Science.gov

Sample records for hplc

  1. HPLC: Early and Recent Perspectives.

    ERIC Educational Resources Information Center

    Karger, Barry L.

    1997-01-01

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

  2. [Determination of amanitatoxins by HPLC].

    PubMed

    Nishizawa, Chiemi; Yamaura, Yoshio

    2003-10-01

    High-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous determination of alpha-amanitin, beta-amanitin and phalloidin in serum. Three toxins were extracted by reflux in a water bath at 80 degrees C for one hour and purified by Sep-Pak Plus tC18 cartridges. The HPLC assay was performed under gradient conditions using Develosil RP AQUEOUS column. The moble phase consisted with a mixture of acetonitorile containing 0.01 M ammonium acetate(pH 5.0). The column effluence was monitored at 295 nm, 302 nm and 230 nm for 35 min. Detection limit of three toxins in serum were 0.2 microgram/ml respectively. High recovery yields in the range of 81.5-88.1% for toxins were obtained by using this method. PMID:14740566

  3. HPLC for quality control of polyimides

    NASA Technical Reports Server (NTRS)

    Young, P. R.; Sykes, G. F.

    1979-01-01

    High Pressure Liquid Chromatography (HPLC) as a quality control tool for polyimide resins and prepregs are presented. A data base to help establish accept/reject criteria for these materials was developed. This work is intended to supplement, not replace, standard quality control tests normally conducted on incoming resins and prepregs. To help achieve these objectives, the HPLC separation of LARC-160 polyimide precursor resin was characterized. Room temperature resin aging effects were studied. Graphite reinforced composites made from fresh and aged resin were fabricated and tested to determine if changes observed by HPLC were significant.

  4. Determination of zinc pyrithione in shampoos by HPLC and HPLC-MS/MS.

    PubMed

    Gu, Yu-Xiang; Wang, Qing-He; Zhou, Ze-Lin; Lv, Qing; Mai, Cheng-Hua

    2014-01-01

    Methods have been developed for the determination of zinc pyrithione (ZPT) in shampoos using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS). Samples were washed by water first to remove surfactant and water-soluble impurities, then ultrasonic-extracted by acetonitrile-methanol for 30 min, and finally analyzed by MG C18 column (250 mm x 4.6 mm, 5 μm) or RP-18e (100 mm x 3 mm, 2 μm) plus APCI-MS/MS. Limits of detection were determined as 0.015% (HPLC) and 0.003% (HPLC-MS/MS), with a limit of quantization of 0.05% and 0.01%, respectively. The recoveries were 85.8-104% (HPLC) and 87.6-107% (HPLC-MS/MS). A good linear relationship was obtained from 3.20 μg·ml(-1) to 200 μg·ml(-1) (HPLC) and 1.00 μg·ml(-1) to 200 μg·ml(-1) (HPLC-MS/MS). The proposed methods have been successfully applied to the analysis of ZPT in many shampoos. The established two methods were rapid and reproducible with low interference.

  5. Determination of zinc pyrithione in shampoos by HPLC and HPLC-MS/MS.

    PubMed

    Gu, Yu-Xiang; Wang, Qing-He; Zhou, Ze-Lin; Lv, Qing; Mai, Cheng-Hua

    2014-01-01

    Methods have been developed for the determination of zinc pyrithione (ZPT) in shampoos using high-performance liquid chromatography (HPLC) and high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS). Samples were washed by water first to remove surfactant and water-soluble impurities, then ultrasonic-extracted by acetonitrile-methanol for 30 min, and finally analyzed by MG C18 column (250 mm x 4.6 mm, 5 μm) or RP-18e (100 mm x 3 mm, 2 μm) plus APCI-MS/MS. Limits of detection were determined as 0.015% (HPLC) and 0.003% (HPLC-MS/MS), with a limit of quantization of 0.05% and 0.01%, respectively. The recoveries were 85.8-104% (HPLC) and 87.6-107% (HPLC-MS/MS). A good linear relationship was obtained from 3.20 μg·ml(-1) to 200 μg·ml(-1) (HPLC) and 1.00 μg·ml(-1) to 200 μg·ml(-1) (HPLC-MS/MS). The proposed methods have been successfully applied to the analysis of ZPT in many shampoos. The established two methods were rapid and reproducible with low interference. PMID:25682618

  6. Determination of CMPO using HPLC -UV

    SciTech Connect

    Gracy Elias; Gary S. Groenewold; Bruce J. Mincher; Stephen P. Mezyk

    2012-06-01

    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 system precision. CMPO-nitric acid complexes were also identified, that account for the apparent loss of CMPO in acidic environment, independent of irradiation.

  7. HPLC determination of naproxen in plasma.

    PubMed

    Tashtoush, B M; Al-Taani, B M

    2003-09-01

    An assay method using isocratic HPLC with fluorometric detection for the determination of naproxen sodium in plasma is presented. A reverse phase Microbondapack column was used with a mobile phase consisting of 42% acetonitrile and 58% water adjusted to pH 3 using phosphoric acid. The fluorometric detector with an excitation wavelength of 270 nm and emission wavelength of 340 nm provided high sensitivity and no interferences from plasma constituents. Plasma samples were injected to HPLC without any extraction. The method was precise and reproducible as was demonstrated by replicate analysis of pooled plasma sample containing 0.5-80 microg/ml naproxen sodium.

  8. An Inexpensive Digital Gradient Controller for HPLC.

    ERIC Educational Resources Information Center

    Brady, James E.; Carr, Peter W.

    1983-01-01

    Use of gradient elution techniques in high performance liquid chromatography (HPLC) is often essential for direct separation of complex mixtures. Since most commercial controllers have features that are of marginal value for instructional purposes, a low-cost controller capable of illustrating essential features of gradient elution was developed.…

  9. Methods and applications of HPLC-AMS

    NASA Astrophysics Data System (ADS)

    Buchholz, Bruce A.; Dueker, Stephen R.; Lin, Yumei; Clifford, Andrew J.; Vogel, John S.

    2000-10-01

    Pharmacokinetics of physiologic doses of nutrients, pesticides, and herbicides can easily be traced in humans using a 14C-labeled compound. Basic kinetics can be monitored in blood or urine by measuring the elevation in the 14C 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. Unknowns are tentatively identified by co-elution with known standards and chemical tests that reveal functional groupings. Metabolites are quantified with the 14C 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 14C per HPLC fraction are typically achieved. Baselines are maintained by limiting injected 14C activity <0.17 Bq (4.5 pCi) on the HPLC column.

  10. Arsenic speciation in chinese seaweeds using HPLC-ICP-MS and HPLC-ES-MS.

    PubMed

    Van Hulle, Marijn; Zhang, Chao; Zhang, Xinrong; Cornelis, Rita

    2002-05-01

    Three common Chinese edible seaweeds, one brown (Laminaria japonica) and two red (Porphyra crispata and Eucheuma denticulatum), were examined for their total arsenic content. The As species were extracted with yields of 76.4, 69.8 and 25.0%, respectively. Anion-exchange and cation-exchange high-performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were used for the separation of the different arsenic species in two of the three seaweed extracts (Laminaria and Porphyra). The main arsenic species in the algal extracts are arseno sugars, although it has been shown that the Laminaria seaweed contains significant amounts of dimethylarsinic acid (DMA). HPLC was coupled with electrospray mass spectrometry (ES-MS) for structural confirmation of the arsenic species. The mass spectrometer settings for the arseno sugars were optimised using standards. The conclusions drawn on the basis of HPLC-ICP-MS were confirmed by the HPLC-ES-MS data. The HPLC-ES-MS method is capable of determining both arseno sugars and DMA in the seaweeds. The unknown compounds seen in the HPLC-ICP-MS chromatogram of Laminaria could not be ascribed to trimethylarsenic oxide or tetramethylarsonium ion. PMID:12081041

  11. [Determination of azoxystrobin in tea by HPLC].

    PubMed

    Chonan, T

    2001-08-01

    A determination method has been developed for azoxystrobin in tea by HPLC. Azoxystrobin was extracted from a sample with acetone, and the extract was passed through an alumina column to remove tannin. The eluate was concentrated to ca. 25 mL and passed through a Sep-Pak Vac tC18 to remove pigments. The eluate was cleaned-up by using liquid-liquid partition, and Florisil and silica-gel columns. The HPLC analysis for azoxystrobin was carried out on a C18 column with acetonitrile-water (9:11) as the mobile phase, with ultraviolet detection at 260 nm. The recovery of azoxystrobin fortified at the level of 0.4 microgram/g was 90.2% and the limit of determination was 0.2 microgram/g.

  12. Retinoid quantification by HPLC/MS(n)

    NASA Technical Reports Server (NTRS)

    McCaffery, Peter; Evans, James; Koul, Omanand; Volpert, Amy; Reid, Kevin; Ullman, M. David

    2002-01-01

    Retinoic acid (RA) mediates most of the biological effects of vitamin A that are essential for vertebrate survival. It acts through binding to receptors that belong to the nuclear receptor transcription factor superfamily (Mangelsdorf et al. 1994). It is also a highly potent vertebrate teratogen. To determine the function and effects of endogenous and exogenous RA, it is important to have a highly specific, sensitive, accurate, and precise analytical procedure. Current analyses of RA and other retinoids are labor intensive, of poor sensitivity, have limited specificity, or require compatibility with RA reporter cell lines (Chen et al. 1995. BIOCHEM: Pharmacol. 50: 1257-1264; Creech Kraft et al. 1994. BIOCHEM: J. 301: 111-119; Lanvers et al. 1996. J. Chromatogr. B Biomed. Appl. 685: 233-240; Maden et al. 1998. DEVELOPMENT: 125: 4133-4144; Wagner et al. 1992. DEVELOPMENT: 116: 55-66). This paper describes an HPLC/mass spectrometry/mass spectrometry product ion scan (HPLC/MS(n)) procedure for the analysis of retinoids that employs atmospheric pressure chemical ionization MS. The retinoids are separated by normal-phase column chromatography with a linear hexane-isopropanol-dioxane gradient. Each retinoid is detected by a unique series of MS(n) functions set at optimal collision-induced dissociation energy (30% to 32%) for all MS(n) steps. The scan events are divided into three segments, based on HPLC elution order, to maximize the mass spectrometer duty cycle. The all-trans, 9-cis, and 13-cis RA isomers are separated, if desired, by an isocratic hexane-dioxane-isopropanol mobile phase. This paper describes an HPLC/MS(n) procedure possessing high sensitivity and specificity for retinoids.

  13. HPLC Determination of Taurine in Sports Drinks

    NASA Astrophysics Data System (ADS)

    Orth, Dale L.

    2001-06-01

    The amino acid taurine (2-aminoethanesulfonic acid) is present as a nutritional supplement in many sports drinks. An experiment, suitable for a junior-senior level instrumental analysis course, is described to measure the amount of taurine in these sports drinks. A pre-column derivatization with Sanger's reagent, 2,4-dinitrofluorobenzene, is followed by an HPLC separation utilizing a gradient elution, and detection at 360 nm.

  14. An Investigation Into HPLC Data Quality Problems

    NASA Technical Reports Server (NTRS)

    Hooker, Stanford B.; VanHeukelem, Laurie

    2011-01-01

    This report summarizes the analyses and results produced by a five-member investigative team of Government, university, and industry experts, established by NASA HQ. The team examined data quality problems associated with high performance liquid chromatography (HPLC) analyses of pigment concentrations in seawater samples produced by the San Diego State University (SDSU) Center for Hydro-Optics and Remote Sensing (CHORS). This report shows CHORS did not validate the methods used before placing them into service to analyze field samples for NASA principal investigators (PIs), even though the HPLC literature contained easily accessible method validation procedures, and the importance of implementing them, more than a decade ago. In addition, there were so many sources of significant variance in the CHORS methodologies, that the HPLC system rarely operated within performance criteria capable of producing the requisite data quality. It is the recommendation of the investigative team to a) not correct the data, b) make all the data that was temporarily sequestered available for scientific use, and c) label the affected data with an appropriate warning, e.g., "These data are not validated and should not be used as the sole basis for a scientific result, conclusion, or hypothesis--independent corroborating evidence is required."

  15. Optimizing Chromatographic Separation: An Experiment Using an HPLC Simulator

    ERIC Educational Resources Information Center

    Shalliker, R. A.; Kayillo, S.; Dennis, G. R.

    2008-01-01

    Optimization of a chromatographic separation within the time constraints of a laboratory session is practically impossible. However, by employing a HPLC simulator, experiments can be designed that allow students to develop an appreciation of the complexities involved in optimization procedures. In the present exercise, a HPLC simulator from "JCE…

  16. HPLC and HPLC/MS analysis of red ink on counterfeit 100-yuan notes.

    PubMed

    Xu, Ying-jian; Zhou, Xin-xin; Shi, Xiao-fan

    2016-02-01

    Counterfeiting is a significant problem for most major currencies and has high social and economic costs. Chemical and physical identifiers that are unique to counterfeit currency are critical to forensic analysis. The 100-yuan Chinese note is mostly red. Here, we analyzed the red ink used in 100-yuan Chinese notes and developed a method to extract and analyze these dyes via high performance liquid chromatography (HPLC) and HPLC/mass spectrometry (MS). We used this approach to analyze the chemical structures of the adulterated colorants from 46 counterfeit 100-yuan notes seized from different locations. The results showed that a variety of inks were found among the seized counterfeit notes from different sources. The chromatographic data signature could be used to clearly discriminate authentic from counterfeit notes, but could also potentially be used to trace the notes to the counterfeiter. To the best of our knowledge, this is the first report to use HPLC/MS to profile red dyes in Chinese currency with important implications for the forensics and law enforcement communities. PMID:26750988

  17. Efficient and economic HPLC performance qualification.

    PubMed

    Kaminski, Lukas; Degenhardt, Matthias; Ermer, Joachim; Feussner, Claus; Höwer-Fritzen, Heidemarie; Link, Peter; Renger, Bernd; Tegtmeier, Martin; Wätzig, Hermann

    2010-02-01

    Analytical instrument qualification (AIQ) is a prerequisite for any analytical method validation and thus must be considered as a vital basis of analytical data integrity and quality in pharmaceutical analysis. There is a well-established system of qualification phases-Design Qualification, Installation Qualification (IQ), Operational Qualification (OQ) and Performance Qualification (PQ). As HPLC systems are "off the shelf" equipment, Design Qualification may be disregarded here. IQ establishes that the instrument is received as designed and that it is properly installed. OQ is carried out modularly with the intention to ensure that the specific modules of the system and the whole system are operating according to the defined specifications. PQ as the last step of the initial qualification is supposed to ensure continued satisfactory performance of an instrument under actual running conditions over the anticipated working range during daily use. However, PQ is not a one time exercise, but is currently repeated regularly independently from routine use of the analytical system using standard reference test condition. But this approach, which is time consuming and expensive only provides a snapshot of system performance. As HPLC procedures generally require a system suitability test (SST) prior and/or after test, it might be far more reasonable and robust to use these SST data for a continuous PQ. The work presented here demonstrates that, under certain circumstances, satisfactory instrument performance assessment can be derived from system suitability tests and performance data from daily use as well. A generally accepted qualification list, consisting of only twelve critical parameters, was compiled in a first step. Some parameters such as injector or thermostatting accuracy were considered redundant while others were successfully incorporated in the proposed holistic approach. System suitability test data as well as OQ/PQ data were provided from different sources

  18. Comprehensive quality evaluation of Chishao by HPLC.

    PubMed

    Zaiyou, Jian; Wenquan, Wang; Guifang, Xu; Li, Meng; Junling, Hou

    2013-01-01

    Objetivo: El propósito de este artículo es la evaluación cualitativa extensa de Chishao. Métodos: En el experimento de este trabajo, se establecen los espectros de identificación de Chishao en todas las localizaciones mediante RP-HPLC y el método del análisis de componentes principales con las áreas pico de RPHPLC. Resultados: La calidad de Chishao en el norte de China o del procedente de Paeonia lactiflora es mejor que la de otras localizaciones o procedente de P. obovata, P. mairei y P. anomala. Los resultados son congruentes con la impresión tradicional de la calidad de esta planta. Estos resultados indican que el análisis de los componentes principales (ACP) puede utilizarse como método eficaz y económico para evaluar la calidad de Chishao y podría aplicarse a otras plantas medicinales chinas. Conclusiones: Dada la complicada base de la eficacia de la Medicina tradicional china (MTC), un método como el ACP para diversos componentes químicos parece ser más adecuado para la evaluación de la calidad de la MTC en comparación con la determinación de un único o unos pocos agentes químicos.

  19. Postcolumn HPLC Detection of Mono- and Oligosaccharides with a Chemosensor

    PubMed Central

    Kim, Kyu Kwang; Escobedo, Jorge O.; St. Luce, Nadia N.; Rusin, Oleksandr; Wong, Douglas; Strongin, Robert M.

    2012-01-01

    Novel chromophoric compound 1 promotes the HPLC postcolumn detection of mono- and oligosaccharides. The detection of chromatographic peaks in the visible region for glucose, fructose, maltodextrins, sialic acid, and a ganglioside can be accomplished with a standard UV–vis detector. The use of selective, reversible binding agents in automated HPLC assays should allow for improved monitoring of specific analytes as well as material recovery. PMID:14682751

  20. Analysis of munitions constituents in IMX formulations by HPLC and HPLC-MS.

    PubMed

    Russell, A L; Seiter, J M; Coleman, J G; Winstead, B; Bednar, A J

    2014-10-01

    The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents, such as 2,4,6-trinitrotolene (TNT), for improved safety. The IMX formulations are more stable and therefore less prone to accidental detonation while designed to match the performance of legacy materials. Two formulations, IMX 101 and 104 are being investigated as a replacement for TNT in artillery rounds and composition B Army mortars, respectively. The chemical formulations of IMX-101 and 104 are comprised of four constituents;2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1-nitroguanidine (NQ), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) which are mixed in various ratios to achieve the desired performance. The current work details the analysis of the IMX constituents by single column HPLC-UV-ESI-MS. Detection limits determined are in agreement with similar HPLC analysis of compounds, ranging from 7 to 9μg/L. Gradient mobile phases are used to allow separation of the 4 target compounds in more complex mixture of other concomitant compounds. Mass spectra are used to confirm analyte identity with chromatographic retention time. PMID:25059196

  1. Analysis of munitions constituents in IMX formulations by HPLC and HPLC-MS.

    PubMed

    Russell, A L; Seiter, J M; Coleman, J G; Winstead, B; Bednar, A J

    2014-10-01

    The use of Insensitive Munitions eXplosives (IMX) is increasing as the Army seeks to replace certain conventional munitions constituents, such as 2,4,6-trinitrotolene (TNT), for improved safety. The IMX formulations are more stable and therefore less prone to accidental detonation while designed to match the performance of legacy materials. Two formulations, IMX 101 and 104 are being investigated as a replacement for TNT in artillery rounds and composition B Army mortars, respectively. The chemical formulations of IMX-101 and 104 are comprised of four constituents;2,4-dinitroanisole (DNAN), 3-nitro-1,2,4-triazol-5-one (NTO), 1-nitroguanidine (NQ), and Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) which are mixed in various ratios to achieve the desired performance. The current work details the analysis of the IMX constituents by single column HPLC-UV-ESI-MS. Detection limits determined are in agreement with similar HPLC analysis of compounds, ranging from 7 to 9μg/L. Gradient mobile phases are used to allow separation of the 4 target compounds in more complex mixture of other concomitant compounds. Mass spectra are used to confirm analyte identity with chromatographic retention time.

  2. Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry.

    PubMed

    McCoy, Leslie F; Scholl, Peter F; Sutcliffe, Anne E; Kieszak, Stephanie M; Powers, Carissa D; Rogers, Helen S; Gong, Yun Yun; Groopman, John D; Wild, Christopher P; Schleicher, Rosemary L

    2008-07-01

    Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B(1). In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.

  3. Identification of monofloral honeys using HPLC-ECD and chemometrics.

    PubMed

    Zhao, Jing; Du, Xiaojing; Cheng, Ni; Chen, Lanzhen; Xue, Xiaofeng; Zhao, Jing; Wu, Liming; Cao, Wei

    2016-03-01

    A total of 77 jujube, longan and chaste honey samples were collected from 18 different areas of China. Thirteen types of phenolic acids in the honey samples were analysed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Moreover, HPLC-ECD fingerprints of the monofloral honey samples were established. From the analysis of the HPLC-ECD fingerprints, common chromatography peak information was obtained, and principal component analysis and discriminant analysis were performed using selected common chromatography peak areas as variables. By comparing with phenolic acids as variables, using a chemometric analysis which is based on the use of common chromatography peaks as variables, 36 honey samples and 41 test samples could be correctly identified according to their floral origin.

  4. Study of rat hypothalamic proteome by HPLC/ESI ion trap and HPLC/ESI-Q-TOF MS.

    PubMed

    Iqbal, Javed; Li, Wang; Ullah, Kaleem; Hasan, Murtaza; Linna, Guo; Awan, Umer; Zhang, Yongqian; Batool, Sajida; Qing, Hong; Deng, Yulin

    2013-08-01

    The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI-ion trap and HPLC/ESI-quadrupole-TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI-ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI-quadrupole-TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole-TOF while 62 were found with the HPLC/ESI-ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used.

  5. A new HPLC method to determine Donepezil hydrochloride in tablets.

    PubMed

    Pappa, Horacio; Farrú, Romina; Vilanova, Paula Otaño; Palacios, Marcelo; Pizzorno, María Teresa

    2002-01-01

    A HPLC stability-indicating assay for Donepezil hydrochloride in tablets was developed and validated. Donepezil hydrochloride is a reversible inhibitor of acetylcholinesterase, indicated for the treatment of mild to moderate dementia of the Alzheimer's type. The HPLC method was performed with a reversed phase C18 column, detection at 268 nm and a mixture of methanol, phosphate buffer 0.02 M and triethylamine (50:50:0.5) as mobile phase. Typical retention time for Donepezil was 9 min. The method was statistically validated for linearity, accuracy, precision and selectivity following ICH recommendations. Due to its simplicity and accuracy, the method can be used for routine quality control analysis.

  6. Analysis of limette and bergamot distilled essential oils by HPLC.

    PubMed

    Buiarelli, Francesca; Cartoni, Giampaolo; Coccioli, Franco; Jasionowska, Renata; Mazzarino, Monica

    2002-04-01

    This work examines the distilled essential oils of limette and bergamot in order to assess the presence of low volatile substances such as coumarins (bergapten) which, being toxic, must be eliminated before using these oils in the food industry. The quantitative determination of coumarins was carried out by spectrofluorimetric detection. The substances present in the chromatograms, obtained by HPLC with UV detection at 254 nm, were then identified. Moreover, a new coumarin that is present in small quantities was identified using HPLC-MS.

  7. HPLC ANALYSIS OF VINCLOZOLIN AND ITS METABOLITES IN SERUM

    EPA Science Inventory


    HPLC ANALYSIS OF VINCLOZOLIN AND ITS METABOLITES IN SERUM. A Sierra-Santoyo1,2, H A Barton1 and M F Hughes1. 1US EPA, ORD, NHEERL, ETD, RTP, NC; 2Toxicology Section, CINVESTAV-IPN, Mexico City, Mexico.

    The fungicide vinclozolin (V) is used predominantly for treatment...

  8. Separation of kafirins on surface porous RP-HPLC columns

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Surface porous HPLC columns were investigated for the separation of kafarins, storage proteins of grain sorghum. Kafirins were successfully separated using C3, C8 and C18 surface porous stationary phases in less than 17 min. Separations using a monolithic C18 stationary phase were also developed ...

  9. Lipid analysis via HPLC with a charged aerosol detector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most lipid extracts are a mixture of saturated and unsaturated molecules. Therefore, the most successful HPLC detectors for the quantitative analysis of lipids have involved the use of “universal” or “mass” detectors such as flame ionization detectors (FID) and evaporative light scattering detectors...

  10. Methods and applications of HPLC-AMS (WBio 5)

    SciTech Connect

    Bucholz, B A; Clifford, A J; Duecker, S R; Lin, Y; Vogel, J S

    1999-09-29

    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. 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.

  11. Capillary electrophoresis as an orthogonal technique in HPLC method validation.

    PubMed

    Jimidar, M Ilias; De Smet, Maurits; Sneyers, Rudy; Van Ael, Willy; Janssens, Willy; Redlich, Dirk; Cockaerts, Paul

    2003-01-01

    High-performance liquid chromatography is usually used to assay the main compound and organic impurity content of drug substance and drug product during pharmaceutical development. A crucial validation parameter of these methods is specificity--the ability to unequivocally assess the analyte in the presence of component expected to be present. Typically, these include impurities, degradation products, and matrices. Besides adequate chromatographic separation with sufficient selectivity, additional 2- or 3-D spectroscopic or chromatographic tools are frequently necessary for this purpose. In our current practice, HPLC is used with ultraviolet photodiode array detection and on-line mass spectrometry (LC-UVDAD-MS) during the assessment of specificity. Although this approach is very powerful and can solve the majority of problems, separation of isomers of the main compound is still difficult. Since HPLC usually cannot offer the required selectivity and because of the similar molecular weights, structural isomers are not specifically detected using LC-MS. Capillary electrophoresis, on the other hand, offers high separation efficiency and can be applied as an adjunct to HPLC. Therefore, a set of highly selective CE methods is used orthogonally in the specificity assessment of HPLC methods.

  12. Extraction, Separation, and Identification of Phenolic Compounds in Virgin Olive Oil by HPLC-DAD and HPLC-MS

    PubMed Central

    Tasioula-Margari, Maria; Tsabolatidou, Eleftheria

    2015-01-01

    The aim of this study was to evaluate the recovery of individual phenolic compounds extracted from virgin olive oil (VOO), from different Greek olive varieties. Sufficient recoveries (90%) of all individual phenolic compounds were obtained using methanol as an extraction solvent, acetonitrile for residue solubilization, and two washing steps with hexane. Moreover, in order to elucidate structural characteristics of phenolic compounds in VOO, high performance liquid chromatography with a diode array detector (HPLC-DAD) at 280 and 340 nm and HPLC coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS) in the negative-ion mode were performed. The most abundant phenolic compounds were oleuropein derivatives with m/z 319 and 377 and ligstroside derivatives with m/z 303, 361. Lignans, such as 1-acetoxypinoresinol and pinoresinol were also present in substantial quantities in the phenolic fraction. However, pinoresinol was co-eluted with dialdehydic form of ligstroside aglycone (DAFLA) and it was not possible to be quantified separately. The phenolic extracts, obtained from different VOO samples, yielded similar HPLC profiles. Differences, however, were observed in the last part of the chromatogram, corresponding to isomers of the aldehydic form of ligstroside aglycone. Oxidized phenolic products, originating from secoiridoids, were also detected. PMID:26783843

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. HPLC determination of imidazole antimycotis in antidandruff cosmetic products.

    PubMed

    Gagliardi, Luigi; De Orsi, Daniela; Chimenti, Paola; Porra', Rita; Tonelli, Domenica

    2003-08-01

    A simple HPLC method for the determination of imidazole antimycotics in cosmetic antidandruff formulations has been developed. HPLC was carried out on a Discovery RP-Amide C16 column and spectrophotometric detection was performed at 220 nm. The initial mobile phase was a mixture of acetonitrile and aqueous 10(-3) M NaClO4 (pH 3.0) in the ratio of 15:85 (v/v); then a linear gradient up to 46% acetonitrile in 70 min, and up to 50% in 80 min. The extraction procedure has been validated by analyzing samples of shampoo and lotion spiked with 1% of the active principles. The recoveries were greater than 95% and the reproducibility was within 3%.

  15. Analysis of Biomass Sugars Using a Novel HPLC Method

    SciTech Connect

    Agblevor, F. A.; Hames, B. R.; Schell, D.; Chum, H. L.

    2007-01-01

    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 and 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).

  16. Cholecalciferol sulfate identification in human milk by HPLC.

    PubMed

    Boulch, N L; Cancela, L; Miravet, L

    1982-04-01

    Synthetic vitamin D3 sulfate was prepared by reacting cholecalciferol with sulfamic acid in pyridine. Vitamin D3 sulfate ammonium salt was purified by crystallisation and transformed in sulfate sodium salt. Homogeneity was controlled by reverse phase high pressure liquid chromatography (HPLC). Purified synthetic vitamin D3 sulfate sodium salt was used as a reference. Milk whey was obtained after protein precipitation by adding ethanol. Vitamin D3 sulfoconjugate was identified in supernatant (lyophylized) after purification by Sephadex LH 20 and HPLC. Milk whey purified fraction obtained exhibited the same ultra-violet absorption (UV) as synthetic vitamin D3 sulfate; after solvolysis, cholecalciferol was liberated from natural and synthetic sulfoconjugate. The results confirmed that vitamin D3 sulfate was present in human milk. PMID:6294930

  17. HPLC-ICP atomic emission spectrometry in environmental analysis

    SciTech Connect

    Frame, E.M.S.

    1996-10-01

    Trace concentrations of many compounds are difficult to determine directly by traditional chromatographic techniques. Organosilicon compounds, for example, have no chromophoric groups. Size exclusion (SEC) and high performance liquid chromatography (HPLC) can separate high and low molecular weight organosilicon compounds, but routinely employ detectors with insufficient sensitivity for trace concentrations in environmental samples. Elemental analysis methods such as inductively coupled plasma optical emission spectrometry (ICP) provide excellent element sensitivity but no speciation information. Interfacing of a chromatographic method with ICP has been shown to provide quantitative determination of trace compounds while retaining the chemical speciation information. This work will describe the power of the HPLC-ICP combination for trace compounds in environmental samples to identify and determine ppm levels of organometallics.

  18. Gradient Scouting in Reversed-Phase HPLC Revisited

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  19. Surface confined ionic liquid as a stationary phase for HPLC

    SciTech Connect

    Wang, Qian; Baker, Gary A; Baker, Sheila N; Colon, Luis

    2006-01-01

    Trimethoxysilane ionosilane derivatives of room temperature ionic liquids based on alkylimidazolium bromides were synthesized for attachment to silica support material. The derivatives 1-methyl-3-(trimethoxysilylpropyl)imidazolium bromide and 1-butyl-3-(trimethoxysilylpropyl)imidazolium bromide were used to modify the surface of 3 {micro}m diameter silica particles to act as the stationary phase for HPLC. The modified particles were characterized by thermogravimetric analysis (TGA) and {sup 13}C and {sup 29}Si NMR spectroscopies. The surface modification procedure rendered particles with a surface coverage of 0.84 {micro}mol m{sup -2} for the alkylimidazolium bromide. The ionic liquid moiety was predominantly attached to the silica surface through two siloxane bonds of the ionosilane derivative (63%). Columns packed with the modified silica material were tested under HPLC conditions. Preliminary evaluation of the stationary phase for HPLC was performed using aromatic carboxylic acids as model compounds. The separation mechanism appears to involve multiple interactions including ion exchange, hydrophobic interaction, and other electrostatic interactions.

  20. [Validation of a HPLC method for ochratoxin A determination].

    PubMed

    Bulea, Delia; Spac, A F; Dorneanu, V

    2011-01-01

    Ochratoxin A is a mycotoxin produced by various species of Aspergillus and Penicillium. Ochratoxin A has been detected in cereals and cereal products, coffee beans, beer, wine, spices, pig's kidney and cow's milk. For ochratoxin A, a HPLC method was developed and validated. Ochratoxin A was determined by RP-HPLC, using a liquid chromatograph type HP 1090 Series II, equiped with a fluorescence detector. The analysis was performed with a Phenomenex column, type Luna C18(2) 100A (150 x 4.6 mm; 5 microm) with a mobile phase consisting of a mixture of acetonitrile/water/acid acetic (99/99/2), a flow of 0.7 mL/min. For detection, the wavelenght of excitation was 228 nm and wavelenght of emision was 423 nm. The calibration graph was linear in 6.25-50 ng/mL concentration range (r2 = 0,9991). The detection limits was 1.6 ng/mL and the quantification limit was 4.9 ng/mL. The method precision (RSD = 2.4975%) and the accuracy (recovery was 100.1%) were studied. The HPLC method was applyed for ochratoxin A from food samples with good results. PMID:21870763

  1. Rapid screening of enzyme inhibitors using profiling of enzyme-metabolite assay by HPLC (PREMA-HPLC).

    PubMed

    Vasantha, K Y; Murugesh, C S; Sattur, A P

    2012-02-01

    A number of isolates from different ecosystems were screened for their ability to inhibit tyrosinase resulting in the selection of isolate CFR 101, which showed an inhibition of 72%. The metabolites present in the crude extract of the selected isolate was profiled through high-performance liquid chromatography (HPLC) before the enzyme inhibition assay to reveal a 66% decrease in area of the peak at room temperature for 13.9 min, after the assay. Upon purification, this peak was identified as kojic acid, a known inhibitor of tyrosinase. This unique technique of combining a reaction assay mixture with HPLC profile wherein inhibitors can be rapidly pinpointed in crude extracts addresses the drawback of rapid chemical high-throughput screening (HTS) systems, which is limited to the chemical nature of metabolites without any evidence of their biological activities.

  2. Qualitative and quantitative analysis of chemical constituents of Centipeda minima by HPLC-QTOF-MS & HPLC-DAD.

    PubMed

    Chan, Chi-On; Jin, Deng-Ping; Dong, Nai-Ping; Chen, Si-Bao; Mok, Daniel Kam Wah

    2016-06-01

    A high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS) method in both positive and negative ion modes was established to investigate the major constituents in the ethanolic extract of Centipeda minima (EBSC). Twelve common components including flavones and their glycosides, phenolic and polyphenolic acids, and sesquiterpene lactone were identified in ten batches of samples based on comparison with the retention time and accurate mass of external standards (mass accuracy within 3ppm) or the fragmentation patterns of tandem MS. Meanwhile, a simple, accurate and reliable HPLC-DAD method was also developed to determine the content of 10 chemical markers simultaneously. Results obtained from method validations including linearity, accuracy and precision showed that this new method is reliable and robust. Isochlorogenic acid A and brevilin A were found to be the most abundant in the ethanol extract of EBSC and could be served as markers for quality control of EBSC.

  3. Analysis of a simulated heroin distribution chain by HPLC.

    PubMed

    Zelkowicz, Avraham; Magora, Amir; Ravreby, Mark D; Levy, Rina

    2005-07-01

    A heroin distribution chain was simulated by taking three different seizures and preparing four additional samples from each seizure by adding a paracetamol-caffeine mixture in varying amounts, resulting in three different batches each composed of five samples. All of the samples from the three batches were analyzed using HPLC with a UV-PDA detector at a wavelength of 230 nm. The area ratio of various opium alkaloids, acetylation products and components were compared. From the results of the UV area ratios, the fifteen samples could readily be separated into three batches of five samples, with each batch of five samples having a common origin.

  4. Coral Pigments: Quantification Using HPLC and Detection by Remote Sensing

    NASA Technical Reports Server (NTRS)

    Cottone, Mary C.

    1995-01-01

    Widespread coral bleaching (loss of pigments of symbiotic dinoflagellates), and the corresponding decline in coral reef health worldwide, mandates the monitoring of coral pigmentation. Samples of the corals Porites compressa and P. lobata were collected from a healthy reef at Puako, Hawaii, and chlorophyll (chl) a, peridinin, and Beta-carotene (Beta-car) were quantified using reverse-phase high performance liquid chromatography (HPLC). Detailed procedures are presented for the extraction of the coral pigments in 90% acetone, and the separation, identification, and quantification of the major zooxanthellar pigments using spectrophotometry and a modification of the HPLC system described by Mantoura and Llewellyn (1983). Beta-apo-8-carotenal was found to be inadequate as in internal standard, due to coelution with chl b and/or chl a allomer in the sample extracts. Improvements are suggested, which may result in better resolution of the major pigments and greater accuracy in quantification. Average concentrations of peridinin, chl a, and Beta-car in corals on the reef were 5.01, 8.59, and 0.29, micro-grams/cm(exp 2), respectively. Average concentrations of peridinin and Beta-car did not differ significantly between the two coral species sampled; however, the mean chl a concentration in P. compressa specimens (7.81 ,micro-grams/cm(exp 2) was significantly lower than that in P. lobata specimens (9.96 11g/cm2). Chl a concentrations determined spectrophotometrically were significantly higher than those generated through HPLC, suggesting that spectrophotometry overestimates chl a concentrations. The average ratio of chl a-to-peridinin concentrations was 1.90, with a large (53%) coefficient of variation and a significant difference between the two species sampled. Additional data are needed before conclusions can be drawn regarding average pigment concentrations in healthy corals and the consistency of the chl a/peridinin ratio. The HPLC pigment concentration values

  5. Determination of Sinomenine in Cubosome Nanoparticles by HPLC Technique

    PubMed Central

    Zhou, Yanfang; Guo, Chunlian; Chen, Hongying; Zhang, Yudai; Peng, Xinsheng; Zhu, Ping

    2015-01-01

    We applied HPLC technique to quantitatively analyze sinomenine in cubosome nanoparticles. The chromatographic method was performed by using an isocratic system. The mobile phase was composed of methanol-PBS(pH6.8)-triethylamine (50 : 50 : 0.1%) with a flow rate of 1 mL/min; the detection wavelength was at 265 nm. Sinomenine can be successfully separated with good linearity (the regression equation is A = 10835C + 1058; R2 = 1.0) and perfect recovery (102.2%). The chromatograph technique was proper for quality control of sinomenine in cubosome nanoparticles. PMID:25734024

  6. Determination of pindolol in human serum by HPLC.

    PubMed

    Chmielowiec, D; Schuster, D; Gengo, F

    1991-01-01

    A method for determining pindolol from human serum by high-performance liquid chromatography (HPLC) is presented. Pindolol is extracted into methylene chloride from 1 mL of alkalinized serum with a recovery of 87%. The organic layer is evaporated and the residue is reconstituted in mobile phase for injection into the column. Samples are eluted from a 5-micron C18 column (250 x 4.6 mm) with acetonitrile-water containing 0.1% triethylamine, pH adjusted to 3.5 with phosphoric acid (20:80 v/v). Samples as low as 2 ng/mL have been detected.

  7. [Analysis of HPLC and NIRS fingerprints of Chrysanthemum indicum of different processing methods].

    PubMed

    Hu, Xiao-li; Bai, Yan; Lei, Jing-wei; Zhang, Di-wen; Hao, Min

    2015-10-01

    This paper studied the HPLC and NIRS fingerprints of Chrysanthemum with different processing methods, including directly drying, drying after steamed, and drying after fried. The method of discriminant analysis of TQ software was used to analysis the NIRS fingerprint of Chrysanthemum with three different processing methods, and the results were consistent with HPLC fingerprint similarity analysis. NIRS and HPLC fingerprints were of different characteristics, and the combination of the two methods can quickly and accurately identify Chrysanthemum with different processing methods. PMID:26975109

  8. 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.

  9. 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. PMID:15158993

  10. Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC-ESI-MS-MS.

    PubMed

    Stecher, G; Huck, C W; Popp, M; Bonn, G K

    2001-09-01

    To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL(-1)-50 microg mL(-1)). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL(-1)-50 microg mL(-1)) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC-ESI-MS method was highly suitable and an essential improvement compared

  11. Determination of capsinoids by HPLC-DAD in capsicum species.

    PubMed

    Singh, Satyavan; Jarret, Robert; Russo, Vincent; Majetich, George; Shimkus, Joel; Bushway, Rodney; Perkins, Brian

    2009-05-13

    Capsicum fruits contain a newly discovered phytochemical called capsinoids. Because little is known about the quantities of these compounds in both sweet and pungent pepper fruits, a high-performance liquid chromatography (HPLC) method was developed to identify and quantify the capsinoids (naturally present E-capsiate and dihydrocapsiate) utilizing fruit obtained from a variety of Capsicum spp. in the U.S. Department of Agriculture's Capsicum germplasm collection. Capsinoids were extracted with acetonitrile, filtered, and analyzed using an HPLC system equipped with a C(18) monolithic column, gradient pump, and diode array detector. The elution solvents were acetonitrile and water (60:40) with an isocratic flow rate of 1.0 mL/min. Forty-nine samples representing distinct morphotypes of four cultivated species ( C. annuum var. annuum, C. annuum var. glabriusculum, C. baccatum , C. chinense , and C. frutescens ) contained detectable levels (11-369 microg/g) of E-capsiate quantified at a wavelength of 280 nm. Nine of the E-capsiate-containing samples also had dihydrocapsiate (18-86 micro/g). Gas chromatography with a mass spectrometry detector (GC-MS) confirmed the presence of these compounds in the Capsicum spp.

  12. Development of SPME-HPLC methodology for detection of nitroexplosives

    NASA Astrophysics Data System (ADS)

    Peña-Luengas, Sandra L.; Jerez-Rozo, Jackeline I.; Correa, Sandra N.; Peña, Nelson E.; Hernández-Rivera, Samuel P.

    2007-04-01

    Solid phase microextraction (SPME) has been coupled with liquid chromatography to widen its range of application to nonvolatile and thermally unstable compounds, generally limited for SPME-GC. A method for analysis of nitroaromatic explosives and its degradations products was developed by coupling SPME and high performance liquid chromatography with ultraviolet detection (HPLC/UV), introducing a modified interface that ensure accuracy, precision, repeatability, high efficiency, unique selectivity and high sensitive to detection and quantification of explosives from surface soil samples and increased chromatographic efficiency. A pretreatment step was introduced for the soil samples which extracted the target compounds into an aqueous phase. Several parameters that affect the microextraction were evaluated, such as: fiber coating, adsorption and desorption time and stirring rate. The effect of salting out (NaCl) on analyte extraction and the role of various solvents on SPME fiber were also evaluated. Carbowax-templated resin (CW/TPR) and Polydimethilsiloxane-divinilbenzene (PDMS-DVB) fibers were used to extract the analytes from the aqueous samples. Explosives were detected at low μg/mL concentrations. This study demonstrates that SPME-HPLC is a very promising method of analysis of explosives from aqueous samples and has been successfully applied to the determination of nitroaromatic compounds, such as TNT.

  13. HPLC analysis as a tool for assessing targeted liposome composition.

    PubMed

    Oswald, Mira; Platscher, Michael; Geissler, Simon; Goepferich, Achim

    2016-01-30

    Functionalized phospholipids are indispensable materials for the design of targeted liposomes. Control over the quality and quantity of phospholipids is thereby key in the successful development and manufacture of such formulations. This was also the case for a complex liposomal preparation composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), Cholesterol (CHO), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000). To this end, an RP-HPLC method was developed. Detection was done via evaporative light scattering (ELS) for liposomal components. The method was validated for linearity, precision, accuracy, sensitivity and robustness. The liposomal compounds had a non-linear quadratic response in the concentration range of 0.012-0.42 mg/ml with a correlation coefficient greater than 0.99 with an accuracy of method confirmed 95-105% of the theoretical concentration. Furthermore, degradation products from the liposomal formulation could be identified. The presented method was successfully implemented as a control tool during the preparation of functionalized liposomes. It underlined the benefit of HPLC analysis of phospholipids during liposome preparation as an easy and rapid control method for the functionalized lipid at each preparation step as well as for the quantification of all components.

  14. [Determination of furbenicillin sodium and its related substances by HPLC].

    PubMed

    Chang, Yan; Tian, Ye; Ma, Yue; Hu, Chang-qin

    2015-12-01

    Furbenicillin is a broad-spectrum semisynthetic penicillin with strong antibacterial activity against Gram-negative bacteria. Furbenicillin sodium is determined by volumetric method in current criteria. However, the criteria does not contain an assay of related substances of furbenicillin sodium. In this study, we established a method for detection and analysis of furbenicillin sodium and its related substances by HPLC. The analysis was performed with a C18 column under a gradient elution, the detection wavelength was 225 nm, and the column temperature was 35 degrees C. The reliability and accuracy of established method was validated in this study. Pure samples of furbenicillin sodium and its related substances were prepared. The structures, biological activities, and chromatographic retention behaviors of furbenicillin sodium and its related substances were identified using NMR, CLSI agar dilution method, and HPLC. All results in the current study provide ample evidence that this method is able to determine the reasonable limits in the quality-control protocol for furbenicillin sodium. PMID:27169288

  15. HPLC assisted Raman spectroscopic studies on bladder cancer

    NASA Astrophysics Data System (ADS)

    Zha, W. L.; Cheng, Y.; Yu, W.; Zhang, X. B.; Shen, A. G.; Hu, J. M.

    2015-04-01

    We applied confocal Raman spectroscopy to investigate 12 normal bladder tissues and 30 tumor tissues, and then depicted the spectral differences between the normal and the tumor tissues and the potential canceration mechanism with the aid of the high-performance liquid chromatographic (HPLC) technique. Normal tissues were demonstrated to contain higher tryptophan, cholesterol and lipid content, while bladder tumor tissues were rich in nucleic acids, collagen and carotenoids. In particular, β-carotene, one of the major types of carotenoids, was found through HPLC analysis of the extract of bladder tissues. The statistical software SPSS was applied to classify the spectra of the two types of tissues according to their differences. The sensitivity and specificity of 96.7 and 66.7% were obtained, respectively. In addition, different layers of the bladder wall including mucosa (lumps), muscle and adipose bladder tissue were analyzed by Raman mapping technique in response to previous Raman studies of bladder tissues. All of these will play an important role as a directive tool for the future diagnosis of bladder cancer in vivo.

  16. Spectrophotometric and HPLC determination of secnidazole in pharmaceutical tablets.

    PubMed

    El Wallily, A F; Abdine, H H; Razak, O A; Zamel, S

    2000-07-01

    Simple and accurate spectrophotometric and HPLC methods were developed for the determination of secnidazole in tablets dosage form. The first spectrophotometric method depends on the reduction of secnidazole molecule with zinc dust and hydrochloric acid followed by condensation with either p-dimethylaminobenzaldehyde or anisaldehyde to give colored chromogens having absorbance at 494 and 398 nm, respectively. The second method was based on the reaction of the drug with sodium nitroprusside in the presence or absence of hydroxylammonium hydrochloride. The formed colored chromogens were measured at 584 and 508 nm, respectively. The experimental conditions were optimized and Beer's law was obeyed over the applicable concentration ranges. The application of HPLC procedures depended on using either a conventional or microbore reverse-phase (C18) column along with mobile phases consisting of water and methanol (30:70), at pH of 3.5. Both techniques were applied successfully for the analysis of secnidazole in tablets form. The results obtained from both procedures were statistically compared using the Student's-t and F-variance ratio tests. PMID:10857557

  17. 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.

  18. Evaluation of TEGDMA leaching from four resin cements by HPLC

    PubMed Central

    Altintas, Subutay Han; Usumez, Aslihan

    2012-01-01

    Objective The aim of this study was to evaluate the elution of TEGDMA from dual cured resin cements, used for bonding of ceramic restoration by high performance liquid chromatography (HPLC). Methods: Forty freshly extracted caries and restoration free molar teeth used in this study. Standardized Class I preparations were prepared in all teeth. Ceramic inlays were cemented with one of the dual cured resin cements (Variolink II, Rely X ARC, Rely X Unicem and Resilute). After cementation, specimens were stored in 75% ethanol solution. HPLC was used to analyze the amounts of TEGDMA in different time intervals. Two-way ANOVA and Tukey HSD tests were used to evaluate the results (P<.05). Results: The amount of TEGDMA eluted from Resilute was the highest and the amount of TEG-DMA eluted from Rely X Unicem was the lowest (P<.05). The total amount of monomers was the highest after 21 days (P<.05). Conclusion: In the case of resin cements, elution of TEGDMA was the highest in Resilute and lowest in Rely X Unicem. The amount of TEGDMA eluted from resin cements was influenced by the time. PMID:22904653

  19. Determination of anthocyanins from camu-camu (Myrciaria dubia) by HPLC-PDA, HPLC-MS, and NMR.

    PubMed

    Zanatta, Cinthia Fernanda; Cuevas, Elyana; Bobbio, Florinda O; Winterhalter, Peter; Mercadante, Adriana Z

    2005-11-30

    Camu-camu [Myrciaria dubia (HBK) McVaugh] is a small fruit native to the Amazonian rain forest. Its anthocyanin profile has now been investigated for the first time. Fruits from two different regions of the São Paulo state, Brazil, were analyzed. The major anthocyanins were isolated by high-speed countercurrent chromatography. HPLC-PDA, HPLC-MS/MS, and 1H NMR were used to confirm the identity of the main anthocyanins of camu-camu. Cyanidin-3-glucoside was identified as the major pigment in the fruits from both regions, representing 89.5% in the fruits produced in Iguape and 88.0% in those from Mirandópolis, followed by the delphinidin-3-glucoside, ranging between 4.2 and 5.1%, respectively. Higher total anthocyanin contents were detected in the fruits from Iguape (54.0 +/- 25.9 mg/100 g) compared to those from Mirandópolis (30.3 +/- 6.8 mg/100 g), most likely because of the lower temperatures in the Iguape region. PMID:16302773

  20. Characterization and quantitation of anthocyanins in purple-fleshed sweet potatoes cultivated in Korea by HPLC-DAD and HPLC-ESI-QTOF-MS/MS.

    PubMed

    Lee, Mi Jin; Park, Jeong Seob; Choi, Dong Seong; Jung, Mun Yhung

    2013-03-27

    The characterization and quantitative analysis of anthocyanins in four purple-fleshed sweet potato varieties (Borami, Mokpo 62, Shinzami, and Zami) cultivated in Korea were carried out by HPLC/diode array detector (DAD), HPLC-TOF/MS, and HPLC-MS/MS analyses. For the identification of anthocyanins, molecular formulas were first calculated by using the exact mass data of the molecular ions ([M](+)). The patterns of isotope ions of M(+) were also monitored to confirm the assignment of the molecular formulas. HPLC-MS(2) analysis was further conducted for elucidating their molecular structures. Twenty-seven different anthocyanins were tentatively identified in the sweet potatoes. Six of them are the first reported in sweet potatoes roots. The quantity and profiles of anthocyanins in sweet potatoes varied greatly with variety. Borami was found, for the first time, to be a rare sweet potato variety with an exceptionally high quantity of pelargonidin-based anthocyanins.

  1. Characterization and quantitation of anthocyanins in purple-fleshed sweet potatoes cultivated in Korea by HPLC-DAD and HPLC-ESI-QTOF-MS/MS.

    PubMed

    Lee, Mi Jin; Park, Jeong Seob; Choi, Dong Seong; Jung, Mun Yhung

    2013-03-27

    The characterization and quantitative analysis of anthocyanins in four purple-fleshed sweet potato varieties (Borami, Mokpo 62, Shinzami, and Zami) cultivated in Korea were carried out by HPLC/diode array detector (DAD), HPLC-TOF/MS, and HPLC-MS/MS analyses. For the identification of anthocyanins, molecular formulas were first calculated by using the exact mass data of the molecular ions ([M](+)). The patterns of isotope ions of M(+) were also monitored to confirm the assignment of the molecular formulas. HPLC-MS(2) analysis was further conducted for elucidating their molecular structures. Twenty-seven different anthocyanins were tentatively identified in the sweet potatoes. Six of them are the first reported in sweet potatoes roots. The quantity and profiles of anthocyanins in sweet potatoes varied greatly with variety. Borami was found, for the first time, to be a rare sweet potato variety with an exceptionally high quantity of pelargonidin-based anthocyanins. PMID:23464823

  2. Ellagitannin Composition of Blackberry As Determined by HPLC-ESI-MS and MALDI-TOF-MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apache blackberries (Rubus sp.) were evaluated by HPLC-MS and MALDI-TOF-MS to identify ellagitannins present in the flesh, torus (receptacle tissue), and seeds. Most ellagitannins were only present or detectable in seed tissues. Ellagitannins identified by HPLC-MS in the seeds included pedunculagi...

  3. Size exclusion HPLC of proteins for evaluation of durum wheat quality

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The present research aimed to assess size exclusion HPLC (SE-HPLC) in protein molecular weight distribution determination for quality evaluation of durum semolina. Semolina samples were milled from 13 durum genotypes grown at 7 locations in 2009 and 2010 in ND. Sodium dodecyl sulfate (SDS) buffer ...

  4. Chemical Profiling (HPLC-NMR & HPLC-MS), Isolation, and Identification of Bioactive Meroditerpenoids from the Southern Australian Marine Brown Alga Sargassum paradoxum

    PubMed Central

    Brkljača, Robert; Urban, Sylvia

    2014-01-01

    A phytochemical investigation of a southern Australian marine brown alga, Sargassum paradoxum, resulted in the isolation and identification of four new (5, 9, 10, and 15) and nine previously reported (1, 2, 6–8, and 11–14) bioactive meroditerpenoids. HPLC-NMR and HPLC-MS were central to the identification of a new unstable compound, sargahydroquinal (9), and pivotal in the deconvolution of eight (1, 2, 5–7, and 10–12) other meroditerpenoids. In particular, the complete characterization and identification of the two main constituents (1 and 2) in the crude dichloromethane extract was achieved using stop-flow HPLC-NMR and HPLC-MS. This study resulted in the first acquisition of gHMBCAD NMR spectra in the stop-flow HPLC-NMR mode for a system solely equipped with a 60 μL HPLC-NMR flow cell without the use of a cold probe, microcoil, or any pre-concentration. PMID:25551779

  5. 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.

  6. Quantitative analysis of PMR-15 polyimide resin by HPLC

    NASA Technical Reports Server (NTRS)

    Roberts, Gary D.; Lauver, Richard W.

    1987-01-01

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

  7. Validated HPLC method for quantifying permethrin in pharmaceutical formulations.

    PubMed

    García, E; García, A; Barbas, C

    2001-03-01

    An isocratic HPLC method for permethrin determination in raw material and pharmaceutical presentations as lotion and shampoo has been developed and validated following ICH recommendations. Cis and trans- isomers, impurities and degradation products are well separated. The chromatographic analysis were performed on a 4 microm particle C-18 Nova-Pak (Waters, Madrid, Spain) column (15 x 0.39 cm) kept in a Biorad column oven at 35 degrees C. Mobile phase consisted of methanol--water (78:22, v/v) at a flow rate of 1 ml/min. UV detection was performed at 272 nm and peaks were identified with retention times as compared with standards and confirmed with characteristic spectra using the photodiode array detector.

  8. [Determination of cefixime blood plasma levels by HPLC].

    PubMed

    Pisarev, V V; Zaĭtseva, K V; Smirnova, L B; Belolipetskaia, V G; Kibal'chich, D A; Koltunov, I E

    2009-01-01

    For comparative study of the pharmacokinetics of Cemidexor (capsules of 100 mg) and Suprax (capsules of 400 mg), a method of HPLC with quantitative determination of cefixime (the active substance in the drugs) in the blood plasma of patients with UV detection was developed. The data teproducibility with an account of the admissibility criterion was observed within the interval of all the concentrations (0.06-10 mcg/ml). The accuracy and correctness of the method also corresponded to the admissibility criteria. The lower limit of the quantitative determimation of the cefexime blood plasma levels was 0.06 mcg/ml. The pharmacokinetics was studied with the open crossed randomized method. The results were used for calculation of the pharmacokinetic parameters required for estimation of the bioequivalence of the drugs. The statistical analysis of the pharmacokinetic parameters showed that Cemidoxor and Suprax were bioequivalent.

  9. In vitro monitoring of ciprofloxacin antacids interactions by UV & HPLC.

    PubMed

    Sultana, Najma; Arayne, M Saeed; Hussain, Fida

    2005-10-01

    Ciprofloxacin or 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl]-3-quinoline carboxylic acid is a fluorinated quinolone antibacterial agent extensively used worldwide. There are number of drug interactions reported for this antibacterial agent. In present studies, in vitro release of ciprofloxacin hydrochloride in presence of various antacids like sodium bicarbonate, calcium hydroxide, calcium carbonate, aluminum hydroxide, magnesium hydroxide, magnesium carbonate, magnesium trisilicate and magaldrate has been studied on BP 2002 dissolution test apparatus. Drug in each case was analyzed either by measuring the absorbance of aliquots at 278 and 316 nm on a UV/VIS spectrophotometer, or by reversed-phase high-performance liquid chromatographic (RP-HPLC) method. These studies were carried out in simulated gastric and intestinal juices for three hours at 37 degrees C. The availability of ciprofloxacin was found to be markedly retarded in presence of all the antacids studied.

  10. Quantitative and qualitative HPLC analysis of thermogenic weight loss products.

    PubMed

    Schaneberg, B T; Khan, I A

    2004-11-01

    An HPLC qualitative and quantitative method of seven analytes (caffeine, ephedrine, forskolin, icariin, pseudoephedrine, synephrine, and yohimbine) in thermogenic weight loss preparations available on the market is described in this paper. After 45 min the seven analytes were separated and detected in the acetonitrile: water (80:20) extract. The method uses a Waters XTerra RP18 (5 microm particle size) column as the stationary phase, a gradient mobile phase of water (5.0 mM SDS) and acetonitrile, and a UV detection of 210 nm. The correlation coefficients for the calibration curves and the recovery rates ranged from 0.994 to 0.999 and from 97.45% to 101.05%, respectively. The qualitative and quantitative results are discussed. PMID:15587578

  11. Characterization of nutraceuticals and functional foods by innovative HPLC methods.

    PubMed

    Corradini, Claudio; Galanti, Roberta; Nicoletti, Isabella

    2002-04-01

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

  12. Contamination in HPLC quantified benzaldehyde is from polypropylene microtubes.

    PubMed

    Shemesh, Tomer; Karschimkus, Connie; Rowley, Kevin G

    2005-11-01

    Semicarbazide-sensitive amine oxidase (SSAO; EC 1.4.3.6) is a copper-containing enzyme predominantly expressed by vascular smooth muscle cells. SSAO deaminates primary amines to produce aldehydes and oxygen peroxides, and may thus play a role in vascular damage. SSAO activity can be quantified by assaying benzaldehyde production using fluorescent derivatisation and separation by HPLC. We performed the derivatisation step in polypropylene or borosilicate glass tubes over 45 min at 95 degrees C. High and obstructing background levels of benzaldehyde were found in one batch of polypropylene vials, as opposed to its alternatives. Treatment and handling of product shipment into the country did not account for introduction of contaminant into packaged vials nor did any reagent used in the assay. We conclude that the source of contamination was most likely due to variation in the commercial production process. Use of borosilicate vials for assays based on aldehyde production and derivatisation is recommended.

  13. HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis

    PubMed Central

    Srinivasan, G. V.; Unnikrishnan, K. P.; Rema Shree, A. B.; Balachandran, Indira

    2008-01-01

    A high-performance liquid chromatographic method for the estimation of berberine in the stem of Tinospora cordifolia (Willd.) Miers. ex Hook.f. and Thoms. and Tinospora sinensis (Lour.) Merrill is described. The dried stems of T. cordifolia and T. sinensis were defatted with petroleum ether (60-80°). The marc was dried and further extracted with methanol. The concentration of berberine in methanol extract was determined using a C-18 reverse phase column with a mobile phase of acetonitrile:water (10:90 v/v) at a flow rate of 0.6 ml/min and with UV detection at 266 nm. TLC and HPLC comparison of both the species revealed significant variation in the chemical constitution of the two species. This observation becomes important in the context of the use of T. sinensis in place of the genuine drug T. cordifolia. PMID:20390090

  14. HPLC Estimation of berberine in Tinospora cordifolia and Tinospora sinensis.

    PubMed

    Srinivasan, G V; Unnikrishnan, K P; Rema Shree, A B; Balachandran, Indira

    2008-01-01

    A high-performance liquid chromatographic method for the estimation of berberine in the stem of Tinospora cordifolia (Willd.) Miers. ex Hook.f. and Thoms. and Tinospora sinensis (Lour.) Merrill is described. The dried stems of T. cordifolia and T. sinensis were defatted with petroleum ether (60-80 degrees ). The marc was dried and further extracted with methanol. The concentration of berberine in methanol extract was determined using a C-18 reverse phase column with a mobile phase of acetonitrile:water (10:90 v/v) at a flow rate of 0.6 ml/min and with UV detection at 266 nm. TLC and HPLC comparison of both the species revealed significant variation in the chemical constitution of the two species. This observation becomes important in the context of the use of T. sinensis in place of the genuine drug T. cordifolia. PMID:20390090

  15. Validated HPLC and Ultra-HPLC Methods for Determination of Dronedarone and Amiodarone Application for Counterfeit Drug Analysis.

    PubMed

    El-Bagary, Ramzia I; Elkady, Ehab F; Mowaka, Shereen; Attallah, Maria

    2015-01-01

    Two simple, accurate, and precise chromatographic methods have been developed and validated for the determination of dronedarone (DRO) HCl and amiodarone (AMI) HCl either alone or in binary mixtures due to the possibility of using AMI as a counterfeit of DRO because of its lower price. First, an RP-HPLC method is described for the simultaneous determination of DRO and AMI. Chromatographic separation was achieved on a BDS Hypersil C18 column (150×4.6 mm, 5 μm). Isocratic elution based on potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (10+90, v/v) at a flow rate of 2 mL/min with UV detection at 254 nm was performed. The second method is RP ultra-HPLC in which the chromatographic separation was achieved on an AcclaimTM RSLC 120 C18 column (100×2.1 mm, 2.2 μm) using isocratic elution with potassium dihydrogen phosphate buffer with 0.1% triethylamine pH 6-methanol (5+95, v/v) at a flow rate of 1 mL/min with UV detection at 254 nm. Linearity, accuracy, and precision of the two methods were found to be acceptable over the concentration ranges of 5-80 μg/mL for both DRO and AMI. The results were statistically compared using one-way analysis of variance. The optimized methods were validated and proved to be specific, robust, precise, and accurate for the QC of the drugs in their pharmaceutical preparations. PMID:26651561

  16. Identification of major and minor constituents of Harpagophytum procumbens (Devil's claw) using HPLC-SPE-NMR and HPLC-ESIMS/APCIMS.

    PubMed

    Clarkson, Cailean; Staerk, Dan; Hansen, Steen Honoré; Smith, Peter J; Jaroszewski, Jerzy W

    2006-09-01

    The HPLC-SPE-NMR technique, supported by HPLC-MS measurements, was used to determine structures of major as well as some minor constituents of ethanol and petroleum ether extracts of Harpagophytum procumbens (Devil's claw) roots. This method was also shown to be applicable for rapid and precise on-line identification of secondary metabolites present in commercial herbal products of H. procumbens. A total of 15 compounds (1-14 and 17) were identified from the ethanol and petroleum ether extracts, including a novel Diels-Alder dimer 14. Optimization of the HPLC-SPE-NMR experiments included quantitative (1)H NMR measurements, determination of trapping and elution efficiency, effect of multiple trapping of analytes, use of various deuterated solvents for SPE cartridge elution, and effect of post-column dilution ratio of eluent with water. Linear accumulation of apolar and relatively polar analytes was demonstrated for at least 8-10 repeated trappings, resulting in greatly improved signal-to-noise ratios in NMR spectra and reduced acquisition times. Thus, the HPLC-SPE-NMR technique provides an efficient means of identification of multiple components of crude extracts. By allowing on-line generation of high-quality 2D NMR data without traditional purification of extract components, the HPLC-SPE-NMR methodology represents a paradigm shift in natural products research with respect to structure elucidation. PMID:16989520

  17. Development of HPLC/ESI-MS and HPLC/1H NMR methods for the identification of photocatalytic degradation products of iodosulfuron.

    PubMed

    Sleiman, Mohamad; Ferronato, Corinne; Fenet, Bernard; Baudot, Robert; Jaber, Farouk; Chovelon, Jean-Marc

    2006-05-01

    In the present study, HPLC/ESI-MS and stopped-flow HPLC/1H NMR methods were developed and applied to separate and characterize the byproducts arising from TiO2-catalyzed photodegradation of the herbicide iodosulfuron methyl ester (IOME) in aqueous solution under UV irradiation. Prior to identification, irradiated solutions of IOME (200 and 1000 mg.L(-1)) were concentrated by solid-phase extraction using two cartridges: Isolute C18 and Isolute ENV+. Analytical separation was achieved on a C18 reversed-phase column with ACN/H2O (HPLC/MS) or ACN/D2O (HPLC/NMR) as mobile phase and a linear gradient with a chromatographic run time of 35 min. The combination of UV and MS data allowed the structural elucidation of more than 20 degradation products, whereas 1H NMR data permitted an unequivocal confirmation of the identities of major products and the differentiation of several positional isomers, in particular, the hydroxylation isomers. The obtained results permitted us to propose a possible degradation scheme and to put in evidence the presence of privileged sites for the attack of OH radicals. This work shows, for the first time, the application of combined HPLC with UV, MS, and NMR detection for complete structural elucidation of photocatalytic degradation products, and it will be of particular value in studies on the elimination of pollutants in aqueous solutions by photocatalysis.

  18. [Establishment of hepatocyte extraction combined with HPLC(HE-HPLC) and application in analysis of active components in the fruits of Gardenia jasminoides extract].

    PubMed

    Hong, Min; Ma, Hongyu; Zhu, Quan

    2009-02-01

    To screen effective principles from traditional Chinese medicine, a method named hepatocyte extraction combined with HPLC (HE-HPLC) was establish in this study. The active principles in the fruits of Gardenia jasminoides Ellis extract were combined with the hepatocytes under imitated physiological environments. Then the unattached substances were washed off by PBS with pH 7.4. After that the conjugated compounds were eluted by PBS with pH 4.0. These compounds released from target sites were collected and handled through SPE to be condensed, and analyzed by HPLC. The results indicated that two characteristic active compounds in the fruits of G. jasminoides extract binded to the hepatocytes. One of them is geniposide. The other is continued to be identified. It is showed that active principles which could bind to hepotocyte (through receptors, Channels, enzymes, etc.) could be detected, at least partly, by HE-HPLC analysis. There was a significant correlation between the retention properties of the active compounds which was obtained by HE-HPLC and their pharmacological effects on hepotocytes. PMID:19459310

  19. Ultra-fast HPLC-ICP-MS analysis of oxaliplatin in patient urine.

    PubMed

    Koellensperger, Gunda; Hann, Stephan

    2010-05-01

    A novel method for rapid HPLC-ICP-MS analysis of oxaliplatin in human urine was developed implementing a stationary HPLC phase with a particle size of 1.8 microm. The method allowed a cycle time of <1 min at a HPLC flow rate of 0.9 mL min(-1). Procedural limits of detection of 0.05 microg L(-1) oxaliplatin (150 fg on column) were obtained. Analysis of oxaliplatin in patient urine showed that accurate quantification of the intact drug demanded for storage at -80 degrees C and rapid measurement after thawing.

  20. Electrochemically Pretreated Carbon Microfiber Electrodes as Sensitive HPLC-EC Detectors

    PubMed Central

    Bartosova, Zdenka; Riman, Daniel; Jakubec, Petr; Halouzka, Vladimir; Hrbac, Jan; Jirovsky, David

    2012-01-01

    The paper focuses on the analysis and detection of electroactive compounds using high-performance liquid chromatography (HPLC) combined with electrochemical detection (EC). The fabrication and utilization of electrochemically treated carbon fiber microelectrodes (CFMs) as highly sensitive amperometric detectors in HPLC are described. The applied pretreatment procedure is beneficial for analytical characteristics of the sensor as demonstrated by analysis of the model set of phenolic acids. The combination of CFM with separation power of HPLC technique allows for improved detection limits due to unique electrochemical properties of carbon fibers. The CFM proved to be a promising tool for amperometric detection in liquid chromatography. PMID:22654586

  1. Simultaneous determination of synthetic colorants in yogurt by HPLC.

    PubMed

    de Araújo Siqueira Bento, Waleska; Lima, Bruno Parente; Paim, Ana Paula S

    2015-09-15

    This article reports on a method to determine synthetic dyes in yogurts and milk drinks. Initially a method for extraction of artificial dyes was developed to pretreat samples in order to extract most of the artificial colorants. Then, the colorants were analyzed by HPLC-PAD using gradient elutions. The method was linear in the range of 0.5-25mgL(-1) colorants (0.9991

  2. Quality evaluation of Carthami Flos by HPLC-UV.

    PubMed

    Li, Ying; Piao, Donggen; Zhang, Haiyan; Kim, Taein; Lee, Seung-Ho; Chang, Hyeun Wook; Woo, Mi-Hee; Son, Jong Keun

    2015-01-01

    A HPLC-DAD method was developed for simultaneous determination of four marker compounds, kaempferol-3-O-rutinoside, safflomin A, safflomin B and bidenoside C, in the extract of the flowers of Carthamus tinctorius Linne. Natural samples were extracted in 50% aqueous methanol by ultra-sonication for 60 min. Marker compounds were separated on a C18 column by two-step gradient elution of mobile phase (acetonitrile/water) at a flow rate of 0.75 mL/min and detected at 210 nm. The retention times of safflomin A and safflomin B were shifted according to the pH of the mobile phase. The optimized analytical method was validated to confirm linearity, precision, accuracy and stability of marker compounds. The method was successfully employed to analyze 17 natural samples from different regions, and the data matrix was monitored and visualized by principal component analysis. The assay method could be applied for quality control of the flowers of C. tinctorius Linne.

  3. HPLC-Fingerprints and Antioxidant Constituents of Phyla nodiflora

    PubMed Central

    Yen, Feng-Lin; Chen, Pei-Chun; Wang, Moo-Chin; Lin, Chun-Nan; Lee, Chiang-Wen; Ko, Horng-Huey

    2014-01-01

    Phyla nodiflora is a creeping perennial herb, widely distributed in the most tropical and subtropical regions. It has been used as a folk medicine, herbal beverage, or folk cosmetic. For these usages, the development of a chemical quality control method of this plant is necessary. In the present study, ten compounds, namely, 3,7,4′,5′-tetrahydroxy-3′-methoxyflavone (1), nodifloretin (2), 4′-hydroxywogonin (3), onopordin (4), cirsiliol (5), 5,7,8,4′-tetrahydroxy-3′-methoxyflavone (6), eupafolin (7), hispidulin (8), larycitrin (9), and β-sitosterol were isolated from the methanolic extract of the aerial part of P. nodiflora (PNM) and their structures were identified by 1D-NMR comparing their spectra with the literature. The antioxidant activities of these compounds were evaluated by free radical scavenging activity and tyrosinase inhibitory effect in cell-free systems. Compounds 4, 5, and 7 showed strong antioxidant activity. To control the quality of P. nodiflora, a simple and reliable method of high-performance liquid chromatography combined with ultraviolet detector (HPLC-UV) was established for both the fingerprint analysis and the quantitative determination of two selected active compounds, onopordin (4) and eupafolin (7). Statistical analysis of the obtained data demonstrated that our method achieved the desired linearity, precision, and accuracy. The results indicated that the developed method can be used as a quality evaluation method for PNM. PMID:25140335

  4. Simplified micromethod for the HPLC measurement of diclofenac in plasma.

    PubMed

    Santos, S R; Donzella, H; Bertoline, M A; Pereira, M D; Omosako, C E; Porta, V

    1992-01-01

    A simple and sensitive micromethod based on HPLC is described for the measurement of diclofenac in 200 microliters plasma. A single extraction with dichloromethane in acidic medium was an essential clean-up step. Diclofenac and its internal standard (cyclohexendiphenyl propionic acid) was eluted at 3.3 and 6.5 min from a 4-micron C18 reverse-phase column using a mobile phase consisting of 0.75 M sodium acetate buffer, pH 5.0, and acetonitrile (55:45, v/v) at a flow rate of 0.9 ml/min with detection at 282 nm. The method, validated on the basis of parameters evaluated for the confidence limits of diclofenac measurements in spiked plasma, presented 1 ng/ml sensitivity, 10-10,000 ng/ml linearity, and 3.5% and 5.7% intra- and interassay precision, respectively. Peak plasma diclofenac levels ranging from 177 to 841 ng/ml and from 276 to 1008 ng/ml were obtained for two slow-release formulations. A wide range (1 ng/ml-3 micrograms/ml) was observed for plasma diclofenac levels of volunteers during a 24-h study period.

  5. Simultaneous determination of synthetic colorants in yogurt by HPLC.

    PubMed

    de Araújo Siqueira Bento, Waleska; Lima, Bruno Parente; Paim, Ana Paula S

    2015-09-15

    This article reports on a method to determine synthetic dyes in yogurts and milk drinks. Initially a method for extraction of artificial dyes was developed to pretreat samples in order to extract most of the artificial colorants. Then, the colorants were analyzed by HPLC-PAD using gradient elutions. The method was linear in the range of 0.5-25mgL(-1) colorants (0.9991

  6. Dissipation of hexythiozox on beans pods by HPLC-DAD.

    PubMed

    Abd-Alrahman, Sherif H

    2013-04-01

    An effective analytical method for the residue analysis of a novel acaricide hexythiozox and its dissipation in beans pods were studied. Hexythiozox residues were extracted from beans pods samples and the extract was cleaned up according to QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method and determined by high-performance liquid chromatography with photodiode array detector (HPLC-DAD). At fortification levels of 0.1, 0.5, and 1.0 mg kg(-1) in Beans Pods, it was shown that recoveries ranged from 82.4 % to 89.6 % with relative standard deviation (RSD) of 6 %-9 %. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.02 and 0.06 mg kg(-1), respectively. The dissipation half-life time of hexythiozox residues in beans pods was 12.04 days. According to maximum residue limit (MRL) 0.5 mg kg(-1), the preharvest interval (PHI) of hexythiozox on beans pods was 10 days after the treatment. Based on the results of this study and the relevant residue regulation, hexythiozox residue levels will be acceptable when applied to beans pods in Egypt.

  7. HPLC retention thermodynamics of grape and wine tannins.

    PubMed

    Barak, Jennifer A; Kennedy, James A

    2013-05-01

    The effect of grape and wine tannin structure on retention thermodynamics under reversed-phase high-performance liquid chromatography conditions on a polystyrene divinylbenzene column was investigated. On the basis of retention response to temperature, an alternative retention factor was developed to approximate the combined temperature response of the complex, unresolvable tannin mixture. This alternative retention factor was based upon relative tannin peak areas separated by an abrupt change in solvent gradient. Using this alternative retention factor, retention thermodynamics were calculated. Van't Hoff relationships of the natural log of the alternative retention factor against temperature followed Kirchoff's relationship. An inverse quadratic equation was fit to the data, and from this the thermodynamic parameters for tannin retention were calculated. All tannin fractions exhibited exothermic, spontaneous interaction, with enthalpy-entropy compensation observed. Normalizing for tannin size, distinct tannin compositional effects on thermodynamic parameters were observed. The results of this study indicate that HPLC can be valuable for measuring the thermodynamics of tannin interaction with a hydrophobic surface and provides a potentially valuable alternative to calorimetry. Furthermore, the information gathered may provide insight into understanding red wine astringency quality. PMID:23565723

  8. [Determation of strychnine and brucine in Strychnos by HPLC].

    PubMed

    Gu, Z P; Zhang, S M; Wang, C L; Lian, W Y; Xiao, P G; Chen, J M

    1997-10-01

    This paper describes the determination of strychnine and brucine in the seeds, root, stem and leaves of Strychnos species by HPLC. The analytical column used was ZY110 YNG-C18. The mobile phase was KH2PO4(0.01 mol.L-1)--MeOH(73:27), pH2.5, regulated by 10% H3PO4. Flow rate was 1.0 ml.min-1. The detection wavelength was 264 nm. The linear ranges of strychmine and brucine were 0.18-7.26 micrograms and 0.11-4.32 micrograms, respectively. The recoveries of strychnine and brucine were 98.27% and 98.04%, respectively. The analytical results showed that the contents of strychnine and brucine in samples showed great difference between different species. The contents of strychnine in the seeds of Strychnos wallichiana and S. ignatii were 5.6% and 3.9%, respectively. These results show that the two Strychnos species may be developed as the resources of strychnine. PMID:11596226

  9. Determination of heterocyclic aromatic amines in food products: automation of the sample preparation method prior to HPLC and HPLC-MS quantification.

    PubMed

    Fay, L B; Ali, S; Gross, G A

    1997-05-12

    Heat-processing protein-rich foods may cause the formation of heterocyclic aromatic amines (HAAs), all of which have mutagenic and some also carcinogenic potential. Accurately measuring HAA levels in food products is therefore a necessary to realistically assess this risk factor. A solid-phase extraction method for quantitative HAA analysis has been developed by us over the last few years. This method has recently been automated using a robotic workstation and now allows almost unattended sample preparation, a process which saves a human operator about five hours of benchwork. Cleaned-up samples were analyzed by high performance liquid chromatography (HPLC) and ultraviolet (UV) or mass spectrometric (MS) detection. While HPLC-UV remains the daily tool to quantify HAAs, we found HPLC-electrospray-MS to be an alternative detection method with unique advantages, suited for both HAA identification and quantification.

  10. 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.

  11. Analysis of flavonoids and iridoids in Vitex negundo by HPLC-PDA and method validation.

    PubMed

    Roy, Somendu K; Bairwa, Khemraj; Grover, Jagdeep; Srivastava, Amit; Jachak, Sanjay M

    2013-09-01

    The leaves of Vitex negundo have been reported to contain various bioactive constituents including iridoids and flavonoids. This is the first report on the simultaneous determination of iridoids and flavonoids by HPLC in three different samples of V. negundo leaves collected from three regions of India. Separation of iridoids and flavonoids was accomplished by HPLC and further elaborated for their quantification in V. negundo leaves using a C-18 column with detection at 254 and 330 nm, respectively. The developed HPLC method showed good linearity (r2 > or = 0.999), high precision (RSD < 5%) and a good recovery (99.3-103.0%) of the compounds. All the validation parameters of the developed HPLC were found to be within the permissible limits according to the ICH guidelines. The developed method was robust, accurate and reliable for the quality control of V. negundo leaves.

  12. HPLC-MTT assay: anticancer activity of aqueous garlic extract is from allicin.

    PubMed

    Lee, Jenny; Gupta, Shalini; Huang, Jin-Sheng; Jayathilaka, Lasanthi P; Lee, Bao-Shiang

    2013-05-15

    A strategy using reversed-phase high-performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrometry (MS), nuclear magnetic resonance (NMR), chemical synthesis, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay to identify allicin as the active anticancer compound in aqueous garlic extract (AGE) is described. Changing the pH of AGE from 7.0 to 5.0 eliminated interfering molecules and enabled a clean HPLC separation of the constituents in AGE. MTT assay of the HPLC fractions identified an active fraction. Further analysis by TLC, MS, and NMR verified the active HPLC fraction as allicin. Chemically synthesized allicin was used to provide further confirmation. The results clearly identify the active compound in AGE as allicin.

  13. HPLC SEPARATION OF CHIRAL ORGANOPHOSPHORUS PESTICIDES ON POLYSACCHARIDE CHIRAL STATIONARY PHASES

    EPA Science Inventory

    High-performance liquid chromatographic separation of the individual enantiomers of 12 organophosphorus pesticides (OPs) were obtained on polysaccharide chiral HPLC columns using an alkane-alcohol mobile phase. The OP pesticides were crotoxyphos, dialifor, dyfonate, fenamiphos, ...

  14. Characterization and quantitative determination of impurities in piperaquine phosphate by HPLC and LC/MS/MS.

    PubMed

    Dongre, Vaijanath G; Karmuse, Pravin P; Ghugare, Pradeep D; Gupta, Mukesh; Nerurkar, Bipin; Shaha, Chirag; Kumar, Ashok

    2007-01-01

    Four impurities in piperaquine phosphate bulk drug substance were detected by a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. These impurities were identified by LC/MS/MS. The structures of impurities were confirmed by spectroscopic studies (NMR and IR) conducted using synthesized authentic compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. The system suitability of HPLC analysis established the validity of the separation. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. Forced degradation studies were also performed for piperaquine phosphate bulk drug samples to demonstrate the stability indicating power of the newly developed HPLC method. PMID:16916594

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

    NASA Astrophysics Data System (ADS)

    Gupta, Lokesh Kumar

    2012-11-01

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

  16. Fenofibrate raw materials: HPLC methods for assay and purity and an NMR method for purity.

    PubMed

    Lacroix, P M; Dawson, B A; Sears, R W; Black, D B; Cyr, T D; Ethier, J C

    1998-11-01

    HPLC methods for drug content and HPLC and NMR methods for related compounds in fenofibrate raw materials were developed. The HPLC methods resolved 11 known and six unknown impurities from the drug. The HPLC system was comprised of a Waters Symmetry ODS column (100 x 4.6 mm, 3.5 microm), a mobile phase consisting of acetonitrile water trifluoroacetic acid 700/300/l (v/v/v) at a flow rate of 1 ml min(-1). and a UV detector set at 280 nm. Minimum quantifiable amounts were about 0.1% for three of the compounds and less than 0.05% for the other eight. Individual impurities in 14 raw materials ranged from trace levels to 0.25%, and total impurities from 0.04 to 0.53% (w/w). Six unknown impurities were detected by HPLC, all at levels below 0.10%, assuming the same relative response as fenofibrate. An NMR method for related compounds was also developed and it was suitable for 12 known and several unknown impurities. It requires an NMR of 400 MHz, or greater, field strength. Individual impurities in the raw materials analyzed ranged from trace levels to 0.24%, and total impurities from trace levels to 0.59%. Several lots contained small amounts of unknown impurities at trace levels. Three lots, all from the same manufacturer, contained an unknown impurity, not detectable by HPLC, which was not present in the other raw materials. It was estimated to be present at a level greater than 0.2%. The results for related compounds by the two techniques were consistent. The main differences stem from the low sensitivity of the HPLC method for some of the related compounds at 280 nm, or from the higher limits of quantitation by the NMR method for several other impurities using the conditions specified. A fifteenth raw material was not homogeneous in its content of impurity VI, a synthetic intermediate and possible degradation product. The HPLC/MS results provided information on the peak purity (number of components) for minor HPLC peaks, as well as structural data such as the

  17. HPLC-DAD determination of benzo(a) pyrene in waste gas

    SciTech Connect

    Lehotay, J.; Halmo, F.; Brandsteterova, E.; Oktavec, D. )

    1993-01-01

    Diode array detection (DAD) was used in HPLC trace determination of benzo(a)pyrene in gas emission. The linearity and detection limit was calculated. It was found that DAD - HPLC combination is selective and sensitive enough to be used for the monitoring of the benzo(a)pyrene concentration in gas emission where its presence is presumed. The limit of the determination of the suggested method is 1/[mu]g/m[sup 3].

  18. Determination of organic nitro compounds using HPLC-UV-PAED

    NASA Astrophysics Data System (ADS)

    Marple, Ronita L.; LaCourse, William R.

    2004-12-01

    High-performance liquid chromatography with ultra violet and photo-assisted electrochemical detection (HPLC-UV-PAED) has been applied to the sensitive and selective determination of organic nitro compounds. The system was first developed for the determination of nitro explosives, and PAED has shown superior sensitivity over UV detection for these compounds (i.e., <1 part-per-trillion for HMX). The system also shows enhanced selectivity over the traditional UV method in that two detectors can be used for improved analyte identification. Also, having two detectors permits chemometric resolution of overlapping peaks, and this is not addressed in the UV method. Because this method is applicable to a wide range of nitro explosives, it was predicted that PAED would show the same sensitivity and selectivity toward other types of nitro compounds. Since its development, the system's use has been expanded to include the determination of nitro-containing pharmaceuticals and glycosylated nitro compounds in biological matrices. Model compounds were chosen, specifically nitroglycerin and related compounds and nitrophenyl-glucoside, to represent these classes. PAED showed superior detection limits over low wavelength UV detection for nitroglycerin (PAED = 0.3ppb, UV at 220nm = 48ppb), demonstrating PAED"s applicability to determining nitro-pharmaceuticals. Conversely, UV detection at 220nm proved to be more sensitive than PAED for nitrophenyl-glucoside (UV at 220 = 0.6ppb, PAED = 3.6ppb). However, when nitrophenyl-glucoside was spiked into urine, PAED determination resulted in 99+0.3% recovery, while UV at 220nm resulted in 116+0.2% recovery, suggesting that UV determination may suffer from matrix interference.

  19. HPLC-DAD protein kinase inhibitor analysis in human serum.

    PubMed

    Dziadosz, Marek; Lessig, Rüdiger; Bartels, Heidemarie

    2012-04-15

    We here describe an HPLC-DAD method to analyse different protein kinase inhibitors. Potential applications of this method are pharmacokinetic studies and therapeutic drug monitoring. Optimised chromatography conditions resulted in a very good separation of seven inhibitors (vatalanib, bosutinib, canertinib, tandutinib, pazopanib, dasatinib - internal standard and erlotinib). The good sensitivity makes this method competitive with LC/MS/MS. The separation was performed with a Lichrospher 100-5 RP8, 250 mm × 4 mm column maintained at 30 ± 1 °C, and with a mobile phase of 0.05 M H(3)PO(4)/KH(2)PO(4) (pH=2.3)-acetonitrile (7:3, v/v) at a flow rate of 0.7 mL/min. A simple and fast sample preparation sequence with liquid-liquid extraction led to good recoveries (73-90%) of all analytes. The recovery hardly reached 50% only for pazopanib. This method can also be used for targeted protein kinase inhibitor quantification. A perfect linearity in the validated range (20-10,000 ng/mL) and an LOQ of 20 ng/mL were achieved. The relative standard deviations and accuracies of all examined drug concentrations gave values much lower than 15% both for between- and within-batch calculations. All analysed PKIs were stable for 6 months in a 1mg/mL dimethyl sulfoxide stock solution. Vatalanib, bosutinib and erlotinib were also stable in human serum in the whole examined concentration range. PMID:22425385

  20. The rapid isolation of highly purified cadmium metallothioneins using HPLC

    SciTech Connect

    Vella, G.; Bylander, J.; Hazen-martin, D.; O'Connor, K.; Phoebe, C. Millipore Corp., Milford, MA )

    1991-03-11

    The central role of the proximal tubule in the renal toxicity due to cadmium (Cd) poisoning has been well documented, however, the mechanisms leading to the onset of this nephrotoxic effect are presently not well understood. The most likely mechanism for the initiation of toxicity involves the accumulation of Cd in the liver where it induces the formation of, and is complexed with the metallothioneins (MT-I and MT-II) followed by release from the liver and subsequent absorption by the proximal tubule. Methods for obtaining MTs for specific RIA and immunohistochemistry have been hampered by difficulties in removing impurities. To obtain highly purified Cd-MTs for use in RIA as well as to assay the relative affects of exposure to Cd-MT and ionic Cd in vitro on human proximal tubule cells, Cd-MT-I and Cd-MT-II were prepared from rat liver for use in these studies using a rapid two step chromatographic procedure employing gel filtration and high performance anion exchange chromatography. Repeated HPLC separations revealed a time dependent conversion of a non-metallothionein protein component to form which coeluted with Cd-MT-I during the ion exchange separation. This was preventable by cooling the sample or by adding proteinase inhibitors. This method demonstrated the importance of speed in the purification which permitted the isolation of highly purified Cd-MTs in high yields. Due to the extreme sensitivity, this method may also be amenable as an alternative for a non-radioactive assay of Cd-MT formation and breakdown in toxicological studies.

  1. Identifying Phytoplankton Classes In California Reservoirs Using HPLC Pigment Analysis

    NASA Astrophysics Data System (ADS)

    Siddiqui, S.; Peacock, M. B.; Kudela, R. M.; Negrey, K.

    2014-12-01

    Few bodies of water are routinely monitored for phytoplankton composition due to monetary and time constraints, especially the less accessible bodies of water in central and southern California. These lakes and estuaries are important for economic reasons such as tourism and fishing. This project investigated the composition of phytoplankton present using pigment analysis to identify dominant phytoplankton groups. A total of 28 different sites with a wide range of salinity (0 - 60) in central and southern California were examined. These included 13 different bodies of water in central California: 6 in the Sierras, 7 in the San Francisco Bay Estuary, and 15 from southern California. The samples were analyzed using high-performance liquid-chromatography (HPLC) to quantify the pigments present (using retention time and the spectral thumbprint). Diagnostic pigments were used to indicate the phytoplankton class composition, focusing on diatoms, dinoflagellates, cryptophytes, and cyanobacteria - all key phytoplankton groups indicative of the health of the sampled reservoir. Our results indicated that cyanobacteria dominated four of the seven bodies of central California water (Mono Lake, Bridgeport Reservoir, Steamboat Slough, and Pinto Lake); cryptophytes and nannoflagellates dominated two of the central California bodies of water (Mare Island Strait and Topaz Lake); and diatoms and dinoflagellates dominated one central California body of water, Oakland Inner Harbor, comprising more than 70% of the phytoplankton present. We expect the bodies of water from Southern California to be as disparate. Though this data is only a snapshot, it has significant implications in comparing different ecosystems across California, and it has the potential to provide valuable insight into the composition of phytoplankton communities.

  2. Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development.

    PubMed

    Cuyckens, Filip; Balcaen, Lieve I L; De Wolf, Kenny; De Samber, Bjorn; Van Looveren, Cis; Hurkmans, Rob; Vanhaecke, Frank

    2008-04-01

    A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The (79)Br/(81)Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like "bromatograms". The combination of accurate mass measurements and the fragmentation behavior in the MS(2) spectra obtained using the Q-Tof Ultima mass spectrometer or MS(n) spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0-24 h post-dose.

  3. Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS.

    PubMed

    Chandra, A; Rana, J; Li, Y

    2001-08-01

    A method has been established and validated for identification and quantification of individual, as well as total, anthocyanins by HPLC and LC/ES-MS in botanical raw materials used in the herbal supplement industry. The anthocyanins were separated and identified on the basis of their respective M(+) (cation) using LC/ES-MS. Separated anthocyanins were individually calculated against one commercially available anthocyanin external standard (cyanidin-3-glucoside chloride) and expressed as its equivalents. Amounts of each anthocyanin calculated as external standard equivalent were then multiplied by a molecular-weight correction factor to afford their specific quantities. Experimental procedures and use of a molecular-weight correction factors are substantiated and validated using Balaton tart cherry and elderberry as templates. Cyanidin-3-glucoside chloride has been widely used in the botanical industry to calculate total anthocyanins. In our studies on tart cherry and elderberry, its use as external standard followed by use of molecular-weight correction factors should provide relatively accurate results for total anthocyanins, because of the presence of cyanidin as their major anthocyanidin backbone. The method proposed here is simple and has a direct sample preparation procedure without any solid-phase extraction. It enables selection and use of commercially available anthocyanins as external standards for quantification of specific anthocyanins in the sample matrix irrespective of their commercial availability as analytical standards. It can be used as a template and applied for similar quantification in several anthocyanin-containing raw materials for routine quality control procedures, thus providing consistency in analytical testing of botanical raw materials used for manufacturing efficacious and true-to-the-label nutritional supplements.

  4. 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.

  5. [Determination of ethychlozate and its degradation product in fruits by HPLC and LC/MS].

    PubMed

    Takatsuki, Satoshi; Nemoto, Satoru; Sasaki, Kumiko; Toyoda, Masatake

    2002-02-01

    A method was developed for the analysis of ethychlozate (CIE) and its decomposition compound, 5-chloro-3(1H)-indazolylacetic acid (CIA) in fruits by HPLC and LC/MS. The sample was homogenized with 1 mol/L HC1, and CIE and CIA were extracted with 5 mol/L HCl and acetone. They were extracted from the acetone extract with diethylether-n-hexane (2:1). CIE was hydrolyzed to CIA with methanol-4 mol/L KOH (1:1). The solution was made acidic, and CIA was extracted with diethylether-n-hexane (2:1). The extract was cleaned up on a silica gel column. CIA was determined by HPLC-UV and LC/MS (Scan or SIR). Four fruits were spiked with CIE or CIA at 0.5 microgram/g and analyzed by the proposed method with HPLC. The average recoveries were 77.2-83.2% for CIE and 71.2-89.2% for CIA. The concentrations determined by LC/MS were 10-25% higher than the values by HPLC. The limit of detection (LOD) of CIA standard solution by HPLC corresponds to 0.015 microgram/g of CIE in the sample. In the same way, the LOD of CIA by LC/MS (SIR) corresponds to 0.009 microgram/g of CIE in the sample.

  6. Ion pair-reversed phase HPLC approach facilitates subcloning of PCR products and screening of recombinant colonies.

    PubMed

    Shaw-Bruha, C M; Lamb, K A

    2000-04-01

    The isolation of a single DNA molecule for cloning is technically difficult, and the subsequent screening of colonies for recombinant DNA clones is time consuming. Ion pair-reversed phase HPLC (IP-RP-HPLC) analysis of nucleic acids improves the resolution and isolation of PCR products to be cloned. We demonstrate that PCR products analyzed and collected using the IP-RP-HPLC approach (WAVE DNA Fragment Analysis System) can be cloned directly into a plasmid vector. In addition, we demonstrate that when IP-RP-HPLC analysis is extended to the colony screening process, the time required for these procedures is reduced.

  7. PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC AND PREP-HPLC.

    PubMed

    Wang, Xiaohong; Liang, Yong; Peng, Cuilin; Xie, Huichun; Pan, Man; Zhang, Tianyou; Ito, Yoichiro

    2011-01-01

    Combined with medium-pressure liquid chromatography (MPLC) and preparative high-pressure liquid chromatography (Prep-HPLC), high-speed countercurrent chromatography (HSCCC) was successfully applied for separation and purification of isoflavonoids from the extract of belamcanda. HSCCC separation was performed on a two-phase solvent system composed of methyl tert-butyl ether -ethyl acetate - n-butyl alcohol - acetonitrile -0.1% aqueous trifluoroacetic acid at a volume radio of 1:2:1:1:5. Semi-purified peak fractions from HSCCC separation were further purified by Prep-HPLC. Nine well-separated fractions were analyzed by HPLC-UV absorption spectrometry to determine their purities and characterized with ESI-MS(n). Except for peaksland VII (unknown) seven compounds were identified as apocynin (peak II), mangiferin (peak III), 7-O-methylmangiferin (peak IV), hispidulin (peak V), 3'-hydroxyltectoridin (peak VI), iristectorin B (peak VII), isoiridin (peak IX).

  8. Chemical fingerprint analysis for quality control and identification of Ziyang green tea by HPLC.

    PubMed

    He, Xiaoye; Li, Jianke; Zhao, Wei; Liu, Run; Zhang, Lin; Kong, Xianghong

    2015-03-15

    A simple and reliable HPLC fingerprint method was developed and validated for the quality control and identification of Ziyang green tea. Ten batches of Ziyang green tea collected from different plantations in Shaanxi Ziyang of China were used to establish the fingerprint. The feasibility and advantages of the used HPLC fingerprint were verified for its similarity evaluation by systematically comparing chromatograms with professional analytical software recommended by State Food and Drug Administration (SFDA) of China. The similarities of the fingerprints of 10 batches of tea samples were all more than 0.981. Additionally, simultaneous quantification of 10 major bioactive ingredients in the tea samples was conducted to interpret the consistency of the quality test. The results indicated that the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation and quantification analysis can be successfully used to assess the quality and to identify the authenticity of Ziyang green tea.

  9. TLC and HPLC characteristics of desacetylmatricarin, leucodin, achillin and their 8alpha-angeloxy-derivatives.

    PubMed

    Glasl, S; Mucaji, P; Werner, I; Jurenitsch, J

    2003-07-01

    Five guaianolides, including two pairs of isomers, from a Hungarian taxon of the Achillea millefolium group were characterized analytically. Different chromatographic systems on TLC and HPLC were developed for the analysis of these compounds. TLC of leucodin, 8alpha-angeloxy-leucodin, achillin, 8alpha-angeloxy-achillin and desacetylmatricarin was performed on silica gel using dichloromethaneacetone and cyclohexane-ethylacetate mixtures as mobile phases. HPLC on stationary phases as LiChrospher RP2, LiChrospher RP8, LiChrospher RP18e, Hypersil BDS C18 and Aquasil C18 required isocratic and gradient systems with different methanol-water mixtures as mobile phases. The presented RF values and retention times allow the identification of the respective 2-oxo-guaianolides which are marker substances for certain non-proazulene containing species. Their TLC and HPLC fingerprints are compared to those of proazulene containing species and are relevant for quality control.

  10. Using HPLC pigment analysis to investigate phytoplankton taxonomy: the importance of knowing your species

    NASA Astrophysics Data System (ADS)

    Irigoien, Xabier; Meyer, Bettina; Harris, Roger; Harbour, Derek

    2004-04-01

    Phytoplankton microscopic enumerations and HPLC analyses of their pigments were performed weekly for a complete year at a coastal station in the English Channel. The taxonomic composition of the phytoplankton community was assessed using the HPLC results combined with the mathematical tool CHEMTAX in two different ways. Firstly, without using the species level taxonomic information obtained at the microscopic level (blind analyses), and secondly by including the information from the microscopic taxonomic analysis (directed analyses). The results indicate that, due to the particular pigment composition of some species (for example, the dinoflagellate, Karenia mikimotoi and the haptophyte, Phaeocystis pouchetii), a blind analysis would result in very significant errors in the taxonomic determination of the bloom events at this station. Major blooms of Karenia mikimotoi and P. pouchetii were mistaken for blooms of diatoms on the basis of a blind HPLC-CHEMTAX analysis. Only with the information from the microscopic observations was it possible to obtain an accurate representation of the phytoplankton community.

  11. 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.

  12. An RP-HPLC determination of 5-hydroxymethylfurfural in honey The case of strawberry tree honey.

    PubMed

    Spano, Nadia; Casula, Lucia; Panzanelli, Angelo; Pilo, Maria I; Piu, Paola C; Scanu, Roberta; Tapparo, Andrea; Sanna, Gavino

    2006-02-15

    The use of the RP-HPLC official method of the International Honey Commission (IHC) for the determination of 5-hydroxymethylfurfural (HMF) in strawberry tree honey (Arbutus unedo, a typical Sardinian honey) has brought to light a specific and heavy chromatographic interference that prevents accurate quantification. The interference has been identified as homogentisic acid (HA), i.e. the marker of the botanical origin of the honey. For this reason, an alternative RP-HPLC method is proposed. The bias-free method allows a complete separation of HMF from HA to the baseline level and is faster and more precise than the RP-HPLC official method: the detection and quantification limits are 1.9 and 4.0mgkg(-1), respectively, whereas the repeatability is ca. 2% in the HMF concentration range of 5-140mgkg(-1). PMID:18970477

  13. Improved Method for HPLC Analysis of Polyamines, Agmatine and Aromatic Monoamines in Plant Tissue

    PubMed Central

    Slocum, Robert D.; Flores, Hector E.; Galston, Arthur W.; Weinstein, Leonard 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 (Daucus carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues. Images Figure 4 Figure 5 PMID:11537449

  14. 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.

  15. 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.

  16. A uHPLC-MS mathematical modeling approach to dry powder inhaler single agglomerate analysis.

    PubMed

    Pennington, Justin; Lena, John; Medendorp, Joseph; Ewing, Gary

    2011-10-01

    Demonstration of content uniformity (CU) is critical toward the successful development of dry powder inhalers (DPIs). Methods for unit dose CU determination for DPI products are well-established within the field of respiratory science. Recent advances in the area include a uHPLC-MS method for high-throughput uniformity analysis, which allows for a greater understanding of blending operations as the industry transitions to a quality-by-design approach to development. Further enhancements to this uHPLC-MS method now enable it to determine CU and sample weight at the single agglomerate level, which is roughly 50× smaller than a unit dose. When coupled with optical microscopy-based agglomerate sizing, the enhanced uHPLC-MS method can also predict the density and porosity of individual agglomerates. Expanding analytical capabilities to the single agglomerate level provides greater insights and confidence in the DPI manufacturing process.

  17. Analysis and stability of the constituents of Curcuma longa and Harpagophytum procumbens tinctures by HPLC-DAD and HPLC-ESI-MS.

    PubMed

    Karioti, Anastasia; Fani, Elisa; Vincieri, Franco Francesco; Bilia, Anna Rita

    2011-06-01

    In the present study two methods based on liquid chromatography with diode array detection (HPLC-DAD) coupled to an electrospray ionisation (ESI-MS) interface were developed for the determination of constituents in the tinctures (60%, v/v, DER 1:5) of turmeric (Curcuma longa L.) and Devil's claw (Harpagophytum procumbens L.). The developed simple and effective assays permitted the quality control of both tinctures. The aim of this work was to assess the qualitative and quantitative profile of the constituents of two widely marketed commercial preparations and to evaluate chemical stability of their marker constituents during accelerated thermal stability test by HPLC analysis. Characteristic constituents of C. longa rhizomes are the curcuminoids, whereas characteristic constituents of H. procumbens are acylated iridoid glycosides and phenylethylalcohols. Constituents of Devil's claw tincture (mainly iridoids) were more stable than curcuminoids of turmeric. PMID:21420820

  18. Nonanthocyanin secondary metabolites of black raspberry (Rubus occidentalis L.) fruits: identification by HPLC-DAD, NMR, HPLC-ESI-MS, and ESI-MS/MS analyses.

    PubMed

    Paudel, Liladhar; Wyzgoski, Faith J; Scheerens, Joseph C; Chanon, Ann M; Reese, R Neil; Smiljanic, Danijela; Wesdemiotis, Chrys; Blakeslee, Joshua J; Riedl, Kenneth M; Rinaldi, Peter L

    2013-12-11

    Nonanthocyanin secondary metabolites potentially contributing to the antiproliferative bioactivity of black raspberry ( Rubus occidentalis L.) fruits were extracted in ethyl acetate and isolated by semipreparative and analytical HPLC and analyzed by NMR, HPLC-ESI-MS, and ESI-MS/MS techniques. Here we present complete and partial structures of a variety of the chemical entities such as quercetin 3-glucoside, quercetin 3-rutinoside, myricetin glucoside, dihydrokaempferol glucoside, benzoic acid β-d-glucopyranosyl ester, 3,4-dihydroxybenzoic acid, epicatechin, caffeic acid, p-coumaric acid, p-coumaryl glucoside, p-coumaryl sugar ester, ellagic acid, methyl ellagic acid acetylpentose, methyl ellagic acid valerylpentose, trans-piceid, phloretin glucoside (phloridzin), dihydrosinapic acid, salicylic acid β-d-glucopyranosyl ester, a salicylic acid derivative without attached sugar, p-alkylphenyl glucoside, and a citric acid derivative. To our knowledge, 15 of these compounds were not previously reported in black raspberry fruits.

  19. Analysis of flame retardant additives in polymer fractions of waste of electric and electronic equipment (WEEE) by means of HPLC-UV/MS and GPC-HPLC-UV.

    PubMed

    Schlummer, Martin; Brandl, Fritz; Mäurer, Andreas; van Eldik, Rudi

    2005-01-28

    An HPLC-UV/MS method has been developed to identify and quantify flame retardants in post-consumer plastics from waste of electric and electronic equipment (WEEE). Atmospheric pressure chemical ionisation spectra of 15 brominated and phosphate-based flame retardants were recorded and interpreted. The method was applied to detect flame retardant additives in polymer extracts obtained from pressurised liquid extraction of solid polymers. In addition, a screening method was developed for soluble styrene polymers to isolate a flame retardant fraction through the application of gel permeation chromatography (GPC). This fraction was transferred to an online-coupled HPLC column and detected by UV spectroscopy, which allowed a reliable qualitative and quantitative analysis of brominated flame retardants in the polymer solutions.

  20. Comparison of piracetam measured with HPLC-DAD, HPLC-ESI-MS, DIP-APCI-MS, and a newly developed and optimized DIP-ESI-MS.

    PubMed

    Lenzen, Claudia; Winterfeld, Gottfried A; Schmitz, Oliver J

    2016-06-01

    The direct inlet probe-electrospray ionization (DIP-ESI) presented here was based on the direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) developed by our group. It was coupled to an ion trap mass spectrometer (MS) for the detection of more polar compounds such as degradation products from pharmaceuticals. First, the position of the ESI tip, the gas and solvent flow rates, as well as the gas temperature were optimized with the help of the statistic program Minitab® 17 and a caffeine standard. The ability to perform quantitative analyses was also tested by using different concentrations of caffeine and camphor. Calibration curves with a quadratic calibration regression of R (2) = 0.9997 and 0.9998 for caffeine and camphor, respectively, were obtained. The limit of detection of 2.5 and 1.7 ng per injection for caffeine and camphor were determined, respectively. Furthermore, a solution of piracetam was used to compare established analytical methods for this drug and its impurities such as HPLC-diode array detector (DAD) and HPLC-ESI-MS with the DIP-APCI and the developed DIP-ESI. With HPLC-DAD and 10 μg piracetam on column, no impurity could be detected. With HPLC-ESI-MS, two impurities (A and B) were identified with only 4.6 μg piracetam on column, while with DIP-ESI, an amount of 1.6 μg piracetam was sufficient. In the case of the DIP-ESI measurements, all detected impurities could be identified by MS/MS studies. Graphical Abstract Scheme of the DIP-ESI principle. PMID:27071761

  1. Development and evaluation of a new method for the determination of the carotenoid content in selected vegetables by HPLC and HPLC-MS-MS.

    PubMed

    Huck, C W; Popp, M; Scherz, H; Bonn, G K

    2000-10-01

    Epidemologic studies have shown inverse correlation between the consumption of carotenoid-rich vegetables and the incidence of cancer. Therefore, analytical techniques for the quantitative determination of carotenoids in complex sample matrices are important. The most used method is reversed-phase (RP)-high-performance liquid chromatography (HPLC). In this study, seventeen mobile-phase systems described in the literature and six RP-HPLC columns with differences in particle size and porosity are evaluated. Derived from these results, a new mobile-phase (acetonitrile, methanol, chloroform, and n-heptane) including solvent modifiers is presented, which allows an improved and more efficient separation of carotenoids. From all columns tested, the best chromatographic parameters are found using a silica C18 column (250 x 2 mm, 5 microm, 100 A). As was found, absorbance detection at 450 nm allows the determination of the carotenoids down to the picogram range with good linearity (R2 > 0.98). For the identification and quantitation of carotenoids in complex sample matrices (containing additionally other ultraviolet-absorbing compounds), the optimized RP chromatographic system is coupled to a mass spectrometer (MS) using an atmospheric pressure ionization interface. The calibration plots show high linearity (R2 > 0.99), and the detection limit is found in the lower nanogram range. Furthermore, collision-induced dissociation in the ion source allows for the identification of carotenoids by their characteristic fragmentation pathways. In this study, a total of nine species of vegetables commonly consumed in Central Europe are analyzed for their contents of carotenoids (namely lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene) by RP-HPLC and RP-HPLC-MS-MS. It is found that good sources for lutein are spinach, kale, and broccoli, and sources for beta-carotene are broccoli, spinach, kale, carrots, and tomatoes. This new method is an improvement for the identification and

  2. Identification and determination of major constituents in a traditional Chinese medicine compound recipe Xiongdankaiming tablet using HPLC-PDA/ESI-MSn and HPLC-UV/ELSD* #

    PubMed Central

    Wang, Shu-fang; Leng, Jing; Xu, Yi-min; Feng, Mei-ling

    2013-01-01

    Xiongdankaiming tablet (XDKMT), a well-known compound in traditional Chinese medicine, is widely used for the treatment of acute iridocyclitis and primary open-angle glaucoma. In this paper, accurate and reliable methods were developed for the identification of 20 constituents using high-performance liquid chromatography with photo-diode array and electron spray ionization-mass spectrometry (HPLC-PDA/ESI-MSn), and determination of nine of the constituents (chlorogenic acid, gentiopicroside, isochlorogenic acid B, diosmetin-7-O-β-D-glucopyranoside, apigenin, diosmetin, tauroursodeoxycholic acid, acacetin, and taurochenodeoxycholic acid) was developed using HPLC with ultraviolet absorption detector and evaporative light scattering detector (HPLC-UV/ELSD) for the first time. The best results were obtained on a Zorbax SB-C18 column with gradient elution using water (0.1% formic acid) (A) and methanol (0.1% formic acid) (B) at a flow rate of 0.7 ml/min. Tauroursodeoxycholic acid and taurochenodeoxycholic acid, owing to their low UV absorption, were detected by ELSD. The other seven compounds were analyzed by HPLC-UV with variable wavelengths. The calibration curves of all nine constituents showed good linear regression (R 2>0.9996) within the linearity ranges. The limits of detection and quantification were in the ranges of 0.0460–9.90 μg/ml and 0.115–24.8 μg/ml, respectively. The accuracy, in terms of recovery, varied from 95.3% to 104.9% with relative standard deviations (RSDs) less than 4.4%. Precision (with the intra- and inter-day variations less than 4.4%) was also suitable for its intended use. The developed method was successfully applied for the analysis of major components in XDKMT, which provides an appropriate method for the quality control of XDKMT. PMID:23825146

  3. Identification and determination of major constituents in a traditional Chinese medicine compound recipe Xiongdankaiming tablet using HPLC-PDA/ESI-MS(n) and HPLC-UV/ELSD.

    PubMed

    Wang, Shu-Fang; Leng, Jing; Xu, Yi-Min; Feng, Mei-Ling

    2013-07-01

    Xiongdankaiming tablet (XDKMT), a well-known compound in traditional Chinese medicine, is widely used for the treatment of acute iridocyclitis and primary open-angle glaucoma. In this paper, accurate and reliable methods were developed for the identification of 20 constituents using high-performance liquid chromatography with photo-diode array and electron spray ionization-mass spectrometry (HPLC-PDA/ESI-MS(n)), and determination of nine of the constituents (chlorogenic acid, gentiopicroside, isochlorogenic acid B, diosmetin-7-O-β-d-glucopyranoside, apigenin, diosmetin, tauroursodeoxycholic acid, acacetin, and taurochenodeoxycholic acid) was developed using HPLC with ultraviolet absorption detector and evaporative light scattering detector (HPLC-UV/ELSD) for the first time. The best results were obtained on a Zorbax SB-C18 column with gradient elution using water (0.1% formic acid) (A) and methanol (0.1% formic acid) (B) at a flow rate of 0.7 ml/min. Tauroursodeoxycholic acid and taurochenodeoxycholic acid, owing to their low UV absorption, were detected by ELSD. The other seven compounds were analyzed by HPLC-UV with variable wavelengths. The calibration curves of all nine constituents showed good linear regression (R(2)>0.9996) within the linearity ranges. The limits of detection and quantification were in the ranges of 0.0460-9.90 μg/ml and 0.115-24.8 μg/ml, respectively. The accuracy, in terms of recovery, varied from 95.3% to 104.9% with relative standard deviations (RSDs) less than 4.4%. Precision (with the intra- and inter-day variations less than 4.4%) was also suitable for its intended use. The developed method was successfully applied for the analysis of major components in XDKMT, which provides an appropriate method for the quality control of XDKMT.

  4. Comparison of piracetam measured with HPLC-DAD, HPLC-ESI-MS, DIP-APCI-MS, and a newly developed and optimized DIP-ESI-MS.

    PubMed

    Lenzen, Claudia; Winterfeld, Gottfried A; Schmitz, Oliver J

    2016-06-01

    The direct inlet probe-electrospray ionization (DIP-ESI) presented here was based on the direct inlet probe-atmospheric pressure chemical ionization (DIP-APCI) developed by our group. It was coupled to an ion trap mass spectrometer (MS) for the detection of more polar compounds such as degradation products from pharmaceuticals. First, the position of the ESI tip, the gas and solvent flow rates, as well as the gas temperature were optimized with the help of the statistic program Minitab® 17 and a caffeine standard. The ability to perform quantitative analyses was also tested by using different concentrations of caffeine and camphor. Calibration curves with a quadratic calibration regression of R (2) = 0.9997 and 0.9998 for caffeine and camphor, respectively, were obtained. The limit of detection of 2.5 and 1.7 ng per injection for caffeine and camphor were determined, respectively. Furthermore, a solution of piracetam was used to compare established analytical methods for this drug and its impurities such as HPLC-diode array detector (DAD) and HPLC-ESI-MS with the DIP-APCI and the developed DIP-ESI. With HPLC-DAD and 10 μg piracetam on column, no impurity could be detected. With HPLC-ESI-MS, two impurities (A and B) were identified with only 4.6 μg piracetam on column, while with DIP-ESI, an amount of 1.6 μg piracetam was sufficient. In the case of the DIP-ESI measurements, all detected impurities could be identified by MS/MS studies. Graphical Abstract Scheme of the DIP-ESI principle.

  5. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS.

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

    NASA Technical Reports Server (NTRS)

    Morales, W.

    1986-01-01

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

  7. 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.

  8. Identification and quantification of components in extracts of Uncaria tomentosa by HPLC-ES/MS.

    PubMed

    Montoro, P; Carbone, V; Quiroz, J de Dioz Zuniga; De Simone, F; Pizza, C

    2004-01-01

    The two main classes of secondary metabolites, alkaloids and quinovic acid glycosides, of Uncaria tomentosa (Willd.) DC. (Rubiaceae), a Peruvian plant commonly known as 'uña de gato', have been analysed. Separation of the alkaloidal fraction was achieved using a solid phase extraction method based on cationic exchange, and an analytical method employing HPLC-ES/MS has been developed. Quantitative data for commercial wild bark, cultivated bark and leaves are reported. The analysis of quinovic acid glycosides was performed directly on the crude extract using both a fast analytical method based on flow injection ES/MS, and a more complete analytical technique using HPLC-MS. PMID:14979528

  9. [HPLC determination of troxerutin in plasma and urine following oral administration in man].

    PubMed

    Dittrich, P; Ostrowski, J; Beubler, E; Schraven, E; Kukovetz, W

    1985-01-01

    A high-performance liquid chromatographic method (HPLC) is described for the analysis of 3',4',7-tri-O-(beta-hydroxy-ethyl)-rutoside (troxerutin) in human plasma and urine. After separation of interfering substances on XAD-2 trihydroxyethylrutoside is converted to tetrahydroxyethylrutoside by 2-chlorethanol in alkaline medium. After HPLC-separation tetrahydroxyethylrutoside is quantified by fluorescence detection. The pharmacokinetics of troxerutin were measured in plasma after oral administration to man. The relative bioavailability of the drug from Venelbin was 97.8 +/- 37.1% compared to an aqueous standard solution.

  10. HPLC and HPLC/MS/MS Studies on Stress, Accelerated and Intermediate Degradation Tests of Antivirally Active Tricyclic Analog of Acyclovir.

    PubMed

    Lesniewska, Monika A; Dereziński, Paweł; Klupczyńska, Agnieszka; Kokot, Zenon J; Ostrowski, Tomasz; Zeidler, Joanna; Muszalska, Izabela

    2015-01-01

    The degradation behavior of a tricyclic analog of acyclovir [6-(4-MeOPh)-TACV] was determined in accordance with International Conference on Harmonization guidelines for good clinical practice under different stress conditions (neutral hydrolysis, strong acid/base degradation, oxidative decomposition, photodegradation, and thermal degradation). Accelerated [40±2°C/75%±5% relative humidity (RH)] and intermediate (30±2°C/65%±5% RH) stability tests were also performed. For observation of the degradation of the tested compound the RP-HPLC was used, whereas for the analysis of its degradation products HPLC/MS/MS was used. Degradation of the tested substance allowed its classification as unstable in neutral environment, acidic/alkaline medium, and in the presence of oxidizing agent. The tested compound was also light sensitive and was classified as photolabile both in solution and in the solid phase. However, the observed photodegradation in the solid phase was at a much lower level than in the case of photodegradation in solution. The study showed that both air temperature and RH had no significant effect on the stability of the tested substance during storage for 1 month at 100°C (dry heat) as well as during accelerated and intermediate tests. Based on the HPLC/MS/MS analysis, it can be concluded that acyclovir was formed as a degradation product of 6-(4-MeOPh)-TACV.

  11. High performance liquid chromatography (HPLC): Water and environmental samples. (Latest citations from the EI compendex*plus database). Published Search

    SciTech Connect

    1996-01-01

    The bibliography contains citations concerning analyses of water by high pressure, high speed, high performance liquid chromatographic (HPLC). Environmental-related samples such as wastewaters, sewage sludge, and sediment are also covered. HPLC techniques are discussed and equipment such as detectors and columns is evaluated. (Contains 50-250 citations and includes a subject term index and title list.) (Copyright NERAC, Inc. 1995)

  12. 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.

  13. Quantification of the molecular species of diacylglycerols,triacylglycerols and tetraacylglycerols in lesquerella (Physaria fendleri) oil by HPLC and MS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ten diacylglycerols (DAG), 74 triacylglycerols (TAG) and 13 tetraacylglycerols in the seed oil of Physaria fendleri were recently identified by HPLC and MS. These acylglycerols (AG) were quantified by HPLC with evaporative light scattering detector and electrospray ionization mass spectrometry of th...

  14. HPLC analysis of linear furocoumarins (psoralens) in healthy celery (Apium graveolens).

    PubMed

    Beier, R C; Ivie, G W; Oertli, E H; Holt, D L

    1983-04-01

    Four linear furocoumarins (psoralen, bergapten, xanthotoxin, and isopimpinellin) were isolated from three varieties of healthy, commercially grown celery (Apium graveolens). Psoralen has not previously been reported to occur in celery. Combined levels of these photomutagenic and photocarcinogenic furocoumarins measured by normal-phase HPLC did not exceed 1.3 ppm in any of the celery varieties studied.

  15. 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 ...

  16. Melanin determination by high performance liquid chromatography (HPLC) for K. marxianus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ultraviolet light (UV) mutated K. marxianus was found to turn dark brown during a growth assay. This brown color was hypothesized to be melanin overproduction influenced by the UV exposure. Cell cultures were oxidized and HPLC analyzed to determine melanin concentrations. The resulting melanin con...

  17. Prediction of peptide retention at different HPLC conditions from multiple linear regression models.

    PubMed

    Baczek, Tomasz; Wiczling, Paweł; Marszałł, Michał; Heyden, Yvan Vander; Kaliszan, Roman

    2005-01-01

    To quantitatively characterize the structure of a peptide and to predict its gradient retention time at given HPLC conditions three structural descriptors are used: (i) logarithm of the sum of retention times of the amino acids composing the peptide, log SumAA, (ii) logarithm of the van der Waals volume of the peptide, log VDW(Vol), (iii) and the logarithm of the peptide's calculated n-octanol-water partition coefficient, clog P. The log SumAA descriptor is obtained from empirical data for 20 natural amino acids, determined in a given HPLC system. The two other descriptors are calculated from the peptides' structural formulas using molecular modeling methods. The quantitative structure-retention relationships (QSRR), build by multiple linear regression, describe HPLC retention of peptide on a given chromatographic system on which the retention of the 20 amino acids was predetermined. A structurally diversified series of 98 peptides was employed. The predicted gradient retention times on several chromatographic systems were in good agreement with the experimental data. The QSRR equations, derived for a given system operated at variable gradient times and temperatures allowed for the prediction of peptide retention in that system. Matching the experimental HPLC retention to the theoretically predicted for a presumed peptide could facilitate original protein identification in proteomics. In conjunction with MS data, prediction of the retention time for a given peptide might be used to improve the confidence of peptide identifications and to increase the number of correctly identified peptides.

  18. 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. PMID:26353956

  19. Using HPLC-Mass Spectrometry to Teach Proteomics Concepts with Problem-Based Techniques

    ERIC Educational Resources Information Center

    Short, Michael; Short, Anne; Vankempen, Rachel; Seymour, Michael; Burnatowska-Hledin, Maria

    2010-01-01

    Practical instruction of proteomics concepts was provided using high-performance liquid chromatography coupled with a mass selective detection system (HPLC-MS) for the analysis of simulated protein digests. The samples were prepared from selected dipeptides in order to facilitate the mass spectral identification. As part of the prelaboratory…

  20. Functional region identification in proteins by accumulative-quantitative peptide mapping using RP-HPLC-MS.

    PubMed

    Kuipers, Bas J H; Bakx, E J; Gruppen, Harry

    2007-11-14

    A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or the peptide mixture is too complex to purify and characterize each peptide individually. In the present work, aggregating peptides obtained by proteolytic hydrolysis of soy glycinin were used as a case study. The aggregating peptides are isolated and subsequently further degraded with trypsin to result in peptides with a mass <5000 Da to enable sequence identification using RP-HPLC-MS in combination with MS/MS. Prior to RP-HPLC the peptides are fractionated using anion and cation exchange chromatography. The fractions obtained are analyzed with RP-HPLC-MS. The peptides, with identified sequences, were quantified using the peak areas of the RP-HPLC chromatograms measured at 214 nm. Next, the peak areas were corrected for the molar extinction coefficient of the individual peptides, followed by accumulative-quantitative peptide mapping. The results show that in complex systems, based on the method described, the regions in the parental protein from which the functional peptides originate can be properly identified.

  1. A confirmatory HPLC-MS/MS method for ten synthetic corticosteroids in bovine urines.

    PubMed

    Savu, S R; Silvestro, L; Haag, A; Sörgel, F

    1996-12-01

    In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

  2. Extraction and Purification of Glucoraphanin by Preparative High-Performance Liquid Chromatography (HPLC)

    ERIC Educational Resources Information Center

    Lee, Iris; Boyce, Mary C.

    2011-01-01

    A student activity that focuses on the isolation of glucoraphanin from broccoli using preparative high-performance liquid chromatography (HPLC) is presented here. Glucoraphanin is a glucosinolate, whose byproducts are known to possess anticancer properties. It is present naturally at high levels in broccoli and other "Brassica" vegetables. This…

  3. Characterization and classification of stationary phases in HPLC and SFC – a review.

    PubMed

    Galea, Charlene; Mangelings, Debby; Vander Heyden, Yvan

    2015-07-30

    Packed column supercritical fluid chromatography (pSFC) is an attractive technique in drug discovery related analysis because it offers several advantages over the more commonly used high-performance liquid chromatography (HPLC) technique. The environmental-friendly CO2 mobile phase, the high-throughput capacity, the increased efficiency and the lower operational costs give SFC additional benefits over HPLC in analysis related to drug development. The latter technique is well established and has been used for decades in the pharmaceutical industry. On the other hand, SFC is still in its infancy, even though the technique has been known for decades and researchers are still discovering the possibilities and limitations of this technique. Column characterization aims to obtain a quantitative understanding of the properties of a column that influence the selectivity of a separation. Columns have been extensively characterized in HPLC using several approaches. However, limited column characterization has been done in SFC. Determining column properties allows the rapid selection of appropriate columns for method development for a particular application. Hence, they also show which columns will provide similar or dissimilar separations. The aim of this review is to compare and contrast approaches used to characterize stationary phases in HPLC and SFC, and to highlight points that still may need to be researched further. PMID:26320631

  4. Evaluation of Haemagglutinin Content by RP-HPLC to Generate Pandemic Influenza Vaccine

    PubMed Central

    Kang, Hyunkyung; Roh, Hang Sik; Song, Hyemin; Lee, Kwangmoon; Chung, Seung-Tae; Ban, Sang-ja; Mo, In Pil; An, Beum-Soo; Ahn, Chi-Young

    2016-01-01

    The potency of influenza vaccine is determined based on its hemagglutinin (HA) content. In general, single radial immunodiffusion (SRID) assay has been utilized as the standard method to measure HA content. However, preparation of reagents for SRID such as antigen and antibody takes approximately 2~3 months, which causes delays in the development of influenza vaccine. Therefore, quantification of HA content by other alternative methods is required. In this study, we measured HA contents of H1N1 antigen and H1N1 influenza vaccine by reverse phase-high performance liquid chromatography (RP-HPLC) methods. The presence of HA1 and HA2 was investigated by silver staining and Western blot assay. In addition, accuracy and repeatability of HA measurement by RP-HPLC were evaluated. Comparison of HA concentration by SRID and RP-HPLC revealed a precise correlation between the two methods. Our results suggest that RP-HPLC assay can replace SRID in the event of a pandemic flu outbreak for rapid vaccine development.

  5. Differentiation of Cirsium japonicum and C. setosum by TLC and HPLC-MS.

    PubMed

    Ganzera, Markus; Pöcher, Astrid; Stuppner, Hermann

    2005-01-01

    The Chinese Pharmacopoeia indicates the use of field thistle (Cirsium setosum) and Japanese field thistle (C. japonicum) in the treatment of bleeding and inflammation. In the absence of an analytical method for the differentiation and analysis of these two species, TLC and HPLC-MS methods have been developed for this purpose. Both species could be readily distinguished by their flavonoid pattern as revealed by TLC on silica gel layers eluted with ethyl acetate:formic acid:acetic acid:water. The quantitative determination of four flavonoids, namely hispidulin-7-neohesperidoside, linarin, pectolinarin and luteolin, was possible using HPLC. Their optimum separation was achieved on a C12 column eluted with water and 0.025% trifluoroacetic acid in acetonitrile. HPLC-MS experiments were performed to confirm peak identity. In samples of C. japonicum, pectolinarin was the major flavonoid (0.32-2.00%), followed by linarin, hispidulin-7-neohesperidoside and luteolin; the total flavonoid content varied from 0.81 to 3.67%. In C. setosum only one flavonoid (linarin; 1.36-2.83%) was assignable. The HPLC method was validated for linearity, limit of detection (< or = 1.7 ng on-column), peak purity, repeatability (< or = 2.3%) and accuracy (recovery rates of spiked samples were between 99.2 and 101.6%).

  6. HPLC pigment analysis of marine phytoplankton during a red tide occurrence in Tolo Harbour, Hong Kong.

    PubMed

    Wong, C Kwan; Wong, C Kim

    2003-09-01

    A red tide was detected in the inner parts of Tolo Harbour, Hong Kong, in November 2000. Water samples were collected from a fixed station at the centre of the red tide patch for microscopic analysis of phytoplankton community composition and high performance liquid chromatography (HPLC) analysis of phytoplankton pigments. At the peak of the red tide on 24 November 2000, phytoplankton was dominated by the dinoflagellate Scrippsiella trochoidea. The red tide began to decline at the end of November and, by 1 December 2000, the phytoplankton was dominated by diatoms. Chlorophylls and carotenoids in water samples were analysed using HPLC pigment separation technique. Dinoflagellates were indicated by the signature pigment peridinin. Significant correlation (r=0.999) was found between the peridinin concentration and dinoflagellate density. A decrease in peridinin and an increase in fucoxanthin, a major carotenoid in diatoms, marked the shift in phytoplankton composition at the end of the red tide. HPLC analysis also revealed the occurrence of minor phytoplankton groups that are difficult to identify by light microscopy. Red tide monitoring and study of red tide dynamics in Hong Kong have been based on cell counting and spectrophotometric or fluorometric measurement of chlorophyll a. HPLC pigment analysis provides an effective alternative for investigating phytoplankton dynamics during red tide and other algal blooms.

  7. SEPARATION OF OCTYLPHENOL POLYETHER ALCOHOLS SURFACTANTS BY CAPILLARY COLUMN SFC AND HPLC

    EPA Science Inventory

    Separation of nonionic octylphenol polyether alcohols (OPA) by supercritical fluid chromatography (SFC) and HPLC is described. Using a density programming and a 50-μm i.d. capillary column, a total of 18 group oligomers was separated. The effects of the operating parameters, such...

  8. Simultaneous HPLC quantitative analysis of active compounds in leaves of Moringa oleifera Lam.

    PubMed

    Vongsak, Boonyadist; Sithisarn, Pongtip; Gritsanapan, Wandee

    2014-08-01

    Moringa oleifera Lam. has been used as a traditional medicine for the treatment of numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) analysis was developed and validated for the determination of the contents of crypto-chlorogenic acid, isoquercetin and astragalin, the primary antioxidative compounds, in M. oleifera leaves. HPLC analysis was successfully conducted by using a Hypersil BDS C18 column, eluted with a gradient of methanol-1% acetic acid with a flow rate of 1 mL/min, and detected at 334 nm. Parameters for the validation included linearity, precision, accuracy and limits of detection and quantitation. The developed HPLC method was precise, with relative standard deviation < 2%. The recovery values of crypto-chlorogenic acid, isoquercetin and astragalin in M. oleifera leaf extracts were 98.50, 98.47 and 98.59%, respectively. The average contents of these compounds in the dried ethanolic extracts of the leaves of M. oleifera collected from different regions of Thailand were 0.081, 0.120 and 0.153% (w/w), respectively. The developed HPLC method was appropriate and practical for the simultaneous analysis of crypto-chlorogenic acid, isoquercetin and astragalin in the leaf extract of M. oleifera. This work is valuable as guidance for the standardization of the leaf extracts and pharmaceutical products of M. oleifera. PMID:23828911

  9. H.p.l.c. analysis of di- and tri-carboxylic porphyrins in porphyric patients.

    PubMed Central

    Jackson, A H; Rao, K R; Smith, S G; Lash, T D

    1985-01-01

    New h.p.l.c. methods have been developed for the quantitative determination of di- and tri-carboxylic porphyrin methyl esters, and applied to the analysis of faecal extracts from patients with four different types of porphyria. PMID:3994688

  10. [Determination of myristicin and safrol in volatile oil of nutmeg and its processed products by HPLC].

    PubMed

    Jia, T; Sha, M; Wang, Z; Cao, A

    1997-07-01

    The contents of myristicin and safrol in the volatile oil of nutmeg and its processed products were determined by HPLC. This method is fast, accurate and gives good resolution. It has also been confirmed that myristicin tends to decrease in varying degrees after the nutmeg has been processed. PMID:11038899

  11. 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...

  12. 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. PMID:24912723

  13. Preparation of (+)-trans-isoalliin and its isomers by chemical synthesis and RP-HPLC resolution.

    PubMed

    Jayathilaka, Lasanthi; Gupta, Shalini; Huang, Jin-Sheng; Lee, Jenny; Lee, Bao-Shiang

    2014-09-01

    Naturally occurring (+)-trans-isoalliin, (R(C)R(S))-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (-)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (-)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC.

  14. Preparation of (+)-Trans-Isoalliin and Its Isomers by Chemical Synthesis and RP-HPLC Resolution

    PubMed Central

    Jayathilaka, Lasanthi; Gupta, Shalini; Huang, Jin-Sheng; Lee, Jenny; Lee, Bao-Shiang

    2014-01-01

    Naturally occurring (+)-trans-isoalliin, (RCRS)-(+)-trans-S-1-propenyl-L-cysteine sulfoxide, is a major cysteine sulfoxide in onion. The importance of producing it synthetically to support further research is very well recognized. The (+)-trans-isoalliin is prepared by chemical synthesis and reversed-phase (RP)-HPLC. First, S-2-propenyl-L-cysteine (deoxyalliin) is formed from L-cysteine and allyl bromide, which is then isomerized to S-1-propenyl-L-cysteine (deoxyisoalliin) by a base-catalyzed reaction. A mixture of cis and trans forms of deoxyisoalliin is formed and separated by RP-HPLC. Oxidation of the trans form of deoxyisoalliin by H2O2 produces a mixture of (−)- and (+)-trans-isoalliin. Finally, RP-HPLC is used successfully in separating (−)- and (+)-trans-isoalliin, and hence, (+)-trans-isoalliin is synthesized for the first time in this study. In addition, the (±) diastereomers of cis-isoalliin are also separated and purified by RP-HPLC. PMID:25187757

  15. Simultaneous HPLC quantitative analysis of active compounds in leaves of Moringa oleifera Lam.

    PubMed

    Vongsak, Boonyadist; Sithisarn, Pongtip; Gritsanapan, Wandee

    2014-08-01

    Moringa oleifera Lam. has been used as a traditional medicine for the treatment of numerous diseases. A simultaneous high-performance liquid chromatography (HPLC) analysis was developed and validated for the determination of the contents of crypto-chlorogenic acid, isoquercetin and astragalin, the primary antioxidative compounds, in M. oleifera leaves. HPLC analysis was successfully conducted by using a Hypersil BDS C18 column, eluted with a gradient of methanol-1% acetic acid with a flow rate of 1 mL/min, and detected at 334 nm. Parameters for the validation included linearity, precision, accuracy and limits of detection and quantitation. The developed HPLC method was precise, with relative standard deviation < 2%. The recovery values of crypto-chlorogenic acid, isoquercetin and astragalin in M. oleifera leaf extracts were 98.50, 98.47 and 98.59%, respectively. The average contents of these compounds in the dried ethanolic extracts of the leaves of M. oleifera collected from different regions of Thailand were 0.081, 0.120 and 0.153% (w/w), respectively. The developed HPLC method was appropriate and practical for the simultaneous analysis of crypto-chlorogenic acid, isoquercetin and astragalin in the leaf extract of M. oleifera. This work is valuable as guidance for the standardization of the leaf extracts and pharmaceutical products of M. oleifera.

  16. HPLC imprinted-stationary phase prepared by precipitation polymerisation for the determination of thiabendazole in fruit.

    PubMed

    Turiel, E; Tadeo, J L; Cormack, P A G; Martin-Esteban, A

    2005-12-01

    A molecularly imprinted polymer (MIP) tailored for the HPLC determination of the fungicide thiabendazole (TBZ) has been synthesised in one single preparative step by precipitation polymerisation in an acetonitrile/toluene co-solvent, using TBZ as template molecule, methacrylic acid as functional monomer and divinylbenzene-80 as crosslinker. The imprinted polymer particulates obtained were characterised by scanning electron microscopy and nitrogen sorption porosimetry. These analyses showed clearly that spherical polymer particulates (polymer microspheres) with narrow size distributions (average particle diameter approximately 3.5 microm) and well-developed pore structures had been produced. The imprinted microspheres were packed into a stainless steel HPLC column (50 x 4.6 mm id) and evaluated as an imprinted stationary phase. The imprinting effect was demonstrated clearly, i.e., the column was observed to bind TBZ selectively, and the effect of different chromatographic parameters (e.g., temperature, flow-rate and elution solvents) on TBZ retention/elution studied. Under optimised conditions, the TBZ-imprinted column was used for the HPLC-fluorescence (HPLC-F) determination of TBZ directly from orange (both whole fruit and juice), lemon, grape and strawberry extracts at low concentration levels in less than 15 min, without any need for a clean-up step in the analytical protocol. PMID:16284658

  17. PHYTOPLANKTON PIGMENT ANALYSIS BY HPLC FOR ASSESSING COMMUNITY COMPOSITION IN THE LAURENTIAN GREAT LAKES

    EPA Science Inventory

    A technique to rapidly assess phytoplankton dynamics is being evaluated for its utility in the Great Lakes. Comparison to traditional microscopic techniques and to more recent in-situ FluoroProbe technology will allow us to determine if HPLC pigment analysis can provide unique a...

  18. HPLC monitoring of spontaneous non-linear peptidization dynamics of selected amino acids in solution.

    PubMed

    Godziek, Agnieszka; Maciejowska, Anna; Sajewicz, Mieczysław; Kowalska, Teresa

    2015-03-01

    This is our new study in a series of publications devoted to exploration of applicability of high-performance liquid chromatography (HPLC) to providing answers to difficult questions from the area of the reaction kinetics and mechanisms with non-linear reactions. Although an excellent analytical performance of HPLC is an indisputable fact, so far its performance as a tool in the kinetic and mechanistic studies has been tested to a lesser extent. In our earlier studies, spontaneous peptidization dynamics of amino acids in solution was demonstrated by means of HPLC upon a few amino acid examples, and on that basis a theoretical model has been developed, anticipating an interdependence of dynamics on chemical structures of amino acids involved. In order to expand the spectrum of experimentally investigated amino acid cases, in this study we present the results valid for three novel amino acids of significant life sciences importance, which differ in terms of peptidization dynamics. Experimental evidence originates from the achiral HPLC with the evaporative light scattering detection and MS detection. A conclusion is drawn that different spontaneous peptidization dynamics of amino acids may significantly influence chemical composition of proteins encountered in living organisms. Hence, a need emerges for systematic physicochemical studies on spontaneous non-linear peptidization dynamics of proteinogenic amino acids in liquid abiotic (but also in the biotic) systems.

  19. Estimation of phytoplankton biomass using HPLC pigment analysis in the southwestern Black Sea

    NASA Astrophysics Data System (ADS)

    Ediger, D.; Soydemir, N.; Kideys, A. E.

    2006-08-01

    The phytoplankton population of the southwestern Black Sea in May 2001 was studied by taxonomic analysis using microscopic examination and by pigment analyses using high-performance liquid chromatography (HPLC). Pigment data, which identified phytoplankton assemblages dominated by dinoflagellates, diatoms and coccolithophores in May 2001, were compared to phytoplankton cell counts and biomass. There were significant ( p<0.002-0.01, r=0.56-0.67) relationships between the taxon-specific pigment concentrations and the taxon-specific cell numbers during this sampling period. The ratios of chlorophyll- a to the dominant accessory pigments calculated by multiple linear regressions were 1.2 (chlorophyll- a: peridinin) in dinoflagellates, 1.8 (chlorophyll- a: fucoxanthin) in diatoms, and 2.66 (chlorophyll- a: 19'-hexonoyloxyfucoxanthin) in coccolithophores. HPLC-determined chlorophyll- a biomass correlated well with the sum of the group-specific pigment biomass ( p<0.001, r2=0.95). The phytoplankton assemblage as revealed by the microscopic and HPLC analyses was thus made up of common Black Sea groups showing that HPLC pigment analysis can be used to quantify phytoplankton assemblages in the Black Sea based on simple ratios.

  20. 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...

  1. HPLC imprinted-stationary phase prepared by precipitation polymerisation for the determination of thiabendazole in fruit.

    PubMed

    Turiel, E; Tadeo, J L; Cormack, P A G; Martin-Esteban, A

    2005-12-01

    A molecularly imprinted polymer (MIP) tailored for the HPLC determination of the fungicide thiabendazole (TBZ) has been synthesised in one single preparative step by precipitation polymerisation in an acetonitrile/toluene co-solvent, using TBZ as template molecule, methacrylic acid as functional monomer and divinylbenzene-80 as crosslinker. The imprinted polymer particulates obtained were characterised by scanning electron microscopy and nitrogen sorption porosimetry. These analyses showed clearly that spherical polymer particulates (polymer microspheres) with narrow size distributions (average particle diameter approximately 3.5 microm) and well-developed pore structures had been produced. The imprinted microspheres were packed into a stainless steel HPLC column (50 x 4.6 mm id) and evaluated as an imprinted stationary phase. The imprinting effect was demonstrated clearly, i.e., the column was observed to bind TBZ selectively, and the effect of different chromatographic parameters (e.g., temperature, flow-rate and elution solvents) on TBZ retention/elution studied. Under optimised conditions, the TBZ-imprinted column was used for the HPLC-fluorescence (HPLC-F) determination of TBZ directly from orange (both whole fruit and juice), lemon, grape and strawberry extracts at low concentration levels in less than 15 min, without any need for a clean-up step in the analytical protocol.

  2. The Mysterious Death: An HPLC Lab Experiment. An Undergraduate Forensic Lab

    ERIC Educational Resources Information Center

    Beussman, Douglas J.

    2007-01-01

    A high-performance liquid chromatography (HPLC) laboratory experiment based on the separation of four prescription drugs (disopyramide, lidocaine, procainamide, and quinidine) is presented. The experiment is set within the forensic science context of the discovery of a patient's mysterious death where a drug overdose is suspected. Each lab group…

  3. [Determination of aucubin in Plantago asiatica L., P. major L. and P. depressa Willd. by HPLC].

    PubMed

    Guo, Y; Cha, M; Chao, A; Yuan, C

    1991-12-01

    A HPLC method for the determination of aucubin in P. asiatica, P. major and P. depressa was established. The mobile phase is MeOH-H2O (17:83). Good in producibility and high in rate of recovery, this method provides an effective way for the study of Plantago.

  4. Retention of [(18)F]fluoride on reversed phase HPLC columns.

    PubMed

    Ory, Dieter; Van den Brande, Jeroen; de Groot, Tjibbe; Serdons, Kim; Bex, Marva; Declercq, Lieven; Cleeren, Frederik; Ooms, Maarten; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy

    2015-01-01

    As [(18)F]fluoride is a starting reagent in the radiosynthesis of most fluorine-18 labeled positron emission tomography (PET) tracers, its chromatographic behavior on reversed phase (RP) HPLC columns is important for the purification performance and accuracy of RP HPLC quality control methods. We have investigated the chromatographic behavior and recovery of [(18)F]fluoride as a function of the type and brand of RP HPLC column, the pH and the composition of the mobile phase. Elution and elution profile of [(18)F]fluoride from six RP-HPLC columns (Waters XBridge C18 3 mm × 100 mm 3.5 μm; Grace Platinum EPS C18 4.6 mm × 100 mm, 3 μm; Waters XTerra C18 4.6 mm × 250 mm, 5 μm; Phenomenex C18 4.6 mm × 150 mm, 5 μm; Hamilton PRP-1 column 4.1 mm × 150 mm, 5 μm; Merck KGaA Chromolith Performance C18 3 mm × 100 mm) eluted with mobile phase composed of phosphate or acetate buffers (pH 2, 3, 4, 5, 7.3 and 9) and acetonitrile or ethanol as organic modifier were characterized. The elution profile was determined by on-line radioactivity measurement in the column eluate and recovery was calculated by comparison of radioactivity eluted with the HPLC column present or absent in the chromatographic flow path. Interestingly, [(18)F]fluoride recovery increased with increasing pH. At pH 3 all packed silica-based columns showed significant retention of fluorine-18, whereas almost no retention was observed on a polymeric PRP-1 column. However at pH 5, [(18)F]fluoride recovery was above 90% for each tested column. In addition, small differences were observed when changing the composition of the mobile phase. We therefore recommend to use a mobile phase with pH > 5 for silica based C18 columns for both quality control and semi-preparative HPLC of fluorine-18 labeled PET radiopharmaceuticals. If required a lower pH can be used in combination with a polymer based HPLC column.

  5. HPLC/ICP-MS in combination with "reverse" online isotope dilution in drug metabolism studies.

    PubMed

    Meermann, Björn; Hulstaert, Anne; Laenen, Aline; Van Looveren, Cis; Vliegen, Maarten; Cuyckens, Filip; Vanhaecke, Frank

    2012-03-01

    During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel ((14)C or (3)H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisible, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach. However, the method lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with "reverse" online isotope dilution ("reverse" online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a (81)Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and "mixed" species, based on their isotopic "fingerprint". A good repeatability (relative standard deviation of 4.2%) and limit of detection (0.35 mg of drug compound L(-1) of feces extract), of the same order of magnitude as those observed for "normal" online ID HPLC/ICP-MS and HPLC/radiodetection, were achieved.

  6. Shape matters: size-exclusion HPLC for the study of nucleic acid structural polymorphism

    PubMed Central

    Largy, Eric; Mergny, Jean-Louis

    2014-01-01

    In recent years, an increasing number of reports have been focused on the structure and biological role of non-canonical nucleic acid secondary structures. Many of these studies involve the use of oligonucleotides that can often adopt a variety of structures depending on the experimental conditions, and hence change the outcome of an assay. The knowledge of the structure(s) formed by oligonucleotides is thus critical to correctly interpret the results, and gain insight into the biological role of these particular sequences. Herein we demonstrate that size-exclusion HPLC (SE-HPLC) is a simple yet surprisingly powerful tool to quickly and effortlessly assess the secondary structure(s) formed by oligonucleotides. For the first time, an extensive calibration and validation of the use of SE-HPLC to confidently detect the presence of different species displaying various structure and/or molecularity, involving >110 oligonucleotides forming a variety of secondary structures (antiparallel, parallel, A-tract bent and mismatched duplexes, triplexes, G-quadruplexes and i-motifs, RNA stem loops), is performed. Moreover, we introduce simple metrics that allow the use of SE-HPLC without the need for a tedious calibration work. We show that the remarkable versatility of the method allows to quickly establish the influence of a number of experimental parameters on nucleic acid structuration and to operate on a wide range of oligonucleotide concentrations. Case studies are provided to clearly illustrate the all-terrain capabilities of SE-HPLC for oligonucleotide secondary structure analysis. Finally, this manuscript features a number of important observations contributing to a better understanding of nucleic acid structural polymorphism. PMID:25143531

  7. Determination of cadmium and lead species and phytochelatins in pea (Pisum sativum) by HPLC-ICP-MS and HPLC-ESI-MSn.

    PubMed

    Barałkiewicz, Danuta; Kózka, Małgorzata; Piechalak, Aneta; Tomaszewska, Barbara; Sobczak, Paweł

    2009-07-15

    An analytical approach based on hyphenated techniques was used for studying the speciation of cadmium and lead in Pisum sativum. Proper preservation conditions were employed to avoid the oxidation of -SH groups and corresponding decomposition of metal-binding complexes. SEC column was washed with 5 mM beta-mercaptoethanol and then samples were analysed using ICP-MS as a detector. Results showed that cadmium is the inhibitor of lead uptake. HPLC-ESI-MS(n) assays revealed fragmentation pathways of phytochelatins. PMID:19559910

  8. Determination of cadmium and lead species and phytochelatins in pea (Pisum sativum) by HPLC-ICP-MS and HPLC-ESI-MSn.

    PubMed

    Barałkiewicz, Danuta; Kózka, Małgorzata; Piechalak, Aneta; Tomaszewska, Barbara; Sobczak, Paweł

    2009-07-15

    An analytical approach based on hyphenated techniques was used for studying the speciation of cadmium and lead in Pisum sativum. Proper preservation conditions were employed to avoid the oxidation of -SH groups and corresponding decomposition of metal-binding complexes. SEC column was washed with 5 mM beta-mercaptoethanol and then samples were analysed using ICP-MS as a detector. Results showed that cadmium is the inhibitor of lead uptake. HPLC-ESI-MS(n) assays revealed fragmentation pathways of phytochelatins.

  9. [Estrogens determination of livestock dung based on UE-SPE-HPLC/FLD].

    PubMed

    Fu, Yin-Jie; Ling, Wan-Ting; Dong, Chang-Xun; Liu, Juan; Gao, Yan-Zheng; Pan, Yu-Lan

    2013-11-01

    A method for detecting the estrogens estriol, 17beta-estradiol, ethinyl estradiol, and bisphenol A in livestock dung was established by the combination of ultrasonic extraction (UE), solid phase extraction (SPE) purification, and high performance liquid chromatography (HPLC) with fluorescence detector (FLD). The dung samples were extracted with ethyl acetate ultrasonication for 30 min, and purified with C18 solid phase extraction column and related solvents. The test four estrogens in the dung samples were isolated with Inertsil ODS-SP-C18 reversed-phase columns (150 mm x 4.6 mm, 5 microm), and the isolated estrogens were detected with HPLC/FLD. The mobile phase of HPLC for the detection was methanol/acetonitrile/water (volume ratio of 20:30:50), with a flow rate of 0.8 mL x min(-1). The excitation and emission wavelengths of FLD were 280 and 310 nm, respectively, the HPLC column temperature was 40 degrees C, and the injection volume was 20 microL. Good linearity (correlation coefficient greater than 0.9995) was observed by the HPLC/FLD detection when the test four estrogens concentrations were in the range of 1.00-1000.00 microg x L(-1). The detection limit of estriol, bisphenol A, 17beta-estradiol, and ethinyl estradiol was 3.35, 5.01, 2.13, and 1.12 microg x kg(-1), respectively. When the added estrogens concentrations of pig, cow, and chicken dung samples were 0.05, 0.40, and, 1.00 microg x kg(-1), the average recovery of the four estrogens was 75.1%-91.1%, 78.4%-117.0%, and 78.6%-97.8%, respectively, with the relatively standard deviations (RSD, n = 6) all less than 6%. By adopting the established SPE-HPLC/FLD method to detect the estrogens in real pig, cow, and chicken dung samples from parts of the large-scale livestock raising farms in Nanjing of East China, the detection reproducibility was high, and the detection limit was low, being available and effective for the detection of the estrogens in livestock dung. PMID:24564161

  10. Phytochemical analysis of Rosa hybrida cv. 'Jardin de Granville' by HPTLC, HPLC-DAD and HPLC-ESI-HRMS: polyphenolic fingerprints of six plant organs.

    PubMed

    Riffault, Ludivine; Destandau, Emilie; Pasquier, Laure; André, Patrice; Elfakir, Claire

    2014-03-01

    The Rosa hybrida cultivar 'Jardin de Granville', a delicate clear pink flower, is here investigated through a progressive analytical strategy using complementary phytochemical screening methods in order to characterize the polyphenol content of several parts of the plant. The microwave hydro-ethanolic extract analysis of six different parts of the plant, carried out by High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) enabled initial identification of the polar molecular families present in each organ, namely tannins and flavonoids (quercetin and kaempferol derivatives). The HPLC fingerprints displayed different profiles for each organ, attesting to the original composition and potential valuation of the different plant parts. More detailed analyses of the extracts were then carried out by High Performance Liquid Chromatography coupled with electrospray ionization (ESI) mass spectrometry with a Q-TOF analyzer (ESI-HR-Q-TOF). Around 60 compounds were identified, mainly gallo-tannins, ellagi-tannins, catechin derivatives and glycoside derivatives of quercetin and kaempferol. Some compounds such as hyperoside or ellagic acid appeared to be ubiquitous and were found in abundance in each plant part. Woods were the richest organ in catechin and proanthocyanidin derivatives while kaempferol derivatives were more numerous and abundant in bud and flower parts. PMID:24461781

  11. Phytochemical analysis of Rosa hybrida cv. 'Jardin de Granville' by HPTLC, HPLC-DAD and HPLC-ESI-HRMS: polyphenolic fingerprints of six plant organs.

    PubMed

    Riffault, Ludivine; Destandau, Emilie; Pasquier, Laure; André, Patrice; Elfakir, Claire

    2014-03-01

    The Rosa hybrida cultivar 'Jardin de Granville', a delicate clear pink flower, is here investigated through a progressive analytical strategy using complementary phytochemical screening methods in order to characterize the polyphenol content of several parts of the plant. The microwave hydro-ethanolic extract analysis of six different parts of the plant, carried out by High Performance Thin Layer Chromatography (HPTLC) and High Performance Liquid Chromatography coupled with a Diode Array Detector (HPLC-DAD) enabled initial identification of the polar molecular families present in each organ, namely tannins and flavonoids (quercetin and kaempferol derivatives). The HPLC fingerprints displayed different profiles for each organ, attesting to the original composition and potential valuation of the different plant parts. More detailed analyses of the extracts were then carried out by High Performance Liquid Chromatography coupled with electrospray ionization (ESI) mass spectrometry with a Q-TOF analyzer (ESI-HR-Q-TOF). Around 60 compounds were identified, mainly gallo-tannins, ellagi-tannins, catechin derivatives and glycoside derivatives of quercetin and kaempferol. Some compounds such as hyperoside or ellagic acid appeared to be ubiquitous and were found in abundance in each plant part. Woods were the richest organ in catechin and proanthocyanidin derivatives while kaempferol derivatives were more numerous and abundant in bud and flower parts.

  12. Analysis of the constituents and quality control of Viola odorata aqueous preparations by HPLC-DAD and HPLC-ESI-MS.

    PubMed

    Karioti, Anastasia; Furlan, Claudia; Vincieri, Franco Francesco; Bilia, Anna Rita

    2011-02-01

    In the present study, a method based on liquid chromatography with diode array detection (HPLC-DAD) coupled to an electrospray ionisation (ESI) interface was developed for the determination of the constituents in the aqueous preparations of Viola odorata L. flowering tops. The developed assay was fast, simple and effective and permitted the quality control of the preparations. The aim of this work was to assess the qualitative and quantitative profile of the investigated preparations, which find until today wide applications in food and cosmetic industry, and to propose a validated method for their quality control. Characteristic constituents of V. odorata flowers are considered to be the anthocyanins; however, a detailed literature research showed that data concerning their chemical content are scarce. HPLC-DAD-ESI-MS analyses supported by extensive preparative chromatographic investigations and 2D NMR analyses revealed the predominance of complex flavonol glycosides and permitted the complete characterisation of the content of V. odorata preparations. This is the first report of detailed analysis of the chemical composition of V. odorata flowers.

  13. Validated stability indicating RP-HPLC for quantitation of nitazoxanide in presence of its alkaline degradation products and their characterization by HPLC-tandem mass spectrometry.

    PubMed

    Hegazy, Maha; Kessiba, Amira; El Gindy, Ahmed Emad; Abdelkawy, Mohamed

    2014-10-01

    A simple and sensitive stability indicating HPLC method was developed and validated for quantitative determination of Nitazoxanide (NTZ), a new antiprotozoal drug, in presence of degradation products generated under forced alkaline hydrolysis. Chromatographic separation was achieved on Inertsil C8-3 column (150 × 4.6 mm i.d.) using a mobile phase composed of acetonitrile: 50 mM ammonium acetate buffer (50:50, v/v, pH 5.0 adjusted with acetic acid) at a flow rate of 1 mL/min. Quantification was achieved with UV detection at 298 nm based on relative peak area. The method was linear over the concentration range of 0.8-50 µg/mL (r = 0.9999) with a limit of detection and quantification 0.0410 and 0.1242 µg/mL, respectively. The developed method has the requisite accuracy, selectivity, sensitivity and precision to assay NTZ in presence of its degradation products either in bulk powder or in pharmaceutical formulations. The degradation products were then identified by HPLC-MS/MS analysis using an electrospray ionization source and an ion trap analyzer.

  14. Retention of ionizable compounds on HPLC. 12. The properties of liquid chromatography buffers in acetonitrile-water mobile phases that influence HPLC retention.

    PubMed

    Espinosa, Sonia; Bosch, Elisabeth; Rosés, Marti

    2002-08-01

    The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for ace tate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 60% in acetonitrile (v/v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estimation of the retention of an acid/base compound in a particular acetonitrile-water buffered mobile phase.

  15. 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.

  16. Analysis and stability of the constituents of artichoke and St. John's wort tinctures by HPLC-DAD and HPLC-MS.

    PubMed

    Bilia, A R; Bergonzi, M C; Mazzi, G; Vincieri, F F

    2002-05-01

    In continuing our investigations on tinctures, which represent both herbal drug preparations and herbal medicinal products, 40% and 60% v/v tinctures of artichoke and St. John's wort were investigated. Artichoke is largely used in hepatic disorders, while St. John's wort is an anti-inflammatory, antidepressant, and healing agent. Both herbal drugs contain various constituents, although the compounds responsible for the main effects have not yet been completely identified. However, caffeoylquinic acids and flavones seem to be of crucial importance for the activity of artichoke, as well as flavonoids, naphthodianthrones, and phloroglucinol derivatives for St. John's wort, and they are used as marker constituents. Thus, quantification of all these constituents was performed using high-performance liquid chromatography-diode array detection (HPLC-DAD) and HPLC--mass spectrometry (MS) analyses with rutin as external standard. In addition the stability of the constituents of these tinctures from accelerated and long-term testing was also evaluated. From the results it was evidenced that constituent content depends on the solvent used for the extraction. The stability was also shown to be very different and seems to be related to the water content of the tinctures.

  17. Measurement of methyl mercury (I) and mercury (II) in fish tissues and sediments by HPLC-ICPMS and HPLC-HGAAS.

    PubMed

    Jagtap, Rajani; Krikowa, Frank; Maher, William; Foster, Simon; Ellwood, Michael

    2011-07-15

    A procedure for the extraction and determination of methyl mercury and mercury (II) in fish muscle tissues and sediment samples is presented. The procedure involves extraction with 5% (v/v) 2-mercaptoethanol, separation and determination of mercury species by HPLC-ICPMS using a Perkin-Elmer 3 μm C8 (33 mm×3 mm) column and a mobile phase 3 containing 0.5% (v/v) 2-mercaptoethanol and 5% (v/v) CH(3)OH (pH 5.5) at a flow rate 1.5 ml min(-1) and a temperature of 25°C. Calibration curves for methyl mercury (I) and mercury (II) standards were linear in the range of 0-100 μgl(-1) (r(2)=0.9990 and r(2)=0.9995 respectively). The lowest measurable mercury was 0.4 μgl(-1) which corresponds to 0.01 μgg(-1) in fish tissues and sediments. Methyl mercury concentrations measured in biological certified reference materials, NRCC DORM - 2 Dogfish muscle (4.4±0.8 μgg(-1)), NRCC Dolt - 3 Dogfish liver (1.55±0.09 μgg(-1)), NIST RM 50 Albacore Tuna (0.89±0.08 μgg(-1)) and IRMM IMEP-20 Tuna fish (3.6±0.6 μgg(-1)) were in agreement with the certified value (4.47±0.32μgg(-1), 1.59±0.12 μgg(-1), 0.87±0.03 μgg(-1), 4.24±0.27 μgg(-1) respectively). For the sediment reference material ERM CC 580, a methyl mercury concentration of 0.070±0.002 μgg(-1) was measured which corresponds to an extraction efficiency of 92±3% of certified values (0.076±0.04 μgg(-1)) but within the range of published values (0.040-0.084 μgg(-1); mean±s.d.: 0.073±0.05 μgg(-1), n=40) for this material. The extraction procedure for the fish tissues was also compared against an enzymatic extraction using Protease type XIV that has been previously published and similar results were obtained. The use of HPLC-HGAAS with a Phenomenox 5 μm Luna C18 (250 mm×4.6 mm) column and a mobile phase containing 0.06 moll(-1) ammonium acetate (Merck Pty Limited, Australia) in 5% (v/v) methanol and 0.1% (w/v) l-cysteine at 25°C was evaluated as a complementary alternative to HPLC-ICPMS for the measurement of

  18. Measurement of methyl mercury (I) and mercury (II) in fish tissues and sediments by HPLC-ICPMS and HPLC-HGAAS.

    PubMed

    Jagtap, Rajani; Krikowa, Frank; Maher, William; Foster, Simon; Ellwood, Michael

    2011-07-15

    A procedure for the extraction and determination of methyl mercury and mercury (II) in fish muscle tissues and sediment samples is presented. The procedure involves extraction with 5% (v/v) 2-mercaptoethanol, separation and determination of mercury species by HPLC-ICPMS using a Perkin-Elmer 3 μm C8 (33 mm×3 mm) column and a mobile phase 3 containing 0.5% (v/v) 2-mercaptoethanol and 5% (v/v) CH(3)OH (pH 5.5) at a flow rate 1.5 ml min(-1) and a temperature of 25°C. Calibration curves for methyl mercury (I) and mercury (II) standards were linear in the range of 0-100 μgl(-1) (r(2)=0.9990 and r(2)=0.9995 respectively). The lowest measurable mercury was 0.4 μgl(-1) which corresponds to 0.01 μgg(-1) in fish tissues and sediments. Methyl mercury concentrations measured in biological certified reference materials, NRCC DORM - 2 Dogfish muscle (4.4±0.8 μgg(-1)), NRCC Dolt - 3 Dogfish liver (1.55±0.09 μgg(-1)), NIST RM 50 Albacore Tuna (0.89±0.08 μgg(-1)) and IRMM IMEP-20 Tuna fish (3.6±0.6 μgg(-1)) were in agreement with the certified value (4.47±0.32μgg(-1), 1.59±0.12 μgg(-1), 0.87±0.03 μgg(-1), 4.24±0.27 μgg(-1) respectively). For the sediment reference material ERM CC 580, a methyl mercury concentration of 0.070±0.002 μgg(-1) was measured which corresponds to an extraction efficiency of 92±3% of certified values (0.076±0.04 μgg(-1)) but within the range of published values (0.040-0.084 μgg(-1); mean±s.d.: 0.073±0.05 μgg(-1), n=40) for this material. The extraction procedure for the fish tissues was also compared against an enzymatic extraction using Protease type XIV that has been previously published and similar results were obtained. The use of HPLC-HGAAS with a Phenomenox 5 μm Luna C18 (250 mm×4.6 mm) column and a mobile phase containing 0.06 moll(-1) ammonium acetate (Merck Pty Limited, Australia) in 5% (v/v) methanol and 0.1% (w/v) l-cysteine at 25°C was evaluated as a complementary alternative to HPLC-ICPMS for the measurement of

  19. [Determination of 7 bio-active alkaloids in Stephania plants by RP-HPLC].

    PubMed

    Huang, J; Guo, J; Duan, G

    1998-07-01

    Seven bio-active alkaloids (stepholidine, sinoacutine, isocorydine, l-tetrahydropalmatine, crebanine, fanchinoline and tetrandrine) in Stephania plants were determined by RP-HPLC, using UV detection (282 nm) and gradient elution. The reversed phase system consisted of ODS column and methanol-water-triethylamine as mobile phase. The flow rate was 1.0 ml.min-1. Good linearity between peak heights and concentrations of the alkaloids was obtained in the concentration range. The HPLC method proved accurate, precise and sensitive. The results showed that there were some differences in the occurrence and content of the alkaloids between various species and between the same species from different habitats and collecting time. Based on the results, some species with high content of the 7 bio-active alkaloids were selected. The study provided some useful information for the utilization of medicinal plant resources in the genus Stephania. PMID:12016887

  20. Chemical fingerprint and quantitative analysis for quality control of polyphenols extracted from pomegranate peel by HPLC.

    PubMed

    Li, Jianke; He, Xiaoye; Li, Mengying; Zhao, Wei; Liu, Liu; Kong, Xianghong

    2015-06-01

    A simple and efficient HPLC fingerprint method was developed and validated for quality control of the polyphenols extracted from pomegranate peel (PPPs). Ten batches of pomegranate collected from different orchards in Shaanxi Lintong of China were used to establish the fingerprint. For the fingerprint analysis, 15 characteristic peaks were selected to evaluate the similarities of 10 batches of the PPPs. The similarities of the PPPs samples were all more than 0.968, indicating that the samples from different areas of Lintong were consistent. Additionally, simultaneous quantification of eight monophenols (including gallic acid, punicalagin, catechin, chlorogenic acid, caffeic acid, epicatechin, rutin, and ellagic acid) in the PPPs was conducted to interpret the consistency of the quality test. The results demonstrated that the HPLC fingerprint as a characteristic distinguishing method combining similarity evaluation and quantitative analysis can be successfully used to assess the quality and to identify the authenticity of the PPPs.

  1. [ILs-HPLC simultanesous determination of five alkaloids in phellodenddri chinensis cortex].

    PubMed

    Jiang, Xin-Yi; Zhang, Hui-Fen; Wang, Sheng-Nan; Chen, Xiao-Hui

    2014-10-01

    A RP-HPLC method was established for simultaneous determination of phellodendrine hydrochloride (PH1), magnoflorine hydrochloride (MH), jatrorrhizine hydrochloride (JH), palmatine hydrochloride (PH2) and berberine hydrochloride (BH) in Phellodendri Chinensis Cortex by using ionic liquids as mobile phase additives. The separation was performed on a Kromasil C18 (4.6 mm x 250 mm, 5 μm) coupled with ultraviolet (UV) detection. The effect of extraction solvent, detection wavelength, length of alkyl chain on different imidazolium ionic liquids and concentration of ionic liquids on the separation and determination of alkaloids were investigated. Ionic liquid, [BMIm] BF4, can obviously improve the resolution and peak shape. This ILs-HPLC method is simple, rapid, and reliable, which can be used for determination of alkaloids in Phellodenddri Chinensis Cortex. PMID:25612445

  2. Simultaneous analysis of neuroendocrine tumor markers by HPLC-electrochemical detection.

    PubMed

    Manickum, T

    2009-12-15

    A validated, high pressure liquid chromatographic (HPLC) method for simultaneous quantitation of urinary catecholic acids 4-hydroxy-3-methoxymandelic acid (HMMA) (vanylmandelic acid) (VMA), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and 5-hydroxyindole-3-acetic acid (5-HIAA) was developed. Sample preparation involved liquid-liquid extraction of acidified urine, containing iso-HMMA (IS) as internal standard, with ether, evaporation of the organic extract, followed by reconstitution of the residue in phosphate buffer at pH 3.3. After reversed-phase HPLC at 35 degrees C and separation on a Licrospher 125mmx4mm C(18) column (5microm particle size) with phosphate buffer (pH 3.5)-acetone (950:50, v/v) as eluent, quantitation is achieved by electrochemical detection using coulometric detection at a potential of +350mV. The method was successfully applied to routine diagnosis of neuroblastoma, carcinoid syndrome and pheochromocytoma. PMID:19926540

  3. [Preparation of ferulic acid, senkyunolide I and senkyunolide H from Ligusticum chuanxiong by preparative HPLC].

    PubMed

    Xiong, Yao-Kun; Liang, Shuang; Hong, Yan-Long; Yang, Xiu-Juan; Shen, Lan; Du, Yan; Feng, Yi

    2013-06-01

    Preparative HPLC was used to prepare ferulic acid, senkyunolide I and senkyunolide H from Ligusticum chuanxiong. The separation was conducted on a Shim-Pack Prep-ODS (20.0 mm x 250 mm, 5 microm) column with the mobile phase of methanol-0.2% glacial acetic acid (50:50)at the flow rate of 5 mL x min(-1). The detection wavelength was 278 nm, and the purity of each compound was detected by HPLC analysis. Spectral data analyses including UV, ESI-MS and NMR were used to identify their structures. This method is simple, fast, which is suitable for preparation of standard reference of ferulic acid, senkyunolide I and senkyunolide H from L. chuanxiong and can meet the requirement of new drug research and development. PMID:24066590

  4. Quantitative metabolite profiling of edible onion species by NMR and HPLC-MS.

    PubMed

    Soininen, Tuula H; Jukarainen, Niko; Auriola, Seppo O K; Julkunen-Tiitto, Riitta; Karjalainen, Reijo; Vepsäläinen, Jouko J

    2014-12-15

    Allium genus is a treasure trove of valuable bioactive compounds with potentially therapeutically important properties. This work utilises HPLC-MS and a constrained total-line-shape (CTLS) approach applied to (1)H NMR spectra to quantify metabolites present in onion species to reveal important inter-species differences. Extensive differences were detected between the sugar concentrations in onion species. Yellow onion contained the highest and red onion the lowest amounts of amino acids. The main flavonol-glucosides were quercetin 3,4'-diglucoside and quercetin 4'-glucoside. In general, the levels of flavonols were, higher in yellow onions than in red onions, and garlic and leek contained a lower amount of flavonols than the other Allium species. Our results highlight how (1)H NMR together with HPLC-MS can be useful in the quantification and the identification of the most abundant metabolites, representing an efficient means to pinpoint important functional food ingredients from Allium species.

  5. Validation of simultaneous volumetric and HPLC methods for the determination of pridinol mesylate in raw material.

    PubMed

    Simionato, Laura D; Ferello, Leonardo; Stamer, Sebastián; Zubata, Patricia D; Segall, Adriana I

    2013-01-01

    Simple, sensitive, and economical simultaneous volumetric and HPLC methods for the determination of pridinol mesylate in raw material have been developed. The volumetric method is based on the reaction of pridinol with sodium lauryl sulphate in diluted sulphuric acid. Dimethyl yellow was used as indicator to detect the end point of the titration in aqueous/organic layer. The HPLC method for the determination of pridinol mesylate employs a reverse phase C18 column at ambient temperature with a mobile phase consisting of acetonitrile: 0.05 M potassium dihydrogen phosphate, pH adjusted to 5.0 (1 : 2, v/v). The flow rate was 0.8 mL/min. Quantitation was achieved with UV detection at 258 nm based on peak area. Both methods were found to be suitable for the quality control of pridinol mesylate in raw material.

  6. GlycoBase and autoGU: resources for interpreting HPLC-glycan data.

    PubMed

    Campbell, Matthew P; Royle, Lousie; Rudd, Pauline M

    2015-01-01

    The biological relevance of protein glycosylation has made glycomics, the comprehensive study to identify all glycans in an organism, indispensable in many research fields. Determining the structure and functional relationship of glycoproteins requires the comprehensive characterization of glycan structures by a range of analytical methods. High performance liquid chromatography (HPLC) is a well-established technology commonly used for the complete structural elucidation of N- and O-linked glycans; however, the analysis of data is a major bottleneck and robust bioinformatic solutions are required. This chapter describes the availability of databases and tools, GlycoBase and autoGU developed in conjunction with the EUROCarbDB initiative, to assist the interpretation of HPLC-glycan data collections.

  7. Effect of oxotremorine, physostigmine, and scopolamine on brain acetylcholine synthesis: a study using HPLC

    SciTech Connect

    Bertrand, N.; Beley, A. )

    1990-11-01

    The synthesis rate of brain acetylcholine (ACh) was estimated in mice following i.v. administration of ({sup 3}H)choline (Ch). The measurements were performed 1 min after the tracer injection, using the ({sup 3}H)ACh/({sup 3}H)Ch specific radioactivity ratio as an index of ACh synthesis rate. Endogenous and labeled Ch and ACh were quantified using HPLC methodology. Oxotremorine and physostigmine (0.5 mg/kg, i.p.) increased the steady state concentration of brain ACh by + 130% and 84%, respectively and of Ch by + 60% (oxotremorine); they decreased ACh synthesis by 62 and 55%, respectively. By contrast, scopolamine (0.7 mg/kg, i.p.) decreased the cerebral content of Ch by - 26% and of ACh by - 23% without enhancing the synthesis of ACh. The results show the utility of HPLC methodology in the investigation of ACh turnover.

  8. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions. PMID:16001238

  9. HPLC-PDA-MS fingerprinting to assess the authenticity of pomegranate beverages.

    PubMed

    Borges, Gina; Crozier, Alan

    2012-12-01

    HPLC with fluorescence, PDA and mass spectrometric detection were used to analyse the (poly)phenol content of a Bordeaux red wine, POM Wonderful pomegranate juice and three other beverages advertised as being made from 100% pomegranate juice. The red wine and POM Wonderful juice contained characteristic anthocyanin profiles with the latter also being characterised by the presence of ellagitannins and the former by flavan-3-ols monomers and procyanidin dimers and trimers. The three other pomegranate products all contained the expected ellagitannins but their anthocyanin profiles were a mixture of red wine and pomegranate anthocyanins. They also contained flavan-3-ol monomers and procyanidin dimers and trimers, components not usually detected in 100% pomegranate beverages. The HPLC-based procedures, therefore, provide a straight-forward means of accessing the authenticity of pomegranate-based products with the ready detection of constituents derived from red grapes.

  10. Identification of collagen types in tissues using HPLC-MS/MS.

    PubMed

    Pataridis, Statis; Eckhardt, Adam; Mikulíková, Katerina; Sedláková, Pavla; Miksík, Ivan

    2008-10-01

    A method for the determination and quantification of collagen types I-V in rat tissues has been developed. This method is based on collagen fragmentation by cyanogen bromide followed by trypsin digestion. After that, HPLC-MS/MS (HPLC coupled to an IT mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Specific peptides for each collagen type were selected. According to online databases, these peptides are present in human, bovine, and rat collagens. As a result, this method can be potentially applied to other species' tissues as well, such as human tissues, and provides a universal and simple method of quantifying collagen types. The applicability of this method for analyzing collagen types was demonstrated on rat tissues (skin, tendon, and aorta).

  11. HPLC study on the stability of bendamustine hydrochloride immobilized onto polyphosphoesters.

    PubMed

    Pencheva, Ivanka; Bogomilova, Anita; Koseva, Neli; Obreshkova, Danka; Troev, Kolio

    2008-12-01

    Novel water soluble polymer complexes of bendamustine hydrochloride, a bifunctional alkylating agent with antimetabolic and cytotoxic activity, were developed using biodegradable polymer carriers-poly(oxyethylene H-phosphonate), poly(methyloxyethylene phosphate) and poly(hydroxyoxyethylene phosphate). Bendamustine hydrochloride was immobilized onto polyphosphoesters via covalent, ionic and hydrogen bonding. The structure of the complexes formed was elucidated by (1)H, (13)C, (31)P NMR and FT-IR spectroscopy. The chemical stability of bendamustine hydrochloride in the novel complexes was studied by HPLC analysis based on a validated method with appointed analytical parameters such as specificity, repeatability, limit of quantitation, limit of detection and linearity. The results from the HPLC indicate that in neutral (pH 7) and alkaline (pH 9) media bendamustine hydrochloride in the polymer complexes is more stable than the pure bendamustine hydrochloride. The enhanced stability of the immobilized drug is explained with the drug interaction with the polymer carriers or their degradation products.

  12. New method for speciation analysis of aluminium fluoride complexes by HPLC-FAAS hyphenated technique.

    PubMed

    Frankowski, M; Zioła-Frankowska, A; Siepak, J

    2010-03-15

    Speciation analysis of aluminium in the presented system of HPLC-FAAS hyphenated technique lasts 4min. Using the bifunctional column in model analysis and using the calculation methods for modelling using the Mineql program enabled the authors to presume that particular forms will be subjected to elution in the following order: (1) AlF(2)(+) and AlF(4)(-), (2) AlF(2+) and AlF(3)(0) and (3) Al(3+). Based on the obtained results for model solutions, the presented method enables the determination of aluminium fluoride complexes and Al(3+) speciation form. The study compares the tendency of occurrence variability of aluminium fluoride complexes and Al(3+) form, determined based on the results obtained using the HPLC-FAAS hyphenated technique with the trend defined based on the Mineql program calculation method. The method was successfully applied to soil samples. PMID:20152461

  13. Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC

    NASA Astrophysics Data System (ADS)

    Zhang, Jingjing; Zhang, Quanbin; Wang, Jing; Shi, Xuelian; Zhang, Zhongshan

    2009-09-01

    We developed an HPLC method for analysis of the monosaccharide composition of fucoidans. The fucoidan was hydrolyzed into monosaccharides with 2 mol/L trifluoroacetic acid. Using ribose as the internal standard, the monosaccharide derivatives, obtained with 1-Phenyl-3-methyl-5-pyrazolone (PMP), were separated by reverse-phase HPLC using a gradient elution process, and monitored by ultraviolet detection at 245 nm. In the concentration range of 0.1-2.0 mmol/L, the peak area of each monosaccharide had a good linear relationship with its concentration ( r 2>0.998). The average recoveries of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and fucose were 86.2%, 95.1%, 62.5%, 102.0%, 94.8%, 66.6%, and 105.1%, respectively. This method was accurate and had good reproducibility and could be used to determine the monosaccharide contents of fucoidans.

  14. Quantification of phytochelatins in plants by reversed-phase HPLC-ESI-MS-MS.

    PubMed

    El-Zohri, M H A; Cabala, R; Frank, H

    2005-08-01

    An on-line HPLC-ESI-MS-MS method has been developed for determination of glutathione and phytochelatins (PC) in plant tissues. For sample pretreatment, dithiothreitol (DTT) must be added at the very beginning, as an anti-oxidant. Optimization of instrumental conditions i.e. composition of HPLC mobile phase, ionization efficiency of the electrospray interface, and MS-MS detection in the multiple ion-monitoring mode, are the central aspects of this work. A polystyrene-packed column was found to be superior to a standard silica-packed reversed-phase column. A concave quadratic gradient of ammonium formate buffer and acetonitrile was found to be optimum. The limits of quantitation were 0.2 micromol kg(-1) plant tissue for glutathione and PC. The method has been applied to analysis of tissue samples from Vicia faba grown in Cd-containing nutrient solutions.

  15. Isolation, Identification, and Characterization of One Degradation Product in Ambroxol by HPLC-Hyphenated Techniques

    PubMed Central

    Thummala, Veera Raghava Raju; Ivaturi, Mrutyunjaya Rao; Nittala, Someswara Rao

    2014-01-01

    This study details the isolation, identification, and characterization of ambroxol’s unknown impurity. One unknown impurity of ambroxol was formed in the formulated drug under stress conditions [40°C /75% relative humidity (RH) for 6 months] with the relative retention time (RRT) 0.68 in RP-HPLC. The impurity was enriched by exposing it to heat and it was isolated by using preparative HPLC. The enriched impurity was purified and characterized using the following sophisticated techniques: 2D NMR (gDQ-COSY, gHSQC, and gHMBC), FTIR, and LC-MS/MS. On the basis of the spectral data, the impurity was characterized as trans-4-(6,8-dibromoquinazolin-3(4H)-yl)cyclohexanol. PMID:24959402

  16. Photo- and thermal-stability studies on benzimidazole anthelmintics by HPLC and GC-MS.

    PubMed

    Ragno, Gaetano; Risoli, Antonella; Ioele, Giuseppina; De Luca, Michele

    2006-06-01

    Photo- and thermal-stability of the anthelmintics Albendazole, Mebendazole and Fenbendazole as in solid as in solution form has been investigated, by using a Xenon arc lamp as a radiation source, according to the ICH guideline for the drug stability tests. The degradation process was monitored by a HPLC method. All drugs showed high photosensitivity in solution but a reliable stability in solid form and when exposed to a temperature up to 50 degrees C. Two main degradation products from hydrolysis of the carbamic groups were identified by GC-MS. Validation studies demonstrated high accuracy (recovery 94 to 106%) and precision (RSD under 4.6%) of the HPLC method. The analytical procedure was successfully applied to the control of the drugs in the respective pharmaceutical formulations. PMID:16755047

  17. Monitoring of aminophenol isomers in surface water samples using a new HPLC method.

    PubMed

    Badea, Irinel Adriana; Axinte, Lacramioara; Vladescu, Luminita

    2013-03-01

    A new HPLC method was developed for the simultaneous determination of aminophenol isomers by means of a mixed-mode stationary phase containing both SCX and C18 moieties. All factors influencing the separation were discussed and optimized. The chromatographic conditions for the separation of aminophenols are the stationary phase duet SCX/C18, the mobile phase of aqueous phosphate buffer (pH 4.85):methanol = 85:15 (v/v) delivered with a flow rate of 1 mL/min and a detection at 285 nm. The method proposed was validated in terms of linearity, limits of detection and quantification, accuracy and precision. The HPLC method elaborated here was applied with good results on river water samples. In order to survey the quality of surface rivers entered in treatment plants which deliver water for Bucharest, two major rivers were included in a monitoring program which last more than 1 year.

  18. Fingerprint analysis of polysaccharides from different Ganoderma by HPLC combined with chemometrics methods.

    PubMed

    Sun, Xiaomei; Wang, Haohao; Han, Xiaofeng; Chen, Shangwei; Zhu, Song; Dai, Jun

    2014-12-19

    A fingerprint analysis method has been developed for characterization and discrimination of polysaccharides from different Ganoderma by high performance liquid chromatography (HPLC) coupled with chemometrics means. The polysaccharides were extracted under ultrasonic-assisted condition, and then partly hydrolyzed with trifluoroacetic acid. Monosaccharides and oligosaccharides in the hydrolyzates were subjected to pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone and HPLC analysis, which will generate unique fingerprint information related to chemical composition and structure of polysaccharides. The peak data were imported to professional software in order to obtain standard fingerprint profiles and evaluate similarity of different samples. Meanwhile, the data were further processed by hierarchical cluster analysis and principal component analysis. Polysaccharides from different parts or species of Ganoderma or polysaccharides from the same parts of Ganoderma but from different geographical regions or different strains could be differentiated clearly. This fingerprint analysis method can be applied to identification and quality control of different Ganoderma and their products.

  19. Development of an HPLC post-column antioxidant assay for Solidago canadensis radical scavengers.

    PubMed

    Marksa, Mindaugas; Radušienė, Jolita; Jakštas, Valdas; Ivanauskas, Liudas; Marksienė, Rūta

    2016-01-01

    The aim of this work was to modify and validate the post-column high-performance liquid chromatography (HPLC)-ABTS and DPPH methods for evaluating the antioxidant activity of the methanolic extracts of Solidago canadensis (Canadian goldenrod) leaves and flowers. Separation of the analytes was performed via the HPLC-PDA method on a YMC analytical column using a gradient elution program. Three compounds with antioxidant properties - chlorogenic acid, rutin and isoquercitrin - and two unidentified antioxidants were established. The research showed that the coil temperature regimes and loop length combinations influence the optimised post-column assay method for detecting the antioxidant activity of goldenrod radical scavengers. Investigations established that the temperature in the reaction coil was a substantial factor contributing to the signal strength of the analytes after reacting with the DPPH and ABTS radicals.

  20. Development of an HPLC post-column antioxidant assay for Solidago canadensis radical scavengers.

    PubMed

    Marksa, Mindaugas; Radušienė, Jolita; Jakštas, Valdas; Ivanauskas, Liudas; Marksienė, Rūta

    2016-01-01

    The aim of this work was to modify and validate the post-column high-performance liquid chromatography (HPLC)-ABTS and DPPH methods for evaluating the antioxidant activity of the methanolic extracts of Solidago canadensis (Canadian goldenrod) leaves and flowers. Separation of the analytes was performed via the HPLC-PDA method on a YMC analytical column using a gradient elution program. Three compounds with antioxidant properties - chlorogenic acid, rutin and isoquercitrin - and two unidentified antioxidants were established. The research showed that the coil temperature regimes and loop length combinations influence the optimised post-column assay method for detecting the antioxidant activity of goldenrod radical scavengers. Investigations established that the temperature in the reaction coil was a substantial factor contributing to the signal strength of the analytes after reacting with the DPPH and ABTS radicals. PMID:25835071

  1. A Rapid, Robust and Ultra-Sensitive HPLC Enantioseparation of β-Amino Alcohols.

    PubMed

    Dubey, Rituraj; Bhushan, Ravi

    2015-09-01

    Enantioseparation of amino acid analogues racemic β-amino alcohols has hitherto been thought impossible without prior derivatization. Method developers of chromatographic enantioseparation often face detection challenge due to low ultraviolet (UV) absorbance of these molecules. A new chiral derivatizing reagent, benzimidazole-(S)-naproxen amide, was synthesized to provide UV detectable chiral moiety for detection of respective diastereomers of eight β-amino alcohols (dl-alaninol, dl-leucinol, dl-prolinol, dl-phenylalaninol, dl-phenylglycinol, dl-valinol, dl-homophenylalaninol and dl-methioninol). An HPLC method has been developed and validated and both the diastereomers were separated within 31 min runtime. Excellent resolution to a pmol detection limit level was achieved. The developed HPLC method for enantioseparation of β-amino alcohols is the first-ever report of its own kind having such a high detection sensitivity. PMID:25824571

  2. Validation of Simultaneous Volumetric and HPLC Methods for the Determination of Pridinol Mesylate in Raw Material

    PubMed Central

    Simionato, Laura D.; Ferello, Leonardo; Stamer, Sebastián; Zubata, Patricia D.; Segall, Adriana I.

    2013-01-01

    Simple, sensitive, and economical simultaneous volumetric and HPLC methods for the determination of pridinol mesylate in raw material have been developed. The volumetric method is based on the reaction of pridinol with sodium lauryl sulphate in diluted sulphuric acid. Dimethyl yellow was used as indicator to detect the end point of the titration in aqueous/organic layer. The HPLC method for the determination of pridinol mesylate employs a reverse phase C18 column at ambient temperature with a mobile phase consisting of acetonitrile: 0.05 M potassium dihydrogen phosphate, pH adjusted to 5.0 (1 : 2, v/v). The flow rate was 0.8 mL/min. Quantitation was achieved with UV detection at 258 nm based on peak area. Both methods were found to be suitable for the quality control of pridinol mesylate in raw material. PMID:24224103

  3. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC.

  4. Non-extraction HPLC method for the simultaneous measurement of theophylline and caffeine in human serum.

    PubMed

    Rainbow, S J; Dawson, C M; Tickner, T R

    1989-11-01

    A non extraction HPLC method is described for the simultaneous measurement of theophylline and caffeine in human serum using a Pinkerton ISRP column and u.v. detection at 275 nm. The method is suitable for therapeutic monitoring of theophylline levels in adults and, in particular, quantitation of both theophylline and caffeine in premature neonates where as little as 10 microL of sample can be used. Comparison of theophylline levels obtained by this method with EMIT analysis show a correlation coefficient of 0.97 (n = 37) in adults and 0.79 (n = 16) in premature neonates. There was no correlation between serum theophylline and caffeine levels in premature neonates receiving theophylline therapy. No interference was encountered from endogenous plasma components or other drugs in the 53 patients studied. Precision of the assays compares well with reported values for extraction HPLC and immunochemical analyses.

  5. Ultratraces of carotenes in tomato purées: HPLC-TLS study

    NASA Astrophysics Data System (ADS)

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

    2003-01-01

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

  6. Enantiomerization of Allylic Trifluoromethyl Sulfoxides Studied by HPLC Analysis and DFT Calculations.

    PubMed

    Bailly, Laetitia; Petit, Emilie; Maeno, Mayaka; Shibata, Norio; Trapp, Oliver; Cardinael, Pascal; Chataigner, Isabelle; Cahard, Dominique

    2016-02-01

    Enantiomerization of allylic trifluoromethyl sulfoxides occurs spontaneously at room temperature through the corresponding allylic trifluoromethanesulfenates via a [2,3]-sigmatropic rearrangement. Dynamic enantioselective high-performance liquid chromatography (HPLC) analysis revealed the stereodynamics of these sulfoxides ranging from chromatographic resolution to peak coalescence at temperatures between 5 and 53 °C. The rate constant of enantiomerization and activation parameters were determined and compared with Density Functional Theory (DFT) calculations.

  7. Efficient HPLC purification of endohedral metallofullerenes on a porphyrin-silica stationary phase

    SciTech Connect

    Xiao, Jie; Savina, M.R.; Martin, G.B.; Francis, A.H.; Meyerhoff, M.E. )

    1994-10-05

    Endohedral metallofullrenes are among the most fascinating materials to emerge from ongoing efforts to produce and characterize closed-cage carbon compounds. In this paper, we describe the use of a new tetraphenylporphyrin-silica support material for efficient HPLC purification of the metallofullerenes from crude soot extract. The true single-stage separation from pyridine extracts demonstrates the unique chemical selectivity exhibited by employing [pi]-electron-rich tetraphenylporphyrin-silica stationary phases in chemical purifications. 18 refs., 3 figs.

  8. HPLC-methods for determination of lipoic acid and its reduced form in human plasma.

    PubMed

    Teichert, J; Preiss, R

    1992-11-01

    A method has been developed for the HPLC analysis of lipoic acid and its reduced form (dihydrolipoic acid) in biological samples. Both substances are released from the samples by enzymatic hydrolysis and extracted by solid phase column. The extracts, after evaporation, were chromatographed and quantified by electrochemical detection. The basic level was in the range 1-25 ng/ml for lipoic acid and 33-145 ng/ml for dihydrolipoic acid (6 healthy volunteers).

  9. Determination of atropisomeric configurations of macrocyclic bisbibenzyls by HPLC-CD/UV and quantum chemical calculations.

    PubMed

    Wang, Li-Ning; Xie, Chun-Feng; Zhu, Xiao-Song; Fan, Pei-Hong; Lou, Hong-Xiang

    2011-04-01

    Isoriccardin C (1) and riccardin D (2), isolated from the liverwort Reboulia hemisphaerica, were first characterized to be a mixture of two enantiomeric atropisomers by online chiral high-performance liquid chromatography-circular dichroism (HPLC-CD) analysis. Exemplarily for bisbibenzyls of the diarylether-biphenyl type, the absolute atropisomeric configurations of compunds 1 and 2 were determined by the analysis of their CD data coupled with quantum chemical CD calculations.

  10. Investigation of 4,5-epoxymorphinan degradation during analysis by HPLC.

    PubMed

    Quarry, Mary Ann; Sebastian, Dolores S; Diana, Frank

    2002-08-22

    Compounds of the 4,5-epoxymorphinan series have been shown to degrade in solution to the corresponding 2,2'-dimers when stored in amber glass HPLC vials. A colorant in the glass has been shown to catalyze the degradation. Although amber glass is routinely used to protect solutions from light degradation, it should not be used without evaluating its effect on sample stability.

  11. HPLC determination of eight polyphenols in the leaves of Crataegus pinnatifida Bge. var. major.

    PubMed

    Ying, Xixiang; Wang, Rongxiang; Xu, Jing; Zhang, Wenjie; Li, Haibo; Zhang, Chaoshen; Li, Famei

    2009-03-01

    A simple high-performance liquid chromatographic (HPLC) assay using the internal standard method is developed for the simultaneous determination of eight polyphenols. The analyzed compounds isolated from the leaves of Crataegus pinnatifida Bge. var. major include chlorogenic acid, vitexin-4"-O-glucoside, vitexin-2"-O-rhamnoside, vitexin, rutin, hyperoside, isoquercitrin, and quercetin. HPLC analysis is performed on a Diamonsil C18 analytical column (150 x 4.6 mm, i.d., 5-microm) using solvent (A) acetonitrile-tetrahydrofuran (95:5, v/v) and (B) 1% aqueous phosphoric acid as the mobile phase with UV absorption at 270 nm. The calibration curves of the eight polyphenols are linear (r(2) > 0.9992) over the concentration range of 0.0894-120.0 microg/mL. The mean recoveries are 95.4% to 98.1%. The results indicate that the HPLC method developed can easily be applied to the determination of eight polyphenols in the leaves of Crataegus pinnatifida Bge. var. major.

  12. An HPLC chromatographic reactor approach for investigating the hydrolytic stability of a pharmaceutical compound.

    PubMed

    Skrdla, Peter J; Abrahim, Ahmed; Wu, Yan

    2006-06-01

    The solution-phase hydrolysis kinetics of the Aprepitant (Emend) prodrug, Fosaprepitant Dimeglumine, were investigated using an HPLC chromatographic reactor approach. The term 'chromatographic reactor' refers to the use of an analytical-scale column as both a flow-through reactor and, simultaneously, as separation medium for the reactant(s) and product(s). Recently, we reported a novel mathematical treatment for the kinetic data obtained from chromatographic reactors, which we believe is superior to other treatments in terms of its accuracy, robustness and ease of implementation. In this work, we demonstrate that our treatment may be applied equally well to HPLC reactors, as previously we studied only GC reactors. It is found that the hydrolysis of Fosaprepitant Dimeglumine (FD) has an apparent activation energy of 107 kJ/mol when the reaction is investigated on-column, using the gradient elution conditions of the validated HPLC impurity profile method for this compound. For comparison, the activation energy determined for the same reaction occurring in a quiescent solution consisting of a fixed ratio of acetonitrile-0.1% v/v aqueous H3PO4 (50:50, v/v) is 91 kJ/mol, calculated using direct application of the Arrhenius equation. The data presented show that, when used as a screening tool, chromatographic reactors may be feasible for use in the pharmaceutical industry to quickly gauge the relative stabilities of various compounds with similar degradation pathways. PMID:16524680

  13. New HPLC evidence on endogenous tauret in retina and pigment epithelium.

    PubMed

    Petrosian, A M; Haroutounian, J E; Gundersen, T E; Blomhoff, R; Fugelli, K; Kanli, H

    2000-01-01

    This investigation was improve the separation for tauret (retinylidene taurine) and to compare its content in the retina under dark and light adaptation. To prevent tauret hydrolysis, retinal samples were quickly frozen and lyophilized. Methanol extracts of dried retina and pigment epithelium from both dark- or light-adapted frogs, Rana ridibunda, were injected onto HPLC. Synthetic standard tauret appeared at 4.7 min after the solvent front. At the same time, an endogenous substance was eluted from the mixed retinal and pigment epithelial samples. The UV spectra of this endogenous compound matched with the spectra of synthetic tauret obtained under identical conditions, with lambda(max) = 446 nm at peak. We conclude that the HPLC system used permitted full separation of tauret from the methanol extracts of the retina and pigment epithelium. TLC and further HPLC analysis have shown that tauret quantities were several times higher in the retina and pigment epithelium of the frogs adapted to dark compared with those light-adapted (about 4 h under 1000 1x illumination). Tauret based vitamin A transport is probably involved in other systems as well, where along with its other known beneficial effects taurine probably is necessary to facilitate vitamin A transport.

  14. A rapid HPLC method for indirect quantification of β-lactamase activity in milk.

    PubMed

    Zhou, Shuang; Wang, Dan; Zhao, Yunfeng; Wu, Yongning

    2015-04-01

    To circumvent the strictly regulated limits of antibiotics in milk, illegal addition of β-lactamase to lower the antibiotic levels in milk has been reported recently in China. Herein, we describe a fast, sensitive, and robust HPLC-UV method for the determination of β-lactamase activity in milk, based on an indirect quantification strategy. The test milk sample was mixed with a known amount of penicillin G, a specific substrate of β-lactamase. After incubation, an aliquot of the mixture was injected into the HPLC-UV system to quantify the remaining penicillin G in less than 10 min. Comparative analysis of the amount of penicillin G before and after incubation was used to indirectly deduce the activity of β-lactamase in the test sample. This method was successfully applied to milk products with different fat percentages, resulting in a detection limit of 0.6 U/mL. Good recoveries, ranging from 94 to 105%, were obtained from blank milk samples spiked with β-lactamase at levels of 2 to 50 U/mL, with relative standard deviations <6%. A good correlation was demonstrated between the HPLC method and the conventional culture-based assay. Using this method, the activity changes in β-lactamase during milk pasteurization, sterilization, and storage were investigated. The advantages of low-cost, accurate quantification and easily accessible instrumentation make the proposed method an ideal alternative for high-throughput routine analysis in the dairy industry.

  15. [Establishment and application of HPLC-QAMS for quality evaluation of Chuanxiong Rhizoma].

    PubMed

    Qiao, Feng-xian; Cai, Hao; Tu, Peng-fei; Pei, Ke; Song, Xiao-qing

    2015-06-01

    A quantitative analysis method of multi-components with a single marker (QAMS) for simultaneous determination of six marker compounds (one from phenolic acids and five from phthalides) in Chuanxiong Rhizoma was established by applying HPLC and using butylidenephthalide as the internal reference substance. And also the feasibility and accuracy of the established method for quality evaluation and application of Chuanxiong Rhizoma were investigated and validated. The analysis was performed with the mobile phase consisting of acetonitrile - 0.2% aqueous formic acid. The flow rate was 1.0 mL . min-1 and the column temperature was maintained at 30 °C. The detection wavelengths were set at 252 nm (for ferulic acid, Z-ligustilide, and butylidenephthalide) and 266 nm (for senkyunolide I, senkyunolide A, and coniferyl ferulate), separately, and 20 µL was injected for analysis with gradient elution. The results showed that there were no significant differences observed between the HPLC-QAMS method and the external standard method (RSD <5%). The relative correction factors were credible (RSD < 5%) in changed chromatographic conditions. The established HPLC-QAMS method can be accurately used for simultaneously evaluating and controlling the quality of Chuanxiong Rhizoma with multi-components. PMID:26521448

  16. 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.

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

    PubMed

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

    2016-03-11

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

  18. Development of an HPLC method for determination of pentachloronitrobenzene, hexachlorobenzene and their possible metabolites

    PubMed Central

    2011-01-01

    Background Pentachloronitrobenzene (PCNB) and hexachlorobenzene (HCB) are highly toxic and widespread in every environmental compartment. Some of metabolic products such as amino/nitro containing chlorinated aromatic compounds can be determined by gas chromatography coupled with electron capture detector (GC-ECD). However, it is difficult to identify some of chlorophenolic and chloroquinolic intermediates produced from PCNB and HCB by the above mentioned technique. Therefore, for analysis of these compounds and their metabolites, we have developed a high performance liquid chromatography (HPLC) based method. Results The extraction of PCNB and HCB from soil and minimal salt medium was carried out with ethyl acetate and hexane respectively with good recoveries (98% for PCNB and 97% for HCB). The validation of the proposed extraction and HPLC method was done by analysis of PCNB and HCB biodegradation and their metabolites identification from anaerobic enriched soil samples. Conclusion A rapid, sensitive and simple HPLC based analytical method was developed for the analysis of PCNB, HCB and their possible intermediates. PMID:22112041

  19. Comparative antioxidant activity and HPLC profiles of some selected Korean thistles.

    PubMed

    Jeong, Da Mi; Jung, Hyun Ah; Choi, Jae Sue

    2008-01-01

    As yet, no comparative analyses have been conducted regarding the comparative antioxidant activities and HPLC profiles of thistles distributed in Korea. Thus, this study was performed in order to evaluate the antioxidant potentials of seven Korean thistles: Cirsium lineare, Cirsium chanroenicum, Cirsium setidens, Cirsium japonicum var. ussuriense, Cirsium nipponicum, Cirslum pendulum and Carduus crispus, via peroxynitrite and DPPH free radical assays. Among seven Korean thistles, Carduus crispus exhibited the most significant antioxidant activity in both DPPH assay and peroxynitrite. In order to characterize the compounds contained in Korean thistles, we conducted HPLC analyses on the following ten flavonoids: luteolin-5-glucoside (1), luteolin-7-glucoside (2), apigenin-7-glucoside (3), hispidulin-7-neohesperidoside (4), apigenin-7-glucuronide (5), cirsimarin (6), pectolinarin (7), luteolin (8), apigenin (9) and acacetin (10). The results of our HPLC analyses indicated the presence of pectolinarin in the whole plants of C. setidens, C. lineare, C. nipponicum, C. pendulum, the aerial and underground parts of C. japonicum var. ussuriense, and the aerial parts of C. chanroenicum. Moreover, we were able to identify hispidulin-7-neohesperidoside and luteolin-7-glucoside in the whole plants of Carduus crispus, acacetin in the aerial parts of C. chanroenicum, cirsimarin in C. lineare.

  20. Quantitative analysis of eugenol in clove extract by a validated HPLC method.

    PubMed

    Yun, So-Mi; Lee, Myoung-Heon; Lee, Kwang-Jick; Ku, Hyun-Ok; Son, Seong-Wan; Joo, Yi-Seok

    2010-01-01

    Clove (Eugenia caryophyllata) is a well-known medicinal plant used for diarrhea, digestive disorders, or in antiseptics in Korea. Eugenol is the main active ingredient of clove and has been chosen as a marker compound for the chemical evaluation or QC of clove. This paper reports the development and validation of an HPLC-diode array detection (DAD) method for the determination of eugenol in clove. HPLC separation was accomplished on an XTerra RP18 column (250 x 4.6 mm id, 5 microm) with an isocratic mobile phase of 60% methanol and DAD at 280 nm. Calibration graphs were linear with very good correlation coefficients (r2 > 0.9999) from 12.5 to 1000 ng/mL. The LOD was 0.81 and the LOQ was 2.47 ng/mL. The method showed good intraday precision (%RSD 0.08-0.27%) and interday precision (%RSD 0.32-1.19%). The method was applied to the analysis of eugenol from clove cultivated in various countries (Indonesia, Singapore, and China). Quantitative analysis of the 15 clove samples showed that the content of eugenol varied significantly, ranging from 163 to 1049 ppb. The method of determination of eugenol by HPLC is accurate to evaluate the quality and safety assurance of clove, based on the results of this study.

  1. Size-exclusion HPLC as a sensitive and calibrationless method for complex peptide mixtures quantification.

    PubMed

    Bodin, Alice; Framboisier, Xavier; Alonso, Dominique; Marc, Ivan; Kapel, Romain

    2015-12-01

    This work describes an original methodology to quantify complex peptide mixtures by size-exclusion high-performance liquid chromatography (SE-HPLC). The methodology was first tested on simulated elutions of peptide mixtures. For this set of experiments, a good estimation of the total peptide concentration was observed (error less than 10 %). Then 30 fractions obtained by ultrafiltration of hydrolysates from two different sources were titrated by Kjeldahl or BCA analysis and analysed by SE-HPLC for an experimental validation of the methodology. Very good matchs between methods were obtained. The linear working range depends on the hydrolysate but is generally between 0.2 and 4gL(-1) (i.e. between 10 and 200μg). Moreover, the presence of organic solvents or salts in samples does not impact the accuracy of the methodology contrary to common quantification methods. Hence, the findings of this study show that total concentration of complex peptide mixture can be efficiently determinate by the proposed methodology using simple SE-HPLC analysis.

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

    PubMed

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

    2007-01-01

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

  3. Simultaneous Fe, S and P Speciation Using HPLC=HR-ICPMS

    NASA Astrophysics Data System (ADS)

    Hamester, M.; Lindemann, T.; Rottmann, L.; Douthitt, C. B.

    2004-12-01

    Iron, sulphur and phosphorus-containing biomolecules play important roles in biochemistry and proteomics and are part of the metallome of an organism. Analysis of HPLC peaks by ICPMS for these metals is a difficult analytical challenge because of polyatomic interferences: carbon, oxygen and nitrogen based interferences (NO+, NOH+, COH+, H3CO+, O2+) are the principal limitation in P and S detection and ArO+ is the principal interference on Fe. Polyatomic interferences formed by the elements in the different mobile phases can be separated from P and S in the molecules of interest, allowing the use of gradient elution. With high resolution ICPMS (HR-ICPMS), P and S can be completely resolved from these interferences even in 100 % acetonitrile. Protein phosphorylation can be determined accurately by HR-ICPMS, either on HPLC peaks or by laser ablation of gel electrophoresis spots. The high power magnet field regulator of the Finnigan ELEMENT2 enables rapid mass scanning, e.g. a duty cycle of 99% is obtained for the simultaneous detection of P and S. In order to show the potential of HR-ICPMS for metallomics, mixtures of deoxyribonucleotides (dAMP, dTMP, dGMP, dCMP) and peptides have been separated by HPLC with simultaneous on-line quantitation of P and S, and hemoglobin and myoglobin were analyzed for Fe/S ratios and quantitative determination of Fe.

  4. Validation of HPLC and CE methods for determination of organic acids in sour cassava starch wastewater.

    PubMed

    de Sena Aquino, Ana Carolina Moura; Azevedo, Mônia Stremel; Ribeiro, Deise Helena Baggio; Costa, Ana Carolina Oliveira; Amante, Edna Regina

    2015-04-01

    Fast and efficient analytical methods to determine the concentrations of lactic, acetic, propionic and butyric acids in sour cassava starch wastewater using reversed-phase high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), were developed and validated. Good linearity (R(2) > 0.999) and significance with F > 25,000 for all acids was showed. The matrix effect was not detected. The coefficient of variation values indicated good repeatability. The limits of detection (LOD) ranged from 1.0 to 3.7 and 2.0 to 3.0, and the limits of quantification (LOQ) from 3.1 to 12.2, and 8.0 to 12.5mg/L for HPLC and CE, respectively. The quantification of the samples did not reveal significant differences between the methods for all compounds analyzed. However, the benefits of CE in relation to HPLC, such as lower costs and less waste generation, along with shorter analysis times, need to be taken into consideration.

  5. Comparative antioxidant activity and HPLC profiles of some selected Korean thistles.

    PubMed

    Jeong, Da Mi; Jung, Hyun Ah; Choi, Jae Sue

    2008-01-01

    As yet, no comparative analyses have been conducted regarding the comparative antioxidant activities and HPLC profiles of thistles distributed in Korea. Thus, this study was performed in order to evaluate the antioxidant potentials of seven Korean thistles: Cirsium lineare, Cirsium chanroenicum, Cirsium setidens, Cirsium japonicum var. ussuriense, Cirsium nipponicum, Cirslum pendulum and Carduus crispus, via peroxynitrite and DPPH free radical assays. Among seven Korean thistles, Carduus crispus exhibited the most significant antioxidant activity in both DPPH assay and peroxynitrite. In order to characterize the compounds contained in Korean thistles, we conducted HPLC analyses on the following ten flavonoids: luteolin-5-glucoside (1), luteolin-7-glucoside (2), apigenin-7-glucoside (3), hispidulin-7-neohesperidoside (4), apigenin-7-glucuronide (5), cirsimarin (6), pectolinarin (7), luteolin (8), apigenin (9) and acacetin (10). The results of our HPLC analyses indicated the presence of pectolinarin in the whole plants of C. setidens, C. lineare, C. nipponicum, C. pendulum, the aerial and underground parts of C. japonicum var. ussuriense, and the aerial parts of C. chanroenicum. Moreover, we were able to identify hispidulin-7-neohesperidoside and luteolin-7-glucoside in the whole plants of Carduus crispus, acacetin in the aerial parts of C. chanroenicum, cirsimarin in C. lineare. PMID:18277604

  6. Estimation of color of durum wheat. Comparison of WSB, HPLC, and reflectance colorimeter measurements.

    PubMed

    Fratianni, Alessandra; Irano, Mario; Panfili, Gianfranco; Acquistucci, Rita

    2005-04-01

    Color is an important parameter involved in the definition of semolina and pasta quality. This character is mainly due to natural pigments (carotenoids) that are present at different levels in cereals and cereal products, due to botanical origin, growing conditions, distribution in the kernel, and technological processes. In food industries, color measurements are usually performed by means of automatic instruments that are rapid and safe, as alternatives to the chemical extraction methods. In this study, automatic measurements (CIE, color-space system L, a, b), water-saturated butanol (WSB), and HPLC determinations have been applied to evaluate the carotenoid content in whole meals and respective semolina samples produced from wheat cultivated in the years 2001 and 2002. In whole meals, total carotenoids, determined by HPLC, were about 3.0 microg/g (2001) and 3.5 microg/g (2002) calculated on dry weight (dw) and about 3.0 and 3.2 microg/g dw in corresponding semolina samples. The b values for the same period were 19.78 and 15.75, respectively, in raw materials and 20.03-21.67 in semolina. Results have confirmed lutein and beta-carotene as the main components mainly responsible for the yellow color in wheat grains. The ability of the index b to express natural dyeing was dependent on sample characteristics as demonstrated by the relationships found between this index and pigments, although the best correlation resulted between HPLC and WSB. PMID:15796565

  7. [Determination method of ultra-high-intensity sweetener, advantame, in processed foods by HPLC and LC-MS/MS].

    PubMed

    Kobayashi, Miki; Terada, Hisaya; Nakajima, Masahiro

    2015-01-01

    A simple method using HPLC and LC-MS/MS was developed for the determination of ultra-high-intensity sweetener, advantame, in processed foods. Advantame was extracted by dialysis, and cleaned up on a Sep-Pak Plus C18 cartridge, then determined by HPLC and LC-MS/MS. The recoveries from 5 kinds of processed foods fortified at the levels of 0.001 g/kg and 0.01 g/kg were 64.1-89.9% (RSD 0.9-6.9%) by HPLC and 68.8-99.9% (RSD 0.8-4.9%) by LC-MS/MS. The quantitation limit was 0.0004 g/kg by HPLC and 0.00004 g/kg by LC-MS/MS.

  8. Improved HPLC Method Using 2,3-naphthalenedicarboxaldehyde as Fluorescent Labeling Agent for Quantification of Histamine in Human Immunoglobulin Preparations

    PubMed Central

    Kim, Jung-Hwan; Shin, In Soo; Lee, Yoo Kyoung; Oh, Ho Jung; Ban, Sang Ja

    2011-01-01

    Objectives To develop and optimize quantitative HPLC method using 2,3-naphthalenedicarboxaldehyde (NDA) after simple and efficient solid phase extraction to determine the histamine in a biopharmaceutical (Histobulin™). Methods The HPLC method was established using NDA-induced Histobulin and compared with the recently reported HPLC method using o-phthaldehyde (OPA). The validated NDA-applied HPLC method was adjusted to 15 lots of Histobulin and compared by the current lot-release-test method using fluorimetry in recovery of histamine and reproducibility. Results Analyses of six HPLC chromatograms using NDA and OPA each were compared. NDA produced a more stable chromatogram baseline than OPA, and showed better stability. The HPLC analysis was validated in accuracy (91–103%), precision (interday/intraday assay CV ≤2.30%), and linearity of dose–response curve (R2 ≥ 0.9919). The detection limit was 0.0076 μg/mL and the quantitative limit was 0.0229 μg/mL. The amount of histamine per 12 mg of immunoglobulin was determined to be 0.17 ± 0.016 μg by the HPLC and 0.025 ± 0.013 μg by the current lot-release-test method using fluorimetry. Conclusion NDA derivatization showed better stability compared with the OPA method. Therefore the newly established NDA-derivatizated HPLC method may be more suitable than the fluorimetric method in lot-release-tests of biopharmaceuticals. PMID:24159462

  9. Quantification of lipoic acid from skin samples by HPLC using ultraviolet, electrochemical and evaporative light scattering detectors.

    PubMed

    Campos, Patrícia Mazureki; Praça, Fabíola Silva Garcia; Bentley, Maria Vitória Lopes Badra

    2016-04-15

    Lipoic acid (LA) is an endogenous organosulfur compound with potent antioxidant property. LA is often used as a drug for the treatment of skin disorders. For the accomplishment of topical applications of LA appropriate drug quantification methods are essential. Thus far, no HPLC methods have been reported for the measurement of LA extracted from skin. In this article we report on the development and validation of three sensitive and specific HPLC methods for LA and dihydrolipoic acid (DHLA) using ultraviolet (UV), electrochemical (EC) or evaporative light scattering (ELS) detection. These methods demonstrate different linearity ranges. The chromatographic separations were performed by RP-HPLC (250 × 4 mm, 5 μm) with isocratic elution using an acidic mobile phase for the three detection techniques. The lower limits of detection and quantification were 0.04 and 0.08 ng LA, respectively, for HPLC coupled to ELS, an innovative detector for LA with high sensitivity. The extraction of LA from skin samples showed recoveries greater than 71%. The recovered LA concentrations from stratum corneum and epidermis+dermis layers were: 5.41 ± 0.56 and 4.92 ± 0.33 μg/mL, respectively for HPLC/UV and 6.52 ± 0.49 and 5.01 ± 0.41 μg/mL, respectively, for HPLC/EC for the added LA concentration (6.67 μg/mL), and 8.88 ± 0.46 and 8.95 ± 0.08 μg/mL, respectively, for HPLC/ELS for the added LA concentration (10 μg/mL). These three optimized HPLC methods allowed for a simple, rapid and reliable determination of LA in human skin. They should be useful for the development of drug delivery systems for topical applications of LA.

  10. Optical spectroscopic and reverse-phase HPLC analyses of Hg(II) binding to phytochelatins.

    PubMed

    Mehra, R K; Miclat, J; Kodati, V R; Abdullah, R; Hunter, T C; Mulchandani, P

    1996-02-15

    Optical spectroscopy and reverse-phase HPLC were used to investigate the binding of Hg(II) to plant metal-binding peptides (phytochelatins) with the structure (gammaGlu-Cys)2Gly, (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly. Glutathione-mediated transfer of Hg(II) into phytochelatins and the transfer of the metal ion from one phytochelatin to another was also studied using reverse-phase HPLC. The saturation of Hg(II)-induced bands in the UV/visible and CD spectra of (gammaGlu-Cys)2Gly suggested the formation of a single Hg(II)-binding species of this peptide with a stoichiometry of one metal ion per peptide molecule. The separation of apo-(gammaGlu-Cys)2Gly from its Hg(II) derivative on a C18 reverse-phase column also indicated the same metal-binding stoichiometry. The UV/visible spectra of both (gammaGlu-Cys)3Gly and (gammaGlu-Cys)4Gly at pH 7.4 showed distinct shoulders in the ligand-to-metal charge-transfer region at 280-290 mm. Two distinct Hg(II)-binding species, occurring at metal-binding stoichiometries of around 1.25 and 2.0 Hg(II) ions per peptide molecule, were observed for (gammaGlu-Cys)3Gly. These species exhibited specific spectral features in the charge-transfer region and were separable by HPLC. Similarly, two main Hg(II)-binding species of (gammaGlu-Cys)4Gly were observed by UV/visible and CD spectroscopy at metal-binding stoichiometries of around 1.25 and 2.5 respectively. Only a single peak of Hg(II)-(gammaGlu-Cys)4Gly complexes was resolved under the conditions used for HPLC. The overall Hg(II)-binding stoichiometries of phytochelatins were similar at pH 2.0 and at pH 7.4, indicating that pH did not influence the final Hg(II)-binding capacity of these peptides. The reverse-phase HPLC assays indicated a rapid transfer of Hg(II) from glutathione to phytochelatins. These assays also demonstrated a facile transfer of the metal ion from shorter- to longer-chain phytochelatins. The strength of Hg(II) binding to glutathione and phytochelatins followed the

  11. Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS

    SciTech Connect

    Kubachka, Kevin M.; Kohan, Michael C.; Herbin-Davis, Karen; Creed, John T. Thomas, David J.

    2009-09-01

    Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of {sup 34}S labeled dimethylthioarsinic acid ({sup 34}S-DMTA{sup V}) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched {sup 34}S-DMTA{sup V} made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS{sup V}). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, {sup 34}S-DMTA{sup V} underwent several transformations. Labile {sup 34}S was exchanged with more abundant {sup 32}S to produce {sup 32}S-DMTA{sup V}, a thiol group was added to yield DMDTA{sup V}, and a methyl group was added to yield {sup 34}S-TMAS{sup V}. Because incubation of {sup 34}S-DMTA{sup V} resulted in the formation of {sup 34}S-TMAS{sup V}, the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS{sup V} from dimethylarsinic acid (DMA{sup V}) would proceed via a dimethylarsinous acid (DMA{sup III}) intermediate and would necessitate the loss of {sup 34}S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA{sup V} to TMAS{sup V}. Additionally, the detection of

  12. Exploring the in vitro formation of trimethylarsine sulfide from dimethylthioarsinic acid in anaerobic microflora of mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS.

    PubMed

    Kubachka, Kevin M; Kohan, Michael C; Herbin-Davis, Karen; Creed, John T; Thomas, David J

    2009-09-01

    Although metabolism of arsenicals to form methylated oxoarsenical species has been extensively studied, less is known about the formation of thiolated arsenical species that have recently been detected as urinary metabolites. Indeed, their presence suggests that the metabolism of ingested arsenic is more complex than previously thought. Recent reports have shown that thiolated arsenicals can be produced by the anaerobic microflora of the mouse cecum, suggesting that metabolism prior to systemic absorption may be a significant determinant of the pattern and extent of exposure to various arsenic-containing species. Here, we examined the metabolism of 34S labeled dimethylthioarsinic acid (34S-DMTA(V)) by the anaerobic microflora of the mouse cecum using HPLC-ICP-MS and HPLC-ESI-MS/MS to monitor for the presence of various oxo- and thioarsenicals. The use of isotopically enriched 34S-DMTA(V) made it possible to differentiate among potential metabolic pathways for production of the trimethylarsine sulfide (TMAS(V)). Upon in vitro incubation in an assay containing anaerobic microflora of mouse cecum, 34S-DMTA(V) underwent several transformations. Labile 34S was exchanged with more abundant 32S to produce 32S-DMTA(V), a thiol group was added to yield DMDTA(V), and a methyl group was added to yield 34S-TMAS(V). Because incubation of 34S-DMTA(V) resulted in the formation of 34S-TMAS(V), the pathway for its formation must preserve the arsenic-sulfur bond. The alternative metabolic pathway postulated for formation of TMAS(V) from dimethylarsinic acid (DMA(V)) would proceed via a dimethylarsinous acid (DMA(III)) intermediate and would necessitate the loss of 34S label. Structural confirmation of the metabolic product was achieved using HPLC-ESI-MS/MS. The data presented support the direct methylation of DMTA(V) to TMAS(V). Additionally, the detection of isotopically pure 34S-TMAS(V) raises questions about the sulfur exchange properties of TMAS(V) in the cecum material

  13. The metabolism of 4-bromoaniline in the bile-cannulated rat: application of ICPMS (79/81Br), HPLC-ICPMS & HPLC-oaTOFMS

    PubMed Central

    Duckett, Catherine; McCullagh, Michael; Smith, Christopher; Wilson, Ian D

    2015-01-01

    Abstract 1. An excretion balance study was performed following i.p. administration of 4-bromoaniline (50 mg kg−1) to bile-cannulated rats, using bromine-detected (79/81Br) ICPMS for quantification. Approximately 90% of the dose was recovered in urine (68.9 ± 3.6%) and bile (21.4 ± 1.4%) by 48 h post-administration. 2. HPLC-ICPMS (79/81Br) was used to selectively detect and profile the major urinary and biliary-excreted metabolites and determined that the 0–12 h urine contained at least 21 brominated metabolites with 19 bromine-containing peaks observed in the 6–12 h bile samples. 3. The urinary and biliary metabolites were subsequently profiled using HPLC-oaTOFMS. By exploiting the distinctive bromine isotope pattern ca. 60 brominated metabolites were detected in the urine in negative electrospray ionisation (ESI) mode while bile contained ca. 21. 4. While a large number of bromine-containing metabolites were detected, the profiles were dominated by a few major components with the bulk of the 4-bromoaniline-related material in urine accounted for by 4-bromoanaline O-sulfate (∼75% of the total by ICPMS, 84% by TOFMS). In bile a hydroxylated N-acetyl compound was the major metabolite detected, forming some ∼65% of the 4-bromoaniline-related material by ICPMS (37% by TOFMS). PMID:25837688

  14. The metabolism of 4-bromoaniline in the bile-cannulated rat: application of ICPMS ((79/81)Br), HPLC-ICPMS & HPLC-oaTOFMS.

    PubMed

    Duckett, Catherine; McCullagh, Michael; Smith, Christopher; Wilson, Ian D

    2015-01-01

    1. An excretion balance study was performed following i.p. administration of 4-bromoaniline (50 mg kg(-1)) to bile-cannulated rats, using bromine-detected ((79/81)Br) ICPMS for quantification. Approximately 90% of the dose was recovered in urine (68.9 ± 3.6%) and bile (21.4 ± 1.4%) by 48 h post-administration. 2. HPLC-ICPMS ((79/81)Br) was used to selectively detect and profile the major urinary and biliary-excreted metabolites and determined that the 0-12 h urine contained at least 21 brominated metabolites with 19 bromine-containing peaks observed in the 6-12 h bile samples. 3. The urinary and biliary metabolites were subsequently profiled using HPLC-oaTOFMS. By exploiting the distinctive bromine isotope pattern ca. 60 brominated metabolites were detected in the urine in negative electrospray ionisation (ESI) mode while bile contained ca. 21. 4. While a large number of bromine-containing metabolites were detected, the profiles were dominated by a few major components with the bulk of the 4-bromoaniline-related material in urine accounted for by 4-bromoanaline O-sulfate (∼75% of the total by ICPMS, 84% by TOFMS). In bile a hydroxylated N-acetyl compound was the major metabolite detected, forming some ∼65% of the 4-bromoaniline-related material by ICPMS (37% by TOFMS).

  15. Aflatoxins contamination in Pakistani brown rice: a comparison of TLC, HPLC, LC-MS/MS and ELISA techniques.

    PubMed

    Iqbal, Javed; Asghar, Muhammad Asif; Ahmed, Aftab; Khan, Mobeen Ahmed; Jamil, Khalid

    2014-12-01

    Advancement in the field of analytical food-chemistry has explored various experimental techniques for aflatoxins (AFs) quantification. The present study was aimed to compare four different techniques; thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the analysis of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) in brown rice (n = 120) being collected from Karachi, Pakistan. All the four assays provide precised, accurate and comparable results. However, some differences were observed. For instance, TLC, HPLC and LC-MS/MS methodologies offered the advantage of the quantification of individual toxins in contrast to ELISA technique. The contamination ranges of AFB₁/AFB₂ as determined by TLC, HPLC and LC-MS/MS were 1.18-9.97/0.59-1.52, 0.16-10.54/0.26-1.35 and 0.11-10.88/0.38-1.48 µg/kg, respectively. However, AFG₁ and AFG₂ were not detected in any tested samples. Furthermore, owing to low-detection limit and sensitivity, HPLC and LC-MS/MS methodologies have identified greater number of contaminated samples in comparison to TLC and ELISA techniques. The overall average results of total AFs as provided by HPLC (3.79 µg/kg) and LC-MS/MS (3.89 µg/kg) were found higher in comparison to TLC (3.68 µg/kg) and ELISA (3.70 µg/kg). On the basis of achieved results, it was concluded that TLC, HPLC, LC-MS/MS and ELISA techniques are valuable tool for the quantification of AFs in cereals and grains. Furthermore, HPLC and LC-MS/MS techniques offer an added advantage for the detection of AFs in diminutive levels.

  16. A rapid and reliable technique for N-nitrosodimethylamine analysis in reclaimed water by HPLC-photochemical reaction-chemiluminescence.

    PubMed

    Fujioka, Takahiro; Takeuchi, Haruka; Tanaka, Hiroaki; Nghiem, Long D; Ishida, Kenneth P; Kodamatani, Hitoshi

    2016-10-01

    A fast and reliable analytical technique was evaluated and validated for determination of N-nitrosodimethylamine (NDMA) formation and rejection by reverse osmosis (RO) membranes in potable water reuse applications. The analytical instrument used in this study is high-performance liquid chromatography (HPLC), photochemical reaction (PR) and chemiluminescence (CL) - namely HPLC-PR-CL. Results reported here show that HPLC-PR-CL can be used to measure NDMA with a similar level of accuracy compared to conventional and more time-consuming techniques using gas chromatography and tandem mass spectrometry detection in combination with solid phase extraction. Among key residual chemicals (i.e. monochloramine, hydrogen peroxide and hypochlorite) in reclaimed wastewater, hypochlorite was the only constituent that interfered with the determination of NDMA by HPLC-PR-CL. However, hypochlorite interference was eliminated by adding ascorbic acid as a reducing agent. Direct injection of ultrafiltration (UF)-treated wastewater samples into HPLC-PR-CL also resulted in an underestimation of the NDMA concentration possibly due to interference by organic substances in the UF-treated wastewater. Accurate determination of NDMA concentrations in UF-treated wastewater was achieved by reducing the sample injection volume from 200 to 20 μL, though this increased the method detection limit from 0.2 to 2 ng/L. In contrast, no interference was observed with RO permeate. These results suggest that RO membranes could remove part of substances that interfere with the NDMA analysis by HPLC-PR-CL. In addition, RO treatment experiments demonstrated that HPLC-PR-CL was capable of evaluating near real-time variation in NDMA rejection by RO. PMID:27423127

  17. Determination of log P values of new cyclen based antimalarial drug leads using RP-HPLC.

    PubMed

    Rudraraju, A V; Amoyaw, P N A; Hubin, T J; Khan, M O F

    2014-09-01

    Lipophilicity, expressed by log P, is an important physicochemical property of drugs that affects many biological processes, including drug absorption and distribution. The main purpose of this study to determine the log P values of newly discovered drug leads using reversed-phase high-performance liquid chromatography (RP-HPLC). The reference standards, with varying polarity ranges, were dissolved in methanol and analyzed by RP-HPLC using a C18 column. The mobile phase consisted of a mixture of acetonitrile, methanol and water in a gradient elution mode. A calibration curve was plotted between the experimental log P values and obtained log k values of the reference standard compounds and a best fit line was obtained. The log k values of the new drug leads were determined in the same solvent system and were used to calculate the respective log P values by using the best fit equation. The log P vs. log k data gave a best fit linear curve that had an R2 of 0.9786 with Pvalues of the intercept and slope of 1.19 x 10(-6) and 1.56 x 10(-10), respectively, at 0.05 level of significance. Log P values of 15 new drug leads and related compounds, all of which are derivatives of macrocyclic polyamines and their metal complexes, were determined. The values obtained are closely related to the calculated log P (Clog P) values using ChemDraw Ultra 12.0. This experiment provided efficient, fast and reasonable estimates of log P values of the new drug leads by using RP-HPLC.

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

    NASA Technical Reports Server (NTRS)

    2005-01-01

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

  19. An HPLC method for the determination of bromadiolone plasma kinetics and its residues in hen eggs.

    PubMed

    Giorgi, Mario; Mengozzi, Grazia

    2010-10-01

    Cereal-based bromadiolone anticoagulant is often used for rodent control, and because these baits are attractive for poultry they may be accidentally ingested. Thus, the aim of this study was to develop a new high-performance liquid chromatography (HPLC) method for the determination of bromadiolone residues in hens' eggs and its plasma kinetics. Laying hens (n = 48) were divided into four groups of 12 animals each. Groups I and II received orally a single dose of bromadiolone 10 mg/kg, group III received a single dose of bromadiolone 60 mg/kg, and group IV was the control. Eggs were collected from groups I, III, and IV, whereas plasma was collected from groups II and IV. The HPLC method developed was reproducible, sensitive, accurate, and linear within the range 0.1-20 μg/g. The final HPLC conditions were as follows: mobile phase MeOH-ammonium acetate (0.5 M) triethylamine buffer (pH 5, 51:49, v/v); analytical column Luna C₁₈ ODS2; wavelength 260 nm; flow rate of 1.5 mL/min; and warfarin as internal standard (5 μg/mL). Recoveries for bromadiolone were in the range of 72-80% with RSD lower than 10%. Pharmacokinetic behavior of bromadiolone in hens results faster than that reported in other animals and humans. Following 10 and 60 mg/kg treatment bromadiolone was not detected in albumen but was present in yolk from day 4 to 5 and from day 2 to 9. In conclusion, the bromadiolone amount found in eggs was well below the toxic dose of this anticoagulant for humans, and no anticoagulant effect should be observed. PMID:20875232

  20. Determination of azide impurity in sartans using reversed-phase HPLC with UV detection.

    PubMed

    Gričar, Maja; Andrenšek, Samo

    2016-06-01

    A simple and robust, gradient HPLC method was developed for determination of azide ppm (μgg(-1)) levels in different sartans (irbesartan, candesartan, valsartan). The sartan was dissolved in 0.1M NaOH. Then pH was adjusted to 4.5 with 20% H3PO4 followed by dilution with water. Precipitated API was removed by filtration using 0.45μm membrane PVDF (Polyvinylidene Fluoride) membrane filter, and supernatant was analyzed by gradient elution HPLC at room temperature with Hydro RP HPLC 250×4.6mm, 4μm column and UV detection at 205nm. The best sensitivity was achieved by UV detection cell with 60mm optical path length: LOD 0.17μgg(-1) and LOQ 0.84μgg(-1) for azide. The USP requirement for maximum azide content in irbesartan is 10μgg(-1). The analytical method was validated as per International Conference on Harmonization (ICH) guidelines with respect to system precision, intraday precision (repeatability), intermediate precision (ruggedness), linearity, quantitation limit, detection limit, accuracy, standard and sample solution stability, robustness and selectivity/specificity. The method was linear in the range from LOQ (0.84μgg(-1)) to 101μgg(-1) of azide. The correlation coefficient was 0.9999 and bias on y-axis for 84μgg(-1) test concentration was 0.33%. The accuracy of the method was established based on the recovery obtained between 94.0% and 103.0% for azide. Standard and sample solutions were stable for at least 48h at room temperature and in refrigerator. The method was found to be robust for variation in column temperature (±5°C) and mobile phase flow rate (±0.2mLmin(-1)) and selective for anions such as bromide, nitrate, nitrite, formate and acetate. PMID:26999319

  1. Evaluation of tamoxifen and metabolites by LC-MS/MS and HPLC methods.

    PubMed

    Heath, D D; Flat, S W; Wu, A H B; Pruitt, M A; Rock, C L

    2014-01-01

    Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and its metabolites, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxytamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam), is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and its metabolites. This fully automated analytical method is specific, accurate and sensitive. The LC-MS/MS automated technique has now become a widely accepted reference method. This study analysed a randomly selected batch of blood samples from participants enrolled in a breast cancer study to compare results from this reference method in 40 samples with those obtained from a recently developed high-performance liquid chromatography (HPLC) method with fluorescence detection. The mean (SD) concentrations for the LC-MS/MS method (endoxifen 12.6 [7.5] ng/mL, tamoxifen 105 [44] ng/mL, 4-HT 1.9 [1.0] ng/mL, ND-tam 181 [69] ng/mL) and the HPLC method (endoxifen 13.1 [7.8] ng/mL, tamoxifen 108 [55] ng/mL, 4-HT 1.8 [0.8] ng/mL, ND-tam 184 [81] ng/mL) did not show any significant differences. The results confirm that the HPLC method offers an accurate and comparable alternative for the quantification of tamoxifen and tamoxifen metabolites. PMID:24693573

  2. A lectin HPLC method to enrich selectively-glycosylated peptides from complex biological samples.

    PubMed

    Johansen, Eric; Schilling, Birgit; Lerch, Michael; Niles, Richard K; Liu, Haichuan; Li, Bensheng; Allen, Simon; Hall, Steven C; Witkowska, H Ewa; Regnier, Fred E; Gibson, Bradford W; Fisher, Susan J; Drake, Penelope M

    2009-01-01

    Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls-fucosylated and sialylated human lactoferrin glycopeptides-and negative controls-high mannose glycopeptides from Saccharomyces cerevisiae-that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include

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

    PubMed

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

    2015-10-01

    Authentication and quality assessment of Cordyceps sinensis, a precious and pricey natural product that offers a variety of health benefits, is highly significant. To identify effective chemical markers, authentic C. sinensis was thoroughly screened by using HPLC-MS/MS. In addition to many previously reported ingredients, two glycosides, i.e., cyclo-Ala-Leu-rhamnose and Phe-o-glucose, were detected for the first time in this material. Six ingredients detected, including cordycepin, D-mannitol, Phe, Phe-o-glucose, cyclo-Gly-Pro, and cyclo-Ala-Leu-rhamnose, were selected as a collection of chemical markers. An HPLC-MS/MS method was developed to simultaneously quantify them with sensitivity and specificity. The method had limits of detection ranging from 0.008 μg mL(-1) for cordycepin to 0.75 μg mL(-1) for cyclo-Gly-Pro. Recovery was found between 96 and 103 % in all tests. To evaluate the effectiveness of the marker collection proposed, five authentic C. sinensis samples and five samples of its substitutes were analyzed. Cordycepin, D-mannitol, and Phe were found present in all samples. The contents ranged from 0.0076 to 0.029 % (w/w) for cordycepin, 0.33 to 18.9 % for mannitol, and 0.0013 to 0.642 % for Phe. Interestingly, the two glycosides, Phe-o-glucose and cyclo-Ala-Leu-rhamnose, were detected only in authentic C. sinensis samples. These results indicated that the proposed protocol based on HPLC-MS/MS quantification of the markers might have a great potential in authentication and quality assessment of C. sinensis. Graphical abstract Chemical markers of C. sinensis identified in this work. PMID:26302964

  4. A comparative study for PSP toxins quantification by using MBA and HPLC official methods in shellfish.

    PubMed

    Ben-Gigirey, B; Rodríguez-Velasco, M L; Otero, A; Vieites, J M; Cabado, A G

    2012-10-01

    Commission Regulation (EC) N° 2074/2005 recognises the biological method as the reference method for Paralytic Shellfish Poisoning (PSP) toxins detection in molluscs. It was amended by Commission Regulation (EC) N° 1664/2006 that accepted the so-called Lawrence method as an alternative to the reference method. The goal of this study was to compare AOAC Official Methods of Analysis 959.08 (Biological method) and 2005.06 (Prechromatographic Oxidation and Liquid Chromatography with fluorescence detection) in samples with different toxin profiles. The influence of extraction solvent in the total samples toxicity was also evaluated. A total of 40 samples including mussels, clams, scallops, razor-clams, cockles, oysters and barnacles were analysed by both official methods. Samples were selected with Alexandrium and Gymnodinium toxic profiles, from different origin and including several presentations: fresh, frozen, canned and boiled. Acetic and hydrochloric acid extractions were performed in all samples and the extracts were simultaneously analysed by both methods. Most samples were naturally contaminated and two samples were spiked. Comparison of both official methods, mouse bioassay (MBA) with HCl extraction and Liquid Chromatography with fluorescence detection (HPLC-FLD) with acetic acid extraction, led to an 85% of consistent results regarding compliance with legal limit, including samples below and above it. The linear correlation coefficient was r² = 0.69 and the paired t test (two tails, α = 0.05) indicated that there were not significant differences among both sets of data. Nevertheless, toxicity differences were found in several samples. In 15 out of 18 shellfish with a Gymnodinium toxic profile, higher toxicity levels were obtained by MBA. This fact was more evident in 7 samples, partially related to the lack of standards and the impossibility of analysing dc-NEO, C1, 2 and GTX6 at the beginning of the study. However, other factors concerning the extraction

  5. Stability indicating HPLC-method for the determination of econazole nitrate in cream and lotion formulations.

    PubMed

    Christinat, R; Zulliger, H W

    1984-01-01

    A simple, fast HPLC-method for the determination of econazole nitrate in cream (Pevaryl, Pevisone) and lotion formulations (based on polyethylenic oleic glycerides and mono/di-stearic esters of ethylene- and polyethylene glycol) is described. The method is stability indicating as well as linear (range 5-15 mg econazole nitrate/g) and shows a good recovery (98.7-100.2%) and a good reproducibility (cv less than 1%, n = 10). The chromatographic separation is achieved on a RP-18 column using methanol/aqueous ammoniumcarbonate solution/tetrahydrofurane as the mobile phase. Quantification of the chromatograms is done by internal standard method using peak areas.

  6. [Rapid determination of saccharin sodium in ice cream by high performance liquid chromatography (HPLC)].

    PubMed

    Zhang, X; Zhang, W; Dai, J

    1999-07-01

    This is a method for the rapid determination of saccharin sodium in ice cream by HPLC. A radial compression C18 cohumn, 100 mm x 8 mm i.d., and ultraviolet detector at 220 nm were applied to the determination without extraction. The solution of samples would be determined after they were treated with the solution of copper sulfate and sodium hydroxide in water. The detectable amount was 0.38 mg/kg, the coefficient of variation CV was 1.72%, and the recovery was 96.25%-101.75%.

  7. Design and Prototype of an Automated Column-Switching HPLC System for Radiometabolite Analysis.

    PubMed

    Vasdev, Neil; Collier, Thomas Lee

    2016-01-01

    Column-switching high performance liquid chromatography (HPLC) is extensively used for the critical analysis of radiolabeled ligands and their metabolites in plasma. However, the lack of streamlined apparatus and consequently varying protocols remain as a challenge among positron emission tomography laboratories. We report here the prototype apparatus and implementation of a fully automated and simplified column-switching procedure to allow for the easy and automated determination of radioligands and their metabolites in up to 5 mL of plasma. The system has been used with conventional UV and coincidence radiation detectors, as well as with a single quadrupole mass spectrometer. PMID:27548189

  8. Identification of compounds in wine by HPLC-tandem mass spectrometry.

    PubMed

    Bevilacqua, Lucio; Buiarelli, Francesca; Coccioli, Franco; Jasionowska, Renata

    2004-01-01

    In this work several compounds were detected in wines by HPLC-tandem mass spectrometry. In particular cinnamic and benzoic acids, tyrosol, apigenin-7-glucoside and luteolin-7-glucoside were identified and quantified in Italian wines. Red wines show bigger amount of cinnamic and benzoic acids than white wines. tyrosol is in bigger amount with respect to two flavones: luteolin-7-glucoside and apigenin-7-glucoside. These last two flavones are only in some wine, but it can be important to detect the presence of different substances in small amount to be able to characterize a wine.

  9. Design and Prototype of an Automated Column-Switching HPLC System for Radiometabolite Analysis

    PubMed Central

    Vasdev, Neil; Collier, Thomas Lee

    2016-01-01

    Column-switching high performance liquid chromatography (HPLC) is extensively used for the critical analysis of radiolabeled ligands and their metabolites in plasma. However, the lack of streamlined apparatus and consequently varying protocols remain as a challenge among positron emission tomography laboratories. We report here the prototype apparatus and implementation of a fully automated and simplified column-switching procedure to allow for the easy and automated determination of radioligands and their metabolites in up to 5 mL of plasma. The system has been used with conventional UV and coincidence radiation detectors, as well as with a single quadrupole mass spectrometer. PMID:27548189

  10. Atropisomeric determination of chiral hydroxylated metabolites of polychlorinated biphenyls using HPLC-MS

    PubMed Central

    2013-01-01

    Background Polychlorinated biphenyls (PCBs) are a group of environmental persistent organic pollutants, which can be metabolized into a series of metabolites, including hydroxylated metabolites (OH-PCBs) in biota. Nineteen of 209 PCB congeners can form chiral stable isomers. However, atropisomeric determination of the hydroxylated metabolites of these chiral PCBs has never been reported by LC methods. In this work, a novel HPLC-MS method was developed to detect five chiral OH-PCBs (4OH-PCB91, 5OH-PCB91, 4OH-PCB95, 5OH-PCB95 and 5OH-PCB149) using HPLC-MS without a derivatization step. Results The influences of column-type, column temperature, flow rate and ratio of the mobile phase on the atropisomeric separation were investigated in detail. In the final method, calibration curves, based on peak areas against concentration, were linear in a range of 1–100 ng mL-1 of five chiral OH-PCBs with correlation coefficients ranging from 0.9996 to 0.9999 for all atropisomers of OH-PCBs. The relative standard deviations measured at the 10.0 ng mL-1 level for atropisomers of five chiral OH-PCBs were in the range of 0.60-7.55% (n = 5). Calculated detection limits (S/N = 3) of five chiral OH-PCBs were between 0.31 and 0.60 ng mL-1 for all OH-PCB atropisomers. Conclusion This HPLC-MS method was developed to detect chiral OH-PCBs and further successfully applied to measure OH-PCB atropisomer levels and enantiomeric fractions (EFs) in rat liver microsomal samples. The results from LC-MS method were highly consistent with those from GC-ECD method. It is the first time to report these OH-PCB atropisomers detected in microsomes by HPLC-MS. The proposed method might be applied also to detect chiral OH-PCBs in environmental samples and for metabolites of PCBs in vivo. PMID:24360245

  11. Determination of the Scoville Heat Value for Hot Sauces and Chilies: An HPLC Experiment

    NASA Astrophysics Data System (ADS)

    Batchelor, James D.; Jones, Bradley T.

    2000-02-01

    A laboratory experiment for a junior- or senior-level college instrumental analysis course has been designed and tested. The student will isolate the capsainoids from commercial food products (chilies and sauces) using a simple extraction technique. The identity and concentration of the capsainoids are determined by high-performance liquid chromatography (HPLC). The concentrations are then used to determine the pungency (Scoville heat value) of the foods. Taste tests can be used to verify the relative pungency of the products. The experiment is designed to be completed in less than four hours. A letter from Paul Vorndam in our April 2000 issue addresses the above.

  12. Chemical Constituents from Daphne giraldii Nitsche and Their Contents Simultaneous Determination by HPLC.

    PubMed

    Huyiligeqi; Dong, Xiaoxv; Yang, Chunjing; Xu, Guanling; Cao, Sali; Fu, Jing; Lin, Longfei; Ni, Jian

    2016-01-01

    Daphne giraldii Nitsche (Thymelaeaceae) is widely distributed in the Chinese provinces of Shaanxi, Gansu, and Qinghai, which has been used in Chinese folk medicine to treat ache and rheumatism. Pharmacologic tests have revealed that the plant has anti-inflammatory, analgesic, and anticancer activities. However, there is still not enough systemic investigation on the chemical constituents and the method for the contents simultaneous determination in D. giraldii. Therefore, the isolation and characterization of the compounds from the stem barks of this plant were reported. Moreover, a facile, accurate, and reliable method has been developed and validated for their simultaneous determination using HPLC-DAD. PMID:27190543

  13. Chemical Constituents from Daphne giraldii Nitsche and Their Contents Simultaneous Determination by HPLC

    PubMed Central

    Huyiligeqi; Dong, Xiaoxv; Yang, Chunjing; Xu, Guanling; Cao, Sali; Fu, Jing; Lin, Longfei; Ni, Jian

    2016-01-01

    Daphne giraldii Nitsche (Thymelaeaceae) is widely distributed in the Chinese provinces of Shaanxi, Gansu, and Qinghai, which has been used in Chinese folk medicine to treat ache and rheumatism. Pharmacologic tests have revealed that the plant has anti-inflammatory, analgesic, and anticancer activities. However, there is still not enough systemic investigation on the chemical constituents and the method for the contents simultaneous determination in D. giraldii. Therefore, the isolation and characterization of the compounds from the stem barks of this plant were reported. Moreover, a facile, accurate, and reliable method has been developed and validated for their simultaneous determination using HPLC-DAD. PMID:27190543

  14. Identification of compounds in wine by HPLC-tandem mass spectrometry.

    PubMed

    Bevilacqua, Lucio; Buiarelli, Francesca; Coccioli, Franco; Jasionowska, Renata

    2004-01-01

    In this work several compounds were detected in wines by HPLC-tandem mass spectrometry. In particular cinnamic and benzoic acids, tyrosol, apigenin-7-glucoside and luteolin-7-glucoside were identified and quantified in Italian wines. Red wines show bigger amount of cinnamic and benzoic acids than white wines. tyrosol is in bigger amount with respect to two flavones: luteolin-7-glucoside and apigenin-7-glucoside. These last two flavones are only in some wine, but it can be important to detect the presence of different substances in small amount to be able to characterize a wine. PMID:15506618

  15. Analysis of multiple sweeteners and their degradation products in lassi by HPLC and HPTLC plates.

    PubMed

    George, V; Arora, S; Wadhwa, B K; Singh, A K

    2010-08-01

    A solid phase extraction method using C18 cartridges was standardized for the isolation of multiple sweeteners (aspartame, acesulfame-K and saccharin) and their degradation products (diketopiperazine, Lphenylalanine, acetoacetamide and 2-sulfobenzoic acid) from lassi. Analytical conditions for HPLC were standardized over C18 column using UV detector for the simultaneous separation and estimation of multiple sweeteners and their degradation products in lassi sample isolates. A simple cartridge free method was developed for the isolation of sucralose from lassi. Method was also standardized for qualitative detection and quantitative estimation of sucralose over amino and silica gel plates of HPTLC.

  16. Screening of Panamanian Plant Extracts for Pesticidal Properties and HPLC-Based Identification of Active Compounds

    PubMed Central

    Guldbrandsen, Niels; De Mieri, Maria; Gupta, Mahabir; Seiser, Tobias; Wiebe, Christine; Dickhaut, Joachim; Reingruber, Rüdiger; Sorgenfrei, Oliver; Hamburger, Matthias

    2015-01-01

    A library of 600 taxonomically diverse Panamanian plant extracts was screened for fungicidal, insecticidal, and herbicidal activities. A total of 19 active extracts were submitted to HPLC-based activity profiling, and extracts of Bocconia frutescens, Miconia affinis, Myrcia splendens, Combretum aff. laxum, and Erythroxylum macrophyllum were selected for the isolation of compounds. Chelerythrine (2), macarpine (3), dihydrosanguinarine (5), and arjunolic acid (8) showed moderate-to-good fungicidal activity. Myricetin-3-O-(6’’-O-galloyl)-β-galactopyranoside (13) showed moderate insecticidal activity, but no compound with herbicidal activity was identified. PMID:26839818

  17. HPLC Determination Of Polycyclic Aromatic Compounds By Fluorescence Detected With A Charge-Coupled Device

    NASA Astrophysics Data System (ADS)

    Jalkian, Rafi D.; Denton, M. Bonner

    1989-05-01

    A solid-state two-dimensional charge-coupled device (CCD) is used to quantitate polycyclic aromatic compounds by native fluorescence. The mixture is separated by reverse phase high performance liquid chromatography (HPLC) and the fluorescence emission from each peak is integrated by the detector. A new technique, charge-dependent variable binning, applied to one-dimensional spectroscopy leads to outstanding dynamic range. Excellent linearity and limits of detection are obtained for several priority pollutants. Synchronous and derivative techniques can be applied to the two-dimensional fluorescence data obtained to resolve overlapping peaks. The analyzed mixture consists of fluorene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, and perylene.

  18. Structural characterization of low level degradants in aztreonam injection and an innovative approach to aid HPLC method validation.

    PubMed

    Ye, Qingmei; Ding, Wei; Rinaldi, Frank; Huang, Yande; Miller, Scott A; Bolgar, Mark

    2016-05-30

    Three new degradants have been identified from drug product and active pharmaceutical ingredient stability samples of aztreonam, a marketed synthetic monocyclic beta-lactam antibiotic. The degradants were detected following the implementation of a new, more selective HPLC method for the determination of impurities and degradants. The new method was developed in response to changes in the regulatory requirement for mature products. Two of the new unknown Degradants (I and II) were observed in chromatograms from stability samples of aztreonam injection. The third new Degradant (III) was observed during a stability study of the aztreonam active pharmaceutical ingredient. These degradants were structurally characterized. A small amount (ca. 1-3mg) of each degradant was isolated via preparative HPLC for structure elucidation using accurate MS, one and two-dimensional NMR spectroscopy. The small amount of each NMR sample was then reused as a standard for HPLC purity/impurity method validation. Their exact concentrations were determined using quantitative NMR which enabled the execution of the quantitative elements of the HPLC method validation. This innovative approach eliminated the need to isolate or synthesize larger quantities of markers for HPLC/UV method validation, thus saving significant time and reducing costs. PMID:26991056

  19. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay.

    PubMed

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-07-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  20. Real-time separation of natural products by ultrafast 2D NMR coupled to on-line HPLC.

    PubMed

    Queiroz, Luiz H K; Queiroz, Darlene P K; Dhooghe, Liene; Ferreira, Antonio G; Giraudeau, Patrick

    2012-05-21

    Hyphenated HPLC-NMR is an extremely efficient analytical tool, which makes it possible to perform on-flow experiments where 1D NMR spectra are obtained in real time as the analytes are separated and eluted from the chromatographic column. However, it is incompatible with multidimensional NMR methods that form an indispensible tool for the study of complex mixtures. Recently, Frydman and co-workers have proposed an ultrafast 2D NMR approach, where a complete 2D NMR correlation can be recorded in a single scan, thus providing a solution to the irreversibility of hyphenated techniques. This paper presents the first implementation of on-line ultrafast HPLC-NMR. Ultrafast COSY spectra are acquired every 12 s in the course of a chromatographic run performed on a mixture of natural aromatic compounds. The results, obtained on a commercial HPLC-NMR setup, highlight the generality of the ultrafast HPLC-NMR methodology, thus opening the way to a number of applications in the numerous fields in which HPLC-NMR forms a routine analytical tool.

  1. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S

    PubMed Central

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-01-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  2. Chiral HPLC versus chiral SFC: evaluation of long-term stability and selectivity of Chiralcel OD using various eluents.

    PubMed

    Lynam, K G; Nicolas, E C

    1993-01-01

    The long-term stability of Chiralcel OD columns under varying HPLC and SFC conditions was evaluated. Two new columns from the same supplier lot were procured, and one was installed in an HPLC system, and the other was installed in an SFC system. Enantiomeric mixtures of trans-stilbene oxide and carbobenzyloxy phenylalaninol were repeatedly injected in both systems over several days. For HPLC, hexane was the primary solvent used, along with IPA, EtOH, MeOH/IPA, or EtOH/TFA modifier. Carbon dioxide was the primary SFC solvent, together with the similar modifiers as the HPLC. Column performance was monitored by measuring resolution, theoretical plate number, and alpha between enantiomeric peaks. It was observed that, when the eluent strengths were adjusted to provide comparable retention times, the Chiralcel OD gave superior enantiomeric resolution when used in the SFC than the HPLC. Equilibration in between solvents was faster with SFC. The columns were demonstrated to be quite stable in both systems. PMID:8123734

  3. Application of Continuous-Flow HPLC-Proton-Nuclear Magnetic Resonance Spectroscopy and HPLC-Thermospray-Mass Spectroscopy for the Structural Elucidation of Phototransformation Products of 2,4,6-Trinitrotoluene.

    PubMed

    Godejohann, M; Astratov, M; Preiss, A; Levsen, K; Mügge, C

    1998-10-01

    An aqueous solution of 2,4,6-trinitrotoluene (TNT) was irradiated by natural sunlight for a period of 1 month to generate phototransformation products of this compound. After solid-phase extraction on a poly(styrene-divinylbenzene) copolymer at pH 1, the structures of several acidic nitroaromatic compounds were identified by means of continuous-flow HPLC/(1)H NMR and HPLC/TSP-MS measurements of this extract. By interpretation of both NMR and MS spectra, it was even possible to characterize noncommercially available phototransformation products of TNT. The results obtained by continuous-flow HPLC/(1)H NMR were compared with those obtained by the investigation of a groundwater sample of a former ammunition site near Elsnig, Germany. The results show that several identified phototransformation products of TNT are also present in this groundwater sample.

  4. Mass spectrometry and NMR spectroscopy: modern high-end detectors for high resolution separation techniques--state of the art in natural product HPLC-MS, HPLC-NMR, and CE-MS hyphenations.

    PubMed

    Seger, Christoph; Sturm, Sonja; Stuppner, Hermann

    2013-07-01

    Current natural product research is unthinkable without the use of high resolution separation techniques as high performance liquid chromatography or capillary electrophoresis (HPLC or CE respectively) combined with mass spectrometers (MS) or nuclear magnetic resonance (NMR) spectrometers. These hyphenated instrumental analysis platforms (CE-MS, HPLC-MS or HPLC-NMR) are valuable tools for natural product de novo identification, as well as the authentication, distribution, and quantification of constituents in biogenic raw materials, natural medicines and biological materials obtained from model organisms, animals and humans. Moreover, metabolic profiling and metabolic fingerprinting applications can be addressed as well as pharmacodynamic and pharmacokinetic issues. This review provides an overview of latest technological developments, discusses the assets and drawbacks of the available hyphenation techniques, and describes typical analytical workflows.

  5. Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

    PubMed

    Shibukawa, Masami; Miyake, Ayaka; Eda, Sayaka; Saito, Shingo

    2015-09-15

    Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water. PMID:26320351

  6. Determination of the cis-trans isomerization barriers of L-alanyl-L-proline in aqueous solutions and at water/hydrophobic interfaces by on-line temperature-jump relaxation HPLC and dynamic on-column reaction HPLC.

    PubMed

    Shibukawa, Masami; Miyake, Ayaka; Eda, Sayaka; Saito, Shingo

    2015-09-15

    Proline cis-trans isomerization is known to play a key role in the rate-determining steps of protein folding. It is thus very important to understand the influence of environments, not only bulk solutions but also microenvironments such as interfaces, on the isomerization reaction of proline peptides. Here we present two HPLC methods for measurements of kinetic and equilibrium parameters for the isomerization reactions in bulk solutions and at liquid/solid interfaces. On-line temperature-jump relaxation HPLC (T-jump HPLC) allows the determination of forward and reverse rate constants of the isomerization in a bulk solution by monitoring the whole time course of conversion of pure isomers from both sides of the reaction, in contrast to other HPLC and capillary zone electrophoresis as well as spectrometric and calorimetric methods, which use a mixture of the isomers. We can then determine cis-trans isomerization barriers of the peptide at liquid/solid interfaces from the kinetic data obtained by dynamic on-column reaction HPLC and T-jump HPLC. We observed that the interconversion around the peptide bond for l-alanyl-l-proline (Ala-Pro) in water is accelerated at the surfaces of an alkyl-bonded silica and a poly(styrene-divinylbenzene) copolymer resin, and this is caused by a remarkable decrease in the enthalpy of activation. The molecular structures of the cis and trans forms of Ala-Pro estimated by quantum mechanics calculation reveal that an equilibrium shift toward the cis form as well as the rapid isomerization of Ala-Pro at the water/hydrophobic interfaces can be attributed to the lower polarity of the interfacial water at the surfaces of the hydrophobic materials compared to that of bulk water.

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

    NASA Astrophysics Data System (ADS)

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

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

  8. Determination of mitragynine in urine matrices by bar adsorptive microextraction and HPLC analysis.

    PubMed

    Neng, N R; Ahmad, S M; Gaspar, H; Nogueira, J M F

    2015-11-01

    Bar adsorptive microextraction combined with liquid desorption followed by high performance liquid chromatography with diode array detection (BAµE-LD/HPLC-DAD) is proposed for the determination of the psychoactive alkaloid mitragynine (MG) in human urine matrices. By using a modified N-vinylpyrrolidone polymer (P2) sorbent phase, high selectivity and efficiency is achieved. Assays performed by BAµE(P2)-LD/HPLC-DAD on 25 mL water samples spiked at the 8.0 µg L(-1) level yielded average recoveries around 100% of MG, under optimized experimental conditions. The analytical performance showed good precision (RSD<15%), appropriated detection limits of 0.10 µg L(-1) and linear dynamic ranges (0.6-24.0 μg L(-1)) with convenient determination coefficients of 0.9924. By using the standard addition method, the application of the present methodology for the determination of MG in human urine matrices after Kratom consumer, allowed very good performances. The proposed methodology proved to be a suitable alternative to monitor MG in biological fluid matrices, showing to be easy to implement, reliable, sensitive and requiring low sample volumes, when compared with other sorbent-based methods. PMID:26452798

  9. HPLC analysis of polyphenols in the fruits of Rubus idaeus L. (Rosaceae).

    PubMed

    Sparzak, B; Merino-Arevalo, M; Vander Heyden, Y; Krauze-Baranowska, M; Majdan, M; Fecka, I; Głód, D; Bączek, T

    2010-11-01

    The separation of anthocyanins present in the fruits of 11 varieties of red raspberries (Rubus idaeus L.) was performed by high performance liquid chromatography (HPLC) with a diode-array detector and evaporative light scattering detection (ELSD). The ELSD parameters--drift tube temperature, nebulising gas flow rate and gain value--were optimised to get the best detection and identification of the anthocyanins. The varieties Heritage and Willamette had the simplest anthocyanin sets consisting of only two predominant anthocyanins--cyanidin-3-O-sophoroside (1) and cyanidin-3-O-glucoside (3), while in the other varieties two other predominant compounds were also present, cyanidin-3-O-rutinoside (4) and cyanidin-3-O-(2(G)-O-glucosylrutinoside) (2). Moreover, using ELSD, simultaneous analysis of anthocyanins and sanguiin H-6 (5), an ellagitannin, was performed. The contents of anthocyanins and sanguiin H-6 (5) were estimated by HPLC with ultraviolet-visible (UV-vis) light detection. The determined concentrations of anthocyanins varied from 76.22 to 277.06 mg per 100 g of dry weight (d.w.). The content of sanguiin H-6 (5) was in the range from 135.04 to 547.48 mg per 100 g of d.w.

  10. Analysis of bioactivities and chemical composition of Ziziphus joazeiro Mart. using HPLC-DAD.

    PubMed

    Brito, Sharlene M O; Coutinho, Henrique D M; Talvani, Andre; Coronel, Cathia; Barbosa, Andreza G R; Vega, Celeste; Figueredo, Fernando G; Tintino, Saulo R; Lima, Luciene F; Boligon, Aline A; Athayde, Margareth L; Menezes, Irwin R A

    2015-11-01

    The aim of this study was to evaluate the chemical profile and antioxidant, antimicrobial and antiparasitic activities of the hydroalcoholic extract of the leaves of Ziziphus joazeiro Mart. (HELZJ). The antioxidant DPPH and FRAP assays and chemical profile were determined by colorimetric methods and HPLC/DAD. The antiparasitic, antibiotic and antibiotic-modifying activity were evaluated by microdilution assays. The HPLC-DAD assay showed the presence of mostly tannins and flavonoids, such as caffeic acid and quercetin. The levels of polyphenols and flavonoids were 183.136 mg/g extract and 7.37 mg/g extract, respectively. DPPH and FRAP showed low antioxidant activity for the extract. The antibacterial and antifungal activities were not of clinical relevance, showing MIC>1024 μg/mL. However, synergism was observed between HELZJ and the antibiotics amikacin and gentamicin, which resulted in decreased bacterial drug resistance. EHFZJ showed low toxicity in fibroblasts in vitro, while antiparasitic results against Trypnosoma cruzi, Leishmania braziliensis and Leishmania infantum were not clinically relevant. Thus, our results indicate that Z. joazeiro Mart. (HELZJ) could be a source of plant-derived natural products that could lead to the development of promising new antibiotic compounds for infectious diseases.

  11. Ultrasound-assisted extraction, HPLC analysis, and antioxidant activity of polyphenols from unripe apple.

    PubMed

    Yue, Tianli; Shao, Dongyan; Yuan, Yahong; Wang, Zhouli; Qiang, Chunyan

    2012-08-01

    The polyphenols were extracted from the unripe apple assisted by a highly efficient and simple method of the ultrasound. Response surface methodology was used to investigate the effects of processing parameters, including ultrasound power, extraction time, temperature, and ethanol concentration on total polyphenols yield and polyphenols composition was analyzed by HPLC. Antioxidant activity of the polyphenols was evaluated as 2, 2-diphenyl-1-picrylhydracyl scavenging activity and inhibition activity of lipid peroxidation. The results showed that 10-100 times higher total polyphenols yield was obtained from the unripe apple than those from the reported apple pomace. The optimum extraction conditions were ultrasonic power of 519.39 W, extraction time of 30 min, extraction temperature 50°C, ethanol concentration of 50% gave the total polyphenols yield of 13.26 ± 0.56 mg GAE/g. HPLC analysis indicated that (-)-epicatechin, procyanidin B2, chlorogenic acid, and procyanidin B1 were the predominant polyphenols in unripe apple, which contributed to the higher antioxidant activity to 2, 2-diphenyl-1-picrylhydracyl of unripe apple polyphenols than other apple polyphenols. The extracted polyphenols had higher ability to inhibit lipid peroxidation than butylated hydroxy toluene, which demonstrated that the unripe apple polyphenols have the potential to be used as a substitute of some synthetic antioxidants.

  12. Preparative HPLC method for the purification of sulforaphane and sulforaphane nitrile from Brassica oleracea.

    PubMed

    Matusheski, N V; Wallig, M A; Juvik, J A; Klein, B P; Kushad, M M; Jeffery, E H

    2001-04-01

    An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-microm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties.

  13. 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.

  14. Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

    PubMed

    Pavlović, Bojana; Cvijetić, Nataša; Dragačević, Luka; Ivković, Branka; Vujić, Zorica; Kuntić, Vesna

    2016-01-01

    One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories.

  15. HPLC analyses of cultures of Phoma spp.: differentiation among groups and species through secondary metabolite profiles.

    PubMed

    Pedras, M S; Biesenthal, C J

    2000-08-01

    The metabolite profiles of 26 isolates of the blackleg fungus (Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.), obtained from diverse parts of the world (part of the International Blackleg Crucifer Network collection), were studied utilizing specific culture conditions, HPLC analysis, and a set of chemical markers. This fungus is the causative agent of blackleg disease of brassica oilseeds; a virulent strain of the pathogen has caused significant rapeseed (Brassica napus L., and B. rapa L.) and canola (B. napus L., and B. rapa L.) losses in Canada, and is also considered a serious agricultural problem worldwide. Effective surveys of blackleg epidemics require simple and reliable analytical methodology to differentiate among the diverse groups of isolates. The chemical analysis of phytotoxins and related secondary metabolites is perhaps one of the most discriminating and the least ambiguous methods for differentiation of Phoma blackleg isolates. Following HPLC analyses, the 26 isolates could be placed in three main groups, irrespective of country of origin: isolates producing phomamide and sirodesmins, isolates producing indolyl dioxopiperazines, and isolates producing polyketides. Discussion of the implications of our findings and suggestions for species reclassification are provided.

  16. HPLC analysis of 5H-benzo[a]carbazole with antifungal activity.

    PubMed

    Segall, A I; Vitale, M F; Perez, V L; Pizzorno, M T

    2003-04-01

    A sensitive and simple high-performance liquid chromatographic (HPLC) method for the assay of 6,11-dihydro-2-methoxy-5H-benzo[a]carbazole (1) and 6,11-dihydro-2-methoxy-11-[2-(1-piperidinyl)]ethyl-5H-benzo[a]carbazole (2) was developed. The procedure is based on the use of the reversed-phase high-performance liquid chromatographic (RP-HPLC) method with UV detector. Each analysis required no longer than 11 min. A linear relationship between the concentration of both the drugs and the UV absorbance at 254 nm was obtained. This linearity was maintained over the concentration ranged from 5 to 80 microg/ml. The detection limits were found to be 1.6 and 0.7 ng for compounds 1 and 2. The quantitation limits were found to be 5.3 and 2.5 ng for compounds 1 and 2, respectively. For recovery studies, several determinations were carried out. Recovery values ranged from 98 to 102.1% for compound 1 and from 98.4 to 101.6% for compound 2. Method precision was also evaluated and RSD% found was less than 2%. This method was applied without any interference from degradation products.

  17. Development and validation of an HPLC method for tetracycline-related USP monographs.

    PubMed

    Hussien, Emad M

    2014-09-01

    A novel reversed-phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4-epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H3 PO4 as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80-120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50-150% of the acceptance criteria specified in the individual USP monographs for 4-epianhydrotetracycline hydrochloride. The limit of quantification for 4-epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust.

  18. HPLC Analysis, Optimization of Extraction Conditions and Biological Evaluation of Corylopsis coreana Uyeki Flos.

    PubMed

    Seo, Ji-Hye; Kim, Jung-Eun; Shim, Jung-Hyun; Yoon, Goo; Bang, Mi-Ae; Bae, Chun-Sik; Lee, Kyung-Jin; Park, Dae-Hun; Cho, Seung-Sik

    2016-01-15

    A method for the separation and quantification of three flavonoids and one isocoumarin by reverse-phase high performance liquid chromatography (HPLC) has been developed and validated. Four constituents present in a crude ethanolic extract of the flowers of Coryloposis coreana Uyeki, were analyzed. Bergenin, quercetin, quercitrin and isosalipurposide were used as calibration standards. In the present study, an excellent linearity was obtained with an r² higher than 0.999. The chromatographic peaks showed good resolution. In combination with other validation data, including precision, specificity, and accuracy, this method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of bergenin, quercetin, quercitrin and isosalipurposide in the crude ethanolic extract of C. coreana Uyeki flos. Furthermore, the plant extracts were analyzed with HPLC to determine the four constituents and compositional differences in the extracts obtained under different extraction conditions. Several extracts of them which was dependent on the ethanol percentage of solvent were also analyzed for their antimicrobial and antioxidant activities. One hundred % ethanolic extract from C. coreana Uyeki flos showed the best antimicrobial activity against the methicillin-resistant Staphylococcus aureus (MRSA) strain. Eighty % ethanolic extract showed the best antioxidant activity and phenolic content. Taken of all, these results suggest that the flower of C. coreana Uyeki flos may be a useful source for the cure and/or prevention of septic arthritis, and the validated method was useful for the quality control of C. coreana Uyeki.

  19. Antioxidant, DNA protective efficacy and HPLC analysis of Annona muricata (soursop) extracts.

    PubMed

    George, V Cijo; Kumar, D R Naveen; Suresh, P K; Kumar, R Ashok

    2015-04-01

    Annona muricata is a naturally occurring edible plant with wide array of therapeutic potentials. In India, it has a long history of traditional use in treating various ailments. The present investigation was carried out to characterize the phytochemicals present in the methanolic and aqueous leaf extracts of A. muricata, followed by validation of its radical scavenging and DNA protection activities. The extracts were also analyzed for its total phenolic contents and subjected to HPLC analysis to determine its active metabolites. The radical scavenging activities were premeditated by various complementary assays (DRSA, FRAP and HRSA). Further, its DNA protection efficacy against H2O2 induced toxicity was evaluated using pBR322 plasmid DNA. The results revealed that the extracts were highly rich in various phytochemicals including luteolin, homoorientin, tangeretin, quercetin, daidzein, epicatechin gallate, emodin and coumaric acid. Both the extracts showed significant (p < 0.05) radical scavenging activities, while methanolic extract demonstrated improved protection against H2O2-induced DNA damage when compared to aqueous extract. A strong positive correlation was observed for the estimated total phenolic contents and radical scavenging potentials of the extracts. Further HPLC analysis of the phyto-constituents of the extracts provides a sound scientific basis for compound isolation.

  20. A global seasonal surface ocean climatology of phytoplankton types based on CHEMTAX analysis of HPLC pigments

    NASA Astrophysics Data System (ADS)

    Swan, Chantal M.; Vogt, Meike; Gruber, Nicolas; Laufkoetter, Charlotte

    2016-03-01

    Much advancement has been made in recent years in field data assimilation, remote sensing and ecosystem modeling, yet our global view of phytoplankton biogeography beyond chlorophyll biomass is still a cursory taxonomic picture with vast areas of the open ocean requiring field validations. High performance liquid chromatography (HPLC) pigment data combined with inverse methods offer an advantage over many other phytoplankton quantification measures by way of providing an immediate perspective of the whole phytoplankton community in a sample as a function of chlorophyll biomass. Historically, such chemotaxonomic analysis has been conducted mainly at local spatial and temporal scales in the ocean. Here, we apply a widely tested inverse approach, CHEMTAX, to a global climatology of pigment observations from HPLC. This study marks the first systematic and objective global application of CHEMTAX, yielding a seasonal climatology comprised of ~1500 1°×1° global grid points of the major phytoplankton pigment types in the ocean characterizing cyanobacteria, haptophytes, chlorophytes, cryptophytes, dinoflagellates, and diatoms, with results validated against prior regional studies where possible. Key findings from this new global view of specific phytoplankton abundances from pigments are a) the large global proportion of marine haptophytes (comprising 32±5% of total chlorophyll), whose biogeochemical functional roles are relatively unknown, and b) the contrasting spatial scales of complexity in global community structure that can be explained in part by regional oceanographic conditions. The results are publically accessible via

  1. Determination of glucosamine and its derivatives released from photocrosslinked gelatin hydrogels using HPLC.

    PubMed

    Suo, Hairui; Xu, Kedi; Zhang, Hengyi; Zheng, Xiaoxiang

    2016-02-01

    A simple, accurate and validated reverse-phase high-performance liquid chromatography (HPLC)/UV method is developed for the determination of glucosamine hydrochloride (GlcN), N-acetyl-glucosamine (NAG) and N-acryloyl-glucosamine (AGA) released from photocrosslinked gelatin hydrogels. The HPLC separation was achieved on a Shimadzu InertSustain amino column (250 × 4.6 mm, 5 µm particle size) at room temperature using a mobile phase of acetonitrile-phosphate buffer (75:25, v/v, pH 6.0) at a flow rate of 1.0 mL/min and UV detection of 194 nm. The method was validated for specificity, linearity, limit of detection and quantification, accuracy, precision, extraction recovery and solution stability. The calibration curves were with excellent linearity, with correlation coefficients (R(2)) >0.999 for all three drugs. The intra- and inter-day variation was <3.10% and the relative error was between -1.43 and 1.78%. The extraction recovery results ranged from 94.62 to 99.33%, demonstrating the absence of matrix effect. The sample and standard solutions were stable for more than 2 months. The method was successfully used for the analysis of released properties of drugs physically encapsulated and chemically crosslinked in the gelatin hydrogels.

  2. Validation of a HPLC method for simultaneous determination of five sunscreens in lotion preparation.

    PubMed

    Kedor-Hackmann, E R M; De Lourdes Pérez González, M L; Singh, A K; Santoro, M I R M

    2006-06-01

    The aim of this research was to develop and validate a high-performance liquid chromatographic (HPLC) method for simultaneous determination of five sunscreens, namely benzophenone-3 (B-3), butyl methoxydibenzoylmethane (BM), octyl methoxycinnamate (OM), octyl salicylate (OS) and homosalate (HS). The separation and quantitative determination was made by HPLC at 40 +/-1 degrees C with a gradient elution from 10% to 100% mobile phase B in mobile phase A. The gradient liquid chromatographic system constituted of mobile phase A [acetonitrile : water (10 : 90 v/v)] and mobile phase B [acetonitrile : water (90 : 10 v/v)], at a flow rate of 1.0 mL min(-1) and ultraviolet detection at 310 nm. The separation was obtained with two Waters reversed phase columns: Novapack C-18 and Symmetry((R)) C-18 connected in series. All sunscreens were efficiently separated within 17 min. The coefficient of correlation and average recovery for B-3, BM, OM, OS and HS were 0.9798 and 98.5%, 0.9672 and 98.8%, 0.9922 and 99.1%, 0.9961 and 98.9% and 0.9909 and 99.4% respectively. The relative standard deviations obtained were between 1.07% and 2.44%. The excipients did not interfere in the analysis. The results showed that the proposed method could be used for rapid and simultaneous determination of B-3, BM, OM, OS and HS in sunscreen lotions with precision, accuracy and specificity.

  3. Direct UV Spectrophotometry and HPLC Determination of Triton X-100 in Split Virus Influenza Vaccine.

    PubMed

    Pavlović, Bojana; Cvijetić, Nataša; Dragačević, Luka; Ivković, Branka; Vujić, Zorica; Kuntić, Vesna

    2016-01-01

    One of the most commonly used surfactants in the production of split virus influenza vaccine is nonionic surfactant Triton X-100. After splitting of the virus is accomplished, Triton X-100 is removed from the vaccine by subsequent production steps. Because of toxicity of Triton X-100, which remains in the vaccine in residual amounts, a sufficiently sensitive method for its detection and quantification needs to be defined. Two methods for determination of Triton X-100 residuals were developed: the UV-spectrophotometry and HPLC methods. For both methods, preparation of vaccine samples and removal of proteins and virus particles were crucial: samples were treated with methanol (1:1) and then centrifuged at 25 000 × g for 30 min. After such treatment, the majority of vaccine components that interfered in the UV region were removed, and diluted samples could be directly measured. The chromatographic system included C18 column, step methanol gradient, and detection at 225 nm with a single peak of Triton X-100 at 12.6 min. Both methods were validated and gave satisfactory results for accuracy, precision, specificity, linearity, and robustness. LOQ was slightly lower for the HPLC method. Hence, it was shown that both methods are suitable for analysis of residual amounts of Triton X-100, with the advantages of the UV method being its simplicity and availability in most laboratories. PMID:26960682

  4. Quantitative Determination of Catechin as Chemical Marker in Pediatric Polyherbal Syrup by HPLC/DAD.

    PubMed

    Sheikh, Zeeshan A; Siddiqui, Zafar A; Naveed, Safila; Usmanghani, Khan

    2016-09-01

    Vivabon syrup is a balanced composition of dietary ingredients of phytopharmaceutical nature for maintaining the physique, vigor, vitality and balanced growth of children. The herbal ingredients of pediatric syrup are rich in bioflavonoid, proteins, vitamins, glycosides and trace elements. Vivabon is formulated with herbal drugs such as Phoenix sylvestris, Emblica officinalis, Withania somnifera, Centella asiatica, Amomum subulatum, Zingiber officinalis, Trigonella foenum-graecum, Centaurea behen and Piper longum Catechins are flavan-3-ols that are found widely in the medicinal herbs and are utilized for anti-inflammatory, cardio protective, hepato-protective, neural protection and other biological activities. In general, the dietary intake of flavonoids has been regarded traditionally as beneficial for body growth. Standardization of Vivabon syrup dosage form using HPLC/DAD has been developed for quantitative estimation of Catechin as a chemical marker. The method was validated as per ICH guidelines. Validation studies demonstrated that the developed HPLC method is quite distinct, reproducible as well as quick and fast. The relatively high recovery and low comparable standard deviation confirm the suitability of the developed method for the determination of Catechin in syrup. PMID:27165575

  5. HPLC/QTOF-MS/MS application to investigate phenolic constituents from Ficus pandurata H. aerial roots.

    PubMed

    Zhang, Xiaoping; Lv, Huiqing; Li, Zuguang; Jiang, Kezhi; Lee, Maw-Rong

    2015-06-01

    Ficus pandurata H. aerial roots are used as a traditional Chinese medicine for the treatment of uarthritis, indigestion and hyperuricemia. However, the bioactive constituents responsible for the pharmacological effects of F. pandurata H. are unclear. A simple and efficient HPLC/QTOF-MS/MS (high-performance liquid chromatography/electrospray ionization with quadrupole time-of-flight tandem mass spectrometry) method was established to detect and identify active constituents in the n-butanol extract of F. pandurata H. aerial roots. Chemical constituents were separated and investigated by HPLC/QTOF-MS/MS in the negative-ion mode. Thirty-seven compounds, including hydroxycinnamic acid derivatives, hydroxybenzoic acid derivatives, hydroquinone glycosides, flavonoid glycosides, etc., were identified or tentatively characterized in the n-butanol extract of F. pandurata H. aerial roots by comparing the UV spectra, accurate mass spectra and fragmentation pathways and retrieving the reference literatures. Moreover, the flavonoid trisaccharides and hydroxybenzoic acid derivatives were tentatively characterized in F. pandurata H. for the first time. The analytical tool used here is very valuable in the rapid separation and identification of the multiple and minor constituents in the n-butanol extract of F. pandurata H. aerial roots.

  6. Quantitative HPLC Analysis of an Analgesic/Caffeine Formulation: Determination of Caffeine

    NASA Astrophysics Data System (ADS)

    Ferguson, Glenda K.

    1998-04-01

    A modern high performance liquid chromatography (HPLC) laboratory experiment which entails the separation of acetaminophen, aspirin, and caffeine and the quantitative assay of caffeine in commercial mixtures of these compounds has been developed. Our HPLC protocol resolves these compounds in only three minutes with a straightforward chromatographic apparatus which consists of a C-18 column, an isocratic mobile phase, UV detection at 254 nm, and an integrator; an expensive, sophisticated system is not required. The separation is both repeatable and rapid. Moreover, the experiment can be completed in a single three-hour period. The experiment is appropriate for any chemistry student who has completed a minimum of one year of general chemistry and is ideal for an analytical or instrumental analysis course. The experiment detailed herein involves the determination of caffeine in Goody's Extra Strength Headache Powders, a commercially available medication which contains acetaminophen, aspirin, and caffeine as active ingredients. However, the separation scheme is not limited to this brand of medication nor is it limited to caffeine as the analyte. With only minor procedural modifications, students can simultaneously quantitate all of these compounds in a commercial mixture. In our procedure, students prepare a series of four caffeine standard solutions as well as a solution from a pharmaceutical analgesic/caffeine mixture, chromatographically analyze each solution in quadruplicate, and plot relative average caffeine standard peak area versus concentration. From the mathematical relationship that results, the concentration of caffeine in the commercial formulation is obtained. Finally, the absolute standard deviation of the mean concentration is calculated.

  7. Measurement of Microcystin -LR in Water Samples Using Improved HPLC Method

    PubMed Central

    Shamsollahi, Hamid Reza; Alimohammadi, Mahmood; Nabizadeh, Ramin; Nazmara, Shahrokh; Mahvi, Amir Hossein

    2015-01-01

    Microcystins are a group of toxic compounds produced by freshwater cyanobacteria and cause diseases. World Health Organization has recommended a concentration of 1 µg/l for Microcystin-LR (MC-LR) in potable water as guideline value. The high performance liquid chromatography (HPLC) followed by C18 analytical column and ultra violet detector for detection of MC-LR. In this regard, 5 different concentrations of MC-LR solutions were injected into HPLC. MC-LR was detected in 5.33 minute retention time and Calibration curve was achieved with R2=0.988. Detection limit for this method was obtained by using acetonitrile solutions (32% and 55%) as a gradient run and a high silanol activity column equal to 0.02 µg/mL. Despite no acidic organic modifier being used in the mixture of solvents, the sensitivity of this method was appropriate for detection of MC-LR. Because of short retention time, reduction in number of solvents and high resolution and suitable sensitivity, this method is affordable and is fast for detection and determination of MC-LR in potable water. PMID:25716387

  8. HPLC and chemometric methods for the simultaneous determination of cyproheptadine hydrochloride, multivitamins, and sorbic acid.

    PubMed

    el-Gindy, Alaa; el-Yazby, Fawzy; Mostafa, Ahmed; Maher, Moustafa M

    2004-06-29

    Three methods are presented for the simultaneous determination of cyproheptadine hydrochloride (CP), thiamine hydrochloride (B1), riboflavin-5-phosphate sodium dihydrate (B2), nicotinamide (B3), pyridoxine hydrochloride (B6), and sorbic acid (SO). The chromatographic method depends on a high performance liquid chromatographic (HPLC) separation on a reversed-phase, RP 18 column. Elution was carried out with 0.1% methanolic hexane sulphonic acid sodium salt (solvent A) and 0.01 M phosphate buffer containing 0.1% hexane sulphonic acid sodium salt, adjusted to an apparent pH of 2.7 (solvent B). Gradient HPLC was used with the solvent ratio changed from 20:80 to 70:30 (over 9 min), then to 80:20 (over 11 min) for solvent A:B, respectively. Quantitation was achieved with UV detection at 220 and 288 nm based on peak area. The other two chemometric methods applied were principal component regression (PCR) and partial least squares (PLS). These approaches were successfully applied to quantify each drug in the mixture using the information included in the UV absorption spectra of appropriate solutions in the range 250-290 nm with the intervals Deltalambda = 0.4 nm at 100 wavelengths. The chemometric methods do not require any separation step. The three methods were successfully applied to a pharmaceutical formulation and the results were compared with each other.

  9. 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%.

  10. Determination of glutamate uptake by high performance liquid chromatography (HPLC) in preparations of retinal tissue.

    PubMed

    Moraes, Edinaldo Rogério da Silva; Grisolia, Alan Barroso Araújo; Oliveira, Karen Renata Matos; Picanço-Diniz, Domingos Luiz Wanderley; Crespo-López, Maria Elena; Maximino, Caio; Batista, Evander de Jesus Oliveira; Herculano, Anderson Manoel

    2012-10-15

    The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated with known concentrations of glutamate (50-2000 μM) for 10 min, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (K(m)=8.2 and V(max)=9.8 nmol/mg protein/min). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration-dependent decrease in glutamate uptake, demonstrating the specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of minute alterations in glutamate transport related with central nervous system injury.

  11. Identification and determination of flavonoids, carotenoids and chlorophyll concentration in Cynodon dactylon (L.) by HPLC analysis.

    PubMed

    Muthukrishnan, Saradha Devi; Kaliyaperumal, Ashokkumar; Subramaniyan, Annapoorani

    2015-01-01

    Cynodon dactylon (L.) is a potent medicinal plant in the traditional and current Indian medicinal systems. The objective of this research was to find out the levels of flavonoids, carotenoids and chlorophyll b in C. dactylon leaves by high-performance liquid chromatography (HPLC) equipped with a diode array detector. HPLC analysis revealed that total carotenoid and total flavonoid concentration were 62 mg/100 g and 249.1 μg/g, respectively. The mean chlorophyll b was 85.1 mg/100 g in C. dactylon. Among the flavonoids, quercetin (164.7 μg/g) was the major flavonoid followed by kaempferol (48.2 μg/g), rutin (18.4 μg/g), catechin (12.1 μg/g) and myricetin (5.7 μg/g). Of the carotenoids, β-carotene (35.2 mg/100 g) was predominant followed by lutein (17.0 mg/100 g), violaxanthin (5.8 mg/100 g) and zeaxanthin (4.2 mg/100 g). Chlorophyll b concentration was 85.1 mg/100 g in C. dactylon. The results of this investigation should be useful information for further pharmacological studies. PMID:25495959

  12. Simultaneous quantification of 19 ginsenosides in black ginseng developed from Panax ginseng by HPLC-ELSD.

    PubMed

    Sun, Bai-Shen; Gu, Li-Juan; Fang, Zhe-Ming; Wang, Chun-yan; Wang, Zhen; Lee, Mi-Ra; Li, Zheng; Li, Jing-Jie; Sung, Chang-Keun

    2009-08-15

    A high-performance liquid chromatographic method with evaporative light scattering detection (HPLC-ELSD) has been developed to identify and quantify 19 ginsenosides (Rg(1), Re, Rf, Rb(1), Rc, Rb(2), Rd, F(4), Rg(6), Rk(3), Rh(4), 20(S)-, 20(R)-Rg(3), 20(S)-, 20(R)-Rs(3), Rk(1), Rg(5), Rs(4), and Rs(5)) in black ginseng (BG, Korean white ginseng that was subjected to nine cycles of steam treatment). Ultrasonication is employed for sample preparation, and the analysis is achieved on a Discovery C(18) column using gradient elution of CH(3)CN-H(2)O-CH(3)COOH without buffer in 40min. The method was validated by linearity (r(2)> or =0.9994), precision (92.0-107.5%), intra- and inter-day accuracy (R.S.D.<3.21%), and limit of detection (LOD< or =93ng). The quantification method was applied to analyze the composition of ginsenosides in Korean white, red, and black ginsengs. During the preparatory process of BG, ginsenosides transform into constituents of low polarity by hydrolysis, isomerization, and dehydration at C-20, and hydrolysis also occurs at C-3 or C-6. The validated HPLC method is expected to provide the basis for the quality assessment of ginseng products.

  13. Identification and determination of flavonoids, carotenoids and chlorophyll concentration in Cynodon dactylon (L.) by HPLC analysis.

    PubMed

    Muthukrishnan, Saradha Devi; Kaliyaperumal, Ashokkumar; Subramaniyan, Annapoorani

    2015-01-01

    Cynodon dactylon (L.) is a potent medicinal plant in the traditional and current Indian medicinal systems. The objective of this research was to find out the levels of flavonoids, carotenoids and chlorophyll b in C. dactylon leaves by high-performance liquid chromatography (HPLC) equipped with a diode array detector. HPLC analysis revealed that total carotenoid and total flavonoid concentration were 62 mg/100 g and 249.1 μg/g, respectively. The mean chlorophyll b was 85.1 mg/100 g in C. dactylon. Among the flavonoids, quercetin (164.7 μg/g) was the major flavonoid followed by kaempferol (48.2 μg/g), rutin (18.4 μg/g), catechin (12.1 μg/g) and myricetin (5.7 μg/g). Of the carotenoids, β-carotene (35.2 mg/100 g) was predominant followed by lutein (17.0 mg/100 g), violaxanthin (5.8 mg/100 g) and zeaxanthin (4.2 mg/100 g). Chlorophyll b concentration was 85.1 mg/100 g in C. dactylon. The results of this investigation should be useful information for further pharmacological studies.

  14. [Study on HPLC-eLSD fingerprint of total steroid saponins in herbs of Dioscorea zingiberensis].

    PubMed

    Zhang, Xin-Xin; Liang, Jin-Ru; Zhao, Ye; Sun, Wen-Ji

    2013-10-01

    To establish a HPLC-ELSD fingerprint for total steroid saponins in herbs of Dioscorea zingiberensis. Welchrom C,8 (4. 6 mm x 250 mm,5 microm) chromatographic column was adopted and eluted with the mobile phase of acetonitrile (A)-water (B) at the flow rate of 1.0 mL min-1. The column temperature was room temperature. The ELSD conditions were as follows: the temperature of drift tube was 90.0 degreeC, the flow rate of carrier gas (N2) was 2. 8 L min-1, and the injection volume was 10 microL. After the detection of 10 batches of samples,the common mode of HPLC-ELSD fingerprint for total steroid saponins in herbs of D. zingiberensis was established for the first time,and 25 common peaks were determined. Among them, 10 peaks were identified by comparing with reference substances. The similarities of 10 batches of herbs were evaluated in the common mode. All of them were higher than 0. 80. This method is so accurate, reliable and highly repeatable that it can provide scientific basis for evaluating and controlling the quality of total steroid saponins in herbs of D. zingiberensis.

  15. [Determination of ellagic acid, flavonoids and goshonoside-F5 in Rubi Fructus by HPLC].

    PubMed

    He, Jian-Ming; Sun, Nan; Wu, Wen-Dan; Fan, Li-Jiao; Guo, Mei-Li

    2013-12-01

    High-performance liquid chromatographic coupled with variable wavelength detection (HPLC-VWD) has been developed for simultaneous determination of 5 analytes including ellagic acid, quercetin, kaempferol-3-O-beta-D-rutinoside, tiliroside and kaempferol, and high-performance liquid chromatographic with an evaporative light scattering detector (HPLC-ELSD) has been established to determine goshonoside-F5 in extract of Rubi Fructus. Chromatographic separations were carried out on an Agilent ZORBAX SB-C18 column (4.6 mm x 250 mm, 5.0 microm). All calibration curves of reference standards revealed good linearity (R2 > 0.999 5) within the concentration ranges tested. The method limits of detection ranged 0.297-90.144 ng and the method limits ofquantitation ranged 0.990-300.480 ng, respectively. Recoveries of 6 analytes were from 97.11% to 101.7%, with RSD less than 2.1%. The result shows that amounts of the 6 analytes in the samples from 16 localities were found to be different. The higher latitude of growing environment, the more ellagic acid in herb. The content of total flavonoids in sample from east localities were higher than that in middle and west localities, and the content of goshonoside-F5 in Bozhou, Anhui province was higher than others. This method was found to be simple, accurate, sensitive with good repeatability. Those results might serve as a sound foundation for further study, quality control and application of Rubi Fructus.

  16. Determination of cytotoxic compounds of Thai traditional medicine called Benjakul using HPLC.

    PubMed

    Itharat, Arunporn; Sakpakdeejaroen, Intouch

    2010-12-01

    Benjakul is a Thai traditional medicine preparation, used for balanced health. From selective interviews of folk doctors in southern Thailand, it was used as the adaptogen drug for cancer patients. In our previous study, the ethanolic extract of Benjakul preparation exhibited high cytotoxic activity against lung cancer cell lines (COR-L23). Piperine has been identified as the main compound in the extract. In addition, plumbagin was found as the most cytotoxic compound. In this study, a reversed-phase high performance liquid chromatography (HPLC) method for quality control such as chemical fingerprint, quantification and stability of the ethanolic extract of Benjakul preparation was developed. The reversed-phase HPLC was performed with a gradient mobile phase composed of water and acetronitrile, and peaks were detected at 256 nm. Based on validation results, this analytical method is precise, accurate and stable for quantitative determination of piperine and plumbagin which are cytotoxic compounds isolated from the ethanolic extract of Benjakul preparation. This method could be suitable for analysis of Benjakul extract. PMID:21294415

  17. HPLC-DAD-ESI-MS Analysis of Flavonoids from Leaves of Different Cultivars of Sweet Osmanthus.

    PubMed

    Wang, Yiguang; Fu, Jianxin; Zhang, Chao; Zhao, Hongbo

    2016-01-01

    Osmanthus fragrans Lour. has traditionally been a popular ornamental plant in China. In this study, ethanol extracts of the leaves of four cultivar groups of O. fragrans were analyzed by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) and high-performance liquid chromatography with electrospray ionization and mass spectrometry (HPLC-ESI-MS). The results suggest that variation in flavonoids among O. fragrans cultivars is quantitative, rather than qualitative. Fifteen components were detected and separated, among which, the structures of 11 flavonoids and two coumarins were identified or tentatively identified. According to principal component analysis (PCA) and hierarchical cluster analysis (HCA) based on the abundance of these components (expressed as rutin equivalents), 22 selected cultivars were classified into four clusters. The seven cultivars from Cluster III ('Xiaoye Sugui', 'Boye Jingui', 'Wuyi Dangui', 'Yingye Dangui', 'Danzhuang', 'Foding Zhu', and 'Tianxiang Taige'), which are enriched in rutin and total flavonoids, and 'Sijigui' from Cluster II which contained the highest amounts of kaempferol glycosides and apigenin 7-O-glucoside, could be selected as potential pharmaceutical resources. However, the chemotaxonomy in this paper does not correlate with the distribution of the existing cultivar groups, demonstrating that the distribution of flavonoids in O. fragrans leaves does not provide an effective means of classification for O. fragrans cultivars based on flower color. PMID:27649119

  18. UPLC and HPLC of caffeoyl esters in wild and cultivated Arctium lappa L.

    PubMed

    Haghi, Ghasem; Hatami, Alireza; Mehran, Mehdi

    2013-05-01

    Analytical methods including ultra-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) with photodiode array (PDA) detector were developed for the analysis of caffeoylquinic acid derivatives in seeds, leaves and roots of Arctium lappa L. Separation was performed on C(18) column utilising 5% (v/v) acetic acid in water and acetonitrile at 330 nm. Both methodologies were validated in terms of linearity, precision, and recovery. The results showed that the major advantages of UPLC, over HPLC were the fast analysis, narrow peaks, high sensitivity, and reduction of solvent consumption. Subsequently the methods were applied for the identification and quantification of chlorogenic acid (5-CQA) and 1,5-dicaffeoylquinic acid (1,5-DCQA) as main compounds in samples. The total phenolic content of samples ranged from 3.93 to 14.13 g of 5-CQA equivalent/100g dry weight (DW). There was a significant variability from 89 to 571 mg/100g for 5-CQA and 48 to 486 mg/100g for 1,5-DCQA in dry material.

  19. Determination of fructooligosaccharides in burdock using HPLC and microwave-assisted extraction.

    PubMed

    Li, Jing; Liu, Xiaomei; Zhou, Bin; Zhao, Jing; Li, Shaoping

    2013-06-19

    The root of burdock ( Arctium lappa L.) is a commonly used vegetable in Asia. Fructooligosaccharides (FOS) are usually considered as its main bioactive components. Thus, quantitative analysis of these components is very important for the quality control of burdock. In this study, an HPLC-ELSD and microwave-assisted extraction method was developed for the simultaneous determination of seven FOS with degrees of polymerization (DP) between 3 and 9, as well as fructose, glucose, and sucrose in burdock from different regions. The separation was performed on a Waters XBridge Amide column (4.6 × 250 mm i.d., 3.5 μm) with gradient elution. All calibration curves for investigated analytes showed good linear regression (r > 0.9990). Their LODs and LOQs were lower than 3.63 and 24.82 μg/mL, respectively. The recoveries ranged from 99.2 to 102.6%. The developed method was successfully applied to determination of ten sugars in burdock from different locations of Asia. The results showed that the contents of FOS in different samples of burdock collected at appropriate times were similar, and the developed HPLC-ELSD with microwave-assisted extraction method is helpful to control the quality of burdock.

  20. Determination of fructooligosaccharides in burdock using HPLC and microwave-assisted extraction.

    PubMed

    Li, Jing; Liu, Xiaomei; Zhou, Bin; Zhao, Jing; Li, Shaoping

    2013-06-19

    The root of burdock ( Arctium lappa L.) is a commonly used vegetable in Asia. Fructooligosaccharides (FOS) are usually considered as its main bioactive components. Thus, quantitative analysis of these components is very important for the quality control of burdock. In this study, an HPLC-ELSD and microwave-assisted extraction method was developed for the simultaneous determination of seven FOS with degrees of polymerization (DP) between 3 and 9, as well as fructose, glucose, and sucrose in burdock from different regions. The separation was performed on a Waters XBridge Amide column (4.6 × 250 mm i.d., 3.5 μm) with gradient elution. All calibration curves for investigated analytes showed good linear regression (r > 0.9990). Their LODs and LOQs were lower than 3.63 and 24.82 μg/mL, respectively. The recoveries ranged from 99.2 to 102.6%. The developed method was successfully applied to determination of ten sugars in burdock from different locations of Asia. The results showed that the contents of FOS in different samples of burdock collected at appropriate times were similar, and the developed HPLC-ELSD with microwave-assisted extraction method is helpful to control the quality of burdock. PMID:23745967

  1. UPLC and HPLC of caffeoyl esters in wild and cultivated Arctium lappa L.

    PubMed

    Haghi, Ghasem; Hatami, Alireza; Mehran, Mehdi

    2013-05-01

    Analytical methods including ultra-performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC) with photodiode array (PDA) detector were developed for the analysis of caffeoylquinic acid derivatives in seeds, leaves and roots of Arctium lappa L. Separation was performed on C(18) column utilising 5% (v/v) acetic acid in water and acetonitrile at 330 nm. Both methodologies were validated in terms of linearity, precision, and recovery. The results showed that the major advantages of UPLC, over HPLC were the fast analysis, narrow peaks, high sensitivity, and reduction of solvent consumption. Subsequently the methods were applied for the identification and quantification of chlorogenic acid (5-CQA) and 1,5-dicaffeoylquinic acid (1,5-DCQA) as main compounds in samples. The total phenolic content of samples ranged from 3.93 to 14.13 g of 5-CQA equivalent/100g dry weight (DW). There was a significant variability from 89 to 571 mg/100g for 5-CQA and 48 to 486 mg/100g for 1,5-DCQA in dry material. PMID:23265494

  2. Simultaneous determination of prenylflavonoid and hop bitter acid in beer lee by HPLC-DAD-MS.

    PubMed

    Kao, T H; Wu, G Y

    2013-11-15

    An HPLC-DAD-MS method with high accuracy and precision was developed for determination of prenylflavonoids and hop bitter acids in beer lee, a by-product from beer brewing process. Four prenylflavonoids and nine hop bitter acids can be simultaneously separated in 29 min using a Thermo HyPURITY C18 column in combination with diode array dectector and mass spectrometer with HPLC solvent gradient system of phosphoric acid aqueous solution at pH 1.6 and acetonitrile at a flow rate of 1.5 mL/min and detection wavelength at 314 nm. Beer lee is found to contain isoxanthohumol (36.2 μg/g), xanthohumol (29.6 μg/g), 8-prenylnaringenin (7.84 μg/g), 6-prenylnaringenin (19.2 μg/g), cohumulone (44.7 μg/g), humulone (123 μg/g), adhumulone (21.8 μg/g), colupulone (44.2 μg/g), lupulone (33.2 μg/g), and adlupulone (5.76 μg/g).

  3. Quality control of Gardeniae Fructus by HPLC-PDA fingerprint coupled with chemometric methods.

    PubMed

    Yin, Fangzhou; Wu, Xiaoyan; Li, Lin; Chen, Yan; Lu, Tuling; Li, Weidong; Cai, Baochang; Yin, Wu

    2015-01-01

    The ripe fruits of Gardenia jasminoides Ellis have been used as traditional Chinese medicine to treat diseases for a long history. Lines of evidence demonstrate that multiple active constituents are responsible for the therapeutic effects of this herbal medicine. However, effective methods for quality control of this herbal medicine are still lacking. In this study, a high-performance liquid chromatography (HPLC) fingerprint analysis was performed on a SinoChrom ODS-BP C18 column (4.6 mm × 250 mm, 5 μm) at 30°C with mobile phase of aqueous solution with 0.1% formic acid and acetonitrile. On the basis of the chromatographic data from 32 batches samples, the HPLC fingerprint pattern containing 27 common peaks was obtained. Among these common peaks, seven peaks were identified by the electrospray ionization-mass spectrometry as geniposidic acid, genipin-1-β-gentiobioside, chlorogenic acid, geniposide, rutin, crocin-1 and crocin-2 and the contents of these seven compounds were simultaneously determined. Finally, chemometric methods including hierarchical clustering analysis and principal component analysis were successfully applied to differentiate the samples from six producing regions. In sum, the data, as described in this study, offer valuable information for the quality control and proper use of Gardeniae Fructus. PMID:26071608

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

    PubMed

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

    2015-07-01

    The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC. PMID:26023813

  5. Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry

    PubMed Central

    Su, Dan; Chan, Clement T.Y.; Gu, Chen; Lim, Kok Seong; Chionh, Yok Hian; McBee, Megan E.; Russell, Brandon S.; Babu, I. Ramesh; Begley, Thomas J.; Dedon, Peter C.

    2015-01-01

    Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions, and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress response mechanism involving selective translation of codon-biased mRNA for critical proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-coupled mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA or other RNA species. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS using dynamic multiple reaction monitoring. This approach enables quantification of modified ribonucleosides in several micrograms of tRNA, or other RNA, in a 15-minute LC-MS run. By comparison, traditional methods for detecting modified ribonucleosides are labor and time intensive, require larger RNA quantities, are modification-specific, or require radioactive labeling. PMID:24625781

  6. Automated modular preparative HPLC-MS purification laboratory with enhanced efficiency.

    PubMed

    Guth, Oliver; Krewer, Dietmar; Freudenberg, Björn; Paulitz, Christian; Hauser, Manfred; Ilg, Kerstin

    2008-01-01

    Automated parallel synthesis as tool to increase productivity in chemical synthesis is well-established. However, even more time-consuming than the synthesis process is the following purification of the resulting crude products. To enhance efficiency of the lead optimization process at Bayer CropScience, a high-throughput HPLC/MS-laboratory for the purification of up to 48 crude products per day in the range of 200-400 mg each in one injection per sample has been set up. The use of Covaris technology for HPLC sample preparation, automated aliquotation during fractionation, and a novel evaporation process by combination with freeze-drying are new key technologies applied successfully for the first time in this purification unit facilitating to achieve the targeted efficiency. The whole process is supported by a specially designed IT-landscape covering each step of the workflow. Both the technical instruments used within the laboratory and the workflow and IT platform are described in this article.

  7. Evaluation of ⁶⁰Co-gamma radiosterilization on Chinese medicines with HPLC/FTIR.

    PubMed

    Huang, Chiung-Hua; Lee, Shih-Chang; Chen, Yueh-Sheng; Yao, Chun-Hsu

    2010-11-01

    The aim of this study was to evaluate the effect of radiosterilization on 30 Chinese medicines using γ-rays from the isotope ⁶⁰Co. Two groups of Chinese medicines, non-treated and dry samples, were treated using a ⁶⁰Co irradiation source at the doses 0, 3, 6 and 9  kGy. After storage for 3 months, characterizations of chemical compounds and functional groups were performed by high-performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy. The results of radiosterlization showed that nearly all of the medicines were decontaminated under the dose of 9  kGy. In most samples, chemical compounds and functional groups were not altered by the irradiation treatment. However, minor changes were found in the molecular structures of 14 medicines under the reported 'safety dose' (10  kGy). The drying process before irradiation could decrease the chemical changes caused by γ-rays to 50%. The HPLC analysis of nine medicines revealed minor changes at a dose of 3  kGy. The findings in this study provide important information that may suggest the need for a re-evaluation of the reported safety dose. Therefore, further investigation may be warranted to insure the safety of γ-radiosterlization of Chinese medicines.

  8. Simultaneous Determination of Matrine and Tinidazole in Compound Lotion by RH-HPLC Method.

    PubMed

    Yin, Zhikui; Ma, Suying; Wang, Jincai; Shang, Xiaojun

    2013-01-01

    A simple, sensitive, and accurate RP-HPLC coupled with UV detector method was developed and validated for simultaneous determination of matrine and tinidazole in compound lotion. The chromatographic separation of the two compounds was carried out with a SinoChoom ODS-BP C18 column (5  μ m, 4.6 mm × 200 mm) analytical column, using a mobile phase consisting of 0.025 mol/L potassium dihydrogen phosphate (containing triethylamine 0.05%, v/v) and acetonitrile (80 : 20, v/v) at a flow rate of 1.0 mL/min. The detection was monitored at 210 and 310 nm for matrine and tinidazole, respectively. Total run time was 12 min, and the column was maintained at 25°C. The excipients in the compound lotion did not interfere with the drug peaks. The calibration curves of matrine and tinidazole were fairly linear over the concentration ranges of 10.0-100.0  μ g/mL (r = 0.9954) and 20.0-200.0  μ g/mL (r = 0.9968), respectively. The RSD of both the intraday and interday variations was below 1.5% for matrine and tinidazole. The proposed HPLC method was validated according to International Conference on Harmonisation and proved to be suitable for the simultaneous determination of matrine and tinidazole in compound lotion.

  9. 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

  10. A gradient based facile HPLC method for simultaneous estimation of antioxidants extracted from tea powder.

    PubMed

    Nanjegowda, Shankara H; Papanna, Manasa G; Achar, Raghu Ram; Rangappa, Kanchugarakoppal S; Mallu, Puttaswamappa; Swamy, Shivananju Nanjunda

    2016-05-01

    A new simple, rapid and precise RP-HPLC method was developed for the extraction and quantitative estimation of caffeine (C), (-)-epigallocatechin gallate (EGCG), (+)-catechin(Ct), (-)-epicatechin(EC), and (-)-epicatechin gallate (ECG) (collectively named as Tea Powder Bioactives TPBAs) extracted from tea powder using different ratios of ethanol: water. The simultaneous determination of TPBAs was performed using the UV spectrophotometric method which employs the absorbance at 205 nm (λmax of caffeine and polyphenols). This method is a gradient based HPLC method with a flow rate of 0.8 mL/min using Inertsil ODS 100 × 4.6 mm, 3 μm column with methanol and ammonium dihydrogen phosphate (pH-2.8) as mobile phase. The method was validated in terms of specificity, precision, linearity, accuracy, limit of quantification (LOQ), and limit of detection (LOD). The linearity of the proposed method was investigated for concentration ranging between 0.5-60 μg/mL with regression co-efficient, R(2) = 0.999-1.0. This method estimates all the TPBAs simultaneously with enhanced precision and linearity as per the ICH guidelines. Also, to confirm the individual TPBA, the antioxidant property of the each TPBA was analyzed which was commensurate with that of the previous reports. PMID:27407191

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

    PubMed

    Makino, Yukiko

    2012-03-01

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

  12. Quantitative analysis of citric acid/sodium hypophosphite modified cotton by HPLC and conductometric titration.

    PubMed

    Ye, Tao; Wang, Bijia; Liu, Jian; Chen, Jiangang; Yang, Yiqi

    2015-05-01

    Isocratic HPLC was used in conjunction with conductometric titration to quantitatively examine the modification of cotton cellulose by citric acid (CA)/sodium hypophosphite (SHP). CA/SHP had been extensively used as a green crosslinking agent for enhancement of cellulose and other carbohydrate polymers without in-depth understanding of the mechanisms. The current study investigated all identifiable secondary polycarboxylic acids from CA decomposition in the CA/SHP-cellulose system under various curing conditions. It was found that CA decomposition was more sensitive to temperature compared with the desirable esterification reaction. Two crosslinking mechanisms, namely ester crosslinking and SHP crosslinking were responsible for the observed improvement in crease resistance of CA/SHP treated cotton fabrics. An oligomer of citraconic acid (CCA) and/or itaconic acid (IA) was identified as a possible contributor to fabric yellowing. Finally, the crease resistance of fabrics correlated strongly with CA preservation in polyol-added CA/SHP crosslinking systems. The dosage of polyol should be held below an inflexion point to keep the undesirable competition against cellulose minimum. The combination of HPLC and conductometric titration was demonstrated to be useful in studying the CA/SHP-cellulose crosslinking system. The findings have implications for better application of CA/SHP in polysaccharide modifications in general.

  13. HPLC/PDA/ESI-MS evaluation of saffron (Crocus sativus L.) adulteration.

    PubMed

    Sabatino, Leonardo; Scordino, Monica; Gargano, Maria; Belligno, Adalgisa; Traulo, Pasqualino; Gagliano, Giacomo

    2011-12-01

    The present study evaluated the reliability of the ISO/TS 3632-2 UV-Vis spectrometric method for saffron classification, making experiments on saffron samples to which were added increasing concentrations of common saffron spice adulterants (safflower, marigold and turmeric). The results showed that the ISO/TS 3632-2 method is not able to detect addition of up to 10-20%, w/w, of saffron adulterants. For additions from 20 to 50%, w/w, of the three adulterants, saffron was classified in a wrong category; addition of higher than 50%, w/w, determined variations in the investigated parameters that did not allow identification of the product as "saffron". In all cases, the method did not permit the recognition of the nature of the adulterant. On the contrary, the specificity of the HPLC/PDA/MS technique allowed the unequivocal identification of adulterant characteristic marker molecules that could be recognized by the values of absorbance and mass. The selection of characteristic ions of each marker molecule has revealed concentrations of up to 5%, w/w, for safflower and marigold and up to 2% for turmeric. In addition, the high dyeing power of turmeric allowed the determination of 2%, w/w, addition using exclusively the HPLC/PDA technique.

  14. HPLC determination and clinical significance of serum prednisone in patients with nephrotic syndrome

    PubMed Central

    Chen, Chun-Mei; Xia, Yun-Cheng; Zhang, Xu-Guang; Peng, Can-Hui; Liu, Fu-You; Peng, You-Ming; Sun, Lin

    2014-01-01

    Aim: A rapid protocol is necessary to determine the serum concentrations of prednisone. Methods: The HP1100 high-performance liquid chromatographic (HPLC) system was employed. The HP Lichrosphere C8 column (250 mm × 4 mm, i.d., 5 μm particle size) was used. The mobile phase was methanol, tetrahydrofuran and water in the ratio 25:25:50. The flow rate was 1.0 ml/min. The sample was monitored by UV absorbance at 240 nm. Acetanilide was used as the internal standard, and methanol was added into the serum for depositing the protein. Results: The chromatography was effective and was not interfered with by the serum components. Good linearity was observed, within the range of 10-500 μg/L for prednisone, and the detection limit was 5 μg/L. The serum concentrations of prednisone between the nephrotic syndrome (NS) group and the control group were significantly different (P < 0.05), while there was no significant difference between the females and males of the NS group (P > 0.05). The serum ncentration of prednisone in the steroid-resistant group was lower than that in the steroid-sensitive group (P < 0.05). Conclusions: HPLC is a practical and reliable method to determine the serum concentration of prednisone with high accuracy, precision, linearity and repeatability. PMID:25664064

  15. Determination of trimebutine and desmethyl-trimebutine in human plasma by HPLC.

    PubMed

    Lavit, M; Saivin, S; Boudra, H; Michel, F; Martin, A; Cahiez, G; Labaune, J P; Chomard, J M; Houin, G

    2000-07-01

    A simple and sensitive HPLC method has been developed to measure trimebutine (CAS 39133-31-8, maleate: CAS 34140-59-5) and its main metabolite desmethyl-trimebutine in human plasma. The method was validated according to the Washington Consensus Conference on the Validation of Analytical Methods. It involved extraction of the plasma with n-hexane containing 2-pentanol, followed by reversed-phase HPLC using a Partisil ODS2 10 microns column and UV detection at 265 nm. The retention times of the internal standard (procaine), desmethyl-trimebutine and trimebutine were 2.4, 4.3 and 6.5 min, respectively. The standard curves were linear from 20 ng.ml-1 (limit of quantitation) to 5000 ng.ml-1 for both compounds. The coefficient of variation for all the criteria of validation were less than 15%. The extraction recoveries obtained for trimebutine and desmethyl-trimebutine were about 90%. Both compounds were very stable upon storage in plasma. The method was tested by measuring the plasma concentrations following oral administration to humans during a bioequivalence study and was shown suitable for pharmacokinetic studies. PMID:10965422

  16. Separation and quantification of water buffalo milk protein fractions and genetic variants by RP-HPLC.

    PubMed

    Bonfatti, Valentina; Giantin, Mery; Rostellato, Roberta; Dacasto, Mauro; Carnier, Paolo

    2013-01-15

    A RP-HPLC method, developed for the separation and quantification of the most common genetic variants of bovine milk proteins, was successfully applied to the analysis of water buffalo milk. All the most common buffalo casein and whey proteins fractions, as well as their genetic variants, were detected and separated simultaneously in 40 min. Purified buffalo proteins were used as calibration standards and a total of 536 individual milk samples were analysed for protein composition. α(S1)-, α(S2)-, βγ-, and κ-casein were 32.2%, 15.8%, 36.5%, and 15.5%, respectively, of total casein content, whereas content of β-Lactoglobulin was approximately 1.3 times as high as that of α-Lactalbumin. The existence of a polymorphism of κ-casein was demonstrated in Mediterranean water buffalo and α(S1)- and κ-casein genetic variants were successfully detected by RP-HPLC. PMID:23122071

  17. HPLC method development, validation, and impurity characterization of a potent antitumor indenoisoquinoline, LMP776 (NSC 725776).

    PubMed

    Wang, Jennie; Liu, Mingtao; Yang, Chun; Wu, Xiaogang; Wang, Euphemia; Liu, Paul

    2016-05-30

    An HPLC method for the assay of a DNA topoisomerase inhibitor, LMP776 (NSC 725776), has been developed and validated. The stress testing of LMP776 was carried out in accordance with International Conference on Harmonization (ICH) guidelines Q1A (R2) under acidic, alkaline, oxidative, thermolytic, and photolytic conditions. The separation of LMP776 from its impurities and degradation products was achieved within 40 min on a Supelco Discovery HS F5 column (150 mm × 4.6 mm i.d., 5 μm) with a gradient mobile phase comprising 38-80% acetonitrile in water, with 0.1% trifluoroacetic acid in both phases. LC/MS was used to obtain mass data for characterization of impurities and degradation products. One major impurity was isolated through chloroform extraction and identified by NMR. The proposed HPLC assay method was validated for specificity, linearity (concentration range 0.25-0.75 mg/mL, r = 0.9999), accuracy (recovery 98.6-100.4%), precision (RSD ≤ 1.4%), and sensitivity (LOD 0.13 μg/mL). The validated method was used in the stability study of the LMP776 drug substance in conformance with the ICH Q1A (R2) guideline. PMID:26970596

  18. 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.

  19. Antioxidant capacity and phenolic compounds of Lonicerae macranthoides by HPLC-DAD-QTOF-MS/MS.

    PubMed

    Hu, Xin; Chen, Lin; Shi, Shuyun; Cai, Ping; Liang, Xuejuan; Zhang, Shuihan

    2016-05-30

    Lonicerae macranthoides with strong antioxidant activity is commonly used in traditional Chinese medicine and folk tea/beverage. However, detailed information about its antioxidant activity and bioactive compounds is limited. Then at first, we comparatively evaluated total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of water extract, petroleum ether, ethyl acetate and n-butanol fractions of L. macranthoides. Ethyl acetate fraction exhibited the highest level of TPC (207.38 mg GAE/g DW), TFC (53.06 mg RE/g DW) and the best DPPH scavenge activity and reducing power. n-Butanol fraction showed the best ABTS(+) and O2(-) scavenging activities. Interestingly, water extract, ethyl acetate and n-butanol fractions showed stronger antioxidant activities than positive control, butylated hydroxytoluene (BHT). After that, thirty-one antioxidant phenolic compounds, including twenty-two phenolic acids and nine flavonoids, were screened by DPPH-HPLC experiment and then identified using HPLC-DAD-QTOF-MS/MS. It is noted that twenty-one compounds (1, 3-4, 6-17, 19, 23, 26, 28-29, and 31), as far as was known, were discovered from L. macranthoide for the first time, and eleven of them (3-4, 10-17, and 23) were reported in Lonicera species for the first time. Results indicated that L. macranthoides could serve as promising source of rich antioxidants in foods, beverages and medicines for health promotion. PMID:26970594

  20. A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells.

    PubMed

    Appala, Raju N; Chigurupati, Sridevi; Appala, Raju V V S S; Krishnan Selvarajan, Kesavanarayanan; Islam Mohammad, Jahidul

    2016-01-01

    A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1-20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells. PMID:27127683

  1. Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens.

    PubMed

    Biró, Krisztina; Solti, László; Barna-Vetró, Ildikó; Bagó, György; Glávits, Róbert; Szabó, Erzsébet; Fink-Gremmels, Johanna

    2002-04-01

    Ochratoxin A is a common feed contaminant, which may impair animal health and may lead to residues in edible tissues of slaughter animals. To simulate field conditions, broiler chicks were exposed to a total of 0.5 mg ochratoxin A per week for each of 4 weeks. Plasma toxin levels and tissue residues were measured by high-performance liquid chromatography (HPLC) and microplate enzyme-linked immunosorbent assay (ELISA). Results indicate an accumulation in plasma and wide distribution into all organs, with high levels in the liver and the kidney. Microscopical changes that could primarily be associated with toxin exposure were glomerulonephrosis, tubulonephrosis, focal tubular epithelial cell proliferation and multiple, adenoma-like structures in the renal parenchyma. The HPLC and ELISA methods gave similar results for both tissue distribution and depletion. Differences in absolute tissue toxin concentrations obtained by the two methods might be attributed to the different extraction and clean-up procedures, along with antibody specificity. The findings indicate that the dose applied causes subclinical tissue lesions and measurable tissue residues. PMID:12396358

  2. Safety of polyethylene terephthalate food containers evaluated by HPLC, migration test, and estimated daily intake.

    PubMed

    Park, H J; Lee, Y J; Kim, M R; Kim, K M

    2008-08-01

    A comparative high-pressure liquid chromatography (HPLC) analysis of monomers, terephthalic acid (TPA), isophthalic acid (IPA), and dimethyl terephthalate (DMT) from polyethylene terephthalate (PET) food containers was conducted. Monomer linearities and sensitivities were calibrated between established and novel HPLC analyses. Safety of PET containers was evaluated with newly established detection methods for TPA, IPA, and DMT. Migration of the 3 monomers into food simulants (water, 4% acetic acid, 20% alcohol, and n-heptane) from 56 PET containers collected from open markets was monitored. Migrated monomers were not detected over 0.1 ppm of detection limit. The corresponding estimated daily intake was measured to confirm the safety of these publicly available PET containers and to permit comparison to the specific migration limit of the European Union. The estimated daily intake of 3 monomers migrating from PET was 0.0384 mg/kg each. This represented only 0.6% of the European Union's specific migration limit, confirming the safety of the examined containers. PMID:19241591

  3. HPLC-DAD-ESI-MS Analysis of Flavonoids from Leaves of Different Cultivars of Sweet Osmanthus.

    PubMed

    Wang, Yiguang; Fu, Jianxin; Zhang, Chao; Zhao, Hongbo

    2016-09-14

    Osmanthus fragrans Lour. has traditionally been a popular ornamental plant in China. In this study, ethanol extracts of the leaves of four cultivar groups of O. fragrans were analyzed by high-performance liquid chromatography coupled with diode array detection (HPLC-DAD) and high-performance liquid chromatography with electrospray ionization and mass spectrometry (HPLC-ESI-MS). The results suggest that variation in flavonoids among O. fragrans cultivars is quantitative, rather than qualitative. Fifteen components were detected and separated, among which, the structures of 11 flavonoids and two coumarins were identified or tentatively identified. According to principal component analysis (PCA) and hierarchical cluster analysis (HCA) based on the abundance of these components (expressed as rutin equivalents), 22 selected cultivars were classified into four clusters. The seven cultivars from Cluster III ('Xiaoye Sugui', 'Boye Jingui', 'Wuyi Dangui', 'Yingye Dangui', 'Danzhuang', 'Foding Zhu', and 'Tianxiang Taige'), which are enriched in rutin and total flavonoids, and 'Sijigui' from Cluster II which contained the highest amounts of kaempferol glycosides and apigenin 7-O-glucoside, could be selected as potential pharmaceutical resources. However, the chemotaxonomy in this paper does not correlate with the distribution of the existing cultivar groups, demonstrating that the distribution of flavonoids in O. fragrans leaves does not provide an effective means of classification for O. fragrans cultivars based on flower color.

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

    PubMed

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

    2015-07-01

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

  5. A Simple HPLC-UV Method for the Determination of Glutathione in PC-12 Cells

    PubMed Central

    Appala, Raju N.; Appala, Raju V. V. S. S.

    2016-01-01

    A highly sensitive and simple HPLC-UV method was developed and validated for the assay of glutathione (GSH) in PC-12 cells. Glutathione is a major intracellular antioxidant having multiple biological effects, best known for its cytoprotective effects against cell damage from reactive oxygen species and toxic reactive metabolites and regulating the cellular redox homeostasis. Due to its own sulfhydryl (SH) group, GSH readily reacts with Ellman's reagent to form a stable dimer which allows for quantitative estimation of GSH in biological systems by UV detection. The separation was achieved using a C8 column with a mobile phase consisting of phosphate buffer adjusted to pH 2.5 (mobile phase A) and acetonitrile (mobile phase B), running in a segmented gradient manner at a flow rate of 0.8 mL/min, and UV detection was performed at 280 nm. The developed HPLC-UV method was validated with respect to precision, accuracy, robustness, and linearity within a range of 1–20 μg/mL. Limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.1 μg/mL, respectively. Furthermore, the method shows the applicability for monitoring the oxidative stress in PC-12 cells. PMID:27127683

  6. Production of L-(1-/sup 11/C)valine by HPLC resolution

    SciTech Connect

    Washburn, L.C.; Sun, T.T.; Byrd, B.L.; Callahan, A.P.

    1982-01-01

    Based on a recently developed analytical technique, preparative high-performance liquid chromatographic (HPLC) resolution of DL-(1-/sup 11/C)valine has been achieved. A conventional reverse-phase HPLC column and a chiral mobile phase (aqueous solution of L-proline, cupric acetate, and sodium acetate) were used. The copper can be removed from the L-valine fraction by precipitation as the sulfide, and final purification by cation-exchange chromatography yields L-(1-/sup 11/C)valine in a form that is acceptable for clinical positron tomographic studies. This purification method does not remove the L-proline introduced in the resolution process, but added L-proline did not affect the tissue distribution of L-(1-/sup 14/C)valine in rats. We have produced up to 60 mCi of L-(1-/sup 11/C)valine in an overall synthesis and resolution time of 50 min. This procedure should be adaptable to the rapid resolution of other C-/sup 11/-labeled amino acid racemates.

  7. Production of L-(1-/sup 11/C)valine by HPLC resolution

    SciTech Connect

    Washburn, L.C.; Sun, T.T.; Byrd, B.L.; Callahan, A.P.

    1982-01-01

    Based on a recently developed analytical technique, preparative high-performance liquid chromatographic (HPLC) resolution of DL-(1-/sup 11/C)valine has been achieved. A conventional reverse-phase HPLC column and a chiral mobile phase (aqueous solution of L-proline, cupric acetate, and sodium acetate) were used. The copper can be removed from the L-valine fraction by precipitation as the sulfide, and final purification by cation-exchange chromatography yields L-(1-/sup 11/C)valine in a form that is acceptable for clinical positron tomographic studies. This purification method does not remove the L-proline introduced in the resolution process, but added L-proline did not affect the tissue distribution of L-(1-/sup 14/C)valine in rats. We have produced up to 60 mCi of L-(1-/sup 11/C)valine in an overall synthesis and resolution time of 50 min. This procedure should be adapable to the rapid resolution of other C-11-labeled amino acid racemates.

  8. A method for the measurement of atmospheric HONO based on DNPH derivatization and HPLC analysis

    SciTech Connect

    Zhou, X.; Qiao, H.; Deng, G.; Civerolo, K.

    1999-10-15

    A simple measurement technique was developed for atmospheric HONO based on aqueous scrubbing using a coil sampler followed by 2,4-dinitrophenylhydrazine (DNPH) derivatization and high-performance liquid chromatographic (HPLC) analysis. Quantitative sampling efficiency was obtained using a 1 mM phosphate buffer, pH 7.0, as the scrubbing solution at a gas sampling flow rate of 2 L min{sup {minus}1} and a liquid flow rate of 0.24 mL min{sup {minus}1}. Derivation of the scrubbed nitrous acid by DNPH was fast and was completed within 5 min in a derivatization medium containing 300 {micro}M DNPH and 8 mM HCI at 45 C. The azide derivative was separated from DNPH reagent and carbonyl derivatives by reverse-phase HPLC and was detected with an UV detector at 309 nm. The detection limit is {le}5 pptv and may be lowered to 1 pptv with further DNPH purification. Interferences from NO, NO{sub 2} PAN, O{sub 3}, HNO{sub 3}, and HCHO were studied and found to be negligible. Ambient HONO concentration was measured simultaneously in downtown Albany, NY, by this method and by an ion chromatographic technique after sampling using a fritted bubbler. The results, from 70 pptv during the day to 1.7 ppbv in the early morning, were in very good agreement from the two techniques, within {+-} 20%.

  9. Optimization of a CUPRAC-Based HPLC Postcolumn Assay and Its Applications for Ginkgo biloba L. Extracts.

    PubMed

    Rimkiene, Laura; Ivanauskas, Liudas; Kubiliene, Asta; Vitkevicius, Konradas; Kiliuviene, Guoda; Jakstas, Valdas

    2015-01-01

    The aim of the present work was to improve and validate the HPLC-CUPRAC postcolumn method for the evaluation of active antioxidant markers from the acetonic extracts of Ginkgo biloba leaves. Improvement of the HPLC online assay was performed by evaluating the suitable loop temperature, the reaction loop length, and the impact of flow rate. Separation of the analytes was performed by the HPLC method on an ACE C18 analytical column using a gradient elution program. The separated antioxidant markers in the extracts reacted with copper(II)-neocuproine (Cu(II)-Nc) reagent in the postcolumn reaction coil. The reagent was reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate with a maximum absorption at 450 nm. Validation experiments confirmed sufficient precision, sensitivity, and effectiveness of the corresponding method, which could be used for further evaluations of active antioxidant compounds in similar plant materials.

  10. Optimization of a CUPRAC-Based HPLC Postcolumn Assay and Its Applications for Ginkgo biloba L. Extracts

    PubMed Central

    Rimkiene, Laura; Ivanauskas, Liudas; Kubiliene, Asta; Vitkevicius, Konradas; Kiliuviene, Guoda; Jakstas, Valdas

    2015-01-01

    The aim of the present work was to improve and validate the HPLC-CUPRAC postcolumn method for the evaluation of active antioxidant markers from the acetonic extracts of Ginkgo biloba leaves. Improvement of the HPLC online assay was performed by evaluating the suitable loop temperature, the reaction loop length, and the impact of flow rate. Separation of the analytes was performed by the HPLC method on an ACE C18 analytical column using a gradient elution program. The separated antioxidant markers in the extracts reacted with copper(II)-neocuproine (Cu(II)-Nc) reagent in the postcolumn reaction coil. The reagent was reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate with a maximum absorption at 450 nm. Validation experiments confirmed sufficient precision, sensitivity, and effectiveness of the corresponding method, which could be used for further evaluations of active antioxidant compounds in similar plant materials. PMID:26236538

  11. On-line (HPLC-NMR) and off-line phytochemical profiling of the Australian plant, Lasiopetalum macrophyllum.

    PubMed

    Timmers, Michael; Urban, Sylvia

    2011-11-01

    On-line (HPLC-NMR) and off-line (HPLC, NMR and MS) methodologies were used to profile the constituents present in the crude extract of Lasiopetalum macrophyllum. On-flow and stop-flow HPLC-NMR supported the presence of trans-tiliroside and permitted partial identification of cis-tiliroside and 4'-methoxy-trans-tiliroside. Off-line isolation led to the unequivocal identification of four flavanoid glycosides including a new structural derivative, 4'-methoxy-cis-tiliroside. This is the first report of flavonoid glycosides occurring in this plant genus. In addition, a number of structure revisions have been proposed for previously reported flavonoid glycosides that were incorrectly assigned.

  12. On-line (HPLC-NMR) and off-line phytochemical profiling of the Australian plant, Lasiopetalum macrophyllum.

    PubMed

    Timmers, Michael; Urban, Sylvia

    2012-05-01

    On-line (HPLC-NMR) and off-line (HPLC, NMR and MS) methodologies were used to profile the constituents present in the crude extract of Lasiopetalum macrophyllum. On-flow and stop-flow HPLC-NMR supported the presence of trans-tiliroside and permitted partial identification of cis-tiliroside and 4'-methoxy-trans-tiliroside. Off-line isolation led to the unequivocal identification of four flavanoid glycosides including a new structural derivative, 4'-methoxycis-tiliroside. This is the first report of flavonoid glycosides occurring in this plant genus. In addition, a number of structure revisions have been proposed for previously reported flavonoid glycosides that were incorrectly assigned.

  13. 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.

  14. Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts.

    PubMed

    Urakova, Irina N; Pozharitskaya, Olga N; Shikov, Alexander N; Kosman, Vera M; Makarov, Valery G

    2008-02-01

    Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.

  15. Single-run HPLC/ESI-LITMS profiling of ginsenosides in plant extracts and ginseng based products.

    PubMed

    Stavrianidi, Andrey; Rodin, Igor; Braun, Arkady; Stekolshchikova, Elena; Shpigun, Oleg

    2015-06-01

    A rapid single-run analytical approach suitable to achieve a comprehensive characterization of ginsenosides - the main bioactive compounds present in plant materials from Panax species and ginseng-based products - was developed. The method is based on high-performance liquid chromatography coupled with electrospray positive ionization linear ion trap mass spectrometry (HPLC/ESI-LITMS). The main ions in the ESI-LITMS spectra were attributed to molecular adducts with sodium and potassium and fragments corresponding to cleavage of the glycosidic bonds. The simplicity of the approach allows laborious sample preparation and sophisticated spectral information-dependent acquisition to be avoided, and provides an opportunity for rapid screening. The method may replace existing HPLC-DAD profiling approaches. The results of this study indicate that HPLC/ESI-LITMS is applicable for quality control purposes on processed products and allows the rapid and direct identification of ginsenosides in crude plant extracts.

  16. HPLC/APCI Mass Spectrometry of Saturated and Unsaturated Hydrocarbons by Using Hydrocarbon Solvents as the APCI Reagent and HPLC Mobile Phase

    NASA Astrophysics Data System (ADS)

    Gao, Jinshan; Owen, Benjamin C.; Borton, David J.; Jin, Zhicheng; Kenttämaa, Hilkka I.

    2012-05-01

    Saturated and unsaturated, linear, branched, and cyclic hydrocarbons, as well as polyaromatic and heteroaromatic hydrocarbons, were successfully ionized by atmospheric pressure chemical ionization (APCI) using small hydrocarbons as reagents in a linear quadrupole ion trap (LQIT) mass spectrometer. Pentane was proved to be the best reagent among the hydrocarbon reagents studied. This ionization method generated different types of abundant ions (i.e., [M + H]+, M+•, [M - H]+ and [M - 2H]+ •), with little or no fragmentation. The radical cations can be differentiated from the even-electron ions by using dimethyl disulfide, thus facilitating molecular weight (MW) determination. While some steroids and lignin monomer model compounds, such as androsterone and 4-hydroxy-3-methoxybenzaldehyde, also formed abundant M+• and [M + H]+ ions, this was not true for all of them. Analysis of two known mixtures as well as a base oil sample demonstrated that each component of the known mixtures could be observed and that a correct MW distribution was obtained for the base oil. The feasibility of using this ionization method on the chromatographic time scale was demonstrated by using high-performance liquid chromatography (HPLC) with hexane as the mobile phase (and APCI reagent) to separate an artificial mixture prior to mass spectrometric analysis.

  17. Genetic, epigenetic, and HPLC fingerprint differentiation between natural and ex situ populations of Rhodiola sachalinensis from Changbai Mountain, China.

    PubMed

    Zhao, Wei; Shi, Xiaozheng; Li, Jiangnan; Guo, Wei; Liu, Chengbai; Chen, Xia

    2014-01-01

    Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R2 = 0.98 for genetic and epigenetic distance; R2 = 0.90 for DNA methylation level and altitude; R2 = -0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis.

  18. Genetic, Epigenetic, and HPLC Fingerprint Differentiation between Natural and Ex Situ Populations of Rhodiola sachalinensis from Changbai Mountain, China

    PubMed Central

    Zhao, Wei; Shi, Xiaozheng; Li, Jiangnan; Guo, Wei; Liu, Chengbai; Chen, Xia

    2014-01-01

    Rhodiola sachalinensis is an endangered species with important medicinal value. We used inter-simple sequence repeat (ISSR) and methylation-sensitive amplified polymorphism (MSAP) markers to analyze genetic and epigenetic differentiation in different populations of R. sachalinensis, including three natural populations and an ex situ population. Chromatographic fingerprint was used to reveal HPLC fingerprint differentiation. According to our results, the ex situ population of R. sachalinensis has higher level genetic diversity and greater HPLC fingerprint variation than natural populations, but shows lower epigenetic diversity. Most genetic variation (54.88%) was found to be distributed within populations, and epigenetic variation was primarily distributed among populations (63.87%). UPGMA cluster analysis of ISSR and MSAP data showed identical results, with individuals from each given population grouping together. The results of UPGMA cluster analysis of HPLC fingerprint patterns was significantly different from results obtained from ISSR and MSAP data. Correlation analysis revealed close relationships among altitude, genetic structure, epigenetic structure, and HPLC fingerprint patterns (R2 = 0.98 for genetic and epigenetic distance; R2 = 0.90 for DNA methylation level and altitude; R2 = –0.95 for HPLC fingerprint and altitude). Taken together, our results indicate that ex situ population of R. sachalinensis show significantly different genetic and epigenetic population structures and HPLC fingerprint patterns. Along with other potential explanations, these findings suggest that the ex situ environmental factors caused by different altitude play an important role in keeping hereditary characteristic of R. sachalinensis. PMID:25386983

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

    SciTech Connect

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

    1994-07-01

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

  20. Synthesis and characterization of a multimode stationary phase: Congo red derivatized silica in nano-flow HPLC.

    PubMed

    Zhang, Yi; Zhang, Yan; Wang, Guan; Chen, Wujuan; He, Pingang; Wang, Qingjiang

    2016-02-01

    A novel Congo red (CR) derivatized silica stationary phase was prepared and packed into a fused silica capillary tube for nano-flow HPLC. A variety of analytes including poly-aromatic hydrocarbons, parabens, acids, sulfonamides, bases, and nucleosides were successfully separated using the CR. In comparison with commercial ODS columns, this new stationary phase has a different separation mechanism (hydrophobically-assisted ion-exchange), which was evident in the separation of benzoic acid derivatives and sulfonamides. The successful application of CR-bonded silica stationary phase in the HILIC and PALC modes demonstrates the effectiveness of this potential chromatographic material in nano flow HPLC.

  1. Evaluation of Pressure Stable Chip-to-Tube Fittings Enabling High-Speed Chip-HPLC with Mass Spectrometric Detection.

    PubMed

    Lotter, Carsten; Heiland, Josef J; Stein, Volkmar; Klimkait, Michael; Queisser, Marco; Belder, Detlev

    2016-08-01

    Appropriate chip-to-tube interfacing is an enabling technology for high-pressure and high-speed liquid chromatography on chip. For this purpose, various approaches, to connect pressure resistant glass chips with HPLC pumps working at pressures of up to 500 bar, were examined. Three side-port and one top-port connection approach were evaluated with regard to pressure stability and extra column band broadening. A clamp-based top-port approach enabled chip-HPLC-MS analysis of herbicides at the highest pressure and speed. PMID:27397738

  2. Synthesis and characterization of a multimode stationary phase: Congo red derivatized silica in nano-flow HPLC.

    PubMed

    Zhang, Yi; Zhang, Yan; Wang, Guan; Chen, Wujuan; He, Pingang; Wang, Qingjiang

    2016-02-01

    A novel Congo red (CR) derivatized silica stationary phase was prepared and packed into a fused silica capillary tube for nano-flow HPLC. A variety of analytes including poly-aromatic hydrocarbons, parabens, acids, sulfonamides, bases, and nucleosides were successfully separated using the CR. In comparison with commercial ODS columns, this new stationary phase has a different separation mechanism (hydrophobically-assisted ion-exchange), which was evident in the separation of benzoic acid derivatives and sulfonamides. The successful application of CR-bonded silica stationary phase in the HILIC and PALC modes demonstrates the effectiveness of this potential chromatographic material in nano flow HPLC. PMID:26646316

  3. Evaluation of Pressure Stable Chip-to-Tube Fittings Enabling High-Speed Chip-HPLC with Mass Spectrometric Detection.

    PubMed

    Lotter, Carsten; Heiland, Josef J; Stein, Volkmar; Klimkait, Michael; Queisser, Marco; Belder, Detlev

    2016-08-01

    Appropriate chip-to-tube interfacing is an enabling technology for high-pressure and high-speed liquid chromatography on chip. For this purpose, various approaches, to connect pressure resistant glass chips with HPLC pumps working at pressures of up to 500 bar, were examined. Three side-port and one top-port connection approach were evaluated with regard to pressure stability and extra column band broadening. A clamp-based top-port approach enabled chip-HPLC-MS analysis of herbicides at the highest pressure and speed.

  4. Application of a new HPLC-ICP-MS method for simultaneous determination of Al(3+) and aluminium fluoride complexes.

    PubMed

    Zioła-Frankowska, Anetta; Kuta, Jan; Frankowski, Marcin

    2015-10-01

    The paper presents the new HPLC-ICP-MS method used for conducting speciation analysis of aluminum as free Al(3+) and aluminum fluoride complexes during one analysis. In the study, 5% HNO3 was used as a derivative reagent in order to minimize the possibility of clogging the torch in ICP-MS. Using the new HPLC-ICP-MS method, speciation analysis of aluminum and aluminum fluoride complexes was conducted on the basis of model solutions and real samples (soil-water extracts and groundwater samples). The analysis in the presented analytical system lasts only 4 min. PMID:27441221

  5. Determination of telaprevir in plasma of HCV-infected patients by HPLC-UV.

    PubMed

    Tempestilli, Massimo; Milano, Elisa; D'Offizi, Gianpiero; Montalbano, Marzia; D'Avolio, Antonio; Gasperi, Tecla; Narciso, Pasquale; Ascenzi, Paolo; Pucillo, Leopoldo P

    2013-09-01

    Telaprevir is a direct acting antiviral agent, used with pegylated interferon and ribavirin for the management of chronic hepatitis C virus (HCV) genotype 1 infection, in patients not responding to therapy with pegylated interferon and ribavirin only. Although 75% of patients achieve a sustained virological response after treatment with telaprevir, adverse drug-drug interactions and undesirable events often occur. Therefore, telaprevir monitoring is pivotal to improve the management of HCV infection. Here, the first High-Performance Liquid Chromatography-Ultraviolet (HPLC-UV) method to quantify telaprevir in human plasma of HCV-genotype 1-infected patients is reported. The volume of the plasma sample was 700 μL. This method involved automated solid-phase extraction with Oasis HLB Cartridge 1 cc (divinylbenzene and N-vinylpyrrolidone). The extracted samples were reconstituted with 150 μL of 60/40 water/acetonitrile. Thirty microliters of these samples was injected into a HPLC-UV system, and the analytes were eluted on a X Terra(®) RP18 column (250 mm × 4.6 mm i.d.) with a particle size of 5 μm. The mobile phase (ammonium acetate buffer, 150 mM, pH 8.0, and methanol:acetonitrile 50:50) was delivered at 1.0 mL/min with linear gradient elution. The total run time for a single analysis was 16 min; telaprevir was detected by UV at 276 and 286 nm. The calibration curve was linear from 312.5 to 20,000 ng/mL (r(2) > 0.996). The absolute recovery of telaprevir ranged between 89 and 93% at concentrations of quality control samples of 800, 4,000, 8,000, and 16,000 ng/mL. Both precision and accuracy were always <15%. The HPLC-UV method reported here: (i) has been validated, (ii) is currently applied to monitor telaprevir in plasma of HCV-infected patients, and (iii) appears useful in a routine laboratory. ,

  6. Phytochemical Composition, Antioxidant Activity and HPLC Fingerprinting Profiles of Three Pyrola Species from Different Regions

    PubMed Central

    Wang, Dongmei; He, Fengyuan; Lv, Zhenjiang; Li, Dengwu

    2014-01-01

    The present study was performed to investigate the variation of phytochemical composition, antioxidant activity and High Performance Liquid Chromatography (HPLC) fingerprinting profiles of three Pyrola species. Thirteen samples (eight P. decorata, three P. calliantha and two P. renifolia) were collected from different regions in China. The tannin, hyperoside and quercetin contents of all samples were determined by reverse-phase HPLC and varied within the range 9.77–34.75, 0.34–2.16 and 0.062–0.147 mg/g dry weigh, respectively. Total flavonoid content was evaluated and varied within the range 16.22–37.82 mg/g dry weight. Antioxidant activity was determined by DPPH assay, with IC50 ranging from 7.96 to 50.33 µg/ml, ABTS•+ and FRAP assay, within the range 612.66–1021.05 and 219.64–398.12 µmol equiv. Trolox/g, respectively. These results revealed that there were significant variations in phytochemical profiles and antioxidant activity among all samples. Due to the higher phytochemical content and significant antioxidant activity, P. calliantha was selected as the most valuable species, and the P. calliantha sample from Left banner of Alxa even possessed the strongest antioxidant activity among all the thirteen samples. Futhermore, Emei Mountain was proved to be the most suitable region for producing P. decorata. Moreover, in order to further evaluate the diversities and quality of Pyrola, HPLC fingerprint analysis coupled with hierarchical cluster and discrimination analyses were introduced to establish a simple, rapid and effective method for accurate identification, classification and quality assessment of Pyrola. Thirteen samples were divided into three groups consistent with their morphological classification. Two types of discriminant functions were generated and the ratio of discrimination was 100%. This method can identify different species of Pyrola and the same species from different regions of origin. Also, it can be used to compare and

  7. HPLC of flavanones and chalcones in different species and clones of Salix.

    PubMed

    Krauze-Baranowska, Mirosława; Pobłocka-Olech, Loretta; Głód, Daniel; Wiwart, Marian; Zieliński, Jerzy; Migas, Piotr

    2013-01-01

    The SPE-HPLC method was developed to determine an isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6) in the bark of eight taxa (I-VIII) belonging to three species of the genus Salix and originating from a natural habitat or cultivated for pharmaceutical purposes. The chalcones were separated by HPLC under gradient elution with the concentration of ACN increasing from 20% to 50% in 0.1% aqueous H3PO, (tG 15 min). The content of both compounds was determined by an external standardization with the use of isoliquiritigenin (7) as a reference substance - a commercially available chalcone, and also isosalipurposide (5) and its derivative, 6"-O-p-coumaroyl ester (6). The latter compound was isolated from the bark of Salix daphnoides (IV) by a CC and semi-preparative HPLC and its structure was elucidated by MS and NMR spectra. It was stated that 6"-O-p-coumaroylisosalipurposide (6), in addition to isosalipurposide (5), is a characteristic flavonoid for the S. daphnoides species. Moreover, the presence of these two chalcones was confirmed in the bark of S. acutifolia (I). Differences were observed in the results obtained from a quantitative analysis due to the type of reference substance used. The content of chalcones was varied and dependent on the species selected for analysis, namely from 22.01/21.08 mg/g in S. daphnoides clone 1095 (III) to 2.47/2.44 mg/g in S. daphnoides (II), collected from a natural habitat. Isosalipurposide (5) was determined in all the investigated species and clones of Salix, besides a number of naringenin derivatives. Separation of all flavonoids: flavanones - naringenin (1), naringenin (+)-5-O-glucoside (2), (-)-5-O-glucoside (3), 7-O-glucoside (4) and chalcones (5 and 6) was performed under gradient elution with the same solvents and changes in ACN concentration from 2% to 37% (tG 60 min). The total amount of flavanones ranged from 4.69 mg/g in S. purpurea clone 1132 (VII) to 41.93 mg/g in S. purpuea (VIII) from Herbapol Wrocław.

  8. HPLC Quantification of Phenolic Acids from Vetiveria zizanioides (L.) Nash and Its Antioxidant and Antimicrobial Activity

    PubMed Central

    Prajna, Jha; Richa, Jindal; Dipjyoti, Chakraborty

    2013-01-01

    Extraction procedure was standardized and for the soluble, glycoside, and wall-bound fractions of phenolic acids from Vetiveria zizanioides. The water soluble alkaline extract which represents the cell wall-bound fraction contained the highest amount of phenolic acids (2.62 ± 1.2 μM/g fwt GA equivalents). Increased phenolic content in the cell wall indicates more lignin deposition which has an important role in plant defense and stress mitigation. Antioxidant property expressed as percentage TEAC value obtained by ABTS assay was correlated with the amount of phenolic acids and showed a Pearson's coefficient 0.988 (significant at 0.01 level). The compounds p-coumaric acid, p-dihydroxybenzoic acid, and ferulic acid were detected in the acidic extracts by HPLC analysis. The plant extracts exhibited considerable antimicrobial activity against tested bacterial and fungal strains. PMID:26555971

  9. Analysis of peptides using an integrated microchip HPLC-MS/MS system.

    SciTech Connect

    Kirby, Brian J.; Chirica, Gabriela S.; Reichmuth, David S.

    2004-06-01

    Hyphendated LC-MS techniques are quickly becoming the standard tool for protemic analyses. For large homogeneous samples, bulk processing methods and capillary injection and separation techniques are suitable. However, for analysis of small or heterogeneous samples, techniques that can manipulate picoliter samples without dilution are required or samples will be lost or corrupted; further, static nanospray-type flowrates are required to maximize SNR. Microchip-level integration of sample injection with separation and mass spectrometry allow small-volume analytes to be processed on chip and immediately injected without dilution for analysis. An on-chip HPLC was fabricated using in situ polymerization of both fixed and mobile polymer monoliths. Integration of the chip with a nanospray MS emitter enables identification of peptides by the use of tandem MS. The chip is capable of analyzing of very small sample volumes (< 200 pl) in short times (< 3 min).

  10. HPLC-DPPH Screening Method for Evaluation of Antioxidant Compounds in Corylus Species.

    PubMed

    Riethmüller, Eszter; Könczöl, Arpad; Szakál, Dorottya; Végh, Krisztina; Balogh, György T; Kéry, Ágnes

    2016-05-01

    Corylus avellana L., C. colurna L. and C. maxima Mill. have been used in traditional medicine for a long time for the treatment of various diseases, such as phlebitis, varicose veins, haemorrhoidal symptoms and eczema. Our previous studies indicated the presence of flavonol-3-O-glycosides, diarylheptanoids and caffeic acid derivatives in the bark and leaf extracts of the three species mentioned above. Comparison of in vitro DPPH scavenging activity of the Corylus extracts prepared with ethyl acetate and methanol to those of well-known antioxidant phenolics was performed. The contribution of certain compounds to the total antioxidant activity of the extracts was investigated by developing a HPLC method coupled to the DPPH radical scavenging assay.

  11. Pressurized liquid extraction and HPLC quantification of folic acid in fortified wheat flours.

    PubMed

    Araújo, Michel Mozeika; Marchioni, Eric; Villavicencio, Anna Lucia Casañas Haasis; Zhao, Minjie; Zimmermann, Pierre; El-Khoury, Etienne; Bergaentzle, Martine

    2012-08-01

    A pressurized liquid extraction (PLE) method using phosphate buffer as solvent was applied for folic acid (FA) extraction from fortified wheat flours and was compared to a standard solid-liquid extraction (SLE) method. Extracted FA was quantified by reverse phase high-performance liquid chromatography (RP-HPLC) hyphenated with a phenyl column and an absorption photometric detector (λ = 280 nm). Detection and quantification limits were 0.12 and 0.4 ng, respectively, corresponding to 0.06 and 0.2 μg g(-1) of analyzed wheat flour. Equivalent FA contents were found by both extraction methods, but a single PLE allowed a total recovery of FA content, whereas at least three successive SLEs were needed to achieve a total recovery of FA. The obtained results indicated that PLE is a rapid and efficient technique for FA extraction from fortified wheat flour.

  12. Screening and HPLC-Based Activity Profiling for New Antiprotozoal Leads from European Plants

    PubMed Central

    Zimmermann, Stefanie; Thomi, Semira; Kaiser, Marcel; Hamburger, Matthias; Adams, Michael

    2012-01-01

    Based on a survey of remedies used in Renaissance Europe to treat malaria, we prepared and screened a library of 254 extracts from 61 plants for antiplasmodial activity in vitro. HPLC-based activity profiling was performed for targeted identification of active constituents in extracts. One of the most remarkable results was the identification of onopordopicrin, a germacranolide sesquiterpene lactone isolated from Arctium nemorosum as a potent inhibitor of P. falciparum with an IC50 of 6.9 μM. It was tested similarly against Trypanosoma brucei rhodesiense, the parasite which causes African sleeping sickness. With an IC50 of 0.37 μM, onopordopicrin was one of the most potent natural products reported so far. Cytotoxicity was determined against rat myoblast L6 cells (IC50: 3.06). PMID:22396915

  13. Stress degradation studies on ezetimibe and development of a validated stability-indicating HPLC assay.

    PubMed

    Singh, Saranjit; Singh, Baljinder; Bahuguna, Rakesh; Wadhwa, Lalit; Saxena, Rahul

    2006-06-01

    Ezetimibe was subjected to different ICH prescribed stress conditions. Degradation was found to occur in hydrolytic and to some extent in photolytic conditions, while the drug was stable to oxidative and thermal stress. The drug was particularly labile under neutral and alkaline hydrolytic conditions. A stability-indicating HPLC method was developed for analysis of the drug in the presence of the degradation products. It involved a C-8 column and a mobile phase composed of ammonium acetate buffer (0.02 M, pH adjusted to 7.0 with ammonium hydroxide) and acetonitrile, which was pushed through the column in a gradient mode. The detection was carried out at 250 nm. The method was validated for linearity, range, precision, accuracy, specificity, selectivity and intermediate precision.

  14. Thiamin analysis in red wine by fluorescence reverse phase-HPLC.

    PubMed

    Liddicoat, Callum; Hucker, Barry; Liang, Hao; Vriesekoop, Frank

    2015-06-15

    The derivatization of thiamin vitamers to their respective thiochrome by ferricyanide to facilitate fluorescence detection following separation by HPLC provides a powerful analytical tool. However the polyphenolic compounds in red wine readily interact with ferricyanide, reducing the effectiveness of ferricyanide oxidation in the derivatization of thiamin. We describe a method to facilitate the removal of polyphenolic compounds that interfere with the ferricyanide derivatization of thiamin. Polyvinylpolypyrrolidone afforded the total removal of phenolic compounds from red wines and allowed a spike recovery of thiamin vitamers (101% for thiamin; 104% for TMP; and 100% for TDP) in a wide range of red wines. This research found that Merlot styles of red wine contained the highest concentration of total thiamin (29.01 ng/mL) while Pinot Noir wines contained the lowest total concentration (8.27 ng/mL).

  15. HPLC profiling of antimicrobial and antioxidant phyco sugars isolated from the South West coast of India.

    PubMed

    Kailas, Abhilash Parameswaran; Nair, Sukumaran Muraleedharan

    2016-10-20

    Phyco sugars isolated from the South West coast of India are commercially and therapeutically interesting due to their various biological activities. These sugars were isolated from six (three red and three green) seaweed species using water at 100°C followed by precipitation using ethanol. The physical, chemical and biological characteristics were explored with specific highlights onto the specific rotation, HPLC-RI sugar speciation, antioxidant and antimicrobial activities. The biological properties were evaluated based on multiple methods and standards, such that the actual nature of the sugar is understood. G. corticata var. cylindrica (31.66%) had the highest sugar content. All the sugars had levo rotatory specific rotation and interesting chemical characteristics. Sugars isolated had competitive biological activities and had the presence of seven monosaccharides and one disaccharide. The results highlighted the socio-economic importance of seaweeds which could be developed as a potential source of bioactive compounds in the upcoming future. PMID:27474603

  16. Laboratory Detection and Analysis of Organic Compounds in Rocks Using HPLC and XRD Methods

    NASA Technical Reports Server (NTRS)

    Dragoi, D.; Kanik, I.; Bar-Cohen, Y.; Sherrit, S.; Tsapin, A.; Kulleck, J.

    2004-01-01

    In this work we describe an analytical method for determining the presence of organic compounds in rocks, limestone, and other composite materials. Our preliminary laboratory experiments on different rocks/limestone show that the organic component in mineralogical matrices is a minor phase on order of hundreds of ppm and can be better detected using high precision liquid chromatography (HPLC). The matrix, which is the major phase, plays an important role in embedding and protecting the organic molecules from the harsh Martian environment. Some rocks bear significant amounts of amino acids therefore, it is possible to identify these phases using powder x-ray diffraction (XRD) by crystallizing the organic. The method of detection/analysis of organics, in particular amino acids, that have been associated with life will be shown in the next section.

  17. Resonance light scattering determination of 6-mercaptopurine coupled with HPLC technique

    NASA Astrophysics Data System (ADS)

    Li, Ai Ping; Peng, Jing Dong; Zhou, MingQiong; Zhang, Jin

    2016-02-01

    A simple, fast, costless, sensitive and selective method of resonance light scattering coupled with HPLC was established for the determination of 6-mercaptopurine in human urine sample. In a Britton-Robinson buffer solution of pH 5.5, the formation of coordination complex between 6-mercaptopurine and metal palladium (II) led to enhance the RLS intensity of the system. The RLS signal was detected by fluorescence detector at λex = λem = 315 nm. The analytical parameters were provided by the coupled system, the linear of 6-mercaptopurine response from 0.0615 to 2.40 μg L- 1 and the limit of detection (S/N = 3) was 0.05 μg L- 1. The presented method has been applied to determine 6-mercaptopurine in human urine samples which obtained satisfactory results. Moreover, the reaction mechanism and possible reasons for enhancement of RLS were fully discussed.

  18. [Identification of bufadienolides profiling in cinobufacino by HPLC-DAD-FT-ICR-MS method].

    PubMed

    Liu, Jun-Qiu; Si, Nan; Yang, Jian; Zhao, Hai-Yu; Bian, Bao-Lin; Wang, Hong-Jie

    2014-02-01

    Cinobufacino injection is a significant anti-tumor medicine for the treatment of various tumors in clinic, which was made from water extraction of the skin of Bufo bufo gargarizans. In present paper, HPLC-DAD-FT-ICR-MS method was used to identify the major bufadienolides in cinobufacino for the first time. Solid-phase extraction with dichloromethane and silica was used to enrich the total bufadienolides in cinobufacino. Based on the UV and high resolution MS/MS data, 33 bufadienolides were analyzed and characterized. Among them, eight compounds were identified by comparing with standard references unambiguously. This study elucidated the major bufadienolides in cinobufacino, which provided material foundation of cinobufacino and will be benefit for the further pharmacological research. PMID:24761616

  19. Aqueous acetonitrile extraction for pesticide residue analysis in agricultural products with HPLC-DAD.

    PubMed

    Watanabe, Eiki; Kobara, Yuso; Baba, Koji; Eun, Heesoo

    2014-07-01

    To reduce hazardous organic solvent consumption during sample preparation procedures as much as possible, an extraction method of smallest feasible sample volume (5g) using aqueous acetonitrile (MeCN) was developed to extract pesticide residues from agricultural samples prior to HPLC-DAD determination. Extraction with MeCN/water (1:1, v/v), and adjustment of the MeCN concentration by diluting with water after extraction recovered successfully most pesticides showing various physicochemical properties. The matrix effects of tested samples on the proposed method developed herein were generally negligibly-small. The average recoveries were in the range 70-120% for all pesticides with the coefficient of variation values below 20%. The reduction rate of organic solvents used for the proposed sample preparation method was up to approximately 60% compared with the Japanese authorised official method for pesticide residue analyses. These results demonstrate the feasibility of the proposed method for pesticides with diverse properties.

  20. Determination of some polyphenolic compounds from Allium species by HPLC-UV-MS.

    PubMed

    Parvu, Marcel; Toiu, Anca; Vlase, Laurian; Alina Parvu, Elena

    2010-09-01

    Five Allium species (Allium obliquum L., A. senescens L. subsp. montanum (Fries) Holub, A. schoenoprasum L. subsp. schoenoprasum, A. fistulosum L. and A. ursinum L.) were analysed in order to determine the presence of 19 polyphenolic compounds through an HPLC method coupled with UV and mass spectrometry detection. The pattern of phenol carboxylic acids indicates the presence of p-coumaric and ferulic acids in all species. Isoquercitrin was found in A. obliquum, A. schoenoprasum and A. fistulosum, rutin in A. senescens subsp. montanum and A. schoenoprasum, whereas quercitrin was found only in A. fistulosum, so they represent potential taxonomic markers that differentiate the four plants. Luteolin and apigenin were identified before and after hydrolysis only in A. obliquum. The amounts of all polyphenols were higher in hydrolysed samples, suggesting that these substances are present both as unbonded and bonded glycosides and/or esters. Our study showed large differences between the five Allium species, both qualitative and quantitative. PMID:20419559

  1. Pentaerythritol Tetranitrate (PETN) Surveillance by HPLC-MS: Instrumental Parameters Development

    SciTech Connect

    Harvey, C A; Meissner, R

    2005-11-04

    Surveillance of PETN Homologs in the stockpile here at LLNL is currently carried out by high performance liquid chromatography (HPLC) with ultra violet (UV) detection. Identification of unknown chromatographic peaks with this detection scheme is severely limited. The design agency is aware of the limitations of this methodology and ordered this study to develop instrumental parameters for the use of a currently owned mass spectrometer (MS) as the detection system. The resulting procedure would be a ''drop-in'' replacement for the current surveillance method (ERD04-524). The addition of quadrupole mass spectrometry provides qualitative identification of PETN and its homologs (Petrin, DiPEHN, TriPEON, and TetraPEDN) using a LLNL generated database, while providing mass clues to the identity of unknown chromatographic peaks.

  2. Extraction and identification of flavonoids from parsley extracts by HPLC analysis

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  3. Acrylamide in Romanian food using HPLC-UV and a health risk assessment.

    PubMed

    Oroian, Mircea; Amariei, Sonia; Gutt, Gheorghe

    2015-01-01

    The aim of this study was to investigate the level of acrylamide from coffee, potato chips and French fries in Romanian food. According to the European Food Safety Authority, coffee beans, potato chips and French fries have the highest levels of acrylamide. For this survey, 50 samples of coffee beans, 50 samples of potato chips and 25 samples of French fries were purchased from different producers from the Romanian market. Acrylamide levels have been quantified using high-performance liquid chromatography with a diode array detector (HPLC-DAD) method, using water as mobile phase. Health risk assessment was achieved by computing the average daily intake, hazard quotient, cumulative risk, carcinogenic risk and cancer risk. For coffee, potato chips and French fries, acrylamide was not shown to pose a health risk in Romanian food. PMID:25753750

  4. Determination of Lutein from Fruit and Vegetables Through an Alkaline Hydrolysis Extraction Method and HPLC Analysis.

    PubMed

    Fratianni, Alessandra; Mignogna, Rossella; Niro, Serena; Panfili, Gianfranco

    2015-12-01

    A simple and rapid analytical method for the determination of lutein content, successfully used for cereal matrices, was evaluated in fruit and vegetables. The method involved the determination of lutein after an alkaline hydrolysis of the sample matrix, followed by extraction with solvents and analysis by normal phase HPLC. The optimized method was simple, precise, and accurate and it was characterized by few steps that could prevent loss of lutein and its degradation. The optimized method was used to evaluate the lutein amounts in several fruit and vegetables. Rich sources of lutein were confirmed to be green vegetables such as parsley, spinach, chicory, chard, broccoli, courgette, and peas, even if in a range of variability. Taking into account the suggested reference values these vegetables can be stated as good sources of lutein. PMID:26540023

  5. Rapid reverse phase-HPLC assay of HMG-CoA reductase activity

    PubMed Central

    Mozzicafreddo, Matteo; Cuccioloni, Massimiliano; Eleuteri, Anna Maria; Angeletti, Mauro

    2010-01-01

    Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP+) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP+, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively. PMID:20418539

  6. Chemical fingerprinting of Gardenia jasminoides Ellis by HPLC-DAD-ESI-MS combined with chemometrics methods.

    PubMed

    Han, Yan; Wen, Jun; Zhou, Tingting; Fan, Guorong

    2015-12-01

    A fingerprint analysis method has been developed for characterization and discrimination of Gardenia jasminoides Ellis from different areas. The chemometrics methods including similarity evaluation, principal components analysis (PCA) and hierarchical clustering analysis (HCA) were introduced to identify more useful chemical markers for improving the quality control standard of dried ripe fruits of G. jasminoides Ellis. Then the selected chemical markers were analyzed by high performance liquid chromatography-diode array detection-electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) qualitatively and quantitatively. 23 characteristic peaks were assigned while 19 peaks of them were identified by comparing retention times, UV and MS spectra with authentic compounds or literature data. Moreover, 14 of them were determined quantitatively which could effectively evaluate the quality of G. jasminoides Ellis. This study was expected to provide comprehensive information for the quality evaluation of G. jasminoides Ellis, which would be a valuable reference for further study and development of this herb and related medicinal products. PMID:26041243

  7. Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD-ESI-MS.

    PubMed

    Bergonzi, M C; Righeschi, C; Isacchi, B; Bilia, A R

    2012-09-15

    A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C(18) column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins. The analytical method was validated and the quantification of active compounds, namely iridoids, was performed. Linearity, precision, repeatability, stability, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were also reported. This assay was successfully applied for qualitative and quantitative analysis of five commercial samples of G. jasminoides Ellis. PMID:23107748

  8. [Ion pair-HPLC of some aromatic amino- and hydroxycarboxylic acids].

    PubMed

    Jira, T; Beyrich, T; Reinhardt, K

    1988-06-01

    Various factors influencing the ion-pair-HPLC separation of some aromatic amino- and hydroxycarbon acids were described and discussed. Distinct effects of the ion pair formation of organic acids with quarternary alkylammonium salts (CTAB) are recognized on condition that the carboxylic group is not blocked by intramolecular H-bonding or partial betain structure. If the carboxylic group is unconnected the retention time increases depending on the pka of the acid with increasing pH if no complete ionization exists. In order to separate similar compounds at low retention time with distinct resolution an organic modifier of suitable strength and high selectivity have to be added to the mobile phase. PMID:3212029

  9. 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.

  10. Anthocyanins and antioxidant capacities of six Chilean berries by HPLC-HR-ESI-ToF-MS.

    PubMed

    Ramirez, Javier E; Zambrano, Ricardo; Sepúlveda, Beatriz; Kennelly, Edward J; Simirgiotis, Mario J

    2015-06-01

    The HPLC profiles of six fruits endemic of the VIII region of Chile were investigated using high resolution mass analysis (HR-ToF-ESI-MS). The anthocyanin fingerprints generated for the fruits were compared and the antioxidant capacities measured by the scavenging of the DPPH radical, the ferric reducing antioxidant power (FRAP), the superoxide anion scavenging activity assay (SA), and correlated with the inhibition of lipid peroxidation in human erythrocytes (LP) and total content of phenolics, flavonoids and anthocyanins measured by spectroscopic methods. Several anthocyanins were identified, including 3-O-glycosides derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin. Three phenolic acids (feruloyl-quinic acid, chlorogenic acid, and neochlorogenic acid) and five flavonols (hyperoside, isoquercitrin, quercetin, rutin, myricetin and isorhamnetin) were also identified. Calafate fruits showed the highest antioxidant activity. However, the highest LP activity was found for Chilean blueberries (>95%) followed by calafate fruits (91.27%) and luma (83.4%). PMID:25624212

  11. Screening of NOS activity and selectivity of newly synthesized acetamidines using RP-HPLC.

    PubMed

    Fantacuzzi, Marialuigia; Maccallini, Cristina; Di Matteo, Mauro; Ammazzalorso, Alessandra; Bruno, Isabella; De Filippis, Barbara; Giampietro, Letizia; Mollica, Adriano; Amoroso, Rosa

    2016-02-20

    Nitric Oxide Synthase (NOS) inhibitors could play a powerful role in inflammatory and neurodegenerative diseases. In this work, novel acetamidine derivatives of NOS were synthesized and the inhibitor activity was evalued. To screen the activity and selectivity, the l-citrulline residue, after the enzymatic NOS assay, was derivatized with o-phthaldialdehyde/N-acetyl cysteine (OPA/NAC) and then evaluated by RP-HPLC method with fluorescence detection. All compounds did not affect the activity of endothelial and neuronal isoforms, while nine of them possessed a percentage of iNOS activity at 10μM lower than 50%, and were selected for IC50 evaluation. Among them, a compound emerged as a very potent (IC50 of 53nM) and selective iNOS inhibitor. PMID:26689740

  12. Comparative HPLC-MSn analysis of canine and human meibomian lipidomes: many similarities, a few differences

    NASA Astrophysics Data System (ADS)

    Butovich, Igor A.; Borowiak, Anna M.; Eule, J. Corinna

    2011-06-01

    The aim of this study was to evaluate the lipidome of meibomian gland secretions in canines (cMGS) - a common pet and laboratory animal - and to compare it with that of human MGS (hMGS), to determine whether canines could be used as a valid experimental animal model in studies of the biochemistry and physiology of the human ocular surface in general, and of the Meibomian glands in particular. The MGS of both species were evaluated using HPLC in combination with atmospheric pressure chemical ionization ion trap mass spectrometry. The main lipid classes found in cMGS were very long chain cholesteryl esters, wax esters, (O-acyl)-omega-hydroxy fatty acids (OAHFA), and cholesteryl esters of OAHFA. The lipidomes of cMGS and hMGS were found to be qualitatively similar, which implies similar biosynthetic and biodegradation pathways in canines and humans. However, some quantitative differences between the two were observed.

  13. Determination of metrafenone in vegetables by matrix solid-phase dispersion and HPLC-UV method.

    PubMed

    Li, Jianjun; Li, Yangyang; Xu, Dongliang; Zhang, Jingyu; Wang, Yuxi; Luo, Chao

    2017-01-01

    A simple method for determination of metrafenone in vegetables by matrix solid-phase dispersion (MSPD) and HPLC was developed. All vegetable samples were extracted with dichloromethane, and then the extracts were directly separated on a reversed-phase column with isocratic elution without a cleanup step. The linearity of metrafenone was good with the concentration between 0.005 and 5mg/kg, and the limit of detection (LOD) of the metrafenone was 0.002mg/kg. The recoveries ranged from 86.5% to 104.8% with the relative standard deviations (RSDs) in the range of 2.1-7.9% (n=6). The results indicated that the method was simple, rapid, highly sensitive and suitable for the determination of metrafenone in vegetables. PMID:27507450

  14. Diphenylamine and derivatives as predictors of gunpowder age by means of HPLC and statistical models.

    PubMed

    López-López, María; Bravo, J Carlos; García-Ruiz, Carmen; Torre, Mercedes

    2013-01-15

    The gunpowder age is information of great importance that could help to establish safety regulations related to the propellants use and manipulation. In this work, a forced aging treatment (65°C for 120 days) was applied to four gunpowders stabilized with diphenylamine (DPA). The evolution of DPA and derivatives (N-nitroso-DPA, 2-nitro-DPA, 4-nitro-DPA, and 4-4'-dinitro-DPA) concentration during the days was leaded by High Performance Liquid Chromatography (HPLC). The variation with time of the peak areas of these compounds was used to construct different statistical models that could predict the gunpowders age. These models were validated using nitrocellulose-based gunpowders of known manufacture date. Models that best predicted the gunpowder age provided prediction errors lower than 6, 4, and 2 years for single-base gunpowders with dinitrotoluene (≥ 10%(m/m)), single-base gunpowders and double-base gunpowders, respectively.

  15. Determination of Lutein from Fruit and Vegetables Through an Alkaline Hydrolysis Extraction Method and HPLC Analysis.

    PubMed

    Fratianni, Alessandra; Mignogna, Rossella; Niro, Serena; Panfili, Gianfranco

    2015-12-01

    A simple and rapid analytical method for the determination of lutein content, successfully used for cereal matrices, was evaluated in fruit and vegetables. The method involved the determination of lutein after an alkaline hydrolysis of the sample matrix, followed by extraction with solvents and analysis by normal phase HPLC. The optimized method was simple, precise, and accurate and it was characterized by few steps that could prevent loss of lutein and its degradation. The optimized method was used to evaluate the lutein amounts in several fruit and vegetables. Rich sources of lutein were confirmed to be green vegetables such as parsley, spinach, chicory, chard, broccoli, courgette, and peas, even if in a range of variability. Taking into account the suggested reference values these vegetables can be stated as good sources of lutein.

  16. Reverse-phase HPLC of benzylpropionitrile dithiocarbamate complexes for the determination of priority pollutant metals

    SciTech Connect

    Park, Y.J.

    1990-01-01

    A new dithiocarbamate, benzylpropionitrile dithiocarbamate (BPDTC), has been synthesized for use in metal analysis. The HPLC behavior of metal chelates of BPDTC has been investigated for the simultaneous determination of antimony, cadmium, chromium, copper, mercury, nickel, lead, selenium, thallium, and zinc, all of which are on the Environmental Protection Agency's list of priority pollutant metals. Metals are extracted into dichloromethane as BPDTC chelates, and then separated on a C-18 column. Cobalt is added as an internal standard. The effects of pH and of three organic modifiers (methanol, acetonitrile, tetrahydrofuran) of the mobile phase on retention time have been investigated. Addition of dichloromethane to the mobile phase increases solubility and chelate stability, and improves the separation of metal BPDTC complexes. BPDTC is added to the aqueous mobile phase to reduce on-column dissociation of the complexes. Detection limits at 260 nm are in the range of 0.1 to 3 ppb using a 1 liter sample.

  17. Recent advances in nonpolar and polar organic monoliths for HPLC and CEC

    PubMed Central

    Jonnada, Murthy; Rathnasekara, Renuka; Rassi, Ziad El

    2015-01-01

    This article is aimed at providing a review of the progress made in the field over the period 2011 to present in order to expand in parts on two previous reviews (S. Karenga and Z. El Rassi, Electrophoresis, 2011, 32, 90-104; D. Gunasena and Z. El Rassi, Electrophoresis, 2012, 33, 251-261). In brief, this review article describes progress made in nonpolar and polar monoliths used in reversed phase HPLC and CEC (RPC/RP-CEC) and in hydrophilic interaction liquid chromatography/CEC (HILIC/HI-CEC), respectively. This article is by no means an exhaustive review of the literature; it is rather a survey of the recent progress made in the field with 69 references published on nonpolar and polar polymeric monoliths. PMID:25266173

  18. HPLC-DPPH Screening Method for Evaluation of Antioxidant Compounds in Corylus Species.

    PubMed

    Riethmüller, Eszter; Könczöl, Arpad; Szakál, Dorottya; Végh, Krisztina; Balogh, György T; Kéry, Ágnes

    2016-05-01

    Corylus avellana L., C. colurna L. and C. maxima Mill. have been used in traditional medicine for a long time for the treatment of various diseases, such as phlebitis, varicose veins, haemorrhoidal symptoms and eczema. Our previous studies indicated the presence of flavonol-3-O-glycosides, diarylheptanoids and caffeic acid derivatives in the bark and leaf extracts of the three species mentioned above. Comparison of in vitro DPPH scavenging activity of the Corylus extracts prepared with ethyl acetate and methanol to those of well-known antioxidant phenolics was performed. The contribution of certain compounds to the total antioxidant activity of the extracts was investigated by developing a HPLC method coupled to the DPPH radical scavenging assay. PMID:27319139

  19. HPLC-Profiles of Tocopherols, Sugars, and Organic Acids in Three Medicinal Plants Consumed as Infusions.

    PubMed

    Roriz, Custódio Lobo; Barros, Lillian; Carvalho, Ana Maria; Ferreira, Isabel C F R

    2014-01-01

    Pterospartum tridentatum (L.) Willk, Gomphrena globosa L., and Cymbopogon citratus (DC.) Stapf are medicinal plants that require a more detailed chemical characterization, given the importance of their consumption as infusions. Therefore, the individual profiles in tocopherols, free sugars, and organic acids were obtained by high performance liquid chromatography (HPLC) coupled to different detectors (fluorescence, refraction index, and photodiode array, resp.). C. citratus revealed the highest content of α-, and total tocopherols, glucose, sucrose, succinic, and ascorbic acids. P. tridentatum presented the highest fructose and total sugars content. Otherwise, G. globosa showed the highest organic acids concentration. As far as we know, this is the first study reporting the mentioned chemical compounds in G. globosa and C. citratus.

  20. HPLC-Profiles of Tocopherols, Sugars, and Organic Acids in Three Medicinal Plants Consumed as Infusions

    PubMed Central

    Roriz, Custódio Lobo; Barros, Lillian; Carvalho, Ana Maria; Ferreira, Isabel C. F. R.

    2014-01-01

    Pterospartum tridentatum (L.) Willk, Gomphrena globosa L., and Cymbopogon citratus (DC.) Stapf are medicinal plants that require a more detailed chemical characterization, given the importance of their consumption as infusions. Therefore, the individual profiles in tocopherols, free sugars, and organic acids were obtained by high performance liquid chromatography (HPLC) coupled to different detectors (fluorescence, refraction index, and photodiode array, resp.). C. citratus revealed the highest content of α-, and total tocopherols, glucose, sucrose, succinic, and ascorbic acids. P. tridentatum presented the highest fructose and total sugars content. Otherwise, G. globosa showed the highest organic acids concentration. As far as we know, this is the first study reporting the mentioned chemical compounds in G. globosa and C. citratus. PMID:26904623

  1. Quantification of Caffeoylquinic Acids in Coffee Brews by HPLC-DAD

    PubMed Central

    Moeenfard, Marzieh; Rocha, Lígia; Alves, Arminda

    2014-01-01

    The influence of different brewing conditions on the concentration of the main caffeoylquinic acids (3-caffeoylquinic acid (3-CQA), 4-caffeoylquinic acid (4-CQA), and 5-caffeoylquinic acid (5-CQA)) was investigated. For this purpose, twenty-four coffee brews were extracted and analyzed using HPLC-DAD at 325 nm. Our findings demonstrate the great impact of brewing techniques on the caffeoylquinic acids (CQAs) content. The major isomer was 3-CQA, accounting for about 50% of the total CQAs, followed by 5-CQA and 4-CQA, accounting for about 24–36% for each one. The total content of CQAs was in the range of 45.79 to 1662.01 mg/L, found in iced cappuccino and pod espresso, respectively. In conclusion, this study demonstrates that coffee brews, in particular those prepared using pressurized methods, can be considered as the potential sources of antioxidants such as CQAs. PMID:25587489

  2. On-site comprehensive analysis of explosives using HPLC-UV-PAED

    NASA Astrophysics Data System (ADS)

    Marple, Ronita L.; LaCourse, William R.

    2004-03-01

    High-performance liquid chromatography with ultra violet and photo-assisted electrochemical detection (HPLC-UV-PAED) has been developed for the sensitive and selective detection of explosives in ground water and soil extracts. Fractionation and preconcentration of explosives is accomplished with on-line solid phase extraction (SPE), which minimizes sample pretreatment and enables faster and more accurate on-site assessment of a contaminated site. Detection limits are equivalent or superior (i.e., <1 part-per-trillion for HMX) to those achieved using the Environmental Protection Agency (EPA) Method 8330. This approach is more broadly applicable, as it is capable of determining a wider range of organic nitro compounds. Soil samples are extracted using pressurized fluid extraction (PFE), and this technique is automatable, field-compatible, and environmentally friendly, adding to the overall efficiency of the methodology.

  3. Determination of polynuclear aromatic hydrocarbons in water by flotation enrichment and HPLC.

    PubMed

    Bo-Xing, X; Yu-Zhi, F

    1988-11-01

    A method of concentration and determination of several polynuclear aromatic hydrocarbons (PAHs) in water by flotation enrichment and HPLC is described. Triton X-100 was used as the foaming agent to extract the PAHs from water by passage of nitrogen. Reversed-phase liquid chromatography with coupled fluorescence detection was applied to separate and determine these PAHs. Various factors which may affect the recovery of PAHs from water, including pH, temperature and the concentration of Triton X-100 added, are discussed. This simplified method of concentrating PAHs from water has been applied to determine PAHs in water from Lake Erie. The method has practical value for the determination of PAHs in large volumes of water.

  4. Determination of active substances in binary mixture antiparasitic veterinary formulations by HPLC.

    PubMed

    Kulik, Anna; Szczotkowska, Aleksandra; Białecka, Wanda; Podolska, Marzena; Kwiatkowska-Puchniarz, Barbara; Mazurek, Aleksander

    2011-01-01

    The purpose of the study was to develop a simple, versatile HPLC method for the identification and quantification of praziquantel and ivermectin (in Equimax) or praziquantel and abamectin (in Abamitel Plus). A satisfactory separation was obtained using the Supelcosil LC-ABZ+ column in gradient system with a mobile phase A: acetonitrile / water in 40:60 ratio and phase B: acetonitrile. The UV detection was set at 245 nm. The correlation coefficient values (> or = 0,998) for all active substances confirmed that the calibration curves (peak area vs. concentration) are linear. The results of the quantification and the statistical evaluation confirmed that the method is accurate and precise. It can also be applied to confirm the identity of benzyl alcohol, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate in Abamitel Plus formulation. PMID:21796928

  5. Simultaneous RP-HPLC Estimation of Cefpodoxime Proxetil and Clavulanic Acid in Tablets

    PubMed Central

    Malathi, S.; Dubey, R. N.; Venkatnarayanan, R.

    2009-01-01

    A new, simple, precise, rapid and accurate RP-HPLC method has been developed for the simultaneous estimation of cefpodoxime proxetil and clavulanic acid from pharmaceutical dosage forms. The method was carried out on a Zorbax Eclipse XDB 5 μ C 18 (150×4.6 mm) column with a mobile phase consisting of acetonitrile:50 mM potassium dihydrogen phosphate buffer (pH 3.0, 70:30 v/v) at a flow rate of 1.0 ml/min. Detection was carried out at 228 nm. Aspirin was used as an internal standard. The retention time of clavulanic acid, cefpodoxime proxetil and aspirin was 4.43, 6.44 and 5.6 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification and solution stability. The proposed method can be used for the estimation of these drugs in combined dosage forms. PMID:20177474

  6. Acrylamide in Romanian food using HPLC-UV and a health risk assessment.

    PubMed

    Oroian, Mircea; Amariei, Sonia; Gutt, Gheorghe

    2015-01-01

    The aim of this study was to investigate the level of acrylamide from coffee, potato chips and French fries in Romanian food. According to the European Food Safety Authority, coffee beans, potato chips and French fries have the highest levels of acrylamide. For this survey, 50 samples of coffee beans, 50 samples of potato chips and 25 samples of French fries were purchased from different producers from the Romanian market. Acrylamide levels have been quantified using high-performance liquid chromatography with a diode array detector (HPLC-DAD) method, using water as mobile phase. Health risk assessment was achieved by computing the average daily intake, hazard quotient, cumulative risk, carcinogenic risk and cancer risk. For coffee, potato chips and French fries, acrylamide was not shown to pose a health risk in Romanian food.

  7. The Fourth SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE-4)

    NASA Technical Reports Server (NTRS)

    Hooker, Stanford B.; Thomas, Crystal S.; van Heukelem, Laurie; Schlueter, louise; Russ, Mary E.; Ras, Josephine; Claustre, Herve; Clementson, Lesley; Canuti, Elisabetta; Berthon, Jean-Francois; Perl, Jason; Normandeau, Claire; Cullen, John; Kienast, Markus; Pinckney, James L.

    2010-01-01

    Ten international laboratories specializing in the determination of marine pigment concentrations using high performance liquid chromatography (HPLC) were intercompared using in situ samples and a mixed pigment sample. Although prior Sea-viewing Wide Field-of-view Sensor (SeaWiFS) High Performance Liquid Chromatography (HPLC) Round-Robin Experiment (SeaHARRE) activities conducted in open-ocean waters covered a wide dynamic range in productivity, and some of the samples were collected in the coastal zone, none of the activities involved exclusively coastal samples. Consequently, SeaHARRE-4 was organized and executed as a strictly coastal activity and the field samples were collected from primarily eutrophic waters within the coastal zone of Denmark. The more restrictive perspective limited the dynamic range in chlorophyll concentration to approximately one and a half orders of magnitude (previous activities covered more than two orders of magnitude). The method intercomparisons were used for the following objectives: a) estimate the uncertainties in quantitating individual pigments and higher-order variables formed from sums and ratios; b) confirm if the chlorophyll a accuracy requirements for ocean color validation activities (approximately 25%, although 15% would allow for algorithm refinement) can be met in coastal waters; c) establish the reduction in uncertainties as a result of applying QA procedures; d) show the importance of establishing a properly defined referencing system in the computation of uncertainties; e) quantify the analytical benefits of performance metrics, and f) demonstrate the utility of a laboratory mix in understanding method performance. In addition, the remote sensing requirements for the in situ determination of total chlorophyll a were investigated to determine whether or not the average uncertainty for this measurement is being satisfied.

  8. [Quantitative determination of 5 active ingredients in different harvest periods of Ligusticum chuanxiong by HPLC].

    PubMed

    Liu, Jin-Liang; Fan, Qiao-Jia; Zheng, Shun-Lin; Tan, Jie; Zhou, Juan; Yuan, Ji-Chao; Yang, Shi-Min; Kong, Fan-Lei

    2014-05-01

    A simple and quick method is described for the determination of ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A and ligustilide in rhizomes of Ligusticum chuanxiong. The 5 active ingredients in the sample was extracted using 40% ethanol and analyzed by reversed-phase high performance liquid chromatography (HPLC). Chromatography separation was performed using Agilent 1100 series HPLC system with a Symmetry C18 column and gradient elution with a mixture of three solvents : solvent A, acetonitrile, solvent B, methanol and solvent C, 1% aqueous acetic acid, 0 min to 5 min A: B: C 20: 40: 40, 5 min to 30 min A: B: C 60 to 100 : 0 : 40 to 0. The effluent was monitored using a VWD detector set at 321 nm (0-4.3 min) and 275 nm (4.31-30 min). The flow rate was set at 1 mL x min(-1) and the injection volume was 10 microL. The column temperature was maintained at 35 degrees C. The calibration curve was linear (r > or = 0.99) over the tested ranges. The average recovery was 94.44%-103.1% (n = 6). The method has been successfully applied to the analysis in different harvest periods of L. chuanxiong samples. In this paper, single-factor randomized block design to study the 5 components content of L. chuanxiong on ten collecting stages. For the L. chuanxiong collected from April 15th to May 30rd, the content of 5 ingredients increased primarily, and then decreased. Determine the appropriate harvest time has important significance to the promotion of the quality of L. chuanxiong. PMID:25095378

  9. Biodistribution study of free and microencapsulated 6-methylcoumarin in Wistar rats by HPLC.

    PubMed

    Hernández, Aura Rocío; Ospina, Luis Fernando; Aragón, Diana Marcela

    2015-02-01

    A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6-methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C18 column was used with UV detection at 321 nm and a gradient system consisting of methanol-deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r(2)  > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4-12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC-free and 6MC-loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC-free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate. PMID:24854221

  10. Phosphorus speciation by coupled HPLC-ICPMS: low level determination of reduced phosphorus in natural materials

    NASA Astrophysics Data System (ADS)

    Atlas, Zachary; Pasek, Matthew; Sampson, Jacqueline

    2015-04-01

    Phosphorus is a geologically important minor element in the Earth's crust commonly found as relatively insoluble apatite. This constraint causes phosphorus to be a key limiting nutrient in biologic processes. Despite this, phosphorus plays a direct role in the formation of DNA, RNA and other cellular materials. Recent works suggest that since reduced phosphorus is considerably more soluble than oxidized phosphorus that it was integrally involved in the development of life on the early Earth and may continue to play a role in biologic productivity to this day. This work examines a new method for quantification and identification of reduced phosphorus as well as applications to the speciation of organo-phosphates separated by coupled HPLC - ICP-MS. We show that reduced phosphorus species (P1+, P3+ and P5+) are cleanly separated in the HPLC and coupled with the ICPMS reaction cell, using oxygen as a reaction gas to effectively convert elemental P to P-O. Analysis at M/Z= 47 producing lower background and flatter baseline chromatography than analyses performed at M/Z = 31. Results suggest very low detection limits (0.05 μM) for P species analyzed as P-O. Additionally we show that this technique has potential to speciate at least 5 other forms of phosphorus compounds. We verified the efficacy of method on numerous materials including leached Archean rocks, suburban retention pond waters, blood and urine samples and most samples show small but detectible levels of reduced phosphorus and or organo-phaospates. This finding in nearly all substances analyzed supports the assumption that the redox processing of phosphorus has played a significant role throughout the history of the Earth and it's presence in the present environment is nearly ubiquitous with the reduced oxidation state phosphorus compounds, phosphite and hypophosphite, potentially acting as significant constituents in the anaerobic environment.

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

    PubMed

    Porel, A; Haty, Sanjukta; Kundu, A

    2011-01-01

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

  12. Biodistribution study of free and microencapsulated 6-methylcoumarin in Wistar rats by HPLC.

    PubMed

    Hernández, Aura Rocío; Ospina, Luis Fernando; Aragón, Diana Marcela

    2015-02-01

    A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6-methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C18 column was used with UV detection at 321 nm and a gradient system consisting of methanol-deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r(2)  > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4-12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC-free and 6MC-loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC-free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate.

  13. Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC-ELSD.

    PubMed

    Ma, Chunmei; Sun, Zhen; Chen, Changbao; Zhang, Lili; Zhu, Shuhua

    2014-02-15

    A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was optimised for simultaneous determination of fructose, sorbitol, glucose and sucrose in fruits. The analysis was carried out on a Phenomenex Luna 5u NH₂ 100A column (250 mm × 4.60mm, 5 micron) with isocratic elution of acetonitrile:water (82.5:17.5, v/v). Drift tube temperature of the ELSD system was set to 82 °C and nitrogen flow rate was 2.0 L min⁻¹. The regression equation revealed good linear relationship (R = 0.9967-0.9989) within test ranges. The limits of detection (LOD) and quantification (LOQ) for four analytes (peach, apple, watermelon, and cherry fruits) were in the range of 0.07-0.27 and 0.22-0.91 mg L⁻¹, respectively. The proposed HPLC-ELSD method was validated for quantification of sugars in peach, apple, watermelon, and cherry fruits, and the results were satisfactory. The results showed that the contents of the four sugars varied among fruits. While fructose (5.79-104.01 mg g⁻¹) and glucose (9.25-99.62 mg g⁻¹) emerged as common sugars in the four fruits, sorbitol (8.70-19.13 mg g⁻¹) were only found in peach, apple and cherry fruits, and sucrose (15.82-106.39 mg g⁻¹) were in peach, apple and watermelon. There was not detectable sorbitol in watermelon and sucrose in cherry fruits, respectively.

  14. Determination of dithiothreitol in complex protein mixtures by HPLC-MS.

    PubMed

    Cindrić, Mario; Cepo, Tina; Marinc, Sabina; Paskvan, Ivan; Mijić, Ivana; Bindila, Laura; Peter-Katalinić, Jasna

    2008-10-01

    Dithiothreitol (DTT) and other reducing agents are typically used in refolding processes of recombinant human proteins during their purification from inclusion bodies. Due to its toxicity, it is essential to monitor the clearance of DTT throughout the analytical flow from the refolding phase to the final formulated product. Here we report a direct, simple, and fast liquid chromatography method using UV and tandem mass spectrometry (MS/MS) detection for DTT evaluation in complex protein mixtures. In aqueous solution DTT exists as an equilibrium mixture of the oxidized and the reduced form (H(2)DTT --> DTT(ox)) and the quantitation tools should therefore be applicable to both forms in a single step or in multiple steps. Oxidation of DTT with aqueous copper(II) nitrate trihydrate solution was introduced to determine a single oxidized compound, i. e. DTT(ox). Proteins and other components of high molecular masses were separated from DTT(ox) by ultrafiltration. Consequently, efficient separation of the DTT(ox )from other flow-through mixture components (sugars, polymers, salts, protein stabilizers) was achieved on an Atlantis dC(18) column. After chromatographic separation, DTT(ox) was selectively identified by UV absorbance at 285 nm or by selected reaction monitoring, measuring signal transition between m/z 151 --> 105. The method was validated in terms of specificity, accuracy, precision, linearity, and limit of quantification and detection. A reversed-phase HPLC separation method with atmospheric pressure chemical ionization and MS/MS detection in negative ion mode is highlighted as a viable alternative to currently existing quantitation methods involving DTT derivatization and HPLC fluorescence detection. The described approach offers simple, straightforward, selective, and high-throughput DTT quantitation in protein mixtures.

  15. HPLC-DAD-MS analysis of dyes identified in textiles from Mount Athos.

    PubMed

    Mantzouris, Dimitrios; Karapanagiotis, Ioannis; Valianou, Lemonia; Panayiotou, Costas

    2011-03-01

    Organic colorants contained in 30 textiles (16th to early 20th century) from the monastery of Simonos Petra (Mount Athos) have been investigated using high-performance liquid chromatography equipped with diode-array detection and mass spectrometry (HPLC-DAD-MS). The components of natural dyes identified in samples treated by the standard HCl dyestuff extraction method were: alizarin, apigenin, butein, carminic acid, chrysoeriol, dcII, dcIV, dcVII, ellagic acid, emodin, fisetin, flavokermesic acid, fustin, genistein, haematein derivative (Hae'), indigotin, indirubin, isoliquiritigenin, isorhamnetin, kaempferide, kaempferol, kermesic acid, luteolin, naringenin, purpurin, quercetin, rhamnazin, rhamnetin, sulfuretin, and type B and type C compounds (last two are markers for Caesalpinia trees). Early, semi-synthetic dyes, for example indigo carmine, fuchsin components, and rhodamine B were identified in objects dated late 19th to early 20th century. A dyestuff extraction method which involves use of TFA, instead of HCl, was applied to selected historical samples, showing that the mild method enables efficient extraction of weld (Reseda luteola L.) and dyer's broom (Genista tinctoria L.) glycosides. The marker compound (Hae') for logwood (Haematoxylum campechianum L.) identification after treatment with HCl was investigated by liquid chromatography coupled to mass spectrometry (LC-MS) in negative electrospray ionization (LC-MS-ESI(-)) mode. LC-MS in negative atmospheric pressure chemical ionization (LC-MS-APCI(-)) mode was used, probably for the first time, to investigate cochineal (Dactylopius coccus Costa) samples. Positive electrospray ionization (LC-MS-ESI(+)) mode was used for identification of fuchsin components. Detailed HPLC-DAD studies were performed on young fustic (Cotinus coggygria Scop.) and Persian berries (Rhamnus trees). PMID:21271239

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

    PubMed Central

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

    2011-01-01

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

  17. A Stability-Indicating RP-HPLC Assay Method for 5-Fluorouracil

    PubMed Central

    Sinha, V. R.; Kumar, R. V.; Bhinge, J. R.

    2009-01-01

    The present study describes the development of a validated RP-HPLC method for the determination of 5-fluorouracil in presence of its degradation products or other pharmaceutical excipients. Stress studies were performed on 5-fluorouracil and it was found that it degrades sufficiently in alkaline conditions, while negligible degradation was observed in acidic, neutral, oxidative and photolytic conditions. The peaks of the degradation products were not observed in the chromatogram due to the nonchromophoric nature of the degradation moiety formed. The separations were carried out on a C-18 reversed phase column (Phenomenex; Prodigy ODS3V, 250×4.6 mm, 5 μ) using 50mM KH2PO4 (pH, 5.0) as mobile phase at a flow rate of 1.2 ml/min and temperature of 30°. The wavelength of detection was 254 nm. A retention time of nearly 6 minutes was obtained. Analytical validation parameters such as specificity and selectivity, linearity, accuracy and precision were evaluated. The calibration curve for 5-fluorouracil was linear (r2=0.999±0.0005) from range of 10 μg/ml to 100 μg/ml. Relative standard deviation values for all the key parameters, was less than 2.0 %. The recovery of the drug after standard addition to the degraded sample was found to be 104.69%. Thus, the developed RP-HPLC method was found to be suitable for the determination of 5-fluorouracil in bulk as well as stability samples of the pharmaceutical dosage forms containing various excipients. PMID:20376215

  18. Single dose pharmacokinetics of atorvastatin oral formulations using a simple HPLC-UV method.

    PubMed

    Sohail, Muhammad; Ahmad, Mahmood; Minhas, Muhammad Usman

    2016-07-01

    The study was aimed to assess pharmacokinetics of atorvastatin (40 mg) in healthy fasted human subjects by a simple and inexpensive high performance liquid chromatography. Experimental design of the study was a randomized, two way, two periods, crossover study (single dose in fasted conditions). Eighteen (18) healthy male volunteers were enrolled according to FDA guidelines. The plasma samples were assayed using an isocratic High Performance Liquid Chromatography (HPLC) system of Agilent technologies USA consisted of an isocratic pump with column of Thermo Electron Corporation USA (ODS hypersil C(18) 4.6 mm x 250 mm), a UV-visible detector set at λ(max) 237 nm. Maximum plasma concentrations (C(max)) of atorvastatin (Mean ± SEM) for the reference product (A) found to be 13.739±0.210ng/ml & 13.374±0.145ng/ml for test product (B). T(max) values (Mean±SEM) of atorvastatin were 1.222 ±0.060 hours and 1.167±0.057 hours for reference and test products, respectively. The values of AUC(0-oo) (Mean ± SEM) for the reference (A) and test product (B) were 73.955 ± 1.715ng.h/ml and 77.773 ± 1.858ng. h/ml, respectively. Other pharmacokinetic parameters of both products were also determined. A statistical non-significant difference between pharmacokinetic parameters has been found and both brands of atorvastatin showed the same rate and extent of absorption in healthy fasted human volunteers after single dose. A simple and cost effective HPLC method was developed and applied. PMID:27393428

  19. Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns?

    PubMed

    Jones, Andrew; Pravadali-Cekic, Sercan; Dennis, Gary R; Shalliker, R Andrew

    2015-08-19

    Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL. A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance. Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column. PMID:26343427

  20. Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

    PubMed

    Kumar, G Pradeep; Srivastava, Vishal; Khandelwal, Kiran; Kumar, Rajesh; Hiriyanna, S G; Kumar, Ajay; Kumar, Pramod

    2016-09-01

    Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6 mm, 5 μm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 μg/mL, while the LOQ was 0.90 μg/mL. The calibration curves were linear in the range of 0.9-7.5 μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc. PMID:27563753

  1. Simple isocratic method for simultaneous determination of caffeine and catechins in tea products by HPLC.

    PubMed

    Fernando, Chamira Dilanka; Soysa, Preethi

    2016-01-01

    Tea is a popular beverage almost all over the world. Many studies show that tea consumption is closely associated with positive health impact. Most of the HPLC methods used for the determination of tea constituents include gradient elution systems which involve expensive instrumentation. The objective of this study was to develop a simple, rapid precise and low cost HPLC method for the separation and quantification of catechins and caffeine in tea (Camellia sinensis). The method utilizes a phenyl column (2.1 × 150 mm) with a UV-detector (280 nm) where excellent chromatographic separation of tea components i.e. gallic acid (GA), caffeine (Caf), epicatechin (EC) and (-)-epigallocatechin gallate (EGCG) was achieved. The isocratic elution system of acetonitrile, glacial acetic acid and deionized water (8:1:91 v/v/v) at a flow rate of 0.5 mL/min was involved. This method produced excellent accuracy and precision. Within run and between run precision was less than 7.5 %. The equations for calibration curves were y = 0.117 (±0.010)x + 0.173 (±0.024), y = 0.100 (±0.003)x + 0.045 (±0.019), y = 0.016 (±0.001)x + 0.006 (±0.004), y = 0.025 (±0.001)x-0.025 (±0.007) for GA, Caf, EC and EGCG respectively. The method validation parameters prove that the method is efficient, a simple and adequate for the quantitative determination of principal components in tea samples. PMID:27429880

  2. Analysis of alkylresorcinols in wheat germ oil and barley germ oil via HPLC and flourescence detection: Cochromatography with tocols

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alkylresorcinols are long chain phenolic compounds that have been reported to be localized in the outer layers of the kernels of wheat, rye, barley and other grains. A sensitive HPLC method with fluorescence detection was recently reported for the quantitative analysis of alkylresorcinols in cereal...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  4. AN IMPROVED HPLC-MS/MS METHOD FOR DETERMINATION OF ISOXAFLUTOLE (BALANCE) AND ITS METABOLITES IN SOILS AND FORAGE PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An analytical method using turbo-spray and heat-nebulizer high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for the analysis of isoxaflutole (IXF) and its two metabolites, diketonitrile (DKN) and the benzoic acid metabolite (BA), at sub 'g/kg levels in soil a...

  5. 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.

  6. Anion Exchange HPLC Isolation of High-Density Lipoprotein (HDL) and On-Line Estimation of Proinflammatory HDL

    PubMed Central

    Ji, Xiang; Xu, Hao; Zhang, Hao; Hillery, Cheryl A.; Gao, Hai-qing; Pritchard, Kirkwood A.

    2014-01-01

    Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL. PMID:24609013

  7. HPLC Determination and MS Confirmation of Malachite Green, Gentian Violet, and Their Leuco Metabolites in Catfish Muscle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Residues of malachite green (MG), gentian violet (GV), and their leuco metabolites in catfish muscle were individually determined by HPLC using visible and fluorescence detectors. This detection scheme obviated a PbO2 column that converts leuco forms to chromatic forms for visible detection, thus el...

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  9. 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…

  10. FISH BILIARY POLYCYCLIC AROMATIC HYDROCARBON METABOLITES ESTIMATED BY FIXED-WAVELENGTH FLUORESCENCE: COMPARISON WITH HPLC-FLUORESCENT DETECTION

    EPA Science Inventory

    Fixed wavelength fluorescence (FF) was compared to high-performance liquid chromatography with fluorescence detection (HPLC-F) as an estimation of polycyclic aromatic hydrocarbon (PAH) exposure to fish. Two excitation/emission wavelength pairs were used to measure naphthalene- an...

  11. [Study on limit detection of flavones in diterpene ginkgolides meglumine injection materials by LC-MS and HPLC-DAD].

    PubMed

    Bi, Sen; Li, Yan-jing; Huang, Wen-zhe; Kang, Dan-yu; Ding, Gang; Xiao, Wei

    2015-08-01

    Limit test of flavones in diterpene ginkgolides meglumine injection materials by UV-Vis and HPLC-DAD method was studied in this essay. The HPLC-DAD method has lower LOD (about 1% of the UV-Vis), that is, the sensitivity is higher than UV-Vis method. Through the analysis of the kinds of flavonoids ingredients in the samples by LC-MS, the three compounds with highest contents are kaempferol, quercetin and isorhamnetin. Kaempferol, quercetin and isorhamnetin were chosen as reference compounds for HPLC analysis, and the HPLC separation analysis was carried on an Agilent Eclipse plus C18 column (4.6 mm x 250 mm, 5 μm) with methanol and water containing 0.4% phosphoric acid (50: 50) as mobile phase, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 360 nm. This method has good specificity, precision and reproducibility. The LODs of quercetin, kaempferide and isorhamnetin were 27.6, 22.3, 29.5 μg x L(-1). The average recovery was 87.9% (RSD 3.3%), 91.7% (RSD 3.1%), 88.3 (RSD 1.3%) for quercetin, kaempferide and isorhamnetin, respectively. Based on the 10 batches of sample results and sensitivity of different HPLC, the content of total flavonoids ingredients of diterpene ginkgolides meglumine injection materials was limited no more than 2 x 10(-5). This method is simple, quick and has good maneuverability, and could be used to the limit test of flavonoids in the diterpene ginkgolides meglumine injection materials.

  12. Retention of ionizable compounds on HPLC. 4. Mobile-phase pH measurement in methanol/water

    PubMed

    Canals; Portal; Bosch; Roses

    2000-04-15

    The different procedures used in HPLC to measure the pH of a mobile phase are evaluated in terms of the rigorous IUPAC definition of pH. The three procedures evaluated are as follows: measurement of the pH of the aqueous HPLC buffer before mixing it with the organic modifier, measurement of the pH of the HPLC buffer after mixing it with the organic modifier using a pH electrode system calibrated with aqueous buffers, and measurement of the pH of the HPLC buffer after mixing it with the organic modifier but calibrating the electrode system with reference buffers prepared in the same mixed solvent used as mobile phase. Following IUPAC definitions and recommendations, the three pH values can be related with the pH scales: w(w)pH, s(w)pH, and s(s)pH, respectively. The relationships between these three pH scales are also presented. The retention of several compounds with acid/base behavior in a C-18 and a polymeric column with buffered methanol/water as mobile phase is related to the mobile phase pH value measured in the three pH scales. It is demonstrated that the s(w)pH and s(s)pH scales give better relationships than the w(w)pH scale (pH measured in the aqueous buffer before mixing it with the organic modifier), commonly used on HPLC. The s(w)pH scale is specially recommended because of its simplicity of measurement: the pH is measured after mixing the aqueous buffer with the organic modifier, but the pH calibration is performed with the common aqueous reference buffers.

  13. [Study on limit detection of flavones in diterpene ginkgolides meglumine injection materials by LC-MS and HPLC-DAD].

    PubMed

    Bi, Sen; Li, Yan-jing; Huang, Wen-zhe; Kang, Dan-yu; Ding, Gang; Xiao, Wei

    2015-08-01

    Limit test of flavones in diterpene ginkgolides meglumine injection materials by UV-Vis and HPLC-DAD method was studied in this essay. The HPLC-DAD method has lower LOD (about 1% of the UV-Vis), that is, the sensitivity is higher than UV-Vis method. Through the analysis of the kinds of flavonoids ingredients in the samples by LC-MS, the three compounds with highest contents are kaempferol, quercetin and isorhamnetin. Kaempferol, quercetin and isorhamnetin were chosen as reference compounds for HPLC analysis, and the HPLC separation analysis was carried on an Agilent Eclipse plus C18 column (4.6 mm x 250 mm, 5 μm) with methanol and water containing 0.4% phosphoric acid (50: 50) as mobile phase, and the flow rate was 1.0 mL x min(-1). The detection wavelength was set at 360 nm. This method has good specificity, precision and reproducibility. The LODs of quercetin, kaempferide and isorhamnetin were 27.6, 22.3, 29.5 μg x L(-1). The average recovery was 87.9% (RSD 3.3%), 91.7% (RSD 3.1%), 88.3 (RSD 1.3%) for quercetin, kaempferide and isorhamnetin, respectively. Based on the 10 batches of sample results and sensitivity of different HPLC, the content of total flavonoids ingredients of diterpene ginkgolides meglumine injection materials was limited no more than 2 x 10(-5). This method is simple, quick and has good maneuverability, and could be used to the limit test of flavonoids in the diterpene ginkgolides meglumine injection materials. PMID:26790294

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

    NASA Astrophysics Data System (ADS)

    Karsten, Ulf; Escoubeyrou, Karine; Charles, François

    2009-09-01

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

  15. Carbon-Isotopic Analysis of Individual Pigments by HPLC-Moving Wire-IRMS

    NASA Astrophysics Data System (ADS)

    Sessions, A. L.; Keely, B. J.; Hayes, J. M.

    2003-12-01

    We have developed a method for directly analyzing the carbon isotope ratios of individual pigments, including chlorophyll (chl) and its derivatives, by coupling a high-performance liquid chromatograph (HPLC) to an isotope-ratio mass spectrometer (IRMS) via a novel `moving-wire' interface. Pigments were separated on a reversed-phase C18 column, using a binary gradient modified from Airs et al. (2001, J. Chrom. A 917, 167-177). The HPLC effluent was dried onto a continuously-spooling nickel wire, and the involatile sample residue was combusted to CO2 and transferred to the IRMS for isotopic analysis. Replicate analyses of a standard solution yield precision for delta13C of better than 0.2‰ for injections containing ~5 μ g of chl-a. A five-fold improvement in sensitivity should be attainable using capillary HPLC to further reduce solvent volumes. The biomarker potential of tetrapyrrole pigments, combined with the geochemical information recorded by isotopic compositions, makes this combination a potent tool for biogeochemical studies. As a demonstration, we analyzed chlorophyll degradation products in sediments from a lake and a salina. First, compounds derived from bacteriochlorophylls (bchl)-c and -d were extracted from sediment cores taken at Kirisjes Lake (Larsmann Hills, Antarctica). These pigments are products of green sulfur bacteria and indicate the presence of an anoxic photic zone. The δ 13C values of bchl-related compounds are near -25‰ . Using published fractionations for Chlorobium species to extrapolate, dissolved CO2 in Kirisjes Lake probably had a δ 13C value of -12 to -21‰ and was strongly influenced by the recycling of organic carbon, possibly including methane. Second, compounds derived from chl-a, bchl-c, and bchl-d were isolated from sediments taken below a living microbial mat in the hypersaline les Salines de la Trinital (South Catalonia, Spain). The sediments contain visible remnants of past microbial mats and pigment distributions

  16. 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

  17. HPLC-DAD phenolic profile, cytotoxic and anti-kinetoplastidae activity of Melissa officinalis.

    PubMed

    Cunha, Francisco; Tintino, Saulo R; Figueredo, Fernando; Barros, Luiz; Duarte, Antonia E; Vega Gomez, Maria Celeste; Coronel, Cathia Cecilia; Rolón, Mírian; Leite, Nadghia; Sobral-Souza, Celestina E; Brito, S V; Waczuc, Emily Pansera; Boligon, Aline Augusti; Athayde, Margareth; Kamdem, Jean Paul; Coutinho, Henrique Douglas Melo; Franco, Jéferson

    2016-09-01

    Context Melissa officinalis subsp. inodora Bornm. (Lamiaceae) has been used since ancient times in folk medicine against various diseases, but it has not been investigated against protozoa. Objective To evaluate the activities of M. officinalis against Leishmania braziliensis, Leishmania infantum and Trypanosoma cruzi as well as its cytotoxicity in fibroblast cell line. Materials and methods The fresh leaves were chopped into 1 cm(2) pieces, washed and macerated with 99.9% of ethanol for 72 h at room temperature. Antiparasitic activity of M. officinalis was accessed by direct counting of cells after serial dilution, while the cytotoxicity of M. officinalis was evaluated in fibroblast cell line (NCTC929) by measuring the reduction of resazurin. The test duration was 24 h. High-performance liquid chromatography (HPLC) was used to characterise the extract. Results The extract at concentrations of 250 and 125 μg/mL inhibited 80.39 and 54.27% of promastigote (LC50  value = 105.78 μg/mL) form of L. infantum, 80.59 and 68.61% of L. brasiliensis (LC50 value  = 110.69 μg/mL) and against epimastigote (LC50 value  = 245.23 μg/mL) forms of T. cruzi with an inhibition of 54.45 and 22.26%, respectively, was observed. The maximum toxicity was noted at 500 μg/mL with 95.41% (LC50  value = 141.01 μg/mL). The HPLC analysis identified caffeic acid and rutin as the major compounds. Discussion The inhibition of the parasites is considered clinically relevant (< 500 μg/mL). Rutin and caffeic acids may be responsible for the antiprotozoal effect of the extract. Conclusion The ethanol extract of M. officinalis can be considered a potential alternative source of natural products with antileishmania and antitrypanosoma activities.

  18. 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

  19. Improved quantification of pyrogenic carbon in soils and sediments by a HPLC-DAD method

    NASA Astrophysics Data System (ADS)

    Wiedemeier, D. B.; Hilf, M. D.; Smittenberg, R. H.; Schmidt, M. W. I.

    2012-04-01

    Fire-derived (pyrogenic) carbon (PyC) is produced by the incomplete combustion of biomass, for example during wildfires. It can persist in the environment for a long time due to its relative resistance against biological and chemical breakdown. Its accurate quantification in soils and sediments is of great interest because the slow turn-over of PyC has implications for the global carbon cycle and carbon budget calculations. Moreover, PyC in pedological and sedimentological records can be used to reconstruct wildfire history or to investigate historical periods like the industrialization. A whole suite of PyC quantification methods exists because PyC is not a defined chemical structure but rather a continuum of thermally altered biomass. The benzene polycarboxylic acids (BPCA) analysis is a molecular marker method that was shown to give conservative estimates of PyC quantity in soils. In addition, it yields qualitative information about the degree of aromaticity and condensation of PyC. The commonly used BPCA method consists in digesting samples with nitric acid that breaks down the PyC into a suite of BPCAs, which are cleaned, derivatized and finally analyzed by gas chromatography-flame ionization detection (GC-FID). Here, we present a modified BPCA method for soils and sediments that uses a high performance liquid chromatography system coupled to diode array detection (HPLC-DAD). We demonstrate that this method greatly enhances the reproducibility of PyC quantification in soil and sediment samples while significantly reducing analysis time. Moreover, much less sample material is needed for precise PyC quantification and we show that the HPLC-DAD method yields consistently higher PyC contents than the GC-FID method. Additionally, the modified method also facilitates δ13C and 14C measurements of the PyC fraction in these complex matrix samples. The isotopic information further improves the assessment of PyC budgets in the environment and the reconstruction of past

  20. 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

  1. 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

  2. Determination of commonly used polar herbicides in agricultural drainage waters in Australia by HPLC.

    PubMed

    Tran, Anh T K; Hyne, Ross V; Doble, Philip

    2007-03-01

    The present study describes the application of different extraction techniques for the preconcentration of ten commonly found acidic and non-acidic polar herbicides (2,4-D, atrazine, bensulfuron-methyl, clomazone, dicamba, diuron, MCPA, metolachlor, simazine and triclopyr) in the aqueous environment. Liquid-liquid extraction (LLE) with dichloromethane, solid-phase extraction (SPE) using Oasis HLB cartridges or SBD-XC Empore disks were compared for extraction efficiency of these herbicides in different matrices, especially water samples from contaminated agricultural drainage water containing high concentrations of particulate matter. Herbicides were separated and quantified by high performance liquid chromatography (HPLC) with an ultraviolet detector. SPE using SDB-XC Empore disks was applied to determine target herbicides in the Murrumbidgee Irrigation Area (NSW, Australia) during a two-week survey from October 2005 to November 2005. The daily aqueous concentrations of herbicides from 24-h composite samples detected at two sites increased after run-off from a storm event and were in the range of: 0.1-17.8 microg l(-1), < 0.1-0.9 microg l(-1) and 0.2-17.8 microg l(-1) at site 1; < 0.1-3.5 microg l(-1), < 0.1-0.2 microg l(-1) and < 0.2-3.2 microg l(-1) at site 2 for simazine, atrazine and diuron, respectively.

  3. Octadecylamine-modified poly (glycidylmethacrylate-divinylbenzene) stationary phase for HPLC determination of N-nitrosamines.

    PubMed

    Liu, Junwei; Wang, Yong; Li, Weixia; Zhang, Peimin; Wang, Nani; Wu, Shuchao; Zhu, Yan

    2016-11-01

    Poly (glycidylmethacrylate-divinylbenzene) (Poly (GMA-DVB)) microspheres were prepared by the two-staged swelling and polymerization method, and modified with octadecylamine (ODA) to obtain ODA-poly (GMA-DVB) stationary phase for HPLC. The new material was characterized by scanning electron microscope, nitrogen adsorption-desorption measurement, Fourier transform infrared spectrum, elemental analysis and thermogravimetric analysis. The results showed that poly (GMA-DVB) microspheres had good monodispersity, porosity and ball shapes. The diameters and specific surface area of the microspheres were about 6µm and 396m(2)g(-1), respectively. ODA-poly (GMA-DVB) stationary phase had good thermal stability. Furthermore, the chromatographic performance of the stationary phase was illustrated by separating n-alkylbenzenes, mono-substituted benzenes and N-nitrosamines. Auxiliary quantum chemistry calculation was also carried out to evaluate the interaction mechanism. According to the evaluation, ODA-poly (GMA-DVB) stationary phase exhibited good hydrophobicity and hydrophobic selectivity, strong stereo-selectivity, polar interaction and π-π interaction. The multi-interaction mechanisms could very likely guarantee its excellent chromatographic performance for the analysis of complex samples. Finally, the column was successfully applied in the determination of N-nitrosamines in pickles sample. PMID:27591617

  4. HPLC analysis of phenolics compounds and antioxidant capacity of leaves of Vitex megapotamica (Sprengel) Moldenke.

    PubMed

    de Brum, Thiele Faccim; Zadra, Marina; Piana, Mariana; Boligon, Aline Augusti; Fröhlich, Janaina Kieling; de Freitas, Robson Borba; Stefanello, Sílvio Terra; Froeder, Amanda Luana Forbrig; Belke, Bianca Vargas; Nunes, Letícia Teixeira; da Silva Jesus, Roberta; Machado, Michel Mansur; da Rocha, João Batista Teixeira; Soares, Félix Alexandre Antunes; Athayde, Margareth Linde

    2013-07-16

    Vitex megapotamica (Sprengel) Moldenke belongs to the Verbenaceae family and is popularly known as "tarumã". The antioxidant capacity of fractions and crude extract from the leaves of V. megapotamica were determined in this study through the capacity to remove reactive species and phenolic compounds were quantified in the various fractions. The IC50 (DPPH) ranged from 14.17 ± 0.76 to 37.63 ± 0.98 µg/mL. The ethyl acetate fraction might contain the strongest lipid peroxidation inhibitory compounds with an IC50 of 16.36 ± 5.09 µg/mL, being also the one with the highest content of polyphenols (522.4 ± 1.12 mg/g), flavonoids (220.48 ± 0.30 mg/g) and condensed tannins (3.86 ± 0.53 mg/g). Compounds quantified by HPLC/DAD in the crude extract and fractions were chlorogenic and rosmarinic acids. Higher dosages of the extracts were more effective in reducing levels of plasma protein carbonyls and were also shown to be able to remove reactive species by a 2',7'-dichlorofluorescein diacetate assay, reducing oxidative stress in all tested fractions. Results obtained indicated that V. megapotamica exhibits good potential to prevent diseases caused by the overproduction of free radicals and it might also be used as a potential source of natural antioxidant agents.

  5. Enzyme-assisted extraction for the HPLC determination of aflatoxin M1 in cheese.

    PubMed

    Pietri, Amedeo; Fortunati, Paola; Mulazzi, Annalisa; Bertuzzi, Terenzio

    2016-02-01

    The extraction of aflatoxin M1 (AFM1) from cheese is generally carried out using chlorinated organic solvents. In this study, two innovative methods were developed, based on an enzyme-assisted (EA) extraction using proteolytic enzymes (pepsin or pepsin-pancreatin). After purification through an immunoaffinity column, AFM1 is determined by HPLC-FLD. A comparison between the new EA methods and an established chloroform (CH) method was carried out on 24 cheese samples. The results showed that the extraction efficiency of the EA methods was independent of ripening time of cheese, whereas the CH method was not able to fully recover AFM1 from ripened cheeses. The simpler (pepsin) of the two methods has been adopted by our laboratory for routine analysis of AFM1 in cheese. In comparison with the CH method, the pepsin-HCl (P-HCl) method is simpler, avoiding solvent evaporation, dissolution and partition in a separating funnel; moreover, it gives higher recoveries, comparable LOD and LOQ and more accurate results.

  6. An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus.

    PubMed

    Ramirez-Malule, Howard; Junne, Stefan; López, Carlos; Zapata, Julian; Sáez, Alex; Neubauer, Peter; Rios-Estepa, Rigoberto

    2016-02-20

    Clavulanic acid (CA) is an important secondary metabolite commercially produced by cultivation of Streptomyces clavuligerus (Sc). It is a potent inhibitor of bacterial β-lactamases. In this work, a specific and improved high performance liquid chromatography (HPLC) method, using a C-18 reversed phase column, diode array detector and gradient elution for CA quantification in fermentation broths of Sc, was developed and successfully validated. Samples were imidazole-derivatized for the purpose of creating a stable chromophore (clavulanate-imidazole). The calibration curve was linear over a typical range of CA concentration between 0.2 and 400mg/L. The detection and quantification limits were 0.01 and 0.02mg/L, respectively. The precision of the method was evaluated for CA spiked into production media and a recovery of 103.8%, on average, was obtained. The clavulanate-imidazole complex was not stable when the samples were not cooled during the analysis. The recovery rate was 39.3% on average. This assay was successfully tested for CA quantification in samples from Sc fermentation, using both, a chemically defined and a complex medium.

  7. Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC.

    PubMed

    Borrego, C M; Garcia-Gil, L J

    1994-07-01

    A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.

  8. [Multiresidue determination of quinolones in animal and fishery products by HPLC].

    PubMed

    Chonan, Takao; Fujimoto, Toru; Inoue, Maki; Tazawa, Teijiro; Ogawa, Hiroshi

    2008-06-01

    A simple and rapid multiresidue method was developed for the determination of twelve quinolones (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, norfloxacin, ofloxacin, orbifloxacin, oxolinic acid and sarafloxacin) in muscle, liver, chicken eggs, milk, prawn and rainbow trout. The quinolones were extracted from a sample with acetonitrile-water (95 : 5). A fifth part of the filtered extract was diluted with water to keep the acetonitrile ratio at ca. 60%, and passed through a C18 mini-column. The eluate was evaporated to dryness, and the residues were dissolved in methanol-water (30 : 70) for HPLC analysis. The quinolones were separated on a Inertsil ODS-3V column (4.6 mm i.d.x250 mm) with a gradient system of 0.1% phosphoric acid-acetonitrile as the mobile phase, with fluorescence detection.No interfering peak was found on the chromatograms of animal and fishery products, except for milk. The recoveries of the quinolones were over 60% from the animal and fishery products fortified at 0.1 microg/g, and the quantification limits of the quinolones were 0.005 microg/g. This proposed method was found to be effective and suitable for the screening of the quinolones in animal and fishery products.

  9. [Application of HPLC-UV method for aripiprazole determination in serum].

    PubMed

    Synowiec, Anna; Gomółka, Ewa; Zyss, Tomasz; Zieba, Andrzej; Florek, Ewa; Piekoszewski, Wojciech

    2012-01-01

    Aripiprazole is a new drug applied in schizophrenia treatment. There are not strict indications for aripiprazole therapeutic drug monitoring. Despite, serum aripiprazole measuring would help control the drug doses effectiveness. The drug monitoring can eliminate overdosing, adverse effects and let control proper drug ingestion. The aim of the paper was to develop a simple method for aripiprazole determination in serum for therapeutic drug monitoring. High performance liquid chromatography with spectrophotometric detection (HPLC-UV) was used. Resolution was performed on LC-8 column; moving phase was solution 0,025M trimethylammonium buffer: acetonitrile (62:38). Isocratic flow was 1,2 ml/min; internal standard (IS) was promazine; monitored wavelength was lambda=214 nm. The validation parameters were: limits of linearity (LOL) 100-800 ng/ml, limit of detection (LOD) 10 ng/ml, limit of quantity (LOQ) 100 ng/ml. Coefficient of variation (CV) describing accuracy and precision didn't cross 10%. The method was useful for therapeutic drug monitoring in serum of patients treated with aripiprazole. PMID:23421079

  10. Investigation of red natural dyes used in historical objects by HPLC-DAD-MS.

    PubMed

    Karapanagiotis, Ioannis; Chryssoulakis, Yannis

    2006-01-01

    High performance liquid chromatography (HPLC) with UV-Vis Diode Array Detection (DAD) and electrospray mass spectrometric (ESI-MS) method was utilized for the identification of coloring components of madder, Armenian and Mexican cochineal, lac dye, brazilwood, safflower and dragon blood--probably the most important red natural dyestuffs found in objects of the cultural heritage. UV-Vis detection limits in the range of 0.2-0.6 ng for carminic acid, alizarin and purpurin were achieved using a gradient elution of H2O-0.01% TFA and CH3CN-0.01% TFA. ESI mass spectrometer was also used, as a supportive detection method to the standard DAD, for further analysis of the tested materials, with the ability to analyze dyestuffs as small as one milligram. The presence of madder was revealed in two historical (Hellenistic and Roman period) samples, found in the Mediterranean area, by identifying purpurin in both of them. Munjistin was also identified in one of the samples (Hellenistic period) while alizarin was not detected, raising questions regarding the exact madder type, utilized in the historical samples. PMID:16736555

  11. Use of ESR and HPLC to follow the anaerobic reaction catalysed by lipoxygenases.

    PubMed

    Brandicourt, Stéphanie; Nicolas, Jacques; Boussard, Aline; Riquet, Anne-Marie

    2015-02-01

    The measurement of the 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) consumption by using ESR allows to follow the anaerobic reaction between linoleic acid (LH) and its 13-hydroperoxide (LOOH) catalysed by lipoxygenase. During this reaction, two types of radicals are initially obtained, alkyl (L) and alkoxyl (LO) radicals which formed two types of adducts (LT and OLT) with TEMPOL as characterised by HPLC. The stoichiometry of the adduct formation is two mole of TEMPOL consumed for one mole of LH and one mole of LOOH. Using ESR, the kinetic parameters and the mechanism of the anaerobic reaction have been determined at pH 6.5 for three different lipoxygenases, soybean, horse bean and wheat and compared to the values obtained at pH 9 for soybean lipoxygenase. Wheat lipoxygenase is very weakly active compared to the other enzymes. An uncompetitive inhibition of the anaerobic reaction catalysed by soybean and horse bean lipoxygenases was observed with 2,6-di-tert-butyl-4-methylphenol (BHT).

  12. Absorbance detector based on a deep UV light emitting diode for narrow-column HPLC.

    PubMed

    Bui, Duy Anh; Bomastyk, Benjamin; Hauser, Peter C

    2013-10-01

    A detector for miniaturized HPLC based on deep UV emitting diodes and UV photodiodes was constructed. The measurement is accomplished by the transverse passage of the radiation from the light-emitting diode (LED) through fused-silica tubing with an internal diameter of 250 μm. The optical cell allows flexible alignment of the LED, tubing, and photodiode for optimization of the light throughput and has an aperture to block stray light. A beam splitter was employed to direct part of the emitted light to a reference photodiode and the Lambert-Beer law was emulated with a log-ratio amplifier circuitry. The detector was tested with two LEDs with emission bands at 280 and 255 nm and showed noise levels as low as 0.25 and 0.22 mAU, respectively. The photometric device was employed successfully in separations using a column of 1 mm inner diameter in isocratic as well as gradient elution. Good linearities over three orders of magnitude in concentration were achieved, and the precision of the measurements was better than 1% in all cases. Detection down to the low micromolar range was possible. PMID:23893947

  13. Antinociceptive activity of the HPLC- and MS-standardized hydroethanolic extract of Pterodon emarginatus Vogel leaves.

    PubMed

    Negri, Giuseppina; Mattei, Rita; Mendes, Fúlvio Rieli

    2014-01-01

    Several studies have demonstrated the analgesic and anti-inflammatory effects of fruit and seed extracts from Pterodon emarginatus Vogel (Fabaceae). The objective of this study was to evaluate the antinociceptive activity of the hydroethanolic extract of P. emarginatus leaves in mice and characterize its chemical composition using HPLC coupled to UV-vis diode array detection and mass spectrometry with electrospray ionization. Our results showed that the doses of 500 and 1000 mg/kg produced an antinociceptive effect, as observed in the hot plate test and writhing induced by acetic acid. The chromatographic profile and spectral mass data suggest the presence of di-C-glycosylflavones (e.g., vicenin-2 and schaftoside), C,O-glycosylflavones (e.g., chrysoeriol-8-C-glucosyl-2″-O-glucuronide-6-C-arabinoside) and luteolin-7-O-rutinoside as the main constituents. Lower levels of oleanane-type saponins, such as soyasaponin Bb and Be, and the saponin derivatives hederagenin and aglycone B, which are typical of Fabaceae family, were also found. From this study, it is suggested that the analgesic effect observed is not due to the terpenoids previously reported from fruit and seed extracts, but could be attributed to flavones and the hederagenin derivatives that were identified as main constituents of the hydroethanolic extract from the leaves.

  14. A selective determination of azide by ion-interaction reversed-phase HPLC

    SciTech Connect

    Gennaro, M.C.; Abrigo, C.; Marengo, E.; Liberatori, A.

    1993-01-01

    A method is presented for the analysis of sodium azide, based on the use of ion-interaction reversed-phase HPLC chromatography. A C-18 reversed-phase is the stationary phase and octylammonium ortho-phosphate at different pH values is the interaction reagent. Spectrophotometric detection at 230 nm is employed. The analysis is free from interference by acetate, carbonate, chloride, fluoride, sulfite, hydrazine, hydroxylamine, nitrate, bromide, iodide, sulfide, thiocyanate and nitrite. A good correlation (r[sup 2] = 0.9782) is obtained between peak area and concentration in the range between 1 and 250 ppb. Samples of tap water spiked with sodium azide (in the range within 25 and 250 ppb) gave per cent average recovery of 98%. The method sensitivity, expressed as signal-to-noise ratio equal to 3, is 50 ppb when the pH of the interaction reagent is equal to 3.0, 30 ppb for pH 6.4 and 10 ppb at pH 8.0.

  15. HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure.

    PubMed

    Mercolini, Laura; Addolorata Saracino, Maria; Bugamelli, Francesca; Ferranti, Anna; Malaguti, Marco; Hrelia, Silvana; Raggi, Maria Augusta

    2008-04-01

    An original analytical method has been developed for the determination of the antioxidants trans-resveratrol (t-RSV) and cis-resveratrol (c-RSV) and of melatonin (MLT) in red and white wine. The method is based on HPLC coupled to fluorescence detection. Separation was obtained by using a RP column (C8, 150 mm x 4.6 mm id, 5 mum) and a mobile phase composed of 79% aqueous phosphate buffer at pH 3.0 and 21% ACN. Fluorescence intensity was monitored at lambda = 386 nm while exciting at lambda = 298 nm, mirtazapine was used as the internal standard. A careful pretreatment of wine samples was developed, using SPE with C18 cartridges (100 mg, 1 mL). The calibration curves were linear over the following concentration ranges: 0.03-5.00 ng/mL for MLT, 3-500 ng/mL for t-RSV and 1-150 ng/mL for c-RSV. The LOD values were 0.01 ng/mL for MLT, 1 ng/mL for t-RSV and 0.3 ng/mL for c-RSV. Precision data, as well as extraction yield and sample purification results, were satisfactory. Thus, the method seems to be suitable for the analysis of MLT and resveratrol isomers in wine samples. Moreover, wine total polyphenol content and antioxidant activity were evaluated.

  16. [Determination of docosyl ferulate in extract of Pygeum africanton Hook. by high performance liquid chromatography (HPLC)].

    PubMed

    You, Y; Cheng, G

    1997-05-01

    This paper reports a method for the determination of docosyl ferulate in the extract of bark of pygeum africanum Hook. by HPLC. After the sample was pretreated, the docosyl ferulate was well separated and determined on a Spherisorb C18 column (250 x 4.6mm, 5microm) using a mobile phase of methanol with a flow rate of 1mL/min. The column temperature was selected at 40 degrees C to avoid tailing of peak. UV detection was performed at 326nm. In order to confirm the docosyl ferulate separated from sample, the peak apex at 10.4 minute was scanned from 195nm to 360nm by photodiode array detector. Its spectrum showed the maxium absorption peak at 240nm and 326nm corresponding with the spectrum of docosyl ferulate. The linear correlation was observed from the 10mg/L to 100mg/L of docosyl ferulate (r = 0.9995). The average recovery was 98.4% +/- 1.98%. Three batches of sample were determined.

  17. HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

    USGS Publications Warehouse

    Yun, S.-S.; Scott, A.P.; Bayer, J.M.; Seelye, J.G.; Close, D.A.; Li, W.

    2003-01-01

    Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20pg-10ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96??g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys. ?? 2003 Elsevier Inc. All rights reserved.

  18. Application of statistical design for the optimization of amino acid separation by reverse-phase HPLC.

    PubMed

    Gheshlaghi, R; Scharer, J M; Moo-Young, M; Douglas, P L

    2008-12-01

    Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 2(5-1) fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluant, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile concentration ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of utmost importance for accurate analysis of a subset of amino acids.

  19. Assessment of enrofloxacin and ciprofloxacin accumulation in pig and calf hair by HPLC and fluorimetric detection.

    PubMed

    Gratacós-Cubarsí, Marta; García-Regueiro, José-Antonio; Castellari, Massimo

    2007-03-01

    Enrofloxacin is a synthetic bacteriostatic administered in veterinary therapy. It can also be used illegally as a growth promoter to enhance feed efficiency and weight gain. This practice is banned in several countries due to its potential negative effects on the environment and human health. A suitable method for extracting and quantifying enrofloxacin (ENR) and its main metabolite ciprofloxacin (CPR) in cattle and pig hair by high-performance liquid chromatography-fluorimetric detection (HPLC-FLD) had been proposed. ENR and CPR were extracted from hair samples with methanol acidified with trifluoroacetic acid for 24 h at 70 degrees C. The extracts were evaporated and redissolved in the mobile phase before injection. This simplified procedure enabled the detection of both CPR and ENR at ng g-1 levels (limit of detection 4-5 ng g-1) without further purification. Detectable residues of ENR were found in calf and pig hairs after the pharmacological treatment was started. Mean concentrations of quinolone (ENR, CPR) in contaminated hairs ranged from 20 to 2,518 ng g-1 in calves and from 152 to 1,140 ng g-1 in pigs. Hair pigmentation enhanced quinolone accumulation significantly. Hair analysis seems to increase the time window available for the retrospective detection of illegal ENR administration compared to edible tissue analysis. PMID:17186229

  20. Derivatization procedure and HPLC determination of 2-ethoxyethanol in cosmetic samples.

    PubMed

    Mariani, E; Villa, C; Neuhoff, C; Dorato, S

    1999-06-01

    Ethylene glycol monoethyl ether or 2-ethoxyethanol finds a wide industrial application as a solvent for lacquers, inks, dyes, household products and as a surfactant. It is also found in cosmetics such as nail products, face cleansers, liquid soaps, oral care products, hair colours and fixatives. The potential hazard to human health of 2-ethoxyethanol following inhalation and dermal exposure has been recently reviewed and the European Cosmetic, Toiletry and Perfumery Association (COLIPA) has issued recommendations suggesting its non-use as a cosmetic ingredient. Therefore a simple and fast monitoring method is necessary for routine control to identify and quantify 2-ethoxyethanol in raw materials and finished cosmetics. We have developed a sensitive and selective method to determine 2-ethoxyethanol in complex matrices by precolumn derivatization with 1-naphthyl isocyanate and RP-HPLC analysis. Four laboratory-made cosmetic formulations (a nail lacquer remover, a baby oil, a skin lotion and an emollient O/W emulsion) containing three known amounts of 2-ethoxyethanol (0.1%, 2.0%, 5.0%) have been studied. The obtained results show that this chromatographic procedure provides a good estimate of the true concentration of 2-ethoxyethanol in complex matrices and it is reliable for routine analyses in quality control.

  1. The Third SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE-3)

    NASA Technical Reports Server (NTRS)

    Hooker, Stanford B.; VanHeukelem, Laurei; Thomas, Crystal S.; Claustre, Herve; Ras, Josephine; Schluter, Louise; Clementson, Lesley; vanderLinde, Dirk; Eker-Develi, Elif; Berthon, Jean-Francois; Barlow, Ray; Sessions, Heather; Ismail, Hassan; Perl, Jason

    2009-01-01

    Seven international laboratories specializing in the determination of marine pigment concentrations using high performance liquid chromatography (HPLC) were intercompared using in situ samples and a mixed pigment sample. The field samples were collected primarily from oligotrophic waters, although mesotrophic and eutrophic waters were also sampled to create a dynamic range in chlorophyll concentration spanning approximately two orders of magnitude (0.020 1.366 mg m^{-3}) The intercomparisons were used to establish the following: a) the uncertainties in quantitating individual pigments and higher-order variables (sums, ratios, and indices); b) the reduction in uncertainties as a result of applying quality assurance (QA) procedures; c) the importance of establishing a properly defined referencing system in the computation of uncertainties; d) the analytical benefits of performance metrics, and e) the utility of a laboratory mix in understanding method performance. In addition, the remote sensing requirements for the in situ determination of total chlorophyll a were investigated to determine whether or not the average uncertainty for this measurement is being satisfied.

  2. Rapid Analysis of Glibenclamide Using an Environmentally Benign Stability-Indicating RP-HPLC Method

    PubMed Central

    Haq, Nazrul; Alanazi, Fars Kaed; Alsarra, Ibrahim Abdullah; Shakeel, Faiyaz

    2014-01-01

    An environmentally benign RP-HPLC approach for rapid analysis of glibenclamide in pure form, developed nanoemulsion and commercial tablets was developed and validated in present investigation. The green chromatographic identification was performed on Lichrosphere 250 X 4.0 mm RP C8 column having a 5 μm packing as a stationary phase using a combination of ethanol: methanol (50:50 % v/v) as a mobile phase, at a flow rate of 1.0 mL/min with UV detection at 245 nm. The proposed method was validated for linearity, selectivity, accuracy, precision, robustness, sensitivity and specificity as per international conference on harmonization (ICH) guidelines. The utility of proposed method was verified by assay of glibenclamide in developed nanoemulsion and commercial tablets. The proposed method was found to be satisfactory in terms of selectivity, precision, accuracy, robustness, sensitivity and specificity. The content of glibenclamide in developed nanoemulsion and commercial tablets was found to be 100.50 % and 99.15 % respectively. The proposed method successfully resoled glibenclamide peak in the presence of its all type of degradation products which indicated stability-indicating property of the proposed method. These results indicated that the green chromatographic method could be successfully employed for routine analysis of glibenclamide in pure drug and various commercial formulations. PMID:25276186

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

    SciTech Connect

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

    2012-01-01

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

  4. Novel separation method for highly sensitive speciation of cancerostatic platinum compounds by HPLC-ICP-MS.

    PubMed

    Hann, S; Stefánka, Zs; Lenz, K; Stingeder, G

    2005-01-01

    A high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method is presented for analysis of cisplatin, monoaquacisplatin, diaquacisplatin, carboplatin, and oxaliplatin in biological and environmental samples. Chromatographic separation was achieved on pentafluorophenylpropyl-functionalized silica gel. For cisplatin, carboplatin, and oxaliplatin limits of detection of 0.09, 0.10, and 0.15 microg L(-1), respectively, were calculated at m/z 194, using aqueous standard solutions. (3 microL injection volume). The method was utilized for model experiments studying the stability of carboplatin and oxaliplatin at different chloride concentrations simulating wastewater and surface water conditions. It was found that a high fraction of carboplatin is stable in ultrapure water and in solutions containing 1.5 mol L(-1) Cl-, whereas oxaliplatin degradation was increased by increasing the chloride concentration. In order to support the assessment of oxaliplatin eco-toxicology, the method was tested for speciation of patient urine. The urine sample contained more than 17 different reaction products, which demonstrates the extensive biotransformation of the compound. In a second step of the study the method was successfully evaluated for monitoring cancerostatic platinum compounds in hospital waste water.

  5. Evaluation of extraction methods for ochratoxin A detection in cocoa beans employing HPLC.

    PubMed

    Mishra, Rupesh K; Catanante, Gaëlle; Hayat, Akhtar; Marty, Jean-Louis

    2016-01-01

    Cocoa is an important ingredient for the chocolate industry and for many food products. However, it is prone to contamination by ochratoxin A (OTA), which is highly toxic and potentially carcinogenic to humans. In this work, four different extraction methods were tested and compared based on their recoveries. The best protocol was established which involves an organic solvent-free extraction method for the detection of OTA in cocoa beans using 1% sodium hydrogen carbonate (NaHCO3) in water within 30 min. The extraction method is rapid (as compared with existing methods), simple, reliable and practical to perform without complex experimental set-ups. The cocoa samples were freshly extracted and cleaned-up using immunoaffinity column (IAC) for HPLC analysis using a fluorescence detector. Under the optimised condition, the limit of detection (LOD) and limit of quantification (LOQ) for OTA were 0.62 and 1.25 ng ml(-1) respectively in standard solutions. The method could successfully quantify OTA in naturally contaminated samples. Moreover, good recoveries of OTA were obtained up to 86.5% in artificially spiked cocoa samples, with a maximum relative standard deviation (RSD) of 2.7%. The proposed extraction method could determine OTA at the level 1.5 µg kg(-)(1), which surpassed the standards set by the European Union for cocoa (2 µg kg(-1)). In addition, an efficiency comparison of IAC and molecular imprinted polymer (MIP) column was also performed and evaluated.

  6. Quantification of lipid alkyl radicals trapped with nitroxyl radical via HPLC with postcolumn thermal decomposition.

    PubMed

    Koshiishi, Ichiro; Tsuchida, Kazunori; Takajo, Tokuko; Komatsu, Makiko

    2005-11-01

    Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spin-trapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N-oxyl (CmdeltaP) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting CmdeltaP-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 degrees C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2'-azobis(2,4-dimethyl-valeronitrile).

  7. HPLC-pDA simultaneous determination and protective effect of Anemarrhena asphodeloides against acute renal failure.

    PubMed

    Seo, Chang-Seob; Ha, Hyekyung; Kim, Young-Jung; Jungb, Ju-Young

    2014-06-01

    We investigated the protective effects against acute renal failure (ARF) of Anemarrhena asphodeloides (AA) and performed simultaneous analysis of three compounds, neomangiferin (1), mangiferin (2), and isomangiferin (3) in AA using a high-performance liquid chromatography-photodiode array. To measure the protective effect of ARF, the levels of reactive oxygen species (ROS) and glutathione depletion were determined using a kit. HPLC analysis was performed using a Gemini Cia column at 40 degrees C. The mobile phase used gradient elution with 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The flow rate was 1.0 mL/min. In our assay, AA extract inhibits cisplatin-induced production of intracellular ROS. Pre-incubation of AA extracts (10-200 microg/mL) markedly maintained cell viability compared with controls in the noncisplatin-treated cells. Calibration curves of all compounds showed good linearity (r2 > or = 0.9992). Recoveries of the three compounds were 98.9-103.4%. The relative standard deviations of intra- and inter-day precision were 0.07-1.73% and 0.12-1.49%, respectively. The amounts of the three components were 1.22-20.63 mg/g. The AA extract has potential as a therapeutic agent for treatment of ARF. In addition, the established method will help to improve quality control of AA.

  8. HPLC analysis of ADMA and other methylated L-arginine analogs in biological fluids.

    PubMed

    Teerlink, Tom

    2007-05-15

    Post-translational methylation of arginine residues in proteins leads to generation of N(G)-monomethylarginine (MMA) and both symmetric and asymmetric dimethylarginine (SDMA and ADMA), that are released into the cytosol upon proteolysis. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Plasma concentrations of ADMA and SDMA are very low, typically between 0.3 and 0.8 microM, making their quantification by HPLC an analytical challenge. Sample preparation usually involves a cleanup step by solid-phase extraction on cation-exchange columns followed by derivatization of amino acids into fluorescent adducts. Because ADMA and SDMA concentrations in healthy subjects show a very narrow distribution, with a between-subject variability of 13% for ADMA and 19% for SDMA, very low imprecision is an essential assay feature. Procedures for sample cleanup, derivatization, and chromatographic separation of arginine and its methylated analogs are the main topics of this review. In addition, important aspects of method validation, pre-analytical factors, and reference values are discussed. PMID:16931194

  9. Submerged fermentation production and characterization of intracellular triterpenoids from Ganoderma lucidum using HPLC-ESI-MS.

    PubMed

    Cui, Mei-lin; Yang, Huan-yi; He, Guo-qing

    2015-12-01

    As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% (0.041 g/ml) and yeast extract 1.89% (0.0189 g/ml), pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H2O and CO2, as well as cleavage of the D-ring. PMID:26642183

  10. Development and Validation of Stability-indicating HPLC Method for Simultaneous Estimation of Cefixime and Linezolid.

    PubMed

    Patel, Nidhi S; Tandel, Falguni B; Patel, Yogita D; Thakkar, Kartavya B

    2014-01-01

    A stability-indicating reverse phase high performance liquid chromatography method was developed and validated for cefixime and linezolid. The wavelength selected for quantitation was 276 nm. The method has been validated for linearity, accuracy, precision, robustness, limit of detection and limit of quantitation. Linearity was observed in the concentration range of 2-12 μg/ml for cefixime and 6-36 μg/ml for linezolid. For RP-HPLC, the separation was achieved by Phenomenex Luna C18 (250×4.6 mm) 5 μm column using phosphate buffer (pH 7):methanol (60:40 v/v) as mobile phase with flow rate 1 ml/min. The retention time of cefixime and linezolid were found to be 3.127 min and 11.986 min, respectively. During force degradation, drug product was exposed to hydrolysis (acid and base hydrolysis), H2O2, thermal degradation and photo degradation. The % degradation was found to be 10 to 20% for both cefixime and linezolid in the given condition. The method specifically estimates both the drugs in presence of all the degradants generated during forced degradation study. The developed methods were simple, specific and economic, which can be used for simultaneous estimation of cefixime and linezolid in tablet dosage form.

  11. A simple HPLC method for the simultaneous analysis of insulin and ovomucoid.

    PubMed

    Vuppugalla, R; Agarwal, V; Khan, M A

    2003-11-01

    An analytical HPLC method is reported for the simultaneous determination of insulin and its enzyme inhibitor, chicken ovomucoid. Verapamil was used as an internal standard. The elution was achieved using a gradient technique (10-15% B for 4 min, 15-35% B from 5th to 11th min and 35-10% B from 12th to 22nd min). The mobile phase used was 0.05% v/v trifluoroacetic acid (TFA) in water and 0.05% v/v TFA in acetonitrile with a flow rate of 1.2 ml/min. The analytes were detected at 210 nm after resolution using a reversed phase C-18 column. Insulin, ovomucoid and verapamil (IS) were eluted at 11.9, 14.2, and 18 min, respectively, free from any interfering endogenous peaks during a run time of 22 min. Linear relationships were observed between the detector response and the concentrations of the analytes (0.05-1 I.U/ml for insulin (r2 = 0.9975) and 5-100 microg/ml for the chicken ovomucoid (r2 = 0.9993)). The assay was found to be highly selective and sensitive due to the absence of any interfering peaks. The lower C.V and % error values of the assay indicates that the assay could accurately and precisely quantitate both insulin and ovomucoid in the examined concentration range. This method can be used for the simultaneous quantitation of insulin and chicken ovomucoid.

  12. [Simultaneous determination of 6 neonicotinoid residues in soil using DLLME-HPLC and UV].

    PubMed

    Sun, Bao-li; Shan, Hong; Li, Yan-hua; Zeng, Ya-ling; Shen, Xiu-li; Tong, Cheng-feng

    2013-09-01

    A simple, cheap and rugged method was developed for simultaneous deter mination of 6 neonicotinoid residues in soil, including imidacloprid, acetamiprid, thiamethoxam, thiacloprid, clothianidin and nitenpyram. The soil sample was produced by dispersive liquid-liquid micro-extraction (DLLME) after extracted by the mixed solution of acetonitrile and CH2Cl2 (2:1, phi). The analytes were separated by HPLC with Alltima C18 column (4.6 mm x 250 mm, 5 microm) and detected by PDA at 260 nm. External standard method was used for quantification. The results showed that good linearity was obtained with correlation coefficients between 0.9982 and 0.9999 in the range of 0.5-200 microg x L(-1). The limits of detection (LODs) were in the range between 0.0005 and 0.003 microg x mL(-1) (S/N = 3). The method was validated with five soil samples spiked at three fortification levels (0.05, 0.1, 1.0 mg x kg(-1)) and recoveries were in the range of 55.3%-95.6% with RSD of 1.4%-7.0%. The effect of clean-up was evaluated by UV spectra and demonstrated that the method established is effective. In conclusion, this method is competent for the simultaneous analysis of 6 neonicotinoid residues in soil. PMID:24369670

  13. Determination of Saponin Content in Hang Maidong and Chuan Maidong via HPLC-ELSD Analysis

    PubMed Central

    Li, Xian-En; Wang, Yu-Xia; Sun, Peng; Liao, Deng-Qun

    2016-01-01

    Zhejiang and Sichuan are regarded as two genuine producing areas of Ophiopogonis radix in China. To study the difference in the quality of Ophiopogonis radix from these two places, the contents of three reported bioactive saponins, that is, ophiopogonins B, D, and D′, in tubers and fibrous roots of Ophiopogon japonicus from Cixi city of Zhejiang and Santai county of Sichuan were quantified using HPLC-ELSD method and compared. Ophiopogonin B and ophiopogonin D′ content in tubers of HMD were higher than those in radix of CMD, whereas ophiopogonin D in HMD was about twice lower than that of CMD. Three ophiopogonins were all detected in fibrous roots of both HMD and CMD. Their averaged content in fibrous roots of CMD was about twice higher than that in tubers of CMD. Ophiopogonin D in fibrous roots of CMD was about five times higher than that of HMD. Our biochemical assay revealed that fibrous roots and tubers of CMD can be of an important saponin source, especially for ophiopogonin D. PMID:26925295

  14. [Application and improvement of aflatoxin analysis in foods using a multifunctional column and HPLC].

    PubMed

    Goda, Y; Akiyama, H; Otsuki, T; Fujii, A; Toyoda, M

    2001-02-01

    In an earlier report, we developed a rapid, sensitive and clean method consisting of non-chloroform extraction, clean-up on a commercial multifunctional cartridge column and HPLC with fluorescence detection for the analyses of aflatoxins. In this report, we applied this method to analyze aflatoxins in nuts, giant corn, cereals, spice and black teas. The method was effective for macadamia nuts, walnuts, hazelnuts, brazil nuts, giant corn, rice, wheat and buckwheat, and the recoveries of aflatoxins B1, B2, G1 and G2 spiked in them at the level of 10 ng/g were 85-106%. However, in the chromatograms of spices and black tea, many background peaks were observed. Therefore, we added a purification step with an affinity column to the clean-up of these samples with the multifunctional cartridge column. After the additional purification, most of the background peaks were gone. The recoveries of aflatoxins B1, B2 and G1 spiked at the level of 10 ng/g were 71-112% except for the case of B2 in white pepper (48%). The recoveries of G2 were 49-95%.

  15. Isolation of 163Ho from dysprosium target material by HPLC for neutrino mass measurements

    DOE PAGESBeta

    Mocko, Veronika; Taylor, Wayne  A.; Nortier, Francois M.; Engle, Jonathan  W.; Barnhart, Todd  E.; Nickles, Robert  J.; Pollington, Anthony  D.; Kunde, Gerd  J.; Rabin, Michael  W.; Birnbaum, Eva  R.

    2015-04-29

    The rare earth isotope 163Ho is of interest for neutrino mass measurements. This report describes the isolation of 163Ho from a proton-irradiated dysprosium target and its purification. A Dy metal target was irradiated with 16 MeV protons for 10 h. After target dissolution, 163Ho was separated from the bulk Dy via cation-exchange high performance liquid chromatography using 70 mmol dm–3 α-hydroxyisobutyric acid as the mobile phase. Subsequent purification of the collected Ho fraction was performed to remove the α-hydroxyisobutyrate chelating agent and to concentrate the Ho in a low ionic strength aqueous matrix. The final solution was characterized by MC-ICP-MSmore » to determine the 163Ho/165Ho ratio, 163Ho and the residual Dy content. The HPLC purification process resulted in a decontamination factor 1.4E5 for Dy. As a result, the isolated Ho fraction contained 24.8 ±1.3 ng of 163Ho corresponding to holmium recovery of 72 ± 3%.« less

  16. Simultaneous HPLC determination of 22 components of essential oils; method robustness with experimental design.

    PubMed

    Porel, A; Sanyal, Y; Kundu, A

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil-II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R(2) >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil.

  17. Simultaneous determination of 15 nitroimidazoles in cosmetics by HPLC coupled with electrospray ionization- tandem mass spectrometry.

    PubMed

    Meng, Xian-Shuang; Bai, Hua; Zhang, Qing; Lv, Qing; Chen, Yun-Xia; Ma, Hui-Juan; Li, Jing-Rui; Ma, Qiang

    2014-01-01

    A sensitive and reliable analytical method based on HPLC/MSIMS has been developed for the simultaneous determination of 15 nitroimidazoles in cosmetics. A diversity of cosmetic samples, including powder, lotion, shampoo, and cream were collected. The samples were ultrasonically extracted with aqueous methanol, and the extracts were then subjected to cleanup bySPE using an Oasis HLB cartridge followed by filtration with a 0.20 pm membrane filter. Afterwards, chromatographic separation was performed on an XSelect CSH C18 column (2.1 x 150 mm, 3.5 pm) maintained at 30°C within 15 min by a gradient of acetonitrile-0.1% aqueous formic acid solution at a flow rate of 0.25 mL/min. The mass spectrometric detection was carried, out using electrospray positive ionization under the multiple reaction monitoring mode. A good linearity was observed over the concentration range from 0.5 to 500 ng/mL. The intraday and interday precisions, which were investigated by determining all target compounds in cosmetics seven times/day and on 7 consecutive days, were below 5.00%. The mean recoveries at three spiked levels ranged from 80.42 to 100.83% with the RSDs from 0.45 to 9.02%. The LOQs were determined to be between 0.01 and 0.1 mg/kg. The method was sufficiently rapid, reliable, and sensitive for the determination of 15 nitroimidazoles in cosmetics. PMID:25632431

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

    PubMed

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

    2015-01-01

    Nicotinic acid, N-methylpyridinium ion, and trigonelline are well studied nutritional biomarkers present in coffee, and they are indicators of thermal decomposition during roasting. However, no method is yet available for their simultaneous determination. This paper describes a rapid and validated HPLC-diode array detector method for the simultaneous quantitation of caffeine, trigonelline, nicotinic acid, N-methylpyridinium ion, 5-caffeoylquinic acid, and 5-hydroxymethyl furfural that is applicable to three coffee matrixes: green, roasted, and instant. Baseline separation among all compounds was achieved in 30 min using a phenyl-hexyl RP column (250×4.6 mm, 5 μm particle size), 0.3% aqueous formic buffer (pH 2.4)-methanol mobile phase at a flow rate of 1 mL/min, and a column temperature at 30°C. The method showed good linear correlation (r2>0.9985), precision (less than 3.9%), sensitivity (LOD=0.023-0.237 μg/mL; LOQ=0.069-0.711 μg/mL), and recovery (84-102%) for all compounds. This simplified method is amenable for a more complete routine evaluation of coffee in industry. PMID:25857885

  19. Comparative peptide mapping of adrenergic and cholinergic neutrotransmitter receptors by reverse-HPLC

    SciTech Connect

    Kerlavage, A.R.; Fraser, C.M.; Venter, J.C.; Shreeve, S.M.

    1986-05-01

    The authors have developed a methodology for unambiguously identifying neutrotransmitter receptor proteins and comparing structural features of related receptors as well as those in different classes. These techniques have been applied to the study of the ..cap alpha..- and ..beta..-adrenergic receptors as well as the muscarinic and nicotinic cholinergic receptors. The method involves comparative peptide mapping of total proteolytic digests of receptor proteins by microbore reverse-phase HPLC in conjunction with covalent modification by specific receptor ligands or (/sup 125/I)-labeling. Femtomole amounts of receptor can be analyzed. The maps of all the (/sup 125/I)-labeled receptors contain between 20 and 25 peaks and each receptor has a unique profile although all are similar in the very hydrophobic region of the map. The ..cap alpha../sub 2/-adrenergic receptor from human platelets has a higher total percentage of hydrophilic peaks than either the guinea pig lung ..beta../sub 2/-adrenergic receptor or the porcine atria muscarinic receptor. Two forms of the muscarinic receptor have very similar but clearly distinct profiles. The nicotinic receptor subunits show a higher degree of homology by this method than was revealed by previous mapping studies which utilized SDS-PAGE or thin-layer techniques.

  20. Improved analysis of dissolved carbohydrates in stream water with HPLC-PAD.

    PubMed

    Cheng, X; Kaplan, L A

    2001-02-01

    This paper describes improvements made to the determination of dissolved carbohydrates in stream water with high-performance liquid chromatography and pulsed amperometric detection (HPLC-PAD). We eliminated interference from dissolved oxygen, separated xylose and mannose along with other molecules, reduced the chromatographic peak shift associated with carbonate accumulation in the column to less than 1% for all samples, and achieved an 8% increase in recovery of hydrolyzed carbohydrates by replacing ion-exchange desalting cartridges with evaporative removal of HCl under N2. These modifications lowered detection limits to less than or equal to 0.4 nM for 10 different monosaccharides and enhanced our ability to follow the dynamics of these molecules in stream ecosystems. In the determination of dissolved free monosaccharides, average relative precision was 1.3% and recovery ranged from 92 to 109%. For dissolved total saccharides, average relative precision was 3.3%. Concentrations were stable when filtered (0.2 microm) stream water was stored for 1 day at 22 degrees C, 1 week at 4 degrees C, or 1.5 months at -20 degrees C. PMID:11217746

  1. Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate.

    PubMed

    Xie, Rui; Tu, Maobing; Wu, Yonnie; Adhikari, Sushil

    2011-04-01

    5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase. The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30 °C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45 °C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.

  2. Simultaneous Quantification of 13 Compounds in Guanxin Shutong Capsule by HPLC Method.

    PubMed

    Zhou, Feifei; Zhang, Lunhui; Gu, Liqiang; Zhang, Yuanyuan; Hou, Chenzhi; Bi, Kaishun; Chen, Xiaohui; Zhang, Huifen

    2016-07-01

    Guanxin Shutong (GXST) capsule, which is frequently used in clinical therapy, has a certain and positive therapeutic effect against coronary heart disease. However, the existing quality standard of GXST capsule is inadequate to control the quality of GXST capsule. In this paper, a new high-performance liquid chromatographic (HPLC) method for simultaneous determination of 13 compounds (gallic acid, danshensu, protocatechuic acid, procatechuic aldehyde, ellagic acid, rosmarinic acid, salvianolic acid A and salvianolic acid B, eugenol, dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA) in GXST capsule was developed and validated. The analytes were successfully separated and quantified with an Agilent TC-C18 column (250 × 4.6 mm, 5 µm) by gradient elution using 0.05% phosphoric acid and acetonitrile as mobile phase. The flow rate was 1 mL/min and the detection wavelength was set at 280 nm. All the compounds showed good linearity (r > 0.9991) in a relatively wide concentration range. The intra-and the inter-day variability were in the range of 0.85-2.68 and 1.47-2.86%, respectively. The recoveries of the selected compounds were in the range of 95.24-104.75%. This method was successfully applied to quantify the 13 components in GXST capsule and was conducive to controlling the quality of GXST capsule. PMID:26969683

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

    PubMed

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

    2011-01-15

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

  4. Quantification of bufadienolides in the poisons of Rhinella marina and Rhaebo guttatus by HPLC-UV.

    PubMed

    Kerkhoff, Jacqueline; Noronha, Janaina da C; Bonfilio, Rudy; Sinhorin, Adilson P; Rodrigues, Domingos de J; Chaves, Mariana H; Vieira, Gerardo Magela

    2016-09-01

    Bufadienolides are the main active compounds in the Bufonidae family of frogs. Recent studies have demonstrated cytotoxic and/or antitumor activity in these molecules. A HPLC-DAD method was developed and validated to quantify three bufadienolides (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of the cane toad poison frogs and smooth-sided toad. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 × 4.6 mm, 5 μm), using gradient elution with acetonitrile and water, at a flow rate of 1.0 mL min(-1) and detection at 296 nm. The method showed linearity (r > 0.999) and adequate recovery values (86%-111%). The limits of quantification of bufadienolides were 7.4 μg mL(-1) for telocinobufagin, 4.2 μg mL(-1) for marinobufagin and 4.0 μg mL(-1) for bufalin. Intraday and interday values of the method were evaluated and presented standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of the cane toad, which showed a content of 60% of marinobufagin. The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise, requiring the development of a technique that presents a greater selectivity. PMID:27390041

  5. The RP-HPLC method for simultaneous estimation of esomeprazole and naproxen in binary combination

    PubMed Central

    Jain, Deepak Kumar; Jain, Nitesh; Charde, Rita; Jain, Nilesh

    2011-01-01

    Objective: A simple, precise, reliable, rapid, sensitive and validated RP-HPLC method has been developed to determine esomeprazole magnesium trihydrate (ESO) and naproxen (NAP) in synthetic mixture form. Materials and Methods: Chromatographic separation achieved isocratically on Phenomenex, Luna C18 column (5 μm, 150mm × 4.60mm) and acetonitrile: phosphate buffer (pH 7.0) in the ratio of 50:50 (v/v) as the mobile phase, at a flow rate of 0.5 ml/min. Detection was carried out at 300 nm. The retention times for NAP and ESO was found to be 2.67 ±0.014 and 5.65 ±0.09 min respectively. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the ICH guidelines. Results: The method was linear in the concentration range of 50-250 μg/ml for NAP and 2-10 μg/ml for ESO with correlation coefficient of 0.999 and 0.998 respectively. The mean recoveries obtained for NAP and ESO were 100.01% and 97.76 % respectively and RSD was less than 2. The correlation coefficients for all components are close to 1. Conclusions: Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of NAP and ESO. PMID:23781450

  6. Simultaneous determination of 15 nitroimidazoles in cosmetics by HPLC coupled with electrospray ionization- tandem mass spectrometry.

    PubMed

    Meng, Xian-Shuang; Bai, Hua; Zhang, Qing; Lv, Qing; Chen, Yun-Xia; Ma, Hui-Juan; Li, Jing-Rui; Ma, Qiang

    2014-01-01

    A sensitive and reliable analytical method based on HPLC/MSIMS has been developed for the simultaneous determination of 15 nitroimidazoles in cosmetics. A diversity of cosmetic samples, including powder, lotion, shampoo, and cream were collected. The samples were ultrasonically extracted with aqueous methanol, and the extracts were then subjected to cleanup bySPE using an Oasis HLB cartridge followed by filtration with a 0.20 pm membrane filter. Afterwards, chromatographic separation was performed on an XSelect CSH C18 column (2.1 x 150 mm, 3.5 pm) maintained at 30°C within 15 min by a gradient of acetonitrile-0.1% aqueous formic acid solution at a flow rate of 0.25 mL/min. The mass spectrometric detection was carried, out using electrospray positive ionization under the multiple reaction monitoring mode. A good linearity was observed over the concentration range from 0.5 to 500 ng/mL. The intraday and interday precisions, which were investigated by determining all target compounds in cosmetics seven times/day and on 7 consecutive days, were below 5.00%. The mean recoveries at three spiked levels ranged from 80.42 to 100.83% with the RSDs from 0.45 to 9.02%. The LOQs were determined to be between 0.01 and 0.1 mg/kg. The method was sufficiently rapid, reliable, and sensitive for the determination of 15 nitroimidazoles in cosmetics.

  7. Pink berry grape (Vitis vinifera L.) characterization: Reflectance spectroscopy, HPLC and molecular markers.

    PubMed

    Rustioni, Laura; De Lorenzis, Gabriella; Hârţa, Monica; Failla, Osvaldo

    2016-01-01

    Color has a fundamental role for the qualitative evaluation and cultivar characterization of fruits. In grape, a normally functional pigment biosynthesis leads to the accumulation of a high quantity of anthocyanins. In this work, 28 Vitis vinifera L. cultivars accumulating low anthocyanins in berries were studied to characterize the biosynthetic dysfunctions in both a phenotypic and genotypic point of view. Reflectance spectroscopy, HPLC profiles and molecular markers related to VvMybA1 and VvMybA2 genes allowed a detailed description of the pigment-related characteristics of these cultivars. Data were consistent concerning the heterozygosity of the non-functional allele in both investigated genes, resulting in a low colored phenotype as described by reflectance. However, the variability in berry colour among our samples was not fully explained by MybA locus, probably due to specific interferences among the biosynthetic pathways, as suggested by the anthocyanin profile variations detected among our samples. The results presented in this work confirmed the importance of the genetic background: grapes accumulating high levels of cyanidin-3-O-glucosides (di-substituted anthocyanin) are generally originated by white cultivar retro-mutations and they seem to preserve the anomalies in the flavonoid hydroxylases enzymes which negatively affect the synthesis of tri-substituted anthocyanins. PMID:26687319

  8. HPLC determination of calcium pantothenate and two preservatives in topical cream.

    PubMed

    Havlíková, L; Matysová, L; Nováková, L; Solich, P

    2006-05-01

    A RP-HPLC method for simultaneous determination of calcium pantothenate and two preservatives methylparaben and propylparaben present in topical cream was developed. Different analytical columns with various stationary phases were tested. During method development, Supelco Discovery C18 column (125 mmx4.0 mm, 5 microm) and Zorbax SB-CN column (150 mmx4.6 mm, 5 microm) were tested. Both were not convenient for analytical separation because of the co-elution of calcium pantothenate with dead volume, and problems with the peak-shape of all components. Good separation was achieved using Zorbax TSM (250 mmx4.6 mm, 5 microm) and Hypersil ODS column (250 mmx4.6 mm, 5 microm), the latter was finally used for the analysis. The analysis time was 12 min, at flow rate 0.7 ml min-1. Chromatography was performed using binary mobile phase composed of methanol and phosphoric acid, pH 2.5, 65:35 (v/v). UV detection was accomplished at 214 nm. The method was validated according to ICH guideline recommendations. The method is suitable for practical routine analysis of commercially produced topical pharmaceutical preparations. PMID:16473491

  9. [Simultaneous determination of rosmarinic acid, oridonin and chrysoplenetin in Isodon rubescens by HPLC].

    PubMed

    Yuan, Xing-Li; Yan, Li-Hua; Zhang, Qi-Wei; Wang, Zhi-Min

    2013-07-01

    An HPLC method was developed for simultaneous quantitation of rosmarinic acid, oridonin and chrysoplenetin in the aerial parts of Isodon rubescens. Samples were analyzed on an Ultimate C18 column (4.6 mm x 250 mm, 5 microm) with methanol and water containing 0.1% formic acid as mobile phases in a linear gradient mode. The flow rate was 1.0 mL x min(-1) and the temperature was set at 30 degrees C. The PDA detector wavelengths were set at 338 nm for rosmarinic and chrysoplenetin and at 242 nm for oridonin. The linear ranges were 0.222-2.78, 0.227-2.84 and 0.005-0.071 microg for rosmarinic acid, oridonin and chrysoplenetin, respectively. The average recoveries of the three constituents were 102.9% (RSD 1.9%), 99.6% (RSD 1.1%) and 102.5% (RSD 0.94%), respectively. This method was proved to be accurate and repeatable, and can be used for quality control of the aerial parts of I. rubescens. PMID:24199569

  10. Quantititative determination of lycorine and galanthamine in Galanthus trojanus and G. cilicicus by HPLC-DAD.

    PubMed

    Kaya, Gulen Irem; Polat, Derya Cicek; Sarikaya, Buket; Onur, Mustafa Ali; Somer, Nehir Unver

    2014-08-01

    Lycorine and galanthamine have various biological activities. A reliable HPLC method coupled with DAD detection was developed and validated for the determination of galanthamine and lycorine in Galanthus trojanus and G. cilicicus. A simple method for the extraction of the alkaloids in low-mass plant samples was employed utilizing columns pre-packed with diatomaceous earth (Extrelut). This method was applied to the aerial parts and bulbs of G. trojanus and G. cilicicus (Amaryllidaceae) collected during the flowering season. The chromatographic separation was performed using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:92.5:7.5) applied at a flow rate of 1 mL min(-1) and using a diode array detector. Validation procedures showed that the method was specific, accurate and precise. The highest amount of lycorine (0.012%) was detected in the bulbs of G. trojanus collected from Can (Canakkale), whereas the aerial parts of this species collected from Bayramiç (Canakkale) was not found to contain this alkaloid. In G. cilicicus samples, lycorine was only determined in the bulbs, giving yields of 0.004%; galanthamine yields were between 0.015-0.016%, but none of the G. trojanus samples contained this latter alkaloid.

  11. Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry.

    PubMed

    Weber, Allan; Ni, Jinsong; Ling, Kah-Hiing John; Acheampong, Andrew; Tang-Liu, Diane D-S; Burk, Robert; Cravatt, Benjamin F; Woodward, David

    2004-04-01

    We investigated the formation of PGF(2alpha) 1-ethanolamide, PGE(2) 1-ethanolamide, and PGD(2) 1-ethanolamide (prostamides F(2alpha), E(2), and D(2), respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH -/-) male mice. One group of three normal mice was not dosed (naïve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH -/- mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F(2alpha), prostamide E(2), prostamide D(2), and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F(2alpha) was detected in tissues in FAAH -/- mice after administration of anandamide. Concentrations of anandamide, prostamide E(2), and prostamide D(2) in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH -/- mice than those of the anandamide-treated control mice. This report demonstrates that prostamides, including prostamide F(2alpha), were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking. PMID:14729864

  12. Quantification of Hydrazine in Human Urine by HPLC-MS-MS.

    PubMed

    Isenberg, Samantha L; Carter, Melissa D; Crow, Brian S; Graham, Leigh Ann; Johnson, Darryl; Beninato, Nick; Steele, Kandace; Thomas, Jerry D; Johnson, Rudolph C

    2016-05-01

    Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels.

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

    PubMed Central

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

    2013-01-01

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

  14. Propolis extract release evaluation from topical formulations by chemiluminescence and HPLC.

    PubMed

    Marquele, Franciane D; Oliveira, Anderson R M; Bonato, Pierina S; Lara, Marilisa G; Fonseca, Maria José V

    2006-05-01

    Propolis is a resinous bee hive product that has many biological activities. Among these activities, the antioxidant activity deserves special interest since it suggests propolis could be successfully applied topically to prevent and treat skin damages. The skin is continuously exposed to free radicals generated in the aging process and by external stimuli such as sunlight. Thus, the development of topical formulations added with propolis extract is justified. However, it raises the necessity of being concerned about the methodologies that could be used to evaluate the propolis extract release from these formulations. So, p-coumaric acid content using HPLC and the antioxidant activity using chemiluminescence were used to assess the release of propolis extract from topical formulations. A low fat content formulation (F1) and a high fat content formulation (F2) were evaluated and they showed that after 6 h, 4.6 microg/cm2 (F1) and 2.75 microg/cm2 (F2) of the p-coumaric acid was released, while it was found that both formulations released about 0.85 microL/cm2 of the antioxidant activity as propolis extract equivalent (AAPEE). Thus, once the antioxidant activity of propolis extract may be the result of the synergic action of several compounds, the obtained results indicate that a release study would be more conclusive if the antioxidant activity was evaluated, besides the measurement of a marker compound content.

  15. Identification of 4-deoxythreonic acid present in human urine by combining HPLC and NMR techniques

    PubMed Central

    Appiah-Amponsah, Emmanuel; Shanaiah, Narasimhamurthy; Nagana Gowda, G. A.; Owusu-Sarfo, Kwadwo; Ye, Tao; Raftery, Daniel

    2010-01-01

    The 1H NMR spectrum of urine exhibits a large number of detectable and quantifiable metabolites and hence urine metabolite profiling is potentially useful for the study of systems biology and the discovery of biomarkers for drug development or clinical applications. While a number of metabolites (50–100) are readily detectable in urine by NMR, a much larger number is potentially available if lower concentration species can be detected unambiguously. Lower concentration metabolites are thought to be more specific to certain disease states and thus it is important to detect these metabolites with certainty. We report the identification of 4-deoxythreonic acid, a relatively low concentration endogenous metabolite that has not been previously identified in the 1H NMR spectrum of human urine. The complimentary use of HPLC and NMR spectroscopy facilitated the unequivocal and non-invasive identification of the molecule in urine which is complicated by extensive peak overlap and multiple, similar resonances from other metabolites such as 3-hydroxybutanoic acid. High-resolution detection and good sensitivity were achieved by the combination of multiple chromatographic fraction collection, sample pre-concentration, and the use of a cryogenically cooled NMR probe. PMID:19615840

  16. [Determination of magnesium stearate in pharmaceutical preparations using derivatization with 2-nitrophenylhydrazine and HPLC].

    PubMed

    Arai, Takashi; Hosoi, Yasue

    2005-03-01

    In general, spectrophotometric methods (inductively coupled plasma or atomic absorption spectrophotometry) are used for the assay of magnesium stearate (Mg-St). In this study, a new rapid, selective assay method was developed for Mg-St in pharmaceutical formulations. The method was based on isocratic reverse-phase liquid chromatography using a mobile phase of acetonitrile-water (80:20, v/v) after precolumn derivatization with 2-nitrophenyl hydrazine for sensitive UV detection. Margaric acid was used as an internal standard and the substances were detectable at 230 nm or 400 nm. Using a short (2 cm) HPLC column reduced the analytical time to 5 min. Validation of the newly developed method was performed in accordance with the International Conference on Harmonization guidelines. The linearity range for Mg-St was 0.00-0.04 mg/ml (as the concentration of injected sample solution) and their correlation factor was 0.9998. The determination and detection limits for Mg-St were 6 microg and 2 microg, respectively. The proposed method was successfully applied to the determination of trace amounts of Mg-St in commercially available tablets with a high recovery percentage, good accuracy, and precision.

  17. A rapid and simple HPLC method for the analysis of propofol in biological fluids.

    PubMed

    Cussonneau, Xavier; De Smet, Els; Lantsoght, Kristof; Salvi, Jean-Paul; Bolon-Larger, Magali; Boulieu, Roselyne

    2007-07-27

    A selective and sensitive high-performance liquid chromatographic method for the analysis of propofol in biological samples was developed. Propofol and thymol (internal standard) were analysed on a Purospher RP-18 endcapped (75 mmx4 mm, 3 microm) stationary phase using acetonitrile and water (65:35, v/v) as eluents at a flow rate of 0.6 mL/min. The excitation and emission wavelengths were 276 and 310 nm, respectively. Sample treatment consisted of deproteinization by acetonitrile containing the internal standard and direct injection of the supernatant. Mean analytical recovery were 105% (CV 2.0%) at concentrations ranging from 0.05 to 10 mg/L. The quantification limit was 3 ng/mL for a 500 microL sample plasma volume and 5 ng/mL for a 500 microL blood sample. The intra-day and inter-day precisions were lower than 5.5% for three concentrations assessed (0.05, 1.0 and 10.0 mg/L). Considering the column size and the flow rate, the separation was achieved with an analysis time less than 6 min with a reduced consumption of solvent. This rapid HPLC method using a simple treatment procedure is sensitive enough for monitoring propofol in human biological samples. PMID:17129698

  18. Quantification of Hydrazine in Human Urine by HPLC-MS-MS.

    PubMed

    Isenberg, Samantha L; Carter, Melissa D; Crow, Brian S; Graham, Leigh Ann; Johnson, Darryl; Beninato, Nick; Steele, Kandace; Thomas, Jerry D; Johnson, Rudolph C

    2016-05-01

    Currently used on F-16 fighter jets and some space shuttles, hydrazine could be released at toxic levels to humans as a result of an accidental leakage or spill. Lower-level exposures occur in industrial workers or as a result of the use of some pharmaceuticals. A method was developed for the quantitation of hydrazine in human urine and can be extended by dilution with water to cover at least six orders of magnitude, allowing measurement at all clinically significant levels of potential exposure. Urine samples were processed by isotope dilution, filtered, derivatized and then quantified by HPLC-MS-MS. The analytical response ratio was linearly proportional to the urine concentration of hydrazine from 0.0493 to 12.3 ng/mL, with an average correlation coefficientRof 0.9985. Inter-run accuracy for 21 runs, expressed as percent relative error (% RE), was ≤14%, and the corresponding precision, expressed as percent relative standard deviation (% RSD), was ≤15%. Because this method can provide a quantitative measurement of clinical samples over six orders of magnitude, it can be used to monitor trace amounts of hydrazine exposure as well as industrial and environmental exposure levels. PMID:26977107

  19. Stability indicating RP-HPLC method for simultaneous determination of piroxicam and ofloxacin in binary combination.

    PubMed

    John, Peter; Azeem, Waqar; Ashfaq, Muhammad; Khan, Islam Ullah; Razzaq, Syed Naeem

    2015-09-01

    A simple and precise RP-HPLC method was developed for simultaneous determination of piroxicam and ofloxacin in pharmaceutical formulations and human serum. Optimum separations of piroxicam, ofloxacin and stress-induced degradation products were achieved by use of Hypersil BDS C8 column (250 x 4.6mm, 5 μm). The mobile phase was a mixture of acetonitrile: 0.012M K2HPO4: 0.008M sodium citrate (both buffers mixed and pH adjusted to 2.8) (50:25:25 v/v/v) delivered at flow rate of 1.5 mL min⁻¹ using DAD at 254 nm. Response was linear function of concentration over the ranges of 70-130 mg mL⁻¹ for piroxicam and ofloxacin (r² ≥ 0.999). The method efficiently separated the analytical peaks from degradation products with acceptable tailing and resolution. The developed method was successfully used for concurrent analysis of piroxicam and ofloxacin in pharmaceutical formulations, human serum and in vitro drug interaction studies.

  20. A stability-indicating HPLC method for simultaneous determination of morphine and naltrexone.

    PubMed

    Jafari-Nodoushan, Milad; Barzin, Jalal; Mobedi, Hamid

    2016-02-01

    This study developed a stability-indicating reversed-phase HPLC method for the simultaneous determination of morphine sulfate and naltrexone hydrochloride content in bulk, Solid dosage forms and in-vitro dissolution samples to support product development and quality control efforts. Chromatographic separation of the pharmaceutical compound was achieved on a perfectSil™ MZ C18 column (250×4.6mm, 5μm) with an isocratic mobile phase composed of a mixture of acetate buffer (10mM, pH 4, containing 0.1% of 1-heptanesulfonic acid sodium salt) and acetonitrile with 80/20 at a flow rate of 1.5mlmin(-1). Both analytes were quantified using a photodiode array detector set at a wavelength of 280nm and column temperature was set to 30°C. naltrexone, morphine and a mixture of the two were subjected to thermal, peroxide, acid, base and photolytic degradation and their peak homogeneity was obtained using a photodiode array detector, demonstrating the specificity of method. These pharmaceuticals were spiked in biological fluid to examine method selectivity. The method was validated for system suitability, linearity, accuracy, precision, detection and quantification limits and robustness and was found it is acceptable in range of 2-250μgml(-1) for morphine and 4-100μgml(-1) for naltrexone.

  1. Development and application of a HPLC method for eight sunscreen agents in suncare products.

    PubMed

    Peruchi, L M; Rath, S

    2012-06-01

    This work describes the development, validation and application of a simple and fast high-performance liquid chromatography-with diode array dectection (HPLC-DAD) method for the determination of eight sunscreen agents: benzophenone-3, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, homosalate (used in two isomeric forms), butyl methoxydibenzoylmethane, 4-methylbenzylidene camphor and ethylhexyl dimethyl PABA in sunscreen formulations. The separation of the eight sunscreen compounds was achieved using an ACE C18 column (250 × 4.6 mm, 5 μm), with a column temperature 20°C, and a mobile phase of 88 : 12 (v/v) methanol-water with isocratic elution. Column temperature strongly influences the retention time and resolution of the compounds. The flow rate was 1.0 mL min(-1) and quantitation was performed by external calibration at the maximum wavelength of each compound. The sample preparation was simple and consisted basically of sample dilution with methanol, centrifugation and filtration in syringe filters before quantitation. Total run time was 18 min. The method was validated according to the parameters: linear range, linearity, selectivity, intra-day and inter-day precision and accuracy. Ten samples of sunscreen emulsions commercially available in Brazil (SPF 30) from different manufacturers were analysed using the proposed method. The number of the sunscreen agents varied between one and five in a single sample. The concentrations of all compounds were in the range of 0.9-10% (w/w) and were in accordance with the current Brazilian legislation.

  2. Absorbance detector based on a deep UV light emitting diode for narrow-column HPLC.

    PubMed

    Bui, Duy Anh; Bomastyk, Benjamin; Hauser, Peter C

    2013-10-01

    A detector for miniaturized HPLC based on deep UV emitting diodes and UV photodiodes was constructed. The measurement is accomplished by the transverse passage of the radiation from the light-emitting diode (LED) through fused-silica tubing with an internal diameter of 250 μm. The optical cell allows flexible alignment of the LED, tubing, and photodiode for optimization of the light throughput and has an aperture to block stray light. A beam splitter was employed to direct part of the emitted light to a reference photodiode and the Lambert-Beer law was emulated with a log-ratio amplifier circuitry. The detector was tested with two LEDs with emission bands at 280 and 255 nm and showed noise levels as low as 0.25 and 0.22 mAU, respectively. The photometric device was employed successfully in separations using a column of 1 mm inner diameter in isocratic as well as gradient elution. Good linearities over three orders of magnitude in concentration were achieved, and the precision of the measurements was better than 1% in all cases. Detection down to the low micromolar range was possible.

  3. Simultaneous HPLC Determination of 22 Components of Essential Oils; Method Robustness with Experimental Design

    PubMed Central

    Porel, A.; Sanyal, Y.; Kundu, A.

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil–II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R2 >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil. PMID:24799735

  4. Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC.

    PubMed

    Djurdjevic, Predrag; Ciric, Andrija; Djurdjevic, Aleksandra; Stankov, Milena Jelikic

    2009-09-01

    A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C(18) XTerra column using a mobile phase of (water+triethylamine (2%, v/v)): acetonitrile=90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 degrees C. The resolution between the two least resolved impurity peaks was in average, R(s,min) > 1.5. Method validation parameters indicate linear dynamic range 0.2-2.0 microg/ml with LOQ ca. 0.20 microg/ml and LOD ca. 0.05 microg/ml for all analytes. The method was applied for the impurities determination in drug tablets and infusion (Avelox), Bayer AG) and for degradation products determination in a stability study of moxifloxacin. The impurity content in the tablets and infusion was quantified as 0.1% of total drug. Two degradation products were noted under hydrolytic conditions. The method can also be used for rapid and accurate quantification of moxifloxacin hydrochloride in its tablets during stability testing.

  5. Identification of ellagic acid conjugates and other polyphenolics in muscadine grapes by HPLC-ESI-MS.

    PubMed

    Lee, Joon-Hee; Johnson, Jodie V; Talcott, Stephen T

    2005-07-27

    Ellagic acid, ellagic acid glycosides, and ellagitannins found in various fruits and nuts, including muscadine grape, are reported to have potential health-promoting benefits and antioxidant properties. This study isolated and identified several ellagic acid derivatives present in muscadine grapes and determined their relative antioxidant properties (AOX). Compounds were extracted from grape skins and pulp using methanol, and the solvent was evaporated. Isolates were dissolved in citric acid buffer (pH 3.5) and absorbed onto C18 cartridges. Nonretained polyphenolics were collected separately and again partitioned from Sephadex LH-20, whereas retained polyphenolics were first eluted with ethyl acetate followed by methanol. Ellagic acid derivatives were identified on the basis of UV and mass spectra, and the presence of ellagitannins was confirmed by a significant increase in free ellagic acid with HPLC followed by acid hydrolysis. Muscadine grapes contained phenolic acids, flavonols, anthocyanins, ellagic acid, and numerous ellagic acid derivatives. AOX varied in the order ethyl acetate > methanol > C18 nonretained fractions; each correlated to both total phenolics (r = 0.90) and total ellagic acid (r = 0.99) contents. Results of this study revealed previously unidentified ellagic acid derivatives in muscadine grapes.

  6. [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. PMID:25011249

  7. Submerged fermentation production and characterization of intracellular triterpenoids from Ganoderma lucidum using HPLC-ESI-MS*

    PubMed Central

    Cui, Mei-lin; Yang, Huan-yi; He, Guo-qing

    2015-01-01

    As the main bioactive metabolites of Ganoderma lucidum, triterpenoids have various pharmacological effects. In this paper, the nutritional requirements and culture conditions of a submerged culture of G. lucidum were optimized using the response surface methodology; maximum mycelia biomass and intracellular triterpenoid production reached 1.87 g/100 ml and 93.21 mg/100 ml, respectively, for a culture consisting of wort 4.10% (0.041 g/ml) and yeast extract 1.89% (0.0189 g/ml), pH 5.40. For the first time, we established that wort, which is cheap and abundant, can replace the more commonly used glucose as the sole source of carbohydrate. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), 10 major ganoderic acids were tentatively identified based on the predominant fragmentation pathways with the elimination of H2O and CO2, as well as cleavage of the D-ring. PMID:26642183

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

    PubMed

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

    2012-01-01

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

  9. Study on the precipitation reaction between baicalin and berberine by HPLC.

    PubMed

    Yi, Li; Xu, Xu

    2004-10-15

    The solubility product equilibrium constant for the precipitation between baicalin and berberine was investigated because of the current interest in decocting process of complex prescription of Chinese herbal medicine. HPLC was used for determining two compounds' equilibrium concentrations at different precipitate conditions to calculate thermodynamic constants and study kinetic process. The analysis was performed on a Kromasil C(18) column with TEA-adjusted 0.02 mol/L H(3)PO(4) (pH 4.82)-acetonitrile (75:25) as mobile phase at a flow-rate of 1.0 ml/min, with detection at 254 nm. According to the experiment result, the molar ratio of baicalin and berberine in precipitate is about 1:1. The experimental K(sp) values are (1.01 +/- 0.12) x 10(-9)mol(2)/L(2) at 20 degrees C in 0.02 mol/L NaH(2)PO(4)-Na(2)HPO(4) (pH 4.82), and (3.20 +/- 0.46) x 10(-9)mol(2)/L(2) at 40 degrees C in the same buffer. The precipitate reaction is an exothermic process and occurs immediately, even though the precipitate cannot be observed in time because the precipitate is light, yellow, flocculous and suspending in the yellow solution.

  10. Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding

    PubMed Central

    Staggs, C.G.; Sealey, W.M.; McCabe, B.J.; Teague, A.M.; Mock, D.M.

    2006-01-01

    Assessing dietary biotin content, biotin bioavailability, and resulting biotin status are crucial in determining whether biotin deficiency is teratogenic in humans. Accuracy in estimating dietary biotin is limited both by data gaps in food composition tables and by inaccuracies in published data. The present study applied sensitive and specific analytical techniques to determine values for biotin content in a select group of foods. Total biotin content of 87 foods was determined using acid hydrolysis and the HPLC/avidin-binding assay. These values are consistent with published values in that meat, fish, poultry, egg, dairy, and some vegetables are relatively rich sources of biotin. However, these biotin values disagreed substantially with published values for many foods. Assay values varied between 247 times greater than published values for a given food to as much as 36% less than the published biotin value. Among 51 foods assayed for which published values were available, only seven agreed within analytical variability (720%). We conclude that published values for biotin content of foods are likely to be inaccurate. PMID:16648879

  11. Determination of EDTA in feed and premix formulations by HPLC-DAD.

    PubMed

    Chiumiento, Francesco; D'Aloise, Antonio; Marchegiani, Francesca; Melai, Valeria

    2015-05-15

    A simple analytical method for the quantitative determination of ethylenediaminetetraacetic acid (EDTA) in feed and premix formulations was developed and validated. The method involves an extraction with an acidic ferric chloride solution, to quantitatively convert EDTA species in the samples into the Fe(III)-EDTA complex, and its subsequent detection by Ion-Pair-Reversed Phase-High Performance Liquid Chromatography-Diode Array Detection (IP-RP-HPLC-DAD). A robust validation procedure was performed according to the Decision 2002/657/EC at concentrations ranging from 25 to 100 mg kg(-1) on sample. Good recoveries (85.6-92.8%) were obtained; repeatability of the method was in the range of 1.3-8.0%, with an intermediate precision ranging from 6.0% to 8.6%, both of them expressed as relative standard deviation (RSD). No interfering species hindered the straightforward detection of EDTA. Hence, the proposed method can be adopted for an effective and rapid routine analysis of products for livestock.

  12. Derivatization Strategy for the Comprehensive Characterization of Endogenous Fatty Aldehydes Using HPLC-Multiple Reaction Monitoring.

    PubMed

    Tie, Cai; Hu, Ting; Jia, Zhi-Xin; Zhang, Jin-Lan

    2016-08-01

    Fatty aldehydes are crucial substances that mediate a wide range of vital physiological functions, particularly lipid peroxidation. Fatty aldehydes such as acrolein and 4-hydroxynonenal (4-HNE) are considered potential biomarkers of myocardial ischemia and dementia, but analytical techniques for fatty aldehydes are lacking. In the present study, a comprehensive characterization strategy with high sensitivity and facility for fatty aldehydes based on derivatization and high-performance liquid chromatography-multiple reaction monitoring (HPLC-MRM) was developed. The fatty aldehydes of a biosample were derivatized using 2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine under mild and efficient reaction conditions at 37 °C for 15 min. The limit of detection (LOD) of the fatty aldehydes varied from 0.1 to 1 pg/mL, depending on the structures of these molecules. General MRM parameters were forged for the analysis of endogenous fatty aldehydes. "Heavy" derivatization reagents with 20 deuterium atoms were synthesized for both the discovery and comprehensive characterization of fatty aldehydes. More than 80 fatty aldehydes were detected in the biosamples. The new strategy was successfully implemented in global fatty aldehyde profiling of plasma and brain tissue of the bilateral common carotid artery (2VO) dementia rat model. Dozens of fatty aldehydes were significantly changed between the control and model groups. These findings further highlight the importance of endogenous fatty aldehydes. PMID:27397858

  13. Simultaneous HPLC determination of 22 components of essential oils; method robustness with experimental design.

    PubMed

    Porel, A; Sanyal, Y; Kundu, A

    2014-01-01

    The aim of the present study was the development and validation of a simple, precise and specific reversed phase HPLC method for the simultaneous determination of 22 components present in different essential oils namely cinnamon bark oil, caraway oil and cardamom fruit oil. The chromatographic separation of all the components was achieved on Wakosil-II C18 column with mixture of 30 mM ammonium acetate buffer (pH 4.7), methanol and acetonitrile in different ratio as mobile phase in a ternary linear gradient mode. The calibration graphs plotted with five different concentrations of each component were linear with a regression coefficient R(2) >0.999. The limit of detection and limit of quantitation were estimated for all the components. Effect on analytical responses by small and deliberate variation of critical factors was examined by robustness testing with Design of Experiment employing Central Composite Design and established that this method was robust. The method was then validated for linearity, precision, accuracy, specificity and demonstrated to be applicable to the determination of the ingredients in commercial sample of essential oil. PMID:24799735

  14. Determination of Antiproliferative Activities of Volatile Contents and HPLC Profiles of Dicranum scoparium (Dicranaceae, Bryophyta).

    PubMed

    Abay, Gökhan; Altun, Muhammed; Koldaş, Serkan; Tüfekçi, Ali Rıza; Demirtas, Ibrahim

    2015-01-01

    The aim of this study was to examine the anticancer activities and phytochemical profiles of Dicranum scoparium against HeLa cell lines. The bio-guided fractionation studies of dichloromethane extract have high antiproliferative activities. Fractions 7, 9, 19, 20 are rich source of unsaturated fatty acids, and- in the case of Fr-19 may improve the antiproliferative activities as well as increase the unsaturated fatty acid content. The effect of proliferative activities in hexane extract can be attributed to the saturated fatty acid composition of D. scoparium. The Fr-9 exhibited strong antiproliferative activity at concentrations of 100 and 50 μg mL(-1) compared to 5-FU. The fractions of 7, 9, 19 and 20 from dichloromethane extracts exhibited antiproliferative activities at a concentration of 100 μg mL(-1). The HPLC-TOF/MS studies gave nine compounds from the most active fraction of dichloromethane at concentrations of 250 and 100 μg mL(-1). The lower activities were obtained from the fractions including steroid derivatives.

  15. HPLC-DAD-ESI/MS(n) profiling of phenolic compounds from Lathyrus cicera L. seeds.

    PubMed

    Ferreres, F; Magalhães, S C Q; Gil-Izquierdo, A; Valentão, P; Cabrita, A R J; Fonseca, A J M; Andrade, P B

    2017-01-01

    Lathyrus cicera L. seeds are of interest for food and feed purposes. Despite the recognized antioxidant activity of the seeds, arising from the phenolic fraction, their phenolic compounds have not been studied in depth yet. Therefore, to determine the phenolics profile of these seeds, a target analysis was performed using high-performance liquid chromatography coupled to photodiode-array detection and electrospray ionization/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)). Thirty-seven glycosylated flavonoids were identified for the first time in the seeds of this species and, according to their MS fragmentation, clustered in flavonol-3-O-di-/tri-glycosides-7-O-rhamnosides and other flavonol-glycosides, and flavonol-3-O-(cinnamoyl)glycoside-7-O-rhamnosides, flavonol-3-O-(dihydrophaseoyl, cinnamoyl)glycoside-7-O-rhamnosides and flavonol-3-O-(malonyl)glycoside-7-O-rhamnosides. Glycosides of kaempferol were the main flavonoids found (10 non-acylated and 21 acylated), followed by those of quercetin (3) and those of isorhamnetin, apigenin and luteolin (1). The most abundant flavonols were identified as kaempferol-3-O-(2-hexosyl)hexoside-7-O-rhamnosides. The methodology used allowed to increase the knowledge on a relevant phytochemical class of seeds from L. cicera. PMID:27507525

  16. Determination of S- and R-warfarin enantiomers by using modified HPLC method.

    PubMed

    Qayyum, Aisha; Najmi, Muzammil Hasan; Khan, Abdul Muqeet; Abbas, Mateen; Naveed, Abdul Khaliq; Jameel, Ahsan

    2015-07-01

    Warfarin is a commonly prescribed anticoagulant existing in two enantiomeric forms S- and R-warfarin. Many techniques have been used to analyze warfarin in plasma but less frequently for enantiomeric analysis. One of the HPLC method employed was further simplified and made economical. Method was validated according to ICH guidelines and was found to be sensitive, reliable and less time consuming. For both enantiomers, LLOQ was 12.5 ng/mL. The CV% and accuracy for method were in the range of 0.8-14.6% and 92-107% respectively. The recoveries for both enantiomers were in the range of 86-103.8%. Blood samples were collected from 170 stable patients taking warfarin and S- and R-warfarin levels were determined by this method. Majority of subjects were found to have S/R-warfarin ratio of about 1:2 as reported in previous studies due to rapid clearance of S-enantiomer than R-enantiomer. However individual subjects data was suggestive of presence of slow metabolizers of S-warfarin leading to altered S/R ratio. Previous studies have also pointed out CYP2C9 polymorphism being responsible for such inter-individual differences in S-warfarin metabolism. So plasma warfarin S/R ratio may serve as a useful phenotypic test for CYP2C9 polymorphism.

  17. New HPLC and fluorometric methods for the determination of pyriproxyfen and pyridalyl insecticide residues in tomatoes.

    PubMed

    Farouk, Maha; Hussein, Lobna A; El Azab, Noha F

    2014-01-01

    Two sensitive, selective, and precise methods for the determination of pyriproxyfen and pyridalyl insecticide residues in tomatoes have been developed. The first method is HPLC with UV detection in which pyriproxyfen and pyridalyl were extracted with ethyl acetate and acetone, respectively, followed by cleanup using column chromatography. The recoveries ranged from 86.03 to 94.55 for pyriproxyfen and 95.08 to 99.38% for pyridalyl in tomato samples. The LOD of the method was 0.217 ppm for pyriproxyfen and 0.1866 ppm for pyridalyl. The second method depends on direct fluorometric determination of pyriproxyfen and pyridalyl in acetic and sulfuric acid at excitation and emission wavelengths of 320 and 646 nm, respectively. The recoveries of pyriproxyfen and pyridalyl in tomato samples ranged from 88 to 98% and 86 to 93%, respectively. The LOD of the method was 0.146 ppm for pyriproxyfen and 0.078 ppm for pyridalyl. Both methods were applied successfully to determine residues and rate of disappearance of pyriproxyfen and pyridalyl from tomatoes. PMID:24672877

  18. HPLC-DAD-ESI/MS(n) profiling of phenolic compounds from Lathyrus cicera L. seeds.

    PubMed

    Ferreres, F; Magalhães, S C Q; Gil-Izquierdo, A; Valentão, P; Cabrita, A R J; Fonseca, A J M; Andrade, P B

    2017-01-01

    Lathyrus cicera L. seeds are of interest for food and feed purposes. Despite the recognized antioxidant activity of the seeds, arising from the phenolic fraction, their phenolic compounds have not been studied in depth yet. Therefore, to determine the phenolics profile of these seeds, a target analysis was performed using high-performance liquid chromatography coupled to photodiode-array detection and electrospray ionization/ion trap mass spectrometry (HPLC-DAD-ESI/MS(n)). Thirty-seven glycosylated flavonoids were identified for the first time in the seeds of this species and, according to their MS fragmentation, clustered in flavonol-3-O-di-/tri-glycosides-7-O-rhamnosides and other flavonol-glycosides, and flavonol-3-O-(cinnamoyl)glycoside-7-O-rhamnosides, flavonol-3-O-(dihydrophaseoyl, cinnamoyl)glycoside-7-O-rhamnosides and flavonol-3-O-(malonyl)glycoside-7-O-rhamnosides. Glycosides of kaempferol were the main flavonoids found (10 non-acylated and 21 acylated), followed by those of quercetin (3) and those of isorhamnetin, apigenin and luteolin (1). The most abundant flavonols were identified as kaempferol-3-O-(2-hexosyl)hexoside-7-O-rhamnosides. The methodology used allowed to increase the knowledge on a relevant phytochemical class of seeds from L. cicera.

  19. Analysis of Tetracyclines in Medicated Feed for Food Animal Production by HPLC-MS/MS

    PubMed Central

    Gavilán, Rosa Elvira; Nebot, Carolina; Miranda, Jose Manuel; Martín-Gómez, Yolanda; Vázquez-Belda, Beatriz; Franco, Carlos Manuel; Cepeda, Alberto

    2015-01-01

    The use of medicated feed is a common practice in animal food production to improve animal health. Tetracyclines and β-Lactams are the groups that are most frequently added to this type of feed. The measurement of the concentration of the analytes in these types of samples is sometimes due to the matrix characteristic, and manufacturers are demanding fast, precise and reproducible methods. A rapid confirmatory method based on a simple extraction protocol using acidified methanol and followed by high performance liquid chromatography coupled to a tandem mass spectrometer for the quantification of four tetracyclines in feed is presented. Validation was performed following the guidelines of Decision 2002/657/EC. Results indicated that the four tetracyclines can be identified and quantified in a concentration range between 50 and 500 mg/kg with recoveries between 84% and 109% and RSD for precision under reproducible conditions between 12% and 16%. Satisfactory results were also obtained with interlaboratory studies and by comparing the method with an HPLC-Fluorescent method. PMID:27025516

  20. Analysis of Tetracyclines in Medicated Feed for Food Animal Production by HPLC-MS/MS.

    PubMed

    Gavilán, Rosa Elvira; Nebot, Carolina; Miranda, Jose Manuel; Martín-Gómez, Yolanda; Vázquez-Belda, Beatriz; Franco, Carlos Manuel; Cepeda, Alberto

    2015-12-24

    The use of medicated feed is a common practice in animal food production to improve animal health. Tetracyclines and β-Lactams are the groups that are most frequently added to this type of feed. The measurement of the concentration of the analytes in these types of samples is sometimes due to the matrix characteristic, and manufacturers are demanding fast, precise and reproducible methods. A rapid confirmatory method based on a simple extraction protocol using acidified methanol and followed by high performance liquid chromatography coupled to a tandem mass spectrometer for the quantification of four tetracyclines in feed is presented. Validation was performed following the guidelines of Decision 2002/657/EC. Results indicated that the four tetracyclines can be identified and quantified in a concentration range between 50 and 500 mg/kg with recoveries between 84% and 109% and RSD for precision under reproducible conditions between 12% and 16%. Satisfactory results were also obtained with interlaboratory studies and by comparing the method with an HPLC-Fluorescent method.