A: It meets the requirements under Thin-Layer Chromatographic Identification Test 201BNP.

B: The retention time of the major peak for hydrocortisone 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)

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

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 0.15 mg per mL.

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

Chromatographic system (see Chromatography 621)— 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 2 mL per minute. Chromatograph five replicate injections of the Standard preparation and record the peak responses as directed under Procedure: the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay 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 the Standard preparation is about 0.6 full-scale. Record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg per g, of C21H30O5 in the Ophthalmic Ointment taken by the formula: in which C is the concentration, in mg per mL, of USP Hydrocortisone RS in the Standard preparation; W is the weight, in g, of the portion of Ophthalmic Ointment taken; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.