In this study, we propose for the first time the use of solid-phase microextraction (SPME) in combination with liquid chromatography−mass spectrometry for untargeted metabolomic profiling of ...biological fluids. To achieve this goal, we first systematically evaluated 42 different SPME coatings for the extraction of 36 metabolites from different chemical classes and of widely varying polarities (log P range of −7.9 to 7.4) in order to identify SPME coatings which are the most suitable for metabolomic studies and to improve the extraction of polar metabolites over the existing commercial SPME devices. Three types of SPME coatings (mixed-mode coatings, polar-enhanced polystyrene−divinylbenzene, and phenylboronic acid) performed the best for simultaneous extraction of both hydrophilic and hydrophobic metabolites at physiological conditions, thus making them suitable for untargeted metabolomic profiling applications. A rapid and simple SPME method was then developed with single-use biocompatible mixed-mode coating for the metabolomic profiling of human plasma in combination with liquid chromatography−high-resolution mass spectrometry on a benchtop Orbitrap system. This optimized SPME method was evaluated versus ultrafiltration and solvent precipitation in terms of metabolite coverage and method precision. SPME detected 1592−3320 features versus 2082−3245 features detected by solvent precipitation methods and 2093−2686 detected for ultrafiltration using the same pooled human plasma sample. Method precision of SPME ranged between 11% and 18% (expressed as median relative standard deviation (RSD) of n = 7 replicates) versus 8−19% for solvent precipitation and 20−22% for ultrafiltration. The results demonstrate that the proposed SPME methodology reduces ionization suppression, provides free concentration information for hydrophobic analytes which are not detected by ultrafiltration methods, and can improve metabolite coverage over existing methodologies.
The primary objective of this review is to discuss recent technological developments in the field of solid-phase microextraction that have enhanced the utility of this sample preparation technique in ...the field of bioanalysis. These developments include introduction of various new biocompatible coating phases suitable for bioanalysis, such as commercial prototype
in vivo SPME devices, as well as the development of sampling interfaces that extend the use of this methodology to small animals such as mice. These new devices permit application of
in vivo SPME to a variety of analyses, including pharmacokinetics, bioaccumulation and metabolomics studies, with good temporal and spatial resolution. New calibration approaches have also been introduced to facilitate
in vivo studies and provide fast and quantitative results without the need to achieve equilibrium. In combination with the drastic improvement in the analytical sensitivity of modern liquid chromatography–tandem mass spectrometry instrumentation, full potential of
in vivo SPME as a sample preparation tool in life sciences can finally be explored. From the instrumentation perspective, SPME was successfully automated in 96-well format for the first time. This opens up new opportunities for high-throughput applications (>1000 samples/day) such as for the determination of unbound and total drug concentrations in complex matrices such as whole blood with no need for sample pretreatment, studies of distribution of drugs in various compartments and/or determination of plasma protein binding and other ligand–receptor binding studies, and this review will summarize the progress in this research area to date.
The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding ...of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.
•Sulfobetaine ZIC-HILIC improved the detection of metabolite standards over the silica HILIC column.•5 mM ammonium acetate was selected for mobile phase based on peak shape and retention.•ZIC-HILIC ...provided higher quality peak shapes but comparable metabolite coverage in human plasma.•Ammonium phosphate addition to plasma samples is not beneficial for either silica or ZIC-HILIC.
To increase metabolome coverage in global LC–MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59–776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.
The ratio between reduced and oxidized thiols, mainly glutathione and oxidized glutathione, is one of the biomarkers for the evaluation of oxidative stress. The accurate measurement of thiol ...concentrations is challenging because reduced thiols are easily oxidized during sample manipulation. Derivatization is commonly used to protect thiols from oxidation. The objective of this work was to systematically compare two cell-permeable derivatizing agents:
N
-ethyl maleimide (NEM) and (
R
)-(+)-
N
-(1-phenylethyl)maleimide (NPEM) in terms of derivatization efficiency, ionization enhancement, side product formation, reaction selectivity for thiols, pH dependence of the reaction, and derivative stability. All thiol measurements and the characterization of side products were performed using a biphenyl reversed phase liquid chromatography–high-resolution mass spectrometry (LC-HRMS). Four thiols, cysteine (CYS), homocysteine,
N
-acetylcysteine (NAC), and glutathione (GSH), were used for the evaluation. Using 1:10 ratio of thiol:derivatizing agent, complete derivatization was obtained within 30 min for both agents tested with the exception of CYS-NEM, where 97% efficiency was obtained. The more hydrophobic NPEM provided better ionization of the thiols, with enhancement ranging from 2.1x for GSH to 5.7x for CYS in comparison to NEM. NPEM derivatization led to more extensive side reactions, such as double derivatization and ring opening, which hindered the accurate measurement of the thiol concentrations. Both NEM and NPEM also showed poor stability of CYS derivative due to its time-dependent conversion to cyclic cysteine-maleimide derivative. Both reagents also showed significant reactivity with amine-containing metabolites depending on the pH used during derivatization, but overall NEM was found to be more selective towards thiol group than NPEM. Taking into account all evaluation criteria, NEM was selected as a more suitable reagent for the thiol protection and derivatization, but strict control of pH 7.0 is recommended to minimize the side reactions. This work illustrates the importance of the characterization of side products and derivative stability during the evaluation of thiol derivatizing agents and contributes fundamental understanding to improve the accuracy of thiol determinations. The key sources of errors during maleimide derivatization include the derivatization of amine-containing metabolites, poor derivative stability of certain thiols (CYS and NAC), and the side reactions especially if ring opening of the reagent is not minimized.
Graphical abstract
This Feature Article highlights some of the key challenges within the field of metabolomics and examines what role separation and analytical sciences can play to improve the use of metabolomics in ...biomarker discovery and personalized medicine. Recent progress in four key areas is highlighted: (i) improving metabolite coverage, (ii) developing accurate methods for unstable metabolites including in vivo global metabolomics methods, (iii) advancing inter-laboratory studies and reference materials and (iv) improving data quality, standardization and quality control of metabolomics studies.
Autophagy, which targets cellular constituents for degradation, is normally inhibited in metabolically replete cells by the metabolic checkpoint kinase mTOR. Although autophagic degradation of ...invasive bacteria has emerged as a critical host defense mechanism, the signals that induce autophagy upon bacterial infection remain unclear. We find that infection of epithelial cells with Shigella and Salmonella triggers acute intracellular amino acid (AA) starvation due to host membrane damage. Pathogen-induced AA starvation caused downregulation of mTOR activity, resulting in the induction of autophagy. In Salmonella-infected cells, membrane integrity and cytosolic AA levels rapidly normalized, favoring mTOR reactivation at the surface of the Salmonella-containing vacuole and bacterial escape from autophagy. In addition, bacteria-induced AA starvation activated the GCN2 kinase, eukaryotic initiation factor 2α, and the transcription factor ATF3-dependent integrated stress response and transcriptional reprogramming. Thus, AA starvation induced by bacterial pathogens is sensed by the host to trigger protective innate immune and stress responses.
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► Intracellular bacteria trigger host amino acid (AA) starvation due to membrane damage ► AA starvation inhibits mTOR, which in-turn activates anti-bacterial autophagy ► Salmonella controls mTOR signaling to escape autophagy ► AA starvation activates GCN2-, eIF2α-, and ATF3-dependent responses
The automation of solid-phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was accomplished using a 96 multiwell plate format, a SPME multifiber device, ...two orbital shakers, and a three-arm robotic system. Extensive optimization of the proposed setup was performed including coating selection, optimization of the fiber coating procedure, confirmation of uniform agitation in all wells, and the selection of the optimal calibration method. The system allows the use of pre-equilibrium extraction times with no deterioration in method precision due to reproducible timing of extraction and desorption steps and reproducible positioning of all fibers within the wells. The applicability of the system for the extraction of several common drugs is demonstrated. The optimized multifiber SPME-LC-MS/MS was subsequently fully validated for the high-throughput analysis of diazepam, lorazepam, nordiazepam, and oxazepam in human whole blood. The proposed method allowed the automated sample preparation of 96 samples in 100 min, which represents the highest throughput of any SPME technique to date, while achieving excellent accuracy (87−113%), precision (≤20% RSD), and sensitivity (limit of quantitation 4 ng/mL). Automated SPME provides unique advantages over automated solid-phase extraction (SPE) including lower cost, the ability to quantitatively determine free and total drug concentrations in a single biofluid sample, and the ability to directly process whole blood samples with absolutely no sample pretreatment required.
A new configuration of C18 thin film extraction phase designed for high sample throughput has been developed and applied to the analysis of benzodiazepines in spiked urine samples using high ...performance liquid chromatography coupled with tandem mass spectrometry. The high throughput analysis was achieved with the use of a robotic autosampler which enabled parallel analyte extraction in a 96-well plate format. Factors affecting data reproducibility, extraction kinetics, sample throughput, and reliability of the system were investigated and optimized. The intrawell reproducibility was 4.5−7.3%, while interwell reproducibility was 7.0−11% in urine and PBS samples. The limits of detection and quantitation were 0.05−0.15 ng/mL and 0.2−2.0 ng/mL for all analytes, respectively. By comparison with optimized automated multifiber SPME relying on rod geometry, the C18 thin films showed higher extraction rates (approximate 2-fold increase) and hence higher sample throughput because of the improved configuration and more effective agitation/mass transfer. In addition, this new configuration provided an extraction phase with greater surface area to volume ratio and greater extraction phase volume, which resulted in approximately 2-fold increase in the extraction capacity for diazepam compared with the extractions with automated multifiber SPME rod geometry. The results of this investigation demonstrated the advantages of using thin films to improve extraction kinetics and sensitivity of automated SPME methods for high performance liquid chromatography.
This protocol describes how to perform automated solid-phase microextraction (SPME) and thin-film microextraction (TFME) in a 96-well plate format for high-throughput analysis of drugs, metabolites ...and any other analytes of interest in biological fluids using liquid chromatography-electrospray tandem mass spectrometry. Sample preparation time required is typically 1 min per sample; hence, the throughput achievable with automated SPME/TFME is comparable with automated 96-well liquid-liquid extraction and solid-phase extraction methods, but greater than most online solid-phase extraction methods. The technique is applicable to complex samples such as whole blood without additional pretreatment. The amount of analyte extracted by SPME/TFME is proportional to the free (unbound) concentration of the analyte; hence, SPME/TFME can be used to determine both total and free concentrations of analytes from a single biofluid sample and to perform automated ligand-receptor binding studies in order to determine binding affinity and/or overall extent of ligand binding to a complex biofluid.
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