柱前衍生化LC-MS/MS法同时测定人血浆中炔雌醇和依托孕烯及其药动学研究

Simultaneous Determination of Ethinyl Estradiol and Etonogestrel in Human Plasma by Pre-column Derivatization Combined with LC-MS/MS and Their Pharmacokinetics

目的:应用柱前衍生化的LC-MS/MS方法,同时测定人血浆中炔雌醇和依托孕烯的含量,并研究中国健康女性受试者口服去氧孕烯炔雌醇片[炔雌醇(EE)和依托孕烯(ENG)]后的药代动力学特征。方法:血浆样品经过甲基叔丁基醚-正己烷(50∶50)液液萃取吹干后,固体物溶解在碳酸钠溶液中,并与丹磺酰氯丙酮溶液在60℃水浴反应10 min,以水-乙腈-甲酸(50∶50∶0.2)复溶进行LC-MS/MS分析。以ZORBAX SB C_(18)(Narrow Bore RR 2.1 mm×100 mm,3.5μm)为分析柱,流动相为含0.1%甲酸水溶液-含0.1%甲酸乙腈溶液,梯度洗脱。炔雌醇衍生物和依托孕烯的检测离子分别为m/z 530.2→171.1和m/z 325.2→257.3,炔雌醇-2,4,16,16-d_(4)和依托孕烯-d_(6)作为内标辅助定量。结果与结论:炔雌醇在2~500 pg·mL^(-1),依托孕烯在50~10000 pg·mL^(-1)范围内线性关系良好。受试者口服受试制剂后炔雌醇C_(max)为(400.3±5.1)pg·mL^(-1),tmax为(2.5±0.5)h,AUC_(0-t)(1998±830)pg·h·mL^(-1);依托孕烯C_(max)为(4790.1±10.1)pg·mL^(-1),t_(max)为(3.0±0.5)h,AUC_(0-t)(42593.9±1570.9)pg·h·mL^(-1)。此为所得炔雌醇与依托孕烯的药动学特征参数。本方法精密度好(<9%),灵敏度高,炔雌醇达到2 pg·mL^(-1),依托孕烯达到50 pg·mL^(-1),所测依托孕烯的LOQ浓度很低。

Objective:An pre-column derivatization liquid chromatography-tandem mass spectrometry method(LC-MS/MS)for the simultaneous determination of ethinyl estradiol(EE)and etonogestrel(ENG)in human plasma was developed.The method was validated and applied to a pharmacokinetic study in healthy Chinese female volunteers after oral administration of the combination tablets containing EE and ENG.Methods:The plasma samples were extracted with a mixture of methyl tert-butyl ether and n-Hexane(50∶50).The extracts were dried and the residues were dissolved in a solution of sodium carbonate,then derived with dansyl chloride at 60℃ for 10 min.The obtained derivatives were then analyzed by the developed LC-MS/MS method.Chromatographic separation was performed on a ZORBAX SB-C_(18)(Narrow Bore RR 2.1 mm×100 mm,3.5μm)column with a gradient elution system of water containing 0.1%acetic acid-acetonitrile containing 0.1%formic acid.The ion transitions recorded in a multiple reaction monitoring mode were m/z 530.2→171.1 for derived ethinyl estradiol and m/z 325.2→257.3 for derived ENG,respectively.Deuterated ethinyl estradiol and deuterated etonogestrel were used as internal standards to assist quantification.Results and Conclusion:The method was validated over the concentration ranges of 2-500 pg·mL^(-1)for EE and 0.01-2 ng·mL^(-1)for ENG,which showed excellent linearities.The LC-MS/MS method was accurate(the CV were less than 9%)and sensitive(the LOQ were 2 pg·mL^(-1)for EE and 50 pg·mL^(-1)for ENG).After the subjects took the test preparation orally,the main pharmacokinetic parameters of EE and ENG were as follows:the C_(max) were(400.3±5.1)pg·mL^(-1)and(4790.1±10.1)pg·mL^(-1),the t_(max) were(2.5±0.5)h and(3.0±0.5)h,the AUC_(0-t) were(1998±830)pg·mL^(-1)and(42593.9±1570.9)pg·h·mL^(-1),respectively.