Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil.
Anal Bioanal Chem. 2011 Mar;399(7):2435-43. doi: 10.1007/s00216-010-4635-2. Epub 2011 Jan 19.
An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak(®) AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min(-1) was used. The column was kept at 23 ± 2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL(-1) and was linear over the concentration range of 50-5,000 and 50-2,500 ng mL(-1) for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.
建立了一种手性高效液相色谱法，采用双相中空纤维液相微萃取（HF-LPME-HPLC），用于测定大鼠肝微粒体中异拉定（ISR）对映异构体及其主要代谢物（异拉定的吡啶衍生物，PDI）。采用双相 HF-LPME 法，以乙酸己酯为接受相，萃取 30 分钟，样品以 1500rpm 搅拌，从 1ml 微粒体介质中提取分析物。首次实现了 ISR 对映异构体和 PDI 的分离。对于这种分离，使用 Chiralpak(®) AD 柱，以正己烷/2-丙醇/乙醇（94:04:02，v/v/v）为流动相，流速为 1.5ml/min。柱温保持在 23±2°C。药物和代谢物检测波长为 325nm，内标奥昔布宁检测波长为 225nm。PDI 的回收率为 23%，每个 ISR 对映异构体的回收率为 19%。该方法的检测限（LOQ）为 50ng/ml，PDI 和每个 ISR 对映异构体的浓度范围分别为 50-5000ng/ml 和 50-2500ng/ml 时均呈线性。该验证方法用于大鼠肝微粒体的 ISR 体外生物转化研究，表明（+）-（S）-ISR 优先发生生物转化。
