identification and quantitation of analytical markers …aoaceurope.com/2011pr/file 07.pdf•...

Identification and quantitation of analytical markers for

herbal medicinal products by UHPLC-DAD/MS

Dr. Melanie Kerst

HWI ANALYTIK GmbH

2

• Regulatory Background• Method development• Identification of analytical markers• Database• Application of the method

– Diaphoretica tea– Rooibos tea– Stomach therapeutic agent

Analytical markers for herbal medicinal products

3

Regulatory Background - Production

• Guideline on Good Agricultural and Collection Practice (GACP) for StartingMaterials of Herbal Origin (EMA/HMPC/246816/2005)– Reproducible quality of plant material requires quality assurance

concerning collection / cultivation / harvesting / first production steps– With respect to hygenic production / low microbial contamination /

careful treatment– Herbal drug: identification, geographic origin and agricultural conditions

• Eudralex: Volume 4 Medicinal Products für Human and Veterinary Use: Good Manufacturing Practice – Annex7: Manufacture of Herbal MedicinalProducts– Collection / cultivation / harvesting– Cutting / drying– Extraction / fractionation / purification …– Further manufacturing process into a dosage form– Detailed description of production process– Specifications for in-process controls

4

Regulatory Background - Quality

• Guideline on Quality of Herbal Medicinal Products/Traditional HerbalMedicinal Products (CPMP/QWP/2819/00 Rev. 1)

• Guideline on Specifications: Test Producedures and AcceptanceCriteria for Herbal Substances, Herbal Preparations and HerbalMedicinal Products/Traditional Herbal Medicinal Products(CPMP/QWP/2820/00 Rev.1)

• Guideline on Declaration of Herbal Substances and HerbalPreparations in Herbal Medicinal Products/Traditional HerbalMedicinal Products in the SPC (EMA/HMPC/287539/2005)

• Guideline on Quality of Combination Herbal MedicinalProducts/Traditional Hebal Medicinal Products(EMA/HMPC/214869/2006)

• Reflection Paper on Markers used for Quantitative and Qualitative Analysis of Herbal Medicinal Products and Traditional HerbalMedicinal Products (EMA/HMPC/253629/2007)

5

Regulatory Background - Quality

• Herbal drug– Complete monograph required– Specifications and analytical methods– Batch results– Including microbiological quality, pesticides, aflatoxins, radioactivity,

heavy metals, impurities• Herbal preparation

– Complete monograph required– Specifications and analytical methods– Batch results– Characteristics, identification, purity, assay (chromatographic methods)

• Herbal medicinal product– Qualitative and quantitative determination of drug substances (herbal

preparations) – Determination of constituents with known therapeutic activity or

analytical markers– Stability testing including fingerprint chromatograms (HPLC, TLC)

6

Assay - Concepts

• Constituents with known therapeutic activity– Chemically defined substances or groups of substances– Generally accepted to contribute substantially to the therapeutic activity

• Markers– Chemically defined substances or groups of substances whtich are of

interest for control purposes independent whether they have anytherapeutic acitivity

– Serve to calculate the quantity of the herbal substance or the herbalpreparation in the herbal medicinal product

– Active marker are generally accepted to contribute to the therapeuticacitivity.

– Analytical marker are determined for analytical purposes only.

7

Extracts

• Standardised extracts are adjusted within an acceptable tolerance to a given content of constituents with known therapeutic activity;standardisation is achieved by adjustment of the extract with inert material or by blending batches of extracts.

• Quantified extracts are adjusted to a defined range of constituents; adjustments are made by blending batches of extracts.

• Other extracts are essentially defined by their production process (state of the herbal drug or animal matter to be extracted, solvent, extraction conditions) and their specifications.

8

Method development - Requirements

• Analytical marker– Stable during processing and storage– Characteristic and selective for the herbal drug => batch specific control– Identification– Sufficient concentration in the herbal drug/preparation/medicinal product– Selective in the presence of the product matrix– Easy to determine– Reference material commercially available

• Analytical method– Suitable for the individual purpose– Reproducible– Straight and simple, no complex detection (MS)– Easy, fast and robust method => „Time is money“– Suitable for batch release and stabiltiy testing

9


• Combination products– One herbal drug preparation is already a mixture of thousands of

compounds– Selective analytical markers / characteristic markers– Reference standards– Markers in low concentrations– Complex matrices/background– Complex sample preparations

• Combination products, examples– Herbal tea with different drugs– Dragee containing dry extract preparations of different drugs– Liquidum containing ethanolic extracts of several drugs (some as

drug substances, some as further ingredients < 5%)

10


• Choice of the stationary phase– Optimisation time consuming– Automated column switching useful– Depends on chemical structure of the analyte (screening

process necessary)

• Optimisation of the mobile phase system– Depends on purpose (assay, fingerprint)– Optimisation of sample preparation must be considered

• Suitable detection including optimisation of detection parameters– Different wavelenths necessary for different markers– Different selectivity with respect to detection wavelength possible– MS detection offers more selectivity

11

Method development - Simplification

• Development of a generic method for screeningpurposes

• Determination of a huge variety of plant secondarymetabolites

• Build-up of a library containing approx. 300 compounds– Retention times– UV spectra– MS and MS/MS spectra– HRMS spectra

12

Method development - Substance classes

• Main substance classes involved during method development– Flavonoids (109)– Terpenes (30) – Alkaloids (26)– Anthranoids (21) – Essential oils (18) – Cumarines (14), tanning agents (14), hydroxy cinnamonic

acids (13), saponines (12), iridoids (6)• Futher substance classes investigated

– Glycosides, amino acids, glukosinolates, bitterns, salicinderivatives, fats, fruit acids, kavapyrones, phenolic carbonic acid, carbonic acid esters, fatty acids, curcumines, furochromes, kavalactones, naphthochinones, phytosterines, polyketides, xanthines

13

Method development - Flavonoids

Silymarines: silybin, isosilybin, silichristin, silydianin

Apigenin, luteolin, caempferol, quercetin, isorhamnetin, amentoflavon, ginkgetin, ginkgolid A,B,C, terpenes

Hyperosid, rutosid, Spiraeosid, vitexin, (-) catechin / (-) epi-catechin

Milk thistleGingko leavesHawthorn

14

Method development – further substance classes

Cumarines, e.g. umbelliferon

Essential oils, e.g. carvon

Anthranoids, e.g. emodin

Alkaloids, e.g. theobromin

Terpenes, e.g. valerenic acid

Iridoids, e.g. gentiopikrin

Saponines, e.g. primulaverin

Hydroxy cinna-monic acids, e.g. chlorogenic acid

Tanning agents, e.g. (-) epica-techin

15

Chromatographic development

UPLC HSS T3 RP 18 endcapped 1,8 µm2,1x100 mm

UPLC HSS C18 RP 18 endcapped 1,8 µm2,1x100 mm

UPLC BEH Shield RP 18 1,7 µm, 2,1x100 mm

(-)Epica

tech

inga

llat -

5,2

27 5,56

1

Isoq

uerc

etin -

5,69

2

Ros

mar

insä

ure

- 6,8

80

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00

(-)E

pica

tech

inga

llat -

5,6

92

5,87

4

Isoq

uerc

etin

- 6,

018

Ros

mar

insä

ure

- 7,4

62

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00

(-)Epica

tech

inga

llat -

6,7

00

Rut

osid -

7,05

3

Hyp

eros

id -

7,25

1Is

oque

rcet

in -

7,35

7

Ros

mar

insä

ure

- 7,9

53

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

5,00 5,50 6,00 6,50 7,00 7,50 8,00 8,50 9,00

16

Chromatographic development

• Mobile Phase – Eluent A: Water/ 0.05% TFA– Eluent B: Acetonitrile/methanol 70/30 (v/v) / 0.05% TFA

• Gradient

• Flow: 0.45 mL/min• Column temperature: 40 °C• Injection volume: 2 µL

1001005025201055Eluent B [%]373522168520t [min]

17

Application to reference standard mixtures

• Gallic acid, chlorogenic acid, caffeic acid, umbelliferon, epicatechingallat, hyperosid, rutosid, isoquercitrin, rosmarinic acid, quercetindihydrate, anethol, rhein, emodin, valerenic acid, glycyrrhicic acid

Gal

luss

äure

- 1,

704

Chl

orog

ensä

ure

- 6,4

07K

affe

esäu

re -

6,76

6

Um

belli

fero

n - 1

0,20

9(-)

Epi

cate

chin

galla

t - 1

0,94

7R

utos

id -

11,7

65H

yper

osid

- 11

,876

Isoq

uerc

etin

- 12

,095

Ros

mar

insä

ure

- 12,

990

Que

rcet

in D

ihyd

rat -

19,

135

Ane

thol

- 22

,080

Rhe

in -

22,3

49

Em

odin

- 25

,017

Val

eren

säur

e - 2

5,74

8

Gly

cyrrh

etin

säur

e - 2

7,39

4

AU

0,00

0,10

0,20

0,30

0,40

0,50

0,60

Minutes0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,0

18

Orie

ntin

- 9,

541 Is

oorie

ntin

- 9,

757

Isov

itexi

n - 1

1,50

0

Rut

osid

- 12

,440

Hyp

eros

id -

12,7

13Is

oque

rcitr

in -

12,9

81

Nar

inge

nin

- 19,

995 Q

uerc

etin

Dih

ydra

t - 2

0,20

7S

ilybi

n - 2

0,89

7

Api

geni

n - 2

1,92

8Is

orha

mne

tin -

22,0

82K

ämpf

erol

- 22

,220

Am

ento

flavo

n - 2

4,55

4

Gin

kget

in -

27,1

6727

,208

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

Minutes0,00 2,00 4,00 6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00 22,00 24,00 26,00 28,00 30,00

Application to reference standard mixtures

• Orientin, isoorientin, isovitexin, rutosid, hyperosid, isoquercitrin, naringenin, quercetin-dihydrate, silybin, apigenin, isorhamnetin, caempferol, amentoflavon, ginkgetin

19

Detection development

• Detection using ESI+ with respectto literature data of flavonoids

700 L/h700 L/hDesolvation Gas (Ar)

25 L/h25 L/hCone gas (N2)

300 °C300 °CDesolvation temperature

100 °C100 °CSource temperature

1,0 V2,0 VExtraction Cone

50 V60 VSampling Cone

3,5 kV6 kVCapillary

ESI -ESI +

20

Applications - Diaphoretica tea

• (1) lime blossom, (2) elderflower, (3) meadowsweet blossom, (4) chamomile blossom, (5) mallow blossom

(1) (2)

(3)

(4) (5)

(1) (2)

(3)

(4) (5)

21


• Main components

22


• Main components

23

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00

Marker der Kamillenblüten

Marker der Mädesüßblüten

Marker der Holunderblüten Marker der Lindenblüten

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00

Marker der Kamillenblüten

Marker der Mädesüßblüten

Marker der Holunderblüten Marker der Lindenblüten


320 nm

lime blossomelder flower

chamomile

meadowsweet

24


Mar

ker d

er K

amill

enbl

üten

- 12

,636

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

0,090

0,100

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

12,638 Peak 1200,8

266,8

337,1

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker d

er K

amill

enbl

üten

- 12

,636

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

0,090

0,100

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

12,638 Peak 1200,8

266,8

337,1

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

nm200,00 250,00 300,00 350,00 400,00 450,00

Markerchamomile

25


1/89/6 15:28:0707-Jul-2011YCA137Kamillenblueten

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100Entwicklung_Phyto_0328 2221 (12.479) Cm (2200:2222-2235:2277) 1: TOF MS ES+

6.72e4271.0610

138.3626182.5264

272.0634

433.1137273.0657397.1870

347.1567556.2805434.1208 579.1620 1096.4473890.1795

865.2097665.4574

810.0192686.2034 951.3950 1077.6508

1138.5767

18:54:4807-Jul-2011YCA137Kamillenblueten

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0

100Entwicklung_Phyto_0333 2479 (13.583) Cm (2477:2480-2489:2592) 1: TOF MS ES-

75.1690.7354

365.3552

143.2422

261.9296

618.4816

517.6545

474.0170

641.3214

1161.2317

929.4293

1051.4409

1167.3527

Apigenin

Apigenin-7-6“-malonyl-glucoside

26


Mar

ker d

er M

ädes

üßbl

üten

- 13

,746

13,9

60

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

13,749 Peak 1202,0

252,7 365,8

AU

0,00

0,05

0,10

0,15

0,20

0,25

0,30

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker d

er M

ädes

üßbl

üten

- 13

,746

13,9

60

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

13,749 Peak 1202,0

252,7 365,8

AU

0,00

0,05

0,10

0,15

0,20

0,25

0,30

nm200,00 250,00 300,00 350,00 400,00 450,00

Spiraeosid

Markermeadowsweet

27


Mar

ker

der

Lin

denb

lüte

n - 2

0,14

4

AU

-0,002

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

0,016

0,018

0,020

Minutes15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

20,175 Peak 2199,6

266,8

314,8

434,9 462,3 489,7

AU

0,000

0,005

0,010

0,015

0,020

nm200,00 250,00 300,00 350,00 400,00 450,00

MarkerLime blossom

28


1/89/3 16:09:2307-Jul-2011YCA137Lindenblueten

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0


676287.0543

215.8230

126.5462 154.5304 249.1772

617.1235

288.0602

309.0958

318.7343

354.1691

394.8332 459.1043

452.9057 554.3755497.2331

559.8870

649.0809

1188.9913

1129.00701089.1062762.0126

742.4511 883.4445780.5224

984.6065

19:36:1207-Jul-2011YCA137Lindenblueten

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0


1.03e5593.1051

164.4900 330.7456179.1554 232.5752358.0602

541.4050511.5008463.7626

594.1088

707.0953

595.1100

596.1154

708.0967

873.9998845.0396709.1012943.3757 1067.4470

972.05211154.3850

Tilirosid

29


Mar

ker

der

Holu

nder

blüt

en -

16,7

30

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

0,24

0,26

0,28

0,30

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

16,719 Peak 1

218,4234,9

319,8

444,9 479,8

AU

0,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

nm200,00 250,00 300,00 350,00 400,00 450,00

Markerelderflower

30


1/89/8 16:50:4407-Jul-2011YCA137Holunderblueten

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150

%

0


3.59e4364.2236

177.0555

171.1489

188.1766346.2113

198.1505

562.2526

365.2263

540.2710

366.2333453.1761 481.2626

578.2266

579.2296

614.3640 692.19931077.6841987.9611

756.7689844.1667

889.09971132.2266

1162.6813

31


479256/35512.5miquelianin(quercetin-3-O-ß-D-glucuronid)

465.1255/35511.5isoquercitrin

465.1255/35411.3hyperosid

611.1255/35611.1rutosid

meadowsweetblossom

271267/33820.2apigenin

449.1256/35510.8luteolin-7-glucoside

177217/3239.8herniarin

chamomile

449.1265/34812.9astragalin (caempferol-3-O-glucosid)

433.1266/34812.7afzelin (caempferol-3-rhamnosid)


611.1255/35611.1rutosid

355.1218/3306.4chlorogenic acid

lime blossom

361242/32912.0rosmarinic acid


611.1255/35611.1rutosid


181217/3235.8caffeic acid

elderflower

[M+H]UV maxima [nm]RT [min]analytical marker

32

Applications – Rooibos tea

• Main components

• as well as flavonols, flavons like quercetin, luteolin, apigenin and corresponding glycosides

33


Erio

dict

yol-I

-glu

cosi

d - 5

,885

Erio

dict

yol-I

I-glu

cosi

d - 6

,852

Asp

alat

hin

- 8,3

06

Vite

xin

- 8,6

98

Orie

ntin

/Isoo

rient

in -

9,46

89,

633 N

otho

fagi

n - 1

0,78

110

,938

Isov

itexi

n - 1

1,37

8

Rut

in -

11,9

04

Hyp

eros

id -

12,3

41Is

oque

rcitr

in -

12,4

01

Asp

acat

- 12

,781

AU

0,00

0,20

0,40

0,60

0,80

1,00

1,20

Minutes5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00

34


740.028612.8Aspacat465.1256/35512.4Isoquercitrin465.1256/35512.3Hyperosid611.1256/35511.9Rutin433.1270/33711.3Isovitexin437.5225/28610.8Nothofagin449.4269/3509.5Isoorientin433.1267/3498.6Vitexin453.5227/2868.3Aspalathin452.12876.8Eriodictyol-II-glucosid452.12875.9Eriodictyol-I-glucosid[M+H]UV maximaRT [min]marker

35

Applications – Stomach therapeutic agent

• (1) blessed thistle, (2) liquorice root, (3) silverweed, (4) wormwood, (5) chamomile, (6) angelica root

36


• Main components

37


Mar

ker

des

Wer

mut

krau

ts -

22,3

37

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,341 Peak 1203,2

255,7

347,7

451,7 492,8AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker

des

Wer

mut

krau

ts -

22,3

37

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,341 Peak 1203,2

255,7

347,7

451,7 492,8AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

nm200,00 250,00 300,00 350,00 400,00 450,00

Artemetin348 nm

wor

mw

ood

38


Mar

ker

der

Ang

elik

awur

zel -

22,

550

AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0,045

0,050

0,055

0,060

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,554 Peak 2203,2

257,6

322,9

AU0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker

der

Ang

elik

awur

zel -

22,

550

AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0,045

0,050

0,055

0,060

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,554 Peak 2203,2

257,6

322,9

AU0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

nm200,00 250,00 300,00 350,00 400,00 450,00

Osthol323 nm

mar

kera

ngel

ica

root

39


Mar

ker d

er S

üßho

lzw

urze

l - 2

2,92

5

AU

0,00

0,20

0,40

0,60

0,80

1,00

1,20

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,929 Peak 1

199,6

251,4

329,1

AU

0,00

0,20

0,40

0,60

0,80

1,00

1,20

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker d

er S

üßho

lzw

urze

l - 2

2,92

5

AU

0,00

0,20

0,40

0,60

0,80

1,00

1,20

Minutes10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00 21,00 22,00 23,00 24,00 25,00

22,929 Peak 1

199,6

251,4

329,1

AU

0,00

0,20

0,40

0,60

0,80

1,00

1,20

nm200,00 250,00 300,00 350,00 400,00 450,00

glycyrrhicic acid251 nm

mar

kerl

iquo

rice

root

40


Mar

ker d

er K

amill

enbl

üten

- 7,

804

AU

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

0,55

0,60

Minutes5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

7,804 Peak 1

198,4

215,4

236,7

319,2

AU

0,00

0,10

0,20

0,30

0,40

0,50

nm200,00 250,00 300,00 350,00 400,00 450,00M

arke

r der

Kam

illen

blüt

en -

7,80

4

AU

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

0,45

0,50

0,55

0,60

Minutes5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

7,804 Peak 1

198,4

215,4

236,7

319,2

AU

0,00

0,10

0,20

0,30

0,40

0,50

nm200,00 250,00 300,00 350,00 400,00 450,00

319 nm

mar

kerc

ham

omile

41


cone = 60 V1/97/10 20:16:1327-Jul-2011YCA137Kamillenblueten TE LC20641

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0


6.64e5177.0542

195.0649

379.0999

196.0679

197.0727

380.1026713.2277

556.2313 751.1781 1107.2854880.3423

ferulic acid ester

42


Mar

ker d

es G

änse

finge

rkra

uts

- 7,3

50

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

0,24

0,26

0,28

0,30

Minutes5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

7,351 Peak 1

218,4

242,9

329,7

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

0,24

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker d

es G

änse

finge

rkra

uts

- 7,3

50

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

0,24

0,26

0,28

0,30

Minutes5,00 6,00 7,00 8,00 9,00 10,00 11,00 12,00 13,00 14,00 15,00 16,00 17,00 18,00 19,00 20,00

7,351 Peak 1

218,4

242,9

329,7

AU

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0,16

0,18

0,20

0,22

0,24

nm200,00 250,00 300,00 350,00 400,00 450,00

330 nm

mar

kers

ilver

wee

d

43


cone = 60 V1/97/9 19:34:5227-Jul-2011YCA137Gaensefingerkraut TE LC20647

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0


1.73e4163.0390

377.0483

186.0557

220.1692

303.0517

556.2783393.0218

465.1054

731.1085

557.2822

558.2762732.1190

1101.1495759.1953 984.3804

cone = 50 V1/97/9 00:24:3328-Jul-2011YCA137Gaensefingerkraut TE LC20647

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0


1.23e5173.0081

191.0190

353.0497

192.0223

324.9488

707.1089

354.0553

549.0485491.0036

565.0150

729.0914

757.1799

761.0263871.1669

1111.2271

mono substitutedcaffeoyl quinic acid

44


Mar

ker d

es B

ened

ikte

nkra

uts

- 18,

74

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

0,090

0,100

Minutes6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00

18,749 Peak 1

218,4

244,1

330,9

456,7474,2

AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0,045

nm200,00 250,00 300,00 350,00 400,00 450,00

Mar

ker d

es B

ened

ikte

nkra

uts

- 18,

74

AU

0,000

0,010

0,020

0,030

0,040

0,050

0,060

0,070

0,080

0,090

0,100

Minutes6,00 8,00 10,00 12,00 14,00 16,00 18,00 20,00

18,749 Peak 1

218,4

244,1

330,9

456,7474,2

AU

0,000

0,005

0,010

0,015

0,020

0,025

0,030

0,035

0,040

0,045

nm200,00 250,00 300,00 350,00 400,00 450,00

bles

sed

this

tle

45


cone = 60 V1/97/12 21:38:5427-Jul-2011YCA137Benediktenkraut TE LC20640

m/z100 200 300 400 500 600 700 800 900 1000 1100

%

0


4.51e3556.2776

250.9719

122.6828492.2043269.2727

557.2757

613.3107 731.1469934.1755

817.7351 1052.2111 1135.1193

46


217222/31018.3bergapten

1633256.1umbelliferon


181249/2955.3caffeic acidangelica root


181217/3235.3caffeic acid

153.0 ([M-H])259/2952.2protocatechic acidblessed thistle


181217/3235.3caffeic acidwormwood

465.1263/35012.8myricitrin

611.1255/35611.0Rutosid

181217/3235.3caffeic acidsilver weed

[M+H]UV maxima [nm]RT [min]analytical marker

47

Identification of analytical markers / Database

• Evaluation of UV data => compound classes• High resolution MS data => exact molecular masses

– Elemental composition– Fragmentation– Interpretation of MS spectra– Comparison with literature data

• Retention times• Advantages

– Easy screening method for herbal medicinal products– Fast method optimisation– Database for identification of markers

48

Company Group

Laboratory Services, Reference Standards, Drug Safety & Non-Clinical Assessment

Regulatory Affairs

Laboratory Services

Pharmaceutical R&D, Product Development

Thank you very much

for your attention!

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