Separation and quantitative determination of 6 Delta a-hydroxycortisol and 6 Delta b-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection

• Published in: Analytical and bioanalytical chemistry Vol. 402; no. 9; pp. 2945 - 2952
• Main Authors: Shibasaki, Hiromi, Okamoto, Sawako, Inoue, Risako, Okita, Misato, Yokokawa, Akitomo, Furuta, Takashi
• Format: Journal Article
• Language: English
• Published: 01.03.2012
• Subjects: Absorbance; Cartridges; Elution; Human; Liquid chromatography; Metabolites; Separation; Urine
• ISSN: 1618-2642
• DOI: 10.1007/s00216-012-5714-3

The present study developed an high-performance liquid chromatography (HPLC) method for the simultaneous determination of urinary metabolites of endogenous cortisol, 6 Delta *a-hydroxycortisol (6 Delta *a-OHF) and 6 Delta *b-hydroxycortisol (6 Delta *b-OHF), in human urine, using 6 Delta *a-hydroxycorticosterone as internal standard. 6 Delta *a-OHF and 6 Delta *b-OHF were extracted from urine with ethyl acetate by using a Sep-Pak C18 plus cartridge. Separation of the stereoisomers was achieved on a reversed-phase hybrid column by a gradient elution of (A) 0.05 M KH2PO4--0.01 M CH3COOH (pH 3.77) and (B) 0.05 M KH2PO4--0.01 M CH3COOH/acetonitrile (2:3, v/v). 6 Delta *a-OHF and 6 Delta *b-OHF were well separated on an XTerra MS C18 5 Delta *mm column using two types of stepwise gradient elution program (programs 2 and 3). Resolutions of 6 Delta *a-OHF and 6 Delta *b-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 6 Delta *a-OHF and 6 Delta *b-OHF were 0.80 ng per injection (s/n=ca. 8), and the lower limits of quantification were 5.02 ng/ml for 6 Delta *a-OHF and 41.08 ng/ml for 6 Delta *b-OHF, respectively. The within-day reproducibilities in the amounts of 6 Delta *a-OHF and 6 Delta *b-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 6 Delta *a-OHF and 6 Delta *b-OHF, respectively. The inter-assay precisions (RSDs) for 6 Delta *a-OHF and 6 Delta *b-OHF were less than 1.99% (gradient 2) and 2.61% (gradient 3), respectively. The present HPLC method was applied to the measurement of 6 Delta *a-OHF and 6 Delta *b-OHF in urine to evaluate the time courses of 6 Delta *a-hydroxylation and 6 Delta *b-hydroxylation clearances of cortisol during 40 days for phenotyping CYP3A in a healthy subject.