Improved method for the on-line metal chelate affinity chromatography-high-performance liquid chromatographic determination of tetracycline antibiotics in animal products.

An improved on-line metal chelate affinity chromatography-high-performance liquid chromatography (MCAC-HPLC) method for the determination of tetracycline antibiotics in animal tissues and egg has been developed. Extraction was carried out with ethyl acetate. The extract was then evaporated to dryness and reconstituted in methanol prior to on-line MCAC clean-up and HPLC-UV determination. Recoveries of tetracycline, oxytetracycline, demeclocycline and chlortetracycline in the range 42% to 101% were obtained from egg, poultry, fish and venison tissues spiked at 25 micrograms kg-1. Limits of detection less than 10 microgram kg-1 were estimated for all four analytes. This method has higher throughput, higher recovery and lower limits of detection than a previously reported on-line MCAC-HPLC method which involved aqueous extraction and solid-phase extraction clean-up.

Determination of 19-nortestosterone and trenbolone in animal tissues by high-performance liquid chromatography with immunoaffinity clean-up.

A method for the simultaneous determination of residues of 17 beta-trenbolone and 17 beta, 19-nortestosterone and their epimers in animal tissues is described, involving immunoaffinity chromatography clean-up and high-performance liquid chromatography with dual-wavelength UV detection. The method has been validated at 2 micrograms/kg in pig and cattle liver and corned beef with recoveries of 41% upwards. The method has been applied to the determination of incurred residues of 19-nortestosterone and trenbolone. Various alternative extraction steps for incurred trenbolone have been investigated, including direct extraction, protease digestion, heating and ultrasonic probe treatment. Glucuronidase digestion has been shown to be the most effective method for this analyte.

The effect of cooking on veterinary drug residues in food: 7. Ivermectin.

The stability to heat and cooking of ivermectin was investigated. The drug was insufficiently soluble in water to allow the effect of heating in simple aqueous solutions to be studied. The effect of a range of cooking processes was investigated on pig muscle and liver, cattle muscle and liver and salmon muscle. The drug was found to be stable to the effect of cooking. Some leaching of ivermectin with juices as they exuded from the foods as they were cooked was observed; in one case this amounted to about 50% of the total residue.

Production of CTC-containing porcine reference materials.

The European Commission (EC) established the Standards, Measurements and Testing programme for the preparation of Reference Materials (RMs) as an aid to harmonise testing for veterinary drug residues throughout the European Union (EU). The production of chlortetracycline (CTC)-free and CTC-incurred pig tissues as candidate RMs is described. High performance liquid chromatography (HPLC) with fluorescence detection of CTC and 4-epi-CTC was used for all tissue analyses. A pilot study revealed that incurred CTC residues were stable in pig kidney, liver and muscle lyophilised powders during storage for 10 weeks at -70, -20 and +37 degrees C, obviating the need for addition of a stabiliser (thimerosal). In the main study, 500 vials each of CTC-free and CTC-incurred kidney, liver and muscle were produced. Target concentrations in the CTC-incurred lyophilised tissue powders were 750-1500, 500-1000 and 300-600 micrograms kg-1 for kidney, liver and muscle, respectively. Following lyophilisation, the mean +/- s concentrations of CTC in the incurred positive RMs were 1,315 +/- 56.9, 765 +/- 35.3 and 378 +/- 16.8 micrograms kg-1 for kidney, liver and muscle respectively. Residual moisture in the RMs ranged from 1.6 +/- 0.53% for muscle to 3.0 +/- 0.50% for liver. Between-vial homogeneity for incurred powders was determined for 20 vials of each material, which had been removed at regular intervals during the filling process. Relative standard deviations (RSDs) for kidney, liver and muscle were 4.3, 4.6 and 4.4% respectively, being within the interassay RSD of the method and indicating that mixing was effective. Stability of powders stored at -18, 4, 20 and 37 degrees C was assessed over a period of 79 weeks. No measurable degradation occurred over this time period at any of the storage temperatures. It is concluded that these candidate RMs are homogenous, stable and are suitable for certification.

Screening and confirmation of triphenylmethane dyes and their leuco metabolites in trout muscle using HPLC-vis and ESP-LC-MS.

An extraction and clean-up protocol for the determination of Malachite Green and Crystal Violet and the corresponding leuco compounds in trout muscle has been developed. Final determination is by HPLC with visible (screening) or ESP-MS (confirmation) detection. In both cases lead(IV) oxide was used on-line to oxidise the leuco compounds back to the parent after chromatographic separation and prior to detection. The procedure was validated down to 2 micrograms kg-1. Intra- and inter-batch precision was measured at 3 levels for all compounds. Recoveries were in the range 66-116% with RSD of 1-17% for determination by HPLC with visible detection. For LC-MS determination, recoveries were in the range 61-94% with RSD of 4-15%. Limited surveillance data indicated that Malachite Green usage was more effectively monitored by including the leuco compound as well as the parent (9 positives for the leuco compound as opposed to 1 for Malachite Green out of 31 samples analysed).

Aspects of extraction, spiking and distribution in the determination of incurred residues of chloramphenicol in animal tissues.

Studies of distribution, extraction procedures and spiking protocols in the determination of incurred chloramphenicol residues in animal tissues have been carried out. An extraction procedure involving glucuronidase enzyme digestion was found to extract 10 times more incurred chloramphenicol from pig kidney than direct extraction without digestion. However, neither protease digestion nor ultrasonic probe treatment resulted in improved chloramphenicol extraction. Chloramphenicol was found to be inhomogeneously distributed within kidney from a treated pig. Highest concentrations were detected in the renal medulla. Muscle tissues from the same animal were found to contain a lower concentration of chloramphenicol residues, but no chloramphenicol residues were detectable in the liver. Chloramphenicol recovery from spiked pig liver was found to be lower than that from kidney, but was improved by the addition of piperonyl butoxide before extraction. This additive had no effect on recovery from spiked pig or cattle kidney. The implications of these results for regulatory surveillance of animal tissue for chloramphenicol residues are discussed.

Effects of extraction and spiking procedures on the determination of incurred residues of oxytetracycline in cattle kidney.

The effects of different extraction and spiking procedures on the determination of incurred oxytetracycline residues in animal tissues have been investigated. The extraction procedures investigated--direct aqueous or organic solvent extraction, enzymic digestion or sonication--all gave similar results for incurred oxytetracycline concentration in cattle kidney after correction for spike recovery. There was therefore no evidence for binding or conjugation of oxytetracycline in this tissue. Highest recovery from spiked tissue was obtained using ethyl acetate as extractant. The effects of spiking procedure (spike contact time, spike solvent and tissue state) on recovery from spiked cattle kidney were also small, indicating that added oxytetracycline spike does not interact with the tissue.

Determination of penicillins in animal tissues at trace residue concentrations: II. Determination of amoxicillin and ampicillin in liver and muscle using cation exchange and porous graphitic carbon solid phase extraction and high-performance liquid chromatography.

A method for the determination of the basic penicillins, amoxicillin and ampicillin, in animal tissue is described. The method used aqueous extraction with tungstate to remove proteins followed by cation exchange solid phase extraction (SPE) clean-up. This extract was further purified using porous graphitic carbon (PGC) SPE. Extracted residues were derivatized with acetic anhydride followed by triazole/mercuric chloride prior to reversed phase HPLC with UV determination at 325 nm. At the Maximum Residue Limit of 50 micrograms/kg in fortified cattle muscle validation gave mean recoveries for amoxicillin of 57% and 50% on two different days with relative standard deviations of 19% and 15% respectively. For ampicillin the recoveries were 72% and 59% with relative standard deviations of 15% and 19% respectively.

The effect of cooking on veterinary drug residues in food; 5. Oxfendazole.

The heat stability of the anthelmintic oxfendazole in water, cooking oil and as incurred residues in cattle liver was investigated. Some evidence of instability was found in boiling water after 3 h. This degradation was associated with the formation of a product which was identified by mass spectrometry as the amine formed by hydrolysis of the carbamate functional group on the oxfendazole molecule. In hot cooking oil at 150 degrees C and 180 degrees C the half-life of oxfendazole was 15 min and 6 min respectively. The amine was also shown to form in oil as the concentration of oxfendazole depleted. The effects of cooking on incurred residues of oxfendazole in cattle liver were inconclusive from this study. Several variable factors were found to be in place including: an unstable equilibrium between oxfendazole, oxfendazole sulphone and fenbendazole in incurred tissue; the overall instability of these compounds in tissue during frozen storage for the duration of the project; the distribution of the residues within the tissue used for the study and the effect of protein binding on extractability of residues from the tissue. It was nevertheless found that: (i) cooking did not destroy residues although it may affect the point of equilibrium between oxfendazole, oxfendazole sulphone, fenbendazole and some other metabolites in incurred tissue; (ii) the amine derivative was not observed in raw incurred tissue; (iii) residues were not evenly distributed in raw incurred tissue; (iv) storage time affected the measured residue concentration either because of losses or protein binding or a combination of both.

The effect of cooking on veterinary drug residues in food. Part 8. Benzylpenicillin.

The stability of benzylpenicillin to heat and cooking was studied. Stability of this compound in water (at 100 degrees C and 65 degrees C), 5% ethanol, 5% sodium bicarbonate, pH 5.5 buffer at 100 degrees C and in hot cooking oil at 140 degrees C and 180 degrees C was established. Benzylpenicillin was stable at 65 degrees C but not stable at higher temperatures with half-life times varying between 15 and 60 min in the solutions investigated. This drug was not stable to cooking, losses being proportional to the harshness of the cooking regime. Where fluids were released during the cooking process, sometimes over half of the residue passed from the solid tissue into the cooking medium.

The effect of cooking on veterinary drug residues in food: 4. Oxytetracycline.

The heat stability of oxytetracycline (OTC) in water and vegetable oil was investigated. Results showed that the drug was unstable in water at 100 degrees C with a half-life of about 2 min, but more stable in oil at 180 degrees C where the half-life was about 8 min. The effect of a range of cooking processes including microwaving, boiling, roasting, grilling, braising and frying on OTC residues in incurred animal tissues was investigated. Substantial net reductions in OTC of 35-94% were observed, with temperature during cooking having the largest impact on the loss. Migration from the tissue into the surrounding liquid or meat juices was observed during the cooking processes. Diode-array analysis of heat-treated OTC standard solutions indicated that no individual closely related compound such as 4-epioxytetracycline, alpha- or beta-apooxytetracycline formed a significant proportion of the breakdown products. OTC was not evenly distributed throughout the tissue, but the effects of this were minimized by selecting adjacent samples for cooking and for the raw control. The findings of this investigation showed that the effect of cooking on residues of OTC should be considered before data obtained from measurements on raw tissue are used for consumer exposure estimates and dietary intake calculations.

The effect of cooking on veterinary drug residues in food: 3. Sulphamethazine (sulphadimidine).

The heat stability of sulphamethazine was investigated. The drug was shown to be stable in boiling water at 100 degrees C. In cooking oil at 260 degrees C, losses were observed, indicating a half-life of about 5 min. At 180 degrees C in cooking oil, sulphamethazine was unstable with a half-life of about 2 h. The effect of a range of cooking processes (boiling, roasting, grilling, frying, pressure cooking and microwaving) on sulphamethazine residues in incurred animal tissue was studied. Once allowance for weight loss during cooking had been made no net unaccountable change in concentration of sulphamethazine was observed in any of the cooking processes investigated. During frozen storage, sulphamethazine residues were found to be stable over a period of 3 months. It was found during this investigation that the method used for analysis which involved acid extraction converted the N4-metabolites to parent sulphamethazine, and hence only sulphamethazine was measured. Sulphamethazine was found to be evenly distributed in the raw incurred tissue used for analysis. Migration from the tissue into the surrounding liquid or meat juices was observed during the cooking process. The findings of this investigation show that surveillance data obtained from measurements on raw tissue are applicable for use in consumer exposure and dietary intake calculations, but only if an acidic extraction method which converts the N4-metabolites to parent sulphamethazine is used for the surveillance. This may not however conform to current Maximum Residue Limit legislation which refers to total parent sulphonamides. Different methods of analysis for sulphonamides are likely to give rise to inter-laboratory variation.

Development of multi-residue methodology for the HPLC determination of veterinary drugs in animal tissues.

The application of solid-phase extraction and HPLC with UV-diode array detection to the multi-residue determination of veterinary drugs is described. A two-stage SPE clean-up was employed, using C18 and silica cartridges. HPLC analysis was carried out on a base-deactivated C8 column using gradient elution systems at two pH values. The procedure establishes the basis of a method for routine screening of pig kidney samples for some sulphonamides, benzimidazoles, nitroimidazoles and nitrofurans at concentrations at or below the UK Maximum Residue Limits (MRLs). Limits of detection of 2-18 micrograms/kg could be achieved for these analytes at recoveries of 40-70%. UV spectra measured on-line were used for confirmation of peak identities at these concentrations. The possibility of extension of this procedure to a wider range of analyses is discussed.

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.

The effect of cooking on veterinary drug residues in food: 1. Clenbuterol.

The heat stability of clenbuterol was investigated. The drug was shown to be stable in boiling water at 100 degrees C. In cooking oil at 260 degrees C, losses were observed, indicating a half-life of about 5 min. The effect of a range of cooking processes (boiling, roasting, frying, microwaving) on clenbuterol residues in fortified and incurred tissue was studied. No net change in the amount of clenbuterol was observed in any of the cooking processes investigated except for deep frying using extreme conditions. There was little observed migration from the tissue into the surrounding liquid or meat juices. Clenbuterol residues were found not to be evenly distributed in the incurred raw tissue used for the investigation. The findings of this investigation show that data obtained from measurements on raw tissue are applicable for use in consumer exposure estimates and dietary intake calculations.

Analysis of trace residues of tetracyclines in animal tissues and fluids using metal chelate affinity chromatography/HPLC.

A novel method of analysis for the trace residue determination of tetracyclines in animal tissues and fluids has been developed. Clean-up of sample extracts is based upon the specific ability of tetracyclines to chelate with divalent metal ions (metal chelate affinity chromatography, MCAC) and determination made by high-performance liquid chromatography. The method has been tested for the determination of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) in porcine kidney and muscle, ovine kidney, bovine kidney and milk, and trout muscle. Recoveries at the 0.05 mg/kg level for OTC, TC and CTC respectively were 75%, 63%, 73% in porcine kidney, 77%, 79%, 76% in porcine muscle, 85%, 54%, 53% in bovine kidney, 78%, 63%, 57% in ovine kidney, 75%, 58%, 56% in fish (trout) muscle, and 80%, 59%, 59% in bovine milk. At this level both within- and between-batch precision, as measured by the coefficient of variation (CV), was less than 10%. Determination to the 0.01 mg/kg level was carried out in all cases, although the method becomes less precise. The method has been used for several months and found to be both reliable and sufficiently rapid for use as a routine quantitative screening procedure. When coupled with liquid chromatography-mass spectrometry (LC-MS) it is suitable for use as a confirmatory method. Analysis of animals treated with tetracyclines has been carried out.

A method for the analysis of lincomycin in porcine and bovine kidney.

A method of analysis has been developed for the estimation of lincomycin in porcine and bovine kidney. The method employed high performance liquid chromatography and Sep-Pak clean-up of methanolic tissue extracts and nitrogen specific gas chromatographic detection. Using spiked (0.1 mg kg-1) extracts recoveries in the range 40-50% were obtained. Analysis at the 0.05 mg kg-1 level is possible. Fifty four samples of kidney destined for UK sale were analysed for the presence of this drug. No samples were found to contain lincomycin.