Given the intricate organization of the brain, tissue sampling for chemical profiling studies have always been a challenging task. It is often exceptionally difficult to obtain homogeneous samples for in vitro/ex vivo experiments without altering or losing valuable information. The obvious approach has been to develop in vivo analytical methods that may cause minimal perturbation to this complex chemical network so as to improve overall reliability of acquired information. Methods such as biosensors and microdialysis (MD) are among sampling methods applied to in vivo brain chemical profiling studies despite their unique challenges. MD is a well-established in vivo analytical sampling method used over the years for monitoring often low-molecular-weight hydrophilic compounds from the interstitial space. The successful application of the method to neuroscience, especially monitoring of neurotransmitters, led to its expansion to a wider range of analytes, including drugs, [1] metabolites, [2] and peptides. [3] A major challenge, however, associated with MD is its difficulty in sampling hydrophobic compounds. Hydrophobic compounds are often highly protein-bound and bind to the MD probe and tubing, thereby affecting relative recovery. The addition of modifiers, such as bovine serum albumin, glycerol in water, [4] or cyclodextrin, [5] is among the approaches that have been used to prevent hydrophobic interactions and to improve relative recoveries. But these techniques often may complicate the pharmacology of the neurological analytes, as the additives are known to interact with the tissue surrounding the probe. [6] Thus, in typical global metabolomics studies, for example, the composition of a measured metabolome can be significantly affected by the analytical procedure, leaving the analysts with results which likely do not adequately reflect accurate composition of the metabolome during sampling. [7] In effect, it will compromise the already challenging efforts in diagnosis, prognostics, and searching for potential biomarkers for therapeutic purposes. Herein, we demonstrate a novel application of solid-phase microextraction (SPME) for in vivo sampling for brain study. For the first time an application of in vivo SPME as a complementary method to MD for braintissue bioanalysis has been presented. Our technique was first validated against MD in targeted analysis of selected neurotransmitters. Their complementary nature was subsequently shown in global profiling of the brain metabolome. From the profiling study, SPME detected groups of lipids such as gangliosides, fatty acids, and lysophospholipids, which are of particular interest in relation to neurodegenerative diseases. SPME derives its selectivity from the extracting sorbent type. Thus, SPME provides the needed flexibility to analysts to tailor investigations to specific biologically hydrophilic/ hydrophobic compounds. For a global study of the metabolome, however, the sorbent choice is one of low selectivity; that is, the sorbent chemical property must enhance simu...

The main objective of metabolomics is the analysis of all lowmolecular-weight compounds present in a particular living system. Metabolomics data is complementary to proteomics, genomics, and transcriptomics data and provides a better understanding of dynamic processes occurring in living systems.[1] The processes of sampling and sample preparation can significantly affect the composition of the measured metabolome, so the analytical results may not adequately reflect the true metabolome composition at the time of sampling. [2][3][4] This is due primarily to poor efficiency (or even complete omission) of metabolism quenching step and multistep handling procedures, which contribute to inadvertent metabolite loss and/or degradation.Herein we introduce in vivo solid-phase microextraction (SPME) as a new sample preparation method for global metabolomics studies of living systems using liquid chromatography-mass spectrometry (LC-MS). SPME is a nonexhaustive sample preparation procedure in which the amount of analyte extracted is governed by the distribution coefficient of the analyte between the SPME coating and sample matrix if the equilibrium is reached or the rate of mass transfer if a short sampling time is used.[5] In vivo SPME allows accurate extraction of the metabolome directly in the tissue or blood of freely moving animals without the need to withdraw a representative biological sample for analysis, under conditions of negligible depletion where the amount of analyte extracted by SPME is independent of the sample volume. [5][6][7] The blood-draw-free nature of the sampling method facilitates multiple sampling of the same living system and can capture unstable or short-lived metabolites.Large biomolecules are not extracted by the specially selected biocompatible SPME coating, so the need for a metabolism quenching step is eliminated. The amount of metabolites extracted is proportional to the biologically active unbound concentration. For metabolomics studies, in vivo SPME provides the simplest and most rapid sample preparation tool available to date to study living systems in a format directly compatible with LC-MS detection. Although SPME was successfully applied to metabolomics studies using GC-MS primarily in headspace mode, [8][9][10][11][12] its capability to provide instantaneous metabolism quenching directly during the sampling process to capture true metabolome of blood or tissue has not been previously evaluated.First, we developed a successful in vivo SPME workflow for direct sampling of metabolome, and applied it to mice as a model system (Figure 1). In this approach, a coated SPME fiber is housed inside hypodermic needle, [13] which is used to pierce the sampling interface containing circulating blood. The fiber is exposed to blood for a pre-set short sampling time of 2 min. During the sampling, analytes are extracted directly into the SPME coating. The key aspect of developing SPME device for metabolomics was selection of the chemical nature of the coating to ensure simultaneous extraction of both...

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Intracerebral microdialysis was utilized to investigate the effect of P-glycoprotein (a drug efflux transporter) induction at the mouse blood-brain barrier (BBB) on brain extracellular fluid concentrations of quinidine, an established substrate of P-glycoprotein. Induction was achieved by treating male CD-1 mice for 3 days with 5 mg/kg/day dexamethasone (DEX), a ligand of the nuclear receptor, pregnane X receptor, and a P-glycoprotein inducer. Tandem liquid chromatography mass spectrometric method was used to quantify analytes in dialysate, blood and plasma. P-glycoprotein, pregnane X receptor and Cyp3a11 (metabolizing enzyme for quinidine) protein expression in capillaries and brain homogenates was measured by immunoblot analysis. Following quinidine i.v. administration, the average ratio of unbound quinidine concentrations in brain extracellular fluid (determined from dialysate samples) to plasma at steady state (375-495 min) or K p, uu, ECF/Plasma in the DEX-treated animals was 2.5-fold lower compared with vehicle-treated animals. In DEX-treated animals, P-glycoprotein expression in brain capillaries was 1.5-fold higher compared with vehicle-treated animals while Cyp3a11 expression in brain capillaries was not significantly different between the two groups. These data demonstrate that P-gp induction mediated by DEX at the BBB can significantly reduce quinidine brain extracellular fluid concentrations by decreasing its brain permeability and further suggest that drug-drug interactions as a result of P-gp induction at the BBB are possible.