Spray reagent— Dissolve 2.0g of ceric ammonium sulfate in 100mLof water with heating and stirring,and slowly add 100mLof phosphoric acid.Filter if necessary.

Procedure— Dissolve a sufficient quantity in water to obtain a solution containing 25mg per mL.Further dilute the solution to 10mg per mLwith methanol,and mix.Also prepare a 10-mg-per-mLsolution of USP Vincristine Sulfate RSin a mixture of dichloromethane and methanol (3:1),and mix.Use a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture (see Chromatography á621ñ).Develop it in a methanol prewash tank,and dry it,for maximum sensitivity,not more than 2hours before use.Score it about 15cm above the points of application.Apply 20µLof each solution at points about 2.5cm from the lower edge of the plate,and dry thoroughly (a current of cool air may be used to help dry the spots).Prepare the developing solvent system consisting of a mixture of fresh ether,methanol,and methylamine solution (2in 5)(95:10:5)immediately prior to development.Place the plate in the nonequilibrated developing chamber that contains a paper liner around the back and sides and developing solvent to a depth of about 2cm.Remove the plate when the solvent moves to the scored line (about 80minutes),and discard the solvent system.Dry the plate in a fume hood at room temperature,heat on a metal plate on a steam bath for about 15minutes,and spray the plate while still hot with Spray reagent.Continue heating the plate for 15minutes to stabilize the spots:the RFvalue and the color of the principal spot obtained from the test specimen correspond to those obtained from the Reference Standard.

Procedure for content uniformity—

Buffer solution— Dissolve 6.3g of ammonium formate in about 900mLof water in a 1000-mLvolumetric flask,adjust with formic acid to a pHof 5.0while stirring,dilute with water to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Vincristine Sulfate RSin Buffer solution,and dilute quantitatively and stepwise with Buffer solutionto obtain a solution having a known concentration of about 40µg per mL.

Test preparation— Dissolve the contents of 1container of Vincristine Sulfate for Injection in an accurately measured volume of Buffer solutionto obtain a solution having a concentration between 40and 50µg per mL.

Procedure— Concomitantly determine the absorbances of the Test preparationand the Standard preparationin 1-cm cells at the wavelength of maximum absorbance at about 262nm versus the Buffer solutionas the blank.Calculate the quantity,in mg,of C46H56N4O10·H2SO4in the container taken by the formula: in which Cis the concentration,in µg per mL,of USP Vincristine Sulfate RS(corrected for loss in weight)in the Standard preparation,Vis the volume,in mL,to which the contents of the container are diluted,and AUand ASare the absorbances of the Test preparationand the Standard preparation,respectively.

Mobile phase,Standard preparation,System suitability preparation,andChromatographic system— Prepare as directed in the Assayunder Vincristine Sulfate.

Assay preparation— Pipet a suitable volume of water into a container of Vincristine Sulfate for Injection to obtain a solution having a concentration of about 1mg of vincristine sulfate per mL.Insert the stopper,and shake to mix.

Procedure— Proceed as directed for Procedurein the Assayunder Vincristine Sulfate.Calculate the quantity,in mg,of vincristine sulfate (C46H56N4O10·H2SO4)in the portion of Vincristine Sulfate for Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Vincristine Sulfate RScorrected for loss in weight in the Standard preparation;and rUand rSare the peak responses for vincristine sulfate obtained from the Assay preparationand the Standard preparation,respectively.