Abstract:

Objective　To establish a method to determine steroids in dried blood spots specimens by liquid chromatography-tandem mass spectrometry（LC-MS/MS） assay. Methods　Two dry blood spots（DBS） of diameter of 3 mm with different concentration of androstenedione，testosterone，17α-hydroxyprogesterone，progesterone，11-deoxycortisol，21-deoxycortisol and cortisol，were punched. Then the two DBS were extracted with 1∶1 methanol/acetonitrile solution，which was labeled with stable isotope internal standards. The extraction of DBS was dried under nitrogen. After reconstituted，the steroids were analyzed by LC-MS/MS. The linearity，precision and accuracy were evaluated. Seven DBS samples of 21-hydroxylase deficiency patients and 23 DBS samples of normal infants were collected，and then the steroids for these samples were analyzed by LC-MS/MS. Results　The intra-day and inter-day precisions were 3.3%～10.9%，and the intra-day and inter-day accuracies were 100.4%～116.5%. The lowest limits of detection（LLOD） of these seven steroids were 0.03～0.25 μg/L. The lowest limits of quantitation（LLOQ） of these seven steroids were 0.125～1.00 μg/L. The linearities of different steroids were 0.997～0.999 respectively. The results of non-parametric test about steroids concentration in infants and 21-OHD patients showed that，the concentration of androstenedione，17-hydroxyprogesterone，progesterone，21-deoxycortisol and cortisol in the 21-OHD patients were significantly higher than those steroids in normal infants，P＜0.05. Conclusion　A method for measurement of the steroids in DBS by LC-MS/MS has been established，which shows good precision，accuracy and linearity. This method can be applied to clinical testing and follow-up therapy of CAH patients，and it is expected to be used as a second-tier test for CAH newborn screening.

Key words: liquid chromatography-tandem mass spectrometry, dried blood spots, steroid

摘要：

目的　建立液相色谱-串联质谱技术（liquid chromatographytandem mass spectrometry，LC-MS/MS）检测干血滤纸片（dried blood spots，DBS）中类固醇激素谱的方法。方法　取含不同浓度类固醇激素（包括雄烯二酮、睾酮、17-羟孕酮、孕酮、11-脱氧皮质醇、21-脱氧皮质醇、皮质醇）的2个DBS（直径3 mm），用含已知类固醇激素内标的1∶1甲醇/乙腈萃取，孵育震荡后氮气吹干，复溶后LC-MS/MS检测。评价方法的线性、精确度、准确度。收集7例21-羟化酶缺乏症患儿及23例正常婴幼儿DBS样本，LC-MS/MS测定样本中类固醇激素浓度。结果　7种类固醇激素批内和批间精确度为3.3%～10.9%，准确度为100.4%～116.5%，最低检测限为0.03～0.25 μg/L，最低定量限为0.125～1.000 μg/L；不同浓度的类固醇激素实测浓度与加入浓度之间的相关系数为0.997～0.999。21-羟化酶缺乏症患儿DBS样本中雄烯二酮、17-羟孕酮、孕酮、21-脱氧皮质醇、皮质醇浓度均显著高于正常男性婴幼儿，具有统计学意义（P＜0.05）。结论　本研究建立了LC-MS/MS测定DBS中类固醇激素谱的方法，具有较好的精确度、准确度及浓度线性关系，可用于临床类固醇激素检测，有助于先天性肾上腺皮质增生症的临床诊断及治疗效果评价，并有望用于此病的新生儿筛查。

关键词: 液相色谱-串联质谱, 干血滤纸片, 类固醇激素