UPLC-MS/MS法测定人血浆中左乙拉西坦浓度及其药代动力学研究

目的:建立专属、快速的超高效液相色谱-串联质谱(UPLC-MS/MS)的分析方法测定人血浆中左乙拉西坦的浓度,进行药代动力学研究。方法:以3-氨基-2-萘甲酸为内标,0.2 mL血浆样品加入280μL甲醇除蛋白后,涡旋提取,取上清液3μL进样。色谱柱:Acquity UPLC BEH C₁₈(2.1 mm×50 mm,1.7μm),流动相:乙腈和0.01 mol·L^-1甲酸铵(pH 3.6),梯度洗脱,流速0.3 mL·min^-1,分析时间4 min;采用电喷雾MS检测,正离子检测模式。结果:左乙拉西坦质量浓度在0.5~128.0μg·mL^-1范围内呈线性,日内、日间精密度分别小于13.1%和11.7%,基质效应影响较小,相对回收率在95.9%~106.8%之间。口服左乙拉西坦片剂后达峰浓度和达峰时间分别为18.23μg·mL^-1和1.31 h。结论:该方法可用于微量血浆测定,并可应用于成人或新生儿癫痫发作,给予左乙拉西坦后对其临床药动学的研究。

Objective: To develop a specific and rapid method for the determination of levetiracetam in plasma using ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), and study pharmacokinetics of levetiracetam.Methods: A plasma aliquot of 0.2 mL was deproteinized by addition of 280 μL methanol which contained 3-amino-2-naphthoic acid as an internal standard.After centrifugation, 3 μL of supernatant was injected into the UPLC-system.Compounds were separated on an Acquity UPLC BEH C₁₈(2.1 mm×50 mm, 1.7 μm) column using gradient elution with mobile phase acetonitrile and 0.01 mol·L^-1 ammonium formate (pH=3.6) with a flow rate of 0.3 mL·min^-1 and a total runtime of 4.0 min.Levetiracetam and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry(ESI-MS/MS). Results: The assay allowed quantification of levetiracetam plasma concentrations in the range of 0.5-128 μg·mL^-1. Intra-assay accuracy was below 13.11% and inter-assay precision was less than 11.7%.Matrix effects were minor, and the recovery of levetiracetam was between 95.9% and 106.8%.The maximum concentration(C_max) were 18.23 μg·mL^-1, and the time to reach this concentration(T_max) was 1.31 h.Conclusion: The developed method required less sample volume of plasma.The method has been applied in a clinical pharmacokinetic study in which adult/neonates received levetiracetam for the treatment of seizures.