Quantification of eight clinically relevant serum adrenal steroids in infants by UHPLC-APCI-MS/MS

• A new UHPLC-APCI-MS/MS-based method was validated to determine eight serum steroids. • It requires 4-fold less sample volume than traditional immunoassays. • It was successfully applied to infants under three months of age. • It has large impact on the assessment of paediatric adrenal disorders in Argentina. There is growing interest in replacing steroid immunoassays with liquid chromatography–tandem mass spectrometry-based methods in many clinical and research applications. The aim of the present work is to develop and validate an ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based method using atmospheric pressure chemical ionisation (APCI) to quantify cortisol, androstenedione, testosterone, pregnenolone, progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, and dehydroepiandrosterone in paediatric serum samples. Sample preparation was performed by protein precipitation using 100 µL of sample . The analytical method was validated in accordance with FDA and EMA international guidelines. Commercial quality control materials and a reference material (NIST®SRM®1950) were analysed for external assessment. Clinical applicability was tested in 61 infants under three months of age (50 healthy infants, eight preterm neonates, and three patients with confirmed adrenal disorders). The LLOQs were lower than 8 nmol/L; within- and between-run CVs were <12 %. Accuracy, in terms of recovery, was 89–111 %. Serum cortisol levels varied widely in healthy infants, and only testosterone levels exhibited sexual dimorphism ( p < 0.0001). Androstenedione and 17-hydroxyprogesterone levels were significantly higher in the preterm group compared with babies born at term. Patients with confirmed adrenal pathologies exhibited abnormal steroid profiles. A novel, accurate, and sensitive UHPLC-APCI-MS/MS-based method for the simultaneous analysis of eight steroids in serum was developed, validated and successfully applied to infants younger than three months of age. The method enabled detection of abnormal values of precursors and steroid hormones in patients with adrenal disorders, with the potential for a large impact on paediatric patient care.