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

摘要

Background. 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-2 and 25(OH) D-3 in serum and to compare two automated immunoassays with the LC-MS/MS method. Methods. Samples were prepared by protein precipitation with ethanol including 25(OH) D-3-d(6), 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 r assay and ADVIA Centaur (R) assay. Results. The LC-MS/MS method had a limit of quantitation of 1 ng/mL for the 25(OH) D-2 and 25(OH) D-3 with linear responses between 1 and 100 ng/mL. Intra-and inter-assay precision were < 8.8% and < 13.2%, respectively. It also showed a smallest mean difference (+ 0.9%) for the SRM level 1 to 3, compared to the two immunoassays. Compared to the LC-MS/MS, the mean biases of the RIAISON and ADVIA were + 2.4 and + 7.9 ng/mL, respectively. Also, the agreement of the LC-MS/ MS with the RIAISON was better than that with the ADVIA. Conclusion. This study suggests that the LC-MS/MS method traceable to the SRM can be reliably applied in routine quantification of 25(OH) D-2 and 25(OH) D-3.