© Benaki Phytopathological Institute
Kasiotis & Machera
38
ing method since it exhibited acceptable re-
covery for 6-CNA. Confirmatory LC-MS was
applied only to positive samples. Samples
prior to chromatography were ultra-filtrated
to remove proteins. LOD for the IC method
was 0.4 mg/L
urine
.
Regarding the LC-MSmethodology, stan-
dard reversed phase separation was per-
formed. Selected ion monitoring (SIM) was
used to quantify 6-CNA, using the [M+1]
+
as
quantitation ion. The LOD of the method
was 2 μg/L
urine
. Maximum 6-CNA concentra-
tions ranged from 7.5 to 84.8 μg/L
urine
. The
origin of 6-CNA was attributed to the ex-
cessive intake of tea beverages and conven-
tionally grown fruits. Nevertheless, the au-
thors did not present an analytical study on
NNDs’ and metabolites’ detection in food
commodities, so as to strengthen the men-
tioned statement.
The same group two years later investi-
gated NNDs metabolites in human urine of
3 patients suspected of subacute exposure
to NNDs (Taira
et al
., 2013). This work came
to fill gaps of the previous work, such as the
non-inclusion of other metabolites of key
members of NNDs family (showing select-
ed metabolites of IMI, ACET, and CLOTH). To
proceed to this study, a qualitative step was
adapted aiming to unveil possible metabol-
ic products. The latter was accomplished
by liquid chromatography time-of-flight
mass spectrometry (LC-TOF/MS). TOF/MS is
known for its inherent advantages, which
are: sensitivity, high mass range, and high-
speed analysis. TOF/MS functioned using a
database of nominal molecular weights of
57 known metabolites of the 3 NNDs of the
study. For ACET, the dominant metabolite
was
N
-desmethyl-acetamiprid (N-desAC-
ET) that until this work was reported only in
rats’ biological fluids. Subsequently, quan-
titation of TOF/MS identified compounds
was performed with LC-MS/MS. Human
urine samples were solid-phase extracted,
and 10-folds concentrated. After loading
urine samples in preconditioned cartridges,
a wash step with H
2
O was applied, and an-
alytes were extracted with ACN. Acidic and
basic extraction was also used utilizing 1.25
μL of formic acid and 10 μL of an ammonium
hydroxide solution, respectively. Data anal-
ysis was based on data mining approach,
which is a computerized procedure used to
unveil patterns in large data sets. An almost
50% of nominated compounds were de-
tected in positive controls by this screening
methodology. Acidic SPE conditions exhibit-
ed the highest retentionwith only two unde-
tectable substances. The qualitative TOF/MS
analysis of human urine confirmed the pres-
ence of 8 metabolites, including N-desAC-
ET and 5-OH-IMI. The highlight of this work
was the first report on N-desACET detection
and quantitation in human urine at 3.2 ng/
mL (one sample) that indicated human ex-
posure to ACET. Taira has also reviewed the
suspected health effects as a consequence
of NNDs exposure in Japan, focusing on in-
halation and oral exposure (Taira, 2014).
Nomura
et al.
(2013) published work on
the detection and quantitation of NNDs’
metabolites in human urine using GC-MS.
Metabolites studied were 6-CNA, 2-CTCA
and 3-FA. The method was validated after
optimizing particular parameters. More spe-
cifically, a hydrolysis step was applied in or-
der deconjugation to take place. Deconju-
gation was tested under acidic and basic
conditions. It was found that addition of 50
μL of sulfuric acid was the optimum condi-
tion for deconjugation of the 3 NNDs. Then
SPE was applied, by eluting compounds
from preconditioned cartridge with metha-
nol after column washing with 0.5 mL of 2%
formic acid. SPE was performed on polymer-
ic strong cation exchange column, also char-
acterized by its non-polar retention mecha-
nism. Methanol was chosen instead of ACN
due to the observed minimization of inter-
ferences in the chromatogram. Division of
the eluate was performed in order to pro-
ceed in separate analysis for 2-CTCA, and
3-FA and 6-CNA respectively. Derivatization
of the analytes with trimethylsilyl group (a
typical group used for such reactions) using
BSTFA-TMCS proceeded smoothly for all tar-
get compounds. For the assessment of re-
covery, spiking was conducted at two dif-
ferent stages: initially at the beginning of
