Dacarbazine for Injection
»Dacarbazine for Injection is a sterile,freeze-dried mixture of Dacarbazine and suitable buffers or diluents.It contains not less than 90.0percent and not more than 110.0percent of the labeled amount of C6H10N6O.
Caution—Great care should be taken to prevent inhaling particles of Dacarbazine for Injection and exposing the skin to it.
Packaging and storage— Preserve in single-dose or in multiple-dose Containers for Sterile Solidsas described under Injections á1ñ,preferably of Type Iglass,protected from light.
Completeness of solution— When dissolved as directed in the labeling,it yields a clear,pale yellow to yellow solution.
Constituted solution— At the time of use,it meets the requirements for Constituted Solutionsunder Injections á1ñ.
Identification—
A: Dissolve a suitable quantity of Dacarbazine for Injection in water to obtain a solution having a concentration of 10mg of dacarbazine per mL.Apply separately 1µLof the freshly prepared solution and 1µLof an aqueous solution,containing 10mg each of USP Dacarbazine RSand citric acid per mL,to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Develop the chromatogram in a solvent system consisting of a mixture of isopropyl alcohol and 1Nammonium hydroxide (3:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,and allow the solvent to evaporate.Spray the plate evenly with a freshly prepared solution containing 1%of ferric chloride and 1%of potassium ferricyanide (prepared by mixing 5mLof a 10%aqueous solution of ferric chloride with 5mLof a 10%aqueous solution of potassium ferricyanide and diluting with water to 50mL).Dacarbazine appears as an intense blue spot on a light yellow background:the RFvalue of the spot obtained from the test solution corresponds to that obtained from the Standard solution.
B: To 1mLof a solution (1in 100)in a test tube add a few crystals of periodic acid and 4drops of methanol.Shake,and after 1minute add 5mLof a 0.2%acetylacetone reagent solution (prepared by mixing 15.0g of ammonium acetate,0.30mLof glacial acetic acid,and 0.20mLof acetylacetone in a 100-mLvolumetric flask,adding water to volume,and mixing).Shake,and place in a water bath maintained at a temperature of 60:an intense yellow color develops in a few minutes (presence of mannitol).
C: To 2drops of an aqueous solution (1in 100)in a 15-mLtest tube add 10mLof a solution prepared by mixing 10mLof acetic anhydride with 30mLof pyridine:an intense yellow color is produced immediately and after a few minutes becomes red-violet (presence of citric acid).
Bacterial endotoxins á85ñ— It contains not more than 0.52USP Endotoxin Unit per mg of dacarbazine.
pHá791ñ: between 3.0and 4.0,in a solution containing an amount of Dacarbazine for Injection equivalent to about 1g of dacarbazine in 100mLof water.
Water,Method Iá921ñ: not more than 1.5%.
Limit of 2-azahypoxanthine— [NOTE—The Mobile phase employed in this procedure is corrosive.The system should be rinsed well with methanol following completion of analysis.]
Mobile phase— Transfer 2.2g of docusate sodium to a 1000-mLvolumetric flask,dissolve in a mixture of 100mLof water and 15mLof glacial acetic acid,and dilute with water to volume.Filter the solution through a 0.5-µm porosity filter.Prepare this solution fresh daily.
Standard solution— Prepare a solution of USP Dacarbazine Related Compound B RSto contain 0.04mg per mL.
Test solution— Constitute the contents of 1vial of Dacarbazine for Injection.Using the contents of the constituted vial,dilute quantitatively with water to obtain a solution containing 4mg of dacarbazine per mL.
Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.2mLper minute.Chromatograph five replicate injections of the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%.
Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph by means of a suitable sampling valve or high-pressure microsyringe.Measure the peak responses at corresponding retention times obtained from the Standard solutionand the Test solution,and calculate the quantity,in mg,of 2-azahypoxanthine monohydrate in the dacarbazine taken by the formula:
(CV)(rU/rS),
in which Cis the concentration,in mg per mL,of USP Dacarbazine Related Compound B RSin the Standard solution;Vis the final volume,in mL,of the Test solution;and rUand rSare the peak responses obtained from the Test solutionand the Standard solution,respectively:not more than 1.0%is found.
Other requirements— It meets the requirements for Sterility Tests á71ñ,Uniformity of Dosage Units á905ñand Labelingunder Injections á1ñ.
Assay— Dissolve the contents of not fewer than 10containers of Dacarbazine for Injection in 0.1Nhydrochloric acid.Transfer and combine the solutions quantitatively rinsing as necessary with 0.1Nhydrochloric acid.Dilute quantitatively,and stepwise if necessary,with 0.1Nhydrochloric acid to obtain a solution containing about 0.4mg per mL.Transfer 2.0mLof this solution to a 250-mLvolumetric flask,dilute with 0.1Nhydrochloric acid to volume,and mix.Dissolve an accurately weighed quantity of USP Dacarbazine RSin 0.1Nhydrochloric acid,and dilute quantitatively and stepwise with the same solvent to obtain a Standard solution having a known concentration of about 3.2µg per mL.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 323nm,with a suitable spectrophotometer,using 0.1Nhydrochloric acid as the blank.Calculate the average quantity,in mg,of C6H10N6Oin each container of Dacarbazine for Injection taken by the formula:
125(VC/N)(AU/AS),
in which Vis the volume,in mL,of the solution produced by dilution of the combined container contents to a concentration of 0.4mg per mLtaking account of dilution factors in the case of stepwise dilution;Cis the concentration,in mg per mL,of USP Dacarbazine RSin the Standard solution;Nis the number of vials taken;and AUand ASare the absorbances of the solution of Dacarbazine for Injection and the Standard solution,respectively.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(PA6)Pharmaceutical Analysis 6
USP28–NF23Page 569
Pharmacopeial Forum:Volume No.28(6)Page 1784
Phone Number:1-301-816-8389