Limit of tretinoin—
Mobile phase—
Prepare a suitable filtered and degassed mixture of isooctane, isopropyl alcohol, and glacial acetic acid (99.65:0.25:0.1).
Standard stock solution—
Dissolve an accurately weighed quantity of
USP Tretinoin RS in a minimum quantity of methylene chloride, add an amount of isooctane to obtain a solution having a known concentration of about 250 µg per mL, and mix.
Standard solution—
Pipet 1 mL of Standard stock solution into a 100-mL volumetric flask, add isooctane to volume, and mix.
Test solution—
Transfer about 25 mg of Isotretinoin, accurately weighed, to a 100-mL volumetric flask, dissolve in a minimum quantity of methylene chloride, add isooctane to volume, and mix.
System suitability stock solution—
Dissolve a quantity of
USP Isotretinoin RS in a minimum amount of methylene chloride, add an amount of isooctane to obtain an isotretinoin concentration of about 250 µg per mL, and mix.
System suitability solution—
Pipet 1 mL of Standard stock solution into a 100-mL volumetric flask, add System suitability stock solution 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 5-µm packing L3. The flow rate is about 1 mL per minute. Chromatograph about 20 µL of the
System suitability solution, and record the peak responses as directed for
Procedure: the relative retention times for isotretinoin and tretinoin are about 0.84 and 1.00, respectively; the resolution,
R, between isotretinoin and tretinoin is not less than 2.0; and the relative standard deviation determined from the tretinoin peak response in replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard solution and the
Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of tretinoin by the formula:
10(C / W)(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Tretinoin RS in the
Standard solution; W is the weight, in mg, of Isotretinoin taken; and
rU and
rS are the tretinoin peak responses obtained from the
Test solution and the
Standard solution, respectively. The content of tretinoin is not more than 1.0%.
;)
