Identification—
A:
Proceed as directed in the following thin-layer chromatographic procedure.
Naphazoline hydrochloride standard solution—
Dissolve a quantity of
USP Naphazoline Hydrochloride RS in water to obtain a solution containing about 1.5 mg per mL.
Pheniramine maleate standard solution—
Dissolve a quantity of
USP Pheniramine Maleate RS in water to obtain a solution containing about 6.0 mg per mL.
Test solution—
Dilute, if necessary, a volume of Ophthalmic Solution with water to obtain a solution containing about 0.25 mg of naphazoline hydrochloride per mL and 3 mg of pheniramine maleate per mL.
Procedure—
Separately apply 5 µL of
Naphazoline hydrochloride standard solution, 10 µL of
Pheniramine maleate standard solution, and 30 µL of the
Test solution to a 20-cm × 20-cm thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of silica gel. Allow the spots to dry, then place the plate in a saturated chromatographic chamber, and develop in a solvent system consisting of methanol, water, and acetic acid (8:1:1) until the solvent front has moved to about 1.5 cm from the top of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow to air-dry. Spray with ninhydrin TS, and place in an oven at 105
to visualize the spots. Both the naphazoline and pheniramine spots are purplish grey in color. The
RF values of the spots obtained from the
Test solution correspond to those obtained from the
Naphazoline hydrochloride standard solution and the
Pheniramine maleate standard solution.
B:
The retention times of the major peaks in the chromatogram of the Assay preparation correspond to those of the Standard preparation, as obtained in the Assay.
Assay—
Buffer solution—
Dissolve 14.2 g of anhydrous dibasic sodium phosphate and 20 mL of triethylamine in 1900 mL of water, adjust with phosphoric acid to a pH of 5.6 ± 0.1, dilute with water to make 2000 mL of solution, and mix.
Mobile phase—
Prepared a filtered and degassed mixture of
Buffer and acetonitrile (80:20). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Naphazoline hydrochloride stock standard solution—
Dissolve an accurately weighed quantity of
USP Naphazoline Hydrochloride RS in
Mobile phase to obtain a solution having a known concentration of about 0.75 mg per mL.
Pheniramine maleate stock standard solution—
Dissolve an accurately weighed quantity of
USP Pheniramine Maleate RS in
Mobile phase to obtain a known concentration of about 3.00 mg per mL.
Standard preparation—
Transfer 1.0 mL of Naphazoline hydrochloride stock standard solution and 3.0 mL of Pheniramine maleate stock standard solution to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix to obtain a solution having known concentrations of naphazoline hydrochloride and pheniramine maleate of 0.03 and 0.36 mg per mL, respectively.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 0.75 mg of naphazoline hydrochloride and 9.0 mg of pheniramine maleate, to a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 270-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the resolution,
R, between the naphazoline peak and the pheniramine peak is not less than 2; the column efficiency, determined from the naphazoline and pheniramine peaks, is not less than 750 theoretical plates; the tailing factor is not greater than 2.5 for pheniramine; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 25 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the peaks. Calculate the quantity, in mg, of naphazoline hydrochloride (C
14H
14N
2·HCl) in each mL of the Ophthalmic Solution taken by the formula:
25(C/V)(rU / rS),
in which
C is the concentration in mg per mL of
USP Naphazoline Hydrochloride RS in the
Standard preparation; V is the volume, in mL, of Ophthalmic solution taken; and
rU and
rS are the naphazoline peak responses obtained from the
Assay preparation and the
Standard preparation, respectively. Calculate the quantity, in mg, of pheniramine maleate (C
16H
20N
2·C
4H
4O
4) in each mL of the Ophthalmic Solution taken by the same formula, changing the terms to refer to pheniramine maleate.
