§ 440.10 Benzylpenicilloyl-polylysine

concentrate.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and Benzylpenicilloyl

purity.

polylysine concentrate is a pale yellow to dark yellow aqueous solution of benzylpenicilloyl e substituted poly-Llysine. It contains one or more suitable and harmless buffers. It is so purified that:

(i) It contains not less than 50 percent and not more than 70 percent benzylpenicilloyl substitution on the polylysine.

(ii) The benzylpenicilloyl concentration is not less than 1.25x10- 2M and not more than 2.0x10- 2M.

(iii) The penamaldate concentration is not more than 6.0×10 - 4M.

(iv) The penicillenate concentration is not more than 2.0x10-4M.

(v) Its pH is not less than 6.5 and not more than 8.5.

(2) Labeling. It shall be labeled in accordance with the requirements of § 432.5 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 percent benzylpenicilloyl substitution, benzylpenicilloyl content, penamaldate content, penicillenate content, and pH.

(ii) Samples required: 2 vials, each containing not less than 5 milliliters.

(b) Tests and methods of assay-(1) Percent benzylpenicilloyl substitution—(i) Lysine content—(a) Equipment. Amino acid analyzer capable of:

(1) Separating the hydrolysis products of benzylpenicilloyl polylysine into discrete components by means of an ion-exchange column.

(2) Mixing the separated amino acid components with a ninhydrin reagent and promoting the reaction in a coil at elevated temperatures.

(3) Quantitating the ninhydrin positive materials by means of a suitable colorimeter and recorder.

(b) Reagents-(1) Citrate buffer: Dissolve and dilute 19.69 grams of sodium citrate dihydrate, 16.5 milliliters of hydrochloric acid, 0.1 milliliter of pentachlorophenol, 5 milliliters of thiodiglycol in 900 milliliters of dis

tilled water; adjust to a pH of 2.2 and dilute to 1 liter with distilled water.

(2) Calibration mixture: Dissolve and dilute equal molar amounts of ammonia, and the L form of lysine in the citrate buffer to result in final concentrations of 2.5 × 10− 4M for each.

(c) Preparation of standard and sample solutions (1) Standard solution (standard lysine solution (2.5× 10-4 M)). Transfer an accurately weighed portion of 54.8 milligrams of lysine dihydrochloride to a 100-milliliter volumetric flask. Dissolve and dilute to mark with citrate buffer. Make an accurate tenfold dilution of this solution with citrate buffer. The resulting standard solution is 2.5x10-4 M with respect to lysine.

(2) Sample solution. Dilute 1 milliliter of the benzylpenicilloyl-polylysine concentrate to 10 milliliters with distilled water. Mix 1 milliliter of the diluted solution with 1.5 milliliters of 6.0N hydrochloric acid and seal in an ampule under nitrogen. Hydrolyze the solution for 22 hours at 110° C. Transfer the contents of the ampule quantitatively into a 50-milliliter round bottom flask and dry by rotary evaporation. Wash the contents and evaporate to dryness three times using 5-milliliter portions of distilled water. Dissolve the hydrolysate in 10 milliliters of citrate buffer.

(d) Procedure. Standardize the procedure for use of the amino acid analyzer with the calibration mixture. Apply 0.5 milliliter of the lysine standard solution to the amino acid analyzer and determine the area of the lysine peak. Apply 0.5 milliliter of the sample solution to the amino acid analyzer and determine the area of the lysine peak.

(e) Calculations. Calculate the lysine content by the following formula: Molar concentration of lysine Ax×2.5 in the benzylpenicilloylpolylysine concentrate

where:

BXC

chloride in 500 milliliters of distilled water.

(2) Saline phosphate buffer, pH 7.6: Dissolve 9 grams of sodium chloride and 1.38 grams monobasic sodium phosphate in 900 milliliters of distilled water, adjust to pH 7.6 and dilute to 1 liter with distilled water.

(b) Preparation of sample solution. Transfer 1 milliliter of the benzylpenicilloyl-polylysine concentrate into a 500-milliliter volumetric flask and dilute to volume with saline phosphate buffer, pH 7.6.

a

(c) Procedure. Transfer 3 milliliters of the sample solution into spectrophotometric cell. Using a suitable spectrophotometer and the saline phosphate buffer, pH 7.6, as a blank, determine the initial absorbance at 282 nanometers. Thereafter, react the diluted benzylpenicilloyl-polylysine solution with 0.02-milliliter portions of the mercuric chloride solution. Determine the absorbance at 282 nanometers at 1 and 3 minutes after each addition of mercuric chloride solution. The increased absorbance at 282 nanometers is used in calculating the benzylpenicilloyl content. Calculate the benzylpenicilloyl content by means of the following formula:

§ 440.11 Carbenicillin indanyl sodium.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Carbenicillin indanyl sodium is the monosodium salt of N-(2carboxy-3,3-dimethyl-7-oxo-4-thia-1azabicyclo [3.2.0] hept-6-yl)-2-phenylmalonamic acid, 1-(5-indanyl) ester. It is so purified and dried that:

(1) Its potency is not less than 659 micrograms and not more than 769 micrograms of carbenicillin per milligram on an anhydrous basis at the time of certification, and not less than 630 micrograms of carbenicillin per milligram on an anhydrous basis at any time during the expiration period. (ii) [Reserved]

(iii) Its moisture content is not more than 2.0 percent.

(iv) Its pH in an aqueous solution containing 100 milligrams per milliliter is not less than 5.0 nor more than 8.0.

(v) It gives a positive result to the identity test for carbenicillin indanyl sodium.

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

(3) Moisture. Proceed as directed in § 436.201 of this chapter.

(4) pH. Proceed as directed in § 436.202 of this chapter, using an aqueous solution containing 100 milligrams per milliliter.

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

[39 FR 18976, May 30, 1974, as amended at 50 FR 19918, May 13, 1985]

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

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

(i) Results of tests and assays on the batch for potency, sterility, pyrogens, moisture, pH, and identity.

(ii) Samples required:

(a) If the batch is packaged for repacking or for use in the manufacture of another drug:

(1) For all tests except sterility: 10 packages, each containing approximately 300 milligrams; and 5 packages, each containing approximately 1 gram.

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

(b) If the batch is packaged for dispensing:

(1) For all tests except sterility: A minimum of 15 immediate containers.

(2) For sterility testing: 20 immediate containers, collected at regular intervals throughout each filling operation.

(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 1.0 percent potassium phosphate buffer, pH 6.0 (solution 1), to give a stock solution of convenient concentration; and also if it is packaged for dispensing, reconstitute as directed in the labeling. Then, using a suitable hypodermic needle and syringe, remove all of the withdrawable contents if it is represented as a single-dose container; or if the labeling specifies the amount of potency in a given volume of the resultant preparation, remove an accurately measured representative portion from each container. If it is a single dose container, use a separate needle and syringe for each container. Dilute with sufficient solution 1 to give a stock solution of convenient concentration. Further dilute the stock solution with solution 1 to the reference concentration of 20.0 micrograms of carbenicillin 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) Pyrogens. Proceed as directed in § 436.32(b) of this chapter, using a solution containing 200 milligrams of carbenicillin per milliliter.

(4) [Reserved]

(5) Moisture. Proceed as directed in § 436.201 of this chapter.

(6) pH. Proceed as directed in § 436.202 of this chapter, using an aqueous solution containing 10 milligrams of carbenicillin per milliliter (or if packaged for dispensing, use a solution prepared as directed for reconstitution in the labeling).

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

[39 FR 18976, May 30, 1974, as amended at 42 FR 59857, Nov. 22, 1977; 45 FR 22921, Apr. 4, 1980; 50 FR 19918, May 13, 1985; 51 FR 27532, Aug. 1, 1986]

(iii) Its moisture content is not less than 3 percent and not more than 5 percent.

(iv) Its pH in an aqueous solution containing 10 milligrams per milliliter is not less than 4.5 nor more than 7.5. (v) Its cloxacillin content is not less than 82.5 percent.

(vi) It passes the identity test.

(vii) It is crystalline.

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

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

(i) Results of tests and assays on the batch for potency, moisture, pH,

cloxacillin content, identity, and crystallinity.

(ii) Samples required: 10 packages, each containing approximately 300 milligrams.

(b) Tests and methods of assay-(1) Potency. Use any of the following methods; however, the results obtained from the microbiological agar diffusion assay shall be conclusive.

(i) Microbiological agar diffusion assay. Proceed as directed in §436.105 of this chapter, preparing the sample for assay as follows: Dissolve an accurately weighed portion of the sample in sufficient 1 percent potassium phosphate buffer, pH 6.0 (solution 1), to give a stock solution of convenient concentration. Further dilute an aliquot of the stock solution with solution 1 to the reference concentration of 5 micrograms of cloxacillin per milliliter (estimated).

(ii) Iodometric assay. Proceed as directed in § 436.204 of this subchapter.

(iii) Hydroxylamine colorimetric assay. Proceed as directed in §436.205 of this subchapter.

(2) [Reserved]

(3) Moisture. Proceed as directed in § 436.201 of this subchapter.

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

(5) Cloxacillin content. Accurately weigh approximately 100 milligrams of the sample and dissolve in sufficient 5N sodium hydroxide to give a total volume of 25 milliliters. Place in a boiling water bath for 30 minutes. Cool, acidify 1 milliliter with 1 milliliter of dilute sulfuric acid (1 in 2), add 8 milliliters of water, and extract with two 25-milliliter portions of ethyl ether. Combine the ether extractives and extract with 25-milliliter portions of 0.1N sodium hydroxide. Combine the alkaline extractives and dilute to 100 milliliters with carbon dioxide-free water. Treat a portion of the cloxacillin working standard in the same manner. Using a suitable spectrophotometer, determine the absorbance of the solution in a 1centimeter cell at the absorption peaks at 257±3 nanometers and at 282±3 nanometers compared with a reagent blank. Determine the percent

cloxacillin in the sample by means of the following calculation:

Percent cloxacillin

where:

A1-Difference in absorbance for the sample between 257 nanometers and 282 nanometers:

A2 Difference in absorbance for the cloxacillin working standard, similarly treated.

(6) Identity. Proceed as directed in § 436.211 of this subchapter, using the 0.5 percent potassium bromide disc described in paragraph (b)(1) of that section.

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

[39 FR 18976, May 30, 1974, as amended at 42 FR 59857, Nov. 22, 1977; 50 FR 19918, May 13, 1985]

§ 440.17 Cyclacillin.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Cyclacillin is 6-(1aminocyclohexanecarboxamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]

heptane-2-carboxylic acid. It is a white to off-white powder. It is so purified and dried that:

(1) It contains not less than 900 micrograms and not more than 1,050 micrograms of cyclacillin per milligram.

(ii) [Reserved]

(iii) Its moisture content is not more than 1.0 percent.

(iv) Its pH in an aqueous solution containing 10 milligrams per milliliter is not less than 4.0 and not more than 6.5.

(v) Its cyclacillin content is not less than 90 percent on an anhydrous basis.

(vi) The acid-base titration concordance is such that the difference between the percent cyclacillin content when determined by nonaqueous acid titration and nonaqueous base titration is not more than six. The potencyacid titration concordance is such that

the difference between the potency value divided by 10 and the percent cyclacillin content of the sample determined by the nonaqueous acid titration is not more than six. The potency base titration concordance is such that the difference between the potency value divided by, 10 and the percent cyclacillin content of the sample determined by the nonaqueous base titration is not more than six.

(vii) It is crystalline.

(viii) It gives a positive identity test for cyclacillin.

(2) Labeling. In addition to the labeling requirements of § 432.5 of this chapter, each package shall bear on its outside wrapper or container and the immediate container the following statement, "For use in the manufacture of nonparenteral drugs only."

(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, moisture, pH, cyclacillin content, concordance, crystallinity, and identity.

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

(b) Tests and methods of assay—(1) Potency. Assay for potency by any of the following methods; however, the results obtained from the iodometric assay shall be conclusive.

(i) Microbiological agar diffusion assay. Proceed as directed in § 436.105 of this chapter, preparing the sample for assay as follows: Dissolve an accurately weighed portion of the sample in sufficient sterile distilled water to give a stock solution containing 1 milligram of cyclacillin per milliliter (estimated). Further dilute an aliquot of the stock