A: Infrared Absorption 197K.

B: A solution (1 in 10) responds to the tests for Sulfate 191.

Solvent A— Prepare a filtered and degassed mixture of water and diethylamine (985:15), adjusted with phosphoric acid to a pH of 7.5.

Solvent B— Use methanol.

High load test preparation— Prepare as directed for Assay preparation in the Assay.

Low load test preparation— Pipet 1 mL of High load test preparation into a 25-mL volumetric flask, dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)—Use the liquid chromatograph equipped as directed in the Assay. The mobile phase is maintained at a flow rate of about 2 mL per minute, with an initial gradient of 62% of Solvent B and 38% of Solvent A for 12 minutes, then changed to increase Solvent B at a rate of 2% per minute, so that after 15 minutes it will comprise 92% of the mixture, then changed to decrease Solvent B at a rate of 15% per minute, so that after 2 minutes it will again comprise 62% of the mixture, then maintained at this ratio for 5 minutes.

Procedure— Separately inject equal volumes (about 200 µL) of the Low load test preparation and of the High load test preparation into the chromatograph, record the chromatograms, and measure the peak responses, ri, of any related substances appearing after the solvent peak in the chromatogram of the High load test preparation. Calculate the total percentage of responses due to related substances taken by the formula: in which rt is the sum of the ri responses, and rv is the vincristine peak response in the chromatogram of the Low load test preparation. Not more than 4.0% is found. Calculate the percentage response of each related substance taken by the formula: Not more than 1.0% of response due to any individual related substance is found.

Diethylamine solution— Mix 5 mL of diethylamine with 295 mL of water, and adjust with phosphoric acid to a pH of 7.5.

Mobile phase— Prepare a filtered and degassed mixture of methanol and Diethylamine solution (70:30). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Vincristine Sulfate RS in water to obtain a solution having a known concentration of about 1 mg per mL.

Assay preparation— Equilibrate a portion of Vincristine Sulfate for 30 minutes with the ambient humidity, transfer about 10 mg, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Using another portion of the equilibrated specimen, determine the loss in weight as directed for USP Vincristine Sulfate RS.

System suitability preparation— Transfer 5 mg of USP Vincristine Sulfate RS and 5 mg of Vinblastine Sulfate RS, each accurately weighed, to a 5-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 297-nm detector, a precolumn packed with porous silica gel, a 2- to 5-cm guard column containing packing L1, and a 4.6-mm × 25-cm analytical column that contains packing L7. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%. Similarly chromatograph 10 µL of the System suitability preparation, and record the peak responses: the resolution, between vincristine sulfate and vinblastine sulfate is not less than 4.0. [note—For a particular column, the resolution may be increased by increasing the proportion of water in the Mobile phase.]

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C46H56N4O10·H2SO4 in the portion of Vincristine Sulfate taken, corrected for loss in weight in the Assay preparation, 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 obtained from the Assay preparation and the Standard preparation, respectively.