A: It meets the requirements under Thin-Layer Chromatographic Identification Test á201BNPñ.

B: The retention time of the major peak for hydrocortisone in the chromatogram of theAssay preparationcorresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay for hydrocortisone.

Mobile phase— Prepare a suitable solution of about 500volumes of methanol,500volumes of water,and 1volume of glacial acetic acid,such that the retention time of hydrocortisone is between 6and 10minutes.

Standard preparation— Dissolve a suitable quantity of USP Hydrocortisone RS,accurately weighed,in methanol and water (1:1)to obtain a solution having a known concentration of about 0.15mg per mL.

Assay preparation— Transfer to a separator about 1.5g,accurately weighed,of Ophthalmic Ointment.Add 3mLofn-hexane,and warm gently on a steam bath with mild agitation until dissolved.Add 7mLofn-hexane,mix by swirling,and extract with four 15-mLportions of methanol and water (1:1).Collect the extracts in a 100-mLvolumetric flask,dilute with methanol and water (1:1)to volume,and mix.Filter the solution,rejecting the first 10mLof the filtrate.

Chromatographic system(see Chromatography á621ñ)— The chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph five replicate injections of theStandard preparation and record the peak responses as directed underProcedure:the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of theStandard preparation and theAssay preparation into the chromatograph by means of a suitable microsyringe or sampling valve,adjusting the specimen size and other operating parameters such that the peak obtained from theStandard preparation is about 0.6full-scale.Record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg per g,of C21H30O5in the Ophthalmic Ointment taken by the formula: in whichCis the concentration,in mg per mL,of USP Hydrocortisone RSin theStandard preparation;Wis the weight,in g,of the portion of Ophthalmic Ointment taken;andrUandrSare the peak responses obtained from theAssay preparation and theStandard preparation,respectively.