Quantification of the 3α and 3β epimers of 25-hydroxyvitamin D3 in dried blood spots by LC-MS/MS using artificial whole blood calibration and chemical derivatization.

While the biological function of the 3α epimer of 25-hydroxyvitamin D3 (25(OH)D3) remains unknown, its presence needs to be accurately captured and separated from the main 3β epimer, to avoid positive bias in vitamin D status analyses. Several recent LC-MS/MS assays for 25(OH)D3 successfully separate the 3α and 3β epimers by chromatography. Unfortunately, none of the existing LC-MS/MS assays, which utilize dried blood spots (DBS) as sampling/storage vessels, is able to quantify the individual epimers. DBS are often used for analysis of infant blood, however, and these samples are particularly likely to contain significant levels of interfering 3α epimer. Furthermore, proper calibration of DBS samples is much more difficult to achieve than for liquid serum or plasma samples. We addressed this important issue by creating an artificial vitamin D-free whole blood for calibration and then quantified 3α- and 3β-25(OH)D3 levels from DBS. After chemical derivatization, the vitamin D epimers were separated on a PFP column and concentrations determined by electrospray ionization LC-MS/MS on a triple quadrupole mass spectrometer. Calibration with artificial whole blood showed improved precision over standard addition (7.6 versus 31.5% RSD for 3β-25(OH)D3). The limits of quantification for 3β-25(OH)D3 and for 3α-25(OH)D3 were 1.0 and 0.1ng/mL, respectively. Excellent intra/interday precisions between 2.1 and 2.2% CV (intra) and 4.4-5.3% CV (inter) were established for 3β-25(OH)D3 and 3α-25(OH)D3. For 3β-25(OH)D3, only small concentration-independent bias and deviation of <3.3ng/mL were seen between serum LC-MS/MS and DBS-LC-MS/MS measurements; analyses of 3α-25(OH)D3 showed deviations of <0.8ng/mL in all experiments.