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

PubMed

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

2017-01-01

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

[Studies on HPLC fingerprint chromatogram of Folium Fici Microcarpa].

PubMed

Fang, Zhi-Jian; Dai, Zhen; Li, Shu-Yuan

2008-10-01

To establish a sensitive and specific method for quality control of Folium Fici Microcarpa, HPLC method was applied for studies on the fingerprint chromatogram of Folium Fici Microcarpa. Isovitexin was used as reference substance to evaluate the chromatogram of 10 samples from different regions and 12 samples collected in different months. The result revealed that all the chromatographic peaks were seperated efficiently. There were 17 common peaks showed in the fingerprint chromatogram. The method of fingerprint chromatogram with characteristic and specificity will be used to identify the quality and evaluate different origins and collection period of Folium Fici Microcarpa.

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.

Analytical capabilities of high performance liquid chromatography - Atmospheric pressure photoionization - Orbitrap mass spectrometry (HPLC-APPI-Orbitrap-MS) for the trace determination of novel and emerging flame retardants in fish.

PubMed

Zacs, D; Bartkevics, V

2015-10-22

A new analytical method was established and validated for the analysis of 27 brominated flame retardants (BFRs), including so called "emerging" and "novel" BFRs (EBFRs and NBFRs) in fish samples. High performance liquid chromatography (HPLC) coupled to Orbitrap mass spectrometry (Orbitrap-MS) employing atmospheric pressure photoionization (APPI) interface operated in negative mode was used for the identification/quantitation of contaminants. HPLC-Orbitrap-MS analysis provided a fast separation of selected analytes within 14 min, thus demonstrating a high throughput processing of samples. The developed methodology was tested by intralaboratory validation in terms of recovery, repeatability, linear calibration ranges, instrumental and method limits of quantitation (i-LOQ and m-LOQ), and where possible, trueness was verified by analysis of certified reference materials (CRMs). Recoveries of analytes were between 80 and 119%, while the repeatability in terms of relative standard deviations (RSDs) was in the range from 1.2 to 15.5%. The measured values for both analyzed CRMs agreed with the provided consensus values, revealing the recovery of reference concentrations in 72-119% range. The elaborated method met the sensitivity criterion according to Commission Recommendation 2014/118/EU on monitoring of BFRs in food products for majority of the compounds. The concentrations of polybrominated diphenyl ethers (PBDEs) in real samples determined by HPLC-APPI-Orbitrap-MS method and validated gas chromatography-high-resolution mass spectrometry (GC-HRMS) method were found to be in a good agreement. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

Bandoniene, Donata; Murkovic, Michael

2002-04-24

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

Analysis of Thiodiglycol: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS777

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

Owens, J; Koester, C

The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method for the analysis of thiodiglycol, the breakdown product of the sulfur mustard HD, in water by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), titled Method EPA MS777 (hereafter referred to as EPA CRL SOP MS777). This draft standard operating procedure (SOP) was distributed to multiple EPA laboratories and to Lawrence Livermore National Laboratory, which was tasked to serve as a reference laboratory for EPA's Environmental Reference Laboratory Network (ERLN) and to develop and validate analytical procedures. The primary objective of this study was to verifymore » the analytical procedures described in MS777 for analysis of thiodiglycol in aqueous samples. The gathered data from this study will be used to: (1) demonstrate analytical method performance; (2) generate quality control acceptance criteria; and (3) revise the SOP to provide a validated method that would be available for use during a homeland security event. The data contained in this report will be compiled, by EPA CRL, with data generated by other EPA Regional laboratories so that performance metrics of Method EPA MS777 can be determined.« less

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

PubMed

Rácz, Anita; Héberger, Károly; Fodor, Marietta

2016-09-01

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

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.

A novel strategy with standardized reference extract qualification and single compound quantitative evaluation for quality control of Panax notoginseng used as a functional food.

PubMed

Li, S P; Qiao, C F; Chen, Y W; Zhao, J; Cui, X M; Zhang, Q W; Liu, X M; Hu, D J

2013-10-25

Root of Panax notoginseng (Burk.) F.H. Chen (Sanqi in Chinese) is one of traditional Chinese medicines (TCMs) based functional food. Saponins are the major bioactive components. The shortage of reference compounds or chemical standards is one of the main bottlenecks for quality control of TCMs. A novel strategy, i.e. standardized reference extract based qualification and single calibrated components directly quantitative estimation of multiple analytes, was proposed to easily and effectively control the quality of natural functional foods such as Sanqi. The feasibility and credibility of this methodology were also assessed with a developed fast HPLC method. Five saponins, including ginsenoside Rg1, Re, Rb1, Rd and notoginsenoside R1 were rapidly separated using a conventional HPLC in 20 min. The quantification method was also compared with individual calibration curve method. The strategy is feasible and credible, which is easily and effectively adapted for improving the quality control of natural functional foods such as Sanqi. Copyright © 2013 Elsevier B.V. All rights reserved.

Simultaneous determination of dextromethorphan HBr and bromhexine HCl in tablets by first-derivative spectrophotometry.

PubMed

Tantishaiyakul, V; Poeaknapo, C; Sribun, P; Sirisuppanon, K

1998-06-01

A rapid, simple and direct assay procedure based on first-derivative spectrophotometry, using a zero-crossing and peak-to-base measurement at 234 and 324 nm, respectively, has been developed for the specific determination of dextromethorphan HBr and bromhexine HCl in tablets. Calibration graphs were linear with the correlation coefficients of 0.9999 for both analytes. The limit of detections were 0.033 and 0.103 microgram ml-1 for dextromethorphan HBr and bromhexine HCl, respectively. A HPLC method has been developed as the reference method. The results obtained by the first-derivative spectrophotometry were in good agreement with those found by the HPLC method.

Direct determination of neonicotinoid insecticides in an analytically challenging crop such as Chinese chives using selective ELISAs.

PubMed

Watanabe, Eiki; Miyake, Shiro

2018-06-05

Easy-to-use commercial kit-based enzyme-linked immunosorbent assays (ELISAs) have been used to detect neonicotinoid dinotefuran, clothianidin and imidacloprid in Chinese chives, which are considered a troublesome matrix for chromatographic techniques. Based on their high water solubility, water was used as an extractant. Matrix interference could be avoided substantially just diluting sample extracts. Average recoveries of insecticides from spiked samples were 85-113%, with relative standard deviation of <15%. The concentrations of insecticides detected from the spiked samples with the proposed ELISA methods correlated well with those by the reference high-performance liquid chromatography (HPLC) method. The residues analyzed by the ELISA methods were consistently 1.24 times that found by the HPLC method, attributable to loss of analyte during sample clean-up for HPLC analyses. It was revealed that the ELISA methods can be applied easily to pesticide residue analysis in troublesome matrix such as Chinese chives.

Identification, characterization, synthesis and HPLC quantification of new process-related impurities and degradation products in retigabine.

PubMed

Douša, Michal; Srbek, Jan; Rádl, Stanislav; Cerný, Josef; Klecán, Ondřej; Havlíček, Jaroslav; Tkadlecová, Marcela; Pekárek, Tomáš; Gibala, Petr; Nováková, Lucie

2014-06-01

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2017-01-01

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

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.

Automated HPLC assay for urinary collagen cross-links: effect of age, menopause, and metabolic bone diseases.

PubMed

Kraenzlin, Marius E; Kraenzlin, Claude A; Meier, Christian; Giunta, Cecilia; Steinmann, Beat

2008-09-01

The pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD) are established markers of bone resorption. We evaluated the analytical and clinical performance of a commercially available PYD HPLC assay and established reference intervals in children and adults. We used a commercially available reagent set (Chromsystems Instruments & Chemicals) to measure PYD and DPD in 319 healthy controls (156 premenopausal women, 80 healthy men, and 83 healthy children age 1 month to 14 years) and 397 patients with metabolic bone diseases (postmenopausal osteoporosis, n = 175; male osteoporosis, n = 176; hyperparathyroidism, n = 17; hyperthyroidism, n = 19; Paget disease, n = 10). The mean intraassay and interassay CVs were <6% and <8% for both PYD and DPD, respectively. The reference interval was constant for premenopausal women in the age group 20-49 years. In men, cross-link values peaked at 20-29 years and decreased thereafter. Women with postmenopausal osteoporosis had significantly higher PYD (51%) and DPD (58%) values compared to premenopausal women. Similar results were found in osteoporotic men. In children the highest values were found in the first weeks and months after birth, followed by a decrease of 50%-60% at age 11-14 years. In metabolic bone diseases cross-link concentrations were significantly increased. The DPD:PYD ratio (mean value approximately 0.2) was remarkably constant in all populations evaluated. The automated HPLC assay is a precise and convenient method for PYD and DPD measurement. We established reference intervals for adult women and men and for children up to 14 years old. The cross-link concentrations we determined by use of this HPLC method confirm its clinical value in enabling identification of increased bone resorption in patients with metabolic bone diseases.

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

PubMed

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

2009-06-01

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

The establishment of tocopherol reference intervals for Hungarian adult population using a validated HPLC method.

PubMed

Veres, Gábor; Szpisjak, László; Bajtai, Attila; Siska, Andrea; Klivényi, Péter; Ilisz, István; Földesi, Imre; Vécsei, László; Zádori, Dénes

2017-09-01

Evidence suggests that decreased α-tocopherol (the most biologically active substance in the vitamin E group) level can cause neurological symptoms, most likely ataxia. The aim of the current study was to first provide reference intervals for serum tocopherols in the adult Hungarian population with appropriate sample size, recruiting healthy control subjects and neurological patients suffering from conditions without symptoms of ataxia, myopathy or cognitive deficiency. A validated HPLC method applying a diode array detector and rac-tocol as internal standard was utilized for that purpose. Furthermore, serum cholesterol levels were determined as well for data normalization. The calculated 2.5-97.5% reference intervals for α-, β/γ- and δ-tocopherols were 24.62-54.67, 0.81-3.69 and 0.29-1.07 μm, respectively, whereas the tocopherol/cholesterol ratios were 5.11-11.27, 0.14-0.72 and 0.06-0.22 μmol/mmol, respectively. The establishment of these reference intervals may improve the diagnostic accuracy of tocopherol measurements in certain neurological conditions with decreased tocopherol levels. Moreover, the current study draws special attention to the possible pitfalls in the complex process of the determination of reference intervals as well, including the selection of study population, the application of internal standard and method validation and the calculation of tocopherol/cholesterol ratios. Copyright © 2017 John Wiley & Sons, Ltd.

Simultaneous Determination of Multiple Ginsenosides in Panax ginseng Herbal Medicines with One Single Reference Standard.

PubMed

Wu, Chunwei; Guan, Qingxiao; Wang, Shumei; Rong, Yueying

2017-01-01

Root of Panax ginseng C. A. Mey (Renseng in Chinese) is a famous Traditional Chinese Medicine. Ginsenosides are the major bioactive components. However, the shortage and high cost of some ginsenoside reference standards make it is difficult for quality control of P. ginseng . A method, single standard for determination of multicomponents (SSDMC), was developed for the simultaneous determination of nine ginsenosides in P. ginseng (ginsenoside Rg 1 , Re, Rf, Rg 2 , Rb 1 , Rc, Rb 2 , Rb 3 , Rd). The analytes were separated on Inertsil ODS-3 C18 (250 mm × 4.6 mm, 5 μm) with gradient elution of acetonitrile and water. The flow rate was 1 mL/min and detection wavelength was set at 203 nm. The feasibility and accuracy of SSDMC were checked by the external standard method, and various high-performance liquid chromatographic (HPLC) instruments and chromatographic conditions were investigated to verify its applicability. Using ginsenoside Rg 1 as the internal reference substance, the contents of other eight ginsenosides were calculated according to conversion factors (F) by HPLC. The method was validated with linearity ( r 2 ≥ 0.9990), precision (relative standard deviation [RSD] ≤2.9%), accuracy (97.5%-100.8%, RSD ≤ 1.6%), repeatability, and stability. There was no significant difference between the SSDMC method and the external standard method. New SSDMC method could be considered as an ideal mean to analyze the components for which reference standards are not readily available. A method, single standard for determination of multicomponents (SSDMC), was established by high-performance liquid chromatography for the simultaneous determination of nine ginsenosides in Panax ginseng (ginsenoside Rg1, Re, Rf, Rg2, Rb1, Rc, Rb2, Rb3, Rd)Various chromatographic conditions were investigated to verify applicability of FsThe feasibility and accuracy of SSDMC were checked by the external standard method. Abbreviations used: DRT: Different value of retention time; F: Conversion factor; HPLC: High-performance Liquid Chromatography; LOD: Limit of detection; LOQ: Limit of quantitation; PD: Percent difference; PPD: 20(S)-protopanaxadiol; PPT: 20(S)-protopanaxatriol; RSD: Relative standard deviation; SSDMC: Single Standard for Determination of Multicomponents; TCM: Traditional Chinese Medicine.

Simultaneous Determination of Ursodeoxycholic Acid and Chenodeoxycholic Acid in Pharmaceutical Dosage Form by HPLC-UV Detection.

PubMed

Khairy, Mostafa A; Mansour, Fotouh R

2017-01-01

A reversed-phase HPLC method was developed for the simultaneous determination of ursodeoxycholic acid (UDCA) and the epimeric isomer, chenodeoxycholic acid (CDCA), in their synthetic mixtures and in tablet dosage form. The proposed HPLC method uses a C18 column and mobile phase consisting of an acetonitrile-phosphate buffer mixture (pH 2.3, 100 mM; 50 + 50, v/v) at a flow rate of 2.0 mL/min with UV detection at 210 nm. The method was validated according to the International Conference on Harmonization guidelines; and linearity, range, accuracy, precision, robustness, and system suitability were studied. The LOD and LOQ were also calculated and found to be 1.23 and 3.73 μg/mL for UDCA and 0.83 and 2.52 μg/mL for CDCA, respectively. The method was adapted for UHPLC, in which baseline separation was achieved in <2.5 min. The assay results of Ursomix tablets by the developed method were statistically compared with those obtained by the reference method using t- and F-tests, and no significant differences were observed.

Development and certification of a coal fly ash certified reference material for selected polycyclic aromatic hydrocarbons.

PubMed

Cao, X; Xu, X; Cui, W; Xi, Z

2001-08-01

The development and certification of a coal fly ash certified reference material (CRM) for polycyclic aromatic hydrocarbons (PAH) is described; this is the first natural matrix CRM for organic environmental analysis in China. The homogeneity and stability of this material have been tested by HPLC. The concentrations of several PAH were determined by use of two independent, different methods--solvent extraction-HPLC analysis with UV detection coupled with fluorescence detection (FLD) and solvent extraction, isolation with a silica column, and GC analysis with flame ionization detection (FID). Five certified values were determined: phenanthrene 7.1 +/- 2.6 microg g(-1), anthracene 2.0 +/- 0.8 microg g(-1), fluoranthene 7.4 +/- 1.9 microg g(-1), pyrene 7 +/- 2 microg g(-1), and benzo[a]pyrene 1.3 +/- 0.3 microg g(-1). Reference values for several other PAH are also suggested.

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

USGS Publications Warehouse

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

2002-01-01

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

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

PubMed

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

2002-10-09

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

Simultaneous determination of nine saponins from Panax notoginseng using HPLC and pressurized liquid extraction.

PubMed

Wan, J B; Lai, C M; Li, S P; Lee, M Y; Kong, L Y; Wang, Y T

2006-04-11

A HPLC and pressurized liquid extraction (PLE) method was developed for simultaneous determination of nine saponins, including notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3 and Rd in Panax notoginseng. The analysis was performed on C18 column with water-acetonitrile gradient elution and the investigated saponins were authenticated by comparing retention time and mass spectra with their reference compounds. Several methods including PLE, ultrasonication, soxhlet extraction and immersion were used for sample preparation and their extraction efficiency was compared. The results showed that PLE has the highest extraction efficiency and repeatability, which would be valuable on standardization of sample preparation for quality control of Chinese medicines. The developed HPLC and PLE is an effective approach for simultaneously quantitative determination of sapoinins in P. notoginseng, which could be used for quality control of P. notoginseng and its preparations.

Chemical fingerprint and metabolic profile analysis of Citrus reticulate 'Chachi' decoction by HPLC-PDA-IT-MS(n) and HPLC-Quadrupole-Orbitrap-MS method.

PubMed

Ye, Xiaolan; Cao, Di; Zhao, Xin; Song, Fenyun; Huang, Qinghua; Fan, Guorong; Wu, Fuhai

2014-11-01

A method incorporating HPLC-PDA-IT-MS(n) with HPLC-Quadrupole-Orbitrap-MS was developed for the investigation of chemical fingerprint of Citrus reticulate 'Chachi' decoction (CRCD) and metabolic profile of SD rat plasma sample after oral administration of CRCD (1.5 g herb/kg). A total of 27 chemical constituents of CRCD were identified from their MW, UV spectra, MS(n) data and retention behavior by comparing the results with those of the reference standards or literature. And 43 compounds were detected in dosed SD rat plasma samples, including 9 prototypes which were identified as hesperetin, isosinensetin, sinensetin, tetramethyl-O-isoscutellarein, nobiletin, tetramethyl-O-scutellarein, HMF (3,5,6,7,8,3',4'-heptamethoxyflavone), tangeretin and 5-demethylnobiletin and 34 metabolites underwent metabolic process of demethylation, glucuronide conjugation, sulfate conjugation or mixed modes. This is the first research for the metabolic profile of CRCD in SD rats, which could lay a foundation for the further studies of CRC or its formulation. Copyright © 2014 Elsevier B.V. All rights reserved.

A Simple and Rapid HPLC-PDA MS Method for the Profiling of Citrus Peels and Traditional Italian Liquors.

PubMed

Guccione, Clizia; Bergonzi, Maria Camilla; Piazzini, Vieri; Bilia, Anna Rita

2016-07-01

A chromatographic method for the qualitative and quantitative characterization of peels and preparations based on different species of Citrus was developed in order to obtain a complete profile of the constituents, including flavonoids and protoalkaloids. Commercial peels of sweet orange, lemon, mandarin, and grapefruit were analyzed. Seventeen constituents including flavanones, flavones, polymethoxyflavones, and protoalkaloids were identified by HPLC-PDA, HPLC-MS, and HPLC-MS/MS using a comparison of retention times and UV-Vis and MS spectra with reference standards and literature data. The total amount of flavanones [neoeriocitrin (5), naringin (8) and hesperidin (9)] and polymethoxflavones [sinensetin (12), nobiletin (14), 3,5,6,7,8,3',4'-heptamethoxyflavone (15), and tangeretin (16)] was determined and expressed as naringin (8) or hesperidin (9), and sinensetin (12), respectively. The protoalkaloid synephrine was detected in all samples, except in grapefruit, but its content was lower than the limit of quantification. Qualitative and quantitative chemical profiles of three different Italian aromatic liquors ("Limoncello", "Arancello", and "Mandarinetto"), prepared according to traditional recipes, were also analyzed. Georg Thieme Verlag KG Stuttgart · New York.

[Research on chemical fingerprint chromatograms of Sinopodophyllum hexandrum].

PubMed

Wang, Ai-Hua; Liu, Guang-Xue; Xu, Feng; Shang, Ming-Ying; Cai, Shao-Qing

2013-10-01

To study the HPLC fingerprint of Sinopodophylli Fructus, compare the major chemical differences in fruit of Sinopodophyllum hexandrum as well as the roots and rhizomas of S. hexandrum, and provide scientific evidence for clinical application and quality control. HPLC fingerprint method was used to analyze 12 fruits of S. hexandrum. A total of 20 common peaks were confirmed, and 12 peaks in the HPLC fingerprint were identified. Furthermore, similarity evaluation method and clustering analysis method were introduced to compare HPLC chromatograms, between fruits and underground parts. In this paper, quercetin-3-O-beta-D-glucopyranosid, kaempferol-3-O-beta-D-glucopyranoside, 8-prenylquercetin and 8-prenylquercetin 3-methyl ether were firstly reported in Sinopodophylli Fructus. Among the existing fingerprint research, a total of 11 peaks were identified for the first time, containing 9 flavonoids and 2 lignans. The chemical constituents differed significantly in different medicinal parts of S. hexandrum. Prenylflavonoid compounds were the main constituents of Sinopodophylli Fructus. However, podophyllotoxin, flavonoids with simple substituent groups and flavonoid glycosides were the major ingredients in the roots and rhizomas of S. hexandrum. This method can be used for the quality control of Sinopodophylli Fructus and the roots and rhizomas of S. hexandrum. It has provided a reference for the pharmacodynamic differences of the two different parts.

Automated solid-phase extraction coupled online with HPLC-FLD for the quantification of zearalenone in edible oil.

PubMed

Drzymala, Sarah S; Weiz, Stefan; Heinze, Julia; Marten, Silvia; Prinz, Carsten; Zimathies, Annett; Garbe, Leif-Alexander; Koch, Matthias

2015-05-01

Established maximum levels for the mycotoxin zearalenone (ZEN) in edible oil require monitoring by reliable analytical methods. Therefore, an automated SPE-HPLC online system based on dynamic covalent hydrazine chemistry has been developed. The SPE step comprises a reversible hydrazone formation by ZEN and a hydrazine moiety covalently attached to a solid phase. Seven hydrazine materials with different properties regarding the resin backbone, pore size, particle size, specific surface area, and loading have been evaluated. As a result, a hydrazine-functionalized silica gel was chosen. The final automated online method was validated and applied to the analysis of three maize germ oil samples including a provisionally certified reference material. Important performance criteria for the recovery (70-120 %) and precision (RSDr <25 %) as set by the Commission Regulation EC 401/2006 were fulfilled: The mean recovery was 78 % and RSDr did not exceed 8 %. The results of the SPE-HPLC online method were further compared to results obtained by liquid-liquid extraction with stable isotope dilution analysis LC-MS/MS and found to be in good agreement. The developed SPE-HPLC online system with fluorescence detection allows a reliable, accurate, and sensitive quantification (limit of quantification, 30 μg/kg) of ZEN in edible oils while significantly reducing the workload. To our knowledge, this is the first report on an automated SPE-HPLC method based on a covalent SPE approach.

Two-dimensional multi-heart cutting centrifugal partition chromatography-liquid chromatography for the preparative isolation of antioxidants from Edelweiss plant.

PubMed

Marlot, Léa; Batteau, Magali; Escofet, Marie-Claire; Nuccio, Sylvie; Coquoin, Véronique; De Vaumas, René; Faure, Karine

2017-06-30

The Edelweiss plant has been recognized as a very valuable source of anti-aging principles due to its composition of antioxidants compounds: leontopodic acid A and 3,5-dicaffeoylquinic acid. In this work, off-line multi-heart cutting CPC-LC separation was set up at industrial scale in order to isolate and produce new high quality reference material of these two antioxidants from Edelweiss. For this purpose, CPC and HPLC methods were developed and optimized at laboratory scale and a comprehensive CPCxHPLC analysis of the crude extract was established. Thereby, the CPC method led to a first separation of the target compounds according to their partition coefficient in the solvent system and the HPLC method was performed on the recovered fractions to lead to a second separation. A 2D CPCxHPLC plot was established in order to know the fractions to select at the industrial scale. Then, the CPC and HPLC methods were transferred at industrial scale and the multi-heart cutting CPC-LC was performed in off-line mode. Using CPC with methyl ter-butyl ether-water 1:1 (v/v) solvent system and LC with Denali C18 column, 2g of crude extract sample were injected and leontopodic acid A and 3,5-dicaffeoylquinic acid were recovered with purity over 97%. The compounds were identified by MS and NMR. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs.

PubMed

Serrano, Ana; van Bommel, Maarten; Hallett, Jessica

2013-11-29

An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41-61% resolution and a decrease of 91-422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds' retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2013-01-01

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

Analysis of Carbamate Pesticides: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS666

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

Owens, J; Koester, C

The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method for analysis of aldicarb, bromadiolone, carbofuran, oxamyl, and methomyl in water by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), titled Method EPA MS666. This draft standard operating procedure (SOP) was distributed to multiple EPA laboratories and to Lawrence Livermore National Laboratory, which was tasked to serve as a reference laboratory for EPA's Environmental Reference Laboratory Network (ERLN) and to develop and validate analytical procedures. The primary objective of this study was to validate and verify the analytical procedures described in MS666 for analysis of carbamatemore » pesticides in aqueous samples. The gathered data from this validation study will be used to: (1) demonstrate analytical method performance; (2) generate quality control acceptance criteria; and (3) revise the SOP to provide a validated method that would be available for use during a homeland security event. The data contained in this report will be compiled, by EPA CRL, with data generated by other EPA Regional laboratories so that performance metrics of Method EPA MS666 can be determined.« less

A comparative uncertainty study of the calibration of macrolide antibiotic reference standards using quantitative nuclear magnetic resonance and mass balance methods.

PubMed

Liu, Shu-Yu; Hu, Chang-Qin

2007-10-17

This study introduces the general method of quantitative nuclear magnetic resonance (qNMR) for the calibration of reference standards of macrolide antibiotics. Several qNMR experimental conditions were optimized including delay, which is an important parameter of quantification. Three kinds of macrolide antibiotics were used to validate the accuracy of the qNMR method by comparison with the results obtained by the high performance liquid chromatography (HPLC) method. The purities of five common reference standards of macrolide antibiotics were measured by the 1H qNMR method and the mass balance method, respectively. The analysis results of the two methods were compared. The qNMR is quick and simple to use. In a new medicine research and development process, qNMR provides a new and reliable method for purity analysis of the reference standard.

Pregnancy and variations of carbohydrate-deficient transferrin levels measured by the candidate reference HPLC method.

PubMed

Bianchi, Vincenza; Ivaldi, Alessandra; Raspagni, Alessia; Arfini, Carlo; Vidali, Matteo

2011-01-01

Contrasting data are available on the diagnostic accuracy of carbohydrate-deficient transferrin (CDT) during pregnancy. These differences may depend in part on how CDT was evaluated and expressed. Here, we report on variations of CDT levels in pregnant women using the high performance liquid chromatography (HPLC) candidate reference method. Alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, mean corpuscular volume, serum transferrin, urine and serum ethyl glucuronide and CDT were measured in 64 women, self-reporting as non-alcohol abusers (age: median 34, IQR: 28-38), at different stages of normal pregnancy (gestational weeks: median 28, IQR: 8-33). CDT was expressed as percentage of disialotransferrin to total transferrin (%CDT). Transferrin was associated with both %CDT (r = 0.66; P < 0.001) and gestational week (r = 0.68; P < 0.001). Interestingly, %CDT was highly correlated with gestational week (r = 0.77; P < 0.001), even after controlling for the effect of transferrin. Moreover, statistically significant differences in %CDT were also evident between women grouped for pregnancy trimester (first trimester: mean 1.01% (SD 0.19); second trimester: 1.30% (SD 0.14); third trimester: 1.53% (SD 0.22); ANOVA P < 0.001). Trend analysis confirmed a proportional increase of %CDT along with pregnancy trimesters (P < 0.001). %CDT, measured with the HPLC candidate reference method, is independently associated with gestational week. Differently from what has been previously reported or expected, the relationship between pregnancy and CDT could be more complex. The diagnostic accuracy of CDT for detecting alcohol abuse in a legal context may be limited in pregnant women and the effect of gestational age should be considered.

Quality evaluation and pattern recognition analyses of marker compounds from five medicinal drugs of Rutaceae family by HPLC/PDA.

PubMed

Zhao, Bing Tian; Kim, Eun Jung; Son, Kun Ho; Son, Jong Keun; Min, Byung Sun; Woo, Mi Hee

2015-08-01

To establish a standard of quality control and to identify different origins for the Rutaceae family [Citri Unshiu Peel (CU), Citri Unshiu Immature Peel (CI), Ponciri Immature Fructus (PI), Aurantii Immature Fructus (AI), and Aurantii Fructus (AU)], 13 standards including rutin (1), narirutin (2), naringin (3), hesperidin (4), neohesperidin (5), neoponcirin (6), poncirin (7), naringenin (8), isosinensetin (9), sinensetin (10), nobiletin (11), heptamethoxyflavone (12), and tangeretin (13) were determined by high performance liquid chromatography (HPLC)/photo-diode array (PDA) analysis. A YMC ODS C18 (250 × 4.6 mm, 5 µm) column was used and the ratio of mobile phases of water (A) and acetonitrile (B) delivered to the column for gradient elution was applied. This method was fully validated with respect to linearity, accuracy, precision, stability, and robustness. The HPLC/PDA method was applied successfully to quantify 13 major compounds in the extracts of CU, CI, PI, AI, and AU. The pattern recognition analysis combined with LC chromatographic data was performed by repeated analysis of 27 reference samples in the above five Rutaceae oriental medicinal drugs. The established HPLC method was rapid and reliable for quantitative analysis and quality control of multiple components in five Rutaceae species with different origins.

Quality Evaluation and Chemical Markers Screening of Salvia miltiorrhiza Bge. (Danshen) Based on HPLC Fingerprints and HPLC-MSn Coupled with Chemometrics.

PubMed

Liang, Wenyi; Chen, Wenjing; Wu, Lingfang; Li, Shi; Qi, Qi; Cui, Yaping; Liang, Linjin; Ye, Ting; Zhang, Lanzhen

2017-03-17

Danshen, the dried root of Salvia miltiorrhiza Bge., is a widely used commercially available herbal drug, and unstable quality of different samples is a current issue. This study focused on a comprehensive and systematic method combining fingerprints and chemical identification with chemometrics for discrimination and quality assessment of Danshen samples. Twenty-five samples were analyzed by HPLC-PAD and HPLC-MS n . Forty-nine components were identified and characteristic fragmentation regularities were summarized for further interpretation of bioactive components. Chemometric analysis was employed to differentiate samples and clarify the quality differences of Danshen including hierarchical cluster analysis, principal component analysis, and partial least squares discriminant analysis. Consistent results were that the samples were divided into three categories which reflected the difference in quality of Danshen samples. By analyzing the reasons for sample classification, it was revealed that the processing method had a more obvious impact on sample classification than the geographical origin, it induced the different content of bioactive compounds and finally lead to different qualities. Cryptotanshinone, trijuganone B, and 15,16-dihydrotanshinone I were screened out as markers to distinguish samples by different processing methods. The developed strategy could provide a reference for evaluation and discrimination of other traditional herbal medicines.

Rapid discrimination and quantification of alkaloids in Corydalis Tuber by near-infrared spectroscopy.

PubMed

Lu, Hai-yan; Wang, Shi-sheng; Cai, Rui; Meng, Yu; Xie, Xin; Zhao, Wei-jie

2012-02-05

With the application of near-infrared spectroscopy (NIRS), a convenient and rapid method for determination of alkaloids in Corydalis Tuber extract and classification for samples from different locations have been developed. Five different samples were collected according to their geographical origin, 2-Der with smoothing point of 17 was applied as the spectral pre-treatment, and the 1st to scaling range algorithm was adjusted to be optimal approach, classification model was constructed over the wavelength range of 4582-4270 cm⁻¹, 5562-4976 cm⁻¹ and 7000-7467 cm⁻¹ with a great recognition rate. For prediction model, partial least squares (PLS) algorithm was utilized referring to HPLC-UV reference method, the optimum models were obtained after adjustment. Pre-processing methods of calibration models were COE for protopine and min-max normalization for palmatine and MSC for tetrahydropalmatine, respectively. The root mean square errors of cross-validation (RMSECV) for protopine, palmatine, tetrahydropalmatine were 0.884, 1.83, 3.23 mg/g. The correlation coefficients (R²) were 99.75, 98.41 and 97.34%. T test was applied, in the model of tetrahydropalmatine; there is no significant difference between NIR prediction and HPLC reference method at 95% confidence interval with t=0.746

Determination of total carbohydrates in wine and wine-like beverages by HPLC with a refractive index detector: First Action 2013.12.

PubMed

Kupina, Steve; Roman, Mark

2014-01-01

An international collaborative study was conducted of an HPLC-refractive index (RI) detector method for the determination of the combined amounts of sugars, glycerol, organic acids, and phenolic compounds in wines and wine-like beverages. Nine collaborating laboratories representing major winery, contract laboratories, and government laboratories tested eight different materials as blind duplicates using the proposed method. Sample materials included red and white wines, port, wine cooler, and nonalcoholic wine. One material was a negative control, and one material was a reference material. Samples were either treated with an ion-exchange resin to remove interfering organic acids prior to analysis or left untreated to include organic acids and phenolics. Red wine samples were treated with polyvinylpolypyrrolidone to remove potential interferences from phenolics prior to analysis. The HPLC analyses were performed on a Bio-Rad Fast Acid Analysis Column using RI detection. Reproducibility (RSD(R)) for untreated samples (sugars + phenolics + organic acids) ranged from 6.6% for Titrivin AA4 reference material to 11.0% for dry red wine. RSD(R) for treated samples (sugars only) ranged from 6.8% for white zinfandel to 18.9% for dry white wine. RSD(R) for treated samples (sugars only) + glycerol ranged from 6.4% for white zinfandel to 19.8% for dry red wine. Based on these results, the method was adopted as Official First Action status for determination of total carbohydrates in wine and wine-like beverages.

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.

Fluorescent adduct formation with terbium: a novel strategy for transferrin glycoform identification in human body fluids and carbohydrate-deficient transferrin HPLC method validation.

PubMed

Sorio, Daniela; De Palo, Elio Franco; Bertaso, Anna; Bortolotti, Federica; Tagliaro, Franco

2017-02-01

This paper puts forward a new method for the transferrin (Tf) glycoform analysis in body fluids that involves the formation of a transferrin-terbium fluorescent adduct (TfFluo). The key idea is to validate the analytical procedure for carbohydrate-deficient transferrin (CDT), a traditional biochemical serum marker to identify chronic alcohol abuse. Terbium added to a human body-fluid sample produced TfFluo. Anion exchange HPLC technique, with fluorescence detection (λ exc 298 nm and λ em 550 nm), permitted clear separation and identification of Tf glycoform peaks without any interfering signals, allowing selective Tf sialoforms analysis in human serum and body fluids (cadaveric blood, cerebrospinal fluid, and dried blood spots) hampered for routine test. Serum samples (n = 78) were analyzed by both traditional absorbance (Abs) and fluorescence (Fl) HPLC methods and CDT% levels demonstrated a significant correlation (p < 0.001 Pearson). Intra- and inter-runs CV% was 3.1 and 4.6%, respectively. The cut-off of 1.9 CDT%, related to the HPLC Abs proposed as the reference method, by interpolation in the correlation curve with the present method demonstrated a 1.3 CDT% cut-off. Method comparison by Passing-Bablok and Bland-Altman tests demonstrated Fl versus Abs agreement. In conclusion, the novel method is a reliable test for CDT% analysis and provides a substantial analytical improvement offering important advantages in terms of types of body fluid analysis. Its sensitivity and absence of interferences extend clinical applications being reliable for CDT assay on body fluids usually not suitable for routine test. Graphical Abstract The formation of a transferrin-terbium fluorescent adduct can be used to analyze the transferrin glycoforms. The HPLC method for carbohydrate-deficient transferrin (CDT%) measurement was validated and employed to determine the levels in different body fluids.

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.

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

PubMed

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

2005-12-01

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

IFCC approved HPLC reference measurement procedure for the alcohol consumption biomarker carbohydrate-deficient transferrin (CDT): Its validation and use.

PubMed

Schellenberg, François; Wielders, Jos; Anton, Raymond; Bianchi, Vincenza; Deenmamode, Jean; Weykamp, Cas; Whitfield, John; Jeppsson, Jan-Olof; Helander, Anders

2017-02-01

Carbohydrate-deficient transferrin (CDT) is used as a biomarker of sustained high alcohol consumption. The currently available measurement procedures for CDT are based on various analytical techniques (HPLC, capillary electrophoresis, nephelometry), some differing in the definition of the analyte and using different reference intervals and cut-off values. The Working Group on Standardization of CDT (WG-CDT), initiated by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), has validated an HPLC candidate reference measurement procedure (cRMP) for CDT (% disialotransferrin to total transferrin based on peak areas), demonstrating that it is suitable as a reference measurement procedure (RMP) for CDT. Presented is a detailed description of the cRMP and its calibration. Practical aspects on how to treat genetic variant and so-called di-tri bridge samples are described. Results of method performance characteristics, as demanded by ISO 15189 and ISO 15193, are given, as well as the reference interval and measurement uncertainty and how to deal with that in routine use. The correlation of the cRMP with commercial CDT procedures and the performance of the cRMP in a network of laboratories are also presented. The performance of the CDT cRMP in combination with previously developed commutable calibrators allows for standardization of the currently available commercial measurement procedures for CDT. The cRMP has recently been approved by the IFCC and will be from now on be known as the IFCC-RMP for CDT, while CDT results standardized according to this RMP should be indicated as CDT IFCC . Copyright © 2016 Elsevier B.V. All rights reserved.

[Identification of impurity peaks in the HPLC chromatogram by LC-MS and two-dimensional chromatographic correlation spectroscopy].

PubMed

Chen, Zhen-Zhen; Zhang, Dou-Sheng; Wang, Nan; Feng, Fang; Hu, Chang-Qin

2012-04-01

A novel qualitative analytical method by using two-dimensional chromatographic correlation spectroscopy techniques for recognizing impurity peaks of HPLC methods of quality control and LC-MS chromatographic system was established. The structures of major degradation products of ceftizoxime and cefdinir were identified by LC-MS and MassWorks application; the standard chromatographic and spectral data of the degradation impurities were obtained by high-performance liquid chromatography with diode array detection. The impurity peaks of two-dimensional chromatography were matched by comparison of spectra and calculating correlation coefficients. Peaks in chromatography can be identified accurately and rapidly in different chromatographic systems such as column and mobile phase changed. The method provides a new way and thought to identify the peaks in quality control of impurities without reference impurity substances.

Near-Infrared Spectroscopy Assay of Key Quality-Indicative Ingredients of Tongkang Tablets.

PubMed

Pan, Wenjie; Ma, Jinfang; Xiao, Xue; Huang, Zhengwei; Zhou, Huanbin; Ge, Fahuan; Pan, Xin

2017-04-01

The objective of this paper is to develop an easy and fast near-infrared spectroscopy (NIRS) assay for the four key quality-indicative active ingredients of Tongkang tablets by comparing the true content of the active ingredients measured by high performance liquid chromatography (HPLC) and the NIRS data. The HPLC values for the active ingredients content of Cimicifuga glycoside, calycosin glucoside, 5-O-methylvisamminol and hesperidin in Tongkang tablets were set as reference values. The NIRS raw spectra of Tongkang tablets were processed using first-order convolution method. The iterative optimization method was chosen to optimize the band for Cimicifuga glycoside and 5-O-methylvisamminol, and correlation coefficient method was used to determine the optimal band of calycosin glucoside and hesperidin. A near-infrared quantitative calibration model was established for each quality-indicative ingredient by partial least-squares method on the basis of the contents detected by HPLC and the obtained NIRS spectra. The correlation coefficient R 2 values of the four models of Cimicifuga glycoside, calycosin glucoside, 5-O-methylvisamminol and hesperidin were 0.9025, 0.8582, 0.9250, and 0.9325, respectively. It was demonstrated that the accuracy of the validation values was approximately 90% by comparison of the predicted results from NIRS models and the HPLC true values, which suggested that NIRS assay was successfully established and validated. It was expected that the quantitative analysis models of the four indicative ingredients could be used to rapidly perform quality control in industrial production of Tongkang tablets.

A clinical assay for the measurement of milrinone in plasma by HPLC mass spectrometry.

PubMed

Chihoho, B; Sage, A B; Smolenski, R T; Vazir, A; Rose, M L; Banner, N R; Leaver, N V

2012-05-01

Milrinone is a bipyridine phosphodiesterase inhibitor with positive inotropic and vasodilatory effects. As interest in longer term use of intravenous therapy increases, it becomes essential to monitor its plasma concentration owing to a narrow therapeutic range, an increased half-life in renal failure and toxicity associated with high levels. A high-performance liquid chromatography (HPLC) method with mass (MS) detection using a triple quadrupole mass spectrometer is presented. The method was compared with the UV/HPLC method and validated according to current international guidelines. Coefficients of variation of less than 7.5% were obtained across the therapeutic range and 18.3% at 2.4 ng/mL, the lower limit of quantitation. Plasma from 13 cardiac surgery patients receiving standard intravenous doses of milrinone were measured. Eight patients achieved therapeutic milrinone levels within 3-4 h post start of infusion, one was borderline sub-therapeutic and four patients achieved levels that were above the upper limit of the therapeutic range and potentially toxic. This method offers high sensitivity, is rapid, easy to use and requires minimal amount of sample. We believe this method could become the reference procedure for clinical monitoring of milrinone and help to improve the safety of the use of this drug in patients with cardiac failure. Copyright © 2011 John Wiley & Sons, Ltd.

Accuracy of noninvasive quantification of brain NAA concentrations using PRESS sequence: verification in a swine model with external standard.

PubMed

Wu, R H; Lin, R; Li, H; Xiao, Z W; Rao, H B; Luo, W H; Guo, G; Huang, K; Zhang, X G; Lang, Z J

2005-01-01

The metabolite ratios had been employed in the field of MR spectroscopy (MRS) for a long period. The main drawback of metabolite ratio is that ratio results are not comparable with absolute metabolite concentration in vivo. The purpose of this study was to examine the accuracy of noninvasive quantification of brain N-acetylaspartate (NAA) concentrations using previously reported MR external standard method. Eight swine were scanned on a GE 1.5 T scanner with a standard head coil. The external standard method was utilized with a sphere filled with NAA, GABA, glutamine, glutamate, creatine, choline chloride, and myo-inositol. The position resolved spectroscopy (PRESS) sequence was used with TE=135 msec, TR=1500 msec, and 128 scan averages. The analysis of MRS was done with SAGE/IDL program. In vivo NAA concentration was obtained using the equation S=N * e(-TE/T₂) * [1-e(-TR/T₁). In vitro NAA concentration was measured by high performance liquid chromatography (HPLC). In the MRS group, the mean concentration of NAA was 10.03 plusmn 0.74 mmol/kg. In the HPLC group, the mean concentration of NAA was 9.22 plusmn 0.55 mmol/kg. There was no significant difference between the two groups (p = 0.46). However, slightly higher value was observed in the MRS group (7/8 swine), compared with HPLC group. The range of differences was between 0.02~2.05 mmol/kg. MRS external reference method could be more accurate than internal reference method. ¹H MRS does not distinguish between N-acetyl resonance frequencies and other N-acetylated amino acids.

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

PubMed

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

2018-02-23

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

[HPLC specific chromatogram of Dendrobium officinale].

PubMed

Yan, Mei-Qiu; Chen, Su-Hong; Lv, Gui-Yuan; Zhou, Gui-Fen; Liu, Xia

2013-02-01

To establish the method of specific chromatogram analysis of ether extract of Dendrobium officinale for identification of D. officinale. Chromatographic separation was carried out at 30 degrees C on an Ultimate C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol and water containing 0.2% phosphoric acid in a gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 280 nm. The similarity evaluation system for chromatographic fingerprint of NPC (National Pharmacopoeia Committee) was adopted to specific chromatogram construction. The HPLC specific chromatogram of D. officinale was constructed with 6 common specific chromatographic peaks including naringenin as a reference peak. The method shows good precision and repeatability of relative retention time. It can be used to identify D. officinale.

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

Quality control and identification of steroid saponins from Dioscorea zingiberensis C. H. Wright by fingerprint with HPLC-ELSD and HPLC-ESI-Quadrupole/Time-of-fight tandem mass spectrometry.

PubMed

Zhang, Xinxin; Liang, Jinru; Liu, Jianli; Zhao, Ye; Gao, Juan; Sun, Wenji; Ito, Yoichiro

2014-03-01

In this study, a fingerprint of steroid saponins, the major bioactive constituents in the crude extracts from Dioscorea zingiberensis C. H. Wright (DZW), has been established for the first time by combined use of the following two methods: high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) and the simultaneous characterization of the steroid saponins by high-performance liquid chromatography coupled with electrospray ionization-mass spectrometry and quadrupole tandem time-of-fight mass analyzers detection (HPLC-ESI-Q/TOF). All HPLC analyses were carried out on a Welchrom C18 column (250mm×4.6mm I.D., 5μm) with a mobile phase composed of water and acetonitrile under gradient elution. There were 68 common characteristic peaks in the fingerprints, in which 12 of them were confirmed by comparing their mass spectra and retention times with those of the reference compounds. In order to identify other unknown peaks, their fragmentation behaviors characteristic of the major groups of steroid saponins from DZW with six types of aglycone skeletons were discussed in detail, and possible MS/MS fragmentation pathways were proposed for aiding the structural identification of these components. According to the summarized fragmentation patterns, these peaks were tentatively assigned by matching their empirical molecular formula with those of the published compounds, or by elucidating their quasi-molecular ions and fragment ions referring to available literature information when the reference standards were unavailable. As a result, 22 new steroid saponins were found in DZW for the first time. In addition, the quantitative analysis of the nine (except for the reference compounds A, B, and C) known peaks was accomplished at the same time which indicated that there was a great variability in the amount of these active compounds in different batches in the crude extracts. This approach could demonstrate that the fingerprint could be considered to be a suitable tool to comprehensively improve the quality control of DZW. The identification and structural elucidation of the peaks in the fingerprint may provide important experimental data for further pharmacological and clinical researches. Copyright © 2014. Published by Elsevier B.V.

HPLC and TLC methods for analysis of [18F]FDG and its metabolites from biological samples.

PubMed

Rokka, Johanna; Grönroos, Tove J; Viljanen, Tapio; Solin, Olof; Haaparanta-Solin, Merja

2017-03-24

The most used positron emission tomography (PET) tracer, 2-[ 18 F]fluoro-2-deoxy-d-glucose ([ 18 F]FDG), is a glucose analogue that is used to measure tissue glucose consumption. Traditionally, the Sokoloff model is the basis for [ 18 F]FDG modeling. According to this model, [ 18 F]FDG is expected to be trapped in a cell in the form of [ 18 F]FDG-6-phosphate ([ 18 F]FDG-6-P). However, several studies have shown that in tissues, [ 18 F]FDG metabolism goes beyond [ 18 F]FDG-6-P. Our aim was to develop radioHPLC and radioTLC methods for analysis of [ 18 F]FDG metabolites from tissue samples. The radioHPLC method uses a sensitive on-line scintillation detector to detect radioactivity, and the radioTLC method employs digital autoradiography to detect the radioactivity distribution on a TLC plate. The HPLC and TLC methods were developed using enzymatically in vitro-produced metabolites of [ 18 F]FDG as reference standards. For this purpose, three [ 18 F]FDG metabolites were synthesized: [ 18 F]FDG-6-P, [ 18 F]FD-PGL, and [ 18 F]FDG-1,6-P2. The two methods were evaluated by analyzing the [ 18 F]FDG metabolic profile from rodent ex vivo tissue homogenates. The HPLC method with an on-line scintillation detector had a wide linearity in a range of 5Bq-5kBq (LOD 46Bq, LOQ 139Bq) and a good resolution (Rs ≥1.9), and separated [ 18 F]FDG and its metabolites clearly. The TLC method combined with digital autoradiography had a high sensitivity in a wide range of radioactivity (0.1Bq-2kBq, LOD 0.24Bq, LOQ 0.31Bq), and multiple samples could be analyzed simultaneously. As our test and the method validation with ex vivo samples showed, both methods are useful, and at best they complement each other in analysis of [ 18 F]FDG and its radioactive metabolites from biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Hepatoprotective effect against CCl4-induced acute liver damage in mice and High-performance liquid chromatography mass spectrometric method for analysis of the constituents of extract of Rubus crataegifolius.

PubMed

Sun, Yongjiao; Jia, Lingyun; Huang, Zhanbo; Wang, Jing; Lu, Jincai; Li, Jing

2017-11-01

This study is an attempt to evaluate the hepatoprotective activity of Rubus Crataegifolius against carbon tetrachloride-induced liver injury in mice. 70% ethanolic, ethyl acetate and n-BuOH extract of R. crataegifolius were administered daily for 14 days in experimental animals before they were treated with CCl 4 . The hepatoprotective activity of the extracts in this study was compared with the reference drug silymarin. A high-performance liquid chromatography mass spectrometric (HPLC-EIS-MS/MS) method was developed for the determination of the constituents of the extracts. According to the data of HPLC-EIS-MS/MS, the chemical structures of the largely 14 constituents of R. crataegifolius were identified online without time-consuming isolation. Ethyl acetate extracts of R. crataegifolius showed strong antioxidant activities and significant protective effect against acute hepatotoxicity induced by CCl 4 . According to the data of HPLC-EIS-MS/MS, Oleanic acid, Phlorizin dehydrate and Quercetin-3-rhamnoside are considered as the main hepatoprotective factor in ethyl acetate extract.

[HPLC Fingerprint of QingGuangAn and Determination of the Main Components].

PubMed

Wang, Min; Shen, Bing-bing; Luo, Juan; Chen, Yang; Yang, Yu-pei; Chen, Sheng-huang

2015-10-01

To establish an HPLC fingerprint of ethanol extract of QingGuangAn, and to determine the contents of paeoniflorin and calycosin-7-glucosid. HPLC analysis was performed on an Agilent 1260 Infinity LC system and carried out at 35 degrees C on a column of GRACE Alltima C18 (250 mm x 4.6 mm, 5 μm). A binary gradient elution system was composed of acetonitrile (phase A) and water solution (phase B). Detection was performed at the wavelength of 254 nm, the mobile flow rate was 0.8 mL/min. A matrix including 20 variations (characteristic peaks area) and 10 samples was constructed for similarity evaluation. The results showed that the collected samples had a good similarity. A specificity fingerprint was produced and 20 characteristic peaks were designated. The content of paeoniflorin and calycosin-7-glucosid was 0.368 and 0.049 mg/g, respectively. It is a reliable, available and quick method for quality control of QingGuangAn,which provides some reference for the comparison of different extracting methods of QingGuangAn and the differences of pharmacodynamic.

[Studies on the identification of psychotropic substances. VIII. Preparation and various analytical data of reference standard of some stimulants, amfepramone, cathinone, N-ethylamphetamine, fenethylline, fenproporex and mefenorex].

PubMed

Shimamine, M; Takahashi, K; Nakahara, Y

1992-01-01

The Reference Standards for amfepramone, cathinone, N-ethylamphetamine, fenethylline, fenproporex and mefenorex were prepared. Their purities determined by HPLC were more than 99.5%. For the identification and determination of these six drugs, their analytical data were measured and discussed by TLC, UV, IR, HPLC, GC/MS and NMR.

Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

PubMed Central

Jacqz-Aigrain, E; Bessa, E; Medard, Y; Mircheva, Y; Vilmer, E

1994-01-01

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively). PMID:7946931

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.

Estimation of biological variation and reference change value of glycated hemoglobin (HbA(1c)) when two analytical methods are used.

PubMed

Ucar, Fatma; Erden, Gonul; Ginis, Zeynep; Ozturk, Gulfer; Sezer, Sevilay; Gurler, Mukaddes; Guneyk, Ahmet

2013-10-01

Available data on biological variation of HbA1c revealed marked heterogeneity. We therefore investigated and estimated the components of biological variation for HbA1c in a group of healthy individuals by applying a recommended and strictly designed study protocol using two different assay methods. Each month, samples were derived on the same day, for three months. Four EDTA whole blood samples were collected from each individual (20 women, 9 men; 20-45 years of age) and stored at -80°C until analysis. HbA1c values were measured by both high performance liquid chromatography (HPLC) (Shimadzu, Prominence, Japan) and boronate affinity chromatography methods (Trinity Biotech, Premier Hb9210, Ireland). All samples were assayed in duplicate in a single batch for each assay method. Estimations were calculated according to the formulas described by Fraser and Harris. The within subject (CV(I))-between subject (CV(G)) biological variations were 1.17% and 5.58%, respectively for HPLC. The calculated CV(I) and CV(G) were 2.15% and 4.03%, respectively for boronate affinity chromatography. Reference change value (RCV) for HPLC and boronate affinity chromatography was 5.4% and 10.4% respectively and individuality index of HbA(1c) was 0.35 and 0.93 respectively. This study for the first time described the components of biological variation for HbA1c in healthy individuals by two different assay methods. Obtained findings showed that the difference between CV(A) values of the methods might considerably affect RCV. These data regarding biological variation of HbA(1c) could be useful for a better evaluation of HbA(1c) test results in clinical interpretation. Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Characterizing a Full Spectrum of Physico-Chemical Properties of Ginsenosides Rb1 and Rg1 to Be Proposed as Standard Reference Materials

PubMed Central

Kim, Il-Woung; Hong, Hee-Do; Choi, Sang Yoon; Hwang, Da-Hye; Her, Youl; Kim, Si-Kwan

2011-01-01

Good manufacturing practice (GMP)-based quality control is an integral component of the common technical document, a formal documentation process for applying a marketing authorization holder to those countries where ginseng is classified as a medicine. In addition, authentication of the physico-chemical properties of ginsenoside reference materials, and qualitative and quantitative batch analytical data based on validated analytical procedures are prerequisites for certifying GMP. Therefore, the aim of this study was to propose an authentication process for isolated ginsenosides Rb1 and Rg1 as reference materials (RM) and for these compounds to be designated as RMs for ginseng preparations throughout the world. Ginsenoside Rb1 and Rg1 were isolated by Diaion HP-20 adsorption chromatography, silica gel flash chromatography, recrystallization, and preparative HPLC. HPLC fractions corresponding to those two ginsenosides were recrystallized in appropriate solvents for the analysis of physico-chemical properties. Documentation of the isolated ginsenosides was made according to the method proposed by Gaedcke and Steinhoff. The ginsenosides were subjected to analyses of their general characteristics, identification, purity, content quantitation, and mass balance tests. The isolated ginsenosides were proven to be a single compound when analyzed by three different HPLC systems. Also, the water content was found to be 0.940% for Rb1 and 0.485% for Rg1, meaning that the net mass balance for ginsenoside Rb1 and Rg1 were 99.060% and 99.515%, respectively. From these results, we could assess and propose a full spectrum of physicochemical properties for the ginsenosides Rb1 and Rg1 as standard reference materials for GMP-based quality control. PMID:23717096

An application of second-order UV-derivative spectrophotometry for study of solvolysis of a novel fluocinolone acetonide ester

NASA Astrophysics Data System (ADS)

Markovic, Bojan; Vladimirov, Sote; Cudina, Olivera; Savic, Vladimir; Karljikovic-Rajic, Katarina

2010-02-01

A novel topical corticosteroid FA-21-PhP, 2-phenoxypropionate ester of fluocinolone acetonide, has been synthesized in order to investigate the possibility of decreasing systemic side effects. In this study model system for in vitro solvolytic reaction of FA-21-PhP has been analyzed in ethanol/water (90:10, v/v) with excess of sodium hydrogen carbonate. The selected conditions have been used as in vitro model for activation of corticosteroid C-21 ester prodrug. The second-order derivative spectrophotometric method (DS) using zero-crossing technique was developed for monitoring ternary mixture of solvolysis. Fluocinolone acetonide (FA) as a solvolyte was determined in the mixture in the concentration range 0.062-0.312 mM using amplitude 2D 274.96. Experimentally determined LOD value was 0.0295 mM. The accuracy of proposed DS method was confirmed with HPLC referent method. Peak area of parent ester FA-21-PhP was used for solvolysis monitoring to ensure the initial stage of changes. Linear relationship in HPLC assay for parent ester was obtained in the concentration range 0.054-0.54 mM, with experimentally determined LOD value of 0.0041 mM. Investigated solvolytic reaction in the presence of excess of NaHCO 3 proceeded via a pseudo-first-order kinetic with significant correlation coefficients 0.9891 and 0.9997 for DS and HPLC, respectively. The values of solvolysis rate constant calculated according to DS and HPLC methods are in good accordance 0.038 and 0.043 h -1, respectively.

Screening and Analysis of the Marker Components in Ganoderma lucidum by HPLC and HPLC-MSn with the Aid of Chemometrics.

PubMed

Wu, Lingfang; Liang, Wenyi; Chen, Wenjing; Li, Shi; Cui, Yaping; Qi, Qi; Zhang, Lanzhen

2017-04-06

Ganoderma triterpenes (GTs) are the major secondary metabolites of Ganoderma lucidum , which is a popularly used traditional Chinese medicine for complementary cancer therapy. The present study was to establish a fingerprint evaluation system based on Similarity Analysis (SA), Cluster Analysis (CA) and Principal Component Analysis (PCA) for the identification and quality control of G. lucidum . Fifteen samples from the Chinese provinces of Hainan, Neimeng, Shangdong, Jilin, Anhui, Henan, Yunnan, Guangxi and Fujian were analyzed by HPLC-PAD and HPLC-MS n . Forty-seven compounds were detected by HPLC, of which forty-two compounds were tentatively identified by comparing their retention times and mass spectrometry data with that of reference compounds and reviewing the literature. Ganoderic acid B, 3,7,15-trihydroxy-11,23-dioxolanost-8,16-dien-26-oic acid, lucidenic acid A, ganoderic acid G, and 3,7-oxo-12-acetylganoderic acid DM were deemed to be the marker compounds to distinguish the samples with different quality according to both CA and PCA. This study provides helpful chemical information for further research on the anti-tumor activity and mechanism of action of G. lucidum . The results proved that fingerprints combined with chemometrics are a simple, rapid and effective method for the quality control of G. lucidum .

Quantification of methionine and selenomethionine in biological samples using multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS).

PubMed

Vu, Dai Long; Ranglová, Karolína; Hájek, Jan; Hrouzek, Pavel

2018-05-01

Quantification of selenated amino-acids currently relies on methods employing inductively coupled plasma mass spectrometry (ICP-MS). Although very accurate, these methods do not allow the simultaneous determination of standard amino-acids, hampering the comparison of the content of selenated versus non-selenated species such as methionine (Met) and selenomethionine (SeMet). This paper reports two approaches for the simultaneous quantification of Met and SeMet. In the first approach, standard enzymatic hydrolysis employing Protease XIV was applied for the preparation of samples. The second approach utilized methanesulfonic acid (MA) for the hydrolysis of samples, either in a reflux system or in a microwave oven, followed by derivatization with diethyl ethoxymethylenemalonate. The prepared samples were then analyzed by multiple reaction monitoring high performance liquid chromatography tandem mass spectrometry (MRM-HPLC-MS/MS). Both approaches provided platforms for the accurate determination of selenium/sulfur substitution rate in Met. Moreover the second approach also provided accurate simultaneous quantification of Met and SeMet with a low limit of detection, low limit of quantification and wide linearity range, comparable to the commonly used gas chromatography mass spectrometry (GC-MS) method or ICP-MS. The novel method was validated using certified reference material in conjunction with the GC-MS reference method. Copyright © 2018. Published by Elsevier B.V.

An Exploration on the Suitability of Airborne Carbonyl Compounds Analysis in relation to Differences in Instrumentation (GC-MS versus HPLC-UV) and Standard Phases (Gas versus Liquid)

PubMed Central

Kim, Ki-Hyun; Szulejko, Jan E.; Kim, Yong-Hyun; Lee, Min-Hee

2014-01-01

The relative performance figure of merits was investigated for the two most common analytical methods employed for carbonyl compounds (CC), for example, between high performance liquid chromatography (HPLC)-UV detector (with 2,4-dinitrophenylhydrazine (DNPH) derivatization) and thermal desorption (TD)-gas chromatography (GC)-mass spectrometry (MS) (without derivatization). To this end, the suitability of each method is assessed by computing the relative recovery (RR) between the gas- and liquid-phase standards containing a suite of CC such as formaldehyde (FA), acetaldehyde (AA), propionaldehyde (PA), butyraldehyde (BA), isovaleraldehyde (IA), and valeraldehyde (VA) along with benzene (B) as a recovery reference for the GC method. The results confirm that a TD-GC-MS is advantageous to attain the maximum recovery for the heavier CCs (i.e., with molecular weights (MW) above BA−MW ≥ 74). On the other hand, the HPLC-UV is favorable for the lighter CCs (like FA and AA) with the least bias. Such compound-specific responses for each platform are validated by relative ordering of CCs as a function of response factor (RF), method detection limit (MDL), and recovery pattern. It is thus desirable to understand the advantages and limitations of each method to attain the CC data with the least experimental bias. PMID:24719571

Comparison of high-pressure liquid chromatography and microbiological assay for determination of ciprofloxacin tablets in human plasma employed in bioequivalence and pharmacokinetics study.

PubMed

Khan, Muhammad Khalid; Khan, Muhammad Farid; Mustafa, Ghulam; Sualah, Mohammed

2012-01-01

Ciprofloxacin was given orally to 28 healthy male volunteers for single oral dose of 500mg; Plasma samples were collected at different time's interval between 0 and 12h and analyzed both by high pressure liquid chromatography and by a microbiological assay. The detection limits (LOD) were 0.02μg/ml and 0.1μg/ml, for both methods respectively. For each method, coefficients of variation (R(2)) were 0.9995 and 0.9918 in plasma and limit of quantitation (LOQ).02 and 0.5μg/ml. The Comparison of means maximum concentration 2.68 μg/ml at 1.5 hr for test and 2.43 μg/ml are attain in HPLC method of Reference at 2hrs respectively. The plasma concentrations measured by microbiological assay of reference tablet are 3.95μg/ml (mean ± SE) at 1 hour and 3.80μg/ml (mean ± SE) at 1 hour. The concentrations in plasma measured by microbiological method were markedly higher than the high-pressure liquid chromatography values which indicates the presence of antimicrobially active metabolites. The mean ± SE values of pharmacokinetic parameters calculated by HPLC method, for total area under the curve (AUC 0-oo) were 13.11, and 11.91 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 44.91 and 48.42 respectively. The elimination rate constant Kel [l/h] showed 0.17 l/h for test and 0.15 l/h reference tablets and likewise, absorption half-life expressed in hours shown 0.67 h for test and 1.04 h for reference respectively. The Mean Residence Time for test is 5.48 h and 5.49 h for reference. The mean ± SE values of pharmacokinetic parameters (Microbiological assay) for total area under the curve (AUC 0-oo) were 22.11 and 19.33 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 29.02 and 31.63 respectively. The elimination rate constant Kel [l/h] showed 0.21 l/h for test and 0.20 l/h reference tablets and likewise, absorption half-life expressed in hours shown 0.86h for test and 0.56 h for reference respectively. The Mean Residence Time for test is 5.27 h and 4.67 h for reference. Significant difference observed between two methods.

Analysis of mexicanolide- and phragmalin-type limonoids from Heynea trijuga using high-performance liquid chromatography/electrospray tandem mass spectrometry.

PubMed

Yang, Wei; Fang, Dong-Mei; He, Hong-Ping; Hao, Xiao-Jiang; Wu, Zhi-Jun; Zhang, Guo-Lin

2013-06-15

Limonoids, a class of tetranortriterpenoids, exhibit various biological effects, including acting as potent antifeedants and insect growth-regulators against various pests. The analysis of phragmalin- and mexicanolide-type limonoids by collision-induced dissociation tandem mass spectrometry (CID-MS/MS) has not been reported. A high-performance liquid chromatography/electrospray ionization (HPLC/ESI)-MS/MS method was developed to investigate the fragmentation patterns of [M+NH4 ](+) ions for nine reference phragmalin- and mexicanolide-type limonoids. The method was also used in the identification of limonoid compounds in botanic extracts of Heynea trijuga. The losses of side chains and the furan part were the major fragmentation patterns. However, there was variation in the relative abundances of product ions resulting from the same fragmentation pathways. A total of 89 phragmalin- and mexicanolide-type limonoids in botanic extracts of Heynea trijuga were detected and 50 of these compounds were identified or tentatively characterized based on elemental constituents, fragmentation pathways, and the profile of the major product ions of reference compounds. In addition, the isomers could be tentatively distinguished. An HPLC/ESI-MS/MS method was developed and could be used to simultaneously identify and screen phragmalin- and mexicanolide-type limonoids in botanic extracts of Heynea trijuga. Copyright © 2013 John Wiley & Sons, Ltd.

Infrared microspectroscopic determination of collagen cross-links in articular cartilage

NASA Astrophysics Data System (ADS)

Rieppo, Lassi; Kokkonen, Harri T.; Kulmala, Katariina A. M.; Kovanen, Vuokko; Lammi, Mikko J.; Töyräs, Juha; Saarakkala, Simo

2017-03-01

Collagen forms an organized network in articular cartilage to give tensile stiffness to the tissue. Due to its long half-life, collagen is susceptible to cross-links caused by advanced glycation end-products. The current standard method for determination of cross-link concentrations in tissues is the destructive high-performance liquid chromatography (HPLC). The aim of this study was to analyze the cross-link concentrations nondestructively from standard unstained histological articular cartilage sections by using Fourier transform infrared (FTIR) microspectroscopy. Half of the bovine articular cartilage samples (n=27) were treated with threose to increase the collagen cross-linking while the other half (n=27) served as a control group. Partial least squares (PLS) regression with variable selection algorithms was used to predict the cross-link concentrations from the measured average FTIR spectra of the samples, and HPLC was used as the reference method for cross-link concentrations. The correlation coefficients between the PLS regression models and the biochemical reference values were r=0.84 (p<0.001), r=0.87 (p<0.001) and r=0.92 (p<0.001) for hydroxylysyl pyridinoline (HP), lysyl pyridinoline (LP), and pentosidine (Pent) cross-links, respectively. The study demonstrated that FTIR microspectroscopy is a feasible method for investigating cross-link concentrations in articular cartilage.

Quantity and quality of black carbon molecular markers as obtained by two chromatographic methods (GC-FID and HPLC-DAD) - How do results compare?

NASA Astrophysics Data System (ADS)

Schneider, M. P. W.; Smittenberg, R. H.; Dittmar, T.; Schmidt, M. W. I.

2009-04-01

Chars produced by wildfires are an important source of black carbon (BC) in the environment. After their deposition on the soil surface they can be distributed into rivers, marine waters and sediments. The analysis of benzenepolycarboxylic acids (BPCAs) as a quantitative measure for black carbon (BC) in soil and sediment samples is a well-established method (Glaser et al., 1998; Brodowski et al., 2005). Briefly, the nitric acid oxidation of fused aromatic ring systems in BC forms eight molecular markers (BPCAs), which can be assigned to BC, and which subsequently can be quantified by GC-FID (gas chromatography with flame ionization detector). Recently, this method was modified for the quantification of BC in seawater samples using HPLC-DAD (High performance liquid chromatography with diode array detector) for the determination of individual BPCAs (Dittmar, 2008). A direct comparison of both analytical techniques is lacking but would be important for future data comparison aimed at the calculation of global BC budgets. Here we present a systematic comparison of the two BPCA quantification methods. We prepared chars under well-defined laboratory conditions. In order to cover a broad spectrum of char properties we used two sources of biomass and a wide range of pyrolysis temperatures. Chestnut hardwood chips (Castanea sativa) and rice straw (Oryza sativa) were pyrolysed at temperatures between 200 and 1000°C under a constant N2 stream. The maximum temperatures were held constant for 5 hours (Hammes et al., 2006). The BC contents of the chars have been analysed using the BPCA extraction method followed by either GC-FID or HPLC-DAD quantification. Preliminary results suggest that both methods yield similar total quantities of BPCA, and also the proportions of the individual markers are similar. Ongoing experiments will allow for a more detailed comparison of the two methods. The BPCA composition of chars formed at different temperatures and from different precursor biomass is being used for this purpose. We seek to establish a conversion factor between both methods, if required. Results show that both the GC and the HPLC method can be used for organic samples containing some silica, such as grass char. Further tests include silica-rich materials, such as soils. Ongoing methodological work aims at carbon isotope analysis (13C and 14C) on individual BPCAs isolated via HPLC. At present the HPLC method employs tetrabutyl ammonium bromide (TBAB) as a modifier for the liquid phase. TBAB is not volatile and contains carbon, it therefore prevents carbon isotopic analysis on isolated BPCAs. References Brodowski, S., Rodionov, A., Haumeier, L., Glaser, B., Amelung, W. (2005) Revised black carbon assessment using benzene polycarboxylic acids. Organic Geochemistry, 36(9), 1299-1310. Dittmar, T. (2008) The molecular level determination of black carbon in marine dissolved organic matter. Organic Geochemistry, 39(4). 396-407. Glaser, B., Haumeier, L., Guggenberger, G., Zech, W. (1998) Black carbon in soils: the use of benzenecarboxylic acids as specific markers. Organic Geochemistry, 29(4), 811-819. Hammes, K. Smernik, R. J., Skjemstad, J. O., Herzog, A., Vogt, U. F., Schmidt, M. W. I. (2006) Synthesis and characterisation of laboratory-charred grass straw (Oryza saliva) and chestnut wood (Castanea sativa) as reference materials for black carbon quantification Organic Geochemistry 37(11). 1629-1633

Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

PubMed

Jönsson, K H; Daas, A; Buchheit, K H; Terao, E

2016-01-01

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

[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.

Monitoring multiple components in vinegar fermentation using Raman spectroscopy.

PubMed

Uysal, Reyhan Selin; Soykut, Esra Acar; Boyaci, Ismail Hakki; Topcu, Ali

2013-12-15

In this study, the utility of Raman spectroscopy (RS) with chemometric methods for quantification of multiple components in the fermentation process was investigated. Vinegar, the product of a two stage fermentation, was used as a model and glucose and fructose consumption, ethanol production and consumption and acetic acid production were followed using RS and the partial least squares (PLS) method. Calibration of the PLS method was performed using model solutions. The prediction capability of the method was then investigated with both model and real samples. HPLC was used as a reference method. The results from comparing RS-PLS and HPLC with each other showed good correlations were obtained between predicted and actual sample values for glucose (R(2)=0.973), fructose (R(2)=0.988), ethanol (R(2)=0.996) and acetic acid (R(2)=0.983). In conclusion, a combination of RS with chemometric methods can be applied to monitor multiple components of the fermentation process from start to finish with a single measurement in a short time. Copyright © 2013 Elsevier Ltd. All rights reserved.

Determination of the purity of pharmaceutical reference materials by 1H NMR using the standardless PULCON methodology.

PubMed

Monakhova, Yulia B; Kohl-Himmelseher, Matthias; Kuballa, Thomas; Lachenmeier, Dirk W

2014-11-01

A fast and reliable nuclear magnetic resonance spectroscopic method for quantitative determination (qNMR) of targeted molecules in reference materials has been established using the ERETIC2 methodology (electronic reference to access in vivo concentrations) based on the PULCON principle (pulse length based concentration determination). The developed approach was validated for the analysis of pharmaceutical samples in the context of official medicines control, including ibandronic acid, amantadine, ambroxol and lercanidipine. The PULCON recoveries were above 94.3% and coefficients of variation (CVs) obtained by quantification of different targeted resonances ranged between 0.7% and 2.8%, demonstrating that the qNMR method is a precise tool for rapid quantification (approximately 15min) of reference materials and medicinal products. Generally, the values were within specification (certified values) provided by the manufactures. The results were in agreement with NMR quantification using an internal standard and validated reference HPLC analysis. The PULCON method was found to be a practical alternative with competitive precision and accuracy to the classical internal reference method and it proved to be applicable to different solvent conditions. The method can be recommended for routine use in medicines control laboratories, especially when the availability and costs of reference compounds are problematic. Copyright © 2014 Elsevier B.V. All rights reserved.

An improved independent component analysis model for 3D chromatogram separation and its solution by multi-areas genetic algorithm.

PubMed

Cui, Lizhi; Poon, Josiah; Poon, Simon K; Chen, Hao; Gao, Junbin; Kwan, Paul; Fan, Kei; Ling, Zhihao

2014-01-01

The 3D chromatogram generated by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) has been researched widely in the field of herbal medicine, grape wine, agriculture, petroleum and so on. Currently, most of the methods used for separating a 3D chromatogram need to know the compounds' number in advance, which could be impossible especially when the compounds are complex or white noise exist. New method which extracts compounds from 3D chromatogram directly is needed. In this paper, a new separation model named parallel Independent Component Analysis constrained by Reference Curve (pICARC) was proposed to transform the separation problem to a multi-parameter optimization issue. It was not necessary to know the number of compounds in the optimization. In order to find all the solutions, an algorithm named multi-areas Genetic Algorithm (mGA) was proposed, where multiple areas of candidate solutions were constructed according to the fitness and distances among the chromosomes. Simulations and experiments on a real life HPLC-DAD data set were used to demonstrate our method and its effectiveness. Through simulations, it can be seen that our method can separate 3D chromatogram to chromatogram peaks and spectra successfully even when they severely overlapped. It is also shown by the experiments that our method is effective to solve real HPLC-DAD data set. Our method can separate 3D chromatogram successfully without knowing the compounds' number in advance, which is fast and effective.

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

PubMed

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

2009-01-01

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

Simultaneous qualification and quantification of baccharane glycosides in Impatientis Semen by HPLC-ESI-MSD and HPLC-ELSD.

PubMed

Li, Hui-Jun; Yu, Jun-Jie; Li, Ping

2011-03-25

This study presents a high performance liquid chromatography (HPLC) with electrospray ionization mass spectrometric detection (ESI-MSD) and evaporative light scattering detection (ELSD) method for the simultaneous qualification and quantification of eight major baccharane glycosides, namely hosenlosides A, B, C, F, G, K, L, and M in Impatientis Semen, a Chinese herbal medicine derived from the seeds of Impatiens balsamina L. In order to achieve optimum performance, several extraction parameters (including extraction solvent, extraction mode, extraction time) were optimized. The baccharane glycosides were separated on a Shim-pack CLC-ODS column with gradient elution of water and methanol. Temperature for the ELSD drift tube was set at 98°C and the nitrogen flow rate was 2.7l/min. The unambiguous identities of the analytes were realized by comparing retention times and mass data with those of reference compounds. The developed method was fully validated in terms of linearity, sensitivity, precision, repeatability, recovery as well as robustness, and subsequently applied to evaluate the quality of 14 batches of Impatientis Semen commercial samples from different collections. Copyright © 2010 Elsevier B.V. All rights reserved.

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

PubMed

Wahlen, Raimund

2004-04-01

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

77 FR 67282 - Dinotefuran; Pesticide Tolerances for Emergency Exemptions

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-09

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

Convergent synthesis of 13N-labelled Peptidic structures using aqueous [13N]NH3.

PubMed

Blower, Julia E; Cousin, Samuel F; Gee, Antony D

2017-01-01

Nitrogen-13 has a 10-min half-life which places time constraints on the complexity of viable synthetic methods for its incorporation into PET imaging agents. In exploring ways to overcome this limitation, we have used the Ugi reaction to develop a rapid one-pot method for radiolabelling peptidic molecules using [ 13 N]NH 3 as a synthetic precursor. Carrier-added [ 13 N]NH 3 (50 μL) was added to a solution of carboxylic acid, aldehyde, and isocyanide in 2,2,2-TFE (200 μL). The mixture was heated in a microwave synthesiser at 120 °C for 10 min. Reactions were analysed by radio-HPLC and radio-LCMS. Isolation of the target 13 N-labelled peptidic Ugi compound was achieved via semi-preparative radio-HPLC as demonstrated for Ugi 1. Radio-HPLC analysis of each reaction confirmed the formation of radioactive products co-eluting with their respective reference standards with radiochemical yields of the crude products ranging from 11% to 23%. Two cyclic γ-lactam structures were also achieved via intra-molecular reactions. Additional radioactive by-products observed in the radio-chromatogram were identified as 13 N-labelled di-imines formed from the reaction of [ 13 N]NH 3 with two isocyanide molecules. The desired 13 N-labelled Ugi product was isolated using semi-preparative HPLC. We have developed a one-pot method that opens up new routes to radiolabel complex, peptidic molecules with 13 N using aqueous [ 13 N]NH 3 as a synthetic precursor.

Application of curve resolution algorithms in the study of drug photodegradation kinetics -- the example of moclobemide.

PubMed

Skibiński, Robert; Komsta, Łukasz

2012-01-01

The photodegradation of moclobemide was studied in methanolic media. Ultra-HPLC (UHPLC)/MS/MS analysis proved decomposition to 4-chlorobenzamide as a major degradation product and small amounts of Ro 16-3177 (4-chloro-N-[2-[(2-hydroxyethyl)amino] ethyl]benzamide) and 2-[(4-chlorobenzylidene)amino]-N-[2-ethoxyethenyl]ethenamine. The methanolic solution was investigated spectrophotometrically in the UV region, registering the spectra during 30 min of degradation. Using reference spectra and a multivariate chemometric method (multivariate curve resolution-alternating least squares), the spectra were resolved and concentration profiles were obtained. The obtained results were in good agreement with a quantitative approach, with UHPLC-diode array detection as the reference method.

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

PubMed

Robitaille, Line; Hoffer, L John

2016-04-21

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

High-performance liquid chromatography-tandem mass spectrometry as a reference for analysis of tacrolimus to assess two immunoassays in patients with liver and renal transplants.

PubMed

Salm, P; Taylor, P J; Clark, A; Balderson, G A; Grygotis, A; Norris, R L; Lynch, S V; Shaw, L M; Pond, S M

1997-12-01

The accuracy and imprecision of three assays used for therapeutic monitoring of tacrolimus were tested using blood-containing weighed-in amounts of the drug, an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA I), and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) assay. Accuracy was acceptable for the HPLC-MS2 assay at all concentrations tested (< 10% deviation) and for the ELISA at 1.0 and 4.0 microg/l. Accuracy was not acceptable for the ELISA at 15.0 and 50.0 microg/l or for the MEIA I at all concentrations tested. Imprecision was acceptable for the HPLC-MS2 assay at all concentrations tested (coefficient of variation < 10%), for the ELISA at 15.0 and 50.0 microg/l, and for the MEIA I at 15.0 and 50.0 microg/l. Imprecision was not acceptable for the ELISA at 1.0 and 4.0 microg/l or for the MEIA I at 1.0 and 4.0 microg/l. This assessment with weighed-in amounts of tacrolimus verified the HPLC-MS2 assay as a reference method. The performance of the two immunoassays with HPLC-MS2 was then compared in the clinical setting using blood from patients with liver (n = 30) and renal (n = 37) transplants. In the liver transplant group (127 samples), the range of tacrolimus concentrations measured by HPLC-MS2, ELISA, and MEIA I was 1.9 to 31.8, 2.1 to 35.0, and less than 0.1 to 36.5 mg/l, respectively. In the renal transplant group (129 samples), the ranges were 1.7 to 26.1, 1.9 to 24.4, and 0.9 to 28.5 microg/l, respectively. Compared with the HPLC-MS2, the ELISA had minimal bias (0.1 to 0.2 microg/l) but unacceptable variability in values (SD > 13%). The MEIA I had unacceptable bias (1.7-1.8 microg/l) and variability (SD > 23%). These data indicated that neither the ELISA nor MEIA I is interchangeable with HPLC-MS2. Moreover, in view of the current trend to reduce the therapeutic dose of tacrolimus, quantitative results using the MEIA I would not be obtainable during therapeutic drug monitoring in some patients in whom effective therapeutic concentrations can be less than 5.0 microg/l.

Assessment of phytoplankton class abundance using fluorescence excitation-emission matrix by parallel factor analysis and nonnegative least squares

NASA Astrophysics Data System (ADS)

Su, Rongguo; Chen, Xiaona; Wu, Zhenzhen; Yao, Peng; Shi, Xiaoyong

2015-07-01

The feasibility of using fluorescence excitation-emission matrix (EEM) along with parallel factor analysis (PARAFAC) and nonnegative least squares (NNLS) method for the differentiation of phytoplankton taxonomic groups was investigated. Forty-one phytoplankton species belonging to 28 genera of five divisions were studied. First, the PARAFAC model was applied to EEMs, and 15 fluorescence components were generated. Second, 15 fluorescence components were found to have a strong discriminating capability based on Bayesian discriminant analysis (BDA). Third, all spectra of the fluorescence component compositions for the 41 phytoplankton species were spectrographically sorted into 61 reference spectra using hierarchical cluster analysis (HCA), and then, the reference spectra were used to establish a database. Finally, the phytoplankton taxonomic groups was differentiated by the reference spectra database using the NNLS method. The five phytoplankton groups were differentiated with the correct discrimination ratios (CDRs) of 100% for single-species samples at the division level. The CDRs for the mixtures were above 91% for the dominant phytoplankton species and above 73% for the subdominant phytoplankton species. Sixteen of the 85 field samples collected from the Changjiang River estuary were analyzed by both HPLC-CHEMTAX and the fluorometric technique developed. The results of both methods reveal that Bacillariophyta was the dominant algal group in these 16 samples and that the subdominant algal groups comprised Dinophyta, Chlorophyta and Cryptophyta. The differentiation results by the fluorometric technique were in good agreement with those from HPLC-CHEMTAX. The results indicate that the fluorometric technique could differentiate algal taxonomic groups accurately at the division level.

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

PubMed

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

2013-01-01

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

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

PubMed

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

2018-10-15

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

Qualitative and quantitative analysis of the diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS.

PubMed

Zhao, Ying-Yong; Zhao, Ye; Zhang, Yong-Min; Lin, Rui-Chao; Sun, Wen-Ji

2009-06-01

Polyporus umbellatus is a widely used anti-aldosteronic diuretic in Traditional Chinese medicine (TCM). A new, sensitive and selective high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS/MS) method for quantitative and qualitative determination of ergosta-4,6,8(14),22-tetraen-3-one(ergone), which is the main diuretic component, was provided for quality control of P. umbellatus crude drug. The ergone in the ethanolic extract of P. umbellatus was unambiguously characterized by HPLC-APCI, and further confirmed by comparing with a standard compound. The trace ergone was detected by the sensitive and selective HPLC-FLD. Linearity (r2 > 0.9998) and recoveries of low, medium and high concentration (100.5%, 100.2% and 100.4%) were consistent with the experimental criteria. The limit of detection (LOD) of ergone was around 0.2 microg/mL. Our results indicated that the content of ergone in P. umbellatus varied significantly from habitat to habitat with contents ranging from 2.13 +/- 0.02 to 59.17 +/- 0.05 microg/g. Comparison among HPLC-FLD and HPLC-UV or HPLC-APCI-MS/MS demonstrated that the HPLC-FLD and HPLC-APCI-MS/MS methods gave similar quantitative results for the selected herb samples, the HPLC-UV methods gave lower quantitative results than HPLC-FLD and HPLC-APCI-MS/MS methods. The established new HPLC-FLD method has the advantages of being rapid, simple, selective and sensitive, and could be used for the routine analysis of P. umbellatus crude drug.

An improved independent component analysis model for 3D chromatogram separation and its solution by multi-areas genetic algorithm

PubMed Central

2014-01-01

Background The 3D chromatogram generated by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) has been researched widely in the field of herbal medicine, grape wine, agriculture, petroleum and so on. Currently, most of the methods used for separating a 3D chromatogram need to know the compounds' number in advance, which could be impossible especially when the compounds are complex or white noise exist. New method which extracts compounds from 3D chromatogram directly is needed. Methods In this paper, a new separation model named parallel Independent Component Analysis constrained by Reference Curve (pICARC) was proposed to transform the separation problem to a multi-parameter optimization issue. It was not necessary to know the number of compounds in the optimization. In order to find all the solutions, an algorithm named multi-areas Genetic Algorithm (mGA) was proposed, where multiple areas of candidate solutions were constructed according to the fitness and distances among the chromosomes. Results Simulations and experiments on a real life HPLC-DAD data set were used to demonstrate our method and its effectiveness. Through simulations, it can be seen that our method can separate 3D chromatogram to chromatogram peaks and spectra successfully even when they severely overlapped. It is also shown by the experiments that our method is effective to solve real HPLC-DAD data set. Conclusions Our method can separate 3D chromatogram successfully without knowing the compounds' number in advance, which is fast and effective. PMID:25474487

Implementation of AICAR analysis by GC-C-IRMS for anti-doping purposes.

PubMed

Buisson, C; Frelat, C; Mongongu, C; Martinat, N; Audran, M

2017-11-01

AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), is a naturally occurring substance which is part to the World Anti-Doping Agency (WADA) Prohibited List. It is claimed to improve physical performance when administered as a supplement. As for other endogenous compounds such as steroids, the gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis remains an efficient tool to differentiate endogenous substances from exogenous ones. A protocol was described in the literature for the analysis of AICAR by GC-C-IRMS. The aim of the present study was to implement this protocol in our laboratory and to propose solutions to avoid the difficulties encountered. The first point discussed in this study is the derivatization step. Due to the structure of the AICAR molecule, conventional derivatization for GC-C-IRMS such as acetylation could not be applied and silylation was preferred. The improvement of the derivatives stability was achieved thanks to several derivatization conditions tested. This adjustment led to a reproducible derivatization pattern with the 3-TMS form as major derivative product. The second point discussed in this study is the diminution of extracts' background noise. Indeed, the implementation of the published protocol was not easy due to high performance liquid chromatography (HPLC) problems encountered when concentrated urine was injected into our system. Also, too many interferences in the endogenous reference compound fractions were observed. The addition of both a wash step before the HPLC purification and a HPLC purification step for the endogenous reference compound (ERC) fraction allowed us to increase the robustness of the method. This study presents the modified protocol compared to the original protocol as well as the evaluation of the whole method performances. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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.

Chemicals Compositions, Antioxidant and Anti-Inflammatory Activity of Cynara scolymus Leaves Extracts, and Analysis of Major Bioactive Polyphenols by HPLC

PubMed Central

Ben Salem, Maryem; Athmouni, Khaled; Ksouda, Kamilia; Dhouibi, Raouia; Sahnoun, Zouheir; Hammami, Serria; Zeghal, Khaled Mounir

2017-01-01

Objective. Artichoke (Cynara scolymus L.) was one of the plant remedies for primary health care. The present study was focused on the determination of chemical composition, antioxidant activities, and anti-inflammatory activity and on analyzing its major bioactive polyphenols by HPLC. Methods. Artichoke Leaves Extracts (ALE) were analyzed for proximate analysis and phytochemical and antioxidant activity by several methods such as DDPH, ABTS, FRAP, and beta-carotene bleaching test. The carrageenan (Carr) model induced paw oedema in order to investigate the anti-inflammatory activity. Identification and quantification of bioactive polyphenols compounds were done by HPLC method. The oxidative stress parameters were determined; CAT, SOD, GSH, MDA, and AOPP activities and the histopathological examination were also performed. Results. It was noted that EtOH extract of ALE contained the highest phenolic, flavonoid, and tannin contents and the strongest antioxidants activities including DDPH (94.23%), ABTS (538.75 mmol), FRAP assay (542.62 umol), and β-carotene bleaching (70.74%) compared to the other extracts of ALE. Administration of EtOH extract at dose 400 mg/kg/bw exhibited a maximum inhibition of inflammation induced by Carr for 3 and 5 hours compared to reference group Indomethacin (Indo). Conclusion. ALE displayed high potential as natural source of minerals and phytochemicals compounds with antioxidant and anti-inflammatory properties. PMID:28539965

Chemicals Compositions, Antioxidant and Anti-Inflammatory Activity of Cynara scolymus Leaves Extracts, and Analysis of Major Bioactive Polyphenols by HPLC.

PubMed

Ben Salem, Maryem; Affes, Hanen; Athmouni, Khaled; Ksouda, Kamilia; Dhouibi, Raouia; Sahnoun, Zouheir; Hammami, Serria; Zeghal, Khaled Mounir

2017-01-01

Objective . Artichoke ( Cynara scolymus L.) was one of the plant remedies for primary health care. The present study was focused on the determination of chemical composition, antioxidant activities, and anti-inflammatory activity and on analyzing its major bioactive polyphenols by HPLC. Methods . Artichoke Leaves Extracts (ALE) were analyzed for proximate analysis and phytochemical and antioxidant activity by several methods such as DDPH, ABTS, FRAP, and beta-carotene bleaching test. The carrageenan (Carr) model induced paw oedema in order to investigate the anti-inflammatory activity. Identification and quantification of bioactive polyphenols compounds were done by HPLC method. The oxidative stress parameters were determined; CAT, SOD, GSH, MDA, and AOPP activities and the histopathological examination were also performed. Results . It was noted that EtOH extract of ALE contained the highest phenolic, flavonoid, and tannin contents and the strongest antioxidants activities including DDPH (94.23%), ABTS (538.75 mmol), FRAP assay (542.62 umol), and β -carotene bleaching (70.74%) compared to the other extracts of ALE. Administration of EtOH extract at dose 400 mg/kg/bw exhibited a maximum inhibition of inflammation induced by Carr for 3 and 5 hours compared to reference group Indomethacin (Indo). Conclusion . ALE displayed high potential as natural source of minerals and phytochemicals compounds with antioxidant and anti-inflammatory properties.

Rapid and simple immunochemical screening combined with hand-shaking extraction for thiamethoxam residue in agricultural products.

PubMed

Watanabe, Eiki; Kobara, Yuso; Miyake, Shiro

2013-06-01

With the aim of expanding the applicability of a kit-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide thiamethoxam, the ELISA was newly applied to three kinds of agricultural samples (green pepper, eggplant and spinach). To offer the ELISA as a screening analysis for thiamethoxam residues, a rapid and simple method of extraction by hand-shaking was used, and speed-up and simplification of the sample treatment before the ELISA analysis were examined. Finally, the validity of the ELISA combined with the proposed extraction method was verified against a reference high-performance liquid chromatography (HPLC) method using real-world agricultural samples. There were no marked matrix effects derived from green pepper and eggplant extracts. On the other hand, although the effect due to a pigment in spinach extract on the assay performance was significant, it was effectively avoided by increasing the dilution level of the spinach extract. For thiamethoxam-spiked samples, acceptable recoveries of 97.9-109.1% and coefficients of variation of 0.3-11.5% were obtained. Inspection of the validity of the ELISA by comparison with the reference HPLC method showed that the two analytical results were very similar, and a high correlation was found between them (r>0.997). The evaluated ELISA combined with hand-shaking extraction provided a rapid and simple screening analysis that was quantitative and reliable for the detection of thiamethoxam in complex agricultural products. © 2012 Society of Chemical Industry.

Effect of fungal mycelia on the HPLC-UV and UV-vis spectrophotometric assessment of mycelium-bound epoxide hydrolase using glycidyl phenyl ether.

PubMed

Dolcet, Marta M; Torres, Mercè; Canela, Ramon

2016-06-25

The use of mycelia as biocatalysts has technical and economic advantages. However, there are several difficulties in obtaining accurate results in mycelium-catalysed reactions. Firstly, sample extraction, indispensable because of the presence of mycelia, can bring into the extract components with a similar structure to that of the analyte of interest; secondly, mycelia can influence the recovery of the analyte. We prepared calibration standards of 3-phenoxy-1,2-propanediol (PPD) in the pure solvent and in the presence of mycelia (spiked before or after extraction) from five fungi (Aspergillus niger, Aspergillus tubingensis, Penicillium aurantiogriseum, Penicillium sp. and Aspergillus terreus). The quantification of PPD was carried out by HPLC-UV and UV-vis spectrophotometry. The manuscript shows that the last method is as accurate as the HPLC method. However, the colorimetric method led to a higher data throughput, which allowed the study of more samples in a shorter time. Matrix effects were evaluated visually from the plotted calibration data and statistically by simultaneously comparing the intercept and slope of calibration curves performed with solvent, post-extraction spiked standards and pre-extraction spiked standards. Significant differences were found between the post- and pre-extraction spiked matrix-matched functions. Pre-extraction spiked matrix-matched functions based on A. tubingensis mycelia, selected as the reference, were validated and used to compensate for low recoveries. These validated functions were successfully applied to the quantification of PPD achieved during the hydrolysis of glycidyl phenyl ether by mycelium-bound epoxide hydrolases and equivalent hydrolysis yields were determined by HPLC-UV and UV-vis spectrophotometry. This study may serve as starting point to implement matrix effects evaluation when mycelium-bound epoxide hydrolases are studied. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2010-01-01

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

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

PubMed

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

2016-02-20

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

Determination of alkylphenols and alkylphenol mono- and diethoxylates in environmental samples by high-performance liquid chromatography

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

Ahel, M.; Giger, W.

1985-07-01

A routine method is described for the quantitative determination of 4-nonylphenol (NP) and 4-nonylphenol mono-(NP1EO) and diethoxylate (NP2EO) in samples from wastewater and sludge treatment and from the aquatic environment. An exhaustive steam-distillation/solvent-extraction procedure was employed to enrich the analytes from aqueous and solid samples. Quantitative determinations were performed by normal-phase high-performance liquid chromatography (HP-LC) using aminosilica columns. Relative standard deviations were 3.0-4.4% in a river water containing 3.9 ..mu..g/L NP, 23.4 ..mu..g/L NP1EO, and 9.4 ..mu..g/L NP2EO. A digested sewage sludge with 1.6 g of NP/kg of dry matter was analyzed with a relative standard deviation of 3.7%. Recoveriesmore » were higher than 80%, and the estimated detection limit in water samples was 0.5 ..mu..g/L. Reversed-phase HPLC on octylsilica provided complementary qualitative data, particularly on homologous alkylphenolic compounds. Good agreement was found between quantitative determinations by HPLC and by high-resolution gas chromatography with flame ionization detection and directly coupled mass spectrometry. Municipal wastewater effluents, sewage sludges, and natural waters were analyzed to demonstrate the method's broad applicability. 19 references, 4 tables, 4 figures.« less

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

PubMed

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

2010-10-01

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

Analysis of lipoprotein profiles of healthy cats by gel-permeation high-performance liquid chromatography

PubMed Central

MIZUTANI, Hisashi; SAKO, Toshinori; OKUDA, Hiroko; ARAI, Nobuaki; KURIYAMA, Koji; MORI, Akihiro; YOSHIMURA, Itaru; KOYAMA, Hidekazu

2016-01-01

Density gradient ultracentrifugation (DGUC) and gel electrophoresis are conventionally used to obtain lipoprotein profiles of animals. We recently applied high-performance liquid chromatography with a gel permeation column (GP-HPLC) and an on-line dual enzymatic system to dogs for lipoprotein profile analysis. We compared the GP-HPLC with DGUC as a method to obtain a feline lipoprotein profile. The lipoprotein profiles showed large and small peaks, which corresponded to high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively, whereas very low-density lipoprotein (VLDL) and chylomicron (CM) were only marginally detected. This profile was very similar to that of dogs reported previously. Healthy cats also had a small amount of cholesterol-rich particles distinct from the normal LDL or HDL profile. There was no difference in lipoprotein profiles between the sexes, but males had a significantly larger LDL particle size (P=0.015). This study shows the feasibility of GP-HPLC for obtaining accurate lipoprotein profiles with small sample volumes and provides valuable reference data for healthy cats that should facilitate diagnoses. PMID:27170431

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

PubMed

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

2018-01-01

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

Validity and reliability of a field technique for sweat Na+ and K+ analysis during exercise in a hot‐humid environment

PubMed Central

Baker, Lindsay B.; Ungaro, Corey T.; Barnes, Kelly A.; Nuccio, Ryan P.; Reimel, Adam J.; Stofan, John R.

2014-01-01

Abstract This study compared a field versus reference laboratory technique for extracting (syringe vs. centrifuge) and analyzing sweat [Na+] and [K+] (compact Horiba B‐722 and B‐731, HORIBA vs. ion chromatography, HPLC) collected with regional absorbent patches during exercise in a hot‐humid environment. Sweat samples were collected from seven anatomical sites on 30 athletes during 1‐h cycling in a heat chamber (33°C, 67% rh). Ten minutes into exercise, skin was cleaned/dried and two sweat patches were applied per anatomical site. After removal, one patch per site was centrifuged and sweat was analyzed with HORIBA in the heat chamber (CENTRIFUGE HORIBA) versus HPLC (CENTRIFUGE HPLC). Sweat from the second patch per site was extracted using a 5‐mL syringe and analyzed with HORIBA in the heat chamber (SYRINGE HORIBA) versus HPLC (SYRINGE HPLC). CENTRIFUGE HORIBA, SYRINGE HPLC, and SYRINGE HORIBA were highly related to CENTRIFUGE HPLC ([Na+]: ICC = 0.96, 0.94, and 0.93, respectively; [K+]: ICC = 0.87, 0.92, and 0.84, respectively), while mean differences from CENTRIFUGE HPLC were small but usually significant ([Na+]: 4.7 ± 7.9 mEql/L, −2.5 ± 9.3 mEq/L, 4.0 ± 10.9 mEq/L (all P < 0.001), respectively; [K+]: 0.44 ± 0.52 mEq/L (P < 0.001), 0.01 ± 0.49 mEq/L (P = 0.77), 0.50 ± 0.48 mEq/L (P < 0.001), respectively). On the basis of typical error of the measurement results, sweat [Na+] and [K+] obtained with SYRINGE HORIBA falls within ±15.4 mEq/L and ±0.68 mEq/L, respectively, of CENTRIFUGE HPLC 95% of the time. The field (SYRINGE HORIBA) method of extracting and analyzing sweat from regional absorbent patches may be useful in obtaining sweat [Na+] when rapid estimates in a hot‐humid field setting are needed. PMID:24793982

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

PubMed

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

2017-04-15

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

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

PubMed Central

Bilaničovà, Dagmar; Marcomini, Antonio

2014-01-01

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

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

PubMed

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

2015-01-01

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

Clonazepam quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry in a bioequivalence study.

PubMed

Cavedal, Luiz E; Mendes, Fabiana D; Domingues, Claudia C; Patni, Anil K; Monif, Tausif; Reyar, Simrit; Pereira, Alberto Dos S; Mendes, Gustavo D; De Nucci, Gilberto

2007-01-01

A rapid, sensitive and specific method for quantifying clonazepam in human plasma using diazepam as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid-liquid extraction using a hexane/diethylether (20 : 80, v/v) solution. The extracts were analysed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed on a Jones Genesis C8 4 microm analytical column (100 x 2.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 0.5-50 ng/ml (r2 > 0.9965). The limit of quantification was 0.5 ng/ml. This HPLC/MS/MS procedure was used to assess the bioequivalence of two clonazepam 2 mg tablet formulations (clonazepam test formulation from Ranbaxy Laboratories Ltd and Rivotril from Roche Laboratórios Ltda as standard reference formulation). Copyright 2006 John Wiley & Sons, Ltd.

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

PubMed

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

2014-01-01

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

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

PubMed

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

1997-05-01

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

Ultra-trace levels analysis of microcystins and nodularin in surface water by on-line solid-phase extraction with high-performance liquid chromatography tandem mass spectrometry.

PubMed

Balest, Lydia; Murgolo, Sapia; Sciancalepore, Lucia; Montemurro, Patrizia; Abis, Pier Paolo; Pastore, Carlo; Mascolo, Giuseppe

2016-06-01

An on-line solid phase extraction coupled with high-performance liquid chromatography in tandem with mass spectrometry (on-line SPE/HPLC/MS-MS) method for the determination of five microcystins and nodularin in surface waters at submicrogram per liter concentrations has been optimized. Maximum recoveries were achieved by carefully optimizing the extraction sample volume, loading solvent, wash solvent, and pH of the sample. The developed method was also validated according to both UNI EN ISO IEC 17025 and UNICHIM guidelines. Specifically, ten analytical runs were performed at three different concentration levels using a reference mix solution containing the six analytes. The method was applied for monitoring the concentrations of microcystins and nodularin in real surface water during a sampling campaign of 9 months in which the ELISA method was used as standard official method. The results of the two methods were compared showing good agreement when the highest concentration values of MCs were found. Graphical abstract An on-line SPE/HPLC/MS-MS method for the determination of five microcystins and nodularin in surface waters at sub μg L(-1) was optimized and compared with ELISA assay method for real samples.

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.

Electrochemical Detectors in HPLC and Ion Chromatography.

PubMed

Horvai, George; Pungor, ErnÕ

1989-01-01

Back in 1952, the renowned Polish electrochemist Wiktor Kemula introduced chromato-polarography, 1 i.e., polaro-graphic detection for liquid chromatography. This technique continued to develop slowly until the early 1970s (for a review see Reference 2) when modem high-performance liquid chromatography (HPLC) emerged. This new, highly efficient chromatographc method could only be. used with detectors ensuring low dispersion. It was not easy to modify the dropping mercury electrode cells to satisfy this requirement. However, at the same time, electroanalytical chemists, who already had much experience in using carbon-based electrodes for oxidative detection in flow analysis, put forward the idea of oxidative amperometric detection in liquid chromatography. 3,4 In this technique, solid or quasi-solid (paste) electrodes were used and this made possible the construction of miniaturized cells with just a few microliter volume.

High-pressure liquid chromatography analysis of antibiotic susceptibility disks.

PubMed Central

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

1979-01-01

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

Standardization, evaluation and early-phase method validation of an analytical scheme for batch-consistency N-glycosylation analysis of recombinant produced glycoproteins.

PubMed

Zietze, Stefan; Müller, Rainer H; Brecht, René

2008-03-01

In order to set up a batch-to-batch-consistency analytical scheme for N-glycosylation analysis, several sample preparation steps including enzyme digestions and fluorophore labelling and two HPLC-methods were established. The whole method scheme was standardized, evaluated and validated according to the requirements on analytical testing in early clinical drug development by usage of a recombinant produced reference glycoprotein (RGP). The standardization of the methods was performed by clearly defined standard operation procedures. During evaluation of the methods, the major interest was in the loss determination of oligosaccharides within the analytical scheme. Validation of the methods was performed with respect to specificity, linearity, repeatability, LOD and LOQ. Due to the fact that reference N-glycan standards were not available, a statistical approach was chosen to derive accuracy from the linearity data. After finishing the validation procedure, defined limits for method variability could be calculated and differences observed in consistency analysis could be separated into significant and incidental ones.

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.

Simple equation for calculation of plasma clearance for evaluation of renal function without urine collection in rats.

PubMed

Liu, Xiang; Peng, Dejun; Tian, Hao; Lu, Chengyu

2017-01-01

To develop an equation for the evaluation of renal function in rats using three dilutions of plasma samples and to validate this method by comparison with a reference method. The investigation was conducted in Sprague-Dawley (SD) rats after delivery of three doses of iohexol, with blood samples collected before and after dosage using a quantitative blood collection method. Plasma iohexol concentrations were detected by high performance liquid chromatography (HPLC). The extraction recovery of iohexol from plasma was >97.30% and the calibration curve was linear (r 2  = 0.9997) over iohexol concentrations ranging from 10 to 1000 µg/mL. The method had an RE of <9.310 and intra- and inter-day RSD of <5.137% and <3.693%, respectively. The plasma clearance values obtained from the equation correlated closely (r = 0.763) with those obtained using the reference method. The relatively correlation in the results obtained using the method under investigation and the reference method indicate that this new equation can be used for preliminary assessment of renal function in rats. © 2016 Asian Pacific Society of Nephrology.

[Study on HPLC fingerprint of Oldenlandia diffusa].

PubMed

Chen, Yan; Yao, Zhi-Hong; Dai, Yi; Cheng, Hong; Wen, Li-Rong; Zhou, Guang-Xiong; Yao, Xin-Sheng

2012-06-01

To establish the HPLC fingerprint chromatogram of Oldenlandia diffusa coupled with chemometrics means for the quality control of multi-batches of medicinal material. The separation was developed on C18 column(4.6 mm x 250 mm, 5 microm) by gradient elution with acetonitrile-water(both containing 0.1 per thousand (V/V) ocetic acid) as mobile phase at a flow rate of 0.8 mL/min, the detection wavelength at 238 nm and column temperature at 30 degrees C. The HPLC fingerprint chromatogram of Oldenlandia diffusa was set up and the main characteristic peaks were identified by comparing with chemical reference substance. The quality of 22 batches of medicinal material was evaluated by similarity assay as well as principal component analysis (PCA) and cluster analysis. The established HPLC fingerprint chromatogram of Oldenlandia diffusa was specific, precise, reproducible and stable. 11 peaks were chemically identified. The similarity of 17 batches of Oldenlandia diffusa was obviously higher than 5 batches of adulterants. PCA showed that 17 batches of Oldenlandia diffusa were in a domain and 5 batches of adulterants were far apart from the domain. The cluster analysis of the 22 batches of medicinal material showed that 17 batches of Oldenlandia diffusa were in a cluster while 5 batches of adulterants were excluded. Further cluster analysis was carried out for the quality consistency of 17 batches of Oldenlandia diffusa and accordingly they were devided into 4 clusters. With the combination of chemometrics means, the HPLC fingerprint chromatogram provides a method for evaluation of authenticity and quality control of Oldenlandia diffusa, which is favorable to improve overall quality control of Oldenlandia diffusa.

Survey of Alternaria toxin contamination in food from the German market, using a rapid HPLC-MS/MS approach.

PubMed

Hickert, Sebastian; Bergmann, Marian; Ersen, Seyma; Cramer, Benedikt; Humpf, Hans-Ulrich

2016-02-01

A HPLC-MS/MS-based method for the quantification of nine mycotoxins produced by fungi of the genus Alternaria in various food matrices was developed. The method relies on a single-step extraction, followed by dilution of the raw extract and direct analysis. In combination with an analysis time per sample of 12 min, the sample preparation is cost-effective and easy to handle. The method covers alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), altenuene (ALT), iso-altenuene (isoALT), tentoxin (TEN), altertoxin-I (ATX-I), and the AAL toxins TA1 and TA2. Some Alternaria toxins which are either not commercially available or very expensive, namely AOH, AME, ALT, isoALT, and ATX-I, were isolated as reference compounds from fungal cultures. The method was extensively validated for tomato products, bakery products, sunflower seeds, fruit juices, and vegetable oils. AOH, AME, TeA, and TEN were found in quantifiable amounts and 92.1% of all analyzed samples (n = 96) showed low level contamination with one or more Alternaria toxins. Based on the obtained results, the average daily exposure to Alternaria toxins in Germany was calculated.

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

PubMed

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

2017-11-01

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

Utility of Experimental Design in Pre-Column Derivatization for the Analysis of Tobramycin by HPLC-Fluorescence Detection: Application to Ophthalmic Solution and Human Plasma.

PubMed

El-Zaher, Asmaa A; Mahrouse, Marianne A

2013-01-01

A novel, selective, and sensitive reversed phase high-performance liquid chromatography (HPLC) method coupled with fluorescence detection has been developed for the determination of tobramycin (TOB) in pure form, in ophthalmic solution and in spiked human plasma. Since TOB lacks UV absorbing chromophores and native fluorescence, pre-column derivatization of TOB was carried out using fluorescamine reagent (0.01%, 1.5 mL) and borate buffer (pH 8.5, 2 mL). Experimental design was applied for optimization of the derivatization step. The resulting highly fluorescent stable derivative was chromatographed on C18 column and eluted using methanol:water (60:40, v/v) at a flow rate of 1 mL min(-1). A fluorescence detector (λex 390 and λem 480 nm) was used. The method was linear over the concentration range 20-200 ng mL(-1). The structure of the fluorescent product was proposed, the method was then validated and applied for the determination of TOB in human plasma. The results were statistically compared with the reference method, revealing no significant difference.

[Determination of oxaprozin in human plasma with high performance liquid chromatography (HPLC) and its application].

PubMed

Mao, Mian; Wang, Ling; Jiang, Xuehua; Yang, Lin

2013-06-01

The present research was aimed to develop a high performance liquid chromatography (HPLC) method to determine oxaprozin in plasma and to evaluate the bioavailability of two oxaprozin enteric coated tablets. A C18 column was used to separate the plasma after protein precipitation and the mobile phase was methanol-12. 5mmol/L ammonium acetate buffer solution (pH=3.0)(71:29). The calibration curve was linear in the concentration range of 0. 50-70. 56 microg . mL-1, and the intra and inter-day RSDs were less than 12. 33% and 10. 42% respectively. A single dose of 0. 4 g reference preparation or test preparation of oxaprozin enteric coated tablets was administered to 20 healthy volunteers according to a randomized crossover study. AUC0-->264h were (4 917. 44 +/- 629. 57) microg . h . mL-1 and (4 604. 30+/-737. 83) microg . h . mL-1, respectively; Cmax were (52. 34+/-7. 68) microg . mL-1 and (48. 66+/-4. 87) microg . mL-1, respectively; Tmax were (18. 70+/-2.27) h and (19. 30+/-1. 63) h, respectively; The relative bioavailability of test preparation was 94.0% +/- 13. 7%. The method is simple, rapid and selective for oxaprozin determination. There is no significant difference in the main pharmacokinetic parameters between the test formulation and reference formulation and the two formulations are in bioequivalence.

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

USGS Publications Warehouse

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

2000-01-01

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

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

PubMed

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

2015-01-01

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

Critical assessment of three high performance liquid chromatography analytical methods for food carotenoid quantification.

PubMed

Dias, M Graça; Oliveira, Luísa; Camões, M Filomena G F C; Nunes, Baltazar; Versloot, Pieter; Hulshof, Paul J M

2010-05-21

Three sets of extraction/saponification/HPLC conditions for food carotenoid quantification were technically and economically compared. Samples were analysed for carotenoids alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, lycopene, and zeaxanthin. All methods demonstrated good performance in the analysis of a composite food standard reference material for the analytes they are applicable to. Methods using two serial connected C(18) columns and a mobile phase based on acetonitrile, achieved a better carotenoid separation than the method using a mobile phase based on methanol and one C(18)-column. Carotenoids from leafy green vegetable matrices appeared to be better extracted with a mixture of methanol and tetrahydrofuran than with tetrahydrofuran alone. Costs of carotenoid determination in foods were lower for the method with mobile phase based on methanol. However for some food matrices and in the case of E-Z isomer separations, this was not technically satisfactory. Food extraction with methanol and tetrahydrofuran with direct evaporation of these solvents, and saponification (when needed) using pyrogallol as antioxidant, combined with a HPLC system using a slight gradient mobile phase based on acetonitrile and a stationary phase composed by two serial connected C(18) columns was the most technically and economically favourable method. 2010. Published by Elsevier B.V.

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.

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

PubMed

Klimczak, Inga; Gliszczyńska-Świgło, Anna

2015-05-15

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

[Comparative study on specific chromatograms and main nucleosides of cultivated and wild Cordyceps sinensis].

PubMed

Zan, Ke; Huang, Li-Li; Guo, Li-Nong; Liu, Jie; Zheng, Jian; Ma, Shuang-Cheng; Qian, Zheng-Ming; Li, Wen-Jia

2017-10-01

This study is to establish the HPLC specific chromatogram and determine four main nucleosides of wild and cultivated Cordyceps sinensis. Uridine, inosine, guanosine and adenosine were selected as reference substance. HPLC analysis was performed on a Waters XSelect HSS T3 C₁₈ (4.6 mm×250 mm, 5 μm), with a mobile phase consisting of water(A)-acetonitrile (B) at a flow rate of 0.6 mL•min⁻¹ (0-5 min,0% B;5-15 min,0%-10% B, 15-30 min,10%-20% B, 30-33 min, 20%-50% B, 33-35 min, 50%-0% B, 35-40 min, 0% B). The detection wavelength was 260 nm and the column temperature was controlled at 30 ℃, and the injection volume was 5 μL. HPLC specific chromatogram of wild and cultivated C. sinensis was established and four main nucleosides were simultaneously determined by the above method. Specific chromatograms and contents of four main nucleosides showed no significant differences between cultivated and wild C. sinensis. These results can provide scientific evidences for further development and utilization of cultivated C. sinensis. Copyright© by the Chinese Pharmaceutical Association.

Quantitative determination of the major saponin mixture bacoside A in Bacopa monnieri by HPLC.

PubMed

Deepak, M; Sangli, G K; Arun, P C; Amit, A

2005-01-01

Bacoside A, the putative bioactive component of the Indian medicinal plant Bacopa monnieri, was found to be a mixture of saponins with bacoside A3 (1), bacopaside II (2), jujubogenin isomer of bacopasaponin C (3) and bacopasaponin C (4) as major constituents. An HPLC method together with an optimised extraction procedure was developed for the estimation of 1-4 in B. monnieri to enable standardisation of the latter. Concentration ranges of the analytes in samples of B. monnieri collected from different regions of India were 0.14-0.85% (w/w) (1), 0.12-0.69% (2), 0.05-0.72% (3) and 0.05-0.44% (4). The importance of using bacoside A, with known concentrations of 1-4, as a reference standard for the routine analysis of B. monnieri is highlighted. Two common flavonoids, luteolin and apigenin, were present in all samples of B. monnieri.

AMT (3-(2-aminopropyl)indole) and 5-IT (5-(2-aminopropyl)indole): an analytical challenge and implications for forensic analysis.

PubMed

Elliott, Simon P; Brandt, Simon D; Freeman, Sally; Archer, Roland P

2013-03-01

5-(2-Aminopropyl)indole (5-IT) and 3-(2-aminopropyl)indole (α-methyltryptamine, AMT) are isomeric substances and their differentiation can be a challenge under routine analytical conditions, especially when reference material is unavailable. 5-IT represents a very recent addition to the battery of new psychoactive substances that are commercially available from online retailers. This report illustrates how subtle differences observed under mass spectral and UV conditions can help to facilitate the differentiation between the two isomers. Analyses included (1)  H and (13) C NMR, GC-EI/CI ion trap MS, applications of several U/HPLC-DAD and HPLC-MS methods. Investigations currently underway also highlight the confirmation that AMT was detected in a number of fatal intoxications. These findings also demonstrate that there is a potential risk of misidentification when dealing with both substances. Copyright © 2012 John Wiley & Sons, Ltd.

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

Development of High-purity Certified Reference Materials for 17 Proteinogenic Amino Acids by Traceable Titration Methods.

PubMed

Kato, Megumi; Yamazaki, Taichi; Kato, Hisashi; Eyama, Sakae; Goto, Mari; Yoshioka, Mariko; Takatsu, Akiko

2015-01-01

To ensure the reliability of amino acid analyses, the National Metrology Institute of Japan of the National Institute of Advanced Industrial Science and Technology (NMIJ/AIST) has developed high-purity certified reference materials (CRMs) for 17 proteinogenic amino acids. These CRMs are intended for use as primary reference materials to enable the traceable quantification of amino acids. The purity of the present CRMs was determined based on two traceable methods: nonaqueous acidimetric titration and nitrogen determination by the Kjeldahl method. Since neither method could distinguish compounds with similar structures, such as amino acid-related impurities, impurities were thoroughly quantified by combining several HPLC methods, and subtracted from the obtained purity of each method. The property value of each amino acid was calculated as a weighted mean of the corrected purities by the two methods. The uncertainty of the property value was obtained by combining measurement uncertainties of the two methods, a difference between the two methods, the uncertainty from the contribution of impurities, and the uncertainty derived from inhomogeneity. The uncertainty derived from instability was considered to be negligible based on stability monitoring of some CRMs. The certified value of each amino acid, property value with uncertainty, was given for both with or without enantiomeric separation.

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

USGS Publications Warehouse

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

2000-01-01

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

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

PubMed

Sheldon, E M; Downar, J B

2000-08-15

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

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

PubMed

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

2004-06-01

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

[HPLC specific chromatogram of Lamiophlomis Herba and its counterfeit and determination of four effective components].

PubMed

Zan, Ke; Jiao, Xing-Ping; Guo, Li-Nong; Zheng, Jian; Ma, Shuang-Cheng

2016-06-01

This study is to establish the HPLC specific chromatogram and determine four main effective components of Lamiophlomis Herba and its counterfeit.Chlorogenic acid, forsythoside B, acteoside and luteoloside were reference substance.HPLC analysis was performed on a Waters XSelect C₁₈ column (4.6 mm×250 mm,5 μm).The mobile phase was acetonitrile-0.5% phosphoric acid solution (18∶82) with isocratic elution.The flow rate was 1.0 mL•min⁻¹, the detection wavelength was 332 nm and the column temperature was 30 ℃.Chemometrics software Chempattern was employed to analyze the research data.HPLC specific chromatogram of Lamiophlomis Herba from different samples were of high similarity, but the similarity of the HPLC specific chromatogram of its counterfeit were less than 0.65.Both of cluster and principal component analysis can distinguish certified products and adulterants.The HPLC specific chromatogram and contents of four effective components can be used for the quality control of Lamiophlomis Herba and its preparations.It provided scientific basis to standardize the use of the crude drug. Copyright© by the Chinese Pharmaceutical Association.

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

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

PubMed

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

2014-01-01

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

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

PubMed Central

2014-01-01

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

Development of an Ion-Pairing Reagent and HPLC-UV Method for the Detection and Quantification of Six Water-Soluble Vitamins in Animal Feed.

PubMed

Kim, Ho Jin

2016-01-01

A novel and simple method for detecting six water-soluble vitamins in animal feed using high performance liquid chromatography equipped with a photodiode array detector (HPLC/PDA) and ion-pairing reagent was developed. The chromatographic peaks of the six water-soluble vitamins were successfully identified by comparing their retention times and UV spectra with reference standards. The mobile phase was composed of buffers A (5 mM PICB-6 in 0.1% CH3COOH) and B (5 mM PICB-6 in 65% methanol). All peaks were detected using a wavelength of 270 nm. Method validation was performed in terms of linearity, sensitivity, selectivity, accuracy, and precision. The limits of detection (LODs) for the instrument employed in these experiments ranged from 25 to 197 μg/kg, and the limits of quantification (LOQs) ranged from 84 to 658 μg/kg. Average recoveries of the six water-soluble vitamins ranged from 82.3% to 98.9%. Method replication resulted in intraday and interday peak area variation of <5.6%. The developed method was specific and reliable and is therefore suitable for the routine analysis of water-soluble vitamins in animal feed.

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

PubMed

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

2011-09-16

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

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

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-07

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

Quality Analysis of Chlorogenic Acid and Hyperoside in Crataegi fructus

PubMed Central

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

2016-01-01

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

Analysis of black carbon molecular markers by two chromatographic methods (GC-FID and HPLC-DAD)

NASA Astrophysics Data System (ADS)

Schneider, Maximilian P. W.; Smittenberg, Rienk H.; Dittmar, Thorsten; Schmidt, Michael W. I.

2010-05-01

The analysis of benzenepolycarboxylic acids (BPCA) as a quantitative measure for black carbon (BC) in soil and sediment samples is a well-established method [1, 2]. Briefly, the oxidation of polycondensated BC molecules forms seven molecular markers, which can be assigned to BC, and which subsequently can be quantified by GC-FID (gas chromatography with flame ionization detector). Recently this method has been refined for BC quantification in seawater samples measuring BPCA on HPLC-DAD (High performance liquid chromatography with diode array detector) [3]. However, a systematic comparison of BC as determined by both analytical techniques would be essential to the calculation of global BC budgets, but is lacking. Here we present data for the systematic comparison of the two BPCA methods, both for quantity and quality. We prepared chars under well-defined laboratory conditions. Chestnut hardwood chips and rice straw were pyrolysed at temperatures between 200 and 1000°C under constant N2 stream. The BC contents of the chars have been analysed using the BPCA extraction method followed by either GC-FID or HPLC-DAD quantification [4]. It appears that the GC-FID method yields systematically lower concentrations of BPCA in the chars compared to the HPLC-DAD method. Possible reasons for the observed difference are i) higher losses of sample material during preparation for GC-FID; ii) different quality of the linear regression used for quantification; iii) incomplete derivatisation of B5CA and B6CA, which is needed for GC-FID analysis. In a next step, we will test different derivatisation procedures (methylation with dimethyl sulfate or diazomethane, and silylation) for their influence on the GC-FID results. The aim of this study is to test if black carbon can be quantified in soil, sediment and water samples using one single method - a crucial step when attempting a global BC budget. References: [1] Brodowski, S., Rodionov, A., Haumeier L., Glaser, B., Amelung, W. (2005) Org. Geochem. 36, 1299-1310. [2] Glaser, B., Haumeier, L., Guggenberger, G., Zech, W. (1998) Org. Geochem. 29, 811-819. [3] Dittmar, T. (2008) Org. Geochem. 39. 396-407. [4] Schneider, M.P.W., Hilf, M., Vogt, U.F., Schmidt, M.W.I., Org. Geochem. (submitted)

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

PubMed

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

2004-01-01

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

Comparison of HbA1c Measurements using 3 Methods in 75 Patients Referred to One Outpatient Department.

PubMed

Roth, Johannes; Müller, Nicolle; Lehmann, Thomas; Böer, Klas; Löbel, Sven; Pum, Joachim; Müller, Ulrich Alfons

2018-01-01

HbA 1c is the most important surrogate parameter to assess the quality of diabetes care and is also used for the diagnosis of diabetes mellitus (DM) since 2010. We investigated the comparability of 3 HbA 1c methods in the city of Jena (Germany). The HbA 1c determination was carried out in 50 healthy subjects and 24 people with DM (age 51.2±16.3 years, HbA 1c 6.8±2.2%) with 3 different hemoglobin A 1c testing methods at 4 locations in one city. Our laboratory (HPLC method) served as a reference for comparing the results. All methods are IFCC standardized and all devices are certified by the interlaboratory test. The mean HbA 1c of people without diabetes was: laboratory A (TOSOH G8, HPLC) 5.7±0.3%; laboratory B (TOSOH G8, HPLC) 5.5±0.3%, laboratory C (VARIANT II) 5.2±0.3%; laboratory D (COBAS INT.) 5.6±0.3%. All differences are significant (p=0.001).The mean HbA 1c of patients with mild to moderate elevated HbA 1c was: Laboratory A 7.5±0.9%; B 7.3±1.0%; C 7.0±0.9%; D 7.5±1.1%. Differences are significant (p=0.001) except between laboratory A and D (p=0.8).The mean HbA 1c of patients with massively increased HbA 1c was: laboratory A 11.5±1.8%; laboratory B 11.4±1.8%; laboratory C 10.8±1.6%; laboratory D 11.5±1.5%. Differences between laboratory A and C, as well as between C and D were significant (p=0.001). The mean IFCC standardized HbA 1c from 75 people differs by up to 0.5% absolute between 4 laboratories. This difference is clinically significant and may lead to misdiagnosis and wrong treatment decisions, while HbA 1c value from one patient were analyzed in different laboratories within a short time. © Georg Thieme Verlag KG Stuttgart · New York.

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.

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

PubMed

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

2007-01-01

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

[Simultaneous determination of 4 diterpenoids in Rabdosia japonica var.glaucocalyx by HPLC-ESI-MS/MS and cluster analysis].

PubMed

Tian, Ting-Ting; Ma, Ying-Hua; Xie, Wei-Wei; Jin, Yi-Ran; Xu, Hui-Jun; Zhang, Lan-Tong; Du, Ying-Feng

2016-01-01

A quick HPLC-ESI-MS/MS method was established for simultaneous determination of four major diterpenoids in Rabdosia japonica var.glaucocalyx, including glaucocalyxin A, oridonin, hebeirubesensin and enmenol. Analysis was performed on an Agilent ZORBAX SB-C18(4.6 mm×250 mm, 5 μm ) column eluted in a gradient program with methanol and water. The flow rate was 0.8 mL•min⁻¹. Multiple reaction monitoring (MRM) scanning mode was performed in negative ion switching mode to apply for the quantitative determination. The calibration curves for the above four compounds were linear in corresponding injection amount. The average recoveries of the compounds ranged from 92.40% to 105.9%, with RSDs of 1.7%-6.5%. The method is simple, rapid, accurate with good repeatability, which can provide a reference for overcalling evaluation the quality of R. japonica var.glaucocalyx. The result of cluster analysis- showed that the quality of R. japonica glaucocalyx var. greatly varied between areas and parts. Copyright© by the Chinese Pharmaceutical Association.

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.

Quantitative analysis of valsartan by two-dimensional liquid chromatography (2D-HPLC) and its application in a bioequivalence study in Chinese volunteers
.

PubMed

Zhang, Min; Deng, Yang; Cai, Hua-Lin; Fang, Ping-Fei; Yan, Miao; Zhang, Bi-Kui; Wu, Yan-Qin

2017-04-01

To develop a sensitive, two-dimensional liquid chromatography (2D-LC) method for determination of valsartan, applied to investigate bioequivalence of two valsartan tablets in Chinese volunteers under fasting condition. A full automatic 2D-HPLC system was used to quantify valsartan in human plasma. The analytes were extracted by protein precipitation, using telmisartan as internal standard. The analytical method was applied in a randomized, crossover bioequivalence study of valsartan tablets; the study enrolled 18 Chinese volunteers (12 were men and 6 were women). The subjects received a single 160-mg dose of test or reference preparation with 7-days of washout under fasting state. Plasma samples were collected, pharmacokinetic parameters were obtained and the bioequivalence was evaluated. The calibration range was 9.2 - 4213.8 ng×mL-1. Inter- and intraprecision was less than 7.0%, and accuracies ranged from 99.5 to 103.8%. The extraction recovery for valsartan varied between 89.3 and 97.8%, and the stability in all conditions was excellent. The 90% CI of AUC_0→36h and C_max were 96.5 - 109.4% and 94.2 - 108.6%, respectively. The relative bioavailability was 103.9 ± 15.7%. No gender difference was observed in pharmacokinetic parameters. A sensitive 2D-HPLC method was established for the estimation of valsartan in human plasma and successfully applied in a bioequivalence study of valsartan, which suggests that these two formulations can be assumed to be bioequivalent.
.

Optimization of Robust HPLC Method for Quantitation of Ambroxol Hydrochloride and Roxithromycin Using a DoE Approach.

PubMed

Patel, Rashmin B; Patel, Nilay M; Patel, Mrunali R; Solanki, Ajay B

2017-03-01

The aim of this work was to develop and optimize a robust HPLC method for the separation and quantitation of ambroxol hydrochloride and roxithromycin utilizing Design of Experiment (DoE) approach. The Plackett-Burman design was used to assess the impact of independent variables (concentration of organic phase, mobile phase pH, flow rate and column temperature) on peak resolution, USP tailing and number of plates. A central composite design was utilized to evaluate the main, interaction, and quadratic effects of independent variables on the selected dependent variables. The optimized HPLC method was validated based on ICH Q2R1 guideline and was used to separate and quantify ambroxol hydrochloride and roxithromycin in tablet formulations. The findings showed that DoE approach could be effectively applied to optimize a robust HPLC method for quantification of ambroxol hydrochloride and roxithromycin in tablet formulations. Statistical comparison between results of proposed and reported HPLC method revealed no significant difference; indicating the ability of proposed HPLC method for analysis of ambroxol hydrochloride and roxithromycin in pharmaceutical formulations. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

PubMed

Rakete, Stefan; Glomb, Marcus A

2013-04-24

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

A Comprehensive Characterisation of Rosemary tea Obtained from Rosmarinus officinalis L. Collected in a sub-Humid Area of Tunisia.

PubMed

Achour, Mariem; Mateos, Raquel; Ben Fredj, Maha; Mtiraoui, Ali; Bravo, Laura; Saguem, Saad

2018-01-01

Rosemary (Rosmarinus officinalis L.) is an aromatic plant common in Tunisia and it is widely consumed as a tea in traditional cuisine and in folk medicine to treat various illnesses. Currently, most research efforts have been focused on rosemary essential oil, alcoholic and aqueous extracts, however, little is reported on rosemary infusion composition. To investigate compounds present in rosemary tea obtained from Rosmarinus officinalis L. collected in a sub-humid area of Tunisia in order to assess whether the traditional rosemary tea preparation method could be considered as a reference method for rosemary's compounds extraction. Qualitative characterisation of Rosmarinus officinalis tea obtained after rosemary infusion in boiled water was determined by high performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). Quantitative analysis relies on high performance liquid chromatography with diode array detector (HPLC-DAD). Forty-nine compounds belonging to six families, namely flavonoids, phenolic acids, phenolic terpenes, jasmonate, phenolic glycosides, and lignans were identified. To the best of the authors' knowledge eucommin A is characterised for the first time in rosemary. Rosmarinic acid (158.13 μg/g dried rosemary) was the main compound followed then by feruloylnepitrin (100.87 μg/g) and luteolin-3'-O-(2″-O-acetyl)-β-d-glucuronide (44.04 μg/g). Among quantified compounds, luteolin-7-O-rutinoside was the compound with the lowest concentration. The infusion method allows several polyphenols present in rosemary tea to be extracted, therefore it could be a reference method for rosemary's compounds extraction. Moreover, traditional Tunisian Rosmarinus officinalis tea consumption is of interest for its rich phenolic content. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

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

PubMed Central

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

2010-01-01

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

Development and validation of a simple and robust method for arsenic speciation in human urine using HPLC/ICP-MS.

PubMed

Sen, Indranil; Zou, Wei; Alvaran, Josephine; Nguyen, Linda; Gajek, Ryszard; She, Jianwen

2015-01-01

In order to better distinguish the different toxic inorganic and organic forms of arsenic (As) exposure in individuals, we have developed and validated a simple and robust analytical method for determining the following six As species in human urine: arsenous (III) acid (As-III), As (V) acid, monomethylarsonic acid, dimethylarsinic acid, arsenobetaine (AsB), and arsenocholine. In this method, human urine is diluted using a pH 5.8 buffer, separation is performed using an anion exchange column with isocratic HPLC, and detection is achieved using inductively coupled plasma-MS. The method uses a single mobile phase consisting of low concentrations of both phosphate buffer (5 mM) and ammonium nitrate salt (5 mM) at pH 9.0; this minimizes the column equilibration time and overcomes challenges with separation between AsB and As-III. In addition, As-III oxidation is prevented by degassing the sample preparation buffer at pH 5.8, degassing the mobile phase online at pH 9.0, and by the use of low temperature (-70 °C) and flip-cap airtight tubes for long term storage of samples. The method was validated using externally provided reference samples. Results were in agreement with target values at varying concentrations and successfully passed external performance test criteria. Internal QC samples were prepared and repeatedly analyzed to assess the method's long-term precision, and further analyses were completed on anonymous donor urine to assess the quality of the method's baseline separation. Results from analyses of external reference samples agreed with target values at varying concentrations, and results from precision studies yielded absolute CV values of 3-14% and recovery from 82 to 115% for the six As species. Analysis of anonymous donor urine confirmed the well-resolved baseline separation capabilities of the method for real participant samples.

The application of artificial neural networks and support vector regression for simultaneous spectrophotometric determination of commercial eye drop contents

NASA Astrophysics Data System (ADS)

Valizadeh, Maryam; Sohrabi, Mahmoud Reza

2018-03-01

In the present study, artificial neural networks (ANNs) and support vector regression (SVR) as intelligent methods coupled with UV spectroscopy for simultaneous quantitative determination of Dorzolamide (DOR) and Timolol (TIM) in eye drop. Several synthetic mixtures were analyzed for validating the proposed methods. At first, neural network time series, which one type of network from the artificial neural network was employed and its efficiency was evaluated. Afterwards, the radial basis network was applied as another neural network. Results showed that the performance of this method is suitable for predicting. Finally, support vector regression was proposed to construct the Zilomole prediction model. Also, root mean square error (RMSE) and mean recovery (%) were calculated for SVR method. Moreover, the proposed methods were compared to the high-performance liquid chromatography (HPLC) as a reference method. One way analysis of variance (ANOVA) test at the 95% confidence level applied to the comparison results of suggested and reference methods that there were no significant differences between them. Also, the effect of interferences was investigated in spike solutions.

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

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

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

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

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

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

PubMed

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

2015-03-25

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

[Rapid identification of micro-constituents in monoammonium glycyrrhizinate raw materials by high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry].

PubMed

Yang, Xue-Dong; Tang, Xu-Yan; Sang, Lin

2012-11-01

To establish a method for rapid identification of micro-constituents in monoammonium glycyrrhizinate by high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry. HPLC preparative chromatograph was adopted for determining the optimal method for high-pressure solid phase extraction under optimal conditions. 5C18-MS-II column (20.0 mm x 20.0 mm) was used as the extraction column, with 35% acetonitrile-acetic acid solution (pH 2. 20) as eluent at the speed of 16 mL x min(-1). The sample size was 0.5 mL, and the extraction cycle was 4.5 min. Then, extract liquid was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) after being concentrated by 100 times. Under the optimal condition of high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry, 10 components were rapidly identified from monoammonium glycyrrhizinate raw materials. Among them, the chemical structures of six micro-constituents were identified as 3-O-[beta-D-glucuronopyranosyl-beta-D-glucuronopyranosyl]-30-0-beta-D-apiopyranosylglycyrrhetic/3-O- [P-D-glucuronopyranosyl-beta-D-glucuronopyranosyl]-30-O-beta-D-arabinopyranosylglycyrrhetic, glycyrrhizic saponin F3, 22-hydroxyglycyrrhizin/18alpha-glycyrrhizic saponin G2, 3-O-[beta-D-rhamnopyranosyl]-24-hydroxyglycyrrhizin, glycyrrhizic saponin J2, and glycyrrhizic saponin B2 by MS(n) spectra analysis and reference to literatures. Four main chemical components were identified as glycyrrhizic saponin G2, 18beta-glycyrrhizic acid, uralglycyrrhizic saponin B and 18alpha-glycyrrhizic acid by liquid chromatography, MS(n) and ultraviolet spectra information and comparison with reference substances. The method can be used to identify chemical constituents in monoammonium glycyrrhizinate quickly and effectively, without any reference substance, which provides basis for quality control and safe application of monoammonium glycyrrhizinate-related products.

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

USGS Publications Warehouse

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

2001-01-01

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

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.

Analysis of formononetin from black cohosh (Actaea racemosa).

PubMed

Jiang, B; Kronenberg, F; Balick, M J; Kennelly, E J

2006-07-01

Black cohosh has been widely used as an herbal medicine for the treatment of symptoms related to menopause in America and Europe during the past several decades, but the bioactive constituents are still unknown. Formononetin is an isoflavone with known estrogen-like activity. This compound was first reported to be isolated from black cohosh in 1985, but subsequent research in 2002 using HPLC-PDA and LC-MS revealed no evidence to show the presence of formononetin in 13 populations of American black cohosh. A more recent report published in 2004 claimed to detect formononetin in an extract of black cohosh rhizomes using a TLC-fluorescent densitometry method. To further resolve these conflicting reports, we analyzed black cohosh roots and rhizomes for the presence of formononetin, using a combined TLC, HPLC-PDA and LC-MS method. We examined both methanolic and aqueous methanolic black cohosh extracts by HPLC-PDA and LC-MS methods, and did not detect formononetin in any extracts. We further determined the limits of detection of formononetin by HPLC-PDA and LC-MS. Our experimental results indicated that the sensitivity and accuracy of the HPLC-PDA and LC-MS methods for the analysis of formononetin were slightly higher than those of the reported fluorescent method, suggesting that the HPLC-PDA and LC-MS methods were reliable for the analysis of formononetin from black cohosh. We also repeated the reported TLC method to concentrate two fractions from a modern black cohosh sample and an 86-year-old black cohosh sample, respectively, and then analyzed these two fractions for formononetin using the HPLC-PDA and LC-MS method instead of the fluorescent method. Formononetin was not detected by HPLC-PDA or LC-MS. From the results of the present study it is not reasonable to attribute the estrogen-like activity of black cohosh extracts to formononetin.

At-line determination of pharmaceuticals small molecule's blending end point using chemometric modeling combined with Fourier transform near infrared spectroscopy

NASA Astrophysics Data System (ADS)

Tewari, Jagdish; Strong, Richard; Boulas, Pierre

2017-02-01

This article summarizes the development and validation of a Fourier transform near infrared spectroscopy (FT-NIR) method for the rapid at-line prediction of active pharmaceutical ingredient (API) in a powder blend to optimize small molecule formulations. The method was used to determine the blend uniformity end-point for a pharmaceutical solid dosage formulation containing a range of API concentrations. A set of calibration spectra from samples with concentrations ranging from 1% to 15% of API (w/w) were collected at-line from 4000 to 12,500 cm- 1. The ability of the FT-NIR method to predict API concentration in the blend samples was validated against a reference high performance liquid chromatography (HPLC) method. The prediction efficiency of four different types of multivariate data modeling methods such as partial least-squares 1 (PLS1), partial least-squares 2 (PLS2), principal component regression (PCR) and artificial neural network (ANN), were compared using relevant multivariate figures of merit. The prediction ability of the regression models were cross validated against results generated with the reference HPLC method. PLS1 and ANN showed excellent and superior prediction abilities when compared to PLS2 and PCR. Based upon these results and because of its decreased complexity compared to ANN, PLS1 was selected as the best chemometric method to predict blend uniformity at-line. The FT-NIR measurement and the associated chemometric analysis were implemented in the production environment for rapid at-line determination of the end-point of the small molecule blending operation. FIGURE 1: Correlation coefficient vs Rank plot FIGURE 2: FT-NIR spectra of different steps of Blend and final blend FIGURE 3: Predictions ability of PCR FIGURE 4: Blend uniformity predication ability of PLS2 FIGURE 5: Prediction efficiency of blend uniformity using ANN FIGURE 6: Comparison of prediction efficiency of chemometric models TABLE 1: Order of Addition for Blending Steps

Steroids

NASA Astrophysics Data System (ADS)

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

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

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

PubMed

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

2010-03-01

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

76 FR 38033 - Cloquintocet-mexyl; Pesticide Tolerances

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-29

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

Development of a percutaneous penetration predictive model by SR-FTIR.

PubMed

Jungman, E; Laugel, C; Rutledge, D N; Dumas, P; Baillet-Guffroy, A

2013-01-30

This work focused on developing a new evaluation criterion of percutaneous penetration, in complement to Log Pow and MW and based on high spatial resolution Fourier transformed infrared (FTIR) microspectroscopy with a synchrotron source (SR-FTIR). Classic Franz cell experiments were run and after 22 h molecule distribution in skin was determined either by HPLC or by SR-FTIR. HPLC data served as reference. HPLC and SR-FTIR results were compared and a new predictive criterion based from SR-FTIR results, named S(index), was determined using a multi-block data analysis technique (ComDim). A predictive cartography of the distribution of molecules in the skin was built and compared to OECD predictive cartography. This new criterion S(index) and the cartography using SR-FTIR/HPLC results provides relevant information for risk analysis regarding prediction of percutaneous penetration and could be used to build a new mathematical model. Copyright © 2012 Elsevier B.V. All rights reserved.

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.

Nontargeted quantitation of lipid classes using hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry with single internal standard and response factor approach.

PubMed

Cífková, Eva; Holčapek, Michal; Lísa, Miroslav; Ovčačíková, Magdaléna; Lyčka, Antonín; Lynen, Frédéric; Sandra, Pat

2012-11-20

The identification and quantitation of a wide range of lipids in complex biological samples is an essential requirement for the lipidomic studies. High-performance liquid chromatography-mass spectrometry (HPLC/MS) has the highest potential to obtain detailed information on the whole lipidome, but the reliable quantitation of multiple lipid classes is still a challenging task. In this work, we describe a new method for the nontargeted quantitation of polar lipid classes separated by hydrophilic interaction liquid chromatography (HILIC) followed by positive-ion electrospray ionization mass spectrometry (ESI-MS) using a single internal lipid standard to which all class specific response factors (RFs) are related to. The developed method enables the nontargeted quantitation of lipid classes and molecules inside these classes in contrast to the conventional targeted quantitation, which is based on predefined selected reaction monitoring (SRM) transitions for selected lipids only. In the nontargeted quantitation method described here, concentrations of lipid classes are obtained by the peak integration in HILIC chromatograms multiplied by their RFs related to the single internal standard (i.e., sphingosyl PE, d17:1/12:0) used as common reference for all polar lipid classes. The accuracy, reproducibility and robustness of the method have been checked by various means: (1) the comparison with conventional lipidomic quantitation using SRM scans on a triple quadrupole (QqQ) mass analyzer, (2) (31)P nuclear magnetic resonance (NMR) quantitation of the total lipid extract, (3) method robustness test using subsequent measurements by three different persons, (4) method transfer to different HPLC/MS systems using different chromatographic conditions, and (5) comparison with previously published results for identical samples, especially human reference plasma from the National Institute of Standards and Technology (NIST human plasma). Results on human plasma, egg yolk and porcine liver extracts are presented and discussed.

Comparative bioavailability study of clonazepam after oral administration of two tablet formulations.

PubMed

Chauhan, B L; Sane, S P; Revankar, S N; Rammamurthy, L; Doshi, B; Bhatt, A D; Bhate, V R; Kulkarni, R D

2000-10-01

To assess the bioavailability of clonazepam from two brands of 2 mg tablet formulations--Epitril and reference brand. A two-way randomised cross-over bioavailability study was carried out in 12 healthy male volunteers. Coded plasma samples were analysed for levels of clonazepam by high performance liquid chromatography (HPLC) method. The mean Cmax, Tmax t1/2 beta and AUC (0-48) for Epitril were: 16.31 +/- 3.07 ng/mL, 1.63 +/- 0.48 h, 46.97 +/- 12.26 h and 207.70 +/- 57.07 ng/ml.h; for reference brand were 19.75 +/- 5.95 ng/mL, 1.42 +/- 0.29 h, 46.88 +/- 11.29 h and 215.70 +/- 50.89 ng/ml.h respectively. These were comparable and the differences were not statistically significant. Based on above pharmacokinetic parameters, Epitril was bioequivalent to reference brand.

Quantification of lycopene in the processed tomato-based products by means of the light-emitting diode (LED) and compact photoacoustic (PA) detector

NASA Astrophysics Data System (ADS)

Bicanic, D.; Skenderović, H.; Marković, K.; Dóka, O.; Pichler, L.; Pichler, G.; Luterotti, S.

2010-03-01

The combined use of a high power light emitting diode (LED) and the compact photoacoustic (PA) detector offers the possibility for a rapid (no extraction needed), accurate (precision 1.5%) and inexpensive quantification of lycopene in different products derived from the thermally processed tomatoes. The concentration of lycopene in selected products ranges from a few mg to several tens mg per 100 g fresh weight. The HPLC was used as the well established reference method.

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

PubMed

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

2012-07-01

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

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

PubMed

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

2011-01-15

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

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

USGS Publications Warehouse

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

2003-01-01

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

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

PubMed

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

2011-04-01

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

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

PubMed

Gobbo-Neto, Leonardo; Lopes, Norberto P

2008-02-27

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

[HPLC Specific Fingerprint of Alcohol Extract of Andrographis paniculata].

PubMed

Liu, Fei-fei; Fan, Chun-lin; Huang, Xiao-jun; Wang, Gui-yang; Wang, Ying; Ye, Wen-cai

2015-07-01

To establish the specific fingerprint of alcohol extract of Andrographis paniculata by HPLC. The analysis was performed on Cosmosil 5C18 -MS-II (250 mm x 4. 6 mm, 5 µm) column, with gradient phase consisting of acetonitrile and water at the flow rate of 1. 0 mL/min. The UV detection wavelength was set at 225 nm, and column temperature was 30 °C. The specific fingerprint chromatogram was established and seven common peaks were identified by comparison with the reference standards and LC-MS. The relative retention times were 1. 00 (No. 1, andrographolide), 1. 04 (No. 2, deoxyandrographoside), 1. 07 (No. 3, isoandrographolide), 1. 10 (No. 4, 14-deoxy-11, 12-didehydroandrographoside), 1. 50 (No. 5, neoandrographolide), 1. 75 ( No. 6, deoxyandrographolide) and 1. 79 (No. 7, dehydroandrographolide). The method is simple and reproducible with high precision, which can provide the basis for the quality control and evaluation of alcohol extract of Andrographis paniculata and its preparations.

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

NASA Technical Reports Server (NTRS)

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

1989-01-01

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

HPLC and UPLC methods for the determination of zearalenone in noodles, cereal snacks and infant formula.

PubMed

Ok, Hyun Ee; Choi, Sung-Wook; Kim, Meehye; Chun, Hyang Sook

2014-11-15

High-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were compared to validate a method for determination of zearalenone (ZON) in noodles, cereal snacks, and infant formulas. The limits of detection and quantification in HPLC and UPLC were found to be 4.0 and 13.0 μg kg(-1) and 2.5 and 8.3 μg kg(-1), respectively. The average recoveries of ZON by HPLC and UPLC ranged from 79.1% to 105.3% and from 85.1% to 114.5%, respectively. The measurement uncertainties of the two methods for ZON determination were within the maximum standard uncertainty. The two methods showed that the levels of ZON in 163 naturally contaminated samples ranged from 4.3 to 8.3 μg kg(-1) by HPLC and 3.1 to 17.6 μg kg(-1) by UPLC. These findings indicate that either method is suitable for the determination of ZON in noodles, cereal snacks, and infant formulas, but UPLC gives faster results with better sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quality control and identification of steroid saponins in crude extracts from Dioscorea zingiberensis C. H. Wright by fingerprint with HPLC-ELSD and HPLC-ESI-Quadrupole/Time-of-fight tandem mass spectrometry

PubMed Central

Zhang, Xinxin; Liang, Jinru; Liu, Jianli; Zhao, Ye; Gao, Juan; Sun, Wenji; Ito, Yoichiro

2014-01-01

In this study, a fingerprint of steroid saponins, the major bioactive constituents in the crude extracts from Dioscorea zingiberensis C. H. Wright (DZW), has been established for the first time by high-performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD) and the simultaneous characterization of the steroid saponins by high-performance liquid chromatography coupled with electrospray ionization-mass spectrometry and quadrupole tandem time-of-fight mass analyzers detection (HPLC-ESI-Q/TOF). These HPLC analyses were both carried out on a Welchrom C18 column (250 mm × 4.6 mm I.D., 5 μm) with a mobile phase composed of water and acetonitrile under gradient elution. There were 68 common characteristic peaks in the fingerprints, in which 12 of them were confirmed by comparing their mass spectra and retention times with those of the reference compounds. In order to identify the other unknown peaks, their fragmentation behaviors characteristic for the major groups of steroid saponins from DZW with six types of aglycone skeletons were discussed in detail, and possible MS/MS fragmentation pathways were proposed for aiding the structural identification of these components. According to the summarized fragmentation patterns, these peaks were tentatively assigned by matching their empirical molecular formula with those of the published compounds, or by elucidating their quasi-molecular ions and fragment ions referring to available literature information when the reference standards were unavailable. As a result, 22 steroid saponins were found in DZW for the first time. In addition, the quantitative analysis of the 12 known peaks was accomplished at the same time which indicated that there was a great variability in the amount of these active compounds in different batches in the crude extracts. This approach could demonstrate that the fingerprint could be considered to be a suitable tool to comprehensively improve the quality control of DZW, and the identification and structural elucidation of the peaks in the fingerprint may provide important experimental data for further pharmacological and clinical researches. PMID:24418811

An effective identification and quantification method for Ginkgo biloba flavonol glycosides with targeted evaluation of adulterated products.

PubMed

Ma, Yuan-Chun; Mani, Ana; Cai, Yaling; Thomson, Jaclyn; Ma, Jie; Peudru, Flavie; Chen, Sarah; Luo, Mai; Zhang, Junzeng; Chapman, Robert G; Shi, Zhen-Tuo

2016-04-15

Ginkgo biloba L. (Ginkgoaceae) leaf extract is one of the most popular herbal products on the market, as it contains flavone glycosides (≥ 24%) and terpene lactones (≥ 6%), which are proposed to have significant physiological effects. Unfortunately, the challenging financial climate has resulted in a natural health product market containing adulterated ginkgo products. 42 ginkgo samples were analyzed to establish an HPLC profile for authentic ginkgo and common ginkgo adulterants, and to develop a method capable of easily detecting adulteration in ginkgo commercial products. In this study an efficient and targeted HPLC analysis method was established that is capable of distinguishing flavonol glycosides and aglycones simultaneously for the evaluation of ginkgo powdered extracts (PEs) and finished products in a single, 13 min run. Thirteen ginkgo leaf samples, fifteen standardized powdered extracts, and fourteen commercially available ginkgo products have been analyzed using this new HPLC method. Chromatograms were compared to six standard reference materials: one flavonol glycoside (rutin), three aglycones (quercetin, kaempferol and isorhamnetin), and two isoflavones (genestin and genistein). The quantitative chromatographic data was interpreted by principal component analysis (PCA), which assisted in the detection of unexpected chromatographic features in various adulterated botanical products. Only three of the commercially available ginkgo finished products tested in this study were determined to be authentic, with flavonol glycoside rutin, and aglycones quercetin, kaempferol, and isorhamnetin found to be common adulterants in the ginkgo powdered extract and finished product samples. Despite evidence of adulteration in most of the samples, each of the samples discussed herein met most of the current pharmacopeial standards. It is therefore critical that a preliminary evaluation be utilized to detect adulteration in commercial ginkgo products, prior to the acid hydrolysis procedure utilized in the current testing methods. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

PubMed

Llambias, E B; Luo, J

1996-01-01

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

Fingerprint of Hedyotis diffusa Willd. by HPLC-MS.

PubMed

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

2008-01-01

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

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

PubMed

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

2017-04-15

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

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

PubMed

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

2016-10-15

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

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

PubMed

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

1995-10-01

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

Analysis of Ethanolamines: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS888

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

Owens, J; Vu, A; Koester, C

The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method titled 'Analysis of Diethanolamine, Triethanolamine, n-Methyldiethanolamine, and n-Ethyldiethanolamine in Water by Single Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS): EPA Method MS888'. This draft standard operating procedure (SOP) was distributed to multiple EPA laboratories and to Lawrence Livermore National Laboratory, which was tasked to serve as a reference laboratory for EPA's Environmental Reference Laboratory Network (ERLN) and to develop and validate analytical procedures. The primary objective of this study was to validate and verify the analytical procedures described in 'EPA Method MS888' for analysis of themore » listed ethanolamines in aqueous samples. The gathered data from this validation study will be used to: (1) demonstrate analytical method performance; (2) generate quality control acceptance criteria; and (3) revise the SOP to provide a validated method that would be available for use during a homeland security event. The data contained in this report will be compiled, by EPA CRL, with data generated by other EPA Regional laboratories so that performance metrics of 'EPA Method MS888' can be determined.« less

Highlight on Bottlenecks in Food Allergen Analysis: Detection and Quantification by Mass Spectrometry.

PubMed

Planque, Mélanie; Arnould, Thierry; Renard, Patricia; Delahaut, Philippe; Dieu, Marc; Gillard, Nathlie

2017-07-01

Food laboratories have developed methods for testing allergens in foods. The efficiency of qualitative and quantitative methods is of prime importance in protecting allergic populations. Unfortunately, food laboratories encounter barriers to developing efficient methods. Bottlenecks include the lack of regulatory thresholds, delays in the emergence of reference materials and guidelines, and the need to detect processed allergens. In this study, ultra-HPLC coupled to tandem MS was used to illustrate difficulties encountered in determining method performances. We measured the major influences of both processing and matrix effects on the detection of egg, milk, soy, and peanut allergens in foodstuffs. The main goals of this work were to identify difficulties that food laboratories still encounter in detecting and quantifying allergens and to sensitize researchers to them.

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

PubMed

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

2015-06-01

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

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

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

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

PubMed

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

2009-10-01

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

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

PubMed

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

2008-12-01

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

Comparison of spectrofluorometric and HPLC methods for the characterization of fecal porphyrins in river otters.

PubMed

Taylor, C; Duffy, L K; Plumley, F G; Bowyer, R T

2000-09-01

A spectrofluorometric method (B. Grandchamp et al., 1980, Biochem. Biophys. Acta 629, 577-586) developed for the determination of amounts of uroporphyrin I (Uro I), coproporphyrin III (Copro III), and protoporphyrin IX (Proto IX) in skin fibroblasts was compared with a high-performance liquid chromatography (HPLC) method for the analysis of porphyrins in fecal samples of river otters (Lutra canadensis). Heptacarboxylate porphyrin I and coproporphyrin I, two porphyrins determined to be critical in defining the porphyrin profile in fecal samples of river otters with the HPLC method, contributed substantially to the calculation of the concentrations of Uro I and Copro III, respectively, in standard solutions of porphyrins with the spectrofluorometric method. Fluorescent components of the fecal matrix complicated the determination of the concentrations of Uro I, Copro III, and Proto IX with the spectrofluorometric method and resulted in erroneous values for the concentrations of these porphyrins compared with values determined with the HPLC method. These results indicate that the complexity of the sample, particularly with regard to the potential presence of interfering fluorescent compounds, as well as porphyrins additional to Uro I, Copro III, and Proto IX, should be considered prior to the application of the spectrofluorometric method. An alternative HPLC method developed for the rapid characterization of porphyrin profiles in fecal samples of river otters is described. Copyright 2000 Academic Press.

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

PubMed

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

2012-12-15

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

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

PubMed

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

2015-01-01

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

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

USGS Publications Warehouse

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

2003-01-01

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

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

PubMed

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

2011-10-01

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

Electrochemical Determination of the Antioxidant Potential of Some Less Common Fruit Species

PubMed Central

Gazdik, Zbynek; Krska, Boris; Adam, Vojtech; Saloun, Jan; Pokorna, Tunde; Reznicek, Vojtech; Horna, Ales; Kizek, Rene

2008-01-01

Various berries and fruit types of less common fruit species are known to contain antioxidants. Consumption of high amounts of antioxidant flavonoids, which display a variety of biological properties, including antiproliferative and anti-inflammatory activity, may have a positive impact on human health, particularly for the prevention of cancer and other inflammatory diseases. In these studies, based on the hypothesis that the fruit extract with the highest content would possess significantly higher health benefits, flavonoid-rich extracts were obtained from some less common fruit species – Blue Honeysuckles (Lonicera Kamtschatica and Lonicera edulis, Turcz. ex. Freyn), Saskatoon berry (Amelanchier alnifolia Nutt.) and Chinese Hawthorn (Crataegus pinnatifida BUNGE) – grown from germplasm held at the Mendel University of Agriculture and Forestry in Brno, Czech Republic and then characterized in terms of biological value based on the results from a relative antioxidant capacity assessment. The antioxidant content evaluation was based on the total flavonoid amount, determined by liquid chromatography with electrochemical detection (HPLC-ED). A DPPH• test was applied as a reference. The antioxidant content measured in Chinese Hawthorn fruit extract identified it as a potent source of flavonoid antioxidants, with a content 9-fold higher than that seen in Amelanchier fruit. The multifunctional HPLC-ED array method coupled with a DPPH• reference appears to be the optimal analytical progress, accurately reflecting the nutritive-therapeutic properties of a fruit. PMID:27873945

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

[The Raman Spectroscopy (RS): A new tool for the analytical quality control of injectable in health settings. Comparison of RS technique versus HPLC and UV/Vis-FTIR, applied to anthracyclines as anticancer drugs].

PubMed

Bourget, P; Amin, A; Moriceau, A; Cassard, B; Vidal, F; Clement, R

2012-12-01

The study compares the performances of three analytical methods devoted to Analytical Quality Control (AQC) of therapeutic solutions formed into care environment, we are talking about Therapeutics Objects(TN) (TOs(TN)). We explored the pharmacological model of two widely used anthracyclines i.e. adriamycin and epirubicin. We compared the performance of the HPLC versus two vibrational spectroscopic techniques: a tandem UV/Vis-FTIR on one hand and Raman Spectroscopy (RS) on the other. The three methods give good results for the key criteria of repeatability, of reproducibility and, of accuracy. A Spearman and a Kendall correlation test confirms the noninferiority of the vibrational techniques as an alternative to the reference method (HPLC). The selection of bands for characterization and quantification by RS is the results of a gradual process adjustment, at the intercept of matrix effects. From the perspective of a AQC associated to release of TOs, RS displays various advantages: (a) to decide quickly (~2min), simultaneously and without intrusion or withdrawal on both the nature of a packaging than on a solvant and this, regardless of the compound of interest; it is the founder asset of the method, (b) to explore qualitatively and quantitatively any kinds of TOs, (c) operator safety is guaranteed during production and in the laboratory, (d) the suppression of analytical releases or waste contribute to protects the environment, (e) the suppression.of consumables, (f) a negligible costs of maintenance, (g) a small budget of technicians training. These results already show that the SR technology is potentially a strong contributor to the safety of the medication cycle and fight against the iatrogenic effects of drugs. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Screening and confirmation of steroids and nitroimidazoles in urine, blood, and food matrices: Sample preparation methods and liquid chromatography tandem mass spectrometric separations.

PubMed

Tölgyesi, Ádám; Barta, Enikő; Simon, Andrea; McDonald, Thomas J; Sharma, Virender K

2017-10-25

Veterinary drugs containing synthetic anabolic steroid and nitroimidazole active agents are not allowed for their applications in livestock of the European Union (EU). This paper presents analyses of twelve selected steroids and six nitroimidazole antibiotics at low levels (1.56μg/L-4.95μg/L and 0.17μg/kg-2.14μg/kg, respectively) in body fluids and egg incurred samples. Analyses involved clean-up procedures, high performance liquid chromatography (HPLC) separation, and tandem mass spectrometric screening and confirmatory methods. Target steroids and nitroimidazoles in samples were cleaned by two independent supported liquid extraction and solid phase extraction procedures. Separation of the selected compounds was conducted on Kinetex XB C-18 HPLC column using gradient elution. The screening methods utilised supported liquid extraction that enabled fast and cost effective clean-up. The confirmatory methods were improved by extending the number of matrices and compounds, and by introducing an isotope dilution mass spectrometry for nitroimidazoles. The new methods were validated according to the recommendation of the European Union Reference Laboratories and the performance characteristics evaluated met fully the criteria. The methods were applied to incurred samples in the proficiency tests. The obtained results of Z-scores demonstrated the applicability of developed protocols of the methods to real samples. The confirmatory methods were applied to the national monitoring program and natural contamination of prednisolone could be detected in urine at low concentration in few samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Determination of arsenic species in marine samples by HPLC-ICP-MS.

PubMed

Hirata, Shizuko; Toshimitsu, Hideki; Aihara, Masato

2006-01-01

Arsenic speciation analysis in marine samples was performed using high performance liquid chromatography (HPLC) with ICP-MS detection. The separation of eight arsenic species viz. arsenite (As(III)), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenate (As(V)), arsenobetaine, trimethylarsine oxide (TMAO), arsenocholine and tetramethylarsonium ion (TeMAs) was achieved on a Shiseido Capcell Pak C18 column by using an isocratic eluent (pH 3.0), in which condition As(III) and MMA were co-eluted. The entire separation was accomplished in 15 min. The detection limits for 8 arsenic species by HPLC/ICP-MS were in the range of 0.02 - 0.10 microg L(-1) based on 3sigma of blank response (n=9). The precision was calculated to be 3.1-7.3% (RSD) for all eight species. The method then successfully applied to several marine samples e.g., oyster, scallop, fish, and shrimps. For the extraction of arsenic species from seafood products, the low power microwave digestion was employed. The extraction efficiency was in the range of 52.9 - 112.3%. Total arsenic concentrations were analyzed by using the microwave acid digestion. The total arsenics in the certified reference materials (DORM-2 and TORT-2) were analyzed and agreed with the certified values. The concentrations of arsenics in marine samples were in the range 6.6 - 35.1 microg g(-1).

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.

Determination of CMPO using HPLC -UV

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

Gracy Elias; Gary S. Groenewold; Bruce J. Mincher

Octyl(phenyl)-N,N-diisobutylcarbamoylmethylphosphine oxide (CMPO) is an extractant proposed for selective separation of radionuclide metals from used nuclear fuel solutions using solvent extraction. Radiolysis reactions can degrade CMPO and reduce separation performance and hence methods for measuring concentration of CMPO and identifying degradation products are needed. A novel high performance liquid chromatography (HPLC) method employing ultraviolet detection (UV) was developed to detect and quantitate CMPO in dodecane. Some radiolysis products in gamma and alpha irradiated CMPO solutions were identified using HPLC/electrospray ionization-mass spectrometry (ESI-MS). Validation data indicated that the HPLC-UV method for CMPO determination provided good linearity, sensitivity, procedure accuracy and systemmore » precision. CMPO-nitric acid complexes were also identified, that account for the apparent loss of CMPO in acidic environment, independent of irradiation.« less

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

NASA Astrophysics Data System (ADS)

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

2017-02-01

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

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

PubMed

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

2008-09-10

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

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC).

PubMed

Grosser, Katharina; van Dam, Nicole M

2017-03-15

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

PubMed Central

Grosser, Katharina; van Dam, Nicole M.

2017-01-01

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here. PMID:28362416

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

PubMed

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

2014-11-01

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

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

Analyses of procyanidins in foods using Diol phase HPLC

USDA-ARS?s Scientific Manuscript database

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

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.

Development of NIRS method for quality control of drug combination artesunate–azithromycin for the treatment of severe malaria

PubMed Central

Boyer, Chantal; Gaudin, Karen; Kauss, Tina; Gaubert, Alexandra; Boudis, Abdelhakim; Verschelden, Justine; Franc, Mickaël; Roussille, Julie; Boucher, Jacques; Olliaro, Piero; White, Nicholas J.; Millet, Pascal; Dubost, Jean-Pierre

2012-01-01

Near infrared spectroscopy (NIRS) methods were developed for the determination of analytical content of an antimalarial-antibiotic (artesunate and azithromycin) co-formulation in hard gelatin capsule (HGC). The NIRS consists of pre-processing treatment of spectra (raw spectra and first-derivation of two spectral zones), a unique principal component analysis model to ensure the specificity and then two partial least-squares regression models for the determination content of each active pharmaceutical ingredient. The NIRS methods were developed and validated with no reference method, since the manufacturing process of HGC is basically mixed excipients with active pharmaceutical ingredients. The accuracy profiles showed β-expectation tolerance limits within the acceptance limits (±5%). The analytical control approach performed by reversed phase (HPLC) required two different methods involving two different preparation and chromatographic methods. NIRS offers advantages in terms of lower costs of equipment and procedures, time saving, environmentally friendly. PMID:22579599

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

PubMed

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

2017-07-01

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

Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry

PubMed Central

Harju, Kirsi; Rapinoja, Marja-Leena; Avondet, Marc-André; Arnold, Werner; Schär, Martin; Burrell, Stephen; Luginbühl, Werner; Vanninen, Paula

2015-01-01

Saxitoxin (STX) and some selected paralytic shellfish poisoning (PSP) analogues in mussel samples were identified and quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sample extraction and purification methods of mussel sample were optimized for LC-MS/MS analysis. The developed method was applied to the analysis of the homogenized mussel samples in the proficiency test (PT) within the EQuATox project (Establishment of Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk). Ten laboratories from eight countries participated in the STX PT. Identification of PSP toxins in naturally contaminated mussel samples was performed by comparison of product ion spectra and retention times with those of reference standards. The quantitative results were obtained with LC-MS/MS by spiking reference standards in toxic mussel extracts. The results were within the z-score of ±1 when compared to the results measured with the official AOAC (Association of Official Analytical Chemists) method 2005.06, pre-column oxidation high-performance liquid chromatography with fluorescence detection (HPLC-FLD). PMID:26610567

Development and validation of polar RP-HPLC method for screening for ectoine high-yield strains in marine bacteria with green chemistry.

PubMed

Chen, Jun; Chen, Jianwei; Wang, Sijia; Zhou, Guangmin; Chen, Danqing; Zhang, Huawei; Wang, Hong

2018-04-02

A novel, green, rapid, and precise polar RP-HPLC method has been successfully developed and screened for ectoine high-yield strain in marine bacteria. Ectoine is a polar and extremely useful solute which allows microorganisms to survive in extreme environmental salinity. This paper describes a polar-HPLC method employed polar RP-C18 (5 μm, 250 × 4.6 mm) using pure water as the mobile phase and a column temperature of 30 °C, coupled with a flow rate at 1.0 mL/min and detected under a UV detector at wavelength of 210 nm. Our method validation demonstrates excellent linearity (R 2  = 0.9993), accuracy (100.55%), and a limit of detection LOQ and LOD of 0.372 and 0.123 μgmL -1 , respectively. These results clearly indicate that the developed polar RP-HPLC method for the separation and determination of ectoine is superior to earlier protocols.

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

PubMed

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

2012-01-01

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

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

PubMed Central

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

2012-01-01

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

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

PubMed

Nestola, Marco; Thellmann, Andrea

2015-01-01

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

Method performance and multi-laboratory assessment of a normal phase high pressure liquid chromatography-fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa and chocolate containing samples.

PubMed

Robbins, Rebecca J; Leonczak, Jadwiga; Johnson, J Christopher; Li, Julia; Kwik-Uribe, Catherine; Prior, Ronald L; Gu, Liwei

2009-06-12

The quantitative parameters and method performance for a normal-phase HPLC separation of flavanols and procyanidins in chocolate and cocoa-containing food products were optimized and assessed. Single laboratory method performance was examined over three months using three separate secondary standards. RSD(r) ranged from 1.9%, 4.5% to 9.0% for cocoa powder, liquor and chocolate samples containing 74.39, 15.47 and 1.87 mg/g flavanols and procyanidins, respectively. Accuracy was determined by comparison to the NIST Standard Reference Material 2384. Inter-lab assessment indicated that variability was quite low for seven different cocoa-containing samples, with a RSD(R) of less than 10% for the range of samples analyzed.

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.

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

USDA-ARS?s Scientific Manuscript database

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

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

ERIC Educational Resources Information Center

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

2007-01-01

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

Development of a low-cost method of analysis for the qualitative and quantitative analysis of butyltins in environmental samples.

PubMed

Bangkedphol, Sornnarin; Keenan, Helen E; Davidson, Christine; Sakultantimetha, Arthit; Songsasen, Apisit

2008-12-01

Most analytical methods for butyltins are based on high resolution techniques with complicated sample preparation. For this study, a simple application of an analytical method was developed using High Performance Liquid Chromatography (HPLC) with UV detection. The developed method was studied to determine tributyltin (TBT), dibutyltin (DBT) and monobutyltin (MBT) in sediment and water samples. The separation was performed in isocratic mode on an ultra cyanopropyl column with a mobile phase of hexane containing 5% THF and 0.03% acetic acid. This method was confirmed using standard GC/MS techniques and verified by statistical paired t-test method. Under the experimental conditions used, the limit of detection (LOD) of TBT and DBT were 0.70 and 0.50 microg/mL, respectively. The optimised extraction method for butyltins in water and sediment samples involved using hexane containing 0.05-0.5% tropolone and 0.2% sodium chloride in water at pH 1.7. The quantitative extraction of butyltin compounds in a certified reference material (BCR-646) and naturally contaminated samples was achieved with recoveries ranging from 95 to 108% and at %RSD 0.02-1.00%. This HPLC method and optimum extraction conditions were used to determine the contamination level of butyltins in environmental samples collected from the Forth and Clyde canal, Scotland, UK. The values obtained severely exceeded the Environmental Quality Standard (EQS) values. Although high resolution methods are utilised extensively for this type of research, the developed method is cheaper in both terms of equipment and running costs, faster in analysis time and has comparable detection limits to the alternative methods. This is advantageous not just as a confirmatory technique but also to enable further research in this field.

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

PubMed

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

2015-10-01

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

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

PubMed Central

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

2014-01-01

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

Current HPLC Methods for Assay of Nano Drug Delivery Systems.

PubMed

Tekkeli, Serife Evrim Kepekci; Kiziltas, Mustafa Volkan

2017-01-01

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

Characteristic component profiling and identification of different Uncaria species based on high-performance liquid chromatography-photodiode array detection tandem ion trap and time of flight mass spectrometry coupled with rDNA ITS sequence.

PubMed

Zhao, Bingqiang; Huang, Yanjun; Chen, Qiulan; Chen, Qizhao; Miao, Hui; Zhu, Shuang; Zeng, Changqing

2018-03-01

Uncaria is a multi-source herb and its species identification has become a bottleneck in quality control. To study the identification method of different Uncaria species herbs through HPLC-MS coupled with rDNA Internal Transcribed Spacer (rDNA ITS) sequence, both plant morphological traits and molecular identification were used to determine the species of every collected Uncaria herb. The genetic analysis of different Uncaria species was performed using their rDNA ITS sequence as a molecular marker. Meanwhile, the phylogenetic relationships of 22 samples from six Uncaria species were divided and classified clearly. By optimizing the chromatographic conditions, a practical HPLC method to differentiate various varieties of Uncaria herbs was set up based on a set of characteristic components across each species. A high-performance liquid chromatography-photodiode array detector tandem ion trap and time of flight mass spectrometry technique combined with reference substances was utilized to derive 21 characteristic compounds containing six groups of six Uncaria species in China. Thus, this study provides a feasible method to solve the current problem of confusion in Uncaria species, and makes a significant step forward in the appropriate clinical use, in-depth research and further utilization of different Uncaria species. Copyright © 2017 John Wiley & Sons, Ltd.

Qualitative and quantitative analyses of Epimedium wushanense by high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem mass spectrometry.

PubMed

Li, Hui-fang; Guan, Xiang-yu; Ye, Min; Xiang, Cheng; Lin, Chang-hu; Sun, Chao; Guo, De-an

2011-06-01

A new HPLC-DAD-ESI-MS(n) method was developed for rapid separation, characterization and quantitation of flavonoids in Epimedium wushanense, a popular Chinese herbal medicine. For qualitative identification, a total of 37 compounds were characterized from the underground and aerial parts of E. wushanense. Among them, 28 compounds were prenylated flavonoids, and 23 were confirmed by comparing with reference standards. For quantitative analysis, 12 major flavonoids including kaempferol glycosides, desmethylicaritin glycosides, and icaritin glycosides were simultaneously determined by HPLC/UV. Samples were separated on a Waters Symmetry C(18) column at 35 °C eluted with a gradient three-component mobile phase of acetonitrile, methanol, and water containing 0.03% v/v formic acid. All the flavonoids showed good linearity (r(2) ≥0.9997). The recoveries varied from 92.6 to 106.1% at three concentration levels. This method was applied to the determination of 20 samples of different geographical sources, harvesting time, and plant parts. Contents of the predominant flavonoid, epimedin C, ranged from 1.4 to 5.1% in aerial parts and 1.0 to 2.8% in underground parts. The methods established in this paper were simple and reliable and could be used for the quality control of E. wushanense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assay of fluoxetine hydrochloride by titrimetric and HPLC methods.

PubMed

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

2000-01-01

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

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

PubMed

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

2017-08-30

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

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

PubMed

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

2005-01-01

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

Stability-Indicating TLC-Densitometric and HPLC Methods for the Simultaneous Determination of Piracetam and Vincamine in the Presence of Their Degradation Products.

PubMed

Ahmed, Amal B; Abdelrahman, Maha M; Abdelwahab, Nada S; Salama, Fathy M

2016-11-01

Newly established TLC-densitometric and RP-HPLC methods were developed and validated for the simultaneous determination of Piracetam (PIR) and Vincamine (VINC) in their pharmaceutical formulation and in the presence of PIR and VINC degradation products, PD and VD, respectively. The proposed TLC-densitometric method is based on the separation and quantitation of the studied components using a developing system that consists of chloroform-methanol-glacial acetic acid-triethylamine (8 + 2 + 0.1 + 0.1, v/v/v/v) on TLC silica gel 60 F254 plates, followed by densitometric scanning at 230 nm. On the other hand, the developed RP-HPLC method is based on the separation of the studied components using an isocratic elution of 0.05 M KH2PO4 (containing 0.1% triethylamine adjusted to pH 3 with orthophosphoric acid)-methanol (95 + 5, v/v) on a C8 column at a flow rate of 1 mL/min with diode-array detection at 230 nm. The developed methods were validated according to International Conference on Harmonization guidelines and demonstrated good accuracy and precision. Moreover, the developed TLC-densitometric and RP-HPLC methods are suitable as stability-indicating assay methods for the simultaneous determination of PD and VD either in bulk powder or pharmaceutical formulation. The results were statistically compared with those obtained by the reported RP-HPLC method using t- and F-tests.

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

PubMed Central

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

2007-01-01

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

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

PubMed

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

2013-10-01

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

Determination of synthetic by-products and an intermediate in the colour additives D&C Red Nos 27 and 28 (phloxine B) and their lakes using conventional HPLC.

PubMed

Weisz, Adrian; Krantz, Zoe B

2014-01-01

Specifications in the US Code of Federal Regulations (CFR) for the colour additives D&C Red No. 27 (R27) and D&C Red No. 28 (R28) limit the levels of the synthetic by-products 2,3,4,5-tetrachloro-6-(3´,5´-dibromo-2´,4´-dihydroxybenzoyl)benzoic acid (SBBA) and "brominated resorcinol" and of the intermediate 3,4,5,6-tetrachlorophthalic acid (TCPA). The present study reports the development and application of a conventional HPLC method for the quantitative determination of these impurities in R27, R28 and their lakes. Because the CFR-listed "brominated resorcinol" refers to a group of synthetic by-products, six variously brominated resorcinols were examined as possible impurities. Due to their rapid decomposition in the presence of the dye, the existence of any brominated resorcinols in R27 and R28 is highly unlikely, and this supposition is supported by the results obtained in this study. SBBA, 2,4,6-tribromoresorcinol (Br3R, the brominated resorcinol most likely to be found as an impurity) and TCPA were quantified by using five-point calibration curves with data points (w/w) that ranged from 0.066% to 0.820% for SBBA, from 0.050% to 0.499% for Br3R, and from 0.061% to 1.410% for TCPA. The HPLC method was applied to the analysis of test portions from batches of R27, R28 and their lakes submitted to the USFDA for certification by domestic and foreign manufacturers.

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

PubMed

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

2018-01-01

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

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

PubMed

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

2016-03-01

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

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

EPA Science Inventory

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

Determination of Urine Albumin by New Simple High-Performance Liquid Chromatography Method.

PubMed

Klapkova, Eva; Fortova, Magdalena; Prusa, Richard; Moravcova, Libuse; Kotaska, Karel

2016-11-01

A simple high-performance liquid chromatography (HPLC) method was developed for the determination of albumin in patients' urine samples without coeluting proteins and was compared with the immunoturbidimetric determination of albumin. Urine albumin is important biomarker in diabetic patients, but part of it is immuno-nonreactive. Albumin was determined by high-performance liquid chromatography (HPLC), UV detection at 280 nm, Zorbax 300SB-C3 column. Immunoturbidimetric analysis was performed using commercial kit on automatic biochemistry analyzer COBAS INTEGRA ® 400, Roche Diagnostics GmbH, Manheim, Germany. The HLPC method was fully validated. No significant interference with other proteins (transferrin, α-1-acid glycoprotein, α-1-antichymotrypsin, antitrypsin, hemopexin) was found. The results from 301 urine samples were compared with immunochemical determination. We found a statistically significant difference between these methods (P = 0.0001, Mann-Whitney test). New simple HPLC method was developed for the determination of urine albumin without coeluting proteins. Our data indicate that the HPLC method is highly specific and more sensitive than immunoturbidimetry. © 2016 Wiley Periodicals, Inc.

Development and Optimisation of an HPLC-DAD-ESI-Q-ToF Method for the Determination of Phenolic Acids and Derivatives

PubMed Central

Restivo, Annalaura; Degano, Ilaria; Ribechini, Erika; Colombini, Maria Perla

2014-01-01

A method for the HPLC-MS/MS analysis of phenols, including phenolic acids and naphtoquinones, using an amide-embedded phase column was developed and compared to the literature methods based on classical C18 stationary phase columns. RP-Amide is a recently developed polar embedded stationary phase, whose wetting properties mean that up to 100% water can be used as an eluent. The increased retention and selectivity for polar compounds and the possibility of working in 100% water conditions make this column particularly interesting for the HPLC analysis of phenolic acids and derivatives. In this study, the chromatographic separation was optimised on an HPLC-DAD, and was used to separate 13 standard phenolic acids and derivatives. The method was validated on an HPLC-ESI-Q-ToF. The acquisition was performed in negative polarity and MS/MS target mode. Ionisation conditions and acquisition parameters for the Q-ToF detector were investigated by working on collision energies and fragmentor potentials. The performance of the method was fully evaluated on standards. Moreover, several raw materials containing phenols were analysed: walnut, gall, wine, malbec grape, French oak, red henna and propolis. Our method allowed us to characterize the phenolic composition in a wide range of matrices and to highlight possible matrix effects. PMID:24551158

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2016-10-14

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

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

PubMed

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

2008-01-01

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

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

PubMed

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

2018-03-15

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

HPLC-MS/MS investigation of biochemical markers for the disclosure of erythropoietin abuse in sports

NASA Astrophysics Data System (ADS)

Appolonova, S. A.; Dikunets, M. A.; Rodchenkov, G. M.

2009-04-01

The polypeptide hormone erythropoietin (EPO), which is a forbidden doping drug, was determined by high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The hypothesis about the influence of EPO on the asymmetric dimethylarginine (ADMA)-dimethylargininedime-thylaminohydrolase (DDAH)-NO-synthase system was verified. Changes in this system can serve as indirect biochemical markers of the presence of the forbidden EPO drug in the organism. In the test group, the concentrations of biochemical markers varied from 10 to 40 μg/ml for ADMA and symmetrical DMA (SDMA) and from 0.5 to 10 μg/ml for arginine and citrulline. A single intravenous administration of r-HuEPO (Epocrin, 2000 ME/day) for two volunteers reliably increased ADMA, SDMA, arginine, and citrulline concentrations to 40-270 μg/ml, 40-240μg/ml, 10-60 μg/ml, and 12-140 μg/ml, respectively, with respect to the reference values. The simultaneous increase in arginine, methylarginines, and citrulline contents could be an indirect marker of EPO abuse. The method is recommended for fast screening analysis.

Measurement of menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

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

HPLC method for urinary theobromine determination: Effect of consumption of cocoa products on theobromine urinary excretion in children.

PubMed

Rodriguez, Adrian; Costa-Bauza, Antonia; Saez-Torres, Concepcion; Rodrigo, Dolores; Grases, Felix

2015-11-01

To validate a simple method of urinary theobromine determination, to assess urinary theobromine levels in 80 healthy children and to relate these levels to consumption of cocoa products. Urine samples were diluted, directly injected into an HPLC system, separated by gradient elution on a C18 column, and detected by UV spectrometry. The method was validated for linearity, limits of detection and quantification, imprecision, accuracy, recovery and interferences. The proposed method was used to assess 12-h day and 12-h night urinary theobromine excretion by 80 healthy children, divided into four groups based on consumption of cocoa products. In addition, urinary excretion of magnesium and oxalate, also present in cocoa, was measured in these four groups. The method was linear to a theobromine concentration of 278μmol/L (50mg/L). LOD and LOQ for urine samples, diluted 1:5 (vol/vol) with water, were 1.1 and 3.6μmol/L respectively. Within-run and between-run imprecisions (CV) were each <2%. Average recovery was 99%, and analysis of a certified reference sample showed an error <2.5%. Theobromine excretion levels were significantly higher in healthy children with higher consumption of cocoa products (p<0.001), but oxalate (p=0.098) and magnesium (p=0.068) excretion levels did not differ significantly. This validated method resulted in urinary theobromine determination with 100% recovery, without sample pretreatment. Urinary theobromine levels in healthy children were directly related to their consumption of cocoa products. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Estimation of the quantification uncertainty from flow injection and liquid chromatography transient signals in inductively coupled plasma mass spectrometry

NASA Astrophysics Data System (ADS)

Laborda, Francisco; Medrano, Jesús; Castillo, Juan R.

2004-06-01

The quality of the quantitative results obtained from transient signals in high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) and flow injection-inductively coupled plasma mass spectrometry (FI-ICPMS) was investigated under multielement conditions. Quantification methods were based on multiple-point calibration by simple and weighted linear regression, and double-point calibration (measurement of the baseline and one standard). An uncertainty model, which includes the main sources of uncertainty from FI-ICPMS and HPLC-ICPMS (signal measurement, sample flow rate and injection volume), was developed to estimate peak area uncertainties and statistical weights used in weighted linear regression. The behaviour of the ICPMS instrument was characterized in order to be considered in the model, concluding that the instrument works as a concentration detector when it is used to monitorize transient signals from flow injection or chromatographic separations. Proper quantification by the three calibration methods was achieved when compared to reference materials, although the double-point calibration allowed to obtain results of the same quality as the multiple-point calibration, shortening the calibration time. Relative expanded uncertainties ranged from 10-20% for concentrations around the LOQ to 5% for concentrations higher than 100 times the LOQ.

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.

Levothyroxine sodium revisited: A wholistic structural elucidation approach of new impurities via HPLC-HRMS/MS, on-line H/D exchange, NMR spectroscopy and chemical synthesis.

PubMed

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

2017-02-20

The structural elucidation of unknown pharmaceutical impurities plays an important role in the quality control of newly developed and well-established active pharmaceutical ingredients (APIs). The United States Pharmacopeia (USP) monograph for the API Levothyroxine Sodium, a synthetic thyroid hormone, features two high pressure liquid chromatography (HPLC) methods using UV-VIS absorption detection to determine organic impurities in the drug substance. The impurity profile of the first USP method ("Procedure 1") has already been extensively studied, however for the second method ("Procedure 2"), which exhibits a significantly different impurity profile, no wholistic structural elucidation of impurities has been performed yet. Applying minor modifications to the chromatographic parameters of USP "Procedure 2" and using various comprehensive structural elucidation methods such as high resolution tandem mass spectrometry with on-line hydrogen-deuterium (H/D) exchange or two-dimensional nuclear magnetic resonance spectroscopy (NMR) we gained new insights about the complex impurity profile of the synthetic thyroid hormone. This resulted in the characterization of 24 compounds previously unknown to literature and the introduction of two new classes of Levothyroxine Sodium impurities. Five novel compounds were unambiguously identified via isolation or synthesis of reference substances and subsequent NMR spectroscopic investigation. Additionally, Collision-Induced Dissociation (CID)-type fragmentation of identified major impurities as well as neutral loss fragmentation patterns of many characterized impurities were discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of newfound natural luminescent properties of melamine, and development and validation of a method of high performance liquid chromatography with fluorescence detection for its determination in kitchen plastic ware.

PubMed

Finete, Virginia de Lourdes Mendes; Gouvêa, Marcos Martins; Marques, Flávia Ferreira de Carvalho; Netto, Annibal Duarte Pereira

2014-06-01

Experimental studies of the natural photoluminescence of melamine in aqueous solutions showed that its fluorescence intensity (at 250/365 nm) was appropriated for analytical purposes. The exploitation of such melamine property provided the basis of development of a new, simple, precise and accurate method based on high performance liquid chromatography with fluorescence detection (HPLC-Fluo) to determine melamine in kitchen plastic ware following aqueous extraction using a microwave oven. Optimization of analytical parameters such as solvent composition, pH and extraction conditions led to limits of detection and quantification of melamine of 0.0081 and 0.027 μg mL(-1), respectively, with a linear range up to 10 μg mL(-1). Sample extracts fortified with melamine at three concentration levels produced an average recovery of 98±6%, which was in agreement with the results achieved with a reference HPLC-UV method. Different samples of kitchen plastic ware analyzed by the developed and optimized method showed melamine concentrations in the aqueous extract up to 17 µg mL(-1), which corresponded to 86.0 mg kg(-1) in these utensils. The results obtained indicate that the use of kitchen plastic ware made of melamine can contaminate food with this compound after heating in a microwave oven. Copyright © 2014. Published by Elsevier B.V.

Parabens determination in cosmetic and personal care products exploiting a multi-syringe chromatographic (MSC) system and chemiluminescent detection.

PubMed

Rodas, Melisa; Portugal, Lindomar A; Avivar, Jessica; Estela, José Manuel; Cerdà, Víctor

2015-10-01

Parabens are widely used in dairy products, such as in cosmetics and personal care products. Thus, in this work a multi-syringe chromatographic (MSC) system is proposed for the first time for the determination of four parabens: methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) in cosmetics and personal care products, as a simpler, practical, and low cost alternative to HPLC methods. Separation was achieved using a 5mm-long precolumn of reversed phase C18 and multi-isocratic separation, i.e. using two consecutive mobile phases, 12:88 acetonitrile:water and 28:72 acetonitrile:water. The use of a multi-syringe buret allowed the easy implementation of chemiluminescent (CL) detection after separation. The chemiluminescent detection is based on the reduction of Ce(IV) by p-hydroxybenzoic acid, product of the acid hydrolysis of parabens, to excite rhodamine 6G (Rho 6G) and measure the resulting light emission. Multivariate designs combined with the concepts of multiple response treatments and desirability functions have been employed to simultaneously optimize and evaluate the responses. The optimized method has proved to be sensitive and precise, obtaining limits of detection between 20 and 40 µg L(-1) and RSD <4.9% in all cases. The method was satisfactorily applied to cosmetics and personal care products, obtaining no significant differences at a confidence level of 95% comparing with the HPLC reference method. Copyright © 2015 Elsevier B.V. All rights reserved.

[Data fusion and multi-components quantitative analysis for identification and quality evaluation of Gentiana rigescens from different geographical origins].

PubMed

Wang, Qin-Qin; Shen, Tao; Zuo, Zhi-Tian; Huang, Heng-Yu; Wang, Yuan-Zhong

2018-03-01

The accumulation of secondary metabolites of traditional Chinese medicine (TCM) is closely related to its origins. The identification of origins and multi-components quantitative evaluation are of great significance to ensure the quality of medicinal materials. In this study, the identification of Gentiana rigescens from different geographical origins was conducted by data fusion of Fourier transform infrared (FTIR) spectroscopy and high performance liquid chromatography (HPLC) in combination of partial least squares discriminant analysis; meanwhile quantitative analysis of index components was conducted to provide an accurate and comprehensive identification and quality evaluation strategy for selecting the best production areas of G. rigescens. In this study, the FTIR and HPLC information of 169 G. rigescens samples from Yunnan, Sichuan, Guangxi and Guizhou Provinces were collected. The raw infrared spectra were pre-treated by multiplicative scatter correction, standard normal variate (SNV) and Savitzky-Golay (SG) derivative. Then the performances of FTIR, HPLC, and low-level data fusion and mid-level data fusion for identification were compared, and the contents of gentiopicroside, swertiamarin, loganic acid and sweroside were determined by HPLC. The results showed that the FTIR spectra of G. rigescens from different geographical origins were different, and the best pre-treatment method was SNV+SG-derivative (second derivative, 15 as the window parameter, and 2 as the polynomial order). The results showed that the accuracy rate of low- and mid-level data fusion (96.43%) in prediction set was higher than that of FTIR and HPLC (94.64%) in prediction set. In addition, the accuracy of low-level data fusion (100%) in the training set was higher than that of mid-level data fusion (99.12%) in training set. The contents of the iridoid glycosides in Yunnan were the highest among different provinces. The average content of gentiopicroside, as a bioactive marker in Chinese pharmacopoeia, was 47.40 mg·g⁻¹, and the maximum was 79.83 mg·g⁻¹. The contents of loganic acid, sweroside and gentiopicroside in Yunnan were significantly different from other provinces ( P <0.05). In comparison of total content of iridoid glycosides in G. rigescens with different geographical origins in Yunnan, it was found that the amount of iridoid glycosides was higher in Eryuan Dali (68.59 mg·g⁻¹) and Yulong Lijiang (66.68 mg·g⁻¹), significantly higher than that in Wuding Chuxiong (52.99 mg·g⁻¹), Chengjiang Yuxi (52.29 mg·g⁻¹) and Xundian Kunming (46.71 mg·g⁻¹) ( P <0.05), so these two places can be used as a reference region for screening cultivation and excellent germplasm resources of G. rigescens. A comprehensive and accurate method was established by data fusion of HPLC-FTIR and quantitative analysis of HPLC for identification and quality evaluation of G. rigescens, which could provide a support for the development and utilization of G. rigescens. Copyright© by the Chinese Pharmaceutical Association.

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

PubMed

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

2013-05-01

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

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

PubMed

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

2011-10-12

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

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

PubMed

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

2015-04-01

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

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

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

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

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

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

PubMed

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

2014-01-01

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

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

PubMed

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

2018-01-01

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

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

PubMed

Leung, Elvis M K; Chan, Wan

2014-02-01

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

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.

Screening and determination of polycyclic aromatic hydrocarbons in seafoods using QuEChERS-based extraction and high-performance liquid chromatography with fluorescence detection.

PubMed

Gratz, Samuel R; Ciolino, Laura A; Mohrhaus, Angela S; Gamble, Bryan M; Gracie, Jill M; Jackson, David S; Roetting, John P; McCauley, Heather A; Heitkemper, Douglas T; Fricke, Fred L; Krol, Walter J; Arsenault, Terri L; White, Jason C; Flottmeyer, Michele M; Johnson, Yoko S

2011-01-01

A rapid, sensitive, and accurate method for the screening and determination of polycyclic aromatic hydrocarbons (PAHs) in edible seafood is described. The method uses quick, easy, cheap, effective, rugged, and safe (QuEChERS)-based extraction and HPLC with fluorescence detection (FLD). The method was developed and validated in response to the massive Deepwater Horizon oil spill in the Gulf of Mexico. Rapid and highly sensitive PAH screening methods are critical tools needed for oil spill response; they help to assess when seafood is safe for harvesting and consumption. Sample preparation involves SPE of edible seafood portions with acetonitrile, followed by the addition of salts to induce water partitioning. After centrifugation, a portion of the acetonitrile layer is filtered prior to analysis via HPLC-FLD. The chromatographic method uses a polymeric C18 stationary phase designed for PAH analysis with gradient elution, and it resolves 15 U.S. Environmental Protection Agency priority parent PAHs in fewer than 20 min. The procedure was validated in three laboratories for the parent PAHs using spike recovery experiments at PAH fortification levels ranging from 25 to 10 000 microg/kg in oysters, shrimp, crab, and finfish, with recoveries ranging from 78 to 99%. Additional validation was conducted for a series of alkylated homologs of naphthalene, dibenzothiophene, and phenanthrene, with recoveries ranging from 87 to 128%. Method accuracy was further assessed based on analysis of National Institute of Standards and Technology Standard Reference Material 1974b. The method provides method detection limits in the sub to low ppb (microg/kg) range, and practical LOQs in the low ppb (microg/kg) range for most of the PAH compounds studied.

Analysis of Phosphonic Acids: Validation of Semi-Volatile Analysis by HPLC-MS/MS by EPA Method MS999

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

Owens, J; Vu, A; Koester, C

The Environmental Protection Agency's (EPA) Region 5 Chicago Regional Laboratory (CRL) developed a method titled Analysis of Diisopropyl Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid, and Pinacolyl Methylphosphonic Acid in Water by Multiple Reaction Monitoring Liquid Chromatography/Tandem Mass Spectrometry: EPA Version MS999. This draft standard operating procedure (SOP) was distributed to multiple EPA laboratories and to Lawrence Livermore National Laboratory, which was tasked to serve as a reference laboratory for EPA's Environmental Reference Laboratory Network (ERLN) and to develop and validate analytical procedures. The primary objective of this study was to validate and verify the analytical procedures describedmore » in EPA Method MS999 for analysis of the listed phosphonic acids and surrogates in aqueous samples. The gathered data from this validation study will be used to: (1) demonstrate analytical method performance; (2) generate quality control acceptance criteria; and (3) revise the SOP to provide a validated method that would be available for use during a homeland security event. The data contained in this report will be compiled, by EPA CRL, with data generated by other EPA Regional laboratories so that performance metrics of EPA Method MS999 can be determined.« less

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

PubMed

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

2015-05-15

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

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

PubMed

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

2013-04-01

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

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

NASA Astrophysics Data System (ADS)

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

2007-11-01

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

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

EPA Science Inventory

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

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

Code of Federal Regulations, 2013 CFR

2013-07-01

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

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

PubMed

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

2017-02-01

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

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-09-08

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

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

DTIC Science & Technology

2008-03-06

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

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

PubMed Central

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

2015-01-01

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

Determination of vitamin B6 by means of differential spectrophotometry in pharmaceutical preparations in the presence of magnesium compounds.

PubMed

Muszalska, Izabela; Puchalska, Marta; Sobczak, Agnieszka

2011-01-01

The content of pyridoxine hydrochloride in two-component pharmaceutical preparations containing various magnesium compounds was examined. The UV differentiation spectrophotometry was devised and compared with the reference method of high performance liquid chromatography (HPLC). The analysis of the absorbance spectra (A) and its first (D1) and second (D2) derivatives made it possible to establish the appropriate analytical wavelengths (A: 290 nm; D1: 302 nm; D2: 308 nm). It was proved that spectrum differentiation significantly corrects errors resulting from overlapping background especially when the magnesium hydroaspartate, lactate or magnesium lactogluconate is present together with vitamin B6.

Bioavailability of ambroxol sustained release preparations. Part II: Single and multiple oral dose studies in man.

PubMed

Janssen, T J; Guelen, P J; Vree, T B; Botterblom, M H; Valducci, R

1988-01-01

The bioavailability of a new ambroxol sustained release preparation (75 mg) based on a dialyzing membrane for controlled release was studied in healthy volunteers after single and multiple oral dose in comparison with a standard sustained release formulation in a cross-over study under carefully controlled conditions. Plasma concentrations of ambroxol were measured by means of a HPLC method. Based on AUC data both preparations are found to be bioequivalent, but show different plasma concentration profiles. The test preparation showed a more pronounced sustained release profile than the reference preparation (single dose) resulting in significantly higher steady state plasma levels.

Simultaneous identification of natural dyes in the collection of drawings and maps from The Royal Chancellery Archives in Granada (Spain) by CE.

PubMed

López-Montes, Ana; Blanc García, Rosario; Espejo, Teresa; Huertas-Perez, José F; Navalón, Alberto; Vílchez, José Luis

2007-04-01

A simple and rapid capillary electrophoretic method with UV detection (CE-UV) has been developed for the identification of five natural dyes namely, carmine, indigo, saffron, gamboge and Rubia tinctoria root. The separation was performed in a fused-silica capillary of 64.5 cm length and 50 microm id. The running buffer was 40 mM sodium tetraborate buffer solution (pH 9.25). The applied potential was 30 kV, the temperature was 25 degrees C and detections were performed at 196, 232, 252, 300 and 356 nm. The injections were under pressure of 50 mbar during 13 s. The method was applied to the identification of carminic acid, gambogic acid, crocetin, indigotin, alizarin and purpurin in the collection of drawings and maps at the Royal Chancellery Archives in Granada (Spain). The method was validated by using HPLC as a reference method.

Near-infrared Spectroscopy as a Process Analytical Technology Tool for Monitoring the Parching Process of Traditional Chinese Medicine Based on Two Kinds of Chemical Indicators.

PubMed

Li, Kaiyue; Wang, Weiying; Liu, Yanping; Jiang, Su; Huang, Guo; Ye, Liming

2017-01-01

The active ingredients and thus pharmacological efficacy of traditional Chinese medicine (TCM) at different degrees of parching process vary greatly. Near-infrared spectroscopy (NIR) was used to develop a new method for rapid online analysis of TCM parching process, using two kinds of chemical indicators (5-(hydroxymethyl) furfural [5-HMF] content and 420 nm absorbance) as reference values which were obviously observed and changed in most TCM parching process. Three representative TCMs, Areca ( Areca catechu L.), Malt ( Hordeum Vulgare L.), and Hawthorn ( Crataegus pinnatifida Bge.), were used in this study. With partial least squares regression, calibration models of NIR were generated based on two kinds of reference values, i.e. 5-HMF contents measured by high-performance liquid chromatography (HPLC) and 420 nm absorbance measured by ultraviolet-visible spectroscopy (UV/Vis), respectively. In the optimized models for 5-HMF, the root mean square errors of prediction (RMSEP) for Areca, Malt, and Hawthorn was 0.0192, 0.0301, and 0.2600 and correlation coefficients ( R cal ) were 99.86%, 99.88%, and 99.88%, respectively. Moreover, in the optimized models using 420 nm absorbance as reference values, the RMSEP for Areca, Malt, and Hawthorn was 0.0229, 0.0096, and 0.0409 and R cal were 99.69%, 99.81%, and 99.62%, respectively. NIR models with 5-HMF content and 420 nm absorbance as reference values can rapidly and effectively identify three kinds of TCM in different parching processes. This method has great promise to replace current subjective color judgment and time-consuming HPLC or UV/Vis methods and is suitable for rapid online analysis and quality control in TCM industrial manufacturing process. Near-infrared spectroscopy.(NIR) was used to develop a new method for online analysis of traditional Chinese medicine.(TCM) parching processCalibration and validation models of Areca, Malt, and Hawthorn were generated by partial least squares regression using 5.(hydroxymethyl) furfural contents and 420.nm absorbance as reference values, respectively, which were main indicator components during parching process of most TCMThe established NIR models of three TCMs had low root mean square errors of prediction and high correlation coefficientsThe NIR method has great promise for use in TCM industrial manufacturing processes for rapid online analysis and quality control. Abbreviations used: NIR: Near-infrared Spectroscopy; TCM: Traditional Chinese medicine; Areca: Areca catechu L.; Hawthorn: Crataegus pinnatifida Bge.; Malt: Hordeum vulgare L.; 5-HMF: 5-(hydroxymethyl) furfural; PLS: Partial least squares; D: Dimension faction; SLS: Straight line subtraction, MSC: Multiplicative scatter correction; VN: Vector normalization; RMSECV: Root mean square errors of cross-validation; RMSEP: Root mean square errors of validation; R cal : Correlation coefficients; RPD: Residual predictive deviation; PAT: Process analytical technology; FDA: Food and Drug Administration; ICH: International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use.

75 FR 22240 - Cyprodinil; Pesticide Tolerances

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-28

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

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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

EPA Science Inventory

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

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

PubMed

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

2012-01-01

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

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

PubMed

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

2018-06-15

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

Analysis of Biomass Sugars Using a Novel HPLC Method

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

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

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

A quick inexpensive laboratory method in acute paracetamol poisoning could improve risk assessment, management and resource utilization

PubMed Central

Senarathna, S.M.D.K. Ganga; Ranganathan, Shalini S.; Buckley, Nick; Soysa, S.S.S.B.D. Preethi; Fernandopulle, B. M. Rohini

2012-01-01

Objectives: Acute paracetamol poisoning is an emerging problem in Sri Lanka. Management guidelines recommend ingested dose and serum paracetamol concentrations to assess the risk. Our aim was to determine the usefulness of the patient's history of an ingested dose of >150 mg/kg and paracetamol concentration obtained by a simple colorimetric method to assess risk in patients with acute paracetamol poisoning. Materials and Methods: Serum paracetamol concentrations were determined in 100 patients with a history of paracetamol overdose using High Performance Liquid Chromatography (HPLC); (reference method). The results were compared to those obtained with a colorimetric method. The utility of risk assessment by reported dose ingested and colorimetric analysis were compared. Results: The area under the receiver operating characteristic curve for the history of ingested dose was 0.578 and there was no dose cut-off providing useful risk categorization. Both analytical methods had less than 5% intra- and inter-batch variation and were accurate on spiked samples. The time from blood collection to result was six times faster and ten times cheaper for colorimetry (30 minutes, US$2) than for HPLC (180 minutes, US$20). The correlation coefficient between the paracetamol levels by the two methods was 0.85. The agreement on clinical risk categorization on the standard nomogram was also good (Kappa = 0.62, sensitivity 81%, specificity 89%). Conclusions: History of dose ingested alone greatly over-estimated the number of patients who need antidotes and it was a poor predictor of risk. Paracetamol concentrations by colorimetry are rapid and inexpensive. The use of these would greatly improve the assessment of risk and greatly reduce unnecessary expenditure on antidotes. PMID:23087506

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

PubMed Central

Duarte, Ana Joana; Vieira, Luis

2013-01-01

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

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

PubMed

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

2010-01-20

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

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

Code of Federal Regulations, 2014 CFR

2014-07-01

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

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

Code of Federal Regulations, 2013 CFR

2013-10-01

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

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

Code of Federal Regulations, 2012 CFR

2012-10-01

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

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

Code of Federal Regulations, 2014 CFR

2014-10-01

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

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

PubMed

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

2016-11-01

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

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.

Can the Afinion HbA1c Point-of-Care instrument be an alternative method for the Tosoh G8 in the case of Hb-Tacoma?

PubMed

Lenters-Westra, Erna; Strunk, Annuska; Campbell, Paul; Slingerland, Robbert J

2017-02-01

Hb-variant interference when reporting HbA1c has been an ongoing challenge since HbA1c was introduced to monitor patients with diabetes mellitus. Most Hb-variants show an abnormal chromatogram when cation-exchange HPLC is used for the determination of HbA1c. Unfortunately, the Tosoh G8 generates what appears to be normal chromatogram in the presence of Hb-Tacoma, yielding a falsely high HbA1c value. The primary aim of the study was to investigate if the Afinion HbA1c point-of-care (POC) instrument could be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma. Whole blood samples were collected in K 2 EDTA tubes from individuals homozygous for HbA (n = 40) and heterozygous for Hb-Tacoma (n = 20). Samples were then immediately analyzed with the Afinion POC instrument. After analysis, aliquots of each sample were frozen at -80 °C. The frozen samples were shipped on dry ice to the European Reference Laboratory for Glycohemoglobin (ERL) and analyzed with three International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and National Glycohemoglobin Standardization Program (NGSP) Secondary Reference Measurement Procedures (SRMPs). The Premier Hb9210 was used as the reference method. When compared to the reference method, samples with Hb-Tacoma yielded mean relative differences of 31.8% on the Tosoh G8, 21.5% on the Roche Tina-quant Gen. 2 and 16.8% on the Afinion. The Afinion cannot be used as an alternative method for the Tosoh G8 when testing for HbA1c in the presence of Hb-Tacoma.

Methods for Quantitative Creatinine Determination.

PubMed

Moore, John F; Sharer, J Daniel

2017-04-06

Reliable measurement of creatinine is necessary to assess kidney function, and also to quantitate drug levels and diagnostic compounds in urine samples. The most commonly used methods are based on the Jaffe principal of alkaline creatinine-picric acid complex color formation. However, other compounds commonly found in serum and urine may interfere with Jaffe creatinine measurements. Therefore, many laboratories have made modifications to the basic method to remove or account for these interfering substances. This appendix will summarize the basic Jaffe method, as well as a modified, automated version. Also described is a high performance liquid chromatography (HPLC) method that separates creatinine from contaminants prior to direct quantification by UV absorption. Lastly, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described that uses stable isotope dilution to reliably quantify creatinine in any sample. This last approach has been recommended by experts in the field as a means to standardize all quantitative creatinine methods against an accepted reference. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

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

PubMed Central

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

2013-01-01

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

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

USDA-ARS?s Scientific Manuscript database

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

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2008-01-01

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

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

PubMed

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

2015-05-01

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

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

PubMed Central

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

1989-01-01

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

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

PubMed

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

2012-11-01

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

[Spectrophotometric and HPLC evaluation of ceftazidime stability].

PubMed

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

2010-01-01

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

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

PubMed

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

2011-03-01

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

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

PubMed Central

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

2011-01-01

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

Quality Assessment of Kumu Injection, a Traditional Chinese Medicine Preparation, Using HPLC Combined with Chemometric Methods and Qualitative and Quantitative Analysis of Multiple Alkaloids by Single Marker.

PubMed

Wang, Ning; Li, Zhi-Yong; Zheng, Xiao-Li; Li, Qiao; Yang, Xin; Xu, Hui

2018-04-09

Kumu injection (KMI) is a common-used traditional Chinese medicine (TCM) preparation made from Picrasma quassioides (D. Don) Benn. rich in alkaloids. An innovative technique for quality assessment of KMI was developed using high performance liquid chromatography (HPLC) combined with chemometric methods and qualitative and quantitative analysis of multi-components by single marker (QAMS). Nigakinone (PQ-6, 5-hydroxy-4-methoxycanthin-6-one), one of the most abundant alkaloids responsible for the major pharmacological activities of Kumu, was used as a reference substance. Six alkaloids in KMI were quantified, including 6-hydroxy- β -carboline-1-carboxylic acid (PQ-1), 4,5-dimethoxycanthin-6-one (PQ-2), β -carboline-1-carboxylic acid (PQ-3), β -carboline-1-propanoic acid (PQ-4), 3-methylcanthin-5,6-dione (PQ-5), and PQ-6. Based on the outcomes of twenty batches of KMI samples, the contents of six alkaloids were used for further chemometric analysis. By hierarchical cluster analysis (HCA), radar plots, and principal component analysis (PCA), all the KMI samples could be categorized into three groups, which were closely related to production date and indicated the crucial influence of herbal raw material on end products of KMI. QAMS combined with chemometric analysis could accurately measure and clearly distinguish the different quality samples of KMI. Hence, QAMS is a feasible and promising method for the quality control of KMI.

Hydrophilic interaction liquid chromatography of anthranilic acid-labelled oligosaccharides with a 4-aminobenzoic acid ethyl ester-labelled dextran hydrolysate internal standard.

PubMed

Neville, David C A; Alonzi, Dominic S; Butters, Terry D

2012-04-13

Hydrophilic interaction liquid chromatography (HILIC) of fluorescently labelled oligosaccharides is used in many laboratories to analyse complex oligosaccharide mixtures. Separations are routinely performed using a TSK gel-Amide 80 HPLC column, and retention times of different oligosaccharide species are converted to glucose unit (GU) values that are determined with reference to an external standard. However, if retention times were to be compared with an internal standard, consistent and more accurate GU values would be obtained. We present a method to perform internal standard-calibrated HILIC of fluorescently labelled oligosaccharides. The method relies on co-injection of 4-aminobenzoic acid ethyl ester (4-ABEE)-labelled internal standard and detection by UV absorption, with 2-AA (2-aminobenzoic acid)-labelled oligosaccharides. 4-ABEE is a UV chromophore and a fluorophore, but there is no overlap of the fluorescent spectrum of 4-ABEE with the commonly used fluorescent reagents. The dual nature of 4-ABEE allows for accurate calculation of the delay between UV and fluorescent signals when determining the GU values of individual oligosaccharides. The GU values obtained are inherently more accurate as slight differences in gradients that can influence retention are negated by use of an internal standard. Therefore, this paper provides the first method for determination of HPLC-derived GU values of fluorescently labelled oligosaccharides using an internal calibrant. Copyright © 2012 Elsevier B.V. All rights reserved.

Advantages of soft versus hard constraints in self-modeling curve resolution problems. Alternating least squares with penalty functions.

PubMed

Gemperline, Paul J; Cash, Eric

2003-08-15

A new algorithm for self-modeling curve resolution (SMCR) that yields improved results by incorporating soft constraints is described. The method uses least squares penalty functions to implement constraints in an alternating least squares algorithm, including nonnegativity, unimodality, equality, and closure constraints. By using least squares penalty functions, soft constraints are formulated rather than hard constraints. Significant benefits are (obtained using soft constraints, especially in the form of fewer distortions due to noise in resolved profiles. Soft equality constraints can also be used to introduce incomplete or partial reference information into SMCR solutions. Four different examples demonstrating application of the new method are presented, including resolution of overlapped HPLC-DAD peaks, flow injection analysis data, and batch reaction data measured by UV/visible and near-infrared spectroscopy (NIR). Each example was selected to show one aspect of the significant advantages of soft constraints over traditionally used hard constraints. Incomplete or partial reference information into self-modeling curve resolution models is described. The method offers a substantial improvement in the ability to resolve time-dependent concentration profiles from mixture spectra recorded as a function of time.

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

PubMed

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

2017-11-08

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

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

PubMed

Zhou, Jue; Qu, Fan

2011-01-01

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

Direct injection analysis of fatty and resin acids in papermaking process waters by HPLC/MS.

PubMed

Valto, Piia; Knuutinen, Juha; Alén, Raimo

2011-04-01

A novel HPLC-atmospheric pressure chemical ionization/MS (HPLC-APCI/MS) method was developed for the rapid analysis of selected fatty and resin acids typically present in papermaking process waters. A mixture of palmitic, stearic, oleic, linolenic, and dehydroabietic acids was separated by a commercial HPLC column (a modified stationary C(18) phase) using gradient elution with methanol/0.15% formic acid (pH 2.5) as a mobile phase. The internal standard (myristic acid) method was used to calculate the correlation coefficients and in the quantitation of the results. In the thorough quality parameters measurement, a mixture of these model acids in aqueous media as well as in six different paper machine process waters was quantitatively determined. The measured quality parameters, such as selectivity, linearity, precision, and accuracy, clearly indicated that, compared with traditional gas chromatographic techniques, the simple method developed provided a faster chromatographic analysis with almost real-time monitoring of these acids. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative determination of flavonoids by column high-performance liquid chromatography with mass spectrometry and ultraviolet absorption detection in Artemisia afra and comparative studies with various species of Artemisia plants.

PubMed

Avula, Bharathi; Wang, Yan-Hong; Smillie, Troy J; Mabusela, Wilfred; Vincent, Leszek; Weitz, Frans; Khan, Ikhlas A

2009-01-01

A simple and specific analytical method for the quantitative determination of flavonoids from the aerial parts of the Artemisia afra plant samples was developed. By column high-performance liquid chromatography (HPLC) with UV absorption and mass spectrometry (MS) detection, separation was achieved on a reversed-phase octadecylsilyl (C18) column with water, methanol, and acetonitrile, all containing 0.1% acetic acid, as the mobile phase. These methods were used to analyze various species of Artemisia plant samples. The wavelength used for quantification of flavonoids with the diode array detector was 335 nm. The limits of detection (LOD) by HPLC/MS were found to be 7.5, 7.5, 10, 2.0, and 2.0 ng/mL; and by LC-UV the LODs were 500, 500, 500, 300, and 300 ng/mL for apigenin, chrysoeriol, tamarixetin, acacetin, and genkwanin, respectively. The HPLC/MS method was found to be 50-150 times more sensitive than the HPLC-UV method. HPLC/MS coupled with an electrospray ionization interface is described for the identification and quantification of flavonoids in various plant samples. This method involved the use of the [M+H]+ ions of the compounds at mass-to-charge ratio of 1.0606, 301.0712, 317.0661, 285.0763, and 285.0763 (calculated mass), respectively, in the positive ion mode with extractive ion monitoring.

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

PubMed

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

2014-01-01

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

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

PubMed

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

2012-01-01

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

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

PubMed

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

1992-12-01

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

Evaluation of a next generation direct whole blood enzymatic assay for hemoglobin A1c on the ARCHITECT c8000 chemistry system.

PubMed

Teodoro-Morrison, Tracy; Janssen, Marcel J W; Mols, Jasper; Hendrickx, Ben H E; Velmans, Mathieu H; Lotz, Johannes; Lackner, Karl; Lennartz, Lieselotte; Armbruster, David; Maine, Gregory; Yip, Paul M

2015-01-01

The utility of HbA1c for the diagnosis of type 2 diabetes requires an accurate, precise and robust test measurement system. Currently, immunoassay and HPLC are the most popular methods for HbA1c quantification, noting however the limitations associated with some platforms, such as imprecision or interference from common hemoglobin variants. Abbott Diagnostics has introduced a fully automated direct enzymatic method for the quantification of HbA1c from whole blood on the ARCHITECT chemistry system. Here we completed a method evaluation of the ARCHITECT HbA1c enzymatic assay for imprecision, accuracy, method comparison, interference from hemoglobin variants and specimen stability. This was completed at three independent clinical laboratories in North America and Europe. The total imprecision ranged from 0.5% to 2.2% CV with low and high level control materials. Around the diagnostic cut-off of 48 mmol/mol, the total imprecision was 0.6% CV. Mean bias using reference samples from IFCC and CAP ranged from -1.1 to 1.0 mmol/mol. The enzymatic assay also showed excellent agreement with HPLC methods, with slopes of 1.01 and correlation coefficients ranging from 0.984 to 0.996 compared to Menarini Adams HA-8160, Bio-Rad Variant II and Variant II Turbo instruments. Finally, no significant effect was observed for erythrocyte sedimentation or interference from common hemoglobin variants in patient samples containing heterozygous HbS, HbC, HbD, HbE, and up to 10% HbF. The ARCHITECT enzymatic assay for HbA1c is a robust and fully automated method that meets the performance requirements to support the diagnosis of type 2 diabetes.

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 quick inexpensive laboratory method in acute paracetamol poisoning could improve risk assessment, management and resource utilization.

PubMed

Senarathna, S M D K Ganga; Ranganathan, Shalini S; Buckley, Nick; Soysa, S S S B D Preethi; Fernandopulle, B M Rohini

2012-01-01

Acute paracetamol poisoning is an emerging problem in Sri Lanka. Management guidelines recommend ingested dose and serum paracetamol concentrations to assess the risk. Our aim was to determine the usefulness of the patient's history of an ingested dose of >150 mg/kg and paracetamol concentration obtained by a simple colorimetric method to assess risk in patients with acute paracetamol poisoning. Serum paracetamol concentrations were determined in 100 patients with a history of paracetamol overdose using High Performance Liquid Chromatography (HPLC); (reference method). The results were compared to those obtained with a colorimetric method. The utility of risk assessment by reported dose ingested and colorimetric analysis were compared. The area under the receiver operating characteristic curve for the history of ingested dose was 0.578 and there was no dose cut-off providing useful risk categorization. Both analytical methods had less than 5% intra- and inter-batch variation and were accurate on spiked samples. The time from blood collection to result was six times faster and ten times cheaper for colorimetry (30 minutes, US$2) than for HPLC (180 minutes, US$20). The correlation coefficient between the paracetamol levels by the two methods was 0.85. The agreement on clinical risk categorization on the standard nomogram was also good (Kappa = 0.62, sensitivity 81%, specificity 89%). History of dose ingested alone greatly over-estimated the number of patients who need antidotes and it was a poor predictor of risk. Paracetamol concentrations by colorimetry are rapid and inexpensive. The use of these would greatly improve the assessment of risk and greatly reduce unnecessary expenditure on antidotes.

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

PubMed Central

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

2013-01-01

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

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

PubMed

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

2013-01-01

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

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

PubMed

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

2018-06-01

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

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

PubMed

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

2015-05-15

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

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

PubMed

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

2008-06-09

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

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

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

PubMed

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

2011-03-23

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

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography-triple quadrupole mass spectrometry with novel shell-type column.

PubMed

Du, Gang; Zhao, Haiyu; Song, Yuelin; Zhang, Qingwen; Wang, Yitao

2011-10-01

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An improved HPLC-DAD method for quantitative comparisons of triterpenes in Ganoderma lucidum and its five related species originating from Vietnam.

PubMed

Ha, Do Thi; Loan, Le Thi; Hung, Tran Manh; Han, Le Vu Ngoc; Khoi, Nguyen Minh; Dung, Le Viet; Min, Byung Sun; Nguyen, Nguyen Phuong Dai

2015-01-09

An HPLC-DAD method for the quality control of wild and cultivated Ganoderma lucidum (Linhzhi) and related species samples was developed and validated. The quantitative determination of G. lucidum and its related species using 14 triterpene constituents, including nine ganoderma acids (compounds 4-12), four alcohols (compounds 13-16), and one sterol (ergosterol, 17) were reported. The standard curves were linear over the concentration range of 7.5-180 µg/mL. The LOD and LOQ values for the analyses varied from 0.34 to 1.41 µg/mL and from 1.01 to 4.23 µg/mL, respectively. The percentage recovery of each reference compound was found to be from 97.09% to 100.79%, and the RSD (%) was less than 2.35%. The precision and accuracy ranged from 0.81%-3.20% and 95.38%-102.19% for intra-day, and from 0.43%-3.67% and 96.63%-103.09% for inter-day, respectively. The study disclosed in detail significant differences between the quantities of analyzed compounds in different samples. The total triterpenes in wild Linhzhi samples were significantly higher than in cultivated ones. The total constituent contents of the five related Linhzhi samples were considerably lower than that in the G. lucidum specimens, except for G. australe as its constituent content outweighed wild Linhzhi's content by 4:1.

Enrichment and purification of six Aconitum alkaloids from Aconiti kusnezoffii radix by macroporous resins and quantification by HPLC-MS.

PubMed

Liu, Jingjing; Li, Qing; Liu, Ran; Yin, Yidi; Chen, Xiaohui; Bi, Kaishun

2014-06-01

Aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine are six main Aconitum alkaloids from traditional Chinese medicine, Aconiti kusnezoffii radix, which possess highly bioactive as well as highly toxic character for medicinal use. In the present study, for the purpose of better utilizing the toxic herbal material, the performance characteristics of NKA-II, D101, X-5, AB-8, S-8, HPD722 and HPD750 macroporous resins for the enrichment and purification of these six Aconitum alkaloids were critically evaluated. Results showed that NKA-II offered the best adsorption and desorption capacities for six Aconitum alkaloids among the seven macroporous resins tested, which were affected significantly by the pH value. Subsequently, dynamic adsorption and desorption experiments had been carried out with the column packed by NKA-II resin to optimize the separation process of six Aconitum alkaloids. After one run treatment with NKA-II resin, the content of total six Aconitum alkaloids were increased from 5.87% to 60.3%, the recovery was 75.8%. Meanwhile, a validated HPLC-MS method had been developed to qualitative and quantitative these six Aconitum alkaloids. This method would provide scientific references to the large-scale production of six Aconitum alkaloids from Aconiti kusnezoffii radix or other plants and might also expand the secure application of these highly toxic components for pharmacy. Copyright © 2014 Elsevier B.V. All rights reserved.

Preliminary Phytochemical Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan.

PubMed

Gul, Rahman; Jan, Syed Umer; Faridullah, Syed; Sherani, Samiullah; Jahan, Nusrat

2017-01-01

The aim of this study was to evaluate the antioxidant activity, screening the phytogenic chemical compounds, and to assess the alkaloids present in the E. intermedia to prove its uses in Pakistani folk medicines for the treatment of asthma and bronchitis. Antioxidant activity was analyzed by using 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. Standard methods were used for the identification of cardiac glycosides, phenolic compounds, flavonoids, anthraquinones, and alkaloids. High performance liquid chromatography (HPLC) was used for quantitative purpose of ephedrine alkaloids in E. intermedia . The quantitative separation was confirmed on Shimadzu 10AVP column (Shampack) of internal diameter (id) 3.0 mm and 50 mm in length. The extract of the solute in flow rate of 1 ml/min at the wavelength 210 nm and methanolic extract showed the antioxidant activity and powerful oxygen free radicals scavenging activities and the IC50 for the E. intermedia plant was near to the reference standard ascorbic acid. The HPLC method was useful for the quantitative purpose of ephedrine (E) and pseudoephedrine (PE) used for 45 samples of one species collected from central habitat in three districts (Ziarat, Shairani, and Kalat) of Balochistan. Results showed that average alkaloid substance in E. intermedia was as follows: PE (0.209%, 0.238%, and 0.22%) and E (0.0538%, 0.0666%, and 0.0514%).

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2008-10-01

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

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

PubMed

Shumow, Laura; Bodor, Alison

2011-07-05

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

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

PubMed

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

2015-10-23

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

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

PubMed

Liu, Rui; Cai, Zongwei; Xu, Baojun

2017-09-22

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

[HPLC-fingerprint-based quality evaluation on a Tibetan medicine Phyllanthus emblica and its tannin parts].

PubMed

Sun, Xue-Fei; Zhang, Hong-Yan; Xia, Qing; Zhao, Hai-Juan; Wu, Ling-Fang; Zhang, Lan-Zhen; Shi, Ren-Bing

2014-04-01

This study is to establish the fingerprint for Phyllanthus emblica and their tannin parts from different habitats by HPLC for its quality control. The determination was carried out on a Diamonsil C18 (4.6 mm x 250 mm, 5 microm) column, with methanol-0.2% glacial acetic acid as mobile phase with gradient elution at a flow rate of 1 mL x min(-1). The temperature was maintained at 30 degrees C and the detected wavelength is 260 nm, Thirteen chromatographic peaks were extracted as the common peaks of the fingerprint of P. emblica, and eleven as the common peaks of P. emblica tannin parts, and five peaks were identified by comparing with referent samples. The fingerprints of 8 samples were compared and classified by similarity evaluation, cluster analysis and principal component analysis (PCA). The similarity degrees of eight P. emblica were between 0.763 and 0.993, while tannin parts were between 0.903 and 0.991. All the samples of P. emblica and their tannin parts were classified into 3 categories. The method was so highly reproducible, simple and reliable that it could provide basis for quality control and evaluation of P. emblica from different habitats.

Carotenoids of two freshwater amphipod species (Gammarus pulex and G. roeseli) and their common acanthocephalan parasite Polymorphus minutus.

PubMed

Gaillard, Maria; Juillet, Cédrik; Cézilly, Frank; Perrot-Minnot, Marie-Jeanne

2004-09-01

Carotenoid compositions of two freshwater Gammarus species (Crustacea: Amphipoda) and of their common acanthocephalan parasite Polymorphus minutus were characterized. The effect of carotenoid uptake by the parasite was addressed by comparing the carotenoid content of uninfected and infected female hosts. Using high-pressure liquid chromatography (HPLC), co-chromatography of reference pigments and electron ionization mass spectrometry of collected HPLC fractions (EI-MS), several xanthophylls and non-polar compounds were identified. Seven kinds of carotenoids, mainly xanthophylls, were identified in gammarids. Astaxanthin was predominant, amounting to 40 wt.% of total carotenoid in both uninfected G. pulex and G. roeseli. By contrast, we found only non-polar compounds with a predominance of esterified forms of astaxanthin in P. minutus larvae. No significant effect of infection on carotenoid content was evidenced in G. pulex and G. roeseli females. Our study highlights the use of a Matrix Solid Phase Dispersion as an efficient extraction method of both xanthophylls and non-polar pigments in small samples, including lipid-rich ones as P. minutus parasite. We discuss on the presumptive pathway leading to the formation of free astaxanthin in gammarids via hydroxy compounds, and on the accumulation of esters of astaxanthin in parasites.

Development of a near-infrared spectroscopy method (NIRS) for fast analysis of total, indolic, aliphatic and individual glucosinolates in new bred open pollinating genotypes of broccoli (Brassica oleracea convar. botrytis var. italica).

PubMed

Sahamishirazi, Samira; Zikeli, Sabine; Fleck, Michael; Claupein, Wilhelm; Graeff-Hoenninger, Simone

2017-10-01

This study describes the development of near-infrared spectroscopy (NIRS) calibration to determine individual and total glucosinolates (GSLs) content of 12 new-bred open-pollinating genotypes of broccoli (Brassica oleracea convar. botrytis var. italica). Six individual GSLs were identified using high-performance-liquid chromatography (HPLC). The NIRS calibration was established based on modified partial least squares regression with reference values of HPLC. The calibration was analyzed using coefficient of determination in prediction (R 2 ) and ratio of preference of determination (RPD). Large variation occurred in the calibrations, R 2 and RPD due to the variability of the samples. Derived calibrations for total-GSLs, aliphatic-GSLs, glucoraphanin and 4-methoxyglucobrassicin were quantitative with a high accuracy (RPD=1.36, 1.65, 1.63, 1.11) while, for indole-GSLs, glucosinigrin, glucoiberin, glucobrassicin and 1-methoxyglucobrassicin were more qualitative (RPD=0.95, 0.62, 0.67, 0.81, 0.56). Overall, the results indicated NIRS has a good potential to determine different GSLs in a large sample pool of broccoli quantitatively and qualitatively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Heat-assisted aqueous extraction of rice flour for arsenic speciation analysis.

PubMed

Narukawa, Tomohiro; Chiba, Koichi

2010-07-28

A versatile heat-assisted pretreatment aqueous extraction method for the analysis of arsenic species in rice was developed. Rice flour certified reference materials NIST SRM1568a and NMIJ CRM 7503-a and a flour made from polished rice were used as samples, and HPLC-ICP-MS was employed for the determination of arsenic species. Arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) were detected in NIST SRM, and As(III), As(V) and DMAA were found in NMIJ CRM and the prepared polished rice flour. The sums of the concentrations of all species in each rice flour sample were 97-102% of the total arsenic concentration in each sample.

Chemometric simultaneous determination of Sofosbuvir and Ledipasvir in pharmaceutical dosage form

NASA Astrophysics Data System (ADS)

Khalili, Mahsa; Sohrabi, Mahmoud Reza; Mirzabeygi, Vahid; Torabi Ziaratgahi, Nahid

2018-04-01

Partial least squares (PLS), different families of continuous wavelet transform (CWT), and first derivative spectrophotometry (DS) techniques were studied for quantification of Sofosbuvir (SFB) and Ledipasvir (LDV) simultaneously without separation step. The components were dissolved in Acetonitrile and the spectral behaviors were evaluated in the range of 200 to 400 nm. The ultraviolet (UV) absorbance of LDV exhibits no interferences between 300 and 400 nm and it was decided to predict the LDV amount through the classic spectrophotometry (CS) method in this spectral region as well. Data matrix of concentrations and calibrated models were developed, and then by applying a validation set the accuracy and precision of each model were studied. Actual concentrations versus predicted concentrations plotted and good correlation coefficients by each method resulted. Pharmaceutical dosage form was quantified by developed methods and the results were compared with the High Performance Liquid Chromatography (HPLC) reference method. Analysis Of Variance (ANOVA) in 95% confidence level showed no significant differences among methods.

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

PubMed

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

2010-05-01

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

Quantification of astaxanthin in shrimp waste hydrolysate by HPLC.

PubMed

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

2006-10-01

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

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2014-01-01

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

Probabilistic classification method on multi wavelength chromatographic data for photosynthetic pigments identification

NASA Astrophysics Data System (ADS)

Prilianti, K. R.; Setiawan, Y.; Indriatmoko, Adhiwibawa, M. A. S.; Limantara, L.; Brotosudarmo, T. H. P.

2014-02-01

Environmental and health problem caused by artificial colorant encourages the increasing usage of natural colorant nowadays. Natural colorant refers to the colorant that is derivate from living organism or minerals. Extensive research topic has been done to exploit these colorant, but recent data shows that only 0.5% of the wide range of plant pigments in the earth has been exhaustively used. Hence development of the pigment characterization technique is an important consideration. High-performance liquid chromatography (HPLC) is a widely used technique to separate pigments in a mixture and identify it. In former HPLC fingerprinting, pigment characterization was based on a single chromatogram from a fixed wavelength (one dimensional) and discard the information contained at other wavelength. Therefore, two dimensional fingerprints have been proposed to use more chromatographic information. Unfortunately this method leads to the data processing problem due to the size of its data matrix. The other common problem in the chromatogram analysis is the subjectivity of the researcher in recognizing the chromatogram pattern. In this research an automated analysis method of the multi wavelength chromatographic data was proposed. Principal component analysis (PCA) was used to compress the data matrix and Maximum Likelihood (ML) classification was applied to identify the chromatogram pattern of the existing pigments in a mixture. Three photosynthetic pigments were selected to show the proposed method. Those pigments are β-carotene, fucoxanthin and zeaxanthin. The result suggests that the method could well inform the existence of the pigments in a particular mixture. A simple computer application was also developed to facilitate real time analysis. Input of the application is multi wavelength chromatographic data matrix and the output is information about the existence of the three pigments.

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

PubMed Central

Congote, L. F.; Hamilton, E. F.; Chow, J. C.; Perry, T. B.

1982-01-01

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells. PMID:7139502

Optimization of ultra-performance liquid chromatography (UPLC) with fluorescence detector (FLD) method for the quantitative determination of selected neurotransmitters in rat brain.

PubMed

Stragierowicz, Joanna; Daragó, Adam; Brzeźnicki, Sławomir; Kilanowicz, Anna

2017-07-26

Glutamate (Glu) and γ-aminobutyric acid (GABA) are the main neurotransmitters in the central nervous system for excitatory and inhibitory processes, respectively. Monitoring these neurotransmitters is an essential tool in establishing pathological functions, among others in terms of occupational exposure to toxic substances. We present modification of the HPLC (high-performance liquid chromatography) to the UPLC (ultra-performance liquid chromatography) method for the simultaneous determination of glutamate and γ-aminobutyric acid in a single injection. The isocratic separation of these neurotransmitter derivatives was performed on Waters Acquity BEH (ethylene bridged hybrid) C18 column with particle size of 1.7 μm at 35°C using a mobile phase consisting of 0.1 M acetate buffer (pH 6.0) and methanol (60:40, v/v) at a flow rate of 0.3 ml/min. The analytes were detected with the fluorescence detector (FLD) using derivatization with o-phthaldialdehyde (OPA), resulting in excitation at 340 nm and emission at 455 nm. Several validation parameters including linearity (0.999), accuracy (101.1%), intra-day precision (1.52-1.84%), inter-day precision (2.47-3.12%), limit of detection (5-30 ng/ml) and quantification (100 ng/ml) were examined. The developed method was also used for the determination of these neurotransmitters in homogenates of selected rat brain structures. The presented UPLC-FLD is characterized by shorter separation time (3.5 min), which is an adaptation of the similar HPLC methods and is an alternative for more expensive references techniques such as liquid chromatography coupled with tandem mass-spectrometry (LC-MS/MS) methods. Med Pr 2017;68(5):583-591. This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Application of LC-MS to the analysis of dyes in objects of historical interest

NASA Astrophysics Data System (ADS)

Zhang, Xian; Laursen, Richard

2009-07-01

High-performance liquid chromatography (HPLC) with photodiode array and mass spectrometric detection permits dyes extracted from objects of historical interest or from natural plant or animal dyestuffs to be characterized on the basis of three orthogonal properties: HPLC retention time, UV-visible spectrum and molecular mass. In the present study, we have focused primarily on yellow dyes, the bulk of which are flavonoid glycosides that would be almost impossible to characterize without mass spectrometric detection. Also critical for this analysis is a method for mild extraction of the dyes from objects (e.g., textiles) without hydrolyzing the glycosidic linkages. This was accomplished using 5% formic acid in methanol, rather than the more traditional 6 M HCl. Mass spectroscopy, besides providing the molecular mass of the dye molecule, sometimes yields additional structural data based on fragmentation patterns. In addition, coeluting compounds can often be detected using extracted ion chromatography. The utility of mass spectrometry is illustrated by the analysis of historical specimens of silk that had been dyed yellow with flavonoid glycosides from Sophora japonica (pagoda tree) and curcumins from Curcuma longa (turmeric). In addition, we have used these techniques to identify the dye type, and sometimes the specific dyestuff, in a variety of objects, including a yellow varnish from a 19th century Tibetan altar and a 3000-year-old wool mortuary textiles, from Xinjiang, China. We are using HPLC with diode array and mass spectrometric detection to create a library of analyzed dyestuffs (>200 so far; mostly plants) to serve as references for identification of dyes in objects of historical interest.

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

PubMed

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

2008-10-19

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

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

PubMed

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

2017-02-01

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

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

PubMed Central

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

2013-01-01

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

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

Treesearch

Rakesh Minocha; Stephanie Long

2004-01-01

The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl...

Conventional high-performance liquid chromatography versus derivative spectrophotometry for the determination of 1,3,6-pyrenetrisulfonic acid trisodium salt and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt in the color additive D&C Green No. 8 (Pyranine).

PubMed

Jitian, Simion; White, Samuel R; Yang, H-H Wendy; Weisz, Adrian

2014-01-10

Specifications in the U.S. Code of Federal Regulations for the color additive D&C Green No. 8 (Colour Index No. 59040) limit the levels of the subsidiary colors 1,3,6-pyrenetrisulfonic acid trisodium salt (P3S) and 1,3,6,8-pyrenetetrasulfonic acid tetrasodium salt (P4S). The present paper describes a comparative study of two possible methods to replace the currently used multi-step TLC/spectrophotometry method of separating and quantifying the minor components P3S and P4S in G8. One of the new approaches uses conventional high-performance liquid chromatography (HPLC) and the other, derivative spectrophotometry. While the derivative spectrophotometric method was shown to be inadequate for the analysis of minor components overwhelmed by components of much higher concentration, the HPLC method was proven highly effective. The closely related, very polar compounds P3S and P4S were separated by the new HPLC method in less than 4 min using a conventional HPLC instrument. P3S and P4S were quantified by using five-point calibration curves with data points that ranged from 0.45 to 7.63% and from 0.13 to 1.82%, by weight, for P3S and P4S, respectively. The HPLC method was applied to the analysis of test portions from 20 batches of D&C Green No. 8 submitted to the U.S. Food and Drug Administration for certification. Published by Elsevier B.V.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

PubMed

Kang, Kyo Bin; Ryu, Jayoung; Cho, Youngwoong; Choi, Sang-Zin; Son, Miwon; Sung, Sang Hyun

2017-05-01

DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative HPLC Analysis of Rosmarinic Acid in Extracts of "Melissa officinalis" and Spectrophotometric Measurement of Their Antioxidant Activities

ERIC Educational Resources Information Center

Canelas, Vera; da Costa, Cristina Teixeira

2007-01-01

The students prepare tea samples using different quantities of lemon balm leaves ("Melissa officinalis") and measure the rosmarinic acid contents by an HPLC-DAD method. The antioxidant properties of the tea samples are evaluated by a spectrophotometric method using a radical-scavenging assay with DPPH. (2,2-diphenyl-1-picrylhydrazyl). Finally the…

Quantitative determination of a-Arbutin, ß-Arbutin, Kojic acid, nicotinamide, hydroquinone, resorcinol, 4-methoxyphenol, 4-ethoxyphenol and ascorbic acid from skin whitening Products by HPLC-UV

USDA-ARS?s Scientific Manuscript database

Development of an analytical method for the simultaneous determination of multifarious skin whitening agents will provide an efficient tool to analyze skin whitening cosmetics. An HPLC-UV method was developed for quantitative analysis of six commonly used whitening agents, a-arbutin, ß-arbutin, koji...

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

DTIC Science & Technology

2012-05-01

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

Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

PubMed

Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

2016-06-01

Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of new impurities of enalapril maleate on oxidation in the presence of magnesium monoperoxyphthalate.

PubMed

Toporisic, Rebeka; Mlakar, Anita; Hvala, Jernej; Prislan, Iztok; Zupancic-Kralj, Lucija

2010-06-05

Stress stability testing and forced degradation were used to determine the stability of enalapril maleate (EM) and to find a degradation pathway for the drug. The degradation impurities, formed under different stressed conditions, were investigated by HPLC and UPLC-MS methods. HPLC analysis showed several degradation impurities of which several were already determined, but on oxidation in the presence of magnesium monoperoxyphthalate (MMPP) several impurities of EM were observed which were not yet characterized. The HPLC methods for determination of EM were validated. The linearity of HPLC method was established in the concentration range between 0.5 and 10 microg/mL with correlation coefficient greater than 0.99. The LOD of EM was 0.2 microg/mL and LOQ was 0.5 microg/mL. The validated HPLC method was used to determine the degradation impurities in samples after stress stability testing and forced degradation of EM. In order to identify new degradation impurities of EM after forced degradation UPLC-MS/MS(n), Orbitrap has been used. It was found that new impurities are oxidation products: (S)-1-((S)-2-((S)-1-ethoxy-4-(o,m,p-hydroxyphenyl)-1-oxobutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid, (2S)-1-((2S)-2-((2S)-1-ethoxy-4-hydroxy-1-oxo-4-phenylbutan-2-ylamino)propanoyl)pyrrolidine-2-carboxylic acid. (S)-2-(3-phenylpropylamino)-1-(pyrrolidin-1-yl)propan-1-one was identified as a new degradation impurity. Copyright (c) 2010. Published by Elsevier B.V.

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

PubMed

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

2013-09-01

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

Simultaneous determination of three polyphenols in rat plasma after orally administering hawthorn leaves extract by the HPLC method.

PubMed

Ying, Xixiang; Meng, Xiansheng; Wang, Siyuan; Wang, Dong; Li, Haibo; Wang, Bing; Du, Yang; Liu, Xun; Zhang, Wenjie; Kang, Tingguo

2012-01-01

A simple and sensitive HPLC method was developed to simultaneously determine three active compounds, vitexin-4″-O-glucoside (VG), vitexin-2″-O-rhamnoside (VR) and hyperoside (HP), in rat plasma after administering the hawthorn leaves extract (HLE). An HPLC assay with baicalin as the internal standard was carried out using a Phenomsil C₁₈ analytical column with UV detection at 332 nm. The mobile phase consisted of methanol-acetonitrile-tetrahydrofuran-1% glacial acetic acid (6 : 1.5 : 18.5 : 74, v/v/v/v). The calibration curves were linear over the range of 2.5-500, 0.2-25 and 0.25-12.5 µg mL⁻¹ for VG, VR and HP, respectively. The method was reproducible and reliable, with relative standard deviations of the intra- and inter-day precision between 1.2% and 13.2% for the analysis of the three analytes. The validated HPLC method herein described was successfully applied to the pharmacokinetic study of VG, VR and HP after oral administration of HLE to rats over the dose range of 2.5-10  mL kg⁻¹.

On-line MSPD-SPE-HPLC/FLD analysis of polycyclic aromatic hydrocarbons in bovine tissues.

PubMed

Gutiérrez-Valencia, Tania M; García de Llasera, Martha P

2017-05-15

A fast method was optimized and validated for simultaneous trace determination of four polycyclic aromatic hydrocarbons: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene and benzo[a]pyrene in bovine tissues. The determination was performed by matrix solid-phase dispersion (MSPD) coupled on-line to solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with fluorescence detection (FLD). The sample was dispersed on C 18 silica sorbent and then the on-line MSPD-SPE-HPLC/FLD method was applied. Several parameters were optimized: cleaning and elution sequences applied to the MSPD cartridge, the flow rate and dilution of extract used for SPE loading. The on-line method was validated over a concentration range of 0.1-0.6ngg -1 obtaining good linearity (r⩾0.998) and precision (RSD)⩽10%. Recovery ranged from 96 to 99% and the limits of detection were 0.012ngg -1 . This methodology was applied to liver samples from unhealthy animals. The results demonstrate that MSDP-SPE-HPLC/FLD method provides reliable, sensitive, accurate and fast data to the food control. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ultrafast UPLC-ESI-MS and HPLC with monolithic column for determination of principal flavor compounds in vanilla pods.

PubMed

Sharma, Upendra K; Sharma, Nandini; Sinha, Arun K; Kumar, Neeraj; Gupta, Ajai P

2009-10-01

In this study, two novel chromatographic methods based on monolithic column high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were developed for the ultrafast determination of principal flavor compounds namely vanillin, vanillic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde in ethanolic extracts of Vanilla planifolia pods. Good separation was achieved within 2.5 min using Chromolith RP18e column (100 mm x 4.6 mm) for HPLC and Acquity BEH C-18 (100 mm x 2.1 mm, 1.7 microm) column for UPLC. Both methods were compared in terms of total analysis time, mobile phase consumption, sensitivity, and validation parameters like precision, accuracy, LOD, and LOQ. Further, system suitability test data including resolution, capacity factor, theoretical plates, and tailing factor was determined for both the methods by ten replicate injections. Monolithic column based HPLC gave better results for most of the selected parameters while UPLC was found to be more eco-friendly with low mobile phase consumption and better sensitivity. Both methods may be used conveniently for the high throughput analysis of large number of samples in comparison to traditional particulate column.

Inter-laboratory validation of an inexpensive streamlined method to measure inorganic arsenic in rice grain.

PubMed

Chaney, Rufus L; Green, Carrie E; Lehotay, Steven J

2018-05-04

With the establishment by CODEX of a 200 ng/g limit of inorganic arsenic (iAs) in polished rice grain, more analyses of iAs will be necessary to ensure compliance in regulatory and trade applications, to assess quality control in commercial rice production, and to conduct research involving iAs in rice crops. Although analytical methods using high-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) have been demonstrated for full speciation of As, this expensive and time-consuming approach is excessive when regulations are based only on iAs. We report a streamlined sample preparation and analysis of iAs in powdered rice based on heated extraction with 0.28 M HNO 3 followed by hydride generation (HG) under control of acidity and other simple conditions. Analysis of iAs is then conducted using flow-injection HG and inexpensive ICP-atomic emission spectroscopy (AES) or other detection means. A key innovation compared with previous methods was to increase the acidity of the reagent solution with 4 M HCl (prior to reduction of As 5+ to As 3+ ), which minimized interferences from dimethylarsinic acid. An inter-laboratory method validation was conducted among 12 laboratories worldwide in the analysis of six shared blind duplicates and a NIST Standard Reference Material involving different types of rice and iAs levels. Also, four laboratories used the standard HPLC-ICP-MS method to analyze the samples. The results between the methods were not significantly different, and the Horwitz ratio averaged 0.52 for the new method, which meets official method validation criteria. Thus, the simpler, more versatile, and less expensive method may be used by laboratories for several purposes to accurately determine iAs in rice grain. Graphical abstract Comparison of iAs results from new and FDA methods.

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

PubMed

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

2015-01-01

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

Chiral separation of the β2-sympathomimetic fenoterol by HPLC and capillary zone electrophoresis for pharmacokinetic studies.

PubMed

Ullrich, Thomas; Wesenberg, Dirk; Bleuel, Corinna; Krauss, Gerd-Joachim; Schmid, Martin G; Weiss, Michael; Gübitz, Gerald

2010-10-01

The development of methods for the separation of the enantiomers of fenoterol by chiral HPLC and capillary zone electrophoresis (CZE) is described. For the HPLC separation precolumn fluorescence derivatization with naphthyl isocyanate was applied. The resulting urea derivatives were resolved on a cellulose tris(3,5-dimethylphenylcarbamate)-coated silica gel column employing a column switching procedure. Detection was carried out fluorimetrically with a detection limit in the low ng/mL range. The method was adapted to the determination of fenoterol enantiomers in rat heart perfusates using liquid-liquid extraction. As an alternative a CE method was used for the direct separation of fenoterol enantiomers comparing different cyclodextrin derivatives as chiral selectors. Copyright © 2010 John Wiley & Sons, Ltd.

Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection †

PubMed Central

Karr, Dale B.; Waters, James K.; Emerich, David W.

1983-01-01

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-β-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis. PMID:16346443

Quantitative determination of limonin in citrus juices by HPLC using computerized solvent optimization.

PubMed

Shaw, P E; Wilson, C W

1988-09-01

The commercially available computer program, Drylab, for optimization of separations by high-performance liquid chromatography (HPLC) using binary solvent mixtures is used to improve an HPLC method for separation of the bitter principle, limonin, in grapefruit and navel orange juices. Best conditions for separation of limonin in a reasonable time are 30 to 32% acetonitrile in water at 0.9 mL/min using a 5-micron C18 column 10 cm long. These conditions are used to analyze grapefruit and navel orange juice samples, and these HPLC results are compared with values determined by enzyme immunoassay or thin-layer chromatography (TLC) on the same samples.

Degradation of components in drug formulations: a comparison between HPLC and DSC methods.

PubMed

Ceschel, G C; Badiello, R; Ronchi, C; Maffei, P

2003-08-08

Information about the stability of drug components and drug formulations is needed to predict the shelf-life of the final products. The studies on the interaction between the drug and the excipients may be carried out by means of accelerated stability tests followed by analytical determination of the active principle (HPLC and other methods) and by means of the differential scanning calorimetry (DSC). This research has been focused to the acetyl salicylic acid (ASA) physical-chemical characterisation by using DSC method in order to evaluate its compatibility with some of the most used excipients. It was possible to show, with the DSC method, the incompatibility of magnesium stearate with ASA; the HPLC data confirm the reduction of ASA concentration in the presence of magnesium stearate. With the other excipients the characteristic endotherms of the drug were always present and no or little degradation was observed with the accelerated stability tests. Therefore, the results with the DSC method are comparable and in good agreement with the results obtained with other methods.

Comparison of cyclosporine determinations in whole blood by three different methods. HPLC, /sup 125/I RIA and /sup 3/H RIA

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

Huang, W.Y.; Lipsey, A.I.; Cheng, M.H.

1987-04-01

The authors have analyzed and compared the cyclosporine concentrations in whole blood specimens from pediatric renal transplant patients using three different methods: high-performance liquid chromatography (HPLC) (5u C18 reverse-phase column), /sup 3/H radioimmunoassay (RIA), and /sup 125/I RIA (substituted /sup 3/H-tracer in Sandoz Kit with /sup 125/I tracer. Results obtained by the /sup 125/I RIA correlated well with results obtained by the /sup 3/H RIA. Both RIA methods had similar correlation with the HPLC method. The /sup 125/I RIA method showed higher sensitivity and greater precision than the /sup 3/H RIA method. The authors conclude that the /sup 125/I RIAmore » method can be used for cyclosporine determination in whole blood specimens. The use of the /sup 125/I RIA provides a simple and rapid method with higher counting efficiency and less background quenching than the /sup 3/H RIA method, which requires cumbersome liquid scintillation counting procedures.« less

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

PubMed

Onal, Armağan

2009-12-01

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

Validation of a method for the quantitation of ghrelin and unacylated ghrelin by HPLC.

PubMed

Staes, Edith; Rozet, Eric; Ucakar, Bernard; Hubert, Philippe; Préat, Véronique

2010-02-05

An HPLC/UV method was first optimized for the separation and quantitation of human acylated and unacylated (or des-acyl) ghrelin from aqueous solutions. This method was validated by an original approach using accuracy profiles based on tolerance intervals for the total error measurement. The concentration range that achieved adequate accuracy extended from 1.85 to 59.30microM and 1.93 to 61.60microM for acylated and unacylated ghrelin, respectively. Then, optimal temperature, pH and buffer for sample storage were determined. Unacylated ghrelin was found to be stable in all conditions tested. At 37 degrees C acylated ghrelin was stable at pH 4 but unstable at pH 7.4, the main degradation product was unacylated ghrelin. Finally, this validated HPLC/UV method was used to evaluate the binding of acylated and unacylated ghrelin to liposomes.

Measurement of H2S in vivo and in vitro by the monobromobimane method

PubMed Central

Shen, Xinggui; Kolluru, Gopi K.; Yuan, Shuai; Kevil, Christopher

2015-01-01

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with RP-HPLC. The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide-dibimane. The resultant fluorescent sulfide-dibimane (SDB) is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels. PMID:25725514

Measurement of H2S in vivo and in vitro by the monobromobimane method.

PubMed

Shen, Xinggui; Kolluru, Gopi K; Yuan, Shuai; Kevil, Christopher G

2015-01-01

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels. © 2015 Elsevier Inc. All rights reserved.

Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

PubMed

Cooper, A D; Stubbings, G W; Kelly, M; Tarbin, J A; Farrington, W H; Shearer, G

1998-07-03

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

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

PubMed

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

2017-09-01

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

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

PubMed

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

2003-09-24

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

Development of Modern Methods for Determination of Stabilizers in Propellants

DTIC Science & Technology

1996-04-01

powder will! . gtve information Jbout the history of this powder and an indication of its future: usefulness. In othdrs words, the determination of...been excluded in Tables I and II. According to Table I, in order to develop an HPLC method for DPA-stabilized powders , the products that should be... powders were determined by each country using its own HPLC method . The results are given in Table XVI. As indicated, the agreement between the two

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

PubMed Central

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

2015-01-01

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

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

DTIC Science & Technology

2012-08-01

subsequent chemical analysis (into acetonitrile for high-performance liquid chromatography [ HPLC ] analysis or hexane for gas chromatography [GC... analysis ) is rapid and complete. In this work, PAHs were analyzed by Waters 2795 HPLC with fluorescent detection (USEPA Method 8310) and PCBs were...detection limits by direct water injection versus SPME with PDMS and coefficient of variation and correlation coefficient for SPME. Analysis by HPLC

Erroneous HbA1c results in a patient with elevated HbC and HbF.

PubMed

Adekanmbi, Joy; Higgins, Trefor; Rodriguez-Capote, Karina; Thomas, Dylan; Winterstein, Jeffrey; Dixon, Tara; Gifford, Jessica L; Krause, Richard; Venner, Allison A; Clarke, Gwen; Estey, Mathew P

2016-11-01

HbA1c is used in the diagnosis and monitoring of diabetes mellitus (DM). Interference from hemoglobin variants is a well-described phenomenon, particularly with HPLC-based methods. While immunoassays may generate more reliable HbA1c results in the presence of some variants, these methods are susceptible to negative interference from high concentrations of HbF. We report a case where an accurate HbA1c result could not be obtained by any available method due to the presence of a compound hemoglobinopathy. HbA1c was measured by HPLC, immunoassay, and capillary electrophoresis. Hemoglobinopathy investigation consisted of a CBC, hemoglobin fractionation by HPLC and electrophoresis, and molecular analysis. HbA1c analysis by HPLC and capillary electrophoresis gave no result. Analysis by immunoassay yielded HbA1c results of 5.9% (Siemens DCA 2000+) and 5.1% (Roche Integra), which were inconsistent with other markers of glycemic control. Hemoglobinopathy investigation showed HbC with the hereditary persistence of fetal hemoglobin-2 Ghana deletion. Reliable HbA1c results may be unobtainable in the presence of some hemoglobinopathies. HPLC and capillary electrophoresis alerted the laboratory to the presence of an unusual hemoglobinopathy. Immunoassays generated falsely low results without warning, which could lead to missed diagnoses and under treatment of patients with DM. Copyright © 2016 Elsevier B.V. All rights reserved.

Identification of phenanthrene derivatives in Aerides rosea (Orchidaceae) using the combined systems HPLC-ESI-HRMS/MS and HPLC-DAD-MS-SPE-UV-NMR.

PubMed

Cakova, Veronika; Urbain, Aurélie; Antheaume, Cyril; Rimlinger, Nicole; Wehrung, Patrick; Bonté, Frédéric; Lobstein, Annelise

2015-01-01

In our continued efforts to contribute to the general knowledge on the chemical diversity of orchids, we have decided to focus our investigations on the Aeridinae subtribe. Following our previous phytochemical study of Vanda coerulea, which has led to the identification of phenanthrene derivatives, a closely related species, Aerides rosea Lodd. ex Lindl. & Paxton, was chosen for investigation. To identify new secondary metabolites, and to avoid isolation of those already known, by means of the combined systems HPLC-DAD(diode-array detector) with high-resolution tandem mass spectrometry (HRMS/MS) and HPLC-DAD-MS-SPE(solid-phase extraction)-UV-NMR. A dereplication strategy was developed using a HPLC-DAD-HRMS/MS targeted method and applied to fractions from A. rosea stem extract. Characterisation of unknown minor compounds was then performed using the combined HPLC-DAD-MS-SPE-UV-NMR system. The dereplication method allowed the characterisation of four compounds (gigantol, imbricatin, methoxycoelonin and coelonin), previously isolated from Vanda coerulea stem extract. The analyses of two fractions permitted the identification of five additional minor constituents including one phenanthropyran, two phenanthrene and two dihydrophenanthrene derivatives. The full set of NMR data of each compound was obtained from microgram quantities. Nine secondary metabolites were characterised in A. rosea stems, utilising HPLC systems combined with high-resolution analytical systems. Two of them are newly described phenanthrene derivatives: aerosanthrene (5-methoxyphenanthrene-2,3,7-triol) and aerosin (3-methoxy-9,10-dihydro-2,5,7-phenanthrenetriol). Copyright © 2014 John Wiley & Sons, Ltd.

A qualitative and quantitative HPTLC densitometry method for the analysis of cannabinoids in Cannabis sativa L.

PubMed

Fischedick, Justin T; Glas, Ronald; Hazekamp, Arno; Verpoorte, Rob

2009-01-01

Cannabis and cannabinoid based medicines are currently under serious investigation for legitimate development as medicinal agents, necessitating new low-cost, high-throughput analytical methods for quality control. The goal of this study was to develop and validate, according to ICH guidelines, a simple rapid HPTLC method for the quantification of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and qualitative analysis of other main neutral cannabinoids found in cannabis. The method was developed and validated with the use of pure cannabinoid reference standards and two medicinal cannabis cultivars. Accuracy was determined by comparing results obtained from the HTPLC method with those obtained from a validated HPLC method. Delta(9)-THC gives linear calibration curves in the range of 50-500 ng at 206 nm with a linear regression of y = 11.858x + 125.99 and r(2) = 0.9968. Results have shown that the HPTLC method is reproducible and accurate for the quantification of Delta(9)-THC in cannabis. The method is also useful for the qualitative screening of the main neutral cannabinoids found in cannabis cultivars.

Targeted liquid chromatography tandem mass spectrometry to quantitate wheat gluten using well-defined reference proteins.

PubMed

Schalk, Kathrin; Koehler, Peter; Scherf, Katharina Anne

2018-01-01

Celiac disease (CD) is an inflammatory disorder of the upper small intestine caused by the ingestion of storage proteins (prolamins and glutelins) from wheat, barley, rye, and, in rare cases, oats. CD patients need to follow a gluten-free diet by consuming gluten-free products with gluten contents of less than 20 mg/kg. Currently, the recommended method for the quantitative determination of gluten is an enzyme-linked immunosorbent assay (ELISA) based on the R5 monoclonal antibody. Because the R5 ELISA mostly detects the prolamin fraction of gluten, a new independent method is required to detect prolamins as well as glutelins. This paper presents the development of a method to quantitate 16 wheat marker peptides derived from all wheat gluten protein types by liquid chromatography tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring mode. The quantitation of each marker peptide in the chymotryptic digest of a defined amount of the respective reference wheat protein type resulted in peptide-specific yields. This enabled the conversion of peptide into protein type concentrations. Gluten contents were expressed as sum of all determined protein type concentrations. This new method was applied to quantitate gluten in wheat starches and compared to R5 ELISA and gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD), which resulted in a strong correlation between LC-MS/MS and the other two methods.

Spectra resolution for simultaneous spectrophotometric determination of lamivudine and zidovudine components in pharmaceutical formulation of human immunodeficiency virus drug based on using continuous wavelet transform and derivative transform techniques.

PubMed

Sohrabi, Mahmoud Reza; Tayefeh Zarkesh, Mahshid

2014-05-01

In the present paper, two spectrophotometric methods based on signal processing are proposed for the simultaneous determination of two components of an anti-HIV drug called lamivudine (LMV) and zidovudine (ZDV). The proposed methods are applied to synthetic binary mixtures and commercial pharmaceutical tablets without the need for any chemical separation procedures. The developed methods are based on the application of Continuous Wavelet Transform (CWT) and Derivative Spectrophotometry (DS) combined with the zero cross point technique. The Daubechies (db5) wavelet family (242 nm) and Dmey wavelet family (236 nm) were found to give the best results under optimum conditions for simultaneous analysis of lamivudine and zidovudine, respectively. In addition, the first derivative absorption spectra were selected for the determination of lamivudine and zidovudine at 266 nm and 248 nm, respectively. Assaying various synthetic mixtures of the components validated the presented methods. Mean recovery values were found to be between 100.31% and 100.2% for CWT and 99.42% and 97.37% for DS, respectively for determination of LMV and ZDV. The results obtained from analyzing the real samples by the proposed methods were compared to the HPLC reference method. One-way ANOVA test at 95% confidence level was applied to the results. The statistical data from comparing the proposed methods with the reference method showed no significant differences. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed Central

2013-01-01

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

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

PubMed

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

2017-01-06

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

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

PubMed Central

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

2012-01-01

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

Detection, characterization and identification of phenolic acids in Danshen using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry.

PubMed

Liu, Ai-Hua; Guo, Hui; Ye, Min; Lin, Yan-Hua; Sun, Jiang-Hao; Xu, Man; Guo, De-An

2007-08-17

By using HPLC-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MS(n)) in negative ion mode, we have analyzed the fragmentation pathways of 11 phenolic acids which were isolated from Danshen. Then the extract of Danshen was analyzed, and a total of 42 phenolic acids, including sixteen new minor constituents, were identified or tentatively identified for the first time. A new solid-phase extraction (SPE) method, new HPLC separation method, new liquid chromatography (LC)-MS and LC-MS(n) (n=3-5) data and proposed fragmentation pathways, LC retention time for phenolic acids are reported.

Analysis and identification of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous by high-performance liquid chromatography.

PubMed

Lu, Mingbo; Zhang, Yang'e; Zhao, Chunfang; Zhou, Pengpeng; Yu, Longjiang

2010-01-01

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3'-dihydroxy-beta,psi-carotene-4,4'-dione (DCD) is produced through gamma-carotene and torulene.

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

PubMed

Rüter, J; Raczek, D I

1992-06-01

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

Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

NASA Astrophysics Data System (ADS)

Barton, Janice S.; Tang, Chung-Fei; Reed, Steven S.

2000-02-01

Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

[Capillary HbA1c determination on type 2 diabetes patients in a primary health centre].

PubMed

Font, María Teresa Carrera; Brichs, María Claustre Solé; Álvarez, María Clara Sala; Olivella, Jose María Navarro; Turó, Josefina Servent; Fernández, María Pilar Felipe

2011-10-01

To determine the reliability and practicability of the point-of-care- test (POCT) analyzer, Afinion, for capillary HbA1c testing. To assess the benefits of its implementation on the intra-annual follow up of type 2 diabetic patients. Descriptive cross-sectional study. Analytical validation of the Afinion reader. Primary Health Care (CAP Carmel and Bon Pastor Clinic Laboratory). A total of 94 type 2 diabetic patients selected according to their previous HbA1c value. We performed one capillary puncture and one venous extraction on each visit. The capillary sample was assessed in real time on the Afinion in the Primary Health Care Centre and the venous sample was sent to Bon Pastor Clinic Laboratory for assessment on an Afinion analyzer and by a high performance liquid chromatrography (HPLC) reference method. Practicability was assesses by both by the operators of the Afinion and the patients using an 11 question questionnaire. The efficiency in terms of process timings was also evaluated. Intra-serial coefficient of variation (CV) was lower than 1% and inter-serial lower than 3%. The regression analysis showed: Afinion capillary sample=0.95 Afinion venous+0.21. No systematic or proportional error was detected in the 95% confidence interval (95% CI). The comparison between venous HPLC and Afinion showed: Afinion capillary sample=0.80 HPLC+1.14. A statistically significant difference was shown for these values at the 95% CI. Practicability was valued by users from 7 to 9.2 (professionals) and from 7.7 to 9.2 (patients). Implementation of the Afinion capillary method for intra-annual testing in follow up of diabetic patients could result in the saving of 600-900 professional hours/year. Afinion seems to be a good choice for the intra-annual determination of HbA1c when compared to the traditional process due to its accessibility, practicability and efficiency. Professionals should know the limitations of the POCT method in order to consider the validity of the results. Copyright © 2010 Elsevier España, S.L. All rights reserved.

Rapid-gradient HPLC method for measuring drug interactions with immobilized artificial membrane: comparison with other lipophilicity measures.

PubMed

Valko, K; Du, C M; Bevan, C D; Reynolds, D P; Abraham, M H

2000-08-01

A fast-gradient high-performance liquid chromatographic (HPLC) method has been suggested to characterize the interactions of drugs with an immobilized artificial membrane (IAM). With a set of standards, the gradient retention times can be converted to Chromatographic Hydrophobicity Index values referring to IAM chromatography (CHI(IAM)) that approximates an acetonitrile concentration with which the equal distribution of compound can be achieved between the mobile phase and IAM. The CHI(IAM) values are more suitable for interlaboratory comparison and for high throughput screening of new molecular entities than the log k(IAM) values (isocratic retention factor on IAM). The fast-gradient method has been validated against the isocratic log k(IAM) values using the linear free energy relationship solvation equations based on the data from 48 compounds. The compound set was selected to provide a wide range and the least cross-correlation between the molecular descriptors in the solvation equation: (2) where SP is a solute property (e.g., logarithm of partition coefficients, reversed-phase (RP)-HPLC retention parameters, such as log k, log k(w), etc.) and the explanatory variables are solute descriptors as follows: R(2) is an excess molar refraction that can be obtained from the measured refractive index of a compound, pi(2)(H) is the solute dipolarity/polarizability, summation operatoralpha(2)(H) and summation operatorbeta(2)(0) are the solute overall or effective hydrogen-bond acidity and basicity, respectively, and V(x) is the McGowan characteristic volume (in cm(3)/100 mol) that can be calculated for any solute simply from molecular structure using a table of atomic constants. It was found that the relative constants of the solvation equation were very similar for the CHI(IAM) and for the log k(IAM). The IAM lipophilicity scale was quite similar to the octanol/water lipophilicity scale for neutral compounds. The effect of charge on the interaction with IAM was studied by varying the mobile phase pH. Copyright 2000 Wiley-Liss, Inc.

Identification of geometrical isomers and comparison of different isomeric samples of astaxanthin.

PubMed

Qiu, Dan; Wu, Yue-Chan; Zhu, Wen-Li; Yin, Hong; Yi, Long-Tao

2012-09-01

A high-performance liquid chromatographic (HPLC) analysis system for isomeric astaxanthin was developed. The separation system consisted of a C(30) column and an elution system of methanol/MTBE/water/dichloromethane (77:13:8:2, v/v/v/v). Using the combination of HPLC diode array detector and HPLC atmospheric pressure chemical ionization mass spectrometry, 11 geometrical isomers and 4 epoxides of astaxanthin were successfully identified. Referred to crystal, only isomerization with different degrees was found for solvent dissolving and iodine catalysis, while melting of astaxanthin caused isomerization, slight oxidation, and more noticeable polymerization confirmed by gel permeation chromatography. Chemical changes in isomeric samples all caused a decrease in UV content. The vibrational spectra (infrared and Raman) showed that epoxide was the only new functional group generated for melting. Changes of several key bands and formations of new bands were found in iodine catalysis and melting samples because of isomerization. Practical Application:  Eleven geometrical isomers and 4 epoxides, which were normally generated for solvent dissolving, iodine catalysis, and melting of astaxanthin, have been identified by C(30) -HPLC-MS technology. Furthermore, different samples were measured by gel permeation chromatography, UV, infrared, and Raman, based on the analysis of messages, the effect of each processing was well understood. © 2012 Institute of Food Technologists®

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

Determination of blood sirolimus concentrations in liver and kidney transplant recipients using the Innofluor® fluorescence polarization immunoassay: Comparison with the microparticle enzyme immunoassay and high-performance liquid chromatography-ultraviolet method

PubMed Central

Bouzas, Lorena; Hermida, Jesús

2009-01-01

Background Although high-performance liquid chromatography (HPLC) is the method of choice for blood sirolimus determination, the microparticle enzyme immunoassay (MEIA) run on the IMx® analyser is widely used in therapeutic monitoring of this immunosuppressant agent. The aim of our study was to evaluate the possible determination of sirolimus using the fluorescence polarization immunoassay (FPIA) commercialized for everolimus quantification. Methods Sirolimus concentrations were determined in whole-blood samples from liver and kidney transplant recipients using the Innofluor® Certican® FPIA (Seradyn Inc.) run on a TDx® analyser (Abbott Laboratories), Sirolimus MEIA run on an IMx® analyser (Abbott Laboratories), and HPLC (UV detection) methods. Results The Innofluor® FPIA has a similar cross-reactivity with everolimus and sirolimus, and the within- and between-run coefficients of variation obtained for sirolimus determination were 2.7%–13.3%. In analysing different blood samples from liver and kidney transplant patients the linear regressions obtained were: FPIA = 1.12 HPLC + 0.43 (n=104, r=0.874), MEIA = 1.14 HPLC (n=146, r=0.892), and FPIA = 1.00 MEIA + 0.29 (n=106, r=0.941). Better correlation coefficients were obtained between the methods in the liver transplant samples (r≥0.900) than in the kidney transplant samples (r≥0.849). No significant effect was found for sirolimus clearance or the blood hematocrit on the relationship between the results produced by both immunoassays and HPLC. Conclusion The Innofluor® FPIA is a valid alternative with an analogous performance to the MEIA for the therapeutic monitoring of sirolimus. PMID:19242874

Application of Attenuated Total Reflectance-Fourier Transformed Infrared (ATR-FTIR) Spectroscopy To Determine the Chlorogenic Acid Isomer Profile and Antioxidant Capacity of Coffee Beans.

PubMed

Liang, Ningjian; Lu, Xiaonan; Hu, Yaxi; Kitts, David D

2016-01-27

The chlorogenic acid isomer profile and antioxidant activity of both green and roasted coffee beans are reported herein using ATR-FTIR spectroscopy combined with chemometric analyses. High-performance liquid chromatography (HPLC) quantified different chlorogenic acid isomer contents for reference, whereas ORAC, ABTS, and DPPH were used to determine the antioxidant activity of the same coffee bean extracts. FTIR spectral data and reference data of 42 coffee bean samples were processed to build optimized PLSR models, and 18 samples were used for external validation of constructed PLSR models. In total, six PLSR models were constructed for six chlorogenic acid isomers to predict content, with three PLSR models constructed to forecast the free radical scavenging activities, obtained using different chemical assays. In conclusion, FTIR spectroscopy, coupled with PLSR, serves as a reliable, nondestructive, and rapid analytical method to quantify chlorogenic acids and to assess different free radical-scavenging capacities in coffee beans.

Assessment of powder blend uniformity: Comparison of real-time NIR blend monitoring with stratified sampling in combination with HPLC and at-line NIR Chemical Imaging.

PubMed

Bakri, Barbara; Weimer, Marco; Hauck, Gerrit; Reich, Gabriele

2015-11-01

Scope of the study was (1) to develop a lean quantitative calibration for real-time near-infrared (NIR) blend monitoring, which meets the requirements in early development of pharmaceutical products and (2) to compare the prediction performance of this approach with the results obtained from stratified sampling using a sample thief in combination with off-line high pressure liquid chromatography (HPLC) and at-line near-infrared chemical imaging (NIRCI). Tablets were manufactured from powder blends and analyzed with NIRCI and HPLC to verify the real-time results. The model formulation contained 25% w/w naproxen as a cohesive active pharmaceutical ingredient (API), microcrystalline cellulose and croscarmellose sodium as cohesive excipients and free-flowing mannitol. Five in-line NIR calibration approaches, all using the spectra from the end of the blending process as reference for PLS modeling, were compared in terms of selectivity, precision, prediction accuracy and robustness. High selectivity could be achieved with a "reduced" approach i.e. API and time saving approach (35% reduction of API amount) based on six concentration levels of the API with three levels realized by three independent powder blends and the additional levels obtained by simply increasing the API concentration in these blends. Accuracy and robustness were further improved by combining this calibration set with a second independent data set comprising different excipient concentrations and reflecting different environmental conditions. The combined calibration model was used to monitor the blending process of independent batches. For this model formulation the target concentration of the API could be achieved within 3 min indicating a short blending time. The in-line NIR approach was verified by stratified sampling HPLC and NIRCI results. All three methods revealed comparable results regarding blend end point determination. Differences in both mean API concentration and RSD values could be attributed to differences in effective sample size and thief sampling errors. This conclusion was supported by HPLC and NIRCI analysis of tablets manufactured from powder blends after different blending times. In summary, the study clearly demonstrates the ability to develop efficient and robust quantitative calibrations for real-time NIR powder blend monitoring with a reduced set of powder blends while avoiding any bias caused by physical sampling. Copyright © 2015 Elsevier B.V. All rights reserved.

Validation of a multi-analyte HPLC-DAD method for determination of uric acid, creatinine, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid and 2-methylhippuric acid in human urine.

PubMed

Remane, Daniela; Grunwald, Soeren; Hoeke, Henrike; Mueller, Andrea; Roeder, Stefan; von Bergen, Martin; Wissenbach, Dirk K

2015-08-15

During the last decades exposure sciences and epidemiological studies attracts more attention to unravel the mechanisms for the development of chronic diseases. According to this an existing HPLC-DAD method for determination of creatinine in urine samples was expended for seven analytes and validated. Creatinine, uric acid, homovanillic acid, niacinamide, hippuric acid, indole-3-acetic acid, and 2-methylhippuric acid were separated by gradient elution (formate buffer/methanol) using an Eclipse Plus C18 Rapid Resolution column (4.6mm×100mm). No interfering signals were detected in mobile phase. After injection of blank urine samples signals for the endogenous compounds but no interferences were detected. All analytes were linear in the selected calibration range and a non weighted calibration model was chosen. Bias, intra-day and inter-day precision for all analytes were below 20% for quality control (QC) low and below 10% for QC medium and high. The limits of quantification in mobile phase were in line with reported reference values but had to be adjusted in urine for homovanillic acid (45mg/L), niacinamide 58.5(mg/L), and indole-3-acetic acid (63mg/L). Comparison of creatinine data obtained by the existing method with those of the developed method showing differences from -120mg/L to +110mg/L with a mean of differences of 29.0mg/L for 50 authentic urine samples. Analyzing 50 authentic urine samples, uric acid, creatinine, hippuric acid, and 2-methylhippuric acid were detected in (nearly) all samples. However, homovanillic acid was detected in 40%, niacinamide in 4% and indole-3-acetic acid was never detected within the selected samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and validation of a GC-C-IRMS method for the confirmation analysis of pseudo-endogenous glucocorticoids in doping control.

PubMed

de la Torre, Xavier; Curcio, Davide; Colamonici, Cristiana; Molaioni, Francesco; Cilia, Marta; Botrè, Francesco

2015-01-01

Glucocorticoids are included in the S9 section of the World Anti-doping Agency (WADA) prohibited list international standard. Some among them are pseudo-endogenous steroids, like cortisol and cortisone, which present the same chemical structure as endogenously produced steroids. We are proposing an analytical method based on gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) which allows discrimination between endogenous and synthetic origin of the urinary metabolites of the pseudo-endogenous glucocorticoids. A preliminary purification treatment by high-performance liquid chromatography (HPLC) of the target compounds (TC) (i.e., cortisol, tetrahydrocortisone (THE) 5α-tetrahydrocortisone (aTHE), tetrahydrocortisol (THF), and 5α-tetrahydrocortisol (aTHF)) allows collection of extracts with adequate purity for the subsequent analysis by IRMS. A population of 40 urine samples was analyzed for the TC and for the endogenous reference compounds (ERC: i.e., 11-desoxy-tetrahydrocortisol (THS) or pregnanediol). For each sample, the difference between the delta values of the ERCs and TCs (Δδ values) were calculated and based on that, some decision limits for atypical findings are proposed. The limits are below 3% units except for cortisol. The fit to purpose of the method has been confirmed by the analysis of urine samples collected in two patients under treatment with 25 mg of cortisone acetate (p.o). The samples showed Δδ values higher than 3 for at least 24 h following administration depending on the TC considered. The method can easily be integrated into existing procedures already used for the HPLC purification and IRMS analysis of pseudo-endogenous steroids with androgenic/anabolic activity. Copyright © 2015 John Wiley & Sons, Ltd.

Structural characterization of constituents with molecular diversity in fractions from Lysidice brevicalyx by liquid chromatography/diode-array detection/electrospray ionization tandem mass spectrometry and liquid chromatography/nuclear magnetic resonance.

PubMed

Qu, Jing; Hu, You-cai; Li, Jian-bei; Wang, Ying-hong; Zhang, Jin-lan; Abliz, Zeper; Yu, Shi-shan; Liu, Yun-bao

2008-01-01

A combination of electrospray ionization tandem mass spectrometry with high-performance liquid chromatography (HPLC/ESI-MSn), and hyphenation of liquid chromatography to nuclear magnetic resonance spectroscopy (HPLC/NMR), have been extensively utilized for on-line analysis of natural products, analyzing metabolite and drug impurity. In our last paper, we reported an on-line analytical method for structural identification of trace alkaloids in the same class. However, the structural types of the constituents in plants were various, such as flavanoids, terpenoids and steroids. It is important to establish an effective analytical method for on-line structural identification of constituents with molecular diversity in extracts of plants. So, in the present study, the fragmentation patterns of some isolated stilbenes, phloroglucinols and flavanoids from Lysidice rhodostegia were investigated by ESI-MSn. Their fragmentation rules and UV characteristics are summarized, and the relationship between the spectral characteristics, rules and the structures is described. According to the fragmentation rules, NMR and UV spectral characteristics, 24 constituents of different types in the fractions from L. brevicalyx of the same genus were structurally characterized on the basis of HPLC/HRMS, HPLC-UV/ESI-MSn, HPLC/1H NMR and HPLC/1H-1H COSY rapidly. Of these, six (10, 13, 14, 16, 17 and 23) are new compounds and all of them are reported from L. brevicalyx for the first time. The aim is to develop an effective analytical method for on-line structural identification of natural products with molecular diversity in plants, and to guide the rapid and direct isolation of novel compounds by chemical screening.

Development of a Thiolysis HPLC Method for the Analysis of Procyanidins in Cranberry Products.

PubMed

Gao, Chi; Cunningham, David G; Liu, Haiyan; Khoo, Christina; Gu, Liwei

2018-03-07

The objective of this study was to develop a thiolysis HPLC method to quantify total procyanidins, the ratio of A-type linkages, and A-type procyanidin equivalents in cranberry products. Cysteamine was utilized as a low-odor substitute of toluene-α-thiol for thiolysis depolymerization. A reaction temperature of 70 °C and reaction time of 20 min, in 0.3 M of HCl, were determined to be optimum depolymerization conditions. Thiolytic products of cranberry procyanidins were separated by RP-HPLC and identified using high-resolution mass spectrometry. Standards curves of good linearity were obtained on thiolyzed procyanidin dimer A2 and B2 external standards. The detection and quantification limits, recovery, and precision of this method were validated. The new method was applied to quantitate total procyanidins, average degree of polymerization, ratio of A-type linkages, and A-type procyanidin equivalents in cranberry products. Results showed that the method was suitable for quantitative and qualitative analysis of procyanidins in cranberry products.

Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC. 2. Application for the analysis of Loy Yang coal oxidation products

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

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

In order to develop separation processes and analytical methods for aromatic carboxylic acids for the coal oxidation products, the separation behavior of aromatic carboxylic acids on a reversed-phase HPLC using eluent containing quaternary ammonium salt was optimized using the solvent gradient method. This method was applied for the analysis of Loy Yang coal oxidation products. It was confirmed that the analytical data using this method were consistent with those determined using gas chromatography.

Stir bar sorptive extraction combined with high performance liquid chromatography-ultraviolet/inductively coupled plasma mass spectrometry for analysis of thyroxine in urine samples.

PubMed

Fan, Wenying; Mao, Xiangju; He, Man; Chen, Beibei; Hu, Bin

2013-11-29

tIn this work, polyethyleneglycol (PEG)/hydroxyl polydimethylsiloxane (OH-PDMS)/γ -mercaptopropyltrimethoxysilane (γ -MPTS) coated stir bar was prepared by sol–gel process and its extraction performance for the extraction of amphoteric thyroxines (3,3',5,5'-tetraiodothyronin, T(4); 3,3',5-triiodothyronine, T(3); reversed-3,3',5-triiodothyronine, rT(3)) and their metabolite (3,5-diiodothyronine,T2) was studied. The preparation reproducibility of PEG/OH-PDMS/γ -MPTS coated stir bar was investigated, and the relative standard deviations (RSDs) in the same batch and among different batches were 3.3–14.3% (n = 5) and 7.7–16.6% (n = 3), respectively. The prepared PEG/OH-PDMS/γ -MPTS coated stir bar could be reused for more than 20 times. Based on this fact, a novel method of stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV)and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of target thyroxinesin human urine samples was developed. The influencing factors of SBSE, such as sample pH, extraction time, stirring rate, salt effect, desorption solution and desorption time, were studied in detail, and the analytical performance of the proposed method was evaluated under the optimized conditions. The enrichment factors (EFs) of the developed method for four target thyroxines were in the range of 14.9–70.4(theoretical enrichment factor was 100). The RSDs were ranging from 4.0% to 13.8% for SBSE-HPLC-UV (c = 25 μg/L, n = 6) and from 3.7% to 6.1% for SBSE-HPLC-ICP-MS (c = 0.5 μg/L, n = 5). The linear range obtained by SBSE-HPLC-UV was 2–500 μg/L for T(2)and 5–500 μg/L for rT3, T(3)and T(4), with correlation coefficients (r) ranging from 0.9957 to 0.9998, respectively, while the linear range obtained by SBSE-HPLC-ICP-MS was 0.05–500 μg/L for T(2) and rT(3), 0.10–200 μg/L for T(3) and 0.05–200 μg/L for T(4)with r ranging from 0.9979 to 0.9998, respectively. The limits of detection (LODs) for the target thyroxines were 0.60–2.20 μg/L for SBSE-HPLC-UV and 0.0071–0.0355 μg/L SBSE-HPLC-ICP-MS, respectively. The developed method was applied for the determination of target thyroxines in urine samples, and the recovery for the spiking samples obtained by SBSE-HPLC-UV was in the range of 81.6–137.6% for human urine,while the recovery for the spiking urine samples obtained by SBSE-HPLC-ICP-MS were in the range of 72.0–121.5%.

Qualitative and quantitative determination of yohimbine in authentic yohimbe bark and in commercial aphrodisiacs by HPLC-UV-API/ MS methods.

PubMed

Zanolari, Boris; Ndjoko, Karine; Ioset, Jean-Robert; Marston, Andrew; Hostettmann, Kurt

2003-01-01

The development and validation of a rapid qualitative and quantitative method based on an HPLC-UV-MS technique with atmospheric pressure chemical ionisation and electrospray ionisation for the analysis of yohimbine in a number of commercial aphrodisiac products is reported. HPLC with multiple-stage mass spectrometry experiments allowed the identification of the target compound and increased the selectivity of complex analyses such as those involved with multi-botanical preparations. The precision and the robustness of the method were improved by the use of two internal standards: codeine for UV detection and deuterium-labelled yohimbine for MS detection. Twenty commercial aphrodisiac preparations were analysed and the amount of yohimbine measured and expressed as the maximal dose per day suggested on product labels ranged from 1.32 to 23.16 mg.

[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.

New model for prediction binary mixture of antihistamine decongestant using artificial neural networks and least squares support vector machine by spectrophotometry method

NASA Astrophysics Data System (ADS)

Mofavvaz, Shirin; Sohrabi, Mahmoud Reza; Nezamzadeh-Ejhieh, Alireza

2017-07-01

In the present study, artificial neural networks (ANNs) and least squares support vector machines (LS-SVM) as intelligent methods based on absorption spectra in the range of 230-300 nm have been used for determination of antihistamine decongestant contents. In the first step, one type of network (feed-forward back-propagation) from the artificial neural network with two different training algorithms, Levenberg-Marquardt (LM) and gradient descent with momentum and adaptive learning rate back-propagation (GDX) algorithm, were employed and their performance was evaluated. The performance of the LM algorithm was better than the GDX algorithm. In the second one, the radial basis network was utilized and results compared with the previous network. In the last one, the other intelligent method named least squares support vector machine was proposed to construct the antihistamine decongestant prediction model and the results were compared with two of the aforementioned networks. The values of the statistical parameters mean square error (MSE), Regression coefficient (R2), correlation coefficient (r) and also mean recovery (%), relative standard deviation (RSD) used for selecting the best model between these methods. Moreover, the proposed methods were compared to the high- performance liquid chromatography (HPLC) as a reference method. One way analysis of variance (ANOVA) test at the 95% confidence level applied to the comparison results of suggested and reference methods that there were no significant differences between them.

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

PubMed

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

2012-07-01

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

MOF-5(Zn)-Fe2O4 nanocomposite based magnetic solid-phase microextraction followed by HPLC-UV for efficient enrichment of colchicine in root of colchicium extracts and plasma samples.

PubMed

Bahrani, Sonia; Ghaedi, Mehrorang; Dashtian, Kheibar; Ostovan, Abbas; Mansoorkhani, Mohammad Javad Khoshnood; Salehi, Amin

2017-11-01

In present work, facile method is developed for determination of colchicine in human plasma sample, autumn and spring root of colchicium extracts by ultrasound assisted dispersive magnetic solid phase microextraction followed by HPLC-UV method (UAD-MSPME-HPLC-UV). Magnetic (Fe 2 O 4 -nanoparticles) metal organic framework-5, (MOF-5(Zn)-Fe 2 O 4 NPs) was synthesized by dispersing MOF-5 and Fe(NO 3 ) 3 .9H 2 O in ethylene glycol (as capping agent) and NaOH (pH adjustment agent) by hydrothermal method. The prepared sorbent was characterized via XRD and SEM analysis and applied as magnetic solid phase in UAD-MSPME-HPLC-UV method. In this method, colchicine molecules were sorbed on MOF-5(Zn)-Fe 2 O 4 NPs sorbent by various mechanisms like ion exchange, hydrogen bonding and electrostatic, ᴨ-ᴨ, hard-hard and dipole-ion interaction followed by exposing sonication waves as incremental mass transfer agent and then the sorbent was separated from the sample matrix by an external magnetic fields. Subsequently, accumulated colchicine were eluted by small volume of desorption organic solvent. Influence of operational variables such as MOF-5(Zn)-Fe 2 O 4 NPs mass, volume of extracting solvent and sonication time on response property (recovery) were studied and optimized by central composite design (CCD) combined with desirability function (DF) approach. Under optimum condition, the method has wide linear calibration rang (0.5-1700ngmL -1 ) with reasonable detection limit (0.13ngmL -1 ) and R 2 =0.9971. Finally, the UAD-MSPME-HPLC-UV method was successfully applied for determination of colchicine autumn and spring root of colchicium extracts and plasma samples. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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

2002-10-15

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

The integrated quality assessment of Chinese commercial dry red wine based on a method of online HPLC-DAD-CL combined with HPLC-ESI-MS.

PubMed

Yu, Hai-Xiang; Sun, Li-Qiong; Qi, Jin

2014-07-01

To apply an integrated quality assessment strategy to investigate the quality of multiple Chinese commercial dry red wine samples. A comprehensive method was developed by combining a high performance liquid chromatography-diode array detector-chemiluminescence (HPLC-DAD-CL) online hyphenated system with an HPLC-ESI-MS technique. Chromatographic and H2O2-scavenging active fingerprints of thirteen batches of different, commercially available Chinese dry red wine samples were obtained and analyzed. Twenty-five compounds, including eighteen antioxidants were identified and evaluated. The dominant and characteristic antioxidants in the samples were identified. The relationships between antioxidant potency and the cultivated variety of grape, producing area, cellaring period, and trade mark are also discussed. The results provide the feasibility for an integrated quality assessment strategy to be efficiently and objectively used in quality (especially antioxidant activity) assessment and identification of dry red wine. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2007-09-01

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

[HPLC method to determine DEHP released into blood from a disposable extracorporeal circulation tube].

PubMed

Liu, Xi; Yu, Jingjing; Li, Shen; Wang, Hong; Liu, Jiaxin

2013-08-01

We used blood as leaching medium, simulating clinical operation under maximum condition, to develop Liquid-phase extraction- High Performance Liquid Chromatography (HPLC) method for determination of plasticizer Di-(2-ethylhexyl)phthalate (DEHP) released from Disposable Extracorporeal Circulation Tube in order to lay the foundation of risk analysis of this product. The characteristic wavelength of DEHP in methanol was detected. Acetonitrile was added to the leaching blood in proportion and extracted DEHP from blood. The methodology for HPLC to quantify DEHP was established and the DEHP amount released from this disposable extracorporeal circulation tube was measured. The experiments showed good results as follows. The characteristic wavelength of DEHP was 272nm. The concentration of DEHP (5-250 microg/mL) kept good linear relationship with peak area (r=0.9999). Method sensitivity was 1 microg/mL. Precisions showed RSD<5%. The adding standard extraction Recovery Rates of 25, 100 and 250 microg DEHP standard were 61.91 +/- 3.32)%, (69.38 +/- 0.55)% and (68.47 +/- 1.15)%. The DEHP maximum amounts released from 3 sets of this disposable extracorporeal circulation tube were 204.14, 106.30 and 165.34 mg/set. Our Liquid-phase Extraction-HPLC method showed high accuracy and precision, and relatively stable recovery rate. Its operation was also convenient.

Evaluation of phosphorus characterization in broiler ileal digesta, manure, and litter samples: (31)P-NMR vs. HPLC.

PubMed

Leytem, A B; Kwanyuen, P; Plumstead, P W; Maguire, R O; Brake, J

2008-01-01

Using 31-phosphorus nuclear magnetic resonance spectroscopy ((31)P-NMR) to characterize phosphorus (P) in animal manures and litter has become a popular technique in the area of nutrient management. To date, there has been no published work evaluating P quantification in manure/litter samples with (31)P-NMR compared to other accepted methods such as high performance liquid chromatography (HPLC). To evaluate the use of (31)P-NMR to quantify myo-inositol hexakisphosphate (phytate) in ileal digesta, manure, and litter from broilers, we compared results obtained from both (31)P-NMR and a more traditional HPLC method. The quantification of phytate in all samples was very consistent between the two methods, with linear regressions having slopes ranging from 0.94 to 1.07 and r(2) values of 0.84 to 0.98. We compared the concentration of total monoester P determined with (31)P-NMR with the total inositol P content determined with HPLC and found a strong linear relationship between the two measurements having slopes ranging from 0.91 to 1.08 and r(2) values of 0.73 to 0.95. This suggests that (31)P-NMR is a very reliable method for quantifying P compounds in manure/litter samples.

Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

PubMed

Katharopoulos, Efstathios; Touloupi, Katerina; Touraki, Maria

2016-08-01

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Simultaneous Speciation of Arsenic, Selenium, and Chromium by HPLC-ICP-MS

USGS Publications Warehouse

Wolf, Ruth E.; Morman, Suzette A.; Morrison, Jean M.; Lamothe, Paul J.

2008-01-01

An adaptation of an analytical method developed for chromium speciation has been utilized for the simultaneous determination of As(III), As(V), Se(IV), Se(VI), Cr(III), and Cr(VI) species using high performance liquid chromatography (HPLC) separation with ICP-MS detection. Reduction of interferences for the determination of As, Se, and Cr by ICP-MS is a major consideration for this method. Toward this end, a Dynamic Reaction Cell (DRC) ICP-MS system was used to detect the species eluted from the chromatographic column. A variety of reaction cell gases and conditions may be utilized, and the advantages and limitations of the gases tested to date will be presented and discussed. The separation and detection of the As, Se, and Cr species of interest can be achieved using the same chromatographic conditions in less than 2 minutes by complexing the Cr(III) with EDTA prior to injection on the HPLC column. Practical aspects of simultaneous speciation analysis will be presented and discussed, including issues with HPLC sample vial contamination, standard and sample contamination, species stability, and considerations regarding sample collection and preservation methods. The results of testing to determine the method's robustness to common concomitant element and anion effects will also be discussed. Finally, results will be presented using the method for the analysis of a variety of environmental and geological samples including waters, soil leachates and simulated bio-fluid leachates.

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.

Evaluation of multiple reaction monitoring cubed for the analysis of tachykinin related peptides in rat spinal cord using a hybrid triple quadrupole-linear ion trap mass spectrometer.

PubMed

Pailleux, Floriane; Beaudry, Francis

2014-02-01

Targeted peptide methods generally use HPLC-MS/MRM approaches. Although dependent on the instrumental resolution, interferences may occur while performing analysis of complex biological matrices. HPLC-MS/MRM(3) is a technique, which provides a significantly better selectivity, compared with HPLC-MS/MRM assay. HPLC-MS/MRM(3) allows the detection and quantitation by enriching standard MRM with secondary product ions that are generated within the linear ion trap. Substance P (SP) and neurokinin A (NKA) are tachykinin peptides playing a central role in pain transmission. The objective of this study was to verify whether HPLC-MS/MRM(3) could provide significant advantages over a more traditional HPLC-MS/MRM assay for the quantification of SP and NKA in rat spinal cord. The results suggest that reconstructed MRM(3) chromatograms display significant improvements with the nearly complete elimination of interfering peaks but the sensitivity (i.e. signal-to-noise ratio) was severely reduced. The precision (%CV) observed was between 3.5% and 24.1% using HPLC-MS/MRM and in the range of 4.3-13.1% with HPLC-MS/MRM(3), for SP and NKA. The observed accuracy was within 10% of the theoretical concentrations tested. HPLC-MS/MRM(3) may improve the assay sensitivity to detect difference between samples by reducing significantly the potential of interferences and therefore reduce instrumental errors. Copyright © 2014 Elsevier B.V. All rights reserved.

Simultaneous determination of wogonin, oroxylin a, schisandrin, paeoniflorin and emodin in rat serum by HPLC-MS/MS and application to pharmacokinetic studies.

PubMed

Chen, Minting; Wei, Suying; Luo, Chaohua; Chen, Feilong; Song, Shuai; Shen, Qun; Mo, Zhixian; Wei, Fenghuan

2017-10-01

Wogonin and oroxylin A in Scutellariae Radix, schisandrin in Chinensis Fructus, paeoniflorin in Moutan Cortex and emodin in Polygoni Cuspidate Rhizome et Radix are anti-inflammatory active compounds. A method for simultaneous determination of the five compounds in rat was developed and validated using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The separation was performed on a Symmetry C 18 column (4.6 × 50 mm, 3.5 μm) with acetonitrile and 0.1% formic acid aqueous solution as the mobile phases. The detection was performed using multiple-reaction monitoring with electrospray ionization source in positive-negative ion mode. The calibration curves showed good linearity (r ≥ 0.9955). The lower limit of quantification (LLOQ) was 5 ng/mL for wogonin and schisandrin, 10 ng/mL for oroxylin A and emodin, and 15 ng/mL for paeoniflorin, respectively. The relative standard deviations of intraday and interday precisions were <11.49 and 14.28%, respectively. The extraction recoveries and matrix effects were acceptable. The analytes were stable under the experiment conditions. The validated method has been successfully applied to pharmacokinetic studies of the five compounds in rats after oral administration of Hu-gan-kan-kang-yuan capsule. This paper would be a valuable reference for pharmacokinetic studies of Chinese medicine preparations containing the five compounds. Copyright © 2017 John Wiley & Sons, Ltd.

Preliminary Phytochemical Screening, Quantitative Analysis of Alkaloids, and Antioxidant Activity of Crude Plant Extracts from Ephedra intermedia Indigenous to Balochistan

PubMed Central

Jan, Syed Umer; Faridullah, Syed; Sherani, Samiullah; Jahan, Nusrat

2017-01-01

The aim of this study was to evaluate the antioxidant activity, screening the phytogenic chemical compounds, and to assess the alkaloids present in the E. intermedia to prove its uses in Pakistani folk medicines for the treatment of asthma and bronchitis. Antioxidant activity was analyzed by using 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. Standard methods were used for the identification of cardiac glycosides, phenolic compounds, flavonoids, anthraquinones, and alkaloids. High performance liquid chromatography (HPLC) was used for quantitative purpose of ephedrine alkaloids in E. intermedia. The quantitative separation was confirmed on Shimadzu 10AVP column (Shampack) of internal diameter (id) 3.0 mm and 50 mm in length. The extract of the solute in flow rate of 1 ml/min at the wavelength 210 nm and methanolic extract showed the antioxidant activity and powerful oxygen free radicals scavenging activities and the IC50 for the E. intermedia plant was near to the reference standard ascorbic acid. The HPLC method was useful for the quantitative purpose of ephedrine (E) and pseudoephedrine (PE) used for 45 samples of one species collected from central habitat in three districts (Ziarat, Shairani, and Kalat) of Balochistan. Results showed that average alkaloid substance in E. intermedia was as follows: PE (0.209%, 0.238%, and 0.22%) and E (0.0538%, 0.0666%, and 0.0514%). PMID:28386582

Determination of pKa values of some antipsychotic drugs by HPLC--correlations with the Kamlet and taft solvatochromic parameters and HPLC analysis in dosage forms.

PubMed

Sanli, Senem; Akmese, Bediha; Altun, Yuksel

2013-01-01

In this study, ionization constant (pKa) values were determined by using the dependence of the retention factor on the pH of the mobile phase for four ionizable drugs, namely, risperidone (RI), clozapine (CL), olanzapine (OL), and sertindole (SE). The effect of the mobile phase composition on the pKa was studied by measuring the pKa at different acetonitrile-water mixtures in an HPLC-UV method. To explain the variation of the pKa values obtained over the whole composition range studied, the quasi-lattice quasi-chemical theory of preferential solvation was applied. The pKa values of drugs were correlated with the Kamlet and Taft solvatochromic parameters. Kamlet and Taft's general equation was reduced to two terms by using combined factor analysis and target factor analysis in these mixtures: the independent term and the hydrogen-bond donating ability a. The HPLC-UV method was successfully applied for the determination of RI, OL, and SE in pharmaceutical dosage forms. CL was chosen as an internal standard. Additionally, the repeatability, reproducibility, selectivity, precision, and accuracy of the method in all media were investigated and calculated.

Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection.

PubMed

Matsutomo, Toshiaki; Kodera, Yukihiro

2016-02-01

Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC. The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE). We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions. Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period. We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE. © 2016 American Society for Nutrition.

PPM mixtures of formaldehyde in gas cylinders: Stability and analysis

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

Wong, K.C.; Miller, S.B.; Patterson, L.M.

1999-07-01

Scott Specialty Gases has been successful in producing stable calibration gases of formaldehyde at low concentration. Critical to this success has been the development of a treatment process for high pressure aluminum cylinders. Formaldehyde cylinders having concentrations of 20ppm and 4ppm were found to show only small decline in concentrations over a period of approximately 12 months. Since no NIST traceable formaldehyde standards (or Standard Reference Material) are available, all Scott's formaldehyde cylinders were originally certified by traditional impinger method. This method involves an extremely tedious purification procedure for 2,4-dinitrophenylhydrazine (2,4-DNPH). A modified version of the impinger method has beenmore » developed and does not require extensive reagent purification for formaldehyde analysis. Extremely low formaldehyde blanks have been obtained with the modified method. The HPLC conditions in the original method were used for chromatographic separations. The modified method results in a lower analytical uncertainty for the formaldehyde standard mixtures. Consequently, it is possible to discern small differences between analytical results that are important for stability study.« less

Utility of eosin Y as a complexing reagent for the determination of citalopram hydrobromide in commercial dosage forms by fluorescence spectrophotometry.

PubMed

Azmi, Syed Najmul Hejaz; Al-Fazari, Ahlam; Al-Badaei, Munira; Al-Mahrazi, Ruqiya

2015-12-01

An accurate, selective and sensitive spectrofluorimetric method was developed for the determination of citalopram hydrobromide in commercial dosage forms. The method was based on the formation of a fluorescent ion-pair complex between citalopram hydrobromide and eosin Y in the presence of a disodium hydrogen phosphate/citric acid buffer solution of pH 3.4 that was extractable in dichloromethane. The extracted complex showed fluorescence intensity at λem = 554 nm after excitation at 259 nm. The calibration curve was linear over at concentrations of 2.0-26.0 µg/mL. Under optimized experimental conditions, the proposed method was validated as per ICH guidelines. The effect of common excipients used as additives was tested and the tolerance limit calculated. The limit of detection for the proposed method was 0.121 μg/mL. The proposed method was successfully applied to the determination of citalopram hydrobromide in commercial dosage forms. The results were compared with the reference RP-HPLC method. Copyright © 2015 John Wiley & Sons, Ltd.

[HPLC-ESI-MS(n) analysis of the water soluble extracts of Fructus Choerospondiatis].

PubMed

Shi, Run-ju; Dai, Yun; Fang, Min-feng; Zhao, Xin; Zheng, Jian-bin; Zheng, Xiao-hui

2007-03-01

To establish an HPLC-ESI-MS(n) method for analyzing the chemical ingredients in the water soluble extracts of Fructus Choerospondiatis. Water-solvable extracts of Fructus Choerospondiatis are obtained by heating recirculation. Multi-stage reaction mode (MRM) of the HPLC-ESI-MS(n) was used to determine the content of Gallic acid, the MS(n) technology was used to obtain the information of characteristic multistage fragment ions so as to identify the chemical structure of peaks in the total current spectrum. Eleven compounds were identified, and one of them is a new unknown ingredient. The method, which has high recovery and specificity, can offer the experimental evidences for the further research of the chemical ingredients extracted from the Fructus Choerospondiatis.

Measurement of Menadione in urine by HPLC

USDA-ARS?s Scientific Manuscript database

Menadione may be an important metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method with a C30 column, fluorescence detection and post-column zinc reduction was developed to measure menadione in urine. The mobile phase was composed of 95% methanol...

Quantitative fundus autofluorescence in mice: correlation with HPLC quantitation of RPE lipofuscin and measurement of retina outer nuclear layer thickness.

PubMed

Sparrow, Janet R; Blonska, Anna; Flynn, Erin; Duncker, Tobias; Greenberg, Jonathan P; Secondi, Roberta; Ueda, Keiko; Delori, François C

2013-04-17

Our study was conducted to establish procedures and protocols for quantitative autofluorescence (qAF) measurements in mice, and to report changes in qAF, A2E bisretinoid concentration, and outer nuclear layer (ONL) thickness in mice of different genotypes and age. Fundus autofluorescence (AF) images (55° lens, 488 nm excitation) were acquired in albino Abca4(-/-), Abca4(+/-), and Abca4(+/+) mice (ages 2-12 months) with a confocal scanning laser ophthalmoscope (cSLO). Gray levels (GLs) in each image were calibrated to an internal fluorescence reference. The bisretinoid A2E was measured by quantitative high performance liquid chromatography (HPLC). Histometric analysis of ONL thicknesses was performed. The Bland-Altman coefficient of repeatability (95% confidence interval) was ±18% for between-session qAF measurements. Mean qAF values increased with age (2-12 months) in all groups of mice. qAF was approximately 2-fold higher in Abca4(-/-) mice than in Abca4(+/+) mice and approximately 20% higher in heterozygous mice. HPLC measurements of the lipofuscin fluorophore A2E also revealed age-associated increases, and the fold difference between Abca4(-/-) and wild-type mice was more pronounced (approximately 3-4-fold) than measurable by qAF. Moreover, A2E levels declined after 8 months of age, a change not observed with qAF. The decline in A2E levels in the Abca4(-/-) mice corresponded to reduced photoreceptor cell viability as reflected in ONL thinning beginning at 8 months of age. The qAF method enables measurement of in vivo lipofuscin and the detection of genotype and age-associated differences. The use of this approach has the potential to aid in understanding retinal disease processes and will facilitate preclinical studies.

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

PubMed

Remeur, Camille; Le Borgne, Erell; Gauthier, Léa; Grougnet, Raphaël; Deguin, Brigitte; Poullain, Cyril; Litaudon, Marc

2017-05-01

Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Profiling indomethacin impurities using high-performance liquid chromatography and nuclear magnetic resonance.

PubMed

Hess, S; Teubert, U; Ortwein, J; Eger, K

2001-12-01

The anti-inflammatory drug indomethacin was investigated regarding new related impurities. Therefore, related substances 2-9 were prepared by independent synthesis and physicochemically characterized. To determine indomethacin and its related substances, a new HPLC-UV method was developed and validated. Indomethacin and its impurities were eluted on a C(18) column with a mobile phase consisting of methanol and an aqueous solution of 0.2% phosphoric acid at a flow rate of 1.5 ml/min and were quantified by UV detection at 320 nm. Overall, the HPLC-UV method was simple and reliable for the detection of eight impurities in indomethacin. In addition to the HPLC-UV method, 1H nuclear magnetic resonance (NMR) was used to investigate indomethacin regarding impurities. For that purpose, related substances 2-9 were systematically added to indomethacin and investigated. The NMR method was found to be very useful for the identification of impurities in bulk substance without prior separation. Both HPLC-UV and NMR were used to analyze 38 batches of indomethacin available on the European market. The outcome was that 42% of the batches did not meet the compendial requirements although they met the specifications of current compendial methods. Some batches contained the previously undescribed impurity 8, while other batches contained by-products from two distinct synthetic routes. The methods presented herein are important contributions to the ongoing efforts to reduce impurities and therefore the risk of adverse side-effects in drugs that are no longer under patent protection.

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

PubMed

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

2013-06-30

Bioavailability of essential lipophilic micronutrients and carotenoids is of utmost interest for human health, as the consumption of these compounds may help alleviate major nutritional deficiencies, cardiovascular disease, and cancer. High-performance liquid chromatography/photo-diode array detection (HPLC-PDA) and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) were compared for the quantitative analysis of α- and β-carotene, β-cryptoxanthin, lutein, lycopene, α-tocopherol, phylloquinone, and several retinyl esters from chylomicron-containing triglyceride rich lipoprotein (TRL) fractions of human plasma obtained from two clinical trials. After selecting an efficient extraction method for the analytes, both the HPLC/PDA and the HPLC/MS/MS methods were developed and several parameters validated using an HP 1200 series HPLC system interfaced with a HP 1200 series diode-array detector (Agilent Technologies, Santa Clara, CA, USA) and a QTRAP 5500 (AB Sciex, Foster City, CA, USA) via an atmospheric pressure chemical ionization (APCI) probe operated in positive ion mode. For lycopene, α- and β-carotene, HPLC/MS/MS was up to 37 times more sensitive than HPLC-PDA. PDA detection was shown to be up to 8 times more sensitive for lutein. MS/MS signals were enhanced by matrix components for lutein and β-cryptoxanthin, as determined by referencing to the matrix-independent PDA signal. In contrast, matrix suppression was observed for retinyl palmitate, α-carotene, and β-carotene. Both detectors showed similar suitability for α-tocopherol, lycopene and retinyl palmitate (representing ~73% of total retinyl esters). MS/MS exclusively allowed the quantitation of minor retinyl esters, phylloquinone, and (Z)-lycopene isomers. HPLC/MS/MS was more sensitive than HPLC-PDA for six of the eight analytes and represents a powerful tool for the analysis of chylomicron samples and potentially other biological samples of limited sample size. When internal standards are available for the target carotenoid, employing MS/MS detection may reduce the necessary blood sample volume, which is particularly advantageous for minimizing risk and discomfort to human subjects during clinical studies. Copyright © 2013 John Wiley & Sons, Ltd.

Rapid characterisation and identification of compounds in Saposhnikoviae Radix by high-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry.

PubMed

Chen, Luxiao; Chen, Xiangyang; Su, Lei; Jiang, Yanyan; Liu, Bin

2018-04-01

Saposhnikoviae Radix (SR), the dried root of Saposhnikovia divaricata (Turcz.) Schischk. (Umbelliferae), is commonly used as a traditional Chinese medicine. In this study, a rapid and accurate method was firstly, developed for the qualitative analysis of SR by high-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS/MS). A total of 45 compounds were identified or tentatively characterised, including 13 chromones, 28 coumarins and four others. Among them, 16 compounds were identified from SR for the first time. In addition, six chromones reference standards, including two isolated compounds of 3'-O-angeloylhamaudol and norcimifugin from the extraction of SR, were used to study the fragmentation pathways of chromones. The developed method was effective for characterising the compounds of SR, and the results of the study enriched the understanding of the chemical connotation.

Comparison between cachaça and rum using pattern recognition methods.

PubMed

Cardoso, Daniel R; Andrade-Sobrinho, Luiz G; Leite-Neto, Alexandre F; Reche, Roni V; Isique, William D; Ferreira, Marcia M C; Lima-Neto, Benedito S; Franco, Douglas W

2004-06-02

The differentiation between cachaça and rum using analytical data referred to alcohols (methanol, propanol, isobutanol, and isopentanol), acetaldehyde, ethyl acetate, organic acids (octanoic acid, decanoic acid, and dodecanoic acid), metals (Al, Ca, Co, Cu, Cr, Fe, Mg, Mn, Ni, Na, and Zn), and polyphenols (protocatechuic acid, sinapaldehyde, syringaldehyde, ellagic acid, syringic acid, gallic acid, (-)-epicatechin, vanillic acid, vanillin, p-coumaric acid, coniferaldehyde, coniferyl alcohol, kaempferol, and quercetin) is described. The organic and metal analyte contents were determined in 18 cachaça and 21 rum samples using chromatographic methods (GC-MS, GC-FID, and HPLC-UV-vis) and inductively coupled plasma atomic emission spectrometry, respectively. The analytical data of the above compounds, when treated by principal component analysis, hierarchical cluster analysis, discriminant analysis, and K-nearest neighbor analysis, provide a very good discrimination between the two classes of beverages.

Validated spectrophotometric methods for determination of sodium valproate based on charge transfer complexation reactions.

PubMed

Belal, Tarek S; El-Kafrawy, Dina S; Mahrous, Mohamed S; Abdel-Khalek, Magdi M; Abo-Gharam, Amira H

2016-02-15

This work presents the development, validation and application of four simple and direct spectrophotometric methods for determination of sodium valproate (VP) through charge transfer complexation reactions. The first method is based on the reaction of the drug with p-chloranilic acid (p-CA) in acetone to give a purple colored product with maximum absorbance at 524nm. The second method depends on the reaction of VP with dichlone (DC) in dimethylformamide forming a reddish orange product measured at 490nm. The third method is based upon the interaction of VP and picric acid (PA) in chloroform resulting in the formation of a yellow complex measured at 415nm. The fourth method involves the formation of a yellow complex peaking at 361nm upon the reaction of the drug with iodine in chloroform. Experimental conditions affecting the color development were studied and optimized. Stoichiometry of the reactions was determined. The proposed spectrophotometric procedures were effectively validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. Calibration curves of the formed color products with p-CA, DC, PA and iodine showed good linear relationships over the concentration ranges 24-144, 40-200, 2-20 and 1-8μg/mL respectively. The proposed methods were successfully applied to the assay of sodium valproate in tablets and oral solution dosage forms with good accuracy and precision. Assay results were statistically compared to a reference pharmacopoeial HPLC method where no significant differences were observed between the proposed methods and reference method. Copyright © 2015 Elsevier B.V. All rights reserved.

Validated spectrophotometric methods for determination of sodium valproate based on charge transfer complexation reactions

NASA Astrophysics Data System (ADS)

Belal, Tarek S.; El-Kafrawy, Dina S.; Mahrous, Mohamed S.; Abdel-Khalek, Magdi M.; Abo-Gharam, Amira H.

2016-02-01

This work presents the development, validation and application of four simple and direct spectrophotometric methods for determination of sodium valproate (VP) through charge transfer complexation reactions. The first method is based on the reaction of the drug with p-chloranilic acid (p-CA) in acetone to give a purple colored product with maximum absorbance at 524 nm. The second method depends on the reaction of VP with dichlone (DC) in dimethylformamide forming a reddish orange product measured at 490 nm. The third method is based upon the interaction of VP and picric acid (PA) in chloroform resulting in the formation of a yellow complex measured at 415 nm. The fourth method involves the formation of a yellow complex peaking at 361 nm upon the reaction of the drug with iodine in chloroform. Experimental conditions affecting the color development were studied and optimized. Stoichiometry of the reactions was determined. The proposed spectrophotometric procedures were effectively validated with respect to linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. Calibration curves of the formed color products with p-CA, DC, PA and iodine showed good linear relationships over the concentration ranges 24-144, 40-200, 2-20 and 1-8 μg/mL respectively. The proposed methods were successfully applied to the assay of sodium valproate in tablets and oral solution dosage forms with good accuracy and precision. Assay results were statistically compared to a reference pharmacopoeial HPLC method where no significant differences were observed between the proposed methods and reference method.

An evaluation of the Diamat HPLC analyser for simultaneous determination of haemoglobins A2 and F

PubMed Central

Carpinelli, Assunta; Majavacca, Rita; Cantu'-Rajnoldi, Angelo; Garatti, Massimo; Paleari, Renata; Ferrari, Maurizio; Agape, Vittorio; Maccioni, Liliana; Pisano, Sandra; Galanello, Renzo

1989-01-01

The authors describe a modification of the instrumental parameters of the Diamat fully automated HPLC system for Hb A2 assay (Bio-Rad Laboratories, Milan, Italy) in order to obtain simultaneous determination of Hb A2 and Hb F. Hb A2 and Hb F measurements are reproducible (within-run CV 2.6%, with Hb A22.7%; 5.1%, with Hb F 1.3%) and accurate (from a comparison with two microchromatographic techniques for Hb A2: r = 0.9639 and 0.9755; with two alkali denaturation procedures for Hb F: r = 0.9990 and 0.9952; with radial immunodiffusion, r = 0.9877). Assay linearity has been confirmed for Hb A2 concentrations between 0 and 6.0%, and for Hb F between 0 and 60%. The data obtained from the analysis of some pathological samples for Hb Bart's, Hb H, Hb J Sardegna, Hb Lepore and Hb S are in agreement with cellulose acetate electrophoresis analysis. The Hb A2 reference intervals for normals (N = 597) and Beta-thalassemia carriers (N = 200) are respectively (95% limits) 2.02-3.27 and 3.92-5.90 in % units. Hb F values measured in normals (N = 968), in β-thal carriers (N = 302) and in δβ-thal carriers (N =3) have been found to be consistent with the usual diagnostic parameters. Some minor limitations emerged: the most relevant concerns Hb A1c, which is overestimated with respect to a reference method (y = 1.217x + 0.16; N = 79; r = 0.9235). A probable interference from labile fractions is responsible for this Hb A1c inaccuracy. PMID:18925255

Rifaximin Stability: A Look at UV, IR, HPLC, and Turbidimetry Methods.

PubMed

Kogawa, Ana Carolina; Salgado, Hérida Regina Nunes

2018-03-01

The study of the stability of medicines is mandated by the International Conference on Harmonization and the World Health Organization. Rifaximin, an antimicrobial marketed in the form of tablets, has no record of stability studies. Thus, the objective of the present work was to investigate the behavior and stability of rifaximin tablets for 6 months under simultaneous conditions of temperature and humidity by UV, IR, HPLC, and turbidimetry techniques. After 6 months of stability study, rifaximin tablets were shown to obey zero-order kinetics when analyzed by physicochemical methods and second-order kinetics when analyzed by a microbiological method. However, the UV method was not suitable for the evaluation of rifaximin. IR, HPLC, and turbidimetry methods can already be used to evaluate the stability of rifaximin tablets. It is important to analyze products with more than one type of method before releasing results mainly in the case of antimicrobial products in which the association of physicochemical and microbiological techniques must be a rule. Rifaximin tablets can be considered stable after 6 months under conditions of 40 ± 2°C and 75 ± 5% relative humidity.

On-line monitoring the extract process of Fu-fang Shuanghua oral solution using near infrared spectroscopy and different PLS algorithms

NASA Astrophysics Data System (ADS)

Kang, Qian; Ru, Qingguo; Liu, Yan; Xu, Lingyan; Liu, Jia; Wang, Yifei; Zhang, Yewen; Li, Hui; Zhang, Qing; Wu, Qing

2016-01-01

An on-line near infrared (NIR) spectroscopy monitoring method with an appropriate multivariate calibration method was developed for the extraction process of Fu-fang Shuanghua oral solution (FSOS). On-line NIR spectra were collected through two fiber optic probes, which were designed to transmit NIR radiation by a 2 mm flange. Partial least squares (PLS), interval PLS (iPLS) and synergy interval PLS (siPLS) algorithms were used comparatively for building the calibration regression models. During the extraction process, the feasibility of NIR spectroscopy was employed to determine the concentrations of chlorogenic acid (CA) content, total phenolic acids contents (TPC), total flavonoids contents (TFC) and soluble solid contents (SSC). High performance liquid chromatography (HPLC), ultraviolet spectrophotometric method (UV) and loss on drying methods were employed as reference methods. Experiment results showed that the performance of siPLS model is the best compared with PLS and iPLS. The calibration models for AC, TPC, TFC and SSC had high values of determination coefficients of (R2) (0.9948, 0.9992, 0.9950 and 0.9832) and low root mean square error of cross validation (RMSECV) (0.0113, 0.0341, 0.1787 and 1.2158), which indicate a good correlation between reference values and NIR predicted values. The overall results show that the on line detection method could be feasible in real application and would be of great value for monitoring the mixed decoction process of FSOS and other Chinese patent medicines.

Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents

DTIC Science & Technology

1997-11-01

status can sometimes be reflected in the infectious potential or drug resistance of those pathogens. For example, in Mycobacterium tuberculosis ... Mycobacterium tuberculosis , its antibiotic resistance and prediction of pathogenicity amongst Mycobacterium spp. based on signature lipid biomarkers ...TITLE AND SUBTITLE Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents 5a. CONTRACT NUMBER 5b

Development and validation of a reversed-phase fluorescence HPLC method for determination of bucillamine in human plasma using pre-column derivatization with monobromobimane.

PubMed

Lee, Kang Choon; Chun, Young Goo; Kim, Insoo; Shin, Beom Soo; Park, Eun-Seok; Yoo, Sun Dong; Youn, Yu Seok

2009-07-15

A simple, specific and sensitive derivatization with monobromobimane (mBrB) and the corresponding HPLC-fluorescence quantitation method for the analysis of bucillamine in human plasma was developed and validated. The analytical procedure involves a simple protein precipitation, pre-column fluorescence derivatization, and separation by reversed-phase high performance liquid chromatography (RP-HPLC). The calibration curve showed good linearity over a wide concentration range (50 ng/mL to 10 microg/mL) in human plasma (r(2)=0.9998). The lower limit of quantitation (LLOQ) was 50 ng/mL. The average precision and accuracy at LLOQ were within 6.3% and 107.6%, respectively. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (300 mg) of bucillamine to 20 healthy Korean volunteers.

Measurement of Menadione in Urine by HPLC

PubMed Central

Rajabi, Ala Al; Peterson, James; Choi, Sang Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-01-01

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C30 column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H2O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R2) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3 pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Research support: USDA ARS Cooperative Agreement 58-1950-7-707. PMID:20719580

Measurement of menadione in urine by HPLC.

PubMed

Al Rajabi, Ala; Peterson, James; Choi, Sang-Woon; Suttie, John; Barakat, Susan; Booth, Sarah L

2010-09-15

Menadione is a metabolite of vitamin K that is excreted in urine. A high performance liquid chromatography (HPLC) method using a C(30) column, post-column zinc reduction and fluorescence detection was developed to measure urinary menadione. The mobile phase was composed of 95% methanol with 0.55% aqueous solution and 5% DI H(2)O. Menaquinone-2 (MK-2) was used as an internal standard. The standard calibration curve was linear with a correlation coefficient (R(2)) of 0.999 for both menadione and MK-2. The lower limit of quantification (LLOQ) was 0.3pmole menadione/mL urine. Sample preparation involved hydrolysis of menadiol conjugates and oxidizing the released menadiol to menadione. Using this method, urinary menadione was shown to increase in response to 3 years of phylloquinone supplementation. This HPLC method is a sensitive and reproducible way to detect menadione in urine. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Selective identification and quantification of saccharin by liquid chromatography and fluorescence detection.

PubMed

Bruno, Sergio N F; Cardoso, Carlos R; Maciel, Márcia Mosca A; Vokac, Lidmila; da Silva Junior, Ademário I

2014-09-15

High-pressure liquid chromatography with ultra-violet detection (HPLC-UV) is one of the most commonly used methods to identify and quantify saccharin in non-alcoholic beverages. However, due to the wide variety of interfering UV spectra in saccharin-containing beverage matrices, the same method cannot be used to measure this analyte accurately. We have developed a new, highly effective method to identify and quantify saccharin using HPLC with fluorescence detection (HPLC-FLD). The excitation wavelength (250 nm) and emission wavelength (440 nm) chosen increased selectivity for all matrices and ensured few changes were required in the mobile phase or other parameters. The presence of saccharin in non-diet beverages - a fraud commonly used to replace more expensive sucrose - was confirmed by comparing coincident peaks as well as the emission spectra of standards and samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

Comparison of capillary electrophoresis and high performance liquid chromatography for detection and quantification of hemoglobin New York.

PubMed

You-Qiong, Li; Hui-Ping, Huang; Zhi-Zhong, Chen; Lin, Zhao; Liang, Liang; Gui-Fang, Qin; Yun, Mo

2016-01-01

Hemoglobin (Hb) New York [β113 (G15) Val→Glu, GTG→GAG], also known as Hb Kaohsiung, is one of the most common Hb variants in South China. Currently, most used screening methods for hemoglobinopathies in South China are high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). However, there is no study comparing the performance of CE and HPLC in the detection of Hb New York. In total 15 samples (including 13 adult blood samples and 2 cord blood samples) with heterozygous Hb New York were analyzed by CE and HPLC. Levels of Hb New York, HbA2, Hb, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were compared within the two groups. All 15 cases (100%) were detected by CE, whereas none was detected by HPLC. Mean values of Hb New York and HbA2 in adult heterozygous group were (43.82±2.47)% and (2.85±0.31)%, respectively; mean values for cord blood group were (7.95±0.78)% and (0.30±0.14)%. CE allows the detection of Hb New York, while this variant is not separated from Hb A on HPLC. CE may be the preferred method for hemoglobinopathy screening in areas with high prevalence of Hb New York.

Metabolite profiling with HPLC-ICP-MS as a tool for in vivo characterization of imaging probes.

PubMed

Boros, Eszter; Pinkhasov, Omar R; Caravan, Peter

2018-01-01

Current analytical methods for characterizing pharmacokinetic and metabolic properties of positron emission tomography (PET) and single photon emission computed tomography (SPECT) probes are limited. Alternative methods to study tracer metabolism are needed. The study objective was to assess the potential of high performance liquid chromatography - inductively coupled plasma - mass spectrometry (HPLC-ICP-MS) for quantification of molecular probe metabolism and pharmacokinetics using stable isotopes. Two known peptide-DOTA conjugates were chelated with nat Ga and nat In. Limit of detection of HPLC-ICP-MS for 69 Ga and 115 In was determined. Rats were administered 50-150 nmol of Ga- and/or In-labeled probes, blood was serially sampled, and plasma analyzed by HPLC-ICP-MS using both reverse phase and size exclusion chromatography. The limits of detection were 0.16 pmol for 115 In and 0.53 pmol for 69 Ga. Metabolites as low as 0.001 %ID/g could be detected and transchelation products identified. Simultaneous administration of Ga- and In-labeled probes allowed the determination of pharmacokinetics and metabolism of both probes in a single animal. HPLC-ICP-MS is a robust, sensitive and radiation-free technique to characterize the pharmacokinetics and metabolism of imaging probes.

Methods and applications of HPLC-AMS (WBio 5)

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

Bucholz, B A; Clifford, A J; Duecker, S R

Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a {sup 14}C-labelled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the {sup 14}C content above the control predose tissue and converting to equivalents of the parent compound. High Performance Liquid Chromatography (HPLC) is an excellent method for the chemical separation of complex mixtures whose profiles afford estimation of biochemical pathways of metabolism. Compounds elute from the HPLC systems with characteristic retention times and can be collected in fractions that can then be graphitized for AMS measurement.more » Unknowns are identified by coelution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the {sup 14}C signal. Thoroughly accounting for the carbon inventory in the LC solvents, ion-pairing agents, samples, and carriers adds some complexity to the analysis. In most cases the total carbon inventory is dominated by carrier. Baseline background and stability need to be carefully monitored. Limits of quantitation near 10 amol of {sup 14}C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected {sup 14}C activity <0.17 Bq (4.5 pCi) on the HPLC column.« less

Analysis of the constituents of aqueous preparations of Stachys recta by HPLC-DAD and HPLC-ESI-MS.

PubMed

Karioti, Anastasia; Bolognesi, Letizia; Vincieri, Franco Francesco; Bilia, Anna Rita

2010-09-21

In the present study a method based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface for the simultaneous determination of phenolic constituents in three aqueous preparations of the herbal medicinal drug Stachys recta. The developed assay was simple and effective and permitted the quality control of S. recta decoctions and infusion. Overall, 30 constituents were detected and identified, belonging mainly to three classes of compounds: caffeoylquinic acids, phenylethanol glycosides and flavonoids. 15 of them were quantified having a lower limit not less than 0.02% of the lyophilized extracts. Only seven of them were previously reported in this species, while 23 were identified for the first time as constituents of S. recta. HPLC-DAD-ESI-MS analysis provided evidence for the certain identification of the main constituents and in some cases of their isomers. Eight constituents were isolated and their structure elucidated by HPLC-ESI-MS and 1D- and 2D-NMR spectroscopy. Among the investigated preparations, the infusion seems to be the best method to extract the native constituents of the plant, while decoction is a more aggressive treatment and causes partial degradation of some acylated flavonoids. Copyright 2010 Elsevier B.V. All rights reserved.

Two-dimensional fingerprinting approach for comparison of complex substances analysed by HPLC-UV and fluorescence detection.

PubMed

Ni, Yongnian; Liu, Ying; Kokot, Serge

2011-02-07

This work is concerned with the research and development of methodology for analysis of complex mixtures such as pharmaceutical or food samples, which contain many analytes. Variously treated samples (swill washed, fried and scorched) of the Rhizoma atractylodis macrocephalae (RAM) traditional Chinese medicine (TCM) as well as the common substitute, Rhizoma atractylodis (RA) TCM were chosen as examples for analysis. A combined data matrix of chromatographic 2-D HPLC-DAD-FLD (two-dimensional high performance liquid chromatography with diode array and fluorescence detectors) fingerprint profiles was constructed with the use of the HPLC-DAD and HPLC-FLD individual data matrices; the purpose was to collect maximum information and to interpret this complex data with the use of various chemometrics methods e.g. the rank-ordering multi-criteria decision making (MCDM) PROMETHEE and GAIA, K-nearest neighbours (KNN), partial least squares (PLS), back propagation-artificial neural networks (BP-ANN) methods. The chemometrics analysis demonstrated that the combined 2-D HPLC-DAD-FLD data matrix does indeed provide more information and facilitates better performing classification/prediction models for the analysis of such complex samples as the RAM and RA ones noted above. It is suggested that this fingerprint approach is suitable for analysis of other complex, multi-analyte substances.

Determination of Trace Level Triclosan in Water by Online Preconcentration and HPLC-UV Diode Array

EPA Science Inventory

An online high performance liquid chromatography (HPLC) method for the detection and quantification of trace levels of triclosan in water is discussed. Triclosan, an anti-bacterial agent, and related compounds have been shown to reach municipal waste waters through the disposal ...

[Study on HPLC fingerprint of Alpinia officinarum].

PubMed

Deng, Yi-Feng; Feng, Li-Na; Luo, Hui

2011-09-01

To establish the chromatography fingerprint of Alpinia officinarum by HPLC. An optimum HPLC conditions which were obtained under the assessment of LC-MS were as follows: Shim-pack VP-ODS column (2.0 mm x 250 mm, 5 microm), 0.1% HAc aqueous solution as phase A, 15% Acetonitrile: 40% Methanol: 45% Tetrafuran as phase B, the flow rate was 0.20 mL/min, column temperature was 35 degrees C and UV detector was set at 280 nm. The HPLC fingerprint of Alpinia officinarum was established, the consensus 10 peaks and their relative retention times along with the ranges of relative area were determined. The method is reliable and stable and can be used for the quality control and identification of Alpinia officinarum.

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

PubMed

Fang, Xin-sheng; Tan, Xiao-mei

2005-09-01

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

Chip-based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids.

PubMed

Chang, Yuwei; Zhao, Chunxia; Wu, Zeming; Zhou, Jia; Zhao, Sumin; Lu, Xin; Xu, Guowang

2012-08-01

In this work a chip-based nano HPLC coupled MS (HPLC-chip/MS) method with a simple sample preparation procedure was developed for the flavonoid profiling of soybean. The analytical properties of the method including the linearity (R(2) , 0.992-0.995), reproducibility (RSD, 1.50-7.66%), intraday precision (RSD, 1.41-5.14%) and interday precision (RSD, 2.76-16.90%) were satisfactory. Compared with the conventional HPLC/MS method, a fast extraction and analysis procedure was applied and more flavonoids were detected in a single run. Additionally, 13 flavonoids in soybean seed were identified for the first time. The method was then applied to the profiling of six varieties of soybean sowed at the same place. A clear discrimination was observed among different cultivars, three isoflavones, accounting for nearly 80% of total flavonoid contents, were found increased in the spring soybeans compared with the summer cultivars. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simultaneous quantitative analysis of main components in linderae reflexae radix with one single marker.

PubMed

Wang, Li-Li; Zhang, Yun-Bin; Sun, Xiao-Ya; Chen, Sui-Qing

2016-05-08

Establish a quantitative analysis of multi-components by the single marker (QAMS) method for quality evaluation and validate its feasibilities by the simultaneous quantitative assay of four main components in Linderae Reflexae Radix. Four main components of pinostrobin, pinosylvin, pinocembrin, and 3,5-dihydroxy-2-(1- p -mentheneyl)- trans -stilbene were selected as analytes to evaluate the quality by RP-HPLC coupled with a UV-detector. The method was evaluated by a comparison of the quantitative results between the external standard method and QAMS with a different HPLC system. The results showed that no significant differences were found in the quantitative results of the four contents of Linderae Reflexae Radix determined by the external standard method and QAMS (RSD <3%). The contents of four analytes (pinosylvin, pinocembrin, pinostrobin, and Reflexanbene I) in Linderae Reflexae Radix were determined by the single marker of pinosylvin. This fingerprint was the spectra determined by Shimadzu LC-20AT and Waters e2695 HPLC that were equipped with three different columns.

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

PubMed

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

2018-06-01

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

An HPLC method for determination of azadirachtin residues in bovine muscle.

PubMed

Gai, María Nella; Álvarez, Christian; Venegas, Raúl; Morales, Javier

2011-04-01

A high-performance liquid chromatography (HPLC) method for the determination of azadirachtin (A and B) residues in bovine muscle has been developed. Azadirachtin is a neutral triterpene and chemotherapeutic agent effective in controlling some pest flies in horses, stables, horns and fruit. The actual HPLC method uses an isocratic elution and UV detection. Liquid-liquid extraction and solid-phase purification was used for the clean-up of the biological matrix. The chromatographic determination of these components is achieved using a C18 analytical column with water-acetonitrile mixture (27.5:72.5, v/v) as mobile phase, 1 mL/min as flow rate, 45 °C column temperature and UV detector at 215 nm. The azadirachtin peaks are well resolved and free of interference from matrix components. The extraction and analytical method developed in this work allows the quantitation of azadirachtin with precision and accuracy, establishing a lower limit of quantitation of azadirachtin, extracted from the biological matrix.

HPLC-Based Method to Evaluate Kinetics of Glucosinolate Hydrolysis by Sinapis alba Myrosinase1

PubMed Central

Vastenhout, Kayla J.; Tornberg, Ruthellen H.; Johnson, Amanda L.; Amolins, Michael W.; Mays, Jared R.

2014-01-01

Isothiocyanates (ITCs) are one of several hydrolysis products of glucosinolates, plant secondary metabolites which are substrates for the thioglucohydrolase myrosinase. Recent pursuits toward the development of synthetic, non-natural ITCs have consequently led to an exploration of generating these compounds from non-natural glucosinolate precursors. Evaluation of the myrosinase-dependent conversion of select non-natural glucosinolates to non-natural ITCs cannot be accomplished using established UV-Vis spectroscopic methods. To overcome this limitation, an alternative HPLC-based analytical approach was developed where initial reaction velocities were generated from non-linear reaction progress curves. Validation of this HPLC method was accomplished through parallel evaluation of three glucosinolates with UV-Vis methodology. The results of this study demonstrate that kinetic data is consistent between both analytical methods and that the tested glucosinolates respond similarly to both Michaelis–Menten and specific activity analyses. Consequently, this work resulted in the complete kinetic characterization of three glucosinolates with Sinapis alba myrosinase, with results that were consistent with previous reports. PMID:25068719

Performance evaluation of the Arkray Adams HA-8160 HbA1c analyser.

PubMed

Thevarajah, T Malathi; Nani, Nordin; Chew, Y Y

2008-12-01

HbA1c measurement is currently routinely used to predict long term outcome of diabetes, thus playing a fundamental role in the management of diabetes. The relationship between HbA1c value and long term diabetic complications has been established by a randomised control Diabetes Control and Complications Trial (DCCT) which used high performance liquid chromatography (HPLC) as a reference method for HbA1c assay. To ensure that HbA1c results from a variety HbA1c assay methods are similar to the DCCT values, the American Diabetes Association (ADA) recommended that all laboratories should use methods certified by the National Glycohemoglobin Standardization Programme (NGSP) with interassay coefficient variation (CV) of < 5% (ideally < 3%). The International Federation of Clinical Chemistry (IFCC) working group on HbA1c standardisation has set a CV < 2.5% as a criteria for its reference laboratories. To evaluate the performance of Arkray Adams HA-8160 HbA1c analyser which uses a cation exchange HPLC method and its correlation to HbA1c assay on Cobas Integra 800 which is an immunoturbidimetric method. For the imprecision study, patient samples and control material of two levels were analysed on HA-8160 analyser 20 times in a single run (within-run imprecision) and twice a day on five consecutive days (between-run imprecision). For the recovery study, two samples each with high and low values were selected and mixed in ratios of 1:3, 1:1 and 3:1, and were analysed by HA-8160. Sixty samples were analysed by both Cobas Integra 800 and HA-8160 for method comparison study. Ten uraemic samples and ten thalassaemic samples were assayed on Cobas Integra 800 and HA 8160 for interference study. Within-run CVs were 0.6% and 0.7% for medium and high value samples respectively, 0.6% and 0.7% for low and high level controls respectively. Between-run CVs were 0.5% and 0.4% for medium and high value samples respectively, 0.5% and 0.6% for low and high level controls respectively. The mean recovery was 100.1%. A good correlation between the 2 methods (Adams = 1.00 Cobas - 0.11, r = 0.98) was observed. The Akray Adams HA-8160 HbA1c analyser performed within the target CV of < 2.5% and showed a good correlation with the Cobas Integra 800.

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

PubMed

Fanali, Chiara; Dugo, Laura; D'Orazio, Giovanni; Lirangi, Melania; Dachà, Marina; Dugo, Paola; Mondello, Luigi

2011-01-01

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determining Vitamin D Status: A Comparison between Commercially Available Assays

PubMed Central

Snellman, Greta; Melhus, Håkan; Gedeborg, Rolf; Byberg, Liisa; Berglund, Lars; Wernroth, Lisa; Michaëlsson, Karl

2010-01-01

Background Vitamin D is not only important for bone health but can also affect the development of several non-bone diseases. The definition of vitamin D insufficiency by serum levels of 25-hydroxyvitamin D depends on the clinical outcome but might also be a consequence of analytical methods used for the definition. Although numerous 25-hydroxyvitamin D assays are available, their comparability is uncertain. We therefore aim to investigate the precision, accuracy and clinical consequences of differences in performance between three common commercially available assays. Methodology/Principal Findings Serum 25-hydroxyvitamin D levels from 204 twins from the Swedish Twin Registry were determined with high-pressure liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS), a radioimmunoassay (RIA) and a chemiluminescent immunoassay (CLIA). High inter-assay disagreement was found. Mean 25-hydroxyvitamin D levels were highest for the HPLC-APCI-MS technique (85 nmol/L, 95% CI 81–89), intermediate for RIA (70 nmol/L, 95% CI 66–74) and lowest with CLIA (60 nmol/L, 95% CI 56–64). Using a 50-nmol/L cut-off, 8% of the subjects were insufficient using HPLC-APCI-MS, 22% with RIA and 43% by CLIA. Because of the heritable component of 25-hydroxyvitamin D status, the accuracy of each method could indirectly be assessed by comparison of within-twin pair correlations. The strongest correlation was found for HPLC-APCI-MS (r = 0.7), intermediate for RIA (r = 0.5) and lowest for CLIA (r = 0.4). Regression analyses between the methods revealed a non-uniform variance (p<0.0001) depending on level of 25-hydroxyvitamin D. Conclusions/Significance There are substantial inter-assay differences in performance. The most valid method was HPLC-APCI-MS. Calibration between 25-hydroxyvitamin D assays is intricate. PMID:20644628

Preparative Isolation and Purification of Flavone C-Glycosides from the Leaves of Ficus microcarpa L. f by Medium-Pressure Liquid Chromatography, High-Speed Countercurrent Chromatography, and Preparative Liquid Chromatography

PubMed Central

Wang, Xiaohong; Liang, Yong; Zhu, Licai; Xie, Huichun; Li, Hang; He, Junting; Pan, Man; Zhang, Tianyou; Ito, Yoichiro

2009-01-01

Combined with medium-pressure liquid chromatography (MPLC) and preparative high-performance liquid chromatography (perp-HPLC), high-speed countercurrent chromatography (HSCCC) was applied for separation and purification of flavone C-glycosides from the crude extract of leaves of Ficus microcarpae L. f. HSCCC separation was performed on a two-phase solvent system composed of methyl tert- butyl ether - ethyl acetate – 1-butanol – acetonitrile – 0.1% aqueous trifluoroacetic acid at a volume ratio of 1:3:1:1:5. Partially resolved peak fractions from HSCCC separation were further purified by preparative HPLC. Four well-separated compounds were obtained and their purities were determined by HPLC. The purities of these peaks were 97.28%, 97.20%, 92.23%, and 98.40%.. These peaks were characterized by ESI-MSn. According to the reference, they were identified as orientin (peak I), isovitexin-3″-O-glucopyranoside (peak II), isovitexin (peak III), and vitexin (peak IV), yielded 1.2 mg, 4.5 mg, 3.3 mg, and 1.8 mg, respectively. PMID:20190866

HPLC Separation of Vitamin E and Its Oxidation Products and Effects of Oxidized Tocotrienols on the Viability of MCF-7 Breast Cancer Cells in Vitro.

PubMed

Drotleff, Astrid M; Büsing, Anne; Willenberg, Ina; Empl, Michael T; Steinberg, Pablo; Ternes, Waldemar

2015-10-14

Tocotrienols, a vitamin E subgroup, exert potent anticancer effects, but easily degrade due to oxidation. Eight vitamin E reference compounds, α-, β-, γ-, or δ-tocopherols or -tocotrienols, were thermally oxidized in n-hexane. The corresponding predominantly dimeric oxidation products were separated from the parent compounds by diol-modified normal-phase HPLC-UV and characterized by mass spectroscopy. The composition of test compounds, that is, α-tocotrienol, γ-tocotrienol, or palm tocotrienol-rich fraction (TRF), before and after thermal oxidation was determined by HPLC-DAD, and MCF-7 cells were treated with both nonoxidized and oxidized test compounds for 72 h. Whereas all nonoxidized test compounds (0-100 μM) led to dose-dependent decreases in cell viability, equimolar oxidized α-tocotrienol had a weaker effect, and oxidized TRF had no such effect. However, the IC50 value of oxidized γ-tocotrienol was lower (85 μM) than that of nonoxidized γ-tocotrienol (134 μM), thereby suggesting that γ-tocotrienol oxidation products are able to reduce tumor cell viability in vitro.

Detection of Free Polyamines in Plants Subjected to Abiotic Stresses by High-Performance Liquid Chromatography (HPLC).

PubMed

Gong, Xiaoqing; Liu, Ji-Hong

2017-01-01

High-performance liquid chromatography (HPLC) is a sensitive, rapid, and accurate technique to detect and characterize various metabolites from plants. The metabolites are extracted with different solvents and eluted with appropriate mobile phases in a designed HPLC program. Polyamines are known to accumulate under abiotic stress conditions in various plant species and thought to provide protection against oxidative stress by scavenging reactive oxygen species. Here, we describe a common method to detect the free polyamines in plant tissues both qualitatively and quantitatively.

Determination of pyridostigmine bromide and its metabolites in biological samples.

PubMed

Zhao, Bin; Moochhala, Shabbir M; Lu, Jia; Tan, Donna; Lai, Mui Hoon

2006-01-01

Pyridostigmine bromide (PB) is a quartenary ammonium compound that inhibits the hydrolysis of acetylcholine by competitive reversible binding to acetylcholinesterase. PB is used for the symptomatic treatment of myasthenia gravis and has been applied as a prophylaxis against nerve agents. Many studies on PB have involved the reliance on techniques that extract and quantify PB in biological samples. This article presents an overview of the currently applied methodologies for the determination of PB and its metabolites in various biological samples. Articles published from January 1975 to the July 2005 were taken into consideration for the discussion of the metabolism and analytical method of PB. HPLC and GC methods have been used and discussed in most of the references cited in this review. Other methods such as RIA and CE that have been recently reported are also mentioned in this article. Basic information about the type of sample used for analysis, sample preparation, chromatographic column, mobile phase, detection mode and validation data are summarized in a table.

Study on Quality Standard of Processed Curcuma Longa Radix

PubMed Central

Zhao, Yongfeng; Quan, Liang; Zhou, Haiting; Cao, Dong; Li, Wenbing; Yang, Zhuo

2017-01-01

To control the quality of Curcuma Longa Radix by establishing quality standards, this paper increased the contents of extract and volatile oil determination. Meanwhile, the curcumin was selected as the internal marker, and the relative correlation factors (RCFs) of demethoxycurcumin and bisdemethoxycurcumin were established by high performance liquid chromatography (HPLC). The contents of multicomponents were calculated based on their RCFs. The rationality and feasibility of the methods were evaluated by comparison of the quantitative results between external standard method (ESM) and quantitative analysis of multicomponents by single-marker (QAMS). Ethanol extracts ranged from 9.749 to 15.644% and the mean value was 13.473%. The volatile oil ranged from 0.45 to 0.90 mL/100 g and the mean value was 0.66 mL/100 g. This method was accurate and feasible and could provide a reference for further comprehensive and effective control of the quality standard of Curcuma Longa Radix and its processed products. PMID:29375640

Phenolic acids in the flowers of Althaea rosea var. nigra.

PubMed

Dudek, Marlena; Matławska, Irena; Szkudlarek, Maurycy

2006-01-01

Distribution of phenolic acids in the flowers of Althaea rosea var. nigra has been studied by 2D-TLC and HPLC methods. The phenolic acids occurring in these fractions have been identified as ferulic, vanillic, syringic, p-coumaric, p-hydroxybenzoic, p-hydroxyphenylacetic and caffeic acids. By means of the HPLC methods the contents of major phenolic acids were estimated. From among the phenolic acids analyzed the syringic, p-hydroxybenzoic and p-coumaric acids are dominant. Total content of phenolic acids was determined by the Arnov's method.

An Immunoassay to Rapidly Measure Acetaminophen Protein Adducts Accurately Identifies Patients with Acute Liver Injury or Failure

PubMed Central

Roberts, Dean W.; Lee, William M.; Hinson, Jack A.; Bai, Shasha; Swearingen, Christopher J.; Stravitz, R. Todd; Reuben, Adrian; Letzig, Lynda; Simpson, Pippa M.; Rule, Jody; Fontana, Robert J.; Ganger, Daniel; Reddy, K. Rajender; Liou, Iris; Fix, Oren; James, Laura P.

2017-01-01

Background & Aims A rapid, reliable point-of-care assay to detect acetaminophen protein adducts in serum of patients with acute liver injury could improve diagnosis and management. AcetaSTAT is a competitive immunoassay used to measure acetaminophen protein adducts formed by toxic metabolites in serum samples from patients. We compared the accuracy of AcetaSTAT vs high-pressure liquid chromatography with electrochemical detection (HPLC-EC, a sensitive and specific quantitative analytical assay) to detect acetaminophen protein adducts. Methods We collected serum samples from 19 healthy individuals (no liver injury, no recent acetaminophen use), 29 patients without acetaminophen-associated acute liver injury, and 33 patients with acetaminophen-associated acute liver injury participating in the Acute Liver Failure Study Group registry. Each serum sample was analyzed by AcetaSTAT (reported as test band amplitude) and HPLC-EC (the reference standard). We also collected data on patient age, sex, weight, level of alanine aminotransferase on test day and peak values, concentration of acetaminophen, diagnoses (by site investigator and causality review committee), and outcome after 21 days. Differences between groups were analyzed using Fisher’s Exact for categorical variables and Kruskal-Wallis Test or Rank-Sum test for continuous variables. Results AcetaSTAT discriminated between patients with and without acetaminophen-associated acute liver injury; the median (and range) AcetaSTAT test band amplitude for patients with acetaminophen-associated acute liver injury was 584 (range, 222–1027) vs 3678 (range, 394–8289) for those without (P<.001). AcetaSTAT identified patients with acetaminophen-associated acute liver injury with 100% sensitivity, 86.2% specificity, a positive-predictive value of 89.2%, and a negative-predictive value of 100%. Results from AcetaSTAT were positive in 4 subjects who received a causality review committee diagnosis of non-acetaminophen–associated acute liver injury; HPLC-EC and biochemical profiles were consistent with acetaminophen-associated acute liver injury in 3 of these cases. Conclusion The competitive immunoassay AcetaSTAT demonstrates a high degree of concordance with HPLC-EC results in identifying patients with acetaminophen-associated acute liver injury. This rapid and simple assay could increase early detection of this disorder and aid clinical management. PMID:27641661

High performance liquid chromatographic determination of caffeine in decaffeinated coffee, tea, and beverage products.

PubMed

Ashoor, S H; Seperich, G J; Monte, W C; Welty, J

1983-05-01

A method was developed for determining caffeine in decaffeinated coffee, tea, and beverage products by high performance liquid chromatography (HPLC). The HPLC system consisted of a Bio-Sil ODS-5S C18 column, methanol-water (25 + 75) mobile phase at 1 mL/min, and a UV detector. The method is simple and specific. Caffeine recoveries were 93.8-98.3% and coefficients of variation were 0.90-2.25%.

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.

Semi-preparative HPLC preparation and HPTLC quantification of tetrahydroamentoflavone as marker in Semecarpus anacardium and its polyherbal formulations.

PubMed

Aravind, S G; Arimboor, Ranjith; Rangan, Meena; Madhavan, Soumya N; Arumughan, C

2008-11-04

Application of modern scientific knowledge coupled with sensitive analytical technique is important for the quality evaluation and standardization of polyherbal formulations. Semecarpus anacardium, an important medicinal plant with wide medicinal properties, is frequently used in a large number of traditional herbal preparations. Tetrahydroamentoflavone (THA), a major bioactive biflavonoid was selected as a chemical marker of S. anacardium and RP-semi-preparative HPLC conditions were optimized for the isolation of tetrahydroamentoflavone. HPTLC analytical method was developed for the fingerprinting of S. anacardium flavonoids and quantification of tetrahydroamentoflavone. The method was validated in terms of their linearity, LOD, LOQ, precision and accuracy and compared with RP-HPLC-DAD method. The methods were demonstrated for the chemical fingerprinting of S. anacardium plant parts and some commercial polyherbal formulations and the amount of tetrahydroamentoflavone was quantified. HPTLC analysis showed that S. anacardium seed contained approximately 10 g kg(-1) of tetrahydroamentoflavone. The methods were able to identify and quantify tetrahydroamentoflavone from complex mixtures of phytochemicals and could be extended to the marker-based standardization of polyherbal formulations, containing S. anacardium.

A single extraction and HPLC procedure for simultaneous analysis of phytosterols, tocopherols and lutein in soybeans.

PubMed

Slavin, Margaret; Yu, Liangli Lucy

2012-12-15

A saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for simultaneous analysis of phytosterols, tocopherols and lutein (a carotenoid) in soybeans. Separation was achieved on a phenyl column with a ternary, isocratic solvent system of acetonitrile, methanol and water (48:22.5:29.5, v/v/v). Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol, stigmasterol, campesterol, and α-, δ- and γ-tocopherols, while lutein was quantified with visible light absorption at 450 nm. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (R(2)>0.99) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. Also, the accuracy of results of four soybeans using the described saponification and HPLC analytical method was validated against existing methods. This method offers a more efficient alternative to individual methods for quantifying lutein, tocopherols and sterols in soybeans. Copyright © 2012 Elsevier Ltd. All rights reserved.

A validated solid-liquid extraction method for the HPLC determination of polyphenols in apple tissues Comparison with pressurised liquid extraction.

PubMed

Alonso-Salces, Rosa M; Barranco, Alejandro; Corta, Edurne; Berrueta, Luis A; Gallo, Blanca; Vicente, Francisca

2005-02-15

A solid-liquid extraction procedure followed by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a photodiode array detector (DAD) for the determination of polyphenols in freeze-dried apple peel and pulp is reported. The extraction step consists in sonicating 0.5g of freeze-dried apple tissue with 30mL of methanol-water-acetic acid (30:69:1, v/v/v) containing 2g of ascorbic acid/L, for 10min in an ultrasonic bath. The whole method was validated, concluding that it is a robust method that presents high extraction efficiencies (peel: >91%, pulp: >95%) and appropriate precisions (within day: R.S.D. (n = 5) <5%, and between days: R.S.D. (n = 5) <7%) at the different concentration levels of polyphenols that can be found in apple samples. The method was compared with one previously published, consisting in a pressurized liquid extraction (PLE) followed by RP-HPLC-DAD determination. The advantages and disadvantages of both methods are discussed.

Pungency Quantitation of Hot Pepper Sauces Using HPLC

NASA Astrophysics Data System (ADS)

Betts, Thomas A.

1999-02-01

A class of compounds known as capsaicinoids are responsible for the "heat" of hot peppers. To determine the pungency of a particular pepper or pepper product, one may quantify the capsaicinoids and relate those concentrations to the perceived heat. The format of the laboratory described here allows students to collectively develop an HPLC method for the quantitation of the two predominant capsaicinoids (capsaicin and dihydrocapsaicin) in hot-pepper products. Each small group of students investigated one of the following aspects of the method: detector wavelength, mobile-phase composition, extraction of capsaicinoids, calibration, and quantitation. The format of the lab forced students to communicate and cooperate to develop this method. The resulting HPLC method involves extraction with acetonitrile followed by solid-phase extraction clean-up, an isocratic 80:20 methanol-water mobile phase, a 4.6 mm by 25 cm C-18 column, and UV absorbance detection at 284 nm. The method developed by the students was then applied to the quantitation of capsaicinoids in a variety of hot pepper sauces. Editor's Note on Hazards in our April 2000 issue addresses the above.

Stability-indicating HPLC-DAD methods for determination of two binary mixtures: Rabeprazole sodium-mosapride citrate and rabeprazole sodium-itopride hydrochloride.

PubMed

El-Fatatry, Hamed M; Mabrouk, Mokhtar M; Hewala, Ismail I; Emam, Ehab H

2014-08-01

Two selective stability-indicating HPLC methods are described for determination of rabeprazole sodium (RZ)-mosapride citrate (MR) and RZ-itopride hydrochloride (IO) mixtures in the presence of their ICH-stress formed degradation products. Separations were achieved on X-Bridge C18 column using two mobile phases: the first for RZ-MR mixture consisted of acetonitrile: 0.025 M KH 2 PO 4 solution: TEA (30:69:1 v/v; pH 7.0); the second for RZ-IO mixture was at ratio of 25:74:1 (v/v; pH 9.25). The detection wavelength was 283 nm. The two methods were validated and validation acceptance criteria were met in all cases. Peak purity testing using contrast angle theory, relative absorbance and log  A versus the wavelengths plots were presented. The % recoveries of the intact drugs were between 99.1% and 102.2% with RSD% values less than 1.6%. Application of the proposed HPLC methods indicated that the methods could be adopted to follow the stability of their formulations.

Inulin determination for food labeling.

PubMed

Zuleta, A; Sambucetti, M E

2001-10-01

Inulin and oligofructose exhibit valuable nutritional and functional attributes, so they are used as supplements as soluble fiber or as macronutrient substitutes. As classic analytical methods for dietary fiber measurement are not effective, several specific methods have been proposed. These methods measure total fructans and are based on one or more enzymatic sample treatments and determination of released sugars. To determine inulin for labeling purposes, we developed an easy and rapid anion-exchange high-performance liquid chromatography (HPLC) method following water extraction of inulin. HPLC conditions included an Aminex HPX- 87C column (Bio-Rad), deionized water at 85 degrees C as the mobile phase and a refractive index detector. The tested foods included tailor-made food products containing known amounts of inulin and commercial products (cookies, milk, ice creams, cheese, and cereal bars). The average recovery was 97%, and the coefficient of variation ranged from 1.1 to 5% in the food matrixes. The obtained results showed that this method provides an easier, faster and cheaper alternative than previous techniques for determining inulin with enough accuracy and precision for routine labeling purposes by direct determination of inulin by HPLC with refractive index detection.

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%.

A sensitive determination method for carbendazim and thiabendazole in apples by solid-phase microextraction-high performance liquid chromatography with fluorescence detection.

PubMed

Hu, Yanxue; Yang, Xiumin; Wang, Chun; Zhao, Jin; Li, Weining; Wang, Zhi

2008-03-01

A new analytical method for the determination of carbendazim (MBC) and thiabendazole (TBZ) in apples is reported, based on solid-phase microextraction (SPME) coupling HPLC with fluorescence detection. The main SPME and HPLC experimental conditions were optimized. The apples were first blended and centrifuged. Then, an aliquot of the resulting solution was subjected to SPME on a 60 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre for 35 min at room temperature with the solution being stirred at 1100 rev min(-1). The extracted pesticides on the SPME fibre were desorbed in the mobile phase into the SPME/HPLC interface for HPLC analysis. The method was linear over the range 0.01-1 mg kg(-1) in apples for both MBC and TBZ, with detection limits of 0.005 and 0.003 mg kg(-1) and correlation coefficients of 0.9995 and 0.9998, respectively. The average recoveries for MBC and TBZ were 91.5 and 92.3% with the relative standard deviations (RSD) of 4.7 and 4.1% at the 0.1 mg kg(-1) level, and 94.6 and 96.1% with RSD of 3.3 and 3.8% at the 0.5 mg kg(-1) level, respectively. The method is simple, sensitive, organic solvent-free and is suitable for the determination of MBC and TBZ in apples.

Topiramate: A Review of Analytical Approaches for the Drug Substance, Its Impurities and Pharmaceutical Formulations.

PubMed

Pinto, Eduardo Costa; Dolzan, Maressa Danielli; Cabral, Lucio Mendes; Armstrong, Daniel W; de Sousa, Valéria Pereira

2016-02-01

An important step during the development of high-performance liquid chromatography (HPLC) methods for quantitative analysis of drugs is choosing the appropriate detector. High sensitivity, reproducibility, stability, wide linear range, compatibility with gradient elution, non-destructive detection of the analyte and response unaffected by changes in the temperature/flow are some of the ideal characteristics of a universal HPLC detector. Topiramate is an anticonvulsant drug mainly used for the treatment of different types of seizures and prophylactic treatment of migraine. Different analytical approaches to quantify topiramate by HPLC have been described because of the lack of chromophoric moieties on its structure, such as derivatization with fluorescent moieties and UV-absorbing moieties, conductivity detection, evaporative light scattering detection, refractive index detection, chemiluminescent nitrogen detection and MS detection. Some methods for the determination of topiramate by capillary electrophoresis and gas chromatography have also been published. This systematic review provides a description of the main analytical methods presented in the literature to analyze topiramate in the drug substance and in pharmaceutical formulations. Each of these methods is briefly discussed, especially considering the detector used with HPLC. In addition, this article presents a review of the data available regarding topiramate stability, degradation products and impurities. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Method development in high-performance liquid chromatography for high-throughput profiling and metabonomic studies of biofluid samples.

PubMed

Pham-Tuan, Hai; Kaskavelis, Lefteris; Daykin, Clare A; Janssen, Hans-Gerd

2003-06-15

"Metabonomics" has in the past decade demonstrated enormous potential in furthering the understanding of, for example, disease processes, toxicological mechanisms, and biomarker discovery. The same principles can also provide a systematic and comprehensive approach to the study of food ingredient impact on consumer health. However, "metabonomic" methodology requires the development of rapid, advanced analytical tools to comprehensively profile biofluid metabolites within consumers. Until now, NMR spectroscopy has been used for this purpose almost exclusively. Chromatographic techniques and in particular HPLC, have not been exploited accordingly. The main drawbacks of chromatography are the long analysis time, instabilities in the sample fingerprint and the rigorous sample preparation required. This contribution addresses these problems in the quest to develop generic methods for high-throughput profiling using HPLC. After a careful optimization process, stable fingerprints of biofluid samples can be obtained using standard HPLC equipment. A method using a short monolithic column and a rapid gradient with a high flow-rate has been developed that allowed rapid and detailed profiling of larger numbers of urine samples. The method can be easily translated into a slow, shallow-gradient high-resolution method for identification of interesting peaks by LC-MS/NMR. A similar approach has been applied for cell culture media samples. Due to the much higher protein content of such samples non-porous polymer-based small particle columns yielded the best results. The study clearly shows that HPLC can be used in metabonomic fingerprinting studies.

Chromatographic methods for determination of S-substituted cysteine derivatives--a comparative study.

PubMed

Kubec, Roman; Dadáková, Eva

2009-10-09

A novel HPLC method for determination of a wide variety of S-substituted cysteine derivatives in Allium species has been developed and validated. This method allows simultaneous separation and quantification of S-alk(en)ylcysteine S-oxides, gamma-glutamyl-S-alk(en)ylcysteines and gamma-glutamyl-S-alk(en)ylcysteine S-oxides in a single run. The procedure is based on extraction of these amino acids and dipeptides by methanol, their derivatization by dansyl chloride and subsequent separation by reversed phase HPLC. The main advantages of the new method are simplicity, excellent stability of derivatives, high sensitivity, specificity and the ability to simultaneously analyze the whole range of S-substituted cysteine derivatives. This method was critically compared with other chromatographic procedures used for quantification of S-substituted cysteine derivatives, namely with two other HPLC methods (derivatization by o-phthaldialdehyde/tert-butylthiol and fluorenylmethyl chloroformate), and with determination by gas chromatography or capillary electrophoresis. Major advantages and drawbacks of these analytical procedures are discussed. Employing these various chromatographic methods, the content and relative proportions of individual S-substituted cysteine derivatives were determined in four most frequently consumed alliaceous vegetables (garlic, onion, shallot, and leek).

Olive oil polyphenols: A quantitative method by high-performance liquid-chromatography-diode-array detection for their determination and the assessment of the related health claim.

PubMed

Ricciutelli, Massimo; Marconi, Shara; Boarelli, Maria Chiara; Caprioli, Giovanni; Sagratini, Gianni; Ballini, Roberto; Fiorini, Dennis

2017-01-20

In order to assess if an extra virgin olive oil (EVOO) can be acknowledged with the health claim related to olive oil polyphenols (Reg. EU n.432/2012), a new method to quantify these species in EVOO, by means of liquid-liquid extraction followed by HPLC-DAD/MS/MS of the hydroalcoholic extract, has been developed and validated. Different extraction procedures, different types of reverse-phase analytical columns (Synergi Polar, Spherisorb ODS2 and Kinetex) and eluents have been tested. The chromatographic column Synergi Polar (250×4.6mm, 4μm), never used before in this kind of application, provided the best results, with water and methanol/isopropanol (9/1) as eluents. The method allows the quantification of the phenolic alcohols tyrosol and hydroxytyrosol, the phenolic acids vanillic, p-coumaric and ferulic acids, secoiridoids derivatives, the lignans, pinoresinol and acetoxypinoresinol and the flavonoids luteolin and apigenin. The new method has been applied to 20 commercial EVOOs belonging to two different price range categories (3.78-5.80 euros/L and 9.5-25.80 euros/L) and 5 olive oils. The obtained results highlight that acetoxypinoresinol, ferulic acid, vanillic acid and the total non secoiridoid phenolic substances resulted to be significantly higher in HEVOOs than in LEVOOs (P=0.0026, 0.0217, 0.0092, 0.0003 respectively). For most of the samples analysed there is excellent agreement between the results obtained by applying the HPLC method adopted by the International Olive Council and the results obtained by applying the presented HPLC method. Results obtained by HPLC methods have been also compared with the ones obtained by the colorimetric Folin-Ciocalteu method. Copyright © 2016 Elsevier B.V. All rights reserved.

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.