Development and Validation of a Sonication-Assisted Dispersive Liquid-Liquid Microextraction Procedure and an HPLC-PDA Method for Quantitative Determination of Zolpidem in Human Plasma and Its Application to Forensic Samples.

The use of z-drugs has increased worldwide since its introduction. Although the prescribing patterns of hypnotics differ among countries, zolpidem is the most widely used z-drug in the world. Zolpidem may be involved in poisoning and deaths. A simple and fast HPLC-PDA method was developed and validated. Zolpidem and the internal standard chloramphenicol were extracted from plasma using a sonication-assisted dispersive liquid-liquid microextraction procedure. The method was validated including selectivity, linearity, precision, accuracy, and recovery. The calibration range (0.15-0.6 µg/mL) covers therapeutic and toxic levels of zolpidem in plasma. The limit of quantification was set at 0.15 µg/mL. Intra- and interday accuracy and precision values were lower than 15% at the concentration levels studied. Excellent recovery results were obtained for all concentrations. The proposed method was successfully applied to ten real postmortem plasma samples. In our series, multiple substances (alcohol and/or other drugs) were detected in most cases of death involving zolpidem. Our analytical method is suitable for routine toxicological analysis.

Determination of lamotrigine in human plasma by HPLC-PDA. Application to forensic samples.

PURPOSE: Therapeutic drug monitoring of plasma lamotrigine (LTG) has customarily been carried out in order to prevent some its adverse effects. For forensic purposes, determination of LTG in plasma is an useful tool in cases of accidental overdose or suicidal attempts. Currently, there are several analytical methods available including some based on LC tandem mass spectrometry techniques, but simple and accessible LC-UV methods still can be useful for the purpose. Here we report on a new high-performance liquid chromatography method for the determination of lamotrigine in human plasma which has been developed and validated including selectivity, sensitivity, accuracy, precision and recovery studies. METHODS: Lamotrigine and the internal standard chloramphenicol were extracted from plasma using liquid-liquid extraction using small volumes of buffer and ethylacetate. Detection was monitored at 305.7 and 276.0 nm for lamotrigine and chloramphenicol, respectively. RESULTS: The method was linear concentration dependence within the range of 0.1-10 µg/ml, with a mean coefficient of correlation r = 0.993. The limit of detection (LOD) was 0.04 µg/ml and the limit of quantification (LOQ) was 0.1 µg/ml. Intra and interday precision values were lower than 9.0% at all concentrations studied. The intra and interday accuracy values ranged from - 7.6 to 10.1%. Recovery was found to be 98.9% or higher. The method here described was successfully applied to 11 postmortem blood samples received at the Forensic Sciences Institute of Santiago de Compostela (Spain). CONCLUSION: A new HPLC method for the determination of lamotrigine in human plasma was developed and validated. A liquid-liquid extraction using small volumes of buffer and ethylacetate was optimized. The proposed method is suitable for forensic toxicological analysis.

Evaluation of an Oral Fluid Collection Device and a Solid-Phase Extraction Method for the Determination of Coca Leaf Alkaloids by Gas Chromatography-Mass Spectrometry.

Some South American countries have ancient traditions that may pose legal problems, such as the consumption of coca leaves, as this can provide positive results for cocaine use after the analysis of biological samples. For this reason, it is necessary to find specific markers that help differentiate legal from illegal consumption, such as tropacocaine, cinnamoylcocaine, and especially hygrine and cuscohygrine. In this work, two techniques for collecting biological samples are compared: the Quantisal® Oral Fluid collection device and passive drooling. Once the samples were collected, they were subjected to solid-phase extraction for subsequent injection into GC-MS. Different validation parameters included in international guides have been studied to evaluate whether the proposed method is valid for the defined purpose, placing special emphasis on the study of the matrix effect and little value on GC-MS analyses. With respect to this parameter, an increase in the signal was found for CUS and t-CIN, but it was not significant for the rest of the substances studied. The recoveries have varied significantly depending on the way of working, being higher when working with standardized areas. After carrying out work with the oral fluid samples collected from laboratory volunteers, the method was applied to two real samples. The results obtained support the need for further research to overcome certain limitations presented by the device.

Alcohol consumption in Menière's disease patients.

Objectives: Dietary changes are useful in the management of Menière's disease; regarding alcohol, many clinicians recommend to avoid or reduce its consumption. However, there are no researches aimed to evaluate whether habitual alcohol consumption is more prevalent and/or more intense in patients with Menière's disease.Methods: Cross-sectional, observational, case-control study, including three groups: patients with Menière's disease, patients with vertigo of other origins, and control subjects. Alcohol consumption was compared between these three groups. Participants in this study were grouped according to alcohol consumption as follows: categorization A1 (nonalcohol vs. alcohol consumers), categorization A2 (nonalcohol, low, moderate, and high alcohol consumers), and categorization A3 (light alcohol consumers: nonconsumers plus low consumers; heavy alcohol consumers: moderate plus high consumers).Results: A total of 180 subjects were included in this study (72 in group A, 72 in group B, and 36 in control group); 117 were women. The mean age was 52.7 years. Mean alcohol consumption was 41.22 g/week. Average consumption of alcohol in group A (50.42 g/week) was higher than in other two groups (36.53 g/week in B and 32.22 g/week in C), but differences were not statistically significant. In Menière's group, light alcohol consumers showed age at onset of symptoms (49.39 years) lower than heavy alcohol consumers (55.51 years). No relationship was observed between alcohol consumption and uni or bilateral affectation.Discussion: It is possible that alcohol consumption delays the age at onset of Menière's disease. A hypothetical explanation is the inhibitory effect of alcohol on hypothalamic production of vasopressin. A reduced release of this neurohormone would increase diuresis and decrease endolymphatic pressure.

Caffeine intake and Menière's disease: Is there relationship?

OBJECTIVES: Although it is commonly recognized that dietary restrictions may improve the clinical course of Menière's disease, their effectiveness has not been definitely demonstrated. The aim of this study was to examine whether caffeine consumption could be involved in Menière's disease. METHODS: Cross-sectional, observational, case-control study, comparing caffeine consumption (intake of coffee, tea, kola-type beverages, energy drinks, and chocolate-containing beverages or foods) between patients with Menière's disease (group A) and patients affected by vertigo with other origins (group B) and/or control subjects (group C). PATIENTS: 180 subjects (72 in group A, 72 in group B, and 36 in group C). Caffeine intake was categorized in four levels: very low (0-25 mg/day), low (26-100 mg/day), moderate (101-300 mg/day), and high (≥301 mg/day). Very low and low intake were considered light consumption, and moderate and high intake, heavy consumption. RESULTS: Mean daily caffeine intake was 175.8 mg. Menière's disease patients showed a daily caffeine intake (222 mg) greater than those not affected by this disease (145 mg). Excluding in group B migraine patients, differences in caffeine intake are significant among the three groups (P = 0.021). There were significantly more heavy-consumers in group A than in other two groups jointed (P = 0.024; OR = 1.301, IC95% (1.015;1.668)). In group A, the age at onset of symptoms in caffeine consumers (49.7 years) was lower than in non-consumers (55.9 years). DISCUSSION: It should be recommended to reduce caffeine intake in those population groups with higher risk of Menière's disease (e.g. subjects with family members suffering from this disease).

Instability Due to Drug-Induced Vestibulotoxicity.

OBJECTIVE: The therapeutic use of ototoxic drugs is relatively common, particularly in patients with severe diseases. It is likely that disturbances of balance in these patients are underestimated by clinicians. MATERIALS AND METHODS: The purpose of this study was to identify drugs involved in the vestibulotoxic origin of instability in a group of 18 patients. RESULTS: Six patients showed both cochlear and vestibular damage, while 12 were affected only by posterior labyrinthine damage. Four groups of drugs were identified: antibiotics (nine patients), cytostatics (four), anti-tuberculosis medicinal products (three), and other drugs (two). Cytostatics were involved in many cases studied, a fact scarcely reported before. CONCLUSION: It is important to ensure an early diagnosis to prevent ototoxic effects induced by drugs. We propose that patients receiving potential ototoxic drugs undergo cochlear and vestibular assessments. Further, we recommend that patients with instability undergo vestibular rehabilitation.

Disability perception in Menière's disease: when, how much and why?

The purpose of the study was to evaluate self-perceived handicap in patients with definite Menière's disease (MD). A cross-sectional study was conducted. To examine the self-perception of disability, participants completed a DHI (Dizziness Handicap Inventory). Parameters compared with DHI scores: sex, age, unilateral/bilateral affectation, time elapsed since the onset of symptoms, pure-tone average (PTA), stages of MD, audiometric change (last 6 months), PTA in low frequencies (PTAl) and audiometric change in PTAl, subjective perception of fluctuating hearing threshold, tinnitus between attacks, number of vertiginous episodes (last 6 months), time elapsed since last attack, subjective perception of instability intercrises and Tumarkin attacks. 90 patients were included; they completed a total of 104 questionnaires. DHI scores ranged from 2 to 100 (average: 47.08, SD 24.45). In 29 cases (27.9 %) the disability perception was mild, in 43 (41.3 %) moderate, and in 32 (30.8 %) severe. Correlation between disability perception and some vestibular symptoms was found: number of typical attacks (last 6 months), time elapsed since last attack, instability intercrises and Tumarkin attacks. No relationship was found with the rest of variables. Disability perception in patients with MD depends primarily on vestibular symptoms (particularly, instability and frequency of attacks). So, we suggest to design a new staging system of MD taking into account both auditory criteria and also vestibular symptoms.

Are the three canals equally susceptible to benign paroxysmal positional vertigo?

A prospective study of patients diagnosed with 'single-canal' benign paroxysmal positional vertigo (BPPV) was performed. Demographic, etiological and prognostic differences between patients with posterior, horizontal and superior canal BPPV were studied. A total of 614 patients diagnosed in a period of 11 years and with a follow-up period of at least 1 year were included in the study. The posterior semicircular canal was affected in 543 casas (88.4%), the horizontal in 39 (6.4%) and the superior canal in 32 (5.2%). Final status at the end of the follow-up period was better in posterior canal BPPV (95% cure) than in horizontal or superior canal BPPV (87% cure in both cases). This may be explained by two facts: a poorer initial response to repositioning maneuvers in anterior canal BPPV than in BPPV involving the other two canals, and a poorer response to maneuvers in recurrences of horizontal canal BPPV.

Antioxidant properties of dimethyl sulfoxide and its viability as a solvent in the evaluation of neuroprotective antioxidants.

INTRODUCTION: Dimethyl sulfoxide is an amphiphilic compound whose miscibility with water and its ability to dissolve lipophilic compounds make it an appreciated solvent in biomedical research. However, its reported antioxidant properties raise doubts about its use as a solvent in evaluating new antioxidants. The goal of this investigation was to evaluate its antioxidant properties and carry out a comparative study on the antioxidant properties of some known neuroprotective antioxidants in the presence and absence of dimethyl sulfoxide. METHODS: The antioxidant properties of dimethyl sulfoxide were studied in rat brain homogenates by determining its ability to reduce both lipid peroxidation (TBARS formation) and protein oxidation (increase in protein carbonyl content and decrease in free thiol content) induced by ferrous chloride/hydrogen peroxide. Its ability to reduce the production of hydroxyl radicals by 6-hydroxydopamine autoxidation was also estimated. The same study was also performed with three known antioxidants (α-phenyl-N-tert-butylnitrone; 2-methyl-2-nitrosopropane; 5,5-dimethyl-1-pyrroline N-oxide) in the presence and absence of dimethyl sulfoxide. RESULTS: Our results showed that dimethyl sulfoxide is able to reduce both lipid peroxidation and protein carbonyl formation induced by ferrous chloride/hydrogen peroxide in rat brain homogenates. It can also reduce the production of hydroxyl radicals during 6-hydroxydopamine autoxidation. However, it increases the oxidation of protein thiol groups caused by ferrous chloride/hydrogen peroxide in rat brain homogenate. DISCUSSION: Despite the here reported antioxidant and pro-oxidant properties of dimethyl sulfoxide, the results obtained with α-phenyl-N-tert-butylnitrone, 2-methyl-2-nitrosopropane, and 5,5-dimethyl-1-pyrroline N-oxide corroborate the antioxidant properties attributed to these compounds and support the potential use of dimethyl sulfoxide as a solvent in the study of the antioxidant properties of lipophilic compounds. CONCLUSION: Dimethyl sulfoxide is a very useful solvent that may be used at relatively low concentrations in the development of new antioxidants with neuroprotective properties.

Brain oxidative stress and selective behaviour of aluminium in specific areas of rat brain: potential effects in a 6-OHDA-induced model of Parkinson's disease.

The ability of aluminium to affect the oxidant status of specific areas of the brain (cerebellum, ventral midbrain, cortex, hippocampus, striatum) was investigated in rats intraperitoneally treated with aluminium chloride (10 mg Al3+/kg/day) for 10 days. The potential of aluminium to act as an etiological factor in Parkinson's disease (PD) was assessed by studying its ability to increase oxidative stress in ventral midbrain and striatum and the striatal dopaminergic neurodegeneration induced by 6-hydroxydopamine in an experimental model of PD.The results showed that aluminium caused an increase in oxidative stress (TBARS, protein carbonyl content, and protein thiol content) for most of the brain regions studied, which was accompanied by a decrease in the activity of some antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase). However, studies in vitro confirmed the inability of aluminium to affect the activity of those enzymes. The reported effects exhibited a regional-selective behaviour for all the cerebral structures studied. Aluminium also enhanced the ability of 6-hydroxydopamine to cause oxidative stress and neurodegeneration in the dopaminergic system, which confirms its potential as a risk factor in the development of PD.

Study on the ability of 1,2,3,4-tetrahydropapaveroline to cause oxidative stress: Mechanisms and potential implications in relation to parkinson's disease.

Tetrahydropapaveroline (THP) is a compound derived from dopamine monoamine oxidase-mediated metabolism, particularly present in the brain of parkinsonian patients receiving L-dopa therapy, and is capable of causing dopaminergic neurodegeneration. The aim of this work was to evaluate the potential of THP to cause oxidative stress on mitochondrial preparations and to gain insight into the molecular mechanisms responsible for its neurotoxicity. Our data show that THP autoxidation occurs with a continuous generation of hydroxyl radicals (*OH) and without the involvement of the Fenton reaction. The presence of ascorbate enhances this process by establishing a redox cycle, which regenerates THP from its quinolic forms. It has been shown that the production of *OH is not affected by the presence of either ferrous or ferric iron. Although THP does not affect lipid peroxidation, it is capable of reducing the high levels of thiobarbituric acid-reactive substances obtained in the presence of ascorbate and/or iron. However, THP autoxidation in the presence of ascorbate causes both an increase in protein carbonyl content and a reduction in protein-free thiol content. THP also increases protein carbonyl content when the autoxidation occurs in the presence of iron. The remarkable role played by ascorbate in the production of oxidative stress by THP autoxidation is of particular interest.