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

Determination for endogenous 5-androstenediol by GC-MS in rat plasma and its pharmacokinetic study

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-d_6(DHEA-d_6)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×0.25μm×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(r^2=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T_(1/2)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×0.25μm×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 （ r^2=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 pharmaeokinetic 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 pharmacokinetie study.