pH9.0Buffer— Dissolve 34.8g of dibasic potassium phosphate in 900mLof water,and adjust to a pHof 9.0,determined electrometrically,by the addition of 3Mhydrochloric acid or 1Msodium hydroxide,as necessary,with mixing.

Internal standard solution— [NOTE—Prepare fresh daily.]Transfer about 25mg of homatropine hydrobromide to a 50-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Standard preparation— [NOTE—Prepare fresh daily.]Dissolve an accurately weighed quantity of USP Atropine Sulfate RSin water,and dilute quantitatively,and stepwise if necessary,with water to obtain a solution having a known concentration of about 0.1mg per mL.Pipet 10mLof this solution into a separator,and proceed as directed for the Assay preparation,beginning with “Add 2.0mLof Internal standard solution.”

Assay preparation— Transfer an accurately measured volume of Ophthalmic Solution,equivalent to about 10mg of Atropine Sulfate,to a 100-mLvolumetric flask,dilute with water to volume,and mix.Pipet 10mLof this solution and treat as follows.Add 2.0mLof Internal standard solutionand 5.0mLof pH9.0Buffer,and adjust the solution in the separator with 1Msodium hydroxide to a pHof 9.0.Extract with two 10-mLportions of methylene chloride,filter the methylene chloride extracts through 1g of anhydrous sodium sulfate supported by a small cotton plug in a funnel into a 50-mLbeaker,and evaporate under a stream of nitrogen to near-dryness.Dissolve the residue in 2.0mLof methylene chloride.

Chromatographic system(see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 2-mm ×1.8-m glass column packed with a 3%phase G3on support S1AB.The carrier gas is nitrogen,flowing at a rate of 25mLper minute.The column temperature is maintained at 225.The injection port and detector temperatures are maintained at 250.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,is not less than 4.0;the tailing factor is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 1µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of atropine sulfate [(C17H23NO3)2·H2SO4·H2O]in each mLof Ophthalmic Solution taken by the formula: in which 694.85and 676.83are the molecular weights of atropine sulfate monohydrate and anhydrous atropine sulfate,respectively;Wis the weight,in mg,of USP Atropine Sulfate RSin the Standard preparation;Vis the volume,in mL,of Ophthalmic Solution taken;and RUand RSare the peak area ratios of atropine sulfate to homatropine hydrobromide obtained from the Assay preparationand the Standard preparation,respectively.