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

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Abstract

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 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). 6α-OHF and 6β-OHF were well separated on an XTerra MS C18 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.

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Authors and Affiliations

  1. Department of Medicinal Chemistry and Clinical Pharmacy, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi Hachioji, Tokyo, 192-0392, Japan

    Hiromi Shibasaki, Sawako Okamoto, Risako Inoue, Misato Okita, Akitomo Yokokawa & Takashi Furuta

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  1. Hiromi Shibasaki
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  2. Sawako Okamoto
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  3. Risako Inoue
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  4. Misato Okita
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  5. Akitomo Yokokawa
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  6. Takashi Furuta
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Correspondence to Takashi Furuta.

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Shibasaki, H., Okamoto, S., Inoue, R. et al. Separation and quantitative determination of 6α-hydroxycortisol and 6β-hydroxycortisol in human urine by high-performance liquid chromatography with ultraviolet absorption detection. Anal Bioanal Chem 402, 2945–2952 (2012). https://doi.org/10.1007/s00216-012-5714-3

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  • DOI: https://doi.org/10.1007/s00216-012-5714-3

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