APPLICATION NOTE

Limit test for rutin and quercetin in Ginkgo leaf dried

extract (Ginkgo biloba L.)
A-130.1

Keywords

Ginkgo biloba L., Ginkgo leaf dried extract, quercetin, rutin

Introduction

Adulteration of ginkgo products with rutin and quercetin or plants rich in

those flavonoids is still a common practice in the market [1]. Comprehensive
HPTLC fingerprinting can provide information beyond identification, avoiding
additional chromatographic tests for detection of adulteration [2].
This limit test for added rutin and quercetin in ginkgo leaf extract is based on
the HPTLC method for identification. The acceptances criteria are taken from
the USP monograph for ginkgo extract [3].

Scope

This method detects adulteration of ginkgo leaf extract (Ginkgo biloba L.) with
levels ≥ 0.5% of quercetin and ≥ 4% of rutin based on the HPTLC fingerprint
of flavonoids.

Recommended devices

Automatic TLC Sampler (ATS 4), Automatic Developing Chamber (ADC 2),
Chromatogram Immersion Device 3, TLC Visualizer, visionCATS software

Samples

Dissolve Ginkgo leaf extract in methanol at a concentration of 10 mg/mL

NOTE: The presented results are to be regarded as examples only!

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Standards

Reference solution for rutin limit test: dissolve 2 mg of rutin in 5 mL of

methanol (0.4 mg/mL). Reference solution for quercetin limit test: weigh
an amount of ginkgo reference extract to yield a solution (in methanol)
representing 0.5% of quercetin in the extract.

Chromatography

Stationary phase HPTLC Si 60 F254 (Merck)

Sample application 3 µL of each standard and sample solution are applied as bands with ATS 4,
15 tracks, band length 8 mm, track distance 11.4 mm, distance from left edge
20 mm, distance from lower edge 8 mm.

Developing solvent Ethyl acetate, water, anhydrous formic acid, and glacial acetic acid
100:26:11:11 (v/v/v/v)

Development In the ADC 2 with chamber saturation (20 minutes). Plates are activated at
33% relative humidity for 10 min using a saturated solution of magnesium
chloride (MgCl2).

Developing 70 mm (from the lower edge)

distance

Plate drying Drying 5 min in the ADC 2

Derivatization Reagent name: Natural products (NP) reagent

Reagent preparation (dipping): 1 g of 2-aminoethyl diphenylborinate in

200 mL of ethyl acetate.

Reagent use: Heat plate at 100°C for 3 min, dip (time 0, speed 3) the warm
plate in NP reagent.

Documentation UV 254 nm prior to derivatization and under UV 366 nm after derivatization

NOTE: The presented results are to be regarded as examples only!

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Results

Chromatograms of reference solution, ginkgo leaf extract and test solutions (products)
under UV 254 nm

Fig. 1) Rutin (track 1), ginkgo extract (track 2), and ginkgo products (containing leaf extract) (tracks 3-5) prior to
derivatization under UV 254 nm. Image of the chromatogram (left) and peak profile of the image (right).

Chromatograms of reference solution, ginkgo leaf extract and test solutions (products)
under UV 366 nm after derivatization

Fig. 2) Rutin (track 1), ginkgo extract (representing 0.5% of quercetin in the extract) (track 2) and ginkgo products
(containing leaf extract) (tracks 3-5) after derivatization under UV 366 nm. Image of the chromatogram (left) and
peak profile of the image (right).

NOTE: The presented results are to be regarded as examples only!

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APPLICATION NOTE

Limit test for rutin from Fig. 1 Limit test for quercetin from Fig. 2

Fig. 3) Peak heights at the position of rutin in the ref- Fig. 4) Peak heights at the position of quercetin in the
erence and test solution. Rutin reference solution @ extract and test solution). Ginkgo extract (represent-
0.4 mg/mL (red bar), ginkgo extract (orange bar) and ing 0.5% of quercetin in the extract, orange bar) and
ginkgo products (dark blue, gray and light blue bars). ginkgo products (dark blue, gray and light blue bars).

Literature

[1] Gafner, S., 2018. Adulteration of Ginkgo biloba leaf extract. Botanical Adulteration Bulletin, 1-8.
[2] Frommenwiler, D.A., Kim, J., Yook, C.S., Tran, T.T.T., Cañigueral, S., Reich, E., 2018. Compre-
hensive HPTLC fingerprinting for quality control of an herbal drug - the case of Angelica gigas root.
Planta Med 84, 465-474.
[3] The United States Pharmacopoeia, 2015b. Monograph for powdered ginkgo extract. In: Pharma-
copoeial Convention 38–NF33, Rockville, MD, USA pp. 6064-6067.

NOTE: The presented results are to be regarded as examples only!

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