Lowering the concentration limits of detection by on-line solid-phase extraction-capillary electrophoresis-electrospray mass spectrometry

The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C(18) derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100muL). The captured analytes were eluted in a small volume of an appropriate solution (20-50nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25mM of formic acid in MeCN):H(2)O and a sheath liquid of 20mM of acetic acid in 50:50 (v/v) methanol:H(2)O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.     

On-line coupling of SPE and CE-MS for peptide analysis

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.     

Recent approaches in sensitive enantioseparations by CE

The latest strategies and instrumental improvements for enhancing the detection sensitivity in chiral analysis by CE are reviewed in this work. Following the previous reviews by García-Ruiz et al. (Electrophoresis 2006, 27, 195-212) and Sánchez-Hernández et al. (Electrophoresis 2008, 29, 237-251; Electrophoresis 2010, 31, 28-43), this review includes those papers that were published during the period from June 2009 to May 2011. These works describe the use of offline and online sample treatment techniques, online sample preconcentration techniques based on electrophoretic principles, and alternative detection systems to UV-Vis to increase the detection sensitivity. The application of the above-mentioned strategies, either alone or combined, to improve the sensitivity in the enantiomeric analysis of a broad range of samples, such as pharmaceutical, biological, food and environmental samples, enables to decrease the limits of detection up to 10?12 M. The use of microchips to achieve sensitive chiral separations is also discussed.     

Design and applications of coupled SPE-CE

CE suffers from an inherent low concentration sensitivity. Analyte detection limits can be improved by combining CE with SPE. This paper presents an overview of coupled SPE-CE systems that have been reported in literature. Attention is paid to fundamental aspects of coupling SPE and CE, as well as to important SPE requirements. Interfaces for inline and online coupling with CE are critically discussed, and their mode of operation is outlined. Advantages and limitations of the interfaces are discussed and typical examples are selected. Finally, some future developments are discussed.     

The current state of two-dimensional electrophoresis with immobilized pH gradients

The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Gorg et al., Electrophoresis 1988, 9, 531-546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.     

CE and CEC analysis of phytochemicals in herbal medicines

CE and CEC, due to their versatility and high efficiency, have attracted great interest in the analysis of phytochemicals in herbs and their preparations. Previously, we reviewed the analysis of phytochemical bioactive compounds by CE in 2006 (Electrophoresis 2006, 27, 4808-4819) or CEC in 2010 (Electrophoresis 2010, 31, 260-277). This review followed the previous studies and covered the literature published since 2006 for CE and 2009 for CEC (excluding those mentioned in the two previous reviews), which emphasized the development of CE and CEC techniques in phytochemical analysis. In addition, sample preparation and detection were also discussed.     

Improving the sensitivity of the determination of ceftiofur by capillary electrophoresis in environmental water samples: in-line solid phase extraction and sample stacking techniques

This paper describes two different approaches for increasing the sensitivity for the analysis of ceftiofur by capillary electrophoresis (CE). Two different techniques based on the introduction of an enlarged volume of sample, namely large volume sample stacking (LVSS) and in-line solid phase extraction (SPE) were studied and compared. LVSS allowed the on-column electrophoretic preconcentration of ceftiofur without modification of the separation capillary. In-line SPE-CE was developed by using a home-made microcartridge that was filled with a reversed-phase sorbent (C₁₈). The microcartridge was coupled in-line near the inlet of the separation capillary. LVSS and in-line SPE-CE allowed automated operation and improved sensitivity for the analysis of ceftiofur with respect to conventional CE. When environmental water samples were analyzed, an additional pretreatment step based on off-line SPE was necessary in both cases to further decrease the detection limits. In terms of sensitivity for the determination of ceftiofur in river water samples, the combination of off-line SPE with in-line SPE-CE was found the most sensitive with a detection limit of 10 ng L⁻¹, whereas the method based on the use of off-line SPE with LVSS presented a detection limit of 100 ng L⁻¹.     

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2008-2010)

Capillary electrophoresis has been alive for over two decades now; yet, its sensitivity is still regarded as being inferior to that of more traditional methods of separation such as HPLC. As such, it is unsurprising that overcoming this issue still generates much scientific interest. This review continues to update this series of reviews, first published in Electrophoresis in 2007, with an update published in 2009 and covers material published through to June 2010. It includes developments in the fields of stacking, covering all methods from field-amplified sample stacking and large volume sample stacking, through to ITP, dynamic pH junction and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.     

Recent advances in amino acid analysis by capillary electrophoresis

This paper describes the most important articles that have been published on amino acid analysis using CE during the period from June 2009 to May 2011 and follows the format of the previous articles of Smith (Electrophoresis 1999, 20, 3078-3083), Prata et al. (Electrophoresis 2001, 22, 4129-4138) and Poinsot et al. (Electrophoresis 2003, 24, 4047-4062; Electrophoresis 2006, 27, 176-194; Electrophoresis 2008, 29, 207-223; Electrophoresis 2010, 31, 105-121). We present new developments in amino acid analysis with CE, which are reported describing the use of lasers or light emitting diodes for fluorescence detection, conductimetry electrochemiluminescence detectors, mass spectrometry applications, and lab-on-a-chip applications using CE. In addition, we describe articles concerning clinical studies and neurochemical applications of these techniques.     

Recent developments in on-line electrochemical stripping analysis--an overview of the last 12 years

This paper presents an overview of the field of electrochemical stripping analysis in flow systems covering developments in the last 12 years (since 1998). The review discusses the flow schemes utilized in stripping analysis, techniques for on-line sample pre-treatment, the main pre-concentration and stripping/detection modes, the most important flow-through cell configurations used and the different types of working electrodes. Finally, applications in inorganic and organic analysis are discussed. Special emphasis is given to different novel approaches developed over the last few years that hold some promise for the future such as the use of the lab-on-a valve (LOV) configuration, microfluidic manifolds, flow-probes for remote sensing, environmentally friendly electrode materials and hyphenation with spectroscopic techniques.     

UV light-emitting diode-induced fluorescence detection combined with online sample concentration techniques for capillary electrophoresis.

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.     

On-line coupling of extraction with gas chromatography

A recent trend in the development of analytical methodologies is the integration of the sample preparation step directly with the chromatographic system. In integrated on-line systems, extraction, clean-up, separation and detection are connected with each other and the whole analytical procedure takes place in a closed, usually automated system. In this review, a critical overview of the principles and benefits of on-line coupling of extraction with gas chromatography is presented. Techniques suitable for the extraction and analysis of various types of compounds in both solid and liquid samples are presented. The potential of on-line techniques is discussed and illustrated with selected examples.     

Use of large-volume sample stacking in on-line solid-phase extraction-capillary electrophoresis for improved sensitivity

We present a new system for the sensitive analysis of cephalosporins by CE using both on-line SPE and large-volume sample stacking (LVSS). Sample volumes of 250 muL were loaded onto the SPE microcolumn which was then desorbed with 426 nL of ACN. The SPE elution plug was injected into the CE system via an in-line valve interface filling approximately 60% of the volume of the separation capillary. Subsequently, LVSS was performed by applying a voltage of -5 kV, which resulted in the simultaneous removal of the elution solvent and the preconcentration of the analytes in a narrow zone. This way the amount of analyte loaded into the capillary could be considerably increased without serious loss of CE separation efficiency. LODs for cefoperazone and ceftiofur were in the ng/L range which represents an improvement of a factor of 8450 and 11 450 when compared with direct CE injection. The cephalosporin test compounds presented a good linear response (corrected peak area) between 0.5 and 10 mug/L with correlation coefficients higher than 0.995. The final method is compared with previously reported LVSS-CE and SPE-CE systems for the analysis of cephalosporins.     

Recent developments in microcolumn liquid chromatography.

An overview of the most recent developments in microcolumn liquid chromatography (LC) is presented. A short theoretical discussion on chromatographic dilution and extracolumn bandbroadening is given and also the recent progress and advances in column technology and instrumentation are reviewed. However, the emphasis of this review is on miniaturized sample clean-up, sample introduction techniques and on both established and more recent detection techniques for microcolumn LC. The hyphenation of miniaturized LC columns with other techniques, specifically on multidimensional chromatography and the coupling of microcolumn LC to mass spectrometry is discussed in detail. Both the on-line and automated off-line interfacing to other separation and detection techniques will also be addressed. Finally, a number of typical microcolumn LC applications are presented in order to demonstrate the potential of microcolumn LC methods in a variety of scientific areas.     

Flow-injection systems for determination of trace manganese in various salts by catalytic photometric detection

Two flow-injection analysis (FIA) systems for the determination of trace manganese in salts are presented using highly sensitive catalytic detection based on the oxidation of 3,4-dihydroxybenzoic acid by hydrogen peroxide. Two different approaches, the use of a large sample volume injection in a usual FIA mode (system A) and on-line coupling of a cation-exchange separation column with detection in a continuous flow system (system B), have proved very effective for eliminating the blank peak problem and thus affording direct injection of a sample solution containing a large concentration of salt. The limits of determinations are 0.04 ppm and 0.01 ppm for systems A and B respectively, when a 5 g sample is used for preparing the 100 ml sample solution. The proposed FIA systems were satisfactorily applied to the determination of manganese at 0.03-1.59 ppm in solar salts (salts made by exposing brine to the sun) with good precision.     

Determination of alkyl- and alkanolamines in drinking and natural waters by capillary electrophoresis with isotachophoretic on-line preconcentration

A procedure is developed for the determination of several amines in drinking and natural waters by capillary electrophoresis with isotachophoretic on-line preconcentration without sample preparation. A background electrolyte based on acridine as an absorbing ion is proposed for analysis with isotachophoretic on-line preconcentration and indirect photometric detection. The sample was injected in the hydrodynamic mode. The procedure was tested on drinking and natural water samples. The accuracy of data obtained was confirmed by the added–found method. The analytical range was from 0.25 to 5 mg/L. The time of one analysis was 5–6 min.     

Microfluidics with MALDI analysis for proteomics—A review

Various microfluidic devices have been developed for proteomic analyses and many of these have been designed specifically for mass spectrometry detection. In this review, we present an overview of chip fabrication, microfluidic components, and the interfacing of these devices to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. These devices can be directly coupled to the mass spectrometer for on-line analysis in real-time, or samples can be analyzed on-chip or deposited onto targets for off-line readout. Several approaches for combining microfluidic devices with analytical functions such as sample cleanup, digestion, and separations with MALDI mass spectrometry are discussed.     

On-line infrared detection in aqueous micro-volume systems

Mid infrared spectroscopy is a non-destructive technique that can provide detailed information on important, molecule-specific features such as the conformation and functional groups of a large range of compounds. Infrared spectroscopy is now an established and frequently used technique for qualitative analysis, i.e. the identification of chemical constituents in a sample. In addition, its use for quantitative purposes has grown dramatically in recent years. It is important to realise that the analytical problem defines the mode of operation and implementation of the FTIR technique. This Highlight article focuses on the advantages and scope of on-line FTIR detection strategies. However, in common with all techniques, on-line FTIR detection has a number of potential shortcomings, which are also discussed.     

Staggered multistep elution solid-phase extraction capillary electrophoresis/tandem mass spectrometry: a high-throughput approach in protein analysis

An approach based on staggered multistep elution solid-phase extraction (SPE) capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) was developed in the analysis of digested protein mixtures. On-line coupling of SPE with CE/MS was achieved using a two-leveled two-cross polydimethylsiloxane (PDMS)-based interface. Multistep elution SPE was used prior to CE to provide an additional dimension of separation, thus extending the separation capacity for the peptide mixture analysis. By decreasing in the number of co-eluting peptides, problems stemming from ionization suppression and finite MS/MS duty cycle were reduced. As a result, sequence coverage increased significantly using multistep elution SPE-CE/MS/MS compared to one-step elution SPE-CE/MS/MS in the analysis of a single protein tryptic digest (49% vs. 18%) and a six protein tryptic digest (22-71% vs. 10-44%). A staggered CE method was incorporated to increase the throughput. The electropherograms of consecutive CE runs were partially overlapped by injecting the sample plug at a fixed time interval. With the use of a 5 min injection interval, slightly poor results were obtained in comparison with the sequential CE method while the total analysis time was reduced to 28%.