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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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