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A simplified high-performance liquid chromatographic method for determination of vitamin d, 25-hydroxyvitamin d and 25-hydroxyvitamin d in human serum

Scandinavian Journal of Clinical and Laboratory Investigation, ISSN: 0036-5513, Vol: 52, Issue: 3, Page: 177-182
1992
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Article Description

Measurement of the serum level of 25-hydroxyvitamin D is the most useful parameter in evaluating vitamin D status. The serum level of vitamin D is a useful parameter in studying short time effects after exposure to ultraviolet light and absorption of the vitamin after oral administration. A method for simultaneous determinations of vitamin D 25-hydroxyvitamin D and 25-hydroxyvitamin D is described. Serum or plasma was extracted by methanol-isopropanol (90:10, v/v) and hexane. The hexane layer was injected in to a reversed-phase (C) high-performance liquid chromatographic (HPLC) system. 25-hydroxyvitamin D and 25-hydroxyvitamin D were eluted by methanol-water (85:15, v/v), and vitamin D by a linear gradient of methanol-water (85:15) and methanolisopropanol-water (87.5:10:2.5), and detected by u.v. absorption. This method gave separate determinations of the D and D forms of 25-hydroxyvitamin D, but owing to an interfering peak the method does not measure vitamin D. The assay was very sensitive with a detection limit of 5 nmol l- for 25-hydroxyvitamin D and D and vitamin D by using 0.5 ml serum or plasma for analysis, so that for low vitamin D levels more than 1 ml of serum is desirable. © 1992 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

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