150 / 302
2
There are a series of regulations issued by the European Union (EU) related to marine biotoxins.
Regulation (EC) No 853/2004
1
concerns the control of lipophilic toxins, establishing maximum
levels for lipophilic toxins in bivalve mollusks destined to the market for human consumption:
• For okadaic acid, dinophysistoxins and pectenotoxins together, 160 micrograms of okadaic
acid equivalent per kilogram
• For yessotoxin, 1 milligram of yessotoxin equivalent per kilogram
• For azaspiracids, 160 micrograms of azaspiracids equivalent per kilogram
In the past, aside from bioassays on mice, most analytical techniques developed for the determina-
tion of marine biotoxins in bivalve mollusks have been based on offline methodologies, i.e. methods
involving solid phase extraction (SPE) or liquid-liquid extraction (LLE) followed by high-pressure
liquid chromatography (HPLC) with fluorometric or UV-diode array detection, or detection by
liquid chromatography coupled with mass spectrometry (LC-MS).
The EU Commission Regulation (EC) No 15/2011,
2
amending Regulation (EC) No 2074/2005
about the testing methods for detecting marine biotoxins in bivalve molluscs, describes an
LC-MS/MS procedure as the reference method for the quantification of lipophilic marine biotoxins,
namely okadaic acid, pectenotoxin 2, azaspiracid 1, and yessotoxin. Moreover, dinophysistoxin 1
(DTX-1) and dinophysistoxin 2 (DTX-2) can be quantified by the calibration curve of okadaic
acid, pectenotoxin 1 by the calibration of pectenotoxin 2, azaspiracid 2 and 3 by the calibration
of azaspiracid 1 and 45-OH-, and 45-homo-OH-yessotoxin by the calibration of yessotoxin.
The purpose of this document is to describe the possible alternatives for analysis of marine
biotoxins in various samples, applying LC-MS based either on triple quadrupole or high-resolution
accurate-mass (HRAM) mass spectrometry (MS).
From Mouse Bioassay to Techniques of the 21st Century
Besides the mouse bioassay test, triple quadrupole LC-MS/MS methods have been the most
commonly used detection techniques for marine biotoxins, providing high sensitivity and selectivity.
However, this technique requires detection of compounds that are pre-selected and fine tuning of
system parameters in order to detect low concentrations in complex matrices. Despite the lack of
capabilities to screen for larger number of compounds, unknown toxins or new metabolites of
known substances, LC and UHPLC-MS/MS have been used successfully in many routine labs.
Recently, HRAM MS has introduced new advantages to residual analysis. Due to the fact the data
acquisition is always performed in the full-scan mode, no special parameter setup or tuning for
specific compounds is needed. The selectivity and sensitivity of the method is achieved via post-
processing of the data using extraction of accurate mass data from the full scan records. By doing
so, selectivity comparable to or better than with triple quadrupole LC-MS/MS technology is
obtained.
3
In addition, post-processing allows for retrospective analysis in order to search for new,
emerging toxins or their metabolites. It also helps to eliminate false positive and false negative
results by providing additional, confirmatory information.
Several types of mass spectrometers operating at high resolving power and providing accurate mass
information have been introduced in the past. The most recent and most advanced instruments are
based on the Thermo Scientific
™
Orbitrap
™
mass analyzer, originally developed by Makarov and
colleagues.
4
The improvements introduced by Orbitrap technology, such as ease of use, mass axis
accuracy and stability, and ultra-high resolving power, have encouraged the adoption of HRAM
systems even in routine laboratories.