paragaph (b)(3) of this section. F=appropriate dilution factor depending on the dilution made in the preparation of the solution for assay.

The content of buffered crystalline penicillin in the batch is satisfactory when determined by the method described in this paragraph if it is not less than 85 percent of that which it is represented to contain.

(c) Procaine penicillin. The procaine penicillin content of the batch is the difference between the total potency determined by the method described in paragraph (a) or (d) of this section and the content of the buffered crystalline penicillin determined by the method described in paragraph (b) of this section. The procaine penicillin content of the batch is satisfactory when determined by the method described in this 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 § 440.80a (b)(5)(iv)(a) of this chapter. Obtain the total potency by adding the number of units found in this solution (units per milliliter × volume) to the number of units found (units per milliliter × volume) in the solution assayed in accordance with paragraph (b)(2) of this section.

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 § 448.510a(b)(1) 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 § 436.201.

[39 FR 18944, May 30, 1974, as amended at 40 FR 13497, Mar. 27, 1975]

(a) Potency—(1) Content of penicillin, streptomycin, and dihydrostreptomycin. Proceed as directed in §536.501(a) of this chapter.

(2) Bacitracin content. Proceed as directed in § 448.510a(b)(1) of this chapter, except that:

(i) Sufficient penicillinase is added to the sample under test to completely inactivate the penicillin present.

(ii) Use as the test organism the streptomycin dihydrostreptomycin resistant strain of either Micrococcus flavus (ATCC 10240A) 1 or Sarcina subflava (ATCC 7468/d),1 grown and (ATCC_7468/d), maintained in media containing 500 micrograms of streptomycin or dihydrostreptomycin per milliliter of media, or calculate from the quantity of streptomycin or dihydrostreptomycin found, using the method prescribed by paragraph (a)(1) of this section, the quantity that would be present when the sample is diluted to contain one unit of bacitracin (labeled potency) per milliliter. Prepare the bacitracin standard curve by adding the calculated quantity of streptomycin or dihydrostreptomycin to each concentration of bacitracin used for

1Available from: American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852.

the curve. Use this standard curve to calculate the bacitracin content of the sample.

(3) Bacitracin methylene disalicylate content. Proceed as directed in paragraph (a)(2) of this section, except prepare the sample as follows: Place a representative portion of the sample (usually approximately 1 gram, accurately weighed) or the entire contents of a single-dose container in blending jar, add 99 milliliters of a 2.0-percent aqueous solution of sodium bicarbonate and 1 milliliter of a 10-percent aqueous solution of polysorbate 80 and blend for 3 minutes in a high-speed blender. Allow the foam to subside, remove an aliquot of the solution, and dilute to 1 unit per milliliter with 1.0-percent phosphate buffer, pH 6.0.

(b) Moisture. Proceed as directed in § 436.201.

[39 FR 18944, May 30, 1974, as amended at 40 FR 13497, Mar. 27, 1975]

§ 436.506 Benzathine penicillin G and buffered crystalline penicillin for aqueous injection.

(a) Total potency (except in single-dose containers). Proceed as directed in § 440.80a(b)(1) of this chapter, except if the bioassay method is used prepare the sample by diluting 1.0 milliliter of the drug suspension with sufficient dimethyl formamide, formamide, or methyl alcohol to dissolve the benzathine penicillin. Make to 100 milliliters with buffer. Shake well and dilute to 1.0 unit per milliliter. If the iodometric method is used, proceed as directed in §440.55a(b) of this chapter, except in preparing the blank solution dilute 1.0 milliliter of the drug suspension to 250 milliliters with 1-percent phosphate buffer at pH 6.0. In preparing the solution for inactivation dissolve 1.0 milliliter of the drug suspension in approximately 20 milliliters of 0.5 N NaOH. Allow to stand for 15 minutes. Dilute to 250 milliliters with distilled water. Pipette a 2.0-milliliter aliquot into a 125-milliliter glass-stoppered Erlenmeyer flask and add 2.0 milliliters 1.2 N HCl and 10 milliliters 0.01 N iodine.

(b) Buffered crystalline penicillin content. Place 1.0 milliliter of the drug suspension in a 10-milliliter volumetric flask and add 20 percent sodium sulfate

er

to make 10 milliliters. Shake well and centrifuge to obtain a clear, or reason- . ably clear, solution. Dilute a 5.0-milliliter aliquot to 50 milliliters with buffand proceed as directed in § 440.80a(b)(1) of this chapter to determine the number of units per milliliter of this solution, and from this value calculate the number of units per milliliter of the drug. The content of buffered crystalline penicillin is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(c) Benzathine penicillin G content. The benzathine penicillin G content of the batch is the difference between the total potency as described in paragraph (a) or (d) of this section and the content of buffered crystalline penicillin determined by the method prescribed in paragraph (b) of this section. The content of benzathine penicillin G is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(d) Total potency of a single-dose container. Add sufficient distilled water to the material remaining in the 10-milliliter volumetric flask referred to in paragraph (b) of this section to bring the volume back to 10 milliliters and determine the number of units per milliliter of this suspension. If the iodometric method is used, 2.0-milliliter aliquots are placed in 50-milliliter volumetric flasks (one blank and one to be inactivated). Obtain the total potency by adding the number of units found in the 10-milliliter volumetric flask to one-half the content of buffered crystalline penicillin found in paragraph (b) of this section.

(e) Sterility. Proceed as directed in § 436.20 using the method described in paragraph (e)(2) of that section, except use medium C in lieu of medium A, and medium F in lieu of medium E. During the period of incubation, shake the tubes at least once daily.

(f) Moisture. Proceed as directed in § 440.74a(b)(5) of this chapter.

(g) Pyrogens. Proceed as directed in § 436.500.

(h) Toxicity. Proceed as directed in § 440.55a(b)(3) of this chapter.

(i) pH. Proceed as directed in § 440.80a(b)(5)(ii) of this chapter, using the suspension resulting when the

product is reconstituted as directed in the labeling.

§ 436.507 Benzathine

procaine

buffered crystalline penicillins for aqueous injection.

(a) Potency-(1) Total potency. Proceed as directed in §440.80a(b)(1) of this chapter, except if the bioassay method is used prepare the sample by diluting one dose of the drug suspension with sufficient dimethyl formamide or formamide or methyl alcohol to dissolve the benzathine penicillin G. Make to 100 milliliters with 1-percent phosphate buffer, pH 6.0. Shake well, and dilute to 1.0 unit per milliliter with buffer. If the iodometric method of assay is used, add the indicated amount of distilled water to the contents of a vial of the sample, shake well, and proceed as follows (except for single-dose containers):

(i) Using a standardized hypodermic syringe, withdraw one dose and dilute with 1-percent phosphate buffer, pH 6.0, to give a concentration of approximately 2,000 units per milliliter. Use 2.0 milliliters of this suspension as the blank in the iodometric assay procedure described in § 440.80a(b)(5)(iv)(a) of this chapter.

(ii) Using a standardized hypodermic syringe, withdraw another dose, place in a flask, and add 20 milliliters of 0.5 N NaOH for each 300,000 units of benzathine penicillin, mix well, being sure that all penicillin is in solution, and allow to stand for 15 minutes. Add 1 milliliter of 1.2 N HCl for each 2 milliliters of 0.5 N NaOH, mix, and dilute with distilled water to the same volume as was used in paragraph (a)(1)(i) of this section. Place 2.0 milliliters in a 125-milliliter glass-stoppered Erlenmeyer flask, add 10 milliliters of 0.01 N iodine, allow to stand for 15 minutes, and titrate with 0.01 N sodium thiosulfate as directed in the iodometric assay procedure in § 440.80a (b)(5)(iv)(a) of this chapter. The total potency of the batch is satisfactory if it contains not less than 85 percent of that which it is represented to contain.

(2) Procaine penicillin content (except for single-dose containers). Make suitable dilutions of the solution prepared in paragraph (a)(1)(ii) of this section to obtain approximately 60 units of pro

caine penicillin per milliliter. Determine the procaine penicillin content by the colorimetric procedure described in § 436.503(b)(3). The 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.

(3) Buffered crystalline penicillin content—(i) Preparation of the solution for assay. (a) 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 with a hypodermic syringe and place in a 10milliliter volumetric flask. Add 20-percent sodium sulfate solution almost to the mark, centrifuge sufficiently to see the meniscus, make to volume with 20percent sodium sulfate solution, shake well, and centrifuge to obtain a clear or reasonably clear solution; or

(b) If the original product contains more than 600,000 units, place it in a 50milliliter volumetric flask, add 20-percent sodium sulfate to the mark, shake well, place a 10-milliliter portion in a centrifuge tube, and centrifuge to obtain a reasonably clear solution.

(c) Dilute a 5.0-milliliter aliquot of the clear solution obtained in paragraph (a) (3)(i) (a) or (b) of this section with 1-percent phosphate buffer, pH 6.0, to give a solution for assay of approximately 2,000 units per milliliter.

(ii) Iodometric assay for total penicillin in the solution for assay. Determine the total quantity of penicillin in the solution for assay by the iodometric assay procedure described in § 440.80a (b)(5)(iv)(a) of this chapter.

(iii) Colorimetric determination of procaine penicillin in the solution for assay. Proceed as directed in §436.503 (b)(3). The content of procaine penicillin in the batch is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(iv) The buffered crystalline penicillin in one dose of the product is calculated as follows:

where:

A=(B-C)F,

A=the buffered crystalline penicillin content of the product.

B=the number of units of penicillin per milliliter as determined in paragraph (a)(3)(ii) of this section.

C=the number of units of procaine penicillin per milliliter as determined in paragraph (a)(3)(iii) of this section. F=the appropriate dilution factor depending on the dilutions made in the preparation of the solution for assay.

The content of buffered crystalline penicillin is satisfactory if the batch contains 85 percent of the number of units per milliliter that it is represented to contain.

(4) Benzathine penicillin content. The sum of the procaine penicillin content determined under paragraph (a)(2) or (6) of this section and the buffered crystalline penicillin content determined under paragraph (a)(3) of this section, subtracted from the total potency determined in paragraph (a)(1) or (5) of this section, represents the benzathine penicillin G content. The benzathine penicillin G content is satisfactory if it is not less than 85 percent of the number of units that it is represented to contain.

(5) Total potency of a single-dose container. Wash out the material remaining in the volumetric flask referred to in paragraph (a)(3)(i)(a) of this section, or combine the contents remaining in the 50-milliliter volumetric flask and in the centrifuge tube referred to in paragraph (a)(3)(i)(b) of this section. Dissolve the material by adding 10 milliliters of 1 N NaOH for each 300,000 units of benzathine penicillin and allow to stand 15 minutes. Add 1 milliliter of 1.2 N HCl for each milliliter of 1 N NaOH and then dilute with distilled water to give a concentration of approximately 2,000 units per milliliter. Place 2.0 milliliters in a 125-milliliter glass-stoppered Erlenmeyer flask, add 10 milliliters of 0.01 N iodine, allow to stand for 15 minutes, and then titrate with 0.01 N sodium thiosulfate as directed in §440.80a (b)(5)(iv)(a) of this chapter. For the blank determination prepare a separate sample as directed in paragraph (a)(3)(i) (a) or (b) of this section and in the first sentence of this paragraph (a)(5), then dilute with 1 percent phosphate buffer, pH 6.0, to give a concentration of approximately 2,000 units per milliliter. The total potency of the one-dose container is equal to the sum of the number of units found in this assay (units per milliliterxvolume) and the number of units

found (units per milliliter xvolume) in the solution for assay in paragraph (a)(3)(ii) of this section.

(6) Procaine penicillin content of a single-dose container. Make suitable dilutions of the NaOH-inactivated solution prepared in paragraph (a)(5) of this section to obtain approximately 60 units of procaine penicillin per milliliter. Determine the procaine penicillin content (units per milliliterx volume) of this solution by the colorimetric procedure described under §436.503(b)(3). To this value add per procaine penicillin content (unit per milliliterxvolume) of the solution for assay, as found in paragraph (a)(3)(iii) of this section, to obtain the procaine penicillin content of the one-dose container. The content of procaine penicillin in the batch is satisfactory if it is not less than 85 percent of that which it is represented to contain.

(b) Sterility. Proceed as directed in § 436.20, using the method described in paragraph (e)(2) of that section, except use medium C in lieu of medium A, and medium F in lieu of medium E. During the period of incubation, shake the tubes at least once daily.

(c) Pyrogens. Proceed as directed in § 440.55a(b)(4) of this chapter. (d) Toxicity. Proceed as directed in § 440.55a(b)(3) of this chapter. (e) Moisture. Proceed as directed in § 440.74a(b)(5) of this chapter.

(f) pH. Proceed as directed in § 440.80a(b)(5)(ii) of this chapter, using the suspension resulting when the product is reconstituted as directed in the labeling.

(b) Moisture. Proceed as directed in § 436.201.

$436.509 Procaine penicillin-streptomycin-polymyxin in oil; procaine penicillin-dihydrostreptomycinpolymyxin in oil; procaine penicillin-streptomycin-polymyxin ointment; procaine penicillin - dihydrostreptomycin - polymyxin ointment. (a) Potency—(1) Penicillin content. Proceed as directed in § 540.380a(b)(1) of this chapter. Its content of penicillin is satisfactory if it contains not less than 85 percent of the number of units per milliliter or per gram that it is represented to contain.

(2) Streptomycin content. Proceed as directed in §544.373(b)(1)(1) of this chapter, except inactivate the penicillin in the combined extractives with sufficient penicillinase at 37° C. for 30 minutes. Its content of streptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams per milliliter or per gram that it is represented to contain.

(3) Dihydrostreptomycin content. Proceed as directed in paragraph (a)(2) of this section, 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 milliliter or per gram that it is represented to contain.

(4) Polymyxin content. Proceed as directed in §444.170a(b)(2)(1) of this chapter, with the following exceptions:

(i) In lieu of the directions for the preparation of the sample described in § 444.170a(b)(2)(i)(g) of this chapter, prepare the sample by one of the following techniques:

(a) Extraction. Place a convenientsized representative quantity of the sample in a separatory funnel containing approximately 50 milliliters of peroxide-free ether. Shake the sample and ether until homogeneous. Add 25 milliliters of 10-percent potassium phosphate buffer (pH 6.0) and shake. Remove the buffer layer and repeat the extraction with 25-milliliter portions of buffer at least three times and any additional times that may be necessary to insure complete extraction of the antibiotic. Combine the extractives. Inactivate the penicillin with suffi

cient penicillinase at 37° C. for 30 minutes. Make the proper estimated dilutions in 10-percent potassium phosphate buffer (pH 6.0) to give a concentration of 10 units per milliliter (estimated).

(b) Blending. Place a convenient-sized representative quantity of the sample in a blending jar containing 1.0 milliliter of polysorbate 80 and sufficient 1percent phosphate buffer (pH 6.0) to give a final volume of 200 milliliters. If the sample consists of substantially more than 1 gram, use sufficient buffer to give a final volume of 500 milliliters. If the concentration of polymyxin in the blend is less than 200 units per milliliter, 10-percent phosphate buffer (pH 6.0) should be used in lieu of 1-percent phosphate buffer (pH 6.0). Using a highspeed blender, blend the mixture for 2 minutes. Inactivate the penicillin with sufficient penicillinase at 37° C. for 30 minutes and make the proper estimated dilutions in 10-percent phosphate buffer (pH 6.0) to give a concentration of 10 units per milliliter (estimated).

(ii) The standard curve is prepared in the following concentrations: 6.4, 8.0, 10.0, 12.5, and 15.6 units per milliliter in 10-percent potassium phosphate buffer, pH 6.0. The 10 units per milliliter concentration is used as the reference point. Its content of polymyxin is satisfactory if it contains not less than 85 percent of the number of units per milliliter or per gram that it is represented to contain.

(b) Moisture. Proceed as directed in § 436.201.