A Metrologically Traceable Candidate Reference Measurement Procedure (cRMP) for Dehydroepiandrosterone Sulfate in Human Serum Based on Isotope Dilution‐Liquid Chromatography–Tandem Mass Spectrometry (ID‐LC–MS/MS)

ABSTRACT Dehydroepiandrosterone sulfate (DHEAS), a crucial steroid hormone for adrenal function and pubertal development, is highly associated with related diseases. This study aims to develop and validate an isotope dilution liquid chromatography tandem mass spectrometry (ID‐LC–MS/MS)‐based candidate reference measurement procedure (cRMP) for quantifying serum DHEAS. Serum samples were prepared by protein precipitation with acetonitrile and separated on a reversed phase column. Assay validation was conducted under the guidance of standard documents, including C62‐A, EP6‐A, EP10‐A3, and C50‐P endorsed by the Clinical and Laboratory Standards Institute (CLSI). The cRMP was established and proven to be highly specific without significant matrix effect and able to accurately quantify DHEAS in human serum. The intra‐assay and inter‐assay imprecision ranged from < 0.1 to 1.0% and from 1.0 to 1.1%, respectively. Trueness was assessed by recovery rate from 99.6 to 101.2%. The limit of detection (LoD) was 0.270 nmol/L, and the lower limit of the measuring interval (LLMI) was 12.1 nmol/L. A linear correlation ranged from 7.60 to 41 858 nmol/L was observed with a correlation coefficient > 0.999. R 2 value of linear regression analysis between this method and clinical immunoassays was ≥ 0.990. The relative expanded uncertainty was 1.5%–2.5% over the concentration range of 12.1–33 043 nmol/L. This study developed an ID‐LC–MS/MS‐based cRMP, which provided high specificity, trueness, and precision for serum DHEAS quantification, contributing to the DHEAS measurement standardization and traceability.