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Clinical Chemistry 43: 1386-1391, 1997;
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(Clinical Chemistry. 1997;43:1386-1391.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Determination of urinary free cortisol by HPLC

Ursula Turpeinen1,a, Helene Markkanen1, Matti Välimäki2 and Ulf-Håkan Stenman1

1 Helsinki University Central Hospital, Laboratory, Haartmaninkatu 2, 00290 Helsinki, Finland, and
2 University of Helsinki, Department of Medicine.
a Author for correspondence. Fax +358-9-471 4804; e-mail ursula. turpeinen{at}hyks.mailnet.fi

We here report a reversed-phase HPLC method for the determination of free cortisol in human urine, using methylprednisolone as the internal standard. Before chromatography, samples were extracted with a C18 solid-phase extraction column and the steroids were separated on a LiChrospher 100 C18 column with a mobile phase of methanol/acetonitrile/water (43/3/54 by vol). Linearity, precision, and accuracy of the method were established. The detection limit was 10 pmol of cortisol, and total CVs were <8%. With various solid-phase extraction columns the recovery of cortisol was 36–97%; recovery of the internal standard was 43–85%. Study of interference by 6 other steroids and metabolites and 24 drugs showed that carbamazepine and digoxin partly overlapped with cortisol, but this interference could be reduced by modification of the mobile phase. The HPLC method was compared with an RIA and an automated immunoassay method. The results obtained by HPLC averaged 40% of the RIA values.


Key Words: indexing terms: hydrocortisone • sample preparation • method comparison • steroids • chromatography, reversed-phase




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