A: Infrared Absorption á197Mñ.

B: The retention times of the two major peaks in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Solution A,Solution B,and Mobile phase— Proceed as directed in the Assay.

Test solution— Prepare as directed for Assay preparationin the Assay.

Chromatographic system— Proceed as directed in the Assay.To evaluate the system suitability requirements,use the Standard preparationprepared as directed in the Assay.

Procedure— Inject a volume (about 20µL)of the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the percentage of each related compound in the portion of Verteporfin taken by the formula: in which riis the individual peak response of each related compound;and rsis the sum of the responses of all the peaks.Not more than 0.6%of the peak having a retention time of about 0.56relative to that of the first verteporfin isomer peak is found;not more than 0.8%of any other individual related compound is found;and the sum of all impurities is not more than 4.0%.

Standard solution: 50µg of methylene chloride per mLin dimethylformamide.

Test solution: 10mg of Verteporfin per mLin dimethylformamide.

Limit: not more than 5000µg per g of Verteporfin (0.5%).

Solution A— Prepare a filtered and degassed mixture of 1%(w/v)aqueous ammonium sulfate,acetonitrile,glacial acetic acid,and 3.6Msulfuric acid (10:10:1:0.027).

Solution B— Prepare a filtered and degassed mixture of 1%(w/v)aqueous ammonium sulfate,tetrahydrofuran,glacial acetic acid,and 3.6Msulfuric acid (10:10:1:0.034).

Mobile phase— Use variable mixtures of Solution Aand Solution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— To a suitable volumetric flask,transfer an accurately weighed quantity of USP Verteporfin RSsufficient to make a 0.25mg per mLsolution.Add a volume of a mixture of acetonitrile and tetrahydrofuran (1:1)equivalent to 60%of the flask volume,and dissolve.Dilute with water to volume,and mix.[NOTE—Protect the solution from light.]

Assay preparation— Transfer about 25mg of Verteporfin,accurately weighed,to a 100-mLvolumetric flask.Add 60mLof a mixture of acetonitrile and tetrahydrofuran (1:1),and dissolve.Dilute with water to volume,and mix.[NOTE—Protect the solution from light.]

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 410-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is 1.5mLper minute.The column temperature is maintained at 30.The chromatograph is programmed as follows. Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the two verteporfin peaks is not less than 2.5;the tailing factor is not more than 1.3;and the relative standard deviation for replicate injections is not more than 2.0%.

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 verteporfin peaks.Calculate the quantity,in mg,of C41H42N4O8in the portion of Verteporfin taken by the formula: in which Cis the concentration,in mg per mL,of USP Verteporfin RSin theStandard preparation;and rUand rSare the sums of the peak responses of the two verteporfin regioisomer peak responses obtained from the Assay preparationand the Standard preparation,respectively.