Internal standard solution— Dissolve a suitable quantity of USP Norethindrone RSin methanol to obtain a solution containing about 850µg per mL.

Diluted internal standard solution— Transfer 5.0mLof Internal standard solutionto a 250-mLflask.Dilute with methanol to volume,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Fluocinolone Acetonide RSin acetonitrile to obtain a solution having a known concentration of about 200µg per mL.Transfer 10.0mLof this solution and 2.0mLof Internal standard solutionto a 100-mLvolumetric flask.Dilute with methanol to volume,and mix.The concentration of USP Fluocinolone Acetonide RSin the Standard preparationis 20µg per mL.

Mobile solvent— Prepare a mixture of acetonitrile and water (1:1).Adjust the ratio as necessary to obtain suitable chromatographic performance.

Assay preparation— Transfer an accurately weighed portion of Ointment,equivalent to about 0.7mg of fluocinolone acetonide,to a 50-mL,round-bottom centrifuge tube.Add 35.0mLof Diluted internal standard solution,emulsify using an ultrasonic probe,and centrifuge to bring the insoluble matter to the bottom.The clear supernatant is the Assay preparation.

Apparatus— Use a suitable high-pressure liquid chromatograph (see Chromatography á621ñ)of the general type equipped with a detector for monitoring UVabsorbance at about 254nm,and capable of providing a flow rate of about 2mLper minute for the Mobile solvent.Use a 50-cm ×4-mm column that contains packing L1so as to provide a resolution factor,R(see Chromatography á621ñ),of at least 2.0between peaks for norethindrone and fluocinolone acetonide.Three replicate injections of the Standard preparationshow a relative standard deviation of not more than 1.5%.

Procedure— Chromatograph equal volumes of the Assay preparationand the Standard preparation,adjusting the system as necessary to obtain peaks of between about 50%and 90%of full-scale.Calculate the quantity,in mg,of C24H30F2O6in the portion of Ointment taken by the formula: in which Cis the concentration,in µg per mL,of USP Fluocinolone Acetonide RSin the Standard preparation;and RUand RSare the ratios of the peak areas of fluocinolone acetonide and the internal standard obtained from the Assay preparationand the Standard preparation,respectively.