A fast and reliable method of liquid chromatography and ultraviolet detection of sulfaguanidine, sulfadiazine, sulfamethazine, sulfamethizole, and sulfamethoxazole in feedingstuffs was described. The method involves THE procedure of preparation of spiked samples, and extraction of sulphonamides from the matrix using a mixture of methanol and acetonitrile, followed by drying the extract and dissolving it in a phosphate buffer. The analysis uses octadecyl (C18) analytical column with UV detection at λ = 260 nm and a gradient programme of mobile phase composition. The analytical procedure has been successfully adopted and validated for quantitative determination of the sulfonamides in feedingstuff samples. Validation included sensitivity, specificity, linearity, repeatability, and intra-laboratory reproducibility. The mean recovery of sulfonamides was 84%, within the working range of 200-2000 mg/kg. Direct, simple sample preparation and HPLC-UV analysis allow the method to be successfully included in the scope of routine analyses. The presented results could be an answer to a need of simple and easy method for sulfonamide determination applicable in medicated feedingstuffs analysis.

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A multiresidue method for the determination of seven nitroimidazoles and their hydroxy metabolites in milk was developed. Milk samples were extracted with acetonitrile and cleaned up on strong cation-exchange solid phase extraction cartridges. Evaporated to dryness first, the extracts obtained were then reconstituted in 0.1% formic acid and injected onto a liquid chromatography-tandem mass spectrometer (LC-MS/MS). The separation of analytes was achieved on gradient mode using a C18 column and a mobile phase consisting of 0.1% formic acid in acetonitrile and 0.1% formic acid in water. Multiple reaction monitoring mode was set for the MS/MS with positive electrospray ionisation. For quantification, the isotope dilution method was used with isotopically labeled analogues of the target analytes. The method was evaluated entirely in accordance with EU Commission Decision 2002/657/EC and all validation criteria were in the required ranges. The method can easily detect and confirm metronidazole, dimetridazole, ronidazole, ipronidazole, and their hydroxy metabolites below the recommended concentration level of 3 µg/kg. The decision limits and detection capabilities ranged from 0.11 µg/kg to 0.22 µg/kg and from 0.19 µg/kg to 0.37 µg/kg respectively. The overall recoveries were between 96.6% and 105.2% with a good coefficient of variation, less than 8.7% under within-laboratory reproducibility conditions.

IntroductionAmitraz is a formamide exhibiting both acaricidal and insecticidal activity and is frequently used by beekeepers to protect honeybee colonies against Varroa destructor mites. The aim of this apiary trial was to evaluate the impact of honeybee colony fumigation with amitraz on the level of contamination of honey stored in combs.Material and MethodsExperimental colonies were fumigated four times every four days with one tablet of Apiwarol per treatment. Honey was sampled from combs of brood chambers and combs of supers one day after each amitraz application and from harvested honey. Amitraz marker residues (as a total of amitraz and metabolites containing parts of molecules with properties specific to the 2,4-DMA group, expressed as amitraz) were evaluated in honey.ResultsAll analysed samples were contaminated with amitraz metabolites. 2,4-DMA and DMPF were the most frequently determined compounds. The average concentration of amitraz marker residue in honey from groups where a smouldering tablet was located directly in beehives was significantly higher than that of residue in honey from groups with indirect smoke generation. No significant effect on the honey contamination deriving from the place where it was exposed to smoke (combs of brood chambers and supers) was noted. Amitraz marker residues exceeded the MRL in 10% of honey samples from combs.ConclusionFumigation of beehives with amitraz results in contamination of honey stored in combs.

Nitroimidazoles are not authorised for the treatment of honey bees in the European Union. However, they can be found in honey largely because they are illegally used in apiculture for the treatment of Nosema. The aim of the study was to examine the possible transfer of nitroimidazoles (metronidazole, ronidazole, dimetridazole and ipronidazole) from contaminated beeswax to honey. The wax foundations fortified with a mixture of four nitroimidazoles at three concentration levels (1000, 10,000 and 100,000 μg kg) were placed in beehives to let the honeybees (Apis mellifera L.) draw out the contaminated wax foundations to honeycombs. At 1 month from the start, the frames filled with capped honey were removed from the hives for a first sampling of honey. Next, the honeycombs were further incubated for 5 months in the laboratory at 35°C and sampled monthly. In the sampled honey, the concentrations of nitroimidazoles and their main metabolites (hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, hydroxyipronidazole) were determined by LC-MS/MS and compared with those determined in the nitroimidazole-containing wax foundations. Each of the tested nitroimidazoles could migrate from beeswax to honey kept in the contaminated combs at each tested concentration level. Higher maximum concentrations of residues in honey sampled from contaminated combs at 1000, 10,000 and 100,000 μg kg were observed for metronidazole (28.9, 368.5 and 2589.4 μg kg respectively) and ronidazole (27.4, 232.9 and 2351.2 μg kg respectively), while lower maximum concentrations were measured for dimetridazole (0.98, 8.4 and 67.7 μg kg) and ipronidazole (0.9, 7.9 and 35.7 μg kg respectively). When we took into account that a frame completely filled with honey on both sides of the comb contained 110 g of beeswax and 2488 g of honey, and that this ratio was constant, then maximum amounts of initial metronidazole, ronidazole, dimetridazole and ipronidazole that migrated from contaminated wax foundations to honey could be calculated: 65-89%, 55-63%, 1.7-2.7% and 1.4-2.3%, respectively.