paragraph if it is not less than 85 percent of that which it is represented to contain.

(d) Total potency of a one-dose container. Wash out the material remaining in the 10-milliliter volumetric flask referred to in paragraph (b) (1) of this section with 1-percent phosphate buffer, pH 6.0. Dilute to give a concentration of approximately 2,000 units per milliliter, and assay by the iodometric method described in § 141a.5 (d) (1). Obtain the total potency by adding the number of units found in this solution (units per milliliter X volume) to the number of units found (units per milliliter X volume) in the solution assayed in accordance with paragraph (b) (2) of this section.

§ 141a.33 Buffered penicillin powder. Proceed as directed in §§ 141a.1 and 141a.5 (a).

§ 141a.34 Procaine pencillin and crystalline penicillin in oil-(a) Total potency. Proceed as directed in § 147a.27 (a) or by the iodometric assay procedure described in § 141a.5 (d) (1), using in the latter procedure a 0.5-milliliter aliquot of the solution prepared as follows: Introduce 1 milliliter of the well-shaken sample, by means of a hypodermic syringe, into a 50-milliliter volumetric flask. Make to 50 milliliters with chloroform-absolute alcohol (1+1) solvent and shake well.

(b) Crystalline penicillin. Proceed as directed in § 141a.32 (b), except prepare the sample as follows: Introduce 1 milliliter of the well-shaken sample, by means of a hypodermic syringe, into a 30-milliliter centrifuge tube equipped with a screw cap. Add 10.0 milliliters of chloroform and 10.0 milliliters of a 20 percent sodium sulfate solution, shake well for about 1 minute and centrifuge to obtain a clear upper layer.

(c) Procaine penicillin. The difference between the total penicillin as determined by paragraph (a) of this section and the crystalline penicillin as determined by paragraph (b) of this section represents the amount of procaine penicillin present.

(d) The procaine penicillin and the crystalline penicillin content of the batch are satisfactory when assayed by the methods described in this section if each is not less than 85 percent of that which it is represented to contain.

(e) Sterility. Proceed as directed in § 141a.7 (b), except in the case of multiple-dose containers use approximately 400 milligrams (activity) from each container, in lieu of 300 milligrams.

(f) Moisture. Proceed as directed in § 141a.7 (c).

§ 141a.35 Penicillin-streptomycin ointment, penicillin - dihydrostreptomycin ointment-(a) Potency-(1) Penicillin content. Proceed as directed in § 141a.8 (a). Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units per gram of ointment that it is represented to contain.

(2) Streptomycin content. Proceed as directed in § 141b.101 of this chapter, except paragraph (k) of that section, and in lieu of the directions in § 141b.101 (e) and (j) (3), test a representative portion of the sample (usually approximately 1 gram, accurately weighed) or the entire contents of a single-dose container prepared by one of the following methods:

(i) To assay by the cup-plate method. Use either extraction or blending.

(a) Extraction. Place the sample in a separatory funnel containing approximately 50 milliliters of peroxide-free ether. If the sample consists of substantially more than 1 gram, use 100 milliliters of ether. Shake the sample and ether until homogeneous. Add 20 milliliters of 0.1 M potassium phosphate buffer, pH 8.0, and shake. If the sample consists of substantially more than 1 gram, use 50 milliliters of buffer. Allow the layers to separate. Remove the buffer layer and repeat the extraction with new portions of buffer at least three times and any additional times necessary to ensure complete extraction of the antibiotic. Combine the extractives and make up to an appropriate measured volume with buffer. To a suitable aliquot add sufficient penicillinase and let stand for 30 minutes at 37° C. to inactivate the penicillin. After inactivation, make the proper estimated dilution with buffer at pH 8.0.

(b) Blending. Place the sample in a blending jar containing 1.0 milliliter of 10-percent aqueous solution of polysorbate 80 and sufficient 0.1 M potassium phosphate buffer, pH 8.0, to give a final volume of 500 milliliters. Using a highspeed blender, blend the mixture for 3

minutes. To a suitable aliquot, add sufficient penicillinase and let stand for 30 minutes at 37° C. to inactivate the penicillin. After inactivation, make the proper estimated dilutions with buffer at pH 8.0.

(ii) To assay by the turbidimetric method. Place the sample in a separatory funnel containing approximately 50 milliliters of peroxide-free ether. If the sample consists of substantially more than 1 gram, use 100 milliliters of ether. Shake the sample and ether until homogeneous. Add 20 milliliters of distilled water, and shake. If the sample consists of substantially more than 1 gram, use 50 milliliters of water. Allow the layers to separate. Remove the aqueous layer and repeat the extraction with new portions of water at least three times and any additional times necessary to ensure complete extraction of the antibiotic. Combine the extractives, and make to an appropriate measured volume with water. Remove the aliquot and, if the ratio of the content of penicillin to the content of streptomycin is equal to or greater than one unit for each microgram, add sufficient penicillinase and let stand for 30 minutes at 37° C. to inactivate the penicillin. Make the proper estimated dilutions with distilled water. Its content of streptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(3) Dihydrostreptomycin content. Proceed as directed in subparagraph (2) of this paragraph, using the dihydrostreptomycin working standard as a standard of comparison. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams per gram of ointment that it is represented to contain. (b) Moisture. Proceed as airected in § 141a.8 (b).

§ 141a.36 Penicillin-streptomycin bougies, penicillin-dihydrostreptomycin bougies-(a) Potency-(1) Penicillin content. Proceed as directed in § 141a.9 (a), except the last sentence of that paragraph. Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units that it is represented to contain.

(2) Streptomycin content. Using 12 bougies, proceed as directed in § 141b.101 of this chapter, except paragraph (k) of that section, and if the cup-plate method

is used, use potassium phosphate buffer (pH 7.8-8.0) for dissolving the sample in lieu of sterile distilled water as directed in § 141b.101 (e) of this chapter and add sufficient penicillinase to the solution under test to completely inactivate the penicillin present. If the turbidimetric method is used, inactivatiion with penicillinase is not necessary unless the ratio of the content of penicillin to the content of streptomycin is equal to or greater than 1.0 unit for each microgram. Its content of streptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(3) Dihydrostreptomycin content. Proceed as directed in subparagraph (2) of this paragraph, using the dihydrostreptomycin working standard as a standard of comparison. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams it is represented to contain.

(b) Moisture. Proceed as directed in § 141a.22 (b).

§ 141a.37 Penicillin bacitracin ointment (a) Potency-(1) Penicillin content. Proceed as directed in §141a.8 (a), except the last sentence of that paragraph. Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units it is represented to contain.

(2) Bacitracin content. Proceed as directed in § 141e.402 (a) of this chapter, except that sufficient penicillinase is added to the sample under test to completely inactivate the penicillin present. Its content of bacitracin is satisfactory if it contains not less than 85 percent of the number of units it is represented to contain.

(b) Moisture. Proceed as directed in § 141a.8 (b).

§ 141a.38 Procaine penicillin and streptomycin in oil, procaine penicillin and dihydrostreptomycin in oil—(a) Potency-(1) Penicillin content. Proceed as directed in § 141a.27 (a) except the last sentence thereof. Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units per milliliter that it is represented to contain.

(2) Streptomycin content. Using 1 milliliter as the test sample proceed as directed in § 141a.35 (a) (2). Its content of streptomycin is satisfactory if it

contains not less than 85 percent of the number of milligrams per milliliter that it is represented to contain.

(3) Dihydrostreptomycin content. Using 1 milliliter as the test sample proceed as directed in § 141a.35 (a) (3). Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams per milliliter that it is represented to contain.

(b) Moisture. Using 1 milliliter as the test sample proceed as directed in § 141a.7 (c).

§ 141a.39 Penicillin and streptomycin, penicillin and dihydrostreptomycin—(a) Potency (1) Sodium or potassium penicillin content. Proceed as directed in § 141a.32 (b), except prepare the sample as follows: Add the indicated amount of distilled water to the contents of a vial of the sample and shake well. Withdraw one dose of the suspension or solution with a hypodermic syringe and place in a 10-milliliter volumetric flask. Also, with the further exception that in the iodometric assay, one drop of 1.2 N HCl is added to the blank immediately before the addition of the 0.01 N I2. The sodium or potassium penicillin content is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(2) Total penicillin content. Proceed as directed in § 141a.29 (a) or § 141a.32 (d), except that in the iodometric assay one drop of 1.2 N HCl is added to the blank immediately before the addition of the 0.01 N 12.

Pro

(3) Procaine penicillin content. ceed as directed in § 141a.32 (c). The procaine penicillin content is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(4) Streptomycin content. Proceed as directed in § 141b.101 (j) and (k) of this chapter.

(5) Dihydrostreptomycin content. Proceed as directed in § 141b.108 (a) of this chapter.

(b) Sterility. Use the entire contents of single-dose containers or the equivalent of approximately 0.5 gram (combined activity) for each multiple-dose container, and proceed as directed in § 141a.2, except that no control tube is used in the test for bacteria.

(c) Toxicity. Proceed as directed in § 141a.4, using as a test dose 0.5 milliliter of a solution of the sample containing

1.0 milligram of streptomycin or dihydrostreptomycin per milliliter.

(d) Pyrogens. Proceed as directed in § 141a.3, using as a test dose 2 milliliters per kilogram of a solution containing 5 milligrams of streptomycin or dihydrostreptomycin per milliliter.

(e) Moisture. Proceed as directed in § 141a.26 (e).

(f) pH. Proceed as directed in § 141a.5 (b), using the solution or suspension resulting when the amount of diluent recommended in the labeling is added.

§ 141a.40 Penicillin tooth powder(a) Potency. Proceed as directed in § 141a.1.

(b) Moisture. Proceed as directed in § 141a.7 (c), but in lieu of the directions for preparing the sample in subparagraph (3) thereof prepare the sample and calculate as follows: Accurately weigh about 1 gm. of the sample into a dry titrating vessel. Add an excess of the Karl Fischer reagent and back titrate immediately with water-methanol solution until the end point is reached. (The entire operation from the addition of the Karl Fischer reagent until the end point is reached should not exceed 1 minute.) (V1-V2ƒ) × e x 100

Percent moisture=

Ws

where W, weight of sample in milligrams. Penicillin § 141a.41 bacitracin troches-(a) Potency-(1) Penicillin content. Proceed as directed in § 141a.12 (a). Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units per troche that it is represented to contain.

(2) Bacitracin content. Proceed as directed in § 141e.403 (a) of this chapter, except that sufficient penicillinase is added to the solution under test to completely inactivate the penicillin present. Its content of bacitracin is satisfactory if it contains not less than 85 percent of the number of units per troche that it is represented to contain.

(b) Moisture. Proceed as directed in § 141a.5 (a).

(2) Content of bacitracin. Proceed as directed in § 141e.401 (a) of this chapter, except that sufficient penicillinase is added to the sample under test to completely inactivate the penicillin present. Its content of bacitracin is satisfactory if it contains not less than 85 percent and not more than 115 percent of the number of units it is represented to contain.

(b) Sterility. Proceed as directed in § 141a.39 (b).

(c) Toxicity. Proceed as directed in § 141a.4 except use physiological salt solution as the diluent.

(d) Pyrogens. Proceed as directed in § 141a.3 using as a test dose 1.0 milliliter per kilogram of a solution containing 300 units of bacitracin per milliliter. Use physiological salt solution as the diluent.

(e) Moisture. Proceed as directed in § 141a.5 (a).

(f) pH. Proceed as directed in § 141a.5 (b), using a solution prepared as directed in the labeling for the drug.

§ 141a.43 l-Ephenamine penicillin G— (a) Potency. Proceed as directed in § 141a.1, except in lieu of paragraph (d) dissolve the sample in sufficient methanol before diluting with sterile distilled water to make an appropriate stock solution.

(b) Sterility. Proceed as directed in § 141a.2.

(c) Pyrogens. Proceed as directed in § 141a.3, except use physiological salt solution as the diluent and inject 2 milliliters per kilogram of a solution containing 800 units per milliliter.

(d) Toxicity. Proceed as directed in § 141a.4, except use physiological salt solution as the diluent and inject, in a 10-second interval, 1.0 milliliter of a solution containing 800 units per milliliter.

(e) Moisture. Proceed as directed in § 141a.5 (a).

(f) pH. Proceed as directed in §. 141a.5 (b), using a saturated aqueous solution prepared by adding 300 milligrams per milliliter.

(g) Microscopical test for crystallinity. Proceed as directed in § 141a.5 (c).

(h) Heat stability. Proceed as directed in § 141a.5 (d) (1), except prepare the sample as follows: Dissolve the sample in 5 milliliters of redistilled methanol. Further dilute this solution with sufficient 1-percent phosphate buffer pH 6.0 to give a concentration of 2.0 milligrams per milliliter.

(j) Specific rotation. Accurately weigh approximately 125 milligrams of the sample in a 25-milliliter glass-stoppered volumetric flask and dissolve in about 15 milliliters of water-acetone (1+1) at 20° C. Dilute to 25 milliliters with water-acetone (1+1) at 20° C. and mix thoroughly. Transfer the solution to a 200-millimeter tube, determine the angular rotation in a suitable polarimeter, using sodium light or a 5,893 Angstrom filter, and calculate the specific rotation. The determination must be completed within 2 hour from the time the solution is prepared.

§ 141a.44 1-Ephenamine penicillin G in oil-(a) Potency. Proceed as directed in § 141a.27 (a), except the last sentence thereof. If it is represented to contain less than 300,000 units per milliliter, its potency is satisfactory if it contains not

N-ethyl piperidine penicillin precipitate × 251 Weight of sample in milligrams

ouraud medium. After sterilization, add sufficient penicillinase to each tube of Sabouraud medium to completely inactivate the penicillin used in the test. During the period of incubation, shake the tubes once daily until solubilization is achieved.

(c) Moisture. Proceed as directed in §141a.5 (a).

(d) Pyrogens. Proceed as directed in § 141a.3, except use physiological salt solution as the diluent and inject 2 milliliters per kilogram of a solution containing 800 units per milliliter.

(e) Toxicity. Proceed as directed in § 141a.4, except use physiological salt solution as the diluent and inject, in a 10-second interval, 1.0 milliliter of a solution containing 800 units per milliliter.

(f) pH-(1) Dry mixture of the drug. Proceed as directed in § 141a.5 (b), using a saturated aqueous solution prepared by adding 300 milligrams per milliliter.

(2) Aqueous suspension of the drug. Proceed as directed in § 141a.5 (b), using the undiluted aqueous suspension.

§ 141a.46 Procaine penicillin in streptomycin sulfate solution, procaine penicillin in dihydrostreptomycin sulfate solution (a) Potency-(1) Procaine penicillin content. Proceed as directed in § 141a.1, except paragraph (i) thereof. If the iodometric assay is used, 1 drop of the 1.2 N HCl is added to the blank immediately before the addition of the 0.01 N I2. Its content of procaine penicillin is satisfactory if it contains not less than 85 percent of the number of units that it is represented to contain.

(2) Streptomycin sulfate content. Proceed as directed in § 141b.101 (j) and (k) of this chapter.

(3) Dihydrostreptomycin sulfate content. Proceed as directed in § 141b.108 (a) of this chapter.

(b) Sterility. Proceed as directed in § 141a.39 (b).

(c) Toxicity. Proceed as directed in § 141a.4, using as a test dose 0.5 milliliter of a solution of the sample containing 1.0 milligram of streptomycin or dihydrostreptomycin per milliliter.

(d) Pyrogens. Proceed as directed in § 141a.39 (d).

(e) pH. Proceed as directed in § 141a.5 (b), using the undiluted aqueous suspension.

§ 141a.47 Benzathine penicillin G— (a) Potency. Proceed as directed in § 141a.1, except if the bioassay method is used dissolve the sample to be tested in sufficient dimethyl formamide or formamide prior to diluting with phosphate buffer solution. If the iodometric method is used proceed as directed in § 141a.5 (d), except in preparing the blank solution weigh 30-60 milligrams of the sample and make a suspension of 2 milligrams per milliliter in 1-percent phosphate buffer at pH 6.0. Shake well, pipette 2.0 milliliters into a 125-milliliter glass-stoppered Erlenmeyer flask, add 10 milliliters 0.01 N iodine and immediately titrate with 0.01 N Na2S2O3. In preparing the solution for inactivation dissolve a weighed sample (30-60 milligrams) in sufficient 1 N NaOH to give 2.0 milligrams per milliliter. Pipette a 2.0-milliliter aliquot into a 125-milliliter glass-stoppered Erlenmeyer flask, and after 15 minutes add 2.0 milliliters 1.2 N HCl and 10 milliliters 0.01 N iodine. Allow to stand for 15 minutes and titrate the excess iodine with 0.01 N Na2S2O3. In calculating the results regard the difference in titers multiplied by 661.8, divided by 4, as the units per milligram.

(b) Sterility. Proceed as directed in § 141a.2, except prior to sterilization add 0.5 milliliter of polysorbate 80 to each tube of thioglycolate and Sabouraud's medium, and after sterilization add sufficient penicillinase to each tube of Sabouraud's medium to completely inactivate the penicillin used in the test. During the period of incubation, shake the tubes at least once daily.

(c) Toxicity. Proceed as directed in § 141a.4, except use physiological salt solution as the diluent and inject 0.25 milliliter of a suspension containing 4,000 units per milliliter.

(d) Pyrogens. Proceed as directed in § 141a.3, except use physiological salt solution as the diluent and inject 0.5 milliliter per kilogram of rabbit of a suspension containing 4,000 units per milliliter. (e) Moisture. Proceed as directed in § 141a.26 (e).

(f) pH. Proceed as directed in § 141a.5 (b), using a saturated aqueous solution prepared by adding 5 milligrams per milliliter.

(g) Microscopical test for crystallinity. Proceed as directed in § 141a.5 (c).

(h) Penicillin G content. Dissolve 50 milligrams of the sample, accurately