A dilute-and-shoot liquid chromatography–tandem mass spectrometry method for urinary 18-hydroxycortisol quantification and its application in establishing reference intervals

Abstract

Background: Eighteen-hydroxycortisol (18-OHF) is a potential biomarker for differential diagnosis of the two major primary aldosteronism subtypes, aldosterone-producing adenoma, and idiopathic hyperaldosteronism. Methods: Urine samples were processed, and the 18-OHF in urine samples were successfully quantified by in-house established dilute-and-shoot liquid chromatography–tandem mass spectrometry (LC–MS/MS) method. Separation was accomplished on a Sigma Ascentis Express C18 column with a gradient mixture of phase (A) 0.2% formic acid in water and phase (B) 0.2% formic acid in methanol at a flow rate of 0.4 ml/min. Mass spectrometric detection was performed in positive electrospray ionization mode via a mass spectrometer. Results: The linearity of urinary 18-OHF ranged from 4.28 to 8.77 × 103 nmol/L, with a lower limit of quantification at 4.28 nmol/L. The intra- and inter-precision were both below 3%. The range of analytical recovery was 97.8%–109.2%. The validated dilute-and-shoot LC–MS/MS method was compared with the SPE LC–MS/MS method modified from the one reported in 2013. The results by Passing–Bablok regression analysis and Bland–Altman plotting demonstrated a good agreement between the two methods. The presented method was then applied to establish sex-specific reference intervals from 62 males and 62 females, respectively. The calculated 2.5%–97.5% reference intervals for 24-h urinary 18-OHF were 113–703 nmol/day for males and 71.2–450 nmol/day for females. Conclusion: The presented dilute-and-shoot LC–MS/MS method for 18-OHF quantification showed a good performance in the clinical application. Furthermore, the sex-specific reference intervals for 24-h urinary 18-OHF were first established and quite important for its application in primary aldosteronism subtyping.