An isotope dilution-liquid chromatography-tandem mass spectrometry-based candidate reference measurement procedure for the quantification of dehydroepiandrosterone sulfate in human serum and plasma.

OBJECTIVES

Dehydroepiandrosterone sulfate (DHEAS), the sulfate ester of dehydroepiandrosterone, is one of the most common steroid hormones in the human body and the precursor of several other androgens. It is primarily used as a diagnostic or prognostic indicator in adrenal and reproductive disorders. Present immunoassays for DHEAS lack sensitivity and specificity, being vulnerable to cross-reactivity with endogenous interferences. Therefore, an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed to quantify DHEAS in human serum/plasma.

METHODS

We ensured traceability to the International System of Units by using quantitative nuclear magnetic resonance to characterize a commercially available DHEAS reference material used for assay calibration. To mitigate matrix effects and prevent interference co-elution, a two-dimensional heart-cut LC method was employed for LC-MS/MS, in combination with a solid phase extraction sample preparation protocol. Selectivity was determined by spiking the prepared internal standard with the interferences testosterone, epi-testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, and estrone, in analyte free matrix. A post-column infusion experiment and comparison of standard line slopes were performed to evaluate matrix effects. Precision and accuracy were assessed via a multi-day validation experiment, and variability components estimated using analysis of variance-based variance-components analysis. Measurement uncertainty (MU) was evaluated in compliance with current guidelines.

RESULTS

This RMP was suitable for analyzing DHEAS within the 0.800 to 8,400 ng/mL (2.17-22,800 nmol/L) range, demonstrating selectivity, sensitivity, and matrix-independence. Trueness and accuracy assessment revealed a relative bias (n=6) between -1.9 and 0.3 % for surrogate matrix samples (except for 5.9 % at the lowest level), -2.3 to 3.6 % for Li-heparin plasma samples and sample dilutions, and an overall bias between 0.7 and 1.8 % (n=60), indicating no statistically significant bias. The measurement process resulted in standard measurement uncertainties (MUs) ranging from 4.0 to 5.6 % for the low range and 3.5-4.2 % for the high range. At a 95 % confidence level (k=2), these uncertainties expanded to 7.9-11.1 % and 7.1-8.3 %, respectively. Reference values, determined from six measurements over multiple days (n=6), had standard MUs between 1.6 and 2.1 % for the low range and 0.9-1.7 % for the high range, with expanded MUs of 3.2-4.3 % and 1.9-3.5 %.

CONCLUSIONS

This RMP exhibited high analytical performance for DHEAS quantification and met requirements for measurement uncertainty. Additionally, it enabled differentiation between the DHEAS and other androgens. Consequently, this RMP is suitable for routine assay standardization and clinical sample evaluation.