Test solution¡ª Dissolve a quantity of Extract in methanol to obtain a solution having a concentration of about 25mg per mL.

Application volume: 5µL.

Developing solvent system: a mixture of methylene chloride,methanol,glacial acetic acid,and water (80:15:2:2).

Spray reagent¡ª Prepare a 5%ferric chloride solution in methanol.

Test solution¡ª Use theTest solutionprepared as directed forIdentificationtestB.

Standard solution¡ª Use the Standard solution 1prepared as directed forIdentificationtestB.

Application volume: 5µL.

Developing solvent system: a mixture of ethyl acetate,formic acid,and water (100:10:6).

Spray reagent: a mixture of phosphoric acid and alcohol (1:1),containing 1%of vanillin.

Procedure¡ª Proceed as directed in the chapter,except to spray the plate with theSpray reagent and heat at 110for 10minutes.Three red bands appear in the middle third of the chromatogram of theStandard solutioncorresponding to two dimeric procyanidins and catechin.The chromatogram of theStandard solutionalso exhibits a blue band between the upper band due to upper dimeric procyanidins and the band due to catechin.The chromatogram of theTest solutioncontains bands that correspond to those found in the chromatogram of theStandard solution.

Solution A¡ª Use filtered and degassed methanol.

Solution B¡ª Carefully weigh 1g of phosphoric acid,and dilute with water.Transfer to a 1000-mLvolumetric flask,dilute with water to volume,and mix.

Mobile phase¡ª Use variable mixtures of Solution AandSolution Bas directed for Chromatographic system.Make adjustments if necessary (see System Suitabilityunder Chromatography ¨¢621ñ).

Standard solution¡ª Dissolve an accurately weighed quantity of USP Maritime Pine Extract RSinSolution Ato obtain a solution having a known concentration of about 2mg per mL.Pass through a membrane having a 0.45-µm or finer porosity.

Test solution¡ª Weigh about 20mg of Extract.Add 10mLof Solution A,and sonicate for 10minutes.Pass through a membrane having a 0.45-µm or finer porosity,discarding the first 4mLof the filtrate.

Chromatographic system(see Chromatography ¨¢621ñ)¡ª The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ¡Á15-cm column that contains base-deactivated packing L7,having less than 5-µm particle size.The column temperature is maintained at 40.The flow rate is about 1.0mLper minute.The chromatograph is programmed as follows.
Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the chromatogram obtained is similar to the Reference Chromatogram provided with the USP Maritime Pine Extract RS;the resolution,R,between taxifolin and ferulic acid is not less than 3.0;and the tailing factor for taxifolin is not more than 2.0.

Procedure¡ª Separately inject equal volumes (about 10µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas for catechin,caffeic acid,taxifolin,and ferulic acid,identifying the peaks by comparison of the chromatogram of theStandard solution with the Reference Chromatogram:the chromatogram of theTest solutionexhibits peaks for catechin,caffeic acid,taxifolin,and ferulic acid at the retention times corresponding to those in the chromatogram of theStandard solution.