Cinnamaldehyde Characterization as an Antibacterial Agent toward E. coli Metabolic Profile Using 96-Blade Solid-Phase Microextraction Coupled to Liquid Chromatography–Mass Spectrometry

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Proteome Research, 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.jproteome.5b00992Sampling and sample preparation plays an important role in untargeted analysis as it influences final composition of the analyzed extract and consequently reflection of the metabolome. In the current work, mechanism of bactericidal action of cinnamaldehyde (CA) against Escherichia coli (E. coli) during bacteria growth applying high-throughput solid-phase microextraction in direct immersion mode coupled to a high-performance liquid chromatography–mass spectrometry system was investigated. Numerous discriminant metabolites due to CA addition to the bacteria culture were mapped in the E. coli metabolic pathways. We propose new metabolic pathways confirming that CA acts as an oxidative stress agent against E. coli. The results of the current research have successfully demonstrated that CA changes the bacterial metabolism through interactions with different biochemical families such as proteins, nucleic acids, lipids, and carbohydrates, which needs further validation by proteomics and transcriptomics studies. The results presented here show the great potential of the novel approach in drug discovery and food safety.Natural Sciences and Engineering Research Council (NSERC) of Canad

Application of Solid Phase Microextraction for Quantitation of Polyunsaturated Fatty Acids in Biological Fluids

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/ac502627wDevelopment of a straightforward strategy for simultaneous quantitative analysis of nonesterified fatty acids (NEFA) species in biofluids is a challenging task because of the extreme complexity of fatty acid distribution in biological matrices. In this study, we present a direct immersion solid phase microextraction method coupled to a liquid chromatography–mass spectrometry platform (DI-SPME- HPLC-ESI -MS) for determination of unconjugated fatty acids (FA) in fish and human plasma. The proposed method was fully validated according to bioanalytical method validation guidelines. The LOD and LOQ were in the range of 0.5–2 and 5–12 ng/mL, respectively, with a linear dynamic range of 100 fold for each compound. Absolute and relative matrix effects were comprehensively evaluated and found to be in the acceptable range of 91–116%. The affinity constant (Ka) of individual FAs to protein albumin was determined to be 9.2 × 104 to 4.3 × 105 M–1. The plasma protein binding (PPB%) was calculated and found to be in the range of 98.0–99.7% for different polyunsaturated fatty acids (PUFAs). The PUFAs under study were found at a high concentration range in fish plasma, whereas only a few were within quantification range in control human plasma. The method was successfully applied for monitoring PUFA changes during the operation in plasma samples obtained from patients undergoing cardiac surgery with the use of cardiopulmonary bypass (CPB). The most significant contribution induced by surgery was noticed in the concentration level of α-linolenic acid (18:3, ALA), arachidonic acid (20:4, AA), and docosahexanoic acid (22:6, DHA) soon after administration of CPB in all cases.Natural Sciences and Engineering Research Council of Canada (NSERC) Supelc

A new strategy for brain tumour metabolomic analysis

Introduction: Nowadays, diagnosis of brain tumours is mainly carried out via neuroimaging techniques. The most widespread methods for routine analysis include computer tomography and magnetic resonance imaging. While such methods are useful to localise tumours, they are unable to offer a conclusive diagnosis of the tumour type. A final diagnosis can only be made via a histological examination of tissue after tumour resection, or, in cases where the location of the tumour is not amenable to resection, after a biopsy of the tumour is carried out. Untargeted metabolite analysis is a relatively new approach to diagnostics, capable of establishing wide characterisation of endogenous metabolites of a given system, a method that can be applied to improve identification of tumour types via biomarker discovery. In this regard, sample collection and preparation can be said to be the most important step in metabolomic studies. Material and methods: In the current study, a solid phase microextraction (SPME) protocol for metabolo-mics, which has been successfully applied towards metabolite analysis in various biological materials in the last few years, was optimised for brain tumour tissue metabolomic analysis. In the current study, the described approach was applied to human brain tumours. Aiming to incur minimal tissue damage, the probes used for sampling were of diameter ca. 0.2 mm. Aiming to optimise the method towards enhanced recovery of the extracted metabolites, various desorption solvents were tested in an optimisation study. The final protocol was used for analysis of a pilot cohort of patients with glioma and meningioma tumours. Results: The results showed that a protocol where chemical biopsy was performed directly from resected tumour with 7-mm-long coating SPME probe and desorption was done using 0.3 mL of a mixture of acetonitrile and water 80:20 v/v was superior to other tested protocols. The optimised method allowed for successful differentiation between the two types of brain tumours studied: meningioma and glioma. Despite the relatively small cohort group involved in the study, several compounds were tentatively identified as statistically significant metabolites responsible for this differentiation. Conclusions: The presented preliminary data demonstrate a potential of the proposed method as a low invasive diagnostic tool for on-site analysis

Solid Phase Microextraction Devices Prepared on Plastic Support as Potential Single-Use Samplers for Bioanalytical Applications

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/full/10.1021/acs.analchem.5b01849This study presents new thin-film solid phase microextraction (SPME) devices prepared on plastic as potential single-use samplers for bioanalysis. Polybutylene terephthalate (PBT) was selected as a support due to its well-known chemical resistance, low cost, and suitability as a material for different medical grade components. The herein proposed samplers were prepared by applying a hydrophilic–lipophilic balanced (HLB)-polyacrylonitrile (PAN) coating on rounded and flat PBT pieces previously sanded with regular sandpaper. SPME devices prepared on PBT were evaluated in terms of robustness, chemical stability, and possible interferences upon exposure to different solvents and matrixes. Rewarding results were found when these samplers were employed for the quantitative analysis of multiple doping substances in common biological matrixes such as urine, plasma, and whole blood. Finally, the proposed thin-film SPME devices made on a PBT were evaluated by conducting multiple extractions from whole blood and plasma using the Concept 96 system. Results showed that more than 20 extractions from plasma and whole blood can be performed without observed decreases in coating performance or peeling of the extraction phase from the plastic surface. These findings demonstrate the robustness of PAN-based coatings applied on such polymeric substrate and open up the possibility of introducing new alternatives and cost-effective materials as support to manufacture SPME biocompatible devices for a wide range of applications, particularly in the clinical field.the Natural Sciences and Engineering Research Council (NSERC) of Canada

High throughput solid phase microextraction: A new alternative for analysis of cellular lipidome?

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.jchromb.2016.09.034 © 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/A new SPME method for untargeted lipidomic study of cell line cultures was proposed for the first time. In this study the feasibility to monitor changes in lipid profile after external stimuli was demonstrated and compared to the conventional Bligh & Dyer method. The human hepatocellular carcinoma (HCC) cell line was used as a model. The obtained results provided a list of up-regulated and down-regulated lipids through a comparison between control (non-stimulated) cells versus the group of cells treated with polyunsaturated fatty acid (20:5). Use of the SPME technique yielded a list of 77 lipid species whose concentrations were recognized to be significantly different between control and treated cells, from which 63 lipids were up-regulated in treated cells. In general, the list was comparable to the peer list obtained by the Bligh & Dyer method. However, more diversity of lipid classes and subclasses such as LPC, sphingomyelins, ceramides, and prenol lipids were observed with the application of the SPME method. Method precision for the SPME approach was within the acceptable analytical range (5-18% RSD) for all detected lipids, which was advantageous over solvent extraction applied. The evaluation of ionization efficiency indicated no matrix effect for the SPME technique, while Bligh" & Dyer presented significant ionization suppression for low abundant species such as LysoPC, PG, ceramides, and sphingomyelins, and ionization enhancement for high abundant phospholipids such as PE.Natural Sciences and Engineering Research Council of Canada (NSERC

Equilibrium ex vivo calibration of homogenized tissue for in vivo SPME quantitation of doxorubicin in lung tissue

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.talanta.2018.02.049 © 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/The fast and sensitive determination of concentrations of anticancer drugs in specific organs can improve the efficacy of chemotherapy and minimize its adverse effects. In this paper, ex vivo solid-phase microextraction (SPME) coupled to LC-MS/MS as a method for rapidly quantitating doxorubicin (DOX) in lung tissue was optimized. Furthermore, the theoretical and practical challenges related to the real-time monitoring of DOX levels in the lung tissue of a living organism (in vivo SPME) are presented. In addition, several parameters for ex vivo/in vivo SPME studies, such as extraction efficiency of autoclaved fibers, intact/homogenized tissue differences, critical tissue amount, and the absence of an internal standard are thoroughly examined. To both accurately quantify DOX in solid tissue and minimize the error related to the lack of an internal standard, a calibration method at equilibrium conditions was chosen. In optimized ex vivo SPME conditions, the targeted compound was extracted by directly introducing a 15 mm (45 µm thickness) mixed-mode fiber into 15 g of homogenized tissue for 20 min, followed by a desorption step in an optimal solvent mixture. The detection limit for DOX was 2.5 µg g−1 of tissue. The optimized ex vivo SPME method was successfully applied for the analysis of DOX in real pig lung biopsies, providing an averaged accuracy and precision of 103.2% and 12.3%, respectively. Additionally, a comparison between SPME and solid-liquid extraction revealed good agreement. The results presented herein demonstrate that the developed SPME method radically simplifies the sample preparation step and eliminates the need for tissue biopsies. These results suggest that SPME can accurately quantify DOX in different tissue compartments and can be potentially useful for monitoring and adjusting drug dosages during chemotherapy in order to achieve effective and safe concentrations of doxorubicin.Natural Sciences and Engineering Research Council (NSERC) of CanadaCanadian Institutes of Health Research (CIHR) [grant 355935]SK CIHR (grant 190953)GL2 (grant GL2-01-019

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

This is the peer reviewed version of the following article: Piri-Moghadam, H., Ahmadi, F., Gómez-Ríos, G. A., Boyacı, E., Reyes-Garcés, N., Aghakhani, A., … Pawliszyn, J. (2016). Fast Quantitation of Target Analytes in Small Volumes of Complex Samples by Matrix-Compatible Solid-Phase Microextraction Devices. Angewandte Chemie International Edition, 55(26), 7510–7514., which has been published in final form at https://doi.org/10.1002/anie.201601476. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.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 mu L). 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.National Sciences and Engineering Research Council of Canada (NSERC

The impact of normothermic and hypothermic preservation methods on kidney lipidome—comparative study using chemical biopsy with microextraction probes

IntroductionNormothermic ex vivo kidney perfusion (NEVKP) is designed to replicate physiological conditions to improve graft outcomes. A comparison of the impact of hypothermic and normothermic preservation techniques on graft quality was performed by lipidomic profiling using solid-phase microextraction (SPME) chemical biopsy as a minimally invasive sampling approach.MethodsDirect kidney sampling was conducted using SPME probes coated with a mixed-mode extraction phase in a porcine autotransplantation model of the renal donor after cardiac death, comparing three preservation methods: static cold storage (SCS), NEVKP, and hypothermic machine perfusion (HMP). The lipidomic analysis was done using ultra-high-performance liquid chromatography coupled with a Q-Exactive Focus Orbitrap mass spectrometer.ResultsChemometric analysis showed that the NEVLP group was separated from SCS and HMP groups. Further in-depth analyses indicated significantly (p &lt; 0.05, VIP &gt; 1) higher levels of acylcarnitines, phosphocholines, ether-linked and longer-chain phosphoethanolamines, triacylglycerols and most lysophosphocholines and lysophosphoethanolamines in the hypothermic preservation group. The results showed that the preservation temperature has a more significant impact on the lipidomic profile of the kidney than the preservation method’s mechanical characteristics.ConclusionHigher levels of lipids detected in the hypothermic preservation group may be related to ischemia-reperfusion injury, mitochondrial dysfunction, pro-inflammatory effect, and oxidative stress. Obtained results suggest the NEVKP method’s beneficial effect on graft function and confirm that SPME chemical biopsy enables low-invasive and repeated sampling of the same tissue, allowing tracking alterations in the graft throughout the entire transplantation procedure

Metabolic profiling of plasma from cardiac surgical patients concurrently administered with tranexamic acid: DI-SPMEâLCâMS analysis

A metabolic profile of plasma samples from patients undergoing heart surgery with the use of cardiopulmonary bypass (CPB) and concurrent administration of tranexamic acid was determined. Direct immersion solid phase microextraction (DI-SPME), a new sampling and sample preparation tool for metabolomics, was used in this study for the first time to investigate clinical samples. The results showed alteration of diverse compounds involved in different biochemical pathways. The most significant contribution in changes induced by surgery and applied pharmacotherapy was noticed in metabolic profile of lysophospholipids, triacylglycerols, mediators of platelet aggregation, and linoleic acid metabolites. Two cases of individual response to treatment were also reported. Keywords: Metabolomics, Heart surgery, Cardiopulmonary bypass, Tranexamic acid, Direct immersion solid phase microextraction, LC/M