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Clin Chim Acta. 2008 May;391(1-2):6-12. doi: 10.1016/j.cca.2008.01.017. Epub 2008 Jan 24.

Measurement of 25-hydroxyvitamin D3 (25OHD3) and 25-hydroxyvitamin D2 (25OHD2) in human serum using liquid chromatography-tandem mass spectrometry and its comparison to a radioimmunoassay method.

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  • 1Centers for Disease Control and Prevention, Atlanta, GA, USA.



Measurement of vitamin D molecules are important in the management of patients with bone disease. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 25OHD(3) and 25OHD(2) in human serum and compared it to the traditionally used DiaSorin radioimmunoassay (RIA).


Serum samples (200 microl) were treated with acetonitrile and centrifuged to remove protein. An online solid-phase extraction procedure was used. Calibration solutions (5-100 ng/ml) of 25OHD(2) and 25OHD(3) were prepared using 4% albumin in phosphate-buffered saline. Chromatography: C18 column, isocratic ethanol/water (83/17, v/v). Mass spectrometry system: atmospheric pressure chemical ionization in the positive ion mode. Transitions: 25OHD(3), m/z 401.4-->383.4; 25OHD(2), m/z 413.4-->395.4; and the internal standard hexadeuterated-25OHD(3), m/z 407.7-->389.7.


Detection limits were 0.49 ng/ml (25OHD(3)) and 1.86 ng/ml (25OHD(2)). Intra- and inter-assay coefficients of variation (CV) were <7% and <11%, respectively, for 25OHD(3) and <9% and <16%, respectively, for 25OHD(2). Recovery averaged (SD) 99% (2%) for 25OHD(3) and 95% (0.8%) for 25OHD(2). In a method comparison of 551 specimens from the National Health and Nutrition Examination Survey, the LC-MS/MS method gave values that were on average 13% higher (95%CI: 11-15%) than RIA results.


This high throughput candidate reference method requires minimal sample preparation and is suitable for routine use for analysis of vitamin D status.

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