APPLICATION NOTES

HPTLC Identification of Ginkgo (Ginkgo biloba) F-16A.1

Ginkgolides

Scope:
This method is suitable for the identification of
Gingko biloba dried leaf and dry extract based on the
HPTLC fingerprint of ginkgolides.

Sample:
In a 25 mL flask 1 g powdered raw material is
refluxed with 10 mL methanol for 10 min. After
cooling the mixture is filtered.
0.1 g of dry extract is sonicated with 10 mL methanol
for 10 min and filtered.
The supernatant is used as test solution.

Standards (optional):
1 mg each of ginkgolide A, ginkgolide B,
ginkgolide C, and bilobalide is individually dissolved
in 1 mL of methanol.

Plate impregnation with sodium acetate solution:

8 g of sodium acetate is dissolved in 200 mL of
ethanol water (3:2). HPTLC plates are immersed
into the solution for 2 seconds and allowed to dry at
room temperature in the hood for 5 min. The plates
are then heated at 90 C for 30 min.

Derivatization reagent:
Acetic anhydride is directly used for spraying.

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Chromatographic conditions:
Stationary phase: HPTLC plates silica gel 60 F254 (Merck), 10x10 cm or
20x10 cm, impregnated with sodium acetate (see
above).
Mobile phase: Toluene, ethyl acetate, acetone, methanol
(20:10:10:1.2)
Sample application: 5 L test solution and 3 L 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 for 20 min (filter paper), 5 mL (respectively
10 mL) developing solvent per trough, developing
distance 60 mm from lower edge of plate. The plate
is then dried with a hair dryer (cold air) for 5 min.
Detection: The plate is sprayed evenly with acetic anhydride
and heated at 180 C for 10 min Examination under
a) UV 254 nm and b) UV 366 nm.

Results:
Compare to the chromatograms below:

Detection of ginkgolides and bilobalide in Ginkgo leaf extracts

a) UV 254 nm,after derivatization b) UV 366 nm, after derivatization

1 2 3 4 5 6 1 2 3 4 5 6
Track 1: Ginkgolide A Track 5: Ginkgolide C
Track 2: Ginkgolide B Track 6: Bilobalide
Track 3, 4: Ginkgo biloba dried leaf

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Comparison of different Gingko biloba leaves and extracts
a) UV 254 nm,after derivatization

b) UV 366 nm, after derivatization

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Track 1, 2: Ginkgo leaf

Track 3: Ginkgo leaf capsule (freeze dried; 1.2-1.8 % flavonoids; US)
Track 4: Ginkgo leaf extract powder (Italy)
Track 5: Ginkgo leaf extract powder (China)
Track 6: Ginkgo leaf extract powder (France)
Track 7: Ginkgo leaf extract powder (China)
Track 8: Ginkgolide A, B, C, bilobalide (with increasing Rf)
Track 9: Ginkgo leaf extract capsule (60 mg) w/gotu kola (US)
Track 10: Ginkgo leaf extract capsule (60 mg; US)
Track 11: Ginkgo leaf extract tablet (yielding 9 mg flavone glycosides; Switzerland)
Track 12: Ginkgo leaf extract tablet (120 mg; US)
Track 13: Ginkgo leaf extract tablet (120 mg; US)
Track 14: Ginkgo tincture (1:5 dry leaf; US)
Track 15: Ginkgo tincture (1:1 fresh leaf; US)
Track 16: Ginkgo tincture (1:10 fresh leaf, Switzerland: current batch)
Track 17: Ginkgo tincture (1:10 fresh leaf, Switzerland: 2 years past expiration date)

References:
American Herbal Pharmacopoeia, 2003

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