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

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 D₂ and D₃, epi-25-hydroxyvitamin D₃ and 24,25-dihydroxyvitamin D₃.

Methods: Sample preparation was based on precipitation and centrifugation of 100μL 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 <6.0 and 6.4% and mean recoveries were within 95-104%. LOQ's were 0.5μg/L for 24,25(OH)₂D₃, 1.1μg/L for 25(OH)D₃ and epi-25(OH)D₃ and 1.7μg/L for 25(OH)D₂. 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.