Methylmalonic acid (MMA) is a functional biomarker of vitamin B12 deficiency. Measurement of plasma MMA is challenging due to its small molecular weight and hydrophilic nature. Several liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been developed for measuring plasma MMA. However, these methods involve lengthy sample preparation, long chromatographic run time, inadequate sensitivity, or interference from succinic acid (SA). Here we report a novel LC-MS/MS method for quantitation of underivatized MMA in serum or heparinized plasma with high sensitivity and selectivity. Sample preparation involved only strong anion exchange solid phase extraction. The extract was purified by online turbulent flow and analyzed on an Organic Acids column. MS/MS analysis was performed in negative electrospray mode, and the analytical time was 6 min. The use of ion ratio confirmation in combination with chromatographic resolution from SA greatly enhanced the selectivity. No interference was observed. This method was linear from 26.2 to 26,010.0 nM with an accuracy of 98–111 %. Total coefficient of variation was less than 4.6 % for three concentration levels tested. Comparison with a reference laboratory LC-MS/MS method using leftover patient serum specimens (n = 48) showed a mean bias of −2.3 nM (−0.61 %) with a Deming regression slope of 1.016, intercept of −6.6 nM, standard error of estimate of 25.3 nM, and a correlation coefficient of 0.9945. In conclusion, this LC-MS/MS method offers highly sensitive and selective quantitation of MMA in serum and plasma with simple sample preparation.
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The authors thank Dustin Bunch for his critical reading of this manuscript.
The Cleveland Clinic institutional review board's approval was obtained for collecting leftover patient samples used in this work.
All authors confirmed they have contributed to the intellectual content of this paper and have met the following three requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
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No authors declared any potential conflicts of interest.
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Yuan, C., Gabler, J., El-Khoury, J.M. et al. Highly sensitive and selective measurement of underivatized methylmalonic acid in serum and plasma by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 404, 133–140 (2012). https://doi.org/10.1007/s00216-012-6099-z
- Methylmalonic acid
- Liquid chromatography
- Mass spectrometry