LC-MS/MS analysis of vitamin D₃ metabolites in human serum using a salting-out based liquid-liquid extraction and...

LC-MS/MS has recently emerged as the best-practice for simultaneous analysis of vitamin D metabolites. We have developed and validated an LC-MS/MS method for simultaneous quantification of 25(OH)D₃, 24,25(OH)₂D₃, and 3-epi-25(OH)D₃ in human serum. These three metabolites were extracted from 50 μL of serum by acetonitrile protein precipitation followed by salting-out of acetonitrile. DAPTAD (4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione) was used to derivatize the extracted metabolites and their deuterated isotope internal standards. Chromatographic separation was achieved on a UPLC C18 column (Waters® ACQUITY 100 × 2.1 mm, 1.7 µm) utilizing 0.1% formic acid and acetonitrile as mobile phases. Limits of quantification were 1 ng/mL for 25(OH)D₃ and 0.1 ng/mL for 24,25(OH)D₃ and 3-epi-25(OH)D₃. In-house and external Vitamin D External Quality Assessment Scheme (DEQAS) quality control sample analysis revealed satisfactory method accuracy. Within-analytical batch and between analytical batches precision were <15%. Extraction recovery for the three analytes were all ˃ 85% and all showed adequate autosampler, bench-top and freeze-thaw stability. Inter-methodological comparison of 25(OH)D₃ results in patient serum samples revealed systematic and proportional differences between our method and DiaSorin® Liaison immunoassay, however a good agreement with an independent LC-MS/MS method was found.