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

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 µl) 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 3 and 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.