FORMATION IN AN AQUEOUS MATRIX AND PROPERTIES AND CHROMATOGRAPHIC BEHAVIOR OF 1-PYRENYLDIAZOMETHANE DERIVATIVES OF METHYLMALONIC ACID AND OTHER SHORT-CHAIN DICARBOXYLIC-ACIDS

Methylmalonic acid (MMA) and some short-chain dicarboxylic acids were derivatized with the fluorescent labeling reagent 1-pyrenyldiazomethane (PDAM) in an aqueous matrix. The derivatization of MMA and ethylmalonic acid (EMA) proceeded and leveled off rapidly at low pH, whereas at alkaline pH, the fluorescent derivative was progressively formed over a period of more than 24 h, and higher fluorescence yield was obtained. The PDAM esters of MMA and EMA had an excitation maximum at 340 mm and emission maxima at 376 and 395 mm. They were stable for days in aqueous media at room temperature. The MMA derivative was identified as 1-pyrenylmethyl methylmalonate monoester by mass spectrometry, and formation of 1-pyrenylmethyl methylmalonate diester could not be demonstrated. The free carboxylic acid moiety and the ionization of this group may explain the unique chromatographic properties of the MMA derivative, i.e. a marked increase in the capacity factor in a reversed-phase system by decreasing the pH of the mobile phase. The MMA derivative was separated from PDAM esters of several other short-chain dicarboxylic acids by a acetonitrile gradient in formate buffer, pH 2.5. These data may form the basis for the construction of automated MMA assays involving derivatization in aqueous media followed by liquid chromatography.