3-BROMOMETHYL-7-METHOXY-1,4-BENZOXAZIN-2-ONE AS A HIGHLY SENSITIVE FLUORESCENCE DERIVATIZATION REAGENT FOR CARBOXYLIC-ACIDS IN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A fluorescence labelling reagent, 3-bromomethyl-7-methoxy-1,4-benzoxazin-2-one (BrMB), was synthesized from 2-amino-5-methoxyphenol and ethyl pyruvate by the Wislecenus reaction followed by bromination. Picomole to nanomole amounts of saturated aliphatic fatty acids were converted into the corresponding fluorogenic esters in the presence of anhydrous potassium carbonate and a crown ether as catalysts. BrMB derivatives of fatty acids were separated on a reversed-phase column and were sensitively detected fluorimetrically at 440 nm with excitation at 345 nm. Quantitative studies revealed that n-caproic acid was esterified completely under mild conditions and with sufficient reproducibility. The detection limit of the BrMB derivative of n-caproic acid was just below 2 fmol.