A: Evaporate 3mLof the aqueous layer on a steam bath,under a stream of nitrogen,to dryness.Dissolve the residue,warming if necessary,in 1mLof alcohol.Add 5mLof chloroform,filter,evaporate the filtrate under a stream of nitrogen to dryness,and dry the residue at 105for 30minutes:the IRabsorption spectrum of a potassium bromide dispersion of the residue of neostigmine bromide so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Neostigmine Bromide RS.

B: Aportion of the aqueous layer responds to the tests for Bromide á191ñ.

Medium: water;500mL.

Apparatus 2: 50rpm.

Time: 45minutes.

Procedure— At the specified time interval,withdraw 30mLof the solution under test,and filter.Pipet 10mLeach of the filtered test solution,a Standard solution having a known concentration of USP Neostigmine Bromide RS,and water to provide a blank,into respective 125-mLseparators.Proceed as directed for Procedurein the Assay,beginning with “Add 15mLof a solution.”

Tolerances— Not less than 75%(Q)of the labeled amount of C12H19BrN2O2is dissolved in 45minutes.

Standard preparation— Dissolve a suitable quantity of USP Neostigmine Bromide RS,accurately weighed,in water,and dilute quantitatively and stepwise with water to obtain a solution having a concentration of about 40µg per mL.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 50mg of neostigmine bromide,to a 100-mLvolumetric flask,add about 50mLof water,shake by mechanical means for about 30minutes,add water to volume,mix,and filter.Pipet 4mLof the clear filtrate into a 50-mLvolumetric flask,add water to volume,and mix.

Procedure— Pipet 10mLeach of Assay preparationand Standard preparationinto respective 125-mLseparators,and treat each solution as follows.Add 15mLof a solution prepared by dissolving 25mg of hexanitrodiphenylamine in methylene chloride to make 250mL,without grinding the solid or heating the solution.Then add 10mLof 5Nsodium hydroxide,and shake vigorously for 30seconds.Collect the methylene chloride layer in a 100-mLvolumetric flask,and extract the aqueous layer with three 15-mLportions of methylene chloride,collecting the methylene chloride extracts in each respective flask.Add methylene chloride to volume,and mix.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 420nm,with a suitable spectrophotometer,using methylene chloride as the blank.Calculate the quantity,in mg,of C12H19BrN2O2in the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Neostigmine Bromide RSin the Standard preparation,and AUand ASare the absorbances of the solutions from the Assay preparationand the Standard preparation,respectively.