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.

B: The lanthanide selectivity test detects gadolinium (III) in 0.1 N nitric acid with arsenazo (III). Prepare 1.5 × 10–4 M arsenazo (III) solution by dissolving 30 mg of arsenazo (III) and 160 mg of urea in 100 mL water, adding 1.6 mL of nitric acid, and diluting with water to 250 mL. Add 10 mg of Gadoversetamide to 1.0 mL of the 1.5 × 10–4 M arsenazo (III) solution, and mix. The color changes from a wine red to green-blue, indicating the presence of gadolinium.

MES buffer— Dissolve 97.6 g of 2-morpholinoethanesulfonic acid (MES) in about 950 mL of water, and mix. Adjust with 20% sodium hydroxide to a pH of 6, dilute with water to 1000 mL, and mix.

Edetate titrant: 0.002 M edetate disodium VS.

0.003 M Gadolinium (III) titrant— Transfer 0.790 g of gadolinium chloride to a 1000-mL volumetric flask, dilute with water to volume, and mix.

Test solution— Transfer about 5 g of Gadoversetamide, accurately weighed, into a 250-mL flask, and add 20 mL of water and 2 mL of hydrochloric acid. Stir, and heat to boiling. Rinse the sides of the flask with water. Add 50 mL of MES buffer and 100 to 150 µL of xylenol orange TS to impart a light yellow color. Heat to boiling, adjust with ammonium hydroxide to a pH of 6, and continue boiling for 2 minutes. If the solution is yellow, proceed as directed under Uncomplexed chelatable material. If the color is red-violet, proceed as directed under Free gadolinium (III).

Uncomplexed chelatable material— Continue boiling, and titrate with 0.003 M Gadolinium (III) titrant to a red-violet endpoint that undergoes no further color change upon addition of more titrant. Record the volume of titrant used to reach the endpoint.

Free gadolinium (III)— Continue boiling and titrate with Edetate titrant to a yellow or yellow-orange endpoint that undergoes no further color change upon addition of more titrant. Record the volume of titrant used to reach the endpoint.

Calculations— If the Test solution was titrated with Edetate titrant, calculate the percentage of Free gadolinium (III) in the portion of Gadoversetamide taken by the formula: in which W is the weight, in g, of Gadoversetamide taken; VEU is the volume, in mL, of Edetate titrant used to titrate the Test solution; and ME is the molarity of the Edetate titrant. If the sample was titrated with 0.003 M Gadolinium (III) titrant, calculate the percentage of Uncomplexed chelatable material in the portion of Gadoversetamide taken by the formula: in which W is as defined herein; VGU is the volume, in mL, of 0.003 M Gadolinium (III) titrant used to titrate the Test solution; and MG is the molarity of the 0.003 M Gadolinium (III) titrant. Not more than 0.05% of free gadolinium III and not more than 0.1% of uncomplexed chelatable material, both calculated on the anydrous basis, are found.

Mobile phase— Add 2 mL of 5 M phosphoric acid to 550 mL of water, mix, and adjust with 10% (w/w) ammonium hydroxide to a pH of 5.0. Add 450 mL of acetonitrile, mix, filter, and degas.

0.4 M Borate buffer— Add 12.4 g of boric acid to 300 mL of water, and swirl to suspend. Add 100 mL of 1 N potassium hydroxide, and mix. Adjust with about 60 mL of 1 N potassium hydroxide to a pH of 10.0, dilute with water to 500 mL, and mix. Test the pH, and adjust if necessary.

o-Phthalaldehyde reagent— Dissolve 25 mg of o-phthalaldehyde in 0.75 mL of methanol, add 25 mL of 0.4 M Borate buffer having a pH of 10.0 and 25 µL of 2-mercaptoethanol, and mix. [note—Protect from light. Discard after 3 days.]

Standard solutions— Prepare aqueous solutions of 2-methoxyethylamine having known concentrations of about 1, 20, and 50 µg per mL, respectively. Derivatize by adding an equal volume of o-Phthalaldehyde reagent to each solution immediately before injection.

Test solution— Transfer about 250 mg of Gadoversetamide, accurately weighed, to a 5-mL volumetric flask, and dissolve in and dilute with water to volume. Derivatize the solution by combining equal volumes of o-Phthalaldehyde reagent and Test solution immediately before injection.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 335-nm detector and a 250-mm × 4.6-mm column that contains 5-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard solutions, and record the chromatograms as directed for Procedure: the relative retention times of o-phthalaldehyde and 2-methoxyethylamine are about 0.6 and 1.0, respectively. Plot the concentration of 2-methoxyethylamine in each Standard solution versus its peak area, and perform a regression analysis to obtain a slope and an intercept. The correlation coefficient, r, is not less than 0.995, and the relative standard deviation for replicate injections of the 50 µg per mL Standard solution is not more than 5%.

Procedure— Separately inject equal volumes (about 50 µL) of the Test solution and the Standard solutions into the chromatograph, record the chromatograms, and measure the peak responses. Determine the concentration, in µg per mL, of 2-methoxyethylamine in the Test solution from the standard response line. Calculate the percentage of 2-methoxyethylamine by the formula: in which C is the concentration, in µg per mL, of 2-methoxyethylamine obtained from the Standard response line; and W is the weight, in mg, of Gadoversetamide taken. Not more than 0.10% (w/w) of 2-methoxyethylamine is present, calculated on the anhydrous basis.

Internal standard solution— Dilute butyl alcohol with water (3:5000).

Standard solutions— To four separate 5-mL volumetric flasks, transfer the following designated compositions: Dilute each flask with water to volume, and mix. The resulting Standard solutions contain about 5, 20, 50, and 100 µg of isopropyl alcohol and acetonitrile per mL.

Test solution— Transfer about 500 mg of Gadoversetamide, accurately weighed, to a 5-mL volumetric flask. Add 1.0 mL of Internal standard solution, dissolve in and dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The gas chromatograph is equipped with a flame-ionization detector and a 0.53-mm × 30-m capillary column with a 1.0-µm thickness of phase G35. Helium is used as the carrier gas, at a flow rate of about 5 mL per minute. The column temperature is maintained at 35 for 5 minutes, then increased at a rate of 15 per minute to 110. The injection port temperature is maintained at 150, and the detector temperature is maintained at 300. Chromatograph the Standard solutions, and record the peak area ratios as directed for Procedure: the relative retention times are about 0.5 for isopropyl alcohol, 0.7 for acetonitrile, and 1.0 for butyl alcohol. Plot the concentration for each standard versus its peak area ratio, and perform a regression analysis. The correlation coefficient, r, is not less than 0.995 for each analyte; and the relative standard deviation for replicate injections of the 100 µg per mL Standard solution is not more than 5%.

Procedure— Separately inject equal volumes (about 2 µL) of the Test solution and the Standard solutions into the chromatograph, record the chromatograms, and measure the peak area ratios of the standard peak to the internal standard peak. Determine the concentration of isopropyl alcohol and acetonitrile from the respective standard response lines. Calculate the percentage (w/w) of each solvent in the portion of Gadoversetamide taken by the formula: in which C is the concentration, in µg per mL (obtained from the respective standard response line) of isopropyl alcohol and acetonitrile in the Test solution; and W is the weight (anhydrous), in mg, of the portion of Gadoversetamide taken: not more than 0.1% (w/w) of isopropyl alcohol is found; and not more than 0.025% (w/w) of acetonitrile is found. The total residual solvent content (sum of the % w/w isopropyl alcohol and the % w/w acetonitrile) does not exceed 0.1% w/w.

Solution A— Dissolve 2.06 g of monobasic potassium phosphate and 18.6 mL of 20% w/w tetraethylammonium hydroxide in 950 mL of water. Adjust with phosphoric acid to a pH of 7, dilute with water to make 1000 mL, mix, filter, and degas.

Solution B— Prepare a filtered and degassed mixture of Solution A and acetonitrile (475:25).

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

Standard stock solution— Transfer about 50 mg each of USP Gadoversetamide Related Compound A RS and USP Gadodiamide Related Compound B RS, accurately weighed, to a 50-mL volumetric flask, dissolve in and dilute with water to volume, and mix. [note—The solution may be stored for a week.]

Standard solutions— Prepare aqueous solutions of diluted Standard stock solution containing about 25, 150, and 250 µg of each Reference Standard per mL.

Test solution— Transfer about 250 mg of Gadoversetamide, accurately weighed, to a 10-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 205-nm detector and a metal-free 4.6- × 150-mm column that contains 5-µm packing L1. The flow rate is 1 mL per minute. The chromatograph is programmed to pump a mixture of Solution A to Solution B (97:3). The column temperature is maintained at about 25. The relative retention times are about 0.6 for gadodiamide related compound B and 0.7 for gadoversetamide related compound A; the resolution, R, between gadodiamide related compound B and gadoversetamide related compound A is not less than 1.0; and the relative standard deviation for replicate injections is not more than 5% for the 250 µg per mL Standard solution.

Procedure— Separately inject equal volumes (about 50 µL) of the Test solution and the Standard solutions into the chromatograph. Allow 1 hour between injections to remove slow-eluting impurities. Determine the quantities, in µg per mL, of gadoversetamide related compound A and gadodiamide related compound B from the respective Standard response lines. Calculate the percentage of gadoversetamide related compound A in the portion of Gadoversetamide taken by the formula: in which C is the concentration of gadoversetamide related compound A, in µg per mL, obtained from the Standard response line; and V is the concentration of gadoversetamide, in µg per mL, in the Test solution. Not more than 1.0% (w/w) of gadoversetamide related compound A is found, calculated on the anhydrous basis. Calculate the percentage of gadodiamide related compound B in the portion of Gadoversetamide taken by the following formula: in which C is the concentration of gadodiamide related compound B, in µg per mL, obtained from the Standard response line; and V is as described herein. Not more than 0.5% (w/w) of gadodiamide related compound B is found, calculated on the anhydrous basis.

Mobile phase— Dissolve 1.5 g of boric acid in about 950 mL of water, and mix. Adjust with ammonium hydroxide to a pH of 6.8, add 15 mL of acetonitrile, dilute with water to make 1000 mL, mix, filter, and degas.

Standard preparations— Prepare solutions of USP Gadoversetamide RS in Mobile phase having known concentrations of about 1.2, 1.0, and 0.8 mg per mL.

Assay preparation— Transfer about 100 mg of Gadoversetamide, 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 205-nm detector and a metal-free 4.6- × 250-mm column that contains 5-µm packing L1. The column temperature is maintained at about 50. The flow rate is about 1 mL per minute. Chromatograph the Standard preparations, and record the peak responses as directed for Procedure. Plot the concentration of each Standard versus its peak area and perform a regression analysis to obtain a slope and intercept for the Standard response line. The correlation coefficient, r, is not less than 0.995; and the relative standard deviation for replicate injections of the 1.0 mg per mL Standard preparation is not more than 2%.

Procedure— Separately inject equal volumes (about 10 µL) of the Assay preparation and Standard preparations into the chromatograph, record the chromatograms, and measure the area of the gadoversetamide peak. Determine the quantity, in mg per mL, of gadoversetamide from the Standard response line. Calculate the quantity, in % (w/w), of C20H34GdN5O10 in the portion of Gadoversetamide taken by the formula: in which C is the concentration, in mg per mL, obtained from the Standard response line; and W is the weight (anhydrous), in mg, of the portion of Gadoversetamide taken to prepare the Assay preparation.