The first fluorescent sensor for the detection of closantel in meat.

Closantel is widely used in the management of parasitic infestation in livestock, but is contraindicated in humans due to its high toxic to human retina. Thus, development of a fast and selective method for the detection of closantel residues in animal products is highly needed yet still challenging. In the present study, we report a supramolecular fluorescent sensor for closantel detection through a two-step screening process. The fluorescent sensor can detect closantel with a fast response (<10 s), high sensitivity, and high selectivity. The limit of detection is 0.29 ppm, which is much lower than the maximum residue level set by government. Moreover, the applicability of this sensor has been demonstrated in commercial drugs tablets, injection fluids, and real edible animal products (muscle, kidney, and liver). This work provides the first fluorescence analytical tool for accurate and selective determination of closantel, and may inspire more sensor design for food analysis.

Quantitative Analysis of Abamectin, Albendazole, Levamisole HCl and Closantel in Q-DRENCH Oral Suspension Using a Stability-Indicating HPLC-DAD Method.

Combination therapy of many anthelmintic drugs has been used to achieve fast animal curing. Q-DRENCH is an oral suspension, containing four different active drugs against GIT worms in sheep, commonly used in Australia and New Zeeland. The anti-parasitic drugs are Albendazole (ALB), Levamisole HCl (LEV), Abamectin (ABA), and Closantel (CLO). The main purpose of this study is to present a new simultaneous stability-indicting HPLC-DAD method for the analysis of the four drugs. The recommended liquid system was 1 mL of Triethylamine/L water, adjusting the pH to 3.5 by glacial acetic acid: acetonitrile solvent (20:80, v/v). Isocratic elusion achieved the desired results of separation at a 2 mL/min flow rate using Zorbax C-18 as a stationary phase. Detection was performed at 210 nm. The linearity ranges were 15.15 to 93.75 µg/mL for ALB, 25 to 150 µg/mL for LEV, 30 to 150 µg/mL for ABA, and 11.7 to 140.63 µg/mL for CLO. Moreover, the final greenness score was 0.62 using the AGREE tool, which reflects the eco-friendly nature. Moreover, the four drugs were determined successfully in the presence of their stressful degradation products. This work presents the first chromatographic method for simultaneous analysis for Q-DRENCH oral suspension drugs in the presence of their stressful degradation products.

Simultaneous Determination of Abamectin and Closantel in Veterinary Formulation by Validated HPLC Method.

A rapid and accurate high-performance liquid chromatographic method was developed for the determination of both abamectin and closantel in the veterinary formulation. The chromatographic separation was conducted on an Agilent 1200 with a UV detector using Waters C18 (4.6 mm × 50 mm; 2.7 µm). The mobile phase consisted of acetonitrile:water (80:20 v/v) adjusts pH 3.0 using diluted phosphoric acid. The flow rate of 1.5 mL min-1 was used. An injection volume of 10 µL was used The calibration curve of abamectin B1b was linear with a correlation coefficient (r2) = 0.9996; over a concentration range of 2.0-8.0 µg/mL, abamectin B1a was linear with a correlation coefficient (r2) = 0.9997; over a concentration range of 8.0-32.0 µg/mL; with a retention time of 2.18 and 3.72 minutes for avermectin B1b and avermectin B1a, respectively. While the calibration curve of closantel was linear with a correlation coefficient (r2) = 0.99929; over a concentration range of 250.0-1,000.0 µg/mL for; with a retention time of 5.84 minutes. Correlation coefficient was r2 ≥ 0.999. The relative standard deviation was found to be ≤ 2. The proposed method was validated and successfully applied for the simultaneous determination of abamectin and closantel in the veterinary formulation.

Comparative Study of Univariate Spectrophotometry and Multivariate Calibration for the Determination of Levamisole Hydrochloride and Closantel Sodium in a Binary Mixture.

Six simple, accurate, reproducible, and selective derivative spectrophotometric and chemometric methods have been developed and validated for the determination of levamisole HCl (Lev) either alone or in combination with closantel sodium (Clo) in the pharmaceutical dosage form. Lev was determined by first-derivative, first-derivative ratio, and mean-centering methods by measuring the peak amplitude at 220.8, 243.8, and 210.4 nm, respectively. The methods were linear over the concentration range 2.0-10.0 µg/mL Lev. The methods exhibited a high accuracy, with recovery data within ±1.9% and RSD <1.3% (n = 9) for the determination of Lev in the presence of Clo. Fortunately, Lev showed no significant UV absorbance at 370.6 nm, which allowed the determination of Clo over the concentration range 16.0-80.0 µg/mL using zero-order spectra, with a high precision (RSD <1.5%, n = 9). Furthermore, principal component regression and partial least-squares with optimized parameters were used for the determination of Lev in the presence of Clo. The recovery was within ±1%, with RSD <1.0% (n = 9) and root mean square error of prediction ≤1.0. The proposed methods were validated according to the International Conference on Harmonization guidelines. The proposed methods were used in the determination of Lev and Clo in a binary mixture and a pharmaceutical formulation, with high accuracy and precision.

Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta.

Closantel (CLS) is currently used in programs for the strategic control of gastrointestinal nematodes. CLS is extralabel used in different dairy goat production systems. From available data in dairy cows, it can be concluded that residues of CLS persist in milk. The current work evaluated the concentration profiles of CLS in plasma and milk from lactating orally treated dairy goats to assess the residues pattern in dairy products such as cheese and ricotta. Six (6) female Saanen dairy goats were treated orally with CLS administered at 10 mg/kg. Blood and milk samples were collected between 0 and 36 days post-treatment. The whole milk production was collected at 1, 4, 7, and 10 days post-treatment to produce soft cheese and ricotta. CLS concentrations in plasma, milk, cheese, whey, and ricotta were determined by HPLC. The concentrations of CLS measured in plasma were higher than those measured in milk at all sampling times. However, the calculated withdrawal time for CLS in milk was between 39 and 43 days postadministration to dairy goats. CLS residual concentrations in cheese (between 0.93 and 1.8 µg/g) were higher than those measured in the milk used for its production. CLS concentrations in ricotta were sixfold higher than those in the milk and 20-fold higher than those in the whey used for its production. The persistent and high residual concentrations of CLS in the milk and in the cheese and ricotta should be seriously considered before issuing any recommendation on the extralabel use of CLS in dairy goat farms.

Comparative method validation for closantel determination in cattle and sheep milk according to European Union Volume 8 and Veterinary International Conference on Harmonization guidelines.

A specific and sensitive LC-MS/MS method was developed for quantitative determination of closantel in bovine and ovine colostrum and tank milk. Sample preparation consisted of extracting milk samples with acetonitrile/acetone (80/20, v/v) followed by SPE clean-up with Oasis mixed anion exchange columns. After elution with 5% formic acid in acetonitrile and evaporation to dryness, the residue was reconstituted in acetonitrile and water. HPLC separation was achieved on a Zorbax Eclipse Plus C18 column and a gradient elution program with 1mM ammonium acetate in water and acetonitrile. For closantel determination in bovine milk, the method was validated according to EU Volume 8 guidelines whereas for ovine milk both EU Volume 8 and VICH GL49 criteria were applied. The linear range of the method is between 10 and 2000 µg/kg, the limit of quantification 10 µg/kg and limit of detection is 0.63 and 0.32 µg/kg for sheep colostrum and tank milk and 1.27 and 1.24 µg/kg for cattle colostrum and tank milk, respectively. Both guidelines cover a similar set of parameters (linearity, accuracy, precision, limit of detection and limit of quantification), although the acceptance criteria might differ (accuracy and precision) or no specific acceptability ranges are specified in neither guidelines (LOD and LOQ). For some parameters, only one of the guidelines indicates acceptance criteria: EU Volume 8 for applicability, practicability and susceptibility and VICH GL 49 for linearity, specificity and analyte stability.

Investigation of the persistence of closantel residues in bovine milk following lactating-cow and dry-cow treatments and its migration into dairy products.

Closantel is a veterinary drug used to treat liver fluke in cattle and sheep. A provisional maximum residue limit (MRL) of 45 µg/kg in milk has been set by the European Union. The purpose of this study was to investigate the persistence of closantel residues in milk and the migration of residues into milk products. Following dry-cow treatment, residues ranged from undetectable to 8.7 µg/kg at the first milking. Following lactating-cow treatment, residues detected ranged from 278 to 482 µg/kg at day 1 post-treatment and were detectable above the MRL for 52 days and detectable for 198 days. At day 2 and day 23 post-treatment, the milk was collected and dairy products manufactured. Closantel residues concentrated in the cheese, butter, and skim milk powder. The results indicate that closantel is best used as a dry-cow treatment.

[Determination of phenolic and salicylanilide anthelmintics in liquid milk by high performance liquid chromatography].

An analytical method for the determination of nitroxynil, oxyclozanide, closantel and rafoxanide in liquid milk by high performance liquid chromatography (HPLC) has been established. The milk sample was extracted with acetonitrile containing 1% (v/v) triethylamine. The supernatant was purified by an anion exchange solid phase extraction column. The analyte was detected by ultraviolet detector after the HPLC separation on a C18 RP column. The mobile phase was composed of acetonitrile-0.02 mol/L ammonium acetate solution with pH 4.0. The linear ranges of the four drugs in the spiked blank milk samples were 5-500 microg/kg, and the correlation coefficients were higher than 0.99. The limits of detection (LOD) were 3 microg/kg, and the limits of quantification (LOQ) were 5 microg/kg. The average recoveries and relative standard deviations (RSDs) of nitroxynil, oxyclozanide, closantel and rafoxanide at the spiked levels of 1/2MRL (maximum residue limit), MRL and 2MRL ranged from 92.20% to 96.13%, 5.55% to 16.30%; 87.40% to 94.74%, 5.40% to 12.21%; 86.97% to 91.09%, 2.67% to 8.17%; and 77.86% to 95.36%, 5.02% to 13.15% respectively. The method is simple and sensitive for the quantification of phenolic and salicylanilide anthelmintics in liquid milk.

Emergency slaughter of casualty cattle increases the prevalence of anthelmintic drug residues in muscle.

The ProSafeBeef project studied the prevalence of residues of anthelmintic drugs used to control parasitic worms and fluke in beef cattle in Ireland. Injured (casualty) cattle may enter the human food chain under certain conditions, verified by an attending veterinarian and the livestock keeper. An analytical survey was conducted to determine if muscle from casualty cattle contained a higher prevalence of anthelmintic drug residues than healthy (full slaughter weight) cattle as a result of possible non-observance of complete drug withdrawal periods. A validated analytical method based on matrix solid-phase dispersive extraction (QuEChERS) and ultra-performance liquid chromatography-tandem mass spectrometry was used to quantify 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCα, 0.15-10.2 µg kg⁻¹). Of 199 control samples of beef purchased in Irish shops, 7% contained detectable anthelmintic drug residues but all were compliant with European Union Maximum Residue Limits (MRL). Of 305 muscle samples from injured cattle submitted to abattoirs in Northern Ireland, 17% contained detectable residues and 2% were non-compliant (containing either residues at concentrations above the MRL or residues of a compound unlicensed for use in cattle). Closantel and ivermectin were the most common residues, but a wider range of drugs was detected in muscle of casualty cattle than in retail beef. These data suggest that specific targeting of casualty cattle for testing for anthelmintic residues may be warranted in a manner similar to the targeted testing for antimicrobial compounds often applied in European National Residues Surveillance Schemes.

Determination of closantel and rafoxanide in animal tissues by online anionic mixed-mode solid-phase extraction followed by isotope dilution liquid chromatography tandem mass spectrometry.

A rapid and high-throughput isotope dilution LC-MS/MS method with online sample pre-concentration and clean-up using anionic mixed-mode SPE was described for the determination of closantel and rafoxanide in edible bovine and ovine tissues. Tissue samples were extracted with acetonitrile and acetone mixture (60:40, v/v). Sample pre-concentration, clean-up and analysis were completed simultaneously with the online MAX SPE LC-MS/MS system. Closantel-(13) C(6) and rafoxanide-(13) C(6) were used as the internal standards to improve the precision of the method. The method was validated with edible ovine and bovine tissues (muscle, kidney and liver) fortified at three different levels. The accuracy and RSD were 86-106% and ≤14%, respectively. This high-throughput method was suitable for routine quantitative analysis of closantel and rafoxanide in food safety surveillance samples.

Quantitative analysis of closantel and rafoxanide in bovine and ovine muscles by high-performance liquid chromatography with fluorescence detection.

An HPLC method with a fluorescence detector (HPLC-FLD) was described for the quantitative determination of closantel and rafoxanide in bovine and ovine muscles. A structural analog closely related to rafoxanide, viz., N-[4-(4-chlorophenoxy)phenyl]-2-hydroxy-3,5-diiodobenzamide, was synthesized as an internal standard. Bovine and ovine muscles were extracted with acetonitrile-acetone (60 + 40, v/v) followed by cleanup on mixed mode anionic exchange SPE cartridges. After evaporation and reconstitution with the mobile phase, the sample was analyzed by HPLC-FLD using internal standard calibration. The method was validated by using fortified bovine and ovine muscles at 15, 30, and 60 microg/kg. The accuracy and RSD were 70-110% and < or =10%, respectively.

Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry.

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.

Screening of closantel and rafoxanide in animal muscles by HPLC with fluorescence detection and confirmation using MS.

A sensitive and selective LC-FLD method was described for determination of closantel and rafoxanide in bovine and ovine muscles. Bovine and ovine muscles were extracted with ACN and acetone mixture (80:20, v/v). After cleanup with Oasis MAX SPE cartridges, the sample was analyzed by LC-FLD using the control point approach. No false-negative result was observed at or below maximum residue limits and the false-positive rate was below 5%. Suspected positive sample was confirmed by LC-MS/MS. This method was suitable for screening of large batch of samples and hence considerably reduced the time and cost required for quantitation and confirmatory analyses.

Multi-residue determination of phenolic and salicylanilide anthelmintics and related compounds in bovine kidney by liquid chromatography-tandem mass spectrometry.

This paper describes an analytical method for four phenolic and salicylanilide anthelmintics authorised for use within the EU (nitroxinil, oxyclozanide, rafoxanide and closantel) in bovine kidney, and the extension of this procedure to include a number of related compounds; ioxynil, niclosamide, salicylanide and 3-trifluoromethyl-4-nitrophenol (TFM). The method comprises a solvent extraction with 1% acetic acid in acetone and clean-up using a mixed-mode anion-exchange solid phase extraction column. Determination is by reversed phase LC-MS/MS. The method was validated to the latest EU requirements (Commission Decision 2002/657/EC) using both spiked and incurred tissues and was subject to second laboratory evaluation.

Determination of closantel residues in milk and animal tissues by HPLC with fluorescence detection and SPE with oasis MAX cartridges.

A liquid chromatographic method for the determination of closantel residues in milk and tissues is developed and validated. An acetonitrile-acetone solution (80:20, v/v) is used for the extraction of closantel residues from milk and animal tissues, and the extract is purified by solid-phase extraction with Oasis MAX cartridges and a mixture of formic acid-acetonitrile (5:95, v/v) as the elution solution. A C(18) bonded silica column is used for chromatographic separation. The mobile phase consists of acetonitrile-water (85:15, v/v) containing 0.05% triethylamine at pH 2.5, adjusted with phosphoric acid with the flow-rate set at 1.0 mL/min. Using the fluorescence emission of closantel at lambda(ex) = 335 nm and lambda(ex) = 510 nm, the calibration curve is linear, with a correlation coefficient of 0.9999 over the concentration range of 10-5000 microg/kg for the tissue sample and 10-5000 microg/L for the milk sample. The detection limit (s/n = 3) is 3 microg/kg for tissue sample and 3 microg/L for milk sample. The intra- and inter-day repeatabilities are between 3.35-7.66% and 4.04-8.67%, respectively. The proposed method enables the quantitative determination of closantel residues at levels as low as 10 microg/kg in animal tissue samples and 10 microg/L in milk samples.

Validated method for determination of ultra-trace closantel residues in bovine tissues and milk by solid-phase extraction and liquid chromatography-electrospray ionization-tandem mass spectrometry.

A liquid chromatographic-electrospray ionisation-tandem mass spectrometry method (LC-ESI-MS/MS) with solid extraction was developed and validated for the detection and determination of closantel residues in bovine tissues and milk. An acetonitrile-acetone mixture (80:20, v/v) was used for one-stage extraction of closantel residues in bovine tissues and milk samples, and the extract was cleaned by solid phase extraction with Oasis MAX cartridges. The mass spectrometer was operated in multiple reactions monitoring mode with negative electrospray interface. The limits of detection in different matrices were in the range of 0.008-0.009 microg/kg. The overall recoveries for bovine muscle, liver, kidney and milk samples spiked at four levels including MRL were in the range of 76.0-94.3%. The overall relative standard deviations were in the range of 3.57-8.61%. The linearity is satisfactory with a correlation coefficient (r(2)) of 0.9913-0.9987 at both concentration ranges of 0.02-100 microg/kg and 200-5000 microg/kg. The method is capable of identifying closantel residues at > or =0.02 microg/kg levels and was applied in the determination of closantel residues in animal origin foods.

Assessment of the solid-state composition of an active salicylanilide compound by FT-Raman spectroscopy.

A biologically active salicylanilide compound currently appears in three known solid-state forms: polymorph I (Pol I), polymorph II (Pol II) and the amorphous form (Amorph). The obtained FT-Raman spectra revealed several regions of interest (ROIs) qualitatively distinguishing the different forms, allowing samples with an unknown polymorphic composition to be quantitatively analysed by FT-Raman spectroscopy. The Markov-transformed peak areas of the Raman-bands in the ROIs from the samples were determined and compared with the transformed peak areas obtained for the reference solid-state forms. A constrainted linear regression model estimated the contribution of each reference to the different samples. The applicability of this approach was demonstrated by analysing commercially available batches.

[Concentration in plasma and excretion in milk of lactating cows after oral administration of tribromsalan, oxyclozanide and bromofenofos].

The fasciolicides tribromsalan (TBS), oxyclozanide (OCZ) and bromofenofos (BFF) were orally administered to three lactating cows. The concentrations of TBS, OCZ and the BFF metabolite dephosphate bromofenofos (DBFF) in plasma, and the excretion of these compounds in milk were determined by high-performance liquid chromatography. In plasma, the concentrations of TBS, OCZ and DBFF reached maximum at about 1.0 day and the compounds remained detectable until 5.7, 7.4 and 15.1 days after administration, respectively. The detection limits of these compounds in plasma were 10, 2 and 2 ppb, respectively. In milk, the concentrations of TBS, OCZ and DBFF reached maximum at about 24 hours and the compounds remained detectable until 30-47, 30-47 and 78-119 hours after administration, respectively. The detection limits of these compounds in milk were 5.1 and 1 ppb, respectively. The residence times of TBS and BFF were very close to the withdrawal times of the fasciolicides.

Quantitative determination of Closantel residues in milk by high-performance liquid chromatography with fluorescence detection.

A HPLC method with fluorescence detection for quantitative determination of Closantel residues in milk has been developed and validated. The proposed cleaning procedure with acetonitrile and acetone extraction, and solid-phase clean-up with Florisil enables concentrations of Closantel below 50 micrograms/l to be determined. The method was shown to be sufficient, precise, accurate, selective and rugged. The method was applied in the regular monitoring of Closantel residues in milk and of the pharmacokinetic behavior of Closantel in sheep.

Determination of closantel residues in plasma and tissues by high-performance liquid chromatography with fluorescence detection.

The influence of the pH of the mobile phase with some modifiers on the chromatographic behavior and fluorescence properties of closantel have been investigated. At acidic pH values (2-6), the benzamide moiety of the closantel forms a six-membered ring by hydrogen bonding and possesses a native fluorescence. Using the fluorescence emission of closantel at lambda(ex) = 335 nm, lambda(em) = 510 nm, and pH 2.5 of the mobile phase, a linear calibration curve was estimated over a concentration range of about two orders of magnitude with a correlation coefficient larger than 0.992. The limit of the fluorescence detection was 10 microg/kg. This value was at least 10 times lower than that using UV detection. The method was applied to the determination of closantel in plasma and tissue samples, purified by a solid-phase extraction with C18 cartridges.