Biomedical Chromatography

Williamson, Leah N.; Bartlett, Michael G.

doi: 10.1002/bmc.844pmid: 17474074

Over the last decade, time‐of‐flight (TOF) instruments have increasingly been used as quantitation tools. In addition, because of their high resolving power, they can be used for verification of empirical formulas. Historically, TOF instruments have had limited quantitation capabilities because of their narrow dynamic range. However, recent advances have improved these limitations. This review covers the rationale for using TOF for LC detection, and describes the many methods currently in the literature for the quantitation of pharmaceuticals, environmental pollutants, explosives and many phytochemicals. Copyright © 2007 John Wiley & Sons, Ltd.

Yu, Zhiguo; Gao, Xiaoxia; Zhao, Yunli; Bi, Kaishun

doi: 10.1002/bmc.764pmid: 17385809

A high‐performance liquid chromatographic method was developed for the simultaneous determination and pharmacokinetic studies of safflor yellow A, puerarin, 3′‐methoxyl‐puerarin, and puerarinapioside in the plasma and tissues of rats that had been administered with the traditional Chinese medicine (TCM) preparation Naodesheng via the caudal vein. Samples taken from rats were subjected to protein precipitation with acetone. Separation of these four compounds was accomplished on a Kromisil C18 stationary phase using a mobile phase of acetonitrile–0.1% phosphoric acid–tetrahydrofuran (8:92:2, v/v/v) at a flow‐rate of 1.0 mL/min. The detection wavelength was set at 250 nm. The calibration curves of the four components were linear in the given concentration ranges. The intra‐ and inter‐day precisions in plasma and tissues were less than 15% and the extraction recoveries were higher than 60%. The lower limits of quantitation of four components were low enough to determine the four components. These four components all exhibited kinetics that fitted a two‐compartment model in rats. The elimination half‐life was 1.19 h for safflor yellow A, 2.69 h for puerarin, 2.94 h for 3′‐methoxyl‐puerarin, and 0.87 h for puerarinapioside, respectively. Following administration of a single injection of Naodesheng, the concentration (C) of the four components in the tissues showed Ckidney > Clung, Cliver > Cspleen, Cstomach, Cheart, approximately. The method is a reliable tool for performing studies of safflor yellow A and three puerarin isoflavones in different biological material. Copyright © 2007 John Wiley & Sons, Ltd.

Samtani, Mahesh N.; Jusko, William J.

doi: 10.1002/bmc.788pmid: 17385808

A sensitive, specific, accurate and precise LC/MS/MS method was developed for the simultaneous measurement of dexamethasone and corticosterone in rat plasma. The method was extended to dexamethasone analysis in rat plasma ultrafiltrate and fetal tissues. Samples were processed using SPE involving Oasis HLB cartridges, which offered complete extraction recovery for the analytes. Samples were subsequently analyzed using LC/MS/MS. A structurally related corticosteroid, prednisolone, was used as the internal standard. Using a 500 µL plasma sample, limits of quantification of 0.2 and 2.0 ng/mL were achievable for dexamethasone and corticosterone. This level of sensitivity allowed characterization of maternal/fetal dexamethasone profiles after administration of multiple doses of dexamethasone sodium phosphate to rats. However, this sensitivity was not satisfactory for corticosterone during pharmacokinetic studies involving dexamethasone due to its strong adrenosuppressive effect. This led us to investigate the suitability of a commercially available radioimmunoassay kit, which through extensive testing and minor modifications was found to offer extremely sensitive, specific, accurate and precise analysis of corticosterone. Knowledge of the steroid profiles captured using these highly sensitive analytical tools may potentially help in the optimization of corticosteroid therapy during pregnancy. Copyright © 2007 John Wiley & Sons, Ltd.

Hou, Yuan‐Yuan; Peng, Jia‐Min; Chao, Ruo‐Bing

doi: 10.1002/bmc.791pmid: 17326051

In order to research the pharmacokinetics of salvianolic acid A (SalA), a herbal ingredient isolated from Salvia miltiorrhiza Bunge, after intravenous administration to rats, a specific and accurate high‐performance liquid chromatography (HPLC) was developed. The assay procedure involved simple liquid–liquid extraction of SalA and internal standard (IS, ethyl‐p‐hydroxybenzoate) from plasma into ethyl acetate. The organic layer was separated and evaporated under reduced pressure at 40°C. The residue was reconstituted in the mobile phase and analyzed on an Inertsil C8 column, monitored at 285 nm. The mobile phase, which consisted of methanol–acetonitrile–water–formic acid (10:20:70:0.4, by vol), was used at a flow rate of 1.0 mL/min. The ratio of the peak area of the analyte to IS was applied to quantify the plasma samples. The standard curve for SalA was linear (r2 = 0.9999) in the concentration range of 0.75–150 µg/mL. The limit of quantitation (LOQ) of SalA was 0.75 µg/mL. The intra‐ and inter‐day precisions (RSD) of the quality control (QC) samples were in the ranges of 2.17–3.29 and 1.24–5.28%, respectively. Accuracy in the measurement of QC samples ranged from 94.7 to 101.1%. This method was validated for specificity, accuracy and precision and was successfully applied to the pharmacokinetic study of SalA in rat plasma after intravenous administration of Danshen injection. Copyright © 2007 John Wiley & Sons, Ltd.

Cho, Soon‐Kil; Abd El‐Aty, A. M.; Park, Young‐Seok; Choi, Jeong‐Heui; Khay, Sathya; Kang, Cheol‐Ah; Park, Byung‐Jun; Kim, Sun‐Ju; Shim, Jae‐Han

doi: 10.1002/bmc.792pmid: 17385804

An analytical procedure using accelerated solvent extraction and gas chromatography with an electron capture detector has been optimized to simultaneously determine the residue of two insecticides (diazinon and EPN) and one fungicide (isoprothiolane) in polished rice and was confirmed by GC–mass spectrometry. Several parameters, including temperature, pressure, solvent ratio, cell size and cell cycle, were thoroughly investigated to find the optimal extraction conditions. The average recoveries of the three pesticides were between 82.7 and 126.4% at spiking levels of 0.1 and 0.5 ppm. The relative standard deviations were less than 7% for all of the recovery tests. The optimum accelerated solvent extraction operating conditions were 100°C, 1500 atm, acetone–n‐hexane (20:80 v/v) as the extraction solvent, two cycles, and a cell size of 33 ml. The total extraction time was approximately 20 min. The optimized procedure has also been applied to the determination of diazinon, isoprothiolane and EPN in real rice samples. In conclusion, accelerated solvent extraction was used for the first time for the analysis of diazinon, isoprothiolane and EPN in polished rice and offers the possibility of a fast and simple process for obtaining a quantitative extraction of the studied pesticides. Copyright © 2007 John Wiley & Sons, Ltd.

Zheng, Ren‐Chao; Zheng, Yu‐Guo; Shen, Yin‐Chu

doi: 10.1002/bmc.793pmid: 17340564

A simple and sensitive method employing gas chromatography was developed for the enantioseparation and determination of 2,2‐dimethylcyclopropanecarboxamide and its acid in the bioconversion broth. Samples were recovered in high yield by extracting the broth with equal volumes of ethyl acetate twice. Separation of the four enantiomers was performed on a gamma‐cyclodextrin based chiral column BGB‐175. The effect of column flow rate and temperature on the retention and resolution of the enantiomers was investigated. The proposed method exhibited good linearity, repeatability and precision, and was successfully used to monitor and control the bioconversion process of 2,2‐dimethylcyclopropanecarboxamide. Copyright © 2007 John Wiley & Sons, Ltd.

Wada, Mitsuhiro; Nishiwaki, Junichiro; Yamane, Tomoko; Ohwaki, Yuichi; Aboul‐Enein, Hassan Y.; Nakashima, Kenichiro

doi: 10.1002/bmc.794pmid: 17340563

The present study aims to investigate the possibility of interaction of aspirin (Asp) or clopidogrel (CG) on donepezil (DP) hydrochloride in rats by HPLC‐fluorescence detection. The separation of DP was achieved in ca. 13 min without interference of Asp and CG on the chromatogram. DP levels in rat plasma with a single administration of DP (5 mg/kg, i.p., group I) and those with a co‐administration of Asp (200 mg/kg, p.o., group II or 200 mg/kg, i.p., group III) or CG (5 mg/kg, p.o., group IV) were monitored. The DP concentrations determined in rat plasma ranged from 25.0 to 336.1 ng/mL. Pharmacokinetic parameters for these groups were calculated and compared with one another. No significant difference was observed on the comparison of group I with other groups except for the mean resident time of group IV (p = 0.012). These basic findings may help clinical inference when DP is co‐administered with Asp and CG to human. Copyright © 2007 John Wiley & Sons, Ltd.

Sparidans, Rolf W.; Prins, Jan M.; Schellens, Jan H.M.; Beijnen, Jos H.

doi: 10.1002/bmc.797pmid: 17340566

A liquid chromatography–tandem mass spectrometric assay for the determination of the antiretroviral nucleoside emtricitabine in human plasma was developed and validated using a simple sample pre‐treatment procedure. After addition of 5′‐deoxy‐5‐fluorocytidine as the internal standard and protein precipitation with acetonitrile, the supernatant was directly injected in the isocratic chromatographic system using a polar embedded reversed‐phase column and formic acid in water–methanol as the eluent. The eluate was completely led into an electrospray interface with positive ionization and the analytes were quantified using triple quadrupole mass spectrometry. The assay was validated in the range 5–5000 ng/mL. Intra‐day precisions were ≤7% and inter‐day precisions were ≤10%. Accuracies between 92 and 99% were found. The analytes were chemically stable under all relevant conditions and the assay was applied in the analysis of plasma samples of HIV‐infected patients treated with the drug. Copyright © 2007 John Wiley & Sons, Ltd.

Chen, Yunyun; Xing, Dongming; Wang, Wei; Ding, Yi; Du, Lijun

doi: 10.1002/bmc.798pmid: 17385810

The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post‐ischemic recovery. An IP‐HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro‐2a cell line. The chromatographic conditions were: a Zorbax SB‐C18 reversed‐phase column; mobile phase 100 mm KH2PO4, 1 mm tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 m), filtered through a 0.45 µm Millipore filter and degassed prior to use. The flow‐rate was 1.0 mL/min. The injection volume was 20 µL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 ± 1°C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4°C) in 1 mL of an ice‐cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform. Copyright © 2007 John Wiley & Sons, Ltd.