Determination of methylmalonic acid by high-performance liquid chromatography

An isocratic reverse-phase C18 high-performance liquid chromatography (HPLC) technique for methylmalonic acid (MMA) with fluorescence detection is described. MMA is extracted from an acidified sample (plasma, urine, etc.) with ethyl acetate. The extract is dried and derivatized with monodansylcadaverine and dicyclohexylcarbodiimide prior to injection on the HPLC. Ethylmalonic acid is used as an internal standard. Evaluation of the technique was with ovine blood plasma, liver, rumen fluid, and urine. The technique can quantitate as little as 0.2 mumol/liter MMA in samples (based on 10 times the detection limit) and is therefore sufficiently sensitive to measure levels in normal blood plasma. The method is rapid, with up to 60 samples per day throughput, and so is suitable as a routine diagnostic test for the detection of vitamin B12 deficiency.