Identification of hormone esters in injection site in muscle tissues by LC/MS/MS.

The detection of hormone abuse for growth promotion in food animal production is a global concern. Initial testing for hormones in Canada was directed at the compounds approved for use in beef cattle, melengestrol acetate, trenbolone acetate and zeranol, and the banned compound diethylstilbestrol (DES). No hormonal growth promoters are approved for use in veal production in Canada. However, instances of use of trenbolone and clenbuterol were detected in Canada in the 1990s. During the development of a new analytical method for testosterone and progesterone, there were reports of suspicious injection sites being found in veal calves. Upon implementation of the method, analysis of investigative samples revealed significant residues of testosterone in some injection sites. To prove that the source of these residues was exogenous, a fully validated method for hormone esters was developed to confirm the presence of exogenous hormones in these injection sites. The QUECHERS model was employed in methods development and resulted in a simple, effective extraction technique that consisted of sample pre-homogenization, liquid/liquid partitioning, extract dilution, filtration and use of LC/MS/MS to provide detection selectivity. The result was an adaptable MS/MS confirmation technique that meets the needs of Canadian regulatory authorities to confirm the misuse of injectable testosterone, and potentially other hormones, in food animal production.

Determination of neomycin in animal tissues by liquid chromatography.

Tissue samples are digested under hot alkaline conditions after initial conditioning at room temperature with phosphate-buffered saline. The cooled digest is deproteinated with concentrated perchloric acid. After centrifugation and pH adjustment, the clear supernatant is applied to an ion-exchange cartridge, and after the cartridge is washed, the neomycin is eluted with dilute perchloric acid. This eluate is derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a wide-pore spherical silica C4 column and fluorescence detection. Recovery and repeatability are calculated from tissue extract standard calibration curves produced from the same assay. Recoveries ranged from 80 to 120% for fortifications of 0.25-1.00 mg/kg for muscle tissue and from 80 to 100% for fortifications of 0.50-10.0 mg/kg for kidney tissue. Limits of quantitation were 0.25 and 0.50 mg/kg, respectively, for muscle and kidney tissues. Limits of detection were 0.125 and 0.20 mg/kg, respectively, for muscle and kidney tissues.

Depletion of penicillin G residues in tissues, plasma and injection sites of market pigs injected intramuscularly with procaine penicillin G.

Procaine penicillin G was administered by intramuscular (i.m.) injection to groups of healthy 100 kg market pigs at the approved label dose (15,000 IU/kg body weight), once daily for three consecutive days; or an extra-label dose (66,000 IU/kg body weight), once daily for five consecutive days. Penicillin G residue depletion was followed in plasma, tissue and injection sites using a liquid chromatographic method. Groups of pigs were killed 1, 2, 3, 4, 5 and 8 days after the last injection with the label dose. Penicillin G was not detected in liver after 1 day of withdrawal, in muscle and fat after 2 days of withdrawal, in plasma after 4 days of withdrawal, in skin after 5 days of withdrawal, or in kidney and the injection sites after 8 days of withdrawal. Other groups of pigs were killed 1, 2, 3, 5 and 7 days after injection with the extra-label dose. In these pigs penicillin G was not found in liver after 2 days of withdrawal, in fat after 3 days of withdrawal, or in the muscle, skin, plasma and injection sites after 7 days of withdrawal. Penicillin G was found at all times in the kidneys of the groups of pigs that received the high dose. The technique used for neck injections was critical to obtain intramuscular rather than intermuscular injections. The Bureau of Veterinary Drugs, Health Protection Branch, Health Canada calculated that the appropriate withdrawal period for pigs was 8 days for a dose of 15,000 IU procaine penicillin G/kg body weight and 15 days for a dose of 66,000 IU/kg.

Bacterial inhibition tests used to screen for antimicrobial veterinary drug residues in slaughtered animals.

Bacterial inhibition tests used to screen milk, tissues, blood, and urine for antimicrobial veterinary drug residues must be high volume, quick, rugged, inexpensive, and sensitive. Bacterial inhibition tests--such as the Swab Test on Premises (STOP), the Calf Antibiotic and Sulfa Test (CAST), the Fast Antibiotic Screen Test (FAST), the Charm Farm Test (CFT), the Antimicrobial Inhibition Monitor 96 (AIM-96) assay, the German Three Plate Test, the European Union Four Plate Test and the New Dutch Kidney Test--have been used to screen tissues for antimicrobial activity. The CFT and the Brilliant Black Reduction Test (BBRT) also have been used to screen plasma. The Live Animal Swab Test (LAST) was developed to screen urine. This review examines the use and limitations of these screening tests for regulatory control and avoidance of veterinary drug residues in meat. The ideal bacterial inhibition test for screening antimicrobial residues in slaughtered animals does not exist. Each of the current and potential tests has limitations.

Residue depletion in tissues and fluids from swine fed sulfamethazine, chlortetracycline and penicillin G in combination.

Twenty-four hogs were fed a ration for 14 days containing three times the recommended label dose of a combination drug which included sulfamethazine, chlortetracycline and penicillin G. Groups of six hogs were slaughtered 0, 2, 4, or 8 days after withdrawal. Six untreated control hogs were slaughtered 5 days before the first group, of six treated hogs, were slaughtered. Residue concentrations were determined in kidney, liver, muscle, serum and urine. At zero withdrawal the kidney from one hog contained 0.018 mg penicillin G per kg and the serum from the same hog contained 0.016 mg penicillin G per litre. Penicillin G was not detected in any other samples that were analysed. Chlortetracycline concentrations in tissues at zero withdrawal time were below accepted Canadian Maximum Residue Limits (MRL) for chlortetracycline of 1 mg/kg in muscle, 2 mg/kg in liver and 4 mg/kg in kidney and were below the limit of quantitation in all tissues 4 days after withdrawal. Sulfamethazine persisted in the tissues longer than penicillin G or chlortetracycline. Sulfamethazine concentrations were above the Canadian MRL of 0.1 mg/kg at zero withdrawal time and did not decrease to below the MRL until 8 days after withdrawal. Our results suggest that, if the label withdrawal period of 10 days is observed, an increase in the dosage of up to three times the recommended rate is unlikely to increase significantly the risk that residues would occur in the tissues of treated hogs at concentrations which exceed MRLs. Sulfamethazine concentrations in all matrices decreased after storage at -76 degrees C for 6 months.

Chlortetracycline, oxytetracycline, and tetracycline in edible animal tissues, liquid chromatographic method: collaborative study.

Thirteen laboratories analyzed samples of edible animal tissues for tetracycline residues. The method included extraction of analytes into buffer, elution from a C18 solid-phase extraction (SPE) cartridge, and reversed-phase liquid chromatographic (LC) analysis, including use of a confirmation column. An additional laboratory, using an alternative LC assay based on a different sample cleanup, also analyzed the samples. Results showed the 2 methods are comparable. The LC method for determination of cholortetracycline, oxytetracycline, and tetracycline in edible animal tissues has been adopted by AOAC INTERNATIONAL. Results from 13 laboratories indicate that the method under study provides generally better results at the higher concentrations tested than at concentrations near the detection limit and that there is less problem with interferences in muscle tissue than in kidney. The method can achieve reliable results for analytes and matrixes studied at concentrations from 0.1 to 0.6 ppm and above, depending on the analyte-matrix combination, with generally better performance to be expected with muscle than with kidney. The poorer performance for fortified samples, particularly kidney, was attributed to additional homogenization steps required to prepare these samples. Recovery of analytes from different lots of solid-phase extraction (SPE) cartridges was an important variable.

Long-term trends and sources of organochlorine contamination in Canadian tundra Peregrine Falcons, Falco peregrinus tundrius.

Levels of eggshell thinning, and organochlorine residues in egg contents, blood plasma of adults and juveniles, tissue samples, and prey species were determined for a population of migratory Peregrine Falcons (Falco peregrinus tundrius) breeding in the Canadian Arctic. Temporal trends were assessed by comparing data collected during 1991-1994, with data from 1982-1986, for the same population. Shells (n=54) from 1991-1994 averaged 15% thinner than eggs produced prior to the introduction of DDT. No improvement in shell thickness was detected between decades. Mean DDE residue levels in eggs showed a decline from 7.6 mg kg (1982-1986) to 4.5 mg kg (1991-1994), but there was no significant change in SigmaPCB residues. Moreover, the proportion of clutches with eggs exceeding critical SigmaPCB, DDE, and dieldrin residue levels (10%) did not change between decades. Relative to Greenland and Alaskan populations, F. p. tundrius at Rankin Inlet show high levels of organochlorine contamination and little reduction in residues over the last decade. These Tundra Peregrines continue to be exposed to organochlorines in Latin America; however, results also link relatively high levels in the study population with waterfowl species that do not leave Canada in winter.

Comparison of four commercially available rapid test kits with liquid chromatography for detecting penicillin G residues in bovine plasma.

Four commercially available rapid tests (Brilliant Black reduction test, LacTek test, Charm Farm test, and Charm Test II receptor assay) were compared with a liquid chromatographic (LC) method (lowest quantitatable level of 5 ng/mL) in their efficiency, reliability, and sensitivity to detect penicillin G in bovine plasma. Samples were obtained from 16 steers treated with procaine penicillin G alone or in combination with its long-acting form, benzathine penicillin G. The steers were injected intramuscularly with penicillin G doses ranging from label dose to about 9 times label dose. When results of the Brilliant Black reduction, LacTek, Charm Test II, and Charm Farm tests for penicillin G in plasma (with detection sensitivities of 5, 10, 20, and 30 ng/mL, respectively) were compared with results of LC, none of the rapid tests gave false-positive results. Each rapid test elicited a positive response when used to test bovine plasma containing penicillin G residues at concentrations above the test's detection sensitivity. The simplicity, selectivity, and sensitivity of the rapid tests, coupled with rapidity with which results are obtained, make them suitable for use in large-volume preslaughter screening of penicillin-treated cattle.

Investigation of Charm Test II receptor assays for the detection of antimicrobial residues in suspect meat samples.

Charm Test II receptor assays for beta-lactams, sulfonamides, (dihydro)streptomycin and erythromycin were applied to 257 bovine muscle and kidney samples, and 215 porcine muscle and kidney samples collected from animals suspected to contain antimicrobial residues. The assays were run in conjunction with Agriculture and Agri-food Canada's routine diagnostic confirmation analyses for suspect samples collected at federally inspected packing plants. All samples were subjected to the Charm Test II receptor assays and thin layer chromatography-bioautography (TLC-BA). Selected samples were quantitatively analysed using a liquid chromatographic method for penicillin G and a thin layer chromatography-fluorescence densitometry (TLC-FD) method for sulfonamides. The Charm Test II assays for beta-lactams, (dihydro)streptomycin and erythromycin were an acceptable alternative to the TLC-BA screen for laboratory confirmation of the presence of these compounds, with enhanced sensitivity for (dihydro)streptomycin and erythromycin. In addition, the Charm Test II provided a sensitive screen for sulfonamides as confirmed by the standard TLC-FD procedure. The analysis time, laboratory space and analyst time required to complete the Charm Test II assays is less than that for TLC-BA. Operating costs are similar for both analyses, but the Charm Test II does require capital expenditure for a scintillation counter.

Disposition of penicillin G after administration of benzathine penicillin G, or a combination of benzathine penicillin G and procaine penicillin G in cattle.

Plasma concentration of penicillin G was evaluated in beef steers after administration of either a combination of benzathine penicillin G and procaine penicillin G in a 1:1 mixture at a dosage of 9,000 U/kg of body weight, IM (n = 5), 24,000 U/kg, IM (n = 5), or 8,800 U/kg, SC (n = 5), or benzathine penicillin G alone at a dosage of 12,000 U/kg, IM (n = 7). Plasma concentration of penicillin G was measured by use of a high-performance liquid chromatography assay that had a limit of determination of 0.005 microgram/ml. At a dosage for this combination of 9,000 U/kg IM, and 8,800 U/kg, SC, which are approved label recommendations in Canada, and the United States, respectively, mean (+/- SEM) peak plasma concentration was 0.58 (+/- 0.15) and 0.44 (+/- 0.02) microgram/ml, respectively. Although plasma penicillin concentration was quantifiable for 7 days in the steers that received 9,000 U/kg, IM, and for 4 days in the steers that received 8,800 U/kg, SC, the concentration was < 0.1 microgram/ml in both groups after the first 12 hours. After administration of the combination at dosage of 24,000 U/kg, IM, there was an initial peak plasma concentration at approximately 2 hours; thereafter, plasma concentration decreased slowly, with half-life of 58 hours. Although plasma penicillin G concentration was quantifiable for 12 days at this dosage, concentration was < 0.1 microgram/ml after the first 48 hours. After the initial 48 hours, plasma concentration of penicillin was of similar magnitude and decreased at similar rate for the combination at dosage of 24,000 U/kg and for 12,000 U/kg of benzathine penicillin G alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of penicillin G in milk by liquid chromatography.

A liquid chromatographic (LC) method that was previously developed for penicillin G residues in animal tissues has been adapted to milk and milk products. After protein precipitation with sodium tungstate, samples are applied to a C18 solid-phase extraction cartridge, from which penicillin is eluted, derivatized with 1,2,4-triazole-mercuric chloride solution, and analyzed by isocratic liquid chromatography (LC) on a C18 column with UV detection at 325 nm. Quantitation is done with reference to penicillin V as an internal standard. Penicillin G recoveries were determined to be > 70% on standards fortified at 3-60 ppb. Accuracy approached 100% using the penicillin V internal standard. The detection limit for penicillin G residues was 3 ppb in fluid milk. Samples may be confirmed by thermospray/LC at concentrations approaching the detection limit of the UV method.

Analysis of streptomycin and dihydrostreptomycin in milk by liquid chromatography.

A method developed for the determination of the aminoglycoside antibiotics streptomycin and dihydrostreptomycin in tissues was applied to the analysis of fluid milk. Samples are extracted with 3.6% perchloric acid, and then injected onto a trace enrichment column, from which they are eluted onto a reversed-phase analytical column. The analytes are detected by fluorescence following postcolumn derivatization with 1,2-naphthoquinone-4-sulfonic acid. Recovery of analytes was in the range of 50-65% for skim or partially defatted fluid milk, while recoveries for homogenized whole milk were lower. Limits of quantitation were 10 ppb for streptomycin and 20 ppb for dihydrostreptomycin.

Determination of streptomycin and dihydrostreptomycin in animal tissue by on-line sample enrichment liquid chromatography.

A method for the determination of streptomycin and dihydrostreptomycin in pork and bovine muscle and kidney was developed. Dilute perchloric acid solution is used to precipitate proteins and extract the analytes from the tissue. The extract is loaded onto a cation-exchange, solid-phase extraction column, and the drugs are eluted with pH 8 phosphate buffer. The eluant is chromatographed by using an on-line column enrichment liquid chromatographic system with postcolumn derivatization using 1,2-naphthoquinone-4-sulfonic acid and detection by fluorescence. The recoveries were 61.1% (coefficient of variation [CV], 7.3%) for streptomycin and 55.3% (CV, 8.2%) for dihydrostreptomycin. The detection limits were 10 ppb for streptomycin and 20 ppb for dihydrostreptomycin.

Depletion of penicillin G residues in tissues and injection sites of yearling beef steers dosed with benzathine penicillin G alone or in combination with procaine penicillin G.

The contribution of benzathine penicillin G to residues in tissues and injection sites of yearling beef steers was assessed by treating seven groups of five to seven steers with either benzathine and procaine penicillin G together or benzathine penicillin G alone. Steers were injected with a commercial combination of benzathine and procaine penicillin G according to the Canadian (intramuscular) or United States (subcutaneous) label dosages of 8600 and 8800 IU penicillin G/kg body weight, respectively. They were killed 14 or 30 days after the intramuscular injections, and 30 days after the subcutaneous injections. At the label withdrawal times, Canadian 14 days and United States 30 days, the levels in the injection sites for all of the treatments were 30-60 times above the Canadian and United States' Maximum Residue Limit of 50 micrograms/kg, while liver, kidney and gluteal muscle levels were below the Maximum Residue Limit. Other steers were injected intramuscularly with 24,000 IU benzathine/procaine penicillin G/kg body weight and slaughtered 8, 14 or 50 days after injection. Fifty-day injection site residues were 24 times the Maximum Residue Limit. Another group of steers was injected intramuscularly with benzathine penicillin G alone at 12,000 IU/kg body weight and slaughtered 14 days later. Penicillin G levels in the injection sites were 156 times the Maximum Residue Limit. The persistence of penicillin G residues at the injection sites in all the treatment groups appears to be attributable primarily to benzathine penicillin G. Visual inspection of muscle surfaces did not reliably reveal all injection site lesions in the underlying musculature.

Depletion of intramuscularly and subcutaneously injected procaine penicillin G from tissues and plasma of yearling beef steers.

Withdrawal periods required when doses of 24,000 IU and 66,000 IU of procaine penicillin G/kg body weight were administered to yearling beef steers by intramuscular injection daily for five consecutive days were investigated. These dosages are in excess of product label recommendations, but are in the range of procaine penicillin G dosages that have been administered for the treatment of some feedlot bacterial diseases. The approved dose in Canada is 7,500 IU/kg body weight intramuscularly, once daily, with a withdrawal period of five days. Based on the tissue residue data from this study, the appropriate withdrawal period is ten days for the 24,000 IU/kg body weight dose and 21 days for the 66,000 IU/kg body weight dose when administered intramuscularly to yearling beef steers. In a related study, 18 yearling beef steers received 66,000 IU of procaine penicillin G/kg body weight administered by subcutaneous injection, an extra-label treatment in terms of both dose and route of administration, typical of current practice in some circumstances. Deposits of the drug were visible at subcutaneous injection sites up to ten days after injection, with more inflammation and hemorrhage observed than for intramuscular injections of the same dose. These results suggest that procaine penicillin G should not be administered subcutaneously at high doses; and therefore a withdrawal period was not established for subcutaneous injection.

A study of the disposition of procaine penicillin G in feedlot steers following intramuscular and subcutaneous injection.

The disposition of an aqueous suspension of procaine penicillin G (300,000 U/mL) was studied in feedlot steers. Four groups of three steers were used. Steers in groups 1 and 2 received procaine penicillin G once daily for 5 days intramuscularly (i.m.) at a dose of 24,000 U/kg (group 1) or of 66,000 U/kg (group 2). The injection on the last day was administered in the gluteal muscle. Steers in group 3 (i.m. neck injection) and group 4 [subcutaneous (s.c.) injection] each received a single dose of procaine penicillin G at a dose of 66,000 U/kg. From every animal, after the last injection in groups 1 and 2 and following the single injection in groups 3 and 4, a series of blood samples was taken at fixed time intervals. The plasma from these samples was analysed for penicillin G by a high performance liquid chromatography (HPLC) assay in order to determine the disposition of penicillin. The maximum plasma concentration (Cmax) and the area under the curve (AUC) were significantly different between groups 1 and 2, but we found no difference in the disappearance rate constant between these two groups. Group 4 single s.c. injections produced a lower mean Cmax (1.85 +/- 0.27 microgram/mL) than the mean Cmax (4.24 +/- 1.08 micrograms/mL) produced in group 3 by i.m. injections into the neck muscle or the mean Cmax (2.63 +/- 0.27 microgram/mL) produced in group 2 by i.m. injections into the gluteal muscle. However the mean Cmax produced by i.m. injections into the neck muscles (group 3) was higher than the mean Cmax produced by i.m. injections into the gluteal muscle (group 2). Additionally, the disappearance t1/2 was longer (18.08 h) in group 4 following the s.c. injection and shorter (8.85 h) in group 3 following the i.m. neck injection, than the t1/2 following administration of the same dose i.m. into the gluteal muscle (15.96 h) in group 2. In this study, when procaine penicillin G was injected into the gluteal muscle, doses of 66,000 U/kg were necessary to produce plasma concentrations that were above a minimum inhibitory concentration (MIC) for penicillin G of 1.0 microgram/mL as compared to doses of 24,000 U/kg.

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization.

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C18 solid-phase extraction column. After elution with 500 microliters of elution solution, the penicillins are derivatized with 500 microliters of 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. The penicillin-mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C18 column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.

Determination of penicillin G residues in edible animal tissues by liquid chromatography.

An improved method has been developed for the determination of benzyl penicillin in animal tissues. Tissues are fortified with a known amount of penicillin V (internal standard) and extracted with water. The extract is deproteinized with sulfuric acid and sodium tungstate, filtered, and concentrated on a conditioned C18 solid phase extraction column. Penicillin V and benzyl penicillin are then eluted from the column with 1 mL 60% acetonitrile-35% water-5% 0.2M phosphate buffer solution and derivatized with 1 mL 1,2,4-triazole-mercuric chloride solution at 65 degrees C for 30 min. An aliquot of this sample is analyzed by reverse phase liquid chromatography with UV detection at 325 nm. The limit of detection is 5 micrograms/kg (ppb) penicillin G (8.4 IU/kg) in liver, kidney, and muscle tissues).