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
OH
O
O
NH
O
O
OH
O
OH
Azaspiracid
Dinophysis spp.OkadaicAcid
O
OH
O
OH
OH
O O
O
O
H
H
OH
OH
O
O
Diarrhetic Shellfish Poisoning
DomoicAcid
H
CH3
CH 3
N
H
COOH
COOH
COOH
Amnesic Shellfish Poisoning
Pseudonitzschia spp..
O
O
NH2
N
NNH2
+ NH
NH
NH2
+
OH
OH
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
CH
3
H
O
H
O
CH
3
H H
H
CH
3
H
H
CH3
H
O
CH3
H CH
3
H
H
H
H
H
H
O
CH
3
OH
R
O
O
O O
O
O
O
O
CH
3
H
O
H
O
CH
3
H H
H
CH
3
H
H
CH3
H
O
CH3
H CH
3
H
H
H
H
H
H
O
CH
3
OH
R
Azaspiracid Shellfish Poisoning
O
O
O
O
OH
OH
O
O O
O
O
OH O
O
Dinophysis acuminata
Pectenotoxins
Yessotoxins
O
O
O
O
O
O
O
O
O
O
O
NaO3SO
OH
HO
NaO3SO
Protoceratium reticulatum

O O
O
O
O
NH
O
O
HO
O
OH
H
H
H
H
H
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
DA
GYM
SPX
YTX
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
%RelativeIntensity
20
40
60
80
100
97
671 855
967
925
559349293237
181
743
713
631
475
x10
489
799
405
1075
657
687
O
O
O
O
O
O
O
O
O
O
O
O
HO
OH
OHH
H
H
H
H
H H
H
H
H
S
O
O
-
O
H
H
H
H
H
H
H
H
855
925
967
799
713
687
631
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
OA
DTX2
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
O
HO
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
O
O
O
O
OH
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 R3
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 = saxitoxin
NEO = neosaxitoxin
GTX = gonyautoxins
1
3
7
9
10 12
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
OA
DTX2
PTX2
AZA3
DTX1
AZA1
AZA2
PTX1
(- SRM) (+ SRM)
Matrix CRMsAccurately 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

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

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

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

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.

Typical Mix of
Spanish Bee Pollen
Bee Pollen of
Echium vulgare

Pyrrolizidine Alkaloids (PAs)
Heliotridine
N
OH
HOH
Retronecine
N
OH
OH H
Most PAs share common
structural features

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

Isomers
N
HOH
O
O OH
OH
Lycopsamine
N
HOH
O
O
OH
OH
Intermedine
N
HOH
O
O
OH
OH
Indicine
N
HOH
O
O
OH
OH
Echinatine
N
H
O
O
OH
OH
OH
Rinderine
Retronecine -based
Heliotridine-based

Isomers
N
HOH
O
O OH
OH
Lycopsamine
N
HOH
O
O
OH
OH
Intermedine
N
HOH
O
O
OH
OH
Indicine
N
HOH
O
O
OH
OH
Echinatine
N
H
O
O
OH
OH
OH
Rinderine
(-)-viridifloric acid
(-)-trachelanthic acid
(+)-trachelanthic acid

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

PAs in Forget-me-not

Plant Material
(Plant Parts, Pollen, Nectar)
Food
(Honey, Herbs, Leafy Vegetable, Flour)
Feed
Insects
Milk Eggs Meat?

Natternkopf
(Echium vulgare)
N
H
O
O
CH3
HOH
OH CH3
CH3
OH
O
O
CH3
CH3
H
Echimidin

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

Festphasenextraktion
(Solid Phase Extraction, SPE)
Starker Kationentauscher
- Konditionierung mit 0,05M H2SO4
- Probenaufgabe
- Waschen
- Elution mit ammoniakalischem MeOH

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

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

Wiederfindung und Linearität
Wiederfindung
von Lycopsamin in
Polyflora- und
Wald-Honig
0
20
40
60
80
100
120
140
1 2,5 5 7,5 10 15 1 2,5 5 7,5 10 15
Lycopsamine added [µg/kg]
Recovery[%]
Polyfloral Honey Honey Dew Honey
RSD = 5,2% RSD = 12,3 %
Linearität von
Lycopsamin in
Polyflora-Honig
R
2
= 0,9958
0E+00
1E+06
2E+06
3E+06
4E+06
0 2 4 6 8 10 12 14 16
Lycopsamine [µg/kg]
Response
[areacounts]

PA in Honig aus Chile
IS

Thank You for Your
Attention!
Arne Dübecke
Quality Services International GmbH
Flughafendamm 9a
D-28199 Bremen, Germany
 +49 421 59 47 70
 info@qsi-q3.de
 www.qsi-q3.de

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

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