Limit of isotretinoin—
Mobile phase—
Prepare a suitable filtered and degassed mixture of isooctane, isopropyl alcohol, and glacial acetic acid (99.65:0.25:0.1), making adjustments if necessary (see
System Suitability under
Chromatography 
621
).
System suitability solution—
Dissolve a quantity of
USP Tretinoin RS in a minimum amount of methylene chloride, add a suitable amount of isooctane to obtain a solution having a tretinoin concentration of about 250 µg per mL, and mix.
Standard solution—
Dissolve an accurately weighed quantity of
USP Isotretinoin RS in a minimum quantity of methylene chloride, and add isooctane to obtain a solution having a known concentration of about 250 µg per mL.
System suitability preparation—
Pipet 5 mL of Standard solution into a 100-mL volumetric flask, add System suitability solution to volume, and mix.
Standard preparation—
Pipet 5 mL of Standard solution into a 100-mL volumetric flask, add isooctane to volume, and mix.
Test preparation—
Transfer about 25 mg of Tretinoin, accurately weighed, to a 100-mL volumetric flask, dissolve in a minimum quantity of methylene chloride, add isooctane to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 352-nm detector and a 4.0-mm × 25-cm column containing packing L3. The flow rate is about 1 mL per minute. Chromatograph about 20 µL of
System suitability preparation, and record the peak responses. The relative retention times for isotretinoin and tretinoin are about 0.84 and 1.00, respectively. The relative standard deviation of the isotretinoin peak response in replicate injections is not more than 2.0%, and the resolution,
R, of isotretinoin and tretinoin is not less than 2.0.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Test preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of isotretinoin taken by the formula:
10(C / W)(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Isotretinoin RS in the
Standard preparation,
W is the weight, in mg, of Tretinoin taken, and
rU and
rS are the peak responses of the isotretinoin peaks obtained from the
Test preparation and the
Standard preparation, respectively. The content of isotretinoin is not more than 5.0%.
;)
