A: Infrared Absorption á197Kñ.

B: Prepare a mixture of the Standard preparationand the Assay preparation(1:1),and chromatograph the mixture as directed in the Assay:the chromatogram thus obtained exhibits a single major peak due to acebutolol.

C: It responds to the tests for Chloride á191ñ,when tested as directed for alkaloidal hydrochlorides.

Standard solution— Prepare a solution of USP Acebutolol Hydrochloride RSin methanol containing 1.0mg per mL.

Test solution 1— Prepare a solution of Acebutolol Hydrochloride in methanol containing 10mg per mL.

Test solution 2— Mix 1mLof Test solution 1and 9mLof methanol.

Reference solution 1— Transfer 3.0mLof the Standard solutionto a 100-mLvolumetric flask,dilute with methanol to volume,and mix.

Reference solution 2— Mix 5.0mLof Reference solution 1and 10.0mLof methanol.

Procedure— Apply separate 20-µLportions of the Standard solution,Test solution 1,Test Solution 2,Reference solution 1,and Reference solution 2to a suitable thin-layer chromatographic plate (see Thin-Layer Chromatographyunder Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatograms in a solvent system consisting of the upper layer of a mixture of water,butyl alcohol,and glacial acetic acid (50:40:10)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.Examine the plate under short-wavelength UVlight:the chromatograms from Test solution 2and the Standard solutionshow principal spots at about the same RFvalue.No secondary spot in the chromatogram from Test solution 1,excluding the area at the point of application,is more intense than the principal spot obtained from Reference solution 1(0.3%),and not more than two secondary spots in the chromatogram from Test solution 1are more intense than the principal spot obtained from Reference solution 2(0.1%),and the total of all impurities detected in the chromatogram of Test solution 1is not more than 0.5%.

Mobile phase— Prepare a filtered and degassed mixture of methanol,a 0.3%aqueous solution of sodium dodecyl sulfate,and glacial acetic acid (675:325:20).Make adjustments if necessary to achieve a retention time for acebutolol of between 4minutes and 7minutes (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Quantitatively dissolve an accurately weighed quantity of USP Acebutolol Hydrochloride RSin water to obtain a solution having a known concentration of about 0.14mg per mL.

Assay preparation— Transfer about 35mg of Acebutolol Hydrochloride,accurately weighed,to a 250-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 1500theoretical plates;the tailing factor is not more than 2.5;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C18H28N2O4·HCl in the portion of Acebutolol Hydrochloride taken by the formula: in which Cis the concentration,in mg per mL,of USP Acebutolol Hydrochloride RSin the Standard preparation;and rUand rSare the acebutolol peak responses obtained from the Assay preparationand the Standard preparation,respectively.