Separation of antidepressants by capillary electrophoresis with in-line solid-phase extraction using a novel monolithic adsorbent

The separation of three selective serotonin reuptake inhibitors (SSRIs) by capillary electrophoresis (CE) with fully integrated solid-phase extraction (SPE) is described. Polymeric monolithic SPE modules were prepared in situ within a fused silica capillary from either butyl methacrylate-co-ethylene dimethacrylate or 3-sulfopropyl methacrylate-co-butyl methacrylate-co-ethylene dimethacrylate. Using a 1 cm SPE module placed at the inlet of the capillary, a mixture of sertraline, fluoxetine and fluvoxamine was extracted from aqueous solution by applying a simple pressure rinse. Under pressure-driven conditions, efficient elution was possible from both SPE materials investigated using 50 mM phosphate buffer, pH 3.5 in acetonitrile (20/80, v/v). Two different strategies were investigated for the efficient elution and subsequent CE separation. Injection of an aqueous sample plug directly into the non-aqueous elution/separation buffer was found to be unsuitable with poor elution profiles observed in the electrodriven mode. Alternatively, a sample plug equivalent to several capillary volumes could be injected by pressure followed by filling the capillary with the non-aqueous elution/separation buffer from the outlet end using a combination of pressure and electrodriven flow. Using a neutral monolith, efficient elution/separation was not possible due to an unstable electroosmotic flow (EOF), however, by adding the ionisable monomer, 3-sulfopropyl methacrylate to the SPE module to increase and stabilise the EOF, it was possible to achieve efficient elution from the SPE module, followed by baseline separation by CE using a 200 mM acetate buffer, pH 3.5 in acetonitrile (10/90, v/v). With enrichment factors of over 500 achieved for each of the analytes this demonstrates the potential of in-line SPE-CE for the sensitive analysis of these drugs.     

Capillary electrophoresis of neurotransmitters using in-line solid-phase extraction and preconcentration using a methacrylate-based weak cation-exchange monolithic stationary phase and a pH step gradient

A novel approach for in-line solid-phase extraction capillary electrophoresis (SPE-CE) for basic analytes was developed. The method is based on the use of a weak cation-exchange monolith synthesised in situ in the front end of the CE capillary via photoinitiated polymerization to form poly(methacrylic acid-co-ethylene glycol dimethacrylate), which was used to create the SPE phase in-line with the CE separation capillary. The monolithic SPE material exhibited a surface area of 23.1 m2/g and a capacity of 403 nM for dopamine. Adsorption of the analytes as protonated, cationic species onto the SPE phase was achieved using an electrolyte of 6 mM phosphate and 12 mM sodium ion, buffered at pH 7.0, which is above the pKa of the monolith but below the pKa of the analytes. Elution of the analytes from the SPE phase was achieved using an electrolyte with a pH below that of the pKa of the monolith, namely 12 mM phosphate and 12 mM sodium ion, buffered at pH 3.0. Due to the discontinuous electrolyte combination, analytes were simultaneously eluted and focused as the electrophoretically mobilised pH step boundary moved through the SPE monolith, after which the analytes were separated by conventional CZE in the remainder of the capillary. Quantitative extraction from a solution of 0.5 microg/ml dopamine and epinephrine was achieved when flushing up to 15 column volumes of sample through the capillary. The limits of detection (S/N=3) for dopamine and epinephrine were 3.7 and 4.3 ng/ml, and this method provided a sensitivity enhancement for dopamine of 462 times compared to CZE using hydrodynamic injection. The developed method was used to preconcentrate a test mixture of neurotransmitters comprising dopamine, epinephrine, 5-hydroxytryptamine, metanephrine and also histamine. The applicability of this approach to real life samples was demonstrated by using a urine sample from a healthy person to detect dopamine at sub-ppm levels.     

On-line sequential preconcentration of inorganic anions by anion-selective exhaustive injection and base-stacking in capillary zone electrophoresis

A sequential electrostacking method based on anion-selective exhaustive injection (ASEI) and base-stacking (BS) is presented for the preconcentration and determination of inorganic anions by capillary zone electrophoresis (CZE) in this paper. Tetradecyltrimethylammonium bromide as an electroosmotic flow (EOF) modifier was added into the buffer to suppress EOF of the capillary. Firstly, a water plug was hydrodynamically injected into the capillary. During ASEI under negative high voltage, the sample anions migrated quickly towards the boundary between the water plug and buffer in the capillary. Then an alkaline zone was injected electrokinetically to concentrate the anions further. With the sequential electrostacking method, the preconcentration factor of (0.8-1.3) x 10(5) was obtained compared with the conventionally electrokinetic injection and the relative standard deviation of peak area was 3.3-5.3% (n = 5). The detection limits of ASEI-BS-CZE for six inorganic anions were 6-14 ng/L. The proposed method has been adopted to analyze six anions in cigarette samples successfully.     

On-line preconcentration methods for capillary electrophoresis

The limits of detection (LOD) for capillary electrophoresis (CE) are constrained by the dimensions of the capillary. For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary. In addition, the reduced pathlength hinders common optical detection methods such as UV detection. Many different techniques have been developed to improve the LOD for CE. In general these techniques are designed to compress analyte bands within the capillary, thereby increasing the volume of sample that can be injected without loss of CE efficiency. This on-line sample preconcentration, generally referred to as stacking, is based on either the manipulation of differences in the electrophoretic mobility of analytes at the boundary of two buffers with differing resistivities or the partitioning of analytes into a stationary or pseudostationary phase. This article will discuss a number of different techniques, including field-amplified sample stacking, large-volume sample stacking, pH-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, and sweeping.     

Polymeric monolithic stationary phases for capillary electrochromatography

This review summarizes the contributions of a number of groups working in the rapidly growing area of monolithic columns for capillary electrochromatography (CEC), with a focus on those prepared from synthetic polymers. Monoliths have quickly become a well-established stationary phase format in the field of CEC. The simplicity of their in situ preparation method as well as the good control over their porous properties and surface chemistries make the monolithic separation media an attractive alternative to capillary columns packed with particulate materials. A wide variety of approaches as well as materials used for the preparation of the monolithic stationary phases are detailed. Their excellent chromatographic performance is demonstrated by numerous separations of different analytes.     

On-line sample concentration of organic anions in capillary zone electrophoresis by micelle to solvent stacking

Micelle to solvent stacking (MSS) is a new on-line sample concentration technique for charged analytes in capillary zone electrophoresis (CZE). Sample concentration in MSS mainly relies on the reversal in the effective electrophoretic mobility of the analyte at the boundary zone between the sample solution (S) and CZE background solution (BGS) inside the capillary. The basic condition for MSS is that the S is prepared in a matrix that contains an additive (i.e., micelles) which interacts with and has an opposite charge compared to the analytes. In addition, the BGS must contain a sufficient percentage of organic solvent. MSS was first reported for organic cations using anionic dodecyl sulfate micelles as additive in the S and methanol or acetonitrile as organic solvent in the BGS. Here, theoretical and experimental studies on MSS are described for organic anions using cationic cetyltrimethyl ammonium micelles as additive in the S and methanol as organic solvent in the BGS. Up to an order of magnitude improvement in concentration sensitivity was obtained for the test hypolipidaemic drugs using MSS in CZE with UV detection. The optimized method was also evaluated to the analysis of a spiked wastewater sample that was subjected to a simple extraction step.     

Sensitive determination of anions in saliva using capillary electrophoresis after transient isotachophoretic preconcentration

A transient isotachophoresis-capillary electrophoresis (tITP-CE) system for the determination of minor inorganic anions in saliva is described. The complete separation and quantification of bromide, iodide, nitrate, nitrite, and thiocyanate has been achieved with only centrifugation and dilution of the saliva sample. In-line tITP preconcentration conditions, created by introduction of the plugs of 5 mM dithionic acid (leading electrolyte) and 10 mM formic acid (terminating electrolyte) before and after the sample zone, respectively, allowed the limits of direct UV absorption detection (at 200 nm) to be up to 50-fold improved as compared with CE without tITP. As a result, nitrate and thiocyanate were still detectable at 4.6 and 3.8 μg l⁻¹, respectively, in 1000 times diluted saliva. The daily variations of anionic concentrations in saliva samples taken from a smoking health volunteer were discussed based on the results of tITP-CE analysis. It was confirmed that the thiocyanate concentration in saliva noticeably increased after smoking. This is apparently the first report on simultaneous quantification of more than four anionic salivary constituents using CE.     

Ion exchange-based preconcentration for the determination of anions by capillary electrophoresis

In the present paper a capillary zone electrophoresis (CZE)-compatible preconcentration technique for anions, based on ion exchange, is described. The described preconcentration approach has found limited use until recently because of the inherent elution step that leads to contamination of the sample with eluent components. In this paper, we describe an improved anion exchange-based preconcentration technique in which contamination of the sample with the eluent constituents, which occurs during anion elution from the preconcentration column, is eliminated by on-line chemical suppression on a packed-bed suppressor column. In the present communication, the basic principles of the proposed anion enrichment system are presented. The system was optimized, resulting in a minimal additional dilution of the eluted sample plug. This was achieved by the use of a computer-controlled, sensing/switching system. The effectiveness of the developed method was later tested on the determination of some anions in a synthetic sample using CE apparatus.     

On-line preconcentration strategies for trace analysis of metabolites by capillary electrophoresis

Analysis of low concentrations of metabolites is required for new fields of biological research, such as metabolomics. In this review, recent work in our laboratory aimed at developing improved strategies for on-line sample preconcentration of metabolites by capillary electrophoresis (CE) is presented. Dynamic pH junction, sweeping and dynamic pH junction-sweeping represent three complementary methods for electrokinetic focusing of large volumes of sample directly on-capillary. Focusing selectivity and focusing efficiency are two factors that can be used to assess the suitability of each method for different classes of metabolites. Buffer properties can be selected to enhance the focusing of specific types of metabolites based on knowledge of the analyte physicochemical properties. The application of on-line preconcentration CE for trace analysis of metabolites in real samples of interest, such as biological fluids and cellular extracts, is also demonstrated. Under optimum conditions, up to three orders of magnitude increase in concentration sensitivity can be realized for several classes of metabolites, including catecholamines, purines, nucleosides, nucleotides, amino acids, steroids and coenzymes. Recent work on hyphenating on-line preconcentration with multiplexed CE is highlighted as a promising platform for sensitive and high-throughput analyses of metabolites.     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

On-line cation-exchange preconcentration and capillary electrophoresis coupled by tee joint interface

An on-line preconcentration method based on ion exchange solid phase extraction was developed for the determination of cationic analytes in capillary electrophoresis (CE). The preconcentration-separation system consisted of a preconcentration capillary bonded with carboxyl cation-exchange stationary phase, a separation capillary for zone electrophoresis and a tee joint interface of the capillaries. Two capillaries were connected closely inside a 0.3 mm i.d. polytetrafluoroethylene tube with a side opening and fixed together by the interface. The preparations of the preconcentration capillaries and interface were described in detail in this paper. The on-line preconcentration and separation procedure of the analysis system included washing and conditioning the capillaries, loading analytes, filling with buffer solution, eluting analytes and separating by capillary zone electrophoresis (CZE). Several analysis parameters, including sample loading flow rate and time, eluting solution and volume, inner diameter and length of preconcentration capillary etc., were investigated. The proposed method enhanced the detection sensitivity of CE-UV about 5000 times for propranolol and metoprolol compared with normally electrokinetic injection. The detection limits of propranolol and metoprolol were 0.02 and 0.1 microg/L with the proposed method respectively, whereas those were 0.1 and 0.5 mg/L with conventional electrokinetic injection. The experiment results demonstrate that the proposed technique can increase the preconcentration factor evidently.     

On-line sample preconcentration by sweeping with dodecyltrimethylammonium bromide in capillary zone electrophoresis

On-line sample preconcentration of oligonucleotides with a new sweeping carrier was developed by using dodecyltrimethylammonium bromide (DTAB) below the critical micelle concentration (CMC). The sweeping results with DTAB below and above the CMC were compared. The use of DTAB below the CMC benefits the preconcentration of the oligonucleotides, while the use of DTAB above the CMC is good for hydrophobic small molecules. The factors affecting the sweeping results were optimized and this method was evaluated by constructing calibration curves for thrombin aptamers. The sweeping scheme produced a 112-fold sensitivity enhancement for the oligonucleotides relative to that run in a running buffer without DTAB. The sweeping method developed here can be a good reinforcement of the preconcentration scheme by sweeping when less-hydrophobic analytes or large negatively-charged molecules need to be preconcentrated.     

On-line sample preconcentration in capillary electrophoresis. Fundamentals and applications

On-line preconcentration is one of the aspects of analytical method development using capillary electrophoretic techniques. The choice of the sample matrix alone can significantly alter both method sensitivity and separation efficiency. The recent trend to detect samples in narrower separation vessels also necessitates the need to improve detection sensitivity. The desire to detect very low levels of analytes using limited amounts of sample from biological specimens and the high separation efficiency obtainable using very large injections compared to classical small size injections also adds to this list. Indeed, one of the rich areas of research in the capillary electrophoresis field is on on-line sample preconcentration. More than 400 published research articles gathered from the http://www.webofscience.com from the year 2000 described a form of on-line preconcentration in capillary electrophoresis. This review provides a comprehensive table listing the applications of on-line preconcentration in capillary electrophoresis.     

On-line preconcentration of perfluorooctanoic acid and perfluorooctanesulfonic acid by nonaqueous capillary electrophoresis

Separation of major environmental pollutants as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) by capillary electrophoresis is reported for the first time. It is not possible to resolve the solutes in an aqueous media. However, the use of methanol and acetonitrile as the background electrolyte (BGE) solvents allowed their rapid separation in an uncoated capillary. A major effort was put into BGE optimization in respect to both separation efficiency and detection for further on‐line preconcentration. 5 mmol.L^?1 naphthalene‐1‐sulfonic acid and 10 mmol.L^?1 triethylamine dissolved in ACN/MeOH (50:50 v/v) provided best separation and detection conditions. Next, the large‐volume sample stacking and the field‐amplified sample injection were applied and compared. Large‐volume sample stacking improved limits of detection (LODs) with regard to the standard injection by 69 times for PFOA and 143 times for PFOS with LODs of 280 and 230 nmol.L^?1, respectively. Field‐amplified sample injection improved LODs 624 times for PFOAand 806 times for PFOS with LODs 31 and 40 nmol.L^?1, respectively. Both preconcentration methods showed repeatabilities of migration times less than 1.2% RSD intraday and 6.6% RSD interday. The method was applied on PFOA and PFOS analysis in a sample of river water treated with solid‐phase extraction, which further improved LOD toward 5.6 × 10^?10 mol.L^?1 for PFOS and 6.4 × 10^?10 mol.L^?1 for PFOA and allows the method to be used for river water contamination screening or decomposition studies.     

Electrochromatography in poly(dimethyl)siloxane microchips using organic monolithic stationary phases

This paper shows the in situ synthesis of an hexyl acrylate monolith in PDMS microfluidic devices and its subsequent use as stationary phase for electrochromatography on chip. To overcome the ability of PDMS material to absorb organic monomers, surface modification of the enclosed channels was realized by UV-mediated graft polymerization. This grafting procedure is based on the preliminary adsorption of a photoinitiator onto the PDMS surface and polymerization of charged monomers. Next, hexyl acrylate monoliths were cast in situ using photopolymerization process. The chromatographic behavior of the monolithic column was confirmed by the successful separation of derivatized catecholamines in the PDMS device using a 30 mm effective separation length (100 microm x 100 microm section). Efficiencies reached up to 200,000 plates per meter.     

On-line sample preconcentration of cationic analytes by dynamic pH junction in capillary electrophoresis

To improve detection sensitivity of cationic analytes, a dynamic pH junction technique was examined. Dynamic pH junction is an on-line focusing method in capillary electrophoresis (CE) based on the difference in the analyte's mobility between the background electrolyte (BGE) and sample matrix. The effects of pH values and concentrations of the BGE and the sample matrix on dynamic pH junction were examined. Optimization of analyte focusing resulted in enhanced detection responses of about 100-160-fold in terms of peak heights for some anilines in comparison to conventional injections. In particular, the concentration limits of detection (LOD) (S/N = 3) for the test anilines obtained with dynamic pH junction were from 1.9 to 3.7 ppb with UV detection without any pretreatment procedure.     

On-line preconcentration capillary electrophoresis for purity profiling of synthetic peptides

In this work, an on-line preconcentration capillary electrophoresis method was optimized and evaluated for the purity control of the biologically active synthetic peptide fas-F (a 28-residue long fragment of fasciculin-1) and applied for the purity profiling of angiotensin I, oxytocin, bradykinin and luteinizing hormone releasing hormone. The laboratory-made device of the analyte concentrator cartridge consisted of a fused-silica capillary piece (150 μm i.d.×8 mm in length) packed with silica-based C18 reversed-phase chromatographic material and coupled on-line near the inlet of the separation capillary (bare fused-silica capillary, 75 μm from the concentrator to the detector). Separation of impurities present in assayed samples was achieved by using 25 mM potassium dihydrogen phosphate, pH 3.5, as running buffer and a mixture of acetonitrile: running buffer, 75:25 (v/v), as elution buffer. The intra-day relative standard deviation (R.S.D.) values for migration times ranged from 3.4 to 4.2 and 2.2-2.6% for peak areas. The inter-day R.S.D. values were 5.6-7.1 and 4.6-5.1% for migration times and peak areas, respectively. The impurity profiles obtained for each peptide were compared by CZE and on-line preconcentration CE. The proposed method allowed the preconcentration and separation of impurities with greater selectivity and higher sensitivity (100-200-fold) with respect to capillary zone electrophoresis without on-line preconcentration.     

Poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases in capillary electrochromatography

In this study, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases, which were prepared by single step in situ polymerization of divinylbenzene and various alkyl methacrylates (butyl-, octyl-, or lauryl-methacrylate), were developed as separation columns of benzophenone compounds for capillary electrochromatography (CEC). In addition to the presence of plenty of benzene moieties, the stationary phases contained long and flexible alkyl groups on the surface. With an increase in the molecular length of alkyl methacrylate, the polymeric monolith, which had higher hydrophobicity, effectively reduced the peak tailing of benzophenones, but a weaker retention was observed. The unusual phenomenon was likely due to the π–π interaction between the aromatic compound and the polymeric material. The usage of longer alkyl methacrylate as reaction monomer limited the retention of aromatic compounds on the stationary phase surface, thus the π–π interaction between them was possibly reduced. Consequently, the retention time of aromatic compounds was markedly decreased with an increase in carbon length of alkyl methacrylate that was carried on the polymeric monolith. Compared to previous reports on polystyrene-based columns in which the peak-tailing problem was reduced by decreasing the benzene moieties on the stationary phase, this study demonstrated that the undesirable retention (peak-tailing) could also be improved by the inclusion of long alkyl methacrylate to the polystyrene-based columns.     

Determination of urea-derived pesticides in fruits and vegetables by solid-phase preconcentration and capillary electrophoresis

A multiresidue analytical method based on solid-phase extraction (SPE) enrichment combined with capillary electrophoresis (CE), using micellar electrokinetic capillary chromatography (MEKC), was developed to determine ten substituted urea pesticides in orange and tomato samples. Several factors such as pH, composition and concentration of the buffer, concentration of surfactant, addition of organic solvent, and working voltage were optimized to obtain the best compound separation in the shortest time. Separation can be achieved in 7 min using a micellar aqueous pH 9 buffer composed of 4 mM borate and 35 mM sodium dodecyl sulfate. After an SPE procedure, which provided a 10-fold enrichment, the limit of detection was about 0.05 mg kg(-1), which is in the order of the maximum residue limits (MRLs) established by the European Union (EU) for most of these compounds. Increasing the enrichment factor by using a larger amount of sample is difficult in oranges due to the matrix interferences, but is possible in tomatoes, which gave cleaner extracts and easily reached a 25-fold enrichment factor. The procedure involving SPE and CE provided acceptable recoveries (ranged 42-118%) and relative standard deviations (RSDs; < 19%) at levels between 0.3 and 5 mg kg(-1).     

Investigation of the ion-exchange properties of methacrylate-based mixed-mode monolithic stationary phases employed as stationary phases in capillary electrochromatography

The potential of methacrylate-based mixed-mode monolithic stationary phases bearing sulfonic acid groups for the separation of positively charged analytes (alkylanilines, amino acids, and peptides) by capillary electrochromatography (CEC) is investigated. The retention mechanism of protonated alkylanilines as positively charged model solutes on these negatively charged mixed-mode stationary phases is investigated by studying the influence of mobile phase and stationary phase parameters on the corrected retention factor which was calculated by taking the electrophoretic mobility of the solutes into consideration. It is shown that both solvophobic and ion-exchange interactions contribute to the retention of these analytes. The dependence of the corrected retention factor on (1) the concentration of the counter ion ammonium and (2) the number of methylene groups in the alkyl chain of the model analytes investigated shows clearly that a one-site model (solvophobic and ion-exchange interactions take place simultaneously at a single type of site) has to be taken to describe the retention behaviour observed. Comparison of the CEC separation of these charged analytes with electrophoretic mobilities determined by open-tubular capillary electrophoresis shows that mainly chromatographic interactions (solvophobic and ion-exchange interactions) are responsible for the selectivity observed in CEC, while the electrophoretic migration of these analytes plays only a minor role.