Lindner Johanna M, Vogeser Michael, Grimm Stefanie H
Institute of Laboratory Medicine, Hospital of the Ludwig-Maximilians-University Munich, Marchioninistraße 15, 81377 Munich, Germany.
Institute of Laboratory Medicine, Hospital of the Ludwig-Maximilians-University Munich, Marchioninistraße 15, 81377 Munich, Germany.
J Pharm Biomed Anal. 2017 Aug 5;142:66-73. doi: 10.1016/j.jpba.2017.04.020. Epub 2017 Apr 19.
The measurement of steroid hormones and their corticoid precursors is an important aspect in endocrinology since these analytes are biomarkers for several endocrine disorders. Over the last few years, HPLC-MS/MS has become the method of choice to analyze these compounds. There are already several methods using stationary phases modified with C18 groups. However, since these columns sometimes do not enable sufficient separation of some isobaric steroids, we investigated the potential of a different RP modification using biphenyl groups for the separation of challenging isobars such as corticosterone, 11- and 21-deoxycortisol. The aim of our work was the development of an isotope dilution UHPLC-MS/MS assay for clinical research that combines simple and effective sample preparation with a powerful MS method quantifying a broad steroid panel (aldosterone, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-OH-progesterone, progesterone, and testosterone) in human serum. After a manual protein precipitation step using zinc trifluoroacetate (ZnTFA) in methanol, the supernatants were directly injected into the UHPLC-MS system. Chromatographic baseline separation of all isobaric compounds (corticosterone↔11-deoxycortisol↔21-deoxycortisol, 17-OH-progesterone↔11-deoxycorticosterone, and aldosterone↔cortisone) was achieved using a Kinetex Biphenyl column (150×2.1mm, 1.7μm) with a mobile phase consisting of 0.2mM ammonium fluoride in water and methanol. The total run time was 10min. For detection we used a Xevo TQ-S mass spectrometer operating in the ESI positive and negative modes. The method was validated according to the EMA guideline for bioanalytical method validation. The results for accuracy (within-run: 92.3%-115%, between-run: 92.4 %-113%) and imprecision (within-run: 0.80%-9.05%, between-run: 1.98 %-15.2%) were satisfying. The recovery ranged from 95% to 111%. The matrix effect was between 93% and 112% and an excellent linearity with R>0.99 for all analytes was achieved. It was demonstrated that biphenyl based columns are a powerful tool for comprehensive, MS based steroid assays including various isobaric substances. Additionally, we could evince that ZnTFA is a convenient precipitation agent suitable for steroid analysis.
甾体激素及其皮质激素前体的测定是内分泌学中的一个重要方面，因为这些分析物是多种内分泌疾病的生物标志物。在过去几年中，高效液相色谱-串联质谱法（HPLC-MS/MS）已成为分析这些化合物的首选方法。已经有几种使用C18基团修饰的固定相的方法。然而，由于这些色谱柱有时无法充分分离某些等压甾体，我们研究了使用联苯基团进行不同反相修饰以分离具有挑战性的等压物（如皮质酮、11-和21-脱氧皮质醇）的潜力。我们工作的目的是开发一种用于临床研究的同位素稀释超高效液相色谱-串联质谱法（UHPLC-MS/MS）测定方法，该方法将简单有效的样品制备与强大的质谱方法相结合，可对人血清中的多种甾体进行定量分析（醛固酮、皮质酮、皮质醇、可的松、11-脱氧皮质酮、11-脱氧皮质醇、21-脱氧皮质醇、脱氢表雄酮、脱氢表雄酮硫酸盐、17-羟孕酮、孕酮和睾酮）。在使用甲醇中的三氟乙酸锌（ZnTFA）进行手动蛋白沉淀步骤后，将上清液直接注入UHPLC-MS系统。使用Kinetex联苯色谱柱（150×2.1mm，1.7μm），流动相由水中的0.2mM氟化铵和甲醇组成，实现了所有等压化合物（皮质酮↔11-脱氧皮质醇↔21-脱氧皮质醇、17-羟孕酮↔11-脱氧皮质酮和醛固酮↔可的松）的色谱基线分离。总运行时间为10分钟。检测使用在电喷雾电离（ESI）正模式和负模式下运行的Xevo TQ-S质谱仪。该方法根据欧洲药品管理局（EMA）生物分析方法验证指南进行了验证。准确度（批内：92.3%-115%，批间：92.4%-113%）和精密度（批内：0.80%-9.05%，批间：1.98%-15.
