A. Area of the amikacin peak in the chromatogram of the sample (at a retention time equal to that observed for the standard);

A, Area of the amikcacin peak in the chromatogram of the amikacin working standard;

P, Amikacin activity in the amikacin working standard solution in micrograms per milliliter;

Cu-Milligrams of the sample per milliliter of sample solution; and

m=Percent loss on drying of the sample.

(2) Loss on drying. Proceed as directed in § 436.200(c) of this chapter.

(3) pH. Proceed as directed in § 436.202 of this chapter, using an aqueous solution containing 10 milligrams per milliliter.

(4) Identity. Proceed as directed in § 436.318 of this chapter.

(5) Residue on ignition. Proceed as directed in § 436.207(a) of this chapter.

(6) Specific rotation. Proceed as directed in § 436.210 of this chapter, using an aqueous solution containing 20 milligrams of amikacin sulfate per milliliter, and a 1.0 decimeter polarimeter tube. Calculate the specific rotation on the anhydrous basis.

(7) Crystallinity. Proceed as directed in § 436.203(a) of this chapter.

[55 FR 38676, Sept. 20, 1990]

§ 444.10a Dihydrostreptomycin sulfate, crystalline

dihydrostreptomycin

sulfate, dihydrostreptomycin hydrochloride.

(a) Requirements for certification—(1) Dihydrostreptomycin sulfate is the hydrogenated sulfate salt of a kind of streptomycin or a mixture of two or more such salts; crystalline dihydrostreptomycin sulfate is the hydro

genated crystalline sulfate salt of a kind of streptomycin or a mixture of two or more such salts; dihydrostreptomycin hydrochloride is the hydrogenated hydrochloride salt of a kind of streptomycin or a mixture of two or more such salts. Each such drug conforms to all requirements prescribed by § 444.70a(a) for streptomycin sulfate and streptomycin hydrochloride, and is subject to all procedures prescribed by § 444.70a(a) for streptomycin sulfate and streptomycin hydrochloride, except

that:

(i) Its potency is not less than 650 micrograms per milligram, except that if it is crystalline dihydrostreptomycin sulfate its potency is not less than 725 micrograms per milligram.

(ii) Its content of streptomycin sulfate or streptomycin hydrochloride is not more than 3.0 percent when calculated as streptomycin base, except that if it is crystalline dihydrostreptomycin sulfate its content of streptomycin sulfate is not more than 1.0 percent.

(iii) Its labeling shall conform to the requirements of § 444.70a(a)(3)(iii).

(b) Tests and methods of assay—(1) Potency. Using the dihydrostreptomycin working standard as a standard of comparison, proceed as directed in § 444.70a(b)(1). Its potency is satisfactory if it contains not less than 90 percent of the number of milligrams that it is represented to contain.

(2) Content of streptomycin sulfate or streptomycin hydrochloride—(i) Reagents. (a) 10 percent ferric chloride stock solution. Dissolve 5 grams of FeCl3•6H2O in 50 milliliters 0.1N HCl.

(b) 0.25 percent ferric chloride solution. Dilute 2.5 milliliters of 10 percent ferric chloride in 0.1N HC1 to 100 milliliters with 0.01N MC1. Prepare the solution fresh daily.

(ii) Standard curve. Keep the working standard (obtained from the Food and Drug Administration) at -20° C. in tightly stoppered containers which in turn are kept in larger stoppered vials containing a suitable desiccant. Dry an appropriate amount of the working standard at 100° C. and a pressure of 5 millimeters or less for 4 hours. Prepare a stock aqueous solution containing 1.0 milligram of streptomycin base per milliliter. Store this standard solution

in a refrigerator and use for no longer than 2 weeks. Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 milliliters of this standard solution and 10 milliliters of distilled water to each of six 25-milliliter volumetric flasks. Add 9.0, 8.0, 7.0, 6.0, and 5.0 milliliters of distilled water to the five tubes, respectively, to give each a total volume of 10 milliliters. To each add 2.0 milliliters of 1N NaOH and then heat the flasks in a boiling water bath for 10 minutes. Cool the flasks in ice water for 3 minutes and acidify the solutions with 2.0 milliliters of 1.2N HCl. To each flask add 5.0 milliliters of 0.25 percent ferric chloride reagent, make to volume with distilled water, and mix thoroughly. Transfer the colored solutions to 2.0-centimeter absorption cells and measure the percent light transmission at 530 mu in a suitable photoelectric colorimeter. Set the colorimeter at 100 percent light transmission for the zero concentration and then obtain the percent light transmission of the sample. Prepare a standard curve on semilog paper, plotting the percent light transmission on the logarithmic ordinate scale and the concentration of streptomycin base on the abscissa.

(iii) Procedure. Dilute the contents of a vial or a sufficient amount of bulk material to give a concentration of approximately 20 milligrams per milliliter. From the amount of streptomycin obtained, calculate the percent streptomycin as follows:

Percent streptomycin=(Milligrams of streptomycin-100)/(Milligrams of dihydrostreptomycin found in the sample used)

(3) Sterility. Proceed as directed in § 436.20 of this chapter, using the method described in paragraph (e)(1) of that section.

(4) Toxicity, pyrogens, histamine, moisture, pH, crystallinity. Proceed as directed in §§ 444.70a(b) (3), (4), (5), (6) and 440.80a(b)(5)(iii) of this chapter.

§ 444.20 Gentamicin sulfate.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Gentamicin sulfate is the sulfate salt of a kind of gentamicin or a mixture of two or more such salts. It is a powder, white to buff in color. It is readily soluble in water but insoluble

in ethanol. It is so purified and dried that:

(i) Its potency is not less than 590 micrograms of gentamicin per milligram on an anhydrous basis.

(ii) [Reserved]

(iii) Its loss on drying is not more than 18.0 percent.

(iv) Its pH in an aqueous solution containing 40 milligrams per milliliter is not less than 3.5 and not more than 5.5.

(v) Its specific rotation in an aqueous solution containing 10 milligrams per milliliter at 25°C. is not less than +107° and not more than + 121°.

(vi) Its content of gentamicin C1 is not less than 25 nor more than 50 percent; of gentamicin Cia, not less than 15 nor more than 40 percent; and of gentamicin C2, not less than 20 nor more than 50 percent.

(vii) It gives a positive identity test for gentamicin sulfate.

(2) Labeling. It shall be labeled in accordance with the requirements of § 432.5(b) of this chapter.

(3) Requests for certification; samples. In addition to complying with the requirements of § 431.1 of this chapter, each such request shall contain:

(i) Results of tests and assays on the batch for potency, loss on drying, pH, specific rotation, content of gentamicins C1, Cia, and C2, and identity.

(ii) Samples required. 10 packages, each containing approximately 500 milligrams.

(b) Tests and methods of assay—(1) Potency. Proceed as directed in § 436.105 of this chapter, preparing the sample for assay as follows: Dissolve an accurately weighed sample in sufficient 0.1M potassium phosphate buffer, pH 8.0 (solution 3), to give a stock solution of convenient concentration. Further dilute the stock solution with solution 3 to the reference concentration of 0.1 microgram of gentamicin per milliliter (estimated).

(2) [Reserved]

(3) Loss on drying. Proceed as directed in § 436.200(c) of this chapter.

(4) pH. Proceed as directed in §436.202 of this chapter, using an aqueous solution containing 40 milligrams of gentamicin per milliliter.

(5) Specific rotation. Accurately weigh the sample to be tested in a volumetric flask and dilute with sufficient distilled water to give a solution containing approximately 10 milligrams per milliliter. Proceed as directed in § 436.210 of this chapter, using a 1.0-decimeter polarimeter tube and calculate the specific rotation on an anhydrous basis.

(6) Content of gentamicins C1, C1a, and C2. Proceed as directed in § 444.20a(b)(8).

(7) Identity. Proceed as directed in § 436.211 of this chapter, using a 0.5 percent mixture of the sample in a potassium bromide disc prepared as described in paragraph (b)(1) of that section.

[39 FR 19046, May 30, 1974, as amended at 50 FR 19919, May 13, 1985]

§ 444.20a Sterile gentamicin sulfate.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Sterile gentamicin sulfate is the sulfate salt of a kind of gentamicin or a mixture of two or more such salts. It is a powder, white to buff in color. It is readily soluble in water but insoluble in ethanol. It is so purified and dried that:

(1) Its potency is not less than 590 micrograms of gentamicin per milligram on an anhydrous basis.

(ii) It is sterile.

(iii) [Reserved]

(iv) It is nonpyrogenic.

(v) Its loss on drying is not more than 18.0 percent.

(vi) Its pH in an aqueous solution containing 40 milligrams per milliliter is not less than 3.5 and not more than 5.5.

(vii) Its specific rotation in an aqueous solution containing 10 milligrams per milliliter at 25° C. is not less than +107° and not more than +121°.

1

(viii) Its content of gentamicin C1 is not less than 25 nor more than 50 percent; of gentamicin Cia, not less than 15 nor more than 40 percent; and of gentamicin C2, not less than 20 nor more than 50 percent.

(ix) It gives a positive identity test for gentamicin sulfate.

(2) Labeling. It shall be labeled in accordance with the requirements of § 432.5(b) of this chapter.

(3) Requests for certification; samples. In addition to complying with the re

quirements of §431.1 of this chapter, each such request shall contain:

(1) Results of tests and assays on the batch for potency, sterility, pyrogens, loss on drying, pH, specific rotation, content of gentamicins C1, Cia, and C2, and identity.

(ii) Samples required:

(a) For all tests except sterility: 10 packages, each containing approximately 500 milligrams.

(b) For sterility testing: 20 packages, each containing approximately 300 milligrams.

(b) Tests and methods of assay—(1) Potency. Proceed as directed in § 436.105 of this chapter, preparing the sample for assay as follows: Dissolve an accurately weighed sample in sufficient 0.1M potassium phosphate buffer, pH 8.0 (solution 3), to give a stock solution of convenient concentration. Further dilute the stock solution with sufficient solution 3 to give a reference concentration of 0.1 microgram of gentamicin per milliliter (estimated). (2) Sterility. Proceed as directed in § 436.20 of this chapter, using the method described in paragraph (e)(1) of that section.

(3) [Reserved]

(4) Pyrogens. Proceed as directed in § 436.32(a) of this chapter, using a solution containing 10.0 milligrams of gentamicin per milliliter.

(5) Loss on drying. Proceed as directed in § 436.200(c) of this chapter.

(6) pH. Proceed as directed in § 436.202 of this chapter, using an aqueous solution containing 40 40 milligrams of gentamicin per milliliter.

(7) Specific rotation. Accurately weigh the sample to be tested in a volumetric flask and dilute with sufficient distilled water to give a solution containing approximately 10 milligrams per milliliter. Proceed as in directed § 436.210 of this chapter, using a 1-decimeter polarimeter tube and calculate the specific rotation on an anhydrous basis.

(8) Content of gentamicins C1, Cia, and C2-(i) Equipment-(a) Chamber (chromatographic). Use a suitable chromatography jar with a tightly fitting, ground glass contact top for descending chromatography.

(b) Sheets (chromatographic). Cut a 57 × 46-centimeter sheet of Whatman No. 2

filter paper, or chromatographic paper that will produce similar results, into four strips of about 14.25 x 46 centimeters. Draw a starting line 9 centimeters from one end and mark two dots on this line, each 4 centimeters from each edge.

(ii) Reagents. Use reagent grade solvents and chemicals.

(iii) Solvent system. In each of two separators, equilibrate 200 milliliters of chloroform and 100 milliliters of methanol with 100 milliliters of 17 percent (9 molar) ammonium hydroxide. Without allowing the phases of one to separate, add the entire mixture to the chromatography jar and allow 24 hours for saturation. Allow the second separator to stand until the phases separate and use the lower phase only chromatographic solvent.

as the

(iv) Ninhydrin reagent. To 1 gram of ninhydrin and 0.1 gram of cadmium acetate, add 3 milliliters of water and 1.5 milliliters of gaacial acetic acid and shake. Add 100 milliliters of n-propanol and shake until solution is complete. Keep this solution in a brown bottle under refrigeration.

(v) Procedure. Prepare an aqueous solution containing 40 milligrams of the sample per milliliter. Apply 5 microliters of this solution to each dot on the sheet. Prepare two such sheets and place them in the tank so that elution

will take place from separate troughs. Fill the two troughs with the chromatographic solvent. Develop the sheets in a descending manner until the solvent front reaches the bottom of the paper (approximately 31⁄2 hours at 25° C.). Remove the sheets and dry in a hood for 30 minutes. Cut each sheet in half, lengthwise. Spray one half with ninhydrin reagent and place the sprayed strip in a drying oven at 100° C. for 1 minute. The gentamicin fractions appear as reddish zones. The zone furthest from the origin is gentamicin C1, the one closest is gentamicin Cia, and the middle zone is gentamicin C2. Cut the corresponding zones out of the other unsprayed half of the sheet. Cut each portion of the sheet thus obtianed into small strips and put those from each zone into a separate 125-milliliter glass-stoppered flask. Add 50 milliliters of 0.1M potassium phosphate buffer, pH 8, to each flask and swirl the flask mechanically for 30 minutes. Decant the solution from each flask into separate test tubes and allow the paper to settle. Pipet 4 milliliters of each clear solution into a 25-milliliter volumetric flask and make to volume with the pH 8 buffer. Assay these solutions as directed in paragraph (b)(1) of this section.

(vi) Calculations.

(9) Identity. Proceed as directed in § 436.211 of this chapter, using a 0.5 percent mixture of the sample in a potassium bromide disc prepared as described in paragraph (b)(2) of that section.

[39 FR 19046, May 30, 1974, as amended at 50 FR 19919, May 13, 1985]

§ 444.30 Kanamycin sulfate.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Kanamycin sulfate is the sulfate salt of a kind of kanamycin or a mixture of two or more such salts. It is so purified and dried that:

(i) Its potency on an anhydrous basis is not less than 750 micrograms of kanamycin per milligram.

(ii) [Reserved]

(iii) Its loss on drying is not more than 4 percent.

(iv) Its pH is an aqueous solution containing 10 milligrams per milliliter is not less than 6.5 and not more than 8.5

(v) Its residue on ignition is not more than 1.0 percent.

(vi) It gives a positive identity test for kanamycin.

(vii) It contains not more than 5.0 percent kanamycin B.

(viii) It is crystalline.

(2) Labeling. It shall be labeled in accordance with the requirements of § 432.5(b) of this chapter.

(3) Requests for certification; samples. In addition to complying with the requirements of §431.1 of this chapter, each such request shall contain:

(i) Results of tests and assays on the batch for potency, loss on drying, pH, residue on ignition, identity, kanamycin B content, and crystallinity.

(ii) Samples required on the batch: 10 packages, each containing approximately 500 milligrams.

(b) Tests and methods of assay—(1) Potency. Proceed as directed in § 436.106 of this chapter, preparing the sample for assay as follows: Dissolve an accurately weighed sample in sufficient sterile distilled water to give a stock solution of convenient concentration. Further dilute an aliquot of the stock solution with sterile distilled water to the reference concentration of 10

micrograms of kanamycin per milliliter (estimated).

(2) [Reserved]

(3) Loss on drying. Proceed as directed in § 436.200(b) of this chapter.

(4) pH. Proceed as directed in § 436.202 of this chapter, using a solution containing 10 milligrams per milliliter.

(5) Residue on ignition. Proceed as directed in § 436.207(a) of this chapter.

(6) Identity. Dissolve about 10 milligrams of kanamycin sulfate in 1 milliliter of water, and add 1 milliliter of a 1:500 solution of triketohydrindene hydrate in normal butyl alcohol; then add 0.5 milliliter of pyridine. Heat in a steam bath for 5 minutes and add 10 milliliters of water; a deep-purple color is produced.

(7) Kanamycin B content—(i) Cylinders (cups). Use cylinders described under § 440.80a(b)(1)(i) of this chapter.

(ii) Culture medium. Use ingredients that conform to the standards prescribed by the U.S.P. or N.F. Make agar for the base and seed layers as follows:

(iii) Working standard. Dissolve a suitable quantity of the kanamycin sulfate working standard, accurately weighed, in 0.1M potassium phosphate buffer, pH 8.0, to give a concentration equivalent to 1.0 milligram of kanamycin per milliliter.

(iv) Preparation of sample. To 100 milligrams, accurately weighed, of kanamycin sulfate in a suitable container (such as a 7.5-milliliter serum vial) add 5.0 milliliters of 6N hydrochloric acid, and tightly close the container. Heat in a water bath at 100° C. for 1 hour and cool. Add 4 milliliters of 6N sodium hydroxide, then dilute with sterile 0.1M potassium phosphate buffer, pH 8.0, to obtain a concentration of the equivalent of 1 microgram of kanamycin per milliliter (estimated).