Identification—
The retention time of the major peak in the chromatogram of the
Assay preparation corresponds to that in the chromatogram of the
Standard preparation obtained as directed in the
Assay.
Assay—
[NOTE—Avoid exposure to strong light, and use low-actinic glassware in the performance of the following procedure. Use stabilized tetrahydrofuran in the preparation of the
Standard preparation and the
Assay preparation.
]
Dilute phosphoric acid—
Dilute 10 mL of phosphoric acid with water to 100 mL.
Phosphate buffer—
Dissolve 1.38 g of monobasic sodium phosphate in 1000 mL of water, adjust with Dilute phosphoric acid to a pH of 3.0, and mix.
Diluting solution—
Prepare a mixture of water and Dilute phosphoric acid (9:1).
Mobile phase—
[NOTE—Phosphate buffer and tetrahydrofuran may be filtered and degassed separately
before mixing.
] Prepare a filtered and degassed mixture of
Phosphate buffer and tetrahydrofuran (58:42). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Tretinoin RS in tetrahydrofuran to obtain a solution having a known concentration of about 0.4 mg per mL. Dilute a known volume of this solution, quantitatively and stepwise if necessary, with a mixture of tetrahydrofuran and
Diluting solution (3:2) to obtain a solution having a known concentration of about 4 µg per mL.
Assay preparation—
Transfer an accurately weighed quantity of Cream, equivalent to 1.0 mg of tretinoin, to a 50-mL volumetric flask, and add 20.0 mL of tetrahydrofuran. Shake the flask to disperse the cream, dilute with tetrahydrofuran to volume, mix, and filter, if necessary. Transfer 5.0 mL of this solution to a 25-mL volumetric flask, dilute with a mixture of tetrahydrofuran and Diluting solution (3:2) to volume, mix, and filter.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 365-nm detector and a 3.9-mm × 15-cm column that contains 4-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 25 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 tretinoin (C
20H
28O
2) in the portion of Cream taken by the formula:
0.250C(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Tretinoin RS in the
Standard preparation, and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
