ether. Add 20 milliliters of 0.1 M potassium phosphate buffer (pH 8.0) and shake. Remove the buffer layer and repeat the extraction with three additional 20-milliliter portions of the buffer. Place the buffer solution in a second separatory funnel and wash with three 30-milliliter portions of ether. Discard the ether washes. Remove an aliquot of the buffer solution and proceed as directed in §440.80a(b) (1) of this chapter, except $440.80a(b)(1)(iv) and (ix) of this chapter. If the iodometric chemical assay is used, proceed as directed in § 440.80a(b)(5)(iv)(a) of this chapter, except prepare the sample as directed in §536.501(a)(1) of this chapter. 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 an aliquot of the buffer solution prepared as directed in paragraph (a)(1) of this section, proceed as directed in §444.70a (b)(1) through (9) of this chapter, except add sufficient penicillinase to completely inactivate the penicillin present. 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 paragraph (a)(2) of this section, using the dihydrostreptomycin working standard as the standard of comparison. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(4) Erythromycin content. Proceed as directed in §444.570b(b)(1)(i)(b) of this chapter, except prepare the sample as follows: Place a representative sample (usually approximately 1.0 gram, accurately weighed), in a glass blending jar containing 99 milliliters of 0.1 M potassium phosphate buffer, pH 8.0, and 1 milliliter of polysorbate 80. Using a high-speed blender, blend for 2 to 3 minutes. Add 100 milliliters of 0.1 M potassium phosphate buffer, pH 8.0, and blend for an additional 2 to 3 minutes. Prepare an intermediate dilution by diluting an aliquot of the filtrate with 0.1 M potassium phosphate buffer (pH 8.0), and add sufficient penicillinase to inactivate the penicillin. Then further di

lute with buffer to give an erythromycin content of 1.0 microgram per milliliter (estimated). Its content of erythromycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is presented to contain.

(b) Moisture. Proceed as directed in § 436.500(c).

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

§ 436.511 Penicillin-streptomycin-bacitracin methylene disalicylate-neomycin ointment; penicillin-dihydrostreptomycin-bacitracin methylene disalicylate-neomycin ointment.

(a) Potency—(1) Penicillin content. Proceed as directed in §540.380a(b)(1) of this chapter. Its penicillin content is satisfactory if it contains not less than 85 percent of the number of units that it is represented to contain.

(2) Streptomycin content. Proceed as directed in §436.105 of this chapter. 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 § 436.105 of this chapter. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain. (4) Bacitracin methylene disalicylate content. Proceed as directed in § 436.505(a)(3). Its potency is satisfactory if it contains not less than 85 percent of the equivalent number of units of bacitracin that it is represented to contain.

(5) Neomycin content. Proceed as directed in § 436.105 of this chapter. Its content of neomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that 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]

novobiocin-resistant strain of Staphylococcus aureus (ATCC 12692),1 except prepare the sample as follows: Place the equivalent of one dose of sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 1 percent potassium phosphate buffer, pH 6.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender and make appropriate dilutions, using 1 percent potassium phosphate buffer, pH 6.0. Its content of penicillin G is satisfactory if it contains not less than 85 percent of the number of units that it is represented to contain.

(2) Novobiocin content. Proceed as directed in §440.180d(b)(3)(i), with the following exceptions:

(i) Prepare the sample as follows: Place the equivalent of one dose of sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 10 percent potassium phosphate buffer, pH 6.0 (solution 6) to give a final concentration of 0.5 microgram novobiocin per milliliter (estimated), and allow to stand for 2-hour at 37° C. before filling the plates.

(ii) Aseptically add to the seed agar used for this assay, at the time the bacterial suspension is added, a slurry of Dowex 50 WX-4, Na+ type 200-400 mesh, sufficient to make a total concentration of 2 percent. Prepare the slurry by adding 50 grams of the resin to 30 milliliters of distilled water and sterilize for 15 minutes at 15 pounds pressure. Mix the slurry thoroughly before adding. Its content of novobiocin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(3) Neomycin content. Proceed as directed in §436.517(b)(1) of this chapter, using the Staphylococcus epidermidis (ATCC 12228)1 procedure, except:

(i) Prepare the sample as follows: Place the equivalent of one dose of

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

sample in a blending jar, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 0.1M potassium phosphate buffer, pH 8.0, to give a final concentration of 1.0 microgram neomycin per milliliter (estimated), and allow to stand for 2-hour at 37° C. before filling the plates.

(ii) Aseptically add to the seed agar used for this assay, at the time the bacterial suspension is added, a slurry of Dowex 1-X8, Cl type 200-400 mesh, to make a total concentration of 1 percent. Prepare the slurry by adding 50 grams of the resin to 30 milliliters of distilled water and sterilize for 15 minutes at 15 pounds pressure. Mix the slurry thoroughly before adding. Its content of neomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(4) Dihydrostreptomycin content. Proceed as directed in §436.105 except prepare the sample by placing the equivalent of one dose in a blender, add 1.0 milliliter of polysorbate 80 and a quantity of 0.1M potassium phosphate buffer, pH 8.0, sufficient to make a total of 500 milliliters. Blend for 5 minutes with a high-speed blender. To an aliquot, add sufficient penicillinase to inactivate the penicillin, further dilute with 0.1M potassium phosphate buffer, pH 8.0, to give a final concentration of 1.0 microgram dihydrostreptomycin per milliliter (estimated), and allow to stand for 2-hour at 37° C. before filling the plates. Its content of dihydrostreptomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(b) Moisture. Proceed as directed in § 436.500(c).

[39 FR 18944, May 30, 1974, as amended at 41 FR 10886, Mar. 15, 1976]

chlortetracycline hydrochloride troches proceed as directed in § 446.10a(b)(1) of this chapter, except § 446.10a(b)(1)(x), and in lieu of the directions in § 446.10a(b)(1)(iv) and (viii)(c) of this chapter prepare the sample as follows: Place 12 troches in a glass blending jar containing 500 milliliters of 0.01N HCl. Using a high-speed blender, blend for 3 to 5 minutes and then make the proper estimated dilutions in the buffer solution. The average potency of the troches is satisfactory if they contain not less than 85 percent of the number of milligrams they are represented to contain.

(b) Moisture. Proceed as directed in § 440.80a(b)(5)(1) of this chapter.

§ 436.514 Chlortetracycline

hydro

chloride powder topical; tetracycline hydrochloride powder topical.

(a) Potency-(1) Dry powder. Using a 3.0-gram sample or the entire contents of the immediate container for each determination, prepare the sample as follows: Using a high-speed blender, blend a 3.0-gram sample in a glass blending jar containing 500 milliliters of 0.01 N HCl (use 0.1 N HCl if it is tetracycline), or reconstitute in the immediate container as directed in the labeling of the drug. Transfer an appropriate aliquot of 1.0 milliliter to 5.0 milliliters to a 100-milliliter volumetric flask and make to mark with 0.01 N HCl (use 0.1 N HCl if it is tetracycline). Withdraw an aliquot from the volumetric flask, and if it is chlortetracycline hydrochloride dilute to 0.06 μ g. per milliliter, using 0.1 M potassium phosphate buffer, pH 4.5, and proceed as directed in §446.10a(b)(1) of this chapter. If it is tetracycline hydrochloride, dilute to 0.24 μ g. per milliliter, using 0.1 M potassium phosphate buffer, pH 4.5, and proceed as directed in § 446.81a(b)(1) of this chapter. The average potency is satisfactory if it contains not less than 85 percent of the number of milligrams of chlortetracycline hydrochloride or tetracycline hydrocloride per gram or per immediate container that it is represented to contain.

(2) Powder packaged with inert gases. Spray, as directed in the labeling, the entire contents of each container to be tested into a separate 2-liter Erlen

meyer flask, held in a horizontal position. Add 500 milliliters of 0.1 N HCl and shake to dissolve the contents. Immediately remove aliquots of this solution and, using 0.1 M potassium phosphate buffer, pH 4.5, for further dilutions, proceed as directed in § 446.10a(b)(1) of this chapter if it is chlortetracycline hydrochloride powder or § 446.81a(b)(1) of this chapter if it is tetracycline hydrochloride powder. Calculate the average total amount of antibiotic expelled from the containers. The total potency is satisfactory if it contains not less than 85 percent of the number of milligrams of chlortetracycline hydrochloride or tetracycline hydrochloride that it is represented to contain.

(b) Moisture. Proceed as directed in § 440.80a(b)(5)(i) of this chapter, except if it is packaged with inert gases proceed as directed in §536.513(c) of this chapter.

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

§ 436.515 Capsules tetracycline

and

oleandomycin phosphate; capsules tetracycline and troleandomycin; capsules tetracycline hydrochloride and oleandomycin phosphate; capsules tetracycline hydrochloride

and troleandomycin.

(a) Potency—(1) Tetracycline or tetracycline hydrochloride content by turbidimetric assay-(i) Test culture and media. Maintain the test organism Escherichia coli (ATCC 10536)1 on the agar described in § 440.80a(b)(1) (ii)(a) of this chapter. For use in the assay, prepare a suspension of the organism every 2 weeks, as follows: Transfer the organism to a fresh agar slant and incubate at 37°C. overnight. Wash the growth from the slant with the aid of 2 milliliters of sterile distilled water and sterile glass beads into a Roux bottle containing 300 milliliters of the maintenance medium. Incubate overnight at 37° C. and then harvest the growth with 50 milliliters of sterile distilled water and sterile glass beads. Standardize this suspension by determining the dilution that will permit 40-percent light

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

transmission in a photoelectric colorimeter using a 650-millimicron filter and an 18-millimeter diameter test tube as an absorption cell. Prepare the daily inoculum by adding 10 milliliters of that dilution to each liter of nutrient broth, prepared as directed in § 440.80a (b)(1)(ii)(c) of this chapter, needed for the test.

(ii) Working standard and solutions. Dissolve an appropriate amount of the working standard in sufficient 0.1 N HCl to give a concentration of 1,000 micrograms per milliliter. This stock solution may be kept in the refrigerator for 1 week. Make daily dilutions of the stock solution with 0.1 M potassium phosphate buffer (pH 4.5) to obtain concentrations of 0.146, 0.187, 0.240, 0.308, and 0.395 micrograms per milliliter. Add 1.0 milliliter of each such concentration to each of three 16 millimeters x 125 millimeters test tubes.

(iii) Preparation of sample. Dissolve the contents of a representative number of capsules in sufficient 0.1 N HC1 to give a stock solution of convenient concentration. Further dilute the stock solution with 0.1 M potassium phosphate buffer (pH 4.5) to obtain a final concentration of 0.24 microgram per milliliter (estimated). Add 1.0-milliliter of this dilution to each of three 16 millimeters x 125 millimeters test tubes.

(iv) Procedure. To each of the 16 millimeters x 125 millimeters test tubes prepared in paragraph (a)(1)(ii) and (iii) of this section, add 9.0 milliliters of the inoculated nutrient broth described in paragraph (a)(1)(i) of this section and place immediately in a 37° C. water bath for 3 to 4 hours. After incubation, add 0.5 milliliter of a 12-percent formaldehyde solution to each tube and read the absorbance values in a suitable photoelectric colorimeter using a wavelength of 530 millimicrons. Set the instrument at zero absorbance with clear uninoculated broth prepared as described in §440.80a(b)(1)(ii)(c) of this chapter.

(v) Estimation of potency. Plot the average values for each concentration of the standard on arithmetic graph paper with absorbance values on the ordinate and tetracycline or tetracycline hydrochloride concentrations on the abscissa. Construct the best straightline

Plot the values obtained for L and H and connect the points with a straight line. Average the absorbance values for the sample and read the tetracycline or tetracycline hydrochloride concentration from the standard curve. Multiply the concentration by appropriate dilution factors to obtain the tetracycline or tetracycline hydrochloride content of the sample. Its potency is satisfactory if it contains the equivalent of not less than 85 percent of the number of milligrams of tetracycline hydrochloride that it is represented to contain.

(2) Oleandomycin content. (i) If oleandomycin phosphate is used, proceed as directed in paragraph (c)(1) of this section, except prepare the sample as follows: Dissolve the contents of a representative number of capsules in sufficient 0.1 M potassium phosphate buffer (pH 8.0) to give a stock solution of convenient concentration. Further dilute with 0.1 M potassium phosphate buffer (pH 8.0) to obtain a final concentration of 5.0 μ g. of oleandomycin activity per milliliter (estimated).

(ii) If troleandomycin is used, proceed as follows: Dissolve the contents of a representative number of capsules in chloroform to give a stock solution of 1.0 milligram of oleandomycin activity per milliliter. Transfer 30 milliliters of the chloroform solution to a glass-stoppered test tube (200 millimeters x 22 millimeters) and add 20 milliliters of 1 N sodium hydroxide. Shake for 1 minute and centrifuge briefly to aid in the separation of the layers. With the aid of a syringe and needle, remove and discard the aqueous layer. Repeat the washing procedure with two more 20-milliter portions of 1 N sodium hydroxide solution. Filter the chloroform layer through a pledget of cotton.

179-070 0—98———16

Dilute an aliquot of this solution with chloroform to give a solution containing approximately 25 μ g. of oleandomycin per milliliter. Transfer a 5.0 milliliter aliquot to a 40 milliliter glass-stoppered centrifuge tube, dilute to 20 milliliters, with chloroform, and determine the oleandomycin content as directed in paragraph (d)(1)(i) of this section.

Its content of oleandomycin is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(b) Moisture. Proceed as directed in § 440.80a(b)(5)(1) of this chapter.

(c) Oleandomycin phosphate used in making the capsules—(1) Potency-(i) Cylinders (cups). Used cylinders described in § 440.80a(b)(1)(i) of this chapter.

(ii) Culture media. (a) Use the nutrient agar described in § 440.80a (b)(1)(ii)(a) of this chapter for the seed layer and base layer, except that its pH after sterilization is 7.8 to 8.0.

(b) Use the nutrient agar described in § 440.80a(b)(1)(ii)(a) of this chapter for maintaining the test organism.

(iii) Working standard. Dissolve a suitable weighed quantity (usually 25 milligrams or less) of the working standard (obtained from the Food and Drug Administration) in 2 milliliters of ethanol, then add sufficient 0.1 M potassium phosphate buffer, pH 8.0, to give a concentration of 1,000 micrograms of oleandomycin base per milliliter. This stock solution may be kept in the refrigerator for 3 days.

(iv) Preparation of sample. Dissolve the sample in sufficient 0.1 M potassium phosphate buffer (pH 8.0) to give a convenient stock solution. Further dilute in 0.1 M potassium phosphate buffer (pH 8.0) to give a final concentration of 5.0 micrograms per milliliter (estimated).

(v) Preparation of test organism. The test organism is Staphylococcus epidermidis (ATCC 12228)1 which is maintained on slants or agar described under paragraph (c)(1)(ii)(a) of this section. Wash the organism from the agar slant with 3 milliliters of sterile phys

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

iological saline solution onto a large agar surface such as that provided by a Roux bottle containing 300 milliliters of the agar described in paragraph (c)(1)(ii)(a) of this section. Spread the suspension of organisms over the entire agar surface with the aid of sterile glass beads. Incubate for 4 hours at 32° C. and then wash the resulting growth from the agar surface with about 30 milliliters of sterile physiological saline solution. Standardize the suspension by determining the dilution that will give 80-percent light transmission, using a suitable photoelectric colorimeter with a 650-millimicron filter and an 18-millimeter-diameter test tube as an absorption cell. Run test plates to determine the quantity of the diluted suspension (usually 1.5 milliliters) that should be added to each 100 milliliters of agar to give clear, sharp zones of inhibition of appropriate size.

(vi) Preparation of plates. Add 21 milliliters of the agar prepared as described in paragraph (c)(1)(ii)(a) of this section to each Petri dish (20 millimeters x 100 millimeters). Distribute the agar evenly in the plates and allow it to harden. Use the plates the same day they are prepared. Melt a sufficient amount of the agar described in paragraph (c)(1)(ii)(a) of this section, cool to 48° C., add the proper amount of the test organism as described in paragraph (c)(1)(v) of this section and mix thoroughly. Add 4 milliliters of this inoculated agar to each Petri dish. Distribute the agar evenly in the plates, cover with porcelain covers glazed on the outside, and allow to harden. After the agar has hardened, place 6 cylinders on the agar surface so that they are at approximately 60° intervals on a 2.8-centimeter radius.

(vii) Standard curve. Prepare the daily standard curve by further diluting the 1,000 micrograms per milliliter stock solution in 0.1 M potassium phosphate buffer (pH 8.0) to obtain concentrations of 3.2, 4.0, 5.0, 6.25 and 7.80 micrograms per milliliter. Use 3 plates for the determination of each point on the curve, except the 5.0 micrograms per milliliter concentration, a total of 12 plates. On each of 3 plates fill 3 cylinders with the 5.0 micrograms per milliliter standard, and the other 3 cylinders with the concentration under test. Thus, there