Su Min, Drotleff Bernhard, Janker Tamara, Bürger Zoé, Kimmig Ann-Christin S, Derntl Birgit, Lämmerhofer Michael
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tuebingen, 72076 Tuebingen, Germany.
Metabolomics Core Facility, EMBL Heidelberg, 69117 Heidelberg, Germany.
Anal Chem. 2025 Jul 1;97(25):13496-13503. doi: 10.1021/acs.analchem.5c01912. Epub 2025 Jun 20.
Quantifying endogenous and exogenous steroids at low concentrations in biological matrices remains a major analytical challenge. Immunoassay-based diagnostics are limited by cross-reactivity, particularly at low levels, prompting a shift toward (ultra)high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) for clinical applications. A key limitation for endogenous hormone quantification is the absence of a true blank matrix for external calibration. To address this, we developed a surrogate calibration method employing 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) derivatization for estrogens, enabling sensitive and selective quantification alongside nonderivatized steroids. Stable isotope-labeled surrogate calibrants and internal standards were used to achieve matrix-matched quantification within a clinically relevant range. Parallelism between analytes and surrogate calibrants was systematically verified in plasma across multiple calibration levels. The method was further optimized through the use of narrow-bore UHPLC columns and refined chromatographic conditions to enhance sensitivity and resolution for a broad analyte panel. Combined with efficient protein precipitation and 96-well plate-based solid-phase extraction, the developed assay achieves pg/mL-level quantification in human plasma with high precision and accuracy. This integrated approach uniquely combines surrogate calibration for endogenous steroids with external calibration for exogenous contraceptives, including sensitive DMIS-based derivatization for estrogens, enabling comprehensive hormonal profiling in a single run. Beyond its analytical scope, the method outlines a structured validation strategy, which is aligned with regulatory principles, and may therefore serve as a practical reference for future LC-MS/MS assays employing surrogate calibration.
在生物基质中对低浓度的内源性和外源性类固醇进行定量分析仍然是一项重大的分析挑战。基于免疫分析的诊断方法受到交叉反应性的限制，尤其是在低水平时，这促使临床应用向（超）高效液相色谱-串联质谱法（（U）HPLC-MS/MS）转变。内源性激素定量分析的一个关键限制是缺乏用于外部校准的真实空白基质。为了解决这个问题，我们开发了一种替代校准方法，采用1,2-二甲基咪唑-5-磺酰氯（DMIS）对雌激素进行衍生化，能够在对非衍生化类固醇进行灵敏和选择性定量的同时，对雌激素进行定量。使用稳定同位素标记的替代校准物和内标物，在临床相关范围内实现基质匹配定量。在多个校准水平的血浆中系统地验证了分析物与替代校准物之间的平行性。通过使用窄内径超高效液相色谱柱和优化的色谱条件进一步优化该方法，以提高对广泛分析物的灵敏度和分辨率。结合高效的蛋白质沉淀和基于96孔板的固相萃取，所开发的分析方法能够在人血浆中以高精度和准确性实现皮克/毫升级别的定量。这种综合方法独特地将内源性类固醇的替代校准与外源性避孕药的外部校准相结合，包括基于DMIS的灵敏雌激素衍生化，能够在一次运行中实现全面的激素分析。除了其分析范围外，该方法还概述了一种符合监管原则的结构化验证策略，因此可为未来采用替代校准的液相色谱-串联质谱分析提供实用参考。
