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.     

Fast quantification of amino acids by microchip electrophoresis–mass spectrometry

A fast microchip electrophoresis–nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 μM. Limits of detection were found to be 0.37 μM for Asp and 0.33 μM for Glu (S/N?=?3). Assay repeatability (relative standard deviation, n?=?6) was 4.2 and 4.5 %, for Asp and Glu at 5.0 μM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.     

Alignment of capillary electrophoresis–mass spectrometry datasets using accurate mass information

Capillary electrophoresis–mass spectrometry (CE–MS) is a powerful technique for the analysis of small soluble compounds in biological fluids. A major drawback of CE is the poor migration time reproducibility, which makes it difficult to combine data from different experiments and correctly assign compounds. A number of alignment algorithms have been developed but not all of them can cope with large and irregular time shifts between CE–MS runs. Here we present a genetic algorithm designed for alignment of CE–MS data using accurate mass information. The utility of the algorithm was demonstrated on real data, and the results were compared with one of the existing packages. The new algorithm showed a significant reduction of elution time variation in the aligned datasets. The importance of mass accuracy for the performance of the algorithm was also demonstrated by comparing alignments of datasets from a standard time-of-flight (TOF) instrument with those from the new ultrahigh resolution TOF maXis (Bruker Daltonics).     

Online dynamic pH junction–sweeping for the determination of benzoic and sorbic acids in food products by capillary electrophoresis

A simple, low-cost, and efficient online focusing method that combines a dynamic pH junction and sweeping by capillary electrophoresis with polymer solutions was developed and optimized for the simultaneous determination of benzoic acid (BA) and sorbic acid (SA). A sample solution consisting of 2.5 mM phosphate at pH 3.0 and a buffer solution containing 15 mM tetraborate (pH 9.2), 40 mM sodium dodecyl sulfate, and 0.100 % (w/v) poly(ethylene oxide) were utilized to realize dynamic pH junction–sweeping for BA and SA. Under the optimized conditions, the entire analysis process was completed in 7 min, and a 900-fold sensitivity enhancement was achieved with limits of detection (S/N?=?3) as low as 8.2 and 6.1 nM for BA and SA, respectively. The linear ranges were between 20 nM and 20 μM for BA and 20 nM and 10 μM for SA, with correlation coefficients greater than 0.992. The recoveries of the proposed method ranged from 90 to 113 %. These satisfactory results indicate that this method has the potential to be an effective analytical tool for the rapid screening of BA and SA in different food products.

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Figure An online focusing strategy combining dynamic pH junction and sweeping for sensitive determination of benzoic and sorbic acid in food products using capillary electrophoresis wit polymer solutions

     

Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides

Fast capillary electrophoresis–mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1–6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm⁻¹. Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.     

On-line sample stacking of peptides in capillary electrophoresis for the study of prebiotic reactions between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides

The reaction between α,α-dialkylated amino acids and amino acid N-carboxyanhydrides is slow leading to low concentrations of products (peptides). The detection by capillary electrophoresis of the analytes contained in such samples is therefore a challenging issue. In this work, on-line sample pre-concentration methods based on field-amplified sample stacking have been implemented and compared. Because of the high ionic strength present in the sample matrix, samples were diluted with an organic solvent prior to analysis to decrease the sample conductivity. Different modes of sample injection (field amplified sample injection (FASI), hydrodynamic normal sample stacking (NSS) or large volume sample stacking (LVSS)) were compared. Pre-concentration factors of 20 for FASI, about 30–40 for NSS and 60 for LVSS were obtained for the analysis of (l,l) dipeptide of valine in a large excess of isovaline and 0.2 M of ionic strength. For LVSS application and resolution optimisation, a new non-covalent coating based on the partial modification of the capillary surface was used to tune the electroosmotic flow magnitude and to pump the sample matrix out of the capillary. This on-line sample pre-concentration step allowed confirming that oligopeptides including α,α-dialkylated amino acids are formed during the reaction between α,α-dialkylated amino acids and N-carboxyanhydride amino acids.     

Metabolomic analysis of amino acids and organic acids in aging mouse eyes using gas chromatography–tandem mass spectrometry

This is a metabolomics study for monitoring altered amino acid (AA) and organic acid (OA) metabolism of in eyes from aging an mouse model at 8 and 18 weeks and 18 months. Simultaneous metabolic profiling analysis of OAs and AAs was performed as ethoxycarbonyl/methoxime/tert-butyldimethylsilyl derivatives by gas chromatography–tandem mass spectrometry. A total of 42 metabolites—24 AAs and 18 OAs—were determined and their composition values were normalized to the corresponding mean values of 8-week-old mice as the control group. Then their normalized values were plotted as star graphs, which were distorted and readily distinguishable for each age-related group. Among the 42 metabolites, 18 AAs and 11 OAs were age dependent and significantly different (p < 0.05). Principal component analysis and partial least squares discriminant analysis showed unclear separation between 8- and 18-week-old mice but clear separation between these and 18-month-old mice. In particular, the variable importance in projection scores of 4-hydroxyproline, cis-aconitic acid, glycine, isocitric acid, leucine, pipecolic acid and lysine from partial least-squares–discriminant analysis were higher than 1.3. A heatmap for the classification and visualization of 42 metabolites showed differences in metabolite changes with aging. Altered AA and OA profiles were monitored, which may explain the metabolic disturbance of AA and OA. These findings are related to mitochondrial dysfunctions related to energy metabolism and the impaired antioxidant system in the aging eye. Therefore, the present metabolomics results of the association between physiological states and altered metabolism of AA and OA will be useful for understanding the aging eye and related diseases.     

Characterization and quantification of metallothionein isoforms by capillary electrophoresis–inductively coupled plasma–isotope-dilution mass spectrometry

In a new approach to the characterization and quantification of metallothionein isoforms an on-line isotope-dilution method in combination with the coupling of capillary electrophoresis (CE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) is reported. Metallothionein (MT) isoforms are separated by CE and the elements Cu, Zn, Cd, and S are detected simultaneously by use of ICP-SFMS in the medium resolution mode. On-line isotope dilution is performed by continuous introduction of an isotopically enriched, species-unspecific spike solution after the separation step. MT from rabbit liver and a further purified MT-1 isoform were quantified by determination of sulfur, and the stoichiometric compositions of the metalloprotein complexes are characterized by determination of their sulfur-to-metal ratios.     

Trace analysis of free and combined amino acids in atmospheric aerosols by gas chromatography–mass spectrometry

The analysis of amino acids by gas chromatography mass spectrometry (GC–MS) after their derivatization with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide was investigated as an alternative approach for the determination of free (FAA) and combined amino acids (CAA) in aerosols. This technique showed excellent linearity with r² values ranging from 0.9029 to 0.9995 and instrumental limits of detection ranging from 0.3 to 46 pg for the different amino acids. The quality of water used for sample extraction was found to be of utmost importance for achieving low blank levels of FAA and CAA. The addition of isopropanol during the extraction of aerosols was also shown to minimize the coextraction of inorganic salts that interfered with the analysis of FAA, Moreover, the ascorbic acid was found to be the most effective reagent for preventing the oxidative destruction of CAA during the hydrolysis process. By the analysis of spiked aerosol samples, the average recoveries determined for FAA and CAA were higher than 60% and the associated relative standard deviation was lower than 10% for the majority of amino acids. The application of the adopted method in background aerosols of the eastern Mediterranean enabled the unambiguous identification and quantification of 20 amino acids. The total concentration of FAA and CAA in aerosols ranged from 13 to 34 ng m⁻³ and from 29 to 79 ng m⁻³, respectively. The GC–MS based method is proposed to overcome several analytical difficulties usually encountered with the conventional HPLC-fluoresence technique.     

Preparation and evaluation of an immunoaffinity sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

In this study, we explored a procedure for the preparation of an immunoaffinity (IA) sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). We followed a site-specific antibody immobilization approach based on the covalent attachment of the oxidized antibodies through their carbohydrate moieties to hydrazide silica particles, using a polyclonal antibody against Endomorphin 1 and 2 (End1 and End2). The main features of the IA sorbent were studied, such as the amount of hydrazide groups and antibodies attached onto oxidized diol silica particles. Once the procedure was optimized, standard solutions of End1 and End2 were used in order to establish the IA-SPE-CE–MS methodology. Acceptable repeatability, reproducibility and linearity range values were obtained for the proposed methodology. The limits of detection (LODs) of 1 ng mL⁻¹ were approximately 100-fold better than those obtained by CE–MS. Selectivity of the IA sorbent was good but some cross-reactivity against Dynorphin A (1–7) was observed when a mixture of several opioid peptides was analyzed. Human plasma samples spiked with End1 and End2 were also analyzed and both peptides could be detected down to 100 ng mL⁻¹.     

Isoform differentiation of intact AGP from human serum by capillary electrophoresis–mass spectrometry

Human AGP is an acidic glycoprotein mainly produced by liver that presents a high degree of heterogeneity. It can present different amino acid sequences and has five N-glycosylation sites leading to a wide range of different protein isoforms. AGP structure and composition has been widely studied due to its drug-binding behavior and relation with disease. However, so far, the characterization has been performed only on protein fragments, i.e., the peptide or glycan level. Here, the analysis of intact human AGP purified from human serum is performed by capillary electrophoresis–time-of-flight mass spectrometry. In this way, it is possible to characterize more than 150 human AGP isoforms, differing both in the amino acid sequence and in the glycosylation. The detected masses could be attributed unequivocally to an overall composition based on the combination of the analysis of the released glycans and the characterization of the deglycosylated protein. Different AGP samples purified from human serum were characterized and compared. High inter-individual variability among AGP isoforms expression was observed. The presented method enables for the first time clinical studies based on detailed isoform distribution of intact glycoproteins.     

Anionic metabolic profiling of urine from antibiotic-treated rats by capillary electrophoresis–mass spectrometry

A recently developed capillary electrophoresis (CE)-negative-ionisation mass spectrometry (MS) method was used to profile anionic metabolites in a microbial-host co-metabolism study. Urine samples from rats receiving antibiotics (penicillin G and streptomycin sulfate) for 0, 4, or 8 days were analysed. A quality control sample was measured repeatedly to monitor the performance of the applied CE-MS method. After peak alignment, relative standard deviations (RSDs) for migration time of five representative compounds were below 0.4 %, whereas RSDs for peak area were 7.9–13.5 %. Using univariate and principal component analysis of obtained urinary metabolic profiles, groups of rats receiving different antibiotic treatment could be distinguished based on 17 discriminatory compounds, of which 15 were downregulated and 2 were upregulated upon treatment. Eleven compounds remained down- or upregulated after discontinuation of the antibiotics administration, whereas a recovery effect was observed for others. Based on accurate mass, nine compounds were putatively identified; these included the microbial-mammalian co-metabolites hippuric acid and indoxyl sulfate. Some discriminatory compounds were also observed by other analytical techniques, but CE-MS uniquely revealed ten metabolites modulated by antibiotic exposure, including aconitic acid and an oxocholic acid. This clearly demonstrates the added value of CE-MS for nontargeted profiling of small anionic metabolites in biological samples.     

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by high-performance liquid chromatography–mass spectrometry and capillary zone electrophoresis–mass spectrometry

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by various oxo-compounds (glucose, ribose, glyoxal and glutardialdehyde) have been investigated using high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Both of these methods used mass spectrometric (MS) detection. Three enzymes (trypsin, pepsin, proteinase K) were used to digest glycated BSA. The extent of modification depended on the selected oxo-compound. Reactivity increased progressively from glucose to glutardialdehyde (glucose < ribose < glyoxal < glutardialdehyde). Carboxymethylation of lysine (CML) was the main type of modification detected. The HPLC/MS method achieved higher coverage and a larger amount of CML was identified compared to CZE/MS.     

Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples

Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.     

Microchip capillary electrophoresis–electrospray ionization–mass spectrometry of intact proteins using uncoated Ormocomp microchips

We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis–electrospray ionization (CE–ESI) microchips. The microchips are fabricated fully of commercial inorganic–organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp–Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8–9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10⁴ theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6–5.9% RSD, n = 4) and intact proteins (1.3–7.5% RSD, n = 3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.     

Identification of phenolic compounds from pollen extracts using capillary electrophoresis–electrospray time-of-flight mass spectrometry

In this work, a new, easy and rapid method of analyzing phenolic compounds in pollen extract, based on capillary electrophoresis coupled with electrospray ionization time-of-flight-mass spectrometry (CE–ESI–TOF–MS), has been developed. A systematic investigation of separation parameters has been performed with respect to resolution, sensitivity, analysis time and peak shape. The electrophoretic parameters and electrospray conditions must be optimized to obtain reproducible analyses. Using this method, several important phenolic compounds such as acetin-glucoside, 7-O-methylherbacetin-3-sophoroside, galloyl-glucose, quercetin-3-sophoroside, apigenin-6,8-di-C-glycoside, quercetin-3-rutinoside, genistein-7-O-β-D-glucoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside and 2′,4′,6′-trihydroxy-3′-formyldihydrochalcone have been determined directly from pollen extract. The efficiency, the rapidity, the small amounts of sample required, and the high resolution of CE coupled with the sensitivity, the selectivity, the accurate masses and the true isotopic patterns obtained using TOF-MS point to the potential of this approach for identifying the phenolic compounds present in pollen.     

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometry for determination of drugs of abuse in human urine

In-line solid-phase extraction–capillary electrophoresis coupled with mass spectrometric detection (SPE–CE–MS) has been used for determination of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), codeine (COD), hydrocodeine (HCOD), and 6-acetylmorphine (6AM) in urine. The preconcentration system consists of a small capillary filled with Oasis HLB sorbent and inserted into the inlet section of the electrophoresis capillary. The SPE–CE–MS experimental conditions were optimized as follows: the sample (adjusted to pH 6.0) was loaded at 930 mbar for 60 min, elution was performed with methanol at 50 mbar for 35 s, 60 mmol L⁻¹ ammonium acetate at pH 3.8 was used as running buffer, the separation voltage was 30 kV, and the sheath liquid at a flow rate of 5.0 μL min⁻¹ was isopropanol–water 50:50 (v/v) containing 0.5% acetic acid. Analysis of urine samples spiked with the four drugs and diluted 1:1 (v/v) was studied in the linear range 0.08–10 ng mL⁻¹. Detection limits (LODs) (S/N = 3) were between 0.013 and 0.210 ng mL⁻¹. Repeatability (expressed as relative standard deviation) was below 7.2%. The method developed enables simple and effective determination of these drugs of abuse in urine samples at the levels encountered in toxicology and doping.     

Analysis of recombinant human erythropoietin and novel erythropoiesis stimulating protein digests by immunoaffinity capillary electrophoresis–mass spectrometry

In this work, we demonstrate that detection of a specific peptide marker by immunoaffinity capillary electrophoresis–mass spectrometry (IA-CE–MS) could be used to confirm the presence of recombinant human erythropoietin (rhEPO) in solution. Besides the carbohydrate content, the amino acid sequence of novel erythropoiesis stimulating protein (NESP) differs from human erythropoietin (hEPO) at five positions (Ala30Asn, His32Thr, Pro87Val, Trp88Asn, and Pro90Thr). After digesting both glycoproteins in solution by trypsin and PNGase F, two specific proteotypic peptides, EPO (77–97) and NESP (77–97) which differ in three amino acids, were selected as rhEPO and NESP markers, respectively. Both digests and their mixtures were analyzed by IA-CE–MS. The IA stationary phase was prepared from a custom made polyclonal anti-EPO (81–95) antibody immobilized on a solid support of CNBr-Sepharose 4B and was packed in a microcartridge near the inlet of the separation capillary. As the antibody was directed to a synthetic peptide EPO (81–95), only the proteotypic peptide EPO (77–97) was retained. The retained peptide was eluted, separated by electrophoresis and detected by MS. The method was specific to confirm the presence of rhEPO in solution. Although the limits of detection for the peptide marker were similar to those obtained with CE–MS (a few mg/L), these results show the potential of this novel approach to detect in the future rhEPO and its analogues selectively and unambiguously at the levels expected in biological fluids.     

Ultra-sensitive quantification of lysozyme based on element chelate labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry

In this study, an ultra-sensitive method for the quantification of lysozyme based on the Gd³⁺ diethylenetriamine-N,N,N′,N″,N″-pentaacetic acid labeling and capillary electrophoresis–inductively coupled plasma mass spectrometry (CE–ICP–MS) was described. The Gd³⁺-tagged lysozyme was effectively separated by capillary electrophoresis (CE) and sensitively determined by inductively coupled plasma mass spectrometry (ICP–MS). Based on the gadolinium-tagging and CE–ICP–MS, the lysozyme was determined within 12 min with an extremely low detection limit of 3.89 attomole (3.89 × 10⁻¹¹ mol L⁻¹ for 100 nL of sample injection) and a RSD < 6% (n = 5). The proposed method has been successfully used to detect lysozyme in saliva samples with a recovery of 91–106%, suggesting that our method is sensitive and reliable. The success of the present method provides a new potential for the biological assays and sensitive detection of low-abundant proteins.     

Simulation of oxidative stress of guanosine and 8-oxo-7,8-dihydroguanosine by electrochemically assisted injection–capillary electrophoresis–mass spectrometry

Oxidative stress plays a crucial role in DNA and RNA damage within biological cells. As a consequence, mutations of DNA can occur, leading to disorders like cancer and neurodegenerative and cardiovascular diseases. The oxidative attack of guanosine and 8-oxo-7,8-dihydroguanosine is simulated by electrochemistry coupled to capillary electrophoresis–mass spectrometry. The electrochemical conversion of the compound of interest is implemented in the injection protocol termed electrochemically assisted injection (EAI). In this way, oxidation products of guanosine can be generated electrochemically, separated by capillary electrophoresis, and detected by electrospray ionization time-of-flight mass spectrometry (EAI–CE–MS). A fully automated laboratory-made EAI cell with an integrated buffer reservoir and a compartment holding screen-printed electrodes is used for the injection. In this study, parameters like pH of the sample solution and the redox potential applied during the injection were investigated in terms of corresponding formation of well-known markers of DNA damage. The important product species, 8-oxo-7,8-dihydroguanosine, was investigated in a separate study to distinguish between primary and secondary oxidation products. A comparison of product species formed under alkaline, neutral, and acidic conditions is presented. To compare real biological systems with an analytical approach for simulation of oxidative stress, it is desirable to have a well-defined control over the redox potential and to use solutions, which are close to physiological conditions. In contrast to typical HPLC–MS protocols, the hyphenation of EAI, CE, and MS enables the generation and separation of species involved without the use of organic solvents. Thus, information of the electrochemical behavior of the nucleoside guanosine as well as the primary oxidation product 8-oxo-7,8-dihydroguanosine can be characterized under conditions close to the physiological situation. In addition, the migration behavior found in CE separations of product species can be used to identify compounds if several possible species have the same mass-to-charge values determined by MS detection.

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