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

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.