Part I—

Spray reagent— Dissolve 300 mg of ninhydrin in a mixture of 100 mL of butyl alcohol and 3 mL of glacial acetic acid.

Standard preparation A— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of USP Trientine Hydrochloride RS in methanol to obtain a solution containing 10 mg per mL.

Standard preparation B— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of diethylenetriamine in methanol to obtain a solution containing 1.0 mg per mL. Transfer 3.0 mL of this solution to a 100-mL volumetric flask, dilute with methanol to volume, and mix.

Standard preparation C— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of 1-(2-aminoethyl)piperazine in methanol to obtain a solution containing 1.0 mg per mL. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with methanol to volume, and mix.

Standard preparation D— [NOTE—Use low-actinic glassware.] Transfer 5.0 mL of Standard preparation C to a 10-mL volumetric flask, dilute with methanol to volume, and mix.

Test preparation— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of Trientine Hydrochloride in methanol to obtain a solution containing 10 mg per mL.

Procedure— Apply separately 3 µL each of the Test preparation, of Standard preparation B, and of Standard preparation C to a suitable unwashed, high performance thin-layer chromatographic plate (see Chromatography 621) having a 1.5-cm preadsorbent zone and coated with a 0.15-mm layer of chromatographic silica gel mixture. To a fourth spot, apply 3 µL each of Standard preparations A, B, and C. To a fifth spot, apply 3 µL each of Standard preparations A, B, and D. Allow the spots to dry, place the plate in a chromatographic chamber, and develop the chromatograms in a solvent system consisting of a mixture of isopropyl alcohol and ammonium hydroxide (3:2) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and dry the plate with the aid of a current of air. Spray the plate with Spray reagent, dry at 105 for 5 minutes, and observe the plate under long-wavelength UV light. Determine the locus of the diethylenetriamine and the 1-(2-aminoethyl)piperazine spots from the chromatograms of Standard preparations B and C, respectively. Determine the concentration of diethylenetriamine in the Test preparation by comparing the size and intensity of any secondary spot from the chromatogram of the Test preparation having an RF value corresponding to the RF value of diethylenetriamine with the diethylenetriamine spots obtained from the chromatograms of the Standard preparation mixtures. Determine the concentration of any other observed impurities in the Test preparation by comparing the size and intensity of any other secondary spots from the chromatogram of the Test preparation with the 1-(2-aminoethyl)piperazine spots obtained from the chromatograms of the Standard preparation mixtures.

Part II—

Spray reagent— Dissolve 200 mg of ninhydrin in 100 mL of alcohol.

Tris (2-aminoethyl)amine stock solution—[NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of tris(2-aminoethyl)amine in methanol to obtain a solution containing 1.0 mg per mL.

Standard preparation A— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of USP Trientine Hydrochloride RS in methanol to obtain a solution containing 10 mg per mL.

Standard preparation B— [NOTE—Use low-actinic glassware.] Transfer 1.0 mL of Tris(2-aminoethyl)amine stock solution to a 10-mL volumetric flask, dilute with methanol to volume, and mix.

Standard preparation C— [NOTE—Use low-actinic glassware.] Transfer 0.5 mL of Tris(2-aminoethyl)amine stock solution to a 10-mL volumetric flask, dilute with methanol to volume, and mix.

Test preparation— [NOTE—Use low-actinic glassware.] Dissolve an accurately weighed quantity of Trientine Hydrochloride in methanol to obtain a solution containing 10 mg per mL.

Procedure— Apply separately 3 µL each of the Test preparation and of Standard preparation A to a suitable thin-layer chromatographic plate (see Chromatography 621) coated with a 0.25-mm layer of chromatographic silica gel mixture and previously washed with methanol. To a third spot apply 3 µL each of Standard preparations A and B. To a fourth spot, apply 3 µL each of Standard preparations A and C. Allow the spots to dry, place the plate in a chromatographic chamber, and develop the chromatograms in a solvent system consisting of a mixture of ammonium hydroxide and alcohol (2:1) at a temperature of 2 to 6 until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and dry the plate with the aid of a current of air. Spray the plate with Spray reagent, dry at 105 for 5 minutes, and observe the plate under long-wavelength UV light. Determine the concentration of tris(2-aminoethyl)amine in the Test preparation by comparing the size and intensity of any secondary spot from the chromatogram of the Test preparation having an RF value corresponding to the RF value of tris(2-aminoethyl)amine with the tris(2-aminoethyl)amine spots obtained from the chromatograms of the Standard preparation mixtures.

(Official January 1, 2007)