Development of an innovative analytical method for forensic detection of cocaine, antidepressants, and metabolites in postmortem blood using magnetic nanoparticles.

Cocaine and antidepressants rank high globally in substance consumption, emphasizing their impact on public health. The determination of these compounds and related substances in biological samples is crucial for forensic toxicology. This study focused on developing an innovative analytical method for the determination of cocaine, antidepressants, and their related metabolites in postmortem blood samples, using unmodified commercial Fe3O4 nanoparticles as a sorbent for dispersive magnetic solid-phase extraction (m-d-SPE), coupled with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. An aliquot of 100 µL of whole blood and 5 µL of the internal standard pool were added to 30 mg of nanoparticles. The nanoparticles were separated from the sample using a neodymium magnet inserted into a 3D-printed microtube rack. The liquid was then discarded, followed by desorption with 300 µL of 1/1/1 acetonitrile/methanol/ethyl acetate. The sample was vortexed and separated, and 1.5 µL of the organic supernatant was injected into the LC-MS/MS. The method was acceptably validated and successfully applied to 263 postmortem blood samples. All samples evaluated in this study were positive for at least one substance. The most frequent analyte was benzoylecgonine, followed by cocaine and cocaethylene. The most common antidepressants encountered in the analyzed samples were citalopram and fluoxetine, followed by fluoxetine's metabolite norfluoxetine. This study describes the first report of this sorbent in postmortem blood analysis, demonstrating satisfactory results for linearity, precision, accuracy, and selectivity for all compounds. The method's applicability was confirmed, establishing it as an efficient and sustainable alternative to traditional techniques for forensic casework.

A validated dilute-and-shoot LC-MS/MS urine screening for the analysis of 95 illicit drugs and medicines: insights from clinical and forensic Brazilian cases.

Urine toxicological analysis is a relevant tool in both clinical and forensic scenarios, enabling the diagnosis of acute poisonings, elucidation of deaths, verification of substance use in the workplace, and identification of drug-facilitated crimes. For these analyses, the dilute-and-shoot technique associated with liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) is a promising alternative since it has demonstrated satisfactory results and broad applicability. This study developed and validated a comprehensive LC-MS-MS screening method to analyze 95 illicit drugs and medicines in urine samples and application to clinical and forensic Brazilian cases. The dilute-and-shoot protocol was defined through multivariate optimization studies and was set using 100 µL of sample and 300 µL of solvent. The total chromatographic run time was 7.5 minutes. The method was validated following the recommendations of the ANSI/ASB Standard 036 Guideline. The lower limits of quantification varied from 20 to 100 ng/mL. Within-run and between-run precision CVs% were lower than 20%, and bias was within ± 20%. Only four of the 95 analytes presented significant ionization suppression or enhancement (> 25%). As proof of applicability, 839 urine samples from in vivo and postmortem cases were analyzed. In total, 90.9% of the analyzed samples were positive for at least one substance, and 78 of the 95 analytes were detected. The most prevalent substances were lidocaine (40.2%), acetaminophen (38.0%), and benzoylecgonine (31.5%). The developed method proved to be an efficient and simplified alternative for analyzing 95 therapeutic and illicit drugs in urine samples. Additionally, the results obtained from sample analysis are essential to understanding the profile of Brazilian substance use, serving as a valuable database for the promotion of health and safety public policies.

A partial agonist of PPARγ prevents paclitaxel-induced peripheral neuropathy in mice, by inhibiting neuroinflammation.

BACKGROUND AND PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of paclitaxel, affecting 30-50% of patients. Increased survival and concern with patients' quality of life have encouraged the search for new tools to prevent paclitaxel-induced neuropathy. This study presents the glitazone 4-[(Z)-(2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl]-N-phenylbenzene-sulfonamide (TZD-A1) as a partial agonist of peroxisome proliferator-activated receptor γ (PPARγ), its toxicological profile and effects on paclitaxel-induced CIPN in mice. EXPERIMENTAL APPROACH: Interactions of TZD-A1 with PPARγ were analysed using in silico docking and in vitro reporter gene assays. Pharmacokinetics and toxicity were evaluated using in silico, in vitro and in vivo (C57Bl/6 mice) analyses. Effects of TZD-A1 on CIPN were investigated in paclitaxel-injected mice. Axonal and dorsal root ganglion damage, mitochondrial complex activity and cytokine levels, brain-derived neurotrophic factor (BDNF), nuclear factor erythroid 2-related factor 2 (Nrf2) and PPARγ, were also measured. KEY RESULTS: Docking analysis predicted TZD-A1 interactions with PPARγ compatible with partial agonism, which were corroborated by in vitro reporter gene assays. Good oral bioavailability and safety profile of TZD-A1 were shown in silico, in vitro and in vivo. Paclitaxel-injected mice, concomitantly treated with TZD-A1 by i.p. or oral administration, exhibited decreased mechanical and thermal hypersensitivity, effects apparently mediated by inhibition of neuroinflammation and mitochondrial damage, through increasing Nrf2 and PPARγ levels, and up-regulating BDNF. CONCLUSION AND IMPLICATIONS: TZD-A1, a partial agonist of PPARγ, provided neuroprotection and reduced hypersensitivity induced by paclitaxel. Allied to its safety profile and good bioavailability, TZD-A1 is a promising drug candidate to prevent and treat CIPN in cancer patients.

Cork sheet as an efficient biosorbent for forensic toxicology: Application to vitreous humor analysis.

There is an increasing number of people affected worldwide by mental health disorders, such as depression and anxiety. One of the main courses of treatment, along with psychotherapy, is the use of psychoactive medications, like antidepressants and benzodiazepines. Also, the unprescribed use of these substances is a concerning public health issue. Hence, the analysis of psychotropic medications is mandatory in postmortem toxicology and various biological samples can be used for this detection, among them the vitreous humor (VH) stands out. Also, there is a demand for more sustainable and more efficient extraction methodologies according to green chemistry. An example is solid phase microextraction techniques (SPME), which use a solid sorbent and small solvent amounts. Biosorbents are substances of natural origin with sorptive properties, and they have been successfully used in SPME in environmental toxicology for water analysis, mainly. This study aimed to develop a sustainable, fast, cheap and simple SPME methodology using cork sheet strips as a biosorbent, to extract antidepressants, benzodiazepines and others from VH samples by liquid chromatography coupled to tandem mass spectrometry. The extraction was conducted in a 96-well plate using 200 µL of VH and optimization of relevant parameters for extraction was performed. For solvent optimization, two simplex-centroid experiments were planned for extraction and desorption and to evaluate time and pH, a Doehlert design experiment was performed. The analytical method for the determination and quantification of 17 substances was validated. The quantification limits were 5 ng/mL for all analytes and the calibration curves were linear between 5 and 30 ng/mL. This study was able to develop an efficient, cheap, simple and fast microextraction method for 17 analytes in VH, using strips of cork sheet for extraction and a 96-well plate as a container. Furthermore, this approach system could be automated for routine toxicology laboratories.

A new approach with dried plasma spots: a rapid determination of antidepressants and antipsychotics by fast GC-MS.

Background: The increasing prevalence of poisoning cases related to antidepressants and antipsychotics has raised concerns. Methods: To address this issue, a new adaptation of the dried plasma spot technique was developed using a 24-well plate and fast gas chromatography-mass spectrometry. The method involves the optimization of extraction variables and sample preparation, and was successfully validated. Results: The limits of quantitation ranged from 20 to 60 ng/ml, and accuracy ranged from 87.8% to 112.2%. The technique was applied to 102 human plasma samples from suspected poisoning cases, with positivity of 90.2%. Conclusion: This method provides a cheap, easy to implement and fast approach, making it ideal for toxicological emergency laboratories and promoting valuable support for healthcare professionals managing poisoning cases involving antidepressants and antipsychotics.

An affordable and semiautomated approach as a novel strategy for the extraction of DNA using magnetic ionic liquids followed by real time-polymerase chain reaction.

This technical note describes a novel and straightforward experimental strategy for the extraction/capture of DNA using magnetic ionic liquid (MIL) followed by real time-polymerase chain reaction (qPCR) analysis. An affordable and low-cost magneto-based multiwell platform was first examined for capturing DNA allowing for simultaneous extractions that increased the analysis throughput of the experimental workflow. This configuration was composed of a series of neodymium rod magnets attached to a multiwell device in which a magneto-active extraction phase (MIL) was suspended for a single drop microextraction (SDME) approach. In this configuration, up to 32 extractions were able to be performed simultaneously, and DNA was successfully extracted from aqueous samples. Furthermore, as a proof-of-concept, this affordable and simple experimental strategy proved to be efficient for the extraction/capture of DNA from challenging samples such as whole blood without any pretreatment. This fact also consists of important feature compared to previous methodologies that required additional steps of sample preparation.

Cannabidiol treatment improves metabolic profile and decreases hypothalamic inflammation caused by maternal obesity.

Introduction: The implications of maternal overnutrition on offspring metabolic and neuroimmune development are well-known. Increasing evidence now suggests that maternal obesity and poor dietary habits during pregnancy and lactation can increase the risk of central and peripheral metabolic dysregulation in the offspring, but the mechanisms are not sufficiently established. Furthermore, despite many studies addressing preventive measures targeted at the mother, very few propose practical approaches to treat the damages when they are already installed. Methods: Here we investigated the potential of cannabidiol (CBD) treatment to attenuate the effects of maternal obesity induced by a cafeteria diet on hypothalamic inflammation and the peripheral metabolic profile of the offspring in Wistar rats. Results: We have observed that maternal obesity induced a range of metabolic imbalances in the offspring in a sex-dependant manner, with higher deposition of visceral white adipose tissue, increased plasma fasting glucose and lipopolysaccharides (LPS) levels in both sexes, but the increase in serum cholesterol and triglycerides only occurred in females, while the increase in plasma insulin and the homeostatic model assessment index (HOMA-IR) was only observed in male offspring. We also found an overexpression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNFα), interleukin (IL) 6, and interleukin (IL) 1ß in the hypothalamus, a trademark of neuroinflammation. Interestingly, the expression of GFAP, a marker for astrogliosis, was reduced in the offspring of obese mothers, indicating an adaptive mechanism to in utero neuroinflammation. Treatment with 50 mg/kg CBD oil by oral gavage was able to reduce white adipose tissue and revert insulin resistance in males, reduce plasma triglycerides in females, and attenuate plasma LPS levels and overexpression of TNFα and IL6 in the hypothalamus of both sexes. Discussion: Together, these results indicate an intricate interplay between peripheral and central counterparts in both the pathogenicity of maternal obesity and the therapeutic effects of CBD. In this context, the impairment of internal hypothalamic circuitry caused by neuroinflammation runs in tandem with the disruptions of important metabolic processes, which can be attenuated by CBD treatment in both ends.

Development and validation of a dried plasma spot LC-MS/MS method for therapeutic monitoring of vancomycin and comparison with enzyme-multiplied immunoassay.

Vancomycin is used as an antimicrobial agent for the treatment of severe gram-positive infections. The importance of therapeutic monitoring of antimicrobials has led to the development of more specific sample preparation techniques capable of identifying with accuracy the concentration of this substance in the organism. An aliquot of 10 µl of plasma was transferred to Whatman 903 paper and dried at room temperature. The extraction method was performed by cutting and transferring the paper to a microtube and adding sodium phosphate buffer and internal standard. The mixture was shaken and centrifuged, and a 5-µl aliquot was injected into the analytical system. The optimization of the main parameters that can influence the extraction efficiency was performed using multivariate approaches to obtain the best conditions. The method developed was validated, providing coefficients of determination higher than 0.994 and a lower limit of quantification of 1 mg/L. Within- and between-run precision ranged from 11.4 to 17.30% and from 6.65 to 13.51%, respectively. This method was successfully applied to 75 samples of patients undergoing vancomycin therapy. The method was rapid, simple, and environmentally friendly with satisfactory analytical performance and was advantageous over the laborious and time-consuming methodologies used in therapeutic drug monitoring routine analyses.

A multi-analyte LC-MS/MS method for the determination of 57 pharmaceuticals and illicit drugs in plasma, and its application to poisoning cases.

The diagnostic methods in an emergency scenario must be simple, fast, and efficient to provide an effectiveness and efficient treatment, thus reducing the consequences of exposure. Considering the sample analysis, the protein precipitation combined with LC-MS/MS has been shown to be a good strategy for the simultaneous determination of compounds of toxicological interest, such as medicines and drugs of abuse. In this study, a rapid and simple multi-analyte method was developed and validated for the quantification of 57 pharmaceuticals and illicit drugs in plasma samples. Sample pre-treatment consists of protein precipitation of 50 µL of the sample with 240 µL of organic solvent mixture (MeOH:ACN, 3:1, v/v), centrifugation, and injection into the LC-MS/MS, with a chromatographic run time of 7 min. The method was validated considering lower limit of quantification (LLOQ), interferences, linearity, precision, accuracy, dilution integrity, carryover, and matrix effect. The LLOQs ranged from 5 to 20 ng/mL and all analytes were linear (r2>0.99) in the tested concentration ranges. The method proved to be precise and accurate, presenting QC concentrations for all analytes within acceptable limits by the guideline used (CV % ≤20 % and bias ± 20 %). The developed method was successfully applied in 470 plasma samples of real cases of poisoning. A total of 80 % of the samples were positive for at least one substance, with acetaminophen (32.1 %), diazepam (25.1 %), and lidocaine (18.9 %) being the most detected. The most prevalent exposure circumstance among the cases was suicide attempt. The most frequent age groups were young adults between 20 and 29 years old and children under 5 years old. The methodology developed proved to be efficient in the simultaneous determination of 57 substances of toxicological interest, contributing to a correct diagnosis and, consequently, to the most appropriate management and treatment of the intoxicated patient. Furthermore, it is possible to observe the most commonly involved toxic agents in the Rio Grande do Sul, southern Brazil, helping to trace a profile of the poisoning patient, important in toxicovigilance actions.

Zinc Supplementation Partially Decreases the Harmful Effects of a Cafeteria Diet in Rats but Does Not Prevent Intestinal Dysbiosis.

Zinc (Zn) plays an important role in metabolic homeostasis and may modulate neurological impairment related to obesity. The present study aimed to evaluate the effect of Zn supplementation on the intestinal microbiota, fatty acid profile, and neurofunctional parameters in obese male Wistar rats. Rats were fed a cafeteria diet (CAF), composed of ultra-processed and highly caloric and palatable foods, for 20 weeks to induce obesity. From week 16, Zn supplementation was started (10 mg/kg/day). At the end of the experiment, we evaluated the colon morphology, composition of gut microbiota, intestinal fatty acids, integrity of the intestinal barrier and blood-brain barrier (BBB), and neuroplasticity markers in the cerebral cortex and hippocampus. Obese rats showed dysbiosis, morphological changes, short-chain fatty acid (SCFA) reduction, and increased saturated fatty acids in the colon. BBB may also be compromised in CAF-fed animals, as claudin-5 expression is reduced in the cerebral cortex. In addition, synaptophysin was decreased in the hippocampus, which may affect synaptic function. Our findings showed that Zn could not protect obese animals from intestinal dysbiosis. However, an increase in acetate levels was observed, which suggests a partial beneficial effect of Zn. Thus, Zn supplementation may not be sufficient to protect from obesity-related dysfunctions.

Liquid chromatography-tandem mass spectrometry method for simultaneous quantification of neurotransmitters in rat brain tissue exposed to 4'-fluoro-α-PHP.

The combination of different advanced analytical techniques makes it possible to determine the concentrations of neurotransmitters in various biological matrices, providing a complex and comprehensive study of the effects of psychoactive substances. The present study aimed to develop and validate a fast and simple analytical method for the determination of acetylcholine, serotonin, γ-aminobutyric acid, glutamate, dopamine and metabolites in rats brain tissue by liquid chromatography coupled to tandem mass spectrometry. The brain was homogenized and aliquots of the sample, dopamine-d4 , and acetone were added to a tube and then vortexed and centrifuged. The supernatant was collected and dried. The residue was reconstituted and injected. The LLOQ ranged from 0.001 to 1 µg/g; the intra-run precision ranged from 0.47 to 11.52%; the inter-run precision ranged from 0.68 to 17.54%; and the bias ranged from 89.10 to 109.60%. As proof of concept, the method was applied to animals exposed to the synthetic cathinone 4'-fuoro-α-pyrrolidinohexanophenone (300 mg/kg). In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of neurotransmitters. This analytical tool can be used to support the investigation of the changes in the neurochemical profile for the characterization of the mechanism of action of psychoactive substances, as well as both neurological and psychiatric diseases.

Toxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor inhalation on rheumatoid arthritis.

Tobacco combustion exposure worsens rheumatoid arthritis (RA). Non-combustible tobacco devices, as heat-not-burn tobacco (HNBT), are emerging as harm reduction to smokers by releasing nicotine and lower combustible tobacco products. Nevertheless, HNBT toxicity remains unclear. Hence, here we investigated the impacts of the tobacco combustible product (cigarette smoke; CS) or HNBT vapor exposures on antigen-induced arthritis (AIA) in C57BL/6 mice. Animals were exposed to airflow, HNBT vapor, or CS during 1 h/twice a day, under the Health Canada Intense (HCI) smoking regime, between days 14 to 20 after the first immunization. At day 21, 16 h after the last exposures, mice were i.a. challenged and the AIA effects were evaluated 24 h later. CS- or HNBT-exposed mice presented equivalent blood nicotine levels. CS exposure worsened articular symptoms, pulmonary inflammation, and expression of lung metallothioneins. Nevertheless, CS or HNBT exposures reduced lymphoid organs' cellularity, splenocyte proliferation and IL-2 secretion. Additional in vitro CS or HNBT exposures confirmed the harmful effects on splenocytes, which were partially mediated by the activation of nicotine/α7nAchR pathway. Associated, data demonstrate the toxic mechanisms of CS or HNBT inhalation at HCI regime on RA, and highlight that further investigations are fundamental to assure the toxicity of emerging tobacco products on the immune system during specific challenges.

Decreased Native T1 Values and Impaired Myocardial Contractility in Anabolic Steroid Users.

Anabolic androgenic steroid (AAS) abuse leads to myocardial toxicity. Human studies are conflicting about the myocardial fibrosis in AAS users. We evaluated cardiac tissue characterization, left ventricle (LV) function, and cardiac structure by cardiovascular magnetic resonance (CMR). Twenty strength-trained AAS users (AASU) aged 29±5 yr, 20 strength-trained AAS nonusers (AASNU), and 7 sedentary controls (SC) were enrolled. Native T1 mapping, late-gadolinium enhancement (LGE), extracellular volume (ECV), and myocardial strain were evaluated. AASU showed lower Native T1 values than AASNU (888±162 vs. 1020±179 ms p=0.047). Focal myocardial fibrosis was found in 2 AASU. AASU showed lower LV radial strain (30±8 vs. 38±6%, p<0.01), LV circumferential strain (-17±3 vs. -20±2%, p<0.01), and LV global longitudinal strain (-17±3 vs. -20±3%, p<0.01) than AASNU by CMR. By echocardiography, AASU demonstrated lower 4-chamber longitudinal strain than AASNU (-15±g3 vs. -18±2%, p=0.03). ECV was similar among AASU, AASNU, and SC (28±10 vs. 28±7 vs. 30±7%, p=0.93). AASU had higher LV mass index than AASNU and SC (85±14 vs. 64±8 vs. 58±5 g/m2, respectively, p<0.01). AAS abuse may be linked to decreased myocardial native T1 values, impaired myocardial contractility, and focal fibrosis. These alterations may be associated with maladaptive cardiac hypertrophy in young AAS users.

Application of Homogeneous Liquid-Liquid Microextraction With Switchable Hydrophilicity Solvents to the Determination of MDMA, MDA and NBOMes in Postmortem Blood Samples.

Synthetic drugs for recreational purposes are in constant evolution, and their consumption promotes a significant increase in intoxication cases, resulting in damaging public health. The development of analytical methodologies to confirm the consumption of illicit drugs in biological matrices is required for the control of these substances. This work exploited the development of an extraction method based on homogenous liquid-liquid microextraction with switchable hydrophilicity solvent (SHS) as extraction phase for the determination of the synthetic drugs 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and N-methoxybenzyl-methoxyphenylethylamine derivates (25B, 25C and 25I) in postmortem blood, followed by liquid chromatography coupled to mass spectrometry in tandem. The optimized sample preparation conditions consisted of using 250 µL of ZnSO4 10% and 50 µL of NaOH 1 mol/L in the protein precipitation step; N,N-dimethylcyclohexylamine was used as SHS, 650 µL of a mixture of SHS:HCl 6 mol/L (1:1 v/v), 500 µL of whole blood, 500 µL of NaOH 10 mol/L and 1 min of extraction time. The proposed method was validated, providing determination coefficients higher than 0.99 for all analytes; limit of detection and limit of quantitation ranged from 0.1 to 10 ng/mL; intra-run precision from 2.16% to 9.19%; inter-run precision from 2.39% to 9.59%; bias from 93.57% to 115.71% and matrix effects from 28.94% to 51.54%. The developed method was successfully applied to four authentic postmortem blood samples from synthetic drugs users, and it was found to be reliable with good selectivity.

Multivariate analysis applied in dataset of Poison Control Center of São Paulo, Brazil.

Multivariate analysis techniques could be used to identify possible intercorrelations in intoxications cases. The statistical analyses used were a multiple logistic regression, multiple correspondence analysis, principal component and hierarchical cluster analysis. Of the 320 samples analysed, 192 samples were positive for some of the investigated toxic agents, of which 100 were positive for ethanol and 131 were positive for other substances. It was possible to group the patients into 3 clusters, which appears 66.5% of this information in the three first factorial axes. On the first axis, the male patients were separated from the female patients. Patients exposed to drugs, between 30 and 39 years old were grouped in the same cluster. On the second factorial axis, patients who were intoxicated with ethanol and who became intoxicated with diazepam were grouped. This work contributed to the mapping of intoxication cases at the Poison Control Centre of the São Paulo city, Brazil (CCI-SP) and serves as an initial study for the creation of a database that could be updated constantly and thus could provide a toxicovigilance system for educational policies.

A straightforward and semiautomated membrane-based method as efficient tool for the determination of cocaine and its metabolites in urine samples using liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry.

In this study, a novel and straightforward analytical methodology was proposed for the determination of cocaine (COC) and its main metabolites benzoylecgonine (BZE) cocaethylene (CE) and hydroxy­cocaine (COCOH) in urine samples. This approach consisted of a high-throughput and semiautomated configuration based on hollow-fiber renewal liquid membrane extraction (HFRLM) coupled to a 96-well plate system, which was proposed for the first time to analyze complex biological samples such as urine. The analytical determinations were performed using ultra-high performance liquid chromatography coupled to quadrupole time-of-flight-mass spectrometry (LC-ESI-QTOF-MS). The analytical methodology was fully optimized through Doehlert and simplex-centroid designs, and univariate approaches. Polypropylene membranes of 1 cm length were inserted in the pins of an extraction blade combined with a 96-well plate system and its pores were filled with hexane:dichloromethane:ethyl acetate (1:1:1 v/v/v) for 180 s; moreover, 20 µL of this mixture was added to the sample to allow for a renewable liquid membrane. The extraction step was carried out by keeping the blades immersed in vials containing 1.5 mL of diluted urine adjusted at pH 10 with 10% (w/v) of Na2CO3 during 20 min, followed by liquid desorption with 100 µL of acetonitrile. Finally, the extract was dried under N2 stream and resuspended with 20 µL of ultrapure water. Satisfactory analytical performance was obtained with coefficients of determination ranging from 0.9875 for BZE to 0.9986 for CE; intra-day precision ranged from 1.6 to 13.5%, and inter-day precision varied from 2.2 to 17.5%. Limits of detection ranged from 1.5 to 15.1 ng mL-1, and limits of quantification varied from 5 to 50 ng mL-1, with relative recoveries varied from 70.7 to 124.1%.

Stability in clinical use and stress testing of meropenem antibiotic by direct infusion ESI-Q-TOF: Quantitative method and identification of degradation products.

An ESI-MS/MS method through direct infusion was validated for quantitative analysis of meropenem powder for injection. The validation parameters were established in a rapid analysis of 30 s. Drug stability was studied through the submission to stress testing, resulting on four degradation products. Under hydrolytic conditions, in acid, neutral and alkaline media, the major degradation product was formed through the cleavage of the ß-lactam ring. Oxidation of the drug using H2O2 (3%) showed the formation of two degradation products from a decarboxylation reaction and N-oxide formation. Under high humidity conditions, there was detected a dimer product. The stability of meropenem after reconstitution was studied in conditions that simulate its clinical use. In samples reconstituted and diluted in infusion fluids, an extensive degradation was observed. At room temperature meropenem maintained its content > 90% for up to 4 h when prepared in 5% glucose and for up to 12 h when prepared in 0.9% NaCl. Through ESI-MS/MS analyzes it was observed a degradation product formed by ß-lactam ring cleavage, detected in all conditions studied. It was also identified a degradation product formed only in 5% glucose, generated by the hydrolysis of ß-lactam followed by the attachment of a glucose molecule to the nitrogen of the pyrrolidine ring. In general, all the results obtained in the stability studies contribute to the knowledge about this antibiotic and future candidates of this class.

Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study.

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets.