Quantitation of 25-hydroxyvitamin D in dried blood spots by 2D LC-MS/MS without derivatization and correlation with serum in adult and pediatric studies

Background: Demand for measurement of 25-hydroxyvitamin D (25OHD) is growing and dried blood spot (DBS) sampling is attractive as samples are easier to collect, transport and store. Methods: A 2D LC-MS/MS assay without derivatization was developed. DBS punches (3.2 mm) were ultrasonicated with d(6)-250HD(3) in 70% methanol followed by hexane extraction, dry-down and reconstitution. The assay was validated and applied to two studies comparing whole blood adult DBS with serum samples (n = 40) and neonatal whole blood DBS with cord serum samples (n = 80). Results: The assay was validated in whole blood DBS over the range 13-106 nmol/L 25OHD(3) and 11-91 nmol/L 25OHD(2) with a limit of detection of 3 nmol/L. Intra-and inter-day imprecision was < 13% CV and bias < 12%. The assay had high recovery and minimal matrix effects. Triplicate DBS study samples had a mean CV of 13% for 25OHD(3). No 25OHD(2) was detected. DBS calculated serum 25OHD(3) concentrations correlated strongly with serum concentrations in the adult DBS/serum study (r = 0.94) and moderately in the neonatal DBS/cord serum study (r = 0.69). Conclusions: Direct quantitation of 25OHD in DBS by 2D LC-MS/MS without derivatization was found to be an alternative to serum quantitation applicable to clinical research studies on adult DBS samples.