Journal of Chromatography B, 814 (2005) 75–81 Analysis of benidipine enantiomers in human plasma by liquid chromatography–mass spectrometry using a macrocyclic antibiotic (Vancomycin) chiral stationary phase column Wonku Kang a , Dong-Jun Lee a , Kwang-Hyeon Liu a , Yu Eun Sunwoo a , Kwang-il Kwon b , In-June Cha a , Jae-Gook Shin a,∗ a Department of Pharmacology and Pharmacogenomics Research Center, College of Medicine, Inje University, Busan 614-735, Republic of Korea b College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea Received 12 May 2004; accepted 4 October 2004 Abstract We used a novel chromatographic method to rapidly and simply characterize the pharmacokinetics of benidipine enantiomers in human plasma. The stereoisomers of benidipine were extracted from plasma using diethylether under alkaline conditions. After evaporating the organic layer, the residue was reconstituted in the mobile phase (methanol:acetic acid:triethylamine, 100:0.01:0.0001, v/v/v). The enantiomers in the extract were separated on a macrocyclic antibiotic (Vancomycin) chiral stationary phase column. The mobile phase was eluted at 1ml/min and was split by an interface. One-fifth of the eluent was used to quantify both isomers in a tandem mass spectrometer in multiple reaction- monitoring mode. The coefficient of variation of the precision of the assay was less than 8%, the assay accuracy was between 93.4 and 113.3%, and the limit of detection was 0.05 ng/ml for 1 ml of plasma. The method described above was used to measure the concentration of both benidipine enantiomers in plasma from healthy subjects who received a single oral dose of a racemate of 8 mg benidipine. The C max and AUC inf values of (+)-alpha benidipine were higher than those of (-)-alpha benidipine by 1.96- and 1.85-fold, respectively (p < 0.001), whereas, the T max and t 1/2 for each of the benidipine stereoisomers were not significantly different. © 2004 Elsevier B.V. All rights reserved. Keywords: Benidipine; Enantiomers; Chiral stationary phase; Tandem mass spectrometry 1. Introduction Benidipine is a dihydropyridine calcium antagonist that is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. In animal studies, the kinetic behavior and dynamic efficacies, respectively, of the benidipine stereoisomers were different [1–4]. Because the hepatic intrinsic clearance rate for (-)-alpha benidipine is greater than that for (+)-alpha benidipine, plasma con- centrations of (+)-alpha benidipine are greater than those of (-)-alpha benidipine following the administration of ∗ Corresponding author. Tel.: +82 51 890 6709; fax: +82 51 893 1232. E-mail address: phshinjg@inje.ac.kr (J.-G. Shin). a benidipine racemate to rats [5]. In addition, the hy- potensive effect of (+)-alpha benidipine was between 30- and 100-fold the effect of (-)-alpha benidipine in spon- taneously hypertensive rats [1]. The aforementioned ob- servations suggest that the competitive metabolism of the stereoisomers of benidipine that follows the oral adminis- tration of the benidipine racemate gives rise to differential pharmacokinetics for the different enantiomers of benidip- ine. Although several different methods have been used to measure benidipine concentrations in plasma [6–9], the enan- tioselective pharmacokinetics of the stereoisomers of beni- dipine in humans has not been examined. Therefore, we de- veloped a novel chiral chromatography method to determine 1570-0232/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2004.10.006