Source: MOBOT, Tropicos.org.^[1]

Source: MOBOT, Tropicos.org.^[2]

Source: MOBOT, Tropicos.org.^[3]

Source: Royal Botanic Gardens, Kew.^[4]

UPLC

Camellia sinensis (leaf) UPLC

Extraction Solvent: Acetone & water (80:20)

Diluent: 0.5% formic acid in water

Test Sample Preparation: Transfer 1 g of ground plant material into a screw cap bottle, add 50 ml of Extraction Solvent, tightly cap, and shake for 4 h in a mechanical shaker at room temperature. Filter about 10 ml of extract using a 0.20 um PTFE membrane filter. Dilute 2.0 ml of filtered solution to 10 mL with Diluent.

Column: 100 mm x 2.1 mm, 1.7 um, Waters Acquity BEH C18

Mobile Phase: 0.5% formic acid in acetonitrile (Solution A) and 0.5% formic acid in water (Solution B)

Elution: Gradient, see Table below

Column Temperature: 30°C

Flow rate: See Table below

Detection: UV, 274 nm

Injection volume: 1.0 uL, maintained at 10°C

Needle wash: Acetonitrile

Source: Indena S.p.A. ^[19]

Table: Gradient program

HPLC

Camellia sinensis (leaf) HPLC

Extraction Solvent: Acetone, water (80:20)

Diluent: 0.05% formic acid in water

Test Sample Preparation: Transfer 1 g of ground plant material into a screw cap bottle, add 50 ml of Extraction Solvent, tightly cap, and shake for 4 h in a mechanical shaker at room temperature. Filter about 10 ml of extract using a 0.20 um PTFE membrane filter. Dilute 2.0 ml of filtered solution to 10 mL with Diluent.

Column: 15-cm x 4.6-mm, 3 um, YMC-Pack ODS-A

Mobile Phase: 0.05% formic acid in water (Solution A), 0.05% formic acid in methanol (Solution B), and acetonitrile (Solution C)

Elution: Gradient, see Tables below

Column Temperature: 40°C

Flow rate: 1.0 mL/min

Detection: UV, 274 nm

Injection volume: 10 uL

Source: Indena S.p.A. ^[20]

Table for HPLC systems with dwell volume ˂ 2.0 mL

Table for HPLC systems with dwell volume > 4.0 mL

Camellia sinensis HPTLC ID - Vanillin Sulfuric Acid UV 365 nm

Green Tea (leaf) (Camellia sinensis)

Lane Assignments Lanes, from left to right (Track, Volume, Sample):

1. 2 μL Epicatechin ~0.1% in Methanol
2. 3 μL Camellia sinensis-1 (herb)
3. 3 μL Camellia sinensis-2 (leaf)
4. 3 μL Camellia sinensis-3 (herb)
5. 3 μL Camellia sinensis-3 (herb)
6. 4 μL Camellia sinensis-4 (leaf)
7. 2 μL Camellia sinensis-5 (leaf)
8. 2 μL Epigallocatechin Gallate ~0.1% in Methanol

Reference materials used here have been authenticated by macroscopic, microscopic &/or TLC studies according to the reference source cited below held at Alkemists Laboratories, Costa Mesa, CA. 

Stationary Phase Silica gel 60, F254, 10 x 10 cm HPTLC plates 

Mobile Phase CHCl3: ethyl formate: HCOOH [5/4/1] 

Sample Preparation Method 0.3 g + 3ml 70% grain EtOH sonicated + heated @ 50° C ~ 1 hr 

Detection Method Vanillin/H2SO4 Reagent -> 110° C 5 min -> UV 365 nm 

Reference see Herbal Drugs and Phytopharmaceuticals, Wichtl, M., 1994

Source: Elan M. Sudberg, Alkemist Laboratories ^[21]

Green Tea (leaf) HPTLC ID - Fast Blue salt B reagent, white RT

Green Tea (leaf) (Camellia sinensis)

Lane Assignments Lanes, from left to right (Track, Volume, Sample):

1. 5 µL (-)-Epigallocatechin-3-O-gallate
2. 5 µL (-)-Epigallocatechin
3. 5 µL (-)-Epicatechin gallate
4. 5 µL (-)-Epicatechin
5. 1 µL Green Tea leaf 1
6. 1 µL Green Tea leaf 2
7. 1 µL Green Tea leaf 3
8. 1 µL Green Tea leaf 4 

Reference Sample(s) Reference:Individually dissolve 1 mg of (-)-epigallocatechin and 1 mg of (-)-epicatechin gallate each in 20 mL of methanol; Optional: Individually dissolve 1 mg of (-)-epigallo-catechin-3-O-gallate and 1 mg of (-)-epicatechin each in 20 mL of methanol; Store all solutions at -20°C. 

Stationary Phase Stationary phase, i.e. Silica gel 60, F254 

Mobile Phase Toluene, acetone, formic acid 9:9:2 (v/v/v) 

Sample Preparation Method Sample: Mix 100 mg of powdered sample with 10 mL of methanol, water 4:1 and sonicate for 10 minutes, then centrifuge or filter the solutions and use the supernatants / filtrates as test solutions.

Derivatization reagent: Fast Blue salt B reagent; Preparation: dissolve 140 mg of Fast Blue salt B in 10 mL of water and add 140 mL of methanol and 50 mL of dichloromethane. Store reagent in the dark at 4°C; Use: preheat the plate to 100°C for 2 min, then dip (time 0, speed 5), dry for 5 min in the fume hood. 

Detection Method Unsaturated chamber; developing distance 60 mm from lower edge; relative humidity 33% 

Other Notes Images presented in this entry are examples and are not intended to be used as basis for setting specifications for quality control purposes. System suitability test: (-)-Epigallocatechin: brown zone at Rf ~ 0.46; (-)-Epicatechin gallate: brown zone at Rf ~ 0.52

Identification: Compare result with reference images. The fingerprint of the test solution is similar to that of the corresponding botanical reference sample. Additional weak zones may be present. The chromatogram of the test solution shows four brownish-orange zones corresponding to reference substance epigallocatechin-3-O-gallate (Rf ~ 0.37), (-)-epigallocatechin (Rf ~ 0.46), (-)-epicatechin gallate (Rf ~ 0.52), and (-)-epicatechin (Rf ~ 0.62). The lowest zone is the most intense and the upper zone is the faintest. The two zones in between are clearly separated (black arrows).

Source: HPTLC Association ^[22]