Brain Vascular Expression of Monomeric C-Reactive Protein Is Blocked by C10M Following Intraperitoneal Injection in an ApoE-/- Murine Model of Dyslipidemia: An Immunohistochemical Analysis.

Introduction The neurovascular unit (NVU), comprising vascular and glial cells along with neurons, is vital for maintaining the blood-brain barrier (BBB) and cerebral homeostasis. Dysfunction of the NVU is implicated in key neurodegenerative disorders such as Alzheimer's disease (AD). Monomeric C-reactive protein (mCRP), the dissociated form of native, pentameric C-reactive protein (pCRP), is associated with enhanced pro-inflammatory responses in the vascular system, leading to increased permeability and potential NVU disruption. Methods This study utilized ApoE-/- mice receiving a high-fat diet which were injected intraperitoneally with either mCRP or mCRP together with a small molecule inhibitor (C10M) and investigated the deposition of mCRP and CD105 expression in the brain parenchyma and its localization within the microvasculature. Results Histological analysis revealed significant mCRP deposition in brain microvessels and neurons, indicating potential disruption of the BBB and neuronal damage. Moreover, co-administration of C10M effectively blocked mCRP accumulation in the brain parenchyma, suggesting its potential as a therapeutic agent for effectively inhibiting inflammation-associated degenerative changes. Immunohistochemical staining demonstrated co-localization of mCRP with CD105, indicating potential angiogenic activation and increased susceptibility to inflammatory insult. Discussion These findings provide evidence supporting the potential role of mCRP as a contributor to neuroinflammation in individuals with chronic systemic inflammation. Conclusion Further studies in human subjects should help validate the efficacy of C10M in preventing or halting neurodegeneration in conditions such as AD and stroke-associated dementia.

Drug Repurposing of Metformin for the Treatment of Haloperidol-Related Behavior Disorders and Oxidative Stress: A Preliminary Study.

A particular attribute of the brain lies in the ability to learn, acquire information from the environment, and utilize the learned information. Previous research has noted that various factors (e.g., age, stress, anxiety, pathological issues), including antipsychotic medications, affect the brain and memory. The current study aimed to reveal the effects of chronic metformin treatment on the cognitive performance of rats and on commonly measured markers for oxidative stress. Wistar male rats (n = 40) were randomly divided into four groups: CTR (n = 10)-control group, METF (n = 10)-animals receiving metformin 500 mg/kg, HAL (n = 10)-animals receiving haloperidol 2 mg/kg, and HALMETF (n = 10)-animals receiving haloperidol 2 mg/kg and metformin 500 mg/kg. The medication was administered daily by oral gavage for 40 days. Memory and learning were assessed using the Morris Water Maze (MWM) test. At the end of the MWM, the rodents were decapitated under anesthesia, and the brain and blood samples were assayed by liquid chromatography for markers of oxidative stress (malondialdehyde, MDA, reduced/oxidized glutathione ratio, GSH/GSSG). The quantification of brain-derived neurotrophic factor (BDNF) was performed using the conventional sandwich ELISA technique. In the HALMETF group, metformin attenuated the negative effects of haloperidol. Brain and plasma MDA levels increased in the HAL group. Brain and plasma GSH/GSSG ratios and BDNF levels did not reveal any differences between groups. In conclusion, metformin treatment limits the deleterious cognitive effects of haloperidol. The effect on oxidative stress markers may also point toward an antioxidant-like effect of metformin, but this needs further tests for confirmation.

Relationship between Serum 25-Hydroxyvitamin D Levels and Hormonal Status in Infertile Women: A Retrospective Study.

(1) Background: Infertility is a prevalent issue that affects 10-18% of couples worldwide, and up to 30% in Eastern Europe. Infertility of unknown etiology is one of the most challenging forms of infertility. Vitamin D has recently been extensively studied and researchers are investigating its possible role in ovulation and pregnancy. While the evidence suggests a positive association between vitamin D and ovulation, more research is needed to understand the role that vitamin D plays in ovulation. (2) Methods: In this study, 86 infertile patients were studied retrospectively in a single center by analyzing their anthropometric (body mass index), biochemical (total cholesterol, insulin resistance) and hormonal data in correlation with their vitamin D status. (3) Results: It was found that the mid-luteal progesterone level was significantly related to the 25-hydroxy vitamin D level in a multivariate linear regression model. An analysis of subgroups of ovulatory and anovulatory patients suggests that serum 25-hydroxy vitamin D levels of approximately 20 ng/mL may be necessary to trigger ovulation or to support progesterone secretion. (4) Conclusions: Vitamin D deficiency should be considered when infertility of unknown etiology and progesterone-related disorders occur.

Anticonvulsant Action and Long-Term Effects of Chronic Cannabidiol Treatment in the Rat Pentylenetetrazole-Kindling Model of Epilepsy.

Cannabidiol (CBD) showed anticonvulsant action in several preclinical models and is currently approved by regulatory agencies to treat childhood epilepsy syndromes. However, CBD treatment has limited benefits, and its long-term effects on cognition are not fully understood yet. This study aimed to examine the impact of long-term CBD treatment in the pentylenetetrazole (PTZ)-kindling model of epilepsy. Adult male Wistar rats (N = 24) received PTZ (35 mg/kg intraperitoneally) every other day until two consecutive generalized seizures occurred. CBD (60 mg/kg body weight) was administered daily by the oral route until the kindled state was achieved (n = 12). To confirm that the formulation and administration techniques were not of concern, liquid chromatography-mass spectrometry was performed to test the brain penetration of the CBD formula. As a result of CBD treatment, a lower mortality rate and significantly prolonged generalized seizure latency (925.3 ± 120.0 vs. 550.1 ± 69.62 s) were observed, while the frequency and duration of generalized seizures were not influenced. The CBD-treated group showed a significant decrease in vertical exploration in the open field test and a significant decrease in the discrimination index in the novel object recognition (NOR) test (-0.01 ± 0.17 vs. 0.57 ± 0.15, p = 0.04). The observed behavioral characteristics may be connected to the decreased thickness of the stratum pyramidale or the decreased astrogliosis observed in the hippocampus. In conclusion, CBD treatment did not prevent kindling, nor did it affect seizure frequency or duration. However, it did increase the latency to the first seizure and decreased the prolonged status epilepticus-related mortality in PTZ-kindled rats. The cognitive impairment observed in the NOR test may be related to the high dose used in this study, which may warrant further investigation.

Vitamin D Status Assessment: Lack of Correlation between Serum and Hair 25-Hydroxycholecalciferol Levels in Healthy Young Adults.

Vitamin D deficiency has been linked to numerous health problems, including those resulting from disturbed calcium-phosphorus homeostasis, and neuropsychiatric and autoimmune disorders. Nearly one-third of the global population has suboptimal levels of vitamin D, according to epidemiological data. Vitamin D status is usually determined by measuring serum 25(OH)D, but, for decades, serum 25(OH)D measurement has been hampered by a lack of standardization. There have been many recent initiatives to develop reference substances and methods for measuring vitamin D and its metabolites, and re-evaluating the optimal values. It was also suggested that alternative biological samples could also be used, such as hair, since it has been established that lipophilic substances, such as corticosteroids, can also be found in hair. The purpose of this study was to determine the correlation between 25(OH)D3 concentrations in serum and hair, and other demographic features in 26 healthy Caucasian young adult volunteers. The determination of 25(OH)D3 and cholecalciferol was carried out using liquid chromatography coupled with mass spectrometry (LC-MS) from blood and hair samples taken at two timepoints separated by nine weeks. In the hair samples of 18 out of 26 subjects, 25(OH)D was detected at a mean (±SEM) concentration of 17.07 ± 5.375 pg/mg at the first sampling time, and 58.90 ± 25.97 pg/mg at the second sampling time. A multiple linear regression analysis revealed no effects of gender, body mass index, supplementation, or sun exposure on hair 25(OH)D3 concentrations, but supplementation and sun exposure significantly increased serum 25(OH)D3 concentrations. In addition, serum and hair 25(OH)D3 concentrations did not correlate; however, there was a strong correlation between the two sampling times for serum 25(OH)D3 concentrations. In conclusion, this study confirmed that 25(OH)D3 could be detected in human hair, but its use as a biomarker warrants further investigations since no link was found between serum 25(OH)D3 concentrations, supplementation, sun exposure, and hair 25(OH)D3 concentrations levels.

Role of Vitamin D in Cognitive Dysfunction: New Molecular Concepts and Discrepancies between Animal and Human Findings.

PURPOSE OF REVIEW: increasing evidence suggests that besides the several metabolic, endocrine, and immune functions of 1alpha,25-dihydroxyvitamin D (1,25(OH)2D), the neuronal effects of 1,25(OH)2D should also be considered an essential contributor to the development of cognition in the early years and its maintenance in aging. The developmental disabilities induced by vitamin D deficiency (VDD) include neurological disorders (e.g., attention deficit hyperactivity disorder, autism spectrum disorder, schizophrenia) characterized by cognitive dysfunction. On the other hand, VDD has frequently been associated with dementia of aging and neurodegenerative diseases (e.g., Alzheimer's, Parkinson's disease). RECENT FINDINGS: various cells (i.e., neurons, astrocytes, and microglia) within the central nervous system (CNS) express vitamin D receptors (VDR). Moreover, some of them are capable of synthesizing and catabolizing 1,25(OH)2D via 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1) and 25-hydroxyvitamin D 24-hydroxylase (CYP24A1) enzymes, respectively. Both 1,25(OH)2D and 25-hydroxyvitamin D were determined from different areas of the brain and their uneven distribution suggests that vitamin D signaling might have a paracrine or autocrine nature in the CNS. Although both cholecalciferol and 25-hydroxyvitamin D pass the blood-brain barrier, the influence of supplementation has not yet demonstrated to have a direct impact on neuronal functions. So, this review summarizes the existing evidence for the action of vitamin D on cognitive function in animal models and humans and discusses the possible pitfalls of therapeutic clinical translation.

Short-Term Amygdala Low-Frequency Stimulation Does not Influence Hippocampal Interneuron Changes Observed in the Pilocarpine Model of Epilepsy.

Temporal lobe epilepsy (TLE) is characterized by changes in interneuron numbers in the hippocampus. Deep brain stimulation (DBS) is an emerging tool to treat TLE seizures, although its mechanisms are not fully deciphered. We aimed to depict the effect of amygdala DBS on the density of the most common interneuron types in the CA1 hippocampal subfield in the lithium-pilocarpine model of epilepsy. Status epilepticus was induced in male Wistar rats. Eight weeks later, a stimulation electrode was implanted to the left basolateral amygdala of both pilocarpine-treated (Pilo, n = 14) and age-matched control rats (n = 12). Ten Pilo and 4 control animals received for 10 days 4 daily packages of 50 s 4 Hz regular stimulation trains. At the end of the stimulation period, interneurons were identified by immunolabeling for parvalbumin (PV), neuropeptide Y (NPY), and neuronal nitric oxide synthase (nNOS). Cell density was determined in the CA1 subfield of the hippocampus using confocal microscopy. We found that PV+ cell density was preserved in pilocarpine-treated rats, while the NPY+/nNOS+ cell density decreased significantly. The amygdala DBS did not significantly change the cell density in healthy or in epileptic animals. We conclude that DBS with low frequency applied for 10 days does not influence interneuron cell density changes in the hippocampus of epileptic rats.

Oral Anticoagulant Treatment in Patients with Atrial Fibrillation and Chronic Kidney Disease.

Over the past few decades, a series of innovative medicines have been developed in order to optimize anticoagulation therapy for atrial fibrillation (AF). As a result, a number of nonvitamin K antagonist oral anticoagulants (NOAC) that directly target the enzymatic activity of factor II and factor Xa have been successfully licensed providing a more predictable effect and better safety profile compared to conventional anticoagulants (heparins and vitamin K antagonists (VKAs)). However, comparative efficacy and safety data is limited in patients with advanced chronic kidney disease (i.e., CKD stage 4/5 and end stage renal disease) because patients with estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73 m2 were actively excluded from landmark trials, thus representing a major clinical limitation for the currently available agents. However, the renal function of AF patients can be altered over time. On the other hand, patients with CKD have an increased risk of developing AF. This review article will provide an overview of current concepts and recent evidence guiding the clinical use of NOACs in patients with CKD requiring chronic anticoagulation, and the associated risks and benefits of treatment in this specific patient population.

Amygdala Low-Frequency Stimulation Reduces Pathological Phase-Amplitude Coupling in the Pilocarpine Model of Epilepsy.

Temporal-lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy and warrants the development of new therapies, such as deep-brain stimulation (DBS). DBS was applied to different brain regions for patients with epilepsy; however, the mechanisms of action are not fully understood. Therefore, we tried to characterize the effect of amygdala DBS on hippocampal electrical activity in the lithium-pilocarpine model in male Wistar rats. After status epilepticus (SE) induction, seizure patterns were determined based on continuous video recordings. Recording electrodes were inserted in the left and right hippocampus and a stimulating electrode in the left basolateral amygdala of both Pilo and age-matched control rats 10 weeks after SE. Daily stimulation protocol consisted of 4 × 50 s stimulation trains (4-Hz, regular interpulse interval) for 10 days. The hippocampal electroencephalogram was analyzed offline: interictal epileptiform discharge (IED) frequency, spectral analysis, and phase-amplitude coupling (PAC) between delta band and higher frequencies were measured. We found that the seizure rate and duration decreased (by 23% and 26.5%) and the decrease in seizure rate correlated negatively with the IED frequency. PAC was elevated in epileptic animals and DBS reduced the pathologically increased PAC and increased the average theta power (25.9% ± 1.1 vs. 30.3% ± 1.1; p < 0.01). Increasing theta power and reducing the PAC could be two possible mechanisms by which DBS may exhibit its antiepileptic effect in TLE; moreover, they could be used to monitor effectiveness of stimulation.

Effects of Chronic Cannabidiol Treatment in the Rat Chronic Unpredictable Mild Stress Model of Depression.

Several neuropharmacological actions of cannabidiol (CBD) due to the modulation of the endocannabinoid system as well as direct serotonergic and gamma-aminobutyric acidergic actions have recently been identified. The current study aimed to reveal the effect of a long-term CBD treatment in the chronic unpredictable mild stress (CUMS) model of depression. Adult male Wistar rats (n = 24) were exposed to various stressors on a daily basis in order to induce anhedonia and anxiety-like behaviors. CBD (10 mg/kg body weight) was administered by daily intraperitoneal injections for 28 days (n = 12). The effects of the treatment were assessed on body weight, sucrose preference, and exploratory and anxiety-related behavior in the open field (OF) and elevated plus maze (EPM) tests. Hair corticosterone was also assayed by liquid chromatography-mass spectrometry. At the end of the experiment, CBD-treated rats showed a higher rate of body weight gain (5.94% vs. 0.67%) and sucrose preference compared to controls. A significant increase in vertical exploration and a trend of increase in distance traveled in the OF test were observed in the CBD-treated group compared to the vehicle-treated group. The EPM test did not reveal any differences between the groups. Hair corticosterone levels increased in the CBD-treated group, while they decreased in controls compared to baseline (+36.01% vs. -45.91%). In conclusion, CBD exerted a prohedonic effect in rats subjected to CUMS, demonstrated by the increased sucrose preference after three weeks of treatment. The reversal of the effect of CUMS on hair corticosterone concentrations might also point toward an anxiolytic or antidepressant-like effect of CBD, but this needs further confirmation.

Role of Lacosamide in Preventing Pentylenetetrazole Kindling-Induced Alterations in the Expression of the Gamma-2 Subunit of the GABAA Receptor in Rats.

BACKGROUND: Epilepsy remains challenging to treat still no etiologic treatment has been identified, however, some antiepileptic drugs (AEDs) are able to modify the pathogenesis of the disease. Lacosamide (LCM) has been shown to possess complex anticonvulsant and neuroprotective actions, being an enhancer of the slow inactivation of voltage-gated sodium channels, and it has the potential to prevent epileptogenesis. Recent evidence has shown that LCM indirectly improves the function of GABAA receptors. Receptors at most GABAergic synapses involve the gamma-2 subunit, which contributes to both phasic and tonic inhibition, and its presence assures benzodiazepine sensitivity. Moreover, mutant gamma-2 subunits were associated with generalized epilepsy syndromes. In animal models, the expression of the gamma-2 subunit of the gamma-aminobutyric acid A receptor (GABAAg2) was shown to be increased in pentylenetetrazole (PTZ)-induced chemical kindling in Wistar rats. OBJECTIVE: This study hypothesized that LCM might affect the kindling process by influencing the expression of GABAA receptors in the hippocampus. METHODS: The gene and protein expression levels of the GABAAg2 were studied using RT-qPCR and immunofluorescent staining. RESULTS: It was found that LCM treatment (10 mg/kg i.p. daily for 57 days) reduced the maximal intensity of the PTZ-induced seizures but did not prevent kindling. On the other hand, LCM treatment reverted the increase of mRNA expression of GABAAg2 in the hippocampus and prevented the decrease of GABAAg2 protein in the hippocampal CA1 region. CONCLUSION: LCM could exhibit modulatory effects on the GABAergic system of the hippocampus that may be independent of the anticonvulsant action.

Effects of Cariprazine, Aripiprazole, and Olanzapine on Mouse Fibroblast Culture: Changes in Adiponectin Contents in Supernatants, Triglyceride Accumulation, and Peroxisome Proliferator-Activated Receptor-γ Expression.

Background and Objectives: The use of the dopamine-partial agonist subclass (also termed dopamine stabilizers) of atypical antipsychotics for the treatment of negative schizophrenia symptoms and some mood disorders has increased recently. Similar to other second-generation antipsychotics (SGAs), aripiprazole (ARI) and cariprazine (CAR) also influence food intake, but the peripheral effects of these drugs on adipose-tissue homeostasis, including adipokine secretion as well as lipo- and adipogenesis, are not fully elucidated. In this study, we explored the adipocyte-related mechanisms induced by second-generation antipsychotics (SGAs), leading to changes in peripheral signals involved in energy homeostasis. Materials and Methods: CAR, a new SGA, was compared with ARI and olanzapine (OLA), using cell cultures to study adipogenesis, and the expression levels of peroxisome proliferator-activated receptor-γ (PPAR-γ) was measured in adipocytes derived from mouse fibroblasts, by western blotting on days 7, 14, and 21 postinduction. The triglyceride (TG) content of the cells was also evaluated on day 15 using Oil Red O staining, and the adiponectin (AN) content in the cell culture supernatants was quantified on days 7 and 15 by enzyme-linked immunosorbent assay. Cells were treated with two concentrations of ARI (0.5 and 20 µg/mL), OLA (1 and 20 µg/mL), and CAR (0.1 and 2 µg/mL). Results: Both concentrations of ARI and OLA, as well as the lower concentration of CAR, significantly increased the TG contents. The AN levels in the supernatants were significantly increased by the higher concentration of ARI on days 7 and 15 (p < 0.05). Although PPAR-γ levels were not significantly affected by ARI and OLA, the lower concentration of CAR induced a significant time-dependent decrease in PPAR-γ expression (p < 0.05). Conclusions: The in vitro adipogenesis considered from TG accumulation, AN secretion, and PPAR-γ expression was differently influenced by ARI, CAR, and OLA. Understanding the adipocyte-related mechanisms of antipsychotics could contribute to understanding their weight-influencing effect.

Chronic fluoxetine treatment induces lipid accumulation but does not alter the expression of Pref-1 in rat adipose tissue.

This study aims to investigate the effects of chronic fluoxetine (FLX) treatment on preadipocyte factor-1 (Pref-1) expression in subcutaneous, visceral and brown adipose tissues, and on the size of vacuoles in a dipocytes obtained from the perirenal regions in rats. Twenty-eight Wistar rats were treated with FLX at two different doses and fourteen animals received vehicle. After 40 days of treatment, the subcutaneous, perirenal and interscapular adipose tissues were collected. Pref-1 expression was examined using an immunohistochemical method and the vacuolar area was measured in stained sections. In the low dose FLX group, the size of vacuoles increased both in male and female animals. The high dose of FLX also induced a significant increase of vacuole size, but only in male animals. Neither of the two doses of FLX has significantly affected the Pref-1 expression in any type of adipose tissue.

Distribution of lacosamide in the rat brain assessed by in vitro slice technique.

Lacosamide is a newer anticonvulsant and is the only one that enhances the slow inactivation of voltage gated sodium channels. It is also claimed to have disease-modifying potential, but its pharmacokinetic properties have been much less discussed in the literature. In rats, lacosamide shows restricted distribution to tissues, and the brain-to-plasma partition coefficient (Kp) is only 0.553. In this study, the brain disposition of lacosamide was evaluated in rat brains, and its neuropharmacokinetic parameters (i.e., protein binding and intracellular accumulation) were assessed using in vitro methods. Brain slice experiments and brain homogenate binding studies were performed for several drugs acting on the central nervous system, and drugs were assayed by using a liquid chromatography-mass spectrometry system. By applying a combined approach, it was found that (1) the unbound volume of distribution in the brain for lacosamide (Vu,brain = 1.37) was lower than that of other classical anticonvulsants; (2) the unbound fraction of lacosamide in the brain (0.899) was slightly lower than its unbound fraction in plasma (0.96); (3) the unbound intracellular-to-extracellular concentration ratio of lacosamide was 1.233, meaning that lacosamide was accumulated in the intracellular space because of its physicochemical properties and zwitterionic structure; and (4) the unbound brain-to-plasma concentration ratio of lacosamide was lower than the total brain-to-plasma concentration ratio (Kp,uu,brain = 0.42 vs. Kp = 0.553). In conclusion, the limited brain distribution of lacosamide is not related to its nonspecific protein-binding capacity; rather, an active transport mechanism across the blood-brain barrier may be involved, which reduces the anticonvulsant and/or antiepileptogenic actions of this drug.

Differential effects of sodium channel blockers on in vitro induced epileptiform activities.

Antiepileptic drugs act on voltage gated sodium channels in many different ways: rufinamide is thought to influence the fast inactivation, so its anticonvulsant action could be similar to carbamazepine, whereas lacosamide enhances the slow inactivation; however some antidepressants were also described to act in the same way. Rufinamide, lacosamide, carbamazepine, fluoxetine and imipramine were tested using in vitro models of epileptiform activities. Extracellular local field potentials were recorded using hippocampal slices from immature rats and the pattern of epileptiform activities was analyzed. Seizure-like events (SLE), but not interictal bursts were sensitive to AEDs' action. Rufinamide increased interictal periods by prolonging preictal phase and reducing SLE duration, and was the only tested AED which reduced SLE frequency. Lacosamide's effect resembled that of fluoxetine in the low-Mg2+ model: both drugs reduced markedly the SLE duration, but increased their frequency. Imipramine and fluoxetine irreversibly suppressed SLE in all slices. Some proconvulsive type of action on SLEs such as increasing preictal neuronal activity by rufinamide and increasing SLE frequency by lacosamide, fluoxetine and carbamazepine, were also observed. Newer drugs were more efficient than carbamazepine, and the anticonvulsant action of antidepressants on in vitro epileptiform activities may seem somewhat surprising.

Dose-dependent pharmacokinetics and brain penetration of rufinamide following intravenous and oral administration to rats.

Rufinamide is a third-generation antiepileptic drug, approved recently as an orphan drug for the treatment of Lennox-Gastaut syndrome. Although extensive research was conducted, its pharmacokinetics in rats was not described. This work addresses that area by describing in a rapid pharmacokinetic study the main pharmacokinetic properties of rufinamide at three different doses of 1 mg/kg body weight (bw), 5 mg/kg bw, and 20 mg/kg bw. Furthermore, total brain concentrations of the drug were determined in order to characterize its brain-to-plasma partition coefficient. Adult Wistar male rats, weighing 200-450 g, were administered rufinamide by intravenous and oral routes. Rufinamide concentrations from plasma samples and brain tissue homogenate were determined using a liquid chromatography-mass spectrometric method and pharmacokinetic parameters were calculated. The mean half-life was between 7 and 13 h, depending on route of administration--intravenously administered drug was eliminated faster than orally administered drug. Mean (S.E.M.) total plasma clearance was 84.01 ± 3.80 ml/h/kg for intravenous administration, while the apparent plasma clearance for oral administration was 95.52 ± 39.45 ml/h/kg. The mean (S.E.M.) maximum plasma concentration reached after oral administration of 1 mg/kg bw and 5 mg/kg bw was 0.89 ± 0.09 µg/ml and 3.188 ± 0.71 µg/ml, respectively. The median (range) time to reach maximum plasma concentration (t(max)) was 4 (2-8)h. Mean (S.E.M.) brain-to-plasma concentration ratio of rufinamide was 0.514 ± 0.036, consistent with the brain-to-plasma ratio calculated from the area under curves (AUC(0-t)) of 0.441 ± 0.047. No influence of dose, route of administration, or post-dosing time was observed on brain-to-plasma ratio.

Rapid LC-MS/MS method for determination of drotaverine in a bioequivalence study.

A liquid chromatography coupled with tandem mass spectrometry method for the quantification of the antispasmodic drug drotaverine in human plasma was developed and validated according to the current bioanalytical guidelines. The internal standard used was imipramine. The separation was performed on a Kinetex C18 50×3mm, 2.6µm column under isocratic conditions using a mobile phase of 65:35 (v/v) formic acid 0.2% (v/v) in water and acetonitrile at 40°C with a flow rate of 0.4ml/min. The detection of drotaverine and the internal standard was performed in multiple reaction monitoring (MRM) mode using an ion trap mass spectrometer with electrospray ionization, operating in positive mode. The human plasma samples (0.24ml) were deproteinized with methanol and aliquots of 4µl from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r(2)>0.997), precision (CV<6.3%) and accuracy (bias<5.4%) over the range of 2.24-448ng/ml drotaverine in plasma. The recovery was between 91 and 98%. The limit of quantification was 2.24ng/ml. The analysis required only a 3.0min run. The developed and validated method for the determination of drotaverine in human plasma was successfully applied in a bioequivalence study, for analyzing approximately 1000 subject's samples.

Liquid chromatography-mass spectrometric determination of rufinamide in low volume plasma samples.

Quantification of rufinamide in plasma was achieved using a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method. The chromatographic separation was achieved on a reversed phase column (Zorbax SB-C18 100mm×3mm, 3.5µm) under isocratic conditions. The mobile phase consisted of a mixture of water containing 0.1% formic acid and methanol (50:50, v/v). The mass spectrometric detection of the analyte was in multiple reaction monitoring mode (MRM) using an electrospray positive ionization (ESI positive). The monitored ions were 127m/z derived from 239m/z rufinamide and 108m/z derived from 251m/z the internal standard (lacosamide). Protein precipitation with methanol was applied for sample preparation using only 50µl aliquots. The concentration range was 40-2000ng/ml for rufinamide in plasma. The limit of detection was 1.25ng/ml and the lower limit of quantification was established at 5ng/ml rufinamide concentration. Selectivity and matrix effect was verified using individual human, rat and rabbit plasma samples. Short-term, post-preparative and freeze-thaw stability was also investigated. The proposed method provides accuracy, precision and high-throughput (short runtime 4.5min) for quantitative determination of rufinamide in plasma. This is the first reported liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for analysis of rufinamide from low volume plasma samples. The LC-MS/MS method was validated according to the current official guidelines and can be applied to accurately measure rufinamide level of large number of plasma samples from clinical studies or therapeutic drug monitoring.