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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 and sensitive GC-MS derivatization method for the determination of 5-androstenediol in plasma of rats using SIM mode. Methods: Dehydroepiandrosterone-2,2,3,4,4,6-d_6 (DHEA-d_6) was used as an internal standard. The plasma samples were extracted with MTBE and centrifuged. The supernatant was air-dried with nitrogen and derivatized with BSTFA-TMCS (99: 1) for GC-MS analysis. RESTEK column: Rxi ~ -5 ms (30 m × 0.25 μm × 0.25 mm); temperature programmed: the initial temperature was 60 ℃, maintained for 1 min, the temperature was raised to 220 ℃ at 40 ℃ · min -1, Then ramped to 300 ℃ for 5 min at the rate of 5 ℃ · min ~ (-1). Using selective ion scan mode (SIM), the qualitative ions of 5-androstenediol were m / z 434 and 344, As m / z 344, DHEA-d 6 as m / z 366,276 and m / z 276 for quantified ion. Results: The linearity of the concentration of 5-androstenediol in plasma ranged from 80 to 5 000 ng · mL -1 (r 2 = 0.999 3). The accuracy of low, medium and high concentrations (N = 4) ranged from 75.96% to 104.01%; low and high concentrations of plasma samples were placed at room temperature for 12 h, 4 ℃ refrigerator for 24 h, -20 ℃ for 30 d, freeze-thaw repeated four times, and the derivatized samples were placed at room temperature for 4,8,12,24 h can remain stable. The drug-time curve of subcutaneous injection of 5-androstenediol suspension in rats accorded with two-compartment model. The pharmacokinetic parameters were T 1/2 (26.45 ± 8.40) h, AUC 0-t (2 887.46 ± 1 033.70) μg · h -1 · L -1. Conclusion: This method is validated by methodology and can be used as a method for the determination of 5-androstenediol in plasma of rats. It is suitable for pharmacokinetic studies.