Establishing a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for both 18‐hydroxycorticosterone and its interference: Revisiting the specificity characteristic of LC‐MS/MS in the steroid measurement

Abstract Isomeric interference is essential in mass spectrometry (MS) analysis of steroid hormones. This study aims to investigate the specificity of liquid chromatography‐tandem MS (LC‐MS/MS) for measuring 18‐hydroxycorticosterone (18‐OHB) and to develop a validated LC‐MS/MS method. To identify the interfering substance on 18‐OHB quantification in plasma, high‐performance liquid chromatography‐tandem time‐of‐flight mass spectrometry was used for the retention time comparison and the fragment characteristics matching. 18‐OHB, cortisone, and cortisol could be chromatographically separated using water containing 0.025 mM ammonium fluoride and acetonitrile on the UPLC BEH C8 column. The interference of 18‐OHB with similar ion transitions was finally identified as 20β‐dihydrocortisone (20α‐DHE), instead of cortisone, cortisol, or 20β‐dihydrocortisone. By using optimized chromatographic conditions, 18‐OHB, and 20α‐DHE were separated well and the linearities were ≥0.999. The recovery rates ranged from 94.59% to 105.27% for 18‐OHB and 85.21%–101.23% for 20α‐DHE. The total coefficient of variations in the precision evaluation was ≤4.6% for 18‐OHB and ≤4.3% for 20α‐DHE. The medians of 18‐OHB and 20α‐DHE levels in patients with primary aldosteronism were 0.48 ng/mL and 0.14 ng/mL, respectively. This study explored the potential risks associated with isomeric interference in LC‐MS/MS for 18‐OHB quantification and established a robust LC‐MS/MS method for detecting plasma 18‐OHB and 20α‐DHE. Comprehensively assessing the effect of the underlying isomers when using LC‐MS/MS for hormone measurements is recommended.