Determination Of Residues Of Trimethoprim And Diaveridine In Food Of Animal Origin

Trimethoprim (TMP) and diaveridine (DVD) are members of the2,4-diamino pyrimidine class ofdihydrofolate reductase inhibitors. In veterinary clinic, trimethoprim or diaveridine is widely used incombination with sulfonamides or antibiotics for treatment of pullorum disease, fowl cholera andrespiratory tract secondary bacterial infection. Due to widely and improper use of the two drugs, theresidues in the food of animal origin may be harmful to human health. Therefore, Ministry ofAgriculture of China and EU have reported their recommended maximum residue limits (MRLs)50μgkg-1for TMP in muscle, liver, kidney, and fat+skin tissues of porcine and poultry as well as muscle+skin tissues of fin fish, besides China set TMP as a marker residue. And Japan has reported theirrecommended maximum residue limits (MRLs)50μg kg-1for DVD in chicken tissues. So it isparticularly important to ensure human health and food safety by detecting the residues of trimethoprimand diaveridine in the food of animal origin.The objective of this study was to develop a sensitive, rapid method which has wide applicabilityfor detecting trimethoprim and diaveridine in muscle, liver, kidney, and fat+skin tissues of porcine andchicken as well as muscle+skin tissues of fin fish, by using high performance liquid chromatography-tandem mass spectrometry. The results are as the following:1A high performance liquid chromatography-tandem mass spectrometry method for thedetermination of trimethoprim and Diaveridine in muscle, liver, kidney, and fat+skin tissues of porcineand chicken as well as muscle+skin tissues of fin fish. Analytes are extracted from samples usingacetonitrile by liquid-liquid extraction. After dehydration and be evaporated to dryness, extracts ispurified by Oasis MCX cartridges. Chromatographic separation was performed on a hydrophilicinteraction liquid chromatography (HILIC) column.The analytes were determined usingtriple-quadrupole mass spectrometry in positive electrospray ionization and multiple reactionmonitoring mode. Trimethoprim-D3is described for use as an internal standard for quantitative analysis.And results show that trimethoprim and diaveridine showed a good linearity in a range between2～200μg kg-1and1～100μg kg-1in nine tissues, respectively. The recoveries of nine tissues from spikedsamples ranged from76.0%to112.2%, with the relative standard deviations being below19.2%. Limitsof detection for trimethoprim in tissues ranged from0.6to1μg kg-1, and limits of detection fordiaveridine in tissues ranged from0.3to0.5μg kg-1, Limits of quantification for trimethoprim anddiaveridine are2μg kg-1and1μg kg-1, respectively.2This method is applied to detect the two drugs in the samples that from supermarket and treatinganimals. The results of detecting real sample from supermarket show that no residue beyond the MRLhas been detected and results of detecting real sample from animal treating experiment show that theresidue concentrations of trimethoprim and diaveridine in tissues reduced with increasing withdrawal.And results further verify the feasibility of method for the determination of trimethoprim anddiaveridine in tissues. 3The draft of national standard that―Determination of residues of trimethoprim and diaveridine infood of animal origin—LC-MS/MS method‖related to this study is drafted.In conclusion, a high performance liquid chromatography-tandem mass spectrometry method forsimultaneous determination of trimethoprim and diaveridine in muscle, liver, kidney, and fat+skintissues of porcine and chicken as well as muscle+skin tissues of fin fish. This method is of highsensitivity, good reproducibility, widely applicability in animal tissues, and the accuracy and precisionof it can satisfy the requirements of the documents related residue of veterinary drug of ChineseAgriculture Ministry. Thus, the analytical method can be employed to detect trimethoprim anddiaveridine in real samples as routine analysis.