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

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Pediatric Diagnostics

Background:

• Accurate quantification of serum/plasma methylmalonic acid (MMA) is crucial for diagnosing and managing methylmalonic acidemia in pediatric patients.
• Existing methods may require complex sample preparation, posing challenges for routine clinical laboratories.

Purpose of the Study:

• To develop and validate a simplified liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA analysis.
• To establish a robust assay for monitoring methylmalonic acidemia in pediatric populations.

Main Methods:

• Methylmalonic acid (MMA) and its stable isotope-labeled analog (d3-MMA) were extracted using supported liquid extraction (SLE).
• Assay performance characteristics including imprecision, bias, linearity, recovery, and carryover were evaluated.
• Correlation with a national reference laboratory's LC-MS/MS assay and the relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) were assessed.

Main Results:

• The developed LC-MS/MS method achieved baseline separation of MMA and succinic acid within 7 minutes.
• The assay demonstrated linearity from 0.1 to 500 μM, with intra-day and inter-day imprecision CVs ranging from 4.1% to 15.7%.
• Recovery was between 93-125%, carryover was <0.04%, and a strong correlation (Deming regression: 1.026) was observed with a reference method.

Conclusions:

• This study presents the first LC-MS/MS method utilizing SLE for MMA extraction, offering a simpler approach.
• The method is validated for accurate MMA quantification across a wide concentration range, suitable for clinical diagnostics.
• Challenges in developing SLE-based LC-MS/MS assays for analytes with large concentration spans are highlighted, providing guidance for implementation.