Identification—
Apply 10-µL portions of the
Assay preparation and the
Standard preparation prepared as directed in the
Assay to a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow to dry, develop the chromatogram in a solvent system consisting of equal volumes of acetone, butyl alcohol, glacial acetic acid, toluene, and water 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 allow the solvent to evaporate. Locate the spots by staining the plate with iodine vapor for about 50 minutes, then view the plate under short-wavelength UV light: the
RF value of the principal spot obtained from the
Assay preparation corresponds to that obtained from the
Standard preparation.
Limit of methyldopa-glucose reaction product [TO BE DETERMINED IF SUCROSE IS PRESENT]—
Mobile phase—
Prepare as directed in the Assay.
Standard solution—
Transfer about 25 mg of
USP Methyldopa RS, accurately weighed, to a 25-mL volumetric flask, add 1.0 mL of
Solution A, dilute with 0.1 N sulfuric acid to volume, and mix. The
Standard solution has a known concentration of about 18 µg of
USP Methyldopa-glucose Reaction Product RS per mL.
Test solution—
Prepare as directed for Assay preparation in the Assay.
Chromatographic system—
Use the system described under Chromatographic system in the Assay. The relative retention times for methyldopa and methyldopa-glucose reaction product are about 1.0 and 0.8, respectively. Chromatograph three replicate injections of the Standard solution: the resolution factor, R, between methyldopa and methyldopa-glucose reaction product is not less than 2.0. The relative standard deviations for three replicate injections of the Standard solution are not more than 2.0% and 3.0% for methyldopa and methyldopa-glucose reaction product, respectively.
Procedure—
Proceed as directed for
Procedure in the
Assay. Calculate the quantity, in µg, of methyldopa equivalent to the methyldopa-glucose reaction product in each mL of the Oral Suspension taken by the formula:
(211.22/373.35)(250)(CD/W)(rU / rS),
in which 211.22 and 373.35 are the molecular weights of anhydrous methyldopa and methyldopa-glucose reaction product, respectively;
C is the concentration, in µg per mL, of
USP Methyldopa-glucose Reaction Product RS in the
Standard solution; rU and
rS are the peak responses of the methyldopa-glucose reaction product obtained from the
Test solution and the
Standard solution, respectively; and the other terms are as defined therein. The limit is 10.0%, based on the methyldopa content of the Oral Suspension as determined in the
Assay.
Assay—
Mobile phase—
To 6.8 g of monobasic potassium phosphate add 750 mL of water, and stir until solution is complete. Adjust with 1 M phosphoric acid to a pH of 3.5, dilute with water to 1000 mL, mix, and pass through a filter having a 10-µm or finer porosity.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Methyldopa RS in 0.1 N sulfuric acid to obtain a solution having a known concentration of about 1 mg of anhydrous methyldopa per mL.
Assay preparation—
Transfer an accurately measured volume of Oral Suspension, freshly mixed, equivalent to about 250 mg of methyldopa, to a 250-mL volumetric flask, dilute with 0.1 N sulfuric acid to volume, and mix to dissolve the methyldopa. Pass the solution through a 0.45-µm membrane filter before using.
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 280-mm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph three replicate injections of the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 50 µL) of the
Standard preparation and the
Assay preparation into the chromatograph by means of a suitable microsyringe or sampling valve, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
10H
13NO
4 in each mL of the Oral Suspension taken by the formula:
250(C/V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Methyldopa RS in the
Standard preparation; V is the volume, in mL, of Oral Suspension taken; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
.