Method Development and Validation of Methylmalonic Acid by Liquid   Chromatography / Mass Spectrometry

Muhammad Hamid Awais; Muhammad  Aamir; Sobia Irum  Kirmani; Hafiza Monaza  Batool; Nisar  Ahmed; Sadiq  Ali

doi:10.51253/pafmj.v75iSUPPL-5.7623

Authors

Muhammad Hamid Awais Department of Chemical Pathology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan
Muhammad Aamir Department of Chemical Pathology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan
Sobia Irum Kirmani Department of Chemical Pathology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan
Hafiza Monaza Batool Department of Toxicology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan
Nisar Ahmed Department of Toxicology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan
Sadiq Ali Department of Toxicology, Armed Forces Institute of Pathology/National University of Medical Sciences (NUMS), Rawalpindi Pakistan

DOI:

https://doi.org/10.51253/pafmj.v75iSUPPL-5.7623

Keywords:

Liquid Chromatography-Mass Spectrometry, Methylmalonic Acid, Validation.

Abstract

Objective: To develop and validate an accurate and cost-effective method for analyzing methylmalonic acid (MMA) in serum on liquid chromatography/mass spectrometry.

Study Design: Cross sectional (validation) study.

Place and Duration of Study: Department of Chemical Pathology and Endocrinology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Jul to Dec 2020.

Methodology: Method development was initiated to detect MMA using commercially available standards to achieve analyte signal. Liquid phase extraction was performed to extract the MMA from serum by using methanol, acetonitrile, and Formic Acid as an extracting solvent. Separation of the compound was achieved with Agilent SB-C18 column (4.6x150mm, 1.8 µm) using an isocratic elution having 20% mobile phase A (0.1% Formic Acid in water) and 80% mobile phase B (0.1% Formic Acid in Acetonitrile) at a flow rate of 0.7 mL/min.

Results: The AMR of MMA was 33-4227 nmol/L with LOD of 15 nmol/L. The lower limit of quantification was validated at 33 nmol/L. The calculated bias was -12.727. The within and between day imprecision at four concentrations levels were 0.7-7.5%. The method was found stable after the storage & freeze-thaw cycle. The integrity of the diluted sample was maintained for each dilution factor.

Conclusion: The presented method for MMA determination is accurate, cost-effective, specific, and has good clinical correlation. This method can be used in routine to accurately estimate MMA levels with good time management and less financial burden.

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References

Tecleab A, Schofield R, Ramanathan L, Carlow DC. A simple and sensitive method for quantitative measurement of methylmalonic acid by turbulent flow chromatography and tandem mass spectrometry. Journal of chromatography & separation techniques 2016; 7(5).

2. Magera MJ, Helgeson JK, Matern D, Rinaldo P. Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry. Clinical chemistry 2000; 46(11): 1804-1810.

3. Herrmann W, Obeid R. Ursachen und frühzeitige Diagnostik von Vitamin-B12-Mangel. Dtsch Arztebl 2008; 105(40): 680-685.

4. Klee GG. Cobalamin and folate evaluation: measurement of methylmalonic acid and homocysteine vs vitamin B12 and folate. Clinical chemistry 2000; 46(8): 1277-1283.

5. Plessl T, Bürer C, Lutz S, Yue WW, Baumgartner MR, Froese DS. Protein destabilization and loss of protein‐protein interaction are fundamental mechanisms in cblA‐type methylmalonic aciduria. Human mutation 2017; 38(8): 988-1001.

6. Green R, Allen LH, Bjørke-Monsen A-L, Brito A, Guéant J-L, Miller JW, et al. Vitamin B 12 deficiency. Nature reviews Disease primers 2017; 3(1): 1-20.

7. Sloan JL, Carrillo N, Adams D, Venditti CP. Disorders of intracellular cobalamin metabolism. GeneReviews®[Internet] 2018.

8. Pandor A, Eastham J, Beverley C, Chilcott J, Paisley S. Clinical effectiveness and cost-effectiveness of neonatal screening for inborn errors of metabolism using tandem mass spectrometry: a systematic review. NIHR Health Technology Assessment programme: Executive Summaries 2004.

9. Ozben T. Expanded newborn screening and confirmatory follow-up testing for inborn errors of metabolism detected by tandem mass spectrometry. Clinical Chemistry and Laboratory Medicine (CCLM) 2013; 51(1): 157-176.

10. Cox E, White A. Methyl-malonic acid excretion: an index of vitamin-B12 deficiency. Lancet 1962; 2: 853-856.

11. Fenton W, Gravel R, Rosenblatt D. Disorders of Propionate and Methylmalonate Metabolism in The Metabolic and Molecular Bases for Inhertited Disease. Edited by: Scriver CR, Beaudet AL, Sly WS, Valle D. McGraw-Hill, Inc., New York; 2001.

12. Kushnir MM, Nelson GJ, Frank EL, Rockwood AL. High-throughput analysis of methylmalonic acid in serum, plasma, and urine by LC-MS/MS. Method for analyzing isomers without chromatographic separation. Clinical Applications of Mass Spectrometry in Biomolecular Analysis: Springer 2016: 159-173.

13. Mak J, Wong BT, Kwan T, Le A, Cowan TM. Rapid underivatized method for quantitative methylmalonic acid by liquid chromatography–tandem mass spectrometry. The Journal of Applied Laboratory Medicine 2018; 3(3): 408-417.

14. Carvalho VM, Kok F. Determination of serum methylmalonic acid by alkylative extraction and liquid chromatography coupled to tandem mass spectrometry. Analytical biochemistry 2008; 381(1): 67-73.

15. Schmedes A, Brandslund I. Analysis of methylmalonic acid in plasma by liquid chromatography–tandem mass spectrometry. Clinical chemistry 2006; 52(4): 754-757.

16. FDA U. Guidance for industry, bioanalytical method validation (2018). 2001.

17. Pfeiffer C, Gunter E, editors. Automated assay for methylmalonic acid (MMA) in plasma. Clinical chemistry; 1999: Amer Assoc Clinical Chemistry.

18. Straczek J, Felden F, Dousset B, Gueant JL, Belleville F. Quantification of methylmalonic acid in serum measured by capillary gas chromatography—mass spectrometry as tert.-butyldimethylsilyl derivatives. Journal of Chromatography B: Biomedical Sciences and Applications 1993; 620(1): 1-7.

19. Mineva EM, Zhang M, Rabinowitz DJ, Phinney KW, Pfeiffer CM. An LC-MS/MS method for serum methylmalonic acid suitable for monitoring vitamin B12 status in population surveys. Analytical and bioanalytical chemistry 2015; 407(11): 2955-2964.