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

Featured Application: Methylmalonic Acid (MMA) on Force C18

  • 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.

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.

To overcome these obstacles, we developed a simplified method for methylmalonic acid analysis. 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.

PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.68117.373.1
2.Methyl-D3-malonic acid2.02120.276.2
3.Methylmalonic acid2.03117.373.1
ColumnForce C18 (cat.# 963431E)
Dimensions:100 mm x 3.0 mm ID
Particle Size:3 µm
Pore Size:100 Å
Guard Column:Force C18 EXP guard column cartridge 3 mm ID, (cat.# 963450253)
Temp.:35 °C
Conc.: 13 ng/mL Methylmalonic acid (endogenous) in double charcoal stripped human plasma
Inj. Vol.:3 µL
Mobile Phase
A:0.5% Formic acid in water
B:0.5% Formic acid in methanol
Time (min)Flow (mL/min)%A%B
Ion Mode:ESI-
Sample Preparation100 μL of sample (double charcoal stripped human plasma containing 13 ng/mL of endogenous methylmalonic acid) was aliquoted for extraction. 5 μL of internal standard (2500 ng/mL MMA-D3 in water) was added to the sample. The sample was precipitated using 300 μL of 0.5% formic acid in methanol followed by a 10 second vortex at 3000 rpm. The sample was then centrifuged at 4000 rpm for 10 minutes at 10 °C. 250 μL of the supernatant was filtered using a Thomson SINGLE StEP standard filter vial (PVDF, 0.2 μm, Restek cat.# 25895) prior to analysis.