Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.     

HPLC method for determination of verapamil in human plasma after solid-phase extraction

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm × 4 mm I.D., 5 μm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH₂PO₄ with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10–500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.     

Determination of diarylheptanoids from Alpinia officinarum (Lesser Galangal) by HPLC with photodiode array and electrochemical detection

Normal-phase column chromatography followed by semi-preparative reversed-phase HPLC has been used to isolate, from the rhizomes of Alpinia officinarum, five diarylheptanoids identified as 5-hydroxy-7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone, 5-methoxy-7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone, 7-(4"-hydroxyphenyl)-1-phenylhept-4-en-3-one, 7-(4"-hydroxy-3"-methoxyphenyl)-1-phenyl-hept-4-en-3-one, 1,7-diphenylhept-4-en-3-one. The levels of these five diarylheptanoids in root material were determined quantitatively by HPLC with UV detection and the assay methods so developed were simple, rapid and accurate. Four of the diarylheptanoids could also be detected by HPLC with electrochemical detection (ECD) in the oxidative mode, and ECD was found to have a higher sensitivity than photodiode array detection.     

The determination of homocysteine-thiolactone in human plasma

The thioester homocysteine-thiolactone, a reactive metabolite of homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of homocysteine-thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma homocysteine-thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma homocysteine-thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma homocysteine-thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o-phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, homocysteine-thiolactone concentrations in plasma from normal healthy human subjects (n=60) were found to vary from zero to 34.8 nM, with an average of 2.82+/-6.13 nM. In 29 of the 60 human plasma samples analyzed, homocysteine-thiolactone levels were below the detection limit. Homocysteine-thiolactone represented from 0 to 0.28%, on average 0.023+/-0.05%, of plasma total homocysteine.     

Determining the pharmacokinetics of psilocin in rat plasma using ultra-performance liquid chromatography coupled with a photodiode array detector after orally administering an extract of Gymnopilus spectabilis

This study established ultra-performance liquid chromatography coupled with a photodiode array detector for determining psilocin and its pharmacokinetics in rat plasma after orally administering an extract of Gymnopilus spectabilis. The extract was separated on an ODS C18 column (2.3 μm, 100 mm × 2.1 mm I.D.) by gradient elution with (A) water containing 50mM AcONH(4) and (B) acetonitrile. The wavelength was set at 265 nm and the injection volume was 10 μL. Under these conditions, the calibration curve was linear over the concentration range 0.2-20 μg/mL with a correlation coefficient of r(2)=0.9992. The inter- and intraday precision levels were less than 7% and the accuracies (%) were within the range 92.0-102.5%. The method was sufficiently valid to be applied to a pharmacokinetics study of psilocin in rat plasma. The pharmacokinetic parameters of psilocin in rat plasma after the oral administration of a G. spectabilis extract were as follows: C(max), 0.43 ± 0.12 μg/mL; T(max), 90 ± 2.1 min; AUC(0→t), 1238.3 ± 96.4 (μg/mL) min; and T(1/2), 117.3 ± 40.3 min.     

Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector

A new method based on fluorescence derivatization with 5‐(dimethylamino) naphthalene‐1‐sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high‐performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C₁₈ column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o‐phosphoric acid, 1.2 ml/min. The injection volume was 20 μl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emission 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27–70.99 and 18.81–212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24–0.59 and 0.35–0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13–0.51 and 0.04–0.15, respectively.     

Determination of solifenacin in human plasma by liquid chromatography-tandem mass spectrometry

A liquid chromatography-electrospray tandem mass spectrometry method was developed and validated to quantitate solifenacin in human plasma. The assay was based on protein precipitation with methanol and liquid chromatography performed on a pentafluorophenylpropylsilica column (50×4mm, 3μm particles), the mobile phase consisted of methanol - 100mM ammonium acetate containing 1% of formic acid (90:10, v/v). Quantification was through positive-ion mode and selected reaction monitoring at m/z 363→193 and 368→198 for solifenacin and the internal standard solifenacin-D(5), respectively. The lower limit of quantitation was 0.47ng/ml using 0.25ml of plasma and linearity was demonstrated up to 42ng/ml. Intra-assay and inter-assay precision expressed by relative standard deviation was less than 11% and inaccuracy did not exceed 11% at all levels. The assay was applied to the analysis of samples from a pharmacokinetic study.     

Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection

A reversed-phase HPLC method for the simultaneous analysis of nine structurally similar withanolides, namely, 27-hydroxy withanone, 17-hydroxy withaferin A, 17-hydroxy-27-deoxy withaferin A, withaferin A, withanolide D, 27-hydroxy withanolide B, withanolide A, withanone and 27-deoxywithaferin A, has been developed using a linear binary gradient solvent system comprising methanol and water containing 0.1% acetic acid. Both photodiode array and evaporative light scattering detection were used to profile the extract compositions and to quantify the withanolides therein. Homogeneity and purity of each peak was ascertained by comparative evaluation of the on-line UV spectra of the eluted compounds with those of the reference compounds. The method has been validated with respect to various parameters of performance quality including computation regression analysis based on calibration curves, peak resolution factor, asymmetry factor, tailing factor, RSD (%) of retention time and peak area response, limit of quantivation, limit of detection, precision and recovery. The developed method has been applied to the analysis of leaf and root tissues of Withania somnifera for withanolide content.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Determination of the enantiomers of citalopram,its demethylated and propionic acid metabolites in human plasma by chiral HPLC

A stereoselective HPLC assay has been developed to analyze the enantiomers of citalopram and of its three main metabolites in plasma after their separation on a Chiracel OD column. Using a fluorescence detector, the limit of quantification in plasma samples was 15, 4, 5, and 2 ng/ml for the enantiomers of citalopram (CIT), desmethylcitalopram (DCIT), didesmethylcitalopram (DDCIT), and for the citalopram propionic acid derivative (CIT-PROP), respectively. Except for CIT, all metabolites were derivatized with achiral reagents. Identification of the enantiomers was realized with an optical rotation detector which showed that the enantiomers invert their rotation depending on the polarity and nature of the solvent. Under varying conditions, a racemization study has shown that the pure enantiomers of CIT and its demethylated metabolites are configurationally stable. Preliminary results obtained with five patients treated with CIT show a mean S/R ratio of 0.7 for both CIT and its active metabolite DCIT and of 3.6 for CIT-PROP in plasma. This suggests that the pharmacologically relevant (+)-(S)-isomers of CIT and DCIT could be preferentially and steroselectively metabolized to CIT-PROP. © 1995 Wiley-Liss, Inc.     

Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

Determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry

A fast and robust method for the determination of platinum in human subcellular microsamples by inductively coupled plasma mass spectrometry was developed, characterized, and validated. Samples of isolated DNA and exosome fractions from human ovarian (2008) and melanoma (T289) cancer cell lines were used. To keep the sample consumption to approximately 10 microl and obtain a high robustness of the system, a flow injection sample introduction system with a 4.6-microl sample loop was used in combination with a conventional pneumatic nebulizer and a spray chamber. The system was optimized with respect to signal/noise ratio using a multivariate experimental design. The system proved to be well suited for routine analysis of large sample series, and several hundreds of samples could be analyzed without maintenance or downtime. The detection limit of the method was 0.12 pg (26 pg/g) platinum. To avoid systematic errors from nonspectral interferences, it was necessary to use reagent matched calibration standards or isotope dilution analysis. An uncertainty budget was constructed to estimate the total expanded uncertainty of the method, giving a quantification limit of 2.3 pg (0.5 ng/g) platinum in DNA samples. The uncertainty was sufficiently low to study quantitative differences in the formation of Pt-DNA adducts after treatment with cisplatin using different exposure times and concentrations.     

高效液相色谱法测定大鼠血浆中来那度胺的浓度及其药动学* 龚

摘要目的：建立高效液相色谱法测定大鼠血浆中来那度胺的浓度。方法：色谱柱采用Venusil ASB C18 column (4.6 mm× 250 mm, 5 μm) 购自于博纳艾杰尔科技有限公司。样品采用梯度洗脱,流动相为乙腈和0.05%甲酸溶液,流速为1.0 mL/min,检测波长为 254 nm,以沙利度胺为内标。结果：来那度胺血药浓度的线性范围为0.1-5 滋g/mL,最低检测限为0.1 μg/mL,三个浓度的QC 样品 (0.2, 1 and 5μg/mL)的提取回收率分别为78.8± 3.8, 80.1 ± 3.2 and 79.1 ± 7.6 %。结论：此方法准确、简便、灵敏度高,对来那度胺 的血药浓度测定和药物代谢动力学研究极具价值。     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.     

高压液相荧光检测法检测血浆同型半胱氨酸

目的:建立高压液相荧光检测法测定血浆中同型半胱氨酸(homocysteine,Hcy)的方法.方法:应用Symmetry ShieldTMRP18色谱柱,0.08 mol/L醋酸钠1%甲醇为流动相,与巯基特异结合的荧光物质SBD-F衍生巯基来检测血浆中的同型半胱氨酸浓度.结果:该法的平均回收率为95.8～100.8%,相对标准差为1.2～2.0%.结论:本检测法方法准确,可用于实验室样品的测定.     

Rapid identification of elaiophylin and geldanamycin inStreptomyces fermentation broths using CPC coupled with a photodiode array detector and LC-MS methodologies

During the course of screening microbial broth extracts in various high through-put bioassays (eg receptor binding or enzyme inhibition), several actinomycete cultures were discovered to produce active metabolites. The natural products elaiophylin and/or geldanamycin are produced by severalStreptomyces violaceusniger strains, and the bioactivity of the extracts from these cultures was frequently associated with the fractions containing these metabolites. CPC coupled to a photodiode array detector and LC-MS techniques were applied to these broth extracts to ascertain rapidly when these natural products were present. These methodologies allowed us to identify the metabolites quickly in the crude extract, and the application demonstrated further the utility of CPC-photodiode array detection and LC-MS as powerful, initial analytical tools in analyses of the complex metabolite profiles produced by microorganisms.     

Simultaneous quantification of capsaicin and dihydrocapsaicin in rat plasma using HPLC coupled with tandem mass spectrometry

A rapid, simple and sensitive HPLC–ESI–MS/MS method was developed for the simultaneous determination of capsaicin and dihydrocapsaicin in rat plasma. Plasma samples containing capsaicin, dihydrocapsaicin and phenacetin (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with mobile phase of acetonitrile/water (55:45, v/v) containing 0.1% formic acid (v/v) at a flow rate of 0.2 mL/min with an operating temperature of 25 °C. Quantification was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source by selected reaction monitoring (SRM) of the transitions at m/z 306–137 for capsaicin, m/z 308–137 for dihydrocapsaicin and m/z 180–110 for the IS. Linear detection responses were obtained for capsaicin and dihydrocapsaicin ranging from 1 to 500 ng/mL and the lower limits of quantitation (LLOQs) for the two compounds were 1 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 9.79% for the two analytes, while the deviations of assay accuracies were within ±10.63%. The average recoveries of the analytes were greater than 89.88%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of capsaicin and dihydrocapsaicin in rats after subcutaneous administration of capsaicin (natural, containing 65% capsaicin and 35% dihydrocapsaicin).     

Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection

A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from ?15.0 to ?0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.     

Analysis of 12 beta-lactam antibiotics in human plasma by HPLC with ultraviolet detection

A simple and economical high performance liquid chromatography method was developed and validated for routine analysis of 12 Penicillin, Cephalosporin and Carbapenem antibiotics in 200 μL of human plasma. Antibiotics determined were Ceftazidime, Meropenem, Ceftriaxone, Ampicillin, Cefazolin, Ertapenem, Cephalothin, Benzylpenicillin, Flucloxacillin, Dicloxacillin, Piperacillin and Ticarcillin. There was a common sample preparation approach involving precipitation of proteins with acetonitrile and removal of lipid-soluble components by a chloroform wash. Separations were performed on a Waters X-bridge C18 column with, depending on analytes, one of three acetonitrile–phosphate buffer mobile phases. Detection was by UV at 210, 260 and 304 nm. Validation has demonstrated the method to be linear, accurate and precise. The method has been used in a pathology laboratory for therapeutic drug monitoring (TDM) of beta-lactams in critically ill patients.     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.