Phosphate solution— Dissolve 38g of monobasic sodium phosphate and 2g of anhydrous dibasic sodium phosphate in water to make 1000mL.Adjust the pH,if necessary,by slight variation of the ratio of the two ingredients,to 5.3±0.1.

Bromocresol green solution— Dissolve 250mg of bromocresol green sodium salt in 250mLof Phosphate solution.

Standard preparation— Dissolve a suitable quantity of USP Pyridostigmine Bromide RS,accurately weighed,in water,to obtain a solution having a known concentration of about 1.2mg per mL.

Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 120mg of pyridostigmine bromide,to a 100-mLvolumetric flask,add water to volume,and mix.

Procedure— Transfer 10mLeach,accurately measured,of the Assay preparationand the Standard preparationto separate 125-mLseparators.Into each separator pipet 10mLof water,20mLof Phosphate solution,and 5mLof Bromocresol green solution.Extract each solution with six 15-mLportions of chloroform,collecting the chloroform extracts in respective 100-mLvolumetric flasks,then add chloroform to volume in each flask,and mix.Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 415nm,with a suitable spectrophotometer,using chloroform as the blank.Calculate the quantity,in mg,of pyridostigmine bromide (C9H13BrN2O2)in the portion of Oral Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Pyridostigmine Bromide RSin the Standard preparation;and AUand ASare the absorbances of the solutions obtained from the Assay preparationand the Standard preparation,respectively.