Headspace versus Direct Immersion Solid Phase Microextraction in Complex Matrixes: Investigation of Analyte Behavior in Multicomponent Mixtures

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b01850This work aims to investigate the behavior of analytes in complex mixtures and matrixes with the use of solid-phase microextraction (SPME). Various factors that influence analyte uptake such as coating chemistry, extraction mode, the physicochemical properties of analytes, and matrix complexity were considered. At first, an aqueous system containing analytes bearing different hydrophobicities, molecular weights, and chemical functionalities was investigated by using commercially available liquid and solid porous coatings. The differences in the mass transfer mechanisms resulted in a more pronounced occurrence of coating saturation in headspace mode. Contrariwise, direct immersion extraction minimizes the occurrence of artifacts related to coating saturation and provides enhanced extraction of polar compounds. In addition, matrix-compatible PDMS-modified solid coatings, characterized by a new morphology that avoids coating fouling, were compared to their nonmodified analogues. The obtained results indicate that PDMS-modified coatings reduce artifacts associated with coating saturation, even in headspace mode. This factor, coupled to their matrix compatibility, make the use of direct SPME very practical as a quantification approach and the best choice for metabolomics studies where wide coverage is intended. To further understand the influence on analyte uptake on a system where additional interactions occur due to matrix components, ex vivo and in vivo sampling conditions were simulated using a starch matrix model, with the aim of mimicking plant-derived materials. Our results corroborate the fact that matrix handling can affect analyte/matrix equilibria, with consequent release of high concentrations of previously bound hydrophobic compounds, potentially leading to coating saturation. Direct immersion SPME limited the occurrence of the artifacts, which confirms the suitability of SPME for in vivo applications. These findings shed light into the implementation of in vivo SPME strategies in quantitative metabolomics studies of complex plant-based systems.The Natural Sciences and Engineering Research Council of Canada (NSERC) Sigma-Aldrich Corporatio

Advances in solid-phase microextraction as sample preparation method for food analysis.

Within all steps involved in the analytical process, sample preparation is considered the most time-consuming step. Therefore, substantial efforts have focused on the search for automated sample preparation strategies that minimize sample handling and errors associated with human interference. Solid phase microextraction (SPME) addresses well the necessity for simple and automated sample preparation, with the integration of sampling, extraction, clean up and instrumental introduction into a single step. In SPME, selective extraction of compounds takes place based on the degree of distribution of the analyte between the SPME coating and the sample matrix. For this reason, the correct choice of SPME coating for a given application has great influence on the acquisition of reliable analytical data. In spite of its great potential, the implementation of SPME in the analysis of complex matrices, such as food, has been hindered by the lack of suitable SPME coatings that possess compatibility with complex matrices while maintaining sufficient sensitivity for trace applications. The main problem resides in the fact that the most matrix compatible coating, PDMS, has limited extraction efficiency towards less hydrophobic analytes, whereas the coating that exhibits best extraction efficiency towards pesticides, in general, is PDMS/DVB. PDMS/DVB as a solid coating suffers from the attachment of matrix components onto the coating surface, known as fouling. Fouling does not only considerably shorten coating reusability, but it also causes significant changes in extraction efficiency, skewing the reliability of the data obtained. Therefore, in this thesis, a new approach to fabricate a matrix-compatible SPME coating for GC-based analysis of food matrices is presented. The developed matrix-compatible coating was evaluated for its reusability in complex matrices, namely grape pulp and Concord grape juice, as well as for its extraction capabilities towards various analytes bearing different physicochemical properties. First, a method to impart matrix-compatibility to commercially available solid SPME coatings was developed. The method consists of applying a thin layer of PDMS onto the solid coating, in this case PDMS/DVB. The main premise behind this approach was to create a coating that presents the matrix compatibility of PDMS, while maintaining the sensitivity obtained with PDMS/DVB. The reusability of the obtained PDMS-modified coating was evaluated in grape pulp, and rewarding results were obtained since the coating could be reused for over 100 extractions. Moreover, the PDMS-modified coating presented a similar extraction efficacy to that presented by the original PDMS/DVB coating towards the triazole pesticides, used as model analytes. The developed PDMS-modified coating was then employed to develop a simple and fast DI-SPME-GC-ToFMS method for determination of ten triazole fungicides in grapes and strawberries. The method was successfully validated, and the figures of merit obtained with the SPME method were compared to those obtained with the QuEChERS method. The limits of quantitation reached by SPME were at least one order of magnitude lower than those achieved by the QuEChERS method, whereas precision and accuracy were comparable for both methods. Subsequently, given the vast option of commercial PDMS blends available, different types of PDMS were compared for their reusability in complex matrices, and parameters associated with the PDMS-overcoated fiber fabrication were investigated in regards to their effect on fiber longevity. Results showed that the long-term reusability of such coatings is a function of the coating’s fabrication process, such as achievement of smooth and uniform PDMS surface, and sealing of both fiber ends by PDMS layer. Regarding PDMS type, best results were obtained with Sylgard ® 184. Since one of the most important branches of food analysis involves the simultaneous analysis of pesticides with a wide range of polarities and from different classes, the PDMS-modified coating was evaluated for the extraction of analytes of different polarities (log P = 1.43 to 6) from water samples in order to understand the mass transfer of analytes within the PDMS outer layer during the mass uptake process. Results showed that for hydrophobic analytes, the kinetics of extraction of the PDMS-modified coating are quite similar to that of the original PDMS/DVB. However, for more polar analytes, the rate-limiting step is the diffusion through the coating; therefore, the PDMS layer affects the kinetic uptake. The main implication of these results is quite evident if a method aiming at simultaneous determinations of both polar and non-polar analytes is to be developed, such as is the case in multiclass pesticide analysis, since the sensitivity of the method at too short extraction times might not be enough for polar analytes. Finally, once the PDMS-overcoated fibers were proven to be robust and compatible for use in fruit pulp, the DI-SPME-ToFMS method for multiresidue pesticide determination in grapes was developed and SPME parameters that can affect extraction efficiency were optimized via multivariate methods. Despite a thorough investigation during optimization, the most polar pesticides, acephate and omethoate, could not be detected. Next, a careful evaluation of internal standards was presented and attentively discussed. The results showed that two pairs of internal standards, interchangeable amongst them (i.e. only two internal standards were needed) were sufficient to ensure reliable, precise and accurate analytical data. Interestingly, only two internal standards at the time were needed, and among the choices presented, the use of non-deuterated compounds presents an affordable, cost-effective solution for the method. Next, the method was fully validated for 40 pesticides in compliance to EU/SANCO requirements (R2 > 0.995, RSD < 20%, and 80% < accuracy < 120%). The validated method exhibited excellent performance for pesticides such as chlorothalonil, dicofol, and folpet, which are considered the weak link in QuEChERS-based multiresidue methods. Pyrethroid pesticides were not validated due to their non-specific adsorption onto the vial walls. For pyrethroids, a solvent pre-extraction step should be incorporated in order to avoid losses due to the interaction of these compounds with glassware. Overall, despite the challenges and limitations encountered, it is evident that the practical aspects of the PDMS-modified coating demonstrated in this thesis create new opportunities for SPME applied in food analysis.1 yea

Capturing Plant Metabolome with Direct-Immersion in Vivo Solid Phase Microextraction of Plant Tissues

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/acs.analchem.5b03684 .For the first time, an in vivo sampling mode of direct immersion–solid phase microextraction (DI-SPME) was employed to capture the metabolome of living plant specimens, using apple (Malus × domestica Borkh.) as a model system. Metabolites were extracted from apple tissues and introduced by thermal desorption into a comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry instrument. The feasibility of this sampling approach, based on exploitation of microextraction principles, including negligible depletion of free analyte concentrations, solventless sampling and sample preparation, and on-site compatibility, was determined in global metabolite analysis. Rather than adopting an approach of traditional sample preparation, requiring metabolism quenching and laborious sample preparation, the objective of the study was to capture the metabolome in vivo, evaluate the feasibility of the approach to provide unbiased extraction coverage, and compare analytical precision when different SPME sampling modes are employed. The potential of in vivo DI-SPME in quantitative plant metabolomics was assessed by evaluating changes in metabolic fingerprints in response to fruit maturation. The in vivo SPME sampling approach has been demonstrated as capable of sampling living systems with high reproducibility, considering that nearly 50% of hundreds of evaluated compounds included in the determination of analytical performance met the 15% RSD FDA criterion. Esters were extracted with high repeatability (% RSD for hexyl butanoate and butyl butanoate of 16.5 and 5.9, respectively, from 9 determinations in 3 apples) and found to be upregulated in response to apple fruit maturation.Natural Sciences and Engineering Research Council of Canada (NSERC

Methodical evaluation and improvement of matrix compatible PDMS-overcoated coating for direct immersion solid phase microextraction gas chromatography (DI-SPME-GC)-based applications

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.aca.2016.03.015 © 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/The main quest for the implementation of direct SPME to complex matrices has been the development of matrix compatible coatings that provide sufficient sensitivity towards the target analytes. In this context, we present here a thorough evaluation of PDMS-overcoated fibers suitable for simultaneous extraction of different polarities analytes, while maintaining adequate matrix compatibility. For this, eleven analytes were selected, from various application classes (pesticides, industrial chemicals and pharmaceuticals) and with a wide range of log P values (ranging from 1.43 to 6). The model matrix chosen was commercial Concord grape juice, which is rich in pigments such as anthocyanins, and contains approximately 20% of sugar (w/w). Two types of PDMS, as well as other intrinsic factors associated with the PDMS-overcoated fiber fabrication are studied. The evaluation showed that the PDMS-overcoated fibers considerably slowed down the coating fouling process during direct immersion in complex matrices of high sugar content. Longevity differences could be seen between the two types of PDMS tested, with a proprietary Sylgard (R) giving superior performance because of lesser amount of reactive groups and enhanced hydrophobicity. Conversely, the thickness of the outer layer did not seem to have a significant effect on the fiber lifetime. We also demonstrate that the uniformity of the overcoated PDMS layer is paramount to the achievement of reliable data and extended fiber lifetime. Employing the optimum overcoated fiber, limits of detection (LOD) in the range of 0.2-1.3 ng/g could be achieved. Additional improvement is attainable by introducing washing of the coatings after desorption, so that any carbon build-up (fouling) left on the coating surface after thermal desorption can be removed.Natural Sciences and Engineering Research Council of Canada (NSERC

Coupling solid phase microextraction to complementary separation platforms for metabotyping of E. coli metabolome in response to natural antibacterial agents

The final publication is available at Springer via http://dx.doi.org/10.1007/s11306-016-1111-9Introduction Essential oils are known to possess antimicrobial activity; thus, their use has played an important role over the years in medicine and for food preservation purposes. Objective The effect of clove oil and its major constituents as bactericidal agents on the global metabolic profiling of E. coli bacteria was assessed by means of metabolic alterations, using solid phase microextraction (SPME) as a sample preparation method coupled to complementary analytical platforms. Method E. Coli cultures treated with clove oil and its major individual components were sampled by HS-SPME-GCxGC-ToF/MS and SPME-UPLC-MS. Full factorial design was applied in order to estimate the most effective antibacterial agent towards E. coli. Central composite design and factorial design were applied to investigate parameters influencing metabolite coverage and efficiency by SPME. Results The metabolic profile, including 500 metabolites identified by LC-MS and 789 components detected by GCxGC-ToF/MS, 125 of which were identified as dys-egulated metabolites, revealed changes in the metabolome provoked by the antibacterial activity of clove oil, and in particular its major constituent eugenol. Analyses of individual components selected using orthogonal projections to latent structures discriminant analysis showed a neat differentiation between control samples in comparison to treated samples in various sets of metabolic pathways. Conclusions The combination of a sample preparation method capable of providing cleaner extracts coupled to different analytical platforms was successful in uncovering changes in metabolic pathways associated with lipids biodegradation, changes in the TCA cycle, amino acids, and enzyme inhibitors in response to antibacterial treatment.Natural Sciences and Engineering Research Council (NSERC) of CanadaCNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Brazi

Exploiting the tunable selectivity features of polymeric ionic liquid-based SPME sorbents in food analysis

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.talanta.2018.06.011 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/In this work, the performances of polymeric ionic liquid (PIL) based solid-phase microextraction (SPME) coatings were assessed for applications concerning food safety and quality. Two different polymeric ionic liquid coatings, namely poly(1–4-vinylbenzyl-3-hexadecylimidazolium) bis[(trifluoromethyl)sulfonyl] imide (poly([VBHDIM][NTf2]), PIL 1, and N,N-didecyl-N-methyl-d-glucaminium poly(2-methyl-acrylic acid 2-[1-(3-{2-[2-(3-trifluoromethanesulfonylamino-propoxy)-ethoxy]-ethoxy}-propylamino)-vinylamino]-ethyl ester) (poly([DDMGlu][MTFSI]), PIL 2, were evaluated. The PIL-based coatings were compared to commercially available SPME coatings in terms of their performance toward extraction of pesticides and fruit metabolites. The partition coefficients (Kfs) of the tested coatings were calculated, with PIL 1 demonstrating similar or better performance compared to the commercial coatings. Design of experiment (DoE) was applied to optimize the parameters that most influenced SPME extraction, and a quantitative method for determination of 5 organophosphorus pesticides was developed by using PIL-based coatings and commercial SPME fibers. Despite the thin layer of the sorbent coating, PIL 1 achieved limits of quantitation at the low part-per-billion level. Moreover, in a comparative investigation of analyte coverage carried out via HS-SPME-GCxGC-ToF/MS with grape homogenate as model matrix, excellent performances were observed for the PIL-based coatings toward the determination of fruit metabolites, demonstrating their capability towards broad extractive coverage of analytes characterized by various physicochemical properties.Natural Sciences and Engineering Research Council of Canada ["IRCPJ 184412-15"]National Science Foundation ["CHE-1709372"]

In Vitro and In Vivo Investigation of the Efficacy of Arylimidamide DB1831 and Its Mesylated Salt Form - DB1965 - against Trypanosoma cruzi Infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA - DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T.cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC50 value/48 h of 5–40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T.cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T.cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole

Many Labs 5:Testing pre-data collection peer review as an intervention to increase replicability

Replication studies in psychological science sometimes fail to reproduce prior findings. If these studies use methods that are unfaithful to the original study or ineffective in eliciting the phenomenon of interest, then a failure to replicate may be a failure of the protocol rather than a challenge to the original finding. Formal pre-data-collection peer review by experts may address shortcomings and increase replicability rates. We selected 10 replication studies from the Reproducibility Project: Psychology (RP:P; Open Science Collaboration, 2015) for which the original authors had expressed concerns about the replication designs before data collection; only one of these studies had yielded a statistically significant effect (p < .05). Commenters suggested that lack of adherence to expert review and low-powered tests were the reasons that most of these RP:P studies failed to replicate the original effects. We revised the replication protocols and received formal peer review prior to conducting new replication studies. We administered the RP:P and revised protocols in multiple laboratories (median number of laboratories per original study = 6.5, range = 3?9; median total sample = 1,279.5, range = 276?3,512) for high-powered tests of each original finding with both protocols. Overall, following the preregistered analysis plan, we found that the revised protocols produced effect sizes similar to those of the RP:P protocols (?r = .002 or .014, depending on analytic approach). The median effect size for the revised protocols (r = .05) was similar to that of the RP:P protocols (r = .04) and the original RP:P replications (r = .11), and smaller than that of the original studies (r = .37). Analysis of the cumulative evidence across the original studies and the corresponding three replication attempts provided very precise estimates of the 10 tested effects and indicated that their effect sizes (median r = .07, range = .00?.15) were 78% smaller, on average, than the original effect sizes (median r = .37, range = .19?.50)