Pre-column fluorescence derivatization using leucine-coumarnylamide for HPLC determination of mono- and dicarboxylic acids in plasma.

A sensitive HPLC assay utilising a simple fluorescence pre-column labelling technique was developed for plasma level monitoring of different types of mono- and dicarboxylic acids. Carboxylic acids form mixed anhydrides with ethyl chloroformate in the presence of triethylamine; the mixed anhydrides further react with L-leucine-4-methyl-7-coumarinylamide, forming highly fluorescent and stable amides. Drugs with no chromophore (azelaic acid, and its longer carbon chain analogues) or week UV absorption (artelinic acid, enalaprilat) were used as model compounds. The plasma samples were extracted using ion exchange solid phase cartridges. The separation was performed on an Axxiom C18 (5 microns, 4.6 x 250 mm) column. The detector wavelengths were set at 330 nm for excitation and 390 nm for emission.