Identification—
Dilute a suitable quantity of Ophthalmic Solution with water to obtain a solution having a concentration of about 20 µg of timolol per mL: the UV absorption spectrum of the solution so obtained exhibits maxima and minima at the same wavelengths as that of a similar preparation of
USP Timolol Maleate RS, concomitantly measured.
Assay—
pH 2.8 phosphate buffer—
Dissolve 11.1 g of monobasic sodium phosphate in 1000 mL of water, adjust with phosphoric acid to a pH of 2.8 ± 0.05, filter, and degas.
Diluent—
Prepare a mixture of acetonitrile and pH 2.8 phosphate buffer (2:1).
Mobile phase—
Prepare a mixture of
pH 2.8 phosphate buffer and methanol (65:35). Make adjustments if necessary (see
System Suitability under
Chromatography 
621
).
[NOTE—Minimize the time the Reference Standard, the Ophthalmic Solution, the standard stock solution, the
Standard preparation, and the
Assay preparation are exposed to direct light.
]
Standard preparation—
Transfer about 34 mg of
USP Timolol Maleate RS, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Transfer 5.0 mL of this stock solution to a 50-mL volumetric flask, add 15 mL of
Diluent, dilute with water to volume, and mix.
Assay preparation—
Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 5 mg of timolol, to a 50-mL volumetric flask, add 15 mL of Diluent, dilute with water to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 295-nm detector and a 4.6-mm × 15-cm column that contains 5-µm packing L1. The column temperature is maintained at 40
, and the flow rate is about 1.2 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the tailing factor is not more than 2.0, the column efficiency is not less than 3600 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 peak area responses for the major peaks. Calculate the quantity, in mg, of timolol (C
13H
24N
4O
3S) in each mL of Ophthalmic Solution taken by the formula:
(316.43 / 432.49)(50C / V)(rU / rS),
in which 316.43 and 432.49 are the molecular weights of timolol and timolol maleate, respectively,
C is the concentration, in mg per mL, of
USP Timolol Maleate RS in the
Standard preparation,
V is the volume, in mL, of Ophthalmic Solution taken, and
rU and
rS are the peak area responses of the timolol peaks obtained from the
Assay preparation and the
Standard preparation, respectively.
