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06/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-15

APPLICATION NOTES

HPTLC Identification of Goldenseal (Hydrastis canadensis)

Sample:In a test tube 0.25 g powdered drug are extracted with 4 mLmethanol - water (8:2) for 30 min in an ultrasonic bath atroom temperature. The mixture is filtered and the residuewashed twice with 2 mL methanol each. In a 20 mLvolumetric flask filtrate and washings are combined and filledup to the mark with methanol. This is the test solution.

Note: Samples are sensitive to light and heat. Store in therefrigerator in colored vials.

Standards (optional):

In a 50 mL volumetric flask, 25 mg (1R,9S)--hydrastine HCl,1 mg hydrastinine HCl, 1.2 mg palmatine chloride and 1.2 mgberberine chloride are dissolved in methanol and filled up tothe mark.

Derivatization reagent (optional):

Ninhydrin reagent: 0.6 g ninhydrin are dissolved in 190 mLisopropanol, then 5 mL acetic acid are added.

Chromatographic conditions:

Stationary phase: HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or20x10cm.

Mobile Phase: Ethyl acetate, methanol, formic acid, water (50:10:6:3)

Sample application: 5 L test solution and standard are applied as 8 mm bands,min. 2 mm apart, 8 mm from lower edge of plate.

Development: 10x10 cm or 20x10 cm Twin Trough Chamber, saturated for20 min (filter paper), 5 mL (respectively 10 mL) developingsolvent per trough, developing distance 60 mm from loweredge of plate. The plate is then dried with a hair dryer (coldair) for 5 min.

Detection: a) UV 254 nm

b) UV 366 nm

c) Ninhydrin reagent: the plate is immersed in reagent for1 s, dried in stream of cold air, then heated at 110C for2 min. Examination in white light.

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APPLICATION NOTES

Results:

Compare to the chromatograms below:

1 a UV 254 nm 1 b UV 366 nm

1 c Ninhydrin reagent / white light:

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HPTLC of golden seal

Track 1: HydrastinineTrack 2: HydrastineTrack 3: Golden seal (extract)

Track 4: Palmatine chlorideTrack 5: Berberine chlorideTrack 6: Mixture of standards

Note: Palmatine must not be seen in the sample!

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APPLICATION NOTES

Comparison of Goldenseal and common adulterants:

2a

2b

2c

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06/03 Please contact CAMAG for more application notes and products for analysis of herbals! F-15

APPLICATION NOTES

2 b UV 366 nm overexposed

Caption Figures 2a-c

Track 1: Standard mixture: hydrastinine, hydrastine, palmatine, berberine (with increasing Rf) 5 L

Track 2: Rumex crispus

Track 3: Mahonia aquafolium

Track 4: Coptis chinensis

Track 5: Xanthorrhizasp. leaves

Track 6: Hydrastis canadensis(root) authenticated

Track 7: Xanthorrhizasp. steam

Track 8: Xanthorrhizaroot

Track 9: Berberis nervosa

Track 10: Berberis vulgaris

Track 11: Standard mixture: hydrastinine, hydrastine, palmatine, berberine (with increasing Rf) 3 L

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