ISOLATION OF

MARINE TOXINE
Safrina Dyah Hardiningtyas
 CLASSIFICATION OF BIOTOXINS
Marine Toxins

Poisons Venoms Secondary

• Domoic acid (ASP)
metabolites
• Proteineous
• Saxitoxins (PSP) • Terpenes
• Brevetoxins (NSP)
• Okadoic acid • Alkaloid
(DSP) • Flavonoid
EXTRACTION
Extraction in chemistry is a separation process
consisting in the separation of a substance from a
matrix.
 Extraction is a process whereby the prepared marine organisms
tissue (i.e. shellfish) is converted into liquid containing released toxins.

Prinsip dasar separasi Teknik separasi

Ukuran Filtrasi, dialisis, kromatografi esklusi
Densitas dan massa Sentrifugasi
Perubahan keadaan fisik Distilasi, sublimasi, rekristalisasi
Perubahan keadaan Pengendapan, pertukaran ion,
kimia elektrodeposisi, volatisasi
Partisi antar fasa Ekstraksi, kromatografi
EXTRACTION
There exist several types of extraction, including:
 liquid–liquid extraction: the compounds are separate
according to their relative solubility in two different
immiscible liquid phases
 solid-phase extraction, Example : Thin Layer
chromatography, column chromatography
 acid-base extraction.
 CLASSIFICATION OF BIOTOXINS
Marine Toxins
According to chemical property, marine
Poisons Venoms
poisons Secondary
classified on 2 group:
• Hydrophilic toxins metabolites
• Phytoplankton: • Proteineous
Domoic acid (ASP)
dinoflagelata
Saxitoxins (PSP) • Terpenes
• Shellfish • Alkaloid
• Lipophilic toxins
Brevetoxins (NSP) • Flavonoid
Okadoic acid (DSP)
alkaloid
EXTRACTION OF MARINE POISONS
▪Acid-based Extraction
Acid hydrolysis: method for extract of hydrophilic
toxins (ASP and PSP)
Alkaline hydrolysis: method for extract of
lipophilic toxins (NSP, DSP)
▪Solvent extraction: can be use for extract hydrophilic
and/or lipophilic toxins. Examples: Methanol, acetone
EXTRACTION FOR HYDROPHILIC
TOXINS
Extraction method:
Acidic extraction : acetate acid, HCl,
Methanol extraction: usually using 50% methanol
PHYTOPLANKTON EXTRACTION

This extraction method can be

used with the PSP and ASP Rapid
tests.
MINI AOAC METHOD
• The Association of Official Analytical Chemists (AOAC) is
the approved method of extraction used by regulatory
agencies.
• The Mini AOAC Extraction is a scaled-down version of the
official method, based on a 10 g sub sample from 100g of
shellfish puree and was validated by Jellett Biotek Ltd.
(Toxicon 40 [2002] 1407-1425
• The Mini AOAC extraction method can be used with PSP
and ASP Rapid tests

.
METHANOL EXTRACTION

This extraction method can be

used with the ASP Rapid tests.
EXTRACTION FOR LIPOPHILIC
TOXINS
Extraction method:
Methanol extraction and Alkaline hydrolysis
Methanol extraction and dichloromethane extraction
Acetone extraction
 CLASSIFICATION OF BIOTOXINS
Marine Toxins

Poisons Venoms Secondary

• Domoic acid (ASP)
metabolites
• Proteineous
• Saxitoxins (PSP) • Terpenes
• Brevetoxins (NSP)
• Okadoic acid • Alkaloid
(DSP) • Flavonoid
EXTRACTION OF MARINE VENOMS
Cone snail

Stone fish Sea snake

EXTRACTION OF MARINE VENOMS
METHOD OF ANALYSIS FOR
MARINE POISONS
▪Biology: Mouse bioassay, Daphnia magna assay, fish assay
▪Chemical :
▪ fluorometric and colorimetric techniques (UV-Vis spectrometer and Fluorescence
spectrometer),
▪ Chromatography techniques (HPLC, LC-MS, LC-FLD (fluorescence detection),
electrophoresis technique

▪Biochemical : ELISA, radioimmunoassay (RIA), stick rapid

test
 BIOLOGY METHOD

Daphnia magna Artemia salina rainbow trout (Oncorhynchus mykiss) or

carp (Cyprinus carpio).
MOUSE BIOASSAY
▪The mouse bioassay is a semiquantitative assay that provides
sufficient reproducibility and detection limit for regulatory
purposes.
▪Its major advantage is that it measures biological toxicity
directly and as such should be maintained as a reference
method.
▪The drawbacks of this method are
▪costly and time-consuming and cannot be automatized,
and
▪ its detection limit (320–380 μg STX equivalents/kg
edible parts) is very near to the maximum permitted level
of 800 μg STX equivalents/kg (AOAC 2000; Wekell et al.
2004).
▪Poor sensitivity
▪Poor correlation with human oral toxixity
▪the mouse bioassay is the controversial use of live animals.
ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAYS)
ELISA: based on enzymatic color-reaction
Type enzyme for ELISA
➢Horseradish peroxidase : product of reaction : Yellow colour
➢Alkaline Phosphatase: product of reaction : Blue colour (Abs 590 nm)

The advantage ELISA:

▪more sensitive than LC and
▪more specific than the mouse bioassay, and
▪faster than LC
▪less expensive and more practical.
Type of ELISA
ELISA
ELISA was optimized for maximum sensitivity using
standard procedures:
▪The direct ELISAs were optimized by titrating antibody coating
concentration against increasing concentrations of toxin–enzyme
conjugate with, and without, free toxin as competitor
ELISA
The indirect ELISA, checkerboard titrations of antibody and plate-
coating conjugate were performed. These were followed by titration
of second antibody and competition studies using free toxin to
determine the reagent preparation giving greatest sensitivity,
accuracy, assay robustness, and precision;

Reference toxin solutions were diluted with ethanol–PBS to ensure maximum ethanol
concentration in the assay of 10% (v/v) for all assays except the DSP (okadaic acid)
ELISA in which alcohol concentrations of 18% were used.
ELISA FOR MARINE TOXINS
Enzyme-linked immunosorbent assay (ELISA) test kits have
been developed and are commercially available.
 The DSP-Check® ELISA test kit from UBE Industries, Tokyo, Japan has
been used throughout the world for screening OA and DTX1 at a claimed
detection limit of 20 ng/g. The monoclonal antibody in the DSP-Check®
test kit cross-reacts with DTX1 at a level comparable to OA but PTXs and
YTXs are not reactive (Hallegraeff et al., 1995).
 The Rougier Bio-Tech® ELISA test kit utilizes an anti-OA monoclonal
antibody and an antiidiotypic antibody which competes with OA for
binding sites on the anti-OA antibody. The antibody in this test kit exhibits
a much higher sensitivity (10-20 fold) for OA than either DTX1or DTX2,
and methyl-, diol- and alcohol derivatives of OA will also bind to the
antibody, whereas DTX3 and brevetoxin-1 do not cross-react at all.

Garthwaite et al. (2001) developed an integrated ELISA

screening system for ASP, NSP, PSP and DSP toxins
(including yessotoxin). The system detects suspected
shellfish samples. Thereafter the suspected samples have
to be analysed by methods approved by international
regulatory authorities. Alcohol extraction gave good
recovery of all toxin groups.
A METHOD COMBINING A KIT WITH BIONIC E-EYE
FOR RAPID DETECTION OF DIARRHETIC SHELLFISH
POISONING ON SITE DOI: 10.1039/C8AY00676H

450 nm

the kit reached a very low limit of detection 0.19 μg/L and the
linear detection range was 0.2-10 μg/L
SELF-ASSEMBLED MONOLAYER-BASED
IMMUNOASSAYS FOR OKADAIC ACID
DETECTION IN SEAWATER AS
MONITORING TOOLS
https://doi.org/10.1016/j.marenvres.2017.11.004
https://doi.org/10.1016/j.toxicon.2015.01.015
Diarrhoeic Shellfish Poisoning (DSP) kit
The assay is based on the inhibition of the protein
phosphatase (PP2A) by DSP toxins (okadaic acid and
dinophysistoxin :OA and DTXs), which is the next
generation assay principal to ELISA and mass
spectrometry.
This principal is same as in-vivo toxicity process of
DSP, so this assay measure not only DSP but also
entire toxicity, that is ideal evaluation methods for
DSP.
Minimum detection limit is 0.036ug/g, and minimum
quantification limit is 0.056ug/g

Assay Principle & Procedure

PP2A can hydrolyze a colorless artificial substrate, p-
nitrophenyl phosphate (p-NPP), and produces the
yellow color of p-nitrophenol (p-NP) in the alkaline
solution. The intensity of the color is proportional to
the enzyme activity and the absorbance is measured
at 405 nm. The concentration of DSP toxins in the
sample is calculated from the standard curve
produced using known concentrations of OA.
Lateral Flow Immunoassay (LFIA) for the Rapid
Screening of Paralytic Shellfish Toxins (PSTs) from
Shellfish Extracts DOI: 10.1021/acs.analchem.5b00608

A single-step lateral flow immunoassay

(LFIA) was developed and validated
for the rapid screening of paralytic
shellfish toxins (PSTs) from a variety of
shellfish species, at concentrations
relevant to regulatory limits of 800 μg
STX-diHCl equivalents/kg shellfish
meat. A simple aqueous extraction
protocol was performed within several
minutes from sample homogenate. The
qualitative result was generated after
a 5 min run time using a portable
reader which removed subjectivity
from data interpretation.
TOXINS RAPID TEST
TERIMA KASIH