Vincristine Sulfate
C46H56N4O10·H2SO4 923.04

Vincaleukoblastine,22-oxo-,sulfate (1:1)(salt).
Leurocristine sulfate (1:1)(salt) [2068-78-2].
»Vincristine Sulfate contains not less than 95.0percent and not more than 105.0percent of C46H56N4O10·H2SO4,corrections being applied for loss in weight.
[Caution—Handle Vincristine Sulfate with great care since it is a potent cytotoxic agent. ]
Packaging and storage— Preserve in tight,light-resistant containers,and store in a freezer.
USP Reference standards á11ñ— USP Vincristine Sulfate RS.USP Vinblastine RS.[NOTE—No Loss on drying determination is needed.]
Identification—
A: Infrared Absorption á197Kñ.
B: Asolution (1in 10)responds to the tests forSulfate á191ñ.
pHá791ñ: between 3.5and 4.5,in a solution (1in 1000).
Loss on drying (see Thermal Analysis á891ñ)— [NOTE—In this procedure,perform weighings rapidly with minimum exposure of the substances to air.]Determine the percentage of volatile substances by thermogravimetric analysis on an appropriately calibrated instrument,using about 10mg of Vincristine Sulfate,accurately weighed.Heat the specimen at the rate of 5per minute between ambient temperature and 200in an atmosphere of nitrogen at a flow rate of 40mLper minute.From the thermogram,determine the accumulated loss in weight between ambient temperature and a point on the plateau before decomposition is indicated (at about 160):it loses not more than 12.0%of its weight.
Related compounds—
Solvent A— Prepare a filtered and degassed mixture of water and diethylamine (985:15),adjusted with phosphoric acid to a pHof 7.5.
Solvent B— Use methanol.
High load test preparation— Prepare as directed for Assay preparationin the Assay.
Low load test preparation— Pipet 1mLof High load test preparationinto a 25-mLvolumetric flask,dilute with water to volume,and mix.
Chromatographic system (see Chromatography á621ñ)—Use the liquid chromatograph equipped as directed in theAssay.The mobile phase is maintained at a flow rate of about 2mLper minute,with an initial gradient of 62%of Solvent Band 38%of Solvent Afor 12minutes,then changed to increase Solvent Bat a rate of 2%per minute,so that after 15minutes it will comprise 92%of the mixture,then changed to decrease Solvent Bat a rate of 15%per minute,so that after 2minutes it will again comprise 62%of the mixture,then maintained at this ratio for 5minutes.
Procedure— Separately inject equal volumes (about 200µL)of the Low load test preparationand of theHigh load test preparationinto the chromatograph,record the chromatograms,and measure the peak responses,ri,of any related substances appearing after the solvent peak in the chromatogram of theHigh load test preparation.Calculate the total percentage of responses due to related substances taken by the formula:
100rt/(rt+25rv),
in which rtis the sum of the riresponses,and rvis 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:
100ri/(rt+25rv).
Not more than 1.0%of response due to any individual related substance is found.
Assay—
Diethylamine solution— Mix 5mLof diethylamine with 295mLof water,and adjust with phosphoric acid to a pHof 7.5.
Mobile phase— Prepare a filtered and degassed mixture of methanol andDiethylamine solution(70:30).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).
Standard preparation— Dissolve an accurately weighed quantity of USP Vincristine Sulfate RSin water to obtain a solution having a known concentration of about 1mg per mL.
Assay preparation— Equilibrate a portion of Vincristine Sulfate for 30minutes with the ambient humidity,transfer about 10mg,accurately weighed,to a 10-mLvolumetric 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 5mg of USP Vincristine Sulfate RSand 5mg of Vinblastine Sulfate RS,each accurately weighed,to a 5-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.
Chromatographic system (see Chromatography á621ñ)—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 1.5mLper 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 10µLof theSystem 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 theMobile phase.]
Procedure— Separately inject equal volumes (about 10µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C46H56N4O10·H2SO4in the portion of Vincristine Sulfate taken,corrected for loss in weight in theAssay preparation,by the formula:
10C(rU/rS),
in which Cis the concentration,in mg per mL,ofUSP Vincristine Sulfate RS,corrected for loss in weight in theStandard preparation;andrUandrSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.
Auxiliary Information— Staff Liaison:Lawrence Evans,III,Ph.D.,Scientist
Expert Committee:(PA6)Pharmaceutical Analysis 6
USP28–NF23Page 2025
Phone Number:1-301-816-8389