A simple analytical procedure to replace HPLC for monitoring treatment concentrations of chloramine-T on fish culture facilities

USGS Publications Warehouse

Dawson, Verdel K.; Meinertz, Jeffery R.; Schmidt, Larry J.; Gingerich, William H.

2003-01-01

Concentrations of chloramine-T must be monitored during experimental treatments of fish when studying the effectiveness of the drug for controlling bacterial gill disease. A surrogate analytical method for analysis of chloramine-T to replace the existing high-performance liquid chromatography (HPLC) method is described. A surrogate method was needed because the existing HPLC method is expensive, requires a specialist to use, and is not generally available at fish hatcheries. Criteria for selection of a replacement method included ease of use, analysis time, cost, safety, sensitivity, accuracy, and precision. The most promising approach was to use the determination of chlorine concentrations as an indicator of chloramine-T. Of the currently available methods for analysis of chlorine, the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method best fit the established criteria. The surrogate method was evaluated under a variety of water quality conditions. Regression analysis of all DPD colorimetric analyses with the HPLC values produced a linear model (Y=0.9602 X+0.1259) with an r2 value of 0.9960. The average accuracy (percent recovery) of the DPD method relative to the HPLC method for the combined set of water quality data was 101.5%. The surrogate method was also evaluated with chloramine-T solutions that contained various concentrations of fish feed or selected densities of rainbow trout. When samples were analyzed within 2 h, the results of the surrogate method were consistent with those of the HPLC method. When samples with high concentrations of organic material were allowed to age more than 2 h before being analyzed, the DPD method seemed to be susceptible to interference, possibly from the development of other chloramine compounds. However, even after aging samples 6 h, the accuracy of the surrogate DPD method relative to the HPLC method was within the range of 80–120%. Based on the data comparing the two methods, the U.S. Food and Drug Administration has concluded that the DPD colorimetric method is appropriate to use to measure chloramine-T in water during pivotal efficacy trials designed to support the approval of chloramine-T for use in fish culture.

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.

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

Method transfer from high-pressure liquid chromatography to ultra-high-pressure liquid chromatography. I. A thermodynamic perspective.

PubMed

Åsberg, Dennis; Leśko, Marek; Samuelsson, Jörgen; Kaczmarski, Krzysztof; Fornstedt, Torgny

2014-10-03

This is the first investigation in a series that aims to enhance the scientific knowledge needed for reliable analytical method transfer between HPLC and UHPLC using the quality by design (QbD) framework. Here, we investigated the differences and similarities from a thermodynamic point of view between RP-LC separations conducted with 3.5μm (HPLC) and 1.7μm (UHPLC) C18 particles. Three different model solutes and one pharmaceutical compound were used: the uncharged cycloheptanone, the cationic benzyltriethylammonium chloride, the anionic sodium 2-naphatlene sulfonate and the pharmaceutical compound omeprazole, which was anionic at the studied pH. Adsorption data were determined for the four solutes at varying fractions of organic modifier and in gradient elution in both the HPLC and UHPLC system, respectively. From the adsorption data, the adsorption energy distribution of each compound was calculated and the adsorption isotherm model was estimated. We found that the adsorption energy distribution was similar, with only minor differences in degree of homogeneity, for HPLC and UHPLC stationary phases. The adsorption isotherm model did not change between HPLC and UHPLC, but the parameter values changed considerably especially for the ionic compounds. The dependence of the organic modifier followed the same trend in HPLC as in UHPLC. These results indicates that the adsorption mechanism of a solute is the same on HPLC and UHPLC stationary phases which simplifies design of a single analytical method applicable to both HPLC and UHPLC conditions within the QbD framework. Copyright © 2014. Published by Elsevier B.V.

Detection of honey adulteration with starch syrup by high performance liquid chromatography.

PubMed

Wang, Shaoqing; Guo, Qilei; Wang, Linlin; Lin, Li; Shi, Hailiang; Cao, Hong; Cao, Baosen

2015-04-01

According to saccharide profile comparison between starch syrups and pure honeys analysed through high performance liquid chromatography (HPLC), a characteristic peak was found at 15.25 min retention time in HPLC chromatogram of syrup, but no peak was observed at the same retention time in chromatogram of pure honeys. This characteristic peak for syrup was identified as an overlapping peak of oligosaccharides with more than 5 degree of polymerisation (DP) based on HPLC chromatogram comparison between starch syrup and a series of standard mono-, di- and oligosaccharides of 3-7 DP. Additionally syrup content correlated linearly with the height of the characteristic peak of syrup under different slope in two ranges 2.5-7.5% and 10-100%, respectively. Therefore, the characteristic peak at 15.25 min retention time can serve as a syrup indicator in HPLC analysis of the adulterated honeys. This new HPLC method for honey adulteration detection was further applied in an authenticity inspection on more than 100 commercial honeys. In addition to the improved accuracy of honey adulteration detection, the proposed HPLC method was simple, low cost and easy practice for honey product quality control by government department considering the popularity of HPLC device and technology. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quality evaluation of Yin Chen Hao Tang extract based on fingerprint chromatogram and simultaneous determination of five bioactive constituents.

PubMed

Wang, Xijun; Lv, Haitao; Sun, Hui; Jiang, Xingang; Wu, Zeming; Sun, Wenjun; Wang, Ping; Liu, Lian; Bi, Kaishun

2008-01-01

A completely validated method based on HPLC coupled with photodiode array detector (HPLC-UV) was described for evaluating and controlling quality of Yin Chen Hao Tang extract (YCHTE). First, HPLC-UV fingerprint chromatogram of YCHTE was established for preliminarily elucidating amount and chromatographic trajectory of chemical constituents in YCHTE. Second, for the first time, five mainly bioactive constituents in YCHTE were simultaneously determined based on fingerprint chromatogram for furthermore controlling the quality of YCHTE quantitatively. The developed method was applied to analyze 12 batches of YCHTE samples which consisted of herbal drugs from different places of production, showed acceptable linearity, intraday (RSD <5%), interday precision (RSD <4.80%), and accuracy (RSD <2.80%). As a result, fingerprint chromatogram determined 15 representative general fingerprint peaks, and the fingerprint chromatogram resemblances are all better than 0.9996. The contents of five analytes in different batches of YCHTE samples do not indicate significant difference. So, it is concluded that the developed HPLC-UV method is a more fully validated and complete method for evaluating and controlling the quality of YCHTE.

Comparative HPLC/ESI-MS and HPLC/DAD study of different populations of cultivated, wild and commercial Gentiana lutea L.

PubMed

Mustafa, Ahmed M; Caprioli, Giovanni; Ricciutelli, Massimo; Maggi, Filippo; Marín, Rosa; Vittori, Sauro; Sagratini, Gianni

2015-05-01

The root of Gentiana lutea L., famous for its bitter properties, is often used in alcoholic bitter beverages, food products and traditional medicine to stimulate the appetite and improve digestion. This study presents a new, fast, and accurate HPLC method using HPLC/ESI-MS and HPLC/DAD for simultaneous analysis of iridoids (loganic acid), secoiridoids (gentiopicroside, sweroside, swertiamarin, amarogentin) and xanthones (isogentisin) in different populations of G.lutea L., cultivated in the Monti Sibillini National Park, obtained wild there, or purchased commercially. Comparison of HPLC/ESI-MS and HPLC/DAD indicated that HPLC/ESI-MS is more sensitive, reliable and selective. Analysis of twenty samples showed that gentiopicroside is the most dominant compound (1.85-3.97%), followed by loganic acid (0.11-1.30%), isogentisin (0.03-0.48%), sweroside (0.05-0.35%), swertiamarin (0.08-0.30%), and amarogentin (0.01-0.07%). The results confirmed the high quality of the G.lutea cultivated in the Monti Sibillini National Park. Copyright © 2014 Elsevier Ltd. All rights reserved.

HPLC-MS/MS methods for the determination of 52 perfluoroalkyl and polyfluoroalkyl substances in aqueous samples.

PubMed

Gremmel, Christoph; Frömel, Tobias; Knepper, Thomas P

2017-02-01

Two quantitative methods using high-performance liquid chromatography (HPLC) combined with triple quadrupole tandem mass spectrometry (MS/MS) were developed to determine perfluoroalkyl and polyfluoroalkyl substances (PFASs) in aqueous samples. The first HPLC-MS/MS method was applied to 47 PFASs of 12 different substance classes with acidic characteristics such as perfluoroalkyl carboxylic acids (PFCAs) and perfluoroalkane sulfonic acids (PFSAs), as well as precursor substances and biotransformation intermediates (e.g., unsaturated fluorotelomer carboxylic acids). In addition, 25 13 C-, 18 O-, and 2 H-labeled PFASs were used as internal standards in this method. The second HPLC-MS/MS method was applied to fluorotelomer alcohols (FTOHs) and perfluorooctane sulfonamidoethanols as these compounds have physicochemical properties different from those of the previous ones. Accuracy between 82% and 110% and a standard deviation in the range from 2% to 22% depending on the substances were determined during the evaluation of repeatability and precision. The method quantification limit after solid-phase extraction ranged from 0.3 to 199 ng/L depending on the analyte and matrix. The HPLC-MS/MS methods developed were suitable for the determination of PFASs in aqueous samples (e.g., wastewater treatment plant effluents or influents after solid-phase extraction). These methods will be helpful in monitoring campaigns to evaluate the relevance of precursor substances as indirect sources of perfluorinated substances in the environment. In one exemplary application in an industrial wastewater treatment plant, FTOHs were found to be the major substance class in the influent; in particular, 6:2-FTOH was the predominant compound in the industrial samples and accounted for 74% of the total PFAS concentration. The increase in the concentration of the transformation products of FTOHs in the corresponding effluent, such as fluorotelomer carboxylic acids, unsaturated fluorotelomer carboxylic acids, n:3 polyfluorinated saturated carboxylic acids (n indicates the number of nonfluorinated carbon atoms), and PFCAs, indicated biotransformation of FTOHs or their derivatives during wastewater treatment. However, only 33 mol% of the total amount of PFASs present in the influent was quantified in the corresponding effluent. Graphical abstract Method development of an HPLC-MS/MS multi-method for the determination of PFASs in aqueos samples.

Characterization of nonstarch polysaccharides content from different edible organs of some vegetables, determined by GC and HPLC: comparative study.

PubMed

Villanueva-Suárez, M J; Redondo-Cuenca, A; Rodríguez-Sevilla, M D; de las Heras Martínez, M

2003-09-24

Content and composition of dietary fiber as nonstarch polysaccharides (NSP) was determined in vegetables belonging to different types of edible organs, using GC and HPLC. Samples analyzed were subterranean organs (radish and leek), leaves (celery, swiss chard, and lettuce), stalks (celery, swiss chard, and asparagus), inflorescence (broccoli), and fruits (tomato, green pepper, and marrow). The results indicate that though the monomeric profile is similar in all these samples quantitative differences were found for neutral sugars and uronic acids among samples of the same type of vegetal organ. The NSP values determined using CG method were in good agreement with HPLC method (R(2) = 0.9005). However, arabinose, mannose, and galactose plus rhamnose are more influenced by the analytical method used than the rest of the monomers in nearly all the samples analyzed. Final values of NSP depend on the method used in celery stalks, broccoli, and green pepper.

Development and validation of a rapid stability indicating HPLC-method using monolithic stationary phase and two spectrophotometric methods for determination of antihistaminic acrivastine in capsules

NASA Astrophysics Data System (ADS)

Gouda, Ayman A.; Hashem, Hisham; Jira, Thomas

2014-09-01

Simple, rapid and accurate high performance liquid chromatographic (HPLC) and spectrophotometric methods are described for determination of antihistaminic acrivastine in capsules. The first method (method A) is based on accurate, sensitive and stability indicating chromatographic separation method. Chromolith® Performance RP-18e column, a relatively new packing material consisting of monolithic rods of highly porous silica, was used as stationary phase applying isocratic binary mobile phase of ACN and 25 mM NaH2PO4 pH 4.0 in the ratio of 22.5:77.5 at flow rate of 5.0 mL/min and 40 °C. A diode array detector was used at 254 nm for detection. The elution time of acrivastine was found to be 2.080 ± 0.032. The second and third methods (methods B and C) are based on the oxidation of acrivastine with excess N-bromosuccinimide (NBS) and determination of the unconsumed NBS with, metol-sulphanilic acid (λmax: 520 nm) or amaranth dye (λmax: 530 nm). The reacted oxidant corresponds to the drug content. Beer’s law is obeyed over the concentration range 1.563-50, 2.0-20 and 1.0-10 μg mL-1 for methods A, B and C, respectively. The limits of detection and quantitation were 0.40, 0.292 and 0.113 μg mL-1 and 0.782, 0.973 and 0.376 μg mL-1 for methods A, B and C, respectively. The HPLC method was validated for system suitability, linearity, precision, limits of detection and quantitation, specificity, stability and robustness. Stability tests were done through exposure of the analyte solution for four different stress conditions and the results indicate no interference of degradants with HPLC-method. The proposed methods was favorably applied for determination of acrivastine in capsules formulation. Statistical comparison of the obtained results from the analysis of the studied drug to those of the reported method using t- and F-tests showed no significant difference between them.

Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)

PubMed Central

Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

2017-01-01

Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels. PMID:28170419

[The qualitative analysis and quantitative analysis of purification of salvianolic acids by macroreticular resin].

PubMed

Fang, Xin-sheng; Tan, Xiao-mei

2005-09-01

To purify salvianolic acids by macroreticular resin,then mensurate the contents of salvianolic acids and analyse the chromatogram with HPLC. Make salvianolic acids with macroreticular resin; mensurate the content of Salvianolic acids with UV spestrophotometry: the control compound is protocaechuic aldehyde, and the wavelength is 281 nm. Analysis the chromatogram with HPLC, and compare the chromatogram in different technics: zorbax ODS column (4.6 mm x 250 mm, 5 microm), mobilephase: 1% aceticacid-water and methanol in different proportions, the wavelength is 281 nm. The contents of salvianolic acids is 53.8%; HPLC chromatogram indicate that the method is reasonable to make salvianolic acids. Determination of contents and HPLC chromatogram can control the quality of Salvianolic acids more accurately.

A Stability-Indicating HPLC Method for the Determination of Fingolimod in Pharmaceutical Dosage Forms.

PubMed

Souri, Effat; Zargarpoor, Mohammad; Mottaghi, Siavash; Ahmadkhaniha, Reza; Kebriaeezadeh, Abbas

2015-01-01

Fingolimod is an immunosuppressive agent which is used for the prophylaxis of organ transplantation rejection or multiple sclerosis treatment. In this study, systematic forced degradation studies on fingolimod bulk powder were performed to develop a stability-indicating HPLC method. Separation of fingolimod and its degradation products was achieved on a Nova-Pak C8 column. The mobile phase was a mixture of potassium dihydrogenphosphate 50 mM (pH 3.0) and acetonitrile (45:55, v/v) at a flow rate of 1 ml/min. The proposed method was linear in the range of 0.125-20 μg mL(-1). The within-day and between-day coefficients of variation were in the range of 0.6-1.2%. The developed method was successfully applied for the determination of the fingolimod amount in pharmaceutical dosage forms.

A simple analytical procedure to replace HPLC for monitoring treatment concentrations of chloramine-T on fish culture facilities

USGS Publications Warehouse

Dawson, V.K.; Meinertz, J.R.; Schmidt, L.J.; Gingerich, W.H.

2003-01-01

Concentrations of chloramine-T must be monitored during experimental treatments of fish when studying the effectiveness of the drug for controlling bacterial gill disease. A surrogate analytical method for analysis of chloramine-T to replace the existing high-performance liquid chromatography (HPLC) method is described. A surrogate method was needed because the existing HPLC method is expensive, requires a specialist to use, and is not generally available at fish hatcheries. Criteria for selection of a replacement method included ease of use, analysis time, cost, safety, sensitivity, accuracy, and precision. The most promising approach was to use the determination of chlorine concentrations as an indicator of chloramine-T. Of the currently available methods for analysis of chlorine, the DPD (N,N-diethyl-p-phenylenediamine) colorimetric method best fit the established criteria. The surrogate method was evaluated under a variety of water quality conditions. Regression analysis of all DPD colorimetric analyses with the HPLC values produced a linear model (Y=0.9602 X+0.1259) with an r2 value of 0.9960. The average accuracy (percent recovery) of the DPD method relative to the HPLC method for the combined set of water quality data was 101.5%. The surrogate method was also evaluated with chloramine-T solutions that contained various concentrations of fish feed or selected densities of rainbow trout. When samples were analyzed within 2 h, the results of the surrogate method were consistent with those of the HPLC method. When samples with high concentrations of organic material were allowed to age more than 2 h before being analyzed, the DPD method seemed to be susceptible to interference, possibly from the development of other chloramine compounds. However, even after aging samples 6 h, the accuracy of the surrogate DPD method relative to the HPLC method was within the range of 80-120%. Based on the data comparing the two methods, the U.S. Food and Drug Administration has concluded that the DPD colorimetric method is appropriate to use to measure chloramine-T in water during pivotal efficacy trials designed to support the approval of chloramine-T for use in fish culture. ?? 2003 Elsevier Science B.V. All rights reserved.

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.

Spectrophotometric and HPLC determinations of anti-diabetic drugs, rosiglitazone maleate and metformin hydrochloride, in pure form and in pharmaceutical preparations.

PubMed

Onal, Armağan

2009-12-01

In this study, three spectrophotometric methods and one HPLC method were developed for analysis of anti-diabetic drugs in tablets. The two spectrophotometric methods were based on the reaction of rosiglitazone (RSG) with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and bromocresol green (BCG). Linear relationship between the absorbance at lambda(max) and the drug concentration was found to be in the ranges 6.0-50.0 and 1.5-12 microg ml(-1) for DDQ and BCG methods, respectively. The third spectrophotometric method consists of a zero-crossing first-derivative spectrophotometric method for simultaneous analysis of RSG and metformin (MTF) in tablets. The calibration curves were linear within the concentration ranges of 5.0-50 microg ml(-1) for RSG and 1.0-10.0 microg ml(-1) for MTF. The fourth method is a rapid stability-indicating HPLC method developed for the determination of RSG. A linear response was observed within the concentration range of 0.25-2.5 microg ml(-1). The proposed methods have been successfully applied to the tablet analysis.

HPLC analysis and standardization of Brahmi vati - An Ayurvedic poly-herbal formulation.

PubMed

Mishra, Amrita; Mishra, Arun K; Tiwari, Om Prakash; Jha, Shivesh

2013-09-01

The aim of the present study was to standardize Brahmi vati (BV) by simultaneous quantitative estimation of Bacoside A3 and Piperine adopting HPLC-UV method. BV very important Ayurvedic polyherbo formulation used to treat epilepsy and mental disorders containing thirty eight ingredients including Bacopa monnieri L. and Piper longum L. An HPLC-UV method was developed for the standardization of BV in light of simultaneous quantitative estimation of Bacoside A3 and Piperine, the major constituents of B. monnieri L. and P. longum L. respectively. The developed method was validated on parameters including linearity, precision, accuracy and robustness. The HPLC analysis showed significant increase in amount of Bacoside A3 and Piperine in the in-house sample of BV when compared with all three different marketed samples of the same. Results showed variations in the amount of Bacoside A3 and Piperine in different samples which indicate non-uniformity in their quality which will lead to difference in their therapeutic effects. The outcome of the present investigation underlines the importance of standardization of Ayurvedic formulations. The developed method may be further used to standardize other samples of BV or other formulations containing Bacoside A3 and Piperine.

Stability-Indicating Related Substances HPLC Method for Droxidopa and Characterization of Related Substances Using LC-MS and NMR.

PubMed

Kumar, Thangarathinam; Ramya, Mohandass; Arockiasamy Xavier, S J

2016-11-01

Stress degradation studies using high-performance liquid chromatography (HPLC) was performed and validated for Droxidopa (L-DOPS). Droxidopa was susceptible to acid hydrolysis (0.1 N HCl), alkaline hydrolysis (0.15 N NaOH) and thermal degradation (105°C). It was found to be resistant to white light, oxidation and UV light exposure (72 h). The thermal, acid and alkali degradation impurities were detected with the retention time (RT) of 12.7, 19.25 and 22.95 min. Our HPLC method detected process impurities (2R,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropionic acid (Impurity H), N-Hydroxypthalimide (Impurity N), (2R,3S)-2-amino-3-(benzo[d][1,3]dioxol-5-yl)-3-hydroxypropionic acid (Impurity L) and L-threo n-phthaloyl-3-(3, 4-dihydroxyphenyl)-serine (Intermediate) with RTs of 3.48, 15.5, 25.76 and 28.0 min. The related substances were further characterized and confirmed by liquid chromatography-mass spectroscopy (LC-MS), and nuclear magnetic resonance spectroscopy analysis. Our HPLC method detected up to 0.05 µg/mL of Droxidopa with S/N > 3.0 and quantified up to 0.10 µg /mL of Droxidopa with S/N ratio > 10.0. Droxidopa was highly stable for 12 h after its preparation for HPLC analysis. Our newly developed HPLC method was highly precise, specific, reliable and accurate for the analysis of Droxidopa and its related substances. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative and pattern recognition analyses of magnoflorine, spinosin, 6'''-feruloyl spinosin and jujuboside A by HPLC in Zizyphi Semen.

PubMed

Kim, Won Il; Zhao, Bing Tian; Zhang, Hai Yan; Lee, Je Hyun; Son, Jong Keun; Woo, Mi Hee

2014-01-01

Two rapid and simple HPLC methods with UV detector to determine three main compounds (magnoflorine, spinosin and 6'''-feruloyl spinosin) and evaporative light scattering detector (ELSD) to determine jujuboside A were developed for the chemical analyses of Zizyphi Semen. Magnoflorine, spinosin, and 6'''-feruloyl spinosin were separated with an YMC J'sphere ODS-H80 column (250 mm × 4.6 mm, 4 μm) by the gradient elution followed by the isocratic elution using methanol with 0.1 % formic acid and water with 0.1 % formic acid as the mobile phase. The flow rate was 1.0 mL/min. Jujuboside A was separated by HPLC-ELSD with YoungJinBioChrom Aegispak C18-L column (250 mm × 4.6 mm, 5 μm) column in a gradient elution using methanol with 0.1 % formic acid (A) and water with 0.1 % formic acid as the mobile phase. These two methods were fully validated with respect to linearity, precision, accuracy, stability, and robustness. These HPLC methods were applied successfully to quantify four compounds in a Zizyphi Semen extract. The HPLC analytical methods were validated for pattern recognition analysis by repeated analysis of 91 seed samples corresponding to 48 Zizyphus jujuba var. spinosa (J01-J48) and 43 Zizyphus mauritiana (M01-M43). The results indicate that these methods are suitable for a quality evaluation of Zizyphi Semen.

Analysis Study of Stevioside and Rebaudioside A from Stevia rebaudiana Bertoni by Normal Phase SPE and RP-HPLC

NASA Astrophysics Data System (ADS)

Martono, Y.; Rohman, A.; Riyanto, S.; Martono, S.

2018-04-01

Solid Phase Extraction (SPE) method using silica as sorbent for stevioside and rebaudiosida A analysis in Stevia rebaudiana Bertoni leaf have not been performed. The aim of this study is to develop SPE method using silica as sorbent for Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) analysis of stevioside and rebaudiosida A in S. rebaudiana leaf. The results of this study indicate that the optimal conditions for normal phase SPE (silica) are conditioned with 3.0 mL of hexane. The sample loading volume is 0.1 mL. Cartridge is eluted with 1.0 mL acetonitrile: water (80: 20, v/v) to separate both analytes. The cartridge is washed with chloroform and water of 0.3 mL respectively. The developed SPE sample preparation method meets the accuracy and precision test and can be used for the analysis of stevioside and rebaudioside A by RP-HPLC.

Comparison of Peak-area Ratios and Percentage Peak Area Derived from HPLC-evaporative Light Scattering and Refractive Index Detectors for Palm Oil and its Fractions.

PubMed

Ping, Bonnie Tay Yen; Aziz, Haliza Abdul; Idris, Zainab

2018-01-01

High-Performance Liquid Chromatography (HPLC) methods via evaporative light scattering (ELS) and refractive index (RI) detectors are used by the local palm oil industry to monitor the TAG profiles of palm oil and its fractions. The quantitation method used is based on area normalization of the TAG components and expressed as percentage area. Although not frequently used, peak-area ratios based on TAG profiles are a possible qualitative method for characterizing the TAG of palm oil and its fractions. This paper aims to compare these two detectors in terms of peak-area ratio, percentage peak area composition, and TAG elution profiles. The triacylglycerol (TAG) composition for palm oil and its fractions were analysed under similar HPLC conditions i.e. mobile phase and column. However, different sample concentrations were used for the detectors while remaining within the linearity limits of the detectors. These concentrations also gave a good baseline resolved separation for all the TAGs components. The results of the ELSD method's percentage area composition for the TAGs of palm oil and its fractions differed from those of RID. This indicates an unequal response of TAGs for palm oil and its fractions using the ELSD, also affecting the peak area ratios. They were found not to be equivalent to those obtained using the HPLC-RID. The ELSD method showed a better baseline separation for the TAGs components, with a more stable baseline as compared with the corresponding HPLC-RID. In conclusion, the percentage area compositions and peak-area ratios for palm oil and its fractions as derived from HPLC-ELSD and RID were not equivalent due to different responses of TAG components to the ELSD detector. The HPLC-RID has a better accuracy for percentage area composition and peak-area ratio because the TAG components response equally to the detector.

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.

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9-12 Years Children.

PubMed

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9-11 years were determined by two methods of CPBA and HPLC. Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes.

A Stability-Indicating HPLC Method for the Determination of Fingolimod in Pharmaceutical Dosage Forms

PubMed Central

Souri, Effat; Zargarpoor, Mohammad; Mottaghi, Siavash; Ahmadkhaniha, Reza; Kebriaeezadeh, Abbas

2015-01-01

Fingolimod is an immunosuppressive agent which is used for the prophylaxis of organ transplantation rejection or multiple sclerosis treatment. In this study, systematic forced degradation studies on fingolimod bulk powder were performed to develop a stability-indicating HPLC method. Separation of fingolimod and its degradation products was achieved on a Nova-Pak C8 column. The mobile phase was a mixture of potassium dihydrogenphosphate 50 mM (pH 3.0) and acetonitrile (45:55, v/v) at a flow rate of 1 ml/min. The proposed method was linear in the range of 0.125–20 μg mL−1. The within-day and between-day coefficients of variation were in the range of 0.6–1.2%. The developed method was successfully applied for the determination of the fingolimod amount in pharmaceutical dosage forms. PMID:26839803

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.

Study on the Multi-marker Components Quantitative HPLC Fingerprint of the Compound Chinese Medicine Wuwei Changyanning Granule

PubMed Central

Yang, Xian; Yang, Shui-Ping; Zhang, Xue; Yu, Xiao-Dong; He, Qi-Yi; Wang, Bo-Chu

2014-01-01

The aim of this paper is to develop a rapid and highly sensitive quantitative HPLC fingerprint method with multiple indicators by using the Compound Chinese Medicine Wuwei Changyanning granule and 5 herbs in the prescription. The quantitative fingerprint chromatogram with multiple indicators was investigated. і)6 compositions included rutin, gallic acid, chlorogenic acid, atractylenolide Ⅰ, pachymic acid and apigenin, which originated from 5 herbs respectively, were selected as quantitative compositions, and their contents were determined using HPLC from 11 batches granules and the corresponding 5 medicinal materials. ⅱ) The precision, stability and repeatability of fingerprinting were investigated. In addition, common peaks number, the percentage of non-common peaks and similarity were also studied. Among them, 21 common peaks in the granule could find the source of peaks from the 5 herbs, among of 10 peaks from Niuerfeng, 9 peaks from Laliao, 3 peaks from Baishu, 3 peaks from Fuling and 5 peaks from Guanghuoxiang. The results showed that the identification method of fingerprinting was reliable. PMID:25587307

Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children

PubMed Central

Zahedi Rad, Maliheh; Neyestani, Tirang Reza; Nikooyeh, Bahareh; Shariatzadeh, Nastaran; Kalayi, Ali; Khalaji, Niloufar; Gharavi, Azam

2015-01-01

Background: The most reliable indicator of Vitamin D status is circulating concentration of 25-hydroxycalciferol (25(OH) D) routinely determined by enzyme-immunoassays (EIA) methods. This study was performed to compare commonly used competitive protein-binding assays (CPBA)-based EIA with the gold standard, high-pressure liquid chromatography (HPLC). Methods: Concentrations of 25(OH) D in sera from 257 randomly selected school children aged 9–11 years were determined by two methods of CPBA and HPLC. Results: Mean 25(OH) D concentration was 22 ± 18.8 and 21.9 ± 15.6 nmol/L by CPBA and HPLC, respectively. However, mean 25(OH) D concentrations of the two methods became different after excluding undetectable samples (25.1 ± 18.9 vs. 29 ± 14.5 nmol/L, respectively; P = 0.04). Based on predefined Vitamin D deficiency as 25(OH) D < 12.5 nmol/L, CPBA sensitivity and specificity were 44.2% and 60.6%, respectively, compared to HPLC. In receiver operating characteristic curve analysis, the best cut-offs for CPBA was 5.8 nmol/L, which gave 82% sensitivity, but specificity was 17%. Conclusions: Though CPBA may be used as a screening tool, more reliable methods are needed for diagnostic purposes. PMID:26330983

[Determination of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla by HPLC].

PubMed

Yang, Xiu-Juan; Hong, Yan-Long; Wu, Fei; Ruan, Ke-Feng; Feng, Yi

2013-03-01

To explore an HPLC method for determination of rhnchophylline and isorhnchophylline in Uncaria rhnchophylla. An HPLC method has been developed for determination of rhnchophylline and isorhnchophylline. The transformation of rhnchophylline and isorhnchophylline after heating was also studied by HPLC-ESI-MS. Good linearities of rhynchophylline and isorhynchophylline were 0.064-5.100, 0.064-5.110 mg, respectively. The average recoveries were from 87.51% to 88.83% for rhynchophylline and from 107.9% to 113.9% for isorhynchophylline. The recoveries of rhynchophylline and isorhnchophylline reference solutions after extraction were 12.60% and 40.00% in the reflux extraction procedure, respectively. While in the ultrasonic extraction procedure, the average recoveries of rhynchophylline and isorhynchophylline was from 99.48% to 103.2% and from 97.00% to 99.59%, resepectively. The recoveries of rhynchophylline and isorhnchophylline reference solutions after extraction were 47.08% and 51.03%, respectively. The unqualified recovery could be elucidated by HPLC-ESI-MS analysis, indicating that trhynchophylline could be transformed mostly into isorhynchophylline and a little amount of unkown composition, while isorhynchophylline could be transformed into rhynchophylline isocorynoxeine, corynoxeine and 22-O-beta-D-glucopyranosyl isocorynoxeinic acid during the extraction procedure. Ultrasonic extraction procedure was more sutble for HPLC determination of the content of rhynchophylline and isorhynchophylline in U. rhnchophylla, however, the recovery problems should be paid attention to when it comes to the determination.

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

PubMed

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

2009-01-01

Although there are various determination methods for gamma -oryzanol contained in rice bran oil by absorptiometry, normal-phase HPLC, and reversed-phase HPLC, their accuracies and the correlations among them have not been revealed yet. Chloroform-containing mixed solvents are widely used as mobile phases in some HPLC methods, but researchers have been apprehensive about its use in terms of safety for the human body and the environment.In the present study, a simple and accurate determination method was developed by improving the reversed-phase HPLC method. This novel HPLC method uses methanol/acetonitrile/acetic acid (52/45/3 v/v/v), a non-chlorinated solvent, as the mobile phase, and shows an excellent linearity (y = 0.9527x + 0.1241, R(2) = 0.9974) with absorptiometry. The mean relative errors among the existing 3 methods and the novel method, determined by adding fixed amounts of gamma-oryzanol into refined rice salad oil, were -4.7% for the absorptiometry, -6.8% for the existing normal-phase HPLC, +4.6% for the existing reversed-phase HPLC, and -1.6% for the novel reversed-phase HPLC method. gamma -Oryzanol content in 12 kinds of crude rice bran oils obtained from different sources were determined by the four methods. The mean content of those oils were 1.75+/-0.18% for the absorptiometry, 1.29+/-0.11% for the existing normal-phase HPLC, 1.51+/-0.10% for the existing reversed-phase HPLC, and 1.54+/-0.19% for the novel reversed-phase HPLC method.

77 FR 67282 - Dinotefuran; Pesticide Tolerances for Emergency Exemptions

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

... performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method is available. For the determination of residues of dinotefuran only, an HPLC/ultraviolet (UV) detection method is available. For the determination of only the metabolites (DN and UF), HPLC/MS and HPLC/MS/MS methods are available. These methods...

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.

[HPLC fingerprint of the antiarrhythmic fraction of Valeriana officinalis].

PubMed

Duan, Xue-Yun; Gong, Zhan-Feng; Chen, Shu-He; Fang, Ying; Liu, Yan-Wen

2009-06-01

To establish HPLC fingerprints of the Antiarrhythmic fraction of Valeriana officinalis. Agilent C18 (250 mm x 4.6 mm, 5 microm) column was used and the acetonitrile-water was chosen as the mobile phase in a gradient mode. The column temperature was 380 degrees C and the detection wavelength was 218 nm. The detection time was 70 min, and the flow rate was 1.0 mL/ min. Fifteen characteristic peaks were indicated in HPLC fingerprints. The relative retention time and the ranges of relative areas of the common peaks were also determined. This method is simple and accurate with a good reproducibility and provides a reference standard for the quality control of Valeriana officinalis.

Stability indicating HPLC-UV method for detection of curcumin in Curcuma longa extract and emulsion formulation.

PubMed

Syed, Haroon Khalid; Liew, Kai Bin; Loh, Gabriel Onn Kit; Peh, Kok Khiang

2015-03-01

A stability-indicating HPLC-UV method for the determination of curcumin in Curcuma longa extract and emulsion was developed. The system suitability parameters, theoretical plates (N), tailing factor (T), capacity factor (K'), height equivalent of a theoretical plate (H) and resolution (Rs) were calculated. Stress degradation studies (acid, base, oxidation, heat and UV light) of curcumin were performed in emulsion. It was found that N>6500, T<1.1, K' was 2.68-3.75, HETP about 37 and Rs was 1.8. The method was linear from 2 to 200 μg/mL with a correlation coefficient of 0.9998. The intra-day precision and accuracy for curcumin were ⩽0.87% and ⩽2.0%, while the inter-day precision and accuracy values were ⩽2.1% and ⩽-1.92. Curcumin degraded in emulsion under acid, alkali and UV light. In conclusion, the stability-indicating method could be employed to determine curcumin in bulk and emulsions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Simultaneous Estimation of Withaferin A and Z-Guggulsterone in Marketed Formulation by RP-HPLC.

PubMed

Agrawal, Poonam; Vegda, Rashmi; Laddha, Kirti

2015-07-01

A simple, rapid, precise and accurate high-performance liquid chromatography (HPLC) method was developed for simultaneous estimation of withaferin A and Z-guggulsterone in a polyherbal formulation containing Withania somnifera and Commiphora wightii. The chromatographic separation was achieved on a Purosphere RP-18 column (particle size 5 µm) with a mobile phase consisting of Solvent A (acetonitrile) and Solvent B (water) with the following gradients: 0-7 min, 50% A in B; 7-9 min, 50-80% A in B; 9-20 min, 80% A in B at a flow rate of 1 mL/min and detection at 235 nm. The marker compounds were well separated on the chromatogram within 20 min. The results obtained indicate accuracy and reliability of the developed simultaneous HPLC method for the quantification of withaferin A and Z-guggulsterone. The proposed method was found to be reproducible, specific, precise and accurate for simultaneous estimation of these marker compounds in a combined dosage form. The HPLC method was appropriate and the two markers are well resolved, enabling efficient quantitative analysis of withaferin A and Z-guggulsterone. The method can be successively used for quantitative analysis of these two marker constituents in combination of marketed polyherbal formulation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Cholesterol concentrations in lipoprotein fractions separated by anion-exchange-high-performance liquid chromatography in healthy dogs and dogs with hypercholesterolemia.

PubMed

Oda, Hitomi; Mori, Akihiro; Hirowatari, Yuji; Takoura, Toshie; Manita, Daisuke; Takahashi, Tomoya; Shono, Saori; Onozawa, Eri; Mizutani, Hisashi; Miki, Yohei; Itabashi, Yukiko; Sako, Toshinori

2017-10-01

Anion-exchange (AEX)-high-performance liquid chromatography (HPLC) for measurement of cholesterol can be used to separate serum lipoproteins (high-density lipoprotein (HDL); low-density lipoprotein (LDL); intermediate-density lipoprotein (IDL); very-low-density lipoprotein (VLDL)) in humans. However, AEX-HPLC has not been applied in veterinary practice. We had three objectives: (i) the validation of AEX-HPLC methods including the correlation of serum cholesterol concentration in lipoprotein fraction measured by AEX-HPLC and gel permeation-HPLC (GP-HPLC) in healthy dogs and those with hypercholesterolemia was investigated; (ii) the reference intervals of lipoprotein fractions measured by AEX-HPLC from healthy dogs (n=40) was established; (iii) lipoprotein fractions from the serum of healthy dogs (n=12) and dogs with hypercholesterolemia (n=23) were compared. Analytic reproducibility and precision of AEX-HPLC were acceptable. Positive correlation between serum concentrations of total cholesterol (Total-Chol), HDL cholesterol (HDL-Chol), LDL cholesterol (LDL-Chol)+IDL cholesterol (IDL-Chol), and VLDL cholesterol (VLDL-Chol) was noted for AEX-HPLC and GP-HPLC in healthy dogs and dogs with hypercholesterolemia. Reference intervals measured by AEX-HPLC for serum concentrations of Total-Chol, HDL-Chol, and LDL-Chol were determined to be 2.97-9.32, 2.79-6.57, 0.16-3.28mmol/L (2.5-97.5% interval), respectively. Furthermore, there was significant difference in lipoprotein profiles between healthy and dogs with hypercholesterolemia. These results suggest that AEX-HPLC can be used to evaluate lipoprotein profiles in dogs and could be a new useful indicator of hyperlipidemia in dogs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ensuring repeatability and robustness of poly(glycidyl methacrylate-co-ethylene dimethacrylate) HPLC monolithic columns of 3 mm id through covalent bonding to the column wall.

PubMed

Laaniste, Asko; Kruve, Anneli; Leito, Ivo

2013-08-01

Two different methods to reinforce the poly(glycidyl methacrylate-co-ethylene dimethacrylate) HPLC monolithic columns of 3 mm id in a glass column reservoir were studied: composite columns with polymeric particles in the monolith and surface treatment of the reservoir wall. Of the two methods used to counter the mechanical instability and formation of flow channels (composite columns and column wall surface treatment), we demonstrated that proper column wall surface treatment was sufficient to solve both problems. Our study also indicated that no surface treatment is efficient, and of the methods studied silanization in acidified ethanol solution and constant renewal of the reaction mixture (dynamic mode) proved to be the most effective. As a result of this study, we have been able to prepare repeatable and durable methacrylate HPLC columns with good efficiencies. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ultratraces of carotenes in tomato purées: HPLC-TLS study

NASA Astrophysics Data System (ADS)

Luterotti, S.; Marković, K.; Franko, M.; Bicanic, D.; Vahčić, N.; Doka, O.

2003-01-01

The present study was designed to provide information about (i) the profile of carotene pigments and (ii) trace quantities of lycopene and β-carotene left in tomato purées. The ultrasensitive method comprising HPLC and thermal lens spectrometric (TLS) detection enabled us to detect as low as 0.3 and 1.1 ng ml-1 lycopene and β-carotene in purée extracts, respectively. Total concentration of β-carotene and lycopene (varying from 3 to 170 ng g-1) in the examined tomato purées may serve as an indicator of the carotene-specific antioxidative capacity of these products. Although conventional spectrophotometry can be used to rapidly assess the quality of products derived from tomatoes, a highly sensitive and selective method such as HPLC-TLS is needed for reliable analyses of samples such as, for example, those subjected to inappropriate storage and/or handling.

Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection.

PubMed

Colombo, Renata; Lanças, Fernando M; Yariwake, Janete H

2006-01-20

A high-performance liquid chromatography (HPLC) method with photo-diode array (DAD) detection was developed to separate and quantify flavonoids in sugarcane leaves and bagasse (= the crushed sugarcane refuse from juice extraction), and in sugarcane juice. Sugarcane flavonoids consist of a complex mixture of aglycones and glycosides (including flavonolignan glycosides), and the HPLC-UV method herein proposed is suitable for their quantification as total flavonoids. This method was applied to analyze samples of cultivated sugarcane, commercial juice and transgenic sugarcane leaves. Sugarcane leaves proved a promising source of flavonoids: an average of 1.10 mg of total flavonoids/g plant material was found in fresh leaves. Moreover, the flavonoid content of sugarcane juice (0.6 mg/mL) is comparable to other food sources of flavonoids previously reported. Transgenic sugarcane leaves ("Bowman-Birk" and "Kunitz") were compared with non-modified ("control") plant samples using the proposed HPLC-UV method, which indicated that the content of total flavonoids in transgenic plants is different from that in non-modified sugarcane.

High performance liquid chromatography used for quality control of Achyranthis Radix.

PubMed

Zhao, Bing Tian; Jeong, Su Yang; Moon, Dong Cheul; Son, Kun Ho; Son, Jong Keun; Woo, Mi Hee

2012-08-01

To establish a standard of quality control and to identify reliable Achyranthis Radix, three phytoecdysones including ecdysterone (1), 25R-inokosterone (2) and 25S-inokosterone (3) were determined by quantitative HPLC/UV analysis. Three phytoecdysones were separated with an YMC J'sphere ODS C(18) column (250 mm × 4.6 mm, 4 μm) by isocratic elution using 0.1% formic acid in water and acetonitrile (85:15, v/v%) as the mobile phase. The flow rate was 1.0 mL/min and the UV detector wavelength was set at 245 nm. The standards were quantified by HPLC/UV from Achyranthes bidentata Blume and Achyranthes japonica Nakai, as well as Cyathula capitata Moq. and Cyathula officinalis Kuan, which are of a different genus but are comparative herbs. The method was successfully used in the analysis of Achyranthis Radix of different geographical origin or genera with relatively simple conditions and procedures, and the assay results were satisfactory for linearity, recovery, precision, accuracy, stability and robustness. The HPLC analytical method for pattern recognition analysis was validated by repeated analysis of eighteen A. bidentata Blume samples and ten A. japonica Nakai samples. The results indicate that the established HPLC/UV method is suitable for quantitation and pattern recognition analyses for quality evaluation of Achyranthis Radix.

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.

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.

Updated folate data in the Dutch Food Composition Database and implications for intake estimates

PubMed Central

Westenbrink, Susanne; Jansen-van der Vliet, Martine; van Rossum, Caroline

2012-01-01

Background and objective Nutrient values are influenced by the analytical method used. Food folate measured by high performance liquid chromatography (HPLC) or by microbiological assay (MA) yield different results, with in general higher results from MA than from HPLC. This leads to the question of how to deal with different analytical methods in compiling standardised and internationally comparable food composition databases? A recent inventory on folate in European food composition databases indicated that currently MA is more widely used than HPCL. Since older Dutch values are produced by HPLC and newer values by MA, analytical methods and procedures for compiling folate data in the Dutch Food Composition Database (NEVO) were reconsidered and folate values were updated. This article describes the impact of this revision of folate values in the NEVO database as well as the expected impact on the folate intake assessment in the Dutch National Food Consumption Survey (DNFCS). Design The folate values were revised by replacing HPLC with MA values from recent Dutch analyses. Previously MA folate values taken from foreign food composition tables had been recalculated to the HPLC level, assuming a 27% lower value from HPLC analyses. These recalculated values were replaced by the original MA values. Dutch HPLC and MA values were compared to each other. Folate intake was assessed for a subgroup within the DNFCS to estimate the impact of the update. Results In the updated NEVO database nearly all folate values were produced by MA or derived from MA values which resulted in an average increase of 24%. The median habitual folate intake in young children was increased by 11–15% using the updated folate values. Conclusion The current approach for folate in NEVO resulted in more transparency in data production and documentation and higher comparability among European databases. Results of food consumption surveys are expected to show higher folate intakes when using the updated values. PMID:22481900

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog.

PubMed

Dunphy, Michael J; Sysel, Annette M; Lupica, Joseph A; Griffith, Kristie; Sherrod, Taylor; Bauer, Joseph A

2014-04-01

Nitrosylcobalamin (NO-Cbl), a novel vitamin B 12 analog and anti-tumor agent, functions as a biologic 'Trojan horse', utilizing the vitamin B 12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis ® RP-amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min -1 ) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B 12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl.

A Stability-Indicating HPLC Method for the Determination of Nitrosylcobalamin (NO-Cbl), a Novel Vitamin B12 Analog

PubMed Central

Dunphy, Michael J.; Sysel, Annette M.; Lupica, Joseph A.; Griffith, Kristie; Sherrod, Taylor

2014-01-01

Nitrosylcobalamin (NO-Cbl), a novel vitamin B12 analog and anti-tumor agent, functions as a biologic ‘Trojan horse’, utilizing the vitamin B12 transcobalamin II transport protein and cell surface receptor to specifically target cancer cells. a stability-indicating HPLC method was developed for the detection of NO-Cbl during forced degradation studies. This method utilized an ascentis® RP-amide (150 mm × 4.6 mm, 5 μm) column at 35 °C with a mobile phase (1.0 mL min−1) combining a gradient of methanol and an acetate buffer at pH 6.0. Detection wavelengths of 450 and 254 nm were used to detect corrin and non-corrin-based products, respectively. NO-Cbl, synthesized from hydroxocobalamin and pure nitric oxide gas, was subjected to degradative stress conditions including oxidation, hydrolysis and thermal and radiant energy challenge. The method was validated by assessing linearity, accuracy, precision, detection and quantitation limits and robustness. The method was applied successfully for purity assessment of synthesized NO-Cbl and for the determination of NO-Cbl during kinetic studies in aqueous solution and in solid-state degradation assessments. This HPLC method is suitable for the separation of cobalamins in aqueous and methanolic solutions, for routine detection of NO-Cbl and for purity assessment of synthesized NO-Cbl. additionally, this method has potential application in identification and monitoring of diseases involving altered nitric oxide homeostasis where vitamin B12 therapy is utilized to scavenge excess nitric oxide, subsequently resulting in the in vivo production of NO-Cbl. PMID:24855323

Antifungal and antibacterial activity and chemical composition of polar and non-polar extracts of Athrixia phylicoides determined using bioautography and HPLC

PubMed Central

2013-01-01

Background Athrixia phylicoides DC. (Asteraceae) is used medicinally in South Africa to treat a plethora of ailments, including heart problems, diabetes, diarrhoea, sores and infected wounds. It is also prepared in the form of a tea (hot decoction) taken as a refreshing, pleasant-tasting beverage with commercialization potential. Methods Extracts of the dried ground aerial parts were prepared using organic solvents (diethyl ether, dichloromethane/methanol, ethyl acetate and ethanol) and water. These extracts were subjected to HPLC, TLC and bioautography analysis with the aim of linking a range of peaks visualized in HPLC chromatography profiles to antibacterial and antifungal activity of the same extracts. Results HPLC revealed a group of compounds extracted by more than one solvent. Compounds identified include inositol, caffeic acid, quercetin, kaempferol, apigenin, hymenoxin and oleanolic acid. The organic extracts displayed similar TLC profiles, and bioautography indicated approximately five antibacterial compounds, but only two antifungal compounds in these extracts. Bioautography indicated that cold water extracted the least antimicrobial compounds. Conclusions Several previously unknown compounds were identified in Athrixia phylicoides extracts, and bioautography indicated a number of antibacterial and antifungal compounds. There were notable differences in chemical composition and bioactivity between the organic and aqueous extracts. Further research is necessary to fully characterize the active components of the extracts. PMID:24330447

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.

Comparison of HPLC & spectrophotometric methods for estimation of antiretroviral drug content in pharmaceutical products.

PubMed

Hemanth Kumar, A K; Sudha, V; Swaminathan, Soumya; Ramachandran, Geetha

2010-10-01

Simple and reliable methods to estimate drugs in pharmaceutical products are needed. In most cases, antiretroviral drug estimations are performed using a HPLC method, requiring expensive equipment and trained technicians. A relatively simple and accurate method to estimate antiretroviral drugs in pharmaceutical preparations is by spectrophotometric method, which is cheap and simple to use as compared to HPLC. We undertook this study to standardise methods for estimation of nevirapine (NVP), lamivudine (3TC) and stavudine (d4T) in single tablets/capsules by HPLC and spectrophotometry and to compare the content of these drugs determined by both these methods. Twenty tablets/capsules of NVP, 3TC and d4T each were analysed for their drug content by HPLC and spectrophotometric methods. Suitably diluted drug solutions were run on HPLC fitted with a C18 column using UV detection at ambient temperature. The absorbance of the diluted drug solutions were read in a spectrophotometer at 300, 285 and 270 nm for NVP, 3TC and d4T respectively. Pure powders of the drugs were used to prepare calibration standards of known drug concentrations, which was set up with each assay. The inter-day variation (%) of standards for NVP, 3TC and d4T ranged from 2.5 to 6.7, 2.1 to 7.7 and 6.2 to 7.7, respectively by HPLC. The corresponding values by spectrophotometric method were 2.7 to 4.7, 4.2 to 7.2 and 3.8 to 6.0. The per cent variation between the HPLC and spectrophotometric methods ranged from 0.45 to 4.49 per cent, 0 to 4.98 per cent and 0.35 to 8.73 per cent for NVP, 3TC and d4T,respectively. The contents of NVP, 3TC and d4T in the tablets estimated by HPLC and spectrophotometric methods were similar, and the variation in the amount of these drugs estimated by HPLC and spectrophotometric methods was below 10 per cent. This suggests that the spectrophotometric method is as accurate as the HPLC method for estimation of NVP, 3TC and d4T in tablet/capsule. Hence laboratories that do not have HPLC equipment can also undertake these drug estimations using spectrophotometer.

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.

Separation methods applicable to urinary creatine and creatinine.

PubMed

Smith-Palmer, Truis

2002-12-05

Urinary creatinine has been analyzed for many years as an indicator of glomerular filtration rate. More recently, interest in studying the uptake of creatine as a result of creatine supplementation, a practice increasingly common among bodybuilders and athletes, has lead to a need to measure urinary creatine concentrations. Creatine levels are of the same order of magnitude as creatinine levels when subjects have recently ingested creatine, while somewhat elevated urinary creatine concentrations in non-supplementing subjects can be an indication of a degenerative disease of the muscle. Urinary creatine and creatinine can be analyzed by HPLC using a variety of columns. Detection methods include absorption, fluorescence after post-column derivatization, and mass spectrometry, and some methods have been automated. Capillary zone electrophoresis and micellar electrokinetic capillary chromatography have also been used to analyze urinary creatine and creatinine. Creatine and creatinine have also been analyzed in serum and tissue using HPLC and CE, and many of these separations could also be applicable to urinary analysis.

Development and Validation of a Stability-Indicating Assay of Etofenamate by RP-HPLC and Characterization of Degradation Products

PubMed Central

Peraman, Ramalingam; Nayakanti, Devanna; Dugga, Hari Hara Theja; Kodikonda, Sudhakara

2013-01-01

A validated stability-indicating RP-HPLC method for etofenamate (ETF) was developed by separating its degradation products on a C18 (250 mm × 4.6 mm 5 μm) Qualisil BDS column using a phosphate buffer (pH-adjusted to 6.0 with orthophosphoric acid) and methanol in the ratio of 20:80 % v/v as the mobile phase at a flow rate of 1.0 mL/min. The column effluents were monitored by a photodiode array detector set at 286 nm. The method was validated in terms of specificity, linearity, accuracy, precision, detection limit, quantification limit, and robustness. Forced degradation of etofenamate was carried out under acidic, basic, thermal, photo, and peroxide conditions and the major degradation products of acidic and basic degradation were isolated and characterized by 1H-NMR, 13C-NMR, and mass spectral studies. The mass balance of the method varied between 92–99%. PMID:24482770

Comparative evaluation of an ISO 3632 method and an HPLC-DAD method for safranal quantity determination in saffron.

PubMed

García-Rodríguez, M Valle; López-Córcoles, Horacio; Alonso, Gonzalo L; Pappas, Christos S; Polissiou, Moschos G; Tarantilis, Petros A

2017-04-15

The aim of this work was a comparison of the ISO 3632 (2011) method and an HPLC-DAD method for safranal quantity determination in saffron. Samples from different origins were analysed by UV-vis according to ISO 3632 (2011) and by HPLC-DAD. Both methods were compared, and there was no correlation between the safranal content obtained by UV-vis and HPLC-DAD. An over-estimation in the UV-vis experiment was observed, which was related to the cis-crocetin esters content, as well as other compounds. The results demonstrated that there was no relationship between ISO quality categories and safranal content using HPLC-DAD. Therefore, HPLC-DAD might be preferable to UV-vis for determining the safranal content and the classification of saffron for commercial purposes. In addition, HPLC-DAD was adequate for determining the three foremost parameters that define the quality of saffron (crocetin esters, picrocrocin and safranal); therefore, this approach could be included in the ISO 3632 method (2011). Copyright © 2016 Elsevier Ltd. All rights reserved.

Fluorescent HPLC for the detection of specific protein oxidation carbonyls - α-aminoadipic and γ-glutamic semialdehydes - in meat systems.

PubMed

Utrera, Mariana; Morcuende, David; Rodríguez-Carpena, Javier-Germán; Estévez, Mario

2011-12-01

Precise methodologies for the routine analysis of particular protein carbonyls are required in order to progress in this topic of increasing interest. The present paper originally describes the application of an improved method for the detection of α-aminoadipic and γ-glutamic semialdehydes in a meat system by using a derivatization procedure with p-amino-benzoic acid (ABA) followed by fluorescent high-performance liquid chromatography (HPLC). The method development comprises i) the description of a simple HPLC program which allows the efficient separation of the ABA and the key standard compounds and ii) the optimization of the procedure for the preparation of a meat sample in order to maximize the fluorescent signal for both protein carbonyls. Furthermore, the suitability of this method is evaluated by applying the technique to porcine burger patties. The present procedure enables an accurate and relatively fast analysis of both semialdehydes in meat samples in which they could play an interesting role as reliable indicators of protein oxidation. Copyright © 2011 Elsevier Ltd. All rights reserved.

The use of capillary electrophoresis as part of a specificity testing strategy for mitoguazone dihydrochloride HPLC methods.

PubMed

Thomson, C E; Gray, M R; Baxter, M P

1997-05-01

Capillary electrophoresis (CE) has been used as part of a validation experiment designed to prove the specificity of high performance liquid chromatography (HPLC) methods used for analysis of mitoguazone dihydrochloride drug substance. Data regarding accuracy, precision and sensitivity of the CE methods are presented as well as a comparison of results obtained from CE, HPLC and thin-layer chromatography (TLC) analysis of samples stressed under a variety of conditions. It was concluded that, not only were the HPLC methods being investigated specific, but that CE could potentially be used to replace HPLC for the routine assay of mitoguazone dihydrochloride.

[Continual improvement of quantitative analytical method development of Panax notogineng saponins based on quality by design].

PubMed

Dai, Sheng-Yun; Xu, Bing; Shi, Xin-Yuan; Xu, Xiang; Sun, Ying-Qiang; Qiao, Yan-Jiang

2017-03-01

This study is aimed to propose a continual improvement strategy based on quality by design (QbD). An ultra high performance liquid chromatography (UPLC) method was developed to accomplish the method transformation from HPLC to UPLC of Panax notogineng saponins (PNS) and achieve the continual improvement of PNS based on QbD, for example. Plackett-Burman screening design and Box-Behnken optimization design were employed to further understand the relationship between the critical method parameters (CMPs) and critical method attributes (CMAs). And then the Bayesian design space was built. The separation degree of the critical peaks (ginsenoside Rg₁ and ginsenoside Re) was over 2.0 and the analysis time was less than 17 min by a method chosen from the design space with 20% of the initial concentration of the acetonitrile, 10 min of the isocratic time and 6%•min⁻¹ of the gradient slope. At last, the optimum method was validated by accuracy profile. Based on the same analytical target profile (ATP), the comparison of HPLC and UPLC including chromatograph method, CMA identification, CMP-CMA model and system suitability test (SST) indicated that the UPLC method could shorten the analysis time, improve the critical separation and satisfy the requirement of the SST. In all, HPLC method could be replaced by UPLC for the quantity analysis of PNS. Copyright© by the Chinese Pharmaceutical Association.

Stability of extemporaneously prepared preservative-free prochlorperazine nasal spray.

PubMed

Yellepeddi, Venkata K

2018-01-01

The stability of an extemporaneously prepared preservative-free prochlorperazine 5-mg/mL nasal spray was evaluated. The preservative-free prochlorperazine nasal spray was prepared by adding 250 mg of prochlorperazine edisylate to 50 mL of citrate buffer in a low-density polyethylene nasal spray bottle. A stability-indicating high-performance liquid chromatography (HPLC) method was developed and validated using the major degradant prochlorperazine sulfoxide and by performing forced-degradation studies. For chemical stability studies, 3 100-μL samples of the preservative-free prochlorperazine from 5 nasal spray bottles stored at room temperature were collected at days 0, 20, 30, 45, and 60 and were assayed in triplicate using the stability-indicating HPLC method. Microbiological testing involved antimicrobial effectiveness testing based on United States Pharmacopeia ( USP ) chapter 51 and quantitative microbiological enumeration of aerobic bacteria, yeasts, and mold based on USP chapter 61. Samples for microbiological testing were collected at days 0, 30, and 60. The stability-indicating HPLC method clearly identified the degradation product prochlorperazine sulfoxide without interference from prochlorperazine. All tested solutions retained over 90% of the initial prochlorperazine concentration for the 60-day study period. There were no detectable changes in color, pH, and viscosity in any sample. There was no growth of bacteria, yeast, and mold for 60 days in all samples tested. An extemporaneously prepared preservative-free nasal spray solution of prochlorperazine edisylate 5 mg/mL was physically, chemically, and microbiologically stable for 60 days when stored at room temperature in low-density polyethylene bottles. Copyright © 2018 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Multiple fingerprinting analyses in quality control of Cassiae Semen polysaccharides.

PubMed

Cheng, Jing; He, Siyu; Wan, Qiang; Jing, Pu

2018-03-01

Quality control issue overshadows potential health benefits of Cassiae Semen due to the analytic limitations. In this study, multiple-fingerprint analysis integrated with several chemometrics was performed to assess the polysaccharide quality of Cassiae Semen harvested from different locations. FT-IR, HPLC, and GC fingerprints of polysaccharide extracts from the authentic source were established as standard profiles, applying to assess the quality of foreign sources. Analyses of FT-IR fingerprints of polysaccharide extracts using either Pearson correlation analysis or principal component analysis (PCA), or HPLC fingerprints of partially hydrolyzed polysaccharides with PCA, distinguished the foreign sources from the authentic source. However, HPLC or GC fingerprints of completely hydrolyzed polysaccharides couldn't identify all foreign sources and the methodology using GC is quite limited in determining the monosaccharide composition. This indicates that FT-IR/HPLC fingerprints of non/partially-hydrolyzed polysaccharides, respectively, accompanied by multiple chemometrics methods, might be potentially applied in detecting and differentiating sources of Cassiae Semen. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of solid-phase microextraction in the investigation of protein binding of pharmaceuticals.

PubMed

Theodoridis, Georgios

2006-01-18

Protein-drug interactions of seven common pharmaceuticals were studied using solid-phase microextraction (SPME). SPME can be used in such investigations on the condition that no analyte depletion occurs. In multi-compartment systems (e.g. a proteinaceous matrix) only the free portion of the analyte is able to partition into the SPME fiber. In addition if no sample depletion occurs, the bound drug-free drug equilibria are not disturbed. In the present study seven pharmaceuticals (quinine, quinidine, naproxen, ciprofloxacin, haloperidol, paclitaxel and nortriptyline) were assayed by SPME. For quantitative purposes SPME was validated first in the absence of proteins. Calibration curves were constructed for each drug by HPLC-fluorescence and HPLC-UV analysis. SPME was combined to HPLC off-line, desorption occurring in HPLC inserts filled with 200 microL methanol. Binding of each drug to human serum albumin was studied independently. Experimental results were in agreement with literature data and ultrafiltration experiments, indicating the feasibility of the method for such bioanalytical purposes.

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.

High-pressure liquid chromatography analysis of antibiotic susceptibility disks.

PubMed Central

Hagel, R B; Waysek, E H; Cort, W M

1979-01-01

The analysis of antibiotic susceptibility disks by high-pressure liquid chromatography (HPLC) was investigated. Methods are presented for the potency determination of mecillinam, ampicillin, carbenicillin, and cephalothin alone and in various combinations. Good agreement between HPLC and microbiological data is observed for potency determinations with recoveries of greater than 95%. Relative standard deviations of lower than 2% are recorded for each HPLC method. HPLC methods offer improved accuracy and greater precision when compared to the standard microbiological methods of analysis for susceptibility disks. PMID:507793

Profiling of alpha-dicarbonyl content of commercial honeys from different botanical origins: identification of 3,4-dideoxyglucoson-3-ene (3,4-DGE) and related compounds.

PubMed

Marceau, Eric; Yaylayan, Varoujan A

2009-11-25

The alpha-dicarbonyl contents of commercial honey samples from different botanical origins were analyzed as their quinoxaline derivatives using HPLC-DAD, HPLC-MS, HPLC-MS/MS, and HPLC-TOF-MS. A total of nine such compounds were detected, of which five were previously reported in honey (glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal, and 2,3-butanedione) and three were reported only from sources other than honey [3-deoxypentulose, 1,4-dideoxyhexulose, and 3,4-dideoxyglucoson-3-ene (3,4-DGE)]. An unknown alpha-dicarbonyl compound was also tentatively identified as an oxidation product of 3,4-DGE and was termed 3,4-dideoxyglucosone-3,5-diene (3,4-DGD). Only glyoxal (0.3-1.3 mg/kg), methylglyoxal (0.8-33 mg/kg), and 2,3-butanedione (0-4.3 mg/kg) were quantified in all honey samples. Furthermore, analysis of the alpha-dicarbonyl profile of various honey samples indicated that certain alpha-dicarbonyl compounds are found in specific honey samples in much higher proportions relative to the average amounts. The free radical scavenging activity as measured by DPPH method has also indicated that the darker honey samples such as buckwheat, manuka, blueberry, and eucalyptus had higher antioxidant properties compared to lighter-colored samples.

A Stability Indicating HPLC Method for the Determination of Fluvoxamine in Pharmaceutical Dosage Forms.

PubMed

Souri, Effat; Donyayi, Hassan; Khaniha, Reza Ahmad; Barazandeh Tehrani, Maliheh

2015-01-01

Fluvoxamine maleate is a selective serotonin reuptake inhibitor, which is used for the treatment of different types of depressive disorders. In the present study, a stability indicating HPLC method was developed and validated for the determination of fluvoxamine maleate. The chromatographic separation was carried out using a Nova-Pak CN column and a mixture of K2HPO4 50 mM (pH 7.0) and acetonitrile (60: 40, v/v) as the mobile phase. Target compounds were detected using a UV detector set at 235 nm. The developed method was linear over the concentration range of 1-80 μg/ml with acceptable precision (CV values < 2.0%) and accuracy (error values < 1.6%). The degradation studies showed that fluvoxamine maleate is relatively unstable under acidic, basic and oxidative conditions and also when exposed to UV radiation. On the other hand, the bulk powder of fluvoxamine maleate was relatively stable when exposed to visible light or heat. The proposed method was successfully applied for the determination of active ingredient of fluvoxamine dosage form without any interference from tablet excipients.

Purification and characterization of selenium-containing phycocyanin from selenium-enriched Spirulina platensis.

PubMed

Chen, Tianfeng; Wong, Yum-Shing; Zheng, Wenjie

2006-11-01

A fast protein liquid chromatographic method for purification of selenium-containing phycocyanin (Se-PC) from selenium-enriched Spirulina platensis was described in this study. The purification procedures involved fractionation by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange chromatography and Sephacry S-300 size exclusion chromatography. The purity ratio (A620/A280) and the separation factor (A620/A655) of the purified Se-PC were 5.12 and 7.92, respectively. The Se concentration of purified Se-PC was 496.5 microg g(-1) protein, as determined by ICP-AES analysis. The purity of the Se-PC was further characterized by UV-VIS and fluorescence spectrometry, SDS-PAGE, RP-HPLC and gel filtration HPLC. The apparent molecular mass of the native Se-PC determined by gel filtration HPLC was 109 kDa, indicating that the protein existed as a trimer. SDS-PAGE of the purified Se-PC yielded two major bands corresponding to the alpha and beta subunits. A better separation of these two subunits was obtained by RP-HPLC. Identification of the alpha and beta subunits separated by SDS-PAGE and RP-HPLC was achieved by peptide mass fingerprinting (PMF) using MALDI-TOF-TOF mass spectrometry.

Methods of analysis by the U.S. Geological Survey Organic Geochemistry Research Group; determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

USGS Publications Warehouse

Zimmerman, L.R.; Hostetler, K.A.; Thurman, E.M.

2000-01-01

Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: acetochlor ethanesulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The mean HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.50, and 2.0 mg/L (micrograms per liter) ranged from 84 to 112 percent, with relative standard deviations of 18 percent or less. The mean HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.20, and 2.0 mg/L ranged from 81 to 125 percent, with relative standard deviations of 20 percent or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 mg/L, whereas the LOQ using the HPLC/MS method was 0.05 mg/L. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water.

A novel approach for quantitation of nonderivatized sialic acid in protein therapeutics using hydrophilic interaction chromatographic separation and nano quantity analyte detection.

PubMed

Chemmalil, Letha; Suravajjala, Sreekanth; See, Kate; Jordan, Eric; Furtado, Marsha; Sun, Chong; Hosselet, Stephen

2015-01-01

This paper describes a novel approach for the quantitation of nonderivatized sialic acid in glycoproteins, separated by hydrophilic interaction chromatography, and detection by Nano Quantity Analyte Detector (NQAD). The detection technique of NQAD is based on measuring change in the size of dry aerosol and converting the particle count rate into chromatographic output signal. NQAD detector is suitable for the detection of sialic acid, which lacks sufficiently active chromophore or fluorophore. The water condensation particle counting technology allows the analyte to be enlarged using water vapor to provide highest sensitivity. Derivatization-free analysis of glycoproteins using HPLC/NQAD method with PolyGLYCOPLEX™ amide column is well correlated with HPLC method with precolumn derivatization using 1, 2-diamino-4, 5-methylenedioxybenzene (DMB) as well as the Dionex-based high-pH anion-exchange chromatography (or ion chromatography) with pulsed amperometric detection (HPAEC-PAD). With the elimination of derivatization step, HPLC/NQAD method is more efficient than HPLC/DMB method. HPLC/NQAD method is more reproducible than HPAEC-PAD method as HPAEC-PAD method suffers high variability because of electrode fouling during analysis. Overall, HPLC/NQAD method offers broad linear dynamic range as well as excellent precision, accuracy, repeatability, reliability, and ease of use, with acceptable comparability to the commonly used HPAEC-PAD and HPLC/DMB methods. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Determination of GABA and vigabatrin in human plasma by a rapid and simple HPLC method: correlation between clinical response to vigabatrin and increase in plasma GABA.

PubMed

Löscher, W; Fassbender, C P; Gram, L; Gramer, M; Hörstermann, D; Zahner, B; Stefan, H

1993-03-01

The novel antiepileptic drug vigabatrin (Sabril) acts by inhibiting degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), increasing the GABA concentrations in the brain. Because the GABA degrading enzyme GABA aminotransferase (GABA-T) is also present in peripheral tissues, including blood platelets, measurement of plasma GABA levels might be a useful indication of the pharmacological response to vigabatrin during therapeutic monitoring. However, because of the very low concentrations of GABA in plasma, the few methods available for plasma GABA analysis are time-consuming, difficult to perform and/or not selective enough because of potential interference with other plasma constituents. In the present study, a rapid, selective and sensitive amino acid analysis HPLC method has been developed for plasma GABA determination with fluorescence detection, using o-phthaldialdehyde as a precolumn derivatizing agent. By employing a 3 microns particle size reversed-phase column and a multi-step gradient system of two solvents, the very low endogenous concentration of GABA in human plasma could be reproducibly quantitated without interference of other endogenous compounds. Incubation of human plasma samples with GABA degrading enzyme(s) resulted in an almost total loss of the GABA peak, thus demonstrating the specificity of the method for GABA analysis. In addition to GABA and other endogenous amino acids, the HPLC method could be used to quantitate plasma levels of vigabatrin. Thus, this improved HPLC amino acid assay might be used to examine whether concomitant monitoring of plasma GABA and vigabatrin is useful for clinical purposes. This was examined in 20 epileptic patients undergoing chronic treatment with vigabatrin. The average plasma GABA level of these 20 patients did not differ significantly from non-epileptic controls. However, when epileptic patients were subdivided according to their clinical response to vigabatrin, vigabatrin responders had significantly higher GABA levels than nonresponders or controls. In contrast to the difference in plasma GABA, vigabatrin responders and nonresponders did not differ in dose or plasma level of vigabatrin. These data may indicate that determination of plasma GABA is a valuable non-invasive method for therapeutic monitoring in patients on medication with vigabatrin.

Determination of 3-hydroxypropylmercapturic acid in urine by three column-switching high-performance liquid chromatography with electrochemical detection using a diamond electrode.

PubMed

Higashi, Kyohei; Shibasaki, Mana; Kuni, Kyoshiro; Uemura, Takeshi; Waragai, Masaaki; Uemura, Kenichi; Igarashi, Kazuei; Toida, Toshihiko

2017-09-29

A three column-switching high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was established to determine 3-hydroxypropylmercapturic acid (3-HPMA) in urine. An extracted urine sample was consecutively fractionated using a strong anion-exchange column (first column) and a C8 column (second column) via a switching valve before application on an Octa Decyl Silyl (ODS) column (third column), followed by ECD analysis. The% recovery of 3-HPMA standard throughout the three-column process and limit of detection (LOD) were 94±1% and 0.1pmol, respectively. A solid phase extraction step is required for the sensitive analysis of 3-HPMA in urine by column-switching HPLC-ECD despite a decreased% recovery (55%) of urine sample spiked with 100pmol of 3-HPMA. To test the utility of our column-switching HPLC-ECD method, 3-HPMA levels of 27 urine samples were determined, and the correlation between HPLC-ECD and LC-Electrospray ionization (ESI)-MS/MS method was examined. As a result, the median values of μmol 3-HPMA/g Creatinine (Cre) in urine obtained by column-switching HPLC-ECD and LC-MS/MS were 2.19±2.12μmol/g Cre and 2.13±3.38μmol/g Cre, respectively, and the calibration curve (y=1.5171x-1.007) exhibited good linearity within a defined range (r 2 =0.907). These results indicate that the combination of column-switching HPLC and ECD is a powerful tool for the specific, reliable detection of 3-HPMA in urine. Copyright © 2017 Elsevier B.V. All rights reserved.

Spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of Curcuma aromatica.

PubMed

Liu, Meiqiong; Wu, Youjiao; Huang, Shushi; Liu, Huagang; Feng, Jie

2018-02-23

Curcuma aromatica is used as a traditional Chinese medicine, and it is mainly distributed in Guangxi, China. In this study, 10 batches of C. aromatica were collected from different origins in Guangxi. The fingerprints were established by HPLC technique to investigate the quality stability of C. aromatica. The spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of C. aromatica was assessed by similarity analysis, gray relational analysis and multiple linear regression analysis. From the results, the similarity values between each batch of C. aromatica and reference fingerprint were >0.880, indicating the good quality stability of the 10 batches of C. aromatica. Twenty common peaks were selected as the fingerprints to evaluate the quality and hypolipidemic effect of C. aromatica. The results of spectrum-effect relationship showed that peaks 10, 18, 13, 15 and 17 in the fingerprints were closely related to hypolipidemic effect. This study successfully established the spectrum-effect relationship between HPLC fingerprints and hypolipidemic effect of C. aromatica, which provided methods for quality control and more effectively studies on bioactive compounds of C. aromatica. It could also provide a new simple and effective method for utilizing the fingerprints to optimize the Chinese prescription and develop traditional Chinese medicine. Copyright © 2018 John Wiley & Sons, Ltd.

High pressure liquid chromatographic method for the separation and quantitation of water-soluble radiolabeled benzene metabolites

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

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

1988-05-01

The glucuronide and sulfate conjugates of benzene metabolite as well as muconic acid and pre-phenyl- and phenylmercapturic acids were separated by ion-pairing HPLC. The HPLC method developed was suitable for automated analysis of a large number of tissue or excreta samples. p-Nitrophenyl (/sup 14/C)glucuronide was used as an internal standard for quantitation of these water-soluble metabolites. Quantitation was verified by spiking liver tissue with various amounts of phenylsulfate or glucuronides of phenol, catechol, or hydroquinone and analyzing by HPLC. Values determined by HPLC analysis were within 10% of the actual amount with which the liver was spiked. The amount ofmore » metabolite present in urine following exposure to (/sup 3/H)benzene was determined using p-nitrophenyl (/sup 14/C)glucuronide as an internal standard. Phenylsulfate was the major water-soluble metabolite in the urine of F344 rats exposed to 50 ppm (/sup 3/H)benzene for 6 h. Muconic acid and an unknown metabolite which decomposed in acidic media to phenylmercapturic acid were also present. Liver, however, contained a different metabolic profile. This indicates that urinary metabolite profiles may not be a true reflection of what is seen in individual tissues.« less

HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts.

PubMed

Pellati, Federica; Orlandini, Giulia; Pinetti, Diego; Benvenuti, Stefania

2011-07-15

In this study, the composition of polyphenols (phenolic acids and flavonoids) in propolis extracts was investigated by HPLC-DAD and HPLC-ESI-MS/MS by comparing the performance of ion trap and triple quadrupole mass analyzers. The analyses were carried out on an Ascentis C(18) column (250mm×4.6mm I.D., 5μm), with a mobile phase composed by 0.1% formic acid in water and acetonitrile. Overall, the UV spectra, the MS and MS/MS data allowed the identification of 40 compounds. In the case of flavonoids, the triple quadrupole mass analyzer provided more collision energy if compared with the ion trap, originating product ions at best sensitivity. The HPLC method was validated in agreement with ICH guidelines: the correlation coefficients were >0.998; the limit of detection was in the range 1.6-4.6μg/ml; the recovery range was 96-105%; the intra- and inter-day %RSD values for retention times and peak areas were found to be <0.3 and 1.9%, respectively. The developed technique was applied to the analysis of hydroalcoholic extracts of propolis available on the Italian market. Although the chromatographic profile of the analyzed samples was similar, the quantitative analysis indicated that there is a great variability in the amount of the active compounds: the content of total phenolic acids ranged from 0.17 to 16.67mg/ml and the level of total flavonoids from 2.48 to 41.10mg/ml. The proposed method can be considered suitable for the phytochemical analysis of propolis extracts used in phytotherapy. Copyright © 2011 Elsevier B.V. All rights reserved.

Comparison of UPLC and HPLC methods for determination of vitamin C.

PubMed

Klimczak, Inga; Gliszczyńska-Świgło, Anna

2015-05-15

Ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) methods for determination of ascorbic acid (AA) and total AA (TAA) contents (as the sum of AA and dehydroascorbic acid (DHAA) after its reduction to AA) in fruit beverages and in pharmaceutical preparations were compared. Both methods are rapid: total time of analysis was 15 and 6 min for HPLC and UPLC methods, respectively. The methods were validated in terms of linearity, instrument precision, limits of detection (LOD) and quantification (LOQ), accuracy and recovery. Intra- and inter-day instrument precisions for fruit juices, expressed as RSD, were 2.2% and 2.4% for HPLC, respectively, and 1.7% and 1.9% for UPLC, respectively. For vitamin C tablets, inter- and intra-day precisions were 0.4% and 0.5%, respectively (HPLC), and 0.5% and 0.3%, respectively (UPLC). Both methods were sensitive: LOD was 0.049 μg/mL for HPLC and 0.024 μg/mL for UPLC while LOQs were 0.149 and 0.073 μg/mL for HPLC and UPLC, respectively. These methods could be useful in the routine qualitative and quantitative analysis of AA or TAA in pharmaceutical preparations or fruit beverages. However, UPLC method is more sensitive, faster and consumes less eluent. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of primary standards for use in the HPLC analysis of the procyanidin content of cocoa and chocolate containing products.

PubMed

Hurst, William J; Stanley, Bruce; Glinski, Jan A; Davey, Matthew; Payne, Mark J; Stuart, David A

2009-10-15

This report describes the characterization of a series of commercially available procyanidin standards ranging from dimers DP = 2 to decamers DP = 10 for the determination of procyanidins from cocoa and chocolate. Using a combination of HPLC with fluorescence detection and MALDI-TOF mass spectrometry, the purity of each standard was determined and these data were used to determine relative response factors. These response factors were compared with other response factors obtained from published methods. Data comparing the procyanidin analysis of a commercially available US dark chocolate calculated using each of the calibration methods indicates divergent results and demonstrate that previous methods may significantly underreport the procyanidins in cocoa-containing products. These results have far reaching implications because the previous calibration methods have been used to develop data for a variety of scientific reports, including food databases and clinical studies.

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

Speciation of Bio-Available Iodine in Abalone (Haliotis discus hannai) by High-Performance Liquid Chromatography Hyphenated with Inductively Coupled Plasma-Mass Spectrometry Using an In Vitro Method.

PubMed

Doh, Han Sol; Park, Hyun Jin

2018-06-01

Abalone is one of the most valuable marine products found in East Asia because it is rich in nutritious substances including iodine. In this study, the in vitro dialyzability approach was used to assess the bio-available iodine species in abalone. Iodide, iodate, 3-iodo-L-tyrosine (MIT), and 3,5-diiodo-L-tyrosine (DIT) were separated by high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). To assure the consistency, reliability, and accuracy of the data, the method was validated. Comparison of the total iodine in abalone muscle and viscera indicated that abalone muscle showed greater digestion/absorption efficiency than abalone viscera (digestion efficiency: 68.13 ± 2.59% and 47.88 ± 5.76% and absorption efficiency: 59.78 ± 2.93% and 35.12 ± 1.43% for abalone viscera and muscle, respectively). However, evaluation of the sum of the analyzed iodine species targeted in this study by HPLC-ICP-MS indicated that abalone muscle showed lower digestion efficiency and similar absorption efficiency compared to that of abalone viscera (digestion efficiency: 35.52 ± 5.41% and 28.84 ± 1.83%; absorption efficiency: 23.56 ± 4.38% and 27.56 ± 1.51% for abalone viscera and muscle, respectively). The main forms of iodine detected in abalone muscle were iodide and MIT, whereas iodide was the major form in abalone viscera. The bio-available iodine in abalone was quantified via an in vitro method employing HPLC-ICP-MS. The results of this study indicated that abalone is feasible as a new iodine source and may prospectively find application in iodine-fortified foods. © 2018 Institute of Food Technologists®.

Determination of chloroacetanilide herbicide metabolites in water using high-performance liquid chromatography-diode array detection and high-performance liquid chromatography/mass spectrometry

USGS Publications Warehouse

Hostetler, K.A.; Thurman, E.M.

2000-01-01

Analytical methods using high-performance liquid chromatography-diode array detection (HPLC-DAD) and high-performance liquid chromatography/mass spectrometry (HPLC/MS) were developed for the analysis of the following chloroacetanilide herbicide metabolites in water: alachlor ethanesulfonic acid (ESA); alachlor oxanilic acid; acetochlor ESA; acetochlor oxanilic acid; metolachlor ESA; and metolachlor oxanilic acid. Good precision and accuracy were demonstrated for both the HPLC-DAD and HPLC/MS methods in reagent water, surface water, and ground water. The average HPLC-DAD recoveries of the chloroacetanilide herbicide metabolites from water samples spiked at 0.25, 0.5 and 2.0 ??g/l ranged from 84 to 112%, with relative standard deviations of 18% or less. The average HPLC/MS recoveries of the metabolites from water samples spiked at 0.05, 0.2 and 2.0 ??g/l ranged from 81 to 118%, with relative standard deviations of 20% or less. The limit of quantitation (LOQ) for all metabolites using the HPLC-DAD method was 0.20 ??g/l, whereas the LOQ using the HPLC/MS method was at 0.05 ??g/l. These metabolite-determination methods are valuable for acquiring information about water quality and the fate and transport of the parent chloroacetanilide herbicides in water. Copyright (C) 2000 Elsevier Science B.V.

Development and validation of a single robust HPLC method for the characterization of a pharmaceutical starting material and impurities from three suppliers using three separate synthetic routes.

PubMed

Sheldon, E M; Downar, J B

2000-08-15

Novel approaches to the development of analytical procedures for monitoring incoming starting material in support of chemical/pharmaceutical processes are described. High technology solutions were utilized for timely process development and preparation of high quality clinical supplies. A single robust HPLC method was developed and characterized for the analysis of the key starting material from three suppliers. Each supplier used a different process for the preparation of this material and, therefore, each suppliers' material exhibited a unique impurity profile. The HPLC method utilized standard techniques acceptable for release testing in a QC/manufacturing environment. An automated experimental design protocol was used to characterize the robustness of the HPLC method. The method was evaluated for linearity, limit of quantitation, solution stability, and precision of replicate injections. An LC-MS method that emulated the release HPLC method was developed and the identities of impurities were mapped between the two methods.

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.

Evaluation of a novel semi-automated HPLC procedure for whole blood cyclosporin A confirms equivalence to adjusted monoclonal values from Abbott TDx.

PubMed

Roberts, Norman B; Dutton, John; Higgins, Gerald; Allars, Lesley

2005-01-01

The problem in the measurement of cyclosporin (CyA) is that the widely used immuno-based assays suffer from interference by metabolites present in unpredictable excess. To resolve this, the consensus view has been to develop more specific and robust procedures for the measurement of CyA alone in order to give values similar to those obtained by HPLC. We developed an alternative strategy based on Abbott poly- and monoclonal assays to derive an adjusted monoclonal value as an equivalent measurement to HPLC. We have now evaluated a recently developed semi-automated HPLC procedure and used it to test the validity of the adjusted monoclonal value. The automated HPLC procedure with online clean-up was optimised for the separation of CyA and internal standard CyD. The assay was simple to use, precise and gave good recovery of cyclosporin from whole blood. Comparisons with the more specific immunoassays Abbott AxSym and EMIT showed close agreement, whereas Abbott monoclonal values indicated up to 20% positive bias. In contrast, the adjusted monoclonal values gave good agreement with HPLC. Data obtained from HPLC linked to tandem mass spectrometry (MS) indicated closer agreement with Abbott monoclonal values than expected, suggesting some positive bias with MS. The benefit of using an adjusted monoclonal value is that a result equivalent to HPLC is obtained, as well as an indication of the concentration of metabolites from the Abbott polyclonal measurement.

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.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant.

PubMed

Gaber, Yasser; Akerman, Cecilia Orellana; Hatti-Kaul, Rajni

2014-01-01

N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation.

Environmentally evaluated HPLC-ELSD method to monitor enzymatic synthesis of a non-ionic surfactant

PubMed Central

2014-01-01

Background N-Lauroyl-N-methylglucamide is a biodegradable surfactant derived from renewable resources. In an earlier study, we presented an enzymatic solvent-free method for synthesis of this compound. In the present report, the HPLC method developed to follow the reaction between lauric acid/methyl laurate and N-methyl glucamine (MEG) and its environmental assessment are described. Results Use of ultraviolet (UV) absorption or refractive index (RI) detectors did not allow the detection of N-methyl glucamine (MEG). With Evaporative light scattering detector ELSD, it was possible to apply a gradient elution, and detect MEG with a limit of detection, LOD = 0.12 μg. A good separation of the peaks: MEG, lauric acid, product (amide) and by-product (amide-ester) was achieved with the gradient program with a run time of 40 min. The setting of ELSD detector was optimized using methyl laurate as the analyte. LC-MS/MS was used to confirm the amide and amide-ester peaks. We evaluated the greenness of the developed method using the freely available software HPLC-Environmental Assessment Tool (HPLC-EAT) and the method got a scoring of 73 HPLC-EAT units, implying that the analytical procedure was more environmentally benign compared to some other methods reported in literature whose HPLC-EAT values scored up to 182. Conclusion Use of ELSD detector allowed the detection and quantification of the substrates and the reaction products of enzymatic synthesis of the surfactant, N-lauroyl-N-methylglucamide. The developed HPLC method has acceptable environmental profile based on HPLC-EAT evaluation. PMID:24914404

Simultaneous determination of chromones and coumarins in Radix Saposhnikoviae by high performance liquid chromatography with diode array and tandem mass detectors.

PubMed

Kim, Min Kyung; Yang, Dong-Hyug; Jung, Mihye; Jung, Eun Ha; Eom, Han Young; Suh, Joon Hyuk; Min, Jung Won; Kim, Unyong; Min, Hyeyoung; Kim, Jinwoong; Han, Sang Beom

2011-09-16

Methods using high performance liquid chromatography with diode array detection (HPLC-DAD) and tandem mass spectrometry (HPLC-MS/MS) were developed and validated for the simultaneous determination of 5 chromones and 6 coumarins: prim-O-glucosylcimifugin (1), cimifugin (2), nodakenin (3), 4'-O-β-d-glucosyl-5-O-methylvisamminol (4), sec-O-glucosylhamaudol (5), psoralen (6), bergapten (7), imperatorin (8), phellopterin (9), 3'-O-angeloylhamaudol (10) and anomalin (11), in Radix Saposhnikoviae. The separation conditions for HPLC-DAD were optimized using an Ascentis Express C18 (4.6 mm×100 mm, 2.7 μm particle size) fused-core column. The mobile phase was composed of 10% aqueous acetonitrile (A) and 90% acetonitrile (B) and the elution was performed under a gradient mode at a flow rate of 1.0 mL/min. The detection wavelength was set at 300 nm. The HPLC-DAD method yielded a base line separation of the 11 components in 50% methanol extract of Radix Saposhnikoviae with no interfering peaks detected. The HPLC-DAD method was validated in terms of linearity, accuracy and precision (intra- and inter-day), limit of quantification (LOQ), recovery, and robustness. Specific determination of the 11 components was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) source. This HPLC-MS/MS method was also validated by determining the linearity, limit of quantification, accuracy, and precision. Quantification of the 11 components in 51 commercial Radix Saposhnikoviae samples was successfully performed using the developed HPLC-DAD method. The identity, batch-to-batch consistency, and authenticity of Radix Saposhnikoviae were successfully monitored by the proposed HPLC-DAD and HPLC-MS/MS methods. Copyright © 2011 Elsevier B.V. All rights reserved.

76 FR 55329 - Receipt of Several Pesticide Petitions Filed for Residues of Pesticide Chemicals in or on Various...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-07

... (HPLC) method was validated for determination of dinotefuran, DN and UF in or on tomatoes and peppers... quantified after HPLC separation by tandem mass spectrometry (MS/MS) detection. Contact: Sidney Jackson, (703..., group 9 at 0.03 ppm. Adequate analytical methods (HPLC-fluorescence methods) are available for...

HPLC determination of flavonoid glycosides in Mongolian Dianthus versicolor Fisch. (Caryophyllaceae) compared with quantification by UV spectrophotometry.

PubMed

Obmann, Astrid; Purevsuren, Sodnomtseren; Zehl, Martin; Kletter, Christa; Reznicek, Gottfried; Narantuya, Samdan; Glasl, Sabine

2012-01-01

Dianthus versicolor is used in traditional Mongolian medicine against liver impairment. Fractions enriched in flavone-di- and triglycosides were shown to enhance bile secretion. Therefore, reliable and accurate analytical methods are needed for the determination of these flavonoids in the crude drug and extracts thereof. To provide a validated HPLC-DAD (diode array detector) method especially developed for the separation of polar flavonoids and to compare the data obtained with those evaluated by UV spectrophotometry. Separations were carried out on an Aquasil® C₁₈-column (4.6 mm × 250.0 mm, 5 µm) with a linear gradient of acetonitrile and water (adjusted to pH 2.8 with trifluoroacetic acid) as mobile phase. Rutoside was employed as internal standard with linear behavior in a concentration range of 0.007-3.5 mg/mL. Accuracy was determined by spiking the crude drug with saponarin resulting in recoveries between 92% and 102%. The method allows the quantification of highly polar flavonoid glycosides and the determination of their total content. For saponarin a linear response was evaluated within the range 0.007-3.5 mg/mL (R²  > 0.9999). It was proven that threefold sonication represents a time-saving, effective and cheap method for the extraction of the polar flavonoid glycosides. The contents determined by HPLC were shown to be in agreement with those obtained employing UV spectrophotometry. The study has indicated that the newly developed HPLC method represents a powerful technique for the quality control of D. versicolor. Ultraviolet spectrophotometry may be used alternatively provided that the less polar flavonoids are removed by purification. Copyright © 2011 John Wiley & Sons, Ltd.

Development and optimization of SPE-HPLC-UV/ELSD for simultaneous determination of nine bioactive components in Shenqi Fuzheng Injection based on Quality by Design principles.

PubMed

Wang, Lu; Qu, Haibin

2016-03-01

A method combining solid phase extraction, high performance liquid chromatography, and ultraviolet/evaporative light scattering detection (SPE-HPLC-UV/ELSD) was developed according to Quality by Design (QbD) principles and used to assay nine bioactive compounds within a botanical drug, Shenqi Fuzheng Injection. Risk assessment and a Plackett-Burman design were utilized to evaluate the impact of 11 factors on the resolutions and signal-to-noise of chromatographic peaks. Multiple regression and Pareto ranking analysis indicated that the sorbent mass, sample volume, flow rate, column temperature, evaporator temperature, and gas flow rate were statistically significant (p < 0.05) in this procedure. Furthermore, a Box-Behnken design combined with response surface analysis was employed to study the relationships between the quality of SPE-HPLC-UV/ELSD analysis and four significant factors, i.e., flow rate, column temperature, evaporator temperature, and gas flow rate. An analytical design space of SPE-HPLC-UV/ELSD was then constructed by calculated Monte Carlo probability. In the presented approach, the operating parameters of sample preparation, chromatographic separation, and compound detection were investigated simultaneously. Eight terms of method validation, i.e., system-suitability tests, method robustness/ruggedness, sensitivity, precision, repeatability, linearity, accuracy, and stability, were accomplished at a selected working point. These results revealed that the QbD principles were suitable in the development of analytical procedures for samples in complex matrices. Meanwhile, the analytical quality and method robustness were validated by the analytical design space. The presented strategy provides a tutorial on the development of a robust QbD-compliant quantitative method for samples in complex matrices.

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

A simple high performance liquid chromatography method for analyzing paraquat in soil solution samples.

PubMed

Ouyang, Ying; Mansell, Robert S; Nkedi-Kizza, Peter

2004-01-01

A high performance liquid chromatography (HPLC) method with UV detection was developed to analyze paraquat (1,1'-dimethyl-4,4'-dipyridinium dichloride) herbicide content in soil solution samples. The analytical method was compared with the liquid scintillation counting (LSC) method using 14C-paraquat. Agreement obtained between the two methods was reasonable. However, the detection limit for paraquat analysis was 0.5 mg L(-1) by the HPLC method and 0.05 mg L(-1) by the LSC method. The LSC method was, therefore, 10 times more precise than the HPLC method for solution concentrations less than 1 mg L(-1). In spite of the high detection limit, the UC (nonradioactive) HPLC method provides an inexpensive and environmentally safe means for determining paraquat concentration in soil solution compared with the 14C-LSC method.

A method for the preparation of curcumin by ultrasonic-assisted ammonium sulfate/ethanol aqueous two phase extraction.

PubMed

Xu, Guangkuan; Hao, Changchun; Tian, Suyang; Gao, Feng; Sun, Wenyuan; Sun, Runguang

2017-01-15

This study investigated a new and easy-to-industrialized extracting method for curcumin from Curcuma longa rhizomes using ultrasonic extraction technology combined with ammonium sulfate/ethanol aqueous two-phase system (ATPS), and the preparation of curcumin using the semi-preparative HPLC. The single-factor experiments and response surface methodology (RSM) were utilized to determine the optimal material-solvent ratio, ultrasonic intensity (UI) and ultrasonic time. The optimum extraction conditions were finally determined to be material-solvent rate of 3.29:100, ultrasonic intensity of 33.63W/cm 2 and ultrasonic time of 17min. At these optimum conditions, the extraction yield could reach 46.91mg/g. And the extraction yields of curcumin remained stable in the case of amplification, which indicated that scale-up extraction was feasible and efficient. Afterwards, the semi-preparative HPLC experiment was carried out, in which optimal preparation conditions were elected according to the single factor experiment. The prepared curcumin was obtained and the purity could up to 85.58% by the semi-preparative HPLC. Copyright © 2016 Elsevier B.V. All rights reserved.

Simultaneous determination of potassium guaiacolsulfonate, guaifenesin, diphenhydramine HCl and carbetapentane citrate in syrups by using HPLC-DAD coupled with partial least squares multivariate calibration.

PubMed

Dönmez, Ozlem Aksu; Aşçi, Bürge; Bozdoğan, Abdürrezzak; Sungur, Sidika

2011-02-15

A simple and rapid analytical procedure was proposed for the determination of chromatographic peaks by means of partial least squares multivariate calibration (PLS) of high-performance liquid chromatography with diode array detection (HPLC-DAD). The method is exemplified with analysis of quaternary mixtures of potassium guaiacolsulfonate (PG), guaifenesin (GU), diphenhydramine HCI (DP) and carbetapentane citrate (CP) in syrup preparations. In this method, the area does not need to be directly measured and predictions are more accurate. Though the chromatographic and spectral peaks of the analytes were heavily overlapped and interferents coeluted with the compounds studied, good recoveries of analytes could be obtained with HPLC-DAD coupled with PLS calibration. This method was tested by analyzing the synthetic mixture of PG, GU, DP and CP. As a comparison method, a classsical HPLC method was used. The proposed methods were applied to syrups samples containing four drugs and the obtained results were statistically compared with each other. Finally, the main advantage of HPLC-PLS method over the classical HPLC method tried to emphasized as the using of simple mobile phase, shorter analysis time and no use of internal standard and gradient elution. Copyright © 2010 Elsevier B.V. All rights reserved.

Effect of Nanoparticle Surface on the HPLC Elution Profile of Liposomal Nanoparticles.

PubMed

Itoh, Naoki; Yamamoto, Eiichi; Santa, Tomofumi; Funatsu, Takashi; Kato, Masaru

2016-06-01

Nanoparticles have been used in diverse areas, and even broader applications are expected in the future. Since surface modification can influence the configuration and toxicity of nanoparticles, a rapid screening method is important to ensure nanoparticle quality. We examined the effect of the nanoparticle surface morphology on the HPLC elution profile using two types of 100-nm liposomal nanoparticles (AmBisome(Ⓡ) and DOXIL(Ⓡ)). These 100-nm-sized nanoparticles eluted before the holdup time (about 4 min), even when a column packed with particles with a relatively large pore size (30 nm) was used. The elution time of the nanoparticles increased with pegylation of the nanoparticles and protein adsorption to the nanoparticles; however, the nanoparticles still eluted before the holdup time. The results of this study indicate that HPLC is a suitable tool for rapid evaluation of the surface of liposomal nanoparticles.

Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS.

PubMed

Hager, Tiffany J; Howard, Luke R; Liyanage, Rohana; Lay, Jackson O; Prior, Ronald L

2008-02-13

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

A novel approach for the quantitation of carbohydrates in mash, wort, and beer with RP-HPLC using 1-naphthylamine for precolumn derivatization.

PubMed

Rakete, Stefan; Glomb, Marcus A

2013-04-24

A novel universal method for the determination of reducing mono-, di-, and oligosaccharides in complex matrices on RP-HPLC using 1-naphthylamine for precolumn derivatization with sodium cyanoborhydride was established to study changes in the carbohydrate profile during beer brewing. Fluorescence and mass spectrometric detection enabled very sensitive analyses of beer-relevant carbohydrates. Mass spectrometry additionally allowed the identification of the molecular weight and thereby the degree of polymerization of unknown carbohydrates. Thus, carbohydrates with up to 16 glucose units were detected. Comparison demonstrated that the novel method was superior to fluorophore-assisted carbohydrate electrophoresis (FACE). The results proved the HPLC method clearly to be more powerful in regard to sensitivity and resolution. Analogous to FACE, this method was designated fluorophore-assisted carbohydrate HPLC (FAC-HPLC).

Validation and Uncertainty Estimation of an Ecofriendly and Stability-Indicating HPLC Method for Determination of Diltiazem in Pharmaceutical Preparations

PubMed Central

Sadeghi, Fahimeh; Navidpour, Latifeh; Bayat, Sima; Afshar, Minoo

2013-01-01

A green, simple, and stability-indicating RP-HPLC method was developed for the determination of diltiazem in topical preparations. The separation was based on a C18 analytical column using a mobile phase consisted of ethanol: phosphoric acid solution (pH = 2.5) (35 : 65, v/v). Column temperature was set at 50°C and quantitation was achieved with UV detection at 240 nm. In forced degradation studies, the drug was subjected to oxidation, hydrolysis, photolysis, and heat. The method was validated for specificity, selectivity, linearity, precision, accuracy, and robustness. The applied procedure was found to be linear in diltiazem concentration range of 0.5–50 μg/mL (r 2 = 0.9996). Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. The recoveries obtained (99.25%–101.66%) ensured the accuracy of the developed method. The degradation products as well as the pharmaceutical excipients were well resolved from the pure drug. The expanded uncertainty (5.63%) of the method was also estimated from method validation data. Accordingly, the proposed validated and sustainable procedure was proved to be suitable for routine analyzing and stability studies of diltiazem in pharmaceutical preparations. PMID:24163778

Industrial application of green chromatography - II. Separation and analysis of preservatives in skincare products using subcritical water chromatography.

PubMed

Yang, Y; Kapalavavi, B; Gujjar, L; Hadrous, S; Marple, R; Gamsky, C

2012-10-01

Several high-temperature liquid chromatography (HTLC) and subcritical water chromatography (SBWC) methods have been successfully developed in this study for separation and analysis of preservatives contained in Olay skincare creams. Efficient separation and quantitative analysis of preservatives have been achieved on four commercially available ZirChrom and Waters XBridge columns at temperatures ranging from 100 to 200°C. The quantification results obtained by both HTLC and SBWC methods developed for preservatives analysis are accurate and reproducible. A large number of replicate HTLC and SBWC runs also indicate no significant system building-up or interference for skincare cream analysis. Compared with traditional HPLC separation carried out at ambient temperature, the HTLC methods can save up to 90% methanol required in the HPLC mobile phase. However, the SBWC methods developed in this project completely eliminated the use of toxic organic solvents required in the HPLC mobile phase, thus saving a significant amount of money and making the environment greener. Although both homemade and commercial systems can accomplish SBWC separations, the SBWC methods using the commercial system for preservative analysis are recommended for industrial applications because they can be directly applied in industrial plant settings. © 2012 The Authors ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

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.

High-performance liquid chromatography method for the determination of hydrogen peroxide present or released in teeth bleaching kits and hair cosmetic products.

PubMed

Gimeno, Pascal; Bousquet, Claudine; Lassu, Nelly; Maggio, Annie-Françoise; Civade, Corinne; Brenier, Charlotte; Lempereur, Laurent

2015-03-25

This manuscript presents an HPLC/UV method for the determination of hydrogen peroxide present or released in teeth bleaching products and hair products. The method is based on an oxidation of triphenylphosphine into triphenylphosphine oxide by hydrogen peroxide. Triphenylphosphine oxide formed is quantified by HPLC/UV. Validation data were obtained using the ISO 12787 standard approach, particularly adapted when it is not possible to make reconstituted sample matrices. For comparative purpose, hydrogen peroxide was also determined using ceric sulfate titrimetry for both types of products. For hair products, a cross validation of both ceric titrimetric method and HPLC/UV method using the cosmetic 82/434/EEC directive (official iodometric titration method) was performed. Results obtained for 6 commercialized teeth whitening products and 5 hair products point out similar hydrogen peroxide contain using either the HPLC/UV method or ceric sulfate titrimetric method. For hair products, results were similar to the hydrogen peroxide content using the cosmetic 82/434/EEC directive method and for the HPLC/UV method, mean recoveries obtained on spiked samples, using the ISO 12787 standard, ranges from 100% to 110% with a RSD<3.0%. To assess the analytical method proposed, the HPLC method was used to control 35 teeth bleaching products during a market survey and highlight for 5 products, hydrogen peroxide contents higher than the regulated limit. Copyright © 2015 Elsevier B.V. All rights reserved.

Methods of Analysis by the U.S. Geological Survey Organic Geochemistry Research Group-Update and Additions to the Determination of Chloroacetanilide Herbicide Degradation Compounds in Water Using High-Performance Liquid Chromatography/Mass Spectrometry

USGS Publications Warehouse

Lee, E.A.; Kish, J.L.; Zimmerman, L.R.; Thurman, E.

2001-01-01

An analytical method using high-performance liquid chromatography/mass spectrometry (HPLC/MS) was developed by the U.S. Geological Survey in 1999 for the analysis of selected chloroacetanilide herbicide degradation compounds in water. These compounds were acetochlor ethane sulfonic acid (ESA), acetochlor oxanilic acid (OXA), alachlor ESA, alachlor OXA, metolachlor ESA, and metolachlor OXA. The HPLC/MS method was updated in 2000, and the method detection limits were modified accordingly. Four other degradation compounds also were added to the list of compounds that can be analyzed using HPLC/MS; these compounds were dimethenamid ESA, dimethenamid OXA, flufenacet ESA, and flufenacet OXA. Except for flufenacet OXA, good precision and accuracy were demonstrated for the updated HPLC/MS method in buffered reagent water, surface water, and ground water. The mean HPLC/MS recoveries of the degradation compounds from water samples spiked at 0.20 and 1.0 ?g/L (microgram per liter) ranged from 75 to 114 percent, with relative standard deviations of 15.8 percent or less for all compounds except flufenacet OXA, which had relative standard deviations ranging from 11.3 to 48.9 percent. Method detection levels (MDL's) using the updated HPLC/MS method varied from 0.009 to 0.045 ?g/L, with the flufenacet OXA MDL at 0.072 ?g/L. The updated HPLC/MS method is valuable for acquiring information about the fate and transport of the parent chloroacetanilide herbicides in water.

A bioanalytical HPLC method for coumestrol quantification in skin permeation tests followed by UPLC-QTOF/HDMS stability-indicating method for identification of degradation products.

PubMed

Bianchi, Sara E; Teixeira, Helder F; Kaiser, Samuel; Ortega, George G; Schneider, Paulo Henrique; Bassani, Valquiria L

2016-05-01

Coumestrol is present in several species of the Fabaceae family widely distributed in plants. The estrogenic and antioxidant activities of this molecule show its potential as skin anti-aging agent. These characteristics reveal the interest in developing analytical methodology for permeation studies, as well as to know the stability of coumestrol identifying the major degradation products. Thus, the present study was designed, first, to develop and validate a versatile liquid chromatography (HPLC) method to quantify coumestrol in a hydrogel formulation in different porcine skin layers (stratum corneum, epidermis, and dermis) in permeation tests. In the stability-indicating test coumestrol samples were exposed to stress conditions: temperature, UVC light, oxidative, acid and alkaline media. The degradation products, as well as the constituents extracted from the hydrogel, adhesive tape or skin were not eluted in the retention time of the coumestrol. Hence, the HPLC method showed to be versatile, specific, accurate, precise and robust showing excellent performance for quantifying coumestrol in complex matrices involving skin permeation studies. Coumestrol recovery from porcine ear skin was found to be in the range of 97.07-107.28 μg/mL; the intra-day precision (repeatability) and intermediate precision (inter-day precision), respectively lower than 4.71% and 2.09%. The analysis using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight high definition mass spectrometry detector (UPLC-QTOF/HDMS) suggest the MS fragmentation patterns and the chemical structure of the main degradation products. These results represent new and relevant findings for the development of coumestrol pharmaceutical and cosmetic products. Copyright © 2016 Elsevier B.V. All rights reserved.

Stability-indicating HPLC Method for Simultaneous Determination of Terbutaline Sulphate, Bromhexine Hydrochloride and Guaifenesin

PubMed Central

Porel, A.; Haty, Sanjukta; Kundu, A.

2011-01-01

The aim of the present study was the development and subsequent validation of a simple, precise and stability-indicating reversed phase HPLC method for the simultaneous determination of guaifenesin, terbutaline sulphate and bromhexine hydrochloride in the presence of their potential impurities in a single run. The photolytic as well as hydrolytic impurities were detected as 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]-ethanone from terbutaline, 2-methoxyphenol and an unknown impurity identified as (2RS)-3-(2-hydroxyphenoxy)-propane-1,2-diol from guaifenesin. The chromatographic separation of all the three active components and their impurities was achieved on Wakosil II column, using phosphate buffer (pH 3.0) and acetonitrile as mobile phase which was delivered initially in the ratio of 80:20 (v/v) for 18 min, then changed to 60:40 (v/v) for next 12 min, and finally equilibrated back to 80:20 (v/v) for 10 min. Other HPLC parameters were: Flow rate at 1.0 ml/min, detection wavelengths 248 and 280 nm, injection volume 10 μl. The calibration graphs plotted with five concentrations of each component were linear with a regression coefficient R2 >0.9999. The limit of detection and limit of quantitation were estimated for all the five impurities. The established method was then validated for linearity, precision, accuracy, and specificity and demonstrated to be applicable to the determination of the active ingredients in commercial and model cough syrup. No interference from the formulation excipients was observed. These results suggest that this LC method can be used for the determination of multiple active ingredients and their impurities in a cough and cold syrup. PMID:22131621

Stability-indicating HPLC Method for Simultaneous Determination of Terbutaline Sulphate, Bromhexine Hydrochloride and Guaifenesin.

PubMed

Porel, A; Haty, Sanjukta; Kundu, A

2011-01-01

The aim of the present study was the development and subsequent validation of a simple, precise and stability-indicating reversed phase HPLC method for the simultaneous determination of guaifenesin, terbutaline sulphate and bromhexine hydrochloride in the presence of their potential impurities in a single run. The photolytic as well as hydrolytic impurities were detected as 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde, 1-(3,5-dihydroxyphenyl)-2-[(1,1-dimethylethyl) amino]-ethanone from terbutaline, 2-methoxyphenol and an unknown impurity identified as (2RS)-3-(2-hydroxyphenoxy)-propane-1,2-diol from guaifenesin. The chromatographic separation of all the three active components and their impurities was achieved on Wakosil II column, using phosphate buffer (pH 3.0) and acetonitrile as mobile phase which was delivered initially in the ratio of 80:20 (v/v) for 18 min, then changed to 60:40 (v/v) for next 12 min, and finally equilibrated back to 80:20 (v/v) for 10 min. Other HPLC parameters were: Flow rate at 1.0 ml/min, detection wavelengths 248 and 280 nm, injection volume 10 μl. The calibration graphs plotted with five concentrations of each component were linear with a regression coefficient R(2) >0.9999. The limit of detection and limit of quantitation were estimated for all the five impurities. The established method was then validated for linearity, precision, accuracy, and specificity and demonstrated to be applicable to the determination of the active ingredients in commercial and model cough syrup. No interference from the formulation excipients was observed. These results suggest that this LC method can be used for the determination of multiple active ingredients and their impurities in a cough and cold syrup.

Steroids

NASA Astrophysics Data System (ADS)

Frey, Felix J.; Frey, Brigitte M.; Benet, Leslie Z.

If a radioimmunoassay, a protein binding method, or a colorimetric assay for the assessment of a steroid level is replaced by high performance liquid chromatography (HPLC), the cost for the determination of a steroid level increases at least initially because one must acquire the new HPLC equipment. Therefore, if an older method provides the same results as the new, "advanced" HPLC method, the only advantage resulting from the introduction of a high performance chromatographic assay is that gained by the manufacturer in terms of greater sales. Thus, justification for the assessment of steroids by HPLC is only obtained if the quality and/or quantity of information gained is significantly increased as compared to that provided by the conventional methods. But this evidential relation, that more and better information justifies a higher price in any case, is no longer true in health care, with the birth some years ago of the categoric imperative for the reduction of costs in the medical sector. That is, each new technology introduced for health maintenance should demonstrate at least a stabilizing impact on total medical expenditures. Therefore, after reviewing the presently available HPLC methods for the clinically important steroids, we will consider whether HPLC analyses for these steroids can be recommended without violating this vox populi.

High-Performance Liquid Chromatography (HPLC) Quantification of Liposome-Delivered Doxorubicin in Arthritic Joints of Collagen-Induced Arthritis Rats.

PubMed

Niu, Hongqing; Xu, Menghua; Li, Shuangtian; Chen, Junwei; Luo, Jing; Zhao, Xiangcong; Gao, Chong; Li, Xiaofeng

2017-04-14

BACKGROUND Neoangiogenesis occurring in inflamed articular synovium in early rheumatoid arthritis (RA) is characterized by enhanced vascular permeability that allows nanoparticle agents, including liposomes, to deliver encapsulated drugs to arthritic joints and subsequently improve therapeutic efficacy and reduce adverse effects. However, the targeting distribution of liposomes in arthritic joints during RA has not been quantitatively demonstrated. We performed this study to evaluate the targeting distribution of PEGylated doxorubicin liposomes in the arthritic joints of collagen-induced arthritis (CIA) rats by high-performance liquid chromatography (HPLC). MATERIAL AND METHODS Two doxorubicin formulations were administered to CIA rats via tail intravenous injection at a single dose (50 mg/m²). CIA rats were sacrificed and the tissues of the inflamed ankle joints were collected. The content of doxorubicin in the arthritic joints was analyzed by a validated and reproducible HPLC method. A two-way ANOVA for 2×5 factorial design was used for statistical analysis. RESULTS The developed HPLC method was sensitive, precise, and reproducible. The method was successfully applied to quantify doxorubicin content in arthritic tissues. At each time point (6, 12, 24, 48, and 72 h), doxorubicin content in the arthritic joints of the doxorubicin liposome group (DOX-LIP group) was higher than in the free doxorubicin group (DOX group) (P<0.05). In the DOX-LIP group, doxorubicin levels in the arthritic joints increased gradually and significantly in the interval of 6-72 h post-administration. CONCLUSIONS PEGylated doxorubicin liposomes were targeted to, accumulated, and retained in the arthritic joints of CIA rats. The present study indicates that liposome encapsulation increases the therapeutic efficacy of antirheumatic drugs, presenting a promising therapeutic strategy for RA.

A validated stability indicating RP-HPLC method for estimation of Armodafinil in pharmaceutical dosage forms and characterization of its base hydrolytic product.

PubMed

Venkateswarlu, Kambham; Rangareddy, Ardhgeri; Narasimhaiah, Kanaka; Sharma, Hemraj; Bandi, Naga Mallikarjuna Raja

2017-01-01

The main objective of present study was to develop a RP-HPLC method for estimation of Armodafinil in pharmaceutical dosage forms and characterization of its base hydrolytic product. The method was developed for Armodafinil estimation and base hydrolytic products were characterized. The separation was carried out on C18 column by using mobile phase as mixture of water and methanol (45:55%v/v). Eluents were detected at 220nm at 1ml/min. Stress studies were performed with milder conditions followed by stronger conditions so as to get sufficient degradation around 20%. A total of five degradation products were detected and separated from analyte. The linearity of the proposed method was investigated in the range of 20-120µg/ml for Armodafinil. The detection limit and quantification limit was found to be 0.01183μg/ml and 0.035µg/ml respectively. The precision % RSD was found to be less than 2% and the recovery was between 98-102%. Armodafinil was found to be more sensitive to the base hydrolysis and yielded its carboxylic acid as degradant. The developed method was stability indicating assay, suitable to quantify Armodafinil in presence of possible degradants. The drug was sensitive to acid, base &photolytic stress and resistant to thermal &oxidation.

Determination and confirmation of isopropyl p-toluenesulfonate in cosmetics by HPLC-diode array detector method and GC-MS.

PubMed

Tay, B Y P; Yung, S C; Teoh, T Y

2016-12-01

Isopropyl p-toluenesulfonate (IPTS) is a potentially genotoxic by-product formed during the esterification of palm oil-based palmitic and palm kernel oil-based myristic acid with isopropanol to produce isopropyl palmitate or isopropyl myristate. There are no methods described for the analysis of IPTS in cosmetic products. In this work, we have established a simple, precise and accurate method to determine the presence and level of IPTS in various finished cosmetic products which contain palm-based esters in their formulations. An Agilent 1200 series high-performance liquid chromatography (HPLC) unit using a diode-array detector (DAD) has been employed and optimized to detect IPTS in cosmetic products. For the separation, a reverse-phase Hypersil Gold C8 column (5 μm, 4.6 mm i.d. 250 mm) 5 mM tetrabutylammonium phosphate buffer 50 : 50, (v/v) solution in acetonitrile as mobile phase, in isocratic mode and a flow rate of 0.8 mL min -1 were used. A second method using a gas chromatography/mass selective detector GC-MSD was also developed to confirm the IPTS identity in the cosmetic products. Recoveries of IPTS from cosmetic matrices such as a lotion, cleansing milk and a cream ranged from 94.0% to 101.1% with <5% relative standard deviation (%RSD) showing good accuracy and repeatability of the method. The six-point calibration curves (determined over the range 0.5-50 μg mL -1 ) have a correlation coefficient of 0.9999 (based on HPLC peak area) and 0.9998 (based on HPLC peak height). The intra- and interday precisions (measured by the %RSD) of the method were <2% and <5%, respectively, indicating that the developed method is reliable, precise and reproducible. The detection and quantification limit of the method were found to be 0.5 μg mL -1 and 1.6 μg mL -1 , respectively. Analyses of 83 commercial cosmetics showed no presence of IPTS. The validation data indicated that this method was suitable for the quantitative analysis of IPTS in commercial cosmetics. This method is applicable for analyses of trace levels of IPTS in cosmetics and has the advantage of using only simple sample preparation steps. © 2016 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis.

PubMed

Matuszewski, B K

2006-01-18

A simple experimental approach for studying and identifying the relative matrix effect (for example "plasma-to-plasma" and/or "urine-to-urine") in quantitative analyses by HPLC-MS/MS is described. Using as a database a large number of examples of methods developed in recent years in our laboratories, the relationship between the precision of standard line slopes constructed in five different lots of a biofluid (for example plasma) and the reliability of determination of concentration of an analyte in a particular plasma lot (or subject) was examined. In addition, the precision of standard line slopes was compared when stable isotope-labeled analytes versus analogs were used as internal standards (IS). Also, in some cases, a direct comparison of standard line slopes was made when different HPLC-MS interfaces (APCI versus ESI) were used for the assay of the same compound, using the same IS and the same sample preparation and chromatographic separation conditions. In selected cases, the precision of standard line slopes in five different lots of a biofluid was compared with precision values determined five times in a single lot. The results of these studies indicated that the variability of standard line slopes in different lots of a biofluid [precision of standard line slopes expressed as coefficient of variation, CV (%)] may serve as a good indicator of a relative matrix effect and, it is suggested, this precision value should not exceed 3-4% for the method to be considered reliable and free from the relative matrix effect liability. Based on the results presented, in order to assess the relative matrix effect in bioanalytical methods, it is recommended to perform assay precision and accuracy determination in five different lots of a biofluid, instead of repeat (n=5) analysis in the same, single biofluid lot, calculate standard line slopes and precision of these slopes, and to use <3-4% slope precision value as a guide for method applicability to support clinical studies. It was also demonstrated that when stable isotope-labeled analytes were used as internal standards, the precision of standard line slopes in five different lots of a biofluid was

Quantification of quercetin glycosides in 6 onion cultivars and comparisons of hydrolysis-HPLC and spectrophotometric methods in measuring total quercetin concentrations.

PubMed

Yoo, Kil Sun; Lee, Eun Jin; Patil, Bhimanagouda S

2010-03-01

This study was performed to purify and quantify quercetin glycosides (QG) and aglycone (free) quercetin (Q) in 6 selected onion cultivars and to compare analytical approaches based on high-performance liquid chromatography (HPLC) and spectrophotometry for the quantification of total quercetin (TQ) concentrations. Individual mono- and di-glycoside Q compounds were purified using a semipreparative HPLC and identified by comparing spectral data and by confirming corresponding peaks of QG and Q after incomplete enzyme-hydrolysis. Purified QG were quantified as Q by enzyme-hydrolysis/HPLC. TQ concentrations obtained from 20 onion bulbs with enzyme-hydrolysis/HPLC, no-hydrolysis/HPLC, and a spectrophotometric method without prior hydrolysis were significantly correlated (r(2)= 0.99) and were about 15% higher, identical, or 10% less than those concentrations by a standard acid-hydrolysis/HPLC method, respectively. During enzyme-hydrolysis of onion extracts, progressive reduction of the QG and formation of the corresponding mono-glycosides and Q were monitored using an analytical HPLC. TQ ranged from 83 to 330 microg/g F.W. in 6 selected cultivars of long-day or short-day onions. Q3,4'G and Q4'G were the 2 major compounds and comprised approximately between 94% and 97% of TQ in onions.

Evaluation of subsurface exploration, sampling, and water-quality-analysis methods at an abandoned wood-preserving plant site at Jackson, Tennessee

USGS Publications Warehouse

Parks, W.S.; Carmichael, J.K.; Mirecki, J.E.

1993-01-01

Direct Push Technology (DPT) and a modified-auger method of sampling were used at an abandoned wood-preserving plant site at Jackson, Tennessee, to collect lithologic data and ground-water samples in an area known to be affected by a subsurface creosote plume. The groundwater samples were analyzed using (1) gas chromatography with photo-ionization detection (GS/PID), (2) high- performance liquid chromatography (HPLC), (3) colonmetric phenol analysis, and (4) toxicity bioassay. DPT piezocone and cone-penetrometer-type tools provided lithologic data and ground-water samples at two onsite stations to a depth of refusal of about 35 feet below land surface. With the assistance of an auger rig, this depth was extended to about 65 feet by pushing the tools in advance of the augers. Following the DPT work, a modified-auger method was tested by the USGS. This method left doubt as to the integrity of the samples collected once zones of contamination were penetrated. GC/PID and HPLC methods of water-quality analysis provided the most data concerning contaminants in the ground-water and proved to be the most effective in creosote plume detection. Analyses from these methods showed that the highest concentrations of contaminants were detected at depths less than about 35 feet below land surface. Phenol analyses provided data supplemental to the HPLC analyses. Bioassay data indicated that toxicity associated with the plume extended to depths of about 55 feet below land surface.

Short communication: Determination of withdrawal time for oxytetracycline in different types of goats for milk consumption.

PubMed

Attaie, Rahmat; Bsharat, Mohammed; Mora-Gutierrez, Adela; Woldesenbet, Sela

2015-07-01

Antibiotics are widely used in animal husbandry and the presence of antibiotics in milk is a health hazard. The objective of this study was to determine residual amounts of oxytetracycline in fresh, aged, and pasteurized milk of 3 breeds of goats using HPLC analysis. It was also essential to determine the safe withdrawal period of oxytetracycline in lactating goats. The quantitative results obtained using the HPLC system were compared with the tolerance limit of oxytetracycline in milk in the United States. Fifteen milking does, 5 Nubians, 5 Alpines, and 5 LaManchas were randomly selected from the milking herd at the International Goat Research Center at Prairie View A&M University. A simple sample preparation and isocratic HPLC method using ultraviolet detection was used for analysis of milk samples. The HPLC results indicated that the withdrawal period of oxytetracycline in treated Alpine does was 82h (7 milking), whereas for Nubian does the period was 58h (5 milking), and for LaManchas the period was 72h (6 milking) after drug administration. The overall withdrawal period for all the treated goats of 3 breeds was 72h. Although these results indicated that the depletion rate of this antibiotic was faster in goats than the reported data for cows, the 96-h withdrawal period that is currently used for lactating cows is still necessary for these 3 breeds of goats. Additionally, our results indicated that oxytetracycline is not stable in goat milk at refrigeration temperature or during pasteurization and will decrease significantly. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

76 FR 38033 - Cloquintocet-mexyl; Pesticide Tolerances

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-29

... enforcement methods are the High Performance Liquid Chromatography with Ultraviolet Detection (HPLC/UV) method REM 138.01 for determination of cloquintocet-mexyl (parent) and the HPLC/UV Method REM 138.10 for...

Development and validation of stability indicating method for the quantitative determination of venlafaxine hydrochloride in extended release formulation using high performance liquid chromatography

PubMed Central

Kaur, Jaspreet; Srinivasan, K. K.; Joseph, Alex; Gupta, Abhishek; Singh, Yogendra; Srinivas, Kona S.; Jain, Garima

2010-01-01

Objective: Venlafaxine,hydrochloride is a structurally novel phenethyl bicyclic antidepressant, and is usually categorized as a serotonin–norepinephrine reuptake inhibitor (SNRI) but it has been referred to as a serotonin–norepinephrine–dopamine reuptake inhibitor. It inhibits the reuptake of dopamine. Venlafaxine HCL is widely prescribed in the form of sustained release formulations. In the current article we are reporting the development and validation of a fast and simple stability indicating, isocratic high performance liquid chromatographic (HPLC) method for the determination of venlafaxine hydrochloride in sustained release formulations. Materials and Methods: The quantitative determination of venlafaxine hydrochloride was performed on a Kromasil C18 analytical column (250 × 4.6 mm i.d., 5 μm particle size) with 0.01 M phosphate buffer (pH 4.5): methanol (40: 60) as a mobile phase, at a flow rate of 1.0 ml/min. For HPLC methods, UV detection was made at 225 nm. Results: During method validation, parameters such as precision, linearity, accuracy, stability, limit of quantification and detection and specificity were evaluated, which remained within acceptable limits. Conclusions: The method has been successfully applied for the quantification and dissolution profiling of Venlafaxine HCL in sustained release formulation. The method presents a simple and reliable solution for the routine quantitative analysis of Venlafaxine HCL. PMID:21814426

Fast and accurate determination of arsenobetaine in fish tissues using accelerated solvent extraction and HPLC-ICP-MS determination.

PubMed

Wahlen, Raimund

2004-04-01

A high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) method has been developed for the fast and accurate analysis of arsenobetaine (AsB) in fish samples extracted by accelerated solvent extraction. The combined extraction and analysis approach is validated using certified reference materials for AsB in fish and during a European intercomparison exercise with a blind sample. Up to six species of arsenic (As) can be separated and quantitated in the extracts within a 10-min isocratic elution. The method is optimized so as to minimize time-consuming sample preparation steps and allow for automated extraction and analysis of large sample batches. A comparison of standard addition and external calibration show no significant difference in the results obtained, which indicates that the LC-ICP-MS method is not influenced by severe matrix effects. The extraction procedure can process up to 24 samples in an automated manner, yet the robustness of the developed HPLC-ICP-MS approach is highlighted by the capability to run more than 50 injections per sequence, which equates to a total run-time of more than 12 h. The method can therefore be used to rapidly and accurately assess the proportion of nontoxic AsB in fish samples with high total As content during toxicological screening studies.

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

Kawamura, M.; Yoshida, K.; Akabane, S.

We measured endogenous angiotensins (ANGs) I, IIandIII using a system of extraction by Sep-Pak column followed by high performance liquid chromatography (HPLC) combined with radioimmunoassay (RIA). An excellent separation of ANGs was obtained by HPLC. The recovery of ANGs I, IIandIII was 80-84%, when these authentic peptides were added to 6 ml of plasma. The coefficient of variation of the ANGs was 0.04-0.09 for intra-assay and 0.08-0.13 for inter-assay, thereby indicating a good reproducibility. Plasma ANGs I, IIandIII measured by this method in 5 normal volunteers were 51,4.5 and 1.2 pg/ml. In the presence of captopril, ANGs IIandIII decreased bymore » 84% and 77%, respectively, while ANG I increased 5.1 times. This method is therefore useful to assess the precise levels of plasma ANGs.« less

A simple HPLC-DAD method for simultaneous detection of two organophosphates, profenofos and fenthion, and validation by soil microcosm experiment.

PubMed

Mahajan, Rishi; Chatterjee, Subhankar

2018-05-05

Indiscriminate use of two broad spectrum pesticides, profenofos and fenthion, in agricultural system, often results in their accumulation in a non-target niche and leaching into water bodies. The present study, therefore, aims at developing a simple and rapid HPLC method that allows simultaneous extraction and detection of these two pesticides, especially in run-off water. Extraction of the two pesticides from spiked water samples using dichloromethane resulted in recovery ranging between 80 and 90%. An HPLC run of 20 min under optimized chromatographic parameters (mobile phase: methanol (75%) and water (25%); flow rate of 0.8 ml min -1 ; diode array detector at wavelength 210 nm) resulted in a significant difference in retention times of two pesticides (4.593 min) which allows a window of opportunity to study any possible intermediates/transformants of the parent compounds while evaluating run-off waters from agricultural fields. The HPLC method developed allowed simultaneous detection of profenofos and fenthion with a single injection into the HPLC system with 0.0328 mg l -1 (32.83 ng ml -1 ) being the limit of detection (LOD) and 0.0995 mg l -1 (99.5 ng ml -1 ) as the limit of quantification (LOQ) for fenthion; for profenofos, LOD and LOQ were 0.104 mg l -1 (104.50 ng ml -1 ) and 0.316 mg l -1 (316.65 ng ml -1 ), respectively. The findings were further validated using the soil microcosm experiment that allowed simultaneous detection and quantification of profenofos and fenthion. The findings indicate towards the practical significance of the methodology developed as the soil microcosm experiment closely mimics the agricultural run-off water under natural environmental conditions.

Identification of a Panax ginseng fruit fingerprint by HPLC-ESI-MS.

PubMed

Zhao, H F; Xu, F F; Guo, Y T; Mi, H

2016-03-11

Over many years, parts of Panax ginseng (root and rhizome) have been identified and applied for medical purposes as traditional Chinese herbal medicine. Recently, research has indicated that ginseng fruit also contains similar compounds and is as rich as the other parts of the ginseng. This discovery may dramatically improve the efficient of outputs derived from ginseng products. Here, a new technique combining high-performance liquid chromatography (HPLC) with electrospray ionization tandem mass spectrometry (ESI-MS) was employed to identify the fingerprint of P. ginseng fruit. Using HPLC, compounds that are important for medical purposes were extracted and purified. Combined with ESI-MS, the characteristic peaks (nine common peaks) of those compounds were identified, and the accuracy was confirmed by analysis using the Chromatographic Fingerprint Similarity Evaluation System (2004A edition). Overall, 15 batches of ginseng fruit had a similarity of more than 0.80, 13 batches of samples had a similarity between 0.97 and 0.99, and two batches had a similarity less than 0.90. The test solution and mobile phase selection was discussed. The HPLC-ESI-MS method can produce repeatable and reliable results and can be applied in the quality control of P. ginseng fruit.

Construction of a hydrazone-linked chiral covalent organic framework-silica composite as the stationary phase for high performance liquid chromatography.

PubMed

Zhang, Kai; Cai, Song-Liang; Yan, Yi-Lun; He, Zi-Hao; Lin, Hui-Mei; Huang, Xiao-Ling; Zheng, Sheng-Run; Fan, Jun; Zhang, Wei-Guang

2017-10-13

Covalent organic frameworks (COFs), as an emerging class of crystalline porous organic polymers, have great potential for applications in chromatographic separation owning to their fascinating crystalline structures and outstanding properties. However, development of COF materials as novel stationary phases in high performance liquid chromatography (HPLC) is just in its infancy. Herein, we report the design and construction of a new hydrazone-linked chiral COF, termed BtaMth COF, from a chiral hydrazide building block (Mth) and present a one-pot synthetic method for the fabrication of BtaMth@SiO 2 composite for HPLC separation of isomers. The as-synthesized BtaMth chiral COF displays good crystallinity, high porosity, as well as excellent chemical stability. Meanwhile, the fabricated HPLC column by using BtaMth@SiO 2 composite as the new stationary phase exhibits high resolution performances for the separation of positional isomers including nitrotoluene and nitrochlorobenzene, as well as cis-trans isomers including beta-cypermethrin and metconazole. Additionally, some effects such as the composition of the mobile phase and column temperature for HPLC separations on the BtaMth@SiO 2 packed column also have been studied in detail. The successful applications indicate the great potentials of hydrazone-linked chiral COF-silica composite as novel stationary phase for the efficient HPLC separation. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of very low levels of 5-(hydroxymethyl)-2-furaldehyde (HMF) in natural honey: comparison between the HPLC technique and the spectrophotometric white method.

PubMed

Truzzi, Cristina; Annibaldi, Anna; Illuminati, Silvia; Finale, Carolina; Rossetti, Monica; Scarponi, Giuseppe

2012-07-01

In this work we compared 2 official methods for the determination of HMF in honey, the spectrophotometric White method and the HPLC method (International Honey Commission) for the determination of HMF in unifloral honey and honeydew samples with a very low HMF content (<4 mg/kg), which is the most critical determination in terms of accuracy and precision of methods. In honey solutions, the limits of quantification for HPLC and White methods are 0.83 mg/L and 0.67 mg/L, respectively, and the linearity range is confirmed up to 20 mg/L for the HPLC method and up to 5 mg/L for the White method. In honeys with HMF >5 mg/kg, the molar extinction coefficient is 15369, lower than the literature value of 16830, and should be used for HMF determination. For samples with HMF content in the range 1-4 mg/kg the accuracy of the 2 methods is comparable both for unifloral and honeydew samples, whereas as regards precision, the HPLC method gives better results (3.5% compared with 6.4% for the White method). So, in general, the HPLC method seems to be more appropriate for the determination of HMF in honey in the range 1-4 mg/kg thanks to its greater precision, but for samples with a HMF content of less than 1 mg/kg the analyses are inaccurate for both methods. This work can help governmental and private laboratories that perform food analyses to choose the best method for the determination of HMF at very low levels in unifloral honey and honeydew samples. © 2012 Institute of Food Technologists®

Chemical characteristics combined with bioactivity for comprehensive evaluation of Panax ginseng C.A. Meyer in different ages and seasons based on HPLC-DAD and chemometric methods.

PubMed

Shan, Si-Ming; Luo, Jian-Guang; Huang, Fang; Kong, Ling-Yi

2014-02-01

Panax ginseng C.A. Meyer has been known as a valuable traditional Chinese medicines for thousands years of history. Ginsenosides, the main active constituents, exhibit prominent immunoregulation effect. The present study first describes a holistic method based on chemical characteristic and lymphocyte proliferative capacity to evaluate systematically the quality of P. ginseng in thirty samples from different seasons during 2-6 years. The HPLC fingerprints were evaluated using principle component analysis (PCA) and hierarchical clustering analysis (HCA). The spectrum-efficacy model between HPLC fingerprints and T-lymphocyte proliferative activities was investigated by principal component regression (PCR) and partial least squares (PLS). The results indicated that the growth of the ginsenosides could be grouped into three periods and from August of the fifth year, P. ginseng appeared significant lymphocyte proliferative capacity. Close correlation existed between the spectrum-efficacy relationship and ginsenosides Rb1, Ro, Rc, Rb2 and Re were the main contributive components to the lymphocyte proliferative capacity. This comprehensive strategy, providing reliable and adequate scientific evidence, could be applied to other TCMs to ameliorate their quality control. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of the BioRad Variant and Primus Ultra2 high-pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins.

PubMed

Gosselin, R C; Carlin, A C; Dwyre, D M

2011-04-01

Hemoglobin variants are a result of genetic changes resulting in abnormal or dys-synchronous hemoglobin chain production (thalassemia) or the generation of hemoglobin chain variants such as hemoglobin S. Automated high-pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies. In this study, we evaluated the performance of two HPLC methods used in the detection of common hemoglobin variants: Variant and Ultra2. There were 377 samples tested, 26% (99/377) with HbS, 8.5% (32/377) with HbC, 20.7% (78/377) with other hemoglobin variant or thalassemia, and 2.9% with increased hemoglobin A(1) c. The interpretations of each chromatograph were compared. There were no differences noted for hemoglobins A(0), S, or C. There were significant differences between HPLC methods for hemoglobins F, A(2), and A(1) c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). Both Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A(2), and between hemoglobins S and F using the Ultra2 HPLC method. © 2010 Blackwell Publishing Ltd.

Development of a sensitive high-performance liquid chromatographic method with simple extraction for simultaneous determination of huperzine A and huperzine B in the species containing lycopodium alkaloids.

PubMed

Zhang, Yanqing; Xie, Junbo; Chen, Wen-Qian; Zhou, Tian-Yan; Lu, Wei

2009-01-01

A sensitive HPLC method with simple extraction was developed for simultaneous determination of huperzine A (HupA) and huperzine B (HupB) in Huperzia serrata, H. crispata, H. miyoshiana, and Lycopodiastrum casuarinoides. In order to avoid conventional multiple-step and time-consuming sample preparation methods, direct reflux extraction with alkaline chloroform was adopted. The quantitative determination was conducted by reversed-phase HPLC with a photodiode array detector set at 308 nm. Separation was performed on a Luna C18 column (250 x 4.6 mm id, 5 microm) with methanol-0.2% aqueous acetic acid (18 + 82, v/v) mobile phase. The method was validated for accuracy, reproducibility, precision, and limits of detection and quantification. Quantification of the two active compounds in the samples was performed by this newly developed method, and the content of HupA and HupB varied substantially among four different species. The satisfactory results indicated that the developed method can readily be utilized for quality control of the species of Huperziaceae and Lycopodiaceae containing the two compounds.

Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.

PubMed

Carbonell, Pablo; Turpin, María C; Torres-Moreno, Daniel; Molina-Martínez, Irene; García-Solano, José; Perez-Guillermo, Miguel; Conesa-Zamora, Pablo

2011-09-01

The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffin-embedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent (κ = 0.9411 and κ = 0.8988, respectively); for HRM, the concordances were good (κ = 0.7973 and κ = 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in archival paraffin-embedded tissues. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

[Study on arsenic speciation changes in crude and processed traditional Chinese medicines by HPLC-ICP-MS].

PubMed

Jin, Peng-fei; Wu, Xue-jun; Zou, Ding; Kuang, Yong-mei; Hu, Xin; Jiang, Wen-qing; Sun, Chun-hua

2011-03-01

A HPLC-ICP-MS method for simultaneous determination of As(III), As(V), MMA and DMA in traditional Chinese medicines (TCMs) was established, and the contents of As(III), As(V), MMA and DMA in a TCM with high total arsenic content (Cordyceps) and 5 crude and processed TCMs (Radix Astragali, Radix et Rhizoma Rhei, Radix Scutellariae, Radix Polygoni Multiflori and Radix Rehmanniae) were determined and analyzed. The method validation indicated that the correlative coefficients (r) for all speciations were bigger than 0.9984; the limits of quantitation (LOQ) were from 0.8 to 1.0 microg x L(-1); the reproducibility and stability were satisfactory with all RSDs less than 10%; the spiked recoveries ranged from 82.40% to 119.5%. The results of samples analysis showed that the inorganic arsenic (As(III) and As(V)) was the dominating speciation in the tested TCMs; MMA and DMA were not found in all plant resourced TCMs, but MMA was found in Cordyceps; all the tested TCMs indicated a content increasing of inorganic arsenic after processing.

A Stability-Indicating HPLC Method for the Determination of Memantine Hydrochloride in Dosage Forms through Derivatization with 1-Fluoro-2,4-dinitrobenzene

PubMed Central

Jalalizadeh, Hassan; Raei, Mahdi; Tafti, Razieh Fallah; Farsam, Hassan; Kebriaeezadeh, Abbas; Souri, Effat

2014-01-01

Memantine is chemically a tricyclic amine and is used for Parkinson’s disease and movement disorders. Although several HPLC methods with different derivatization reagents have been developed for the determination of memantine in biological fluids, there are some complications which limit the use of these methods in routine analysis of memantine in in vitro tests. We established a simple, sensitive, precise, and accurate HPLC method for the quantification of memantine in dosage forms. Pre-column derivatization of memantine was performed with 1-fluoro-2,4-dinitrobenzene and the reaction product was separated on a Nova-Pak C18 column. A mixture of acetonitrile and sodium dihydrogenphosphate (pH 2.5; 0.05 M) (70: 30, v/v) was used as the mobile phase. UV detection was performed at 360 nm. Forced degradation studies were performed on a powdered tablet sample of memantine hydro-chloride using acidic (0.1 M hydrochloric acid), basic (0.1 M sodium hydroxide), oxidative (10% hydrogen peroxide), thermal (105°C), photolytic, and humidity conditions. Good linearity (r2=0.999) was obtained over the range of 1–12 μg mL−1 of memantine hydrochloride with acceptable within-day and between-day precision values in the range of 0.05–0.95%. The proposed method was used for the assay determination and dissolution rate study of memantine dosage forms with excellent specificity. PMID:24959398

Development and Validation of a Precise, Single HPLC Method for the Determination of Tolperisone Impurities in API and Pharmaceutical Dosage Forms.

PubMed

Raju, Thummala Veera Raghava; Seshadri, Raja Kumar; Arutla, Srinivas; Mohan, Tharlapu Satya Sankarsana Jagan; Rao, Ivaturi Mrutyunjaya; Nittala, Someswara Rao

2013-01-01

A novel, sensitive, stability-indicating HPLC method has been developed for the quantitative estimation of Tolperisone-related impurities in both bulk drugs and pharmaceutical dosage forms. Effective chromatographic separation was achieved on a C18 stationary phase with a simple mobile phase combination delivered in a simple gradient programme, and quantitation was by ultraviolet detection at 254 nm. The mobile phase consisted of a buffer and acetonitrile delivered at a flow rate 1.0 ml/min. The buffer consisted of 0.01 M potassium dihydrogen phosphate with the pH adjusted to 8.0 by using diethylamine. In the developed HPLC method, the resolution between Tolperisone and its four potential impurities was found to be greater than 2.0. Regression analysis showed an R value (correlation coefficient) of greater than 0.999 for the Tolperisone impurities. This method was capable of detecting all four impurities of Tolperisone at a level of 0.19 μg/mL with respect to the test concentration of 1000 μg/mL for a 10 µl injection volume. The tablets were subjected to the stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation. Considerable degradation was found to occur in base hydrolysis, water hydrolysis, and oxidation. The stress samples were assayed against a qualified reference standard and the mass balance was found to be close to 100%. The established method was validated and found to be linear, accurate, precise, specific, robust, and rugged.

Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis.

PubMed

Gobbo-Neto, Leonardo; Lopes, Norberto P

2008-02-27

Lychnophora ericoides Mart. (Asteraceae, Vernonieae) is a plant, endemic to Brazil, with occurrence restricted to the "cerrado" biome. Traditional medicine employs alcoholic and aqueous-alcoholic preparations of leaves from this species for the treatment of wounds, inflammation, and pain. Furthermore, leaves of L. ericoides are also widely used as flavorings for the Brazilian traditional spirit "cachaça". A method has been developed for the extraction and HPLC-DAD analysis of the secondary metabolites of L. ericoides leaves. This analytical method was validated with 11 secondary metabolites chosen to represent the different classes and polarities of secondary metabolites occurring in L. ericoides leaves, and good responses were obtained for each validation parameter analyzed. The same HPLC analytical method was also employed for online secondary metabolite identification by HPLC-DAD-MS and HPLC-DAD-MS/MS, leading to the identification of di- C-glucosylflavones, coumaroylglucosylflavonols, flavone, flavanones, flavonols, chalcones, goyazensolide, and eremantholide-type sesquiterpene lactones and positional isomeric series of chlorogenic acids possessing caffeic and/or ferulic moieties. Among the 52 chromatographic peaks observed, 36 were fully identified and 8 were attributed to compounds belonging to series of caffeoylferuloylquinic and diferuloylquinic acids that could not be individualized from each other.

Development and validation of stability indicating HPLC methods for related substances and assay analyses of amoxicillin and potassium clavulanate mixtures.

PubMed

Bellur Atici, Esen; Yazar, Yücel; Ağtaş, Çağan; Ridvanoğlu, Nurten; Karlığa, Bekir

2017-03-20

Antibacterial combinations consisting of the semisynthetic antibiotic amoxicillin (amox) and the β-lactamase inhibitor potassium clavulanate (clav) are commonly used and several chromatographic methods were reported for their quantification in mixtures. In the present work, single HPLC method for related substances analyses of amoxicillin and potassium clavulanate mixtures was developed and validated according to international conference on harmonization (ICH) guidelines. Eighteen amoxicillin and six potassium clavulanate impurities were successfully separated from each other by using triple gradient elution using a Thermo Hypersil Zorbax BDS C18 (250 mm×4.6mm, 3μm) column with 50μL injection volumes at a wavelength of 215nm. Commercially unavailable impurities were formed by degradation of amoxicillin and potassium clavulanate, identified by LC-MS studies and used during analytical method development and validation studies. Also, process related amoxicillin impurity-P was synthesized and characterized by using nuclear magnetic resonance (NMR) and mass spectroscopy (MS) for the first time. As complementary of this work, an assay method for amoxicillin and potassium clavulanate mixtures was developed and validated; stress-testing and stability studies of amox/clav mixtures was carried out under specified conditions according to ICH and analyzed by using validated stability-indicating assay and related substances methods. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Effects of muffin processing on fumonisins from 14C-labeled toxins produced in cultured corn kernels.

PubMed

Avantaggiato, Giuseppina; De La Campa, Regina; Miller, J David; Visconti, Angelo

2003-10-01

The persistence of fumonisins during cooking is known to be affected by several factors, including thermal degradation and the presence of various ingredients in corn-based food recipes that can react with the toxin. A method for the production of corn kernels containing 14C-fumonisins was developed. The corn kernels were colonized by Fusarium verticillioides MRC 826 and supplemented with 1,2-14C-sodium acetate. The specific activity of 14C-FB1 produced made the study of its fate in cornmeal muffins possible. The double-extraction acetonitrile-water-methanol/immunoaffinity column/o-phthaldialdehyde high-performance liquid chromatography (HPLC) method was used to determine FB1 levels in cornmeal muffins. Reductions in FB1 levels in muffins spiked with 14C-labeled and unlabeled FB1 (43 and 48%, respectively) were similar, indicating that the extraction method was efficient and consistent with previous reports. However, with the labeled corn kernel material, recovery levels based on the 14C counts for the eluate from an immunoaffinity column were much higher (90%). This finding indicates that some fumonisin-related compounds other than FB1 that were present in the cornmeal were recognized by the antibodies but not by the HPLC method.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Flores, H. E.; Galston, A. W.; Weinstein, L. H.

1989-01-01

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

A high-performance liquid chromatography method for the serotonin release assay is equivalent to the radioactive method.

PubMed

Sono-Koree, N K; Crist, R A; Frank, E L; Rodgers, G M; Smock, K J

2016-02-01

The serotonin release assay (SRA) is considered the gold standard laboratory test for heparin-induced thrombocytopenia (HIT). The historic SRA method uses platelets loaded with radiolabeled serotonin to evaluate platelet activation by HIT immune complexes. However, a nonradioactive method is desirable. We report the performance characteristics of a high-performance liquid chromatography (HPLC) SRA method. We validated the performance characteristics of an HPLC-SRA method, including correlation with a reference laboratory using the radioactive method. Serotonin released from reagent platelets was quantified by HPLC using fluorescent detection. Results were expressed as % release and classified as positive, negative, or indeterminate based on previously published cutoffs. Serum samples from 250 subjects with suspected HIT were tested in the HPLC-SRA and with the radioactive method. Concordant classifications were observed in 230 samples (92%). Sera from 41 healthy individuals tested negative. Between-run imprecision studies showed standard deviation of <6 (% release) for positive, weak positive, and negative serum pools. Stability studies demonstrated stability after two freeze-thaw cycles or up to a week of refrigeration. The HPLC-SRA has robust performance characteristics, equivalent to the historic radioactive method, but avoids the complexities of working with radioactivity. © 2015 John Wiley & Sons Ltd.

A simple method for HPLC retention time prediction: linear calibration using two reference substances.

PubMed

Sun, Lei; Jin, Hong-Yu; Tian, Run-Tao; Wang, Ming-Juan; Liu, Li-Na; Ye, Liu-Ping; Zuo, Tian-Tian; Ma, Shuang-Cheng

2017-01-01

Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately. But the reference substances are costly. Thus, the relative retention (RR) method has been widely adopted in pharmacopoeias and literatures for characterizing HPLC behaviors of those reference substances unavailable. The problem is it is difficult to reproduce the RR on different columns due to the error between measured retention time (t R ) and predicted t R in some cases. Therefore, it is useful to develop an alternative and simple method for prediction of t R accurately. In the present study, based on the thermodynamic theory of HPLC, a method named linear calibration using two reference substances (LCTRS) was proposed. The method includes three steps, procedure of two points prediction, procedure of validation by multiple points regression and sequential matching. The t R of compounds on a HPLC column can be calculated by standard retention time and linear relationship. The method was validated in two medicines on 30 columns. It was demonstrated that, LCTRS method is simple, but more accurate and more robust on different HPLC columns than RR method. Hence quality standards using LCTRS method are easy to reproduce in different laboratories with lower cost of reference substances.

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.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

PubMed

Llambias, E B; Luo, J

1996-01-01

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

PubMed

Yang, Ting; Yang, Yi-Hua; Yang, Ju-Yun; Chen, Ben-Mei; Duan, Ju-Ping; Yu, Shu-Yi; Ouyang, Hong-Tao; Cheng, Jun-Ping; Chen, Yu-Xiang

2008-01-01

A HPLC-MS fingerprint method has been developed based on the consistent chromatographic features of the major chemical constituents among 10 batches of Hedyotis diffusa Willd. Chromatographic separation was conducted on a Hypersil-Keystone Hypurity C(18) column using methanol:water:acetic acid as the mobile phase. Major compounds, including oleanolic acid, ursolic acid and ferulic acid, were analysed by HPLC-MS. Their analysis was ascertained by comparison with data derived from the standard compounds. The HPLC-MS fingerprint was successfully applied to analyse and differentiate samples from different geographical origins, or processing methods. H. diffusa was well distinguished from Hedyotis chrysotricha by HPLC-MS. Therefore the establishment of fingerprint of H. diffusa is critical in assessing and controlling its overall quality.

Characterization of organic and conventional sweet basil leaves using chromatographic and flow-injection mass spectrometric (FIMS) fingerprints combined with principal component analysis

PubMed Central

Lu, Yingjian; Gao, Boyan; Chen, Pei; Charles, Denys; Yu, Liangli (Lucy)

2014-01-01

Sweet basil, Ocimum basilicum., is one of the most important and wildly used spices and has been shown to have antioxidant, antibacterial, and anti-diarrheal activities. In this study, high performance liquid chromatographic (HPLC) and flow-injection mass spectrometric (FIMS) fingerprinting techniques were used to differentiate organic and conventional sweet basil leaf samples. Principal component analysis (PCA) of the fingerprints indicated that both HPLC and FIMS fingerprints could effectively detect the chemical differences in the organic and conventional sweet basil leaf samples. This study suggested that the organic basil sample contained greater concentrations of almost all the major compounds than its conventional counterpart on a per same botanical weight basis. The FIMS method was able to rapidly differentiate the organic and conventional sweet basil leaf samples (1 min analysis time), whereas the HPLC fingerprints provided more information about the chemical composition of the basil samples with a longer analytical time. PMID:24518341

Characterisation of organic and conventional sweet basil leaves using chromatographic and flow-injection mass spectrometric (FIMS) fingerprints combined with principal component analysis.

PubMed

Lu, Yingjian; Gao, Boyan; Chen, Pei; Charles, Denys; Yu, Liangli Lucy

2014-07-01

Sweet basil, Ocimum basilicum, is one of the most important and wildly used spices and has been shown to have antioxidant, antibacterial, and anti-diarrheal activities. In this study, high performance liquid chromatographic (HPLC) and flow-injection mass spectrometric (FIMS) fingerprinting techniques were used to differentiate organic and conventional sweet basil leaf samples. Principal component analysis (PCA) of the fingerprints indicated that both HPLC and FIMS fingerprints could effectively detect the chemical differences in the organic and conventional sweet basil leaf samples. This study suggested that the organic basil sample contained greater concentrations of almost all the major compounds than its conventional counterpart on a per same botanical weight basis. The FIMS method was able to rapidly differentiate the organic and conventional sweet basil leaf samples (1min analysis time), whereas the HPLC fingerprints provided more information about the chemical composition of the basil samples with a longer analytical time. Copyright © 2014 Elsevier Ltd. All rights reserved.

A rapid stability-indicating, fused-core HPLC method for simultaneous determination of β-artemether and lumefantrine in anti-malarial fixed dose combination products

PubMed Central

2013-01-01

Background Artemisinin-based fixed dose combination (FDC) products are recommended by World Health Organization (WHO) as a first-line treatment. However, the current artemisinin FDC products, such as β-artemether and lumefantrine, are inherently unstable and require controlled distribution and storage conditions, which are not always available in resource-limited settings. Moreover, quality control is hampered by lack of suitable analytical methods. Thus, there is a need for a rapid and simple, but stability-indicating method for the simultaneous assay of β-artemether and lumefantrine FDC products. Methods Three reversed-phase fused-core HPLC columns (Halo RP-Amide, Halo C18 and Halo Phenyl-hexyl), all thermostated at 30°C, were evaluated. β-artemether and lumefantrine (unstressed and stressed), and reference-related impurities were injected and chromatographic parameters were assessed. Optimal chromatographic parameters were obtained using Halo RP-Amide column and an isocratic mobile phase composed of acetonitrile and 1mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 μl injection volume. Quantification was performed at 210 nm and 335 nm for β-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®. Results Both β-artemether and lumefantrine were separated from each other as well as from the specified and unspecified related impurities including degradants. A complete chromatographic run only took four minutes. Evaluation of the method, including a Plackett-Burman robustness verification within analytical QbD-principles, and real-life samples showed the method is suitable for quantitative assay purposes of both active pharmaceutical ingredients, with a mean recovery relative standard deviation (± RSD) of 99.7 % (± 0.7%) for β-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified β-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to β-artemether active pharmaceutical ingredient (API) itself. Conclusions A rapid, robust, precise and accurate stability-indicating, quantitative fused-core isocratic HPLC method was developed for simultaneous assay of β-artemether and lumefantrine. This method can be applied in the routine regulatory quality control of FDC products. The in-silico toxicological investigation using Derek Nexus® indicated that the overall toxicity risk for β-artemether-related impurities is comparable to that of β-artemether API. PMID:23631682

Rapid, cost-effective and accurate quantification of Yucca schidigera Roezl. steroidal saponins using HPLC-ELSD method.

PubMed

Tenon, Mathieu; Feuillère, Nicolas; Roller, Marc; Birtić, Simona

2017-04-15

Yucca GRAS-labelled saponins have been and are increasingly used in food/feed, pharmaceutical or cosmetic industries. Existing techniques presently used for Yucca steroidal saponin quantification remain either inaccurate and misleading or accurate but time consuming and cost prohibitive. The method reported here addresses all of the above challenges. HPLC/ELSD technique is an accurate and reliable method that yields results of appropriate repeatability and reproducibility. This method does not over- or under-estimate levels of steroidal saponins. HPLC/ELSD method does not require each and every pure standard of saponins, to quantify the group of steroidal saponins. The method is a time- and cost-effective technique that is suitable for routine industrial analyses. HPLC/ELSD methods yield a saponin fingerprints specific to the plant species. As the method is capable of distinguishing saponin profiles from taxonomically distant species, it can unravel plant adulteration issues. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Detection of malondialdehyde in processed meat products without interference from the ingredients.

PubMed

Jung, Samooel; Nam, Ki Chang; Jo, Cheorun

2016-10-15

Our aim was to develop a method for accurate quantification of malondialdehyde (MDA) in meat products. MDA content of uncured ground pork (Control); ground pork cured with sodium nitrite (Nitrite); and ground pork cured with sodium nitrite, sodium chloride, sodium pyrophosphate, maltodextrin, and a sausage seasoning (Mix) was measured by the 2-thiobarbituric acid (TBA) assay with MDA extraction by trichloroacetic acid (method A) and two high-performance liquid chromatography (HPLC) methods: i) HPLC separation of the MDA-dinitrophenyl hydrazine adduct (method B) and ii) HPLC separation of MDA (method C) after MDA extraction with acetonitrile. Methods A and B could not quantify MDA accurately in groups Nitrite and Mix. Nevertheless, MDA in groups Control, Nitrite, and Mix was accurately quantified by method C with good recovery. Therefore, direct MDA quantification by HPLC after MDA extraction with acetonitrile (method C) is useful for accurate measurement of MDA content in processed meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development and validation of chromatographic methods (HPLC and GC) for the determination of the active components (benzocaine, tyrothricin and menthol) of a pharmaceutical preparation.

PubMed

Ortiz-Boyer, F; Tena, M T; Luque de Castro, M D; Valcárcel, M

1995-10-01

Methods are reported for the determination of tyrothricin and benzocaine by HPLC and menthol by GC in the analysis of throat lozenges (tablets) containing all three compounds. After optimization of the variables involved in both HPLC and GC the methods have been characterized and validated according to the guidelines of the Spanish Pharmacopoeia, and applied to both the monitoring of the manufacturing process and the quality control of the final product.

Stability indicating simplified HPLC method for simultaneous analysis of resveratrol and quercetin in nanoparticles and human plasma.

PubMed

Kumar, Sandeep; Lather, Viney; Pandita, Deepti

2016-04-15

Resveratrol and quercetin are well-known polyphenolic compounds present in common foods, which have demonstrated enormous potential in the treatment of a wide variety of diseases. Owing to their exciting synergistic potential and combination delivery applications, we developed a simple and rapid RP-HPLC method based on isosbestic point detection. The separation was carried out on phenomenex Synergi 4μ Hydro-RP 80A column using methanol: acetonitrile (ACN): 0.1% phosphoric acid (60:10:30) as mobile phase. The method was able to quantify nanograms of analytes simultaneously on a single wavelength (269 nm), making it highly sensitive, rapid as well as economical. Additionally, forced degradation studies of resveratrol and quercetin were established and the method's applicability was evaluated on PLGA nanoparticles and human plasma. The analytes peaks were found to be well resolved in the presence of degradation products and excipients. The simplicity of the developed method potentializes its suitability for routine in vitro and in vivo analysis of resveratrol and quercetin. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

Screening of extraction methods for glycoproteins from jellyfish ( Rhopilema esculentum) oral-arms by high performance liquid chromatography

NASA Astrophysics Data System (ADS)

Ren, Guoyan; Li, Bafang; Zhao, Xue; Zhuang, Yongliang; Yan, Mingyan; Hou, Hu; Zhang, Xiukun; Chen, Li

2009-03-01

In order to select an optimum extraction method for the target glycoprotein (TGP) from jellyfish ( Rhopilema esculentum) oral-arms, a high performance liquid chromatography (HPLC)-assay for the determination of the TGP was developed. Purified target glycoprotein was taken as a standard glycoprotein. The results showed that the calibration curves for peak area plotted against concentration for TGP were linear ( r = 0.9984, y = 4.5895 x+47.601) over concentrations ranging from 50 to 400 mgL-1. The mean extraction recovery was 97.84% (CV2.60%). The fractions containing TGP were isolated from jellyfish ( R. esculentum) oral-arms by four extraction methods: 1) water extraction (WE), 2) phosphate buffer solution (PBS) extraction (PE), 3) ultrasound-assisted water extraction (UA-WE), 4) ultrasound-assisted PBS extraction (UA-PE). The lyophilized extract was dissolved in Milli-Q water and analyzed directly on a short TSK-GEL G4000PWXL (7.8 mm×300 mm) column. Our results indicated that the UA-PE method was the optimum extraction method selected by HPLC.

The Second SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE-2)

NASA Technical Reports Server (NTRS)

2005-01-01

Eight international laboratories specializing in the determination of marine pigment concentrations using high performance liquid chromatography (HPLC) were intercompared using in situ samples and a variety of laboratory standards. The field samples were collected primarily from eutrophic waters, although mesotrophic waters were also sampled to create a dynamic range in chlorophyll concentration spanning approximately two orders of magnitude (0.3 25.8 mg m-3). The intercomparisons were used to establish the following: a) the uncertainties in quantitating individual pigments and higher-order variables (sums, ratios, and indices); b) an evaluation of spectrophotometric versus HPLC uncertainties in the determination of total chlorophyll a; and c) the reduction in uncertainties as a result of applying quality assurance (QA) procedures associated with extraction, separation, injection, degradation, detection, calibration, and reporting (particularly limits of detection and quantitation). In addition, the remote sensing requirements for the in situ determination of total chlorophyll a were investigated to determine whether or not the average uncertainty for this measurement is being satisfied. The culmination of the activity was a validation of the round-robin methodology plus the development of the requirements for validating an individual HPLC method. The validation process includes the measurements required to initially demonstrate a pigment is validated, and the measurements that must be made during sample analysis to confirm a method remains validated. The so-called performance-based metrics developed here describe a set of thresholds for a variety of easily-measured parameters with a corresponding set of performance categories. The aggregate set of performance parameters and categories establish a) the overall performance capability of the method, and b) whether or not the capability is consistent with the required accuracy objectives.

Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

PubMed

Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

2015-06-01

A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC-ESI-MS/MS.

PubMed

Park, Ah Yeon; Park, So-Young; Lee, Jaehyun; Jung, Mihye; Kim, Jinwoong; Kang, Sam Sik; Youm, Jeong-Rok; Han, Sang Beom

2009-10-01

Rapid, simple and reliable HPLC/UV and LC-ESI-MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C(30) column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC-ESI-MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC-ESI-MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC-ESI-MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra- and inter-day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC-ESI-MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright (c) 2009 John Wiley & Sons, Ltd.

A Simple and Specific Stability- Indicating RP-HPLC Method for Routine Assay of Adefovir Dipivoxil in Bulk and Tablet Dosage Form.

PubMed

Darsazan, Bahar; Shafaati, Alireza; Mortazavi, Seyed Alireza; Zarghi, Afshin

2017-01-01

A simple and reliable stability-indicating RP-HPLC method was developed and validated for analysis of adefovir dipivoxil (ADV).The chromatographic separation was performed on a C 18 column using a mixture of acetonitrile-citrate buffer (10 mM at pH 5.2) 36:64 (%v/v) as mobile phase, at a flow rate of 1.5 mL/min. Detection was carried out at 260 nm and a sharp peak was obtained for ADV at a retention time of 5.8 ± 0.01 min. No interferences were observed from its stress degradation products. The method was validated according to the international guidelines. Linear regression analysis of data for the calibration plot showed a linear relationship between peak area and concentration over the range of 0.5-16 μg/mL; the regression coefficient was 0.9999and the linear regression equation was y = 24844x-2941.3. The detection (LOD) and quantification (LOQ) limits were 0.12 and 0.35 μg/mL, respectively. The results proved the method was fast (analysis time less than 7 min), precise, reproducible, and accurate for analysis of ADV over a wide range of concentration. The proposed specific method was used for routine quantification of ADV in pharmaceutical bulk and a tablet dosage form.

Comparison of on-site field measured inorganic arsenic in rice with laboratory measurements using a field deployable method: Method validation.

PubMed

Mlangeni, Angstone Thembachako; Vecchi, Valeria; Norton, Gareth J; Raab, Andrea; Krupp, Eva M; Feldmann, Joerg

2018-10-15

A commercial arsenic field kit designed to measure inorganic arsenic (iAs) in water was modified into a field deployable method (FDM) to measure iAs in rice. While the method has been validated to give precise and accurate results in the laboratory, its on-site field performance has not been evaluated. This study was designed to test the method on-site in Malawi in order to evaluate its accuracy and precision in determination of iAs on-site by comparing with a validated reference method and giving original data on inorganic arsenic in Malawian rice and rice-based products. The method was validated by using the established laboratory-based HPLC-ICPMS. Statistical tests indicated there were no significant differences between on-site and laboratory iAs measurements determined using the FDM (p = 0.263, ά = 0.05) and between on-site measurements and measurements determined using HPLC-ICP-MS (p = 0.299, ά = 0.05). This method allows quick (within 1 h) and efficient screening of rice containing iAs concentrations on-site. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparison of high-pressure liquid chromatography (HPLC) and Griess reagent-spectroscopic methods for the measurement of nitrate in serum from healthy individuals in the Nordic countries.

PubMed

Larsen, Tine Lise; Nilsen, Valentina; Andersen, Dag Olav; Francis, George; Rustad, Pål; Mansoor, Mohammad Azam

2008-12-01

Bioavailability of NO can be estimated by measuring the concentration of nitrate (NO(3)) in serum. However, the methods used for the measurement NO(3) in plasma or serum show a great degree of variation. Therefore, we compared two analytical methods for the measurement of NO(3) in serum. The concentration of NO(3) in 600 serum samples collected from healthy individuals was determined by the HPLC and by the Griess reagent-spectroscopic method. The concentration of NO(3) in the samples was 29.4+/-16.1 micromol/L and 26.2+/-14.0 micromol/L (mean+/-SD) measured by HPLC and Griess reagent-spectroscopic method respectively (p<0.0001). We detected a significant correlation between the two methods (R=0.81, p<0.0001). A significant correlation between the two methods may suggest that either method can be used for the measurement of NO(3) in serum, however the Griess reagent-spectroscopic method measures lower concentrations of NO(3) than the HPLC method.

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa

PubMed Central

Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

2015-01-01

Background: The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. Objectives: In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. Materials and Methods: A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. Results: The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Conclusions: Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the growth of P. aeruginosa PTCC 1430. The antimicrobial potency of this combination can be useful for designing and developing alternative therapeutic strategies against P. aeruginosa infections. PMID:26034539

Simultaneous column chromatographic extraction and purification of abscisic acid in peanut plants for direct HPLC analysis.

PubMed

Zhang, Ya-Wen; Fan, Wei-Wei; Li, Hui; Ni, He; Han, Han-Bing; Li, Hai-Hang

2015-10-01

Abscisic acid (ABA), a universal signaling molecule, plays important roles in regulating plant growth, development and stress responses. The low contents and complex components in plants make it difficult to be accurately analyzed. A novel one-step sample preparation method for ABA in plants was developed. Fresh peanut (Arachis hypogaea) plant materials were fixed by oven-drying, microwave drying, boiling or Carnoy's fixative, and loaded onto a mini-preparing column. After washed the impurities, ABA was eluted with a small amount of solvent. ABA in plant materials was completely extracted and purified in 2mL solution and directly analyzed by HPLC, with a 99.3% recovery rate. Multiple samples can be simultaneously prepared. Analyses using this method indicated that the endogenous ABA in oven-dried peanut leaves increased 20.2-fold from 1.01 to 20.37μgg(-1) dry weight within 12h and then decreased in 30% polyethylene glycol 6000 treated plants, and increased 3.34-fold from 0.85 to 2.84μgg(-1) dry weight in 5 days and then decreased in soil drought treated plants. The method combined the column chromatographic extraction and solid-phase separation technologies in one step and can completely extracted plant endogenous ABA in a purified and highly concentrated form for direct HPLC analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography.

PubMed

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-11-01

To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 °C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 μm) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min(-1) at 210 nm and 230 nm detection. The injection volume was 10 μL, and the separation was carried out isothermally at 30 °C in a heated chamber. The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants.

Analysis of aldehydes in human exhaled breath condensates by in-tube SPME-HPLC.

PubMed

Wang, ShuLing; Hu, Sheng; Xu, Hui

2015-11-05

In this paper, polypyrrole/graphene (PPy/G) composite coating was prepared by a facile electrochemical polymerization strategy on the inner surface of a stainless steel (SS) tube. Based on the coating tube, a novel online in-tube solid-phase microextraction -high performance liquid chromatography (IT-SPME-HPLC) was developed and applied for the extraction of aldehydes in the human exhaled breath condensates (EBC). The hybrid PPy/G nanocomposite exhibits remarkable chemical and mechanical stability, high selectivity, and satisfactory extraction performance toward aldehyde compounds. Moreover, the proposed online IT-SPME-HPLC method possesses numerous superiorities, such as time and cost saving, process simplicity, high precision and sensitivity. Some parameters related to extraction efficiency were optimized systematically. Under the optimal conditions, the recoveries of the aldehyde compounds at three spiked concentration levels varied in the range of 85%-117%. Good linearity was obtained with excellent correlation coefficients (R(2)) being larger than 0.994. The relative standard deviations (n = 5) of the method ranged from 1.8% to 11.3% and the limits of detection were between 2.3 and 3.3 nmol L(-1). The successful application of the proposed method in human EBC indicated that it is a promising approach for the determination of trace aldehyde metabolites in complex EBC samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Screening natural antioxidants in peanut shell using DPPH-HPLC-DAD-TOF/MS methods.

PubMed

Qiu, Jiying; Chen, Leilei; Zhu, Qingjun; Wang, Daijie; Wang, Wenliang; Sun, Xin; Liu, Xiaoyong; Du, Fangling

2012-12-15

Peanut shell, a byproduct in oil production, is rich in natural antioxidants. Here, a rapid and efficient method using DPPH-HPLC-DAD-TOF/MS was used for the first time to screen antioxidants in peanut shell. The method is based on the hypothesis that upon reaction with 1, 1-diphenyl-2-picrylhydrazyl (DPPH), the peak areas of compounds with potential antioxidant activities in the HPLC chromatogram will be significantly reduced or disappeared, and the identity confirmation could be achieved by HPLC-DAD-TOF/MS technique. With this method, three compounds possessing potential antioxidant activities were found abundantly in the methanolic extract of peanut shell. They were identified as 5,7-dihydroxychromone, eriodictyol, and luteolin. The contents of these compounds were 0.59, 0.92, and 2.36 mg/g, respectively, and luteolin possessed the strongest radical scavenging capacity. DPPH-HPLC-DAD-TOF/MS assay facilitated rapid identification and determination of natural antioxidants in peanut shell, which may be helpful for value-added utilization of peanut processing byproducts. Copyright © 2012 Elsevier Ltd. All rights reserved.

Repeatability Assessment by ISO 11843-7 in Quantitative HPLC for Herbal Medicines.

PubMed

Chen, Liangmian; Kotani, Akira; Hakamata, Hideki; Tsutsumi, Risa; Hayashi, Yuzuru; Wang, Zhimin; Kusu, Fumiyo

2015-01-01

We have proposed an assessment methods to estimate the measurement relative standard deviation (RSD) of chromatographic peaks in quantitative HPLC for herbal medicines by the methodology of ISO 11843 Part 7 (ISO 11843-7:2012), which provides detection limits stochastically. In quantitative HPLC with UV detection (HPLC-UV) of Scutellaria Radix for the determination of baicalin, the measurement RSD of baicalin by ISO 11843-7:2012 stochastically was within a 95% confidence interval of the statistically obtained RSD by repetitive measurements (n = 6). Thus, our findings show that it is applicable for estimating of the repeatability of HPLC-UV for determining baicalin without repeated measurements. In addition, the allowable limit of the "System repeatability" in "Liquid Chromatography" regulated in a pharmacopoeia can be obtained by the present assessment method. Moreover, the present assessment method was also successfully applied to estimate the measurement RSDs of quantitative three-channel liquid chromatography with electrochemical detection (LC-3ECD) of Chrysanthemi Flos for determining caffeoylquinic acids and flavonoids. By the present repeatability assessment method, reliable measurement RSD was obtained stochastically, and the experimental time was remarkably reduced.

Determination of Four Major Saponins in Skin and Endosperm of Seeds of Horse Chestnut (Aesculus Hippocastanum L.) Using High Performance Liquid Chromatography with Positive Confirmation by Thin Layer Chromatography

PubMed Central

Abudayeh, Zead Helmi Mahmoud; Al Azzam, Khaldun Mohammad; Naddaf, Ahmad; Karpiuk, Uliana Vladimirovna; Kislichenko, Viktoria Sergeevna

2015-01-01

Purpose: To separate and quantify four major saponins in the extracts of the skin and the endosperm of seeds of horse chestnut (Aesculus hippocastanum L.) using ultrasonic solvent extraction followed by a high performance liquid chromatography-diode array detector (HPLC-DAD) with positive confirmation by thin layer chromatography (TLC). Methods: The saponins: escin Ia, escin Ib, isoescin Ia and isoescin Ib were extracted using ultrasonic extraction method. The optimized extraction conditions were: 70% methanol as extraction solvent, 80 °C as extraction temperature, and the extraction time was achieved in 4 hours. The HPLC conditions used: Zorbax SB-ODS-(150 mm × 2.1 mm, 3 μm) column, acetonitrile and 0.10% phosphoric acid solution (39:61 v/v) as mobile phase, flow rate was 0.5 mL min−1 at 210 nm and 230 nm detection. The injection volume was 10 μL, and the separation was carried out isothermally at 30 °C in a heated chamber. Results: The results indicated that the developed HPLC method is simple, sensitive and reliable. Moreover, the content of escins in seeds decreased by more than 30% in endosperm and by more than 40% in skin upon storage for two years. Conclusion: This assay can be readily utilized as a quality control method for horse chestnut and other related medicinal plants. PMID:26819933

Comparison of a specific HPLC determination of toxic aconite alkaloids in processed Radix aconiti with a titration method of total alkaloids.

PubMed

Csupor, Dezso; Borcsa, Botond; Heydel, Barbara; Hohmann, Judit; Zupkó, István; Ma, Yan; Widowitz, Ute; Bauer, Rudolf

2011-10-01

In traditional Chinese medicine, Aconitum (Ranunculaceae) roots are only applied after processing. Nevertheless, several cases of poisoning by improperly processed aconite roots have been reported. The aim of this study was to develop a reliable analytical method to assess the amount of toxic aconite alkaloids in commercial aconite roots, and to compare this method with the commonly used total alkaloid content determination by titration. The content of mesaconitine, aconitine, and hypaconitine in 16 commercial samples of processed aconite roots was determined by an HPLC method and the total alkaloid content by indirect titration. Five samples were selected for in vivo toxicological investigation. In most of the commercial samples, toxic alkaloids were not detectable, or only traces were found. In four samples, we could detect >0.04% toxic aconite alkaloids, the highest with a content of 0.16%. The results of HPLC analysis were compared with the results obtained by titration, and no correlation was found between the two methods. The in vivo results reassured the validity of the HPLC determination. Samples with mesaconitine, aconitine, and hypaconitine content below the HPLC detection limit still contained up to 0.2% alkaloids determined by titration. Since titration of alkaloids gives no information selectively on the aconitine-type alkaloid content and toxicity of aconite roots this method is not appropriate for safety assessment. The HPLC method developed by us provides a quick and reliable assessment of toxicity and should be considered as a purity test in pharmacopoeia monographs.

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

High-speed counter-current chromatography coupled online to high performance liquid chromatography-diode array detector-mass spectrometry for purification, analysis and identification of target compounds from natural products.

PubMed

Liang, Xuejuan; Zhang, Yuping; Chen, Wei; Cai, Ping; Zhang, Shuihan; Chen, Xiaoqin; Shi, Shuyun

2015-03-13

A challenge in coupling high-speed counter-current chromatography (HSCCC) online with high performance liquid chromatography (HPLC) for purity analysis was their time incompatibility. Consequently, HSCCC-HPLC was conducted by either controlling HPLC analysis time and HSCCC flow rate or using stop-and-go scheme. For natural products containing compounds with a wide range of polarities, the former would optimize experimental conditions, while the latter required more time. Here, a novel HSCCC-HPLC-diode array detector-mass spectrometry (HSCCC-HPLC-DAD-MS) was developed for undisrupted purification, analysis and identification of multi-compounds from natural products. Two six-port injection valves and a six-port switching valve were used as interface for collecting key HSCCC effluents alternatively for HPLC-DAD-MS analysis and identification. The ethyl acetate extract of Malus doumeri was performed on the hyphenated system to verify its efficacy. Five main flavonoids, 3-hydroxyphloridzin (1), phloridzin (2), 4',6'-dihydroxyhydrochalcone-2'-O-β-D-glucopyranoside (3, first found in M. doumeri), phloretin (4), and chrysin (5), were purified with purities over 99% by extrusion elution and/or stepwise elution mode in two-step HSCCC, and 25mM ammonium acetate solution was selected instead of water to depress emulsification in the first HSCCC. The online system shortened manipulation time largely compared with off-line analysis procedure and stop-and-go scheme. The results indicated that the present method could serve as a simple, rapid and effective way to achieve target compounds with high purity from natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

The determination of antipyrine elimination in saliva by liquid chromatography.

PubMed

Gartzke, J; Jäger, H

1991-01-01

A simple, fast and reliable liquid chromatographic method for the determination of antipyrine in saliva is described. The elimination of antipyrine is a good indicator for general evaluation of the liver function for dispositional purposes for example in occupational and environmental medicine. The described LC method was compared with a more extensive photometric procedure. The results obtained from both methods show very good correlation. Only one measurement is necessary to determine the antipyrine clearance. Furthermore the antipyrine dosage can be minimized, because of the sensitivity of the HPLC-method.

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

PubMed Central

Yang, Yi; Zhao, Yongxin; Gu, Dongyu; Ayupbek, Amatjan; Huang, Yun; Dou, Jun; Ito, Yoichiro; Zhang, Tianyou; Aisa, Haji Akber

2010-01-01

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4′-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, 1H NMR and 13C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC. PMID:21494318

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

An in vitro AChE inhibition assay combined with UF-HPLC-ESI-Q-TOF/MS approach for screening and characterizing of AChE inhibitors from roots of Coptis chinensis Franch.

PubMed

Zhao, Hengqiang; Zhou, Siduo; Zhang, Minmin; Feng, Jinhong; Wang, Shanshan; Wang, Daijie; Geng, Yanling; Wang, Xiao

2016-02-20

In this study, an in vitro acetylcholinesterase (AChE) inhibition assay based on microplate reader combined with ultrafiltration high performance liquid chromatography-electrospray quadrupole time of flight mass (UF-HPLC-ESI-Q-TOF/MS) was developed for the rapid screening and identification of acetylcholinesterase inhibitors (AChEI) from roots of Coptis chinensis Franch. Incubation conditions such as enzyme concentration, incubation time, incubation temperature and co-solvent was optimized so as to get better screening results. Five alkaloids including columbamine, jatrorrhizine, coptisine, palmatine and berberine were found with AChE inhibition activity in the 80% ethanol extract of C. chinensis Franch. The screened compounds were identified by HPLC-DAD-ESI-Q-TOF/MS compared with the reference stands and literatures. The screened results were verified by in vitro AChE inhibition assays, palmatine showed the best AChE inhibitory activities with IC50 values of 36.6μM among the five compounds. Results of the present study indicated that the combinative method using in vitro AChE inhibition assay and UF-HPLC-ESI-Q-TOF/MS could be widely applied for rapid screening and identification of AChEI from complex TCM extract. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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.

Development of an isocratic HPLC method for catechin quantification and its application to formulation studies.

PubMed

Li, Danhui; Martini, Nataly; Wu, Zimei; Wen, Jingyuan

2012-10-01

The aim of this study was to develop a simple, rapid and accurate isocratic HPLC analytical method to qualify and quantify five catechin derivatives, namely (+)-catechin (C), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG). To validate the analytical method, linearity, repeatability, intermediate precision, sensitivity, selectivity and recovery were investigated. The five catechin derivatives were completely separated by HPLC using a mobile phase containing 0.1% TFA in Milli-Q water (pH 2.0) mixed with methanol at the volume ratio of 75:25 at a flow rate of 0.8 ml/min. The method was shown to be linear (r²>0.99), repeatable with instrumental precision<2.0 and intra-assay precision<2.5 (%CV, percent coefficient of variation), precise with intra-day variation<1 and inter-day variation<2.5 (%CV, percent coefficient of variation) and sensitive (LOD<1 μg/mL and LOQ<3 μg/mL) over the calibration range for all five derivatives. Derivatives could be fully recovered in the presence of niosomal formulation (recovery rates>91%). Selectivity of the method was proven by the forced degradation studies, which showed that under acidic, basic, oxidation temperature and photolysis stresses, the parent drug can be separated from the degradation products by means of this analytical method. The described method was successfully applied in the in vitro release studies of catechin-loaded niosomes to manifest its utility in formulation characterization. Obtained results indicated that the drug release from niosomal formulations was a biphasic process and a diffusion mechanism regulated the permeation of catechin niosomes. Copyright © 2012 Elsevier B.V. All rights reserved.

Identification and Structure Elucidation of Forced Degradation Products of the Novel Propionic acid Derivative Loxoprofen: Development of Stability-Indicating Chromatographic Methods Validated as per ICH Guidelines.

PubMed

Eissa, Maya S; Abd El-Sattar, Osama I

2017-04-01

Loxoprofen sodium (LOX) is a recently developed novel propionic acid derivative. Owing to its instability under both hydrolytic and oxidative conditions, the development of simple, rapid and sensitive methods for its determination in the presence of its possible forced degradation products becomes essential. Two simple chromatographic methods, high-performance thin layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC), were developed associated with ultraviolet (UV) detection. In HPTLC-densitometric method, the separation of LOX from its degradation products was achieved using silica gel F254 plates and toluene:acetone:acetic acid (1.8:1.0:0.1, v/v/v) as the developing system followed by densitometric scanning at 220 nm. In the HPLC-UV method, the separation was performed using isocratic elution system with acetonitrile: 0.15% triethylamine (pH 2.2) (50:50, v/v) on C18 analytical column. The flow rate was optimized at 1.0 mL·min-1 and UV detection was achieved at 220 nm. Validation was performed in accordance with the International Conference on Harmonization guidelines and the method was perfectly applied for determination of LOX in its pharmaceutical preparation. The results obtained were statistically compared to those obtained after application of the official HPLC method, where no significant difference was found incompliance with precision and accuracy. Identification and characterization of the possible hydrolytic degradation product under alkaline conditions and that produced during oxidative degradation using hydrogen peroxide were structurally elucidated using infrared and mass spectrometry analyses. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Quantitative determination and classification of energy drinks using near-infrared spectroscopy.

PubMed

Rácz, Anita; Héberger, Károly; Fodor, Marietta

2016-09-01

Almost a hundred commercially available energy drink samples from Hungary, Slovakia, and Greece were collected for the quantitative determination of their caffeine and sugar content with FT-NIR spectroscopy and high-performance liquid chromatography (HPLC). Calibration models were built with partial least-squares regression (PLSR). An HPLC-UV method was used to measure the reference values for caffeine content, while sugar contents were measured with the Schoorl method. Both the nominal sugar content (as indicated on the cans) and the measured sugar concentration were used as references. Although the Schoorl method has larger error and bias, appropriate models could be developed using both references. The validation of the models was based on sevenfold cross-validation and external validation. FT-NIR analysis is a good candidate to replace the HPLC-UV method, because it is much cheaper than any chromatographic method, while it is also more time-efficient. The combination of FT-NIR with multidimensional chemometric techniques like PLSR can be a good option for the detection of low caffeine concentrations in energy drinks. Moreover, three types of energy drinks that contain (i) taurine, (ii) arginine, and (iii) none of these two components were classified correctly using principal component analysis and linear discriminant analysis. Such classifications are important for the detection of adulterated samples and for quality control, as well. In this case, more than a hundred samples were used for the evaluation. The classification was validated with cross-validation and several randomization tests (X-scrambling). Graphical Abstract The way of energy drinks from cans to appropriate chemometric models.

Simultaneous determination of loxoprofen and its diastereomeric alcohol metabolites in human plasma and urine by a simple HPLC-UV detection method.

PubMed

Choo, K S; Kim, I W; Jung, J K; Suh, Y G; Chung, S J; Lee, M H; Shim, C K

2001-06-01

A simple, reliable HPLC-UV detection method was developed for the simultaneous determination of loxoprofen and its metabolites (i.e. trans- and cis-alcohol metabolites), in human plasma and urine samples. The method involves the addition of a ketoprofen (internal standard) solution in methanol, zinc sulfate solution and acetonitrile to plasma and urine samples, followed by centrifugation. An aliquot of the supernatant was evaporated to dryness, and the residue reconstituted in a mobile phase (acetonitrile:water=35:65 v/v, pH 3.0). An aliquot of the solution was then directly injected into the HPLC system. Separations were performed on octadecylsilica column (250x4.5 mm, 5 microm) with a guard column (3.2x1.5 cm, 7 microm) at ambient temperature. Loxoprofen and the metabolites in the eluent were monitored at 220 nm (a.u.f.s. 0.005). Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 10 and over 96%, respectively, in the 200 approximately 15000 ng ml(-1) range for plasma and 500 approximately 50000 ng ml(-1) range for urine. Calibration curves for all the compounds in the plasma and urine were linear over the above-mentioned concentration ranges with a common correlation coefficient of 0.999. The detection limit of the present method was 100 ng for all the compounds. These results indicate that the present method is very simple and readily applicable to routine bioavailability studies of these compounds with an acceptable sensitivity.

Evaluation of hemoglobin A1c measurement from filter paper using high-performance liquid chromatography and immunoturbidimetric assay.

PubMed

Wu, Yonghua; Yang, Xu; Wang, Haining; Li, Zhenrong; Wang, Tiancheng

2017-04-01

Glycated hemoglobin (HbA 1c ) measurement from whole blood (WB) samples is inconvenient for epidemic surveillance and self-monitoring of glycemic level. We evaluated HbA 1c measurement from WB blotted on filter paper (FP), which can be easily transported to central laboratories, with high-performance liquid chromatography (HPLC) and immunoturbidimetric assay (ITA). WB was applied to Whatman filter paper. By using HPLC and WB samples as reference methods, these FP samples were evaluated on HPLC and ITA. Inter- and intra-assay variation, WB vs. FP agreement and sample stability at 20-25 °C and -70 °C were assessed by statistical analysis. Results showed that the coefficient of variation (CV, %) of FP samples for HPLC and ITA were 0.44-1.02% and 1.47-2.72%, respectively (intra-assay); 2.13-3.56% and 3.21-4.82%, respectively (inter-assay). The correlation of WB HPLC with FP analyzed using HPLC and ITA are both significant (p < 0.001). Sample stability showed that FP method up to 5 days at 20-25 °C and 5 weeks at -70 °C is accurate and reproducible. In conclusion, FP samples analyzed by HPLC and ITA can both provide an alternative to WB for HbA 1c measurement, supporting the use of FP method in epidemic surveillance and healthcare units.

Validation of blood vitamin A concentrations in cattle: comparison of a new cow-side test (iCheck™ FLUORO) with high-performance liquid chromatography (HPLC).

PubMed

Raila, Jens; Kawashima, Chiho; Sauerwein, Helga; Hülsmann, Nadine; Knorr, Christoph; Myamoto, Akio; Schweigert, Florian J

2017-05-10

Plasma concentration of retinol is an accepted indicator to assess the vitamin A (retinol) status in cattle. However, the determination of vitamin A requires a time consuming multi-step procedure, which needs specific equipment to perform extraction, centrifugation or saponification prior to high-performance liquid chromatography (HPLC). The concentrations of retinol in whole blood (n = 10), plasma (n = 132) and serum (n = 61) were measured by a new rapid cow-side test (iCheck™ FLUORO) and compared with those by HPLC in two independent laboratories in Germany (DE) and Japan (JP). Retinol concentrations in plasma ranged from 0.033 to 0.532 mg/L, and in serum from 0.043 to 0.360 mg/L (HPLC method). No significant differences in retinol levels were observed between the new rapid cow-side test and HPLC performed in different laboratories (HPLC vs. iCheck™ FLUORO: 0.320 ± 0.047 mg/L vs. 0.333 ± 0.044 mg/L, and 0.240 ± 0.096 mg/L vs. 0.241 ± 0.069 mg/L, lab DE and lab JP, respectively). A similar comparability was observed when whole blood was used (HPLC vs. iCheck™ FLUORO: 0.353 ± 0.084 mg/L vs. 0.341 ± 0.064 mg/L). Results showed a good agreement between both methods based on correlation coefficients of r 2  = 0.87 (P < 0.001) and Bland-Altman blots revealed no significant bias for all comparison. With the new rapid cow-side test (iCheck™ FLUORO) retinol concentrations in cattle can be reliably assessed within a few minutes and directly in the barn using even whole blood without the necessity of prior centrifugation. The ease of the application of the new rapid cow-side test and its portability can improve the diagnostic of vitamin A status and will help to control vitamin A supplementation in specific vitamin A feeding regimes such as used to optimize health status in calves or meat marbling in Japanese Black cattle.

High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than one year.

PubMed

Khalil, M I; Sulaiman, S A; Gan, S H

2010-01-01

5-Hydroxymethylfurfural (HMF) content is an indicator of the purity of honey. High concentrations of HMF in honey indicate overheating, poor storage conditions and old honey. This study investigated the HMF content of nine Malaysian honey samples, as well as the correlation of HMF formation with physicochemical properties of honey. Based on the recommendation by the International Honey Commission, three methods for the determination of HMF were used: (1) high performance liquid chromatography (HPLC), (2) White spectrophotometry and (3) Winkler spectrophotometry methods. HPLC and White spectrophotometric results yielded almost similar values, whereas the Winkler method showed higher readings. The physicochemical properties of honey (pH, free acids, lactones and total acids) showed significant correlation with HMF content and may provide parameters that could be used to make quick assessments of honey quality. The HMF content of fresh Malaysian honey samples stored for 3-6 months (at 2.80-24.87 mg/kg) was within the internationally recommended value (80 mg/kg for tropical honeys), while honey samples stored for longer periods (12-24 months) contained much higher HMF concentrations (128.19-1131.76 mg/kg). Therefore, it is recommended that honey should generally be consumed within one year, regardless of the type. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

[Quality evaluation of Artemisiae Argyi Folium based on fingerprint analysis and quantitative analysis of multicomponents].

PubMed

Guo, Long; Jiao, Qian; Zhang, Dan; Liu, Ai-Peng; Wang, Qian; Zheng, Yu-Guang

2018-03-01

Artemisiae Argyi Folium, the dried leaves of Artemisia argyi, has been widely used in traditional Chinese and folk medicines for treatment of hemorrhage, pain, and skin itch. Phytochemical studies indicated that volatile oil, organic acid and flavonoids were the main bioactive components in Artemisiae Argyi Folium. Compared to the volatile compounds, the research of nonvolatile compounds in Artemisiae Argyi Folium are limited. In the present study, an accurate and reliable fingerprint approach was developed using HPLC for quality control of Artemisiae Argyi Folium. A total of 10 common peaks were marked，and the similarity of all the Artemisiae Argyi Folium samples was above 0.940. The established fingerprint method could be used for quality control of Artemisiae Argyi Folium. Furthermore, an HPLC method was applied for simultaneous determination of seven bioactive compounds including five organic acids and two flavonoids in Artemisiae Argyi Folium and Artemisiae Lavandulaefoliae Folium samples. Moreover, chemometrics methods such as hierarchical clustering analysis and principal component analysis were performed to compare and discriminate the Artemisiae Argyi Folium and Artemisiae Lavandulaefoliae Folium based on the quantitative data of analytes. The results indicated that simultaneous quantification of multicomponents coupled with chemometrics analysis could be a well-acceptable strategy to identify and evaluate the quality of Artemisiae Argyi Folium. Copyright© by the Chinese Pharmaceutical Association.

Development of high performance liquid chromatography method for miconazole analysis in powder sample

NASA Astrophysics Data System (ADS)

Hermawan, D.; Suwandri; Sulaeman, U.; Istiqomah, A.; Aboul-Enein, H. Y.

2017-02-01

A simple high performance liquid chromatography (HPLC) method has been developed in this study for the analysis of miconazole, an antifungal drug, in powder sample. The optimized HPLC system using C8 column was achieved using mobile phase composition containing methanol:water (85:15, v/v), a flow rate of 0.8 mL/min, and UV detection at 220 nm. The calibration graph was linear in the range from 10 to 50 mg/L with r 2 of 0.9983. The limit of detection (LOD) and limit of quantitation (LOQ) obtained were 2.24 mg/L and 7.47 mg/L, respectively. The present HPLC method is applicable for the determination of miconazole in the powder sample with a recovery of 101.28 % (RSD = 0.96%, n = 3). The developed HPLC method provides short analysis time, high reproducibility and high sensitivity.

Comparison of HPLC, UV spectrophotometry and potentiometric titration methods for the determination of lumefantrine in pharmaceutical products.

PubMed

da Costa César, Isabela; Nogueira, Fernando Henrique Andrade; Pianetti, Gérson Antônio

2008-09-10

This paper describes the development and evaluation of a HPLC, UV spectrophotometry and potentiometric titration methods to quantify lumefantrine in raw materials and tablets. HPLC analyses were carried out using a Symmetry C(18) column and a mobile phase composed of methanol and 0.05% trifluoroacetic acid (80:20), with a flow rate of 1.0ml/min and UV detection at 335nm. For the spectrophotometric analyses, methanol was used as solvent and the wavelength of 335nm was selected for the detection. Non-aqueous titration of lumefantrine was carried out using perchloric acid as titrant and glacial acetic acid/acetic anhydride as solvent. The end point was potentiometrically determined. The three evaluated methods showed to be adequate to quantify lumefantrine in raw materials, while HPLC and UV methods presented the most reliable results for the analyses of tablets.

[HPLC fingerprint analysis of flavonoids of phyllanthi fructus from different habitats].

PubMed

Wang, Fei; Wang, Shuai; Meng, Xian-sheng; Bao, Yong-rui; Zhu, Ying-huan

2014-11-01

To establish the HPLC fingerprint of flavonoids of Phyllanthi Fructus from different habitats. HPLC method was adopted. The flavonoids composition of Phyllanthi Fructus from 10 different habitats was determined on an Agilent C, chromatographic column with 0. 5% formic acid water (A)-acetonitrile (B) as the mobile phase in gradient elution under the wavelength of 254 nm. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus were established to evaluate the qualitiy of them. The HPLC fingerprints of flavonoids composition of Phyllanthi Fructus from 10 different habitats were established. 18 common peaks were found and the similarities of them were more than 0. 90 except the ones from Guangxi and Guangdong. The method is simple, accurate and repeatable. It can be used for research and quality control of the effective components in Phyllanthi Fructus.

Simultaneous qualitative and quantitative analysis of phenolic acids and flavonoids for the quality control of Apocynum venetum L. leaves by HPLC-DAD-ESI-IT-TOF-MS and HPLC-DAD.

PubMed

An, Haijuan; Wang, Hong; Lan, Yuexiang; Hashi, Yuki; Chen, Shizhong

2013-11-01

A reliable method based on high performance liquid chromatography-diode array detector-electrospray ionization-ion trap-time of flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS) was developed for the identification of phenolic acids and flavonoids in Apocynum venetum L. leaves and its adulterant, Pocynum hendersonii (Hook. f.) Woodson leaves. A total of 21 compounds were identified or tentatively identified, including 4 phenolic acids and 17 flavonoids. 3-O-caffeoylquinic acid (3-CQA) and caffeic acid were detected for the first time in A. venetum leaves; 4-O-caffeoylquinic acid (4-CQA), 3-CQA, caffeic acid, quercetin-3-O-(6"-O-malonyl)-galactoside, quercetin-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-malonyl)-glucoside, kaempferol-3-O-(6"-O-acetyl)-glucoside, and kaempferol-3-O-dihexoside were detected for the first time in P. hendersonii leaves. Cluster analysis was employed to analyze 24 batches of A. venetum leaves and 5 batches of P. hendersonii leaves collected from various regions in China. The analysis, which was based on the 21 compounds, indicated that profiles of these compounds were distinct between the two species, and among A. venetum leaf samples from different origins. 18 of these 21 compounds were selected as the markers and simultaneously analyzed by HPLC-DAD for the first time. The quantitative analytical method was validated and subsequently applied to the comprehensive quality evaluation of 24 batches of A. venetum leaves. © 2013 Elsevier B.V. All rights reserved.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

Quantitative Clinical Diagnostic Analysis of Acetone in Human Blood by HPLC: A Metabolomic Search for Acetone as Indicator

PubMed Central

Akgul Kalkan, Esin; Sahiner, Mehtap; Ulker Cakir, Dilek; Alpaslan, Duygu; Yilmaz, Selehattin

2016-01-01

Using high-performance liquid chromatography (HPLC) and 2,4-dinitrophenylhydrazine (2,4-DNPH) as a derivatizing reagent, an analytical method was developed for the quantitative determination of acetone in human blood. The determination was carried out at 365 nm using an ultraviolet-visible (UV-Vis) diode array detector (DAD). For acetone as its 2,4-dinitrophenylhydrazone derivative, a good separation was achieved with a ThermoAcclaim C18 column (15 cm × 4.6 mm × 3 μm) at retention time (t R) 12.10 min and flowrate of 1 mL min−1 using a (methanol/acetonitrile) water elution gradient. The methodology is simple, rapid, sensitive, and of low cost, exhibits good reproducibility, and allows the analysis of acetone in biological fluids. A calibration curve was obtained for acetone using its standard solutions in acetonitrile. Quantitative analysis of acetone in human blood was successfully carried out using this calibration graph. The applied method was validated in parameters of linearity, limit of detection and quantification, accuracy, and precision. We also present acetone as a useful tool for the HPLC-based metabolomic investigation of endogenous metabolism and quantitative clinical diagnostic analysis. PMID:27298750

Analyses of procyanidins in foods using Diol phase HPLC

USDA-ARS?s Scientific Manuscript database

Separation of procyanidins using silica-based HPLC suffered from poor resolution for higher oligomers and low sensitivity due to the fluorescence quenching effects of methylene chloride in the mobile phase. Optimization of a published Diol-phase HPLC method resulted in near baseline separation for p...

Dual Wavelength RP-HPLC Method for Simultaneous Determination of Two Antispasmodic Drugs: An Application in Pharmaceutical and Human Serum.

PubMed

Hasan, Najmul; Chaiharn, Mathurot; Khan, Sauleha; Khalid, Hira; Sher, Nawab; Siddiqui, Farhan Ahmed; Siddiqui, Muhammad Zain

2013-01-01

A reverse phase stability indicating HPLC method for simultaneous determination of two antispasmodic drugs in pharmaceutical parenteral dosage forms (injectable) and in serum has been developed and validated. Mobile phase ingredients consist of Acetonitrile : buffer : sulfuric acid 0.1 M (50 : 50 : 0.3 v/v/v), at flow rate 1.0 mL/min using a Hibar μ Bondapak ODS C18 column monitored at dual wavelength of 266 nm and 205 nm for phloroglucinol and trimethylphloroglucinol, respectively. The drugs were subjected to stress conditions of hydrolysis (oxidation, base, acid, and thermal degradation). Oxidation degraded the molecule drastically while there was not so much significant effect of other stress conditions. The calibration curve was linear with a correlation coefficient of 0.9999 and 0.9992 for PG and TMP, respectively. The drug recoveries fall in the range of 98.56% and 101.24% with 10 pg/mL and 33 pg/mL limit of detection and limit of quantification for both phloroglucinol and trimethylphloroglucinol. The method was validated in accordance with ICH guidelines and was applied successfully to quantify the amount of trimethylphloroglucinol and phloroglucinol in bulk, injectable form and physiological fluid. Forced degradation studies proved the stability indicating abilities of the method.

Quantification of Methylated Selenium, Sulfur, and Arsenic in the Environment

PubMed Central

Vriens, Bas; Ammann, Adrian A.; Hagendorfer, Harald; Lenz, Markus; Berg, Michael; Winkel, Lenny H. E.

2014-01-01

Biomethylation and volatilization of trace elements may contribute to their redistribution in the environment. However, quantification of volatile, methylated species in the environment is complicated by a lack of straightforward and field-deployable air sampling methods that preserve element speciation. This paper presents a robust and versatile gas trapping method for the simultaneous preconcentration of volatile selenium (Se), sulfur (S), and arsenic (As) species. Using HPLC-HR-ICP-MS and ESI-MS/MS analyses, we demonstrate that volatile Se and S species efficiently transform into specific non-volatile compounds during trapping, which enables the deduction of the original gaseous speciation. With minor adaptations, the presented HPLC-HR-ICP-MS method also allows for the quantification of 13 non-volatile methylated species and oxyanions of Se, S, and As in natural waters. Application of these methods in a peatland indicated that, at the selected sites, fluxes varied between 190–210 ng Se·m−2·d−1, 90–270 ng As·m−2·d−1, and 4–14 µg S·m−2·d−1, and contained at least 70% methylated Se and S species. In the surface water, methylated species were particularly abundant for As (>50% of total As). Our results indicate that methylation plays a significant role in the biogeochemical cycles of these elements. PMID:25047128

In Vitro Evaluation of Eslicarbazepine Delivery via Enteral Feeding Tubes.

PubMed

Reindel, Kristin; Zhao, Fang; Hughes, Susan; Dave, Vivek S

2017-12-01

Purpose: The feasibility of preparing an eslicarbazepine acetate suspension using Aptiom tablets for administration via enteral feeding tubes was evaluated. Methods: Eslicarbazepine acetate suspension (40 mg/mL) was prepared using Aptiom tablets after optimizing the tablet crushing methods and the vehicle composition. A stability-indicating high-performance liquid chromatography (HPLC) method was developed to monitor the eslicarbazepine stability in the prepared suspension. Three enteric feeding tubes of various composition and dimensions were evaluated for the delivery of the suspensions. The suspension was evaluated for the physical and chemical stability for 48 hours. Results: The reproducibility and consistency of particle size reduction was found to be best with standard mortar/pestle. The viscosity analysis and physical stability studies showed that ORA-Plus:water (50:50 v/v) was optimal for suspending ability and flowability of suspension through the tubes. The developed HPLC method was found to be stability indicating and suitable for the assay of eslicarbazepine acetate in the prepared suspension. The eslicarbazepine concentrations in separately prepared suspensions were within acceptable range (±3%), indicating accuracy and reproducibility of the procedure. The eslicarbazepine concentrations in suspensions before and after delivery through the enteric feeding tubes were within acceptable range (±4%), indicating absence of any physical/chemical interactions of eslicarbazepine with the tubes and a successful delivery of eslicarbazepine dosage via enteric feeding tubes. The stability study results showed that eslicarbazepine concentration in the suspension remained unchanged when stored at room temperature for 48 hours. Conclusion: The study presents a convenient procedure for the preparation of a stable suspension of eslicarbazepine acetate (40 mg/mL) using Aptiom tablets, for administration via enteral feeding tubes.

Simultaneous Determination of First-Line 4-FDC Antituberculosis Drugs by UHPLC-UV and HPLC-UV: A Comparative Study.

PubMed

Franco, Pedro H C; Chellini, Paula R; Oliveira, Marcone A L; Pianetti, Gerson A

2017-07-01

Tuberculosis is the second most deadly infectious disease, surpassed only by HIV/AIDS, and has resulted in over 1 billion deaths in the last 200 years. The World Health Organization estimates that in 2014, 9.6 million people were infected by this disease and 1.5 million had died. First-choice treatment consists of fixed-dose combination tablets containing rifampicin, isoniazid, pyrazinamide, and ethambutol hydrochloride (4-FDC). There are pharmacopeial protocols available to test 4-FDC, but they are prolonged, two-step methods. One single-step method in the literature performs the simultaneous determination by HPLC, but requires a long acquisition time. In this context, an ultra-HPLC (UHPLC) method was developed based on the HPLC method with the objective of reducing analysis time. A C18 column (1.9 µm particle size) was used with UV-diode-array detection at 238 and 282 nm. The method was found to be selective, linear, exact, precise, and robust. Samples from two batches were analyzed and the results compared with those obtained by the HPLC method, with no statistically significant differences observed (P > 0.05). This UHPLC method reduced the analysis time from 17 to 4 min, with a more than 90% reduction in sample and reagent consumption and a financial economy of almost 50-fold.

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

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.

Determination of vitamins D2 and D3 in selected food matrices by online high-performance liquid chromatography-gas chromatography-mass spectrometry (HPLC-GC-MS).

PubMed

Nestola, Marco; Thellmann, Andrea

2015-01-01

An online normal-phase liquid chromatography-gas chromatography-mass spectrometry (HPLC-GC-MS) method was developed for the determination of vitamins D2 and D3 in selected food matrices. Transfer of the sample from HPLC to GC was realized by large volume on-column injection; detection was performed with a time-of-flight mass spectrometer (TOF-MS). Typical GC problems in the determination of vitamin D such as sample degradation or sensitivity issues, previously reported in the literature, were not observed. Determination of total vitamin D content was done by quantitation of its pyro isomer based on an isotopically labelled internal standard (ISTD). Extracted ion traces of analyte and ISTD showed cross-contribution, but non-linearity of the calibration curve was not determined inside the chosen calibration range by selection of appropriate quantifier ions. Absolute limits of detection (LOD) and quantitation (LOQ) for vitamins D2 and D3 were calculated as approximately 50 and 150 pg, respectively. Repeatability with internal standard correction was below 2 %. Good agreement between quantitative results of an established high-performance liquid chromatography with UV detection (HPLC-UV) method and HPLC-GC-MS was found. Sterol-enriched margarine was subjected to HPLC-GC-MS and HPLC-MS/MS for comparison, because HPLC-UV showed strong matrix interferences. HPLC-GC-MS produced comparable results with less manual sample cleanup. In summary, online hyphenation of HPLC and GC allowed a minimization in manual sample preparation with an increase of sample throughput.

A New Improved RP-HPLC Method for Assay of Rosuvastatin Calcium in Tablets

PubMed Central

Kaila, H. O.; Ambasana, M. A.; Thakkar, R. S.; Saravaia, H. T.; Shah, A. K.

2010-01-01

A reliable and sensitive isocratic stability indicating RP-HPLC method has been developed and validated for assay of rosuvastatin calcium in tablets and for determination of content uniformity. An isocratic separation of rosuvastatin calcium was achieved on YMC C8, 150×4.6 mm i.d., 5 μm particle size columns with a flow rate of 1.5 ml/min and using a photodiode array detector to monitor the eluate at 242 nm. The mobile phase consisted of acetonitrile: water (40:60, v/v) pH 3.5 adjusted with phosphoric acid. The drug was subjected to oxidation, hydrolysis, photolysis and thermal degradation. All degradation products in an overall analytical run time of approximately 10 min with the parent compound rosuvastatin eluting at approximately 5.2 min. Response was a linear function of drug concentration in the range of 0.5-80 μg/ml (r2= 0.9993) with a limit of detection and quantification of 0.1 and 0.5 μg/ml respectively. Accuracy (recovery) was between 99.6 and 101.7%. Degradation products resulting from the stress studies did not interfere with the detection of rosuvastatin and the assay is thus stability-indicating. PMID:21694991

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.

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.

Effects of hemoglobin variants HbJ Bangkok, HbE, HbG Taipei, and HbH on analysis of glycated hemoglobin via ion-exchange high-performance liquid chromatography.

PubMed

Zhang, Xiu-Ming; Wen, Dong-Mei; Xu, Sheng-Nan; Suo, Ming-Huan; Chen, Ya-Qiong

2018-01-01

To explore the effects of HbJ Bangkok, HbE, HbG Taipei, and α-thalassemia HbH on the results of HbA1c assessment using ion-exchange high-performance liquid chromatography (IE-HPLC). We enrolled five patients in which the results of the IE-HPLC HbA1c assay were inconsistent with the average levels of FBG. We performed hemoglobin capillary (Hb) electrophoresis using whole-blood samples. We also sequenced the genes encoding Hb using dideoxy-mediated chain termination and analyzed HbA1c using borate affinity HPLC (BA-HPLC) and turbidimetric inhibition immunoassay (TINIA). Two patients had the HbJ Bangkok variant. Hb genotypes of these patients were β 41-42 /β J Bangkok and β N /β J Bangkok , and the content of HbJ Bangkok was 93.9% and 52.4%, respectively. The remaining three patients had the following: HbE (β N /β E Hb genotype, 23.6% HbE content), HbG Taipei (β N /β G Taipei Hb genotype, 39.4% HbG Taipei content), and α-thalassemia HbH (6.1% HbH content, 2.8% Hb Bart's content). In the patients with β-thalassemia and HbJ Bangkok variants, the presence of the variants interfered with the results of HbA1c analyses using IE-HPLC and TINIA; in the remaining four patients, there was interference with the results of HbA1c IE-HPLC but not with the TINIA assay. There was no interference with BA-HPLC HbA1c results. HbJ Bangkok, HbE, HbG Taipei Hb, and α-thalassemia HbH disease cause varying degrees of interference with the analysis of HbA1c using IE-HPLC. In these patients, we suggest using methods free from such interference for the analysis of HbA1c and other indicators to monitor blood glucose levels. © 2017 Wiley Periodicals, Inc.

Selenium speciation analysis of Misgurnus anguillicaudatus selenoprotein by HPLC-ICP-MS and HPLC-ESI-MS/MS

USDA-ARS?s Scientific Manuscript database

Analytical methods for selenium (Se) speciation were developed using high performance liquid chromatography (HPLC) coupled to either inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionization tandem mass spectrometry (ESI-MS/MS). Separations of selenomethionine (Se-Met) and sel...

Competitive Nitration of Benzene-Fluorobenzene and Benzene-Toluene Mixtures: Orientation and Reactivity Studies Using HPLC

ERIC Educational Resources Information Center

Blankespoor, Ronald L.; Hogendoorn, Stephanie; Pearson, Andrea

2007-01-01

The reactivity and orientation effects of a substituent are analyzed by using HPLC to determine the competitive nitration of the benzene-toluene and benzene-fluorobenzene mixtures. The results have shown that HPLC is an excellent instrumental method to use in analyzing these mixtures.

Characterization of Compounds in Psoralea corylifolia Using High-Performance Liquid Chromatography Diode Array Detection, Time-of-Flight Mass Spectrometry and Quadrupole Ion Trap Mass Spectrometry.

PubMed

Tan, Guangguo; Yang, Tiehong; Miao, Huayan; Chen, Hao; Chai, Yifeng; Wu, Hong

2015-10-01

High-performance liquid chromatography with diode array detection (HPLC-DAD), time-of-flight mass spectrometry (HPLC-TOFMS) and quadrupole ion trap mass spectrometry (HPLC-QITMS) were used for separation and identification of multi-components in Psoralea corylifolia. Benefiting from combining the accurate mass measurement of HPLC-TOFMS to generate elemental compositions, the complementary multilevel structural information provided by HPLC-QITMS and the characteristic UV spectra obtained from HPLC-DAD, 24 components in P. corylifolia were identified. The five groups of isomers were differentiated based on the fragmentation behaviors in QITMS and UV spectra. It can be concluded that an effective method based on the combination of HPLC-DAD, HPLC-TOFMS and HPLC-QITMS for identification of chemical components in P. corylifolia was established. The results provide essential data for further pharmacological and clinical studies of P. corylifolia and facilitate the rapid quality control of the crude drug. © Crown copyright 2015.

An HPLC/UV method for the determination of RGH-1756 in dog and rat plasma.

PubMed

Terjéki, E; Kapás, M

2001-03-01

RGH-1756 (1-(2-methoxy-phenyl)-4-(4-[4-(6-imidazo[2,1-b]-thiazolyl)-phenoxy]-butyl)-piperazine dimethansulphonate) is a novel atypical antipsychotic candidate of Gedeon Richter Ltd. A new HPLC method has been developed and validated for the quantitative determination of RGH-1756 in dog and rat plasma. The compound and the internal standard are extracted from the biological samples by a simple and fast liquid--liquid extraction method, using 1-chlorobutane. The recovery for RGH-1756 is about 90%. The extracts are analyzed by reversed phase HPLC (column: Supelcosil-LC-18-DB 250*4.6 mm, 5 microm, eluent:acetonitrile:methanol:0.2 molar ammonium-acetate 40:25:35, lambda=254 nm). The assay is specific for RGH-1756. The standard curves are linear in the range between 10 and 2000 ng ml(-1). The overall precision (expressed as CV%) and accuracy (expressed as bias%) of quality controls and calibration standards are within 15%. The validated lower limit of quantification is 10 ng/ml. No indications have been found for possible instabilities of RGH-1756 in plasma, in the extraction solvent, or after repeated thawing-freezing cycles. The method has been succesfully applied for the bioavailability studies of RGH-1756 in the two animal species. In these studies results of the inprocess method validation have shown the reliability of the method, too. CV% of quality controls in the rat study has been found between 7.4 and 10.0%, in the dog study between 4.1 and 12.5%. The bias has ranged from 0.4 to 3.8% and from -4.5 to 1.2% in the rat and dog study, respectively.

A simple and accurate HPLC method for fecal bile acid profile in healthy and cirrhotic subjects: validation by GC-MS and LC-MS[S

PubMed Central

Kakiyama, Genta; Muto, Akina; Takei, Hajime; Nittono, Hiroshi; Murai, Tsuyoshi; Kurosawa, Takao; Hofmann, Alan F.; Pandak, William M.; Bajaj, Jasmohan S.

2014-01-01

We have developed a simple and accurate HPLC method for measurement of fecal bile acids using phenacyl derivatives of unconjugated bile acids, and applied it to the measurement of fecal bile acids in cirrhotic patients. The HPLC method has the following steps: 1) lyophilization of the stool sample; 2) reconstitution in buffer and enzymatic deconjugation using cholylglycine hydrolase/sulfatase; 3) incubation with 0.1 N NaOH in 50% isopropanol at 60°C to hydrolyze esterified bile acids; 4) extraction of bile acids from particulate material using 0.1 N NaOH; 5) isolation of deconjugated bile acids by solid phase extraction; 6) formation of phenacyl esters by derivatization using phenacyl bromide; and 7) HPLC separation measuring eluted peaks at 254 nm. The method was validated by showing that results obtained by HPLC agreed with those obtained by LC-MS/MS and GC-MS. We then applied the method to measuring total fecal bile acid (concentration) and bile acid profile in samples from 38 patients with cirrhosis (17 early, 21 advanced) and 10 healthy subjects. Bile acid concentrations were significantly lower in patients with advanced cirrhosis, suggesting impaired bile acid synthesis. PMID:24627129

Current HPLC Methods for Assay of Nano Drug Delivery Systems.

PubMed

Tekkeli, Serife Evrim Kepekci; Kiziltas, Mustafa Volkan

2017-01-01

In nano drug formulations the mechanism of release is a critical process to recognize controlled and targeted drug delivery systems. In order to gain high bioavailability and specificity from the drug to reach its therapeutic goal, the active substance must be loaded into the nanoparticles efficiently. Therefore, the amount in biological fluids or tissues and the remaining amount in nano carriers are very important parameters to understand the potential of the nano drug delivery systems. For this aim, suitable and validated quantitation methods are required to determine released drug concentrations from nano pharmaceutical formulations. HPLC (High Performance Liquid Chromatography) is one of the most common techniques used for determination of released drug content out of nano drug formulations, in different physical conditions, over different periods of time. Since there are many types of HPLC methods depending on detector and column types, it is a challenge for the researchers to choose a suitable method that is simple, fast and validated HPLC techniques for their nano drug delivery systems. This review's goal is to compare HPLC methods that are currently used in different nano drug delivery systems in order to provide detailed and useful information for researchers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Assay of fluoxetine hydrochloride by titrimetric and HPLC methods.

PubMed

Bueno, F; Bergold, A M; Fröehlich, P E

2000-01-01

Two alternative methods were proposed to assay Fluoxetine Hydrochloride: a titrimetric method and another by HPLC using as mobile phase water pH 3.5: acetonitrile (65:35). These methods were applied to the determination of Fluoxetine as such or in formulations (capsules). The titrimetric method is an alternative for pharmacies and small industries. Both methods showed accuracy and precision and are an alternative to the official methods.

Development and Validation of HPLC-DAD and UHPLC-DAD Methods for the Simultaneous Determination of Guanylhydrazone Derivatives Employing a Factorial Design.

PubMed

Azevedo de Brito, Wanessa; Gomes Dantas, Monique; Andrade Nogueira, Fernando Henrique; Ferreira da Silva-Júnior, Edeildo; Xavier de Araújo-Júnior, João; Aquino, Thiago Mendonça de; Adélia Nogueira Ribeiro, Êurica; da Silva Solon, Lilian Grace; Soares Aragão, Cícero Flávio; Barreto Gomes, Ana Paula

2017-08-30

Guanylhydrazones are molecules with great pharmacological potential in various therapeutic areas, including antitumoral activity. Factorial design is an excellent tool in the optimization of a chromatographic method, because it is possible quickly change factors such as temperature, mobile phase composition, mobile phase pH, column length, among others to establish the optimal conditions of analysis. The aim of the present work was to develop and validate a HPLC and UHPLC methods for the simultaneous determination of guanylhydrazones with anticancer activity employing experimental design. Precise, exact, linear and robust HPLC and UHPLC methods were developed and validated for the simultaneous quantification of the guanylhydrazones LQM10, LQM14, and LQM17. The UHPLC method was more economic, with a four times less solvent consumption, and 20 times less injection volume, what allowed better column performance. Comparing the empirical approach employed in the HPLC method development to the DoE approach employed in the UHPLC method development, we can conclude that the factorial design made the method development faster, more practical and rational. This resulted in methods that can be employed in the analysis, evaluation and quality control of these new synthetic guanylhydrazones.

Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS.

PubMed

Liu, Shiming; Chen, Kaoshan; Schliemann, Willibald; Strack, Dieter

2005-01-01

A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.

A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

PubMed Central

Zhu, Hao-Jie; Wang, Jun-Sheng; Patrick, Kennerly S.; Donovan, Jennifer L.; DeVane, C. Lindsay; Markowitz, John S.

2007-01-01

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (λex: 330 nm, λem: 460 nm). The lower limit of determination was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were ≤ 9.10 % and ≤ 7.58 %, respectively. The accuracy ranged between 92.59 % and 103.06 %. The method was successfully applied to the pharmacokinetic study of a subject who received a single oral dose (0.3 mg/kg) of immediate-release MPH and yielded consistent results with that of the GC-MS method. This method is the first HPLC assay with non-MS detection providing sufficient reliability and sensitivity for both pre-clinical and clinical studies of MPH. PMID:17804308

Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong) leaves using HSCCC guided by DPPH-HPLC experiment.

PubMed

Dai, Xingping; Huang, Qiong; Zhou, Boting; Gong, Zhicheng; Liu, Zhaoqian; Shi, Shuyun

2013-08-15

Seven antioxidants were purified from Eucommia ulmoides Oliv. leaves using HSCCC guided by DPPH-HPLC experiment. HSCCC was successfully used to separate target antioxidants by three runs with different solvent systems after D101 column chromatography fractionation. Ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected as the optimum solvent system to purify geniposidic acid. Ethyl acetate-ethanol-water (4:1:5, v/v/v) was used to isolate caffeic acid, chlorogenic acid and ferulic acid. While three flavonoids, quercetin-3-O-sambubioside, rutin and isoquercitrin were purified by petroleum ether-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v). The structures were identified by MS and NMR. Antioxidant activities were assessed, and compounds 2-7 showed strong antioxidant activities. This is the first report about separation of antioxidants from E. ulmoides leaves by HSCCC. The results indicated that the combinative methods using DPPH-HPLC and HSCCC could be widely applied for screening and isolation of antioxidants from complex extracts. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Identification of chemical constituents in Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn].

PubMed

Wang, Ai-Hua; Ma, Li-Man; Fan, Shan-Shan; Liu, Guang-Xue; Xu, Feng; Shang, Ming-Ying; Cai, Shao-Qing

2018-01-01

This experiment was performed to analyze and identify the chemical constituents of Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn. The analysis was performed on an Agilent Zorbax SB-C₁₈ (4.6 mm×250 mm, 5 μm) column.The mobile phase consisted of 0.1% formic acid was used for gradient at a flow rate of 1.0 mL·min⁻¹. Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. The results indicated that 54 compounds consisted of 18 lignans and 36 flavonoids from Xiaoyelian had been detected by their HRMS data, the information of literature and reference substance. Among them, 27 compounds were reported in Sinopodophylli Fructus for the first time. In conclusion, an HPLC-DAD-ESI-IT-TOF-MSn method was established to qualitative analysis of Xiaoyelian in this study, which will provide the evidence for evaluating the quality of Xiaoyelian herbs, clarifying the mechanism, and guiding the development of pharmacological active ingredients. Copyright© by the Chinese Pharmaceutical Association.

Production of the antimicrobial peptides Caseicin A and B by Bacillus isolates growing on sodium caseinate.

PubMed

Kent, R M; Guinane, C M; O'Connor, P M; Fitzgerald, G F; Hill, C; Stanton, C; Ross, R P

2012-08-01

The aim of this study was to identify Bacillus isolates capable of degrading sodium caseinate and subsequently to generate bioactive peptides with antimicrobial activity. Sodium caseinate (2.5% w/v) was inoculated separately with 16 Bacillus isolates and allowed to ferment overnight. Protein breakdown in the fermentates was analysed using gel permeation-HPLC (GP-HPLC) and screened for peptides (<3-kDa) with MALDI-TOF mass spectrometry. Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR), two previously characterized antimicrobial peptides, were identified in the fermentates of both Bacillus cereus and Bacillus thuringiensis isolates. The caseicin peptides were subsequently purified by RP-HPLC and antimicrobial assays indicated that the peptides maintained the previously identified inhibitory activity against the infant formula pathogen Cronobacter sakazakii. We report a new method using Bacillus sp. to generate two previously characterized antimicrobial peptides from casein. This study highlights the potential to exploit Bacillus sp. or the enzymes they produce for the generation of bioactive antimicrobial peptides from bovine casein. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

PubMed

Dave, Mihika B; Dherai, Alpa J; Udani, Vrajesh P; Hegde, Anaita U; Desai, Neelu A; Ashavaid, Tester F

2018-01-01

Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects. © 2017 Wiley Periodicals, Inc.

HPLC & NMR-based forced degradation studies of ifosfamide: The potential of NMR in stability studies.

PubMed

Salman, D; Peron, J-M R; Goronga, T; Barton, S; Swinden, J; Nabhani-Gebara, S

2016-03-01

The aim of this study is to conduct a forced degradation study on ifosfamide under several stress conditions to investigate the robustness of the developed HPLC method. It also aims to provide further insight into the stability of ifosfamide and its degradation profile using both HPLC and NMR. Ifosfamide solutions (20mg/mL; n=15, 20mL) were stressed in triplicate by heating (70°C), under acidic (pH 1 & 4) and alkaline (pH 10 & 12) conditions. Samples were analysed periodically using HPLC and FT-NMR. Ifosfamide was most stable under weakly acidic conditions (pH 4). NMR results suggested that the mechanism of ifosfamide degradation involves the cleavage of the PN bond. For all stress conditions, HPLC was not able to detect ifosfamide degradation products that were detected by NMR. These results suggest that the developed HPLC method for ifosfamide did not detect the degradation products shown by NMR. It is possible that degradation products co-elute with ifosfamide, do not elute altogether or are not amenable to the detection method employed. Therefore, investigation of ifosfamide stability requires additional techniques that do not suffer from the aforementioned shortcomings. Copyright © 2015 Académie Nationale de Pharmacie. Published by Elsevier Masson SAS. All rights reserved.

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...

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

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

Development of an Advanced HPLC-MS/MS Method for the Determination of Carotenoids and Fat-Soluble Vitamins in Human Plasma.

PubMed

Hrvolová, Barbora; Martínez-Huélamo, Miriam; Colmán-Martínez, Mariel; Hurtado-Barroso, Sara; Lamuela-Raventós, Rosa Maria; Kalina, Jiří

2016-10-14

The concentration of carotenoids and fat-soluble vitamins in human plasma may play a significant role in numerous chronic diseases such as age-related macular degeneration and some types of cancer. Although these compounds are of utmost interest for human health, methods for their simultaneous determination are scarce. A new high pressure liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the quantification of selected carotenoids and fat-soluble vitamins in human plasma was developed, validated, and then applied in a pilot dietary intervention study with healthy volunteers. In 50 min, 16 analytes were separated with an excellent resolution and suitable MS signal intensity. The proposed HPLC-MS/MS method led to improvements in the limits of detection (LOD) and quantification (LOQ) for all analyzed compounds compared to the most often used HPLC-DAD methods, in some cases being more than 100-fold lower. LOD values were between 0.001 and 0.422 µg/mL and LOQ values ranged from 0.003 to 1.406 µg/mL, according to the analyte. The accuracy, precision, and stability met with the acceptance criteria of the AOAC (Association of Official Analytical Chemists) International. According to these results, the described HPLC-MS/MS method is adequately sensitive, repeatable and suitable for the large-scale analysis of compounds in biological fluids.

Automated measurement of carbohydrate-deficient transferrin using the Bio-Rad %CDT by the HPLC test on a Variant HPLC system: evaluation and comparison with other routine procedures.

PubMed

Schellenberg, François; Mennetrey, Louise; Girre, Catherine; Nalpas, Bertrand; Pagès, Jean Christophe

2008-01-01

In this study, we evaluated the new %CDT by the HPLC method (Bio-Rad, Germany) on a Varianttrade mark HPLC system (Bio-Rad), checked the correlation with well-known methods and calculated the diagnostic value of the test. Intra-run and day-to-day precision values were calculated for samples with extreme serum transferrin concentrations, high trisialotransferrin and interfering conditions (haemolysed, lactescent and icteric samples). The method was compared with two routine procedures, the %CDT TIA (Bio-Rad, Hercules, CA, USA) and the Capillarystrade mark CDT (Sebia, France). A total of 350 clinical sera samples were used for a case-control study. Precision values were better in high CDT and medium CDT pools than in low CDT pools. The serum transferrin concentration had no effect on CDT measurement, except in samples with serum transferrin <1 g/L. Haemolysis was the only interfering situation. The method showed high correlation (r(2) > 0.95) with the two other methods (%CDT TIA and CZE %CDT). The global predictive value of the test was >0.90 at 1.9% cut-off. These results demonstrate that the %CDT by the HPLC test is suitable for CDT routine measurement; the results from the high-throughput Varianttrade mark system are well correlated with other methods and are of high diagnostic value.

Comparative evaluation of three different ELISA assays and HPLC-ESI-ITMS/MS for the analysis of Nε-carboxymethyl lysine in food samples.

PubMed

Gómez-Ojeda, Armando; Jaramillo-Ortíz, Sarahi; Wrobel, Katarzyna; Wrobel, Kazimierz; Barbosa-Sabanero, Gloria; Luevano-Contreras, Claudia; de la Maza, Maria Pia; Uribarri, Jaime; Del Castillo, Ma Dolores; Garay-Sevilla, Ma Eugenia

2018-03-15

N ε -carboxymethyl-lysine (CML) is measured in food, but there is a controversy concerning the most convenient yet reliable method(s) for this task. This work compares three different ELISA assays and HPLC-ESI-ITMS/MS for the analysis of CML in several food items. The four methods showed the same decreasing order of CML concentration: beef, bacon>chicken > fish>dairy products>grain products>fruits/vegetables. HPLC-ESI-ITMS/MS results highly correlated with those obtained by ELISA performed with monoclonal CML-antibody (β=0.98, p<0.0001) whereas My Bio Source® kit results were not correlated with those provided by Lamider®. Small differences of CML concentrations in food items prepared by different culinary treatment were clearly distinguished by HPLC-ESI-ITMS/MS, but could not always be detected by ELISA. This work demonstrates a reasonable relationship between CM determined by ELISA and HPLC-ESI-ITMS/MS and therefore supports the implementation of ELISA in food CML/AGEs screening. Copyright © 2017. Published by Elsevier Ltd.

New Stability-Indicating RP-HPLC Method for Determination of Diclofenac Potassium and Metaxalone from their Combined Dosage Form

PubMed Central

Panda, Sagar Suman; Patanaik, Debasis; Ravi Kumar, Bera V. V.

2012-01-01

A simple, precise and accurate isocratic RP-HPLC stability-indicating assay method has been developed to determine diclofenac potassium and metaxalone in their combined dosage forms. Isocratic separation was achieved on a Hibar-C18, Lichrosphere-100® (250 mm × 4.6 mm i.d., particle size 5 μm) column at room temperature in isocratic mode, the mobile phase consists of methanol: water (80:20, v/v) at a flow rate of 1.0 ml/min, the injection volume was 20 μl and UV detection was carried out at 280nm. The drug was subjected to acid and alkali hydrolysis, oxidation, photolysis and heat as stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and system suitability. The method was linear in the drug concentration range of 2.5–30 μg/ml and 20–240 μg/ml for diclofenac potassium and metaxalone, respectively. The precision (RSD) of six samples was 0.83 and 0.93% for repeatability, and the intermediate precision (RSD) among six-sample preparation was 1.63 and 0.49% for diclofenac potassium and metaxalone, respectively. The mean recoveries were between 100.99–102.58% and 99.97–100.01% for diclofenac potassium and metaxalone, respectively. The proposed method can be used successfully for routine analysis of the drug in bulk and combined pharmaceutical dosage forms. PMID:22396909

Validated HPLC-Diode Array Detector Method for Simultaneous Evaluation of Six Quality Markers in Coffee.

PubMed

Gant, Anastasia; Leyva, Vanessa E; Gonzalez, Ana E; Maruenda, Helena

2015-01-01

Nicotinic acid, N-methylpyridinium ion, and trigonelline are well studied nutritional biomarkers present in coffee, and they are indicators of thermal decomposition during roasting. However, no method is yet available for their simultaneous determination. This paper describes a rapid and validated HPLC-diode array detector method for the simultaneous quantitation of caffeine, trigonelline, nicotinic acid, N-methylpyridinium ion, 5-caffeoylquinic acid, and 5-hydroxymethyl furfural that is applicable to three coffee matrixes: green, roasted, and instant. Baseline separation among all compounds was achieved in 30 min using a phenyl-hexyl RP column (250×4.6 mm, 5 μm particle size), 0.3% aqueous formic buffer (pH 2.4)-methanol mobile phase at a flow rate of 1 mL/min, and a column temperature at 30°C. The method showed good linear correlation (r2>0.9985), precision (less than 3.9%), sensitivity (LOD=0.023-0.237 μg/mL; LOQ=0.069-0.711 μg/mL), and recovery (84-102%) for all compounds. This simplified method is amenable for a more complete routine evaluation of coffee in industry.

Measurement of menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

Mammals convert phylloquinone to MK-4, with menadione as a possible intermediate. We developed and validated a method measuring urinary menadione. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed. The mobile...

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

PubMed Central

Zhu, Qinchang; Yu, Zhiqiang; Kabashima, Tsutomu; Yin, Sheng; Dragusha, Shpend; El-Mahdy, Ahmed F. M.; Ejupi, Valon; Shibata, Takayuki; Kai, Masaaki

2015-01-01

Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance. PMID:25988960

HPLC: Early and Recent Perspectives.

ERIC Educational Resources Information Center

Karger, Barry L.

1997-01-01

Provides a perspective on what it was like in the early days of high-performance liquid chromatography (HPLC) and several of the key developments. Focuses on the advances in HPLC generally, and more specifically for the biological sciences, that were necessary for the method to reach the preeminent stage of today. Contains 20 references. (JRH)

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.

High-performance liquid chromatography (HPLC) as a tool for monitoring the fate of fluazinam in soil.

PubMed

Hakala, Kati P; Tuomainen, Päivi M; Yli-Halla, Markku J; Hartikainen, Helinä

2014-01-01

Fluazinam is a widely used pesticide employed against the fungal disease late blight in potato cultivation. A specific, repeatable, and rapid high-performance liquid chromatography (HPLC) method utilizing a diode array detector (DAD) was developed to determine the presence of fluazinam in soil. The method consists of acetonitrile (ACN) extraction, clean-up with solid-phase extraction (SPE), and separation using a mobile phase consisting of 70% ACN and 30% water (v/v), including 0.02% acetic acid. HPLC was performed with a C18 column and the detection wavelength was 240 nm. The method was successfully applied to an incubation experiment and to soil samples taken from potato fields where fluazinam had been applied two to three times during the on-going growing season. In the 90-day incubation experiment, analytical standard fluazinam and the commercial fungicide Shirlan(®) were added to soil samples that had never been treated with fluazinam, and were then extracted with ACN and 0.01 M calcium chloride (CaCl2). Fluazinam was not extractable with CaCl2, indicating that it does not leach to watercourses in the dissolved form. Recovery with ACN extraction for sandy soils was 72-95% immediately after application and 53-73% after 90 days of incubation. Out of the eight potato field soil samples, fluazinam was found in two samples at concentrations of 2.1 mg kg(-1) and 1.9 mg kg(-1), well above the limit of quantification (0.1 mg kg(-1)).

Multi-step infrared macro-fingerprint features of ethanol extracts from different Cistanche species in China combined with HPLC fingerprint

NASA Astrophysics Data System (ADS)

Xu, Rong; Sun, Suqin; Zhu, Weicheng; Xu, Changhua; Liu, Yougang; Shen, Liang; Shi, Yue; Chen, Jun

2014-07-01

The genus Cistanche generally has four species in China, including C. deserticola (CD), C. tubulosa (CT), C. salsa (CS) and C. sinensis (CSN), among which CD and CT are official herbal sources of Cistanche Herba (CH). To clarify the sources of CH and ensure the clinical efficacy and safety, a multi-step IR macro-fingerprint method was developed to analyze and evaluate the ethanol extracts of the four species. Through this method, the four species were distinctively distinguished, and the main active components phenylethanoid glycosides (PhGs) were estimated rapidly according to the fingerprint features in the original IR spectra, second derivative spectra, correlation coefficients and 2D-IR correlation spectra. The exclusive IR fingerprints in the spectra including the positions, shapes and numbers of peaks indicated that constitutes of CD were the most abundant, and CT had the highest level of PhGs. The results deduced by some macroscopic features in IR fingerprint were in agreement with the HPLC fingerprint of PhGs from the four species, but it should be noted that the IR provided more chemical information than HPLC. In conclusion, with the advantages of high resolution, cost effective and speediness, the macroscopic IR fingerprint method should be a promising analytical technique for discriminating extremely similar herbal medicine, monitoring and tracing the constituents of different extracts and even for quality control of the complex systems such as TCM.

Monitoring of monosaccharides, oligosaccharides, ethanol and glycerol during wort fermentation by biosensors, HPLC and spectrophotometry.

PubMed

Monošík, Rastislav; Magdolen, Peter; Stredanský, Miroslav; Šturdík, Ernest

2013-05-01

The aim of the present study was to analyze sugar levels (namely maltose, maltotriose, glucose and fructose) and alcohols (ethanol and glycerol) during the fermentation process in wort samples by amperometric enzymatic biosensors developed by our research group for industrial application, HPLC and spectrophotometry, and to compare the suitability of the presented methods for determination of individual analytes. We can conclude that for the specific monitoring of maltose or maltotriose only the HPLC method was suitable. On the other hand, biosensors and spectrophotometry reflected a decrease in total sugar concentration better and were able to detect both glucose and fructose in the later stages of fermentation, while HPLC was not. This can be attributed to the low detection limits and good sensitivity of the proposed methods. For the ethanol and glycerol analysis all methods proved to be suitable. However, concerning the cost expenses and time analysis, biosensors represented the best option. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rapid NMR method for the quantification of organic compounds in thin stillage.

PubMed

Ratanapariyanuch, Kornsulee; Shen, Jianheng; Jia, Yunhua; Tyler, Robert T; Shim, Youn Young; Reaney, Martin J T

2011-10-12

Thin stillage contains organic and inorganic compounds, some of which may be valuable fermentation coproducts. This study describes a thorough analysis of the major solutes present in thin stillage as revealed by NMR and HPLC. The concentration of charged and neutral organic compounds in thin stillage was determined by excitation sculpting NMR methods (double pulse field gradient spin echo). Compounds identified by NMR included isopropanol, ethanol, lactic acid, 1,3-propanediol, acetic acid, succinic acid, glycerophosphorylcholine, betaine, glycerol, and 2-phenylethanol. The concentrations of lactic and acetic acid determined with NMR were comparable to those determined using HPLC. HPLC and NMR were complementary, as more compounds were identified using both methods. NMR analysis revealed that stillage contained the nitrogenous organic compounds betaine and glycerophosphorylcholine, which contributed as much as 24% of the nitrogen present in the stillage. These compounds were not observed by HPLC analysis.

Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry.

PubMed

Gamboa da Costa, Gonçalo; Marques, M Matilde; Beland, Frederick A; Freeman, James P; Churchwell, Mona I; Doerge, Daniel R

2003-03-01

The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts; the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial. The detection and quantitation of tamoxifen DNA adducts have relied primarily upon (32)P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry, being used to a much lesser extent. To expand the range of available analytical methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). alpha-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg alpha-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD(3))(2)]-alpha-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/10(9) nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The method was validated over the range of 8-1 520,000 adducts/10(8) nucleotides using 100 microg samples of DNA modified in vitro. Analysis of liver DNA from female Sprague-Dawley rats treated by gavage with seven daily doses of 20 mg tamoxifen/kg body weight gave a value of 496 +/- 16 adducts/10(8) nucleotides for (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen and 626 +/- 18 adducts/10(8) nucleotides for (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen. These data indicate that the HPLC-ES-MS/MS methodology has sufficient sensitivity and precision to be useful in the analysis of tamoxifen DNA adducts formed in vivo in experimental models and may be able to detect tamoxifen DNA adduct formation in human tissue samples.

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.

Stability indicating HPLC method for the estimation of oxycodone and lidocaine in rectal gel.

PubMed

Gebauer, M G; McClure, A F; Vlahakis, T L

2001-07-31

An HPLC method for the quantification of oxycodone and lidocaine in a gel matrix is described. The mobile phase consisted of methanol--water--acetic acid (35:15:1 v/v/v) and was delivered at 1.5 ml/min through a 4.6 x 250 mm Zorbax SB-C8 column. Oxycodone was detected at 285 nm and lidocaine at 264 nm. Linear calibration curves were obtained for oxycodone in the range of 0.05--1.5% (w/w) and for lidocaine in the range of 0.1--5.0% (w/w). Oxycodone and lidocaine were treated with hydrogen peroxide and the oxidation products were readily separated on the column. The method was applied to assess the stability of a gel containing oxycodone hydrochloride (0.3% w/w) and lidocaine (1.5% w/w). The gel was stored under refrigeration in ready-to-use syringes and under these conditions oxycodone and lidocaine were stable for at least 1 year. The gel is useful in the management of tenesmus in rectal cancer.

Determination of Trace Elements in Uranium by HPLC-ID-ICP-MS: NTNFC Final Report

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

Manard, Benjamin Thomas; Wylie, Ernest Miller II; Xu, Ning

This report covers the FY 16 effort for the HPLC-ID-ICP-MS methodology 1) sub-method validation for the group I&II elements, 2) sub-method stood-up and validation for REE, 3) sub-method development for the transition element, and 4) completion of a comprehensive SOP for three families of elements.

Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method.

PubMed

Çelik, S Esin; Özyürek, Mustafa; Güçlü, Kubilay; Çapanoğlu, Esra; Apak, Reşat

2014-01-01

Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. The separation and UV detection of polyphenols were performed on a C18 -column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (µg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower. Copyright © 2014 John Wiley & Sons, Ltd.

Harmful Algae Records in Venice Lagoon and in Po River Delta (Northern Adriatic Sea, Italy)

PubMed Central

Bilaničovà, Dagmar; Marcomini, Antonio

2014-01-01

A detailed review of harmful algal blooms (HAB) in northern Adriatic Sea lagoons (Po River Delta and Venice lagoon) is presented to provide “updated reference conditions” for future research and monitoring activities. In the study areas, the high mollusc production requires the necessity to identify better methods able to prevent risks for human health and socioeconomical interests. So, an integrated approach for the identification and quantification of algal toxins is presented by combining microscopy techniques with Liquid Chromatography coupled with High Resolution Time of Flight Mass Spectrometry (HPLC-HR-TOF-MS). The method efficiency was first tested on some samples from the mentioned coastal areas, where Dinophysis spp. occurred during summer in the sites directly affected by seawaters. Although cell abundance was always <200 cells/L, the presence of Pectenotoxin-2 (PTX2), detected by HPLC-HR-TOF-MS, indicated the potential release of detectable amounts of toxins even at low cell abundance. PMID:24683360

Optical waveguide lightmode spectroscopy technique-based immunosensor development for aflatoxin B1 determination in spice paprika samples.

PubMed

Majer-Baranyi, Krisztina; Zalán, Zsolt; Mörtl, Mária; Juracsek, Judit; Szendrő, István; Székács, András; Adányi, Nóra

2016-11-15

Optical waveguide lightmode spectroscopy (OWLS) technique has been applied to label-free detection of aflatoxin B1 in a competitive immunoassay format, with the aim to compare the analytical goodness of the developed OWLS immunosenor with HPLC and enzyme-linked immunosorbent assay (ELISA) methods for the detection of aflatoxin in spice paprika matrix. We have also assessed applicability of the QuEChERS method prior to ELISA measurements, and the results were compared to those obtained by traditional solvent extraction followed by immunoaffinity clean-up. The AFB1 content of sixty commercial spice paprika samples from different countries were measured with the developed and optimized OWLS immunosensor. Comparing the results from the indirect immunosensor to that obtained by HPLC or ELISA provided excellent correlation (with regression coefficients above 0.94) indicating that the competitive OWLS immunosensor has a potential for quick determination of aflatoxin B1 in paprika samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

Antioxidant capacities and total phenolic contents increase with gamma irradiation in two types of Malaysian honey.

PubMed

Hussein, Saba Zuhair; Yusoff, Kamaruddin Mohd; Makpol, Suzana; Yusof, Yasmin Anum Mohd

2011-07-27

Two types of monofloral Malaysian honey (Gelam and Nenas) were analyzed to determine their antioxidant activities and total phenolic and flavonoid contents, with and without gamma irradiation. Our results showed that both types of honey can scavenge free radicals and exhibit high antioxidant-reducing power; however, Gelam honey exhibited higher antioxidant activity (p < 0.05) than Nenas honey, which is in good correlation (r = 0.9899) with its phenolic contents. Interestingly, we also noted that both irradiated honeys have higher antioxidant activities and total phenolic and flavonoid contents compared to nonirradiated honeys by Folin-Ciocalteu and UV-spectrophotometry methods, respectively. However, HPLC analysis for phenolic compounds showed insignificant increase between irradiated and nonirradiated honeys. The phenolic compounds such as: caffeic acid, chlorogenic acid, ellagic acid, p- coumaric acid, quercetin and hesperetin as indicated by HPLC method were found to be higher in Gelam honey versus Nenas honey. In conclusion, irradiation of honey causes enhanced antioxidant activities and flavonoid compounds.

Rapid estimation of concentration of aromatic classes in middistillate fuels by high-performance liquid chromatography

NASA Technical Reports Server (NTRS)

Otterson, D. A.; Seng, G. T.

1985-01-01

An high performance liquid chromatography (HPLC) method to estimate four aromatic classes in middistillate fuels is presented. Average refractive indices are used in a correlation to obtain the concentrations of each of the aromatic classes from HPLC data. The aromatic class concentrations can be obtained in about 15 min when the concentration of the aromatic group is known. Seven fuels with a wide range of compositions were used to test the method. Relative errors in the concentration of the two major aromatic classes were not over 10 percent. Absolute errors of the minor classes were all less than 0.3 percent. The data show that errors in group-type analyses using sulfuric acid derived standards are greater for fuels containing high concentrations of polycyclic aromatics. Corrections are based on the change in refractive index of the aromatic fraction which can occur when sulfuric acid and the fuel react. These corrections improved both the precision and the accuracy of the group-type results.

N1, N14-diferuloylspermine as an antioxidative phytochemical contained in leaves of Cardamine fauriei.

PubMed

Abe, Keima; Matsuura, Hideyuki; Ukai, Mitsuko; Shimura, Hanako; Koshino, Hiroyuki; Suzuki, Takashi

2017-10-01

Most Brassicaceae vegetables are ideal dietary sources of antioxidants beneficial for human health. Cardamine fauriei (Ezo-wasabi in Japanese) is a wild, edible Brassicaceae herb native to Hokkaido, Japan. To clarify the main antioxidative phytochemical, an 80% methanol extraction from the leaves was fractionated with Diaion® HP-20, Sephadex® LH-20, and Sep-Pak® C18 cartridges, and the fraction with strong antioxidant activity depending on DPPH method was purified by HPLC. Based on the analyses using HRESIMS and MS/MS, the compound might be N 1 , N 14 -diferuloylspermine. This rare phenol compound was chemically synthesized, whose data on HPLC, MS and 1 H NMR were compared with those of naturally derived compound from C. fauriei. All results indicated they were the same compound. The radical-scavenging properties of diferuloylspermine were evaluated by ORAC and ESR spin trapping methods, with the diferuloylspermine showing high scavenging activities of the ROO · , O 2 ·- , and HO · radicals as was those of conventional antioxidants.

Discrimination of red and white rice bran from Indonesia using HPLC fingerprint analysis combined with chemometrics.

PubMed

Sabir, Aryani; Rafi, Mohamad; Darusman, Latifah K

2017-04-15

HPLC fingerprint analysis combined with chemometrics was developed to discriminate between the red and the white rice bran grown in Indonesia. The major component in rice bran is γ-oryzanol which consisted of 4 main compounds, namely cycloartenol ferulate, cyclobranol ferulate, campesterol ferulate and β-sitosterol ferulate. Separation of these four compounds along with other compounds was performed using C18 and methanol-acetonitrile with gradient elution system. By using these intensity variations, principal component and discriminant analysis were performed to discriminate the two samples. Discriminant analysis was successfully discriminated the red from the white rice bran with predictive ability of the model showed a satisfactory classification for the test samples. The results of this study indicated that the developed method was suitable as quality control method for rice bran in terms of identification and discrimination of the red and the white rice bran. Copyright © 2016 Elsevier Ltd. All rights reserved.

Harmful algae records in Venice lagoon and in Po River Delta (northern Adriatic Sea, Italy).

PubMed

Facca, Chiara; Bilaničovà, Dagmar; Pojana, Giulio; Sfriso, Adriano; Marcomini, Antonio

2014-01-01

A detailed review of harmful algal blooms (HAB) in northern Adriatic Sea lagoons (Po River Delta and Venice lagoon) is presented to provide "updated reference conditions" for future research and monitoring activities. In the study areas, the high mollusc production requires the necessity to identify better methods able to prevent risks for human health and socioeconomical interests. So, an integrated approach for the identification and quantification of algal toxins is presented by combining microscopy techniques with Liquid Chromatography coupled with High Resolution Time of Flight Mass Spectrometry (HPLC-HR-TOF-MS). The method efficiency was first tested on some samples from the mentioned coastal areas, where Dinophysis spp. occurred during summer in the sites directly affected by seawaters. Although cell abundance was always <200 cells/L, the presence of Pectenotoxin-2 (PTX2), detected by HPLC-HR-TOF-MS, indicated the potential release of detectable amounts of toxins even at low cell abundance.

Pharmacokinetic Comparative Study of Gastrodin and Rhynchophylline after Oral Administration of Different Prescriptions of Yizhi Tablets in Rats by an HPLC-ESI/MS Method

PubMed Central

Ge, Zhaohui; Liang, Qionglin; Wang, Yiming; Luo, Guoan

2014-01-01

Pharmacokinetic characters of rhynchophylline (RIN), gastrodin (GAS), and gastrodigenin (p-hydroxybenzyl alcohol, HBA) were investigated after oral administration of different prescriptions of Yizhi: Yizhi tablets or effective parts of tianma (total saponins from Gastrodiae, EPT) and gouteng (rhynchophylla alkaloids, EPG). At different predetermined time points after administration, the concentrations of GAS, HBA, and RIN in rat plasma were determined by an HPLC-ESI/MS method, and the main pharmacokinetic parameters were investigated. The results showed that the pharmacokinetic parameters C max and AUC0–∞ (P < 0.05) were dramatically different after oral administration of different prescriptions of Yizhi. The data indicated that the pharmacokinetic processes of GAS, HBA, and RIN in rats would interact with each other or be affected by other components in Yizhi. The rationality of the compatibility of Uncaria and Gastrodia elata as a classic “herb pair” has been verified from the pharmacokinetic viewpoint. PMID:25610474

Simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps and traditional Chinese medicine Radix angelicae pubescentis using excitation-emission matrix fluorescence coupled with second-order calibration method

NASA Astrophysics Data System (ADS)

Wang, Li; Wu, Hai-Long; Yin, Xiao-Li; Hu, Yong; Gu, Hui-Wen; Yu, Ru-Qin

2017-01-01

A chemometrics-assisted excitation-emission matrix (EEM) fluorescence method is presented for simultaneous determination of umbelliferone and scopoletin in Tibetan medicine Saussurea laniceps (SL) and traditional Chinese medicine Radix angelicae pubescentis (RAP). Using the strategy of combining EEM fluorescence data with second-order calibration method based on the alternating trilinear decomposition (ATLD) algorithm, the simultaneous quantification of umbelliferone and scopoletin in the two different complex systems was achieved successfully, even in the presence of potential interferents. The pretreatment is simple due to the "second-order advantage" and the use of "mathematical separation" instead of awkward "physical or chemical separation". Satisfactory results have been achieved with the limits of detection (LODs) of umbelliferone and scopoletin being 0.06 ng mL- 1 and 0.16 ng mL- 1, respectively. The average spike recoveries of umbelliferone and scopoletin are 98.8 ± 4.3% and 102.5 ± 3.3%, respectively. Besides, HPLC-DAD method was used to further validate the presented strategy, and t-test indicates that prediction results of the two methods have no significant differences. Satisfactory experimental results imply that our method is fast, low-cost and sensitive when compared with HPLC-DAD method.

A thin film degradation study of a fluorinated polyether liquid lubricant using an HPLC method

NASA Technical Reports Server (NTRS)

Morales, W.

1986-01-01

A High Pressure Liquid Chromatography (HPLC) separation method was developed to study and analyze a fluorinated polyether fluid which is promising liquid lubricant for future applications. This HPLC separation method was used in a preliminary study investigating the catalytic effect of various metal, metal alloy, and ceramic engineering materials on the degradation of this fluid in a dry air atmosphere at 345 C. Using a 440 C stainless steel as a reference catalytic material it was found that a titanium alloy and a chromium plated material degraded the fluorinated polyether fluid substantially more than the reference material.

Determination of Bortezomib in API Samples Using HPLC: Assessment of Enantiomeric and Diastereomeric Impurities.

PubMed

Kamalzadeh, Zahra; Babanezhad, Esmaeil; Ghaffari, Solmaz; Mohseni Ezhiyeh, Alireza; Mohammadnejad, Mahdieh; Naghibfar, Mehdi; Bararjanian, Morteza; Attar, Hossein

2017-08-01

A new, normal phase high performance liquid chromatography (NP-HPLC) method was developed for separation of Bortezomib (BZB) enantiomers and quantitative determination of (1S,2R)-enantiomer of BZB in active pharmaceutical ingredient (API) samples. The developed method was validated based on International Conference on Harmonisation (ICH) guidelines and it was proved to be accurate, precise and robust. The obtained resolution (RS) between the enantiomers was more than 2. The calibration curve for (1S,2R)-enantiomer was found to be linear in the concentration range of 0.24-5.36 mg/L with regression coefficient (R2) of 0.9998. Additionally, the limit of detection (LOD) and limit of quantification (LOQ) were 0.052 and 0.16 mg/L, respectively. Also, in this study, a precise, sensitive and robust gradient reversed-phase HPLC (RP-HPLC) method was developed and validated for determination of BZB in API samples. The detector response was linear over the concentration range of 0.26-1110.5 mg/L. The values of R2, LOD and LOQ were 0.9999, 0.084 and 0.25 mg/L, respectively. For both NP-HPLC and RP-HPLC methods, all of the RSD (%) values obtained in the precision study were <1.0%. System suitability parameters in terms of tailing factor (TF), number of theoretical plates (N) and RS were TF < 2.0, N > 2,000 and RS > 2.0. The performance of two common integration methods of valley to valley and drop perpendicular for drawing the baseline between two adjacent peaks were investigated for the determination of diastereomeric impurity (Imp-D) in the BZB-API samples. The results showed that the valley to valley method outperform the drop perpendicular method for calculation of Imp-D peak areas. Therefore, valley to valley method was chosen for peak integration. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

HepG2 cells biospecific extraction and HPLC-ESI-MS analysis for screening potential antiatherosclerotic active components in Bupeuri radix.

PubMed

Liu, Shuqiang; Tan, Zhibin; Li, Pingting; Gao, Xiaoling; Zeng, Yuaner; Wang, Shuling

2016-03-20

HepG2 cells biospecific extraction method and high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) analysis was proposed for screening of potential antiatherosclerotic active components in Bupeuri radix, a well-known Traditional Chinese Medicine (TCM). The hypothesis suggested that when cells are incubated together with the extracts of TCM, the potential bioactive components in the TCM should selectively combine with the receptor or channel of HepG2 cells, then the eluate which contained biospecific component binding to HepG2 cells was identified using HPLC-ESI-MS analysis. The potential bioactive components of Bupeuri radix were investigated using the proposed approach. Five compounds in the saikosaponins of Bupeuri radix were detected as these components selectively combined with HepG2 cells, among these compounds, two potentially bioactive compounds namely saikosaponin b1 and saikosaponin b2 (SSb2) were identified by comparing with the chromatography of the standard sample and analysis of the structural clearance characterization of MS. Then SSb2 was used to assess the uptake of DiI-high density lipoprotein (HDL) in HepG2 cells for antiatherosclerotic activity. The results have showed that SSb2, with indicated concentrations (5, 15, 25, and 40 μM) could remarkably uptake dioctadecylindocarbocyanine labeled- (DiI) -HDL in HepG2 cells (Vs control group, *P<0.01). In conclusion, the application of HepG2 biospecific extraction coupled with HPLC-ESI-MS analysis is a rapid, convenient, and reliable method for screening potential bioactive components in TCM and SSb2 may be a valuable novel drug agent for the treatment of atherosclerosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of chemical markers in Cordyceps sinensis by HPLC-MS/MS.

PubMed

Hu, Hankun; Xiao, Ling; Zheng, Baogen; Wei, Xin; Ellis, Alexis; Liu, Yi-Ming

2015-10-01

Authentication and quality assessment of Cordyceps sinensis, a precious and pricey natural product that offers a variety of health benefits, is highly significant. To identify effective chemical markers, authentic C. sinensis was thoroughly screened by using HPLC-MS/MS. In addition to many previously reported ingredients, two glycosides, i.e., cyclo-Ala-Leu-rhamnose and Phe-o-glucose, were detected for the first time in this material. Six ingredients detected, including cordycepin, D-mannitol, Phe, Phe-o-glucose, cyclo-Gly-Pro, and cyclo-Ala-Leu-rhamnose, were selected as a collection of chemical markers. An HPLC-MS/MS method was developed to simultaneously quantify them with sensitivity and specificity. The method had limits of detection ranging from 0.008 μg mL(-1) for cordycepin to 0.75 μg mL(-1) for cyclo-Gly-Pro. Recovery was found between 96 and 103 % in all tests. To evaluate the effectiveness of the marker collection proposed, five authentic C. sinensis samples and five samples of its substitutes were analyzed. Cordycepin, D-mannitol, and Phe were found present in all samples. The contents ranged from 0.0076 to 0.029 % (w/w) for cordycepin, 0.33 to 18.9 % for mannitol, and 0.0013 to 0.642 % for Phe. Interestingly, the two glycosides, Phe-o-glucose and cyclo-Ala-Leu-rhamnose, were detected only in authentic C. sinensis samples. These results indicated that the proposed protocol based on HPLC-MS/MS quantification of the markers might have a great potential in authentication and quality assessment of C. sinensis. Graphical abstract Chemical markers of C. sinensis identified in this work.

Reduction of iodate in iodated salt to iodide during cooking with iodine as measured by an improved HPLC/ICP-MS method.

PubMed

Liu, Liejun; Li, Xiuwei; Wang, Haiyan; Cao, Xiaoxiao; Ma, Wei

2017-04-01

Iodate is a strong oxidant, and some animal studies indicate that iodate intake may cause adverse effects. A key focus of the safety assessment of potassium iodate as a salt additive is determining whether iodate is safely reduced to iodide in food. To study the reduction of iodate in table salt to iodide and molecular iodine during cooking. Fifteen food samples cooked with and without iodated salt were prepared in duplicate. The iodine in the cooked food was extracted with deionized water. The iodine species in the extracts were determined by using an improved high-performance liquid chromatography/inductively coupled plasma-mass spectrometry (HPLC/ICP-MS). The cooking temperature and the pH of the food were determined. The conversion rate of iodate in iodated salt to iodide and molecular iodine was 96.4%±14.7% during cooking, with 86.8%±14.5% of the iodate converted to iodide ions and 9.6% ±6.2% converted to molecular iodine to lose. The limit of detection, limit of quantification, relative standard deviation and recovery rate of the method HPLC/ICP-MS were 0.70 μg/L for I - (0.69 μg/L for IO 3 - ), 2.10 μg/L for I - (2.06 μg/L for IO 3 - ), 2.6% and 101.6%±2.6%, respectively. Almost all iodate added to food was converted into iodide and molecular iodine during cooking. The improved HPLC/ICP-MS was reliable in the determination of iodine species in food extracts. Copyright © 2017 Elsevier Inc. All rights reserved.

Antioxidant components of Viburnum opulus L. determined by on-line HPLC-UV-ABTS radical scavenging and LC-UV-ESI-MS methods.

PubMed

Karaçelik, Ayça Aktaş; Küçük, Murat; İskefiyeli, Zeynep; Aydemir, Sezgin; De Smet, Seppe; Miserez, Bram; Sandra, Patrick

2015-05-15

Antioxidant activity of the juice and seed and skin extracts prepared with methanol, acetonitrile, and water of Viburnum opulus L. grown in Eastern Black Sea Region were studied with an on-line HPLC-ABTS method and off-line antioxidant methods, among which a linear positive correlation was observed. The fruit extracts were analysed with the HPLC-UV method optimised with 14 standard phenolics. Identification of the phenolic components in the juice was made using an HPLC-UV-ESI-MS method. Nineteen phenolic compounds in juice were identified by comparing the retention times and mass spectra with those of the standards and the phenolics reported in the literature. The major peaks in the juice belonged to coumaroyl-quinic acid, chlorogenic acid, procyanidin B2, and procyanidin trimer. Quite different antioxidant composition profiles were obtained from the extracts with the solvents of different polarities. The antioxidant activities of the seed extracts were higher than those of the skin extracts in general. Copyright © 2014 Elsevier Ltd. All rights reserved.

Determination and removal of antibiotics in secondary effluent using a horizontal subsurface flow constructed wetland.

PubMed

Zhang, Chunhui; Ning, Ke; Zhang, Wenwen; Guo, Yuanjie; Chen, Jun; Liang, Chen

2013-04-01

Increased attention is currently being directed towards the potential negative effects of antibiotics and other PPCPs discharged into the aquatic environment via municipal WWTP secondary effluents. A number of analytical methods, such as high performance liquid chromatography technologies, including a high performance liquid chromatography-fluorescence method (HPLC-FLD), high performance liquid chromatography-UV detection method (HPLC-UV) and high performance liquid chromatography-mass spectrometry method (HPLC-MS), have been suggested as determination technologies for antibiotic residues in water. In this study, we implement a HPLC-MS/MS combined method to detect and analyze antibiotics in WWTP secondary effluent and apply a horizontal subsurface flow constructed wetland (CW) as an advanced wastewater treatment for removing antibiotics in the WWTP secondary effluent. The results show that there were 2 macrolides, 2 quinolones and 5 sulfas in WWTP secondary effluent among all the 22 antibiotics considered. After the CW advanced treatment, the concentration removal efficiencies and removal loads of 9 antibiotics were 53-100% and 0.004-0.7307 μg m(-2) per day, respectively.

A PLS-based extractive spectrophotometric method for simultaneous determination of carbamazepine and carbamazepine-10,11-epoxide in plasma and comparison with HPLC

NASA Astrophysics Data System (ADS)

Hemmateenejad, Bahram; Rezaei, Zahra; Khabnadideh, Soghra; Saffari, Maryam

2007-11-01

Carbamazepine (CBZ) undergoes enzyme biotransformation through epoxidation with the formation of its metabolite, carbamazepine-10,11-epoxide (CBZE). A simple chemometrics-assisted spectrophotometric method has been proposed for simultaneous determination of CBZ and CBZE in plasma. A liquid extraction procedure was operated to separate the analytes from plasma, and the UV absorbance spectra of the resultant solutions were subjected to partial least squares (PLS) regression. The optimum number of PLS latent variables was selected according to the PRESS values of leave-one-out cross-validation. A HPLC method was also employed for comparison. The respective mean recoveries for analysis of CBZ and CBZE in synthetic mixtures were 102.57 (±0.25)% and 103.00 (±0.09)% for PLS and 99.40 (±0.15)% and 102.20 (±0.02)%. The concentrations of CBZ and CBZE were also determined in five patients using the PLS and HPLC methods. The results showed that the data obtained by PLS were comparable with those obtained by HPLC method.

ANALYSIS OF SELECTED PYRETHROID PESTICIDES USING REVERSE PHASE HIGH PRESSURE LIQUID CHROMATOGRAPHY/UV

EPA Science Inventory

This research was conducted in cooperation with EPA Region 4 in Athens, GA to develop a method to analyze selected pyrethroid pesticides using Reverse Phase-High Pressure Liquid Chromatography (HPLC). This HPLC method will aid researchers in separating and identifying these py...

40 CFR Appendix A to Subpart C of... - Alternative Testing Methods Approved for Analyses Under the Safe Drinking Water Act

Code of Federal Regulations, 2013 CFR

2013-07-01

... Chromatography/Mass Spectrometry (GC/MS) 525.3 24 Carbofuran High-performance liquid chromatography (HPLC) with... (HPLC) with Post-Column Derivatization and Fluorescence Detection 6651 B 6651 B 6651 B-00. Heptachlor... Spectrometry (GC/MS) 525.3 24 Oxamyl High-performance liquid chromatography (HPLC) with post-column...

Determination of Three Corticosteroids in the Biologic Matrix of Vitreous Humor by HPLC-tandem Mass Spectrometry: Method Development and Validation.

PubMed

Prieto, Esther; Vispe, Eugenio; Otín-Mallada, Sofía; Garcia-Martin, Elena; Polo-Llorens, Vicente; Fraile, José M; Pablo, Luis E; Mayoral, José A

2017-02-01

To develop a simple, specific, and rapid method to determine corticosteroid concentrations in vitreous humor. An analytical method based on high-pressure liquid chromatography-tandem mass spectrometry (HPLC-MS) with a simple extraction procedure was developed. New Zealand albino rabbits (n = 54) received a single (0.1 mL) intravitreal injection of dexamethasone (DXM, 0.1 mg), methylprednisolone (MP, 2 mg), or triamcinolone acetonide (TA, 10 mg). Eyes were enucleated and mean vitreous steroid levels were quantified at 12 h and 1, 2, 3, 7, and 14 days. Corticosteroids were extracted from the vitreous with acetonitrile, and TA was extracted with ethyl acetate, yielding high protein precipitation and clean solution samples. Vitreous samples were analyzed by isocratic HPLC-MS with mobile phase comprising acetonitrile and 2 mM ammonium formate buffer in water, pH 3.5. The linear range was 50-100,000 ng/g with a lower quantification limit of 45 ng/g for DXM and MP, and 50 ng/g for TA. Vitreous levels of DXM and MP were not detectable 14 days post-administration. Vitreous levels of TA were positive and stable throughout the study in both injected and control eyes. The HPLC-MS analytical method is an alternative to HPLC-MS/MS methods, sensitive enough for identifying and quantifying steroids in vitreous humor at a therapeutic dosage scale.

Chemical Differentiation of Dendrobium officinale and Dendrobium devonianum by Using HPLC Fingerprints, HPLC-ESI-MS, and HPTLC Analyses

PubMed Central

Ye, Zi; Dai, Jia-Rong; Zhang, Cheng-Gang; Lu, Ye; Wu, Lei-Lei; Gong, Amy G. W.; Wang, Zheng-Tao

2017-01-01

The stems of Dendrobium officinale Kimura et Migo (Dendrobii Officinalis Caulis) have a high medicinal value as a traditional Chinese medicine (TCM). Because of the limited supply, D. officinale is a high priced TCM, and therefore adulterants are commonly found in the herbal market. The dried stems of a closely related Dendrobium species, Dendrobium devonianum Paxt., are commonly used as the substitute; however, there is no effective method to distinguish the two Dendrobium species. Here, a high performance liquid chromatography (HPLC) method was successfully developed and applied to differentiate D. officinale and D. devonianum by comparing the chromatograms according to the characteristic peaks. A HPLC coupled with electrospray ionization multistage mass spectrometry (HPLC-ESI-MS) method was further applied for structural elucidation of 15 flavonoids, 5 phenolic acids, and 1 lignan in D. officinale. Among these flavonoids, 4 flavonoid C-glycosides were firstly reported in D. officinale, and violanthin and isoviolanthin were identified to be specific for D. officinale compared with D. devonianum. Then, two representative components were used as chemical markers. A rapid and reliable high performance thin layer chromatography (HPTLC) method was applied in distinguishing D. officinale from D. devonianum. The results of this work have demonstrated that these developed analytical methods can be used to discriminate D. officinale and D. devonianum effectively and conveniently. PMID:28769988

HPLC-based quantification of bacterial housekeeping nucleotides and alarmone messengers ppGpp and pppGpp.

PubMed

Varik, Vallo; Oliveira, Sofia Raquel Alves; Hauryliuk, Vasili; Tenson, Tanel

2017-09-08

Here we describe an HPLC-based method to quantify bacterial housekeeping nucleotides and the signaling messengers ppGpp and pppGpp. We have replicated and tested several previously reported HPLC-based approaches and assembled a method that can process 50 samples in three days, thus making kinetically resolved experiments feasible. The method combines cell harvesting by rapid filtration, followed by acid extraction, freeze-drying with chromatographic separation. We use a combination of C18 IPRP-HPLC (GMP unresolved and co-migrating with IMP; GDP and GTP; AMP, ADP and ATP; CTP; UTP) and SAX-HPLC in isocratic mode (ppGpp and pppGpp) with UV detection. The approach is applicable to bacteria without the requirement of metabolic labelling with 32P-labelled radioactive precursors. We applied our method to quantify nucleotide pools in Escherichia coli BW25113 K12-strain both throughout the growth curve and during acute stringent response induced by mupirocin. While ppGpp and pppGpp levels vary drastically (40- and ≥8-fold, respectively) these changes are decoupled from the quotients of the housekeeping pool and guanosine and adenosine housekeeping nucleotides: NTP/NDP/NMP ratio remains stable at 6/1/0.3 during both normal batch culture growth and upon acute amino acid starvation.

Quantitative Determination of Fluorinated Caffeic Acid Phenethyl Ester Derivative From Rat Blood Plasma by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

DTIC Science & Technology

2008-03-06

Caffeic acid phenethyl ester (CAPE), a plant-derived polyphenolic compound (Fig. 1), is a component of bee propolis . Propolis has been used as a folk...analytical methods have been documented. These include an HPLC-UV determination of CAPE from a propolis -containing gel [13], HPLC-ESI-MS measurement...of CAPE from crude propolis [14], and HPLC- ESI-MS/MS analysis of CAPE in biological samples [15]. In this paper, we developed a method using ultra

Quantification of maltol in Korean ginseng (Panax ginseng) products by high-performance liquid chromatography-diode array detector

PubMed Central

Jeong, Hyun Cheol; Hong, Hee-Do; Kim, Young-Chan; Rhee, Young Kyoung; Choi, Sang Yoon; Kim, Kyung-Tack; Kim, Sung Soo; Lee, Young-Chul; Cho, Chang-Won

2015-01-01

Background: Maltol, as a type of phenolic compounds, is produced by the browning reaction during the high-temperature treatment of ginseng. Thus, maltol can be used as a marker for the quality control of various ginseng products manufactured by high-temperature treatment including red ginseng. For the quantification of maltol in Korean ginseng products, an effective high-performance liquid chromatography-diode array detector (HPLC-DAD) method was developed. Materials and Methods: The HPLC-DAD method for maltol quantification coupled with a liquid-liquid extraction (LLE) method was developed and validated in terms of linearity, precision, and accuracy. An HPLC separation was performed on a C18 column. Results: The LLE methods and HPLC running conditions for maltol quantification were optimized. The calibration curve of the maltol exhibited good linearity (R2 = 1.00). The limit of detection value of maltol was 0.26 μg/mL, and the limit of quantification value was 0.79 μg/mL. The relative standard deviations (RSDs) of the data of the intra- and inter-day experiments were <1.27% and 0.61%, respectively. The results of the recovery test were 101.35–101.75% with an RSD value of 0.21–1.65%. The developed method was applied successfully to quantify the maltol in three ginseng products manufactured by different methods. Conclusion: The results of validation demonstrated that the proposed HPLC-DAD method was useful for the quantification of maltol in various ginseng products. PMID:26246746

Stability-indicating methods for the determination of piretanide in presence of the alkaline induced degradates.

PubMed

Youssef, Nadia F

2005-10-04

Stability-indicating high performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and first-derivative of ratio spectra (1DD) methods are developed for the determination of piretanide in presence of its alkaline induced degradates. HPLC method depends on separation of piretanide from its degradates on mu-Bondapak C18 column using methanol:water:acetic acid (70:30:1, v/v/v) as a mobile phase at flow rate 1.0 ml/min and UV detector at 275 nm. TLC densitometic method is based on the difference in Rf-values between the intact drug and its degradates on thin-layer silica gel. Iso-propanol:ammonia 33% (8:2, v/v) was used as a developing mobile phase and the chromatogram was scanned at 275 nm. The derivative of ratio spectra method (1DD) depends on the measurement of the absorbance at 288 nm in the first-derivative of ratio spectra for the determination of the cited drug in the presence of its degradates. Calibration graphs of the three suggested methods are linear in the concentration ranges 0.02-0.3 microg/20 microl, 0.5-10 microg/spot and 5-50 microg/ml, with mean percentage recovery 99.27+/-0.52, 99,17+/-1.01 and 99.65+/-1.01%, respectively. The three proposed methods were successfully applied for the determination of piretanide in bulk powder, laboratory-prepared mixtures and pharmaceutical dosage form with good accuracy and precision. The results were statistically analyzed and compared with those obtained by the official method. Validation of the method was determined with favourable specificity, linearity, precision, and accuracy was assessed by applying the standard addition technique.

Development of RP-HPLC, Stability Indicating Method for Degradation Products of Linagliptin in Presence of Metformin HCl by Applying 2 Level Factorial Design; and Identification of Impurity-VII, VIII and IX and Synthesis of Impurity-VII.

PubMed

Jadhav, Sushant B; Reddy, P Sunil; Narayanan, Kalyanaraman L; Bhosale, Popatrao N

2017-06-27

The novel reverse phase-high performance liquid chromatography (RP-HPLC), stability indicating method was developed for determination of linagliptin (LGP) and its related substances in linagliptin and metformin HCl (MET HCl) tablets by implementing design of experiment to understand the critical method parameters and their relation with critical method attributes; to ensure robustness of the method. The separation of nine specified impurities was achieved with a Zorbax SB-Aq 250 × 4.6 mm, 5 µm column, using gradient elution and a detector wavelength of 225 nm, and validated in accordance with International Conference on Harmonization (ICH) guidelines and found to be accurate, precise, reproducible, robust, and specific . The drug was found to be degrading extensively in heat, humidity, basic, and oxidation conditions and was forming degradation products during stability studies. After slight modification in the buffer and the column, the same method was used for liquid chromatography-mass spectrometry (LC-MS) and ultra-performance liquid chromatography -time-of-flight/mass spectrometry UPLC-TOF/MS analysis, to identify m/z and fragmentation of maximum unspecified degradation products i.e., Impurity-VII ( 7 ), Impurity-VIII ( 8 ), and Impurity-IX ( 9 ) formed during stability studies. Based on the results, a degradation pathway for the drug has been proposed and synthesis of Impurity-VII ( 7 ) is also discussed to ensure an in-depth understanding of LGP and its related degradation products and optimum performance during the lifetime of the product.

Characterisation of a major phytoplankton bloom in the River Thames (UK) using flow cytometry and high performance liquid chromatography.

PubMed

Moorhouse, H L; Read, D S; McGowan, S; Wagner, M; Roberts, C; Armstrong, L K; Nicholls, D J E; Wickham, H D; Hutchins, M G; Bowes, M J

2018-05-15

Recent river studies have observed rapid phytoplankton dynamics, driven by diurnal cycling and short-term responses to storm events, highlighting the need to adopt new high-frequency characterisation methods to understand these complex ecological systems. This study utilised two such analytical methods; pigment analysis by high performance liquid chromatography (HPLC) and cell counting by flow cytometry (FCM), alongside traditional chlorophyll spectrophotometry and light microscopy screening, to characterise the major phytoplankton bloom of 2015 in the River Thames, UK. All analytical techniques observed a rapid increase in chlorophyll a concentration and cell abundances from March to early June, caused primarily by a diatom bloom. Light microscopy identified a shift from pennate to centric diatoms during this period. The initial diatom bloom coincided with increased HPLC peridinin concentrations, indicating the presence of dinoflagellates which were likely to be consuming the diatom population. The diatom bloom declined rapidly in early June, coinciding with a storm event. There were low chlorophyll a concentrations (by both HPLC and spectrophotometric methods) throughout July and August, implying low biomass and phytoplankton activity. However, FCM revealed high abundances of pico-chlorophytes and cyanobacteria through July and August, showing that phytoplankton communities remain active and abundant throughout the summer period. In combination, these techniques are able to simultaneously characterise a wider range of phytoplankton groups, with greater certainty, and provide improved understanding of phytoplankton functioning (e.g. production of UV inhibiting pigments by cyanobacteria in response to high light levels) and ecological status (through examination of pigment degradation products). Combined HPLC and FCM analyses offer rapid and cost-effective characterisation of phytoplankton communities at appropriate timescales. This will allow a more-targeted use of light microscopy to capture phytoplankton peaks or to investigate periods of rapid community succession. This will lead to greater system understanding of phytoplankton succession in response to biogeochemical drivers. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Fruit Antioxidants during Vinegar Processing: Changes in Content and in Vitro Bio-Accessibility

PubMed Central

Bakir, Sena; Toydemir, Gamze; Boyacioglu, Dilek; Beekwilder, Jules; Capanoglu, Esra

2016-01-01

Background: Vinegars based on fruit juices could conserve part of the health-associated compounds present in the fruits. However, in general very limited knowledge exists on the consequences of vinegar-making on different antioxidant compounds from fruit. In this study vinegars derived from apple and grape are studied. Methods: A number of steps, starting from the fermentation of the fruit juices to the formation of the final vinegars, were studied from an industrial vinegar process. The effect of each of the vinegar processing steps on content of antioxidants, phenolic compounds and flavonoids was studied, by spectroscopic methods and by high-performance liquid chromatography (HPLC). Results: The major observation was that spectrophotometric methods indicate a strong loss of antioxidant phenolic compounds during the transition from fruit wine to fruit vinegar. A targeted HPLC analysis indicates that metabolites such as gallic acid are lost in later stages of the vinegar process. Conclusion: The major conclusion of this work is that major changes occur in phenolic compounds during vinegar making. An untargeted metabolite analysis should be used to reveal these changes in more detail. In addition, the effect of vinegar processing on bio-accessibility of phenolic compounds was investigated by mimicking the digestive tract in an in vitro set up. This study is meant to provide insight into the potential of vinegar as a source of health-related compounds from fruit. PMID:27690020

75 FR 22240 - Cyprodinil; Pesticide Tolerances

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-28

... chromatography with ultraviolet detector (HPLC/UV)) is available to enforce the tolerance expression on plant commodities. In addition, a high performance liquid chromatography with mass spectrometry (HPLC/MS) method...

HPLC determination of strychnine and brucine in rat tissues and the distribution study of processed semen strychni.

PubMed

Chen, Jun; Hou, Ting; Fang, Yun; Chen, Zhi-peng; Liu, Xiao; Cai, Hao; Lu, Tu-lin; Yan, Guo-jun; Cai, Bao-chang

2011-01-01

A simple and low-cost HPLC method with UV absorbance detection was developed and validated to simultaneously determine strychnine and brucine, the most abundant alkaloids in the processed Semen Strychni, in rat tissues (kidney, liver, spleen, lung, heart, stomach, small intestine, brain and plasma). The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. The LOQs were in the range of 0.039-0.050 µg/ml for different tissue or plasma samples. The extraction recoveries varied from 71.63 to 98.79%. The linear range was 0.05-2 µg/ml with correlation coefficient of over 0.991. The intra- and inter-day precision was less than 15%. Then the method was used to measure the tissue distribution of strychnine and brucine after intravenous administration of 1 mg/kg crude alkaloids fraction (CAF) extracted from the processed Semen Strychni. The results revealed that strychnine and brucine possessed similar tissue distribution characterization. The highest level was observed in kidney, while the lowest level was found in brain. It was indicated that kidney might be the primary excretion organ of prototype strychnine and brucine. It was also deduced that strychnine and brucine had difficulty in crossing the blood-brain barrier. Furthermore, no long-term accumulation of strychnine and brucine was found in rat tissues.

HPLC determination of olanzapine and carbamazepine in their nicotinamide cocrystals and investigation of the dissolution profiles of cocrystal tablet formulations.

PubMed

Renkoğlu, Pelin; Çelebier, Mustafa; Arıca-Yegin, Betül

2015-05-01

Cocrystals have recently gained importance in the pharmaceutical industry. In this study, olanzapine and carbamazepine cocrystals were synthesized by using nicotinamide as cocrystal forming agent to achieve improvements in the physicochemical characteristics of the active ingredients. An HPLC method was developed to determine the amount, thus, the stoichiometric ratios of olanzapine and carbamazepine in the synthesized cocrystals. Olanzapine:nicotinamide and olanzapine tablet formulations were prepared and the developed HPLC method was applied successfully in order to compare the dissolution profiles of these formulations. An ACE 5 CN, 25 cm × 4.6 mm, 5 µm column was used and a gradient elution program was performed for simultaneous determination of olanzapine, carbamazepine and nicotinamide. Phosphate buffer (pH 5.0, 25 mM) and methanol was used in a ratio from 80:20 to 70:30 while the flow rate was 1 mL min(-1) for the elution of the compounds within 12 min. In conclusion, two different aims were achieved, the first one was to indicate the stoichiometric ratios of the active ingredients olanzapine and carbamazepine with nicotinamide in their cocrystals, and the second one was the comparison of the dissolution profiles of the olanzapine and olanzapine:nicotinamide cocrystal formulations. It was found that the cocrystal formulation with nicotinamide improved the dissolution profile of olanzapine.

DETERMINATION OF CHLOROPHEONIS, NITROPHENOIS AND METHYLPHENOIS IN GROUND-WATER SAMPLES USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

EPA Science Inventory

A high performance liquid chromatography (HPLC) method was developed to quantitatively determine phenolic compounds and their isomers in aqueous samples. The HPLC method can analyze a mixture of 15 contaminants in the same analytical run with an analysis time of 25 minutes. The...

(PRESENT AT NCCU) ANALYSIS OF SELECTED PYRETHROID PESTICIDES USING REVERSE PHASE HIGH LIQUID CHROMATOGRAPHY

EPA Science Inventory

This research was conducted in cooperation with EPA Region 4 in Athens, GA to develop a method to analyze selected pyrethroid pesticides using Reverse Phase-High Pressure Liquid Chromatography (HPLC). This HPLC method will aid researchers in separating and identifying these pyre...

A SIMPLE HPLC METHOD FOR DETECTING CARBARYL AND 1-NAPHTHOL IN BIOLOGICAL TISSUES.

EPA Science Inventory

Carbamates are a class of pesticide used in both agricultural and residential applications. A simple HPLC method for detecting Carb and its metabolite 1-naphthol (Naph) in tissues was developed to try to correlate tissue levels of carbaryl (Carb) (a prototypical carbamate) with c...

DETERMINATION OF CHLOROPHENOLS, NITROPHENOLS, AND METHYLPHENOLS IN GROUND-WATER SAMPLES USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

EPA Science Inventory

A high performance liquid chromatography (HPLC) method was developed to quantitatively determine phenolic compounds and their isomers in aqueous samples. The HPLC method can analyze a mixture of 15 contaminants in the same analytical run with an analysis time of 25 minutes. The...

Comparative study of fourteen alkaloids from Uncaria rhynchophylla hooks and leaves using HPLC-diode array detection-atmospheric pressure chemical ionization/MS method.

PubMed

Qu, Jialin; Gong, Tianxing; Ma, Bin; Zhang, Lin; Kano, Yoshihiro; Yuan, Dan

2012-01-01

The purpose of the study is to compare alkaloid profile of Uncaria rhynchophylla hooks and leaves. Ten oxindole alkaloids and four glycosidic indole alkaloids were identified using HPLC-diode array detection (DAD) or LC-atmospheric pressure chemical ionization (APCI)-MS method, and a HPLC-UV method for simultaneous quantification of major alkaloids was validated. The hooks are characterized by high levels of four oxindole alkaloids rhynchophylline (R), isorhynchophylline (IR), corynoxeine (C) and isocorynoxeine (IC), while the leaves contained high level of two glycosidic indole alkaloids vincoside lactam (VL) and strictosidine (S). The presented methods have proven its usefulness in chemical characterization of U. rhynchophylla hooks and leaves.

Development and comparison of HPLC-MS/MS and UPLC-MS/MS methods for determining eight coccidiostats in beef.

PubMed

Zhao, Xia; Wang, Bo; Xie, Kaizhou; Liu, Jianyu; Zhang, Yangyang; Wang, Yajuan; Guo, Yawen; Zhang, Genxi; Dai, Guojun; Wang, Jinyu

2018-06-15

A high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for determining eight coccidiostat (halofuginone, lasalocid, maduramicin, monensin, narasin, nigericin, robenidine and salinomycin) residues in beef were developed and compared. Samples were extracted with a mixture of acetic acid, acetonitrile and ethyl acetate and were then purified on a C 18 solid-phase extraction (SPE) column. The purified samples were analyzed by HPLC-MS/MS and UPLC-MS/MS, using 0.1% formic acid-water solution (A) and pure methanol (B) as the mobile phase. The samples were fractionated on a C 18 column using different gradient elution procedures, followed by qualitative analysis using a mass spectrometer operated in multiple reaction monitoring (MRM) mode with positive electrospray ionization; the external standard method was used for quantitation. At spiked levels that ranged from the limit of quantification (LOQ) to 100 μg/kg, the average recoveries were 71.96%-100.32% and 71.24%-89.24%, with relative standard deviations (RSDs) of 2.65%-12.38% and 2.98%-14.86% for UPLC-MS/MS and HPLC-MS/MS, respectively. The limits of detection (LODs) and LOQs of the eight coccidiostats were 0.14-0.32 μg/kg and 0.43-1.21 μg/kg, respectively, for UPLC-MS/MS analysis and 0.16-0.58 μg/kg and 0.53-1.92 μg/kg, respectively, for HPLC-MS/MS analysis. Both methods had good accuracy and precision, but UPLC-MS/MS had higher sensitivity than HPLC-MS/MS. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of Biomass Sugars Using a Novel HPLC Method

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

Agblevor, F. A.; Hames, B. R.; Schell, D.

The precise quantitative analysis of biomass sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time consuming but the alternative high-performance liquid chromatography (HPLC) method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively andmore » there is excellent baseline resolution of all the sugars. This method was demonstrated for standard sugars, pretreated corn stover liquid and solid fractions. Our method can also be used to analyze dimeric sugars (cellobiose and sucrose).« less

Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa.

PubMed

Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

2015-04-01

The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the growth of P. aeruginosa PTCC 1430. The antimicrobial potency of this combination can be useful for designing and developing alternative therapeutic strategies against P. aeruginosa infections.

Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

PubMed Central

Duarte, Ana Joana; Vieira, Luis

2013-01-01

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. PMID:27335677

Metabolite fingerprinting of Camptotheca acuminata and the HPLC-ESI-MS/MS analysis of camptothecin and related alkaloids.

PubMed

Montoro, Paola; Maldini, Mariateresa; Piacente, Sonia; Macchia, Mario; Pizza, Cosimo

2010-01-20

The major phytochemical constituents, namely, alkaloids, flavonoids and ellagic acid derivatives, of leaves of Camptotheca acuminata were identified using high performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ESI-MS) in extracts of plants cultivated in Italy and collected at different growth stages. Alkaloids related to camptothecin were identified and quantified by HPLC coupled with ESI-tandem mass spectrometry (MS/MS) employing, respectively, an ion trap and a triple quadrupole mass analyser. The fragmentation patterns of alkaloids related to camptothecin were analysed and a specific Multiple Reaction Monitoring HPLC-MS/MS method was developed for the quantitative determination of these constituents. The described method provides high sensitivity and specificity for the characterisation and quantitative determination of the alkaloids in C. acuminata.

A validated stability-indicating HPLC method for determination of varenicline in its bulk and tablets

PubMed Central

2011-01-01

A simple, sensitive and accurate stability-indicating HPLC method has been developed and validated for determination of varenicline (VRC) in its bulk form and pharmaceutical tablets. Chromatographic separation was achieved on a Zorbax Eclipse XDB-C8 column (150 mm × 4.6 mm i.d., particle size 5 μm, maintained at ambient temperature) by a mobile phase consisted of acetonitrile and 50 mM potassium dihydrogen phosphate buffer (10:90, v/v) with apparent pH of 3.5 ± 0.1 and a flow rate of 1.0 ml/min. The detection wavelength was set at 235 nm. VRC was subjected to different accelerated stress conditions. The degradation products, when any, were well resolved from the pure drug with significantly different retention time values. The method was linear (r = 0.9998) at a concentration range of 2 - 14 μg/ml. The limit of detection and limit of quantitation were 0.38 and 1.11 μg/ml, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 2%. The accuracy of the method was proved; the mean recovery of VRC was 100.10 ± 1.08%. The proposed method has high throughput as the analysis involved short run-time (~ 6 min). The method met the ICH/FDA regulatory requirements. The proposed method was successfully applied for the determination of VRC in bulk and tablets with acceptable accuracy and precisions; the label claim percentages were 99.65 ± 0.32%. The results demonstrated that the method would have a great value when applied in quality control and stability studies for VRC. PMID:21672253

40 CFR Appendix A to Subpart C of... - Alternative Testing Methods Approved for Analyses Under the Safe Drinking Water Act

Code of Federal Regulations, 2014 CFR

2014-07-01

... Spectrometry (GC/MS) 525.3 24 Carbofuran High-performance liquid chromatography (HPLC) with post-column... (HPLC) with Post-Column Derivatization and Fluorescence Detection 6651 B 6651 B 6651 B-00, B-05... Chromatography/Mass Spectrometry (GC/MS) 525.3 24 Oxamyl High-performance liquid chromatography (HPLC) with post...

46 CFR Appendix D to Subpart C of... - Sampling and Analytical Methods for Benzene Monitoring-Measurement Procedures

Code of Federal Regulations, 2013 CFR

2013-10-01

... samples are analyzed directly by high performance liquid chromatography (HPLC). Detection limits: 0.01% by... proper selection of HPLC parameters. 2.4. Samples must be free of any particulates that may clog the... clarification kit. 3. Apparatus 3.1. Liquid chromatograph equipped with a UV detector. 3.2. HPLC Column that...

46 CFR Appendix D to Subpart C of... - Sampling and Analytical Methods for Benzene Monitoring-Measurement Procedures

Code of Federal Regulations, 2012 CFR

2012-10-01

... samples are analyzed directly by high performance liquid chromatography (HPLC). Detection limits: 0.01% by... proper selection of HPLC parameters. 2.4. Samples must be free of any particulates that may clog the... clarification kit. 3. Apparatus 3.1. Liquid chromatograph equipped with a UV detector. 3.2. HPLC Column that...

46 CFR Appendix D to Subpart C of... - Sampling and Analytical Methods for Benzene Monitoring-Measurement Procedures

Code of Federal Regulations, 2014 CFR

2014-10-01

... samples are analyzed directly by high performance liquid chromatography (HPLC). Detection limits: 0.01% by... proper selection of HPLC parameters. 2.4. Samples must be free of any particulates that may clog the... clarification kit. 3. Apparatus 3.1. Liquid chromatograph equipped with a UV detector. 3.2. HPLC Column that...

Ion chromatography for the precise analysis of chloride and sodium in sweat for the diagnosis of cystic fibrosis.

PubMed

Doorn, J; Storteboom, T T R; Mulder, A M; de Jong, W H A; Rottier, B L; Kema, I P

2015-07-01

Measurement of chloride in sweat is an essential part of the diagnostic algorithm for cystic fibrosis. The lack in sensitivity and reproducibility of current methods led us to develop an ion chromatography/high-performance liquid chromatography (IC/HPLC) method, suitable for the analysis of both chloride and sodium in small volumes of sweat. Precision, linearity and limit of detection of an in-house developed IC/HPLC method were established. Method comparison between the newly developed IC/HPLC method and the traditional Chlorocounter was performed, and trueness was determined using Passing Bablok method comparison with external quality assurance material (Royal College of Pathologists of Australasia). Precision and linearity fulfill criteria as established by UK guidelines are comparable with inductively coupled plasma-mass spectrometry methods. Passing Bablok analysis demonstrated excellent correlation between IC/HPLC measurements and external quality assessment target values, for both chloride and sodium. With a limit of quantitation of 0.95 mmol/L, our method is suitable for the analysis of small amounts of sweat and can thus be used in combination with the Macroduct collection system. Although a chromatographic application results in a somewhat more expensive test compared to a Chlorocounter test, more accurate measurements are achieved. In addition, simultaneous measurements of sodium concentrations will result in better detection of false positives, less test repeating and thus faster and more accurate and effective diagnosis. The described IC/HPLC method, therefore, provides a precise, relatively cheap and easy-to-handle application for the analysis of both chloride and sodium in sweat. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Development and validation of a stability-indicating RP-HPLC method for assay of betamethasone and estimation of its related compounds.

PubMed

Fu, Qiang; Shou, Minshan; Chien, Dwight; Markovich, Robert; Rustum, Abu M

2010-02-05

Betamethasone (9alpha-fluoro-16beta-methylprednisolone) is one of the members of the corticosteriod family of active pharmaceutical ingredient (API), which is widely used as an anti-inflammatory agent and also as a starting material to manufacture various esters of betamethasone. A stability-indicating reverse-phase high performance liquid chromatography (RP-HPLC) method has been developed and validated which can separate and accurately quantitate low levels of 26 betamethasone related compounds. The stability-indicating capability of the method was demonstrated through adequate separation of all potential betamethasone related compounds from betamethasone and also from each other that are present in aged and stress degraded betamethasone stability samples. Chromatographic separation of betamethasone and its related compounds was achieved by using a gradient elution at a flow rate of 1.0mL/min on a ACE 3 C18 column (150mmx4.6mm, 3microm particle size, 100A pore size) at 40 degrees C. Mobile phase A of the gradient was 0.1% methanesulfonic acid in aqueous solution and mobile phase B was a mixture of tert-butanol and 1,4-dioxane (7:93, v/v). UV detection at 254nm was employed to monitor the analytes. For betamethasone 21-aldehyde, the QL and DL were 0.02% and 0.01% respectively. For betamethasone and the rest of the betamethasone related compounds, the QL and DL were 0.05% and 0.02%. The precision of betamethasone assay is 0.6% and the accuracy of betamethasone assay ranged from 98.1% to 99.9%.

Monitoring the chemical production of citrus-derived bioactive 5-demethylnobiletin using surface enhanced Raman spectroscopy

PubMed Central

Zheng, Jinkai; Fang, Xiang; Cao, Yong; Xiao, Hang; He, Lili

2013-01-01

To develop an accurate and convenient method for monitoring the production of citrus-derived bioactive 5-demethylnobiletin from demethylation reaction of nobiletin, we compared surface enhanced Raman spectroscopy (SERS) methods with a conventional HPLC method. Our results show that both the substrate-based and solution-based SERS methods correlated with HPLC method very well. The solution method produced lower root mean square error of calibration and higher correlation coefficient than the substrate method. The solution method utilized an ‘affinity chromatography’-like procedure to separate the reactant nobiletin from the product 5-demthylnobiletin based on their different binding affinity to the silver dendrites. The substrate method was found simpler and faster to collect the SERS ‘fingerprint’ spectra of the samples as no incubation between samples and silver was needed and only trace amount of samples were required. Our results demonstrated that the SERS methods were superior to HPLC method in conveniently and rapidly characterizing and quantifying 5-demethylnobiletin production. PMID:23885986

Multi-wavelength spectrophotometric analysis for detection of xanthochromia in cerebrospinal fluid and accuracy for the diagnosis of subarachnoid hemorrhage.

PubMed

Smith, Andrew; Wu, Alan H B; Lynch, Kara L; Ko, Nerissa; Grenache, David G

2013-09-23

Cerebrospinal fluid (CSF) was examined for bilirubin, an important indicator for diagnosis of subarachnoid hemorrhage (SAH). A multi-wavelength (340, 415, and 460 nm) spectrophotometric assay was developed for the quantitative measurement of bilirubin in CSF, enabling the mathematical correction for absorbance of hemoglobin and proteins. Bilirubin and hemoglobin results were correlated to HPLC and a standard colorimetric assay, respectively. A subset of samples was sent for an absorbance reading at 450 nm following baseline correction. The multi-wavelength bilirubin assay was validated on 70 patients with confirmed SAH and 70 patients with neurologic symptoms who ruled out for SAH. The multi-wavelength spectrophometric assay demonstrated no interferences due to proteins (albumin) up to 30 g/l or oxyhemoglobin up to 260 mg/l. The assay limit of detection was 0.2 mg/l, linear to 20 mg/l, and CVs ranged from 1 to 6% at bilirubin concentrations of 0.84 and 2.1mg/l. The spectrophotometric assay correlated to HPLC and the colorimetric assay for bilirubin and hemoglobin, respectively. Results also correlated to the absorbance method (with removal of samples with high hemoglobin and proteins). The area under the ROC curve for diagnosis of SAH was 0.971 and 0.954 for the HPLC and spectrophotometric assay, respectively. At a cutoff of 0.2mg/l, the clinical specificity was 100% for both assays, and the clinical sensitivity was 94.3% and 88.6% for SAH for the HPLC and spectrophotometric asays, respectively. The multi-wavelength spectrophotometric assay is an objective alternative to visual inspection, HPLC, and absorbance for CSF bilirubin. Copyright © 2013 Elsevier B.V. All rights reserved.

Diversity oriented high-throughput screening of 1,3,4-oxadiazole modified chlorophenylureas and halogenobenzamides by HPLC with peptidomimetic calixarene-bonded stationary phases.

PubMed

Bazylak, Grzegorz; Malak, Anna; Ali, Imran; Borowiak, Teresa; Dutkiewicz, Grzegorz

2008-06-01

Retention profiles in series of the neutral and highly hydrophobic 1,3,4-oxadiazoles containing chlorophenylurea and halogenobenzamide moiety and indicating analgesic activity were determined in the isocratic standard- and narrow-bore HPLC systems employing, respectively, various octadecylsilica and different calixarene bonded stationary phases. When acetonitrile - 2.65 mM phosphoric acid (55 : 45, %, v/v), pH* 3.25, mobile phase was applied retention of these compounds increased with decline of their overall hydrophobicity according to the general preference of more polar compounds by calixarene cavity in time of its non-specific host-guest supramolecular interactions with halogenated substances. The size of calixarene nanocavity and its upper-rim substitution did not change the observed retention order, resolution and selectivity of separation for oxadiazoles. Compared to the retention on the non-end-capped and the highly-end-capped octadecylsilica HPLC column a most improved separation of some regioisomers of halogenated 1,3,4-oxadiazoles were observed on both used calixarene-type HPLC supports. In addition, preliminary data on the self-assembled supramolecular crystal structure of exemplary 1,3,4-oxadiazolchlorophenylurea with cis-elongated conformation was reported and formation of the monovalent inclusion host-guest complexes between 1,3,4-oxadiazoles and each calixarene-type stationary phase was studied with molecular modelling MM+ and AM1 methods. The structural, isomeric and energetic factors leading to the hydrogen bond stabilized inclusion complexes between these species were considered and used for explanation of observed retention sequence and selectivity of 1,3,4-oxadiazoles separation in applied calixarene-based HPLC systems. All these data would be useful in future development of optimized procedures enabling encapsulation of 1,3,4-oxadiazolurea-type drugs with calixarenes.

A simple HPLC method for the comprehensive analysis of cis/trans (Z/E) geometrical isomers of carotenoids for nutritional studies

USDA-ARS?s Scientific Manuscript database

Geometrical isomers of carotenoids behave differently in aspects like stability towards oxidants, bioavailability, vitamin A activity and specificity for enzymes. The availability of HPLC methods for their detailed profiling is therefore advisable to expand our knowledge on their metabolism and biol...

Improved method for reliable HMW-GS identification by RP-HPLC and SDS-PAGE in common wheat cultivars

USDA-ARS?s Scientific Manuscript database

The accurate identification of alleles for high-molecular weight glutenins (HMW-GS) is critical for wheat breeding programs targeting end-use quality. RP-HPLC methods were optimized for separation of HMW-GS, resulting in enhanced resolution of 1By and 1Dx subunits. Statistically significant differe...

On-line HPLC-DPPH screening method for evaluation of radical scavenging phenols extracted from apples (Malus domestica L.).

PubMed

Bandoniene, Donata; Murkovic, Michael

2002-04-24

An on-line HPLC-DPPH screening method for phenolic antioxidants in apple methanol/water (80:20, v/v) extracts was applied. The determination of antioxidants was based on a decrease in absorbance at 515 nm after postcolumn reaction of HPLC-separated antioxidants with the 2,2'-diphenyl-1-picrylhydrazyl radicals (DPPH*). Each of the antioxidants separated by the HPLC column was observed as a negative peak corresponding to its antioxidative activity. The on-line method was applied for quantitative analysis of the antioxidants. A linear dependence of negative peak area on concentration of the reference antioxidants was observed. For validation of the on-line method the limit of detection, LOD (microg/mL), and the limit of quantification, LOQ (microg/mL), of the phenolic compounds were determined. Comparison of the UV and DPPH radical quenching chromatograms with authentic compounds identified catechin, chlorogenic acid, caffeic acid, epicatechin, and phloridzin in the apple cultivars (Lobo, Golden Delicious, and Boskoop), and the distribution of total antioxidant activity was calculated.

Simultaneous quantification by HPLC of the phenolic compounds for the crude drug of Prunus serotina subsp. capuli.

PubMed

Rivero-Cruz, Blanca

2014-08-01

Prunus serotina Ehrenb. subsp. capuli (Cav.) McVaugh (Rosaceae), commonly known as "capulin", is a native North American tree, commercialized and used in folk medicine for the treatment of the hypertension, gastrointestinal illnesses, and cough. This work developed a suitable HPLC method for quantifying the major active constituents of the infusion of P. serotina, the most important preparation consumed by populations around the world. The analytical method was performed using a Fortis-RP column (150 mm × 4.6 mm; film thickness 5 µm). The mobile phase consisted of an isocratic acetate buffer solution (pH 2.7; A) and methanol (B) (65:35 v/v) at a flow rate of 1.0 mL min(-1). The proposed method was applied to the quantification of 1-3 in several samples of the leaves of P. serotina. The results indicated that amounts of 1-3 in the samples analyzed are uniform, and greater amounts of chlorogenic acid (2; 479.9 ± 33.6 µg g(-1), dry matter) along with hyperoside (1; 185.7 ± 55.3 µg g(-1), dry matter) were present. On the other hand, benzaldehyde (3; 118.2 ± 12.1 µg g(-1) dry matter) was found to be in lower concentration. A simple, sensitive, precise, and reproducible HPLC method for the simultaneous quantification of 1-3 in P. serotina was developed and validated. This is the first report on the quantification of 1-3 as active principles, and compound 1 was selected as a marker of P. serotina, which could be useful to guarantee the quality of the crude drug and herbal products.

Cobalamin Concentrations in Fetal Liver Show Gender Differences: A Result from Using a High-Pressure Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry as an Ultratrace Cobalt Speciation Method.

PubMed

Bosle, Janine; Goetz, Sven; Raab, Andrea; Krupp, Eva M; Scheckel, Kirk G; Lombi, Enzo; Meharg, Andrew A; Fowler, Paul A; Feldmann, Jörg

2016-12-20

Maternal diet and lifestyle choices may affect placental transfer of cobalamin (Cbl) to the fetus. Fetal liver concentration of Cbl reflects nutritional status with regards to vitamin B12, but at these low concentration current Cbl measurement methods lack robustness. An analytical method based on enzymatic extraction with subsequent reversed-phase-high-pressure liquid chromatography (RP-HPLC) separation and parallel ICPMS and electrospray ionization (ESI)-Orbitrap-MS to determine specifically Cbl species in liver samples of only 10-50 mg was developed using 14 pig livers. Subsequently 55 human fetal livers were analyzed. HPLC-ICPMS analysis for cobalt (Co) and Cbl gave detection limits of 0.18 ng/g and 0.88 ng/g d.m. in liver samples, respectively, with a recovery of >95%. Total Co (Co t ) concentration did not reflect the amount of Cbl or vitamin B12 in the liver. Cbl bound Co contributes only 45 ± 15% to Co t . XRF mapping and μXANES analysis confirmed the occurrence of non-Cbl cobalt in pig liver hot spots indicating particular Co. No correlations of total cobalt nor Cbl with fetal weight or weeks of gestation were found for the human fetal livers. Although no gender difference could be identified for total Co concentration, female livers were significantly higher in Cbl concentration (24.1 ± 7.8 ng/g) than those from male fetuses (19.8 ± 7.1 ng/g) (p = 0.04). This HPLC-ICPMS method was able to quantify total Co t and Cbl in fetus liver, and it was sensitive and precise enough to identify this gender difference.

Performance characteristics of two bioassays and high-performance liquid chromatography for determination of flucytosine in serum.

PubMed Central

St-Germain, G; Lapierre, S; Tessier, D

1989-01-01

We compared the accuracy and precision of two microbiological methods and one high-pressure liquid chromatography (HPLC) procedure used to measure the concentrations of flucytosine in serum. On the basis of an analysis of six standards, all methods were judged reliable within acceptable limits for clinical use. With the biological methods, a slight loss of linearity was observed in the 75- to 100-micrograms/ml range. Compared with the bioassays, the HPLC method did not present linearity problems and was more precise and accurate in the critical zone of 100 micrograms/ml. On average, results obtained with patient sera containing 50 to 100 micrograms of flucytosine per ml were 10.6% higher with the HPLC method than with the bioassays. Standards for the biological assays may be prepared in serum or water. PMID:2802566

Development and validation of a new method to simultaneously quantify triazoles in plasma spotted on dry sample spot devices and analysed by HPLC-MS.

PubMed

Baietto, Lorena; D'Avolio, Antonio; Marra, Cristina; Simiele, Marco; Cusato, Jessica; Pace, Simone; Ariaudo, Alessandra; De Rosa, Francesco Giuseppe; Di Perri, Giovanni

2012-11-01

Therapeutic drug monitoring (TDM) of triazoles is widely used in clinical practice to optimize therapy. TDM is limited by technical problems and cost considerations, such as sample storage and dry-ice shipping. We aimed to develop and validate a new method to analyse itraconazole, posaconazole and voriconazole in plasma spotted on dry sample spot devices (DSSDs) and to quantify them by an HPLC system. Extraction from DSSDs was done using n-hexane/ethyl acetate and ammonia solution. Samples were analysed using HPLC with mass spectrometry (HPLC-MS). Accuracy and precision were assayed by inter- and intra-day validation. The stability of triazoles in plasma spotted on DSSDs was investigated at room temperature for 1 month. The method was compared with a validated standard HPLC method for quantification of triazoles in human plasma. Mean inter- and intra-day accuracy and precision were <15% for all compounds. Triazoles were stable for 2 weeks at room temperature. The method was linear (r(2) > 0.999) in the range 0.031-8 mg/L for itraconazole and posaconazole, and 0.058-15 mg/L for voriconazole. High sensitivity was observed; limits of detection were 0.008, 0.004 and 0.007 mg/L for itraconazole, posaconazole and voriconazole, respectively. A high degree of correlation (r(2) > 0.94) was obtained between the DSSD method and the standard method of analysis. The method that we developed and validated to quantify triazoles in human plasma spotted on DSSDs is accurate and precise. It overcomes problems related to plasma sample storage and shipment, allowing TDM to be performed in a cheaper and safer manner.

[Spectrophotometric and HPLC evaluation of ceftazidime stability].

PubMed

Palade, B; Cioroiu, B; Lazăr, Doina; Corciovă, Andreia; Lazăr, M I

2010-01-01

In this paper we followed up the stability of ceftazidime, raw material used in drug industry. Matherials and methods: We used three spectrophotometric methods based on ceftazidime property to form complexes with p-chloranilic acid (ac. p-CA), 3-methylbenzothiazolin-2-on hydrazone (MBTH) and N-(1-naphtil) etilendiamine (NEDA) and a chromatographic method (HPLC). Our results revealed that the substances analyzed maintained minimum content allowable.

Development of validated stability indicating assay method for simultaneous estimation of metformin hydrochloride and vildagliptin by RP-HPLC.

PubMed

Satheeshkumar, N; Pradeepkumar, M; Shanthikumar, S; Rao, V J

2014-03-01

A simple, precise and stability-indicating HPLC method was developed and validated for the simultaneous determination of metformin hydrochloride (MET) and vildagliptin (VLG) in pharmaceutical dosage forms. The method involves use of easily available inexpensive laboratory reagents. The separation was achieved on Grace Cyano column (250 mm×4.6 mm) 5 µm with isocratic flow. The mobile phase was pumped at a flow rate of 1.0 mL/min, consisted of 25 mM ammonium bicarbonate buffer and acetonitrile (65:35, v/v). The UV detection was carried out at 207 nm. A linear response was observed over the concentration range of 25-125 µg/mL for MET and 50-250 µg/mL for VLG respectively. Limit of detection and limit of quantification for MET were 0.36 µg/mL and 1.22 µg/mL, and for VLG were 0.75 µg/mL and 2.51 µg/mL respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for specificity, linearity, accuracy, precision, robustness, and system suitability. Individual drugs (MET and VLG) were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions. The resultant stressed samples were analyzed by the proposed method. The method gave high resolution among the degradation products and the analytes. The peak purity of analyte peak in the stressed samples was confirmed by photo diode array detector. The proposed method was successfully applied for the quantitative analysis of MET and VLG in tablet dosage form, which will help to improve quality control and contribute to stability studies of pharmaceutical tablets containing these drugs. © Georg Thieme Verlag KG Stuttgart · New York.

[Isolation and purification of seven catechin compounds from fresh tea leaves by semi-preparative liquid chromatography].

PubMed

Gong, Zhihong; Chen, Si; Gao, Jiangtao; Li, Meihong; Wang, Xiaxia; Lin, Jun; Yu, Xiaomin

2017-11-08

An effective and simple method was established to simultaneously purify seven tea catechins (gallocatechin (GC), epigallocatechin (EGC), catechin (C), epigallocatechin-3- O -gallate (EGCG), epicatechin (EC), epigallocatechin-3- O -(3- O -methyl)-gallate (EGCG3"Me) and epicatechin-3- O -gallate (ECG)) from fresh tea leaves by semi-preparative high performance liquid chromatography (HPLC). Fresh leaves of Tieguanyin tea were successively extracted with methanol and chloroform. Then crude catechins were precipitated from the aqueous fraction of chloroform extraction by adding lead subacetate. Crude catechins were used for the isolation of the seven target catechin compounds by semi-preparative HPLC. Methanol-water and acetonitrile-water were sequentially used as mobile phases. After two rounds of semi-preparative HPLC, all target compounds were achieved with high purities (>90%). The proposed method was tested on two additional tea cultivars and showed similar results. This method demonstrated a simple and efficient strategy based on solvent extraction, ion precipitation and semi-preparative HPLC for the preparation of multiple catechins from tea leaves.

Analysis of the extracts of Isatis tinctoria by new analytical approaches of HPLC, MS and NMR.

PubMed

Zhou, Jue; Qu, Fan

2011-01-01

The methods of extraction, separation and analysis of alkaloids and indole glucosinolates (GLs) ofIsatis tinctoria were reviewed. Different analytical approaches such as High-pressure Liquid Chromatography (HPLC), Liquid Chromatography with Electrospray Ionization Mass Spectrometry (LC/ESI/MS), Electrospray Ionization Time-Of-Flight Mass Spectrometry (ESI-TOF-MS), and Nuclear Magnetic Resonance (NMR) were used to validate and identity of these constituents. These methods provide rapid separation, identification and quantitative measurements of alkaloids and GLs of Isatis tinctoria. By connection with different detectors to HPLC such as PDA, ELSD, ESI- and APCI-MS in positive and negative ion modes, complicated compounds could be detected with at least two independent detection modes. The molecular formula can be derived in a second step of ESI-TOF-MS data. But for some constituents, UV and MS cannot provide sufficient structure identification. After peak purification, NMR by semi-preparative HPLC can be used as a complementary method.

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.

Quantitative determination of cucurbitane-type triterpenes and triterpene glycosides in dietary supplements containing bitter melon (Momordica charantia) by HPLC-MS/MS.

PubMed

Ma, Jun; Krynitsky, Alexander J; Grundel, Erich; Rader, Jeanne I

2012-01-01

Momordica charantia L. (Cucurbitaceae), commonly known as bitter melon, is widely cultivated in many tropical and subtropical areas of the world. It is a common food staple; its fruits, leaves, seeds, stems, and roots also have a long history of use in traditional medicine. In the United States, dietary supplements labeled as containing bitter melon can be purchased over-the-counter and from Internet suppliers. Currently, no quantitative analytical method is available for monitoring the content of cucurbitane-type triterpenes and triterpene glycosides, the major constituents of bitter melon, in such supplements. We investigated the use of HPLC-electrospray ionization (ESI)-MS/MS for the quantitative determination of such compounds in dietary supplements containing bitter melon. Values for each compound obtained from external calibration were compared with those obtained from the method of standard additions to address matrix effects associated with ESI. In addition, the cucurbitane-type triterpene and triterpene glycoside contents of two dietary supplements determined by the HPLC-ESI-MS/MS method with standard additions were compared with those measured by an HPLC method with evaporative light scattering detection, which was recently developed for quantification of such compounds in dried fruits of M. charantia. The contents of five cucurbitane-type triterpenes and triterpene glycosides in 10 dietary supplements were measured using the HPLC-ESI-MS/MS method with standard additions. The total contents of the five compounds ranged from 17 to 3464 microg/serving.

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.

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.

Comparison of high-performance liquid chromatography/tandem mass spectrometry and high-performance liquid chromatography/photo-diode array detection for the quantitation of carotenoids, retinyl esters, α-tocopherol and phylloquinone in chylomicron-rich fractions of human plasma

PubMed Central

Kopec, Rachel E.; Schweiggert, Ralf M.; Riedl, Ken M.; Carle, Reinhold; Schwartz, Steven J.

2013-01-01

Rationale Bioavailability of essential lipophilic micronutrients and carotenoids is of utmost interest for human health, as the consumption of these compounds may help alleviate major nutritional deficiencies, cardiovascular disease, and cancer. High-performance liquid chromatography/photo-diode array detection (HPLC-PDA) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) were compared for the quantitative analysis of α- and β-carotene, β-cryptoxanthin, lutein, lycopene, α-tocopherol, phylloquinone, and several retinyl esters from chylomicron-containing triglyceride rich lipoprotein (TRL) fractions of human plasma obtained from two clinical trials. Methods After selecting an efficient extraction method for the analytes, both the HPLC/PDA and the HPLC/MS/MS methods were developed and several parameters validated using an HP 1200 series HPLC system interfaced with a HP 1200 series diode-array detector (Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 5500 (AB Sciex, Foster City, CA, USA) via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. Results For lycopene, α- and β-carotene, HPLC/MS/MS was up to 37 times more sensitive than HPLC-PDA. PDA detection was shown to be up to 8 times more sensitive for lutein. MS/MS signals were enhanced by matrix components for lutein and β-cryptoxanthin, as determined by referencing to the matrix-independent PDA signal. In contrast, matrix suppression was observed for retinyl palmitate, α-carotene, and β-carotene. Both detectors showed similar suitability for α-tocopherol, lycopene and retinyl palmitate (representing ~73% of total retinyl esters). MS/MS exclusively allowed the quantitation of minor retinyl esters, phylloquinone, and (Z)-lycopene isomers. Conclusions HPLC/MS/MS was more sensitive than HPLC-PDA for six of the eight analytes and represents a powerful tool for the analysis of chylomicron samples and potentially other biological samples of limited sample size. When internal standards are available for the target carotenoid, employing MS/MS detection may reduce the necessary blood sample volume, which is particularly advantageous for minimizing risk and discomfort to human subjects during clinical studies. PMID:23681818

Determination of isoxaflutole (balance) and its metabolites in water using solid phase extraction followed by high-performance liquid chromatography with ultraviolet or mass spectrometry

USGS Publications Warehouse

Lin, Chung-Ho; Lerch, Robert N.; Thurman, E. Michael; Garrett , Harold E.; George, Milon F.

2002-01-01

Balance (isoxaflutole, IXF) belongs to a new family of herbicides referred to as isoxazoles. IXF has a very short soil half-life (<24 h), degrading to a biologically active diketonitrile (DKN) metabolite that is more polar and considerably more stable. Further degradation of the DKN metabolite produces a nonbiologically active benzoic acid (BA) metabolite. Analytical methods using solid phase extraction followed by high-performance liquid chromatography−UV (HPLC-UV) or high-performance liquid chromatography−mass spectrometry (HPLC-MS) were developed for the analysis of IXF and its metabolites in distilled deionized water and ground water samples. To successfully detect and quantify the BA metabolite by HPLC-UV from ground water samples, a sequential elution scheme was necessary. Using HPLC-UV, the mean recoveries from sequential elution of the parent and its two metabolites from fortified ground water samples ranged from 68.6 to 101.4%. For HPLC-MS, solid phase extraction of ground water samples was performed using a polystyrene divinylbenzene polymer resin. The mean HPLC-MS recoveries of the three compounds from ground water samples spiked at 0.05−2 μg/L ranged from 100.9 to 110.3%. The limits of quantitation for HPLC-UV are approximately 150 ng/L for IXF, 100 ng/L for DKN, and 250 ng/L for BA. The limit of quantitation by HPLC-MS is 50 ng/L for each compound. The methods developed in this work can be applied to determine the transport and fate of Balance in the environment.

Determination of isoxaflutole (balance) and its metabolites in water using solid phase extraction followed by high-performance liquid chromatography with ultraviolet or mass spectrometry.

PubMed

Lin, Chung-Ho; Lerch, Robert N; Thurman, E Michael; Garrett, Harold E; George, Milon F

2002-10-09

Balance (isoxaflutole, IXF) belongs to a new family of herbicides referred to as isoxazoles. IXF has a very short soil half-life (<24 h), degrading to a biologically active diketonitrile (DKN) metabolite that is more polar and considerably more stable. Further degradation of the DKN metabolite produces a nonbiologically active benzoic acid (BA) metabolite. Analytical methods using solid phase extraction followed by high-performance liquid chromatography-UV (HPLC-UV) or high-performance liquid chromatography-mass spectrometry (HPLC-MS) were developed for the analysis of IXF and its metabolites in distilled deionized water and ground water samples. To successfully detect and quantify the BA metabolite by HPLC-UV from ground water samples, a sequential elution scheme was necessary. Using HPLC-UV, the mean recoveries from sequential elution of the parent and its two metabolites from fortified ground water samples ranged from 68.6 to 101.4%. For HPLC-MS, solid phase extraction of ground water samples was performed using a polystyrene divinylbenzene polymer resin. The mean HPLC-MS recoveries of the three compounds from ground water samples spiked at 0.05-2 microg/L ranged from 100.9 to 110.3%. The limits of quantitation for HPLC-UV are approximately 150 ng/L for IXF, 100 ng/L for DKN, and 250 ng/L for BA. The limit of quantitation by HPLC-MS is 50 ng/L for each compound. The methods developed in this work can be applied to determine the transport and fate of Balance in the environment.

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.

Detection of β-Lactams and Chloramphenicol Residues in Raw Milk-Development and Application of an HPLC-DAD Method in Comparison with Microbial Inhibition Assays.

PubMed

Karageorgou, Eftychia; Christoforidou, Sofia; Ioannidou, Maria; Psomas, Evdoxios; Samouris, Georgios

2018-06-01

The present study was carried out to assess the detection sensitivity of four microbial inhibition assays (MIAs) in comparison with the results obtained by the High Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) method for antibiotics of the β-lactam group and chloramphenicol in fortified raw milk samples. MIAs presented fairly good results when detecting β-lactams, whereas none were able to detect chloramphenicol at or above the permissible limits. HPLC analysis revealed high recoveries of examined compounds, whereas all detection limits observed were lower than their respective maximum residue limits (MRL) values. The extraction and clean-up procedure of antibiotics was performed by a modified matrix solid phase dispersion procedure using a mixture of Plexa by Agilent and QuEChERS as a sorbent. The HPLC method developed was validated, determining the accuracy, precision, linearity, decision limit, and detection capability. Both methods were used to monitor raw milk samples of several cows and sheep, obtained from producers in different regions of Greece, for the presence of examined antibiotic residues. Results obtained showed that MIAs could be used effectively and routinely to detect antibiotic residues in several milk types. However, in some cases, spoilage of milk samples revealed that the kits' sensitivity could be strongly affected, whereas this fact does not affect the effectiveness of HPLC-DAD analysis.

Optimisation of solid-phase microextraction coupled to HPLC-UV for the determination of organochlorine pesticides and their metabolites in environmental liquid samples.

PubMed

Torres Padrón, M E; Sosa Ferrera, Z; Santana Rodríguez, J J

2006-09-01

A solid-phase microextraction (SPME) procedure using two commercial fibers coupled with high-performance liquid chromatography (HPLC) is presented for the extraction and determination of organochlorine pesticides in water samples. We have evaluated the extraction efficiency of this kind of compound using two different fibers: 60-mum polydimethylsiloxane-divinylbenzene (PDMS-DVB) and Carbowax/TPR-100 (CW/TPR). Parameters involved in the extraction and desorption procedures (e.g. extraction time, ionic strength, extraction temperature, desorption and soaking time) were studied and optimized to achieve the maximum efficiency. Results indicate that both PDMS-DVB and CW/TPR fibers are suitable for the extraction of this type of compound, and a simple calibration curve method based on simple aqueous standards can be used. All the correlation coefficients were better than 0.9950, and the RSDs ranged from 7% to 13% for 60-mum PDMS-DVB fiber and from 3% to 10% for CW/TPR fiber. Optimized procedures were applied to the determination of a mixture of six organochlorine pesticides in environmental liquid samples (sea, sewage and ground waters), employing HPLC with UV-diode array detector.

HPLC and LC-MS/MS methods for determination of sodium benzoate and potassium sorbate in food and beverages: performances of local accredited laboratories via proficiency tests in Turkey.

PubMed

Gören, Ahmet C; Bilsel, Gökhan; Şimşek, Adnan; Bilsel, Mine; Akçadağ, Fatma; Topal, Kevser; Ozgen, Hasan

2015-05-15

High Performance Liquid Chromatography LC-UV and LC-MS/MS methods were developed and validated for quantitative analyses of sodium benzoate and potassium sorbate in foods and beverages. HPLC-UV and LC-MS/MS methods were compared for quantitative analyses of sodium benzoate and potassium sorbate in a representative ketchup sample. Optimisation of the methods enabled the chromatographic separation of the analytes in less than 4 min. A correlation coefficient of 0.999 was achieved over the measured calibration range for both compounds and methods (HPLC and LC-MS/MS). The uncertainty values of sodium benzoate and potassium sorbate were found as 0.199 and 0.150 mg/L by HPLC and 0.072 and 0.044 mg/L by LC-MS/MS, respectively. Proficiency testing performance of Turkish accredited laboratories between the years 2005 and 2013 was evaluated and reported herein. The aim of the proficiency testing scheme was to evaluate the performance of the laboratories, analysing benzoate and sorbate in tomato ketchup. Copyright © 2014 Elsevier Ltd. All rights reserved.

A stability indicating HPLC method for determination of mebeverine in the presence of its degradation products and kinetic study of its degradation in oxidative condition

PubMed Central

Souri, E.; Aghdami, A. Negahban; Adib, N.

2014-01-01

An HPLC method for determination of mebeverine hydrochloride (MH) in the presence of its degradation products was developed. The degradation of MH was studied under hydrolysis, oxidative and photolysis stress conditions. Under alkaline, acidic and oxidative conditions, degradation of MH was observed. The separation was performed using a Symmetry C18 column and a mixture of 50 mM KH2PO4, acetonitrile and tetrahydrfuran (THF) (63:35:2; v/v/v) as the mobile phase. No interference peaks from degradation products in acidic, alkaline and oxidative conditions were observed. The linearity, accuracy and precision of the method were studied. The method was linear over the range of 1-100 μg/ml MH (r2>0.999) and the CV values for intra-day and inter-day variations were in the range of 1.0-1.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method were 1.0 and 0.2 μg/ml, respectively. Determination of MH in pharmaceutical dosage forms was performed using the developed method. Furthermore the kinetics of the degradation of MH in the presence of hydrogen peroxide was investigated. The proposed method could be a suitable method for routine quality control studies of mebeverine dosage forms. PMID:25657790

A stability indicating HPLC method for determination of mebeverine in the presence of its degradation products and kinetic study of its degradation in oxidative condition.

PubMed

Souri, E; Aghdami, A Negahban; Adib, N

2014-01-01

An HPLC method for determination of mebeverine hydrochloride (MH) in the presence of its degradation products was developed. The degradation of MH was studied under hydrolysis, oxidative and photolysis stress conditions. Under alkaline, acidic and oxidative conditions, degradation of MH was observed. The separation was performed using a Symmetry C18 column and a mixture of 50 mM KH2PO4, acetonitrile and tetrahydrfuran (THF) (63:35:2; v/v/v) as the mobile phase. No interference peaks from degradation products in acidic, alkaline and oxidative conditions were observed. The linearity, accuracy and precision of the method were studied. The method was linear over the range of 1-100 μg/ml MH (r(2)>0.999) and the CV values for intra-day and inter-day variations were in the range of 1.0-1.8%. The limit of quantification (LOQ) and the limit of detection (LOD) of the method were 1.0 and 0.2 μg/ml, respectively. Determination of MH in pharmaceutical dosage forms was performed using the developed method. Furthermore the kinetics of the degradation of MH in the presence of hydrogen peroxide was investigated. The proposed method could be a suitable method for routine quality control studies of mebeverine dosage forms.

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.

HPLC analysis and standardization of Brahmi vati – An Ayurvedic poly-herbal formulation

PubMed Central

Mishra, Amrita; Mishra, Arun K.; Tiwari, Om Prakash; Jha, Shivesh

2013-01-01

Objectives The aim of the present study was to standardize Brahmi vati (BV) by simultaneous quantitative estimation of Bacoside A3 and Piperine adopting HPLC–UV method. BV very important Ayurvedic polyherbo formulation used to treat epilepsy and mental disorders containing thirty eight ingredients including Bacopa monnieri L. and Piper longum L. Materials and methods An HPLC–UV method was developed for the standardization of BV in light of simultaneous quantitative estimation of Bacoside A3 and Piperine, the major constituents of B. monnieri L. and P. longum L. respectively. The developed method was validated on parameters including linearity, precision, accuracy and robustness. Results The HPLC analysis showed significant increase in amount of Bacoside A3 and Piperine in the in-house sample of BV when compared with all three different marketed samples of the same. Results showed variations in the amount of Bacoside A3 and Piperine in different samples which indicate non-uniformity in their quality which will lead to difference in their therapeutic effects. Conclusion The outcome of the present investigation underlines the importance of standardization of Ayurvedic formulations. The developed method may be further used to standardize other samples of BV or other formulations containing Bacoside A3 and Piperine. PMID:24396246

A bridging study for oxytetracycline in the edible fillet of rainbow trout: Analysis by a liquid chromatographic method and the official microbial inhibition assay

USGS Publications Warehouse

Stehly, G.R.; Gingerich, W.H.; Kiessling, C.R.; Cutting, J.H.

1999-01-01

Oxytetracycline (OTC) is a drug approved by the U.S. Food and Drug Administration (FDA) to control certain diseases in salmonids and catfish. OTC is also a likely control agent for diseases of other fish species and for other diseases of salmonids and catfish not currently on the label. One requirement for FDA to extend and expand the approval of this antibacterial agent to other fish species is residue depletion studies. The current regulatory method for OTC in fish tissue, based on microbial inhibition, lacks sensitivity and specificity. To conduct residue depletion studies for OTC in fish with a liquid chromatographic method, a bridging study was required to determine its relationship with the official microbial inhibition assay. Triplicate samples of rainbow trout fillet tissue fortified with OTC at 0.3, 0.6, 1.2, 2.4, 4.8, and 9.6 ppm and fillet tissue with incurred OTC at approximately 0.75, 1.5, and 3.75 ppm were analyzed by high-performance liquid chromatography (HPLC) and the microbial inhibition assay. The results indicated that the 2 methods are essentially identical in the tested range, with mean coefficients of variation of 1.05% for the HPLC method and 3.94% for the microbial inhibition assay.

Biomarkers of Exposure to Toxic Substances Volume 7: Identification of Potential Serum Protein Biomarkers Indicative of Low Level Kidney Degradation in Response to Toxin Exposures

DTIC Science & Technology

2009-05-01

equilibrated for 4 min with Buffer A with a flow rate of 1 mL/min at room temperature. Once the HPLC lines and MARS column were flushed and equilibrated...ul 4 ) FT mouse control HPLC 10 ul 9) E mouse control Spin Column 10 ul 5) E mouse control HPLC 10 ul 10) Blue MW Standard The distinct...of Low Level Kidney Degradation in Response to Toxin Exposures Christopher L. Woolard Camilla A. Mauzy Biosciences and Protection

Turbine Engine Lubricant Reclamation.

DTIC Science & Technology

1983-06-01

results indicate no differences in additive/degradation product removal by varying the basic material level or between Ca(OH)2 and MgO. Data indicates...0H) 2 ɘ.09 .o difference in removal by changing Ca(OH) 2 treat- . ment level by HPLC and TLC. Thin Layer Chromatography Oil + adsorbents Oil + Ca(0H...2.1%) 0.17 3% by weight 0.17 4% by weight 0.16 No difference in removal by changing Ca(OH)2 treat- ment level by HPLC and TLC. Thin Layer

Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: models in glyphosate-resistant and -susceptible crops.

PubMed

Zelaya, Ian A; Anderson, Jennifer A H; Owen, Micheal D K; Landes, Reid D

2011-03-23

Endogenous shikimic acid determinations are routinely used to assess the efficacy of glyphosate in plants. Numerous analytical methods exist in the public domain for the detection of shikimic acid, yet the most commonly cited comprise spectrophotometric and high-pressure liquid chromatography (HPLC) methods. This paper compares an HPLC and two spectrophotometric methods (Spec 1 and Spec 2) and assesses the effectiveness in the detection of shikimic acid in the tissues of glyphosate-treated plants. Furthermore, the study evaluates the versatility of two acid-based shikimic acid extraction methods and assesses the longevity of plant extract samples under different storage conditions. Finally, Spec 1 and Spec 2 are further characterized with respect to (1) the capacity to discern between shikimic acid and chemically related alicyclic hydroxy acids, (2) the stability of the chromophore (t1/2), (3) the detection limits, and (4) the cost and simplicity of undertaking the analytical procedure. Overall, spectrophotometric methods were more cost-effective and simpler to execute yet provided a narrower detection limit compared to HPLC. All three methods were specific to shikimic acid and detected the compound in the tissues of glyphosate-susceptible crops, increasing exponentially in concentration within 24 h of glyphosate application and plateauing at approximately 72 h. Spec 1 estimated more shikimic acid in identical plant extract samples compared to Spec 2 and, likewise, HPLC detection was more effective than spectrophotometric determinations. Given the unprecedented global adoption of glyphosate-resistant crops and concomitant use of glyphosate, an effective and accurate assessment of glyphosate efficacy is important. Endogenous shikimic acid determinations are instrumental in corroborating the efficacy of glyphosate and therefore have numerous applications in herbicide research and related areas of science as well as resolving many commercial issues as a consequence of glyphosate utilization.

Analytical methods for determination of alkaloids and saponins from roots of Caulophyllum thalictroids (L) Michx using UPLC HPLC and HPTLC

USDA-ARS?s Scientific Manuscript database

A comparison study of analytical methods including HPLC, UPLC and HPTLC are presented in this paper for the determination of major alkaloid and triterpene saponins from the roots of Caulophyllum thalictroides (L.) Michx. (blue cohosh) and dietary supplements claiming to contain blue cohosh. The meth...

Determination of sulfonamides and trimethoprim using high temperature HPLC with simultaneous temperature and solvent gradient.

PubMed

Giegold, Sascha; Teutenberg, Thorsten; Tuerk, Jochen; Kiffmeyer, Thekla; Wenclawiak, Bernd

2008-10-01

A fast HPLC method for the analysis of eight selected sulfonamides (SA) and trimethoprim has been developed with the use of high temperature HPLC. The separation could be achieved in less than 1.5 min on a 50 mm sub 2 microm column with simultaneous solvent and temperature gradient programming. Due to the lower viscosity of the mobile phase and the increased mass transfer at higher temperatures, the separation could be performed on a conventional HPLC system obtaining peak widths at half height between 0.6 and 1.3 s.

Comparison of high-performance liquid chromatography and supercritical fluid chromatography using evaporative light scattering detection for the determination of plasticizers in medical devices.

PubMed

Lecoeur, Marie; Decaudin, Bertrand; Guillotin, Yoann; Sautou, Valérie; Vaccher, Claude

2015-10-23

Recently, interest in supercritical fluid chromatography (SFC) has increased due to its high throughput and the development of new system improving chromatographic performances. However, most papers dealt with fundamental studies and chiral applications and only few works described validation process of SFC method. Likewise, evaporative light scattering detection (ELSD) has been widely employed in liquid chromatography but only a few recent works presented its quantitative performances hyphenated with SFC apparatus. The present paper discusses about the quantitative performances of SFC-ELSD compared to HPLC-ELSD, for the determination of plasticizers (ATBC, DEHA, DEHT and TOTM) in PVC tubing used as medical devices. After the development of HPLC-ELSD, both methods were evaluated based on the total error approach using accuracy profile. The results show that HPLC-ELSD was more precise than SFC-ELSD but lower limits of quantitation were obtained by SFC. Hence, HPLC was validated in the ± 10% acceptance limits whereas SFC lacks of accuracy to quantify plasticizers. Finally, both methods were used to determine the composition of plasticized-PVC medical devices. Results demonstrated that SFC and HPLC both hyphenated with ELSD provided similar results. Copyright © 2015 Elsevier B.V. All rights reserved.

Fingerprint chromatogram analysis of Pseudostellaria heterophylla (Miq.) Pax root by high performance liquid chromatography.

PubMed

Han, Chao; Chen, Junhui; Chen, Bo; Lee, Frank Sen-Chun; Wang, Xiaoru

2006-09-01

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the fingerprinting of extracts from the root of Pseudostellaria heterophylla (Miq.) Pax. HPLC with gradient elution was performed on an authentic reference standard of powdered P. heterophylla (Miq.) Pax root and 11 plant samples of the root were collected from different geographic locations. The HPLC chromatograms have been standardized through the selection and identification of reference peaks and the normalization of retention times and peak intensities of all the common peaks. The standardized HPLC fingerprints show high stability and reproducibility, and thus can be used effectively for the screening analysis or quality assessment of the root or its derived products. Similarity index calculations based on cosine angle values or correlation methods have been performed on the HPLC fingerprints. As a group, the fingerprints of the P. heterophylla (Miq.) Pax samples studied are highly correlated with closely similar fingerprints. Within the group, the samples can be further divided into subgroups based on hierarchical clustering analysis (HCA). Sample grouping based on HCA coincides nicely with those based on the geographical origins of the samples. The HPLC fingerprinting techniques thus have high potential in authentication or source-tracing types of applications.

Determination of trace amounts of rare-earth elements in highly pure neodymium oxide by sector field inductively coupled plasma mass spectrometry (ICP-SFMS) and high-performance liquid chromatography (HPLC) techniques

NASA Astrophysics Data System (ADS)

Pedreira, W. R.; Sarkis, J. E. S.; da Silva Queiroz, C. A.; Rodrigues, C.; Tomiyoshi, I. A.; Abrão, A.

2003-02-01

Recently rare-earth elements (REE) have received much attention in fields of geochemistry and industry. Rapid and accurate determinations of them are increasingly required as industrial demands expand. Sector field inductively coupled plasma mass spectrometry (ICP-SFMS) with high-performance liquid chromatography (HPLC) has been applied to the determination of REE. HR ICP-MS was used as an element-selective detector for HPLC in highly pure materials. The separation of REE with HPLC helped to avoid erroneous analytical results due to spectral interferences. Sixteen elements (Sc, Y and 14 lanthanides) were determined selectively with the HPLC/ICP-SFMS system using a concentration gradient methods. The detection limits with the HPLC/ICP-SFMS system were about 0.5-10 pg mL-1. The percentage recovery ranged from 90% to 100% for different REE. The %RSD of the methods varying between 2.5% and 4.5% for a set of five (n=5) replicates was found for the IPEN's material and for the certificate reference sample. Determination of trace REEs in two highly pure neodymium oxides samples (IPEN and Johnson Matthey Company) were performed. In short, the IPEN's materials which are highly pure (>99.9%) were successfully analyzed without spectral interferences.

Characterization and quantification of flavonoids and saponins in adzuki bean (Vigna angularis L.) by HPLC-DAD-ESI-MSn analysis.

PubMed

Liu, Rui; Cai, Zongwei; Xu, Baojun

2017-09-22

Bioactive activities of adzuki bean have been widely reported, however, the phytochemical information of adzuki bean is incomplete. The aim of this study was to characterize and quantify flavonoids and saponins in adzuki bean. High performance liquid chromatography with diode array detection and electro spray ionization-tandem multi-stage mass spectrometry (HPLC-DAD-ESI-MS n ) were applied to do qualitative and quantitative analyses. A total of 15 compounds from adzuki bean were identified by HPLC-DAD-ESI-MS n . Among 15 compounds identified, four flavonoids (catechin, vitexin-4″-O-glucoside, quercetin-3-O-glucoside, and quercetin-3-O-rutinoside) and six saponins (azukisaponin I, II, III, IV, V, and VI) in adzuki bean were further quantified by external calibration method using HPLC-MS with the program of time segment and extract ion chromatogram (EIC) analysis. Current qualitative and quantitative method based on HPLC and MS technique provides a scientific basis for in vitro and in vivo pharmacological study in the future. Graphical abstract Isolation and characterization of flavonoids and saponins from adzuki bean.

Stability indicating HPLC-DAD method for analysis of Ketorolac binary and ternary mixtures in eye drops: Quantitative analysis in rabbit aqueous humor.

PubMed

El Yazbi, Fawzy A; Hassan, Ekram M; Khamis, Essam F; Ragab, Marwa A A; Hamdy, Mohamed M A

2017-11-15

Ketorolac tromethamine (KTC) with phenylephrine hydrochloride (PHE) binary mixture (mixture 1) and their ternary mixture with chlorpheniramine maleate (CPM) (mixture 2) were analyzed using a validated HPLC-DAD method. The developed method was suitable for the in vitro as well as quantitative analysis of the targeted mixtures in rabbit aqueous humor. The analysis in dosage form (eye drops) was a stability indicating one at which drugs were separated from possible degradation products arising from different stress conditions (in vitro analysis). For analysis in aqueous humor, Guaifenesin (GUF) was used as internal standard and the method was validated according to FDA regulation for analysis in biological fluids. Agilent 5 HC-C18(2) 150×4.6mm was used as stationary phase with a gradient eluting solvent of 20mM phosphate buffer pH 4.6 containing 0.2% triethylamine and acetonitrile. The drugs were resolved with retention times of 2.41, 5.26, 7.92 and 9.64min for PHE, GUF, KTC and CPM, respectively. The method was sensitive and selective to analyze simultaneously the three drugs in presence of possible forced degradation products and dosage form excipients (in vitro analysis) and also with the internal standard, in presence of aqueous humor interferences (analysis in biological fluid), at a single wavelength (261nm). No extraction procedure was required for analysis in aqueous humor. The simplicity of the method emphasizes its capability to analyze the drugs in vivo (in rabbit aqueous humor) and in vitro (in pharmaceutical formulations). Copyright © 2017 Elsevier B.V. All rights reserved.

[Application of fingerprint chromatogram in quality assessment of apple cider].

PubMed

Xu, Kangzhen; Song, Jirong; Ren, Yinghui; Ma, Haixia; Huang, Jie; Du, Xiaodan

2007-01-01

Fingerprints of 14 apple cider samples from different manufacturers were studied using high performance liquid chromatography (HPLC) with an electrochemical detector (ECD). The analysis was carried out on a Zorbax SB-C18 column at 30 degrees C with 2% (v/v) methanol aqueous solution-4% (v/v) acetic acid aqueous solution as mobile phase at a flow rate of 0.8 mL/min. The electrochemical detector was set at 0.7 V. By calculating the relative retention times of certain peaks with chlorogenic acid as the reference standard, 8 common peaks in the samples were analyzed. Relative retention times for the common peaks of various samples were calculated, and the similarities of all the samples were figured out through each peak area with the vectorial angle cosine method and correlative coefficient method. The results indicated that apple cider products of the same manufacturer have good similarity, with the similarities greater than 92.7%. According to this experiment, effectual microcosmic information for apple cider analysis was gained through HPLC and ECD. Moreover, this test method will help the analysis and the control of product quality, the development of new products and the establishment of trade standard.

Graphene oxide-SiO2 nanocomposite as the adsorbent for extraction and preconcentration of plant hormones for HPLC analysis.

PubMed

Zhang, Xiaona; Niu, Jiahua; Zhang, Xiaoting; Xiao, Rui; Lu, Minghua; Cai, Zongwei

2017-03-01

In this research, a modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method based on graphene oxide@SiO 2 (SiO 2 @GO) nanocomposite as adsorbent of dispersive solid-phase extraction (dSPE) combined with high performance liquid chromatography (HPLC) for the analysis of four plant hormones in different plants was established. The as-prepared SiO 2 @GO was characterized by scanning electron microscopy, transmission electron microscopy and infrared spectroscopy. The experimental conditions for dSPE, including the ratio of material to liquid, pH of sample, adsorption and desorption time, desorption temperature as well as desorption solution, were investigated. The detection limits for the analysis of indole-3-acetic acid, indole-3-butyric acid, 1-naphthylacetic acid and abscisic acid were achieved below 0.05μgmL -1 . The established method was applied to the analysis of the plant hormones in fruits, vegetables and other food samples. The obtained results indicated that the method was sensitive, accurate, convenient and quick, which provided an alternative analytical approach for plant hormones in complex matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of intracellular glutathione and cysteine using HPLC with a monolithic column after derivatization with monobromobimane.

PubMed

Conlan, Xavier A; Stupka, Nicole; McDermott, Geoffrey P; Francis, Paul S; Barnett, Neil W

2010-05-01

An optimized high-performance liquid chromatography (HPLC) method is used to show that, as myoblasts differentiate into multinucleated muscle fibers, there is a shift to a more oxidized cell redox state. The HPLC method incorporated derivatization with monobromobimane for the determination of the reduced (GSH) and oxidized (GSSG) forms of glutathione and the reduced (Cys) and oxidized (CysSS) forms of cysteine. The derivatization was optimized to improve the sensitivity of the approach; the limits of detection for glutathione and cysteine were 3 x 10(-8) and 5 x 10(-8) M, respectively. 2009 John Wiley & Sons, Ltd.

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.

Pharmacokinetic study of arctigenin in rat plasma and organ tissue by RP-HPLC method.

PubMed

He, Fan; Dou, De-Qiang; Hou, Qiang; Sun, Yu; Kang, Ting-Guo

2013-01-01

A high-performance liquid chromatography (HPLC) technique was developed for the determination of arctigenin in plasma and various organs of rats after the oral administration of 30, 50 and 70 mgkg(-1) of arctigenin to the Sprague-Dawley rats. Results showed that the validated HPLC method was simple, fast, reproducible and suitable to the determination of arctigenin in rat plasma and organ tissue and one-compartmental model with zero-order absorption process can well describe the changes of arctigenin concentration in the plasma. The concentration of compound was highest in the spleen, less in the liver and the least in the lung.

Adulteration and cultivation region identification of American ginseng using HPLC coupled with multivariate analysis

PubMed Central

Yu, Chunhao; Wang, Chong-Zhi; Zhou, Chun-Jie; Wang, Bin; Han, Lide; Zhang, Chun-Feng; Wu, Xiao-Hui; Yuan, Chun-Su

2014-01-01

American ginseng (Panax quinquefolius) is originally grown in North America. Due to price difference and supply shortage, American ginseng recently has been cultivated in northern China. Further, in the market, some Asian ginsengs are labeled as American ginseng. In this study, forty-three American ginseng samples cultivated in the USA, Canada or China were collected and 14 ginseng saponins were determined using HPLC. HPLC coupled with hierarchical cluster analysis and principal component analysis was developed to identify the species. Subsequently, an HPLC-linear discriminant analysis was established to discriminate cultivation regions of American ginseng. This method was successfully applied to identify the sources of 6 commercial American ginseng samples. Two of them were identified as Asian ginseng, while 4 others were identified as American ginseng, which were cultivated in the USA (3) and China (1). Our newly developed method can be used to identify American ginseng with different cultivation regions. PMID:25044150

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.

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.

Estimation of Koc values for deuterated benzene, toluene, and ethylbenzene, and application to ground water contamination studies.

PubMed

Poulson, S R; Drever, J I; Colberg, P J

1997-11-01

Sorption partition coefficients between water and organic carbon (Koc) for deuterated benzene, toluene, and ethylbenzene have been estimated by measuring values of the octanol-water partition coefficient (Kow) and HPLC retention factors (k1), which correlate closely to values of Koc. Measured values of log Kow for non-deuterated and deuterated toluene are 2.77 (+/- 0.02) and 2.78 (+/- 0.04), respectively, indicating that within experimental error, log Koc for deuterated and non-deuterated toluene are the same. The HPLC method provides greater precision, and yields values of delta log Koc (= log Koc [deuterated]-log Koc [non-deuterated]) of -0.021 (+/- 0.001) for benzene, -0.028 (+/- 0.002) for toluene, and -0.035 (+/- 0.003) for ethylbenzene. The small values of delta log Koc demonstrates that deuterated compounds are excellent tracers for the hydrologic behavior of ground water contaminants.

Determination of Phenols Isomers in Water by Novel Nanosilica/Polydimethylsiloxane-Coated Stirring Bar Combined with High Performance Liquid Chromatography-Fourier Transform Infrared Spectroscopy.

PubMed

Zheng, Bei; Li, Wentao; Liu, Lin; Wang, Xin; Chen, Chen; Yu, Zhiyong; Li, Hongyan

2017-08-18

A novel nanosilica/polydimethylsiloxane (SiO 2 /PDMS) coated stirring bar was adopted in the sorption extraction (SBSE) of phenols in water, and the high performance liquid chromatography-fourier transform infrared spectroscopy (HPLC-FTIR) was subsequently used to determination of phenol concentration. The SiO 2 /PDMS coating was prepared by sol-gel method and characterized with respect to morphology and specific surface area. The results of field-emission scanning electron microscope (FE-SEM) and N 2 adsorption-desorption as well as phenol adsorption experiments denoted that SiO 2 /PDMS has larger surface area and better adsorption capacity than commercial PDMS. The extraction efficiency of phenol with SiO 2 /PDMS coated stirring bar was optimized in terms of ion strength, flow rate of phenol-involved influent, type of desorption solvent and desorption time. More than 75% of phenol desorption efficiency could be kept even after 50 cycles of extraction, indicating the high stability of the SiO 2 /PDMS coated stirring bar. Approximately 0.16 mg/L 2, 5-dimethylphenol (2, 5-DMP), which was 34-fold more toxic than phenol, was detected in water through HPLC-FTIR. However, 2, 5-DMP could be oxidized to 5-methy-2-hydroxy benzaldehyde after disinfection in drinking water treatment process. Therefore, the proposed method of SiO 2 /PDMS-SBSE-HPLC-FTIR is successfully applied in the analysis of phenols isomers in aqueous environment.

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).

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine.

PubMed

Makino, Yukiko

2012-03-01

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d-methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d-methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C18 MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH2 PO4-acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d-methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high-purity d-methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high-purity methamphetamine profiling. Copyright © 2011 John Wiley & Sons, Ltd.

Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration–HPLC-MS

PubMed Central

Chen, Guilin; Guo, Mingquan

2017-01-01

Gymnema sylvestre R. Br. (Asclepiadaceae) has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control) at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS) was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre. In this way, 9 compounds with higher enrichment factors (EFs) were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS) platform in the early anti-diabetic drug discovery stage. PMID:28496409

Rapid Screening for α-Glucosidase Inhibitors from Gymnema sylvestre by Affinity Ultrafiltration-HPLC-MS.

PubMed

Chen, Guilin; Guo, Mingquan

2017-01-01

Gymnema sylvestre R. Br. (Asclepiadaceae) has been known to posses potential anti-diabetic activity, and the gymnemic acids were reported as the main bioactive components in this plant species. However, the specific components responsible for the hypoglycemic effect still remain unknown. In the present study, the in vitro study revealed that the extract of G. sylvestre exhibited significant inhibitory activity against α-glucosidase with IC 50 at 68.70 ± 1.22 μg/mL compared to acarbose (positive control) at 59.03 ± 2.30 μg/mL, which further indicated the potential anti-diabetic activity. To this end, a method based on affinity ultrafiltration coupled with liquid chromatography mass spectrometry (UF-HPLC-MS) was established to rapidly screen and identify the α-glucosidase inhibitors from G. sylvestre . In this way, 9 compounds with higher enrichment factors (EFs) were identified according to their MS/MS spectra. Finally, the structure-activity relationships revealed that glycosylation could decrease the potential antisweet activity of sapogenins, and other components except gymnemic acids in G. sylvestre could also be good α-glucosidase inhibitors due to their synergistic effects. Taken together, the proposed method combing α-glucosidase and UF-HPLC-MS presents high efficiency for rapidly screening and identifying potential inhibitors of α-glucosidase from complex natural products, and could be further explored as a valuable high-throughput screening (HTS) platform in the early anti-diabetic drug discovery stage.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method.

PubMed

Zhang, Pei-Ting; Pan, Bi-Yan; Liao, Qiong-Feng; Yao, Mei-Cun; Xu, Xin-Jun; Wan, Jin-Zhi; Liu, Dan; Xie, Zhi-Yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth.

Simultaneous Quantification of Limonin, Two Indolequinazoline Alkaloids, and Four Quinolone Alkaloids in Evodia rutaecarpa (Juss.) Benth by HPLC-DAD Method

PubMed Central

Zhang, Pei-ting; Pan, Bi-yan; Liao, Qiong-feng; Yao, Mei-cun; Xu, Xin-jun; Wan, Jin-zhi; Liu, Dan; Xie, Zhi-yong

2013-01-01

A simple and efficient HPLC-DAD (225 nm) method was developed and validated for the simultaneous determination of limonin and six key alkaloids (evodiamine, rutaecarpine, 1-methyl-2-undecyl-4(1H)-quinolone, evocarpine, 1-methy-2-[(6Z,9Z)]-6,9-pentadecadienyl-4-(1H)-quinolone, and dihydroevocarpine) in Evodia rutaecarpa (Juss.) Benth, which has been widely used as one of the Traditional Chinese Medicines. The chromatographic separation was carried out on a Hypersil BDS C18 column, and gradient elution was employed with a mobile phase containing acetonitrile and water. Contents of the analytes in 18 batches of samples were analyzed by ultrasonic extraction with ethanol and water mixture (80 : 20, v/v) followed by HPLC analysis. Separation of the seven analytes was achieved within 60 min with good linearity (r > 0.999). The RSD of both the intraday and interday precision was below 1.85%. The accuracy at different concentrations was within the range of 97.91 to 100.49%. Hierarchical clustering analysis was performed to differentiate and classify the samples based on the contents of the seven constituents. This study indicated that the quality control of E. rutaecarpa could be simplified to the measurement of four constituents, and that limonin, 1-methyl-2-undecyl-4(1H)-quinolone, and dihydroevocarpine should also be served as the chemical markers together with evodiamine for the quality control of Evodia rutaecarpa (Juss.) Benth. PMID:23738236

Combined HPLC-CUPRAC (cupric ion reducing antioxidant capacity) assay of parsley, celery leaves, and nettle.

PubMed

Yildiz, Leyla; Başkan, Kevser Sözgen; Tütem, Esma; Apak, Reşat

2008-10-19

This study aims to identify the essential antioxidant compounds present in parsley (Petroselinum sativum) and celery (Apium graveolens) leaves belonging to the Umbelliferae (Apiaceae) family, and in stinging nettle (Urtica dioica) belonging to Urticaceae family, to measure the total antioxidant capacity (TAC) of these compounds with CUPRAC (cupric ion reducing antioxidant capacity) and ABTS spectrophotometric methods, and to correlate the TAC with high performance liquid chromatography (HPLC) findings. The CUPRAC spectrophotometric method of TAC assay using copper(II)-neocuproine (2,9-dimethyl-1,10-phenanthroline) as the chromogenic oxidant was developed in our laboratories. The individual antioxidant constituents of plant extracts were identified and quantified by HPLC on a C18 column using a modified mobile phase of gradient elution comprised of MeOH-0.2% o-phosphoric acid and UV detection for polyphenols at 280 nm. The TAC values of HPLC-quantified antioxidant constituents were found, and compared for the first time with those found by CUPRAC. The TAC of HPLC-quantified compounds accounted for a relatively high percentage of the observed CUPRAC capacities of plant extracts, namely 81% of nettle, 60-77% of parsley (in different hydrolyzates of extract and solid sample), and 41-57% of celery leaves (in different hydrolyzates). The CUPRAC total capacities of the 70% MeOH extracts of studied plants (in the units of mmol trolox g(-1)plant) were in the order: celery leaves>nettle>parsley. The TAC calculated with the aid of HPLC-spectrophotometry did not compensate for 100% of the CUPRAC total capacities, because all flavonoid glycosides subjected to hydrolysis were either not detectable with HPLC, or not converted to the corresponding aglycons (i.e., easily detectable and quantifiable with HPLC) during the hydrolysis step.

Rapid, Standardized Method for Determination of Mycobacterium tuberculosis Drug Susceptibility by Use of Mycolic Acid Analysis▿

PubMed Central

Parrish, Nicole; Osterhout, Gerard; Dionne, Kim; Sweeney, Amy; Kwiatkowski, Nicole; Carroll, Karen; Jost, Kenneth C.; Dick, James

2007-01-01

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates. PMID:17913928

Application of quality by design concept to develop a dual gradient elution stability-indicating method for cloxacillin forced degradation studies using combined mixture-process variable models.

PubMed

Zhang, Xia; Hu, Changqin

2017-09-08

Penicillins are typical of complex ionic samples which likely contain large number of degradation-related impurities (DRIs) with different polarities and charge properties. It is often a challenge to develop selective and robust high performance liquid chromatography (HPLC) methods for the efficient separation of all DRIs. In this study, an analytical quality by design (AQbD) approach was proposed for stability-indicating method development of cloxacillin. The structures, retention and UV characteristics rules of penicillins and their impurities were summarized and served as useful prior knowledge. Through quality risk assessment and screen design, 3 critical process parameters (CPPs) were defined, including 2 mixture variables (MVs) and 1 process variable (PV). A combined mixture-process variable (MPV) design was conducted to evaluate the 3 CPPs simultaneously and a response surface methodology (RSM) was used to achieve the optimal experiment parameters. A dual gradient elution was performed to change buffer pH, mobile-phase type and strength simultaneously. The design spaces (DSs) was evaluated using Monte Carlo simulation to give their possibility of meeting the specifications of CQAs. A Plackett-Burman design was performed to test the robustness around the working points and to decide the normal operating ranges (NORs). Finally, validation was performed following International Conference on Harmonisation (ICH) guidelines. To our knowledge, this is the first study of using MPV design and dual gradient elution to develop HPLC methods and improve separations for complex ionic samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Validated stability-indicating HPLC method for paricalcitol in pharmaceutical dosage form according to ICH guidelines: application to stability studies.

PubMed

Ortega, Raquel; Navas, Natalia; Salmerón, Antonio; Cabeza, José; Capitán-Vallvey, Luís F

2011-01-01

A stability-indicating HPLC method with diode array detection for the determination of paricalcitol, a synthetic vitamin D2 analog, was developed. Analytical parameters were studied according to International Conference on Harmonization guidelines. A C18 column (250 x 4.6 mm, 5 microm particle size) maintained at 25 degrees C was used as the stationary phase, and acetonitrile-water (70 + 30, v/v) as the mobile phase. Chromatograms were recorded at 250 nm. In forced degradation studies, the effects of acid, base, oxidation, temperature, and UV light were investigated and showed no interference with the drug peak. The method was found to be linear (r = 0.9989) at concentrations ranging from 0.6 to 10.0 mg/L paricalcitol, precise (repeatability and intermediate precision estimated as RSD less than 3.5%), accurate (recoveries higher than 95%), specific, and robust. The LOD and LOQ were 0.6 and 0.2 mg/L, respectively. The validated method was used for paricalcitol determination in a formal stability study of its pharmaceutical dosage form in preloaded syringes. The stability of a diluted solution of its pharmaceutical form in Viaflo bags was also tested. The results showed that paricalcitol was stable in preloaded syringes during a period of 30 days from preparation in the different storage conditions tested (room temperature without protection from daylight and 4.4 degrees C with protection from daylight). On the contrary, paricalcitol was quickly lost when stored in Viaflo bags by adsorption onto the walls of the container.

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.

Stability of aspartame and neotame in pasteurized and in-bottle sterilized flavoured milk.

PubMed

Kumari, Anuradha; Choudhary, Sonika; Arora, Sumit; Sharma, Vivek

2016-04-01

Analytical high performance liquid chromatography (HPLC) conditions were standardized along with the isolation procedure for separation of aspartame and neotame in flavoured milk (pasteurized and in-bottle sterilized flavoured milk). The recovery of the method was approximately 98% for both aspartame and neotame. The proposed HPLC method can be successfully used for the routine determination of aspartame and neotame in flavoured milk. Pasteurization (90 °C/20 min) resulted in approximately 40% loss of aspartame and only 8% of neotame was degraded. On storage (4-7°C/7 days) aspartame and neotame content decreased significantly (P<0.05) from 59.70% to 44.61% and 91.78% to 87.18%, respectively. Sterilization (121 °C/15 min) resulted in complete degradation of aspartame; however, 50.50% of neotame remained intact. During storage (30 °C/60 days) neotame content decreased significantly (P<0.05) from 50.36% to 8.67%. Results indicated that neotame exhibited better stability than aspartame in both pasteurized and in-bottle sterilized flavoured milk. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of fumonisin contamination using HPLC and ELISA methods in bt and near-isogenic maize hybrids infested with European corn borer or western bean cutworm.

PubMed

Bowers, Erin; Hellmich, Richard; Munkvold, Gary

2014-07-09

Field trials were conducted from 2007 to 2010 to compare grain fumonisin levels among non-Bt maize hybrids and Bt hybrids with transgenic protection against manual infestations of European corn borer (ECB) and Western bean cutworm (WBC). HPLC and ELISA were used to measure fumonisin levels. Results of the methods were highly correlated, but ELISA estimates were higher. Bt hybrids experienced less insect injury, Fusarium ear rot, and fumonisin contamination compared to non-Bt hybrids. WBC infestation increased fumonisin content compared to natural infestation in non-Bt and hybrids expressing Cry1Ab protein in five of eight possible comparisons; in Cry1F hybrids, WBC did not impact fumonisins. These results indicate that WBC is capable of increasing fumonisin levels in maize. Under WBC infestation, Cry1F mitigated this risk more consistently than Cry1Ab or non-Bt hybrids. Transgenically expressed Bt proteins active against multiple lepidopteran pests can provide broad, consistent reductions in the risk of fumonisin contamination.

LC-NMR Technique in the Analysis of Phytosterols in Natural Extracts

PubMed Central

Horník, Štěpán; Sajfrtová, Marie; Sýkora, Jan; Březinová, Anna; Wimmer, Zdeněk

2013-01-01

The ability of LC-NMR to detect simultaneously free and conjugated phytosterols in natural extracts was tested. The advantages and disadvantages of a gradient HPLC-NMR method were compared to the fast composition screening using SEC-NMR method. Fractions of free and conjugated phytosterols were isolated and analyzed by isocratic HPLC-NMR methods. The results of qualitative and quantitative analyses were in a good agreement with the literature data. PMID:24455424

Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

Treesearch

Rakesh Minocha; Stephanie Long

2004-01-01

The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

Conventional high-performance liquid chromatography versus derivative spectrophotometry for the determination of 1,3,6-pyrenetrisulfonic acid trisodium salt and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt in the color additive D&C Green No. 8 (Pyranine).

PubMed

Jitian, Simion; White, Samuel R; Yang, H-H Wendy; Weisz, Adrian

2014-01-10

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Green No. 8 (Colour Index No. 59040) limit the levels of the subsidiary colors 1,3,6-pyrenetrisulfonic acid trisodium salt (P3S) and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (P4S). The present paper describes a comparative study of two possible methods to replace the currently used multi-step TLC/spectrophotometry method of separating and quantifying the minor components P3S and P4S in G8. One of the new approaches uses conventional high-performance liquid chromatography (HPLC) and the other, derivative spectrophotometry. While the derivative spectrophotometric method was shown to be inadequate for the analysis of minor components overwhelmed by components of much higher concentration, the HPLC method was proven highly effective. The closely related, very polar compounds P3S and P4S were separated by the new HPLC method in less than 4 min using a conventional HPLC instrument. P3S and P4S were quantified by using five-point calibration curves with data points that ranged from 0.45 to 7.63% and from 0.13 to 1.82%, by weight, for P3S and P4S, respectively. The HPLC method was applied to the analysis of test portions from 20 batches of D&C Green No. 8 submitted to the U.S. Food and Drug Administration for certification. Published by Elsevier B.V.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

PubMed

Kang, Kyo Bin; Ryu, Jayoung; Cho, Youngwoong; Choi, Sang-Zin; Son, Miwon; Sung, Sang Hyun

2017-05-01

DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative HPLC Analysis of Rosmarinic Acid in Extracts of "Melissa officinalis" and Spectrophotometric Measurement of Their Antioxidant Activities

ERIC Educational Resources Information Center

Canelas, Vera; da Costa, Cristina Teixeira

2007-01-01

The students prepare tea samples using different quantities of lemon balm leaves ("Melissa officinalis") and measure the rosmarinic acid contents by an HPLC-DAD method. The antioxidant properties of the tea samples are evaluated by a spectrophotometric method using a radical-scavenging assay with DPPH. (2,2-diphenyl-1-picrylhydrazyl). Finally the…

Quantitative determination of a-Arbutin, ß-Arbutin, Kojic acid, nicotinamide, hydroquinone, resorcinol, 4-methoxyphenol, 4-ethoxyphenol and ascorbic acid from skin whitening Products by HPLC-UV

USDA-ARS?s Scientific Manuscript database

Development of an analytical method for the simultaneous determination of multifarious skin whitening agents will provide an efficient tool to analyze skin whitening cosmetics. An HPLC-UV method was developed for quantitative analysis of six commonly used whitening agents, a-arbutin, ß-arbutin, koji...

Applicability of Screening Tests for Oxytetracycline in the Milk of Three Breeds of Goats.

PubMed

Attaie, Rahmat; Bsharat, Mohammed; Mora-Gutierrez, Adela

2016-06-01

Antibiotics are widely used in animal husbandry, and the presence of antibiotic residues in milk is a health hazard. The objectives of this study were to determine residual amounts of oxytetracycline in the milk of three breeds of goats using high-pressure liquid chromatography (HPLC) analysis and screening tests. It was also essential to explore the safe withdrawal period of oxytetracycline in lactating goats and examine the applicability of Charm ROSA and SNAP screening tests. The qualitative results of these tests were compared with the quantitative results of the HPLC method. Fifteen milking does, five each from the Alpine, LaMancha, and Nubian breeds, were selected from the herd at Prairie View A&M University. Milk samples containing antibiotic residues were deproteinized by HCl and acetonitrile, and then oxytetracycline was extracted from the supernatant. The residues of oxytetracycline in goat's milk up to 110 h after injection were qualitatively detected using the Charm ROSA test. Similarly, the SNAP test detected the antibiotic residues in milk up to 110 h after treatment. The HPLC results indicated that oxytetracycline residues in milk from Alpine goats were below the tolerance level (300 ng/ml) 82 h after drug treatment (72 h for LaManchas, 58 h for Nubians); however, the results of the screening tests would indicate longer withdrawal periods for milk from the breeds of goats studied, which would result in economic losses to goat's milk producers. The results of this study also indicated that oxytetracycline was not stable in raw goat's milk at refrigeration temperature or during pasteurization and that the concentrations decreased significantly. Commercial goat's milk is usually exposed to several hours of refrigeration and then to pasteurization. The results of this study indicated that, if oxytetracycline was present in raw goat's milk, the concentration would decrease significantly before it was marketed.

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

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.

Two-dimensional multi-heart cutting centrifugal partition chromatography-liquid chromatography for the preparative isolation of antioxidants from Edelweiss plant.

PubMed

Marlot, Léa; Batteau, Magali; Escofet, Marie-Claire; Nuccio, Sylvie; Coquoin, Véronique; De Vaumas, René; Faure, Karine

2017-06-30

The Edelweiss plant has been recognized as a very valuable source of anti-aging principles due to its composition of antioxidants compounds: leontopodic acid A and 3,5-dicaffeoylquinic acid. In this work, off-line multi-heart cutting CPC-LC separation was set up at industrial scale in order to isolate and produce new high quality reference material of these two antioxidants from Edelweiss. For this purpose, CPC and HPLC methods were developed and optimized at laboratory scale and a comprehensive CPCxHPLC analysis of the crude extract was established. Thereby, the CPC method led to a first separation of the target compounds according to their partition coefficient in the solvent system and the HPLC method was performed on the recovered fractions to lead to a second separation. A 2D CPCxHPLC plot was established in order to know the fractions to select at the industrial scale. Then, the CPC and HPLC methods were transferred at industrial scale and the multi-heart cutting CPC-LC was performed in off-line mode. Using CPC with methyl ter-butyl ether-water 1:1 (v/v) solvent system and LC with Denali C18 column, 2g of crude extract sample were injected and leontopodic acid A and 3,5-dicaffeoylquinic acid were recovered with purity over 97%. The compounds were identified by MS and NMR. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

PubMed

Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

2016-06-01

Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate.

PubMed

Toporisic, Rebeka; Mlakar, Anita; Hvala, Jernej; Prislan, Iztok; Zupancic-Kralj, Lucija

2010-06-05

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC-MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 microg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 microg/mL and LOQ was 0.5 microg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC-MS/MS(n), Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity. Copyright (c) 2010. Published by Elsevier B.V.

Simultaneous determination of three polyphenols in rat plasma after orally administering hawthorn leaves extract by the HPLC method.

PubMed

Ying, Xixiang; Meng, Xiansheng; Wang, Siyuan; Wang, Dong; Li, Haibo; Wang, Bing; Du, Yang; Liu, Xun; Zhang, Wenjie; Kang, Tingguo

2012-01-01

A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C₁₈ analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6 : 1.5 : 18.5 : 74, v/v/v/v). The calibration curves were linear over the range of 2.5-500, 0.2-25 and 0.25-12.5 µg mL⁻¹ for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5-10  mL kg⁻¹.

On-line MSPD-SPE-HPLC/FLD analysis of polycyclic aromatic hydrocarbons in bovine tissues.

PubMed

Gutiérrez-Valencia, Tania M; García de Llasera, Martha P

2017-05-15

A fast method was optimized and validated for simultaneous trace determination of four polycyclic aromatic hydrocarbons: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in bovine tissues. The determination was performed by matrix solid-phase dispersion (MSPD) coupled on-line to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection (FLD). The sample was dispersed on C 18 silica sorbent and then the on-line MSPD-SPE-HPLC/FLD method was applied. Several parameters were optimized: cleaning and elution sequences applied to the MSPD cartridge, the flow rate and dilution of extract used for SPE loading. The on-line method was validated over a concentration range of 0.1-0.6ngg -1 obtaining good linearity (r⩾0.998) and precision (RSD)⩽10%. Recovery ranged from 96 to 99% and the limits of detection were 0.012ngg -1 . This methodology was applied to liver samples from unhealthy animals. The results demonstrate that MSDP-SPE-HPLC/FLD method provides reliable, sensitive, accurate and fast data to the food control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ultrafast UPLC-ESI-MS and HPLC with monolithic column for determination of principal flavor compounds in vanilla pods.

PubMed

Sharma, Upendra K; Sharma, Nandini; Sinha, Arun K; Kumar, Neeraj; Gupta, Ajai P

2009-10-01

In this study, two novel chromatographic methods based on monolithic column high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were developed for the ultrafast determination of principal flavor compounds namely vanillin, vanillic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde in ethanolic extracts of Vanilla planifolia pods. Good separation was achieved within 2.5 min using Chromolith RP18e column (100 mm x 4.6 mm) for HPLC and Acquity BEH C-18 (100 mm x 2.1 mm, 1.7 microm) column for UPLC. Both methods were compared in terms of total analysis time, mobile phase consumption, sensitivity, and validation parameters like precision, accuracy, LOD, and LOQ. Further, system suitability test data including resolution, capacity factor, theoretical plates, and tailing factor was determined for both the methods by ten replicate injections. Monolithic column based HPLC gave better results for most of the selected parameters while UPLC was found to be more eco-friendly with low mobile phase consumption and better sensitivity. Both methods may be used conveniently for the high throughput analysis of large number of samples in comparison to traditional particulate column.

Quantitative and qualitative analysis of the novel antitumor 1,3,4-oxadiazole derivative (GLB) and its metabolites using HPLC-UV and UPLC-QTOF-MS

PubMed Central

Li, Pu; Wang, Xin; Li, Jian; Meng, Zhi-Yun; Li, Shu-Chun; Li, Zhong-Jun; Lu, Ying-Yuan; Ren, Hong; Lou, Ya-Qing; Lu, Chuang; Dou, Gui-Fang; Zhang, Guo-Liang

2015-01-01

Fructose-based 3-acetyl-2,3-dihydro-1,3,4-oxadiazole (GLB) is a novel antitumor agent and belongs to glycosylated spiro-heterocyclic oxadiazole scaffold derivative. This research first reported a simple, specific, sensitive and stable high performance liquid chromatography -ultraviolet detector (HPLC-UV) method for the quantitative determination of GLB in plasma. In this method, the chromatographic separation was achieved with a reversed phase C18 column. The calibration curve for GLB was linear at 300 nm. The lower limit of quantification was 10 ng/mL. The precision, accuracy and stability of the method were validated adequately. This method was successfully applied to the pharmacokinetic study in rats for detection of GLB after oral administration. Moreover, the structures of parent compound GLB and its two major metabolites M1 and M2 were identified in plasma using an ultra performance liquid chromatography- electrospray ionization-quadrupole-time of flight- mass spectrometry (UPLC-ESI-QTOF-MS) method. Our results indicated that the di-hydroxylation (M1) and hydroxylation (M2) of GLB are the major metabolites. In conclusion, the present study provided valuable information on an analytical method for the determination of GLB and its metabolites in rats, can be used to support further developing of this antitumor agent. PMID:26148672

A validated RP-HPLC method for simultaneous determination of propranolol and valsartan in bulk drug and gel formulation

PubMed Central

Imam, Syed Sarim; Ahad, Abdul; Aqil, Mohammed; Sultana, Yasmin; Ali, Asgar

2013-01-01

Objective: A simple, precise, and stability indicating high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of propranolol hydrochloride and valsartan in pharmaceutical dosage form. Materials and Methods: The method involves the use of easily available inexpensive laboratory reagents. The separation was achieved on Hypersil ODS C-18 column (250*4.6 mm, i.d., 5 μm particle size) with isocratic flow with UV detector. The mobile phase at a flow rate of 1.0 mL/min consisted of acetonitrile, methanol, and 0.01 M disodium hydrogen phosphate (pH 3.5) in the ratio of 50:35:15 v/v. Results: A linear response was observed over the concentration range 5-50 μg/mL of propranolol and the concentration range 4-32 μg/mL of valsartan. Limit of detection and limit of quantitation for propranolol were 0.27 μg/mL and 0.85 μg/mL, and for valsartan were 0.45 μg/mL and 1.39 μg/mL, respectively. The method was successfully validated in accordance to ICH guidelines acceptance criteria for linearity, accuracy, precision, specificity, robustness. Conclusion: The analysis concluded that the method was selective for simultaneous estimation of propranolol and valsartan can be potentially used for the estimation of these drugs in combined dosage form. PMID:23559826

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.

[Simultaneous determination of six fluorescent whitening agents in plastic and paper packaging materials by high performance liquid chromatography].

PubMed

Zhang, Juzhou; Ji, Shuilin; Cai, Huimei; Li, Jing; Wang, Yongxin; Wang, Jingqiu

2017-11-08

A novel analytical method was developed for the simultaneous determination of six fluorescent whitening agents (FWAs:FWA 135, FWA 184, FWA 185, FWA 199, FWA 378 and FWA 393) in paper and plastic food packaging materials by high performance liquid chromatography with fluorescence detection (HPLC-FLD). The sample was extracted with mixed solution of chloroform and acetonitrile (3:7, v/v), then cleaned up by HLB solid phase extraction column. Qualitative and quantitative analyses were carried out by HPLC. The sample was separated on a Phenomenex C18 column using acetonitrile and 5 mmol/L ammonium acetate aqueous solution as mobile phases. The results indicated that the linear range of FWA393 was 15-1500 μg/L and the linear ranges of the other five FWAs were 5-500 μg/L with correlation coefficients greater than 0.999. The recoveries in spiked samples were between 80.4% and 125.0% with RSDs ( n =6) of 1%-13%. Furthermore, this method was applied to analyze 12 samples in the market to verify the practicality of the method. The method showed the advantages of simplicity, high recovery and good precision, and is suitable for the detection of the six fluorescent whitening agents in food packaging materials.

Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

PubMed

Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

2014-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites.

Chiral separation of the β2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

PubMed

Ullrich, Thomas; Wesenberg, Dirk; Bleuel, Corinna; Krauss, Gerd-Joachim; Schmid, Martin G; Weiss, Michael; Gübitz, Gerald

2010-10-01

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

PubMed

Douša, Michal; Srbek, Jan; Rádl, Stanislav; Cerný, Josef; Klecán, Ondřej; Havlíček, Jaroslav; Tkadlecová, Marcela; Pekárek, Tomáš; Gibala, Petr; Nováková, Lucie

2014-06-01

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection †

PubMed Central

Karr, Dale B.; Waters, James K.; Emerich, David W.

1983-01-01

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. PMID:16346443

Quantitative determination of limonin in citrus juices by HPLC using computerized solvent optimization.

PubMed

Shaw, P E; Wilson, C W

1988-09-01

The commercially available computer program, Drylab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-micron C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

New analytical technique for carbon dioxide absorption solvents

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

Pouryousefi, F.; Idem, R.O.

2008-02-15

The densities and refractive indices of two binary systems (water + MEA and water + MDEA) and three ternary systems (water + MEA + CO{sub 2}, water + MDEA + CO{sub 2}, and water + MEA + MDEA) used for carbon dioxide (CO{sub 2}) capture were measured over the range of compositions of the aqueous alkanolamine(s) used for CO{sub 2} absorption at temperatures from 295 to 338 K. Experimental densities were modeled empirically, while the experimental refractive indices were modeled using well-established models from the known values of their pure-component densities and refractive indices. The density and Gladstone-Dale refractive indexmore » models were then used to obtain the compositions of unknown samples of the binary and ternary systems by simultaneous solution of the density and refractive index equations. The results from this technique have been compared with HPLC (high-performance liquid chromatography) results, while a third independent technique (acid-base titration) was used to verify the results. The results show that the systems' compositions obtained from the simple and easy-to-use refractive index/density technique were very comparable to the expensive and laborious HPLC/titration techniques, suggesting that the refractive index/density technique can be used to replace existing methods for analysis of fresh or nondegraded, CO{sub 2}-loaded, single and mixed alkanolamine solutions.« less

Degradation of components in drug formulations: a comparison between HPLC and DSC methods.

PubMed

Ceschel, G C; Badiello, R; Ronchi, C; Maffei, P

2003-08-08

Information about the stability of drug components and drug formulations is needed to predict the shelf-life of the final products. The studies on the interaction between the drug and the excipients may be carried out by means of accelerated stability tests followed by analytical determination of the active principle (HPLC and other methods) and by means of the differential scanning calorimetry (DSC). This research has been focused to the acetyl salicylic acid (ASA) physical-chemical characterisation by using DSC method in order to evaluate its compatibility with some of the most used excipients. It was possible to show, with the DSC method, the incompatibility of magnesium stearate with ASA; the HPLC data confirm the reduction of ASA concentration in the presence of magnesium stearate. With the other excipients the characteristic endotherms of the drug were always present and no or little degradation was observed with the accelerated stability tests. Therefore, the results with the DSC method are comparable and in good agreement with the results obtained with other methods.

Comparison of cyclosporine determinations in whole blood by three different methods. HPLC, /sup 125/I RIA and /sup 3/H RIA

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

Huang, W.Y.; Lipsey, A.I.; Cheng, M.H.

1987-04-01

The authors have analyzed and compared the cyclosporine concentrations in whole blood specimens from pediatric renal transplant patients using three different methods: high-performance liquid chromatography (HPLC) (5u C18 reverse-phase column), /sup 3/H radioimmunoassay (RIA), and /sup 125/I RIA (substituted /sup 3/H-tracer in Sandoz Kit with /sup 125/I tracer. Results obtained by the /sup 125/I RIA correlated well with results obtained by the /sup 3/H RIA. Both RIA methods had similar correlation with the HPLC method. The /sup 125/I RIA method showed higher sensitivity and greater precision than the /sup 3/H RIA method. The authors conclude that the /sup 125/I RIAmore » method can be used for cyclosporine determination in whole blood specimens. The use of the /sup 125/I RIA provides a simple and rapid method with higher counting efficiency and less background quenching than the /sup 3/H RIA method, which requires cumbersome liquid scintillation counting procedures.« less

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

Validation of a method for the quantitation of ghrelin and unacylated ghrelin by HPLC.

PubMed

Staes, Edith; Rozet, Eric; Ucakar, Bernard; Hubert, Philippe; Préat, Véronique

2010-02-05

An HPLC/UV method was first optimized for the separation and quantitation of human acylated and unacylated (or des-acyl) ghrelin from aqueous solutions. This method was validated by an original approach using accuracy profiles based on tolerance intervals for the total error measurement. The concentration range that achieved adequate accuracy extended from 1.85 to 59.30microM and 1.93 to 61.60microM for acylated and unacylated ghrelin, respectively. Then, optimal temperature, pH and buffer for sample storage were determined. Unacylated ghrelin was found to be stable in all conditions tested. At 37 degrees C acylated ghrelin was stable at pH 4 but unstable at pH 7.4, the main degradation product was unacylated ghrelin. Finally, this validated HPLC/UV method was used to evaluate the binding of acylated and unacylated ghrelin to liposomes.

Measurement of H2S in vivo and in vitro by the monobromobimane method

PubMed Central

Shen, Xinggui; Kolluru, Gopi K.; Yuan, Shuai; Kevil, Christopher

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 RP-HPLC. The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide-dibimane. The resultant fluorescent sulfide-dibimane (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. PMID:25725514

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.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

PubMed

Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

1998-07-03

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

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.

Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum by selective solid phase extraction and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry.

PubMed

Hu, Xin; Zhao, Huading; Shi, Shuyun; Li, Hui; Zhou, Xiaoling; Jiao, Feipeng; Jiang, Xinyu; Peng, Dongming; Chen, Xiaoqin

2015-08-10

The complexity of natural products always leads to the co-elution of interfering compounds with bioactive compounds, which then has a detrimental effect on structural elucidation. Here, a new method, based on selective solid phase extraction combined with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking and high performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (HPLC-DAD-QTOF-MS/MS), is described for sensitive screening, selective extraction and identification of polyphenolic antioxidants in Polygonatum odoratum. First, 25 polyphenolic antioxidants (1-25) were screened by DPPH spiking with HPLC. Second, polydopamine coated Fe3O4 microspheres (Fe3O4@PDA) were prepared to selectively extract target antioxidants with extraction efficiency from 55% to 100% when the amount of Fe3O4@PDA, extraction time, desorption solvent and time were 10mg, 20 min, acetonitrile, and 5 min. Third, 25 antioxidants (10 cinnamides and 15 homoisoflavanones) were identified by HPLC-DAD-QTOF-MS/MS. Furthermore, the DPPH scavenging activities of purified compounds (IC50, 1.6-32.8 μg/mL) validated the method. Among the identified antioxidants, four of them (12, 13, 18 and 19) were new compounds, four of them (2, 4, 8 and 14) were first obtained from family Liliaceae, five of them (1, 3, 5, 7 and 9) were first reported in genus Polygonatum, while one compound (24) was first identified in this species. The results indicated that the proposed method was an efficient and sensitive approach to explore polyphenolic antioxidants from complex natural products. Copyright © 2015 Elsevier B.V. All rights reserved.

Superparamagnetic graphene oxide-based dispersive-solid phase extraction for preconcentration and determination of tamsulosin hydrochloride in human plasma by high performance liquid chromatography-ultraviolet detection.

PubMed

Pashaei, Yaser; Ghorbani-Bidkorbeh, Fatemeh; Shekarchi, Maryam

2017-05-26

In the present study, superparamagnetic graphene oxide-Fe 3 O 4 nanocomposites were successfully prepared by a modified impregnation method (MGO mi ) and their application as a sorbent in the magnetic-dispersive solid phase extraction (M-dSPE) mode to the preconcentration and determination of tamsulosin hydrochloride (TMS) in human plasma was investigated by coupling with high performance liquid chromatography-ultraviolet detection (HPLC-UV). The structure, morphology and magnetic properties of the prepared nanocomposites were characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and vibrating sample magnetometry (VSM). Some factors affecting the extraction efficiency, including the pH value, amount of sorbent, extraction time, elution solvent and its volume, and desorption time were studied and optimized. Magnetic nanocomposites plasma extraction of TMS following HPLC analyses showed a linear calibration curve in the range of 0.5-50.0ngmL -1 with an acceptable correlation coefficient (R 2 =0.9988). The method was sensitive, with a low limit of detection (0.17ngmL -1 ) and quantification (0.48ngmL -1 ). Inter- and intra-day precision expressed as relative standard deviation (n=3) and the preconcentration factor, were found to be 5.6-7.2%, 2.9-4.2% and 10, respectively. Good recoveries (98.1-101.4%) with low relative standard deviations (4.2-5.0%) indicated that the matrices under consideration do not significantly affect the extraction process. Due to its high precision and accuracy, the developed method may be a HPLC-UV alternative with M-dSPE for bioequivalence analysis of TMS in human plasma. Copyright © 2017 Elsevier B.V. All rights reserved.

Chemometrics enhanced HPLC-DAD performance for rapid quantification of carbamazepine and phenobarbital in human serum samples.

PubMed

Vosough, Maryam; Ghafghazi, Shiva; Sabetkasaei, Masoumeh

2014-02-01

This paper describes development and validation of a simple and efficient bioanalytical procedure for simultaneous determination of phenobarbital and carbamazepine in human serum samples using high performance liquid chromatography with photodiode-array detection (HPLC-DAD) regarding a fast elution methodology in less than 5 min. Briefly, this method consisted of a simple deproteinization step of serum samples followed by HPLC analysis on a Bonus-RP column using an isocratic mode of elution with acetonitrile/K2HPO4 (pH=7.5) buffer solution (45:55). Due to the presence of serum endogenous components as non-calibrated components in the sample, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS), has been applied on a set of absorbance matrices collected as a function of retention time and wavelengths. Acceptable resolution and quantification results were achieved in the presence of matrix interferences and the second-order advantage was fully exploited. The average recoveries for carbamazepine and phenobarbital were 89.7% and 86.1% and relative standard deviation values were lower than 9%. Additionally, computed elliptical joint confidence region (EJCR) confirmed the accuracy of the proposed method and indicated the absence of both constant and proportional errors in the predicted concentrations. The developed method enabled the determination of the analytes in different serum samples in the presence of overlapped profiles, while keeping experimental time and extraction steps at minimum. Finally, the serum concentration levels of carbamazepine in three time intervals were reported for morphine-dependents who had received carbamazepine for treating their neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

Development, Validation and Application of a Stability Indicating HPLC Method to Quantify Lidocaine from Polyethylene-co-Vinyl Acetate (EVA) Matrices and Biological Fluids.

PubMed

Bhusal, Prabhat; Sharma, Manisha; Harrison, Jeff; Procter, Georgina; Andrews, Gavin; Jones, David S; Hill, Andrew G; Svirskis, Darren

2017-09-01

An efficient and cost-effective quantification procedure for lidocaine by HPLC has been developed to estimate lidocaine from an EVA matrix, plasma, peritoneal fluid and intra-articular fluid (IAF). This method guarantees the resolution of lidocaine from the degradation products obtained from alkaline and oxidative stress. Chromatographic separation of lidocaine was achieved with a retention time of 7 min using a C18 column with a mobile phase comprising acetonitrile and potassium dihydrogen phosphate buffer (pH 5.5; 0.02 M) in the ratio of 26:74 at a flow rate of 1 mL min-1 with detection at 230 nm. Instability of lidocaine was observed to an oxidizing (0.02% H2O2) and alkaline environments (0.1 M NaOH). The calibration curve was found to be linear within the concentration range of 0.40-50.0 μg/mL. Intra-day and inter-day accuracy ranged between 95.9% and 99.1%, with precision (% RSD) below 6.70%. The limit of quantification and limit of detection were 0.40 μg/mL and 0.025 μg/mL, respectively. The simple extraction method described enabled the quantification of lidocaine from an EVA matrix using dichloromethane as a solvent. The assay and content uniformity of lidocaine within an EVA matrix were 103 ± 3.60% and 100 ± 2.60%, respectively. The ability of this method to quantify lidocaine release from EVA films was also demonstrated. Extraction of lidocaine from plasma, peritoneal fluid and IAF followed by HPLC analysis confirmed the utility of this method for ex vivo and in vivo studies where the calibration plot was found to be linear from 1.60 to 50.0 μg/mL. © Crown copyright 2017.

Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods

PubMed Central

Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.

2015-01-01

Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabolites of tamoxifen. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. We analyzed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentration for the LC-MS/MS (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108[55]ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL), the methods did not show any significant differences. Our results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

Analysis of a spacecraft instrument ball bearing assembly lubricated by a perfluoroalkylether

NASA Technical Reports Server (NTRS)

Morales, W.; Jones, W. R., Jr.; Buckley, D. H.

1986-01-01

An analysis of a spacecraft instrument ball bearing assembly, subjected to a scanning life test, was performed to determine the possible case of rotational problems involving these units aboard several satellites. The analysis indicated an ineffective transfer of a fluorinated liquid lubricant from a phenolic retainer to the bearing balls. Part of the analysis led to a novel HPLC separation method employing a fluorinated mobile phase in conjunction with silica based size exclusion columns.

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

Erroneous HbA1c results in a patient with elevated HbC and HbF.

PubMed

Adekanmbi, Joy; Higgins, Trefor; Rodriguez-Capote, Karina; Thomas, Dylan; Winterstein, Jeffrey; Dixon, Tara; Gifford, Jessica L; Krause, Richard; Venner, Allison A; Clarke, Gwen; Estey, Mathew P

2016-11-01

HbA1c is used in the diagnosis and monitoring of diabetes mellitus (DM). Interference from hemoglobin variants is a well-described phenomenon, particularly with HPLC-based methods. While immunoassays may generate more reliable HbA1c results in the presence of some variants, these methods are susceptible to negative interference from high concentrations of HbF. We report a case where an accurate HbA1c result could not be obtained by any available method due to the presence of a compound hemoglobinopathy. HbA1c was measured by HPLC, immunoassay, and capillary electrophoresis. Hemoglobinopathy investigation consisted of a CBC, hemoglobin fractionation by HPLC and electrophoresis, and molecular analysis. HbA1c analysis by HPLC and capillary electrophoresis gave no result. Analysis by immunoassay yielded HbA1c results of 5.9% (Siemens DCA 2000+) and 5.1% (Roche Integra), which were inconsistent with other markers of glycemic control. Hemoglobinopathy investigation showed HbC with the hereditary persistence of fetal hemoglobin-2 Ghana deletion. Reliable HbA1c results may be unobtainable in the presence of some hemoglobinopathies. HPLC and capillary electrophoresis alerted the laboratory to the presence of an unusual hemoglobinopathy. Immunoassays generated falsely low results without warning, which could lead to missed diagnoses and under treatment of patients with DM. Copyright © 2016 Elsevier B.V. All rights reserved.

Using milk fat to reduce the irritation and bitter taste of ibuprofen

PubMed Central

Bennett, Samantha M.; Zhou, Lisa; Hayes, John E.

2012-01-01

Bitterness and irritation elicited by pharmaceutically active molecules remain problematic for pediatric medications, fortified foods and dietary supplements. Few effective methods exist for reducing these unpalatable sensations, negatively impacting medication compliance and intake of beneficial phytonutrients. A physicochemical approach to masking these sensations may be the most successful approach for generalizability to a wide range of structurally and functionally unique compounds. Here, solutions of the non-steroidal anti- inflammatory drug, ibuprofen, were prepared in milk products with varying fat content. Our hypothesis, based on other reports of similar phenomena, was that increasing the fat content would cause ibuprofen to selectively partition into the fat phase, thereby reducing interaction with sensory receptors and decreasing adversive sensations. Quantification of the aqueous concentration of ibuprofen was performed using an isocratic HPLC method coupled with an external standard curve. Sensory testing showed a modest but significant decrease (~20%) in irritation ratings between the skim milk (0% fat) and the half-and-half (11% fat) samples, indicating that increased fat may contribute to a reduced sensory response. Bitterness was not reduced, remaining constant over all fat levels. The HPLC results indicate a constant amount of ibuprofen remained in the aqueous phase regardless of fat level, so a simple partitioning hypothesis cannot explain the reduced irritancy ratings. Association of ionized ibuprofen with continuous phase solutes such as unabsorbed protein should be explored in future work. PMID:23527314

High-performance liquid chromatography-tandem mass spectrometry as a reference for analysis of tacrolimus to assess two immunoassays in patients with liver and renal transplants.

PubMed

Salm, P; Taylor, P J; Clark, A; Balderson, G A; Grygotis, A; Norris, R L; Lynch, S V; Shaw, L M; Pond, S M

1997-12-01

The accuracy and imprecision of three assays used for therapeutic monitoring of tacrolimus were tested using blood-containing weighed-in amounts of the drug, an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA I), and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) assay. Accuracy was acceptable for the HPLC-MS2 assay at all concentrations tested (< 10% deviation) and for the ELISA at 1.0 and 4.0 microg/l. Accuracy was not acceptable for the ELISA at 15.0 and 50.0 microg/l or for the MEIA I at all concentrations tested. Imprecision was acceptable for the HPLC-MS2 assay at all concentrations tested (coefficient of variation < 10%), for the ELISA at 15.0 and 50.0 microg/l, and for the MEIA I at 15.0 and 50.0 microg/l. Imprecision was not acceptable for the ELISA at 1.0 and 4.0 microg/l or for the MEIA I at 1.0 and 4.0 microg/l. This assessment with weighed-in amounts of tacrolimus verified the HPLC-MS2 assay as a reference method. The performance of the two immunoassays with HPLC-MS2 was then compared in the clinical setting using blood from patients with liver (n = 30) and renal (n = 37) transplants. In the liver transplant group (127 samples), the range of tacrolimus concentrations measured by HPLC-MS2, ELISA, and MEIA I was 1.9 to 31.8, 2.1 to 35.0, and less than 0.1 to 36.5 mg/l, respectively. In the renal transplant group (129 samples), the ranges were 1.7 to 26.1, 1.9 to 24.4, and 0.9 to 28.5 microg/l, respectively. Compared with the HPLC-MS2, the ELISA had minimal bias (0.1 to 0.2 microg/l) but unacceptable variability in values (SD > 13%). The MEIA I had unacceptable bias (1.7-1.8 microg/l) and variability (SD > 23%). These data indicated that neither the ELISA nor MEIA I is interchangeable with HPLC-MS2. Moreover, in view of the current trend to reduce the therapeutic dose of tacrolimus, quantitative results using the MEIA I would not be obtainable during therapeutic drug monitoring in some patients in whom effective therapeutic concentrations can be less than 5.0 microg/l.

Identification of phenanthrene derivatives in Aerides rosea (Orchidaceae) using the combined systems HPLC-ESI-HRMS/MS and HPLC-DAD-MS-SPE-UV-NMR.

PubMed

Cakova, Veronika; Urbain, Aurélie; Antheaume, Cyril; Rimlinger, Nicole; Wehrung, Patrick; Bonté, Frédéric; Lobstein, Annelise

2015-01-01

In our continued efforts to contribute to the general knowledge on the chemical diversity of orchids, we have decided to focus our investigations on the Aeridinae subtribe. Following our previous phytochemical study of Vanda coerulea, which has led to the identification of phenanthrene derivatives, a closely related species, Aerides rosea Lodd. ex Lindl. & Paxton, was chosen for investigation. To identify new secondary metabolites, and to avoid isolation of those already known, by means of the combined systems HPLC-DAD(diode-array detector) with high-resolution tandem mass spectrometry (HRMS/MS) and HPLC-DAD-MS-SPE(solid-phase extraction)-UV-NMR. A dereplication strategy was developed using a HPLC-DAD-HRMS/MS targeted method and applied to fractions from A. rosea stem extract. Characterisation of unknown minor compounds was then performed using the combined HPLC-DAD-MS-SPE-UV-NMR system. The dereplication method allowed the characterisation of four compounds (gigantol, imbricatin, methoxycoelonin and coelonin), previously isolated from Vanda coerulea stem extract. The analyses of two fractions permitted the identification of five additional minor constituents including one phenanthropyran, two phenanthrene and two dihydrophenanthrene derivatives. The full set of NMR data of each compound was obtained from microgram quantities. Nine secondary metabolites were characterised in A. rosea stems, utilising HPLC systems combined with high-resolution analytical systems. Two of them are newly described phenanthrene derivatives: aerosanthrene (5-methoxyphenanthrene-2,3,7-triol) and aerosin (3-methoxy-9,10-dihydro-2,5,7-phenanthrenetriol). Copyright © 2014 John Wiley & Sons, Ltd.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

PubMed

Sorio, Daniela; De Palo, Elio Franco; Bertaso, Anna; Bortolotti, Federica; Tagliaro, Franco

2017-02-01

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

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.

Chromatographic methods for metabolite profiling of virus- and phytoplasma-infected plants of Echinacea purpurea.

PubMed

Pellati, Federica; Epifano, Francesco; Contaldo, Nicoletta; Orlandini, Giulia; Cavicchi, Lisa; Genovese, Salvatore; Bertelli, Davide; Benvenuti, Stefania; Curini, Massimo; Bertaccini, Assunta; Bellardi, Maria Grazia

2011-10-12

This study was focused on the effects of virus and phytoplasma infections on the production of Echinacea purpurea (L.) Moench secondary metabolites, such as caffeic acid derivatives, alkamides, and essential oil. The identification of caffeic acid derivatives and alkamides was carried out by means of high-performance liquid chromatography-diode array detection (HPLC-DAD), HPLC-electrospray ionization-mass spectrometry (ESI-MS), and MS(2). Quantitative analysis of these compounds was carried out using HPLC-DAD. The results indicated that the presence of the two pathogens significantly decreases (P < 0.05) the content of cichoric acid, the main caffeic acid derivative. Regarding the main alkamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, a significant decrease (P < 0.05) in the content of this secondary metabolite was observed in virus-infected plants in comparison with healthy plants, while in the phytoplasma-infected sample the variation of this secondary metabolite was not appreciable. The % relative area of the E/Z isomers of this alkamide was also found to change in infected samples. The gas chromatography (GC) and GC-MS analysis of E. purpurea essential oil enabled the identification of 30 compounds. The main significant differences (P < 0.05) in the semiquantitative composition were observed for three components: limonene, cis-verbenol, and verbenone. The results indicate that the presence of virus and phytoplasma has an appreciable influence on the content of E. purpurea secondary metabolites, which is an important issue in defining the commercial quality, market value, and therapeutic efficacy of this herbal drug.

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.

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites.

PubMed

Xie, Li; Chen, Liqin; Gu, Pan; Wei, Lanlan; Kang, Xuejun

2018-03-01

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry.

PubMed

Liu, Ai-Hua; Guo, Hui; Ye, Min; Lin, Yan-Hua; Sun, Jiang-Hao; Xu, Man; Guo, De-An

2007-08-17

By using HPLC-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)) in negative ion mode, we have analyzed the fragmentation pathways of 11 phenolic acids which were isolated from Danshen. Then the extract of Danshen was analyzed, and a total of 42 phenolic acids, including sixteen new minor constituents, were identified or tentatively identified for the first time. A new solid-phase extraction (SPE) method, new HPLC separation method, new liquid chromatography (LC)-MS and LC-MS(n) (n=3-5) data and proposed fragmentation pathways, LC retention time for phenolic acids are reported.

Analysis and identification of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous by high-performance liquid chromatography.

PubMed

Lu, Mingbo; Zhang, Yang'e; Zhao, Chunfang; Zhou, Pengpeng; Yu, Longjiang

2010-01-01

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3'-dihydroxy-beta,psi-carotene-4,4'-dione (DCD) is produced through gamma-carotene and torulene.

[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%.

Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

NASA Astrophysics Data System (ADS)

Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

2000-02-01

Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

Comparative evaluation of ICP sample introduction systems to be used in the metabolite profiling of chlorine-containing pharmaceuticals via HPLC-ICP-MS.

PubMed

Klencsár, Balázs; Sánchez, Carlos; Balcaen, Lieve; Todolí, José; Lynen, Frederic; Vanhaecke, Frank

2018-05-10

A systematic evaluation of four different ICP sample introduction systems to be used in the context of metabolite profiling of chlorine-containing pharmaceuticals via HPLC-ICP-MS was carried out using diclofenac and its major metabolite, 4'-hydroxy-diclofenac, as model compounds. The strict requirements for GMP validation of chromatographic methods in the pharmaceutical industry were adhered to in this context. The final aim of this investigation is an extension of the applicability and validatability of HPLC-ICP-MS in the field of pharmaceutical R&D. Five different gradient programmes were tested while the baseline peak width (w b ), peak capacity (P), USP tailing factor (A s ) and USP signal-to-noise ratio (USP S/N) were determined as major indicators of the chromatographic performance and the values obtained were compared to the corresponding FDA recommendations (if applicable). Four different ICP-MS sample introductions systems were investigated involving two units typically working at higher flow rates (∼1.0 mL min -1 ) and another two systems working at lower flow rates (∼0.1 mL min -1 ). Optimal conditions with potential for applicability under GMP conditions were found at a mobile phase flow rate of 1.0 mL min -1 by using a pneumatic micro-flow LC nebulizer mounted onto a Peltier-cooled cyclonic spray chamber cooled to -1 °C for sample introduction. Under these conditions, HPLC-ICP-MS provided a chromatographic performance similar to that of HPLC with UV detection. The peak shape (USP tailing factor = 1.1-1.4) was significantly improved compared to that obtained with the Peltier-cooled Scott-type spray chamber. Two alternative sample introduction systems - a POINT ® and a High-Temperature Torch-Integrated Sample Introduction System (hTISIS) - were also tested at a flow rate of 0.1 mL min -1 using a chromatographic column with 1.0 mm ID. Although these systems allowed the peak shape to be improved compared to that obtained with the traditional Scott-type spray chamber, the limits of detection and of quantification achievable were strongly compromised due to the significantly lower sensitivity observed for Cl. In addition to a comparison of the aforementioned sample introduction systems, also the effect of spray chamber temperature was evaluated and it was demonstrated that proper temperature control plays an essential role in the optimization of HPLC-ICP-MS methods. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Identification of phanerosporic acid in birch degraded by Phanerochaete chrysosporium

Treesearch

Michael D. Mozuch; Philip J. Kersten

2003-01-01

Extracts of Phanerochaete chrysosporium cultures grown on birch or on a malt extract-peptone-glucose agar medium were analysed by HPLC. A major component from the two sources appears to be identical by HPLC and UV- visible spectrometry. The product isolated from agar-grown cultures was purified to apparent homogeneity and structure analysis by NMR indicates that the...

Determination of blood sirolimus concentrations in liver and kidney transplant recipients using the Innofluor® fluorescence polarization immunoassay: Comparison with the microparticle enzyme immunoassay and high-performance liquid chromatography-ultraviolet method

PubMed Central

Bouzas, Lorena; Hermida, Jesús

2009-01-01

Background Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx® analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification. Methods Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor® Certican® FPIA (Seradyn Inc.) run on a TDx® analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx® analyser (Abbott Laboratories), and HPLC (UV detection) methods. Results The Innofluor® FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%–13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r≥0.900) than in the kidney transplant samples (r≥0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC. Conclusion The Innofluor® FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus. PMID:19242874

HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora

PubMed Central

Yen, Feng-Lin; Chen, Pei-Chun; Wang, Moo-Chin; Lin, Chun-Nan; Lee, Chiang-Wen; Ko, Horng-Huey

2014-01-01

Phyla nodiflora is a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of this plant is necessary. In the present study, ten compounds, namely, 3,7,4′,5′-tetrahydroxy-3′-methoxyflavone (1), nodifloretin (2), 4′-hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4′-tetrahydroxy-3′-methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), and β-sitosterol were isolated from the methanolic extract of the aerial part of P. nodiflora (PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds 4, 5, and 7 showed strong antioxidant activity. To control the quality of P. nodiflora, a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4) and eupafolin (7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM. PMID:25140335

Characterization of chemical constituents in Rhodiola Crenulate by high-performance liquid chromatography coupled with Fourier-transform ion cyclotron resonance mass spectrometer (HPLC-FT-ICR MS).

PubMed

Han, Fei; Li, Yanting; Mao, Xinjuan; Xu, Rui; Yin, Ran

2016-05-01

In this work, an approach using high-performance liquid chromatography coupled with diode-array detection and Fourier-transform ion cyclotron resonance mass spectrometer (HPLC-FT-ICR MS) for the identification and profiling of chemical constituents in Rhodiola crenulata was developed for the first time. The chromatographic separation was achieved on an Inertsil ODS-3 column (150 mm × 4.6 mm,3 µm) using a gradient elution program, and the detection was performed on a Bruker Solarix 7.0 T mass spectrometer equipped with electrospray ionization source in both positive and negative modes. Under the optimized conditions, a total of 48 chemical compounds, including 26 alcohols and their glycosides, 12 flavonoids and their glycosides, 5 flavanols and gallic acid derivatives, 4 organic acids and 1 cyanogenic glycoside were identified or tentatively characterized. The results indicated that the developed HPLC-FT-ICR MS method with ultra-high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents in R. crenulata. And it provides a helpful chemical basis for further research on R. crenulata. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Occupational exposure to rubber vulcanization products during repair of rubber conveyor belts in a brown coal mine.

PubMed

Gromiec, Jan P; Wesołowski, Wiktor; Brzeźnicki, Sławomir; Wróblewska-Jakubowska, Krystyna; Kucharska, Małgorzata

2002-12-01

Several hundred chemical compounds were found in workroom environments in the rubber industry, but most of the published exposure data relate to the production of tyres; information from the "non-tyre" sections are very limited, if any. This study was carried out to identify chemical substances and measure their air concentrations in the repair shop of a brown coal mine in which damaged rubber conveyor belts were repaired. GC-MS and HPLC analysis of stationary air samples resulted in identification of aliphatic and aromatic hydrocarbons to C12, PAHs, alcohols, phenols, ketones, heterocyclic nitrogen and sulfur compounds. Quantitative evaluation of occupational exposure included determination of organic compound vapours collected on charcoal (GC-MSD), polycyclic aromatic hydrocarbons (HPLC), N-nitrosoamines and other amines (GC-NPD) and DNPH derivatives of aldehydes (HPLC) in the breathing zone of workers representing all job titles. The concentrations of investigated compounds were very low. Carcinogenic substances: N-nitrosoamines, benzene, PAHs were not present in workroom air in concentrations exceeding limits of detection of the analytical methods being applied; concentrations of methylisobutylketone, tetrachloroethylene, naphtha, aromatic hydrocarbons, phthalates and aldehydes were much lower than the respective occupational exposure limit values. The results indicate much lower exposure than that reported in the production of tyres and other fabricated rubber products.

Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance.

PubMed

Qu, Jing; Hu, You-cai; Li, Jian-bei; Wang, Ying-hong; Zhang, Jin-lan; Abliz, Zeper; Yu, Shi-shan; Liu, Yun-bao

2008-01-01

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.

Direct determination of neonicotinoid insecticides in an analytically challenging crop such as Chinese chives using selective ELISAs.

PubMed

Watanabe, Eiki; Miyake, Shiro

2018-06-05

Easy-to-use commercial kit-based enzyme-linked immunosorbent assays (ELISAs) have been used to detect neonicotinoid dinotefuran, clothianidin and imidacloprid in Chinese chives, which are considered a troublesome matrix for chromatographic techniques. Based on their high water solubility, water was used as an extractant. Matrix interference could be avoided substantially just diluting sample extracts. Average recoveries of insecticides from spiked samples were 85-113%, with relative standard deviation of <15%. The concentrations of insecticides detected from the spiked samples with the proposed ELISA methods correlated well with those by the reference high-performance liquid chromatography (HPLC) method. The residues analyzed by the ELISA methods were consistently 1.24 times that found by the HPLC method, attributable to loss of analyte during sample clean-up for HPLC analyses. It was revealed that the ELISA methods can be applied easily to pesticide residue analysis in troublesome matrix such as Chinese chives.

Development of a Thiolysis HPLC Method for the Analysis of Procyanidins in Cranberry Products.

PubMed

Gao, Chi; Cunningham, David G; Liu, Haiyan; Khoo, Christina; Gu, Liwei

2018-03-07

The objective of this study was to develop a thiolysis HPLC method to quantify total procyanidins, the ratio of A-type linkages, and A-type procyanidin equivalents in cranberry products. Cysteamine was utilized as a low-odor substitute of toluene-α-thiol for thiolysis depolymerization. A reaction temperature of 70 °C and reaction time of 20 min, in 0.3 M of HCl, were determined to be optimum depolymerization conditions. Thiolytic products of cranberry procyanidins were separated by RP-HPLC and identified using high-resolution mass spectrometry. Standards curves of good linearity were obtained on thiolyzed procyanidin dimer A2 and B2 external standards. The detection and quantification limits, recovery, and precision of this method were validated. The new method was applied to quantitate total procyanidins, average degree of polymerization, ratio of A-type linkages, and A-type procyanidin equivalents in cranberry products. Results showed that the method was suitable for quantitative and qualitative analysis of procyanidins in cranberry products.

Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC. 2. Application for the analysis of Loy Yang coal oxidation products

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

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

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 was optimized using the solvent gradient method. This method was applied for the analysis of Loy Yang coal oxidation products. It was confirmed that the analytical data using this method were consistent with those determined using gas chromatography.

Stir bar sorptive extraction combined with high performance liquid chromatography-ultraviolet/inductively coupled plasma mass spectrometry for analysis of thyroxine in urine samples.

PubMed

Fan, Wenying; Mao, Xiangju; He, Man; Chen, Beibei; Hu, Bin

2013-11-29

tIn this work, polyethyleneglycol (PEG)/hydroxyl polydimethylsiloxane (OH-PDMS)/γ -mercaptopropyltrimethoxysilane (γ -MPTS) coated stir bar was prepared by sol–gel process and its extraction performance for the extraction of amphoteric thyroxines (3,3',5,5'-tetraiodothyronin, T(4); 3,3',5-triiodothyronine, T(3); reversed-3,3',5-triiodothyronine, rT(3)) and their metabolite (3,5-diiodothyronine,T2) was studied. The preparation reproducibility of PEG/OH-PDMS/γ -MPTS coated stir bar was investigated, and the relative standard deviations (RSDs) in the same batch and among different batches were 3.3–14.3% (n = 5) and 7.7–16.6% (n = 3), respectively. The prepared PEG/OH-PDMS/γ -MPTS coated stir bar could be reused for more than 20 times. Based on this fact, a novel method of stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV)and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of target thyroxinesin human urine samples was developed. The influencing factors of SBSE, such as sample pH, extraction time, stirring rate, salt effect, desorption solution and desorption time, were studied in detail, and the analytical performance of the proposed method was evaluated under the optimized conditions. The enrichment factors (EFs) of the developed method for four target thyroxines were in the range of 14.9–70.4(theoretical enrichment factor was 100). The RSDs were ranging from 4.0% to 13.8% for SBSE-HPLC-UV (c = 25 μg/L, n = 6) and from 3.7% to 6.1% for SBSE-HPLC-ICP-MS (c = 0.5 μg/L, n = 5). The linear range obtained by SBSE-HPLC-UV was 2–500 μg/L for T(2)and 5–500 μg/L for rT3, T(3)and T(4), with correlation coefficients (r) ranging from 0.9957 to 0.9998, respectively, while the linear range obtained by SBSE-HPLC-ICP-MS was 0.05–500 μg/L for T(2) and rT(3), 0.10–200 μg/L for T(3) and 0.05–200 μg/L for T(4)with r ranging from 0.9979 to 0.9998, respectively. The limits of detection (LODs) for the target thyroxines were 0.60–2.20 μg/L for SBSE-HPLC-UV and 0.0071–0.0355 μg/L SBSE-HPLC-ICP-MS, respectively. The developed method was applied for the determination of target thyroxines in urine samples, and the recovery for the spiking samples obtained by SBSE-HPLC-UV was in the range of 81.6–137.6% for human urine,while the recovery for the spiking urine samples obtained by SBSE-HPLC-ICP-MS were in the range of 72.0–121.5%.

Qualitative and quantitative determination of yohimbine in authentic yohimbe bark and in commercial aphrodisiacs by HPLC-UV-API/ MS methods.

PubMed

Zanolari, Boris; Ndjoko, Karine; Ioset, Jean-Robert; Marston, Andrew; Hostettmann, Kurt

2003-01-01

The development and validation of a rapid qualitative and quantitative method based on an HPLC-UV-MS technique with atmospheric pressure chemical ionisation and electrospray ionisation for the analysis of yohimbine in a number of commercial aphrodisiac products is reported. HPLC with multiple-stage mass spectrometry experiments allowed the identification of the target compound and increased the selectivity of complex analyses such as those involved with multi-botanical preparations. The precision and the robustness of the method were improved by the use of two internal standards: codeine for UV detection and deuterium-labelled yohimbine for MS detection. Twenty commercial aphrodisiac preparations were analysed and the amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg.

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.

MOF-5(Zn)-Fe2O4 nanocomposite based magnetic solid-phase microextraction followed by HPLC-UV for efficient enrichment of colchicine in root of colchicium extracts and plasma samples.

PubMed

Bahrani, Sonia; Ghaedi, Mehrorang; Dashtian, Kheibar; Ostovan, Abbas; Mansoorkhani, Mohammad Javad Khoshnood; Salehi, Amin

2017-11-01

In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe 2 O 4 -nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe 2 O 4 NPs) was synthesized by dispersing MOF-5 and Fe(NO 3 ) 3 .9H 2 O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe 2 O 4 NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, ᴨ-ᴨ, hard-hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe 2 O 4 NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5-1700ngmL -1 ) with reasonable detection limit (0.13ngmL -1 ) and R 2 =0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and characterization of a bactericidal withanolide from Physalis virginiana

PubMed Central

Gibson, Kathleen A.; Reese, R. Neil; Halaweish, Fathi T.; Ren, Yulin

2012-01-01

Background: Physalis virginiana (Virginia Groundcherry) is a member of the family Solenaceae. Several species of the Physalis genus have been used traditionally by American Indians as medicinal treatments. Materials and Methods: This study investigated the antibacterial activity of chemicals extracted from P. virginiana through antibacterial disc and cytotoxicity assays. Isolation and purification of an antimicrobial compound was achieved through flash chromatography and preparative HPLC. Finally, identification of chemical structure was determined from 1H and 13C NMR and MS. Results: Disc assays showed that crude ethanol extracts were effective antibacterial agents against one gram-negative and seven gram-positive bacterial strains. Cytotoxicity assays indicated that it is less toxic than gentamicin controls. Isolation of the active component showed it to be a relatively polar compound. 1H and 13C NMR chemical shifts together with HRMS indicated a similar structure to withanolides previously identified from Physalis angulata. HRMS analysis showed a molecular mass of 472.2857 which corresponds to a molecular formula C28H40O6. Conclusion: An antibacterial withanolide was isolated from P. virginiana using flash chromatography and HPLC separations. The chemical structure was determined by NMR and MS to be the withanolide physagulin V. PMID:22438659

The integrated quality assessment of Chinese commercial dry red wine based on a method of online HPLC-DAD-CL combined with HPLC-ESI-MS.

PubMed

Yu, Hai-Xiang; Sun, Li-Qiong; Qi, Jin

2014-07-01

To apply an integrated quality assessment strategy to investigate the quality of multiple Chinese commercial dry red wine samples. A comprehensive method was developed by combining a high performance liquid chromatography-diode array detector-chemiluminescence (HPLC-DAD-CL) online hyphenated system with an HPLC-ESI-MS technique. Chromatographic and H2O2-scavenging active fingerprints of thirteen batches of different, commercially available Chinese dry red wine samples were obtained and analyzed. Twenty-five compounds, including eighteen antioxidants were identified and evaluated. The dominant and characteristic antioxidants in the samples were identified. The relationships between antioxidant potency and the cultivated variety of grape, producing area, cellaring period, and trade mark are also discussed. The results provide the feasibility for an integrated quality assessment strategy to be efficiently and objectively used in quality (especially antioxidant activity) assessment and identification of dry red wine. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2007-09-01

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

[Research on chemical fingerprint chromatograms of Sinopodophyllum hexandrum].

PubMed

Wang, Ai-Hua; Liu, Guang-Xue; Xu, Feng; Shang, Ming-Ying; Cai, Shao-Qing

2013-10-01

To study the HPLC fingerprint of Sinopodophylli Fructus, compare the major chemical differences in fruit of Sinopodophyllum hexandrum as well as the roots and rhizomas of S. hexandrum, and provide scientific evidence for clinical application and quality control. HPLC fingerprint method was used to analyze 12 fruits of S. hexandrum. A total of 20 common peaks were confirmed, and 12 peaks in the HPLC fingerprint were identified. Furthermore, similarity evaluation method and clustering analysis method were introduced to compare HPLC chromatograms, between fruits and underground parts. In this paper, quercetin-3-O-beta-D-glucopyranosid, kaempferol-3-O-beta-D-glucopyranoside, 8-prenylquercetin and 8-prenylquercetin 3-methyl ether were firstly reported in Sinopodophylli Fructus. Among the existing fingerprint research, a total of 11 peaks were identified for the first time, containing 9 flavonoids and 2 lignans. The chemical constituents differed significantly in different medicinal parts of S. hexandrum. Prenylflavonoid compounds were the main constituents of Sinopodophylli Fructus. However, podophyllotoxin, flavonoids with simple substituent groups and flavonoid glycosides were the major ingredients in the roots and rhizomas of S. hexandrum. This method can be used for the quality control of Sinopodophylli Fructus and the roots and rhizomas of S. hexandrum. It has provided a reference for the pharmacodynamic differences of the two different parts.

[HPLC method to determine DEHP released into blood from a disposable extracorporeal circulation tube].

PubMed

Liu, Xi; Yu, Jingjing; Li, Shen; Wang, Hong; Liu, Jiaxin

2013-08-01

We used blood as leaching medium, simulating clinical operation under maximum condition, to develop Liquid-phase extraction- High Performance Liquid Chromatography (HPLC) method for determination of plasticizer Di-(2-ethylhexyl)phthalate (DEHP) released from Disposable Extracorporeal Circulation Tube in order to lay the foundation of risk analysis of this product. The characteristic wavelength of DEHP in methanol was detected. Acetonitrile was added to the leaching blood in proportion and extracted DEHP from blood. The methodology for HPLC to quantify DEHP was established and the DEHP amount released from this disposable extracorporeal circulation tube was measured. The experiments showed good results as follows. The characteristic wavelength of DEHP was 272nm. The concentration of DEHP (5-250 microg/mL) kept good linear relationship with peak area (r=0.9999). Method sensitivity was 1 microg/mL. Precisions showed RSD<5%. The adding standard extraction Recovery Rates of 25, 100 and 250 microg DEHP standard were 61.91 +/- 3.32)%, (69.38 +/- 0.55)% and (68.47 +/- 1.15)%. The DEHP maximum amounts released from 3 sets of this disposable extracorporeal circulation tube were 204.14, 106.30 and 165.34 mg/set. Our Liquid-phase Extraction-HPLC method showed high accuracy and precision, and relatively stable recovery rate. Its operation was also convenient.

Evaluation of phosphorus characterization in broiler ileal digesta, manure, and litter samples: (31)P-NMR vs. HPLC.

PubMed

Leytem, A B; Kwanyuen, P; Plumstead, P W; Maguire, R O; Brake, J

2008-01-01

Using 31-phosphorus nuclear magnetic resonance spectroscopy ((31)P-NMR) to characterize phosphorus (P) in animal manures and litter has become a popular technique in the area of nutrient management. To date, there has been no published work evaluating P quantification in manure/litter samples with (31)P-NMR compared to other accepted methods such as high performance liquid chromatography (HPLC). To evaluate the use of (31)P-NMR to quantify myo-inositol hexakisphosphate (phytate) in ileal digesta, manure, and litter from broilers, we compared results obtained from both (31)P-NMR and a more traditional HPLC method. The quantification of phytate in all samples was very consistent between the two methods, with linear regressions having slopes ranging from 0.94 to 1.07 and r(2) values of 0.84 to 0.98. We compared the concentration of total monoester P determined with (31)P-NMR with the total inositol P content determined with HPLC and found a strong linear relationship between the two measurements having slopes ranging from 0.91 to 1.08 and r(2) values of 0.73 to 0.95. This suggests that (31)P-NMR is a very reliable method for quantifying P compounds in manure/litter samples.

Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

PubMed

Katharopoulos, Efstathios; Touloupi, Katerina; Touraki, Maria

2016-08-01

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC. Copyright © 2016 Elsevier B.V. All rights reserved.

A Study of Method Development, Validation, and Forced Degradation for Simultaneous Quantification of Paracetamol and Ibuprofen in Pharmaceutical Dosage Form by RP-HPLC Method

PubMed Central

Jahan, Md. Sarowar; Islam, Md. Jahirul; Begum, Rehana; Kayesh, Ruhul; Rahman, Asma

2014-01-01

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R2) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R2 > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0–103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5–20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition. PMID:25452691

A Study of Method Development, Validation, and Forced Degradation for Simultaneous Quantification of Paracetamol and Ibuprofen in Pharmaceutical Dosage Form by RP-HPLC Method.

PubMed

Jahan, Md Sarowar; Islam, Md Jahirul; Begum, Rehana; Kayesh, Ruhul; Rahman, Asma

2014-01-01

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R (2)) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R (2) > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0-103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5-20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition.

Simultaneous determination of seven lignans in Justicia procumbens by high performance liquid chromatography-photodiode array detection using relative response factors.

PubMed

Luo, Zuliang; Kong, Weijun; Qiu, Feng; Yang, Meihua; Li, Qian; Wei, Riwei; Yang, Xiaoli; Qin, Jieping

2013-02-01

A simple and sensitive HPLC coupled with photodiode array (HPLC-PDA) method was developed for simultaneous determination of seven lignans in Justicia procumbens using relative response factors (RRFs). The chromatographic separation was performed on a Shiseido Capcell Pak C(18) column (250 × 4.6 mm id, 5 μm), a gradient elution of acetonitrile/water, and a photodiode array detector. The column temperature was maintained at 35°C and the detection wavelength was set at 256 nm. Chinensinaphthol methyl ether was selected as the reference compound for calculating the relative response factors of the lignans. It has shown that the RRFs for lignans are quite similar at 256 nm of detection under different analytical conditions (different columns and HPLC instruments). Using RRFs, not every lignan is needed as a reference standard, making the method ideal for rapid, routine analysis, especially for those laboratories where lignans standards are not readily available. An economic and practicable HPLC method using RRFs was established for the determination of seven lignans in J. procumbens. This method not only can determine multiple indexes in traditional Chinese medicines (TCMs) simultaneously, but also resolve the problem of lacking of chemical standards. It will be a good quality evaluation method and pattern for TCMs. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous Speciation of Arsenic, Selenium, and Chromium by HPLC-ICP-MS

USGS Publications Warehouse

Wolf, Ruth E.; Morman, Suzette A.; Morrison, Jean M.; Lamothe, Paul J.

2008-01-01

An adaptation of an analytical method developed for chromium speciation has been utilized for the simultaneous determination of As(III), As(V), Se(IV), Se(VI), Cr(III), and Cr(VI) species using high performance liquid chromatography (HPLC) separation with ICP-MS detection. Reduction of interferences for the determination of As, Se, and Cr by ICP-MS is a major consideration for this method. Toward this end, a Dynamic Reaction Cell (DRC) ICP-MS system was used to detect the species eluted from the chromatographic column. A variety of reaction cell gases and conditions may be utilized, and the advantages and limitations of the gases tested to date will be presented and discussed. The separation and detection of the As, Se, and Cr species of interest can be achieved using the same chromatographic conditions in less than 2 minutes by complexing the Cr(III) with EDTA prior to injection on the HPLC column. Practical aspects of simultaneous speciation analysis will be presented and discussed, including issues with HPLC sample vial contamination, standard and sample contamination, species stability, and considerations regarding sample collection and preservation methods. The results of testing to determine the method's robustness to common concomitant element and anion effects will also be discussed. Finally, results will be presented using the method for the analysis of a variety of environmental and geological samples including waters, soil leachates and simulated bio-fluid leachates.

Evaluation of multiple reaction monitoring cubed for the analysis of tachykinin related peptides in rat spinal cord using a hybrid triple quadrupole-linear ion trap mass spectrometer.

PubMed

Pailleux, Floriane; Beaudry, Francis

2014-02-01

Targeted peptide methods generally use HPLC-MS/MRM approaches. Although dependent on the instrumental resolution, interferences may occur while performing analysis of complex biological matrices. HPLC-MS/MRM(3) is a technique, which provides a significantly better selectivity, compared with HPLC-MS/MRM assay. HPLC-MS/MRM(3) allows the detection and quantitation by enriching standard MRM with secondary product ions that are generated within the linear ion trap. Substance P (SP) and neurokinin A (NKA) are tachykinin peptides playing a central role in pain transmission. The objective of this study was to verify whether HPLC-MS/MRM(3) could provide significant advantages over a more traditional HPLC-MS/MRM assay for the quantification of SP and NKA in rat spinal cord. The results suggest that reconstructed MRM(3) chromatograms display significant improvements with the nearly complete elimination of interfering peaks but the sensitivity (i.e. signal-to-noise ratio) was severely reduced. The precision (%CV) observed was between 3.5% and 24.1% using HPLC-MS/MRM and in the range of 4.3-13.1% with HPLC-MS/MRM(3), for SP and NKA. The observed accuracy was within 10% of the theoretical concentrations tested. HPLC-MS/MRM(3) may improve the assay sensitivity to detect difference between samples by reducing significantly the potential of interferences and therefore reduce instrumental errors. Copyright © 2014 Elsevier B.V. All rights reserved.

HPLC and TLC methods for analysis of [18F]FDG and its metabolites from biological samples.

PubMed

Rokka, Johanna; Grönroos, Tove J; Viljanen, Tapio; Solin, Olof; Haaparanta-Solin, Merja

2017-03-24

The most used positron emission tomography (PET) tracer, 2-[ 18 F]fluoro-2-deoxy-d-glucose ([ 18 F]FDG), is a glucose analogue that is used to measure tissue glucose consumption. Traditionally, the Sokoloff model is the basis for [ 18 F]FDG modeling. According to this model, [ 18 F]FDG is expected to be trapped in a cell in the form of [ 18 F]FDG-6-phosphate ([ 18 F]FDG-6-P). However, several studies have shown that in tissues, [ 18 F]FDG metabolism goes beyond [ 18 F]FDG-6-P. Our aim was to develop radioHPLC and radioTLC methods for analysis of [ 18 F]FDG metabolites from tissue samples. The radioHPLC method uses a sensitive on-line scintillation detector to detect radioactivity, and the radioTLC method employs digital autoradiography to detect the radioactivity distribution on a TLC plate. The HPLC and TLC methods were developed using enzymatically in vitro-produced metabolites of [ 18 F]FDG as reference standards. For this purpose, three [ 18 F]FDG metabolites were synthesized: [ 18 F]FDG-6-P, [ 18 F]FD-PGL, and [ 18 F]FDG-1,6-P2. The two methods were evaluated by analyzing the [ 18 F]FDG metabolic profile from rodent ex vivo tissue homogenates. The HPLC method with an on-line scintillation detector had a wide linearity in a range of 5Bq-5kBq (LOD 46Bq, LOQ 139Bq) and a good resolution (Rs ≥1.9), and separated [ 18 F]FDG and its metabolites clearly. The TLC method combined with digital autoradiography had a high sensitivity in a wide range of radioactivity (0.1Bq-2kBq, LOD 0.24Bq, LOQ 0.31Bq), and multiple samples could be analyzed simultaneously. As our test and the method validation with ex vivo samples showed, both methods are useful, and at best they complement each other in analysis of [ 18 F]FDG and its radioactive metabolites from biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi.

PubMed

Watanabe, Makoto; Ohata, Masahiko; Hayakawa, Sumio; Isemura, Mamoru; Kumazawa, Shigenori; Nakayama, Tsutomu; Furugori, Michiyo; Kinae, Naohide

2003-03-01

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

Simultaneous determination of five free and total flavonoids in rat plasma by ultra HPLC-MS/MS and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats.

PubMed

Wang, Xiaofan; Zhao, Xu; Gu, Liqiang; Lv, Chunxiao; He, Bosai; Liu, Zhenzhen; Hou, Pengyi; Bi, Kaishun; Chen, Xiaohui

2014-03-15

A simple and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) method has been developed for the simultaneous determination of five free flavonoids (amentoflavone, isorhamnetin, naringenin, kaempferol and quercetin) and their total (free and conjugated) forms, and to compare the pharmacokinetics of these active ingredients in normal and hyperlipidemic rats. The free and total forms of these flavonoids were extracted by liquid-liquid extraction with ethyl acetate. The conjugated flavonoids were deconjugated by the enzyme β-Glucuronidase and Sulfatase. Chromatographic separation was accomplished on a ZORBAX Eclipse XDB-C8 USP L7 column using gradient elution. Detection was performed on a 4000Q uHPLC-MS/MS system from AB Sciex using negative ion mode in the multiple reaction monitoring (MRM) mode. The lower limits of quantification were 2.0-5.0ng/mL for all the analytes. Intra-day and inter-day precision were less than 15% and accuracy ranged from -9.3% to 11.0%, and the mean extraction recoveries of analytes and internal standard (IS) from rat plasma were all more than 81.7%. The validated method was successfully applied to a comparative pharmacokinetic study of five free and total analytes in rat plasma. The results indicated that the absorption of five total flavonoids in hyperlipidemia group were significantly higher than those in normal group with similar concentration-time curves. Copyright © 2014 Elsevier B.V. All rights reserved.

Pharmacokinetics of isochlorgenic acid C in rats by HPLC-MS: Absolute bioavailability and dose proportionality.

PubMed

Huang, Li Hua; Xiong, Xiao Hong; Zhong, Yun Ming; Cen, Mei Feng; Cheng, Xuan Ge; Wang, Gui Xiang; Zang, Lin Quan; Wang, Su Jun

2016-06-05

Isochlorgenic acid C (IAC), one of the bioactive compounds of Lonicera japonica, exhibited diverse pharmacological effects. However, its pharmacokinetic properties and bioavailability remained unresolved. To determine the absolute bioavailability in rats and the dose proportionality on the pharmacokinetics of single oral dose of IAC. A validated HPLC-MS method was developed for the determination of IAC in rat plasma. Plasma concentration versus time data were generated following oral and intravenous dosing. The pharmacokinetic analysis was performed using DAS 3.0 software analysis. Absolute bioavailability in rats was determined by comparing pharmacokinetic data after administration of single oral (5, 10 and 25mgkg(-1)) and intravenous (5mgkg(-1)) doses of IAC. The dose proportionality of AUC(0-∞) and Cmax were analyzed by linear regression. Experimental data showed that absolute oral bioavailability of IAC in rats across the doses ranged between 14.4% and 16.9%. The regression analysis of AUC(0-∞) and Cmax at the three doses (5, 10 and 25mgkg(-1)) indicated that the equations were y=35.23x+117.20 (r=0.998) and y=121.03x+255.74 (r=0.995), respectively. A new HPLC-MS method was developed to determine the bioavailability and the dose proportionality of IAC. Bioavailability of IAC in rats was poor and both Cmax and AUC(0-∞) of IAC had a positive correlation with dose. Evaluation of the pharmacokinetics of IAC will be useful in assessing concentration-effect relationships for the potential therapeutic applications of IAC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

[Study on HPLC fingerprint of flavonoids from Houttuynia cordata by comparing with fingerprint reference].

PubMed

Zhang, Ting-Ting; Wu, Yi; Hang, Tai-Jun

2009-05-01

To establish a stable and repeatable HPLC fingerprint standard and evaluate the flavonoids from Houttuynia cordata qualitatively and quantitatively. HPLC separation was performed on a C18 column with methanol-0.1% phosphoric acid mixed solution as mobile phase in gradient elution mode. The fingerprint reference was determined as one of the most typical chromatograms and used to be compared with other samples through Cosine and Relative Euclid Distance methods, thus the chromatographic fingerprints of flavonoids from Houttuynia cordata were evaluated by constitutes and contents, respectively. Fourteen mutual peaks were fixed in the HPLC fingerprint of flavonoids from Houttaynia cordata. It showed good results in validation tests in which the quercitrin's peak was set as the reference peak to calculate relative retention time and area of other peaks in the chromatograms, and the RSD were less than 0.2% and 5.0%, respectively. The linear ranges for quercitrin was 1.07-83.4 microg/mL (r=0.9999) and the average recovery was 100.3%. The method shows good repeatability, ruggedness and reliability. Comparing with the established reference fingerprint, the evaluation system including Cosine and Relative Euclid Distance methods lays dependable foundation for controlling the quality of Houttuynia cordata.

Determination of pKa values of some antipsychotic drugs by HPLC--correlations with the Kamlet and taft solvatochromic parameters and HPLC analysis in dosage forms.

PubMed

Sanli, Senem; Akmese, Bediha; Altun, Yuksel

2013-01-01

In this study, ionization constant (pKa) values were determined by using the dependence of the retention factor on the pH of the mobile phase for four ionizable drugs, namely, risperidone (RI), clozapine (CL), olanzapine (OL), and sertindole (SE). The effect of the mobile phase composition on the pKa was studied by measuring the pKa at different acetonitrile-water mixtures in an HPLC-UV method. To explain the variation of the pKa values obtained over the whole composition range studied, the quasi-lattice quasi-chemical theory of preferential solvation was applied. The pKa values of drugs were correlated with the Kamlet and Taft solvatochromic parameters. Kamlet and Taft's general equation was reduced to two terms by using combined factor analysis and target factor analysis in these mixtures: the independent term and the hydrogen-bond donating ability a. The HPLC-UV method was successfully applied for the determination of RI, OL, and SE in pharmaceutical dosage forms. CL was chosen as an internal standard. Additionally, the repeatability, reproducibility, selectivity, precision, and accuracy of the method in all media were investigated and calculated.

Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection.

PubMed

Matsutomo, Toshiaki; Kodera, Yukihiro

2016-02-01

Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC. The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE). We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions. Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period. We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE. © 2016 American Society for Nutrition.

[HPLC-ESI-MS(n) analysis of the water soluble extracts of Fructus Choerospondiatis].

PubMed

Shi, Run-ju; Dai, Yun; Fang, Min-feng; Zhao, Xin; Zheng, Jian-bin; Zheng, Xiao-hui

2007-03-01

To establish an HPLC-ESI-MS(n) method for analyzing the chemical ingredients in the water soluble extracts of Fructus Choerospondiatis. Water-solvable extracts of Fructus Choerospondiatis are obtained by heating recirculation. Multi-stage reaction mode (MRM) of the HPLC-ESI-MS(n) was used to determine the content of Gallic acid, the MS(n) technology was used to obtain the information of characteristic multistage fragment ions so as to identify the chemical structure of peaks in the total current spectrum. Eleven compounds were identified, and one of them is a new unknown ingredient. The method, which has high recovery and specificity, can offer the experimental evidences for the further research of the chemical ingredients extracted from the Fructus Choerospondiatis.

Measurement of Menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

Menadione may be an important metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed to measure menadione in urine. The mobile phase was composed of 95% methanol...

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

PubMed

Remeur, Camille; Le Borgne, Erell; Gauthier, Léa; Grougnet, Raphaël; Deguin, Brigitte; Poullain, Cyril; Litaudon, Marc

2017-05-01

Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Profiling indomethacin impurities using high-performance liquid chromatography and nuclear magnetic resonance.

PubMed

Hess, S; Teubert, U; Ortwein, J; Eger, K

2001-12-01

The anti-inflammatory drug indomethacin was investigated regarding new related impurities. Therefore, related substances 2-9 were prepared by independent synthesis and physicochemically characterized. To determine indomethacin and its related substances, a new HPLC-UV method was developed and validated. Indomethacin and its impurities were eluted on a C(18) column with a mobile phase consisting of methanol and an aqueous solution of 0.2% phosphoric acid at a flow rate of 1.5 ml/min and were quantified by UV detection at 320 nm. Overall, the HPLC-UV method was simple and reliable for the detection of eight impurities in indomethacin. In addition to the HPLC-UV method, 1H nuclear magnetic resonance (NMR) was used to investigate indomethacin regarding impurities. For that purpose, related substances 2-9 were systematically added to indomethacin and investigated. The NMR method was found to be very useful for the identification of impurities in bulk substance without prior separation. Both HPLC-UV and NMR were used to analyze 38 batches of indomethacin available on the European market. The outcome was that 42% of the batches did not meet the compendial requirements although they met the specifications of current compendial methods. Some batches contained the previously undescribed impurity 8, while other batches contained by-products from two distinct synthetic routes. The methods presented herein are important contributions to the ongoing efforts to reduce impurities and therefore the risk of adverse side-effects in drugs that are no longer under patent protection.

Comparison of high-performance liquid chromatography/tandem mass spectrometry and high-performance liquid chromatography/photo-diode array detection for the quantitation of carotenoids, retinyl esters, α-tocopherol and phylloquinone in chylomicron-rich fractions of human plasma.

PubMed

Kopec, Rachel E; Schweiggert, Ralf M; Riedl, Ken M; Carle, Reinhold; Schwartz, Steven J

2013-06-30

Bioavailability of essential lipophilic micronutrients and carotenoids is of utmost interest for human health, as the consumption of these compounds may help alleviate major nutritional deficiencies, cardiovascular disease, and cancer. High-performance liquid chromatography/photo-diode array detection (HPLC-PDA) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) were compared for the quantitative analysis of α- and β-carotene, β-cryptoxanthin, lutein, lycopene, α-tocopherol, phylloquinone, and several retinyl esters from chylomicron-containing triglyceride rich lipoprotein (TRL) fractions of human plasma obtained from two clinical trials. After selecting an efficient extraction method for the analytes, both the HPLC/PDA and the HPLC/MS/MS methods were developed and several parameters validated using an HP 1200 series HPLC system interfaced with a HP 1200 series diode-array detector (Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 5500 (AB Sciex, Foster City, CA, USA) via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. For lycopene, α- and β-carotene, HPLC/MS/MS was up to 37 times more sensitive than HPLC-PDA. PDA detection was shown to be up to 8 times more sensitive for lutein. MS/MS signals were enhanced by matrix components for lutein and β-cryptoxanthin, as determined by referencing to the matrix-independent PDA signal. In contrast, matrix suppression was observed for retinyl palmitate, α-carotene, and β-carotene. Both detectors showed similar suitability for α-tocopherol, lycopene and retinyl palmitate (representing ~73% of total retinyl esters). MS/MS exclusively allowed the quantitation of minor retinyl esters, phylloquinone, and (Z)-lycopene isomers. HPLC/MS/MS was more sensitive than HPLC-PDA for six of the eight analytes and represents a powerful tool for the analysis of chylomicron samples and potentially other biological samples of limited sample size. When internal standards are available for the target carotenoid, employing MS/MS detection may reduce the necessary blood sample volume, which is particularly advantageous for minimizing risk and discomfort to human subjects during clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.

Rapid Identification of unstable acyl glucoside flavonoids of Oxytropis racemosa Turcz by high-performance liquid chromatography-diode array detection-electrospray ionisation/multi-stage mass spectrometry.

PubMed

Song, Shuang; Zheng, Xiu-Ping; Liu, Wei-Dong; Du, Rui-Fang; Feng, Zi-Ming; Zhang, Pei-Cheng; Bi, Li-Fu

2013-02-01

Oxytropis racemosa Turcz is an important minority medicine that is used mainly to improve children's indigestion, especially in inner Mongolia and Tibet. Previous studies indicated that the characteristic constituents of this plant are acylated flavonoids. Rapidly identify the characteristic chemical constituents of O. racemosa by high-performance liquid chromatography-diode array detection-electrospray ionisation/multi-stage mass spectrometry (HPLC-DAD-ESI/MS(n) ) and suggest a useful method to control the quality of this medicinal plant. In the HPLC fingerprint, 32 flavonoids were tentatively identified by a detailed analysis of their mass spectra, UV spectra and retention times. Furthermore, 13 flavonoids were confirmed by comparison with previously isolated compounds obtained from O. racemosa. In total, 32 flavonoids, including 13 flavonoids with 3-hydroxy-3-methylglutaric acid (HMG) moieties and four flavonoids with 3-malonyl moieties, were identified in the extract of O. racemosa. Among the compounds identified, 10 were characterised as new compounds for their particular acylated sugar moieties. The method described is effective for obtaining a comprehensive phytochemical profile of plants containing unstable acylated flavonoids. The method is also useful for constructing the chromatographic fingerprint of the minority medicine -O. racemosa Turcz for quality control. Copyright © 2012 John Wiley & Sons, Ltd.

Development and in vitro/in vivo Evaluation of a Silastic Intravaginal Ring for Mifepristone Delivery.

PubMed

Duan, X; Ning, M

2015-01-01

Preparation and in vitro/in vivo evaluation of mifepristone intravaginal ring formulations were investigated. In the present study, it is reported that a mifepristone intravaginal ring of reservoir design comprising of a mifepristone silicone elastomer core enclosed in a silicone layer. During the preparation of intravaginal ring solid dispersion method was employed which improved the release rate of drug from the intravaginal ring. In vitro release studies performed under sink conditions and the released drug amounts were estimated using UV spectrometry at 310 nm. In addition, the in vivo release profile of in-house devices was evaluated in female New Zealand white rabbits. The rabbit plasma samples were processed and analyzed using a validated HPLC-MS method. Norgestrel was used as internal standard, and plasma samples contained mifepristone and internal standard were deproteinized, and then subjected to HPLC-MS analysis under condition of electrospray ionization in the selected ion monitoring mode. The drug release from intravaginal ring made in house was constant for 21 days in rabbits, which suggested the mifepristone intravaginal ring release system would be useful in clinical practice in the future. The result indicated the in vitro/in vivo correlation is perfect, which explained in vitro release analysis method developed was feasible.

Effect of associated bacteria on the growth and toxicity of Alexandrium catenella.

PubMed

Uribe, Paulina; Espejo, Romilio T

2003-01-01

Saprophytic bacteria in cultures of the marine dinoflagellate Alexandrium catenella were removed to assess their effect on growth and paralytic shellfish poisoning toxin production of this dinoflagellate. The actual axenic status was demonstrated by the lack of observable bacteria both immediately after treatment and following extended incubation in the absence of antibiotics. Bacteria were measured by counting CFU and also by epifluorescence microscopy and PCR amplification of bacterial 16S-23S spacer ribosomal DNA to detect noncultivable bacteria. Removal of bacteria did not have any effect on the growth of the dinoflagellate except for the inhibition of A. catenella disintegration after reaching the stationary phase. Toxicity was determined in dinoflagellate cell extracts by different methods: high-performance liquid chromatography (HPLC); an electrophysiological test called the Electrotest, which measures the inhibition of saxitoxin-sensitive Na(+) channels expressed in a cell line; and a mouse bioassay, which measures the toxic effect on the whole mammal neuromuscular system. A lower toxicity of the dinoflagellates in axenic culture was observed by these three methods, though the difference was significant only by the mouse bioassay and HPLC methods. Altogether the results indicate that axenic cultures of A. catenella are able to produce toxin, though the total toxicity is probably diminished to about one-fifth of that in nonaxenic cultures.

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.

Rifaximin Stability: A Look at UV, IR, HPLC, and Turbidimetry Methods.

PubMed

Kogawa, Ana Carolina; Salgado, Hérida Regina Nunes

2018-03-01

The study of the stability of medicines is mandated by the International Conference on Harmonization and the World Health Organization. Rifaximin, an antimicrobial marketed in the form of tablets, has no record of stability studies. Thus, the objective of the present work was to investigate the behavior and stability of rifaximin tablets for 6 months under simultaneous conditions of temperature and humidity by UV, IR, HPLC, and turbidimetry techniques. After 6 months of stability study, rifaximin tablets were shown to obey zero-order kinetics when analyzed by physicochemical methods and second-order kinetics when analyzed by a microbiological method. However, the UV method was not suitable for the evaluation of rifaximin. IR, HPLC, and turbidimetry methods can already be used to evaluate the stability of rifaximin tablets. It is important to analyze products with more than one type of method before releasing results mainly in the case of antimicrobial products in which the association of physicochemical and microbiological techniques must be a rule. Rifaximin tablets can be considered stable after 6 months under conditions of 40 ± 2°C and 75 ± 5% relative humidity.

HPLC-DAD-ELSD Combined Pharmacodynamics and Serum Medicinal Chemistry for Quality Assessment of Huangqi Granule

PubMed Central

Zhao, Yang; Gong, Xiao-Jian; He, Yan; Ma, Feng-Wei; Zhou, Mei; Zhao, Chao; Niu, Yi; Deng, Jie

2015-01-01

Objective To more scientifically and reasonably control the quality of Huangqi Granules, preliminary studies on the pharmacodynamics and serum pharmacochemistry of this medicine were performed. DPPH and MTT experiments showed that water extracts of Huangqi Granules had good antioxidant activity and increased immunity. Timed blood samples collected 5 min, 15 min, and 30 min after oral administration of a set amount of Huangqi Granules were collected and tested using UPLC-ESI-MS/MS. As a result, calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, and formononetin were found to exist in rat blood after dosing, indicating that the five chemical compounds might have pharmacological activity, and based on this result, they were designated biomarkers for quality control of Huangqi Granules. Consequently, a simple, rapid and efficient method was developed in the present study for the simultaneous determination of the five characteristic compounds in Huangqi Granules using HPLC-DAD-ELSD. Materials and Methods The separation was performed using an Agilent Hypersil ODS column (4.6 × 250 mm, 5 μm) at 30 ℃. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B) with a flow rate of 1 mL/min. The drift tube temperature of the ELSD system was set to 85 ℃, and the nitrogen pressure was 3.5 bar. Results All five characteristic compounds had good linear behavior with r2 values greater than 0.9972. The recoveries varied from 96.31% to 101.22%. Subsequently, the developed method was applied to evaluate the quality of Huangqi Granules from different batches, and hierarchical clustering analysis (HCA) was used to analyze the classification of the samples based on the values of the five compounds. Conclusion The established HPLC method combined with HCA proved to be effective to evaluate the quality of Huangqi Granules. PMID:25915040

Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents

DTIC Science & Technology

1997-11-01

status can sometimes be reflected in the infectious potential or drug resistance of those pathogens. For example, in Mycobacterium tuberculosis ... Mycobacterium tuberculosis , its antibiotic resistance and prediction of pathogenicity amongst Mycobacterium spp. based on signature lipid biomarkers ...TITLE AND SUBTITLE Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents 5a. CONTRACT NUMBER 5b

Development and validation of a reversed-phase fluorescence HPLC method for determination of bucillamine in human plasma using pre-column derivatization with monobromobimane.

PubMed

Lee, Kang Choon; Chun, Young Goo; Kim, Insoo; Shin, Beom Soo; Park, Eun-Seok; Yoo, Sun Dong; Youn, Yu Seok

2009-07-15

A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50 ng/mL to 10 microg/mL) in human plasma (r(2)=0.9998). The lower limit of quantitation (LLOQ) was 50 ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300 mg) of bucillamine to 20 healthy Korean volunteers.

Measurement of Menadione in Urine by HPLC

PubMed Central

Rajabi, Ala Al; Peterson, James; Choi, Sang Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-01-01

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C30 column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H2O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R2) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Research support: USDA ARS Cooperative Agreement 58-1950-7-707. PMID:20719580

Measurement of menadione in urine by HPLC.

PubMed

Al Rajabi, Ala; Peterson, James; Choi, Sang-Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-09-15

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Selective identification and quantification of saccharin by liquid chromatography and fluorescence detection.

PubMed

Bruno, Sergio N F; Cardoso, Carlos R; Maciel, Márcia Mosca A; Vokac, Lidmila; da Silva Junior, Ademário I

2014-09-15

High-pressure liquid chromatography with ultra-violet detection (HPLC-UV) is one of the most commonly used methods to identify and quantify saccharin in non-alcoholic beverages. However, due to the wide variety of interfering UV spectra in saccharin-containing beverage matrices, the same method cannot be used to measure this analyte accurately. We have developed a new, highly effective method to identify and quantify saccharin using HPLC with fluorescence detection (HPLC-FLD). The excitation wavelength (250 nm) and emission wavelength (440 nm) chosen increased selectivity for all matrices and ensured few changes were required in the mobile phase or other parameters. The presence of saccharin in non-diet beverages - a fraud commonly used to replace more expensive sucrose - was confirmed by comparing coincident peaks as well as the emission spectra of standards and samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Description and evaluation of a peracetic acid air sampling and analysis method.

PubMed

Nordling, John; Kinsky, Owen R; Osorio, Magdalena; Pechacek, Nathan

2017-12-01

Peracetic acid (PAA) is a corrosive chemical with a pungent odor, which is extensively used in occupational settings and causes various health hazards in exposed workers. Currently, there is no US government agency recommended method that could be applied universally for the sampling and analysis of PAA. Legacy methods for determining airborne PAA vapor levels frequently suffered from cross-reactivity with other chemicals, particularly hydrogen peroxide (H 2 O 2 ). Therefore, to remove the confounding factor of cross-reactivity, a new viable, sensitive method was developed for assessment of PAA exposure levels, based on the differential reaction kinetics of PAA with methyl p-tolylsulfide (MTS), relative to H 2 O 2 , to preferentially derive methyl p-tolysulfoxide (MTSO). By quantifying MTSO concentration produced in the liquid capture solution from an air sampler, using an internal standard, and utilizing the reaction stoichiometry of PAA and MTS, the original airborne concentration of PAA is determined. After refining this liquid trap high-performance liquid chromatography (HPLC) method in the laboratory, it was tested in five workplace settings where PAA products were used. PAA levels ranged from the detection limit of 0.013 parts per million (ppm) to 0.4 ppm. The results indicate a viable and potentially dependable method to assess the concentrations of PAA vapors under occupational exposure scenarios, though only a small number of field measurements were taken while field testing this method. However, the low limit of detection and precision offered by this method makes it a strong candidate for further testing and validation to expand the uses of this liquid trap HPLC method.

Comparison of capillary electrophoresis and high performance liquid chromatography for detection and quantification of hemoglobin New York.

PubMed

You-Qiong, Li; Hui-Ping, Huang; Zhi-Zhong, Chen; Lin, Zhao; Liang, Liang; Gui-Fang, Qin; Yun, Mo

2016-01-01

Hemoglobin (Hb) New York [β113 (G15) Val→Glu, GTG→GAG], also known as Hb Kaohsiung, is one of the most common Hb variants in South China. Currently, most used screening methods for hemoglobinopathies in South China are high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). However, there is no study comparing the performance of CE and HPLC in the detection of Hb New York. In total 15 samples (including 13 adult blood samples and 2 cord blood samples) with heterozygous Hb New York were analyzed by CE and HPLC. Levels of Hb New York, HbA2, Hb, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were compared within the two groups. All 15 cases (100%) were detected by CE, whereas none was detected by HPLC. Mean values of Hb New York and HbA2 in adult heterozygous group were (43.82±2.47)% and (2.85±0.31)%, respectively; mean values for cord blood group were (7.95±0.78)% and (0.30±0.14)%. CE allows the detection of Hb New York, while this variant is not separated from Hb A on HPLC. CE may be the preferred method for hemoglobinopathy screening in areas with high prevalence of Hb New York.

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.

Methods and applications of HPLC-AMS (WBio 5)

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

Bucholz, B A; Clifford, A J; Duecker, S R

Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a {sup 14}C-labelled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the {sup 14}C content above the control predose tissue and converting to equivalents of the parent compound. High Performance Liquid Chromatography (HPLC) is an excellent method for the chemical separation of complex mixtures whose profiles afford estimation of biochemical pathways of metabolism. Compounds elute from the HPLC systems with characteristic retention times and can be collected in fractions that can then be graphitized for AMS measurement.more » Unknowns are identified by coelution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the {sup 14}C signal. Thoroughly accounting for the carbon inventory in the LC solvents, ion-pairing agents, samples, and carriers adds some complexity to the analysis. In most cases the total carbon inventory is dominated by carrier. Baseline background and stability need to be carefully monitored. Limits of quantitation near 10 amol of {sup 14}C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected {sup 14}C activity <0.17 Bq (4.5 pCi) on the HPLC column.« less

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC-DAD and HPLC-ESI-MS.

PubMed

Karioti, Anastasia; Bolognesi, Letizia; Vincieri, Franco Francesco; Bilia, Anna Rita

2010-09-21

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC-DAD-ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC-ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids. Copyright 2010 Elsevier B.V. All rights reserved.

Two-dimensional fingerprinting approach for comparison of complex substances analysed by HPLC-UV and fluorescence detection.

PubMed

Ni, Yongnian; Liu, Ying; Kokot, Serge

2011-02-07

This work is concerned with the research and development of methodology for analysis of complex mixtures such as pharmaceutical or food samples, which contain many analytes. Variously treated samples (swill washed, fried and scorched) of the Rhizoma atractylodis macrocephalae (RAM) traditional Chinese medicine (TCM) as well as the common substitute, Rhizoma atractylodis (RA) TCM were chosen as examples for analysis. A combined data matrix of chromatographic 2-D HPLC-DAD-FLD (two-dimensional high performance liquid chromatography with diode array and fluorescence detectors) fingerprint profiles was constructed with the use of the HPLC-DAD and HPLC-FLD individual data matrices; the purpose was to collect maximum information and to interpret this complex data with the use of various chemometrics methods e.g. the rank-ordering multi-criteria decision making (MCDM) PROMETHEE and GAIA, K-nearest neighbours (KNN), partial least squares (PLS), back propagation-artificial neural networks (BP-ANN) methods. The chemometrics analysis demonstrated that the combined 2-D HPLC-DAD-FLD data matrix does indeed provide more information and facilitates better performing classification/prediction models for the analysis of such complex samples as the RAM and RA ones noted above. It is suggested that this fingerprint approach is suitable for analysis of other complex, multi-analyte substances.

Determination of Trace Level Triclosan in Water by Online Preconcentration and HPLC-UV Diode Array

EPA Science Inventory

An online high performance liquid chromatography (HPLC) method for the detection and quantification of trace levels of triclosan in water is discussed. Triclosan, an anti-bacterial agent, and related compounds have been shown to reach municipal waste waters through the disposal ...

[Study on HPLC fingerprint of Alpinia officinarum].

PubMed

Deng, Yi-Feng; Feng, Li-Na; Luo, Hui

2011-09-01

To establish the chromatography fingerprint of Alpinia officinarum by HPLC. An optimum HPLC conditions which were obtained under the assessment of LC-MS were as follows: Shim-pack VP-ODS column (2.0 mm x 250 mm, 5 microm), 0.1% HAc aqueous solution as phase A, 15% Acetonitrile: 40% Methanol: 45% Tetrafuran as phase B, the flow rate was 0.20 mL/min, column temperature was 35 degrees C and UV detector was set at 280 nm. The HPLC fingerprint of Alpinia officinarum was established, the consensus 10 peaks and their relative retention times along with the ranges of relative area were determined. The method is reliable and stable and can be used for the quality control and identification of Alpinia officinarum.

Chip-based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids.

PubMed

Chang, Yuwei; Zhao, Chunxia; Wu, Zeming; Zhou, Jia; Zhao, Sumin; Lu, Xin; Xu, Guowang

2012-08-01

In this work a chip-based nano HPLC coupled MS (HPLC-chip/MS) method with a simple sample preparation procedure was developed for the flavonoid profiling of soybean. The analytical properties of the method including the linearity (R(2) , 0.992-0.995), reproducibility (RSD, 1.50-7.66%), intraday precision (RSD, 1.41-5.14%) and interday precision (RSD, 2.76-16.90%) were satisfactory. Compared with the conventional HPLC/MS method, a fast extraction and analysis procedure was applied and more flavonoids were detected in a single run. Additionally, 13 flavonoids in soybean seed were identified for the first time. The method was then applied to the profiling of six varieties of soybean sowed at the same place. A clear discrimination was observed among different cultivars, three isoflavones, accounting for nearly 80% of total flavonoid contents, were found increased in the spring soybeans compared with the summer cultivars. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous quantitative analysis of main components in linderae reflexae radix with one single marker.

PubMed

Wang, Li-Li; Zhang, Yun-Bin; Sun, Xiao-Ya; Chen, Sui-Qing

2016-05-08

Establish a quantitative analysis of multi-components by the single marker (QAMS) method for quality evaluation and validate its feasibilities by the simultaneous quantitative assay of four main components in Linderae Reflexae Radix. Four main components of pinostrobin, pinosylvin, pinocembrin, and 3,5-dihydroxy-2-(1- p -mentheneyl)- trans -stilbene were selected as analytes to evaluate the quality by RP-HPLC coupled with a UV-detector. The method was evaluated by a comparison of the quantitative results between the external standard method and QAMS with a different HPLC system. The results showed that no significant differences were found in the quantitative results of the four contents of Linderae Reflexae Radix determined by the external standard method and QAMS (RSD <3%). The contents of four analytes (pinosylvin, pinocembrin, pinostrobin, and Reflexanbene I) in Linderae Reflexae Radix were determined by the single marker of pinosylvin. This fingerprint was the spectra determined by Shimadzu LC-20AT and Waters e2695 HPLC that were equipped with three different columns.

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.

An HPLC method for determination of azadirachtin residues in bovine muscle.

PubMed

Gai, María Nella; Álvarez, Christian; Venegas, Raúl; Morales, Javier

2011-04-01

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.

HPLC-Based Method to Evaluate Kinetics of Glucosinolate Hydrolysis by Sinapis alba Myrosinase1

PubMed Central

Vastenhout, Kayla J.; Tornberg, Ruthellen H.; Johnson, Amanda L.; Amolins, Michael W.; Mays, Jared R.

2014-01-01

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites which are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic, non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established UV-Vis spectroscopic methods. To overcome this limitation, an alternative HPLC-based analytical approach was developed where initial reaction velocities were generated from non-linear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV-Vis methodology. The results of this study demonstrate that kinetic data is consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports. PMID:25068719

Validation of a simple HPLC-UV method for rifampicin determination in plasma: Application to the study of rifampicin arteriovenous concentration gradient.

PubMed

Goutal, Sébastien; Auvity, Sylvain; Legrand, Tiphaine; Hauquier, Fanny; Cisternino, Salvatore; Chapy, Hélène; Saba, Wadad; Tournier, Nicolas

2016-05-10

In clinical practice, rifampicin exposure is estimated from its concentration in venous blood samples. In this study, we hypothesized that differences in rifampicin concentration may exist between arterial and venous plasma. An HPLC-UV method for determining rifampicin concentration in plasma using rifapentine as an internal standard was validated. The method, which requires a simple protein precipitation procedure as sample preparation, was performed to compare venous and arterial plasma kinetics after a single therapeutic dose of rifampicin (8.6 mg/kg i.v, infused over 30 min) in baboons (n=3). The method was linear from 0.1 to 40 μg mL(-1) and all validation parameters fulfilled the international requirements. In baboons, rifampicin concentration in arterial plasma was higher than in venous plasma. Arterial Cmax was 2.1±0.2 fold higher than venous Cmax. The area under the curve (AUC) from 0 to 120 min was ∼80% higher in arterial plasma, indicating a significant arteriovenous concentration gradient in early rifampicin pharmacokinetics. Arterial and venous plasma concentrations obtained 6h after rifampicin injection were not different. An important arteriovenous equilibration delay for rifampicin pharmacokinetics is reported. Determination in venous plasma concentrations may considerably underestimate rifampicin exposure to organs during the distribution phase. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

PubMed

Fanali, Chiara; Dugo, Laura; D'Orazio, Giovanni; Lirangi, Melania; Dachà, Marina; Dugo, Paola; Mondello, Luigi

2011-01-01

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determining Vitamin D Status: A Comparison between Commercially Available Assays

PubMed Central

Snellman, Greta; Melhus, Håkan; Gedeborg, Rolf; Byberg, Liisa; Berglund, Lars; Wernroth, Lisa; Michaëlsson, Karl

2010-01-01

Background Vitamin D is not only important for bone health but can also affect the development of several non-bone diseases. The definition of vitamin D insufficiency by serum levels of 25-hydroxyvitamin D depends on the clinical outcome but might also be a consequence of analytical methods used for the definition. Although numerous 25-hydroxyvitamin D assays are available, their comparability is uncertain. We therefore aim to investigate the precision, accuracy and clinical consequences of differences in performance between three common commercially available assays. Methodology/Principal Findings Serum 25-hydroxyvitamin D levels from 204 twins from the Swedish Twin Registry were determined with high-pressure liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), a radioimmunoassay (RIA) and a chemiluminescent immunoassay (CLIA). High inter-assay disagreement was found. Mean 25-hydroxyvitamin D levels were highest for the HPLC-APCI-MS technique (85 nmol/L, 95% CI 81–89), intermediate for RIA (70 nmol/L, 95% CI 66–74) and lowest with CLIA (60 nmol/L, 95% CI 56–64). Using a 50-nmol/L cut-off, 8% of the subjects were insufficient using HPLC-APCI-MS, 22% with RIA and 43% by CLIA. Because of the heritable component of 25-hydroxyvitamin D status, the accuracy of each method could indirectly be assessed by comparison of within-twin pair correlations. The strongest correlation was found for HPLC-APCI-MS (r = 0.7), intermediate for RIA (r = 0.5) and lowest for CLIA (r = 0.4). Regression analyses between the methods revealed a non-uniform variance (p<0.0001) depending on level of 25-hydroxyvitamin D. Conclusions/Significance There are substantial inter-assay differences in performance. The most valid method was HPLC-APCI-MS. Calibration between 25-hydroxyvitamin D assays is intricate. PMID:20644628

Application of electrolysis for inactivation of an antiviral drug that is one of possible selection pressure to drug-resistant influenza viruses.

PubMed

Kobayashi, Toyohide; Hirose, Jun; Wu, Hong; Sano, Kouichi; Katsumata, Takahiro; Tsujibo, Hiroshi; Nakano, Takashi

2013-12-01

The recent development of antiviral drugs has led to concern that the release of the chemicals in surface water due to expanded medical use could induce drug-resistant mutant viruses in zoonosis. Many researchers have noted that the appearance of an oseltamivir (Tamiflu(®))-resistant avian influenza mutant virus, which may spread to humans, could be induced by oseltamivir contamination of surface water. Although past studies have reported electrolysis as a possible method for degradation of antineoplastics and antibacterials in water, the validity of the method for treatment of antiviral drugs is unknown. In this study, electrolysis was used to degrade an antiviral prodrug, oseltamivir, and a stable active form, oseltamivir carboxylate, and the degradation process was monitored with HPLC-UV and the neuraminidase inhibitory assay. HPLC-UV-detectable oseltamivir and oseltamivir carboxylate were decomposed by electrolysis within 60 min, and inhibitory activity of neuraminidase decreased below the detection limit of the assay used. Cytotoxic and genotoxic activity were not detected in electrolyzed fluid. These results indicate that electrolysis is a possible treatment for inactivation of the antiviral drug oseltamivir. Copyright © 2013 Elsevier B.V. All rights reserved.

Analysis of pharmaceutical impurities using multi-heartcutting 2D LC coupled with UV-charged aerosol MS detection.

PubMed

Zhang, Kelly; Li, Yi; Tsang, Midco; Chetwyn, Nik P

2013-09-01

To overcome challenges in HPLC impurity analysis of pharmaceuticals, we developed an automated online multi-heartcutting 2D HPLC system with hyphenated UV-charged aerosol MS detection. The first dimension has a primary column and the second dimension has six orthogonal columns to enhance flexibility and selectivity. The two dimensions were interfaced by a pair of switching valves equipped with six trapping loops that allow multi-heartcutting of peaks of interest in the first dimension and also allow "peak parking." The hyphenated UV-charged aerosol MS detection provides comprehensive detection for compounds with and without UV chromophores, organics, and inorganics. It also provides structural information for impurity identification. A hidden degradation product that co-eluted with the drug main peak was revealed by RP × RP separation and thus enabled the stability-indicating method development. A poorly retained polar component with no UV chromophores was analyzed by RP × hydrophilic interaction liquid chromatography separation with charged aerosol detection. Furthermore, using this system, the structures of low-level impurities separated by a method using nonvolatile phosphate buffer were identified and tracked by MS in the second dimension. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of Free Polyamines in Plants Subjected to Abiotic Stresses by High-Performance Liquid Chromatography (HPLC).

PubMed

Gong, Xiaoqing; Liu, Ji-Hong

2017-01-01

High-performance liquid chromatography (HPLC) is a sensitive, rapid, and accurate technique to detect and characterize various metabolites from plants. The metabolites are extracted with different solvents and eluted with appropriate mobile phases in a designed HPLC program. Polyamines are known to accumulate under abiotic stress conditions in various plant species and thought to provide protection against oxidative stress by scavenging reactive oxygen species. Here, we describe a common method to detect the free polyamines in plant tissues both qualitatively and quantitatively.

Phenolic acids in the flowers of Althaea rosea var. nigra.

PubMed

Dudek, Marlena; Matławska, Irena; Szkudlarek, Maurycy

2006-01-01

Distribution of phenolic acids in the flowers of Althaea rosea var. nigra has been studied by 2D-TLC and HPLC methods. The phenolic acids occurring in these fractions have been identified as ferulic, vanillic, syringic, p-coumaric, p-hydroxybenzoic, p-hydroxyphenylacetic and caffeic acids. By means of the HPLC methods the contents of major phenolic acids were estimated. From among the phenolic acids analyzed the syringic, p-hydroxybenzoic and p-coumaric acids are dominant. Total content of phenolic acids was determined by the Arnov's method.

Partial Characterization of Venom from the Colombian Spider Phoneutria Boliviensis (Aranae:Ctenidae)

PubMed Central

Estrada-Gomez, Sebastian; Vargas Muñoz, Leidy Johana; Lanchero, Paula; Segura Latorre, Cesar

2015-01-01

We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors. PMID:26264023

Stability of dronabinol capsules when stored frozen, refrigerated, or at room temperature.

PubMed

Wempe, Michael F; Oldland, Alan; Stolpman, Nancy; Kiser, Tyree H

2016-07-15

Results of a study to determine the 90-day stability of dronabinol capsules stored under various temperature conditions are reported. High-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was used to assess the stability of dronabinol capsules (synthetic delta-9-tetrahydrocannabinol [Δ9-THC] mixed with high-grade sesame oil and other inactive ingredients and encapsulated as soft gelatin capsules) that were frozen, refrigerated, or kept at room temperature for three months. The dronabinol capsules remained in the original foil-sealed blister packs until preparation for HPLC-UV assessment. The primary endpoint was the percentage of the initial Δ9-THC concentration remaining at multiple designated time points. The secondary aim was to perform forced-degradation studies under acidic conditions to demonstrate that the HPLC-UV method used was stability indicating. The appearance of the dronabinol capsules remained unaltered during frozen, cold, or room-temperature storage. Regardless of storage condition, the percentage of the initial Δ9-THC content remaining was greater than 97% for all evaluated samples at all time points over the three-month study. These experimental data indicate that the product packaging and the sesame oil used to formulate dronabinol capsules efficiently protect Δ9-THC from oxidative degradation to cannabinol; this suggests that pharmacies can store dronabinol capsules in nonrefrigerated automated dispensing systems, with a capsule expiration date of 90 days after removal from the refrigerator. Dronabinol capsules may be stored at room temperature in their original packaging for up to three months without compromising capsule appearance and with minimal reduction in Δ9-THC concentration. Copyright © 2016 by the American Society of Health-System Pharmacists, Inc. All rights reserved.

Dissemination of the highly expressed Bx7 glutenin subunit (Glu-B1al allele) in wheat as revealed by novel PCR markers and RP-HPLC.

PubMed

Butow, B J; Gale, K R; Ikea, J; Juhász, A; Bedö, Z; Tamás, L; Gianibelli, M C

2004-11-01

Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called 'over-expressing' allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization.

Isolation and Characterization of a Novel, Highly Selective Astaxanthin-Producing Marine Bacterium.

PubMed

Asker, Dalal

2017-10-18

A high-throughput screening approach for astaxanthin-producing bacteria led to the discovery of a novel, highly selective astaxanthin-producing marine bacterium (strain N-5). Phylogenetic analysis based on partial 16S rRNA gene and phenotypic metabolic testing indicated it belongs to the genus Brevundimonas. Therefore, it was designated as Brevundimonas sp. strain N-5. To identify and quantify carotenoids produced by strain N-5, HPLC-DAD and HPLC-MS methods were used. The culture conditions including media, shaking, and time had significant effects on cell growth and carotenoids production including astaxanthin. The total carotenoids were ∼601.2 μg g -1 dry cells including a remarkable amount (364.6 μg g -1 dry cells) of optically pure astaxanthin (3S, 3'S) isomer, with high selectivity (∼60.6%) under medium aeration conditions. Notably, increasing the culture aeration enhanced astaxanthin production up to 85% of total carotenoids. This is the first report that describes a natural, highly selective astaxanthin-producing marine bacterium.

An improved HPLC method for the analysis of citrus limonoids in culture media.

PubMed

Tian, Qingguo; Miller, Edward G; Jayaprakasha, G K; Patil, Bhimanagouda S

2007-02-01

Recent studies have shown that citrus limonoids have potential health benefits. However, information on the absorption and metabolism of limonoids in human gastrointestinal (GI) tract is limited. In the present study we have investigated the metabolism of limonin glucoside (LG), the predominant limonoid in citrus by four microorganisms (Enterococcus fecalis, Escherichia coli, Lactobacillus salivarius, and Candida albican) widely present in the human lower GI tract. LG and metabolites in the culture medium were purified using solid phase extraction and analyzed using HPLC using UV detection at 210nm. The identity of LG was further confirmed by electrospray ionization mass spectrometry (ESI-MS). Significant metabolic activity of Escherichia coli and Candida albican on LG was observed. Several unidentified metabolites were also found in the medium. The results of the present study indicated that LG may be metabolized in the intestine by some microbes. Further studies are needed to establish the possible route of LG metabolism in the human system.

A reexamination of amino acids in lunar soil

NASA Technical Reports Server (NTRS)

Brinton, K. L. F.; Bada, J. L.; Arnold, J. R.

1993-01-01

Amino acids in lunar soils provide an important indicator of the level of prebiotic organic compounds on the moon. The results provide insight into the chemistry of amino acid precursors, and furthermore, given the flux of carbonaceous material to the moon, we can evaluate the survival of organics upon impact. The amino acid contents of both hydrolyzed and unhydrolyzed hot-water extracts of Apollo 17 lunar soil were determined using ophthaldialdehyde/N-acetyl cysteine (OPA/NAC) derivatization followed by HPLC analysis. Previous studies of lunar amino acids were inconclusive, as the technique used (derivatization with ninhydrin followed by HPLC analysis) was unable to discriminate between cosmogenic amino acids and terrestrial contaminants. Cosmogenic amino acids are racemic, and many of the amino acids found in carbonaceous meteorites such as Murchison, i.e., alpha-amino-i-butyric acid (aib), are extremely rare on Earth. The ninhydrin method does not distinguish amino acid enantiomers, nor does it detect alpha-alkyl amino acids such as aib, whereas the OPA/NAC technique does both.

The Third SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE-3)

NASA Technical Reports Server (NTRS)

Hooker, Stanford B.; VanHeukelem, Laurei; Thomas, Crystal S.; Claustre, Herve; Ras, Josephine; Schluter, Louise; Clementson, Lesley; vanderLinde, Dirk; Eker-Develi, Elif; Berthon, Jean-Francois;

A Jerte Valley Cherry-Based Product as a Supply of Tryptophan

PubMed Central

Garrido, María; Espino, Javier; Toribio-Delgado, Antonio F.; Cubero, Javier; Maynar-Mariño, Juan I.; Barriga, Carmen; Paredes, Sergio D.; Rodríguez, Ana B.

2012-01-01

L-Tryptophan (tryptophan) is an essential amino acid in humans. It has important roles as a precursor of different bioactive compounds. Based on previous studies in which tryptophan has been shown to be present in fresh cherries, the aim of the present work was to analyze the tryptophan content of a Jerte Valley cherry-based product. A previously optimized method of analysis of tryptophan was used, ie, high-performance liquid chromatography with fluorescence detection (HPLC/FL). As expected, HPLC/FL technique permitted to detect and quantify the tryptophan content in a different matrix rather than fresh cherries. In fact, the Jerte Valley cherry-based product contained 69.54 ± 10.64 ppm of tryptophan, thereby showing that this product is a good source of tryptophan. In summary, it has been proven that the Jerte Valley cherry-based product is rich in tryptophan and may be indicated as a supply of this essential amino acid as well as having potential health benefits for conditions where tryptophan is necessary. PMID:22553424

Separation of cordycepin from Cordyceps militaris fermentation supernatant using preparative HPLC and evaluation of its antibacterial activity as an NAD+-dependent DNA ligase inhibitor

PubMed Central

Zhou, Xiaofeng; Cai, Guoqiang; He, Yi; Tong, Guotong

2016-01-01

Cordycepin exhibits various bio-activities, including anticancer, antibacterial, antiviral and immune regulation activities, and is a significant focus of research. However, the preparation of high-purity cordycepin remains challenging. Also, the molecular target with which cordycepin interacts to cause an antibacterial effect remains unknown. In the present study, cordycepin was prepared by preparative high-performance liquid chromatography (prep-HPLC) and the purity obtained was 99.6%, indicating that this technique may be useful for the large-scale isolation of cordycepin in the future. The results of computational molecular docking analysis indicated that the interaction energy between cordycepin and NAD+-dependent DNA ligase (LigA) was lower than that between cordycepin and other common antibacterial targets. The highly pure cordycepin obtained by prep-HPLC demonstrated inhibitory activity against LigA from various bacteria in vitro. In conclusion, cordycepin may be useful as a broad-spectrum antibiotic targeting LigA in various bacteria. PMID:27588098

Ingredient Consistency of Commercially Available Polyphenol and Tocopherol Nutraceuticals

PubMed Central

Remsberg, Connie M.; Good, Renee L.; Davies, Neal M.

2010-01-01

Label claims of vitamin E succinate and polyphenolic nutraceuticals are assessed. A validated HPLC method was utilized to assess vitamin E succinate products. Three novel LC/MS methods were used to assess the polyphenols, pterostilbene, phloretin, and myricetin, in dietary supplements. The amount of vitamin E succinate varied from 0-130% of the stated label content with two products containing vitamin E acetate rather than vitamin E succinate. Expected polyphenols were found in 7 of the 8 supplement products. None of the polyphenol supplements contained content within 100-120% of label claims. The present study indicates a lack of uniformity in nutraceutical products. PMID:27721342

High performance liquid chromatographic determination of caffeine in decaffeinated coffee, tea, and beverage products.

PubMed

Ashoor, S H; Seperich, G J; Monte, W C; Welty, J

1983-05-01

A method was developed for determining caffeine in decaffeinated coffee, tea, and beverage products by high performance liquid chromatography (HPLC). The HPLC system consisted of a Bio-Sil ODS-5S C18 column, methanol-water (25 + 75) mobile phase at 1 mL/min, and a UV detector. The method is simple and specific. Caffeine recoveries were 93.8-98.3% and coefficients of variation were 0.90-2.25%.

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

Analysis of antibiotic fungicide kasugamycin in irrigation water by high performance liquid chromatography.

PubMed

Sheu, Ceshing; Chen, Shu-Chuan; Lo, Chi-Chu

2010-07-01

A high performance liquid chromatographic (HPLC) analysis method with an ultraviolet (UV) detector and an Aqua C18 (250 x 4.6 mm, Phenomenex) column were applied to analyze the antibiotic fungicide kasugamycin in water. An aromatic sulfonic acid spe column (Backerbond, J. T. Backer) was used to remove the interfering materials from irrigation water. A good linear relation existed between the concentration of the fungicide and the peak area, and correlation coefficient of linearity from 0.1 to 10.2 microg/mL was 0.998. The accuracies expressed as the recoveries of kasugamycin from irrigation water ranged from 112.2 to 111.7 %. The precisions expressed as relative standard deviations (RSD) were found to be below 7.0 %. The quantitative detection limit (LOQ) of kasugamycin in irrigation water was set at 2.2 microg/mL which was 2-times higher than the method detection limit (MDL) 1.03 microg/mL. Electrospray ionization-mass (ESI-MS) and fast-atom bombardment-mass (FAB-MS) were applied to compare the ability of identifying the component of the eluent peak from HPLC, and the result indicated that electrospray ionization-mass (ESI-MS) was more sensitive than fast-atom bombardment-mass (FAB-MS) in the detection of kasugamycin. There was no kasugamycin residue detected in irrigation water samples collected from paddyfields at Wufong, indicated that the residues of kasugamycin in water were less than 2.2 microg/mL, and the risk of water contamination was very low.

Amino Acid profile as a feasible tool for determination of the authenticity of fruit juices.

PubMed

Asadpoor, Mostafa; Ansarin, Masoud; Nemati, Mahboob

2014-12-01

Fruit juice is a nutrient rich food product with a direct connection to public health. The purpose of this research was to determine the amino acid profile of juices and provide a quick and accurate indicator for determining their authenticity. The method of analysis was HPLC with fluorescence detector and pre-column derivatization by orthophtaldialdehyde (OPA). Sixty-six samples of fruit juices were analyzed, and fourteen amino acids were identified and determined in the sampled fruit juices. The fruit samples used for this analysis were apples, oranges, cherry, pineapple, mango, apricot, pomegranate, peach and grapes. The results showed that 32% of samples tested in this study had a lower concentrate percentage as compared to that of their labels and/or other possible authenticity problems in the manufacturing process. The following samples showed probable adulteration: four cherry juice samples, two pomegranate juice samples, one mango, three grape, four peach, seven orange, two apple and one apricot juice samples. In general, determining the amount of amino acids and comparing sample amino acids profiles with the standard values seems to be an indicator for quality control. This method can provide the regulatory agencies with a tool, to help produce a healthier juice. The aim of this study is the analytical control of the fruit juice composition is becoming an important issue, and HPLC can provide an important and essential tool for more accurate research as well as for routine analysis.

Semi-preparative HPLC preparation and HPTLC quantification of tetrahydroamentoflavone as marker in Semecarpus anacardium and its polyherbal formulations.

PubMed

Aravind, S G; Arimboor, Ranjith; Rangan, Meena; Madhavan, Soumya N; Arumughan, C

2008-11-04

Application of modern scientific knowledge coupled with sensitive analytical technique is important for the quality evaluation and standardization of polyherbal formulations. Semecarpus anacardium, an important medicinal plant with wide medicinal properties, is frequently used in a large number of traditional herbal preparations. Tetrahydroamentoflavone (THA), a major bioactive biflavonoid was selected as a chemical marker of S. anacardium and RP-semi-preparative HPLC conditions were optimized for the isolation of tetrahydroamentoflavone. HPTLC analytical method was developed for the fingerprinting of S. anacardium flavonoids and quantification of tetrahydroamentoflavone. The method was validated in terms of their linearity, LOD, LOQ, precision and accuracy and compared with RP-HPLC-DAD method. The methods were demonstrated for the chemical fingerprinting of S. anacardium plant parts and some commercial polyherbal formulations and the amount of tetrahydroamentoflavone was quantified. HPTLC analysis showed that S. anacardium seed contained approximately 10 g kg(-1) of tetrahydroamentoflavone. The methods were able to identify and quantify tetrahydroamentoflavone from complex mixtures of phytochemicals and could be extended to the marker-based standardization of polyherbal formulations, containing S. anacardium.

A single extraction and HPLC procedure for simultaneous analysis of phytosterols, tocopherols and lutein in soybeans.

PubMed

Slavin, Margaret; Yu, Liangli Lucy

2012-12-15

A saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for simultaneous analysis of phytosterols, tocopherols and lutein (a carotenoid) in soybeans. Separation was achieved on a phenyl column with a ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v). Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol, stigmasterol, campesterol, and α-, δ- and γ-tocopherols, while lutein was quantified with visible light absorption at 450 nm. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (R(2)>0.99) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. Also, the accuracy of results of four soybeans using the described saponification and HPLC analytical method was validated against existing methods. This method offers a more efficient alternative to individual methods for quantifying lutein, tocopherols and sterols in soybeans. Copyright © 2012 Elsevier Ltd. All rights reserved.

A validated solid-liquid extraction method for the HPLC determination of polyphenols in apple tissues Comparison with pressurised liquid extraction.

PubMed

Alonso-Salces, Rosa M; Barranco, Alejandro; Corta, Edurne; Berrueta, Luis A; Gallo, Blanca; Vicente, Francisca

2005-02-15

A solid-liquid extraction procedure followed by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector (DAD) for the determination of polyphenols in freeze-dried apple peel and pulp is reported. The extraction step consists in sonicating 0.5g of freeze-dried apple tissue with 30mL of methanol-water-acetic acid (30:69:1, v/v/v) containing 2g of ascorbic acid/L, for 10min in an ultrasonic bath. The whole method was validated, concluding that it is a robust method that presents high extraction efficiencies (peel: >91%, pulp: >95%) and appropriate precisions (within day: R.S.D. (n = 5) <5%, and between days: R.S.D. (n = 5) <7%) at the different concentration levels of polyphenols that can be found in apple samples. The method was compared with one previously published, consisting in a pressurized liquid extraction (PLE) followed by RP-HPLC-DAD determination. The advantages and disadvantages of both methods are discussed.

Pungency Quantitation of Hot Pepper Sauces Using HPLC

NASA Astrophysics Data System (ADS)

Betts, Thomas A.

1999-02-01

A class of compounds known as capsaicinoids are responsible for the "heat" of hot peppers. To determine the pungency of a particular pepper or pepper product, one may quantify the capsaicinoids and relate those concentrations to the perceived heat. The format of the laboratory described here allows students to collectively develop an HPLC method for the quantitation of the two predominant capsaicinoids (capsaicin and dihydrocapsaicin) in hot-pepper products. Each small group of students investigated one of the following aspects of the method: detector wavelength, mobile-phase composition, extraction of capsaicinoids, calibration, and quantitation. The format of the lab forced students to communicate and cooperate to develop this method. The resulting HPLC method involves extraction with acetonitrile followed by solid-phase extraction clean-up, an isocratic 80:20 methanol-water mobile phase, a 4.6 mm by 25 cm C-18 column, and UV absorbance detection at 284 nm. The method developed by the students was then applied to the quantitation of capsaicinoids in a variety of hot pepper sauces. Editor's Note on Hazards in our April 2000 issue addresses the above.

Inulin determination for food labeling.

PubMed

Zuleta, A; Sambucetti, M E

2001-10-01

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.