Quantitation of 25-hydroxyvitamin D2 and D3 in serum and plasma by LCMS/MS

Objective: We developed and validated an analytical method for quantifying 25-hydroxyvitamin D-2 (25OHD(2)) and 25-hydroxyvitamin D-3 (25OHD(3)) in serum and plasma. Methods: Samples, pretreated with zinc sulfate and methanol, were extracted with hexane. Separation was achieved via UHPLC and 25OHD quantification was accomplished by a triple quadrupole mass spectrometer. Results: Imprecision was 3.6-15.1%CV and bias 88.0-126.0%. Extraction efficiency was 76.5-110.5%, whereas the matrix effect ranged from -46.7 to -32.0%. The method was applied to authentic specimens. The results showed no significant difference between serum and plasma; strong correlation with paired values from an external laboratory; and analyte stability for 15 days. Conclusion: This method provides reliable and accurate measurement of 25OHD for use in clinical practice.