Professional Documents
Culture Documents
com
Abstract
A method for the chiral separation of propiconazole using cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC)
with hydroxypropyl-␥-cyclodextrin (HP-␥-CD) as chiral selector is reported. The use of a mixture of 30 mM HP-␥-CD, 50 mM SDS,
methanol–acetonitrile 10%:5% (v/v) in 25 mM phosphate buffer solution was able to separate two enantiomeric pairs of propiconazole. Stacking-
and sweeping-CD-MEKC under neutral pH (pH 7) and under acidic condition (pH 3.0) were used as two on-line preconcentration methods to
increase detection sensitivity of propiconazole. Good repeatabilities in the migration time, peak area and peak height were obtained in terms of
relative standard deviation (RSD). A sensitivity enhancement factor of 100-fold was achieved using sweeping-CD-MEKC at acidic pH. This is
the first report on the separation of two pairs of propiconazole enantiomers and all the enantiomers of fenbuconazole and tebuconazole using
sweeping-CD-MEKC. The limit of detection (S/N = 3) for the three triazole fungicides ranged from 0.09 to 0.1 g/mL, which is well below the
maximum residue limits (MRL) set by Codex Alimentarius Commission (CAC). Combination of solid-phase extraction (SPE) pretreatment and
sweeping-CD-MEKC procedure was applied to the determination of selected triazole fungicides in grapes samples spiked at concentration 10–40
times lower than the MRL established by the CAC. The average recoveries of the selected fungicides in spiked grapes samples were good, ranging
from 73% to 109% with RSD of 9–12% (n = 3).
© 2007 Elsevier B.V. All rights reserved.
Keywords: Propiconazole; Chiral triazole fungicides; Enantiomer separation; Capillary electrophoresis; CD-MEKC; On-line preconcentration; Stacking; Sweeping
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2007.09.003
108 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113
sample preconcentration techniques, sample stacking and two pairs of propiconazole enantiomers and all the enantiomers
sweeping are known to be effective techniques for enhancement of fenbuconazole and tebuconazole by CD-MEKC. The opti-
of the concentration sensitivity in MEKC [19–21]. mized on-line sample preconcentration-CD-MEKC procedure
Triazole fungicides represent the most important category of combined with SPE pretreatment was applied to the determi-
fungicides to date. They have excellent protective, curative and nation of selected triazole fungicides in grapes samples spiked
eradicant power towards a wide spectrum of crop diseases. Chi- at levels 10–40 times lower than the maximum residue limits
rality is expected to play a crucial role in the bioactivities of (MRL) set by Codex Alimentarius Commission (CAC) [26].
triazole fungicides. The enantiomeric separations of triazole-
type fungicides have been performed using CE procedures 2. Experimental
[22–25]. Mixtures of uniconazole and diniconazole enantiomers
were baseline resolved using 50 mM phosphate buffer (pH 6.5) 2.1. Chemicals and reagents
containing 5 mM carboxymethyl-␥-CD and a temperature of
50 ◦ C in less than 5 min [22]. Simultaneous chiral separation Propiconazole, tebuconazole and fenbuconazole were
of triadimefon and triadimenol [23] and chiral separation of obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany),
14 triazole fungicides (including propiconazole, tebuconazole, 2-hydroxypropyl-␥-cyclodextrin (HP-␥-CD) was obtained from
without fenbuconazole) [24] were successfully achieved using Sigma (St. Louis, MO, USA), sodium dodecyl sulfate (SDS)
sulfated--cyclodextrin-mediated CE under acidic conditions. was obtained from Fisher Scientific (Loughborough, UK), and
However, the limit of detections (LODs) were not discussed. disodium hydrogen phosphate 12-hydrate was obtained from
Cyclodextrin-modified micellar electrokinetic chromatography Riedel-de Haen (Seelze, Germany). All other chemicals and sol-
(CD-MEKC) has also been employed to the enantiomeric vents were common brands of analytical-reagent grade or better,
and isomeric separation of seven commonly used pesticides and were used as received. Water was collected from a Millipore
(including propiconazole). However, only two diastereomers of Water Purification System (Molsheim, France). Fresh grapes
propiconazole were baseline separated in this study [25]. It is were obtained from local market in Skudai, Johor, Malaysia.
therefore of interest to study the CD-MEKC method for the enan- The stock solutions of the individual triazole fungi-
tiomeric separation of two pairs of propiconazole enantiomers cides were prepared in methanol in the concentration range
and the capabilities of two on-line preconcentration methods, i.e. 2000–4000 mg/L. Final dilutions (concentrations in the figures)
stacking- and sweeping-CD-MEKC were evaluated. Sweeping- were prepared by diluting the stock solution with various sam-
CD-MEKC at acidic pH was found to give the highest sensitivity ple solvents mentioned at the respective places. Sample solutions
(100-fold) and the method was applied to the chiral separation were prepared by diluting the stock solution with pure water for
of propiconazole enantiomers followed by the chiral separation normal stacking mode-CD-MEKC and with a buffer/separation
of two other triazole fungicides, i.e. fenbuconazole and tebu- solution without micellar phase for sweeping-CD-MEKC. The
conazole (Fig. 1). This is the first ever reported separation of separation solutions for CD-MEKC were prepared by dissolving
appropriate amounts of SDS, HP-␥-CD, methanol and acetoni- the calibration curve along with the signal-to-noise ratio (S/N)
trile in phosphate buffers and adjusting the pH of the buffer as 3.
with phosphoric acid solution. All running buffers were filtered
through a 0.45 m nylon syringe filter from Whatman (Clifton, 3. Results and discussion
NJ, USA).
3.1. Optimization of the MEKC conditions
2.2. Instrumentation
The chiral separation of propiconazole enantiomers was first
All electropherograms were obtained with the Agilent explored by MEKC using bile salts (sodium cholate and sodium
capillary electrophoresis system (Agilent Technologies, Wald- deoxycholate) as chiral surfactants. Effects of different bile salt
bronn, Germany), equipped with temperature control and diode types and concentrations on the enantiomeric separation of prop-
array detection (DAD). Separations were performed using an iconazole were explored to resolve the four enantiomers of
untreated fused silica capillary of 64.5 cm × 50 m i.d. (with an propiconazole in this initial study. However, only two isomers
effective length of 56 cm to the detector window) obtained from of propiconazole were observed in these experiments (electro-
Polymicro Technologies (Phoenix, AZ, USA). Sample injec- pherogram not shown). Since the separation of the propiconazole
tion was performed hydrodynamically for 1–120 s at 50 mbar. enantiomers was not satisfactory, the use of an achiral ionic sur-
The detection wavelength used was 200 nm and the capillary factant, SDS, with HP-␥-CD as chiral selector was then explored.
temperature was maintained at 20 ◦ C. Data were collected and As the previous work by Wu et al. [24] and Penmetsa [25] has
processed on computer using ChemStation software (Agilent not been successful in separating the four stereoisomers of prop-
Technologies). The new capillary was conditioned by pass- iconazole, our work focused on the optimization process and
ing 1 M NaOH solution for 10 min followed by washing with applying the optimum conditions to two on-line preconcentra-
deionized water for 10 min and finally equilibrating with an tion methods, i.e. stacking and sweeping.
appropriate running buffer for 10 min. Between runs, the cap- The HP-␥-CD concentration was first varied in order to
illary was washed with 0.1 M NaOH, water, and run buffer for achieve the best separation of the two pairs of propiconazole
2 min each. All sample injections were performed in triplicate. enantiomers by CD-MEKC. Separation was obtained for two
pairs of propiconazole enantiomers using HP-␥-CD in the con-
2.3. Extraction procedure centration range 10–40 mM (with 50 mM SDS and 15% (v/v) of
methanol in 25 mM phosphate buffer solution, pH 7.0, the run-
A representative portion of sample (150 g of grapes) was ning voltage 30 kV and 20 ◦ C). It was found that the migration
chopped and homogenized. Five-gram portions of sample was times of the propiconazole enantiomers gradually shortened with
spiked with 250 ng each of fenbuconazole, tebuconazole and stepwise increase in the concentration of HP-␥-CD. This obser-
propiconazole, then placed into an Erlenmeyer flask and homog- vation is expected since with increasing HP-␥-CD concentration,
enized with 5 mL of methanol and 5 mL of water by sonication more of the analyte will be included in the HP-␥-CD phase and
for 15 min. The resulting suspension was filtered through a will be transported quickly towards the detector. The best reso-
Buchner funnel and the cake filter was washed twice with 10 mL lutions (Rs > 1.50) and peak efficiency (N > 200 000) were found
of deionized water. The filtrate was adjusted to a volume of to be at 30 mM HP-␥-CD. Based on this data, optimal concen-
100 mL with deionized water and passed under vacuum through tration for HP-␥-CD was set at 30 mM. This concentration was
a C18 solid-phase column, which was preconditioned with 10 mL employed in all subsequent investigations.
of methanol and 10 mL of deionized water. Fungicides were The effect of phosphate buffer concentration on the resolu-
eluted with 2× 5 mL of dichloromethane. The eluate was con- tion and efficiency of the propiconazole enantiomers was also
centrated under stream of nitrogen to dryness. Then it was evaluated at the optimal HP-␥-CD concentration (other condi-
reconstituted with 0.5 mL of buffer solution (without micellar tions were kept constant). An increase in the concentration of
phase). buffer from 10 to 50 mM caused a significant increase in the
migration time of all the propiconazole enantiomers. A 25 mM
2.4. Validation procedure phosphate buffer was selected for optimal concentration since it
yielded very good peak efficiencies (N > 200 000) with all Rs val-
The performance of the system was examined in terms of the ues greater than 1.50. This buffer concentration was employed
linearity, repeatability, increase in detection sensitivity or sensi- in all subsequent investigations.
tivity enhancement factor (SEF), and limit of detection (LOD). The effect of buffer pH on the chiral separation of propi-
The linearity of the optimized method was assessed by construct- conazole by MEKC was also investigated at basic/neutral pH,
ing the calibration curve of average peak areas (n = 3) against the ranging from 9.4 to 7.0. Resolution of all propiconazole enan-
concentration of standards (at the linear range). The repeatabil- tiomers was found to be greater than 1.20. The best separation
ity in the optimized method concerning the relative standard was found to be at pH 7.0 (Rs > 1.50). Based on this data, pH 7.0
deviation for peak area (RSD%, n = 3) was examined for the was employed in normal mode MEKC separation.
injection of the 1 g/mL sample. SEF was calculated by com- The effect of SDS concentration was also evaluated in this
paring (peak area obtained with sweeping/peak area with usual study, ranging from 10 to 40 mM. At 10 and 25 mM SDS, prop-
MEKC injection) × dilution factor. The LOD was determined by iconazole enantiomers were only partly separated, while at 50
110 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113
Table 1
Linearity, repeatability, LODs (S/N = 3) and sensitivity enhancement factor (SEF) for propiconazole enantiomers in the optimized CD-MEKC and on-line-CD-MEKC
methods under neutral condition (pH 7.0)
CD-MEKC Stacking-CD-MEKC Sweeping-CD-MEKC
Analysis conditions as in Fig. 2. SEFarea : (peak area obtained with on-line-CD-MEKC/peak area with usual MEKC injection) × dilution factor. SEFLOD :
(LODCD-MEKC /LODon-line-CD-MEKC ).
W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113 111
(r2 > 0.996) and RSDs for migration time, peak height and peak
area were less than 6.0%. There is a 5-fold increase in the detec-
tion sensitivity for each propiconazole enantiomer in the normal
stacking mode CD-MEKC method (LOD = 2.0 g/mL, S/N = 3)
and about 25-fold increase in the sweeping-CD-MEKC method
(LOD = 0.4 g/mL) compared to CD-MEKC method at pH 7.0
(LOD = 10 g/mL). Although these values are modest for the
analysis of real samples, the results clearly show the possibility
of improving the detectability by using stacking and sweeping
in CD-MEKC method at neutral pH.
Table 2
Linearity, repeatability, sensitivity enhancement factor (SEF) and LODs (S/N = 3) of the optimized sweeping-CD-MEKC method under acidic condition (pH 3.0)
Fungicide Peaka Regression equationb r2 RSD peak area (%, n = 3) SEFarea c LODs (g/mL)
area : (peak area obtained with sweeping/peak area with usual MEKC injection) × dilution factor.
c SEF
112 W.A. Wan Ibrahim et al. / J. Chromatogr. A 1170 (2007) 107–113
Table 3
Percent recovery and repeatability of three selected triazole fungicides spiked in
grapes sample (amount of sample processed 5 g) by SPE-sweeping-CD-MEKC
method (pH 3.0) and MRL established by the Codex Alimentarius Commission
Fungicide Fortified Average RSD MRL
control (mg/kg) recovery (%) (%, n = 3) (mg/kg)
4. Conclusion
[10] B. Chankvetadze, J. Chromatogr. A 792 (1997) 269. [19] J.B. Kim, J.P. Quirino, K. Otsuka, S. Terabe, J. Chromatogr. A 916 (2001)
[11] S.H. Edwards, S.A. Shamsi, Electrophoresis 23 (2002) 1320. 123.
[12] D. Iqbal, S.A.A. Rizvi, C. Akbay, S.A. Shamsi, J. Chromatogr. A 1043 [20] J.B. Kim, S. Terabe, J. Pharm. Biomed. Anal. 30 (2003) 1625.
(2004) 291. [21] J. Palmer, N.J. Munro, J.P. Landers, Anal. Chem. 71 (1999) 1679.
[13] J.J.B. Nevado, C.G. Cabanillas, M.J.V. Llerena, V.R. Robledo, J. Chro- [22] Y.M. Biosca, C.G. Ruiz, M.L. Marina, Electrophoresis 21 (2000)3240.
matogr. A 1072 (2005) 249. [23] Y.S. Wu, H.K. Lee, S.F.Y. Li, Electrophoresis 21 (2000) 1611.
[14] K. Otsuka, S. Terabe, J. Chromatogr. A 875 (2000) 163. [24] Y.S. Wu, H.K. Lee, S.F.Y. Li, J. Chromatogr. A 912 (2001) 171.
[15] J. Jiang, C.A. Lucy, J. Chromatogr. A 966 (2002) 239. [25] K.V. Penmetsa, Ph.D. Dissertation, North Carolina State University,
[16] M. Molina, S.K. Wiedmer, M. Jussila, M. Silva, M.L. Riekkola, J. Chro- Raleigh, NC, 1997. UMI Number: 9804241.
matogr. A 927 (2001) 191. [26] http://www.codexalimentarious.net/mrls/pestdes/jsp checked on June
[17] R.C. Martınez, E.R. Gonzalo, P.R. Ruiz, J.D. Alvarez, J. Chromatogr. A 2007.
990 (2003) 291. [27] P. Wang, S. Jiang, D. Liu, P. Wang, Z. Zhou, J. Biochem. Biophys. Methods
[18] A.J. Garcia, Y. Pico, G. Font, J. Chromatogr. A 1073 (2005) 229. 62 (2005) 219.