A: Dissolve a quantity,equivalent to about 12mg of echothiophate iodide,in 10mLof water.To 1mLof this solution add 0.2mLof 2Nhydrochloric acid and 0.2mLof 30percent hydrogen peroxide:the color turns brown.Add a few drops of solvent hexane,and shake:the solvent hexane acquires a pink color.

B: To 5mLof the solution prepared for Identificationtest Aadd 0.5mLof sodium hydroxide solution (1in 2),heat at 50for 2minutes,cool to room temperature,then add 1mLof sodium nitroferricyanide TS:a deep-red color is produced.

Dry alcohol— Wash about 150g of 8-to 12-mesh type 3Amolecular sieve with several portions of dehydrated alcohol to remove the fine particles.Place the washed molecular sieve in a shallow borosilicate glass tray,heat in an oven at 350for 2hours,and cool in a dry box.Transfer the dry molecular sieve to a 1-liter conical flask,add about 700mLof dehydrated alcohol,insert the stopper,mix,and allow to stand for not less than 48hours before using.

Internal standard solution— [NOTE—Prepare this solution fresh daily.]Place 0.17mLof methanol in a 100-mLvolumetric flask,add Dry alcoholto volume,and mix.

Standard preparations— [NOTE—Prepare these solutions fresh daily.]Into three 25-mLvolumetric flasks,each containing about 15mLof Internal standard solution,transfer 5µL,40µL,and 75µLof water,respectively.Dilute with Internal standard solutionto volume,and mix.

Test preparation— Carefully remove the protective retainer and cap from 5vials of Echothiophate Iodide for Ophthalmic Solution without removing the elastomeric septum closure.Discard the separated parts,and weigh accurately each closed vial and contents.Inject through the septum of each vial 400µLof Internal standard solution,accurately measured,using a suitable gas-tight syringe,and allow to stand for 1hour,swirling occasionally to dissolve the residue.After 1hour,using a gas-tight syringe,remove 300µLof solution from each vial,transfer to a dry small-volume sample-collecting vial equipped with a sampling valve system,*and mix the combined solutions.

Chromatographic system (see Chromatography á621ñ)—The chromatograph is equipped with a thermal conductivity detector and a 2-mm ×1.8-m silylated glass column packed with 80-to 100-mesh surface-silanized packing S3.The column is maintained at a temperature of about 115,the injection port and detector block are maintained at temperatures of about 200and 225,respectively,and dry helium is used as the carrier gas at a flow rate of about 45mLper minute.Chromatograph a sufficient number of injections of a Standard preparation,and record the peak responses as directed for Procedure:the resolution factor between the water and methanol peaks is not less than 2.0;and the relative standard deviation is not more than 5.0%.

Procedure— Inject a portion (3µLto 4µL)of each Standard preparationinto the chromatograph,record the chromatogram,and measure the responses for the first (water)and second (methanol)major peaks obtained for each.Plot the ratios of the peak responses of water to methanol versus the concentration,in mg per mL,of water in each Standard preparation.[NOTE—If the plot is not linear,discard it,and repeat the chromatography on additional portions of the Standard preparations.]Similarly inject a portion of the Test preparation,record the chromatogram,and measure the responses for the two major peaks.By comparison with the linear standard plot,determine the concentration,in mg per mL,of water in the Test preparationas that corresponding to the ratio of the peak responses of water to methanol from the Test preparation.Remove the elastomeric septum closure from each test vial,discard the contents,and rinse each vial and closure with several portions of methanol.Dry the vials and the closures in a stream of dry nitrogen,weigh accurately,and subtract this weight from that of the closed vials and contents obtained as directed under Test preparation.Calculate the water content,in percentage,taken by the formula: in which Cis the concentration,in mg per mL,of water in the Test preparation,Vis the volume,in mL,of Internal standard solutionadded to each test specimen vial as directed under Test preparation,and Wis the average weight,in mg,of the test specimens in the vials:not more than 2.0%is found.