Rapid Underivatized Method for Quantitative Methylmalonic Acid by Liquid Chromatography-Tandem Mass Spectrometry

Increased methylmalonic acid (MMA) levels can aid in assessing vitamin B12 deficiency or abnormal propionate metabolism. MMA analysis by LC-MS/MS is challenging because of both the nanomolar reference range and potential interference from succinic acid, an endogenous isomer. We show that ultrafiltration followed by gradient chromatography permits rapid, sensitive, and selective quantification that is essentially devoid of matrix effects.

Fifty microliters of serum or plasma were mixed with 50 μL of MMA-d3 and deproteinized by ultrafiltration. Filtrates were analyzed by reversed-phase LC-MS/MS. The clinical performance of the MMA assay was validated using guidelines from both the College of American Pathologists and the Clinical and Laboratory Standards Institute. Matrix effects were examined by postcolumn infusion, phospholipid analysis, and peak area comparisons.

The analytical measurement range was 0.05 to 100 μmol/L. The resolution between physiological succinic acid and MMA was >2.3. Recovery of MMA averaged 92%, and MMA eluted away from ion suppressants. Direct correlation with our earlier method and with consensus data from external proficiency testing yielded an R2 ≥ 0.9409 and average biases less than ±5%. In the production environment, ongoing correlation with external proficiency testing yielded an R2 of 0.9980 and a mean bias of 0.36%. Over 1.7 years, the imprecision of 2 quality control levels was <6.4%.

We combined ultrafiltration, a simple sample extraction method, with gradient chromatography to exclude matrix effects to accurately and precisely quantify MMA.