Apostolou Constantinos, Kousoulos Constantinos, Dotsikas Yannis, Soumelas Georgios-Stefanos, Kolocouri Filomila, Ziaka Afroditi, Loukas Yannis L
Laboratory of Pharmaceutical Analysis and Bioequivalence Services (GLP Compliant), Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Panepistimioupoli Zografou GR 157 71, Athens, Greece.
J Pharm Biomed Anal. 2008 Mar 13;46(4):771-9. doi: 10.1016/j.jpba.2007.12.001. Epub 2007 Dec 8.
A fully automated high-throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of simvastatin (SV) and simvastatin acid (SVA) in human plasma. Plasma samples were treated by acetonitrile (ACN) addition for protein precipitation (PP) and subsequent two-step liquid-liquid extraction (LLE) in 96-deepwell plates, using methyl t-butyl ether (MTBE) as the organic solvent. ACN addition step was proven to enhance method sensitivity, as well as producing cleaner samples for injection. Lovastatin (LV) and lovastatin acid (LVA) were used as internal standards (IS) for SV and SVA quantification respectively. A relatively small plasma volume (300 microL) was employed and all procedure liquid transfer steps were performed automatically, by the use of robotic liquid handling workstations. Both electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources were applied and compared for LC-MS/MS sample analysis, with ESI proven to be more sensitive for the specific analytes. Polarity switch (from negative to positive ionization mode) was performed during the same analytical run, so as for the simultaneous SV and SVA determination to be possible. The method had a short sample preparation time, as well as a chromatographic run time of just 1.9 min, the shortest so far reported for SV determination. It was validated and fulfilled all preset criteria for sensitivity, specificity, linearity (0.100-40.0 ng/mL), inter- and intra-accuracy and precision for both molecules. The proposed method was applied to the rapid and reliable simultaneous determination of SV and SVA in a bioequivalence study, after per os administration of a SV tablet (80 mg).
建立了一种全自动高通量液相色谱/串联质谱（LC-MS/MS）方法，用于同时定量测定人血浆中的辛伐他汀（SV）和辛伐他汀酸（SVA）。血浆样品通过加入乙腈（ACN）进行蛋白沉淀（PP），随后在96孔深孔板中进行两步液液萃取（LLE），使用甲基叔丁基醚（MTBE）作为有机溶剂。已证明加入ACN步骤可提高方法灵敏度，并产生更纯净的进样样品。洛伐他汀（LV）和洛伐他汀酸（LVA）分别用作SV和SVA定量的内标（IS）。采用相对较小的血浆体积（300 μL），所有程序中的液体转移步骤均通过使用机器人液体处理工作站自动进行。同时应用电喷雾（ESI）和大气压化学电离（APCI）源进行LC-MS/MS样品分析，结果证明ESI对特定分析物更灵敏。在同一分析运行过程中进行极性切换（从负离子模式到正离子模式），以便能够同时测定SV和SVA。该方法样品制备时间短，色谱运行时间仅为1.9分钟，是迄今为止报道的测定SV最短的时间。该方法经过验证，满足了所有预设的灵敏度、特异性、线性（0.100 - 40.0 ng/mL）、分子间和分子内准确度及精密度标准。所提出的方法应用于口服辛伐他汀片（80 mg）后的生物等效性研究中SV和SVA的快速可靠同时测定。
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