Powdered Decaffeinated Green Tea Extract
DEFINITION
Powdered Decaffeinated Green Tea Extract is prepared from the young, unfermented leaf and leaf buds of Camellia sinensis (L.) Kuntze (Fam. Theaceae), also known as Thea sinensis L., using suitable solvents such as alcohol, methanol, acetone, or water or mixtures of these solvents; the caffeine has been removed. The ratio of the starting crude plant material to Powdered Extract is 6:1–10:1. It contains NLT 60.0% of polyphenols, calculated as (–)-epigallocatechin-3-O-gallate, NLT 40.0% of (–)-epigallocatechin-3-O-gallate, and NMT 0.1% of caffeine, calculated on the anhydrous basis.
IDENTIFICATION
•  A. Thin-Layer Chromatographic Identification Test
Standard solution:  4 mg/mL of USP Powdered Decaffeinated Green Tea Extract RS in a mixture of alcohol and water (4:1), sonicate for 10 min, and centrifuge. Use the clear supernatant. [Note—Prepare fresh. Store below 20, if storage is needed. ]
Sample solution:  4 mg/mL of Powdered Decaffeinated Green Tea Extract in a mixture of alcohol and water (4:1), sonicate for 10 min, and centrifuge. Use the clear supernatant.
Chromatographic system 
Adsorbent:  Use a chromatographic silica gel mixture with an average particle size of 5 µm (HPTLC plates).
Application volume:  1 µL
Developing solvent system:  Toluene, acetone, and formic acid (9:9:2)
Immersion reagent:  Dissolve 140 mg of fast blue B salt in 10 mL of water, and add 140 mL of methanol and 50 mL of dichloromethane. [Note—Prepare weekly, and store at 4 in the dark. ]
Analysis 
Samples:  Standard solution and Sample solution
Use an unsaturated chamber, and condition the plate to a relative humidity of 30% using a suitable device. Develop the chromatograms, dry the plate at 100, dip in the Immersion reagent, dry, and immediately examine the plate under visible light. [Note—The chromatogram is stable up to 30 min; afterward, the plate's background darkens significantly. ]
Acceptance criteria:  The chromatogram of the Sample solution exhibits main bands similar in position and color to the main bands in the chromatogram of the Standard solution. The chromatogram of the Sample solution exhibits four main brownish orange bands with RF values of approximately 0.38, 0.48, 0.52, and 0.62 corresponding to (–)-epigallocatechin-3-O-gallate, (–)-epigallocatechin, (–)-epicatechin-3-O-gallate, and (–)-epicatechin, respectively. The most intense band is the one for (–)-epigallocatechin-3-O-gallate. The least intense band is the one for (–)-epicatechin.
•  B. HPLC Identification Test
Analysis:  Proceed as directed in the test for Content of Polyphenols.
Acceptance criteria:  The chromatogram of the Sample solution exhibits 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 at retention times corresponding to those in the chromatogram of Standard solution B.
COMPOSITION
•  Content of Polyphenols
Solution A:  Methanol, phosphoric acid 85%, and water (50: 3.5: 946.5)
Solution B:  Acetonitrile and methanol (95:5)
Mobile phase:  See Table 1.
Table 1
Time
(min)
Solution A
(%)
Solution B
(%)
0 94 6
20 94 6
50 78 22
70 38 62
75 94 6
90 94 6
Standard solution A:  0.1 mg/mL of USP (–)-Epigallocatechin-3-O-gallate RS in Solution A
Standard solution B:  0.4 mg/mL of USP Powdered Decaffeinated Green Tea Extract RS in Solution A. Mix, and centrifuge.
Sample solution:  0.4 mg/mL of Powdered Decaffeinated Green Tea Extract in Solution A. Mix, and centrifuge.
Chromatographic system 
Mode:  LC
Detector:  UV 278 nm
Column:  4.6-mm 6 25-cm; 5-µm packing L1
Column temperature:  25 ± 1
Flow rate:  0.8 mL/min
Injection size:  15 µL
System suitability 
Samples:  Standard solution A and Standard solution B
Suitability requirements 
Chromatogram similarity:  The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Powdered Decaffeinated Green Tea Extract RS being used.
Resolution:  NLT 1 between the (–)-epigallocatechin-3-O-gallate peak and the preceding peak, Standard solution B
Tailing factor:  0.8–2.0 for the (–)-epigallocatechin-3-O-gallate peak, Standard solution A
Relative standard deviation:  NMT 2.0% for the (–)-epigallocatechin-3-O-gallate peak, Standard solution A
Analysis 
Samples:  Standard solution A, Standard solution B, and Sample solution
Record the chromatograms, and measure the areas of the analyte peaks. Using the chromatogram of Standard solution B 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 Table 2.
Table 2
Analyte Relative
Retention
Time
(–)-Epigallocatechin 0.56
(+)-Catechin 0.68
(–)-Epicatechin 0.98
(–)-Epigallocatechin-3-O-gallate 1.00
(–)-Gallocatechin-3-O-gallate 1.09
(–)-Epigallocatechin-3-O-(3¢-O-methyl)-gallate 1.19
(–)-Epicatechin-3-O-gallate 1.27
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:
Result = (rU/rS) × (CS/CU) × 100
rU== peak area of each of the polyphenols in the Sample solution
rS== peak area of (–)-epigallocatechin-3-O-gallate in Standard solution A
CS== concentration of USP (–)- Epigallocatechin-3-O-gallate RS in Standard solution A (mg/mL)
CU== concentration of Powdered Decaffeinated Green Tea Extract in the Sample solution (mg/mL)
Add the percentages calculated for the individual polyphenols.
Acceptance criteria:  NLT 40.0% of (–)-epigallocatechin-3-O-gallate and NLT 60.0% of polyphenols, calculated as (–)-epigallocatechin-3-O-gallate on the anhydrous basis
CONTAMINANTS
•  Heavy Metals, Method II 231: NMT 20 µg/g
•  Microbial Enumeration Test 2021: The total aerobic microbial count does not exceed 104 cfu/g, and the total combined yeasts and molds count does not exceed 103 cfu/g.
•  Absence of Specified Microorganisms 2022: It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.
SPECIFIC TESTS
•  Limit of Gallic Acid
Solution A, Solution B, Mobile phase, and Chromatographic system:  Proceed as directed in the test for Content of Polyphenols.
Standard solution:  0.2 mg/mL of gallic acid in Solution A
Sample solution:  20 mg/mL of Powdered Extract in Solution A. Mix, and centrifuge.
Analysis 
Samples:  Standard solution and Sample solution
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:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of gallic acid in the Standard solution (mg/mL)
CU== concentration of Powdered Decaffeinated Green Tea Extract in the Sample solution (mg/mL)
Acceptance criteria:  NMT 1.0%
•  Limit of Caffeine
Solution A:  Methanol, tetrahydrofuran, phosphoric acid 85%, and water (50:10:3.5:936.5)
Solution B:  Acetonitrile, methanol, and phosphoric acid 85% (946.5: 50: 3.5)
Mobile phase:  See Table 3.
Table 3
Time
(min)
Solution A
(%)
Solution B
(%)
0 100 0
30 100 0
35 0 100
40 0 100
45 100 0
55 100 0
Standard solution:  1 µg/mL of USP Caffeine RS in methanol
Sample solution:  1 mg/mL of Powdered Extract in methanol
Chromatographic system 
Mode:  LC
Detector:  UV 272 nm
Column:  4.6-mm × 25-cm; 5-µm packing L601
Column temperature:  25 ± 1
Flow rate:  1 mL/min
Injection size:  15 µL
System suitability 
Sample:  Standard solution
Suitability requirements 
Relative standard deviation:  NMT 2.0% determined from the caffeine peak
Analysis 
Samples:  Standard solution and Sample solution
Record the chromatograms, and measure the areas of the caffeine peaks.
Separately calculate the percentages of caffeine in the portion of Powdered Extract taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of USP Caffeine RS in the Standard solution (mg/mL)
CU== concentration of Powdered Decaffeinated Green Tea Extract in the Sample solution (mg/mL)
Acceptance criteria:  NMT 0.1%
•  Water Determination, Method Ia 921: NMT 6.0%, determined on a 0.5 g
•  Residue on Ignition 281: NMT 0.5%, determined on 1.0 g
•  Other Requirements: It meets the requirements of the test for Residual Solvents in Botanical Extracts 565, General Pharmacopeial Requirements.
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in well-closed containers, protected from light and moisture, and store at controlled room temperature.
•  Labeling: The label states the Latin binomial and, following the official name, the part of the plant from which the article was derived. It meets other labeling requirements in Botanical Extracts 565.
•  USP Reference Standards 11
USP Caffeine RS Click to View Structure
USP (–)-Epigallocatechin-3-O-gallate RS
USP Powdered Decaffeinated Green Tea Extract RS

1  Endcapped packing L1 columns can also be used in this test.
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