Application of capillary zone electrophoresis with large-volume sample stacking to the

Transcription

4060
Jorge J. Soto-Chinchilla
Ana M. García-Campaña
Laura Gámiz-Gracia
Carmen Cruces-Blanco
Department of Analytical Chemistry,
Faculty of Sciences,
University of Granada,
Granada, Spain
Received March 21, 2006
Revised May 6, 2006
Accepted May 21, 2006
Electrophoresis 2006, 27, 4060–4068
Research Article
Application of capillary zone electrophoresis
with large-volume sample stacking to the
sensitive determination of sulfonamides in
meat and ground water
A CZE method with UV-Vis detection has been established and validated for the
determination of nine sulfonamides: sulfapyridine, sulfamethazine, sulfamerazine, sulfamether, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfachlorpyridazine, and
sulfamethizole. Optimum separation was obtained on a 64.5 cm675 mm bubble cell
capillary using a buffer containing 45 mM sodium phosphate and 10% methanol at
pH 7.3, with temperature and voltage of 277C and 25 kV, respectively. p-Aminobenzoic
acid was used as an internal standard . Taking into account the lack of sensitivity of the
UV-Vis detection, the application of an on-line preconcentration methodology, such as
large-volume sample stacking with polarity switching has been proposed. This procedure combined with a solvent extraction/SPE method applied for off-line preconcentration and cleanup provides a significant improvement in the LODs, ranging
from 2.59 to 22.95 mg/L for the studied compounds; the quantification of these residues being possible below the levels established by EU legislation in animal food
products, such as meat. Satisfactory recoveries were also obtained in the analysis of
these compounds in ground water.
Keywords: Capillary zone electrophoresis / Large-volume sample stacking / Meat /
Solid phase extraction / Sulfonamides / Water
DOI 10.1002/elps.200600166
1 Introduction
Many veterinary drugs are used for the treatment of foodproducing animals. Among them, there is a class of antibiotics named sulfonamides which are antibacterial and
anti-infective drugs, commonly used for the treatment of
diseases in medicine and veterinary practice, such as
gastrointestinal and respiratory infections, being
administrated orally or mixed with animal feed with therapeutic and prophylactic purposes. Because of their
Correspondence: Professor Ana M. García-Campaña, Department
of Analytical Chemistry, Faculty of Sciences, University of Granada,
Avd. Fuente Nueva s/n, E-18071 Granada, Spain
E-mail: [email protected]
Fax: 134-958-249510
Abbreviations: HLB, hydrophilic–lipophilic balance; IS, internal
standard; LVSS, large-volume sample stacking; MRL, maximum residue level; NSM, normal stacking mode; PABA, p-aminobenzoic acid;
SCP, sulfachlorpyridazine; SDM, sulfadimethoxine; SDZ, sulfadiazine; SMI, sulfamethizole; SPD, sulfapyridine; SMR, sulfamerazine;
SMT, sulfamether; SMX, sulfamethoxazole; SMZ, sulfamethazine
© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
broad spectrum of activity and their low costs [1], they are
widely used nowadays, but the problem is that sometimes they are applied without respecting safety recommendations which results in undesirable residues in animal tissues, meat, or biofluids such as milk. As it has been
observed that most of the sulfonamides are suspected to
be carcinogenic, and produce thyroid tumors in rodents
[2], affecting also human [3], the use of these compounds
is of great public health concern and their residues have
to be controlled. Due to the fact that these analytes may
be present in minute concentrations, and may pose a
health threat to consumers, the European Union (EU) has
adopted a maximum sulfonamide residue level (MRL) of
100 mg/kg [4], and their determination in multiple animal
tissues, meat and other animal by-products such as milk
or eggs, have drawn great attention in the last years. As a
consequence of nonadequate treatment of human and
animal excretions, antibiotic residues can be present in
the environment for a long period of time, leading to the
appearance of antimicrobial resistance [5]. Sulfonamides
can be used against human diseases; they spread to the
surface water through urban waste water because the
www.electrophoresis-journal.com
actual procedure for waste water treatment cannot completely remove these compounds [5]. Also, because of
their use in veterinary care, they are found in soils, ground,
and surface waters due to the use of animal excretions as
manure.
There are several methods for the determination of
sulfonamides, mainly using chromatographic techniques,
especially HPLC with UV, fluorescence, electrochemical
detector, and MS detectors [7–16]. Recently, chemiluminescence detection has been proposed as sensitive
detection for the HPLC analysis of sulfonamides of veterinary use in milk [17]. Among all of these detection systems employed with HPLC, diode-array detection is the
most widely used and it has been applied for the determination of sulfonamides in milk [18, 19] but, sometimes,
it presents a lack of selectivity, being necessary the
application of efficient matrix cleanup procedures [14] or
a very selective detector. Also, HPLC-MS has been used
for the establishment of multimethods for the analysis of
pharmaceutical residues in environmental samples,
including sulfonamides [20, 21]. GC coupled to MS (GCMS) is relatively sensitive and selective, but routine residue analyses are not feasible because multiple purification steps are required prior to the analysis of thermally
labile and nonvolatile sulfonamides. However, several
methods involving GC-MS have been developed for
detecting sulfonamides after derivatization [22–24].
CE is a popular and powerful separation technique
nowadays, mainly because of many advantageous features of this technique, including high resolution, great
efficiency, rapid analysis, and small consumption of
both sample and solvent in comparison with HPLC. The
development of CE methods to separate diverse analytical samples has been growing very rapidly over the
past decade [25–29], and the technique has demonstrated its efficiency in many applications [30, 31],
including the determination of antibiotics [32–34]. Despite of these, CE is not very common for the determination of sulfonamides. Some papers have been published, using mainly UV [35–37] or amperometric detection [38], applying the CZE mode [39–43] or MEKC [41,
44–46] in different matrices such as pharmaceutical
compounds, biological fluids, or food of animal origin.
The coupling of CE to MS for the determination of sulfonamides is gaining in importance in the last years. A
first approach for their determination in food by CE-MS
was carried out [47], and the characterization of isomeric sulfonamides using CE coupled with the MS/MS
was proposed to identify residues in milk extracts.
Recently, a new methodology for the screening and
analytical confirmation of seven sulfonamide residues in
milk using CE-MS has been published [48].
CE and CEC
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The first use of CE for the analysis of seven sulfonamides
in meat was reported by Ackermans et al. [39], studying
the corresponding migration times and the influence of
the meat extract depending on the running buffer nature.
A simple sample pretreatment consisting on extraction
with ACN and centrifugation was applied, obtaining
detection limits of 2–9 mg/mL. Due to the complex nature
of meat, sample pretreatments are often required to
remove proteins, fat, and potential interferences. Also, an
important problem in the application of CE coupled to UVVis detection is the relatively high detection limits, which
are an important drawback for its application in the control of drug residues in food, according to European
recommendations. In this sense, solvent extraction and
an SPE method were employed for sample cleanup and
preconcentration, prior to the quantitative CE determination of eight sulfonamides in meat, with detection limits
ranging 5–10 mg/kg [35].
Several on-column sample preconcentration methods
can also be combined with CE to increase the amount of
the analyte introduced into the capillary obtaining an
improvement in sensitivity. Some reviews have been
published about different sample stacking techniques
[49–54]. Among the different modes, the simplest one is
normal stacking mode (NSM), which is done by dissolving
the sample in a low conductivity matrix and by injecting
the resulting sample solution hydrodynamically. Focusing
happens at the interface between the low conductivity
matrix and the buffer, due to the abrupt change in electrophoretic velocity. A limitation of NSM is the short optimum sample plug length that can be injected into the
capillary without loss of separation efficiency or resolution
[50]. However, large-volume sample stacking (LVSS) [55]
implies the introduction of a volume, greater than that
found optimum in NSM. In this case, the sample matrix
must be pumped out from the capillary in order to preserve separation efficiency. Pumping is carried out with
external pressure or with EOF, with the direction of
pumping always opposite to that of the electrophoretic
movement of charged solutes, and its velocity lower than
the electrophoretic velocity of the charged solutes. Using
this strategy, only positive or negative solutes can be
effectively concentrated at one time. Concentration factors of more than 100 are reported for LVSS, improving
LOD from two orders of magnitude. In LVSS two modalities exist, with or without change of polarity. For the
case of anions, LVSS with polarity switching is a mode
that permits to control the EOF in CZE separations, involving high EOF conditions to carry the separate analytes to the detector. This is done by introducing hydrodynamically, a large plug of low conductivity sample into
the capillary, and applying negative voltage at the injection end. The large solvent plug is then electroosmotically
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J. J. Soto-Chinchilla et al.
pushed out of the capillary while the negative species
stack-up at the boundary between the sample zone and
the BGE. Once the main part of the low-conductivity zone
has been pushed out of the capillary, the positive voltage
is applied to carry out the separation.
In this paper, we have developed a new method for the
extraction and determination of nine sulfonamides of
veterinary use (sulfapyridine (SPD), sulfamethazine
(SMZ), sulfamerazine (SMR), sulfamether (SMT), sulfadiazine (SDZ), sulfadimethoxine (SDM), sulfamethoxazole
(SMX), sulfachlorpyridazine (SCP), and sulfamethizole
(SMI)), whose maximum residue levels (MRLs) are regulated by the EU [4]. Their chemical structures are shown in
Fig. 1. For the first time, an on-line preconcentration step
has been developed and applied for the analysis of these
compounds by CE, improving significantly the detection
limits. The combination of an adequate SPE procedure
with the selection and optimization of the LVSS procedure
permits the quantitative determination of these sulfonamides with satisfactory accuracy. The SPE-LVSS-CZE
method has shown its usefulness in the simultaneous
monitoring of these residues in meat and water, at the
very low mg/L levels.
Figure 1. Chemical structures
of the studied sulfonamides.
CE and CEC
2 Materials and methods
2.1 Chemicals
All reagents were analytical reagent grade. Solvents
were HPLC grade and sulfonamides were analytical
standard grade. ACN, methanol, sodium dihydrogen
phosphate, disodium hydrogen phosphate, and
sodium hydroxide were obtained from Panreac-Química (Madrid, Spain). Ultrapure water (Milli-Q plus
system, Millipore, Bedford, MA, USA) was used
throughout the work. A stock standard solution of
100 mg/L of each sulfonamide (SPD, Riedel-de-Haën
and SMZ, SMR, SMT, SDZ, SDM, SMX, SCP, and SMI,
Vetranal, Sigma-Aldrich Química, Madrid, Spain) was
prepared by dissolving 10 mg of the product in 100 mL
of methanol (E. Merck, Darmstadt, Germany) in a calibrated flask. The solutions were stable for at least
2 months, stored in the dark at 47C. Working standard
solutions containing all the sulfonamides were freshly
prepared by dilution of the stock solutions with imidazole solution (10 mM, pH 9.8) in the presence of
methanol (10%) (Sigma-Aldrich Química). p-Aminobenzoic acid (PABA) (Fluka, Sigma-Aldrich Química)
was used as internal standard (IS). A stock solution of
100 mg/L of PABA was prepared by dissolving 10 mg
of the product in 100 mL of water. The solution was
stable for at least 1 month.
Extraction cartridges containing an Oasis® hydrophilic–
liphophilic balance (HLB; 60 mg, 3 cc; Waters, Milford,
MA, USA) and Alumina N (500 mg, 3 cc; E. Merck) in
homemade cartridges, were used.
2.2 Instrumentation
CE experiments were carried out with an HP3D CE
instrument (Agilent Technologies, Waldbronn, Germany)
equipped with a diode array detector (DAD). Data were
collected using the software provided with the HP
ChemStation version A.09.01. Separation was carried
out in a bared fused-silica capillary (64.5 cm675 mm id,
effective length 56 cm) with an optical path length of
200 mm (bubble cell capillary from Agilent Technologies).
For pH measurements, a pH meter (Crison model
pH 2000, Barcelona, Spain) was employed with a resolution of 60.01 pH unit. The extraction and preconcentration process was achieved with a vacuum
manifold system from Supelco (Bellafonte, PA, USA)
coupled with a vacuum pump (Büchi model B-169,
Switzerland). Rotavapor (Büchi RE 121, Büchi Laboratoriums-Technik AG, Flawil, Switzerland), was used for
sample preparation.
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2.3 Electrophoretic procedure
Before the first use, the capillary was conditioned by
flushing with 1 M NaOH for 10 min at 607C, then with
water for 5 min, and finally with the BGE solution for
20 min. A pressure of 1 bar was applied. At the beginning
of each day, the capillary was prewashed with an N2
pressure of 7 bar for 1 min with 0.1 M NaOH, 1 min with
water and 2 min with running buffer. After each run, the
capillary was postwashed with 7 bar for 0.75 min
0.1 M NaOH, 0.5 min with deionized water, and 1 min with
buffer to maintain an adequate reproducibility of run-torun injections. The electrophoretic separation was
achieved with a voltage of 25 kV (normal mode). The running buffer was an aqueous solution of sodium dihydrogen phosphate/disodium hydrogen phosphate 45 mM
adjusted to pH 7.3 with orthophosphoric acid and containing 10% v/v methanol. All the sulfonamides were
monitored at 265 nm with a bandwidth of 16 nm. The
temperature of the capillary was kept constant at 277C.
Injection of the sample occurred using the following LVSS
procedure.
2.4 LVSS procedure
Samples containing the analytes were dissolved in imidazole solution (pH 9.8, 10 mM, methanol 10%) and
loaded with a pressure of 7 bar for 0.5 min. In this way, the
whole capillary column was filled with the sample solution. After sample injection, a negative voltage (228 kV)
was applied. Sample matrix removal from the capillary
was indicated by monitoring the electric current, which
progressively increased to its normal value as the lowconductivity injected zone was eliminated from the capillary. At this stage the stacking process could be considered complete. The high voltage was then switched
from negative to positive (25 kV).
2.5 Sample treatment
2.5.1 Meat samples
Pork meat samples purchased from a local market were
used in this study. For extraction, preconcentration, and
cleanup, we have developed a sample treatment with
some modifications from one previously proposed [35],
based on the use of ACN for sample extraction and
SPE cartridges for sample cleanup. Meat (3 g) was
spiked with different concentration levels of sulfonamides. After spiking and homogenizing, 20 mL of ACN
was added and the samples were vortexed for 2 min.
ACN extracted a lot of endogenous substance from
meat, therefore, two different SPE cartridges were
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needed in order to cleanup and concentrate the samples.
The first one, Alumina N, is a polar sorbent SPE cartridge. The extract was percolated through the cartridge
at a flow rate of approximately 1 mL/min. Then, a mixture of ACN/water (80:20 v/v) was used as a washing
solution. The loading and washing solutions were collected, dried off with a rotavapor and redissolved with
10 mL of water by vortexing for 2 min. The water extract
was passed through the second cartridge, an Oasis
HLB, to remove potential interferences and to cleanup.
The cartridge was previously activated with 2 mL of
methanol and 2 mL of water. After sample percolation,
final elution was carried out with methanol. This methanol solution was evaporated to dryness under a gentle
nitrogen current at 507C, and then reconstituted with the
imidazole solution containing PABA (150 mg/L). Detailed
sample preparation steps of the procedure are shown in
Fig. 2.
Figure 2. Extraction procedure for meat samples.
2.5.2 Water samples
Different samples of ground water were collected near a
cow farm in Santa Fe (Granada, Spain). In this case, an
HLB-SPE cartridge was used in order to cleanup and to
preconcentrate the samples. Water (10 mL) was spiked
with different concentration levels of sulfonamides. The
water extracts were loaded through the cartridge after
conditioning with 3 mL of methanol and 3 mL of acetic
acid (50%, pH 3). Methanol was used to elute the
compounds. This methanol solution was evaporated to
dryness under nitrogen gas at 507C and then reconstituted with imidazole solution containing PABA
(150 mg/L).
3 Results and discussion
3.1 Optimization of CE experimental conditions
Preliminary studies were carried out in order to optimize
the experimental parameters affecting the CE separation
of the target compounds by UV-Vis detection. The UV-Vis
spectra of the analytes were registered choosing 265 nm
with a bandwidth of 16 nm for monitoring the selecting
sulfonamides. To find a running buffer suitable for the
separation of the analytes we checked different buffers in
a pH range of 2–10. Finally, a pH of 7.3 was selected, because the best resolution was obtained at this value,
using sodium dihydrogen phosphate/disodium hydrogen
phosphate as a suitable buffer. A significant improvement
in resolution was observed with the addition of methanol
to the electrophoretic buffer, demanding a study using
different percentages (5, 10, and 15%). An important
interaction was observed between the pH and the methanol percentage, achieving the best results for a pH 7.3
and 10% methanol (adjusting the pH after the addition of
methanol), with a resolution higher than 1.5 for all the
peaks. A voltage of 25 kV was applied as optimum so as
to achieve a good compromise between the running time
and the electric current.
Different buffer concentrations were tested (40, 45, and
50 mM); 45 mM was selected as the optimum concentration, in order to obtain the best resolution with an
adequate electric current. The effect of temperature on
the separation was investigated in the range of 25–307C,
as lower or higher values did not provide an adequate
resolution for all the analytes. A capillary temperature of
277C provided the separation of all the sulfonamides with
satisfactory resolutions, thus this temperature was
selected as optimum. The summary of the optimized
experimental conditions is shown in Table 1.
3.2 Optimization of the LVSS with polarity
switching procedure
Considering the poor sensitivity of CE using the UV-Vis
detection, the direct use of this technique for the direct
analysis of sulfonamides would not be appropriate for
its monitoring in foodstuff of animal origin, obtaining
LODs above their MRL values imposed by legislation.
For this reason, in the present work we have combined
the SPE with an on-line preconcentration protocol,
providing a simple and inexpensive methodology for
Table 1. Summary of the optimized CE experimental
conditions
Capillary
Fused-silica capillary
64.5 cm675 mm id,
optical path length of 200 mm
Separation buffer
Sodium phosphate, 45 mM
with 10% methanol
pH
7.3
Separation voltage
25 kV
Temperature
277C
LVSS solution
Imidazole 10 mM with
10% methanol
LVSS voltage
228 kV
improving sensitivity. The buffer conditions and polarity
allowed the use of LVSS with polarity switching for anions,
considering that all the molecules are negatively charged.
At the start, a large volume of sample prepared in a low
conductivity matrix is injected and a voltage at negative
polarity is applied for focusing of zones and removal of
sample matrix. When the anions are completely focused
and most of the sample matrix is removed, voltage is
stopped, and polarity is reversed. This occurs when the
current reached 95–99% of its value. Finally, a voltage at
positive polarity is applied in order to separate and detect
the focused zones.
In LVSS we have optimized three significant parameters:
sample solvent, size of plug sample, and voltage. The
most relevant variable in this type of preconcentration
methodology is the sample solvent, because it requires a
very low conductivity to obtain the best focusing of the
analytes. Different sample solvents were studied: water,
methanol, and an imidazole solution (10 mM, pH 9.8,
10% methanol), considering their low conductivity and a
suitable pH in which all the sulfonamides are charged in
the negative form [56]. Diluted BGE was not tested as
sample solvent, as it does not provide a suitable pH for all
the sulfonamides to be charged, and it provides higher
conductivity than the other tested solvents, decreasing
the effectiveness of the LVSS. Only imidazole medium
allows us to apply the LVSS process, obtaining satisfactory results. Using methanol, it was not possible to carry
out the stacking procedure because the capillary cannot
be filled with the separation buffer. In the case of water, it
was not possible to carry out the stacking for SPD, because this compound was not charged. In order to
increase the efficiency of LVSS, we studied the effect of
the presence of organic solvent in the imidazole solution.
With this purpose, different 10 mM imidazole solutions,
containing increasing percentages of methanol (0, 5, 10,
CE and CEC
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and 25% v/v) were tested as sample solvent. The best
results were obtained in the presence of 10% methanol.
Below a 10%, lower signals were obtained and above this
percentage, peaks were significantly overlapped. For the
optimization of the size of plug sample, different percentages (50, 70, 90, and 100%) of the capillary were filled in
each experience. These percentages were estimated,
based on the diameter and length of the capillary and the
applied pressure. The results showed that it was possible
to fill the whole volume of the capillary, being achieved by
applying a pressure of 7 bar for 0.5 min. The negative
voltage was studied in the interval of 225 to 230 kV.
Finally, 228 kV was selected as optimum, as this value
permits a rapid filling of the capillary in a stable system.
Values higher than 228 kV frequently produce the capillary rupture. Once all these optimum values were selected, the whole LVSS process was completed in around
3.5 min, getting an improvement of the sensitivity of
around 15 times.
Electropherograms of a meat sample spiked with 100 mg/
kg, and of a water sample spiked with 50 mg/L of each
sulfonamide, respectively, obtained after application of
the corresponding SPE procedures, and using the optimized LVSS at the selected CE experimental conditions
are shown in Fig. 3. No interferences from the matrix were
observed in water samples. In meat sample one interfering peak appeared at the same migration time of SPD, so
its detection was not possible in this matrix.
3.3 Validation of the electrophoretic procedure
3.3.1 Linearity, LOD, and LOQ
The linearity of the response was established from eight
calibration levels corresponding to 10, 25, 50, 75, 100,
150, 200, and 250 mg/L of each sulfonamide except for
SPD. For this compound, calibration levels were 50, 75,
100, 125, 150, 200, 250, and 300 mg/L. In all cases,
150 mg/L of PABA was added as IS.
Each concentration level was injected by triplicate. Calibration curves were established by considering the relative corrected peak areas (as the ratio analyte peak per
migration time to IS peak per migration time) as a function
of the analyte standard concentration. The statistic parameters calculated by least-square regression, and the
performance characteristics are presented in Table 2.
The satisfactory determination coefficients confirm that
sulfonamide responses were linear over the studied
range. LODs and LOQs have been calculated using the
S/N ratio and have been calculated considering the analytical procedure without taking into account the previous
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Figure 3. (A) Pork meat sample spiked with 100 mg/kg of each sulfonamide. (B) Ground water sample spiked with 50 mg/L of each sulfonamide. Separation conditions as indicated in Table 1.
Table 2. Statistics and performance characteristics of
the proposed method (concentration vs. relative
corrected peak areas)
Analyte
Linearity Intercept Slope
range
(mg/L)
R2
LOD
(mg/L)
LOQ
(mg/L)
SPD
SMZ
SMR
SMT
SDZ
SDM
SMX
SCP
SMI
23–300
10–250
11–250
11–250
6–250
13–250
5–250
9–250
12–250
0.9919
0.9863
0.9949
0.9939
0.9920
0.9906
0.9936
0.9926
0.9922
22.95
7.59
8.33
7.93
3.68
11.19
2.59
6.50
10.00
25.83
10.39
10.85
10.51
5.87
13.48
4.78
9.30
12.40
20.076
20.023
20.029
20.026
20.013
20.045
20.008
20.019
20.038
0.0035
0.0036
0.0040
0.0039
0.0046
0.0044
0.0046
0.0036
0.0042
treatment of the sample, which obviously implies a preconcentration step allowing to quantify the analytes at the
levels found in real matrixes.
3.3.2 Recovery studies
In order to check the applicability of the proposed
methodology, trueness of this method was examined
using recovery studies by adding sulfonamides in sulfonamide-free pork meat and ground water samples.
Pork meat samples were spiked with a mixture of the
eight sulfonamides (SMZ, SMR, SMT, SDZ, SDM, SMX,
SCP, and SMI) at different levels (50, 100, and 150 mg/kg
for each one), selected according to the recommendations of the European Commission concerning the
monitoring of drugs [57]. Each level was prepared by
triplicate and it was injected three times. The identification of each sulfonamide was based on both their
migration times and absorption spectra. The results are
shown in Table 3 and as can be seen, the proposed
method provides good results in terms of both trueness
and precision.
CE and CEC
Table 3. Recovery for each sulfonamide at different
spiked levels in pork meat samples
SMZ SMR SMT SDZ SDM SMX SCP SMI
Added (50 mg/L)
R (%)
RSD
83.9 94.8 83.8 98.7 90.2 93.3 96.2 87.1
6.0 4.9 3.9 4.8 8.6 7.6 3.7 4.7
Added (100 mg/L)
R (%)
RSD
95.8 98.0 94.0 99.3 96.3 95.1 98.0 81.2
3.5 2.9 4.8 3.6 3.8 5.0 3.8 9.4
Added (150 mg/L)
R (%)
RSD
98.0 97.3 97.6 98.0 93.0 93.2 90.9 96.3
3.7 2.5 4.0 3.6 8.6 6.2 6.5 8.4
Mean value (n = 9).
Table 4. Recovery for each sulfonamide at different
spiked levels in ground water samples
SPD SMZ SMR SMT SDZ SDM SMX SCP SMI
Added (5 mg/L)
R (%)
RSD
98.0 96.1 99.4 98.8 98.6 95.9 100.3 96.9 96.7
3.0 5.0 2.4 2.8 3.1 3.0 1.3 2.6 3.5
Added (50 mg/L)
R (%)
RSD
and for the first time, on-line preconcentration using LVSS
has been carried out, improving the sensitivity of the
detection and making it suitable for the monitoring of
these residues in foodstuffs of animal origin (pork meat)
and in environmental samples (ground waters). The
separation takes place in less than 13 min and in the case
of meat samples, this new combined method provides
good recoveries, ranging from 81.2 to 99.3%. The LODs
obtained are low enough for quantifying these residues in
meat below the legislated MRL established by the EU,
being among the fastest and more sensitive methods for
the analysis of sulfonamides. In the case of water samples, at this moment, no established levels exist for these
emerging pollutants, but the method shows its usefulness
for their satisfactory detection in environmental samples.
The National Institute of Agricultural and Food Research
and Technology (INIA, Ministerio de Agricultura, Pesca y
Alimentación, Project Ref. CAL03-087-C2-1) and EU
funds (FEDER) supported this work. LGG is grateful to the
Plan Propio of the University of Granada for a research
contract. JJST is grateful to “Fundación La Caixa” for a
predoctoral grant.
89.2 91.9 92.8 97.4 94.7 90.7 93.6 98.2 75.6
8.8 7.7 7.6 3.4 8.6 7.2 4.7 4.5 6.8
Added (20 mg/L)
R (%)
RSD
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98.9 98.8 98.5 98.7 99.1 99.0 99.3 98.9 98.5
1.0 1.5 0.9 2.1 1.4 1.8 1.1 1.3 1.5
Mean value (n = 9).
Ground water samples were spiked with a mixture of nine
sulfonamides (SPD, SMZ, SMR, SMT, SDZ, SDM, SMX,
SCP, and SMI) at different levels (5, 20, and 50 mg/L for
each one) selected in relation to the low concentrations,
possibly found in this kind of samples. Each level was
prepared by triplicate and it was injected three times. The
results are shown in Table 4. Accuracy was demonstrated
considering the satisfactory results of this study.
4 Concluding remarks
A sensitive and rapid electrophoretic method has been
developed and validated for the analysis of sulfonamides.
For the first time, to the best of our knowledge, SPD has
been quantified by CZE. Previously to the CE analysis, a
solvent extraction/SPE procedure has been applied for
extraction, off-line preconcentration and sample cleanup,
5 References
[1] Brugere, H., Bruñere-Picux, J., Villenin, P., Rec. Med. Vet.
1985, 161, 1241–1246.
[2] Littlefield, N. A., Sheldon, W. G., Allen, R., Gaylor, D. W.,
Food Chem. Toxicol. 1990, 28, 157–167.
[3] Van Koten-Vermeulen, J. E. M., Woulters, M. F. A., Van
Leeuwen, F. X. R., WHO Food Additives Series, World Health
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Application of capillary zone electrophoresis with large-volume sample stacking to the

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