U.R. Tjaden

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellmans reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

Isotachophoresis as an on-line concentration pretreatment technique in capillary electrophoresis

One of the major disadvantages of capillary electrophoresis (CE) is its limited loadability. Therefore, the on-line coupling of isotachophoresis (ITP) and CE was studied with regard to its potential for the improvement of the minimum concentration that can be measured by CE. Based on the concentrating and separating power of ITP, detection limits could be lowered by at least two orders of magnitude. Especially for biological samples containing proteins, it appeared that in non-treated capillaries the electromigration characteristics are hardly influenced when isotachophoretic pretreatment is applied. The potential of ITP-CE coupling is illustrated by the analysis of o-phthaldialdehyde and fluorescein isothiocyanate derivatives of a number of amino acids.

Rapid Communication: Neuropeptide Expression and Processing as Revealed by Direct Matrix-Assisted Laser Desorption Ionization Mass Spectrometry of Single Neurons

Abstract: Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI‐MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI‐ MS study of direct peptide fingerprinting in the single neuron these experients demonstrate that MALDI‐MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single‐cell studies in vertebrates, including humans.

Capillary electrophoresis-mass spectrometry

Abstract The developments and state-of-the-art in capillary electrophoresis—mass spectrometry (CE—MS) are reviewed and evaluated. Attention is paid to interfaces for CE—MS, the coupling of CE to the interface, applications of CE—MS in both qualitative and quantitative analysis, CE buffer composition and the coupling of other electromigration techniques to MS. The state-of-the-art is critically reviewed. Special attention is paid to the achievable concentration detection limits, because the present limits in the low-ppm range prohibit the broad analytical use of the CE—MS technique. Current approaches to solving or removing these problems are discussed.

Pseudo-electrochromatography-mass spectrometry : a new alternative

Abstract Pseudo-electrochromatography (pEC) is a separation method that is based on the combination of the chromatographic and electrophoretic behaviour of compounds. This combination enables tuning of the selectivity without changing the composition of the mobile phase. The loadability of pEC is considerably higher than for capillary electrophoresis, which makes the coupling to mass spectrometry very attractive. The applicability of the method was examined for some nucleotides, alkaloids and an antiviral drug as model compounds. The method appeared to be able to replace a modifier gradient elution in reversed-phase system, thus circumventing the use of an expensive gradient system. pEC has been combined with continuous-flow fast atom bombardment mass spectrometry, as is demonstrated with some examples showing the improvement in the performance of the total system.

Automated isotachophoretic analyte focusing for capillary zone electrophoresis in a single capillary using hydrodynamic back-pressure programming

Abstract An automated isotachophoretic (ITP) analyte focusing procedure prior to capillary zone electrophoresis (CZE)` is described. The ITP focusing step is carried out in the same capillary as the CZE. Hydrodynamic back-pressure programming resulted in a reduction in the effects of the electroosmotic flow-rate during the ITP step and allowed the removal of terminating buffer before the CZE run was started. The characteristics of this on-line focusing procedure were studied for several anionic test compounds.

Liquid chromatography-mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with liquid chromatography-mass spectrometry (LC-MS), for the analysis of biological samples is described. The system was tested with cortisol and prednisolone for plasma analysis and arachidonic acid for urine analysis. A precolumn packed with a 25-micron C18 alkyl-diol support is used for direct plasma or urine injection. Using column-switching techniques, the analytes enriched on the precolumn are eluted to the analytical column without transfer loss. An on-line heart-cut technique was employed and only the analyte-containing fraction eluting from the LC column is directed to the MS to protect the LC-MS interface and ion-source from contamination. The whole system is operated in a parallel mode, that is, sample pre-treatment and LC-MS analysis are performed simultaneously to provide the shortest possible analysis time. The only off-line sample pre-treatment step required was centrifugation to remove particulate matter. With the fully automated system, total analysis times of 5 and 9.5 min were achieved for cortisol in serum and arachidonic acid in urine, respectively. Cortisol and related compounds were quantitatively recovered from plasma with a detection limit for prednisolone (direct injection of 100 microliters on restricted-access precolumn) of 2 ng/ml.

Pseudo-electrochromatography—negative-ion electrospray mass spectrometry of aromatic glucuronides and food colours

Pseudo-electrochromatography is a combination of liquid chromatography and an electromigration technique, especially directed at the separation of ionic compounds prior to mass spectrometric detection with a mobile phase composition compatible with mass spectrometry. The application of pseudo-electrochromatography to the separation of food colours and aromatic glucuronides is described. An example of selectivity tuning by applying voltages of differing polarity during the chromatographic run is given. The coupling of pseudo-electrochromatography with electrospray mass spectrometry is demonstrated. Differences in the effects of the axial potential over the column between silica-based and polymeric packing materials are discussed.

Analyte focusing in capillary electrophoresis using on-line isotachophoresis

Abstract The on-line coupling of isotachophoresis with capillary electrophoresis as presented earlier was improved with respect to recovery, reproducibility and ease of operation by developing a new system. Owing to the isotachophoretic sample pretreatment resulting in analyte focusing, the corresponding determination limits in capillary electrophoresis could be enhanced by three orders of magnitude. It is shown that both reproducibility and selectivity are improved considerably owing to the two-dimensional electrophoretic approach. The potential of one-line coupled isotachophoresis-electrophoresis was demonstrated using a fluorescein isothiocyanate derivative of the peptide angiotensin III as test compound.

A free‐flow electrophoresis chip device for interfacing capillary isoelectric focusing on‐line with electrospray mass spectrometry

When electrospray ionisation mass spectrometry (ESI-MS) is used on-line with capillary isoelectric focusing (CIEF), the presence of the carrier ampholytes creating the IEF pH gradient is not desirable. With the purpose of removing these ampholytes, we have developed a free-flow electrophoresis (FFE) device and coupled it to CIEF. The different parameters inherent to the resulting CIEF/FFE system were optimised using ultraviolet absorbance (UV) detection. The on-line coupling of this system with ESI-MS was successfully realised for three model proteins (myoglobin, carbonic anhydrase I and beta-lactoglobulin B).