中国临床药学杂志

目的建立一种灵敏、快速、稳定的超高效液相色谱串联质谱(UPLC-MS/MS)法测定人血清中苯妥英的浓度,并探讨其在苯妥英治疗药物监测(TDM)中的应用。方法在血清添加同位素内标苯妥英-D₁₀,经乙腈沉淀蛋白后,以Shim-pack XR-ODSⅢ(50 mm×2.0 mm,1.6μm)为分析柱进行反相色谱分离,以0.1%乙酸水溶液、乙腈为流动相进行梯度洗脱,流速为0.3 mL·min^-1;以电喷雾离子化串联四级杆质谱、正离子多反应监测进行定量检测,监测离子反应分别为m/z 253.3→182.2(苯妥英),m/z 263.3→192.3(内标苯妥英-D₁₀)。用建立的方法参加2018年上半年卫生部临床检验中心茶碱TDM室间质量评价。结果 UPLC-MS/MS法检测苯妥英的线性范围为0.016～2.051 mg·L^-1,批内和批间精密度分别为1.68%～4.98%和1.49%～3.82%,准确度均为85%～115%。样本的提取回收率≥93%,基质效应≥97%。室温放置6 h、自动进样器放置18 h的稳定性均良好。UPLC-MS/MS法测定结果参与室间质评样本均合格。结论该UPLC-MS/MS法采用同位素内标法,具有前处理简单、检测快速、特异性好和灵敏度高的特点,测定结果符合全国室间质量评价要求,可用于苯妥英的临床TDM。

AIM To establish a sensitive, rapid and stable ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) assay to quantify phenytoin in human serum, and to investigate the application significance in the therapeutic drug monitoring(TDM) for phenytoin. METHODS After being added with isotopic phenytoin-D₁₀ as internal standard, serum samples were processed by protein precipitation using acetonitrile. Chromatography was performed on a Shim-pack XR-ODSⅢ analytical column(50 mm×2.0 mm,1.6 μm) with a binary mobile phase composed of 0.1% acetic acid in water and acetonitrile, the flow rate was 0.3 mL·min^-1 phenytoin and internal standard were monitored by a positive electrospray iontandem mass spectrometry system. MS detection was used in a multiple reaction monitoring mode with MS/MS ion transitions at m/z 253.3→182.2(phenytoin), m/z 263.3→192.3(internal standard, IS). The proficiency performance of UPLC-MS/MS was tested by participation to external quality assessment of phenytoin TDM for the National Center for Clinical Laboratory. RESULTS The linear range of UPLC-MS/MS for the determination of phenytoin was 0.016-2.051 mg·L^-1. The accuracy was 85%-115% with within-run and between-run precisions of 1.68%-4.98% and 1.49%-3.82% respectively. The extract recovery of sample was ≥93%, and the matrix effect was ≥97%. Stabilities were good under room temperature for 6 h and autosampler for 18 h. The external quality assessment proficiency test showed good. CONCLUSION By the isotopic internal standard method, the UPLC-MS/MS is simple, rapid, specific and sensitive for the determination of serum phenytoin. The measurement results meet the requirements of the external quality assessment proficiency test, and it is suitable for phenytoin TDM in clinical practice.