» Levodopa Capsules contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of levodopa (C9H11NO4).
Packaging and storage Preserve in tight, light-resistant containers, in a dry place, and prevent exposure to excessive heat.
USP Reference standards 11
USP Levodopa Related Compound A RS
Identification Shake a quantity of the contents of the Capsules, equivalent to about 500 mg of levodopa, with 25 mL of 3 N hydrochloric acid, and filter. Adjust the acidity of the filtrate with 6 N ammonium hydroxide to a pH of 3, added dropwise with stirring, and allow to stand, protected from light, for several hours. Filter, wash the precipitate with water, and dry at 105: the residue meets the requirements for Identification test A under Levodopa.
Medium: 0.01 N hydrochloric acid; 900 mL.
Apparatus 1: 100 rpm.
Time: 30 minutes.
Procedure Determine the amount of C9H11NO4 dissolved by employing UV absorption at the wavelength of maximum absorbance at about 280 nm on filtered portions of the solution under test, suitably diluted with Medium, in comparison with a Standard solution having a known concentration of USP Levodopa RS in the same Medium.
Tolerances Not less than 75% (Q) of the labeled amount of C9H11NO4 is dissolved in 30 minutes.
Uniformity of dosage units 905: meet the requirements.
Ferric chloridepotassium ferricyanide solution Just prior to use, mix 2 volumes of ferric chloride solution (1 in 10) with 1 volume of potassium ferricyanide solution (1 in 20) to obtain about 100 mL of solution.
Sodium metabisulfite solution Dissolve 100 mg of sodium metabisulfite in 10 mL of 1.2 N hydrochloric acid, and dilute with acetone to 100 mL.
Standard solution A [noteUse low-actinic glassware. ] Dissolve 2.5 mg of USP Levodopa Related Compound A RS in Sodium metabisulfite solution to make 25.0 mL. Pipet 1 mL of this solution into a 10-mL volumetric flask containing 100 mg of USP Levodopa RS, and dilute with Sodium metabisulfite solution to volume. Mix this solution just prior to application.
Standard solution B [noteUse low-actinic glassware. ] Dissolve 2.5 mg of USP 3-Methoxytyrosine RS in Sodium metabisulfite solution to make 25.0 mL. Pipet 5 mL of this solution into a 10-mL volumetric flask, and dilute with Sodium metabisulfite solution to volume. Mix this solution just prior to application.
Test solution [noteUse low-actinic glassware. ] Just prior to application, dissolve 100 mg of the residue obtained in the Identification test in 10.0 mL of Sodium metabisulfite solution.
Developing solvent Prepare a mixture of 150 mL of butyl alcohol, 75 mL of glacial acetic acid, 75 mL of water, and 15 mL of methanol.
Chromatographic plates Use suitable thin-layer chromatographic plates (see Chromatography 621) coated with a 0.25-mm layer of microcrystalline cellulose. Predevelop a plate in the Developing solvent until the solvent front has moved not less than 18 cm from the origin. Remove the plate from the chamber, and dry in a current of air for about 10 minutes. [noteThe plate may be developed overnight: solvent overflow during predevelopment is of no consequence. ]
Procedure Apply 10 µL each of the Test solution and Standard solutions A and B to separate points about 3 cm from the bottom of the plate. Dry the spots in a stream of nitrogen, and develop the chromatogram in a suitable low-actinic chamber equilibrated for 5 minutes with a freshly mixed portion of Developing solvent, until the solvent front has moved about 15 cm from the line of application. Remove the plate from the chamber, mark the solvent front, and dry in a current of air for about 10 minutes. Spray the plate with Ferric chloridepotassium ferricyanide solution. The levodopa related compound A produces a spot at an RF of about 0.25, and the 3-methoxytyrosine produces a spot at an RF of about 0.5. Any spots from the Test solution, occurring at RF values 0.25 and 0.5, are not greater in size or intensity than the spots at the respective RF values produced by Standard solutions A and B, corresponding to not more than 0.1% of levodopa related compound A and not more than 0.5% of 3-methoxytyrosine, respectively. (A bleached spot, which is an artifact resulting from the development of the Sodium metabisulfite solution, may appear at an RF value of about 0.6.)
Assay Weigh the contents of not fewer than 20 Capsules, mix, and transfer an accurately weighed portion of the powder, equivalent to about 175 mg of levodopa, to a 100-mL volumetric flask. Add 50 mL of 0.1 N hydrochloric acid, and shake the mixture by mechanical means for 5 minutes. Add 0.1 N hydrochloric acid to volume, mix, and filter, discarding the first 20 mL of the filtrate. Dilute 2.0 mL of the subsequent filtrate with 0.1 N hydrochloric acid to 100 mL. Concomitantly determine the absorbances of this solution and a similarly prepared Standard solution of USP Levodopa RS, having a known concentration of about 35 µg per mL, in 1-cm cells at the wavelength of maximum absorbance at about 280 nm with a suitable spectrophotometer, using 0.1 N hydrochloric acid as the blank. Calculate the quantity, in mg, of levodopa (C9H11NO4) in the portion of Capsule contents taken by the formula:
5C(AU / AS)in which C is the concentration, in µg per mL, of USP Levodopa RS in the Standard solution; and AU and AS are the absorbances of the solution from the Capsule contents and the Standard solution, respectively.
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USP35NF30 Page 3669Pharmacopeial Forum: Volume No. 29(3) Page 633