A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparationas obtained in the Assay.

B: Shake a quantity of finely powdered Tablets,equivalent to about 100mg of pyridostigmine bromide,with 20mLof water for 5minutes,and filter the mixture:the filtrate responds to the tests for Bromide á191ñ.

Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 60minutes.

Procedure— Determine the amount of C9H13BrN2O2dissolved from UVabsorbances at the wavelength of maximum absorbance at about 270nm of filtered portions of the solution under test,suitably diluted with water,in comparison with a Standard solution having a known concentration of USP Pyridostigmine Bromide RSin the same medium.

Tolerances— Not less than 80%(Q)of the labeled amount of C9H13BrN2O2is dissolved in 60minutes.

Buffer solution— Mix 11.2g of phosphoric acid with 500mLof water,and adjust with a 50%solution of sodium hydroxide in water to a pHof 7.0.Dilute with water to 1000mL.

Mobile phase— Dissolve 1g of sodium 1-heptanesulfonate in 500mLof water in a 1000-mLvolumetric flask,and add 5.0mLof triethylamine and 100mLof acetonitrile.Dilute with water to volume,and mix.Adjust with phosphoric acid to a pHof 3.0.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Pyridostigmine Bromide RSin Buffer solution,and dilute quantitatively,and stepwise if necessary,to obtain a solution having a known concentration of 0.25mg 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 pyridostigmine bromide,to a 200-mLvolumetric flask,add 100mLof Buffer solution,and shake for 30minutes.Dilute with Buffer solutionto volume,mix,and centrifuge.Use a portion of the supernatant as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 270-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.5,and the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C9H13BrN2O2in the portion of Tablets taken by the formula: in which Cis the concentration,in mg per mL,of USP Pyridostigmine Bromide RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.