Journal of Chromatography A, 1216 (2009) 4539–4552

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage:www.elsevier.com/locate/chroma

Method validation and comparison of acetonitrile and acetone extraction for the analysis of
169 pesticides in soya grain by liquid chromatography–tandem mass spectrometry

a,∗ b b c c
Ionara R. Pizzutti , André de Kok , Maurice Hiemstra , Cristine Wickert , Osmar D. Prestes
aFederal University of Santa Maria, Chemistry Department, Center of Research and Analysis of Residues and Contaminants (CEPARC), Santa Maria, RS, Brazil
b VWA – Food and Consumer Product Safety Authority, Chemistry Laboratory, R&D Group, National Reference Laboratory (NRL) for Pesticide and Mycotoxin Analysis in Food,
Amsterdam, The Netherlands
c
Federal University of Santa Maria, Chemistry Department, Laboratory of Pesticide Residue Analysis (LARP), Santa Maria, RS, Brazil

article info abstract

Article history: An acetonitrile-based extraction method for the analysis of 169 pesticides in soya grain, using liquid chromatography–tandem
Received 20 October 2008 mass spectrometry (LC–MS/MS) in the positive and negative electrospray ion-ization (ESI) mode, has been optimized and
Received in revised form 23 March 2009 validated. This method has been compared with our earlier published acetone-based extraction method, as part of a
Accepted 24 March 2009 Available online 28
comprehensive study of both extraction meth-ods, in combination with various gas chromatography–(tandem) mass
March 2009
spectrometry [GC–MS(/MS)] and LC–MS/MS techniques, using different detection modes. Linearity of calibration curves,
instrument lim-its of detection (LODs) and matrix effects were evaluated by preparing standards in solvent and in the two
Keywords:
soya matrix extracts from acetone and acetonitrile extractions, at seven levels, with six replicate injections per level. Limits of
Pesticides
Soya grain detection were calculated based on practically realized repeatability rela-tive standard deviations (RSDs), rather than based on
Multi-residue method (extrapolated) signal/noise ratios. Accuracies (as
Extraction
LC–MS/MS % recoveries), precision (as repeatability of recovery experiments) and method limits of quantification (LOQs) were
compared. The acetonitrile method consists of the extraction of a 2-g sample with 20 mL of acetonitrile (containing 1% acetic
acid), followed by a partitioning step with magnesium sulphate and a subsequent buffering step with sodium acetate. After
mixing an aliquot with methanol, the extract can be injected directly into the LC–MS/MS system, without any cleanup.
Cleanup hardly improved selectivity and appeared to have minor changes of the matrix effect, as was earlier noticed for the
−1 2
acetone method. Good linearity of the calibration curves was obtained over the range from 0.1 or 0.25 to 10 ng mL , with r
−1
≥ 0.99. Instrument LOD values generally varied from 0.1 to 0.25 ng mL , for both methods. Matrix effects were not
significant or negligible for nearly all pesticides. Recoveries were in the range 70–120%, with RSD ≤ 20%. If not, they were
−1
still mostly in the 50–70% range, with good precision (RSD ≤ 20%). The method LOQ values were most often 10 g kg for
almost all pesticides, with good repeatability RSDs. Apart from some minor pros and cons, both compared methods are fast,
efficient and robust, with good method performances. The two methods were applied successfully in a routine analysis
environment, during surveys in 2007 and 2008.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Nowadays, consumers tend to change their food consumption habits
towards a preference for healthy fruits and vegetables pro-viding also the
right nutritional value. The benefits of soybean [Glycine max (L.) Merrill] and
the importance to include this legume and its products in the daily diet have
been very well known by the Chinese people since more than 2000 years B.C.
In the beginning of the 20th century, the main properties of soya, the high
content
∗ grain in the world, after China.
Corresponding author. Tel.: +5555 3220 9458; fax: +5555 3220 9458. E-mail address:
pizzutti@quimica.ufsm.br (I.R. Pizzutti).
of oils and proteins, called the attention of the occidental people that started to
use this grain not just for consumption, but also for industrial and technical
applications, such as amongst others, in the areas of cosmetics, textiles, inks,
disinfectants, fertilizers and bio-fuel. However, the applications of soybean for
food and feed are still the most important [1].
Soybean belongs, together with wheat, rice, corn and potatoes, to the five
most important food and feed crops in the world. Brazil is the second biggest
producer of soya grain, but the first exporter. Seventy-five percent of the total
Brazilian production of soybean is exported, mainly to China and the
European Union [2]. The Nether-lands is the second biggest importer of soya

0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2009.03.064
 4540 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552

Taking into account the continuous increase in consumption of all

products originating from soybean and other grains, legislation was
established recently [3] in order to keep pesticide residues at the lowest
possible levels, and guarantee food and feed safety. Since 1 September 2008,
a new European Union regulation became into force setting around 145,000
harmonised maximum residue levels (MRLs) for the tens of thousands
commodity–pesticide combina-tions [4]. Compliance with these MRLs
requires an efficient control via regulatory monitoring programs and
enforcement, analysis of raw and processed food and feed [5], risk
assessment, field-application trials, organic food verification [6] and border
control for international trade of food and feeding stuffs [7].

The development of sensitive, selective and reproducible analyt-ical

methods and techniques has always been a prerequisite for the achievement of
high-quality results in enforcement and monitor-ing programmes. Nowadays,
other characteristics of an analytical quantitative method are also requested,
such as usage of the small-est possible amount of sample and chemicals, more
environmental friendly approaches, safer and less hazardous chemicals to the
ana-lysts, cost-effective, less laborious and faster methods [8–10].

Until recently, most improvements in multi-residue methods for pesticides

have been achieved for crops with a high water con-tent, like fruits and
vegetables. In the last years, research has now also been directed towards
analysis of pesticide residues in more complex matrices, such as grains and its
derived products, and matrices with a high fat and sugar content. These latter
matrices are now gradually becoming less difficult, thanks to the newest, more
advanced chromatographic detection techniques [11–15].

Classical analytical methods based on acetone [16], acetonitrile

[17] and ethyl acetate [18] extraction have been continuously opti-mized and
modified in such a way to improve considerably their performances and
applications for pesticide residues analysis, also for the more complex
matrices mentioned above.
Recent versions of the acetone [12,13] and acetonitrile [19] methods and
its many modifications and applications [20–24] and also methanol-based
[25] and ethyl acetate-based [26] methods have achieved the state-of-the-art
stage of multi-residue methods for pesticide residues.
As follow-up of our previous study [12] on the development of an
acetone-based extraction method, this study aimed at the opti-mization and
validation of an acetonitrile-based extraction method
[27] for the analysis of 169 pesticides in soya grain by LC–MS/MS using
both the positive and negative ESI ionization mode.
The results for the validation parameters, such as matrix effect, accuracy,
precision, linearity range, detection and quantitation lim-its, sensitivity and
selectivity, were compared for the two methods.
2. Experimental
In this study, soya extract 1 means the extract obtained by the acetone
extraction [1,12] method (Fig. 1) and soya extract 2 means the extract
obtained by the acetonitrile extraction method described in Section 2.5 of this
paper and also shown schematically in Fig. 2. For both methods cited, no
clean up procedure was applied for analysis by LC–MS/MS.
2.1. Chemicals and reagents
Fig. 1. Scheme of the acetone extraction method for the determination of pesticides in soya
grain.
(Philadelphia, PA, USA), Cyanamid (Princeton, NJ, USA) and DuPont
(Wilmington, DE, USA).
2.2. Standard solutions
Standard stock solutions were prepared at 1.00 mg mL
−1
mainly in
toluene. However, for those compounds with solubility problems in toluene,
methanol or acetone were used. A stock standard mix-ture solution containing
−1
all 169 pesticides studied, was prepared at 1 g mL of each pesticide, in
methanol (0.1% acetic acid). This solution was used as spiking solution and
also to prepare the stan-dard solutions to obtain the calibration curves from the
pesticides in solvent.
Blank soya extracts (1 and 2) were used for preparation of standard
solutions in matrix. Hence, all standard solutions were prepared in solvent, in
soya extract 1 (acetone extraction) and soya extract 2 (acetonitrile extraction)
in order to evaluate the matrix effect for each extraction method. The
standards in matrix extract (matrix-matched standards) were used for the
calculation of recov-eries.

Methanol, toluene, acetone, acetonitrile (pesticide grade), and anhydrous
magnesium sulphate and sodium acetate (analytical reagent grade) were
purchased from Merck (Darmstadt, Germany). Pesticides reference standards
(purity > 97%) were obtained from Dr. Ehrenstorfer (Augsburg, Germany);
Riedel de Haën (Seelze, Germany); Hayashi (Osaka, Japan), Bayer
(Leverkusen, Germany); Zeneca (Wilmington, DE, USA); Janssen (Beerse,
Belgium), Novartis (Basel, Switzerland), Agrevo (Frankfurt, Germany),
Rohm and Haas
2.3. Analytical instrumentation
The chromatographic system consisted of a Waters Alliance 2695
separation module (Milford, MA, USA), equipped with a qua-ternary solvent
delivery system, degasser, autosampler and column heater. An Alltima C 18
analytical column (150 mm × 3.2 mm I.D., 5 m particle size) from Alltech
(Deerfield, IL, USA), fitted with a C18 guard column (4 mm × 3.0 mm I.D.)
purchased from Phenomenex
 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552
Fig. 2. Scheme of the acetonitrile extraction method for the determination of pes-ticides in soya
grain.
(Torrance, CA, USA) and kept in a column heater (at a constant temperature
◦
of 35 C), were applied to perform chromatographic separations.
A Waters Quattro Ultima tandem mass spectrometer (Manch-ester, UK),
equipped with an electrospray ionization (ESI) interface (Z-spray), operating
in the positive or negative ion mode, was the detection system used.
2.4. Chromatographic and mass spectrometric conditions
For the ESI negative ionization mode, an isocratic run was per-formed
during a total time of 10 min, using a mobile phase (flow rate at 0.3 mL
−1 −1
min ) constituted of 90% methanol in 5 mmol L ammonium formate. A
divert valve was placed between the ana-lytical column outlet and the mass
spectrometer inlet, and during the first 1.0 min of the chromatographic run the
flow was diverted to waste.
For the ESI positive ionization run mode, the gradient program started at
−1
25% methanol in 5 mmol L ammonium formate solu-tion and was directly
−1
ramped linearly over the first 15 min to 95% methanol in 5 mmol L
ammonium formate solution. This compo-sition was held for a further 10 min
before returning to the initial condition in 0.1 min. While the elution and
acquisition was con-tinued until 31 min, the column was re-equilibrated at the
initial mobile phase composition until 32.5 min, which was also the total run
−1
time. The flow (0.3 mL min ) was diverted to waste during the first 2.5 min
of the chromatographic run. The autosampler was flushed with methanol
between analytical runs to avoid carry over. The injection volume of each
sample was 5 L.
4541
The mass spectrometer ion source parameters were: capillary voltage 2.0
kV; sample cone voltage 35 V (constant for all pesti-cides); source
◦ ◦
temperature 100 C and desolvation gas temperature 350 C. The flow rates
−1
for desolvation gas and cone gas (N 2 ) were set at 100 and 600 L h ,
respectively. Multiple reaction monitoring (MRM) experiments were
conducted with a dwell time and inter channel delay time of 20 and 10 ms,
respectively, for all pesticides. Ions were collisionally fragmented with argon
−3
at 3.5 × 10 mbar. Optimization of the collision energy (CE) for each
individual pes-ticide was done by infusion of the pesticide directly into the LC
effluent using a syringe pump (Harvard, Kent, UK). For instrument control,
data acquisition and processing, MassLynx and QuanLynx software 4.0 was
used. Precursor and product ions monitored, time windows/functions and
optimized collision energies, for both ESI positive and negative ionization
modes were described in our pre-vious paper about this comprehensive study
[1,12].
2.5. Acetone and acetonitrile extractions
The acetone extraction procedure was already described in part 1 of this
study [12] and is just shown schematically in Fig. 1. To per-form the
acetonitrile extraction (Fig. 2), an amount (2.0 ± 0.02 g) of soya grain (milled
and homogenized) was weighed into a 50 mL PTFE centrifuge tube and 4.0 g
of water was added, allowing soak-ing for 1 h. A volume (20 mL) of
acetonitrile containing 1% of acetic acid was added to the tube, and manual
shaking was performed during ±4.5 s. Then, 2.0 g anhydrous magnesium
sulphate and 2.5 g sodium acetate, were added to each tube and immediate
vigor-ous, manual shaking was performed, in order to avoid coagulation,
which could have a negative influence on the partitioning process. Besides,
buffering was applied at this step, which provides a bet-ter stability for some
pH-dependent pesticides. The acetonitrile extracts (upper layer) were decanted
into other tubes, containing 2.0 g of anhydrous magnesium sulphate. The
tubes were shaken manually for 20 s. After centrifugation at 3000 rpm for 1
min, an aliquot of 0.5 mL extract was diluted with 0.5 mL of methanol into an
autosampler vial. The final extract before injection into the LC–MS/MS
−1
system corresponds with a matrix-equivalent concen-tration of 50 mg mL .
2.6. Method validation study
One single chromatographic run was performed to analyse 155 pesticides
by LC–MS/MS in the ESI positive mode and another run for the 14 pesticides
analysed in the ESI negative mode.
2.6.1. Calibration curves, linearity, LOD and LOQ
The evaluation of the calibration curves’ linearity was done based on
injections of the standard solutions prepared in organic solvent
(acetonitrile/methanol, 1:1) and also in blank soya extract, at the
−1
concentrations 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 and 10.0 ng mL . These solutions
were each injected six times (n = 6). The range of pesticide concentrations in
the matrix extract corresponds with a range of residue levels in the soya bean
−1
samples from 2 to 200 g kg .
Calculations were performed of the average peak areas, rela-tive standard
deviations (RSDs) and calibration curve equations, and also the determination
2
coefficients (r ) and linear ranges were determined for each pesticide
analysed.
Both from the calibration curves and the repeatability (RSD) data, at the
lowest concentration levels of each individual pesti-cide of the in total 169
pesticides studied, the instrument limits of detection (LOD) and limits of
quantification (LOQ) (LODi and LOQi , respectively) and also the method
LOD and LOQ (LODm and LOQm , respectively) were estimated.
4542 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552

The LODi was defined as the minimum concentration of an analyte that

can be identified, measured and reported, with 99% confidence that the
analyte concentration is greater then zero. The LODi was calculated from the
RSD% of the average detec-tor responses (peak areas) of six replicate
injections at the four lowest, detectable concentration levels, via the formula:
−1
LODi (ng mL ) = 3 × RSD × concentration (provided that the response of
the blank was zero). From these calculated values, a best estimated, rounded
LODi value was established. As a consequence, this con-centration should
always have been really injected and detectable repeatedly (with a signal-to-
noise ratio >2) all six times at that level, while the RSD should not have
exceeded 33%.

The estimated LOQi was defined as the minimum concentra-tion of an

analyte that can be identified and quantified with 99% confidence. The
(estimated) LOQi was calculated via the formula: LOQ i = 10 × RSD ×
concentration, that is LOQi = 3.3 × LODi .
The real LOQm was based on the accuracy and precision data, obtained
via the recovery determinations and was defined as the lowest validated spike
level meeting the requirements of a recovery within the range 70–120% and a
RSD ≤ 20% [28].
2.6.2. Matrix effect evaluation and comparison
The matrix effect was evaluated by comparing the peak areas from
standard solutions (n = 6) of the 169 pesticides in solvent
(ace-tonitrile/methanol, 1:1) with the peak areas obtained from standard
solutions, of the same pesticides, prepared either in blank soya extract 1 or
blank soya extract 2, at all seven concentrations men-tioned in Section 2.6.1.
This way, it was possible to calculate and compare the positive or negative
matrix effect, that is an increase or decrease of the detector response,
respectively, for both extrac-tion methods. The matrix effect was calculated
via the formula:

(X2 − X1 )
Matrix effect (%) = × 100 (%)
X1
where X1 = average area of the pesticide standard in solvent
(ace-tonitrile/methanol, 1:1), at a specific concentration; X2 = average area of
the pesticide standard in blank soya extract 1 or 2, at the same concentration.

When the average matrix effect exceeds circa 20%, it can gener-ally be
considered to having a significant effect on the quantitative analytical results
[29], but it is also necessary to take the repeatabil-ity (expressed as RSD
values) of the average peak areas into account [30].

2.6.3. Accuracy and precision

The accuracy and precision of both the acetone and acetonitrile extraction
method were evaluated through recovery experiments by spiking pesticides to
−1
a blank soya sample at three different lev-els (10, 50 and 100 g kg ), six
replicates at each level (n = 6). The spiking procedure was performed by
adding the standard mix-ture solution containing 169 pesticides (Section 2.2)
to milled and homogenized soya grain that had been previously soaked with
water during 1 h, before applying either the acetone (Fig. 1) or acetonitrile
extraction method (Fig. 2). The contact time of the pes-ticides with the wetted
sample before addition of extraction solvent was kept at a minimum. Each
blank soya extract (1 and 2) were also analysed six times.

In order to check whether the evaporation procedure (a gen-eral step

applied in many methods for pesticide residues analysis) could cause losses of
−1
some pesticides, the spiking procedure (only at 100 g kg ) was also done
after the extraction and before the evaporation steps. The blank soya extract 1
(10 mL) was thereto placed into a 25 mL tube and the evaporation was
◦
conducted in a water bath, with a bath temperature starting at 45 C and
◦
contin-uing to 62 C, until near dryness. The remaining residue of solvent
O
D
L

25 or
tra blank

or

25 or
164
soy
aex

%(
%(
(g

5.

50
0.
79

0
.
 pesticideswit hacert ain
r stand ard solutio nsprep ared
Table 1Percentage of 2, linear range of the calibration curves, estimated instrumentandmethodLOD i and LOD m ), derived from (LOD in solvent, in blank ct1and

performedbyLC-ESI–
soya extract 2 Analysis was MS/MS inthepositiveandnegativemodes.

Percentageofpesticides

LC-ESI–MS/MSpositive(numberofpesticides:155) LC-ESI–MS/MSnegative(number ofpesticides:14)

2 Linear Range r i −1 ), LOD m LOD 2 r LinearRange i −1 ),LOD
) −1 (gkg ) −1 kg
Solvent 90%(140/155)0.or1≥90%(140/155)0.999 184%(130/155)0.or ≥93%(13/14)0.999 170%(10/14)0.or 11/14)0.or1

0.ngmL25–
10 0.n gmL25–10
−1 2. or0 ), 2.0 or 0.(LOD25 −1 2.0 or ), 2.0 (LOD25
−1 5.gkg0–200 ) m 5.(LOD0 −1 5.gkg0–200 ) m (LOD0
%(9/14)0.or
Soya extract 1 2590%(140/155)0. ≥50%(7/14)0.999

−1 –10ngmL ), 02. 0.(LOD

(L
OD
−1 5.gkg0–200 ≥100%(14/14)0.99 ) m 0
Soya extract 2 ≥95%(147/155)0,99993%(144/155)0.or1 91%(141/155)or ≥80%(11/14)0.999 80%(11/14)0.or1 7/14)0.or1

25–
100.ngmL 25 –1 00 .ng mL
−1 02. or ), 02. or 250.(LOD −1 02. or ), 02. 0.(LOD

−1 0–2005.gkg ) m 05.(LOD −1 0–2005.gkg ) m 5.(LOD

 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552 4543

Table 2
Average matrix effect (in %, n = 6) for a selected number of pesticides, analysed by LC–MS/MS (ESI positive mode). Soya extract 1 and 2 are obtained from acetone and acetonitrile
extraction, respectively.
*
Pesticide Matrix effect (%)
Soya extract 1 Soya extract 2
−1 −1
Concentration (ng mL ) Concentration (ng mL )

5 2.5 5 2.5
Acephate −2 4 −74 −70
Methamidophos −22 −18 −77 −75
Aldicarb sulfoxide 28 14 −70 −76
Azamethiphos −23 −12 0 13
Boscalid −25 −10 1 −6
Butocarboxim sulfoxide 7 2 −52 −52
Omethoate −5 −1 −75 −75
Fenoxycarb −36 −29 −36 −41
Fenpyroximate −25 −13 −21 −10
Flusilazole −26 −22 −12 −8
Mepanipyrim −42 −35 −17 −24
Paclobutrazole −39 −41 −16 −14
Spinosad A/D −26 −22 −25 −5
Tebufenozide −36 −47 −22 −23
Vamidothion sulfoxide 0 1 −76 −66
*
Matrix effect (%) = [(X2 − X1 )/X1 ] × 100 (%), where X1 and X2 = average peak area of the pesticide standard solution in solvent and soya extract, respectively.

was allowed to evaporate in the air (in a fume hood) at room tem-perature.
The extract reconstitution was done by adding 1.0 mL of solvent or standard
−1
mixture solution (50 ng mL ). The blank soya extract 2 (5 mL) was
transferred to a 25 mL tube and the evapo-ration was conducted in a
◦ range with equivalent pesticide residue levels in the sample of 2 or 5 to 200 g
RapidVap System (speed: 50%; temp.: 50 C; time: 20 min; vacuum: 160
mbar). The residue was reconsti-tuted in 1.0 mL of methanol or 1.0 mL of a
−1
standard mixture solution (50 ng mL ).
Then, the obtained recoveries and RSD data were evaluated and
compared, considering the different stages of spiking.
3. Results and discussion
3.1. Calibration curves, linearity, LOD and LOQ
In Table 1, the summarized results for the linearity of the calibra-tion
curves are given for the pesticides standard solutions prepared in solvent and
those prepared either in soya extract 1 or 2. The coefficient of determination
2 those from soya extract 1. However, the reverse situation can be observed for
(r ) and linear range of the calibration curves, both for pesticides analysed by
ESI positive and negative modes, are shown. Approximately 90% (140
pesticides) and 95% (147 pesticides), respectively, of the pesticides in solvent
or in soya
2
extract 2, analysed by ESI positive mode, showed r ≥ 0.999 and a linear
−1
range between 0.1 or 0.25 and 10.0 ng mL . The same results were obtained
in the ESI negative mode for 93% (13) and 80% (11) of the pesticides in
solvent and soya extract 2, respectively. This corre-sponds with an application
−1
kg .
The estimated, rounded instrument LOD i values, based on the RSD of
repeatedly injected solutions (n = 6) at that concentration, were 0.1 or 0.25 ng
−1
mL for 91% and 50% of the pesticides in matrix extract 2, detected in the
positive and negative mode, respectively, compared with corresponding
percentages of 84% and 79% for the pesticides in pure solvent. Based on the
−1
LODi , corresponding LOQm values could be estimated of 2 or 5 g kg ,
−1
being near the target method LOQ of 10 g kg , which was therefore also
selected as the lowest spike level for the recovery studies.
When comparing the results of soya extract 2 with those of soya extract 1,
it can be noticed from Table 1 that for the 155 pesticides determined by the
positive mode, the results obtained from soya extract 2 are slightly better than
the pesticides analysed by the negative mode, which means that the soya
extract 1 seems slightly better.

−1
Fig. 3. Comparison of number and percentage of pesticides with a LOQ m of 10, 50 or 100 g kg , not detected (n.d.) or not qualifying for the quantitation criteria (n.q.), when performing the
acetonitrile and acetone extraction method and LC–MS/MS analysis in the ESI positive and negative modes.
4544 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552

Table 3
−1
Average recovery and RSD (in %, n = 6) and method LOQm , obtained by acetonitrile extraction of soya samples, spiked at 10, 50 and 100 g kg , and analysed by LC–MS/MS (ESI positive mode).

−1 −1
Pesticide Spike levels ( g kg ) LOQm ( g kg )
100 50 10

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)
Acephate 78 6.0 84 11.8 66 9.3 50
Methamidophos 73 8.4 67 8.4 64 14.8 100
Acetamiprid 92 3.0 88 5.3 81 6.0 10
Aldicarb sulfoxide 79 8.3 75 2.4 69 11.6 50
Aldicarb sulfone 92 4.6 88 5.2 81 6.0 10
Azaconazole 101 1.1 96 2.9 81 8.9 10
Azamethiphos 93 2.4 93 7.0 83 2.6 10
Azinphos-methyl 89 7.0 81 12.0 91 12.7 10
Azoxystrobin 95 3.1 89 5.1 77 5.6 10
Bitertanol 99 6.9 93 7.9 88 27.1 50
Boscalid 94 4.1 89 1.7 84 10.1 10
Bromuconazole 95 4.9 94 1.0 87 17.1 10
Bupirimate 86 5.8 88 9.8 81 5.4 10
Ethirimol 61 2.7 49 19.1 50 12.4 n.q.
Buprofezin 88 4.4 84 4.8 68 11.0 50
Butocarboxim sulfoxide 76 5.8 75 9.9 79 22.0 50
Butocarboxim sulfone 88 2.5 80 3.0 80 3.0 10
Carbaryl 92 5.4 89 7.9 85 6.7 10
Carbendazim 84 3.1 80 7.9 77 5.4 10
Carbofuran 95 1.9 93 4.6 82 2.3 10
Carbofuran, 3-hydroxy 92 3.8 83 4.0 86 3.9 10
Carpropamid 94 3.8 93 0.9 88 6.8 10
Chlorbromuron 85 5.9 89 6.1 69 20.2 50
Clofentezine 90 10.3 88 3.6 69 18.2 50
Clothianidin 88 2.9 85 5.3 80 3.4 10
Cyazofamid 76 6.2 81 5.7 76 5.8 10
Cymoxanil 80 0.9 82 7.0 70 17.5 10
Cyproconazole 102 4.6 102 2.5 77 21.3 50
Cyprodinil 95 1.6 93 2.1 85 5.1 10
Demeton-O-sulfoxide 96 2.0 88 5.7 81 4.6 10
Oxydemeton-methyl 90 2.0 86 3.3 77 5.2 10
Demeton-S-methyl. sulfone 88 2.7 86 3.7 80 3.8 10
Desmedipham 45 9.2 57 13.0 48 12.4 n.q.
Dichlofluanid 24 14.2 34 20.1 25 29.4 n.q.
Dichlorvos 83 4.2 98 8.1 79 3.2 10
Trichlorphon 11 33.0 26 38.5 14 48.0 n.q.
Dicrotophos 90 4.0 91 9.9 78 18.0 10
Diethofencarb 94 2.1 90 5.0 84 3.7 10
Difenoconazole 153 7.4 153 1.2 148 9.5 n.q.
Dimethoate 95 2.3 91 5.8 79 9.0 10
Omethoate 69 6.7 75 6.5 72 12.3 10
Dimethomorph 97 2.2 94 4.0 87 8.0 10
Dimoxystrobin 100 3.5 91 4.5 70 5.8 10
Diniconazole 98 9.7 98 4.7 90 23.4 50
Diuron 88 4.5 83 6.8 80 7.7 10
Dodemorph 69 4.7 88 5.9 70 6.9 10
Epoxiconazole 103 3.6 94 6.2 89 7.0 10
Ethiofencarb sulfoxide 78 4.3 78 5.5 75 4.7 10
Ethiofencarb sulfone 68 6.6 69 4.9 65 7.5 n.q.
Ethiprole 83 4.2 85 3.6 92 35.0 50
Etofenprox 60 35.0 109 8.2 66 12.5 n.q.
Famoxadone 95 9.0 94 12.1 91 33.7 50
Fenamidone 98 4.7 95 6.1 78 9.0 10
Fenamiphos 76 3.2 83 17.1 75 8.9 10
Fenarimol 95 3.9 92 8.2 67 9.2 50
Fenazaquin 66 28.2 99 11.0 86 5.6 10
Fenbuconazole 97 4.0 90 10.1 83 8.1 10
Fenhexamid 94 7.2 83 4.7 67 9.8 50
Fenoxycarb 99 4.3 95 4.5 79 4.8 10
Fenpropidin 80 9.2 76 4.2 63 10.6 50
Fepropimorph 77 15.9 109 7.7 85 8.0 10
Fenpyroximate 79 16.5 74 27.0 75 15.2 10
Fenthion 108 9.6 112 11.6 127 12.6 50
Fenthion sulfoxide 91 2.8 91 4.5 82 6.4 10
Flufenacet 101 8.7 91 9.5 89 17.0 10
Fluquinconazole 97 6.8 97 6.7 102 23.5 50
Flusilazole 95 8.5 92 5.4 80 13.2 10
Flutolanil 104 2.8 98 4.4 85 3.2 10
Flutriafol 97 2.2 94 2.9 79 3.7 10
Fosthiazate 93 2.6 89 6.0 83 5.9 10
Furathiocarb 89 7.6 88 2.5 73 11.6 10
Halofenozide 100 5.8 92 9.9 76 11.5 10
 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552 4545

Table 3 (Continued )

−1 −1
Pesticide Spike levels ( g kg ) LOQm ( g kg )
100 50 10

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)
Hexaconazole 105 4.3 101 4.9 48 33.3 50
Hexythiazox 72 8.3 72 17.6 48 23.0 50
Imazalil 77 3.3 65 10.9 67 20.8 100
Imidaclopride 83 6.1 79 2.9 71 10.0 10
Indoxacarb 73 10.8 70 15.1 53 28.2 50
Iprovalicarb 71 4.4 61 9.8 48 16.4 100
Isoprothiolane 76 2.1 72 6.9 54 22.9 50
Isoxaflutole 74 3.9 66 11.7 54 27.2 100
Isoxathion 70 9.0 72 11.0 54 20.1 50
Kresoxim-methyl 68 8.4 69 19.7 47 24.6 n.q.
Linuron 72 7.0 70 4.7 61 21.3 50
Malathion 75 9.9 77 8.6 49 27.4 50
Mefenacet 75 3.7 70 6.5 55 16.2 50
Mepanipyrim 71 7.5 70 8.6 59 23.7 50
Mephosfolan 87 4.2 80 3.8 78 3.2 10
Mepronil 70 4.6 62 9.4 53 25.4 100
Metalaxyl 82 4.1 75 3.5 68 6.2 50
Metconazole 68 6.3 63 9.3 57 31.6 n.q.
Methidathion 78 6.2 74 9.4 57 18.5 50
Methiocarb 79 3.7 72 4.3 63 16.4 50
Methiocarb sulfoxide 83 5.5 77 5.2 81 7.3 10
Methiocarb sulfone 83 4.2 76 6.4 75 7.1 10
Methomyl 102 10.8 89 9.3 107 4.7 10
Thiodicarb 55 14.0 57 17.0 33 11.7 n.q.
Metobromuron 79 4.2 74 2.1 62 11.5 50
Metoxuron 63 7.3 60 4.5 60 11.7 n.q.
Methoxyfenozide 68 5.9 57 5.2 46 27.4 n.q.
Monocrotophos 86 4.3 78 9.9 86 10.8 10
Monolinuron 82 2.9 79 5.9 70 7.3 10
Myclobutanil 66 10.5 61 15.2 55 33.5 n.q.
Nitenpyran 82 6.1 81 7.5 86 14.7 10
Nuarimol 74 5.6 70 4.8 63 15.3 50
Oxadixyl 78 18.6 70 16.4 70 8.5 10
Oxamyl 79 7.4 70 5.6 76 12.9 10
Oxamyl-oxime 76 8.1 70 6.1 82 8.8 10
Oxycarboxin 82 4.3 82 5.7 81 5.7 10
Paclobutrazole 72 10.1 63 21.6 n.d. 100
Penconazole 75 2.2 69 12.3 56 29.2 100
Pencycuron 8 9.6 8 13.6 n.d. n.q.
Phenmedipham 67 3.7 58 9.8 50 21.3 n.q.
Phosphamidon 88 4.8 83 6.0 84 5.2 10
Picoxystrobin 71 4.2 66 9.9 45 17.8 100
Piperonyl butoxide 75 5.3 71 13.0 51 22.7 50
Pirimicarb 86 5.0 75 4.1 69 5.7 50
Pirimicarb, desmethyl 84 5.1 78 4.0 78 7.3 10
Prochloraz 72 4.8 64 8.6 48 21.3 100
Profenofos 73 4.5 72 11.1 51 24.9 50
Propiconazole 75 3.8 70 8.4 n.d. 50
Propoxur 88 3.7 84 4.3 83 3.2 10
Propyzamide 72 5.4 68 7.3 51 17.5 100
Pymetrozine 65 6.3 63 4.3 63 12.9 n.q.
Pyraclostrobin 69 4.9 64 12.8 49 17.0 n.q.
Pyridaben 73 7.6 66 19.9 46 20.1 100
Pyridaphenthion 75 5.9 71 6.0 52 24.8 50
Pyrifenox 75 4.3 77 6.2 163 30.9 50
Pyrimethanil 77 3.8 73 5.4 60 10.7 50
Pyriproxyfen 68 6.7 67 14.6 50 15.4 n.q.
Spinosad A/D 64 11.9 75 10.9 n.d. n.q.
Spirodiclofen 73 6.6 72 15.4 55 14.9 50
Spiroxamine 56 12.3 62 7.7 n.d. n.q.
Tebuconazole 71 5.4 65 6.4 53 28.9 100
Tebufenozide 58 13.2 49 13.8 39 16.9 n.q.
Tebufenpyrad 71 5.1 66 11.5 48 27.8 100
Tetraconazole 65 10.0 82 5.9 169 8.4 n.q.
Thiabendazole 86 5.3 82 2.7 75 8.0 10
Thiacloprid 81 6.5 79 6.5 76 5.6 10
Thiamethoxam 81 6.3 83 6.2 81 5.5 10
Thiofanox sulfoxide 82 5.0 75 6.6 74 24.8 50
Thiofanox sulfone 94 5.8 63 14.5 n.d. 100
Thiometon sulfoxide 85 5.0 80 2.6 81 5.2 10
Tiometom-sulfona 82 4.9 79 4.0 69 11.8 10
Tolclophos methyl 64 6.5 72 15.4 58 18.0 n.q.
Tolylfluanid 85 4.1 86 5.0 129 20.1 50
Triadimefon 75 4.5 67 7.9 56 16.5 100
4546 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552

Table 3 (Continued )

−1 −1
Pesticide Spike levels ( g kg ) LOQm ( g kg )
100 50 10

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)
Triadimenol 66 15.7 55 22.2 n.d. n.q.
Tricyclazole 85 6.5 81 5.6 78 3.0 10
Trifloxystrobin 68 6.0 66 11.5 52 16.6 n.q.
Triflumizole 74 4.5 64 7.9 42 26.5 50
Triforine 60 6.8 56 8.6 n.d. n.q.
Triticonazole 71 10,8 63 11.1 58 22.7 100
Vamidothion sulfoxide 73 7.4 73 2.6 68 10.8 50
Vamidothion sulfone 80 6.6 78 6.2 79 6.3 10
Zoxamide 74 12.3 64 9.9 50 24.1 100
n.d.: not detected; n.q.: not qualifying for the quantitation criteria for at least one spike level.

3.2. Matrix effect of soya extracts
Matrix effect is the observed effect of an increase (enhancement) or
decrease in detector response (a positive or negative matrix effect,
respectively) of a pesticide present in a matrix extract com-pared with the
same pesticide present in just solvent. The matrix effect in LC–MS is usually
caused by extracted matrix components, eluting at the same retention time as
the pesticide and thereby influencing the ionization process occurring in the
ion source. This results in a decreased (“ion suppression”) or increased
number of ions formed.
Based on practical experience in routine analysis of pesticide residues in
food, a matrix effect ≥20% can generally be considered as significant and has
to be taken into account when reporting quanti-tative results accompanied
with a typical measurement uncertainty of 50% [28]. Matrix effects are
always variable with time and condi-tion of instrument used and it should thus
be realized that the data obtained during validation, are just indicative and
should therefore always be continuously monitored for the actual figures
during rou-tine application of a method. The matrix effect comparison
between soya extract 1 and soya extract 2, for the pesticides analysed in the
ESI positive ionization mode, is shown in the Table 2. The matrix effects are
−1
shown for two concentrations (5 and 2.5 ng mL ), only for those selected
pesticides which showed a significant matrix effect for at least one or both of
the 2 extracts. It can be concluded that the matrix effect for most of the 155
pesticides studied in the positive ESI mode is negligible or not significant.
Even though the extracts have not been cleaned up, the LC–MS interface can
apparently handle these relatively dirty extracts. Less than 10% of the total
number of pesticides shows a significant negative matrix
Table 4
Average recovery and RSD (in %, n = 6) and method LOQm , obtained by acetonitrile extraction of soya samples, spiked at 10, 50 and 100 g kg
effect for both extraction methods. However, it is striking to see that for some
specific pesticides, there is a difference for which extrac-tion method the
significant matrix effect is observed. For the most polar pesticides, that is
those eluting first in the LC–MS (typically retention time range 4–7 min), the
negative matrix effect is higher for acetonitrile than for acetone extracts: e.g.
methamidophos, acephate, butocarboxim sulfoxide, omethoate, aldicarb
sulfoxide and vamidothion sulfoxide. This could be explained by the more
efficient extraction of polar matrix interferences from the soya matrix by
acetonitrile compared to acetone, causing more ion sup-pression. For
fenoxycarb, fenpyroximate and spinosad, the matrix effects are rather similar
for both extraction methods. For some more apolar (late eluting) pesticides,
the negative matrix effect appears to be higher for acetone than for acetonitrile
extracts: e.g. azamethiphos, boscalid, paclobutrazole, flusilazole, tebufenozide
and mepanipyrim. This indicates that acetone is probably extracting some
non-polar matrix components more efficiently than acetonitrile, causing more
ion suppression for non-polar pesticides.
All pesticides tested and detected in the negative ESI negative mode
showed no significant matrix effect for both the acetone and acetonitrile
extraction methods.
3.3. Accuracy, precision, method LOQ and selectivity
The method performance evaluation was done according to the validation
criteria for quantitative methods for pesticide analysis in food and feed, laid
down in the SANCO document 2007/3131 [28]. Typically a recovery within
the range of 70–120% and a repeata-bility RSD ≤ 20% is required. Some
recoveries below 70% are still
−1
, and analysed by LC–MS/MS (ESI-negative mode).

−1 −1
Pesticide Spike levels ( g kg ) LOQm ( g kg )
100 50 10

Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%)
2,4-D n.d. n.d. n.d. n.d.
Chlorfluazuron 92 11.6 87 12.9 107 20.1 50
Diflubenzuron 124 25.6 82 9.5 122 20.6 n.q.
Fipronil 80 11.4 86 10.2 95 16.7 10
Fluazinan 79 8.1 91 7.5 101 7.0 10
Flucycloxuron 164 36.9 n.d. n.d. n.q.
Fludioxanil 93 12.6 88 12.8 102 5.6 10
Flufenoxuron 103 11.7 104 12.1 n.d. 50
Flusulfamide n.d. n.d. n.d. n.d.
Hexaflumuron 90 2.9 91 7.6 109 20.6 50
Lufenuron 82 3.9 88 9.5 91 10.4 10
Novaluron 92 4.9 88 7.1 106 5.0 10
Teflubenzuron 83 6.5 94 5.6 97 6.8 10
Triflumuron 122 27.4 81 4.4 98 9.0 n.q.
n.d.: not detected; n.q.: not qualifying for the quantitation criteria for at least one spike level.
 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552
Fig. 4. Total ion chromatograms obtained by LC–MS/MS ESI positive and negative modes of (A) blank soya extract; soya spiked at (B) 10 g kg
extraction method.
acceptable for a multi-residue method including a large number of pesticides,
provided that a good precision (RSD ≤ 20%) is obtained. Correction for
recovery during routine application would then be recommended.
The summarized results of the recoveries (%) and RSD (%) of the
−1
pesticides spiked to the blank soya samples at 10, 50 and 100 g kg and
extracted by the acetonitrile-based method, are shown in Tables 3 and 4 for
the LC–MS/MS analysis in the posi-tive and negative ionization mode,
respectively. The real (validated) method LOQ m , defined as the lowest
validated spike level meeting the requirements mentioned above, are included
in Tables 3 and 4 for each individual pesticide. When both of the two criteria
were not met for all the spike levels tested, it was concluded that the pesticide
could not be quantified reliably. When at least one of the two criteria was not
met for all the spike levels, the pesticide can just be determined semi-
quantitatively or qualitatively.
In total, 70 of the 155 pesticides analysed in the positive mode had a
−1
LOQm of 10 g kg . Those pesticides with higher LOQ values, had either a
recovery <70% or a RSD > 20%, or both, at one or two of the spike levels,
but were always detectable. All pesticides analysed in the positive mode
−1
could at least be detected at levels of 50 and 100 g kg . for acetonitrile extracts of blank soybean and spiked
4547
−1 −1
and (C) 100 g kg (10× attenuated), by acetonitrile
The number and percentage of pesticides within each recov-ery range or
−1
not detected (n.d.) at the 10, 50 and 100 g kg spike levels, are shown in Fig.
3. Most of the pesticides anal-ysed by LC-ESI–MS/MS positive mode (Table
3) showed good recoveries. Significantly low recoveries were observed for
ethiri-mol, desmedipham, dichlofluanid, trichlorphon, pencycuron and
tebufenozide. This may have been likely caused rather by degra-dation of the
pesticide in the acetonitrile solvent.
From the 14 pesticides analysed by LC-ESI–MS/MS negative mode
(Table 4), two pesticides, 2,4-D and flusulfamide, could not be extracted and
were not detected at all. For comparison, it is interest-ing to note, that with the
acetone extraction, flusulfamide could not be extracted either, but on the other
hand, 2,4-D was at least partly extractable with acetone. The different
behavior is probably caused by an expected different pH (that is, a higher pH
with acetonitrile) during the extraction step. Flucycloxuron is only detectable
at the highest spike level, but very irreproducible. For the other pesticides,
recoveries and RSD values were mostly acceptable.
Selectivity of the method is best illustrated with figures of chro-
matograms. The total ion chromatograms for 155 pesticides in the ESI
positive mode and 14 pesticides in the ESI negative mode are shown in Fig. 4
4548 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552

Fig. 5. Multiple reaction monitoring chromatograms for azoxystrobin and tebuconazole (LC–MS/MS ESI positive mode), (A) 1st transition and (B) 2nd transition for (C) blank soya extract; (D)
−1 −1
standard solution 5 ng mL in blank soya extract and (E) extract of soya spiked at 10 g kg , by acetonitrile extraction method.

−1
at 10 and 100 g kg . The good sensitivity of this method can be typically
seen from the high responses of the pesticides in the eight overlapping scan
−1
functions at the 10 g kg spike level, com-pared with the 10× attenuated
−1
total ion chromatograms at the 100 g kg spike level. The selectivity of the
detection method is also clearly visible from the chromatograms of the blank
extracts, which show hardly any interfering matrix peaks.
Even more illustrative for the selectivity are the multiple reac-tion
monitoring chromatograms for the individual pesticides, as shown in Fig. 5,
for example, for the two transitions of azoxystrobin,
−1
404 → 372 and 404 → 344, measured in the same run. Even at the 10 g kg
spike level, for both the first (quantitation) and the sec-ond (confirmation)
transition, the very good signal-to-noise (S/N) ratio is clearly visible.
Another example is also given in Fig. 5 for the two MRM transitions of
tebuconazole (308 → 70 and 435 → 125), also show-ing a good S/N ratio for
the first MRM transition. However, in this case, due to a very unfavorable ion
ratio, the peak from the second (confirmation) MRM chromatogram is below
−1
the detection limit and therefore the 10 g kg level can be con-
 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552 4549

Fig. 6. Multiple reaction monitoring chromatograms for lufenuron and fludioxonil (LC–MS/MS ESI-negative mode), (A) 1st transition and (B) 2nd transition for (C) blank soya extract; (D) standard
−1 −1
solution 5 ng mL in blank soya extract and (E) extract of soya spiked at 10 g kg , using the acetonitrile extraction method.

sidered as the validated LOQ, but not as a (confirmed) limit of identification
(LOI), because the ion ratio of the two MRM transitions cannot be confirmed
within the acceptable range [28,31].
The LC-ESI–MS/MS negative mode also shows similar selec-tivity, as
can be seen in Fig. 6 for the MRM chromatograms of lufenuron, an
insecticide belonging to the class of benzoylurea insecticides, which can be
detected more sensitively in the neg-ative mode than in the positive one.
−1
Lufenuron has a LOQm of 10 g kg based on the 1st MRM transition MRM
(509 → 339) and also the LOI is at the same level, as can be concluded from
the S/N ratio of the 2nd MRM transition (509 → 326) at this concentration.
In the example of fludioxonil (Fig. 6), it is illustrated that, despite the good
selectivity of the MS/MS detection method, the LC separa-tion is also very
important. The 1st MRM transition (247 → 180) is distinctly selective, but the
2nd MRM transition (247 → 126) gives not only a significant response at the
retention time of fludioxonil, but also at a retention time around 2.9 min. Due
to the good resolu-tion, the identification can be confirmed via the ion ratio
still being within the acceptable range relative to that of the matrix-matched
standard solution.
4550 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552
Fig. 7. Comparison of number and percentage of pesticides within each recovery range or not detected (n.d.) at 10, 50 and 100 g kg spike levels, when performing the acetone and acetonitrile
extraction methods and LC–MS/MS analysis in the positive (A) and negative (B) ionization mode. Number of pesticides is depicted next to the bars.
3.4. Comparison of the acetonitrile and acetone extraction
methods performances
In this study, an acetonitrile-based extraction method, which comprises in
fact the recently AOAC International collaboratively studied and validated,
buffered QuEChERS (acronym for quick, easy, cheap, efficient, rugged and
safe) method [27], however, with-out applying dispersive solid-phase
extraction (SPE) as cleanup, was compared with an earlier developed, very
fast and sim-ple one-step acetone extraction/dichloromethane-light petroleum
◦
(b.p. 40–60 C) partitioning method [1,12], without any buffering or cleanup.
The performance characteristics for the majority of the 169 pesticides studied
were acceptable for both methods. In Figs. 3 and 7, the comparison between
the acetonitrile and acetone extraction methods is illustrated via bar graphs,
for the obtained LOQm and recovery data, respectively
The number of pesticides with a LOQm at the target level of
−1
10 g kg and acceptable RSDs is slightly higher for the acetoni-trile method
than the acetone method. It should be kept in mind, however, that this is not
caused by the sensitivity of the LC–MS/MS detection method, which is
essentially the same for both methods, but by the differences in recoveries and
RSDs at the lowest spike level.
−1
On an average, the recoveries were 10–15% lower and/or the RSDs were
somewhat higher at the lowest levels with the acetone method, causing that in
the latter method less pesticides were ful-filling the strict requirements of both
a recovery range 70–120% and RSD ≤ 20%. For both methods, detection at 50
−1
and 100 g kg was always achieved.
To prove whether the somewhat lower recoveries of the acetone method
might have been caused by possible pesticide losses during the evaporation
step of the acetone method, a separate experiment was performed for the
determination of the recoveries from spiked soya extracts (corresponding with
−1
100 g kg ). It can be concluded from the data, that the evaporation step does
not lead to any sig-nificant losses, and therefore, the extraction step is
responsible for the deviation from the 100% recovery.
The speed and user friendlyness of the acetone extraction in real practice
are slightly better than for the acetonitrile method, despite the extra
evaporation step, which can be easily performed in batch and offers the
additional advantage of reconstitution in the optimal solvent methanol for LC
analysis of the very early eluting pesticides, without any further dilution of the
original extract. On the other hand, the acetonitrile extract, after dilution with
methanol, can be directly injected, thus allowing faster sample extract
introduction in the LC–MS/MS instrument.
 I.R. Pizzutti et al. / J. Chromatogr. A 1216 (2009) 4539–4552 4551

Table 5
Surveys of pesticide residues in soya products using acetone (2007) and acetonitrile (2008) extraction and LC–MS/MS analysis.

2007 2008
−1 −1
Soya Product Pesticide Residue level ( g kg ) Soya Product Pesticide Residue level ( g kg )
Soya beans Piperonyl butoxide 3 Soya beans (organic) Carbofuran <2
*
Soya beans (11×) n.d. Piperonyl butoxide <2
Soya pellets n.d. Soya beans (organic) Piperonyl butoxide <2
Soya pellets Piperonyl butoxide 6 Soya beans (organic) Tetraconazole <2
Soya proteins Chloorpyrifos <2 Piperonyl butoxide <2
Soya proteins Tebuconazole <2 Soya beans (organic) Clofentezine <2
Soya proteins Chloorpyrifos 2 Piperonyl butoxide <2
Cyproconazole 4 Soya beans (organic) Piperonyl butoxide <2
Epoxiconazole 7 Carbofuran <2
Flutriafol 2 Soya beans (5×) n.d.
Myclobutanil 2 Soybean meat (frozen) n.d.
Tebuconazole <2 Soya (minced) n.d.
Tetraconazole 2 Soya beans for tofu n.d.
Soya proteins Epoxiconazole 7 Soya pellets n.d.
Soya proteins Chloorpyrifos <2 Soya chunks Pyrimethanil 2
Flutriafol <2 Methoxyfenozide <2
Piperonyl butoxide 2 Carbendazin 8
Propoxur 8
Soya proteins Piperonyl butoxide 4
Soya proteins Cyproconazole <2
Tebuconazole 2
Soya proteins (4×) n.d.
Soya sauce Thiabendazole 4
* n.d.: no residues detected.

Another typical difference is the use of the Polytron homoge-nizer for
acetone extraction in the open 250-mL PTFE tubes, which allows the
extraction of 5 g soya sample amounts with 100 mL extraction solvent. Using
the typical 50 mL tubes of the QuEChERS method, with manual shaking for
the acetonitrile extraction, the soya sample amount has to be limited to not
more than 2 g and due to the voluminous matrix, not more than 20 mL
extraction solvent can be used. The extraction ratios for the acetone and
acetonitrile methods were thus 20:1 and 10:1, respectively. The lower solvent
use and exclusion of chlorinated solvents is a clear environmen-tal advantage
of the acetonitrile method. Upscaling the acetonitrile method to a 5-g sample
and a 50-mL extraction volume in the open 250-mL PTFE tubes is an
alternative, but the advantage the origi-nal low-solvent usage and safety
aspects of closed extraction tubes would be lost then.
extraction/dichloromethane-light petroleum partitioning method, without any
buffering or cleanup. The method performance was evaluated against the
criteria for method validation of the most recent EU guidelines, SANCO
document 3131/2007 [28].
The performance characteristics for the majority of the 169 pesticides
studied were acceptable for both methods. The num-ber of pesticides with a
−1
LOQm at the target level of 10 g kg and acceptable RSDs is slightly higher
for the acetonitrile method than for the acetone method. However, the speed
and user friendlyness of the acetone extraction in real practice is slightly better
than for the acetonitrile method. Both methods have been proved to be
successful as a real quantitative, multi-residue method for pesticide residues
analysis in soya grain samples and some derived products during application
in routine surveys.

We have successfully applied both methods in the routine lab-oratory in

surveys during 2007 and 2008, using the acetone and acetonitrile extraction
method, respectively. The residues detected in the soya samples are shown in
Table 5. It is striking that almost all residue levels detected are within the
−1
range of 1–10 g kg . This could be explained by the fact that samples were
mostly taken from big shipments, representing usually large, mixed lots or
consignments. Besides, a relatively high percentage of organi-cally grown
samples were taken for the 2008 survey. For a future project, we are planning
to critically compare both extraction methods side-by-side for a number of
representative, positive sam-ples with a sufficiently high residue level, in
order to be able to draw conclusions about significant differences in
extraction effi-ciencies of incurred residues. Unfortunately, from the samples
analysed so far, only a limited number would qualify for such an aim.
Acknowledgements
We gratefully acknowledge VWA – Food and Consumer Product Safety
Authority, The Netherlands and the European Commission (Alfa II
Programme B – Project EUROLANTRAP No. AML/B7-311/97/0666/II0461-
FA-FCD-FI) for funding this project.
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4. Conclusions
Two fast and efficient extraction methods for the determi-nation of 169
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optimized and validated. An acetonitrile-based extraction method (buffered
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