PubMed

Călinescu, Octavian; Badea, Irinel A; Vlădescu, Luminiţa; Meltzer, Viorica; Pincu, Elena

2012-04-01

Determination of acetaminophen and its main impurities: 4-nitrophenol, 4'-chloroacetanilide, as well as 4-aminophenol and its degradation products, p-benzoquinone and hydroquinone has been developed and validated by a new high-performance liquid chromatography method. Chromatographic separation has been obtained on a Hypersil Duet C18/SCX column, using gradient elution, with a mixture of phosphate buffer (pH = 4.88) and methanol as a mobile phase. Analysis time did not exceed 14.5 min and good resolutions, peak shapes and asymmetries have resulted. The linearity of the method has been tested in the range of 5.0-60 µg/mL for acetaminophen and 0.5-6 µg/mL for the other compounds. The limits of detection and quantification have been also established to be lower than 0.1 µg/mL and 0.5 µg/mL, respectively. The method has been successfully applied for the analysis of commercial acetaminophen preparations. © The Author [2012]. Published by Oxford University Press. All rights reserved.

PROFILING GLYCOL-SPLIT HEPARINS BY HPLC/MS ANALYSIS OF THEIR HEPARINASE-GENERATED OLIGOSACCHARIDES1

PubMed Central

Alekseeva, Anna; Casu, Benito; Torri, Giangiacomo; Pierro, Sabrina; Naggi, Annamaria

2012-01-01

Glycol-split (gs) heparins, obtained by periodate oxidation / borohydride reduction of heparin currently used as anticoagulant and antithrombotic drug, are arousing increasing interest in anti-cancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs-heparins has not been studied yet in detail. In this work, an ion-pair reversed-phase chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs-heparins usually prepared from porcine mucosal heparin. The method has been also found very effective in analyzing glycol-split derivatives obtained from heparins of different animal and tissue origin. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs-heparins mainly contain tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs-heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues has been shown to be a marker of the gs-modified AT binding site within heparin chains. PMID:23201389

Characterization of bioactive compounds in Tunisian bitter orange (Citrus aurantium L.) peel and juice and determination of their antioxidant activities.

PubMed

Jabri Karoui, Iness; Marzouk, Brahim

2013-01-01

Citrus aurantium peel and juice aroma compounds were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), whereas phenolic compounds analysis was performed by reversed-phase high-performance liquid chromatography (RP-HPLC). Limonene was the major volatile compound of bitter orange peel (90.25%) and juice (91.61%). HPLC analysis of bitter orange peel and juice methanolic extracts indicated that phenolic acids constitute their main phenolic class representing 73.80% and 71.25%, respectively, followed by flavonoids (23.02% and 23.13%, resp.). p-Coumaric and ferulic acids were the most abundant phenolic compounds representing 24.68% and 23.79%, respectively, in the peel, while the juice contained 18.02% and 19.04%, respectively. The antioxidant activities of bitter orange peel and juice methanolic extracts have been evaluated using four in vitro assays, and the results were compared with the standard antioxidants (BHT, BHA, and ascorbic acid). Our findings demonstrated that Citrus aurantium peel and juice possess antioxidant activities which were less effective than those of antioxidant standards. Both extracts may be suggested as a new potential source of natural antioxidant.

Progressing the analysis of Improvised Explosive Devices: Comparative study for trace detection of explosive residues in handprints by Raman spectroscopy and liquid chromatography.

PubMed

Zapata, Félix; de la Ossa, Mª Ángeles Fernández; Gilchrist, Elizabeth; Barron, Leon; García-Ruiz, Carmen

2016-12-01

Concerning the dreadful global threat of terrorist attacks, the detection of explosive residues in biological traces and marks is a current need in both forensics and homeland security. This study examines the potential of Raman microscopy in comparison to liquid chromatography (ion chromatography (IC) and reversed-phase high performance liquid chromatography (RP-HPLC)) to detect, identify and quantify residues in human handmarks of explosives and energetic salts commonly used to manufacture Improvised Explosive Devices (IEDs) including dynamite, ammonium nitrate, single- and double-smokeless gunpowders and black powder. Dynamite, ammonium nitrate and black powder were detected through the identification of the energetic salts by Raman spectroscopy, their respective anions by IC, and organic components by RP-HPLC. Smokeless gunpowders were not detected, either by Raman spectroscopy or the two liquid chromatography techniques. Several aspects of handprint collection, sample treatment and a critical comparison of the identification of compounds by both techniques are discussed. Raman microscopy and liquid chromatography were shown to be complementary to one another offering more comprehensive information for trace explosives analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Development and single-laboratory validation of an HPLC method for the determination of cyclamate sweetener in foodstuffs.

PubMed

Scotter, M J; Castle, L; Roberts, D P T; Macarthur, R; Brereton, P A; Hasnip, S K; Katz, N

2009-05-01

A method for the determination of cyclamate has been developed and single-laboratory validated for a range of foodstuffs including carbonated and fruit-juice drinks, fruit preserves, spreads, and dairy desserts. The method uses the peroxide oxidation of cyclamate to cyclohexylamine followed by derivatization with trinitrobenzenesulfonic acid and analysis by a modified reversed-phase high-performance liquid chromatography-ultraviolet light (HPLC-UV). Cycloheptylamine is used as an internal standard. The limits of detection were in the range 1-20 mg kg(-1) and the analysis was linear up to 1300 mg kg(-1) cyclamic acid in foods and up to 67 mg l(-1) in beverages. Analytical recovery was between 82% and 123%, and results were recovery corrected. Precision was within experimentally predicted levels for all of the matrices tested and Horrat values for the combined standard uncertainty associated with the measurement of cyclamate between 0.4 (water-based drinks) and 1.7 (spreads). The method was used successfully to test three soft drink samples for homogeneity before analytical performance assessment. The method is recommended for use in monitoring compliance and for formal testing by collaborative trial.

Characterization of Bioactive Compounds in Tunisian Bitter Orange (Citrus aurantium L.) Peel and Juice and Determination of Their Antioxidant Activities

PubMed Central

Jabri karoui, Iness; Marzouk, Brahim

2013-01-01

Citrus aurantium peel and juice aroma compounds were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), whereas phenolic compounds analysis was performed by reversed-phase high-performance liquid chromatography (RP-HPLC). Limonene was the major volatile compound of bitter orange peel (90.25%) and juice (91.61%). HPLC analysis of bitter orange peel and juice methanolic extracts indicated that phenolic acids constitute their main phenolic class representing 73.80% and 71.25%, respectively, followed by flavonoids (23.02% and 23.13%, resp.). p-Coumaric and ferulic acids were the most abundant phenolic compounds representing 24.68% and 23.79%, respectively, in the peel, while the juice contained 18.02% and 19.04%, respectively. The antioxidant activities of bitter orange peel and juice methanolic extracts have been evaluated using four in vitro assays, and the results were compared with the standard antioxidants (BHT, BHA, and ascorbic acid). Our findings demonstrated that Citrus aurantium peel and juice possess antioxidant activities which were less effective than those of antioxidant standards. Both extracts may be suggested as a new potential source of natural antioxidant. PMID:23841062

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

PubMed

Krungkrai, J; Wutipraditkul, N; Prapunwattana, P; Krungkrai, S R; Rochanakij, S

2001-12-15

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes. (c)2001 Elsevier Science.

Measurement of H2S in vivo and in vitro by the monobromobimane method.

PubMed

Shen, Xinggui; Kolluru, Gopi K; Yuan, Shuai; Kevil, Christopher G

2015-01-01

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels. © 2015 Elsevier Inc. All rights reserved.

Simultaneous Determination of Piperine, Capsaicin, and Dihydrocapsaicin in Korean Instant-Noodle (Ramyun) Soup Base Using High-Performance Liquid Chromatography with Ultraviolet Detection.

PubMed

Shim, You-Shin; Kim, Jong-Chan; Jeong, Seung-Weon

2016-01-01

A simultaneous analytical method for piperine, capsaicin, and dihydrocapsaicin in Korean instant-noodle soup base using HPLC was validated in terms of precision, accuracy, sensitivity, and linearity. The HPLC separation was performed on a reversed-phase C18 column (5 μm particle size, 4.6 mm id, 250 mm length) using a UV detector fixed at 280 nm. The LOD and LOQ of the HPLC analyses ranged from 0.25 to 1.03 mg/kg. The intraday and interday precisions of the individual piperine, capsaicin, and dihydrocapsaicin were <10.55%, and the recovery values ranged from 85.43 to 94.68%. The calibration curves exhibited good linearity (r(2) = 0.999) within the tested ranges. These results suggest that the analytical method in this study can be used to classify Korean instant noodles based on their levels of spiciness.

Isolation and Characterization of Precise Dye/Dendrimer Ratios

PubMed Central

Dougherty, Casey A.; Furgal, Joseph C.; van Dongen, Mallory A.; Goodson, Theodore; Banaszak Holl, Mark M.; Manono, Janet; DiMaggio, Stassi

2014-01-01

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne `click' ligand, separation into sample containing precisely defined `click' ligand/particle ratios using reverse-phase high performance liquid chromatography (rp-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using rp-HPLC. These materials were characterized using 1H and 19F NMR, rp-HPLC, UV-Vis and fluorescence spectroscopy, lifetime measurements, and MALDI. PMID:24604830

Determination of apalcillin and its metabolites in human body fluids by high-pressure liquid chromatography.

PubMed Central

Borner, K; Lode, H; Elvers, A

1982-01-01

We describe two methods for the quantitative analysis of apalcillin and its metabolites in serum and urine by reverse-phase high-pressure liquid chromatography (HPLC), a fast isocratic method for the parent drug, and a gradient method that allows the simultaneous assay of two metabolites. Serum was deproteinized with acetonitrile, and urine was diluted with buffer solution. The detection limit was about 0.5 micrograms/ml at a detection wavelength of 254 nm and 1.5 micrograms/ml at 310 nm. Within-batch precision (coefficient of variation) varied from 10.2 to 1.1% for concentrations of 7.8 and 185.3 micrograms/ml of serum, respectively. Recovery rates of 95.1 and 97.7% were found in spiked sera. Results obtained by HPLC correlated well with those from a standard microbiological assay (agar diffusion test); the resulting bivariate regression equation for serum was y-bioassay = 2.5 micrograms/ml + 0.992 X xHPLC, and that for urine was ybioassay = 12.0 micrograms/ml + 1.009 X xHPLC. At a detection wavelength of 315 nm, no interferences were observed in 10 healthy volunteers. Healthy subjects who were given 2 g of apalcillin intravenously excreted 18% of the parent drug within 24 h in the urine. Two inactive compounds were furthermore identified in urine as the isomeric forms of the penicilloic acids. Their excretion within 24 h amounted to 6.9 and 11.2% of the dose. PMID:6818901

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites

PubMed Central

Kolářová, L.; Nobilis, M.

2008-01-01

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]+ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MSn analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well. PMID:18345532

Identification of the anti-oxidant components in a two-step solvent extract of bovine bile lipid: Application of reverse phase HPLC, mass spectrometry and fluorimetric assays.

PubMed

Singh, Namrata; Bhattacharyya, Debasish

2016-04-15

An ether extract of nine different bacterial metabolites in combination with two solvent extract (ether followed by ethanol) of bile lipids from ox gall bladder is used as an immune stimulator drug. Over the years bile acids are discussed regarding their anti-oxidant and lipid peroxidation properties. Since some of the bile acids are known to be potent antioxidants, presence of similar activity in the solvent extract of ox bile lipid was investigated using TLC and reverse phase HPLC systems. Fractions from HPLC were analyzed with mass spectrometry using electrospray ionization. The presence of twelve different bile acids along with other substances in small proportions including fatty acids, sulfate conjugates and bile pigments were confirmed. The twelve separated peaks had similar retention times as those of tauroursodeoxycholic acid, glycoursodeoxycholic acid, taurocholic acid, glycocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid. Subsequently, all fractions were tested for their anti-oxidative property on HepG2 cells exposed to H2O2 that served as an oxidative injury model. Four fluorescent dyes H2DCF DA, MitoSOX red, Amplex red and DAF-2 DA were used for estimation of reactive radicals in the HepG2 cells. Among the separated bile acids, tauroursodeoxycholic acid, glycoursodeoxycholic acid and ursodeoxycholic acid prevented the HepG2 cells from H2O2-induced oxidative stress. Copyright © 2015 Elsevier B.V. All rights reserved.

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry.

PubMed

Kakimoto, Kensaku; Toriba, Akira; Ohno, Takanori; Ueno, Mariko; Kameda, Takayuki; Tang, Ning; Hayakawa, Kazuichi

2008-05-15

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C(18) reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated (r(2)=0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.

Development and validation of a reversed-phase HPLC method for simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet dosage form.

PubMed

Shaikh, K A; Patil, S D; Devkhile, A B

2008-12-15

A simple, precise and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous estimation of ambroxol hydrochloride and azithromycin in tablet formulations. The chromatographic separation was achieved on a Xterra RP18 (250 mm x 4.6 mm, 5 microm) analytical column. A Mixture of acetonitrile-dipotassium phosphate (30 mM) (50:50, v/v) (pH 9.0) was used as the mobile phase, at a flow rate of 1.7 ml/min and detector wavelength at 215 nm. The retention time of ambroxol and azithromycin was found to be 5.0 and 11.5 min, respectively. The validation of the proposed method was carried out for specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness. The linear dynamic ranges were from 30-180 to 250-1500 microg/ml for ambroxol hydrochloride and azithromycin, respectively. The percentage recovery obtained for ambroxol hydrochloride and azithromycin were 99.40 and 99.90%, respectively. Limit of detection and quantification for azithromycin were 0.8 and 2.3 microg/ml, for ambroxol hydrochloride 0.004 and 0.01 microg/ml, respectively. The developed method can be used for routine quality control analysis of titled drugs in combination in tablet formulation.

Secondary Organic Aerosol Formation from Glyoxal: photochemical versus dark uptake and reversible versus irreversible SOA formation

NASA Astrophysics Data System (ADS)

Waxman, E.; Slowik, J. G.; Kampf, C. J.; Timkovsky, J.; Noziere, B.; Praplan, A. P.; Pfaffenberger, L.; Holzinger, R.; Hoffmann, T.; Dommen, J.; Prevot, A. S.; Baltensperger, U.; Volkamer, R.

2011-12-01

Glyoxal forms secondary organic aerosol (SOA) by partitioning to the aerosol aqueous phase according to Henry's law. The subsequent processing by heterogeneous and multiphase reactions shifts the partitioning towards aerosols. Currently it is not well understood whether these reactions result in reversible or irreversible SOA formation, and what parameters influence the rate limiting step of multiphase processing. We conducted a series of simulation chamber experiments at PSI in April and May 2011 to investigate processing under dark conditions, UV and/or visible light irradiated conditions, and in the presence and absence of OH radicals. Experiments used ammonium sulfate or ammonium sulfate/fulvic acid mixtures as seed aerosols, and were conducted between 50% and 85% relative humidity at approximately constant RH over the course of any given experiment. Glyoxal was produced photochemically from acetylene, using HONO photolysis as the OH radical source. Gas-phase glyoxal was measured by the CU LED-Cavity Enhanced-DOAS. The Thermal-Desorption Proton-Transfer-Reaction Mass Spectrometer (TD-PTR-MS) and Ion Chromatography Mass Spectrometer (IC-MS) monitored both gas and aerosol-phase organic reaction products. Particle composition was monitored by High-Resolution Time-of-Flight Aerosol Mass Spectrometry (HR-ToF-AMS), and HPLC-ESI MS/MS and LC-MS analysis of filter samples.

Secondary Organic Aerosol Formation from Glyoxal: photochemical versus dark uptake and reversible versus irreversible SOA formation

NASA Astrophysics Data System (ADS)

Waxman, E.; Slowik, J.; Kampf, C.; Timkovsky, J.; Noziere, B.; Praplan, A.; Pffafenberger, L.; Holzinger, R.; Hoffmann, T.; Dommen, J.; Prevot, A.; Baltensperger, U.; Volkamer, R.

2012-04-01

Glyoxal forms secondary organic aerosol (SOA) by partitioning to the aerosol aqueous phase according to Henry's law. The subsequent processing by heterogeneous and multiphase reactions shifts the partitioning towards aerosols. Currently it is not well understood whether these reactions result in reversible or irreversible SOA formation, and what parameters influence the rate limiting step of multiphase processing. We conducted a series of simulation chamber experiments at PSI in April and May 2011 to investigate processing under dark conditions, UV and/or visible light irradiated conditions, and in the presence and absence of OH radicals. Experiments used ammonium sulfate or ammonium sulfate/fulvic acid mixtures as seed aerosols, and were conducted between 50% and 85% relative humidity at approximately constant RH over the course of any given experiment. Glyoxal was produced photochemically from acetylene, using HONO photolysis as the OH radical source. Gas-phase glyoxal was measured by the CU LED-Cavity Enhanced-DOAS. The Thermal-Desorption Proton-Transfer-Reaction Mass Spectrometer (TD-PTR-MS) and Ion Chromatography Mass Spectrometer (IC-MS) monitored both gas and aerosol-phase organic reaction products. Particle composition was monitored by High-Resolution Time-of-Flight Aerosol Mass Spectrometry (HR-ToF-AMS), and HPLC-ESI MS/MS and LC-MS analysis of filter samples.

Salting-Out Assisted Liquid-Liquid Extraction for Quantification of Febuxostat in Plasma Using RP-HPLC and Its Pharmacokinetic Application.

PubMed

Tandel, Devang; Shah, Purvi; Patel, Kalpana; Thakkar, Vaishali; Patel, Kirti; Gandhi, Tejal

2016-11-01

A rapid and sensitive reversed-phase high-performance liquid chromatography (HPLC) method using novel salting-out assisted liquid-liquid extraction technique has been developed for the quantitative determination of febuxostat (FEB), used for the treatment of gout, in rat plasma. The method was validated according to US FDA guideline. Separation was achieved using a Phenomenex Luna-C 18 (250 × 4.60 mm, 5 µm) column and mobile phase composed of potassium dihydrogen orthophosphate buffer 25 mM, adjusted to pH 6.8 with triethylamine:methanol in a ratio of 35:65 (v/v) showing retention time 5.56 and 8.86 min for FEB and internal standard, respectively. The optimal salting-out parameters; 1 mL of acetonitrile and 200 µL of 2 M ammonium acetate salt showed extraction recovery >90% for FEB from plasma. This extraction procedure afforded clear samples resulting in convenient and cost-saving procedure and showed good linear relationship (r > 0.9997) between peak area ratio and concentration from 0.3 to 20 µg/mL. The results of pharmacokinetic study showed that absorption profile of spherical agglomerate of FEB compared to marketed formulation was higher indicating greater systemic absorption. In conclusion, the developed SALLE-HPLC method with simple ultraviolet detection offered a number of advantages including good quantitative ability, wide linear range, high recovery, short analysis time as well as low cost. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Triacylglycerol Analysis in Human Milk and Other Mammalian Species: Small-Scale Sample Preparation, Characterization, and Statistical Classification Using HPLC-ELSD Profiles.

PubMed

Ten-Doménech, Isabel; Beltrán-Iturat, Eduardo; Herrero-Martínez, José Manuel; Sancho-Llopis, Juan Vicente; Simó-Alfonso, Ernesto Francisco

2015-06-24

In this work, a method for the separation of triacylglycerols (TAGs) present in human milk and from other mammalian species by reversed-phase high-performance liquid chromatography using a core-shell particle packed column with UV and evaporative light-scattering detectors is described. Under optimal conditions, a mobile phase containing acetonitrile/n-pentanol at 10 °C gave an excellent resolution among more than 50 TAG peaks. A small-scale method for fat extraction in these milks (particularly of interest for human milk samples) using minimal amounts of sample and reagents was also developed. The proposed extraction protocol and the traditional method were compared, giving similar results, with respect to the total fat and relative TAG contents. Finally, a statistical study based on linear discriminant analysis on the TAG composition of different types of milks (human, cow, sheep, and goat) was carried out to differentiate the samples according to their mammalian origin.

Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected beta-blockers.

PubMed

Flieger, J

2010-01-22

The addition of the homologous series of perfluorinated acids-trifluoroacetic acid (TFAA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) to mobile phases for reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-blockers was tested. Acidic modifiers were responsible for acidification of mobile phase (pH 3) ensuring the protonation of the beta-blockers and further ion pairs creation. The effect of the type and concentration of mobile phase additives on retention parameters, the efficiency of the peaks, their symmetry and separation selectivity of the beta-blockers mixture were all studied. It appeared that at increasing acid concentration, the retention factor, for all compounds investigated, increased to varying degrees. It should be stressed that the presence of acids more significantly affected the retention of the most hydrophobic beta-blockers. Differences in hydrophobicity of drugs can be maximized through variation of the hydrophobicity of additives. Thus, the relative increase in the retention depends on either concentration and hydrophobicity of the anionic mobile phase additive or hydrophobicity of analytes. According to QSRR (quantitative structure retention relationship) methodology, chromatographic lipophilicity parameters: isocratic log k and log k(w) values (extrapolated retention to pure water) were correlated with the molecular (log P(o/w)) and apparent (log P(app)) octanol-water partition coefficients obtained experimentally by countercurrent chromatography (CCC) or predicted by Pallas software. The obtained, satisfactory retention-hydrophobicity correlations indicate that, in the case of the basic drugs examined in RP-HPLC systems modified with perfluorinated acids, the retention is mainly governed by their hydrophobicity. Copyright 2009 Elsevier B.V. All rights reserved.

Uptake and speciation of selenium in garlic cultivated in soil amended with symbiotic fungi (mycorrhiza) and selenate.

PubMed

Larsen, Erik H; Lobinski, Ryszard; Burger-Meÿer, Karin; Hansen, Marianne; Ruzik, Rafal; Mazurowska, Lena; Rasmussen, Peter Have; Sloth, Jens J; Scholten, Olga; Kik, Chris

2006-07-01

The scope of the work was to investigate the influence of selenate fertilisation and the addition of symbiotic fungi (mycorrhiza) to soil on selenium and selenium species concentrations in garlic. The selenium species were extracted from garlic cultivated in experimental plots by proteolytic enzymes, which ensured liberation of selenium species contained in peptides or proteins. Separate extractions using an aqueous solution of enzyme-deactivating hydroxylamine hydrochloride counteracted the possible degradation of labile selenium species by enzymes (such as alliinase) that occur naturally in garlic. The selenium content in garlic, which was analysed by ICP-MS, showed that addition of mycorrhiza to the natural soil increased the selenium uptake by garlic tenfold to 15 microg g(-1) (dry mass). Fertilisation with selenate and addition of mycorrhiza strongly increased the selenium content in garlic to around one part per thousand. The parallel analysis of the sample extracts by cation exchange and reversed-phase HPLC with ICP-MS detection showed that gamma-glutamyl-Se-methyl-selenocysteine amounted to 2/3, whereas methylselenocysteine, selenomethionine and selenate each amounted to a few percent of the total chromatographed selenium in all garlic samples. Se-allyl-selenocysteine and Se-propyl-selenocysteine, which are selenium analogues of biologically active sulfur-containing amino acids known to occur in garlic, were searched for but not detected in any of the extracts. The amendment of soil by mycorrhiza and/or by selenate increased the content of selenium but not the distribution of detected selenium species in garlic. Finally, the use of two-dimensional HPLC (size exclusion followed by reversed-phase) allowed the structural characterisation of gamma-glutamyl-Se-methyl-selenocysteine and gamma-glutamyl-Se-methyl-selenomethionine in isolated chromatographic fractions by quadrupole time-of-flight mass spectrometry.

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples.

PubMed

Mohammadpour, Amir Hooshang; Ramezani, Mohammad; Tavakoli Anaraki, Nasim; Malaekeh-Nikouei, Bizhan; Amel Farzad, Sara; Hosseinzadeh, Hossein

2013-01-01

The present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. The HPLC method was carried out by using a C18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. The UV detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C18 (200mg) cartridges or with direct precipitation using acetonitrile. The calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. The intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. The inter day coefficients of variation were 1.69 - 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. The validated direct precipitation method for HPLC satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study.

Development and Validation of HPLC Method for Determination of Crocetin, a constituent of Saffron, in Human Serum Samples

PubMed Central

Mohammadpour, Amir Hooshang; Ramezani, Mohammad; Tavakoli Anaraki, Nasim; Malaekeh-Nikouei, Bizhan; Amel Farzad, Sara; Hosseinzadeh, Hossein

2013-01-01

Objective(s): The present study reports the development and validation of a sensitive and rapid extraction method beside high performance liquid chromatographic method for the determination of crocetin in human serum. Materials and Methods: The HPLC method was carried out by using a C18 reversed-phase column and a mobile phase composed of methanol/water/acetic acid (85:14.5:0.5 v/v/v) at the flow rate of 0.8 ml/min. The UV detector was set at 423 nm and 13-cis retinoic acid was used as the internal standard. Serum samples were pretreated with solid-phase extraction using Bond Elut C18 (200mg) cartridges or with direct precipitation using acetonitrile. Results: The calibration curves were linear over the range of 0.05-1.25 µg/ml for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction. The mean recoveries of crocetin over a concentration range of 0.05-5 µg/ml serum for direct precipitation method and 0.5-5 µg/ml for solid-phase extraction were above 70 % and 60 %, respectively. The intraday coefficients of variation were 0.37- 2.6% for direct precipitation method and 0.64 - 5.43% for solid-phase extraction. The inter day coefficients of variation were 1.69 – 6.03% for direct precipitation method and 5.13-12.74% for solid-phase extraction, respectively. The lower limit of quantification for crocetin was 0.05 µg/ml for direct precipitation method and 0.5 µg/ml for solid-phase extraction. Conclusion: The validated direct precipitation method for HPLC satisfied all of the criteria that were necessary for a bioanalytical method and could reliably quantitate crocetin in human serum for future clinical pharmacokinetic study. PMID:23638292

A method for the measurement of atmospheric HONO based on DNPH derivatization and HPLC analysis

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

Zhou, X.; Qiao, H.; Deng, G.

1999-10-15

A simple measurement technique was developed for atmospheric HONO based on aqueous scrubbing using a coil sampler followed by 2,4-dinitrophenylhydrazine (DNPH) derivatization and high-performance liquid chromatographic (HPLC) analysis. Quantitative sampling efficiency was obtained using a 1 mM phosphate buffer, pH 7.0, as the scrubbing solution at a gas sampling flow rate of 2 L min{sup {minus}1} and a liquid flow rate of 0.24 mL min{sup {minus}1}. Derivation of the scrubbed nitrous acid by DNPH was fast and was completed within 5 min in a derivatization medium containing 300 {micro}M DNPH and 8 mM HCI at 45 C. The azide derivativemore » was separated from DNPH reagent and carbonyl derivatives by reverse-phase HPLC and was detected with an UV detector at 309 nm. The detection limit is {le}5 pptv and may be lowered to 1 pptv with further DNPH purification. Interferences from NO, NO{sub 2} PAN, O{sub 3}, HNO{sub 3}, and HCHO were studied and found to be negligible. Ambient HONO concentration was measured simultaneously in downtown Albany, NY, by this method and by an ion chromatographic technique after sampling using a fritted bubbler. The results, from 70 pptv during the day to 1.7 ppbv in the early morning, were in very good agreement from the two techniques, within {+-} 20%.« less

DETERMINATION OF VENLAFAXINE, VILAZODONE AND THEIR MAIN ACTIVE METABOLITES IN HUMAN SERUM BY HPLC-DAD AND HPLC-MS.

PubMed

Petruczynik, Anna; Wroblewski, Karol; Szultka-Mlynska, Malgorzata; Buszewsk, Boguslaw; Karakula-Juchnowicz, Hanna; Gajewski, Jacek; Morylowska-Topolska, Justyna; Waksmundzka-Hajnosi, Monika

2017-05-01

A high performance liquid chromatography (HPLC) method for simultaneous analysis of venlafaxine and its major metabolite 0-desmethylvenlafaxine and vilazodone and its methabolite M10 have been devel- oped and validated. Chromatography was performed on the Phenyl-Hexyl column with mobile phase containing methanol, acetate buffer at pH 3.5 and diethylamine. The application of stationary phase with 7r-7c moieties and mobile phase containing diethylamine as silanol blocker lets to obtain double protection against silanols and thus very high theoretical plate numbers were obtained. The good separation selectivity, good peaks' symmetry and very high systems efficiency for all investigated compounds were obtained in applied chromatographic system. The method is very efficient and suitable for the analysis of investigated drugs and their metabolites in human serum for patients' pharmacotherapy control.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

PubMed

Kramer, S; Blaschke, G

2001-02-10

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

Development of a method for the analysis of nucleotides from the mantle tissue of the mussel Mytilus galloprovincialis.

PubMed

Blanco López, S L; Moal, J; San Juan Serrano, F

2000-09-01

Reversed-phase HPLC was applied to obtain a sensitive and efficient means for quantitating nucleotides in the mussel Mytilus galloprovincialis. We obtained a good separation of adenylic, guanylic, uridylic and cytidylic nucleotides. Adenine nucleotides play a critical role in the regulation and integration of cellular metabolism; particularly in the mantle tissue in the mussel, they are involved in the regulation of the enzyme glycogen phosphorylase, a key enzyme in the transfer of bioenergetic reserves (glycogen) to gametogenic development; it is of great importance to have a measure of the concentrations in vivo during the reproductive cycle of the organism. Different elution conditions were tested: isocratic versus step gradient elution, different mobile phase pH and the type and proportion of ion-pairing agent added to the mobile phase. The best method was selected and the separation and accurate determination of adenine, citidine, guanine and uridine nucleotides was accomplished within a 20-min run, with UV-Vis detection (254 nm).

Determination of 4-nonylphenol and 4-octylphenol in human blood samples by high-performance liquid chromatography with multi-electrode electrochemical coulometric-array detection.

PubMed

Inoue, K; Yoshimura, Y; Makino, T; Nakazawa, H

2000-11-01

Alkylphenols can affect human health because they disrupt the endocrine system. In this study, an analytical method for determining trace amounts of 4-nonylphenol (NP) and 4-octylphenol (OP) in human blood samples was developed. Reversed-phase HPLC with multi-electrode electrochemical coulometric-array detection was used for the determination of NP and OP in plasma and serum samples prepared with a solid-phase extraction method. The separation was achieved using an isocratic mobile phase of 0.7% phosphoric acid-acetonitrile with a C18 reversed phase column. The detection limits of NP and OP were 1.0 and 0.5 ng ml-1, respectively. The recoveries of NP and OP added to human plasma samples were above 70.0% with a relative standard deviation of less than 15.5%. The method was found to be applicable to the determination of NP and OP in various human blood samples such as serum and plasma.

Direct injection analysis of fatty and resin acids in papermaking process waters by HPLC/MS.

PubMed

Valto, Piia; Knuutinen, Juha; Alén, Raimo

2011-04-01

A novel HPLC-atmospheric pressure chemical ionization/MS (HPLC-APCI/MS) method was developed for the rapid analysis of selected fatty and resin acids typically present in papermaking process waters. A mixture of palmitic, stearic, oleic, linolenic, and dehydroabietic acids was separated by a commercial HPLC column (a modified stationary C(18) phase) using gradient elution with methanol/0.15% formic acid (pH 2.5) as a mobile phase. The internal standard (myristic acid) method was used to calculate the correlation coefficients and in the quantitation of the results. In the thorough quality parameters measurement, a mixture of these model acids in aqueous media as well as in six different paper machine process waters was quantitatively determined. The measured quality parameters, such as selectivity, linearity, precision, and accuracy, clearly indicated that, compared with traditional gas chromatographic techniques, the simple method developed provided a faster chromatographic analysis with almost real-time monitoring of these acids. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of theanine, GABA, and other amino acids in green, oolong, black, and Pu-erh teas with dabsylation and high-performance liquid chromatography.

PubMed

Syu, Kai-Yang; Lin, Chih-Li; Huang, Hsiu-Chen; Lin, Jen-Kun

2008-09-10

Dabsyl chloride (dimethylaminoazobenzene sulfonyl chloride), a useful chromophoric labeling reagent for amino acids and amines, was developed in this laboratory in 1975. Although several methods have been developed to determine various types of amino acids, a quick and easy method of determining theanine, GABA, and other amino acids has not been developed in one HPLC system. In this paper are analyzed the free amino acid contents of theanine and GABA in different teas (green tea, black tea, oolong tea, Pu-erh tea, and GABA tea) with a dabsylation and reverse phase high-performance liquid chromatography (HPLC) system coupled with a detector at 425 nm absorbance. Two reverse phase columns, Hypersil GOLD and Zorbax ODS, were used and gave different resolutions of dabsyl amino acids in the gradient elution program. The data suggest that the tea source or the steps of tea-making may contribute to the theanine contents variations. High theanine contents of high-mountain tea were observed in both green tea and oolong tea. Furthermore, the raw (natural fermented) Pu-erh tea contained more theanine than ripe (wet fermented) Pu-erh tea, and the GABA contents in normal teas were generally lower than that in GABA tea.

Alternative measures of lipophilicity: from octanol-water partitioning to IAM retention.

PubMed

Giaginis, Costas; Tsantili-Kakoulidou, Anna

2008-08-01

This review describes lipophilicity parameters currently used in drug design and QSAR studies. After a short historical overview, the complex nature of lipophilicity as the outcome of polar/nonpolar inter- and intramolecular interactions is analysed and considered as the background for the discussion of the different lipophilicity descriptors. The first part focuses on octanol-water partitioning of neutral and ionisable compounds, evaluates the efficiency of predictions and provides a short description of the experimental methods for the determination of distribution coefficients. A next part is dedicated to reversed-phase chromatographic techniques, HPLC and TLC in lipophilicity assessment. The two methods are evaluated for their efficiency to simulate octanol-water and the progress achieved in the refinement of suitable chromatographic conditions, in particular in the field of HPLC, is outlined. Liposomes as direct models of biological membranes are examined and phospolipophilicity is compared to the traditional lipophilicity concept. Difficulties associated with liposome-water partitioning are discussed. The last part focuses on Immobilised Artificial Membrane (IAM) chromatography as an alternative which combines membrane simulation with rapid measurements. IAM chromatographic retention is compared to octanol-water and liposome-water partitioning as well as to reversed-phase retention and its potential to predict biopartitioning and biological activities is discussed.

A float mechanism of retention in reversed-phase chromatography

NASA Astrophysics Data System (ADS)

Deineka, V. I.; Deineka, L. A.; Saenko, I. I.; Chulkov, A. N.

2015-07-01

A float mechanism of retention in reversed-phase HPLC is proposed as an alternative to the known mechanisms of the distribution and hydrophobic expulsion of sorbate to the surface of a sorbent. Experimental data that the sorption of a flavylium structure is poorly sensitive to the position of OH groups, and that the retention of anthocyanins depends on the length of bonded alkyl radicals of reversed phase, form the basis of the proposed hypothesis. It is noted that the retention of anthocyanins depends on the orientation of hydroxyl groups in carbohydrate radicals, due to which the chromatographic behavior of anthocyanins is different for glucosides and galactosides, for arabinosides and xylosides, and so on. In other words, retention is a reliable indicator of the composition of a carbohydrate fragment. It is concluded that carbohydrate radicals serve as unique floats, while flat flavilic ions penetrate into the bonded phase. The existence of floats is the main reason for the lower efficiency (of the number of theoretical plates) of the peaks of anthocyanins. It is shown that if two carbohydrate radicals are present at different sites of aglycone (a two-float sorbate), the peaks of the substance are characterized by substantial additional broadening.

Leishmanicidal and cytotoxic activity from plants used in Tacana traditional medicine (Bolivia).

PubMed

Arévalo-Lopéz, Diandra; Nina, Nélida; Ticona, Juan C; Limachi, Ivan; Salamanca, Efrain; Udaeta, Enrique; Paredes, Crispin; Espinoza, Boris; Serato, Alcides; Garnica, David; Limachi, Abigail; Coaquira, Dayana; Salazar, Sarah; Flores, Ninoska; Sterner, Olov; Giménez, Alberto

2018-04-24

Thirty-eight Tacana medicinal plant species used to treat skin problems, including leishmania ulcers, skin infections, inflammation and wound healing, were collected in the community of Buena Vista, Bolivia, with the Tacana people. Twenty two species are documented for the first time as medicinal plants for this ethnic group living in the northern area of the Department of La Paz. To evaluate the leishmanicidal effect (IC 50 ) and cytotoxicity (LD 50 ) of the selected plants. To carry out bioguided studies on the active extracts. To assess the potential of Bolivian plant biodiversity associated with traditional knowledge in the discovery of alternative sources to fight leishmaniasis. Seventy three ethanol extracts were prepared from 38 species by maceration and were evaluated in vitro against promastigotes of Leishmania amazonensis and L. braziliensis. Active extracts (IC 50 ≤ 50 μg/mL) were fractionated by chromatography on Silica gel column and the fractions were assessed against the two Leishmania strains. The most active fractions and the crude extracts were evaluated against reference strains of L. amazonensis, L. braziliensis, L. aethiopica, two native strains (L. Lainsoni and L. braziliensis) and for cytotoxicity against HeLa cells. The chromatographic profile of the active fractions was obtained by reverse phase chromatography using HPLC. From the 73 extracts, 39 extracts (53.4%) were inactive and 34 showed activity. Thirteen species were sselected for bioguided studies. The crude extracts and their 36 fractions were evaluated against two Leishmania strains. The most active fraction were tested in a panel of five leishmania strains and for cytotoxicity. The Selective Index (SI = LD 50 /IC 50 ) was calculated, and were generally low. Retention time and UV spectra were recorded for the active fractions by HPLC-DAD using a reverse phase column. Profiles were very different from each other, showing the presence of different compounds. Bolivian traditional knowledge from the Tacanba was useful to identify plants with effect on Leishmania promastigotes. Chromatographic bioguided studies showed stronger leishmanicidal and cytotoxic activity for the medium polar fraction. HPLC analysis showed different chromatographic profiles of the active fractions. Copyright © 2018 Elsevier B.V. All rights reserved.

Diazonium modification of porous graphitic carbon with catechol and amide groups for hydrophilic interaction and attenuated reversed phase liquid chromatography.

PubMed

Iverson, Chad D; Zhang, Ya; Lucy, Charles A

2015-11-27

Porous graphitic carbon (PGC) is an increasingly popular and attractive phase for HPLC on account of its chemical and thermal stability, and its unique separation mechanism. However, native PGC is strongly hydrophobic and in some instances excessively retentive. As part of our effort to build a library of hydrophilic covalently modified PGC phases, we functionalized PGC with catechol and amide groups by means of aryl diazonium chemistry to produce two new phases. Successful grafting was confirmed by X-ray photoelectron spectroscopy (XPS). Under HILIC conditions, the Catechol-PGC showed up to 5-fold increased retention relative to unmodified PGC and selectivity that differed from four other HILIC phases. Under reversed phase conditions, the Amide-PGC reduced the retentivity of PGC by almost 90%. The chromatographic performance of Catechol-PGC and Amide-PGC is demonstrated by separations of nucleobases, nucleosides, phenols, alkaline pharmaceuticals, and performance enhancing stimulants. These compounds had retention factors (k) ranging from 0.5 to 13. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of etoxazole residues in fruits and vegetables by SPE clean-up and HPLC-DAD.

PubMed

Malhat, Farag; Badawy, Hany; Barakat, Dalia; Saber, Ayman

2013-01-01

A method for determination of etoxazole residues in apples, strawberries and green beans was developed and validated. The analyte was extracted with acetonitrile from foodstuff and a charcoal-celite cartridge was used for clean-up of raw extracts. Reversed phase high performance liquid chromatography with photodiode array detector (HPLC-DAD) was used for the determination and quantification of etoxazole residues in the studied samples. The calibration graphs of etoxazole in a solvent or three blank matrixes were linear within the tested intervals 0.01-2 mg L(-1), with correlation coefficient of determination >0.999. The combined solid phase extraction (SPE) clean-up and the chromatographic method steps were sensitive and reliable for simultaneous determination of etoxazole residues in the studied samples. The average recoveries of etoxazole in the tested foodstuffs were between 93.4 to 102% at spiking levels of 0.01, 0.10, and 0.50 mg kg(-1), with relative standard deviations ranging from 2.8 to 4.7%, in agreement with directives for method validation in residue analyses. The limit of detection (LOD) of the HPLC-DAD system was 100 pg. The limit of quantification of the entire method was 0.01 mg kg(-1).

Development and validation of RP HPLC method to determine nandrolone phenylpropionate in different pharmaceutical formulations.

PubMed

Mukherjee, Jayanti; Das, Ayan; Chakrabarty, Uday Sankar; Sahoo, Bijay Kumar; Dey, Goutam; Choudhury, Hira; Pal, Tapan Kumar

2011-01-01

This study describes development and subsequent validation of a reversed phase high performance liquid chromatographic (RP-HPLC) method for the estimation of nandrolone phenylpropionate, an anabolic steroid, in bulk drug, in conventional parenteral dosage formulation and in prepared nanoparticle dosage form. The chromatographic system consisted of a Luna Phenomenex, CN (250 mm x 4.6 mm, 5 microm) column, an isocratic mobile phase comprising 10 mM phosphate buffer and acetonitrile (50:50, v/v) and UV detection at 240 nm. Nandrolone phenylpropionate was eluted about 6.3 min with no interfering peaks of excipients used for the preparation of dosage forms. The method was linear over the range from 0.050 to 25 microg/mL in raw drug (r2 = 0.9994). The intra-day and inter-day precision values were in the range of 0.219-0.609% and 0.441-0.875%, respectively. Limits of detection and quantitation were 0.010 microg/mL and 0.050 microg/mL, respectively. The results were validated according to International Conference on Harmonization (ICH) guidelines in parenteral and prepared nanoparticle formulation. The validated HPLC method is simple, sensitive, precise, accurate and reproducible.

Purification, characterization, and sequencing of antimicrobial peptides, Cy-AMP1, Cy-AMP2, and Cy-AMP3, from the Cycad (Cycas revoluta) seeds.

PubMed

Yokoyama, Seiya; Kato, Kouji; Koba, Atsuko; Minami, Yuji; Watanabe, Keiichi; Yagi, Fumio

2008-12-01

Novel antimicrobial peptides (AMP), designated Cy-AMP1, Cy-AMP2, and Cy-AMP3, were purified from seeds of the cycad (Cycas revoluta) by a CM cellulofine column, ion-exchange HPLC on SP COSMOGEL, and reverse-phase HPLC. They had molecular masses of 4583.2 Da, 4568.9 Da and 9275.8 Da, respectively, by MALDI-TOF MS analysis. Half of the amino acid residues of Cy-AMP1 and Cy-AMP2 were cysteine, glycine and proline, and their sequences were similar. The sequence of Cy-AMP3 showed high homology to various lipid transfer proteins. For Cy-AMP1 and Cy-AMP2, the concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic fungi, Gram-positive and Gram-negative bacteria were 7.0-8.9 microg/ml. The Cy-AMP3 had weak antimicrobial activity. The structural and antimicrobial characteristics of Cy-AMP1 and Cy-AMP2 indicated that they are a novel type of antimicrobial peptide belonging to a plant defensin family.

A high-performance liquid chromatography-based radiometric assay for acyl-CoA:alcohol transacylase from jojoba.

PubMed

Garver, W S; Kemp, J D; Kuehn, G D

1992-12-01

Acyl-CoA:alcohol transacylase catalyzes the final step in the biosynthesis of storage liquid wax esters from acyl-CoA fatty acids and fatty alcohols in a limited number of microbes, algae, and Simmondsia chinensis Link (jojoba). An improved and automated method of enzyme assay for this catalyst from cotyledons of jojoba is described. The assay method uses reversed-phase C18 high performance liquid chromatography (HPLC) to separate the labeled C30:1 liquid wax product, [14C]-dodecanyl-octadecenoate, from the unreacted substrate, [14C]octadecenoyl-CoA (oleyl-CoA), and other components produced from enzymes present in the crude homogenate of jojoba cotyledons, including [14C]-octadecenoic acid (oleic acid) and [14C]octadecenol (oleyol). Methods are also described for microscale chemical synthesis in one vessel of 14C-radiolabeled substrates and products for the transacylase. These labeled reagents are required to confirm the HPLC separations of reaction products. The radioactive components are quantitated using an on-line flow-through scintillation detector enabling sensitive and precise analysis of the reaction products.

A rapid HPLC method for simultaneous determination of tretinoin and isotretinoin in dermatological formulations.

PubMed

Tashtoush, Bassam M; Jacobson, Elaine L; Jacobson, Myron K

2007-02-19

A rapid method using an isocratic high-pressure liquid chromatography and UV detection for determination of both all-trans retinoic acid (tretinoin) and 13-cis retinoic acid (isotretinoin) in dermatological preparations is presented. Tretinoin and isotretinoin samples were extracted with acetonitrile by a procedure that can be completed in less than 10 min. Subsequent separation and quantification of amounts as low as 10 pmol was accomplished in less than 15 min using reversed-phase HPLC with isocratic elution with 0.01% trifluoroacetic acid (TFA)/acetonitrile (15:85, v/v). Validation experiments confirmed the precision and accuracy of the method. When applied to commercial tretinoin samples, recoveries of 104.9% for cream formulations and 107.7% for gel formulations were obtained. Application of the method for analysis of a tretinoin cream exposed to solar simulated light (SSL) demonstrated detection of the major photoisomerization product isotretinoin as well as 9-cis retinoic acid, demonstrating the utility of the method for studies of tretinoin photostability. The method should also facilitate studies of the formulation compatibility and photocompatibility of tretinoin with agents that may improve its clinical tolerability.

Measurement of stable isotopic enrichment and concentration of long-chain fatty acyl-carnitines in tissue by HPLC-MS.

PubMed

Sun, Dayong; Cree, Melanie G; Zhang, Xiao-Jun; Bøersheim, Elisabet; Wolfe, Robert R

2006-02-01

We have developed a new method for the simultaneous measurements of stable isotopic tracer enrichments and concentrations of individual long-chain fatty acyl-carnitines in muscle tissue using ion-pairing high-performance liquid chromatography-electrospray ionization quadrupole mass spectrometry in the selected ion monitoring (SIM) mode. Long-chain fatty acyl-carnitines were extracted from frozen muscle tissue samples by acetonitrile/methanol. Baseline separation was achieved by reverse-phase HPLC in the presence of the volatile ion-pairing reagent heptafluorobutyric acid. The SIM capability of a single quadrupole mass analyzer allows further separation of the ions of interest from the sample matrixes, providing very clean total and selected ion chromatograms that can be used to calculate the stable isotopic tracer enrichment and concentration of long-chain fatty acyl-carnitines in a single analysis. The combination of these two separation techniques greatly simplifies the sample preparation procedure and increases the detection sensitivity. Applying this protocol to biological muscle samples proves it to be a very sensitive, accurate, and precise analytical tool.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

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.

Behavior of new complexes of tetrakis(4-methoxylphenyl)porphyrin with heavy rare earth elements in reversed-phase high performance liquid chromatography.

PubMed

Zhang, Jun-Feng; Wang, Hong; Hou, An-Xin; Wang, Chang-Fa; Zhang, Hua-Shan

2004-08-01

An HPLC method has been developed for the separation of new complexes of tetrakis(4-methoxylphenyl)porphyrin (TMOPP) with four heavy rare earth elements (RE = Y, Er, Tm, and Yb). The function of amine and acid in the mobile phase has been investigated and a reasonable explanation is presented. Successful separation of the RE-TMOPP-Cl complexes is accomplished in 10 min with a mobile phase consisting of methanol-water-acetic acid-triethanolamine. The detection limits (S/N= 3) for the four complexes are 0.01 microg/mL. This method is rapid, sensitive, and simple.

Quality Evaluation of Juniperus rigida Sieb. et Zucc. Based on Phenolic Profiles, Bioactivity, and HPLC Fingerprint Combined with Chemometrics

PubMed Central

Liu, Zehua; Wang, Dongmei; Li, Dengwu; Zhang, Shuai

2017-01-01

Juniperus rigida (J. rigida) which is endemic to East Asia, has traditionally been used as an ethnomedicinal plant in China. This study was undertaken to evaluate the quality of J. rigida samples derived from 11 primary regions in China. Ten phenolic compounds were simultaneously quantified using reversed-phase high-performance liquid chromatography (RP-HPLC), and chlorogenic acid, catechin, podophyllotoxin, and amentoflavone were found to be the main compounds in J. rigida needles, with the highest contents detected for catechin and podophyllotoxin. J. rigida from Jilin (S9, S10) and Liaoning (S11) exhibited the highest contents of phenolic profiles (total phenolics, total flavonoids and 10 phenolic compounds) and the strongest antioxidant and antibacterial activities, followed by Shaanxi (S2, S3). A similarity analysis (SA) demonstrated substantial similarities in fingerprint chromatograms, from which 14 common peaks were selected. The similarity values varied from 0.85 to 0.98. Chemometrics techniques, including hierarchical cluster analysis (HCA), principal component analysis (PCA), and discriminant analysis (DA), were further applied to facilitate accurate classification and quantification of the J. rigida samples derived from the 11 regions. The results supported HPLC data showing that all J. rigida samples exhibit considerable variations in phenolic profiles, and the samples were further clustered into three major groups coincident with their geographical regions of origin. In addition, two discriminant functions with a 100% discrimination ratio were constructed to further distinguish and classify samples with unknown membership on the basis of eigenvalues to allow optimal discrimination among the groups. Our comprehensive findings on matching phenolic profiles and bioactivities along with data from fingerprint chromatograms with chemometrics provide an effective tool for screening and quality evaluation of J. rigida and related medicinal preparations. PMID:28469573

A comparative evaluation of different ionic liquids for arsenic species separation and determination in wine varietals by liquid chromatography - hydride generation atomic fluorescence spectrometry.

PubMed

Castro Grijalba, Alexander; Fiorentini, Emiliano F; Martinez, Luis D; Wuilloud, Rodolfo G

2016-09-02

The application of different ionic liquids (ILs) as modifiers for chromatographic separation and determination of arsenite [As(III)], arsenate [As(V)], dimethylarsonic acid (DMA) and monomethylarsonic acid (MMA) species in wine samples, by reversed-phase high performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry detection (RP-HPLC-HG-AFS) was studied in this work. Several factors influencing the chromatographic separation of the As species, such as pH of the mobile phase, buffer solution concentration, buffer type, IL concentration and length of alkyl groups in ILs were evaluated. The complete separation of As species was achieved using a C18 column in isocratic mode with a mobile phase composed of 0.5% (v/v) 1-octyl-3-methylimidazolium chloride ([C8mim]Cl) and 5% (v/v) methanol at pH 8.5. A multivariate methodology was used to optimize the variables involved in AFS detection of As species after they were separated by HPLC. The ILs showed remarkable performance for the separation of As species, which was obtained within 18min with a resolution higher than 0.83. The limits of detection for As(III), As(V), MMA and DMA were 0.81, 0.89, 0.62 and 1.00μg As L(-1). The proposed method was applied for As speciation analysis in white and red wine samples originated from different grape varieties. Copyright © 2016 Elsevier B.V. All rights reserved.

A validated solid-phase extraction HPLC method for the simultaneous determination of the citrus flavanone aglycones hesperetin and naringenin in urine.

PubMed

Kanaze, Feras Imad; Kokkalou, Eugene; Georgarakis, Manolis; Niopas, Ioannis

2004-09-21

A simple, specific, precise, accurate, and robust HPLC assay for the simultaneous analysis of hesperetin and naringenin in human urine was developed and validated. Urine samples were incubated with beta-glucuronidase/sulphatase and the analytes were isolated by solid-phase extraction using C18 cartridges and separated on a C8 reversed phase column using a mixture of methanol/water/acetic acid (40:58:2, v/v/v) at 45 degrees C. The method was found to be linear in the 50-1200 ng/ml concentration range for both hesperetin and naringenin (r > 0.999). The accuracy of the method was greater than 94.8%, while the intra- and inter-day precision for hesperetin was better than 4.9 and 8.2%, respectively and for naringenin was better than 5.3 and 7.8%, respectively. Recovery for hesperetin, naringenin and internal standard 7-ethoxycoumarin was greater than 70.9%. The method has been applied for the determination of hesperetin and naringenin in urine samples obtained from a male volunteer following a single 300 mg oral dose of each of the corresponding flavanone glycosides hesperidin and naringin. The intra- and inter-day reproducibility through enzyme hydrolysis was less than 3.9% for both total (free + conjugated) hesperetin and naringenin. Stability studies showed urine quality control samples to be stable for both hesperetin and naringenin through three freeze-thaw cycles and at room temperature for 24 h (error < or = 3.6%).

Isolation and identification of human metabolites from a novel anti-tumor candidate drug 5-chlorogenic acid injection by HPLC-HRMS/MSn and HPLC-SPE-NMR.

PubMed

Ren, Tiankun; Wang, Yanan; Wang, Caihong; Zhang, Mengtian; Huang, Wang; Jiang, Jiandong; Li, Wenbin; Zhang, Jinlan

2017-12-01

A novel anti-tumor candidate drug, 5-chlorogenic acid (5-CQA) injection, was used for the treatment of malignant glioma in clinical trial (phase I) in China. The isolation and identification of the metabolites of 5-CQA injection in humans were investigated in the present study. Urine and feces samples obtained after intramuscular administration of 5-CQA injection to healthy adults have been analyzed by high-performance liquid chromatography coupled with high-resolution mass and multiple-stage mass spectrometry (HPLC-HRMS/MS n ). No metabolite was detected in human feces; however, in human urine, a total of six metabolites were identified including isomerized 5-CQA (P1 and P2), hydrolyzed 5-CQA (M1and M2), and methylated 5-CQA (M3 and M4). Among them, M3 and M4 were the main metabolites and target analytes for human mass balance study. Additionally, the structure of M3 and M4 was characterized by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance (HPLC-SPE-NMR), and the results demonstrated that the methoxy group of M3 and M4 was exclusively attributed to C-3' and C-4', respectively. Due to the unavailability of commercial reference, the pure products of M3 and M4 were synthesized by 5-CQA methylation and followed by isolation and purification. Moreover, the potential activity of M3 and M4 on malignant glioma was predicted using a reverse molecular docking analysis on eight malignant glioma-related pathways. The results showed that M3 and M4 had various interactions against malignant glioma-related targets. Our study provides an insight into the metabolism of 5-CQA injection in humans and supports the clinical human mass balance study. Graphical abstract ᅟ.

One-pot synthesized functionalized mesoporous silica as a reversed-phase sorbent for solid-phase extraction of endocrine disrupting compounds in milks.

PubMed

Gañán, Judith; Morante-Zarcero, Sonia; Pérez-Quintanilla, Damián; Marina, María Luisa; Sierra, Isabel

2016-01-08

A new procedure for the determination of 12 naturally occurring hormones and some related synthetic chemicals in milk, commonly used as growth promoters in cattle, is reported. The method is based on liquid-liquid extraction followed by solid-phase extraction (SPE) using a new one-pot synthesized ordered mesoporous silica (of the SBA-15 type) functionalized with octadecyl groups (denoted as SBA-15-C18-CO) as reversed-phase sorbent. The analytes were eluted with methanol and then submitted to HPLC with diode array detection. Under optimal conditions, the method quantification limit for the analytes ranged from 0.023 to 1.36μg/mL. The sorbent affored the extraction of estrone, 17β-estradiol, estriol, progesterone, hexestrol, diethylstilbestrol, 4-androstene-3,17-dione, ethinylestradiol, 17α-methyltestosterone, nandrolone, prednisolone and testosterone with mean recoveries ranging from 72% to 105% (except for diethylstilbestrol) with RSD<11%. These results were comparable and, in some cases, even better than those obtained with other extraction methods, therefore SBA-15-C18-CO mesoporous silica possess a high potential as a reversed-phase sorbent for SPE of the 12 mentioned endocrine disrupting compounds in milk samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor with HPLC and electrochemical detection.

PubMed

Kuhlmann, O; Stoldt, G; Struck, H G; Krauss, G J

1998-09-01

A sensitive and selective bioanalytical method for simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor using reversed-phase HPLC and electrochemical detection is described. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microns; 150 x 4.6 mm I.D.). This support is coated with a hydrophilic polyoxyethylenepolymer. It allows protein-containing samples to be injected directly onto the column. The electrochemical detector permit a detection limit of 500 pg diclofenac per ml (daily relative standard deviation 6.3%) and 50 ng oxybuprocaine per ml (daily R.S.D. 2.6%), respectively. Results of administered and measured drug-concentrations in time dependent decrease are presented.

Multiple forms of statherin in human salivary secretions.

PubMed

Jensen, J L; Lamkin, M S; Troxler, R F; Oppenheim, F G

1991-01-01

Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SV1, statherin SV2, and statherin SV3. SV1 is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene.

Isolation and characterization of an anticoagulant present in the salivary glands of the bont-legged tick, Hyalomma truncatum.

PubMed

Joubert, A M; Crause, J C; Gaspar, A R; Clarke, F C; Spickett, A M; Neitz, A W

1995-02-01

A low molecular mass anticoagulant (17 kDa) was isolated from the salivary glands of prefed female Hyalomma truncatum ticks by means of reverse phase and anion-exchange HPLC. Trypsin digestion and amino acid analysis confirmed the protein nature of the anticoagulant. The inhibitor appears to be uncompetitive with a Ki of 6.9 x 10(-10)M. The target of the anticoagulant is factor Xa at the junction of the extrinsic and intrinsic pathways. This may be crucial for the survival of the tick, making it feasible to investigate the possibility of vaccination with this antihaemostatic against tick feeding. In addition, tick anticoagulants may possibly have therapeutic application in controlling thrombosis.

Preparation of stationary phases for reversed-phase high-performance liquid chromatography using thermal treatments at high temperature.

PubMed

Vigna, Camila R M; Bottoli, Carla B G; Collins, Kenneth E; Collins, Carol H

2007-07-13

Batches of poly(methyloctylsiloxane) (PMOS)-loaded silica were prepared by deposition from a solution of PMOS into the pores of HPLC silica. Portions of PMOS-loaded silica were subjected to a thermal treatment at 100 degrees C for 24h (condition 1) in a tube furnace under a nitrogen atmosphere. After that, the material was heated for 4h at higher temperatures (150-400 degrees C) (condition 2). Heating at higher temperatures produces polymer bilayers. Non-immobilized and thermally treated stationary phases were characterized by percent carbon, (29)Si cross-polarization magic angle spinning nuclear magnetic resonance spectroscopy and reversed-phase chromatographic performance. The results show that thermal treatment between 150 and 300 degrees C accelerates the immobilization process, possibly due to some bond breaking of the polysiloxane, with formation of strong linkages to the surface of the support, resulting in more complete coverage of the silica. The chromatographic results show an improvement of efficiency with the increase of the temperature of condition 2 up to 300 degrees C and an increase in the resolution of the components, mainly for the phase heated at 300 degrees C. Such results demonstrate that a two-step thermal treatment (100 degrees C then 150-300 degrees C) produces stationary phases with good properties for use in reversed-phase high-performance liquid chromatography.

Novel Pathways of Nitroaromatic Metabolism: Hydroxylamine Formation, Reactivity and Potential for Ring Fission for Destruction of TNT-CU1214

DTIC Science & Technology

2005-08-15

phase was terminated when HPLC analysis showed that the initial TNT was in the form of DHA6NT and aminophenols , products that have been reported...terminated when HPLC analysis showed that the initial TNT was in the form of DHA6NT and aminophenols , products that have been reported previously (2...Department of Defense HPLC High Performance Liquid Chromatography IPTG Isopropyl-β-D-1-thiogalactopyranoside LB Luria-Bertani Broth NB Nitrobenzene

Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

PubMed

Bhushan, Ravi; Nagar, Hariom

2015-03-01

Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.

A Fast and Validated Reversed-Phase HPLC Method for Simultaneous Determination of Simvastatin, Atorvastatin, Telmisartan and Irbesartan in Bulk Drugs and Tablet Formulations

PubMed Central

Alhazmi, Hassan A.; Alnami, Ahmed M.; Arishi, Mohammed A. A.; Alameer, Raad K.; Al Bratty, Mohammed; Rehman, Zia ur; Javed, Sadique A.; Arbab, Ismail A.

2017-01-01

The aim of this study was to develop and validate a fast and simple reversed-phase HPLC method for simultaneous determination of four cardiovascular agents—atorvastatin, simvastatin, telmisartan and irbesartan in bulk drugs and tablet oral dosage forms. The chromatographic separation was accomplished by using Symmetry C18 column (75 mm × 4.6 mm; 3.5 μ) with a mobile phase consisting of ammonium acetate buffer (10 mM; pH 4.0) and acetonitrile in a ratio 40:60 v/v. Flow rate was maintained at 1 mL/min up to 3.5 min, and then suddenly changed to 2 mL/min till the end of the run (7.5 min). The data was acquired using ultraviolet detector monitored at 220 nm. The method was validated for linearity, precision, accuracy and specificity. The developed method has shown excellent linearity (R2 > 0.999) over the concentration range of 1–16 µg/mL. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.189–0.190 and 0.603–0.630 µg/mL, respectively. Inter-day and intra-day accuracy and precision data were recorded in the acceptable limits. The new method has successfully been applied for quantification of all four drugs in their tablet dosage forms with percent recovery within 100 ± 2%. PMID:29257120

Development and optimisation of an HPLC-DAD-ESI-Q-ToF method for the determination of phenolic acids and derivatives.

PubMed

Restivo, Annalaura; Degano, Ilaria; Ribechini, Erika; Colombini, Maria Perla

2014-01-01

A method for the HPLC-MS/MS analysis of phenols, including phenolic acids and naphtoquinones, using an amide-embedded phase column was developed and compared to the literature methods based on classical C18 stationary phase columns. RP-Amide is a recently developed polar embedded stationary phase, whose wetting properties mean that up to 100% water can be used as an eluent. The increased retention and selectivity for polar compounds and the possibility of working in 100% water conditions make this column particularly interesting for the HPLC analysis of phenolic acids and derivatives. In this study, the chromatographic separation was optimised on an HPLC-DAD, and was used to separate 13 standard phenolic acids and derivatives. The method was validated on an HPLC-ESI-Q-ToF. The acquisition was performed in negative polarity and MS/MS target mode. Ionisation conditions and acquisition parameters for the Q-ToF detector were investigated by working on collision energies and fragmentor potentials. The performance of the method was fully evaluated on standards. Moreover, several raw materials containing phenols were analysed: walnut, gall, wine, malbec grape, French oak, red henna and propolis. Our method allowed us to characterize the phenolic composition in a wide range of matrices and to highlight possible matrix effects.

Simultaneous quantification of metronidazole, tinidazole, ornidazole and morinidazole in human saliva.

PubMed

Wang, Yongqing; Zhang, Peipei; Jiang, Ningling; Gong, Xiaojian; Meng, Ling; Wang, Dewang; Ou, Ning; Zhang, Haibo

2012-06-15

The aim of this study was to develop a rapid and sensitive method for the simultaneous quantification of metronidazole (MEZ), tinidazole (TNZ), ornidazole (ONZ) and morinidazole (MNZ) in human saliva. A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection at 318 nm was carried out on a C18 column, using a mixture of potassium dihydrogen phosphate buffer, acetonitrile, and methanol (55:15:30, v/v/v) as a mobile phase with a flow rate of 1.0 ml/min. The saliva samples (100 μl) were firstly deproteinized by precipitation with methanol (400 μl), after which they were centrifuged and the supernatants were directly injected into the HPLC system. This method produced linear responses in the concentration ranges of 25.2-5040.0, 23.9-4790.0, 25.4-5080.0, 25.0-5000.0 ng/ml with detection limits of 6.0, 17.6, 10.0 and 11.3 ng/ml for MEZ, TNZ, ONZ and MNZ (S/N=3), respectively. The methods were validated in terms of intra- and inter-batch precision (within 7.3% and 9.1%, respectively), accuracy, linearity, recovery and stability. The study proved that HPLC is both sensitive and selective for the simultaneous quantification of MEZ, TNZ, ONZ and MNZ in human saliva using a single mobile phase. Copyright © 2012 Elsevier B.V. All rights reserved.

A green ultrasonic-assisted liquid-liquid microextraction based on deep eutectic solvent for the HPLC-UV determination of ferulic, caffeic and cinnamic acid from olive, almond, sesame and cinnamon oil.

PubMed

Khezeli, Tahere; Daneshfar, Ali; Sahraei, Reza

2016-04-01

A simple, inexpensive and sensitive ultrasonic-assisted liquid-liquid microextraction method based on deep eutectic solvent (UALLME-DES) was used for the extraction of three phenolic acids (ferulic, caffeic and cinnamic) from vegetable oils. In a typical experiment, deep eutectic solvent as green extraction solvent was added to n-hexane (as a typical oil medium) containing target analytes. Subsequently, the extraction was accelerated by sonication. After the extraction, phase separation (DES rich phase/n-hexane phase) was performed by centrifugation. DES rich phase (lower phase) was withdrawn by a micro-syringe and submitted to isocratic reverse-phase HPLC with UV detection. Under optimum conditions obtained by response surface methodology (RSM) and desirability function (DF), the method has good linear calibration ranges (between 1.30 and 1000 µg L(-1)), coefficients of determination (r(2)>0.9949) and low limits of detection (between 0.39 and 0.63 µg L(-1)). This procedure was successfully applied to the determination of target analytes in olive, almond, sesame and cinnamon oil samples. The relative mean recoveries ranged from 94.7% to 104.6%. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection.

PubMed

Xie, Kaizhou; Jia, Longfei; Yao, Yilin; Xu, Dong; Chen, Shuqing; Xie, Xing; Pei, Yan; Bao, Wenbin; Dai, Guojun; Wang, Jinyu; Liu, Zongping

2011-08-01

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

[Determination of triterpenoic acids in fruits of Ziziphus jujuba using HPLC-MS with polymeric ODS column].

PubMed

Zhang, Yong; Zhou, An; Xie, Xiao-Mei

2013-03-01

A simple and sensitive method has been developed to simultaneously determine betunilic acid, oleanolic acid and ursolic acid in the fruits of Ziziphus jujuba from different regions by HPLC-MS. This HPLC assay was performed on PAH polymeric C18 bonded stationary phase column with mobile phase contained acetonitrile-water (90: 10) and with negative ESI detection mode. The developed approach was characterized by short time consumption for chromatographic separation, high sensitivity and good reliability so as to meet the requirements for rapid analysis of large-batch fruits of Z. jujuba from different habitats.

Development and validation of a novel stability-indicating HPLC method for the simultaneous assay of betamethasone-17-valerate, fusidic acid, potassium sorbate, methylparaben and propylparaben in a topical cream preparation.

PubMed

Byrne, Jonathan; Velasco-Torrijos, Trinidad; Reinhardt, Robert

2014-08-05

A novel stability-indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous assay of betamethasone-17-valerate, fusidic acid and potassium sorbate as well as methyl- and propylparaben in a topical cream preparation has been developed. A 100mm×3.0mm ID. Ascentis Express C18 column maintained at 30°C and UV detection at 240nm were used. A gradient programme was employed at a flow-rate of 0.75ml/min. Mobile phase A comprised of an 83:17 (v/v) mixture of acetonitrile and methanol and mobile phase B of a 10g/l solution of 85% phosphoric acid in purified water. The method has been validated according to current International Conference on Harmonisation (ICH) guidelines and applied during formulation development and stability studies. The procedure has been shown to be stability-indicating for the topical cream. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous analysis of thiamphenicol and its prodrug thiamphenicol glycinate in human plasma and urine by high performance liquid chromatography: application to pharmacokinetic study.

PubMed

Chen, Xijing; Yang, Bing; Ni, Liang; Wang, Guangji

2006-06-07

A simple and sensitive method for simultaneous determination of the active compound, thiamphenicol (TAP) and its prodrug, thiamphenicol glycinate (TG) in human plasma and urine is described. The procedure involved extraction of TG and TAP with ethyl acetate (plasma) or 100-fold dilution with the mobile phase (urine) followed by determination by reversed-phase high performance liquid chromatography (HPLC) with UV detection at 224 nm. Separation of the compounds was achieved on a column packed with Hypersil ODS2. The mobile phase consisted of acetonitrile-water containing 0.003 M tetrabutyl ammonium bromide and 0.056 M ammonium acetate (87:13, v/v) with a flow rate of 1.0 ml/min. The chromatograms did not contain interfering peaks due to the suitable extraction procedure and chromatographic conditions. The calibration curves of TG and TAP were linear ranging from 0.78 to 100 microg/ml in plasma and in urine. The intra-day and inter-day relative standard deviations (S.D.) were less than 10%. The recoveries of TG and TAP in plasma and urine were above 80%. TG was not stable in plasma samples and after extraction at ambient temperature or in freeze-thaw cycles, and hence the samples for injection on HPLC column should be stored in refrigerator or under ice cooling prior to analysis, and the plasma samples should not experience the freeze-thaw cycle more than one time. Unlike TAP, TG could not be detected in most urine samples. Application of this method demonstrated that it was feasible for the clinical pharmacokinetic study.

Identification and characterization of potential impurities of donepezil.

PubMed

Krishna Reddy, K V S R; Moses Babu, J; Kumar, P Anil; Chandrashekar, E R R; Mathad, Vijayavitthal T; Eswaraiah, S; Reddy, M Satyanarayana; Vyas, K

2004-09-03

Five unknown impurities ranging from 0.05 to 0.2% in donepezil were detected by a simple isocratic reversed-phase high performance liquid chromatography (HPLC). These impurities were isolated from crude sample of donepezil using isocratic reversed-phase preparative high performance liquid chromatography. Based on the spectral data (IR, NMR and MS), the structures of these impurities were characterised as 5,6-dimethoxy-2-(4-pyridylmethyl)-1-indanone (impurity I), 4-(5,6-dimethoxy-2,3-dihydro-1H-2-indenylmethyl) piperidine (impurity II), 2-(1-benzyl-4-piperdylmethyl)-5,6-dimethoxy-1-indanol (impurity III) 1-benzyl-4(5,6-dimethoxy-2,3-dihydro-1H-2-indenylmethyl) piperidine (impurity IV) and 1,1-dibenzyl-4(5,6-dimethoxy-1-oxo-2,3-dihydro-2H-2-indenylmethyl)hexahydropyridinium bromide (impurity V). The synthesis of these impurities and their formation was discussed.

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51.

PubMed

Messaoudi, Soumaya; Kergourlay, Gilles; Dalgalarrondo, Michèle; Choiset, Yvan; Ferchichi, Mounir; Prévost, Hervé; Pilet, Marie-France; Chobert, Jean-Marc; Manai, Mohamed; Dousset, Xavier

2012-10-01

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C(18) column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Thermodynamics of the Sorption of Benzimidazoles on Octadecyl Silica Gel from Water-Methanol Eluents

NASA Astrophysics Data System (ADS)

Shafigulin, R. V.; Bulanova, A. V.

2018-02-01

The standard enthalpy and entropy component of transferring benzimidazoles from water-methanol solutions to surfaces of octadecyl silica gel are determined using reversed-phase high-performance liquid chromatography (RP HPLC). The dependences between the enthalpy and polarizability of the molecules of the studied benzimidazoles, the enthalpy and the entropy factor are studied, and the influence of the quantitative composition of the water-methanol solution on the enthalpy are studied.

Sarcoplasmic calcium-binding protein: identification as a new allergen of the black tiger shrimp Penaeus monodon.

PubMed

Shiomi, Kazuo; Sato, Yuichiro; Hamamoto, Shohei; Mita, Hajime; Shimakura, Kuniyoshi

2008-01-01

Tropomyosin and arginine kinase have been identified as crustacean allergens. During purification of arginine kinase from black tiger shrimp Penaeus monodon, we found a new allergen of 20-kDa. A 20-kDa allergen was purified from the abdominal muscle of black tiger shrimp by salting-out, anion-exchange HPLC and reverse-phase HPLC. Following digestion of the 20-kDa allergen with lysyl endopeptidase, peptide fragments were isolated by reverse-phase HPLC, and 2 of them were sequenced. The 20-kDa allergen, together with tropomyosin and arginine kinase purified from black tiger shrimp, was evaluated for IgE reactivity by ELISA. Five species of crustaceans (kuruma shrimp, American lobster, pink shrimp, king crab and snow crab) were surveyed for the 20-kDa allergen by immunoblotting. The 20-kDa allergen was purified from black tiger shrimp and identified as a sarcoplasmic calcium-binding protein (SCP) based on the determined amino acid sequences of 2 enzymatic fragments. Of 16 sera from crustacean-allergic patients, 8 and 13 reacted to SCP and tropomyosin, respectively; the reactivity to arginine kinase was weakly recognized with 10 sera. In immunoblotting, an IgE-reactive 20-kDa protein was also detected in kuruma shrimp, American lobster and pink shrimp but not in 2 species of crab. Preadsorption of the sera with black tiger shrimp SCP abolished the IgE reactivity of the 20-kDa protein, suggesting the 20-kDa protein to be an SCP. SCP is a new crustacean allergen, and distribution of IgE-reactive SCP is probably limited to shrimp and crayfish. (c) 2008 S. Karger AG, Basel.

Modified HPLC-ESI-MS Method for Glycated Hemoglobin Quantification Based on the IFCC Reference Measurement Procedure and Its Application for Quantitative Analyses in Clinical Laboratories of China.

PubMed

Song, Zhixin; Xie, Baoyuan; Ma, Huaian; Zhang, Rui; Li, Pengfei; Liu, Lihong; Yue, Yuhong; Zhang, Jianping; Tong, Qing; Wang, Qingtao

2016-09-01

The level of glycated hemoglobin (HbA1c ) has been recognized as an important indicator of long-term glycemic control. However, the HbA1c measurement is not currently included as a diagnostic determinant in China. Current study aims to assess a candidate modified International Federation of Clinical Chemistry reference method for the forthcoming standardization of HbA1c measurements in China. The HbA1c concentration was measured using a modified high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method. The modified method replaces the propylcyanide column with a C18 reversed-phase column, which has a lower cost and is more commonly used in China, and uses 0.1% (26.5 mmol/l) formic acid instead of trifluoroacetic acid. Moreover, in order to minimize matrix interference and reduce the running time, a solid-phase extraction was employed. The discrepancies between HbA1c measurements using conventional methods and the HPLC-ESI-MS method were clarified in clinical samples from healthy people and diabetic patients. Corresponding samples were distributed to 89 hospitals in Beijing for external quality assessment. The linearity, reliability, and accuracy of the modified HPLC-ESI-MS method with a shortened running time of 6 min were successfully validated. Out of 89 hospitals evaluated, the relative biases of HbA1c concentrations were < 8% for 74 hospitals and < 5% for 60 hospitals. Compared with other conventional methods, HbA1c concentrations determined by HPLC methods were similar to the values obtained from the current HPLC-ESI-MS method. The HPLC-ESI-MS method represents an improvement over existing methods and provides a simple, stable, and rapid HbA1c measurement with strong signal intensities and reduced ion suppression. © 2015 Wiley Periodicals, Inc.

Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus.

PubMed

Barrientos, Leticia; Herrera, Christian L; Montenegro, Gloria; Ortega, Ximena; Veloz, Jorge; Alvear, Marysol; Cuevas, Alejandro; Saavedra, Nicolás; Salazar, Luis A

2013-01-01

Propolis is a non-toxic natural substance with multiple pharmacological properties including anti-cancer, antioxidant, fungicidal, antibacterial, antiviral, and anti-inflammatory among others. The aim of this study was to determine the chemical and botanical characterization of Chilean propolis samples and to evaluate their biological activity against the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Twenty propolis samples were obtained from beekeeping producers from the central and southern regions of Chile. The botanical profile was determined by palynological analysis. Total phenolic contents were determined using colorimetric assays. Reverse phase HPLC and HPLC-MS were used to determine the chemical composition. The minimum inhibitory concentration (MIC) was determined on S. mutans and S. sobrinus. All propolis samples were dominated by structures from native plant species. The characterization by HPLC/MS, evidenced the presence of quercetin, myricetin, kaempferol, rutine, pinocembrin, coumaric acid, caffeic acid and caffeic acid phenethyl ester, that have already been described in these propolis with conventional HPLC. Although all propolis samples inhibited the mutans streptococci growth, it was observed a wide spectrum of action (MIC 0.90 to 8.22 μg mL(-1)). Given that results it becomes increasingly evident the need of standardization procedures, where we combine both the determination of botanical and the chemical characterization of the extracts. Research conducted to date, describes a promising effectiveness of propolis in the prevention of caries and other diseases of the oral cavity, making it necessary to develop studies to identify and understand the therapeutic targets or mechanisms of molecular action of the various compounds present on them.

Anti-chikungunya activity of luteolin and apigenin rich fraction from Cynodon dactylon.

PubMed

Murali, Krishnan Saravana; Sivasubramanian, Srinivasan; Vincent, Savariar; Murugan, Shanmugaraj Bala; Giridaran, Bupesh; Dinesh, Sundaram; Gunasekaran, Palani; Krishnasamy, Kaveri; Sathishkumar, Ramalingam

2015-05-01

To obtain luteolin and apigenin rich fraction from the ethanolic extract of Cynodon dactylon (L.) (C. dactylon) Pers and evaluate the fraction's cytotoxicity and anti-Chikungunya potential using Vero cells. The ethanolic extract of C. dactylon was subjected to silica gel column chromatography to obtain anti-chikungunya virus (CHIKV) fraction. Reverse phase-HPLC and GC-MS studies were carried out to identify the major phytochemicals in the fraction using phytochemical standards. Cytotoxicity and the potential of the fraction against CHIKV were evaluated in vitro using Vero cells. Reduction in viral replication was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) after treating the viral infected Vero cells with the fraction. Reverse Phase-HPLC and GC-MS studies confirmed the presence of flavonoids, luteolin and apigenin as major phytochemicals in the anti-CHIKV ethanolic fraction of C. dactylon. The fraction was found to exhibit potent viral inhibitory activity (about 98%) at the concentration of 50 µg/mL as observed by reduction in cytopathic effect, and the cytotoxic concentration of the fraction was found to be 250 µg/mL. RT-PCR analyses indicated that the reduction in viral mRNA synthesis in fraction treated infected cells was much higher than the viral infected control cells. Luteolin and apigenin rich ethanolic fraction from C. dactylon can be utilized as a potential therapeutic agent against CHIKV infection as the fraction does not show cytotoxicity while inhibiting the virus. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Carotenoids from Foods of Plant, Animal and Marine Origin: An Efficient HPLC-DAD Separation Method.

PubMed

Strati, Irini F; Sinanoglou, Vassilia J; Kora, Lintita; Miniadis-Meimaroglou, Sofia; Oreopoulou, Vassiliki

2012-12-19

Carotenoids are important antioxidant compounds, present in many foods of plant, animal and marine origin. The aim of the present study was to describe the carotenoid composition of tomato waste, prawn muscle and cephalothorax and avian (duck and goose) egg yolks through the use of a modified gradient elution HPLC method with a C 30 reversed-phase column for the efficient separation and analysis of carotenoids and their cis -isomers. Elution time was reduced from 60 to 45 min without affecting the separation efficiency. All- trans lycopene predominated in tomato waste, followed by all- trans -β-carotene, 13- cis -lutein and all- trans lutein, while minor amounts of 9- cis -lutein, 13- cis -β-carotene and 9- cis -β-carotene were also detected. Considering the above findings, tomato waste is confirmed to be an excellent source of recovering carotenoids, especially all- trans lycopene, for commercial use. Xanthophylls were the major carotenoids of avian egg yolks, all- trans lutein and all- trans zeaxanthin in duck and goose egg yolk, respectively. In the Penaeus kerathurus prawn, several carotenoids (zeaxanthin, all- trans -lutein, canthaxanthin, cryptoxanthin, optical and geometrical astaxanthin isomers) were identified in considerable amounts by the same method. A major advantage of this HPLC method was the efficient separation of carotenoids and their cis -isomers, originating from a wide range of matrices.

Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-glucoside, Safety and Anti-Inflammatory Effect

PubMed Central

Miguel, Felipe Galeti; Spinola, Nathália Favaretto; Ribeiro, Diego Luis; Barcelos, Gustavo Rafael Mazzaron; Antunes, Lusânia Maria Greggi; Hori, Juliana Issa; Marquele-Oliveira, Franciane; Rocha, Bruno Alves; Berretta, Andresa Aparecida

2015-01-01

Chamomile is a medicinal plant, which presents several biological effects, especially the anti-inflammatory effect. One of the compounds related to this effect is apigenin, a flavonoid that is mostly found in its glycosylated form, apigenin-7-glucoside (APG), in natural sources. However, the affectivity and safety of this glycoside have not been well explored for topical application. In this context, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC-DAD) method to quantify APG in chamomile preparations. Additionally, the safety and the anti-inflammatory potential of this flavonoid were verified. The RP-HPLC-DAD method was developed and validated with linearity at 24.0–36.0 μg/mL range (r = 0.9994). Intra- and interday precision (RSD) were 0.27–2.66% and accuracy was 98.27–101.21%. The validated method was applied in the analysis of chamomile flower heads, glycolic extract, and Kamillen cream, supporting the method application in the quality control of chamomile preparations. Furthermore, the APG safety was assessed by MTT cytotoxicity assay and mutagenic protocols and the anti-inflammatory activity was confirmed by a diminished TNF-α production showed by mice macrophages treated with APG following LPS treatment. PMID:26421053

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

PubMed Central

Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P.

2014-01-01

Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. PMID:24323699

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

PubMed

Iwamoto, T; Grove, A; Montal, M O; Montal, M; Tomich, J M

1994-06-01

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Quantitative analysis of triacylglycerol regioisomers in fats and oils using reversed-phase high-performance liquid chromatography and atmospheric pressure chemical ionization mass spectrometry.

PubMed

Fauconnot, Laëtitia; Hau, Jörg; Aeschlimann, Jean-Marc; Fay, Laurent-Bernard; Dionisi, Fabiola

2004-01-01

Positional distribution of fatty acyl chains of triacylglycerols (TGs) in vegetable oils and fats (palm oil, cocoa butter) and animal fats (beef, pork and chicken fats) was examined by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to atmospheric pressure chemical ionization using a quadrupole mass spectrometer. Quantification of regioisomers was achieved for TGs containing two different fatty acyl chains (palmitic (P), stearic (S), oleic (O), and/or linoleic (L)). For seven pairs of 'AAB/ABA'-type TGs, namely PPS/PSP, PPO/POP, SSO/SOS, POO/OPO, SOO/OSO, PPL/PLP and LLS/LSL, calibration curves were established on the basis of the difference in relative abundances of the fragment ions produced by preferred losses of the fatty acid from the 1/3-position compared to the 2-position. In practice the positional isomers AAB and ABA yield mass spectra showing a significant difference in relative abundance ratios of the ions AA(+) to AB(+). Statistical analysis of the validation data obtained from analysis of TG standards and spiked oils showed that, under repeatability conditions, least-squares regression can be used to establish calibration curves for all pairs. The regression models show linear behavior that allow the determination of the proportion of each regioisomer in an AAB/ABA pair, within a working range from 10 to 1000 microg/mL and a 95% confidence interval of +/-3% for three replicates. Copyright 2003 John Wiley & Sons, Ltd.

Optimized HPLC method for analysis of polar and nonpolar heterocyclic amines in cooked meat products.

PubMed

Gibis, Monika

2009-01-01

A simple, precise, and specific column high-performance liquid chromatographic (HPLC) method with UV absorption diode array and fluorescence detection has been developed by optimizing a previously described method for the simultaneous quantification of 15 polar and nonpolar heterocyclic amines (HAs) in fried meat products. The HPLC determination could be improved due to the application of a silica-based reversed-phase column with octadecyl groups (TSK-gel Super ODS) and a particle size of 2 microm. The separation of HAs in the complex meat matrix was performed with a 21 min mobile phase gradient. The method was validated for instrumental precision, repeatability, and selectivity and compared with a previously published method. After liquid adsorption of the basic sample mixture on diatomaceous earth, HAs were extracted with ethyl acetate. For cleanup, solid-phase extraction (silica propylsulfonic acid and octadecyl cartridges) and different washing steps were applied. Both nonpolar and polar HAs were determined in one fraction. The calibration curves of all HAs were linear for the applied detection system (correlation coefficient = 0.990-0.995). The recoveries, with the exception of 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), were between 42 and 98% from meat samples spiked in a range of 1.5 to 3.3 ng/g for fluorescence-active and 4.3 to 8 ng/g for UV-active HAs. For quantification of HAs, the standard addition method was used for adjustment of different characteristics of HAs in the extraction. In fried meat samples (chicken breast and beef patties), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline(4,8-DiMelQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), norharmane, and harmane were found in a concentration range of 0.02 to 14.3 ng/g.

Concurrent estimation of amlodipine besylate, hydrochlorothiazide and valsartan by RP-HPLC, HPTLC and UV-spectrophotometry.

PubMed

Sharma, Manish; Kothari, Charmy; Sherikar, Omkar; Mehta, Priti

2014-01-01

Accurate, sensitive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC), high-performance thin-layer chromatography (HPTLC) and ultraviolet (UV) spectrophopometric methods were developed for the concurrent estimation of amlodipine besylate (AMLO), hydrochlorothiazide (HCTZ) and valsartan (VALS) in bulk and combined tablet dosage forms. For the RP-HPLC method, separation was achieved on a C18 column using potassium dihydrogen orthophosphate buffer (50 mM, pH 3.7) with 0.2% triethylamine as the modifier and acetonitrile in the ratio of 56:44 (v/v) as the mobile phase. Quantification was achieved using a photodiode array detector at 232 nm over a concentration range of 2-25 µg/mL for AMLO, 5-45 µg/mL for HCTZ and 20-150 µg/mL for VALS. For the HPTLC method, the drugs were separated by using ethyl acetate-methanol-toluene-ammonia (7.5:3:2:0.8, v/v/v/v) as the mobile phase. Quantification was achieved using UV detection at 242 nm over a concentration range of 100-600 ng/spot for AMLO, 150-900 ng/spot for HCTZ and 1,200-3,200 ng/spot for VALS. The UV-spectrophotometric simultaneous equation method was based on the measurement of absorbance at three wavelengths; i.e., at 237.6 nm (λmax of AMLO), 270.2 nm (λmax of HCTZ) and 249.2 nm (λmax of VALS) in methanol. Quantification was achieved over the concentration range of 2-20 µg/mL for AMLO, 5-25 µg/mL HCTZ and 10-50 µg/mL for VALS. All methods were validated according to International Conference on Harmonization guidelines and successfully applied to marketed pharmaceutical formulations. Additionally, the three methods were compared statistically by an analysis of variance test, which revealed no significant difference between the proposed methods with respect to accuracy and precision.

Metabolite profiling with HPLC-ICP-MS as a tool for in vivo characterization of imaging probes.

PubMed

Boros, Eszter; Pinkhasov, Omar R; Caravan, Peter

2018-01-01

Current analytical methods for characterizing pharmacokinetic and metabolic properties of positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes are limited. Alternative methods to study tracer metabolism are needed. The study objective was to assess the potential of high performance liquid chromatography - inductively coupled plasma - mass spectrometry (HPLC-ICP-MS) for quantification of molecular probe metabolism and pharmacokinetics using stable isotopes. Two known peptide-DOTA conjugates were chelated with nat Ga and nat In. Limit of detection of HPLC-ICP-MS for 69 Ga and 115 In was determined. Rats were administered 50-150 nmol of Ga- and/or In-labeled probes, blood was serially sampled, and plasma analyzed by HPLC-ICP-MS using both reverse phase and size exclusion chromatography. The limits of detection were 0.16 pmol for 115 In and 0.53 pmol for 69 Ga. Metabolites as low as 0.001 %ID/g could be detected and transchelation products identified. Simultaneous administration of Ga- and In-labeled probes allowed the determination of pharmacokinetics and metabolism of both probes in a single animal. HPLC-ICP-MS is a robust, sensitive and radiation-free technique to characterize the pharmacokinetics and metabolism of imaging probes.

Linear modeling of the soil-water partition coefficient normalized to organic carbon content by reversed-phase thin-layer chromatography.

PubMed

Andrić, Filip; Šegan, Sandra; Dramićanin, Aleksandra; Majstorović, Helena; Milojković-Opsenica, Dušanka

2016-08-05

Soil-water partition coefficient normalized to the organic carbon content (KOC) is one of the crucial properties influencing the fate of organic compounds in the environment. Chromatographic methods are well established alternative for direct sorption techniques used for KOC determination. The present work proposes reversed-phase thin-layer chromatography (RP-TLC) as a simpler, yet equally accurate method as officially recommended HPLC technique. Several TLC systems were studied including octadecyl-(RP18) and cyano-(CN) modified silica layers in combination with methanol-water and acetonitrile-water mixtures as mobile phases. In total 50 compounds of different molecular shape, size, and various ability to establish specific interactions were selected (phenols, beznodiazepines, triazine herbicides, and polyaromatic hydrocarbons). Calibration set of 29 compounds with known logKOC values determined by sorption experiments was used to build simple univariate calibrations, Principal Component Regression (PCR) and Partial Least Squares (PLS) models between logKOC and TLC retention parameters. Models exhibit good statistical performance, indicating that CN-layers contribute better to logKOC modeling than RP18-silica. The most promising TLC methods, officially recommended HPLC method, and four in silico estimation approaches have been compared by non-parametric Sum of Ranking Differences approach (SRD). The best estimations of logKOC values were achieved by simple univariate calibration of TLC retention data involving CN-silica layers and moderate content of methanol (40-50%v/v). They were ranked far well compared to the officially recommended HPLC method which was ranked in the middle. The worst estimates have been obtained from in silico computations based on octanol-water partition coefficient. Linear Solvation Energy Relationship study revealed that increased polarity of CN-layers over RP18 in combination with methanol-water mixtures is the key to better modeling of logKOC through significant diminishing of dipolar and proton accepting influence of the mobile phase as well as enhancing molar refractivity in excess of the chromatographic systems. Copyright © 2016 Elsevier B.V. All rights reserved.

UV-visible spectroscopy as an alternative to liquid chromatography for determination of everolimus in surfactant-containing dissolution media: a useful approach based on solid-phase extraction.

PubMed

Kamberi, Marika; Tran, Thu-Ngoc

2012-11-01

High-throughput 96-well solid phase extraction (SPE) plate with C-18 reversed phase sorbent followed by UV-visible (UV-Vis) microplate reader was applied to the analysis of hydrophobic drugs in surfactant-containing dissolution media, which are often used to evaluate the in-vitro drug release of drug eluting stents (DES). Everolimus and dissolution medium containing Triton X-405 were selected as representatives, and the appropriate SPE conditions (adsorption, washing and elution) were investigated to obtain a practical and reliable sample clean-up. It was shown that the developed SPE procedure was capable of removing interfering components (Triton X-405 and its impurities), allowing for an accurate automated spectrophotometric analysis to be performed. The proposed UV-Vis spectrophotometric method yielded equivalent results compared to a classical LC analysis method. Linear regression analysis indicated that both methods have the ability to obtain test results that are directly proportional to the concentration of analyte in the sample within the selected range of 1.0-10 μg/ml for everolimus, with a coefficient of correlation (r(2)) value of >0.998 and standard deviation of the residuals (Syx) of <2%. The individual recoveries of everolimus ranged from 97 to 104% for the UV-Vis spectrophotometric method and from 98 to 102 for the HPLC method, respectively. The 95% CI of the mean recovery for the UV-Vis spectrophotometric method was 99-102% and for the HPLC method was 99-101%. No statistical difference was found between the mean recoveries of the methods (p=0.42). Hence the methods are free from interference due to Triton and other chemicals present in the dissolution medium. The variation in the amount of everolimus estimated by UV-Vis spectrophotometric and HPLC methods was ≤3.5%, and the drug release profiles obtained by both methods were found to be equivalent by evaluation with two-one-sided t-test (two-tailed, p=0.62; mean of differences, 0.17; 95% CI, 0.62-0.96) and similarity factor f2 (f2 value, 87). The excellent conformity of the results makes UV-Vis spectrophotometer an ideal tool for analyzing the drugs in the media containing surfactants, after SPE. The 96-well SPE plates in combination with UV-Vis microplate reader provide a high throughput method for the determination of in-vitro drug release profile of DES. Switching from HPLC to UV-Vis spectrophotometer microplate reader assay reduces the solvent consumption and labor required for the sample analyses. This directly impacts the profitability of the laboratory. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid and sensitive method for determination of withaferin-A in human plasma by HPLC.

PubMed

Patial, Pankaj; Gota, Vikram

2011-02-01

To develop and validate a rapid and sensitive high-performance liquid chromatographic method for determination of withaferin-A in human plasma. Withaferin-A, the active molecule of a traditional Indian herb, has demonstrated several biological activities in preclinical models. A validated bioassay is not available for its pharmacokinetic evaluation. The chromatographic system used a reverse-phase C18 column with UV-visible detection at 225 nm. The mobile phase consisted of water and acetonitrile applied in a gradient flow. Withaferin-A was extracted by simple protein-precipitation technique. The calibration curve was linear in the concentration range of 0.05-1.6 µg/ml. The method has the desired sensitivity to detect the plasma concentration range of withaferin-A that is likely to show biological activity based on in vitro data. This is the first HPLC method ever described for the estimation of withaferin-A in human plasma which could be applied for pharmacokinetic studies.

HPLC-based lipophilicity of pyrrolyl-acetic acid ARIs: Relationships with biological activity.

PubMed

Chrysanthakopoulos, Marios; Nicolaou, Ioannis; Demopoulos, Vassilis J; Tsantili-Kakoulidou, Anna

2010-01-01

Reversed phase HPLC was used to assess the lipophilicity of a series pyrrolyl-acetic acid derivatives with aldose reductase inhibitory activity. The pH conditions were adjusted at 3.0 to investigate the behavior of the neutral species and at pH 7.4, at which the ionized form predominates, using phosphate and MOPS buffer. Retention was monitored in absence and in presence of different amounts of n-octanol in the mobile phase in order to explore the chromatographic conditions which best reproduce the octanol-water partition or distribution coefficients. The effect of n-octanol in retention was systematically studied and its role in lipophilicity assessment was evaluated. Nevertheless rather moderate regression equations were obtained, which deviated significantly from the ideal 1:1 correlation. No significant effect of buffer was observed. The appropriateness of retention factors to be used in correlation with aldose reductase inhibitory activity was further evaluated and compared to the efficiency of the corresponding octanol-water logP values.

Individual Phosphatidylcholine Species Analysis by RP-HPLC-ELSD for Determination of Polyenylphosphatidylcholine in Lecithins.

PubMed

Lee, Wei-Ju; Weng, Shun-Hsiang; Su, Nan-Wei

2015-04-22

Polyenylphosphatidylcholine (PPC), a subgroup of the bioactive agents in phosphatidylcholine (PC), has been indicated to possess liver-protective effects. This study aimed to investigate a promising and feasible method to determine PC molecular species with a reverse phase (RP) high-performance liquid chromatograph (HPLC) equipped with an evaporative light scattering detector (ELSD). Chromatography was achieved using a C30 column and an isocratic mobile phase consisting of acetonitrile/methanol/triethylamine (40/58/2, v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80 °C for the drift tube and an air flow rate of 1.8 L/min. To identify individual peaks on the chromatogram, MALDI-TOF-MS was employed for initial detection, and then the results were used to investigate the relationship between the retention time and fatty acyl chains of each PC molecule. A linear correlation was observed between the retention time and theoretical carbon number (TCN) of individual PC species. The compositions of PC molecular species in soybean and sunflower lecithins were similar to each other, and the major PC molecular species were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC), 1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC). The contents of LLPC in soybean PC and sunflower PC were 40.6% and 64.3%, respectively.

Novel HPLC Analysis of Hydrocortisone in Conventional and Controlled-Release Pharmaceutical Preparations.

PubMed

Adi-Dako, Ofosua; Oppong Bekoe, Samuel; Ofori-Kwakye, Kwabena; Appiah, Enoch; Peprah, Paul

2017-01-01

An isocratic sensitive and precise reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination and quantification of hydrocortisone in controlled-release and conventional (tablets and injections) pharmaceutical preparations. Chromatographic separation was achieved on an ODS (C18), 5  μ m, 4.6 × 150 mm, with an isocratic elution using a freshly prepared mobile phase of composition methanol : water : acetic acid (60 : 30 : 10, v/v/v) at a flow rate of 1.0 ml/min. The detection of the drug was successfully achieved at a wavelength of 254 nm. The retention time obtained for the drug was 2.26 min. The proposed method produced linear detectable responses in the concentration range of 0.02 to 0.4 mg/ml of hydrocortisone. High recoveries of 98-101% were attained at concentration levels of 80%, 100%, and 120%. The intraday and interday precision (RSD) were 0.19-0.55% and 0.33-0.71%, respectively. A comparison of hydrocortisone analyses data from the developed method and the official USP method showed no significant difference ( p > 0.05) at a 95% confidence interval. The method was successfully applied to the determination and quantification of hydrocortisone in six controlled-release and fifteen conventional release pharmaceutical preparations.

Novel HPLC Analysis of Hydrocortisone in Conventional and Controlled-Release Pharmaceutical Preparations

PubMed Central

Oppong Bekoe, Samuel; Appiah, Enoch; Peprah, Paul

2017-01-01

An isocratic sensitive and precise reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination and quantification of hydrocortisone in controlled-release and conventional (tablets and injections) pharmaceutical preparations. Chromatographic separation was achieved on an ODS (C18), 5 μm, 4.6 × 150 mm, with an isocratic elution using a freshly prepared mobile phase of composition methanol : water : acetic acid (60 : 30 : 10, v/v/v) at a flow rate of 1.0 ml/min. The detection of the drug was successfully achieved at a wavelength of 254 nm. The retention time obtained for the drug was 2.26 min. The proposed method produced linear detectable responses in the concentration range of 0.02 to 0.4 mg/ml of hydrocortisone. High recoveries of 98–101% were attained at concentration levels of 80%, 100%, and 120%. The intraday and interday precision (RSD) were 0.19–0.55% and 0.33–0.71%, respectively. A comparison of hydrocortisone analyses data from the developed method and the official USP method showed no significant difference (p > 0.05) at a 95% confidence interval. The method was successfully applied to the determination and quantification of hydrocortisone in six controlled-release and fifteen conventional release pharmaceutical preparations. PMID:28660092

Structures of Phytosterols and Triterpenoids with Potential Anti-Cancer Activity in Bran of Black Non-Glutinous Rice

PubMed Central

Suttiarporn, Panawan; Chumpolsri, Watcharapong; Mahatheeranont, Sugunya; Luangkamin, Suwaporn; Teepsawang, Somsuda; Leardkamolkarn, Vijittra

2015-01-01

Structures of some bioactive phytochemicals in bran extract of the black rice cv. Riceberry that had demonstrated anti-cancer activity in leukemic cell line were investigated. After saponification with potassium hydroxide, separation of the unsaponified fraction by reversed-phase high performance liquid chromatography (HPLC) resulted in four sub-fractions that had a certain degree of anti-proliferation against a mouse leukemic cell line (WEHI-3 cell), this being IC50 at 24 h ranging between 2.80–467.11 μg/mL. Further purification of the bioactive substances contained in these four sub-fractions was performed by normal-phase HPLC. Structural characterization by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) resulted in, overall, the structures of seven phytosterols and four triterpenoids. Four phytosterols, 24-methylene-ergosta-5-en-3β-ol, 24-methylene-ergosta-7-en-3β-ol, fucosterol, and gramisterol, along with three triterpenoids, cycloeucalenol, lupenone, and lupeol, were found in the two sub-fractions that showed strong anti-leukemic cell proliferation (IC50 = 2.80 and 32.89 μg/mL). The other sterols and triterpenoids were campesterol, stigmasterol, β-sitosterol and 24-methylenecycloartanol. Together with the data from in vitro biological analysis, we suggest that gramisterol is a significant anti-cancer lead compound in Riceberry bran extract. PMID:25756784

Actinidin enhances protein digestion in the small intestine as assessed using an in vitro digestion model.

PubMed

Kaur, Lovedeep; Rutherfurd, Shane M; Moughan, Paul J; Drummond, Lynley; Boland, Mike J

2010-04-28

This paper describes an in vitro study that tests the proposition that actinidin from green kiwifruit influences the digestion of proteins in the small intestine. Different food proteins, from sources including soy, meat, milk, and cereals, were incubated in the presence or absence of green kiwifruit extract (containing actinidin) using a two-stage in vitro digestion system consisting of an incubation with pepsin at stomach pH (simulating gastric digestion) and then with added pancreatin at small intestinal pH, simulating upper tract digestion in humans. The digests from the small intestinal stage (following the gastric digestion phase) were subjected to gel electrophoresis (SDS-PAGE) to assess loss of intact protein and development of large peptides during the in vitro simulated digestion. Kiwifruit extract influenced the digestion patterns of all of the proteins to various extents. For some proteins, actinidin had little impact on digestion. However, for other proteins, the presence of kiwifruit extract resulted in a substantially greater loss of intact protein and different peptide patterns from those seen after digestion with pepsin and pancreatin alone. In particular, enhanced digestion of whey protein isolate, zein, gluten, and gliadin was observed. In addition, reverse-phase HPLC (RP-HPLC) analysis showed that a 2.5 h incubation of sodium caseinate with kiwifruit extract alone resulted in approximately 45% loss of intact protein.

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).

Identification and characterization of gadolinium(III) complexes in biological tissue extracts.

PubMed

Kahakachchi, Chethaka L; Moore, Dennis A

2010-07-01

The gadolinium species present in a rat kidney following intravenous administration of a gadolinium-based magnetic resonance contrast agent (Optimark™, Gadoversetamide injection) to a rat was examined in the present study. The major gadolinium species in the supernatant of the rat kidney tissue extracts was determined by reversed-phase liquid chromatography with online inductively coupled plasma optical emission spectrometry (HPLC-ICP-OES). The identity of the compound was established by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) detection. The principal gadolinium(III) complex in a rat kidney tissue extract was identified as Gd-DTPA-BMEA 24 Hrs and 7 days after a single intravenous injection of Optimark™ (gadoversetamide; Gd-DTPA-BMEA) at a dose of 5 mmol Gd/kg body weight. The study demonstrated for the first time the feasibility of the use of two complementary techniques, HPLC-ICP-OES and HPLC-ESI-MS to study the in vivo behavior of gadolinium-based magnetic resonance contrast media.

[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).

Purification and stability characterization of a cell regulatory sialoglycopeptide inhibitor

NASA Technical Reports Server (NTRS)

Moos, P. J.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

1995-01-01

Previous attempts to physically separate the cell cycle inhibitory and protease activities in preparations of a purified cell regulatory sialoglycopeptide (CeReS) inhibitor were largely unsuccessful. Gradient elution of the inhibitor preparation from a DEAE HPLC column separated the cell growth inhibitor from the protease, and the two activities have been shown to be distinct and non-overlapping. The additional purification increased the specific biological activity of the CeReS preparation by approximately two-fold. The major inhibitory fraction that eluted from the DEAE column was further analyzed by tricine-SDS-PAGE and microbore reverse phase HPLC and shown to be homogeneous in nature. Two other fractions separated by DEAE HPLC, also devoid of protease activity, were shown to be inhibitory to cell proliferation and most likely represented modified relatives of the CeReS inhibitor. The highly purified CeReS was chemically characterized for amino acid and carbohydrate composition and the role of the carbohydrate in cell proliferation inhibition, stability, and protease resistance was assessed.

Instrumental analysis of terminal-conjugated dienes for reexamination of the sex pheromone secreted by a nettle moth, Parasa lepida lepida.

PubMed

Islam, M D Azharul; Yamakawa, Rei; Do, Nguyen Duc; Numakura, Naoko; Suzuki, Toshiro; Ando, Tetsu

2009-05-01

Conjugated dienyl compounds make one of the main groups of lepidopteran sex pheromones, and GC has been frequently used to determine the configurations of the double bonds. However, the separation of two geometric isomers of a terminal-conjugated diene, such as 7,9-decadien-1-ol secreted by a nettle moth Parasa lepida lepida (Limacodidae), is assumed to be difficult. In order to clarify the chromatographic separation of the terminal dienes, 7,9-decadienyl and 9,11-dodecadienyl compounds (alcohols, acetates, and aldehydes) were analyzed by GC and HPLC. On a capillary GC column, the (E)-isomers flowed out slightly faster than the corresponding (Z)-isomers, but their peaks almost overlapped. On the other hand, HPLC equipped with an ODS column completely separated the two geometric isomers examined and the (Z)-isomers eluted from the column faster than the (E)-isomers without dependence on a functional group. In addition to undergoing direct HPLC analysis without derivatization, the dienyl alcohols were converted into 3,5-dinitrobenzoates and analyzed by LC-ESI-MS operated under the same reversed-phase condition. The two separated geometric isomers were sensitively monitored by negative ions at m/z 211, M, M+1, M+17, and M+31, which were characteristically derived from the benzoates. Based on these results, a pheromone extract of P. l. lepida was examined, and it was confirmed that the female moths exclusively produced the (Z)-isomer of the 7,9-diene. Furthermore, a GC-EAD analysis and a field evaluation with both geometrical isomers indicated that the mating communication of P. l. lepida is predominantly mediated with the (Z)-isomer.

Evaluation of sulfur isotopic enrichment of urine metabolites for the differentiation of healthy and prostate cancer mice after the administration of 34S labelled yeast.

PubMed

Galilea San Blas, Oscar; Moreno Sanz, Fernando; Herrero Espílez, Pilar; Sainz Menéndez, Rosa María; Mayo Barallo, Juan Carlos; Marchante-Gayón, Juan Manuel; García Alonso, José Ignacio

2017-01-01

Sulfur isotopic enrichment of urine metabolites in healthy and prostate cancer mice using 34 S enriched yeast and High Performance Liquid Chromatography coupled to Multicollector Inductively Coupled Plasma Mass Spectrometry (HPLC-MC-ICP-MS) has been evaluated. A 30 weeks experiment (since the eleventh to the fortieth week of life) was carried out collecting the urine of three healthy mice and three transgenic mice with prostate cancer during 24h after a single oral administration of a 34 S enriched yeast slurry. The isotopic enrichment of different sulphur metabolites was monitored by coupling a C18 reverse phase HPLC column with a multicollector ICP-MS using a membrane desolvating system. Quantification of sulfur in the chromatographic peaks was carried out by post-column isotope dilution using a 33 S enriched spike. Differences between the 34 S enrichment in the urine metabolites of healthy and prostate cancer mice were found from the beginning of the disease. Both populations could be differentiated using a principal component analysis (PCA). Finally, 7 unknown mice were correctly classified in each population using a linear discriminant analysis. Copyright © 2016 Elsevier GmbH. All rights reserved.

Fast Gradient Elution Reversed-Phase HPLC with Diode-Array Detection as a High Throughput Screening Method for Drugs of Abuse

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

Peter W. Carr; K.M. Fuller; D.R. Stoll

A new approach has been developed by modifying a conventional gradient elution liquid chromatograph for the high throughput screening of biological samples to detect the presence of regulated intoxicants. The goal of this work was to improve the speed of a gradient elution screening method over current approaches by optimizing the operational parameters of both the column and the instrument without compromising the reproducibility of the retention times, which are the basis for the identification. Most importantly, the novel instrument configuration substantially reduces the time needed to re-equilibrate the column between gradient runs, thereby reducing the total time for eachmore » analysis. The total analysis time for each gradient elution run is only 2.8 minutes, including 0.3 minutes for column reequilibration between analyses. Retention times standard calibration solutes are reproducible to better than 0.002 minutes in consecutive runs. A corrected retention index was adopted to account for day-to-day and column-to-column variations in retention time. The discriminating power and mean list length were calculated for a library of 47 intoxicants and compared with previous work from other laboratories to evaluate fast gradient elution HPLC as a screening tool.« less

Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid-liquid extraction followed by high performance liquid chromatography.

PubMed

Valdivielso, Izaskun; Bustamante, María Ángeles; Ruiz de Gordoa, Juan Carlos; Nájera, Ana Isabel; de Renobales, Mertxe; Barron, Luis Javier R

2015-04-15

Carotenoids and tocopherols from botanical species abundant in Atlantic mountain grasslands were simultaneously extracted using one-step solid-liquid phase. A single n-hexane/2-propanol extract containing both types of compounds was injected twice under two different sets of HPLC conditions to separate the tocopherols by normal-phase chromatography and carotenoids by reverse-phase mode. The method allowed reproducible quantification in plant samples of very low amounts of α-, β-, γ- and δ-tocopherols (LOD from 0.0379 to 0.0720 μg g(-1) DM) and over 15 different xanthophylls and carotene isomers. The simplified one-step extraction without saponification significantly increased the recovery of tocopherols and carotenoids, thereby enabling the determination of α-tocopherol acetate in plant samples. The two different sets of chromatographic analysis provided near baseline separation of individual compounds without interference from other lipid compounds extracted from plants, and a very sensitive and accurate detection of tocopherols and carotenoids. The detection of minor individual components in botanical species from grasslands is nowadays of high interest in searching for biomarkers for foods derived from grazing animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid.

PubMed

Pelà, M; Del Zoppo, L; Allegri, L; Marzola, E; Ruzza, C; Calo, G; Perissutti, E; Frecentese, F; Salvadori, S; Guerrini, R

2014-07-01

The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the lpv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.

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.

High Performance Liquid Chromatography

NASA Astrophysics Data System (ADS)

Talcott, Stephen

High performance liquid chromatography (HPLC) has many applications in food chemistry. Food components that have been analyzed with HPLC include organic acids, vitamins, amino acids, sugars, nitrosamines, certain pesticides, metabolites, fatty acids, aflatoxins, pigments, and certain food additives. Unlike gas chromatography, it is not necessary for the compound being analyzed to be volatile. It is necessary, however, for the compounds to have some solubility in the mobile phase. It is important that the solubilized samples for injection be free from all particulate matter, so centrifugation and filtration are common procedures. Also, solid-phase extraction is used commonly in sample preparation to remove interfering compounds from the sample matrix prior to HPLC analysis.

Method for the determination of chromium in feed matrix by HPLC.

PubMed

Umesh, Balakrishnan; Rajendran, Rajendra Moorthy; Manoharan, Muthu Tamizh

2015-11-01

An improved method for the chromatographic separation and determination of chromium (III) and (VI) [ CRIII AND CRVI: ] in mineral mixtures and feed samples has been developed. The method uses precolumn derivatization using ammonium pyrrolidinedithiocarbamate ( APD: ) followed by reversed-phase liquid chromatography to separate the chromium ions. Both Cr(III) and Cr(VI) species are chelated with ammonium pyrrolidinedithiocarbamate prior to separation by mixing with acetonitrile and 0.5 mmol acetate buffer (pH 4.5). Optimum chromatographic separations were obtained with a polymer-based reversed-phase column (Kinetex, 5 μ, 250 × 4.5 mm, Phenomenex, Torrance, CA) and a mobile phase containing acetonitrile and water (7:3). Both Cr(III) and Cr(VI) ion concentrations were directly determined from the corresponding areas in the chromatogram. The effect of analytical parameters, including pH, concentration of ligand, incubation temperature, and mobile phase, was optimized for both chromium complexes. The range of the procedure was found to be linear for Cr(III) and Cr(VI) concentrations between 0.125 and 4 μg/mL (r² = 0.9926) and 0.1 and 3.0 μg/mL (r² = 0.9983), respectively. Precision was evaluated by replicate analysis in which the percentage relative standard deviation values for chromium complex were found to be below 4.0. The recoveries obtained (85-115%) for both Cr(III) and Cr(VI) complexes indicated the accuracy of the developed method. The degradation products, as well as the excipients, were well resolved from the chromium complex peak in the chromatogram. Finally, the new method proved to be suitable for routine analysis of Cr(III) and Cr(VI) species in raw materials, mineral mixtures, and feed samples. © 2015 Poultry Science Association Inc.

Development and Optimisation of an HPLC-DAD-ESI-Q-ToF Method for the Determination of Phenolic Acids and Derivatives

PubMed Central

Restivo, Annalaura; Degano, Ilaria; Ribechini, Erika; Colombini, Maria Perla

2014-01-01

A method for the HPLC-MS/MS analysis of phenols, including phenolic acids and naphtoquinones, using an amide-embedded phase column was developed and compared to the literature methods based on classical C18 stationary phase columns. RP-Amide is a recently developed polar embedded stationary phase, whose wetting properties mean that up to 100% water can be used as an eluent. The increased retention and selectivity for polar compounds and the possibility of working in 100% water conditions make this column particularly interesting for the HPLC analysis of phenolic acids and derivatives. In this study, the chromatographic separation was optimised on an HPLC-DAD, and was used to separate 13 standard phenolic acids and derivatives. The method was validated on an HPLC-ESI-Q-ToF. The acquisition was performed in negative polarity and MS/MS target mode. Ionisation conditions and acquisition parameters for the Q-ToF detector were investigated by working on collision energies and fragmentor potentials. The performance of the method was fully evaluated on standards. Moreover, several raw materials containing phenols were analysed: walnut, gall, wine, malbec grape, French oak, red henna and propolis. Our method allowed us to characterize the phenolic composition in a wide range of matrices and to highlight possible matrix effects. PMID:24551158

A cytotoxic serine proteinase isolated from mouse submandibular gland.

PubMed

Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

1989-08-01

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

Determination of the Antibiotic Oxytetracycline in Commercial Milk by Solid-Phase Extraction: A High-Performance Liquid Chromatography (HPLC) Experiment for Quantitative Instrumental Analysis

ERIC Educational Resources Information Center

Mei-Ratliff, Yuan

2012-01-01

Trace levels of oxytetracylcine spiked into commercial milk samples are extracted, cleaned up, and preconcentrated using a C[subscript 18] solid-phase extraction column. The extract is then analyzed by a high-performance liquid chromatography (HPLC) instrument equipped with a UV detector and a C[subscript 18] column (150 mm x 4.6 mm x 3.5 [mu]m).…

Contamination of injectable solutions with 2-mercaptobenzothiazole leached from rubber closures.

PubMed

Reepmeyer, J C; Juhl, Y H

1983-11-01

An impurity, discovered in a sample of digoxin injectable solution commercially packaged in a syringe for single-dose delivery, was found to originate from the rubber closure of the syringe and was identified as 2-mercaptobenzothiazole, a common accelerator for rubber vulcanization. Several similarly packaged injectable solutions of a variety of drugs from various manufacturers were examined and over half contained 2-mercaptobenzothiazole. The compound was identified by UV spectrophotometry (including a pH-dependent shift in its absorbance maximum), by mass spectrometry, and by comparison with standard 2-mercaptobenzothiazole using silica gel and reverse-phase high-performance liquid chromatography (HPLC). The presence of this impurity in injectable solutions may have implications with regard to toxicity and may interfere with the assay of digoxin injectable solution by HPLC.

Quantification of Dihydroxyacetone Phosphate (DHAP) in Human Red Blood Cells by HPLC-TripleTOF 5600™ Mass Spectrometer.

PubMed

Deng, Shuang; Scott, David; Myers, Douglas; Garg, Uttam

2016-01-01

Triosephosphate isomerase (TPI) is a glycolytic enzyme which catalyzes the interconversion between glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). TPI deficiency results in accumulation of DHAP in human red blood cells and other tissues. The disease is characterized by congenital hemolytic anemia, and progressive neuromuscular dysfunction. The laboratory diagnosis is generally made by measurement of TPI activity in RBCs. Measurement of DHAP can be useful in further confirmation and follow-up of the disease. We developed HPLC/TOF-MS method for quantitation of DHAP in RBCs. The method involves simple protein precipitation, reverse phase C8 column chromatography, ion pairing with tributylamine, and long run time of 50 min to separate the two isomers (G3P and DHAP).

Recent advances in nonpolar and polar organic monoliths for HPLC and CEC

PubMed Central

Jonnada, Murthy; Rathnasekara, Renuka; Rassi, Ziad El

2015-01-01

This article is aimed at providing a review of the progress made in the field over the period 2011 to present in order to expand in parts on two previous reviews (S. Karenga and Z. El Rassi, Electrophoresis, 2011, 32, 90-104; D. Gunasena and Z. El Rassi, Electrophoresis, 2012, 33, 251-261). In brief, this review article describes progress made in nonpolar and polar monoliths used in reversed phase HPLC and CEC (RPC/RP-CEC) and in hydrophilic interaction liquid chromatography/CEC (HILIC/HI-CEC), respectively. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 69 references published on nonpolar and polar polymeric monoliths. PMID:25266173

HPLC/Fluorometric Detection of Carvedilol in Real Human Plasma Samples Using Liquid-Liquid Extraction.

PubMed

Yilmaz, Bilal; Arslan, Sakir

2016-03-01

A simple, rapid and sensitive high-performance liquid chromatography (HPLC) method has been developed to quantify carvedilol in human plasma using an isocratic system with fluorescence detection. The method included a single-step liquid-liquid extraction with diethylether and ethylacetate mixture (3 : 1, v/v). HPLC separation was carried out by reversed-phase chromatography with a mobile phase composed of 20 mM phosphate buffer (pH 7)-acetonitrile (65 : 35, v/v), pumped at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 240 nm (excitation) and 330 nm (emission). The calibration curve for carvedilol was linear from 10 to 250 ng/mL. Intra- and interday precision values for carvedilol in human plasma were <4.93%, and accuracy (relative error) was better than 4.71%. The analytical recovery of carvedilol from human plasma averaged out to 91.8%. The limits of detection and quantification of carvedilol were 3.0 and 10 ng/mL, respectively. Also, the method was successfully applied to three patients with hypertension who had been given an oral tablet of 25 mg carvedilol. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

[[Chiral separation of five arylpropionic acid drugs and determination of their enantiomers in pharmaceutical preparations by reversed-phase high performance liquid chromatography with cellulose-tris-(4-methylbenzoate) stationary phase

PubMed

Luo, An; Wan, Qiang; Fan, Huajun; Chen, Zhi; Wu, Xuehao; Huang, Xiaowen; Zang, Linquan

2014-09-01

Chromatographic behaviors for enantiomeric separation of arylpropionic acid drugs were systematically developed by reversed phase-high performance liquid chromatography (RP-HPLC) using cellulose-tris-(4-methylbenzoate) (CTMB) as chiral stationary phase (CSP). The effects of the composition of the mobile phase, additives and temperature on chiral separation of flurbiprofen, pranoprofen, naproxen, ibuprofen and loxoprofen were further investigated. The enantiomers had been successfully separated on CSP of CTMB by the mobile phase of methanol-0.1% (v/v) formic acid except naproxen by acetonitrile-0.1% (v/v) formic acid at 25 °C. The mechanisms of the racemic resolution for the above mentioned five drugs are discussed thermodynamically and structurally. The resolutions between respective enantiomers for arylpropionic acid drugs on CTMB had significant differences due to their chromatographic behaviors. The order of resolutions ranked pranoprofen, loxoprofen, flurbiprofen, ibuprofen and naproxen. The method established has been successfully applied to the determination of the enantiomers of the five drugs in commercial preparations under the optimized conditions. It proved that the method is simple, reliable and accurate.

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.

Characterizing Tityus discrepans scorpion venom from a fractal perspective: Venom complexity, effects of captivity, sexual dimorphism, differences among species.

PubMed

D'Suze, Gina; Sandoval, Moisés; Sevcik, Carlos

2015-12-15

A characteristic of venom elution patterns, shared with many other complex systems, is that many their features cannot be properly described with statistical or euclidean concepts. The understanding of such systems became possible with Mandelbrot's fractal analysis. Venom elution patterns were produced using the reversed phase high performance liquid chromatography (HPLC) with 1 mg of venom. One reason for the lack of quantitative analyses of the sources of venom variability is parametrizing the venom chromatograms' complexity. We quantize this complexity by means of an algorithm which estimates the contortedness (Q) of a waveform. Fractal analysis was used to compare venoms and to measure inter- and intra-specific venom variability. We studied variations in venom complexity derived from gender, seasonal and environmental factors, duration of captivity in the laboratory, technique used to milk venom. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography.

PubMed

Chen, Lili; Liao, Linchuan; Zuo, Zhong; Yan, Youyi; Yang, Lin; Fu, Qiang; Chen, Yu; Hou, Junhong

2007-04-11

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 microm Zorbax Dikema C18 column (150 mm x 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine-acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 microg/ml in plasma and 0.007 and 0.14 microg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 microg/ml and nikethamide ranged from 1.25 to 106.8 microg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 microg/ml of lidocaine and 8.0 to 72.4 microg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.

Analysis of the monosaccharide composition of water-soluble polysaccharides from Sargassum fusiforme by high performance liquid chromatography/electrospray ionisation mass spectrometry.

PubMed

Wu, Xiaodan; Jiang, Wei; Lu, Jiajia; Yu, Ying; Wu, Bin

2014-02-15

Sargassum fusiforme (hijiki) is the well-known edible algae, whose polysaccharides have been proved to possess interesting bioactivities like antitumor, antioxidant, antimicrobial and immunomodulatory activities. A facile and sensitive method based on high-performance liquid chromatography method of pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) coupled with electrospray ionisation mass spectrometry (HPLC/ESI-MS) has been established for the analysis of the monosaccharide composition of polysaccharides in S. fusiforme. Monosaccharides have been converted into PMP-labelled derivatives with aqueous ammonia as a catalyst at 70 °C for 30 min. The optimisation of the pre-column derivatization process was studied. The LODs of the monosaccharides were in the range from 0.01 to 0.02 nmol. PMP-labelled mixture of monosaccharides has been well separated by a reverse-phase HPLC and detected by on-line ESI-MS method under optimised conditions. The mobile phase of elution system was chosen as acetonitrile (solvent A) and 20mM aqueous ammonium acetate (solvent B) (pH 3.0) with Zorbax XDB-C18 column at 30 °C for the separation of the monosaccharide derivatives. Identification of the monosaccharides composition was carried out by analysis with mass spectral behaviour and chromatography characteristics of 1-phenyl-3-methyl-5-pyrazolone (PMP) labelled monosaccharides. All PMP-labelled derivatives display high chemical stabilities, whose regular MS fragmentation is specific for reducing labelled sugars. The result showed that the S. fusiforme polysaccharide consisted of mannose, glucose, galactose, xylose, fucose and glucuronic acid or galacturonic acid, or both uronic acids. Copyright © 2013 Elsevier Ltd. All rights reserved.

A rapid monitoring method for inorganic arsenic in rice flour using reversed phase-high performance liquid chromatography-inductively coupled plasma mass spectrometry.

PubMed

Narukawa, Tomohiro; Chiba, Koichi; Sinaviwat, Savarin; Feldmann, Jörg

2017-01-06

A new rapid monitoring method by means of high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) following the heat-assisted extraction was developed for measurement of total inorganic arsenic species in rice flour. As(III) and As(V) eluted at the same retention time and completely separated from organoarsenic species by an isocratic elution program on a reversed phase column. Therefore, neither ambiguous oxidation of arsenite to arsenate nor the integration of two peaks were necessary to determine directly the target analyte inorganic arsenic. Rapid injection allowed measuring 3 replicates within 6min and this combined with a quantitative extraction of all arsenic species from rice flour by a 15min HNO 3 -H 2 O 2 extraction makes this the fastest laboratory based method for inorganic arsenic in rice flour. Copyright © 2016 Elsevier B.V. All rights reserved.

Ultrasound-assisted analyte extraction for the determination of sulfate and elemental sulfur in zinc sulfide by different liquid chromatography techniques.

PubMed

Dash, K; Thangavel, S; Krishnamurthy, N V; Rao, S V; Karunasagar, D; Arunachalam, J

2005-04-01

The speciation and determination of sulfate (SO4(2-)) and elemental sulfur (S degree) in zinc sulfide (ZnS) using ion-chromatography (IC) and reversed-phase liquid chromatography (RPLC) respectively is described. Three sample pretreatment approaches were employed with the aim of determining sulfate: (i) conventional water extraction of the analyte; (ii) solid-liquid aqueous extraction with an ultrasonic probe; and (iii) elimination of the zinc sulfide matrix via ion-exchange dissolution (IED). The separation of sulfate was carried out by an anion-exchange column (IonPac AS17), followed by suppressed conductivity detection. Elemental sulfur was extracted ultrasonically from the acid treated sample solution into chloroform and separated on a reversed phase HPLC column equipped with a diode array detector (DAD) at 264 nm. The achievable solid detection limits for sulfate and sulfur were 35 and 10 microg g(-1) respectively.

Analysis of clonazepam in a tablet dosage form using smallbore HPLC.

PubMed

Spell, J C; Stewart, J T

1998-11-01

A stability indicating, reversed phase high-performance liquid chromatographic method utilizing a smallbore HPLC column has been developed for the determination of clonazepam in a commercial tablet dosage form. The use of a small bore column results in a substantial solvent savings, as well as a greater mass sensitivity, especially in the identification of degradation peaks in a chromatogram. The method involves ultraviolet detection at 254 nm and utilized a 150 x 3.0 mm i.d. column packed with 3 microm octyldecylsilane particles with a mobile phase of water methanol acetonitrile (40:30:30, v/v/v) at a flow rate of 400 microl min(-1) at ambient temperature, with and without the use of 1,2-dichlorobenzene as the internal standard. The current USP method for the analysis of clonazepam using a 300 x 3.9 mm i.d. conventional octyldecylsilane column was utilized as a comparison to the smallbore method. The retention times for clonazepam and the internal standard on the 3.0 mm i.d. column were 4.0 and 12.5 min, respectively. The intra- and interday RSDs on the 3.0 mm i.d. column were < 0.55% (n =4) using the internal standard, and < 0.19% (n = 4) without the internal standard at the lower limit of the standard curve, 50 microg ml(-1) and had a limit of detection of 24 ng ml(-1). The assay using the 3.0 mm i.d. column was shown to be suitable for measuring clonazepam in a tablet dosage form.

Preparation and retention mechanism study of graphene and graphene oxide bonded silica microspheres as stationary phases for high performance liquid chromatography.

PubMed

Zhang, Xiaoqiong; Chen, Sha; Han, Qiang; Ding, Mingyu

2013-09-13

Graphene oxide (GO) bonded stationary phase for high performance liquid chromatography (HPLC) was fabricated by coating GO sheets onto aminosilica microspheres via covalent coupling. Graphene (G) functionalized HPLC stationary phase was then prepared through hydrazine reduction of GO bonded silica (GO@SiO2) composite, which was the first example of using graphene as stationary-phase component for HPLC. Effective separations of the tested neutral and polar compounds on both GO@SiO2 and graphene bonded silica (G@SiO2) columns were achieved under the optimal experimental conditions. Compared with commercial C18 column, the different chromatographic performances of GO and graphene bonded columns were ascribed to their unique retention mechanisms. The polyaromatic scaffold of GO and graphene gives π-π stacking property and hydrophobic effect, and other retention mechanisms, such as π-π electron-donor-acceptor (EDA) interaction for the separation of nitroaromatic compounds and hydrogen bonding for hydroxyl and amino compounds, may also be taken into consideration. Experimental results indicated that the mixed-mode retention mechanism can facilitate the separation of analytes with similar hydrophobicity, which is a unique property compared with C18 column. Additionally, G@SiO2 showed higher affinity to aromatic analytes in contrast with GO@SiO2 and its retention mechanism was not consistent with the typical reversed phase behavior. The separation of aromatic compounds on G@SiO2 column relies primarily on the π-π stacking interaction and then the hydrophobicity, while the two interactions have equal shares on GO@SiO2 column. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and validation of an HPLC method for the determination of dibenzoylmethane in rat plasma and its application to the pharmacokinetic study.

PubMed

Shen, Guoxiang; Hong, Jin-Liern; Kong, Ah-Ng Tony

2007-06-01

A highly sensitive and simple high-performance liquid chromatographic (HPLC) assay has been developed and validated for the quantification of dibenzoylmethane (DBM) in rat plasma. DBM and internal standard (I.S.) 1-(5-chloro-2-hydroxy-4-methylphenyl)-3-phenyl-1,3-propanedione (CHMPP) were extracted from rat plasma by ethyl acetate/methanol (95:5, v/v) and analyzed using reverse-phase gradient elution with a Phenomenex Gemini C18 5-mum column. A gradient of mobile phase (mobile phase A: water/methanol (80:20, v/v) with 0.1% TFA and mobile phase B: acetonitrile with 0.1% TFA) at a flow rate of 0.2 mL/min, and ultraviolet (UV) detection at 335 nm were utilized. The lower limit of quantification (LLOQ) using 50 microL rat plasma was 0.05 microg/mL. The calibration curve was linear over a concentration range of 0.05-20 microg/mL. The mean recoveries were 80.6+/-5.7, 83.4+/-1.6 and 77.1+/-3.4% with quality control (QC) level of 0.05, 1 and 20 microg/mL, respectively. Intra- and inter-day assay accuracy and precision fulfilled US FDA guidance for industry bioanalytical method validation. Stability studies showed that DBM was stable in rat plasma after 4h incubation at room temperature, one month storage at -80 degrees C and three freeze/thaw cycles, as well as in reconstitute buffer for 48 h at 4 degrees C. The utility of the assay was confirmed by the successful analysis of plasma samples from DBM pharmacokinetics studies in the rats after oral and intravenous administrations.

Selectivity mapping of the binding sites of (E)-resveratrol imprinted polymers using structurally diverse polyphenolic compounds present in Pinot noir grape skins.

PubMed

Hashim, Shima N N S; Schwarz, Lachlan J; Danylec, Basil; Potdar, Mahesh K; Boysen, Reinhard I; Hearn, Milton T W

2016-12-01

This investigation describes a general procedure for the selectivity mapping of molecularly imprinted polymers, using (E)-resveratrol-imprinted polymers as the exemplar, and polyphenolic compounds present in Pinot noir grape skin extracts as the test compounds. The procedure is based on the analysis of samples generated before and after solid-phase extraction of (E)-resveratrol and other polyphenols contained within the Pinot noir grape skins using (E)-resveratrol-imprinted polymers. Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation tandem mass spectrometry (ESI MS/MS) was then employed for compound analysis and identification. Under optimised solid-phase extraction conditions, the (E)-resveratrol-imprinted polymer showed high binding affinity and selectivity towards (E)-resveratrol, whilst no resveratrol was bound by the corresponding non-imprinted polymer. In addition, quercetin-3-O-glucuronide and a dimer of catechin-methyl-5-furfuraldehyde, which share some structural features with (E)-resveratrol, were also bound by the (E)-resveratrol-imprinted polymer. Polyphenols that were non-specifically retained by both the imprinted and non-imprinted polymer were (+)-catechin, a B-type procyanidin and (-)-epicatechin. The compounds that did not bind to the (E)-resveratrol molecularly imprinted polymer had at least one of the following molecular characteristics in comparison to the (E)-resveratrol template: (i) different spatial arrangements of their phenolic hydroxyl groups, (ii) less than three or more than four phenolic hydroxyl groups, or (iii) contained a bulky substituent moiety. The results show that capillary RP-HPLC in conjunction with ESI MS/MS represent very useful techniques for mapping the selectivity of the binding sites of imprinted polymer. Moreover, this procedure permits performance monitoring of the characteristics of molecularly imprinted polymers intended for solid-phase extraction of bioactive and nutraceutical molecules from diverse agricultural waste sources. Copyright © 2016 Elsevier B.V. All rights reserved.

High perfomance liquid chromatography fingerprint analysis for quality control of brotowali (Tinospora crispa)

NASA Astrophysics Data System (ADS)

Syarifah, V. B.; Rafi, M.; Wahyuni, W. T.

2017-05-01

Brotowali (Tinospora crispa) is widely used in Indonesia as ingredient of herbal medicine formulation. To ensure the quality, safety, and efficacy of herbal medicine products, its chemical constituents should be continuously evaluated. High performance liquid chromatography (HPLC) fingerprint is one of powerful technique for this quality control process. In this study, HPLC fingerprint analysis method was developed for quality control of brotowali. HPLC analysis was performed in C18 column and detection was performed using photodiode array detector. The optimum mobile phase for brotowali fingerprint was acetonitrile (ACN) and 0.1% formic acid in gradient elution mode at a flow rate of 1 mL/min. The number of peaks detected in HPLC fingerprint of brotowali was 32 peaks and 23 peaks for stems and leaves, respectively. Berberine as marker compound was detected at retention time of 20.525 minutes. Evaluation of analytical performance including precision, reproducibility, and stability prove that this HPLC fingerprint analysis was reliable and could be applied for quality control of brotowali.

Surface confined ionic liquid as a stationary phase for HPLC

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

Wang, Qian; Baker, Gary A; Baker, Sheila N

Trimethoxysilane ionosilane derivatives of room temperature ionic liquids based on alkylimidazolium bromides were synthesized for attachment to silica support material. The derivatives 1-methyl-3-(trimethoxysilylpropyl)imidazolium bromide and 1-butyl-3-(trimethoxysilylpropyl)imidazolium bromide were used to modify the surface of 3 {micro}m diameter silica particles to act as the stationary phase for HPLC. The modified particles were characterized by thermogravimetric analysis (TGA) and {sup 13}C and {sup 29}Si NMR spectroscopies. The surface modification procedure rendered particles with a surface coverage of 0.84 {micro}mol m{sup -2} for the alkylimidazolium bromide. The ionic liquid moiety was predominantly attached to the silica surface through two siloxane bonds of themore » ionosilane derivative (63%). Columns packed with the modified silica material were tested under HPLC conditions. Preliminary evaluation of the stationary phase for HPLC was performed using aromatic carboxylic acids as model compounds. The separation mechanism appears to involve multiple interactions including ion exchange, hydrophobic interaction, and other electrostatic interactions.« less

High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1 in cattle feed supplements

PubMed Central

Mochamad, Lazuardi; Hermanto, Bambang

2017-01-01

Aim: The objective of the current study is to determine the concentration of aflatoxin B1 using high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector. Materials and Methods: Aflatoxin B1 certified reference grade from Trilogy Analytical Laboratory dissolved acetonitrile (ACN) at 10 µg/mL was using standard assessment. HPLC instruments such as ultraviolet-PDA detector used a Shimadzu LC-6AD pump with DGU-20A5 degasser, communication module-20A, and PDA detector SPD-M20A with FRC-10A fraction collector. The HPLC was set isocratic method at 354 nm with a reverse-phase ODS C18 column (LiChrospher® 100 RP-18; diameter, 5 µm) under a 20°C controlled column chamber. Rheodyne® sample loops were performed in 20 µL capacities. The mobile phase was performed at fraction 63:26:11 H2O: methanol:ACN at pH 6.8. A total of 1 kg of feed contained 10% bread crumbs and 30% concentrated, 40% forage, and 20% soybean dregs were using commercials samples. Samples were extracted by ACN and separated with solid phase extraction ODS 1 mL than elution with mobile phase to collect at drying samples performed. The samples were ready to use after added 1 mL mobile phase than injected into the system of HPLC. Results: We found that the retention time of aflatoxin B1 was approximately 10.858 min. Linearity of 0.01-0.08 µg/mL aflatoxin B1 dissolved in mobile phase was obtained at R2=0.9. These results demonstrate that these methods can be used to analyze aflatoxin B1 and gain 89-99% recovery. The limit of detection of this assay was obtained at 3.5 × 10−6 µg/mL. Conclusion: This method was easy to apply and suitable to analyzing at small concentrations of aflatoxin B1 in formulated product of feed cattle. PMID:28919686

High-throughput determination of faropenem in human plasma and urine by on-line solid-phase extraction coupled to high-performance liquid chromatography with UV detection and its application to the pharmacokinetic study.

PubMed

Xie, Rui; Wen, Jun; Wei, Hua; Fan, Guorong; Zhang, Dabing

2010-05-01

An automated system using on-line solid-phase extraction and HPLC with UV detection was developed for the determination of faropenem in human plasma and urine. Analytical process was performed isocratically with two reversed-phase columns connected by a switching valve. After simple pretreatment for plasma and urine with acetonitrile, a volume of 100microl upper layer of the plasma or urine samples was injected for on-line SPE column switching HPLC-UV analysis. The analytes were retained on the self-made trap column (Lichrospher C(18), 4.6mmx37mm, 25microm) with the loading solvent (20mM NaH(2)PO(4) adjusted pH 3.5) at flow rate of 2mlmin(-1), and most matrix materials were removed from the column to waste. After 0.5min washing, the valve was switched to another position so that the target analytes could be eluted from trap column to analytical column in the back-flush mode by the mobile phase (acetonitrile-20mM NaH(2)PO(4) adjusted pH 3.5, 16:84, v/v) at flow rate of 1.5mlmin(-1), and then separated on the analytical column (Ultimate XB-C(18), 4.6mmx50mm, 5microm).The complete cycle of the on-line SPE preconcentration purification and HPLC separation of the analytes was 5min. Calibration curves with good linearities (r=0.9994 for plasma sample and r=0.9988 for urine sample) were obtained in the range 0.02-5microgml(-1) in plasma and 0.05-10microg ml(-1) in urine for faropenem. The optimized method showed good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy. The method was successfully utilized to quantify faropenem in human plasma and urine to support the clinical pharmacokinetic studies. Copyright 2009 Elsevier B.V. All rights reserved.

Simultaneous determination of 16 purine derivatives in urinary calculi by gradient reversed-phase high-performance liquid chromatography with UV detection.

PubMed

Safranow, Krzysztof; Machoy, Zygmunt

2005-05-25

A reversed-phase high-performance liquid chromatography (HPLC) method with ultraviolet detection has been developed for the analysis of purines in urinary calculi. The method using gradient of methanol concentration and pH was able to separate 16 compounds: uric acid, 2,8-dihydroxyadenine, xanthine, hypoxanthine, allopurinol and oxypurinol as well as 10 methyl derivatives of uric acid or xanthine (1-, 3-, 7- and 9-methyluric acid, 1,3-, 1,7- and 3,7-dimethyluric acid, 1-, 3- and 7-methylxanthine). Limits of detection for individual compounds ranged from 0.006 to 0.035 mg purine/g of the stone weight and precision (CV%) was 0.5-2.4%. The method enabled us to detect in human uric acid stones admixtures of nine other purine derivatives: natural metabolites (hypoxanthine, xanthine, 2,8-dihydroxyadenine) and methylated purines (1-, 3- and 7-methyluric acid, 1,3-dimethyluric acid, 3- and 7-methylxanthine) originating from the metabolism of methylxanthines (caffeine, theophylline and theobromine). The method allows simultaneous quantitation of all known purine constituents of urinary stones, including methylated purines, and may be used as a reference one for diagnosing disorders of purine metabolism and research on the pathogenesis of urolithiasis.

Study and development of reversed-phase HPLC systems for the determination of 2-imidazolines in the presence of preservatives in pharmaceutical preparations.

PubMed

Antoniou, Constantinos G; Markopoulou, Catherine K; Kouskoura, Maria G; Koundourellis, John E

2011-01-01

Different HPLC chromatographic systems were investigated on a C18 ACE 5 pm, 150 x 4.6 mm id column for the determination of tymazoline, tramazoline, and antazoline, with either naphazoline or xylometazoline, in commercial preparations. For the development and optimization of the systems, a Response Surface Method (r=0.925-0.980) was used to illustrate the changes in k as a function of pH values and different salt concentrations. The simultaneous separation of 2-imidazolines was accomplished at 40 degrees C with 0.01 M ammonium acetate-methanol (50+50, v/v, pH 6.0) mobile phase at a flow rate of 1.2 mL/min. In order to deal with the usual coexistence of 2-imidazolines with benzethonium and benzalkonium chloride preservatives, it was necessary to use another chromatographic system, 0.01 M ammonium acetate-methanol (50+50, v/v) mobile phase on a cyano ACE 5 pm, 150 x 4.6 mm id column. As part of a more thorough theoretical investigation, a partial least-squares (PLS) technique was used for modeling the RP-HPLC retention data. The model was based on molecular structure descriptors of the analytes' X variables and on their retention time (Log K) Y. The goodness of fit was estimated by the PLS correlation coefficient (r2) and root mean square error of estimation values, which were 0.994 and 0.0479, respectively.

The determination and quantification of photosynthetic pigments by reverse phase high-performance liquid chromatography, thin-layer chromatography, and spectrophotometry.

PubMed

Pocock, Tessa; Król, Marianna; Huner, Norman P A

2004-01-01

Chorophylls and carotenoids are functionally important pigment molecules in photosynthetic organisms. Methods for the determination of chlorophylls a and b, beta-carotene, neoxanthin, and the pigments that are involved in photoprotective cycles such as the xanthophylls are discussed. These cycles involve the reversible de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin, as well as the reversible de-epoxidation of lutein-5,6-epoxide into lutein. This chapter describes pigment extraction procedures from higher plants and green algae. Methods for the determination and quantification using high-performance liquid chromatograpy (HPLC) are described as well as methods for the separation and purification of pigments for use as standards using thin-layer chromatography (TLC). In addition, several spectrophotometric methods for the quantification of chlorophylls a and b are described.

Three phases hollow fiber LPME combined with HPLC-UV for extraction, preconcentration and determination of valerenic acid in Valeriana officinalis.

PubMed

Mirzaei, Mohamad; Dinpanah, Hossein

2011-07-01

In the present work, the applicability of hollow fiber-based liquid phase microextraction (HF-LPME) was evaluated for the extraction and preconcentration of valerenic acid prior to its determination by reversed-phase HPLC/UV. The target drug was extracted from 5.0 mL of aqueous solution with pH 3.5 into an organic extracting solvent (dihexyl ether) impregnated in the pores of a hollow fiber and finally back extracted into 10 μ L of aqueous solution with pH 9.5 located inside the lumen of the hollow fiber. In order to obtain high extraction efficiency, the parameters affecting the HF-LPME, including pH of the donor and acceptor phases, type of organic phase, ionic strength, the volume ratio of donor to acceptor phase, stirring rate and extraction time were studied and optimized. Under the optimized conditions, enrichment factor up to 446 was achieved and the relative standard deviation (RSD) of the method was 4.36% (n = 9). The linear range was 7.5-850 μg L⁻¹ with correlation coefficient (r²=0.999), detection limits was 2.5 μg L⁻¹ and the LOQ was 7.5 μg L⁻¹. The proposed method was evaluated by extraction and determination of valerenic acid in some Iranian wild species of Valerianaceae. Copyright © 2011 Elsevier B.V. All rights reserved.

Development and validation of liquid chromatographic and UV derivative spectrophotometric methods for the determination of famciclovir in pharmaceutical dosage forms.

PubMed

Srinubabu, Gedela; Sudharani, Batchu; Sridhar, Lade; Rao, Jvln Seshagiri

2006-06-01

A high-performance liquid chromatographic method and a UV derivative spectrophotometric method for the determination of famciclovir, a highly active antiviral agent, in tablets were developed in the present work. The various parameters, such as linearity, precision, accuracy, specificity, robustness, limit of detection and limit of quantitation were studied according to International Conference on Harmonization guidelines. HPLC was carried out by using the reversed-phase technique on an RP-18 column with a mobile phase composed of 50 mM monobasic phosphate buffer and methanol (50 : 50; v/v), adjusted to pH 3.05 with orthophosphoric acid. The mobile phase was pumped at a flow rate of 1 ml/min and detection was made at 242 nm with UV dual absorbance detector. The first derivative UV spectrophotometric method was performed at 226.5 nm. Statistical analysis was done by Student's t-test and F-test, which showed no significant difference between the results obtained by the two methods. The proposed methods are highly sensitive, precise and accurate and therefore can be used for its Intended purpose.

STEROL PROFILES IN 18 MACROALGAE OF THE PORTUGUESE COAST(1).

PubMed

Lopes, Graciliana; Sousa, Carla; Bernardo, João; Andrade, Paula B; Valentão, Patrícia; Ferreres, Federico; Mouga, Teresa

2011-10-01

The sterol profiles of dominant macroalgae occurring in the western Portuguese coast were evaluated. An analytical procedure, involving alkaline hydrolysis and extraction followed by separation by reversed-phase HPLC-diode array detection (HPLC-DAD), was optimized for the study of their sterols composition. The validated methodology is short in analysis time (as the compounds are determined in <20 min), sensitive, reproducible, and accurate. It was then successfully applied to the determination of campesterol, cholesterol, desmosterol, ergosterol, fucosterol, stigmasterol, and β-sitosterol in 18 species (three Chlorophyta, five Rhodophyta, and 10 Phaeophyta). The profiles obtained for the several macroalgal species were considerably different. C29 sterols were predominant in Phaeophyta and Chlorophyta (71%-95% of total sterol content), while in Rhodophyta cholesterol content is significantly higher (34%-87%). Among the studied species, Asparagopsis armata Harv. contained the lowest sterol amount (555 mg · kg(-1) dry weight), and Cystoseira tamariscifolia (Huds.) Papenf. the highest one (6,502 mg · kg(-1) dry weight). Data obtained may be helpful in identifying suitable marine sources of sterols, with potential applications in the food and pharmaceutical industries. © 2011 Phycological Society of America.

Preparation of penta-O-galloyl-β-D-glucose from tannic acid and plasma pharmacokinetic analyses by liquid-liquid extraction and reverse-phase HPLC.

PubMed

Li, Li; Shaik, Ahmad Ali; Zhang, Jinhui; Nhkata, Katai; Wang, Lei; Zhang, Yong; Xing, Chengguo; Kim, Sung-Hoon; Lü, Junxuan

2011-02-20

The gallotannin penta-O-galloyl-beta-D-glucose (PGG) has many biological activities including in vivo anti-cancer efficacy. We present in this paper a scaled-up protocol for its preparation in high purity from tannic acid by acidic methanolysis with typical yield of 15%. We also describe a method for the analysis of PGG in mouse plasma by HPLC and its application in preliminary pharmacokinetic studies. A liquid-liquid extraction (LLE) protocol was optimized for the extraction of PGG from mouse plasma. The extraction efficiency for PGG at 1 μg/mL in mouse plasma was 70.0±1.3% (n=5). The limit of detection (LOD) for PGG was approximately 0.2 μg/mL. Preliminary pharmacokinetic parameters of PGG following a single i.p. injection with 5% ethanol/saline vehicle in mice were established. The peak plasma PGG concentrations (C(max)) were approximately 3-4 μM at a dose of 0.5 mg per mouse (∼20 mg/kg) at 2 h post-injection (T(max)). Copyright © 2010 Elsevier B.V. All rights reserved.

Rapid high performance liquid chromatography method development with high prediction accuracy, using 5cm long narrow bore columns packed with sub-2microm particles and Design Space computer modeling.

PubMed

Fekete, Szabolcs; Fekete, Jeno; Molnár, Imre; Ganzler, Katalin

2009-11-06

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cmx2.1mm columns packed with sub-2microm particles and computer simulation (DryLab((R)) package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3-5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.

Identification of the urinary metabolites of 4-bromoaniline and 4-bromo-[carbonyl-13C]-acetanilide in rat.

PubMed

Scarfe, G B; Nicholson, J K; Lindon, J C; Wilson, I D; Taylor, S; Clayton, E; Wright, B

2002-04-01

1. The urinary excretion of 4-bromoaniline and its [carbonyl-(13)C]-labelled N-acetanilide, together with their corresponding metabolites, have been investigated in the rat following i.p. administration at 50 mg kg(-1). 2. Metabolite profiling was performed by reversed-phase HPLC with UV detection, whilst identification was performed using a combination of enzymic hydrolysis and directly coupled HPLC-NMR-MS analysis. The urinary metabolite profile was quantitatively and qualitatively similar for both compounds with little of either excreted unchanged. 3. The major metabolite present in urine was 2-amino-5-bromophenylsulphate, but, in addition, a number of metabolites with modification of the N-acetyl moiety were identified (from both the [(13)C]-acetanilide or produced following acetylation of the free bromoaniline). 4. For 4-bromoacetanilide, N-deacetylation was a major route of metabolism, but despite the detection of the acetanilide following the administration of the free aniline, there was no evidence of reacetylation (futile deacetylation). 5. Metabolites resulting from the oxidation of the acetyl group included a novel glucuronide of an N-glycolanilide, an unusual N-oxanilic acid and a novel N-acetyl cysteine conjugate.