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

• Published in: International journal of molecular sciences Vol. 24; no. 6; p. 5489
• Main Authors: Chhonker, Yashpal S., Ahmed, Nusrat, Johnston, Christine M., Barnabas, Ruanne V., Murry, Daryl J.
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 13.03.2023; MDPI
• Subjects: Alfacalcidol; Biomarkers; Blood; Calcifediol; Chromatography; Chromatography, Liquid - methods; Coronaviruses; COVID-19; Diagnosis; Disease; Formic acid; Health aspects; Humans; Infections; Ions; Medical research; Medicine, Experimental; Metabolites; Organic acids; Pandemics; Physiological aspects; Physiology; Plasma; Reproducibility of Results; Retention; SARS-CoV-2; Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Tandem Mass Spectrometry - methods; Vitamin D; Vitamins; Massachusetts; biomarker; LC–MS/MS; SARS-CoV-2(+); vitamin D
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms24065489

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.