A: Infrared Absorption á197Kñ.

B: It responds to the tests for Sulfate á191ñ.

Dilute nitric acid— Dilute 20mLof nitric acid to 2000mLwith water.

Copper stock solution— Transfer 1.000g of copper to a 1000-mLvolumetric flask,dissolve in 20mLof nitric acid,dilute with Dilute nitric acidto volume,and mix.Store in a polyethylene bottle.This solution contains 1000µg of copper per mL.

Standard preparations— Transfer 5.0mLof Copper stock solutionto a 100-mLvolumetric flask,dilute with Dilute nitric acidto volume,and mix.Transfer 3.0,9.0,and 15.0mL,respectively,of this solution to separate 100-mLvolumetric flasks,dilute the contents of each flask with Dilute nitric acidto volume,and mix.These Standard preparationscontain,respectively,1.5,4.5,and 7.5µg of copper per mL.

Test preparation— Dissolve about 75mg of Bleomycin Sulfate,accurately weighed,in 10.0mLof Dilute nitric acid.

Procedure— Concomitantly determine the absorbances of the Standard preparationsand the Test preparationat the copper emission line at 324.8nm,with a suitable atomic absorption spectrophotometer (see Spectrophotometry and Light-Scattering á851ñ)equipped with a copper hollow-cathode lamp and an air–acetylene flame,using Dilute nitric acidas the blank.Plot the absorbances of the Standard preparationsversus 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: in which Wis the weight,in mg,of Bleomycin Sulfate taken to prepare the Test preparation:not more than 0.1%is found.

Mobile phase— Dissolve 960mg of sodium 1-pentanesulfonate in 1000mLof deaerated 0.08Nacetic acid,adjust with ammonium hydroxide to a pHof 4.3,filter,and degas.[NOTE—1.86g 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 60minutes,and allow chromatography to proceed with the final gradient mixture for a further 20minutes or until demethylbleomycin A2has been eluted.

Test preparation— Dissolve Bleomycin Sulfate in deaerated water to obtain a solution having a concentration of about 2.5Bleomycin Units per mL.Store this solution in a refrigerator until just prior to use.

Chromatographic system (see Chromatography á621ñ)—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 1.2mLper minute.

Procedure— Inject about 10µLof the Test preparationinto 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 B4taken by the formula: in which rfis the peak response corresponding to the particular bleomycin and rtis the total of the responses of all peaks:the content of bleomycin A2is between 55%and 70%;the content of bleomycin B2is between 25%and 32%;the content of bleomycin B4is not more than 1%;and the combined percentage of bleomycin A2and bleomycin B2is not less than 90%.

Assay preparation— Dissolve a suitable quantity of Bleomycin Sulfate,accurately weighed,in Buffer No.16,and quantitatively dilute with Buffer No.16to obtain a solution having a convenient concentration.

Procedure— Proceed as directed under Antibiotics—Microbial Assays á81ñ,using an accurately measured volume of Assay preparationdiluted quantitatively and stepwise with Buffer No.16to yield a Test Dilutionhaving a concentration assumed to be equal to the median dose level of the Standard.