Publication Date

Summer 2020

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Advisor

Daryl Eggers

Keywords

clinical, liquid chromatography, mass spectrometry, methlymalonic acid, quantitation, vitamin B12

Subject Areas

Chemistry; Analytical chemistry; Biochemistry

Abstract

The aim of the study was to develop a quantitative assay for methylmalonic acid (MMA), an important biomarker for clinical diagnosis of vitamin B12 (B12) deficiency. The techniques of liquid chromatography and tandem mass spectrometry (LC-MS/MS) are used for separation and quantitation of MMA, as well as its endogenous isomer succinic acid. The MS/MS method was developed using an infusion set-up and manual tuning experiments. Relevant transitions were detected for the two analytes and MMA-d3, then a preliminary MS/MS method was developed. The preliminary LC method was further optimized. Various gradients, mobile phases (MPs), and additive combinations were used across three-column chemistries. The three-column chemistries chosen were representative of distinct separation modes, C18 for reverse phase, Diamond Hydride (DH) for aqueous normal phase (ANP), and BEH-Amide for hydrophilic interaction liquid chromatography. The chromatographic parameters of peak shape, capacity factor (k), selectivity (α), and resolution (R) were used to judge the quality of separation. In conclusion, the DH column showed the best peak shape, k, and R. The peak width of MMA on DH was 0.13 minutes with a symmetrical shape, and RMMA on DH was 4.7. In contrast, BEH-Amide showed the best α. Additionally, experimental evidence confirmed that the DH column retains by an ANP mechanism, as shown by the increase in kMMA from 3.72 to 4.89, with increased retention when using a low salt MP.

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