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.

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.

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.

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.

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.