A:Ultraviolet Absorption á197Uñ—

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.

Mobile phase— Prepare as directed in the Assay.

Standard stock solution— Dissolve accurately weighed quantities of USP Verteporfin RSand USP Verteporfin Related Compound A RSin a mixture of acetonitrile and tetrahydrofuran (1:1)to obtain a solution having known concentrations of about 0.167mg per mLand 6.67µg per mL,respectively.

Standard solution— Dissolve 3parts of the Standard stock solutionwith 2parts water to obtain a solution having known concentrations of 0.1mg per mLand 4µg per mL,respectively.[NOTE—Protect solution from light.]

Test solution— Use the Assay preparation.

Chromatographic system— The liquid chromatograph is equipped with a 410-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is 1.4mLper minute.The column temperature is maintained at 30.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the resolution,R,between the two verteporfin isomeric 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 of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of verteporfin related compound Ain the portion of Verteporfin for Injection taken by the formula: in which CSis the concentration,in µg per mL,of USP Verteporfin Related Compound A RSin the Standard solution;Lis the labeled quantity,in mg,of verteporfin in Verteporfin for Injection;and rUand rSare the peak responses for verteporfin related compound Ain the Test solutionand the Standard solution,respectively.Not more than 4.0%is found.

Mobile phase— Prepare a filtered and degassed mixture of 1%(w/v)ammonium sulfate solution,acetonitrile,tetrahydrofuran,and acetic acid (20:11:9:2),and adjust with 3.6Msulfuric acid to a pHof 3.0.

Standard preparation— Dissolve an accurately weighed quantity of USP Verteporfin RSin a mixture of acetonitrile and tetrahydrofuran (1:1)to obtain a solution containing 0.167mg per mL.Quantitatively dilute this solution with water to obtain a solution having a known concentration of about 0.1mg per mL.[NOTE—Protect solution from light.]

Assay preparation— Reconstitute 1vial of Verteporfin for Injection with deionized water to obtain an approximate concentration of 2mg per mL,and mix.Quantitatively transfer the contents to a 200-mLvolumetric flask,rinsing the vial with a mixture of water,tetrahydrofuran,and acetonitrile (4:3:3),and dilute with a mixture of water,tetrahydrofuran,and acetonitrile (4:3:3)to volume.[NOTE—Protect 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 5-µm packing L1.The flow rate is 1.4mLper minute.The column temperature is maintained at 30.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the peaks for the two verteporfin isomers 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 peak responses for verteporfin.Calculate the quantity,in mg,of verteporfin (C41H42N4O8)in the portion of Verteporfin for Injection 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 for the two verteporfin isomeric peaks in the Assay preparationand the Standard preparation,respectively.