Identification—
Transfer the finely ground contents of 1Tablet to a test tube,add 10mLof dilute alcohol (1in 2),shake for 5minutes,and centrifuge.Use the clear supernatant as the Test solution.Prepare a solution of alcohol and 0.1Nsodium hydroxide (1:1)containing in each mLabout 10mg of
USP Methyldopa RSand 10mg of
USP Chlorothiazide RS.Apply 20µLof the Test solution on a line parallel to and about 2cm from the bottom edge of a 20-cm ×10-cm thin-layer chromatographic plate (see
Chromatography á621ñ)coated with chromatographic silica gel mixture,and apply 20µLof the Standard solution separately on the starting line.Allow the spots to dry,develop the chromatogram in a solvent system consisting of equal volumes of glacial acetic acid,acetone,butyl alcohol,toluene,and water until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing tank,and allow the solvent to evaporate.View the plate under short-wavelength UVlight:the solution under test exhibits two major spots having
RFvalues corresponding to those of the two major spots obtained with the Standard solution.
Dissolution á711ñ—
PROCEDURE FOR METHYLDOPA
—
Medium:
0.1Nhydrochloric acid;900mL.
Apparatus 2:
75rpm.
Time:
30minutes.
Standard preparation—
Dissolve an accurately weighed quantity of
USP Methyldopa RSin
Dissolution Medium,and dilute quantitatively with the same solvent to obtain a solution having a known concentration of about 275µg of anhydrous methyldopa per mL.
Ferrous tartrate solution—
Dissolve 1g of ferrous sulfate,2g of potassium sodium tartrate,and 100mg of sodium bisulfite in water to make 100mL,and mix.Use a freshly prepared solution.
Buffer solution—
Dissolve 50g of ammonium acetate in 1000mLof dilute alcohol (1in 5).Adjust with 6Nammonium hydroxide to a pHof 8.5.
Procedure—
Filter 35mLof the solution under test,and transfer an aliquot estimated to contain between 2mg and 3mg of methyldopa to a 100-mLvolumetric flask.Adjust the final volume,if necessary,with
Dissolution Mediumto 10mL.To a second 100-mLvolumetric flask add 10.0mLof
Standard preparation,and to a third 100-mLvolumetric flask add 10.0mLof
Dissolution Mediumto provide a blank.Pipet 5.0mLof
Ferrous tartrate solutioninto each flask,dilute with
Buffer solutionto volume,and mix.Concomitantly determine the absorbances of the treated
Standard preparationand test solution at the wavelength of maximum absorbance at about 520nm,with a suitable spectrophotometer,against the reagent blank.Calculate the amount of C
10H
13NO
4dissolved,in mg,taken by the formula:
9(C/V)(AU/AS),
in which
Cis the concentration,in µg of anhydrous methyldopa per mL,of
USP Methyldopa RSin the
Standard preparation;
Vis the volume,in mL,of the aliquot of test solution used;and
AUand
ASare the absorbances of the solutions from the test solution and the
Standard preparation,respectively.
Tolerances—
Not less than 80%(Q)of the labeled amount of C10H13NO4is dissolved in 30minutes.
PROCEDURE FOR CHLOROTHIAZIDE
—
Apparatus 2:
75rpm.
Time:
60minutes.
Procedure—
Determine the amount of C
7H
6ClN
3O
4S
2dissolved from UVabsorbances of the solution under test,suitably diluted with
Dissolution Medium,if necessary,at the wavelength of maximum absorbance at about 317nm in comparison with a Standard solution having a known concentration of
USP Chlorothiazide RSin the same
Medium.
Tolerances—
Not less than 75%(Q)of the labeled amount of C7H6ClN3O4S2is dissolved in 60minutes.
Assay—
Mobile phase—
Prepare a filtered and degassed mixture of 0.08
Mmonobasic sodium phosphate and methanol (95:5).Adjust by the addition of phosphoric acid to a pHof 2.8.Make adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Transfer to a 100-mLvolumetric flask accurately weighed quantities of
USP Methyldopa RSand
USP Chlorothiazide RS,equivalent to one-fifth of their labeled amounts,in mg,per Tablet.Add 15mLof water and 5mLof 1Nhydrochloric acid,and sonicate for about 3minutes.Add 10mLof acetonitrile,and sonicate for 2minutes.Dilute with water to volume,and mix.
Assay preparation—
Weigh and finely powder not less than 10Tablets.Transfer an accurately weighed portion of the powder,equivalent to the weight of 1Tablet,to a 500-mLvolumetric flask.Add 75mLof water and 25mLof 1Nhydrochloric acid,and sonicate for about 5minutes.Add 50mLof acetonitrile,and sonicate for 10minutes.Dilute with water to volume,and mix.Filter through a 0.45-to 2.0-µm membrane filter,discarding the first 10mL.
Chromatographic system
(see
Chromatography á621ñ)—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 2mLper minute.Chromatograph the
Standard preparation,and record the peak responses as directed under
Procedure:the column efficiency determined from the chlorothiazide peak is not less than 1300theoretical plates,the tailing factor for chlorothiazide peak is not more than 2,the resolution,
R,between the chlorothiazide and methyldopa peaks is not less than 7,and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 1.0for methyldopa and 2.5for chlorothiazide.Calculate the quantity,in mg,of chlorothiazide (C
6H
6ClN
3O
4S
2)in the portion of Tablets taken by the formula:
500C(rU/rS),
in which
Cis the concentration,in mg per mL,of
USP Chlorothiazide RSin the
Standard preparation;and
rUand
rSare the peak responses of the chlorothiazide peak obtained from the
Assay preparationand the
Standard preparation,respectively.Calculate the quantity,in mg,of methyldopa (C
10H
13NO
4)taken by the same formula,reading “methyldopa”instead of “chlorothiazide.”