Determine the amounts of chlordiazepoxide hydrochloride and clidinium bromide dissolved using the following method.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Place a glass wool plug in the bottom of a 2.5-cm × 35 ± 5-cm glass chromatographic tube, add 2 g of chromatographic siliceous earth triturated with 1 mL of 1 N hydrochloric acid, and lightly pack with a glass tamping rod. Empty a number of Capsules, equivalent to about 15 mg of clidinium bromide, into a 100-mL beaker, add 3 mL of 1 N hydrochloric acid, and swirl to dissolve. Add 4 g of chromatographic siliceous earth, mix with a spatula, and add the Capsule-siliceous earth mixture to the chromatographic tube. Dry-wash the beaker with an additional 0.5 to 1.0 g of chromatographic siliceous earth, adding the washing to the top of the column. Lightly pack with the tamping rod, and overlay the column with glass wool. Insert the lower exit tube of the column into a 125-mL separator, and elute the column with 100 mL of chloroform previously distilled over 1 N sulfuric acid and saturated with water. Extract the chloroform eluate with 20 mL of freshly prepared ascorbic acid solution (1 in 20), preserving the extract. Extract the eluate with a second 15-mL portion of the ascorbic acid solution, combine the extracts in the separator, and discard the chloroform layer. Neutralize the acid extracts by adding sodium bicarbonate until the solution is slightly alkaline to the pH paper. Extract the slightly alkaline solution with two 25-mL portions of chloroform, combine the chloroform extracts, and pass through dry, fluted filter paper into a 100-mL beaker. Evaporate the chloroform to dryness with the aid of a stream of nitrogen, and transfer the residue to a glass-stoppered 1.0-mL volumetric flask, using methanol to facilitate the transfer. Dilute with methanol to volume.

Reference solution— Dissolve 3.0 mg of USP 3-Quinuclidinyl Benzilate RS in 100 mL of methanol, and mix.

Developing solvent system: methanol.

Procedure— Separately apply 15 µL of the Reference solution and 100 µL of the Test solution to a suitable chromatographic plate, place the plate in a paper-lined, methanol-saturated chromatographic chamber, and proceed as directed for Thin-layer Chromatography under Chromatography 621. Remove the plate, air-dry, spray with potassium iodoplatinate TS, and allow the spots to develop for 10 minutes. Any spot in the chromatogram obtained from the Test solution occurring at an RF value of about 0.3 is not greater in size or intensity than the spot in the chromatogram obtained from the Reference solution: not more than 0.03% of 3-quinuclidinyl benzilate is found.

Adsorbent, Developing solvent system, Chromatographic plates, and Spray reagent— Prepare as directed in the test for Related compounds under Clidinium Bromide.

Extracting solvent mixture: a mixture of dehydrated alcohol and cyclohexane (1:1).

Test solution— Empty a number of Capsules, equivalent to about 25 mg of clidinium bromide, into a glass-stoppered centrifuge tube, and add 5 mL of Extracting solvent mixture. Heat the tube gently, with shaking, to 50, centrifuge, and decant the clear supernatant into a second tube. Repeat the addition of Extracting solvent mixture twice, heating, centrifuging, and decanting as before, and combine the three extracts in a single tube. Gently heating, evaporate the combined extracts under a stream of nitrogen to dryness. Dissolve the residue in 0.5 mL of methanol.

Standard solution— Dissolve 50 mg of USP Clidinium Bromide RS in 1.0 mL of 0.1 N methanolic hydrochloric acid. Prepare this solution at the time of use.

Reference solution— Dissolve 50 mg of USP Clidinium Bromide RS in 1.0 mL of 0.1 N methanolic hydrochloric acid, and add 20 µL of a solution of 25 mg of USP Clidinium Bromide Related Compound A RS in 1.0 mL of methanol. Prepare this solution at the time of use.

Procedure— Separately apply the Standard solution, the Reference solution, and the Test solution as directed in Thin-Layer Chromatography under Chromatography 621. Place the plates in an unsaturated chromatographic chamber containing freshly prepared Developing solvent system, and develop the chromatogram until the solvent front has moved about 15 cm. Remove the plates, and dry at 105 for 10 minutes. Cool to room temperature, and spray with Spray reagent. Any spot in the chromatogram obtained from the Test solution occurring at an RF value of about 0.4 is not greater in size or intensity than the corresponding spot in the chromatogram obtained from the Reference solution; and the Standard solution shows no spot at the RF value corresponding to that of clidinium bromide related compound A: not more than 1.0% of clidinium bromide related compound A is found.

50C(rU / rS),