[Code of Federal Regulations]
[Title 21, Volume 6]
[Revised as of April 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 21CFR556.220]
[Page 351-354]
TITLE 21--FOOD AND DRUGS
CHAPTER I--FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN
SERVICES (CONTINUED)
PART 556_TOLERANCES FOR RESIDUES OF NEW ANIMAL DRUGS IN FOOD--Table of
Subpart B_Specific Tolerances for Residues of New Animal Drugs
Sec. 556.220 3,5-Dinitrobenzamide.
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. Method of analysis--3,5-dinitrobenzamide. A method for 3,5-
dinitrobenzamide (3,5-DNBA) in chicken tissues is described with a
cleanup step that removes most of the interfering materials, thus
allowing uncompensated measurements to be read. The 3,5-DNBA is
extracted from the sample with acetone and chloroform and prepared for
chromatography by removing the aqueous phase in a separatory funnel and
the solvents in a flash evaporator. The extract residue is
chromatographed on alumina to remove several lipid components and
residues of other drugs. The benzamide eluate is passed through a column
of Dowex-50 resin, or equivalent, to remove arylamines; for example, 3-
amino-5-nitrobenzamide. The 3,5-DNBA fraction is reduced, after removal
of alcohol, with TiCl3 in basic solution to an arylamine,
presumably 3,5-diaminobenzamide. The reduced fraction is placed on
another Dowex-50 column, most of the interfering substances are removed
with washings of alcohol and water, and the arylamine residue is eluted
with 4N HCl. Colorimetric measurement is made in a 100-millimeter cell
at 530 millimicrons after reacting the residue with Bratton-Marshall
reagents.
II. Reagents. A. Acetone.
B. Acetyl-(p- nitrophenyl)-sulfanilamide (APNPS) standard--melting
point range 264 [deg]C.-267 [deg]C. Weigh and transfer 10 milligrams of
APNPS to a 100-milliliter flask, dissolve and dilute to volume with
acetone.
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 6N HCl.
3. Heat and stir while on a bath at 80 [deg]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 6N HCl.
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 N-1-naphthyl-ethylenediamine
dihydrochloride per 100 milliliters of water. Refrigerate when not in
use. Prepare fresh weekly.
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 [deg]C.-
186 [deg]C.
J. Ethyl alcohol--absolute, A.C.S.
[[Page 352]]
K. Eluting reagent A. The formula and volume required in procedure
step V-D is dependent on the adsorptive strength of the Al2
O3. For each lot Al2 O3, make the
following test:
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
[deg]C.) until the solvents are evaporated.
6. Add 10 milliliters of 4N HCl to each, cover with watch glasses
and heat (90 [deg]C.) for 30 minutes; cool to room temperature.
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 Al2 O3 should be
used.
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 these are suitable, another lot of
Al2 O3 should be used.
L. Hydrochloric acid, 4N. Add two volumes of water to one volume of
HCl.
M. Diatomaceous earth--Hyflo Super Cel, Johns-Manville Co., or
equivalent substance.
N. N-1-Naphthylethylenediamine dihydrochloride.
O. Sodium hydroxide solution, 10N. Dissolve 100 grams of sodium
hydroxide in water and dilute to 25 milliliters.
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. Special apparatus. A. Absorption cells--Beckman No. 75195
matched set of two cylindrical silica cells with 100 millimeter optical
length, or equivalent cells.
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-o-toluamide (ANOT) in chicken
tissues'' published in ``Journal of Agricultural and Food Chemistry,''
volume 9, pages 201-204 (1961).
H. Glycerol manostat. For regulating pressure on columns: To
Al2 O3 columns, 15-inch head pressure; to ion-
exchange columns, 30-inch head pressure.
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 [deg]C.-50[deg] C.).
N. Water bath (90 [deg]C.).
IV. Standard curve. A. 1. Weigh 100 milligrams of 3,5-DNBA and
transfer to a 1-liter volumetric flask with acetone.
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 [deg]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 TiCl3 and 4 drops of 10N
NaOH. Continue mixing until chalky-white in appearance.
3. Add 2 milliliters of HCl, mix, and allow to stand for 5 minutes.
4. Transfer to 50-milliliter volumetric flask and dilute with 4N HCl
to 40-45 milliliters.
5. Cool to 0 [deg]C.-5 [deg]C. by placing in a freezer or ice bath.
[[Page 353]]
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 4N HCl.
C. Perform colorimetric measurement at 530 millimicrons as follows:
1. Fill two matched 100-millimeter cells with 4N HCl and place into
spectrophotometer.
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. Procedure. A. Extraction. 1. Mince 350 grams of tissue in a 1-
quart blending jar for 3 minutes. Use samples obtained from either
freshly killed or quickly frozen birds. The latter should be analyzed as
soon as thawed. For fibrous meats (for example, muscle, skin) put
through a meat grinder before mincing.
2. Weight 100 0.5 grams of each replicate
sample in a 150-milliliter beaker. Analyze each sample in triplicate and
average the results. Reproducibility of 10 percent
between such analyses has been obtained.
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
[deg]C.-50 [deg]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
Al2O3.
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
[[Page 354]]
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 [deg]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 [deg]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 10N
sodium hydroxide. Continue mixing until greyish color disappears.
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 4N HCl into a 50-milliliter volumetric flask (actinic ware) for 3,5-
DNBA analysis. Avoid direct sunlight. The arylamine has been found to be
photosensitive.
H. Color development and measurement. 1. Cool to 0 [deg]C.-5 [deg]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 4N HCl.
3. Perform colorimetric measurement at 530 millimicrons as follows:
a. Fill two matched 100-millimeter cells with 4N HCl and place into
instrument.
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.