Medium: 0.1Nacetic acid;500mL.

Apparatus 1: 100rpm.

Time: 45minutes.

p-Toluenesulfonic acid solution— Dissolve 1g of p-toluenesulfonic acid in 100mLof glacial acetic acid.

Standard solution— Dissolve an accurately weighed quantity of USP Reserpine RSin glacial acetic acid,and dilute quantitatively,and stepwise if necessary,with glacial acetic acid to obtain a solution having a known concentration of about 0.1µg per mL.

Test solution— Pipet an aliquot of the filtered solution under test,containing about 11µg of reserpine,into a 125-mLseparatory funnel.Extract with three 10-mLportions of chloroform,collecting the extracts in a 100-mLvolumetric flask,dilute with glacial acetic acid to volume,and mix.

Procedure— Into three individual 50-mLtest tubes,pipet 10mLeach of the Standard solution,the Test solution,and glacial acetic acid to provide the blank.Treat each as follows.Add 10mLof p-Toluenesulfonic acid solution,insert a stopper,and mix gently.Place in a steam bath for 10minutes.Remove from the steam bath,and cool.Determine the amount of C33H40N2O9dissolved from the fluorescences of the Test solutionand the Standard solutionusing a suitable spectrophotometer arranged to deliver activation radiation at 390nm and to measure the resultant fluorescence at the emission wavelength of about 480nm.

Tolerances— Not less than 75%(Q)of the labeled amount of C33H40N2O9is dissolved in 45minutes.

Procedure for content uniformity—

Phosphoric acid-methanol solution— Dilute 20mLof phosphoric acid with 200mLof methanol,then dilute with water to 1000mL,and mix.

Vanadium pentoxide reagent— Prepare a saturated solution of vanadium pentoxide in phosphoric acid by shaking by mechanical means for 1hour 100mg of vanadium pentoxide with 100mLof phosphoric acid.Allow undissolved solids to settle overnight,and filter the supernatant through a medium-porosity,sintered-glass funnel.

Standard solution— Using an accurately weighed quantity of USP Reserpine RS,prepare a solution in Phosphoric acid-methanol solutionhaving a known concentration of about 0.002mg per mL.

Test preparation— Place 1Tablet in a suitable container,and add an accurately measured volume of Phosphoric acid-methanol solutionso that the concentration of the final solution is about 0.002mg of reserpine per mL.Shake by mechanical means until the tablet is completely disintegrated.Filter before using.

Procedure— With the Apparatusset as described for Automated Dissolution Test for Reserpine Tabletsunder Automated Methods of Analysis á16ñ,conduct determinations of one Standard per five Tablets.Calculate the quantity,in mg,of reserpine (C33H40N2O9)in the Tablet taken by the formula: in which Cis the concentration,in mg per mL,of USP Reserpine RSin the Standard solution;Dis the dilution factor for the Test preparation;and IUand ISare the fluorescence intensities obtained from the Test preparationand the Standard solution,respectively.

Adsorbent— Use acid-washed chromatographic siliceous earth.

Chromatographic tube— Select a chromatographic tube about 200mm long and about 22mm in internal diameter that is constricted to an outlet at the lower end.Insert at the constriction a small pledget of glass wool,previously washed with chloroform and air-dried.

Chromatographic column— Mix 1g of Adsorbentwith 0.5mLof freshly prepared sodium bicarbonate solution (1in 50)in a 100-mLbeaker until the mixture appears fluffy and uniformly moistened,transfer to the Chromatographic tube,and tamp lightly with a packing rod to a thickness of about 7to 9mm.Mix uniformly 1g of Adsorbentwith 0.5mLof freshly prepared citric acid solution (1in 200),transfer to the Chromatographic tube,and tamp lightly with a packing rod.Mix uniformly 1g of Adsorbentwith 0.5mLof water,transfer to the Chromatographic tube,and tamp lightly with a packing rod.

Blank mixture— Combine 1mLof dimethyl sulfoxide and 2g of Adsorbentin a suitable container,and stir thoroughly until the mass is uniformly wetted and free from lumps.

Blank solution— Transfer the Blank mixturethrough a powder funnel to a prepared Chromatographic column.Scrub the beaker with about 1g of Adsorbent,and add it through the funnel to the tube.Wipe the spatula,beaker,and funnel with a tuft of glass wool,previously washed with chloroform and air-dried.Place the glass wool in the tube,and press it down on the column with the packing rod,so that the overall height of the column is between 55mm and 65mm.Rinse the spatula,beaker,and funnel with the first portion of the chloroform used to elute the specimen.Elute the reserpine with 45mLof chloroform.[NOTE—Aproperly packed column elutes in 4to 8minutes.]Collect the eluate in a 50-mLvolumetric flask containing 14mLof methanol.Rinse the tip of the column with chloroform,add chloroform to volume,and mix.

Standard solution— [NOTE—Use actinic glassware for this solution.]Dissolve 25.0mg of USP Reserpine RS,accurately weighed,in 0.25mLof chloroform,and mix with about 30mLof methanol previously warmed to 50.Transfer the mixture to a 250-mLvolumetric flask with the aid of warm methanol,cool the solution to room temperature,dilute with methanol to volume,and mix.Just before use,pipet 10mLof this solution into a 50-mLvolumetric flask,add 36mLof chloroform,dilute with methanol to volume,and mix.

Test mixture— Weigh and finely powder,to pass a 60-mesh sieve,not fewer than 20Tablets.Weigh accurately a portion of the powder,equivalent to about 1mg of reserpine,but not more than 1g of the powder,and transfer to a 150-mLbeaker.Dry-mix the powder with about 500mg of Adsorbent,then mix with 1mLof dimethyl sulfoxide (immobile solvent).Stir thoroughly until the mass is uniformly wetted and free from lumps,and allow the mixture to stand for 5minutes.Add another 500mg of Adsorbent,and thoroughly work it into the mass.Again add an amount of Adsorbentso that the total amount added is 2g,and disperse it completely in the mass.

Test solution— Transfer the Test mixturethrough a powder funnel to a prepared Chromatographic column.Proceed as directed for Blank solutionbeginning with “Scrub the beaker with about 1g of Adsorbent.”

Procedure— Determine the UVabsorption spectrum of the Test solution,between 255and 350nm,using the Blank solutionin the reference cell.Similarly,determine the UVabsorption spectrum of the Standard solution,using a solution of 3.6volumes of chloroform and 1.4volumes of methanol as the blank.The two spectra are similar,and the ratio,A268/A295,for the Test solutiondoes not differ by more than 4.0%from the corresponding ratio for the Standard solution.Calculate the quantity,in mg,of reserpine (C33H40N2O9)in the portion of Tablets taken by the formula: in which AUand ASare the absorbances of the Test solutionand the Standard solution,respectively,at the absorption maximum at about 268nm.The result so obtained does not differ more than 6.0%from that obtained in the Assay.

Mobile phase,Standard preparation,andChromatographic system— Proceed as directed in the Assayunder Reserpine.

Assay preparation— Weigh and finely powder not fewer than 20Tablets.Transfer an accurately weighed quantity of the powder,equivalent to about 1mg of reserpine,to a 100-mLvolumetric flask.Add Mobile phaseto volume,and mix.Filter a portion through a 0.8-µm or finer membrane disk.

Procedure— Proceed as directed for Procedurein the Assayunder Reserpine,and calculate the quantity,in mg,of reserpine (C33H40N2O9)in the portion of Tablets taken by the formula: in which the terms are as defined therein.