Chromatographic analysis of salicylic compounds in different species of the genus Salix.

PubMed

Pobłocka-Olech, Loretta; van Nederkassel, Anne-Marie; Vander Heyden, Yvan; Krauze-Baranowska, Mirosława; Glód, Daniel; Baczek, Tomasz

2007-11-01

The separation of nine phenol glycosides--salicin, salicortin, 2'-acetylsalicortin, populin, tremulacin, salidroside, triandrin, picein and helicin--by normal phase (NP), reversed phase (RP) HPLC techniques and a coupling of NP and RP monolithic silica columns was studied. Among the above nine compounds only five--salicin, populin, tremulacin, salidroside and triandrin--were resolved in an NP system with a mobile phase comprising hexane/isopropanol/methanol (87:12:1, v/v/v). Optimized separation was performed with two coupled monolithic silica columns of different polarity (bare silica and RP-18). The method was applied to verify the presence of salicylic compounds and other phenolic derivatives in the bark of six species from the genus Salix, namely S. purpurea, S. daphnoides clone 1095, S. alba clone 1100, S. triandra, S. viminalis, and S. herbacea. Gradient elution with a mobile phase composed of acetonitrile and water containing 0.05% of trifluoroacetic acid, with increasing acetonitrile concentration from 3% to 48%, was chosen as optimal. For the selective detection of the salicylic compounds, an evaporative light scattering detector was employed along with a UV detector. The differences in the composition of phenols in the different plant materials were confirmed. Additionally, it must be emphasized that for the first time the presence of 2'-acetylsalicortin was revealed in S. alba clone 1100. Furthermore, an SPE-HPLC method was developed for the rapid analysis of the salicin content, analyzed as free and total fraction, in willow barks. The determined concentrations of total salicin varied from 25.4 mg/g in S. alba clone 1100 to 96.47 mg/g in S. daphnoides clone 1095.

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

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

PubMed

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

2011-01-01

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

New Stability Indicating RP-HPLC Method for the Estimation of Cefpirome Sulphate in Bulk and Pharmaceutical Dosage Forms.

PubMed

Rao, Kareti Srinivasa; Kumar, Keshar Nargesh; Joydeep, Datta

2011-01-01

A simple stability indicating reversed-phase HPLC method was developed and subsequently validated for estimation of Cefpirome sulphate (CPS) present in pharmaceutical dosage forms. The proposed RP-HPLC method utilizes a LiChroCART-Lichrosphere100, C18 RP column (250 mm × 4mm × 5 μm) in an isocratic separation mode with mobile phase consisting of methanol and water in the proportion of 50:50 % (v/v), at a flow rate 1ml/min, and the effluent was monitored at 270 nm. The retention time of CPS was 2.733 min and its formulation was exposed to acidic, alkaline, photolytic, thermal and oxidative stress conditions, and the stressed samples were analyzed by the proposed method. The described method was linear over a range of 0.5-200μg/ml. The percentage recovery was 99.46. F-test and t-test at 95% confidence level were used to check the intermediate precision data obtained under different experimental setups; the calculated value was found to be less than the critical value.

Composition and Molecular Weight Distribution of Carob Germ Proteins Fractions

USDA-ARS?s Scientific Manuscript database

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

[Assay of three hydrolyzable tannins in Fructus Chebulae from different habitats by RP-HPLC].

PubMed

Ding, G; Liu, Y; Wang, W

2000-06-01

Three hydrolyzable tannins chebulinic acid (I), chebulagic acid(II) and 1,3, 6-tri-O-galloyl-beta-D-glucose (III) in Fructus Chebulae from different habitats were determined by RP-HPLC method. The contents of I and II were obviously interrelated with the variety and characteristics of Fructus Chebulae. It's suitable to use I and II as indexes in quality evaluation of the crude drug of Fructus Chebulae.

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

PubMed

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

2016-11-15

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

Tocochromanols composition in kernels recovered from different apricot varieties: RP-HPLC/FLD and RP-UPLC-ESI/MS(n) study.

PubMed

Górnaś, Paweł; Mišina, Inga; Grāvīte, Ilze; Soliven, Arianne; Kaufmane, Edīte; Segliņa, Dalija

2015-01-01

Composition of tocochromanols in kernels recovered from 16 different apricot varieties (Prunus armeniaca L.) was studied. Three tocopherol (T) homologues, namely α, γ and δ, were quantified in all tested samples by an RP-HPLC/FLD method. The γ-T was the main tocopherol homologue identified in apricot kernels and constituted approximately 93% of total detected tocopherols. The RP-UPLC-ESI/MS(n) method detected trace amounts of two tocotrienol homologues α and γ in the apricot kernels. The concentration of individual tocopherol homologues in kernels of different apricots varieties, expressed in mg/100 g dwb, was in the following range: 1.38-4.41 (α-T), 42.48-73.27 (γ-T) and 0.77-2.09 (δ-T). Moreover, the ratio between individual tocopherol homologues α:γ:δ was nearly constant in all varieties and amounted to approximately 2:39:1.

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

NASA Astrophysics Data System (ADS)

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

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

Isolation, Characterization, and RP-HPLC Estimation of P-Coumaric Acid from Methanolic Extract of Durva Grass (Cynodon dactylon Linn.) (Pers.)

PubMed Central

Karthikeyan, Ramadoss; Devadasu, Chapala; Srinivasa Babu, Puttagunta

2015-01-01

P-coumaric acid is a nonflavonoid phenolic acid and is a major constituent of the species Cynodon dactylon Linn. (Pers.). In this study isolation of P-coumaric acid was achieved by preparative TLC and the compound thus isolated was characterised by UV, mass, and H1 NMR spectral analysis. An isocratic RP-HPLC method was developed for the estimation of P-coumaric acid from methanolic extracts of durva grass. The chromatographic separations were achieved by RP-C18 column (250 mm × 4.6 mm, 5 μ), Shimadzu LC-20AT Prominence liquid chromatograph, and a mobile phase composed of water : methanol : glacial acetic acid (65 : 34 : 1 v/v). The flow rate was 1.0 mL/min and the analyses of column effluents were performed using UV-visible detector at 310 nm. Retention time of P-coumaric acid was found to be 6.617 min. This method has obeyed linearity over the concentration range of 2–10 μg/mL and the regression coefficient obtained from linearity plot for P-coumaric acid was found to be 0.999. RP-HPLC method was validated in pursuance of ICH guidelines. PMID:25788944

Simultaneous determination of 12 chemical constituents in the traditional Chinese Medicinal Prescription Xiao-Yao-San-Jia-Wei by HPLC coupled with photodiode array detection.

PubMed

Zhang, Hongmin; Chen, Shiwei; Qin, Feng; Huang, Xi; Ren, Ping; Gu, Xinqi

2008-12-15

An HPLC-photodiode array (PDA) detection method was established for the simultaneous determination of 12 components in Xiao-Yao-San-Jia-Wei (XYSJW): geniposide, puerarin, paeoniflorin, ferulic acid, liquiritin, hesperidin, naringin, paeonol, daidzein, glycyrrhizic acid, honokiol, and magnolol. These were separated in less than 70 min using a Waters Symmetry Shield RP 18 column with gradient elution using (A) acetonitrile, (B) water, and (C) acetic acid at a flow rate of 1 ml/min, and with a PDA detector. All calibration curves showed good linear regression (r(2)>0.9992) within the test ranges. The method was validated for specificity, accuracy, precision, and limits of detection. The proposed method enables in a single run the simultaneous identification and determination for quality control of 12 multi-structural components of XYSJW forming the basis of its therapeutic effect.

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...

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

PubMed

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

2017-07-01

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

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

EPA Science Inventory

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

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

PubMed

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

2012-04-13

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

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.

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

PubMed Central

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC50 value of 2.27 μg mL−1 and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation. PMID:22489129

Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

PubMed

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.

Development and validation of a stability-indicating RP-HPLC method for determination of atomoxetine hydrochloride in tablets.

PubMed

Patel, Sejal K; Patel, Natvarlal J

2010-01-01

This paper describes the development of a stability-indicating RP-HPLC method for the determination of atomoxetine hydrochloride (ATX) in the presence of its degradation products generated from forced decomposition studies. The drug substance was subjected to stress conditions of acid, base, oxidation, wet heat, dry heat, and photodegradation. In stability tests, the drug was susceptible to acid, base, oxidation, and dry and wet heat degradation. It was found to be stable under the photolytic conditions tested. The drug was successfully separated from the degradation products formed under stress conditions on a Phenomenex C18 column (250 x 4.6 mm id, 5 microm particle size) by using acetonitrile-methanol-0.032 M ammonium acetate (55 + 05 + 40, v/v/v) as the mobile phase at 1.0 mL/min and 40 degrees C. Photodiode array detection at 275 nm was used for quantitation after RP-HPLC over the concentration range of 0.5-5 microg/mL with a mean recovery of 100.8 +/- 0.4% for ATX. Statistical analysis demonstrated that the method is repeatable, specific, and accurate for the estimation of ATX. Because the method effectively separates the drug from its degradation products, it can be used as a stability-indicating method.

Development and Validation of a Stability-Indicating RP-HPLC Method for Duloxetine Hydrochloride in its Bulk and Tablet Dosage Form

PubMed Central

Chhalotiya, Usmangani K.; Bhatt, Kashyap K.; Shah, Dimal A.; Baldania, Sunil L.

2010-01-01

The objective of the present work was to develop a stability-indicating RP-HPLC method for duloxetine hydrochloride (DUL) in the presence of its degradation products generated from forced decomposition studies. The drug substance was found to be susceptible to stress conditions of acid hydrolysis. The drug was found to be stable to dry heat, photodegradation, oxidation and basic condition attempted. Successful separation of the drug from the degradation products formed under acidic stress conditions was achieved on a Hypersil C-18 column (250 mm × 4.6 mm id, 5μm particle size) using acetonitrile: 0.01 M potassium dihydrogen phosphate buffer (pH 5.4 adjusted with orthophosphoric acid) (50:50, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Quantification was achieved with photodiode array detection at 229 nm over the concentration range 1–25 μg/ml with range of recovery 99.8–101.3 % for DUL by the RP-HPLC method. Statistical analysis proved the method to be repeatable, specific, and accurate for estimation of DUL. It can be used as a stability-indicating method due to its effective separation of the drug from its degradation products, PMID:21179321

Molecular characterization and bio-functional property determination using SDS-PAGE and RP-HPLC of protein fractions from two Nigella species.

PubMed

Alu'datt, Muhammad H; Rababah, Taha; Alhamad, Mohammad N; Alodat, Moh'd; Al-Mahasneh, Majdi A; Gammoh, Sana; Ereifej, Khalil; Almajwal, Ali; Kubow, Stan

2017-09-01

This study aimed to investigate the molecular and bio-functional properties of protein fractions from Nigella damascena and Nigella arvensis, including the albumin, globulin, glutein-1, glutein-2 and prolamin fractions. Protein subunits were not observed in globulin and prolamin fractions. No peaks appeared in RP-HPLC chromatograms of globulin for either species. Two predominant peaks were observed in the RP-HPLC profiles of all protein fractions. Proteins separated by RP-HPLC have potential inhibitory and antioxidant activities in all fractions. Optimum ACE-inhibitory and antioxidant activities of proteins separated by RP-HPLC were observed in glutein-2 and albumin, respectively, for both species. For pepsin and combined pepsin-trypsin hydrolyses, the highest degree of hydrolysis (DH) was obtained in glutein-2 fraction of Nigella arvensis. Highest ACE-inhibitory activity of hydrolyzed protein fractions was found at 4h via pepsin hydrolysis in globulin fraction of Nigella damascena. Highest antioxidant activities of hydrolyzed protein fractions were found in glutelin-2 for both species. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and validation of an RP-HPLC method for quantitative determination of vanillin and related phenolic compounds in Vanilla planifolia.

PubMed

Sinha, Arun Kumar; Verma, Subash Chandra; Sharma, Upendra Kumar

2007-01-01

A simple and fast method was developed using RP-HPLC for separation and quantitative determination of vanillin and related phenolic compounds in ethanolic extract of pods of Vanilla planifolia. Ten phenolic compounds, namely 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal were quantitatively determined using ACN, methanol, and 0.2% acetic acid in water as a mobile phase with a gradient elution mode. The method showed good linearity, high precision, and good recovery of compounds of interest. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.

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

Reverse-phase HPLC analysis of human alpha crystallin.

PubMed

Swamy, M S; Abraham, E C

1991-03-01

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

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

PubMed

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

2014-03-01

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

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.

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

PubMed

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

2009-07-01

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

[Determination of aristolochic acid A in Guanxinsuhe preparations by RP-HPLC].

PubMed

Li, Lin; Gao, Hui-Min; Wang, Zhi-Min; Wang, Wei-Hao

2006-01-01

To establish a determination method of aristolochic acid A in Guanxisuhe preparations by RP-HPLC. The instrument used was Hewlett-Packard 1100 HPLC with a Alltech C18 column (4.6 mm x 250 mm, 5 microm). The mobile phase was methanol-water-acetic acid (68: 32:1) and the flow rate was 1.0 mL x min(-1). The UV detection wavelength was 390 nm and the column temperature was at 35 degrees C. The extracted solvent for the preparations was methanol solution contained 10% formic acid. The calibration curve was linear (r = 0.999 9) within the range of 0.119-1.89 microg for aristolochic acid A. The average recovery 99.0%, RSD 0.63%. The method with good linear relationship was convenient, quick, accurate, and suitable for the quality control of the aristolochic acid A in Guanxinsuhe and other traditional Chinese medicines containing aristolochic acid A.

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

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

PubMed

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

2018-06-02

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

Validation of a RP-HPLC-DAD Method for Chamomile (Matricaria recutita) Preparations and Assessment of the Marker, Apigenin-7-glucoside, Safety and Anti-Inflammatory Effect

PubMed Central

Miguel, Felipe Galeti; Spinola, Nathália Favaretto; Ribeiro, Diego Luis; Barcelos, Gustavo Rafael Mazzaron; Antunes, Lusânia Maria Greggi; Hori, Juliana Issa; Marquele-Oliveira, Franciane; Rocha, Bruno Alves; Berretta, Andresa Aparecida

2015-01-01

Chamomile is a medicinal plant, which presents several biological effects, especially the anti-inflammatory effect. One of the compounds related to this effect is apigenin, a flavonoid that is mostly found in its glycosylated form, apigenin-7-glucoside (APG), in natural sources. However, the affectivity and safety of this glycoside have not been well explored for topical application. In this context, the aim of this work was to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC-DAD) method to quantify APG in chamomile preparations. Additionally, the safety and the anti-inflammatory potential of this flavonoid were verified. The RP-HPLC-DAD method was developed and validated with linearity at 24.0–36.0 μg/mL range (r = 0.9994). Intra- and interday precision (RSD) were 0.27–2.66% and accuracy was 98.27–101.21%. The validated method was applied in the analysis of chamomile flower heads, glycolic extract, and Kamillen cream, supporting the method application in the quality control of chamomile preparations. Furthermore, the APG safety was assessed by MTT cytotoxicity assay and mutagenic protocols and the anti-inflammatory activity was confirmed by a diminished TNF-α production showed by mice macrophages treated with APG following LPS treatment. PMID:26421053

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.

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

PubMed

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

2017-07-01

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

Identification of Potent ACE Inhibitory Peptides from Wild Almond Proteins.

PubMed

Mirzapour, Mozhgan; Rezaei, Karamatollah; Sentandreu, Miguel Angel

2017-10-01

In this study, the production, fractionation, purification and identification of ACE (angiotensin-I-converting enzyme) inhibitory peptides from wild almond (Amygdalus scoparia) proteins were investigated. Wild almond proteins were hydrolyzed using 5 different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme) and assayed for their ACE inhibitory activities. The degree of ACE inhibiting activity obtained after hydrolysis was found to be in the following order: alcalase > chymotrypsin > trypsin/pepsin > flavourzyme. The hydrolysates obtained from alcalase (IC 50 = 0.8 mg/mL) were fractionated by sequential ultrafiltration at 10 and 3 kDa cutoff values and the most active fraction (<3 kDa) was further separated using reversed phase high-performance liquid chromatography (RP-HPLC). Peptide sequence identifications were carried out on highly potential fractions obtained from RP-HPLC by means of liquid chromatography coupled to electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS). Sequencing of ACE inhibitory peptides present in the fraction 26 of RP-HPLC resulted in the identification of 3 peptide sequences (VVNE, VVTR, and VVGVD) not reported previously in the literature. Sequence identification of fractions 40 and 42 from RP-HPLC, which showed the highest ACE inhibitory activities (84.1% and 86.9%, respectively), resulted in the identification of more than 40 potential ACE inhibitory sequences. The results indicate that wild almond protein is a rich source of potential antihypertensive peptides and can be suggested for applications in functional foods and drinks with respect to hindrance and mitigation of hypertension after in vivo assessment. This study has shown the potential of wild almond proteins as good sources for producing ACE-inhibitory active peptides. According to this finding, peptides with higher ACE inhibitory activities could be released during the gastrointestinal digestion and contribute to the health- promoting activities of this natural protein source. © 2017 Institute of Food Technologists®.

Prediction of soil organic carbon partition coefficients by soil column liquid chromatography.

PubMed

Guo, Rongbo; Liang, Xinmiao; Chen, Jiping; Wu, Wenzhong; Zhang, Qing; Martens, Dieter; Kettrup, Antonius

2004-04-30

To avoid the limitation of the widely used prediction methods of soil organic carbon partition coefficients (KOC) from hydrophobic parameters, e.g., the n-octanol/water partition coefficients (KOW) and the reversed phase high performance liquid chromatographic (RP-HPLC) retention factors, the soil column liquid chromatographic (SCLC) method was developed for KOC prediction. The real soils were used as the packing materials of RP-HPLC columns, and the correlations between the retention factors of organic compounds on soil columns (ksoil) and KOC measured by batch equilibrium method were studied. Good correlations were achieved between ksoil and KOC for three types of soils with different properties. All the square of the correlation coefficients (R2) of the linear regression between log ksoil and log KOC were higher than 0.89 with standard deviations of less than 0.21. In addition, the prediction of KOC from KOW and the RP-HPLC retention factors on cyanopropyl (CN) stationary phase (kCN) was comparatively evaluated for the three types of soils. The results show that the prediction of KOC from kCN and KOW is only applicable to some specific types of soils. The results obtained in the present study proved that the SCLC method is appropriate for the KOC prediction for different types of soils, however the applicability of using hydrophobic parameters to predict KOC largely depends on the properties of soil concerned.

RP-HPLC Determination of Phenylalkanoids and Monoterpenoids in Rhodiola rosea and Identification by LC-ESI-TOF

USDA-ARS?s Scientific Manuscript database

An HPLC method permitting the simultaneous determination of fourteen compounds (phenylalkanoids and monoterpenoids) from the roots of Rhodiola rosea was developed. A separation was achieved within 35 minutes by using C-18 column material, a water/acetonitrile mobile phase, both containing 0.05% phos...

Precolumn Derivatization with Bromine to Improve Separation and Detection Sensitivity of Triacylglycerols in Edible Oil by Reversed-Phase High Performance Liquid Chromatography.

PubMed

Shan, Xiao-Lin; Liu, Xiao-Ting; Gong, Can; Xu, Xu

2018-01-01

The complexity of triacylglycerols (TAGs) in edible oils is largely due to the many similar unsaturated TAG compounds, which makes profiling TAGs difficult. In this study, precolumn derivatization with bromine (Br 2 ) was used to improve the separation and detection sensitivity of TAGs in edible oils by RP-HPLC. Oil samples dissolved in n-hexane and TAGs were derived by reaction with a Br2-CCl 4 (1:1, v/v) solution for 3 h at room temperature. The derivate product solution was stable and was best separated and detected by RP-HPLC using a C18 column, with a mobile phase of methanol-n-hexane (91.5:8.5, v/v) at 25°C. A detection wavelength of 230 nm was used. The results showed that the approach enabled the separation and detection of more similar TAGs by RP-HPLC. The method was applied to profile 20 types of edible oil, and the results presented the differences in the TAG profiles of various edible oils, which may be useful in the identification of edible oils.

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C-18 and RP-amide columns.

PubMed

Kumar, Ashwini; Kumar Malik, Ashok; Kumar Tewary, Dhananjay; Singh, Baldev

2008-02-01

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.

Metallothionein quantification in clams by reversed-phase high-performance liquid chromatography coupled to fluorescence detection after monobromobimane derivatization.

PubMed

Alhama, José; Romero-Ruiz, Antonio; López-Barea, Juan

2006-02-24

In this paper, we describe a highly specific, sensitive and reliable method for total metallothionein (MT) quantification by RP-HPLC coupled to fluorescence detection following reaction with monobromobimane of thiols from metal-depleted MT after heat-denaturation of extracts in the presence of sodium dodecyl sulphate (SDS). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the identity of the peak resolved (t(R)=16.44) with MT: a highly fluorescent protein of approximately 8.3 kDa, in agreement with the high thiol content and low MT size. Other heat-resistant and Cys-containing proteins of 35 kDa were efficiently separated. The new method was successfully used to quantify MT content in digestive gland of clams from southern Spanish coastal sites with different metal levels, and is proposed as a tool for using MTs as biomarker in monitoring programmes.

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

NASA Astrophysics Data System (ADS)

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

1997-02-01

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

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

PubMed

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

2018-01-05

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

[Determination of genkwanin in flos Genkwa by HPLC].

PubMed

Zhang, B; Yuan, S; Xia, K

1996-04-01

In this paper, the method for determining genkwanin in Flos Genkwa was established by HPLC. Detected at 332nm on a Lichrosorb 5 RP-18 column with a mobile phase of methanol-water-acetic acid (65:35:5), the content of genkwanin in Flos Genkwa was determined to be 0.16%. The recovery rate was 95.46% and RSD 1.15%.

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.

Angiotensin-converting enzyme (ACE) inhibitory potential of standardized Mucuna pruriens seed extract.

PubMed

Chaudhary, Sushil Kumar; De, Apurba; Bhadra, Santanu; Mukherjee, Pulok K

2015-01-01

Mucuna pruriens Linn. (Fabaceae) is a tropical legume, traditionally used for controlling blood pressure. Inhibition of angiotensin-converting enzyme (ACE) is one of the successful strategies for controlling hypertension. The present study evaluated the ACE inhibition potential of the standardized extract of M. pruriens seeds. Standardization of the extract and its fractions were carried out by RP-HPLC method [methanol and 1% v/v acetic acid in water (5:95 v/v)] using levodopa as a marker. The ACE inhibition activity of the extract and fractions was evaluated at different concentrations (20, 40, 60, 80, and 100 µg/mL) using the HPLC-DAD and the UV spectrophotometric method. The liberation of hippuric acid (HA) from hippuryl-L-histidyl-L-leucine (HHL) was estimated in the spectrophotometric method and RP-HPLC assay at 228 nm. Methanol extract and aqueous fraction showed a maximum activity with IC50 values of 38.44 ± 0.90 and 57.07 ± 2.90 µg/mL (RP-HPLC), and 52.68 ± 2.02 and 67.65 ± 2.40 µg/mL (spectrophotometry), respectively. The study revealed that the aqueous extract contains the highest amount of levodopa. Eventually the methanol extract showed highest ACE inhibition activity except levodopa alone. It was further observed that the inhibition was altered with respect to the change in the content of levodopa in the extract. Thus, it can be assumed that levodopa may be responsible for the ACE inhibition activity of M. pruriens seeds. It can be concluded that M. pruriens seed is a potential ACE inhibitor can be explored further as an effective antihypertensive agent.

[Determination of content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes by RP-HPLC method].

PubMed

Han, Meihua; Chen, Jing; Chen, Shilin; Wang, Xiangtao

2009-05-01

To establish a RP-HPLC method for content and entrapment efficiency of 20 (S)-protopanaxadiol in pharmacosomes. The separation was performed with a COSMOSIL 5 C18-MS-II column (4.6 mm x 250 mm, 5 mmicrom) using methanol-water (95:5) as the mobile phase and detected at 203 nm. The flow rate was 1.0 mL x min(-1) and 50 microL sample solution was injected for each time. The calibration curve was linear within the range 0.1-0.5 mg x mL(-1) (r = 0. 9999) , the intra-day RSD and inter-day RSD were less than 2% and the average recovery was between 101.44%-103.11% (n = 3). The method is simple, accurate, sensitive and applicable for determination of content and entrapment efficiency of 20 (S)-protopanaxadiol pharmacosomes.

Determination of n-octanol/water partition coefficient for DDT-related compounds by RP-HPLC with a novel dual-point retention time correction.

PubMed

Han, Shu-ying; Qiao, Jun-qin; Zhang, Yun-yang; Yang, Li-li; Lian, Hong-zhen; Ge, Xin; Chen, Hong-yuan

2011-03-01

n-Octanol/water partition coefficients (P) for DDTs and dicofol were determined by reversed-phase high performance liquid chromatography (RP-HPLC) on a C(18) column using methanol-water mixture as mobile phase. A dual-point retention time correction (DP-RTC) was proposed to rectify chromatographic retention time (t(R)) shift resulted from stationary phase aging. Based on this correction, the relationship between logP and logk(w), the logarithm of the retention factor extrapolated to pure water, was investigated for a set of 12 benzene homologues and DDT-related compounds with reliable experimental P as model compounds. A linear regression logP=(1.10±0.04) logk(w) - (0.60±0.17) was established with correlation coefficient R(2) of 0.988, cross-validated correlation coefficient R(cv)(2) of 0.983 and standard deviation (SD) of 0.156. This model was further validated using four verification compounds, naphthalene, biphenyl, 2,2-bis(4-chlorophenyl)-1,1-dichloroethane (p,p'-DDD) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethene (p,p'-DDE) with similar structure to DDT. The RP-HPLC-determined P values showed good consistency with shake-flask (SFM) or slow-stirring (SSM) results, especially for highly hydrophobic compounds with logP in the range of 4-7. Then, the P values for five DDT-related compounds, 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1,1-trichloroethane (o,p'-DDT), 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethane (o,p'-DDD), 2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1-dichloroethene (o,p'-DDE), and 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol) and its main degradation product 4,4'-dichlorobenzophenone (p,p'-DBP) were evaluated by the improved RP-HPLC method for the first time. The excellent precision with SD less than 0.03 proved that the novel DP-RTC protocol can significantly increases the determination accuracy and reliability of P by RP-HPLC. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

PubMed

Hauff, Simone; Vetter, Walter

2010-12-24

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

Development and validation of RP HPLC method to determine nandrolone phenylpropionate in different pharmaceutical formulations.

PubMed

Mukherjee, Jayanti; Das, Ayan; Chakrabarty, Uday Sankar; Sahoo, Bijay Kumar; Dey, Goutam; Choudhury, Hira; Pal, Tapan Kumar

2011-01-01

This study describes development and subsequent validation of a reversed phase high performance liquid chromatographic (RP-HPLC) method for the estimation of nandrolone phenylpropionate, an anabolic steroid, in bulk drug, in conventional parenteral dosage formulation and in prepared nanoparticle dosage form. The chromatographic system consisted of a Luna Phenomenex, CN (250 mm x 4.6 mm, 5 microm) column, an isocratic mobile phase comprising 10 mM phosphate buffer and acetonitrile (50:50, v/v) and UV detection at 240 nm. Nandrolone phenylpropionate was eluted about 6.3 min with no interfering peaks of excipients used for the preparation of dosage forms. The method was linear over the range from 0.050 to 25 microg/mL in raw drug (r2 = 0.9994). The intra-day and inter-day precision values were in the range of 0.219-0.609% and 0.441-0.875%, respectively. Limits of detection and quantitation were 0.010 microg/mL and 0.050 microg/mL, respectively. The results were validated according to International Conference on Harmonization (ICH) guidelines in parenteral and prepared nanoparticle formulation. The validated HPLC method is simple, sensitive, precise, accurate and reproducible.

Selective enrichment and desalting of hydrophilic peptides using graphene oxide.

PubMed

Jiang, Miao; Qi, Linyu; Liu, Peiru; Wang, Zijun; Duan, Zhigui; Wang, Ying; Liu, Zhonghua; Chen, Ping

2016-08-01

The wide variety and low abundance of peptides in tissue brought great difficulties to the separation and identification of peptides, which is not in favor of the development of peptidomics. RP-HPLC, which could purify small molecules based on their hydrophobicity, has been widely used in the separation and enrichment of peptide due to its fast, good reproducibility and high resolution. However, RP-HPLC requires the instrument and expensive C18 column and its sample capacity is also limited. Recently, graphene oxide has been applied to the adsorption of amino acids. However, the enrichment efficiency and selectivity of graphene oxide for peptides remain unclear. In this study, the adsorption efficiency and selectivity of graphene oxide and RP-C18 matrix were compared on trypsinized α-actin and also on tissue extracts from pituitary gland and hippocampus. For α-actin, there exhibit similar elution peaks for total trypsinized products and those adsorpted by GO and C18 matrix. But peptides adsorbed by GO showed the higher hydrophilic peaks than which adsorbed by C18 matrix. The resulted RP-HPLC profile showed that most of peptides enriched by graphene oxide were eluted at low concentration of organic solvent, while peptides adsorbed by RP-C18 matrix were mostly eluted at relatively high concentration. Moreover, mass spectrometry analysis suggested that, in pituitary sample, there were 495 peptides enriched by graphene oxide, 447 peptides enriched by RP-C18 matrix while in hippocampus sample 333 and 243 peptides respectively. The GRAVY value analysis suggested that the graphene oxide has a stronger adsorption for highly hydrophilic peptides compared to the RP-C18 matrix. Furthermore, the combination of these two methods could notably increase the number of identification peptides but also the number of predicted protein precursors. Our study provided a new thought to the role of graphene oxide during the enrichment of peptides from tissue which should be useful for peptidomics study. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

Lacker, T; Strohschein, S; Albert, K

1999-08-27

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

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

PubMed

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

2006-02-03

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

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

PubMed

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

2011-09-01

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

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

RP-HPLC method for simultaneous estimation of vigabatrin, gamma-aminobutyric acid and taurine in biological samples.

PubMed

Police, Anitha; Shankar, Vijay Kumar; Narasimha Murthy, S

2018-02-15

Vigabatrin is used as first line drug in treatment of infantile spasms for its potential benefit overweighing risk of causing permanent peripheral visual field defects and retinal damage. Chronic administration of vigabatrin in rats has demonstrated these ocular events are result of GABA accumulation and depletion of taurine levels in retinal tissues. In vigabatrin clinical studies taurine plasma level is considered as biomarker for studying structure and function of retina. The analytical method is essential to monitor taurine levels along with vigabatrin and GABA. A RP-HPLC method has been developed and validated for simultaneous estimation of vigabatrin, GABA and taurine using surrogate matrix. Analytes were extracted from human plasma, rat plasma, retina and brain by simple protein precipitation method and derivatized by naphthalene 2, 3‑dicarboxaldehyde to produce stable fluorescent active isoindole derivatives. The chromatographic analysis was performed on Zorbax Eclipse AAA column using gradient elution profile and eluent was monitored using fluorescence detector. A linear plot of calibration curve was observed in concentration range of 64.6 to 6458, 51.5 to 5150 and 62.5 to 6258 ng/mL for vigabatrin, GABA and taurine, respectively with r 2  ≥ 0.997 for all analytes. The method was successfully applied for estimating levels of vigabatrin and its modulator effect on GABA and taurine levels in rat plasma, brain and retinal tissue. This RP-HPLC method can be applied in clinical and preclinical studies to explore the effect of taurine deficiency and to investigate novel approaches for alleviating vigabatrin induced ocular toxicity. Copyright © 2018. Published by Elsevier B.V.

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.

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

PubMed

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

1992-02-28

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

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.

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

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

PubMed

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

2006-01-15

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

Determination and identification of hydrophilic and hydrophobic arsenic species in methanol extract of fresh cod liver by RP-HPLC with simultaneous ICP-MS and ESI-Q-TOF-MS detection.

PubMed

Arroyo-Abad, Uriel; Lischka, Susanne; Piechotta, Christian; Mattusch, Jürgen; Reemtsma, Thorsten

2013-12-01

The present study was focused on the determination and identification of arsenic species in methanolic extracts of cod liver. Arsenic species were fractionated and the fractions analysed by RP-HPLC-ICP-MS coupled with ESI-Q-TOF-MS. The total concentration of arsenic in the fresh cod liver was analysed by ICP-MS to be 1.53±0.02 mg As kg(-1)w.w. and the extraction recovery was ca. 100% and the column recovery >93%. Besides polar inorganic and methylated arsenic species (>70%) more hydrophobic arsenic-containing fatty acids and hydrocarbons occurred. Based on the mass spectrometric data proposals for molecular structures were elaborated for 20 of the organic As species included 10 arsenic-containing fatty acids (AsFA) and an arsenic-containing hydrocarbon (AsHC) mentioned for the first time in fresh cod liver. Arsenobetaine was found as main water-soluble arsenic compound in cod liver followed by higher molecular mass arsenic-containing fatty acids and hydrocarbons. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of free thiol variants of an IgG1 by reversed phase ultra high pressure liquid chromatography coupled with mass spectrometry.

PubMed

Liu, Hongbin; Jeong, Justin; Kao, Yung-Hsiang; Zhang, Yonghua Taylor

2015-05-10

RP-HPLC has been demonstrated as a powerful tool to study antibody free thiol and disulfide variants. Recently, the introduction of UHPLC columns with wide pore size (300Å) and small particle size (1.7μm) offered the opportunity to further improve the separation of such variants. This paper describes a systematic evaluation of stationary phases, operating parameters, and mobile phases for a UHPLC based method to separate free thiol variants of a recombinant monoclonal antibody (referred as mAb A), targeting high resolution, high throughput and improved recovery. Among the four different stationary phases evaluated, UHPLC diphenyl columns were found to provide the best separation. Using an optimized UHPLC method, free thiol variants of mAb A were separated in 5min. Importantly, the UHPLC method revealed minor variants that had coeluted in an HPLC based method, and the UHPLC method is also applicable as a platform method for characterization of other mAbs as well. Furthermore, an on-line UHPLC-MS method was developed to characterize the separated variants, and this method can streamline the characterization of fully assembled monoclonal and bispecific therapeutic antibodies. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Standardization of RP-HPLC methods for the detection of the major peanut allergens Ara h 1, Ara h 2 and Ara h 3

USDA-ARS?s Scientific Manuscript database

Crude peanut extract (CPE) was analyzed for three major allergens (Ara h 1, h 2, and h 3) using a C12 and a C18 column at two wavelengths (280 and 220 nm) and under different solvent conditions. HPLC profiles were compared for retention time, resolution, and peak heights. CPE samples were spiked wit...

RP-HPLC-DAD-ESI-QTOF-MS based metabolic profiling of the potential Olea europaea by-product "wood" and its comparison with leaf counterpart.

PubMed

Ammar, Sonda; Contreras, Maria Del Mar; Gargouri, Boutheina; Segura-Carretero, Antonio; Bouaziz, Mohamed

2017-05-01

Olea europaea L. organs such as leaves, stems and roots have been associated with numerous in vivo and in vitro biological activities and used for traditional medicinal purposes. However, tree wood is an untapped resource with little information about their chemical composition. That is why, the objective of this study is to increase the knowledge about phytochemicals from 'Chemlali' olive wood by means of mass spectrometry-based analyses. Its comparison with by-products derived from leaves was also studied. Hydromethanol extracts from wood and leaves with stems of 'Chemlali' olive cultivar were analysed using reversed-phase (RP) high-performance liquid chromatography (HPLC) coupled to two detection systems: diode-array detection (DAD) and quadrupole time-of-flight (QTOF) mass spectrometry (MS) in negative ion mode. Tandem MS experiments were performed to establish the chemical structure of olive phytochemicals. A total of 85 compounds were characterised in the studied olive parts and classified as: sugars (3), organic acids (5), one phenolic aldehyde, simple phenolic acids (6), simple phenylethanoids (5), flavonoids (14), coumarins (3), caffeoyl phenylethanoid derivatives (6), iridoids (5), secoiridoids (32), and lignans (5). To our knowledge, the major part of these metabolites was not previously reported in olive tree wood, and 10 olive chemical constituents were identified for the first time in the Oleaceae family. The results presented here demonstrated the usefulness of the methodology proposed, based on RP-HPLC-DAD-ESI-QTOF-MS and MS/MS, to develop an exhaustive metabolic profiling and to recover new biologically active compounds in olive wood with pharmacologic and cosmetic potential. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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

PubMed

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

2008-12-23

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

Fractionation and proteomic analysis of the Walterinnesia aegyptia snake venom using OFFGEL and MALDI-TOF-MS techniques.

PubMed

Abd El Aziz, Tarek Mohamed; Bourgoin-Voillard, Sandrine; Combemale, Stéphanie; Beroud, Rémy; Fadl, Mahmoud; Seve, Michel; De Waard, Michel

2015-10-01

Animal venoms are complex mixtures of more than 100 different compounds, including peptides, proteins, and nonprotein compounds such as lipids, carbohydrates, and metal ions. In addition, the existing compounds show a wide range of molecular weights and concentrations within these venoms, making separation and purification procedures quite tedious. Here, we analyzed for the first time by MS the advantages of using the OFFGEL technique in the separation of the venom components of the Egyptian Elapidae Walterinnesia aegyptia snake compared to two classical methods of separation, SEC and RP-HPLC. We demonstrate that OFFGEL separates venom components over a larger scale of fractions, preserve respectable resolution with regard to the presence of a given compound in adjacent fractions and allows the identification of a greater number of ions by MS (102 over 134 total ions). We also conclude that applying several separating techniques (SEC and RP-HPLC in addition to OFFGEL) provides complementary results in terms of ion detection (21 more for SEC and 22 more with RP-HPLC). As a result, we provide a complete list of 134 ions present in the venom of W. aegyptia by using all these techniques combined. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Study on HPLC fingerprint of 11 Taraxacum species in northeast of China].

PubMed

Zhu, Dan; Zhao, Xin; Xu, Qiao; Ning, Wei

2011-04-01

To study the RP-HPLC fingerprints of 11 plants in the genus Taraxacum for their quality control. The fingerprints were determined using an Agilent 1100 series instrument system. Chromatographic analyses were performed on a Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) analytical column,eluted with methanol and water containing 0.5% acetic acid as the mobile phases in gradient elution at the flow rate of 1.0 mL x min(-1). The detection wavelength was 323 nm. The temperature of column was 35 degrees C. Eleven species of Taraxacum in northeast of China were detected respectively. Twenty-five common peaks existed in 11 RP-HPLC fingerprints. By comparing the retention time and the on-line UV spectra, peaks No. 10, No. 12, No. 16 and No. 25 were identified as chlorogenic acid, caffeic acid, p-coumaroy acid and luteolin respectively. The analytical method with good precision and reproducibility can be useful in the quality control of Taraxacum plants.

Bioactive compounds, RP-HPLC analysis of phenolics, and antioxidant activity of some Portuguese shrub species extracts.

PubMed

Luís, Angelo; Domingues, Fernanda; Duarte, Ana Paula

2011-12-01

In the ecosystem of Serra Da Estrela, some plant species have the potential to be used as raw material for extraction of bioactive products. The goal of this work was to determine the phenolic, flavonoid, tannin and alkaloid contents of the methanolic extracts of some shrubs (Echinospartum ibericum, Pterospartum tridentatum, Juniperus communis, Ruscus aculeatus, Rubus ulmifolius, Hakea sericea, Cytisus multiflorus, Crataegus monogyna, Erica arborea and Ipomoea acuminata), and then to correlate the phenolic compounds and flavonoids with the antioxidant activity of each extract. The Folin-Ciocalteu's method was used for the determination of total phenols, and tannins were then precipitated with polyvinylpolypyrrolidone (PVPP); a colorimetric method with aluminum chloride was used for the determination of flavonoids, and a Dragendorff's reagent method was used for total alkaloid estimation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene bleaching tests were used to assess the antioxidant activity of extracts. The identification of phenolic compounds present in extracts was performed using RP-HPLC. A positive linear correlation between antioxidant activity index and total phenolic content of methanolic extracts was observed. The RP-HPLC procedure showed that the most common compounds were ferulic and ellagic acids and quercetin. Most of the studied shrubs have significant antioxidant properties that are probably due to the existence of phenolic compounds in the extracts. It is noteworthy to emphasize that for Echinospartum ibericum, Hakea sericea and Ipomoea acuminata, to the best of our knowledge, no phytochemical studies have been undertaken nor their use in traditional medicine been described.

Development and validation of an RP-HPLC method for the quantitation of odanacatib in rat and human plasma and its application to a pharmacokinetic study.

PubMed

Police, Anitha; Gurav, Sandip; Dhiman, Vinay; Zainuddin, Mohd; Bhamidipati, Ravi Kanth; Rajagopal, Sriram; Mullangi, Ramesh

2015-11-01

A simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of odanacatib in rat and human plasma. The bioanalytical procedure involves extraction of odanacatib and itraconazole (internal standard, IS) from a 200 μL plasma aliquot with simple liquid-liquid extraction process. Chromatographic separation was achieved on a Symmetry Shield RP18 using an isocratic mobile phase at a flow rate of 0.7 mL/min. The UV detection wave length was 268 nm. Odanacatib and IS eluted at 5.5 and 8.6 min, respectively with a total run time of 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 50.9-2037 ng/mL (r(2) = 0.994). The intra- and inter-day precisions were in the range of 2.06-5.11 and 5.84-13.1%, respectively, in rat plasma and 2.38-7.90 and 6.39-10.2%, respectively, in human plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.

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

Validation of a HPLC method for determination of hydroxymethylfurfural in crude palm oil.

PubMed

Ariffin, Abdul Azis; Ghazali, H M; Kavousi, Parviz

2014-07-01

For the first time 5-hydroxymethyl-2-furaldehyde (HMF) was separated from crude palm oil (CPO), and its authenticity was determined using an RP-HPLC method. Separation was accomplished with isocratic elution of a mobile phase comprising water and methanol (92:8 v/v) on a Purospher Star RP-18e column (250mm×4.6mm, 5.0μm). The flow rate was adjusted to 1ml/min and detection was performed at 284nm. The method was validated, and results obtained exhibit a good recovery (95.58% to 98.39%). Assessment of precision showed that the relative standard deviations (RSD%) of retention times and peak areas of spiked samples were less than 0.59% and 2.66%, respectively. Further, the limit of detection (LOD) and LOQ were 0.02, 0.05mg/kg, respectively, and the response was linear across the applied ranges. The crude palm oil samples analysed exhibited HMF content less than 2.27mg/kg. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of celiac disease related oat proteins: bases for the development of high quality oat varieties suitable for celiac patients.

PubMed

Giménez, María J; Real, Ana; García-Molina, M Dolores; Sousa, Carolina; Barro, Francisco

2017-02-17

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients.

Characterization of celiac disease related oat proteins: bases for the development of high quality oat varieties suitable for celiac patients

PubMed Central

Giménez, María J.; Real, Ana; García-Molina, M. Dolores; Sousa, Carolina; Barro, Francisco

2017-01-01

Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times. The immunotoxicity of oat varieties for patients with celiac disease (CD) has been tested using a competitive ELISA based on G12 monoclonal antibody. The oat lines show, on average, seven avenin peaks giving profiles with certain similarities. Based on this similarity, most of the accessions have been grouped into avenin patterns. The variability of RP-HPLC profiles of the collection is great, but not sufficient to uniquely identify the different varieties of the set. Overall, the immunogenicity of the collection is less than 20 ppm. However, there is a different distribution of toxicity ranges between the different peak patterns. We conclude that the RP-HPLC technique is useful to establish groups of varieties differing in degree of toxicity for CD patients. PMID:28209962

Linear modeling of the soil-water partition coefficient normalized to organic carbon content by reversed-phase thin-layer chromatography.

PubMed

Andrić, Filip; Šegan, Sandra; Dramićanin, Aleksandra; Majstorović, Helena; Milojković-Opsenica, Dušanka

2016-08-05

Soil-water partition coefficient normalized to the organic carbon content (KOC) is one of the crucial properties influencing the fate of organic compounds in the environment. Chromatographic methods are well established alternative for direct sorption techniques used for KOC determination. The present work proposes reversed-phase thin-layer chromatography (RP-TLC) as a simpler, yet equally accurate method as officially recommended HPLC technique. Several TLC systems were studied including octadecyl-(RP18) and cyano-(CN) modified silica layers in combination with methanol-water and acetonitrile-water mixtures as mobile phases. In total 50 compounds of different molecular shape, size, and various ability to establish specific interactions were selected (phenols, beznodiazepines, triazine herbicides, and polyaromatic hydrocarbons). Calibration set of 29 compounds with known logKOC values determined by sorption experiments was used to build simple univariate calibrations, Principal Component Regression (PCR) and Partial Least Squares (PLS) models between logKOC and TLC retention parameters. Models exhibit good statistical performance, indicating that CN-layers contribute better to logKOC modeling than RP18-silica. The most promising TLC methods, officially recommended HPLC method, and four in silico estimation approaches have been compared by non-parametric Sum of Ranking Differences approach (SRD). The best estimations of logKOC values were achieved by simple univariate calibration of TLC retention data involving CN-silica layers and moderate content of methanol (40-50%v/v). They were ranked far well compared to the officially recommended HPLC method which was ranked in the middle. The worst estimates have been obtained from in silico computations based on octanol-water partition coefficient. Linear Solvation Energy Relationship study revealed that increased polarity of CN-layers over RP18 in combination with methanol-water mixtures is the key to better modeling of logKOC through significant diminishing of dipolar and proton accepting influence of the mobile phase as well as enhancing molar refractivity in excess of the chromatographic systems. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

RP-HPLC Method Development and Validation for Determination of Eptifibatide Acetate in Bulk Drug Substance and Pharmaceutical Dosage Forms.

PubMed

Bavand Savadkouhi, Maryam; Vahidi, Hossein; Ayatollahi, Abdul Majid; Hooshfar, Shirin; Kobarfard, Farzad

2017-01-01

A new, rapid, economical and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column (150 x 4.60 mm i.d., 5 µM particle size) at ambient temperature using acetonitrile (ACN), water and trifluoroacetic acid (TFA) as mobile phase at flow rate of 1 mL/min and UV detection at 275 nm. Eptifibatide acetate exhibited linearity over the concentration range of 0.15-2 mg/mL (r 2 =0.997) with limit of detection of 0.15 mg/mL The accuracy of the method was 96.4-103.8%. The intra-day and inter-day precision were between 0.052% and 0.598%, respectively. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms.

HPLC Determination of Esculin and Esculetin in Rat Plasma for Pharmacokinetic Studies.

PubMed

Rehman, Shaheed Ur; Kim, In Sook; Kang, Ki Sung; Yoo, Hye Hyun

2015-09-01

An optimized, sensitive and validated reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection is described for simultaneous determination of esculin and its aglycone, esculetin, in rat plasma. After addition of internal standard (chrysin), plasma samples were pretreated by solid-phase extraction and introduced into the HPLC system. Analytes were separated on a RP C18 column with a mobile phase of 0.075% acetic acid in water (solvent A) and 90% acetonitrile in solvent A (solvent B) using gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 338 nm. Calibration curves for esculin and esculetin were constructed over a range of 10-1,000 ng/mL. The developed method was found to be specific, precise and accurate. The method was successfully applied to study the pharmacokinetics of esculin and esculetin in rats. After oral administration of 120 mg/kg, the mean Cmax values were 340.3 and 316.5 ng/mL and the AUClast values were 377.3 and 1276.5 h ng/mL for esculin and esculetin, respectively. The bioavailability of esculin was calculated to be 0.62%. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

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

2016-09-01

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

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

PubMed

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

2017-08-05

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

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

PubMed

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

2000-10-27

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

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

PubMed Central

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

2011-01-01

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

Concurrent estimation of amlodipine besylate, hydrochlorothiazide and valsartan by RP-HPLC, HPTLC and UV-spectrophotometry.

PubMed

Sharma, Manish; Kothari, Charmy; Sherikar, Omkar; Mehta, Priti

2014-01-01

Accurate, sensitive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC), high-performance thin-layer chromatography (HPTLC) and ultraviolet (UV) spectrophopometric methods were developed for the concurrent estimation of amlodipine besylate (AMLO), hydrochlorothiazide (HCTZ) and valsartan (VALS) in bulk and combined tablet dosage forms. For the RP-HPLC method, separation was achieved on a C18 column using potassium dihydrogen orthophosphate buffer (50 mM, pH 3.7) with 0.2% triethylamine as the modifier and acetonitrile in the ratio of 56:44 (v/v) as the mobile phase. Quantification was achieved using a photodiode array detector at 232 nm over a concentration range of 2-25 µg/mL for AMLO, 5-45 µg/mL for HCTZ and 20-150 µg/mL for VALS. For the HPTLC method, the drugs were separated by using ethyl acetate-methanol-toluene-ammonia (7.5:3:2:0.8, v/v/v/v) as the mobile phase. Quantification was achieved using UV detection at 242 nm over a concentration range of 100-600 ng/spot for AMLO, 150-900 ng/spot for HCTZ and 1,200-3,200 ng/spot for VALS. The UV-spectrophotometric simultaneous equation method was based on the measurement of absorbance at three wavelengths; i.e., at 237.6 nm (λmax of AMLO), 270.2 nm (λmax of HCTZ) and 249.2 nm (λmax of VALS) in methanol. Quantification was achieved over the concentration range of 2-20 µg/mL for AMLO, 5-25 µg/mL HCTZ and 10-50 µg/mL for VALS. All methods were validated according to International Conference on Harmonization guidelines and successfully applied to marketed pharmaceutical formulations. Additionally, the three methods were compared statistically by an analysis of variance test, which revealed no significant difference between the proposed methods with respect to accuracy and precision.

An Optimized Method for the Measurement of Acetaldehyde by High-Performance Liquid Chromatography

PubMed Central

Guan, Xiangying; Rubin, Emanuel; Anni, Helen

2011-01-01

Background Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase, and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). Methods We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent,, time and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DPN) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison to AcH-DPN standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. Results Derivatization of acetaldehyde was performed at pH 4.0 with a 80-fold molar excess of DNPH. The reaction was completed in 40 min at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-min chromatography program. Acetaldehyde detection was linear up to 80 μM. The recovery of acetaldehyde was >88% in culture media, and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 μM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 μl) plasma sampling. Conclusions An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small volume sampling of culture media and biological fluids. PMID:21895715

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

PubMed

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

2012-11-15

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

Isolation and Characterization of Precise Dye/Dendrimer Ratios

PubMed Central

Dougherty, Casey A.; Furgal, Joseph C.; van Dongen, Mallory A.; Goodson, Theodore; Banaszak Holl, Mark M.; Manono, Janet; DiMaggio, Stassi

2014-01-01

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6-carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne `click' ligand, separation into sample containing precisely defined `click' ligand/particle ratios using reverse-phase high performance liquid chromatography (rp-HPLC), followed by reaction with excess azide-functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using rp-HPLC. These materials were characterized using 1H and 19F NMR, rp-HPLC, UV-Vis and fluorescence spectroscopy, lifetime measurements, and MALDI. PMID:24604830

Rapid determination of flavonoids and phenolic acids in grape juices and wines by RP-HPLC/DAD: Method validation and characterization of commercial products of the new Brazilian varieties of grape.

PubMed

Padilha, Carla Valéria da Silva; Miskinis, Gabriela Aquino; de Souza, Marcelo Eduardo Alves Olinda; Pereira, Giuliano Elias; de Oliveira, Débora; Bordignon-Luiz, Marilde Terezinha; Lima, Marcos Dos Santos

2017-08-01

A method for rapid determination of phenolic compounds by reversed-phase high-performance liquid chromatography (RP-HPLC), using a new column of faster resolution was validated and used to characterize commercial products produced with new grape Brazilian varieties of Northeast of Brazil. The in vitro antioxidant activity was also measured. The method showed linearity (R>0.9995), good precision (CV%<2.78), recovery (91.8-105.1%) and limits of detection (0.04-0.85mgL -1 ) and quantification (0.04-1.41mgL -1 ) according to other methods previously published with the difference of a run time of only 25min. The results obtained in the characterization of the samples differed for juices and wines from other world regions, mainly because of the high values of (-)-epigallocatechin and trans-caftaric acid. The products analyzed showed high antioxidant activity, especially the wine samples with values higher than those from wines of different regions of the world. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analytical strategy for the assessment of the protein glycation status in uremic patients by high-performance liquid chromatography.

PubMed

Floridi, A; Trizza, V; Paolotti, P; Lucarelli, C

1999-06-18

We propose a newly integrated procedure for the analysis of furosine (early glycation product) and pentosidine (glycoxidation end-product) in plasma proteins and the simultaneous assessment of advanced glycation end-product (AGE) peptides and free pentosidine in plasma. In order to determine furosine and protein-linked pentosidine, plasma proteins were hydrolyzed in 8 M HCl and each analyte was purified by solid-phase extraction. Furosine was determined by ion-pair RP-HPLC methodology with isocratic elution and spectrophotometric detection at 280 nm and pentosidine by ion-pair RP-HPLC by using gradient elution and fluorimetric detection at 335/385 nm. To assess free pentosidine concentration and simultaneously evaluate the AGE peptides, an aliquot of plasma sample was diluted and ultrafiltered by using Centricon 10 M(r) < or = 10,000) ultrafiltration membranes. Free pentosidine and AGE peptides were analysed by ion-pair RP-HPLC, by using gradient elution and fluorimetric detection at 385 nm upon excitation at 335 nm. The HPLC methodology has been successfully used for the determination of glycation and glycoxidation protein status in uremic patients.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

PubMed

Parr, Maria Kristina; Wuest, Bernhard; Naegele, Edgar; Joseph, Jan F; Wenzel, Maxi; Schmidt, Alexander H; Stanic, Mijo; de la Torre, Xavier; Botrè, Francesco

2016-09-01

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

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

PubMed

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

2014-08-01

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

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

PubMed Central

Mezzar, Serena; de Schryver, Evelyn; Van Veldhoven, Paul P.

2014-01-01

Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates. PMID:24323699

Cluster analysis of historical and modern hard red spring wheat cultivars based on parentage and HPLC analysis of gluten forming proteins

USDA-ARS?s Scientific Manuscript database

In this study, 30 hard red spring (HRS) wheat cultivars released between 1910 and 2013 were analyzed to determine how they cluster in terms of parentage and protein data, analyzed by reverse-phase HPLC (RP-HPLC) of gliadins, and size-exclusion HPLC (SE-HPLC) of unreduced proteins. Dwarfing genes in...

Rapid Quantitative Analysis of Naringenin in the Fruit Bodies of Inonotus vaninii by Two-phase Acid Hydrolysis Followed by Reversed Phase-high Performance Liquid Chromatography-ultra Violet.

PubMed

Guohua, Xia; Pan, Ruirong; Bao, Rui; Ge, Yanru; Zhou, Cunshan; Shen, Yuping

2017-01-01

Sanghuang is one of mystical traditional Chinese medicines recorded earliest 2000 years ago, that included various fungi of Inonotus genus and was well-known for antitumor effect in modern medicine. Inonotus vaninii is grown in natural forest of Northeastern China merely and used as Sanghuang commercially, but it has no quality control specification until now. This study was to establish a rapid method of two-phase acid hydrolysis followed by reversed phase-high performance liquid chromatography-ultra violet (RP-HPLC-UV) to quantify naringenin in the fruit body of I. vaninii . Sample solution was prepared by pretreatment of raw material in two-phase acid hydrolysis and the hydrolysis technology was optimized. After reconstitution, analysis was performed using RP-HPLC-UV. The method validation was investigated and the naringenin content of sample and comparison were determined. The naringenin was obtained by two-phase acid hydrolysis method, namely, 10.0 g of raw material was hydrolyzed in 200 mL of 1% sulfuric acid aqueous solution (v/v) and 400 mL of chloroform in oil bath at 110°C for 2 h. Good linearity ( r = 0.9992) was achieved between concentration of analyte and peak area. The relative standard deviation (RSD) of precision was 2.47% and the RSD of naringenin contents for repeatability was 3.13%. The accuracy was supported with recoveries at 96.37%, 97.30%, and 99.31%. The sample solution prepared using the proposed method contained higher content of naringenin than conventional method and was stable for 8 h. Due to the high efficiency of sample preparation and high reliability of the HPLC method, it is feasible to use this method for routine analysis of naringenin in the fungus. A convenient two-phase acid hydrolysis was employed to produce naringenin from raw material, and then an efficient and reliable reversed phase-high performance liquid chromatography-ultra violet method was established to monitor naringenin in the fruit bodies of Inonotus vaninii . The newly established method could be used to control the quality of the herb. Abbreviations used: RP-HPLC-UV: Reversed Phase-High Performance Liquid Chromatography-Ultra Violet, RSD: Relative Standard Deviation, EtOAc: Ethyl acetate, ACN: Acetonitrile, MeOH: Methanol, RH: Relative Humility.

An optimized method for the measurement of acetaldehyde by high-performance liquid chromatography.

PubMed

Guan, Xiangying; Rubin, Emanuel; Anni, Helen

2012-03-01

Acetaldehyde is produced during ethanol metabolism predominantly in the liver by alcohol dehydrogenase and rapidly eliminated by oxidation to acetate via aldehyde dehydrogenase. Assessment of circulating acetaldehyde levels in biological matrices is performed by headspace gas chromatography and reverse phase high-performance liquid chromatography (RP-HPLC). We have developed an optimized method for the measurement of acetaldehyde by RP-HPLC in hepatoma cell culture medium, blood, and plasma. After sample deproteinization, acetaldehyde was derivatized with 2,4-dinitrophenylhydrazine (DNPH). The reaction was optimized for pH, amount of derivatization reagent, time, and temperature. Extraction methods of the acetaldehyde-hydrazone (AcH-DNP) stable derivative and product stability studies were carried out. Acetaldehyde was identified by its retention time in comparison with AcH-DNP standard, using a new chromatography gradient program, and quantitated based on external reference standards and standard addition calibration curves in the presence and absence of ethanol. Derivatization of acetaldehyde was performed at pH 4.0 with an 80-fold molar excess of DNPH. The reaction was completed in 40 minutes at ambient temperature, and the product was stable for 2 days. A clear separation of AcH-DNP from DNPH was obtained with a new 11-minute chromatography program. Acetaldehyde detection was linear up to 80 μM. The recovery of acetaldehyde was >88% in culture media and >78% in plasma. We quantitatively determined the ethanol-derived acetaldehyde in hepatoma cells, rat blood and plasma with a detection limit around 3 μM. The accuracy of the method was <9% for intraday and <15% for interday measurements, in small volume (70 μl) plasma sampling. An optimized method for the quantitative determination of acetaldehyde in biological systems was developed using derivatization with DNPH, followed by a short RP-HPLC separation of AcH-DNP. The method has an extended linear range, is reproducible and applicable to small-volume sampling of culture media and biological fluids. Copyright © 2011 by the Research Society on Alcoholism.

A rapid method for isolation and purification of an anticoagulant from Whitmania pigra.

PubMed

Zhong, Shan; Cui, Zheng; Sakura, Naoki; Wang, Dong; Li, Jianlin; Zhai, Yan

2007-05-01

Whitmania pigra is common in China and has been used as a traditional Chinese anticoagulant medicine for years, but its effective components are unknown to scientists. In this article we report a rapid method for isolation and purification of an anticoagulant from W. pigra for the first time. An acetone-water extract of W. pigra was subjected to anion-exchange chromatography on a Sephadex DEAE A-50 column, and gel permeation chromatography on Sephadex G-25 and Sephadex LH-20 columns successively, which afforded a fraction with potent anticoagulant activity. An anticoagulant was isolated and purified from this fraction by reversed-phase high-performance liquid chromatography (RP-HPLC). It was identified as a single pure substance by RP-HPLC and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). This component was named whitmanin and its molecular weight was determined as 8608 Da by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS). (c) 2006 John Wiley & Sons, Ltd.

Acid-Base Chemistry of White Wine: Analytical Characterisation and Chemical Modelling

PubMed Central

Prenesti, Enrico; Berto, Silvia; Toso, Simona; Daniele, Pier Giuseppe

2012-01-01

A chemical model of the acid-base properties is optimized for each white wine under study, together with the calculation of their ionic strength, taking into account the contributions of all significant ionic species (strong electrolytes and weak one sensitive to the chemical equilibria). Coupling the HPLC-IEC and HPLC-RP methods, we are able to quantify up to 12 carboxylic acids, the most relevant substances responsible of the acid-base equilibria of wine. The analytical concentration of carboxylic acids and of other acid-base active substances was used as input, with the total acidity, for the chemical modelling step of the study based on the contemporary treatment of overlapped protonation equilibria. New protonation constants were refined (L-lactic and succinic acids) with respect to our previous investigation on red wines. Attention was paid for mixed solvent (ethanol-water mixture), ionic strength, and temperature to ensure a thermodynamic level to the study. Validation of the chemical model optimized is achieved by way of conductometric measurements and using a synthetic “wine” especially adapted for testing. PMID:22566762

Acid-base chemistry of white wine: analytical characterisation and chemical modelling.

PubMed

Prenesti, Enrico; Berto, Silvia; Toso, Simona; Daniele, Pier Giuseppe

2012-01-01

A chemical model of the acid-base properties is optimized for each white wine under study, together with the calculation of their ionic strength, taking into account the contributions of all significant ionic species (strong electrolytes and weak one sensitive to the chemical equilibria). Coupling the HPLC-IEC and HPLC-RP methods, we are able to quantify up to 12 carboxylic acids, the most relevant substances responsible of the acid-base equilibria of wine. The analytical concentration of carboxylic acids and of other acid-base active substances was used as input, with the total acidity, for the chemical modelling step of the study based on the contemporary treatment of overlapped protonation equilibria. New protonation constants were refined (L-lactic and succinic acids) with respect to our previous investigation on red wines. Attention was paid for mixed solvent (ethanol-water mixture), ionic strength, and temperature to ensure a thermodynamic level to the study. Validation of the chemical model optimized is achieved by way of conductometric measurements and using a synthetic "wine" especially adapted for testing.

Accessible silanol sites - beneficial for the RP-HPLC separation of constitutional and diastereomeric azaspirovesamicol isomers.

PubMed

Wenzel, Barbara; Fischer, Steffen; Brust, Peter; Steinbach, Jörg

2010-12-10

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C(18) and special end-capped C(8) and C(18) phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH(4)OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH(4)OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH(4)OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism. Copyright © 2010 Elsevier B.V. All rights reserved.

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

PubMed

Petrova, Olga E; Sauer, Karin

2017-01-01

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

[HPLC fingerprint chromatogram analysis of some Taraxacum in Henan province].

PubMed

Li, Xi-Feng; Shi, Hui-Min; Xu, Min; Meng, Lu

2008-10-01

To analyze the HPLC fingerprint chromatogram of some Taraxacum in Henan. Samples of different species, producing areas, harvest seasons and medicinal parts were determined by RP-HPLC. The chromatogram was evaluated by software of evaluating similarity. The components of different species in Taraxacum were the same and could be substituted for each other. The contents of coffeic acid and chlorogenic acid in different producing areas were very different,which in fecund soil was better. The period of flowering and fruiting in Spring was the best gather period, and the components in different parts were different. The quality of medicinal materal within Taraxacum should be controlled better by this method.

A novel, simple and inexpensive procedure for the simultaneous determination of iopamidol and p-aminohippuric acid for renal function assessment from plasma samples in awake rats.

PubMed

Rodríguez-Romero, Violeta; González-Villalva, Karla I; Reyes, José L; Franco-Bourland, Rebecca E; Guízar-Sahagún, Gabriel; Castañeda-Hernández, Gilberto; Cruz-Antonio, Leticia

2015-03-25

The purpose of the current study was to design, validate and implement a novel analytical method for the simultaneous plasma measurement of iopamidol and p-aminohippuric acid (PAH) to estimate renal function in awake rats. A reverse-phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous measurement of iopamidol (for glomerular filtration rate estimation, GFR) and PAH (for tubular secretion determination, TS) was designed and validated using a C-18 column, 0.1M acetic acid-10% acetonitrile (90:10, v/v) as mobile phase, at a flow rate of 0.3 ml/min, and UV detection at 270 nm. Iopamidol (244.8 mg/kg) was administered intravenously followed immediately by sodium PAH (100 mg/kg) to healthy female Sprague-Dawley rats. Plasma samples obtained at 2.5, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min after drug administration were deproteinized with 2.5% trichloroacetic acid containing p-aminobenzoic acid as internal standard, and separated by the validated RP-HPLC method described above. The iopamidol and PAH chromatographic data were analyzed using a non-compartmental model. The results demonstrated that the RP-HPLC method was linear in ranges between 15-120 μg/ml and 2.5-120 μg/ml for iopamidol and PAH, respectively. Precision and accuracy were within 15% for both drugs. Recovery of iopamidol and PAH was 92% and 100%, respectively. Plasma iopamidol and PAH clearances in awake rats, estimates for GFR and TS, respectively, were 1.49±0.20 ml/min and 3.73±0.38 ml/min. In conclusion, the method here described is a simple and reliable procedure, for the simultaneous and time-saving determination of GFR and TS from plasma samples in the conscious rat. Copyright © 2014 Elsevier B.V. All rights reserved.

RP-HPLC determination of water-soluble vitamins in honey.

PubMed

Ciulu, Marco; Solinas, Silvia; Floris, Ignazio; Panzanelli, Angelo; Pilo, Maria I; Piu, Paola C; Spano, Nadia; Sanna, Gavino

2011-01-15

The assessment and validation of reliable analytical methods for the determination of vitamins in sugar-based matrices (e.g. honey) are still scarcely explored fields of research. This study proposes and fully validates a simple and fast RP-HPLC method for the simultaneous determination of five water-soluble vitamins (vitamin B(2), riboflavin; vitamin B(3), nicotinic acid; vitamin B(5), pantothenic acid; vitamin B(9), folic acid; and vitamin C, ascorbic acid) in honey. The method provides low detection and quantification limits, very good linearity in a large concentration interval, very good precision, and the absence of any bias. It has been successfully applied to 28 honey samples (mainly from Sardinia, Italy) of 12 different botanical origins. While the overall amount of the analytes in the samples is quite low (always below 40 mg kg(-1)), we have observed a marked dependence of some of their concentrations (i.e. vitamin B(3) and vitamin B(5)) and the botanical origin of the honey. This insight might lead to important characterization features for this food item. Copyright © 2010 Elsevier B.V. All rights reserved.

Achiral-chiral two-dimensional chromatography of free amino acids in milk: A promising tool for detecting different levels of mastitis in cows.

PubMed

Ianni, Federica; Sardella, Roccaldo; Lisanti, Antonella; Gioiello, Antimo; Cenci Goga, Beniamino Terzo; Lindner, Wolfgang; Natalini, Benedetto

2015-12-10

In two-dimensional HPLC (2D-HPLC) "heart-cut" applications, two columns are connected in series via a switching valve and volume fractions from the "primary" column are re-injected on the "secondary" column. The heart-cut 2D-HPLC system here described was implemented by connecting a reversed-phase (RP) column (first dimension) to a chiral column (second dimension) containing a quinidine-based chiral stationary phase. The system was used to evaluate the change in the enantiomeric excess value of dansylated (Dns) amino acids (AAs) in milk samples from two cows with different "California Mastitis Test" scores: negative test for sample 1, positive for sample 2. Apart from the co-elution of Dns-Arg/Dns-Gly and the reduced chemoselectivity for Dns-Leu/Dns-allo-Ile, the optimized achiral RP method distinguished the remaining standard Dns-AAs. Dns-AAs were identified in the chromatograms of the real samples, and in higher concentration Dns-Ala, Dns-Arg, Dns-Asp, Dns-Glu, Dns-Ile, Dns-Leu, Dns-Phe and Dns-Val. Except Dns-Arg, the chiral column enabled the RP enantioseparation of all the other compounds (α and RS values up to 1.65 and 8.63, respectively, for Dns-Phe). In sample 2, the amounts of Dns-d-AAs were rather elevated, in particular for Dns-Ala and Dns-Asp. Instead, for sample 1, D-isomers were detected for Dns-Ala, Dns-Glu and Dns-Leu. The proposed 2D-HPLC method could be useful for the identification of clinical mastitis difficult to be diagnosed. Moreover, the eventual progressive reduction of D-AAs levels with the degree of sub-clinical mastitis could allow the building of mathematical models to use for the diagnosis of early stages of mastitis. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous quantitative determination of six active components in traditional Chinese medicinal preparation Cerebralcare Granule® by RP-HPLC coupled with diode array detection for quality control.

PubMed

Wang, Xiang-yang; Ma, Xiao-hui; Li, Wei; Chu, Yang; Guo, Jia-hua; Zhou, Shui-ping; Zhu, Yong-hong

2014-09-01

A simple, accurate and reliable method for the simultaneous separation and determination of six active components (protocatechuic acid, chlorogenic acid, caffeic acid, paeoniflorin, ferulic acid and rosmarinic acid) in traditional Chinese medicinal preparation Cerebralcare Granule(®) (CG) was developed using reverse-phase high-performance liquid chromatography coupled with diode array detector detection. The chromatographic separation was performed on a Hypersil GOLD C18 column with aqueous formic acid (0.1%, v/v) and acetonitrile as mobile phase at a flow rate of 0.2 ml/min at 30 °C. Because of the different UV characteristics of these components, change detection wavelength method was used for quantitative analysis. All of the analytes showed good linearity (r > 0.9992). The established method showed good precision and relative standard deviations (%) for intra-day and inter-day variations of 0.15-1.81 and 0.11-1.98%, respectively. The validated method was successfully applied to the simultaneously determination of six active components in CG from different batches. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Determination of ambroxol hydrochloride, methylparaben and benzoic acid in pharmaceutical preparations based on sequential injection technique coupled with monolithic column.

PubMed

Satínský, Dalibor; Huclová, Jitka; Ferreira, Raquel L C; Montenegro, Maria Conceição B S M; Solich, Petr

2006-02-13

The porous monolithic columns show high performance at relatively low pressure. The coupling of short monoliths with sequential injection technique (SIA) results in a new approach to implementation of separation step to non-separation low-pressure method. In this contribution, a new separation method for simultaneous determination of ambroxol, methylparaben and benzoic acid was developed based on a novel reversed-phase sequential injection chromatography (SIC) technique with UV detection. A Chromolith SpeedROD RP-18e, 50-4.6 mm column with 10 mm precolumn and a FIAlab 3000 system with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-tetrahydrofuran-0.05M acetic acid (10:10:90, v/v/v), pH 3.75 adjusted with triethylamine, flow rate 0.48 mlmin(-1), UV-detection was at 245 nm. The analysis time was <11 min. A new SIC method was validated and compared with HPLC. The method was found to be useful for the routine analysis of the active compounds ambroxol and preservatives (methylparaben or benzoic acid) in various pharmaceutical syrups and drops.

Cryochemistry: freezing effect on peptide coupling in different organic solutions.

PubMed

Vajda, T; Szókán, G; Hollósi, M

1998-06-01

The freezing effect on peptide coupling in organic solutions of different polarity has been investigated and compared with the results obtained in liquid phase. The model reaction of DCC-activated coupling of Boc-Ala-Phe-OH with H-Ala-OBu(t) has been carried out in dioxane, dimethylsulfoxide and formamide, as well as in mixtures (90%/10%, v/v) of dioxane with acetonitrile, dimethylformamide, dimethylsulfoxide and formamide. The reactions have been traced and evaluated by RP-HPLC analysis. Freezing the reaction mixture resulted in all cases in a significant suppression of the N-dipeptidylurea side-product formation together with a slight decrease of tripeptide epimerization. The coupling yields and the side effects depended on the solvent, with the dioxane and dioxane/acetonitrile mixture produced the best results. The role of freezing and solvent in the improved results is discussed.

RP-HPLC Method Development and Validation for Determination of Eptifibatide Acetate in Bulk Drug Substance and Pharmaceutical Dosage Forms

PubMed Central

Bavand Savadkouhi, Maryam; Vahidi, Hossein; Ayatollahi, Abdul Majid; Hooshfar, Shirin; Kobarfard, Farzad

2017-01-01

A new, rapid, economical and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column (150 x 4.60 mm i.d., 5 µM particle size) at ambient temperature using acetonitrile (ACN), water and trifluoroacetic acid (TFA) as mobile phase at flow rate of 1 mL/min and UV detection at 275 nm. Eptifibatide acetate exhibited linearity over the concentration range of 0.15-2 mg/mL (r2=0.997) with limit of detection of 0.15 mg/mL The accuracy of the method was 96.4-103.8%. The intra-day and inter-day precision were between 0.052% and 0.598%, respectively. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms. PMID:28979304

Understanding Single-Stranded Telomere End Binding by an Essential Protein

DTIC Science & Technology

2000-08-01

BioPharma Inc., 1885 33rd Street, Boulder, CO 80301 Traditional sequential medicinal chemistry methods have been augmented by combinatorial synthesis...on the same wells that were being analyzed in parallel by RP-HPLC/UV for purity. The sampling protocol for purity determination at Array BioPharma is

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

PubMed Central

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

2016-01-01

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

Microcystin in cyanobacterial blooms in a Chilean lake.

PubMed

Campos, V; Cantarero, S; Urrutia, H; Heinze, R; Wirsing, B; Neumann, U; Weckesser, J

1999-05-01

Cyanobacterial blooms dominated by Microcystis sp. occurred in lake Rocuant ("marisma", near Concepción/Chile) in February 1995 and 1996. In the bloom samples collected in both years the hepatotoxin microcystin was detected by RP-HPLC in both samples and in the sample of 1995 also by a toxicity assay using primary rat hepatocytes. In the bloom of 1995, the microcystin content of the dry bloom biomass was determined to be 130 micrograms/g on the basis of the RP-HPLC peak area and 800 micrograms/g on the basis of the rat hepatotoxicity assay, respectively. In the bloom of 1996, RP-HPLC analysis revealed a microcystin content of 8.13 micrograms/g bloom material dry weight. In this year no hepatotoxicity was measured using a concentration range up to 0.8 mg (d. w.) of bloom material per ml in the rat hepatotoxicity assay. This is the first report on the detection of microcystins in Chilean water bodies.

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

PubMed Central

Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

2012-01-01

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications. PMID:22548121

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation.

PubMed

Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

2012-01-01

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

Ultrasound-Assisted Surfactant-Enhanced Emulsification Micro-Extraction Followed by HPLC for Determination of Preservatives in Water, Beverages and Personal Care Products.

PubMed

Jan-E, Sudarat; Santaladchaiyakit, Yanawath; Burakham, Rodjana

2017-01-01

An ultrasound-assisted surfactant-enhanced emulsification micro-extraction (UASEME) procedure has been developed for pre-concentration of benzoic acid (BA) and paraben preservatives, including methylparaben, ethylparaben, propylparaben and butylparaben, prior to high-performance liquid chromatography-ultraviolet (HPLC-UV) analysis. Separations were performed on a Lichrospher RP-18 endcapped 5 µm, using an isocratic mobile phase of 40% acetonitile, at a flow rate of 1 mL min -1 The selected UASEME conditions comprised the use of 10 mL sample extract, 125 µL 1-octanol as extraction solvent and 0.05 mmol L -1 Tween 20 as emulsifier, 0.5% sodium chloride, ultrasonication time of 6 min and centrifugation time of 10 min. Method performance demonstrated wide linear range between 0.5 and 7,000 µg L -1 (R 2  > 0.9903) and limits of detection between 0.03 and 10 µg L -1 , which providing the enrichment factors of 15-184. The method precision (relative standard deviation) was <7%. The developed UASEME coupled with HPLC-UV has been successfully applied to determine four paraben preservatives in various sample matrices such as water, beverages and personal care products. The recoveries in the range of 70-138.1% were obtained. However, BA could not be determined in real sample extracts. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

Simultaneous determination of chloramphenicol, dexamethasone and naphazoline in ternary and quaternary mixtures by RP-HPLC, derivative and wavelet transforms of UV ratio spectra

NASA Astrophysics Data System (ADS)

Hoang, Vu Dang; Hue, Nguyen Thu; Tho, Nguyen Huu; Nguyen, Hue Minh Thi

2015-03-01

The application of chemometrics-assisted UV spectrophotometry and RP-HPLC to the simultaneous determination of chloramphenicol, dexamethasone and naphazoline in ternary and quaternary mixtures is presented. The spectrophotometric procedure is based on the first-order derivative and wavelet transforms of ratio spectra using single, double and successive divisors. The ratio spectra were differentiated and smoothed using Savitzky-Golay filter; whereas wavelet transform realized with wavelet functions (i.e. db6, gaus5 and coif3) to obtain highest spectral recoveries. For the RP-HPLC procedure, the separation was achieved on a ZORBAX SB-C18 (150 × 4.6 mm; 5 μm) column at ambient temperature and the total run time was less than 7 min. A mixture of acetonitrile - 25 mM phosphate buffer pH 3 (27:73, v/v) was used as the mobile phase at a flow rate of 1.0 mL/min and the effluent monitored by measuring absorbance at 220 nm. Calibration graphs were established in the range 20-70 mg/L for chloramphenicol, 6-14 mg/L for dexamethasone and 3-8 mg/L for naphazoline (R2 > 0.990). The RP-HPLC and ratio spectra transformed by a combination of derivative-wavelet algorithms proved to be able to successfully determine all analytes in commercial eye drop formulations without sample matrix interference (mean percent recoveries, 97.4-104.3%).

Gradient RP-HPLC method for the determination of potential impurities in atazanavir sulfate.

PubMed

Chitturi, Sreenivasa Rao; Somannavar, Yallappa Somappa; Peruri, Badarinadh Gupta; Nallapati, Sreenivas; Sharma, Hemant Kumar; Budidet, Shankar Reddy; Handa, Vijay Kumar; Vurimindi, Hima Bindu

2011-04-28

This paper proposes a simple and selective RP-HPLC method for the determination of process impurities and degradation products (degradants) of atazanavir sulfate (ATV) drug substance. Chromatographic separation was achieved on Ascentis(®) Express C8, (150mm×4.6mm, 2.7μm) column thermostated at 30°C under gradient elution by a binary mixture of potassium dihydrogen phosphate (pH 3.5, 0.02M) and ACN at a flow rate of 1.0ml/min. A photodiode array (PDA) detector set at 250nm was used for detection. Stress testing (forced degradation) of ATV was carried out under acidic, alkaline, oxidative, photolytic, thermal and humidity conditions. In presence of alkali, ATV transformed into cyclized products and the order of degradation reaction is determined by the method of initial rates. The unknown process impurities and alkaline degradants are isolated by preparative LC and characterized by ESI-MS/MS, (1)H NMR, and FT-IR spectral data. The developed method is validated with respect to sensitivity (lod and loq), linearity, precision, accuracy and robustness and can be implemented for routine quality control analysis and stability testing of ATV. Copyright © 2011 Elsevier B.V. All rights reserved.

Determination of phenolic compounds derived from hydrolysable tannins in biological matrices by RP-HPLC.

PubMed

Díez, María Teresa; García del Moral, Pilar; Resines, José Antonio; Arín, María Jesús

2008-08-01

An RP-HPLC method for the determination of four phenolic compounds: gallic acid (GA), pyrogallol (PY), resorcinol (RE) and ellagic acid (EA), derived from hydrolysable tannins is reported. Separation was achieved on a SunFire C18 (250 x 4.6 mm id, 5 microm) column at 40 degrees C with gradient elution. UV detection at 280 nm was applied. The developed method was validated in terms of linearity, accuracy and precision. Satisfactory repeatability and between day precision were noticed with RSD values lower than 3%. Recoveries from different biological samples ranged from 91.50 to 105.25%. The LODs were estimated as 1.70 mg/L for PY, 1.68 mg/L for GA, 1.52 mg/L for RE and 0.98 mg/L for EA with a 20 microL injection volume. The method was applied for the determination of these compounds in oak leaves and in ruminal fluid and urine samples taken from beef cattle fed with oak leaves. The proposed method could be used in ruminant nutrition studies to verify the effect that a diet rich in tannins have on ruminal fermentation and to determine the toxicity of these compounds.

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

PubMed

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

2010-06-05

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

HPLC-MS/MS method for the simultaneous determination of MB07133 and its metabolites, cytarabine and arabinofuranosyluracil, in rat plasma.

PubMed

Wang, Dan; Xiao, Qingqing; Yang, Wanqiu; Qian, Wei; Yang, Jin

2016-02-20

MB07133 is an intravenously administered cytarabine mononucleotide (araCMP) prodrug, for the treatment of hepatocellular carcinoma (HCC). A simple, selective and sensitive HPLC-MS/MS method using high pressure liquid chromatography (HPLC) coupled to triple-quadrupole mass spectrometer, was developed and validated for the detection of prodrug MB07133 and its metabolites, cytarabine (araC) and arabinofuranosyluracil (araU) in rat plasma. Protein precipitation using 3% trichloroacetic acid (TCA) was employed to extract analytes from 100μL rat plasma. Adequate separation of araC and araU from their endogenous compounds was achieved on the Synergi(®) fusion-RP column (150mm×4.6mm, 4μm) by a gradient-elution with a mobile phase consisting of ammonium formate (1mM) and methanol at a flow rate of 1mL/min. Multiple reaction monitoring mode (MRM) was applied in the detection of MB07133, araC, araU and Ganciclovir (internal standard) with ion pairs 441.2/330.2, 244.2/112.2, 245.2/113.2 and 256.1/152.2, respectively. The assays were validated with respect to specificity, linearity (100-50000ng/mL for MB07133, 2-1000ng/mL for araC and araU), accuracy and precision, extraction recovery, matrix effect and stability. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of MB07133 in male Sprague-Dawley rats (18mg/kg i.v.). Copyright © 2015 Elsevier B.V. All rights reserved.

Analytical Eco-Scale for Assessing the Greenness of a Developed RP-HPLC Method Used for Simultaneous Analysis of Combined Antihypertensive Medications.

PubMed

Mohamed, Heba M; Lamie, Nesrine T

2016-09-01

In the past few decades the analytical community has been focused on eliminating or reducing the usage of hazardous chemicals and solvents, in different analytical methodologies, that have been ascertained to be extremely dangerous to human health and environment. In this context, environmentally friendly, green, or clean practices have been implemented in different research areas. This study presents a greener alternative of conventional RP-HPLC methods for the simultaneous determination and quantitative analysis of a pharmaceutical ternary mixture composed of telmisartan, hydrochlorothiazide, and amlodipine besylate, using an ecofriendly mobile phase and short run time with the least amount of waste production. This solvent-replacement approach was feasible without compromising method performance criteria, such as separation efficiency, peak symmetry, and chromatographic retention. The greenness profile of the proposed method was assessed and compared with reported conventional methods using the analytical Eco-Scale as an assessment tool. The proposed method was found to be greener in terms of usage of hazardous chemicals and solvents, energy consumption, and production of waste. The proposed method can be safely used for the routine analysis of the studied pharmaceutical ternary mixture with a minimal detrimental impact on human health and the environment.

[Determination of homocysteine level in plasma by high performance liquids chromatography with fluorescence detection].

PubMed

Da, Xu; Qian, Ling-Jia

2005-08-01

To establish a method for detection of plasma total homocysteine with HPLC. The chromatography analysis was carried out using a Symmetry Shield RP18. The mobile phase was sodium acetate (0.08 mol/L) and methanol (1%) and we utilized a HPLC system with fluorescence detection of plasma homocysteine derivatized from reaction with 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). The average recoveries were 95.8 - 100.8% and the relative standard deviations were 1.2-2.0%. The results showed it to be a rapid and accurate method for the determination of homocysteine level in plasma.

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

PubMed

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

2009-12-01

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

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.

Comparative Study of Antioxidant Power, Polyphenols, Flavonoids and Betacyanins of the Peel and Pulp of Three Tunisian Opuntia Forms

PubMed Central

Yeddes, Nizar; Chérif, Jamila K.; Guyot, Sylvain; Sotin, Hélène; Ayadi, Malika T.

2013-01-01

The antioxidant activity and the chemical composition of methanol extracts from peel and pulp belonging to two species of Tunisian prickly pears Opuntia ficus indica (spiny and thornless forms) and Opuntia stricta have been studied. The antioxidant capacity was measured by DPPH radical scavenging activity. The total phenolic compound (TPC) and the total flavonoid content were determined by the Folin–Ciocalteu method and colorimetric method, respectively. The phenolic compounds were identified and quantified by high-performance liquid chromatography (HPLC) coupled with an electrospray ionization mass spectrometry (ESI-MS). The results showed that O. stricta fruits present the best antioxidant activities than the two forms of O. ficus indica, while the TPC was more important in O. ficus indica than in the O. stricta fruits. The peels have higher flavonoids than pulp, and the thornless variety has more flavonoid than the spiny. The RP-HPLC and ESI-MS analysis detected two classes of phenolic compounds and betalain pigments. Isorhamnetin derivatives are the dominant flavonol glycoside identified in O. ficus indica (spiny: 65.25 μg·g−1; thornless: 77.03 μg·g−1) and O. stricta peels (19.22 μg·g−1). PMID:26787622

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.

Quantitative analysis of iridoids, secoiridoids, xanthones and xanthone glycosides in Gentiana lutea L. roots by RP-HPLC and LC-MS.

PubMed

Aberham, Anita; Schwaiger, Stefan; Stuppner, Hermann; Ganzera, Markus

2007-11-05

The here described HPLC-method enables the determination of all major, currently known bioactive compounds in gentian roots. A separation of iridoids (loganic acid), secoiridoids (swertiamarin, gentiopicroside, amarogentin, sweroside), xanthones (gentisin, isogentisin) and two xanthone glycosides (gentiosides) was possible on RP-18 column material, using 0.025% aqueous TFA, acetonitrile and n-propanol as mobile phase. The method is sensitive (LOD

Quantification of 4'-geranyloxyferulic acid (GOFA) in honey samples of different origin by validated RP-HPLC-UV method.

PubMed

Genovese, Salvatore; Taddeo, Vito Alessandro; Fiorito, Serena; Epifano, Francesco

2016-01-05

Natural honey has been employed as a nutraceutical agent with benefits and therapeutic promises for humans for many centuries. It has been largely used as food and medicine by all generations, traditions, and civilizations, both ancient and modern. Several chemicals having beneficial effects for human health have been reported as components of natural honey and these include sugars, organic acids, aminoacids, minerals, and vitamins. Also some important phytochemicals have been described and these comprise tannins, flavonoids, terpenes, saponins, and alkaloids. In this note it is described the successful application of a RP HPLC-UV-vis method for the separation and quantification of 4'-geranyloxyferulic acid (GOFA) in four honey samples of different origin. Concentration values showed a great variation between the four samples tested, being chestnut honey the one richest in GOFA (7.87 mg/g). The findings described herein represent the first example reported in the literature of the characterization of an oxyprenylated phenylpropanoid in honey. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Development of green extraction processes for Nannochloropsis gaditana biomass valorization.

PubMed

Sánchez-Camargo, Andrea Del Pilar; Pleite, Natalia; Mendiola, José Antonio; Cifuentes, Alejandro; Herrero, Miguel; Gilbert-López, Bienvenida; Ibáñez, Elena

2018-04-23

In the present work, the valorization of Nannochloropsis gaditana biomass is proposed within the concept of biorefinery. To this aim, high-pressure homogenization (HPH) was used to break down the strong cell wall and supercritical fluid extraction (SFE) with pure CO 2 was applied as a first step to extract valuable compounds (such as non-polar lipids and pigments). Extraction of the remaining residue for the recovery of bioactive compounds was studied by means of an experimental design based on response surface methodology (RSM) employing pressurized liquid extraction (PLE) with green solvents such as water and ethanol. Optimum extract was achieved with pure ethanol at 170°C for 20 min, providing an important antioxidant capacity (0.72 ± 0.03 mmol trolox eq g -1 extract). Complete chemical characterization of the optimum extract was carried out by using different chromatographic methods such as reverse-phase high-performance liquid chromatography with diode array detection (RP-HPLC-DAD), normal-phase HPLC with evaporative light scattering detection (NP-HPLC-ELSD) and gas chromatography coupled to mass spectrometry detection (GC-MS); carotenoids (e.g. violaxanthin), chlorophylls and polar lipids were the main compounds observed while palmitoleic, palmitic, myristic acids and the polyunsaturated eicosapentanoic (EPA) acid were the predominant fatty acids in all PLE extracts. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of rifampicin in human plasma by high-performance liquid chromatography coupled with ultraviolet detection after automatized solid-liquid extraction.

PubMed

Louveau, B; Fernandez, C; Zahr, N; Sauvageon-Martre, H; Maslanka, P; Faure, P; Mourah, S; Goldwirt, L

2016-12-01

A precise and accurate high-performance liquid chromatography (HPLC) quantification method of rifampicin in human plasma was developed and validated using ultraviolet detection after an automatized solid-phase extraction. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision, accuracy, lower limit of quantification and stability. Chromatographic separation was performed on a Chromolith RP 8 column using a mixture of 0.05 m acetate buffer pH 5.7-acetonitrile (35:65, v/v) as mobile phase. The compounds were detected at a wavelength of 335 nm with a lower limit of quantification of 0.05 mg/L in human plasma. Retention times for rifampicin and 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline used as internal standard were respectively 3.77 and 4.81 min. This robust and exact method was successfully applied in routine for therapeutic drug monitoring in patients treated with rifampicin. Copyright © 2016 John Wiley & Sons, Ltd.

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.

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.

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

A new application of micellar liquid chromatography in the determination of free ampicillin concentration in the drug-human serum albumin standard solution in comparison with the adsorption method.

PubMed

Stępnik, Katarzyna E; Malinowska, Irena; Maciejewska, Małgorzata

2016-06-01

The determination of free drug concentration is a very important issue in the field of pharmacology because only the unbound drug fraction can achieve a pharmacological effect. Due to the ability to solubilize many different compounds in micellar aggregates, micellar liquid chromatography (MLC) can be used for direct determination of free drug concentration. Proteins are not retained on the stationary phase probably due to the formation of protein - surfactant complexes which are excluded from the pores of stationary phase. The micellar method is simple and fast. It does not require any pre-preparation of the tested samples for analysis. The main aim of this paper is to demonstrate a completely new applicability of the analytical use of MLC concerning the determination of free drug concentration in the standard solution of human serum albumin. The well-known adsorption method using RP-HPLC and the spectrophotometric technique was applied as the reference method. The results show that the free drug concentration value obtained in the MLC system (based on the RP-8 stationary phase and CTAB) is similar to that obtained by the adsorption method: both RP-HPLC (95.83μgmL(-1), 79.86% of free form) and spectrophotometry (95.71μgmL(-1), 79.76%). In the MLC the free drug concentration was 93.98μgmL(-1) (78.3%). This indicates that the obtained results are within the analytical range of % of free ampicillin fraction and the MLC with direct sample injection can be treated like a promising method for the determination of free drug concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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 and characterization of allergic polymerized impurities in cephalosporins by 2D-HPSEC×LC-IT-TOF MS.

PubMed

Xu, Yu; Wang, DanDan; Tang, Lan; Wang, Jian

2017-10-25

Eleven unknown allergic impurities in cefodizime, cefmenoxime and cefonicid were separated and characterized by a trap-free two-dimensional high performance size exclusion chromatography (HPSEC) and reversed phase liquid chromatography (RP-HPLC) coupled to high resolution ion trap/time-of-flight mass spectrometry (2D-HPSEC×LC-IT-TOF MS) with positive and negative modes of electrospray ionization method. Separation and characterization the allergic polymerized impurities in β-lactam antibiotics were on the basis of column-switching technique which effectively combined the advantages of HPSEC and the ability of RP-HPLC to identify the special impurities. In the first dimension HPSEC, the column was Xtimate SEC-120 analytical column (7.8mm×30cm, 5μm), and the gradient elution used pH 7.0 buffer-acetonitrile as mobile phase And the second dimension analytical column was ZORBAX SB-C18 (4.6×150mm, 3.5μm) with ammonium formate solution (10mM) and ammonium formate (8mM) in [acetonitrile-water (4:1, v/v)] solution as mobile phase. Structures of eleven unknown impurities were deduced based on the high resolution MS n data with both positive and negative modes, in which nine impurities were polymerized impurities. The forming mechanism of β-lactam antibiotic polymerization in cephalosporins was also studied. The question on incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and online demineralization technique, which was worthy of widespread use and application for the advantages of stability and repeatability. Copyright © 2017. Published by Elsevier B.V.

Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

PubMed

Bhushan, Ravi; Nagar, Hariom

2015-03-01

Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification. Copyright © 2014 John Wiley & Sons, Ltd.

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

PubMed

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

2011-10-01

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

Metal-organic framework MIL-101 as sorbent based on double-pumps controlled on-line solid-phase extraction coupled with high-performance liquid chromatography for the determination of flavonoids in environmental water samples.

PubMed

Liu, Yue; Hu, Jia; Li, Yan; Li, Xiao-Shuang; Wang, Zhong-Liang

2016-10-01

A novel method with high sensitivity for the rapid determination of chrysin, apigenin and luteolin in environment water samples was developed by double-pumps controlled on-line solid-phase extraction (SPE) coupled with high-performance liquid chromatography (HPLC). In the developed technique, metal organic framework MIL-101 was synthesized and applied as a sorbent for SPE. The as-synthesized MIL-101 was characterized by scanning electron microscope, X-ray diffraction spectrometry, thermal gravimetric analysis and micropore physisorption analysis. The MIL-101 behaved as a fast kinetics in the adsorption of chrysin, apigenin and luteolin. On-line SPE of chrysin, apigenin and luteolin was processed by loading a sample solution at a flow rate of 1.0 mL/min for 10 min. The extracted analytes were subsequently eluted into a ZORBAX Bonus-RP analytical column (25 cm long × 4.6 mm i.d.) for HPLC separation under isocratic condition with a mobile phase (MeOH: ACN: 0.02 M H 3 PO 4 = 35:35:30) at a flow rate of 1.0 mL/min. Experimental conditions, including ionic strength, sample pH, sample loading rates, sample loading time and desorption analytes time, were further optimized to obtain efficient preconcentration and high-precision determination of the analytes mentioned above. The method achieved the merits of simplicity, rapidity, sensitivity, wide linear range and high sample throughput. The possible mechanism for the adsorption of flavonoids on MIL-101 was proposed. The developed method has been applied to determine trace chrysin, apigenin and luteolin in a variety of environmental water samples. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensitive determination of nitrophenol isomers by reverse-phase high-performance liquid chromatography in conjunction with liquid-liquid extraction

USDA-ARS?s Scientific Manuscript database

A method for the highly sensitive determination of 2-, 3- and 4- nitrophenols was developed using reverse-phase high-performance liquid chromatography (RP-HPLC) with a UV photodiode array detector. Using a reverse-phase column and 40% aqueous acetonitrile as an eluent (i.e. isocratic elution), the i...

Identification and Characterization of Asulam Impurities in Self Made Bulk Batch Synthesis and Quantification by RP-HPLC Method.

PubMed

Mahaboob Basha, D; Venkata Reddy, G; Gopi Krishna, Y; Kumara Swamy, B E; Vijay, Rajani

2018-04-19

The first approach of this research paper explores the simultaneous characterization and determination of theAsulam active ingredient and its associated nine impurities in bulk batch production by the gradient reverse-phase high-performance liquid chromatographic (RP-HPLC) method. The best separation from its potential impurities and reproducible method was achieved by selecting the Cosmosil C-18 (250 × 4.6 mm, 5 μm particle size) analytical column with a run time of 40 min. The pumping chromatographic mobile phase was composed of 0.1% formic acid in milli-Q water (pH ~2.72) and methanol (80 + 20, v/v). An ambient column-oven temperature and UV detection at 260 nm were used. For this broad resolution, a gradient program was employed at a flow rate of 1.20 mL/min. All potential related substances in Asulam bulk manufacturing were ascertained by mass, proton nuclear magnetic resonance, and infrared spectroscopy. The developed HPLC method was validated with respect to linearity (25.64-151.83 mg/L for Asulam and 0.71-16.29, 1.02-12.26, 1.01-20.29, 0.60-10.01, 1.04-16.65, 0.94-22.47, 0.93-16.60, 1.00-12.45, 1.00-12.45, and 0.71-12.17 mg/L for Impurities A to I with a correlation coefficient 0.999 for Asulam and all the impurities), precision (RSD, % for active analyte Asulam and impurities were ˂2%), accuracy (percent recovery for Asulam at two levels ranged from 99.28 to 99.35%, and for Impurities A to I, it was 93.44 to 101.41%), and specificity. Hence, this simple and reliable HPLC method was able to determine the purity of Asulam active analyte and the level of impurities in bulk batch synthesis. By using this quantified procedure, five self-made production batches were analyzed simultaneously.

Use of a multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods.

PubMed

Bao, Lei; Oles, Carolyn J; White, Kevin D; Sapp, Chelsea; Trucksess, Mary W

2011-01-01

Deoxynivalenol (DON), also known as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium spp. DON, 12, 13-epoxy-3,7 trihydroxytrichothec-9-en-8-one, is one of the most frequently detected mycotoxins in agricultural commodities worldwide. A method consisting of extraction, filtration, column cleanup, and RP-HPLC-UV separation and quantitation was validated for the determination of DON in grains (rice and barley), grain products (whole wheat flour, white flour, wheat germ, and wheat bran), and processed foods (bread, breakfast cereals, and pretzels). A 25 g test portion was extracted with 100 mL acetonitrile-water (84 + 16, v/v). After blending for 3 min, the supernatant was applied to a multifunctional column (MycoSep 225). The purified filtrate (2 mL) was evaporated to dryness and redissolved in the mobile phase. The toxins were then subjected to RP-HPLC-UV analysis. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON added at levels ranging from 0.5 to 1.5 microg/g for all test matrixes were from 75 to 98%. SD and RSD(r) ranged from 0.7 to 11.6% and 0.9 to 12.7%, respectively. Within-laboratory HorRat values were from 0.1 to 0.7 for all matrixes analyzed. The method was found to meet AOAC method performance criteria for grains, grain products, and processed foods. The identity of DON in naturally contaminated test sample extracts was confirmed by HPLC/MS/MS analysis.

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

PubMed

Li, Hui; Lu, Dingqiang; Liu, Weimin

2004-05-01

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

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

PubMed

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

2018-02-01

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

Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m.

PubMed

Fuscaldi, Leonardo Lima; Dos Santos, Daniel Moreira; Pinheiro, Natália Gabriela Silva; Araújo, Raquel Silva; de Barros, André Luís Branco; Resende, Jarbas Magalhães; Fernandes, Simone Odília Antunes; de Lima, Maria Elena; Cardoso, Valbert Nascimento

2016-01-01

Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them with (99m)Tc. Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538) and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC with (99m)Tc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2 (.) 2H2O), and standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA, followed by Tukey test (p-values < 0.05). Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth, whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol(.)L(-1) (S. aureus) and 10.10 μmol(.)L(-1) (E. coli). Thus, only the latter was radiolabeled with (99m)Tc. The radiochemical purity analysis of LyeTx I-K-HYNIC-(99m)Tc showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2 (.) 2H2O; pH = 7; heating for 15 min) yielded a radiochemical purity of 87 ± 1 % (n = 3). However, RP-HPLC data suggested (99m)Tc transchelation from LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria. Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and aseptic inflammation.

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.

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

PubMed

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

2011-04-01

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

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

PubMed

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

2018-01-01

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

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...

Simultaneous determination of atorvastatin calcium and olmesartan medoxomil in a pharmaceutical formulation by reversed phase high-performance liquid chromatography, high-performance thin-layer chromatography, and UV spectrophotometric methods.

PubMed

Soni, Hiral; Kothari, Charmy; Khatri, Deepak; Mehta, Priti

2014-01-01

Validated RP-HPLC, HPTLC, and UV spectrophotometric methods have been developed for the simultaneous determination of atorvastatin calcium (ATV) and olmesartan medoxomil (OLM) in a pharmaceutical formulation. The RP-HPLC separation was achieved on a Kromasil C18 column (250 x 4.6 mm, 5 microm particle size) using 0.01 M potassium dihydrogen o-phosphate (pH 4 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1.5 mL/min. Quantification was achieved by UV detection at 276 nm. The HPTLC separation was achieved on precoated silica gel 60F254 plates using chloroform-methanol-acetonitrile (4 + 2+ 4, v/v/v) mobile phase. Quantification was achieved with UV detection at 276 nm. The UV-Vis spectrophotometric method was based on the simultaneous equation method that involves measurement of absorbance at two wavelengths, i.e., 255 nm (lambda max of OLM) and 246.2 nm (lambda max of ATV) in methanol. All three methods were validated as per International Conference on Harmonization guidelines. The proposed methods were simple, precise, accurate, and applicable for the simultaneous determination of ATV and OLM in a marketed formulation. The results obtained by applying the proposed methods were statistically analyzed and were found satisfactory.

Solid-phase extraction NMR studies of chromatographic fractions of saponins from Quillaja saponaria.

PubMed

Nyberg, Nils T; Baumann, Herbert; Kenne, Lennart

2003-01-15

The saponin mixture QH-B from the tree Quillaja saponaria var. Molina was fractionated by RP-HPLC in several steps. The fractions were analyzed by solid-phase extraction NMR (SPE-NMR), a technique combining the workup by solid-phase extraction with on-line coupling to an NMR flow probe. Together with MALDI-TOF mass spectrometry and comparison with chemical shifts of similar saponins, the structures of both major and minor components in QH-B could be obtained. The procedure described is a simple method to determine the structure of components in a complex mixture. The two major fractions of the mixture were found to contain at least 28 saponins, differing in the carbohydrate substructures. Eight of these have not previously been determined. The 28 saponins formed 14 equilibrium pairs by the migration of an O-acyl group between two adjacent positions on a fucosyl residue.

Physico-chemical behaviour of β irradiated plastic materials currently used as packagings and medical products

NASA Astrophysics Data System (ADS)

Yagoubi, N.; Baillet, A.; Pellerin, F.; Ferrier, D.

1995-11-01

The combined chromatographic technics and thermal analysis constitute an informative methodology for studying the modifications which could occur following a radiotreatment of plastic material at different doses (25 to 100 kGy). Several plastic materials used as packagings (PVC, PE, PS) were investigated. SEC method coupled with UV and DDL detections was applied to document any changes in molecular weight distribution. Reticulation and scission were the main observed degradation phenomena. These structural modifications were supported by TGA data, while the DSC provided information on modifications in crystallinity. In addition, RP-HPLC was carried out for the evaluation of the radiochemical behaviour of the additives and monomers. Firstly we demonstrated the degradation of high molecular weight phenolic antioxidants in BHT within the PEVA. Secondly, the modifications of amino 6 caproic acid and ɛ caprolactam, present in polyamid 6, depend on the irradiation doses.

Coupling HPLC-SPE-NMR with a microplate-based high-resolution antioxidant assay for efficient analysis of antioxidants in food--validation and proof-of-concept study with caper buds.

PubMed

Wiese, Stefanie; Wubshet, Sileshi G; Nielsen, John; Staerk, Dan

2013-12-15

This work describes the coupling of a microplate-based antioxidant assay with a hyphenated system consisting of high-performance liquid chromatography-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HPLC-SPE-NMR/high-resolution antioxidant assay, for the analysis of complex food extracts. The applicability of the microplate-based antioxidant assay for high-resolution screening of common food phenolics as well as parameters related to their trapping efficiency, elution behavior, and recovery on/from SPE cartridges are described. It was found that the microplate-based high-resolution antioxidant assay is an attractive and easy implementable alternative to direct on-line screening methods. Furthermore, it was shown that Resin SH and Resin GP SPE material are superior to RP C18HD for trapping of phenolic compounds. Proof-of-concept study was performed with caper bud extract, revealing the most important antioxidants to be quercetin, kaempferol, rutin, kaempferol-3-O-β-rutinoside and N(1),N(5),N(10)-triphenylpropenoyl spermidine amides. Targeted isolation of the latter, and comprehensive NMR experiments showed them to be N(1),N(10)-di-(E)-caffeoyl-N(5)-p-(E)-coumaroyl spermidine, N(1)-(E)-caffeoyl-N(5),N(10)-di-p-(E)-coumaroyl spermidine, N(10)-(E)-caffeoyl-N(1),N(5)-di-p-(E)-coumaroyl spermidine, and N(1),N(5),N(10)-tri-p-(E)-coumaroyl spermidine amides. Copyright © 2013 Elsevier Ltd. All rights reserved.

Simultaneous Quantitative Determination of 12 Active Components in Yuanhu Zhitong Prescription by RP-HPLC Coupled with Photodiode Array Detection

PubMed Central

Zhang, Xiaokai; Zhang, Song; Yang, Qian; Cao, Wei; Xie, Yanhua; Qiu, Pengcheng; Wang, Siwang

2015-01-01

Background: Yuanhu Zhitong prescription (YZP) is a famous traditional Chinese medicine formula, which is officially recorded in Chinese Pharmacopoeia for the treatment of stomach pain, hypochondriac pain, headache and dysmenorrhea caused by qi-stagnancy and blood stasis. It is the first report for the simultaneous determination of 12 active components in YZP. Objective: A newly, simple, accurate and reliable method for the separation and determination of 12 active components (protopine, α-allocryptopine, coptisine, xanthotol, palmatine, dehydrocorydaline, glaucine, tetrahydropalmatine, tetrahydroberberine, imperatorin, corydaline, isoimperatorin) in YZP was developed and validated using HPLC-PAD. Materials and Methods: The analytes were performed on a Phenomenex Luna-C18 (2) column (250×4.6 mm, 5.0 μm) with a gradient elution program using a mixture of acetonitrile and 0.1% phosphoric acid water solution (adjusted with triethylamine to pH 5.6) as mobile phase. Analytes were performed at 30°C with a flow rate of 1.0 mL/min. Results: The validated method was applied to analyze four major dosage forms of YZP coming from different manufacturers with good linearity (r2, 0.9981~0.9999), precision (RSD, 0.24~2.89%), repeatability (RSD, 0.15~3.34%), stability (RSD, 0.14~3.35%), recovery (91.13~110.81%) of the 12 components. Conclusion: The proposed method enables the separation and determination of 12 active components in a single run for the quality control of YZP. PMID:25709212

Simultaneous quantification of delta-9-THC, THC-acid A, CBN and CBD in seized drugs using HPLC-DAD.

PubMed

Ambach, Lars; Penitschka, Franziska; Broillet, Alain; König, Stefan; Weinmann, Wolfgang; Bernhard, Werner

2014-10-01

An HPLC-DAD method for the quantitative analysis of Δ(9)-tetrahydrocannabinol (THC), Δ(9)-tetrahydrocannabinolic acid-A (THCA-A), cannabidiol (CBD), and cannabinol (CBN) in confiscated cannabis products has been developed, fully validated and applied to analyse seized cannabis products. For determination of the THC content of plant material, this method combines quantitation of THCA-A, which is the inactive precursor of THC, and free THC. Plant material was dried, homogenized and extracted with methanol by ultrasonication. Chromatographic separation was achieved with a Waters Alliance 2695 HPLC equipped with a Merck LiChrospher 60 RP-Select B (5μm) precolumn and a Merck LiChroCart 125-4 LiChrospher 60 RP-Select B (5μm) analytical column. Analytes were detected and quantified using a Waters 2996 photo diode array detector. This method has been accepted by the public authorities of Switzerland (Bundesamt für Gesundheit, Federal Office of Public Health), and has been used to analyse 9092 samples since 2000. Since no thermal decarboxylation of THCA-A occurs, the method is highly reproducible for different cannabis materials. Two calibration ranges are used, a lower one for THC, CBN and CBD, and a higher one for THCA-A, due to its dominant presence in fresh plant material. As provider of the Swiss proficiency test, the robustness of this method has been tested over several years, and homogeneity tests even in the low calibration range (1%) show high precision (RSD≤4.3%, except CBD) and accuracy (bias≤4.1%, except CBN). Copyright © 2014. Published by Elsevier Ireland Ltd.

Isolation and Structural Characterization of Antioxidant Peptides from Degreased Apricot Seed Kernels.

PubMed

Zhang, Haisheng; Xue, Jing; Zhao, Huanxia; Zhao, Xinshuai; Xue, Huanhuan; Sun, Yuhan; Xue, Wanrui

2018-05-03

Background : The composition and sequence of amino acids have a prominent influence on theantioxidant activities of peptides. Objective : A series of isolation and purification experiments was conducted to explore the amino acid sequence of antioxidant peptides, which led to its antioxidation causes. Methods : The degreased apricot seed kernels were hydrolyzed by compound proteases of alkaline protease and flavor protease (3:2, u/u) to prepare apricot seed kernel hydrolysates (ASKH). ASKH were separated into ASKH-A and ASKH-B by dialysis bag. ASKH-B (MW < 3.5 kDa) was further separated into fractions by Sephadex G-25 and G-15 gel-filtration chromatography. Reversed-phase HPLC (RP-HPLC) was performed to separate fraction B4b into two antioxidant peptides (peptide B4b-4 and B4b-6). Results : The amino acid sequences were Val-Leu-Tyr-Ile-Trp and Ser-Val-Pro-Tyr-Glu, respectively. Conclusions : The results suggested that ASKH antioxidant peptides may have potential utility as healthy ingredients and as food preservatives due to their antioxidant activity. Highlights : Materials with regional characteristics were selected to explore, and hydrolysates were identified by RP-HPLC and matrix-assisted laser desorption ionization-time-of-flight-MS to obtain amino acid sequences.

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

C-glucosidic ellagitannins from Lythri herba (European Pharmacopoeia): chromatographic profile and structure determination.

PubMed

Piwowarski, Jakub P; Kiss, Anna K

2013-01-01

Lythri herba, a pharmacopoeial plant material (European Pharmacopoea), is obtained from flowering parts of purple loosestrife (Lythrum salicaria L.). Although extracts from this plant material have been proven to possess some interesting biological activities and its pharmacopoeial standardisation is based on total tannin content determination, the phytochemical characterisation of this main group of compounds has not yet been fully conducted. To isolate ellagitannins from Lythri herba, determine their structures and develop chromatographic methods for their qualitative analysis. Five C-glucosidic ellagitannins - monomeric- vescalagin and castalagin together with new dimeric structures - salicarinins A-C, composed of vescalagin and stachyurin, vescalagin and casuarinin, castalagin and casuarinin units connected via formation of valoneoyl group, were isolated using column chromatography and preparative HPLC. Structures were determined according to (1) H and (13) C-NMR (one- and two-dimensional), electrospray ionisation-time of flight (ESI-TOF), electrospray ionisation-ion trap (ESI-MS(n) ) and circular dichroism (CD) spectra, together with acidic hydrolysis products analysis. HPTLC on RP-18 modified plates and HPLC-DAD-MS(n) on RP-18 column methods were developed for separation of the five main ellagitannins. Copyright © 2012 John Wiley & Sons, Ltd.

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

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.

Counter-current chromatography with off-line detection by ultra high performance liquid chromatography/high resolution mass spectrometry in the study of the phenolic profile of Lippia origanoides.

PubMed

Leitão, Suzana Guimaraes; Leitão, Gilda Guimarães; Vicco, Douglas K T; Pereira, João Paulo Barreto; de Morais Simão, Gustavo; Oliveira, Danilo R; Celano, Rita; Campone, Luca; Piccinelli, Anna Lisa; Rastrelli, Luca

2017-10-20

Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMS n ) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMS n and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMS n studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique's potential. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2011-01-01

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

Biodiversity of Salix spp. honeydew and nectar honeys determined by RP-HPLC and evaluation of their antioxidant capacity.

PubMed

Tuberoso, Carlo I G; Jerković, Igor; Bifulco, Ersilia; Marijanović, Zvonimir

2011-05-01

Rare unifloral willow (Salix spp.) honeys obtained from nectar or honeydew were investigated by direct RP-HPLC-DAD method in order to identify and quantify compounds that can be used as possible markers of their origin. Antioxidant and antiradical activities of willow honeys were evaluated using FRAP (=ferric reducing antioxidant assay) and DPPH (=1,1-diphenyl-2-picrylhydrazyl radical) tests, respectively. Also HMF (=5-(hydroxymethyl)furfural), diastase activity, and CIE L*a*b*C*h* chromatic coordinates were evaluated. Abscisic acids (ABA) are typical of willow nectar honey, with a predominance of (Z,E)-ABA on (E,E)-ABA (98.2 and 31.7 mg/kg, resp.). Kinurenic acid and salicylic acid are useful to mark willow honeydew honey. The proposed HPLC-DAD method proved to be easy and reliable to identify the two different Salix spp. honeys, being not affected from any sample preparation artifact. Total antioxidant activity measured with the FRAP assay ranged from 3.2 to 12.6 mmol Fe(2+) /kg, and the antiradical activity measured with the DPPH assay ranged from 0.6 to 3.0 mmol TEAC (=Trolox equivalent antioxidant capacity)/kg in nectar and honeydew honeys, respectively. Salix spp. nectar and honeydew honeys proved to be two completely different honeys, because, besides color attributes, they show different antioxidant properties and specific compounds. Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.

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.

[Simultaneous determination of five active constitutents in Xiaochaihu Tang by HPLC].

PubMed

Liu, Qingchun; Zhao, Junning; Yan, Liangchun; Yi, Jinhai; Song, Jun

2010-03-01

To establish a HPLC-PDA method for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang. A Symmetry Shield RP18 (4.6 mm x 250 mm, 5.0 microm) was used with a mobile phase of acetonitrile-0.01% H3PO4 in gradient elution. The detection wavelength was 251 nm,the flow rate was 0.45 mL x min(-1) and the column temperature was maintained at 30 degrees C. The accuracy, precision, sensitivity, specificity and linearity of this method met the requirements. The contents of the five effective fractions were determined simultaneously. The method is rapid,simple and accurate and it can be suitable for the determination of baicalin, wogonoside, baicalein, wogonin and glycyrrhizic acid in Xiaochaihu Tang simultaneously.

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.

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.

Development and application of a high-performance liquid chromatography method using monolithic columns for the analysis of ecstasy tablets.

PubMed

Mc Fadden, Kim; Gillespie, John; Carney, Brian; O'Driscoll, Daniel

2006-07-07

A rapid and selective HPLC method using monolithic columns was developed for the separation and quantification of the principal amphetamines in ecstasy tablets. Three monolithic (Chromolith RP18e) columns of different lengths (25, 50 and 100 mm) were assessed. Validation studies including linearity, selectivity, precision, accuracy and limit of detection and quantification were carried out using the Chromolith SpeedROD, RP-18e, 50 mm x 4.6 mm column. Column backpressure and van Deemter plots demonstrated that monolithic columns provide higher efficiency at higher flow rates when compared to particulate columns without the loss of peak resolution. Application of the monolithic column to a large number of ecstasy tablets seized in Ireland ensured its suitability for the routine analysis of ecstasy tablets.

Analytical method development and validation of simultaneous estimation of rabeprazole, pantoprazole, and itopride by reverse-phase high-performance liquid chromatography.

PubMed

Perumal, Senthamil Selvan; Ekambaram, Sanmuga Priya; Raja, Samundeswari

2014-12-01

A simple, selective, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of rabeprazole (RP), pantoprazole (PP), and itopride (IP) has been developed. The compounds were well separated on a Phenomenex C 18 (Luna) column (250 mm × 4.6 mm, dp = 5 μm) with C 18 guard column (4 mm × 3 mm × 5 μm) with a mobile phase consisting of buffer containing 10 mM potassium dihydrogen orthophosphate (adjusted to pH 6.8): acetonitrile (70:30 v/v) at a flow rate of 1.0 mL/min and ultraviolet detection at 288 nm. The retention time of RP, PP, and IP were 5.35, 7.92, and 11.16 minutes, respectively. Validation of the proposed method was carried out according to International Conference on Harmonisation (ICH) guidelines. Linearity range was obtained for RP, PP, and IP over the concentration range of 2.5-25, 1-30, and 3-35 μg/mL and the r 2 values were 0.994, 0.978, and 0.991, respectively. The calculated limit of detection (LOD) values were 1, 0.3, and 1 μg/mL and limit of quantitation (LOQ) values were 2.5, 1, and 3 μg/mL for RP, PP, and IP correspondingly. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of RP, PP, and IP in combined dosage form. Copyright © 2014. Published by Elsevier B.V.

Silica-based monolithic column with evaporative light scattering detector for HPLC analysis of bacosides and apigenin in Bacopa monnieri.

PubMed

Bhandari, Pamita; Kumar, Neeraj; Singh, Bikram; Singh, Virendra; Kaur, Inderjeet

2009-08-01

A high performance liquid chromatographic method using a silica-based monolithic column coupled with evaporative light scattering detector (HPLC-ELSD) was developed and validated for simultaneous quantification of bacosides (bacoside A, bacopaside I, bacoside A(3), bacopaside II, bacopaside X, bacopasaponin C) and apigenin in Bacopa monnieri. The chromatographic resolution was achieved on a Chromolith RP-18 (100x4.6 mm) column with acetonitrile/water (30:70) as mobile phase in isocratic elution at a flow rate of 0.7 mL/min. The drift tube temperature of the ELSD was set to 95 degrees C, and the nitrogen flow rate was 2.0 SLM (standard liter per minute). The calibration curves revealed a good linear relationship (r(2) > 0.9988) within the test ranges. The detection limits (S/N = 3) and the quantification limits (S/N = 10) for the compounds were in the range of 0.54-6.06 and 1.61-18.78 microg/mL, respectively. Satisfactory average recovery was observed in the range of 95.8-99.0%. The method showed good reproducibility for the quantification of these compounds in B. monnieri with intra- and inter-day precision of less than 0.69 and 0.67%, respectively. The validated method was successfully applied to quantify analytes in nine accessions of B. monnieri and thus provides a new basis for overall quality assessment of B. monnieri.

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

PubMed

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

2012-01-01

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

Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake.

PubMed

Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S; Jain, Shalini; Lowell, Bradford B; Krashes, Michael J; Wess, Jürgen

2016-01-08

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity.

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.

Quantification of allantoin in various Zea mays L. hybrids by RP-HPLC with UV detection.

PubMed

Maksimović, Z; Malenović, A; Jancić, B; Kovacević, N

2004-07-01

A RP-HPLC method for quantification of allantoin in silk of fifteen maize hybrids (Zea mays L., Poaceae) was described. Following extraction of the plant material with an acetone-water (7:3, VN) mixture, filtration and dilution, the extracts were analyzed without previous chemical derivatization. Separation and quantification were achieved using an Alltech Econosil C18 column under isocratic conditions at 40 degrees C. The mobile phase flow (20% methanol--80% water with 5 mM sodium laurylsulfate added at pH 2.5, adjusted with 85% orthophosphoric acid; pH of water phase was finally adjusted at 6.0 by addition of triethylamine) was maintained at 1.0 mL/min. Column effluent was monitored at 235 nm. This simple procedure afforded efficient separation and quantification of allantoin in plant material, without interference of polyphenols or other plant constituents of medium to high polarity, or similar UV absorption. Our study revealed that the silk of all investigated maize hybrids could be considered relatively rich in allantoin, covering the concentration range between 215 and 289 mg per 100 g of dry plant material.

[Comparison study of different methods for extracting volatile oil from bergamot].

PubMed

Chen, Fei; Li, Qun-li; Sheng, Liu-qing; Qiu, Jiao-ying

2008-08-01

To test different methods for extracting volatile oil from bergamot. The determination of bergapten was carried out by RP-HPLC. Four different ways of organic solvent extraction, steam-input distillation, distillation of the material mixed with water and press extraction were compared. Bergapten wasnt extracted by ways of steam-input distillation and distillation of the material mixed with water. The steam distillation extraction can be taken to extract volatile oil from bergamot for protecting humans' skins.

Pressurized planar electrochromatography, high-performance thin-layer chromatography and high-performance liquid chromatography--comparison of performance.

PubMed

Płocharz, Paweł; Klimek-Turek, Anna; Dzido, Tadeusz H

2010-07-16

Kinetic performance, measured by plate height, of High-Performance Thin-Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Pressurized Planar Electrochromatography (PPEC) was compared for the systems with adsorbent of the HPTLC RP18W plate from Merck as the stationary phase and the mobile phase composed of acetonitrile and buffer solution. The HPLC column was packed with the adsorbent, which was scrapped from the chromatographic plate mentioned. An additional HPLC column was also packed with adsorbent of 5 microm particle diameter, C18 type silica based (LiChrosorb RP-18 from Merck). The dependence of plate height of both HPLC and PPEC separating systems on flow velocity of the mobile phase and on migration distance of the mobile phase in TLC system was presented applying test solute (prednisolone succinate). The highest performance, amongst systems investigated, was obtained for the PPEC system. The separation efficiency of the systems investigated in the paper was additionally confirmed by the separation of test component mixture composed of six hormones. 2010 Elsevier B.V. All rights reserved.

Derivative spectrum chromatographic method for the determination of trimethoprim in honey samples using an on-line solid-phase extraction technique.

PubMed

Uchiyama, Kazuhisa; Kondo, Mari; Yokochi, Rika; Takeuchi, Yuri; Yamamoto, Atsushi; Inoue, Yoshinori

2011-07-01

A simple, selective and rapid analytical method for determination of trimethoprim (TMP) in honey samples was developed and validated. This method is based on a SPE technique followed by HPLC with photodiode array detection. After dilution and filtration, aliquots of 500 μL honey samples were directly injected to an on-line SPE HPLC system. TMP was extracted on an RP SPE column, and separated on a hydrophilic interaction chromatography column during HPLC analysis. At the first detection step, the noise level of the photodiode array data was reduced with two-dimensional equalizer filtering, and then the smoothed data were subjected to derivative spectrum chromatography. On the second-derivative chromatogram at 254 nm, the limit of detection and the limit of quantification of TMP in a honey sample were 5 and 10 ng/g, respectively. The proposed method showed high accuracy (60-103%) with adequate sensitivity for TMP monitoring in honey samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Metabolic Analysis

NASA Astrophysics Data System (ADS)

Tolstikov, Vladimir V.

Analysis of the metabolome with coverage of all of the possibly detectable components in the sample, rather than analysis of each individual metabolite at a given time, can be accomplished by metabolic analysis. Targeted and/or nontargeted approaches are applied as needed for particular experiments. Monitoring hundreds or more metabolites at a given time requires high-throughput and high-end techniques that enable screening for relative changes in, rather than absolute concentrations of, compounds within a wide dynamic range. Most of the analytical techniques useful for these purposes use GC or HPLC/UPLC separation modules coupled to a fast and accurate mass spectrometer. GC separations require chemical modification (derivatization) before analysis, and work efficiently for the small molecules. HPLC separations are better suited for the analysis of labile and nonvolatile polar and nonpolar compounds in their native form. Direct infusion and NMR-based techniques are mostly used for fingerprinting and snap phenotyping, where applicable. Discovery and validation of metabolic biomarkers are exciting and promising opportunities offered by metabolic analysis applied to biological and biomedical experiments. We have demonstrated that GC-TOF-MS, HPLC/UPLC-RP-MS and HILIC-LC-MS techniques used for metabolic analysis offer sufficient metabolome mapping providing researchers with confident data for subsequent multivariate analysis and data mining.

RP-HPLC method development and validation for simultaneous estimation of atorvastatin calcium and pioglitazone hydrochloride in pharmaceutical dosage form.

PubMed

Peraman, Ramalingam; Mallikarjuna, Sasikala; Ammineni, Pravalika; Kondreddy, Vinod kumar

2014-10-01

A simple, selective, rapid, precise and economical reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for simultaneous estimation of atorvastatin calcium (ATV) and pioglitazone hydrochloride (PIO) from pharmaceutical formulation. The method is carried out on a C8 (25 cm × 4.6 mm i.d., 5 μm) column with a mobile phase consisting of acetonitrile (ACN):water (pH adjusted to 6.2 using o-phosphoric acid) in the ratio of 45:55 (v/v). The retention time of ATV and PIO is 4.1 and 8.1 min, respectively, with the flow rate of 1 mL/min with diode array detector detection at 232 nm. The linear regression analysis data from the linearity plot showed good linear relationship with a correlation coefficient (R(2)) value for ATV and PIO of 0.9998 and 0.9997 in the concentration range of 10-80 µg mL(-1), respectively. The relative standard deviation for intraday precision has been found to be <2.0%. The method is validated according to the ICH guidelines. The developed method is validated in terms of specificity, selectivity, accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for simultaneous estimation of these drugs in marketed dosage forms. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

High-performance liquid chromatographic determination of the beta2-selective adrenergic agonist fenoterol in human plasma after fluorescence derivatization.

PubMed

Kramer, S; Blaschke, G

2001-02-10

A sensitive high-performance liquid chromatographic method has been developed for the determination of the beta2-selective adrenergic agonist fenoterol in human plasma. To improve the sensitivity of the method, fenoterol was derivatized with N-(chloroformyl)-carbazole prior to HPLC analysis yielding highly fluorescent derivatives. The assay involves protein precipitation with acetonitrile, liquid-liquid-extraction of fenoterol from plasma with isobutanol under alkaline conditions followed by derivatization with N-(chloroformyl)-carbazole. Reversed-phase liquid chromatographic determination of the fenoterol derivative was performed using a column-switching system consisting of a LiChrospher 100 RP 18 and a LiChrospher RP-Select B column with acetonitrile, methanol and water as mobile phase. The limit of quantitation in human plasma was 376 pg fenoterol/ml. The method was successfully applied for the assay of fenoterol in patient plasma.

High performance liquid chromatography method for the determination of cinnamyl alcohol dehydrogenase activity in soybean roots.

PubMed

dos Santos, W D; Ferrarese, Maria de Lourdes Lucio; Ferrarese-Filho, O

2006-01-01

This study proposes a simple, quick and reliable method for determining the cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) activity in soybean (Glycine max L. Merr.) roots using reversed-phase high performance liquid chromatography (RP-HPLC). The method includes a single extraction of the tissue and conduction of the enzymatic reaction at 30 degrees C with cinnamaldehydes (coniferyl or sinapyl), substrates of CAD. Disappearance of the substrates in the reaction mixture is monitored at 340 nm (for coniferaldehyde) or 345 nm (for sinapaldehyde) by isocratic elution with methanol/acetic acid through a GLC-ODS (M) column. This HPLC technique furnishes a rapid and reliable measure of cinnamaldehyde substrates, and may be used as an alternative tool to analyze CAD activity in enzyme preparation without previous purification.

Contribution of flavonoids to the overall radical scavenging activity of olive (Olea europaea L.) leaf polar extracts.

PubMed

Goulas, Vlassios; Papoti, Vassiliki T; Exarchou, Vassiliki; Tsimidou, Maria Z; Gerothanassis, Ioannis P

2010-03-24

The contribution of flavonoids to the overall radical scavenging activity of olive leaf polar extracts, known to be good sources of oleuropein related compounds, was examined. Off line and on line HPLC-DPPH(*) assays were employed, whereas flavonoid content was estimated colorimetrically. Individual flavonoid composition was first assessed by RP-HPLC coupled with diode array and fluorescence detectors and verified by LC-MS detection system. Olive leaf was found a robust source of flavonoids regardless sampling parameters (olive cultivar, leaf age or sampling date). Total flavonoids accounted for the 13-27% of the total radical scavenging activity assessed using the on line protocol. Luteolin 7-O-glucoside was one of the dominant scavengers (8-25%). Taking into consideration frequency of appearance the contribution of luteolin (3-13%) was considered important, too. Our findings support that olive leaf, except for oleuropein and related compounds, is also a stable source of bioactive flavonoids.

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

PubMed

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

2018-05-01

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

Gs-coupled GPCR signalling in AgRP neurons triggers sustained increase in food intake

PubMed Central

Nakajima, Ken-ichiro; Cui, Zhenzhong; Li, Chia; Meister, Jaroslawna; Cui, Yinghong; Fu, Ou; Smith, Adam S.; Jain, Shalini; Lowell, Bradford B.; Krashes, Michael J.; Wess, Jürgen

2016-01-01

Agouti-related peptide (AgRP) neurons of the hypothalamus play a key role in regulating food intake and body weight, by releasing three different orexigenic molecules: AgRP; GABA; and neuropeptide Y. AgRP neurons express various G protein-coupled receptors (GPCRs) with different coupling properties, including Gs-linked GPCRs. At present, the potential role of Gs-coupled GPCRs in regulating the activity of AgRP neurons remains unknown. Here we show that the activation of Gs-coupled receptors expressed by AgRP neurons leads to a robust and sustained increase in food intake. We also provide detailed mechanistic data linking the stimulation of this class of receptors to the observed feeding phenotype. Moreover, we show that this pathway is clearly distinct from other GPCR signalling cascades that are operative in AgRP neurons. Our data suggest that drugs able to inhibit this signalling pathway may become useful for the treatment of obesity. PMID:26743492

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions.

PubMed

Satínský, Dalibor; Havlíková, Lucie; Solich, Petr

2013-08-01

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min(-1) and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5% triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min(-1) and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL(-1).

UHPLC-UV method for the determination of flavonoids in dietary supplements and for evaluation of their antioxidant activities.

PubMed

Magiera, Sylwia; Baranowska, Irena; Lautenszleger, Anna

2015-01-01

A simple and rapid ultra-high performance liquid chromatographic (UHPLC) method coupled with an ultraviolet detector (UV) has been developed and validated for the separation and determination of 14 major flavonoids ((±)-catechin, (-)-epicatechin, glycitin, (-)-epicatechin gallate, rutin, quercitrin, hesperidine, neohesperidine, daidzein, glycitein, quercetin, genistein, hesperetin, and biochanin A) in herbal dietary supplements. The flavonoids have been separated on a Chromolith Fast Gradient Monolithic RP-18e column utilizing a mobile phase composed of 0.05% trifluoroacetic acid in water and acetonitrile in gradient elution mode. Under these conditions, flavonoids were separated in a 5 min run. The selectivity of the developed UHPLC-UV method was confirmed by comparison with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis. The validation parameters such as linearity, sensitivity, precision, and accuracy were found to be highly satisfactory. The optimized method was applied to determination of flavonoids in different dietary supplements. Additionally, the developed HPLC-UV method combined with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay was used in the evaluation of antioxidant activity of the selected flavonoids. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel liquid chromatography/tandem mass spectrometry (LC-MS/MS) based bioanalytical method for quantification of ethyl esters of Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) and its application in pharmacokinetic study.

PubMed

Viswanathan, Sekarbabu; Verma, P R P; Ganesan, Muniyandithevar; Manivannan, Jeganathan

2017-07-15

Omega-3 fatty acids are clinically useful and the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are prevalent in fish and fish oils. Omega-3 fatty acid formulations should undergo a rigorous regulatory step in order to obtain United States Food and Drug Administration (USFDA) approval as prescription drug. In connection with that, despite quantifying EPA and DHA fatty acids, there is a need for quantifying the level of ethyl esters of them in biological samples. In this study, we make use of reverse phase high performance liquid chromatography coupled with mass spectrometry (RP-HPLC-MS)technique for the method development. Here, we have developed a novel multiple reaction monitoring method along with optimized parameters for quantification of EPA and DHA as ethyl esters. Additionally, we attempted to validate the bio-analytical method by conducting the sensitivity, selectivity, precision accuracy batch, carryover test and matrix stability experiments. Furthermore, we also implemented our validated method for evaluation of pharmacokinetics of omega fatty acid ethyl ester formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

Investigating biological activity spectrum for novel quinoline analogues 2: hydroxyquinolinecarboxamides with photosynthesis-inhibiting activity.

PubMed

Musiol, Robert; Tabak, Dominik; Niedbala, Halina; Podeszwa, Barbara; Jampilek, Josef; Kralova, Katarina; Dohnal, Jiri; Finster, Jacek; Mencel, Agnieszka; Polanski, Jaroslaw

2008-04-15

Two series of amides based on quinoline scaffold were designed and synthesized in search of photosynthesis inhibitors. The compounds were tested for their photosynthesis-inhibiting activity against Spinacia oleracea L. and Chlorella vulgaris Beij. The compounds lipophilicity was determined by the RP-HPLC method. Several compounds showed biological activity similar or even higher than that of the standard (DCMU). The structure-activity relationships are discussed.

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

PubMed

Can, Nafiz O; Arli, Goksel

2010-01-01

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

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

Use of biopartitioning micellar chromatography and RP-HPLC for the determination of blood-brain barrier penetration of α-adrenergic/imidazoline receptor ligands, and QSPR analysis.

PubMed

Vucicevic, J; Popovic, M; Nikolic, K; Filipic, S; Obradovic, D; Agbaba, D

2017-03-01

For this study, 31 compounds, including 16 imidazoline/α-adrenergic receptor (IRs/α-ARs) ligands and 15 central nervous system (CNS) drugs, were characterized in terms of the retention factors (k) obtained using biopartitioning micellar and classical reversed phase chromatography (log k BMC and log k wRP , respectively). Based on the retention factor (log k wRP ) and slope of the linear curve (S) the isocratic parameter (φ 0 ) was calculated. Obtained retention factors were correlated with experimental log BB values for the group of examined compounds. High correlations were obtained between logarithm of biopartitioning micellar chromatography (BMC) retention factor and effective permeability (r(log k BMC /log BB): 0.77), while for RP-HPLC system the correlations were lower (r(log k wRP /log BB): 0.58; r(S/log BB): -0.50; r(φ 0 /P e ): 0.61). Based on the log k BMC retention data and calculated molecular parameters of the examined compounds, quantitative structure-permeability relationship (QSPR) models were developed using partial least squares, stepwise multiple linear regression, support vector machine and artificial neural network methodologies. A high degree of structural diversity of the analysed IRs/α-ARs ligands and CNS drugs provides wide applicability domain of the QSPR models for estimation of blood-brain barrier penetration of the related compounds.

Development and validation of a new HPLC-UV method for the simultaneous determination of triclabendazole and ivermectin B1a in a pharmaceutical formulation.

PubMed

Shurbaji, Maher; Abu Al Rub, Mohamad H; Saket, Munib M; Qaisi, Ali M; Salim, Maher L; Abu-Nameh, Eyad S M

2010-01-01

A rapid, simple, and sensitive RP-HPLC analytical method was developed for the simultaneous determination of triclabendazole and ivermectin in combination using a C18 RP column. The mobile phase was acetonitrile-methanol-water-acetic acid (56 + 36 + 7.5 + 0.5, v/v/v/v) at a pH of 4.35 and flow rate of 1.0 mL/min. A 245 nm UV detection wavelength was used. Complete validation, including linearity, accuracy, recovery, LOD, LOQ, precision, robustness, stability, and peak purity, was performed. The calibration curve was linear over the range 50.09-150.26 microg/mL for triclabendazole with r = 0.9999 and 27.01-81.02 microg/mL for ivermectin with r = 0.9999. Calculated LOD and LOQ for triclabendazole were 0.03 and 0.08 microg/mL, respectively, and for ivermectin 0.07 and 0.20 microg/mL, respectively. The intraday precision obtained was 98.71% with RSD of 0.87% for triclabendazole and 100.79% with RSD 0.73% for ivermectin. The interday precision obtained was 99.51% with RSD of 0.35% for triclabendazole and 100.55% with RSD of 0.59% for ivermectin. Robustness was also studied, and there was no significant variation of the system suitability of the analytical method with small changes in experimental parameters.

[RP-HPLC method for determination of protopine in plasma and pharmacokinetics in rats].

PubMed

Yang, D L; Huang, X N; Sun, A S; Huang, B; Ye, L; Shi, J S

2001-10-01

To develop a reversed phase high performance liquid chromatographic method (RP-HPLC) for determination of protopine (Pro) in rat plasma and to investigate the pharmacokinetics of Pro in rats. The column was packed with 5 microns C18. The mobile phase (pH 5.6) was a mixture of methanol-water-10% acetic acid (80:20:2). After twice extracted with ether under basic condition, and reextracted with 0.02 mol.L-1 sulfuric acid, protopine in the plasma samples was isolated well. The content of protopine in the plasma sample was measured by UV detector at 285 nm. The lowest limit of detection was 50 ng.mL-1. The intraday and interday precisions were 1.5%-3.0% and 2.1%-6.2%, respectively. The mean recovery was 80.6%-97.6%. A good linear relationship between the peak height and the concentration of protopine in rat plasma was observed. The pharmacokinetics of protopine had been investigated in rats after intravenous administration 10 mg.kg-1. The concentration-time curve of protopine in rat was confirmed to two-compartment open model. The T1/2 alpha, T1/2 beta, Ke, CL, Vd were 0.05 h, 1.85 h, 1.52 h, 6.41 L.h-1 and 17.27 L, respectively. This method is suitable for studies on pharmacokinetics of protopine.

Characterization of taste-active fractions in red wine combining HPLC fractionation, sensory analysis and ultra performance liquid chromatography coupled with mass spectrometry detection.

PubMed

Sáenz-Navajas, María-Pilar; Ferreira, Vicente; Dizy, Marta; Fernández-Zurbano, Purificación

2010-07-19

Five Tempranillo wines exhibiting marked differences in taste and/or astringency were selected for the study. In each wine the non-volatile extract was obtained by freeze-drying and further liquid extraction in order to eliminate remaining volatile compounds. This extract was fractionated by semipreparative C18-reverse phase-high performance liquid chromatography (C18-RP-HPLC) into nine fractions which were freeze-dried, reconstituted with water and sensory assessed for taste attributes and astringency by a specifically trained sensory panel. Results have shown that wine bitterness and astringency cannot be easily related to the bitter and astringent character of the HPLC fractions, what can be due to the existence of perceptual and physicochemical interactions. While the bitter character of the bitterest fractions may be attributed to some flavonols (myricetin, quercetin and their glycosides) the development of a sensitive UPLC-MS method to quantify astringent compounds present in wines has made it possible to demonstrate that proanthocyanidins monomers, dimers, trimers and tetramers, both galloylated or non-galloylated are not relevant compounds for the perceived astringency of the fractions, while cis-aconitic acid, and secondarily vainillic, and syringic acids and ethyl syringate, are the most important molecules driving astringency in two of the fractions (F5 and F6). The identity of the chemicals responsible for the astringency of the third fraction could be assigned to some proanthocyanidins (higher than the tetramer) capable to precipitate with ovalbumin. 2010 Elsevier B.V. All rights reserved.

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

PubMed

G Archana; Dhodapkar, Rita; Kumar, Anupama

2016-09-01

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

[A new HPLC procedure for cyclamate in food with pre-chromatographic derivatization].

PubMed

Schwedt, G; Hauck, M

1988-08-01

A high-pressure liquid chromatography (HPLC) procedure for the detection of cyclamate in liquid and solid samples is presented, which depends on oxidation and the reaction of cyclohexylamine with o-phthaldialdehyde to form a condensation product. The results of the HPLC analysis, using an RP-C 18 separation system with UV detection at 242 nm are reported. Contents, from 2 to 400 mg/l, can be detected in less than 2 h (HPLC analysis within 20 min) with relative standard deviations of 4%. Only for cucumber infusions were incomplete recoveries of 68% obtained.

An optimized high-performance liquid chromatography (HPLC) method for benzoylmesaconine determination in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) and its products

PubMed Central

Xie, Ying; Zhou, Hua; Wong, Yuen Fan; Liu, Zhongqiu; Xu, Hongxi; Jiang, Zhihong; Liu, Liang

2008-01-01

Background Benzoylmesaconine (BMA) is the main Aconitum alkaloid in Radix Aconiti Lateralis Preparata (Fuzi, aconite roots) with potent pharmacological activities, such as analgesia and anti-inflammation. The present study developed a simple and reliable method using BMA as a marker compound for the quality control of processed aconite roots and their products. Methods After extraction, a high-performance liquid chromatography (HPLC) determination of BMA was conducted on a RP-C18 column by gradient elution with acetonitrile and aqueous phase, containing 0.1% phosphoric acid adjusted with triethylamine to pH 3.0. Results A distinct peak profile was obtained and separation of BMA was achieved. Method validation showed that the relative standard deviations (RSDs) of the precision of BMA in all intra-day and inter-day assays were less than 1.36%, and that the average recovery rate was 96.95%. Quantitative analysis of BMA showed that the content of BMA varied significantly in processed aconite roots and their products. Conclusion This HPLC method using BMA as a marker compound is applicable to the quality control of processed aconite roots and their products. PMID:18513409

Assessment of the chromatographic lipophilicity of eight cephalosporins on different stationary phases.

PubMed

Dąbrowska, Monika; Starek, Małgorzata; Komsta, Łukasz; Szafrański, Przemysław; Stasiewicz-Urban, Anna; Opoka, Włodzimierz

2017-04-01

The retention behaviors were investigated for a series of eight cephalosporins in thin-layer chromatography (TLC) using stationary phases of RP-2, RP-8, RP-18, NH 2 , DIOL, and CN chemically bonded silica gel. Additionally, various binary mobile phases (water/methanol and water/acetone) were used in different volume proportions. The retention behavior of the analyzed molecules was defined by R M0 constant. In addition, reversed phase high performance liquid chromatography (RP-HPLC) was performed in lipophilicity studies by using immobilized artificial membrane (IAM) stationary phase. Obtained chromatographic data (R M0 and logk' IAM ) were correlated with the lipophilicity, expressed as values of the log calculated (logP calc ) and experimental (logP exp(shake-flask) ) partition coefficient. Principal component analysis (PCA) was applied in order to obtain an overview of similarity or dissimilarity among the analyzed compounds. Hierarchical cluster analysis (HCA) was performed to compare the separation characteristics of the applied stationary phases. This study was undertaken to identify the best chromatographic system and chromatographic data processing method to enable the prediction of logP values. A comprehensive chromatographic investigation into the retention of the analyzed cephalosporins revealed a similar behavior on RP-18, RP-8 and CN stationary phases. The weak correlations obtained between experimental and certain computed lipophilicity indices revealed that R M0 and PC1/RM are relevant lipophilicity parameters and the RP-8, CN and RP-18 plates are appropriate stationary phases for lipophilicity investigation, whereas computational approaches still cannot fully replace experimentation. Copyright © 2017 Elsevier B.V. All rights reserved.

A simple, rapid and sensitive RP-HPLC-UV method for the simultaneous determination of sorafenib & paclitaxel in plasma and pharmaceutical dosage forms: Application to pharmacokinetic study.

PubMed

Khan, Ismail; Iqbal, Zafar; Khan, Abad; Hassan, Muhammad; Nasir, Fazle; Raza, Abida; Ahmad, Lateef; Khan, Amjad; Akhlaq Mughal, Muhammad

2016-10-15

A simple, economical, fast, and sensitive RP-HPLC-UV method has been developed for the simultaneous quantification of Sorafenib and paclitaxel in biological samples and formulations using piroxicam as an internal standard. The experimental conditions were optimized and method was validated according to the standard guidelines. The separation of both the analytes and internal standard was achieved on Discovery HS C18 column (250mm×4.6mm, 5μm) using Acetonitrile and TFA (0.025%) in the ratio of (65:35V/V) as the mobile phase in isocratic mode at a flow rate of 1ml/min, with a wavelength of 245nm and at a column oven temperature of 25°Cin a short run time of 12min. The limits of detection (LLOD) were 5 and 10ng/ml while the limits of quantification (LLOQ) were 10 and 15ng/ml for sorafenib and paclitaxel, respectively. Sorafenib, paclitaxel and piroxicam (IS) were extracted from biological samples by applying acetonitrile as a precipitating and extraction solvent. The method is linear in the range of 15-20,000ng/ml for paclitaxel and 10-5000ng/ml for sorafenib, respectively. The method is sensitive and reliable by considering both of its intra-day and inter-day co-efficient of variance. The method was successfully applied for the quantification of the above mentioned drugs in plasma. The developed method will be applied towards sorafenib and paclitaxel pharmacokinetics studies in animal models. 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.

Gradient Scouting in Reversed-Phase HPLC Revisited

ERIC Educational Resources Information Center

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

2011-01-01

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

Determination of catechins and caffeine in camillia sinensis raw materials, extracts, and dietary supplements by HPLC-uv: single-laboratory validation.

PubMed

Roman, Mark C

2013-01-01

A rapid method has been developed to quantify seven catechins and caffeine in green tea (Camillia sinensis) raw material and powdered extract, and dietary supplements containing green tea extract. The method utilizes RP HPLC with a phenyl-based stationary phase and gradient elution. Detection is by UV absorbance. The total run time, including column re-equilibration, is 13 min. Single-laboratory validation (SLV) has been performed on the method to determine the repeatability, accuracy, selectivity, LOD, LOQ, ruggedness, and linearity for (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-gallocatechin gallate, (-)-epigallocatechin gallate, and (+)-gallocatechin, as well as caffeine. Repeatability precision and recovery results met AOAC guidelines for SLV studies for all catechins and caffeine down to a level of approximately 20 mg/g. Finished products containing high concentrations of minerals require the use of EDTA to prevent decomposition of the catechins.

Development, Validation and Application of RP-HPLC Method: Simultaneous Determination of Antihistamine and Preservatives with Paracetamol in Liquid Formulations and Human Serum.

PubMed

Hasan, Najmul; Chaiharn, Mathurot; Toor, Umair Ali; Mirani, Zulfiqar Ali; Sajjad, Ghulam; Sher, Nawab; Aziz, Mubashir; Siddiqui, Farhan Ahmed

2016-01-01

In this article we describe development and validation of stability indicating, accurate, specific, precise and simple Ion-pairing RP-HPLC method for simultaneous determination of paracetamol and cetirizine HCl along with preservatives i.e. propylparaben, and methylparaben in pharmaceutical dosage forms of oral solution and in serum. Acetonitrile: Buffer: Sulfuric Acid (45:55:0.3 v/v/v) was the mobile phase at flow rate 1.0 mL min(-1) using a Hibar(®) Lichrosorb(®) C18 column and monitored at wavelength of 230nm. The averages of absolute and relative recoveries were found to be 99.3%, 99.5%, 99.8% and 98.7% with correlation coefficient of 0.9977, 0.9998, 0.9984, and 0.9997 for cetirizine HCl, paracetamol, methylparaben and Propylparaben respectively. The limit of quantification and limit of detection were in range of 0.3 to 2.7 ng mL(-1) and 0.1 to 0.8 ng mL(-1) respectively. Under stress conditions of acidic, basic, oxidative, and thermal degradation, maximum degradation was observed in basic and oxidative stress where a significant impact was observed while all drugs were found almost stable in the other conditions. The developed method was validated in accordance with ICH and AOAC guidelines. The proposed method was successfully applied to quantify amount of paracetamol, cetirizine HCl and two most common microbial preservatives in bulk, dosage form and physiological fluid.

An Experimental Design Approach for Impurity Profiling of Valacyclovir-Related Products by RP-HPLC

PubMed Central

Katakam, Prakash; Dey, Baishakhi; Hwisa, Nagiat T; Assaleh, Fathi H; Chandu, Babu R; Singla, Rajeev K; Mitra, Analava

2014-01-01

Abstract Impurity profiling has become an important phase of pharmaceutical research where both spectroscopic and chromatographic methods find applications. The analytical methodology needs to be very sensitive, specific, and precise which will separate and determine the impurity of interest at the 0.1% level. Current research reports a validated RP-HPLC method to detect and separate valacyclovir-related impurities (Imp-E and Imp-G) using the Box-Behnken design approach of response surface methodology. A gradient mobile phase (buffer: acetonitrile as mobile phase A and acetonitrile: methanol as mobile phase B) was used. Linearity was found in the concentration range of 50–150 μg/mL. The mean recovery of impurities was 99.9% and 103.2%, respectively. The %RSD for the peak areas of Imp-E and Imp-G were 0.9 and 0.1, respectively. No blank interferences at the retention times of the impurities suggest the specificity of the method. The LOD values were 0.0024 μg/mL for Imp-E and 0.04 μg/mL for Imp-G and the LOQ values were obtained as 0.0082 μg/mL and 0.136 μg/mL, respectively, for the impurities. The S/N ratios in both cases were within the specification limits. Proper peak shapes and satisfactory resolution with good retention times suggested the suitability of the method for impurity profiling of valacyclovir-related drug substances. PMID:25853072

Development, Validation and Application of RP-HPLC Method: Simultaneous Determination of Antihistamine and Preservatives with Paracetamol in Liquid Formulations and Human Serum

PubMed Central

Hasan, Najmul; Chaiharn, Mathurot; Toor, Umair Ali; Mirani, Zulfiqar Ali; Sajjad, Ghulam; Sher, Nawab; Aziz, Mubashir; Siddiqui, Farhan Ahmed

2016-01-01

In this article we describe development and validation of stability indicating, accurate, specific, precise and simple Ion-pairing RP-HPLC method for simultaneous determination of paracetamol and cetirizine HCl along with preservatives i.e. propylparaben, and methylparaben in pharmaceutical dosage forms of oral solution and in serum. Acetonitrile: Buffer: Sulfuric Acid (45:55:0.3 v/v/v) was the mobile phase at flow rate 1.0 mL min-1 using a Hibar® Lichrosorb® C18 column and monitored at wavelength of 230nm. The averages of absolute and relative recoveries were found to be 99.3%, 99.5%, 99.8% and 98.7% with correlation coefficient of 0.9977, 0.9998, 0.9984, and 0.9997 for cetirizine HCl, paracetamol, methylparaben and Propylparaben respectively. The limit of quantification and limit of detection were in range of 0.3 to 2.7 ng mL-1 and 0.1 to 0.8 ng mL-1 respectively. Under stress conditions of acidic, basic, oxidative, and thermal degradation, maximum degradation was observed in basic and oxidative stress where a significant impact was observed while all drugs were found almost stable in the other conditions. The developed method was validated in accordance with ICH and AOAC guidelines. The proposed method was successfully applied to quantify amount of paracetamol, cetirizine HCl and two most common microbial preservatives in bulk, dosage form and physiological fluid. PMID:27651840

Resolving Identification Issues of Saraca asoca from Its Adulterant and Commercial Samples Using Phytochemical Markers

PubMed Central

Hegde, Satisha; Hegde, Harsha Vasudev; Jalalpure, Sunil Satyappa; Peram, Malleswara Rao; Pai, Sandeep Ramachandra; Roy, Subarna

2017-01-01

Saraca asoca (Roxb.) De Wilde (Ashoka) is a highly valued endangered medicinal tree species from Western Ghats of India. Besides treating cardiac and circulatory problems, S. asoca provides immense relief in gynecological disorders. Higher price and demand, in contrast to the smaller population size of the plant, have motivated adulteration with other plants such as Polyalthia longifolia (Sonnerat) Thwaites. The fundamental concerns in quality control of S. asoca arise due to its part of medicinal value (Bark) and the chemical composition. Phytochemical fingerprinting with proper selection of analytical markers is a promising method in addressing quality control issues. In the present study, high-performance liquid chromatography of phenolic compounds (gallic acid, catechin, and epicatechin) coupled to multivariate analysis was used. Five samples each of S. asoca, P. longifolia from two localities alongside five commercial market samples showed evidence of adulteration. Subsequently, multivariate hierarchical cluster analysis and principal component analysis was established to discriminate the adulterants of S. asoca. The proposed method ascertains identification of S. asoca from its putative adulterant P. longifolia and commercial market samples. The data generated may also serve as baseline data to form a quality standard for pharmacopoeias. SUMMARY Simultaneous quantification of gallic acid, catechin, epicatechin from Saraca asoca by high-performance liquid chromatographyDetection of S. asoca from adulterant and commercial samplesUse of analytical method along with a statistical tool for addressing quality issues. Abbreviations used: HPLC: High Performance Liquid Chromatography; RP-HPLC: Reverse Phase High Performance Liquid Chromatography; CAT: Catechin; EPI: Epicatechin; GA: Gallic acid; PCA: Principal Component Analysis. PMID:28808391

Evaluation of a Propolis Water Extract Using a Reliable RP-HPLC Methodology and In Vitro and In Vivo Efficacy and Safety Characterisation

PubMed Central

Rocha, Bruno Alves; Bueno, Paula Carolina Pires; Vaz, Mirela Mara de Oliveira Lima Leite; Nascimento, Andresa Piacezzi; Ferreira, Nathália Ursoli; Moreno, Gabriela de Padua; Rodrigues, Marina Rezende; Costa-Machado, Ana Rita de Mello; Barizon, Edna Aparecida; Campos, Jacqueline Costa Lima; de Oliveira, Pollyanna Francielli; Acésio, Nathália de Oliveira; Martins, Sabrina de Paula Lima; Tavares, Denise Crispim; Berretta, Andresa Aparecida

2013-01-01

Since the beginning of propolis research, several groups have studied its antibacterial, antifungal, and antiviral properties. However, most of these studies have only employed propolis ethanolic extract (PEE) leading to little knowledge about the biological activities of propolis water extract (PWE). Based on this, in a previous study, we demonstrated the anti-inflammatory and immunomodulatory activities of PWE. In order to better understand the equilibrium between effectiveness and toxicity, which is essential for a new medicine, the characteristics of PWE were analyzed. We developed and validated an RP-HPLC method to chemically characterize PWE and PEE and evaluated the in vitro antioxidant/antimicrobial activity for both extracts and the safety of PWE via determining genotoxic potential using in vitro and in vivo mammalian micronucleus assays. We have concluded that the proposed analytical methodology was reliable, and both extracts showed similar chemical composition. The extracts presented antioxidant and antimicrobial effects, while PWE demonstrated higher antioxidant activity and more efficacious for the most of the microorganisms tested than PEE. Finally, PWE was shown to be safe using micronucleus assays. PMID:23710228

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

PubMed

An, Meichen; Liu, Ning

2010-02-01

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

Validation and evaluation of an HPLC methodology for the quantification of the potent antimitotic compound (+)-discodermolide in the Caribbean marine sponge Discodermia dissoluta.

PubMed

Valderrama, Katherine; Castellanos, Leonardo; Zea, Sven

2010-08-01

The sponge Discodermia dissoluta is the source of the potent antimitotic compound (+)-discodermolide. The relatively abundant and shallow populations of this sponge in Santa Marta, Colombia, allow for studies to evaluate the natural and biotechnological supply options of (+)-discodermolide. In this work, an RP-HPLC-UV methodology for the quantification of (+)-discodermolide from sponge samples was tested and validated. Our protocol for extracting this compound from the sponge included lyophilization, exhaustive methanol extraction, partitioning using water and dichloromethane, purification of the organic fraction in RP-18 cartridges and then finally retrieving the (+)-discodermolide in the methanol-water (80:20 v/v) fraction. This fraction was injected into an HPLC system with an Xterra RP-18 column and a detection wavelength of 235 nm. The calibration curve was linear, making it possible to calculate the LODs and quantification in these experiments. The intra-day and inter-day precision showed relative standard deviations lower than 5%. The accuracy, determined as the percentage recovery, was 99.4%. Nine samples of the sponge from the Bahamas, Bonaire, Curaçao and Santa Marta had concentrations of (+)-discodermolide ranging from 5.3 to 29.3 microg/g(-1) of wet sponge. This methodology is quick and simple, allowing for the quantification in sponges from natural environments, in situ cultures or dissociated cells.

The red wine extract-induced activation of endothelial nitric oxide synthase is mediated by a great variety of polyphenolic compounds.

PubMed

Auger, Cyril; Chaabi, Mehdi; Anselm, Eric; Lobstein, Annelise; Schini-Kerth, Valérie B

2010-07-01

Phenolic extracts from red wine (RWPs) have been shown to induce nitric oxide (NO)-mediated vasoprotective effects, mainly by causing the PI3-kinase/Akt-dependent activation of endothelial NO synthase (eNOS). RWPs contain several hundreds of phenolic compounds. The aim of the present study was to identify red wine phenolic compounds capable of activating eNOS in endothelial cells using multi-step fractionation. The red wine phenolic extract was fractionated using Sephadex LH-20 and preparative RP-HPLC approaches. The ability of a fraction to activate eNOS was assessed by determining the phosphorylation level of Akt and eNOS by Western blot analysis, and NO formation by electron spin resonance spectroscopy. Tentative identification of phenolic compounds in fractions was performed by MALDI-TOF and HPLC-MS techniques. Separation of RWPs by Sephadex LH-20 generated nine fractions (fractions A to I), of which fractions F, G, H and I caused significant eNOS activation. Fraction F was then subjected to semi-preparative RP-HPLC to generate ten subfractions (subfraction SF1 to SF10), all of which caused eNOS activation. The active fractions and subfractions contained mainly procyanidins and anthocyanins. Isolation of phenolic compounds from SF9 by semi-preparative RP-HLPC lead to the identification of petunidin-O-coumaroyl-glucoside as a potent activator of eNOS.

Conformationally selective biophysical assay for influenza vaccine potency determination.

PubMed

Wen, Yingxia; Han, Liqun; Palladino, Giuseppe; Ferrari, Annette; Xie, Yuhong; Carfi, Andrea; Dormitzer, Philip R; Settembre, Ethan C

2015-10-05

Influenza vaccines are the primary intervention for reducing the substantial health burden from pandemic and seasonal influenza. Hemagglutinin (HA) is the most important influenza vaccine antigen. Subunit and split influenza vaccines are formulated, released for clinical use, and tested for stability based on an in vitro potency assay, single-radial immunodiffusion (SRID), which selectively detects HA that is immunologically active (capable of eliciting neutralizing or hemagglutination inhibiting antibodies in an immunized subject). The time consuming generation of strain-specific sheep antisera and calibrated antigen standards for SRID can delay vaccine release. The limitation in generating SRID reagents was evident during the early days of the 2009 pandemic, prompting efforts to develop more practical, alternative, quantitative assays for immunologically active HA. Here we demonstrate that, under native conditions, trypsin selectively digests HA produced from egg or mammalian cell in monovalent vaccines that is altered by stress conditions such as reduced pH, elevated temperature, or deamidation, leaving native, pre-fusion HA, intact. Subsequent reverse-phase high pressure liquid chromatography (RP-HPLC) can separate trypsin-resistant HA from the digested HA. Integration of the resulting RP-HPLC peak yields HA quantities that match well the values obtained by SRID. Therefore, trypsin digestion, to pre-select immunologically active HA, followed by quantification by RP-HPLC is a promising alternative in vitro potency assay for influenza vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

Intraspecific Variation of Centruroides Edwardsii Venom from Two Regions of Colombia

PubMed Central

Estrada-Gómez, Sebastián; Cupitra, Nelson Ivan; Arango, Walter Murillo; Vargas Muñoz, Leidy Johana

2014-01-01

We report the first description studies, partial characterization, and intraspecific difference of Centruroides edwardsii, Gervais 1843, venom. C. edwardsii from two Colombian regions (Antioquia and Tolima) were evaluated. Both venoms showed hemolytic activity, possibly dependent of enzymatic active phospholipases, and neither coagulant nor proteolytic activities were observed. Venom electrophoretic profile showed significant differences between C. edwardsii venom from both regions. A high concentration of proteins with molecular masses between 31 kDa and 97.4 kDa, and an important concentration close or below 14.4 kDa were detected. RP-HPLC retention times between 38.2 min and 42.1 min, showed bands close to 14.4 kDa, which may correspond to phospholipases. RP-HPLC venom profile showed a well conserved region in both venoms between 7 and 17 min, after this, significant differences were detected. From Tolima region venom, 50 well-defined peaks were detected, while in the Antioquia region venom, 55 well-defined peaks were detected. Larvicidal activity was only detected in the C. edwardsii venom from Antioquia. No antimicrobial activity was observed using complete venom or RP-HPLC collected fractions of both venoms. Lethally activity (carried out on female albino swiss mice) was detected at doses over 19.2 mg/kg of crude venom. Toxic effects included distress, excitability, eye irritation and secretions, hyperventilation, ataxia, paralysis, and salivation. PMID:25025710

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.

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

PubMed

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

1999-09-01

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

Extraction and identification of flavonoids from parsley extracts by HPLC analysis

NASA Astrophysics Data System (ADS)

Stan, M.; Soran, M. L.; Varodi, C.; Lung, I.

2012-02-01

Flavonoids are phenolic compounds isolated from a wide variety of plants, and are valuable for their multiple properties, including antioxidant and antimicrobial activities. In the present work, parsley (Petroselinum crispum L.) extracts were obtained by three different extraction techniques: maceration, ultrasonic-assisted and microwave-assisted solvent extractions. The extractions were performed with ethanol-water mixtures in various ratios. From these extracts, flavonoids like the flavones apigenin and luteolin, and the flavonols quercetin and kaempferol were identified using an HPLC Shimadzu apparatus equipped with PDA and MS detectors. The separation method involved a gradient step. The mobile phase consisted of two solvents: acetonitrile and distilled water with 0.1% formic acid. The separation was performed on a RP-C18 column.

Simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor with HPLC and electrochemical detection.

PubMed

Kuhlmann, O; Stoldt, G; Struck, H G; Krauss, G J

1998-09-01

A sensitive and selective bioanalytical method for simultaneous determination of diclofenac and oxybuprocaine in human aqueous humor using reversed-phase HPLC and electrochemical detection is described. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microns; 150 x 4.6 mm I.D.). This support is coated with a hydrophilic polyoxyethylenepolymer. It allows protein-containing samples to be injected directly onto the column. The electrochemical detector permit a detection limit of 500 pg diclofenac per ml (daily relative standard deviation 6.3%) and 50 ng oxybuprocaine per ml (daily R.S.D. 2.6%), respectively. Results of administered and measured drug-concentrations in time dependent decrease are presented.

Influence of storage conditions on the stability of monomeric anthocyanins studied by reversed-phase high-performance liquid chromatography.

PubMed

Morais, Helena; Ramos, Cristina; Forgács, Esther; Cserháti, Tibor; Oliviera, José

2002-04-25

The effect of light, storage time and temperature on the decomposition rate of monomeric anthocyanin pigments extracted from skins of grape (Vitis vinifera var. Red globe) was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The impact of various storage conditions on the pigment stability was assessed by stepwise regression analysis. RP-HPLC separated well the five anthocyanins identified and proved the presence of other unidentified pigments at lower concentrations. Stepwise regression analysis confirmed that the overall decomposition rate of monomeric anthocyanins, peonidin-3-glucoside and malvidin-3-glucoside significantly depended on the time and temperature of storage, the effect of storage time being the most important. The presence or absence of light exerted a negligible impact on the decomposition rate.

Development and validation of a high performance liquid chromatographic method for simultaneous determination of vitamins A and D3 in fluid milk products.

PubMed

Chen, Yang; Reddy, Ravinder M; Li, Wenjing; Yettlla, Ramesh R; Lopez, Salvador; Woodman, Michael

2015-01-01

An HPLC method for simultaneous determination of vitamins A and D3 in fluid milk was developed and validated. Saponification and extraction conditions were studied for optimum recovery and simplicity. An RP HPLC system equipped with a C18 column and diode array detector was used for quantitation. The method was subjected to a single-laboratory validation using skim, 2% fat, and whole milk samples at concentrations of 50, 100, and 200% of the recommended fortification levels for vitamins A and D3 for Grade "A" fluid milk. The method quantitation limits for vitamins A and D3 were 0.0072 and 0.0026 μg/mL, respectively. Average recoveries between 94 and 110% and SD values ranging from 2.7 to 6.9% were obtained for both vitamins A and D3. The accuracy of the method was evaluated using a National Institute of Standards and Technology standard reference material (1849a) and proficiency test samples.

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

DOE PAGES

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

2015-01-29

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

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

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

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

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

Analysis of iridoids, secoiridoids and xanthones in Centaurium erythraea, Frasera caroliniensis and Gentiana lutea using LC-MS and RP-HPLC.

PubMed

Aberham, Anita; Pieri, Valerio; Croom, Edward M; Ellmerer, Ernst; Stuppner, Hermann

2011-02-20

This study presents a new and validated HPLC method for the simultaneous determination of bioactive compounds in Centaurium erythraea, Frasera caroliniensis and Gentiana lutea. The iridoid loganic acid, four secoiridoids and 29 xanthones were separated on a RP-18 column, using aqueous o-phosphoric acid (0.085%, v/v) and acetonitrile as mobile phase. Phytochemical investigation of C. erythraea herb and F. caroliniensis roots resulted into isolation of 25 xanthones and three secoiridoids the structure of which was elucidated by spectroscopic means (NMR, MS and UV). 1,3,8-Trihydroxy-5,6-dimethoxyxanthone, isolated from C. erythraea, turned out to be a novel xanthone. The stability of the analytes was tested by subjecting samples to light, moisture and different temperatures. After six months of storage, decomposition of gentiopicroside and sweroside was observed. The swertiamarin content was nearly unchanged when stored at room temperature or in the refrigerator, but high temperature conditions reduced the content to 85%. In contrast, xanthones were stable under long-term, refrigerated and accelerated conditions. The established chromatographic method has been successfully applied for the quantification of the bioactive compounds in the three plants. The presence and distribution of polyoxygenated xanthones within the three members of the Gentianaceae family and their significance as analytical markers are discussed. Copyright © 2010 Elsevier B.V. All rights reserved.

Simultaneous determination of multi drug components Theophylline, Etofylline, Guaiphenesine and Ambroxol Hydrochloride by validated RP-HPLC method in liquid dosage form.

PubMed

Jain, Jainendra Kumar; Prakash, M S; Mishra, Rajnish K; Khandhar, Amit P

2008-04-01

The RP-HPLC (reverse phase high performance liquid chromatography) method was developed and validated for simultaneous determination of Multi drug components i.e., Theophylline, Etofylline, Guaiphenesine and Ambroxol Hydrochloride in a liquid dosage form. Chromatographic separation of the four drugs was performed on a Hypersil Phenyl BDS (25cmX4.6mm, 5mm). The mobile phase constituted of triethylamine pH 3.0 buffer: methanol (85:15) v/v was delivered at the flow rate 1.5 mL/min. Detection was performed at 235 nm. The peak purity of Theophylline, Etofylline, Guaiphenesine and Ambroxol Hydrochloride were 0.99970, 0.99979, 0.99986 and 0.99949 respectively. Calibration curves were linear with correlation coefficient between 0.99995 to 0.99997 over a concentration range of 5 to 37 microg/mL for Theophylline, 19 to 140 microg/mL for Etofylline, 20 to 149 microg/mL for Guaiphenesine and 6 to 45 microg/mL for Ambroxol hydrochloride. The relative standard deviation (RSD) was found < 2.0%. The percentage recovery was found between the range of 98.6% and 100.5% at three different levels. Robustness and ruggedness were performed and result found within the RSD of 2%. All the parameters of validation were found in the acceptance range of ICH guideline.

A novel RP-HPLC method for simultaneous determination of potassium sorbate and sodium benzoate in soft drinks using C18-bonded monolithic silica column.

PubMed

Can, Nafiz O; Arli, Goksel; Lafci, Yigit

2011-08-01

Potassium sorbate and sodium benzoate are food additives that are generally employed for prevention of food spoilage originating from bacteria, molds or yeasts. Although these compounds were generally recognized as safe due to their low risk of acute and chronic toxicity, they have limitations of usage to protect human health. Development and validation of a novel RP-HPLC method, in which a C18-bonded monolithic silica column was used as stationary phase to assay these compounds, is described for the first time. Aliquots of 10 μL of samples were injected into chromatograph and eluted using phosphate buffer (0.025 M, pH 2.0)-water-acetonitrile (50:45:5, v/v/v) solution, which was pumped at the rate of 3.0 mL/min. To sharpen the peaks, 10 mM octylamine was added to the mobile phase. Potassium sorbate and sodium benzoate were detected at about 12(th) and 14(th) min, respectively, and quantified at 230 nm using photodiode array detector. A total of 41 samples were prepared by simply filtering through 0.45 μm filters after sonication, and injected into the system without any pre-treatment steps. Applicability of the method was demonstrated by performing total procedure on samples of different brands and types, and their compliance to official regulations was assessed. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

Resin comparison and fast automated stepwise conventional synthesis of human SDF-1alpha.

PubMed

Patel, Hirendra; Chantell, Christina A; Fuentes, German; Menakuru, Mahendra; Park, Jae H

2008-12-01

Human SDF-1alpha contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 x 2 min and coupling times of 2 x 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP-HPLC column and the mass of the final product was confirmed with MALDI-TOF mass spectrometry. Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.

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

PubMed

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

2018-06-01

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

Advantages of automation in plasma sample preparation prior to HPLC/MS/MS quantification: application to the determination of cilazapril and cilazaprilat in a bioequivalence study.

PubMed

Kolocouri, Filomila; Dotsikas, Yannis; Apostolou, Constantinos; Kousoulos, Constantinos; Soumelas, Georgios-Stefanos; Loukas, Yannis L

2011-01-01

An HPLC/MS/MS method characterized by complete automation and high throughput was developed for the determination of cilazapril and its active metabolite cilazaprilat in human plasma. All sample preparation and analysis steps were performed by using 2.2 mL 96 deep-well plates, while robotic liquid handling workstations were utilized for all liquid transfer steps, including liquid-liquid extraction. The whole procedure was very fast compared to a manual procedure with vials and no automation. The method also had a very short chromatographic run time of 1.5 min. Sample analysis was performed by RP-HPLC/MS/MS with positive electrospray ionization using multiple reaction monitoring. The calibration curve was linear in the range of 0.500-300 and 0.250-150 ng/mL for cilazapril and cilazaprilat, respectively. The proposed method was fully validated and proved to be selective, accurate, precise, reproducible, and suitable for the determination of cilazapril and cilazaprilat in human plasma. Therefore, it was applied to a bioequivalence study after per os administration of 2.5 mg tablet formulations of cilazapril.

Comparing monolithic and fused core HPLC columns for fast chromatographic analysis of fat-soluble vitamins.

PubMed

Kurdi, Said El; Muaileq, Dina Abu; Alhazmi, Hassan A; Bratty, Mohammed Al; Deeb, Sami El

2017-06-27

HPLC stationary phases of monolithic and fused core type can be used to achieve fast chromatographic separation as an alternative to UPLC. In this study, monolithic and fused core stationary phases are compared for fast separation of four fat-soluble vitamins. Three new methods on the first and second generation monolithic silica RP-18e columns and a fused core pentafluoro-phenyl propyl column were developed. Application of three fused core columns offered comparable separations of retinyl palmitate, DL-α-tocopheryl acetate, cholecalciferol and menadione in terms of elution speed and separation efficiency. Separation was achieved in approx. 5 min with good resolution (Rs > 5) and precision (RSD ≤ 0.6 %). Monolithic columns showed, however, a higher number of theoretical plates, better precision and lower column backpressure than the fused core column. The three developed methods were successfully applied to separate and quantitate fat-soluble vitamins in commercial products.

High-performance liquid chromatographic assay for the determination of Aloe Emodin in mouse plasma.

PubMed

Zaffaroni, M; Mucignat, C; Pecere, T; Zagotto, G; Frapolli, R; D'Incalci, M; Zucchetti, M

2003-10-25

An isocratic high-performance liquid chromatography (HPLC) method was developed and validated to determine Aloe Emodin (AE) in mouse plasma. The analysis required 0.3 ml of plasma and involves extraction with dichloromethane. The HPLC separation was carried out on Symmetry Shield RP18, a mobile phase of methanol-water-acetic acid (65:35:0.2) and fluorescence detection at lambda(ex)=410 nm and lambda(em)=510 nm. The retention time of AE was 11.7 min. The assay was linear from 10 to 1,000 ng/ml (r2 > or = 0.999), showed intra- and inter-day precision within 7.8 and 4.7%, and accuracy of 87.3-105.7%. Detection limit (LOD) and quantification limit (LOQ) were 4.5 and 5 ng/ml, respectively. The method was applied to determine for the first time the pharmacokinetic of AE in mice.

Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities.

PubMed

Ciric, Andrija; Jelikic-Stankov, Milena; Cvijovic, Milica; Djurdjevic, Predrag

2018-04-01

An isocratic RP-HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin-7-O-glucoside, kaempferol and kaempferol-3-O-glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water-acetonitrile, pH 2.8 (adjusted with formic acid), flow rate 0.5 mL min -1 and column temperature 35°C. The obtained levels of bioflavonoids (mg per 100 mL of juice) were as follows: for quercetin, ca. 0.21-5.12; for kaempferol, ca. 0.05-1.2; for rutin, ca. 0.4-6.5; for luteolin-7-O-glucoside, ca. 5.6-10.2; and for kaempferol-3-O-glucoside, ca. 0.02-0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC 50 values varied from 8.56 to 14.05 mg L -1 . These values are ~2.5-3 times lower than quercetin, ascorbic acid and Trolox®, but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative analysis of PEG-functionalized colloidal gold nanoparticles using charged aerosol detection.

PubMed

Smith, Mackensie C; Crist, Rachael M; Clogston, Jeffrey D; McNeil, Scott E

2015-05-01

Surface characteristics of a nanoparticle, such as functionalization with polyethylene glycol (PEG), are critical to understand and achieve optimal biocompatibility. Routine physicochemical characterization such as UV-vis spectroscopy (for gold nanoparticles), dynamic light scattering, and zeta potential are commonly used to assess the presence of PEG. However, these techniques are merely qualitative and are not sensitive enough to distinguish differences in PEG quantity, density, or presentation. As an alternative, two methods are described here which allow for quantitative measurement of PEG on PEGylated gold nanoparticles. The first, a displacement method, utilizes dithiothreitol to displace PEG from the gold surface. The dithiothreitol-coated gold nanoparticles are separated from the mixture via centrifugation, and the excess dithiothreitol and dissociated PEG are separated through reversed-phase high-performance liquid chromatography (RP-HPLC). The second, a dissolution method, utilizes potassium cyanide to dissolve the gold nanoparticles and liberate PEG. Excess CN(-), Au(CN)2 (-), and free PEG are separated using RP-HPLC. In both techniques, the free PEG can be quantified against a standard curve using charged aerosol detection. The displacement and dissolution methods are validated here using 2-, 5-, 10-, and 20-kDa PEGylated 30-nm colloidal gold nanoparticles. Further value in these techniques is demonstrated not only by quantitating the total PEG fraction but also by being able to be adapted to quantitate the free unbound PEG and the bound PEG fractions. This is an important distinction, as differences in the bound and unbound PEG fractions can affect biocompatibility, which would not be detected in techniques that only quantitate the total PEG fraction.

Investigating biological activity spectrum for novel quinoline analogues.

PubMed

Musiol, Robert; Jampilek, Josef; Kralova, Katarina; Richardson, Des R; Kalinowski, Danuta; Podeszwa, Barbara; Finster, Jacek; Niedbala, Halina; Palka, Anna; Polanski, Jaroslaw

2007-02-01

The lack of the wide spectrum of biological data is an important obstacle preventing the efficient molecular design. Quinoline derivatives are known to exhibit a variety of biological effects. In the current publication, we tested a series of novel quinoline analogues for their photosynthesis-inhibiting activity (the inhibition of photosynthetic electron transport in spinach chloroplasts (Spinacia oleracea L.) and the reduction of chlorophyll content in Chlorella vulgaris Beij.). Moreover, antiproliferative activity was measured using SK-N-MC neuroepithelioma cell line. We described the structure-activity relationships (SAR) between the chemical structure and biological effects of the synthesized compounds. We also measured the lipophilicity of the novel compounds by means of the RP-HPLC and illustrate the relationships between the RP-HPLC retention parameter logK (the logarithm of capacity factor K) and logP data calculated by available programs.

Photoirradiation of Retinyl Palmitate in Ethanol with Ultraviolet Light - Formation of Photodecomposition Products, Reactive Oxygen Species, and Lipid Peroxides

PubMed Central

Xia, Qingsu; Yin, Jun J.; Wamer, Wayne G.; Cherng, Shu-Hui; Boudreau, Mary D.; Howard, Paul C.; Yu, Hongtao; Fu, Peter P.

2006-01-01

We have previously reported that photoirradiation of retinyl palmitate (RP), a storage and ester form of vitamin A (retinol), with UVA light resulted in the formation of photodecomposition products, generation of reactive oxygen species, and induction of lipid peroxidation. In this paper, we report our results following the photoirradiation of RP in ethanol by an UV lamp with approximately equal UVA and UVB light. The photodecomposition products were separated by reversed-phase HPLC and characterized spectroscopically by comparison with authentic standards. The identified products include: 4-keto-RP, 11-ethoxy-12-hydroxy-RP, 13-ethoxy-14-hydroxy-RP, anhydroretinol (AR), and trans- and cis-15-ethoxy-AR. Photoirradiation of RP in the presence of a lipid, methyl linoleate, resulted in induction of lipid peroxidation. Lipid peroxidation was inhibited when sodium azide was present during photoirradiation which suggests free radicals were formed. Our results demonstrate that, similar to irradiation with UVA light, RP can act as a photosensitizer leading to free radical formation and induction of lipid peroxidation following irradiation with UVB light. PMID:16823091

Use of high-performance liquid chromatography with diode array detection coupled to electrospray-Qq-time-of-flight mass spectrometry for the direct characterization of the phenolic fraction in organic commercial juices.

PubMed

Rodríguez-Medina, I C; Segura-Carretero, A; Fernández-Gutiérrez, A

2009-06-05

We have developed a direct method for the qualitative analysis of polyphenols in commercial organic fruit juices. The juices were diluted with water (50/50), filtered and directly injected. The analysis of phenolic compounds was carried out by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to photodiode array detection (DAD) and electrospray ionisation-Qq-time-of-flight mass spectrometry (ESI-Qq-TOF-MS). A unique gradient program has been optimized for the separation of several phenolic classes and the analysis time was only 5 min. The fruit juice samples were successfully analysed in positive and negative ionisation modes. In positive mode the anthocyanins were identified whereas the vast majority of polyphenols were identified using the negative ionisation mode. The sensitivity, together with mass accuracy and true isotopic pattern of the Qq-TOF-MS, allowed the identification of the phenolic compounds. Moreover, the advantage of the proposed method is the combined search of MS and MS/MS spectra, which improves the identification of compounds considerably, reducing ambiguities and false positive hits. Therefore the total fragmentation of the compound ion leading to the aglycone ion or other fragments was corroborated by MS-MS. The method was successfully employed to characterize diverse phenolic families in commercially available organic juices from four different fruits and consequently could be used in the future for the quantification purposes to compare different content of polyphenols in juices.

Analysis of anti-neoplastic drug in bacterial ghost matrix, w/o/w double nanoemulsion and w/o nanoemulsion by a validated 'green' liquid chromatographic method.

PubMed

Youssof, Abdullah M E; Salem-Bekhit, Mounir M; Shakeel, Faiyaz; Alanazi, Fars K; Haq, Nazrul

2016-07-01

The objective of the present investigation was to develop and validate a 'green' reversed phase high-performance liquid chromatography (RP-HPLC) method for rapid analysis of a cytotoxic drug 5-fluorouracil (5-FU) in bulk drug, marketed injection, water-in-oil (w/o) nanoemulsion, double water-in-oil-in-water (w/o/w) nanoemulsion and bacterial ghost (BG) matrix. The chromatography study was carried out at room temperature (25±1°C) using an HPLC system with the help of ultraviolet (UV)-visible detector. The chromatographic performance was achieved with a Nucleodur 150mm×4.6mm RP C8 column filled with 5µm filler as a static phase. The mobile phase consisted of ethyl acetate: methanol (7:3% v/v) which was delivered at a flow rate of 1.0mLmin(-1) and the drug was detected in UV mode at 254nm. The developed method was validated in terms of linearity (r(2)=0.998), accuracy (98.19-102.09%), precision (% RSD=0.58-1.17), robustness (% RSD=0.12-0.53) and sensitivity with satisfactory results. The efficiency of the method was demonstrated by the assay of the drug in marketed injection, w/o nanoemulsion, w/o/w nanoemulsion and BG with satisfactory results. The successful resolution of the drug along with its degradation products clearly established the stability-indicating nature of the proposed method. Overall, these results suggested that the proposed analytical method could be effectively applied to the routine analysis of 5-FU in bulk drug, various pharmaceutical dosage forms and BG. Copyright © 2016 Elsevier B.V. All rights reserved.

A new validated method for the simultaneous determination of benzocaine, propylparaben and benzyl alcohol in a bioadhesive gel by HPLC.

PubMed

Pérez-Lozano, P; García-Montoya, E; Orriols, A; Miñarro, M; Ticó, J R; Suñé-Negre, J M

2005-10-04

A new HPLC-RP method has been developed and validated for the simultaneous determination of benzocaine, two preservatives (propylparaben (nipasol) and benzyl alcohol) and degradation products of benzocaine in a semisolid pharmaceutical dosage form (benzocaine gel). The method uses a Nucleosil 120 C18 column and gradient elution. The mobile phase consisted of a mixture of methanol and glacial acetic acid (10%, v/v) at different proportion according to a time-schedule programme, pumped at a flow rate of 2.0 ml min(-1). The DAD detector was set at 258 nm. The validation study was carried out fulfilling the ICH guidelines in order to prove that the new analytical method, meets the reliability characteristics, and these characteristics showed the capacity of analytical method to keep, throughout the time, the fundamental criteria for validation: selectivity, linearity, precision, accuracy and sensitivity. The method was applied during the quality control of benzocaine gel in order to quantify the drug (benzocaine), preservatives and degraded products and proved to be suitable for rapid and reliable quality control method.

Selective detection of thiosulfate-containing peptides using tandem mass spectrometry.

PubMed

Raftery, Mark J

2005-01-01

Incubation of proteins or peptides containing disulfide bonds (S-S) with sodium sulfite (Na(2)SO(3)) cleaves S-S bonds producing approximately equimolar amounts of free thiols (-SH) and thiosulfates (-S-SO(3)H), a process known as sulfitolysis. Proteins and peptides containing thiosulfates were separated by reverse-phase high-performance liquid chromatography (RP-HPLC) and characterized by mass spectrometry (MS) and peptide mapping. The mass of the thiosulfate-containing peptide formed from oxidized insulin B chain was 3478.02 Da, 80 Da greater than the reduced peptide and corresponding precisely to addition of sulfur trioxide (SO(3)). Disulfide bond cleavage was also observed using RP-HPLC and MS after incubation of the intramolecular homodimer of mouse S100A8 (mass 20614 Da). The mass of HPLC-separated A8-SH was 10308 Da, and 10388 Da for A8-S-SO(3)H. Loss of SO(3) from multiply charged precursor ions was generally observed at elevated declustering potentials in the source region or within q(2) at relatively low collision energies (approximately 20 V). The characteristic loss of SO(3) at low collision energies preceded peptide backbone fragmentations at higher collision energies. Accurate mass measurement and charge-state discrimination, using a hybrid quadrupole time-of-flight mass spectrometer, allowed specific detection of thiosulfate-containing peptides. An information-dependent acquisition method, where the switch criterion was loss of m/z 79.9568, specifically identified 11 thiosulfate-containing peptides using nano-LC/MS from a tryptic digest of bovine serum albumin (BSA).

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

NASA Astrophysics Data System (ADS)

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

2018-07-01

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

Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer.

PubMed

Podust, Vladimir N; Sim, Bee-Cheng; Kothari, Dharti; Henthorn, Lana; Gu, Chen; Wang, Chia-wei; McLaughlin, Bryant; Schellenberger, Volker

2013-11-01

XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.

Chemical Composition and, Cellular Evaluation of the Antioxidant Activity of Desmodium adscendens Leaves.

PubMed

Muanda, François Nsemi; Bouayed, Jaouad; Djilani, Abdelouaheb; Yao, Chunyan; Soulimani, Rachid; Dicko, Amadou

2011-01-01

Desmodium adscendens plant is widely used as juice or tea in various parts of the world against a wide range of diseases. This study determines the quality and the quantity of polyphenols, flavonoids, anthocyanins, and tannins in D. adscendens leaves by UV-spectrophotometry and RP-HPLC methods. In addition, the antioxidant capacity of these phenolic compounds is evaluated by ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic)), DPPH (2,2-diphenyl-1 picrylhydrazyl), and Cellular tests. D. adscendens leaves are mainly composite of flavonoid compounds with 12.8 mg of catechin equivalent (CE)/g dw. The amounts of total polyphenol compounds are 11.1 mg of gallic acid equivalent (GAE)/g dw. The quantity of total anthocyanin and total tannin compounds is not considerable 0.0182 mg CgE/g dw and 0.39 mg CE/g dw, respectively. A direct correlation between phenolic compounds and antioxidant activity is observed (R(2) = 0.96). The RP-HPLC analyses reveal that the main phenolic compound identified in the methanol-water extract is quercetrin dihydrat (2.11 mg/mL). According to the results, it is observed that D. adscendens leaves possess a considerable scavenging antioxidant and antiradical capacity, therefore these antioxidant properties might increase the therapeutic value of this medicinal plant.

Quantification of Aconitum alkaloids in aconite roots by a modified RP-HPLC method.

PubMed

Jiang, Zhi-Hong; Xie, Ying; Zhou, Hua; Wang, Jing-Rong; Liu, Zhong-Qiu; Wong, Yuen-Fan; Cai, Xiong; Xu, Hong-Xi; Liu, Liang

2005-01-01

The three Aconitum alkaloids, aconitine (1), mesaconitine (2) and hypaconitine (3), are pharmacologically active but also highly toxic. A standardised method is needed for assessing the levels of these alkaloids in aconite roots in order to ensure the safe use of these plant materials as medicinal herbs. By optimising extraction, separation and measurement conditions, a reliable, reproducible and accurate method for the quantitative determination of all three Aconitum alkaloids in unprocessed and processed aconite roots has been developed. This method should be appropriate for use in the quality control of Aconitum products. The three Aconitum alkaloids were separated by a modified HPLC method employing a C18 column gradient eluted with acetonitrile and ammonium bicarbonate buffer. Quantification of Aconitum alkaloids, detected at 240 nm, in different batches of samples showed that the content of 1, 2 and 3 varied significantly. In general, the alkaloid content of unprocessed roots was higher than that of processed roots. These variations were considered to be the result of differences in species, processing methods and places of origin of the samples.

Development and validation of an RP-HPLC method for quantification of cinnamic acid derivatives and kaurane-type diterpenes in Mikania laevigata and Mikania glomerata.

PubMed

Bertolucci, Suzan Kelly; Pereira, Ana Bárbara; Pinto, José Eduardo; de Aquino Ribeiro, José Antônio; de Oliveira, Alaíde Braga; Braga, Fernão Castro

2009-02-01

MIKANIA GLOMERATA and MIKANIA LAEVIGATA (Asteraceae) are medicinal plants popularly named 'guaco' in Brazil. The leaves of both species are used to treat respiratory diseases, with coumarin (CO) and kaurane-type diterpenes being regarded as the bioactive constituents. A new and simple RP-HPLC method was developed and validated for the simultaneous quantification of CO, O-coumaric (OC), benzoylgrandifloric (BA), cinnamoylgrandifloric (CA) and kaurenoic (KA) acids in the species. Optimal separation was achieved with an alternating gradient elution of methanol and acetonitrile and detection was carried out by DAD at three different wavelengths: 210 nm for CO, OC, KA; 230 nm for BA; and 270 nm for CA. The extracts showed good stability during 42 hours under normal laboratory conditions (temperature of 23 +/- 2 degrees C). The standard curves were linear over the range 0.5 - 5.0 microg (CO), 0.25 - 4.0 microg (OC), 1.0 - 8.0 microg (BA), 0.5 - 3.0 microg (CA) and 0.8 - 12.0 microg (KA), with R(2) > 0.999 for all compounds. The method showed good precision for intra-day (RSD < 4.6 %) and inter-day assays (RSD < 4.4 %). The recovery was between 99.9 and 105.3 %, except for CO and OC in M. glomerata (73.2 - 91.6 % and 86.3 - 117.4 %, respectively). The limits of quantification and detection were in the range of 0.025 - 0.800 microg and 0.007 - 0.240 microg. The method was tested for new and old columns, temperature variation (26 and 28 degrees C) and by different operators in the same laboratory. The method was successfully applied to samples of both species.

Rapid simultaneous determination of marmelosin, umbelliferone and scopoletin from Aegle marmelos fruit by RP-HPLC.

PubMed

Shinde, P B; Katekhaye, S D; Mulik, M B; Laddha, K S

2014-09-01

The surge of interest in naturally occurring phytochemicals with high therapeutic potential has led to the discovery of many molecules, out of which naturally occuring coumarins such as marmelosin, umbelliferone and scopoletin present in Aegle marmelos (Bael) fruit shows good therapeutic potential. The aim of the present work is to develop and validate Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method for simultaneous determination of marmelosin, umbelliferone and scopoletin in A. marmelos fruit extracts. The chromatographic separation was performed with isocratic elution of 55:45 (%, v/v) methanol-water containing 0.1 % acetic acid as mobile phase. The method used to analyse the extract of A. marmelos showed good resolution with retention time within 12 min. The relative concentrations of above phytoconstituent were determined in A. marmelos fruits. The method was found to give compact peaks for scopoletin, umbelliferone and marmelosin (Rt of 4.6, 6.5 and 11.3 min respectively) and were linear over the range 5-30 μg ml(-1) (R(2) = 0.9655), 2-10 μg ml(-1) (R(2) = 0.9964) and 2-10 μg ml(-1) (R(2) = 0.9862) respectively. The mean recoveries for marmelosin, umbelliferone and scopoletin at three concentrations were in the range of 98.8-102.9, 98.8-101.1 and 94.2-98.3 % respectively. The relative standard deviation of accuracy, precision and repeatability were within 2 %, indicating the method produced highly reproducible results. Therefore this simple, precise and accurate method enables simultaneous separation of this phytoconstituent and hence can be successfully applied in analysis and routine quality control of herbal material and formulation containing A. marmelos.

Simultaneous detection of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry.

PubMed

Sai, Fan; Hong, Miao; Yunfeng, Zhao; Huijing, Chen; Yongning, Wu

2012-02-29

A sensitive method has been developed for the simultaneous determination of residues of 25 β₂-agonists and 23 β-blockers in animal foods by high-performance liquid chromatography coupled with linear ion trap mass spectrometry (HPLC-LIT-MS). This method is based on a new procedure of hydrolysis and extraction by 5% trichloracetic acid, and then cleaned up by mixed strong cation exchange (MCX) cartridges coupled with a novelty cleanup step by methanol. Methanol and 0.1% formic acid were used as mobile phases for gradient elution, while a Supelco Ascentis Express Rp-Amide column was used for LC separation. ESI positive ion scan mode was used with consecutive reaction monitoring (CRM, MS³). Nine β₂-agonists labeled by the deuterium isotope were used as internal standards for quantification. The linear ranges of 48 analytes were from 5 to 200 μg/L; the coefficient of correlation was not less than 0.995. Blank pork muscle, blank liver, and blank kidney were selected as representative matrix for spiked standard recovery test. The recoveries of each compound were in the range of 46.6-118.9%, and the relative standard deviations were in the range of 1.9-28.2%. Decision limits (CCα, α = 0.01) of 48 analytes in muscles, liver, and kidney samples ranged from 0.05 to 0.49 μg/kg, and the detection capability (CCβ, β = 0.05) ranged from 0.13 to 1.64 μg/kg. This method was successfully applied to 110 real animal origin food samples including meat, liver, and kidney of pig and chicken samples.

Determination of partition coefficients n-octanol/water for treosulfan and its epoxy-transformers: an example of a negative correlation between lipophilicity of unionized compounds and their retention in reversed-phase chromatography.

PubMed

Główka, Franciszek K; Romański, Michał; Siemiątkowska, Anna

2013-04-01

For the last decade an alkylating agent treosulfan (TREO) has been successfully applied in clinical trials in conditioning prior to hematopoietic stem cell transplantation. Pharmacological activity of the pro-drug depends on its epoxy-transformers, monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB), which are formed in a non-enzymatic consecutive reaction accompanied by a release of methanesulfonic acid. In the present study partition coefficient n-octanol/water (POW) of TREO as well as its biologically active epoxy-transformers was determined empirically (applying a classical shake-flask method) and in silico for the first time. In vitro the partition was investigated at 37°C in the system composed of the pre-saturated n-octanol and 0.05 M acetate buffer pH 4.4 adjusted with sodium and potassium chloride to ionic strength of 0.16 M. Concentration of the analytes was quantified by reversed-phase high performance liquid chromatography (RP-HPLC) method in which retention time increased from S,S-DEB to TREO. It was shown that neither association nor dissociation of the tested compounds in the applied phases occurred. Calculated logPOW (TREO: -1.58±0.04, S,S-EBDM: -1.18±0.02, S,S-DEB: -0.40±0.03) indicate the hydrophilic character of the all three entities, corresponding to its pharmacokinetic parameters described in the literature. Experimentally determined logPOW of the compounds were best comparable to the values predicted by algorithm ALOGPs. Interestingly, the POW values determined in vitro as well as in silico were inversely correlated with the retention times observed in the endcapped RP-HPLC column. It might be explained by the fact that a cleavage of methansulfonic acid from a small molecule of TREO generates significant changes in the molecular structure. Consequently, despite the common chemical origin, TREO, S,S-EBDM and S,S-DEB do not constitute a 'congeneric' series of compounds. We concluded that this might occur in other low-weight species, therefore measurement of their POW by RP-HPLC had to be applied with a special care. Copyright © 2013 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.

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

The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: 'flipping' peptide charge states for characterization by positive ion mode ESI-MS.

PubMed

Garapati, Sriramya; Burns, Colin S

2014-03-01

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Screening antimutagenic and antiproliferative properties of extracts isolated from Jackfruit pulp (Artocarpus heterophyllus Lam).

PubMed

Ruiz-Montañez, G; Burgos-Hernández, A; Calderón-Santoyo, M; López-Saiz, C M; Velázquez-Contreras, C A; Navarro-Ocaña, A; Ragazzo-Sánchez, J A

2015-05-15

The present focused on the study of the antimutagenic and antiproliferative potential of pulp Jackfruit (Artocarpus heterophyllus Lam) extract, using Salmonella typhimurium tester strains TA98 and TA100 with metabolic activation (S9) and a cancer cell line M12.C3.F6 (murine B-cell lymphoma), respectively. Jackfruit pulp extract was sequentially fractionated by chromatography (RP-HPLC) and each fraction was tested for antimutagenic and antiproliferative activities. The organic extracts obtained from Jackfruit pulp reduced the number of revertants caused by aflatoxin B1 (AFB1) and proliferation of cells M12.C3.F6; a dose-response relationship was showed. Sequential RP-HPLC fractionation of the active extracts produced both antimutagenic and/or antiproliferative fractions. These results suggested that the Jackfruit contained compounds with chemoprotective properties to reduce the mutagenicity of AFB1, also proliferation of a cancer cell line. Copyright © 2014 Elsevier Ltd. All rights reserved.

Sulfanilic acid-modified chitosan mini-spheres and their application for lysozyme purification from egg white.

PubMed

Hirsch, Daniela B; Baieli, María F; Urtasun, Nicolás; Lázaro-Martínez, Juan M; Glisoni, Romina J; Miranda, María V; Cascone, Osvaldo; Wolman, Federico J

2018-03-01

A cation exchange matrix with zwitterionic and multimodal properties was synthesized by a simple reaction sequence coupling sulfanilic acid to a chitosan based support. The novel chromatographic matrix was physico-chemically characterized by ss-NMR and ζ potential, and its chromatographic performance was evaluated for lysozyme purification from diluted egg white. The maximum adsorption capacity, calculated according to Langmuir adsorption isotherm, was 50.07 ± 1.47 mg g -1 while the dissociation constant was 0.074 ± 0.012 mg mL -1 . The process for lysozyme purification from egg white was optimized, with 81.9% yield and a purity degree of 86.5%, according to RP-HPLC analysis. This work shows novel possible applications of chitosan based materials. The simple synthesis reactions combined with the simple mode of use of the chitosan matrix represents a novel method to purify proteins from raw starting materials. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:387-396, 2018. © 2017 American Institute of Chemical Engineers.

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%.

A comparative evaluation of different ionic liquids for arsenic species separation and determination in wine varietals by liquid chromatography - hydride generation atomic fluorescence spectrometry.

PubMed

Castro Grijalba, Alexander; Fiorentini, Emiliano F; Martinez, Luis D; Wuilloud, Rodolfo G

2016-09-02

The application of different ionic liquids (ILs) as modifiers for chromatographic separation and determination of arsenite [As(III)], arsenate [As(V)], dimethylarsonic acid (DMA) and monomethylarsonic acid (MMA) species in wine samples, by reversed-phase high performance liquid chromatography coupled to hydride generation atomic fluorescence spectrometry detection (RP-HPLC-HG-AFS) was studied in this work. Several factors influencing the chromatographic separation of the As species, such as pH of the mobile phase, buffer solution concentration, buffer type, IL concentration and length of alkyl groups in ILs were evaluated. The complete separation of As species was achieved using a C18 column in isocratic mode with a mobile phase composed of 0.5% (v/v) 1-octyl-3-methylimidazolium chloride ([C8mim]Cl) and 5% (v/v) methanol at pH 8.5. A multivariate methodology was used to optimize the variables involved in AFS detection of As species after they were separated by HPLC. The ILs showed remarkable performance for the separation of As species, which was obtained within 18min with a resolution higher than 0.83. The limits of detection for As(III), As(V), MMA and DMA were 0.81, 0.89, 0.62 and 1.00μg As L(-1). The proposed method was applied for As speciation analysis in white and red wine samples originated from different grape varieties. Copyright © 2016 Elsevier B.V. All rights reserved.

Sequential injection-bead injection-lab-on-valve coupled to high-performance liquid chromatography for online renewable micro-solid-phase extraction of carbamate residues in food and environmental samples.

PubMed

Vichapong, Jitlada; Burakham, Rodjana; Srijaranai, Supalax; Grudpan, Kate

2011-07-01

A sequential injection-bead injection-lab-on-valve system was hyphenated to HPLC for online renewable micro-solid-phase extraction of carbamate insecticides. The carbamates studied were isoprocarb, methomyl, carbaryl, carbofuran, methiocarb, promecarb, and propoxur. LiChroprep(®) RP-18 beads (25-40 μm) were employed as renewable sorbent packing in a microcolumn situated inside the LOV platform mounted above the multiposition valve of the sequential injection system. The analytes sorbed by the microcolumn were eluted using 80% acetonitrile in 0.1% acetic acid before online introduction to the HPLC system. Separation was performed on an Atlantis C-18 column (4.6 × 150 mm, 5 μm) utilizing gradient elution with a flow rate of 1.0 mL/min and a detection wavelength at 270 nm. The sequential injection system offers the means of performing automated handling of sample preconcentration and matrix removal. The enrichment factors ranged between 20 and 125, leading to limits of detection (LODs) in the range of 1-20 μg/L. Good reproducibility was obtained with relative standard deviations of <0.7 and 5.4% for retention time and peak area, respectively. The developed method has been successfully applied to the determination of carbamate residues in fruit, vegetable, and water samples. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

[Screening of Active Fractions from Huanglian Jiedu Decoction against Primary Neuron Injury after Oxygen-Glucose Deprivation].

PubMed

Huang, Zhu-yan; Pan, Bei-bei; Huang, Chun-yan; Ye, Yi-lu; Liu, Dan-dan; Yu, Yue-ping; Zhang, Qi

2015-08-01

To observe the protective effect of active fractions of Huanglian Jiedu Decoction (HJD) on primary cortical neuron injury after oxygen-glucose deprivation (OGD)/reperfusion (R) injury. Methods Using macroporous resin method, HJDFE30, HJDFE50, HJDFE75, and HJDFE95 with 30%, 50%, 75%, and 95% alcohol were respectively prepared. Then the content of active components in different HJD fractions was determined with reverse phase high-performance liquid chromatography (RP-HPLC). The OGD/R injury model was induced by sodium dithionite on primary cortical neurons in neonate rats. MTT assay was used to observe the effect of four fractions (HJDFE30, HJDFE50, HJDFE75, and HJDFE95) and seven index components of HJD on the neuron viability. RP-HPLC showed active component(s) contained in HJDFE30 was geniposide; baicalin, palmatine, berberine, and wogonside contained in HJDFE50; baicalin, berberine, baicalein, and wogonin contained in HJDFE75. The neuron viability was decreased after OGD for 20 min and reperfusion for 1 h, (P <0. 01), and significantly increased after administered with HJD, HJDFE30, HJDFE50, and HJDFE75 (P <0. 05, P <0. 01). Geniposide, baicalin, baicalein, palmatine, wogonside, and wogonin could increase the cortical neuron viability (P <0. 05, P <0. 01). HJDFE30, HJDFE50, and HJDFE75, as active fractions of HJD, had protective effect on primary cortical neuron injury after OGD/R. Furthermore, geniposide, baicalin, and baicalein were main active components of HJD.

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

PubMed

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

2015-03-27

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

Comprehensive analysis of proteins of pH fractionated samples using monolithic LC/MS/MS, intact MW measurement and MALDI-QIT-TOF MS

PubMed Central

Yoo, Chul; Patwa, Tasneem H.; Kreunin, Paweena; Miller, Fred R.; Huber, Christian G.; Nesvizhskii, Alexey I.; Lubman, David M.

2012-01-01

A comprehensive platform that integrates information from the protein and peptide levels by combining various MS techniques has been employed for the analysis of proteins in fully malignant human breast cancer cells. The cell lysates were subjected to chromatofocusing fractionation, followed by tryptic digestion of pH fractions for on-line monolithic RP-HPLC interfaced with linear ion trap MS analysis for rapid protein identification. This unique approach of direct analysis of pH fractions resulted in the identification of large numbers of proteins from several selected pH fractions, in which approximately 1.5 μg of each of the pH fraction digests was consumed for an analysis time of ca 50 min. In order to combine valuable information retained at the protein level with the protein identifications obtained from the peptide level information, the same pH fraction was analyzed using nonporous (NPS)-RP-HPLC/ESI-TOF MS to obtain intact protein MW measurements. In order to further validate the protein identification procedures from the fraction digest analysis, NPS-RP-HPLC separation was performed for off-line protein collection to closely examine each protein using MALDI-TOF MS and MALDI-quadrupole ion trap (QIT)-TOF MS, and excellent agreement of protein identifications was consistently observed. It was also observed that the comparison to intact MW and other MS information was particularly useful for analyzing proteins whose identifications were suggested by one sequenced peptide from fraction digest analysis. PMID:17206599

RP-HPLC ANALYSIS OF ACIDIC AND BASIC DRUGS IN SYSTEMS WITH DIETHYLAMINE AS ELUENTS ADDITIVE.

PubMed

Petruczynik, Anna; Wroblewski, Karol; Strozek, Szymon; Waksmundzka-Hajnos, Monika

2016-11-01

The chromatographic behavior of some basic and acidic drugs was studied on Cl 8, Phenyl-Hexyl and Polar RP columns with methanol or acetonitrile as organic modifiers of aqueous mobile phases containing addition of diethylamine. Diethylamine plays a double function of silanol blocker reagent in analysis of basic drugs and ion-pair reagent in analysis of acidic drugs. Most symmetrical peaks and highest system efficiency were obtained on Phenyl-Hexyl and Polar RP columns in tested mobile phase systems compared to results obtained on C18 column. A new rapid, simple, specific and accurate reverse phase liquid chromatographic method was developed for the simultaneous determination of atorvastatin - antihyperlipidemic drug and amlodipine - calcium channel blocker in one pharmaceutical formulation. Atorvastatin is an acidic compounds while amlodipine is a basic substance. The chromatographic separation was carried out on Phenyl-Hexyl column by gradient elution mode with acetonitrile as organic modifier, acetate buffer at pH 3.5 and Q.025 M/L diethylamine. The proposed method was validated for specificity, precision, accuracy, linearity, and robustness. The linearity range of atorvastatin and amlodipine for 5 - 100 μg/mL was obtained with limits of-detection (LOD) 3.2750 gg/mL and 3.2102 μg/mL, respectively. The proposed method made use of DAD as a tool for peak identity and purity confirmation.

Seasonal variation of Brazilian red propolis: Antibacterial activity, synergistic effect and phytochemical screening.

PubMed

Regueira, M S; Tintino, Saulo Relison; da Silva, Ana Raquel Pereira; Costa, Maria do Socorro; Boligon, Aline Augusti; Matias, Edinardo F F; de Queiroz Balbino, Valdir; Menezes, Irwin R A; Melo Coutinho, Henrique Douglas

2017-09-01

The aim of this study was to investigate the effect of the dry and rainy season on the antibacterial activity and chemical composition of the Brazilian red propolis. The samples were collected in rainy (RP-PER) and dry (RP-PED) seasons and analyzed by HPLC-DAD. The extracts were tested alone and in association with antibiotics against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The HPLC analysis identified luteolin and quercetin as the main compounds. Seasonal variation was observed according to concentrations of the compounds. The MIC values against E. coli ranged from 128 μg/mL to 512 μg/mL (EC 06 and EC ATCC). The red propolis showed MIC values of 512 μg/mL against both strains of P. aeruginosa used in our study (PA03 and PA24) and against strains of Gram-positive bacteria S. aureus the MICs ranged from 64 μg/mL to ≥1024 μg/mL (SA10). A synergistic effect was observed when we combined the RP-PED with gentamicin against all the strains tested. When we combined the RP-PED with Imipenem, we only observed synergistic effect against P. aeruginosa. According to our synergistic activity results, the utilization of red propolis collected in the drier periods can be used as an adjuvant against multiresistant bacterial infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

A validated HPLC-PDA method for identification and quantification of two bioactive alkaloids, ephedrine and cryptolepine, in different Sida species.

PubMed

Chatterjee, Arnab; Kumar, Satyanshu; Chattopadhyay, Sunil K

2013-12-01

A simple, rapid, accurate and reproducible reverse-phase HPLC method has been developed for the identification and quantification of two alkaloids ephedrine and cryptolepine in different extracts of Sida species using photodiode array detection. Baseline separation of the two alkaloids was achieved on a Waters RP-18 X-terra column (250 × 4.6 mm, 5 µm) using a solvent system consisting of a mixture of water containing 0.1% Trifluoroacetic acid (TFA) and acetonitrile in a gradient elution mode with detection at 210 and 280 nm for ephedrine and cryptolepine, respectively. The calibration curves were linear in a concentration range of 10-250 µg/mL for both the alkaloids with correlation coefficient values >0.99. The limits of detection and quantification for ephedrine and cryptolepine were 5 and 10 µg/mL and 2.5 and 5 µg/mL, respectively. Relative standard deviation values for intra-day and inter-day precision were 1.22 and 1.04% for ephedrine and 1.71 and 2.06% for cryptolepine, respectively. Analytical recovery ranged from 92.46 to 103.95%. The developed HPLC method was applied to identify and quantify ephedrine and cryptolepine in different extracts of Sida species. Copyright © 2013 John Wiley & Sons, Ltd.

[Influence of combination on the specific chromatogram of Glycyrrhiza in sini decoctions by HPLC].

PubMed

Zhao, Huai-Bin; Hong, Yan-Long; Wang, You-Jie; Shen, Lan; Wu, Fei; Feng, Yi; Ruan, Ke-Feng

2012-04-01

The paper is to report the establishment of an HPLC specific chromatogram of Glycyrrhiza in Sini decoctions and the influence of combination on the specific chromatogram. The RP-HPLC method was used with a Phenomenex Gemini C18 column (250 mm x 4.6 mm ID, 5 microm), and acetonitrile-0.05% trifluoroacetic acid (gradient elution) as mobile phase. Flow rate was 0.8 mL x min(-1) and the detection wavelength was set at 232 nm. The temperature of column was 30 degrees C. The method is stable and reliable with a good reproducibility, it can be used to determine the specific chromatogram of Glycyrrhiza in Sini Decoctions. Twenty peaks were selected as specific peaks in Sini Decoction with liquiritin peak as the reference peak. Six of them were from Glycyrrhiza and the other 6 peaks were from both Glycyrrhiza and Ganjiangfuzi Decoction. The areas of specific peaks of Sini Decoctions were smaller than those in the chromatogram of Glycyrrhiza. The specific chromatogram of Glycyrrhiza in Sini Decoctions is markedly influenced by Radix Aconiti Carmichaeli and Rhizoma Zingiberis. The areas of the specific peaks in Sini Decoctions were reduced obviously. The method is stable and reliable with a good reproducibility, it can be used to determine the specific chromatogram of Glycyrrhiza in Sini Decoctions.

Multivariate optimization and validation of an analytical methodology by RP-HPLC for the determination of losartan potassium in capsules.

PubMed

Bonfilio, Rudy; Tarley, César Ricardo Teixeira; Pereira, Gislaine Ribeiro; Salgado, Hérida Regina Nunes; de Araújo, Magali Benjamim

2009-11-15

This paper describes the optimization and validation of an analytical methodology for the determination of losartan potassium in capsules by HPLC using 2(5-1) fractional factorial and Doehlert designs. This multivariate approach allows a considerable improvement in chromatographic performance using fewer experiments, without additional cost for columns or other equipment. The HPLC method utilized potassium phosphate buffer (pH 6.2; 58 mmol L(-1))-acetonitrile (65:35, v/v) as the mobile phase, pumped at a flow rate of 1.0 mL min(-1). An octylsilane column (100 mm x 4.6mm i.d., 5 microm) maintained at 35 degrees C was used as the stationary phase. UV detection was performed at 254 nm. The method was validated according to the ICH guidelines, showing accuracy, precision (intra-day relative standard deviation (R.S.D.) and inter-day R.S.D values <2.0%), selectivity, robustness and linearity (r=0.9998) over a concentration range from 30 to 70 mg L(-1) of losartan potassium. The limits of detection and quantification were 0.114 and 0.420 mg L(-1), respectively. The validated method may be used to quantify losartan potassium in capsules and to determine the stability of this drug.

Development and Validation of an HPLC Method for Simultaneous Determination of Rifampicin, Isoniazid, Pyrazinamide, and Ethambutol Hydrochloride in Pharmaceutical Formulations.

PubMed

Chellini, Paula R; Lages, Eduardo B; Franco, Pedro H C; Nogueira, Fernando H A; César, Isabela C; Pianetti, Gerson A

2015-01-01

Tuberculosis treatment consists of a fixed dose combination of rifampicin (RIF), isoniazid (INH), pyrazinamide (PYZ), and ethambutol hydrochloride (EMB). The combined treatment using various drugs is necessary for patient curing, without recrudescence, and for prevention of drug-resistant mutants, which may occur during treatment. An HPLC-diode array detector (DAD) method for the simultaneous determination of RIF, INH, PYZ, and EMB in fixed dose combination tablets was developed and validated. Chromatographic experiments were performed on an Agilent 1200 HPLC system, and the separation was carried out on a Purospher STAR RP18e (250×4.6 mm id, 5 μm, Merck) analytical column. Gradient elution was carried out with a mobile phase of 20 mM monobasic sodium phosphate buffer with 0.2% triethylamine (pH 7.0) and acetonitrile at a flow rate of 1.5 mL/min. The total run time was 12 min, and the re-equilibration time was 5 min. EMB detection was performed at 210 nm, and RIF, INH, and PYZ were detected at 238 nm, using a DAD. The method proved to be specific, linear (r2>0.99), precise (RSD<2%), accurate, and robust and may be applied to the QC analysis of pharmaceutical formulations.

Simultaneous estimation of phenolic acids in sea buckthorn (Hippophaë rhamnoides) using RP-HPLC with DAD.

PubMed

Arimboor, Ranjith; Kumar, K Sarin; Arumughan, C

2008-05-12

A RP-HPLC-DAD method was developed and validated for the simultaneous analysis of nine phenolic acids including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, salicylic acid, p-coumaric acid, cinnamic acid, caffiec acid and ferulic acid in sea buckthorn (SB) (Hippophaë rhamnoides) berries and leaves. The method was validated in terms of linearity, LOD, precision, accuracy and recovery and found to be satisfactory. Phenolic acid derivatives in anatomical parts of SB berries and leaves were separated into free phenolic acids, phenolic acids bound as esters and phenolic acids bound as glycosides and profiled in HPLC. Berry pulp contained a total of 1068 mg/kg phenolic acids, of which 58.8% was derived from phenolic glycosides. Free phenolic acids and phenolic acid esters constituted 20.0% and 21.2%, respectively, of total phenolic acids in SB berry pulp. The total phenolic acid content in seed kernel (5741 mg/kg) was higher than that in berry pulp and seed coat (Table 2). Phenolic acids liberated from soluble esters constituted the major fraction of phenolic acids (57.3% of total phenolic acids) in seed kernel. 8.4% and 34.3% of total phenolic acids in seed kernel were, respectively contributed by free and phenolic acids liberated from glycosidic bonds. The total soluble phenolic acids content in seed coat (448 mg/kg) was lower than that in seed kernel and pulp (Table 2). Proportion of free phenolic acids in total phenolic acids in seed coat was higher than that in seed kernel and pulp. Phenolic acids bound as esters and glycosides, respectively contributed 49.1% and 20.3% of total phenolic acids in seed coat. The major fraction (approximately 70%) of phenolic acids in SB berries was found to be concentrated in the seeds. Gallic acid was the predominant phenolic acid both in free and bound forms in SB berry parts and leaves.

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

PubMed Central

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

2011-01-01

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

Simultaneous determination of domperidone and Itopride in pharmaceuticals and human plasma using RP-HPLC/UV detection: Method development, validation and application of the method in in-vivo evaluation of fast dispersible tablets.

PubMed

Khan, Amjad; Iqbal, Zafar; Khadra, Ibrahim; Ahmad, Lateef; Khan, Abad; Khan, Muhammad Imran; Ullah, Zia; Ismail

2016-03-20

Domperidone and Itopride are pro-kinetic agents, regulating the gastric motility and are commonly prescribed as anti emetic drugs. In the present study a simple, rapid and sensitive RP-HPLC/UV method was developed for simultaneous determination of Domperidone and Itopride in pharmaceutical samples and human plasma, using Tenofavir as internal standard. Experimental conditions were optimized and method was validated according to the standard guidelines. Combination of water (pH 3.0) and acetonitrile (65:35 v/v) was used as mobile phase, pumped at the flow rate of 1.5 ml/min. Detector wavelength was set at 210 nm and column oven temperature was 40oC. Unlike conventional liquid-liquid extraction, simple precipitation technique was applied for drug extraction from human plasma using acetonitrile for deprotienation. The method showed adequate separation of both the analytes and best resolution was achieved using Hypersil BDS C8 column (150 mm × 4.6 mm, 5 μm). The method was quite linear in the range of 20-600 ng/ml. Recovery of the method was 92.31% and 89.82% for Domperidone and Itopride, respectively. Retention time of both the analytes and internal standard was below 15 min. The lower limit of detection (LLOD) and lower limit of quantification (LLOQ) for Domperidone were 5 and 10 ng/ml while for Itopride was 12 and 15 ng/ml, respectively. The developed method was successfully applied for in-vivo analysis of fast dispersible tablets of Domperidone in healthy human volunteer. The proposed method was a part of formulation development study and was efficiently applied for determination of the two drugs in various pharmaceutical products and human plasma. Copyright © 2015 Elsevier B.V. All rights reserved.

A method for the low-level (ng/g) determination of perfluorooctanoate in carpet by LC-MS-MS using matrix extracted standards.

PubMed

L'Empereur, Karen; Stadalius, Marilyn; Zhu, Yongdong; Mansoori, Bashir A; Isemura, Tsuguhide; Kaiser, Mary A; Knaup, Wolfgang; Noguchi, Masahiro

2008-08-01

Fluorotelomer-based acrylic polymers are applied to the surface of carpet to impart oil, stain, and water repellence properties. Concerns that fluorotelomer-based polymers are a possible source of "low level" exposure to humans, coupled with their widespread use have prompted the need to develop a method to detect and measure perfluorooctanoate (PFO) in carpet. A liquid chromatography tandem mass spectrometry method for the determination of PFO in carpet using a dual labeled 13C-perfluoroctanoic acid (13C-PFOA) internal standard is successfully developed and validated. Levels of PFO are determined using a gradient, reversed-phase high-performance liquid chromatography (HPLC) method with acetic acid acidified water-methanol, separated on a 50 mm Phenomenex Synergi Polar RP column. Ions monitored are 413 (parent) and 369 (daughter) for PFO and 415 (parent) and 370 (daughter) for dual labeled 13C-PFOA internal standard. Accuracy and precision over three days for 5 to 900 ng/g PFO in carpet ranged from 2.4% to 7.6% and 3.7% to 14.1%, respectively. Overall extraction efficiency for samples (n=30) fortified with 13C-PFOA at 20 ng/g and perfluorooctanoic acid (PFOA) at 5, 50, and 500 ng/g is 98.9%+/-8.1%. Specificity of the method was evaluated with two different carpet samples.

[Studies on HPLC chromatogram of phenolic constituents of Cortex Magnoliae Officinalis].

PubMed

Huang, Wen-hua; Guo, Bao-lin; Si, Jin-ping

2005-07-01

To study the chemical characteristic, to identify the different forms and to establish the new standard for the quality control of Cortex Magnoliae Officinalis. HPLC method was used with acetonitrile-water (63:37) as the mobile phase at room temperature. The chromatographic column was Lichrospher 100 RP-18e (4.6 mm x 250 mm, 5 microm). The flow rate was 1 mL x min(-1), and the detection wavelength was 294 nm. The chromatograms of 45 individuals from 13 seed resources of Cortex Magnolia Officinalis were recorded. The chemical characteristics analysis and comparability' s calculation of seed resources were made. It was proposed that the area ratio of peak 5 to 6 (characteristic I) and the area ratio of peak 5 and 6 to the total peak areas (characteristic II) are the identification characteristics for different seed resources of Cortex Magnoliae Officinalis. This method can be used effectively to identify the high quality seed resource of Cortex Magnoliae Officinalis.

A HPLC method for the quantification of butyramide and acetamide at ppb levels in hydrogeothermal waters

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

Gracy Elias; Earl D. Mattson; Jessica E. Little

A quantitative analytical method to determine butyramide and acetamide concentrations at the low ppb levels in geothermal waters has been developed. The analytes are concentrated in a preparation step by evaporation and analyzed using HPLC-UV. Chromatographic separation is achieved isocratically with a RP C-18 column using a 30 mM phosphate buffer solution with 5 mM heptane sulfonic acid and methanol (98:2 ratio) as the mobile phase. Absorbance is measured at 200 nm. The limit of detection (LOD) for BA and AA were 2.0 {mu}g L{sup -1} and 2.5 {mu}g L{sup -1}, respectively. The limit of quantification (LOQ) for BA andmore » AA were 5.7 {mu}g L{sup -1} and 7.7 {mu}g L{sup -1}, respectively, at the detection wavelength of 200 nm. Attaining these levels of quantification better allows these amides to be used as thermally reactive tracers in low-temperature hydrogeothermal systems.« less

Development and validation of a RP-HPLC method for the quantitation of tofacitinib in rat plasma and its application to a pharmacokinetic study.

PubMed

S, Vijay Kumar; Dhiman, Vinay; Giri, Kalpesh Kumar; Sharma, Kuldeep; Zainuddin, Mohd; Mullangi, Ramesh

2015-09-01

A novel, simple, specific, sensitive and reproducible high-performance liquid chromatography (HPLC) assay method has been developed and validated for the estimation of tofacitinib in rat plasma. The bioanalytical procedure involves extraction of tofacitinib and itraconazole (internal standard, IS) from rat plasma with a simple liquid-liquid extraction process. The chromatographic analysis was performed on a Waters Alliance system using a gradient mobile phase conditions at a flow rate of 1.0 mL/min and C18 column maintained at 40 ± 1 °C. The eluate was monitored using an UV detector set at 287 nm. Tofacitinib and IS eluted at 6.5 and 8.3 min, respectively and the total run time was 10 min. Method validation was performed as per US Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curve was linear over a concentration range of 182-5035 ng/mL (r(2) = 0.995). The intra- and inter-day precisions were in the range of 1.41-11.2 and 3.66-8.81%, respectively, in rat plasma. The validated HPLC method was successfully applied to a pharmacokinetic study in rats. Copyright © 2015 John Wiley & Sons, Ltd.

Sesamin and sesamolin as unexpected contaminants in various cold-pressed plant oils: NP-HPLC/FLD/DAD and RP-UPLC-ESI/MS(n) study.

PubMed

Górnaś, Paweł; Siger, Aleksander; Pugajeva, Iveta; Segliņa, Dalija

2014-04-01

Thirteen cold-pressed oils (Japanese quince seed, black caraway, flaxseed, rapeseed, hemp, peanut, sunflower, pumpkin, hazelnut, poppy, walnut, almond and sesame oil) manufactured by the same company over a 2-year period (2011-12) were assessed for lipophilic compounds. The presence of sesamin and sesamolin, two characteristic lignans of sesame oil, were detected in all tested plant oils. Both lignans were identified by NP-HPLC/FLD/DAD and confirmed by a RP-UPLC-ESI/MS(n) method. The lowest amount of sesamin and sesamolin was found for Japanese quince seed oil (0.10 and 0.27 mg/100 g), and the highest, excluding sesame oil, for almond oil (36.21 and 105.42 mg/100 g, respectively). The highly significant correlation between sesamolin and sesamin concentrations was found in all samples tested (r = 0.9999; p < 0.00001). These results indicate contamination of cold-pressed oils from the same source. This investigation highlights the fact that increasing the range of products manufactured by the same company can contribute to a lesser regard for the quality of the final product. Moreover, less attention paid to the quality of final product can be related to the health risks of consumers especially sensitive to allergens. Therefore, proper cleaning of processing equipment is needed to prevent cross-contact of cold-pressed oils.

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

PubMed

Galeotti, Fabio; Volpi, Nicola

2011-09-01

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

Individual Phosphatidylcholine Species Analysis by RP-HPLC-ELSD for Determination of Polyenylphosphatidylcholine in Lecithins.

PubMed

Lee, Wei-Ju; Weng, Shun-Hsiang; Su, Nan-Wei

2015-04-22

Polyenylphosphatidylcholine (PPC), a subgroup of the bioactive agents in phosphatidylcholine (PC), has been indicated to possess liver-protective effects. This study aimed to investigate a promising and feasible method to determine PC molecular species with a reverse phase (RP) high-performance liquid chromatograph (HPLC) equipped with an evaporative light scattering detector (ELSD). Chromatography was achieved using a C30 column and an isocratic mobile phase consisting of acetonitrile/methanol/triethylamine (40/58/2, v/v/v) at a flow rate of 1 mL/min, and ELSD detection was performed using 80 °C for the drift tube and an air flow rate of 1.8 L/min. To identify individual peaks on the chromatogram, MALDI-TOF-MS was employed for initial detection, and then the results were used to investigate the relationship between the retention time and fatty acyl chains of each PC molecule. A linear correlation was observed between the retention time and theoretical carbon number (TCN) of individual PC species. The compositions of PC molecular species in soybean and sunflower lecithins were similar to each other, and the major PC molecular species were 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LLPC), 1-oleoyl-2-linoleoyl-sn-glycero-3-phosphocholine (OLPC), and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC). The contents of LLPC in soybean PC and sunflower PC were 40.6% and 64.3%, respectively.

HPLC determination of cefprozil in tablets using monolithic and C18 silica columns.

PubMed

Can, Nafiz O

2011-08-01

Cefprozil (CPZ) is a second-generation semi-synthetic cephalosporin antibiotic that commonly exists as the mixture of Z and E diastereoisomers, at the ratio of approximately 9:1. A novel reversed-phase HPLC method for the determination of CPZ in tablets was described. The separation of CPZ diastereoisomers and caffeine (internal standard) was carried out by applying the same analytical and instrumental conditions on two stationary phases, which have different surface chemistries. The columns used in the study were monolithic silica Merck Chromolith Performance RP-18e and conventional C18 silica Phenomenex Synergi Hydro RP columns. In total, 10 μL aliquots of samples were injected into the system and eluted using water-acetonitrile (90:10, v/v) solution, which was pumped through the column at a flow rate of 1.0 mL/min. The analyte peaks were detected at 200 nm using diode array detector with high specificity. CPZ diastereoisomers and caffeine were measured within 13 min using the C18 column, whereas <5 min was required for the monolithic one. Validation studies were performed according to official recommendations. Value of a monolithic column for the assay of diastereoisomers in pharmaceutical tablets was evaluated for the first time and found as a powerful alternative to highly efficient C18 columns. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

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

PubMed

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

2011-10-01

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

Quality aspects of fibrinolytic agents based on biochemical characterization.

PubMed

Werner, R G; Bassarab, S; Hoffmann, H; Schlüter, M

1991-11-01

The purity, composition and in vitro fibrinolytic activity of four commercially available fibrinolytic agents, alteplase (recombinant tissue plasminogen activator, rt-PA, Actilyse; CAS 105857-23-6), streptokinase, urokinase and anistreplase (ansioyl-plasminogen-streptokinase activator-complex, APSAC), have been compared in this investigation. The fibrinolytic activity was measured in an in vitro thrombolytic assay. In this assay a human blood thrombus is dissolved in an environment of human plasma. This assay is representative for the in vivo situation, where plasminogen activation is also a limiting step in thrombolysis. In the in vitro thrombolytic assay alteplase is about 10 times more effective in clot lysis than either streptokinase or urokinase and more than 300 times more active than anistreplase. In addition, the ratio of active ingredient to total protein content in the preparations was analysed by RP-HPLC, SDS-PAGE, GPC-HPLC and amino acid analysis. The portion of active ingredient per total protein was 99.9% for alteplase, 55% for anistreplase, 20% for urokinase and 1% for streptokinase. This demonstrates that alteplase is the only fibrinolytic agent tested which is essentially free of protein additives of human origine and potential contaminants associated therewith. The superior purity of alteplase compared to the other fibrinolytics was confirmed by SDS-PAGE, RP-HPLC, and HPLC-GPC. Significant levels of aggregates were detected in streptokinase and urokinase preparations, whereas alteplase and anistreplase were essentially free of aggregates. These data demonstrate that there are significant differences in composition, purity and in vitro activity between different fibrinolytic agents.

Rapid high performance liquid chromatography method development with high prediction accuracy, using 5cm long narrow bore columns packed with sub-2microm particles and Design Space computer modeling.

PubMed

Fekete, Szabolcs; Fekete, Jeno; Molnár, Imre; Ganzler, Katalin

2009-11-06

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5cmx2.1mm columns packed with sub-2microm particles and computer simulation (DryLab((R)) package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3-5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p>400bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.

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.

Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry.

PubMed

Romarís-Hortas, Vanessa; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

2013-09-27

The combination of reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in edible seaweed. A sample pre-treatment based on ultrasound assisted enzymatic hydrolysis was optimized for the extraction of these iodinated amino acids. Pancreatin was selected as the most adequate type of enzyme, and parameters affecting the extraction efficiency (pH, temperature, mass of enzyme and extraction time) were evaluated by univariate approaches. In addition, extractable inorganic iodine (iodide) was also quantified by anion exchange high performance liquid chromatography (AE-HPLC) coupled with ICP-MS. The proposed procedure offered limits of detection of 1.1 and 4.3ngg(-1) for MIT and DIT, respectively. Total iodine contents in seaweed, as well as total iodine in enzymatic digests were measured by ICP-MS after microwave assisted alkaline digestion with tetramethylamonium hydroxide (TMAH) for total iodine assessment, and also by treating the pancreatin extracts (extractable total iodine assessment). The optimized procedure was successfully applied to five different types of edible seaweed. The highest total iodine content, and also the highest iodide levels, was found in the brown seaweed Kombu (6646±45μgg(-1)). Regarding iodinated amino acids, Nori (a red seaweed) was by far the one with the highest amount of both species (42±3 and 0.41±0.024μgg(-1) for MIT and DIT, respectively). In general, MIT concentrations were much higher than the amounts of DIT, which suggests that iodine from iodinated proteins in seaweed is most likely bound in the form of MIT residues. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and validation of a novel stability-indicating HPLC method for the simultaneous assay of betamethasone-17-valerate, fusidic acid, potassium sorbate, methylparaben and propylparaben in a topical cream preparation.

PubMed

Byrne, Jonathan; Velasco-Torrijos, Trinidad; Reinhardt, Robert

2014-08-05

A novel stability-indicating reversed phase high performance liquid chromatographic (RP-HPLC) method for the simultaneous assay of betamethasone-17-valerate, fusidic acid and potassium sorbate as well as methyl- and propylparaben in a topical cream preparation has been developed. A 100mm×3.0mm ID. Ascentis Express C18 column maintained at 30°C and UV detection at 240nm were used. A gradient programme was employed at a flow-rate of 0.75ml/min. Mobile phase A comprised of an 83:17 (v/v) mixture of acetonitrile and methanol and mobile phase B of a 10g/l solution of 85% phosphoric acid in purified water. The method has been validated according to current International Conference on Harmonisation (ICH) guidelines and applied during formulation development and stability studies. The procedure has been shown to be stability-indicating for the topical cream. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of atorvastatin calcium, ezetimibe, and fenofibrate in a tablet formulation by HPLC.

PubMed

Patel, Archita; Macwana, Chhaya; Parmar, Vishal; Patel, Samir

2012-01-01

An accurate, simple, reproducible, and sensitive HPLC method was developed and validated for the simultaneous determination of atorvastatin calcium, ezetimibe, and fenofibrate in a tablet formulation. The analyses were performed on an RP C18 column, 150 x 4.60 mm id, 5 pm particle size. The mobile phase methanol-acetonitrile-water (76 + 13 + 11, v/v/v), was pumped at a constant flow rate of 1 mL/min. UV detection was performed at 253 nm. Retention times of atorvastatin calcium, ezetimibe, and fenofibrate were found to be 2.25, 3.68, and 6.41 min, respectively. The method was validated in terms of linearity, precision, accuracy, LOD, LOQ, and robustness. The response was linear in the range 2-10 microg/mL (r2 = 0.998) for atorvastatin calcium, 2-10 microg/mL (r2 = 0.998) for ezetimibe, and 40-120 microg/mL (r2 = 0.998) for fenofibrate. The developed method can be used for routine quality analysis of the drugs in the tablet formulation.

Simultaneous determination of vitexin-2"-O-glucoside, vitexin-2"-O-rhamnoside, rutin, and hyperoside in the extract of hawthorn (Crataegus pinnatifida Bge.) leaves by RP-HPLC with ultraviolet photodiode array detection.

PubMed

Cheng, Shan; Qiu, Feng; Huang, Jia; He, Junqi

2007-03-01

RP-HPLC with UV photodiode array detection (UV-DAD) was developed and validated for the simultaneous determination of vitexin-2"-O-glucoside, vitexin-2"-O-rhamnoside, rutin, and hyperoside in the extract of hawthorn (Crataegus pinnatifida Bge.) leaves. The analytes of interest were separated on a Diamonsil C18 column (250 x 4.6 mm id, 5 microm) with the mobile phase consisting of THF/ACN/methanol/ 0.05% phosphoric acid solution (pH 5.0) (18:1:1:80 v/vl/v). The flow rate was set at 1.0 mL/min and the eluent was detected at 340 nm for the four flavonoids. The method was linear over the studied range of 1.00-100 microg/mL for the four analytes of interest with the correlation coefficient for each analyte greater than 0.999. The LOD and LOQwere 0.03 and 0.10 microg/mL, 0.03 and 0.10 microg/mL, 0.05 and 0.15 pg/mL, 0.10 and 0.30 microg/mL for vitexin-2"-O-glucoside, vitexin-2"-0-rhamnoside, rutin, and hyperoside, respectively. The optimized method was successfully applied to the analysis of four important flavonoids in the extract of hawthorn leaves. The total amounts of the four flavonoids were 22.2, 62.3, 4.27, and 8.24 mg/g dry weight for vitexin-2"-O-glucoside, vitexin-2"-O-rhamnoside, rutin, and hyperoside in the extract of hawthorn leaves, respectively.

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

PubMed

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

2012-01-01

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

Roselle Polyphenols Exert Potent Negative Inotropic Effects via Modulation of Intracellular Calcium Regulatory Channels in Isolated Rat Heart.

PubMed

Lim, Yi-Cheng; Budin, Siti Balkis; Othman, Faizah; Latip, Jalifah; Zainalabidin, Satirah

2017-07-01

Roselle (Hibiscus sabdariffa Linn.) calyces have demonstrated propitious cardioprotective effects in animal and clinical studies; however, little is known about its action on cardiac mechanical function. This study was undertaken to investigate direct action of roselle polyphenols (RP) on cardiac function in Langendorff-perfused rat hearts. We utilized RP extract which consists of 12 flavonoids and seven phenolic acids (as shown by HPLC profiling) and has a safe concentration range between 125 and 500 μg/ml in this study. Direct perfusion of RP in concentration-dependent manner lowered systolic function of the heart as shown by lowered LVDP and dP/dt max , suggesting a negative inotropic effect. RP also reduced heart rate (negative chronotropic action) while simultaneously increasing maximal velocity of relaxation (positive lusitropic action). Conversely, RP perfusion increased coronary pressure, an indicator for improvement in coronary blood flow. Inotropic responses elicited by pharmacological agonists for L-type Ca 2+ channel [(±)-Bay K 8644], ryanodine receptor (4-chloro-m-cresol), β-adrenergic receptor (isoproterenol) and SERCA blocker (thapsigargin) were all abolished by RP. In conclusion, RP elicits negative inotropic, negative chronotropic and positive lusitropic responses by possibly modulating calcium entry, release and reuptake in the heart. Our findings have shown the potential use of RP as a therapeutic agent to treat conditions like arrhythmia.

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.

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

Recent trends in ultra-fast HPLC: new generation superficially porous silica columns.

PubMed

Ali, Imran; Al-Othman, Zeid A; Nagae, Norikaju; Gaitonde, Vinay D; Dutta, Kamlesh K

2012-12-01

New generation columns, i.e. packed with superficially porous silica particles are available as trade names with following manufacturers: Halo, Ascentis Express, Proshell 120, Kinetex, Accucore, Sunshell, and Nucleoshell. These provide ultra-fast HPLC separations for a variety of compounds with moderate sample loading capacity and low back pressure. Chemistries of these columns are C(8), C(18), RP-Amide, hydrophilic interaction liquid chromatography, penta fluorophenyl (PFP), F5, and RP-aqua. Normally, the silica gel particles are of 2.7 and 1.7 μm as total and inner solid core diameters, respectively, with 0.5-μm-thick of outer porous layer having 90 Å pore sizes and 150 m(2)/g surface area. This article describes these new generation columns with special emphasis on their textures and chemistries, separations, optimization, and comparison (inter and intra stationary phases). Besides, future perspectives have also been discussed. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates.

PubMed

Chen, Ning; Yang, Hongmei; Sun, Yi; Niu, Jun; Liu, Shuying

2012-12-01

Walnut proteins were hydrolyzed separately using three different proteases to obtain antioxidant peptides. The antioxidant activities of the hydrolysates were measured using 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. Among hydrolysates, pepsin hydrolysate obtained by 3h exhibited the highest antioxidant activities, which could also quench the hydroxyl radical, chelate ferrous ion, exhibit reducing power and inhibit the lipid peroxidation. Then, 3-h pepsin hydrolysates were purified sequentially by ultrafiltration, gel filtration and RP-HPLC. The sequence of the peptide with the highest antioxidative activity was identified to be Ala-Asp-Ala-Phe (423.23 Da) using RP-HPLC-ESI-MS, which was identified for the first time from walnut protein hydrolysates. Last, the inhibition of the peptide on lipid peroxidation was similar with that of reduced glutathione (GSH). These results indicate that the protein hydrolysates and/or its isolated peptides may be effectively used as food additives. Copyright © 2012 Elsevier Inc. All rights reserved.

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.

Improved HPLC method with the aid of chemometric strategy: determination of loxoprofen in pharmaceutical formulation.

PubMed

Venkatesan, P; Janardhanan, V Sree; Muralidharan, C; Valliappan, K

2012-06-01

Loxoprofen belongs to a class of Nonsteroidal anti-inflammatory drug acts by inhibiting isoforms of cyclo-oxygenase 1 and 2. In this study an improved RP-HPLC method was developed for the quantification of loxoprofen in pharmaceutical dosage form. For that purpose an experimental design approach was employed. Factors-independent variables (organic modifier, pH of the mobile phase and flow rate) were extracted from the preliminary study and as dependent variables three responses (loxoprofen retention factor, resolution between loxoprofen probenecid and retention time of probenecid) were selected. For the improvement of method development and optimization step, Derringer's desirability function was applied to simultaneously optimize the chosen three responses. The procedure allowed deduction of optimal conditions and the predicted optimum was acetonitrile: water (53:47, v/v), pH of the mobile phase adjusted at to 2.9 with ortho phosphoric acid. The separation was achieved in less than 4minutes. The method was applied in the quality control of commercial tablets. The method showed good agreement between the experimental data and predictive value throughout the studied parameter space. The optimized assay condition was validated according to International conference on harmonisation guidelines to confirm specificity, linearity, accuracy and precision.

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.

[Simultaneous separation and detection of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate by RP-HPLC and structure confirmation].

PubMed

Zhao, Yan-Yan; Liu, Li-Yan; Han, Yuan-Yuan; Li, Yue-Qiu; Wang, Yan; Shi, Min-Jian

2013-08-01

A simple, fast and sensitive analytical method for the simultaneous separation and detection of 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B by RP-HPLC and drug quality standard was established. The structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate have been confirmed. Reference European Pharmacopoeia EP7.0 version, British Pharmacopoeia 2012 version, National Drug Standards of China (WS 1-XG-2002), domestic and international interrelated literature were referred to select the composition of mobile phase. The experimental parameters including salt concentration, pH, addition quantities of organic solvent, column temperature and flow rate were optimized. Finally, the assay was conducted on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 0.01 mol x mL(-1) ammonium perchlorate (add ammonia to adjust the pH value to 8.2) -methanol (48 : 52) as mobile phase at the flow rate of 0.8 mL x min(-1), and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. The MS, NMR, UV and RP-HPLC were used to confirm the structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate. Under the optimized separation conditions, the calibration curves of 18 alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B showed good linearity within the concentration of 0.50-100 microg x mL(-1) (r = 0.999 9). The detection limits for 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B were 0.15, 0.10, 0.10, 0.15 microg x mL(-1) respectively. The method is sensitive, reproducible and the results are accurate and reliable. It can be used for chiral resolution of 18alpha-glycyrrhizinic acid, 18Pbeta-glycyrrhizinic acid, and detection content of principal component and related substances of raw material drug of ammonium glycyrrhizinate. It is concluded that the separation of principal component isomer of raw material drug of ammonium glycyrrhizinate and the validity of the substance's structure assignments of retention time being 1.2 in the European pharmacopoeia EP7.0 version, British pharmacopoeia 2012 version remains open to question. It may be of practical value for the quality control of raw material drug, preparation, and Chinese herbal medicine of ammonium glycyrrhizinate.

Further aspects of ochratoxin A-cation interactions: complex formation with zinc ions and a novel analytical application of ochratoxin A-magnesium interaction in the HPLC-FLD system.

PubMed

Poór, Miklós; Kuzma, Mónika; Matisz, Gergely; Li, Yin; Perjési, Pál; Kunsági-Máté, Sándor; Kőszegi, Tamás

2014-04-10

Ochratoxin A (OTA) is a mycotoxin produced by different Aspergillus and Penicillium species. Since its mechanism of action is not fully understood yet, it is important to gain further insight into different interactions of OTA at the molecular level. OTA is found worldwide in many foods and drinks. Moreover, it can also be detected in human and animal tissues and body fluids, as well. Therefore, the development of highly sensitive quantitative methods for the determination of OTA is of utmost importance. OTA most likely forms complexes with divalent cations, both in cells and body fluids. In the present study, the OTA-zinc interaction was investigated and compared to OTA-magnesium complex formation using fluorescence spectroscopy and molecular modeling. Our results show that zinc(II) ion forms a two-fold higher stable complex with OTA than magnesium(II) ion. In addition, based on the enhanced fluorescence emission of OTA in its magnesium-bound form, a novel RP-HPLC-fluorescence detector (FLD) method was also established. Our results highlight that the application of magnesium chloride in alkaline eluents results in an approximately two-fold increase in sensitivity using the HPLC-FLD technique.

Bio-assay guided isolation of α-glucosidase inhibitory constituents from Hibiscus mutabilis leaves.

PubMed

Kumar, Deepak; Kumar, Hemanth; Vedasiromoni, J R; Pal, Bikas C

2012-01-01

The increasing demand for natural-product-based medicines and health-care products for the management of diabetes encouraged investigation of this commonly available Indian plant. To establish the anti-diabetic (α-glucosidase inhibitory) activity of H. mutabilis leaf extract, isolate and identify the constituents responsible for the activity, and validate a HPLC method for quantification of the active constituents for standardisation of the extract. The methanolic extract of leaves was partitioned between water, n-butanol and ethyl acetate. Bio-assay guided fractionation, based on inhibition of α-glucosidase, allowed isolation and identification of the active components. The active components were quantified using RP-HPLC-DAD validated for linearity, limit of detection, limit of quantification, precision, accuracy and robustness for this plant extract and the partitioned fractions. Ferulic acid and caffeic acid were identified as the α-glucosidase inhibitors present in H. mutabilis. They were partitioned into an ethyl acetate fraction. The HPLC-DAD calibration curve showed good linearity (r² > 0.99). For the recovery studies the %RSD was less than 2%. The interday and intraday variations were found to be less than 4% RSD for retention time and response. The identification of α-glucosidase inhibition activity in H. mutabilis supports further investigations into the possible use of the plant for the management of diabetes. The HPLC method validated for these extracts will be useful in future research with the plant. Copyright © 2011 John Wiley & Sons, Ltd.

Sorption of benzotriazoles under the conditions of RP HPLC

NASA Astrophysics Data System (ADS)

Dzhabieva, S. A.; Kurbatova, S. V.; Belousova, Z. P.

2016-02-01

The results of a chromatographic study of sorption of several benzotriazole derivatives on octadecyl silica gel were reported. The physicochemical and electronic parameters of benzotriazoles were calculated. The effect of the structure of analyte molecules and eluent composition on chromatographic retention of these substances was analyzed.

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

NASA Astrophysics Data System (ADS)

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

2012-08-01

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

Development of an HPLC method for determination of metabolic compounds in myocardial tissue.

PubMed

Volonté, M G; Yuln, G; Quiroga, P; Consolini, A E

2004-05-28

The determination of adenine nucleotides and creatine compounds has great importance in the characterization of ischemic myocardial injury and post-ischemic recovery. It was developed by an HPLC method for the quantification of creatine (Cr), creatine phosphate (CrP), hypoxanthine (HX), AMP, adenosine (Ad), ADP and ATP in isolated perfused rat hearts. The chromatographic conditions were: RP 18 column; mobile phase composed by KH(2)PO(4) (215 mM), tetrabutylammonium hydrogen sulfate (2.3mM), acetonitrile (4%) and KOH (1M 0.4%); flow rate 1 ml min(-1); temperature 25 degrees C; injection volume 20 microl; detection at 220 nm and height peak (HP) as the integration parameter. The method was validated by means of linearity and sensitivity evaluations, using calibration curves done with five concentration levels of each compound. The limits of quantification (LOQ) were also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. The purity of the peaks was established using enzymatic peak shift analysis with hexokinase and creatine kinase and also comparing HP at various wavelengths. Frozen hearts were homogenized with a mechanical homogenizer for 3 min at 0 degrees C added with 5 ml of 0.4N HCLO(4). After precipitation with 0.8 ml of 2M KOH the extract was shaked for 2 min and later centrifuged at 0 degrees C for 10 min. The supernatant was kept on ice, filtrated and injected into the HPLC system. The results show that the method for the determination of Cr, CrP, HX, AMP, Ad, ADP and ATP by HPLC here described has good linearity, LOQ, precision, specificity and is simple and rapid to perform.

Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair.

PubMed

Zhu, Kevin Y; Leung, K Wing; Ting, Annie K L; Wong, Zack C F; Ng, Winki Y Y; Choi, Roy C Y; Dong, Tina T X; Wang, Tiejie; Lau, David T W; Tsim, Karl W K

2012-03-01

A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.

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

PubMed

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

2012-01-13

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

Development and validation of a fast RP-HPLC method for determination of methotrexate entrapment efficiency in polymeric nanocapsules.

PubMed

Sartori, Tatiane; Seigi Murakami, Fabio; Pinheiro Cruz, Ariane; Machado de Campos, Angela

2008-07-01

A rapid and effective isocratic chromatographic procedure is successfully developed to determinate methotrexate (MTX) entrapment efficiency (EE) in polymeric nanocapsules using reversed-phase high-performance liquid chromatography. The method employed a RP-C(18) Shimadzu Shim-pack CLC-ODS (150 mm x 4.6 mm, 5 microm) column with mobile phase constituted by a mixture of water-acetonitrile-tetrahydrofuran (65:30:5 v/v/v; pH 3.0) at a flow rate of 0.8 mL/min. The eluate is monitored with a UV detector set at 313 nm. The parameters used in the validation process are: linearity, specificity, precision, accuracy, and limit of quantitation (LOQ). The linearity is evaluated by a calibration curve in the concentration range of 10-50 microg/mL and presented a correlation coefficient of 0.9998. The polymers (PLA or PLA-PEG), oil, and surfactants used in the nanocapsule formulation did not interfere with analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.72%. Results are satisfactory, and the method proved to be adequate for the determination of methotrexate in nanocapsules formulations.

Extraction and preliminary chemical characterization of the venom of the spider wasp Pepsis decorata (Hymenoptera: Pompilidae).

PubMed

Nolasco, Matheus; Biondi, Ilka; Pimenta, Daniel C; Branco, Alexsandro

2018-04-26

Arthropod venoms may be considered important sources of bioactive molecules; however, technical difficulties, such as venom extraction and homogeneity may impair the biochemical identification of new molecules. In this context, we have developed a method to maintain wasps in captivity that allows the collection of the venom, without use of chemical, mechanical or electrical stimuli. The crude venom was analyzed by RP-HPLC-ESIQ-ToF and 20 peptides were identified by de novo peptide sequencing, among them Eumenine-Mastoparan and a Ponericin-G2-simile peptide. Copyright © 2018. Published by Elsevier Ltd.

High efficiency liquid chromatography techniques coupled to mass spectrometry for the characterization of mate extracts.

PubMed

Dugo, Paola; Cacciola, Francesco; Donato, Paola; Jacques, Rosângela Assis; Caramão, Elina Bastos; Mondello, Luigi

2009-10-23

There is growing interest related to rapid screening and full characterization of the constituents of plants with medicinal properties; among these, "Mate" or Yerba Maté is a tea-like beverage widely consumed in South America, obtained from the dried leaves of Ilex paraguariensis. The high content in polyphenols accounts for in vitro and in vivo antioxidant activity of the extracts obtained from this plant; on the other hand, the high complexity of the samples extracted, depending on the method employed, may preclude complete resolution by conventional HPLC techniques. For this purpose, a comprehensive two-dimensional liquid chromatography (LC x LC) system, comprised of an RP-Amide first dimension and a partially porous octadecylsilica column in the second dimension, has been compared with a one-dimensional system. The latter was operated using a partially porous octadecylsilica column, with diode array (DAD) and electrospray/ion trap-time of flight (ESI/IT-TOF) detection for the most complex extracts. The employment of the hybrid mass spectrometer allowed unequivocal identification of several compounds in the mate extracts. Using LC x LC-MS(3), it was possible to discriminate between congeners of chlorogenic acids, along with monoacyl- and diacylchlorogenic acid esters.

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

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis.

PubMed

Juang, Lih-Jeng; Sheu, Shuenn-Jyi; Lin, Ta-Chen

2004-06-01

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.

Characterization of Jamaican agro-industrial wastes. Part II, fatty acid profiling using HPLC: precolumn derivatization with phenacyl bromide.

PubMed

Bailey-Shaw, Y A; Golden, K D; Pearson, A G M; Porter, R B R

2012-09-01

This paper describes the determination of fatty acid composition of coffee, citrus and rum distillery wastes using reversed-phase high-performance liquid chromatography (RP-HPLC). Lipid extracts of the waste samples are derivatized with phenacyl bromide and their phenacyl esters are separated on a C8 reversed-phase column by using continuous gradient elution with water and acetonitrile. The presence of saturated and unsaturated fatty acids in quantifiable amounts in the examined wastes, as well as the high percentage recoveries, are clear indications that these wastes have potential value as inexpensive sources of lipids. The HPLC procedures described here could be adopted for further analysis of materials of this nature.

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

PubMed

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

2008-07-11

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

Separation and Measurement of Plant Alkaloid Enantiomers by RP-HPLC Analysis of their Fmoc-Alanine Analogs

USDA-ARS?s Scientific Manuscript database

Plants often synthesize secondary metabolites that are enantiomers. Enantiomers can cause very different physiological responses. Ammodendrine (1) and anabasine (2) are teratogens that can cause congenital malformations in livestock and enantiomeric forms of each have been found in Lupinus spp. an...

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

USDA-ARS?s Scientific Manuscript database

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

Comparison of adsorption coefficient (K[sub oc]) for soils and HPLC retention factors of aromatic hydrocarbons using a chemically immobilized humic acid column in RP-HPLC

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

Szabo, G.; Bulman, R.A.

The determination of soil adsorption coefficients (K[sub oc]) via HPLC capacity factors (k[prime]) has been studied, including the effect of column type and mobile phase composition on the correlation between log K[sub oc] and log k[prime]. K[sub oc] values obtained by procedures other than HPLC correlate well with HPLC capacity factors determined on a chemically immobilized humic acid stationary phase, and it is suggested that this phase is a better model for the sorption onto soil or sediment than the octadecyl-, phenyl- and ethylsilica phases. By using log k[prime][sub w] a theoretical capacity factor has been obtained by extrapolation ofmore » the retention data in a binary solvent system to pure aqueous eluent. There is a better correlation between log K[sub oc] and log k[prime][sub w] than the correlation between log K[sub oc] and log k[prime].« less

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

PubMed

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

2005-09-15

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

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

Determination of piroxicam in pharmaceutical preparations by ultraviolet direct spectrophotometry, ultraviolet difference spectrophotometry and high performance liquid chromatography

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

Hackmann, E.R.M.; Santos Gianotto, E.A. dos; Miritello Santoro, M.I.R.

1993-02-01

Piroxicam in pharmaceutical preparations (capsules (C), tablets (T), oral drops (OD), suppositories (S) and simulated sample (SS)) was determined by UV direct spectrophotometry (UVS) at 333 nm, by UV difference spectrophotometry (UVDS) at 327 nm, and in C and T, by high performance liquid chromatography (HPLC). For UVS, Beer's law was obeyed in the range 3.0-8.5 [mu]g/mL. The coefficient of correlation (CC), absolute precision (AP) and relative precision (RP) were 0.9999, 0.02 and 0.33%, respectively. The coefficient of variation (CV) for C, T, OD, S and SS were 0.48%, 0.35%, 0.48% and 0.19%, respectively. The recovery average (RA) was 100.22%.more » For UVDS, Beer's law was obeyed in the range 5.0-15.0 [mu]g/mL. The CC, AP and RP were respectively 0.9999, 0.05 and 0.47%. The CV for C, T, OD, S and SS were 0.64%, 0.84%, 0.62%, 0.54% and 0.15%, respectively. The RA was 99.02%. In HPLC determination, a LiChrospher[reg sign] 100 RP-18 (5 [mu]m) in LiChroCART[reg sign] 125-4 column at ambient temperature with a mobile phase consisting of methanol: (buffer solution citric acid-dibasic sodium phosphate pH 3.0) (55:45) and UV detection at 254 nm enabled the determination of piroxicam in C and T. The response peak area versus concentration presented linearity in the range 10.0-100.0 [mu]g/mL. The CC, AP and RP were 0.9997, 0.45 and 0.90%, respectively. The CV was 0.51%-0.82% and the RA, 97.13%. 14 refs., 1 fig., 5 tabs.« less

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

PubMed Central

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

2016-01-01

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

Matrix solid-phase dispersion technique for the determination of a new antiallergic drug, bilastine, in rat faeces.

PubMed

Berrueta, L A; Fernández-Armentia, M; Bakkali, A; Gonzalo, A; Lucero, M L; Orjales, A

2001-08-25

A matrix solid-phase dispersion (MSPD) procedure for the isolation and HPLC determination of a new antiallergic agent, bilastine, in rat faeces is presented. The effect on recovery of empirical variables such as nature, pH and volume of the washing and elution liquids and nature of the adsorbent has been tested. The best recoveries were attained using an octadecylsilyl sorbent, 10 ml of a 0.1 M NaHCO3-Na2CO3 aqueous buffer of pH 10.0 as washing solvent and 10 ml of methanol as elution solvent. The extracts were evaporated to dryness and reconstituted in mobile phase before their injection into a HPLC system, equipped with a Discovery RP-amide C16 column and a fluorescence detector. The method allows one to reach recoveries of 95.0% within the concentration range 0.05-10 microg/g, with within-day repeatabilities of less than 5% and between-day repeatabilities of less than 9% within this range. This method has been successfully applied to the excretion studies of bilastine in the rat.

Analysis of 26 amino acids in human plasma by HPLC using AQC as derivatizing agent and its application in metabolic laboratory.

PubMed

Sharma, Gaurav; Attri, Savita Verma; Behra, Bijaylaxmi; Bhisikar, Swapnil; Kumar, Praveen; Tageja, Minni; Sharda, Sheetal; Singhi, Pratibha; Singhi, Sunit

2014-05-01

The present study reports the simultaneous analysis of 26 physiological amino acids in plasma along with total cysteine and homocysteine by high-performance liquid chromatography (HPLC) employing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as precolumn derivatizing reagent. Separations were carried out using Lichrospher 100 RP-18e (5 μm) 250 × 4.0 mm column connected to 100 CN 4.0 × 4.0 mm guard column on a quaternary HPLC system and run time was 53 min. Linearity of the peak areas for different concentrations ranging from 2.5 to 100 pmol/μL of individual amino acids was determined. A good linearity (R (2) > 0.998) was achieved in the standard mixture for each amino acid. Recovery of amino acids incorporated at the time of derivatization ranged from 95 to 106 %. Using this method we have established the normative data of amino acids in plasma, the profile being comparable to the range reported in literature and identified cases of classical homocystinuria, cobalamin defect/deficiency, non-ketotic hyperglycinemia, hyperprolinemia, ketotic hyperglycinemia, urea cycle defect and maple syrup urine disease.

Identification and optimization of parameters for the semi-continuous production of garbage enzyme from pre-consumer organic waste by green RP-HPLC method.

PubMed

Arun, C; Sivashanmugam, P

2015-10-01

Reuse and management of organic solid waste, reduce the environmental impact on human health and increase the economic status by generating valuable products for current and novel applications. Garbage enzyme is one such product produced from fermentation of organic solid waste and it can be used as liquid fertilizer, antimicrobial agents, treatment of domestic wastewater, municipal and industrial sludge treatment, etc. The semi-continuous production of garbage enzyme in large quantity at minimal time period and at lesser cost is needed to cater for treatment of increasing quantities of industrial waste activated sludge. This necessitates a parameter for monitoring and control for the scaling up of current process on semi-continuous basis. In the present study a RP-HPLC (Reversed Phase-High Performance Liquid Chromatography) method is used for quantification of standard organic acid at optimized condition 30°C column oven temperature, pH 2.7, and 0.7 ml/min flow rate of the mobile phase (potassium dihydrogen phosphate in water) at 50mM concentration. The garbage enzyme solution collected in 15, 30, 45, 60, 75 and 90 days were used as sample to determine the concentration of organic acid. Among these, 90th day sample showed the maximum concentration of 78.14 g/l of acetic acid in garbage enzyme, whereas other organic acids concentration got decreased when compare to the 15th day sample. This result confirms that the matured garbage enzyme contains a higher concentration of acetic acid and thus it can be used as a monitoring parameter for semi-continuous production of garbage enzyme in large scale. Copyright © 2015 Elsevier Ltd. All rights reserved.

Method optimization and quality assurance in speciation analysis using high performance liquid chromatography with detection by inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Larsen, Erik H.

1998-02-01

Achievement of optimum selectivity, sensitivity and robustness in speciation analysis using high performance liquid chromatography (HPLC) with inductively coupled mass spectrometry (ICP-MS) detection requires that each instrumental component is selected and optimized with a view to the ideal operating characteristics of the entire hyphenated system. An isocratic HPLC system, which employs an aqueous mobile phase with organic buffer constituents, is well suited for introduction into the ICP-MS because of the stability of the detector response and high degree of analyte sensitivity attained. Anion and cation exchange HPLC systems, which meet these requirements, were used for the seperation of selenium and arsenic species in crude extracts of biological samples. Furthermore, the signal-to-noise ratios obtained for these incompletely ionized elements in the argon ICP were further enhanced by a factor of four by continously introducing carbon as methanol via the mobile phase into the ICP. Sources of error in the HPLC system (column overload), in the sample introduction system (memory by organic solvents) and in the ICP-MS (spectroscopic interferences) and their prevention are also discussed. The optimized anion and cation exchange HPLC-ICP-MS systems were used for arsenic speciation in contaminated ground water and in an in-house shrimp reference sample. For the purpose of verification, HPLC coupled with tandem mass spectrometry with electrospray ionization was additionally used for arsenic speciation in the shrimp sample. With this analytical technique the HPLC retention time in combination with mass analysis of the molecular ions and their collision-induced fragments provide almost conclusive evidence of the identity of the analyte species. The speciation methods are validated by establishing a mass balance of the analytes in each fraction of the extraction procedure, by recovery of spikes and by employing and comparing independent techniques. The urgent need for reference materials certified for elemental species is stressed.

Determination of citrus limonoid glucosides by high performance liquid chromatography coupled to post-column reaction with Ehrlich’s Reagent

USDA-ARS?s Scientific Manuscript database

A method for the identification and quantification of citrus limonoid glucosides in juices based upon high performance liquid chromatography (HPLC) separation coupled to post-column reaction with Ehrlichs’s reagent has been developed. This method utilizes a phenyl stationary phase and an isocratic ...

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

Measurement of lumefantrine and its metabolite in plasma by high performance liquid chromatography with ultraviolet detection.

PubMed

Khalil, Insaf F; Abildrup, Ulla; Alifrangis, Lene H; Maiga, Deogratius; Alifrangis, Michael; Hoegberg, Lotte; Vestergaard, Lasse S; Persson, Ola Per-Eric; Nyagonde, Nyagonde; Lemnge, Martha M; Theander, Thor G; Bygbjerg, Ib C

2011-01-05

Artemether-lumefantrine (ARM-LUM) has in recent years become the first-line treatment for uncomplicated malaria in many Sub-Saharan African countries. Vigorous monitoring of the therapeutic efficacy of this treatment is needed. This requires high-quality studies following standard protocols; ideally, such studies should incorporate measurement of drug levels in the study patients to exclude the possibility that insufficient drug levels explain an observed treatment failure. Several methods for measuring lumefantrine (LUM) in plasma by HPLC are available; however, several of these methods have some limitations in terms of high costs and limited feasibility arising from large required sample volumes and demanding sample preparation. Therefore, we set out to develop a simpler reversed phase high performance liquid chromatography (RP-HPLC) method based on UV detection for simultaneous measurement of LUM and its major metabolite the desbutyl LUM (DL) in plasma. Halofantrine was used as an internal standard. Liquid-liquid extraction of samples was carried out using hexane-ethyl acetate (70:30, v/v). Chromatographic separation was carried out on a Synergi Polar-RP column (250 mm × 300 mm, particle size 4 μm). The mobile phase consisted of acetonitrile-0.1M ammonium acetate buffer adjusted to pH 4.9 (85:15%, v/v). Absorbance of the compounds was monitored at 335 nm using a reference wavelength of 360 nm. Absolute extraction recovery for LUM and DL were 88% and 90%, respectively. Inter- and intraday coefficients of variation for LUM and DL were ≤ 10%. The lower limits of quantification for LUM and DL were 12.5 and 6.5 ng/ml, respectively. After validation, the methodology was transferred to a local laboratory in Tanga Tanzania and samples from a small subset of malaria patients were analysed for LUM. The method appears to be applicable in settings with limited facilities. Copyright © 2010 Elsevier B.V. All rights reserved.

Inorganics in Organics: Tracking down the Intrinsic Equilibriums between Organic Molecules and Trace Elements in Oceanic Waters

NASA Astrophysics Data System (ADS)

Lechtenfeld, O. J.; Koch, B. P.; Kattner, G.

2010-12-01

Recent developments in analytical instrumentation enable to describe biogeochemical processes in oceanic waters on a molecular level. This is the prerequisite to integrate biological and geochemical parameters and to develop chemical cycles on a global perspective. The state-of-the-art Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) applications for dissolved organic matter (DOM) focus mainly on carbon, hydrogen, oxygen and nitrogen isotopes. Implementation of sulfur and especially phosphorus in the molecular formula assignment has been questionable because of ambiguous calculated elemental formulas. On the other hand, many compounds bearing these elements are well known to occur in the dissolved state as part of the permanent recycling processes (e.g. phospholipids, phosphonates) but analytics of dissolved organic phosphorus (DOP) and sulfur (DOS) are often hampered by the large inorganic P and S pools. Even less is known about complexation characteristics of the DOM moieties. Although electrochemical methods provide some information about trace metal speciation, the high amount of organic molecules and its insufficient description as chemical functional classes prevent the assignment of trace metals to ligand classes. Nevertheless, it is undoubtful that a varying but extensive amount of transition metals is bond in form of organic complexes. Hyphenation of reversed phase high performance liquid chromatography (RP-HPLC) with high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) is a valuable tool to study these metal-organic interactions in a qualitative and quantitative approach. We established a desolvation method that allows direct transfer of high organic solvent loads into the plasma. Thus, in combination with internal standardization and external calibration, the investigation of a broad polarity scale was possible. This approach overcomes previous restrictions to non-organic solvent separation techniques like size exclusion chromatography (SEC). We used solid phase extracted DOM (SPE-DOM) from Atlantic and Southern Ocean water samples to show that organic sulfur and phosphorus species can be separated via RP-HPLC and that the partitioning can be correlated to trace metal binding capabilities in the different fractions. A molecular level investigation of these fractions via FT-ICR-MS revealed further details of the complexation features and connects the polarity-based separation on a C18 column to O/C and H/C elemental ratios. With our study, we showed that biologically relevant transition metals (e.g. Fe, Ni, Cu) and uranium are intrinsic constituents of the DOM fractions. Moreover, a comparison between samples from different ecological provinces and diagenetic conditions was performed to highlight the benefits of this approach for future marine biogeochemical research.

Laboratory production of human prolactin from CHO cells adapted to serum-free suspension culture.

PubMed

Arthuso, Fernanda Santos; Bartolini, Paolo; Soares, Carlos Roberto Jorge

2012-08-01

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23 kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40 days, to grow in suspension in serum-free medium. High cell densities of up to 4.0 × 10(6) cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30 days. Two harvesting strategies were set up, 50 or 100 % of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5-7 μg hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.

Different spectrophotometric methods applied for the analysis of simeprevir in the presence of its oxidative degradation product: Acomparative study

NASA Astrophysics Data System (ADS)

Attia, Khalid A. M.; El-Abasawi, Nasr M.; El-Olemy, Ahmed; Serag, Ahmed

2018-02-01

Five simple spectrophotometric methods were developed for the determination of simeprevir in the presence of its oxidative degradation product namely, ratio difference, mean centering, derivative ratio using the Savitsky-Golay filters, second derivative and continuous wavelet transform. These methods are linear in the range of 2.5-40 μg/mL and validated according to the ICH guidelines. The obtained results of accuracy, repeatability and precision were found to be within the acceptable limits. The specificity of the proposed methods was tested using laboratory prepared mixtures and assessed by applying the standard addition technique. Furthermore, these methods were statistically comparable to RP-HPLC method and good results were obtained. So, they can be used for the routine analysis of simeprevir in quality-control laboratories.

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.

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

PubMed

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

2016-06-01

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

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

NASA Astrophysics Data System (ADS)

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

2014-06-01

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

Fingerprint chromatogram analysis of extracts from the leaves of Tripterygium wilfordii Hook. F. by high performance liquid chromatography.

PubMed

Li, Ke; Wang, Shudong

2005-05-01

A simple and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the study of fingerprint chromatograms of extracts from the leaves of Tripterygium wilfordii Hook. F. (TWHF) and for controlling the quality of the herb. HPLC separation of the extracts was performed on a Lichrospher RP-18 column and detected by ultraviolet absorbance at 210 nm. The column temperature was maintained at 35 degrees C. A mobile phase composed of acetonitrile:H2O in the ratio of 39:61 (v/v) was found to be most suitable for this separation at a flow rate of 0.8 mL/min with isocratic elution. Under the chromatographic conditions described, the peak profile of the 10 components collected within 35 min made up the fingerprint of the extracts from leaves of TWHF with universal features. The fingerprint chromatograms had a good stability, precision, and reproducibility. The similarity of the extracts from leaves of TWHF collected in summer and winter was studied with triptolide as a reference peak. The method is suitable for differentiation of extracts from the leaves of TWHF, and can be used as a quality control method for this herb.

Picosecond Control of Photogenerated Radical Pair Lifetimes Using a Stable Third Radical.

PubMed

Horwitz, Noah E; Phelan, Brian T; Nelson, Jordan N; Krzyaniak, Matthew D; Wasielewski, Michael R

2016-05-12

Photoinduced electron transfer reactions in organic donor-acceptor systems leading to long-lived radical ion pairs (RPs) have attracted broad interest for their potential applications in fields as diverse as solar energy conversion and spintronics. We present the photophysics and spin dynamics of an electron donor - electron acceptor - stable radical system consisting of a meta-phenylenediamine (mPD) donor covalently linked to a 4-aminonaphthalene-1,8-dicarboximide (ANI) electron-accepting chromophore as well as an α,γ-bisdiphenylene-β-phenylallyl (BDPA) stable radical. Selective photoexcitation of ANI produces the BDPA-mPD(+•)-ANI(-•) triradical in which the mPD(+•)-ANI(-•) RP spins are strongly exchange coupled. The presence of BDPA is found to greatly increase the RP intersystem crossing rate from the initially photogenerated BDPA-(1)(mPD(+•)-ANI(-•)) to BDPA-(3)(mPD(+•)-ANI(-•)), resulting in accelerated RP recombination via the triplet channel to produce BDPA-mPD-(3*)ANI as compared to a reference molecule lacking the BDPA radical. The RP recombination rates observed are much faster than those previously reported for weakly coupled triradical systems. Time-resolved EPR spectroscopy shows that this process is also associated with strong spin polarization of the stable radical. Overall, these results show that RP intersystem crossing rates can be strongly influenced by stable radicals nearby strongly coupled RP systems, making it possible to use a third spin to control RP lifetimes down to a picosecond time scale.

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection.

PubMed

Xie, Kaizhou; Jia, Longfei; Yao, Yilin; Xu, Dong; Chen, Shuqing; Xie, Xing; Pei, Yan; Bao, Wenbin; Dai, Guojun; Wang, Jinyu; Liu, Zongping

2011-08-01

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

High-performance liquid chromatographic method for the simultaneous determination of 24 fragrance allergens to study scented products.

PubMed

Villa, C; Gambaro, R; Mariani, E; Dorato, S

2007-07-27

The European legislation on cosmetic products has recently required the declaration of 26 compounds (24 volatile chemicals and 2 natural extracts) on the label of final products when exceeding a stipulated cut-off level. In this work a rapid reliable and specific RP-HPLC method coupled with diode array detector (DAD) has been developed for the simultaneous determination and quantification of the 24 volatile chemicals: amyl cinnamal, benzyl alcohol, cinnamyl alcohol, citral, eugenol, hydroxy-citronellal, isoeugenol, amylcinnamyl alcohol, benzyl salicylate, cinnamal, coumarin, geraniol, Lyral (hydroxy-methylpentylcyclohexene carboxaldehyde), anisyl alcohol, benzyl cinnamate, farnesol, Lilial (2-(4-tert-butylbenzyl)propionaldehyde) linalool, benzyl benzoate, citronellol, hexyl cinnamal, limonene, methylheptin carbonate, alpha-isomethyl ionone (3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one). The 24 analytes were appropriately separated over a running time of 40 min, on a C18 column using a simple gradient elution (acetonitrile/water) with flow rate from 0.7 to 1.0 ml/min and UV acquisition at 210, 254 and 280 nm. All calibration curves showed good linearity (r2>0.99) within test ranges. The method was successfully applied to the qualitative and quantitative determination of the potential allergens in four commercial scented products, with satisfactory accuracy and precision. The results indicated that this simple and efficient method can be used for quality assessment of complex matrices such us cosmetic scented products.

Three-step HPLC-ESI-MS/MS procedure for screening and identifying non-target flavonoid derivatives

NASA Astrophysics Data System (ADS)

Rak, Gábor; Fodor, Péter; Abrankó, László

2010-02-01

A three-step HPLC-ESI-MS/MS procedure is designed for screening and identification of non-target flavonoid derivatives of selected flavonoid aglycones. In this method the five commonly appearing aglycones (apigenin, luteolin, myricetin, naringenin and quercetin) were selected. The method consists of three individual mass spectrometric experiments of which the first two were implemented within a single chromatographic acquisition. The third step was carried out during a replicate chromatographic run using the same RP-HPLC conditions. The first step, a multiple reaction monitoring (MRM) scan of the aglycones was performed to define the number of derivatives relating to the selected aglycones. For this purpose the characteristic aglycone parts of the unknowns were used as specific tags of the molecules, which were generated as in-source fragments. Secondly, a full scan MS experiment is performed to identify the masses of the potential derivatives of the selected aglycones. Finally, the third step had the capability to confirm the supposed derivatives. The developed method was applied to a commercially available black currant juice to demonstrate its capability to detect and identify various flavonoid glycosides without any preliminary information about their presence in the sample. As a result 13 compounds were detected and identified in total. Namely, 3 different myricetin glycosides and the myricetin aglycone 2 luteolin glycosides plus the aglycone and 3 quercetin glycosides plus the aglycone could be identified from the tested black currant sample. In the case of apigenin and naringenin only the aglycones could be detected.

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.

Development and validation of micellar liquid chromatographic methods for the determination of antibiotics in different matrixes.

PubMed

Rambla-Alegre, Maria; Esteve-Romero, Josep; Carda-Broch, Samuel

2011-01-01

Antibiotics are the most important bioactive and chemotherapeutic compounds to be produced by microbiological synthesis, and they have proved their worth in a variety of fields, such as medicinal chemistry, agriculture, and the food industry. Interest in antibiotics has grown in parallel with an increasingly high degree of productivity in the field of analytical applications. Therefore, it is necessary to develop chromatographic procedures capable of determining various drugs simultaneously in the shortest possible time. Micellar liquid chromatography (MLC) is an RP-HPLC technique that offers advantages over conventional HPLC as far as sample preparation, selectivity, and versatility are concerned. Its main advantage is that samples can be injected directly into the chromatographic system with no previous preparation step. This paper mainly focuses on the results of the authors' own recent research and reports the chromatographic conditions for determination of various antibiotics (penicillins, quinolones, and sulfonamides) in different matrixes (pharmaceuticals, biological fluids, and food). The work of other authors on MLC-based antibiotic determination has been included.

Analysis of wax esters by silver-ion high-performance liquid chromatography-tandem mass spectrometry.

PubMed

Vrkoslav, Vladimír; Urbanová, Klára; Háková, Matina; Cvačka, Josef

2013-08-09

Wax esters (WEs), esters of long-chain fatty acids and long-chain alcohols, were analysed by Ag-HPLC/APCI-MS/MS. Two ChromSpher Lipids columns connected in series (a total length of 50cm) and hexane-2-propanol-acetonitrile mobile phases were used to achieve good separation of the molecular species. The chromatographic behaviour of WEs was studied under optimised conditions: retention increased with the number of double bonds and with the temperature (15-35°C); retention times were affected by the double-bond position, trans isomers eluted earlier than cis isomers, and the WEs were partially separated depending on the aliphatic-chain length. The WEs provided simple APCI spectra with [M+H](+) ions, the MS/MS spectra showed fragments, which allowed their identification. The method was applied for an analysis of the WE mixtures from jojoba oil and human hair and the results were compared with analogous data from an optimised RP-HPLC system. Copyright © 2013 Elsevier B.V. All rights reserved.

[Determination of aristolochic acid A in Radix Aristolociae and Herba Asari by RP-HPLC].

PubMed

Jiang, Xu; Wang, Zhi-min; You, Li-shuan; Dai, Li-ping; Ding, Guang-zhi

2004-05-01

To develop a HPLC method to determine the contents of aristolochic A in aristolochia debilis and Asarun spp.. Methanol-water-formic acid extracts were separated on an Alltech C18 column with methanol-water-acetic acid (68:32:1) as mobile phase. The flow rate was 1.0 mL x min(-1). UV detection wavelength was 390 nm. Column temperature was 35 degrees C. Aristolochic acid A was separated well. The relationship of injection amounts and peak areas was linear (r = 0.9999) the range of 0.12-1.89 microg x g(-1) and the recovery rate was 101.8% (n = 5). 11 samples of aristolochia debilis which bought from different areas in China were determined, and the contents of aristolochic acid A varied from 0.9 to 2 mg x g(-1). The difference of the contents in Asarum spp. was obvious. The highest is 0.35, and aristolochic acid A couldn't be detected in one sample.

[Study on the optimal extraction process of chaihushugan powder].

PubMed

Wang, Chun-yan; Zhang, Wan-ming; Zhang, Dan-shen; An, Fang; Tian, Jia-ming

2009-11-01

To study the optimal extraction process of chaihushugan powder by orthogonal design. RP-HPLC method was developed for the determination of saikosaponin a, ferulic acid, hesperidin and paeoniflorin in chaihushugan powder. The contents of the components and the extraction yield were selected as assessment indices. Four factors were study by L9 (3(4)), including the alcohol concentration, amount of alcohol, duration of extraction and times of extraction. The optimal extracting condition was 80% alcohol consumed as 10 times of crude herb amount, and extracting two times for 90 min each time. This study supplies theoretical base for the development of chaihushugan powder formulation.

Isolation and purification of two antioxidant peptides from alcalase hydrolysate of Arca subcrenata.

PubMed

Li, Ting-Fei; Ye, Bin; Song, Li-Yan; Yu, Rong-Min

2014-07-01

To investigate the constituents with antioxidant activities from alcalase hydrolysate of Arca subcrenata. The consecutive chromatographic methods were employed,including ion-exchange chromatography, gel filtration chromatography, and reverse phase high-performance liquid chromatography (RP-HPLC). The amino acid sequences of the purified antioxidant peptides were determined by automated Edman degradation. Under the guidance of the assay of scavenging free radicals, two peptides with antioxidant activities, termed as A-Bg1 and A-Bh, were isolated and purified from the alcalase hydrolysate of Arca subcrenata. Constituents from the hydrolysate of Arca subcrenata might be a new potential resource of antioxidants.

Determination of Natamycin in Turkish Yoghurt.

PubMed

Bilgic Alkaya, Dilek; Karalomlu, Ozlem

2016-01-01

This study was aimed at developing RP-HPLC method for determination of natamycin in Turkish yoghurt. Chromatographic separation was achieved on a C8 column (150 mm × 4.6 mm × 5 µm) with a mobile phase of methanol : water : acetic acid (12 : 8 : 1 v/v/v), at 1 mL/min flow rate with a detection of 303 nm. Natamycin was spiked into handmade yoghurt samples and used for validation. The method has been fully validated according to ISO 9233-2, 2007 (IDF 140-2, 2007). It was successfully applied to determination of 28 different Turkish yoghurt products. Findings dealing with the presence of natamycin in cheese samples are presented.

Stability studies of crude plant material of Bacopa monnieri and quantitative determination of bacopaside I and bacoside A by HPLC.

PubMed

Srivastava, Pratibha; Raut, Hema N; Puntambekar, Hemalata M; Desai, Anagha C

2012-01-01

Bacopa monnieri (BM) contains several dammarane-type triterpenoid saponins including bacopaside I and bacoside A. These bioactive compounds may be used as chemical markers for the quality control of different BM products used for promoting mental health and intellect. Quantification of bacopaside I and bacoside A in crude plant material of BM stored under the stability study conditions by HPLC. Crude BM samples were stored at long-term (LS; 30°C and 65% RH), accelerated (AS; 40°C and 75% RH) and real-time (RT) study conditions. HPLC of BM extracts was carried out using a LiChroCART Purospher® STAR RP-18 endcapped column along with a guard column, Purospher STAR RP 18e 4.0 4.0 mm 5 µm using a gradient of acetonitrile (A) and water containing 0.05% (v/v) orthophosphoric acid (B) at a flow rate 1.5 mL/min with UV detection at 205 nm. The linear range of bacopaside I and bacoside A was 0.2 to 1 mg/mL. With the help of a regression equation the coefficient of determination (r²) values for bacopaside I and bacoside A were found to be > 0.999 and > 0.994 respectively. Relative standard deviation (RSD) values were < 4.0 for all the concentrations injected (n = 3). The HPLC study indicated that BM samples kept under LS condition are rich in saponin contents as compared with the samples stored under AS and RT study conditions. The study indicated that BM plant material should be used fresh to obtain maximum concentration of active saponins or it should be stored under LS conditions up to 3 months. Copyright © 2012 John Wiley & Sons, Ltd.

Validated chromatographic and spectrophotometric methods for analysis of some amoebicide drugs in their combined pharmaceutical preparation.

PubMed

Abdelaleem, Eglal Adelhamid; Abdelwahab, Nada Sayed

2013-01-01

This work is concerned with development and validation of chromatographic and spectrophotometric methods for analysis of mebeverine HCl (MEH), diloxanide furoate (DF) and metronidazole (MET) in Dimetrol® tablets - spectrophotometric and RP-HPLC methods using UV detection. The developed spectrophotometric methods depend on determination of MEH and DF in the combined dosage form using the successive derivative ratio spectra method which depends on derivatization of the obtained ratio spectra in two steps using methanol as a solvent and measuring MEH at 226.4-232.2 nm (peak to peak) and DF at 260.6-264.8 nm (peak to peak). While MET concentrations were determined using first derivative (1D) at λ = 327 nm using the same solvent. The chromatographic method depends on HPLC separation on ODS column and elution with a mobile phase consisting water: methanol: triethylamine (25: 75: 0.5, by volume, orthophosphoric acid to pH =4). Pumping the mobile phase at 0.7 ml min-1 with UV at 230 nm. Factors affecting the developed methods were studied and optimized, moreover, they have been validated as per ICH guideline and the results demonstrated that the suggested methods are reproducible, reliable and can be applied for routine use with short time of analysis. Statistical analysis of the two developed methods with each other using F and student's-t tests showed no significant difference.

Content Variation of Catechin Markers, Total Phenolics and Caffeine in Green Tea Dietary Supplements.

PubMed

Abourashed, Ehab A; Roberson, Cindy Leslie A; Elsharkawy, Nancy

2016-01-01

Green tea (Camellia sinensis) preparations are among the top selling products in the United States dietary supplements market. Numerous manufacturers claim different levels of specific catechin markers in their products while many others use total phenolic concentration instead, or not at all. Limited quality control results have been published for green tea dietary supplements over the past seven years. Thus, the goal of this work was to correlate determined levels of phenolics, catechins, and caffeine with manufacturer label claims for selected dietary supplement products (26 total) purchased in the United States. The Folin-Ciocalteu (FC) method was used to determine the total phenolic content while reversed-phase (RP) HPLC was used to quantify the major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG). The developed HPLC method was validated for accuracy and precision. It utilized a C8 column with gradient elution of acetonitrile in 0.1% aqueous formic acid over 11 min total run time. Peak detection was performed at 280 nm. Caffeine was also included in the HPLC method as another non-phenolic alkaloid marker commonly found in green tea. Both methods showed a good correlation between the content of catechins and polyphenolic compounds in the selected products. The ranges of total catechins and polyphenol concentrations were 3.8-70.2% and 3.6-95.8%, respectively, while that of caffeine was 0.8-11.2%. The selected products displayed a wide range of marker levels. A lack of conformity in disclosing the actual levels of marker compounds was also noticed in the labeling of many products.

Development and application of a validated stability-indicating high-performance liquid chromatographic method using photodiode array detection for simultaneous determination of granisetron, methylparaben, propylparaben, sodium benzoate, and their main degradation products in oral pharmaceutical preparations.

PubMed

Hewala, Ismail; El-Fatatry, Hamed; Emam, Ehab; Mabrouk, Mokhtar

2011-01-01

A simple, rapid, and sensitive RP-HPLC method using photodiode array detection was developed and validated for the simultaneous determination of granisetron hydrochloride, 1-methyl-1H-indazole-3-carboxylic acid (the main degradation product of granisetron), sodium benzoate, methylparaben, propylparaben, and 4-hydroxybenzoic acid (the main degradation product of parabens) in granisetron oral drops and solutions. The separation of the compounds was achieved within 8 min on a SymmetryShield RP18 column (100 x 4.6 mm id, 3.5 microm particle size) using the mobile phase acetonitrile--0.05 M KH2PO4 buffered to pH 3 using H3PO4 (3+7, v/v). The photodiode array detector was used to test the purity of the peaks, and the chromatograms were extracted at 240 nm. The method was validated, and validation acceptance criteria were met in all cases. The robust method was successfully applied to the determination of granisetron and preservatives, as well as their degradation products in different batches of granisetron oral drops and solutions. The method proved to be sensitive for determination down to 0.04% (w/w) of granisetron degradation product relative to granisetron and 0.03% (w/w) 4-hydroxybenzoic acid relative to total parabens.

Ultra-High Performance Liquid Chromatography (UHPLC) Method for the Determination of Limonene in Sweet Orange (Citrus sinensis) Oil: Implications for Limonene Stability.

PubMed

Bernart, Matthew W

2015-01-01

The citrus-derived bioactive monoterpene limonene is an important industrial commodity and fragrance constituent. An RP isocratic elution C18 ultra-HPLC (UHPLC) method using a superficially porous stationary phase and photodiode array (PDA) detector has been developed for determining the limonene content of sweet orange (Citrus sinensis) oil. The method is fast with a cycle time of 1.2 min, linear, precise, accurate, specific, and stability indicating, and it satisfies U.S. Pharmacopeia suitability parameters. The method may be useful in its present form for limonene processing, or modified for research on more polar compounds of the terpenome. A forced-degradation experiment showed that limonene is degraded by heat in hydro-ethanolic solution. PDA detection facilitates classification of minor components of the essential oil, including β-myrcene.

Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique

NASA Astrophysics Data System (ADS)

Li, Ai Ping; Peng, Jing Dong; Zhou, MingQiong; Zhang, Jin

2016-02-01

A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH 5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λex = λem = 315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L- 1 and the limit of detection (S/N = 3) was 0.05 μg L- 1. The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed.

Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique.

PubMed

Li, Ai Ping; Peng, Jing Dong; Zhou, MingQiong; Zhang, Jin

2016-02-05

A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λ(ex)=λ(em)=315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L(-1) and the limit of detection (S/N=3) was 0.05 μg L(-1). The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

USDA-ARS?s Scientific Manuscript database

Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The f...

Qualitative and quantitative analyses of flavonoids in Spirodela polyrrhiza by high-performance liquid chromatography coupled with mass spectrometry.

PubMed

Qiao, Xue; He, Wen-ni; Xiang, Cheng; Han, Jian; Wu, Li-jun; Guo, De-an; Ye, Min

2011-01-01

Spirodela polyrrhiza (L.) Schleid. is a traditional Chinese herbal medicine for the treatment of influenza. Despite its wide use in Chinese medicine, no report on quality control of this herb is available so far. To establish qualitative and quantitative analytical methods by high-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) for the quality control of S. polyrrhiza. The methanol extract of S. polyrrhiza was analysed by HPLC/ESI-MS(n). Flavonoids were identified by comparing with reference standards or according to their MS(n) (n = 2-4) fragmentation behaviours. Based on LC/MS data, a standardised HPLC fingerprint was established by analysing 15 batches of commercial herbal samples. Furthermore, quantitative analysis was conducted by determining five major flavonoids, namely luteolin 8-C-glucoside, apigenin 8-C-glucoside, luteolin 7-O-glucoside, apigenin 7-O-glucoside and luteolin. A total of 18 flavonoids were identified by LC/MS, and 14 of them were reported from this herb for the first time. The HPLC fingerprints contained 10 common peaks, and could differentiate good quality batches from counterfeits. The total contents of five major flavonoids in S. polyrrhiza varied significantly from 4.28 to 19.87 mg/g. Qualitative LC/MS and quantitative HPLC analytical methods were established for the comprehensive quality control of S. polyrrhiza. Copyright © 2011 John Wiley & Sons, Ltd.

Identification of intact high molecular weight glutenin subunits from the wheat proteome using combined liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Lagrain, Bert; Brunnbauer, Markus; Rombouts, Ine; Koehler, Peter

2013-01-01

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.

Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

PubMed Central

Lagrain, Bert; Brunnbauer, Markus; Rombouts, Ine; Koehler, Peter

2013-01-01

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction. PMID:23520527

HPLC-ESI-MS/MS validated method for simultaneous quantification of zopiclone and its metabolites, N-desmethyl zopiclone and zopiclone-N-oxide in human plasma.

PubMed

Mistri, Hiren N; Jangid, Arvind G; Pudage, Ashutosh; Shrivastav, Pranav

2008-03-15

A simple, selective and sensitive isocratic HPLC method with triple quadrupole mass spectrometry detection has been developed and validated for simultaneous quantification of zopiclone and its metabolites in human plasma. The analytes were extracted using solid phase extraction, separated on Symmetry shield RP8 column (150 mm x 4.6 mm i.d., 3.5 microm particle size) and detected by tandem mass spectrometry with a turbo ion spray interface. Metaxalone was used as an internal standard. The method had a chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 0.5-150 ng/mL for both zopiclone and N-desmethyl zopiclone and 1-150 ng/mL for zopiclone-N-oxide. The intra-batch and inter-batch accuracy and precision evaluated at lower limit of quantification and quality control levels were within 89.5-109.1% and 3.0-14.7%, respectively, for all the analytes. The recoveries calculated for the analytes and internal standard were > or = 90% from spiked plasma samples. The validated method was successfully employed for a comparative bioavailability study after oral administration of 7.5 mg zopiclone (test and reference) to 16 healthy volunteers under fasted condition.

Anti-cholinesterase activity of the standardized extract of Syzygium aromaticum L.

PubMed

Dalai, Manoj K; Bhadra, Santanu; Chaudhary, Sushil K; Bandyopadhyay, Arun; Mukherjee, Pulok K

2014-04-01

Clove (Syzygium aromaticum) is a well-known culinary spice with strong aroma; contains a high amount of oil known as clove oil. The major phyto-constituent of the clove oil is eugenol. Clove and its oil possess various medicinal uses in indigenous medicine as an antiseptic, anti-oxidant, analgesic and neuroprotective properties. Thus, it draws much attention among researchers from pharmaceutical, food and cosmetic industries. The aim of the present study was to determine the anti-cholinesterase activity of the methanol extract of clove, its oil and eugenol. In vitro anti-cholinesterase activity of S. aromaticum was performed by a thin layer chromatography bio autography, 96 well micro titer plate and kinetic methods. Reverse phase high performance liquid chromatography (RP-HPLC) analysis was carried out to identify the biomarker compound eugenol in clove oil. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition study revealed that eugenol possess better inhibition of the enzymes than extract and oil. Clove extract, its oil and eugenol showed better inhibition of AChE than BChE. Polyphenolic compound eugenol was detected through RP-HPLC analysis. The content of eugenol in essential oil was found to be 0.5 μg/ml. Kinetic analysis of the cholinesterase inhibition study of the extract; clove oil and eugenol have shown that they possess mixed type of inhibition for AChE and non-competitive type of inhibition for BChE. These results might be useful in explaining the effect of clove as anti-cholinesterase agent for the management of cognitive ailments like Alzheimer's disease.

Application of total-reflection X-ray fluorescence spectrometry and high-performance liquid chromatography for the chemical characterization of xylem saps of nickel contaminated cucumber plants

NASA Astrophysics Data System (ADS)

Mihucz, Victor G.; Tatár, Eniko; Varga, Anita; Záray, Gyula; Cseh, Edit

2001-11-01

Total-reflection X-ray fluorescence (TXRF) spectrometry, reversed-phase (RP) and size-exclusion (SE) high-performance liquid chromatography (HPLC) methods were applied for the characterization of low-volume xylem sap of control and nickel contaminated cucumber plants growing in hydroponics containing urea as the sole nitrogen source. In these saps collected for 1 h, Ca, K, Fe, Mn, Ni, Zn, as well as malic, citric and fumaric acids were determined. The SEC measurements showed that macromolecules were not detectable in the samples. Nickel contamination had minimum impact on the organic acid transport, however, the transport of Zn, K and Fe was reduced by 50, 22 and 11%, respectively. This observation supports the results of our earlier experiments when nitrate ions were used as the sole nitrogen form. At the same time, the fresh root weight and the volume of the collected xylem sap increased by 36 and 85%, respectively. Therefore, nickel addition seemed to decrease the urea toxicity of the plants. By pooling the eluting fractions of the SEC column, which were 10-fold concentrated by freeze-drying, the series of the resulted samples were analyzed by the TXRF spectrometry and RP-HPLC. The three organic acids could be identified in only one of the fractions, which contained Fe and, in the case of the contaminated plants, Ni in detectable concentration. However, considerable parts of these two elements and Mn, as well as practically the total amounts of Cu may be transported by unidentified organic compounds in the xylem.

In vitro study on the antioxidant activity of a polyphenol-rich extract from Pinus brutia bark and its fractions.

PubMed

Cretu, Elena; Karonen, Maarit; Salminen, Juha-Pekka; Mircea, Cornelia; Trifan, Adriana; Charalambous, Christiana; Constantinou, Andreas I; Miron, Anca

2013-11-01

A crude hydromethanolic extract from Pinus brutia bark and its fractions (diethyl ether, ethyl acetate, n-butanol, and aqueous fractions) were studied with regard to their phenolic content and antioxidant activities. The total phenolics and proanthocyanidins in each extract were quantified by spectrophotometric methods; the polyphenolic profile was analyzed by RP-HPLC-DAD-ESI-MS. All extracts were tested with regard to their ability to scavenge free radicals (ABTS radical cation, superoxide and hydroxyl radicals), reduce ferric ions, and inhibit 15-lipoxygenase. P. brutia bark extracts had high phenolic contents (303.79±7.34-448.90±1.39 mg/g). Except diethyl ether extract, all other extracts contained proanthocyanidins ranging from 225.79±3.94 to 250.40±1.44 mg/g. Several polyphenols were identified by RP-HPLC-DAD-ESI-MS: taxifolin in diethyl ether extract, a taxifolin-O-hexoside, catechin, procyanidin dimers, and trimers in ethyl acetate extract. Except diethyl ether extract, all other extracts were effective scavengers of superoxide and hydroxyl radicals (EC₅₀=33.5±1.1-54.93±2.85 μg/mL and 0.47±0.06-0.6±0.0 mg/mL, respectively). All extracts had noticeable 15-lipoxygenase inhibitory effects (EC₅₀=22.47±0.75-34.43±2.25 μg/mL). We conclude that P. brutia bark is very promising for the dietary supplements industry due to its high free radical scavenging and 15-lipoxygenase inhibitory effects.

RP-HPLC/MS/MS Analysis of the Phenolic Compounds, Antioxidant and Antimicrobial Activities of Salvia L. Species

PubMed Central

Tohma, Hatice; Köksal, Ekrem; Kılıç, Ömer; Alan, Yusuf; Yılmaz, Mustafa Abdullah; Gülçin, İlhami; Bursal, Ercan; Alwasel, Saleh H.

2016-01-01

The identification and quantification of the phenolic contents of methanolic extracts of three Salvia L. species namely S. brachyantha (Bordz.) Pobed, S. aethiopis L., and S. microstegia Boiss. and Bal. were evaluated using reverse phase high performance liquid chromatography, UV adsorption, and mass spectrometry (RP-HPLC/MS). In order to determine the antioxidant capacity of these species, cupric ions (Cu2+) reducing assay (CUPRAC) and ferric ions (Fe3+) reducing assay (FRAP) were performed to screen the reducing capacity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was employed for evaluation of the radical scavenging activity for both solvents. In further investigation, the antimicrobial activities of Salvia species were tested using the disc diffusion method against three Gram-positive and four Gram-negative microbial species, as well as three fungi species. The results showed that there is a total of 18 detectable phenols, the most abundant of which was kaempferol in S. microstegia and rosmarinic acids in S. brachyantha and S aethiopis. The other major phenols were found to be apigenin, luteolin, p-coumaric acid, and chlorogenic acid. All species tested showed moderate and lower antioxidant activity than standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and ascorbic acid. The ethanolic extracts of Salvia species revealed a wide range of antimicrobial activity. S. brachyantha and S. microstegia showed the highest antimicrobial activities against B. subtilis, whereas S. aethiopis was more effective on Y. lipolytica. None of the extracts showed anti-fungal activity against S. cerevisiae. Thus these species could be valuable due to their bioactive compounds. PMID:27775656

Chemical composition, antioxidant and antimicrobial effects of Tunisian Limoniastrum guyonianum Durieu ex Boiss extracts.

PubMed

Bouzidi, Amel; Benzarti, Anissa; Arem, Amira El; Mahfoudhi, Adel; Hammami, Saoussen; Gorcii, Mohamed; Mastouri, Maha; Hellal, Ahmed Noureddine; Mighri, Zine

2016-07-01

In the present investigation, extracts obtained from L. guyonianum Durieu ex Boiss. aerial parts were used to evaluate total phenolic, flavonoid and tannin contents. A study of antioxidant activities of the prepared samples was carried out on the basis of 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2-2'-azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS+.) and ferric reducing antioxidant power (FRAP) assays. Moreover, the efficiency of methanolic, chloroformic and petroleum ether extracts and the deriving fractions from the methanolic extract was tested against human bacterial and fungal pathogenic strains using micro dilution method in 96 multiwell microtiter plate. Furthermore, leaves and stems extracts were subjected to RP-HPLC for phenolic compounds identification. Results showed that polyphenolic contents and antioxidant activities varied considerably as function of solvent polarity. Moreover, antiradical capacities against DPPH, ABTS(+.) and reducing power were maxima in methanol aerial parts extract which showed the highest polyphenol contents (134mg CE/g DW). The antimicrobial activities showed that methanolic, chloroformic and petroleum ether extracts were found to be most potent against Pseudomonas aeruginosa and Staphylococcus aureus with MIC values of 23 and 46μ.mL(-1), respectively. The fractions F(13) and F(16) have a great antifungal potential against Candida glabrata, Candida krusei and Candida parapsilesis (MIC=39μ.mL(-1)). The RP-HPLC analysis lead the identification of gallic, procatechuic and trans-cinnamic acids, methyl-4-hydroxybenzoate, n-propyl-3,4,5-trihydroxybenzoate, epicatechin, naringin and myricetin in L. guyonianum Durieu ex Boiss. leaves and stems extracts.

Coupling of on-column trypsin digestion-peptide mapping and principal component analysis for stability and biosimilarity assessment of recombinant human growth hormone.

PubMed

Shatat, Sara M; Eltanany, Basma M; Mohamed, Abeer A; Al-Ghobashy, Medhat A; Fathalla, Faten A; Abbas, Samah S

2018-01-01

Peptide mapping (PM) is a vital technique in biopharmaceutical industry. The fingerprint obtained helps to qualitatively confirm host stability as well as verify primary structure, purity and integrity of the target protein. Yet, in-solution digestion followed by tandem mass spectrometry is not suitable as a routine quality control test. It is time consuming and requires sophisticated, expensive instruments and highly skilled operators. In an attempt to enhance the fuctionality of PM and extract multi-dimentional data about various critical quality attributes and comparability of biosimilars, coupling of PM generated using immobilized trypsin followed by HPLC-UV to principal component analysis (PCA) is proposed. Recombinant human growth hormone (rhGH); was selected as a model biopharmaceutical since it is available in the market from different manufacturers and its PM is a well-established pharmacopoeial test. Samples of different rhGH biosimilars as well as degraded samples: deamidated and oxidized were subjected to trypsin digestion followed by RP-HPLC-UV analysis. PCA of the entire chromatograms of test and reference samples was then conducted. Comparison of the scores of samples and investigation of the loadings plots clearly indicated the applicability of PM-PCA for: i) identity testing, ii) biosimilarity assessment and iii) stability evaluation. Hotelling's T 2 and Q statistics were employed at 95% confidence level to measure the variation and to test the conformance of each sample to the PCA model, respectively. Coupling of PM to PCA provided a novel tool to identify peptide fragments responsible for variation between the test and reference samples as well as evaluation of the extent and relative significance of this variability. Transformation of conventional PM that is largely based on subjective visual comparison into an objective statiscally-guided analysis framework should provide a simple and economic tool to help both manufacturers and regulatory authorities in quality and biosimilarity assessment of biopharmaceuticals. Copyright © 2017 Elsevier B.V. All rights reserved.

Adenylate cyclase-stimulating, bone-resorbing and B TGF-like activities in canine apocrine cell adenocarcinoma of the anal sac.

PubMed

Weir, E C; Centrella, M; Matus, R E; Brooks, M L; Wu, T; Insogna, K L

1988-12-01

Canine apocrine cell adenocarcinoma of the anal sac (APO-AS) is a spontaneously occurring tumor that causes humorally mediated hypercalcemia in 90% of cases. To further define the nature of the responsible mediator in APO-AS, we examined tumor extracts from five APO-AS and four control tumors for adenylate cyclase-stimulating activity (ACSA). All extracts from APO-AS contained potent ACSA, whereas the four control tumors did not. The ACSA extracted from one tumor demonstrated a dose response curve parallel to that of synthetic bovinePTH-(1-34) and was 80% inhibited by Nle8,18,Tyr34 bPTH-(3-34)amide at a concentration of 10(-5) M. Extracts from three APO-AS and three control tumors were also examined for in vitro bone-resorbing activity (BRA). All APO-AS contained significant BRA, stimulating resorption 1.47 to 2.13-fold over basal, whereas none of the control tumors stimulated resorption. Purification of one extract using C18 reverse-phase high pressure liquid chromatography (RP-HPLC) resulted in a single sharp peak of ACSA which was 400-fold purified compared with the initial extract. This pool also contained significant bone-resorbing activity, whereas none of the adjacent pools did. Purification of a second extract using sequential CN and C18 RP-HPLC followed by size exclusion HPLC resulted in material that was at least 10,000-fold purified, and showed co-purification of ACSA and B TGF-like activity.

A fully automated and fast method using direct sample injection combined with fused-core column on-line SPE-HPLC for determination of ochratoxin A and citrinin in lager beers.

PubMed

Lhotská, Ivona; Šatínský, Dalibor; Havlíková, Lucie; Solich, Petr

2016-05-01

A new fast and sensitive method based on on-line solid-phase extraction on a fused-core precolumn coupled to liquid chromatography with fluorescence detection has been developed for ochratoxin A (OTA) and citrinin (CIT) determination in lager beer samples. Direct injection of 100 μL filtered beer samples into an on-line SPE-HPLC system enabled fast and effective sample extraction including separation in less than 6 min. Preconcentration of OTA and CIT from beer samples was performed on an Ascentis Express RP C18 guard column (5 × 4.6 mm), particle size 2.7 μm, with a mobile phase of methanol/0.5% aqueous acetic acid pH 2.8 (30:70, v/v) at a flow rate of 2.0 mL min(-1). The flow switch from extraction column to analytical column in back-flush mode was set at 2.0 min and the separation was performed on the fused-core column Ascentis Express Phenyl-Hexyl (100 × 4.6 mm), particle size 2.7 μm, with a mobile phase acetonitrile/0.5% aqueous acetic acid pH 2.8 in a gradient elution at a flow rate of 1.0 mL min(-1) and temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 335/497 nm. The accuracy of the method, defined as the mean recoveries of OTA and CIT from light and dark beer samples, was in the range 98.3-102.1%. The method showed high sensitivity owing to on-line preconcentration; LOQ values were found to be 10 and 20 ng L(-1) for OTA and CIT, respectively. The found values of OTA and CIT in all tested light, dark and wheat beer samples were significantly below the maximum tolerable limits (3.0 μg kg(-1) for OTA and 2000 μg kg(-1) for CIT) set by the European Union.

Detection and quantification of the selective EP4 receptor antagonist CJ-023423 (grapiprant) in canine plasma by HPLC with spectrofluorimetric detection.

PubMed

Vito, Virgina De; Saba, Alessandro; Lee, Hong-Ki; Owen, Helen; Poapolathep, Amnart; Giorgi, Mario

2016-01-25

Grapiprant, a novel pharmacologically active ingredient, acts as a selective EP4 receptor antagonist whose physiological ligand is prostaglandin E2 (PGE2). It is currently under development for use in humans and dogs for the control of pain and inflammation associated with osteoarthritis. The aim of the present study was to develop an easy and sensitive method to quantify grapiprant in canine plasma and to apply the method in a canine patient. Several parameters, both in the extraction and detection method were evaluated. The final mobile phase consisted of ACN:AcONH4 (20 mM) solution, pH 4 (70:30, v/v) at a flow rate of 1 mL/min. The elution of grapiprant and IS (metoclopramide) was carried out in isocratic mode through a Synergi Polar-RP 80A analytical column (150 mm × 4.6 mm). The best excitation and emission wavelengths were 320 and 365 nm, respectively. Grapiprant was extracted from the plasma using CHCl3, which gave a recovery of 88.1 ± 10.22% and a lower limit of quantification (LLOQ) of 10 ng/mL. The method was validated in terms of linearity, limit of detection (LOD), LLOQ, selectivity, accuracy and precision, extraction recovery, stability, and inter-laboratory cross validation, according to international guidelines. The chromatographic runs were specific with no interfering peaks at the retention times of the analyte and IS, as confirmed by HPLC-MS experiments. In conclusion, this was a simple and effective method using HPLC-FL to detect grapiprant in plasma, which may be useful for future pharmacokinetic studies. Copyright © 2015 Elsevier B.V. All rights reserved.

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

HPLC-MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute-and-shoot sample preparation methods, respectively.

PubMed

Liu, Junguo; Song, Jiuxue; Huang, Karen; Michel, Deborah; Fang, Jim

2018-05-01

A high-performance liquid chromatography tandem-mass spectrometry (HPLC-MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP-Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water-1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute-and-shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, cyanidin-3-xyloside and quercetin-3-galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples. Copyright © 2017 John Wiley & Sons, Ltd.

Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana).

PubMed

Grynbaum, Marc David; Hentschel, Petra; Putzbach, Karsten; Rehbein, Jens; Krucker, Manfred; Nicholson, Graeme; Albert, Klaus

2005-09-01

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: a case study of application to Passiflora incarnata L. extract separations.

PubMed

Pietrogrande, Maria Chiara; Dondi, Francesco; Ciogli, Alessia; Gasparrini, Francesco; Piccin, Antonella; Serafini, Mauro

2010-06-25

In this study, a comparative investigation was performed of HPLC Ascentis (2.7 microm particles) columns based on fused-core particle technology and Acquity (1.7 microm particles) columns requiring UPLC instruments, in comparison with Chromolith RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m(tot)), separation efficiency (sigma), peak capacity (n(c)), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis (2.7 microm particle) column and Acquity (1.7 microm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% - relative abundance of the deterministic component - and c(EACF) - similarity index computed on ACVF. Copyright 2010 Elsevier B.V. All rights reserved.

Fast Determination of Toxic Arsenic Species in Food Samples Using Narrow-bore High-Performance Liquid-Chromatography Inductively Coupled Plasma Mass Spectrometry.

PubMed

Terol, Amanda; Marcinkowska, Monika; Ardini, Francisco; Grotti, Marco

2016-01-01

A new method for the speciation analysis of arsenic in food using narrow-bore high-performance liquid-chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) has been developed. Fast separation of arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid was carried out in 7 min using an anion-exchange narrow-bore Nucleosil 100 SB column and 12 mM ammonium dihydrogen phosphate of pH 5.2 as the mobile phase, at a flow rate of 0.3 mL min(-1). A PFA-ST micronebulizer jointed to a cyclonic spray chamber was used for HPLC-ICP-MS coupling. Compared with standard-bore HPLC-ICP-MS, the new method has provided higher sensitivity, reduced mobile-phase consumption, a lower matrix plasma load and a shorter analysis time. The achieved instrumental limits of detection were in the 0.3 - 0.4 ng As mL(-1) range, and the precision was better than 3%. The arsenic compounds were efficiently (>80%) extracted from various food samples using a 1:5 methanol/water solution, with additional ultrasonic treatment for rice products. The applicability of this method was demonstrated by the analysis of several samples, such as seafood (fish, mussels, shrimps, edible algae) and rice-based products (Jasmine and Arborio rice, spaghetti, flour, crackers), including three certified reference materials.

LC-MS and MS/MS in the analysis of recombinant proteins

NASA Astrophysics Data System (ADS)

Coulot, M.; Domon, B.; Grossenbacher, H.; Guenat, C.; Maerki, W.; Müller, D. R.; Richter, W. J.

1993-03-01

Applicability and performance of electrospray ionization mass spectrometry (ESIMS) is demonstrated for protein analysis. ESIMS is applied in conjunction with on-line HPLC (LC-ESlMS) and direct tandem mass spectrometry (positive and negative ion mode ESlMS/MS) to the structural characterization of a recombinant protein (r-hirudin variant 1) and a congener phosphorylated at threonine 45 (RP-1).

Recursion equations in predicting band width under gradient elution.

PubMed

Liang, Heng; Liu, Ying

2004-06-18

The evolution of solute zone under gradient elution is a typical problem of non-linear continuity equation since the local diffusion coefficient and local migration velocity of the mass cells of solute zones are the functions of position and time due to space- and time-variable mobile phase composition. In this paper, based on the mesoscopic approaches (Lagrangian description, the continuity theory and the local equilibrium assumption), the evolution of solute zones in space- and time-dependent fields is described by the iterative addition of local probability density of the mass cells of solute zones. Furthermore, on macroscopic levels, the recursion equations have been proposed to simulate zone migration and spreading in reversed-phase high-performance liquid chromatography (RP-HPLC) through directly relating local retention factor and local diffusion coefficient to local mobile phase concentration. This new approach differs entirely from the traditional theories on plate concept with Eulerian description, since band width recursion equation is actually the accumulation of local diffusion coefficients of solute zones to discrete-time slices. Recursion equations and literature equations were used in dealing with same experimental data in RP-HPLC, and the comparison results show that the recursion equations can accurately predict band width under gradient elution.

Progressing the analysis of Improvised Explosive Devices: Comparative study for trace detection of explosive residues in handprints by Raman spectroscopy and liquid chromatography.

PubMed

Zapata, Félix; de la Ossa, Mª Ángeles Fernández; Gilchrist, Elizabeth; Barron, Leon; García-Ruiz, Carmen

2016-12-01

Concerning the dreadful global threat of terrorist attacks, the detection of explosive residues in biological traces and marks is a current need in both forensics and homeland security. This study examines the potential of Raman microscopy in comparison to liquid chromatography (ion chromatography (IC) and reversed-phase high performance liquid chromatography (RP-HPLC)) to detect, identify and quantify residues in human handmarks of explosives and energetic salts commonly used to manufacture Improvised Explosive Devices (IEDs) including dynamite, ammonium nitrate, single- and double-smokeless gunpowders and black powder. Dynamite, ammonium nitrate and black powder were detected through the identification of the energetic salts by Raman spectroscopy, their respective anions by IC, and organic components by RP-HPLC. Smokeless gunpowders were not detected, either by Raman spectroscopy or the two liquid chromatography techniques. Several aspects of handprint collection, sample treatment and a critical comparison of the identification of compounds by both techniques are discussed. Raman microscopy and liquid chromatography were shown to be complementary to one another offering more comprehensive information for trace explosives analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Lepeophtheirus salmonis: characterization of prostaglandin E(2) in secretory products of the salmon louse by RP-HPLC and mass spectrometry.

PubMed

Fast, M D; Ross, N W; Craft, C A; Locke, S J; MacKinnon, S L; Johnson, S C

2004-01-01

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.

Effect of loop structure of bovine lactoferricin on apoptosis in Jurkat cells.

PubMed

Zhang, Tie-nan; Yang, Wei; Liu, Ning

2010-06-01

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces apoptosis in Jurkat cells. However less is known about the influence of this kind of apoptosis on the intra-cellular ceramide metabolism and the structure-function relationship between the loop structure of LfcinB and its action of inducing apoptosis in Jurkat cells. In the present study, the artificially synthesized LfcinB and LfcinB-derived peptide (Cys 19 residue in LfcinB was replaced by Ala) was added in Jurkat cells, the nucleolus shape was observed by fluorescent microscopy, the ceramide concentration in Jurkat cells was determined by reversed phase high performance liquid chromatography (RP-HPLC). The results of MTT assay showed that LfcinB inhibited proliferation of Jurkat cells, and the inhibition rate was approximately 18.90%. Moreover, the inhibition rate of LfcinB together with MAPP was upto approximately 59.89%. The RP-HPLC result showed that LfcinB improved the ceramide level in Jurkat cells. By using the DNA fragmentation assay and observing the nucleolus shape, the result displayed deficiency of the loop structure could cause LfcinB losing the biological activity of inducing apoptosis in Jurkat cells.

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.

Detection of monohydroxylated polycyclic aromatic hydrocarbons in urine and particulate matter using LC separations coupled with integrated SPE and fluorescence detection or coupled with high-resolution time-of-flight mass spectrometry.

PubMed

Lintelmann, Jutta; Wu, Xiao; Kuhn, Evelyn; Ritter, Sebastian; Schmidt, Claudia; Zimmermann, Ralf

2018-05-01

A high-performance liquid chromatographic (HPLC) method with integrated solid-phase extraction for the determination of 1-hydroxypyrene and 1-, 2-, 3-, 4- and 9-hydroxyphenanthrene in urine was developed and validated. After enzymatic treatment and centrifugation of 500 μL urine, 100 μL of the sample was directly injected into the HPLC system. Integrated solid-phase extraction was performed on a selective, copper phthalocyanine modified packing material. Subsequent chromatographic separation was achieved on a pentafluorophenyl core-shell column using a methanol gradient. For quantification, time-programmed fluorescence detection was used. Matrix-dependent recoveries were between 94.8 and 102.4%, repeatability and reproducibility ranged from 2.2 to 17.9% and detection limits lay between 2.6 and 13.6 ng/L urine. A set of 16 samples from normally exposed adults was analyzed using this HPLC-fluorescence detection method. Results were comparable with those reported in other studies. The chromatographic separation of the method was transferred to an ultra-high-performance liquid chromatography pentafluorophenyl core-shell column and coupled to a high-resolution time-of-flight mass spectrometer (HR-TOF-MS). The resulting method was used to demonstrate the applicability of LC-HR-TOF-MS for simultaneous target and suspect screening of monohydroxylated polycyclic aromatic hydrocarbons in extracts of urine and particulate matter. Copyright © 2018 John Wiley & Sons, Ltd.

Reversed-phase high-performance liquid chromatography purification of methyl esters of C(16)-C(28) polyunsaturated fatty acids in microalgae, including octacosaoctaenoic acid [28:8(n-3)].

PubMed

Mansour, Maged P

2005-12-02

A preparative reversed-phase (RP; C(18)) high-performance liquid chromatography (HPLC) method with gradient elution using acetonitrile (MeCN)-chloroform (CHCl(3)) (or dichloromethane (DCM)) and evaporative light-scattering detection (ELSD) with automatic multiple injection and fraction collection was used to purify milligram quantities of microalgal polyunsaturated fatty acids (PUFA), separated as methyl esters (ME). PUFA-ME purified included methyl esters of docosahexaenoic acid (DHA; 22:6(n-3)), eicosapentaenoic acid (EPA; 20:5(n-3)) and the unusual very long-chain (C(28)) highly unsaturated fatty acid (VLC-HUFA), octacosaoctaenoic acid [28:8(n-3)(4, 7, 10, 13, 16, 19, 22, 25)] from the marine dinoflagellate Scrippsiella sp. CS-295/c. Other PUFA purified from various microalgae using this RP-HPLC method to greater than 95% purity included 16:3(n-4), 16:4(n-3), 16:4(n-1) and 18:5(n-3). The number of injections required was variable and depended on the abundance of the desired PUFA-ME, and resolution from closely eluting PUFA-ME, which determined the maximum loading. The purity of these fatty acids was determined by electron impact (EI) GC-MS and the chain length and location of double bonds was determined by EI GC-MS of 4,4-dimethyl oxazoline (DMOX) derivatives formed using a low temperature method. Advantages over silver-ion HPLC for purifying PUFA-ME is that separation occurs according to chain length as well as degree of unsaturation enabling separation of PUFA-ME with the same degree of unsaturation but different chain length (i.e. between 18:5(n-3) and 20:5(n-3)). In addition, PUFA-ME are not strongly adsorbed, but elute earlier than their more saturated corresponding FAME of the same chain length. This method is robust, simple, and requires only a short re-equilibration time. It is a useful tool for preparing milligram quantities of pure PUFA-ME for bioactive screening (as free fatty acids), although many multiple injections may be required for minor PUFA-ME. It also enabled dose-response and structure-activity studies to be carried out. It can be used for the enrichment of low levels of VLC-HUFA-ME to facilitate elucidation of their chemical structure and so is a useful adjunct to EI GC-MS of DMOX derivatives and other techniques such as NMR, which requires milligram quantities of purified compounds.

Mutant MiRP1 subunits modulate HERG K+ channel gating: a mechanism for pro-arrhythmia in long QT syndrome type 6

PubMed Central

Lu, Yu; Mahaut-Smith, Martyn P; Huang, Christopher L-H; Vandenberg, Jamie I

2003-01-01

Mutations in KCNE2, which encodes the minK-related protein 1 (MiRP1), are associated with an increased risk of arrhythmias; however, the underlying mechanisms are unknown. MiRP1 is thought to associate with many K+ channel α-subunits, including HERG K+ channels, which have a major role in suppressing arrhythmias initiated by premature beats. In this study we have investigated in chinese hamster ovary (CHO) cells at 37 °C the effects of co-expressing HERG K+ channels with either wild-type (WT) MiRP1 or one of three mutant MiRP1 subunits, T8A, Q9E and M54T. The most significant effects of MiRP1 subunits on HERG channels were a more negative steady-state activation for HERG + T8A MiRP1 and a more positive steady-state activation for HERG + M54T MiRP1 compared to either HERG + WT MiRP1 or HERG alone. All three mutants caused a significant slowing of deactivation at depolarised potentials. T8A MiRP1 also caused an acceleration of inactivation and recovery from inactivation compared to HERG + WT MiRP1. During ventricular action potential clamp experiments there was a significant decrease in current in the early phases of the action potential for HERG + WT MiRP1 channels compared to HERG alone. This effect was not as prominent for the mutant MiRP1 subunits. During premature action potential clamp protocols, the T8A and Q9E mutants, but not the M54T mutant, resulted in significantly larger current spikes during closely coupled premature beats, compared to HERG + WT MiRP1. At longer coupling intervals, all three mutants resulted in larger current spikes than HERG alone or HERG + WT MiRP1 channels. It is therefore possible that augmentation of HERG currents in the early diastolic period may be pro-arrhythmic. PMID:12923204

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).

Combining Laser Ablation/Liquid Phase Collection Surface Sampling and High-Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry

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

Ovchinnikova, Olga S; Kertesz, Vilmos; Van Berkel, Gary J

This paper describes the coupling of ambient pressure transmission geometry laser ablation with a liquid phase sample collection method for surface sampling and ionization with subsequent mass spectral analysis. A commercially available autosampler was adapted to produce a liquid droplet at the end of the syringe injection needle while in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collection was followed by either flow injection or a high performance liquid chromatography (HPLC) separation of the extracted components and detection with electrospray ionization mass spectrometry (ESI-MS). To illustrate the analytical utility of thismore » coupling, thin films of a commercial ink sample containing rhodamine 6G and of mixed isobaric rhodamine B and 6G dyes on glass microscope slides were analyzed. The flow injection and HPLC/ESI-MS analysis revealed successful laser ablation, capture and, with HPLC, the separation of the two compounds. The ablated circular area was about 70 m in diameter for these experiments. The spatial sampling resolution afforded by the laser ablation, as well as the ability to use sample processing methods like HPLC between the sample collection and ionization steps, makes this combined surface sampling/ionization technique a highly versatile analytical tool.« less

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

PubMed

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

2014-01-01

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

Impact of sampling parameters on the radical scavenging potential of olive (Olea europaea L.) leaves.

PubMed

Papoti, Vassiliki T; Tsimidou, Maria Z

2009-05-13

The impact of sampling parameters, that is, cultivar, leaf age, and sampling date, on the radical scavenging potential of olive leaf extracts was examined via the DPPH(*) and other assays. Total phenol content was estimated colorimetrically and by fluorometry, whereas phenol composition was assessed by RP-HPLC coupled with diode array, fluorometric, and MS detection systems. Oleuropein was not always the major leaf constituent. Considerable differences noted in individual phenol levels (hydroxytyrosol, oleuropein and other secoiridoids, verbascoside, and flavonoids) among samples were not reflected either in the total phenol content or in the radical scavenging potential of the extracts. It can be suggested that olive leaf is a robust source of radical scavengers throughout the year and that differentiation in the levels of individual components depends rather on sampling period than on cultivar or age. The latter does not present predictable regularity. Exploitation of all types of leaves expected in an olive tree shoot for the extraction of bioactive compounds is feasible.

Effects of Hydrocarbon-Based Grease on Rapid Prototype Material Used for Grease Retention Shrouds

NASA Technical Reports Server (NTRS)

Zakrajsek, Andrew J.; Valco, Daniel J.; Street, Kenneth W., Jr.

2010-01-01

Effects of hydrocarbon-based greases on specific rapid prototype (RP) materials used to fabricate grease retention shrouds (GRS) were explored in this study. Grease retention shrouds are being considered as a way to maintain adequate grease lubrication at the gear mesh in a prototype research transmission system. Due to their design and manufacturing flexibility, rapid prototype materials were chosen for the grease retention shrouds. In order to gain a better understanding of the short and long term effects grease pose on RP materials, research was conducted on the interaction of hydrocarbon-based grease with RP materials. The materials used in this study were durable polyamide (nylon), acrylonitrile butadiene styrene (ABS), and WaterClear 10120. Testing was conducted using Mobilgrease 28 and Syn-Tech 3913G grease (gear coupling grease). These greases were selected due to their regular use with mechanical components. To investigate the effect that grease has on RP materials, the following methods were used to obtain qualitative and quantitative data: Fourier transform infrared spectroscopy (FT-IR), interference profilometer measurements, digital camera imaging, physical shape measurement, and visual observations. To record the changes in the RP materials due to contact with the grease, data was taken before and after the grease application. Results showed that the WaterClear 10120 RP material provided the best resistance to grease penetration as compared to nylon and ABS RP materials. The manufacturing process, and thus resulting surface conditions of the RP material, played a key role in the grease penetration properties and resilience of these materials.

An HPLC method for the determination of selected amino acids in human embryo culture medium.

PubMed

Drábková, Petra; Andrlová, Lenka; Kanďár, Roman

2017-02-01

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra-cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at -80°C. Filtered medium samples were derivatized with ortho-phthalaldehyde (naphthalene-2,3-dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse-phase columns (LichroCART, Purospher STAR RP 18e or Ascentis Express C 18 ) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra-assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous determination of bioactive compounds in Piper nigrum L. and a species comparison study using HPLC-PDA.

PubMed

Rao, Vidadala Rama Subba; Raju, Sagi Satyanarayana; Sarma, Vanka Umamaheswara; Sabine, Fouriner; Babu, Kothapalli Hari; Babu, Katragadda Suresh; Rao, Janaswamy Madhusudana

2011-08-01

Piper nigrum L. is a traditional medicine widely used in India for illnesses such as constipation, diarrhoea, earache, gangrene, heart disease, hernia, hoarseness, indigestion, insect bites, insomnia, joint pain, liver problems, lung disease, oral abscesses, sunburn, tooth decay and toothaches. In this study, six bioactive compounds, namely piperine (1), pellitorine (2), guineensine (3), pipnoohine (4), trichostachine (5) and piperonal (6) were quantified in different extracts of P. nigrum L. and compared with those of P. longum L. and P. chaba Hunter. To evaluate the quality of P. nigrum, a simple, accurate and precise HPLC-PDA method was developed for the simultaneous determination of the above-mentioned six compounds. The separation was achieved by Phenomenex Luna RP C(18) column (150 × 4.6 mm, 5 µm, Phenomenex Inc, CA, USA) with a binary gradient solvent system of water-acetonitrile, at a flow rate of 1.0 mL min(-1) and detected at 210, 232, 262 and 343 nm. All six calibration curves showed good linearity (R (2) > 0.9966). The method was reproducible with intra- and inter-day variations of less than 2% and 5%, respectively. The results demonstrated that this method is simple, reliable and suitable for the quality control of these plants.

[Simultaneous determination of four common nonprotein nitrogen substances in urine by high performance liquid chromatography].

PubMed

Ma, Yuhua; Huang, Dongqun; Zhang, Rui; Xu, Shiru; Feng, Shun

2013-11-01

A high performance liquid chromatographic (HPLC) method was proposed to simultaneously determine four common nonprotein nitrogen substances, including creatine (Cr), creatinine (Cn), uric acid (Ua) and pseudouridine (Pu) in urine. After proteins being removed by acetone precipitation method, freeze drying and redissolving, the urine samples were analyzed by HPLC. Chromatographic separation was performed on a Waters RP18 Column (150 mm x 4.60 mm, 3.5 microm) in gradient elution mode using 10.0 mmol/L KH2PO4 solution (pH 4.78) and acetonitrile as mobile phases at a flow rate of 0.8 mL/min. The samples were detected at 220 nm. Rapid separation was achieved within 7 min. Under the optimized conditions, good linearities of four common nonprotein nitrogen substances were obtained in the range of 0.1-250 mg/L. The detection limits were 9.31 (Cr), 26.19 (Cn), 4.70 (Ua), an 6.30 (Pu) microg/L and the recoveries were in the range of 81%-111% with the relative standar deviations of 0.23%-2.78% (n = 3). The results demonstrate that this method is simple, rapid and accurate with good reproducibility, and can provide early diagnosis and preliminary judgment for type 2 diabetes mellitus (T2DM) patients with renal damage.

[Simultaneous determination of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid with HPLC].

PubMed

Zhang, Yu-Lin; Li, Yu-Ping

2013-10-01

To establish an HPLC method for determining the contents of scopolamine hydrobromide, atropine sulfate, ephedrine hydrochloride and pseudoephedrine hydrochloride in Zhichuanling oral liquid. Agela Durashell RP-C18 (4. 6 mm x250 mm, 5 microm) was adopted, with acetonitrile-sodium phosphate buffer solution (0. 07 mol L-1 sodium phosphate solution with 17.5 mmol L-1 sodium dodecylsulfate adjusted to pH 6.0 with phosphoric acid solution) (30:70) as the mobile phase. The flow rate was 0. 9 mL min -1, the detection wavelength was 207 nm, and the column temperature was 25 degree C. Scopolamine hydrobromide, atropine sulfate, ephedrine hlvdrochloride and pseudoephedrine hydrochloride showed good linear relations with peak areas within the concentration range of 0. 021 21-1. 060 5 pg (r =0. 999 3) , 0. 011 14-0. 557 microg (r = 0. 999 6) , 0. 200 56-10. 028 microg (r =0. 999 7) and 0.070 33-3. 516 5 gg (r =0. 999 6), respectively, with the average recoveries of 101.9% , 99. 80%, 100. 3%, 100. 2% (n=6). The method was so quick, simple, highly reproducible and specific that it could be used as one of quality control methods of Zhichuanling oral liquid.

Development and Validation of an RP-HPLC Method for the Determination of Vinpocetine and Folic Acid in the Presence of a Vinpocetine Alkaline Degradation Product in Bulk and in Capsule Form.

PubMed

Elkady, Ehab F; Tammam, Marwa H; Mohamed, Ayman A

2017-05-01

An alkaline-forced degradation hydrolytic product of vinpocetine was prepared and characterized by 1H-NMR, FTIR spectroscopy, and MS. Subsequently, a simple, selective, and validated reversed-phase HPLC method was developed for the simultaneous estimation of vinpocetine and folic acid in the presence of a vinpocetine alkaline degradation product. Chromatographic separation was achieved using an isocratic mobile phase consisting of acetonitrile-0.02 M KH2PO4 [containing 0.2% (v/v) triethylamine and adjusted to pH 6 with orthophosphoric acid; (80 + 20, v/v)] at a flow rate of 0.9 mL/min at ambient temperature on a Eurospher II C18 (250 × 4.6 mm, 5 μm) column, with UV detection at 280 nm for folic acid and 230 nm for vinpocetine and its alkaline hydrolytic product. Linearity, accuracy, and precision were found to be acceptable over a concentration range of 12.5-200 μg/mL for vinpocetine and 1-16 μg/mL for folic acid. The proposed method was successfully applied for the determination of both drugs and a vinpocetine hydrolysis product in a laboratory-prepared mixture and in a capsule containing both drugs.

Validated Spectrophotometric and RP-HPLC-DAD Methods for the Determination of Ursodeoxycholic Acid Based on Derivatization with 2-Nitrophenylhydrazine.

PubMed

El-Kafrawy, Dina S; Belal, Tarek S; Mahrous, Mohamed S; Abdel-Khalek, Magdi M; Abo-Gharam, Amira H

2017-05-01

This work describes the development, validation, and application of two simple, accurate, and reliable methods for the determination of ursodeoxycholic acid (UDCA) in bulk powder and in pharmaceutical dosage forms. The carboxylic acid group in UDCA was exploited for the development of these novel methods. Method 1 is the colorimetric determination of the drug based on its reaction with 2-nitrophenylhydrazine hydrochloride in the presence of a water-soluble carbodiimide coupler [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride] and pyridine to produce an acid hydrazide derivative, which ionizes to yield an intense violet color with maximum absorption at 553 nm. Method 2 uses reversed-phase HPLC with diode-array detection for the determination of UDCA after precolumn derivatization using the same reaction mentioned above. The acid hydrazide reaction product was separated using a Pinnacle DB C8 column (4.6 × 150 mm, 5 μm particle size) and a mobile phase consisting of 0.01 M acetate buffer (pH 3)-methanol-acetonitrile (30 + 30 + 40, v/v/v) isocratically pumped at a flow rate of 1 mL/min. Ibuprofen was used as the internal standard (IS). The peaks of the reaction product and IS were monitored at 400 nm. Different experimental parameters for both methods were carefully optimized. Analytical performance of the developed methods were statistically validated for linearity, range, precision, accuracy, specificity, robustness, LOD, and LOQ. Calibration curves showed good linear relationships for concentration ranges 32-192 and 60-600 μg/mL for methods 1 and 2, respectively. The proposed methods were successfully applied for the assay of UDCA in bulk form, capsules, and oral suspension with good accuracy and precision. Assay results were statistically compared with a reference pharmacopeial HPLC method, and no significant differences were observed between proposed and reference methods.

Identification and quantification of flavonoids and chromes in Baeckea frutescens by using HPLC coupled with diode-array detection and quadruple time-of-flight mass spectrometry.

PubMed

Jia, Bei-Xi; Huangfu, Qian-Qian; Ren, Feng-Xiao; Jia, Lu; Zhang, Yan-Bing; Liu, Hong-Min; Yang, Jie; Wang, Qiang

2015-01-01

This article marks the first report on high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and quadruple time-of-flight mass spectrometry (Q-TOF/MS) for the identification and quantification of main bioactive constituents in Baeckea frutescens. In total, 24 compounds were identified or tentatively characterised based on their retention behaviours, UV profiles and MS fragment information. Furthermore, a validated method with good linearity, sensitivity, precision, stability, repeatability and accuracy was successfully applied for simultaneous determination of five flavonoids and one chromone in different plant parts of B. frutescens collected at different harvest times, and their dynamic contents revealed the appropriate harvest times. The established HPLC-DAD-Q-TOF/MS using multi-bioactive markers was proved to be a validated strategy for the quality evaluation on both raw materials and related products of B. frutescens.

Novel HPLC Analysis of Hydrocortisone in Conventional and Controlled-Release Pharmaceutical Preparations.

PubMed

Adi-Dako, Ofosua; Oppong Bekoe, Samuel; Ofori-Kwakye, Kwabena; Appiah, Enoch; Peprah, Paul

2017-01-01

An isocratic sensitive and precise reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination and quantification of hydrocortisone in controlled-release and conventional (tablets and injections) pharmaceutical preparations. Chromatographic separation was achieved on an ODS (C18), 5  μ m, 4.6 × 150 mm, with an isocratic elution using a freshly prepared mobile phase of composition methanol : water : acetic acid (60 : 30 : 10, v/v/v) at a flow rate of 1.0 ml/min. The detection of the drug was successfully achieved at a wavelength of 254 nm. The retention time obtained for the drug was 2.26 min. The proposed method produced linear detectable responses in the concentration range of 0.02 to 0.4 mg/ml of hydrocortisone. High recoveries of 98-101% were attained at concentration levels of 80%, 100%, and 120%. The intraday and interday precision (RSD) were 0.19-0.55% and 0.33-0.71%, respectively. A comparison of hydrocortisone analyses data from the developed method and the official USP method showed no significant difference ( p > 0.05) at a 95% confidence interval. The method was successfully applied to the determination and quantification of hydrocortisone in six controlled-release and fifteen conventional release pharmaceutical preparations.

Novel HPLC Analysis of Hydrocortisone in Conventional and Controlled-Release Pharmaceutical Preparations

PubMed Central

Oppong Bekoe, Samuel; Appiah, Enoch; Peprah, Paul

2017-01-01

An isocratic sensitive and precise reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination and quantification of hydrocortisone in controlled-release and conventional (tablets and injections) pharmaceutical preparations. Chromatographic separation was achieved on an ODS (C18), 5 μm, 4.6 × 150 mm, with an isocratic elution using a freshly prepared mobile phase of composition methanol : water : acetic acid (60 : 30 : 10, v/v/v) at a flow rate of 1.0 ml/min. The detection of the drug was successfully achieved at a wavelength of 254 nm. The retention time obtained for the drug was 2.26 min. The proposed method produced linear detectable responses in the concentration range of 0.02 to 0.4 mg/ml of hydrocortisone. High recoveries of 98–101% were attained at concentration levels of 80%, 100%, and 120%. The intraday and interday precision (RSD) were 0.19–0.55% and 0.33–0.71%, respectively. A comparison of hydrocortisone analyses data from the developed method and the official USP method showed no significant difference (p > 0.05) at a 95% confidence interval. The method was successfully applied to the determination and quantification of hydrocortisone in six controlled-release and fifteen conventional release pharmaceutical preparations. PMID:28660092

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

PubMed

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

2017-01-01

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

Simultaneous determination of PPCPs, EDCs, and artificial sweeteners in environmental water samples using a single-step SPE coupled with HPLC-MS/MS and isotope dilution.

PubMed

Tran, Ngoc Han; Hu, Jiangyong; Ong, Say Leong

2013-09-15

A high-throughput method for the simultaneous determination of 24 pharmaceuticals and personal care products (PPCPs), endocrine disrupting chemicals (EDCs) and artificial sweeteners (ASs) was developed. The method was based on a single-step solid phase extraction (SPE) coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and isotope dilution. In this study, a single-step SPE procedure was optimized for simultaneous extraction of all target analytes. Good recoveries (≥ 70%) were observed for all target analytes when extraction was performed using Chromabond(®) HR-X (500 mg, 6 mL) cartridges under acidic condition (pH 2). HPLC-MS/MS parameters were optimized for the simultaneous analysis of 24 PPCPs, EDCs and ASs in a single injection. Quantification was performed by using 13 isotopically labeled internal standards (ILIS), which allows correcting efficiently the loss of the analytes during SPE procedure, matrix effects during HPLC-MS/MS and fluctuation in MS/MS signal intensity due to instrument. Method quantification limit (MQL) for most of the target analytes was below 10 ng/L in all water samples. The method was successfully applied for the simultaneous determination of PPCPs, EDCs and ASs in raw wastewater, surface water and groundwater samples collected in a local catchment area in Singapore. In conclusion, the developed method provided a valuable tool for investigating the occurrence, behavior, transport, and the fate of PPCPs, EDCs and ASs in the aquatic environment. Copyright © 2013 Elsevier B.V. All rights reserved.

Hybridation of different chiral separation techniques with ICP-MS detection for the separation and determination of selenomethionine enantiomers: chiral speciation of selenized yeast.

PubMed

Méndez, S P; González, E B; Sanz-Medel, A

2001-05-01

Enantioseparation and determination of selenomethionine enantiomers in selenized yeast was investigated using chiral separation techniques based on different principles, coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS) for selenium-specific detection. High performance liquid chromatography (HPLC) on a beta-cyclodestrin (beta-CD) column, cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC), gas chromatography (GC) on a Chirasil-L-Val column, and HPLC on a Chirobiotic T column have been investigated as the chiral separation techniques. For HPLC separation on the beta-CD column, and also for CD-MEKC, selenomethionine enantiomers were derivatized with NDA/CN(-). For chiral separation by GC, selenomethionine enantiomers were converted into their N-trifluoroacetyl (TFA)-O-alkyl esters. The developed hybridation methodologies are compared with respect to enantioselectivity, sensitivity and analysis time. The usefulness of the best-suited method [HPLC (Chirobiotic T)-ICP-MS] was demonstrated by its application to the successful chiral speciation of selenium and D-and L-selenomethionine content determination in selenized yeast. Copyright 2001 John Wiley & Sons, Ltd.

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.

Coupling solid-phase microextraction and high-performance liquid chromatography for direct and sensitive determination of halogenated fungicides in wine.

PubMed

Millán, S; Sampedro, M C; Unceta, N; Goicolea, M A; Rodríguez, E; Barrio, R J

2003-05-02

A solid-phase microextraction (SPME) method coupled to high-performance liquid chromatography with diode array detection (HPLC-DAD) for the analysis of six organochlorine fungicides (nuarimol, triadimenol, triadimefon, folpet, vinclozolin and penconazole) in wine was developed. For this purpose, polydimethylsiloxane-divinylbenzene-coated fibers were utilized and all factors affecting throughput, precision, and accuracy of the SPME method were investigated and optimized. These factors include: matrix influence, extraction and desorption time, percentage of ethanol, pH, salt effect and desorption mode. The performed analytical procedure showed detectability ranging from 4 to 27 microg l(-1) and precision from 2.4 to 14.2% (as intra-day relative standard deviation, RSD) and 4.7-25.7% (as inter-day RSD) depending on the fungicide. The results demonstrate the suitability of the SPME-HPLC-DAD method to analyze these organochlorine fungicides in red wine.

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.

Stability-indicating method for simultaneous estimation of olmesartan medoxomile, amlodipine besylate and hydrochlorothiazide by RP-HPLC in tablet dosage form.

PubMed

Jain, P S; Patel, M K; Gorle, A P; Chaudhari, A J; Surana, S J

2012-09-01

A simple, specific, accurate and precise stability-indicating reversed-phase high-performance liquid chromatographic method was developed for simultaneous estimation of olmesartan medoxomile (OLME), amlodipine besylate (AMLO) and hydrochlorothiazide (HCTZ) in tablet dosage form. The method was developed using an RP C18 base deactivated silica column (250 × 4.6 mm, 5 µm) with a mobile phase consisting of triethylamine (pH 3.0) adjusted with orthophosphoric acid (A) and acetonitrile (B), with a timed gradient program of T/%B: 0/30, 7/70, 8/30, 10/30 with a flow rate of 1.4 mL/min. Ultraviolet detection was used at 236 nm. The retention times for OLME, AMLO and HCTZ were found to be 6.72, 4.28 and 2.30, respectively. The proposed method was validated for precision, accuracy, linearity, range, robustness, ruggedness and force degradation study. The calibration curves of OLME, AMLO and HCTZ were linear over the range of 50-150, 12.5-37.5 and 31-93 µg/mL, respectively. The method was found to be sensitive. The limits of detection of OLME, AMLO and HCTZ were determined 0.19, 0.16 and 0.22 µg/mL and limits of quantification of OLME, AMLO and HCTZ were determined 0.57, 0.49 and 0.66, respectively. Forced degradation study was performed according to International Conference on Harmonization guidelines.

Advances in HPLC-ICP-MS interface techniques for metal speciation

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

Hill, S.J.

The relentless demand for lower detection limits is increasingly coupled to the requirement for elemental speciation. This is particularly true in environmental and clinical fields where total levels are often insufficient for mobility and toxicity studies. This demand for both qualitative and quantitative data on the individual species present in complex samples has led to the development of various interfaces to couple some form of chromatography, usually gas chromatography (GC) or high performance liquid chromatography (HPLC) to an element specific detector. Today inductively coupled plasma-mass spectrometry is often employed since it offers excellent detection limits, element specific information (including isotopicmore » data) and the potential for multi-element studies. Ms presentation will concentrate on HPLC couplings although the advantages and disadvantages of both GC and HPLC couplings to ICP-MS will be discussed. Particular attention will be given to the optimization of both the chromatography and detection systems. Details will be presented of several successful HPLC interface designs and ways of facilitating high levels of a range of organic solvents (e.g. methanol and THF) in the HPLC mobile phase will be highlighted. The advantages of using a sheath gas and practical ways of achieving this will also be discussed. Finally the use of isotope dilution analysis in conjunction with HPLC-ICP-MS will be outlined. In all cases the impact of using the most appropriate approach will be demonstrated using both environmental and clinical samples.« less

Nonadiabatic dynamics of photo-induced proton-coupled electron transfer reactions via ring-polymer surface hopping

NASA Astrophysics Data System (ADS)

Shakib, Farnaz; Huo, Pengfei

Photo-induced proton-coupled electron transfer reactions (PCET) are at the heart of energy conversion reactions in photocatalysis. Here, we apply the recently developed ring-polymer surface-hopping (RPSH) approach to simulate the nonadiabatic dynamics of photo-induced PCET. The RPSH method incorporates ring-polymer (RP) quantization of the proton into the fewest-switches surface-hopping (FSSH) approach. Using two diabatic electronic states, corresponding to the electron donor and acceptor states, we model photo-induced PCET with the proton described by a classical isomorphism RP. From the RPSH method, we obtain numerical results that are comparable to those obtained when the proton is treated quantum mechanically. This accuracy stems from incorporating exact quantum statistics, such as proton tunnelling, into approximate quantum dynamics. Additionally, RPSH offers the numerical accuracy along with the computational efficiency. Namely, compared to the FSSH approach in vibronic representation, there is no need to calculate a massive number of vibronic states explicitly. This approach opens up the possibility to accurately and efficiently simulate photo-induced PCET with multiple transferring protons or electrons.

Simultaneous high-throughput determination of clenbuterol, ambroxol and bromhexine in pharmaceutical formulations by HPLC with potentiometric detection.

PubMed

Bazylak, Grzegorz; Nagels, Luc J

2003-08-08

Potentiometric detection of clenbuterol, ambroxol and bromhexine in marketed pharmaceuticals was described in six isocratic HPLC systems. The podant- and macrocyclic-type neutral ionophores, N,N,N',N'-tetracyclohexyl-oxybis(o-phenyleneoxy)diacetamide (TOPA) and hexakis(2,3,6-tri-O-octyl)-alpha-cyclodextrin (OCD), were applied in poly(vinyl)chloride (PVC)-based liquid membrane electrodes. Both types of neutral ionophores improve the sensitivity for all mentioned drugs when compared with a tetrakis(p-chlorophenyl)borate (BOR)-based electrode as well as with single wavelength UV detection. Detection limits (S/N=3) of 2.6 x 10(-10) mol l(-1) (injected concentration) for the highly hydrophobic bromhexine were achieved with the TOPA-based electrode and a cyano reversed-phase (RP)-HPLC with Uptisphere UP5CN-25QS silica column (250 x 4.6 mm i.d.) eluted with acetonitrile (AcN)-ethanol-perchloric acid (1.66 mM) (60:2:38, v/v/v) (pH* 2.45). Comparable result was obtained with OCD-based electrodes and an XTerra RP18 hybrid silica-polymer column eluted with AcN-phosphoric acid (20 mM) (25:75, v/v) (pH* 2.60). In the mobile phases containing 60-75% v/v AcN or methanol, stable and reproducible response of both types of neutral ionophore-based electrodes was observed for at least 3 days. The results of the validated procedure for reliable simultaneous determination of the drugs in fortified representative samples of pharmaceuticals were also presented.

Quantification of penicillin G during labor and delivery by capillary electrophoresis.

PubMed

Thomas, Andrea; Ukpoma, Omon K; Inman, Jennifer A; Kaul, Anil K; Beeson, James H; Roberts, Kenneth P

2008-04-24

In this study, a capillary electrophoresis (CE) method was developed as a means to measure levels of penicillin G (PCN G) in Group B Streptococcus (GBS) positive pregnant women during labor and delivery. Volunteers for this developmental study were administered five million units of PCN G at the onset of labor. Urine, blood, and amniotic fluid samples were collected during labor and post delivery. Samples were semi-purified by solid-phase extraction (SPE) using Waters tC18 SepPak 3cc cartridges with a sodium phosphate/methanol step gradient for elution. Capillary electrophoresis or reversed-phase high-performance liquid chromatography (RP-HPLC) with diode-array absorbance detection were used to separate the samples in less than 30 min. Quantification was accomplished by establishing a calibration curve with a linear dynamic range. The tC18 SPE methodology provided substantial sample clean-up with high recovery yields of PCN G ( approximately 90%). It was found that SPE was critical for maintaining the integrity of the separation column when using RP-HPLC, but was not necessary for sample analysis by CE where no stationary phase is present. Quantification results ranged from millimolar concentrations of PCN G in maternal urine to micromolar concentrations in amniotic fluid. Serum and cord blood levels of PCN G were below quantification limits, which is likely due to the prolonged delay in sample collection after antibiotic administration. These results show that CE can serve as a simple and effective means to characterize the pharmacokinetic distribution of PCN G from mother to unborn fetus during labor and delivery. It is anticipated that similar methodologies have the potential to provide a quick, simple, and cost-effective means of monitoring the clinical efficacy of PCN G and other drugs during pregnancy.

Quality Evaluation of Potentilla fruticosa L. by High Performance Liquid Chromatography Fingerprinting Associated with Chemometric Methods.

PubMed

Liu, Wei; Wang, Dongmei; Liu, Jianjun; Li, Dengwu; Yin, Dongxue

2016-01-01

The present study was performed to assess the quality of Potentilla fruticosa L. sampled from distinct regions of China using high performance liquid chromatography (HPLC) fingerprinting coupled with a suite of chemometric methods. For this quantitative analysis, the main active phytochemical compositions and the antioxidant activity in P. fruticosa were also investigated. Considering the high percentages and antioxidant activities of phytochemicals, P. fruticosa samples from Kangding, Sichuan were selected as the most valuable raw materials. Similarity analysis (SA) of HPLC fingerprints, hierarchical cluster analysis (HCA), principle component analysis (PCA), and discriminant analysis (DA) were further employed to provide accurate classification and quality estimates of P. fruticosa. Two principal components (PCs) were collected by PCA. PC1 separated samples from Kangding, Sichuan, capturing 57.64% of the variance, whereas PC2 contributed to further separation, capturing 18.97% of the variance. Two kinds of discriminant functions with a 100% discrimination ratio were constructed. The results strongly supported the conclusion that the eight samples from different regions were clustered into three major groups, corresponding with their morphological classification, for which HPLC analysis confirmed the considerable variation in phytochemical compositions and that P. fruticosa samples from Kangding, Sichuan were of high quality. The results of SA, HCA, PCA, and DA were in agreement and performed well for the quality assessment of P. fruticosa. Consequently, HPLC fingerprinting coupled with chemometric techniques provides a highly flexible and reliable method for the quality evaluation of traditional Chinese medicines.

Quality Evaluation of Potentilla fruticosa L. by High Performance Liquid Chromatography Fingerprinting Associated with Chemometric Methods

PubMed Central

Liu, Wei; Wang, Dongmei; Liu, Jianjun; Li, Dengwu; Yin, Dongxue

2016-01-01

The present study was performed to assess the quality of Potentilla fruticosa L. sampled from distinct regions of China using high performance liquid chromatography (HPLC) fingerprinting coupled with a suite of chemometric methods. For this quantitative analysis, the main active phytochemical compositions and the antioxidant activity in P. fruticosa were also investigated. Considering the high percentages and antioxidant activities of phytochemicals, P. fruticosa samples from Kangding, Sichuan were selected as the most valuable raw materials. Similarity analysis (SA) of HPLC fingerprints, hierarchical cluster analysis (HCA), principle component analysis (PCA), and discriminant analysis (DA) were further employed to provide accurate classification and quality estimates of P. fruticosa. Two principal components (PCs) were collected by PCA. PC1 separated samples from Kangding, Sichuan, capturing 57.64% of the variance, whereas PC2 contributed to further separation, capturing 18.97% of the variance. Two kinds of discriminant functions with a 100% discrimination ratio were constructed. The results strongly supported the conclusion that the eight samples from different regions were clustered into three major groups, corresponding with their morphological classification, for which HPLC analysis confirmed the considerable variation in phytochemical compositions and that P. fruticosa samples from Kangding, Sichuan were of high quality. The results of SA, HCA, PCA, and DA were in agreement and performed well for the quality assessment of P. fruticosa. Consequently, HPLC fingerprinting coupled with chemometric techniques provides a highly flexible and reliable method for the quality evaluation of traditional Chinese medicines. PMID:26890416

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.

A simple, sensitive and rapid isocratic reversed-phase high-performance liquid chromatography method for determination and stability study of curcumin in pharmaceutical samples

PubMed Central

Amanolahi, Farjad; Mohammadi, Ali; Kazemi Oskuee, Reza; Nassirli, Hooriyeh; Malaekeh-Nikouei, Bizhan

2017-01-01

Objective: This study was designed to develop and validate a new reversed-phase high-performance liquid chromatography (RP-HPLC) method based on Q2 (R1) International Conference on Harmonization (ICH) guideline for determination of curcumin in pharmaceutical samples. Materials and Methods: The HPLC instrument method was optimized with isocratic elution with acetonitrile: ammonium acetate (45:55, v/v, pH 3.5), C18 column (150 mm×4.6 mm×5 µm particle size) and a flow rate of 1 ml/min in ambient condition and total retention time of 17 min. The volume of injection was set at 20 µl and detection was recorded at 425 nm. The robustness of the method was examined by changing the mobile phase composition, mobile phase pH, and flow rate. Results: The method was validated with respect to precision, accuracy and linearity in a concentration range of 2-100 µg/ml. The limit of detection (LOD) and limit of quantification (LOQ) were 0.25 and 0.5 µg/ml, respectively. The percentage of recovery was 98.9 to 100.5 with relative standard deviation (RSD) < 0.638%. Conclusion: The method was found to be simple, sensitive and rapid for determination of curcumin in pharmaceutical samples and had enough sensitivity to detect degradation product of curcumin produced under photolysis and hydrolysis stress condition. PMID:29062806

Fourth-derivative synchronous spectrofluorimetry and HPLC with fluorescence detection as two analytical techniques for the simultaneous determination of itopride and domperidone.

PubMed

Ibrahim, Fawzia; Nasr, Jenny Jeehan

2016-02-01

Two simple, rapid and sensitive methods, namely, fourth-derivative synchronous spectrofluorimetry (method I) and HPLC with fluorescence detection (method II) were developed for the simultaneous analysis of a binary mixture of itopride HCl (ITP) and domperidone (DOM) without prior separation. The first method was based on measuring the fourth derivative of the synchronous fluorescence spectra of the two drugs at Δλ = 40 nm in methanol. The different experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully optimized. Chromatographic separation was performed in < 6.0 min using a RP C18 column (250 mm × 4.6 mm i.d., 5 µm particle size) with fluorescence detection at 344 nm after excitation at 285 nm. A mobile phase composed of a mixture of 0.02 M phosphate buffer with acetonitrile in a ratio of 55 : 45, pH 4.5, was used at a flow rate of 1 mL/min. Linearity ranges were found to be 0.1-2 µg/mL for ITP in both methods, whereas those for DOM were found to be 0.08-2 and 0.05-1.5 µg/mL in methods I and II, respectively. The proposed methods were successfully applied for the determination of the studied drugs in synthetic mixtures and laboratory-prepared tablets. Copyright © 2015 John Wiley & Sons, Ltd.

Qualitative and quantitative analysis of the saponins in Panax notoginseng leaves using ultra-performance liquid chromatography coupled with time-of-flight tandem mass spectrometry and high performance liquid chromatography coupled with UV detector.

PubMed

Liu, Fang; Ma, Ni; He, Chengwei; Hu, Yuanjia; Li, Peng; Chen, Meiwan; Su, Huanxing; Wan, Jian-Bo

2018-04-01

Panax notoginseng leaves (PNL) exhibit extensive activities, but few analytical methods have been established to exclusively determine the dammarane triterpene saponins in PNL. Ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/Q-TOF MS) and HPLC-UV methods were developed for the qualitative and quantitative analysis of ginsenosides in PNL, respectively. Extraction conditions, including solvents and extraction methods, were optimized, which showed that ginsenosides Rc and Rb3, the main components of PNL, are transformed to notoginsenosides Fe and Fd, respectively, in the presence of water, by removing a glucose residue from position C-3 via possible enzymatic hydrolysis. A total of 57 saponins were identified in the methanolic extract of PNL by UPLC/Q-TOF MS. Among them, 19 components were unambiguously characterized by their reference substances. Additionally, seven saponins of PNL-ginsenosides Rb1, Rc, Rb2, and Rb3, and notoginsenosides Fc, Fe, and Fd-were quantified using the HPLC-UV method after extraction with methanol. The separation of analytes, particularly the separation of notoginsenoside Fc and ginsenoside Rc, was achieved on a Zorbax ODS C8 column at a temperature of 35°C. This developed HPLC-UV method provides an adequate linearity ( r 2  > 0.999), repeatability (relative standard deviation, RSD < 2.98%), and inter- and intraday variations (RSD < 4.40%) with recovery (98.7-106.1%) of seven saponins concerned. This validated method was also conducted to determine seven components in 10 batches of PNL. These findings are beneficial to the quality control of PNL and its relevant products.

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

PubMed

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

1997-08-22

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

Detection of Maillard reaction products by a coupled HPLC-Fraction collector technique and FTIR characterization of Cu(II)-complexation with the isolated species

NASA Astrophysics Data System (ADS)

Ioannou, Aristos; Daskalakis, Vangelis; Varotsis, Constantinos

2017-08-01

The isolation of reaction products of asparagine with reducing sugars at alkaline pH and high temperature has been probed by a combination of high performance liquid chromatography (HPLC) coupled with a Fraction Collector. The UV-vis and FTIR spectra of the isolated Maillard reaction products showed structure-sensitive changes as depicted by deamination events and formation of asparagine-saccharide conjugates. The initial reaction species of the Asn-Gluc reaction were also characterized by Density Functional Theory (DFT) methods. Evidence for Cu (II) metal ion complexation with the Maillard reaction products is supported by UV-vis and FTIR spectroscopy.

Analysis of quaternary ammonium and phosphonium ionic liquids by reversed-phase high-performance liquid chromatography with charged aerosol detection and unified calibration.

PubMed

Stojanovic, Anja; Lämmerhofer, Michael; Kogelnig, Daniel; Schiesel, Simone; Sturm, Martin; Galanski, Markus; Krachler, Regina; Keppler, Bernhard K; Lindner, Wolfgang

2008-10-31

Several hydrophobic ionic liquids (ILs) based on long-chain aliphatic ammonium- and phosphonium cations and selected aromatic anions were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) employing trifluoroacetic acid as ion-pairing additive to the acetonitrile-containing mobile phase and adopting a step-gradient elution mode. The coupling of charged aerosol detection (CAD) for the non-chromophoric aliphatic cations with diode array detection (DAD) for the aromatic anions allowed their simultaneous analysis in a set of new ILs derived from either tricaprylmethylammonium chloride (Aliquat 336) and trihexyltetradecylphosphonium chloride as precursors. Aliquat 336 is a mix of ammonium cations with distinct aliphatic chain lengths. In the course of the studies it turned out that CAD generates an identical detection response for all the distinct aliphatic cations. Due to lack of single component standards of the individual Aliquat 336 cation species, a unified calibration function was established for the quantitative analysis of the quaternary ammonium cations of the ILs. The developed method was validated according to ICH guidelines, which confirmed the validity of the unified calibration. The application of the method revealed molar ratios of cation to anion close to 1 indicating a quantitative exchange of the chloride ions of the precursors by the various aromatic anions in the course of the synthesis of new ILs. Anomalies of CAD observed for the detection of some aromatic anions (thiosalicylate and benzoate) are discussed.

Comparative Assessment of the Effect of Hyper-glycosylation on the Pattern and Kinetics of Degradation of Darbepoetin Alfa using a Stability-Indicating Orthogonal Testing Protocol.

PubMed

Moenes, Eman M; Al-Ghobashy, Medhat A; Mohamed, Abeer A; Salem, Maissa Y

2018-01-01

Darbepoetin alfa (DA); hyper-glycosylated Erythropoietin alfa (EPO) is an essential treatment of anemia in patients with chronic kidney failure and cancer. In this study, DA and EPO were subjected to physicochemical stress factors that might be encountered during production, transport and storage (pH, temperature, agitation, repeated freeze-thaw and oxidation). An orthogonal stability-indicating assay protocol comprised of SE-HPLC, RP-HPLC, ELISA and SDS-PAGE was developed and validated to investigate the effect of further glycosylation of DA on the pattern and kinetics of degradation. Results showed a relatively higher stability and lower tendency to form high molecular weight aggregates in the case of DA when compared to EPO, under equivalent stress conditions. Dimers and aggregates were formed for both drugs across the whole pH range and following incubation at temperatures higher than 2-8°C or repeated freeze/thaw. The same observation was noted upon agitation of standard samples prepared in the formulation buffers at high speed and upon oxidation with hydrogen peroxide. The agreement between SE-HPLC, supported with spectral purity data and ELISA confirmed the specificity of both techniques for the intact drugs. Results of RP-HPLC and SDS-PAGE indicated that dimerization occurred through disulfide and bi-tyrosine covalent bonds in the case of pH and oxidation, respectively. It was evident that aggregation was significantly suppressed upon increasing the glycan size and under any of the studied stress factors loss of the glycan has not been observed. These observations supported with the slow kinetics of degradation confirmed the superiority of glyco-engineering over chemical pegylation to enhance the stability of EPO. Formation of such potentially immunogenic product-related impurities at all tested stress factors confirmed the need for orthogonal testing protocols to investigate the complex pattern of degradation of such sensitive products. Copyright © 2017 Elsevier B.V. All rights reserved.

Antioxidant capacity, phenolic acids and caffeine contents of some commercial coffees available on the Romanian market.

PubMed

Trandafir, Ion; Nour, Violeta; Ionica, Mira Elena

2013-03-01

In the present study a simple and highly sensitive RP-HPLC method has been established for simultaneous determination of chlorogenic acid, caffeic acid, vanillic acid and caffeine in coffee samples. The method has been applied to eight different coffees available on the Romanian market which were previously analysed concerning the total polyphenols content and antioxidant capacity. Reduction of the DPPH radical was used to determine the antioxidant capacity of the coffee extracts while the total polyphenols content was determined by spectrophotometry (Folin Ciocalteu's method). The total polyphenols content ranged from 1.98 g GAE/100 g to 4.19 g GAE/100 g while the caffeine content ranged from 1.89 g/100 g to 3.05 g/100 g. A large variability was observed in chlorogenic acid content of the investigated coffee samples which ranged between 0.6 and 2.32 g/100 g.

Sensitive indoor air monitoring of formaldehyde and other carbonyl compounds using the 2,4-dinitrophenylhydrazine method.

PubMed

Sandner, F; Dott, W; Hollender, J

2001-03-01

The toxic potential of formaldehyde and other aliphatic/aromatic carbonyl compounds requires the determination of even low amounts of these compounds in indoor air. The existing DFG-method for workplace monitoring using adsorption at 2,4-dinitrophenylhydrazine (DNPH)-coated sorbents followed by HPLC-UV/DAD analysis of the extract was modified in order to decrease detection limits. The improvement included an increase in volume and rate of the air sampling, testing applicability of different adsorption materials and a decrease of the extraction volume of the hydrazones. 13 DNPH-derivatives could be separated well on a RP18-column followed by UV/DAD-detection at 365 nm. Recovery rates of 70-100% were determined (apart from acetone with 19%) using dynamically produced artifical carbonyl atmospheres. Detection limits of 0.05-0.4 microgram/m3 were reached by this method which are sufficient for indoor air monitoring.

High-performance liquid chromatography ultraviolet-photodiode array detection method for aflatoxin B1 in cattle feed supplements

PubMed Central

Mochamad, Lazuardi; Hermanto, Bambang

2017-01-01

Aim: The objective of the current study is to determine the concentration of aflatoxin B1 using high-performance liquid chromatography (HPLC) with a photodiode array (PDA) detector. Materials and Methods: Aflatoxin B1 certified reference grade from Trilogy Analytical Laboratory dissolved acetonitrile (ACN) at 10 µg/mL was using standard assessment. HPLC instruments such as ultraviolet-PDA detector used a Shimadzu LC-6AD pump with DGU-20A5 degasser, communication module-20A, and PDA detector SPD-M20A with FRC-10A fraction collector. The HPLC was set isocratic method at 354 nm with a reverse-phase ODS C18 column (LiChrospher® 100 RP-18; diameter, 5 µm) under a 20°C controlled column chamber. Rheodyne® sample loops were performed in 20 µL capacities. The mobile phase was performed at fraction 63:26:11 H2O: methanol:ACN at pH 6.8. A total of 1 kg of feed contained 10% bread crumbs and 30% concentrated, 40% forage, and 20% soybean dregs were using commercials samples. Samples were extracted by ACN and separated with solid phase extraction ODS 1 mL than elution with mobile phase to collect at drying samples performed. The samples were ready to use after added 1 mL mobile phase than injected into the system of HPLC. Results: We found that the retention time of aflatoxin B1 was approximately 10.858 min. Linearity of 0.01-0.08 µg/mL aflatoxin B1 dissolved in mobile phase was obtained at R2=0.9. These results demonstrate that these methods can be used to analyze aflatoxin B1 and gain 89-99% recovery. The limit of detection of this assay was obtained at 3.5 × 10−6 µg/mL. Conclusion: This method was easy to apply and suitable to analyzing at small concentrations of aflatoxin B1 in formulated product of feed cattle. PMID:28919686

Quality Evaluation of the Traditional Medicine Majun Mupakhi ELA via Chromatographic Fingerprinting Coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS and the Antioxidant Activity In Vitro

PubMed Central

Reheman, Ayinuer; Ma, Qing Ling; Nijat, Dilaram; Abdulla, Rahima

2018-01-01

By merging a high-performance liquid chromatography diode array detector (HPLC-DAD) method with high-performance thin-layer chromatography (HPTLC), an assay was developed for chemical fingerprinting and quantitative analysis of traditional medicine Majun Mupakhi ELA (MME), and constituent compounds were identified using HPLC coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS method. In addition, the antioxidant capacity of MME was assessed based on the ability of components to scavenge radicals using in vitro method. Using a HPLC-DAD method with HPTLC easily validated the chemical fingerprinting results and quantified three characteristic components, namely, gallic acid (1), daidzein (2), and icariin (3), in commercial MMEs. The three compounds presented excellent regression values (R2 = 0.9999) in the ranges of the test and the method recovery was in the range from 100.49% to 100.68%. The fingerprints had 27 common characteristic peaks, of which 13 were verified by rapid UHPLC-DAD-Q-Orbitrap-MS analysis. In vitro antioxidant assays rapidly assessed and contrasted antioxidant activity or the free radical scavenging activity of the main polyphenolic classes in MMEs, and the antioxidant capacity was mostly affected by the presence of gallic acid. Thus, this study establishes a powerful and meaningful approach for MME quality control and for assessing in vitro antioxidant activity. PMID:29692853

Quality Evaluation of the Traditional Medicine Majun Mupakhi ELA via Chromatographic Fingerprinting Coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS and the Antioxidant Activity In Vitro.

PubMed

Reheman, Ayinuer; Aisa, Haji Akber; Ma, Qing Ling; Nijat, Dilaram; Abdulla, Rahima

2018-01-01

By merging a high-performance liquid chromatography diode array detector (HPLC-DAD) method with high-performance thin-layer chromatography (HPTLC), an assay was developed for chemical fingerprinting and quantitative analysis of traditional medicine Majun Mupakhi ELA (MME), and constituent compounds were identified using HPLC coupled with UHPLC-DAD-Quadrupole-Orbitrap-MS method. In addition, the antioxidant capacity of MME was assessed based on the ability of components to scavenge radicals using in vitro method. Using a HPLC-DAD method with HPTLC easily validated the chemical fingerprinting results and quantified three characteristic components, namely, gallic acid (1), daidzein (2), and icariin (3), in commercial MMEs. The three compounds presented excellent regression values ( R 2 = 0.9999) in the ranges of the test and the method recovery was in the range from 100.49% to 100.68%. The fingerprints had 27 common characteristic peaks, of which 13 were verified by rapid UHPLC-DAD-Q-Orbitrap-MS analysis. In vitro antioxidant assays rapidly assessed and contrasted antioxidant activity or the free radical scavenging activity of the main polyphenolic classes in MMEs, and the antioxidant capacity was mostly affected by the presence of gallic acid. Thus, this study establishes a powerful and meaningful approach for MME quality control and for assessing in vitro antioxidant activity.

Complexity in estimation of esomeprazole and its related impurities' stability in various stress conditions in low-dose aspirin and esomeprazole magnesium capsules.

PubMed

Reddy, Palavai Sripal; Hotha, Kishore Kumar; Sait, Shakil

2013-01-01

A complex, sensitive, and precise high-performance liquid chromatographic method for the profiling of impurities of esomeprazole in low-dose aspirin and esomeprazole capsules has been developed, validated, and used for the determination of impurities in pharmaceutical products. Esomeprazole and its related impurities' development in the presence of aspirin was traditionally difficult due to aspirin's sensitivity to basic conditions and esomeprazole's sensitivity to acidic conditions. When aspirin is under basic, humid, and extreme temperature conditions, it produces salicylic acid and acetic acid moieties. These two byproducts create an acidic environment for the esomeprazole. Due to the volatility and migration phenomenon of the produced acetic acid and salicylic acid from aspirin in the capsule dosage form, esomeprazole's purity, stability, and quantification are affected. The objective of the present research work was to develop a gradient reversed-phase liquid chromatographic method to separate all the degradation products and process-related impurities from the main peak. The impurities were well-separated on a RP8 column (150 mm × 4.6mm, X-terra, RP8, 3.5μm) by the gradient program using a glycine buffer (0.08 M, pH adjusted to 9.0 with 50% NaOH), acetonitrile, and methanol at a flow rate of 1.0 mL min(-1) with detection wavelength at 305 nm and column temperature at 30°C. The developed method was found to be specific, precise, linear, accurate, rugged, and robust. LOQ values for all of the known impurities were below reporting thresholds. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis, and thermal degradation in the presence of aspirin. The developed RP-HPLC method was validated according to the present ICH guidelines for specificity, linearity, accuracy, precision, limit of detection, limit of quantification, ruggedness, and robustness.

RP-HPLC×HILIC chromatography for quantifying ertapenem sodium with a look at green chemistry.

PubMed

Pedroso, Tahisa M; Medeiros, Ana C D; Salgado, Herida R N

2016-11-01

Ertapenem sodium is a polar and ionizable compound; therefore, it has little retention on traditional C18 columns in reverse-phase high-performance liquid chromatography, even using a highly-aqueous mobile phase that can result in dewetting in the stationary phase. Thus, the most coherent process for ERTM is to develop a method for Hydrophilic Interaction Chromatography. However, for the traditional methods in HILIC, the use of a highly organic mobile phase is necessary; usually an amount exceeding 80% acetonitrile is necessary. On the other hand, the RP-HPLC mode is considered for the analysis technique, which is more often used for quantification of substances, and new columns are often introduced to analyze different groups of compounds. Two new analytical methods have been developed for routine analysis. The proposed chromatographic method was adequate and advantageous by presenting simplicity, linearity, precision, accuracy, robustness, detection limits, and satisfactory quantification. Analytical methods are constantly undergoing changes and improvements. Researchers worldwide are rapidly adopting Green Chemistry. The development of new pharmaceutical methods based in Green chemistry has been encouraged by universities and the pharmaceutical industry. Issues related to green chemistry are in evidence and they have been featured in international journals of high impact. The methods described here have economic advantages and they feature an eco-friendly focus, which is discussed in this work. This work was developed with an environmental conscience, always looking to minimize the possible generated organic waste. Therefore, discussion on this aspect is included. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2018-03-05

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

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.

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

PubMed

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

2010-05-15

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

Profile of nucleosides and nucleotides in donkey's milk.

PubMed

Vincenzetti, Silvia; Pucciarelli, Stefania; Nucci, Chiara; Polzonetti, Valeria; Cammertoni, Natalina; Polidori, Paolo

2014-01-01

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkey's milk may be considered a good replacer for cow's milk in feeding children with severe Ig-E mediated cow's milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

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

Sorption of 4-carboxyquinoline derivatives from aqueous acetonitrile solutions on the surface of porous graphitized carbon

NASA Astrophysics Data System (ADS)

Savchenkova, A. S.; Buryak, A. K.; Kurbatova, S. V.

2015-09-01

The sorption of 4-carboxyquinoline derivatives from aqueous acetonitrile solutions on porous graphitized carbon was studied. The effect of the structure of analyte molecules and the eluent composition on the characteristics of retention under the conditions of RP HPLC was analyzed. The effect of pH of the eluent on the shift of equilibrium in aqueous acetonitrile solutions was investigated.

Unique variability of tocopherol composition in various seed oils recovered from by-products of apple industry: rapid and simple determination of all four homologues (α, β, γ and δ) by RP-HPLC/FLD.

PubMed

Górnaś, Paweł

2015-04-01

The tocochromanol profile was studied in seed oils recovered from by-products of fruit industry, five dessert and seven crab apple varieties grown in Eastern Europe (Latvia). The seed oils obtained from dessert apples were characterized by higher contents of tocopherols (191.05-379.08 mg/100g oil) when compared to seed oils recovered from crab apples (130.55-202.54 mg/100g oil). The predominant homologues of tocopherol in all the studied samples were α and β over γ and δ. However, seed oils recovered from the apple cultivars 'Antej' and 'Beforest' had a unique profile of four tocopherol homologues (α:β:γ:δ) 91.41:80.55:72.46:79.03 and 114.55:112.84:78.69:73.00 mg/100g oil, respectively. A single dilution of seed oils in 2-propanol facilitated the direct use samples in the DPPH assay as well as injection into the RP-HPLC system containing a PFP (pentafluorophenyl) column, which resulted in a rapid separation of all four tocopherol homologues with excellent repeatability and reproducibility. Copyright © 2014 Elsevier Ltd. All rights reserved.

Elucidation of the defence mechanism in microalgae Chlorella sorokiniana under mercury exposure. Identification of Hg-phytochelatins.

PubMed

Gómez-Jacinto, Verónica; García-Barrera, Tamara; Gómez-Ariza, José Luis; Garbayo-Nores, Inés; Vílchez-Lobato, Carlos

2015-08-05

Algae and aquatic macrophytes are capable of accumulating heavy metals up to concentrations several orders of magnitude higher than those existing in their surrounding environment. Investigation of mercury toxicology in microalgae is of great interest from ecological point of view, since they could be used as bioindicator to evaluate aquatic ecosystems affected by Hg pollution. In this study, we have performed an exposure experiment focused on the biological response of microalgae Chlorella sorokiniana, a unicellular model organism, to Hg-induced toxicity. The culture was exposed to different concentrations of this element for nine days, namely 0.5, 1, 5 and 10mg L(-1) of HgCl2 (as Hg). To achieve a better understanding of the biological mechanisms triggered by Hg-induced toxicity in this alga a metallomic approach based on SEC-ICP-ORS-MS was applied to survey biomarkers of biological response to mercury contamination in surface water. In addition, the combination of RP-HPLC-ICP-ORS-MS and RP-HPLC-ESI-QqQ-TOF-MS was applied to identify, for the first time, two Hg-binding phytochelatins in this aquatic organism, using cell extracts from microalgae exposed to inorganic mercury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Characterization and quantitation of low and high molecular weight phenolic compounds in apple seeds.

PubMed

Fromm, Matthias; Bayha, Sandra; Carle, Reinhold; Kammerer, Dietmar R

2012-02-08

The phenolic constituents of seeds of 12 different apple cultivars were fractionated by sequential extraction with aqueous acetone (30:70, v/v) and ethyl acetate after hexane extraction of the lipids. Low molecular weight phenolic compounds were individually quantitated by RP-HPLC-DAD. The contents of extractable and nonextractable procyanidins were determined by applying RP-HPLC following thiolysis and n-butanol/HCl hydrolysis, respectively. As expected, the results revealed marked differences of the ethyl acetate extracts, aqueous acetone extracts, and insoluble residues with regard to contents and mean degrees of polymerization of procyanidins. Total phenolic contents in the defatted apple seed residues ranged between 18.4 and 99.8 mg/g. Phloridzin was the most abundant phenolic compound, representing 79-92% of monomeric polyphenols. Yields of phenolic compounds significantly differed among the cultivars under study, with seeds of cider apples generally being richer in phloridzin and catechins than seeds of dessert apple cultivars. This is the first study presenting comprehensive data on the contents of phenolic compounds in apple seeds comprising extractable and nonextractable procyanidins. Furthermore, the present work points out a strategy for the sustainable and complete exploitation of apple seeds as valuable agro-industrial byproducts, in particular as a rich source of phloridzin and antioxidant flavanols.

Effect of sodium chloride and sodium bicarbonate on the physicochemical properties of soft wheat flour doughs and gluten polymerization.

PubMed

Chen, Gengjun; Ehmke, Laura; Miller, Rebecca; Faa, Pierre; Smith, Gordon; Li, Yonghui

2018-06-07

Soft wheat flour doughs were prepared with different levels of salt (NaCl) and/or baking soda (NaHCO3). Oscillation rheology, elongational viscosity, and extensibility of doughs were tested to evaluate the effect of salt and/or baking soda on the physical properties of doughs. Furthermore, a series of physical-biochemical analytical techniques were used to investigate gluten polymerization in doughs, including Zeta potential analyzer, Fourier transform infrared spectroscopy (FTIR), spectrophotometer, and reversed phase high performance liquid chromatography (RP-HPLC). Addition of high levels of NaHCO3 (1.0 % fwb), either by itself or in combination with NaCl, increased dough strength, elongational viscosity, and viscoelasticity. RP-HPLC results demonstrated macromolecular aggregation of gluten proteins in the presence of NaCl and/or NaHCO3. Addition of NaHCO3 or NaCl also decreased both free sulfhydryl content and random coil structure of gluten isolated from the doughs. Overall, NaCl and/or NaHCO3 induced the changes of molecular conformation of gluten, which impacted the physicochemical qualities of soft wheat flour dough. Our study provides a better understanding of salt and baking soda functionality in the formation of soft flour dough, which will support the searching of feasible sodium reduction strategies in soft flour bakery products.

Quickly Screening for Potential α-Glucosidase Inhibitors from Guava Leaves Tea by Bioaffinity Ultrafiltration Coupled with HPLC-ESI-TOF/MS Method.

PubMed

Wang, Lu; Liu, Yufeng; Luo, You; Huang, Kuiying; Wu, Zhenqiang

2018-02-14

Guava leaves tea (GLT) has a potential antihyperglycemic effect. Nevertheless, it is unclear which compound plays a key role in reducing blood sugar. In this study, GLT extract (IC 50 = 19.37 ± 0.21 μg/mL) exhibited a stronger inhibitory potency against α-glucosidase than did acarbose (positive control) at IC 50 = 178.52 ± 1.37 μg/mL. To rapidly identify the specific α-glucosidase inhibitor components from GLT, an approach based on bioaffinity ultrafiltration combined with high performance liquid chromatography coupled to electrospray ionization-time-of-flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS) was developed. Under the optimal bioaffinity ultrafiltration conditions, 11 corresponding potential α-glucosidase inhibitors with high affinity degrees (ADs) were screened and identified from the GLT extract. Quercetin (IC 50 = 4.51 ± 0.71 μg/mL) and procyanidin B3 (IC 50 = 28.67 ± 5.81 μg/mL) were determined to be primarily responsible for the antihyperglycemic effect, which further verified the established screening method. Moreover, structure-activity relationships were discussed. In conclusion, the BAUF-HPLC-ESI-TOF/MS method could be applied to determine the potential α-glucosidase inhibitors from complex natural products quickly.

Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson’s disease

PubMed Central

Samoylenko, Volodymyr; Rahman, Md. Mostafizur; Tekwani, Babu L.; Tripathi, Lalit M.; Wang, Yan-Hong; Khan, Shabana I.; Khan, Ikhlas A.; Miller, Loren S.; Joshi, Vaishali C.; Muhammad, Ilias

2009-01-01

Aim of the study Parkinson’s disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of B. caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism. Materials and methods The aqueous extract of B. caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron®. Structure elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl transferase enzymes, as well as antioxidant and cytotoxic activities of both B. caapi extract and isolated compounds were evaluated. Results An examination of the aqueous extracts of B. caapi cultivar Da Vine yielded two new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing “azepino[1,2-a]tetrahydro-β-carboline” unique carbon framework. One additional new natural tetrahydronorharmine (4), four known β-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two known proanthocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and a new disaccharide β-D-fructofuranosyl-(2→5)-fructopyranose (14) together with known sacharose (15) and β-D-glucose (16) were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10–13, 17, 18) were also prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant human brain monoamine oxidase (MAO) -A and -B enzymes (IC50 2.5 and 2.0 nM, and 25 and 20 µM, respectively), and (−)-epicatechin (8) and (−)-procyanidin B2 (9) showed potent antioxidant and moderate MAO-B inhibitory activities (IC50 <0.13 and 0.57 µg/mL, and 65 and 35 µM). HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7–9) were present in high concentrations in dried bark of large branch. Analysis of regular/commercial B. caapi dried stems showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency. Conclusion Collectively, these results give additional basis to the existing claim of B. caapi stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders. PMID:19879939

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

PubMed

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

2014-03-01

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

Microwave-assisted extraction of herbacetin diglucoside from flax (Linum usitatissimum L.) seed cakes and its quantification using an RP-HPLC-UV system.

PubMed

Fliniaux, Ophélie; Corbin, Cyrielle; Ramsay, Aina; Renouard, Sullivan; Beejmohun, Vickram; Doussot, Joël; Falguières, Annie; Ferroud, Clotilde; Lamblin, Frédéric; Lainé, Eric; Roscher, Albrecht; Grand, Eric; Mesnard, François; Hano, Christophe

2014-03-10

Flax (Linum usitatissimum L.) seeds are widely used for oil extraction and the cold-pressed flaxseed (or linseed) cakes obtained during this process constitute a valuable by-product. The flavonol herbacetin diglucoside (HDG) has been previously reported as a constituent of the flaxseed lignan macromolecule linked through ester bonds to the linker molecule hydroxymethylglutaric acid. In this context, the development and validation of a new approach using microwave-assisted extraction (MAE) of HDG from flaxseed cakes followed by quantification with a reverse-phase HPLC system with UV detection was purposed. The experimental parameters affecting the HDG extraction yield, such as microwave power, extraction time and sodium hydroxide concentration, from the lignan macromolecule were optimized. A maximum HDG concentration of 5.76 mg/g DW in flaxseed cakes was measured following an irradiation time of 6 min, for a microwave power of 150 W using a direct extraction in 0.1 M NaOH in 70% (v/v) aqueous methanol. The optimized method was proven to be rapid and reliable in terms of precision, repeatability, stability and accuracy for the extraction of HDG. Comparison with a conventional extraction method demonstrated that MAE is more effective and less time-consuming.

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection.

PubMed

Ishihara, K; Fukutake, M; Asano, T; Mizuhara, Y; Wakui, Y; Yanagisawa, T; Kamei, H; Ohmori, S; Kitada, M

2001-04-05

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20x3.9 mm I.D.) and analytical column (Symmetry C18, 75x4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile-water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40 degrees C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.

Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: effect of neutrophil depletion on extracellular ascorbyl radical formation.

PubMed

Matsuo, Y; Kihara, T; Ikeda, M; Ninomiya, M; Onodera, H; Kogure, K

1995-11-01

A growing body of experimental data indicate that oxygen radicals may mediate the brain injury during ischemia-reperfusion. One potential source of oxygen radicals is activated neutrophils. To study the role of neutrophils in radical production during cerebral ischemia-reperfusion, we evaluated the effects of depletion of circulating neutrophils by administration of an anti-neutrophil monoclonal antibody (RP3) on radical formation in rats with 1-h middle cerebral artery (MCA) occlusion. In the present study, we employed a new electron spin resonance method coupled with brain microdialysis. The method uses the endogenous ascorbyl radical (AR) concentration as a marker of oxygen radicals and requires no spin-trapping agents. In the vehicle controls, extracellular AR decreased during MCA occlusion. After reperfusion, AR significantly increased at 30 min and 1 h, returned to near basal level until 2 h, and increased again at 24 h after reperfusion. In the rats treated with RP3, AR decreased during MCA occlusion to the same extent as in the vehicle control. However, RP3 treatment completely inhibited the increase in extracellular AR after reperfusion. RP3 treatment exerted no effect on the changes in extracellular ascorbate or tissue PO2 throughout the experimental period. In conclusion, neutrophils are a major source of oxygen radicals during reperfusion after focal cerebral ischemia.

AMPK modulatory activity of olive–tree leaves phenolic compounds: Bioassay-guided isolation on adipocyte model and in silico approach

PubMed Central

Jiménez-Sánchez, Cecilia; Olivares-Vicente, Mariló; Rodríguez-Pérez, Celia; Herranz-López, María; Lozano-Sánchez, Jesús; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto; Encinar, José Antonio; Micol, Vicente

2017-01-01

Scope Olive-tree polyphenols have demonstrated potential for the management of obesity-related pathologies. We aimed to explore the capacity of Olive-tree leaves extract to modulate triglyceride accumulation and AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. Methods Intracellular triglycerides and AMPK activity were measured on the hypertrophic 3T3-L1 adipocyte model by AdipoRed and immunofluorescence microscopy, respectively. Reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) was used for the fractionation of the extract and the identification of the compounds. In-silico molecular docking of the AMPK alpha-2, beta and gamma subunits with the identified compounds was performed. Results Olive-tree leaves extract decreased the intracellular lipid accumulation through AMPK-dependent mechanisms in hypertrophic adipocytes. Secoiridoids, cinnamic acids, phenylethanoids and phenylpropanoids, flavonoids and lignans were the candidates predicted to account for this effect. Molecular docking revealed that some compounds may be AMPK-gamma modulators. The modulatory effects of compounds over the alpha and beta AMPK subunits appear to be less probable. Conclusions Olive-tree leaves polyphenols modulate AMPK activity, which may become a therapeutic aid in the management of obesity-associated disturbances. The natural occurrence of these compounds may have important nutritional implications for the design of functional ingredients. PMID:28278224

Determination of itopride hydrochloride in human plasma by RP-HPLC with fluorescence detection and its use in bioequivalence study.

PubMed

Ma, Jing; Yuan, Li-Hua; Ding, Mei-Juan; Zhang, Jun; Zhang, Qing; Xu, Qun-Wei; Zhou, Xue-Min

2009-03-01

A sensitive, selective and simple method using a precipitation of protein with 10% perchloric acid, followed by high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of itopride hydrochloride in human plasma, using levofloxacin as the internal standard (IS). Chromatographic separation was obtained within 7.0 min using a reverse phase Hypersil BDS C(18) (250 mm x 4.6 mm, 5 microm) column and an isocratic mobile phase, constituting of a mixture of 0.1 mol/l ammonium acetate-methanol (30:70, v/v) flowing at 1.1 ml/min. The excitation and emission wavelengths were set at 304 and 344 nm, respectively. The method was validated over the concentration range of 5 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 5 ng/ml. The extractive recovery of itopride hydrochloride from the biological matrix was more than 80.77%. The intra-day accuracy of the drug containing serum samples was more than 82.94% with a precision of 2.81-4.37%. The inter-day accuracy was 82.91% or more, with a precision of 6.89-9.54%. The limit we have used (70-143%) is based on the local regulatory authority (SFDA). The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

Validation of a high performance liquid chromatography method for the stabilization of epigallocatechin gallate.

PubMed

Fangueiro, Joana F; Parra, Alexander; Silva, Amélia M; Egea, Maria A; Souto, Eliana B; Garcia, Maria L; Calpena, Ana C

2014-11-20

Epigallocatechin gallate (EGCG) is a green tea catechin with potential health benefits, such as anti-oxidant, anti-carcinogenic and anti-inflammatory effects. In general, EGCG is highly susceptible to degradation, therefore presenting stability problems. The present paper was focused on the study of EGCG stability in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) medium regarding the pH dependency, storage temperature and in the presence of ascorbic acid a reducing agent. The evaluation of EGCG in HEPES buffer has demonstrated that this molecule is not able of maintaining its physicochemical properties and potential beneficial effects, since it is partially or completely degraded, depending on the EGCG concentration. The storage temperature of EGCG most suitable to maintain its structure was shown to be the lower values (4 or -20 °C). The pH 3.5 was able to provide greater stability than pH 7.4. However, the presence of a reducing agent (i.e., ascorbic acid) was shown to provide greater protection against degradation of EGCG. A validation method based on RP-HPLC with UV-vis detection was carried out for two media: water and a biocompatible physiological medium composed of Transcutol®P, ethanol and ascorbic acid. The quantification of EGCG for purposes, using pure EGCG, requires a validated HPLC method which could be possible to apply in pharmacokinetic and pharmacodynamics studies. Copyright © 2014. Published by Elsevier B.V.

New validated method for piracetam HPLC determination in human plasma.

PubMed

Curticapean, Augustin; Imre, Silvia

2007-01-10

The new method for HPLC determination of piracetam in human plasma was developed and validated by a new approach. The simple determination by UV detection was performed on supernatant, obtained from plasma, after proteins precipitation with perchloric acid. The chromatographic separation of piracetam under a gradient elution was achieved at room temperature with a RP-18 LiChroSpher 100 column and aqueous mobile phase containing acetonitrile and methanol. The quantitative determination of piracetam was performed at 200 nm with a lower limit of quantification LLQ=2 microg/ml. For this limit, the calculated values of the coefficient of variation and difference between mean and the nominal concentration are CV%=9.7 and bias%=0.9 for the intra-day assay, and CV%=19.1 and bias%=-7.45 for the between-days assay. For precision, the range was CV%=1.8/11.6 in the intra-day and between-days assay, and for accuracy, the range was bias%=2.3/14.9 in the intra-day and between-days assay. In addition, the stability of piracetam in different conditions was verified. Piracetam proved to be stable in plasma during 4 weeks at -20 degrees C and for 36 h at 20 degrees C in the supernatant after protein precipitation. The new proposed method was used for a bioequivalence study of two medicines containing 800 mg piracetam.

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.

Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as Tools in the Identification of Bioactives

PubMed Central

Malherbe, Christiaan J.; de Beer, Dalene; Joubert, Elizabeth

2012-01-01

Biochemical detection (BCD) methods are commonly used to screen plant extracts for specific biological activities in batch assays. Traditionally, bioactives in the most active extracts were identified through time-consuming bio-assay guided fractionation until single active compounds could be isolated. Not only are isolation procedures often tedious, but they could also lead to artifact formation. On-line coupling of BCD assays to high performance liquid chromatography (HPLC) is gaining ground as a high resolution screening technique to overcome problems associated with pre-isolation by measuring the effects of compounds post-column directly after separation. To date, several on-line HPLC-BCD assays, applied to whole plant extracts and mixtures, have been published. In this review the focus will fall on enzyme-based, receptor-based and antioxidant assays. PMID:22489144

Identification of moisture content in tobacco plant leaves using outlier sample eliminating algorithms and hyperspectral data.

PubMed

Sun, Jun; Zhou, Xin; Wu, Xiaohong; Zhang, Xiaodong; Li, Qinglin

2016-02-26

Fast identification of moisture content in tobacco plant leaves plays a key role in the tobacco cultivation industry and benefits the management of tobacco plant in the farm. In order to identify moisture content of tobacco plant leaves in a fast and nondestructive way, a method involving Mahalanobis distance coupled with Monte Carlo cross validation(MD-MCCV) was proposed to eliminate outlier sample in this study. The hyperspectral data of 200 tobacco plant leaf samples of 20 moisture gradients were obtained using FieldSpc(®) 3 spectrometer. Savitzky-Golay smoothing(SG), roughness penalty smoothing(RPS), kernel smoothing(KS) and median smoothing(MS) were used to preprocess the raw spectra. In addition, Mahalanobis distance(MD), Monte Carlo cross validation(MCCV) and Mahalanobis distance coupled to Monte Carlo cross validation(MD-MCCV) were applied to select the outlier sample of the raw spectrum and four smoothing preprocessing spectra. Successive projections algorithm (SPA) was used to extract the most influential wavelengths. Multiple Linear Regression (MLR) was applied to build the prediction models based on preprocessed spectra feature in characteristic wavelengths. The results showed that the preferably four prediction model were MD-MCCV-SG (Rp(2) = 0.8401 and RMSEP = 0.1355), MD-MCCV-RPS (Rp(2) = 0.8030 and RMSEP = 0.1274), MD-MCCV-KS (Rp(2) = 0.8117 and RMSEP = 0.1433), MD-MCCV-MS (Rp(2) = 0.9132 and RMSEP = 0.1162). MD-MCCV algorithm performed best among MD algorithm, MCCV algorithm and the method without sample pretreatment algorithm in the eliminating outlier sample from 20 different moisture gradients of tobacco plant leaves and MD-MCCV can be used to eliminate outlier sample in the spectral preprocessing. Copyright © 2016 Elsevier Inc. All rights reserved.

[Investigation on the chromatogram of diterpenoids in Pteris semipinnata by HPLC-APCI-MS].

PubMed

Deng, Yifeng; Liang, Nianci

2005-04-01

To identify and compare the main peaks of HPLC-APCI-MS FP of the diterpenoids in Pteris semipinnata collected from different region and time, a quadrupole mass spectrometer coupled with atmospheric pressure chemical ionization interface was employed as a detector for HPLC to establish total ion chromatography. HPLC retention time and MS spectrum were used to identify comprehensively. 4F, 5F and 6F were identified from the chromatography comparing with their standards. The saturated state of 6F and glycoside of 4F and 5F were inferred. The content of 5F in samples collected from region of Guangzhou or in Nov. and Dec. were comparatively higher. This method is highly effective and fast,which can be applied to research and develop for diterpenoids in Pteris semipinnata L as new antitumor drug resource.

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

Thermodynamics of the Sorption of Benzimidazoles on Octadecyl Silica Gel from Water-Methanol Eluents

NASA Astrophysics Data System (ADS)

Shafigulin, R. V.; Bulanova, A. V.

2018-02-01

The standard enthalpy and entropy component of transferring benzimidazoles from water-methanol solutions to surfaces of octadecyl silica gel are determined using reversed-phase high-performance liquid chromatography (RP HPLC). The dependences between the enthalpy and polarizability of the molecules of the studied benzimidazoles, the enthalpy and the entropy factor are studied, and the influence of the quantitative composition of the water-methanol solution on the enthalpy are studied.

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

PubMed

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

2005-04-01

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

Characterization of nutraceuticals and functional foods by innovative HPLC methods.

PubMed

Corradini, Claudio; Galanti, Roberta; Nicoletti, Isabella

2002-04-01

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

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection.

PubMed

Wyss, R; Bucheli, F

1997-10-24

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 microm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250x4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3-100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%-94.4% and the mean inter-assay precision was 2.8%-3.2% (range 0.3-100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at -20 degrees C for 4.3 months and at -80 degrees C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.

Non-enzymatic browning in citrus juice: chemical markers, their detection and ways to improve product quality.

PubMed

Bharate, Sonali S; Bharate, Sandip B

2014-10-01

Citrus juices are widely consumed due to their nutritional benefits and variety of pharmacological properties. Non-enzymatic browning (NEB) is one of the most important chemical reactions responsible for quality and color changes during the heating or prolonged storage of citrus products. The present review covers various aspects of NEB in citrus juice viz. chemistry of NEB, identifiable markers of NEB, analytical methods to identify NEB markers and ways to improve the quality of citrus juice. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is one of the promising marker formed during browning process with number of analytical methods reported for its analysis; therefore it can be used as an indicator for NEB process. Amongst analytical methods reported, RP-HPLC is more sensitive and accurate method, which can be used as analytical tool. NEB can be prevented by removal of amino acids/ proteins (via ion exchange treatment) or by targeting NEB reactions (e.g. blockage of furfural/ HMF by sulphiting agent).

RP-HPLC method with electrochemical detection for the determination of metoclopramide in serum and its use in pharmacokinetic studies.

PubMed

Lamparczyk, H; Chmielewska, A; Konieczna, L; Plenis, A; Zarzycki, P K

2001-12-01

A rapid and sensitive reversed-phase high performance liquid chromatographic method has been developed for the determination of metoclopramide in serum. The assay was performed after single extraction with ethyl ether using methyl parahydroxybenzoate as internal standard. Chromatographic separations were performed on C(18) stationary phase with a mobile phase composed of methanol-phosphate buffer pH 3 (30:70 v/v). Analytes were detected electrochemically. The quantification limit for metoclopramide in serum was 2 ng mL(-1). Linearity of the method was confirmed in the range of 5-120 ng mL(-1) (correlation coefficient 0.9998). Within-day relative standard deviations (RSDs) ranged from 0.3 to 5.5% and between-day RSDs from 0.8 to 6.0%. The analytical method was successfully applied for the determination of pharmacokinetic parameters after ingestion of 10 mg dose of metoclopramide. Studies were performed on 18 healthy volunteers of both sexes. Copyright 2001 John Wiley & Sons, Ltd.

Development of new method for simultaneous analysis of piracetam and levetiracetam in pharmaceuticals and biological fluids: application in stability studies.

PubMed

Siddiqui, Farhan Ahmed; Sher, Nawab; Shafi, Nighat; Wafa Sial, Alisha; Ahmad, Mansoor; Mehjebeen; Naseem, Huma

2014-01-01

RP-HPLC ultraviolet detection simultaneous quantification of piracetam and levetiracetam has been developed and validated. The chromatography was obtained on a Nucleosil C18 column of 25 cm×0.46 cm, 10 μm, dimension. The mobile phase was a (70:30 v/v) mixture of 0.1 g/L of triethylamine and acetonitrile. Smooth flow of mobile phase at 1 mL/min was set and 205 nm wavelength was selected. Results were evaluated through statistical parameters which qualify the method reproducibility and selectivity for the quantification of piracetam, levetiracetam, and their impurities hence proving stability-indicating properties. The proposed method is significantly important, permitting the separation of the main constituent piracetam from levetiracetam. Linear behavior was observed between 20 ng/mL and 10,000 ng/mL for both drugs. The proposed method was checked in bulk drugs, dosage formulations, physiological condition, and clinical investigations and excellent outcome was witnessed.

Polyphasic approach for differentiating Penicillium nordicum from Penicillium verrucosum.

PubMed

Berni, E; Degola, F; Cacchioli, C; Restivo, F M; Spotti, E

2011-04-01

The aim of this research was to use a polyphasic approach to differentiate Penicillium verrucosum from Penicillium nordicum, to compare different techniques, and to select the most suitable for industrial use. In particular, (1) a cultural technique with two substrates selective for these species; (2) a molecular diagnostic test recently set up and a RAPD procedure derived from this assay; (3) an RP-HPLC analysis to quantify ochratoxin A (OTA) production and (4) an automated system based on fungal carbon source utilisation (Biolog Microstation™) were used. Thirty strains isolated from meat products and originally identified as P. verrucosum by morphological methods were re-examined by newer cultural tests and by PCR methods. All were found to belong to P. nordicum. Their biochemical and chemical characterisation supported the results obtained by cultural and molecular techniques and showed the varied ability in P. verrucosum and P. nordicum to metabolise carbon-based sources and to produce OTA at different concentrations, respectively.

A simple LC/MRM-MS-based method to quantify free linker-payload in antibody-drug conjugate preparations.

PubMed

Zmolek, Wesley; Bañas, Stefanie; Barfield, Robyn M; Rabuka, David; Drake, Penelope M

2016-10-01

Antibody-drug conjugates represent a growing class of biologic drugs that use the targeted specificity of an antibody to direct the localization of a small molecule drug, often a cytotoxic payload. After conjugation, antibody-drug conjugate preparations typically retain a residual amount of free (unconjugated) linker-payload. Monitoring this free small molecule drug component is important due to the potential for free payload to mediate unintended (off-target) toxicity. We developed a simple RP-HPLC/MRM-MS-based assay that can be rapidly employed to quantify free linker-payload. The method uses low sample volumes and offers an LLOQ of 10nM with 370pg on column. This analytical approach was used to monitor free linker-payload removal during optimization of the tangential flow filtration manufacturing step. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Studies on betaxanthin profiles of vegetables and fruits from the Chenopodiaceae and Cactaceae.

PubMed

Kugler, Florian; Graneis, Stephan; Stintzing, Florian C; Carle, Reinhold

2007-01-01

The present study provides an update on the betaxanthin (bx) compositions of red and yellow beetroots, yellow-coloured Swiss chard petioles, and yellow-orange cactus pear. Applying RP-HPLC coupled with positive ion electrospray mass spectrometry and by comparison with UV-vis and mass spectrometric characteristics as well as retention times of semi-synthesized reference compounds, 24 betaxanthins were identified in red and yellow beetroot hypocotyls. Twenty-five and thirteen betaxanthins were present in yellow Swiss chard petioles and the cactus pear cultivar 'Gialla', respectively. Ethanolamine-bx and threonine-bx were found to be novel betaxanthins in Chenopodiaceae representatives, which to the best of our knowledge have not been reported as genuine pigments so far. Furthermore, aspartic acid-bx (miraxanthin II), lysine-bx, and methionine-bx, hitherto found in other families, were identified in the Chenopodiaceae for the first time. Additionally, tyrosine-bx (portulacaxanthin II) and tryptophan-bx have not been earlier reported to occur in the Cactaceae. These findings provide valuable phytochemical information and may be useful for a better understanding of the functional properties of betaxanthins in plants.

Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS).

PubMed

Choudhury, Nila Roy; Michlewski, Gracjan

2018-06-08

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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%.

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

PubMed

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

2017-05-01

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

Log D versus HPLC derived hydrophobicity: The development of predictive tools to aid in the rational design of bioactive peptoids

DOE PAGES

Bolt, H. L.; Williams, C. E. J.; Brooks, R. V.; ...

2017-01-13

Hydrophobicity has proven to be an extremely useful parameter in small molecule drug discovery programmes given that it can be used as a predictive tool to enable rational design. For larger molecules, including peptoids, where folding is possible, the situation is more complicated and the average hydrophobicity (as determined by RP-HPLC retention time) may not always provide an effective predictive tool for rational design. Herein, we report the first ever application of partitioning experiments to determine the log D values for a series of peptoids. By comparing log D and average hydrophobicities we highlight the potential advantage of employing themore » former as a predictive tool in the rational design of biologically active peptoids.« less

Recent advances in nonpolar and polar organic monoliths for HPLC and CEC

PubMed Central

Jonnada, Murthy; Rathnasekara, Renuka; Rassi, Ziad El

2015-01-01

This article is aimed at providing a review of the progress made in the field over the period 2011 to present in order to expand in parts on two previous reviews (S. Karenga and Z. El Rassi, Electrophoresis, 2011, 32, 90-104; D. Gunasena and Z. El Rassi, Electrophoresis, 2012, 33, 251-261). In brief, this review article describes progress made in nonpolar and polar monoliths used in reversed phase HPLC and CEC (RPC/RP-CEC) and in hydrophilic interaction liquid chromatography/CEC (HILIC/HI-CEC), respectively. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 69 references published on nonpolar and polar polymeric monoliths. PMID:25266173

Log D versus HPLC derived hydrophobicity: The development of predictive tools to aid in the rational design of bioactive peptoids

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

Bolt, H. L.; Williams, C. E. J.; Brooks, R. V.

Hydrophobicity has proven to be an extremely useful parameter in small molecule drug discovery programmes given that it can be used as a predictive tool to enable rational design. For larger molecules, including peptoids, where folding is possible, the situation is more complicated and the average hydrophobicity (as determined by RP-HPLC retention time) may not always provide an effective predictive tool for rational design. Herein, we report the first ever application of partitioning experiments to determine the log D values for a series of peptoids. By comparing log D and average hydrophobicities we highlight the potential advantage of employing themore » former as a predictive tool in the rational design of biologically active peptoids.« less

Preparative isolation of oligomeric procyanidins from Hawthorn (Crataegus spp.).

PubMed

Zumdick, S; Petereit, F; Luftmann, H; Hensel, A

2009-04-01

The oligomeric procyanidins (OPC) from Hawthorn leaves and flowers (Crataegi folium cum flore) are considered to be in part responsible for the cardiotonic clinical activity of the herbal material. Effective methods for rapid isolation of these heterogenous oligomeric clusters with defined molecular weight as reference compounds are not published until now. Therefore the water soluble fraction of an acetone/water (7 + 3) extract of Hawthorn leaves and flowers was fractionated by a combination of MPLC on RP-18 material and preparative HPLC using a diol stationary phase. This procedure resulted in the effective isolation of procyanidins with a distinct degree of polymerization (DP) from dimers DP2 up to tridecamers DP13. Exact mass measurements with negative ESI-TOF/MS were employed to confirm the respective structures of the isolated procyanidins.

Modeling RP-1 fuel advanced distillation data using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry and partial least squares analysis.

PubMed

Kehimkar, Benjamin; Parsons, Brendon A; Hoggard, Jamin C; Billingsley, Matthew C; Bruno, Thomas J; Synovec, Robert E

2015-01-01

Recent efforts in predicting rocket propulsion (RP-1) fuel performance through modeling put greater emphasis on obtaining detailed and accurate fuel properties, as well as elucidating the relationships between fuel compositions and their properties. Herein, we study multidimensional chromatographic data obtained by comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC-TOFMS) to analyze RP-1 fuels. For GC × GC separations, RTX-Wax (polar stationary phase) and RTX-1 (non-polar stationary phase) columns were implemented for the primary and secondary dimensions, respectively, to separate the chemical compound classes (alkanes, cycloalkanes, aromatics, etc.), providing a significant level of chemical compositional information. The GC × GC-TOFMS data were analyzed using partial least squares regression (PLS) chemometric analysis to model and predict advanced distillation curve (ADC) data for ten RP-1 fuels that were previously analyzed using the ADC method. The PLS modeling provides insight into the chemical species that impact the ADC data. The PLS modeling correlates compositional information found in the GC × GC-TOFMS chromatograms of each RP-1 fuel, and their respective ADC, and allows prediction of the ADC for each RP-1 fuel with good precision and accuracy. The root-mean-square error of calibration (RMSEC) ranged from 0.1 to 0.5 °C, and was typically below ∼0.2 °C, for the PLS calibration of the ADC modeling with GC × GC-TOFMS data, indicating a good fit of the model to the calibration data. Likewise, the predictive power of the overall method via PLS modeling was assessed using leave-one-out cross-validation (LOOCV) yielding root-mean-square error of cross-validation (RMSECV) ranging from 1.4 to 2.6 °C, and was typically below ∼2.0 °C, at each % distilled measurement point during the ADC analysis.

Extraction and Determination of Cyproheptadine in Human Urine by DLLME-HPLC Method.

PubMed

Maham, Mehdi; Kiarostami, Vahid; Waqif-Husain, Syed; Abroomand-Azar, Parviz; Tehrani, Mohammad Saber; Khoeini Sharifabadi, Malihe; Afrouzi, Hossein; Shapouri, Mahmoudreza; Karami-Osboo, Rouhollah

2013-01-01

Novel dispersive liquid-liquid microextraction (DLLME), coupled with high performance liquid chromatography with photodiode array detection (HPLC-DAD) has been applied for the extraction and determination of cyproheptadine (CPH), an antihistamine, in human urine samples. In this method, 0.6 mL of acetonitrile (disperser solvent) containing 30 μL of carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5 mL urine sample. After centrifugation, the sedimented phase containing enriched analyte was dissolved in acetonitrile and an aliquot of this solution injected into the HPLC system for analysis. Development of DLLME procedure includes optimization of some important parameters such as kind and volume of extraction and disperser solvent, pH and salt addition. The proposed method has good linearity in the range of 0.02-4.5 μg mL(-1) and low detection limit (13.1 ng mL(-1)). The repeatability of the method, expressed as relative standard deviation was 4.9% (n = 3). This method has also been applied to the analysis of real urine samples with satisfactory relative recoveries in the range of 91.6-101.0%.

Fingerprint analysis of polysaccharides from different Ganoderma by HPLC combined with chemometrics methods.

PubMed

Sun, Xiaomei; Wang, Haohao; Han, Xiaofeng; Chen, Shangwei; Zhu, Song; Dai, Jun

2014-12-19

A fingerprint analysis method has been developed for characterization and discrimination of polysaccharides from different Ganoderma by high performance liquid chromatography (HPLC) coupled with chemometrics means. The polysaccharides were extracted under ultrasonic-assisted condition, and then partly hydrolyzed with trifluoroacetic acid. Monosaccharides and oligosaccharides in the hydrolyzates were subjected to pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone and HPLC analysis, which will generate unique fingerprint information related to chemical composition and structure of polysaccharides. The peak data were imported to professional software in order to obtain standard fingerprint profiles and evaluate similarity of different samples. Meanwhile, the data were further processed by hierarchical cluster analysis and principal component analysis. Polysaccharides from different parts or species of Ganoderma or polysaccharides from the same parts of Ganoderma but from different geographical regions or different strains could be differentiated clearly. This fingerprint analysis method can be applied to identification and quality control of different Ganoderma and their products. Copyright © 2014 Elsevier Ltd. All rights reserved.

Study and development of reversed-phase HPLC systems for the determination of 2-imidazolines in the presence of preservatives in pharmaceutical preparations.

PubMed

Antoniou, Constantinos G; Markopoulou, Catherine K; Kouskoura, Maria G; Koundourellis, John E

2011-01-01

Different HPLC chromatographic systems were investigated on a C18 ACE 5 pm, 150 x 4.6 mm id column for the determination of tymazoline, tramazoline, and antazoline, with either naphazoline or xylometazoline, in commercial preparations. For the development and optimization of the systems, a Response Surface Method (r=0.925-0.980) was used to illustrate the changes in k as a function of pH values and different salt concentrations. The simultaneous separation of 2-imidazolines was accomplished at 40 degrees C with 0.01 M ammonium acetate-methanol (50+50, v/v, pH 6.0) mobile phase at a flow rate of 1.2 mL/min. In order to deal with the usual coexistence of 2-imidazolines with benzethonium and benzalkonium chloride preservatives, it was necessary to use another chromatographic system, 0.01 M ammonium acetate-methanol (50+50, v/v) mobile phase on a cyano ACE 5 pm, 150 x 4.6 mm id column. As part of a more thorough theoretical investigation, a partial least-squares (PLS) technique was used for modeling the RP-HPLC retention data. The model was based on molecular structure descriptors of the analytes' X variables and on their retention time (Log K) Y. The goodness of fit was estimated by the PLS correlation coefficient (r2) and root mean square error of estimation values, which were 0.994 and 0.0479, respectively.

Simultaneous determination of eight major steroids from Polyporus umbellatus by high-performance liquid chromatography coupled with mass spectrometry detections.

PubMed

Zhao, Ying-yong; Cheng, Xian-long; Zhang, Yongmin; Zhao, Ye; Lin, Rui-chao; Sun, Wen-ji

2010-02-01

Polyporus umbellatus is a widely used diuretic herbal medicine. In this study, a high-performance liquid chromatography coupled with atmospheric pressure chemical ionization-mass spectrometric detection (HPLC-APCI-MS) method was developed for qualitative and quantitative analysis of steroids, as well as for the quality control of Polyporus umbellatus. The selectivity, reproducibility and sensitivity were compared with HPLC with photodiode array detection and evaporative light scattering detection (ELSD). Selective ion monitoring in positive mode was used for qualitative and quantitative analysis of eight major components and beta-ecdysterone was used as the internal standard. Limits of detection and quantification fell in the ranges 7-21 and 18-63 ng/mL for the eight analytes with an injection of 10 microL samples, and all calibration curves showed good linear regression (r(2) > 0.9919) within the test range. The quantitative results demonstrated that samples from different localities showed different qualities. Advantages, in comparison with conventional HPLC-diode array detection and HPLC-ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements along with characteristic retention time, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Polyporus umbellatus matrixes. (c) 2009 John Wiley & Sons, Ltd.

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.

A validated HPLC-UV method for the analysis of galloylquinic acid derivatives and flavonoids in Copaifera langsdorffii leaves.

PubMed

Motta, Erick Vicente da Silva; da Costa, Juliana de Carvalho; Bastos, Jairo Kenupp

2017-09-01

Copaifera langsdorffii Desf. (Fabaceae, Caesalpinioideae), popularly known as "copaiba" or "pau d'óleo", is a species of tree that is found throughout Brazil. The leaves of this tree are used in folk medicine to treat kidney stones. Galloylquinic acid derivatives and flavonoids are the main secondary metabolites found in C. langsdorffii leaves and are likely to be responsible for the effectiveness of this treatment. As an attempt to produce a phytotherapic, we have developed a reliable HPLC-UV method for the quality control of C. langsdorffii leaves. Phenolic compounds were extracted from C. langsdorffii leaves using 70% aqueous ethanol as the extraction solvent. HPLC-UV analyses were carried out on a Synergi Polar-RP column (100×3.0mm, 2.5μm), and the mobile phase was made up of formic acid-water (0.1:99.9, solvent A), and isopropanol-methanol-acetonitrile (5:40:60, solvent B). The elution gradient was A:B (90:10 to 85:15) in 8.0min, followed by A:B (85:15 to 64:36) up to 30.0min, using a flow rate of 0.7mL/min, and UV detection at 280nm. This method was used to quantify nine galloylquinic acid derivatives and two flavonoids, which gave a good detection response and linearity in the range of 1.88-110.0μg/mL. Furthermore, the detection and quantification limits ranged from 0.070 to 0.752μg/mL, and 0.211-2.278μg/mL respectively, with a maximum RSD of 4.18%. The method is reliable for the quality control of C. langsdorffii raw material, its hydroethanolic extract, and could potentially be used to quantify these compounds in other Copaifera species. Copyright © 2017 Elsevier B.V. All rights reserved.

Carrier mediated hollow fiber liquid phase microextraction combined with HPLC-UV for preconcentration and determination of some tetracycline antibiotics.

PubMed

Shariati, Shahab; Yamini, Yadollah; Esrafili, Ali

2009-02-01

In the present study, a simple and efficient preconcentration method was developed using carrier mediated three phase liquid phase microextraction prior to HPLC-UV for simultaneous extraction and determination of trace amounts of highly hydrophilic tetracycline antibiotics including tetracycline (TCN), oxytetracycline (OTCN) and doxycycline (DCN) in bovine milk, human plasma and water samples. For extraction, 11.0 mL of the aqueous sample containing TCNs and 0.05 M Na(2)HPO(4) (9.10.995). Finally, applicability of the proposed method was successfully confirmed by extraction and determination of the drugs in water and plasma samples as well as in bovine milk samples with low and high fat contents. Comparing to the traditional methods, the proposed method exhibits high sensitivity and high preconcentration factors as well as good precision. The extraction setup is simple and due to active transport of analytes, high cleanup effect and good selectivity are obtained in extraction process.

Novel and sensitive reversed-phase high-pressure liquid chromatography method with electrochemical detection for the simultaneous and fast determination of eight biogenic amines and metabolites in human brain tissue.

PubMed

Van Dam, Debby; Vermeiren, Yannick; Aerts, Tony; De Deyn, Peter Paul

2014-08-01

A fast and simple RP-HPLC method with electrochemical detection (ECD) and ion pair chromatography was developed, optimized and validated in order to simultaneously determine eight different biogenic amines and metabolites in post-mortem human brain tissue in a single-run analytical approach. The compounds of interest are the indolamine serotonin (5-hydroxytryptamine, 5-HT), the catecholamines dopamine (DA) and (nor)epinephrine ((N)E), as well as their respective metabolites, i.e. 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), 5-hydroxy-3-indoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). A two-level fractional factorial experimental design was applied to study the effect of five experimental factors (i.e. the ion-pair counter concentration, the level of organic modifier, the pH of the mobile phase, the temperature of the column, and the voltage setting of the detector) on the chromatographic behaviour. The cross effect between the five quantitative factors and the capacity and separation factors of the analytes were then analysed using a Standard Least Squares model. The optimized method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). Our human brain tissue sample preparation procedure is straightforward and relatively short, which allows samples to be loaded onto the HPLC system within approximately 4h. Additionally, a high sample throughput was achieved after optimization due to a total runtime of maximally 40min per sample. The conditions and settings of the HPLC system were found to be accurate with high intra and inter-assay repeatability, recovery and accuracy rates. The robust analytical method results in very low detection limits and good separation for all of the eight biogenic amines and metabolites in this complex mixture of biological analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel reverse phase high-performance liquid chromatography method for standardization of Orthosiphon stamineus leaf extracts

PubMed Central

Saidan, Noor Hafizoh; Aisha, Abdalrahim F.A.; Hamil, Mohd Shahrul Ridzuan; Majid, Amin Malik Shah Abdul; Ismail, Zhari

2015-01-01

Background: Orthosiphon stamineus Benth. (Lamiaceae) is a traditional medicinal plant which has been used in treating various ailments such as kidney diseases, bladder inflammation, arthritis and diabetes. The leaves contain high concentration of phenolic compounds, thus, rosmarinic acid (RA), 3’-hydroxy-5, 6, 7, 4’-tetramethoxyflavone (TMF), sinensetin (SIN) and eupatorin (EUP) were chosen as a marker compounds for standardization of various O. stamineus leaf extracts. Objective: The aim was to develop and validate a new high-performance liquid chromatography (HPLC) method for quantification of 4 marker compounds (RA, TMF, SIN, EUP) in various O. stamineus leaf extracts. Materials and Methods: The method was developed and validated using RP-HPLC-diode-array detection at 320 nm for accuracy, precision and limits of detection and was applied for quantification of it markers in five different extracts prepared in solvents with increasing polarity, using a gradient mobile phase 0.1% formic acid: Acetonitrile at a flow rate of 1 ml/min on reverse phase acclaim polar advantage II C18 column (3 μm, 3 × 150 mm) with 18 min separation time. Results: The developed method provided satisfactory precision, and the accuracy of this method was in the range of 90.2% to 105.5%. All of 4 compounds showed good linearity at R2 > 0.999. Conclusion: The developed method is a simple, cost effective with shorter run time (18 min) in comparison to previous methods (30 min) and utilization of environmental-friendly solvents system. Therefore, this method has the potential to replace currently used methods in the routine standardization work of O. stamineus extracts, raw materials and its commercial products. PMID:25598631

High-performance liquid chromatographic characterization of some medical plant extracts used in cosmetic formulas.

PubMed

Schulz, H; Albroscheit, G

1988-06-17

Rapid and reliable methods are presented for the characterization of biologically active and/or characteristic constituents in aqueous extracts of Hamamelis virginiana, Matricaria chamomilla, Achillea millefolium, Thymus vulgaris, Althaea officinalis and Cinchonia spp. Prior to high-performance liquid chromatographic (HPLC) separation a clean-up step was performed using a solid-phase extraction system. The purified extracts were analysed by HPLC coupled with a diode-array detector and a fluorescence detector. In some instances, previously unreported components of the aqueous plant extracts were found.

Gas and liquid chromatography with inductively coupled plasma mass spectrometry detection for environmental speciation analysis — advances and limitations

NASA Astrophysics Data System (ADS)

Szpunar, Joanna; McSheehy, Shona; Połeć, Kasia; Vacchina, Véronique; Mounicou, Sandra; Rodriguez, Isaac; Łobiński, Ryszard

2000-07-01

Recent advances in the coupling of gas chromatography (GC) and high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP MS) and their role in trace element speciation analysis of environmental materials are presented. The discussion is illustrated with three research examples concerning the following topics: (i) development and coupling of multicapillary microcolumn GC with ICP MS for speciation of organotin in sediment and biological tissue samples; (ii) speciation of arsenic in marine algae by size-exclusion-anion-exchange HPLC-ICP MS; and (iii) speciation of cadmium in plant cell cultures by size-exclusion HPLC-ICP MS. Particular attention is paid to the problem of signal identification in ICP MS chromatograms; the potential of electrospray MS/MS for this purpose is highlighted.

Quantification of cortisol in human eccrine sweat by liquid chromatography - tandem mass spectrometry.

PubMed

Jia, Min; Chew, Wade M; Feinstein, Yelena; Skeath, Perry; Sternberg, Esther M

2016-03-21

Cortisol has long been recognized as the "stress biomarker" in evaluating stress related disorders. Plasma, urine or saliva are the current source for cortisol analysis. The sampling of these biofluids is either invasive or has reliability problems that could lead to inaccurate results. Sweat has drawn increasing attention as a promising source for non-invasive stress analysis. A sensitive HPLC-MS/MS method was developed for the quantitation of cortisol ((11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione) in human eccrine sweat. At least one unknown isomer that has previously not been reported and could potentially interfere with quantification was separated from cortisol with mixed mode RP HPLC. Detection of cortisol was carried out using atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) in positive ion mode, using cortisol-9,11,12,12-D4 as internal standard. LOD and LOQ were estimated to be 0.04 ng ml(-1) and 0.1 ng ml(-1), respectively. Linear range of 0.10-25.00 ng ml(-1) was obtained. Intraday precision (2.5%-9.7%) and accuracy (0.5%-2.1%), interday precision (12.3%-18.7%) and accuracy (7.1%-15.1%) were achieved. This method has been successfully applied to the cortisol analysis of human eccrine sweat samples. This is the first demonstration that HPLC-MS/MS can be used for the sensitive and highly specific determination of cortisol in human eccrine sweat in the presence of at least one isomer that has similar hydrophobicity as cortisol. This study demonstrated that human eccrine sweat could be used as a promising source for non-invasive assessment of stress biomarkers such as cortisol and other steroid hormones.

Thermal Stability of Rhodopsin and Progression of Retinitis Pigmentosa

PubMed Central

Liu, Monica Yun; Liu, Jian; Mehrotra, Devi; Liu, Yuting; Guo, Ying; Baldera-Aguayo, Pedro A.; Mooney, Victoria L.; Nour, Adel M.; Yan, Elsa C. Y.

2013-01-01

Over 100 point mutations in the rhodopsin gene have been associated with retinitis pigmentosa (RP), a family of inherited visual disorders. Among these, we focused on characterizing the S186W mutation. We compared the thermal properties of the S186W mutant with another RP-causing mutant, D190N, and with WT rhodopsin. To assess thermal stability, we measured the rate of two thermal reactions contributing to the thermal decay of rhodopsin as follows: thermal isomerization of 11-cis-retinal and hydrolysis of the protonated Schiff base linkage between the 11-cis-retinal chromophore and opsin protein. We used UV-visible spectroscopy and HPLC to examine the kinetics of these reactions at 37 and 55 °C for WT and mutant rhodopsin purified from HEK293 cells. Compared with WT rhodopsin and the D190N mutant, the S186W mutation dramatically increases the rates of both thermal isomerization and dark state hydrolysis of the Schiff base by 1–2 orders of magnitude. The results suggest that the S186W mutant thermally destabilizes rhodopsin by disrupting a hydrogen bond network at the receptor's active site. The decrease in the thermal stability of dark state rhodopsin is likely to be associated with higher levels of dark noise that undermine the sensitivity of rhodopsin, potentially accounting for night blindness in the early stages of RP. Further studies of the thermal stability of additional pathogenic rhodopsin mutations in conjunction with clinical studies are expected to provide insight into the molecular mechanism of RP and test the correlation between rhodopsin's thermal stability and RP progression in patients. PMID:23625926

Immunotherapeutic Vaccine as an Alternative Treatment to Overcome Drug-Resistant Ovarian Cancer

DTIC Science & Technology

2012-07-01

2.7. Fractionation of SCX purified peptides by C-18 chromatography TheSCXpurified iTRAQ labeledpeptidemixturewas fractionated by a Dionex C-18 RP... chromatography as described above for the MHC peptides. All the iTRAQ labeled synthetic peptides were analyzed by a Dionex 3000 nano ultimate HPLC (Sunnyvale CA... chromatography and LC-MS/MS. Proteomics 7:4292-302. 15. Pachuk CJ, Ciccarelli RB, Samuel M, Bayer ME, Troutman RD, Zurawski DV, Schauer JI, Higgins TJ

In vitro angiotensin I converting enzyme inhibition by a peptide isolated from Chiropsalmus quadrigatus Haeckel (box jellyfish) venom hydrolysate.

PubMed

So, Pamela Berilyn T; Rubio, Peter; Lirio, Stephen; Macabeo, Allan Patrick; Huang, Hsi-Ya; Corpuz, Mary Jho-Anne T; Villaflores, Oliver B

2016-09-01

The anti-angiotensin I converting enzyme activity of box jellyfish, Chiropsalmus quadrigatus Haeckel venom hydrolysate was studied. The venom extract was obtained by centrifugation and ultrasonication. Protein concentration of 12.99 μg/mL was determined using Bradford assay. The pepsin and papain hydrolysate was tested for its toxicity by Limit test following the OECD Guideline 425 using 5 female Sprague-Dawley rats. Results showed that the hydrolysate is nontoxic with an LD50 above 2000 mg/kg. In vitro angiotensin I converting enzyme (ACE) inhibitory activity was determined using ACE kit-WST. Isolation of ACE inhibitory peptides using column chromatography with SP-Sephadex G-25 yielded 8 pooled fractions with fraction 3 (86.5%) exhibiting the highest activity. This was followed by reverse phase - high performance liquid chromatography (RP-HPLC) with an octadecyl silica column (Inertsil ODS-3) using methanol:water 15:85 at a flow rate of 1.0 mL/min. Among the 13 fractions separated with the RP-HPLC, fraction 3.5 exhibited the highest ACE inhibitory activity (84.1%). The peptide sequence ACPGPNPGRP (IC50 2.03 μM) from fraction 3.5 was identified using Matrix-assisted laser desorption/ionization with time-of-flight tandem mass spectroscopy analysis (MALDI-TOF/MS). Copyright © 2016 Elsevier Ltd. All rights reserved.

[Analysis of phenylethanoid glycosides of Herba cistanchis by RP-HPLC].

PubMed

Tu, P F; Wang, B; Deyama, T; Zhang, Z G; Lou, Z C

1997-04-01

The Chinese drug "Rou Cong-rong" (Herba Cistanchis) is one of the commonly used drugs in Chinese traditional medicine. It is used to reinforce the vital function of kidney, especially that of the sexual organs and induce laxation, for the treatment of impotence, premature ejaculation in men, infertility, morbid leukorrhea, profuse metrorrhagia in women, and chronic constipation in the aged. This paper deals with the qualitative and quantitative analysis of phenylethanoid glycosides of four species and one variety of Genus Cistanche and 23 lots of commercial crude drugs of Herba Cistanchis by RP-HPLC. The results were as follows: the chemical constituents of Cistanche deserticola Ma, C. salsa (C. A. Mey) G. Beck, C. salsa var. albiflora P. F. Tu et Z. C. Lou and C. tubulosa were similar while those of C. sinensis were different from the others; the contents of echinacoside and acteoside of C. salsa, which were 2.13% and 1.51%, were the highest of the genus Cistanche. An ODS column (Alltima C18, 5 microns, 250 x 4.6 mm) was employed. Linear gradient elution of acetonitrile--1.5% acetic acid was used as mobile phase, and concentration of acetontrile was from 8% to 20% (0-60 min) in the qualitative analysis, and from 11.5 to 20% (0-35 min) in the quantitative analysis. The flow rate was 1.2 ml.min-1. The detection wavelength was set at 335 nm.

Isolation, cloning, and characterization of the 2S albumin: a new allergen from hazelnut.

PubMed

Garino, Cristiano; Zuidmeer, Laurian; Marsh, Justin; Lovegrove, Alison; Morati, Maria; Versteeg, Serge; Schilte, Piet; Shewry, Peter; Arlorio, Marco; van Ree, Ronald

2010-09-01

2S albumins are the major allergens involved in severe food allergy to nuts, seeds, and legumes. We aimed to isolate, clone, and express 2S albumin from hazelnut and determine its allergenicity. 2S albumin from hazelnut extract was purified using size exclusion chromatography and RP-HPLC. After N-terminal sequencing, degenerated and poly-d(T) primers were used to clone the 2S albumin sequence from hazelnut cDNA. After expression in Escherichia coli and affinity purification, IgE reactivity was evaluated by Immunoblot/ImmunoCAP (inhibition) analyses using sera of nut-allergic patients. N-terminal sequencing of a approximately 10 kDa peak from size exclusion chromatography/RP-HPLC gave two sequences highly homologous to pecan 2S albumin, an 11 amino acid (aa) N-terminal and a 10 aa internal peptide. The obtained clone (441 bp) encoded a 147 aa hazelnut 2S albumin consisting of a putative signal peptide (22 aa), a linker peptide (20 aa), and the mature protein sequence (105 aa). The latter was successfully expressed in E. coli. Both recombinant and natural 2S albumin demonstrated similar IgE reactivity in Immunoblot/ImmunoCAP (inhibition) analyses. We confirmed the postulated role of hazelnut 2S albumin as an allergen. The availability of recombinant molecules will allow establishing the importance of hazelnut 2S albumin for hazelnut allergy.