Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,and methanol (58:21:21).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Hydrocortisone RSin alcohol and dilute quantitatively,and stepwise if necessary,with alcohol to obtain a solution having a known concentration of about 0.1mg per mL.Quantitatively dilute a known volume of the final solution with an equal volume of water to obtain the Standard preparation.

Assay preparation— In a tared,100-mLvolumetric flask,weigh 100mLof Lotion that previously has been shaken to ensure homogeneity,allow to stand until the entrapped air rises,and finally invert carefully just prior to transfer to the volumetric flask.Transfer an accurately weighed quantity of Lotion,freshly mixed but free from air bubbles,equivalent to about 10mg of hydrocortisone,to a 40-mLbeaker,and add about 30mLof alcohol.Warm gently until the Lotion is dispersed,and cool to room temperature.Quantitatively transfer the mixture,filter through a pledget of cotton previously moistened with alcohol to a 100-mLvolumetric flask,rinse the beaker with two 20-mLportions of alcohol,and collect the washings in the same volumetric flask.Dilute with alcohol to volume,and mix.Quantitatively dilute a known volume of this solution with an equal volume of water to obtain the Assay preparation.

System suitability preparation— Dissolve about 5mg of propylparaben in 100mLof alcohol.Dilute 1mLof this solution with the Standard preparationto 10mL,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1mLper minute.Chromatograph the Standard preparationand the System suitability preparation,and record the peak responses as directed for Procedure:the column efficiency determined from the analyte peak is not less than 1000theoretical plates,the tailing factor for the analyte peak is not more than 1.2,the resolution,R,between the propylparaben and hydrocortisone peaks is not less than 2.0,and the relative standard deviation for replicate injections of the Standard preparationis not more than 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of hydrocortisone (C21H30O5)in the portion of Lotion taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone RSin the Standard preparation;and rUand the rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.From the observed weight of 100mLof the Lotion,calculate the quantity of C21H30O5in each 100mL.