Abstract

Quantitation of Urinary 6ß-Hydroxycortisol and Free Cortisol by Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry

Jun Han, Shirin Kalyan, Jerilynn C. Prior and Christoph H. Borchers

A new UPLC-MS method that combined 0.01% formic acid/isopropanol as the mobile phase and an ESI-QTOF instrument has been developed for reliable quantitation of urinary 6β-hydroxycortisol (6βHC) and free cortisol (FC). The use of 0.01% formic acid/isopropanol for binary gradient elution on a reversed-phase C18 column resulted in 3.1 and 5.0 times the peak areas for 6βHC and FC, respectively, as compared to 0.1% formic acid/acetonitrile. Liquid-liquid extraction with ethyl acetate at pH 5 was superior to solid-phase extraction for sample preparation. A mass window of 50 ppm was used for quantitative monitoring of 6βHC (m/z 379.212) and FC (m/z 363.217) using full-mass detection, with the on-column limits of detection as 4.0 and 1.4 fmol, and the lower limits of quantitation as 13.6 and 6.9 fmol for 6βHC and FC. The accuracy of quantitation ranged from 93.3% to 102.3% recoveries at three spiking levels, with the maximum intraday and interday %CV being 3.7 and 5.3 for both analytes. This LC-MS method was then applied to the quantitation of 6βHC and FC in the urine pairs collected during the follicular and premenstrual phases of menstrual cycle from each of sixty-one premenopausal women — forty-one with ovulatory cycles and twenty with subclinical anovulatory cycles. Paired two-tailed T-tests showed no significant difference (p>0.05) between the metabolic ratios of urinary 6βHC/FC during the follicular and premenstrual phases in both ovulatory and anovulatory subjects, indicating no correlation of the urinary 6βHC/FC ratios between the two physiological phases of menstrual cycle in these subjects.