A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the major peak in the chromatogram of the Standard preparation,as obtained in the Assay.

B: Apply 2µLof Apraclonidine Ophthalmic Solution and 2µLof a Standard solution of USP Apraclonidine Hydrochloride RSin methanol containing about 11.5mg per mLto a suitable high performance thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.2-mm layer of chromatographic silica gel mixture,or equivalent.Allow the applications to dry,and develop the chromatogram in a solvent system consisting of a mixture of chloroform,methanol,and ammonium hydroxide (74:22:4)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 solvent to evaporate.Locate the spots on the plate by viewing under short-wavelength UVlight.[NOTE—The apraclonidine spot should appear as a blue spot.]Spray the plate with fluorescamine solution,prepared by dissolving about 25mg of fluorescamine in 25mLof acetone.[NOTE—Avoid prolonged or repeated breathing of the aerosol from the fluorescamine spray.Also avoid prolonged or repeated contact with skin.Fluorescamine solution should be sprayed only in a hood.]Examine the plate under normal light and long-wavelength UVlight.[NOTE—The apraclonidine spot should appear as a yellow spot under normal light and as a white spot under long-wavelength UVlight.]The RFvalue and appearance of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.

Phosphate buffer— Prepare as directed in the test for Chromatographic purityunder Apraclonidine Hydrochloride.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate buffer,acetonitrile,and methanol (68:30:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Apraclonidine Hydrochloride RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a Stock standard solutionhaving a known concentration of about 0.23mg per mL.Transfer 2.5mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain a Standard preparationhaving a known concentration of about 11.5µg of USP Apraclonidine Hydrochloride RSper mL(equivalent to about 10µg of apraclonidine per mL).

Resolution solution— Transfer about 1mLof propiophenone to a 100-mLvolumetric flask,dilute with methanol to volume,and mix.Transfer 3.0mLof this solution to a 50-mLvolumetric flask,dilute with methanol to volume,and mix.Transfer 1.0mLof this solution and 5.0mLof the Stock standard solutionto a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 20mg of apraclonidine,to a 100-mLvolumetric flask,dilute with water to volume,and mix.Transfer 2.5mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and an 8-mm ×100-mm column that contains packing L7.The flow rate is about 3mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for apraclonidine and 1.0for propiophenone;the column efficiency determined from the analyte peak is not less than 1000theoretical plates;the tailing factor for the analyte peak is not more than 2.2;the resolution,R,between the analyte and propiophenone peaks is not less than 3.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of apraclonidine (C9H10Cl2N4)in each mLof the Ophthalmic Solution taken by the formula: in which 245.11and 281.57are the molecular weights of apraclonidine and apraclonidine hydrochloride,respectively;Cis the concentration,in µg per mL,of USP Apraclonidine Hydrochloride RSin the Standard preparation;Vis the volume,in mL,of Ophthalmic Solution taken;and rUand rSare the apraclonidine peak responses obtained from the Assay preparationand the Standard preparation,respectively.