Limit of verteporfin related compound A—
Mobile phase—
Prepare as directed in the Assay.
Standard stock solution—
Dissolve accurately weighed quantities of
USP Verteporfin RS and
USP Verteporfin Related Compound A RS in a mixture of acetonitrile and tetrahydrofuran (1:1) to obtain a solution having known concentrations of about 0.167 mg per mL and 6.67 µg per mL, respectively.
Standard solution—
Dissolve 3 parts of the Standard stock solution with 2 parts water to obtain a solution having known concentrations of 0.1 mg per mL and 4 µg 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 1.4 mL per 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 solution and the
Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of verteporfin related compound A in the portion of Verteporfin for Injection taken by the formula:
20(CS / L)(rU / rS),
in which
CS is the concentration, in µg per mL, of
USP Verteporfin Related Compound A RS in the
Standard solution; L is the labeled quantity, in mg, of verteporfin in Verteporfin for Injection; and
rU and
rS are the peak responses for verteporfin related compound A in the
Test solution and the
Standard solution, respectively. Not more than 4.0% is found.
Assay—
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 3.6 M sulfuric acid to a pH of 3.0.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Verteporfin RS in a mixture of acetonitrile and tetrahydrofuran (1:1) to obtain a solution containing 0.167 mg per mL. Quantitatively dilute this solution with water to obtain a solution having a known concentration of about 0.1 mg per mL.
[NOTE—Protect solution from light.
]
Assay preparation—
Reconstitute 1 vial of Verteporfin for Injection with deionized water to obtain an approximate concentration of 2 mg per mL, and mix. Quantitatively transfer the contents to a 200-mL volumetric 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 
621
)—
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 1.4 mL per 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 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses for verteporfin. Calculate the quantity, in mg, of verteporfin (C
41H
42N
4O
8) in the portion of Verteporfin for Injection taken by the formula:
200C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Verteporfin RS in the
Standard preparation; and
rU and
rS are the sums of the peak responses for the two verteporfin isomeric peaks in the
Assay preparation and the
Standard preparation, respectively.
