Determination of Amitraz and Its Metabolites in Whole Blood Using Solid-phase Extraction and Liquid Chromatography-tandem Mass Spectrometry

A method was developed for determination of amitraz and its metabolites, N-[2,4-(dimethylphenyl)-N'-methylformamidine (DMPF), 2,4-dimethylformamidine (DMF), 2,4-dimethylaniline (DMA) in whole blood. The analytes were extracted by solid-phase extraction (SPE) using dichloromethane, acetonitrile and methanol (2:1:1) mixture as elute solution. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the positive ion mode using multiple reaction monitoring (MRM) technique. Collision-induced dissociation (CID) of amitraz at the electrospray source in MS/MS was observed in the analytic conditions. The method was validated in human whole blood spiked at three concentration levels. The low limit of detection (LOD) and the low limit of quantification (LOQ) for all the analytes were below 0.5μg/L and 2μg/L, respectively. Recoveries were between 90.2% and 104.5%, Bias and relative standard deviation (RSD) were below 15% (n=6). The good linear relationships were obtained in certain concentration ranges of amitraz and its metabolites. The results demonstrated the method is exclusive, sensitive and accurate, and can be applied in forensic toxicology.