Bleomycin Sulfate
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This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition
Issued and maintained by the United States Pharmacopeial Convention (USP)
Bleomycin sulfate (salt).
Bleomycin sulfate (salt)
CAS RN: 9041-93-4; UNII: 7DP3NTV15T.
Bleomycin Sulfate is the sulfate salt of bleomycin, a mixture of basic cytotoxic glycopeptides produced by the growth of Streptomyces verticillus, or produced by other means. It has a potency of not less than 1.5 Bleomycin Units and not more than 2.0 Bleomycin Units per mg.
Packaging and storage-Preserve in tight containers.
Labeling-Where it is intended for use in preparing injectable dosage forms, the label states that it is sterile or must be subjected to further processing during the preparation of injectable dosage forms.
USP REFERENCE STANDARDS (11)
USP Bleomycin Sulfate RS
1 Identification
Change to read:
A: Spectroscopic Identification Tests (197), Infrared Spectroscopy: 197K (CN 1-May-2020)
B: It responds to the tests for Sulfate (191).
PH (791): between 4.5 and 6.0, in a solution containing 10 Bleomycin Units per mL
LOSS ON DRYING (731)-Dry it in vacuum at a pressure not exceeding 5 mm of mercury at 60 for 3 hours: it loses not more than 6.0% of its weight.
2 Copper
Dilute nitric acid-Dilute 20 ml. of nitric acid to 2000 mL with water.
Copper stock solution-Transfer 1.000 g of copper to a 1000-mL volumetric flask, dissolve in 20 mL of nitric acid, dilute with Dilute nitric acid to volume, and mix. Store in a polyethylene bottle. This solution contains 1000 µg of copper per mL.
Standard preparations-Transfer 5.0 ml. of Copper stock solution to a 100-mL volumetric flask, dilute with Dilute nitric acid to volume, and mix.
Transfer 3.0, 9.0, and 15.0 mL, respectively, of this solution to separate 100-mL volumetric flasks, dilute the contents of each flask with Dilute nitric acid to volume, and mix. These Standard preparations contain, respectively, 1.5, 4.5, and 7.5 µg of copper per mL.
Test preparation-Dissolve about 75 mg of Bleomycin Sulfate, accurately weighed, in 10.0 mL of Dilute nitric acid.
Procedure-Concomitantly determine the absorbances of the Standard preparations and the Test preparation at the copper emission line at 324.8 nm, with a suitable atomic absorption spectrophotometer (see Atomic Absorption Spectroscopy (852)) equipped with a copper hollow-cathode lamp and an air-acetylene flame, using Dilute nitric acid as the blank. Plot the absorbances of the Standard preparations versus concentration, in µg per ml, of copper, and draw the straight line best fitting the three plotted points. From the graph so obtained, determine the concentration, C, in µg per mL, of copper in the Test preparation. Calculate the percentage of copper in the portion of Bleomycin Sulfate taken by the formula:
C/W
in which W is the weight, in mg, of Bleomycin Sulfate taken to prepare the Test preparation: not more than 0.1% is found.
3 Content of bleomycins
Mobile phase-Dissolve 960 mg of sodium 1-pentane sulfonate in 1000 mL of deaerated 0.08 N acetic acid, adjust with ammonium hydroxide to a pH of 4.3, filter, and degas. [NOTE-1.86 g of edetate disodium may be included if needed to obtain satisfactory chromatography.] Use a linear gradient of 10% to 40% methanol mixed with this solution, with a gradient mixing time of 60 minutes, and allow chromatography to proceed with the final gradient mixture for a further 20 minutes or until demethylbleomycin A2 has been eluted.
Test preparation-Dissolve Bleomycin Sulfate in deaerated water to obtain a solution having a concentration of about 2.5 Bleomycin Units per mL. Store this solution in a refrigerator until just prior to use.
Chromatographic system (see Chromatography (621))-The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm x 250-mm stainless steel column containing packing L1. The flow rate is about 1.2 mL per minute.
Procedure-Inject about 10 µl of the Test preparation into the chromatograph by means of a suitable microsyringe or sampling valve, record the chromatogram, and measure the peak responses for all peaks. The elution order is bleomycinic acid, bleomycin A2 (major peak), bleomycin A5' bleomycin B2 (major peak), bleomycin B4' and demethylbleomycin A2. Calculate the percentage contents of bleomycin A2' , bleomycin B2, and bleomycin B4 taken by the formula:
100rf /rt
in which rf is the peak response corresponding to the particular bleomycin and rt is the total of the responses of all peaks: the content of bleomycin A2 is between 55% and 70%; the content of bleomycin B2 is between 25% and 32%; the content of bleomycin B4 is not more than 1%; and the combined percentage of bleomycin A2 and bleomycin B2 is not less than 90%.
Other requirements-Where the label states that Bleomycin Sulfate is sterile, it meets the requirements for Sterility, and Bacterial endotoxins under Bleomycin for Injection. Where the label states that Bleomycin Sulfate must be subjected to further processing during the preparation of injectable dosage forms, it meets the requirements for Bacterial endotoxins under Bleomycin for Injection.
4 Assay
Assay preparation-Dissolve a suitable quantity of Bleomycin Sulfate, accurately weighed, in Buffer B. 16, and quantitatively dilute with Buffer B. 16 to obtain a solution having a convenient concentration.
Procedure-Proceed as directed under Antibiotics-Microbial Assays (81), using an accurately measured volume of Assay preparation diluted quantitatively and stepwise with Buffer B. 16 to yield a Test Dilution having a concentration assumed to be equal to the median dose level of the Standard.
