HPTLC for identification of botanicals and their adulterants

Dr. Anita Ankli CAMAG Laboratory Sonnenmattstrasse 11 CH-4132 Muttenz

The Team

Overview
What is HPTLC? Reproducibility is our goal Methodology for standardization in HPTLC Some HPTLC methods Identity and natural variability of botanicals, Adulteration with other plant products or APIs Mixtures of plant products Limit test

What is HPTLC?
High Performance Thin-Layer Chromatography
TLC for the 21st century
Instrumental TLC Application Development Documentation Densitometry

A new concept
Suitable instruments

Scientific basis
Standardized methodology Validated methods

Truly plug and play

Fully cGMP compliant

Comparison TLC-HPTLC of flavonoids

TLC plate 20 x 20 cm

HPTLC plate 20 x 10 cm

Comparison HPTLC - TLC

HPTLC Average particle size: 5-7 m TLC 10-15 m

Particle size distribution:

Separation distanze: Running time:

narrow
3 - 20 min

wide
30 -200 min

30 - 70 mm 100 - 150 mm

Solvent consumption:
Detection limit, absorb.:

5 - 10 ml

50 ml
1 - 100 ng

10 - 100 ng 100 - 1000 ng

fluoresc.: 0.1 - 10 ng

Power of HPTLC
Screening rapid , many samples, comparision on one plate Fingerprint for complex mixtures, disposable plates Flexibility kind of detection, multiple detection, mobile phase Simple documentation visual electronic images

Fields of application
Plant Drugs ID, batch conformity, adulterations Pharmaceutical industry Food and cosmetic industry (lipids, colors...)

Environmental analysis (pesticides...)

Forensics

Reproducibility is our goal

Acanthopanax

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Successful standardization
Echinacea
Original image published 2001 June 30, 2005 CSI Laboratory

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Standardization of methodology
Plate setup and handling Sample application (as band)

Chamber geometry and saturation

Humidity control Developing distance

SOP

Derivatization procedure
Documentation (electronic images) Our SOP is in full compliance with PhEur, USP, ChP Available at: www.camag-laboratory.com

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Chamber conditioning
Twin Trough Chamber
precondition saturation unsaturation

Toluene, ethyl acetate, acetic acid = 70 : 33 : 3; HPTLC Silica gel 60 F254,

Left: Schisandra chinensis, right: S. sphenanthera

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Influence of the relative humidity

Green tea: Polyphenoles
15% 30% 47% 60% RH 17%

Test dye
47% 75% RH

Toluene, acetone, formic acid = 4.5 : 4.5 : 1

Toluene

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The basic HPTLC setup

Application Development Derivatization Documentation

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Identity of Ginger rhizome (Zingiber officinalis)

white light (WRT) Toluene, ethyl acetate = 9:1 Deriv.: Anisaldehyde reagent

UV 366nm

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

6-Gingerol 8-Gingerol 10-Gingerol 6-Shogaol Ginger rhizome 1 Ginger rhizome 2 Ginger rhizome 3 Ginger rhizome 4 Ginger rhizome 5 Ginger rhizome 6 Ginger rhizome 7 Alpinia officinarum, rhizome Kaempferia galangal, rhizome Alpinia oxyphylla, fruit Alpinia katsumadai, semen

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Variability of Cimicifuga racemosa

UV 366nm Validated method: Toluene, ethyl formate, formic acid = 5 : 3 : 2 Deriv.: Sulfuric acid reagent

WRT

1: Actein, 2-9: different Cimicifuga racemosa, rhizome

1 2 3 4 5 6 7 8 9 10

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Adulterants of Cimicifuga racemosa

UV 366 nm
UV 254nm, before deriv.

white light

10

11

10

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1 2 3 4 5 6 7 8 9 10 11

Isoferulic acid Norcimifugin Actein 23-epi-26-Deoxyactein Cimifugin Cimicifuga racemosa, rhizone 1 Cimicifuga racemosa, rhizome 2 C. foetida, rhizome C. heracleifolia, rhizome C. dahurica, rhizome C. americana, Yellow cohosh

Toluene, ethyl formate, formic acid = 5:3:2

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Collaborative trial (I) Cimicifuga racemosa with 5 % adulteration

Actein Actein

R1

R2

R3

R4

Appl. volume: 20 l Derivatization with Boric acid/oxalic acid, 120 C during 5 min System suitability test: Actein Rf 0.37 Fluorescent zone with Rf = 0.24 (Rf = 0.06) 5 % C. foetida in C. racemosa

Actein

Actein

G1

G2

G3

G4

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Collaborative trial with 5 % mixtures (II)

Actein

*
Actein

R1

R2

R3

R4

Derivatization with antimony (III) chloride 120 C for 10 min

Actein

Actein

G1

G2

G3

G4

Fluorescent zone with Rf = 0.39 5 % C. dahurica or C. heracleifolia in C. racemosa

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Collaborative trial with 5 % mixtures (III)

*
R1 R2 R3 R4

Evaluation: UV254 nm

G1

G2

G3

G4

Fluorescence quenching zone at Rf=0.30 5 % of C. americana in C. racemosa

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PhEur 7.3 Cimicifuga racemosa

knowledge database of PhEur (www.edqm.eu)

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ID and adulteration of Equisetum arvense

Equisetum palustre Equisetum arvense

Ethyl aceate, water, acetic acid, formic acid = 67 : 18 : 7.5 : 7.5, NP reagent, Standards: rutin, hyperoside, caffeic acid

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Sage oil evidence of an adulteration

GC

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Ylang Ylang
Linolenic acid

Essential oil: toluene, ethyl acetate = 95 : 5, Anisaldehyde reagent

Argan oil

Sunflower oil

Various fatty oils: DCM, acetic acid, acetone = 20 : 40 : 50 Phosphomolybdic acid reagent

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Herbal slimming supplements adulterated with Sibutramin (API)

Swissmedic

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Detection of sibutramin by HPTLC

UV spectrum

UV 225nm UV 254nm

Toluene, methanol = 95 : 5

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Limit of detection of Aristolochic acid

1: Ref (a), 2: Ref (b) (2 ppm AA1), 3: Stephania, 4: Aucklandia, 5: Vladimiria, 6: Aristolochia debilis

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Screening test for Aristolochic acid

PhEur 7.3

Solvent mixture: anhydrous formic acid R, water R, methanol R (1:9:40 V/V/V). Test solution. To 1.0 g of the powdered herbal drug add 10.0 mL of the solvent mixture, sonicate for 10 min and centrifuge. Reference solution (a). Disperse an amount of aristolochia HRS corresponding to 0.10 mg of aristolochic acid I in 20.0 mL of the solvent mixture, sonicate for 10 min and centrifuge. Reference solution (b). Dilute 1.0 mL of reference solution (a) to 25.0 mL with methanol R. Plate: TLC silica gel F254 plate R (2-10 m) = HPTLC Mobile phase: anhydrous formic acid R, water R, ethyl acetate R, toluene R (3:3:30:60 V/V/V/V); use the upper layer. Application: 20 L as bands of 8 mm. Development: over a path of 6 cm. Drying: in a current of cold air for 5 min. Detection: spray with a 100 g/L solution of stannous chloride R in dilute hydrochloric acid R until the plate is slightly wet, heat at 100 C for 1 min and examine in ultraviolet light at 365 nm. System suitability: the chromatogram obtained with reference solution (a) shows 2 greenish-blue zones due to aristolochic acids I and II between RF = 0.35 and RF = 0.55, which may not be completely separated; the chromatogram obtained with reference solution (b) shows at least 1 of these zones (corresponding to 2 ppm of aristolochic acid I). Results: in the chromatogram obtained with the test solution no zone is similar in position and fluorescence to any of the zones due to aristolochic acids in the chromatogram obtained with reference solution (a). If the chromatogram obtained with the test solution shows any zones similar in position and fluorescence to any of the zones due to aristolochic acids I and II in the chromatogram obtained with reference solution (b), apply method B.

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Amarant - a red azo dye - as adulterant of Bilberry extract

Image evaluated under white light enhanced

1-Butanol, water, formic acid = 40 : 15 : 10

1: Bilberry dry extract 2: Bilberry dry extract spiked with amaranth (spiking level 0.25 %) 3. Amaranth

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Sources of methods
European Pharmacopoeia (EP) New monographs feature TLC and HPTLC in parallel

British, French, German, Swiss Pharmacopoeias Offer specific monographs not found in EP

The USP Dietary Supplement Compendium TLC and state of the art HPTLC

Chinese Pharmacopoeia, Japanese Pharmacopoeia, American Herbal Pharmacopoeia, Quality Standards

of Indian Medicinal Plants, Wagner, H. and Bladt, S.

Plant Drug Analysis,

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Thyme leaf plates 1 - 3

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Thyme leaf comparison viewer

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Three channels per track

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Pattern recognition Master thesis, R. Ambhl, UNI Basel

Evaluation by principal component analysis (PCA) and multivariate analysis of variance (MANOVA)

Identification of probe1 () by MANOVA

Dendrogram of group means

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Thank you for your attention

anita.ankli@camag.com
www.camag-laboratory.com