Brain Microdialysis Research Articles

Pharmacokinetic profiles of the five isoflavonoids from Pueraria lobata roots in the CSF and plasma of rats

Ethnopharmacological relevanceTraditional Chinese medicine Radix Puerariae, the roots of Pueraria lobata (Wild.) Ohwi., has been widely used for the treatment of cardiovascular and cerebrovascular diseases in China for centuries. Isoflavonoids are believed the active components of this herb. Aim of this studyThe present study aims to investigate the brain penetration and pharmacokinetics of five active isoflavonoids in the ventricular CSF and plasma of rats after intravenous administration of a Pueraria isoflavonoids (PIF) extract, to better understand the active components of this herb for neuro-activities. Material and methodsUnder anesthesia condition, SD rats (n=6) were successively suffered two surgeries for implanting cannulas at lateral ventricle and right jugular vein for brain microdialysis and blood collection, respectively. After recovery, the rats received intravenous dose of PIF at 80mg/kg and the concentrations of puerarin (PU), 3′-methoxypuerarin (MPU), 3′-hydroxypuerarin (HPU), daidzein (DA) and daidzein-8-C-apiosyl-(1-6)-glycoside (DAC) in the ventricular dialysate and plasma samples were determined using a ultra-fast liquid chromatography tandem mass spectrometry method. ResultsComplete concentration versus time profiles of the five components in plasma and four components except for HPU in ventricular CSF were obtained. After dosing, the average C0 values of PU, MPU, DA, DAC and HPU in plasma were estimated 6.53, 13.72, 1.54, 15.84 and 86.07µg/mL, and PU, MPU, DA and DAC were rapidly penetrated to the brain and reached to their Cmax of 521.52, 415.00, 74.34 and 380.03ng/mL in CSF at about 0.5–0.8h, respectively. The elimination t1/2 of PU, DA and DAC in CSF and plasma were no significant difference, while the t1/2 of MPU in ventricular CSF was longer than that in plasma which may attributable to the different physiological environment of central and peripheral compartments. The brain penetration index (AUCCSF/AUCplasma) was found to be about 9.29, 7.25, 11.96, and 4.21% for PU, MPU, DA, and DAC respectively. ConclusionPU, MPU, DA, DAC can quickly penetrate to the brain through the blood brain barrier (BBB) and might be responsible for the neuro-pharmacological activities of P. lobata.

Neurochemical Mediation of Affiliation and Aggression Associated With Pair-Bonding

BackgroundThe neuropeptides vasopressin and corticotropin-releasing factor facilitate, while serotonin inhibits, aggression. How the brain is wired to coordinate interactions between these functionally opposed neurotransmitters to control behavioral states is poorly understood. MethodsPair-bonded male prairie voles (Microtus ochrogaster) were infused with a retrograde tracer, Fluoro-Gold, and tested for affiliation and aggression toward a female partner or novel female subject. Subsequent immunocytochemical experiments examined neuronal activation using Fos and neurochemical/neuroreceptor profiles on brain areas involved in these social behaviors. Finally, a series of behavioral pharmacologic and real-time in vivo brain microdialysis experiments were performed on male prairie voles displaying affiliation or aggression. ResultsWe localized a subpopulation of excitatory vasopressin neurons in the anterior hypothalamus that may gate corticotropin-releasing factor output from the amygdala to the anterior hypothalamus and then the lateral septum to modulate aggression associated with mate guarding. Conversely, we identified a subset of inhibitory serotonergic projection neurons in the dorsal raphe that project to the anterior hypothalamus and may mediate the spatiotemporal release of neuropeptides and their interactions in modulating aggression and affiliation. ConclusionsTogether, this study establishes the medial extended amygdala as a major neural substrate regulating the switch between positive and negative affective states, wherein several neurochemicals converge and interact to coordinate divergent social behaviors.

Cocktail-Dosing Microdialysis Study to Simultaneously Assess Delivery of Multiple Organic–Cationic Drugs to the Brain

Brain microdialysis is a powerful tool to estimate brain-to-plasma unbound concentration ratio at the steady state (Kp,uu) of compounds by direct measurement of the unbound concentration in brain interstitial fluid. Here, we evaluated a method to estimate Kp,uu values of multiple organic–cationic drugs simultaneously, by means of brain microdialysis combined with cocktail dosing. Five cationic drugs (diphenhydramine, memantine, oxycodone, pyrilamine, and tramadol), substrates of the proton-coupled organic cation antiport system, were selected as model drugs, and compared under single-dosing and cocktail-dosing conditions. We selected doses of the drugs at which no significant drug–drug interaction occurs at the proton-coupled organic cation antiport system in the blood–brain barrier (BBB). This was confirmed by uptake studies in hCMEC/D3 cells, an in vitro BBB model. The Kp,uu values after cocktail administration were in the range of 1.8-5.2, and were in good agreement with those after single administration. These results suggest that the microdialysis method with cocktail dosing is suitable to estimate Kp,uu values of several cationic drugs simultaneously, if there is no drug–drug interaction during BBB transport. The method could be useful for evaluating drug candidates with high Kp,uu values at an early stage in the development of central nervous system-acting drugs.

Maternal Immune Activation Disrupts Dopamine System in the Offspring.

Background:In utero exposure to maternal viral infections is associated with a higher incidence of psychiatric disorders with a supposed neurodevelopmental origin, including schizophrenia. Hence, immune response factors exert a negative impact on brain maturation that predisposes the offspring to the emergence of pathological phenotypes later in life. Although ventral tegmental area dopamine neurons and their target regions play essential roles in the pathophysiology of psychoses, it remains to be fully elucidated how dopamine activity and functionality are disrupted in maternal immune activation models of schizophrenia.Methods:Here, we used an immune-mediated neurodevelopmental disruption model based on prenatal administration of the polyriboinosinic-polyribocytidilic acid in rats, which mimics a viral infection and recapitulates behavioral abnormalities relevant to psychiatric disorders in the offspring. Extracellular dopamine levels were measured by brain microdialysis in both the nucleus accumbens shell and the medial prefrontal cortex, whereas dopamine neurons in ventral tegmental area were studied by in vivo electrophysiology.Results:Polyriboinosinic-polyribocytidilic acid-treated animals, at adulthood, displayed deficits in sensorimotor gating, memory, and social interaction and increased baseline extracellular dopamine levels in the nucleus accumbens, but not in the prefrontal cortex. In polyriboinosinic-polyribocytidilic acid rats, dopamine neurons showed reduced spontaneously firing rate and population activity.Conclusions:These results confirm that maternal immune activation severely impairs dopamine system and that the polyriboinosinic-polyribocytidilic acid model can be considered a proper animal model of a psychiatric condition that fulfills a multidimensional set of validity criteria predictive of a human pathology.

An improved microbore UHPLC method with electrochemical detection for the simultaneous determination of low monoamine levels in in vivo brain microdialysis samples

The simultaneous determination of the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in in vivo microdialysis samples remains challenging because of the low extracellular neurotransmitter levels in different brain regions, specific sample characteristics, and the quest for high temporal resolution and a multi-target strategy in neuropharmacological research. A fast and sensitive microbore (1.0mm i.d. column) UHPLC method coupled to electrochemical detection (ECD) is developed by means of design of experiments with the emphasis on sufficient retention of NA within an acceptable total analysis time. Indeed, NA is the earliest eluting compound and often interferes with the broad solvent front originating from the sample matrix. The sensitive UHPLC-ECD assay (LLOQ of 100pM for NA and 150pM for DA and 5-HT) with an analysis time of 8min for standard solutions and 20min for in vivo microdialysis samples originating from rat hippocampus, prefrontal cortex and striatum, is validated applying accuracy profiles. The combination of in vivo microdialysis and microbore UHPLC-ECD has shown to be particularly suitable for future contributions to neuropharmacological research on the monoaminergic system.

Lipocalin-2 is increased in progressive multiple sclerosis and inhibits remyelination.

Objective:We aimed to examine the regulation of lipocalin-2 (LCN2) in multiple sclerosis (MS) and its potential functional relevance with regard to myelination and neurodegeneration.Methods:We determined LCN2 levels in 3 different studies: (1) in CSF and plasma from a case-control study comparing patients with MS (n = 147) with controls (n = 50) and patients with relapsing-remitting MS (n = 75) with patients with progressive MS (n = 72); (2) in CSF and brain tissue microdialysates from a case series of 7 patients with progressive MS; and (3) in CSF at baseline and 60 weeks after natalizumab treatment in a cohort study of 17 patients with progressive MS. Correlation to neurofilament light, a marker of neuroaxonal injury, was tested. The effect of LCN2 on myelination and neurodegeneration was studied in a rat in vitro neuroglial cell coculture model.Results:Intrathecal production of LCN2 was increased predominantly in patients with progressive MS (p < 0.005 vs relapsing-remitting MS) and displayed a positive correlation to neurofilament light (p = 0.005). Levels of LCN2 in brain microdialysates were severalfold higher than in the CSF, suggesting local production in progressive MS. Treatment with natalizumab in progressive MS reduced LCN2 levels an average of 13% (p < 0.0001). LCN2 was found to inhibit remyelination in a dose-dependent manner in vitro.Conclusions:LCN2 production is predominantly increased in progressive MS. Although this moderate increase does not support the use of LCN2 as a biomarker, the correlation to neurofilament light and the inhibitory effect on remyelination suggest that LCN2 might contribute to neurodegeneration through myelination-dependent pathways.

Sensitive and accurate determination of neurotransmitters from in vivo rat brain microdialysate of Parkinson's disease using in situ ultrasound-assisted derivatization dispersive liquid–liquid microextraction by UHPLC-MS/MS

In situ UA-DDLLME coupled with UHPLC-MS/MS has been developed for simultaneous determination of neurotransmitters and baicalein from Parkinson's disease rats.

Evaluation of a Portable Microchip Electrophoresis Fluorescence Detection System for the Analysis of Amino Acid Neurotransmitters in Brain Dialysis Samples.

A portable fluorescence detection system for use with microchip electrophoresis was developed and compared to a benchtop system. Using this system, six neuroactive amines commonly found in brain dialysate (arginine, citrulline, taurine, histamine, glutamate, and aspartate) were derivatized offline with naphthalene-2,3-dicarboxaldehyde/cyanide, separated electrophoretically, and detected by fluorescence. The limits of detection for the analytes of interest were 50 - 250 nM for the benchtop system and 250 nM - 1.3 μM for the portable system, both of which were adequate for most analyte detection in brain microdialysis samples. The portable system was then demonstrated for the detection of the same six amines in a rat brain microdialysis sample.

Magnesium Concentration in Cerebrospinal and Brain Interstitial Fluid Correlates with Serum Level in Induced Hypermagnesemia

Being a modulator of the N-methyl-D-aspartate (NMDA) receptor function magnesium has been studied for its neuroprotective and vasodilatative properties in acute and delayed brain ischemia due to vasospasm in aneurysmal subarachnoid haemorrhage (aSAH) and stroke. A number of clinical phase II and III studies have correlated serum magnesium concentrations after intravenous continuous application to clinical outcome and have failed to show a positive therapeutic effect. However, no study supported its conclusion by providing evidence for a local increase in magnesium, i.e. in the cerebrospinal fluid (CSF) and the brain parenchyma. The objective of our observational study was to compare magnesium levels in serum, CSF, and brain microdialysis samples (MDS) in patients with aSAH. Seventeen patients with aSAH at World Federation of Neurosurgeons (WFNS) grade IV and V were included. According to our internal standard treatment protocols, all patients received a ventricular catheter, a frontal intracerebral microdialysis probe, and a continuous intravenous application of 80 mmol MgSO4 per 24 hours. Magnesium concentrations of serum, CSF, and MDS were recorded. We found a positive and significant correlation of increased serum levels of magnesium to CSF and MDS magnesium concentrations. These pharmacokinetic findings may serve as a basis for further discussion of the concept of induced hypermagnesemia in patients with aSAH, especially in the context of recent level A evidence of the lack of clinical benefit.

Determination of Histamine by High-Performance Liquid Chromatography After Precolumn Derivatization witho-Phthalaldehyde-Sulfite

A fast and sensitive method was developed for in vivo determination of histamine in the brain microdialysate by reverse ion pair chromatography with electrochemical detection. The microdialysates were derivatized with o-phthalaldehyde and sodium sulfite, and separation was achieved using isocratic elution within 10 min. The separation was performed in an Agilent Eclipse Plus C18 column (3.0 × 150 mm, particle size 3.5 μm), and the mobile phase consisted of 100 mM monosodium phosphate (pH 6.0), 500 mg L(-1) OSA and 20% methanol (v/v). The linearity (R(2)) was found to be >0.999, with a range from 2 to 50 nM and excellent repeatability (relative standard deviation, 2.29-6.04%), and the limit of detection was 0.4 nM. This method was successfully applied to analyze the extracellular concentration of histamine in the hypothalamus of rats, with probe recovery calculated in vivo.

Ethanol and Acetaldehyde After Intraperitoneal Administration to Aldh2-Knockout Mice-Reflection in Blood and Brain Levels.

This paper reports, for the first time, on the analysis of ethanol (EtOH) and acetaldehyde (AcH) concentrations in the blood and brains of Aldh2-knockout (Aldh2-KO) and C57B6/6J (WT) mice. Animals were administrated EtOH (1.0, 2.0 or 4.0g/kg) or 4-methylpyrazole (4-MP, 82mg/kg) plus AcH (50, 100 or 200mg/kg) intraperitoneally. During the blood tests, samples from the orbital sinus of the eye were collected. During the brain tests, dialysates were collected every 5min (equal to a 15µl sample) from the striatum using in vivo brain microdialysis. Samples were collected at 5, 10, 15, 20, 25, 30 and 60min intervals post-EtOH and -AcH injection, and then analyzed by head-space GC. In the EtOH groups, high AcH levels were found in the blood and brains of Aldh2-KO mice, while only small traces of AcH were seen in the blood and brains of WT mice. No significant differences in EtOH levels were observed between the WT and the Aldh2-KO mice for either the EtOH dose. EtOH concentrations in the brain were comparable to the EtOH concentrations in the blood, but the AcH concentrations in the brain were four to five times lower compared to the AcH concentrations in the blood. In the AcH groups, high AcH levels were found in both WT and Aldh2-KO mice. Levels reached a sharp peak at 5min and then quickly declined for 60min. Brain AcH concentrations were almost equal to the concentrations found in the blood, where the AcH concentrations were approximately two times higher in the Aldh2-KO mice than in the WT mice, both in the blood and the brain. Our results suggest that systemic EtOH and AcH administration can cause a greater increase in AcH accumulation in the blood and brains of Aldh2-KO mice, where EtOH concentrations in the Aldh2-KO mice were comparable to the EtOH concentrations in the WT mice. Furthermore, detection of EtOH and AcH in the blood and brain was found to be dose-dependent in both genotypes.

Study on recovery and its influencing factors of ferulic acid and tetramethylpyrazine in cerebral microdialysis probe

To establish a method for detecting microdialysis recovery of tetramethylpyrazine (TMP) and ferulic acid (FA) and investigating the influencing factors, providing the basis for further in vivo microdialysis experiments. The concentration of FA and TMP in dialysates were determined by high pressure liquid chromatography ( HPLC) and probe recovery were calculated respectively. The influence of the flow rates, medium concentration, temperature and in vivo probe stability on the recovery of FA and TMP were investigated by using concentration difference method (incremental method and decrement method). The recovery obtained by incremental method were similar to by decrement method. The in vitro recovery rate of FA and TMP decreased with the increase of 1-2.5 μL min(-1), and increased obviously with the temperature of 25-42 degrees C under the same conditions. The concentration of FA and TMP had no obvious effect on the probe recovery under the same flow rate. In addition, the recovery of TMP and FA remained stable and showed similar trends under the condition of four concentration cycles, indicating that the intra day reproducibility of the concentration difference method was good. The recovery of brain microdialysis probes in vivo 8 h maintained a relatively stable, but certain differences existed between different brain microdialysis probes, demonstrating that each probe was required for recovery correction in vivo experiment. Microdialysis sampling can be used for the local brain pharmacokinetic study of FA and TMP, and retrodialysis method can be used in probe recovery of FA and TMP in vivo.

An LC/MS quantitative and microdialysis method for cyclovirobuxine D pharmacokinetics in rat plasma and brain: The pharmacokinetic comparison of three different drug delivery routes.

To explore the brain-targeting of cyclovirobuxine D(CVB-D) after administered intranasally, the pharmacokinetics of CVB-D via three different drug delivery routes: intragastric (i.g.), intranasal (i.n.), and intravenous (i.v.) in rat brain and blood was compared. Firstly, an in vivo microdialysis method for sampling CVB-D in both plasma and brain of the rat was established. Secondly, a liquid chromatography-tandem mass spectrometry method has been developed and validated for determination of CVB-D in microdialysis samples. For plasma and brain microdialysis samples, liquid-liquid extraction was used and donepezil was chosen as internal standard. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI-MS/MS). Chromatographic separation was achieved on a agilent C18 column with a mobile phase of methanol-water (50:50, v/v) (pH 3.2) containing 0.1% formic acid and 5mM ammonium acetate. Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring (MRM) of the transitions at m/z 403.4→372.3 for CVB-D and m/z 380.2→243.1 for donepezil (IS). Good linearities were obtained in the range of 10-4000ng/mL in rat microdialysates for CVB-D. The lowest limit of quantitation was 5ng/mL, with an extraction recovery >75%, and no significant matrix effects. Intra- and inter-day precisions were all <15% with accuracies of 97.26-116.20%. All of which proved that the established method was successfully applied to the pharmacokinetic study of CVB-D. Simultaneously, brain uptake and pharmacokinetic studies were performed by determination of CVB-D concentration in blood and brain respectively for CVB-D i.g., i.n. and i.v.. Results showed that the intranasal CVB-D could improve brain targeting and had advantages for direct nose to brain transport of CVB-D when compared with injection and oral delivery routes, which indicates that intranasal administration of CVB-D could be a promising approach for the treatment of cerebrovascular disease.

Assessment of Amino Acid Neurotransmitters in Rat Brain Microdialysis Samples by High-Performance Liquid Chromatography with Coulometric Detection

A simple method was developed for simultaneous determination of five amino acid neurotransmitters (taurine, glutamate, aspartate, glycine, and γ-aminobutyric acid) in brain microdialysates by high-performance liquid chromatography with coulometric detection. The microdialysates were derivatized with o-phthalaldehyde in 5 mM sodium sulfite and separated within 17 min using isocratic elution. Detection parameters for these transmitters were linearity (r2) > 0.999 over the concentration range from 0.1 to 15.00 µM (0.01–1.00 µM for GABA); reproducibility 2.88%–11.67% (relative standard deviation); the limits of detection 4.4–17.2 fmol; and recovery 86.66%–103.42%. This method was successfully applied to analyze these neurotransmitters in the brain. The changes of neurotransmitters were also investigated via local administration of potassium chloride and bicuculline, which was a γ-aminobutyric acid receptor antagonist. It was found that the developed method is more efficient as compared to previously reported methods in simultaneously determining five major neurotransmitters because it shows quick separation and sensitive detection, and is easy to operate. This method can be a practical choice for continuous monitoring of the amino acid neurotransmittersin vivo.

Fe3+-functionalized carbon quantum dots: A facile preparation strategy and detection for ascorbic acid in rat brain microdialysates

This study reports an Fe3+-functionalized carbon quantum dots (Fe3+-functionalized CQDs) for the highly sensitive and selective detection of ascorbic acid (AA) in rat brain microdialysates based on the specific redox reaction between iron(III) ions and AA. The carbon quantum dots (CQDs) were synthesized by one-step pyrolysis of a small organic molecules i.e. tris(hydroxymethyl)aminomethane (Tris). Fe3+ can tightly chelate to the surface of CQDs by the hydroxyl group to form Fe3+-functionalized CQDs while the fluorescence of CQDs can be effectively quenched by Fe3+ via Fluorescence resonance energy transfer (FRET). The fluorescence of the Fe3+-functionalized CQDs can be sensitively turned on by AA to give an “on–off–on” fluorescence response through the oxidation–reduction between Fe3+ and AA since the produced Fe2+ has much lower chelating ability to CQDs and the fluorescence of CQDs can be restored. This Fe3+-functionalized CQDs based nanoprobe shows high selective and sensitive response in the concentration of AA ranging from 0.1μM to 50μM with the detection limit as lower as 9.1nM, which is lower than other assays. Finally, the proposed fluorescent probe was successfully applied to direct analysis of AA in biological fluids, i.e. rat brain microdialysates, and may pave a new route to the design of effective carbon quantum dots-based fluorescence probes for other bioassay.

Brain ‘dipstick’ can spot dangerous energy drop

To check a car's oil levels, one can use a dipstick. To check the brain has enough energy, he might be able to do the same, using a probe that can monitor fluid in the brain. The goal is to save brain tissue, says Elham Rostami of the Karolinska Institute in Stockholm Sweden. Rostami and 47 others published guidelines on how and when to use the technique, known as brain microdialysis, in the hope that more hospitals would adopt it.

Permeation of Dopamine Sulfate through the Blood-Brain Barrier.

Dopamine sulfate (DA-3- and DA-4-S) have been determined in the human brain, but it is unclear whether they are locally formed in the central nervous system (CNS), or transported into the CNS from peripheral sources. In the current study, permeation of the blood-brain barrier (BBB) by DA-S was studied by injecting 13C6-labelled regioisomers of DA-S (13DA-3-S and 13DA-4-S) and dopamine (DA) subcutaneously (s.c.) in anesthetized rats, then analyzing brain microdialysis and plasma samples by UPLC-MS/MS. The results in the microdialysis samples demonstrated that brain concentrations of 13DA-S regioisomers clearly increased after the s.c. injections. The concentration of DA did not change, indicating the permeation of DA-S through an intact BBB. The analysis of plasma samples, however, showed that DA-S only permeates the BBB to a small extent, as the concentrations in plasma were substantially higher than in the microdialysis samples. The results also showed that the concentrations of DA-3-S were around three times higher than the concentrations of DA-4-S in rat brain, as well as in the plasma samples after the s.c. injections, indicating that DA-3-S and DA-4-S permeate the BBB with similar efficiency. The fate of 13DA-S in brain was followed by monitoring 13C6-labelled DA-S hydrolysis products, i.e. 13DA and its common metabolites; however, no 13C6-labelled products were detected. This suggests that DA-S either permeates through the BBB back to the peripheral circulation or is dissociated or metabolized by unexpected mechanisms.

Quantitative and antioxidative behavior of Trolox in rats' blood and brain by HPLC-UV and SMFIA-CL methods.

Trolox, a water-soluble vitamin E analogue has been used as a positive control in Trolox equivalent antioxidant capacity and oxygen radical antioxidant capacity assays due to its high antioxidative effect. In this study, the ex vivo antioxidative effects of Trolox and its concentration in blood and brain microdialysates from rat after administration were evaluated by newly established semi-microflow injection analysis, chemiluminescence detection and HPLC-UV. In the administration test, the antioxidative effect of Trolox in blood and brain microdialysates after a single administration of 200 mg/kg of Trolox to rats could be monitored. The antioxidative effects in blood (12.0 ± 2.1) and brain (8.4 ± 2.1, × 10(3) antioxidative effect % × min) also increased. Additionally, the areas under the curve (AUC)s0-360 (n = 3) for blood and brain calculated with quantitative data were 10.5 ± 1.2 and 9.7 ± 2.5 mg/mL × min, respectively. This result indicates that Trolox transferability through the blood-brain barrier is high. The increase in the antioxidative effects caused by Trolox in the blood and brain could be confirmed because good correlations between concentration and antioxidative effects (r ≥ 0.702) were obtained. The fact that Trolox can produce an antioxidative effect in rat brain was clarified.

Clozapine and GABA transmission in schizophrenia disease models: establishing principles to guide treatments.

Schizophrenia disease models are necessary to elucidate underlying changes and to establish new therapeutic strategies towards a stage where drug efficacy in schizophrenia (against all classes of symptoms) can be predicted. Here we summarise the evidence for a GABA dysfunction in schizophrenia and review the functional neuroanatomy of five pathways implicated in schizophrenia, namely the mesocortical, mesolimbic, ventral striopallidal, dorsal striopallidal and perforant pathways including the role of local GABA transmission and we describe the effect of clozapine on local neurotransmitter release. This review also evaluates psychotropic drug-induced, neurodevelopmental and environmental disease models including their compatibility with brain microdialysis. The validity of disease models including face, construct, etiological and predictive validity and how these models constitute theories about this illness is also addressed. A disease model based on the effect of the abrupt withdrawal of clozapine on GABA release is also described. The review concludes that while no single animal model is entirely successful in reproducing schizophreniform symptomatology, a disease model based on an ability to prevent and/or reverse the abrupt clozapine discontinuation-induced changes in GABA release in brain regions implicated in schizophrenia may be useful for hypothesis testing and for in vivo screening of novel ligands not limited to a single pharmacological class.

Quantification of β-hydroxymethylbutyrate and leucine by ultrahigh performance liquid chromatography tandem mass spectrometry at different situations and stages of a rodent life.

The main objective of this work was to develop a method to measure Leucine (Leu) and β-hydroxymethylbutyrate (HMB) at basal levels in serum, urine, milk and brain microdialysates in rats. Ultrahigh performance liquid chromatography-electrospray-tandem mass spectrometry (UHPLC-ESI-MS/MS) was used as analytical technique. The sample treatment was simple and consisted of dilution with methanol and centrifugation for serum and urine, dilution with water and filtration with an Amicon filter for milk, and treatment with formic acid with no further dilution for microdialyzates. The procedures for sampling and the UHPLC-MS/MS parameters were accurately optimized to achieve the highest recoveries and to enhance the analytical characteristics of the method. For chromatographic separation, an Acquity UPLC BEH Amide column using acetonitrile-water gradient with formic acid as additive was used. The total run time was 4min. The analytical characteristics (accuracy, selectivity and sensitivity) of the proposed method were evaluated. The limits of detection (LODs) obtained ranged from 0.4 to 7ngmL(-1) and the limits of quantification (LOQs) from 1 to 22ngmL(-1). Precision, expressed as relative standard deviation (% RSD), was lower than 15% in all cases, and the determination coefficient (R(2)) was equal or higher than 99.0% with a residual deviation for each calibration point lower than ±25%. Mean recoveries were between 85 and 115%. The method was successfully applied to these matrices being able to detect significant differences between physiological situations, strains and stages of life.