Loss on drying (see Thermal Analysis 
891
)—
[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 10 mg of Vincristine Sulfate, accurately weighed. Heat the specimen at the rate of 5
per minute between ambient temperature and 200
in an atmosphere of nitrogen at a flow rate of 40 mL per 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 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:
100rt / (rt + 25rv),
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:
100ri / (rt + 25rv).
Not more than 1.0% of response due to any individual related substance is found.
Assay—
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.
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 C
46H
56N
4O
10·H
2SO
4 in the portion of Vincristine Sulfate taken, corrected for loss in weight in the
Assay preparation, by the formula:
10C(rU / rS),
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.
