© Benaki Phytopathological Institute
Neonicotinoids Biomonitoring: A review
39
the extraction and then before derivatiza-
tion. The calibration curve was constructed
in the range of 0.6 to 10 μg/L, using pooled
urine, exhibiting correlation coefficients for
all analytes above 0.99. In the same context,
within-run precision was determined at five
concentration levels, with acceptable % rel-
ative standard deviation (RSD) values. Be-
tween-run precision was assessed similarly
at 0.6 and 5 μg/L for five consecutive days,
with RSD% below 13%. LOD and LOQ were
calculated using the signal to noise (S/N) ra-
tio of 3 and 10 in respect and were 0.1 and
0.3 μg/L, correspondingly. Application of
the method to real samples unveiled a high
frequency of detection for 3-FA, which is at-
tributed to the frequent use of DINOT in Ja-
pan. Even though the presence of CLOTH is
unambiguous in agricultural commodities
in Japan, its metabolite 2-CTCA displayed
low detection rate. The latter, as the authors
state is unclear. Thus, it can be a challenge
for future endeavors. Overall mean concen-
trations were 1.8 and 2.6 μg/L for 6-CNA and
3-FA, respectively. 2-CTCA was detected
only in one farmer at 0.1 μg/L.
One year later, and as a compendium
of their previous work, Ueyama
et al.
(2014)
dealt with urinary NNDs metabolites. The
main principal of this work was to focus
solely on the urinary metabolites, overcom-
ing overestimation of concentrations result-
ing from dietary intake. Consequently, they
developed a straightforward method for si-
multaneous determination of urinary NNDs
using LC-MS/MS. Sample preparation be-
gan with acidification of urine sample and
addition of internal standard (IS). Then, the
urine sample was incubated and applied to
SPE. After conditioning, and a washing step,
the majority of analytes were eluted with
MeOH. NITEN and the IS were finally eluted
with MeOH:ACN that contained 5% of am-
monia (NH
3
) solution. The use of NH
3
aided
the elution of NITEN that exhibits high ion-
ic binding to the SPE material. LODs varied
from 0.01 to 0.12 μg/L. The concentration re-
sults showed that the Japanese population
was exposed to NNDs. In particular, the de-
tection frequencies were higher than 50%
for all analytes, excepting NITEN. Overall,
the authors pointed out two limitations. The
first regarded the use of only one IS, and the
second the difficulty to identify NNDs peak
near LOD.
Yamamuro
et al.
(2014) developed a nov-
el analytical method, for detecting NNDs
in serum and urine. Until then most works
on NNDs and metabolites were concentrat-
ed either in environmental or food samples
that might contain NNDs or in biological
fluids but with limited number of analytes.
Therefore, this work came to fill this gap
since it dealt with almost all NNDs. In addi-
tion, the authors included three ACET me-
tabolites. The sample preparation step was
simple. A low volume of sample was dilut-
ed with water and then purified, through a
cartridge containing diatomaceous earth.
This step although it seems as an SPE step,
it works via LLE that occurs among the elu-
ate (chloroform: isopropanol, 3:1), and a gel
formed on the diatomaceous earth surface.
Acceptable analytical performance was ob-
tained only when elution was repeated with
ten portions of low volumes (3 mL each) of
the mentioned solvent mixture. The opti-
mum mobile phase was a pH 3-buffered
methanol, which provided substantial sen-
sitivity, except FLON. Linearity of the calibra-
tion curve for each analyte was studied over
a range of concentrations, starting from the
LOQup to 1 μg/mL. All correlation coefficient
values were above 0.99, thus acceptable. Ex-
tensive validation of the method proved its
efficacy and robustness. Sensitivity was sub-
stantial as depicted by the respective LOD
values (serum 0.1-0.2 ng/mL, urine 0.1-1 ng/
mL). It is foreseen that this approach can be-
come a useful vehicle in forensic laborato-
ries, which investigate human poisoning in-
cidents with NNDs.
Jamin
et al.
(2014) published a cutting-
edge work, in an untargeted profiling of
pesticide metabolites in urine from preg-
nant women from a French epidemiological
cohort. To carry out such profiling, the au-
thors generated a pesticide metabolite list
based on the likelihood of pesticide use in
the study area. Analysis was accomplished
