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Literature Content === |
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 test method for St.John's Wort Extract |
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Origin: USP |
Show: 299 |
Time: 2009-2-5 22:50:40 |
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Powdered St.John's Wort Extract
Identification¡ª
A:Thin-Layer Chromatographic Identification Test ¨¢201ñ(presence of hypericin,pseudohypericin,hyperoside,and rutin)¡ª
Test solution¡ª Transfer about 1g of Powdered Extract,accurately weighed,to a 50-mLflask,add 20.0mLof methanol,shake well,and use the clear supernatant.
Standard solution¡ª Transfer about 0.25g of USP Powdered St.John's Wort Extract RS,accurately weighed,to a 25-mLflask,add 5.0mLof methanol,shake well,and use the clear supernatant.
Developing solvent system¡ª Prepare a mixture of ethyl acetate,water,glacial acetic acid,and formic acid (10:2.6:1.1:1.1),and use the upper phase of the mixture.[NOTE¡ªSaturate the chromatographic chamber with the Developing solvent systemvapors prior to the development of the chromatogram.]
Spray reagent A¡ª Prepare a solution of diphenylborinic acid,ethanolamine ester in methanol containing 10mg per mL.
Spray reagent B¡ª Prepare a solution of polyethylene glycol 400in alcohol containing 50mg per mL.
Procedure¡ª Develop the chromatogram until the solvent front has moved not less than 18cm,and dry the plate with the aid of a current of air.Spray the plate withSpray reagent A,then withSpray reagent B,and examine the plate under UVlight at 365nm:the two red zones due to hypericin and pseudohypericin atRFvalues of about 0.85and 0.80,respectively,in the chromatogram of theTest solution,correspond in color andRFvalue to those in the chromatogram of theStandard solution;the two yellow zones due to hyperoside and rutin atRFvalues of about 0.50and 0.35,respectively,in the chromatogram of theTest solution,correspond in color andRFvalue to those in the chromatogram of theStandard solution.Other colored zones of varying intensities may be observed in the chromatogram of theTest solution.
B:Thin-Layer Chromatographic Identification Test ¨¢201ñ(presence of hyperforin)¡ª
Test solution and Standard solution¡ª Proceed as directed forIdentificationtestA.
Developing solvent system¡ª Prepare a mixture of solvent hexane and ethyl acetate (8:2).[NOTE¡ªSaturate the chromatographic chamber with theDeveloping solvent systemvapors prior to the development of the chromatogram.]
Spray reagent¡ª Prepare a solution containing 0.38g of ceric ammonium sulfate and 3.8g of ammonium molybdate in 100-mLof 2Nsulfuric acid.
Procedure¡ª Develop the chromatogram until the solvent front has moved not less than 18cm,and dry the plate with the aid of a current of air.Spray the plate with Spray reagent,heat the plate at 140  for 15minutes,and examine under UVlight:the blue zone due to hyperforin at an RFvalue of about 0.54in the chromatogram of the Test solutioncorresponds in color and RFvalue to that in the chromatogram of the Standard solution.
Microbial enumeration ¨¢2021ñ¡ª It meets the requirements of the tests for absence of Salmonella speciesand Escherichia coli.The total bacterial count does not exceed 10 4cfu per g,and the total combined molds and yeasts does not exceed 1000cfu per g.
Content of hypericin and pseudohypericin¡ª
Solution A,Solution B,Solution C,Mobile phase,Standard solution 1,Standard solution 2,and Chromatographic system¡ª Proceed as directed for the Content of hypericin and pseudohypericin test under St.John's Wort.
Test solution¡ª Transfer about 25mg of Powdered Extract,accurately weighed,to a 25-mLvolumetric flask,add about 10mLof a mixture of methanol and water (9:1),and sonicate.Dilute with a mixture of methanol and water (9:1)to volume,and mix.Pass through a polytef membrane filter having a 0.45-µm or finer porosity,and use the filtrate.
Procedure¡ª Proceed as directed for the Content of hypericin and pseudohypericintest under St.John's Wort.Calculate the percentages of hypericin (C 30H 16O 8)and pseudohypericin (C 30H 16O 9)in the portion of Powdered Extract taken by the formula:
2500(C/FW)(rU/rS), in which Wis the weight,in mg,of Powdered Extract taken to prepare the Test solution;and the other terms are as defined therein.Add the percentages obtained for hypericin and pseudohypericin.
Content of hyperforin¡ª Using the chromatograms obtained in the test for Content of hypericin and pseudohypericin,calculate the percentage of hyperforin (C 35H 52O 4)in the portion of Powdered Extract taken by the formula:
2500(C/W0.46)(rU/rS), in which 0.46is the response factor of hyperforin,relative to that of oxybenzone; rUis the response of the hyperforin peak in the chromatogram of the Test solution;and the other terms are as defined therein. |
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