Enabling insights into the maturation of the renin-angiotensin-aldosterone system in children Development of a low-volume LC-MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite

Introduction: As part of the renin-angiotensin-aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. Methods: A HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight-mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. Results: Using the Sciex TripleTOF 6600, a reliable determination in 50 mu L serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between - 1.21 and - 6.99 % and 2.07 and - 10.22 %, respectively, confirming compliance with international guidelines. Conclusion: A simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 mu L serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.