A: Thin-Layer Chromatographic Identification Test 201—

B: The retention times of the peaks for ()-epigallocatechin, (+)-catechin, ()-epicatechin, ()-epigallocatechin-3-O-gallate, ()-gallocatechin-3-O-gallate, ()-epigallocatechin-3-O-(3¢-O-methyl)-gallate, and ()-epicatechin-3-O-gallate in the chromatogram of the Test solution correspond to those in the chromatogram of Standard solution 2, as obtained in the test for Content of polyphenols.

Solution A, Solution B, Mobile phase, and Chromatographic system —Proceed as directed in the test for Content of polyphenols.

Standard solution— Dissolve an accurately weighed quantity of gallic acid in Solution A to obtain a solution having a known concentration of about 0.2 mg per mL.

Test solution— Transfer about 500 mg of Powdered Extract, accurately weighed, to a 25-mL volumetric flask, dissolve, dilute with Solution A to volume, mix, and centrifuge.

Procedure— Separately inject equal volumes (about 15 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the gallic acid peaks. Separately calculate the percentages of gallic acid in the portion of Powdered Extract taken using the formula: in which C is the concentration, in mg per mL, of gallic acid in the Standard solution; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; and rU and rS are the peak responses obtained for gallic acid in the Test solution and the Standard solution, respectively: not more than 1.0% is found.

Solution A— Prepare a filtered and degassed mixture of water, methanol, tetrahydrofuran, and phosphoric acid 85% (936.5:50:10:3.5).

Solution B— Prepare a filtered and degassed mixture of acetonitrile, methanol, and phosphoric acid 85% (946.5:50:3.5).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Dissolve an accurately weighed quantity of USP Caffeine RS in methanol to obtain a solution having a known concentration of about 0.1 mg per mL. Transfer 1.0 mL of this solution to a 100-mL volumetric flask, dilute with methanol to volume, and mix.

Test solution— Transfer about 10 mg of Powdered Extract, accurately weighed, to a 10-mL volumetric flask, add 5 mL of methanol, dissolve, dilute with methanol to volume, mix, and centrifuge.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 272-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L60.* The flow rate is about 1.0 mL per minute. The column temperature is maintained at 25 ± 1. The chromatograph is programmed as follows. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative standard deviation determined from the caffeine peak for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 15 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas of the caffeine peaks. Separately calculate the percentages of caffeine in the portion of Powdered Extract taken using the formula: in which C is the concentration, in mg per mL, of USP Caffeine RS in the Standard solution; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; and rU and rS are the peak responses obtained for caffeine in the Test solution and the Standard solution, respectively: not more than 0.1% is found.

Solution A— Prepare a filtered and degassed mixture of water, methanol, and phosphoric acid 85% (946.5:50:3.5).

Solution B— Prepare a filtered and degassed mixture of acetonitrile and methanol (95:5).

Mobile phase— Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution 1— Dissolve an accurately weighed quantity of USP ()-Epigallocatechin-3-O-gallate RS in Solution A to obtain a solution having a known concentration of about 1.0 mg per mL. Transfer 1.0 mL of this solution to a 10-mL volumetric flask, dilute with Solution A to volume, and mix.

Standard solution 2— Transfer about 20 mg of USP Powdered Decaffeinated Green Tea Extract RS, accurately weighed, to a 10-mL volumetric flask, add 5 mL of Solution A, dissolve, dilute with the same solvent to volume, and mix. Transfer 2.0 mL of this solution to a 10-mL volumetric flask, dilute with Solution A to volume, mix, and centrifuge.

Test solution— Proceed as directed for Standard solution 2, except to use Powdered Extract.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 278-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L1. The flow rate is about 0.8 mL per minute. The column temperature is maintained at 25 ± 1. The chromatograph is programmed as follows. Chromatograph Standard solution 1, and record the peak responses as directed for Procedure: the tailing factor for the ()-epigallocatechin-3-O-gallate peak is not less than 0.8 and not more than 2.0; and the relative standard deviation determined from the ()-epigallocatechin-3-O-gallate peak for replicate injections is not more than 2.0%. Chromatograph Standard solution 2, and record the peak responses as directed for Procedure: the chromatogram obtained is similar to the Reference chromatogram provided with the lot of USP Powdered Decaffeinated Green Tea Extract RS being used; and the resolution, R, between the ()-epigallocatechin-3-O-gallate peak and the preceding peak is not less than 1.

Procedure— Separately inject equal volumes (about 15 µL) of Standard solution 1, Standard solution 2, and the Test solution into the chromatograph; record the chromatograms; and measure the areas of the analyte peaks. Using the chromatogram of Standard solution 2 and the Reference chromatogram provided with the lot of USP Powdered Decaffeinated Green Tea Extract RS, identify the retention times of the peaks corresponding to the different polyphenols. The approximate relative retention times of the polyphenols are provided in the following table: Separately calculate the percentages of ()-epigallocatechin, (+)-catechin, ()-epicatechin, ()-epigallocatechin-3-O-gallate, ()-gallocatechin-3-O-gallate, ()-epigallocatechin-3-O-(3'-O-methyl)-gallate, and ()-epicatechin-3-O-gallate as ()-epigallocatechin-3-O-gallate in the portion of the Powdered Extract taken using the formula: in which C is the concentration, in mg per mL, of USP ()-Epigallocatechin-3-O-gallate RS in Standard solution 1; W is the weight, in mg, of Powdered Extract taken to prepare the Test solution; rU is the peak response obtained for each of the polyphenols in the Test solution; and rS is the peak response obtained for ()-epigallocatechin-3-O-gallate in Standard solution 1: not less than 40.0% of ()-epigallocatechin-3-O-gallate is found. Add the percentages calculated for the individual analytes: not less than 60.0% of polyphenols, calculated as ()-epigallocatechin-3-O-gallate, is found.