Hptlc for Identification of Botanicals and Their Adulterants
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Transcript of 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