© Benaki Phytopathological Institute
Kasiotis & Machera
36
entails a denaturation stage that is accom-
plished by heating or the use of an organ-
ic solvent. After the solvent addition (usually
acetonitrile (ACN) or methanol), the organ-
ic phase is separated from the protein by
cyclomixing and centrifugation (Kole
et al
.,
2011). Kole
et al.
(2011) have also reviewed re-
cent advances in sample preparation in the
bioanalysis domain including all available
preparation steps prior to analysis.
A last but imperative parameter is the
validation of the analytical methods. In the
bioanalysis field, several validation guide-
lines and protocols (Bioanalytical-Validation;
ICH, 2005) are adopted, which deal with par-
ticular validation parameters such as the
limit of detection and quantification (LOD
and LOQ), accuracy and precision. Matrix ef-
fects are also critical, considering the com-
plexity of matrices, from which the analyst
has to selectively extract the compounds of
interest. A Belgian group reviewed succes-
sively these effects in bioanalytical meth-
ods, and proposed solutions to reduce or
eliminate matrix interferences (Van Eeck-
haut
et al
., 2009).
A critical feature that deserves attention
is the
in vivo
metabolism of NNDs. It is ac-
knowledged that the insertion of chemicals
into humans’ body is accompanied by sev-
eral reactions that occur and usually break-
down the parent compounds to smaller
molecules. Breakdown products should not
be neglected, since it is well reported that
some of these molecules exhibit significant
toxicity in several organisms that can sur-
pass those of parent molecules (Nauen
et
al
., 2001; la Farre
et al
., 2008). Metabolism of
NNDs is extensive, including several meta-
bolic products produced by reactions such
as reduction, demethylation, hydroxylation,
and olefin formation. Hence, it is practically
impossible to incorporate all metabolites in
a targeted analytical method since many of
them are not commercially available. In the
group of NNDs, 6-chloronicotinic acid (6-
CNA) is a common metabolite for IMI, NITEN,
THIAC and ACET, considering that the latter
share the chloropyridinyl moiety in their
structure, and therefore it is widely includ-
ed in analytical methods. CLOTH and THI-
AM contain the chlothiazole core and one
of their key metabolites is 2-chloro-1,3-thi-
azole-5-carboxylic acid (2-CTCA). Finally, DI-
NOT that contains the furanyl moiety is con-
verted to 3-furoic acid (3-FA). Fundamental
in vivo
metabolites of NNDs are depicted in
Figure 2.
Biomonitoring Studies
Of the first works published on NNDs
and their metabolites was that of Uroz
et al.
(2001). The authors developed an analyti-
cal method for the monitoring of 6-CNA in
human urine by gas chromatography tan-
dem mass spectrometry (GC-MS/MS). Sam-
ple preparation consisted of acidification of
urine and heating (for deconjugation to take
place) and then the passage of the resulting
mixture through an Amberlite XAD-4 car-
tridge. Amberlite XAD-4 resin, which is a
polymeric sorbent with adsorbing potency
for hydrophobic molecules, was selected af-
ter comparison with octadecyl carbon chain
material (C18). Clean up was achieved with
water of low pH and hexane. Then, 6-CNA
was eluted with diethyl ether. Recoveries
were optimum with acidic pH, although
very low pH would decrease the resin’s ad-
sorption capacity. Though, 6-CNA is a mol-
ecule that is more compatible with liquid
chromatography (LC), the authors choose
gas chromatography. Hence, the dry sample
was reconstituted in hexane and subjected
to derivatization with a hexafluoroisopro-
panol (HFIP) using a carbodiimide as a cou-
pling agent. After neutralization of the ex-
tract, the latter was injected into the GC-MS/
MS system. Analysis time was short (6 min),
monitoring the precursor ion of derivatized
6-CNA and several daughter ions. LOD of
the method was determined to 16 pg/mL,
which is the lowest reported in the litera-
ture for 6-CNA (LODs of NNDs and metabo-
lites for selected works incorporated in this
review are presented in Table 1). Finally, the
application of the method to five urine sam-
ples of agricultural workers did not disclose
the presence of 6-CNA.
Taira
et al.
(2011) reported 6-CNA pres-
