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

• 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.

	Peaks	tR (min)	Precursor Ion	Product Ion
1.	Succinic acid	1.68	117.3	73.1
2.	Methyl-D3-malonic acid	2.02	120.2	76.2
3.	Methylmalonic acid	2.03	117.3	73.1

Column	Force 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
Standard/Sample
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 0.00 0.7 95 5 0.50 0.7 95 5 3.00 0.7 5 95 3.01 0.7 95 5 5.00 0.7 95 5

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	HPLC
Sample Preparation	100 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.

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