基于UPLC-MS/MS测定鹿茸饮片中13个激素的含量

Determination of 13 hormones in Cervi Cornu Pantotrichum slices based on UPLC-MS/MS

目的:利用UPLC-MS/MS法测定10批鹿茸中4个雄激素(诺龙、雄烯二酮、睾酮、甲睾酮),3个孕激素(炔诺酮、孕酮、甲羟孕酮),4个雌激素(己烯雌酚、己烷雌酚、雌二醇、雌三醇)和2个皮质醇激素(可的松、氢化可的松)的含量,明确鹿茸整支和不同部位饮片(蜡片、粉片、纱片、骨片)中13个激素的含量。方法:样品经甲醇提取,N-丙基乙二胺(PSA)和中性氧化铝净化,经ACQUITY BEH C18色谱柱(100 mm×2.1 mm, 1.7μm)分离,分别在电喷雾正负离子模式下以多反应监测模式(multiple reaction monitoring, MRM)进行测定。结果:鹿茸中雄烯二酮、睾酮、孕酮、己烯雌酚、己烷雌酚、雌二醇、雌三醇、可的松、氢化可的松9个激素均有检出,诺龙、炔诺酮、甲睾酮及甲羟孕酮未检出。其中雄烯二酮含量最少,平均含量为0.29μg·kg^(-1),低于睾酮的平均含量1.95μg·kg^(-1)。4个雌激素检出量较高,占13个激素的50.7%,其中己烯雌酚含量最高,平均含量为3.23μg·kg^(-1)。此外,鹿茸蜡片中13个激素含量显著高于粉片、纱片和骨片,平均含量为34.62μg·kg^(-1)。粉片、纱片和骨片中13个激素的平均含量分别为19.68、14.60、12.72μg·kg^(-1),无显著差异。结论:该检测方法前处理过程简单,灵敏度、准确度及精密度均符合要求,适用于鹿茸中激素的检测。

Objective:To establish a Ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method to detect 4 androgens(nandrolone,4-androstene-3,17-dione,testosterone and methylthstosterone),3 progestins(19-norethindrone,progesterone and medroxyprogesterone),4 estrogen(diethylstilbestrol,hexestrol,estradiol and estriol)and 2 glucocorticoids(cortisone and cortisol)in Cervi Cornu Pantotrichum slices.Contents of 13 hormones were determined in different parts of Cervi Cornu Pantotrichum(wax slices,powder slices,gauze slices and bone slices).Methods:Samples were extracted with methanol and purified by primary secondary amine(PSA)and Al_2O_3.The samples were separated by ACQUITY BEH C_(18)column(100 mm×2.1 mm,1.7μm)and detected by multiple reaction monitoring(MRM)in electrospray positive and negative ion mode respectively.Results:4-androstene-3,17-dione,testosterone,progesterone,diethylstilbestrol,hexestrol,estradiol,estriol,cortisone and cortisol were detected.Nandrolone,19-norethindrone,methyltestosterone and medroxyprogesterone were not detected in Cervi Cornu Pantotrichum.The content of 4-androstene-3,17-dione as androgens was the lowest(0.29μg·kg^(-1))in 10 batches Cervi Cornu Pantotrichum,which was lower than testosterone(1.95μg·kg^(-1)).Four kinds of estrogen were relatively high,accounting for 50.7%of the 13 kinds of hormones,among which diethylstilbestrol was the highest(3.23μg·kg^(-1)).The contents of 13 hormones in wax slices,powder slices,gauze slices and bone slices decreased successively.The average content of 13 hormone was 34.62μg·kg^(-1)in wax slices,which was higher than that in powder slices,gauze slices and bone slices(19.68,14.60 and 12.72μg·kg^(-1),respectively).Conclusion:The sensitivity,accuracy and precision of this method meet the detection requirements.It is suitable for the detection of hormones in Cervi Cornu Pantotrichum.