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II. Reagents. A. Acetone.

B. Acetyl-(p- nitrophenyl)-sulfanilamide (APNPS) standard—melting point range 264 °C.–267 °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 °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 °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 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 °C.) until the solvents are evaporated.

6. Add 10 milliliters of 4N HCl to each, cover with watch glasses and heat (90 °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 °C.–50° C.).

N. Water bath (90 °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 °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 °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 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 °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 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 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 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 °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 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.