A: Infrared Absorption 197K.

B: The retention time of the major peak in the chromatogram of the Test solution corresponds to that in the chromatogram of the Standard solution, as obtained in the test for Chromatographic purity.

C: A solution (1 in 20) responds to the tests for Sulfate 191.

Test solution: 50 mg per mL, in water.

Buffer solution— Dissolve 7.0 g of monobasic potassium phosphate in 1000 mL of water, adjust with 0.05 M phosphoric acid to a pH of 3.3, and mix.

Solution A— Dissolve 3.5 g of sodium dodecyl sulfate in 606 mL of Buffer solution, add 320 mL of acetonitrile, and mix.

Solution B— Use acetonitrile.

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard stock solution— Dissolve an accurately weighed quantity of USP Hyoscyamine Sulfate RS in Solution A to obtain a solution having a known concentration of about 1.2 mg of hyoscyamine sulfate per mL.

Standard solution— Dilute a portion of the Standard stock solution with Solution A to obtain a solution having a known concentration of about 0.24 mg of hyoscyamine sulfate per mL.

Diluted standard solution— Dilute the Standard solution with Solution A to obtain a solution having a known concentration of about 0.24 µg of hyoscyamine sulfate per mL.

System suitability solution— Prepare a solution of USP Hyoscyamine Related Compound A RS in Solution A having a concentration of about 2.4 µg per mL. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, add 10 mL of the Standard stock solution, dilute with Solution A to volume and mix.

Test solution— Transfer about 60 mg of Hyoscyamine Sulfate, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with Solution A to volume, and mix. Transfer 10 mL of this solution to a 50-mL volumetric flask, and dilute with Solution A to volume. This solution contains about 0.24 mg of hyoscyamine sulfate per mL.

Chromatographic system (see Chromatography 621— The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm ×10-cm column that contains 3-µm packing L1. The flow rate is about 1.0 mL per minute. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0, and the relative standard deviation for replicate injections is not more than 1.0%. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the resolution, R, between hyoscyamine related compound A and hyoscyamine is greater than 2.0.

Procedure— Separately inject equal volumes (about 10 µL) of the Diluted standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses of the major peaks. Calculate the percentage of each impurity in the portion of Hyoscyamine Sulfate taken by the formula: in which CS is the concentration, in µg per mL, of hyoscyamine sulfate in the Diluted standard solution; CT is the concentration, in mg per mL, of hyoscyamine sulfate in the Test solution; ri is the peak response for each impurity obtained from the Test solution; and rS is the hyoscyamine peak response obtained from the Diluted standard solution. In addition to not exceeding the limits for each impurity in Table 1, not more than 0.1% of any other individual impurity is found; and not more than 0.5% of total impurities is found.