Use of short chain alkyl imidazolium ionic liquids for on‐line stacking and sweeping of methotrexate,flinic acid and folic acid: Their application to biological fluids

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on‐line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping‐MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping‐MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused‐silica capillary pre‐filled with phosphate buffer containing 3.0 mol/L of 1‐butyl‐3‐methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping‐MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping‐MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π–π interactions. In sweeping‐MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22‐ and 5‐fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.     

Determination of malachite green in fish water samples by cloud‐point extraction coupled to cation‐selective exhaustive injection and sweeping‐MEKC

We have employed a high‐sensitivity off‐line coupled with on‐line preconcentration method, cloud‐point extraction (CPE)/cation‐selective exhaustive injection (CSEI) and sweeping‐MEKC, for the analysis of malachite green. The variables that affect CPE were investigated. The optimal conditions were 250 g/L of Triton X‐100, 10% of Na₂SO₄ (w/v), heat‐assisted at 60°C for 20 min. We monitored the effects of several of the CSEI‐sweeping‐MEKC parameters – including the type of BGE, the concentrations of SDS, the injection length of the high‐conductivity buffer, and the injection time of the sample – to optimize the separation process. The optimal BGE was 50 mM citric acid (pH 2.2) containing 100 mM SDS. In addition, electrokinetic injection of the sample at 15 kV for 800 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancement for malachite green was 1.9×10⁴ relative to CZE; the coefficients of determination exceeded 0.9928. The LOD, based on an S/N of 3:1, of CSEI‐sweeping‐MEKC was 0.87 ng/mL; in contrast, when using off‐line CPE/CSEI‐sweeping‐MEKC the sensitivity increased to 69.6 pg/mL. This proposed method was successfully applied to determine trace amounts of malachite green in fish water samples.     

Determination of cocaine and its metabolites using cation-selective exhaustive injection and sweeping-MEKC

We have employed a high-sensitivity on-line preconcentration method, cation-selective exhaustive injection (CSEI) and sweeping MEKC, for the analysis of cocaine, benzoylecgonine, norcocaine, and cocaethylene. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of SDS and organic modifier, the injection length of the high-conductivity buffer, and the injection time of the sample - to optimize the separation process. The optimal BGE was 100 mM phosphoric acid (pH 1.8) containing 75 mM SDS with 10% 2-propanol and 10% tetrahydrofuran as the organic modifier. In addition, electrokinetic injection of the sample at 15 kV for 900 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancements for cocaine, norcocaine, and cocaethylene ranged from 2.06 x 10(4) to 3.96 x 10(4); for benzoylecgonine it was 1.75 x 10(3); the coefficients of determination exceeded 0.9958. The LODs, based on an S/N ratio of 3:1, of sweeping-MEKC ranged from 33.5 to 52.8 ng/mL; in contrast, when using CSEI-sweeping-MEKC the sensitivity increased to range from 29.7 to 236 pg/mL. Under the optimal conditions, we analyzed cocaine in a human urine sample prepared using off-line SPE to minimize the influence of the matrix. The recovery of the SPE efficiency was satisfactory (ca. 74.9-87.6%). Our experimental results suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used to determine cocaine and its metabolites with high sensitivity in human urine.     

Use of high‐conductivity sample solution with sweeping‐micellar electrokinetic capillary chromatography for trace‐level quantification of paliperidone in human plasma

Paliperidone is a new antipsychotic drug with a relatively low therapeutic concentration of 20–60 ng/mL. We established an accurate and sensitive CE method for the determination of paliperidone concentrations in human plasma in this study. To minimize matrix effect caused by quantification errors, paliperidone was extracted from human plasma using Oasis HLB SPE cartridges with three‐step washing procedure. To achieve sensitive quantification of paliperidone in human plasma, a high‐conductivity sample solution with sweeping‐MEKC method was applied for analysis. The separation is performed in a BGE composed of 75 mM phosphoric acid, 100 mM SDS, 12% acetonitrile, and 15% tetrahydrofuran. Sample solution consisted of 10% methanol in 250 mM phosphoric acid and the conductivity ratio between sample matrix and BGE was 2.0 (γ, sample/BGE). The results showed it able to detect paliperidone in plasma samples at concentration as low as 10 ng/mL (S/N = 3) with a linear range between 20 and 200 ng/mL. Compared to the conventional MEKC method, the sensitivity enhancement factor of the developed sweeping‐MEKC method was 100. Intra‐ and interday precision of peak area ratios were less than 6.03%; the method accuracy was between 93.4 and 97.9%. This method was successfully applied to the analysis of plasma samples of patients undergoing paliperidone treatment.     

In‐capillary microextraction using monolithic polymers: Application to preconcentration of carbamate pesticides prior to their separation by MEKC

In the present work we report a novel procedure for in‐capillary microextraction using a monolithic polymeric sorbent. In the proposed methodology, sample treatment takes place in the CE instrument but in a different capillary from that used for the electrophoretic separation. Polymers based on butyl methacrylate and divinylbenzene formed in situ inside a capillary column were assayed. The best results were found with the divinylbenzene‐based polymers. The usefulness of the proposed procedure was checked for the determination of carbamate pesticides (Methomyl, Asulam, Carbendazim, Aldicarb, Carbetamide, Propoxur, Pirimicarb, Carbaryl, Carbofuran and Methiocarb) and three of their degradation compounds (Aldicarb sulphoxide, 2‐isopropoxyphenol and α‐naphthol) using MEKC. The optimization of the MEKC is reported, a good separation of the 13 analytes being obtained in less than 6 min. The analytical method using in‐capillary microextraction was validated in terms of linearity, repeatability, precision (RSD≤18% for 50 μg/L), and LODs (1–16 μg/L), and it revealed the usefulness of this in‐capillary preconcentration procedure for the determination of analytes of intermediate polarity.     

Automated sample treatment with the injection of large sample volumes for the determination of contaminants and metabolites in urine

This work reports the development of a simple and automated method for the quantitative determination of several contaminants (triazine, phenylurea, and phenoxyacid herbicides; carbamate insecticides and industrial chemicals) and their metabolites in human urine with a simplified sample treatment. The method is based on the online coupling of an extraction column with RP LC separation–UV detection; this coupling enabled fast online cleanup of the urine samples, efficiently eliminating matrix components and providing appropriate selectivity for the determination of such compounds. The variables affecting the automated method were optimized: sorbent type, washing solvent and time, and the sample volume injected. The optimized sample treatment reported here allowed the direct injection of large volumes of urine (1500 μL) into the online system as a way to improve the sensitivity of the method; limits of detection in the 1–10 ng/mL range were achieved for an injected volume of 1500 μL of urine, precision being 10% or better at a concentration level of 20 ng/mL. The online configuration proposed has advantages such as automation (all the steps involved in the analysis – injection of the urine, sample cleanup, analyte enrichment, separation and detection – are carried out automatically) with high precision and sensitivity, reducing manual sample manipulation to freezing and sample filtration.     

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 μm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1?:?1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.     

Determination of chlorophenols in human urine based on the integration of on-line automated clean-up and preconcentration unit with micellar electrokinetic chromatography.

A new method was developed and validated for the determination of chlorophenols in human urine by using micellar electrokinetic chromatography (MEKC) coupled via a mechanic arm to an on-line automatic clean-up and preconcentration unit for urine samples. Separation is accomplished by using a selective buffer consisting of 15 mM borate, 25 mM phosphate and 100 mM sodium dodecyl sulfate (SDS) at pH 9.1 in addition to a positive power supply of 25 kV at 18 degrees C. The proposed capillary electrophoresis (CE) method allows the separation of 11 chlorophenols within 7 min with a reproducibility as relative standard deviation (RSD) between 2.6% and 7.2%, and limits of detection (LODs) between 0.08 and 0.46 microg/mL for all chlorophenols. Urine samples were previously hydrolyzed with 37% HCl at 80 degrees C for 60 min and then cleaned on a C-18 mini-column. Recoveries ranged from 58% to 103%. The preconcentration treatment affords limits of determination between 4 and 12 ng/mL for all chlorophenols except pentachlorophenol and 4-chlorophenol, which could not be determined. The overall analysis time, including on-line clean-up, preconcentration and electrophoretic separation is 20 min per sample.     

On-line preconcentration and determination of ketamine and norketamine by micellar electrokinetic chromatography. Complementary method to gas chromatography/mass spectrometry

We have investigated a rapid, simple, and highly efficient on-line preconcentration method using in micellar electrokinetic chromatography (MEKC) for the analysis of abused drugs. Ketamine is an anesthetic that has been abused as a hallucinogen. We applied the sample sweeping technique first to ketamine and its major metabolite, norketamine, and separated the analytes with MEKC. Several of the sweeping MEKC parameters to effect successful separations, such as the concentration of sodium dodecyl sulfate (SDS), the injection time, and the applied voltage were optimized. The improvements in the number of theoretical plates under the different separation conditions are presented clearly in a three-dimensional representation. The limits of detection were 2.8, 3.4, and 3.3 ng/mL for ketamine, norketamine, and ketamine-D(4), respectively. The enrichment factor for each compound was within the range of 540-800. Experimental results are in agreement with those of analysis conducted by gas chromatography/mass spectroscopy (GC/MS). Therefore, we believe that sweeping, combined with MEKC, represents a suitable complementary method to GC/MS for use in clinical and forensic analyses of ketamine and norketamine.     

Metal complex separation with on-line sample preconcentration in micellar electrokinetic chromatography.

Micellar electrokinetic chromatography (MEKC) using a cationic surfactant as a pseudostationary phase was examined to separate anionic metal cyclohexane-1,2-diaminetetraacetic acid (CDTA) complexes. Cetyltrimethylammonium chloride (CTAC) was employed as the cationic surfactant micelle, its addition leading to EOF reversal. Cu(II), Co(II), Zn(II), Mn(II) and Pb(II) were used as test analytes, and the complete separation was obtained by MEKC. On-line sample preconcentration by sweeping was also examined to improve the detection sensitivity. From 15- to 42-fold increases in the detection sensitivity in terms of the peak heights were obtained by sweeping with a cationic micelle in the presence of high EOF. The limits of detection were in the range (0.6 - 1.8) x 10(-6) M with UV detection without any off-line preconcentration step.     

Determination of cypromazine and its metabolite melamine in milk by cation-selective exhaustive injection and sweeping-capillary micellar electrokinetic chromatography

In this study, we described a high‐sensitive on‐line preconcentration method for cypromazine (CYP) and melamine (MEL) analysis using cation‐selective exhaustive injection (CSEI) combined with sweeping‐MEKC. The optimum conditions of on‐line concentration and separation were discussed. The BGE contained 100 mM SDS, 50 mM phosphoric acid (pH=2.0) and 15% acetonitrile (v/v). The sample was injected at 10 kV for 600 s, separated at ?20 kV, and detected at 210 nm. The sensitivity enhancements were 6222 for CYP and 9179 for MEL. The linear dynamic ranges were 0.4?25 ng/mL for CYP (r=0.9995) and 0.2?12 ng/mL for MEL (r=0.9991). The LODs (signal‐to‐noise ratio, 3) were 43.7 and 23.4 pg/mL for CYP and MEL, respectively. The proposed method was applied to analyze CYP and MEL in dairy products pretreated using off‐line SPE to minimize the influence of the matrix. The recoveries of CYP and MEL were satisfactory (ca. 74–83%). The experimental results suggest that the CSEI‐sweeping‐MEKC method is feasible for the application to simultaneously detect trace levels of CYP and its metabolite MEL in real milk samples.     

Insight into the mechanism of transient trapping in micellar electrokinetic chromatography

Transient trapping is a new mechanism of on-line sample concentration and separation that has recently been presented. It involves the injection of a short length of micellar solution in front of the sample, making it similar to sweeping in partial-filling MEKC. Here, we examine the mechanism of transient trapping by the use of computer simulations and compare it to sweeping in MEKC for the two analytes, sulforhodamine B and 101. The simulation results confirm the mechanism for concentration and separation originally proposed. The mechanism for concentration is similar to sweeping since the analytes are picked and accumulated by the micelles that penetrate the sample zone. The mechanism for separation is however quite unique since the concentrated analytes are trapped for a few seconds on the sample/micelle boundary before they are released as the concentration of micelle is reduced as it undergoes electromigration dispersion and the analytes separate down a micelle gradient. Simulation results suggested that a significant contribution of band broadening arises from the micelle gradient, with shallower gradients resulting in broader peaks. However, this is offset by an increase in selectivity, such that resolution was enhanced even though the peaks are broader. Transient trapping analysis with similar resolution to those obtained by sweeping MEKC could be achieved in 1/10 of the time and 1/4 of the capillary length, which results in a 2-3 times increase in sensitivity.     

On-line concentration and determination of all-trans- and 13-cis- retinoic acids in rabbit serum by application of sweeping technique in micellar electrokinetic chromatography

A simple and rapid micellar electrokinetic chromatography (MEKC) method with UV detection was developed for the simultaneous separation and determination of all-trans- and 13-cis-retinoic acids in rabbit serum by on-line sweeping concentration technique. The serum sample was simply deproteinized and centrifuged. Various parameters affecting sample enrichment and separation were systematically investigated. Under optimal conditions, the analytes could be well separated within 17min, and the relative standard deviations (RSD) of migration times and peak areas were less than 3.4%. Compared with the conventional MEKC injection method, the 18- and 19-fold improvements in sensitivity were achieved, respectively. The proposed method has been successfully applied to the determination of all-trans- and 13-cis-retinoic acids in serum samples from rabbits and could be feasible for the further pharmacokinetics study of all-trans-retinoic acid.     

Concentration and determination of cotinine in serum by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC), combined with on-line concentration techniques, cation-selective exhaustive injection (CSEI) and sweeping, was developed for the analysis of cotinine, the primary biomarker for exposure to secondhand smoke. Experimental parameters including sample matrix, surfactant concentration, injection length and concentration of high-conductivity buffer, and sample electrokinetic injection time were optimized for electrophoretic enrichment and separation processes. Under the optimal conditions, the detection sensitivity of cotinine was enhanced by about 5000-fold using CSEI-sweeping MEKC compared to normal MEKC. The limit of detection for cotinine was found to be 0.2 ng/mL using ultraviolet absorbance detection. Furthermore, the developed method was successfully applied to the detection of cotinine in mouse serum samples.     

Continuous solid-phase extraction and preconcentration of bisphenol A in aqueous samples using molecularly imprinted columns

A bisphenol A (BPA) molecularly imprinted polymer, the composition of which was optimised using a chemometric approach, has been applied to the selective preconcentration of the template from aqueous samples. The selectivity of the polymer toward BPA and related compounds was evaluated chromatographically. The BPA-imprinted polymer was packed in a column and used for continuous on-column solid-phase extraction (MISPE) of aqueous samples followed by subsequent analysis by HPLC with fluorescence detection of the eluted fractions. The composition of the washing solvent applied in the MISPE procedure was optimised to favour the specific interactions of the MIP with BPA and to remove the non-selectively bound matrix components. The MISPE method has proven to be effective for selective preconcentration of BPA in aqueous samples (recoveries >84% obtained in the eluate for 10–100 mL sample volumes) enabling detection and quantification limits of 1.0 and 3.3 ng mL^–1, respectively (based on 25 mL sample size). Analytical recoveries were between 92 and 101% for river water samples spiked with known amounts of BPA (30, 60, and 80 ng mL^–1); relative standard deviations (RSD) were lower than 5.0%.     

Determination of cyanobacterial cyclic peptide hepatotoxins in drinking water using CE

Four cyanobacteria hepatotoxins microcystin LR, microcystin RR, microcystin YR, and nodularin were simultaneously determined in drinking water using CZE and MEKC coupled with UV detection. The toxins were satisfactorily separated in both CZE and MEKC modes. Detection limits were in the range of 0.82–4.81 μg/mL, with R² values of 0.994–0.999. The linearity range tested for the standards was 5–100 μg/mL and RSD percentages were in the range of 1.0–2.5% for retention time and 3.0–10.2% for peak area. When a known amount of standard was spiked into a known volume of water and extracted, recoveries were 90.3% (RR), 101.5% (nodularin), 90.6% (YR), and 88.2% (LR). The use of SPE enabled cleanup and pre‐concentration of a real sample to achieve a 100‐fold concentration factor. Detection limits after SPE of the real sample spiked with microcystins were 0.090 μg/L (RR), 0.076 μg/L (YR), and 0.110 μg/L (LR), with RSD percentage values of 9.9–11.7% for peak area and 2.2–3.3% for retention time. The technique developed provides an alternative method for determining microcystins at levels of concentration that will be able to meet WHO drinking water guidelines for microcystins.     

Quantitative determination of nicotinic acid in micro liter volume of urine sample by drop-to-drop solvent microextraction coupled to matrix assisted laser desorption/ionization mass spectrometry

Drop-to-drop solvent microextraction (DDSME) coupled with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) for quantitative determination of nicotinic acid in one drop of urine sample has been proposed. All parameters, such as type of organic solvent, extraction time, exposure volume solvent, pH of the sample solution that affecting the separation and preconcentration of nicotinic acid were investigated. Under the optimal conditions, the detection limit of the method was 20 ng mL(-1) and the relative standard deviations (RSD) for determination of the nicotinic acid were in the range of 8.0-12.5%. The calculated calibration curves gave linearity in the range of 80-1000 ng mL(-1). The main advantages of the proposed method are simple, fast, and small amount of sample solution is used for separation and preconcentration of nicotinic acid. This method could be also useful for the analysis of other interested analytes in small volume of biological samples, like plasma, saliva and urine, where the availability of samples are limited.     

Determination of nikethamide by micellar electrokinetic chromatography

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)–UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000 g ), separation by MEKC and ultraviolet‐absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 μA) using a standard fused‐silica capillary (effective length 51 cm, 75 μm i.d.). The determined limit of detection for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4–280 μmol/L (0.71–49.90 μg/mL), with R² 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.     

Development of an ultrasensitive stacking technique for 5‐nitroimidazole determination in untreated biological fluids by micellar electrokinetic chromatography

Cation‐selective exhaustive injection and sweeping followed by a MEKC separation is evaluated for the sensitive analysis of 5‐nitroimidazoles in untreated human serum and urine. Deproteinized serum and urine samples were diluted 76 and 143 times, respectively, in a low‐conductivity solvent (5.00 mM orthophosphoric acid containing 5.0% v/v methanol). Samples were electrokinetically injected at 9.8 kV for 632 s in a previously conditioned fused‐silica capillary (65.0 cm × 50 μm id). Separation was performed at –30 kV and 20°C using 44 mM phosphate buffer (pH 2.5), 123 mM SDS, and 8% v/v tetrahydrofurane as BGE. Signals were monitored at 276 nm and peak area was selected as analytical response. Good linearity (R² ≥ 0.988) and LODs lower than 1.5 and 1.8 μg/mL were achieved in serum and urine, respectively.     

Micellar nanotubes dispersed electrokinetic chromatography for the simultaneous determination of antibiotics in bovine milk

A method to determine four antibiotics for veterinary use (ciprofloxacin, enrofloxacin, florfenicol, and chloramphenicol) of different families (fluoroquinolones and amphenicols) in bovine milk was developed. The determination of the analytes was carried out using micellar electrokinetic capillary chromatography (MEKC) with a common sodium borate-SDS buffer solution containing single-walled carbon nanotubes (SWCNTs). In this way, a great improvement in the electrophoretic resolution and the separation efficiency was achieved compared to MEKC. An online reverse electrode polarity-stacking mode (REPSM) was carried out to enhance sensitivity. This step was performed in only 2 min and it allowed a stacked percentage of 103. That means that all the amount of injected analytes is effectively stacked. When this stacking procedure was combined with an off-line preconcentration step, based on SPE, analytes could be detected in lower concentration than the established maximum residue limits (MRLs). The LODs for the four compounds were between 6.8 and 13.8 μg L(-1) and the RSD values were between 1.1% and 6.6%. The whole method was applied to spiked real samples with acceptable precision and satisfactory recoveries.