A: Infrared Absorption 197M.

B: Ultraviolet Absorption 197U—

C: To 10 mg add 0.15 mL of a solution of ninhydrin in sulfuric acid (1 in 250): a dark purple color is produced within 5 to 10 minutes. Add 0.15 mL of water: the color changes to pale brownish yellow.

Test solution: 44 mg per mL, in a solvent that is a solution of aluminum chloride in water (2 in 3) which previously has been treated with activated charcoal, filtered, and adjusted with 0.25 N sodium hydroxide to a pH of 1.5.

Developing solvent— Mix 65 parts by volume of butyl alcohol, 15 parts by volume of glacial acetic acid, and 25 parts by volume of water. Prepare this mixture fresh.

Chromatographic plate— Prepare a thin-layer chromatographic plate with a suitable grade of cellulose, 250 µm thick, prewashed with the Developing solvent. Wash the plate by placing it in a tank containing the solvent system and allowing the solvent to rise to the top of the plate. Dry with the aid of a current of dry air.

Spray solution 1— Dissolve 300 mg of p-nitroaniline in 100 mL of 10 N hydrochloric acid (Solution A). Dissolve 2.5 g of sodium nitrite in 50 mL of water (Solution B). Mix 90 mL of Solution A and 10 mL of Solution B (Spray solution 1). Prepare all solutions fresh, just before spraying.

Spray solution 2— Dissolve 25 g of sodium carbonate in 100 mL of water, and mix.

Test solution— Dissolve 100 mg of Methyldopa in methanol, and dilute with methanol to 10.0 mL.

Standard solution— Dissolve 5.0 mg of USP 3-O-Methylmethyldopa RS in methanol, and dilute with methanol to 50.0 mL to obtain a Standard solution having a known concentration of 100 µg per mL.

Procedure— Apply 20 µL of the Test solution in two 10-µL increments and 10 µL of the Standard solution to the Chromatographic plate, so that the spots are not larger than 0.5 cm in diameter. Develop the chromatogram using the Developing solvent until the solvent front has moved about 10 cm from the origin. Remove the plate from the chamber, and dry with the aid of a current of dry air until no odor of acetic acid is perceptible. Place the plate in a vertical position, and evenly spray with Spray solution 1 until the adsorbent layer is uniformly soaked down to the glass (do not overspray). Place the plate in a horizontal position, and dry as completely as possible with the aid of a current of warm dry air (no odor of hydrochloric acid is perceptible). Place the plate in a vertical position, and evenly spray with Spray solution 2 until the plate is uniformly wet (do not overspray). The major methyldopa spot is black on a pale pink or orange background at an RF value of about 0.50, and the 3-O-methylmethyldopa spot is dark on a similar background at an RF value of about 0.65. The area and intensity of any 3-O-methylmethyldopa spot from the Test solution are not greater than those from the Standard solution (0.5%).