MSACL 2023 Abstract

Vitamin B12 Detection from Self-Collect Devices Using LC-MS/MS

Frances Morris, Kavinda DeSilva, Mariko Nakano, Zane Hauck, Charles Sailey
Molecular Testing Labs

Frances Morris, M.S. (Presenter) Molecular Testing Labs

Relevant Financial Disclosures (within past 24 months, reported on Feb 19, 2023) Salary Molecular Testing Labs Conflict mitigation NOT REQUIRED.

Introduction:
Cobalamin, commonly known as vitamin B12, in an essential vitamin used to maintain biological functions in the human body, including fatty acid and amino acid metabolism. Vitamin B12 is introduced to the body typically through the consumption of animal products including meat, eggs, and dairy but is also available and commonly consumed in supplementary forms and fortified foods. Deficiency of this essential vitamin is commonly observed and can lead to fatigue, anemia, weakness, and more.

Four common forms of cobalamins can be found in human serum including biologically active forms— adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl)— and common supplemental forms— cyanocobalamin (CNCbl) and hydroxocobalamin (OHCbl). Most current clinical diagnostics use immunoassays, which are incapable of distinguishing the different forms of cobalamin. However, these forms absorb differently in the human body and have different bioavailability; therefore, a simple concentration of cobalamins in serum may not adequately indicate vitamin b12 deficiency. LC-MS/MS can distinguish between these compounds both chromatographically and through the different m/z ratios of the forms and thus can provide more specific diagnostic information. Additionally, because of the prevalence of cobalamin deficiency, there is a high demand for accessible testing, such as at-home self-collect devices. Therefore, specific detection of cobalamins from self-collect devices is of interest.

Objectives:
The primary objective of this assay is to determine clinical levels of vitamin B12 from at-home collection devices.

Methods:
Patient samples were collected using dried serum separator cards and correlated to serum collected in serum separator tubes. These patient samples were cleaned up for analysis and injected on an HPLC-MS/MS system (SCIEX 6500). Calibration standards were prepared in stripped human serum and were observed at a linear range from 50.0- 2,000 pg/mL.

Results:
Cobalamin forms were successfully chromatographically separated. Prepared quality control samples at 250 pg/mL and were successfully detected for CNCbl, MeCbl, and AdoCbl (up to 8% CV).

Conclusion:
LC-MS/MS can be used to distinguish different forms of cobalamins at some clinically significant concentration levels. Further testing is needed to validate robustness and stability to ultimately be used to analyze commercially collected samples.