Quantitative analysis of 34 sex (pro)hormones, conjugates and bioactive oxidation products thereof in human plasma by GC- and LC-MS/MS and systematic investigation of overestimations of analyte concentrations not accounted for by method validation

IF 2.1 4区医学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Steroids Pub Date : 2024-05-18 DOI:10.1016/j.steroids.2024.109441

Miriam Raps, Carolin Kleider, Leane Lehmann

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Abstract

When investigating endocrine disorders, it is essential to assess a comprehensive quantitative profile of sex (pro)hormones in plasma including conjugates. Thus, the present study aimed to develop and validate a comprehensive mass spectrometry-based multimethod combining the direct analysis of unconjugated sex (pro)hormones and oxidation products thereof (by GC), as well as their sulfates and glucuronides present in higher concentrations (by LC) with the indirect quantification of glucuronides present in lower concentrations after selective glucuronide hydrolysis (by GC) and its application to plasma derived from ten pre- and postmenopausal women and men each. Even guideline-compliant validation experiments cannot completely reflect overestimation of analyte concentrations due to effects depending on the individual ratio of analytes (i.e. chemical formation of analytes or incomplete removal of interfering analytes). Thus, the extent of processes not accounted for by the calibration strategy were investigated and maximum over- or underestimations of analyte concentrations were assessed for each plasma sample individually. 34 analytes were successfully calibrated, validated (median accuracy 101.1 %, median inter-day precision 8.1 %) and 31 were detected above the detection limit in plasma samples. The sporadic maximum individual over- or underestimation of analyte concentrations amounted to less than 20 %.

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利用气相色谱-质谱（GC）和液相色谱-质谱（LC-MS/MS）对人体血浆中的 34 种性（原）激素、其共轭物和生物活性氧化产物进行定量分析，并对方法验证未考虑的分析物浓度高估情况进行系统调查

在研究内分泌失调时，必须对血浆中的性（原）激素（包括共轭物）进行全面的定量分析。因此，本研究旨在开发和验证一种基于质谱的综合多方法，该方法结合了直接分析未结合的性（原）激素及其氧化产物（气相色谱法）、高浓度的性激素硫酸盐和葡萄糖醛酸苷（液相色谱法），以及选择性水解葡萄糖醛酸苷后间接定量低浓度的葡萄糖醛酸苷（气相色谱法），并将其应用于绝经前和绝经后的十名女性和男性的血浆中。即使是符合指南要求的验证实验，也无法完全反映出分析物浓度的高估，这是因为分析物的个体比例会产生影响（即分析物的化学形成或干扰分析物的未完全去除）。因此，对校准策略未考虑的过程的程度进行了调查，并对每个血浆样本的分析物浓度的最大高估或低估进行了评估。成功校准和验证了 34 种分析物（准确度中位数为 101.1%，日间精确度中位数为 8.1%），31 种分析物在血浆样本中的检测结果超过了检测限。个别分析物浓度的零星最大高估或低估率低于 20%。

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来源期刊

Steroids 医学-内分泌学与代谢

CiteScore

5.10

自引率

3.70%

发文量

120

审稿时长

73 days

期刊介绍： STEROIDS is an international research journal devoted to studies on all chemical and biological aspects of steroidal moieties. The journal focuses on both experimental and theoretical studies on the biology, chemistry, biosynthesis, metabolism, molecular biology, physiology and pharmacology of steroids and other molecules that target or regulate steroid receptors. Manuscripts presenting clinical research related to steroids, steroid drug development, comparative endocrinology of steroid hormones, investigations on the mechanism of steroid action and steroid chemistry are all appropriate for submission for peer review. STEROIDS publishes both original research and timely reviews. For details concerning the preparation of manuscripts see Instructions to Authors, which is published in each issue of the journal.