Development and validation of a method for determining amitraz and its metabolites in honey using HPLC–MS/MS method

Abstract

A selective, easy-to-implement, express method for the simultaneous determination of amitraz and its metabolites residual content in honey in the range of analytes mass fraction from 25 to 250 μg/kg has been developed and validated. The technique is based on the analytes liquid-liquid extraction with dichloromethane from alkalized aqueous solutions of honey to improve the stability of the acid-labile insecticide during sample preparation. The resulting extracts are sufficiently pure and do not require additional purification using solid-phase extraction or its variants. The extractant choice is based on a preliminary study of amitraz, DMFF, DMF and DMA distribution in water-organic solvent (n-hexane, toluene, dichloromethane, chloroform) extraction systems. Quantitative determination is carried out with HPLC–MS/MS in multiple reactions monitoring mode using matrix calibration in the range of the mass fraction of analytes in honey from 25 to 250 µg/kg. Depending on the analyte, the values of the detection limit ranged from 0.2 to 2.3 µg/kg, the limit of determination was from 0.6 to 4.6 µg/kg, the relative standard deviation of the repeatability – from 1.4 to 5.8 %, relative standard deviation of intermediate precision – from 1.4 to 7.7 %, relative expanded uncertainty – from 9 to 22 %. Calibration graphs were linear in the range from 25 to 250 µg/kg. The instrumental limits of detection and determination, the matrix effect, the degree of extraction, the overall efficiency of the process, and the bias were also evaluated in the course of the validation process.