Reaction of 5-bromomethylfluorescein (5-BMF) with cefuroxime and other carboxyl-containing analytes to form derivatives suitable for laser-induced fluorescence detection

Abstract

The aim of this work was to evaluate the reactivity of 5-bromomethylfluorescein (5-BMF), a relatively new absorbance and fluorescence derivatization reagent, toward various classes of carboxylic acids. The model analytes included cefuroxime and benzoic acid (aromatic monocarboxylic acid), oleic acid and prostaglandin (unsaturated carboxylic acids), adipic acid and suberic acid (aliphatic dicarboxylic acids), palmitic acid and γ-cholesteryloxybutyric acid (CBA; aliphatic saturated carboxylic acids). Confirmation of conjugate formation was performed using TLC, HPLC and/or electrospray ionization mass spectrometry (ESI–MS). The yields of example conjugates were maximized using a sequential single factor approach. The conjugate of cefuroxime appeared to form via an intermediate. Palmitic acid produced two distinct conjugates; oleic acid and prostaglandin E₂ produced single derivatives. CBA was derivatized to form a highly hydrophobic conjugate. 5-BMF failed to form a conjugate with benzoic acid, and dicarboxylic acids. This may have been due to the formation of intermolecular complexes or highly polar salts of the acids, which were insoluble in the organic reaction medium. The conjugates were monitored by UV absorbance, fluorescence and argon ion laser-induced fluorescence (LIF). 5-BMF was found to be a suitable reagent for LIF detection of the stated analytes.