A: Transfer a volume of Injection,equivalent to about 2.5mg of hyoscyamine sulfate,to a separator.Render alkaline with 6Nammonium hydroxide,and extract with 25mLof methylene chloride,filtering the extract into a beaker.Evaporate to dryness.Add 2drops of nitric acid to the dry residue,and evaporate on a steam bath to dryness.Add a few drops of alcoholic potassium hydroxide TS:a violet color is produced.

B: Transfer a volume of Injection,equivalent to about 2.5mg of hyoscyamine sulfate,to a separator.Render the solution acidic with 3Nsulfuric acid,and extract with 30mLof methylene chloride.Discard the extract.Render the solution alkaline with 6Nammonium hydroxide,and extract with 30mLof methylene chloride,filtering the extract into a beaker.Evaporate to dryness.Dissolve the residue in a small amount of 0.1Nhydrochloric acid,and add gold chloride TS,with shaking,until a definite precipitate separates.Heat until the precipitate dissolves,and allow the solution to cool:lustrous golden yellow scales are formed.

C: After evaporation to dryness,or appropriate adjustment of concentration,it responds to the tests for Sulfate á191ñ.

D: The angular rotation of the Injection is levorotatory.

Internal standard solution— Dissolve about 25mg of homatropine hydrobromide in water contained in a 50-mLvolumetric flask,add water to volume,and mix.Prepare fresh daily.

Standard preparation— Dissolve about 10mg of USP Hyoscyamine Sulfate RS,accurately weighed,in water contained in a 100-mLvolumetric flask,add water to volume,and mix.Prepare fresh daily.Pipet 10mLof this solution into a separator,add 2.0mLof Internal standard solution,and adjust with 6Nammonium hydroxide to a pHof 9.Extract with three 10-mLportions of methylene chloride,filter the methylene chloride extracts through 1g of anhydrous sodium sulfate supported by a small cotton plug in a funnel into a suitable container,and evaporate on a steam bath with the aid of a current of air to dryness.Do not heat past dryness.Dissolve the residue in 2.0mLof methylene chloride.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 1.0mg of hyoscyamine sulfate,to a separator containing 5mLof water,and add 2.0mLof Internal standard solution.Proceed as directed under Standard preparation,beginning with “adjust with 6Nammonium hydroxide to a pHof 9.”

Chromatographic system— Under typical conditions,the gas chromatograph contains a 2-mm ×1.8-m glass column packed with 3%liquid phase G3on support S1AB,conditioned as directed (see Chromatography á621ñ).Maintain the column temperature at 225,and use nitrogen as the carrier gas.

System suitability— Chromatograph a sufficient number of injections of the Standard preparation,and record peak areas as directed under Procedure.The analytical system is suitable for conducting this assay if the relative standard deviation for the ratio of the peak areas does not exceed 2.0%,the resolution factor is not less than 4.0,and the tailing factor does not exceed 2.0.

Procedure— Inject appropriate portions of the Assay preparationand the Standard preparationsuccessively into the gas chromatograph.Measure the areas under the peaks for hyoscyamine and homatropine in each chromatogram.Calculate the ratio,RU,of the area of the hyoscyamine peak to the area of the internal standard peak in the chromatogram from the Assay preparation,and similarly calculate the ratio RS,in the chromatogram from the Standard preparation.Calculate the quantity,in mg,of hyoscyamine sulfate [(C17H23NO3)2·H2SO4·2H2O]in each mLof the Injection taken by the formula: in which 1.053is the ratio of the molecular weight of hydrated hyoscyamine sulfate to that of anhydrous hyoscyamine sulfate;Wis the weight,in mg,of USP Hyoscyamine Sulfate RStaken for the Standard preparation;and Vis the volume,in mL,of Injection taken.