A: Infrared Absorption 197M.

B: A solution (1 in 100) meets the requirements of the test for Bromide 191.

C: 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.

Adsorbent— Use a suitable high-performance thin-layer chromatographic silica gel mixture.

Test solution— Dissolve about 100 mg of Ipratropium Bromide, accurately weighed, in 5 mL of methanol.

Stock standard solution— Dissolve an accurately weighed quantity of USP Ipratropium Bromide Related Compound A RS in methanol and dilute quantitatively, and stepwise if necessary, with methanol to obtain a solution having a known concentration of about 0.05 mg per mL.

Standard solutions— In separate flasks, dilute 0.5, 1.0, and 2.0 mL of the Stock standard solution with methanol to 5 mL to obtain a set of standard solutions having known concentrations of approximately 0.005, 0.01, and 0.02 mg of ipratropium bromide per mL. These solutions correspond to 0.025% (Standard solution C), 0.05% (Standard solution B), and 0.1% (Standard solution A), respectively, relative to the Test solution.

Application volume: 1 µL.

Developing solvent system— Prepare a mixture of methylene chloride, dehydrated alcohol, water, and formic acid (18:18:3:1).

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. Develop the plate for about 15 minutes, then remove from the tank and dry at 60 for 15 minutes. Spray the plate with the Dragendorff's TS, and allow to dry briefly. Spray the plate with sodium nitrite solution (5 in 100), and immediately cover with a glass plate. The RF for ipratropium bromide related compound A and ipratropium bromide are about 0.15 and 0.36, respectively. Any spot in the chromatogram obtained from the Test solution, except for the principal spot, is not more intense than the spot in the chromatogram obtained from Standard solution A (0.1%).

Phosphate solution, Buffer, Mobile phase, and Chromatographic system— Proceed as directed in the Assay.

System suitability solution— Dissolve suitable quantities of USP Ipratropium Bromide RS and USP Ipratropium Bromide Related Compound B RS in Mobile phase to obtain a solution containing about 0.03 mg per mL and 0.01 mg per mL, respectively.

Standard solution— Dissolve an accurately weighed quantity of USP Ipratropium Bromide RS in Mobile phase and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.03 mg per mL.

Test solution— Transfer about 250 mg of Ipratropium Bromide, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are listed in the accompanying table; the resolution, R, between ipratropium and ipratropium related compound B is not less than 4; the tailing factor of the ipratropium peak is not more than 2.5; and the relative standard deviation for replicate injections is not more than 5%.

Procedure— Separately inject equal volumes (about 5 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of related compounds in the portion of Ipratropium Bromide taken by the formula: in which F is the relative response factor of the related compound relative to ipratropium bromide; CS is the concentration, in mg per mL, of USP Ipratropium Bromide RS in the Standard solution; CT is the concentration, in mg per mL, of Ipratropium Bromide in the Test solution; ri is the individual peak response of the individual related compound; and rS is the peak response of ipratropium in the Standard solution. See the accompanying table for relative retention times, relative response factors, and acceptance criteria.

Phosphate solution— Transfer 8.9 g of dibasic sodium phosphate dihydrate to a 100-mL volumetric flask. Dissolve in and dilute with water to volume, and mix.

Buffer— Transfer 14.3 g of monobasic sodium phosphate dihydrate and 2.0 g of tetrapropylammonium chloride to a 1-L volumetric flask, dissolve in and dilute with water to volume, and mix. Adjust with Phosphate solution to a pH of 5.5 ± 0.2. Pass through a nylon membrane filter having a porosity of 0.45 µm or finer.

Mobile phase— Prepare a filtered and degassed mixture of Buffer and methanol (87:13). [note—Do not use the Mobile phase after 36 hours.] Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Dissolve suitable quantities of USP Ipratropium Bromide RS and USP Ipratropium Related Compound C RS in Mobile phase and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.5 mg per mL and 0.1 mg per mL, respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Ipratropium Bromide RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.5 mg per mL.

Assay preparation— Transfer about 50 mg of Ipratropium Bromide, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 220-nm detector and 3.9-mm × 15-cm column that contains 4-µm packing L1. The flow rate is about 1.5 mL per minute. The column temperature is maintained at 30. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for ipratropium related compound C and 1.0 for ipratropium bromide; the resolution, R, between ipratropium related compound C and ipratropium is not less than 4; the tailing factor of the ipratropium peak is not more than 2.5; and the relative standard deviation for replicate injections is not more than 1%.

Procedure— Separately inject equal volumes (about 5 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 percent, of C20 H30BrNO3·H2O in the portion of Ipratropium Bromide taken by the formula: in which CS is the concentration, in mg per mL, of USP Ipratropium Bromide RS in the Standard preparation; CT is the concentration, in mg per mL, of Ipratropium Bromide in the Assay preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.