Piper methysticum G.Forst.
Source: Images courtesy of the C.V. Starr Virtual Herbarium of the New York Botanical Garden^[1]

Piper methysticum HPTLC ID - Vanillin Sulfuric Acid UV 365 nm

Kava (rhizome) (Piper methysticum)

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

1. 3 μL Kavain
2. 3 μL Piper methysticum-1 (rhizome)
3. 3 μL Piper methysticum-2 (rhizome)
4. 3 μL Piper methysticum-3 (rhizome)
5. 3 μL Piper methysticum-3 (rhizome)
6. 3 μL Piper methysticum-4 (rhizome)
7. 3 μL Piper methysticum-5 (rhizome)
8. 3 μL Kavain

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 H2O: acetonitrile: CH3OH: AcCOOH: >Reverse Phase!!!! [4/3/3/0.01/] 

Sample Preparation Method 0.3g+3mL 70% grain EtOH sonicate/heat @ 50° C ~ 1/2 hr 

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

Reference see Plant Drug Analysis, Wagner, H., 1996

Source: Elan M. Sudberg, Alkemist Laboratories ^[7]

Kava (rhizome) HPTLC ID - Anisaldehyde reagent UV 366 nm

Kava (rhizome) (Piper methysticum)

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

1. 2 µL Kavain
2. 2 µL Dehydrokavain
3. 2 µL Methysticin
4. 2 µL Dehydromethysticin
5. 2 µL Yangonin
6. 2 µL Desmethoxyyangonin
7. 2 µL Kava rhizome 1
8. 2 µL Kava capsules
9. 2 µL Kava dry extract
10. 2 µL Kava root paste
11. 2 µL Kava acetone extract
12. 2 µL Kava liquid extract
13. 2 µL Kava rhizome 2
14. 2 µL Kava rhizome 3
15. 2 µL Kava rhizome 4
16. 2 µL Kava liquid extract 

Reference Sample(s) Reference: Dissolve 1 mg of kavain in 2 mL of toluene. Dissolve 1 mg of desmethoxyyangonin in 2 mL of toluene. Optional: dissolve 1 mg of dihydrokavain, 1 mg of methysticin, 1 mg of dihydromethysticin, and 1 mg of yangonin each in 2 mL of toluene. 

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

Mobile Phase tert-Butyl methyl ether, n-hexane 7:3 (v/v) 

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

Derivatization reagent: Anisaldehyde reagent; Preparation: 170 mL of ice-cooled methanol are mixed with 20 mL of acetic acid, 10 mL of sulfuric acid and 1 mL of anisaldehyde; Use: Dip (time 0, speed 5), heat at 100°C for 4 min. 

Detection Method Unsaturated chamber; developing distance 70 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 (UV 366 nm): Kavain: reddish zone at Rf ~ 0.27; Desmethoxyyangonin: blue zone at Rf ~ 0.30

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. Under UV 366 nm the chromatogram of the test solution shows a green zone (grey zone under white RT) at Rf ~ 0.08, a red zone (purple zone under white RT) at Rf ~ 0.16, another green zone (grayish and weak zone under white RT) at Rf ~ 0.19, another red zone (red and intense zone under white RT) at Rf ~ 0.27 corresponding to reference substance kavain, just above it a faint blue zone at Rf ~ 0.30 corresponding to reference substance desmethoxyyangonin (yellow arrows), and a brown zone (green zone under white RT) at Rf ~ 0.35. Under white light there are several yellow and reddish zones in the upper part of the chromatogram.

Plate preparation: Dissolve 8 g of caffeine in 200 mL of dichloromethane. Use: Dip (time 0, speed 5) plate, dry at room temperature for 5 minutes, then heat at 80°C for 5 minutes.

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  Kava (rhizome) HPTLC ID - Anisaldehyde reagent UV 366 nm

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  Kava (rhizome) HPTLC ID - Anisaldehyde reagent, white RT

Source: HPTLC Association ^[8]