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  • Title: 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.
    Author: Chen H, McCoy LF, Schleicher RL, Pfeiffer CM.
    Journal: Clin Chim Acta; 2008 May; 391(1-2):6-12. PubMed ID: 18279671.
    Abstract:
    BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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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