Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development

• A simple LC-MS/MS assay for serum MMA quantification using supported-liquid extraction (SLE) is described.
• Unexpected challenges encountered during SLE LC-MS/MS method development and their resolutions are discussed.
• Correlation analysis of serum MMA and propionyl acylcarnitine revealed patient-specific trends.

ObjectivesAnalysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation.Design and methodsMMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals.ResultsBaseline separation between MMA and succinic acid was completed in 7 min. The assay was linear from 0.1 to 500 μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526 μM) and 5.0 to 15.7% (0.3 to 233 μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of − 1.335. Carryover was determined to be < 0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine.ConclusionThis report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude.