21 U.S.C. 342, 360b, 371.
(a) Tolerances established in this part are based upon residues of drugs in edible products of food-producing animals treated with such drugs. Consideration of an appropriate tolerance for a drug shall result in a conclusion either that:
(1) Finite residues will be present in the edible products—in which case a finite tolerance is required; or
(2) It is not possible to determine whether finite residues will be incurred but there is reasonable expectation that they may be present—in which case a tolerance for negligible residue is required; or
(3) The drug induces cancer when ingested by man or animal or, after tests which are appropriate for the evaluation of the safety of such drug, has been shown to induce cancer in man or animal; however, such drug will not adversely affect the animals for which it is intended, and no residue of such drug will be found by prescribed methods of analysis in any edible portion of such animals after slaughter or in any food yielded by or derived from the living animal—in which case the accepted method of analysis shall be published or cited, if previously published and available elsewhere, in this part; or
(4) It may or may not be possible to determine whether finite residues will be incurred but there is no reasonable expectation that they may be present—in which case the establishment of a tolerance is not required; or
(5) The drug is such that it may be metabolized and/or assimilated in such form that any possible residue would be indistinguishable from normal tissue constituents—in which case the establishment of a tolerance is not required.
(b) No tolerance established pursuant to paragraph (a)(1) of this section will be set at any level higher than that reflected by the permitted use of the drug.
(c) Any tolerance required pursuant to this section will, in addition to the toxicological considerations, be conditioned on the availability of a practicable analytical method to determine the quantity of residue. Such method must be sensitive to and reliable at the established tolerance level or, in certain instances, may be sensitive at a higher level where such level is also deemed satisfactory and safe in light of the toxicity of the drug residue and of the unlikelihood of such residue's exceeding the tolerance.
A tolerance of 0.1 part per million is established for negligible residues of 2-acetylamino-5-nitrothiazole in the edible tissues of turkeys.
Tolerances are established for combined residues of aklomide (2-chloro-4-nitrobenzamide) and its metabolite (4-amino-2-chlorobenzamide) in uncooked edible tissues of chickens as follows:
(a) 4.5 parts per million in liver and muscle.
(b) 3 parts per million in skin with fat.
(a)
(b)
(2)
A tolerance of 0.01 part per million is established for negligible residues of amoxicillin in milk and in the uncooked edible tissues of cattle.
A tolerance of 0.01 p/m is established for negligible residues of ampicillin in the uncooked edible tissues of swine and cattle and in milk.
Tolerances are established as follows for residues of amprolium (1-(4-amino-2-
(a) In the edible tissues and in eggs of chickens and turkeys:
(1) 1 part per million in uncooked liver and kidney.
(2) 0.5 part per million in uncooked muscle tissue.
(3) In eggs:
(i) 8 parts per million in egg yolks.
(ii) 4 parts per million in whole eggs.
(b) In the edible tissues of calves:
(1) 2.0 parts per million in uncooked fat.
(2) 0.5 part per million in uncooked muscle tissue, liver, and kidney.
(c) In the edible tissues of pheasants:
(1) 1 part per million in uncooked liver.
(2) 0.5 part per million in uncooked muscle.
A tolerance of 0.1 part per million is established for parent apramycin (marker residue) in kidney (target tissue) of swine. The acceptable daily intake (ADI) for total residues of
Tolerances for total residues of combined arsenic (calculated as As) in food are established as follows:
(a) In edible tissues and in eggs of chickens and turkeys:
(1) 0.5 part per million in uncooked muscle tissue.
(2) 2 parts per million in uncooked edible by-products.
(3) 0.5 part per million in eggs.
(b) In edible tissues of swine:
(1) 2 parts per million in uncooked liver and kidney.
(2) 0.5 part per million in uncooked muscle tissue and by-products other than liver and kidney.
(a)
(b)
Tolerances are established for residues of buquinolate as follows:
(a) In edible tissues of chickens:
(1) 0.4 part per million in uncooked liver, kidney, and skin with fat.
(2) 0.1 part per million in uncooked muscle.
(b) In eggs:
(1) 0.5 part per million in uncooked yolk.
(2) 0.2 part per million in uncooked whole eggs.
A tolerance of 30 parts per billion is established for residues of quinoxaline-2-carboxylic acid (marker residue) in liver (target tissue) of swine.
A tolerance of zero is established for residues of carbomycin in the uncooked edible tissues of chickens.
(a)
(b)
(2)
A tolerance of 0.02 parts per million (ppm) is established for residues of cephapirin in the milk and 0.1 ppm in the uncooked edible tissues of dairy cattle.
A tolerance of zero is established for residues of chlorhexidine in the uncooked edible tissues of calves.
A tolerance of zero is established for residues of chlorobutanol in milk from dairy animals.
(a)
(b)
(2) A tolerance is established for residues of chlortetracycline in eggs of 0.4 ppm.
Tolerances for residues of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in food are established as follows:
(a) In cereal grains, vegetables, and fruits: 0.2 part per million.
(b) In chickens and turkeys:
(1) 15 parts per million in uncooked liver and kidney.
(2) 5 parts per million in uncooked muscle.
(c) In cattle, sheep, and goats:
(1) 3 parts per million in uncooked kidney.
(2) 1.5 parts per million in uncooked liver.
(3) 0.2 part per million in uncooked muscle.
(d) In swine: 0.2 part per million in uncooked edible tissues.
(e) In milk: 0.02 part per million (negligible residue).
Tolerances are established for residues of clorsulon in cattle as follows:
(a) The tolerance for clorsulon (market residue) in kidney (target tissue) is 1.0 part per million. A marker residue of 1.0 part per million corresponds to a total residue of 3.0 parts per million in kidney.
(b) The safe concentrations for total clorsulon residues in uncooked edible cattle tissues are: muscle, 1.0 part per million; liver, 2.0 parts per million; kidney, 3.0 parts per million; and fat, 4.0 parts per million.
A tolerance of 0.01 part per million is established for negligible residues of cloxacillin in the uncooked edible tissues of cattle and in milk.
A tolerance for residues of colistimethate in the edible tissues of chickens is not required.
(a)
(b)
(1) 1 part per million (ppm) in skeletal muscle.
(2) 2 ppm in other tissues.
(a)
(b)
(2) [Reserved]
A tolerance of 0.1 part per million is established for negligible residues of dichlorvos (2,2-dichlorovinyl dimethyl phosphate) in the edible tissues of swine.
Tolerances are established for residues of dihydrostreptomycin in uncooked, edible tissues of cattle and swine of 2.0 parts per million (ppm) in kidney and 0.5 ppm in other tissues, and 0.125 ppm in milk.
No residues of 3,5-dinitrobenzamide may be found in the uncooked edible tissues of chickens as determined by the following method of analysis:
I.
II.
B. Acetyl-(
C. Alumina—activated F-20, 80-200 mesh, Aluminum Co. of America, or equivalent substance.
D. Ammonium sulfamate.
E. Ammonium sulfamate solution 1.25 grams of ammonium sulfamate per 100 milliliters of water. Refrigerate when not in use. Prepare fresh weekly.
F. Cation-exchange resin—Dowex 50W-X8, 200-400 mesh, Baker Analyzed Reagent, or equivalent, prepared as follows:
1. Place 500 grams of resin into a 3-liter beaker.
2. Add 2,000 milligrams of 6
3. Heat and stir while on a bath at 80° C. for 6 hours. Discontinue heating and continue stirring overnight.
4. Filter the resin on a Buchner funnel (24 cm.) fitted with Whatman No. 1 paper.
5. Wash the resin bed with four 500-milliliter portions of 6
6. Wash the resin bed with 500-milliliter portions of deionized water until the effluent has a pH of 5 or higher.
7. Wash the resin bed with three 400-milliliter portions of specially denatured alcohol 3A. Drain thoroughly.
8. Make a slurry of resin in 1,250 milliliters of specially denatured alcohol 3A.
G. Chloroform.
H. Coupling reagent—0.25 gram of
I. 3,5-Dinitrobenzamide (3,5-DNBA standard). Add to boiling specially denatured alcohol 3A until a saturated solution is obtained and treat with activated carbon, filtered and crystallize by cooling to room temperature. The 3,5-DNBA therefrom is treated a second time with activated carbon and then recrystallized three more times from specially denatured alcohol 3A. The third crystallization is washed with diethyl ether and dried in a vacuum desiccator, melting point range 185° C.-186° C.
J. Ethyl alcohol—absolute, A.C.S.
K. Eluting reagent A. The formula and volume required in procedure step V-D is dependent on the adsorptive strength of the Al
1. Prepare a column (see procedure step V-D for determining formula and volume to eluting reagent A).
2. Transfer 1 milliliter of APNPS standard (100 micrograms per milliliter) in 75 milliliters of chloroform to the column.
3. Wash the column with 100 milliliters of chloroform and discard the eluate.
4. Pass through 100 milliliters of solution consisting of specially denatured alcohol 3A and ethyl alcohol 1:1 (volume to volume). Collect one 50-milliliter and five 10-milliliter portions; these make up the first, second, third, fourth, fifth, and sixth portions of eluate.
5. Place in beakers under a stream of air on a water bath (90° C.) until the solvents are evaporated.
6. Add 10 milliliters of 4
7. Add the Bratton-Marshall reagents.
8. All fractions show a slight color. Note the portion containing the first significant increase in pink color.
a. If the color increases in the second, third, or fourth portions of eluate, the formula in procedure step V-D is suitable and, depending on the portion, 45, 55, or 65 milliliters, respectively, should be used in procedure step V-D4. Thereby, the APNPS is retained on the column and the benzamides are eluted.
b. If the color increases in the first portion, the eluting strength of the reagent is too strong. Return the test, substituting 1:4 (volume to volume) in procedure step V-D4. If 1:4 (volume to volume) is too strong, rerun with ethyl alcohol in procedure step V-D. If none of these are suitable, another lot of Al
c. If the color increases in the fifth or sixth portion, the eluting strength of the reagent is too weak. Rerun the test, substituting in procedure step V-D4, respectively, 4:1 (volume to volume), specially denatured alcohol 3A: methyl alcohol, 4:1 (volume to volume), until a suitable formula is found. If none of
L. Hydrochloric acid, 4
M. Diatomaceous earth—Hyflo Super Cel, Johns-Manville Co., or equivalent substance.
N.
O. Sodium hydroxide solution, 10
P. Sodium nitrite solution—0.25 grams of sodium nitrite per 100 milliliters of water. Refrigerate when not in use. Prepare fresh weekly.
Q. Specially denatured alcohol, formula 3A-100 parts of 190-proof ethyl alcohol plus 5 parts of commercial methyl alcohol.
R. Titanium(ous) chloride-20 percent solution.
III.
B. Autotransformer—type 500B, or equivalent. To regulate speed of mixer.
C. Centrifuge.
D. Centrifuge tubes—50-milliliter size with glass stopper.
E. Chromatography tubes—Corning No. 38460, 20 millimeters A 400 millimeters and having a tapered 29/42 joint with coarse, fritted disc, or equivalent tubes.
F. Evaporator—vacuum, rotary, thin film.
G. Ion-exchange column—as described by Thiegs et al. in “Determination of 3-amino-5-nitro-
H. Glycerol manostat. For regulating pressure on columns: To Al
I. Motor speed control. For regulating speed on 1-quart blender.
J. Volumetric flasks—50 milliliter size, actinic ware.
K. Mixer—Vortex Jr. Model K-500-1, Scientific Industries, Inc., or equivalent mixer.
L. One-quart blender.
M. Water bath (45° C.-50° C.).
N. Water bath (90° C.).
IV.
2. Dissolve and dilute with acetone to volume.
3. Dilute 1 milliliter to 100 milliliters.
4. Add 5.0 milliliters of water to each of six centrifuge tubes.
5. Add standard to each of the tubes to contain one of the following amounts: 0.0, 1.0, 2.0, 3.0, 5.0, and 10.0 micrograms of 3,5-DNBA.
B. Prepare each tube for colorimetric measurement as follows:
1. Place the tube in a hot water bath (90° C.) until 5.0 milliliters remain. Cool to room temperature.
2. While mixing on Vortex mixer, or equivalent, regulated with an autotransformer, add 2 drops of TiCl
3. Add 2 milliliters of HCl, mix, and allow to stand for 5 minutes.
4. Transfer to 50-milliliter volumetric flask and dilute with 4
5. Cool to 0° C.-5° C. by placing in a freezer or ice bath.
6. Perform the Bratton-Marshall reaction in subdued light as follows:
a. Add 1 milliliter of sodium nitrite reagent, mix, and allow to stand for 1 minute.
b. Add 1 milliliter of ammonium sufamate reagent, mix, and allow to stand for 1 minute.
c. Add 1 milliliter of coupling reagent, mix, and allow to stand for 10 minutes.
d. Dilute to volume with 4
C. Perform colorimetric measurement at 530 millimicrons as follows:
1. Fill two matched 100-millimeter cells with 4
2. Adjust dark current.
3. Adjust to zero absorbance.
4. Replace acid in cell of sample side of compartment with standard to be measured.
5. The standard curve should be run five different times. Plot equivalent concentration in tissue versus mean absorbance at each concentration. If computer is available, a better procedure is to calculate the equation of the standard curve by means of least squares.
V.
2. Weight 100
3. Transfer the sample to a 1-quart blender jar. For kidney and liver tissues, make a slurry with acetone in the weighing beaker. Transfer with several rinses of acetone.
4. Blend the sample for 5 minutes with 250 milliliters of acetone and a 100-milliliter beakerful of diatomaceous earth.
5. Filter through a Buchner funnel containing a wetted Whatman No. 5 filter paper (12.5 cm.) into a 1-liter suction flask.
6. Rinse the blender jar into the funnel with three 25-milliliter portions of acetone.
7. Transfer the pulp and paper from the funnel to the aforementioned blender jar.
8. Add 250 milliliters of chloroform.
9. Blend for 3 minutes.
10. Filter through the aforementioned apparatus of procedure step V-A5. For rapid filtration of skin and blood samples, prepare funnel by adding diatomaceous earth and tamping evenly over paper to a thickness of 3 to 5 millimeters.
11. Rinse the blender jar into the funnel with three 25-milliliter rinses of chloroform.
B. Phasic separation. 1. Pour the combined filtrates into a 1-liter separatory funnel.
2. Rinse the suction flask twice with 25 milliliters of chloroform.
3. Mix the funnel contents by gently rocking and swirling for 30 seconds.
4. Let stand 10 minutes to allow phases to separate.
a. The upper (aqueous) phase (30 to 50 milliliters) is not always emulsion-free. Losses from emulsions have not been significant.
b. If an upper (aqueous) phase does not appear, add an additional 100 milliliters of chloroform and 10 milliliters of water and repeat procedure step V-B3.
5. Withdraw the lower phase into a 1-liter round-bottom flask, and discard upper phase. Withdraw nearly all of the lower phase, let stand for 2 to 3 minutes, then withdraw the remainder.
C. Evaporation. Attach the flask on a thin-film rotary evaporator connected to a vacuum supply, and place in a water bath maintained at 45° C.-50° C. until an oily residue remains. Do not overheat the sample or allow to go to dryness.
D. Adsorption chromatography. 1. Prepare a chromatography column using a column with calibrated etchings to indicate appropriate adsorbent and solvent levels as follows:
a. Fill tube to a depth of 60 millimeters with Al
b. Tap walls gently with hands.
c. Add anhydrous sodium sulfate to an additional depth of 25 millimeters.
d. Wet and wash column with 50 milliliters of chloroform.
i. During chromatography, make each addition to the tube when the liquid level has reached the top of the sodium sulfate layer.
ii. Increase the percolation rates by applying a slight air pressure to the top of the column.
2. Transfer the residue from procedure step V-C to the column with four 15-milliliter rinses of chloroform. Then rinse the walls of the tube and sodium sulfate layer with three 5-milliliter portions of chloroform. Percolation rate: 15 to 25 milliliters per minute. No color from sample should be seen in sodium sulfate layer after final rinse.
3. Wash column with 100 milliliters of chloroform. Discard eluate.
4. Add 75 milliliters of eluting reagent A and collect eluate A in a 250-milliliter beaker for cation-exchange chromatography.
a. Refer to “Eluting reagent A” under “Reagents” (II-K) for determining formula and volume.
b. Percolation rate: 8 to 12 milliliters per minute.
E. Cation-exchange chromatography—No. 1. 1. Prepare an ion-exchange column as follows:
a. Add a uniform slurry of resin to the column to obtain a 4 to 5 centimeter bed depth after settling.
i. Obtain a uniform slurry using a magnetic stirrer. To add the required amount of resin, calibrate the slurry and transfer it with a 10-milliliter pipette to deliver a reproducible volume.
ii. Increase the flow rate to 2 to 4 milliliters per minute by applying air pressure to the column. A glycerol manostat adjusted to 30 inches and attached between an air supply and column provides adequate pressure.
b. Wash the resin with 10 milliliters of eluting reagent A. Discard eluate.
2. Pass eluate A from procedure step V-D4 through the column. Collect in a 250-milliliter beaker.
3. Pass 50 milliliters of specially denatured alcohol 3A through the column. Combine with the eluate of procedure step V-E2.
F. Reduction. 1. Place the eluate A fraction from procedure step V-E3 on a hot water bath (90° C.) and evaporate with a stream of air until 5 to 10 milliliters remain. Do not overheat the sample or allow the sample to go to dryness.
2. Transfer to centrifuge tube and rinse beaker three times with 3 milliliters of specially denatured alcohol 3A.
3. Evaporate on a hot water bath (90° C.) under a stream of air until alcohol has evaporated. Do not overheat the sample or allow the sample to go to dryness.
4. Remove the tube from the water bath and immediately add 5.0 milliliters of water.
5. While mixing, add 2 drops of titanium chloride and 4 drops of 10
a. Mix on Vortex Jr. mixer, or equivalent, regulated with autotransformer.
b. Precipitate of insoluble tissue substances and white titanium salts is present after reduction is complete.
6. Dilute to 50 milliliters with specially denatured alcohol 3A and mix.
7. Centrifuge for 5 minutes at 2,000 r.p.m.
G. Cation-exchange chromatography—No. 2. 1. Prepare resin column by procedure step V-E.
2. Pass the centrifugate of procedure step V-F7 through column. Use three rinses of specially denatured alcohol 3A, each 5 milliliters, to aid in transferring of sample.
3. Pass 50 milliliters of specially denatured alcohol 3A through the column.
4. Pass 50 milliliters of deionized water through the column.
5. Elute arylamine residue from the resin with 40 to 43 milliliters of 4
H. Color development and measurement. 1. Cool to 0° C.-5° C. by placing in a freezer or ice bath.
2. Perform the Bratton-Marshall reaction in subdued light as follows:
a. Add 1 milliliter of sodium nitrite reagent, mix, and allow to stand for 1 minute.
b. Add 1 milliliter of ammonium sulfamate reagent, mix, and allow to stand for 1 minute.
c. Add 1 milliliter of coupling reagent, mix, and allow to stand for 10 minutes.
d. Dilute to volume with 4
3. Perform colorimetric measurement at 530 millimicrons as follows:
a. Fill two matched 100-millimeter cells with 4
b. Adjust dark current.
c. Adjust to zero absorbance.
d. Replace acid in cell of sample side of compartment with sample to be measured.
e. Record absorbance observed.
I. Calculations. Determine parts per billion (observed) from the standard curve.
(a)
(b)
(2)
(a)
(b)
(2) [Reserved]
The acceptable daily intake for enrofloxacin is 3 micrograms per kilogram of body weight per day.
(a)
(b)
Tolerances for residues of erythromycin in food are established as follows:
(a) 0.1 part per million in uncooked edible tissues of beef cattle and swine.
(b) Zero in milk.
(c) 0.025 part per million in uncooked eggs.
(d) 0.125 part per million (negligible residue) in uncooked edible tissues of chickens and turkeys.
No residues of estradiol, resulting from the use of estradiol or any of the related esters, are permitted in excess of the following increments above the concentrations of estradiol naturally present in untreated animals:
(a) In uncooked edible tissues of heifers, steers, and calves:
(1) 120 parts per trillion for muscle.
(2) 480 parts per trillion for fat.
(3) 360 parts per trillion for kidney.
(4) 240 parts per trillion for liver.
(b) In uncooked edible tissues of lambs:
(1) 120 parts per trillion for muscle.
(2) 600 parts per trillion for fat, kidney, and liver.
Tolerance for residues of ethopabate converted to metaphenetidine are established in the edible tissues of chickens as follows:
(a) 1.5 parts per million in uncooked liver and kidney.
(b) 0.5 part per million in uncooked muscle.
A tolerance of zero is established for residues of ethylenediamine in milk.
Tolerances are established for residues of famphur including its oxygen analog in or on meat, fat, or meat byproducts of cattle at 0.1 part per million.
(a)
(b)
(ii)
(iii)
(2)
(ii)
(3)
(ii)
(4)
(ii)
A tolerance for marker residue of fenprostalene in cattle is not needed. The safe concentrations for the total residues of fenprostalene in the uncooked edible tissues of cattle are 10 parts per billion in muscle, 20 parts per billion in liver, 30 parts per billion in kidney, 40 parts per billion in fat, and 100 parts per billion in the injection site. As used in this section “tolerance” refers to a concentration of a marker residue in the target tissue selected to monitor for total residues of the drug in the target animal, and “safe concentrations” refer to the concentrations of total residues considered safe in edible tissues.
(a)
(b)
(a)
(b)
A tolerance of zero is established for residues of furazolidone in the uncooked edible tissues of swine.
(a) A tolerance of 0.1 part per million is established for negligible residues of gentamicin sulfate in the uncooked edible tissues of chickens and turkeys.
(b) Tolerances are established for total residues of gentamicin in edible tissues of swine as follows: 0.1 part per million in muscle, 0.3 part per million in liver, and 0.4 part per million in fat
(a)
(b)
The marker residue selected to monitor for total residues of halofuginone hydrobromide in broilers and turkeys is parent halofuginone hydrobromide and the target tissue selected is liver. A tolerance is established in broilers of 0.16 part per million and in turkeys of 0.13 part per million for parent halofuginone hydrobromide in liver. These marker residue concentrations in liver correspond to total residue concentrations of 0.3 part per million in liver. The safe concentrations for total residues of halofuginone hydrobromide in the uncooked edible tissues of broilers and turkeys are 0.1 part per million in muscle, 0.3 part per million in liver, and 0.2 part per million in skin with adhering fat. As used in this section, “tolerance” refers to a concentration of a marker residue in the target tissue selected to monitor for total residues of the drug in the target animal, and “safe concentrations” refers to the concentrations of total residues considered safe in edible tissues.
A tolerance of 0.1 part per million is established for negligible residues of haloxon (3-chloro-7-hydroxy-4-methyl-coumarin bis(2-chloroethyl) phosphate) in the edible tissues of cattle.
A tolerance is established for negligible residues of hydrocortisone (as hydrocortisone sodium succinate or hydrocortisone acetate) in milk at 10 parts per billion.
A tolerance of zero is established for residues of hygromycin B in or on eggs and the uncooked edible tissues of swine and poultry.
(a)
(b)
(i)
(ii)
(iii)
(iv)
(v)
(2)
(i)
(ii)
(a) [Reserved]
(b)
(2)
(3)
(4)
A tolerance of 0.1 part per million is established for negligible residues of levamisole hydrochloride in the edible tissues of cattle, sheep, and swine.
(a)
(b)
(c)
A tolerance is established for residues of maduramicin ammonium in chickens as follows:
(a) A tolerance for maduramicin ammonium (marker residue) in chickens is 0.38 parts per million in fat (target tissue). A tolerance refers to the concentration of marker residues in the target tissue used to monitor for total drug residues in the target animals.
(b) The safe concentrations for total maduramicin ammonium residues in uncooked edible chicken tissues are: 0.24 parts per million in muscle; 0.72 parts per million in liver; 0.48 parts per million in skin; and 0.48 parts per million in fat. A safe concentration refers to the total residue concentration considered safe in edible tissues.
A tolerance of 25 parts per billion is established for residues of the parent compound, melengestrol acetate, in fat of cattle.
A tolerance of zero is established for residues of methylparaben in milk from dairy animals.
A tolerance is established for negligible residues of methylprednisolone in milk at 10 parts per billion.
A tolerance of 0.02 part per million is established for negligible residues of metoserpate hydrochloride (methyl-
(a)
(b)
(2)
A tolerance of 0.7 part per million is established for
(a)
(b)
(ii)
(iii)
(2) [Reserved]
A tolerance for narasin residues in chickens is not needed. The safe concentrations for total narasin residues in uncooked edible chicken tissues are: 0.6 part per million in muscle; 1.8 parts per million in liver; 1.2 parts per million in skin with adhering fat and fat. A tolerance refers to the concentration of marker residues in the target tissue used to monitor for total drug residues in the target animals. A safe concentration refers to the total residue concentration considered safe in edible tissues.
(a)
(b)
(1)
(2)
(3)
A tolerance of 0.1 part per million is established for negligible residues of nequinate in the uncooked edible tissues of chickens.
A tolerance of 4 parts per million is established for residues of nicarbazin in uncooked chicken muscle, liver, skin, and kidney.
Tolerances for residues of novobiocin are established at 0.1 part per million in milk from dairy animals and 1 part per million in the uncooked edible tissues of cattle, chickens, turkeys, and ducks.
A tolerance of zero is established for residues of nystatin in or on eggs and the uncooked edible tissues of swine and poultry.
Tolerances are established for negligible residues of oleandomycin in uncooked edible tissues of chickens, turkeys, and swine at 0.15 part per million.
(a) [Reserved]
(b)
(a)
(b)
Tolerances are established for residues of penicillin and the salts of penicillin in food as follows:
(a) 0.05 part per million (negligible residue) in the uncooked edible tissues of cattle.
(b) Zero in the uncooked edible tissues of chickens, pheasants, quail, swine, and sheep; in eggs; and in milk or in any processed food in which such milk has been used.
(c) 0.01 part per million in the uncooked edible tissues of turkeys.
A tolerance of 0.1 part per million piperazine base is established for edible tissues of poultry and swine.
(a)
(b)
(ii)
(iii)
(2) [Reserved]
A tolerance of zero is established for residues of prednisolone in milk from dairy animals.
A tolerance of zero is established for residues of prednisone in milk from dairy animals.
No residues of progesterone are permitted in excess of the following increments above the concentrations of progesterone naturally present in untreated animals:
(a) In uncooked edible tissues of steers and calves:
(1) 3 parts per billion for muscle.
(2) 12 parts per billion for fat.
(3) 9 parts per billion for kidney.
(4) 6 parts per billion for liver.
(b) In uncooked edible tissues of lambs:
(1) 3 parts per billion for muscle.
(2) 15 parts per billion for fat, kidney, and liver.
A tolerance of zero is established for residues of propylparaben in milk from dairy animals.
Tolerances are established for residues of pyrantel tartrate in edible tissues of swine as follows:
(a) 10 parts per million in liver and kidney.
(b) 1 part per million in muscle.
(a)
(b)
Tolerances are established for residues of robenidine hydrochloride in edible tissues of chickens as follows:
(a) 0.2 part per million in skin and fat.
(b) 0.1 part per million (negligible residue) in edible tissues other than skin and fat.
A tolerance of zero is established for residues of salicylic acid in milk from dairy animals.
(a)
(b) [Reserved]
A tolerance for residues of sarafloxacin in edible turkey and broiler chickens tissues is not required.
(a)
(b)
(2) [Reserved]
(a)
(b)
(c)
Tolerances are established for residues of streptomycin in uncooked, edible tissues of chickens, swine, and calves of 2.0 parts per million (ppm) in kidney and 0.5 ppm in other tissues.
Tolerances for residues of sulfabromomethazine sodium in food are established as follows:
(a) In the uncooked edible tissues of cattle at 0.1 part per million (negligible residue).
(b) In milk at 0.01 part per million (negligible residue).
A tolerance of zero is established for residues of sodium sulfachloropyrazine monohydrate in the uncooked edible tissues of chickens.
A tolerance of 0.1 part per million is established for negligible residues of sulfachlorpyridazine in uncooked edible tissues of calves and swine.
(a) [Reserved]
(b)
(2) A tolerance of 0.01 ppm is established for negligible residues of sulfadimethoxine in milk.
Tolerances for residues of sulfaethoxypyridazine in food are established as follows:
(a) Zero in the uncooked edible tissues of swine and in milk.
(b) 0.1 part per million (negligible residue) in uncooked edible tissues of cattle.
A tolerance of zero is established for residues of sulfamerazine (N
A tolerance of 0.1 part per million is established for negligible residues of sulfamethazine in the uncooked edible tissues of chickens, turkeys, cattle, and swine.
A tolerance of zero is established for residues of sulfanitran (acetyl(
A tolerance of 0.1 part per million is established for negligible residues of sulfaquinoxaline in the uncooked edible tissues of chickens, turkeys, calves, and cattle.
A tolerance of 0.1 part per million is established for negligible residues of sulfathiazole in the uncooked edible tissues of swine.
A tolerance of zero is established for residues of sulfomyxin (N-sulfomethyl-polymyxin B sodium salt) in uncooked edible tissues from chickens and turkeys.
No residues of testosterone, resulting from the use of testosterone propionate, are permitted in excess of the following increments above the concentrations of testosterone naturally present in untreated animals:
(a) In uncooked edible tissues of heifers:
(1) 0.64 part per billion in muscle.
(2) 2.6 parts per billion in fat.
(3) 1.9 parts per billion in kidney.
(4) 1.3 parts per billion in liver.
(b) [Reserved]
(a)
(b)
Tolerances are established at 0.1 part per million for negligible residues of thiabendazole in uncooked edible tissues of cattle, goats, sheep, pheasants, and swine, and at 0.05 part per million for negligible residues in milk.
(a)
(b)
(2)
A tolerance of 0.6 part per million is established for 8-
(a)
(b)
Tolerances are established for residues of tylosin in edible products of animals as follows:
(a) In chickens and turkeys: 0.2 part per million (negligible residue) in uncooked fat, muscle, liver, and kidney.
(b) In cattle: 0.2 part per million (negligible residue) in uncooked fat, muscle, liver, and kidney.
(c) In swine: 0.2 part per million (negligible residue) in uncooked fat, muscle, liver, and kidney.
(d) In milk: 0.05 part per million (negligible residue).
(e) In eggs: 0.2 part per million (negligible residue).
A tolerance of 200 parts per billion (ppb) is established for residues of tripelennamine in uncooked edible tissues of cattle and 20 ppb in milk.
(a)
(b)
(2)
(a)
(b)
A gas chromatographic method for the determination of the drug in frozen beef tissues is described. Tissue is frozen and stored in a deep freezer until ready for examination. A weighed portion of wet tissue (with exception of fat) is homogenized and lyophilized to dry solid. The drug is recovered from dry tissue by an extraction with methanol in a Soxhlet extractor. The methanol extract is digested in the presence of hydrochloric acid to hydrolyze conjugates should any be present. Elimination of impurities is brought about by liquid partition transfer successively to chloroform to 1
A. Carbon tetrachloride, N.F., Fisher Scientific C-186, or equivalent.
B. Chloroform, N.F., Fisher Scientific C-296, or equivalent.
C. Chromatograph gases, flow rates adjusted to maximize sensitivity for specific chromatograph.
1. Carrier gas, conventional tank helium.
2. Flame makeup gas.
a. Oxygen, conventional tank oxygen.
b. Hydrogen, Linde high purity, or equivalent.
D. Column packing, 3 percent GE SE-52 (Applied Science Laboratories) on P.E. Celite 60-80 mesh (Johns Manville Product No. 154-0048), or equivalent.
E. Ether, anhydrous, Fisher Scientific E-138, or equivalent.
F. Hexamethyldisilazane, Dow-Corning, Peninsular, or equivalent.
G. Hydrochloric acid, analytical reagent grade.
H. Methanol, certified A.C.S., spectranalyzed, Fisher Scientific A-408, or equivalent.
I. Phosphoric acid, analytical reagent grade.
J. Pyridine, anhydrous, A.C.S. reagent grade.
K. Silating reagent mixture: Pipet 8 milliliters each of pyridine and hexamethyldisilazane and 4 milliliters of trimethylchlorosilane into a clean glass vial with a polyethylene cap and mix thoroughly. Let stand overnight and decant supernatant liquid into a vial. Cap and store at room temperature for daily use. If kept dry, the reagent is stable for more than a month. Blanks are scanned by gas chromatography on each new bottle of J, F, and N material used in the silating reagent mixture for possible peak interference in the region of zeranol derivative.
L. Sodium chloride, analytical reagent grade.
M. Sodium hydroxide, analytical reagent grade.
N. Trimethylchlorosilane, Dow-Corning, Peninsular, or equivalent.
O. Water, distilled in glass.
P. Zeranol, primary standard.
Q. Solutions.
1. 2
2. 3
3. 2 percent w/v solium chloride in water.
4. 1
A. Extraction assemblies, Soxhlet, improved, standard taper grindings, Pyrex brand glass, 1,000 milliliters capacity, Sargent Catalog S-31265D, or equivalent.
B. Flasks, freeze drying, widemouth, 1,000 milliliters capacity, 24/40 standard taper grindings, Pyrex brand glass, Sargent Catalog S-28875-20-F, or equivalent.
C. Flasks, homogenizing, 250 milliliters, Sargent Catalog S-61716, or equivalent.
D. Funnels, separatory, Squibb stopper, with Teflon stopcock plug, Pyrex brand glass, 250- and 500-milliliter capacities, Sargent Catalog S-35815-20-F or G, or equivalent.
E. Gas chromatograph, F and M Model 5750 with flame ionization detector, or equivalent.
F. Gas chromatography column: Stainless steel tubing, 6 feet by
1. Prepare a TMS derivative of a 1,000-microgram zeranol standard as described in the procedure section. Inject 1-microliter quantities to determine whether the column is responding to the conditioning. After the column shows a response at the 1,000-microgram level, proceed to smaller quantities to optimize conditions.
2. The column and chromatograph must be conditioned to achieve a minimum sensitivity response so that a peak 5 millimeters in height results from an injection of 5 microliter of standard preparation containing 1 microgram of zeranol in the derivative preparation. This criterion must be met before tissue assay is attempted.
3. The column is brought to 250 °C. after conditioning and held at that temperature for at least 12 hours before making a run.
G. Heating mantle, electric, Glas-Col. Sargent Catalog S-40866H, or equivalent.
H. Hot plate, with gradient rheostat heat control.
I. Meat grinder, manually operated or equivalent.
J. Steam bath.
K. Syringe, Hamilton Micro Syringe Model 701, 10-microliter capacity, or equivalent.
L. Torsion balance, 0.1 gram sensitivity, 500 grams capacity.
M. Vials, 1-dram glass with plastic tops, Owens-Illinois, Opticlear, or equivalent.
N. Virtis freeze drier, Sargent Catalog S-28881-80, or equivalent.
O. Virtis homogenizing mill, macro, Virtis No. 45, Sargent Catalog S-61700, or equivalent.
A. Stock solution A: Accurately weigh 0.1000 gram of zeranol, primary standard, into a 250-milliliter beaker. Dissolve the standard in 80 milliliters of methanol and accurately dilute to 100 milliliters in a volumetric flask with methanol. By preparation, the solution contains 1,000 micrograms per milliliter.
B. Stock solution B: Dilute 10.0 milliliters of stock solution A to 100 milliliters with methanol to provide a standard containing 100 micrograms of the drug per milliliter.
C. Stock solution C: Dilute 5.0 milliliters of stock solution B to 100 milliliters with methanol to provide a standard of 5 micrograms per milliliter.
D. Stock solution D: Dilute 2.0 milliliters of stock solution B to 100 milliliters with methanol to provide a standard of 2 micrograms per milliliter. Transfer 1.0 milliliter of stock solution D to a 1-dram glass vial, evaporate to a dry residue in a vacuum desiccator at reduced pressure. The residue contains 2 micrograms of zeranol to be used as a calibration standard in operation of the gas chromatograph.
A. Preparation of glassware: Glassware should be washed in detergent or chromic acid solution to remove contaminants and rinsed in water to remove traces of cleaning agent. Rinse with methanol before using.
B. Preparation of sample.
1. Collect muscle, liver, kidney, and tripe from a freshly sacrificed animal under the cleanest conditions possible.
2. Grind the fresh tissue in a meat grinder, divide into 100-gram portions, and wrap in aluminum foil. Store wrapped tissue in a deep freeze. Fat should be wrapped in foil and stored in deep freeze.
C. Extraction procedure for muscle, liver, kidney, and tripe.
1. Weight 100 grams of partially thawed tissue into a 250-milliliter homogenizing flask, add 60 milliliters of water, and attach to a Virtis “45” Tissue Mill, or equivalent.
2. Mix the materials at 45,000 r.p.m. for 5 minutes to obtain a thin homogenate.
3. Transfer the homogenate to a 1-liter, widemouth, freeze drying flask using 10-20 milliliters of water for a rinse.
4. Place the flask on its side in a nearly horizontal position in a slurry of dry ice and acetone. Rotate the flask on its side as the homogenate cools to set down a uniform frozen solid layer on the wall of the flask.
5. Mount the flask on a Virtis freeze drier, or equivalent, and lyophilize to dry solids. This operation usually requires 20-24 hours.
6. Transfer the solid cake to a clean sheet of paper and crumble by hand to a size convenient for transfer to an extraction thimble.
7. Transfer the solids to a single thickness 60 x 180 milliliter Soxhlet extraction thimble and compact the solids sufficiently to guarantee complete immersion during solid extraction.
8. Transfer 600 milliliters of methanol to a 1-liter pot of a Soxhlet extraction assembly and place the thimble in the extractor. Mount a large glass funnel in the neck of the extractor with the stem extending into the thimble. Rinse the 1-liter freeze drying flask with three 50-milliliter portions of fresh methanol and transfer the rinses through the funnel into the thimble. Mount the condenser in the extractor and extract the solids for 15 hours. The extractor should be heated with the electric heating mantle so that a fill-empty cycle requires 18-24 minutes.
9. Drain the methanol from the thimble. Composite the methanol from the extractor and pot in an 800-milliliter beaker.
10. Rinse the pot with 10 milliliters of methanol and add to the methanol composite. Transfer 50 milliliters of 2
D. Extraction procedure for fat.
1. Cut fat into
2. Transfer 100 grams of the prepared fat to a 60- x 180-millimeter extraction thimble and extract with 750 milliliters of methanol for 15 hours in the Soxhlet extractor. The extractor should be heated with the electric heating mantle so that a fill-empty cycle requires 18-24 minutes.
3. Drain the methanol from the thimble. Composite the methanol from the extractor and pot into an 800-milliliter beaker.
4. Rinse the pot with 10 milliliters of methanol and add to the methanol composite. Transfer 50 milliliters of 2
E. Solvent partition.
1. Transfer the methanol concentrate to a 500-milliliter separatory funnel, identified by number as 1, with 70 milliliters of chloroform rinse and mix.
2. Add 300 milliliters of water and without shaking allow liquid phases to separate.
3. Withdraw the chloroform layer into a separatory funnel, identified by number as 2, containing 100 milliliters of 2 percent aqueous sodium chloride.
4. Gently mix the contents of funnel 2 horizontally end to end 30 times and allow phases to separate. Usually about 20 minutes are required to obtain maximum chloroform separation.
5. Withdraw the chloroform layer into a beaker.
6. Extract with shaking the contents of funnels 1 and 2 successively with three more 50-milliliter portions of chloroform.
7. Composite the chloroform extracts and concentrate to 125 milliliters by evaporation on a steam bath and cool to room temperature.
8. Transfer the chloroform composite to a 250-milliliter separatory funnel, fitted with a Teflon stopcock, using 10 milliliters of chloroform as a rinse.
9. Extract the chloroform with three separate 20-milliliter portions of 1
10. Perform an extraction by gently inverting the closed funnel and returning the funnel to an upright position.
11. Repeat phase mixing 30 times per extraction.
12. Allow phases to separate for 10 minutes. The time delay allows for gradual dissipation of the emulsion to improve phase separation. The zeranol transfers from the chloroform to the upper sodium hydroxide phase in this operation.
13. Composite the sodium hydroxide extracts.
14. Wash the sodium hydroxide extract with three 50-milliliter portions of chloroform using the technique as in step 9 and the same 10-minute interval for phase separation. Washing the chloroform removes the emulsion and unwanted impurities from the sodium hydroxide phase.
15. Discard the chloroform washes. Transfer the sodium hydroxide extracts to a 250-milliliter beaker. Rinse each separatory funnel with two 5-milliliter portions of water and add to the sodium hydroxide extract. Wash each funnel twice with tap water and twice with distilled water before next use.
16. Neutralize the washed sodium hydroxide extract to pH 8.0 by dropwise addition of 3
17. Transfer the pH 8.0 water extract to a 250-milliliter separatory funnel using 10 to 20 milliliters of water for a rinse.
18. Extract the solution with three separate 50-milliliter portions of carbon tetrachloride. The zeranol transfers to the lower carbon tetrachloride phase. Use the same 30-count phase-mixing technique as in step 9 and allow the mixture to stand 5 minutes for phase separation.
19. Composite the carbon tetrachloride extracts.
20. Extract the carbon tetrachloride composite with two 20-milliliter portions of 1
21. Composite the sodium hydroxide extracts.
22. Wash the extract with two 50-milliliter portions of carbon tetrachloride. Allow the mixture to stand 5 minutes for phase separation. Discard the carbon tetrachloride washes.
23. Transfer the sodium hydroxide extract into a 250-milliliter beaker. Rinse the separatory funnel with two 5-milliliter portions of water and add to the sodium hydroxide extract. Wash each funnel twice with tap water and twice with distilled water before next use. Adjust the sodium hydroxide extract to a pH of 9.5 by dropwise addition of 3
24. Extract the pH 9.5 water solution with three separate 30-milliliter portions of anhydrous ethyl ether. Allow the mixture to stand 5 minutes for phase separation. The zeranol transfers to the upper ether phase.
25. Composite the ether extracts in a 125-milliliter Erlenmeyer flask.
26. Reduce the volume of ether to about 1-2 milliliters by evaporation on a hot plate with low heat while removing vapor from top of flask by vacuum aspiration.
27. Transfer ether residue to a 1-dram glass vial. Rinse down flask side wall with 1-2 milliliters of fresh ether and transfer to the glass vial.
28. Continue evaporation of ether to 0.1 milliliter.
29. Place vial in a vacuum desiccator and evaporate residue at line vacuum and room temperature overnight to dryness.
30. Close vial with a plastic cap and submit ether residue for preparation of TMS derivative and gas chromatographic assay.
F. Gas liquid chromatography.
1. Start the gas chromatography and maintain the following operational conditions:
Carrier gas pressure: 50 p.s.i. at tank.
Carrier gas flow rate: Sufficient to give zeranol derivative peak a retention time of 4-8 minutes.
Electrometer range: 10
Detector temperature: 325 °C.
Injection port temperature: 325 °C.
Column temperature: 250°-280 °C., operate isothermally.
Recorder sensitivity: 1 millivolt.
Recorder chart speed: 1 inch per minute.
Sample size: 1 microliter to 5 microliters as necessary to give desired peak area for quantitative measurement.
Septums: Replace each evening and allow to condition overnight at operational temperature.
Flame assembly: Remove silica ash from the flame assembly each week. The flame assembly is removed; the anode, flame jet, and chimney are cleaned with a nylon bristle brush. Water and acetone are drawn through the jet capillary to remove any foreign material.
2. Add 0.2 milliliter of silating reagent to the sample or to the zeranol standard.
3. Stopper the vial and shake vigorously.
4. Warm the vial at 40°-50° C. for a few minutes, then roll the vial on a horizontal plane to insure that all of the interior surfaces of the vial have been in contact with the reagent.
5. Let vial stand for 4 hours or overnight in a warm area (40 °C.) to allow reaction to reach completion.
6. Place vial in a small padded centrifuge tube and centrifuge to settle the precipitate and insure that all the liquid is at the bottom of the vial.
7. Inject 1.0-5.0 microliters of clear solution into the chromatograph. At the beginning of the day's run, make 3-5 injections of a standard to condition the column for that day before taking quantitative data.
8. Run known mixtures at the beginning, middle, and end of the day's run over the concentration range of samples to be analyzed to compensate for day-to-day sensitivity fluctuations and drift. If four or less
Area values are obtained on known mixtures and samples by multiplying the net peak height by the peak width at half height or by counting squares. Area values obtained on knowns are plotted versus zeranol concentration. Calibration plots indicate a near linear function in the 0-10 microgram range. Area values obtained on samples are converted directly to microgram quantities using the curve. Control tests demonstrated a 70 percent recovery of zeranol from spiked wet beef liver and muscle necessitating a correction factor.
0.7=Correction factor for 70 percent recovery.
W=Grams of tissue examined.
A. Fortification of reagent blank.
1. For those using this method for the first time either for recovery study or tissue assay, a solvent blank and solvent fortified with zeranol should be processed through the entire procedure. This preliminary operation will establish whether or not the procedure is free from contamination arising from solvents and glassware and demonstrate the level of recovery of the standard zeranol. Level of recovery should be in the same range as the samples.
2. Transfer 600 milliliters of methanol to a 1-liter beaker. Add 50 milliliters of 2
3. Transfer 600 milliliters of methanol to a 1-liter beaker. Add 50 milliliters of 2
4. Assay both samples as described in the procedure beginning extraction step V-E1.
B. Fortification of samples.
1. Transfer 100-gram portions of partially thawed tissues into 250-milliliter homogenizing flasks and set half of them aside to serve as tissue blanks.
2. Add to the remaining samples 1 milliliter of stock solution D to serve as fortified samples to which 20 parts per billion zearalanol have been added.
3. Assay both fortified and unfortified tissue as described in the procedure section beginning with V-C1.
Tolerances are established for residues of zoalene (3,5-dinitro-
(a) In edible tissues of chickens:
(1) 6 parts per million in uncooked liver and kidney.
(2) 3 parts per million in uncooked muscle tissue.
(3) 2 parts per million in uncooked fat.
(b) In edible tissues of turkeys: 3 parts per million in uncooked muscle tissue and liver.