Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No.

2, 2014 285
SPECIAL GUEST EDITOR SECTION

Intralaboratory Validation of a Fast and Sensitive

UHPLC/MS/MS Method with Fast Polarity Switching for the
Analysis of Lipophilic Shellfish Toxins
Ana Braa-Magdalena

European Union Reference Laboratory for Marine Biotoxins (EURLMB), Spanish Food Safety and Nutrition Agency, Ministry of
Health, Social Policy and Equality, CITEXVI (Ciudad Tecnolgica de Vigo), Fonte das Abelleiras No. 4, Campus Universitario de
Vigo As Lagoas-Marcosende, 36310, Vigo, Spain

Jos Manuel Leo-Martins

University of Vigo, Chemistry Faculty, Department of Analytical and Food Chemistry, Chemistry Faculty, Campus Universitario
de Vigo As Lagoas Marcosende, 36310, Vigo, Spain

Thomas Glauner

Agilent Technologies Sales & Services GmbH & Co. KG, Waldbronn, Germany

Ana Gago-Martnez1

European Union Reference Laboratory for Marine Biotoxins (EURLMB), Spanish Food Safety and Nutrition Agency, Ministry of
Health, Social Policy and Equality, CITEXVI (Ciudad Tecnolgica de Vigo), Fonte das Abelleiras No. 4, Campus Universitario de
Vigo As Lagoas-Marcosende, 36310 Vigo, Spain; University of Vigo, Department of Analytical and Food Chemistry, Chemistry
Faculty, Campus Universitario de Vigo As Lagoas Marcosende, 36310 Vigo, Spain

This paper shows the results of an intralaboratory

validation of a fast method for the determination
of lipophilic shellfish toxins working under acidic
conditions using ultra-high performance LC
(UHPLC) with MS/MS. Fourteen lipophilic marine
toxins and domoic acid were acquired with fast
polarity switching. Whereas azaspiracids (AZAs),
pecenotoxins, 13-desmethyl spirolide C (SPX1), and
gymnodimine were analyzed in the positive mode,
yessotoxins (YTXs) were measured in negative
mode. The okadaic acid (OA) group compounds were
analyzed in both positive and negative ionization
modes, and the accuracy of the results for both
were compared. When using dynamic multiple
reaction monitoring (MRM) in fast polarity switching,
LODs were lower and reproducibility and linearity
were better compared to static MRM. The UHPLC
separation allowed for higher sample throughput
in routine use. Compared to the previously used
HPLC/MS/MS method, LODs were improved up to
a factor of 10 in mussel extract. Matrix effects were
evaluated by comparing standards prepared in
solvent with matrix-matched calibrations in blank
mussel extract. For accurate quantification matrixmatched calibrations were used when analyzing
reference mussel materials, providing recoveries for
OA, Dynophysis toxins (DTX)1, DTX2, YTX, AZA1,
and SPX1 between 80 and 120% with RSDs below 8%
over a 3-day validation procedure.
Guest edited as a special report on Marine Toxins by
James Hungerford and Ana Gago-Martnez.
1
Corresponding authors e-mail: anagago@uvigo.es
DOI: 10.5740/jaoacint.SGEBrana

ipophilic shellfish toxins (LTs) produced by several

microalgal species are bioaccumulated in filter-feeding
molluscan shellfish such as mussels, scallops, oysters,
cockles, and clams. On the basis of their chemical structure,
LTs are classified in several groups including okadaic acid (OA)
group, yessotoxin (YTX) group, azaspiracid (AZA) group,
pectenotoxin (PTX) group, brevetoxin (BTX) group, and the
cyclic imines group, mainly composed of spirolides (SPXs) and
gymnodimines (GYMs;1). In order to ensure public safety and
long-term viability of commercial shellfish markets, European
Union (EU) legislation has established maximum limits for
several LT groups that should not be exceeded when placing
shellfish products into the market [Commission Regulation
(EC) No. 853/2004;2)].
Mouse bioassay (MBA) was the longstanding method for
the analysis of marine biotoxins, being the official method
for the main groups of marine toxins in most countries.
With regards to the analysis of lipophilic toxins, the EU
Legislation (EC No. 15/2011;3) established multitoxin
LC/MS/MS as the reference method for the analysis of these
toxins since July2011. Besides ethical concerns regarding
animal use, this decision is based on the main drawbacks of
the MBA, such as low sensitivity and reproducibility, lack of
specificity, and limited information on toxin composition (47).
Recent advances in analytical instrumentation allowed the
development of alternative methods, most of them based on
LC/MS/MS. In fact, this technique is presented as the most
promising alternative for detection and quantitation of marine
toxins in shellfish (812). The usability of LC/MS/MS methods
for this application was shown in the interlaboratory validation
study coordinated by the European Union Reference Laboratory
for Marine Biotoxins (EURLMB;13), as well as in some other
independent interlaboratory validation studies carried out in
the EU using different LC/MS/MS methods (14,15). Whereas
different HPLC conditions were used, common for all methods
is the use of positive and negative electrospray ionization

286 Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No. 2, 2014
Table 1. MS/MS conditions used for the MRM acquisition of data for LTs on a 6460 Agilent Technologies mass spectrometer
Analyte

ESI mode

Precursor ion, m/z

Product ion, m/z

Fragmentor, V

Collision energy, V

Cell accelerator

DA

Positive

312.1

266.1

15

10

DA

Positive

312.1

161.1

15

15

OA

Negative

803.5

255.2

295

54

OA

Negative

803.5

113.1

295

74

DTX1

Negative

817.5

255.2

295

54

DTX1

Negative

817.5

113.1

295

74

DTX2

Negative

803.5

255.2

295

54

DTX2

Negative

803.5

113.1

295

74

OA

Positive

827.5

809.5

260

46

OA

Positive

827.5

723.5

260

54

DTX1

Positive

841.5

823.5

260

46

DTX1

Positive

841.5

737.5

260

54

DTX2

Positive

827.5

809.5

260

46

DTX2

Positive

827.5

723.5

260

54

YTX

Negative

1141.6

1061.6

240

37

YTX

Negative

1141.6

855.5

240

90

homo-YTX

Negative

1155.6

1075.6

240

37

homo-YTX

Negative

1155.6

869.5

240

90

45-OH-YTX

Negative

1157.6

1077.6

240

37

45-OH-YTX

Negative

1157.6

871.5

240

90

45-OH-homo- YTX

Negative

1171.6

1091.6

240

37

45-OH-homo- YTX

Negative

1171.6

869.5

240

90

AZA1

Positive

842.6

824.6

220

29

AZA1

Positive

842.6

672.4

220

53

AZA2

Positive

856.5

838.5

220

29

AZA2

Positive

856.5

672.4

220

53

AZA3

Positive

828.5

810.5

220

29

AZA3

Positive

828.5

658.4

220

53

PTX1

Positive

892.5

821.3

200

24

PTX1

Positive

892.5

213.1

200

44

PTX2

Positive

876.5

823.3

200

24

PTX2

Positive

876.5

213.1

200

44

GYM

Positive

508.5

490.5

250

24

GYM

Positive

508.5

392.3

250

40

SPX1

Positive

692.4

444.3

225

40

SPX1

Positive

692.4

164.1

225

54

(ESI) to achieve the lowest LOQs for the determination of all

regulated LTs.
The common procedure includes the extraction of LTs from
the hepatopancreas in contaminated bivalves with methanol,
resulting in a crude extract rich in matrix constituents that might
cause matrix effects in the ESI resulting in signal enhancement
or reduction. The extent of matrix effects depends on the
biological matrix selected for analysis and needs to be evaluated
in analytical procedures in order to establish the correct
protocol for the quantification of LTs in bivalve contaminated
samples(1618).

The introduction of ultra-high performance LC (UHPLC)

allowed for faster analysis times and higher throughput than
conventional HPLC. This is possible due to the use of smaller
particles for the stationary phase, resulting in higher separation
efficiency and improved peak shapes(19). The short run times
and narrow peaks require short cycle times in MS detection
in order to have enough data points across the peak. This may
result in low dwell times especially when fast polarity switching
is employed, required for the detection of all lipophilic toxins
using acidic conditions. The use of up-to-date analytical
equipment and software tools for effective management of the

Figure 1: UHPLC-MS/MS
chromatogram
contaminated
extract
analyzed with
Dynamic
Braa
-Magdalenaofeta al
.: Journal mussel
of aoaC
InternatIonal
Vol
. 97, no. 2, 2014
MRM fast polarity switching. Peak heights were normalized for each chromatogram.

287

Figure 1. UHPLC/MS/MS chromatogram of a contaminated mussel extract analyzed with DMRM fast polarity switching. Peak heights were
normalized for each chromatogram.

duty cycle of the mass spectrometer is required for accurate

quantification and confirmation.
This work shows the application of a new acquisition mode
in LC/MS/MS to the analysis of LTs including the OA, PTX,
YTX, AZA groups, and SPX1 and GYM-A, most of which are
currently regulated in the EU. Acidic conditions were used in
order to extend the scope of method to additional toxins like
domoic acid (DA).
Multitoxin analysis was performed in dynamic multiple
reaction monitoring (DMRM) mode enabled for fast polarity
switching. The DMRM mode optimizes the duty cycle of the
mass spectrometer for a fixed cycle time based on the retention
time and a variable retention time window for each analyte.
The aim of the present study was to provide a UHPLC/MS/MS
method with fast polarity switching for the analysis of the major
lipophilic marine toxins and DA in shellfish samples. Shellfish
samples were extracted according to the EU harmonized
standard operating procedure (SOP) for the determination of
lipophilic marine toxins in molluscs without further cleanup
(EURLMB SOP version 4; 13). Results of the successful inhouse validation for different shellfish matrixes are shown.
Experimental
Chemicals and Standards
Water and acetonitrile (LC/MS grade) were purchased
from Panreac (Barcelona, Spain). Methanol (HPLC grade),
hydrochloric acid (37% purity), and sodium hydroxide (99%
purity) analytical grade were purchased from Merck (Barcelona,
Spain). Formic acid (98100% purity) and ammonium formate
(99% purity) were purchased from Sigma-Aldrich (Madrid,
Spain).
DA (CRM-DA-f), OA (CRM-OA-c,), PTX-2 (CRM-PTX-2),
AZA 1 (CRM-AZA1), YTX (CRM-YTX), 13-SPX1 C (CRMSPX-1), and GYM A (CRM-GYM), as well as certified reference
material including OA and dinophysistoxin 1 (CRM-DSPMUS-b), were purchased from the National Research Council,
Institute for Marine Biosciences (NRC-CNRC), Halifax,
Canada. Prereleased reference material made of a freeze-dried
mussel tissue homogenate of Mytilus edulis (RM-FDMT)
containing DA, OA, Dynophysis toxins (DTX)1-2, AZA-1-3,

PTX-2, YTX, and SPX-1; a blank shellfish tissue (MUS-zero);

and a multitoxin calibration solution (NRC RM-Multi-toxin)
containing OA, DTX1, DTX2, AZA1, AZA2, AZA3, PTX2,
and YTX were kindly donated by Dr. M. A. Quilliam from
NRC-CNRC.
Standards and Sample Preparation
Stock standard solutions with concentrations of 200 ng/mL
for OA, PTX2, AZA1, SPX1, GYM, 500 ng/mL for YTX, and
10 g/mL for DA were prepared in methanol. Working standard
solutions were prepared in methanol and blank mussel tissue
extract (MUS-Zero) in a concentration ranging from 0.3 to
40 ng/mL for OA, PTX2, AZA1, SPX1, and GYMA, for YTX
from 0.75 to 100 ng/mL, and for DA from 0.1 to 1.0 g/mL.
Extraction Procedure
Samples and blank mussel material were extracted using the
procedure described in the EU harmonized SOP (13) published
by the EURLMB. A 2.0 g amount of the homogenized shellfish
sample was accurately weighed into a 50 mL centrifuge tube.
A 9 mL volume of methanol was added, and the samples were
extracted by rigorous shaking on a vortex mixer (Multi Reax;
Table 2. Correlation coefficients derived from a six-point
calibration for OA group toxins using SMRM and DMRM
modes; calibration curves were constructed as the mean of
two sets ranging from 3 to 40 ng/mL
SMRM

DMRM

r2

r2

OA +

0.991

0.998

OA

0.997

0.999

DTX1 +

0.981

0.996

DTX1

0.999

0.998

DTX2 +

0.994

0.998

DTX2

0.998

0.998

Toxin (ESI mode)

Five-point calibration curve.

288 Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No. 2, 2014
Table 3. Experimental LODs (ng/mL) for OA group toxins
with DMRM in the positive and negative ESI modes (n = 4)
Solvent

OA

OA+

DTX1

DTX1+

DTX2

DTX2+

Methanol

>0.43

0.23

>0.5

0.22

>0.38

0.06

MMS

0.16

0.12

0.21

0.19

0.01

0.08

Heidolph Instruments, Schwabach, Germany) for 3min. After

centrifuging for 10min at 20C the supernatant was transferred
to a 20mL volumetric flask, and the remaining tissue pellet was
extracted again by adding 9 mL methanol and using a highspeed homogenizer (Ultra-Turrax T 25 Basic (IKA Werke,
Staufen, Germany). After centrifugation (Eppendorf AG 5810R,
Hamburg, Germany) using the conditions previously described
the supernatant of the second extraction was combined with
the first extract, and the volumetric flask was filled up to the
mark with methanol. Free OA, DTX1, DTX2, PTXs, AZAs,
SPX-1, GYM, and DA were quantified in this crude extract.
In order to determine the total content of OA and DTXs, 1mL
aliquots of the crude extracts were hydrolyzed with 125L
2.5M NaOH solution at 76C for 40min. Afterwards, the
extract was neutralized by adding 125L 2.5M HCl solution
and homogenized by vortex mixing for 0.5min. Both extracts,
with and without hydrolysis, were filtered through a PVDF filter
with a pore size of 0.2m filter (PVDF Membrane; Membrane
Solutions, Millipore, Cork, Ireland) prior analysis.
Preparation CRM-ASP-MUS-d and CRM-DSP-MUS-b
Certified Reference Materials
For DA, 2.0 g CRM-DSP-MUS-d was accurately weighed
into a centrifuge tube and extracted with 8mL methanolwater
(1 + 1, v/v). The sample was homogenized using an Ultraturrax
for 3min and centrifuged for 10min. The supernatant was
filtered through a PVDF filter with a pore size of 0.22m, and
5L was injected into the chromatographic system.
For DSP toxins the extraction was conducted as recommended
by EURLMB SOP (13); 1.9g CRM-DSP-MUS-b was accurately
weighed, transferred into a 50mL centrifuge tube, and extracted
following the procedure described above. Dilutions of 1:50 and
1:6 of the extract were prepared by transferring 400L and
Table 4. Mean slope and RSD for a six-point calibration
curve for all LTs using methanol and MMSs; calibration
curve data is the mean of three sets of standards ranging
from 3 to 40 ng/mL
Methanol

MMS
Slope

RSD, %a

1.6

1172

2.2

0.3

1072

0.7

129625

9.1

50792

3.4

SPX1

38542

3.0

30065

1.9

PTX2

3839

1.0

2550

1.0

Toxin

Slope

RSD, %

OA +

498

OA

335

AZA1

YTX
GYMA
a

n = 3.

325

0.4

240

2.0

79061

2.7

60207

0.5

3.3mL, respectively, of the crude extract into a 20mL volumetric

flask. The flask was filled up to the mark with methanol.
Preparation and Extraction of FDMT
Reconstitution of the FDMT was carried out following the
procedure recommended by the manufacturer (NRC-CNRC).
FDMT (0.35g) was reconstituted in a 50mL centrifuge tube
by adding 1.65mL deionized water, followed by vortex mixing
for 30s and sonication for 1min in an ultrasonic bath (Selectra;
Ultrasonic-OR, Barcelona, Spain). The extraction of the LTs
was carried out according to the method described previously.
UHPLC/MS/MS Analysis
Chromatographic separation was carried out using an
Agilent Technologies (Waghaeusel-Wiesental, Germany)
1290 Infinity UHPLC system consisting of a binary pump, a
high-performance autosampler, and a thermostated column
compartment. DA and lipophilic toxins were separated using an
Agilent ZORBAX SB-octylsilyl (C8) Rapid Resolution High
Definition column (50 2.1 mm id, 1.8 m particle size) at
40C. Mobile phase A was 100% water and B was acetonitrile
water (95 + 5, v/v), both containing 2mM ammonium formate
and 50mM formic acid. A gradient program with a flow rate of
0.4mL/min was run starting with 0.5min isocratic at 12% B,
followed by a linear gradient to 50% B in 2.5min and then a
linear gradient to 90% B in 3.5min. After an isocratic hold time
of 2min at 90% B, a linear gradient was applied to return to the
starting conditions of 12% B in 0.1min. The total run time was
8.5min, and an equilibration time of 1min was allowed prior
to the next injection. A 5L amount of the diluted crude extract
were injected without any further cleanup. The samples in the
autosampler were cooled to 7C.
MS detection was performed using an Agilent G6460A triple
quadrupole mass spectrometer equipped with an Agilent Jet
Stream ESI source (Agilent Technologies). Source and interface
conditions were optimized for the analysis of LTs in both positive
and negative ionization mode and were adjusted to achieve the
best sensitivity for all compounds. The capillary voltage was set
to 4kV in the negative mode and 4.5kV in the positive mode. A
drying gas flow of 7L/min at a temperature of 200C, a nebulizer
gas pressure of 30 psi (Nitrogen Generator System, Zefiro
40, Evry, France), and a sheath gas (nitrogen 99.999% pure,
Airliquide, Porrio, Spain), flow of 11 L/min at a temperature
of 400C were chosen. The collision energy and cell accelerator
voltages were optimized for each analyte in flow injection
analysis using the Mass Hunter Optimizer software (Mass Hunter
Workstation software B.04.01) and are shown in Table1. For
those LTs for which no reference standards were available, the
parameters were adjusted according to those optimized for their
structurally related commercially available toxin. Two MRM
transitions were set up for all toxins. The transition with the
higher intensity was used for quantification, while the transition
with the lower intensity was used for confirmation purposes.
Calibration and Quantitation (QC Criteria)
UHPLC/MS/MS provided satisfactory results for crude
extracts in methanol without further cleanup if QC parameters
described by EURLMB SOP Version4 are strictly followed.

Figure 2: Calibration curves for OA (negative mode) and AZA1 (positive mode) in methanol and matrix

matched standards (MMS) (calibration range 3-40 ng/ml)

Braa-Magdalena et al.: Journal
of AOAC International Vol. 97, No. 2, 2014 289

OA + (MMS)
y = 1172,5x - 566,39

45000

R2 = 0,9984
OA- (MMS)
y = 1072,2x - 1977,5

35000

R2 = 0,9948

30000

OA + (MeOH)
y = 497,83x + 9,6025

25000

R2 = 0,9998

20000
15000
10000

OA - (MeOH)
y = 334,59x - 60,826

5000

R2 = 0,9981

Results and Discussion

10

15

20

25

30

35

40

45

40

45

OA Conc. (ng/mL)

6000000
AZA (MeOH)
y = 129625x - 255082

5000000

Peak Area

A short chromatographic method with a cycle time of 8.5 min

was developed that allowed the analysis of all LTs studied
and DA. The run time was not further reduced in order to
maintain a robust method, suited also for the most challenging
matrixes. With the applied gradient all compounds could be
baseline separated except for YTX and homo-YTX and for
45-hydroxy-YTX and 45-hydroxyhomo-YTX, which could
not be separated by chromatography but have been separated
based on their specific MRM transitions in MS. OA and DTX2,
which share the same MRM transitions, have been separated
with a chromatographic resolution (Rs) > 2. Figure1 shows the
chromatogram of a sample extract containing most of the LTs
and DA. The peak heights of each toxin were normalized to
100% for all toxins except DTX2.
In LC/MS/MS detection the charge state of each toxin depends
on its structure and the mobile phase conditions. Under acidic
conditions, YTX and its analogs were analyzed in the negative
mode, while PTXs, AZAs, GYM, SPX1, and DA were analyzed
in the positive mode. The compounds of the OA group, OA,
DTX1, and DTX2, are ionizable in positive and negative modes
and thus, were measured in both. Table1 summarizes the MRM
transitions used as quantifier and qualifier for the determination
of DA and LTs.
The retention time for DA was 1.2 min using the
chromatographic conditions described previously. Therefore,
the inclusion of DA in the LT multitoxin method is an excellent
tool for laboratories monitoring marine biotoxins. The close
elution of compounds detected in the positive and negative
mode was considered as the major drawback of methods for
LTs using acidic mobile phases for the UHPLC separation.
Several methods were published for the analysis of these
toxins but with two or more separate injections with the MS
instrument operated either in the negative or positive ESI
mode(11,20). However, modern mass spectrometers are
capable of fast polarity switching without loss in performance,
therefore allowing the analysis of all compounds in a single run.
DMRM automatically constructs timetables for multiple
analytes throughout the UHPLC/MS/MS analysis based on
the retention time window for each compound. It allows the
instrument to acquire MRM data only during a stated retention
time window, thus reducing the number of concurrent ion
transitions. DMRM utilizes a constant cycle time to ensure
uniform distribution of data points. This will improve the
sampling of chromatographic peaks, resulting in better peak
symmetry that enables reproducibility in retention time
measurement, peak areas, and accuracy of quantification.
Acquisition of the LT data using conventional MRM with a
single time segment was compared with a method using DMRM.
Although DMRM was developed for large methods covering

50000

40000

Peak Area

The QC criteria include chromatographic resolution (peak

resolution OA/DTX2 1), relative retention time drift (1%),
calibration curve (r2 0.98 derived from at least five calibration
points and constructed as the average of the first and second
set of the calibration curve injections), response drift (<25%
slope variation between the two sets of the calibration curve
injections), blank QC sample (to be injected before and after the
calibration sets, between hydrolyzed samples), and S/N of the
qualifier transition intensity 3.

R2 = 0,9979

4000000
AZA (MMS)
y = 50790x + 54001

3000000

R2 = 0,9925

2000000
1000000
0
0

10

15

20

25

30

35

AZA 1 Conc. (ng/mL)

Figure 2. Calibration curves for OA (negative mode) and AZA1

(positive mode) in methanol (MeOH) and MMSs in the range
340ng/mL.

hundreds of analytes, maximizing the available dwell times for

each individual transition can also be beneficial for methods
with lower numbers of analytes with regards to sensitivity and
reproducibility.
Calibration curves ranging from 3 to 40 ng/mL were prepared
for all LTs with both polarity modes. When comparing the
calibration curves of OA acquired with conventional MRM and
DMRM, the slopes of the calibration curves constructed in the
negative mode were similar, indicating that the peak areas were
not affected by the different dwell times. For DMRM, the dwell
time for OA was 20ms compared to 34ms for the conventional
MRM method. In contrast, the calibration curve acquired in the
positive mode showed a slightly lower slope due to the lower
response of OA for the DMRM method. However, the use of
DMRM resulted in better correlation coefficients than SMRM
(r2; Table2) and better within-day precision, both indicating the
better reproducibility of the DMRM method. In addition, S/N
values of the lowest calibration standards were improved for all
toxins when using DMRM, leading to lower LODs. Compared
to the current HPLC/MS/MS (EURLMB) method, LODs were
reduced by a factor of10(20). Therefore DMRM was selected
as the acquisition mode for the UHPLC/MS/MS method, which
was validated for the analysis of LTs. Table3 shows the LODs
of the OA group toxins based on a S/N of 3 using negative and
positive ESI obtained for solvent standards and matrix matched
standards (MMSs) and based on triplicate injections. LODs
achieved were low for all OA group toxins, particularly for
DTX2 in positive mode. The LODs for the MMSs were better,
indicating either a pronounced enhancement effect in the ESI or,

290 Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No. 2, 2014
Table5. Recoveries obtained for OA and DTX1 for both
ionization modes given as accuracy with RSD for CRMASP-Mus-d and CRM-DSP-Mus-b
Accuracy, %

RSD, %a

DA

77

4.0

OA

109

5.0

OA +

119

7.0

DTX1

98

5.0

DTX1+

103

4.0

NRC-CRM-DSP/ASP-MUS

n = 3.

more likely, lower adsorption of the analyte to active surfaces

due to the occupation of active sites by the matrix.
Matrix Effects
Matrix effects were evaluated for all individually available
certified reference standards including OA, DTX1, DTX2,
PTX2, AZA1, AZA2, AZA3, YTX, GYM, and SPX1 by
comparing standards prepared in methanol and in blank mussel
tissue extract (MUS-Zero; Table4). For the OA group toxins,
enhanced signal intensity was observed in the MMSs in both
ionization modes. Figure2 shows the calibration curves for
OA in the positive and negative modes for a solvent calibration
and MMS. The calibration slopes are increased by a factor of
2.3 and 3.2 for positive and negative ionization, respectively.
As mentioned before, this might be caused by either signal
enhancement in the ESI or reduced adsorption to active sites
in the presence of matrix constituents or a combination of both.
For all other LTs, ion suppression has been observed for the
MMSs by a factor of 1.3 and 2.5 for the positive and negative
ionization modes, respectively.
Accuracy
The accuracy of the method was evaluated by analyzing
a commercially available certified reference material
(CRM-MUS-b) containing OA and DTX1. Due to the high
concentration of OA and DTX1 in this certified material,
1:50 and 1:6 dilutions of the methanolic extract were made
and analyzed. This resulted in an expected concentration of
20.4 ng/mL DTX1 and 19.2 ng/mL OA. DTX1 and OA were
quantitated by external calibration with MMSs to calculate the

accuracy of the method. Table5 shows the apparent recoveries

obtained for DA, OA, and DTX1. DA and DSP toxins were
assessed using positive and both ionization modes, respectively.
Assuming a complete extraction of the toxins, the observed
recoveries correspond to the matrix effects in the ESI.
The recovery of DA in the certified material was 77%;
however, the repeatability was consistently good at 4% RSD.
Further investigations are ongoing for simultaneous extraction
of DA with LTs.
For DSP toxins using both ionization modes, recoveries
of 98 to 119% were observed with RSD values below 10%.
This shows that the matrix-matched calibrations successfully
compensated for the matrix effects in the ESI. Because there
is currently no commercially available CRM for the other
LTs, their accuracy was tested with a new prereleased FDMT
material containing OA, DTX1, DTX2, YTX, AZA1, AZA2,
AZA3, PTX2, and SPX1.
For the determination of the OA group toxins, only free toxin
concentrations in the NRC RM-FDMT were evaluated, since
only those values were assigned by the manufacturer for the
material. Table6 shows the reproducibility and accuracy of
the method for a 3 day validation study by duplicate injection
during each day. For most of the LTs studied, the recoveries
obtained were between 80 and 120% with an interday variation
of less than 8% RSD and are in good agreement with indicative
concentrations of the manufacturer. Larger deviations from the
indicative concentrations were observed for the OA group and
PTX2, which is expected owing to their structure and chemical
properties in acidic conditions. The net charge state of the OA
group and PTX2 at pH3 is neutral(11), which means they can
be measured in positive and negative ESI modes. However,
this neutral charge seems not to be appropriate at this pH to
produce efficient ionization in the ESI probe, and consequently,
quantification is inaccurate. When using matrix matched
calibrations for these toxins, the accuracy for the method
improved in comparison with quantification using external
calibration.
Concentrations for the duplicate injections of the same sample
were in good agreement, with deviations of less than 10%.
This is important to demonstrate that a single sample injection
typically is sufficient to obtain the correct result, which leads to
higher sample throughput in routine use.
In addition to the reference material, different seafood
matrixes (mussels, cockles, clams, and razor clams) were
analyzed. Some of them were naturally contaminated with one
or more different LTs, and some others were spiked at different

Table 6. Accuracy and precision data between days obtained on prereleased FDMT; all concentrations are expressed in g/kg
Toxin

OA a

DTX1 a

DTX2 a

YTX

AZA1

AZA2

AZA3

SPX1

PTX2 a

Day 1

264

158

719

353

548

169

167

344

157

Day 2

310

145

755

371

546

165

156

358

151

Day 3

287

134

786

368

542

160

153

344

158

Average

287

146

753

364

545

164

158

349

155

SD

23,1

12,1

33,5

9,6

3,1

4,5

7,4

8,1

13,4

RSD, %

8,0

8,3

4,5

2,6

0,6

2,7

4,6

2,3

2,4

Indicative concentrations

274

133

595

450

712

197

175

472

119

Accuracy, %

104

109

>120

81

77

83

90

74

>120

Matrix-matched calibration.

Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No. 2, 2014 291
Table 7. Intralaboratory repeatability study carried out on contaminated samples with different toxin profiles and different
biological matrixes
Toxin profile, g/kg
Matrix sample
Donax trunculus

Ensis acuatus

Mytilus edulis

Chamelea gallina

Cerastoderma edule

M. edulis (cooked)
a

OA

DTX1

DTX2

PTX2

YTX

AZA1

AZA2

AZA3

90.5

<LOD

162.9

289.1

<LOD

156.2

<LOQ

<LOD

104.0

<LOD

174.6

274.7

<LOD

141.9

<LOQ

<LOD

68.8

<LOD

68.1

<LOQ

<LOD

76.9

<LOQ

<LOD

65.4

<LOD

62.2

<LOQ

<LOD

75.0

<LOQ

<LOD

63.7

220.0

<LOQ

ND

138.5

548.1

34.9

<LOD

61.4

206.5

<LOQ

ND

134.8

516.1

32.1

LOD

58.1

<LOD

53.7

261.9

<LOD

269.8

30.3

<LOD

57.7

<LOD

52.5

254.4

<LOD

257.7

27.7

<LOD

70.9

<LOD

75.3

<LOD

<LOD

76.5

<LOQ

<LOD

67.1

<LOD

66.4

<LOD

<LOD

64.4

<LOQ

<LOD

224.4

<LOD

44.5

<LOD

<LOD

442.6

29.3

144.5

178.0

<LOD

44.4

<LOD

<LOD

432.5

27.7

142.3

ND = Not detected.

superior to the MBA, due to the higher sensitivity and selectivity

and lower chemical interferences.

concentration levels. The toxin profiles were determined

using duplicate injections following the protocol described in
the collaborative study coordinated by the EURLMB over a

Triggered MRM

3 day period. Sample quantitation was carried out using MMS

Another challenge to overcome in the analysis of LTs are

matrix interferences on the target MRM traces of compounds
quantification based on matrix-matched calibrations provided
having the same precursor mass and fragments and a similar
more accurate results.
polarity. Additional information on the identity of a compound
peak might help to eliminate potential false-positive reporting,
The results of the measurements are shown in Table7. The
especially in high throughput environments. One approach is
determined concentrations are in good agreement with previous
to acquire spectra for the toxins for enhanced confirmation.
measurements. It should be noted that the results for the blind
This can be done via triggered MRM (TMRM), which allows
both accurate quantification and highly sensitive compound
duplicates were very consistent for each biological sample
confirmation with a single injection. In TMRM, one or more
with RSDs typically below 10%. These studies clearly indicate
primary transitions are acquired for a target compound. If
Figure
3: Chromatograms
triggered
MRM
PTX2 in
FDMT.goes
Spectra
shown
in
the of
triggering
transition
above are
a given
threshold
up to
that the
UHPLC/MS/MS
method is a and
powerful
analytical
tool,spectra
calibrations because it has been shown that the RM-FDMT

comparison to the reference library spectrum of PTX2.

PTX2

PTX2

PTX2b

PTX2b

Figure 3. Chromatograms and TMRM spectra of PTX2 in FDMT. Spectra are shown in comparison to the reference library spectrum of PTX2.

292 Braa-Magdalena et al.: Journal of AOAC International Vol. 97, No. 2, 2014
nine additional MRMs are triggered/compound, resulting
in an MRM product ion spectra that can be library searched.
Compared to classical product ion spectra, this acquisition
mode is highly sensitive; each MRM transition can be acquired
with the optimum collision energy and reasonably long dwell
time. An example is shown in Figure3. In the FDMT for
PTX2, there is a second peak on both MRM transitions that is
just 0.5min separated. The ion ratio is slightly higher than the
expected value. When comparing the acquired spectrum with
the spectrum stored in the reference library, all acquired ions
were present for this second peak. However, the resulting score
was 77.9, which does not confirm this compound as PTX2 but
indicates that it might belong to the PTX family. This second
small peak could be PTX2b, which is chemically converted
from PTX2 in acidic conditions(21).
Conclusions
The new UHPLC/MS/MS method developed allowed the
analysis of 14 marine lipophilic toxins with a significant
decrease of the run time compared to classical HPLC methods.
By using DMRM better sensitivity and reproducibility could
2
be achieved, resulting in better r values for the calibration
curves compared to conventional MRM. Matrix effects in the
ESI were observed (signal suppression and enhancement) and
had effects on the quantification of most of the toxins. One way
to compensate for the matrix effects was external calibration
in blank mussel tissue. Good accuracy was observed with the
implemented method when analyzing the reference materials
CRM-DSP-MUS-b and RM-FDMT for most of the lipophilic
toxins, particularly for the OA group toxins. Excellent RSDs
were obtained during a 3consecutive day study of FDMT
and naturally contaminated and spiked shellfish samples with
different toxin profiles.
Acknowledgments
This work has been supported by funds from Directorate
General for Health and Consumers, European Commission (EU,
Brussels, Belgium) and the Spanish Food Safety and Nutrition
Agency. We thank Michael Quilliam (National Research
Council, Canada) for kindly providing the freeze dried material
and MUS-Zero for this study.
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