Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane

Abstract

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-319]. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiazomethane derivatives were detected with a laser-induced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH > 4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the p K a value (5.2–5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary length) were tested, and were largely found to comply with validated theoretical models for capillary zone electrophoresis. In an optimal system, as judged by high number of theoretical plates, high resolution and short run times, the MMA derivative was separated from related 1-pyrenylmethyl monoesters. The laser-induced fluorescence detection afforded a limit of detection of about 40 nmol/1 (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to detect <1 μmol/1 endogenous MMA in human serum.