Hptlc for Identification of Botanicals and Their Adulterants

7/28/2019 Hptlc for Identification of Botanicals and Their Adulterants

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HPTLC for identification of

botanicals and their adulterants

Dr. Anita AnkliCAMAG LaboratorySonnenmattstrasse 11CH-4132 Muttenz


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2 The Team


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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 orAPIs

Mixtures of plant products

Limit test


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What is HPTLC?

TLC for the 21st century Instrumental TLC

Application

Development

Documentation Densitometry

Truly plug and play

Fully cGMP compliant

A new concept

Suitable instruments

Scientific basis

Standardized methodology

Validated methods

High Performance Thin-Layer Chromatography


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Comparison TLC-HPTLC of flavonoids

TLC plate 20 x 20 cm

HPTLC plate 20 x 10 cm


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Comparison HPTLC - TLCHPTLC TLC

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

Particle size distribution: narrow wide

Separation distanze: 30 - 70 mm 100 - 150 mm

Running time: 3 - 20 min 30 -200 min

Solvent consumption: 5 - 10 ml 50 ml

Detection limit, absorb.: 10 - 100 ng 100 - 1000 ng

fluoresc.: 0.1 - 10 ng 1 - 100 ng


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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


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Fields of application

Plant Drugs

ID, batch conformity, adulterations Pharmaceutical industry

Food and cosmetic industry (lipids, colors...)

Environmental analysis (pesticides...)

Forensics


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Reproducibility is our goal

Acanthopanax


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Successful standardizationEchinacea June 30, 2005 CSI Laboratory

Original image

published 2001


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Standardization of methodology Plate setup and handling

Sample application (as band)

Chamber geometry and saturation

Humidity control

Developing distance

Derivatization procedure

Documentation (electronic images)

SOP

Our SOP is in full compliance with PhEur, USP, ChP

Available at: www.camag-laboratory.com


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Chamber conditioningTwin 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

15% 30% 47% 60% RH 17% 47% 75% RH

Green tea: Polyphenoles

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

Test dye


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The basic HPTLC setupApplication Development Derivatization Documentation


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Identity of Ginger rhizome (Zingiber officinalis)Toluene, ethyl acetate

= 9:1

Deriv.: Anisaldehyde

reagent

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

1 6-Gingerol2 8-Gingerol3 10-Gingerol4 6-Shogaol5 Ginger rhizome 1

6 Ginger rhizome 27 Ginger rhizome 38 Ginger rhizome 49 Ginger rhizome 510 Ginger rhizome 611 Ginger rhizome 712 Alpinia officinarum, rhizome13 Kaempferia galangal, rhizome14 Alpinia oxyphylla, fruit15 Alpinia katsumadai, semen

white light

(WRT)

UV 366nm


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Variability ofCimicifuga racemosaValidated method:

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

Deriv.: Sulfuric acid reagent

1: Actein, 2-9: differentCimicifuga racemosa,rhizome

WRT

UV 366nm

1 2 3 4 5 6 7 8 9 10


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Adulterants ofCimicifuga racemosa

1 Isoferulic acid

2 Norcimifugin3 Actein

4 23-epi-26-Deoxyactein

5 Cimifugin

6 Cimicifuga racemosa, rhizone 1

7 Cimicifuga racemosa, rhizome 2

8 C. foetida, rhizome

9 C. heracleifolia, rhizome

10 C. dahurica, rhizome

11 C. americana, Yellow cohosh1 2 3 4 5 6 7 8 9 10 11

white light

UV 254nm, before deriv.

5 6 7 8 9 10 11

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

UV 366 nm


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Collaborative trial (I)

Cimicifuga racemosa with 5 % adulteration

Actein

G1 G2 G3 G4

Actein

Appl. volume: 20 l

Derivatization with

Boric acid/oxalic acid,120 C during 5 min

Fluorescent zone with

Rf = 0.24 (Rf = 0.06) 5 % C. foetida in C.

racemosa

Actein

Actein

R1 R2 R3 R4*

*

System suitability

test:Actein Rf

0.37


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Collaborative trial with 5 % mixtures (II)Derivatization with

antimony (III) chloride

120 C for 10 min

Fluorescent zone withRf = 0.39 5 % C.

dahurica orC. heraclei-

folia in C. racemosa

R1 R2 R3 R4

G1 G2 G3 G4

A

ctein

Actein

Actein

Actein

*

*


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Collaborative trial with 5 % mixtures (III)

Evaluation: UV254 nmR1 R2 R3 R4

G1 G2 G3 G4

Fluorescencequenching zone at

Rf=0.30 5 % of

C. americana in C.

racemosa

*

*


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PhEur 7.3 Cimicifuga racemosaknowledge database of

PhEur (www.edqm.eu)


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ID and adulteration ofEquisetum arvense

Equisetum palustre

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

Equisetum arvense


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Sage oil evidence of an adulteration

GC


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Ylang YlangEssential oil: toluene,

ethyl acetate = 95 : 5,

Anisaldehyde reagent

Various fatty oils:DCM, acetic acid, acetone

= 20 : 40 : 50

Phosphomolybdic acid

reagent

Sunflower

oil

Linolenic

acid

A

rganoil


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Herbal slimming supplements -

adulterated with Sibutramin (API)Swissmedic


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Detection of sibutramin by HPTLC

UV 254nm

UV 225nm

UV spectrum

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

1 2 3 4 5 6


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Screening test for Aristolochic acid PhEur 7.3Solvent 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 andcentrifuge.

Reference solution (a). Disperse an amount of aristolochia HRS corresponding to 0.10 mg of aristolochic acid I in 20.0 mLof 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 ofstannous chloride Rin dilute hydrochloric acid Runtil 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 IIbetween 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 ofaristolochic acid I).

Results: in the chromatogram obtained with the test solution no zone is similar in position and fluorescence to any of thezones 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 thezones 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 extractImage evaluated under white light

1: Bilberry dry extract

2: Bilberry dry extract spiked with

amaranth (spiking level 0.25 %)

3. Amaranth

1 2 3 1 2 3

enhanced

1-Butanol, water, formic acid

= 40 : 15 : 10


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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 [email protected]

www.camag-laboratory.com

Hptlc for Identification of Botanicals and Their Adulterants

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