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

5α-dihydrotestosterone (DHT) is a potent androgen, related to male sexual development and irreversibly synthesized from testosterone via 5α-reductase. Dysfunctions in the 5α-reductase system, locally or globally, can have substantial health impacts; measurement of both DHT levels and the testosterone-DHT ratio are thus important for diagnosis and treatment monitoring. For that reason, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) to quantify DHT in human serum/plasma was developed.

We utilized certified primary reference material for DHT provided by the National Measurement Institute of Australia (NMIA) to calibrate our assay and ensure SI (International System of Units) traceability. To mitigate matrix effects and prevent the co-elution of interferences, two-dimensional heart-cut chromatography was employed for LC-MS/MS, in combination with a solid phase extraction (SPE) sample preparation protocol. Selectivity was determined by spiking the prepared internal standard with possibly interfering substances such as the inactive isomer 5β-DHT and other similar compounds. Comparison of standard line slopes was performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, and variability components estimated using analysis of variance (ANOVA)-based variance component analysis (VCA). Measurement uncertainty (MU) was evaluated in compliance with current guidelines.

This RMP was suitable for analyzing DHT within the range of 0.0160-2.76 ng/mL (0.0551 nmol/L-9.50 nmol/L), demonstrating selectivity, sensitivity and matrix-independence. Intermediate precision was ≤2.1 %, repeatability was ≤1.6 % across all concentration levels, and relative mean bias ranged from -2.2 to 2.5 %, across matrices and concentrations. Expanded MU for reference value assignment (n=6) was ≤2.8 %, irrespective of concentration or sample type.

This RMP exhibited high analytical performance for DHT quantification and met requirements for measurement uncertainty. Additionally, it enabled differentiation between the 5α-DHT and 5β-DHT isomers. Consequently, this RMP is suitable for routine assay standardization and clinical sample evaluation.