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will be 36 five-microgram determinations and 9 determinations for each of the other points on the curve. After incubation, read the diameters of the circles of inhibition in the plates. Average the readings of the 5.0 micrograms per milliliter concentration and the readings of the point tested for each set of 3 plates and average also all 36 readings of the 5.0 micrograms per milliliter concentration. The average of the 36 readings of the 5.0 micrograms per milliliter concentration is the correction point for the curve. Correct the average value obtained for each point to the figure it would be if the 5.0 micrograms per milliliter reading for that set of 3 plates were the same as the correction point. Thus, if in correcting the 4.0-microgram concentration, the average of the 36 readings of the 5.0-microgram concentration were 20.0 millimeters, and the average of the 5.0-microgram concentration of this set of 3 plates were 19.8 millimeters, the correction would be +0.2 millimeter. If the average

reading of the 4.0microgram concentration of these same 3 plates were 19.0 millimeters, the corrected value would be 19.2 millimeters. Plot these corrected values, including the average of the 5.0 micrograms per milliliter concentration, on 2-cycle semilog paper, using the concentration in micrograms per milliliter as the ordinate (the logarithmic scale) and the diameter of the zone of inhibition as the abscissa. Draw the standard curve through these points, either by inspection or by means of the following equations:

with the standard 5.0 micrograms per milliliter solution and 3 cylinders with the 5.0 micrograms per milliliter (estimated) sample, alternating standard and sample. Incubate all plates, including those containing the standard curve, at 32° C.-35° C. overnight, and measure the diameter of each circle of inhibition. To estimate the potency of the sample, average the zone readings of the standard and the zone readings of the sample on the 3 plates used. If the sample gives a larger zone size than the average of the standard, add the difference between them to the 5.0 micrograms per milliliter zone on the standard curve. If the average sample value is lower than the standard value, subtract the difference between them from the 5.0 micrograms per milliliter value on the curve. From the standard curve, read the potencies corresponding to these corrected values of zone sizes.

(2) Toxicity. Proceed as directed in $ 440.80a(b)(4) of this chapter, except use physiological salt solution as the diluent, and inject 0.5 milliliter of a solution containing 8 milligrams per milliliter.

(3) Moisture. Proceed as directed in $ 440.80(b)(5)(i) of this chapter.

(4) pH. Proceed as directed in $ 440.80a(b)(5)(ii) of this chapter, using a solution containing 100 milligrams per milliliter

(5) Crystallinity. Proceed as directed in $ 440.80a(b)(5)(iii) of this chapter.

(d) Troleandomycin used in making the capsules-(1) Potency—(i) Chemical method—(a) Reagents and equipment. (1) Methyl orange reagent: Shake 0.5 M boric acid solution for about 12 hours (to insure saturation) with an excess of methyl orange indicators. An alternative method is to heat the mixture to about 50° C. and shake for about an hour. Then allow to cool. Filter the saturated dye solution and wash three times with chloroform. Store the dye solution over chloroform.

(2) Acid-alcohol solution: Add 2 milliliters of concentrated sulfuric acid to 98 milliliters of absolute methyl alcohol.

(3) Glycerin: Reagent grade.

(4) Centrifuge tubes: 40 milliliters, glass-stoppered.

L=(3a+26+c-e)/5, H=(3e+2d+C-a)/5,

where: L=corrected zone diameter for the lowest

concentration of the standard curve, H=corrected zone diameter for the highest

concentration of the standard curve, c=average zone diameter for 36 readings of

the 5.0 micrograms per milliliter stand

ard. a, b, d, e=corrected average values for the

3.2, 4.0, 6.25, and 7.81 micrograms per milliliter standard solutions, respectively.

Plot the values obtained for L and H and connect with a straight line.

(viii) Assay. Use 3 plates for each sample. Fill 3 cylinders on each plate

(6) Procedure. Prepare a chloroform solution containing 50.0 milligrams activity of standard oleandomycin base in 200 milliliters of solution. Transfer 10.0 milliliters of the solution to a 100milliliter volumetric flask and dilute to volume with chloroform. Transfer 2.0, 4.0, 6.0, and 8.0 milliliters of this solution to glass-stoppered centrifuge tubes (40-milliliter size) and dilute to a total volume of 20.0 milliliters each with chloroform. To the 20.0 milliliters of the solution present in each (40-milliliter size) centrifuge tube add 0.2 milliliter of glacial acetic acid, 0.20 milliliter of glycerin, and 0.40 milliliter of methyl orange reagent. Shake for 5 minutes and centrifuge for 3 minutes. Immediately transfer to another tube a 10.0-milliliter aliquot from the chloroform (lower) layer. Care must be exercised to see that no portion of the dyeglycerin-phase is included with the chloroform aliquot. Add 1.0 milliliter of acid-alcohol solution to this chloroform aliquot, mix well, and read the absorbancy at 535 mu, using a l-centimeter cell and a suitable photometer and using chloroform, similarly treated, as a blank. Prepare a standard curve, plotting the absorbance values of the standard solutions against the concentration expressed in micrograms per aliquot. Accurately weigh the sample to be tested to give 50 milligrams (estimated) of oleandomycin activity, dissolve in chloroform, and make to 200 milliliters with chloroform. Transfer 10.0 milliliters to a 100-milliliter volumetric flask and make to volume with chloroform. Transfer 5.0 milliliters to a glass-stoppered centrifuge tube and proceed as above. Determine the potency of the sample from the standard curve.

(ii) Microbiological assay. Proceed as directed in paragraph (c)(1) of this section, except:

(a) In lieu of the directions in paragraph (c)(1)(ii)(a) of this section, use the nutrient agar described in $ 440.80a(b)(1)(ii)(a) of this chapter for the seed and base layers, except add 2.0 milliliters of polysorbate 80 to each 100 milliliters of agar. Its pH after sterilization is 7.8 to 8.0.

(6) In lieu of the directions in paragraph (c)(1)(iii) of this section, dissolve a suitable weighed quantity (usually 25

milligrams

or

less) of the troleandomycin working standard (obtained from the Food and Drug Administration) in sufficient 80 percent isopropyl alcohol-water solution to give a concentration of 1,000 micrograms per milliliter (estimated). Use the solution the day that it is prepared.

(c) In lieu of the directions in paragraph (c)(1)(iv) of this section, dissolve the sample in sufficient 80 percent isopropyl alcohol-water solution to give a convenient stock solution. Further dilute in 0.2 M potassium phosphate buffer, pH 10.5 (35 grams of dipotassium phosphate plus 2 milliliters of 10 N NaOH, q.s. to 1 liter), to give a final concentration of 15 micrograms per milliliter (estimated).

(d) In lieu of the directions in paragraph (c)(1)(vi) of this section, use the agar described in paragraph (d)(1)(ii)(a) of this section for both layers. Use the plates as soon after seeding as is practical. If they are not to be used shortly after seeding, then they should be refrigerated until ready for use.

(e) In lieu of the directions for preparing the standard curve in paragraph (c)(1)(vii) of this section, prepare the standard curve by diluting the stock solution in 0.2 M potassium phosphate buffer, pH 10.5, to give concentrations of 9.6, 12.0, 15.0, 18.8, and 23.4 micrograms per milliliter. The 15.0 micrograms per milliliter is the reference concentration.

In lieu of the directions in paragraph (c)(1)(viii) of this section, incubate the plates at 37° C. overnight. The concentration of the sample and standard being tested is 15.0 micrograms per milliliter.

(2) Toxicity. Administer orally, by means of a cannula or other suitable device, to each of five mice within the weight range of 18 grams to 25 grams, 0.5 milliliter of a suspension containing 200 milligrams per milliliter in normal saline solution. If no animal dies within 48 hours, the sample is nontoxic. If one or more animals die within 48 hours, repeat the test, using for each test five or more previously unused mice weighing 20 grams (+0.5 gram) each; if the total deaths within 48 hours is no greater than 10 percent of

as

a

the total number of animals tested, including the original test, the sample is nontoxic.

(3) Moisture. Proceed as directed in $ 440.80a(b)(5)(i) of this chapter. (4) PH. Proceed

directed in $ 440.80a(b)(5)(ii) of this chapter, using a saturated aqueous-ethanol (1:1) solution prepared by adding 100 milligrams per milliliter.

(5) Paper chromatograph method–(i) Apparatus

and

Teagents/(a) Chromatographic chamber (cylinder glass-stoppered museum jar 11.5 inches x 3.5 inches).

(6) Chromatographic paper (8 inches x 8 inches Whatman No. 1).

(c) 0.1 N hydrochloric acid.

(d) Resolving solvent: Butyl acetate, benzene, nitromethane, pyridine (5:5:5:1 by volume).

(e) Spray reagent: 15 grams antimony trichloride per 100 milliliters of chloroform.

(ii) Procedure. Dissolve the sample in chloroform to give a solution containing 10 milligrams to 20 milligrams per milliliter, Prepare

sheet of chromatographic paper by drawing a line of origin parallel to and 1 inch from the edge of the paper. Wet the paper thoroughly with the 0.1 N hydrochloric acid and blot it firmly between sheets of absorbent paper. Starting 2 inches in from the edges and at l-inch intervals, apply 3 microliters to 5 microliters of the sample solutions to the starting line. Allow a few minutes for the paper to dry partially. While the paper is still damp, form a cylinder by bringing the outer edges together, allowing about 1-inch overlap, and secure with a paper clip. Stand the paper in the chromatographic chamber, which has been filled to a depth of 1/2inch with the resolving solvent. After the solvent front rises to a height of 4 inches to 5 inches above the origin, remove the paper from the tank and hang it up to air dry. Spray the dried paper with the antimony trichloride reagent. Hang the paper in a 100° C. oven for 3 minutes. A purple spot becomes visible for trioleandomycin at an Rp value of about 0.85. The approximate Rf values for

diacetyloleandomycin, monoacetyloleandomycin,

and oleandomycin are, respectively, 0.72, 0.27, and 0.13.

(6) Acetyl determination (1) Apparatus and reagents. (a) One three-necked Pyrex flask of approximately 45 milliliters capacity, pear-shaped with Tjoints, agar inlet tube, glass-stoppered funnel, glass condenser, and bubble counter.

(6) 50-milliliter Pyrex Erlenmeyer flask.

(c) 10-milliliter burette, calibrated in 0.02 milliliter.

(d) Anhydrous methanol, reagent grade. (e) 2 N sodium hydroxide solution.

Sulfuric acid solution prepared by adding 100 milliliters of concentrated H2SO4 to 200 milliliters of water.

(9)1 N barium chloride solution.

(h) Phenolphthalein solution (1 percent in ethanol).

(i) Water-pumped nitrogen. 6) NaOH solution, 0.015 N.

(ii) Procedure. Weight accurately (to 0.01 milligram) approximately 30 milligrams of the sample into the threenecked acetyl flask. Add 2.0 milliliters of methanol to dissolve the sample, then add slowly with gentle swirling, 1.0 milliliter of NaOH solution. Connect the gas inlet tube with bubble counter attached, and adjust nitrogen flow to about two bubbles a second. Put glassstoppered funnel in centerneck of acetyl flask and put about 5 milliliters of H2O in the funnel. Add a boiling chip to the solution and attach condenser in the refluxing position with water cooling. Adjust burner flame under acetyl flask to reflux solution gently. Reflux for 30 minutes. Cool assembly slightly then rinse down condenser (still in reflux position) with a few milliliters of H20. Reassemble condenser to the distillation position and add water through the funnel to make a total of approximately 5 milliliters of H2O added to acetyl flask. Adjust burner flame so that about 5 milliliters of H2O and methanol is distilled over in approximately 10 minutes. Discard this distillate. Cool acetyl flask slightly. Acidify solution in flask by adding 1 milliliter of the sulfuric acid solution through the funnel. Adjust burner flame and distill over approximately 20 milliliters of distillate into an Erlenmeyer flask in about 20 minutes, adding water through the funnel as necessary. It is important to keep the liquid volume in the acetyl flask around 2 milliliters to 3 milliliters in order to obtain a quantitative recovery of the acetic acid. Collect a second fraction of distillate, about 10 milliliters in volume. As the second fraction is distilling, process the first fraction. Heat the first reaction and boil gently about 20 seconds. Add a few drops of BaCl2 solution to check if any sulfate was distilled over. If the sulfate is present, discard and repeat the whole determination. If the sulfate is absent immediately titrate the solution with the 0.015 N NaOH solution to a faint pink endpoint, using one drop of phenolphthalein solution as the indicator. Repeat the above procedure with the second fraction. If the second fraction requires less than 0.10 milliliter of the 0.015 N NaOH solution and all the acetic acid has been distilled over, the determination is completed. If greater than this, collect a third fraction of approximately 10 milliliters and titrate this as before. Total volumes of NaOH used and calculate results as follows:

$ 436.517(b)(1)(iii) is used to prepare the standard curve, by further diluting with pH 8.0 buffer to final concentrations of 0.64, 0.80, 1.0, 1.25, and 1.56 u g. per milliliter. The 1.0 ug per milliliter solution is the reference concentration. In lieu of the method described in this subparagraph, the neomycin content may also be determined as follows. Using the aqueous solution described, prepare the sample and proceed as directed in 8436.517(b)(1), except use Staphylococcus aureus (American Type Culture Collection 12715)1 as the test organism, which is grown and maintained on agar containing 100 u g. of tetracycline hydrochloride per milliliter of agar. Its neomycin content is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(2) Tetracycline hydrochloride-neomycin sulfate powder--(i) Tetracycline hydrochloride content. Prepare the sample as directed in 8436.514(a)(2). Use an appropriate aliquot and proceed as directed in $446.81a(b)(1) of this chapter. Its tetracycline hydrochloride content is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(ii) Neomycin content. Use an appropriate aliquot of the solution prepared in paragraph (a)(2)(i) of this section and proceed as directed in paragraph (a)(1)(ii) of this section. Its neomycin content is satisfactory if it contains not less than 85 percent of the number of milligrams that it is represented to contain.

(b) Sterility. Thoroughly cleanse with a suitable disinfectant the value (do not flame) of each container to be tested. Into each of two empty, sterile Erlenmeyer flasks stoppered with a cotton plug, spray quantitites sufficient to yield a residue of approximately the equivalent of 50 milligrams from 10 separate cans by removing the plug temporarily and using aseptic technique while spraying; allow propellant to evaporate, add 250 milliliters to 500 milliliters of diluting fluid B in lieu of diluting fluid A, and swirl the flasks to dissolve the contents. Then proceed as

(Milliliters of NaOH YN NaOH X 0.043 x 100)

Weight sample in grams=Percent acetyl.

(7) Crystallinity. Proceed as directed in $440.80a(b)(5)(iii) of this chapter.

$ 436.516 Tetracycline-neomycin com

plex powder topical; tetracycline hydrochloride-neomycin sulfate

powder topical. (a) Potency_(1) Tetracycline-neomycin complex powder-(i) Tetracycline content. Proceed as directed in $436.514(a)(2), except use water in lieu of 0.1 N HCl for dissolving the sample. Its tetracycline content is satisfactory if it contains not less than 85 percent of the equivalent number of milligrams of tetracycline hydrochloride that it is represented to contain.

(ii) Neomycin content. Using 0.1 M potassium phosphate buffer, pH 8.0, dilute an appropriate aliquot of the aqueous solution, prepared as directed in paragraph (a)(1) of this section, to a final concentration of 1 ug. per milliliter (estimated), and proceed as directed in $ 436.515(c)(1), except that the neomycin standard stock solution described

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

directed in $ 436.20 of this chapter using the method described in paragraph (e)(1) of that section.

(c) Moisture. Proceed as directed in $536.513(c) of this chapter.

(d) Tetracycline-neoinycin complex used in making the drug—(1) Potency-(i) Tetracycline content. Dissolve the sample to be tested in sufficient water to give a convenient stock solution. Using an appropriate aliquot, proceed as directed in $446.81a(b)(1).

(ii) Neomycin content. Using an aliquot of the stock solution prepared as directed in paragraph (d)(1)(i) of this paragraph, proceed as directed in paragraph (a)(2) of this section, except the last sentence of that subparagraph.

(2) Toxicity. Proceed as directed in $ 440.80a(b)(4) of this chapter using 0.5 milliliter of a solution prepared by diluting the sample with physiological sodium chloride solution to contain 200 U g. of neomycin per milliliter (estimated).

(3) pH. Using a 1-percent aqueous solution, proceed

directed in $ 440.80a(b)(5)(ii) of this chapter.

as

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

(3) Disintegration time. Proceed as directed in $440.180a(b)(3).

(b) Neomycin used in making the tablets-(1) Potency–(i) Cylinders (cups). Use cylinders described under $ 440.80a(b)(1)(i) of this chapter.

(ii) Culture medium. Use the medium described in $ 440.80a(b)(1) (ii)(a) of this chapter for both the base and seed layers, except its pH after sterilization is 7.8 to 8.0.

(iii) Working standard. Dry the working standard (obtained from the U.S.P. Reference Standards Committee, 46 Park Avenue, New York 16, N.Y.) for 3 hours at 60° C. and a pressure of 5 millimeters or less and weigh out a sufficient quantity to make a convenient stock solution by diluting with a 0.1 M potassium phosphate buffer, pH 7.8 to 8.0. The stock solution, when stored at a temperature of approximately 15° C., or less, may be used for a period not exceeding 1 month.

(iv) Standard curve. Using the stock solution, prepare a daily standard curve as directed in $444.70a(b)(1)(iv) of this chapter, using solutions of the neomycin working standard in 0.1M potassium phosphate buffer, pH 8.0, in concentrations of 6.4, 8.0, 10.0, 12.5, and 15.6 micrograms per milliliter if the test organism Staphylococcus aureus (ATCC 6538P), 1 or in concentrations of 0.64, 0.80, 1.0, 1.25, and 1.56 micrograms per milliliter if the test organism is Staphylococcus epidermidis (ATCC 12228).1 The 10.0 micrograms per milliliter and the 1.0 microgram per milliliter concentrations are used as the reference points.

(V) Preparation of test organism. The test organism is Staphylococcus aureus (ATCC 6538P), 1 which is maintained on agar described in $ 440.80a(b)(1)(ii)(a) of this chapter. From a stock slant inoculate a Roux bottle containing this same agar and incubate for 24 hours at 32° C.-35° C. Wash the resulting growth from the agar surface with about 50 milliliters of sterile sodium chloride solution. Standardize this suspension by determining the dilution that will permit 80 percent light transmission through a filter at 6500 Angstrom units

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

8 436.517 Bacitracin-neomycin tablets;

zinc bacitracin-neomycin tablets; bacitracin methylene disalicylate

neomycin tablets. (a) Tablets (1) Potency–(i) Bacitracin, zinc bacitracin, or bacitracin methylene disalicylate content. Proceed as directed in $448.110a(b)(1). Its content of bacitracin, zinc bacitracin, or bacitracin methylene disalicylate is satisfactory if it contains not less than 85 percent of the number of units per tablet that it is represented to contain.

(ii) Neomycin content. Place 5 tablets in a blending jar and add thereto 200 milliliters of a 500-milliliter quantity of 0.10-percent phosphate buffer pH 8.0. After blending for 1 minute with a high-speed blender, add the remainder of the buffer. Blend again for 1 minute and make the proper estimated dilutions in the buffer and proceed as directed in paragraph (b)(1) of this section. Its content of neomycin is satisfactory if it contains not less than 85 percent of the number of milligrams of activity that it is represented to contain.

1 See footnote 1 to $436.516.

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