Mobile solvent— Prepare a mixture of 2volumes of methanol,2volumes of acetonitrile,and 6volumes of water.

Standard preparation— Dissolve a suitable quantity of USP Hydrocortisone RS,accurately weighed,in alcohol and dichloromethane (75:25)to obtain a solution having a known concentration of about 0.1mg per mL.Dilute this solution with alcohol to a concentration of 10µg per mL.

Assay preparation— Weigh accurately an aliquot of Gel,equivalent to about 10mg of hydrocortisone,and dilute with alcohol and dichloromethane (75:25)to obtain a solution having a concentration of about 0.1mg per mL.Dilute this solution with alcohol to a concentration of 10µg per mL.

Procedure— Introduce equal volumes (between 5µLand 15µL)of the Assay preparationand the Standard preparationinto a high-pressure liquid chromatograph (see Chromatography á621ñ)operated at room temperature,by means of a suitable microsyringe or sampling valve,adjusting the specimen size and other operating parameters such that the peak obtained from the Standard preparationis about 0.6full-scale.Typically,the apparatus is fitted with a 4-mm ×30-cm column that contains packing L1and is equipped with an UVdetector capable of monitoring absorption at 254nm,and a suitable recorder,and is capable of operating at a column pressure of up to 6000psi.In a suitable chromatogram,the coefficient of variation for five replicate injections of the Standard preparationis not more than 3.0%.Determine the ratios of the peak heights,at equivalent retention times,obtained from the Assay preparationand the Standard preparation,and calculate the quantity,in mg,of hydrocortisone (C21H30O5)in the portion of Gel taken by the formula: in which Cis the concentration,in µg per mL,of USP Hydrocortisone RSin the Standard preparation;and HUand HSare the peak heights of the Assay preparationand the Standard preparation,respectively.