Yeap Li-Ling, Lo Yoke-Lin
Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
PLoS One. 2014 Nov 6;9(11):e111544. doi: 10.1371/journal.pone.0111544. eCollection 2014.
A simple liquid chromatography tandem mass spectrometry method was developed and validated according to the guidelines of the US Food and Drug Administration and the European Medicines Agency for a simultaneous quantification of levetiracetam (LEV) and its metabolite, UCB L057 in the plasma of patients. A 0.050 mL plasma sample was prepared by a simple and direct protein precipitation with 0.450 mL acetonitrile (ACN) containing 1 µg/mL of internal standard (IS, diphenhydramine), then vortex mixed and centrifuged. A 0.100 mL of the clear supernatant was diluted with 0.400 mL water and well mixed. A 0.010 mL of the resultant solution was injected into an Agilent Zorbax SB-C18 (2.1 mm×100 mm, 3.5 µm) column with an isocratic elution at 0.5 mL/min using a mixture of 0.1% formic acid in water and ACN (40:60 v/v). Detection was performed using an AB Sciex API 3000 triple quadrupole mass spectrometer, equipped with a Turbo Ion Spray source, operating in a positive mode: LEV at transition 171.1>154.1, UCB L057 at 172.5>126.1, and IS at 256.3>167.3; with an assay run time of 2 minutes. The lower limit of quantification (LLOQ) for both LEV and UCB L057 was validated at 0.5 µg/mL, while their lower limit of detection (LOD) was 0.25 µg/mL. The calibration curves were linear between 0.5 and 100 µg/mL for both analytes. The inaccuracy and imprecision of both intra-assay and inter-assay were less than 10%. Matrix effects were consistent between sources of plasma and the recoveries of all compounds were between 100% and 110%. Stability was established under various storage and processing conditions. The carryovers from both LEV and UCB L057 were less than 6% of the LLOQ and 0.13% of the IS. This assay method has been successfully applied to a population pharmacokinetic study of LEV in patients with epilepsy.
根据美国食品药品监督管理局和欧洲药品管理局的指导方针，开发并验证了一种简单的液相色谱串联质谱法，用于同时定量患者血浆中的左乙拉西坦（LEV）及其代谢物UCB L057。通过用含有1μg/mL内标（IS，苯海拉明）的0.450 mL乙腈（ACN）进行简单直接的蛋白沉淀来制备0.050 mL血浆样品，然后涡旋混合并离心。取0.100 mL清澈上清液用0.400 mL水稀释并充分混合。取0.010 mL所得溶液注入Agilent Zorbax SB-C18（2.1 mm×100 mm，3.5 µm）色谱柱，使用0.1%甲酸水溶液和ACN（40:60 v/v）的混合物以0.5 mL/min的流速进行等度洗脱。使用配备Turbo Ion Spray源的AB Sciex API 3000三重四极杆质谱仪进行检测，以正模式运行：LEV的跃迁为171.1>154.1，UCB L057的跃迁为172.5>126.1，IS的跃迁为256.3>167.3；分析运行时间为2分钟。LEV和UCB L057的定量下限（LLOQ）均验证为0.5μg/mL，而它们的检测下限（LOD）为0.25μg/mL。两种分析物的校准曲线在0.5至100μg/mL之间呈线性。批内和批间的不准确度和不精密度均小于10%。血浆来源之间的基质效应一致，所有化合物的回收率在100%至110%之间。在各种储存和处理条件下确定了稳定性。LEV和UCB L057的残留均小于LLOQ的6%和IS的0.13%。该分析方法已成功应用于癫痫患者中LEV的群体药代动力学研究。
