Two limitations of capillary electrophoresis (CE) are the low sample loadability of the capillary and an incompatibility with high ionic strength samples. Several strategies have been described to preconcentrate and lower the ionic strength of physiological samples prior to CE analysis. These have included both off-capillary and on-capillary approaches. We have previously described a version of on-column field-amplification stacking termed pH-mediated stacking. pH-mediated stacking was initially developed for the separation of cations. In this report, we describe the application of pH-mediated sample stacking to anions. In this method, an electrokinetic injection is used to introduce analyte anions into the CE system and simultaneously replace the sample matrix cations with ammonia from the background electrolyte. Base is then electrokinetically injected to neutralize the sample zone and create a low conductivity region across which the analyte anions will stack. Using this method, a sensitivity enhancement of more than 66-fold was achieved without loss in separation efficiency relative to normal electrokinetic injection. Detection limits of 0.3 μM for four phenolic acids in a physiological sample were achieved using simple UV absorbance detection. The limit to the amount of sample that could be loaded using this technique was the length of the separation capillary. To further increase the amount of sample that could be loaded, a double-capillary system was developed. Using the double-capillary system the sensitivity was increased more than 300-fold and detection limits of 0.06 μM were achieved.Capillary electrophoresis (CE) has become a powerful analytical technique. 1,2 A major limitation of CE analysis is the small sample volume that can be loaded into the separation capillary without compromising the separation. CE is also generally incompatible with high ionic strength samples because of band broadening induced by the weaker electric field across the sample zone relative to the separation electric field. In most cases, physiological samples are subjected to pretreatment, usually involving solid-phase or liquid-liquid extraction, prior to CE analysis. The need for such pretreatment steps increases the difficulty of the analysis and decreases the sample throughput. Several approaches have been described to preconcentrate samples in the CE capillary prior to separation. The most common approach is termed field amplification 3-9 and involves injecting the analytes in a low ionic strength matrix. In this case, the electric field across the sample is stronger than that across the separation capillary. Analyte ions of the proper charge will rapidly migrate across the sample zone and stack at the interface of the sample zone and background electrolyte (BGE). Up to a 100-fold sample preconcentration has been reported using field-amplification stacking. 7 This approach has been modified using polarity reversal to remove the low conductivity sample zone after stacking but prior to CE separation. 10-12 The limi...

Recently, a new ion source, Direct Analysis in Real Time (DART), has been introduced which allows direct biological sample introduction into a mass spectrometry (MS) system. The elimination of conventionally required sample preparation and separation by high-performance liquid chromatography (HPLC) prior to MS analysis represents a remarkable opportunity to reduce assay turn-around time, environmental impact and capital/manpower investment. This new technology initially was used in various qualitative applications to directly detect chemicals on solid surfaces, in liquids and gases. In this study, a DART source operating under ambient pressure with ground potential was installed onto a Sciex 4000 tandem mass spectrometer and employed in the sample analysis of plasma based on direct introduction into the DART-MS/MS system. Reasonable precision and accuracy (%CV and %Error, both <10%) were achieved of a significant number of compounds tested in biological fluids. In addition, the limit of detection for 80% of the tested compounds reached 5 ng/mL or lower which is sufficient for pharmaceutical drug discovery support. Finally, experimental conditions that significantly impacted assay performance were investigated with respect to optimization and limitation. Because of its simplicity, fast data acquisition (3-5 s) and low cost, DART has the potential to significantly impact quantitative pharmaceutical analysis in biological matrices.

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An on-line sample concentration method using sample matrix switching and field amplification peak stacking has been developed. A microbore LC guard column is used to slightly retain the analytes in order to switch from a high ionic strength sample matrix (the physiological fluid) to a low ionic strength matrix (the LC mobile phase). The eluted LC peak is then trapped in a CE system and preconcentrated by field amplification peak stacking. The concentrated sample peak is then analyzed by CE. Compared to normal hydrodynamic injection, the sensitivity was increased by more than 500-fold without loss in resolution. A limit of detection of less than 10 nM for a physiological sample was achieved using UV adsorption detection. This method can be used for negatively or positively charged analytes.A major limitation of capillary electrophoresis (CE) is the poor concentration detection limits achieved using spectrophotometric detectors. To achieve useable detection limits for most biological applications, special detectors such as laser-induced fluorescence or electrochemistry must be used. However, these are limited in the range of compounds that are detectable relative to UV absorbance. Therefore, approaches to improve the concentration detection limits achievable using spectrophotometric detection would dramatically increase the utility of CE.To achieve useable detection limits with UV absorbance, a preconcentration step is typically performed prior to CE analysis. However, one of the strengths of CE is the small sample volume requirement of the technique. Traditional preconcentration approaches, such as solid-phase or liquid-liquid extractions, require sample volumes that eliminate this advantage. Therefore, several on-column approaches to sample preconcentration have been developed. These include field amplification, capillary isotachophoresis, and chromatographic preconcentration.In field amplification stacking, sample analytes are concentrated into a narrow band at the edge of the sample zone. 1-7 This is accomplished by generating a strong electric field across the sample zone by using a sample matrix of significantly lower conductivity than the running buffer. Analytes then migrate much faster in the sample zone than in the running buffer. This approach has been modified using polarity reversal to remove the low-conductivity sample matrix prior to the electrophoretic separation. 8-10 Termed large-volume injection with polarity reversal, the sample is initially injected hydrodynamically; the matrix is electroosmotically removed under reverse polarity conditions while sample anions stack at the boundary of the sample zone and the running buffer. Once the sample matrix is largely removed, the separation is performed under normal polarity. On-column transient capillary isotachophoresis (CITP) * To whom correspondence should be addressed: (phone) 785-864-3072. can be viewed as a sophisticated form of stacking. 11-14 For CITP, the sample zone is sandwiched between a leading electrolyte, with a mobility greater ...