Solution A, Solution B, and Mobile phase
Proceed as directed in the Assay.
Prepare as directed for the Standard preparation in the Assay.
Sensitivity check solution
Dilute the Standard solution with water to obtain a solution having a concentration of 0.25 µg per mL.
Prepare as directed for Assay preparation in the Assay.
Chromatograph the Sensitivity check solution at 410 nm, and record the peak heights: the ratio of the verteporfin peak height to the noise height is not less than 10, the noise height being determined by a suitable procedure. Proceed as directed in the Assay. To evaluate the system suitability requirements, use the Standard preparation prepared as directed in the Assay.
Inject a volume (about 20 µL) of the Test solution
into 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:
100(ri / rs),
in which ri
is the individual peak response of each related compound; and rs
is the sum of the responses of all the peaks. Not more than 0.6% of the peak having a retention time of about 0.56 relative 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%.
Prepare a filtered and degassed mixture of 1% (w/v) aqueous ammonium sulfate, acetonitrile, glacial acetic acid, and 3.6 M sulfuric acid (10:10:1:0.027).
Prepare a filtered and degassed mixture of 1% (w/v) aqueous ammonium sulfate, tetrahydrofuran, glacial acetic acid, and 3.6 M sulfuric acid (10:10:1:0.034).
Use variable mixtures of Solution A
and Solution B
as directed for Chromatographic system.
Make adjustments if necessary (see System Suitability
under Chromatography 621
To a suitable volumetric flask, transfer an accurately weighed quantity of USP Verteporfin RS
sufficient to make a 0.25 mg per mL solution. 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.]
Transfer about 25 mg of Verteporfin, accurately weighed, to a 100-mL volumetric flask. Add 60 mL of a mixture of acetonitrile and tetrahydrofuran (1:1), and dissolve. Dilute with water to volume, and mix. [NOTEProtect the solution from light.]
Chromatographic system (see Chromatography 621)
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 1.5 mL per 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%.
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 verteporfin peaks. Calculate the quantity, in mg, of C41
in the portion of Verteporfin taken by the formula:
100C(rU / rS),
in which C
is the concentration, in mg per mL, of USP Verteporfin RS
in the Standard preparation;
are the sums of the peak responses of the two verteporfin regioisomer peak responses obtained from the Assay preparation
and the Standard preparation,
Calculate the ratio of the peak responses for the two peaks assigned to verteporfin: not less than 0.9 and not more than 1.1.