Endocrine Abstracts

Introduction: The 11-oxygenated androgens are elevated in congenital adrenal hyperplasia (CAH). These steroid biomarkers can be quantified by LC-MS/MS, which has high specificity and allows multi-analyte analysis from a single sample. Dried blood spots (DBS) are minimally invasive and easy to collect, transport and store. They are widely used for monitoring CAH disease control and represent a useful matrix for steroid analysis. Volumetric micro-sampling devices are increasingly available - they collect a fixed volume of blood for analysis and are reported to be more accurate than traditional dried blood spots.

Aim: This study aimed to develop and validate a multiplex LC-MS/MS assay for seven analytes (cortisol, 17-OHP, androstenedione, testosterone, 11-ketotestosterone (11-KT), 11-hydroxyandrostenedione (11-OHA4) and progesterone) from dried blood spots. In addition, we compared W-903 filter paper to the volumetric Capitainer®B10 device.

Method: We developed a LC-MS/MS assay using commercially available standards. The extraction procedure, liquid chromatography method and ionisation conditions were optimised to improve sensitivity. Data were analysed in Skyline and validation metrics calculated in R (version 4.5.1). Standard curves were created over the quantitation range of the assay, to assess linearity, the limits of detection (LOD) and quantitation (LOQ) and the matrix effect. Quality control samples were analysed to determine the accuracy and precision of the assay.

Results: A linear relationship (R²≥0.99) was observed at a concentration range of 0 – 215 ng/ml or 0 – 500 ng/ml depending on the analyte and matrix (Capitainer® or W-903 filter paper). The matrix effect was >100% for all analytes. LODs ranged between 0.06-0.47 ng/ml (Capitainer®) and 0.05 – 0.53 ng/ml (W-903). LOQs ranged between 0.18-1.1 ng/ml (Capitainer®) and 0.12 – 1.35 ng/ml (W-903). Intra-batch and inter-batch precision and accuracy coefficients of variation (CVs) were ≤ 20% for analytes, except 11-OHA4 using W-903 filter paper.

Discussion: We report a multiplex LC-MS/MS method specifically designed for measuring relevant biomarkers, including 11-KT and 11-OHA4, from paediatric CAH patients using DBS samples. The assay achieved a clinically useful LOQ using both Capitainer® and W-903 filter paper. The method is accurate and precise for all analytes using the Capitainer® device. Further studies involving patient samples are planned.