A fast and simple method for simultaneous measurements of 25(OH)D, 24,25(OH)2D and the Vitamin D Metabolite Ratio (VMR) in serum samples by LC-MS/MS

Abstract Background Rapid, easy and reliable measurement of the major vitamin D metabolites is required in order to fulfill the needs of a clinical routine laboratory. To overcome these challenges, we have developed and validated a LC-MS/MS method for the quantification of 25-hydroxyvitamin D 2 and D 3 , epi-25-hydroxyvitamin D 3 and 24,25-dihydroxyvitamin D 3 . Methods Sample preparation was based on precipitation and centrifugation of 100 μL of patient serum, followed by injection into the LC-MS/MS system. Samples from Vitamin D Standardization Program ( n  = 80) and patient samples ( n  = 281) have been compared with a reference LC-MS/MS method. For the analytical validation NIST and Labquality quality control materials were used. Results Mean intra-assay and inter-assay imprecision were 2 D 3 , 1.1 μg/L for 25(OH)D 3 and epi-25(OH)D 3 and 1.7 μg/L for 25(OH)D 2 . A 3% bias obtained between the proposed and the reference method satisfies Vitamin D Standardization Program recommendations. Conclusions We present a rapid, easy, reliable and cost-effective method completely adequate for routine testing, which permits the measurement of the ratio of 24,25-dihydroxyvitamin D to 25-hydroxyvitamin D, Vitamin D Metabolite Ratio (VMR), in serum samples.