Abstract: Carbonyl compounds in mainstream of cigarette smoke were trapped in a mixture solution of acetonitrile and H3PO4 (containing 2,4-dinitrophenylhydrazine), and derivatized simultaneously therein. The derivatized products obtained were taken for HPLC analysis. Merck Lichrosphere RP C18 column was used as separation column, and mixtures of component A, B and C mixed in various ratios were used in the gradient elution. Component A and B are mixtures of water, acetonitrile, tetrahydrofuran and iso-propand with 2 different ratios of (59+30+10+1) and (33+65+1+1) respectively, and component C is acetonitrile. UV-detection with the wavelength of 365 nm was adopted in the determination. Values of detection limit (3S/N) of the method were found in the range of 0.04-0.12 μg·g-1. The proposed method was applied to the analysis of mainstream of cigarette smoke, giving values of recovery in the range of 75.0%-96.0% and RSD′s (n=5) in the range of 2.0%-9.3%.