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 RSin Methylene chloride reagentto obtain a solution having a known concentration of about 0.5mg 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 1µg per mL.

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

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

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,and allow the Test solutionto 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 1.5times the tretinoin response obtained from the Standard solution(1.5%).

Methylene chloride reagent— Transfer 50g of sodium bicarbonate to 1000mLof methylene chloride,shake,and allow to stand overnight.At the time of use,filter suitable portions of this solution,and add 10mg 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 Suitabilityunder Chromatography á621ñ).

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

Assay preparation— Weigh an accurately counted number of Capsules,equivalent to about 200mg 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 5mLof Methylene chloride reagentover each Capsule and rinsing with hexanes.Collect the washings in a 500-mLvolumetric flask,dilute with hexanes to volume,and mix.[NOTE—Reserve a portion of this stock solution for the Chromatographic puritytest.]Transfer 5.0mLof the stock solution to a 200-mLvolumetric flask,dilute with hexanes to volume,and mix to obtain a solution having a concentration of 0.01mg of isotretinoin per mL.

Chromatographic system (see Chromatography á621ñ)— 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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times for isotretinoin and tretinoin are about 0.75and 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 20µL)of the Standard preparationand the Assay preparationinto 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 Cis the concentration,in mg per mL,of USP Isotretinoin RSin the Standard preparation;Nis the number of Capsules taken;and rUand rSare the isotretinoin peak responses obtained from the Assay preparationand the Standard preparation,respectively.