Article pubs.acs.org/JAFC
Distribution of Flunixin Residues in Muscles of Dairy Cattle Dosed with Lipopolysaccharide or Saline and Treated with Flunixin by Intravenous or Intramuscular Injection Weilin L. Shelver,*,† Marilyn J. Schneider,‡,# and David J. Smith† †
Biosciences Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1605 Albrecht Boulevard North, Fargo, North Dakota 58102, United States ‡ Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, United States ABSTRACT: Twenty dairy cows received flunixin meglumine at 2.2 mg/kg bw, administered once daily by either the intravenous (IV) or intramuscular (IM) route for three consecutive days with either intravenous normal saline (NS) or lipopolysaccharide (LPS) providing a balanced design with five animals per group. Cows were sacrificed after a 4 day withdrawal period, and 13 muscle types were collected and assayed for flunixin by LC-MS/MS. After elimination of sample outliers, the main effects of route of administration (IV or IM), treatment (NS or LPS), and tissue type significantly (P < 0.05) affected flunixin residues, with no interaction (P > 0.05). Intramuscular (nonlabel) flunixin administration produced greater (P < 0.05) flunixin residues in muscle than the IV (label) administration, whereas LPS resulted in lower flunixin levels. Differences among the tissue levels indicate it is necessary to specify the tissue to be used for any monitoring of drug levels for consumer protection. KEYWORDS: flunixin, cattle, intravenous, intramuscular, residues
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INTRODUCTION Regulatory agencies set drug residue tolerances for muscle, but often no specific muscle is identified for monitoring or surveillance purposes. Past studies have indicated that for some veterinary drugs there may be differences in residue levels among muscles within an animal. For example, residues of the anticoccidial diclazuril were greater (P < 0.05) in thigh than breast muscle of broilers1 during the first 2 days of withdrawal, whereas enrofloxacin residues were consistently greater (P < 0.05) in breast than in thigh regardless of dose or length of exposure period.2 In both beef calves and cull dairy cows, variations in residue levels of tilmicosin3 and penicillin G4 were observed both between and within individual muscles. Finally, in pork tissue 1−2 h after injection of carazolol, quantifiable residues were present in the diaphragm, but not the gluteal muscle.5 Such results indicate that the choice of sampling for residue monitoring could either positively or negatively bias the likelihood of either detection or quantification. However, whether residues differ between individual muscle types within an animal seems to be compound specific. Flunixin is a nonsteroidal anti-inflammatory drug approved for use in cattle by the U.S. Food and Drug Administration (FDA). Observance of the appropriate preslaughter withdrawal period is important to ensure tissue residues are below tolerance concentrations in products intended for human consumption [25 and 125 parts per billion (ppb) of flunixin free acid in muscle and liver, respectively] (21 CFR 556.286).6 The major source of residue violations for flunixin is with market dairy cows, in which 55−59% of the total flunixin violations occur from the Food Safety and Inspection Service survey.7−9 Flunixin violations might occur after flunixin is given to sick animals,10 which may exhibit prolonged elimination This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society
processes, or if given through an off-label route of administration.10 In this study, flunixin residues in tissues from healthy or lipopolysaccharide (LPS) challenged (to mimic bacterially mediated inflammatory responses of septic animals) dairy cows, dosed intramuscularly (IM, off-label route) or intravenously (IV, label route) with the maximum flunixin meglumine label dose (2.2 mg/kg bw), were compared. The maximum label dosing period of 3 consecutive days was employed, and cattle were slaughtered using the label withdrawal period of 4 days (96 h). Different skeletal muscle groups as well as heart were harvested with the goal of comparing flunixin distribution among muscle groups.
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MATERIALS AND METHODS
Materials. Banamine (flunixin meglumine; Schering-Plough, Summit, NJ, USA) solution for injection was obtained from Stockmen’s Supply (West Fargo, ND, USA). Flunixin reference standard was obtained from USP (Rockville, MD, USA). Flunixin-d3 was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Lipopolysaccharide (LPS; endotoxin) purified from Escherichia coli 0111:B4 by phenol extraction was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). SELECTRASORB CLEAN-UP C18 was purchased from UCT, Inc. Bristol, PA, USA. Whatman Mini-UniPrep syringless filters (PVDF, 0.2 μm) were purchased from Thomson Instrument Co. (Clear Brook, VA, USA). All other reagents were obtained from common chemical suppliers. Tissue Sample Collection. Details of the animal study, including IACUC approval of the study protocol, were described by Smith et al.11 Received: Revised: Accepted: Published: A
October 26, 2016 November 28, 2016 November 28, 2016 November 28, 2016 DOI: 10.1021/acs.jafc.6b04792 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Sample Cleanup. To 1 g of muscle was added 20 μL of a 250 ng/ mL flunixin-d3 solution followed by 8 mL of 80% acetonitrile in water (v/v). The mixture was homogenized using a tissuemizer and the probe rinsed with 2 × 1 mL of 80% acetonitrile. The slurry was centrifuged at 4000g for 10 min. The supernatant was decanted into a 15 mL centrifuge tube containing 500 mg of C18 sorbent and the mixture vortexed for 15 s followed by centrifugation at 4000g for 5 min. An aliquot (5 mL) of the resulting supernatant was transferred to a separate glass tube and the solvent evaporated to 0.70, Table 3). As noted by Kissell et al.,10 who measured plasma data, nonsystemic flunixin administration had a significant (P < 0.05) impact on its terminal plasma halflife, with IM giving higher plasma values than IV administration. Not surprisingly, IM administration also increased muscle residues relative to IV administration (Table 2). Kissell et al.12 showed that mastitisan inflammatory condition of the mammary gland generally attributed to LPS produced by microbesdecreased (P = 0.01) the clearance and increased (P < 0.05) the milk concentrations of flunixin in dairy cows. Our results show a significant (P < 0.05) decrease of muscle flunixin levels in LPS-treated cows (Table 4). Whereas Kissell et al.12 measured plasma kinetic effects over a 24 h time period and milk effects within 12 h of flunixin administration, muscle samples were not removed from our cows until 96 h after C
DOI: 10.1021/acs.jafc.6b04792 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
and supraspinitis were intermediate, with psoas major, vastus lateralis, semimembranosus, semitendinosus, anterior longissiumus, and posterior longissiumus dorsi having the lowest concentrations of flunixin. Regardless of muscle type, none of these values in muscles remotely approached the tolerance level of 25 ng/g established by the U.S. FDA for flunixin in muscle (21 CFR 556.286).6 There was no evidence to support the hypothesis that flunixin levels might differ within a muscle type. That is, flunixin residues did not differ (P > 0.05) between anterior or posterior longissimus dorsi or between dorsal or ventral diaphragm. Previously, Schneider et al.4 discerned numerical, but not statistical, differences in penicillin G levels within muscles. Meenagh et al.5 stated that “in most monitoring programs, diaphragmatic muscle is the muscle of choice for sampling since it is easy to remove and does not devalue the carcass”. Meenagh et al.5 were able to measure carazolol residues in diaphragm but not gluteal muscle; in our study, diaphragm muscle also appears to have higher relative residue concentrations than many of the other muscles. In this study we surveyed a large number of muscle types for flunixin residues and determined that irrespective of route of flunixin administration or treatment with LPS, flunixin residues were well below regulatory limits for muscle flunixin residues (25 ng/g) in dairy cattle. Nevertheless, our data clearly show that the route of drug administration, and whether animals were treated with LPS, clearly influenced drug deposition in muscle and consequent muscle residue levels. Although not having regulatory significance for flunixin, such variables might be significant for analytes for which no tolerance exists, but for
Table 3. Three-Way Analysis of Variance Indexed by Route, Lipopolysaccharide (LPS), and Muscle Types after Elimination of Outlier (P < 0.001) Data (Cow 1, Tail Flunixin Residues) and All of the Data for Cow 18 (Flunixin via IM Administration with LPS) due to Violative Residues source of variation
DF
SS
MS
F
P
route LPS muscle route × LPS route × muscle LPS × muscle route × LPS × muscle residual
1 1 12 1 12 12 12 194
6.378 0.939 6.183 0.0127 0.391 0.143 0.270 17.479
6.378 0.939 0.515 0.013 0.033 0.012 0.023 0.090
70.787 10.419 5.718 0.141 0.361 0.132 0.250
