Development and validation of an LC-MS/MS based method for quantification of 25 hydroxyvitamin D2 and 25 hydroxyvitamin D3 in human serum and plasma

Vitamin D deficiency is increasing in the general population and has become a serious public health risk globally. As a reliable clinical indicator of vitamin status, 25 hydroxyvitamin D (25(OH)D) has been measured by various methods. However, the accuracy of these measurements has been the subject of considerable debate. Here, we report the development and validation of a liquid chromatography-triple quadrupole mass spectrometry based method for the quantification of 25(OH)D2 and 25(OH)D3 in human serum and plasma samples. Samples were first processed by protein precipitation to release the analytes from the vitamin D binding protein (DBP), followed by a liquid-liquid extraction procedure. Analysis was performed on an LC-MS/MS system which utilized an AB Sciex API 3000 mass spectrometer. A six point calibration curve ranging from 2.5 to 100 ng/mL was established for both 25(OH)D2 and 25(OH)D3. A complete method validation was conducted, including intra- and inter-assay accuracy and precision, LLOQ, dilution QC, specificity, recovery, matrix effect, and a thorough stability profile of stock solutions and QC samples. Matching samples of serum and plasma (containing either heparin or EDTA anticoagulant) generated from the same blood samples were tested, and no significant differences in 25(OH)D2 and 25(OH)D3 concentrations were found in these sample matrices. In method comparison, we analyzed 10 serum samples obtained from the Vitamin D External Quality Assessment Scheme (DEQAS), and the total 25(OH)D concentrations measured by our method were very close to the LC-MS/MS Method Mean values provided by DEQAS (average 0.17% bias, R2 = 0.99). However, comparison with the DiaSorin Liaison 25(OH)D TOTAL Assay demonstrated limited correlation between these two methods (R2 = 0.54). In general, concentrations measured by our LC-MS/MS method were roughly 9% higher than those measured by the DiaSorin Liaison assay. The correlation with DiaSorin Liaison measurement was better for samples in the lower concentration range. In summary, we developed and validated an LC-MS/MS based method that can be reliably applied in routine quantification of 25(OH)D2 and 25(OH)D3 in human serum and plasma samples. This method is not suitable for pediatric determinations due to the potential interference of 3-epi 25(OH)D3.