Mobile phase— Mix 300 mL of a 1 in 50 solution of ammonium hydroxide in isopropyl alcohol and 700 mL of n-hexane. Filter through a 0.5-µm filter before using.

Standard preparation— Using an accurately weighed quantity of USP Pilocarpine Hydrochloride RS, prepare a solution having a known concentration of about 1.6 mg per mL.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution, equivalent to about 80 mg of pilocarpine hydrochloride, to a 50-mL volumetric flask. Dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 25-cm column that contains packing L3. The flow rate is about 2 mL per minute. Chromatograph three replicate injections of the Standard preparation, and record the peak responses as directed for 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, record the chromatograms, and measure the responses for the major peaks. The retention time is about 16 minutes for pilocarpine hydrochloride. Calculate the quantity, in mg, of C11H16N2O2·HCl in each mL of the Ophthalmic Solution taken by the formula: in which C is the concentration, in mg per mL, of USP Pilocarpine Hydrochloride RS in the Standard preparation, V is the volume, in mL, of Ophthalmic Solution taken, and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.