Determination of Amitraz and Its Metabolites in Honey by LC-MS/MS

2.00 g of honey sample were taken and 40 ng of the isotopic internal standard were added. The mixture was dissolved and diluted to 10.0 mL with potassium dihydrogen phosphate-sodium hydrogen phasphate buffer solution of pH 7. The solution was extracted twice with acetonitrile (10.0 mL for each extraction and 2.0 g of NaCl were added in the 1st extraction) by centrifuging and swirling for 5 min. The 2 supernatants were combined and its volume was made up to 20.0 mL with acetonitrile. An aliquot of 1.0 mL of the solution was transferred to a centrifuging tube in which 25 mg of PSA, 10 mg of C₁₈ and 30 mg of MgSO₄ were added preliminarily, and the mixture was centrifuged at high speed to perform purification by dispersive SPE. Whole of the supernatant was taken and its volume was made up to 2.0 mL with water. This solution was used for LC-MS/MS analysis. In LC separation, XDB-C₁₈ chromatographic column was used as stationary phase, and mixtures of (A) φ 0.15% formic acid and (B) acetonitrile in various proportions were used as mobile phase in gradient elution. Eluates obtained at various time-intervals were used for MS/MS determination of residual amounts of Amitraz and its 3 main metabolites, i.e., N-2,4-dimethylphenyl-N-methylformamidine, N-(2,4-dimethylphenyl)formamide and 2,4-dimethylaniline in the honey sample. In preparation of working standard curves, mixed standard solutions of the 4 analytes were added to a blank honey sample as matrix, and internal standard was used for quantification simultaneously, interference due to matrix effect was effectively overcome. Linearity ranges for the 4 compounds were in the same range of 0-100 μg·kg^-1, with their lower limits of determination (10S/N) were found as follows 0.10, 0.20, 5.0 2.0 μg·kg^-1. Tests for recovery and precision were performed by addition of standard solutions at 3 concentration levels (5.0, 10 and 20 μg·kg^-1) to a blank honey sample as matrix. Values of recovery found were all greater than 80%, and values of RSD (n=6) found were all less than 10%. It was shown that the proposed method has specialties of easy and rapid in manipulations, and furthermore, values of lower limits of determination for the 4 analytes were found to meet with the requirements of regulations given by our country or abroad.