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

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.