A: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for diphenoxylate hydrochloride.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay for atropine sulfate.

Medium: 0.2 M acetic acid; 500 mL.

Apparatus 1: 150 rpm.

Time: 45 minutes.

Mobile phase— Prepare a suitable degassed mixture of acetonitrile and 0.05 M monobasic potassium phosphate (65:35).

Standard solution— Dissolve an accurately weighed quantity of USP Diphenoxylate Hydrochloride RS in methanol to obtain a solution having a known concentration of about 250 µg per mL. Pipet 10 mL of this solution into a 500-mL volumetric flask, dilute with Dissolution Medium to volume, mix, and filter.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 210-nm detector and a 3.9-mm × 30-cm column that contains packing L11. The flow rate is about 1.0 mL per minute. Chromatograph replicate injections of the Standard solution, and record the peak responses as directed for Procedure: the tailing factor is not more than 1.5; and the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50 µL) of the Standard solution and of filtered portions of the solution under test into the chromatograph, record the chromatograms, measure the response for the major peak, and determine the amount of C30H32N2O2·HCl dissolved.

Tolerances— Not less than 75% (Q) of the labeled amount of C30H32N2O2·HCl is dissolved in 45 minutes.

(Official January 1, 2007)

pH 2.7 Triethylamine phosphate buffer— Transfer approximately 18 mL of triethylamine to a 2000-mL volumetric flask containing about 1000 mL of water, and mix. Add about 11.4 mL of phosphoric acid, mix, and dilute with water to volume.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and pH 2.7 Triethylamine phosphate buffer (55:45). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Diphenoxylate Hydrochloride RS in Mobile phase to obtain a solution having a known concentration of about 0.25 mg per mL.

Assay preparation— Transfer an accurately counted number of Tablets, equivalent to about 25 mg of diphenoxylate hydrochloride, to a 100-mL volumetric flask, add Mobile phase, and shake by mechanical means for about 30 minutes until the Tablets have disintegrated completely. Dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-nm × 15-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for diphenoxylate hydrochloride is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of diphenoxylate hydrochloride (C30H32N2O2·HCl) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Diphenoxylate Hydrochloride RS in the Standard preparation; L is the labeled amount, in mg, of diphenoxylate hydrochloride in each Tablet; D is the concentration, in mg per mL, of diphenoxylate hydrochloride in the Assay preparation; and rU and rS are the peak responses for diphenoxylate obtained from the Assay preparation and the Standard preparation, respectively.

pH 2.7 Triethylamine phosphate buffer— Prepare as directed in the Assay for diphenoxylate hydrochloride.

Mobile phase— Prepare a filtered and degassed mixture of pH 2.7 Triethylamine phosphate buffer, methanol, and acetonitrile (78:18:4). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Atropine Sulfate RS in Mobile phase, and dilute quantitatively and stepwise with Mobile phase to obtain a solution having a known concentration of about 5 µg per mL.

Assay preparation— Transfer an accurately counted number of Tablets, equivalent to about 0.5 mg of atropine sulfate, to a 100-mL volumetric flask, add Mobile phase, and shake by mechanical means for about 30 minutes until the Tablets have disintegrated completely. Dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 206-nm detector and a 4.6-mm × 15-cm column that contains packing L7. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor for the atropine peak is not more than 2.5; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of atropine sulfate [(C17H23NO3)2·H2SO4·H2O] in the portion of Tablets taken by the formula: in which 694.85 and 676.83 are the molecular weights of atropine sulfate monohydrate and anhydrous atropine sulfate, respectively; C is the concentration, in µg per mL, of USP Atropine Sulfate RS in the Standard preparation; L is the labeled amount, in mg, of atropine sulfate in each Tablet; D is the concentration, in µg per mL, of atropine sulfate in the Assay preparation, based on the labeled quantity per Tablet and the extent of dilution; and rU and rS are the peak responses for atropine obtained from the Assay preparation and the Standard preparation, respectively.