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

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.

Simultaneous LC-MS/MS Method for the Quantitation of Probenecid, Albendazole, and Its Metabolites in Human Plasma and Dried Blood Spots

“…DBS collection was used to investigate the simultaneous analysis of seven antimalarial drugs and metabolites in clinical studies [1]. DBS has been used to assess vitamin D status in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [2,3].…”

Vitamin D and Vitamin D Binding Protein in Health and Disease 2.0

“…Unraveling the underlying mechanisms and developing effective interventions could have a transformative impact on sepsis management and patient outcomes. Continuing the exploration of the role of vitamin D in infectious diseases, a subsequent paper titled, “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”, introduces a ground-breaking tool for precisely determining vitamin D concentrations in dried blood spots [ 3 ]. The study was able to precisely measure 25(OH)D 3 , vitamin D 3 , 25(OH)D 2 , and vitamin D 2 in human blood using this method with no discernible matrix effects in subjects who had been tested for SARS-CoV-2.…”