Liquid chromatography-tandem mass spectrometry-based urinary steroid profiling applied to primary aldosteronism diagnosis.

Steroid hormones are essential regulators of physiological homeostasis, which can help in diagnosing primary aldosteronism (PA). However, current methodologies for urinary steroid analysis face critical limitations, including narrow analyte coverage and tedious sample preparation workflows. Since the liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the preferred technique for steroid measurement, there is a great need to establish an LC-MS/MS method combined with simplified and efficient sample pretreatment for multi-steroid profiling. We establish a novel LC-MS/MS method for quantifying 35 steroid hormones within a 25-min runtime. 40 μL of urine samples can be efficiently hydrolyzed at room temperature in just 0.5 h using transgenic β-glucuronidase. Detection is performed on the Waters Acquity I-Class UPLC-tandem with a Waters TQ-S triple quadrupole MS/MS system, and the method performance is systematically evaluated. Linearity is excellent, and the recovery rates range from 80.0 to 120.6%. Acceptable intra-assay and inter-assay precisions are achieved, with the coefficients of variation ranging from 1.86 to 15.10% and 3.91 to 19.75%, respectively. For clinical application, patients with PA (n = 37) exhibit significantly higher levels of aldosterone, tetrahydroaldosterone, 18-hydroxycorticosterone, 18-oxocortisol, and 18-hydroxycortisol compared to non-PA patients (n = 104). Combined steroid profiling demonstrates strong diagnostic performance for PA, yielding an area under the curve of 0.914, with a sensitivity of 0.87 and a specificity of 0.88. In conclusion, this study establishes a technically advanced LC-MS/MS method that integrates efficient enzymatic hydrolysis to enable the accurate quantification of urinary steroids for the diagnosis of endocrine disorders.