Identification—
A:
The retention time of the major peak in the chromatogram of the
Assay preparation corresponds to that of the
Standard preparation as obtained in the
Assay.
C:
Spray reagent—Add 5.6 g of stannous chloride to 10 mL of hydrochloric acid, and stir for 5 minutes. [NOTE—It is not necessary that all of the solids dissolve.] Dissolve 0.2 g of potassium iodide in 90 mL of water. Mix the two solutions together. Disregard any precipitate that is formed. Store in the dark. The solution is usable for at least 1 week.
Procedure—
Prepare a test solution containing 1 mg of Cisplatin per mL and a Standard solution containing 1 mg of
USP Cisplatin RS per mL, both in dimethylformamide. Apply separately 5-µL quantities of each solution to a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture (see
Chromatography 
621
). Place the plate in a suitable chromatographic chamber containing a filter paper lining and equilibrated for 30 minutes with a developer consisting of a mixture of acetone and 1 N nitric acid (180:20). Develop the plate for a distance of about 8 cm from the origin. Remove the plate, and allow it to air-dry. Complete the drying by heating in a forced-air oven at about 100
for 1 minute. Spray the plate with
Spray reagent, heat it in an oven at about 100
for 5 minutes, cool, and spray with a 1 in 50 solution of potassium iodide in water, to bring out the full color of the spots: the principal spot from the test solution corresponds in appearance and
RF value to that produced by the Standard solution.
Limit of trichloroammineplatinate—
Mobile phase—
Transfer 0.8 g of ammonium sulfate to a 2-liter volumetric flask, dissolve in water, and dilute with water to volume. Degas, and filter through a membrane filter prior to use. The pH of this solution is 5.9 ± 0.1. Make adjustments to the ionic strength of the Mobile phase, if necessary, to meet the system suitability requirements.
Standard preparation—
[NOTE—Use low-actinic glassware.
] Dissolve a suitable quantity of
USP Potassium Trichloroammineplatinate RS, accurately weighed, in
saline TS, and dilute quantitatively with
saline TS to obtain a solution having a known concentration of about 6 µg per mL. Use within 4 hours.
Test preparation—
[NOTE—Use low-actinic glassware.
] Transfer about 50 mg of Cisplatin, accurately weighed, to a 100-mL volumetric flask, and dilute with
saline TS to volume. Completely dissolve by stirring by mechanical means for 30 minutes. Use within 4 hours.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 209-nm detector and a 4.6-mm × 25-cm column that contains packing L14. The flow rate is about 2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed under
Procedure: the resolution,
R, between the saline TS peak and the trichloroammineplatinate peak is not less than 2.0, and the relative standard deviation for replicate injections is not more than 3.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Test preparation into the chromatograph, record the chromatograms, and measure the areas for the peaks due to trichloroammineplatinate. The relative retention times are about 1.0 for cisplatin (in the void volume) and 5.0 for trichloroammineplatinate. Calculate the percentage of trichloroammineplatinate taken by the formula:
10(318.48 / 357.58)(rU / rS)(C / W),
in which 318.48 and 357.58 are the formula weights of trichloroammineplatinate and potassium trichloroammineplatinate, respectively;
rU and
rS are the peak areas obtained from the
Test preparation and the
Standard preparation, respectively;
C is the concentration, in µg per mL, of the
Standard preparation; and
W is the weight, in mg, of Cisplatin taken: not more than 1.0% is found.
Limit of transplatin—
Mobile phase—
Prepare a 0.18 M solution in water of monobasic potassium phosphate. Adjust with phosphoric acid to a pH of 3.2, and filter.
Stock standard solution—
Transfer about 10 mg of
USP Transplatin RS, accurately weighed, to a 200-mL volumetric flask, dilute with
saline TS to volume, and dissolve by stirring by mechanical means for 30 minutes.
Working standard solution—
Pipet 5 mL of
Stock standard solution into a 25-mL volumetric flask containing about 12 mg of
USP Cisplatin RS. Dilute with
saline TS to volume, and stir by mechanical means for 30 minutes to dissolve.
Standard preparation—
Pipet 10 mL of
Working standard solution into a 50-mL volumetric flask. Add 5.0 mL of a 1 in 200 solution of thiourea, prepared fresh daily, and 5.0 mL of 1 N hydrochloric acid. Dilute with
saline TS to volume, and mix. Place about 10 mL of this solution in a suitable serum vial, seal with a polytef-lined closure, and heat in a heating block at 60 ± 0.5
for 60 minutes. Remove, and cool to room temperature.
Test solution—
Transfer about 50 mg of Cisplatin, accurately weighed, to a 100-mL volumetric flask, dilute with
saline TS to volume, and dissolve by stirring by mechanical means for 30 minutes.
Test preparation—
Pipet 10 mL of
Test solution into a 50-mL volumetric flask, and proceed as directed for
Standard preparation, beginning with “add 5.0 mL of a 1 in 200 solution of thiourea.”
Resolution solution—
Place about 10 mg of
USP Cisplatin RS in a 200-mL volumetric flask, dilute with
saline TS to volume, and stir by mechanical means for 30 minutes to dissolve. Pipet 10 mL of this solution and 10 mL of
Stock standard solution into a 50-mL volumetric flask, and proceed as directed for
Standard preparation, beginning with “add 5.0 mL of a 1 in 200 solution of thiourea.”
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L9. The column is maintained throughout at a temperature of 45
. The flow rate is about 2.0 mL per minute. Condition the column by pumping
Mobile phase at a flow rate of 2.0 mL per minute for 30 minutes, then at 0.5 mL per minute for 30 minutes, and then again at 2.0 mL per minute for 30 minutes. Chromatograph the
Standard preparation. The retention time of the derivatized transplatin is between 5.0 and 9.0 minutes; or, if it is not, modify the
Mobile phase as necessary, and recondition the column. The column efficiency,
n, is not less than 2500. Chromatograph the
Resolution solution. The resolution,
R, is not less than 1.7. Chromatograph the
Standard preparation as directed for
Procedure: the relative standard deviation for replicate injections is not more than 4.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Test preparation and the
Standard preparation into the chromatograph, record the chromatograms, and measure the areas of the transplatin peaks. The relative retention times are about 1.0 for cisplatin and 1.3 for transplatin. Calculate the percentage of transplatin taken by the formula:
10(C / W)(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Transplatin RS in the
Working standard solution;
W is the weight, in mg, of Cisplatin taken to prepare the
Test solution; and
rU and
rS are the peak areas obtained from the
Test preparation and the
Standard preparation, respectively. Not more than 2.0% is found.
Platinum content—
[NOTE—Thoroughly cleanse all glassware with nitric acid, and rinse with
Purified Water, to prevent “mirroring” of the platinum precipitate.
] Transfer about 0.5 g of Cisplatin, accurately weighed, to a 600-mL beaker. Add 300 mL of 0.1 N hydrochloric acid, and slowly dissolve by heating nearly to boiling on a hot plate covered with an insulating pad, and stirring frequently with a glass stirring rod. When solution is complete, remove the insulating pad, and boil for about 10 minutes. Remove the beaker from the hot plate, allow to cool for 1 minute without stirring, and filter through quantitative, fine-porosity, smooth, dense, ashless filter paper, collecting the filtrate in a 600-mL beaker, completing the transfer to the filter with hot water. Wash the filter with hot water. Place the beaker containing the combined filtrate and washings on a hot plate, and evaporate to a volume of about 300 mL. Place a glass stirring rod in the beaker, and heat the solution to boiling. Slowly add to the center of the beaker, by dropwise additions, 10.0 mL of hydrazine hydrate, 85%.
[Caution—Hydrazine is toxic.
] Add 2 drops of 10 N sodium hydroxide, boil for 10 minutes to coagulate the precipitate for ease of filtration, cool, and filter through quantitative, medium-porosity, smooth, ashless filter paper. Rinse the beaker with hot water, and pour the rinsings onto the filter. Wipe the beaker and the stirring rod with small pieces of the same kind of paper used for this filtration, and place these and the filter containing the precipitate in a No. 1 porcelain crucible, previously ignited to constant weight. Dry on a hot plate covered with an insulating pad, slowly increase the heat to char, and ignite for 1 hour at 800
. Cool in a desiccator, and again weigh: the weight of the platinum so obtained is between 64.42% and 65.22% of the weight of Cisplatin taken, on the anhydrous basis.
Assay—
Mobile phase—
Prepare a suitable solution by mixing ethyl acetate, methanol, dimethylformamide, and degassed water (25:16:5:5), and degas.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Cisplatin RS quantitatively in dimethylformamide to obtain a solution having a known concentration of about 1 mg per mL. Use within 1 hour.
Assay preparation—
Dissolve about 100 mg of Cisplatin, accurately weighed, in dimethylformamide in a 100-mL volumetric flask, dilute with dimethylformamide to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 310-nm detector and a 4.0-mm × 30-cm column that contains packing L8. The flow rate is about 2.0 mL per minute. Chromatograph the
Standard preparation, and record the peak response as directed under
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 40 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 Cl
2H
6N
2Pt in the portion of Cisplatin taken by the formula:
100C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Cisplatin RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
