A: Dilute 5mLwith 10mLof water,and adjust with 1Nsodium hydroxide to a pHof about 7.Add ferric chloride TS:a deep red color is produced,and it is destroyed by the addition of hydrochloric acid.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,both relative to the internal standard obtained as directed in the Assay for acetic acid.

C: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for hydrocortisone.

Internal standard solution— Mix 2.0mLof anisole with methanol to obtain 100mLof solution.

Standard preparation— Quantitatively dilute an accurately weighed quantity of glacial acetic acid with methanol to obtain a solution having a known concentration of about 20mg per mL.Transfer 5.0mLof the resulting solution to a 10-mLvolumetric flask,add 2.0mLof Internal standard solution,dilute with methanol to volume,and mix to obtain a Standard preparationhaving a known concentration of about 10mg of glacial acetic acid per mL.

Assay preparation— Transfer an accurately measured volume of Otic Solution,equivalent to about 100mg of acetic acid,to a 10-mLvolumetric flask,add 2.0mLof Internal standard solution,dilute with methanol to volume,and mix.

Chromatographic system— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm ×1.8-m glass column packed with 20%liquid phase G35on support S1A.The column is maintained at a temperature of 115for 12minutes,programmed to rise at a rate of 35per minute to a temperature of 190,and maintained at 190for 3minutes,nitrogen being used as the carrier gas at a flow rate of about 25mLper minute.The injection port and detector are maintained isothermally at temperatures of about 180and 220,respectively.Chromatograph the Standard preparation,record the chromatogram,and measure the peak responses as directed for Procedure:the resolution,R,between anisole and acetic acid is not less than 1.5,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 4µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses of the major peaks.The retention time of acetic acid is about 1.5relative to that of anisole.Calculate the quantity,in mg,of C2H4O2in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in mg per mL,of glacial acetic acid in the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase— Prepare a solution containing water and acetonitrile (70:30).

Standard preparation— Using an accurately weighed quantity of USP Hydrocortisone RS,prepare a solution in dilute alcohol (1in 2)having a known concentration of about 0.5mg per mL.

Assay preparation— Transfer an accurately measured volume of Otic Solution,equivalent to about 100mg of hydrocortisone,to a 200-mLvolumetric flask.Add dilute alcohol (1in 2)to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid 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 four replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph by means of a suitable sampling valve,record the chromatograms,and measure the responses for the major peak.Calculate the quantity,in mg,of hydrocortisone (C21H30O5)in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.