Quantitation of cortisol and related 3-oxo-4-ene steroids in urine using gas chromatography/mass spectrometry with stable isotope-labeled internal standards

Abstract

A method for the profiling of several important 3-oxo-4-ene urinary steroids is reported. The methodology is combined gas chromatography/mass spectrometry (GC/MS) utilizing stable isotope-labeled internal standards. The following standards were obtained or easily synthesized: [9,11,12,12- 2H 4]cortisol, [1,2- 2H 2] and [9,12,12- 2H 2]cortisone, [1,2- 2H 2]6β-hydroxycortisol, and [1,2- 2H 2]18-hydroxycortisol. We found the following excretions of free steroids for normal adult males and females: cortisol (males mean ± SD, 35 ± 13; females mean ± SD, 23 ± 13), cortisone (males mean ± SD, 58 ± 23; females mean ± SD, 50 ± 22), 6β-hydroxycortisol (males mean ± SD, 164 ± 59; females mean ± SD, 108 ± 55), and 18-hydroxycortisol (males mean ± SD, 148 ± 55; females mean ± SD, 71 ± 30). For 18-hydroxycortisol in particular, the excretions were much higher for males than for females. We found that the larger part of urinary cortisol and cortisone is not free but is released from conjugation by enzymes present in snail digestive juice. Using a pooled urine sample from an equal number of male and female subjects, we found that for cortisol 29% was excreted free, 28% as glucuronide and 43% as other conjugates (probably sulfates). For cortisone 41% was free, 45% β-glucuronide and 14% as other conjugates. Relatively little (3–8%) of the hydroxylated cortisols were excreted conjugated.