Biomedical Chromatography

Lee, Myung‐Jae; Lee, Heon‐Woo; Kang, Jong‐Min; Seo, Ji‐Hyung; Tak, Seong‐Kun; Shim, Wangseob; Yim, Sung‐Vin; Hong, Seung Jae; Lee, Kyung‐Tae

doi: 10.1002/bmc.1392pmid: 20099369

We describe a simple, rapid and sensitive high‐performance liquid chromatography–electrospray ionization tandem mass spectrometric method that was developed for the simultaneous determination of carebastine and pseudoephedrine in human plasma using cisapride as an internal standard. Acquisition was performed in multiple‐reaction monitoring mode by monitoring the transitions: m/z 500.43 > 167.09 for carebastine and m/z 166.04 > 147.88 for pseudoephedrine. The devised method involves a simple single‐step liquid–liquid extraction with ethyl acetate. Chromatographic separation was performed on a C18 reversed‐phase chromatographic column at 0.2 mL/min by isocratic elution with 10 mM ammonium formate buffer–acetonitrile (30:70, v/v; adjusted to pH 3.3 with formic acid). The devised method was validated over 0.5–100 ng/mL of carebastine and 5–1000 ng/mL of pseudoephedrine with acceptable accuracy and precision, and was successfully applied to a bioequivalence study involving a single oral dose (10 mg of ebastine plus 120 mg of pseudoephedrine complex) to healthy Korean volunteers. Copyright © 2010 John Wiley & Sons, Ltd.

Elbashir, Abdalla A.; Aboul‐Enein, Hassan Y.

doi: 10.1002/bmc.1417pmid: 20352651

Conductivity detection, which is universal in capillary electrophoresis (CE), has received considerable attention, since the introduction of the axial capacitively coupled contactless detector C4D in 1998. This detector is made of two electrodes which are placed cylindrically around the CE capillary and connected to the AC oscillator. The distance between the electrodes is the detection gap. In this review, applications of CE and MCE with C4D in pharmaceutical and biological analysis are presented.

Tsakalozou, Eleftheria; Horn, Jamie; Leggas, Mark

doi: 10.1002/bmc.1404pmid: 20853460

AR‐67 (7‐t‐butyldimethylsilyl‐10‐hydroxycamptothecin, DB‐67) is a camptothecin analog currently in early stage clinical trials. The lactone moiety of camptothecins hydrolyzes readily in blood to yield the pharmacologically inactive carboxylate form. However the lactone form of third‐generation lipophilic congeners, such as AR‐67, is more stable, possibly due to partitioning into red cell membranes. This prompted us to develop a reverse‐phase HPLC method with fluorescence detection (excitation 380 nm/emission 560 nm), which could quantitate the concentration of AR‐67 lactone and carboxylate in whole blood. Samples were prepared by red cell lysis, protein precipitation with methanol and centrifugation to remove denatured materials. Recovery was estimated to be >85%. Analytes were eluted isocratically with 0.15 m ammonium acetate buffer containing 10 mm TBAP (pH 6.5) and acetonitrile (65:35, v/v) on a Nova‐Pak C18 column (4 µm; 3.9 × 150 mm). The assay was linear in the ranges 0.5–300 and 2.5–300 ng/mL for carboxylate and lactone, respectively. Accuracy and precision were acceptable. AR‐67 forms were stable in whole blood and in methanolic supernatants. This assay has been successfully applied to measure AR‐67 concentrations in whole blood of patients enrolled in a phase I study. Copyright © 2010 John Wiley & Sons, Ltd.

Suresh, P. S.; Giri, Sanjeev; Husain, Raghib; Mullangi, Ramesh

doi: 10.1002/bmc.1405pmid: 20352615

A highly sensitive, rapid assay method has been developed and validated for the estimation of S‐citalopram (S‐CPM) in rat plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. The assay procedure involves a simple liquid–liquid extraction of S‐CPM and phenacetin (internal standard, IS) from rat plasma with t‐butyl methyl ether. Chromatographic separation was operated with 0.2% formic acid:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a Symmetry Shield RP18 column with a total run time of 3.0 min. The MS/MS ion transitions monitored were 325.26 → 109.10 for S‐CPM and 180.10 → 110.10 for IS. Method validation and pre‐clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 0.5 ng/mL and the linearity was observed from 0.5 to 5000 ng/mL. The intra‐ and inter‐day precisions were in the range of 1.14–5.56 and 0.25–12.3%, respectively. This novel method has been applied to a pharmacokinetic study and to estimate brain‐to‐plasma ratio of S‐CPM in rats. Copyright © 2010 John Wiley & Sons, Ltd.

Inamadugu, Jaswanth Kumar; Damaramadugu, Rajasekhar; Mullangi, Ramesh; Ponneri, Venkateswarlu

doi: 10.1002/bmc.1406pmid: 20853461

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.

Lan, Tian; Bi, Huichang; Xu, Suowen; Le, Kang; Xie, Zhiyong; Liu, Yiwei; Huang, Heqing

doi: 10.1002/bmc.1407pmid: 20352614

Sphingosine kinase (SphK) is a key enzyme in modulating the levels of sphingosine 1‐phosphate (S1P) as well as an important enzyme in numerous biological responses. Using C17‐sphingosine as a substrate, we established a rapid, sensitive and highly efficient method for determination of SphK activity by analyzing the product C17‐sphingosine 1‐phosphate (C17‐S1P) using liquid chromatography–tandem mass spectrometry. The standard curve for C17‐S1P was linear over a wide range (10–1000 ng/mL) with correlation coefficient (r2) greater than 0.999. The lower limit of quantification for C17‐S1P was 10 ng/mL. The Km values for C17‐sphingosine and ATP were determined to be 28.17 and 188.5 mM, respectively. More importantly, the SphK activity dramatically increased in cultured HEK 293 cells expressing wild‐type SphK1 as well as cells treated with tumor necrosis factor‐a, a sphingosine kinase activator. In contrast, the SphK activity decreased in cultured HEK 293 cells treated with dimethylsphngosine, a sphingosine kinase inhibitor. In conclusion, this method was sensitive and rapid in the determination of SphK acitivity, providing striking utilities in exploring the sphingosine kinase signaling pathway and screening active compounds targeting SphK activity. Copyright © 2010 John Wiley & Sons, Ltd.

Singh, Dhruv K.; Maheshwari, Gunjan

doi: 10.1002/bmc.1408pmid: 20853462

A simple, selective and precise thin‐layer chromatographic method has been developed for the analysis of eight cephalosporin antibiotics, namely cephadroxil, cephalexin, cefixime, cefaclor, cefpodoxime proxetil, cefuroxime axetil, cefotaxime sodium and ceftriaxone sodium. The hRF values of these cephalosporins were investigated on silica gel G–zinc ferrocyanide layers. Mixing of zinc ferrocyanide with silica gel G resulted in a decrease in hRF values, removal of tailing and better resolutions. The influence of silica gel G–zinc ferrocyanide ratio and mobile phases on the chromatographic behavior of cephalosporins on thin layers was investigated. Cephalosporins were selectively separated in their binary and ternary synthetic mixtures and pharmaceutical formulations. Quantitative separations of cephalosporins from their synthetic mixtures were also achieved with good recoveries (97.8–100.3%). Copyright © 2010 John Wiley & Sons, Ltd.

Yi, Lixin; Bi, Kaishun; Chen, Xinyang; Zhang, Qiufeng; Che, Shuang; Liu, Wentao; Lu, Dan; Chen, Xiaohui

doi: 10.1002/bmc.1409pmid: 20853463

A simple and sensitive LC‐MS method for the determination of periplocin in rat plasma was developed and validated. The chromatographic separation was carried out using a reverse‐phase Kromasil C18 column(150 × 4.6 mm, i.d., 5 µm) with a mobile phase composed of methanol–water (76:24, v/v). The flow rate of mobile phase was 0.8 mL/min. The calibration curve was linear within the concentration range 1–1000 ng/mL. The intra‐ and inter‐day precisions across three validation days over the entire concentration range was lower than 9.2% in terms of relative standard deviation. Accuracy determined at three quality control concentrations ranged from −2.0 to 6.0% in terms of relative error. The validated method was applied to the pharmacokinetic study of periplocin in rat plasma after intravenous and intramuscular administration. Copyright © 2010 John Wiley & Sons, Ltd.

Guo, Zhi‐Yong; Gai, Pan‐Pan; Duan, Jing; Zhai, Jin‐Xia; Zhao, Sha‐Sha; Wang, Sui; Wei, Dan‐Yi

doi: 10.1002/bmc.1410pmid: 20352652

A gas chromatography–mass spectrometry assay was developed and validated for the simultaneous determination of phthalates and adipates in human serum. The phthalates and adipates studied were dimethyl phthalate, diethyl phthalate, dibutyl phthalate, benzylbutyl phthalate, di‐2‐ethylhexyl phthalate, di‐n‐octyl phthalate, diethyl adipate, dibutyl adipate, diisobutyl adipate, bis(2‐butoxyethyl) adipate and di‐2‐ethylhexyl adipate, with diisooctyl phthalate as internal standard. The extraction and cleaning up procedure was carried out with solid‐phase extraction cartridges containing dimethyl butylamine groups, which showed extraction efficiencies over 88% for each analyte and the internal standard. The calibration curves obtained were linear with correlation coefficients greater than 0.98. For all analytes, the assay gave CV% values for intra‐day precision from 4.9 to 13.3% and mean accuracy values from 91.4 to 108.4%, while inter‐day precision was 5.2–13.4% and mean accuracy 91.0–110.2%. The limits of detection for the assay of phthalates and adipates were in the range 0.7–4.5 ng/mL. The method is simple, sensitive and accurate, and allows for simultaneous determination of nanogram levels of phthalates and adipates in human serum. It was successfully applied to an investigation on the level of phthalates and adipates in a non‐occupationally exposed population.

Hotha, Kishore Kumar; Bharathi, D. Vijaya; Kumar, S. Sirish; Reddy, Y. Narsimha; Chatki, Pankaj; Ravindranath, L. K.; Veera, K. N. Jaya; Mullangi, Ramesh

doi: 10.1002/bmc.1411pmid: 20853464

A highly sensitive, rapid assay method has been developed and validated for the simultaneous estimation of tolmetin (TMT) and MED5 in human plasma with liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive‐ion mode. A simple solid‐phase extraction process was used to extract TMT and MED5 along with mycophenolic acid (internal standard, IS) from human plasma. Chromatographic separation was achieved with 0.2% formic acid–acetonitrile (25:75, v/v) at a flow rate of 0.50 mL/min on an X‐Terra RP18 column with a total run time of 2.5 min. The MS/MS ion transitions monitored were 258.1 → 119.0 for TMT, 315.1 → 119.0 for MED5 and 321.2 → 207.0 for IS. Method validation and clinical sample analysis were performed as per FDA guidelines and the results met the acceptance criteria. The lower limit of quantitation achieved was 20 ng/mL and the linearity was observed from 20 to 2000 ng/mL, for both the anlaytes. The intra‐day and inter‐day precisions were in the range 3.27–4.50 and 5.32–8.18%, respectively for TMT and 4.27–5.68 and 5.32–8.85%, respectively for MED5. This novel method has been applied to a clinical pharmacokinetic study. Copyright © 2010 John Wiley & Sons, Ltd.