Quantitative Analysis of 34 Sex (Pro)hormones, Conjugates, and Bioactive Oxidation Products in Human Plasma by GC- and LC-MS/MS and Systematic Investigation of Overestimations of Analyte Concentrations Not Accounted for by Method Validation

The study developed a comprehensive method using GC- and LC-MS/MS to analyze 34 sex (pro)hormones and their conjugates in human plasma, focusing on potential overestimations of analyte concentrations due to incomplete removal of interfering substances and unintended reactions. The method was validated for accuracy, precision, and detection limits, and applied to plasma samples from 10 premenopausal women, 10 postmenopausal women, and 10 men. It found that 29 analytes were quantifiable in more than 50% of the samples, with accuracy ranging from 81% to 115% and precision between 0.3% to 20%. The study emphasizes the importance of optimizing extraction conditions and calibration strategies to ensure accurate hormone profiling, which is crucial for understanding endocrine disorders.