nal potency when determined as follows: Dilute a weighed sample (approximately 30 mg.) with 1 percent phosphate buffer at pH 6.0 to a concentration of approximately 1.2 mg./ml. (2,000 units/ml.). Add 2.0 ml. aliquots to each of two 125ml. glass stoppered Erlenmeyer or iodine flasks. To one add 2.0 ml. of 1N NaOH and allow to stand at room temperature for 15 minutes. At the end of this time add 2.0 ml. of 1.2 N HCl and add 10.0 ml. of 0.01N I. (prepared from 0.1N I, U. S. P.). (Equal volumes of 1N NaOH and 1.2N HCl when mixed give pH 1.0.) After 15 minutes titrate the excess iodine

Percent loss of potency=

using 0.01N Na2S2O, (prepared from 0.1N Na2SO3) standardized accurately against potassium iodate. Toward the end of the titration add approximately 5 ml. of CCL. Continue the titration by the addition of 0.01 to 0.02 ml. portions of the 0.01N Na2S2O3 shaking vigorously after each addition. The end-point is reached when the CC1 layer becomes colorless. To the second flask add 10 ml. of the 0.01N I, and titrate immediately with 0.01N Na2S2O for the blank determination. Regard the difference in titers divided by 2.52 as the milligrams of penicillin sodium salt.

(f) Crystalline penicillin G (1) Reagents. The reagents described in subdivisions (i), (ii), and (iii) of this subparagraph are freshly prepared every three days and are of such quality that when used in this procedure with a known penicillin G not less than 97 percent of penicillin G is recovered.

(i) Amyl acetate solution. Saturate the amyl acetate with the N-ethyl piperidine salt of penicillin G by adding 2 mg. of the salt for each 1 ml. of the solvent. Cool this solution to 0° to 8° C. and filter by drawing it through a plug of cotton on the tip of a pipette immediately before use.

(ii) Acetone solution. Saturate reagent grade acetone with the N-ethyl piperidine salt of penicillin G using 3 mg. of salt for each 1 ml. of acetone. Cool this solution to 0° to 8° C. and filter by drawing it through a plug of cotton on the tip of a pipette immediately before

use.

(iii) N-ethyl piperidine solution. Nethyl piperidine should be stored in brown bottles in a refrigerator. Dilute 1.0 ml. of this reagent with 4.0 ml. of amyl acetate. Saturate this solution with the N-ethyl piperidine salt of penicillin G using about 3 mg. of the salt for each 1.0 ml. of solution. Cool this solu- . tion to 0° to 8° C. and filter by drawing it through a plug of cotton on the tip of a pipette immediately before use.

(iv) Phosphoric acid solution. Prepare by dissolving 1.0 ml. of reagent grade phosphoric acid (85 percent) in 4.0 ml. of water. Cool to 0° to 8° C. and shake before using.

(v) Sodium sulfate. Use powdered anhydrous reagent grade sodium sulfate.

(2) Procedure. Accurately weigh from 60 to 70 mg. of the sample to be tested in a glass test tube or glass vial of approximately 10 ml. capacity. Add 2.0 ml. of water to dissolve the penicillin and cool the solution to 0° to 5° C. Add 2 ml. of the amyl acetate solution and 0.5 ml. of the phosphoric acid solution, stopper and shake vigorously for approximately 15 seconds. Centrifuge to obtain a clear separation of the two layers (approximately 20 seconds). After centrifuging remove as much of the amyl acetate layer as possible (usually about 1.7 to 1.8 ml.) with a 2 ml. hypodermic syringe equipped with a suitable needle. Place about 0.1 gm. of the sodium sulfate in a micro filter funnel (approximately 10 mm. diameter) having a fritted glass disc of medium porosity and add the amyl acetate solution from the hypodermic syringe. Collect the filtrate by suction in a small test tube placed in a suction flask, which is surrounded by cracked ice. Pipette a 1.0 ml. aliquot of the amyl acetate filtrate into a tared flat bottom glass tube (approximately 15 x 50 mm.) containing 1.0 ml. of the acetone solution and 0.5 ml, of the N-ethyl piperidine solution. The time elapsing between acidification and the addition of the filtrate to the above reagents should not be more than three minutes. Place the glass tube containing this mixture in a large weighing bottle, stopper the bottle and allow to stand for not less than 2 hours in a refrigerator at 0° to 8° C. Remove the liquid from the precipitate by means of a tared micro filter stick and wash with

(g) Penicillin K content. Determine the content of penicillin K by the following method:

Al

Dilute a weighed sample or the contents of a vial with 0.3 M phosphate (Na HPO, and KH,PO.) buffer pH 6.0 to give a solution containing approximately 1,000 units/ml. In the case of calcium penicillin where a precipitate of calcium phosphate occurs, remove the precipitate by filtration and use the clear filtrate. Place a 15.0 ml. aliquot of this solution in a 125 ml. separatory funnel, add 30.0 ml. of chloroform U. S. P. and shake for 1 minute. (Carry out all operations at room temperature.) low the mixture to stand with occasional swirling to settle the droplets of chloroform until the top layer is clear (usually about 10 minutes). Draw off all but about 2 ml. of the lower chloroform layer thru a small pledget of cotton into a glass stoppered flask. Take a 4.0 ml. aliquot of the original solution, a 4.0 ml. aliquot of the buffer solution remaining in the separatory funnel and a 10.0 ml. aliquot of the chloroform solution and determine the mg./ml. of penicillin in each by the iodometric assay procedure described in paragraph (e) of this section using 4.0 ml. of the 1N NaOH and 4.0 ml, of the 1.2N HCl for each of the above aliquots. Make blank determinations on the same size aliquots. Calculate the percent penicillin in the buffer layer and in the chloroform layer as compared to the original solution. The sum of these percentages should be 100% ±2%. The percent penicillin K= (96.92% in chloroform-% in buffer) X1.67. (The factors in the above formula are based on distribution coefficients of penicillin K and G between chloroform and aqueous phosphate buffer at pH 6.0.)

[12 F. R. 2222, 2745, as amended at 12 F. R. 8724]

§ 141.6 Sodium penicillin, calcium penicillin, potassium penicillin; penicillin X. Dissolve the contents of a 100,000

unit ampul in about 20 ml. of ice cold distilled water. Transfer quantitatively to a 100 ml. volumetric flask, rinsing the ampul with small portions of ice cold water and make to 100 ml. Pipette a 50.0 ml. aliquot into a 125 ml. separatory funnel, then add 50.0 ml. of cold chloroform and shake the mixture. Add an amount of approximately 1N H2SO, to bring the pH of the aqueous layer to 2.0. (The amount of 1N H2SO, to be added is calculated by titrating a separate 5.0 ml. aliquot of the 100 ml. dilution to pH 2.0 using a suitable pH meter.) Shake the mixture vigorously for one minute. Allow the layers to separate and filter the chloroform through a small pledget of cotton, moistened with chloroform, into a second 125 ml. separatory funnel. Shake the acid aqueous solution with a second 50.0 ml. of cold chloroform and, when the layers have separated, withdraw the chloroform through the same filter into the second separatory funnel. Immediately neutralize the acid aqueous solution, containing the penicillin X, with 0.1N NaOH to pH 6.5 to 7.0 using the pH meter and make to 100 ml. with water. Make appropriate dilutions in 1 percent phosphate buffer at pH 6.0 and assay as directed in § 141.1 (f) or (h). Shake the combined chloroform extracts, containing any penicillin G, etc., with small successive portions of cold NaHCO3 solution (0.1 percent), until the combined NaHCO, extracts give a pH of 7.0, and make to 100 ml. with water. Make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. Assay these last dilutions as directed in § 141.1 (f) or (h). The potency of the penicillin X fraction plus potency of the penicillin G, etc., fraction should approximate that of the potency of the original solution. All of the above extractions should be carried out in a cold room.

[12 F. R. 2223]

§ 141.7 Penicillin in oil and wax-(a) Potency. Proceed as directed in § 141.1

except paragraph (i) thereof and, in lieu of the directions in paragraph (d), of § 141.1, prepare sample as follows:

Liquefy the sample by warming, thoroughly mix, and withdraw 1.0 ml. using a sterile syringe equipped with an 18 gauge needle. Transfer to a separatory funnel containing approximately 50 ml. of peroxide-free ether. Shake the separatory funnel vigorously to bring about complete mixing of the material with the ether. Shake with a 25 ml. portion of 1 percent phosphate buffer at pH 6.0. Remove the buffer layer and repeat the extraction with three 25-ml. quantities of buffer. Combine the extracts and make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The sample may also be prepared by transferring aseptically 1.0 ml. of the penicillin in oil and wax to a blending jar containing 100 ml. of sterile distilled water. Using a high-speed blender, blend this mixture for 1 minute and then make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. If the label represents the potency of the penicillin in oil and wax as 200,000 units per ml. or less, it is satisfactory if it is 85 percent or more of the potency so represented; if represented as more than 200,000 units per ml., it is satisfactory if it is 90 percent or more of the potency so represented.

(b) Sterility. Proceed as directed in § 141.2, except that sufficient penicillinase is added to the thioglycollate medium to inactivate the penicillin added in the test and, in lieu of the directions in the first three sentences of paragraph (b) of § 141.2, proceed as follows:

Liquefy the sample by warming and add aseptically approximately 1.0 ml. to 9.0 ml. of sterile warm cottonseed oil. Shake vigorously. Transfer 1.0 ml. aseptically to each of four tubes containing 15 ml. of fluid thioglycollate medium with added penicillinase.

(c) Moisture-(1) Reagents—(i) Karl Fischer reagent. Preserve the reagent in glass-stoppered bottles and use from an all glass automatic burette, protecting the solution from the moisture in the air.

(ii) Water-methanol solution. Use methanol containing approximately 1 mg. of water per milliliter. Store the solution in a glass bottle attached to an automatic burette and protect from moisture in the air at all times.

(2) Standardization of Karl Fischer reagent. Add a known volume of the Karl Fischer reagent to a suitable titrat

ing vessel which has been previously dried at 105° C. and cooled in a desiccator. Introduce a mechanical stirrer and two platinum electrodes which are connected to a suitable electrometric apparatus for measurement of the endpoint. Start the stirrer and titrate with the water-methanol solution until the endpoint is reached. Calculate the milliliters of Karl Fischer reagent equivalent to each milliliter of water-methanol.

Add an accurately weighed quantity of water (approximately 50 mg.) to a dry titrating vessel, add an excess of the Karl Fischer reagent and back titrate with the water-methanol solution as above. Calculate the milligrams of water equivalent to each milliliter of the Karl Fischer reagent. Standardize the Karl Fischer reagent in this manner daily.

volume of Karl Fischer reagent used. v1 = volume of methanol used. ƒ=volume ratio of Karl Fischer reagent to water-methanol solution.

(3) Procedure. Transfer 1.0 ml. of the penicillin in oil and wax to a dry titrating vessel, add 10 ml. of dry chloroform and an excess of the Karl Fischer reagent and back titrate with the watermethanol solution until the endpoint is reached. Transfer 10 ml. of the dry chloroform used to a dry titrating vessel, add an excess of Karl Fischer reagent, and titrate with the water-methanol above. Calculate the milliliters of Karl Fischer reagent equivalent to 10 ml. of chloroform.

as

b=milliliters Karl Fischer reagent equivalent to 10 ml. of chloroform s=volume of the sample in milliliters.

(d) Measurement of penicillin particle size. Vigorously shake the container to obtain an even suspension of the penicillin particles and immediately withdraw therefrom approximately 0.5 ml of the drug into a clean, dry, tuberculin syringe using a dry 18 gauge needle. Discard approximately the first 5 drops of the mixture extruded from the needle and then extrude approximately 1 minim of the remaining mixture into a test-tube containing 3 to 4 ml. of light mineral oil. Thoroughly mix the contents of the tube

and by means of a bacteriological loop (2 mm. inside diameter, 22 gauge wire), immediately place one loopful of the suspension on each ruled chamber of a bright line hemocytometer. (It is not necessary to use a cover slip.) Confirm by means of the low power objective of the microscope the even distribution of particles over the ruled areas of both chambers and repeat with another loopful of the suspension if even dispersion is not obtained.

Use a magnification of 430 or 440 diameters and a calibrated ocular micrometer to measure the penicillin particles. For the purposes of measurement and calculation, the predominant type of crystals observed shall be considered to represent the type of crystals present and the thickness and density of all particles shall be considered constant. Center a large penicillin particle in the microscopic field; measure the particle and all other particles in the field and repeat this operation on other fields until at least 200 particles are measured. Particles of less than 5 microns in length are disregarded. The grouping of the particles by length, the midpoint, the ratio of the midpoints, and the square of the ratio of the midpoints for each group are tabulated below:

When examined by the method described in this section not less than 50 percent of the total relative weight of the penicillin in the drug consists of penicillin having a particle size of not less than 50 microns in length.

[12 F. R. 2223, 2745, as amended at 12 F. R. 7997, and 13 F. R. 4186, 4315]

§ 141.8. Penicillin ointment-(a) Potency. Proceed as directed in § 141.1, except paragraph (i) thereof, and, in lieu of the directions in paragraph (d) of § 141.1 prepare the sample as follows:

Accurately weigh the container and contents and place 0.5 to 1.0 gm. into a separatory funnel containing approximately 50 ml. of peroxide-free ether. Reweigh the container to obtain weight of ointment used in the test. Shake ointment and ether until homogeneous. Shake with a 25 ml. portion of 1 percent phosphate buffer at pH 6.0. Remove the buffer layer and repeat the extraction with three 25 ml. quantities of buffer. Combine the extracts and make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The sample may also be prepared by placing an accurately weighed sample consisting of 0.5 to 1.0 gm. of the ointment into a glass blending jar containing 100 ml. of 1 percent phosphate buffer at pH 6.0. Using a high-speed blender, blend the mixture for 2 minutes and make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The potency of penicillin ointment is satisfactory if it contains not less than 85 percent of the number of units per gram it is represented to contain.

(b) Moisture. Proceed as directed in § 141.7 (c), using a weighed sample of 1.0 to 2.0 gm. dissolved in a mixture of 10 ml. of dry chloroform and 10 ml. of carbon tetrachloride, but in lieu of calculating the milliliters of Karl Fischer reagent equivalent to 10 ml. of chloroform, determine the milliliters of reagent equivalent to 20 ml. of the mixture of chloroform and carbon tetrachloride.

(c) Microorganism count. Prepare nutrient agar as directed in § 141.1 (b) (1). Cool to approximately 48° C. and add sufficient sterile penicillinase solution so that each 20 ml. will contain enough to completely inactivate the amount of penicillin contained in the sample under test. Pour 20 ml. of the agar-penicillinase mixture into Petri dishes and allow to harden. The Petri

Re

dishes are warmed to 37° C. just before use. Accurately weigh the container and contents, place in incubator at 37° C. for one hour, then place 0.1 to 0.5 gm. of the ointment onto the agar surface. weigh the container to obtain weight of ointment used in test. Spread the ointment evenly over the surface of the agar with a sterile glass rod, invert, and place in a 37° C. incubator for 48 hours. Count the number of colonies appearing on the plates and calculate therefrom the number of viable microorganisms per gm. of ointment.

[12 F. R. 2223, as amended at 13 F. R. 2950, 4186]

§ 141.9 Tablets buffered penicillin(a) Potency. Proceed as directed in § 141.1, except paragraph (i) thereof and, in lieu of the directions in paragraph (d) of § 141.1, prepare sample as follows:

Place 12 tablets in a mortar and add approximately 20 ml. of 1 percent phosphate buffer at pH 6.0. Disintegrate the tablets by grinding with a pestle. Transfer with the aid of small portions of the buffer solution to a 500 ml. volumetric flask and make to 500 ml. by adding sufficient phosphate buffer. Make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The sample may also be prepared as follows: Place 12 tablets in a blending jar and add thereto approximately 200 ml. of a 500ml. quantity of sterile distilled water. After blending for 1 minute with a highspeed blender add the remainder of the 500 ml. of water. Blend again for 1 minute and make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The average potency of tablets buffered penicillin is satisfactory if it contains not less than 85 percent of the number of units per tablet it is represented to contain.

(b) Moisture. Proceed as described in § 141.5 (a).

(c) Microorganism count. Proceed as directed in § 141.21 (b).

[12 F. R. 2223, as amended at 13 F. R. 4186, and 8737]

§ 141.11 Penicillin with aluminum hydroxide gel-(a) Sodium penicillin, calcium penicillin, potassium penicillin. Proceed as directed in §§ 141.1, 141.2, 141.4, and 141.5 (a) and (b), and if crystalline penicillin, (d), (e), and (g), and if crystalline penicillin G, (f).

(b) Aluminum hydroxide gel. Thoroughly shake the aluminum hydroxide gel and transfer aseptically 1.0 and 0.1 ml. portions in triplicate to sterile Petri dishes. Pour into each Petri dish 20 ml. of nutrient agar, described in § 141.1 (b) (1), which has been melted and cooled to 48° C. Thoroughly mix the aluminum hydroxide and melted agar. Allow the agar to solidify, invert the Petri dishes, and incubate for 48 hours at 37° C. Count the number of colonies appearing on the plates and calculate therefrom the number of viable bacteria per ml. of the aluminum hydroxide gel.

[12 F. R. 2223]

§ 141.12 Penicillin troches-(a) Potency. Proceed as directed in § 141.1 except paragraph (i) thereof and, in lieu of the directions in paragraph (d) of § 141.1, prepare sample as follows:

If the troche does not contain a masticatory substance, proceed as directed in § 141.9 (a). If the troche contains a masticatory substance, place five troches in a separatory funnel containing 75 ml. of n-hexane; shake until the troches are dissolved. Shake with a 25 ml. portion of 1 percent phosphate buffer at pH 6.0. Remove the buffer layer and repeat the extraction with three 25 ml. quantities of buffer. Combine the extracts and make the proper estimated dilutions in 1 percent phosphate buffer at pH 6.0. The average potency of the troche is satisfactory if it contains not less than 85 percent of the number of units per troche it is represented to contain.

(b) Moisture. Proceed as directed in § 141.5 (a), or if it contains a masticatory substance proceed as directed in § 141.7 (c), using 1.0 to 2.0 gm. dissolved in 10 ml. of dry chloroform.

[12 F. R. 2223, as amended at 13 F. R. 4186] § 141.13 Penicillin dental cones-(a) Potency. Proceed as directed in § 141.1, except paragraph (i) thereof and, in lieu of the directions in paragraph (d) of § 141.1, prepare sample using 5 cones as directed in § 141.9 (a). The average potency of the cone is satisfactory if it contains not less than 85 percent of the number of units per cone it is represented to contain.

(b) Microorganism count. Accurately weigh from 3 to 5 cones in a small test tube and add sufficient sterile penicillinase, contained in a total volume of 2 ml. to inactivate the penicillin present. Let stand one hour. Thoroughly shake the