Stacking effect via solvent polarity differences in micellar electrokinetic chromatography with aqueous-organic background electrolyte

In this manuscript, a new stacking mode for micellar electrokinetic chromatography with anionic micelles and neutral analytes was investigated. The variation in the velocity of the analytes between the sample and the separation zones, needed for stacking effects, was obtained by using solvents with different polarities, the concentration of salts and micelles being identical between the two zones. Whereas a pure aqueous solvent was used in the injection zone, up to 30% ACN was used in the separation zone. In such conditions, higher interactions between the analyte and the micelles are obtained in the sample zone than in the separation zone, this due to the presence of the organic solvent. As the velocity of neutral analyte depends on its interactions with the moving micelles, its speed will drop when crossing two distinct zones where in the second zone its interactions with the micelles are lower than in the outgoing zone. This will induce a stacking effect. This approach was validated using a background electrolyte in the separation zone made with 10 mM disodium hydrogen phosphate, 15 mM sodium dodecyl sulphate and 15% ACN. The three triazines (simazine, atrazine, and terbuthylazine), used as test analytes, were dissolved in 10 mM disodium hydrogen phosphate, 15 mM sodium dodecyl sulphate and either 15 or 0% ACN. A stacking factor of around 2 was obtained injecting the sample in 0% ACN rather than 15% ACN. This was in accordance with the theoretical values predicted measuring the effective mobilities in both zonesCOMPETE/QREN/UE and PTDC/QUI-QUI/116156/2009 and SFRH / BPD / 30548 / 200

Mining for peaks in lc-hrms datasets using finnee - a case study with exhaled breath condensates from healthy, asthmatic, and copd patients

This work was financially supported by the projects: (i) UID/ EQU/00511/2019 - Laboratory for Process Engineering, Environment, Biotechnology and Energy − LEPABE funded by national funds through FCT/MCTES (PIDDAC); (ii) POCI-01-0145-FEDER-029702 and POCI-01-0145-FEDER031297 funded by FEDER funds through COMPETE2020 − Programa Operacional Competitividade e Internacionalizaca̧ õ (POCI) and by national funds (PIDDAC) through FCT/ MCTES; (iii) AstraZeneca − Projecto OLDER (CEDOC/ 2015/59); (iv) iNOVA4Health - UID/Multi/04462/2013, financially supported by FCT/Ministerio da Educação e Ciência, and co-funded by FEDER under the PT2020 Partnership Agreement.Separation techniques hyphenated to high-resolution mass spectrometry are essential in untargeted metabolomic analyses. Due to the complexity and size of the resulting data, analysts rely on computer-assisted tools to mine for features that may represent a chromatographic signal. However, this step remains problematic, and a high number of false positives are often obtained. This work reports a novel approach where each step is carefully controlled to decrease the likelihood of errors. Datasets are first corrected for baseline drift and background noise before the MS scans are converted from profile to centroid. A new alignment strategy that includes purity control is introduced, and features are quantified using the original data with scans recorded as profile, not the extracted features. All the algorithms used in this work are part of the Finnee Matlab toolbox that is freely available. The approach was validated using metabolites in exhaled breath condensates to differentiate individuals diagnosed with asthma from patients with chronic obstructive pulmonary disease. With this new pipeline, twice as many markers were found with Finnee in comparison to XCMS-online, and nearly 50% more than with MS-Dial, two of the most popular freeware for untargeted metabolomics analysis.publishersversionpublishe

Metabolomics profile responses to changing environments in a common bean (Phaseolus vulgaris L.) germplasm collection

Metabolomics is one of the most powerful -omics to assist plant breeding. Despite the recognized genetic diversity in Portuguese common bean germplasm, details on its metabolomics profiles are still missing. Aiming to promote their use and to understand the environment’s effect in bean metabolomics profiles, 107 Portuguese common bean accessions, cropped under contrasting environments, were analyzed using spectrophotometric, untargeted and targeted mass spectrometry approaches. Although genotype was the most relevant factor on bean metabolomics profile, a clear genotype × environment interaction was also detected. Multivariate analysis highlighted, on the heat-stress environment, the existence of higher levels of salicylic acid, and lower levels of triterpene saponins. Three clusters were defined within each environment. White accessions presented the lowest content and the colored ones the highest levels of prenol lipids and flavonoids. Sources of interesting metabolomics profiles are now identified for bean breeding, focusing either on local or on broad adaptation.To FCT, Portugal, in BEGEQA project (PTDC/AGR-TEC/3555/2012), E.M. PhD fellowship (SFRH/BD/89287/2012), as well as to R&D unit, UIDB/04551/2020 (GREEN-IT – Bioresources for sustainability) and COST Action FA1403 (STSM-FA1403-290815-063873) for funding. The authors also acknowledge PORTUGAL 2020 to the Portuguese Mass Spectrometry Network, grant number LISBOA-01-0145-FEDER-402-022125. The project NETDIAMOND (SAICTPAC/0047/2015), financially supported by FEEI (Lisboa 2020 and FCT/POCI-01-0145-FEDER-016385), to the iNOVA4Health (UID/Multi/04462/2013), financially supported by FCT and co-funded by FEDER under the PT2020 Partnership Agreement, as well as to POCI-01-0145-FEDER-029702, funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES

Liquid separation techniques coupled with mass spectrometry for chiral analysis of pharmaceuticals compounds and their metabolites in biological fluids.

Determination of the chiral composition of drugs is nowadays a key step in order to determine purity, activity, bioavailability, biodegradation, etc, of pharmaceuticals. In this manuscript, works published for the last 5 years on the analysis of chiral drugs by liquid separation techniques coupled with mass spectrometry are reviewed. Namely, chiral analysis of pharmaceuticals including e.g., antiinflammatories, antihypertensives, relaxants, etc, by liquid chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry are included. The importance and interest of the analysis of the enantiomers of the active compound and its metabolites in different biological fluids (plasma, urine, cerebrospinal fluid, etc) are also discussed.Peer reviewe

Measuring the lenght of hydrodynamicallly injected plugs in capillary electrophoresis using the electrical current monitoring

Although Capillary Electrophoresis (CE) is nowadays a worldwide separation technique, it is generally recognized that one of its main limitations is its poor robustness for quantitative analysis. Although this limitation can partially be surpassed using internal standards, it is well known that to find adequate standards is a very difficult task when too complex mixtures have to be analyzed. In this work, an alternative method to improve quantitation by CE is presented using the electrical current profile monitored during any CE run. Thus, an abrupt step in the current monitoring is observed when a hydrodynamically injected plug of conductivity different from the background electrolyte leaves the capillary under the influence of the electroosmotic flow. It is demonstrated that under these conditions, the relative amplitude of this step can be used to experimentally measure the injection length. This measure can not only be used for calibration, but also to correct variations of the length injected what is demonstrated to significantly improve the quantitative accuracy and reproducibility of CE. Thus, RSD values for inter-day quantification (5 experiments a day for 5 days) were improved from 10.5 % to 4.2 %. Moreover, it is also demonstrated that accuracy of quantitative determinations by CE can greatly be improved by using this procedure. The method can also be implemented in other separation techniques where the electroosmotic flow is used as driving force (e.g., capillary electrochromatography, micellar electrokinetic chromatography or chip based separations). Advantages and limitations of this approach in comparison to the use of internal standards are also discussed.Financial support from a AGL2005-05320-C02-01 project (Ministerio de Educacion y Ciencia, MEC) and a S-505/AGR-0153 project (Comunidad Autonoma de Madrid, CAM) is acknowledged.Peer reviewe

capillary electrophoresis-mass spectrometry of zein proteins from conventional and transgenic maize

In this work, an original capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to analyze the complex zein proteins fraction from maize. A thorough optimization of i) zein proteins extraction, ii) CE separation and iii) electrospray-mass spectrometry (ESI-MS) detection is carried out in order to obtain highly informative CE-MS profiles of this fraction. The developed CE-MS method provides good separation of multiple zein proteins based on their electrophoretic mobilities as well as adequate characterization of these proteins based on their relative molecular mass (Mr). Zein proteins with small Mr differences (below 100 Da) were easily separated and successfully analyzed by CE-MS. Thus, apart of the so-called 15-kDa--zein and 16-kDa--zein, which are demonstrated to be formed by a heterogeneous group of proteins, numerous -zeins belonging to the 19 and 22-kDa fraction were also identified for the first time in this work. The usefulness of this CE-MS method was corroborated by comparing the zein-proteins fingerprint of various maize lines including transgenic and their corresponding non-transgenic isogenic lines cultivated under the same conditions.Authors are grateful to the AGL2005-05320-C02-01 Project (Ministerio de Educacion y Ciencia) and the S-505/AGR-0153 ALIBIRD Project (Comunidad Autonoma de Madrid) for financial support of this work.Peer reviewe

Reproducible and efficient separation of aggregatable zein proteins by capillary zone electrophoresis using a volatile background electrolyte

Zein proteins are a complex mixture of polypetides that belong to the alcohol soluble storage proteins group (prolamines) in corn. These proteins constitute about 50-60% of the total endosperm protein and are classified in different groups on the basis of differences in their solubility and sequence. Among them, zein proteins are considered the majority group showing a high tendency to aggregate what makes very difficult their analysis by any analytical method. Thus, capillary zone electrophoresis (CZE) of these proteins require the use of very complex background electrolytes (BGEs) non-compatible with on-line ESI-MS analysis. The aim of this work was to find a new BGE for the CZE separation of zein protein fully compatible with ESI-MS while providing further light on the complex CZE separation of aggregatable proteins. Thus, it is demonstrated in this work that efficient and reproducible CZE separations of zein proteins can be achieved by using a BGE composed of water, acetonitrile, formic acid and ammonium hydroxide. Besides, it is shown that zeins analysis is significantly improved by including the effect of an ammonium gradient during their separation. It is experimentally verified that the ammonium gradient can easily be achieved in CZE by either working with a sample zone with a low concentration of ammonium and a background electrolyte (BGE) with a high concentration, or conversely, working with a sample zone with high ammonium concentration and a BGE with low concentration of ammonium, giving rise in both cases to a significant improvement in the CZE separation of these proteins. It is demonstrated that this procedure can give rise to efficiency improvements of up to 20-folds in the CZE separation of zein proteins. Under optimized conditions, 20 proteins could be separated with average efficiencies higher than 400000 theoretical plates/m. Some possible explanations of this effect are discussed including stacking, protein-capillary wall adsorption, protein solubility and protein-salt interactions.Authors are grateful to the AGL2005-05320-C02-01 Project (Ministerio de Educacion y Ciencia) and the S-505/AGR-0153 Project (Comunidad Autonoma de Madrid) for financial support of this work.Peer reviewe

New and simplified two-dimensional capillary electrophoresis-mass spectrometry mapping: analysis of proteolytic digests

Capillary electrophoresis-mass spectrometry (CE-MS) has demonstrated to be a very useful hyphenated technique for Proteomic studies. However, the huge amount of data stored in a single CE-MS run makes necessary to account with procedures able to extract all the relevant information made available by CE-MS. In this work, we present a new and easy approach able to generate a simplified two-dimensional map from CE-MS raw data. This new approach provides the automatic detection and characterization of the most abundant ions from the CE-MS data including their m/z values, ion intensities and analysis times. It is demonstrated that visualization of CE-MS data in this simplified 2D format allows (i) an easy and simultaneous visual inspection of large datasets, (ii) an immediate perception of relevant differences in closely related samples, (iii) a rapid monitoring of data quality levels in different samples and (iv) a fast discrimination between comigrating polypeptides and ESI-MS fragmentation ions. The strategy proposed in this work does not rely on an excellent mass accuracy for peak detection and filtering since MS values obtained from an ion trap analyzer are used. Moreover, the methodology developed works directly with the CE-MS raw data, without interference by the user, giving simultaneously a simplified 2D map and a much easier and more complete data evaluation. Besides, this procedure can easily be implemented in any CE-MS laboratory. The usefulness of this approach is validated by studying the very similar trypsin digests from bovine, rabbit and horse cytochrome c. It is demonstrated that this simplified 2D approach allows obtaining in a fast and simple way specific markers for each species.Authors are grateful to the AGL2005-05320-C02-01 Project (Ministerio de Educacion y Ciencia) and the S-505/AGR-0153 Project (Comunidad Autonoma de Madrid, CAM) for financial support of this work.Peer reviewe

Field amplified separation in capillary electrophoresis (FAsCE): a capillary electrophoresis mode

6 pages, 4 figures, 2 tables.-- PMID: 17073426 [PubMed].-- Printed version published Nov 1, 2006.In field amplified injection in capillary electrophoresis (CE), the capillary is filled with two buffering zones of different ionic strength; this induces an amplified electrical-field in the low ionic strength zone and a lower field in the high ionic strength zone, making sample stacking feasible. The electro-osmotic flow (eof) usually observed in CE, however, displaces the low field zone and induces an extra band broadening preventing any CE separation in the field amplified zone. These limitations have originated the restricted use of field amplification in CE only for stacking purposes. For the first time, in this work it is theoretically shown and experimentally corroborated that CE separation speed and efficiency can simultaneously be increased if the whole separation is performed in the field amplified zone, using what we have called Field Amplified Separation in Capillary Electrophoresis (FAsCE). The possibilities of this new CE mode are investigated using a new and simple coating able to provide near zero eof at the selected separation pH. Using FAsCE, improvements of 20 % for separation speed and 40 % for efficiency are achieved. Moreover, a modified FAsCE approach is investigated filling the capillary with the high ionic strength buffer up to the interior of the detection window. Under these conditions, an additional 3-fold increase in sensitivity is also observed. The most interesting results were obtained combining the short-end injection mode and this modified FAsCE approach. Under these conditions, apart of a 3-fold improvement in efficiency and sensitivity, the total analysis time was drastically reduced to 40 s, giving rise to a time reduction of more than 7-fold compared to normal CE. This speed enhancement brings about one of the fastest CE separations achieved using capillaries, demonstrating the great possibilities of FAsCE as a new, sensitive, efficient and fast CE separation mode.G.L.E. thanks the Spanish MEC for a postdoctoral grant. The authors are grateful to the AGL2005-05320-C02-01 Project (Ministerio de Educación y Ciencia) and the S-505/AGR-0153 Project(Comunidad Autonóma de Madrid, CAM) for financial support of this work.Peer reviewe