A: Dilute 5 mL with 10 mL of water, and adjust with 1 N sodium hydroxide to a pH of 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 preparation corresponds 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 preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for hydrocortisone.

(Official January 1, 2007)

Internal standard solution— Mix 2.0 mL of anisole with methanol to obtain 100 mL of 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 20 mg per mL. Transfer 5.0 mL of the resulting solution to a 10-mL volumetric flask, add 2.0 mL of Internal standard solution, dilute with methanol to volume, and mix to obtain a Standard preparation having a known concentration of about 10 mg of glacial acetic acid per mL.

Assay preparation— Transfer an accurately measured volume of Otic Solution, equivalent to about 100 mg of acetic acid, to a 10-mL volumetric flask, add 2.0 mL of 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 G35 on support S1A. The column is maintained at a temperature of 115 for 12 minutes, programmed to rise at a rate of 35 per minute to a temperature of 190, and maintained at 190 for 3 minutes, nitrogen being used as the carrier gas at a flow rate of about 25 mL per minute. The injection port and detector are maintained isothermally at temperatures of about 180 and 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 4 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses of the major peaks. The retention time of acetic acid is about 1.5 relative to that of anisole. Calculate the quantity, in mg, of C2H4O2 in each mL of the Otic Solution taken by the formula: in which C is the concentration, in mg per mL, of glacial acetic acid in the Standard preparation; V is the volume, in mL, of Otic Solution taken; and RU and RS are the peak response ratios obtained from the Assay preparation and 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 (1 in 2) having a known concentration of about 0.5 mg per mL.

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

Chromatographic system (see Chromatography 621)—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 2 mL per 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 20 µL) of the Standard preparation and the Assay preparation into the chromatograph by means of a suitable sampling valve, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of hydrocortisone (C21H30O5) in each mL of the Otic Solution taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard preparation; V is the volume, in mL, of Otic Solution taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.