A Rapid and Simple Liquid-Chromatography-Tandem Mass Spectrometry Method for Measuring 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 in Human Serum: Comparison with Two Automated Immunoassays

Total 25-hydroxyvitamin D (25(OH)D) is well-known to be a reliable biomarker of human vitamin D status, with the recognition of widespread vitamin D insufficiency in general populations. The aims of this study are to validate a fast and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying 25(OH)D and 25(OH)D in serum and to compare two automated immunoassays with the LC-MS/MS method. Samples were prepared by protein precipitation with ethanol including 25(OH)D -d , followed by a liquid-liquid extraction with hexane. The analytes were separated within a total run time of 3 min. Accuracy was evaluated with standard reference materials (SRM) 972a. Using 150 samples, the LC-MS/MS method was compared with the LIAISON assay and ADVIA Centaur assay. The LC-MS/MS method had a limit of quantitation of 1 ng/mL for the 25(OH)D and 25(OH)D with linear responses between 1 and 100 ng/mL. Intra- and inter-assay precision were