反相高效液相色谱法同时测定人血浆中磷苯妥英、苯妥英及其主要代谢产物4’-羟苯妥英的浓度

Simultaneous Rapid Separation and Determination of Fosphenytoin,Phenytoin and Their Main Metabolite 4′-Hydroxyphenytoin in Human Plasma Using Reversed-Phase HPLC

目的建立同时测定人血浆中抗癫痫药物磷苯妥英(FOS)、苯妥英(PHT)及其主要代谢产物4′-羟苯妥英(4′-HPPH)浓度的高效液相色谱法。方法以盐酸普萘洛尔为内标,血浆样品100μL经20%磷酸酸化后以乙酸乙酯1 mL萃取,提取后氮气吹干,加入流动相100μL复溶,取20μL进样测定。色谱柱采用Agilent RX-C8柱(4.6 mm×250 mm,5μm),柱温:40℃;流动相为甲醇-乙腈-0.1%三氟乙酸水溶液(23∶17∶60),流速为1.5 mL·min-1,紫外检测波长210nm。结果血浆内源性杂质和常用合并用药对样品测定无干扰,色谱峰分离良好。FOS、PHT、4′-HPPH的线性范围分别是1～400,0.5～50和0.5～10 mg·L-1,相关系数(r)均大于0.999 1。平均方法回收率为92.79%～107.90%。各组分的日内、日间RSD均小于8%。结论该方法具有良好的准确性、精密性和灵敏性,且操作快速、简便,各组分之间分离良好,适用于临床血药浓度的监测。

OBJECTIVE To develop a simple reversed-phase high-performance liquid chromatography （HPLC） method for the simultaneous determination of the antiepileptie drugs （AEDs） phenytoin （PHT）, its prndrug fosphenytoin （FOS）, and its metabolite 4＇-hydroxyphenytoin （4＇-HPPH） in human plasma. METHODS Plasma sample （100 μL） pre-treatment involved acidification with 20% phosphoric acid, simple extraction with 1 mL of acetic ester containing 10 mg·L ^-1 propranolol hydrochloride as internal standard and evaporation of solvent, followed by injection of reconstituted sample onto a Agilent RX-Cs column （4.6 mm× 250 mm,5 μm）. The mobile phase consisted of methanol-acetonitrile-0. 1% triflouroacetic acid （23：17 60） , and delivered at a 1.5 mL· min ^-1. The analytes were detected at the wavelength of 210 nm. RESULTS The method was found to be linear over the concentration ranges investigated, 1 -400 mg·L^-1 for FOS, 0. 5 -50 mg·L^-1 for PHT and 0. 5 -10 mg·L^-1 for 4＇-HPPH, respectively. The intra- and inter-day reproducibility was good with the RSD of 8% or below and relative recovery was ranged from 92. 79% to 107.90% for all analytes. CONCLUSION This method was proved to be accurate, sensitive and convenient and suitable tot clinical therapeutic drug monitoring.