A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.

Mobile phase— Proceed as directed in the Assay.

Standard solution— Dissolve 2.0 mg each of USP Acitretin RS, USP Acitretin Related Compound A RS, and USP Acitretin Related Compound B RS, accurately weighed, in 2.5 mL of tetrahydrofuran, and dilute quantitatively with alcohol to obtain a solution having known concentrations of 0.8 µg per mL of each. [note—Store the solution at 4 prior to injection.]

Test solution— Transfer about 25 mg of Acitretin, accurately weighed, to a 100-mL volumetric flask, dissolve in 5 mL of tetrahydrofuran, dilute with alcohol to volume, and mix. [note—Store the solution at 4 prior to injection.]

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the resolution, R, between USP Acitretin Related Compound A RS (relative retention time of about 0.78) and USP Acitretin RS is not less than 1.5; the resolution, R, between USP Acitretin Related Compound B RS (relative retention time of about 1.61) and USP Acitretin RS is not less than 1.5; and the relative standard deviation for replicate injections is not more than 10.0% for acitretin related compound A and not more than 10.0% for acitretin related compound B.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of acitretin related compound A and acitretin related compound B in the portion of Acitretin taken by the formula: in which C is the concentration, in µg per mL, of USP Acitretin Related Compound A RS or USP Acitretin Related Compound B RS in the Standard solution; W is the quantity, in mg, of Acitretin taken; and rU and rS are the peak responses of the relevant impurity in the chromatograms of the Test solution and the Standard solution, respectively: not more than 0.3% of acitretin related compound A is found; and not more than 0.3% of acitretin related compound B is found. Calculate the percentage of impurities other than acitretin related compounds A and B in the portion of Acitretin taken by the formula: in which C is the concentration, in µg per mL, of USP Acitretin RS in the Standard solution; W is the quantity, in mg, of Acitretin taken; rU is the peak response of each individual unspecified impurity in the Test solution; and rS is the peak response of USP Acitretin RS in the Standard solution: not more than 0.1% of any individual unspecified impurity is found; not more than 0.4% of total unspecified impurities is found; and not more than 1.0% of total impurities is found.

Mobile phase— Prepare a filtered and degassed mixture of alcohol, water, and glacial acetic acid (92:8:0.3). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability preparation— In a 200-mL volumetric flask, dissolve 2.0 mg each of USP Acitretin RS and USP Tretinoin RS in tetrahydrofuran, dilute with alcohol to volume, and mix. Pipet 5.0 mL of this solution into a 200-mL volumetric flask, dilute quantitatively with alcohol, and mix. [note—Store the solution at 4 prior to injection.]

Standard preparation— Dissolve an accurately weighed quantity of USP Acitretin RS in tetrahydrofuran, and dilute quantitatively with alcohol to obtain a solution having a known concentration of about 0.1 mg per mL. [note—The final concentration of tetrahydrofuran in the preparation will be 2% (v/v). Store the solution at 4 prior to injection.]

Assay preparation— Transfer about 50 mg of Acitretin, accurately weighed, to a 200-mL volumetric flask, dissolve in 10 mL of tetrahydrofuran, dilute with alcohol to volume, and mix. Pipet 20 mL of the solution into a 50-mL volumetric flask, dilute with alcohol to volume, and mix. [note—Store the solution at 4 prior to injection.]

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 360-nm detector and a 4-mm × 25-cm column that contains packing L1. The flow rate is about 0.6 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between USP Tretinoin RS (relative retention time of about 0.84) and USP Acitretin RS is not less than 2.0; and the relative standard deviation for replicate injections of acitretin is not more than 1.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the acitretin peaks. Calculate the quantity, in mg, of C21H26O3 in the portion of Acitretin taken by the formula: in which C is the concentration, in mg per mL, of USP Acitretin RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.