CAPILLARY ZONE ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION FOR ANALYSIS OF METHYLMALONIC ACID AND OTHER SHORT-CHAIN DICARBOXYLIC-ACIDS DERIVATIZED WITH 1-PYRENYLDIAZOMETHANE

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids reac t with 1-pyrenyldiazomethane (PDAM) in aqueous matrices and form stabl e, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-319]. We investigated the migration behavi our of these derivatives in capillary zone electrophoresis in fused-si lica capillaries. The 1-pyrenyldiazomethane derivatives were detected with a laser-induced fluorescence detector that was connected to a hel ium-cadmium laser, delivering light at a wavelength of 325 nm, which e xactly matched an excitation maximum of the 1-pyrenylmethyl monoesters . These esters have one free carboxylic acid group, which carries nega tive charge at pH >4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the pK (a) value (5.2-5.8) of the esters. The effects of the composition of t he running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary l ength) were tested, and were largely found to comply with validated th eoretical models for capillary zone electrophoresis. In an optimal sys tem, as judged by high number of theoretical plates, high resolution a nd short run times, the MMA derivative was separated from related 1-py renylmethyl monoesters. The laser-induced fluorescence detection affor ded a limit of detection of about 40 nmol/l (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to d etect <1 mu mol/l endogenous MMA in human serum.