Alshabrawy, A.K.Bergamin, A.Sharma, D.K.Hickey, S.M.Brooks, D.A.O'Loughlin, P.Wiese, M.D.Anderson, P.H.2022-04-072022-04-072021Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2021; 1173(122654):122654-1-122654-91570-02321873-376Xhttps://hdl.handle.net/2440/134778Available online 12 March 2021LC-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.enCrown Copyright © 2021 Published by Elsevier B.V. All rights reserved.LC-MS/MSVitamin D3 metabolitesDAPTAD derivatizationSalting-outLiquid-liquid extractionJournal article10.1016/j.jchromb.2021.1226542022-04-072-s2.0-85103687582572938Brooks, D.A. [0000-0001-9098-3626]Anderson, P.H. [0000-0002-8685-3252]