A: Infrared Absorption 197M.

B: 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.

Test solution: 10 mg per mL, in aluminum chloride solution (2 in 3) that has been filtered and then adjusted with 0.25 N sodium hydroxide to a pH of 1.5.

Mobile phase, System suitability solution, Standard preparation, Assay preparation, and Chromatographic system— Proceed as directed in the Assay.

Impurity standard preparation— Dissolve accurately weighed quantities of USP Methyldopa RS and USP Carbidopa Related Compound A RS in Mobile phase to obtain a solution having a known concentration of about 2.5 µg of each per mL.

Procedure— Separately inject equal volumes (about 20 µL) of the Impurity standard preparation and the Assay preparation into the chromatograph by means of a suitable sampling valve, and measure the peak responses. The relative retention times of methyldopa, carbidopa, and carbidopa related compound A are about 0.8, 1.0, and 1.8, respectively. Calculate the percentage of methyldopa in the portion of Carbidopa taken by the formula: in which C is the concentration, in µg per mL, of USP Methyldopa RS in the Impurity standard preparation; W is the weight, in mg, of Carbidopa taken for the Assay preparation; and rU and rS are the peak responses for methyldopa obtained from the Assay and the Impurity standard preparation, respectively. The limit is 0.5%. Calculate the percentage of carbidopa related compound A in the portion of Carbidopa taken by the formula: in which C is the concentration, in µg per mL, of USP Carbidopa Related Compound A RS in the Impurity standard preparation; W is the weight, in mg, of carbidopa taken for the Assay preparation; and rU and rS are the peak responses for carbidopa related compound A obtained from the Assay preparation and the Impurity standard preparation, respectively. The limit is 0.5%.

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of 0.05 M monobasic sodium phosphate, adjusted with phosphoric acid to a pH of 2.7, and alcohol (95:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

System suitability solution— Prepare a solution in Mobile phase containing, in each mL, 0.1 mg of USP Carbidopa RS and 0.1 mg of USP Methyldopa RS.

Standard preparation— Dissolve an accurately weighed quantity of USP Carbidopa RS in Mobile phase to obtain a solution having a known concentration of about 0.5 mg per mL, using gentle heat and ultrasonification, if necessary, to aid dissolution.

Assay preparation— Transfer about 50 mg of Carbidopa, accurately weighed, to a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.8 for methyldopa and 1.0 for carbidopa; and the resolution, R, between methyldopa and carbidopa is not less than 0.9. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation is not more than 1.5%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C10H14N2O4·H2O in the portion of Carbidopa taken by the formula: in which C is the concentration, in mg per mL, of USP Carbidopa RS, as the monohydrate, in the Standard preparation, and rU and rS are the responses of the major peaks obtained from the Assay preparation and the Standard preparation, respectively.