Page 30 - Volume 4, Issue 2, July 2011

© Benaki Phytopathological Institute
Thiram Bioaccumulation in Mytilus galloprovincialis 59
sil SPE cartridge (Waters, SEP-PAK® Cartridg-
es), filtered (Whatman, PuradiscTM 25 TF fil-
ters, 0.45 μm), evaporated under a gentle
stream of nitrogen, reconstituted with ac-
etonitrile (0.5 mL) and 10 μL were injected
to Liquid Chromatograph Mass Spectrome-
ter (LC/MS).
Experimental Procedure for Extraction of
Thiram fromWater Samples
An aliquot of 2 mL of water sample was
mixed with 2 mL of ethyl acetate (vortex, a
MS1 Minishaker, IKA) for 2 minutes. Then the
mixture was placed in an appropriate falcon
tube (15 mL) and centrifuged at 4000 rpm
for 5 min, at 4°C. The organic layer was col-
lected, filtered (Whatman, PuradiscTM 25 TF
filters, 0.45 μm), evaporated under a gentle
stream of nitrogen, reconstituted with ace-
tonitrile (0.5 mL) and then an amount of 10
μL was injected for analysis to the Liquid
Chromatograph Mass Spectrometer.
Liquid Chromatography Mass Spectrometry
A Shimadzu (Kyoto, Japan) LCMS-2010 EV
Liquid Chromatograph Mass Spectrometer
instrument was used with the LCMS solution
version 3.0 software consisting of a SIL-20A
prominence autosampler and an SPD-M20A
diode array detector. The latter were cou-
pled in series with a mass selective detec-
tor equipped with an atmospheric pressure
ionization. The LC separation was achieved
working in positive Electron Spray Ioniza-
tion (ESI) mode, on a Shim-Pack XR-ODS 2.2
μm, 100×4.6 mm i.d. chromatographic col-
umn using a gradient system consisting of
methanol and water. The flow rate was set at
0.8 mL min -1 and the column gradient pro-
gram consisted of 40% methanol and 60%
water, ramped linearly over the course of 7.5
min at 70 % methanol. Then methanol re-
turned in the course of 2 min at 60% con-
centration and the mobile phase was held at
that composition from 9.5 min to 15 min.
Validation procedure
For the validation procedure the follow-
ing parameters were determined: linearity,
repeatability, reproducibility, analytical Lim-
it of Detection (LOD) and Limit of Quantifi-
cation (LOQ), recoveries and matrix depen-
dent variations as it is established by the EU
guidelines. Linearity and matrix effect were
assessed by analyzing standard solutions
and matrix matched standards at six points
in the range of 0.20-7.88 μg mL-1 to cov-
er the expected range of concentrations in
samples. Recovery values derived from for-
tified experiments at two levels of concen-
tration and were considered as the measure
of the trueness of the analytical method. For
repeatability and reproducibility studies of
the LC-MS methodology, five replicate de-
terminations on the same day and on five
different days of a standard solution (0.78
μg mL-1 of thiram) were performed. Repeat-
ability and reproducibility is considered ac-
ceptable when relative standard deviation
values (RSD%) are < 20%. Based on the sta-
tistical definition 3.3 (SY/x )/α and 10(SY/x)/α, of
the LOD and LOQ respectively, they were de-
termined at the concentration levels rang-
ing from 0.20 to 7.88 μg mL-1. SY/x represents
the residual standard deviation and α is the
slope of the respective calibration plot.
Tissue preparation for biochemical as-
says
The three main tissues of mussels (gill,
digestive gland and haemolymph) were
extracted from sacrificed animals after the
end of the experiment. Briefly, for gills, the
valves of the mussel were fully opened with
a metallic scalpel and wet gill tissue (< 0.2
g) was extracted. The gill was gently disag-
gregated in HEPES buffered saline (2 mL).
After centrifugation a fraction of the pel-
let was rediluted in saline (100 μL) and kept
on ice until further processing. For haemo-
lymph the valves of the mussel were slight-
ly opened and haemolymph was abstracted
from the posterior adductor muscle accord-
ing to Rank and Jensen (36) with a 23G nee-
dle and a 1 mL syringe (PiC Insumed, Italy).
Approximately 100 μL were abstracted from
each animal and used without further treat-
ment. Digestive gland suspension prepa-
ration was performed according to Birmel-
in et al. (8) with minor modifications. Briefly,