Rawi Ramautar

CE-MS in metabolomics

An overview of the use of CE‐MS in the field of metabolomics is provided. Metabolomics is concerned with the comprehensive analysis of endogenous low‐molecular‐weight compounds in biological samples. CE‐MS has demonstrated to be a powerful technique for the profiling of polar metabolites in biological samples. This review covers the use of various CE separation modes, capillary coatings, MS analyzers, sample preparation techniques, and data analysis methods used in CE‐MS for metabolomics. The applicability of CE‐MS in metabolomics research is illustrated by giving examples of the analysis of bacterial extracts, plant extracts, urine, plasma, and cerebrospinal fluid samples. The relevant CE‐MS metabolomics studies published between 2000 and 2008 are presented in tabular form, including information on sample type and pretreatment and MS detection mode. Future developments with regard to the use of alternative ionization techniques, the use of coupled separation systems and the potential of microchip CE systems for metabolomics are discussed.

Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry

Over the last two decades, coupled capillary electrophoresis (CE)–mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.

CE-MS for proteomics: Advances in interface development and application.

Capillary electrophoresis-mass spectrometry (CE-MS) has emerged as a powerful technique for the analysis of proteins and peptides. Over the past few years, significant progress has been made in the development of novel and more effective interfaces for hyphenating CE to MS. This review provides an overview of these new interfacing techniques for coupling CE to MS, covering the scientific literature from January 2007 to December 2011. The potential of these new CE-MS interfacing techniques is demonstrated within the field of (clinical) proteomics, more specifically bottom-up proteomics, by showing examples of the analysis of various biological samples. The relevant papers on CE-MS for proteomics are comprehensively summarized in tables, including, e.g. information on sample type and pretreatment, interfacing and MS detection mode. Finally, general conclusions and future perspectives are provided.

Recent developments in coupled SPE‐CE

This article presents an overview of coupled SPE‐CE systems that have been reported in the literature between April 2007 and June 2009. The use of in‐line and on‐line SPE‐CE is covered in this review. Special attention is paid to the use of monoliths and molecularly imprinted polymers in coupled SPE‐CE systems. Application‐oriented research is discussed in which in‐line and on‐line SPE‐CE systems have been used in biomedical, pharmaceutical, environmental and food analysis. The SPE‐CE studies are presented in tables, including information on sample type, SPE sorbent, detection and LOD. Finally, some future developments that may increase the applicability of coupled SPE‐CE are highlighted.

Developments in coupled solid-phase extraction-capillary electrophoresis 2013-2015.

This article presents an overview of the design and application of coupled SPE–CE systems that have been reported in the literature between January 2011 and June 2013. The present paper is an update of three previous review papers covering the years 2000–2011 (Electrophoresis 2008, 29, 108–128; Electrophoresis 2010, 31, 44–54; Electrophoresis 2012, 33, 243–250). The use of in‐line and on‐line SPE–CE approaches is described in this review. Emerging technological developments, such as the use of in‐line frit‐free SPE and chip‐based SPE for extraction of sample components prior to CE analysis, are outlined. Selected examples illustrate the applicability of SPE–CE in biomedical, pharmaceutical, and environmental analysis. A complete overview of recent SPE–CE studies is given in table format, providing information on sample type, SPE sorbent, coupling mode, detection mode, and LOD. Finally, some general conclusions and future perspectives are provided.

Capillary electrophoresis-time of flight-mass spectrometry using noncovalently bilayer-coated capillaries for the analysis of amino acids in human urine

A capillary electrophoresis‐time of flight‐mass spectrometry (CE‐TOF‐MS) method for the analysis of amino acids in human urine was developed. Capillaries noncovalently coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) provided a considerable EOF at low pH, thus facilitating the fast separation of amino acids using a BGE of 1u2005M formic acid (pHu20051.8). The PB–PVS coating proved to be very consistent yielding stable CE‐MS patterns of amino acids in urine with favorable migration time repeatability (RSDs <2%). The relatively low sample loading capacity of CE was circumvented by an in‐capillary preconcentration step based on pH‐mediated stacking allowing 100‐nL sample injection (i.e. ca. 4% of capillary volume). As a result, LODs for amino acids were down to 20u2005nM while achieving satisfactory separation efficiencies. Preliminary validation of the method with urine samples showed good linear responses for the amino acids (R2 >0.99), and RSDs for peak areas were <10%. Special attention was paid to the influence of matrix effects on the quantification of amino acids. The magnitude of ion suppression by the matrix was similar for different urine samples. The CE‐TOF‐MS method was used for the analysis of urine samples of patients with urinary tract infection (UTI). Concentrations of a subset of amino acids were determined and compared with concentrations in urine of healthy controls. Furthermore, partial least squares–discriminant analysis (PLS–DA) of the CE‐TOF‐MS dataset in the 50–450u2005m/z region showed a distinctive grouping of the UTI samples and the control samples. Examination of score and loadings plot revealed a number of compounds, including phenylalanine, to be responsible for grouping of the samples. Thus, the CE‐TOF‐MS method shows good potential for the screening of body fluids based on the analysis of endogenous low‐molecular weight metabolites such as amino acids and related compounds.

Evaluation of CE methods for global metabolic profiling of urine.

In this study, the usefulness of noncovalently coated capillaries with layers of charged polymers is investigated to obtain global electrophoretic profiles of urinary metabolites covering a broad range of different compound classes in a highly repeatable way. Capillaries were coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS), or with a triple layer of PB, dextran sulfate (DS) and PB. The bilayer and triple layer coatings were evaluated at acidic (pH 2.0) and alkaline (pH 9.0) separation conditions, thereby providing separation conditions for basic and acidic compounds. A representative metabolite mixture and spiked urine samples were used for the evaluation of the four CE methods. Migration time repeatability (RSD<2%) and plate numbers (N, 100u2009000–400u2009000) were similar for the test compounds in all CE methods, except for some multivalent ions that may exhibit adsorption to oppositely charged coatings. The analysis of cationic compounds with the PB‐DS‐PB CE method at low pH (i.e. after the EOF time) provided a larger separation window and number of separated peaks in urine compared to the analysis with the PB‐PVS CE method at low pH (i.e. before the EOF time). Approximately, 600 molecular features were detected in rat urine by the PB‐DS‐PB CE‐MS method whereas about 300 features were found with the PB‐PVS CE‐MS method. This difference can be attributed to reduced comigration of compounds with the PB‐DS‐PB CE‐MS method and a related decrease of ion suppression. With regard to the analysis of anionic compounds by CE‐MS, in general analyte responses were significantly lower than that for cationic compounds, most probably due to less efficient ionization and to ion suppression effects caused by the background electrolyte. Hence, further optimization is required for the sensitive CE‐MS analysis of anionic compounds in body fluids. It is concluded that the selection of a CE method for profiling of cationic metabolites in urine depends on the purpose of the study. For high‐throughput analyses, the PB‐PVS CE‐MS method is favored whereas the PB‐DS‐PB CE‐MS method provides a more information‐rich metabolic profile, but at the cost of prolonged analysis time.

Metabolic profiling of mouse cerebrospinal fluid by sheathless CE-MS.

The need for sensitive analytical technologies applicable to metabolic profiling of volume-restricted biological samples is high. Here, we demonstrate feasibility of capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (MS) with sheathless nano-electrospray interface for non-targeted profiling of ionogenic metabolites in body fluids of experimental animals. A representative mixture of the metabolites and body fluids of mice such as cerebrospinal fluid (CSF), urine and plasma were used as examples of low-volume biological samples for method evaluation. An injection volume of only 9xa0nL resulted in limits of detection between 0.7 and 12xa0nM for the metabolite mixture. The method allowed the detection of ∼350 molecular features in mouse CSF (an injection volume of ca. 45xa0nL), while ∼400 features were observed in mouse plasma and ∼3,500 features in mouse urine (an injection volume of ca. 9xa0nL). The low-volume body fluid samples were analyzed directly after only 1:1 dilution with water, thereby fully retaining sample integrity, which is of crucial importance for non-targeted metabolic profiling. As little is known about the metabolic composition of mouse CSF, we identified a fraction of the molecular features in mouse CSF using accurate mass information, migration times, MS/MS data, and comparison with authentic standards. We conclude that sheathless CE-MS can be used for sensitive metabolic profiling of volume-restricted biological samples.

Capillary electrophoresis-mass spectrometry using an in-line sol-gel concentrator for the determination of methionine enkephalin in cerebrospinal fluid

In this study, a CE-MS method using a monolithic sol-gel concentrator for in-line solid-phase extraction (SPE) is evaluated for the analysis of methionine enkephalin in biological samples. Operational SPE parameters such as sample pH, loading volume, elution volume and composition have been studied. After optimization of the in-line preconcentration methodology, a 40-fold preconcentration was demonstrated for a methionine enkephalin test solution using a loading volume of 3200 nL. The method was linear in the range from 62.5 to 1000 ng/mL (R(2)>0.99). R.S.D. values for migration times and peak areas were 1.2% and 8.4%, respectively. Finally, the analysis of cerebrospinal fluid samples spiked with methionine enkephalin and deproteinized with perchloric acid (1:1, v/v) showed a detection limit (S/N=3) of approximately 1 ng/mL (ca. 5 nM). The recoveries of methionine enkephalin for three concentration levels (100, 10 and 1 ng/mL) were in the range of 74-91%, demonstrating the promising potential of the methodology for the analysis of biological samples.

Metabolic analysis of body fluids by capillary electrophoresis using noncovalently coated capillaries

The potential of capillaries noncovalently coated with charged polymers for the metabolic analysis of body fluids by CE is illustrated. Firstly, the usefulness of a coating consisting of a triple layer of polybrene-dextran sulfate-polybrene for the fast analysis of organic acids is described. The CE system allowed direct injections of CSF, plasma and urine samples, yielding good separation efficiencies. RSDs for migration times and peak areas of organic acids in plasma were <3% and <5%, respectively. The usefulness of the system is illustrated by the profiling of organic acids in plasma and urine samples. Secondly, a CE system comprising a bilayer coating of polybrene-poly(vinylsulfonate), which provides a considerable EOF at low pH is described. This system was combined with TOF-MS and used for the fast analysis of amino acids in cerebrospinal fluid (CSF) and urine with minimal sample pretreatment. RSDs for migration times and peak areas of amino acids in CSF and urine were <2% and <10%, respectively. The applicability of the system is demonstrated by the profiling of endogenous low-molecular weight metabolites in CSF from a healthy individual and a patient with complex regional pain syndrome.