Fig. 3a and b shows the effect of ACN concentration on the peak
height of the studied OPPs.Anincrease of the organic modifier in the
sample mixture caused a constant peak height decrease for fenamifos,
fonofos and profenofos, while for fensulphothion, isofenfos,
dialifos, sulprofos and prothiofos the reduction was observed at
ACN concentrations higher than 10%. The slightly different behavior
of the five OPPs can be attributed to their relatively low water
solubility (<1g/mL). In fact the response of these compoundswas
lower when 100% of water was used, and was enhanced when the
water amount was reduced to 90%.
Although lower percentages of water increased the analyte solubility,
it must be added that a decrease of the water amount had
a negative effect on focusing. On the contrary, as the percentage
of water in the sample increased, the affinity of the solutes for the
stationary phasewas higher and initial band broadening, due to the
large sample volume injected, was avoided due to the formation of
a narrowplug at the column head. As a consequence, further experiments
were carried out using 90% of water, representing the best
compromise for all the investigated analytes.
Fig. 4 illustrates the different chromatographic profiles attained
changing the sample dilution solvent and injected volumes. A
great difference in the UV response and resolution was obtained
injecting a 1L amount of sample dissolved in 90/10 or in 50/50
H2O/ACN (Fig. 4a and b, respectively). In particular, the band broadening
occurring in the second case was so dramatic that peak
resolution was nullified. Comparing Fig. 4c and a, the sensitivity
enhancement is evident when the injection volume was increased
from 60 nL (without on-column focusing) to 1L (with on-column
focusing).
It must be noted that in order to make this method suitable for
trace analyses, the OPPs concentration in the samplewas decreased
from 50g/mL (concentration considered at the beginning of the
study, as indicated in Fig. 2) to 1g/mL. As can be observed in
Figs. 2 and 4c, a concentration decrease caused a proportional
signal reduction; increasing the injection volume and exploiting
on-column focusing, sensitivitywas enormously improved, providing
limits of detection of about 100-fold lower (see LOD values in
Table 3).
Fig. 4. Effect of the organic solvent and injected volume on the injection plug,
chromatography and sensitivity: (a) mixture of OPPs (1g/mL) diluted in 90/10
H2O/ACN (v/v), injection volume: 1L; (b) mixture of OPPs (1g/mL) diluted in
50/50 H2O/ACN (v/v), injection volume: 1L; (c) mixture of OPPs (1g/mL) diluted
in 50/50 H2O/ACN (v/v), injection volume: 60 nL. Capillary column: PinnacleTM
II Phenyl (100m ID, 25.0cm packed, 25.5cm effective length). Mobile phase:
ACN/H2O, 60/40 (v/v). Flow rate: 360 nL/min. Detection wavelength: 205 nm.
On the basis of the results attained, all further experiments
were performed using the chromatographic conditions reported in
Table 1, viz., injecting 1L sample volumes, and employing a 90/10
H2O/ACN (v/v) mixture.
3.3. Sample preparation
Selected food samples were fortified with the OPP mixture and
subjected to a preliminary extraction procedure; an additional
purification stepwas achievedprior to nano-LCanalysis. The extraction
procedure described in Section 2.4 was carried out by using
EtAc. The choice of this solvent, instead of acetone used in the
original procedure [21], orACNemployed in multi-residue determination
methods [32],was made considering the several advantages
of this solvent over the others. As well known, EtAc is practically
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