Automated enzymatic hydrolysis-magnetic-bead-assisted sequential extraction workflow for LC-MS/MS profiling of urinary aldosterone-pathway steroids.

Primary aldosteronism (PA) requires accurate subtype differentiation to guide targeted treatment. Urinary steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers a promising noninvasive strategy, but the clinical implementation of urinary steroid profiling remains constrained by manual hydrolysis and labor-intensive sample preparation. Here, we developed an automated magnetic-bead-assisted sequential extraction (MBASE) workflow that integrates thermally controlled enzymatic hydrolysis and WCX-based magnetic bead cleanup for simultaneous quantification of urinary aldosterone (ALD), tetrahydroaldosterone (THALD), 18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF), and 18-oxocortisol (18-OXOF). The optimized WCX-MBASE procedure provided balanced extraction and selective cleanup for urinary aldosterone-pathway steroids, while the F5 column enabled effective chromatographic separation of the target analytes from selected endogenous and exogenous interferences. In this programmable workflow, urine hydrolysis (60 min) and extraction were completed in a single automated pretreatment sequence, reducing intermediate handling and manual intervention. The method showed recoveries of 87.14%-109.56%, within-run imprecision of 3.33%-11.53% and total imprecision of 3.86%-12.73%. Compared with the conventional workflow, MBASE completed the automated hydrolysis-extraction cycle within 76 min, and lowered pretreatment cost while maintaining high analytical concordance. Clinical application in 136 hypertensive patients, including 38 with unilateral primary aldosteronism (UPA), 38 with bilateral primary aldosteronism (BPA), and 60 with essential hypertension, showed increased urinary ALD and related steroids in PA. The combined five-steroid panel achieved an apparent AUC of 0.855 for UPA identification, with an optimism-corrected AUC of 0.827 after bootstrap internal validation. This enzymatic hydrolysis-MBASE workflow provides an automation-compatible LC-MS/MS strategy for rapid and reliable profiling of urinary aldosterone-pathway steroids, supporting further evaluation of its potential for noninvasive PA subtype assessment and treatment monitoring.