Smooth esterification of Di- and tricarboxylic acids with methyl and ethyl chloroformates in gas chromatographic profiling of urinary acidic metabolites

Fast esterification of (di)carboxylic acids in aqueous pyridine-containing media with methyl or ethyl chloroformate is presented. The reaction conditions have been optimized to allow also determination of CA (succinic) and C5 (glutaric) dicarboxylic acids and their substituents, i.e. the important urinary metabolites. Treated with chloroformates, these analytes commonly escape determination due to favouring internal cyclization to 5- and 6-membered rings of their parent anhydrides, prone to decomposition in the hot injection liner. To restore the diester formation, an alternative way of sample processing was developed. It was based on adding alcohol in excess and reagent in two portions, with applied time delay or neutralization of the first portion with hydroxide. This way, the di- and tricarboxylic acids of interest could be esterified effectively, some under formation of minor side-products. The novel approach proved to be quite suitable for screening urinary metabolites, without a necessity to preisolate the compounds and to evaporate to dryness.