A: Prepare a solution of it in methanol containing about 0.5mg of cyclosporine per mL(test solution).Prepare a Standard solution containing 0.5mg per mLof USP Cyclosporine RSin methanol.Separately apply 10-µLportions of the test solution and the Standard solution to a suitable thin-layer chromatographic plate (see Chromatography á621ñ)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 5mLof Solution A(340mg of bismuth subnitrate dissolved in 20mLof 20%acetic acid),5mLof Solution B(8g of potassium iodide dissolved in 20mLof water),20mLof glacial acetic acid,and water to make 100mL.Immediately again spray the plate with hydrogen peroxide TS.Cyclosporine appears as a brown spot having an RFvalue of about 0.45on the chromatograms:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.[NOTE—Disregard any spots at the origin.]

B: The chromatogram of the Assay preparationobtained as directed in the Assayexhibits a major peak for cyclosporine,the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparationobtained as directed in the Assay.

Internal standard solution— Mix 3mLof n-propyl alcohol and 50mLof butyl alcohol.

Standard stock solution— Transfer about 1.6g of dehydrated alcohol,accurately weighed,to a 25-mLvolumetric flask,dilute with butyl alcohol to volume,and mix.

Standard preparation— Transfer 5.0mLof Standard stock solutionand 6.0mLof Internal standard solutionto a 25-mLvolumetric flask,dilute with butyl alcohol to volume,and mix.

Test preparation— Transfer an accurately weighed portion of Injection,equivalent to about 320mg of C2H5OH,to a 25-mLvolumetric flask,add 6.0mLof 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 145for 8minutes and is programmed thereafter to rise to 270at a rate of 32per minute.Nitrogen is used as the carrier gas,flowing at a rate of about 35mLper minute.[NOTE—Make 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— [NOTE—Use peak areas where peak responses are indicated.]Inject separate suitable portions (about 1µL)of the Standard preparationand the Test preparationinto 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 C2H5OHin the portion of Injection taken by the formula: in which Cis the concentration,in mg per mL,of C2H5OHin the Standard preparation;and RUand RSare the peak response ratios of the alcohol peak to the n-propyl alcohol internal standard peak obtained from the Test preparationand 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 Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Cyclosporine RSin methanol to obtain a solution having a known concentration of about 0.5mg 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 1container of Injection,and dilute quantitatively with methanol to obtain a solution containing about 0.5mg 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 0.5mg of cyclosporine per mL.Use this solution promptly after preparation.

Chromatographic system (see Chromatography á621ñ)—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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the capacity factor,k¢,is not less than 3and not more than 10;the column efficiency is not less than 700theoretical 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 20µL)of the Standard preparationand the Assay preparationinto 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 mLof the Injection taken by the same formula: in which Lis the labeled quantity,in mg,of cyclosporine in the container or in each mLof Injection;Dis the concentration,in mg of cyclosporine per mL,of Assay preparation 1or Assay preparation 2based on the labeled quantity in the container or in the volume of Injection taken and the extent of dilution,respectively;Cis the concentration,in mg per mL,of USP Cyclosporine RSin the Standard preparation;Pis the purity,in µg per mg,of USP Cyclosporine RS;and rUand rSare the peak responses obtained from Assay preparation 1or Assay preparation 2and the Standard preparation,respectively.