»Levodopa Capsules contain not less than 90.0percent and not more than 110.0percent 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 á11ñ USP Levodopa RS.USP Levodopa Related Compound A RS.USP3-Methoxytyrosine RS.
Identification Shake a quantity of the contents of the Capsules,equivalent to about 500mg of levodopa,with 25mLof 3Nhydrochloric acid,and filter.Adjust the acidity of the filtrate with 6Nammonium hydroxide to a pHof 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 Identificationtest Aunder Levodopa.
Medium: 0.01Nhydrochloric acid;900mL.
Apparatus 1: 100rpm.
Procedure Determine the amount of C9H11NO4dissolved by employing UVabsorption at the wavelength of maximum absorbance at about 280nm 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 RSin the same Medium.
Tolerances Not less than 75%(Q)of the labeled amount of C9H11NO4is dissolved in 30minutes.
Uniformity of dosage units á905ñ: meet the requirements.
Ferric chloridepotassium ferricyanide solution Just prior to use,mix 2volumes of ferric chloride solution (1in 10)with 1volume of potassium ferricyanide solution (1in 20)to obtain about 100mLof solution.
Sodium metabisulfite solution Dissolve 100mg of sodium metabisulfite in 10mLof 1.2Nhydrochloric acid,and dilute with acetone to 100mL.
Standard solution A [NOTEUse low-actinic glassware.]Dissolve 2.5mg of USP Levodopa Related Compound A RSin Sodium metabisulfite solution to make 25.0mL.Pipet 1mLof this solution into a 10-mLvolumetric flask containing 100mg 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 2.5mg of USP3-Methoxytyrosine RSin Sodium metabisulfite solution to make 25.0mL.Pipet 5mLof this solution into a 10-mLvolumetric 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 100mg of the residue obtained in the Identification test in 10.0mLof Sodium metabisulfite solution.
Developing solvent Prepare a mixture of 150mLof butyl alcohol,75mLof glacial acetic acid,75mLof water,and 15mLof methanol.
Chromatographic plates Use suitable thin-layer chromatographic plates (see Chromatography á621ñ)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 18cm from the origin.Remove the plate from the chamber,and dry in a current of air for about 10minutes.[NOTEThe plate may be developed overnight:solvent overflow during predevelopment is of no consequence.]
Procedure Apply 10µLeach of the Test solution and Standard solutions Aand Bto separate points about 3cm 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 5minutes with a freshly mixed portion of Developing solvent,until the solvent front has moved about 15cm from the line of application.Remove the plate from the chamber,mark the solvent front,and dry in a current of air for about 10minutes.Spray the plate with Ferric chloridepotassium ferricyanide solution.The levodopa related compound Aproduces a spot at an RFof about 0.25,and the 3-methoxytyrosine produces a spot at an RFof about 0.5.Any spots from the Test solution,occuring at RFvalues 0.25and 0.5,are not greater in size or intensity than the spots at the respective RFvalues produced by Standard solutions Aand B,corresponding to not more than 0.1%of levodopa related compound Aand not more than 0.5%of 3-methoxytyrosine,respectively.(Ableached spot,which is an artifact resulting from the development of the Sodium metabisulfite solution,may appear at an RFvalue of about 0.6.)
Assay Weigh the contents of not fewer than 20Capsules,mix,and transfer an accurately weighed portion of the powder,equivalent to about 175mg of levodopa,to a 100-mLvolumetric flask.Add 50mLof 0.1Nhydrochloric acid,and shake the mixture by mechanical means for 5minutes.Add 0.1Nhydrochloric acid to volume,mix,and filter,discarding the first 20mLof the filtrate.Dilute 2.0mLof the subsequent filtrate with 0.1Nhydrochloric acid to 100mL.Concomitantly determine the absorbances of this solution and a similarly prepared Standard solution of USP Levodopa RS,having a known concentration of about 35µg per mL,in 1-cm cells at the wavelength of maximum absorbance at about 280nm with a suitable spectrophotometer,using 0.1Nhydrochloric 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 Cis the concentration,in µg per mL,of USP Levodopa RSin the Standard solution;and AUand ASare the absorbances of the solution from the Capsule contents and the Standard solution,respectively.
Auxiliary Information Staff Liaison:Salvador C.Salado,M.S.,Scientist and Latin American Liaison
Expert Committee:(PA3)Pharmaceutical Analysis 3
USP28NF23Page 1122Pharmacopeial Forum:Volume No.29(3)Page 633