A: Infrared Absorption á197Fñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 20µg per mL.

Medium: water.

Test solution: 20mg per mL,in pH6.0phosphate buffer.

Standard chloride solution— Transfer 165mg of sodium chloride to a 100-mLvolumetric flask,and dissolve in and dilute with water to volume.Transfer 25.0mLof this solution to a 1000-mLvolumetric flask,and dilute with water to volume.This solution contains 25µg of chloride per mL.

Test solution— Transfer about 1.0g of Pilocarpine,accurately weighed,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume.

Procedure— Transfer 5.0mLof the Test solutionto a test tube,add 0.6mLof diluted nitric acid and 0.3mLof silver nitrate TS:any opalescence produced is not greater than that produced by an identically treated solution containing 5.0mLof the Standard chloride solution(0.25%).

Standard sulfate solution— Dissolve 148mg of anhydrous sodium sulfate in water,and dilute with water to 100mL.Dilute 10.0mLof this solution with water to 1000mL.This solution contains 10µg of sulfate per mL.

Procedure— To about 1g of Pilocarpine in a test tube add 1mLof 6Nhydrochloric acid and 4mLof water,and mix.For the control,transfer 4.0mLof Standard sulfate solutionto a test tube,add 1mLof 6Nhydrochloric acid,and mix.Adjust both solutions with pHindicator paper by the dropwise addition of 3Nhydrochloric acid or 6Nammonium hydroxide,if necessary,to a pHof between 2and 3.Add water to maintain the same volume in the control and test specimen tubes.To each tube add 1mLof barium chloride TS,and mix:any turbidity produced in the specimen tube after 10minutes'standing is not greater than that produced in the control (0.004%).

Standard preparation— Prepare a solution of USP Pilocarpine Nitrate RSto contain 43µg per mL.This solution contains the equivalent of 10µg of nitrate ion per mL.

Test preparation— Prepare a solution of Pilocarpine to contain 200mg per mL.

Procedure— Transfer 0.5-mLportions of the Test preparationand of the Standard preparation,respectively,to separate test tubes,and to each tube add 1drop of a 1in 100solution of sulfanilic acid in 5Nacetic acid and 1drop of a 3in 1000solution of N-(1-naphthyl)ethylenediamine dihydrochloride in 5Nacetic acid.Adjust the Standard preparationand the Test preparationwith pHindicator paper by the dropwise addition of 3Nhydrochloric acid or 1Nammonium hydroxide,if necessary to a pHof between 2and 3.To each solution add a few granules of acid-washed,nitrate-free zinc.Heat the test tubes in a water bath at a temperature of about 32.Allow 5minutes for the development of a pink color:any pink color observed in the Test preparationis not greater than that observed in the Standard preparation(0.005%).

Buffer solution ,Mobile phase,System suitability preparation,and Chromatographic system—Proceed as directed in the Assay.

Standard solution— Prepare a solution in water of isopilocarpine nitrate to contain 1.5µg per mL.

Test preparation— Prepare as directed for Assay preparationin the Assay.

Procedure— Separately inject equal volumes (about 40µL)of the Standard solutionand the Test preparationinto the chromatograph,record the chromatograms,and measure the responses for all the peaks.Calculate the percentage of isopilocarpine in the portion of Pilocarpine taken by the formula: in which 208.26and 271.27are the molecular weights of pilocarpine and isopilocarpine nitrate,respectively,Cis the concentration,in µg per mL,of isopilocarpine nitrate in the Standard solution,Wis the weight,in mg,of Pilocarpine taken,and rUand rSare the peak responses due to isopilocarpine in the Test preparationand the Standard solution,respectively:not more than 2%of isopilocarpine is found.Calculate the percentage of all other impurities from the chromatogram of the Test preparationtaken by the formula: in which riis the peak response due to the impurity:no one impurity corresponding to one of the four peaks in the System suitability preparationexceeds 3%;no other individual impurity exceeds 0.5%.The sum total of all impurities,including isopilocarpine,is not more than 5.0%.

Buffer solution— Transfer 13.5mLof phosphoric acid to a 1-liter beaker containing 700mLof water.Add 3mLof triethylamine,and dilute with water to 1000mL.Adjust with 20%sodium hydroxide to a pHof 3.0.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solutionand methanol (98:2).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).[NOTE—Do not store this Mobile phase for more than 2days.]

Standard preparation— Prepare a solution in water having an accurately known concentration of about 40µg of USP Pilocarpine Nitrate RSper mL.[NOTE—Use this solution within 24hours of its preparation.]

Assay preparation— Transfer an accurately weighed quantity of about 15mg of Pilocarpine to a 500-mLvolumetric flask.Dilute with water to volume,and mix.[NOTE—Use this solution within 24hours of its preparation.]

System suitability preparation— Transfer accurately weighed quantities of about 30mg each of pilocarpine hydrochloride and isopilocarpine nitrate to a 50-mLvolumetric flask,and dilute with water to volume.Transfer 25mLof this solution to a suitable flask,add 5mLof 1Nsodium hydroxide,and reflux for 1hour.Cool,and adjust the solution with 0.25Mphosphoric acid to a pHof 7.0.Quantitatively transfer this solution to a 50-mLvolumetric flask,dilute with water to volume,and mix.Dilute the remaining original solution with water to volume,and mix.Add 1mLeach of the refluxed and unrefluxed solutions to a 10-mLvolumetric flask,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 215-nm detector and a 4.6-mm ×12.5-cm column that contains 3-µm packing L1.The flow rate is about 1.0mLper minute.Chromatograph 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%.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:four peaks are observed;the resolution,R,between two adjacent peaks is not less than 1.2,the column efficiency determined for the pilocarpine peak is not less than 1500theoretical plates,and the tailing factor,T,for the pilocarpine peak is not greater than 1.5.The relative retention times for the major peaks are about 0.67for isopilocarpine,0.76for pilocarpine,0.85for pilocarpic acid,and 1.0for isopilocarpic acid.

Procedure— Separately inject equal volumes (about 40µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for all the peaks.Calculate the quantity,in mg,of pilocarpine (C11H16N2O2)in the portion of Pilocarpine taken by the formula: in which 208.26and 271.27are the molecular weights of pilocarpine and pilocarpine nitrate,respectively,Cis the concentration,in mg per mL,of USP Pilocarpine Nitrate RSin the Standard preparation,and rUand rSare the peak responses for pilocarpine obtained from the Assay preparationand the Standard preparation,respectively.