Separation and quantitative determination of 6α-hydroxycortisol and 6β-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection.
The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6α-hydroxycortisol (6α-OHF) and 6β-hydroxycortisol (6β-OHF), in human urine, using 6α-hydroxycorticosterone as internal standard. 6α-OHF and 6β-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C(18) plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH (pH 3.77) and (B) 0.05 M KH(2)PO(4)-0.01 M CH(3)COOH/acetonitrile (2:3, v/v). 6α-OHF and 6β-OHF were well separated on an XTerra MS C(18) 5 μm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6α-OHF and 6β-OHF were Rs = 4.41 for program 2 and Rs = 4.60 for program 3. The analysis was performed within 23~26 min, monitored by UV absorbance at 239 nm. The lower limits of detection of 6α-OHF and 6β-OHF were 0.80 ng per injection (s/n = ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6α-OHF and 41.08 ng/ml for 6β-OHF, respectively. The within-day reproducibilities in the amounts of 6α-OHF and 6β-OHF determined were in good agreement with the actual amounts added, the relative errors being -5.37% and -3.73% (gradient 2) and -5.69% and -3.96% (gradient 3) for both 6α-OHF and 6β-OHF, respectively. The inter-assay precisions (RSDs) for 6α-OHF and 6β-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6α-OHF and 6β-OHF in urine to evaluate the time courses of 6α-hydroxylation and 6β-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.
Department of Medicinal Chemistry and Clinical Pharmacy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi Hachioji, Tokyo, Japan.
Hiromi
H
Shibasaki
Sawako
S
Okamoto
Risako
R
Inoue
Misato
M
Okita
Akitomo
A
Yokokawa
Takashi
T
Furuta
D000042:Absorption; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000652:urine; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D055815:Young Adult
C001380:6 beta-hydroxycortisol; D051544:Cytochrome P-450 CYP3A; D006854:Hydrocortisone