grams of the sample "as is" and 15 milligrams of the working standard dried as directed in §436.200(a) of this chapter. Transfer each weighing to separate 100-milliliter volumetric flasks. Dissolve the material and bring to volume with spectrophotometric-grade methyl alcohol. Mix well. Pipette 5.0 milliliters of each solution into separate 25milliliter volumetric flasks, dilute to volume with spectrophotometric-grade methyl alcohol. Mix well. Using a suitable spectrophotometer and 1-centimeter absorption cells, determine the absorbance of the sample solution at the 240-nanometer and at the 445nanometer absorption peaks (the exact position of the peaks should be determined for the particular instrument used). Determine the absorbance of the standard at the 445-nanometer absorption peak.

(ii) Calculations. Calculate the relative absorptivity and the ratio for the absorbances of the sample as follows:

A1

A2

A,=Absorbance at 240 nanometers for the sample;

A2=Absorbance at 445 nanometers for the sample;

A=Absorbance at 445 nanometers for the standard;

M=Percent moisture in the sample.

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

(5) Identity. The high-pressure liquid chromatogram of the sample determined as directed in paragraph (b)(1) of this section compares qualitatively to that of the dactinomycin working standard.

[49 FR 6092, Feb. 17, 1984, as amended at 49 FR 24018, June 11, 1984; 50 FR 19675, May 10, 1985]

§ 450.22 Daunorubicin hydrochloride. (a) Requirements for certification——(1) Standards of identity, strength, quality, and purity. Daunorubicin hydrochloride is the monohydrochloride salt of (1s,3s)3-acetyl-1,2,3,4,6,11-hexahydro-3,5,12trihydroxy-10-methoxy-6,11-dioxo-1naphthacenyl-3-amino-2,3,6-trideoxy-a

L-lyro-hexopyranoside. It is a red-orange, hygroscopic powder. It is so purified and dried that:

(i) Its potency is not less than 842 micrograms and not more than 1,030 micrograms of daunorubicin per milligram.

(ii) Its moisture content is not more than 3.0 percent.

(iii) Its pH in an aqueous solution containing 5 milligrams per milliliter is not less than 4.5 and not more than 6.5.

(iv) It is crystalline.

(v) It passes the identity test for daunorubicin.

(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:

(1) Results of tests and assays on the batch for potency, moisture, pH, crystallinity, and identity.

(ii) Samples required: 14 packages, each containing approximately 40 milligrams.

(b) Tests and methods of assay. Daunorubicin hydrochloride is toxic. It must be handled with care in the laboratory. Transfer all dry powders in a suitable hood. Wear rubber gloves, protective gowns, head coverings, and protective eye goggles when handling dry powders. Avoid inhaling fine particles of powder. Solutions should not be pipetted by mouth. If the substance contacts the skin, promptly wash with soap and water. Dispose of all waste material by dilution with large volumes of sodium hypochlorite solution.

(1) Potency. Use either of the following methods; however, the results obtained from the high-pressure liquid chromatography shall be conclusive.

(i) High-pressure liquid chromatography. Proceed as directed in § 436.322 of this chapter, except in lieu of the mobile phase and pH described in paragraph (b)(2) of that section, use a mixture of water: acetonitrile (62:38) adjusted to pH 2.2±0.2 with phosphoric acid. Prepare the sample and standard solutions and calculate daunorubicin content as follows:

the

W1-Weight of the daunorubicin working standard in milligrams;

W=Weight of the sample in milligrams; M-Moisture content of the sample in percent;

P=Potency of the daunorubicin working standard in micrograms per milligram.

(ii) Microbiological turbidimetric assay for daunorubicin-(a) Preparation of working standard stock solutions and standard response line concentrations. Dissolve an accurately weighed portion of the working standard with sufficient 0.054M sodium phosphate buffer, pH 6.9 (solution 18), as described in

§ 436.101(a)(18) of this chapter, to obtain a stock solution containing 1 milligram of daunorubicin activity per milliliter. The working standard stock solution may be stored under refrigeration for 1 week. Further dilute an aliquot of the stock solution with solution 18 to obtain standard response line concentrations of 4, 8, and 16 micrograms of daunorubicin activity per milliliter. The 8-micrograms-permilliliter concentration is the reference concentration of the assay.

(b) Preparation of sample solution. Dissolve an accurately weighed portion of the sample with sufficient 0.054M sodium phosphate buffer, pH 6.9 (solution 18), as described in §436.101(a)(18) of this chapter, to obtain a stock solution containing 1 milligram of daunorubicin activity per milliliter (estimated). Further dilute an aliquot of the stock solution with solution 18 to the reference concentration of 8 micrograms of daunorubicin activity per milliliter (estimated).

(c) Procedure for assay. Place 1.0 milliliter of each concentration of the standard response line and of the sample solution in each set of replicate tubes (as described in § 436.100(b)(1) of this chapter). Eighteen tubes are used for the three-point standard response line and six for each sample. To each tube, add 9 milliliters of medium 3 (as listed in §436.102(b)(3) of this chapter), inoculated with 2 milliliters of a suspension of test organism I per liter of medium 3. The suspension of test organism I is prepared as described in § 436.103 of this chapter, except incubate the slants and Roux bottle for 16 to 18 hours at 37° C. Place the inoculated tubes immediately in a water bath at 37°C for approximately 3 hours.

The absorbance value for the growth control should be approximately 0.700.75 and the absorbance values for the 16 and 4 micrograms per milliliter standard doses should be approximately 0.25-0.35 and 0.55-0.65, respectively. An adjustment of the inoculum may be necessary in order to obtain absorbance values to these approximate levels in a 3-hour time period. Remove the tubes from the water bath and add 0.5 milliliter of a 12-percent formaldehyde solution to each tube. Determine the absorbance value of each tube in a suitable spectrophotometer, at a wavelength of 530 nanometers. Set the instrument at zero absorbance with an uninoculated blank composed of the same amounts of medium 3, solution 18, and formaldehyde used in the assay.

(d) Estimation of potency. Estimate the potency of the sample as follows: Using the three x values and the three corresponding y values, calculate Σx, Ex2,(Ex)2, Σy and Exy. Calculate b, the slope (regression coefficient), and a, the Y-intercept of the standard response line by the following equations:

a =

16.0 1.20412

8.0 0.90309

4.0 0.60206

Σχ = (Ex) 2 Σχε

3 2.70927 # 7.34014

2.62795

Mean response, Y, of sample solution=0.405.

Calculated concentration, X, sample solution

=

antilog

= 8.32 micrograms per milliliter

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

(3) pH. Proceed as directed in § 436.202 of this chapter, using an aqueous solution containing 5 milligrams per milliliter.

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

(5) Identity. The high-pressure liquid chromatogram of the sample determined as directed in paragraph (b)(1)(i) of this section compares qualitatively to that of the daunorubicin working standard.

[45 FR 75195, Nov. 14, 1980]

§ 450.24 Doxorubicin hydrochloride.

(a) Requirements for certification—(1) Standards of identity, strength, quality, and purity. Doxorubicin hydrochloride is the monohydrochloride salt of (8S, 10.S)-10-[(3-amino-2,3,6-trideoxy-a-L

lyro- hexopyranosyl)oxy]-8-glycoloyl7,8,9,10-tetrahydro-6,8,11-trihydroxy-1methoxy-5,12-naphthacenedione. It is a red-orange, almost completely odorless, hygroscopic powder. It is so purified and dried that:

(i) Its doxorubicin hydrochloride content is not less than 970 micrograms and not more than 1,020 micrograms of doxorubicin hydrochloride per milligram on the anhydrous and solvent free basis.

(ii) Its total solvent residue (as acetone and alcohol) is not more than 2.5 percent.

(iii) It contains no depressor substances.

(iv) Its moisture content is not more than 4.0 percent.

(v) The pH of an aqueous solution containing 5 milligrams per milliliter is not less than 4.0 and not more than 5.5.

(vi) It is crystalline.

(vii) It passes the identity test for doxorubicin.

(viii) The total of any impurities detected by high-pressure liquid chromatography assay is not more than 3.0 percent.

(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 request shall contain:

(i) Results of tests and assays on the batch for doxorubicin hydrochloride content, solvent residue, depressor substances, moisture, pH, crystallinity, identity, and total impurities.

(ii) Samples required: 14 packages, each containing approximately 40 milligrams.

(b) Tests and methods of assay. Doxorubicin hydrochloride is toxic. It must be handled with care in the laboratory. Transfer all dry powders in a suitable hood while wearing rubber gloves. Avoid inhaling fine particles of powder. Solutions should not be pipetted by mouth. If the substance contacts the skin, wash with soap and water. Dispose of all waste material by dilution with large volumes of dilute sodium hypochlorite (bleach) solution. (1) Doxorubicin hydrochloride content (high-performance liquid chromatography).

Proceed as directed in § 436.216 of this chapter, using ambient temperature, an ultraviolet detection system operating at a wavelength of 254 nanometers, a 4.6-millimeter X 25centimeter column packed with microparticulate (5 to 10 micrometers in diameter) packing material, such as trimethylsilane chemically bonded to porous silica, a flow rate of not more than 2.0 milliliters per minute, and a known injection volume of between 10 and 20 microliters. Mobile phase, working standard and sample solutions, resolution test solution, system suitability requirements, and calculations are as follows:

(1) Mobile phase. Prepare a suitable mixture of water, acetonitrile, methanol, and phosphoric acid (540:290:170:2). Dissolve 1 gram of sodium lauryl sulfate in 1,000 milliliters of this solution, adjust with 2N sodium hydroxide to a pH of 3.6±0.1. Filter through a suitable

filter capable of removing particulate matter to 0.5 micron in diameter. Degas the mobile phase just prior to its introduction into the chromatograph.

(ii) Preparation of working standard, sample, and resolution test solutions—(A) Working standard solution. Dissolve an accurately weighed quantity of doxorubicin hydrochloride working standard in mobile phase to obtain a solution having a known concentration of 0.1 milligram of doxorubicin hydrochloride per milliliter.

(B) Sample solution. Transfer approximately 20 milligrams of sample, accurately weighed, to a 200-milliliter volumetric flask, add mobile phase to volume, and mix. This yields a solution containing 0.1 milligram of doxorubicin hydrochloride per milliliter (estimated).

(C) Resolution test solution. Use either of the following preparation methods:

(1) To 2 milliliters of a 1.0 milligram per milliliter solution of doxorubicin hydrochloride, add 20 microliters of 1N hydrochloric acid. Hold for 30 minutes at 95 °C in an oil bath.

(2) Dissolve about 10 milligrams of doxorubicin hydrochloride in 5 milliliters of water, add 5 milliliters of phosphoric acid, and allow to stand for about 30 minutes. Adjust with 2N sodium hydroxide (about 37 milliliters) to a pH of 2.6±0.1, add 15 milliliters of acetonitrile and 10 milliliters of methanol, mix, and filter. (Note: Portions of this solution may be frozen until needed, then thawed and mixed before use.)

(3) The procedures in paragraphs (b)(1)(ii)(C)(1) and (b)(1)(ii)(C)(2) of this section generate doxorubicinone, the aglycone of doxorubicin. Use this solution to determine the resolution requirement for the chromatographic system.

(iii) System suitability requirements— (A) Asymmetry factor. The asymmetry factor (As (for the doxorubicin peak measured at a point 5 percent of the peak height is not less than 0.7 and not more than 1.2.

(B) Efficiency of the column. The absolute column efficiency (h, (is satisfactory if it is not greater than 10.0, equivalent to 2,500 theoretical plates for a 25-centimeter column of 10-micrometer particles.

(C) Resolution. The resolution (R) between the peaks of doxorubicin and doxorubicinone (generated in situ) is satisfactory if it is not less than 5.5.

(D) Capacity factor. The capacity factor (k) for doxorubicin is satisfactory if it is in the range between 1.0 and 5.0.

(E) Coefficient of variation. The coefficient of variation (relative standard of deviation in percent) of 5 replicate injections is satisfactory if it is not more than 1.0 percent. If the system suitability parameters have been met, then proceed as described in §436.216(b) of this chapter.

(iv) Calculations. Calculate the micrograms of doxorubicin hydrochloride per milligram of sample as follows:

Cu

Milligrams of the sample per milliliter of sample solution;

m = Percent moisture content of the sample; and

X = Percent solvent residue determined as directed in paragraph (b)(2) of this section.

(2) Residue solvent (as acetone and alcohol)—(i) Standard preparation. Transfer to a 100-milliliter volumetric flask about 200 milligrams of acetone, 300 milligrams of dehydrated alcohol, and 1,000 milligrams of dioxane, each accurately weighed, and mix. Dilute with water to volume, and mix. Transfer 5.0 milliliters of the resulting solution to a 50-milliliter volumetric flask, dilute with water to volume, and mix. This solution contains about 0.2 milligram of acetone, 0.3 milligram of alcohol, and 1 milligram of dioxane per milliliter.