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In the past, aside from bioassays on mice, most analytical
techniques developed for the determination of marine
biotoxins in bivalve molluscs have been based on offline
methodologies. These include methods involving solid
phase extraction (SPE) or liquid-liquid extraction (LLE)
followed by high pressure liquid chromatography
(HPLC) with fluorimetric or UV-diode array detection,
as well as detection by liquid chromatography coupled
with mass spectrometry (LC-MS).
The EU Commission Regulation (EC) No 15/2011,
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.
2,3
Moreover, dinophysis-
toxin 1 (DTX-1) and dinophysistoxin 2 (DTX-2) can be
quantified by the calibration curve of okadaic acid,
pectenotoxin 1 by calibration of pectenotoxin 2,
azaspiracid 2 and 3 by calibration of azaspiracid 1 and
45-OH-, and 45-homo-OH-yessotoxin by the calibra-
tion of yessotoxin.
In accordance with current European regulations, we
propose a quick, selective, sensitive, and accurate
analytical method for the determination of lipophilic
marine biotoxins in bivalve molluscs using an
LC-MS/MS method.
Goal
Our goal is to validate analytical procedures proposed in
“
EU-Harmonised Standard Operating Procedure for
determination of Lipophilic marine biotoxins in
molluscs by LC-MS/MS – Version 3
” by LC-MS/MS
using offline extraction.
4
Experimental
Sample Preparation
About 1 kg of bivalve molluscs (
Mytilus Galloprovincialis
)
were cleaned with water and put in a solution of NaCl
(3.5 g/L). After opening, the molluscs were washed with
fresh water, their flesh was removed and placed on a
stainless steel net, and they were washed again with
deionized water. The whole collected raw tissue, not less
than 150 g, was chopped and blended by a mixer.
Extraction procedure
9 mL of 100% methanol (gradient quality) were added
to 2.00 ± 0.05 g of blended tissue, put into a centrifuge
tube, and mixed by vortex for 3 minutes at maximum
speed. After centrifugation at 4000 rpm for 10 minutes,
the supernatant solution was transferred into a vial.
A second aliquot of 9 mL of 100% methanol was further
added to the residual tissue pellet and homogenized for
1 minute by Ultra-turrax
®
(
IKA
®
, USA
) at 12,000 rpm and
the mixture was centrifuged at 4000 rpm for 10 minutes.
Then the supernatant solution was transferred and
combined with the first extract and made up to 20 mL
with 100% methanol. When not immediately analyzed,
the solution was stored at -20 °C.
Spikes of toxin standard solutions can be added to the
blended tissue before the extraction procedure.
Purification Procedure
The organic extract was purified by being passed
through a C18 SPE cartridge preliminarily conditioned
with 1 mL of 100% methanol. A 0.45 μm syringe filter
was placed at the end of the cartridge to improve
purification.
LC Conditions for the Thermo Scientific Hypersil
GOLD Column
System
Thermo Scientific Accela UHPLC
Solvent A
100% water with 2 mM ammonium formate
and 50 mM formic acid
Solvent B
95% acetonitrile + 5% water with 2 mM
ammonium formate and 50 mM formic acid
Flow Rate
200 µL/min
Gradient
The mixture started at 30% solvent B
(8.0 min) followed by a linear gradient up to
90% solvent B in 3.0 min. It went up to 30%
of solvent B in 0.5 min. This composition was
maintained for 5.5 min.
Analytical Column Hypersil GOLD
™
; 50
×
2.1 mm, particle
size 1.9 μm, part number 25002-052130
H-ESI II Source Conditions
Ion Source Polarity
Positive Ion Mode Negative Ion Mode
Spray Voltage
3000 V
2700 V
Capillary Temperature
270 °C
270 °C
Vaporizer Temperature
240 °C
240 °C
Sheath Gas Pressure (N
2
)
15 units
15 units
Auxiliary Gas Pressure (N
2
)
5 units
5 units
MS/MS Setup
MS analysis was carried out on a Thermo Scientific TSQ
Quantum Ultra triple quadrupole mass spectrometer
equipped with a heated electrospray ionization probe
(H-ESI II).
Collision Gas (Ar)
1.5 mTorr
Q1/Q3 Peak Resolution
0.7 u (unit mass resolution)
Scan Time
0.100 s
Scan Width
0.500
m/z
Data Acquisition Mode
SRM