KELOMPOK 1

NAMA ANGGOTA : NADIA RAMADHANI

TIKA YUSRI LESTARI
ADISAPUTRA
REVIEW JURNAL PEMISAHAN KIMIA
JUDUL Contribution of Mass Spectrometry to the Advances in Risk
Characterization of Marine Biotoxins: Towards the
Characterization of Metabolites Implied in
Human Intoxications
TUJUAN The development of analytical methods able to evaluate and
PENELITIAN characterize the different toxic analogs involved in the
contamination, Liquid Chromatography coupled to different
detection modes.
METODE The method of choice due to its potential for separation,
PENELITIAN identification, quantitation, Mass Spectrometric.
HASIL PENELITIAN Marine biotoxins are natural contaminants produced during
harmful algal blooms being accumulated in seafood, thus
representing a threat to human health. Significant progress has
been made in the last few years in the development of analytical
methods able to evaluate and characterize the different toxic
analogs involved in the contamination, Liquid Chromatography
coupled to different detection modes, including Mass
Spectrometry, the method of choice due to its potential for
separation, identification, quantitation and even confirmation of
the different above-mentioned analogs. Despite this, the risk
characterization in humans is still limited, due to several reasons,
including the lack of reference materials or even the limited
access to biological samples from humans intoxicated during
these toxic events and episodes, which hampered the advances in
the evaluation of the metabolites responsible for the toxicity in
humans. Mass Spectrometry has been proven to be a very
powerful tool for confirmation, and in fact, it is playing an
important role in the characterization of the new biotoxins
analogs. The toxin metabolization in humans is still uncertain in
most cases and needs further research in which the
implementation of wmethods is critical. This review is focused on
compiling the most relevant information available regarding the
metabolization of several marine biotoxins groups, which were
identified using Mass Spectrometry after the in vitro exposition of
these toxins to liver microsomes and hepatocytes. Information
about the presence of metabolites in human samples, such as
human urine after intoxication, which could also be used as
potential biomarkers for diagnostic purposes, is also presented.
KESIMPULAN The detection of marine biotoxin metabolites in human samples
for diagnosis purposes is still in its early steps. Currently, most of
the studies of metabolization are focused on in vitro studies
combined with the MS detection of metabolization products. The
 availabil- ity of these data would facilitate the investigation of
specific metabolites once samples from humans exposed to
contaminations were available. While targeted approaches, such
as LC–MS/MS in the MRM mode, allowed the identification of
toxin metabolites, the future perspectives might be focused on
untargeted approaches based on LC–HRMS combined with the
use of software supporting and facilitating the LC–MS
identification of marine biotoxin metabolites. In addition, the
availability of reference materials of toxins with limited standards
available, such as ciguatoxins, will be critical to carry out in vivo
experiments to evaluate the metabolization process. The evolution
of MS and the availability of sensitive MS approaches will also
contribute to the identification, confirmation, and even
characterization of the metabolites allowing the identification of
potential biomarkers of food poisonings in human samples.

JUDUL Bivalve Shellfish Safety in Portugal: Variability of Faecal Levels,

Metal Contaminants and Marine Biotoxins during the Last
Decade (2011–2020)
TUJUAN The need to obtain a safe product for human consumption led to
PENELITIAN the implementation of standardised hygiene regulations for
harvesting and marketing bivalve mollusc.
METODE In this study, the seasonal and temporal variability of these
PENELITIAN parameters were analysed using historical data generated by the
monitoring programme during the last decade.
HASIL PENELITIAN The Portuguese Monitoring Programme during the Years 2011 to
2020
The Portuguese monitoring programme has evolved and changed
throughout the last decade to better cope with the demands of
shellfish consumers and producers, and to respond to the EU
regulation that has been implemented throughout the last decade.
This development and upgrade, always focusing on public health
safety, included a noticeable intensification of the sampling
frequency.
The number of samples for microbiological monitoring increased
for the majority of the production areas, namely RIAV1, RIAV2,
L5, L6 and L8 during the studied period. The highest increase in
the number of tested samples was observed in 2014, with rises
ranging between 95% and 800%.
The sampling efforts for metals monitoring in shellfish also
improved during the last decade. From 2013 to the present date, at
least one speciesfrom each production area began to be
systematically analysed.
For marine biotoxins, the sampling effort practicallydoubled. The
exception was L5, where the number of samples received for
analysis presented a 24% decrease. The reported reduction
resulted from the elimination of one non-strategic sampling point.
The shellfish production areas L7 and RIAV1 were the production
sites that presented the highest rise in the number of analysed
 samples, 392% and 503%, respectively. The sampling effort
increase in the L7 production area resulted from the two new
mussel aquacultures installed in the region in 2014.
KESIMPULAN Overall, during the decade of 2011–2020, the monitoring program
of shellfish produc- tion areas underwent a considerable
improvement, with an increase in the number of tested samples
and their representativeness. Concerning classification of shellfish
production areas, flexibility in the system implementation, such as
reflecting clear and consistent season-dependent variations, could
lower the economic burden on the shellfish industry. Additionally,
in view of metal levels consistently being below the EU
regulatory limits throughout the decade, this contamination issue
is unlikely to restrict the shellfish farm- ing/picking activities in
the studied areas. On the other hand, the occurrence of biotoxins
will certainly persist and an increasing trend is indeed foreseen.
Moreover, new and emerg- ing toxins—resulting from changes in
climatic conditions, human pressures and nutrient inputs into the
coastal areas—are expected to occur on the Portuguese coast, and
may represent a threat to seafood safety until they are included in
the monitoring program.
Good communication channels and data sharing among
institutions are important, as several competent authorities are
involved in the management of shellfish waters, production areas
and the official control of live bivalves’.
These competent authorities should implement prevention and
remediation mea- sures, including reduction of sources of faecal
contamination, pollutant emissions, and environmental restoration
when needed, contributing to the socioeconomic development of
primary food business operators, high quality and edibility of
shellfish products and consumer safety.
The existence of robust and available databases regarding
contamination parameters and environmental factors may
contribute to a more efficient approach to the problem, promoting
the development of predictive models to help forecast and
minimise the effects of future contamination events.
The present study can contribute to the development of strategies
aiming to minimise the stakeholders’ economic losses and to
promote an improvement in the management of shellfish
production areas and in shellfish safety on the Portuguese coast.

JUDUL Paralytic and Amnesic Shellfish Toxins Impacts on Seabirds,

Analyses and Management
TUJUAN Collaboration between seabird experts and marine biotoxins
PENELITIAN researchers is needed to identify and report the potential
involvement of HABs and their toxins in the mortality events.
Future studies on the PSTs and ASTs pharmacodynamics,
together with the establishment of lethal doses in various seabird
species, are also necessary.
METODE The analytical techniques commonly employed, the tissues
PENELITIAN selected and the adjustments done in protocols for processing
seabird matrixes are summarized.
HASIL PENELITIAN Once an event happens, the Avian Mortality Event Response Plan
contemplates 4 response steps:
1. Reporting the event. They provide a list with all the contacts in
each area.
2. Collecting basic information about the event: contact details of
the person in the field, date of onset, exact location, etc. The
response team records the exact number, species,
sex and age of the carcasses, samples collected, preservation
method and storage and
symptoms shown in sick animals.
3. Collection, packaging and shipping the carcasses. Contacting
the laboratory to assist
in samples collection, packaging and storage. Collecting the
freshest dead speci- mens that should be representative of all the
affected species. Discarding carcasses appropriately to prevent
scavenging.
4. Communicating the results in a direct an efficient way
involving general public, national agencies, residents, wildlife
hospitals staff, social media, etc.
The lack of human and logistic means is, in most cases, the main
drawback to de-
veloping complete plans for seabirds MMEs in most countries.
Unfortunately, regarding HAB seabird-related mortalities,
biopsies, necropsies, and toxin analyses (in blood, tissues and
feathers) are rarely conducted [32]. Therefore, cause/effect
associations often rely on anecdotal evidence, which is
insufficient for clearly implicating algal toxins as the causative
agent [29,32]. From the previous sections, it can be inferred that
the number of papers where PSTs and/or ASTs analyses have
been conducted in seabird samples from MMEs is very limited.
On top of that, sometimes analytical sample size is not
representative for the total affected population [32]. Most of the
studies consulted in this review mention a high number of death
seabirds as opposed to a minimal number of samples analyzed.
Another issue may be the methods selected for analysis. Early
studies to analyze PSTs in seabirds were conducted with MBA,
which is not sensitive enough to detect the very low PSTs levels
that could cause seabird death [106]. Some of the more recent
studies used ELISA kits that, although useful for screening
purposes, do not cover most PSTs for which standards are
available [107]. Furthermore, most seabird MMEs incidence
reports are likely to severely underestimate the number of affected
individuals [29] and this could be related to the sampling
procedure employed (i.e., collecting only carcasses found in
beaches). Sensitization of the general public in the potential
harmful effects of HABs in seabirds and citizens involvement in
 the management of seabirds MMEs is important and should be
part of a good management plan.
KESIMPULAN There exist difficulties in establishing the relationship between
marine bird mortality events and HABs due to several factors
such as lack of means and financial resources, specific and
expensive laboratory tests, lack of structured and coordinated
action protocols, absence of systematic history records and, in
certain areas, very low interest in common species. In many
remote areas, the lack of observers and difficult
collection/sampling logistics could be an additional handicap. To
better manage and ensurre the future viability of seabird
populations, it is imperative to investigate and incorporate the
risks posed by HABs to seabirds in different world areas. This
could include the proper design of the sampling plans, the
implementation of standardized necropsy protocols in a
representative number of dead seabirds, the collection of non-
invasive samples from symptomatic and healthy birds, the
adoption of protocols for the recovery of ill birds and the
establishment of procedures for the systematic testing of potential
algal biotoxins involved.
In terms of PST and AST analysis in seabird samples, laboratories
should preferably select sensitive methods that cover all the toxins
for which standards are available. It is also required to adapt these
methods (generally developed and validated for bivalve mollusks)
to seabird matrixes and to their small sample sizes. Tissue
selection is also a critical point. In terms of PSTs, the lack of
studies on their metabolism and elimination on seabirds makes it
more difficult to select the right tissue. The consideration of
nonlethal sampling (feathers, blood or fecal analyses) should be
further explored, since it could allow the analysis of marine
biotoxins in more animals. The phytoplankton monitoring of the
areas where MMEs are taking place is also crucial to aid in the
selection of the potential marine biotoxins present in the samples.
It is important to highlight that the investigation of the cause/s
behind MMEs requires a coordinated research effort at clinical,
ecological and analytical levels. Support from governments and
citizens collaboration are also essential. All together would allow
a better understanding of the effects of HABs in wild bird
populations and their implication on ecosystems health,
permitting their use as sentinels in marine environments.

JUDUL Emerging Marine Biotoxins

TUJUAN The effect of microbiological factors on both growth and toxin
PENELITIAN production
METODE A combination of toxicity screening with an in vitro cell test, i.e.,
PENELITIAN the neuroblastoma cell viability test, is often used to screen the
samples. This assay can also be used for effect directed analysis
as that conducted by Estevez et al.
HASIL PENELITIAN Sample extracts from fish collected from Macaronesia were
fractionated. The fractions were screened for toxicity and the
active fractions were analyzed by liquid chromatography and
tandem mass spectrometry (LC-MS/MS) to confirm the
ciguatoxins involved in these contaminations. Caribbean
ciguatoxin-1 (C-CTX-1) has been identified as being mainly
responsible for the CTX contamination in these areas, although
some other C-CTX1 analogues and metabolites have also been
identified. Reis Costa et al. also reported on the presence of C-
CTX1 in fish from the Portuguese area of Madeira and the
Selvagem Islands [3]. It is difficult to conclude if ciguatoxins are
really new to Macaronesia, or if their recent identification has
been due to the increased research and knowledge on this
particular issue. Alternative approaches can be also used for
screening of a wide variety of toxins, based on non-targeted high
resolution mass spectrometry. However, the confirmation of the
suspected toxins by their accurate mass is still challenging, either
due to the lack of certified standards or the lack of sensitivity of
the high resolution mass spectrometer. Despite these limitations,
significant improvements have been made [4]. In addition, the
sensitivity of the MS detection of polytoxins has been
considerably increased by using the so called cationization [5].
Comparable to the ciguatoxins is the occurrence of tetrodotoxin
(TTX), which was recently associated with contaminations in
bivalves, and not only to fish and gastropods as reported in the
past. Furthermore, it was not expected to occur in temperate water
conditions. However, researchers have demonstrated its presence
in shellfish from the UK as well as seasonal occurrence in the
Netherlands [6,7]. In the Dutch situation, an action limit was set at
44 μg TTX/kg shellfish, which is based on the opinion by
European Food Safety [8]. As TTX has the same mode of action
as saxitoxin (STX), Finch et al. investigated the acute toxicity of
TTX and STX/TTX mixtures in mice through various routes of
administration [9]. They found additivity between STX and TTX,
and therefore concluded that TTX could be treated as a member of
the paralytic shellfish toxin group (i.e., STXs).
KESIMPULAN Alternative approaches can be also used for screening of a wide
variety of toxins, based on non-targeted high resolution mass
spectrometry. However, the confirmation of the suspected toxins
by their accurate mass is still challenging, either due to the lack of
certified standards or the lack of sensitivity of the high resolution
mass spectrometer. Despite these limitations, significant
improvements have been made [4]. In addition, the sensitivity of
the MS detection of polytoxins has been considerably increased
by using the so called cationization [5].
Comparable to the ciguatoxins is the occurrence of tetrodotoxin
(TTX), which was recently associated with contaminations in
bivalves, and not only to fish and gastropods as reported in the
past. Furthermore, it was not expected to occur in temperate water
conditions. However, researchers have demonstrated its presence
 in shellfish from the UK as well as seasonal occurrence in the
Netherlands [6,7]. In the Dutch situation, an action limit was set at
44 μg TTX/kg shellfish, which is based on the opinion by
European Food Safety [8]. As TTX has the same mode of action
as saxitoxin (STX), Finch et al. investigated the acute toxicity of
TTX and STX/TTX mixtures in mice through various routes of
administration [9]. They found additivity between STX and TTX,
and therefore concluded that TTX could be treated as a member of
the paralytic shellfish toxin group (i.e., STXs).
Furthermore, not truly marine biotoxins, cyanobacterial toxins
such as microcystins are known to cause problems in drinking
water and might not only end up in shellfish and fish produced in
freshwater conditions, but also in estuaries [10].

JUDUL The Incidence of Marine Toxins and the Associated Seafood

Poisoning Episodes in the African Countries of the Indian Ocean
and the Red Sea
TUJUAN the aim of this review is to evaluate the occurrence of MTs and
PENELITIAN associated poisoning episodes as a contribution to public health
and monitoring programs as an MT risk assessment tool for this
geographic region.

METODE African countries of the Indian Ocean and the Red Sea, to date,
PENELITIAN only South Africa has a specific monitoring program for MTs and
some other countries count only with respect to centers of seafood
poisoning control
HASIL PENELITIAN The main geographical focus of this review is the African Indian
and the Red Sea coasts, including surrounding islands (Figure 14).
The marine environment of this area is understudied due to a lack
of monitoring infrastructure. There is a high rate of poverty in
local communities, and the local population is vulnerable to
natural disasters [including HABs, tropical storms]. The
exponential increase in population accompanied by
industrialization and climate change contributes to eutrophication
in coastal areas [295,296]. This study area is characterized as
subtropical to tropical climate with a water temperature above 20
◦C [297]. Eutrophication and the transportation of cysts [through
maritime traffic] are considered the main factors contributing to
large phytoplankton blooms, including those comprised of HAB
species and/or pathogenic bacteria [295,296]. Countries with
monitoring programs of marine environments related to control of
seafood poisoning are listed in Table 2. A few of these programs
have noted the presence of MTs (Figure 14) and HAB species
[dinoflagellates, cyanobacteria, diatoms], some of which [HAB
species] were detected/confirmed by microscopic techniques and
some confirmed by partial 16 S rRNA genes analysis [12,13,298–
323]. The occurrence of species of phytoplankton including MTs-
producing HABs has been reported in coastal waters of South
Africa through scientific reports and environmental monitoring
 programmes since 2011 [324]. Reported producer species include
cyanobacteria (Microcystisaeruginosa, Oscillatoria sp.,
Trichodesmium sp.), dinoflagellates (Dinophysisacuminata, D.
rotundata, Alexandrium catenella, A. minutum, Gymnodinium
sp., Prorocentrum sp., Gambierdiscustoxicus, Ostreopsis
siamensis, O. ovata, P. lima, P. concavum), diatoms (Pseudo-
nitzschia multiseries) [19,305,309,315,331–333] and bacteria
(Vibrio parahaemolyticus) [298]. Seafood poisoning cases were
also reported in South Africa caused by PSTs, DSPs, PlTXs and
GYM [19,216,309,334] (Table 3) after the consumption of
mussels (Donax serra, Perna perna and Chloromytilus
meridionalis) (Table 4) [37]. To minimize seafood poisoning by
MTs, South Africa has implemented, through the Department of
Agriculture, a program for MT monitoring in molluscan shellfish
on all coasts (South African Molluscan Shellfish Monitoring and
Control Programme) [324] (Table 2). This program was created
based on the regulations of the European Commission (EC)
Regulation, namely: Commission Regulation (EC) No 2074/2005,
No 853/2004 and No 15/2011 where limit values are described for
MTs and analytical techniques are advised to monitor shellfish
[324].
Due to the absence of legislation regarding CTXs, currently, there
is an absence of monitoring programs regarding this group in
South Africa.Since the Indian Ocean is considered an endemic
site of CTXs, this is a matter of major importance.
KESIMPULAN African Indian Ocean and the Red Sea coasts have a subtropical
and tropical climate, considered optimal for the development and
transportation of several HAB-forming species, and consequently,
the production of MTs. Paradoxically, studiesrelated to the
occurrence and incidence of HABs and MTs are very limited,
from Sout h Africa to Egypt. From a few data available in this
zone, most describe only the genus and not the full species,
making it very difficult to evaluate the occurrence of the toxic
species. The most reported HAB phytoplanktons in this region are
cyanobacteria, followed by dinoflagellates, and diatoms as
potential MT producers. Relative to MTs, the most reported and
involved in seafood poisoning episodes include CTXs, PSTs, and
TTXs. The scarcity of the data related to MTs suggests the need
for further studies and the creation of specific monitoring
programs of HABs, particularly for dinoflagellates and diatoms
since these constitute the phytoplankton that produces more fatal
MTs, though in recent years several genera of bacteria have been
described as producers of a potent group of marine toxins, TTXs,
which have already been detected on the African coasts of the
Indian Ocean and Red Sea. The main MTs that must be monitored
in shellfish are presented in Table 5. Analytical techniques such as
LC-MS/MS are advised and recommended as determination and
quantification methods due to their higher reproducibility,
specificity, sensitivity and capacity to discriminate analogs of
given toxins in the sample. The permitted limit of a toxin in
 shellfish can be adopted from other countries as an example to
follow such as the EU region, USA, Japan, Australia, and New
Zealand.

JUDUL Emerging Marine Biotoxins in Seafood from European Coasts:

Incidence and Analytical Challenges
TUJUAN evaluation and characterization of these toxins are considered a
PENELITIAN priority for the European Food Safety Authorities (EFSA), which
also emphasize the search for occurrence data using reliable and
efficient analytical methods.
METODE -
PENELITIAN
HASIL PENELITIAN The best methodology for qualitative identification is the N2a cell
assay, as for CTXs, proposed
TTX [14]. TTX [14].
The best methodology for qualitative identification is the N2a cell
assay, as for CTXs, proposed
by [41,68–70], since TTX blocks the sodium channels, acting as
an antagonist of O/V. Cell survival is by [41,68–70], since TTX
blocks the sodium channels, acting as an antagonist of O/V. Cell
survival is related to the concentration of TTX in a directly
proportional way; the higher the analyte concentration, related to
the concentration of TTX in a directly proportional way; the
higher the analyte
the more cell survival will be observed. concentration, the more
cell survival will be observed.
This cell assay provides information on the total toxicity related to
the sodium channels of
This cell assay provides information on the total toxicity related to
the sodium channels of the
the different samples (Figure 6). The main limitations of the cell
assay are the lack of specificity; different samples (Figure 6). The
main limitations of the cell assay are the lack of specificity;
nevertheless, it is a good approach for screening toxicity based on
similar mechanisms of action, such as nevertheless, it is a good
approach for screening toxicity based on similar mechanisms of
action, such
the blocking of sodium channels. Matrix effects must also be
considered to avoid false positives.
as the blocking of sodium channels. Matrix effects must also be
considered to avoid false positives.
Regarding physicochemical methods, the most widely used are
the chromatographic methods (both gas and liquid
chromatography) coupled to different detection modes, in
particular to mass spectrometry due to its potential for
confirmation. Hydrophilic interaction liquid chromatography
(HILIC-LC) has been widely recommended for chemical
structures like TTXs [25,71–73] due to its ability to effectively
separate small polar compounds on polar stationary phases.
 Therefore, this chromatographic approach has been selected as the
separation method of choice for these particular compounds while
its coupling to mass spectrometry (MS) also plays an important
role for confirmation purposes. HILIC–LC-MS/MS is, therefore,
the method recommended by the European Union Reference
Laboratory for Marine Biotoxins (EURLMB) for the
determination of TTXs. The method has been interlaboratory
validated [74] and transferred to the EU National Reference
Laboratories (NRLs) to help with the evaluation of the occurrence
data of TTX in mollusks from their production areas (Figure 7).
KESIMPULAN The characterization of the risk of the presence of emerging
marine biotoxins in European coastal areas is a priority concern
for the EU Health Authorities. Occurrence data are necessary to
characterize the risk, and the development of analytical methods
for screening and confirmation is strictly required for this
purpose. The development of reference materials is still a pending
issue that is hampering the advance of both risk evaluation and
characterization.

JUDUL Molecular Responses of Mussel Mytilus galloprovincialis

Associated to Accumulation and Depuration of Marine Biotoxins
Okadaic Acid and Dinophysistoxin-1 Revealed by Shotgun
Proteomics
TUJUAN Functional enrichment analysis revealed the up-regulation of
PENELITIAN carboxylic (GO:0046943) and organic acid transmembrane
transporter activity (GO:0005342) pathways after 3 days uptake
for digestive gland.

METODE Toxin Analysis, experimental design , microalgae culture

PENELITIAN
HASIL PENELITIAN The analysis of DSTs in the digestive gland and gills revealed
accumulation of OA and DTX1 only in the former. A significant
increase (p < 0.05) of OA concentration in mussels was detected
between days 3 and 5 of the uptake phase. No significant
differences were detected for DTX1 between the same days. The
maximum amount recorded was 2819.2 ± 522.2 μg OA/kg and
1107.1 ± 267.9 μg DTX1/kg at the end of the uptake phase to the
microalga P. lima. A significant decrease (p < 0.05) in the amount
of OA and DTX1 accumulated in digestive gland with regard to
day 5 (end of uptake period) was detected at days 14 and 20 of the
experiment, during depuration (Figure 1). At the end of the
depuration phase, only 16% and 47% of the total OA and DTX1,
respectively, accumulated during the exposure to P. lima,
remained in the digestive gland tissues. These results suggest that
the elimination of OA is more efficient than DTX1 in this mussel
species. Attending to the levels observed in the digestive gland
and assuming that it generally represents4 12–14% of the mussels
flesh (Blanco et al., 2007), it is estimated that the amounts in the
 whole body have exceeded more than 3 times the maximum limit
of 160 μg OA eq./kg, allowed in shellfish for consumption.
KESIMPULAN In this work, a shotgun proteomics approach was used to
investigate the molecular mechanisms involved in the response of
gills and digestive gland of the mussel M. galloprovincialis to a
DST-producing P. lima strain. The assessment of toxin
accumulation in the wo tissues revealed a substantial
bioaccumulation of OA and DTX1 only in the digestive gland.
The maximum amount recorded was at the end of the uptake
phase and, after depuration, toxin was still detected in the
digestive gland tissues. Proteomic results for both organs
presented similar dynamics which was characterized by a higher
number of DEPs during the exposure phase in comparison with
depuration. Surprisingly, the early uptake phase accounted for
most responses during the exposure period. Furthermore, the
profile of expression of the DEPs in the early uptake phase
diverged from the latter time-points investigated. Results revealed
tissue-specific responses with almost no common DEPs between
both tissues. The hypergeometric distribution significance test
revealed the up-regulation of carboxylic (GO:0046943) and
organic acid transmembrane transporter activity (GO:0005342)
pathways after 3 days uptake for digestive gland. In this last
organ, most of the DEPs found for all phases were related with
“cellular processes and signaling” and involving signal
transduction mechanisms, cytoskeleton and post-translational
modification, protein turnover, chaperone functions. In gills, the
early uptake phase is characterized by a balance between DEPs
related with “cellular processes and signaling” and “metabolism.”
Regarding the former category, most of DEPs are also involved in
the same subcategories found for digestive gland. At this time
point, carbohydrate, amino acid and lipid metabolism assume a
similar importance. During depuration, most of the DEPs found
for gills stand for “metabolism,” involving mainly secondary
metabolites biosynthesis, transport, and catabolism.

JUDUL In vitro Evaluation of Programmed Cell Death in the Immune

System of Pacific Oyster Crassostrea gigas by the Effect of
Marine Toxins
TUJUAN Therefore, marine toxins trigger PCD’s signaling pathways in C.
PENELITIAN gigas hemocytes, depending on the toxin’s nature, which appears
to be highly conserved both structurally and functionally.
METODE Source of Oysters, Toxins and Apoptosis Inducers, Proteinaceous
PENELITIAN Toxins,Hemolymph Extraction,Quantitative Real-Time PCR
HASIL PENELITIAN Cytotoxicity of Hemocytes
To investigate if marine toxins cause hemocyte toxicity, we
measured cell viability after 4h of exposure to proteinaceous
and non-proteinaceous toxins and apoptosis inducers (Figure 1).
Control cells in sterile saline NaCl, 0.9% (Neg) showed negligible
cell viability changes, while hemocytes treated with 10mM
 HCl pH 2 (Pos) showed the expected cell death of 97%.
Staurosporine (STP), camptothecin (CPT), saxitoxin (STX),
gonyautoxin epimers 2 and 3 (GTX2/3), and the mixture of
brevetoxins 2 and 3 (PbTx-2,-3) provoked a dose-dependent
effect, with hemocyte mortality above 50% for the highest
concentrations. The mixture of okadaic acid and dinophysistoxin
1 (AO/DTX-1) was not toxic at the concentration range tested,
and brevetoxin 2 (PbTx-2) exerted minor toxicity only at 5 μg
mL.
KESIMPULAN Unquestionable pathophysiological implications.
Finally, we do not rule out the importance of different cell death
types that could be causing misbalance of hemocytes, such as
necrosis, necroptosis, autophagy, pyroptosis, pironecrosis, etc.
The marine non-proteinaceous STX, GTX2/3, and PbTx-2,-3
trigger signaling pathways that promote apoptotic cell death, as
the apoptotic inducers CPT and STP, but PbTx-2,-3 did not show
oligonucleosomal fragmentation. Vp and Vc extracts, OA/DTX-1,
and PbTx-2 increased pro-inflammatory caspase-1 indicative of
signaling pathways leading to PCD by a pyroptosis- like process;
still, protective mechanisms could influence in the low cell death
observed. The results presented illustrate the complexity of the
hemocyte response to marine toxins. Nevertheless, they are
consistent with the role of PCD to preserve a healthy and balanced
immunity, keeping hemocytes at normal levels for the systematic
and regulated dismantling elimination of damaged
cells in C. gigas.

JUDUL Marine biotoxins: types of poisoning, underlying mechanisms of

action and risk management programmes

TUJUAN These toxins, which exert hepatotoxic or neurotoxic effects, are

PENELITIAN mainly relevant as potential contaminants of drinking water and
food supplements and are not included in this chapter.
METODE Immunochemical methods, Analytical chemical
PENELITIAN methods,Prevention and risk management
HASIL PENELITIAN The common processing and preparation methods of marine foods
in domestic and industrial practice do not inactivate algal toxins
and may, assuming toxins are water-soluble, at best lead to toxins
cross-over to the boiling water and hence have a dilution effect.
On several occasions, methods for detoxification of mussels have
been described (e.g. treatment with ozone or chlorine
compounds), none of which are ready for implementation in
practice. Although many studies have been dedicated to the
decontamination of drinking water and some promising
approaches have been suggested, a satisfactory solution is not yet
available. The risks resulting from the various algal toxins differ
considerably. Whilst Ciguatera poisonings are assumed to add up
to 50,000 to 500,000 cases annually, the incidence of ASP is
limited to one single outbreak, in spite of the wide distribution of
 domoic acid. The risk of exposure is, besides on consumption
habits, heavily dependent on the geographical location and for
Central Europe should consequently be rather low. Although in
the past years some European regions have seen repeated toxic
algal blooms and contaminated marine animals, intensive
monitoring has prevented major outbreaks. For the major algal
toxins, limits have been established. For some of the toxins, such
as TTX, cyclic imines and palytoxins, no limits have been
established at the EU level. However, the European Food Safety
Authority advises a limit of 30 μg palytoxin/kg meat and of 44 μg
TTX/kg meat (Table 3). This is based on a group ARfD of 0.25
g/kg bw applying to TTX and its analogues (EFSA, 2017).
Regulations and surveillance programs differ from one country to
another (Table 3), depending on the type of toxins encountered.
However, due to globalization and increase in imports/ exports,
there is a trend toward harmonized legislation. While the issues
are different in Pacific Islands, i.e. with a high prevalence of CFP,
there were recent intoxications reported in France and Germany
following consumption of seafood harvested in the Pacific Ocean.
Efforts should be put on defining clear regulatory limits to ensure
food safety. For example, some azaspiracids should be monitored,
i.e. AZA-1, AZA-2 and AZA-3 while AZA-17 and AZA-19 are
out of scope (EFSA, 2008b). When looking at the metabolism of
AZA-17 and AZA-19 during cooking, i.e. the conversion of these
toxins into AZA-1, AZA-2 and AZA-3, one clearly sees a gap that
needs to be filled (McCarron et al., 2009). This issue has received
more attention and some countries have already made a step
ahead by cooking mussels prior to analysis (Jorgensen and Jensen,
2004).
Similarly, no clear rules have been established as to how to
sample seafood to protect the consumer in the best manner.
Ensuring the safety of one batch of shellfish based on analysing
the toxin content in a few individuals does not equal to a risk-free
approach.
KESIMPULAN While it looks like there is an increase in seafood poisoning
incidence, part of this could certainly be explained by better
monitoring programmes implemented in some countries.
Currently, promising in vitro alternatives to the in vivo mouse
bioassay have been developed. This not only is a great
improvement because of ethical considerations, but also provide
more specific insights in the mechanisms of action. Although
some in vitro cell-based assays are also already applied in some
countries, a clear interlaboratory validation study still has
to be conducted.

JUDUL Animal Forensics: Determination of Freshwater and Marine

Toxins in Complex Matrices by Liquid Chromatography–Tandem
Mass Spectrometry
 TUJUAN Mode HLB (domoic acid) were used for sample clean-up and
PENELITIAN concentration of the extracts.
METODE Anatoxin-a and Microcystins
PENELITIAN
HASIL PENELITIAN Water sample from a Northern California river tested positive for
MC-LA. The sample was re-analyzed (in triplicate for each point)
to determine if a quick sonication procedure can replace the long
freeze/thaw process. Temperature during the sonication was
measured using a thermocouple. Distilled water spiked with MC-
LA at 10 ppb was subjected to the same experiment to assure
stability of MC- LA during the sonication and freeze/thaw
process. Water samples from another Northern California river
were collected in the fall. Samples were positive for all
microcystins tested. Comparison of microcystin levels using three
different sample preparation techniques was conducted as
described above.
Table 1-Results: Concentration of MCs. Free”=analyzed “as is”,
no cell wall disruption (shake, vigorously, for 30 seconds, remove
an aliquot, filter, analyze). “Total”=analyzed after cell wall
disruption by: S=sonication (5 min), F/T=Freeze/thaw, 5 cycles,
filtered, analyzed. Anatoxin-a Poisonings in Dogs
• South Fork Eel River, Humboldt County, California: 3 dogs had
seizures within 5-30 minutes after swimming and died within 1
hour. Water and stomach contents were positive for anatoxin-a.
• Ontario, Canada: 3 of 11 dogs died unexpectedly within 1 hour
after swimming in a pond for 5 minutes during a supervised walk
around a farm. Water sample from the pond was positive for
anatoxin-a at 1 μg/g. Stomach contents were all positive for
anatoxin-a.
• Reno, Nevada: dog developed acute neurological signs and died
after ingesting scooped off “algae” in a bucket from a backyard
pond. Gastric contents, bucket contents, pond water, bile and
urine were positive for anatoxin-a.
KESIMPULAN -

PEMISAHAN KIMIA
NO JUDUL TUJUAN METODE HASIL KESIMPULA
N
1. New method for Iam to know metodologi Setiap MBTX Penggunaan
the analysis of New method for diterapkan; ditentukan metodologi
lipophilic marine the analysis of yaitu analit secara QuEChERS
biotoxins in fresh lipophilic diekstraksi individual, dikombinasika
and canned marine biotoxins dengan tetapi menurut n dengan
bivalves by liquid in fresh and asetonitril Peraturan UE penerapan
chromatography canned bivalves dan [2,3,5] untuk UHPLC-
coupled to high by liquid pembersiha setiap HRMS dalam
resolution mass chromatography n kelompok mode MS/MS
spectrometry: A coupled to high ekstraknya MBTX,
quick, easy, cheap, resolution mass konsentrasi telah terbukti
efficient, rugged, spectrometry: A total toksin baik
safe approach quick, easy, harus pendekatan
cheap, efficient, dinyatakan langsung dan
rugged, safe setara dengan sangat handal
approach menggunakan untuk analisis
Toxic MBTX
Faktor Setara lipofilik pada
(TEF) bivalvia.
1).Kuantifikasi Di satu sisi,
masing-masing telah
MBTX dibuktikan
individu bahwa
dilakukan ekstraksi
dengan dengan
menggunakan asetonitril
matriks dikombinasika
pengganti n dengan dSPE
sesuai standar sederhana
(SMMS), untuk dengan C18
mengatasi memberikan
efek penekanan tinggi
ion atau pemulihan
peningkatan absolut untuk
ion biasa dalam langkah
analisis MS. ekstraksi dan
Dengan pembersihan.
demikian, Perawatan
sampel kosong sederhana ini
kerang sangat efektif,
dibubuhi dan rangkaian
dengan analit di sampel yang
lima besar dapat
konsentrasi dilakukan
yang berbeda dianalisis
dan 200. L tanpa
larutan perawatan
eprinomektin khusus sistem
(0,5 mg UHPLC-
L-. HRMS.
1), digunakan Di sisi lain,
sebagai standar mode akuisisi
internal (IS), HRMS/MS,
ditambahkan; yang
setelah itu memenuhi
SMMS persyaratan
diserahkan ke undang-
seluruh undang UE,
prosedur adalah alat
analitis; yang ampuh
kurva kalibrasi untuk
untuk setiap menghindari
analit diperoleh matriks
secara linier gangguan.
analisis regresi Data massa
daerah puncak akurat
relatif terhadap diperoleh
daerah IS untuk setiap
terhadap analit, untuk
konsentrasi. baik ion
Kurva kalibrasi molekuler dan
dalam kisaran fragmen yang
25–350 g kg dipilih,
1 untuk memberikan
semua MBTX informasi yang
digunakan. sangat baik
Eprinomectin untuk
dipilih sebagai mengkonfirma
IS karena si identitas
itu tidak ada senyawa
dalam sampel, dan untuk
itu dapat menghindari
terionisasi di risiko hasil
kedua negatif positif palsu.
dan mode Kalibrasi
positif dan dengan SMMS
memiliki memberikan
berat molekul hasil
yang mirip kuantitatif
dengan yang andal,
analit. dan telah
Karena ditunjukkan
undang-undang bahwa kurva
mensyaratkan kalibrasi
analisis racun dibangun dari
dan esternya, kosong
langkah sampel kerang
hidrolisis dapat cocok
tambahan untuk
diperlukan menganalisis
untuk spesies kerang
mengubah ester yang berbeda.
toksin Eprinomectin
kelompok OA telah
dan untuk menunjukkan
mengukur perilaku yang
kandungan tepat untuk
total OA/DTX digunakan
kelompok, sebagai
seperti yang
dijelaskan di standar
atas. internal untuk
Harus disorot MBTX
bahwa standar lipofilik.
tidak tersedia Metode yang
untuk dikembangkan
semua MBTX , yang telah
yang disahkan. berhasil
Untuk divalidasi,
kuantifikasi cocok untuk
MBTX ini, analisis
seperti kuantitatif
PTX 1, 45-OH- konfirmasi
YTX dan 45- lipofilik
OH-hYTX, MBTX dalam
rekomendasi bivalvia segar
dari dan kalengan
Standard pada tingkat
Operational konsentrasi
Procedure yang diatur.
(SOP) kstrak Metode
kerang yang tersebut saat
mengandung ini digunakan
45-OH-YTX dalam LASPB
dan 45-OH- secara rutin
hYTX analisis, dan
disediakan telah masuk
oleh dalam ruang
Laboratorium lingkup
Referensi akreditasi
Eropa untuk mengikuti
Biotoksin Laut persyaratan
dan itu ISO/IEC17025
digunakan
untuk
menetapkan
waktu retensi
dan parameter
MS untuk
senyawa ini.
Senyawa
diidentifikasi
dengan massa
yang akurat
dari ion
molekuler
dengan akurasi
massa lebih
baik dari
5ppmdan
penyimpangan
waktu retensi
<1%. Untuk
mengkonfirmas
i temuan
positif,
dua ion produk
(bila
memungkinkan
) diperoleh dari
setiap
percobaan
produksi dalam
proses yang
sama. Untuk
menghitung
massa eksak
(m/z
dihitung),
massa elektron
telah
diperhitungkan
sebagai
0,00055 Da
. Selain itu,
rasio ion
didefinisikan
sebagai rasio
antara ion
molekuler dan
ion produk
digunakan
sebagai
kriteria
tambahan
untuk
konfirmasi
hasil positif.
Rasio ion
senyawa dalam
sampel harus
sesuai dengan
rasio ion kurva
kalibrasi
SMMS dengan
toleransi
maksimal
dari ±20%.
2. Determination of Iam to know Metode Biotoksin laut Metode LC-
lipophilic marine multitoksin lipofilik HRMS yang
toxins in mussels. terakumulasi sensitif untuk
Quantification dalam kerang kuantifikasi
and confirmation penyaring dan kelompok
criteria using high dapat utama racun
resolution mass berkembang lipofilik laut
spectrometry menjadi risiko telah
keamanan dikembangkan
pangan [1], [2], dan
[3]. Racun ini divalidasi. Met
diproduksi oleh ode ini
beragam dilakukan
mikroorganism dengan sangat
e sebagaimana baik untuk
dirinci dalam parameter
Paz et yang
al. [3]. Racun diselidiki. Rasi
laut lipofilik o ion sebagai
dapat kriteria
diklasifikasikan konfirmasi
menjadi dipelajari
beberapa secara
kelompok: mendalam. Di
asam okadaat amati bahwa
(OA) dan rasio ion
dinyphysistoxi fragmen dan
ns (DTXs), rasio ion
yessotoxins isotop dapat
(YTXs), digunakan
azaspiracids untuk
(AZAs), mengkonfirma
pectenotoxins si hasil positif,
(PTXs), cyclic tetapi untuk
immines dan masing-
brevetoxins masing
[3]. Untuk senyawa satu
penelitian ini, atau yang lain
beberapa racun bisa lebih
telah dipilih; 1 cocok. Penggu
toksin dari naan kriteria
setiap HRMS dapat
kelompok yang membantu
dijelaskan mencegah
dalam hasil yang
Peraturan salah.
853/2004/EC
[10]: asam
okadaat (OA),
yessotoxin
(YTX),
azaspiracid-1
(AZA1) dan
pectenotoxin-2
(PTX2); 1
toksin dari
kelompok yang
dapat diatur
dalam waktu
dekat seperti
yang muncul
dari pendapat
EFSA [4], [5],
[6], [7], [8], [9]:
13-desmethyl
spirolide C (
SPX1) dan
gymnodimine
(GYM);
Selanjutnya,
untuk semua
racun yang
dipilih bahan
referensi
bersertifikat
tersedia
[10]. Level
yang diizinkan
saat ini oleh
undang-undang
dalam kerang
adalah: untuk
jumlah OA,
DTX, dan PTX
160 μg kg −1 s
etara OA,
untuk jumlah
YTX
1000 μg kg −1
YTX setara dan
untuk jumlah
AZA
160 μg kg −1 s
etara AZA1
[11]. Untuk
golongan
cyclic immines
(spirolides dan
gymnodimines)
dan untuk
 golongan
brevetoxins
belum ada
batasan
legalnya. Namu
n, Otoritas
Keamanan
Pangan Eropa
(EFSA)
mengeluarkan
beberapa
pendapat untuk
setiap
kelompok
toksin, yang
merekomendasi
kan revisi batas
legal ini
(menurunkanny
a, kecuali
untuk YTXs)
[4], [5], [6], [7]
, [8], [9].

3. A low cost novel Bertujuan untuk Metode Experiment Initial studies

sensing system for mengetahui A Experimen with DA was on three
detection of low cost novel conducted in different
dangerous marine sensing system the laboratory. chemicals and
biotoxins in for detection of Small amount different
seafood dangerous of DA of 0.1 seafood
marine biotoxins mg was diluted products have
in seafood in 2.0 ml (2.0 shown that the
g) of water to fabricated
give 50 ppm of interdigital
DA. Table 3 sensors can
shows the respond very
particulars of well to each
the prepared samples and
mussel sample. can clearly
The experiment discriminate
was conducted between them.
at the Sensor 1 was
laboratory selected for
temperature of further
22.5 ◦C with analysis with
humidity of real mussels
48%. Mussel due to its
samples were better
injected with sensitivity.
DA from 0.5 g Analysis of
until 5.0 g. results with
Measurement real mussels
data of air, have shown
voltage output that Sensor 1
and digital can detect the
value of sensor presence of
output were proline on the
collected. surface as well
Result in Fig. as injected
24 shows that proline to each
Sensor 1 sample. It can
responded to be said that
the presence of Sensor 1 was
DA after 1.0 g able to detect
of DA injected the presence
into the mussel of different
with minimum amount of
weight of proline
0.0795 g. The injected into
result shows the mussel
that Sensor 1 samples.
was able to Experiment
detect with DA has
approximately shown that
12.6 g/g of DA novel
in mussel meat. interdigital
The regulatory sensor can be
guideline for used to detect
DA is 20 g/g. the small
The sensor has amount of
the potential to domoic acid in
detect an mussels.
amount even Results from
lower than 10 the
g/g of DA in experiments
mussel meat. have shown
The experiment that novel
was repeated interdigital
by injecting sensing
0.01 ml of system has the
water and 0.01 potential to be
ml of DA of one of the
different options to
concentrations assess the
to the samples quality of
having the seafood
same thickness products for in
of 2.4 mm. The situ
amount of monitoring.
water and DA The sensing
injected were system can
increased until detect the
0.05 ml. The presence of
purpose of this DA in
experiment is shellfish
to observe the meats. The
effect of sensor outcomes
sensitivity with from the
water and also experiments
DA of different provide
concentrations. chances of
Results from opportunity
the experiment for further
are shown in research in
Fig. 25, where developing a
the sensor was low cost
able to clearly miniature type
discriminate of sensor with
the water and a reliable
also the DA at sensing
different system for
concentrations. commercial
The experiment use. Further
was repeated experiments
by injecting 1.0 are also
g of DA into conducted to
several mussel study the
samples of effect of other
different parameters on
thickness. The the sensor
experiment was sensitivity.
conducted to Further
find the research can
threshold level also be
of sensor conducted to
sensitivity with access the
respect to quality of
sample other food for
thickness. The example, to
sensitivity is a detect
function of the salmonella
thickness of poisoning in
sample, meat product.
therefore it is There is a
very important strong
to determine possibility of
the threshold using novel
sensitivity level planar
of samples at interdigital
different sensor to
 thickness so detect
that it will be dangerous
easy to use the biological
sensor without agents
much technical (bacteria,
knowledge. viruses or
Data was toxins) on
collected for food, which is
normal samples used for
and also bioterrorism.
samples
injected with
DA of
respective
samples
thickness. Fig.
26 shows that
the threshold
sensitivity level
can be
determined
with respect to
different
thickness of
mussel
samples. There
are three levels
of sensitivity
threshold that
were observed
from the
experiment.
4. Liquid Bertujuan Metode Contamination In this study,
chromatography untuk LC-MS of shellfish SPE and LC-
quadrupole linear mengetahui with marine QqLIT-MS
ion trap mass Liquid biotoxins has were used in
spectrometry for chromatograph become a combination
multiclass y quadrupole major concern to
screening and linear ion trap of food safety simultaneousl
identification of mass authorities, as y and rapidly
lipophilic marine spectrometry these toxins analyze
biotoxins in for multiclass pose a threat to bivalve
bivalve mollusks screening and public health samples for
identification of and the global the presence
lipophilic shellfish of 17
marine industry. Six lipophilic
biotoxins in groups of marine
bivalve compounds biotoxins. The
mollusks [okadaic acid method, which
(OA), involves an
yessotoxins SPE extraction
(YTX), step, is highly
azaspiracids sensitive (with
(AZA), LODs of
pectenotoxins 0.12–
(PTX), cyclic 13.6 μg/kg),
imines (CI), repeatable,
and and selective
brevetoxins for wild
(PbTx)] are samples of
lipophilic shellfish
marine (oyster,
biotoxins that scallop, and
are similar in mussel).
structure to Different LC-
lipid-soluble MS methods
cyclic for
polyether quantifying
compounds individual
(Joint toxins in
FAO/IOC/WH complex
O, 2004). The mixtures
presence of require
these biotoxins specific
has been analytical
frequently standards
reported in because of
Europe, variations in
America,
China, Canada,
New Zealand,
Japan, and
Chile [1], [2],
[3], [4], [5],
[6]. At least ten
shellfish
poisoning
events have
been identified
along the
Chinese coast
using the
traditional
mouse bioassay
(MBA) in the
last decades.
However,
MBA is widely
recognized as
inefficient,
imprecise,
lacking in
specificity, and
ethically
objectionable
[7]. Thus,
better
identification
methods are
badly needed.
Development
of liquid
chromatograph
y tandem mass
spectrometry
(LC-MS/MS)
as a detection
method is
promising, as
AZA1, YTX,
OA, PTX, and
the CI
gymnodimine
(GYM) have
been detected
with this
technique as
part of regular
monitoring [8],
[9] and during
diarrhetic
shellfish
poisoning
episodes in
China [10]. In
2010, European
legislation set a
three-year
transition
period to
replace the
MBA with LC-
MS/MS for use
in monitoring
marine
biotoxins [11],
[12], [13], [14].
Official rules
for
implementation
 of LC-MS/MS
to identify
biotoxins in
seafood
research
include use of
specific
chromatographi
c conditions
[15], [16] and
matrix effects
[17], [18], [19],
inter-laboratory
validation
studies [13],
solid phase
extraction
(SPE)
purification
[14], [20], and
studies of the
stability of
toxins [21],
[22].

5. THE Iam to know Metode The in-house The method

IMPLEMENTATI THE LC-MS validation based on LC-
ON OF LIQUID IMPLEMENTA study relied on MS/MS for
CHROMATOGR TION OF the concepts the
APHY TANDEM LIQUID described in determination
MASS CHROMATOG Taverniers et of lipophilic
SPECTROMETR RAPHY al. [27], the toxins in
Y FOR THE TANDEM guidelines seafood has
OFFICIAL MASS proposed by been accepted
CONTROL OF SPECTROMET the Regulation by the
LIPOPHILIC RY FOR THE (EC) 657/2002 European
TOXINS IN OFFICIAL on performance Union as a
SEAFOOD: CONTROL OF criteria for reliable
SINGLE- LIPOPHILIC analytical technique to
LABORATORY TOXINS IN methods [28], protect public
VALIDATION SEAFOOD: and the health and
UNDER FOUR SINGLE- methodology reduce the use
CHROMATOGR LABORATOR applied by de of animals for
APHIC Y la Iglesia et al. routine
CONDITIONS VALIDATION [29]. The analysis. The
UNDER FOUR accuracy of the EURLMB
CHROMATOG methods was SOP [11]
RAPHIC assessed by the establishes a
CONDITIONS intermediate solid
precision and framework for
the trueness. the
The spikings implementatio
were done on n of the LC-
blank MS/MS
homogenized method but it
tissue instead is not explicit
of on extracts enough
in MeOH to concerning the
make the chromatograp
validation hic conditions
process as and the matrix
comprehensive effect
as possible. correction
The strategy that
intermediate should be
precision was used. The
expressed as current study
the relative is the first
standard work aimed to
deviation (RSD compare the
in %). It was most common
calculated for chromatograp
each matrix hic
(mussels, alternatives
pacific oysters, for the
clams and sea determination
urchins) at of lipophilic
three different toxins in
concentration seafood by
levels of OA, LC-MS/MS in
PTX2, SPX1, terms of
GYMA and functionality,
AZA1 (80 quality
μg/kg, 160 criteria,
μg/kg and 240 validation
μg/kg) and two parameters
concentration and
levels of YTX quantification
(250 μg/kg and strategies
500 μg/kg) under the
spiked in blank same
homogenized experimental
tissues and settings
quantified (extraction
using external and hydrolysis
standard procedures,
calibration chromatograp
curves. Four hic column,
replicates MS instrument
spread over conditions,
four standards and
consecutive reagents, and
days were analyst). We
analyzed by chose the
single injection alkaline
using daily conditions,
fresh mobile EXS
phase. The calibration as
RSD was quantification
transformed to strategy, and
HorRat value recovery
as the ratio correction for
between the OA with
experimental CRM-DSP
RSD and the Mus b to be
predicted RSD implemented
according to as the routine
the Horwitz method.
equation [30] Alkaline
(Equation 2), conditions
which is provide higher
dependent on sample
the throughput,
concentration lower LOQs,
(C) spiked for and the best
the overall
intermediate performance
precision in terms of
assessment. sensitivity and
HorRat = accuracy in
RSD(%) the validation
experimental / study. The
2(1-0.5logC) EXS strategy
Equation 2 combined with
OA recovery
correction by
CRM-DSP
Mus b
demanded less
time and
standard
investment
and provided
satisfactory
results. The
analysis of
YTX was
challenging
and it is still
being
improved in
our laboratory
by increasing
the number of
injections,
participating
in
collaborative
studies and
preparing
internal
reference
standards to
correct YTXs
recoveries.
When
selecting the
best
chromatograp
hic conditions,
factors such as
the
instrumentatio
n available
(regarding
polarity
switching,
limits of
detection, and
sensitivity),
the number of
samples
needed to
analyze, the
toxin profile
and the sample
matrices
should be
considered.
The matrix
effects should
be examined
carefully,
especially
when
including a
new toxin in
the method or
analyzing a
 new matrix. A
proper
selection
process may
be time and
resources
demanding,
but we hope
that this
comparative
study may
serve as
starting point
to other
laboratories
implementing
their own
methods for
lipophilic
toxins
determination
in seafood by
LC-MS/MS
6. Discovery of new Iam to know Metode An aliquot of This study
analogs of the Discovery of LC-MS 0.2 M NaIO4 demonstrated
marine biotoxin new analogs of solution the existence
azaspiracid in blue the marine (50mL) was of five new
mussels (Mytilus biotoxin added to groups of
edulis) by ultra- azaspiracid in methanolic AZAs, viz.:
performance liquid blue mussels shellfish dihydroxy-,
chromatography/ta (Mytilus edulis) extract (100 carboxy-,
ndem mass by ultra- mL) in an carboxy-
spectrometry performance insert vial and hydroxy- and
liquid mixed with a dehydro-
chromatography vortex mixer AZAs, as well
/tandem mass for 20 s. The as methyl
spectrometry extract was left esters of the
at room toxins.
temperature for Product ion
24 h and then spectra were
analysed by sufficient to
LC/MS/ MS. elucidate the
Ultra- structures of
performance the dihydroxy
liquid analogs, but
chromatograph were
y (UPLC) was insufficient to
carried out on unequivocally
an Acquity determine the
UPLC system structures of
(Waters, UK). the carboxy-
A model 1200 and
HPLC system carboxyhydro
(Agilent, Palo xy-AZAs. The
Alto, CA, potential
USA) was also number of
used for some AZAs
experiments. increases to 32
Mobile phase due to these
A was 100%. findings.
Electrospray Further efforts
ionisation are required to
tandem mass isolate and
spectrometry purify these
(ESI-MS/ MS) compounds to
data was enable the
acquired on a acquisition of
QTOF Ultima NMR spectra
(Micromass, and full
Manchester, structural
UK) in positive elucidation.
ion mode. The Concentration
Z-Spray ESI levels of the
source of the novel analogs
instrument was were found to
operated at 2.8 be less than
kV capillary 5% of total
voltage, 50 kV AZAeq and
cone voltage thus are
and 3508C unlikely to
desolvation contribute
temperature. significantly
The cone gas to overall
was set to 50 L toxicity.
h1 and
desolvation gas
to 550 L h1 .
Argon was
used as
collision gas at
a voltage of 50
eV. Scan times
were 0.2 s with
a delay of 0.1 s.
High- and low-
mass resolution
varied between
5.0 and 15.0
depending on
 the
experiments.
7. Development of a Bertujuan untuk Metode Ionspray LC– Azaspiracid is
method for the mengetahui LC-MS- MS is the most the main toxin
identification of Development of MS powerful responsible for
azaspiracid in a method for the technique and a number of
shellfish by liquid identification of FIA–MS–MS recent human
chromatography– azaspiracid in were carried intoxications
tandem mass shellfish by out on toxin in Europe
spectrometry liquid solu- for the resulting from
chromatography identification shellfish
–tandem mass of known and consumption.
spectrometry new toxins The first micro
tions liquid
containing 1 chromatograp
mg/ml of hy–tandem
azaspiracid. mass
differing both spectrometry
in chemical (micro-LC–
structure and MS–MS)
biological method was
Chromatograph developed for
ic separations the
were determination
performed on a of this novel
action [7–28]. shellfish
The very mild poisoning
ionization in toxin in
the mussels. The
microcolumn analyte was
packed with extracted from
Vydac whole mussel
218TP51 meat with
(Sepa- ionspray acetone and
LC technique chromatograp
usually hed on a C
guarantees the reversed-phase
selecration column (1.0
Group, mm I.D.) by
Hesperia, CA, isocratic
USA) (250 elution at 30
mm31 mm, 18 ml/min
tive formation with
of molecule- acetonitrile–
related ions of water (85:15,
the toxin 5 v/v),
mm) at room containing
temperature, 0.03%
under isocratic trifluoroacetic
con- in addition acid. The
to the toxin was
corresponding ionised in an
retention time, ionspray 1
ditions, with a interface
mobile phase operating in
of acetonitrile– the positive
water ion mode,
providing where only the
sensitive and intact
specific protonated
detection of the molecule,
(85:15, v/v), [M1H] , was
containing generated at
0.03% m/z 842. 1
trifluoroacetic This served as
acid analyte for precursor ion
confirmatory for collision-
purposes. induced
Moreover, the dissociation
(TFA) and a and three
flow-rate of 30 product ions,
ml/min. LC– [M1H2nH O]
MS and LC– with n51–3,
MS–MS were 2
approach is identified for
frequently the the
Mass spectral unambiguous
analysis was toxin
performed on a confirmation
PE- only by selected
method for reaction
obtaining monitoring
structural LC–MS–MS
information analysis. A
due SCIEX detection limit
API III plus of 20 pg,
triple- based on a 3:1
quadrupole signal-to-noise
(PE-Sciex, to ratio, was
the small achieved for
amounts of the analyte.
marine natural This LC–MS–
products MS method
Thornhill, was
Canada). The successfully
mass applied to
spectrometer determine
was available azaspiracid in
for analysis. toxic
equipped with cultivated
 an atmospheric shellfish from
pressure two regions of
ionization The Ireland.
aim of this 2000 Elsevier
research was to Science B.V.
investigate for All rights
the (API) reserved.
source and an
ionspray
interface set at
a first time the
suitability of
the ionspray
LC–MS and
voltage of 5500
V. Ultra-high-
pure (UHP)
nitrogen LC–
MS–MS in
order to
unambiguously
detect azaswas
used as the
curtain gas and
nebulizer gas in
the piracid in
shellfish.
8. Scientific Opinion Iam to know Summary PlTX-group The EFSA
on marine Scientific Method toxins are Panel on
biotoxins in Opinion on complex Contaminants
shellfish – marine biotoxins polyhydroxylat in the Food
Palytoxin group. in shellfish – ed compounds Chain
EFSA Panel on Palytoxin group. with both (CONTAM
Contaminants in EFSA Panel on lipophilic and Panel)
the Food Chain Contaminants in hydrophilic assessed the
(CONTAM) the Food Chain areas. The risks to human
(CONTAM) basic molecule health related
consists of a to the presence
long, partially of palytoxin
unsaturated (PlTX)-group
aliphatic toxins in
backbone shellfish.
containing PlTX-group
cyclic ethers, toxins have
64 chiral mainly been
centers, 40-42 detected in
hydroxyl and 2 soft corals of
amide groups the genus
(Figure 1). The Palythoa and
primary amino- in algae of the
group at the C- genus
115 end of the Ostreopsis.
molecule Blooms of
accounts for Ostreopsis
the basicity of spp. have
PlTX-group recently been
toxins reported in
(Katikou, some
2007). The European
molecular countries.
formula and Occurrence of
molecular Ostreopsis
weight of PlTX spp. may
analogues result in
differ contamination
depending on of shellfish
the Palythoa intended for
species from human
which they are consumption.
obtained Currently
(Moore and there are no
Bartolini, regulations on
1981). The PlTX-group
chemical toxins in
formula of shellfish,
PlTX from P. either in the
toxica is European
C129H233N3 Union (EU),
O54, 115 of the or in other
129 carbons regions of the
being in world. The
continuous toxicological
chain. Its database of
molecular PlTX-group
weight is 2,677 toxins is
Da and it was limited,
found to exist comprising
as a dimer in only acute
aqueous toxicity
solution studies for
(Uemura, PlTX and
2006). Due to ostreocin-D
their two via several
chromophores, routes of
PlTX-group administration
toxins exhibit in various
an ultraviolet animal
(UV) species. The
absorption oral route was
spectrum with least sensitive.
a λmax at 233 Acute toxicity
and 263 nm. and deaths
PlTX-group have been
toxins are reported from
insoluble in human
nonpolar outbreaks, but
solvents, there are no
sparingly reliable
soluble in quantitative
methanol and data on acute
ethanol and toxicity in
soluble in humans. In
pyridine, view of the
dimethyl acute toxicity
sulfoxide and and the lack of
water. They are chronic
heat resistant. toxicity data
In addition to for PlTX-
PlTX, other group toxins,
toxins of the the CONTAM
same group are Panel was
produced by only able to
several derive an oral
Ostreopsis acute
species. For reference dose
example O. (ARfD) of 0.2
siamensis µg/kg b.w. for
produces the the sum of
major toxic PlTX and its
constituent analogue
ostreocin-D ostreocin-D.
which is a In order for a
chemical 60 kg adult to
analogue of avoid
PlTX, also exceeding the
referred to as ARfD a 400 g
42-hydroxy- portion of
3,26-dimethyl- shellfish meat
19,44- should not
dideoxypalytox contain more
in. For these than 12 µg of
two the sum of
compounds PlTX and
preparative ostreocin-D,
isolation and corresponding
characterisation to 30 µg/kg
by nuclear shellfish meat.
magnetic The mouse
resonance bioassay
(NMR) has (MBA) has
 allowed been used to
elucidation of detect PlTX-
the planar group toxins,
structure. Five but cell based
further assays have
analogues have been
been found to developed as
co-occur with alternative.
PlTX in P. However,
tuberculosa, e.g positive
homopalytoxin, results require
bishomopalyto confirmation
xin, by chemical
neopalytoxin, methods. High
deopalytoxin performance
and 42- liquid
hydroxypalytox chromatograp
in, but have not hy-
been reported fluorescence
to be produced detection
by Ostreopsis (HPLC-FLD)
spp. (Uemura and liquid
et al., 1985; chromatograp
Ciminiello et hy-tandem
al. 2009). mass
O.ovata spectrometry
produces (LC-MS/MS)
ovatoxin-A, for methods can
which the be valuable
molecular mass tools for the
(2647,5 Da) determination,
and structural but method
evidence from optimisation
mass and validation
spectrometry as well as the
show that it is development
an analogue of of certified
PlTX. reference
Ovatoxin A has materials and
two oxygen standards are
atoms less than necessary
PlTX but it is
not known
from which
part of the
molecule the
oxygen atoms
are missing
9. Multiplex biotoxin Bertujuan untuk Metode Multiplexing A multiplex
surface plasmon menyimpulkan Biosensor was achieved assay has been
resonance method Multiplex using a developed and
for marine biotoxin surface prototype SPR validated for
biotoxins in algal plasmon biosensor in a the semi-
and seawater resonance 4×4 layout, quantitative,
samples method for with four flow simultaneous
marine biotoxins cells each screening of
in algal and containing four three types of
seawater spots, giving a key marine
samples total of 16 biotoxins in
analytes for seawater
simultaneous samples.
detection. Results for
Saxitoxin, PSP, okadaic
okadaic acid acid and
and domoic domoic acid
acid were toxins can be
successfully detected in as
immobilised little as 13 min
onto all four from one
spots on flow seawater
cell 1, 2 and 3, sample (after
respectively, as extraction) in
shown in Fig. real time.
1. In this Method
research, each detection
biotoxin was limits based
immobilised on on IC20
all four spots of values for
one flow cell PSP, okadaic
allowing for acid and
analysis of domoic acid
samples in toxins were
quadruplicate. 0.82, 0.36 and
Spotting of 1.66 ng/ml,
biotoxins this respectively,
way allowed for the
for the prototype
prolonged shelf multiplex SPR
life of the biosensor.
biosensor chip, Using these
if one of the detection
spots became limits, 47, 59
unstable, there and 61 % of
would still be seawater
results from the samples tested
other three positive for
spots PSP, okadaic
remaining. acid and
domoic acid
toxins,
respectively,
with toxic
seawater
samples found
mainly from
the Rias of
Pontevedra,
Arosa, Muros,
Ares-
Betanzos, and
estuary of
Bayona, Spain
and the
Killary, Cork
and Bell
Harbours of
Ireland. The
simplicity of
the assay
means it could
be used as a
first action
screening
method in a
monitoring
laboratory for
the presence
of these
biotoxins in
seawater
samples.
Many
European
countries test
and identify
phytoplankton
in seawater
samples in
shellfish
harvest areas
with many
setting trigger
levels for
certain toxic
phytoplankton
species. In
Northern
Ireland, these
levels are set
at >0 cells/L
for
Alexandrium
species, >100
cells/L for
Dinophysis/
Prorocentrum
species and
>150,000
cells/L for
Pseudonitzschi
a species.
However, it is
often
impossible to
differentiate
between
certain species
of toxic algae;
and if toxic
species are
present at
trigger levels,
this would
initiate further
testing of
shellfish
whether or not
biotoxins are
present. A
viable
alternative
would be to
monitor
seawater
samples for
the presence
of biotoxins.
An early
warning
detection tool
would be
extremely
beneficial to
the
aquaculture
and fisheries
industry to
allow
economic
decisions to be
 made quickly
in relation to
the harvesting
process for
shellfish.
Although this
is a
laboratory-
based method,
it still has
many
advantages
and benefits
over the
current
detection
systems in its
ability for
multiple toxin
group
analysis. The
next step in
this research
would be to
develop a
multiplex
portable assay
for the
submersible
vehicle.
Without
comprehensiv
e
phytoplankton
and shellfish
monitoring
programmes,
there is
currently no
way to ensure
that shellfish
are safe for
human
consumption
10. Solid-Phase Bertujuan untuk Metode The selectivity
A highly
Extraction-Based menyimpulkan Experiment and sensitivity
Ultra-Sensitive Solid-Phase al of LC–MS-MS sensitive
Detection of Four Extraction- analysis not HPLC–MS-
Lipophilic Marine Based Ultra- only depends
Biotoxins in Sensitive on the MS method
Bivalves by High- Detection of ionization
with an SPE
Performance Four Lipophilic technique and
Liquid Marine mass pretreatment
Chromatography– Biotoxins in spectrometer for the
Tandem Mass Bivalves by but also on the
Spectrometry High- conditions of simultaneous
Performance LC technique. determination
Liquid The
Chromatography composition of of four
–Tandem Mass the solvent to lipophilic
Spectrometry dissolve the
sample marine toxins
generally including
affects the peak
shape and peak GYM, SPX1,
intensity of AZA2 and
analytes in LC–
MS-MS. In PTX2 was
previous presented.
published
papers, the During the
researchers experiment,
prepared the
stock solution solvent effect
of marine was observed.
toxins in
absolute By dissolving
methanol the standard
(3, 4, 14). But
multitoxins in
in our
experiment, we 1 : 1 water :
observed that
methanol
GYM exhibited
two peaks in (v/v), solvent
the ion
effect was
extracted
chromatogram avoided and
when prepared sharp,
in absolute
methanol as symmetrical
shown in peaks of GYM
Figure 2, a.
The front peak and SPX1
was broad and were obtained.
distorted,
which was not Applying a
ascribed to an concentration
isomer. It was
caused by the factor of 7.5
solvent effect. with the SPE
The solubility
procedure, the
of absolute
methanol is LOQs for the
much stronger analyzed
than the eluent
composed of toxins were
acetonitrile and determined to
water, which
makes the be 0.037–0.28
analyte peak µg kg−1. The
abnormal.
When the LOQs were
standard much lower
solution of
multitoxins than other
was prepared in published
1 : 1 mobile
phase A : articles
mobile phase (14, 15),
B, a split peak
was observed which are an
for GYM important
(Figure 2, b).
Compared with aspect in view
Figure 2, a, the of a possible
peak shape
improved and lowering of
the front peak the regulatory
disappeared. It
limits. Since
may be due to
the matrix effects
composition
in post-SPE
consistency
between extract were
solvent and
more
mobile phase.
But the organic significant
additives in the than crude
solvent and the
real-time extract
changing of the without a
mobile phase
probably concentration
resulted in the step, matrix
peak splitting.
The above effects in post-
phenomenon SPE extract
seriously
affects the and crude
identification extract were
and
seriously
quantification
of the analytes. investigated. It
It is is shown that
indispensable
to find a matrix effects
suitable solvent do not expand
to eliminate the
solvent effect proportionatel
so that good y as the
peak shape can
be obtained. In concentration
light of the factor. A
arguments
presented concentration
above, an factor >2 is
attempt to
dissolve the helpful to
standard improve
multitoxins in
1 : 1 water : method
methanol (v/v) sensitivity.
was made. An
excellent sharp The present
and method was
symmetrical successfully
peak were applied to
displayed analyze the
(Figure 2, c). target toxins in
The moderate Chinese
solubility and shellfish
neutral harvested
environment from the East
provided by 1 : China Sea. For
1 water : a reliable
methanol quantitative
guarantee the analysis, a
good spiked blank
performance of extract was
the peak. processed in
the same way
as the
unknown
samples and
used for
correction of
matrix effects.
Our study
reports on the
first
occurrence of
AZA2 and
SPX1 in
Chinese
shellfish in
favor of the
high method
sensitivity.
The maximum
levels of
toxins found
in Chinese
shellfish were
lower than the
recommended
safety limit of
AZAs and
PTXs (GYM
and SPXs not
included).
Hence, the
risk of acute
effects
resulting from
human
exposure to
these toxins
due to the
consumption
of shellfish
from Chinese
coast can be
regarded as
low. Although
GYM and
SPX1 were
not currently
included in the
EU regulation,
the data
obtained can
provide a
primary basis
for
establishing
regulatory
control in
China.
Judul Jurnal atau A single run UPLC-MS/MS method for detection of all EU-
Artikel regulated marine toxins.
Tujuan Penelitian Validation studies demonstrated acceptable method
performance characteristics for linearity, and repeatability
between-batch and within-batch. The study demonstrated that
the UPLC-MS/MS method provides an excellent tool to
determinate hydrophilic and lipophilic toxins and therefore it
could be appropriate for routine testing and interlaboratory
validation
Metode Penelitian PSTs and TTX extraction method > The extraction protocol of
Turner et al. (2015) was followed [26]. The efficiency of the
method was studied by analysing the extracts discarded in each
stage of the protocol.
Lipophilic toxins extraction method > Samples were extracted
following the procedure described in the EU-Harmonised
Standard Operating Procedure for determination of Lipophilic
marine biotoxins in molluscs by LC-MS/MS published by the
EU-RL-MB (July 2011).
DA extraction method > Samples were extracted by procedure
described in the EUHarmonised Standard Operating Procedure
for determination of domoic acid in molluscs by UPLC-MS/MS
(February 2010).
LC-MS analysis > Chromatographic separation was carried out
using a 1290 Infinity ultra-high-performance liquid
chromatography system coupled to an Agilent G6460C Triple
Quadrupole mass spectrometer equipped with an Agilent Jet
Stream ESI source (Agilent Technologies, Waldbronn,
Germany).
Detection limits and calibration curve linearity > Limits of
detection (LODs) and limits of quantification (LOQs) were
calculated following the International Union of Pure and
Applied Chemistry (IUPAC) method.
Repeatability > The within-batch repeatability was measured for
three consecutive batches of mixed standards (nine levels) and
the between-batch repeatability was measured in three separate
days, in triplicate.
Matrix effects > In order to determine the interferences due to
matrix, mussel tissues were extracted following extraction
procedures described for each group of toxins. Toxin standards,
nine concentration levels, were diluted in shellfish extracts and
then analysed by LC methods.
Hasil Penelitian The first aim of this work was the development of a new method
to detect all hydrophilic marine toxins with the same LC
(column and mobile phase) and mass spectrometry conditions.
By using an amide column (normal-phase) method 1 was
developed. The total run time was 15 min for this method. The
mobile phase was a binary gradient of 100% water (A) and 98%
MeCN (B) with different ionization additives at constant flux.
The initial gradient of 11 min, starting by 95% of organic phase
 until 5%, was followed by 1 min of 5% of organic phase and
then initial conditions were recovered in 1 min and maintained
for 2 min more. In these conditions, the three groups of
hydrophilic toxins, DA, TTXs and PSTs, can be separated and
identified. As Fig. 1A shows, DA was the first toxin eluted, at
4.2 min, as it is the least polar compound the first being eluted.
Then TTXs and PSTs were eluted. In the case of TTXs, both
4,9-anhTTX and TTX were eluted with good separation. In the
case PSTs, fourteen compounds can be separated and
individually identified, including isomers such as GTX1 and
GTX4, GTX2 and GTX3, dcGTX2 and dcGTX3, and C1 and
C2. The method permits the quantification of hydrophilic toxins
with LOQs between 1.3 nmol/L and 22.9 nmol/L for STXs
group, 4.9 nmol/L for TTXs group and 20 nmol/L for DA and
LODs < 6.8 nmol/L (Table 1). The calibration curves obtained
were linear over the points of calibration range with correlations
coefficients > 0.99 for each toxin. In addition, repeatability
within-batch and between-batch generated from mixed
standards (nine levels) was checked. The mean between-batch
repeatability data were used to calculate HorRat values for each
toxin (Table 1) [38]. All HorRat values were < 0.5, evidencing
acceptable repeatability for this method. Next, method 1 was
tested with spiked mussels. Mussel matrices were extracted
following validated extraction methods for each group of toxins.
That is, for STXs and TTXs groups the AOAC 2005.06
extraction procedure was used including a clean-up step with a
SPE ENVI-Carb cartridge [26,31]. DA was extracted following
the EU-RL-MB procedure for UPLC-MS/MS methods [34]. The
matrix effect was evaluated by the slope of matrix calibration
curves obtained from different toxin concentrations diluted with
non-toxin shellfish extracts and diluted with solvents. As Table
2 shows, different matrix effects were observed depending on
the toxin analysed, with the exception of C1&C2, for which the
matrix effect was 100%, that is no matrix effect. Matrix effect
for dcNEO and NEO were higher than 100% showing a positive
effect due to enhancement of the ionization. While matrix effect
for STX, dcSTX, dcGTX2&3, GTX1-6, TTX, 4,9-anhTTX and
DA were lower than 100%, indicating a negative effect due to
ionic suppression. Repeatability values with spiked extracts
were under acceptable results showing no variability in
measures within-batch.
Kesimpulan A new LC-MS/MS method was developed for the detection and
quantification of lipophilic and hydrophilic toxins with the same
LC and MS conditions. This method allows to identify and
quantify hydrophilic toxins PSPs, DA and TTX and analogues
and regulated lipophilic toxins. LODs and LOQs for each toxin
were significantly improved in comparison to other official and
validated methods. The repeatability was shown to be
acceptable with acceptable HorRat values. The method was
specific and linear over the full calibration range and provides
 excellent determination of sample toxicity and toxins
concentrations for a wide range of toxins. Therefore, the method
shows a good performance permitting quantitative analysis of
marine biotoxins.

Judul Jurnal atau THE IMPLEMENTATION OF LIQUID

Artikel CHROMATOGRAPHY TANDEM MASS SPECTROMETRY
FOR THE OFFICIAL CONTROL OF LIPOPHILIC TOXINS
IN SEAFOOD: SINGLE-LABORATORY VALIDATION
UNDER FOUR CHROMATOGRAPHIC CONDITIONS.
Tujuan Penelitian We performed a comprehensive study to assess the fit for
purpose of four chromatographic conditions for the
determination of six groups of marine lipophilic toxins (okadaic
acid and dinophysistoxins, pectenotoxins, azaspiracids,
yessotoxins, gymnodimine and spirolides) by LC-MS/MS to
select the most suitable conditions as stated by the European
Union Reference Laboratory for Marine Biotoxins (EURLMB).
This comparative study can help other laboratories to choose the
best conditions for the implementation of LC-MS/MS according
to their own necessities.
Metode Penelitian Preparation of extracts > A triple-step extraction with MeOH
was performed on whole tissues according to the procedure
proposed by Gerssen et al. [13], but samples were homogenized
with a hand blender instead of with an Ultra Turrax
homogenizer.
Alkaline hydrolysis > The alkaline hydrolysis of the samples
was performed according to the EURLMB SOP [11] based on
the protocol initially developed by Mountfort et al. [26].
Chromatographic separation > Toxins were separated on a
Waters X-BridgeTM C8 (guard column 2.1 x 10 mm, 3.5 μm
particle size, column 2.1 x 50 mm, 3.5 μm particle size; Waters,
Milford, MA, USA) in an Agilent 1200 LC system (Agilent
Technologies, Santa Clara, CA) consisting of a binary pump
(G1312B), four channel degasser (G1379B), thermostated low
carry-over autosampler (G1367C + G1330B), and column oven
(G1316B).
Mass spectrometry > We used a triple quadrupole 3200
QTRAP® mass spectrometer (MS) equipped with a TurboV
electrospray ion source (Applied Biosystems, Foster City, CA).
The MS was operated in the multiple reaction monitoring
(MRM) mode, selecting two product ions per toxin to allow
quantification (the most intense transition) and confirmation
(two confirmation ions for GYMA).
Quality requirements posed by the EURLMB > We checked in
every batch the quality control criteria stated by the EURLMB
 SOP [11] regarding resolution, limits of quantification (LOQs)
and linearity.
Validation parameters > The in-house validation study relied on
the concepts described in Taverniers et al. [27], the guidelines
proposed by the Regulation (EC) 657/2002 on performance
criteria for analytical methods [28], and the methodology
applied by de la Iglesia et al.
Calibration strategies and matrix effects assessment > The
external standard calibration curves were prepared in MeOH
(LC-MS grade) from an initial multi-toxin stock solution of 400
ng/mL of OA, PTX2, SPX1, GYMA and AZA1, and 625 ng/mL
of YTX. The calibration curves had six levels in the range of 5
to 60 ng/mL of OA, PTX2, SPX1, GYMA and AZA1 and 8 to
94 ng/mL of YTX.
Statistical analysis > Statistical calculations were performed
using SPSS 17.0. The significance tests used to evaluate the
influence of the species in the matrix effect was a One-Way
ANOVA (one test per toxin), supported by a Levene Test of
Homogeneity of Variances, and a Post Hoc Tukey HSD Test
when the ANOVA test showed significant differences in the
mean between groups (species). Alpha was set at 0.05 (95%
confidence) for all tests and experiments.
Hasil Penelitian Implementation of LC-MS/MS methods according to the
EURLB-SOP quality requirements > We expected tR and
elution order of the toxins to change under different
chromatographic conditions [12] (Figure 1), since the charge
state of the toxins is influenced by the pH of the mobile phase.
Under pH 2, YTX coeluted with PTX2, and the “–ESI toxins”
(OA and YTX) eluted in the same time window as the “+ESI
toxins” (GYMA, SPX1, PTX2 and AZA1). The shift from acidic
to almost neutral conditions reduced OA tR and slightly alkaline
conditions increased the tR of the cyclic imines. When pH was
modified from pH 7.9 to pH 11, tR of OA, YTX and AZA1
became shorter, thus the “–ESI toxins” eluted at the beginning
of the chromatogram and “+ESI toxins” eluted afterwards. This
change in the elution order enabled detection windows to be set
with different polarity in our 3200 QTRAP® and analyze all
toxins in the same run.
Methods performance > The alkaline conditions had the best
overall performance in terms of precision (Table 6). For AZA1,
alkaline conditions provided HorRat values below one in all
matrices and concentrations, but other of conditions were also
precise enough in most cases at medium and high
concentrations. The precision in the analysis of GYMA spiked
in mussels was only satisfactory under alkaline conditions, but
acidic conditions had better precision in sea urchins. The HorRat
values for OA (both crude and hydrolyzed) were in general very
high (up to 3.4 in mussels spiked at 0.5 times the MPL analyzed
under acidic conditions after hydrolysis). The precision for
crude OA in mussels under alkaline conditions was good, but in
 sea urchins the acidic conditions would provide better HorRat
values at medium and high concentrations. For PTX2 and SPX1,
alkaline conditions generally gave better results in terms of
precision.
Calibration strategies and matrix effects assessment > Matrix
effects strongly varied depending on the toxin. Signal
enhancement was especially evident for OA in most matrices
and chromatographic conditions. Overall positive matrix effects
were less important for PTX2, while AZA1 mostly tended to
signal suppression. Matrix effects for cyclic imines depended on
chromatographic conditions (Table 4), and generally suffered
from signal suppression under acidic conditions and moderate
signal enhancement at more alkaline pH. The use of different
chromatographic conditions affects matrix effects by altering
the elution order of interferences, but this effect is difficult to
assess, and it had not been systematically studied before.
Kesimpulan The method based on LC-MS/MS for the determination of
lipophilic toxins in seafood has been accepted by the European
Union as a reliable technique to protect public health and reduce
the use of animals for routine analysis. The EURLMB SOP [11]
establishes a solid framework for the implementation of the LC-
MS/MS method but it is not explicit enough concerning the
chromatographic conditions and the matrix effect correction
strategy that should be used. The current study is the first work
aimed to compare the most common chromatographic
alternatives for the determination of lipophilic toxins in seafood
by LC-MS/MS in terms of functionality, quality criteria,
validation parameters and quantification strategies under the
same experimental settings (extraction and hydrolysis
procedures, chromatographic column, MS instrument
conditions, standards and reagents, and analyst). We chose the
alkaline conditions, EXS calibration as quantification strategy,
and recovery correction for OA with CRM-DSP Mus b to be
implemented as the routine method. Alkaline conditions provide
higher sample throughput, lower LOQs, and the best overall
performance in terms of sensitivity and accuracy in the
validation study. The EXS strategy combined with OA recovery
correction by CRM-DSP Mus b demanded less time and
standard investment and provided satisfactory results. The
analysis of YTX was challenging and it is still being improved
in our laboratory by increasing the number of injections,
participating in collaborative studies and preparing internal
reference standards to correct YTXs recoveries. When selecting
the best chromatographic conditions, factors such as the
instrumentation available (regarding polarity switching, limits
of detection, and sensitivity), the number of samples needed to
analyze, the toxin profile and the sample matrices should be
considered. The matrix effects should be examined carefully,
especially when including a new toxin in the method or
analyzing a new matrix. A proper selection process may be time
 and resources demanding, but we hope that this comparative
study may serve as starting point to other laboratories
implementing their own methods for lipophilic toxins
determination in seafood by LC-MS/MS.

Judul Jurnal atau Optimization and Validation of a High Throughput UHPLC-

Artikel MS/MS Method for Determination of the EU Regulated
Lipophilic Marine Toxins and Occurrence in Fresh and
Processed Shellfish
Tujuan Penelitian In this study a modified ultra-high performance liquid
chromatography tandem mass spectrometry (UHPLC-MS/MS)
method has been developed for the identification and
quantification of EU-regulated lipophilic toxins. The method
optimization included a refinement of SPE-C18 clean-up, in
order to reduce matrix interferences.
Metode Penelitian Chemicals and Working Standard Solutions > Water, methanol
(MeOH) and acetonitrile (MeCN) of LC-MS grade and MeOH
of HPLC grade were purchased from Carlo Erba Reagents
(Rodano, Italy). Ammonium hydroxide (NH4OH, 32%), sodium
hydroxide (NaOH, ≥99%) and hydrochloric acid (HCl, 37%)
were from Merck KGaA (Darmstadt, Germany). Ultrapure
water (H2O 18 MΩ/cm; Milli-Q, Millipore, Sigma Aldrich,
Steinheim, Germany) was used in the clean-up phase. Standard
stock solutions of LMTs in MeOH (OA, 8.37 mg L−1 ; DTX1;
8.52 mg L−1 ; DTX2, 3.78 mg L−1 ; PTX2, 4.41 mg L−1 ;
AZA1, 1.30 mg L−1 ; AZA2, 1.22 mg L−1 ; AZA3, 1.18 mg L
−1 ; YTX, 4.92 mg L−1 ; hYTX, 5.79 mg L−1 ) and freeze-dried
mussel Certified Reference Material for Multiple Marine toxins
(CRM-FDMT1, 3 g) were purchased from the National
Research Council Canada NRCC (Halifax, Canada).
LC-MS/MS Analysis Chromatographic separation was
performed on an UHPLC system, an Ultimate 3000 (Thermo
Fisher Scientific, Waltham, MA, USA). Two columns were
compared: Acquity BEH C18 (2.1 mm × 100 mm; 1.7 µm) and
Waters X-Bridge C18 (2.1 mm × 50 mm, 2.5 µm) (Waters,
Milford, MA-USA). The latter column, coupled with a pre-
column Security Guard ULTRA cartridge UPLC C18 for 2.1
mm (Phenomenex, Torrance, CA, USA), gave the best results
(see below). The column heater was kept at 40 ◦C, while the
autosampler compartment temperature was maintained at 15 ◦C.
An injection volume of 5 µL and a flow rate of 0.2 mL min−1
were set. Both mobile phase A (water) and mobile phase B
(acetonitrile—water 90:10, v/v) contained 0.046% v/v of
NH4OH (pH = 11). Measurement of pH of the eluent was made
with a SympHony pH-meter from VWR International (West
Chester, PA, USA) using a combined glass electrode. Different
elution gradients were tested. The optimized elution gradient,
 started with 20% B, was maintained for 0.5 min, and then
followed by a linear increase to 85% B at minute 4.5. Then B
concentration increased to 98% in 1 min and this composition
was kept for 3 min. B concentration was finally lowered to 20%
in 0.5 min, followed by 5 min of column re-equilibration. The
total duration of the instrumental method was 14 min.
Hasil Penelitian The exhaustive extraction of LMTs was usually performed with
MeOH, as suggested by the EU-RLMB SOP. Lower LoDs may
be achieved by further reduction of the solvent volume of the
crude extract. According to literature data, MeOH was the best
solvent in the elution phase of OA and PTX2, while for AZA1
MeOH with 1% of NH4OH is recommended [38,40,42,43]. The
optimization of clean-up conditions required the comparison of
three different elution solutions.
hromatographic Separation and Gradient Optimization Since the
LMTs contain functional groups, such as -SO3H, -COOH, -
N=NH, they can be protonated or deprotonated depending on
the pH of the solvent. Hence, the retention time and the elution
order of the toxins may be greatly affected by pH.
Acquisition Mode
The triple quadrupole mass analyzer offers various acquisition
modes that can be adopted depending on the aim of the method.
Regulations (EU) No 15/2011 and 627/2019 established that
LC-MS/MS is the reference method for the official control of
LMTs [17,47]. For this reason, selection of molecular ion
(precursor) for fragmentation and analysis of the product ions is
mandatory. The selection reaction monitoring (SRM) was
chosen as the acquisition mode, using the product ion with the
highest relative intensity for quantification purposes, and the
second and the third most intense for identification. Figure 2.
Chromatogram of mussel sample naturally contaminated with
YTX toxins group, spiked with other toxins at a concentration
of 160 µg kg−1 . 3.3. MS Parameters Optimization 3.3.1.
Acquisition Mode The triple quadrupole mass analyzer offers
various acquisition modes that can be adopted depending on the
aim of the method. Regulations (EU) No 15/2011 and 627/2019
established that LC-MS/MS is the reference method for the
official control of LMTs [17,47]. For this reason, selection of
molecular ion (precursor) for fragmentation and analysis of the
product ions is mandatory. The selection reaction monitoring
(SRM) was chosen as the acquisition mode, using the product
ion with the highest relative intensity for quantification
purposes, and the second and the third most intense for
identification. Therefore, adopting the identification points
system reported in Commission Decision 2002/657/CE,
monitoring both molecular ion and two product ions, 4
identification points were reached, resulting in an unequivocal
identification for each toxin
Kesimpulan In this work, an optimized, sensitive and high throughput
analytical method for the simultaneous determination of 11
 lipophilic marine toxins and acylated/esterified forms in fresh
and processed shellfish by ultra-high performance liquid
chromatography coupled with tandem mass spectrometry was
developed, refined for reducing solvent consumption, and
validated. The sample preparation procedure consisted in double
methanolic extraction followed by SPE clean-up that minimized
the matrix effect. The ammonia-based gradient elution was
highly optimized to ensure the best separation of the two
isomers, OA/DTX2, and high selectivity. The method was fully
validated in terms of linearity (R 2 = 0.98), LoD (1–3 µg kg−1 )
and LoQ (3–8 µg kg−1 ), selectivity, precision (RSD = 11.8%),
recovery (73–101%), measurement uncertainty (12–20.3%),
matrix effect (−9–19%), and matrix ruggedness (oysters,
cockles, clams, razor clams, scallops and cephalopod molluscs),
in compliance with the most updated guidelines and regulations.
The optimized method was applied for the analysis of 1000
commercial mollusc samples collected during the last three
years (2019–2021). A preliminary monitoring study was also
presented. Since algae proliferation events are increasing and
new studies of important toxic effects of marine toxins are
reported, the continuous surveillance and the implementation of
analytical methods for determination of these toxicants appear
to be mandatory.

Judul Jurnal atau A low cost novel sensing system for detection of dangerous
Artikel marine biotoxins in seafood
Tujuan Penelitian A novel planar interdigital sensor-based sensing system has
been developed for detection of dangerous marine biotoxins in
seafood. Our main objective is to sense the presence of
dangerous contaminated acid in mussels and other seafood by
observing the change of reactive impedance of the planar
interdigital sensors.
Metode Penelitian The existing method of DA detection is based on using
chromatography technique, surface plasmon resonance (SPR),
and immunoassay technique using enzyme-linked
immunosorbent assay (ELISA). Chromatographic techniques
have been widely used for the detection of marine toxins.
Overview of different chromatographic techniques for marine
toxins detection has been reported by Quilliam [18]. Liquid
chromatography (LC) has been used as Association of
Analytical Communities (AOAC) official method for DA in
mussels [19]. Identification of DA in mussels using LC
technique has been reported in [6,20,21]. A new sensitive
determination method of DA using high-performance liquid
chromatography (HPLC) has been reported in [5,10,22,23].
Although chromatographic technique is one of the best methods
for the detection of DA but they require expensive equipments,
trained personnel, sophisticated method of sampling preparation
 and also it is a time consuming method. SPR has been widely
used as detection technique in biosensing system [3]. A rapid
and sensitive immuno-based screening method was reported to
detect DA present in extracts of shellfish species using a surface
plasmon resonance-based optical biosensor [24]. An
immunosensor based on SPR was used for the detection of DA
[25]. A portable SPR biosensor has been used to detect domoic
acid in clam extracts [26]. The detection method based on SPR
is suitable for laboratory analysis and not suitable for in situ
monitoring since the samples need to be prepared accordingly,
analysis may take longer time and the equipment (SPR) is
expensive. Immunoassay techniques are based on the affinity
recognition between antibodies and antigens, and the most
commonly found format is the enzyme-linked immunosorbent
assay (ELISA) [3]. Research of using ELISA to determine DA
has been reported in [7,27–30]. ELISA method normally can be
used to detect only one particular toxin. Only one research work
reported by Garthwaite et al. [29], which integrates ELISA for
screening of DSP, PSP, ASP and NSP toxins. To develop the
ELISA strip and to prepare the samples will need to follow some
laboratory procedures and tedious work. Also there is no
guarantee that the ELISA strip will respond very well to all
samples. Looking at the complexity of the existing methods,
where samples have to be prepared accordingly to certain
laboratory procedures, we have designed and fabricated novel
planar interdigital sensor based sensing system with the purpose
of an easy detection of molecules for DA. The developed
sensing system is easy to be used for the purpose of sampling
inspection and can provide fast analysis of DA within shellfish
meat for in situ monitoring. The prescreening process or
sampling inspection can be conducted at the site and if the
results are suspicious, therefore further analysis of contaminated
chemicals (DA) in the seafood can be done in the laboratory
using expensive techniques. The developed sensing system
should be reliable and cost effective.
Hasil Penelitian The sensor is connected with a series surface mount resistor of 120 k
to measure the current through the sensor. A frequency of 10
kHz with 10 Vpp is applied to the sensor using Agilent Function
Waveform Generator. The electric field will be created by the
sensor in the system under test. The voltage (sensing voltage)
across the series resistance is observed, to measure the current
flowing to the sensor. Fig. 6 shows the signal waveforms of
excitation voltage and sensing voltage available from Sensor 1.
Kesimpulan Initial studies on three different chemicals and different seafood
products have shown that the fabricated interdigital sensors can
respond very well to each samples and can clearly discriminate
between them. Sensor 1 was selected for further analysis with
real mussels due to its better sensitivity. Analysis of results with
real mussels have shown that Sensor 1 can detect the presence
of proline on the surface as well as injected proline to each
 sample. It can be said that Sensor 1 was able to detect the
presence of different amount of proline injected into the mussel
samples. Experiment with DA has shown that novel interdigital
sensor can be used to detect the small amount of domoic acid in
mussels. Results from the experiments have shown that novel
interdigital sensing system has the potential to be one of the
options to assess the quality of seafood products for in situ
monitoring. The sensing system can detect the presence of DA
in shellfish meats. The outcomes from the experiments provide
chances of opportunity for further research in developing a low
cost miniature type of sensor with a reliable sensing system for
commercial use. Further experiments are also conducted to
study the effect of other parameters on the sensor sensitivity.
Further research can also be conducted to access the quality of
other food for example, to detect salmonella poisoning in meat
product. There is a strong possibility of using novel planar
interdigital sensor to detect dangerous biological agents
(bacteria, viruses or toxins) on food, which is used for
bioterrorism.

Judul Jurnal atau Determination of lipophilic marine toxins in mussels by liquid

Artikel chromatography coupled to high resolution mass spectrometry.
Validation study.
Tujuan Penelitian A LC-HRMS method for determination of major groups of
lipophilic toxins has been developed and validated.
The use of HRMS confirmation criteria can help to avoid false
positives.
The identification and confirmation criteria validated in the
present study can contribute to define new parameters to
implement HRMS in complex analysis, such as it is the case for
lipophilic marine toxins in mussels.
Metode Penelitian Liquid Chromatography > The LC system consisted of a
Surveyor MS Plus pump and an Accela Open AS autosampler
kept at 15 ºC (Thermo Fisher Scienti¿c, San Jose, California). A
5 µL injection volume was used. A HypersilGold C18 (50 mm
x 2.1 mm, 1.9 µm) (Thermo Fisher, Scientific, Bremen,
Germany) was used for the separation of toxins at a flow rate of
300 µL min-1 . Mobile phase A was water and B was
acetonitrile/water (90:10), both containing 6.7 mM ammonium
hidroxyde [13]. The gradient started at 20 % of B and was kept
this composition for 3.5 min. Then, it was increased to 90 % of
B in 16 min and kept 3 min, then returns to initial conditions of
20 % of B maintaining it for 11 min for the column equilibration.
The total duration of the method was 30 min.
High Resolution Mass Spectrometry > Mass spectrometry
analyses were carried out with an Orbitrap-Exactive HCD
(Thermo Fisher Scienti¿c, Bremen, Germany) equipped with a
 heated electrospray source (H-ESI II). Nitrogen (purity > 99.999
%) was used as sheath gas, auxiliary gas and collision gas. The
instrument was daily calibrated in both positive and negative
modes. Three time segments were set. First segment (0 min - 3.5
min) working in negative mode without and with all ion
fragmentation (AIF) (HCD 60 eV), second segment (3.5 min –
8.25 min) in positive mode without and with AIF (HCD 50 eV)
and third segment (8.25 min – 30 min) in positive mode without
and with AIF (HCD 20 eV). The mass range was m/z 400-1250
in full scan and m/z 70-1200 in AIF mode.
Hasil Penelitian We propose that liquid chromatography coupled to high-
resolution mass spectrometry (LC-HRMS) can be a very useful
technique for multi-toxin detection and quantification, due to the
capacity to acquire in full scan with good sensitivity and better
selectivity. Accuracy (trueness and precision), linearity,
calibration curve check, limit of quantification (LOQ) and
specificity were the parameters established for the method
validation. The validation was performed at 0.5 times the current
European Union permitted levels. The method performed very
well for the parameters investigated. The trueness, expressed as
recovery, range from 80 to 94 %, the precision, expressed as
intralaboratory reproducibility, range from 5 % to 22 % and the
LOQs range from 0.9 to 4.8 pg on column.
In addition to optimizing the experimental parameters for
efficient toxin ionization, in the development of the method,
there were some critical aspects that had to be addressed.
Kesimpulan A sensitive LC-HRMS method for quantification of major
groups of marine lipophilic toxins has been developed and
validated. The method performed very well for the parameters
investigated. Ion ratios as confirmation criteria were deeply
studied. It was observed, that both fragment ion ratio and isotope
ion ratio 523 can be used to confirm a positive result, but for
each compound one or the other can be more suitable. The use
of the HRMS criteria can help to prevent false results.
Interferences coming from the matrix can be identified because
data is acquired in full scan mode so matrix effects are
minimized. It has been shown that HRMS provides
incomparable confirmatory performances with excellent
quantitative capabilities. Further studies are necessary to include
more toxins of each group studied and more toxin groups.
Moreover, this study can contribute to define new parameters
based on HRMS, for complex matrix analysis, as it is the case
for lipophilic marine toxins in mussels.

Judul Jurnal atau Extended Targeted and Non-Targeted Strategies for the
Artikel Analysis of Marine Toxins in Mussels and Oysters by (LC-
HRMS)
Tujuan Penelitian To assess the potential of the LC-HRMS method to detect
marine toxins as part of a non-targeted analysis, the authors
performed a proof of concept study as a first essential step
toward a reliable characterization of samples naturally
contaminated with unknown marine toxins and the identification
of the toxins.
Metode Penelitian First, the study was carried out in targeted mode to assess the
performance of the method for known toxins with an extended
range of polarities, including lipophilic toxins (okadaic acid,
dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins,
cyclic imines, brevetoxins) and domoic acid. The targeted
method, assessed for 14 toxins, shows good performance both
in mussel and oyster extracts. The non-target potential of the
method was then challenged via suspects and without a priori
screening by blind analyzing mussel and oyster samples spiked
with marine toxins. The data processing was optimized and
successfully identified the toxins that were spiked in the blind
samples.
Hasil Penelitian When considering the geographical expansion of marine toxins,
the emergence of new toxins and the associated risk for human
health, there is urgent need for versatile and efficient analytical
methods that are able to detect a range, as wide as possible, of
known or emerging toxins. Current detection methods for
marine toxins rely on a priori defined target lists of toxins and
are generally inappropriate for the detection and identification
of emerging compounds. The authors describe the
implementation of a recent approach for the non-targeted
analysis of marine toxins in shellfish with a focus on a
comprehensive workflow for the acquisition and treatment of
the data generated after liquid chromatography coupled with
high resolution mass spectrometry (LC-HRMS) analysis. First,
the study was carried out in targeted mode to assess the
performance of the method for known toxins with an extended
range of polarities, including lipophilic toxins (okadaic acid,
dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins,
cyclic imines, brevetoxins) and domoic acid. The targeted
method, assessed for 14 toxins, shows good performance both
in mussel and oyster extracts. The non-target potential of the
method was then challenged via suspects and without a priori
screening by blind analyzing mussel and oyster samples spiked
with marine toxins. The data processing was optimized and
successfully identified the toxins that were spiked in the blind
samples.
Kesimpulan To assess the potential of the LC-HRMS method to detect
marine toxins as part of a non-targeted analysis, the authors
performed a proof of concept study as a first essential step
toward a reliable characterization of samples naturally
contaminated with unknown marine toxins and the identification
of the toxins. Since there are no guidelines for the validation of
a non-targeted method, the LC-HRMS method that was
 developed for the analysis of marine biotoxins was characterized
according to the approach used in the field of water
micropollutants. The method performances were first evaluated
in targeted mode for marine toxins with different polarities
spiked in mussel and oyster samples and were found to be
satisfactory for the criteria tested (LODs, LOQs, specificity,
matrix effects, accuracy, and precision). The performances of
the optimized non-targeted strategy were then evaluated, both
for the suspect screening approach relying on the use of a library
of 821 toxins and for the without a priori screening of unknowns.
The essential steps for the non-targeted procedure have been
detailed and discussed. The overall workflow was tested on
spiked samples that were analyzed blindly and was shown to be
highly efficient in narrowing down the number of potential false
positive and false negative findings. Whatever the approach
selected, the marine toxins spiked in the samples analyzed as
blind for the proof of concept were picked among the features
detected in LC-HRMS. It is important to report that, although
many tasks could be automated in the data treatment, it is
essential to critically and manually review the results that were
obtained to avoid any misinterpretation.

Judul Jurnal atau New method for the analysis of lipophilic marine biotoxins in
Artikel fresh and canned bivalves by liquid chromatography coupled to
high resolution mass spectrometry: A quick, easy, cheap,
efficient, rugged, safe approach
Tujuan Penelitian A new method for the analysis oflipophilicmarine biotoxins
(okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins,
yessotoxins, spirolids) in fresh and canned bivalves has been
developed.
Metode Penelitian QuEChERS extraction method > One g of bivalve sample was
weighed in a glass tube; 1 mL of potassium dihydrogen
phosphate 1 M solution and 0.2 mL of internal standard solution
of 0.5 mg L−1 were added and mixed with the sample and were
allowed to stand for 20 min. 5 mL acetonitrile was then added
to all samples as the extraction solvent. After vortexing (30 s),
sample was mixed with 0.5 g of magnesium sulphate and 0.1 g
of sodium chloride. Tubes were placed in an axial stirrer
(Amplitude: 120 mm; Speed: 2.3 m s; Acceleration: 60 m s−2;
Abruptness: 7; Delay: 0 s; Cycle time: 180 s; Repetitions: 1) and
centrifuged (3500 rpm, 10 min, 5 ◦C). The supernatant was
transferred to conical glass tubes and 150 mg of magnesium
sulphate and 25 mg of Lichroprep RP-18 were added. After
vortexing (30 s), tubes were placed in an axial stirrer (using
above mentioned conditions) and centrifuged (3500 rpm, 10
min, 5 ◦C). 2 mL of supernatant were evaporated to 1 mL under
a N2 stream at 45 ◦C. 3 mL of 6.7 mM ammonia were added.
 After mixing, reconstituted extracts were filtered using Mini-
UniPrep vials, and injected into the chromatographic system.
Calibration > Quantification of each individual MBTX was
performed using surrogate matrix matched standards (SMMS),
to overcome the usual ion suppression or ion enhancement
effects in MS analysis. Thus, mussel blank samples were spiked
with the analytes at five different concentrations and 200 L of
eprinomectin solution (0.5 mg L − 1), used as internal standard
(IS), was added; afterwards the SMMS were submitted to the
whole analytical procedure; the calibration curves for each
analyte were obtained by linear regression analysis of the peak
areas relative to IS areas against concentration. Calibration
curves in the range 25–350 g kg − 1 for all the MBTXs were
used. Eprinomectin was chosen as IS because it is not present in
the samples, it can be ionized in both negative and positive
modes and it has a molecular weight similar to the analytes.
Hasil Penelitian Acidic conditions were tested first because they are normally
used in mass spectrometry; acidic conditions consisted of an
aqueous mobile phase of 0.1% formic acid in water. The
columns tested in these conditions were: Kinetex XB C18 (100
× 2.10) mm, 1.7 m particle diameter (Phenomenex), Hypersil
Gold aQ C18 (100 × 2.10) mm, 1.9 m particle diameter,
(Thermo Scientific) and Syncronis C18 (100 × 2.10) mm, 1.7 m
particle diameter (Thermo Scientific). Columns were evaluated
in terms of their ability to resolve the analytes and obtain good
peak shapes. Kinetex XB C18 gave asymmetric and broad peaks
for AZAs, while Hypersil Gold aQ C18 gave broad tailing peaks
for AZAs in HESI+ and for YTXs in HESI − . Relating to
Syncronis C18 , resolving problems were observed with YTXs
in acidic condition.
The hybrid Q-Orbitrap mass analyzer presents several
acquisition modes (or a combination of them) that can be carried
out depending on the aim of the method. In the present study,
tandem mass spectrometry is used, since legislation establishes
this technique as the reference method for the official control of
lipophilic marine biotoxins [7] . For this reason, selection of a
molecular ion for fragmentation and analysis of the product ions
is mandatory and the targeted MS/MS acquisition mode was the
optimal solution, as the other acquisition modes presented some
limitations [27] . In comparison to triple quadrupole, high
resolution tandem mass spectrometry provides additional
confirmatory information, helping to eliminate false results
because the molecular ion is monitorized. The specificity of the
accurate mass measurement adds more veracity to any
determination.
To optimize the ion source parameters, a dilution of 1 mg L − 1
of each compound was individually infused into the mass
spectrometer. Working in full scan mode, the molecular ion for
each compound was identified and the optimum voltages,
temperatures and gas flow defined.
 QuEChERS was the method of choice because of its high
throughput and in order to establish a simple and robust method
of analysis. This strategy is based on extraction, usually with
acetonitrile, in the presence of salts, and a clean-up step based
on a dispersive SPE.
Kesimpulan The use ofthe QuEChERSmethodology combined with the
application of UHPLC-HRMS in the MS/MS mode has shown
to be both a straightforward and very reliable approach for the
analysis of lipophilic MBTXs in bivalves. On one hand, it has
been demonstrated that the extraction with acetonitrile
combined with a simple dSPE with C18 provides high absolute
recoveries for the extraction and clean-up steps. This simple
treatment is highly effective, and large series of samples can be
analyzed without special maintenance ofthe UHPLC-HRMS
system. Onthe otherhand,theHRMS/MS acquisitionmode,
whichmeets the requirements of EU legislation,is
apowerfultoolto avoidmatrix interferences. Accurate mass data
are obtained for each analyte, for both the molecular ion and the
selected fragment, providing excellent information in order to
confirm the identity of the compound and to avoid the risk of
false positive results. Calibration with SMMS provides reliable
quantitative results, and it has been shown that a calibration
curve built from blank mussel samples can be suitable to analyze
different bivalve species. Eprinomectin has shown an
appropriate behavior to be used as internal standard for
lipophilic MBTXs. The developed method, which has been
successfully validated, is suitable for the confirmatory
quantitative analysis of lipophilic MBTXs in fresh and canned
bivalves at the regulated concentration levels. The method is
currently used in the LASPB in routine analysis, and it has been
included in the scope of the accreditation following
ISO/IEC17025 requirements.

Judul Jurnal atau Current Trends and Challenges for Rapid SMART Diagnostics
Artikel at Point-of-Site Testing for Marine Toxins
Tujuan Penelitian In the past twenty years marine biotoxin analysis in routine
regulatory monitoring has advanced significantly in Europe
(EU) and other regions from the use of the mouse bioassay
(MBA) towards the high-end analytical techniques such as high-
performance liquid chromatography (HPLC) with tandem mass
spectrometry (MS). Previously, acceptance of these advanced
methods, in progressing away from the MBA, was hindered by
a lack of commercial certified analytical standards for method
development and validation.
Metode Penelitian Mouse Bioassay (MBA) > Until recently, for the past 70 years,
bioassays such as the MBA have been the reference methods
worldwide for identification of DSP [48] and PSPs [49]. MBA
methods involve intraperitoneal injection of replicate shellfish
 extracts to mice and measuring either the time of death after
injection, for a direct measure of PSP toxicity, or
mortality/morbidity or clinical signs of toxin exposure for DSP.
Aside from the obvious ethical issues, testing for the presence
of PSP and DSP using the MBA, suffered from low sensitivity,
inaccuracies due to matrix interferences, whilst offering no
information regarding the toxin profile present in the shellfish
extract or the toxin(s) responsible for death. Following
implementation of EU Directive 2010/63 (on the protection of
animals used for scientific purposes) which prohibits use of an
animal test when an accepted alternative exists [50], MBA have
been gradually replaced in recent years in the EU (i.e., in UK
and Ireland MBA is not in use) with alternative non-animal
methods for shellfish toxin detection. Moreover, as a result of
the scientific, technical, and ethical limitations, countries such
as Australia, Canada and New Zealand no longer use the MBA
test for routine toxicity testing of shellfish. However, this
progress in moving away from the MBA was not without its
challenges in method development, validation and translating
toxicity to the MBA, with equivalency factors [41,51].
Following implementation of Commission Regulation (EU)
2017/1980, the MBA is no longer the reference method for
detecting PSP toxins in the EU [32] and is not listed in the
National Shellfish Sanitation Program (NSSP) ‘Guide for the
Control of Molluscan Shellfish’ as a method to test for DSP or
ASP. This change should ensure the complete replacement of
the MBA at the EU level.
Chemical Method > Chemical detection methods incorporating
chromatographic separation and a range of detectors are now the
current diagnostic gold standards for marine toxin detection:
they are accurate, sensitive, and provide information on the
specific toxins for whichever standards are commercially
available. Similarly, sample preparation methods can be
complex whereby after solvent extraction, the crude shellfish
extract may be submitted to solid-phase extraction (SPE).
Hasil Penelitian Going forward, it will be most important to design highly
accurate quantitative solutions that can give shellfish producers
an accurate assessment of contamination levels in shellfish
produce on their site at the time of testing. This would allow
producers to follow immediate trends and make an informed
decision about the state of a current contamination event or even
predict an upcoming event. Together, tools designed for
practical usage on site will save shellfish producers time and
money, protect consumers, boost consumer confidence, and help
ensure the availability of freshly caught, toxin-free shellfish.
However, these diagnostic tools for toxins should not be
considered in isolation: a holistic approach combining satellite
imagery, POST rapid diagnostics and other developing tools
[24] for HAB detection could aid the understanding of our
oceans in relation to climate and environmental conditions
 moving towards early warning systems that allow enhanced
management and control to complement the data generated by
official control testing programmes. Suitable infrastructure and
handling capacity will also be required on-site to allow these
management systems to benefit shellfish farmers.
Kesimpulan Progress in the field of portable toxin testing for industry is rapid
where toxins are available as standards and many promising
technologies have been developed, each with their own
strengths and weaknesses. Many of these assays would benefit
from miniaturization to make them portable and accessible on-
site. Furthermore, many of these assays require further testing in
new matrices e.g., the full range of bivalve species, gastropods
and crustaceans, optimization and validation of protocols to an
appropriate standard, and comparison against the current
reference methods. It will be important to perform collaborative
validation studies that assess interlaboratory performance
characteristics and also verify the test kit components are robust
and viable across a full range of environments and following
transportation. Different shellfish species thrive in different
waters across the globe, from freezing cold brackish lakes and
estuaries to highly saline, warm tropical lagoons, and testing
equipment needs to be able to function under a wide range of
environmental conditions to accommodate the different farmed
species. For example, environmental temperature, humidity,
salinity, and light density are all important conditions that can
influence test results. Moreover, saltwater is extremely
corrosive, and testing equipment should ideally be water- and
corrosion-resistant. It will additionally be vital to consult and
validate the tests to the end user requirements for their ability to
be fit for purpose on site.

Judul Jurnal atau In-house validation of a liquid chromatography tandem mass

Artikel spectrometry method for the analysis of lipophilic marine toxins
in shellfish using matrix-matched calibration
Tujuan Penelitian
Metode Penelitian SPE clean-up > The SPE procedure was carried out as
described by Gerssen et al. [25]. Strata-X cartridges, 30 mg, 1
mL (Phenomenex, Torrance, CA, USA), were conditioned and
equilibrated using 1 mL of methanol and methanol/water (30:70,
v/v), respectively. The methanolic shellfish extracts (1.2 mL)
were diluted with 2.8 mL water. After 4 mL of diluted extract
had been loaded on the cartridge, the cartridge was washed with
1 mL methanol/water (20:80, v/v). Finally, the toxins were
eluted from the cartridge with 1.2 mL methanol containing 0.3%
v/v of a 25% ammonium hydroxide solution in water.
Preparation of extracts for determination of the
performance characteristics > Blank mussel and oyster
 extracts, different from the ones used for the MMS, were spiked.
The extract (1.8 mL) was spiked with 50, 100 and 150 µL (0.5,
1, 1.5 times the permitted level, respectively) of the mixed
standard stock solution. The total volume was made up to 2 mL
by adding 150, 100 and 50 µL methanol, respectively. After the
spiking, an aliquot (1.2 mL) of the extract was purified by SPE
before analysis. The remainder of the extract was analysed
without further cleanup. On a separate occasion, eight different
shellfish extracts (two mussels, two oysters, two cockles, two
clams) were prepared and spiked at 0.5 times the permitted level
to determine the interspecies repeatability.
Hydrolysis > To determine the amount of esters of OA, DTX1
and DTX2 present in the shellfish sample, alkaline hydrolysis
can be performed.
LC-MS/MS analysis > Chromatographic separation was
achieved using a Shimadzu HPLC system (Shimadzu, ‘s-
Hertogenbosch, The Netherlands) consisting of a degasser
(DGU-20A3 ), a binary pump system (LC20-AD), an
autosampler (SIL-HTc) and a column oven (CTO-20A).
Separation was achieved on a Waters X-Bridge™ C18 (150
mm× 3 mm, 5 µm) column.
Validation parameters investigated > The method was
validated using EU Commission Decision 2002/657/EC as a
guideline. Seven replicates, at each of the three spiking levels
(0.5, 1 and 1.5 times the permitted level), were analysed.
Hasil Penelitian EU legislation demands that the validation of an alternative
method for marine toxins should be carried out according to an
internationally recognized protocol [28]. Commission Decision
2002/657/EC describes the performance characteristics of
analytical methods for so-called group A and group B
substances in products of animal origin [29]. As mentioned in
Council Directive 1996/23/EC, group B substances comprise
compounds such as veterinary drugs, environmental
contaminants and mycotoxins [30]. Therefore, we decided to use
Commission Decision 2002/657/EC as the basis for the
validation of the analytical method for lipophilic marine toxins.
Kesimpulan A recently developed LC-MS/MS method was validated, both
in combination with and without SPE purification, using
European Commission Decision 2002/657/EC as a guideline.
MMS were used instead of spiking standards in methanol to
construct calibration curves. The use of MMS largely eliminates
matrix effects (ion suppression/enhancement). The method
performed very well for the parameters investigated. Only minor
differences were observed between the crude extract and the
SPE-purified extract. The largest difference observed was the
change in sensitivity that occurred during analysis of the crude
extracts. For larger series (more than 20 samples) it is advised to
incorporate an SPE clean-up step, although this will lead to a
more time-consuming method. Furthermore, it was shown that
MMS in blank mussel extracts can be used to quantify other
 matrices such as oyster, cockle and clam. The species
differences did not have a significant effect on the method. The
validated method also performed well at low concentrations for
OA and AZA1. Therefore, we recommend the use of this
method for the analysis of lipophilic marine toxins instead of the
currently used, less sensitive and animal unfriendly mouse and
rat bioassays.

Judul Jurnal atau Multiplex biotoxin surface plasmon resonance method for
Artikel marine biotoxins in algal and seawater samples
Tujuan Penelitian Multiplex immunological methods could therefore be used as
early warning monitoring tools for a variety of marine biotoxins
in seawater samples.
Metode Penelitian A multiplex surface plasmon resonance (SPR) biosensor method
for the detection of paralytic shellfish poisoning (PSP) toxins,
okadaic acid (and analogues) and domoic acid was developed.
This method was compared to enzyme-linked immunosorbent
assay (ELISA) methods. Seawater samples (n0256) from around
Europe were collected by the consortia of an EU project
MIcroarrays for the Detection of Toxic Algae (MIDTAL) and
evaluated using each method. A simple sample preparation
procedure was developed which involved lysing and releasing
the toxins from the algal cells with glass beads followed by
centrifugation and filtering the extract before testing for marine
biotoxins by both multi-SPR and ELISA. Method detection
limits based on IC20 values for PSP, okadaic acid and domoic
acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively, for the
prototype multiplex SPR biosensor.
Hasil Penelitian Evaluation by SPR for seawater samples has shown that 47, 59
and 61 % of total seawater samples tested positive (result greater
than the IC20) for PSP, okadaic acid (and analogues) and
domoic acid toxins, respectively. Toxic samples were received
mainly from Spain and Ireland. This work has demonstrated the
potential of multiplex analysis for marine biotoxins in algal and
seawater samples with results available for 24 samples within a
7 h period for three groups of key marine biotoxins.
Kesimpulan A multiplex assay has been developed and validated for the
semi-quantitative, simultaneous screening of three types of key
marine biotoxins in seawater samples. Results for PSP, okadaic
acid and domoic acid toxins can be detected in as little as 13 min
from one seawater sample (after extraction) in real time. Method
detection limits based on IC20 values for PSP, okadaic acid and
domoic acid toxins were 0.82, 0.36 and 1.66 ng/ml, respectively,
for the prototype multiplex SPR biosensor. Using these
detection limits, 47, 59 and 61 % of seawater samples tested
positive for PSP, okadaic acid and domoic acid toxins,
respectively, with toxic seawater samples found mainly from the
Rias of Pontevedra, Arosa, Muros, Ares-Betanzos, and estuary
of Bayona, Spain and the Killary, Cork and Bell Harbours of
Ireland. The simplicity of the assay means it could be used as a
first action screening method in a monitoring laboratory for the
presence of these biotoxins in seawater samples. Many
European countries test and identify phytoplankton in seawater
samples in shellfish harvest areas with many setting trigger
levels for certain toxic phytoplankton species. In Northern
Ireland, these levels are set at >0 cells/L for Alexandrium
species, >100 cells/L for Dinophysis/ Prorocentrum species and
>150,000 cells/L for Pseudonitzschia species. However, it is
often impossible to differentiate between certain species of toxic
algae; and if toxic species are present at trigger levels, this would
initiate further testing of shellfish whether or not biotoxins are
present. A viable alternative would be to monitor seawater
samples for the presence of biotoxins. An early warning
detection tool would be extremely beneficial to the aquaculture
and fisheries industry to allow economic decisions to be made
quickly in relation to the harvesting process for shellfish.
Although this is a laboratory-based method, it still has many
advantages and benefits over the current detection systems in its
ability for multiple toxin group analysis. The next step in this
research would be to develop a multiplex portable assay for the
submersible vehicle. Without comprehensive phytoplankton and
shellfish monitoring programmes, there is currently no way to
ensure that shellfish are safe for human consumption.