A: The retention time exhibited by methoxsalen in the chromatogram of the Assay preparation corresponds to that of methoxsalen in the chromatogram of the Standard preparation as obtained in the Assay.

B: Place one capsule in 50 mL of alcohol contained in a high-speed glass blender jar and blend thoroughly until the shell is completely dispersed. Dilute a portion quantitatively with alcohol to obtain a solution having a concentration of about 4 µg per mL: the UV absorption spectrum of the solution so obtained exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Methoxsalen RS, concomitantly measured.

FOR SOFT GELATIN CAPSULES—

FOR HARD GELATIN CAPSULES—

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (65:35). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Prepare a solution of trioxsalen in alcohol having a known concentration of 0.2 mg per mL.

Standard preparation— Prepare a solution in alcohol having an accurately known concentration of 0.2 mg of USP Methoxsalen RS per mL. Pipet 2.0 mL of this solution into a 100-mL volumetric flask containing 2.0 mL of Internal standard solution, dilute with Mobile phase to volume, and mix.

Assay preparation— Place not less than 10 Capsules in a high-speed glass blender jar containing 100.0 mL of alcohol, and blend thoroughly. Transfer an accurately measured volume of the aliquot from the blender jar, equivalent to about 2 mg of Methoxsalen, to a 50-mL volumetric flask containing 10.0 mL of Internal standard solution, dilute with alcohol to volume, mix, and filter. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, mix, and filter.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the resolution, R, between the analyte peak and internal standard peak is not less than 4.0, 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 responses for the major peaks. The relative retention times are about 0.5 for Methoxsalen and 1.0 for Trioxsalen. Calculate the quantity, in mg, of methoxsalen (C12H8O4) per Capsule taken by the formula: in which C is the concentration, in mg per mL, of USP Methoxsalen RS in the Standard preparation; V is the volume, in mL, of Assay preparation taken; and RU and RS are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.