Application of capillary electrophoresis with laser-induced fluorescence detection for routine determination of methylmalonic acid in human serum.

Abstract

Methylmalonic acid (MMA) in serum is an established marker of cobalamin deficiency. MMA and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane to form stable, highly fluorescent 1-pyrenylmethyl monoesters. We have analyzed these esters in human blood by capillary electrophoresis (CE) combined with laser-induced fluorescence detection, and here we describe our approach to achieve long-term reproducibility, which is a prerequisite for routine clinical application. To stabilize CE performance and to minimize solute adsorption to the capillary wall, we coated capillaries with linear polyacrylamide, used hydroxypropyl methylcellulose and dimethylformamide as buffer additives, and extensively diluted derivatized samples prior to injection. A discontinuous buffer system was used for sample stacking. Separation was performed in Tris-citrate buffer, pH 6.4, under reversed polarity conditions (negative potential at the inlet vial). The assay was linear for serum MMA concentrations in the range 0.1-200 mumol/L, the total run time was 26 min, the sample output was about 50 samples/24 h, and the coefficients of variation ranged between 3 and 12%, depending on the MMA concentration. Comparison of our assay with two established GC/MS methods demonstrated good correlation and measuring agreement.