Transfer of nitroimidazoles from contaminated beeswax to honey.

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-1) 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-1 were observed for metronidazole (28.9, 368.5 and 2589.4 µg kg-1 respectively) and ronidazole (27.4, 232.9 and 2351.2 µg kg-1 respectively), while lower maximum concentrations were measured for dimetridazole (0.98, 8.4 and 67.7 µg kg-1) and ipronidazole (0.9, 7.9 and 35.7 µg kg-1 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.

Validation of a method for the detection and confirmation of nitroimidazoles and the corresponding hydroxy metabolites in pig plasma by high performance liquid chromatography-tandem mass spectrometry.

Nitroimidazoles (Ronidazole, Dimetridazole, Metronidazole, Ipronidazole) and their hydroxy metabolites are banned substances with antibiotic and anticoccidial activity. They are suspected to be carcinogenic and mutagenic. Since nitroimidazoles showed an inhomogeneous distribution and a rapid degradation in incurred muscle samples, plasma is the preferred target matrix for residue analysis. The analytical method of Polzer et al. [J. Polzer, C. Stachel, P. Gowik, Anal. Chim. Acta 521 (2004) 189] was adapted for liquid chromatography-tandem mass spectrometry detection and was validated in house according to the Commission Decision 2002/657/EC. The method is specific for all nitroimidazole except for Ipronidazole and its metabolite, due to interferences at their retention times in chromatograms of blank plasma and reagents samples. The absence of a matrix effect enables the use of a (linear) calibration curve in solution for quantitation. The apparent recovery (obtained after correction with a deuterated internal standard) is between 93% and 123%, except for the metabolite of Metronidazole (58-63%). The repeatability (CVr=2.49-13.39%) and intralaboratory reproducibility (CVRW=2.49-16.38%) satisfy the Horwitz equation. The obtained values for the detection capacity (CCbeta) range from 0.25 to 1 microg L(-1), while values obtained for the decision limit (CCalpha) are below CCbeta.

[Determination of three nitroimidazole residues in royal jelly by high performance liquid chromatography-tandem mass spectrometry].

A method for analysis of trace metronidazole (MTZ), dimetridazole (DMZ) and ronidazole (RNZ) residues in royal jelly was developed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). After samples were dissolved in sodium hydroxide solution to disassociate target analytes from matrix, liquid-liquid extraction methods by ethyl acetate solvent were used. Matrix effects were minimized and good quantitation results were obtained by using highly-selective reaction monitoring (H-SRM) technology when deuterated dimetridazole (dimetridazole-D3) was selected as internal standard. Limits of detection (LODs) were 1.0 microg/kg for DMZ, 0.5 microg/kg for MTZ and RNZ (S/N > 5). Limits of quantitation (LOQs) were 2.0 microg/kg for DMZ, 1.0 microg/kg for MTZ and RNZ (S/N > 10). The linear ranges were 2.0 - 200 microg/L for all target analytes. Recoveries and relative standard deviations (RSDs) were in the ranges of 96.6% - 110.6% and 2.1% -7.4%, respectively. This method is suitable for statutory residue testing in the National Residue Surveillance Plan in China and meets the requirement for export.

Analysis of four 5-nitroimidazoles and their corresponding hydroxylated metabolites in egg, processed egg, and chicken meat by isotope dilution liquid chromatography tandem mass spectrometry.

An isotope dilution liquid chromatography-electrospray ionization-tandem mass spectrometry method is presented for the simultaneous analysis of several 5-nitroimidazole-based veterinary drugs, which are dimetridazole (DMZ), ronidazole (RNZ), metronidazole (MNZ), ipronidazole (IPZ), and their hydroxylated metabolites (DMZOH, MNZOH, and IPZOH), in egg (fresh egg, whole egg powder, and egg yolk powder) and chicken meat. Data acquisition was achieved by applying multiple reaction monitoring, and quantitation was performed by means of five deuterated internal standards (ISs), namely, DMZ-d3, RNZ-d3, IPZ-d3, DMZOH-d3, and IPZOH-d3, whereas MNZ and MNZOH were quantitated using DMZOH-d3. At the lowest fortification levels (i.e., 0.5 microg/kg for fresh egg and chicken meat and 1.0 microg/kg for other egg-based matrices) and for compounds having their own corresponding deuterated analogue used as an IS, acceptable performance data were obtained (corrected recoveries, 88-111%; decision limits, 0.07-0.36 microg/kg; detection capabilities, 0.11-0.60 microg/kg; and within-lab precision, < or = 15%). The method failed to give acceptable quantitative results for MNZ and MNZOH due to the unavailability of the corresponding deuterated ISs. Nevertheless, a reliable identification of these two analytes at levels < or = 1 microg/kg was still feasible.

Liquid chromatographic multicomponent method for determination of residues of ipronidazole, ronidazole, and dimetridazole and some relevant metabolites in eggs, plasma, and feces and its use in depletion studies in laying hens.

A simple, rapid liquid chromatographic (LC) method that uses UV/VIS detection has been developed for the determination in eggs of residues of the histomonostats dimetridazole (DMZ), ronidazole (RON), ipronidazole (IPR), and side-chain hydroxylated metabolites of DMZ and RON. Sample pretreatment includes an aqueous extraction, purification with an Extrelut cartridge, and acid partitioning with isooctane. An aliquot of the final aqueous extract is injected into a reverse-phase LC system; detection is performed at 313 nm. The limits of determination are in the 5-10 microgram/kg range. A UV/VIS spectrum can be obtained at the 10 microgram/kg level by using diode-array UV/VIS detection. Recoveries are between 80 and 98% with a coefficient of variation of about 5%. Some 20 samples can be analyzed per day. A side-chain hydroxylated metabolite of IPR can also be detected with this method, as demonstrated with samples from animal experiments. After a single oral dose of the drugs to laying hens, residues of the parent compound and/or the hydroxylated metabolites could be detected in eggs 5-8 days after dosing. Plasma distribution and excretion in feces were established both with and without deconjugation. DMZ and IPR were extensively metabolized to hydroxylated nitroimidazole metabolites; RON was excreted mainly as the parent compound.

Determination of dimetridazole and ipronidazole in feeds at cross-contamination levels.

A rapid method for the determination of dimetridazole and ipronidazole in feeds is described. The compounds are extracted from a borate buffer (pH 8.65) with benzene, partitioned into 1N HCl, and then partitioned back into benzene from a basic aqueous phase. The benzene extract is concentrated and injected onto a nonpolar (Apiezon L) gas chromatographic column for determination by 63Ni electron-capture detection. Recoveries from feeds of various composition, spiked at 0.2 ppm with both dimetridazole and ipronidazole, ranged from 70 to 115%; for the same feeds spiked at 1 ppm or more, the recoveries were greater than 80%. Carbadox, furazolidone, levamisole, oxytetracycline, chlortetracycline, sulfamethazine, sulfaquinoxaline, arsanilic acid, piperazine, penicillin, and commonly added vitamins and minerals do not interfere. A 2-dimensional thin layer chromatographic system is presented as a means of additional identification.

Rapid liquid chromatographic determination of dimetridazole and ipronidazole in swine feed.

A simple and rapid method is described for the determination of dimetridazole (DMZ) and ipronidazole (IPR) in swine feeds at various levels (0.11-110 ppm). The drugs are released from feed by prewetting with a buffer, followed by extraction with either methanol or methylene chloride, depending on the drug level; if necessary, an acid-base cleanup is used before the liquid chromatographic analysis. The analytes are separated on a C18 column and monitored at 320 nm for detection and quantitation. Recoveries of DMZ from several feed formulations averaged 108% at the 92.8 ppm level with a standard deviation (SD) of 4.00% and a coefficient of variation (CV) of 3.70%, 101% at the 11.2 ppm level with an SD of 11.9% and a CV of 11.8%, and 100% at the 0.112 ppm level with an SD of 9.27% and a CV of 9.25%. Recoveries of IPR averaged 77.1% at the 12.9 ppm level with an SD of 1.75% and a CV of 2.27%; IPR recoveries averaged 35.2% at the 0.129 ppm level with an SD of 3.39% and a CV of 9.63%.

Quantitative confirmation of dimetridazole and ipronidazole in swine feed by capillary gas chromatography/mass spectrometry with multiple ion detection.

Extracts from 4 types of swine feed containing 0.11 ppm each of dimetridazole (DMZ) and ipronidazole (IPR) were analyzed by capillary gas chromatography/mass spectrometry (GC/MS) using multiple ion detection (MID) techniques. We demonstrate in this paper that the quantitative results obtained by capillary GC/MS with MID are comparable for both compounds to results obtained by liquid chromatography and have a lower coefficient of variation for DMZ. Moreover, consistency in the ion ratios (5 ions in DMZ and 6 ions in IPR) permits identification of these compounds by electron ionization MS.