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ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 659 DRUGS, COSMETICS, FORENSIC SCIENCES Determination of Flunixin Residues in Bovine Muscle Tissue by Liquid Chromatography with UV Detection PHILIP A. ASEA, JOHN R. PATTERSON, and GARY O. KORSRUD Canadian Food Inspection Agency, Centre for Veterinary Drug Residues, Health of Animals Laboratory, 116 Veterinary Rd, Saskatoon, Saskatchewan, S7N 2R3, Canada PATRICIA M. DOWLING University of Saskatchewan, Western College of Veterinary Medicine, Department of Veterinary Biological Sciences, Saskatoon, Saskatchewan, S7N 5B4, Canada JOE O. BOISON1 Canadian Food Inspection Agency, Centre for Veterinary Drug Residues, Health of Animals Laboratory, 116 Veterinary Rd, Saskatoon, Saskatchewan, S7N 2R3, Canada A new and sensitive liquid chromatography–ultra violet method with a detection limit of 6 ng/g (ppb) and a limit of quantification of 15 ng/g was developed for the determination of flunixin residues in bovine muscle tissue. Flunixin in homogenized animal tissue was extracted with acetonitrile after enzyme digestion. The tissue digest (extract) was then cleaned up on a solid-phase extraction cartridge and eluted with acidified hexane. After the eluate was evaporated to dryness under nitrogen at 55EC, the residue was reconstituted in 1 mL mobile phase solution and analyzed by reversedphase gradient chromatography with UV detection at 285 nm. The method was then applied in a survey study of slaughter animals to determine whether flunixin is being used in an off-label manner for veal and beef production in Canada. lunixin (2-[2-methyl-3(trifluoromethyl)phenylamino]3-pyridinecarboxylic acid), a nonsteroidal anti-inflammatory drug (NSAID), is one of the few NSAIDs approved for use in veterinary practice. It is approved for use in horses to alleviate inflammation and pain associated with musculoskeletal disorders and alleviation of visceral pain associated with colic. It is approved for use in food animals in England, France, Switzerland, and Germany. It was, however, not approved for use in food animals in Canada and the United States at the beginning of this study, but a survey of 2000 veterinarians in the United States whose practices were devoted to at least 50% dairy and beef cattle revealed that 95, 69, 67, and 70% of them were using flunixin, dipyrone, aspirin, and phenylbutazone, respectively, alone or in combination with antibiotics in their practice (1, 2). This survey suggests that, at least in the United States, some practi- F Received April 28, 2000. Accepted by JS July 28, 2000. 1 Author to whom correspondence should be addressed. tioners were using NSAIDs either alone or in combination with approved antibiotics to treat food animals, even though these NSAIDs had not been approved for that purpose. At the same time, there were suspicions by some Canadian veterinary inspectors that some producers may be using NSAIDs to “mask the apparent lameness of beef cows being shipped to slaughter.” During the course of the study, however, flunixin was approved for use in beef cattle and nonlactating dairy cattle in the United States for control of pyrexia associated with bovine respiratory disease and endotoxemia. It was also indicated for control of inflammation in endotoxemia. It was not approved for use in lactating or dairy cattle, calves intended for veal, or bulls intended for breeding. In the United States, it is recommended that cattle administered flunixin and intended for food must not be slaughtered within 4 days of last treatment (3). Animals arriving for slaughter in federally regulated abattoirs in Canada are randomly sampled for examination by veterinary inspectors. In addition to looking for obvious signs of disease, the inspectors look for visible injection sites that might indicate that the animal has been treated recently with a veterinary drug. Kidney tissues from these randomly selected animals, as well as those deemed “suspect” as a result of veterinary examination, are tested on site for the presence of antimicrobial drugs with the Swab Test On Premises (STOP) or the Calf Antibiotic and Sulfonamide Test (CAST). In both assays, a cotton swab is saturated with kidney tissue fluids from the kidney of the “test/suspect” animal, positioned on an agar seeded with Bacillus subtilis (STOP) or Bacillus megaterium (CAST), and incubated overnight at either 27–29EC for the STOP, or at 44–45EC for the CAST. If antibiotics (STOP and CAST) or sulfa drugs (CAST only) are present in the kidney fluid, they will diffuse out of the swab into the agar and prevent or inhibit the growth of B. subtilis or B. megaterium. Carcasses that elicit a “presumptive positive” test response by inhibiting the growth of the test organisms are detained at the abattoir while muscle, kidney, and liver tissues from the implicated animals are shipped to a regulatory laboratory to confirm test results. If laboratory test results confirm 660 ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Table 1. Gradient conditions used for LC analysis of flunixin residues Solvent A Initial mobile phase compositiona Final mobile phase composition Methanol, 40% Methanol, 30% B Acetonitrile, 30% Acetonitrile, 50% C 0.05M Ammonium acetate buffer (pH 5.0), 30% 0.05M Ammonium acetate buffer (pH 5.0), 20% a Hold initial gradient conditions for 15 min and linearly ramp to final conditions. Under these LC conditions, flunixin elutes at a retention time of 7.6–7.7 min. A number of analytical methods have been developed for the determination of flunixin residues in plasma and other biological fluids of dogs and horses (4–8). A few methods have been applied to study the pharmacokinetics of flunixin in lactating cattle after single and multiple intramuscular and intravenous administration (9, 10) and in milk (11). However, few methods have been published on the determination of flunixin residues in animal tissues (12). The objective of this study, therefore, was to develop a sensitive analytical method for the determination of flunixin residues in bovine muscle tissue, validate the method, and use it to conduct a pilot survey study to assess the prevalence of flunixin residues in Canadian beef cows and veal at slaughter. Experimental the presence of detectable/violative levels of veterinary drugs in the muscle tissue, the detained carcasses are destroyed and prevented from release into the food chain. Because NSAIDs do not exhibit antimicrobial properties, the STOP and CAST currently used for screening veterinary drug residues in North American slaughter establishments cannot detect antibiotics. Reagents (a) Acetonitrile and methanol.—Both LC grade. Obtained from Burdick & Jackson (Muskegon, MI). (b) Ammonium acetate (LC grade), glacial acetic acid, dibasic potassium phosphate, monobasic potassium phos- Figure 1. Chromatograms of (a) control (drug-free) muscle tissue extract containing diclofenac, and (b) control muscle tissue extract fortified with flunixin at a concentration of 100 ng/g and containing diclofenac, the retention time marker. ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 661 (g) $-glucuronidase.—Type H-5, lyophilized powder, (Sigma). $-glucuronidase solution.—Dissolve 100 mg $-glucuronidase powder in 1.5 mL saline phosphate buffer, pH 6.0. Prepare and use fresh. (h) Flunixin meglumine.—99%+ purity. A gift from Schering Canada (Point Claire, Quebec). (1) Flunixin (50 mg/mL) stock solution.—Weigh 8.3 mg flunixin meglumine into 100 mL volumetric flask. Add 80 mL methanol to dissolve and dilute to volume with methanol. Prepare stock solution quarterly and store at room temperature. (2) Flunixin (2.0 mg/mL) working solution.—Dilute 400 µL 50 µg/mL stock solution to 10 mL with methanol. Prepare monthly and store at room temperature. (i) Water.—Obtained from a Barnstead RO/Nanopure ultrafiltration unit (Dubuque, IA). (j) Mobile phase solutions.—(1) Solvent A.—10% Methanol.—Mix 100 mL methanol with 900 mL water and filter through 0.22 µm Nylon 66 membrane filter. (2) Solvent B.—Acetonitrile.—Filter 1000 mL through 0.22 µm Nylon 66 membrane filter. (3) Solvent C.—0.05M ammonium acetate buffer (pH 5.0). Weigh 1.93 g ammonium acetate into 500 mL volumetric flask. Add 400 mL water to dissolve, adjust pH with TFA, and dilute to volume with water. Filter through 0.22 µm Nylon 66 membrane filter. (k) Potassium acetate solution.—0.04M. Dissolve 785 mg potassium acetate in 200 mL water. (l) Sodium phosphate, dibasic.—0.25M. Weigh 35.5 g dibasic sodium phosphate and dissolve in 1000 mL volumetric flask with water. (m) Potassium phosphate.—Monobasic solution (0.25M, pH 7.0). Weigh 34.0 g potassium phosphate and dissolve with water in 1000 mL volumetric flask. Adjust to pH 7.0 with 0.25M dibasic sodium phosphate solution. (n) Elution solution.—Add 10% (v/v) acetic acid in hexane to 60 mL hexane in 100 mL volumetric flask; add 10 mL glacial acetic acid, mix, and dilute to volume with hexane. (o) Dissolving solution.—(40% Solvent A + 30% solvent B + 30% solvent C.) In 100 mL measuring cylinder, add 40 mL mobile phase solvent A, 30 mL solvent B, and 30 mL solvent C; mix, and filter. Table 2. Recoveries of flunixin added to blank (drug-free) muscle tissues at a concentration of 100 ng/g of tissue Detector responses (peak area in arbitrary units) Date of analysis External standard Fortified sample extract Calculated rec., %a 05/6/1998 47.703 32.916 69 26/6/1998 47.670 32.893 69 06/7/1998 44.089 26.013 59 08/7/1998 47.702 37.208 78 27/7/1998 33.703 22.244 66 27/7/1998 33.670 23.232 69 29/7/1998 42.557 29.364 69 a A mean recovery of 68.4 ± 5.6% flunixin from muscle tissue is calculated. phate.—All obtained from Fisher Scientific Co. (Pittsburgh, PA). (c) Trifluoroacetic acid (TFA).—Obtained from Aldrich Chemical Co. (Milwaukee, WI). (d) Hydrochloric acid.—Obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ). (e) Hexane.—Glass distilled (Omnisolv). Obtained from EM Science (Cherry Hill, NJ). (f) Diclofenac sodium.—(2-[(2,6,dichlorophenyl)amino] benzeneacetic acid monosodium), 99%+ purity, obtained from Sigma Chemical Co. (St. Louis, MO), to be used as a retention time marker. (1) Diclofenac (50 mg/mL) stock solution.—Weigh 5.4 mg diclofenac sodium salt into 100 mL volumetric flask. Add 80 mL methanol to dissolve and dilute to volume with methanol. Store at room temperature. Prepare quarterly. (2) Diclofenac (2.0 mg/mL) working solution.—Dilute 400 µL 50 µg/mL diclofenac stock solution to 10 mL with methanol. Store at room temperature and prepare monthly. Table 3. Intra-assay precision of newly developed analytical method Peak areas (in arbitrary units) measured for flunixin at defined concentrations, ppb Sample 10 25 50 100 150 200 1 3.781 7.902 17.290 33.672 50.021 67.811 2 3.684 7.883 17.431 34.551 50.882 67.991 3 3.553 8.142 17.021 33.702 50.954 67.903 4 3.941 9.001 18.503 36.903 52.502 69.201 Mean 3.740 8.232 17.561 34.707 51.089 68.227 SD 0.163 0.526 0.650 1.519 1.032 0.654 RSD, % 4.3 6.3 3.7 4.4 2.0 1.0 662 ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 Table 4. Interassay precision of newly developed analytical method Peak areas (in arbitrary units) measured for flunixin at the defined concentrations, ppb Day 1 2 3 10 25 50 100 150 200 3.951 7.890 17.195 33.052 49.099 66.562 3.867 7.901 17.230 33.123 51.110 67.321 4.015 8.021 17.480 33.599 50.013 67.895 3.985 7.992 17.547 34.127 51.027 67.913 3.687 7.402 16.937 33.874 50.319 68.211 3.578 7.945 17.315 33.283 51.008 69.013 Mean 3.847 7.859 17.284 33.509 50.429 67.819 SD 0.177 0.229 0.219 0.432 0.789 0.827 RSD, % 4.6 2.9 1.3 1.3 1.6 1.2 Apparatus (a) Solid-phase extraction (SPE) cartridges.—Bond Elut Certify II cartridges with C8 plus strong anion exchange capacity (Varian, Harbor City, CA). (b) 12-Port SPE vacuum manifold.—Supelco (Oakville, ON, Canada). (c) Flatbed mechanical shaker.—Eberbach Corp. (Ann Arbor, MI). (d) Refrigerated centrifuge.—Beckman Model CS-6KR. (e) Nitrogen evaporator N-Evap.—Organomation Associates (South Berlin, MA). (f) Liquid chromatograph.—Hewlett-Packard LC Model 1050 with vacuum degasser, quarternary gradient pump, automated sample injector, and variable wavelength detector (set at 285 nm) controlled by 2D Hewlett-Packard Chemstation (Hewlett-Packard, Mississauga, Canada). Chromatographic separation was conducted on a 3.0 × 250 mm Inertsil ODS-3 (5 µm) column (GL Sciences, Inc., Japan) preceded by a 4.0 × 10 mm Inertsil ODS-3 (5 µm) guard cartridge. vortex mix, centrifuge at 3200 × g for 10 min, and combine supernatants from the 3 extractions. Add 5 mL hexane to combined extract, acidify solution with 50 µL concentrated HCl, shake mixture, and centrifuge to separate phases. Aspirate upper hexane fraction to waste. Add 5 mL hexane to solution, shake, centrifuge to separate phases, and aspirate hexane fraction again to waste. Evaporate remaining solution to ca 5 mL using prepurified nitrogen at 55EC. Add 7 mL 0.25M phosphate buffer (pH 7.0), vortex mix, sonicate for 20 s, and centrifuge at 5EC for 15 min at 3200 × g. Cleanup of Tissue Extracts on Bond Elut Certify II SPE Cartridges Condition a Bond Elut Certify II cartridge with 3 mL methanol followed by 3 mL water. Load tissue extract onto conditioned car- Table 5. Estimation of accuracy of analytical method Coded samplea Flunixin added, Flunixin found,b ng/g ng/g Accuracy, % Sample Preparation Accurately weigh 2 g homogenized test samples into separate 50 mL polypropylene centrifuge tubes. Accurately weigh also 2 g homogenized control (drug-free) muscle tissue into 50 mL polypropylene centrifuge tube. Fortify control tissue with 100 µL 2.0 µg/mL flunixin working standard solution. This represents a fortification level of 100 ng flunixin/g tissue and will be used for recovery correction. Let stand for 15 min along with other test samples. Add 5 mL 0.04M potassium acetate solution to each test portion, shake, and let stand 10 min. Adjust pH of each test portion in centrifuge tube to 4.5 with 30 µL glacial acetic acid, vortexmix, and let stand 10 min. Add 70 µL freshly prepared $-glucuronidase solution, mix, let stand 30 min, and digest overnight (16 h) at 37EC. After digestion, add 5 mL acetonitrile to each tube, vortexmix, and centrifuge at 3200 × g for 10 min. Decant supernatant into clean 50 mL centrifuge tube. Extract digested material twice more with 5 mL acetonitrile and 1 mL 0.04M potassium acetate; FX 2000 190 193 +2 FX 2004 190 180 –5 N1010 190 187 –2 N1008 120 115 –4 N1012 120 121 –1 FX 2002 100 107 +7 FX 2003 100 105 +5 N1013 50 49 –2 N1011 50 59 +18 FX 2005 15 13 –4 FX 2001 15 18 +20 0 ND 0 N1009 a b Samples were prepared by the Laboratory’s Quality Assurance and Quality Control Officer, coded, and provided blind for analysis. Analytical results were corrected for recovery. ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 663 tridge under gravity. Wash cartridge successively with 2 × 1.5 mL water, 3 × 2.0 mL methanol, and dry cartridge by drawing air through it for 20 min (>15 psi). Rinse cartridge with 2 × 2 mL hexane and elute retained flunixin with 2 × 2 mL elution solution into 10 mL glass centrifuge tube. Evaporate eluate to dryness under nitrogen at 55EC. At this point, it is optional to add 100 µL 2.0 µg/mL diclofenac working standard solution to serve as a retention time marker for the chromatographic analysis of flunixin. Dissolve residue in 1000 µL dissolving solution, mix, sonicate for 5 s, and pass through 0.22 µm polyvinyledene difluoride (PVDF) Acrodisc (VWR Canlab, Mississauga, Ontario, Canada) filter for LC analysis. Preparation of Standards for Constructing Calibration Curve Pipet 10, 25, 50, 100, 150, and 200 µL 2.0 µg/mL flunixin working standard solution into separate 10 mL glass centrifuge tubes containing 4 mL elution solution. Evaporate to dryness under nitrogen at 55EC. At this point, it is optional to add 100 µL 2.0 µg/mL diclofenac working standard solution to serve as a retention time marker for chromatographic analysis of flunixin. Reconstitute in 1000 µL dissolving solution to prepare equivalent concentrations of 10, 25, 50, 100, 150, and 200 ng/g flunixin in tissue, respectively. LC Analysis Inject 45 µL calibration standards and test samples into LC operated isothermally at 40EC at a flow rate of 0.4 mL/min with the gradient flow conditions shown in Table 1. Measure peak areas of calibration standards and test samples. Figure 1 shows typical chromatograms of a control (drug-free) muscle tissue (Figure 1a), and a control muscle tissue fortified with flunixin at a concentration of 100 ng/g (Figure 1b), extracted and analyzed according to the described method. Recovery and Validation Studies of Newly Developed Analytical Method The selectivity and specificity of the method were demonstrated by analyzing blank (drug-free) muscle tissue samples obtained from 6 different geographical sources. Recoveries of flunixin added to blank muscle tissues were calculated by comparing detector responses for flunixin in fortified control (drug-free) tissues that had been subjected to the extraction analysis procedure described with those of equivalent external standards (Table 2). Intra-assay precision for the analytical method was determined by analyzing 4 sets of control tissue fortified with flunixin at 10, 25, 50, 100, and 200 ppb on the same day (Table 3). Interassay precision was determined by analyzing 2 sets of control tissues fortified with flunixin at 10, 25, 50, 100, and 200 ppb on each of 3 consecutive days (Table 4). To determine whether endogenous and other veterinary drugs likely to be administered alone or in combination with flunixin might interfere with the analysis of flunixin, control tissues fortified with flunixin at 100 ppb were extracted according to the described method and the extracts were then fortified individually with penicillin G, penicillin V, chloramphenicol, tylosin, tilmicosin, erythromycin, dihydrostreptomycin, and spiramycin, to a concentration of 1000 ppb and injected into the LC system. Method accuracy was verified by analysis of control (drug-free) tissues prepared by the Laboratory’s Quality Assurance Manager, coded, and provided blind for analysis by the developed analytical method (Table 5). Application of Developed Analytical Method Suitability of method for determination of flunixin residues in incurred tissues.—A calf known to have no previous antibiotic treatment history was purchased and housed in the large animal facility at the Western College of Veterinary Medicine (WCVM), University of Saskatchewan (Saskatoon, Canada) for experimental administration of flunixin meglumine. Five days after the animal had been allowed to acclimate, it was Table 6. Analytical results to determine suitability of the developed method for determination of flunixin residues in incurred muscle tissues Location of tissue sample analyzed a Left neck (inj. site) Right neck (normal)b Left semi-membranosus/ semi-tendinosus area (inj. site) Right semi-membranosus/ semi-tendinosus area (normal) a b Inj. site = injection site muscle tissue. Normal = normal muscle tissue. Withdrawal days before slaughter Concn (± SD) of flunixin found, ppb Visible injection site reaction at postmortem 3 34 ± 5 (n = 3) Hemorrhage 1 99 ± 6 (n = 3) Massive hemorrhage 3 20 ± 17 (n = 3) 1 33 ± 6 (n = 3) 3 165 ± 49 (n = 3) Hemorrhage, necrosis 1 9899 ± 120 (n = 3) Hemorrhage, necrosis 3 17 ± 7 (n = 3) 1 31 ± 5 (n = 3) 664 ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 weighed and ear tagged, and the hair around the area of the selected injection site was clipped. On the 7th day, the calf was weighed again and then injected twice with flunixin at a dose of 1 mL per 45.4 kg body weight of a 50 mg/mL flunixin meglumine formulation, once in the left neck and once in the left semi-membranosus/semi-tendinosus area (3-day withdrawal). On the 10th day, the doses were repeated on the same side of the calf in the neck and semi-membranosus/semi-tendinosus area but in locations as far as possible from the previous injections (1-day withdrawal). The calf was slaughtered the following day and after the viscera were removed, the carcass was refrigerated overnight. The 4 marked injection sites were examined the next day for drug reactions after which they were removed (220–280 g) and stored at –76EC for chemical analysis. To obtain homogeneous tissue samples for analysis, a minimum sample size of 200 g injection site tissue or a minimum of 100 g normal muscle tissue sample was required for homogenization (13). One day before analysis, the whole injection site sample or normal muscle tissue sample was removed from the freezer and allowed to thaw at room temperature. A 200 g portion injection site sample or 100 g normal muscle tissue was weighed, cut up in pieces, homogenized in a Sunbeam Osaka-Jr. (Hongkong, China) kitchen blender, and split into 3 sets. One of the 3 sets of test samples was analyzed the next day while the others were stored at –76EC for repeat analysis if necessary. Table 4 shows the residual concentrations of flunixin found in the injection and normal muscle tissues (after triplicate analysis of each sample). Also shown in Table 4 are the results of the postmortem examination of injection site reactions resulting from administration of the drug. Survey study of flunixin residues in slaughter animals (beef and veal) in canadian abattoirs.—Between 1995 and 1998, 633 injection site muscle tissues were analyzed for flunixin. These were taken both from carcasses that were found positive by the STOP test and from carcasses found to be negative by the STOP test. The results of the survey are presented in Table 5. In addition, samples of normal muscle tissue were taken from 335 veal carcasses at federally regulated slaughter establishments between June 6, 1998, and June 30, 1998, and analyzed for flunixin residues. The results of the veal survey are also reported in Table 5. Results and Discussion Figures 1a and 1b show typical chromatograms of a blank (drug-free) muscle tissue extract and blank muscle tissue fortified with flunixin at 100 ng/g, respectively, both containing diclofenac as a retention time marker, using the gradient LC conditions in Table 1. Flunixin and the retention time marker, diclofenac, eluted with retention times of 7.67 and 8.70 min, respectively, on this analytical column. They were both resolved from all other tissue co-extractives and there were no endogenous tissue components likely to interfere with the flunixin assay. In addition, other antibiotics including penicillins G and V, chloramphenicol, tylosin, tilmicosin, erythromycin, dihydrostreptomycin, and spiramycin were not detected when they were co-injected with flunixin extracts on the LC system. It was, therefore, concluded that the newly developed analytical method was selective and specific for the determination of flunixin in bovine muscle tissue. Calibration curves plotted from the UV detector response (peak areas, Y) versus the concentration of flunixin standard (XFNX) over the concentration range 10–200 ng/g, gave the predictor linear equation represented below: Y = 0.4385 {± 0.0042}XFNX – 0.2141 ± {0.4806} where Y denotes the predicted value of the detector response, Y, for a given concentration of flunixin, XFNX. A detection limit of 6 ng/g (S/N = 3) and a limit of quantitation of 15 ng/g [signal-to-noise ratio (S/N) = 10] were calculated for the analytical method after recovery correction. This level of quantitation makes the method ideally suitable for the determination of flunixin residues in bovine muscle tissue for which a U.S. tolerance of 25 ppb has now been established (3). Table 2 shows that the method permits the recovery of 68 ± 5.6% flunixin from fortified bovine muscle tissues. The data also demonstrate the repeatability with which the method Table 7. Results of survey study to establish prevalence of flunixin meglumine residues in beef and veal at slaughter in Canadian federally regulated slaughter establishments Year of sample collectiona 1995 Tissue samples analyzed No. of samples analyzed No. of flunixin positives (concn. in ppb) Injection sites 80 1 [13700]b 218 0 101 0 335 0 (STOP +ve and –ve) 1996 Injection sites (STOP +ve and –ve) 1997 Injection sites (STOP +ve and –ve) c 1998 a b c Normal veal muscle These samples were collected over the fiscal year beginning April 1 of the indicated year and ending March 31 of the following year. The presence of flunixin in this sample was confirmed by LC/MS/MS (unpublished data). These samples were collected between June 6 and June 30, 1998. ASEA ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 84, NO. 3, 2001 665 can recover flunixin from fortified tissues. This is attributable to the use of enzyme digestion to free bound flunixin residues from bovine tissue and explains the dramatic contrast to results of earlier recovery studies conducted in the absence of enzyme digestion that showed not only lower recovery data, but also large variabilities in recovery data from day to day. Tables 3 and 4 show that the method provides good within-day and between-day precision over the concentration range analyzed, and Table 5 shows that it can be used to accurately estimate the amount of flunixin physically added to muscle tissue. To verify that the method was sensitive and suitable for the determination of residual concentrations of flunixin in incurred animal tissues, flunixin meglumine was intentionally administered to a cow calf, and tissue samples were collected and analyzed. The results shown in Table 6 indicate that the method has sufficient analytical sensitivity to detect and quantify physiological concentrations of flunixin in animal tissues. The results also demonstrate very clearly that if flunixin meglumine were to be administered intentionally to beef calves in an off-label manner as was done in this study, there would be significant injection site reactions (massive hemorrhage and/or necrosis) to permit an inspector to detain the carcass for further investigation. In addition, the results indicate that edible muscle tissue from such an animal would be contaminated with significantly high concentrations of flunixin drug residues even after 3 days of withdrawal following drug administration. These results also demonstrate that the withdrawal period of 4 days recommended by the U.S. Food and Drug Administration following flunixin drug administration to beef cattle must be rigorously followed for producers to avoid shipping treated animals with violative drug residues to slaughter. The lower, but still measurable concentrations of drug residue found after 3 days of withdrawal compared with the levels found after a 1 day withdrawal, indicate that flunixin, like other veterinary drugs, will deplete to nondetectable concentration levels with time. In addition, the results clearly show that the newly developed analytical method can be used to identify the carcass of an animal that has been treated with flunixin meglumine in an off-label manner and is being shipped prematurely (at least 3 days) to slaughter. Once it was demonstrated that the method was suitable for its intended purpose, it was applied in a study to determine the prevalence of flunixin residues in veal and beef samples processed in federally regulated abattoirs across Canada. Of the several hundreds of injection site and normal muscle samples tested, only one injection site sample was found to be contaminated with flunixin at a concentration estimated to be 13 700 ng/g. This positive result was confirmed by LC/MS/MS on a Micromass (Manchester, United Kingdom) BIO Q tandem mass spectrometer (unpublished data). The re- sults of the study presented in Table 7 indicate that flunixin was not detected in a representative sample population of Canadian slaughter beef and veal. It also clearly demonstrated that there was very limited evidence to support the suspicion regarding the off-label use of flunixin in food animals in Canada. As indicated earlier, this drug has now been approved for use in the United States for beef cattle, and there are indications that its use for beef cattle is being reviewed for approval consideration in Canada. This method can, therefore, be adapted for use in regulatory laboratories for the surveillance and monitoring of flunixin drug residues in animal tissues. Acknowledgments The authors acknowledge the valuable work done by Valerie Martz (Canadian Food Inspection Agency, Saskatoon, Saskatchewan) in providing tissue samples to verify some of the characteristic operational parameters for the analytical method. We also acknowledge Eli Neidert (Canadian Food Inspection Agency, Ottawa, Ontario) for designing the sampling plans. We also thank all the veterinary inspectors in the various federally inspected abattoirs who assisted with the collection of tissue samples and conducted the field screening tests. 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