W.M.A. Niessen

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.

Capillary isotachophoretic analyte focusing for capillary electrophoresis with mass spectrometric detection using electrospray ionization

An improvement in detectability in capillary electrophoresis-mass spectrometry (CE-MS) was realized using isotachophoretic analyte focusing. The practical approach is described for the on-line coupling of capillary isotachophoresis-capillary electrophoresis-mass spectrometry (ITP-CE-MS) using an electrospray interface. An equation was derived for the calculation of the splitting ratio of ITP zones into the CE system. The applicability of analyte focusing is demonstrated for the analysis of a mixture of anthracyclines. Initial experiments showed a 200-fold improvement in concentration detection limit for ITP-CE-MS compared with CE-MS.

Characterization of sugar oligomers by on-line high-performance anion-exchange chromatography-thermospray mass spectrometry.

Abstract The on-line coupling of high-performance anion-exchange chromatography and thermospray mass spectrometry via an anion-micromembrane suppressor and a booster pump is described. The system was applied to the analysis of homologous series of oligosaccharides. Among others, the mass spectrometric detection of β-1,4-xylose oligomers up to degree of polymerization (DP) 25 is demonstrated. Further, the system was used in the analysis of more complex oligosaccharide samples, containing mixed oligomers of hexoses, pentoses and uronic acids. In such samples oligomers up to DP 10 can be tested. The potential use of this approach in the characterization of oligosaccharides obtained from (enzymic) degradation of plant cell wall oligosaccharides is discussed.

Phase-system switching as an on-line sample pretreatment in the bioanalysis of mitomycin C using supercritical fluid chromatography.

One of the problems of the application of supercritical fluid chromatography (SFC) in bioanalysis is the fact that many sample pretreatment procedures deliver the solutes of interest in a polar solvent, which upon injection will dramatically disturb the phase system characteristics of the SFC system. The phase-system switching approach, recently introduced for liquid chromatography-mass spectrometry, can be used to avoid this problem. Plasma samples containing the thermolabile and pH-sensitive cytostatic drug Mitomycin C (MMC) were injected onto a short precolumn. After washing and drying of the precolumn the compound of interest was desorbed using a supercritical fluid and analyzed by SFC. Up to 1 ml of plasma containing 20 ng of MMC has been analyzed in this way.

Development of optimization strategies in thermospray liquid chromatography—mass spectrometry

Abstract In order to optimize the sensitivity and reproducibility of thermospray liquid chromatography—mass spectrometry in (bio)analytical applications, some of the experimental parameters that influence thermospray buffer ionization have been investigated systematically. Attention was paid to the vaporizer temperature, which is especially important in the analysis of thermolabile compounds, the ammonium acetate concentration, the methanol content and the repeller potential. General optimization strategies for theirmospray buffer ionization have been developed. The usefulness of extensive optimization is discussed for qualitative and quantitative analysis. In quantitative target compound analysis optimization on the analyte is necessary. In qualitative analysis, however, usually unknown compounds have to be analysed and no parent compound is available for optimization purposes.

Liquid chromatography-mass spectrometry in bioanalysis: some applications and a new approach for non-volatile buffer systems.

Combined liquid chromatography mass spectrometry (LC/MS) is still a rapidly developing area. The advantages and limitations of the most important interfaces, i.e. the moving belt interface, the thermospray interface, and continuous-flow fast atom bombardment, are discussed from a bioanalytical point of view. The discussion is underlined by two bioanalytical applications of LC/MS using a moving belt interface. The first example is the identification of unknown compounds in alkaline extracts of post-mortem human urine and plasma. The second example is actually concerned with a new approach in LC/MS: phase-system switching using a short column between the LC chromatography and the moving belt interface. The perspectives of the latter are outlined.

Liquid chromatography—mass spectrometry : The need for a multidimensional approach

Abstract Combined liquid chromatography—mass spectrometry is now used routinely in both qualitative and quantitative analysis. Thefore, it is important to consider the integration of this method is the whole analytical procedure. Attention has to be paid to other topics than interface technology. This paper deals with some of those aspects, especially with those important in target compound analysis. Strategies are outlined and discussed in the tunning of the selectivity of the method, in improving the compability of the liquid chromatography, the interface and mass spectrometry, in enhancing the detectability, and with respect to pre- and post-column derivatization techniques.

Specific screening methods for glucosinolates in sprout extracts using on-line thermospray liquid chromatography-tandem mass spectrometry

Abstract Considering the growing importance of the determination and identification of (desulpho-)glucosinolates in extracts of plant materials, rapid screening methods for desulphoglucosinolates were developed, based on thermospray liquid chromatography-mass spectrometry (LC-MS) and LC-MS-MS. In thermospray LC-MS the screening of plant extracts containing various desulphoglucosinolates can be based on the group-specific fragment at m/z 214, but identification of the peaks is not always straightforward owing to the lack of compound-specific fragments in the mass spectra. Using a constant neutral loss of 162 a.m.u., corresponding to the loss of the sugar ring, in thermospray LC-MS-MS the screening and identification of the various desulphoglucosinolates gives better results, especially because the signal in the MS-MS mode is less dependent on the fluctuating thermospray conditions. In some applications direct thermospray MS-MS analysis of mixtures, injected in the flow-injection mode, can give satisfactory results.

Bioanalysis of erythromycin 2'-ethylsuccinate in plasma using phase-system switching continuous-flow fast atom bombardment liquid chromatography-mass spectrometry.

A specific method for the determination of erythromycin 2-ethylsuccinate (EM-ES) in plasma is described. The method involves a liquid-liquid extraction procedure followed by the analysis of extracts using phase-system switching (PSS) continuous-flow fast atom bombardment (CF-FAB) liquid chromatography-mass spectrometry (LC-MS). In PSS EM-ES is enriched after analytical separation on a short trapping column, from which it is desorbed to the LC-MS interface. In this way, favourable mobile phases can be used for the LC separation and for the MS detection. Using the PSS approach a flow-rate reduction from 1.0 ml/min in the LC system to 15 microliters/min going into the mass spectrometer was achieved without splitting. The determination limit for EM-ES was 0.1 microgram/ml.

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.