Simplified Methylmalonic Acid Analysis without Derivatization in Human Plasma by LC-MS/MS

Restek has developed a simplified method to overcome the obstacles of methylmalonic acid analysis.

Vitamin B12 plays an essential role in metabolic energy production, and deficiency can be difficult to diagnose without testing as it can manifest through a wide variety of symptoms. In clinical testing of plasma samples, elevated levels of methylmalonic acid (MMA) can be used to diagnose functional vitamin B12 deficiency as well as methylmalonic acidemia, an inherited metabolic disorder.

Methylmalonic acid determination is a very sensitive test and is more specific than a homocysteine test, but it typically requires extensive sample pre-treatment using liquid-liquid extraction, derivatization, solvent evaporation, and/or SPE. Additionally, chromatographic resolution can be difficult to achieve between methylmalonic acid and its naturally occurring isomer, succinic acid.

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The approach shown here uses a protein crash sample preparation and does not require derivatization. By eliminating derivatization, considerable time and resource savings are realized and a significant source of variation is removed. For analysis, a direct injection of the supernatant is made onto a Force C18 column. LC-MS/MS results show that methylmalonic acid is clearly resolved from succinic acid, which eliminates potential isobaric interference and makes peak identification and quantitation straightforward. In addition, the 5-minute total chromatographic analysis time makes this method suitable as a high-throughput assay for the clinical diagnosis of vitamin B12 deficiency and methylmalonic acidemia.

  • Baseline separation of isomers ensures accurate reporting.
  • Simple protein crash sample preparation, no derivatization required.
  • Fast 5-minute analysis is suitable for high-throughput labs.


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