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
Spectrophotometry and Light-Scattering 851) equipped with a copper hollow-cathode lamp and an airacetylene 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.
Content of bleomycins
Mobile phase
Dissolve 960 mg of sodium 1-pentanesulfonate in 1000 mL of deaerated 0.08 N acetic acid, adjust with ammonium hydroxide to a pH of 4.3, filter, and degas. [NOTE1.86 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 × 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 A
2 (major peak), bleomycin A
5, bleomycin B
2 (major peak), bleomycin B
4, and demethylbleomycin A
2. Calculate the percentage contents of bleomycin A
2, bleomycin B
2, and bleomycin B
4 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 A
2 is between 55% and 70%; the content of bleomycin B
2 is between 25% and 32%; the content of bleomycin B
4 is not more than 1%; and the combined percentage of bleomycin A
2 and bleomycin B
2 is not less than 90%.
Assay
Assay preparation
Dissolve a suitable quantity of Bleomycin Sulfate, accurately weighed, in Buffer No. 16, and quantitatively dilute with Buffer No. 16 to obtain a solution having a convenient concentration.
Procedure
Proceed as directed under
AntibioticsMicrobial Assays 81, using an accurately measured volume of
Assay preparation diluted quantitatively and stepwise with
Buffer No. 16 to yield a
Test Dilution having a concentration assumed to be equal to the median dose level of the Standard.