using lc ms to quantify and identify natural toxins in food and environmental sample dec 2011

Today’s Speakers

Dr. Michael Sulyok Researcher Center for Analytical Chemistry, Department IFA-Tulln, University of Natural Resources and Life Sciences, Vienna Dr. Michael A. Quilliam Principal Research Officer National Research Council of Canada, Institute for Marine Biosciences, Halifax, Canada Mr. Arne Dübecke Technical Project Manager Quality Services International GmbH (QSI), Bremen, Germany

LC-MS Methods for Shellfish Toxin Analysis

using AB Sciex QTRAP Instruments

NRC Institute for Marine Biosciences

Dec 15, 2011 AB Sciex Webinar

Michael A. Quilliam


Shellfish Poisoning – An international Problem

• Hundreds of deaths and thousands of illnesses occur each year globally.

• The estimated global economic impact of shellfish toxins is estimated at 4 billion US dollars per year. http://www.oceansatlas.org/unatlas/uses/uneptextsph/wastesph/2571gs71041health.html


Toxic

Algae People Digestive Gland

Toxic Incident

Shellfish Poisoning

Time

Detoxification

Monitoring can prevent toxic incidents and save loss of product


Marine Algal Toxins

O O

O

O

O H

O

O

N H

O

O

O H

O

O H

Azaspiracid

Dinophysis spp. Okadaic Acid

O

O H

O

O H

O H

O O O

O H

H

O H

O H

O

O

Diarrhetic Shellfish Poisoning

Domoic Acid

H

C H 3

C H 3

N

H

C O O H

C O O H

C O O H

Amnesic Shellfish Poisoning

Pseudonitzschia spp. .

O

O

N H 2

N

N N H 2

+ N H

N H

N H 2

+

O H

O H

H H

Cl -

Cl -

Saxitoxin

Paralytic Shellfish Poisoning

Alexandrium tamarense

Azadinium spinosum

. Neurotoxic Shellfish Poisoning

Brevetoxin Karenia brevis

O

O

O O

O O

O

O

C H 3

H

O H

O

C H 3

H H

H

C H 3

H

H

C H 3

H

O

C H 3

H C H 3

H

H

H

H

H

H

O

C H 3

O H

R

O

O

O O

O O

O

O

C H 3

H

O H

O

C H 3

H H

H

C H 3

H

H

C H 3

H

O

C H 3

H C H 3

H

H

H

H

H

H

O

C H 3

O H

R

Azaspiracid Shellfish Poisoning

O

O O

O O H

O H O

O O

O

O O H O

O

Dinophysis acuminata

Pectenotoxins

Yessotoxins

O

OO

O

OO

O

O

OO

O

NaO3SO

OH

HO

NaO3SO

Protoceratium reticulatum


O O

O

O

O

NH

O

O

HO

O

OH

H

H

H

HH

H

H

R3

HO

O R1

R2

R4

Azaspiracid R1 R2 R3 R4 [M+H]+

AZA1 H H CH3 H 842.5

AZA2 H CH3 CH3 H 856.5

AZA3 H H H H 828.5

AZA4 OH H H H 844.5

AZA5 H H H OH 844.5

AZA6 H CH3 H H 842.5

Azaspiracid Group

Regulatory Level:

160 mg total AZAs per kg edible

tissue

Over 30 structural analogues

have been detected!


The Analytical Challenges

• To provide monitoring of shellfish for a large

number of regulated toxins with a wide range of

structures.

• To detect and identify toxins at low levels in

shellfish before they cause problems.

earliest possible warning required.

• To operate under ISO17025 with fully validated

methods and a high degree of precision and

accuracy.


Traditional Mouse Bioassay for

Shellfish Toxins

• Poor sensitivity

• Serious problems with false positives and negatives.

• Poor correlation with human oral toxicity

• Lack of validation


Alternative Methods

• Multi-toxin methods are most desirable for comprehensive monitoring of a wide range of toxins.

• There are a number of specific assays (e.g., ELISA) and chemical analysis methods (e.g., LC) available.

• Liquid chromatography combined with mass spectrometry (LC-MS) is now the method of choice EU Directive 15/2011

• Reference materials are required

for all analytical methods.


• Calibration of instrumentation and assays

• Method development

• Method validation – determination of trueness

• Traceability of measurements

• Quality assurance and quality control

Why Do We Need Reference Materials?

Pure shellfish toxins are very rare. If they are commercially

available, they are very expensive and of unknown purity.

It is therefore difficult to have accurate calibration.


• Accurate calibration solutions of

pure toxins in flame-sealed

ampoules

Assured stability and accuracy

No hazard from powdered

toxins

• Ready for use as a calibrant:

Appropriate concentrations

Biotoxin Calibration Solutions

NRC’s approach:

Calibration Solution CRMs

First CRMs:

- domoic acid in 1989

- okadaic acid in 1993

- saxitoxin analogs in 1994

Currently 32 CRMs, including

matrix CRMs


Quilliam, M. A.; Thomson, B. A.;

Scott, G. J., and Siu, K. W. M.

Ion-spray mass spectrometry of

marine neurotoxins.

Rapid Commun. Mass Spectrom.

3, 145-150, 1989.

Electrospray ionization (Ion-spray)

on a SCIEX API-III prototype

LC-MS instrument.

Atmospheric Pressure Ionization LC-MS, 1989


Toxin Timeline

• 1989 First use of ESI-LC-MS for marine toxins

• 1997 First report of a multi-toxin LC-MS method

Time (min)

10 15 20 25 30 35

m/z 312.5

m/z 508.3

m/z 692.5

m/z 706.5

m/z 805.5

m/z 819.5

m/z 946.5

m/z 1160.7

m/z 876.5

m/z 894.5

m/z 842.5

DAGYM

SPXYTX

PTX2sa

OA

DTX2

OA-D8:2

PTX2

DTX1

AZA1

Liquid chromatography-mass spectrometry: a

universal method for the analysis of toxins?

MA Quilliam. 8th Int. Conf. Harmful Algae, Vigo,

Spain, June 1997.


Toxin Timeline

• 1989 First use of ESI-LC-MS for marine toxins

• 1997 First report of a multi-toxin LC-MS method

• 2000 NZ implements LC-MS monitoring

• 2001 Canada implements LC-MS monitoring

• 2011 EU directive 15/2011 on LC-MS as reference method

The availability of readily available reference materials for all

the toxin groups was the key factor in acceptance of LC-MS

as a monitoring tool .


QTRAP 4000 LC-MS

Linear Ion Trap


QTRAP 5500 LC-MS

Linear Ion Trap


SRM with EPI using QTRAP Technology

• Scheduled SRM for detection and quantitation of a large number of analytes in a single run.

• QTRAP technology allowing SRM-triggered enhanced product ion (EPI) spectra acquistion (IDA) for compound confirmation

• Rapid polarity switching on the QTRAP 5500

• Joint AB Sciex-NRC project:

AB SCIEX iMethods for toxins

SRM and EPI spectral libraries


Negative Product Ion Spectrum of a New YTX Analogue

m/z

200 400 600 800 1000 1200

% R

ela

tive Inte

nsity

20

40

60

80

100

97671 855

967

925

559349293237181

743

713

631475

x10

489

799

405

1075

657

687

O

OO

O

O

O

O

O

OO

O

O

HO

OH

OHH

H

H

HH

H H

H

H

H

S

O

O

-O

H

H

HH

H

H

H

H

855

925

967

799

713

687631

559

489

405

181

237

293

349

657

[1-SO3]-

41a-Homo-yessotoxin

1155 (-)


QTRAP Product Ion Spectra Acquired for CRM-AZA-Mus

AZA3

AZA1

AZA2


AZA3 = 14 mg/kg

(10x lower than RL)

Library Search of QTRAP Product Ion Spectra

AZA3

Acquired spectrum

Library match: AZA3


LC-MS Methods for Lipophilic Toxins

Three methods have been published using reversed phase columns with various mobile phases:

• Quilliam et al., 1997

• Acidic mobile phase

• Requires rapid polarity switching

• Stobo et al., 2005; McCarron et al., 2011

• Neutral (pH 7) mobile phase

• Positive and negative time segments

• Gerssen et al., 2009

• Basic (pH 11) mobile phase


Neutral LC conditions – group separation

Gemini-NX C18 3 µm, 50 × 2 mm with 300

µL/min water (A) and MeCN (B) each with 5

mM ammonium acetate, 25-100% B over 5

min, hold 3 min.

• Good peak shapes

• Good resolution

• Separation of toxins into periods according to optimum ionization efficiency

• Suitable for use on the QTRAP4000

YTX

OADTX2

PTX2

AZA3

DTX1

AZA1

AZA2

Time (min)

2 3 4 5 6 7

PTX1

(- SRM) (+ SRM)


LC-MS analysis of lipophilic toxins in CRM-FDMT1

856 > 672

842 > 672

828 > 658

876 > 823COOH-YTX

OA

817 > 255

YTX

PTX2

AZA3

AZA6

AZA1

AZA2

(- SRM) (+ SRM)

45-OH-YTX

DTX2

DTX1

1173 > 1093

* *

**

*

*

*

*

*

**

842 > 658

m/z > m/z

1141 > 1061

1157 > 1077

803 > 255

Retention time (min)

2 3 4 5 6 7 8

Freeze-dried mussel

tissue CRM for multi-

toxin analysis

McCarron et al.,

Anal. Bioanal. Chem.

400, 835 (2011).


YTX

OA DTX2

PTX2

AZA3

DTX1

AZA1

AZA2

Time (min)

3 4 5 6 7 8

PTX1

Acidic LC conditions with polarity switching

892 → 821

876 → 823

828 → 658

842 → 672

856 → 672

(+ SRM)

m/z → m/z

1141 → 1061

803 → 255

817 → 255

(- SRM)

m/z → m/z

• Good peak shapes and resolution

• Separation suited to the QTRAP5500 which is capable of rapid polarity switching

C18 3 µm, 50 × 2 mm with 300 µL/min.

water (A) and MeCN (B) each with 50 mM

formic acid, 2 mM ammonium formate.

25-100% B over 5 min, hold 3 min.


Application of an Automated LC-MS Method

DSP incident

on West Coast

(2011)

• 30 people ill

from mussels

• Variety of

toxins

detected

Gymnodimine

Pectenotoxin

PTX2sa

7epi-PTX2sa

PTX2

DTX1

Spirolide

D

SPX-C

SRM detection mode

?

DSP toxin

O

O O

O

O O

OHO

OH

OH

O OH

OH


Library search of MS/MS spectrum

• Library search

identified toxin as

Pinnatoxin-G

• Poisoning incident in

China associated with

Pinna attenuata

shellfish (1990)

N

OO

O

OOH

O

HO

H

Pinnatoxin G

Unknown, M+H = m/z 694


Saxitoxin Group (Paralytic Shellfish Poisoning, PSP)

N

NNH2

+ NH

NH

NH2

+

OH

OH

H

R4

R1

R2 R

3

O

O

NH2

OH

O

O

NHSO3

-

Toxin R1 R2 R3

STX H H H GTX2 H H OSO3

-

GTX3 H OSO3- H

NEO OH H H GTX1 OH H OSO3

-

GTX4 OH OSO3- H

GTX5 H H H C1 H H OSO3

-

C2 H OSO3- H

GTX6 OH H H C3 OH H OSO3

-

C4 OH OSO3- H

dcSTX H H H dcGTX2 H H OSO3

-

dcGTX3 H OSO3- H

dcNEO OH H H dcGTX1 OH H OSO3

-

dcGTX4 OH OSO3- H

STX = saxitoxinNEO = neosaxitoxinGTX = gonyautoxins

1

3

7

9

1012

6

R4

11


412/332 412/314

Time (min)

0 5 10 15 20 25

NEO

STX

B1

GTX3

C2

C1

GTX4

GTX1

GTX2

Ion Sums

380/300 380/282 300/282 300/204

396/316 396/298 316/298 316/220

x2

x4

HILIC-MS of PSP Toxins in Algae

Alexandrium tamarense,

Shelburne, Nova scotia

Hydrophilic

Interaction

LC (HILIC)


Advanced Instrumentation Validated analytical procedures

Time (min)

2 3 4 5 6 7

YTX

OADTX2

PTX2

AZA3

DTX1

AZA1AZA2

PTX1

(- SRM) (+ SRM)

Matrix CRMs Accurately certified calibrants

A Complete Analytical Solution

Accurate and

comprehensive

monitoring of

shellfish for

toxins.

AB Sciex Webinar on Natural Toxins QSI 2011

48

Quantification and Identification of Pyrrolizidine Alkaloids in Honey by using AB Sciex 4000 QTRAP instruments AB Sciex Webinar

15th December 2011 Arne Duebecke

© Quality Services International

AB Sciex Webinar on Natural Toxins

Quality Services International – founded in 1954

Portfolio

Quality Control of Food, Cosmetics, Pharmaceutics

and Commodities

Product Development

Quality Management

Training and Support

QSI 2011

49

AB Sciex Webinar on Natural Toxins

Quality Services International – founded in 1954

Team of over 50 experts

Chemists

Biologists

Food Chemists

Pharmacists

Environmental Scientist

Biological/Chemical Technicians

Marketing & Sales Specialists

QSI 2011

50


51

Pyrrolizidine Alkaloids (PAs)

• PAs are secondary plant metabolites

• PAs are formed by more than 6000 plant species

• PAs are toxic to the liver

• PAs show cancerogenic properties


52

1977 Up to 3.9 mg/kg PAs found in honey derived from Senecio jacobaea L. (Deinzer et al.); confirmed by Kempf et al. (2011)

1988 PAs containing a double bond in the 1,2-position are potentially toxic to the liver and are under suspicion of causing cancer (WHO)

1992 Limit for PAs in Pharmaceutical Preparations (German Federal Institute for Risk Assessment, BfR)

2004 PAs found in honey derived from Echium spec. And Heliotropium spec. (Beales et al.)

Retrospective


53

2004 – 2008 Several PA-projects in the UK (COT), the Netherlands (RIKILT), Australia (FSANZ) and Germany (Universities of Würzburg & Braunschweig, BfR, SKLM)

2011 Codex Alimentarius discussed PA-issue and will evaluate the possibility to develop a code of practice

August 2011 German Federal Institute for Risk Assessment (BfR) recommended a maximum daily intake of 0.007 µg PAs/kg bodyweight

November 2011 EFSA statement on PAs in Food and Feed

Recent Developments


54

The Challenge – Availability of PAs In 2008 about 10 PA-references commercially available In 2011 about 15 to 20 PA-references commercially available

Problem: There are several hundred different PAs out there!!! Project in progress: Extraction of PA-references

In case you have any PA-references and are willing to share them (esp. the ones that are NOT

commercially available), please tell me.


55

Typical Mix of Spanish Bee Pollen

Bee Pollen of Echium vulgare


56

Pyrrolizidine Alkaloids (PAs)

Heliotridine

N

OH

HOH

Retronecine

N

OHOH H

Most PAs share common structural features


57

Typical Fragments Beales et al. 2004, Colegate et al. 2005

N

OH CH2

O

H

OH

H+

m/z 172

N

CH2

O

H

OH

AngO

H+

m/z 254

PA-N-Oxides

N

OH CH2

+

m/z 138

N

CH2

+

m/z 120

Tert. PAs


58

Isomers

N

HOHO

O OH

OH

Lycopsamine

N

HOHO

O

OH

OH

Intermedine

N

HOHO

O

OH

OH

Indicine

N

HOHO

O

OH

OH

Echinatine

N

HO

O

OH

OH

OH

Rinderine

Retronecine -based

Heliotridine-based


59

Isomers

N

HOHO

O OH

OH

Lycopsamine

N

HOHO

O

OH

OH

Intermedine

N

HOHO

O

OH

OH

Indicine

N

HOHO

O

OH

OH

Echinatine

N

HO

O

OH

OH

OH

Rinderine

(-)-viridifloric acid

(-)-trachelanthic acid

(+)-trachelanthic acid


60

PREC 172 – Cynoglossum sp. (Hound‘s Tongue)

m/z 172 typical for e.g. lycopsamine-N-oxide or its isomers

m/z 316 is precursor ion of m/z 172

Exact Mass of [M+H]+ of e.g. Lycopsamine-N-Oxide = 316


61

PAs in Forget-me-not


62

Plant Material (Plant Parts, Pollen, Nectar)

Food (Honey, Herbs, Leafy Vegetable, Flour)

Feed

Insects

Milk Eggs Meat?


63

Natternkopf (Echium vulgare)

N

HO

O

CH3

HOH

OH CH3

CH3

OH

O

O

CH3

CH3

H

Echimidin


64

Probenaufarbeitung (Beales et al. 2004, Betteridge et al. 2005, Colegate et al. 2005)


65

Festphasenextraktion (Solid Phase Extraction, SPE)

Starker Kationentauscher

- Konditionierung mit 0,05M H2SO4

- Probenaufgabe

- Waschen

- Elution mit ammoniakalischem MeOH


66

Probenaufarbeitung (Beales et al. 2004, Betteridge et al. 2005, Colegate et al. 2005)


67

HPLC-MS/MS-Analyse

• LC-MS/MS im positiv Modus

• Multi Reaction Monitoring (MRM)

• 1 Quantifier, 2 Qualifiers

• Zeit/Messung: 10 min.

© Quality Services International


68

Wiederfindung und Linearität

Wiederfindung von Lycopsamin in

Polyflora- und Wald-Honig

020

4060

80100

120140

1 2,5 5 7,5 10 15 1 2,5 5 7,5 10 15

Lycopsamine added [µg/kg]

Rec

ove

ry [

%]

Polyfloral Honey Honey Dew Honey

RSD = 5,2% RSD = 12,3 %

Linearität von Lycopsamin in

Polyflora-Honig

R2 = 0,9958

0E+00

1E+06

2E+06

3E+06

4E+06

0 2 4 6 8 10 12 14 16

Lycopsamine [µg/kg]

Res

po

nse

[are

a co

un

ts]


69

PA in Honig aus Chile

IS


70

Thank You for Your Attention!

Arne Dübecke

Quality Services International GmbH

Flughafendamm 9a

D-28199 Bremen, Germany

+49 421 59 47 70

[email protected]

www.qsi-q3.de

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