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

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.