Simultaneous and Automated LC-MS Analysis of Plasma Methylmalonic Acid and Short Chain Fatty Acids Using a Carbodiimide-based Derivatization Mark Pratt (Presenter) University Medical Center Groningen Presenter Bio: I am a PhD-student at the department of Laboratory Medicine of the University Medical Centre Groningen in the lab of prof. Ido Kema. The laboratory is specialized in the LC-MS/MS analysis of low molecular weight neuroendocrine biomarkers. My focus is on the development of new strategies for the routine analysis of challenging (combinations of) small molecules, as well as neuroendocrine peptides and proteins.

Authors: M.S. Pratt, C.P. van der Ley, I. Wijbenga-van der Kooi, W.H.A. de Jong, M. van Faassen, I.P. Kema
Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, The Netherlands

Methylmalonic acid has long been regarded as the most sensitive and specific functional marker of vitamin B12 status. However, it may not tell the complete story. Bacteria in the small intestine use vitamin B12 as a cofactor for the metabolism of succinic acid to propionic acid. Therefore, if vitamin B12 is utilized by bacterial overgrowth in the ileum, a vitamin B12 deficiency may occur despite sufficient dietary intake. Furthermore, this may result in high concentrations of the cytotoxic propionic acid.
We have developed a novel LC-MS method for the simultaneous quantification of methylmalonic acid, propionic acid, succinic acid and butyric acid using a carbodiimide-based derivatization. This method may provide increased insight into the cause of vitamin B12 deficiencies, allowing for improved treatment strategies.

Introduction

Methylmalonic acid has long been regarded as the most sensitive and specific functional marker of vitamin B12 status. However, it may not tell the complete story. Bacteria in the small intestine use vitamin B12 as a cofactor for the metabolism of succinic acid to propionic acid, whereas humans use vitamin B12 for the converse detoxification reaction of propionic acid to succinate, which then enters the TCA Cycle. Therefore, if vitamin B12 is utilized by bacterial overgrowth in the ileum, a vitamin B12 deficiency may occur despite sufficient dietary intake. Furthermore, this may result in high concentrations of the cytotoxic propionic acid. [1,2]

Methods

We have developed an LC-MS method for the simultaneous quantification of methylmalonic acid, propionic acid, succinic acid and butyric acid. In order to obtain chromatographic retention and specific mass spectrometric m/z transitions we used an aqueous trifluoroethylamine-mediated carbodiimide derivatization [3]. Online solid phase extraction was performed on polydivinylbenzene cartridges, chromatographic separation was achieved on a fully porous C18 column. Mass spectrometric detection was performed using a triple quadrupole mass spectrometer using positive electrospray ionization.

Results

For methylmalonic acid the analytical method showed excellent correlation with a different LC-MS quantification method. Inter-assay CVs were below 5.4% and intra-assay CVs were below 3.7%, mean recoveries were 96-102%, linearity in the calibration range was excellent (r2>0.999) no matrix-effect or carry-over was observed.

Conclusions & Discussion

The novel LC-MS method for the simultaneous quantification of methylmalonic acid and short-chain fatty acids may provide increased insight into the cause of vitamin B12 deficiencies, allowing for the improvement of treatment strategies.

[1] Louis P, Flint HJ, Formation of propionate and butyrate by the human colonic microbiota. Environmental Microbiology 19, 29-41 (2017)

[2] Green R, Allen LH, Bjørke-Monsen A, Brito A, Guéant J, Miller JW et al. Vitamin B12 deficiency, Nature Reviews Disease Primers 3, 17040 (2017)

[3] Ford QL, Burns JM, Ferry JL. Aqueous in situ derivatization of carboxylic acids by an ionic carbodiimide and 2,2,2-trifluoroethylamine for electron-capture detection. Journal of Chromatography A 1145, 241-245 (2007)