Candidate reference measurement procedure based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry for the quantification of androstenedione in human serum and plasma

Objectives Androstenedione (ASD) is a biomarker used in the diagnosis of hyperandrogenism and adrenal hyperplasia. Therefore, accurate measurement of serum ASD is pivotal in clinical settings. We aimed to develop a novel highly selective reference measurement procedure (RMP) based on isotope dilution-two dimensional-liquid chromatography-tandem mass spectrometry (ID-2D-LC-MS/MS) for the quantification of ASD in human serum/plasma.Methods To achieve high selectivity and sensitivity, a two-dimensional heart-cut LC approach for LC-MS/MS and a supported liquid extraction sample preparation protocol were employed. Matrix effects were evaluated through a post-column infusion experiment and comparison of standard line slopes. Precision and accuracy were determined via a multi-day validation experiment. Reproducibility was assessed by comparing results from two independent laboratories, and measurement uncertainty (MU) was evaluated in compliance with current guidelines.Results Our novel RMP proved effective for measuring ASD concentrations ranging from 0.00800 ng/mL to 12.0 ng/mL (0.0279 nmol/L to 41.9 nmol/L) and demonstrated matrix-independence. The relative mean bias varied between 0.6 and 2.2 % across different matrices and concentration levels. Intermediate precision was observed to be between 1.2 and 1.9 %. The expanded measurement uncertainty for ASD target value assignment ranged from 1.7 to 2.5 %, irrespective of sample concentration. Equivalence to the JCTLM-listed RMP was evaluated through a method comparison study, revealing a high degree of agreement (r >= 0.997). Additionally, by comparing results from two independent laboratories, the transferability and reproducibility of the RMP were confirmed.Conclusions This isotope dilution two-dimensional LC-MS/MS method can be used for the evaluation and standardization of routine assays and for measuring individual patient samples, ensuring traceability.