An isotope dilution-liquid chromatography tandem mass spectrometry-based candidate reference measurement procedure...

Dehydroepiandrosterone (DHEA) is a steroid prohormone and important precursor for estrogen and testosterone biosynthesis. For differential diagnosis of sexual development disorders (e.g., polycystic ovary syndrome, congenital adrenal hyperplasia), accurate measurement of DHEA in clinical testing is essential. To address that need for high-quality, reproducible assays, an isotope dilution-liquid chromatography (LC)-tandem mass spectrometry (MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of DHEA was developed and validated.

A certified primary reference material from the National Measurement Institute of Australia (NMIA), was used for DHEA assay calibration and to ensure traceability to the International System of Units (SI). Two-dimensional heart-cut chromatography was employed for LC-MS/MS in combination with a solid phase extraction sample preparation protocol, to mitigate matrix effects and prevent interference coelution. Selectivity was evaluated by spiking either the analyte or internal standard with potential interferents, such as dehydroepiandrosterone-sulfate, testosterone, and similar steroid compounds. For the evaluation of matrix effects, standard line slopes of various matrices were compared. Precision and accuracy were assessed via a multi-day validation experiment, and variability components were estimated using variance component analysis. Measurement uncertainty was evaluated in compliance with current guidelines.

This candidate RMP proved suitable for the analysis of DHEA in the measurement range of 0.0800-36.0 ng/mL (0.277-125 nmol/L). Predefined requirements for sensitivity and selectivity were fully met, and independence from matrix effects was demonstrated successfully. Across all tested concentration levels, intermediate precision was ≤1.5 % and repeatability was ≤1.0 %, while the relative mean bias ranged from -1.1 to 0.1 %. Regardless of DHEA concentration or sample type, the expanded measurement uncertainty for reference value assignment (n=6) was ≤1.7 %.

This isotope dilution-LC-MS/MS-based candidate RMP for DHEA in human serum and plasma met pre-defined analytical performance requirements such as precision, specificity and measurement uncertainty, and showed superior selectivity towards several potential interferents tested. It is suitable for application in clinical sample evaluation and routine assay standardization.