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In a previous work, we explored zone broadening and the achievable plate numbers in linear drift tube ion mobility-mass spectrometry through developing a plate-height model [1]. On the basis of these findings, the present theoretical study extends the model by exploring peak-to-peak resolution and peak capacity in ion mobility separations. The first part provides a critical overview of chromatography-influenced resolution equations, including refinement of existing formulae. Furthermore, we present exact resolution equations for drift tube ion mobility spectrometry based on first principles. Upon implementing simple modifications, these exact formulae could be readily extended to traveling wave ion mobility separations and to cases when ion mobility spectrometry is coupled to mass spectrometry. The second part focuses on peak capacity. The well-known assumptions of constant plate number and constant peak width form the basis of existing approximate solutions. To overcome their limitations, an exact peak capacity equation is derived for drift tube ion mobility spectrometry. This exact solution is rooted in a suitable physical model of peak broadening, accounting for the finite injection pulse and subsequent diffusional spreading. By borrowing concepts from the theoretical toolbox of chromatography, we believe that the present study will help in integrating ion mobility spectrometry into the unified language of separation science.
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In the past decade, ion mobility spectrometry (IMS) in combination with mass spectrometry (IM-MS) became a widely employed technique for the separation and structural characterization of ionic species in the gas phase. Similarly to chromatography, where studies on the mechanism of band broadening and adequate plate-height equations have been aiding method development and promoting advancements in column technology, a suitable resolving power theory of drift tube ion mobility-mass spectrometry (DTIM-MS) is essential to stimulate further progress in this emerging field of separation science. In the present study, therefore, we explore dispersion processes in detail and present a plate-height model of ion mobility-mass spectrometry. We quantify the effects of five major dispersion processes that contribute to zone broadening and determine the resolving power in DTIM-MS: diffusion, Coulomb repulsion, electric field inhomogeneities, the finite initial spread of the ion cloud and dispersion outside the mobility cell. A solution is provided to account for the nonuniform separation field in IM-MS in the presence of multiple compartments. The equations - derived from first principles - serve as the basis for formulating an expression that is similar in nature to van Deemter's plate-height equation for chromatography. A comprehensive set of experiments was performed on a custom-built DTIM-MS instrument to evaluate the accuracy of the plate-height model, resulting in satisfactory agreement between experiment and theory. Building on these findings, the plate-height equation was employed to explore the influence of drift gas pressure, injection pulse-width and the mobility of ions on resolving power from a theoretical point of view. Our findings may aid instrument design and development in the future, as well as the optimization of measurement conditions to improve ion mobility separations. By employing the plate-height concept and the general formalism of differential migration processes to describe zone spreading in IM-MS, we aim to find a common ground between this emerging method and such well-established techniques as HPLC or CZE. We also hope that the work presented here will facilitate a broader acceptance of IMS as a separation method of great potential by the communities of chromatography and electrophoresis, as well as that of mass spectrometry.
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RATIONALE: The wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses. METHODS: Prior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation. RESULTS: For Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 µM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study. CONCLUSIONS: Chimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening.
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Espectrometria de Massas/métodos , Metabolômica/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Metaboloma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
This study of ion accumulation/release behavior relevant to ion mobility-mass spectrometry (IM-MS) as employed for non-targeted metabolomics involves insight from theoretical studies, and controlled reference experiments involving measurement of low and high molecular mass metabolites in varying concentrations within a complex matrix (yeast extracts). Instrumental settings influencing ion trapping (accumulation time) and release conditions in standard and multiplexed operation have been examined, and translation of these insights to liquid chromatography (LC) in combination with drift tube IM-MS measurements has been made. The focus of the application is non-targeted metabolomics using carefully selected samples to allow quantitative interpretations to be made. Experimental investigation of the IM-MS ion utilization efficiency particularly focusing on the use of the Hadamard transform multiplexing with 4-bit pseudo-random pulsing sequence for assessment of low and high molecular mass metabolites is compared with theoretical modeling of gas-phase behavior of small and large molecules in the IM trapping funnel. Increasing the trapping time for small metabolites with standard IM-MS operation is demonstrated to have a deleterious effect on maintaining a quantitative representation of the metabolite abundance. The application of these insights to real-world non-targeted metabolomics assessment of intracellular extracts from biotechnologically relevant production processes is presented, and the results were compared to LC×IM-MS measurements of the same samples. Spiking of a uniformly 13C-labeled yeast extract (as a standard matrix) with varying amounts of natural metabolites is used to assess the linearity and sensitivity according to the instrument mode of operation (i.e., LC-MS, LC×IM-MS, and LC×[multiplexed]IM-MS). When comparing metabolite quantification using standard and multiplexed operation, sensitivity gain factors of 2-8 were obtained for metabolites with m/z below 250. Taken together, the simulation and experimental results of this study provide insight for optimizing measurement conditions for metabolomics and highlight the need for implementation of multiplexing strategies using short trapping times as relative quantification (e.g., in the context with non-targeted differential analysis) with sufficient sensitivity and working range is a requirement in this field of application.
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Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metabolômica , Aminoácidos/metabolismo , Íons , Padrões de ReferênciaRESUMO
Collision cross section (CCS) measurements resulting from ion mobility-mass spectrometry (IM-MS) experiments provide a promising orthogonal dimension of structural information in MS-based analytical separations. As with any molecular identifier, interlaboratory standardization must precede broad range integration into analytical workflows. In this study, we present a reference drift tube ion mobility mass spectrometer (DTIM-MS) where improvements on the measurement accuracy of experimental parameters influencing IM separations provide standardized drift tube, nitrogen CCS values (DTCCSN2) for over 120 unique ion species with the lowest measurement uncertainty to date. The reproducibility of these DTCCSN2 values are evaluated across three additional laboratories on a commercially available DTIM-MS instrument. The traditional stepped field CCS method performs with a relative standard deviation (RSD) of 0.29% for all ion species across the three additional laboratories. The calibrated single field CCS method, which is compatible with a wide range of chromatographic inlet systems, performs with an average, absolute bias of 0.54% to the standardized stepped field DTCCSN2 values on the reference system. The low RSD and biases observed in this interlaboratory study illustrate the potential of DTIM-MS for providing a molecular identifier for a broad range of discovery based analyses.
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Espectrometria de Mobilidade Iônica/métodos , Laboratórios/normas , Espectrometria de Massas/métodos , Calibragem , Lipídeos/química , Estrutura Molecular , Nitrogênio/química , Proteínas/química , Reprodutibilidade dos TestesRESUMO
Reversed-phase LC combined with high-resolution mass spectrometry (HRMS) is one of the most popular methods for cellular metabolomics studies. Due to the difficulties in analyzing a wide range of polarities encountered in the metabolome, 100%-wettable reversed-phase materials are frequently used to maximize metabolome coverage within a single analysis. Packed with silica-based sub-3 µm diameter particles, these columns allow high separation efficiency and offer a reasonable compromise for metabolome coverage within a single analysis. While direct performance comparison can be made using classical chromatographic characterization approaches, a comprehensive assessment of the column's performance for cellular metabolomics requires use of a full LC-HRMS workflow in order to reflect realistic study conditions used for cellular metabolomics. In this study, a comparison of several reversed-phase LC columns for metabolome analysis using such a dedicated workflow is presented. All columns were tested under the same analytical conditions on an LC-TOF-MS platform using a variety of authentic metabolite standards and biotechnologically relevant yeast cell extracts. Data on total workflow performance including retention behavior, peak capacity, coverage, and molecular feature extraction repeatability from these columns are presented with consideration for both nontargeted screening and differential metabolomics workflows using authentic standards and Pichia pastoris cell extract samples.
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Cromatografia de Fase Reversa/métodos , Metabolômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cromatografia Líquida de Alta Pressão/métodos , Metaboloma , Pichia/metabolismo , Reprodutibilidade dos TestesRESUMO
The combination of CE and MS is now a widely used tool that can provide a combination of high resolution separations with detailed structural information. Recently, we highlighted the benefits of an approach to add further functionality to this well-established hyphenated technique, namely the possibility to perform chemical reactions within the sheath-liquid of the CE-MS interface . Apart from using hydrogen/deuterium exchange for online determination of numbers of exchangeable protons, the addition of DPPH⢠(2,2-diphenyl-1-picrylhydrazyl) to the sheath-liquid can be used as a fast screening tool for studying antioxidant characteristics of individual components. Such a CE-MS methodology allows rapid and information-rich analysis with minimal reagent and sample consumption to be performed. In the present work, we demonstrate the applicability of this approach for the characterization of phenolic plant extracts from the Labiatae family, namely Rosmarinus officinalis and Melissa officinalis. Using the described approach, a wide range of compounds (15 and 13 phenolic compounds, respectively) could be confidently identified using a combination of high resolution CE-MS separations with implementation of online deuterium exchange and DPPH⢠reactions. These compounds included polyphenols, phenolic acids, and triterpene acids. In conjunction with online MS/MS experiments, extensive structural information for aglyconic and glycosylated antioxidants present in the extracts could be obtained using simple experimental changes, which can be carried out prior to the purchasing of expensive chemical standards or the time-consuming preparative isolation of individual compounds.
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Eletroforese Capilar/métodos , Fenóis/análise , Extratos Vegetais/análise , Espectrometria de Massas em Tandem/métodos , Antioxidantes/análise , Compostos de Bifenilo/química , Medição da Troca de Deutério , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Melissa/química , Picratos/química , Extratos Vegetais/química , Rosmarinus/química , Espectrofotometria UltravioletaRESUMO
Concerning polymeric monolithic materials utilized in separation science, the structural and mechanical characteristics from the nanoscopic to the macroscopic scale remain of great interest. Suitable analytical tools are urgently required to understand the polymer monolith's constituent structure, particularly in the case of nanoscale polymer properties that tend to develop gel porosity in contact with a mobile phase ultimately affecting the chromatographic performance. Herein described are our first findings from a characterization of commercially available analytical polymer monoliths based on styrene/divinylbenzene and methacrylate chemistries utilizing confocal Raman spectroscopy imaging and atomic force microscopy (AFM). Confocal Raman spectroscopy can be used to generate a three-dimensional representation of monoliths in both dry state and in contact with solvent. AFM force-indentation measurements on individual cross-sectioned globular features permit detailed assessment of mechanical properties of the stationary phase. This approach allowed so far unprecedented insight and identification of a heterogeneous cross-link density distribution of polymer material within individual globular features on a submicrometer scale.
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Polyimides (PIs) are a group of widely used synthetic materials that service a variety of different purposes including microelectronics, insulating films and aerospace applications. Depending on the requirements (defined by the particular final product), the actual composition of PIs may show substantial chemical variation. To study this variation in chemical structure, CE-MS can be employed for the determination of PI composition following chemical degradation of the polymer sample. PI is chemically decomposed to corresponding aromatic diamine and carboxylic acid components using an alkali fusion reaction. Solid polymer samples are fused in a potassium hydroxide melt yielding reaction products that are diluted in acid and can be immediately analysed by CE coupled to a Q/TOF-MS with quantification performed using conventional UV detection. This approach involves a simple and rapid sample preparation yielding both qualitative and quantitative information regarding the chemical composition of the polymer. Application of the CE-MS approach is shown for a range of commercially available PI and poly(amide-imide) materials and the results are used to infer the respective chemical compositions.
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Eletroforese Capilar/métodos , Imidas/química , Espectrometria de Massas/métodos , Polímeros/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Amidas , Ácidos Carboxílicos/análise , Ácidos Carboxílicos/química , Diaminas/química , Imidas/análise , Polímeros/química , Raios UltravioletaRESUMO
In this study we introduce the implementation of rapid temperature pulses for selectivity tuning in capillary liquid chromatography. Short temperature pulses improved resolution in discrete sections of chromatograms, demonstrated for ion-exchange chromatography (IC) and hydrophilic interaction chromatography (HILIC) modes. Using a resistively heated column module capable of accurate and rapid temperature changes, this concept is first illustrated with separations of small anions by IC using a packed capillary column as well as a series of nucleobases and nucleosides by HILIC using a silica monolithic column with zwitterionic functionality (ZIC-HILIC). Both positive (increasing temperature) and negative temperature pulses are demonstrated to produce significant changes in selectivity and are useful approaches for improving resolution between coeluted compounds. The approach was shown to be reproducible over a large number of replicates. Finally, the use of temperature gradients as well as other complex temperature profiles was also examined for both IC and HILIC separations.
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Within recent years there has been an increase in research focused on the design and application of organic polymer monoliths in all areas of separation science. This is largely driven by the theoretical and practical benefits that these materials should be able to provide, particularly in terms of improved biocompatibility and high permeability. This review summarises recent new developments in this field with a focus on new approaches to the design and synthesis of polymeric monolithic materials for analytical separation science. This includes the use of alternative synthetic methodologies such as the development of hyper-crosslinked monoliths, preparation of hybrid materials and incorporation of nanostructures in the polymeric scaffold. New and developing approaches for the structural characterisation of monolithic columns are also included. Finally, we critically discuss the current chromatographic performances achieved with this column technology as well as where future developments in this field may be directed.
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This HPLC tutorial focuses on the preparation and use of kinetic plots to characterise the performance in isocratic and gradient LC. This graphical approach allows the selection of columns (i.e. optimum particle size and column length) and LC conditions (operating pressure and temperature) to generate a specific number of plates or peak capacity in the shortest possible analysis time. Instrument aspects including the influence of extra-column effects (maximum allowable system volume) and thermal operating conditions (oven type) on performance are discussed. In addition, the performance characteristics of porous-shell particle-packed columns and monolithic stationary phases are presented and the potential of future column designs is discussed.
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Uniform field drift tube ion mobility-mass spectrometry (DTIM-MS) has emerged as a valuable tool for a range of analytical applications. In focus here are standardized collisional cross section values from DTIM-MS (DTCCS) as a candidate identification point for various analytical workflows. Of critical importance in establishing this parameter as a valid identification point is a rugged estimation of uncertainties according to the procedures used for their derivation. Relying on the assumption of the zero-field limit, the primary method of measurement for DTCCS values involves experimental determination of arrival times of an ion measured at several different field strengths transiting a drift tube filled with high purity drift gas, while a method using measurements of external calibrants at a single field strength is employed to allow for online measurements of transient signals (e.g., chromatographic peaks). Both approaches are here considered with respect to the uncertainty of input experimental variables (temperature, pressure, voltages, physical constants) and the steps of the calibration function employed. Estimations of uncertainty were performed according to EURACHEM with Monte Carlo simulations and reveal that existing consensus calibration standards from experimental stepped-field IM-MS determinations have estimated expanded uncertainties in the range of 2.7 to 4.6% (k = 2). Application of these standards for calibration considering these input uncertainties reveals uncertainty estimates of 4.7-9.1% (k = 2) for measured values using an established single-field calibration approach. Finally, directions for improving this situation via new experimental efforts toward standard reference and calibration materials are presented.
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This chapter describes the developments in drift-tube ion mobility-mass spectrometry (DTIM-MS) that have driven application development in 'omics analyses. Harnessing the additional, orthogonal separation that DTIM provides increased confidence in compound identifications as the mass spectral complexity can be reduced and mobility-derived parameters (most prominently the collision cross section, CCS) used to support identity confirmation goals for a variety of 'omics application areas. Presented within this contribution is a methodology for improving the transmission and maintaining accurate determination of drift time-derived CCS (DTCCS) for low molecular weight compounds for a typical nontargeted 'omics (metabolomics) workflow using liquid chromatography in combination with DTIM-MS.
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Espectrometria de Mobilidade Iônica , Espectrometria de Massas , Metabolômica , Cromatografia Líquida de Alta Pressão , Biologia Computacional/métodos , Bases de Dados Factuais , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Use of an organic polymeric monolithic stationary phase for high temperature liquid chromatography (HTLC) with a pure water eluent is demonstrated for the first time. Separations of n-alcohols at temperatures exceeding 200 degrees C were achieved with no statistically significant reduction in chromatographic performance evident after continuous operation for 1000 column volumes above 200 degrees C.
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The characterization of wine via MS-based metabolic fingerprinting techniques remains a challenging undertaking due to the large number of phenolic compounds that cannot be confidently annotated and identified within analytical workflows. The combination of high performance liquid chromatography with low-field drift tube ion mobility time-of-flight mass spectrometry (HPLCâ¯×â¯IMS-TOFMS) offers potential for the confident characterization and fingerprinting of wine using a metabolomics-type workflow. In particular, the use of collision cross section values from low-field drift tube IMS using nitrogen as drift gas (DTCCSN2) in addition to retention time and a high resolution mass spectrum for putative compounds allows rugged statistical assessment and identity confirmation using CCS libraries (<0.5% error) to be performed. In the present work, an HPLCâ¯×â¯IMS-TOFMS platform has been utilized for the fingerprinting of 42 traditionally produced red wines emanating from the Republic of Macedonia. After establishing the reliability of DTCCSN2 as an identification point for wine metabolomics in both ionization modes, fingerprinting of wines according to grape variety was undertaken and a full dataset containing retention, accurate mass and DTCCSN2 values used to derive lists of compounds found to be statistically characteristic for each variety. Putative compounds were further assessed by assignment of in-source and post-drift mass fragments aligned according to retention time, drift time, and accurate mass providing up to seven identification points for a single compound when data from both positive and negative mode measurements are combined.
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Cromatografia Líquida/métodos , Espectrometria de Mobilidade Iônica/métodos , Vinho/análise , Controle de QualidadeRESUMO
Non-targeted analysis of metabolites in hypothesis-generating workflows has proven its potential to answer essential questions that arise when dealing with complex biological systems. Nevertheless, tracking changes in perturbed systems via accurate quantification and the identification process itself represent the most critical challenges in these workflows. Recent advances in ion mobility-mass spectrometry have enabled this technique to increase the confidence of metabolite annotation by introducing a complementary conditional molecular descriptor, that is collision cross section.
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Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Calibragem , Espectrometria de Mobilidade Iônica/instrumentação , Espectrometria de Massas/instrumentação , Incerteza , Fluxo de TrabalhoRESUMO
Measuring the exposome remains a challenge due to the range and number of anthropogenic molecules that are encountered in our daily lives, as well as the complex systemic responses to these exposures. One option for improving the coverage, dynamic range and throughput of measurements is to incorporate ion mobility spectrometry (IMS) into current MS-based analytical methods. The implementation of IMS in exposomics studies will lead to more frequent observations of previously undetected chemicals and metabolites. LC-IMS-MS will provide increased overall measurement dynamic range, resulting in detections of lower abundance molecules. Alternatively, the throughput of IMS-MS alone will provide the opportunity to analyze many thousands of longitudinal samples over lifetimes of exposure, capturing evidence of transitory accumulations of chemicals or metabolites. The volume of data corresponding to these new chemical observations will almost certainly outpace the generation of reference data to enable their confident identification. In this perspective, we briefly review the state-of-the-art in measuring the exposome, and discuss the potential use for IMS-MS and the physico-chemical property of collisional cross section in both exposure assessment and molecular identification.
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Biologia Computacional/métodos , Espectrometria de Massas/métodos , Animais , Cromatografia Líquida/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Metaboloma , Fluxo de TrabalhoRESUMO
Fermentation and cell culture biotechnology in the form of so-called "cell factories" now play an increasingly significant role in production of both large (e.g. proteins, biopharmaceuticals) and small organic molecules for a wide variety of applications. However, associated metabolic engineering optimisation processes relying on genetic modification of organisms used in cell factories, or alteration of production conditions remain a challenging undertaking for improving the final yield and quality of cell factory products. In addition to genomic, transcriptomic and proteomic workflows, analytical metabolomics continues to play a critical role in studying detailed aspects of critical pathways (e.g. via targeted quantification of metabolites), identification of biosynthetic intermediates, and also for phenotype differentiation and the elucidation of previously unknown pathways (e.g. via non-targeted strategies). However, the diversity of primary and secondary metabolites and the broad concentration ranges encompassed during typical biotechnological processes means that simultaneous extraction and robust analytical determination of all parts of interest of the metabolome is effectively impossible. As the integration of metabolome data with transcriptome and proteome data is an essential goal of both targeted and non-targeted methods addressing production optimisation goals, additional sample preparation steps beyond necessary sampling, quenching and extraction protocols including clean-up, analyte enrichment, and derivatisation are important considerations for some classes of metabolites, especially those present in low concentrations or exhibiting poor stability. This contribution critically assesses the potential of current sample preparation strategies applied in metabolomic studies of industrially-relevant cell factory organisms using mass spectrometry-based platforms primarily coupled to liquid-phase sample introduction (i.e. flow injection, liquid chromatography, or capillary electrophoresis). Particular focus is placed on the selectivity and degree of enrichment attainable, as well as demands of speed, absolute quantification, robustness and, ultimately, consideration of fully-integrated bioanalytical solutions to optimise sample handling and throughput.
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Biotecnologia , Indústrias , Metabolômica , Manejo de Espécimes , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta PressãoRESUMO
In the domain of liquid phase separations, the quality of separation obtainable is most readily gauged by consideration of classical chromatographic peak capacity theory. Column-based multidimensional strategies for liquid chromatography remain the most attractive and practical route for increasing the number of spatially resolved components in order to reduce stress on necessary mass spectrometric detection. However, the stress placed on a chromatographic separation step as a second dimension in a comprehensive online methodology (i.e. online LC×LC) is rather high. As an alternative to online LC×LC combinations, coupling of HPLC with ion mobility spectrometry hyphenated to mass spectrometry (IMS-MS) has emerged as an attractive approach to permit comprehensive sampling of first dimension chromatographic peaks and subsequent introduction to an orthogonal IMS separation prior to measurement of ions by a mass spectrometer. In the present work, utilization of classical peak capacity and ion mobility theory allows theoretical assessment of the potential of two- (LC×IMS-MS) or even three-dimensional (LC×LC×IMS-MS) experimental setups to enhance peak capacity and, therefore, the number of correctly annotated features within the framework of complex, non-targeted analysis problems frequently addressed using HPLC-MS strategies. Theoretical calculations indicate that newly-available drift tube IMS-MS instrumentation can yield peak capacities of between 10 and 40 using nitrogen drift gas for typical non-targeted metabolomic, lipidomic and proteomic applications according to the expected reduced mobilities of components in the respective samples. Theoretically, this approach can significantly improve the overall peak capacity of conventional HPLC-(MS) methodologies to in excess of 10(4) depending upon the column length and gradient time employed. A more elaborate combination of LC×LC×IMS-MS would improve the ion suppression limitation and possibly allow access to theoretically even higher peak capacities, but such a combination may render the IMS separation practically redundant as well as imparting the well-known dilution problems associated with LC×LC. Finally, some predictions for the separation of co-eluted isobaric compounds can also be made by considering the required peak-to-peak resolution for acceptable IMS separation. The here-described theoretical predication approach can be used to aid method development for HPLC×IMS-MS and is also accompanied by some practical considerations that should be contemplated in associated non-targeted analysis workflows.