» Cyclosporine Injection is a sterile solution of Cyclosporine in a suitable vehicle. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of cyclosporine (C62H111N11O12).
Packaging and storage Preserve in single-dose or multiple-dose containers.
Labeling Label it to indicate that it is to be diluted with a suitable parenteral vehicle prior to intravenous infusion.
A: Prepare a solution of it in methanol containing about 0.5 mg of cyclosporine per mL (test solution). Prepare a Standard solution containing 0.5 mg per mL of USP Cyclosporine RS in methanol. Separately apply 10-µL portions of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry in a current of air, place the plate in a suitable chromatographic chamber, and develop the chromatogram, using ethyl ether as the developing solvent, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and allow it to dry. Place the plate in a second chromatographic chamber, and develop the chromatogram in a solvent system consisting of a mixture of ethyl acetate, methyl ethyl ketone, water, and formic acid (60:40:2:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and allow it to dry. Spray the plate with a freshly prepared mixture of 5 mL of Solution A (340 mg of bismuth subnitrate dissolved in 20 mL of 20% acetic acid), 5 mL of Solution B (8 g of potassium iodide dissolved in 20 mL of water), 20 mL of glacial acetic acid, and water to make 100 mL. Immediately again spray the plate with hydrogen peroxide TS. Cyclosporine appears as a brown spot having an RF value of about 0.45 on the chromatograms: the RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution. [noteDisregard any spots at the origin.]
Bacterial endotoxins 85 Prepare the test specimen as follows, using USP Endotoxin RS. Make a 1:10 dilution of the Injection with Water for Injection. Add 0.1 mL of the resulting suspension and 0.1 mL of appropriately constituted LAL reagent to a suitable pyrogen-free test tube, and mix on a vortex mixer for about 5 seconds: the article under test contains not more than 0.84 USP Endotoxin Unit per mg of cyclosporine.
Sterility 71: meets the requirements.
Alcohol content (where present)
Internal standard solution Mix 3 mL of n-propyl alcohol and 50 mL of butyl alcohol.
Standard stock solution Transfer about 1.6 g of dehydrated alcohol, accurately weighed, to a 25-mL volumetric flask, dilute with butyl alcohol to volume, and mix.
Standard preparation Transfer 5.0 mL of Standard stock solution and 6.0 mL of Internal standard solution to a 25-mL volumetric flask, dilute with butyl alcohol to volume, and mix.
Test preparation Transfer an accurately weighed portion of Injection, equivalent to about 320 mg of C2H5OH, to a 25-mL volumetric flask, add 6.0 mL of Internal standard solution, dilute with butyl alcohol to volume, and mix.
Chromatographic system The gas chromatograph is equipped with a flame-ionization detector and contains a 2-mm × 2-m glass column packed with support S3. The injection port is maintained at a temperature of about 280, the detector is maintained at about 290, and the column is maintained at 145 for 8 minutes and is programmed thereafter to rise to 270 at a rate of 32 per minute. Nitrogen is used as the carrier gas, flowing at a rate of about 35 mL per minute. [noteMake adjustments if necessary to obtain satisfactory chromatography.]
System suitability Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not greater than 2.0%.
Procedure [noteUse peak areas where peak responses are indicated.] Inject separate suitable portions (about 1 µL) of the Standard preparation and the Test preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. The elution order is alcohol, n-propyl alcohol, and butyl alcohol. Calculate the quantity, in mg, of C2H5OH in the portion of Injection taken by the formula:
25C(RU / RS)in which C is the concentration, in mg per mL, of C2H5OH in the Standard preparation; and RU and RS are the peak response ratios of the alcohol peak to the n-propyl alcohol internal standard peak obtained from the Test preparation and the Standard preparation, respectively: it contains between 80.0% and 120.0% of the labeled amount of C2H5OH.
Mobile phase Prepare a filtered and degassed mixture of acetonitrile, water, methanol, and phosphoric acid (550:400:50:0.5), making adjustments if necessary (see System Suitability under Chromatography 621).
Standard preparation Dissolve an accurately weighed quantity of USP Cyclosporine RS in methanol to obtain a solution having a known concentration of about 0.5 mg per mL. Use this solution promptly after preparation.
Assay preparation 1 (where it is represented as being in a single-dose container)Using a suitable hypodermic needle and syringe, withdraw all of the withdrawable contents from 1 container of Injection, and dilute quantitatively with methanol to obtain a solution containing about 0.5 mg of cyclosporine per mL. Use this solution promptly after preparation.
Assay preparation 2 (where the label states the quantity of cyclosporine in a given volume)Dilute an accurately measured volume of Injection quantitatively with methanol to obtain a solution containing about 0.5 mg of cyclosporine per mL. Use this solution promptly after preparation.
Chromatographic system (see Chromatography 621)The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 25-cm analytical column that contains packing L16. The column is maintained at 70. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the capacity factor, k¢, is not less than 3 and not more than 10; the column efficiency is not less than 700 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 1.5%.
Procedure Separately inject equal volumes (about 20 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 cyclosporine (C26H111N11O12) withdrawn from the container or in each mL of the Injection taken by the same formula:
(L / D)(CP / 1000)(rU / rS)in which L is the labeled quantity, in mg, of cyclosporine in the container or in each mL of Injection; D is the concentration, in mg of cyclosporine per mL, of Assay preparation 1 or Assay preparation 2 based on the labeled quantity in the container or in the volume of Injection taken and the extent of dilution, respectively; C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard preparation; P is the purity, in µg per mg, of USP Cyclosporine RS; and rU and rS are the peak responses obtained from Assay preparation 1 or Assay preparation 2 and the Standard preparation, respectively.
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USP32NF27 Page 2046