Dacarbazine for Injection
» Dacarbazine for Injection is a sterile, freeze-dried mixture of Dacarbazine and suitable buffers or diluents. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of C6H10N6O.
[CautionGreat 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 multiple-dose Containers for Sterile Solids as described under Injections 1, preferably of Type I glass, protected from light.
USP Reference standards 11
USP Endotoxin RS
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 Solutions under Injections 1.
A: Dissolve a suitable quantity of Dacarbazine for Injection in water to obtain a solution having a concentration of 10 mg of dacarbazine per mL. Apply separately 1 µL of the freshly prepared solution and 1 µL of an aqueous solution, containing 10 mg each of USP Dacarbazine RS and citric acid per mL, to a suitable thin-layer chromatographic plate (see Chromatography 621) 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 1 N ammonium 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 5 mL of a 10% aqueous solution of ferric chloride with 5 mL of a 10% aqueous solution of potassium ferricyanide and diluting with water to 50 mL). Dacarbazine appears as an intense blue spot on a light yellow background: the RF value of the spot obtained from the test solution corresponds to that obtained from the Standard solution.
B: To 1 mL of a solution (1 in 100) in a test tube add a few crystals of periodic acid and 4 drops of methanol. Shake, and after 1 minute add 5 mL of a 0.2% acetylacetone reagent solution (prepared by mixing 15.0 g of ammonium acetate, 0.30 mL of glacial acetic acid, and 0.20 mL of acetylacetone in a 100-mL volumetric 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 2 drops of an aqueous solution (1 in 100) in a 15-mL test tube add 10 mL of a solution prepared by mixing 10 mL of acetic anhydride with 30 mL of pyridine: an intense yellow color is produced immediately and after a few minutes becomes red-violet (presence of citric acid).
Bacterial endotoxins 85 It contains not more than 0.52 USP Endotoxin Unit per mg of dacarbazine.
pH 791: between 3.0 and 4.0, in a solution containing an amount of Dacarbazine for Injection equivalent to about 1 g of dacarbazine in 100 mL of water.
Water, Method I 921: not more than 1.5%.
Limit of 2-azahypoxanthine [noteThe Mobile phase employed in this procedure is corrosive. The system should be rinsed well with methanol following completion of analysis. ]
Mobile phase Transfer 2.2 g of docusate sodium to a 1000-mL volumetric flask, dissolve in a mixture of 100 mL of water and 15 mL of 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 RS to contain 0.04 mg per mL.
Test solution Constitute the contents of 1 vial of Dacarbazine for Injection. Using the contents of the constituted vial, dilute quantitatively with water to obtain a solution containing 4 mg of dacarbazine per mL.
Chromatographic system (see Chromatography 621) 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 1.2 mL per 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 20 µL) of the Standard solution and the Test solution into 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 solution and 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 C is the concentration, in mg per mL, of USP Dacarbazine Related Compound B RS in the Standard solution; V is the final volume, in mL, of the Test solution; and rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively: not more than 1.0% is found.
Other requirements It meets the requirements for Sterility Tests 71, Uniformity of Dosage Units 905 and Labeling under Injections 1.
Assay Dissolve the contents of not fewer than 10 containers of Dacarbazine for Injection in 0.1 N hydrochloric acid. Transfer and combine the solutions quantitatively rinsing as necessary with 0.1 N hydrochloric acid. Dilute quantitatively, and stepwise if necessary, with 0.1 N hydrochloric acid to obtain a solution containing about 0.4 mg per mL. Transfer 2.0 mL of this solution to a 250-mL volumetric flask, dilute with 0.1 N hydrochloric acid to volume, and mix. Dissolve an accurately weighed quantity of USP Dacarbazine RS in 0.1 N hydrochloric acid, and dilute quantitatively and stepwise with the same solvent to obtain a Standard solution having a known concentration of about 3.2 µg per mL. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 323 nm, with a suitable spectrophotometer, using 0.1 N hydrochloric acid as the blank. Calculate the average quantity, in mg, of C6H10N6O in each container of Dacarbazine for Injection taken by the formula:
125(VC / N)(AU / AS)in which V is the volume, in mL, of the solution produced by dilution of the combined container contents to a concentration of 0.4 mg per mL taking account of dilution factors in the case of stepwise dilution; C is the concentration, in mg per mL, of USP Dacarbazine RS in the Standard solution; N is the number of vials taken; and AU and AS are the absorbances of the solution of Dacarbazine for Injection and the Standard solution, respectively.
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USP35NF30 Page 2802Pharmacopeial Forum: Volume No. 28(6) Page 1784