Journal of Chromatography A, 1194 (2008) 178–183

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

Comparison of stir bar sorptive extraction and membrane-assisted solvent

extraction for the ultra-performance liquid chromatographic determination
of oxazole fungicide residues in wines and juices
Pilar Viñas, Nerea Aguinaga, Natalia Campillo, Manuel Hernández-Córdoba ∗
Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, E-30071 Murcia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The present study compares two new sample preparation methods, stir bar sorptive extraction (SBSE)
Received 27 February 2008 and membrane-assisted solvent extraction (MASE) coupled to the novel technique of ultra-performance
Received in revised form 14 April 2008 liquid chromatography (UPLC) for the sensitive, selective and solvent-free determination of six oxazole
Accepted 17 April 2008
fungicide residues (hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl and famoxadone) in wine
Available online 23 April 2008
and juices. The analytes were separated on a rapid resolution C18 column (50 mm × 4.6 mm, I.D., 1.8 ␮m)
thermostated at 50 ◦ C with isocratic elution using a 50/50 (v/v) water/acetonitrile (ACN) mobile phase
Keywords:
at a flow-rate of 1 mL min−1 and detected by diode-array detection (DAD). The UPLC method rapidly
Ultra-performance liquid chromatography
Stir bar sorptive extraction
separates the fungicides (7 min). The best results as regards sensitivity, repeatability and analyte recovery
Membrane-assisted solvent extraction were obtained using SBSE with a polydimethylsiloxane (PDMS) twister, at 60 ◦ C for 30 min with stirring at
Oxazole fungicides 1700 rpm in the presence of a 0.1 M acetate/acetic acid buffer (pH 5) and 20% (m/v) sodium chloride. Liquid
Wine desorption was performed with 100 ␮L of a 80/20 (v/v) ACN/water solution in a desorption time of 15 min.
Fruit juice With the PDMS polymer, an apolar phase, hymexazol and oxadixyl were not extracted. Consequently, the
SBSE procedure can only be applied to the other four fungicides. Detection limits ranged from 0.05 to
2.5 ␮g L−1 at a signal to noise ratio of 3, depending on the compound. Recoveries obtained for spiked
samples were satisfactory (83–113%) for all compounds. The proposed method was successfully applied
to the analysis of different samples, residues of chlozolinate and drazoxolon being found in samples of
red wine and grape juice, respectively.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Ultra-performance liquid chromatography (UPLC) uses short
columns packed with sub-2 ␮m particles, mobile phases at high
temperature and high linear velocities, together with instrumen-
tation that operates at higher pressures than are used in HPLC.
The result is an improvement in resolution, sensitivity and speed of
analysis. However, the sampling rate must be high and the detector
cell must have minimal volume to avoid dispersion and maintain
separation efficiency [1,2].
The evaluation of fungicide residues in wine, must and fruit
juices are a priority objective for ensuring their quality and to
protect against possible health risks. Hymexazol, drazoxolon, vin-
clozolin, chlozolinate, oxadixyl and famoxadone are included in the
group of oxazole fungicides. The maximum residue limits (MRLs)
of these fungicides in products of plant origin have been estab-
∗ Corresponding author. Fax: +34 968364148.
E-mail address: hcordoba@um.es (M. Hernández-Córdoba).
lished [3]. Grapes and other fruits are frequently fumigated with
oxazole fungicides and residues may be accumulated in derived
products, such as wine and juices. Although, the European Union
has not yet established MLRs for these matrices, several European
Community Directives have stipulated MRLs in the origin products
[4] for hymexazol and chlozolinate (0.05 ␮g g−1 in fresh citrus and
stone fruits and grapes for vinification), vinclozolin (0.05 ␮g g−1
in citrus, 1 ␮g g−1 in stone fruits and 5 ␮g g−1 in grapes), oxadixyl
(0.05 ␮g g−1 in citrus and stone fruits and 1 ␮g g−1 in grapes) and
famoxadone (0.02 ␮g g−1 in citrus and stone fruits and 2 ␮g g−1 in
grapes).
The analytical methods normally used for the determination
of oxazole fungicides are gas chromatography [5–17], liquid chro-
matography [18–24] or both techniques [25–27]. However, these
techniques use different sample pre-treatments, and require large
sample amounts, large volumes of potentially toxic solvents and
a cleaning step before quantification. Modern sample preparation
techniques offer automation and are clean, selective, rapid and effi-
cient; ideally, they should be cheap, simple and solvent-free. Thus,
the presence of oxazole fungicide residues in malt beverages has

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.04.039
 P. Viñas et al. / J. Chromatogr. A 1194 (2008) 178–183
been analyzed using direct immersion solid-phase microextraction
(DI-SPME) coupled to gas chromatography with mass spectrome-
try in the selected ion monitoring mode, GC–MS(SIM) [28]. Another
technique is stir bar sorptive extraction (SBSE), which represents a
solventless method for the extraction of organic compounds from
aqueous matrices into a polymer coating on a magnetic stirring rod
[29]. The coupling of gas chromatography (GC) and SBSE has been
used to determine vinclozolin [30] and several agrochemicals [31]
in wine. Membrane-assisted solvent extraction (MASE) has recently
been introduced [32] and it is based on small-scale liquid–liquid
extraction with a low-density polyethylene membrane separating
the aqueous sample from the organic solvent. Consequently, this
study proposes a comparison of both non-harmful environmen-
tally sample pre-treatment systems for the rapid determination
of hymexazol, drazoxolon, vinclozolin, chlozolinate, oxadixyl and
famoxadone in samples of wine, must and fruit juices using both
SBSE-UPLC and MASE-UPLC hybridizations with diode-array detec-
tion.
2. Experimental
2.1. Reagents
Analytical-reagent grade acetonitrile, methanol, hexane and
cyclohexane were purchased from Lab-Scan (Dublin, Ireland).
Deionized water was obtained from a Milli-Q water purification
system (Millipore, Bedford, MA, USA).
Six commercially available oxazole fungicides were included in
the optimization of the procedure: hymexazol (5-methylisoxazol-
3-ol or 5-methyl-1,2-oxazol-3-ol) from Sigma (Steinheim,
Germany, 90%), drazoxolon ((EZ)-4-(2-chlorophenylhydrazono)-
3-methyl-1,2-oxazol-5(4H)-one, vinclozolin ((RS)-3-(3,5-dichloro-
phenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione), chlozolina-
te (ethyl(RS)-3-(3,5-dichlorophenyl)-5-methyl-2,4-dioxo-1,3-oxa-
zolidine-5-carboxylate), oxadixyl (2-methoxy-N-(2-oxo-1,3-oxa-
zolidin-3-yl)acet-2 ,6 -xylidide) and famoxadone ((RS)-3-anilino-
5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione) all
from Riedel-de-Haën (Steinheim, Germany, >99%). Stock solu-
tions (100 ␮g mL−1 ) were prepared by dissolving the commercial
products, without previous purification, in methanol, except
famoxadone, which was supplied as a 100 ␮g mL−1 solution in
acetonitrile. All were kept at 4 ◦ C in dark bottles sealed with
PTFE/silicone caps. Working standard solutions were prepared
daily by diluting with water. Other reagents used were sodium chlo-
ride (Sigma, St. Louis, MO, USA), sodium acetate (Riedel-de-Haen,
99%) and acetic acid (Fluka, Buchs, Switzerland, 99.8%).
2.2. Instrumentation
The UPLC system consisted of an Agilent 1200 (Agilent, Wald-
bronn, Germany) binary pump (G1312B) operating at a flow-rate
of 1 mL min−1 . The solvents were degassed using an on-line mem-
brane system (Agilent 1100, G1379A). The column was maintained
in a thermostated compartment (Agilent 1200, G1316B). The detec-
tor was an Agilent 1100 (G1315C) set at 200 nm. Separation was
performed on a Zorbax Eclipse XDB-C18 (Agilent) rapid resolution
column (50 mm × 4.6 mm × 1.8 ␮m). The injection was performed
using an autosampler (Agilent 1200, G1367C) with a 20 ␮L sample
loop.
For SBSE, stir bars of 10-mm length coated with a 0.5 mm
layer of polydimethylsiloxane (PDMS) were obtained from Gerstel
(TwisterTM , Gerstel GmbH, Müllheim a/d Ruhr, Germany). Analyses
were performed in 15 mL amber glass vials and the solutions were
stirred with a magnetic stirrer (IKA RH KT/C, Supelco) at 1700 rpm.
179
To prevent analyte evaporation, vials sealed with hole-caps and
PTFE/silicone septa were used. To control the temperature of the
extraction step, a homemade heating system consisting of a drilled
block provided with an electronic temperature control system was
used. The desorption step was carried out in glass micro-inserts
(3 cm length, 6 mm O.D. and 4 mm I.D.) into 2 mL HPLC vials and
aliquots of 20 ␮L were injected into the chromatographic system
using the autosampler.
The MASE device (Gerstel) consisted of a 15 mL amber glass vial
with a membrane insert made of dense polypropylene (4-cm long
with a wall thickness of 0.03 mm and an internal diameter of 6 mm).
Membrane bags were conditioned by extracting three times with
hexane at room temperature.
2.3. Samples
The samples of different types of wine, must and fruit juices
were obtained commercially. Recovery experiments were carried
out using samples spiked with a standard mixture of fungicides.
The samples were left to equilibrate at room temperature for at
least half an hour before starting the extraction procedure.
2.4. Analytical procedure for SBSE-UPLC
The stir bars were maintained in pure ACN before use. The anal-
yses were performed in triplicate in 15 mL vials, into which 5 mL
of wine or juice, 5 mL of 0.2 M acetate/acetic acid buffer (pH 5)
and 2 g sodium chloride were added. A twisterTM was introduced
into the sample, which was stirred at 1700 rpm with an extrac-
tion temperature of 60 ◦ C for 30 min. After extraction, the twister
was rinsed in distilled water and the residual water droplets were
removed with tissue paper. For liquid desorption of the analytes,
the stir bar was put into an HPLC vial with a glass micro-insert
and was extracted with 100 ␮L of a 80/20 (v/v) ACN/water solution
and a desorption time of 15 min. Then, the stir bar was removed
and the liquid sample was analyzed by UPLC by injecting a 20 ␮L
aliquot with the autosampler. The mobile phase used in isocratic
conditions was a 50/50 (v/v) ACN/water solution. The flow-rate
was 1 mL min−1 throughout. Blank runs of the stir bar were made
occasionally before the analysis to avoid memory effects.
2.5. Analytical procedure for MASE-UPLC
The analyses were performed in triplicate in 15 mL vials, which
were filled with 5 mL of the solution containing the standards, 5 mL
of 0.2 M acetate/acetic acid buffer (pH 5) and 2 g sodium chloride.
The membrane bag (4-cm long, 6 mm internal diameter, 0.03 mm
wall thickness) was fixed to the metal funnel using a Teflon ring, and
the funnel was suspended in the opening of the vial. The membrane
bag was filled with 800 ␮L hexane and the vial was sealed with
a metallic crimp cap [32]. The vial was stirred at 1700 rpm with
an extraction temperature of 30 ◦ C for 30 min. After extraction, the
organic extract was removed manually from the membrane bag
using a microlitre syringe and transferred into a 2 mL sampler vial.
The organic solvent was evaporated using a stream of argon and
the sample was reconstituted with 0.5 mL water. The liquid sample
was analyzed by UPLC as above indicated.
3. Results and discussion
3.1. UPLC separation
The optimal separation conditions of oxazole fungicides were
established by UPLC using a column with a 1.8 ␮m particle size.
Mixtures of ACN and water were used as the mobile phase and
180 P. Viñas et al. / J. Chromatogr. A 1194 (2008) 178–183
Fig. 1. Elution profile obtained for a standard fungicide mixture by UPLC-DAD. Peaks
correspond to: (1) oxadixyl (0.5 ␮g mL−1 ); (2) hymexazol (5 ␮g mL−1 ); (3) chlozoli-
nate (1 ␮g mL−1 ); (4) drazoxolon (0.2 ␮g mL−1 ); (5) vinclozolin (0.5 ␮g mL−1 ) and
(6) famoxadone (0.5 ␮g mL−1 ).
the ratio of ACN to water was varied by isocratic elution between
40 and 70% (v/v) to achieve the best separation of the fungicides.
As expected, both retention time and peak width decreased as the
percentage increased. Finally, a 50% (v/v) concentration was cho-
sen since this allowed elution and separation of all the compounds.
The influence of the mobile phase pH was studied by several 5 mM
acetate/acetic acid buffer solutions in the 3–8 range, and since
retention and peak shape were only slightly modified by the buffer
and separation efficiency was not improved, an ACN/water mixture
was selected. The strong influence of column temperature on com-
pound retention was studied and retention factors decreased for
all the analytes as temperature increased, which also had a bene-
ficial effect in decreasing the back pressure of the system. Thus, a
temperature of 50 ◦ C was selected.
Fig. 1 shows the chromatogram obtained by UPLC-DAD for
a mixture of the six standard fungicides under the selected
chromatographic conditions, without using sample pre-treatment
procedures. For quantitation of the fungicides, the external stan-
dard procedure was used and calibration graphs were obtained by
plotting concentration against peak area, following linear regres-
sion analysis. The analytical data for the calibration graphs, linearity
intervals, limits of detection (calculated as three times the signal-
to-noise ratio) and limits of quantitation (calculated as 10 times the
signal-to-noise ratio) are included in Table 1. Although the LODs
obtained were very low, sensitivity could be improved by using
new sample treatment procedures, SBSE or MASE.
3.2. SBSE optimization
The only commercial polymer developed as a coating for stir bars
is PDMS, an apolar phase. However, hymexazol and oxadixyl were
Table 1
Analytical data for oxazole fungicides using UPLC-DAD
Parameter Oxadixyl Hymexazol
Slopea (mA mL ␮g−1 ) 283 ± 7 4.7 ± 0.1
Intercepta 1.4 ± 0.4 −3 ± 1
Correlation coefficient 0.9993 0.9997
Linearity (␮g mL−1 ) 0.03–5 3.5–50
Detection limit (␮g mL−1 ) 0.01 1 0.25
Quantitation limit (␮g mL−1 ) 0.03 3.3
a
Mean ± standard deviation (n = 6).
not extracted by this coating. Consequently, the SBSE procedure
using the direct immersion mode can only be applied to the other
four fungicides.
The most important parameter affecting SBSE was found to be
extraction time, which was varied between 10 and 60 min (Fig. 2A).
The extraction time profiles for the fungicides using the PDMS
stir bar showed that the extraction efficiency increased up to
approximately 30 min and then levelled off, except in the case of
chlozolinate, which required 45 min. A time of 30 min was selected
to decrease the total analysis time. On the other hand, several par-
allel extractions were performed at the same time, allowing an
additional reduction of the overall analysis time. To check whether
the amount of analytes extracted in the stir bar increases with tem-
perature, this effect was checked in the 25–70 ◦ C range. Fig. 2B
shows that the extraction efficiency increased up to 60 ◦ C for all
compounds, and then decreased at higher temperatures, due to
losses caused by the volatility of the compounds. The extraction
temperature selected was 60 ◦ C. The stirring speed was varied
between 0 and 2000 rpm and was also seen to have a notice-
able effect on the extraction efficiency, faster speeds enhancing
the diffusion of analytes from the sample to the stir bar. The sig-
nals continuously increased up to a speed of 1700 rpm, which was
selected.
The pH of the sample can be adjusted to decrease the solubil-
ity of some analytes and to provide better extraction when they
are mainly present in their neutral form. For this reason, the pH
effect was studied in the 3–8 range, using 0.1 M acetate/acetic acid
buffer solutions. As can be seen in Fig. 3A, the extraction efficiency
increased up to pH 5, especially in the case of famoxadone and vin-
clozolin, and then decreased at higher pH values. Since maximal
extraction was obtained at pH 5, this was selected. The effect of
ionic strength on the extraction efficiency was studied by adding
sodium chloride to give 0–35% (m/v) concentrations. Extraction
improved up to 10 or 25% (m/v), depending on the compounds, and
then decreased (Fig. 3B). Therefore, a compromise concentration of
20% (m/v) was selected.
The optimal sample volume/buffer volume ratio in the vial con-
taining an extraction solution volume of 10 mL was studied by
using a wine sample fortified with 100 ng mL−1 for all compounds
(except chlozolinate, 300 ng mL−1 ) at 5:5, 4:6 and 3:7. The signal
was highest for all the analytes at a volume ratio of 5:5, which was
selected.
For liquid desorption of the compounds, the stir bar was put into
a vial with a glass micro-insert and the solvent and desorption time
were optimized. The solvents assayed for the extraction were pure
ACN and different mixtures of ACN/water and ACN/buffer. Maxi-
mal extraction efficiency was achieved with 100 ␮L of a 80/20 (v/v)
ACN/water solution. The desorption time was varied between 5
and 20 min and optimal results were obtained at 15 min, which
achieved the total desorption of analytes. The existence of a mem-
ory effect was investigated by a second liquid desorption of the
bar under the same desorption conditions. The effect was insignif-
icant.
Chlozolinate Drazoxolon Vinclozolin Famoxadone
25.8 ± 0.4 1241 ± 38 135 ± 3 200 ± 8
−6 ± 2 8±3 6±4 0.5 ± 0.8
0.9998 0.9985 0.9994 0.9980
0.8–50 0.01–1 0.05–2 0.05–2
0.0025 0.015 0.015
0.83 0.008 0.05 0.05
 P. Viñas et al. / J. Chromatogr. A 1194 (2008) 178–183 181

Fig. 2. Influence of the extraction time (A) and extraction temperature (B) using SBSE-UPLC. Sample volume, 10 mL; 50 ng mL−1 fungicides (except 150 ng mL−1 chlozolinate).

3.3. MASE optimization
The choice of extraction solvent was based on its polarity. Cyclo-
hexane, hexane, methanol and acetonitrile proved to be optimal
extraction solvents and were tested. The results suggested that nei-
ther methanol nor acetonitrile provided reproducible extractions as
they were water miscible solvents and diffused through the mem-
brane into the aqueous phase, so that the final volume of the organic
phase was reduced after stirring. Cyclohexane led to poor extrac-
tion efficiency for all compounds, especially chlozolinate. Maximal
extraction of drazoxolon, vinclozolin, chlozolinate and famoxadone
was obtained with hexane, which was selected. However, hymex-
azol and oxadixyl were not extracted by MASE, as had occurred
when using the SBSE procedure. The extraction temperature was
fixed at 30 ◦ C to avoid evaporation of the organic solvent. To opti-
mize the extraction yield, the extraction time was maintained at
30 min (the time optimized for SBSE to permit comparison of both
methods) and the highest stirring rate of 1700 rpm was chosen. The
presence of salt and a change in pH can significatively influence
the extraction of analytes. As in the SBSE method, the addition of
sodium chloride at a 20% (m/v) concentration improved the extrac-
tion efficiency, while adjusting the sample to pH 5 using a 0.1 M
acetate/acetic acid buffer solution provided the best extraction
yield.
3.4. Validation and analytical data. Comparison of both sample
pre-treatment methodologies
The method was validated for linearity, detection limit, selec-
tivity, accuracy and precision. Calibration curves using both
SBSE-UPLC-DAD and MASE-UPLC-DAD were obtained by least-
squares linear regression analysis of the peak area versus analyte
concentration using six concentration levels. Quantification was
performed by the external standard procedure. The validation
parameters, range of linearity and the correlation coefficients
for the fungicides are shown in Table 2. The values of r2 were
good (r2 > 0.99) and excellent linearity was obtained for the range
studied. The limits of detection (calculated as three times the
signal-to-noise ratio) and quantitation (calculated as 10 times
the signal-to-noise ratio) are included in Table 2. It can be seen
from the data that the sensitivity and detection limits for all
fungicides using the sample pre-treatment methods were sig-
nificantly better than when the direct UPLC method was used.
Thus, when using SBSE, detection limits were 100 times lower
for chlozolinate, vinclozolin and famoxadone, and 50 times lower
for drazoxolon. To check the repeatability of the method, 10 repli-
cate analyses of the standards were performed at the level of two
times the quantitation limit for each compound, 15 ng mL−1 for
chlozolinate, 0.5 ng mL−1 for drazoxolon and 1 ng mL−1 for vin-

Fig. 3. Influence of the pH (A) and sodium chloride concentration (B) using SBSE-UPLC. Sample volume, 10 mL; 50 ng mL−1 fungicides (except 150 ng mL−1 chlozolinate).
182 P. Viñas et al. / J. Chromatogr. A 1194 (2008) 178–183

Table 2
Analytical data for oxazole fungicides using the UPLC optimized SBSE- and MASE-based methods

Parameter Chlozolinate Drazoxolon Vinclozolin Famoxadone

SBSE-UPLC-DAD method
Slopea (mA mL ng−1 ) 2.11 ± 0.02 78 ± 2 18.7 ± 0.2 19.8 ± 0.2
Intercepta −7 ± 2 10 ± 4 12 ± 5 1±1
Correlation coefficient 0.9999 0.9999 0.9999 0.9999
Linearity (ng mL−1 ) 5–250 0.2–20 0.5–75 0.5–75
Detection limit (ng mL−1 ) 2.5 0.05 0.15 0.15
Quantitation limit (ng mL−1 ) 8.0 0.15 0.50 0.50
Repeatability (RSD, n = 10) 5.3 5.4 6.3 7.9

MASE-UPLC-DAD method
Slopea (mA mL ng−1 ) 0.46 ± 0.01 0.43 ± 0.02 0.85 ± 0.03 0.16 ± 0.01
Intercepta 10.7 ± 2.6 −0.43 ± 0.4 −7.8 ± 3.7 6.5 ± 1.2
Correlation coefficient 0.9991 0.9983 0.9984 0.9966
Linearity (ng mL−1 ) 30–500 30–500 10–225 40–250
Detection limit (ng mL−1 ) 7.5 10.5 2.5 12
Quantitation limit (ng mL−1 ) 25 35 8 40
Repeatability (RSD, n = 10) 8.4 8.7 9.5 9.2
a
Mean ± standard deviation (n = 6).

Table 3
Calibration slopesa for different samples using SBSE-UPLC (mA mL ng−1 )

Compounds Aqueous standards Ethanol standards Red wine White wine Rosé wine Fruit juice

Chlozolinate 2.11 ± 0.02 0.52 ± 0.02 0.46 ± 0.01 0.56 ± 0.02 0.53 ± 0.03 1.85 ± 0.1
Drazoxolon 78 ± 2 41 ± 0.4 43 ± 1 48 ± 1 47 ± 2 81 ± 1
Vinclozolin 18.7 ± 0.2 15.7 ± 0.2 14.3 ± 0.3 15.5 ± 0.6 15.3 ± 0.3 17.6 ± 1
Famoxadone 19.8 ± 0.2 14.4 ± 0.2 13.0 ± 0.3 15.9 ± 0.5 15.5 ± 0.7 17.2 ± 0.8
a
Mean ± standard deviation (n = 5).

clozolin and famoxadone, with RSD <8% in all cases (Table 2). 3.5. Recovery
These values indicate that the precision of the method was
satisfactory for control residue analysis. In general, the SBSE- In order to check the accuracy of the proposed method, a recov-
based method showed better results for sensitivity, repeatability ery study was carried out by fortifying four samples (red wine,
and analyte recovery than MASE. Considering the chromato- white wine, rosé wine and fruit juice) at two concentration lev-
graphic and validation parameters, the SBSE-UPLC method was els corresponding to two and four times the quantitation limits
most appropriate for the analysis of oxazole fungicide residues (Table 4). The recoveries of the fungicides from spiked samples
in wine and fruit juice samples according to the established varied from 83 to 113% with an average recovery ± SD (n = 32) of
MRLs. 99.4 ± 7. The similarity in recoveries obtained for each fungicide
The selectivity of the method was judged from the absence of in the four samples indicates that the matrix had no influence on
interfering peaks at the elution times of the analytes for blank chro- method performance.
matograms of different samples of wine and fruit juice samples
without any spiking. 3.6. Analysis of samples
The matrix effect of the different samples for the studied fungi-
cides was evaluated by comparing the slopes of aqueous standards Fig. 4 shows typical chromatographic profiles obtained using
and standard additions calibration graphs for different matrices, SBSE-UPLC for a wine sample (A) and the same sample fortified
obtained by plotting concentration (at five levels) against peak with the fungicides (B). Similar chromatograms were obtained for
area and following linear regression analysis. As wine samples the other samples. The profiles demonstrated the absence of inter-
contain approximately 14% (v/v) ethanol, calibration was also per- fering peaks. The chromatographic peaks were first identified by
formed for the standards in the presence of 7% (v/v) ethanol (as comparing the retention data obtained for the standards, the sam-
the samples were diluted in a 1/1 ratio). Table 3 shows the data
obtained. As can be seen, slopes in the presence of 7% (v/v) ethanol Table 4
are lower than those for aqueous standards (especially for chlo- Recoveries of fungicides from different spiked samples (n = 32)
zolinate and drazoxolon). A statistical study was carried out to
Compound Spike level Red wine White wine Rosé wine Juice
compare the slope values of the different wine samples and the (ng mL−1 )
ethanol standards and the Kruskal–Wallis one-way analysis of vari-
Chlozolinate 16 101.7 112.3 102.6 95.6
ance on ranks test showed that there was no statistically significant
32 102.3 96.2 94.9 105.0
difference (P = 0.7288). The comparison of slopes for fruit juice
samples and aqueous standards again demonstrated the absence Drazoxolon 0.3 82.6 101.9 103.9 97.0
0.6 102.1 90.6 97.1 99.9
of statistically significant differences (P = 0.6857). Consequently,
the standard additions method was discarded and calibration Vinclozolin 1.0 87.2 107.5 102.8 105.0
2.0 106.8 95.3 98.2 95.0
was carried out directly with aqueous standards for the fruit
juices and using ethanol-containing standards for the wine sam- Famoxadone 1.0 102.3 107.5 94.6 88.9
ples. 2.0 94.9 91.1 104.1 113.2
 P. Viñas et al. / J. Chromatogr. A 1194 (2008) 178–183
Fig. 4. Elution profile obtained for a red wine (A) and the spiked wine (B) by
SBSE-UPLC-DAD. Sample volume, 10 mL; extraction time, 30 min; extraction tem-
perature, 60 ◦ C; desorption time, 15 min. Peaks correspond to: (1) chlozolinate
(50 ng mL−1 ); (2) drazoxolon (1 ng mL−1 ); (3) vinclozolin (0.5 ng mL−1 ) and (4)
famoxadone (1 ng mL−1 ).
ples and the different samples spiked with the standards under
identical conditions. The average values for the retention times of
fungicides pointed to very good agreement between the retention
data obtained in the different samples. The peaks were identified
again using the DAD detector to continuously measure the spec-
trum while the solute passed through the flow-cell, thus confirming
the identity and the purity of the peaks. Good agreement was found
between the UV spectra of the different peaks obtained for the
standards, the samples and the spiked samples.
After identification of the peaks, 10 different samples were ana-
lyzed corresponding to two red wines, two white wines, two rosé
wines, two musts (red and white) and four fruit juices (peach, grape,
strawberry and multifruits) using the SBSE-UPLC procedure. All
samples were analyzed in triplicate. Chlozolinate was found in a
red wine at a concentration of 94 ± 2 ng mL−1 and drazoxolon in a
grape juice at 0.19 ± 0.1 ng mL−1 .
The MRLs fixed for oxazole fungicides in grapes are between 0.05
and 5 mg kg−1 [4], but the Organisation Internationale de la Vigne
et du Vin (OIV) suggests as MRL levels of fungicides in wines about
1/10 of the corresponding MRL set for grapes [33]. Consequently,
taking 0.005–0.5 mg L−1 as the MRLs for fungicides in wine, the lev-
els found in the wine samples were adequately quantified without
sensitivity problems using the SBSE-UPLC procedure.
4. Conclusion
This work is the first reported analytical study showing the
use of two different sample pre-treatment methodologies, SBSE
and MASE, coupled to UPLC with DAD detection for the analysis
of oxazole fungicide residues (hymexazol, drazoxolon, vinclo-
zolin, chlozolinate, oxadixyl and famoxadone) in wine and fruit
183
juices. SBSE and MASE are non-harmful environmentally pre-
concentration systems which avoid the use of organic solvents
and clean-up steps, thus reducing the sample preparation time
and allowing lower detection limits than when the direct UPLC
method is used. The SBSE-based method showed better results
for sensitivity, repeatability and analyte recovery than MASE and
the procedure was successfully applied to determine the fungicide
content of wine, must and fruit juices, using instrumentation acces-
sible for almost every laboratory equipped for carrying out routine
controls.
Acknowledgements
The authors are grateful to Comunidad Autónoma de la Región
de Murcia (CARM, Fundación Séneca, Project 02993/PI/05) and
to the Spanish MEC (Project CTQ2006-08037/BQU) for financial
support. N. Aguinaga acknowledges a fellowship from CajaMurcia
(Spain).
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