Comprehensive Steroid Assay with Non-Targeted Analysis Using Liquid Chromatography Ion Mobility Mass Spectrometry

Abstract

Aldosterone-producing adenomas (APAs) have different steroid profiles in serum, depending on the causative genetic mutation. Ion mobility is a separation technique for gas-phase ions based on their m/z values, shapes, and sizes. Human serum (100 µL) was purified by liquid–liquid extraction using tert-butyl methyl ether/ethyl acetate at 1/1 (v/v) and mixed with deuterium-labeled steroids as the internal standard. The separated supernatant was dried, re-dissolved in water containing 20% methanol, and injected into a liquid chromatography–ion mobility–mass spectrometer (LC/IM/MS). We established a highly sensitive assay system by separating 20 steroids based on their retention time, m/z value, and drift time. Twenty steroids were measured in the serum of patients with primary aldosteronism, essential hypertension, and healthy subjects and were clearly classified using principal component analysis. This method was also able to detect phosphatidylcholine and phosphatidylethanolamine, which were not targeted. LC/IM/MS has a high selectivity for known compounds and has the potential to provide information on unknown compounds. This analytical method has the potential to elucidate the pathogenesis of APA and identify unknown steroids that could serve as biomarkers for APA with different genetic mutations.