Vincristine Sulfate for Injection
DEFINITION
Vincristine Sulfate for Injection is a sterile mixture of Vincristine Sulfate with suitable diluents. It contains NLT 90.0% and NMT 110.0% of the labeled amount of vincristine sulfate (C46H56N4O10·H2SO4).
[Caution—Handle Vincristine Sulfate for Injection with great care because it is a potent cytotoxic agent. ]
IDENTIFICATION
Change to read:
•  A. Thin-Layer Chromatographic Identification Test 201
Standard solution:  10 mg/mL of USP Vincristine Sulfate RS or USP Vincristine Sulfate (Assay) RS(RB 1-Jul-2011) in dichloromethane and methanol (3:1)
Sample stock solution:  25 mg/mL of vincristine sulfate from Vincristine Sulfate for Injection in water
Sample solution:  10 mg/mL of vincristine sulfate from Sample stock solution in methanol
Chromatographic system 
Mode:  TLC
Adsorbent:  0.25-mm layer of chromatographic silica gel mixture
Application volume:  20 µL
Developing solvent system:  Fresh ether, methanol, and methylamine solution (2 in 5) (19:2:1)
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.
Analysis:  Develop the chromatographic plate in a methanol prewash tank; for maximum sensitivity, dry it NMT 2 h before use. Score it about 15 cm above the points of application. Apply the Sample solution and the Standard 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). Place the plate in the nonequilibrated developing chamber that contains a paper liner around the back and sides and Developing solvent system to a depth of about 2 cm. Remove the plate when the solvent moves to the scored line (about 80 min), 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 min, and spray the plate while still hot with Spray reagent. Continue heating the plate for 15 min to stabilize the spots.
Acceptance criteria:  The RF value and the color of the principal spot from the Sample solution correspond to those from the Standard solution.
•  B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
ASSAY
Change to read:
•  Procedure
Solution A:  Diethylamine and water (1:59). Adjust with phosphoric acid to a pH of 7.5.
Mobile phase:  Methanol and Solution A (70:30)
Standard solution:  1.2 mg/mL(RB 1-Jul-2011) of USP Vincristine Sulfate RS or USP Vincristine Sulfate (Assay) RS(RB 1-Jul-2011) in water
System suitability solution:  1 mg/mL of (RB 1-Jul-2011) USP Vinblastine Sulfate RS in the Standard solution(RB 1-Jul-2011)
Sample solution:  1.2 mg/mL(RB 1-Jul-2011) of vincristine sulfate from Vincristine Sulfate for Injection in water. [Note—Shake to mix. ]
Chromatographic system 
Mode:  LC
Detector:  UV 297 nm
Columns 
Precolumn:  Porous silica gel packing
Guard:  2- to 5-cm; packing L1
Analytical:  4.6-mm × 25-cm; packing L7
Flow rate:  1.5 mL/min
Injection size:  10 µL
System suitability 
Samples:  Standard solution and System suitability solution
Suitability requirements 
Resolution:  NLT 4.0 between vincristine sulfate and vinblastine sulfate, System suitability solution. [Note—For a particular column, the resolution may be increased by increasing the proportion of water in the Mobile phase. ]
Relative standard deviation:  NMT 2.0%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of vincristine sulfate (C46H56N4O10·H2SO4) in the portion of Vincristine Sulfate for Injection taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of vincristine sulfate in the Standard solution (mg/mL)
CU== nominal concentration of vincristine sulfate in the Sample solution (mg/mL)
Acceptance criteria:  90.0%–110.0%
PERFORMANCE TESTS
Change to read:
•  Uniformity of Dosage Units 905
Procedure for content uniformity 
Buffer solution:  Dissolve 6.3 g of ammonium formate in about 900 mL of water. Adjust with formic acid to a pH of 5.0 while stirring, and dilute with water to 1000 mL.
Standard solution:  40–50(RB 1-Jul-2011) µg/mL of USP Vincristine Sulfate RS or USP Vincristine Sulfate (Assay) RS(RB 1-Jul-2011) in Buffer solution
Sample solution:  Dissolve the contents of one container of Vincristine Sulfate for Injection in a suitable volume of Buffer solution to obtain a solution containing 40–50 µg/mL of vincristine sulfate.
Instrumental conditions 
Mode:  UV
Cell:  1 cm
Analytical wavelength:  262 nm
Blank:  Buffer solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the quantity, in mg, of vincristine sulfate (C46H56N4O10·H2SO4) in the container of Vincristine Sulfate for Injection taken:
Result = (AU/AS) × CS × V × F
AU== absorbance of the Sample solution
AS== absorbance of the Standard solution
CS== concentration of USP Vincristine Sulfate RS in the Standard solution (µg/mL)
V== final volume of the Sample solution
F== unit conversion factor, 0.001 mg/µg
Acceptance criteria:  Meets the requirements for solids
IMPURITIES
•  Organic Impurities
Solution A:  Diethylamine and water (3:197). Adjust with phosphoric acid to a pH of 7.5.
Solution B:  Methanol
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 38 62
12 38 62
27 8 92
29 38 62
34 38 62
Standard solution, System suitability solution, and System suitability:   Proceed as directed in the Assay.
Sample solution A:  1 mg/mL of vincristine sulfate from Vincristine Sulfate for Injection in water
Sample solution B:  0.04 mg/mL of vincristine sulfate in water from Sample solution A
Chromatographic system:  Proceed as directed in the Assay, except use a Flow rate of 2 mL/min and an Injection size of 200 µL.
Analysis 
Samples:  Sample solution A and Sample solution B
Calculate the percentage of each impurity in the portion of Vincristine Sulfate for Injection taken:
Result = [rUA/(SrUA + 25rUB)] × 100
rUA== peak response of each impurity appearing after the solvent peak from Sample solution A
rUB== peak response of vincristine from Sample solution B
Calculate the percentage of total impurities in the portion of Vincristine Sulfate for Injection taken:
Result = [SrUA/(SrUA + 25rUB)] × 100
rUA== peak response of each impurity appearing after the solvent peak from Sample solution A
rUB== peak response of vincristine from Sample solution B
Acceptance criteria 
Individual impurities:  NMT 2.0%
Total impurities:  NMT 5.0%
SPECIFIC TESTS
•  Sterility Tests 71: Meets the requirements
•  Bacterial Endotoxins Test 85: It contains NMT 100.0 USP Endotoxin Units/mg of vincristine sulfate.
•  Constituted Solution: At the time of use, it meets the requirements for Injections 1, Constituted Solutions.
•  Other Requirements: It meets the requirements in Injections 1, Labeling.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve as described in Injections 1, Containers for Sterile Solids, and store in a refrigerator.
•  Labeling: The label states, “For Intravenous Use Only—Fatal If Given By Other Routes”.
Where labeled as containing more than 2 mg, it must also be labeled as a Pharmacy Bulk Package (see Injections 1). The labeling directs that the drug be dispensed only in containers enclosed in an overwrap labeled as directed below. When packaged in a Pharmacy Bulk Package, it is exempt from the requirement in Injections 1 that the closure be penetrated only one time after constitution with a suitable sterile transfer device or dispensing set, when it contains a suitable substance or mixture of substances to prevent the growth of microorganisms.
When dispensed, the container or syringe (holding the individual dose prepared for administration to the patient) must be enclosed in an overwrap bearing the statement, “Do Not Remove Covering Until Moment of Injection. For Intravenous Use Only—Fatal If Given By Other Routes.”
Change to read:
•  USP Reference Standards 11
USP Endotoxin RS
USP Vinblastine Sulfate RS Click to View Structure
[Note—No loss on drying determination is needed for USP Vinblastine Sulfate RS. ]
USP Vincristine Sulfate RS Click to View Structure
USP Vincristine Sulfate (Assay) RS
(RB 1-Jul-2011)
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USP35–NF30 Page 5025
Pharmacopeial Forum: Volume No. 37(1)