» Dienestrol Cream is Dienestrol in a suitable water-miscible base. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of C18H18O2.
Packaging and storage Preserve in collapsible tubes or tight containers.
USP Reference standards 11
Identification The chromatogram of the Assay preparation employed in the Assay exhibits two peaks, for dienestrol and the internal standard, whose retention times are identical to those exhibited by the Standard preparation.
Minimum fill 755: meets the requirements.
Mobile phase Prepare a suitable degassed solution of methanol (about 3 in 5) such that the retention time of dienestrol is about 8 to 10 minutes and that of methyltestosterone is about 11 to 14 minutes. Make adjustments, if necessary (see System Suitability under Chromatography 621).
Internal standard solution Dissolve methyltestosterone in methanol to obtain a solution having a concentration of about 125 µg per mL.
Standard preparation Accurately weigh a suitable quantity of USP Dienestrol RS, dissolve in methanol, and dilute quantitatively and stepwise with methanol to obtain a concentration of about 50 µg per mL. Pipet 10 mL of this solution, 5 mL of Internal standard solution, and 5 mL of water to a 50-mL volumetric flask, dilute with methanol to volume, and mix. The concentration of dienestrol in the Standard preparation is about 10 µg per mL.
Assay preparation Pipet 20 mL of methanol and 5 mL of Internal standard solution into a 50-mL screw-capped tube. Using a 5-mL plastic syringe, transfer an accurately weighed quantity of Cream, equivalent to about 0.5 mg of dienestrol, to the tube. Cap the tube, and disperse the mixture with a suitable vibrating mixer at high speed for 3 minutes, then in an ultrasonic bath for 5 minutes. Add 20 mL of methanol, continue shaking by mechanical means for another 10 minutes, then chill in an ice bath for 5 minutes. Filter the mixture through paper, discarding the first 5 mL of the filtrate.
Procedure Introduce equal volumes (about 50 µL) of the Assay preparation and the Standard preparation into a high-pressure liquid chromatograph operated at room temperature, by means of a suitable microsyringe or sampling valve. Typically, the apparatus is fitted with a 25-cm × 4.6-mm column containing packing L1 and equipped with an UV detector capable of monitoring absorption at 254 nm and a suitable recorder. The Mobile phase is maintained at a flow rate of about 2 mL per minute. In a suitable chromatographic system, six replicate injections of the Standard preparation show a relative standard deviation of not more than 2.0% and a resolution factor of not less than 2.0 between the peaks for dienestrol and the internal standard. Calculate the quantity, in mg, of dienestrol (C18H18O2) in the portion of Cream taken by the formula:
0.05C(RU / RS)in which C is the concentration, in µg per mL, of USP Dienestrol RS in the Standard preparation, and RU and RS are the peak area ratios of dienestrol to methyltestosterone obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 2892