Steroid Research & Mass Spectrometry Unit, Division of Pediatric Endocrinology & Diabetlogy, Center of Child and Adolescent Medicine, Justus-Liebig-University, Feulgenstrasse 12, 35392 Giessen, Germany.
Analyst. 2013 Jul 7;138(13):3792-801. doi: 10.1039/c3an36817c.
Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.
在德国科学基金会（DFG）的联合研究项目“生殖中的硫酸甾类”中，需要开发一种分析方法，以便从不同的生物基质（如水溶液、细胞裂解物和血清）中以最高的特异性测定硫酸化和未结合的甾体。鉴于这一分析挑战，LC-MS-MS 是首选技术，因为它允许：(1) 分析完整的甾体缀合物，(2) 同时测定多个分析物（分析物谱分析，靶向代谢组学方法），以及 (3) 独立于交叉反应等现象。样品处理包括将样品与内标（氘标记甾体）孵育，然后进行固相萃取。只有血清样品在固相萃取前需要进行蛋白质沉淀步骤。提取物分为两部分：六种甾体硫酸盐（E1S、E2S、AS、16-OH-DHEAS、PREGS、DHEAS）通过 C18aQ-ESI-MS-MS 在负离子模式下进行分析，11 种未结合的甾体（E3、16-OH-DHEA、E1、E2、(4)A、DHEA、T、17-OH-PREG、Prog、An、PREG）通过 C18-APCI-MS-MS 在正离子模式下进行分析。对于甾体硫酸盐，我们发现灵敏度很高，LOQ 值范围为 0.08 至 1ng/mL。未结合的甾体的 LOQ 值在 0.5 至 10ng/mL 之间。校准曲线显示出极好的线性。甾体硫酸盐的平均日内和日间 CV 为 2.4%，未结合的甾体为 6.4%。准确度 - 通过双水平加标实验确定 - 显示甾体硫酸盐的平均相对误差为 5.9%，未结合的甾体为 6.1%。总之，我们描述了一种从各种生物基质中分析六种甾体硫酸盐和 11 种未结合甾体的新型 LC-MS-MS 方法。
