Comparison of different setups for one- and two-dimensional capillary high-performance liquid chromatography and pressurized capillary electrochromatography coupled on-line with mass spectrometry

A comparison of different separation methods (high-performance liquid chromatography (HPLC), capillary HPLC (CHPLC) and pressurized capillary electrochromatography (pCEC)) coupled on-line with mass spectrometry (MS) is undertaken using the separation of a crude extract of ergot fungus (secalis cornuti) as an example. New and simple setups for a two-dimensional CHPLC coupled on-line with electrospray ionization (ESI)-MS (2D-CHPLC-MS) as well as for capillary size-exclusion chromatography performed under pCEC conditions and coupled on-line with ESI-MS (CSEC-pCEC-MS) are shown. In addition, an improved method for column packing is presented.     

Evaluation of the enantioselectivity of carbon nanoparticles‐modified chiral separation systems using dextrin as chiral selector by capillary electrokinetic chromatography

Nanoparticles (NPs) can be used as pseudostationary phases (PSPs) in EKC, which is similar to the use of micelle additives as applied in MEKC. To date, the use of NPs to enhance enantiomeric separation by EKC with β‐CD or its derivative as chiral selector has been reported only in two papers. However, to the best of our knowledge, there has been no prior effort to use NPs for achieving enantioseparation with polysaccharides as chiral selector. This paper describes for the first time the use of carbon nanoparticles (CNPs) as PSPs to modify chiral separation system employing dextrin as chiral selector for the enantioseparations of several basic drugs in capillary EKC. Three different types of CNPs, including carbogenic nanoparticles (NPs), carboxylated single‐walled carbon nanotubes, and carboxylated multiwalled carbon nanotubes, were used as running buffer additives, respectively. The potential of the PSPs and the effects of dextrin concentration, buffer pH, and buffer concentration on the enantioseparations were evaluated. Four pairs of tested enantiomers were successfully resolved in less than 15 min with the resolution values in the range of 1.41–4.52 under optimized conditions. Compared to the buffer without NPs, the introduction of NPs into the buffer enhanced the separation of the enantiomers.     

LC-MS analysis in the aquatic environment and in water treatment – a critical review

LC-MS has become an invaluable technique for trace analysis of polar compounds in aqueous samples of the environment and in water treatment. LC-MS is of particular importance due to the impetus it has provided for research into the occurrence and fate of polar contaminants, and of their even more polar transformation products. Mass spectrometric detection and identification is most widely used in combination with sample preconcentration, chromatographic separation and atmospheric pressure ionization (API). The focus of the first part of this review is directed particularly toward instruments and method development with respect to their applications for detecting emerging contaminants, microorganisms and humic substances (HS). The current status and future perspectives of 1) mass analyzers, 2) ionization techniques to interface liquid chromatography (LC) with mass spectrometry (MS), 3) methods for preconcentration and separation with respect to their application for water analysis are discussed and examples of applications are given. Quadrupole and ion trap mass analyzers with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are already applied in routine analysis. Time-of-flight (TOF) mass spectrometers are of particular interest for accurate mass measurements for identification of unknowns. For non-polar compounds, different ionization approaches have been described, such as atmospheric pressure photoionization (APPI), electrochemistry with ESI, or electron capture ionization with APCI. In sample preconcentration and separation, solid phase extraction (SPE) with different non-selective sorbent materials and HPLC on reversed-phase materials (RP-HPLC) play the dominant role. In addition, various on-line and miniaturized approaches for sample extraction and sample introduction into the MS have been used. Ion chromatography (IC), size-exclusion chromatography (SEC), and capillary electrophoresis (CE) are alternative separation techniques. Furthermore, the issues of compound identification, matrix effects on quantitation, development of mass spectral libraries and the topic of connecting analysis and toxicity bioassays are addressed.     

High-throughput quantitative bioanalysis by LC/MS/MS

This review article discusses the most recent significant advances in the sample preparation and mass spectrometry aspects of high-throughput bioanalysis by LC/MS/MS for the quantitation of drugs, metabolites and endogenous biomolecules in biological matrices. The introduction and implementation of automated 96-well extraction has brought about high-throughput approaches to the biological sample preparation techniques of solid-phase extraction, liquid-liquid extraction and protein precipitation. The fast-flow on-line extraction technique is a different high-throughput approach that has also significantly speeded up analysis by LC/MS/MS. The use of pierceable caps for biological tubes further enhances the analysis speed and improves the safety in handling biological samples. The need for adequate chromatographic separation in order to eliminate interferences due to metabolites and/or matrix effects in LC/MS/MS is discussed. To highlight our limited understanding of atmospheric pressure ionization mass spectrometry, results from recent investigations that appear to be counter-intuitive are presented. Looking ahead to the future, multiplexed LC/MS/MS systems and capillary LC are presented as areas that can bring about further improvements in analysis speed and sensitivity to quantitative bioanalysis by LC/MS/MS.     

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix-assisted laser desorption/ionization and electrospray-mass spectrometry detection

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.     

Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures

In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targets—pre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches.     

Recent progress in the use of ionic polymers as pseudostationary phases for EKC

This review concerns the introduction, characterization, and application of polymeric pseudostationary phases (PSPs) for EKC since 2004. Achiral and chiral polymers and separations are reviewed, as is the application of polymeric PSPs for the combination of EKC with mass spectrometric detection.     

External accumulation of ions for enhanced electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Electrospray ionization (ESI) in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry provides for mass analysis of biological molecules with unrivaled mass accuracy, resolving power and sensitivity. However, ESI FTICR MS performance with on-line separation techniques such as liquid chromatography (LC) and capillary electrophoresis has to date been limited primarily by pulsed gas assisted accumulation and the incompatibility of the associated pump-down time with the frequent ion beam sampling requirement of on-line chromatographic separation. Here we describe numerous analytical advantages that accrue by trapping ions at high pressure in the first rf-only octupole of a dual octupole ion injection system before ion transfer to the ion trap in the center of the magnet for high performance mass analysis at low pressure. The new configuration improves the duty cycle for analysis of continuously generated ions, and is thus ideally suited for on-line chromatographic applications. LC/ESI FTICR MS is demonstrated on a mixture of 500 fmol of each of three peptides. Additional improvements include a fivefold increase in signal-to-noise ratio and resolving power compared to prior methods on our instrument.     

Separation techniques hyphenated to electrospray-tandem mass spectrometry in proteomics: capillary electrophoresis versus nanoliquid chromatography

Liquid chromatography (LC) nanoelectrospray-tandem mass spectrometry (MS/MS) is a key technology for the study of proteomics, with the main benefit to the characterization of sensitive peptides from complex mixtures. Capillary electrophoresis coupled to mass spectrometry (MS) has been taken into consideration sporadically due to the highly efficient separation and ability to handle low sample amount, yet classified as being less sensitive with respect to analyte concentration. The limitation in capillary zone electrophoresis (CZE) injection volumes can be overcome by on-line solid-phase extraction (SPE). Such an on-line SPE-CZE system was explored in combination with an ion trap (IT) mass spectrometer. Thus, it was possible to inject more than 100 microL sample solution on to the CZE capillary. Concentration limits of detection as low as 100 amol/microL were demonstrated for a peptide standard. This SPE-CZE-microelectrospray ionization (ESI)-MS/MS setup was compared directly to nanoLC/nanoESI using the same sample of a tryptic digest of bovine serum albumin (BSA) as a reference standard. Measurements were made on one IT mass spectrometer with identical acquisition parameters. Both chromatography systems enabled the separation and detection of low levels of peptides from a mixture of moderate complexity, with most peptides identified using both techniques; however, specific differences were obvious. The nanoLC-MS is about five times more sensitive than the CZE-MS, yet the difference was less pronounced than expected. The CZE-MS technique showed reduced loss of peptides, especially for larger peptides (missed cleavages) and is about four times faster than the nanoLC-MS approach.     

Fraction collection in capillary electrophoresis for various stand-alone mass spectrometers

A procedure for collecting fractions during capillary electrophoresis for their analysis using various stand-alone instruments is described. The results of a systematic study of the optimization and application of capillary electrophoresis (CE) in conjunction with a reverse-phase high-performance liquid chromatography electrospray ionization quadrupole time of flight-tandem mass spectrometry (RP-HPLC-ESI-Q-TOF-MS/MS) and inductively-coupled mass spectrometry (ICP-MS) to the analysis of the seed extract of the Japanese Pagoda Tree (Sophora japonica) are presented. The off-line coupling of CE to the matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the proteins mixture was applied. The cathode end of the capillary was placed inside a stainless steel needle using a coaxial liquid-sheath-flow configuration. The optimization of experimental parameters resulted in an efficient methodology for MS analysis of fractions. Several components contained in the extract of S. japonica were identified, some not previously known. It was demonstrated that low sensitivity, which is a real problem in off-line CE–MS analysis, could be tolerated because of a more flexible optimization of the CE separation conditions and the choice of independent stand-alone instruments for analysis of separated fractions. The estimated limit of detection for CE-RP-HPLC-ESI-Q-TOF-MS was 50 μM of polyphenols and for CE-ICP-MS, 1–100 μg/l.     

Interfaces for coupled liquid-phase separation/mass spectrometry techniques. An update on recent developments

An update is presented covering the latest developments in the interfacing of liquid-phase separation systems and mass spectrometers. The interfacing devices presented are those developed for continuous-flow matrix-assisted laser desorption/ionization, micro- and nano-liquid chromatography/masspectrometry (MS), capillary electrophoresis/MS and on-chip separation technologies/MS. From the information that can be found in the most recent literature on the topic, it is evident that the trend towards the miniaturization of separation and interface devices is gaining ground. This can be rationalized by the substantial gains in sensitivity for the detection and study of extremely low levels of analytes and especially of high molecular mass biopolymers.     

Automated software-guided identification of new buspirone metabolites using capillary LC coupled to ion trap and TOF mass spectrometry

The identification and structure elucidation of drug metabolites is one of the main objectives in in vitro ADME studies. Typical modern methodologies involve incubation of the drug with subcellular fractions to simulate metabolism followed by LC-MS/MS or LC-MS(n) analysis and chemometric approaches for the extraction of the metabolites. The objective of this work was the software-guided identification and structure elucidation of major and minor buspirone metabolites using capillary LC as a separation technique and ion trap MS(n) as well as electrospray ionization orthogonal acceleration time-of-flight (ESI oaTOF) mass spectrometry as detection techniques.Buspirone mainly underwent hydroxylation, dihydroxylation and N-oxidation in S9 fractions in the presence of phase I co-factors and the corresponding glucuronides were detected in the presence of phase II co-factors. The use of automated ion trap MS/MS data-dependent acquisition combined with a chemometric tool allowed the detection of five small chromatographic peaks of unexpected metabolites that co-eluted with the larger chromatographic peaks of expected metabolites. Using automatic assignment of ion trap MS/MS fragments as well as accurate mass measurements from an ESI oaTOF mass spectrometer, possible structures were postulated for these metabolites that were previously not reported in the literature.     

High performance fourier transform ion cyclotron resonance mass spectrometric detection for capillary electrophoresis

We describe the current state of the on-line combination of capillary electrophoresis (CE) electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS), and discuss aspects of the FTICR technique relevant to its use as a detection scheme for on-line separations. Aspects including sensitivity, mass resolution, duty cycle, and tandem mass spectrometric capabilities are discussed in the context of online separations with examples from the authors' laboratory.     

Performance of capillary gel electrophoretic analysis of oligonucleotides coupled on-line with electrospray mass spectrometry

Synthetic oligonucleotides (ODNs) are routinely analyzed using capillary gel electrophoresis (CGE) for size-sieving based separations as well as electrospray mass spectrometry (ESI-MS) for identification. On-line coupling of these methods is therefore desired in order to combine the analytical capabilities provided by both methods. Performance of on-line CGE-ESI-MS systems is influenced by various parameters, and choice of optimal conditions is crucial for successful coupling experiments. In this study, we explore characteristics of the on-line coupled CGE-ESI-MS system for ODN analysis. Effects of CGE buffer concentration, capillary length, separation and orifice voltage on CGE separation and MS detection of a phosphodiester ODN mixture were examined. Attention was paid to the influence of the interface, such as geometry of capillary alignment, sheath liquid flow-rate and sheath liquid composition on performance of the system.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

On-line capillary electrophoresis-electrospray mass spectrometry for the stereoselective analysis of drugs and metabolites

The on-line combination of partial-filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the enantioseparation of pharmaceutical drugs and metabolites, namely amphetamines, methadone, venlafaxine and selected tropane alkaloids. The partial-filling technique proved to be a suitable and efficient approach to avoid mass spectrometry (MS) source contamination, as well as signal suppression due to nonvolatile additives. To achieve chiral separation, various chiral selectors were applied, including neutral and particularly negatively charged cyclodextrins. Because of the countercurrent contribution, charged cyclodextrins were found more suitable for the on-line MS detection of separated enantiomers. Hyphenation of capillary electrophoresis (CE) with mass spectrometry was found appropriate for the stereoselective analysis of methadone in real serum samples. Moreover, the use of MS in the selected ion monitoring mode resulted in a very high selectivity, as well as improved sensitivity compared to UV detection. Finally, with atropine as a model compound, the quantitative performances of the method were evaluated and showed high sensitivity, as well as good repeatability in terms of migration time and peak area ratio.     

State-of-the-art of the hyphenation of capillary electrochromatography with mass spectrometry

The high separation efficiency and loading capacity of capillary electrochromatography (CEC) make it an attractive separation mode for coupling with mass spectrometry (MS), which has the ability to unambiguously identify analytes with high selectivity and sensitivity. We present an overview of recent advances on both instrumentation and separation columns employed in CEC-MS systems. In particular, the main characteristics of the stationary phases, as well as the configurations of the column outlet that are related with the coupling arrangements of the MS ionization sources, are reported. At present, packed columns and conventional electrospray ionization (ESI) sources are mainly employed in CEC-MS. Nevertheless, the use of monolithic capillary columns and nanoelectrospray sources has the potential for wide acceptance in the next future. Moreover, the main features of several mass analyzers including ion trap, quadrupole, time-of-flight, magnetic sector, and Fourier transform-ion cyclotron resonance are examined. Finally, current applications of this technology, mainly in the pharmaceutical field and proteomics, are reviewed.     

Analysis of paralytic shellfish poisons by capillary electrophoresis

A capillary electrophoresis (CE) method with UV detection is described for the separation and determination of underivatized toxins associated with paralytic shellfish poisoning (PSP). Confirmation of the electrophoretic peaks was facilitated by mass spectrometric (MS) detection using an ionspray CE-MS interface and by high-performance liquid chromatography with fluorescence detection. The determination of PSP toxins, such as saxitoxin and neosaxitoxin, in toxic dinoflagellates and scallops is demonstrated and comparisons are made with existing techniques.