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

Sheng-Ying Lo1, Cindy Gordon2, Katerina Sadilkova2

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|May 22, 2016
PubMed
Summary

A new liquid chromatography tandem mass spectrometry (LC-MS/MS) method simplifies methylmalonic acid (MMA) analysis for diagnosing methylmalonic acidemia in children. This validated assay offers accurate MMA quantification using supported liquid extraction (SLE).

Keywords:
Inborn errors of metabolismLC-MS/MSMethylmalonic acidMethylmalonic acidemiaSupported-liquid extraction

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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.