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Preanalytical evaluation of serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 measurements using LC-MS/MS
Asuka Mochizuki1, Yoshio Kodera, Tatsuya Saito
1Clinical Proteomics Research Center, Chiba University Hospital, Chiba, Japan.
Background:
Vitamin 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.
Methods:
We 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.
Results:
High-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 (r(2)=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.
Conclusions:
This 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.