GC-MS衍生化法测定大鼠血浆中内源性5-雄烯二醇及药动学研究

目的: 采用SIM 模式,建立一种快速、灵敏的GC-MS 衍生化方法测定大鼠血浆中5-雄烯二醇。方法: 以脱氢表雄甾酮-2,2,3,4,4,6-d6(DHEA-d₆)为内标,血浆样品经过甲基叔丁基醚(MTBE)提取,离心,取上清液氮气吹干,通过BSTFA-TMCS(99:1)衍生化后进行GC-MS 分析。RESTEK 色谱柱:Rxi-5 ms(30 m×0.25 μm×0.25 mm);程序升温:初始温度为60 ℃,维持1 min,以40 ℃·min^-1 速率升至220℃,再以5 ℃·min^-1 的速率升至300 ℃,维持5 min;采用选择离子扫描模式(SIM),5-雄烯二醇的定性离子为m/z 434、344,定量离子为m/z 344,DHEA-d₆ 的定性离子为m/z 366、276,定量离子为m/z 276。结果: 血浆中5-雄烯二醇质量浓度在80~5 000 ng·mL^-1 范围内线性关系良好(r²=0.999 3);低、中、高3 个浓度的准确度在70%~103% 之间;日内、日间精密度均小于5%;提取回收率(n=4)在75.96%~104.01%;低、高2 个浓度的血浆样品在室温放置12 h,4 ℃冰箱中放置24 h,-20 ℃保存30 d、反复冻融4 次,以及衍生化后的样品室温放置4、8、12、24 h 均能保持稳定。大鼠皮下注射5-雄烯二醇混悬液的药-时曲线符合二室模型,其药代学参数为T1/2 为(26.45±8.40)h,AUC_(0-t)为(2 887.46±1 033.70)μg·h^-1·L^-1。结论: 本方法经方法学验证,可作为大鼠血浆中5-雄烯二醇含量的测定方法,适合药动学研究。

Objective: To establish a rapid,sensitive method for the determination of 5-androstenediol in rat plasma using capillary gas chromatography coupled to mass spectrometry(GC-MS)with selected ion monitoring (SIM)model. Methods: Dehydroepiandrosterone-2,2,3,4,4,6-d6(DHEA-d₆)was used as an internal standard,and the plasma samples were extracted by methyl tert-butyl ether(MTBE). The supernatant liquid after centrifugation was dried by nitrogen and the 5-androstenediol was derived by BSTFA-TMCS(99:1). The samples were injected into a RESTEK Rxi^®-5ms column(30 m×0.25 μm×0.25 mm). The initial column temperature was 60 ℃,maintained for 1 min,then raised to 220 ℃ at a rate of 40 ℃·min^-1,and finally increased to 300 ℃ at a rate of 5℃·min^-1 and maintained for another 5 min. The qualitative ions were m/z 434,344 and the quantitative ion was m/z 344 of 5-androstenediol,while the qualitative ions were m/z 366,276 and the quantitative ion was m/z 276 of DHEA-d₆ with the SIM model. Results: The method was validated over the concentration range of 80-5 000 ng·mL^-1 for 5-androstenediol in rate plasma,and there was excellent linearity(r²=0.999 3). The accuracy of low,middle and high mass concentrations was between 70% and 103% and the intra-day and inter-day RSD were both below 5% and the recovery was 75.96%-104.01% over the four concentration levels were evaluated. All the analytes of low and high concentrations in serum were stable at room temperature for 12 h,at 4 ℃ for 24 h,at -20 ℃ for 30 days,after four-cycle freeze-thaw and at room temperature for 4,8,12,24 h after derivatization. The pharmacokinetics behavior after subcutaneous injection of 5-androstenediol could be described by a two-compartment model. The main pharmacokinetic parameters were as follows:the half-life was(26.45±8.40)h,AUC_(0-t)was (2 887.46±1 033.70)μg·h^-1·L^-1. Conclusion: The established method could be used as a quality control method for 5-androstenediol in rat plasma after methodology validation,which is also suitable for pharmacokinetic study.