Dose-Dependent Dissociation of Pro-cognitive Effects of Donepezil on Attention and Cognitive Flexibility in Rhesus Monkeys.

Background: Donepezil exerts pro-cognitive effects by nonselectively enhancing acetylcholine (ACh) across multiple brain systems. Two brain systems that mediate pro-cognitive effects of attentional control and cognitive flexibility are the prefrontal cortex and the anterior striatum, which have different pharmacokinetic sensitivities to ACh modulation. We speculated that these area-specific ACh profiles lead to distinct optimal dose ranges for donepezil to enhance the cognitive domains of attention and flexible learning. Methods: To test for dose-specific effects of donepezil on different cognitive domains, we devised a multitask paradigm for nonhuman primates that assessed attention and cognitive flexibility. The nonhuman primates received either vehicle or variable doses of donepezil before task performance. We measured intracerebral donepezil and its strength in preventing the breakdown of ACh within the prefrontal cortex and anterior striatum using solid phase microextraction neurochemistry. Results: The highest administered donepezil dose improved attention and made the subjects more robust against distractor interference, but it did not improve flexible learning. In contrast, only a lower dose range of donepezil improved flexible learning and reduced perseveration, but without distractor-dependent attentional improvement. Neurochemical measurements confirmed a dose-dependent increase of extracellular donepezil and decreases in choline within the prefrontal cortex and the striatum. Conclusions: The donepezil dose for maximally improving attention differed from the dose range that enhanced cognitive flexibility despite the availability of the drug in two major brain systems supporting these functions. These results suggest that in our cohort of adult monkeys, donepezil traded improvements in attention for improvements in cognitive flexibility at a given dose range.

Impact of pesticide formulation excipients and employed analytical approach on relative matrix effects of pesticide determination in strawberries.

Relative matrix effects between an ambient mass spectrometric technique known as coated blade spray (CBS) and liquid chromatographic separation approach when applied to multiresidue pesticide analysis in strawberry samples are explored. Acceptable slope relative standard deviations (RSD <15 %) were observed for the 9 compounds under study for both CBS-MS/MS (2.2-12.6 %) and LC-MS/MS (2.8-12.9 %) approaches. The findings signify both the elimination of relative matrix effects with the sample preparation and matrix match calibration with internal standard correction methods employed along with no matrix effect compromise made when using the direct-to-MS approach. Similarly, slopes of pesticides spiked from commercially available formulations (containing one or two pesticides) were found to not differ significantly from slopes generated with multiresidue pesticide standards (containing 24 additional pesticides besides the target 9 analytes) with either technique, highlighting the resistance of the employed methods to the excipients present in pesticide formulations in large amounts.

Metabolic profile of fish muscle tissue changes with sampling method, storage strategy and time.

Unstable tissue components (metabolites) are not easily captured and evaluated by traditional metabolomics methods. In this study, a comprehensive investigation of various sampling methods and storage conditions on the metabolomic profile of fish muscle was performed based on in vivo and ex vivo sampling. The GlobalStd algorithm and structure/reaction directed analysis under a linear mixed model were used to investigate the distinctive influences of these factors on the metabolomic profiles of fish tissue obtained via untargeted LC-MS analysis. Immediate analysis of samples yielded different metabolomic profiles compared to that of stored samples. Storage time was found to affect the metabolomic profile in a complex way, whereas storage temperature was shown to not substantially change this pattern. At the reaction level, metabolites involved in homologous series with butylation were shown stable during storage. Overall, our findings demonstrate that immediate instrumental analysis after in vivo solid phase microextraction (SPME) sampling and a reverse time series experimental design should be the preferred approaches for metabolomic profiling if unstable compounds are of interest.

Comprehensive Investigation of Metabolic Changes Occurring in the Rat Brain Hippocampus after Fluoxetine Administration Using Two Complementary In Vivo Techniques: Solid Phase Microextraction and Microdialysis.

Fluoxetine is among the most prescribed antidepressant drugs worldwide. Nevertheless, limited information is known about its definitive mechanism. Although in vivo examinations performed directly in related brain structures can provide more realistic, and therefore more insightful, knowledge regarding the mechanisms and efficacy of this drug, only a few techniques are applicable for in vivo monitoring of metabolic alterations in the brain following an inducement. Among them, solid phase microextraction (SPME) and microdialysis (MD) have emerged as ideal in vivo tools for extraction of information from biosystems. In this investigation, we scrutinized the capabilities of SPME and MD to detect ongoing changes in the brain following acute fluoxetine administration. Sequential in vivo samples were collected simultaneously from male rats' hippocampi using SPME and MD before drug administration in order to establish a baseline; then samples were collected again following fluoxetine administration for an investigation of small molecule alterations. Our results indicate that MD provides more comprehensive information for polar compounds, while SPME provides superior information with respect to lipids and other medium level polar molecules. Interestingly, in the lipidomic investigation, all dysregulated features were found to be membrane lipids and associated compounds. Moreover, in the metabolomic investigations, dysregulation of hippocampal metabolite levels associated with fatty acid transportation and purine metabolisms were among the most notable findings. Overall, our evaluation of the obtained data corroborates that, when used in tandem, SPME and MD are capable of providing comprehensive information regarding the effect of fluoxetine in targeted brain structures and further elucidating this drug's mechanisms of action in the brain.

Investigation of Early Death-Induced Changes in Rat Brain by Solid Phase Microextraction via Untargeted High Resolution Mass Spectrometry: In Vivo versus Postmortem Comparative Study.

Analysis of brain samples obtained postmortem remains a standard approach in neuroscience, despite often being suboptimal for inferring roles of small molecules in the pathophysiology of brain diseases. Sample collection and preservation further hinders conclusive interpretation of biomarker analysis in autopsy samples. We investigate purely death-induced changes affecting rat hippocampus in the first hour of postmortem interval (PMI) by means of untargeted liquid chromatography-mass spectrometry-based metabolomics. The unique possibility of sampling the same brain area of each animal both in vivo and postmortem was enabled by employing solid phase microextraction (SPME) probes. Four millimeter probes coated with mixed mode extraction phase were used to sample awake, freely roaming animals, with 2 more sampling events performed after death. Significant changes in brain neurochemistry were found to occur as soon as 30 min after death, further progressing with increasing PMI, evidenced by relative changes in levels of metabolites and lipids. These included species from several distinct groups, which can be classified as engaged in energy metabolism-related processes, signal transduction, neurotransmission, or inflammatory response. Additionally, we perform thorough analysis of interindividual variability in response to death, which provides insights into how this aspect can obscure conclusions drawn from an untargeted study at single metabolite and pathway level. The results suggest high demand for systematic studies examining the PMI time course with in vivo sampling as a starting point to eliminate artifacts in the form of neurochemical changes assumed to occur in vivo.

In†Vivo Solid-Phase Microextraction for Sampling of Oxylipins in Brain of Awake, Moving Rats.

Oxylipins are key lipid mediators of important brain processes, including pain, sleep, oxidative stress, and inflammation. For the first time, an in-depth profile of up to 52 oxylipins can be obtained from the brains of awake moving animals using in vivo solid-phase microextraction (SPME) chemical biopsy tool in combination with liquid chromatography-high resolution mass spectrometry. Among these, 23 oxylipins are detectable in the majority of healthy wildtype samples. This new approach successfully eliminates the changes in oxylipin concentrations routinely observed during the analysis of post-mortem samples, allows time-course monitoring of their concentrations with high spatial resolution in specific brain regions of interest, and can be performed using the same experimental set-up as in vivo microdialysis (MD) thus providing a new and exciting tool in neuroscience and drug discovery.

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.

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.

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.

Human bone marrow as a tissue in post-mortem identification and determination of psychoactive Substances-Screening methodology.

The aim of this study was to apply a new instrumental approach to the analysis of human bone marrow for forensic purposes. A new screening method for the detection of more than 30 psychoactive drugs in bone marrow was developed and applied to case samples. The drugs used in this study belonged to the following groups: benzodiazepines (BZDs, n=16), tricyclic antidepressants (TCAs, n=5), selective serotonin reuptake inhibitors (SSRI, n=4), serotonin-norepinephrine reuptake inhibitors (n=1), anticonvulsants (n=1), imidazopyridines (n=1) and piperazines (n=3). The sample preparation procedure involved microwave-assisted extraction (MAE) and the experimental settings were optimized using the simplex method. Separation and detection was carried out using a UHPLC-TOF system. The method were validated using marrow samples, and further applied in the analysis of three case samples, in which diazepam, nordiazepam, citalopram, doxepin and paroxetine were successfully detected. Finally the presented method is a good example of an assay that could potentially find an application in forensic analysis.

SPME as a promising tool in translational medicine and drug discovery: From bench to bedside.

Solid phase microextraction (SPME) is a technology where a small amount of an extracting phase dispersed on a solid support is exposed to the sample for a well-defined period of time. The open-bed geometry and biocompatibility of the materials used for manufacturing of the devices makes it very convenient tool for direct extraction from complex biological matrices. The flexibility of the formats permits tailoring the method according the needs of the particular application. Number of studies concerning monitoring of drugs and their metabolites, analysis of metabolome of volatile as well as non-volatile compounds, determination of ligand-protein binding, permeability and compound toxicity was already reported. All these applications were performed in different matrices including biological fluids and tissues, cell cultures, and in living animals. The low invasiveness of in vivo SPME, ability of using very small sample volumes and analysis of cell cultures permits to address the rule of 3R, which is currently acknowledged ethical standard in R&D labs. In the current review systematic evaluation of the applicability of SPME to studies required to be conduct at different stages of drug discovery and development and translational medicine is presented. The advantages and challenges are discussed based on the examples directly showing given experimental design or on the studies, which could be translated to the models routinely used in drug development process.

Development of a Biocompatible In-Tube Solid-Phase Microextraction Device: A Sensitive Approach for Direct Analysis of Single Drops of Complex Matrixes.

The aim of the current study is to develop a sensitive solid-phase microextraction (SPME) device for direct and rapid analysis of untreated complex matrixes (i.e., single drop of the samples, V ≤ 2 µL). A thin layer of a biocompatible nanostructured polypyrrole (PPy) was electrochemically deposited inside a medical grade spinal needle, minimizing the matrix effect. Microsampling was facilitated by loading the sample inside the in-tube SPME device (withdraw of sample via plunger), where extraction was performed under static conditions. Two strategies were used for analysis of the compounds including offline desorption and running the extract to the liquid chromatograph-tandem mass spectrometer (LC-MS/MS) or direct coupling of the in-tube SPME device to the MS. Given the high surface-area-to-volume ratio of the coating, a short equilibrium time (i.e., t ≤ 2 min) was obtained. The whole analytical procedure (i.e., extraction, rinsing, desorption, and LC-MS/MS analysis) was performed within 10 min by LC-MS/MS, and 3 min by in-tube-MS/MS. Possible matrix effects for the prepared device were evaluated in whole blood samples at three levels of concentration, and encouraging results were achieved in the range of 83-120%. The obtained results, no matrix effect, are attributed to the smooth surface and small pore size of the biocompatible PPy coating, which was prepared in the presence of cetyltrimethylammonium bromide (CTAB) surfactant. The in-tube SPME device was shown to be very sensitive, with high total recoveries obtained for all compounds in phosphate-buffered saline (PBS) and urine samples owing to the large volume and capacity of the coating. Subnanogram per milliliter levels of detection were achieved for urine samples, and low nanogram per milliliter levels were found in whole blood samples for all studied compounds with a high protein binding index. Rapid analysis of whole blood samples was achieved without need of any pretreatment or manipulation of sample, revealing the developed in-tube SPME device as an ideal probe for forensic application, drug monitoring, and point-of care-diagnosis.