A: Infrared Absorption 197K.

B: The RF value of the principal spot in the chromatogram, developed with the Basic eluant, obtained from the Test solution corresponds to that obtained from the Standard solution in the Ordinary impurities test.

Test solution— Transfer an accurately weighed quantity, about 1.00 g of Iopromide, to a 20-mL volumetric flask, dissolve in and dilute with water to volume, and mix. Pipet 12.0 mL of this solution into a test tube, add 2.0 mL of pH 3.5 Acetate Buffer, and mix.

Standard solution— Pipet 1.0 mL of Standard Lead Solution (10 µg of lead), into a test tube, add 9.0 mL of water, 2.0 mL of the Test solution, and 2.0 mL of pH 3.5 Acetate Buffer, and mix.

Thioacetamide-glycerin base solution color-comparison tubes— Mix 15 mL of 1 N sodium hydroxide and 5 mL of water, and add 20 mL of glycerin. Pipet 1.0 mL of this solution and 0.20 mL of thioacetamide TS into each of two color-comparison test tubes, and heat in a boiling water bath for 20 seconds. Use these tubes immediately.

Procedure— Immediately add the Test solution to one of the Thioacetamide-glycerin base solution color-comparison tubes and the Standard solution to the other. Mix, allow to stand for 2 minutes, and view downward over a white surface: the color of the solution from the Test solution is not darker than that of the solution from the Standard solution, treated in the same manner.

Test solution— Transfer 500 mg of Iopromide to a 25-mL volumetric flask, add 20 mL of water, and mix.

Standard solution— Dissolve a suitable quantity of USP Iopromide Related Compound A RS in water, and dilute with water to obtain a stock solution having a known concentration of 0.25 mg per mL. Transfer 2.0 mL of this stock solution to a 25-mL volumetric flask, add 18.0 mL of water, and mix.

Blank solution— Transfer 20 mL of water to a 25-mL volumetric flask.

Procedure— Treat each flask as follows. Place the flasks in an ice bath, and protect from light. Add slowly 1.0 mL of 8 N hydrochloric acid, mix, and allow to stand for 5 minutes. Add 1.0 mL of sodium nitrite solution (1 in 50), mix, and allow to stand for 5 minutes. Add 0.50 mL of freshly prepared sulfamic acid solution, (8 in 100). Shake each flask vigorously several times within the next 5 minutes, venting off the gas that evolves. [Caution—Considerable pressure is produced. ] Add 1.0 mL of freshly prepared N-(1-naphthyl)ethylenediamine dihydrochloride solution, (1 in 1000) in a mixture of propylene glycol and water (70:30), and shake. Remove the flasks from the ice bath, and allow to stand in a water bath at about 25 for 10 minutes. Dilute with water to volume, mix, and degas with the aid of sonication for 1 minute. Concomitantly determine the absorbances of the Test solution and the Standard solution in 1-cm cells at the wavelength of maximum absorbance at about 495 nm, with a suitable spectrophotometer, using the Blank solution, treated in the same manner. The absorbance of the Standard solution is not less than 0.40. Calculate the percentage of free aromatic amine in the portion of Iopromide taken by the formula: in which WS is the quantity, in mg, of USP Iopromide Related Compound A RS taken to prepare the Standard solution; WU is the quantity, in mg, of the Iopromide taken to prepare the Test solution; and AU and AS are the absorbances of the Test solution and the Standard solution, respectively: not more than 0.1% is found.

Standard solution— Prepare a solution of alcohol in dimethylformamide to obtain a solution having a known concentration of about 0.050 mg of alcohol (C2H5OH) per mL.

Test solution— Dissolve an accurately weighed portion of Iopromide in dimethylformamide to obtain a concentration of about 50 mg per mL.

Blank solution— Use dimethylformamide.

Chromatographic system (see Chromatography 621)—The gas chromatograph is equipped with a headspace injector, a flame-ionization detector, and a 0.25-mm × 30-m capillary column, the internal wall of which is coated with a 1.4-µm film of liquid phase G43. The column temperature is programmed according to the following steps: it is held at 40 for 10 minutes, then increased at a rate of 5 per minute to 70; it is then increased at a rate of 30 per minute to 220. The injector port is maintained at 160; the headspace sampler is maintained at 80; and the detector is maintained at 250. Helium is used as the carrier gas at a flow rate of about 27 cm per second. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the retention time for alcohol is about 3 minutes; and the relative standard deviation for three injections of the Standard solution is not more than 4.0%. Chromatograph the Blank solution, and record the peak responses as directed for Procedure: the chromatogram shows no peak at the retention time for alcohol.

Procedure— [note—Use peak areas where peak responses are indicated.] Transfer 2.0 mL each of the Test solution, the Standard solution, and the Blank solution to separate headspace vials, add 10 µL of 1 N hydrochloric acid to each vial, then seal the vials using a flanged cap so that the cap can no longer be turned. Record the chromatograms, and measure the responses for the alcohol peak. Calculate the concentration of alcohol in the portion of Iopromide taken by the formula: in which C is the concentration, in mg per mL, of alcohol (C2H5OH) in the Standard solution; I is the quantity, in mg per mL, of Iopromide in the Test solution; and rU and rS are the alcohol peak responses in the chromatograms obtained from the Test solution and the Standard solution, respectively: not more than 0.4% of alcohol (C2H5OH) is found. Use the percentage obtained to calculate the Assay result on the solvent-free basis.

20(WB / WI)[(AY1 + AY2) / (RY1 + RY2)]

Test solution: a mixture of methanol and water (1:1).

Standard solutions: a mixture of methanol and water (1:1).

Visualization solution—

Solution A —Dissolve 2.7 g of ferric chloride in 100 mL of 2.4 N hydrochloric acid. Store this solution in a refrigerator.

Solution B —Dissolve 3.5 g of potassium ferricyanide in 100 mL of water. Store this solution in a refrigerator.

Solution C —Dissolve 5.0 g of sodium arsenite in 30 mL of 1 N sodium hydroxide solution that has been cooled to 0. While stirring, mix with 65 mL of 2.4 N hydrochloric acid, and store at room temperature. Use the clear supernatant.

Procedure —Mix 10 mL of Solution A, 10 mL of Solution B, and 2.0 mL of Solution C. Use within 30 minutes.

Basic eluant: a mixture of dioxane, water, and ammonium hydroxide (85:15:4).

Acidic eluant: a mixture of chloroform, methanol, water, and 96 percent formic acid (62:32:6:2).

Procedure— Apply 1 µL and 2 µL of the Test solution and 1 µL of each of the Standard solutions to two separate thin-layer chromatographic plates. Place one plate in a development chamber containing the Acidic eluant, and the second plate in a development chamber containing the Basic eluant. After the chromatograms have developed, remove the plates from the chambers, and allow to dry at room temperature.

Visualization—

Detection 1—Observe both plates under 254-nm UV light.

Detection 2—The plate developed with the Acidic eluant is exposed to ammonia vapors for 10 to 30 minutes and is air dried. Both plates are exposed to unfiltered 254-nm UV light for several minutes until the principal spots appear yellow. Overspray with Visualization solution, and examine the plates under ambient light. Determine the percentage of all secondary spots, except those due to free aromatic amine and to the N-acetyl compound.

Limit— The sum of all secondary spots observed in the chromatograms of the Test solution, except those due to the free aromatic amine and to the N-acetyl compound in addition to the percentage of N-acetyl compound obtained in the test for Limit of N-Acetyl compound, corresponds to not more than 3.0%.

100(rE1 + rZ1) / (rE1 + rE2 + rZ1 + rZ2)

100(rE2 + rZ2) / (rE1 + rE2 + rZ1 + rZ2):

Diluent— Prepare a mixture of methanol and water (1:1).

Mobile phase— [note—Use chloroform, methanol, and water that have been filtered and degassed.] Mix 6 g of chloroform with 59 g of methanol, then add 900 g of water. Store in a sealed container, and do not stir or sparge the Mobile phase during use.

Standard preparation— Transfer an accurately weighed quantity of about 38 mg of USP Iopromide RS, to a 20-mL volumetric flask. Dissolve in and dilute with Diluent to volume, and mix.

Related compound B standard solution— Transfer about 1.9 mg of USP Iopromide Related Compound B RS, accurately weighed, to a 100-mL volumetric flask. Dissolve in and dilute with Diluent to volume, and mix.

Assay preparation— Transfer about 38 mg of Iopromide, accurately weighed, to a 20-mL volumetric flask. Dissolve in and dilute with Diluent to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 1.2 mL per minute. The temperature is maintained at a constant temperature of about 20. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times for iopromide E1-isomer, iopromide E2-isomer, iopromide Z1-isomer, and iopromide Z2-isomer are about 0.70, 0.75, 0.85, and 1.0, respectively; the resolution, R, between iopromide isomers Z1 and Z2 is not less than 2.0; and the relative standard deviation for replicate injections for total iopromide area is not more than 2.0%. Chromatograph the Related compound B standard solution, and measure the area of the peak responses: the relative retention times for the iopromide related compound B Y1- and Y2-isomers are about 0.28 and 0.31, respectively; and the signal-to-noise ratio for the iopromide related compound B Y2-isomer is not less than 20.

Procedure— [note—Use peak areas where peak responses are indicated.] Separately inject equal volumes (about 10 µL) of the Standard preparation, the Related compound B standard solution, and the Assay preparation into the chromatograph, and measure the responses for the major peaks. Allow the Mobile phase to flow for not less than 60 minutes between each injection to prevent interference from late-eluting amine peaks. Calculate the quantity of C18H24I3N3O8 in the portion of Iopromide taken by the formula: in which C is the concentration, in mg per mL, of USP Iopromide RS in the Standard preparation; and rU and rS are the sums of the peak responses for iopromide E1-isomer, iopromide E2-isomer, iopromide Z1-isomer, and iopromide Z2-isomer in the chromatograms obtained from the Assay preparation and the Standard preparation, respectively.