APPLICATION OF CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION FOR ROUTINE DETERMINATION OF METHYLMALONIC ACID IN HUMANSERUM

Methylmalonic acid (MMA) in serum is an established marker of cobalami n deficiency. MMA and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane to form stable, highly fluorescent 1-pyrenylmeth yl monoesters. We have analyzed these esters in human blood by capilla ry electrophoresis (CE) combined with laser-induced fluorescence detec tion, and here we describe our approach to achieve long-term reproduci bility, which is a prerequisite for routine clinical application. To s tabilize CE performance and to minimize solute adsorption to the capil lary wall, we coated capillaries with linear polyacrylamide, used hydr oxypropyl methylcellulose and dimethylformamide as buffer additives, a nd extensively diluted derivatized samples prior to injection. A disco ntinuous buffer system was used for sample stacking. Separation was pe rformed in Tris-citrate buffer, pH 6.4, under reversed polarity condit ions (negative potential at the inlet vial). The assay was linear for serum MMA concentrations in the range 0.1-200 mu mol/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 M MA concentration. Comparison of our assay with two established GC/MS m ethods demonstrated good correlation and measuring agreement.