Test solution— Use the Assay preparation,except to omit addition of the Internal standard solution.

Mobile phase— Mix 55mLof a solution of water in methanol (5in 100)with 1.0mLof glacial acetic acid,dilute with water-washed 1,2-dichloroethane to 1000mL,and mix.Degas before using.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve 200mg of acetaminophen in 4mLof methanol,dilute with water-washed 1,2-dichloroethane to 200mL,and mix.Keep the solution tightly stoppered and protected from light.

Standard preparation— Accurately weigh about 8mg of USP Hydrocortisone RS,add 4mLof methanol and 4.0mLof Internal standard solution,dilute with chloroform to 100.0mL,and mix to obtain a solution having a known concentration of about 0.08mg of USP Hydrocortisone RSper mL.

Assay preparation— Transfer an accurately weighed quantity of Rectal Suspension,equivalent to about 8mg of hydrocortisone,to a separator.Extract with four 20-mLportions of chloroform,filtering each portion through chloroform-washed cotton into a 100-mLvolumetric flask.Add 4mLof methanol and 4.0mLof Internal standard solution,dilute with chloroform to volume,and mix.Pass the extract through a 0.5-µm porosity polytef membrane filter,discarding the first 20mLof the filtrate.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L3.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.3for acetaminophen and 1.0for hydrocortisone;the resolution,R,between the analyte and internal standard is not less than 2.5;the column efficiency determined from the analyte peak is not less than 5000theoretical plates;and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 10µL)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 Rectal Suspension taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydrocortisone RSin the Standard preparation;and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.