Levamisole Residues Research Articles

Concurrent use of two dual-combination drenches containing monepantel/abamectin and oxfendazole/levamisole in sheep: effect on marker residues 21 and 28 days after administration

ABSTRACT Aims To determine the concentration, in comparison with the maximum residue limit (MRL), of anthelmintic marker residues in the target tissues (liver and fat) of sheep treated concurrently with two oral drenches, one containing monepantel and abamectin and the other oxfendazole and levamisole. Methods On day 0 of the study, 12 sheep (six male and six female; 8–9-months old) were dosed according to individual body weight determined the day prior. Zolvix Plus (dual-active oral drench containing 25 g/L monepantel and 2 g/L abamectin) was administered to all animals prior to administration of Scanda (dual-active oral drench containing 80 g/L levamisole hydrochloride and 45.3 g/L oxfendazole). Six sheep (three male and three female) were slaughtered 21 and 28 days after treatment and renal fat and liver samples were collected. Using validated methods, analyses for monepantel sulfone, abamectin, levamisole and oxfendazole (expressed as total fenbendazole sulfone following conversion of the combined concentrations of oxfendazole, fenbendazole and fenbendazole sulfone) were performed on liver samples while renal fat specimens were analysed for monepantel sulfone and abamectin residues only. Detected concentrations were compared to the established MRL in sheep for each analyte determined by the Ministry for Primary Industries. Results All residues detected in samples of liver and fat collected 21 and 28 days after treatment were below the MRL for each analyte. All liver samples collected on day 21 had detectable monepantel sulfone (mean 232 (min 110, max 388) μg/kg) and oxfendazole (mean 98.7 (min 51.3, max 165) μg/kg) residues below the MRL (5,000 and 500 μg/kg, respectively). Monepantel sulfone (mean 644 (min 242, max 1,119) μg/kg; MRL 7,000 μg/kg) residues were detected in 6/6 renal fat samples. Levamisole residues were detected in 3/6 livers (mean 40.0 (min 14.3, max 78.3) μg/kg; MRL 100 μg/kg), and abamectin residues in 1/6 livers (0.795 μg/kg; MRL 25 μg/kg) and 2/6 fat samples, (mean 0.987 (min 0.514, max 1.46) μg/kg; MRL 50 μg/kg) 21 days after treatment. Conclusion and clinical relevance These results suggest that concurrent administration of Zolvix Plus and Scanda to sheep is unlikely to result in an extended residue profile for any of the active ingredients, with all analytes measured being under the approved New Zealand MRL 21 days after treatment. This work was not completed in line with guidance for establishing official residue profiles, nor is it sufficient to propose a new withholding period.

Research on the Removal of Levamisole Residues in Bovine, Ovine, Caprine, Porcine and Poultry Tissues

In this paper, we set the waiting time for the elimination of levamisole residues at a safe level from tissues (muscles and organs) from animals treated with levamisole 10%—oral solution. We studied the depletion of levamisole residues by high performance liquid chromatography with a mass spectrometer (limit of quantification 2 μg/kg) in the tissues of bovine, ovine, caprine, porcine and poultry (chickens and pigeons) after administration of levamisole (10 mg levamisole/kg body weight for cattle, sheep, goats, pigs and 20 mg levamisole/kg body weight for birds). We found that in cattle, sheep and goats at 7 days after treatment, the residues of levamisole in the liver and at 14 days and in the other tissues were below the established limit value. In pigs at 7 days, residues from all tissues were below the set limit value. Residue depletion in chickens treated with levamisole was rapid, such that 3 days after treatment, the residues in all tissues were below the set limit. Residue depletion was slower in turkeys than in chickens.

Computational ionophore selection during optimization of a portable calixarene based sensor for direct assay of levamisole residues in livestock products

• Portable solid-state potentiometric sensor for rapid, direct assay of levamisole. • Experimental and computation optimization of the sensor composition. • Assay of levamisole residues in bovine milk, muscles, kidney, and liver samples. • Assessment of the entire method for greenness. Poor practices in the livestock industry include the misuse of veterinary drugs to promote production and enhance the feed transformation ratio. The drugs accumulate in the edible tissues and transfer to consumers imposing health hazards. For instance, levamisole (LVM) residues lead to health hazards (e.g. hematological toxicity and agranulocytosis). Accordingly, authorities have drawn strict limits for LVM residues in edible tissues. This work introduces the first portable solid-contact potentiometric sensor for direct LVM assay in multiple bovine products. The sensor included a drop-casted membrane cocktail over a polyaniline coated glassy-carbon substrate. An initial study optimized the membrane cocktail composition for maximum sensitivity and selectivity. Molecular docking supported the experimental results for ionophore selection. The optimized sensor engendered a Nernstian response with a LOD of 1.56 × 10 −6 M. The greenness of the developed method was assessed using the National Environmental Method Index pictogram. The method was officially validated; and successfully determined LVM in bovine milk, muscle, kidney, and liver samples. Quality control laboratories can use the portable sensor for LVM overuse surveillance.

Distribution and elimination of levamisole in eggs and tissues after oral administration to laying hens, determined by LC-MS/MS

ABSTRACTLevamisole was administered to laying hens, and concentrations in eggs and tissues (thigh muscle, breast muscle, liver and kidney) were determined by a newly developed liquid chromatography tandem mass spectrometry method, which allowed trace level quantification of levamisole. The adopted analytical method showed good sensitivity, repeatability and percentage of recovery from spiked matrices. Maximum concentrations of levamisole were found on the first day after the administration (531.1 μg/kg in liver, 164.3 μg/kg in egg yolk, 130.7 μg/kg in kidney, 78.0 μg/kg in breast muscle, 70.7 μg/kg in thigh muscle and 64.0 μg/kg in egg white), after which there is a decline. The compound was rapidly eliminated from eggs, with a half-life of 1.3 days. Elimination appeared to be slower in thigh muscle (3.5 days), breast muscle (3.4 days) and liver (3.3 days). According to this experiment, the levamisole withdrawal periods calculated for eggs, liver, kidney, breast muscle and thigh muscle in laying hens were 14.1, 6.1, >4.0, 14.5 and 13.0 days, respectively. The longest time for levamisole residues to be completely released from tissues was seen in liver samples (37.4 days), followed by thigh muscle, breast muscle and kidney. Elimination from eggs was fastest (16.4 days for levamisole residues to drop below the method quantification limit).

The Effect of Various Cooking and Freezing Processes on the Levamisole Residues in Broiler Tissues

The Effect of Various Cooking and Freezing Processes on the Levamisole Residues in Broiler Tissues

Partitioning of nitroxynil, oxyclozanide and levamisole residues from milk to cream, skim milk and skim milk powder

Veterinary drugs are necessary to control fluke in animals, and if not properly used, residues of these drugs may be found in milk. The aim of this study was to determine whether residues of nitroxynil, levamisole and oxyclozanide in milk partition into skim milk powder during processing. Milk targeted to contain high, medium or low levels of residue was obtained following treatment of trial animals. On separation of cream and skim milk, > 90% of the residue partitioned with the skim milk in all cases. During powder processing, the residues were not degraded with almost 90% of the residue detected in the powder.

Investigation of the Persistence of Levamisole and Oxyclozanide in Milk and Fate in Cheese

In this study, dairy cows (n = six) were treated with an oral combination product containing levamisole (5 mg/kg body weight, (bw)) and oxyclozanide (10 mg/kg bw). Animals were milked twice daily up to day 16 post-treatment. Milk samples were subsequently analyzed by ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The highest levels of levamisole (<600 μg/kg) and oxyclozanide (<25 μg/kg) were determined at first and third milking, respectively. Residues of levamisole and oxyclozanide were typically below reporting limits of 0.83 and 1 μg/kg respectively at the 11th and 13th milking, respectively. Soft (3 days ripening), hard (35 days ripening) and whey cheeses were produced from the milk samples collected from the first two milkings. Levamisole residues were found to concentrate in all cheese types. There was a 3-fold concentration effect for levamisole in mature cheese. Oxyclozanide residues were found to occur at lower levels in soft and hard cheese than milk with a 10-fold concentration in whey cheese compared to milk. The results of this study demonstrate that levamisole and oxyclozanide residues are rapidly excreted in dairy cows and milk is compliant after a few days. Oxyclozanide and levamisole residues were shown to be stable during the fermentation process and the whey heat treatment to persist in cheese.

Determination of benzimidazoles and levamisole residues in milk by liquid chromatography–mass spectrometry: Screening method development and validation

The screening method for the determination of residues of 19 benzimidazoles (parent drugs and their metabolites) and levamisole in bovine milk has been developed and validated. Milk samples were extracted with ethyl acetate, sample extracts were cleaned up by liquid–liquid partitioning with hexane and acidic ethanol. Liquid chromatography–single-quadrupole mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine milk, according to the CD 2002/657/EC criteria. An alternative approach to the validation of the method was applied (“sum MRL” substances). The method was successfully verified in CRL proficiency test.

Determination of Levamisole in Sheep Muscle Tissue by High-Performance Liquid Chromatography and Photo Diode Array Detection

A high-performance liquid chromatographic method for the determination of levamisole (LVM) residues in sheep muscle tissue is described. LVM was extracted with ethyl acetate under alkaline conditions and cleanup was performed by liquid-liquid partition between organic-basic and organic-acid medium. Finally, levamisole was back extracted with chloroform carefully transferred into a clean glass vial and evaporated to dryness at 50 °C under a gentle stream of nitrogen. The remaining dry residue was dissolved in the mobile phase used, filtered and an aliquot was injected automatically into the chromatograph for analysis. Chromatography was performed on a Zorbax®SB-C18 column at 50 °C and detection by a PDA detector monitored at λmax 220 nm. The mobile phase was a mixture of 0.1 % trifluoroacetic acid (v/v) pH 2.0 and acetonitrile-methanol 3 : 2 (v/v) in a combination of 30 : 70 (v/v) and a flow rate of 1.0 mL min−1, delivered isocratically. This analytical method was validated by assessing recovery efficiency using spiked muscle tissue samples with standard solutions in methanol at four fortification levels of 1/2 MRL, 1 MRL, 2 MRL and 4 MRL and five times for each concentration (n = 5). Mean recovery (R%) achieved for muscle tissue was 75.65 ± 2.74% with an acceptable Relative Standard Deviation RSD% = 10.4. The same method was used also for the analysis of kidney, liver and fat (perirenal) and the recoveries found were 70.25 ± 1.07% (RSD% = 1.52), 72.37 ± 3.6% (RSD% = 4.97) and 69.44 ± 2.22% (RSD% = 3.19), respectively. The limit of detection (LOD) for muscle tissue was found to be 2.0 µg kg−1 and the limit of quantification (LOQ) 5.0 µg kg−1.

Levamisole residues in chicken tissues and eggs

Levamisole is currently being used to treat capillaria infection in chickens even though there is no published withdrawal information available for levamisole in chickens. Tissue residue withdrawal of levamisole in chickens was studied in 32 healthy broiler breeder chickens at the age of 32 wk (peak of egg production). Levamisole residues in chicken tissues, eggs, and plasma were determined by HPLC with ultraviolet (UV) detection at 225 nm. The highest level of residue and longest withdrawal after oral administration of 40 mg/kg levamisole to chickens was in the liver. On d 3 the level of levamisole were undetectable in the plasma. On d 9, levamisole residue in eggs was 0.096 microg/g and on d 18 it was 0.06 microg/g or less in all the analyzed chicken tissues. Those levels were lower than the recommended maximum residue limit (MRL). The withdrawal time for levamisole in chickens was longer than for other species tested, which is due in part to a larger dose of levamisole being recommended for chickens. In conclusion from this research, 9 d are needed for levamisole in eggs to be less than the MRL, and 18 d of withdrawal are needed before medicated birds are slaughtered if their tissues are to be safe for human consumption.

Detection of levamisole residues in bovine liver and milk by immunobiosensor

Levamisole is widely used for the control of gastro-intestinal parasites in many animal species due to its broad spectrum action. A maximum residue level in liver was established at 100 μg kg −1, however no value has yet been set for residues in milk. An immuno-based screening method was developed to detect levamisole residues in liver and milk using a Biacore ® biosensor. Levamisole was derivatised to aminolevamisole, conjugated to a carrier protein and the immunogen generated was used to raise a polyclonal antibody in a rabbit. The same drug derivative was immobilised onto a CM5 sensor chip. Binding of the antibody to the sensor chip surface was inhibited in the presence of levamisole. The chip surface was stable for many hundreds of regenerations. A single analytical cycle (sample injection, chip regeneration and system wash) took 6 min to complete. Sample analysis (liver and milk) included an extraction with acetonitrile. Liver samples required an additional filtration step prior to analysis. The 3 σ limit of detection (LOD) was calculated as 6.8 ng g −1 for the liver assay and 0.5 ng ml −1 for the milk assay. Interassay variation was calculated as 6.7% at a concentration of 100 ng g −1 in liver and 2.4% at 2 ng ml −1 in milk. Analysis of incurred liver and milk samples showed close agreement with those determined by liquid chromatography (LC)–mass spectrometry (MS) ( r 2=0.998 and 0.985 for liver and milk analysis respectively).

A highly sensitive gas chromatographic determination of levamisole in milk

An efficient extraction and sensitive gas chromatographic method is described for the determination of the anthelminthic drug levamisole in milk. Levamisole was extracted from alkaline milk with ethyl acetate. Clean‐up of the extract was by a series of liquid‐liquid extraction steps. Levamisole residues in the extract were determined by gas chromatography with a nitrogen‐phosphorus detector. This method was satisfactory for determining levamisole residues in milk as low as 0.5 ng/g. Mean recoveries of 0.5–10.0 ng/g fortified milk samples ranged from 84.5 to 95.2%. Five replicate analyses performed on a milk containing incurred levamisole residues yielded a mean of 3.34 ng/g levamisole with a CV of 3.0%.

The effect of cooking on veterinary drug residues in food: 2. levamisole.

The heat stability of levamisole was investigated. Results obtained indicated that the drug was stable in boiling water at 100 degrees C, but unstable at 260 degrees C in cooking oil, with a half-life of about 5 minutes. The effect of cooking (microwaving, boiling, roasting, grilling and frying) on levamisole residues in a range of fortified and incurred tissue was studied. No evidence of instability was obtained in any of the cooking methods investigated. Most observed net changes fell within the limits of the precision of the method once allowance for weight loss during cooking was made to counter an apparent increase in concentration. Roasting was the only method of cooking where a net loss of levamisole was observed. Insufficient juices were produced to permit analysis in this instance. The net loss of levamisole in the cooked tissue was similar to that found with other cooking methods, where the levamisole lost was found in the cooking liquid or juices. An assessment of homogeneity of the incurred tissue used in the investigation was made. The pig muscle was found not to be homogeneous with larger differences seen between different areas of the animal than within the same muscle. The findings of this investigation showed that data obtained from measurements on raw tissue are suitable for use in consumer exposure estimates and dietary intake calculations.

Confirmation of Levamisole Residues in Cattle and Swine Livers by Capillary Gas Chromatography-Electron Impact Mass Spectrometry

Capillary gas chromatography-electron impact mass spectrometry is used to confirm the presence of levamisole in cattle and swine livers at the 0.1 ppm tolerance level. Use of a fused silica capillary column from injector to detector solved chromatographic problems encountered with the analyte. Of the mass spectrometric techniques evaluated, electron impact mass spectrometry provided the most satisfactory data for a confirmatory method. Recoveries from swine and cattle livers fortified at 0.1 ppm averaged 74.9 and 72.7%, respectively, indicating potential utility of this methodology as a quantitative method. Apparent levamisole residues in control livers were less than 0.01 ppm.

Levamisole residues in milk from a herd of cows suffering from lungworms.

A single intramuscular dose of 7 mg levamisole hydrochloride per kg body weight was administered to a herd of 42 milking cows suffering from lungworms. Levamisole was rapidly excreted in the milk, the peak concentration being found 1 h after administration. The half-life of levamisole in milk was about 5 h. Levamisole levels were below the limit of determination (0.04 microgram/ml) in all milk samples 29 h after treatment.

Gas-Liquid Chromatographic Determination of Levamisole Residues in Bovine Milk

A gas-liquid chromatographic (GLC) method is described for determining residues of levamisole, (1-)-(2,3,5,6-tetrahydro-6-phenylimidazo [2,1-b] thiazole), in milk. Levamisole is extracted from an alkaline milk sample with water-saturated ethyl acetate. After co-extractives are removed by various partitionings, the levamisole concentration is measured by GLC by using an alkali (RbSO4) flame ionization detector, vs. an external standard. This method is satisfactory for determining levamisole residues as low as 0.01 ppm in bovine milk. Apparent residues in control milk samples averaged less than 0.001 ppm. Recoveries of 0.01-2.0 ppm added levamisole ranged from 86.5 to 113%. Animals treated with various Tramisol formulations showed apparent levamisole residues below the validated sensitivity of the method (0.01 ppm) at sampling periods exceeding 48 hr.