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

Bromocresol green solution— Dissolve 250 mg of bromocresol green sodium salt in 250 mL of 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 1.2 mg per mL.

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

Procedure— Transfer 10 mL each, accurately measured, of the Assay preparation and the Standard preparation to separate 125-mL separators. Into each separator pipet 10 mL of water, 20 mL of Phosphate solution, and 5 mL of Bromocresol green solution. Extract each solution with six 15-mL portions of chloroform, collecting the chloroform extracts in respective 100-mL volumetric 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 415 nm, 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 C is the concentration, in mg per mL, of USP Pyridostigmine Bromide RS in the Standard preparation; and AU and AS are the absorbances of the solutions obtained from the Assay preparation and the Standard preparation, respectively.