Page 36 - Volume 2,Issue 2, July 2009

© Benaki Phytopathological Institute
Anagnostopoulos et al. 82
criminates efficiently between the analyte
and the matrix signal. Existing quality con-
trol procedures require qualitative confir-
mation of positive results for LC–MS/MS,
preferably via a second MS/MS transition (5).
With the present analytical scope of 56 pes-
ticides, this results in monitoring 112 tran-
sitions. Stacking more transitions within a
time window drastically reduces the num-
ber of data points across a chromatographic
peak leading to unsatisfactory peak shapes.
Therefore, in the first injection, only one
transition is recorded, the quantification one,
and a second injection of the sample is thus
required with both transitions only for the
compounds that give a positive result; in this
way the false positive results are minimized.
The quantification transition, which in all cas-
es is the one with the highest signal to noise
(S/N) ratio, is selected for screening in order
to minimize the false negative results.
2. MS optimization
Individual standard solutions at 0.01 mg/
ml were prepared in methanol/water (30:70
v/v) and infused into the mass spectrometer
in order to obtain the optimum values for
the cone and capillary voltage for each ana-
lyte. The optimum collision cell energy volt-
age was found to vary between 6 and 81 kV,
depending on the analyte. Product ion mass
spectra for the pesticides were obtained in
the positive mode electrospray ionization
using collision induced dissociation (CID).
Variation of the collision energy influenc-
es both sensitivity and fragmentation. The
collision energy was optimized for two se-
lective product ions of each precursor ion.
The optimization values obtained are listed
in Table 2.
The time–scheduled data acquisition
sequence involved five overlapping seg-
ments, with 8 to 29 transitions each. Table 2
shows the distribution of the transitions into
5 time windows based on analyte retention
times. With dwell times of 100 msec the av-
erage scan cycle time for the segments var-
ied between 0.8 and 2.7 sec. Still, a sufficient
number of data points can be acquired over
the chromatographic peak in order to have
enough sensitivity and allow reproducible
peak area integration for good quantitative
results.
3. LC optimization
Regarding the LC separation, the gradi-
ent was optimized in order to have a sepa-
ration of the 56 selected analytes. Using a
dwell time of 100 msec per transition, we
obtained satisfactory peak shape for all an-
alytes under study. By distributing the an-
alytes along five overlapping segments,
the chromatographic peak was allowed to
be centered in the time window, minimiz-
ing the risk of peak loss due to unexpected
slight changes in retention time.
4. Validation
The validation study carried out was
based on the European SANCO guidelines
(5), and the method was evaluated for its
sensitivity, accuracy, precision, linearity and
specificity. This requires performing recov-
ery experiments with spiked blank peach
samples to estimate the accuracy of the
method. A minimum of five replicates is re-
quired to evaluate precision as well as the
reporting limit (to assess sensitivity) and at
least another higher level (5). In our study,
experiments with fortified peach samples
were performed at three fortification lev-
els (0.01, 0.05 and 0.5 mg/kg), with five repli-
cates at each level.
Linearity was studied using matrix-
matched calibration standards at six con-
centration levels within the range of 0.01 to
0.75 μg/ml and three replicates at each level.
The approximate value of uncertainty (S u) in
the predicted value C (mg/kg) due to the
variability in the Area resulting from the use
of the calibration curve was also estimated
by the following equation:
Where
( )
2n
y y
S
2
i
i
C/ Area -
- ∑
=
, n is the number
of the data points in the calibration,( )
y y
i − is the residual for the i th point and
b
S
S C/ Area
u =