Automated assay of methylmalonic acid in serum and urine by derivatization with 1-pyrenyldiazomethane, liquid chromatography, and fluorescence detection

Abstract

Determination of methylmalonic acid (MMA) in serum has been established as an accurate test for the diagnosis of cobalamin deficiency. We describe here the development and performance of a liquid-chromatographic assay of MMA in blood and urine. The assay is based on our recent finding that one of the carboxylic acid moieties of some short-chain dicarboxylic acids reacts with the fluorogenic reagent 1-pyrenyldiazomethane in an aqueous medium, whereas the other remains underivatized (Anal Chem 1992;63:315-9). The pH-dependent ionization of the free carboxylic acid group of 1-pyrenylmethyl monoesters permits retention on anion-exchange columns, which are used for solid-phase extraction. The analysis is done with a cyanopropyl column coupled in series with an octadecyldimethylsilyl column. Solid-phase extraction and sample injection are carried out automatically by a Gilson ASPEC sample processor. The assay response varies linearly with MMA concentration in the range 0.1-1000 μmol/L in serum. The within-day and between-day CVs are 2.8-10.9%, and the detection limit of 5 fmol injected (∼20 nmol/L in serum) is sufficiently low to determine MMA in serum (mean 0.187 μmol/L, SD 0.084, range 0.044-0.431, n = 44) and urine from healthy subjects.