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The urinary 6β-OH-cortisol/cortisol ratio is a specific, non-invasive marker for evaluating inductive or inhibitory effects on cytochrome P450 3A activity. We propose a new quantitative gas chromatographymass spectrometry with isotope dilution (GC-ID-MS) method for the simultaneous determination of urinary free cortisol (UFC) and 6β-OH-cortisol (6β-OHC). The method utilizes the following: (a) addition of internal standard ( 2 H 2  cortisol) to 1 ml of urine; (b) loading on to an Extrelut column and elution with dichloromethane; (c) derivatization to dimethoxime tri-(trimethyl-silyl)-ether (MOX-TMS); (d) separation and identification by GC-ID-MS. The detection limit for cortisol was 22 pg injected (signal-to-noise ratio 10:1) and for 6β-OH-cortisol 123 pg injected (signal-to-noise ratio 10:1). The intra-assay and the inter-assay imprecision were 4.69% and 7.4% for 6β-OHC and 2.44% and 3.53% for cortisol, respectively. We used this method to analyze 57 morning urine samples of healthy volunteers and patients under different conditions. We found that chronic alcoholics had a significantly higher ratio of 6β-OHC/UFC compared to controls (p&lt;0.0001), whereas adults undergoing methadone therapy and patients with acute alcohol intoxication exhibited a significantly lower urinary 6β-OHC/UFC ratio (p&lt;0.05 and p&lt;0.01, respectively). The proposed method allows a rapid and accurate assessment of the 6β-OHC/UFC ratio.

Gas Chromatography-Mass Spectrometry Measurement of 6β-OH-Cortisol/Cortisol Ratio in Human Urine: A Specific Marker of Enzymatic Induction