The concentration detection limit was superior to literature reports for detection of fatty acids. The mass detection limit provided approximately an order of magnitude improvement over conventional fluorescence. The reagent is potentially useful for analysis of carboxyl containing analytes at low concentrations.

This report describes the first application of 5-(bromomethyl)fluorescein (5-BMF) for the quantitation of a pharmaceutically relevant carboxyl-containing analyte in a biological matrix. An analytical method for quantitation of γ-(cholesteryloxy)butyric acid (CBA), a relatively new antitumor agent, in different tissues of Sprague-Dawley rats was developed. 5-BMF was employed to form a stable and spectrally well-characterized conjugate of CBA. The derivatization yield was maximized by optimizing several reaction variables. The conjugate was separated by high-performance liquid chromatography and quantitated by a laboratory-constructed argon ion laser fluorometer. The detection limits for CBA were 4.6 × 10 -9 and 6.34 × 10 -11 M by conventional and laser-induced fluorescence (LIF), respectively. A derivatization limit of detection of 1.85 × 10 -9 M was achieved by LIF for the conjugate. The analytical method was useful for quantitation of CBA in various tissues in the picogram per milliliter range.