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Clinical Chemistry 23: 1700-1704, 1977;
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Clinical Chemistry, Vol 23, 1700-1704, Copyright © 1977 by American Association for Clinical Chemistry

High-performance liquid chromatographic assay for 25-hydroxyvitamin D3 in serum

TJ Gilbertson and RP Stryd

We describe a precise and specific method for measuring 25- hydroxyvitamin D3 in 1 ml of human serum. Extraction with chloroform/methanol followed by chromatography on a 0.5 X 2-cm silica gel column yields a sample that is sufficiently free of extraneous material for high-performance liquid chromatography on a column of microporous silica gel (10 micron average particle diameter). The measurement is not influenced by vitamins D2 or D3, 25-hydroxyvitamin D2, or any of the more hydroxylated metabolites of the vitamin D group. Results by this method correlate well with a competitive protein- binding assay (r = 0.961), but with a negative bias of 6.9 +/- 3.3 microgram/liter. We measured concentrations of 25-hydroxyvitamin D3 in serum drawn during February from 24 persons who were judged normal by physical examinations. The range was 5.5-23.8 microgram/liter (mean, 15.0 +/- 5.2 microgram/liter). The day-to-day CV for the assay was 5.46%.


The following articles in journals at HighWire Press have cited this article:


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M. Vogeser, A. Kyriatsoulis, E. Huber, and U. Kobold
Candidate Reference Method for the Quantification of Circulating 25-Hydroxyvitamin D3 by Liquid Chromatography-Tandem Mass Spectrometry
Clin. Chem., August 1, 2004; 50(8): 1415 - 1417.
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N. Binkley, D. Krueger, C. S. Cowgill, L. Plum, E. Lake, K. E. Hansen, H. F. DeLuca, and M. K. Drezner
Assay Variation Confounds the Diagnosis of Hypovitaminosis D: A Call for Standardization
J. Clin. Endocrinol. Metab., July 1, 2004; 89(7): 3152 - 3157.
[Abstract] [Full Text] [PDF]

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 Clin. Chem.Home page
U. Turpeinen, U. Hohenthal, and U.-H. Stenman
Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay
Clin. Chem., September 1, 2003; 49(9): 1521 - 1524.
[Full Text] [PDF]


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Copyright © 1977 by the American Association for Clinical Chemistry.