1,720,985 research outputs found
Capillary electrophoresis and hydrophilic interaction chromatography coupled to mass spectrometry for anionic metabolic profiling
No full textMetabolomics encompasses the identification and quantification of low-molecular weight endogenous metabolites in biological samples, such as cells, tissues and body fluids. The identities, concentrations and fluxes of metabolites result from gene expression, protein expression and environmental factors (e.g. bacteria, diet, drugs), which together determine the phenotype of an organism. Therefore, profiling of metabolites present in biological samples provides information on biochemical processes and phenotypes of organisms. Various analytical techniques have been developed to analyze different classes of metabolites, including capillary electrophoresis (CE) and hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS), which are eminently suited for profiling highly polar compounds. This thesis describes the optimization, evaluation and comparison of CE–MS and HILIC–MS using negative electrospray ionization for the profiling of anionic metabolites. Particular attention was paid to sensitivity, reproducibility, separation selectivity and metabolite coverage. It is demonstrated that the use of triethylamine in the background electrolyte and sheath liquid in CE–MS is an effective way to improve the limits of detection of anionic metabolites significantly as compared to earlier developed CE–MS methods applying ammonium acetate. The increase in signal intensities allows the detection of low-abundant metabolites, thereby increasing the metabolite coverage. This is of utmost importance in metabolic profiling studies. The optimization of the mobile phase composition, the gradient of the mobile phase and sample solvent in HILIC–MS resulted in a large separation window with good peak shapes and low limits of detection of anionic metabolites. Detection limits obtained with HILIC–MS were up to 80 times lower compared to CE–MS, mainly due to the larger injection volume. Moreover, the migration and retention times of anionic metabolites were considerably different, demonstrating the different separation selectivities of CE–MS and HILIC–MS. The applicability of the optimal CE–MS and HILIC–MS methods has been tested by analyzing urine samples in studies on antibiotic-treated rats and patients with inborn errors of metabolism. Reproducible metabolite profiles were obtained as demonstrated by the repetitive analysis of a quality control sample. In general, more molecular features were obtained with HILIC–MS. However, CE–MS also revealed a large number of metabolite features, which were not detected with HILIC–MS. Using multivariate data analysis, different groups of urine samples could be discerned based on obtained CE–MS and HILIC–MS data, and potential biomarkers were revealed. These discriminatory compounds were putatively identified based on accurate mass and MS/MS experiments. Some of the biomarker candidates were observed with both CE–MS and HILIC–MS. Several anionic metabolites were also uniquely revealed by one of the applied techniques. This demonstrates that these analytical techniques can provide important and complementary information in metabolic profiling studies
Capillary electrophoresis – mass spectrometry using noncovalently coated capillaries for the analysis of biopharmaceuticals
No full textWith efficient methodologies available in biotechnology today, increasing numbers of recombinantly manufactured pharmaceutical peptides and proteins are being commercialized. The assessment of biopharmaceutical quality in terms of identity, content and purity is an important issue during manufacturing. The biotechnological production process may show variability, which can introduce product diversity, isoforms and closely-related degradation products. Clearly, there is an increasing demand for suitable analytical methods that allow not only protein and peptide identification, but also the separation and quantification of impurities and possible degradation products Capillary electrophoresis-mass spectrometry (CE-MS) provides the high separation efficiency and mass-selective detection that can be very useful for biopharmaceutical product characterization. Many protein modifications, like glycosylation and deamidation, may involve changes of net charge of a protein and, thus, also of its electrophoretic mobility. MS detection of high mass accuracy and resolution, such as provided by time-of-flight (TOF) instruments can yield accurate information on the molecular weight of analyzed proteins species. In this thesis, the potential of CE-TOF-MS for the analysis of biopharmaceuticals has been explored. Emphasis was on development of CE methods for basic proteins, paying particular attention to the prevention of protein adsorption, which deteriorates the separation efficiency. A positively charged Polybrene-dextran sulfate-Polybrene (PB-DS-PB) coating was applied for this purpose. These coatings were prepared by successive flushes with solutions of the charged polymers, leading to formation of electrostatically adsorbed layers. The performance and stability of the triple-layer coating was evaluated for basic proteins analyzed at medium and low pH. The suitability of the triple-layer coating for CE-TOF-MS was studied, paying attention to compatibility. CE-MS coupling was achieved via a sheath-liquid interface, and optimum interfacing conditions were investigated. A disadvantage of this type of interfacing is that the sheath liquid dilutes the CE effluent leading to reduced detection sensitivity. In order to avoid this drawback, the performance of a prototype sheathless interface for intact protein analysis by CE-MS was investigated and compared to sheath-liquid CE-MS. The potential of the developed CE-TOF-MS systems was evaluated by analyzing various pharmaceutical proteins, addressing issues such as purity, stability, heterogeneity, and product composition
Capillary electrophoresis coupled to fluorescence spectroscopy for protein characterisation
No full textProteins are essential molecules in all living organisms. Their involvement in numerous biological processes has led to the development of protein-based medicines (biopharmaceuticals). For good understanding of the properties and function of endogenous proteins and biopharmaceuticals, extensive protein characterisation is required. This involves assessment of features such as purity, heterogeneity, stability, activity and conformation of the protein. For instance, an altered protein conformation may influence the protein function and activity, which could have serious health implications. Clearly, there is a demand for suitable analytical methods that allow assessment of protein identity, purity and conformation. Capillary electrophoresis (CE) coupled to fluorescence spectroscopy provides high separation efficiency and selective spectroscopic detection that can be very useful for protein characterisation. Protein modifications, like glycosylation and deamidation, may involve changes in the net charge of a protein and, thus, of its electrophoretic mobility. Moreover, protein conformational changes, like unfolding or aggregation, are often accompanied by a change in protein molecular radius, which is reflected in the electrophoretic mobility as well. Fluorescence spectroscopy can provide information on the conformational state of a protein by monitoring changes in local tryptophan environments. This thesis describes the development and evaluation of novel fluorescence (Flu) and wavelength-resolved fluorescence (wrFlu) detection systems in CE for analysis of intact proteins. In order to accomplish effective Flu detection, a detector set-up was selected that incorporated a lamp as excitation source, a dedicated fluorescence detection cell and a photomultiplier detector. The analytical characteristics of the selected CE-Flu set-up and its suitability for native protein detection were tested using tryptophan and some model proteins. Protein detection limits were 7-33 nM, which was a factor of 25 better than UV absorbance detection at 280 nm, and comparable to UV detection at low-UV wavelengths. In order to obtain conformational information on separated proteins, employment of wavelength-resolved fluorescence detection was required. Therefore, the photomultiplier detector was replaced by a spectrograph equipped with a sensitive charge-coupled device (CCD) as detector. CE-wrFlu of intact proteins allowed acquisition of protein emission spectra ‘on-the-fly’. Detection limits were 6-32 nM, which is comparable to Flu detection. Analysis of model proteins in native (folded) and denatured (unfolded) state showed that protein conformational changes can be monitored via two independent parameters, the position of maximum emission wavelength and the effective electrophoretic mobility. The usefulness of CE-wrFlu for the study of protein unfolding pathways was investigated by measuring non-reduced and reduced β-lactoglobulin B (β-LGB) in several stages of unfolding. Non-reduced β-LGB showed two distinct unfolding pathways, that showed fast and slow interconversion kinetics between folded and unfolded species, respectively. Reduced β-LGB showed only one unfolding pathway, and unfolded at much lower denaturant concentration. These results suggest that disulphide bonds might be a prerequisite for the observed unfolding behaviour of non-reduced β-LGB. The potential applicability of native CE-Flu for the profiling of biopharmaceuticals was evaluated by the analysis of different products of human recombinant erythropoietin. Distinct and repeatable peak profiles were obtained, which allowed discrimination of different products based on the obtained glycoform patterns
Surface plasmon resonance on-line with liquid chromatography – mass spectrometry for the quantification and identification of proteins
No full textAlthough SPR-MS as a system for studying molecular interactions, e.g. ligand fishing, orphan receptor and drug screening, has been in use since 1997, several aspects remain to be improved. Among these are non-specific adsorption (NSA) and surface modification allowing protein coupling. These are related, as they concern sensor-surface coating allowing both the immobilisation of receptor molecules as well as preventing unwanted interactions. In the case of an on-line SPR-LC-MS/MS system, an interface between the SPR and the LC system is required. Additionally, there is need for a means of efficient digestion of isolated proteins in order to allow tandem-MS identification. The topics that were studied in this thesis arose from these subjects. First surface-coating development was performed for both sensors and enzyme reactors. The developed sensors and reactors were characterised. Next, the integration of all necessary instruments was investigated, ultimately evolving into a proof of principle. Lastly, an application was developed in which the isolation, quantification and identity confirmation of β2-microglobulin in urine was performed
Advances in capillary electrophoresis: In-line preconcentration for biomedical analysis. Impurity profiling of heparin
No full textCapillary electrophoresis (CE) has shown to be highly suitable for the analysis of polar and ionogenic compounds in biomedical and pharmaceutical samples. Separation with CE is based on the charge-to-size ratio of analytes. The application of CE for bioanalysis may be hindered by its relatively low sensitivity. Two sample preconcentration techniques have been applied in this thesis, a dynamic pH junction and an in-capillary solid phase extraction column. In-capillary analyte stacking via dynamic pH junction was investigated with underivatised phenylalanine (Phe) and tyrosine (Tyr) as model amino acids. Samples were mixed with acidic background electrolyte prior to injection. A plug of ammonium hydroxide solution was injected before the sample to produce a pH junction. Zwitterionic amino acids have opposing charges in the respecting solvents and will thus get opposing mobilities. The amino acids stack at the interface between the solvents. LODs for Phe and Tyr in urine were 0.054 and 0.019 μM for a sample injection volume of 10% of the capillary. Another technique for preconcentration of analytes employs an in-line frit-free solid-phase extraction (SPE) column. A 2 mm * 150 µm column was packed with mixed-mode sorbent for the preconcentration of the test compounds 2-ethylidene-1,5-dimethyl-3,3-diphenylpirrolidine (EDDP), dihydrocodeine and codeine. The sorbent had a particle diameter of more than 50 µm and was retained between a short inlet capillary and a separation capillary (50 µm id). The injected sample volume was 60 µL (i.e., 30 capillary volumes). Using MS-detection, LODs were 0.22 pg/mL for EDDP, 2.1 pg/mL for dihydrocodeine and 24 pg/mL for codeine. For the analysis of opioid peptides, a similair SPE-column was packed with a C18 sorbent. The column size was 4 mm * 150 µm, and the injected sample volume was 12 µL. Using SPE-CE-MS, the LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed, and the opioid peptides could be detected down to 1.0 ng/mL. In 2008, oversulfated chondroitin sulfate (OSCS) present in certain lots of heparin was identified as the toxic contaminant responsible for severe side effects following intravenous heparin administration. A preliminary CE method published by the US Food and Drugs Administration showed only partial separation of the OSCS contaminant from heparin. In this thesis an improved CE method is described. Enhancement of the OSCS–heparin separation was achieved by using high concentrations of Tris phosphate (pH 3.0) as background electrolyte. Good separations of OSCS, heparin and DS are obtained within 17 min. The method permits OSCS and DS determination in heparin down to the 0.05% and 0.5% (w/w) level, respectively. The CE method was further optimized to achieve shorter analysis times. Using a much shorter capillary, separation of OSCS, heparin and DS was obtained within 7 min. Detection limits for OSCS and DS were well below 0.5 mg/mL. Further reduction of analysis time could be achieved by employing an effective capillary length of 10 cm, providing full separation within 4 min
Comparison of capillary electrophoresis-mass spectrometry and hydrophilic interaction chromatography-mass spectrometry for anionic metabolic profiling of urine
Get PDFIn order to assess the utility of a recently developed capillary electrophoresis-mass spectrometry (CE-MS) method for the study of anionic metabolites in urine, a comparison was made with hydrophilic interaction chromatography-MS (HILIC-MS) using negative electrospray ionization. After optimization of the HILIC conditions, a gradient employing 10mM ammonium acetate (pH 6.8) in acetonitrile-water (5 min 90% acetonitrile followed by 90%-50% acetonitrile in 10 min) was selected, providing baseline separation of five representative anionic test metabolites. Relative standard deviations (RSDs) for HILIC retention times and peak areas were below 0.2% and 7.7%, respectively, and detection limits were in the range 0.04-2.21 μM. Metabolites in rat urine could also be analysed in a reproducible way with retention time and peak area RSDs below 0.6% and 13.6%, respectively. The CE-MS and HILIC-MS methods were compared in terms of reproducibility, sensitivity, selectivity and coverage of the anionic urinary metabolome. In general, peak area RSDs were similar whereas HILIC-MS yielded better retention-time repeatability and up to 80 times lower detection limits (expressed in injected concentration) for test metabolites as compared to CE-MS. Rat urine analysis by HILIC-MS provided detection of 1360 molecular features compared to 347 molecular features revealed with CE-MS. Of these, a number of 144 molecular features were found with both HILIC-MS and CE-MS, which showed on average 10 times higher peak areas in HILIC-MS. The HILIC retention and CE migration times of the common features were clearly not correlated. The HILIC and CE behavior of the test metabolites and 16 putatively identified common features were evaluated involving their physicochemical properties, indicating a markedly different separation selectivity, and thus significant degree of orthogonality of HILIC and CE
Developments in coupled solid-phase extraction-capillary electrophoresis 2011-2013
No full textThis 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
CE-MS for metabolomics: developments and applications in the period 2012-2014
Get PDFIn the field of metabolomics, CE-MS is now regarded as a useful complementary analytical technique for the profiling of (highly) polar ionogenic metabolites in biological samples. Over the past few years, significant advancements have been made in CE-MS approaches for metabolic profiling studies. This paper, which is a follow-up of three previous review papers covering the years 2000-2012 [Electrophoresis 2009, 30, 276-291; Electrophoresis 2011, 32, 52-65; Electrophoresis 2013, 34, 86-98], provides an update of these developments covering the scientific literature from July 2012 to June 2014. Attention will be paid to novel interfacing techniques for coupling CE to MS and their implications for metabolomics studies. The potential of CEC-MS and MEKC-MS are also considered, and CE-MS systems for high-throughput metabolic profiling are discussed. The applicability of CE-MS for metabolomics studies is demonstrated by representative examples in the fields of biomedical, clinical, microbial, plant, environmental, and food metabolomics. An overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings, and MS detection mode. Finally, general conclusions and perspectives are given
Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping
Get PDFCytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6 min. The method was validated from 0.05 to 5 mu g mL(-1) CAF and 0.025-2.5 mu g mL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precision
Evaluation of fritless solid-phase extraction coupled on-line with capillary electrophoresis-mass spectrometry for the analysis of opioid peptides in cerebrospinal fluid
No full textFritless SPE on-line coupled to CE with UV and MS detection (SPE-CE-UV and SPE-CE-MS) was evaluated for the analysis of opioid peptides. A microcartridge of 150 μm id was packed with a C18 sorbent (particle size > 50 μm), which was retained between a short inlet capillary and a separation capillary (50 μm id). Several experimental parameters were optimized by SPE-CE-UV using solutions of dynorphin A (DynA), endomorphin 1 (End1), and methionine-enkephaline (Met). A microcartridge length of 4 mm was selected, sample was loaded for 10 min at 930 mbar and the retained peptides were eluted with 67 nL of an acidic hydro-organic solution. Using SPE-CE-MS, peak area and migration time repeatabilities for the three opioid peptides were 12-27% and 4-5%, respectively. SPE recovery was lower for the less hydrophobic DynA (22%) than for End1 (66%) and Met (78%) and linearity was satisfactory in all cases between 5 and 60 ng/mL. The LODs varied between 0.5 and 1.0 ng/mL which represent an enhancement of two orders of magnitude when compared with CE-MS. Cerebrospinal fluid (CSF) samples spiked with the opioid peptides were analyzed to demonstrate the applicability to biological samples. Peak area and migration time repeatabilities were similar to the standard solutions and the opioid peptides could be detected down to 1.0 ng/mL
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