New Labeling Reagents for Alcohols in Fluorescence High-Performance Liquid Chromatography

7-Methoxycoumarin-3 (and 4)-carbonyl azides (I and II) were synthesized as highly sensitive fluorescent labeling reagents for use in high-performance liquid chromatography (HPLC). Treatment of 7-methoxycoumarin-3 (and 4)-carbonyl chlorides with a suspension of activated sodium azide in dry acetone gave I and II in 56% and 54% yields, respectively. Compounds I and II reacted with alcohols in dichloromethane to give the corresponding highly fluorescent 7-methoxycoumarin-3 (and 4)-carbamic acid esters (IIIa-e and IVa-g). 4-Amino-7-methoxycoumarin (V), isolated as a by-product on labeling with II, was also obtained by heating II in tetrahydrofuran. Mixtures of primary and secondary alcohols were labeled with I or II and chromatographed on a reversed-phase HPLC column (mobile phase : methanol-water and methanol-chloroform) with a fluorescence detector. The detection limits of a test sample, cholesterol, were 50fg and 10ng/100μl on labeling with I and II, respectively.