Abstract: The structures and fragmentation pathways of 25 kinds of 2,4-dinitrophenylhydrazone derivatives of carbonyl compound (CC-DNPH) were studied by high performance liquid chromatography-electrospray ionization-quadrupole-Orbitrap-high resolution mass spectrometry (HPLC-ESI-Q-Orbitrap MS). The information of parent ions and fragment ions for establishing the screening database was obtained at parallel reaction monitoring (PRM) mode. Full MS/dd-MS² mode was used to obtain peak area of the parent ions for quantitation, and retention time and accurate mass of parent ions and daughter ions were used for screening. At ESI- mode, CC-DNPH formed quasi-molecular ions (QMI) of M-H^-, and further disintegrated into fragment ions of m/z 76, 122, 181, 163, 105, 152, etc. According to the accurate mass of fragment ions, the structures of the fragments were identified and the detailed fragmentation paths were studied. The fragmentation characteristics of saturated fatty aldehydes, unsaturated fatty aldehydes, aromatic aldehydes and ketones were compared. The results of 5 ambient air samples of Tianjin showed that 24 of 25 target CC-DNPHs and 12 extra CC-DNPHs were screened out positive. The 24 targets’ concentrations are 0.006-4.45 μg/m³ and the extra 12 CC-DNPHs’ concentrations are 0.007-4.18 μg/m³. This study provides a valuable basis for further study on the fragmentation schemes of small molecular compounds by mass spectrometry.