title: Gas chromatography-mass spectrometry measurement of 6beta-OH-cortisol/cortisol ratio in human urine: a specific marker of enzymatic induction.
abstract: The urinary 6beta-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 chromatography-mass spectrometry with isotope dilution (GC-ID-MS) method for the simultaneous determination of urinary free cortisol (UFC) and 6beta-OH-cortisol (6beta-OHC). The method utilizes the following: (a) addition of internal standard (2H2 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 6beta-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 6beta-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 6beta-OHC/UFC compared to controls (p<0.0001), whereas adults undergoing methadone therapy and patients with acute alcohol intoxication exhibited a significantly lower urinary 6beta-OHC/UFC ratio (p<0.05 and p<0.01, respectively). The proposed method allows a rapid and accurate assessment of the 6beta-OHC/UFC ratio.
authors.affiliation: Laboratory of Clinical Biochemistry, Azienda Ospedaliera Careggi, Firenze, Italy. fluceri@katamail.com
authors.forename: F
authors.initials: F
authors.lastname: Luceri
authors.forename: S
authors.initials: S
authors.lastname: Fattori
authors.forename: C
authors.initials: C
authors.lastname: Luceri
authors.forename: M
authors.initials: M
authors.lastname: Zorn
authors.forename: P
authors.initials: P
authors.lastname: Mannaioni
authors.forename: G
authors.initials: G
authors.lastname: Messeri
mesh_terms: D000328:Adult; D000437:Alcoholism / Q000175:diagnosis / Q000652:urine; D000929:Antidepressive Agents, Tricyclic / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001569:Benzodiazepines / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000652:urine*; D008691:Methadone / Q000494:pharmacology; D015394:Molecular Structure; D009294:Narcotics / Q000494:pharmacology; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D015203:Reproducibility of Results; D019966:Substance-Related Disorders / Q000175:diagnosis / Q000652:urine; D016482:Urinalysis / Q000379:methods
chemical_list: D000929:Antidepressive Agents, Tricyclic; D009294:Narcotics; D001569:Benzodiazepines; C001380:6 beta-hydroxycortisol; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D008691:Methadone; D006854:Hydrocortisone