Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC–MS/MS

BackgroundVitamin D testing is increasing worldwide. Although immunoassays are still widely used in Japan for the measurement of serum 25-hydroxyvitamin D (25OHD) as an indicator of vitamin D status, development of a simple and high-throughput MS-based method is still needed for routine use in clinical laboratories.MethodsWe designed a method using a triple quadrupole mass spectrometer equipped with a two-step separation approach that used the Aria TLX-2 HPLC system in the selected reaction monitoring mode. Analytical performance of the system and effects of various preanalytical factors were tested.ResultsHigh-throughput quantitative analysis of 25OHD3 and D2 at 15 samples/h was achieved using 25 μl of serum/plasma. Intra- and inter-assay CVs for 25OHD3 were 5% and 7%, respectively. Limit of detection for 25OHD3 was 0.31 ng/ml. No significant effects were seen for clotting time, repeated freeze–thaw cycles, anti-coagulants and possible interfering substances. A good correlation (r2 = 0.947) was found between the present system and the DiaSorin radioimmunoassay. Serum 25OHD3 levels in apparently healthy Japanese subjects were 25.5 ± 9.8 ng/ml for men and 20.9 ± 7.1 ng/ml for women.ConclusionsThis high-throughput LC–MS/MS 25-OHD assay has the potential to be used as a routine clinical laboratory assay for assessing vitamin D status.

► We developed a simple MS-based method for serum 25-hydroxyvitamin D measurement.
► Quantitative 25OHD3 and D2 measurements of 350 samples/day could be achieved.
► The results obtained agreed well with those by the widely used radioimmunoassays.
► The effects of various pre-analytical factors such as diet on the assays were tested.
► This method has a potential to be used on a routine basis in clinical laboratory.