(Official January 1, 2007)

Mobile phase— Mix 480 mL of acetonitrile, 3520 mL of a 0.005 M solution of sodium 1-heptanesulfonate in water, and 4 mL of phosphoric acid.

Standard preparation— Accurately weigh about 25 mg of USP Antipyrine RS, about 25 mg of USP Benzocaine RS, and about 25 mg of USP Phenylephrine Hydrochloride RS into a 250-mL volumetric flask. Add 5 mL of a 0.5 mg per mL solution of p-aminobenzoic acid in Mobile phase. Add 150 mL of Mobile phase, and mix to effect solution, sonicating if necessary. Dilute with Mobile phase to volume, and mix.

Assay preparation A— Transfer an accurately measured volume of Otic Solution, equivalent to about 100 mg of antipyrine, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. Pipet 5 mL of this solution into a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Assay preparation B— Transfer an accurately measured volume of Otic Solution, equivalent to about 100 mg of benzocaine, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix. Pipet 5 mL of this solution into a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Assay preparation P— Transfer an accurately measured volume of Otic Solution, equivalent to about 5 mg of phenylephrine hydrochloride, to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 272-nm detector and a 4.6-mm × 30-cm column that contains packing L11. The flow rate is about 1.5 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.19 for p-aminobenzoic acid, 0.26 for phenylephrine, 0.64 for antipyrine, and 1.0 for benzocaine; the resolution, R, between phenylephrine and aminobenzoic acid is not less than 1.5, and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20 or 25 µL) of the Standard preparation and each of the Assay preparations into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of antipyrine (C11H12N2O) in each mL of the Otic Solution taken by the formula: in which C is the concentration, in µg per mL of USP Antipyrine RS in the Standard preparation; V is the volume, in mL, of Otic Solution taken; and rU and rS are the antipyrine peak responses obtained from Assay preparation A and the Standard preparation, respectively. Calculate the quantity, in mg, of benzocaine (C9H11NO2) in each mL of the Otic Solution taken by the formula: in which C is the concentration, in µg per mL, of USP Benzocaine RS in the Standard preparation; V is the volume, in mL, of Otic Solution taken; and rU and rS are the benzocaine peak responses obtained from Assay preparation B and the Standard preparation, respectively. Calculate the quantity, in µg of phenylephrine hydrochloride (C9H13NO2·HCl) in each mL of the Otic Solution taken by the formula: in which C is the concentration, in µg per mL, of USP Phenylephrine Hydrochloride RS in the Standard preparation; V is the volume, in mL, of Otic Solution taken; and rU and rS are the phenylephrine peak responses obtained from Assay preparation P and the Standard preparation, respectively.