May 2008

A high-performance liquid chromatographic (HPLC) method for determining lehmannine (LMN) in rat plasma was developed for application in the pharmacokinetics study. The plasma was deproteinized with acetonitrile that contained an internal standard and was separated from the aqueous layer by adding sodium chloride. The HPLC assay was carried out using a VP-ODS column at 40°C. The mobile phase was acetonitrile-0.02 mol/L ammonium acetate buffer-triethylamine (35:65:0.04, v/v/v). The flow rate was 1.0 mL/min. The detection wavelength was set at 220 nm. The method was used to determine the concentration-time profiles of LMN in the plasma following oral administration or bolus injection of LMN aqueous solution. The pharmacokinetic parameters of LMN were calculated for the first time by Drug and Statistics 1.0 program. Copyright © 2008 John Wiley & Sons, Ltd.

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A method which involves combination of centrifugal ultrafiltration sampling with high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) analysis was established for screening bioactive compounds binding to calf thymus deoxyribonucleic acid (ct-DNA) from the extracts of Lonicera japonica. Four compounds were screened out and identified as rutin, quercetin-3-O-glucoside, luteolin-7-O-glucoside and lonicerin, based on the comparison of retention time, UV spectra and MS data with those of standards. The DNA-binding capabilities of the latter three flavonoids were found for the first time. The binding mechanisms of rutin, quercetin-3-O-glucoside and luteolin-7-O-glucoside with ct-DNA at the molecular level were explored using acridine orange (AO) as a fluorescence probe. Groove binding is the most appropriate binding mode of these three flavonoids to DNA, according to ultraviolet absorption and fluorescence spectra, as well as melting temperature (Tm) curves and viscosity measurements. The binding constants of rutin, quercetin-3-O-glucoside and luteolin-7-O-glucoside with DNA-AO complex were 3.81 × 103, 3.37 × 103 and 5.50 × 103 L/mol, respectively. Copyright © 2008 John Wiley & Sons, Ltd.

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Pharmacokinetics of diphenidol (DPN) is limited due to the lack of analytical methodology. Here, a micro-assay for DPN quantification was developed, by coupling ultra-performance liquid chromatography with tandem mass spectrometry. The procedure involved plasma precipitation and injection of supernatant into UPLC with an AcquityTM C18 column. Detection was in positive electrospray, following transitions of m/z 310.3 [rarr] 292.3 and m/z 275.3 [rarr] 230.2 for DPN and chlorphenamine (internal standard), respectively. The method was linear with a range of 4-400 ng/mL, and a 2 min run time. This method was applied in a switchability trial, where both formulations of DPN were bioequivalent. Copyright © 2008 John Wiley & Sons, Ltd.

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A method for the determination of the organic acids directly in the urine employing derivatization with trimethyloxonium tetrafluoroborate as a methylating agent and sequential extraction by head space and direct immersion/solid phase microextraction is reported. Furoic acid, hippuric acid, methylhippuric acid, mandelic acid, phenylglyoxylic acid and trans, trans muconic acid contained in urine and proposed by the American Conference of Governmental Industrial Hygienists as biological exposure indices were determined after a fast and economically convenient preparation step and sensitive gas chromatography-ion trap-mass spectrometry/tandem mass spectrometry analysis. Urine is rather a complex sample and hence the acquisition method required specific GC-MS instrumentation capable of supporting the changeover, fully automated during a single chromatographic separation, from mass to tandem mass spectrometry and both chemical and electron ionization modes. The automation of the analytical method provides a number of advantages, including reduced analysis time for both routine analysis and method development, and greater reproducibility. The equilibrium and kinetics of this substances vs head space/direct immersion-solid phase microextraction were investigated and evaluated theoretically. Copyright © 2008 John Wiley & Sons, Ltd.

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A simple and specific analytical method was developed and tested for the determination of pharmaceuticals in mollusc samples. A combination of microwave-assisted micellar extraction (MAME) and solid-phase extraction (SPE) using a non-ionic surfactant, polyoxyethylene 10 lauryl ether, was examined to extract and determine simultaneously a group of pharmaceuticals such as carbamazepine, clorfibric acid, ketoprofen, naproxen, bezafibrate and ibuprofen by liquid chromatography using UV-diode array detector. The MAME extraction performance was evaluated by studying various parameters such as the volume and concentration of surfactant and microwave conditions. Finally, an OASIS HLB cartridge was used as an optimum SPE sorbent to clean up the extracts and preconcentrate the selected analytes. The proposed method showed satisfactory linearity and reproducibility (between 3 and 15%), as well as detection limits ranging from 30 to 220 ng/g. Finally, the method was successfully applied to the determination of the target pharmaceuticals in various kinds of mollusc samples. This study has demonstrated that microwave-assisted micellar extraction with solid-phase extraction may be used as a viable alternative to conventional methods for the extraction of pharmaceuticals in this type of matrices. Copyright © 2008 John Wiley & Sons, Ltd.

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An analytical method based on high-performance liquid chromatographic (HPLC) with ultraviolet (UV) detection was developed for determination of scopolin in rat plasma using aesculin as internal standard (IS). After protein precipitation of plasma sample with methanol, the supernatant was directly injected and analyzed. Chromatographic separation was achieved on a C18 column using methanol and distilled water (22:78, v/v) containing 0.2% (v/v) glacial acetic acid as mobile phase with a column temperature of 30°C. The UV detector was set at 338 nm. The calibration curve was linear over the range of 0.105-13.125 µg/mL with a correlation coefficient of 0.9998. The retention times of aesculin and scopolin were 10.4 and 12.8 min, respectively. The recoveries for plasma samples of 0.105, 4.725 and 13.125 µg/mL were 91.08, 95.30 and 96.10%, respectively. The RSD of intra- and inter-day assay variations was less than 7.35%. The lower limit of detection was 0.03 µg/mL .This HPLC assay is a simple, sensitive and accurate and was successfully applied to the pharmacokinetic study of scopolin in rats. Copyright © 2008 John Wiley & Sons, Ltd.

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A bioequivalence study of two formulations containing norfloxacin was used for identification and assay of the metabolite of norfloxacin in human serum samples. The bioequivalence study was based on an analytical method using liquid chromatography with fluorescence detection. The plasmatic profile of metabolite was similar to norfloxacin for both formulations. Three plasma fractions of norfloxacin metabolite from a volunteer were isolated by liquid chromatography and investigated by atmospheric-pressure chemical ionization mass-spectrometry. The structure of norfloxacin metabolite (7-aminoethylenamino-6-fluoro-4-hydroxy quinoline-3-carboxylic acid) was identified taking into account also the mass spectrometry investigations achieved for norfloxacin and ciprofloxacin (used as internal standard for the analytical method). A theoretical procedure based on quantum chemical calculations has been also used to explain the mass fragmentation in molecules of norfloxacin metabolite that differ from the molecule of norfloxacin. Copyright © 2008 John Wiley & Sons, Ltd.

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A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 [rarr] 263.8, and for IS was m/z 349.0 [rarr] 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers. Copyright © 2008 John Wiley & Sons, Ltd.

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A reversed-phase high-performance liquid chromatographic method with diode array detection was established to simultaneously determine the seven bioactive lignans in Herpetospermum caudigerum, namely ent-isolariciresinol (1), dehydrodiconiferyl alcohol (2), herpetrione (3), herpetin (4), herpetetrone (5), herpetotriol (6) amd herpetal (7). The HPLC assay was performed on a Restek Pinnacle DB C18 column (250 × 4.6 mm, 5 µm) with gradient elution of acetonitrile and 0.1% phosphoric acid within 65 min. The detection wavelength was 240 nm. The flow-rate was 1.0 mL/min. All calibration curves showed good linearity (r2 > 0.9998) within test ranges. The method was reproducible with intra- and inter-day variation of less than 1.98%. The method provided good accuracy with recoveries in the range 95.19-102.64% with RSDs less than 1.52%. The method was successfully applied to the quantification of seven constituents in 15 H. caudigerum samples collected from different cities. The results indicated that the developed assay could be considered as a suitable quality control method for H. caudigerum. Copyright © 2008 John Wiley & Sons, Ltd.

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