Chromatographic impurities—
Prepare a solution of it in chloroform containing about 20 mg per mL (
Solution A). Dilute 1.0 mL of it with chloroform to 100.0 mL (
Solution B). Apply 5-µL portions of both solutions at points along a line about 2.5 cm from one edge of a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture and previously dried at 105
for 30 minutes. Develop the plate in a suitable chamber, without previous equilibration, using a mixture of 9 volumes of benzene and 1 volume of ethyl acetate, until the solvent front has moved to about 15 cm above the line of application. Remove the plate from the chamber, air-dry, and observe under long-wavelength UV light: any spot in the chromatogram from
Solution A, other than the principal spot, is not more intense than the spot from
Solution B (1.0%).
Assay—
Mobile phase—
Prepare a solution of acetonitrile in water (35 in 100). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Internal standard preparation—
Dissolve trioxsalen in alcohol to obtain a solution containing about 0.2 mg per mL.
Standard preparation—
Using an accurately weighed quantity of
USP Methoxsalen RS, prepare a solution in alcohol having a known concentration of about 0.2 mg per mL. Transfer 2.0 mL of this solution to a 100-mL volumetric flask, add 2.0 mL of
Internal standard preparation, dilute with
Mobile phase to volume, and mix to obtain a
Standard preparation having a known concentration of about 4 µg of
USP Methoxsalen RS per mL. Pass through a 0.45-µm disk before using.
Assay preparation—
Using 20 mg of Methoxsalen, accurately weighed, proceed as directed for Standard preparation.
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 and internal standard peaks 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 2.1 for trioxsalen and 1.0 for methoxsalen. Calculate the quantity, in mg, of C
12H
8O
4, in the portion of Methoxsalen taken by the formula:
5C(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Methoxsalen RS in the
Standard preparation; and
RU and
RS are the ratios of the peak responses of methoxsalen to the internal standard obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
