Abstract: An aliquot (1.00 g) of the simulated sample was taken, and 3 mL of acetonitrile and 2 mL of water were added. The mixture was vortexed for 5 min, extracted for 20 min by ultrasound, and centrifuged for 5 min. The supernatant was passed through the pre-activated Qasis HLB solid phase extraction column, in which targets were eluted with 5 mL of acetonitrile. The eluate was blown to near dryness by nitrogen at 40 ℃. The residue was dissolved with 1.00 mL of acetonitrile, and filtered through a 0.22 μm organic filter membrane. The filtrate was analyzed by liquid chromatograph-triple quadrupole mass spectrometer. Twenty-six new psychoactive substances of fentanyl and 2 precursors were separated on ZORBAX SB-C₁₈ column by gradient elution with mixed solutions composed of methanol and 0.1% (volume fraction) formic acid solution containing 0.005 mol·L^-1 ammonium acetate at different volume ratios, ionized by electrospray ion source positive ion mode, and detected by multiple reaction monitoring mode. The parent ions and daughter ions of the 28 targets were determined by analyzing the mass spectra, and the targets were divided into 5 groups according to their structures, which possible fracture mechanism of each group of substances to form stabilized daughter ions was deduced. As found by the results of tests for method validation, linear relationships between values of the mass fraction and the corresponding peak area of the 28 targets were kept in the range of 10-100 μg·kg^-1, with detection limits (3S/N) in the range of 0.012-2.320 μg·kg^-1. The recoveries were found in the range of 86.2%-99.4%, and RSDs (n=5) of the determined values ranged from 1.6% to 6.3%.