Development of a sensitive fluorescent derivatization reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydrazine and its application for determination of aldehydes from alcoholic beverage using high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification.

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethoxy-carbonylhydazine (BCEC) followed by high-performance liquid chromatography with fluorescence detection and enhance mass spectrometric identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing agent BCEC. BCEC can easily and quickly label aldehydes. The maximum excitation (333nm) and emission (390nm) wavelengths were essential no change for all the aldehyde derivatives. The fluorescence intensity was substantially affected by the solvents, being higher in organic than protic solvents. Derivatives are sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M+(CH(2))(n)-1](+) (M: molecular mass of BCEC, n: corresponding aldehyde carbon atom numbers) under positive-ion mode. The collision-induced dissociation of protonated molecular ion formed products at m/z=245.7.0, m/z=263.7 and m/z=217.7, and corresponding the cleavage of CH(2)OCO, CH(2)OCO and NCH(2)CH(2) bonds, respectively. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10- to 15-fold molar reagent excess. Separation of the derivatized aldehydes has been optimized on ZORBAX Eclipse XDB-C(8) column with aqueous acetonitrile in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.08-16.65mumol/L with coefficients of >0.9999. Estimated detection limits for the aldehydes, obtained by successive dilution of a derivatized standard solution containing 16.65mumol/L of each aldehyde (at a signal-to-noise ratio=3:1), are from 3.75 to 16.65fmol.