Identification—
Prepare a test solution by diluting a suitable volume of it with water to obtain a solution containing about 2.5 mg of betaxolol per mL. Separately apply 5 µL of the test solution and 5 µL of a Standard solution of
USP Betaxolol Hydrochloride RS in water containing about 2.75 mg per mL to a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of silica gel. Allow the spots to dry, and develop the chromatogram in a chromatographic chamber, using a solvent system consisting of a mixture of chloroform, isopropyl alcohol, and ammonium hydroxide (70:30:2) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and allow the plate to air-dry. Spray the plate with a 1 in 1000 solution of ninhydrin in isopropyl alcohol, and heat the plate at 105
for 10 minutes. Locate the spots on the plate: the
RF value of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.
Assay—
pH 3.0 Buffer—
Dissolve 7.1 g of anhydrous dibasic sodium phosphate in about 800 mL of water, adjust with phosphoric acid to a pH of 3.0, and dilute with water to make 1000 mL of solution.
Mobile phase—
Prepare a suitable filtered and degassed mixture of
pH 3.0 Buffer and acetonitrile (1:1). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Dissolve an accurately weighed quantity of
USP Betaxolol Hydrochloride RS in
pH 3.0 Buffer to obtain a solution having a known concentration of about 0.11 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 10 mg of betaxolol, to a 100-mL volumetric flask. Dilute with pH 3.0 Buffer to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4-mm × 25-cm column that contains packing L1. The flow rate is about 1.1 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the capacity factor,
k¢, for the main betaxolol peak is between 1 and 3; the tailing factor is not less than 0.8 and not more than 2.0; the column efficiency is not less than 750 theoretical plates; and the relative standard deviation for replicate injections 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, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
18H
29NO
3 in each mL of the Ophthalmic Solution taken by the formula:
(307.43 / 343.89)(100C / V)(rU / rS),
in which 307.43 and 343.89 are the molecular weights of betaxolol and betaxolol hydrochloride, respectively;
C is the concentration, in mg per mL, of
USP Betaxolol Hydrochloride RS in the
Standard preparation; V is the volume, in mL, of Ophthalmic Solution taken; and
rU and
rS are the betaxolol peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
