Untargeted metabolomics profiling of skeletal muscle samples from malignant hyperthermia susceptible patients.

PURPOSE: Malignant hyperthermia (MH) is a potentially fatal hypermetabolic condition triggered by certain anesthetics and caused by defective calcium homeostasis in skeletal muscle cells. Recent evidence has revealed impairment of various biochemical pathways in MH-susceptible patients in the absence of anesthetics. We hypothesized that clinical differences between MH-susceptible and control individuals are reflected in measurable differences in myoplasmic metabolites. METHODS: We performed metabolomic profiling of skeletal muscle samples from MH-negative (control) individuals and MH-susceptible patients undergoing muscle biopsy for diagnosis of MH susceptibility. Cellular metabolites were extracted from 33 fresh and 87 frozen human muscle samples using solid phase microextraction and Metabolon® untargeted biochemical profiling platforms, respectively. Ultra-performance liquid chromatography-high resolution mass spectrometry was used for metabolite identification and validation, followed by analysis of differences in metabolites between the MH-susceptible and MH-negative groups. RESULTS: Significant fold-change differences between the MH-susceptible and control groups in metabolites from various pathways were found (P value range: 0.009 to < 0.001). These included accumulation of long chain acylcarnitines, diacylglycerols, phosphoenolpyruvate, histidine pathway metabolites, lysophosphatidylcholine, oxidative stress markers, and phosphoinositols, as well as decreased levels of monoacylglycerols. The results from both analytical platforms were in agreement. CONCLUSION: This metabolomics study indicates a shift from utilization of carbohydrates towards lipids for energy production in MH-susceptible individuals. This shift may result in inefficiency of beta-oxidation, and increased muscle protein turnover, oxidative stress, and/or lysophosphatidylcholine levels.

RéSUMé: OBJECTIF : L'hyperthermie maligne (HM) est une condition hypermétabolique potentiellement mortelle déclenchée par certains agents anesthésiques et causée par une homéostasie calcique perturbée des cellules musculaires squelettiques. Des données probantes récentes ont mis en lumière une atteinte de diverses voies biochimiques chez les patients susceptibles à l'HM en l'absence d'anesthésiques. Nous avons émis l'hypothèse que les différences cliniques entre les individus susceptibles à l'HM et des témoins se refléteraient dans des différences mesurables de métabolites myoplasmiques. MéTHODE : Nous avons réalisé un profilage métabolomique d'échantillons de muscles squelettiques provenant de personnes négatives à l'HM (témoins) et de patients susceptibles à l'HM subissant une biopsie musculaire dans le but de poser un diagnostic de susceptibilité à l'HM. Les métabolites cellulaires ont été extraits de 33 échantillons de muscles humains frais et de 87 échantillons congelés à l'aide d'une microextraction en phase solide et des plateformes de profilage biochimique non ciblées Metabolon®, respectivement. La chromatographie en phase liquide à haute performance et la spectrométrie de masse à haute résolution ont été utilisées pour l'identification et la validation des métabolites, puis suivies d'une analyse des différences dans les métabolites entre les groupes susceptibles à l'HM et les groupes négatifs à l'HM. RéSULTATS : Des différences significatives ont été observées entre les groupes susceptibles à l'HM et les groupes témoins dans les métabolites issus de diverses voies (P : de 0,009 à < 0,001). Ces différences comprenaient l'accumulation d'acylcarnitines à longue chaîne, de diacylglycérols, de phosphoénolpyruvate, de métabolites de la voie d'histidine, de lysophosphatidylcholine, de marqueurs de stress oxydatif, et de phosphoinositols, aussi bien que des taux réduits de monoacylglycérols. Les résultats des deux plateformes analytiques concordaient. CONCLUSION : Cette étude métabolomique indique un changement de l'utilisation des glucides vers les lipides pour la production d'énergie chez les personnes susceptibles à l'HM. Ce changement pourrait entraîner une inefficacité de la bêta-oxydation, ainsi qu'une augmentation du renouvellement des protéines musculaires, du stress oxydatif, et/ou des taux de lysophosphatidylcholine.

Development of a solid-phase microextraction LC-MS/MS method for determination of oxidative stress biomarkers in biofluids.

Recently the connection between oxidative stress and various diseases, including cancer and Alzheimer's, attracts notice as a pathway suitable for diagnostic purposes. 8-Oxo-deoxyguanosine and 8-oxo-deoxyadenosine produced from the interaction of reactive oxygen species with DNA become prominent as biomarkers. Several methods have been developed for their determination in biofluids, including solid-phase extraction and enzyme-linked immunosorbent assays. However, still, there is a need for reliable and fast analytical methods. In this context, solid-phase microextraction offers many advantages such as flexibility in geometry and applicable sample volume, as well as high adaptability to high-throughput sampling. In this study, a solid-phase microextraction method was developed for the determination of 8-oxo-deoxyguanosine and 8-oxo-deoxyadenosine in biofluids. The extractive phase of solid-phase microextraction consisted of hydrophilic-lipophilic balanced polymeric particles. In order to develop a solid-phase microextraction method suitable for the determination of the analytes in saliva and urine, several parameters, including desorption solvent, desorption time, sample pH, and ionic strength, were scrutinized. Analytical figures of merit indicated that the developed method provides reasonable interday and intraday precisions (<15% in both biofluids) with acceptable accuracy. The method provides a limit of quantification for both biomarkers at 5.0 and 10.0 ng/mL levels in saliva and urine matrices, respectively.

Multineuromodulator measurements across fronto-striatal network areas of the behaving macaque using solid-phase microextraction.

Different neuromodulators rarely act independent from each other to modify neural processes but are instead coreleased, gated, or modulated. To understand this interdependence of neuromodulators and their collective influence on local circuits during different brain states, it is necessary to reliably extract local concentrations of multiple neuromodulators in vivo. Here we describe results using solid-phase microextraction (SPME), a method providing sensitive, multineuromodulator measurements. SPME is a sampling method that is coupled with mass spectrometry to quantify collected analytes. Reliable measurements of glutamate, dopamine, acetylcholine, and choline were made simultaneously within frontal cortex and striatum of two macaque monkeys (Macaca mulatta) during goal-directed behavior. We find glutamate concentrations several orders of magnitude higher than acetylcholine and dopamine in all brain regions. Dopamine was reliably detected in the striatum at tenfold higher concentrations than acetylcholine. Acetylcholine and choline concentrations were detected with high consistency across brain areas within monkeys and between monkeys. These findings illustrate that SPME microprobes provide a versatile novel tool to characterize multiple neuromodulators across different brain areas in vivo to understand the interdependence and covariation of neuromodulators during goal-directed behavior. Such data would be important to better distinguish between different behavioral states and characterize dysfunctional brain states that may be evident in psychiatric disorders.NEW & NOTEWORTHY Our paper reports a reliable and sensitive novel method for measuring the absolute concentrations of glutamate, acetylcholine, choline, dopamine, and serotonin in brain circuits in vivo. We show that this method reliably samples multiple neurochemicals in three brain areas simultaneously while nonhuman primates are engaged in goal-directed behavior. We further describe how the methodology we describe here may be used by electrophysiologists as a low-barrier-to-entry tool for measuring multiple neurochemicals.

Solid Phase Microextraction-Based Miniaturized Probe and Protocol for Extraction of Neurotransmitters from Brains in Vivo.

Despite the importance of monitoring and correlating neurotransmitter concentrations in the brain with observable behavior and brain areas in which they act, in vivo measurement of multiple neurochemicals in the brain remains a challenge. Here, we propose an alternative solid phase microextraction-based (SPME) chemical biopsy approach as a viable method for acquirement of quantitative information on multiple neurotransmitters by one device within a single sampling event, with multisite measurement capabilities and minimized invasiveness, as no tissue is removed. The miniaturized SPME probe developed for integrated in vivo sampling/sample preparation has been thoroughly optimized with respect to probe shape, desorption solvent, and extracting phase tailored for extraction of small hydrophilic molecules via synthesis and functionalization of the SPME coating. Experimental evaluations of sampling time and storage strategy led to achieving appropriate temporal resolution versus recovery balance as well as little or no analyte loss, respectively. Validation of the developed SPME-HPLC-MS/MS protocol in a surrogate brain matrix yielded satisfactory accuracies of 80-100%, precision below 17%, as well as linear dynamic range and limits of quantitation suitable for determining neurochemicals at physiologically relevant levels. Finally, we present a proof-of-concept in vivo application in macaque brain, where several target neurotransmitters were extracted simultaneously from three brain areas. The developed probe and protocol are herein presented as a potential powerful addition to the existing in vivo toolbox for measurements of local levels of neurochemicals in multiple brain systems implicated in the neuropathology of psychiatric disorders.

Space-Resolved Tissue Analysis by Solid-Phase Microextraction Coupled to High-Resolution Mass Spectrometry via Desorption Electrospray Ionization.

It is hard to overstate the tremendous utility of desorption electrospray ionization (DESI) and its various configurations for rapid and high-throughput analyses or spatially resolved imaging of heterogeneous systems. However, there have been few attempts to employ this technique in spatially resolved mode with solid substrates featuring extractive and analyte-enrichment properties. This study documents the development of a platform that combines solid-phase microextraction (SPME) with desorption electrospray ionization mass spectrometry (DESI-MS) for unidimensional investigation of the heterogeneous distribution of compounds in semisolid systems (i.e., depth profiling across the fiber axis), with the ultimate end of employing it for brain tissue analysis. To this end, a DESI interface and a custom holder accommodating SPME probes were built in house, with the latter contributing to reduction of mechanical sources of signal instability. The system was evaluated through the quantitative reconstruction of the laminar and radial concentration gradients of xenobiotics introduced in multilayer gel arrangements and surrogate brain tissue models. Good quantitative capability was achieved by employing a strategy that combined signal correction via preloading internal standard onto SPME fibers and signal integration in scan-by-scan mode. The proposed technique's suitability for characterizing more complex systems, such as rat brains ex vivo, was also evaluated. The proposed approach allows for fast and noninvasive probing of three-dimensional objects without the need for their slicing, and the space-resolved mode reduces the number of required probe insertions, allowing in vivo applications. We foresee suitability of this setup for examining the spatial patterns of local drug release in the brain and the extent of the resultant physiological responses.

In Vivo Brain Sampling Using a Microextraction Probe Reveals Metabolic Changes in Rodents after Deep Brain Stimulation.

Brain metabolomics is an emerging field that complements the more traditional approaches of neuroscience. However, typical brain metabolomics workflows require that animals be sacrificed and tend to involve tedious sample preparation steps. Microdialysis, the standard technique to study brain metabolites in vivo, is encumbered by significant limitations in the analysis of hydrophobic metabolites, which are prone to adsorption losses on microdialysis equipment. An alternative sampling method suitable for in vivo brain studies is solid-phase microextraction (SPME). In SPME, a small probe coated with a biocompatible polymer is employed to extract/enrich analytes from biological matrices. In this work, we report the use of SPME and liquid chromatography-mass spectrometry for untargeted in vivo analysis of rodent's brains after deep brain stimulation (DBS). First, metabolite changes occurring in brain hippocampi after application of 3 h of DBS to the animals' prefrontal cortex were monitored with the proposed approach. As SPME allows for nonlethal sampling, the same group of animals was sampled again after 8 days of daily DBS therapy. After acute DBS, we detected changes in a broad range of metabolites, including the amino acid citrulline, which may reflect changes in nitric oxide production, as well as various phospho- and glycosphingolipids. Measurements conducted after chronic DBS showed a decrease in hippocampal corticosterone, indicating that DBS may have a regulatory effect in the hypothalamic-pituitary-adrenal axis. Our findings demonstrate the potential of in vivo SPME as a tool of scientific and clinical interest capable of revealing changes in a wide range of metabolites in brain tissue.

New Generation of Solid-Phase Microextraction Coatings for Complementary Separation Approaches: A Step toward Comprehensive Metabolomics and Multiresidue Analyses in Complex Matrices.

In this work, a new generation of solid-phase microextraction (SPME) coatings based on polytetrafluoroethylene amorphous fluoroplastics (PTFE AF 2400) as a particle binder is presented. The developed coating was tested for thermal and solvent-assisted desorption, demonstrating its compatibility with both gas- and liquid-chromatographic platforms. The incorporation of hydrophilic-lipophilic balance (HLB) adsorptive particles provided optimal extraction coverage for analytes bearing a broad range of hydrophobicities and molecular weights and of varied chemical diversity. The performance of the newly developed coating was compared to already established coatings based on different polymers such as divinylbenzene/carboxen/polydimethylsiloxane (DVB/Car/PDMS) and octadecyl/benzenesulfonic acid/polyacrylonitrile (C18/SCX/PAN) in order to assess the new prototype versus the existing technology. As this is the first documented instance of PTFE AF being used as a particle immobilizer for SPME, an assessment of the analyte uptake rate and extraction capability of the developed coating was carried out in comparison to other conventionally used polymers. Moreover, the new SPME probes were used to validate an analytical method for determination of banned doping substances, achieving limits of quantitation below the minimum required performance limits (MRPLs) set by the World Anti-Doping Agency (WADA) for most compounds. Considering the broad coverage of the coating in terms of analytes extracted and its suitability for both thermal- and solvent-assisted desorption, these new SPME probes will properly suit various metabolomics applications that involve the use of both gas- and liquid-chromatography.

High-Throughput Screening and Quantitation of Target Compounds in Biofluids by Coated Blade Spray-Mass Spectrometry.

Most contemporary methods of screening and quantitating controlled substances and therapeutic drugs in biofluids typically require laborious, time-consuming, and expensive analytical workflows. In recent years, our group has worked toward developing microextraction (µe)-mass spectrometry (MS) technologies that merge all of the tedious steps of the classical methods into a simple, efficient, and low-cost methodology. Unquestionably, the automation of these technologies allows for faster sample throughput, greater reproducibility, and radically reduced analysis times. Coated blade spray (CBS) is a µe technology engineered for extracting/enriching analytes of interest in complex matrices, and it can be directly coupled with MS instruments to achieve efficient screening and quantitative analysis. In this study, we introduced CBS as a technology that can be arranged to perform either rapid diagnostics (single vial) or the high-throughput (96-well plate) analysis of biofluids. Furthermore, we demonstrate that performing 96-CBS extractions at the same time allows the total analysis time to be reduced to less than 55 s per sample. Aiming to validate the versatility of CBS, substances comprising a broad range of molecular weights, moieties, protein binding, and polarities were selected. Thus, the high-throughput (HT)-CBS technology was used for the concomitant quantitation of 18 compounds (mixture of anabolics, ß-2 agonists, diuretics, stimulants, narcotics, and ß-blockers) spiked in human urine and plasma samples. Excellent precision (∼2.5%), accuracy (≥90%), and linearity (R2 ≥ 0.99) were attained for all the studied compounds, and the limits of quantitation (LOQs) were within the range of 0.1 to 10 ng·mL-1 for plasma and 0.25 to 10 ng·mL-1 for urine. The results reported in this paper confirm CBS's great potential for achieving subsixty-second analyses of target compounds in a broad range of fields such as those related to clinical diagnosis, food, the environment, and forensics.

Deposition of a Sorbent into a Recession on a Solid Support To Provide a New, Mechanically Robust Solid-Phase Microextraction Device.

To date, solid-phase microextraction (SPME) fibers used for in vivo bioanalysis can be too fragile and flexible, which limits suitability for direct tissue sampling. As a result, these devices often require a sheathing needle to prepuncture robust sample matrixes and protect the extraction phase from mechanical damage. To address this limitation, a new SPME device is herein presented which incorporates an extraction phase recessed into the body of a solid needle. This device requires no additional support or shielding during puncture events through protective tissue. The presented device was thoroughly tested, being fired at 90 m·s-1 through fish scales, forced through vial septa, and employed in a targeted study of polyunsaturated fatty acids in salmon where the protective outer skin was repetitively punctured during sampling. Finally, the recessed SPME device was applied to an on-site application for the tissue analysis of wild muskellunge. With this advancement, rapid, minimally invasive, and easily executed in vivo SPME is now possible opening the door to near endless sampling opportunities.

Time Weighted Average Concentration Monitoring Based on Thin Film Solid Phase Microextraction.

Time weighted average (TWA) passive sampling with thin film solid phase microextraction (TF-SPME) and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for collection, identification, and quantification of benzophenone-1, benzophenone-2, benzophenone-3, benzophenone-4, 2-phenylbenzimidazole-5-sulfonic acid, octocrylene, octylmethoxycinnamate, butylmethoxydibenzoylmethane, triclocarban and triclosan in the aquatic environment. Two types of TF-SPME passive samplers, including a retracted thin film device using a hydrophilic lipophilic balance (HLB) coating, and an open bed configuration with an octadecyl silica-based (C18)coating, were evaluated in an aqueous standard generation system. Laboratory calibration results indicated that the thin film retracted device using HLB coating is suitable to determine TWA concentrations of polar analytes in water, with an uptake that was linear up to 70 days. In open bed form, a one-calibrant kinetic calibration technique was accomplished by loading benzophenone3-d5 as calibrant on the C18 coating to quantify all nonpolar compounds. The experimental results showed that the one-calibrant kinetic calibration technique can be used for determination of classes of compounds in cases where deuterated counterparts are either not available or expensive. The developed passive samplers were deployed in wastewater-dominated reaches of the Grand River (Kitchener, ON) to verify their feasibility for determination of TWA concentrations in on-site applications. Field trials results indicated that these devices are suitable for long-term and short-term monitoring of compounds varying in polarity, such as UV blockers and biocide compounds in water, and the data were in good agreement with literature data.

Rapid and Concomitant Analysis of Pharmaceuticals in Treated Wastewater by Coated Blade Spray Mass Spectrometry.

The widespread use of pharmaceuticals in both human and animal populations, and the resultant contamination of surface waters from the outflow of water treatment facilities is an issue of growing concern. This has raised the need for analytical methods that can both perform rapid sample analysis and overcome the limitations of conventional analysis procedures, such as multistep workflows and tedious procedures. Coated blade spray (CBS) is a solid-phase microextraction based technique that enables the direct-to-mass-spectrometry analysis of extracted compounds via the use of limited organic solvent to desorb analytes and perform electrospray ionization. This paper documents how CBS can be applied for the concomitant tandem mass spectrometric (MS/MS) analysis of nine pharmaceuticals in treated wastewater. The total analysis times of less than 11 min provided limits of detection lower than 50 ng L-1 for all target compounds in river water. The CBS methodology was then compared to a conventional solid-phase extraction technique for the analysis of the final effluent of six wastewater treatment facilities. The experimental results strongly suggest that CBS offers scientists a viable alternative method for analyzing water samples that is both rapid and relatively solvent-free.

Development of a Carbon Mesh Supported Thin Film Microextraction Membrane As a Means to Lower the Detection Limits of Benchtop and Portable GC/MS Instrumentation.

In this work, a durable and easy to handle thin film microextraction (TFME) device is reported. The membrane is comprised of poly(divinylbenzene) (DVB) resin particles suspended in a high-density polydimethylsiloxane (PDMS) glue, which is spread onto a carbon fiber mesh. The currently presented membrane was shown to exhibit a substantially lesser amount of siloxane bleed during thermal desorption, while providing a statistically similar extraction efficiency toward a broad spectrum of analytes varying in polarity when compared to an unsupported DVB/PDMS membrane of similar shape and size which was prepared with previously published methods. With the use of hand-portable GC-TMS instrumentation, membranes cut with dimensions 40 mm long by 4.85 mm wide and 40 ± 5 µm thick (per side) were shown to extract 21.2, 19.8, 18.5, 18,4, 26.8, and 23.7 times the amount of 2,4 dichlorophenol, 2,4,6 trichlorophenol, phorate D10, fonofos, chloropyrifos, and parathion, respectively, within 15 min from a 10 ppb aqueous solution as compared to a 65 µm DVB/PDMS solid phase microextraction (SPME) fiber. A portable high volume desorption module prototype was also evaluated and shown to be appropriate for the desorption of analytes with a volatility equal to or lesser than benzene when employed in conjunction with TFME membranes. Indeed, the coupling of these TFME devices to hand-portable gas chromatography toroidial ion trap mass spectrometry (GC-TMS) instrumentation was shown to push detection limits for these pesticides down to the hundreds of ppt levels, nearing that which can be achieved with benchtop instrumentation. Where these membranes can also be coupled to benchtop instrumentation it is reasonable to assume that detection limits could be pushed down even further. As a final proof of the concept, the first ever, entirely on-site TFME-GC-TMS analysis was performed at a construction impacted lake. Results had indicated the presence of contaminants such as toluene, ethylbenzene, xylene, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, and tris(1-chloro-2-propyl)phosphate, which stood out from other naturally occurring compounds detected.

Fast Quantitation of Target Analytes in Small Volumes of Complex Samples by Matrix-Compatible Solid-Phase Microextraction Devices.

Herein we report the development of solid-phase microextraction (SPME) devices designed to perform fast extraction/enrichment of target analytes present in small volumes of complex matrices (i.e. V≤10 µL). Micro-sampling was performed with the use of etched metal tips coated with a thin layer of biocompatible nano-structured polypyrrole (PPy), or by using coated blade spray (CBS) devices. These devices can be coupled either to liquid chromatography (LC), or directly to mass spectrometry (MS) via dedicated interfaces. The reported results demonstrated that the whole analytical procedure can be carried out within a few minutes with high sensitivity and quantitation precision, and can be used to sample from various biological matrices such as blood, urine, or Allium cepa L single-cells.

Micelle assisted thin-film solid phase microextraction: a new approach for determination of quaternary ammonium compounds in environmental samples.

Determination of quaternary ammonium compounds (QACs) often is considered to be a challenging undertaking owing to secondary interactions of the analytes' permanently charged quaternary ammonium head or hydrophobic tail with the utilized labware. Here, for the first time, a micelle assisted thin-film solid phase microextraction (TF-SPME) using a zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) as a matrix modifier is introduced as a novel approach for in-laboratory sample preparation of the challenging compounds. The proposed micelle assisted TF-SPME method offers suppression/enhancement free electrospray ionization of analytes in mass spectrometric detection, minimal interaction of the micelles with the TF-SPME coating, and chromatographic stationary phase and analysis free of secondary interactions. Moreover, it was found that the matrix modifier has multiple functions; when its concentration is found below the critical micelle concentration (CMC), the matrix modifier primarily acts as a surface deactivator; above its CMC, it acts as a stabilizer for QACs. Additionally, shorter equilibrium extraction times in the presence of the modifier demonstrated that micelles also assist in the transfer of analytes from the bulk of the sample to the surface of the coating. The developed micelle assisted TF-SPME protocol using the 96-blade system requires only 30 min of extraction and 15 min of desorption. Together with a conditioning step (15 min), the entire method is 60 min; considering the advantage of using the 96-blade system, if all the blades in the brush are used, the sample preparation time per sample is 0.63 min. Moreover, the recoveries for all analytes with the developed method were found to range within 80.2-97.3%; as such, this method can be considered an open bed solid phase extraction. The proposed method was successfully validated using real samples.

Thin-film microextraction coupled to LC-ESI-MS/MS for determination of quaternary ammonium compounds in water samples.

The dual nature of the quaternary ammonium compounds, having permanently charged hydrophilic quaternary ammonium heads and long-chain hydrophobic tails, makes the sample preparation step and analysis of these compounds challenging. A high-throughput method based on thin-film solid-phase microextraction (SPME) and liquid chromatography mass spectrometry was developed for simultaneous quantitative analysis of nine benzylic and aliphatic quaternary ammonium compounds. Chromatographic separation and detection of analytes were obtained in reverse-phase mode in 8 min using a triple quadrupole mass spectrometer. Hydrophilic lipophilic balance particle-coated blades were found to be the most suitable among the different coatings tested in terms of recoveries and carryover on the blades. For desorption solvents, 70/30, v/v (A/B) with 0.1 % formic acid (where A is 10 mM ammonium acetate in acetonitrile/water (95/5 , v/v) and B is 0.1 % (v/v) formic acid in isopropyl alcohol) was shown to be the most efficient solvent for the desorption of the analytes from the SPME sorbent. The SPME method was optimised in terms of extraction, pH, and preconditioning, as well as extraction and desorption times. Optimum conditions were 45 min of extraction time and 15 min of desorption time, all with agitation. The extraction was found to be optimum in a range of pH 6.0 to 8.0, which is consistent with the natural pH of water samples. Wide linear dynamic ranges with the developed method were obtained for each compound, enabling the application of the method for a wide range of concentrations. The developed method was validated according to the Food and Drug Administration criteria. The proposed method is the first SPME-based approach describing the applicability of the high-throughput thin-film SPME in a 96-well system for analysis of such challenging compounds.

Development and functionalization of electrospun fiber coated thin film microextraction devices for rapid mass spectrometric determination of biologically important polar molecules.

Rapid diagnosis of diseases is one of the challenging areas in clinical research. From the analytical chemist's perspective, the main challenges are isolating the compounds from the bio-specimen and lengthy analysis times. In this regard, solid phase microextraction offers a platform to address the abovementioned challenges. Moreover, its sharp tip-thin film geometry, known as coated blade spray (CBS), can enhance the extraction and act as an ionization source in direct mass spectrometric analysis. In this study, a new CBS device specifically designed for polar analytes was prepared and optimized to determine urinary metabolites. For this purpose, polyacrylonitrile (PAN) was selected as a base polymer as it can be electrospun to form a nanofibrous structure, and it can be modified with weak ion exchange moieties to interact with polar analytes. Following the electrospinning of PAN, hydrolysis was optimized, and conditions leading to sufficient extraction enhancement without dissolving the polymer were obtained when probes were treated with 5.0 M of NaOH for 2.5 h. Using the coated blades prepared as explained, the evaluation of various extraction conditions showed that 5 min is sufficient for equilibrium extraction. In addition, the solution's ionic strength and pH significantly affect the extraction. Optimum sorption was obtained at no salt added and pH 7.0 conditions. The CBS-MS optimization showed that 10.0 µL of ACN/MeOH/H2O (40:40:20, v/v/v) with formic acid kept for 15 seconds on the blade before voltage application leads to the highest signal. The limits of quantification of the analytes are between 50 and 100 ng/mL.

Novel electrospun-based extractive probes for rapid determination of clinically important compounds in human plasma.

BACKGROUND: Coated blade spray (CBS) represents an innovative approach that utilizes solid-phase microextraction principles for sampling and sample preparation. When combined with ambient mass spectrometry (MS), it can also serve as an electrospray ionization source. Therefore, it became a promising tool in analytical applications as it can significantly reduce the analysis time. However, the current CBS coatings are based on the immobilization of extractive particles in bulk polymeric glue, which constrains the diffusion of the analytes to reach the extractive phase; therefore, the full reward of the system cannot be taken at pre-equilibrium. This has sparked the notion of developing new CBS probes that exhibit enhanced kinetics. RESULTS: With this aim, to generate a new extractive phase with improved extraction kinetics, poly(divinylbenzene) (PDVB) nanoparticles were synthesized by mini-emulsion polymerization and then immobilized into sub-micrometer (in diameter) sized polyacrylonitrile fibers which were obtained by electrospinning method. Following the optimization and characterization studies, the electrospun-coated blades were used to determine cholesterol, testosterone, and progesterone in plasma spots using the CBS-MS approach. For testosterone and progesterone, 10 ng mL-1 limits of quantification could be obtained, which was 200 ng mL-1 for cholesterol in spot-sized samples without including any pre-treatment steps to samples prior to extraction. SIGNIFICANCE: The comparison of the initial kinetics for dip-coated and electrospun-coated CBS probes proved that the electrospinning process could enhance the extraction kinetics; therefore, it can be used for more sensitive analyses. The total analysis time with this method, from sample preparation to instrumental analysis, takes only 7 min, which suggests that the new probes are promising for fast diagnostic applications.

A molecularly imprinted polymer as solid phase extraction sorbent for ketoprofen determination in water and artificial serum prior to HPLC.

Ketoprofen (KET) is an active pharmaceutical compound that has pain relieving and antipyretic effects. Its determination in body fluids and environmental waters is important due to widespread use of the compound. In this study, a selective and reliable method has been developed for the determination of ketoprofen in water and artificial serum using molecularly imprinted polymers (MIPs) as a solid phase extraction sorbent prior to HPLC-DAD detection. The MIP was synthesized by copolymerization of methacrylic acid (MAA) and trimethylpropane trimethacrylate (TRIM) in the presence of ketoprofen as the template. For the sake of comparison, nonimprinted polymer (NIP) was also synthesized under the same experimental conditions without the addition of ketoprofen under the same experimental conditions. Critical extraction parameters such as sample pH, shaking time and sorbent amount were optimized and adjusted to 8.0, 24 h, and 10.0 mg, respectively, for a sample volume of 10.0 mL. MIP showed higher selectivity than NIP towards ketoprofen in an artificial matrix containing another pain relieving drug, ibuprofen, and a cardiovascular drug, metoprolol. The proposed method was successfully applied for the detection of ketoprofen in spiked drinking water, tap water, and artificial serum samples, and showed satisfactory results with respective recoveries of 96.8 % (± 0.8), 93.7% (± 0.6), 62.2% (± 0.6), and 69.9% (± 0.6).

Assessment of solid phase microextraction as a sample preparation tool for untargeted analysis of brain tissue using liquid chromatography-mass spectrometry.

This work presents an evaluation of solid-phase microextraction (SPME) SPME in combination with liquid chromatography-high resolution mass spectrometry (LC-HRMS) as an analytical approach for untargeted brain analysis. The study included a characterization of the metabolite coverage provided by C18, mixed-mode (MM, with benzene sulfonic acid and C18 functionalities), and hydrophilic lipophilic balanced (HLB) particles as sorbents in SPME coatings after extraction from cow brain homogenate at static conditions. The effects of desorption solvent, extraction time, and chromatographic modes on the metabolite features detected were investigated. Method precision and absolute matrix effects were also assessed. Among the main findings of this work, it was observed that all three tested coating chemistries were able to provide comparable brain tissue information. HLB provided higher responses for polar metabolites; however, as these fibers were prepared in-house, higher inter-fiber relative standard deviations were also observed. C18 and HLB coatings offered similar responses with respect to lipid-related features, whereas MM and C18 provided the best results in terms of method precision. Our results also showed that the use of methanol is essential for effective desorption of non-polar metabolites. Using a reversed-phase chromatographic method, an average of 800 and 1200 brain metabolite features detected in positive and negative modes, respectively, met inter-fibre RSD values below 30% (n=4) after removal of fibre and solvent artefacts from the associated datasets. For features detected using a lipidomics method, a total of 900 and 1800 features detected using C18 fibers in positive and negative mode, respectively, met the same criteria. In terms of absolute matrix effects, the majority of the model metabolites tested showed values between 80 and 120%, which are within the acceptable range. Overall, the findings of this work lay the foundation for further optimization of parameters for SPME-LC-HRMS methods suitable for in vivo and ex vivo brain (and other tissue) untargeted studies, and support the applicability of this approach for non-destructive tissue metabolomics.