Chiral separation of benzoporphyrin derivative mono- and diacids by laser induced fluorescence-capillary electrophoresis

A method for the separation of benzoporphyrin derivative mono- and diacid (BPDMA, BPDDA) enantiomers by laser induced fluorescence-capillary electrophoresis (LIF-CE) has been developed. By using 300 mM borate buffer, pH 9.2, 25 mM sodium cholate and 10% acetronitrile as electrolyte, +10 kV electrokinetic sampling injection of 2 s and an applied +20 kV voltage across the ends of a 37 cm capillary (30 cm to the detector, 50 microm ID), all six BPD stereoisomers were baseline-separated within 20 min. Formation constants, free electrophoretic and complexation mobilities with borate and cholate were determined based on dynamic complexation capillary electrophoresis theory. The BPD enantiomers can be quantitatively determined in the range of 10(-2)-10(-5) mg mL(-1). The correlation coefficients (r2) of the least-squares linear regression analysis of the BPD enantiomers are in the range of 0.9914-0.9997. Their limits of detection are 2.18-3.5 x 10(-3) mg mL(-1). The relative standard deviations for the separation were 2.90-4.64% (n = 10). In comparison with high-performance liquid chromatography (HPLC), CE has better resolution and efficiency. This separation method was successfully applied to the BPD enantiomers obtained from a matrix of bovine serum and from liposomally formulated material as well as from studies with rat, dog and human microsomes.     

Extremely highly efficient on-line concentration and separation of gold nanoparticles using the reversed electrode polarity stacking mode and surfactant-modified capillary electrophoresis

In this study, gold nanoparticles (Au NPs) were separated using the reversed electrode polarity stacking mode (REPSM) of a capillary electrophoresis (CE) system for on-line enhancement prior to performing surfactant-modified CE separation. Under optimized conditions [running electrolyte buffer, sodium dodecyl sulfate (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (10 mM) at pH 10.0; applied voltage, 20 kV; operating temperature, 25°C; REPSM strategy for sample on-line concentration; REPSM applied prior to initializing separation], two parameters were varied to further enhance the concentration and separation of the Au NPs: (i) the rate of polarity switching (from -20 to +20 kV) between the REPSM and surfactant-modified CE separation modes and (ii) the length of the capillary column. At a polarity switching rate of 1333 kV min(-1) and a column length of ca. 83.5 cm, the resolution of the separation of a mixture of 5.3- and 40.1-nm Au NPs was greater than 19; in addition, the numbers of theoretical plates for the 5.3- and 40.1-nm-diameter Au NPs were greater than 15,000 and up to 1.15×10(7), respectively-the latter being extremely high. Thus, this CE-based method for separating Au NPs provided high performance in terms of separation resolution and the number of theoretical plates, both of which were improved by greater than fivefold relative to those published previously. Notably, the sensitivity enhancement factors for the 5.3- and 40.1-nm-diameter Au NPs were improved (by ca. 20- and 500-fold, respectively) relative to those obtained using conventional surfactant-modified CE separation.     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Capillary electrophoretic separation of heparin oligosaccharides under conditions amenable to mass spectrometric detection

A capillary electrophoresis method for the separation of high-molecular-mass heparin oligosaccharides compatible with mass spectral detection was developed. Structurally defined heparin oligosaccharides ranging in size from tetrasaccharide to tetradecasaccharide were used to optimize the conditions. Applying normal and reversed polarity modes, these oligosaccharides were separated by CE under various conditions. Ammonium hydrogencarbonate (30 mM at pH 8.50) used as the running electrolyte system gave good separation efficiency and resolution in the normal polarity mode. Application of this method to the separation of complicated heparin oligosaccharide mixtures required the addition of electrolyte additives. Ammonium hydrogencarbonate (30 mM), containing triethylamine (10 mM), was useful for the separation of complex oligosaccharide mixtures. Run-to-run and day-to-day precision and limits of detection were established for these separations.     

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.     

Identification of inorganic ions in post‐blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.     

Resolution of overlapped peaks of amino acid derivatives in capillary electrophoresis using multivariate curve resolution based on alternating least squares

The application of chemometric techniques to the resolution of overlapped peaks in capillary electrophoresis (CE) is described. When a physical separation can not be completely accomplished, chemometrics might still resolve the determination of the analytes mathematically. CE with diode array detection can provide a large amount of data consisting of spectra registered over time. In this study, the capillary electrophoretic separation of 1,2-naphthoquinone-4-sulfonate derivatives of amino acids is studied. Most of the common amino acid derivatives can be separated at 30 kV in a fused-silica capillary by using a 40 mM sodium tetraborate + isopropanol (3:1 v/v) solution as background electrolyte. However, peaks of certain derivatives (Phe, His, Leu and Ile) still overlap. A multivariate curve resolution method based on an alternating least squares optimization procedure is used for the resolution of the overlapped electrophoretic peaks. The method takes advantage of spectral and electrophoretic differences of analytes to recover their pure electrophoretic and spectral profiles. In addition, each analyte in the mixture can be quantified using the corresponding standards.     

Application of high performance capillary electrophoresis on toxic alkaloids analysis

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.     

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.     

Design and performance of a light-emitting diode detector compatible with a commercial capillary electrophoresis instrument

Indirect photometric detection in capillary electrophoresis (CE) has been predominantly performed in the UV region, in part due to a lack of suitable high-intensity and low-noise light sources in the visible spectral region. A new photometric detector based on light-emitting diodes (LEDs) as light sources and compatible with a commercially available CE instrument has been designed and constructed and its performance evaluated. The utility of this detector was successfully demonstrated by the indirect photometric detection of anions using a dye as probe and absorbance measured in the visible region. The detector exhibited very low baseline noise (around 0.03 mAU), stable output, and improved upper limit of detection linearity (502 mAU) compared with previously used LED detectors. The detector was tested for indirect detection of anions separated with an electrolyte containing 4 mM Orange G as the indirect detection probe, 10 mM histidine as an isoelectric buffer, and 0.05% hydroxypropylmethylcellulose to suppress the electroosmotic flow. Extremely low detection limits were obtained ranging from 0.16-0.36 microM (excluding chloride 0.56 microM), with separation efficiencies in the range of 154,000-274,000 theoretical plates.     

Capillary electrophoresis with on-chip four-electrode capacitively coupled conductivity detection for application in bioanalysis

Microchip capillary electrophoresis (CE) with integrated four-electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip-based analysis, is compatible with miniaturization and on-chip integration. The glass microchip structure consists of a 6 cm etched channel (20 microm x 70 microm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 microM up to 2 mM. Detection limits were at the low microM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.     

Glucose in human serum determined by capillary electrophoresis with glucose micro-biosensor

A glucose micro-biosensor was employed as detector in capillary electrophoresis (CE) for determining the concentration of glucose in human serum. The micro-biosensor was based on the immobilization of the SWNTs-glucose oxidase-chitosan biocomposite at a platinized Au electrode by electrodeposition. The influencing factors including separation voltage, detection potential, pH value, and the concentration of the buffer were studied. Suitable conditions were obtained for the determination of glucose: running buffer, 25 mM PBS (pH 8.0); separation field strength, 250 V cm(-1); detection potential, 0.80 V vs. saturated calomel electrode. Under optimized detection conditions, glucose responded linearly from the range of 5 μM to 1 mM with a correlation coefficient of 0.9986 for the injection voltage of 5.0 kV and injection time of 10 s. The concentration limit of detection of the method was 1 μM (S/N = 3). The micro-biosensor exhibited good stability and durability in the analytical procedures. The relative standard deviation of the migration time and peak current were 1.7% and 2.6%, respectively. Glucose in human serum from two healthy individuals and two diabetics was successfully determined, giving a good prospect for a new clinical diagnostic instrument.     

A rapid CE-potential gradient detection method for determination of quinolones

A rapid CE coupled with potential gradient detection (PGD) for the separation and detection of four quinolones, namely, enoxacin, ofloxacin (OFL), fleroxacin, and pazufloxacin, was described. Separation was performed in a fused-silica capillary (75 microm x 8.5 cm) using a buffer of 30 mM Tris and 4 mM phosphoric acid at pH 8.9. Under the separation voltage of 3 kV, the quinolones were separated within 2.8 min with good linearity (r(2) >or= 0.985). The method was successfully applied in determining OFL in a pharmaceutical formulation. Also, a liquid-liquid extraction (LLE) method was developed and coupled to CE-PGD in determining quinolones that spiked in milk samples. With dichloromethane and hexane for enrichment and purification, the LLE recoveries of the four quinolones were in the range of 77-106%. The detection limits of the quinolones with LLE-CE-PGD were from 23 to 65 ng/mL. The proposed CE-PGD method was validated with an HPLC method, and the results indicated consistency between the two methods.     

Optimisation of probe concentration in indirect photometric detection in capillary electrophoresis using highly absorbing dyes

In indirect photometric detection in capillary electrophoresis, the concentration of the absorbing probe ion in the background electrolyte should be as high as possible in order to increase the dynamic range of the detection method. For relatively low absorptivity probes (epsilon < 2000 L mol(-1)cm(-1)) used under typical conditions (75 microm ID capillary) the maximum probe concentration is normally limited by the separation current. However, for medium (epsilon approximately/= 2000-15000 L mol(-1)cm(-1)) and especially for high (epsilon > 15000 L mol(-1)cm(-1)) absorptivity probes such as dyes, the maximum concentration may be limited by the background absorbance of the electrolyte which must fall within the linearity range of the detector. In this work, it is shown that another practical factor limiting the probe concentration is the adsorption of probe onto the capillary wall at higher concentrations, resulting in unstable baseline and increased noise. Use of a zwitterionic surfactant to suppress adsorption enabled the concentration of a model probe anion (tartrazine) to be increased by a factor of six times (to 3 mM). This resulted in significant improvements in peaks shapes, resolution between peaks, detection sensitivity and linear calibration range for the analyte anions. Baseline separation of a test mixture was maintained up to 7.5 mM total concentration of sample coions injected (13.7 nL) for the 3 mM electrolyte, with detection limits ranging from 0.63 to 0.94 microM. Peak height reproducibility (over 20 consecutive injections) was improved (values ranging from 1.1 to 1.9%) compared with electrolytes containing lower concentrations of the probe. Overall, the optimised, higher concentration probe electrolyte provided the sensitivity benefits of highly absorbing probes with the additional benefits of ruggedness and improved stacking, peak shapes and resolution.     

Separating stereoisomers of di-, tri-, and tetrapeptides using capillary electrophoresis with contactless conductivity detection

The separation and detection of small oligopeptides in CE with contactless conductivity detection were demonstrated. A strongly acidic separation buffer (0.5 M acetic acid) was employed in order to render the species cationic. Separation of the stereoisomers was achieved in typically 10-15 min by using either dimethyl-beta-CD (DM-beta-CD), (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid (18C(6)H(4)), a combination of the two substances, or of histidine, as buffer additives. Calibration curves were determined for isomers of Gly-Asp and H-Pro-Asp-NH(2), in the range of 0.05-0.5 mM and 0.1-1 mM, respectively, and were found to be linear. LODs were determined to be in the order of 1.0 microM. The determination of isomeric impurities down to about 1% was found possible. Species showing good separation could also be successfully determined on an electrophoretic lab-on-chip device, with analysis times of a few minutes.     

On-line hyphenation of capillary electrophoresis with flame-heated furnace atomic absorption spectrometry for trace mercury speciation

Capillary electrophoresis (CE) was directly interfaced to flame-heated furnace atomic absorption spectrometry (FHF-AAS) via a laboratory-made thermospray interface for nanoliter trace element speciation. The CE-FHF-AAS interface integrated the superiorities of stable CE separation, complete sample introduction, and continuous vaporization for AAS detection without the need of extra external heat sources and any post-column derivation steps. To demonstrate the usefulness of the developed hybrid technique for speciation analysis, three environmentally significant and toxic forms of methylmercury (MeHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) were taken as model analytes. Baseline separation of the three mercury species was achieved by CE in a 60 cm long x 75 microm inner diameter fused-silica capillary at 20 kV and using a mixture of 100 mM boric acid and 10% v/v methanol (pH 8.30) as running electrolyte. The precision (relative standard deviation, RSD, n = 7) of migration time, peak area and peak height for the mercury species at 500 microg x L(-1) (as Hg) level were in the range of 0.9-1.2%, 1.5-1.9%, and 1.4-2.0%, respectively. The detection limit (S/N = 3) of three mercury species was 3.0 +/- 0.15 pg (as Hg), corresponding to 50.8 +/- 2.4 microg x L(-1) (as Hg) for 60 nL sample injection, which was almost independent on specific mercury species. The developed hybrid technique was successfully applied to the speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).     

Rapid analysis of antimicrobial metabolites monoacetylphloroglucinol and 2,4-diacetylphloroglucinol using capillary zone electrophoresis

A rapid capillary electrophoretic (CE) method was developed for the determination of phloroglucinol compounds, monoacetylphloroglucinol (MAPG) and 2,4-diacetylphloroglucinol (DAPG), in microbial supernatants of Pseudomonas fluorescens F113 over a 24-h growth cycle. Prior to electrophoretic separation, solid-phase extraction of supernatant samples on octadecylsilica for the purpose of sample cleanup is recommended. The optimum electrophoretic conditions were found to be 25 mM sodium tetraborate running buffer at pH 9.3, temperature at 25 degrees C with an applied voltage of 25 kV. The capillary was an Agilent fused-silica capillary of total length 33 cm x 50 microm inner diameter, 375 microm outer diameter, with effective length 24.5 cm. While MAPG and DAPG were monitored at selected wavelengths in the range of 214-320 nm, analysis at 214 nm was used and a CE separation time of less than 2 min was achieved. A partial method validation study was performed in accordance with European Agency for Evaluation of Medicinal Products (EMEA) guidelines. The method displayed linearity over the investigated range of 10-200 microg/mL, with limits of detection of 1.2 microg/mL for MAPG and 1.3 microg/mL for DAPG.     

Use of 1-alkyl-3-methylimidazolium-based ionic liquids as background electrolytes in capillary electrophoresis for the analysis of inorganic anions

Separation of inorganic anions in CE is often a challenging task because the electrophoretic mobilities of inorganic anions are comparable to or even greater than the EOF mobility. In this study, we present the use of ionic liquids (ILs) as background electrolytes (BGEs) in CE of inorganic anions. The 1-alkyl-3-methylimidazolium-based ILs as BGEs dynamically coated the capillary wall and induced a reversed EOF. This allowed the anions to comigrate with the EOF and yielded a rapid separation. Increasing the alkyl chain length of the ILs and BGE concentration can significantly improve the separation resolution. With 40 mM 1-butyl-3-methylimidazolium tetrafluoroborate as BGE, good separations of five model anions (Br-, I-, NO2(-), NO3(-), and SCN-) were achieved in a range of buffer pH values. The separation efficiency was as high as 34 600-155 000, and the RSDs of the migration times were less than 0.8% (n = 5).     

Using multiple short-end injections to develop fast electrophoretic separations--applications in iodide analysis

The aim of this study was to develop a fast CE separation method by using multiple short-end injections in a capillary coated with quaternary ammonium chitosan (HACC), in order to determine the iodide content of pharmaceutical formulations. The BGE was composed of 20 mM tris(hydroxymethyl)aminomethane and 11 mM hydrochloric acid, at pH 8. The internal standard used was thiocyanate. Separations were performed in a fused silica capillary (32 cm total length, 8.5 cm effective length and 50 μm i.d.) coated with HACC and direct UV detection at 220 nm. EOF was modified by flushing the capillary with polymeric solution, resulting in a semi-permanent coating of controlled and stable EOF. The EOF was anodic at pH 8. Different strategies, using single and multiple injection short-end configurations, were studied to develop a CE method that resulted in a maximum number of iodide samples analyzed per hour: one plug and flush (Sflush) 35 samples/h, one plug without flush (SWflush) 76 samples/h, four plugs and flush (Mflush) 61 samples/h, and four plugs without flush (MWflush) 80 samples/h. Using the multiple injection configuration, it was possible to inject up to four plugs using spacer electrolytes with good separation efficiency and selectivity. The voltage application time needed to separate the eight peaks (iodide and thiocyanate) with MWflush was only 12s. The method was validated and samples were analyzed using MWflush. Good linearity (R(2)>0.999); a limit of detection 0.4 mg L(-1); intermediate precision better than 3.8% (peak area) and recovery in the range of 99-102% were obtained.