A: To one portion of the dry residue add 2 drops of nitric acid, and evaporate on a steam bath to dryness. Cool and add 10 mL of acetone to dissolve the residue. Add a few drops of alcoholic potassium hydroxide TS: a violet color is produced.

B: Dissolve the other portion of the residue in 1 mL of 0.1 N hydrochloric acid, and add gold chloride TS, dropwise with shaking, until a definite precipitate separates. Slowly heat until the precipitate dissolves, and allow the solution to cool: lustrous golden yellow scales are formed.

pH 9.0 Buffer— Dissolve 34.8 g of dibasic potassium phosphate in 900 mL of water, and adjust to a pH of 9.0, determined electrometrically, by the addition of 3 N hydrochloric acid or 1 N sodium hydroxide, as necessary, with mixing.

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

Standard preparation— Dissolve about 10 mg of USP Hyoscyamine Sulfate RS, accurately weighed, in water contained in a 100-mL volumetric flask, add water to volume, and mix. Prepare fresh daily. Pipet 10.0 mL of this solution into a separator, add 2.0 mL of Internal standard solution and 5.0 mL of pH 9.0 Buffer, and adjust with 1 N sodium hydroxide to a pH of 9.0. Extract with two 10-mL portions of methylene chloride, filter the methylene chloride extracts through 1 g of anhydrous sodium sulfate supported by a small cotton plug in a funnel into a 50-mL beaker, and evaporate under nitrogen to dryness. Dissolve the residue in 2.0 mL of methylene chloride.

Assay preparation— Weigh and finely powder not less than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 0.86 mg of hyoscyamine, to a separator containing 5 mL of pH 9.0 Buffer, and add, by pipet, 2.0 mL of Internal standard solution. Proceed as directed under Standard preparation, beginning with “adjust with 1 N sodium hydroxide to a pH of 9.0.”

Chromatographic system— Under typical conditions, the instrument contains a 1.8-m × 2-mm glass column packed with 3% liquid phase G3 on support S1AB, cured as directed (see Gas Chromatography 621). Maintain the column at 225, and use nitrogen as the carrier gas at a flow rate of 25 mL per minute.

System suitability— Chromatograph six to ten injections of the Standard preparation, and record peak areas as directed for 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 5, and the tailing factor does not exceed 2.0.

Procedure— Inject 1-µL portions of the Assay preparation and the Standard preparation successively into the gas chromatograph. Measure the areas under the peaks for hyoscyamine and homatropine in each chromatogram. Calculate the ratio, AU, 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, AS, in the chromatogram from the Standard preparation. Calculate the quantity, in mg, ofhyoscyamine (C17H23NO3) in the portion of Tablets taken by the formula: in which 289.37 and 676.83 are the molecular weights of hyoscyamine and anhydrous hyoscyamine sulfate, respectively, and W is the weight, in mg, of USP Hyoscyamine Sulfate RS taken for the Standard preparation.