Assay—
Solution A—
Prepare a filtered and degassed mixture of water and acetonitrile (13:7).
Solution B—
Prepare a filtered and degassed mixture of acetonitrile and water (7:3).
Mobile phase—
Use variable mixtures of
Solution A and
Solution B as directed for
Chromatographic system. Make adjustments to either
Solution as necessary (see
System Suitability under
Chromatography 
621
).
System suitability solution—
Dissolve suitable quantities of butylparaben and
USP Amcinonide RS in
Solution B to obtain separate solutions containing 12.5 µg per mL and 20 µg per mL, respectively.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Amcinonide RS in
Solution B, and dilute quantitatively, and stepwise if necessary, with
Solution B to obtain a solution having a known concentration of about 0.02 mg per mL.
Assay preparation—
Transfer about 20 mg of Amcinonide, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Solution B to volume, sonicate for 5 minutes, and mix. Transfer 5 mL of this solution to a 50-mL volumetric flask, dilute with Solution B to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains packing L1, and is programmed to provide variable mixtures of
Solution A and
Solution B. The flow rate is about 2 mL per minute. Equilibrate the system with
Solution A. From 2.5 minutes to 10 minutes after injection, linearly increase the amount of
Solution B to 100%. Chromatograph the
System suitability solution, and record the peak responses as directed under
Procedure: the relative retention times are about 0.78 for butylparaben and 1.0 for amcinonide, and the resolution,
R, between butylparaben and amcinonide is not less than 8.0. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 1.5, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation. Record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
28H
35FO
7 in the portion of Amcinonide taken by the formula:
1000C(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Amcinonide RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
,16
)-.
and +94.0