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

Procedure— Dissolve a sufficient quantity in water to obtain a solution containing 25 mg per mL. Further dilute the solution to 10 mg per mL with methanol, and mix. Also prepare a 10-mg-per-mL solution of USP Vincristine Sulfate RS in 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 621). Develop it in a methanol prewash tank, and dry it, for maximum sensitivity, not more than 2 hours before use. Score it about 15 cm above the points of application. Apply 20 µL of each solution at points about 2.5 cm 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 (2 in 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 2 cm. Remove the plate when the solvent moves to the scored line (about 80 minutes), 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 15 minutes, and spray the plate while still hot with Spray reagent. Continue heating the plate for 15 minutes to stabilize the spots: the RF value 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 6.3 g of ammonium formate in about 900 mL of water in a 1000-mL volumetric flask, adjust with formic acid to a pH of 5.0 while stirring, dilute with water to volume, and mix.

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

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

Procedure— Concomitantly determine the absorbances of the Test preparation and the Standard preparation in 1-cm cells at the wavelength of maximum absorbance at about 262 nm versus the Buffer solution as the blank. Calculate the quantity, in mg, of C46H56N4O10·H2SO4 in the container taken by the formula: in which C is the concentration, in µg per mL, of USP Vincristine Sulfate RS (corrected for loss in weight) in the Standard preparation, V is the volume, in mL, to which the contents of the container are diluted, and AU and AS are the absorbances of the Test preparation and the Standard preparation, respectively.

(Official January 1, 2007)

Mobile phase, Standard preparation, System suitability preparation, and Chromatographic system— Prepare as directed in the Assay under 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 1 mg of vincristine sulfate per mL. Insert the stopper, and shake to mix.

Procedure— Proceed as directed for Procedure in the Assay under 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 C is the concentration, in mg per mL, of USP Vincristine Sulfate RS corrected for loss in weight in the Standard preparation; and rU and rS are the peak responses for vincristine sulfate obtained from the Assay preparation and the Standard preparation, respectively.