Simultaneous determination of 6beta-hydroxycortisol and cortisol in human urine by liquid chromatography with ultraviolet absorbance detection for phenotyping the CYP3A activity determined by the cortisol 6beta-hydroxylation clearance.
This study describes a high-performance liquid chromatographic (HPLC) method for the simultaneous determination of 6beta-hydroxycortisol (6beta-OHF) and cortisol in human urine using either methylprednisolone or beclomethasone as internal standard. Separation was achieved on a reversed-phase phenyl column by a gradient elution of 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH with acetonitrile (4:6, v/v). 6beta-Hydroxycortisol and cortisol were monitored by UV absorption at 239 nm. The lower quantitation limits of the present HPLC method were 21.5 ng/ml for 6beta-OHF and 5.0 ng/ml for cortisol in urine. The within-day reproducibilities in the amounts of 6beta-OHF and cortisol determined were in good agreement with the actual amounts added, the relative error being less than 1.59%. The inter-assay precisions (R.S.D. values) were less than 7.91% for 6beta-OHF and cortisol. The method was compared with the GC/MS method by measuring 6beta-OHF in the same urine samples. A good correlation was found between the amounts determined by the two methods. The regression equations for the HPLC (y) and GC/MS (x) methods were: y=1.0701x+17.389 (r=0.9772) for methylprednisolone as internal standard and y=1.0827x+6.1364 (r=0.9794) for beclomethasone as internal standard.
Furuta
Takashi
T
Department of Medicinal Chemistry and Clinical Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, 192-0392 Tokyo, Japan. furutat@ps.toyaku.ac.jp
Mori
Chieko
C
Suzuki
Atsushi
A
Shibasaki
Hiromi
H
Yokokawa
Akitomo
A
Kasuya
Yasuji
Y
D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000493:pharmacokinetics; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017291:Clarithromycin / Q000008:administration & dosage / Q000493:pharmacokinetics; D051544:Cytochrome P-450 CYP3A; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000652:urine*; D006900:Hydroxylation; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010641:Phenotype; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; C001380:6 beta-hydroxycortisol; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D017291:Clarithromycin; D006854:Hydrocortisone