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

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dc.contributor.authorAlshabrawy, A.K.
dc.contributor.authorBergamin, A.
dc.contributor.authorSharma, D.K.
dc.contributor.authorHickey, S.M.
dc.contributor.authorBrooks, D.A.
dc.contributor.authorO'Loughlin, P.
dc.contributor.authorWiese, M.D.
dc.contributor.authorAnderson, P.H.
dc.date.issued2021
dc.descriptionAvailable online 12 March 2021
dc.description.abstractLC-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.
dc.description.statementofresponsibilityAli K.Alshabrawy, Amanda Bergamin, Deepti K.Sharma, Shane M.Hickey, Doug A.Brooks, Peter O'Loughlin, Michael D. Wiese, Paul H. Anderson
dc.identifier.citationJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2021; 1173(122654):122654-1-122654-9
dc.identifier.doi10.1016/j.jchromb.2021.122654
dc.identifier.issn1570-0232
dc.identifier.issn1873-376X
dc.identifier.orcidBrooks, D.A. [0000-0001-9098-3626]
dc.identifier.orcidAnderson, P.H. [0000-0002-8685-3252]
dc.identifier.urihttps://hdl.handle.net/2440/134778
dc.language.isoen
dc.publisherElsevier BV
dc.rightsCrown Copyright © 2021 Published by Elsevier B.V. All rights reserved.
dc.source.urihttps://doi.org/10.1016/j.jchromb.2021.122654
dc.subjectLC-MS/MS
dc.subjectVitamin D3 metabolites
dc.subjectDAPTAD derivatization
dc.subjectSalting-out
dc.subjectLiquid-liquid extraction
dc.titleLC-MS/MS analysis of vitamin D₃ metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization
dc.title.alternativeLC-MS/MS analysis of vitamin D2 metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization
dc.typeJournal article
pubs.publication-statusPublished

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