A Simultaneous Extraction/Derivatization Strategy for Quantitation of Vitamin D in Dried Blood Spots Using LC–MS/MS: Application to Biomarker Study in Subjects Tested for SARS-CoV-2

Yashpal S. Chhonker, Nusrat Ahmed, Christine M. Johnston, Ruanne V. Barnabas, Daryl J. Murry

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Vitamin D plays a critical role in bone development and maintenance, and in other physiological functions. The quantitation of endogenous levels of individual vitamin D and its metabolites is crucial for assessing several disease state conditions. With cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to the coronavirus disease 2019 (COVID-19) pandemic, there are several studies that have associated lower levels of serum vitamin D with severity of infection in COVID-19 patients. In this context, we have developed and validated a robust LC–MS/MS method for simultaneous quantitation of vitamin D and its metabolites in human dried blood spot (DBS) obtained from participants tested for COVID-19. The chromatographic separation for vitamin D and metabolites was performed using an ACE Excel C18 PFP column protected with a C18 guard column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of formic acid in water (0.1% v/v) as mobile phase A and formic acid in methanol (0.1% v/v) as mobile phase B, operated at a flow rate of 0.5 mL/min. Analysis was performed utilizing the LC–MS/MS technique. The method was sensitive with a limit of quantification of 0.78 ng/mL for all analytes, and had a large dynamic range (200 ng/mL) with a total run time of 11 min. The inter- and intraday accuracy and precision values met the acceptance criteria per the US Food and Drug Administration guidelines. Blood concentrations of 25(OH)D3, vitamin D3, 25(OH)D2, and vitamin D2 over a range of 2–195.6, 0.5–121.5, 0.6–54.9, and 0.5–23.9 ng/mL, respectively, were quantified in 909 DBS samples. In summary, our developed LC−MS/MS method may be used for quantification of vitamin D and its metabolites in DBS, and may be applied to investigations of the emerging role of these compounds in various physiological processes.

Original languageEnglish (US)
Article number5489
JournalInternational journal of molecular sciences
Volume24
Issue number6
DOIs
StatePublished - Mar 2023

Keywords

  • LC–MS/MS
  • SARS-CoV-2(+)
  • biomarker
  • vitamin D

ASJC Scopus subject areas

  • Catalysis
  • Molecular Biology
  • Spectroscopy
  • Computer Science Applications
  • Physical and Theoretical Chemistry
  • Organic Chemistry
  • Inorganic Chemistry

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