NOTE—Avoid exposure to strong light, and use low-actinic glassware in the performance of the following procedures.

Methylene chloride reagent and Mobile phase— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of USP Tretinoin RS in Methylene chloride reagent to obtain a solution having a known concentration of about 0.5 mg per mL. Dilute an accurately measured volume of this solution quantitatively, and stepwise if necessary, with hexanes to obtain a solution having a known concentration of about 1 µg per mL.

Test solution— Transfer 50.0 mL of the stock solution retained from the Assay preparation to a 200-mL volumetric flask, dilute with hexanes to volume, and mix to obtain a solution having a concentration of about 0.1 mg of isotretinoin per mL.

Chromatographic system— Proceed as directed in the Assay, using the Standard preparation prepared in the Assay.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and the Test solution into the chromatograph, and allow the Test solution to elute for not less than two times the retention time of isotretinoin. Record the chromatograms, and measure the peak responses: the peak response for any impurity is not more than that of the tretinoin response obtained from the Standard solution (1.0%); and the sum of all the peak responses, excluding that of isotretinoin, obtained from the Test solution, is not more than 1.5 times the tretinoin response obtained from the Standard solution (1.5%).

(Official January 1, 2007)

Methylene chloride reagent— Transfer 50 g of sodium bicarbonate to 1000 mL of methylene chloride, shake, and allow to stand overnight. At the time of use, filter suitable portions of this solution, and add 10 mg of butylated hydroxytoluene per mL.

Mobile phase— Prepare a filtered and degassed mixture of hexanes, ethyl acetate, and glacial acetic acid (970:30:0.1). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve accurately weighed quantities of USP Isotretinoin RS and USP Tretinoin RS in Methylene chloride reagent to obtain a solution having known concentrations of about 1 mg of each Reference Standard per mL. Transfer 1.0 mL of this solution, and dilute quantitatively with hexanes to 100.0 mL to obtain a solution having known concentrations of about 0.01 mg of each Reference Standard per mL.

Assay preparation— Weigh an accurately counted number of Capsules, equivalent to about 200 mg of isotretinoin, and calculate the average weight per Capsule. With a sharp blade, carefully open the Capsules, without loss of material, and transfer the contents by pipetting 5 mL of Methylene chloride reagent over each Capsule and rinsing with hexanes. Collect the washings in a 500-mL volumetric flask, dilute with hexanes to volume, and mix. [NOTE—Reserve a portion of this stock solution for the Chromatographic purity test.] Transfer 5.0 mL of the stock solution to a 200-mL volumetric flask, dilute with hexanes to volume, and mix to obtain a solution having a concentration of 0.01 mg of isotretinoin per mL.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 365-nm detector and a 4.6-mm × 25-cm column containing packing L3. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times for isotretinoin and tretinoin are about 0.75 and 1.00, respectively; the resolution, R, between isotretinoin and tretinoin is not less than 3.0; the tailing factor for the isotretinoin peak is not greater than 2.5; 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. Calculate the quantity, in mg, of isotretinoin (C20H28O2) in each of the Capsules taken by the formula: in which C is the concentration, in mg per mL, of USP Isotretinoin RS in the Standard preparation; N is the number of Capsules taken; and rU and rS are the isotretinoin peak responses obtained from the Assay preparation and the Standard preparation, respectively.