BRAMA Botanical Risk Assessment Training Session VI ......BRAMA Botanical Risk Assessment Training...

Stefan Gafner, PhD

CSO, American Botanical Council

November 18, 2015

Oryx Hotel, Aqaba

BRAMA

Botanical Risk Assessment Training

Session VI

Identity and Purity Testing

Testing Method

Selection criteria

• Raw material form (whole, powdered, extract)• Extract: phytochemical composition

• Type of adulterant (inert filler, pure compound,

extract)

• Targeted vs non-targeted

• Infrastructure

Testing Method

Specificity

Definition: “To unequivocally assess the analyte

in the presence of other expected components”

Specificity: Bilberry Extract

HPLC-UV chromatogram (535 nm) of bilberry extract

according to the European Pharmacopoeia. Image

provided by Indena S.p.A. (Milan, Italy).

UV/Vis spectrum of amaranth dye

Chen P, Lin LZ, Harnly J. J AOAC Int. 2010;93(4):148-154.

Skullcap (Scutellaria lateriflora)History of Adulteration

•King’s American Dispensatory (1898) lists S. incana and S. ovata as

common adulterants

•Hepatotoxic germander (Teucrium) species as adulterants identified

•S. galericulata listed as synonym in certain textbooks

•Other known adulterants: S. alpina, S. baicalensis

•Recent skullcap adulterations documented in 2010

Need for analytical methods

Traditional Uses

•15 medicinal used reported from native Americans

(Appalachian, Cherokee, Iroquois)

•Introduced to mainstream medicine in 1773 to treat

rabies

•Used as a sedative and antispasmodic herb in

subsequent years

•Listed in USP from 1863-1916 (NF until 1947)

Teucrium canadense

Scutellaria lateriflora

Teucrium chamaedrys


Teucrium canadense

Teucrium chamaedrys

a b c

d e f

g h i

Digital Photo-Microscopy

Images courtesy of Alkemist Labs, Costa Mesa, CA

SG4

Diapositiva 9

SG4 Microscopic analysis, according to [9]: S. lateriflora: a: lower epidermis of leaf showing sinuous walls and a large glandular scale; b: cross section of leaf showing fingerlike trichome with warted exine; c: cross section of the stem showing (A) thin walled parenchyma of the pith (B) cortex (C) epidermis showing flattened cells. T. canadense: d: surface of stem showing strap shaped multicellular trichomes; e: bristle like trichomes of the upper epidermis of the leaf; f: cross section of stem showing epidermal trichomes, epidermis, cortex and parenchyma of the hollow pith. T. chamaedrys: g: powdered flower showing club shaped trichome; h: surface of stem showing a multitude of trichomes including glandular scales, multicellular covering trichomes and some club shaped trichomes; i: cross section of the stem showing (A) epidermis (B) cortex (C) pith comprised of thin walled parenchyma.Stefan Gafner; 26/09/2014

Teucrioside (1): R = lyxoseVerbascoside (2): R= H

Baicalin (3): R1 = H, R2 = glucuronic acidLateriflorin (4): R1= OCH3, R2 = glucuronic acidBaicalein (6): R1 = R2 = H

Dihydrobaicalin (5)


Teucrium sp.

Chemistry

O

OH

R2O

O

OH

R1

O

H

HH

H

OH

OH

H OH

COOH

O

OH

O

O

OH

OO

O

OH

OHO

OH

OHO

OOH

OR

OHOH

CH3

OH

Lane 17: mixture of Scutellaria lateriflora : Teucrium canadense (80:20)Lane 18: mixture of Scutellaria lateriflora : Teucrium chamaedrys (80:20)

Stationary phase: 10 x 10 cm silica gel 60 F254 HPTLC plates; Mobile Phase: Ethyl acetate:formic acid:acetic acid:water (15:1:1:2) Detection: Natural products/polyethylene glycol (NP/PEG) reagent, UV at 366 nm.

Image courtesy of Camag AG, Switzerland

Stationary phase: 10 x 10 cm silica gel 60 F254 HPTLC plates; Mobile Phase: Ethyl acetate:formic acid:acetic acid:water (15:1:1:2) Detection: Natural products/polyethylene glycol (NP/PEG) reagent, UV at 366 nm.

Image courtesy of Camag AG, Switzerland

High Performance Thin-Layer Chromatography

1

3

6

2

4 5

3

4

5

6

2

1

Column: Zorbax XDB C-18 (250 x 4.6 mm I.D., 5 µm)Solvent: MeCN (0.05% TFA) - H2O (0.05% TFA) 14 � 58 in 40 min., flow: 1 mL/min.

Teucrium canadense(70% ethanol extract)

Scutellaria lateriflora(70% ethanol extract)

5

1

2

3

45

6

Teucrium canadense


HPLC-UV

S. lateriflora

S. galericulata

S. mollis

S. humilis

S. ovata

S. incana

S I LB

S: scutellarinI: ikonnikoside IB: baicalinL: lateriflorin

Scutellaria Species Comparison by LC-MS

5.05.56.06.57.07.5 ppm

S.ga

leri

cula

taS.

late

rifl

ora

Comparison between of 1H NMR

traces between S. lateriflora and S.

galericulata from various sources

Experiment: 1D-CPMG (Carr Purcell

Meiboom Gill) experiment using an

echo time of 2.56 ms, a sweep width

of 20 ppm, a data size of 64000

points, 64 scans, and a relaxation

delay of 4 s.

Colson KL, Fischer C, Gafner S et al., 52th

Annual Meeting of the American Society of

Pharmacognosy, August 2011, San Diego, CA.

1H-NMR

Ginkgo biloba

Adulteration of Ginkgo biloba extracts

2003: Canada - 9 samples, 1 adulterated (11%)

2005: Canada - 14 samples, 4 likely adulterated (29%)

2006: China - 19 samples, 3 adulterated (16%)

2006: USA - 21 samples, 10 adulterated (48%)

2010: Germany - 10 samples, 7 likely adulterated (70%)


2012: Japan - 22 samples, 3 likely adulterated (14%)


2014: USA - 37 samples, no evidence of adulteration

2014: Australia - 8 samples, 3 adulterated (38%)

2015: UK – 30 samples, 12 adulterated (40%)

2015: USA - 25 samples, 11-19 adulterated (44-76%)

2015: Canada, in press - 14 samples, 11 adulterated (79%)

Analysis of commercial Ginkgo biloba products - HPTLC

Avula B, Sagi S, Gafner S, et al. Anal Bioanal Chem. 2015; 407(25):7733-7746.

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

Gen

iste

inGinkgo leaf

So

ph

ora

fru

it

Ginkgo supplements

So

ph

ora

flo

wer

Analysis of commercial Ginkgo biloba products –

R = Rutin; Qt = Quercitrin; Q = Quercetin; G = Genistein; K = Kaempferol; I = Isorhamnetin

Ginkgo biloba

Image courtesy of Steven Foster

Styphnolobium japonicum

Image courtesy of Steven Foster

Avula B, Sagi S, Gafner S, et al. Anal Bioanal Chem. 2015; 407(25):7733-7746.

RQt Q

KG I

15212: G. biloba

leaf

R

R

R

R

R

R

R

R

R

Q

Q

Q

Q

Q

G

G

K

K

3246: G. biloba

leaf

9151: G. biloba

leaf

Flavonoid Standard

Mix-6

GBP12

GBP6

8592: S. japonica

flowers

GBP25

GBP24

8540: S. japonica fruits

G

UHPLC-UV/MS

Adulteration of Grape (Vitis vinifera) Seed Extract

• In the United States, grape seed extract (GSE) sales ranked #67 in the mass market and #59 in natural channel in 2014 with sales just over $ 2 Mio*

• Positive effects on platelet and endothelial function and blood flow shown in human clinical trials

• GSEs are often standardized to proanthocyanidin (PAC) content

• Costs for GSE extract sourced in China are US$ 30-110/kg and up, while other PAC-rich extracts are cheaper (pine bark extract US$ 20-22/kg, peanut skin extract US$ 10-13/kg)**

*SPINS/ISI data for 2014**Xin Jin oral communication, August 31, 2015

Adulteration of Grape (Vitis vinifera) Seed

Extract

B-type PACs B-type PACs A-type and B-type PACs

Vitis vinifera Arachis hypogaeaPinus ponderosa

Ponderosa pine image by Max Licher;

http://swbiodiversity.org/seinet/imagelib/imgdetails.php?imgid=19951

Analysis of Commercial Grape Seed Extract

• GSE samples (n=21) from a variety of sources were analyzed by HPTLC and HPLC-UV/MS1

• 6 samples (29%) adulterated with peanut skin extract, another 3 samples (14%) contain A type PACs

• Similar results obtained in study using HPTLC from 2014.2

1Villani TS, Reichert W, Ferruzzi MG, Pasinetti GM, Simon JE, Wu Q. Food Chem. 2015;170:271-280.2Sudberg É, Sudberg S, Nguyen J. AHPA (American Herbal Products Association) Botanical Congress, Las Vegas, NV. October 10, 2014.3Sicherer SH, Muñoz-Furlong A, Godbold JH, Sampson HA. J Allerg Clin Immunol. 2010;125(6):1322-1326.

Procyanidin A2 Procyanidin B1

The allergenic potential of peanut skin represents safety risk. Prevalence to peanut skin allergy in USA is estimated to be 1.4%.3

HPTLC Analysis of Commercial Grape Seed Extracts

Comparison among authentic grape seed and peanut skin raw materials, extracts and grape seed extracts in

commercially available products. Detection after derivatization with Fast Blue salt B under visual light. Image provided

by Alkemist Labs (Costa Mesa, CA).

Black Cohosh Adulteration

Chinese cimicifuga • Sheng ma

Actaea dahurica

Xing an sheng ma

Actaea cimicifuga

Sheng ma

Actaea heracleifolia

Da san ye sheng ma

Black Cohosh AdulterationRecent Reports

2006: HPLC-ELSD (n=4): 25% adulterated1

2006: HPLC-MS (n=11): 36% adulterated2

2012: DNA barcoding (n=36): 25% adulterated3

2014: HPLC-MS/MS & DNA sequencing (n=25): 28% adulterated4

2015: FIMS, NMR & DNA (Sanger) sequencing5

Chinese Actaea raw materials (n=11): 85% adulterated

Finished products (n=14): authentication not possible by NMR, FIMS; DNA found in 5 samples: 20% adulterated

1He K, Pauli GF, Zheng B, et al., J Chromatogr A 2006;1112(1-2):241-2542Jiang B, Kronenberg F, Nuntanakorn P. et al., J Agric Food Chem 2006;54(9):3242-32533Baker DA, Stevenson DW, Little DP. J AOAC Int. 2012;95(4):1023-10344Masada-Atsumi S, Kumeta Y, Takahashi Y, et al., Biol. Pharm. Bull. 2014;37(3):454–460 5Harnly JM, Chen P, Sun J et al., Planta Med. 2015; in press

1He K, Pauli GF, Zheng B, et al., J Chromatogr A 2006;1112(1-2):241-2542Jiang B, Kronenberg F, Nuntanakorn P. et al., J Agric Food Chem 2006;54(9):3242-32533Baker DA, Stevenson DW, Little DP. J AOAC Int. 2012;95(4):1023-10344Masada-Atsumi S, Kumeta Y, Takahashi Y, et al., Biol. Pharm. Bull. 2014;37(3):454–460 5Harnly JM, Chen P, Sun J et al., Planta Med. 2015; in press

Black Cohosh AdulterationMultivariate Analysis

Yuk J, Gafner S, Harnly J, et al., 128th Annual Meeting of the AOAC

International , September 2014, Boca Raton, FL25

A. racemosa

A. dahurica A. podocarpa

A. rubra

-3

0

3

-3

0

3

-3

0

3

PC

3 (1

1.3%

Var

ian

ce)

PC2 (15.3% Variance)PC1 (55.9% Variance)

NMR spectra (1H-NMR) 3D PCA scores plot

Adulteration of Saw Palmetto (Serenoa repens)

SG5

Diapositiva 26

SG5 Conveyor beltStefan Gafner; 24/09/2014

Adulteration of Saw Palmetto DNA Barcoding

Little DP, Jeanson ML. Scientific Reports 2013:3; DOI:doi:10.1038/srep03518

http://www.nature.com/srep/2013/131217/srep03518/pdf/srep03518.pdf

SG6

Diapositiva 27

SG6 matK and rbcL regions used to obtain barcodesStefan Gafner; 24/09/2014

Analysis of Commercial Saw Palmetto Products in 2014*

• 57 samples obtained from 9 countries in Asia, Europe and

North America

• 29 contained only saw palmetto, 28 were combined with other

constituents (vitamins, herbal extracts, or minerals)

• Quantitative analysis of 9 fatty acids by GC-MS

• Qualitative analysis by 1H-NMR and subsequent statistical

evaluation

• Fatty acid contents between 0.1 and 4.6 times of levels claimed

on label

• No adulteration detected

*Booker A, Suter A, Krnjic A et al. J Pharm Pharmacol. 2014;66(6):811-822

SG7

Diapositiva 28

SG7 University of LondonStefan Gafner; 24/09/2014

Testing methods: Laboratory Guidance Documents

• Address testing methods for botanical ingredients of

concern

• Comprehensive review of published analytical methods

with regard to suitability to detect adulteration

• Extensively peer-reviewed by experts from academia,

government, industry and independent analytical

laboratories

• Not intended to be a standard-setting document

Testing methods: Laboratory

Guidance Documents

Number of reviewers 21 20 16

Type of test Scutellaria

lateriflora

Actaea

racemosa

Vaccinium

myrtillus

Macroscopic evaluation

Microscopy

Genetic

TLC/HPTLC

HPLC/UHPLC

Direct MS

NMR

UV/Vis

1

1

2

3

13

1

1

0

1

1

2

8

21

2

1

0

1

1

0

7

26

0

0

4

Total 22 36 39

Method Test parameter Applicability Limitations; Not applicable to

Taxonomy - Identity - Whole living plant - Extracts,

- Powdered or cut crude plant material

Macroscopic

evaluation

- Identity

- Contamination (foreign matter)

- Whole or cut crude plant material - Extracts

- Powdered crude plant material

Microscopy - Identity

- Contamination (e.g., starch, sand)

- Whole, cut or powdered crude plant material - Extracts

Genetics (DNA) - Identity - Whole, cut or powdered crude plant material

- Extracts possessing intact DNA from the parent plant

- Extracts without DNA

- Materials processed using prolonged heat, exposure to

UV light, or irradiation

UV/Vis (standalone) - Strength - Extracts

- Whole, cut or powdered crude plant material after extraction

- Analytes with no UV/Vis chromophore (e.g., sugars and

sugar alcohols, many amino acids) without prior

derivatization

FT-IR - Identity - Extracts


- Extracts containing large amounts of carriers, e.g.,

maltodextrin

FT-NIR - Identity - Extracts

- Whole, powdered or cut crude plant material

- Materials with variable moisture content

- Extracts containing large amounts of carriers, e.g.,

maltodextrin

MS (stand-alone) - Identity - Extracts


- Very high molecular weight analytes

- Non-readily ionizable molecules

NMR - Identity

- Strength

- Extracts


- Certain highly polymerized molecules (e.g., high

molecular weight PACs)

HPTLC - Identity

- Strength

- Extracts


- Highly polar compounds

GC-FID - Identity

- Strength

- Contamination

- Extracts


- Non-volatile compounds

GC-MS - Identity

- Strength

- Contamination

- Extracts


- Non-volatile compounds

HPLC-UV(DAD) - Identity

- Strength

- Extracts


- Analytes with no UV/Vis chromophore (e.g., sugars and

sugar alcohols, many amino acids) without prior

derivatization

HPLC-ELSD - Strength - Extracts


- Highly volatile compounds (e.g., essential oils)

HPLC-MS - Identity

- Strength

- Contamination

- Extracts


- Very low and very high molecular weight analytes

- Non-readily ionizable molecules (e.g. , terpenes with

limited presence of functional groups)

ICP-MS - Strength

- Contamination (metals)

- Extracts


- Organic compounds

BRAMA Botanical Risk Assessment Training Session VI ......BRAMA Botanical Risk Assessment Training...

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