Content uploaded by Anke Kremp
Author content
All content in this area was uploaded by Anke Kremp on Jan 11, 2016
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Article
Results of a Saxitoxin Proficiency Test Including
Characterization of Reference Material and
Stability Studies
Kirsi Harju 1, *, Marja-Leena Rapinoja 1, Marc-André Avondet 2, Werner Arnold 2, Martin Schär 2,
Werner Luginbühl 3, Anke Kremp 4, Sanna Suikkanen 4, Harri Kankaanpää 5, Stephen Burrell 6,
Martin Söderström 1and Paula Vanninen 1
Received: 18 June 2015 ; Accepted: 13 August 2015 ; Published: 25 November 2015
Academic Editor: Andreas Rummel
1VERIFIN (Finnish Institute for Verification of the Chemical Weapons Convention),
Department of Chemistry, P.O. Box 55, A. I. Virtasen aukio 1, University of Helsinki,
FI-00014 Helsinki, Finland; marja-leena.rapinoja@helsinki.fi (M.-L.R.);
martin.soderstrom@helsinki.fi (M.S.); paula.vanninen@helsinki.fi (P.V.)
2Federal Department of Defence, Civil Protection and Sport, SPIEZ LABORATORY, Austrasse 1,
CH-3700 Spiez, Switzerland; marc-andre.avondet@babs.admin.ch (M.-A.A.);
werner.arnold@babs.admin.ch (W.A.); martin.schaer@babs.admin.ch (M.S.)
3ChemStat, Aarstrasse 98, CH-3005 Bern, Switzerland; info@chemstat.ch
4Finnish Environment Institute, Marine Research Centre, Erik Palménin aukio 1,
FI-00560 Helsinki, Finland; anke.kremp@ymparisto.fi (A.K.); sanna.suikkanen@ymparisto.fi (S.S.)
5Finnish Environment Institute, Marine Research Centre, Hakuninmaantie 6, FI-00430 Helsinki, Finland;
harri.kankaanpaa@ymparisto.fi
6Marine Institute, Marine Environment and Food Safety Services, Rinville, Oranmore,
County Galway, Ireland; stephen.burrell@marine.ie
*Correspondence: kirsi.harju@helsinki.fi; Tel.: +358-2941-50351; Fax: +358-2941-50437
Abstract: A saxitoxin (STX) proficiency test (PT) was organized as part of the Establishment of
Quality Assurance for the Detection of Biological Toxins of Potential Bioterrorism Risk (EQuATox)
project. The aim of this PT was to provide an evaluation of existing methods and the European
laboratories’ capabilities for the analysis of STX and some of its analogues in real samples.
Homogenized mussel material and algal cell materials containing paralytic shellfish poisoning
(PSP) toxins were produced as reference sample matrices. The reference material was characterized
using various analytical methods. Acidified algal extract samples at two concentration levels were
prepared from a bulk culture of PSP toxins producing dinoflagellate Alexandrium ostenfeldii. The
homogeneity and stability of the prepared PT samples were studied and found to be fit-for-purpose.
Thereafter, eight STX PT samples were sent to ten participating laboratories from eight countries.
The PT offered the participating laboratories the possibility to assess their performance regarding the
qualitative and quantitative detection of PSP toxins. Various techniques such as official Association
of Official Analytical Chemists (AOAC) methods, immunoassays, and liquid chromatography-mass
spectrometry were used for sample analyses.
Keywords: paralytic shellfish poisoning toxins; saxitoxin; proficiency test; dinoflagellate; mussel
1. Introduction
Establishment of Quality Assurance for the Detection of Biological Toxins of Potential
Bioterrorism Risk (EQuATox) was a 36 month project (1 January 2012–31 December 2014) under
the 7th European Union Framework Programme for Research (FP7) coordinated by the Robert
Toxins 2015,7, 4852–4867; doi:10.3390/toxins7124852 www.mdpi.com/journal/toxins
Toxins 2015,7, 4852–4867
Koch-Institut (Berlin, Germany). The project consisted of four separate proficiency tests (PTs) on
four different toxin types: ricin, saxitoxin (STX), staphylococcal enterotoxin B (SEB), and botulinum
neurotoxin (BoNT). The aim of the EQuATox project was to develop methods, procedures, and
protocols for the analysis of selected chemical and biological substances allowing a comparison
of results from different laboratories. The STX PT was organized by the Finnish Institute for
Verification of the Chemical Weapons Convention, University of Helsinki, Finland (VERIFIN). STX
is a highly poisonous non-peptide, low molecular weight toxin at the interface of classical biological
and chemical agents; it is a well-defined substance under both the Chemical Weapons Convention
(CWC, Schedule 1) and the Biological Weapons Convention (BWC). It is produced by some species
of cyanobacteria and marine dinoflagellates e.g., Alexandrium ostenfeldii in the Baltic Sea [1]. STX
accumulates in seafood, and it may also contaminate drinking water, thus causing severe health
problems. Saxitoxin is a neurotoxin which blocks sodium channels, and intoxication may lead to
paralysis and even death. Numerous fatal cases of paralytic shellfish poisoning (PSP) toxins have
been reported globally [2], but the improved monitoring of microalgae and PSP toxins in shellfish
has decreased the risks. Saxitoxin has also been considered to be a potential bioterrorism risk [3,4].
At least 57 structurally different analogues of STX are listed in a recent literature review [5]. The
commercial availability of certified reference material is limited only to STX and about 10 of its
analogues, which are most commonly produced by microalgae and most likely present in seafood.
Toxicity equivalent factors have been applied to calculate detected analogues as STX equivalents
for monitoring purposes using HPLC with fluorescence detection (FLD), though the acute toxicity
of STX and its analogues via oral administration do not always correlate with the mouse bioassay
(MBA) results, and e.g., the toxicity of neosaxitoxin (NEO) by i.p. injection seems to be higher than
calculated from the toxicity equivalent factors [6]. The regulatory limit (800 µg STXeq/kg) for PSP
toxins in seafood has been set on the basis of the MBA. However, MBA is not sensitive enough for
trace analysis of PSP toxins in drinking water and it is not directly applicable to other matrices like
micro algae. While different technologies for STX detection and analysis have been established, only
a few Association of Official Analytical Chemists (AOAC) official methods are accepted; the mouse
bioassay method [7], the pre-column oxidation [8,9], post-column oxidation HPLC-FLD method [10],
and receptor binding assay (RBA) [11]. Human clinical samples are rarely available for testing of PSP
intoxications, but, recently, a pre-column HPLC-FLD method was validated for the analysis of PSP
toxins in clinical samples [12].
Recent interlaboratory validation studies were carried out mainly on mussel samples employing
the official AOAC methods [13–17]. Based on the discussions in the EQuATox kick-off meeting in
Berlin in March 2012, the main focus of STX PT was the identification and quantification of STX in
real samples instead of spiked reference standards. The PT sample material was produced from
naturally contaminated algae or shellfish. We present herein the results of the preparation and
characterization of the PT sample material, homogeneity and stability studies of the PT samples,
and reported PT results of the participating laboratories. In addition to the official AOAC methods,
valuable information on the applicability of enzyme-linked immunoassay (ELISA), lateral flow assay,
and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of PSP
toxins was obtained from the participating laboratories.
2. Results and Discussion
2.1. Preparation and Characterization of STX PT Sample Material
Toxic algal cell sample material was obtained from A. ostenfeldii cultures grown at the Finnish
Environment Institute (SYKE, Helsinki, Finland). This dinoflagellate produces STX, gonyautoxin 2
(GTX2) and gonyautoxin 3 (GTX3). Toxic mussel material was provided by the Marine Institute
(Galway, Ireland). The toxin profile of the mussel material contained a mixture of STX and some
main PSP toxin analogues i.e., decarbamoyl saxitoxin (dcSTX), neosaxitoxin (NEO) and gonyautoxins
4853
Toxins 2015,7, 4852–4867
(GTX1–5). The algal samples, both solid and liquid, contained only STX, GTX2 and GTX3, which
are naturally produced by A. ostenfeldii in the Baltic Sea. The PSP toxins in naturally contaminated
mussel samples were selected so that, in addition to STX, the mussel samples contained various
closely related PSP toxins, which were also commercially available as reference standards and which
had different substituents, such as carbamoyl, N-sulfocarbamoyl, hydroxyl, and sulfate groups. The
selected PSP analogues with different charge states are affected by factors like pH and are thus
challenging in sample preparation and analysis. Some of the PSP analogues can also be chemically
converted from one to another e.g., during sample preparation and this may cause change in toxicity.
The concentration level of PSP toxins in PT samples was fixed to be high enough for all analytical
techniques; an exception was the most diluted STX PT sample (E1) in which the concentration level
(4.7 ng/mL STX, Table 2) was adjusted to be close to the detection limit of chromatographic methods.
Both sample materials were characterized by two or three laboratories by three methods;
pre-column oxidation high performance chromatography with fluorescence detection (HPLC-FLD),
liquid chromatography-tandem mass spectrometry (LC-MS/MS), and enzyme-linked immunoassay
(ELISA). The PSP toxin profiles of the samples are presented in Table 1. The PSP analogues were
compared against the certified PSP reference standards obtained from the National Research Council
(NRC, Canada). The identification of PSP toxins was based on the Organisation for the Prohibition
of Chemical Weapons (OPCW) retention time criteria of |∆rt| ď0.2 min [18] and on the specific
fragmentation pattern of MS spectra when compared to the certified reference standards [19]. The
presence of GTX4 in the mussel sample could not be confirmed before the PT because of the
non-separable epimeric pair of GTX1 and GTX4 in pre-column oxidation method and the low
sensitivity of GTX4 in the LC-MS/MS measurements.
Table 1. Characterization of paralytic shellfish poisoning (PSP) toxins in saxitoxin (STX) proficiency
test (PT) sample material.
Method Algal Sample Material Mussel Sample Material
PSP Analogues Identified PSP Analogues Identified
Ridascreen ELISA PSP positive PSP positive
LC-MS/MS STX, GTX2, GTX3 STX, dcSTX, NEO, GTX1, GTX2, GTX3
pre-column HPLC-FLD STX, GTX2&3 STX, dcSTX, NEO, GTX1&4, GTX2&3, GTX5
The samples were quantified with pre-column oxidation HPLC-FLD [8] and LC-MS/MS method
validated for algal samples [20]. The assigned values for STX in the samples are presented in Table 2.
Table 2. The assigned values and standard deviations for STX analyzed before the PT.
STX PT Sample Sample Type STX Method
A algal cells on filter paper 572 ˘75 ng/mL LC-MS/MS
E1 algal extract (1:50) 4.7 ˘0.6 ng/mL LC-MS/MS
E2 algal extract (1:10) 28 ˘2 ng/mL LC-MS/MS
M mussel sample 126 ˘0.6 ng/g mussel HPLC-FLD
STX was identified in algal and mussel samples with two LC-MS/MS instruments either in
the product ion scan or in the multiple reaction monitoring (MRM) modes. The chromatographic
separation of PSP toxins was performed with hydrophilic interaction liquid chromatography (HILIC).
The identification was based on the retention time and mass fragmentation criteria [18,19]. The
retention times of the analytes were compared to the retention times of the certified reference
standards. The mussel matrix affected the retention times and, thereafter, the presence of STX
was confirmed with the signal increase after the addition of STX reference standard to the mussel
4854
Toxins 2015,7, 4852–4867
matrix. The relative product ion intensities of the monitored fragment ions of the two distinguished
LC-MS/MS methods fulfilled the EU criteria for both the algal and the mussel samples (Table 3).
Table 3. Retention time (rt, min) and relative product ion intensities (q/Q, %) based identification of
STX in algal and mussel samples either with product ion scan or multiple reaction monitoring (MRM)
mode, liquid chromatography-tandem mass spectrometry (LC-MS/MS) at m/z300.
LC-MS/MS STX Standard
(20–80 ng/mL, n= 3 ˆ7)
STX Algal Sample
n= 90
STX Mussel
Sample n= 17 Tolerance d
rt 6.32 ˘0.06 min 6.31 ˘0.06 min c˘0.2 min
Product Ion Scan am/z q/Q (%) q/Q (%) q/Q (%) q/Q (%)
266/282 19 ˘1 19 ˘2 18 ˘3 13–25
204/282 21 ˘1 22 ˘2 19 ˘2 16–26
186/282 9 ˘1 9 ˘1 8 ˘2 4–14
LC-MS/MS STX Standard
(20–80 ng/mL, n= 3 ˆ7)
STX Algal Sample
n= 90
STX Mussel
Sample n= 17 Tolerance d
rt 15.2 ˘0.1 min 15.1 ˘0.1 min c˘0.2 min
MRM bm/z q/Q (%) q/Q (%) q/Q (%) q/Q (%)
138/204 81 ˘4 68 ˘4 71 ˘6 65–97
282/204 96 ˘5 89 ˘3 94 ˘7 77–115
aFinnigan LXQ; bAB Sciex 3200 QTrap; cSTX confirmed with the reference standard addition; dretention time
tolerance |∆rt| ď0.2 min [18]; EU criteria: compare with the relative ion intensity of the standard, q/Q >50%:
tolerance ˘20%; >20% to 50%: tolerance ˘25%; >10% to 20%: tolerance ˘30%; ď10%: tolerance ˘50% [19].
2.2. Preparation of the PT Samples
The summary of the four prepared STX PT samples is given in Table 4. Algal cells of PSP
producing dinoflagellate A. ostenfeldii were filtered using filter paper. These filters represented the
solid algal sample type (A) in the STX PT. Some of the algal samples on filter paper (A) were further
extracted (n= 15), and two samples of algal extracts were prepared by diluting the pooled extracts
1:50 (E1) and 1:10 (E2) in acidified water (4 mM ammonium formate, pH 3.5 adjusted with formic
acid). The fourth PT sample was a homogenized mussel sample (M). The levels of the total toxicity of
algal and mussel samples were chosen to be toxic enough to be analyzed by MBA. Two concentration
levels of the algal extracts were selected so that the higher concentration level of the PSP toxins would
be close to the detection limit of MBA and the lower concentration would be near the detection
limits and quantitation limits of more sensitive methods, respectively. Thus, the capability of various
methods to detect minor amounts of PSP toxins could be assessed.
Table 4. STX PT samples.
STX PT Sample Sample Type nSample Amount
A Toxic freeze-dried algal sample on filter paper 120 ~350,000 cells
E1 Pooled toxic algal extract (diluted 1:50) 47 5.0 mL
E2 Pooled toxic algal extract (diluted 1:10) 28 5.0 mL
M Toxic homogenized mussel sample 100 ~5.3 g
2.2.1. Homogeneity of the Samples
The homogeneity studies of algal samples were performed using the Finnigan LXQ linear ion
trap LC-MS/MS instrument in product ion scan mode. Algal cells on filter paper (A, n= 15) were
extracted with the LC-MS/MS eluent (4 mM ammonium formate–ACN 40:60, pH 3.5 adjusted with
formic acid), and the volume was adjusted to 2.0 mL. Two LC-MS/MS samples were prepared from
the extracts, and STX in the samples was measured three times (n= 15 ˆ6). Ten parallel algal
extract samples (n= 10) were measured twice (E1 and E2). Statistical analyses did not reveal any
outliers (Table 5). Ten mussel samples were extracted in parallel and analyzed for all PSP toxins
4855
Toxins 2015,7, 4852–4867
with pre-column oxidation HPLC-FLD. They were found sufficiently homogeneous with a relative
standard deviation (RSD) of 5% for STX.
Table 5. Homogeneity results of STX PT samples.
STX PT Sample Sample Type nSTX in the Sample RSD (%) Method
A Algal cells on filter paper 15 654 ˘77 ng on filter 12 LC-MS/MS
E1 Algal extract (1:50) 10 4.7 ˘0.6 ng/mL 14 LC-MS/MS
E2 Algal extract (1:10) 10 28 ˘2 ng/mL 8 LC-MS/MS
M Mussel sample 10 130.7 ˘6.2 ng/g mussel 5 HPLC-FLD
2.2.2. Stability of the Samples
Fifteen algal samples (A, E1, and E2) were randomly selected for the stability study of STX in
algal cell samples and algal extracts. Parallel sets of three samples were kept for four weeks at
4˝C and at room temperature and also for six weeks at ´20 ˝C, 4 ˝C, and at room temperature.
The four-week stability samples were transferred to the freezer (´20 ˝C), and kept there for an
additional two weeks prior to the STX analyses together with the six-week stability samples in the
same LC-MS/MS batch. The algal samples had good stability for PT.
The Marine Institute in Ireland carried out a 32-day reverse isochronous stability study at
´20 ˝C, 4 ˝C, and 40 ˝C for mussel samples. The samples were stored at –80 ˝C after preparation
of the samples and before the start of each stability test. Three aliquots (n= 3) were measured
at each temperature after storage time of 32, 11, five and three days, respectively. Analyses of
STX, GTX2>X3, dcSTX, GTX5, GTX1>X4 and NEO were carried out from these samples with
pre-column oxidation HPLC-FLD method. All PSP analytes in mussel samples showed excellent
stability at ´20 ˝C and 4 ˝C for at least 32 days.
An additional stability study was performed during the PT. Statistically, all samples were found
stable on the significance level of α= 0.05 when stored at 4 ˝C for four weeks (Figure 1). This was also
the case when the calculated standard deviations (SD) for proficiency assessment were considered. If
the criterion of ISO 13528 is taken as a basis, the highest σp(relative) was only 5.4% of the mean. One
significant outlier was found in the algal sample batch (A), and this result was excluded (n= 5) from
the calculations.
As a summary, all analytical data of the tested toxic algal samples and mussel samples indicate
the suitability of the toxic freeze-dried algal material, the toxic algal extracts at two different dilutions
and the toxic mussel sample as applicable sample matrices for this STX PT. The relative standard
deviation for proficiency assessment, σp(relative), was set for each sample based on the homogeneity
and stability data.
Toxins 2015, 7 7
the toxic mussel sample as applicable sample matrices for this STX PT. The relative standard deviation
for proficiency assessment, σp (relative), was set for each sample based on the homogeneity and
stability data.
(a) (b) (c) (d)
Figure 1. Four-week stability of the PT samples at 4 °C and −20 °C during the test.
(a) Algal sample, A; (b) Algal extract, E1; (c) Algal extract, E2; (d) Mussel sample, M.
2.3. STX PT Results
Triplicate algal samples (A), triplicate mussel samples (M), and algal extracts at two toxin
concentration levels (5.0 mL of both E1 and E2), altogether eight samples were sent to ten participating
laboratories from eight countries. All STX PT samples were stored in a freezer for about two months at
−20 °C by the test organizer, and analyzed once more just before the shipment. The samples were shipped
to the laboratories under temperature-controlled conditions by express mail. Electronic reporting forms
were provided via email (22 April 2013). The transportation reports indicated that the temperature of all
sample packages has stayed during the whole transportation between 4.8 and 5.4 °C. All laboratories
reported that the received sample packages were intact. The last package arrived at the participating
laboratory in the morning of the third day after dispatch. The deadline for the reporting was within one
month after the sample dispatch (22 May 2013). The main task for the laboratories was to report results
for the presence of STX in all of the eight samples (A, E1–E2, M). All algal samples (A) and algal
extracts (E1–E2) contained three different PSP toxins. The laboratories were asked to identify those
toxins and additionally to quantify them. The mussel samples (M) contained eight different PSP toxins.
The laboratories were asked to identify those toxins and additionally to quantify them. The quantitative
analyses were asked to be carried out in triplicate, if possible. It was also noted that the sample E1
contained PSP toxins that were too low levels to be detected using MBA.
2.3.1. PSP Toxin Results
All participating laboratories (n = 10) reported their results in time. Results of the PSP toxins in the
samples are presented in Table 6. Four laboratories applied more than one method for the sample
analyses. Reliable unambiguous identification of an analyte should be based on at least two independent
analytical methods according to the OPCW. All analyzed samples were reported to be PSP positive by
all participating laboratories.
Figure 1. Four-week stability of the PT samples at 4 ˝C and ´20 ˝C during the test. (a) Algal sample,
A; (b) Algal extract, E1; (c) Algal extract, E2; (d) Mussel sample, M.
4856
Toxins 2015,7, 4852–4867
2.3. STX PT Results
Triplicate algal samples (A), triplicate mussel samples (M), and algal extracts at two toxin
concentration levels (5.0 mL of both E1 and E2), altogether eight samples were sent to ten participating
laboratories from eight countries. All STX PT samples were stored in a freezer for about two months
at ´20 ˝C by the test organizer, and analyzed once more just before the shipment. The samples
were shipped to the laboratories under temperature-controlled conditions by express mail. Electronic
reporting forms were provided via email (22 April 2013). The transportation reports indicated that
the temperature of all sample packages has stayed during the whole transportation between 4.8 and
5.4 ˝C. All laboratories reported that the received sample packages were intact. The last package
arrived at the participating laboratory in the morning of the third day after dispatch. The deadline
for the reporting was within one month after the sample dispatch (22 May 2013). The main task
for the laboratories was to report results for the presence of STX in all of the eight samples (A,
E1–E2, M). All algal samples (A) and algal extracts (E1–E2) contained three different PSP toxins.
The laboratories were asked to identify those toxins and additionally to quantify them. The mussel
samples (M) contained eight different PSP toxins. The laboratories were asked to identify those toxins
and additionally to quantify them. The quantitative analyses were asked to be carried out in triplicate,
if possible. It was also noted that the sample E1 contained PSP toxins that were too low levels to be
detected using MBA.
2.3.1. PSP Toxin Results
All participating laboratories (n= 10) reported their results in time. Results of the PSP toxins
in the samples are presented in Table 6. Four laboratories applied more than one method for the
sample analyses. Reliable unambiguous identification of an analyte should be based on at least two
independent analytical methods according to the OPCW. All analyzed samples were reported to be
PSP positive by all participating laboratories.
Table 6. Summary of PSP toxin positive samples (+) of ten participating laboratories with any method.
STX PT Sample Analyte 1 2 3 4 5 6 7 8 9 10
A PSP toxin + na + + + + + + + na
E1 PSP toxin + na + + + + + + + na
E2 PSP toxin + na + + + + + + + na
M PSP toxin + + + + + + + na + +
Note: na: not analyzed.
2.3.2. Immunoassay Methods
Immunoassay methods were used by four laboratories. All four test samples were found
PSP positive using the Ridascreen ELISA kit and the Abraxis kit (Table 7). The most diluted
sample (E1, algal extract) was negative using a lateral flow assay (Jellett Rapid Test) as expected
on the basis of the approximate detection limit of 300 µg STXeq/kg [21]. Immunoassay methods
are sensitive for measuring PSP toxins at low levels, except for the Jellett test, which cannot be
applied for the drinking water at health alert level. However, all used immunoassay methods
cannot differentiate PSP congeners. The response of the various PSP toxins is dependent on the kit
properties, and the cross-reactivity of PSP toxins can vary. Further, the results cannot be properly
quantified. Immunoassay methods, especially lateral flow tests, have shown poor sensitivity towards
N-hydroxylated toxins such as GTX1, GTX4 and NEO. These methods are specific for PSP toxins and
good for screening, but they are not sufficient for the identification of saxitoxin. Moreover, it is likely
that the total toxicity is underestimated because of the poor sensitivity of some PSP toxins.
4857
Toxins 2015,7, 4852–4867
Table 7. Immunoassay results of STX PT samples, PSP positive (+) or PSP negative (–).
Laboratory 4 5 5 6 7
Sample
Method Ridascreen Ridascreen Jellett Jellett Abraxis
A + + + + +
E1 + + ´ ´ +
E2 + + + + +
M + + + + +
2.3.3. Mouse Bioassay (MBA)
MBA was used only by two laboratories (Table 8). One participating laboratory used MBA for the
analysis of mussel samples (M) and the other applied this method for the analysis of algal samples (A).
Algal extracts (E1, E2) were not analyzed by MBA. MBA participants were not requested to analyze
sample E1 due to toxin levels being below the limit of detection of the MBA method. So far, MBA is
the only method to measure acute toxicity of samples, but this method provides no information on
the toxin profiles present. The official MBA method is applicable only for mussel samples and it has
not been validated for the algal matrix. MBA has also limited sensitivity and accuracy [22].
Table 8. Mouse bioassay (MBA) results of STX PT samples.
Laboratory 9 10
Sample
Method MBA MBA
A + na
E1 no no
E2 na na
M na +
Note: +: PSP positive; na: not analyzed; no: too dilute for MBA.
2.3.4. Chromatographic Methods
Individual PSP analogues can be determined separately with chromatographic techniques;
pre-column and post-column HPLC-FLD and LC-MS. STX was detected and identified with all
chromatographic techniques applied in those samples that were analyzed (Table 9). However, limited
information was received on the STX identification criteria used by the participating laboratories, and
thus further evaluation of the qualitative results was not possible.
Table 9. Summary of qualitative STX results obtained with LC-MS/MS, pre-column HPLC-FLD (pre),
and post-column HPLC-FLD (post) methods.
Laboratory 1 2 3 4 5 6 7 8 9 10
Method LC-
MS/MS pre-ox post-ox LC-
MS/MS
LC-
MS/MS pre-ox - LC-
MS/MS pre-ox -
Sample Analyte
A STX + na + + + + na + + na
E1 STX + na + + + + na + + na
E2 STX + na + + + + na + + na
M STX + + + + + + na na + na
Note: +: STX positive; na: not analyzed.
Other PSP analogues were also screened with chromatographic techniques and the qualitative
results are summarized in Table 10. Six laboratories reported GTX2>X3, either separately or
together in mussel samples. Six laboratories had found dcSTX and five laboratories NEO in mussel
4858
Toxins 2015,7, 4852–4867
samples. GTX1>X4 was reported by four laboratories, and GTX4 was actually reported only by
one laboratory.
As a summary, only few laboratories were able to identify all PSP analogues in the STX PT
samples. The main differences in the results are probably due to the variable sensitivity of the
methods. In addition it is known that the pre-column method cannot differentiate between the
epimeric pairs of GTX2>X3 and GTX1>X4. One laboratory (3) reported GTX4 in the mussel
sample using the post-column oxidation HPLC-FLD method. GTX5 was reported only by two
laboratories (six and nine). A reason for that may be the temporary unavailability of the certified
GTX5 reference standard before and during the test.
Table 10. Summary of qualitative results of STX analogues found with LC-MS/MS, pre-column
HPLC-FLD (pre-ox), and post-column HPLC-FLD (post-ox) in the samples (laboratories 1–10).
Laboratory 1 2 3 4 5 6 7 8 9 10
Method - pre-ox post-ox LC-
MS/MS
LC-
MS/MS pre-ox - LC-
MS/MS pre-ox -
Sample Analyte
A
GTX2 + +
GTX3 + + +
GTX2&3 + + +
E1
GTX2 + +
GTX3 + +
GTX2&3 + + +
E2
GTX2 + +
GTX3 + + +
GTX2&3 + + +
M
dcSTX + + + + + +
GTX2 + + + +
GTX3 + + +
GTX2&3 + +
GTX1 + +
GTX4 +
GTX1&4 + +
NEO + + + + +
GTX5 + +
2.3.5. Quantitative Results
The quantitative results on STX were statistically evaluated according to the recommendations
of “The International Harmonized Protocol for the Proficiency Testing of Analytical Chemistry
Laboratories” [23] and Algorithm A of the International Standard ISO 13528:2005 [24]. The
quantitative results of STX are presented in Table 11 and Figure 2.
All chromatographic techniques applied for quantitative analysis of STX performed well with
respect to trueness (Figure 3). The variation of quantitative results of STX was very small and mean
of z-scores were within the range of ´2 to +2. Moreover, quantitative LC-MS/MS results for STX
were in good accordance with the official AOAC fluorescence methods. The agreement between
laboratories using the same methods was also remarkable as indicated by the error bars.
When the results of the different matrices were compared, a slightly larger variation in the mussel
sample results was noticed and a significantly larger variation and deviation from the assigned value
was detected in the most diluted sample E1 (Figure 4).
4859
Toxins 2015,7, 4852–4867
Table 11. Summary of reported STX concentrations, assigned values Xa(nominal), their standard and
relative standard uncertainties and the standard deviations of the proficiency assessment.
Sample µ σ Unit nCV Xau(Xa) urel(Xa) (%) σp
A1 621.7 276.4 ng STX/filter 8 0.445 572 19.4 3.4 145.9
A2 522.0 217.8 ng STX/filter 8 0.417 572 19.4 3.4 145.9
A3 609.6 309.4 ng STX/filter 8 0.508 572 19.4 3.4 145.9
E1 8.3 4.8 ng STX/mL 7 0.578 4.7 0.2 4.0 1.2
E2 30.0 13.7 ng STX/mL 8 0.455 28 0.6 2.3 7.1
M1 106.9 59.9 ng STX/g of mussel 6 0.560 126 0.3 0.3 32.1
M2 117.5 69.3 ng STX/g of mussel 6 0.590 126 0.3 0.3 32.1
M3 97.4 54.4 ng STX/g of mussel 6 0.559 126 0.3 0.3 32.1
Note: µ= mean of participants’ results as estimated by robust statistics; σ= standard deviation of participants’
means; n= number of mean results; CV = coefficient of variation (σ/µ); Xa= assigned value; u(Xa) = standard
uncertainty of the assigned value; urel(Xa) = relative standard uncertainty of the assigned value, u(Xa)/Xa(%);
σp= estimated standard deviation for proficiency assessment.
Toxins 2015, 7 11
Table 11. Summary of reported STX concentrations, assigned values Xa (nominal),
their standard and relative standard uncertainties and the standard deviations of the
proficiency assessment.
Sample µ σ Unit n CV Xa u(Xa) urel(Xa) (%) σp
A1 621.7 276.4 ng STX/filter 8 0.445 572 19.4 3.4 145.9
A2 522.0 217.8 ng STX/filter 8 0.417 572 19.4 3.4 145.9
A3 609.6 309.4 ng STX/filter 8 0.508 572 19.4 3.4 145.9
E1 8.3 4.8 ng STX/mL 7 0.578 4.7 0.2 4.0 1.2
E2 30.0 13.7 ng STX/mL 8 0.455 28 0.6 2.3 7.1
M1 106.9 59.9 ng STX/g of mussel 6 0.560 126 0.3 0.3 32.1
M2 117.5 69.3 ng STX/g of mussel 6 0.590 126 0.3 0.3 32.1
M3 97.4 54.4 ng STX/g of mussel 6 0.559 126 0.3 0.3 32.1
Note: µ = mean of participants’ results as estimated by robust statistics; σ = standard deviation of participants’
means; n = number of mean results; CV = coefficient of variation (σ/µ); Xa = assigned value; u(Xa) = standard
uncertainty of the assigned value; urel(Xa) = relative standard uncertainty of the assigned value, u(Xa)/Xa (%);
σp = estimated standard deviation for proficiency assessment.
Figure 2. Cont.
Toxins 2015, 7 12
Figure 2. Graphical overview of the quantitative results of STX by normal probability plots
of z-scores for A, E1–E2, and M. Participating laboratories 1–9, methods: LC-MS/MS (L),
HPLC-FLD pre-column (HR) and post-column (HO).
Figure 3. Effect of methods on the average quality of quantitation of STX, all samples, all
methods, as assessed by the z-score means. Error bars represent ± 1 SD. Number of results:
HPLC-FLD/post = 24, HPLD-FLD/pre = 56, LC-MS/MS = 75.
Figure 4. Effect of sample matrix on the average quality of quantitation of STX, all samples,
all methods, as assessed by the z-score means. The dotted lines represent ±2 z-score values
and the error bars represent ±1 standard error.
Figure 2. Graphical overview of the quantitative results of STX by normal probability plots of z-scores
for A, E1–E2, and M. Participating laboratories 1–9, methods: LC-MS/MS (L), HPLC-FLD pre-column
(HR) and post-column (HO).
4860
Toxins 2015,7, 4852–4867
Toxins 2015, 7 12
Figure 2. Graphical overview of the quantitative results of STX by normal probability plots
of z-scores for A, E1–E2, and M. Participating laboratories 1–9, methods: LC-MS/MS (L),
HPLC-FLD pre-column (HR) and post-column (HO).
Figure 3. Effect of methods on the average quality of quantitation of STX, all samples, all
methods, as assessed by the z-score means. Error bars represent ± 1 SD. Number of results:
HPLC-FLD/post = 24, HPLD-FLD/pre = 56, LC-MS/MS = 75.
Figure 4. Effect of sample matrix on the average quality of quantitation of STX, all samples,
all methods, as assessed by the z-score means. The dotted lines represent ±2 z-score values
and the error bars represent ±1 standard error.
Figure 3. Effect of methods on the average quality of quantitation of STX, all samples, all methods, as
assessed by the z-score means. Error bars represent ˘1 SD. Number of results: HPLC-FLD/post = 24,
HPLD-FLD/pre = 56, LC-MS/MS = 75.
Toxins 2015, 7 12
Figure 2. Graphical overview of the quantitative results of STX by normal probability plots
of z-scores for A, E1–E2, and M. Participating laboratories 1–9, methods: LC-MS/MS (L),
HPLC-FLD pre-column (HR) and post-column (HO).
Figure 3. Effect of methods on the average quality of quantitation of STX, all samples, all
methods, as assessed by the z-score means. Error bars represent ± 1 SD. Number of results:
HPLC-FLD/post = 24, HPLD-FLD/pre = 56, LC-MS/MS = 75.
Figure 4. Effect of sample matrix on the average quality of quantitation of STX, all samples,
all methods, as assessed by the z-score means. The dotted lines represent ±2 z-score values
and the error bars represent ±1 standard error.
Figure 4. Effect of sample matrix on the average quality of quantitation of STX, all samples, all
methods, as assessed by the z-score means. The dotted lines represent ˘2z-score values and the
error bars represent ˘1 standard error.
2.3.6. Quantitative Results of other PSP Toxins
The concentrations of other PSP analogues were determined by two laboratories before the test
with the pre-column oxidation HPLC-FLD method and are presented in Table 12. The total toxicity
of the mussel sample had been set to about 1000 µg STXeq/kg, which was above the regulatory
limit. The homogeneity and stability data for other PSP analogues was not thoroughly studied, and
only few quantitative results were obtained for PSP analogues. Therefore, these results could not be
properly statistically evaluated. The results for PSP toxins in mussel and algal samples are presented
in Tables 12–15.
4861
Toxins 2015,7, 4852–4867
Table 12. Concentrations of PSP toxins ng/g in mussel sample M.
ng/g Mussel dcSTX NEO GTX1 GTX4 GTX1&4 GTX2 GTX3 GTX2&3 GTX5
Before the PT
Ref. Lab 1 a169 ˘8 55 ˘3 na na 316 ˘13 na na 346 ˘24 2.9 ˘0.1
Ref. Lab 2 b194 ˘7 nq na na 430 na na 515 ˘31 35
PT results
n15 15 6 3 6 6 6 6 6
mean 191 157 257 68 704 443 88 555 32
SD 66 154 118 3 409 161 15 40 5
CV% 35 98 46 4 58 36 17 7 16
Note: aMarine Institute; bSPIEZ LABORATORY; na: not analyzed; nq: not quantified; n: number of results.
Table 13. Gonyautoxin 2 (GTX2) and gonyautoxin 3 (GTX3) results in algal sample A (reference value
~4000 ng/filter for GTX2>X3 measured with HPLC-FLD).
PSP Analogue GTX2 GTX3 GTX2&3
ng/filter
n6 9 6
mean 1232 3803 3601
SD 415 850 1639
CV% 34 22 46
Table 14. GTX2 and GTX3 results in E1 (reference value ~40 ng/mL for GTX2>X3 measured with
HPLC-FLD).
PSP Analogue GTX2 GTX3 GTX2&3
ng/mL
n2 2 2
mean 37 44 51
SD 13 25 3
CV% 37 58 6
Table 15. GTX2, and GTX3 results in E2 (reference value ~200 ng/mL for GTX2 & GTX3, measured
with HPLC-FLD).
PSP Analogue GTX2 GTX3 GTX2&3
ng/mL
n232
mean 92 216 214
SD 6 75 121
CV% 6 35 57
3. Experimental Section
3.1. Material Preparation
3.1.1. Toxic Freeze-Dried Algal Sample (STX PT Sample A)
Toxic algal samples were prepared at the Finnish Environment Institute. The PSP toxins
producing strain AOF-0919 of A. ostenfeldii was cultivated at 16 ˝C under light-dark cycle of
14L:10D (ca. 60 µmol photons m´2s´2) in filtered, sterilized sea water (FSW) containing f/2-Si
nutrients and having a salinity of 6 [25]. To grow the volume necessary for the analyses, 10 mL
of an exponentially growing culture (AOF-0919) were inoculated into three replicate culture flasks
containing 150 mL growth medium (FSW with f/2-Si nutrients) and grown for about one month.
Subsequently, 7.4 mL of this inoculum culture were added to each of 30 sterile Nunc™ culture
bottles (EasYFlask™ 75 cm2with filter caps) filled with 150 mL of FSW with f/2-Si nutrients. The
4862
Toxins 2015,7, 4852–4867
cell concentration of the inoculum was calculated to correspond to a final cell concentration of
1000 cells/mL in the experimental bottles. The cultivation was continued for about 5 weeks. The
cell suspensions of the 30 culture bottles were combined, and the concentration of dinoflagellate
cells was calculated (10,200 ˘700 cells/mL, n= 3) from this combined sample. Fractions of 35 mL
from the combined cell suspensions were filtered on filter paper (GF/C, glass microfiber, Ø 25 mm,
1822-025, Whatman, Little Chalfont, UK), and washed with MilliQ water. The filter papers containing
the dinoflagellate cells (~350,000 cells) were put in microcentrifuge tubes (conical, 2 mL, Molecular
BioProducts, catalog #3468, Fisher Scientific, Loughborough, UK). The samples were freeze-dried,
and the tubes sealed with caps (O-ring screw caps, Molecular BioProducts, catalog #3471Y, Fisher
Scientific, Loughborough, UK). The produced samples (n= 120) were stored in a freezer at ´20 ˝C.
3.1.2. Toxic Algal Extracts (STX PT Samples E1 and E2)
Fifteen algal samples on filter paper were extracted with 2 ˆ1.0 mL of LC-MS/MS eluent (4 mM
ammonium formate-ACN 40:60, pH 3.5 adjusted with formic acid), and the volume was adjusted to
2.0 mL. A pooled algal extract was prepared from the extracted algal samples (15 ˆ1.7 mL).
Preparation of the algal extract 1:50 (E1). 5.0 mL of pooled algal extract was diluted to 250.0 mL
with 4 mM ammonium formate, pH 3.5 adjusted with formic acid (diluted algal extract, 1:50).
47 ˆ5.0 mL portions of the diluted algal extracts were measured in 8 mL glass vial (GF 61 ˆ16.6,
2411121634), capped with Teflon sealed caps, and stored in a freezer at ´20 ˝C for PT samples.
Preparation of the algal extract 1:10 (E2). 15.0 mL of pooled algal extract was diluted to 150.0 mL
with 4 mM ammonium formate, pH 3.5 adjusted with formic acid (diluted algal extract, 1:10).
28 ˆ5.0 mL portions of the diluted algal extracts were measured in 8 mL glass vial (GF 61 ˆ16.6,
2411121634), capped with Teflon sealed caps, and stored in a freezer at ´20 ˝C for PT samples.
3.1.3. Toxic Mussel Sample (STX PT Sample M)
Homogenized mussel samples (5 g, n= 100) were obtained from Marine Institute (Galway,
Ireland) in October 2012. Toxic Spanish Mytilus galloprovincialis whole flesh tissue (500 g), toxic
Canadian Mytilus edulis whole flesh tissue (7 g), and blank Irish Mytilus edulis mussel whole flesh
tissue (893 g) were blended with stabilizers and antibiotics (0.02% of each ethoxyquin, ampicillin,
erythromycin, and oxytetracycline) and the moisture content was adjusted to ca. 83.5% with
additional water [26]. The mussel tissues were homogenized with Polytronr6100 (Kinematica™
AG, Luzern, Switzerland), and dispensed into 5 mL polypropylene tubes (Teklab Ltd., Sacriston,
Durham, UK) with a peristaltic pump (Manostat, Barrington, IL, USA) adjusted to dispense ca. 5.3 g
aliquots. The tubes were purged with nitrogen and hermetically sealed with aluminium seal closures
(Seal-it-systems Ltd., Accrington, Lancashire, UK). Wadded screw caps were placed on the tubes. The
frozen mussel samples were stored in a freezer at ´20 ˝C.
3.2. ELISA Measurements of the Samples before PT
STX PT samples were analyzed with a competitive enzyme-linked immunoassay kit (Ridascreen
Fast PSP SC, R1905, R-Biopharm, Darmstadt, Germany). The sample extracts were diluted in the
ELISA buffer provided in the kit with two concentration levels so that the level of the PSP toxins
would be between ca. 5–40 ng/mL. The sample analyses were performed with Ridascreen ELISA
according to the kit instructions and specifications. The absorbances of the samples were measured
in duplicate at 450 nm with a microplate spectrophotometer (Multiskan Go, Thermo Scientific, Vantaa,
Finland). The zero standard (the PSP standard 1) was measured in duplicate as such from two
separate wells (n= 4). The obtained average absorbance value of the zero standards was used for
the calculation of corrected absorbances. The amount of the PSP toxins in the sample was calculated
approximately with the RIDArSOFT Win program with a cubic spline fitting of the standard curve.
4863
Toxins 2015,7, 4852–4867
3.3. Extraction of the Algal Samples for LC-MS/MS Analyses
The freeze-dried algal samples were extracted, centrifuged, and filtered. The algal sample on
filter paper was transferred into 15 mL Falcon tubes (polypropylene, conical, 17 ˆ120 mm). The
mass of the filter paper with algal material was measured, and the algal sample was extracted with
extraction solvent (4 mM ammonium formate-ACN 40:60, pH 3.5 adjusted with formic acid). 1.0 mL
of the extraction solvent was added into the Falcon tube, shaken for 2 min, allowed to stay in an ice
bath for 15 min, and centrifuged with 3700 rpm (relative centrifugal force, RCF 2400ˆg) for 10 min at
4˝C. The sample was transferred into a centrifuge filter (PVDF Ultrafree, 0.45 µm, 0.5 mL, Millipore)
and centrifuged with 14,000 rpm (RCF 21,500ˆg) for 5 min at 4 ˝C. The extraction was repeated with
another 1.0 mL of extraction solvent, and the volume of the extract was adjusted to 2.0 mL with the
extraction solvent.
3.4. Extraction of the Mussel Samples for LC-MS/MS
The mussel samples were extracted with the slightly modified AOAC 2011.02 method [10]. The
mussel sample (5 g) was extracted with 4 mL of 1% acetic acid in MilliQ water by shaking the sample
with Multi Reax mixer at room temperature for 30 min. The sample was heated at 95–100 ˝C for 5 min
and mixed with Multi Reax mixer for 5 min at room temperature. The sample was cooled in an ice
bath for 5 min and centrifuged with 5000 rpm (RCF 2400ˆg) for 10 min at 4 ˝C. The supernatant
was decanted and the extraction was repeated with another 4 mL of 1% acetic acid in MilliQ water.
The extraction solvents were combined and the volume was adjusted to 10.0 mL with 1% acetic acid
in MilliQ water. The sample was transferred into a centrifuge filter (PVDF Ultrafree MC, 0.45 µm,
0.5 mL, Millipore, Carrigtwohill, Ireland) and centrifuged with 14,000 rpm (RCF 21,500ˆg) for 5 min
at 4 ˝C. For the LC-MS/MS analyses, the samples were diluted 1:10 with LC-MS/MS eluent (4 mM
ammonium formate-ACN 40:60, pH 3.5 adjusted with formic acid). The precipitate was filtered
through a LCR filter (PTFE, 0.45 µm, Ø 13 mm, Millex, Merck Millipore, Carrigtwohill, Ireland) prior
to the LC-MS/MS analyses. The detailed sample preparation and LC-MS/MS analyses of mussel
extracts are described by Harju et al. [27].
3.5. LC-MS/MS Analyses
3.5.1. LC-MS/MS Using the Product Ion Scan Mode for the Characterization, Homogeneity and
Stability Studies of Algal and Mussel Samples before the PT
LC-MS/MS was performed using a Finnigan LXQ linear ion trap mass spectrometer with
positive mode electrospray ionization (ESI) source interfaced to a Finnigan Surveyor Autosampler
Plus Liquid Chromatograph (ThermoFinnigan, Hemel Hempstead, UK) with a method validated for
algal samples [20]. The chromatographic separation was carried out on a TOSOH Bioscience HILIC
TSK-gel Amide-80rcolumn (150 mm ˆ4.6 mm, 3 µm particle size, Stuttgart, Germany). A mobile
phase was 4 mM ammonium formate-ACN 40:60, and the pH of the eluents was adjusted to 3.5 with
formic acid. The flow rate was 1.0 mL/min with an accurate post-column splitter (1:20) between
LC and MS. Spray voltage of 5 kV was applied and nitrogen was used as sheath gas. Capillary
temperature was set to 350 ˝C and the relative collision energy was 29%. The quantitative analysis
of STX in algal samples was performed with GTX1 (150 ng/mL) as an internal standard. The mussel
samples were analyzed using only an external STX standard. The certified reference standards of PSP
toxins were purchased from the National Research Council, NRC, Halifax, Canada (except GTX5,
which was temporarily unavailable).
3.5.2. LC-MS/MS with MRM for the Characterization of Algal and Mussel Samples before PT
The LC-MS/MS measurements were carried out on an Applied Biosystems 3200QTrap hybrid
quadrupole-linear ion trap mass spectrometer (Applied Biosystems/MDS Sciex, Foster, CA, USA).
The ion source was a “Turbo V”, SCIEX (Toronto, ON, Canada) which was operated in the positive
4864
Toxins 2015,7, 4852–4867
ESI mode. The chromatographic system was an Agilent Series 1200 HPLC, Agilent Technologies
AG (Basel, Switzerland). A Sequant ZIC-HILIC column 150 ˆ2.1 mm, 5 µm particle size
(Merck, Darmstadt, Germany) in combination with a ZIC-HILIC guard column was used. The
chromatographic conditions were chosen according to Turrell et al. [28]. A mobile phase consisting of
two eluents was used: A (100% deionized water) and B (95%, v/v, acetonitrile). Both eluents contained
2 mM ammonium formate and 3.6 mM formic acid. Flow rate was 0.2 mL/min. The column was kept
at 30 ˝C and the injection volume was 5 µL. The gradient was A: 50%–85% within 15 min, hold 5 min
followed by an equilibration step at A: 50% for 5 min.
3.6. Pre-Column Oxidation HPLC-FLD Analyses
3.6.1. HPLC-FLD Method Used for the Characterization of Sample Material before PT
The measurements were based on the procedures given in the AOAC Official Method 2005.06.
An HPLC system from Thermo Fisher Scientific (Reinach, Switzerland) was used with the following
components: UltiMate LPG-3400RS quaternary analytical pump, UltiMate 3000 analytical split-loop
autosampler, UltiMate TCC-3000RS column thermostat and a fluorescence-detector RF-2000 with
excitation wavelength at 340 nm and emission wavelength at 400 nm. The chromatographic
conditions were chosen according to AOAC. A Supelcosil C18 column (150 ˆ4.6 mm, 5 µm particle
size) from Sigma-Aldrich Chemie GmbH (Buchs, Switzerland) was used. The eluent consisted of
two phases: A: 0.1 M ammonium formate (5% acetonitrile), B: 0.1 M ammonium formate. Flow rate
was 2 mL/min. The gradient conditions were: initially 0% A, hold for 1 min, increase to 5% in 4 min,
increase 5%–70% in 5 min, hold for 2 min and return to 0% A and re-equilibrate for 5 min. The column
temperature was kept at 25 ˝C.
3.6.2. HPLC-FLD Method Used for the Homogeneity and Stability Studies of Mussel Samples
before PT
Samples were extracted, oxidized and analyzed closely following OMA AOAC 2005.06. A
Shimadzu (Kyoto, Japan) LC system with a fluorescence (FLD) detector (ex 340 nm, em 395 nm)
(Shimadzu RF-10AXL) and cooled autosampler (Shimadzu SIL-20A) was used. The LC column
was a reverse phase C-18 Supelcosil (150 mm ˆ4.6 mm, 5 µm) fitted with a C-18 Supelguard
cartridge (20 mm). The LC programme followed was a slightly modified gradient elution based
on that published in AOAC 2005.06 using a flow rate of 1.5 mL/min. The gradient followed was
0%–5% mobile phase B over 5 min, 5%–70% B over the next 4 min, back to 0% B over 2 min, then
keeping at this condition for 7 min before the next injection. Toxin concentrations in sample extracts
were quantified against a five-point calibration. All homogeneity and stability study extracts were
analyzed in the same chromatographic sequence to negate day-to-day instrument variations.
3.7. MBA Tests
National authority permissions for the use of MBA were asked from those laboratories, which
used MBA method in the PT.
4. Conclusions
We have shown the suitability of the algal and mussel sample material for the PT. The prepared
reference material was characterized by various methods and the homogeneity and the stability of the
STX in the samples were assessed prior to the test. Data obtained from the participating laboratories
showed that PSP toxins could be detected in all samples, but MBA and immunoassay methods cannot
differentiate PSP congeners. When compared to MBA, immunoassay methods are specific for PSP
toxins but cannot measure acute toxicity of the samples. MBA turned out not to be applicable to
matrices other than mussel tissue. Most participating laboratories performed well with respect to the
identification and quantification of STX, though quantitative STX results on the most diluted algal
4865
Toxins 2015,7, 4852–4867
extract varied and the coefficients of variation were quite high (42%–59%) for all samples. Limited
information was obtained for other PSP toxins, and more work on the quantification of STX analogues
is needed in the view of the fact that the chemical analyses of STX analogues will replace the MBA in
the future. The quantitative LC-MS/MS results for STX were in good agreement with the fluorescence
results measured with HPLC-FLD.
Acknowledgments: All participating laboratories are kindly acknowledged for contribution to the test and
providing the results. Brigitte Dorner (Robert Koch-Institut, Germany) is thanked for the general coordination
and the management of the EQuATox project. The project was funded by the European Community’s Seventh
Framework Programme (FP7/2007-2013) under grant agreement No. 285120. Kirsi Harju has received additional
funding from the Academy of Finland, project 251609.
Author Contributions: K.H., M-L.R., M.S., and P.V. from VERIFIN (Finland) were responsible for the
organization of the STX PT, characterization of STX reference material with LC-MS/MS and ELISA, homogeneity
and stability studies of the samples with LC-MS/MS, and the quality management of the EQuATox project.
M.A., W.A., and M.S. from SPIEZ LABORATORY (Switzerland) identified and quantified the PSP toxins in
the PT samples with HPLC-FLD and LC-MS/MS. A.K., S.S., and H.K. from the Finnish Environment Institute
(Finland) were responsible for the cultivation of algal samples and preliminary studies of the PSP toxins with
HPLC-FLD. S.B. from Marine Institute (Ireland) provided the homogenized mussel sample material and did the
homogeneity and stability studies of mussel samples before the test. W.L. from ChemStat (Switzerland) carried
out the statistical analyses of sample homogeneity and stability studies as well as of the PT results. All authors
approved the finalized version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References and Notes
1. Kremp, A.; Lindholm, T.; Dressler, N.; Erler, K.; Gerdts, G.; Eirtovaara, S.; Leskinen, E. Bloom Forming
Alexandrium Ostenfeldii (Dinophyceae) in Shallow Waters of the Aland Archipelago, Northern Baltic Sea.
Harmful Algae 2009,8, 318–328.
2. Deeds, J.R.; Landsberg, J.H.; Etheridge, S.M.; Pitcher, G.C.; Longan, S.W. Non-Traditional Vectors for
Paralytic Shellfish Poisoning. Mar. Drugs 2008,6, 308–348.
3. Szinicz, L. History of Chemical and Biological Warfare Agents. Toxicology 2005,214, 167–181.
4. Anderson, P.D. Bioterrorism: Toxins as Weapons. J. Pharm. Pract. 2012,25, 121–129.
5. Wiese, M.; D’Agostino, P.M.; Mihali, T.K.; Moffitt, M.C.; Neilan, B.A. Neurotoxic Alkaloids: Saxitoxin and
Its Analogs. Mar. Drugs 2010,8, 2185–2211.
6. Munday, R.; Thomas, K.; Gibbs, R.; Murphy, C.; Quilliam, M.A. Acute Toxicities of Saxitoxin, Neosaxitoxin,
Decarbamoyl Saxitoxin and Gonyautoxins 1&4 and 2&3 to Mice by various Routes of Administration.
Toxicon 2013,76, 77–83.
7. Anon, A. Paralytic Shellfish Poison, biological method, final action, method 959.08. In Methods of Analysis
of the Association of Official Analytical Chemistry, 15th ed.; AOAC: Arlington, VA, USA, 1990.
8. Anon, A. AOAC official method 2005.06. In AOAC Official Methods of Analysis, 18th ed.; AOAC
International: Gaithersburg, MD, USA, 2006.
9. Turner, A.D.; Norton, D.M.; Hatfield, R.G.; Morris, S.; Reese, A.R.; Algoet, M.; Lees, D.N. Refinement and
Extension of AOAC Method 2005.06 to Include Additional Toxins in Mussels: Single-Laboratory Validation.
J. AOAC Int. 2009,92, 190–207.
10. Anon, A. AOAC official method 2011.02. In AOAC Official Methods of Analysis, 19th ed.; AOAC
International: Gaithersburg, MD, USA, 2012.
11. Anon, A. AOAC official method method 2011.27. In AOAC Official Methods of Analysis, 19th ed.; AOAC
International: Gaithersburg, MD, USA, 2012.
12. DeGrasse, S.; Rivera, V.; Roach, J.; White, K.; Callahan, J.; Couture, D.; Simone, K.; Peredy, T.; Poli, M.
Paralytic Shellfish Toxins in Clinical Matrices: Extension of AOAC Official Method 2005.06 to Human Urine
and Serum and Application to a 2007 Case Study in Maine. Deep Sea Res. Part II 2014,103, 368–375.
13. Turner, A.D.; Lewis, A.M.; Rourke, W.A.; Higman, W.A. Interlaboratory Comparison of Two AOAC
Liquid Chromatographic Fluorescence Detection Methods for Paralytic Shellfish Toxin Analysis Through
Characterization of an Oyster Reference Material. J. AOAC Int. 2014,97, 380–390.
4866
Toxins 2015,7, 4852–4867
14. Turner, A.D.; Hatfield, R.G. Refinement of AOAC Official Method 2005.06 Liquid
Chromatography-Fluorescence Detection Method to Improve Performance Characteristics for the
Determination of Paralytic Shellfish Toxins in King and Queen Scallops. J. AOAC Int. 2012,95, 129–142.
15. Ben-Gigirey, B.; Rodríguez-Velasco, M.L.; Otero, A.; Vieites, J.M.; Cabado, A.G. A Comparative Study for
PSP Toxins Quantification by using MBA and HPLC Official Methods in Shellfish. Toxicon 2012,60, 864–873.
16. Ben-Gigirey, B.; Rodriguez-Velasco, M.L.; Gago-Martinez, A. Extension of the Validation of AOAC Official
Method 2005.06 for dc-GTX2,3: Interlaboratory Study. J. AOAC Int. 2012,95, 111–121.
17. Ben-Gigirey, B.; Rodríguez-Velasco, M.L.; Villar-González, A.; Botana, L.M. Influence of the Sample Toxic
Profile on the Suitability of a High Performance Liquid Chromatography Method for Official Paralytic
Shellfish Toxins Control. J. Chromatogr. A 2007,1140, 78–87.
18. Work Instruction for the Reporting of the Results of the OPCW Proficiency Tests, QDOC/LAB/WI/PT04, p. 48.
19. European Commission. Commission Decision of 12 August 2002 implementing Council Directive
96/23/EC concerning the Performance of Analytical Methods and the Interpretation of Results
(2002/657/EC). Off. J. Eur. Communities 2002,45, 8–36.
20. Halme, M.; Rapinoja, M.-L.; Karjalainen, M.; Vanninen, P. Verification and Quantification of Saxitoxin from
Algal Samples using Fast and Validated Hydrophilic Interaction Liquid Chromatography-Tandem Mass
Spectrometry Method. J. Chromatogr. B 2012,880, 50–57.
21. Laycock, M.V.; Donovan, M.A.; Easy, D.J. Sensitivity of Lateral Flow Tests to Mixtures of Saxitoxins and
Applications to Shellfish and Phytoplankton Monitoring. Toxicon 2010,55, 597–605.
22. Alexander, J.; Benford, D.; Cockburn, A.; Cravedi, J.-P.; Dogliotti, E.; di Domenico, A.; Fernández-Cruz, M.L.;
Fink-Gremmels, J.; Fürst, P.; Galli, C.; et al. Scientific Opinion of the Panel on Contaminants in the Food
Chain on a request from the European Commission on Marine Biotoxins in Shellfish-Saxitoxin Group.
EFSA J. 2009,1019, 1–76.
23. Thompson, M.; Ellison, S.L.R.; Wood, R. The International Harmonized Protocol for the Proficiency Testing
of Analytical Chemistry Laboratories: (IUPAC Technical Report). Pure Appl. Chem. 2006,78, 145–196.
24. International Organization for Standardization. ISO 13528: Statistical Methods for Use in Proficiency Testing
by Interlaboratory Comparisons; International Organization for Standardization: Geneva, Switzerland, 2005.
25. Guillard, R.R.L. Culture of Phytoplankton for Feeding Marine Invertebrates. In Culture of Marine Invertebrate
Animals; Smith, W.L., Chanley, M.H., Eds.; Plenum Press: New York, NY, USA, 1975; pp. 26–60.
26. Burrell, S.; Clion, V.; Auroy, V.; Foley, B.; Turner, A.D. Heat Treatment and the Use of Additives to Improve
the Stability of Paralytic Shellfish Poisoning Toxins in Shellfish Tissue Reference Materials for Internal
Quality Control and Proficiency Testing. Toxicon 2015,99, 80–88.
27. Harju, K.; Rapinoja, M.-L.; Avondet, M.-A.; Arnold, W.; Schär, M.; Burrell, S.; Luginbühl, W.; Vanninen, P.A.
Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency
Test Mussel Sample using Liquid Chromatography-Tandem Mass Spectrometry. Toxins 2015,7. [CrossRef]
28. Turrell, E.; Stobo, L.; Lacaze, J.; Piletsky, S.; Piletska, E. Optimization of Hydrophilic Interaction Liquid
Chromatography/Mass Spectrometry and Development of Solid-Phase Extraction for the Determination
of Paralytic Shellfish Poisoning Toxins. J. AOAC Int. 2008,91, 1372–1386.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open
access article distributed under the terms and conditions of the Creative Commons by
Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
4867
