» Reserpine Tablets contain not less than 90.0 percent and not more than 110.0 percent of the labeled amount of reserpine (C33H40N2O9).
Packaging and storage Preserve in tight, light-resistant containers.
USP Reference standards 11
Identification Evaporate about 2 mL of the Test solution, obtained from the test for Other alkaloids, in a test tube to dryness, add to the residue 0.5 mL of the glacial acetic acid, swirl for 1 to 2 minutes, and add 1 mL of a 1 in 50 solution of vanillin in hydrochloric acid: a pink color is produced, and it becomes deep violet-red within a few minutes or as a result of warming the solution for 10 to 20 seconds.
Dissolution 711 [noteDo not substitute membrane filters for filter paper where the filtration of reserpine-containing solutions is indicated. Reserpine has been shown to be adsorbed onto membranes. ]
Medium: 0.1 N acetic acid; 500 mL.
Apparatus 1: 100 rpm.
Time: 45 minutes.
p-Toluenesulfonic acid solution Dissolve 1 g of p-toluenesulfonic acid in 100 mL of glacial acetic acid.
Standard solution Dissolve an accurately weighed quantity of USP Reserpine RS in glacial acetic acid, and dilute quantitatively, and stepwise if necessary, with glacial acetic acid to obtain a solution having a known concentration of about 0.1 µg per mL.
Test solution Pipet an aliquot of the filtered solution under test, containing about 11 µg of reserpine, into a 125-mL separatory funnel. Extract with three 10-mL portions of chloroform, collecting the extracts in a 100-mL volumetric flask, dilute with glacial acetic acid to volume, and mix.
Procedure Into three individual 50-mL test tubes, pipet 10 mL each of the Standard solution, the Test solution, and glacial acetic acid to provide the blank. Treat each as follows. Add 10 mL of p-Toluenesulfonic acid solution, insert a stopper, and mix gently. Place in a steam bath for 10 minutes. Remove from the steam bath, and cool. Determine the amount of C33H40N2O9 dissolved from the fluorescences of the Test solution and the Standard solution using a suitable spectrophotometer arranged to deliver activation radiation at 390 nm and to measure the resultant fluorescence at the emission wavelength of about 480 nm.
Tolerances Not less than 75% (Q) of the labeled amount of C33H40N2O9 is dissolved in 45 minutes.
Uniformity of dosage units 905: meet the requirements.
Adsorbent Use acid-washed chromatographic siliceous earth.
Chromatographic tube Select a chromatographic tube about 200 mm long and about 22 mm 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 1 g of Adsorbent with 0.5 mL of freshly prepared sodium bicarbonate solution (1 in 50) in a 100-mL beaker 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 7 to 9 mm. Mix uniformly 1 g of Adsorbent with 0.5 mL of freshly prepared citric acid solution (1 in 200), transfer to the Chromatographic tube, and tamp lightly with a packing rod. Mix uniformly 1 g of Adsorbent with 0.5 mL of water, transfer to the Chromatographic tube, and tamp lightly with a packing rod.
Blank mixture Combine 1 mL of dimethyl sulfoxide and 2 g of Adsorbent in a suitable container, and stir thoroughly until the mass is uniformly wetted and free from lumps.
Blank solution Transfer the Blank mixture through a powder funnel to a prepared Chromatographic column. Scrub the beaker with about 1 g 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 55 mm and 65 mm. Rinse the spatula, beaker, and funnel with the first portion of the chloroform used to elute the specimen. Elute the reserpine with 45 mL of chloroform. [noteA properly packed column elutes in 4 to 8 minutes. ] Collect the eluate in a 50-mL volumetric flask containing 14 mL of methanol. Rinse the tip of the column with chloroform, add chloroform to volume, and mix.
Standard solution [noteUse actinic glassware for this solution. ] Dissolve 25.0 mg of USP Reserpine RS, accurately weighed, in 0.25 mL of chloroform, and mix with about 30 mL of methanol previously warmed to 50. Transfer the mixture to a 250-mL volumetric flask with the aid of warm methanol, cool the solution to room temperature, dilute with methanol to volume, and mix. Just before use, pipet 10 mL of this solution into a 50-mL volumetric flask, add 36 mL of chloroform, dilute with methanol to volume, and mix.
Test mixture Weigh and finely powder, to pass a 60-mesh sieve, not fewer than 20 Tablets. Weigh accurately a portion of the powder, equivalent to about 1 mg of reserpine, but not more than 1 g of the powder, and transfer to a 150-mL beaker. Dry-mix the powder with about 500 mg of Adsorbent, then mix with 1 mL of dimethyl sulfoxide (immobile solvent). Stir thoroughly until the mass is uniformly wetted and free from lumps, and allow the mixture to stand for 5 minutes. Add another 500 mg of Adsorbent, and thoroughly work it into the mass. Again add an amount of Adsorbent so that the total amount added is 2 g, and disperse it completely in the mass.
Test solution Transfer the Test mixture through a powder funnel to a prepared Chromatographic column. Proceed as directed for Blank solution beginning with Scrub the beaker with about 1 g of Adsorbent.
Procedure Determine the UV absorption spectrum of the Test solution, between 255 and 350 nm, using the Blank solution in the reference cell. Similarly, determine the UV absorption spectrum of the Standard solution, using a solution of 3.6 volumes of chloroform and 1.4 volumes of methanol as the blank. The two spectra are similar, and the ratio, A268/A295, for the Test solution does 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:
AU / ASin which AU and AS are the absorbances of the Test solution and the Standard solution, respectively, at the absorption maximum at about 268 nm. The result so obtained does not differ more than 6.0% from that obtained in the Assay.
Mobile phase, Standard preparation, and Chromatographic system Proceed as directed in the Assay under Reserpine.
Assay preparation Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed quantity of the powder, equivalent to about 1 mg of reserpine, to a 100-mL volumetric flask. Add Mobile phase to volume, and mix. Filter a portion through a 0.8-µm or finer membrane disk.
Procedure Proceed as directed for Procedure in the Assay under Reserpine, and calculate the quantity, in mg, of reserpine (C33H40N2O9) in the portion of Tablets taken by the formula:
0.1C(rU / rS)in which the terms are as defined therein.
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USP35NF30 Page 4534Pharmacopeial Forum: Volume No. 33(3) Page 453