CH2 N S H H COOH O CONH The Effect of Cooking on Veterinary Drug Residues in Food. Part 8. Benzylpenicillin† Martin D. Rose*, John Bygrave, William H. H. Farrington and George Shearer CSL Food Science Laboratory, Norwich Research Park, Colney, Norwich, UK NR4 7UQ The stability of benzylpenicillin to heat and cooking was studied. Stability of this compound in water (at 100 °C and 65 °C), 5% ethanol, 5% sodium bicarbonate, pH 5.5 buffer at 100 °C and in hot cooking oil at 140 °C and 180 °C was established.Benzylpenicillin was stable at 65 °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. Keywords: Benzylpenicillin; thermal stability; cooking; veterinary drug residues; antibiotic; food Benzylpenicillin (penicillin G) is a widely used antibiotic in both veterinary and human medicine.The structure of this compound is shown in Fig. 1. Concern has been shown over the possible presence of residues of this drug in foods of animal origin due to the occurrence of penicillin hypersensitivity in humans. In veterinary medicine it may be used both to treat specific infections and also as a prophylactic. It is widely used by the dairy industry for the treatment of mastitis in cows.Benzylpenicillin is administered as one or more of a variety of salts which are used to prolong the activity of the drug. These can be the soluble sodium or potassium salts or the longer acting procaine and benzathine salts. The maximum residue limit (MRL) for benzylpenicillin is 4 mg kg21 in milk and 50 mg kg21 in any other edible tissue.1 Both the chemical stability of benzylpenicillin and the microbiological condition of the individual food item needs to be addressed when considering the effects of storage and cooking on this residue.The presence of micro-organisms which produce the enzyme penicillinase could be capable of reducing the concentration of penicillin in food during storage and cooking.2 It has been suggested that observed decreases in the incidence of benzylpenicillin residues in milk in recent years may be attributable to the addition of this enzyme or enzyme producing bacteria to the milk.2 It is of interest that benzylpenicillin is reported to be more stable in UHT treated milk than in raw milk.3 A few studies on the effect of storage and heat treatment of benzylpenicillin residues in food have been carried out.4–7 Most of these4–6 used a microbiological screening assay but one study which used TLC and autoradiochromatography identified degradation products.8 In this study small samples of freeze dried meat soaked in a spiking solution were used.Analysis was carried out on liquid squeezed from the cooked or stored samples. The study did not produce fully quantitative data since only a portion of the radioactivity was recovered. Some decrease in antimicrobial activity was seen for storage of kidney tissue at 4 °C. Cooking could also result in some decrease in activity, although significant activity remained from procaine penicillin with ‘rare’ and ‘medium’ cooking conditions in hamburgers, steaks and pork chops.Only a small percentage of original activity was left after ‘well done’ or ‘exceptionally well done’ cooking. It was shown that the major breakdown product formed during both storage and cooking was the lactate ester of penicilloic acid.8 Antimicrobial activity was reduced in the production of different sausage types with a suggestion that inactivation was due to penicillinase-producing microbes.9 The aim of this study was to use HPLC methodology to produce qualitative and quantitative data about the change in concentration of benzylpenicillin residues in food cooked by a variety of methods.Experimental Benzylpenicillin Heat Stability Experiments to determine heat stability in water were performed at 100 °C and 65 °C. Glass crimp-top autosampler vials (2 ml capacity) were filled with 10 mg ml21 benzylpenicillin solution in water and placed in either a 1 l beaker of boiling water on a hot plate for measurement of stability at 100 °C or in a beaker of water previously equilibrated in a thermostatically controlled air-circulating oven at 65 °C.Samples were removed at intervals, cooled rapidly and analysed by HPLC together with controls of unheated benzylpenicillin solution. To determine heat stability in 5% ethanol, vials were filled with 10 mg ml21 benzylpenicillin in 5% ethanol solution to simulate cooking food with wine or beer. They were placed in a thermostatically controlled air-circulating oven at 100 °C. Samples were removed at intervals and treated as above.To determine heat stability in 5% sodium bicarbonate solution, vials were filled with 10 mg ml21 benzylpenicillin in 5% sodium bicarbonate solution (pH 8.2) and placed in a beaker of boiling water to investigate stability at 100 °C under mildly basic cooking conditions. To determine heat stability in acidic buffer, vials were filled with 10 mg ml21 benzylpenicillin solution in 0.15 m phosphate buffer (pH 5.5) and placed in a beaker of boiling water to investigate stability under acidic conditions.This pH was chosen because it approximates to that of meat.10 Two investigations of heat stability in hot cooking oil were carried out. The first was at 140 °C and the second at 180 °C, a temperature typically used for domestic cooking. Sunflower oil (200 g) was placed in a beaker containing a glass-coated magnetic stirring rod and heated using an electric hot plate. † For Part 7, see ref. 12. Fig. 1 Structure of benzylpenicillin.Analyst, October 1997, Vol. 122 (1095–1099) 1095Temperature was monitored using a mercury in glass thermometer. Benzylpenicillin solution (200 ml) at 1 mg ml21 in methanol was added to give a concentration of 1 mg g21 benzylpenicillin in oil. Sub-samples of at least 5 g were removed at intervals over a period of 2 h. These were placed in 20 ml test tubes which were cooled rapidly by immersion in cold water. Exactly 5 g of oil was mixed with 5 ml hexane and extracted into 5 ml 0.01 m phosphate buffer (pH 8.5).Benzylpenicillin was measured by HPLC together with control standards, prepared by extracting unheated oil spiked at an equivalent concentration. The temperatures achieved during each cooking procedure were monitored using a fibre thermometer. The model used was suitable for temperature measurement between 0–200 °C with an accuracy of 0.1 °C. Samples Benzylpenicillin incurred cattle muscle was obtained from the BBSRC Institute for Animal Health, Compton, Berkshire, UK.The cattle liver was taken from an animal of weight 310 kg dosed by injection with 8.2 ml solution containing 300 mg ml21 procaine benzylpenicillin (Norocillin, Norbrook Laboratories, Kidderminster, UK). This animal was slaughtered 48 h after dosing. The muscle tissue from this animal contained insufficient residues of penicillin to measure. Homogenised muscle tissue from the injection site of a different animal treated for another project was used.This was not an ideal matrix to represent consumer use but since minced beef muscle is used in many standard recipes and this was a source of tissue with a measurable amount of incurred residues it was preferable to the use of tissue fortified with penicillin post mortem. The animal from which this tissue was obtained weighed 100 kg and was treated with a double dose (2 injection sites, left and right rump) of 4 ml solution containing 112.5 mg ml21 benzathine benzylpenicillin and 150 mg ml21 procaine benzylpenicillin.Tissue from these animals was stored at 270 °C until required for use since it has been shown that residues of penicillin are not stable at conventional freezer temperatures. Blank tissues for method validation and quality control samples were obtained from local retail outlets. They were analysed prior to use using the method described below under Analytical Method to verify the absence of benzylpenicillin residues. Cooking Procedure A single piece of tissue was used for each cooking investigation. A portion of raw tissue was removed and analysed in duplicate or triplicate and the remainder was cooked as a single sample.Replicate sub-samples of the cooked tissue were analysed for residues of benzylpenicillin. Samples of water used, or exudate formed during cooking were also analysed. Exact composition of the number of replicates of each sample type was dependent upon the availability of sample, and analytical batch size.All raw incurred samples of a particular tissue type used in the cooking investigations were sub-samples originating from the same animal. Cooking Methods Investigated A variety of cooking methods were used to reflect the wide variation in domestic practise. Specific recipes were as described below. Cattle liver and muscle by frying A slice of cattle liver (156 g) was fried with a little sunflower oil in a pan using an electric hob.The liver was cooked for 20 min, turning occasionally. The cooked liver had a ‘well done’ appearance on the outside but was still pink in the centre. A small amount of homogenised cattle muscle (29 g) was fried in a little sunflower oil on low heat from a gas flame for 10 min. Cattle muscle by boiling, microwave and roasting Water and salt were added to homogenised cattle muscle (73 g) and the mixture was brought to the boil. The mixture was simmered for 13 min. Homogenised cattle muscle (31 g) was cooked for 3 min without the addition of water in a casserole dish with glass lid in a 700 W microwave oven set on medium power.Homogenised cattle muscle (84 g) was cooked in a casserole dish with glass lid in a gas oven, mark 6 (205 °C) for 30 min. No water was added. Cattle liver by braising and casserole Cattle liver (59 g) was fried in a little sunflower oil over a gas flame at low heat for 10 min. Water was added and the mixture brought to the boil and simmered for an additional 10 min.Liver (66 g) was placed in a casserole dish with 60 ml salted water. The sample was cooked at gas mark 7 (220 °C) for 28 min. Analytical Method The analytical method used was based on that developed by Boison et al.11 The method used penicillin V as internal standard for quantification and recovery measurements. This was used at concentrations of 1 mg kg21 for liver experiments and 10 mg kg21 for muscle, to reflect the concentration of the residue in each tissue type.Benzylpenicillin was extracted from tissue with acetone and cleaned up using C18 solid phase extraction (SPE). The eluate from the SPE cartridge was derivatized with 1,2,4-triazole–mercuric chloride and the derivative was subject to reversed-phase HPLC. A schematic diagram of the procedure is shown in Fig. 2. Batches consisted normally of between three and six samples and two blank tissues, one fortified with benzylpenicillin at an appropriate concentration in addition to internal standard.The fortified extract was used as a recovery. A calibration curve was constructed using a range of benzylpenicillin standards prepared in mobile phase covering the concentrations found in the incurred tissue extracts. Results and Discussion Method Validation The method used for the analysis of residues of benzylpenicillin was validated for raw and cooked tissue before use. The validation data obtained for the method are included in Table 1.The method has been previously validated in this laboratory with tissue fortified with 10 mg kg21 benzylpenicillin and the limit of detection is estimated at below 5 mg kg21 which agrees with the figure given by Boison et al. in the original description of the method.11 The method is in routine use in our laboratory and had previously been demonstrated to give a linear response for the range of concentrations measured in this investigation. Heat Stability of Benzylpenicillin Plots of benzylpenicillin concentration against time at 100 °C in various aqueous systems are shown in Fig. 3. At 65 °C benzylpenicillin was stable in water as shown in Fig. 4. The half-life time at 100 °C in water and 5% ethanol was approximately 60 min. This reduced to about 40 min in acidic buffer (pH 5.5) and about 15 min in 5% sodium bicarbonate solution (pH 8.2) at the same temperature. Plots of benzylpen- 1096 Analyst, October 1997, Vol. 122icillin concentration against time in oil at 140 °C and 180 °C are shown in Fig. 5; the approximate half-life times were 45 min and 20 min respectively. This suggested that benzylpenicillin was less stable in an aqueous than in a lipid environment, and indicated that degradation was caused by acid or base catalysed hydrolysis. It was previously established that the likely products from heating benzylpenicillin in simple solution are benzylpenillic acid, isomers of benzylpenicilloic acid, isomers of benzylpenilloic acid and penicillenic acid.8 Effect of Cooking on Residue Concentration The results for the effect of cooking on benzylpenicillin residues in food are all corrected for recovery and are shown in Table 2.The amount of penicillin in food decreased as it was cooked, except for homogenised cattle muscle, by boiling for 10 min. No significant changes in concentration were observed for boiling, when compared with the analytical precision of the method. The boiling experiment was repeated to verify this result because it was not consistent with the results from the other cooking methods.The findings were, however, consistent with the prediction based on the model obtained from heating benzylpenicillin standard in water for the same amount of time. The losses seen for other methods were dependent on cooking time and conditions, the greatest losses observed were for processes which used the highest temperatures and longest times; these were homogenised cattle muscle by frying where a 90% loss was found and cattle liver by braising where a 75% loss was found.The maximum temperature in the frying experiment was higher than any of the other processes at 160 °C for the exterior surfaces and the cooking time was 10 min for muscle and 20 min for liver. Braising involved frying the cattle liver for 10 min where the exterior surface reached a fairly high temperature (up to about 120 °C) followed by simmering for 10 min where the sample was cooked at a constant 100 °C.Variations between replicate analyses for liver were greater than for homogenised muscle probably because of the temperature variation between the surface and interior of the sample. For the cattle liver by casserole experiment a loss of 79% is reported. The concentration in raw tissue was averaged from duplicate measurements of 0.94 and 0.45 mg kg21. The range of the loss Fig. 2 Schematic of analytical procedure.Table 1 Method validation data. Recovery (%) from tissue fortified with benzylpenicillin Cattle muscle Cattle liver Raw Cooked Raw Cooked Day 1/ Day 2/ Day 3/ Day 1/ Day 1/ Day 1/ Sample No. 100 mg kg21 100 mg kg21 100 mg kg21 10 mg kg21 200 mg kg21 1 mg kg21 1 45.2 52.0 59.1 54.9 58.5 46.0 2 49.2 54.2 63.6 72.1 61.8 49.2 3 39.3 52.1 63.3 57.7 58.2 64.7 4 50.8 53.8 55.2 64.8 60.1 54.2 5 38.9 50.9 60.8 61.8 6 65.8 54.4 72.4 Mean (%) 44.7 54.8 59.4 64.0 59.8 53.5 sn21 5.49 5.53 3.94 7.27 1.7 8.2 RSD (%) 12.3 10.1 6.63 11.4 2.9 15.2 n 5 6 6 6 4 4 Fig. 3 Benzylpenicillin stability at 100 °C in water, 5% ethanol, 5% sodium bicarbonate and pH 5.5 buffer. Analyst, October 1997, Vol. 122 1097was between 67% and 85% based on these two starting concentrations. The analytical precision can be assessed from the validation data and the difference between replicate analysis of the homogenised raw muscle tissue. The mean loss was in line with predictions that may have been made on the basis of data obtained from the model systems where drug was heated in simple solutions.The variation in recovery for different cooking methods reflected day to day performance of the method as seen when it is applied to the surveillance of raw tissue samples. The benzylpenicillin which was measured after cooking was found to be distributed between the solid cooked tissue and liquids which were used for cooking or which came from the meat as it was cooked.Sometimes (for casserole and boiling homogenised cattle muscle) over half of the total measured residue was present in the fluid. Distribution of Residues Within the Incurred Tissue No assessment of the distribution of residues within the muscle tissue was possible. This muscle tissue was homogenised prior to cooking, and results from the analysis of raw samples associated with each cooking experiment showed that there was a small decrease in the starting concentration of benzylpenicillin in raw tissue during the course of the work.Samples were kept at 270 °C for long term storage, but were kept at 220 °C for the Fig. 4 Benzylpenicillin stability in water at 65 °C. Table 2 Effect of cooking on benzylpenicillin residues in food Homogenised cattle muscle Cattle liver Fried Microwave Casserole Boiled Boiled Fried Braised Casserole Sample No. 10 min 3 min 30 min 13 min (1) 13 min (2) 20 min 20 min 28 min Recovery (%) 76 76 80 87 100 54 66 51 Raw— 1 13.5 13.5 11.9 12.5 9.9 0.33 0.81 0.94 2 16.2 16.2 10.7 14.8 9.7 0.23 0.82 0.45 3 0.37 Mean/mg kg21 14.9 14.9 11.3 13.7 9.8 0.31 0.82 0.69 Cooked— 1 3.1 8.1 1.9 7.1 4.5 0.25 0.09 0.05 2 2.8 8.3 1.9 6.9 4.7 0.23 0.23 0.13 3 2.7 6.9 2.3 8.1 4.8 0.34 0.08 4 1.8 6.6 4.9 0.07 0.05 5 7.8 0.26 0.04 6 0.22 0.15 Mean/mg kg21 2.8 7.8 2.0 7.3 4.7 0.24 0.20 0.08 RSD (%) 7.2 10.2 11.6 8.6 3.4 51.3 55.8 Fluid— 1 Insufficient Insufficient 1.40 9.22 3.2 Insufficient 0.08 0.08 2 sample sample 1.50 9.22 3.1 sample 0.07 0.10 3 1.41 9.36 3.2 0.10 4 1.40 3.2 0.09 Mean/mg kg21 1.43 9.27 3.2 0.07 0.09 Mass/g— Raw 29.1 31.3 84.4 72.8 50.0 156 58.9 66.1 Cooked 15.0 24.3 94.5 74.5 48.1 129 49.5 50.4 Fluid 0 0 177 90.1 94.0 0 28.1 55.6 Total benzylpenicillin/mg— Raw 434 466 954 997 490 48 48 46 Cooked 43 189 186 544 228 31 9.9 4.5 Fluid — — 253 415 297 — 2 5.2 Net change (%) 290 259 254 24 +7 236 275 279 Fig. 5 Benzylpenicillin stability in cooking oil at 140 °C and 180 °C. 1098 Analyst, October 1997, Vol. 122duration of most of the experimental work which took place over a relatively short (2–3 week) time period. This elevation in temperature was sufficient to affect the stabilty of penicillin residues. It is possible that the process of homogenising raw tissue could have an effect on benzylpenicillin. It was nevertheless preferable to use this tissue which originated from a treated animal than to use only tissue fortified by the addition of drug prior to analysis. Results from experiments carried out on this tissue were in line with results for the liver experiments and with the findings from the experiments conducted on model systems.The analytical precision for cooked liver was much poorer than that expected for analytical error alone and may reflect the difference in temperature between the internal and external parts of the sample during cooking. Conclusions Benzylpenicillin was found to be unstable during cooking in these experiments which were performed on muscle tissue from one animal and liver from a second.Much of the residue migrated with juices which exuded from the tissue as it was cooked. No major metabolites or breakdown products were monitored or identified in this study. The findings supported the losses found using different methodology in a previous investigation where the lactate ester of penicilloic acid was identified as the major product formed in cooked food.8 Surveillance data obtained for measurements of benzylpenicillin in raw tissue may not therefore be directly applicable for use in consumer exposure estimates and dietary intake calculations for the cooked product.Exposure to residues of benzylpenicillin may be reduced by discarding any juices which come from the meat as it is cooked. References 1 EEC, 1992. Council Regulation (EEC) No 675/92 Amending Annexes I and III of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin.(OJ No L 73, 19.3.1992, pp. 8–14). 2 Guay, R., Cardinal, P., Bourassa, C., and Brassard, N., Int. J. Food Microbiol., 1987, 4, 187. 3 Haagsma, N., in Proceedings of the Euroresidue II Conference on Residues of Veterinary Drugs in Food, Veldhoven, Netherlands, 3–5 May 1993, ed. Haagsma, N., Ruiter, A., and Czedic-Eysenberg, P. B., pp. 41–49. 4 Katz, S. E., Fassbender, C.A., DePaolis, A. M., and Rosen, J. D., J. Assoc. Off. Anal. Chem., 1978, 61(3), 564. 5 O’Brien, J. J., Campbell, N., and Conaghan, T., J. Hyg., 1981, 87, 511. 6 Pilet, Ch., and Toma, B., Etude sur la thermostabilit�e de quelques antibiotiques. Rec. Med. Vet., 1969, 145(9), 897. 7 Boison, J. O., Korsrud, G. O., MacNeil, J. D., and Yates, W. D. G., J. AOAC Int., 1992, 75(6), 974. 8 DePaolis, A. M., Katz, S. E., and Rosen, J. D., J. Agric. Food Chem., 1978, 25(5), 1112. 9 Scheibner,G., Monatsh. Veterinaermed., 1972, 27, 161. 10 Price, J. F, and Schweigert, B. S., The science of meat and meat products, Freeman, San Francisco, 2nd edn., 1971. 11 Boison, J. O. K., Salisbury, C. D.C., Chan, W., and MacNeil, J. D., J. AOAC Int., 1991, 74, 497. 12 Rose, M., Farrington, W., and Shearer, G., Food Addit. Contam., in the press. Paper 7/02771K Received April 23, 1997 Accepted July 16, 1997 Analyst, October 1997, Vol. 122 1099 CH2 N S H H COOH O CONH The Effect of Cooking on Veterinary Drug Residues in Food.Part 8. Benzylpenicillin† Martin D. Rose*, John Bygrave, William H. H. Farrington and George Shearer CSL Food Science Laboratory, Norwich Research Park, Colney, Norwich, UK NR4 7UQ The stability of benzylpenicillin to heat and cooking was studied. Stability of this compound in water (at 100 °C and 65 °C), 5% ethanol, 5% sodium bicarbonate, pH 5.5 buffer at 100 °C and in hot cooking oil at 140 °C and 180 °C was established.Benzylpenicillin was stable at 65 °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. Keywords: Benzylpenicillin; thermal stability; cooking; veterinary drug residues; antibiotic; food Benzylpenicillin (penicillin G) is a widely used antibiotic in both veterinary and human medicine.The structure of this compound is shown in Fig. 1. Concern has been shown over the possible presence of residues of this drug in foods of animal origin due to the occurrence of penicillin hypersensitivity in humans. In veterinary medicine it may be used both to treat specific infections and also as a prophylactic. It is widely used by the dairy industry for the treatment of mastitis in cows. Benzylpenicillin is administered as one or more of a variety of salts which are used to prolong the activity of the drug.These can be the soluble sodium or potassium salts or the longer acting procaine and benzathine salts. The maximum residue limit (MRL) for benzylpenicillin is 4 mg kg21 in milk and 50 mg kg21 in any other edible tissue.1 Both the chemical stability of benzylpenicillin and the microbiological condition of the individual food item needs to be addressed when considering the effects of storage and cooking on this residue. The presence of micro-organisms which produce the enzyme penicillinase could be capable of reducing the concentration of penicillin in food during storage and cooking.2 It has been suggested that observed decreases in the incidence of benzylpenicillin residues in milk in recent years may be attributable to the addition of this enzyme or enzyme producing bacteria to the milk.2 It is of interest that benzylpenicillin is reported to be more stable in UHT treated milk than in raw milk.3 A few studies on the effect of storage and heat treatment of benzylpenicillin residues in food have en carried out.4–7 Most of these4–6 used a microbiological screening assay but one study which used TLC and autoradiochromatography identified degradation products.8 In this study small samples of freeze dried meat soaked in a spiking solution were used.Analysis was carried out on liquid squeezed from the cooked or stored samples.The study did not produce fully quantitative data since only a portion of the radioactivity was recovered. Some decrease in antimicrobial activity was seen for storage of kidney tissue at 4 °C. Cooking could also result in some decrease in activity, although significant activity remained from procaine penicillin with ‘rare’ and ‘medium’ cooking conditions in hamburgers, steaks and pork chops. Only a small percentage of original activity was left after ‘well done’ or ‘exceptionally well done’ cooking.It was shown that the major breakdown product formed during both storage and cooking was the lactate ester of penicilloic acid.8 Antimicrobial activity was reduced in the production of different sausage types with a suggestion that inactivation was due to penicillinase-producing microbes.9 The aim of this study was to use HPLC methodology to produce qualitative and quantitative data about the change in concentration of benzylpenicillin residues in food cooked by a variety of methods.Experimental Benzylpenicillin Heat Stability Experiments to determine heat stability in water were performed at 100 °C and 65 °C. Glass crimp-top autosampler vials (2 ml capacity) were filled with 10 mg ml21 benzylpenicillin solution in water and placed in either a 1 l beaker of boiling water on a hot plate for measurement of stability at 100 °C or in a beaker of water previously equilibrated in a thermostatically controlled air-circulating oven at 65 °C.Samples were removed at intervals, cooled rapidly and analysed by HPLC together with controls of unheated benzylpenicillin solution. To determine heat stability in 5% ethanol, vials were filled with 10 mg ml21 benzylpenicillin in 5% ethanol solution to simulate cooking food with wine or beer. They were placed in a thermostatically controlled air-circulating oven at 100 °C. Samples were removed at intervals and treated as above. To determine heat stability in 5% sodium bicarbonate solution, vials were filled with 10 mg ml21 benzylpenicillin in 5% sodium bicarbonate solution (pH 8.2) and placed in a beaker of boiling water to investigate stability at 100 °C under mildly basic cooking conditions.To determine heat stability in acidic buffer, vials were filled with 10 mg ml21 benzylpenicillin solution in 0.15 m phosphate buffer (pH 5.5) and placed in a beaker of boiling water to investigate stability under acidic conditions.This pH was chosen because it approximates to that of meat.10 Two investigations of heat stability in hot cooking oil were carried out. The first was at 140 °C and the second at 180 °C, a temperature typically used for domestic cooking. Sunflower oil (200 g) was placed in a beaker containing a glass-coated magnetic stirring rod and heated using an electric hot plate. † For Part 7, see ref. 12. Fig. 1 Structure of benzylpenicillin. Analyst, October 1997, Vol. 122 (1095–1099) 1095Temperature was monitored using a mercury in glass thermometer. Benzylpenicillin solution (200 ml) at 1 mg ml21 in methanol was added to give a concentration of 1 mg g21 benzylpenicillin in oil. Sub-samples of at least 5 g were removed at intervals over a period of 2 h. These were placed in 20 ml test tubes which were cooled rapidly by immersion in cold water. Exactly 5 g of oil was mixed with 5 ml hexane and extracted into 5 ml 0.01 m phosphate buffer (pH 8.5).Benzylpenicillin was measured by HPLC together with control standards, prepared by extracting unheated oil spiked at an equivalent concentration. The temperatures achieved during each cooking procedure were monitored using a fibre thermometer. The model used was suitable for temperature measurement between 0–200 °C with an accuracy of 0.1 °C. Samples Benzylpenicillin incurred cattle muscle was obtained from the BBSRC Institute for Animal Health, Compton, Berkshire, UK.The cattle liver was taken from an animal of weight 310 kg dosed by injection with 8.2 ml solution containing 300 mg ml21 procaine benzylpenicillin (Norocillin, Norbrook Laboratories, Kidderminster, UK). This animal was slaughtered 48 h after dosing. The muscle tissue from this animal contained insufficient residues of penicillin to measure. Homogenised muscle tissue from the injection site of a different animal treated for another project was used.This was not an ideal matrix to represent consumer use but since minced beef muscle is used in many standard recipes and this was a source of tissue with a measurable amount of incurred residues it was preferable to the use of tissue fortified with penicillin post mortem. The animal from which this tissue was obtained weighed 100 kg and was treated with a double dose (2 injection sites, left and right rump) of 4 ml solution containing 112.5 mg ml21 benzathine benzylpenicillin and 150 mg ml21 procaine benzylpenicillin.Tissue from these animals was stored at 270 °C until required for use since it has been shown that residues of penicillin are not stable at conventional freezer temperatures. Blank tissues for method validation and quality control samples were obtained from local retail outlets. They were analysed prior to use using the method described below under Analytical Method to verify the absence of benzylpenicillin residues. Cooking Procedure A single piece of tissue was used for each cooking investigation.A portion of raw tissue was removed and analysed in duplicate or triplicate and the remainder was cooked as a single sample. Replicate sub-samples of the cooked tissue were analysed for residues of benzylpenicillin. Samples of water used, or exudate formed during cooking were also analysed. Exact composition of the number of replicates of each sample type was dependent upon the availability of sample, and analytical batch size.All raw incurred samples of a particular tissue type used in the cooking investigations were sub-samples originating from the same animal. Cooking Methods Investigated A variety of cooking methods were used to reflect the wide variation in domestic practise. Specific recipes were as described below. Cattle liver and muscle by frying A slice of cattle liver (156 g) was fried with a little sunflower oil in a pan using an electric hob. The liver was cooked for 20 min, turning occasionally.The cooked liver had a ‘well done’ appearance on the outside but was still pink in the centre. A small amount of homogenised cattle muscle (29 g) was fried in a little sunflower oil on low heat from a gas flame for 10 min. Cattle muscle by boiling, microwave and roasting Water and salt were added to homogenised cattle muscle (73 g) and the mixture was brought to the boil. The mixture was simmered for 13 min. Homogenised cattle muscle (31 g) was cooked for 3 min without the addition of water in a casserole dish with glass lid in a 700 W microwave oven set on medium power.Homogenised cattle muscle (84 g) was cooked in a casserole dish with glass lid in a gas oven, mark 6 (205 °C) for 30 min. No water was added. Cattle liver by braising and casserole Cattle liver (59 g) was fried in a little sunflower oil over a gas flame at low heat for 10 min. Water was added and the mixture brought to the boil and simmered for an additional 10 min.Liver (66 g) was placed in a casserole dish with 60 ml salted water. The sample was cooked at gas mark 7 (220 °C) for 28 min. Analytical Method The analytical method used was based on that developed by Boison et al.11 The method used penicillin V as internal standard for quantification and recovery measurements. This was used at concentrations of 1 mg kg21 for liver experiments and 10 mg kg21 for muscle, to reflect the concentration of the residue in each tissue type.Benzylpenicillin was extracted from tissue with acetone and cleaned up using C18 solid phase extraction (SPE). The eluate from the SPE cartridge was derivatized with 1,2,4-triazole–mercuric chloride and the derivative was subject to reversed-phase HPLC. A schematic diagram of the procedure is shown in Fig. 2. Batches consisted normally of between three and six samples and two blank tissues, one fortified with benzylpenicillin at an appropriate concentration in addition to internal standard.The fortified extract was used as a recovery. A calibration curve was constructed using a range of benzylpenicillin standards prepared in mobile phase covering the concentrations found in the incurred tissue extracts. Results and Discussion Method Validation The method used for the analysis of residues of benzylpenicillin was validated for raw and cooked tissue before use. The validation data obtained for the method are included in Table 1. The method has been previously validated in this laboratory with tissue fortified with 10 mg kg21 benzylpenicillin and the limit of detection is estimated at below 5 mg kg21 which agrees with the figure given by Boison et al.in the original description of the method.11 The method is in routine use in our laboratory and had previously been demonstrated to give a linear response for the range of concentrations measured in this investigation. Heat Stability of Benzylpenicillin Plots of benzylpenicillin concentration against time at 100 °C in various aqueous systems are shown in Fig. 3. At 65 °C benzylpenicillin was stable in water as shown in Fig. 4. The half-life time at 100 °C in water and 5% ethanol was approximately 60 min. This reduced to about 40 min in acidic buffer (pH 5.5) and about 15 min in 5% sodium bicarbonate solution (pH 8.2) at the same temperature. Plots of benzylpen- 1096 Analyst, October 1997, Vol. 122icillin concentration against time in oil at 140 °C and 180 °C are shown in Fig. 5; the approximate half-life times were 45 min and 20 min respectively.This suggested that benzylpenicillin was less stable in an aqueous than in a lipid environment, and indicated that degradation was caused by acid or base catalysed hydrolysis. It was previously established that the likely products from heating benzylpenicillin in simple solution are benzylpenillic acid, isomers of benzylpenicilloic acid, isomers of benzylpenilloic acid and penicillenic acid.8 Effect of Cooking on Residue Concentration The results for the effect of cooking on benzylpenicillin residues in food are all corrected for recovery and are shown in Table 2.The amount of penicillin in food decreased as it was cooked, except for homogenised cattle muscle, by boiling for 10 min. No significant changes in concentration were observed for boiling, when compared with the analytical precision of the method. The boiling experiment was repeated to verify this result because it was not consistent with the results from the other cooking methods.The findings were, however, consistent with the prediction based on the model obtained from heating benzylpenicillin standard in water for the same amount of time. The losses seen for other methods were dependent on cooking time and conditions, the greatest losses observed were for processes which used the highest temperatures and longest times; these were homogenised cattle muscle by frying where a 90% loss was found and cattle liver by braising where a 75% loss was found.The maximum temperature in the frying experiment was higher than any of the other processes at 160 °C for the exterior surfaces and the cooking time was 10 min for muscle and 20 min for liver. Braising involved frying the cattle liver for 10 min where the exterior surface reached a fairly high temperature (up to about 120 °C) followed by simmering for 10 min where the sample was cooked at a constant 100 °C.Variations between replicate analyses for liver were greater than for homogenised muscle probably because of the temperature variation between the surface and interior of the sample. For the cattle liver by casserole experiment a loss of 79% is reported. The concentration in raw tissue was averaged from duplicate measurements of 0.94 and 0.45 mg kg21. The range of the loss Fig. 2 Schematic of analytical procedure. Table 1 Method validation data.Recovery (%) from tissue fortified with benzylpenicillin Cattle muscle Cattle liver Raw Cooked Raw Cooked Day 1/ Day 2/ Day 3/ Day 1/ Day 1/ Day 1/ Sample No. 100 mg kg21 100 mg kg21 100 mg kg21 10 mg kg21 200 mg kg21 1 mg kg21 1 45.2 52.0 59.1 54.9 58.5 46.0 2 49.2 54.2 63.6 72.1 61.8 49.2 3 39.3 52.1 63.3 57.7 58.2 64.7 4 50.8 53.8 55.2 64.8 60.1 54.2 5 38.9 50.9 60.8 61.8 6 65.8 54.4 72.4 Mean (%) 44.7 54.8 59.4 64.0 59.8 53.5 sn21 5.49 5.53 3.94 7.27 1.7 8.2 RSD (%) 12.3 10.1 6.63 11.4 2.9 15.2 n 5 6 6 6 4 4 Fig. 3 Benzylpenicillin stability at 100 °C in water, 5% ethanol, 5% sodium bicarbonate and pH 5.5 buffer. Analyst, October 1997, Vol. 122 1097was between 67% and 85% based on these two starting concentrations. The analytical precision can be assessed from the validation data and the difference between replicate analysis of the homogenised raw muscle tissue. The mean loss was in line with predictions that may have been made on the basis of data obtained from the model systems where drug was heated in simple solutions.The variation in recovery for different cooking methods reflected day to day performance of the method as seen when it is applied to the surveillance of raw tissue samples. The benzylpenicillin which was measured after cooking was found to be distributed between the solid cooked tissue and liquids which were used for cooking or which came from the meat as it was cooked. Sometimes (for casserole and boiling homogenised cattle muscle) over half of the total measured residue was present in the fluid.Distribution of Residues Within the Incurred Tissue No assessment of the distribution of residues within the muscle tissue was possible. This muscle tissue was homogenised prior to cooking, and results from the analysis of raw samples associated with each cooking experiment showed that there was a small decrease in the starting concentration of benzylpenicillin in raw tissue during the course of the work.Samples were kept at 270 °C for long term storage, but were kept at 220 °C for the Fig. 4 Benzylpenicillin stability in water at 65 °C. Table 2 Effect of cooking on benzylpenicillin residues in food Homogenised cattle muscle Cattle liver Fried Microwave Casserole Boiled Boiled Fried Braised Casserole Sample No. 10 min 3 min 30 min 13 min (1) 13 min (2) 20 min 20 min 28 min Recovery (%) 76 76 80 87 100 54 66 51 Raw— 1 13.5 13.5 11.9 12.5 9.9 0.33 0.81 0.94 2 16.2 16.2 10.7 14.8 9.7 0.23 0.82 0.45 3 0.37 Mean/mg kg21 14.9 14.9 11.3 13.7 9.8 0.31 0.82 0.69 Cooked— 1 3.1 8.1 1.9 7.1 4.5 0.25 0.09 0.05 2 2.8 8.3 1.9 6.9 4.7 0.23 0.23 0.13 3 2.7 6.9 2.3 8.1 4.8 0.34 0.08 4 1.8 6.6 4.9 0.07 0.05 5 7.8 0.26 0.04 6 0.22 0.15 Mean/mg kg21 2.8 7.8 2.0 7.3 4.7 0.24 0.20 0.08 RSD (%) 7.2 10.2 11.6 8.6 3.4 51.3 55.8 Fluid— 1 Insufficient Insufficient 1.40 9.22 3.2 Insufficient 0.08 0.08 2 sample sample 1.50 9.22 3.1 sample 0.07 0.10 3 1.41 9.36 3.2 0.10 4 1.40 3.2 0.09 Mean/mg kg21 1.43 9.27 3.2 0.07 0.09 Mass/g— Raw 29.1 31.3 84.4 72.8 50.0 156 58.9 66.1 Cooked 15.0 24.3 94.5 74.5 48.1 129 49.5 50.4 Fluid 0 0 177 90.1 94.0 0 28.1 55.6 Total benzylpenicillin/mg— Raw 434 466 954 997 490 48 48 46 Cooked 43 189 186 544 228 31 9.9 4.5 Fluid — — 253 415 297 — 2 5.2 Net change (%) 290 259 254 24 +7 236 275 279 Fig. 5 Benzylpenicillin stability in cooking oil at 140 °C and 180 °C. 1098 Analyst, October 1997, Vol. 122duration of most of the experimental work which took place over a relatively short (2–3 week) time period. This elevation in temperature was sufficient to affect the stabilty of penicillin residues. It is possible that the process of homogenising raw tissue could have an effect on benzylpenicillin. It was nevertheless preferable to use this tissue which originated from a treated animal than to use only tissue fortified by the addition of drug prior to analysis. Results from experiments carried out on this tissue were in line with results for the liver experiments and with the findings from the experiments conducted on model systems.The analytical precision for cooked liver was much poorer than that expected for analytical error alone and may reflect the difference in temperature between the internal and external parts of the sample during cooking. Conclusions Benzylpenicillin was found to be unstable during cooking in these experiments which were performed on muscle tissue from one animal and liver from a second.Much of the residue migrated with juices which exuded from the tissue as it was cooked. No major metabolites or breakdown products were monitored or identified in this study. The findings supported the losses found using different methodology in a previous investigation where the lactate ester of penicilloic acid was identified as the major product formed in cooked food.8 Surveillance data obtained for measurements of benzylpenicillin in raw tissue may not therefore be directly applicable for use in consumer exposure estimates and dietary intake calculations for the cooked product. Exposure to residues of benzylpenicillin may be reduced by discarding any juices which come from the meat as it is cooked. References 1 EEC, 1992. Council Regulation (EEC) No 675/92 Amending Annexes I and III of Council Regulation (EEC) No 2377/90 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. (OJ No L 73, 19.3.1992, pp. 8–14). 2 Guay, R., Cardinal, P., Bourassa, C., and Brassard, N., Int. J. Food Microbiol., 1987, 4, 187. 3 Haagsma, N., in Proceedings of the Euroresidue II Conference on Residues of Veterinary Drugs in Food, Veldhoven, Netherlands, 3–5 May 1993, ed. Haagsma, N., Ruiter, A., and Czedic-Eysenberg, P. B., pp. 41–49. 4 Katz, S. E., Fassbender, C. A., DePaolis, A. M., and Rosen, J. D., J. Assoc. Off. Anal. Chem., 1978, 61(3), 564. 5 O’Brien, J. J., Campbell, N., and Conaghan, T., J. Hyg., 1981, 87, 511. 6 Pilet, Ch., and Toma, B., Etude sur la thermostabilit�e de quelques antibiotiques. Rec. Med. Vet., 1969, 145(9), 897. 7 Boison, J. O., Korsrud, G. O., MacNeil, J. D., and Yates, W. D. G., J. AOAC Int., 1992, 75(6), 974. 8 DePaolis, A. M., Katz, S. E., and Rosen, J. D., J. Agric. Food Chem., 1978, 25(5), 1112. 9 Scheibner,G., Monatsh. Veterinaermed., 1972, 27, 161. 10 Price, J. F, and Schweigert, B. S., The science of meat and meat products, Freeman, San Francisco, 2nd edn., 1971. 11 Boison, J. O. K., Salisbury, C. D.C., Chan, W., and MacNeil, J. D., J. AOAC Int., 1991, 74, 497. 12 Rose, M., Farrington, W., and Shearer, G., Food Addit. Contam., in the press. Paper 7/02771K Received April 23, 1997 Accepted July 16, 1997 Analyst, October 1997, Vol. 122 10