LC-MS/MS analysis of vitamin D₃ metabolites in human serum using a salting-out based liquid-liquid extraction and DAPTAD derivatization
Date
2021
Authors
Alshabrawy, A.K.
Bergamin, A.
Sharma, D.K.
Hickey, S.M.
Brooks, D.A.
O'Loughlin, P.
Wiese, M.D.
Anderson, P.H.
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Advisors
Journal Title
Journal ISSN
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Journal article
Citation
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 2021; 1173(122654):122654-1-122654-9
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Ali K.Alshabrawy, Amanda Bergamin, Deepti K.Sharma, Shane M.Hickey, Doug A.Brooks, Peter O'Loughlin, Michael D. Wiese, Paul H. Anderson
Conference Name
Abstract
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.
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Dissertation Note
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Description
Available online 12 March 2021
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Crown Copyright © 2021 Published by Elsevier B.V. All rights reserved.