Toxicological Analysis Research Articles

LC-MS/MS-MS3 for the Determination and Quantification of Δ9-Tetrahydrocannabinol and Metabolites in Blood Samples.

Due to the high prevalence of cannabinoids in forensic toxicology analysis, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation, for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry methodology (LC-MS/MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9- tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH), in blood samples. Chromatographic analysis of THC, THC-OH, and THC-COOH and their deuterated internal standards was preceded by protein precipitation (PPT) of 0.1 mL of blood samples with acetonitrile. Chromatographic separation was achieved by use of an Acquity UPLC® HHS T3 (100 mm x 2.1mm i.d., 1.8 μm) reversed-phase column, using a gradient elution of 2 mM aqueous ammonium formate, 0.1% formic acid, and methanol at a flow rate of 0.4 mL/min, with a run time of 10 minutes. For the MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ triple quadrupole linear ion trap mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction monitoring (MRM) and linear ion trap mode (MS3). The method was validated in accordance with internationally accepted criteria and guidelines, and proved to be selective and linear between 0.5-100 ng/mL (r2>0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were <15%. The mean recoveries were between 88.0-117.2%, for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantification of cannabinoids in blood, using LC-MS/MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.

Determination of benzodiazepines and Z-hypnotic drugs in whole blood samples by GC–MS/MS: Method development, validation and application

Benzodiazepines (BZDs) and Z-drugs are widely used as anxiolytics, sedative hypnotics, anticonvulsants and muscle relaxants. “Designer benzodiazepines” (DBZDs) are a new psychoactive substance class consisting of benzodiazepine derivatives that are not allowed for medical use and are known for being used recreationally. From a toxicologist standpoint, the huge number of such substances implicate a necessity for developing fast and reliable methods to identify and quantify these substances in biological matrices, especially in blood.In this study, a method based on gas chromatography–tandem mass spectrometry (GC–MS/MS) was developed and validated for the determination of 28 benzodiazepine derivatives and 3 Z- drugs. Liquid–liquid extraction requiring the use of a 0.5 mL sample of whole blood and 1 mL of ethyl acetate was applied. No derivatization was necessary to obtain sensitivity and selectivity.All validation data met the established acceptance criteria in accordance with international forensic toxicology guidelines. The method was linear within the tested range of 1–100 ng/mL, 1–200 ng/mL, 1–250 ng/mL, 5–200 ng/mL and 10–250 ng/mL depending on the analyte. The LOD was in the range of 0.09 to 0.66 ng/mL, while the LOQ was between 1 and 10 ng/mL. The calculated mean accuracy ranged between 85.1% and 114.4% for intraday replicates and between 85.2% and 111.2% for interday replicates. Intraday precision (as CVs) ranged from 0.2% to 12.5%, while interday precision ranged from 2.0% to 14.5%. Recovery was between 83.8% and 111.2%.This study was the first attempt to apply GC–MS/MS for the multianalyte determination of benzodiazepines and Z-drugs without analyte derivatization. The developed method proved to be sensitive, specific, simple and fast. The analysis in MRM mode gives high sensitivity and selectivity. Thus, the presented method can be successfully utilized for clinical and forensic toxicology analysis, for interpretation ofresults and for the preparation of forensic toxicological opinions for courts of law.

Anti-Leishmania amazonensis Activity, Cytotoxic Features, and Chemical Profile of Allium sativum (Garlic) Essential Oil.

Human tegumentary leishmaniasis (HTL) is a serious tropical disease caused by Leishmania amazonensis. Developing new leishmanicidal agents can help overcome current treatment challenges, such as drug resistance and toxicity. Essential oils are a source of lipophilic substances with diverse therapeutic properties. This study aimed to determine the anti-L. amazonensis activity, cytotoxicity, and chemical profile of Allium sativum essential oil (ASEO). The effect of ASEO on parasite and mammalian cells viability was evaluated using resazurin and MTT assays, respectively. The oil's effect against intracellular amastigotes was also determined. Transmission electron microscopy was used to assess the ultrastructural changes induced by ASEO. In addition, the chemical constituents of ASEO were identified by gas chromatography-mass spectrometry (GC-MS). The cytotoxic potential was evaluated in vitro and in silico. The oil displayed IC50 of 1.76, 3.46, and 3.77 µg/mL against promastigotes, axenic, and intracellular amastigotes, respectively. Photomicrographs of treated parasites showed plasma membrane disruption, increased lipid bodies, and autophagic-like structures. ASEO chemical profiling revealed 1,2,4,6-tetrathiepane (24.84%) and diallyl disulfide (16.75%) as major components. Computational pharmacokinetics and toxicological analysis of ASEO's major components demonstrated good oral bioavailability and better toxicological endpoints than the reference drugs. Altogether, the results suggest that ASEO could be an alternative drug candidate against HTL.

A new application of the switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction to analyze synthetic cannabinoids in plasma by LC-MS/MS

Synthetic cannabinoids are still a growing trend among drug users and consist of a group of hundreds of highly potent compounds. To investigate the use of such substances, sample preparation of biological matrices is a crucial step prior to instrumental analysis. Although different efficient extraction techniques have been proposed for that aim, they usually do not fit eco-friendly guidelines that have been gaining popularity in recent years, such as Green Analytical Toxicology. This work uses describes for the first time the use of switchable hydrophilicity solvent-based homogenous liquid–liquid microextraction (SHS-HLLME) for synthetic cannabinoids. This is a green technique that replaces highly toxic organic reagents for switchable hydrophilicity solvents (SHS), substances that can be either water-miscible or immiscible depending on their protonation. Thus, by simply adjusting the pH of the system, these SHS can be used as extraction solvents. A full optimization study including type of SHS, volume of protonated SHS, volume of NaOH, salting-out effect, and extraction time was performed. The optimized procedure consisted of precipitating the proteins of 300 µL of plasma with 300 µL of acetonitrile followed by centrifugation; evaporation of the organic solvent under N2 stream; addition of 500 µL of the protonated DPA, DPA-HCl (6 M) (1:1, v/v); addition of 500 µL of NaOH (10 M); and finally centrifugation and evaporation. Validation results showed determination coefficients ≥ 0.99 for the 0.1–10 ng/mL linear range; 0.01–0.08 ng/mL as limit of detection; 0.1 ng/mL as limit of quantitation; accuracy and imprecision were within acceptable ranges; matrix effect, recovery, and process efficiency ranged from −55.6 to 185.9%, 36–56.7%, and 18.5–148.4%, respectively. The SHS-HLLME herein described was fully optimized providing satisfactory recoveries of 31 synthetic cannabinoids at low concentrations requiring only 300 µL of plasma. In addition, the validation results showed that the technique is a reliable eco-friendly alternative for clinical and toxicological analysis.

An Estimation of the Antiviral Activity and Toxicity of Biologically Active Substances Obtained from the Raw Materials of Artemisia cina Berg. In Vitro and In Vivo.

Species of the genus Artemisia are well known for their use as ingredients in ancient medicine. The advantage of using plant extracts compared to individual pharmaceutical ingredients is the rate of adaptation of the pathogenic microorganisms to the drug. Due to the rapid development of multidrug-resistance in microorganisms in the field, it is essential to search for novel, effective drugs with low toxicity. Therefore, the purpose of this study was to isolate and study the biologically active substances obtained from various substances in the raw materials of Artemisia cina Berg. The identification of the main biologically active components was performed using the method of chromato-mass spectrometry. Moreover, the antiviral activity of several extracts was studied using the method of measuring limiting dilutions (the Reed-Mench method), with some modifications. For the first time, the biological activity of extracts from the raw material of Artemisia cina Berg. upon the SARS-CoV-2 virus was confirmed. All the obtained extracts exhibited nontoxic effects in animals, with an LD50 greater than 2 g/kg. Comprehensive toxicological analyses are also presented in the study, such as those of the biochemical parameters of urine after one day and one week of the extracts' administration in mice at a dose of 2 g/kg body weight. In all groups of animals that received extracts of Artemisia cina Berg., a slight increase in the presence of red blood cells in their urine was observed one day following the administration of the extracts. This increase decreased somewhat after a week; however, it remained higher than the levels observed in the control animals. In the three groups, there was also a slight increase in the amount of ketones in the urine. Two weeks following the administration of the extracts to these groups, the internal organs of the animals were examined. The examination showed that the internal organs of the animals that received the extracts were not visibly different from those of the control animals in terms of their size or appearance. The weight of the internal organs of the animals that received the extracts was also similar to the weight of the internal organs of the control animals, illustrating the absence of toxicity.

Fatal concentrations of antidepressant and antipsychotic drugs in post-mortem femoral blood.

Antidepressants and antipsychotics are both an important class of prescription drugs within post-mortem (PM) toxicology because most of the substances are toxic in overdose and the mental disorders being treated may be associated with suicidality. A wide range of antidepressants and antipsychotics are currently included in up-to-date PM toxicology analysis protocols. However, apart from case studies, few reports on fatal concentrations based on large number of cases have been published in the literature. Based on PM investigations in Finland between 2000-2020, this study provides fatal reference concentrations in poisonings due to an antidepressant or an antipsychotic drug assigned as the principal intoxicant. Summary statistics for drug concentrations in PM femoral blood (min, max, mean, 10th, 25th, 50th, 75th, 90thpercentile) were calculated for 17 antidepressant (N = 2007) and for 12 antipsychotic drugs (N = 1161). The proportion of suicide, accident and undetermined manner of death is indicated for each drug. Further, the fatal concentrations obtained in this study were evaluated by comparison with fatal and "normal" PM concentrations reported by two previously published approaches, the grouped causes of death approach and the all causes of death approach, respectively. This study shows that, despite the well-known variation in PM drug concentrations, competently generated fatal concentration results for the drugs studied are consistent to such an extent that they can be used as a reference in the interpretation process.

An Autopsy Case of Drowning Under the Influence of Etizolam: A Case Report

A fatal case of drowning under the influence of etizolam is presented. Quantitative toxicological analysis revealed etizolam concentrations of 0.50 µg/mL and 0.068 µg/mL in femoral venous blood and urine, respectively. According to the autopsy findings, the results of toxicological examinations, and the investigation by the authorities, it is concluded that the cause of death is drowning under the influence of etizolam.

Luminescence Toxicological Analysis of Water Supply Systems in Dispersed Rural Areas: A Case Study in Boyacá, Colombia

The quality of water supply systems is still a major problem in developing countries, especially in rural areas. The acute bioluminescence V. fischeri inhibition assay is widely recognized as a toxicological method that can be used to detect the acute effects of different contaminants. In this study, the physicochemical characteristics and toxicology of 72 water samples collected in 18 rural aqueducts located in Boyacá (Colombia) were evaluated. The primary economic activities identified as potential influencers of water quality in the water supply basins were agriculture (n = 3), livestock (n = 2), and domestic sewage discharge (n = 1). The average luminescence inhibition rate was 66%, with a minimum of 29%, and a maximum of 97%. A total of 85% of the tested samples (n = 61) had “moderate acute hazard”, while 15% (n = 15) had “acute hazard”. A total of 95% of the aqueducts distributed water with high risk. There was a weak positive correlation between the apparent color and the V. fischeri inhibition rate (p &lt; 0.05). The water treatments, including disinfection, and the economic activities had no correlation with the inhibition rate of luminescent bacteria. The results of this investigation can be used by sanitary authorities to incorporate future toxicological monitoring of chemical contaminants, such as humic substances and metals, into water-quality monitoring in rural areas.

Pulmonary capillaritis as a cause of lethal diffuse alveolar hemorrhage.

Lethal pulmonary hemoptysis is a common finding in forensic medicine. Since hemoptysis does not necessarily occur before death and its previous symptoms are usually unspecific, indicative signs or findings at the site of a corpse may be completely absent. If lethal acute alveolar hemorrhage is found in the post-mortem examination, a differential diagnosis should be made for traumatic, substance-related, infectious, or organic causes. Cocaine-associated pulmonary hemorrhage and drug reactions are in the foreground. For organic causes, autoimmune diseases should be considered in addition to cardiac, haematological, and infectious causes. The following two cases show similarities both in the sudden, unexpected deaths and the anamnesis of the two deceased women. One of the deceased received a Corona vaccination a few months earlier. In each case, the post-mortem examination revealed an acute diffuse pulmonary hemorrhage caused by acute inflammation of the lung capillaries. This case presentation demonstrates the necessity of a complete autopsy including toxicological and histological analyses. The documentation and publication of rare causes of death are essential for medical research and practice in order to critically consider and discuss the possibility of to date unknown associations in similar cases.

Oral Cavity Fluid as an Alternative Postmortem Matrix: Comparison to Simultaneously Collected Blood and Urine Specimens.

In postmortem toxicology analysis, a variety of specimens consisting of fluids and tissues are often collected, each with an intrinsic value. Oral cavity fluid (OCF) is emerging as an alternative matrix in forensic toxicology for contributing to a diagnosis in postmortem cases; especially when blood is limited or not available. The aim of this study was to assess the analytical results obtained from OCF and compare them with blood, urine, and other traditional matrices collected from the same postmortem subjects. Of the 62 decedents studied (including 1 stillborn, 1 charred, and 3 decomposed subjects), 56 had quantifiable drugs and metabolites data in the OCF, blood, and urine. Notable findings were benzoylecgonine (24 cases), ethyl sulfate (23 cases), acetaminophen (21 cases), morphine (21 cases), naloxone (21 cases), gabapentin (20 cases), fentanyl (17 cases), and 6-acetylmorphine (15 cases), which were detected more frequently in OCF than in blood (heart, femoral, or body cavity) or urine. This study suggests that OCF is a suitable matrix for detecting and quantifying analytes in postmortem subjects compared with traditional matrices, particularly when other matrices are limited or difficult to collect because of body condition or putrefaction.

Homicide by Hanging: a Comprehensive Review of Published Literature

Homicide by hanging is a rare incident and, in contrast to suicidal hanging cases, only limited number of homicidal hanging cases have been reported. This comprehensive review is conducted to summarize the published forensic pathology evidence in homicide by hanging. A systematic search in five journal databases resulted in 12 case reports. All articles passed the selection criteria and were appraised using JBI critical appraisal worksheet for case reports. Fourteen victims were reported which in most of the cases psychosocial background were reported. Household conflict was the common background. Reef knot was the majority knot being used. The autopsy findings vary in the studies, some of which reported intact larynx, thyroid, and hyoid cartilage. Only one case mentioned thyroid cartilage fracture. In addition to the ligature mark, other violence findings include bruises, abrasions, stab wounds, and sexual violence. Toxicological analysis was limited, only 3 case reports reported alcohol in the victim’s serum. There are no distinguishing features in the external or internal neck injuries attributable to homicidal hanging. A thorough autopsy and holistic approach should be considered. They are including demographic characteristics, location of the victim, social and psychiatric history, ligature’s characteristic, signs of violence and toxicological analysis.

From unknown poisoning to carbamazepine poisoning

In the 2020 annual report of the American Association of Poison Control Centers, 2562 toxic exposures to carbamazepine have been reported, 908 resulted in hospitalization, and among these about 5-6% were life-threatening or resulted in significant disability. A 15-year-old female patient was brought under suspicion of alcohol poisoning. The result of alcoholemia was 11.0mg/dL and the toxicological screening for THC, opiates, methadone, tramadol, amphetamine, MDMA, cocaine, benzodiazepines, buprenorphine was negative. At admission she was comatose (GCS=6), with metabolic acidosis, hypotension, rare short-term partial seizures, isochoric and later dilated pupils, body temperature was 36.4°C, with oxygen saturation from 89 up to 93%. Toxicological analysis were immediately extended. After three hours of admission, a result was obtained where the plasma concentration for carbamazepine was 167mmol/L. The patient was quickly prepared for hemodialysis which was performed for four hours. After 8 hours of admission the patient woke up with stable vital parameters.

A simple approach for determination of plasmatic levels of carbamazepine and phenobarbital in poisoning cases using DLLME and liquid chromatography

Carbamazepine (CBZ) and phenobarbital (PHB) are commonly used antiepileptic medications. Due to their accessibility and narrow therapeutic range, many cases of poisoning cases involving these substances have been reported, whether intentional or unintentional. In managing cases of CBZ and PHB poisoning, monitoring plasma levels are important to choose the best treatment for the patient and staging intoxication cases. The use of dispersive liquid-liquid microextraction (DLLME) has proven to be a good approach for toxicological analysis in biological samples and is increasingly used in the clinical-forensic scenario. This study aims to develop a simple method using DLLME and HPLC-UV for the quantification of CBZ and PHB in plasma samples from poisoning cases. The main variables involved in the DLLME were optimized using multivariate experiments. The procedure is based on plasma deproteinization using a dispersive solvent with subsequent extraction with the extractor solvent. The bioanalytical method was fully validated. The lower limits of quantification were 2 μg/mL for CBZ and 10 μg/mL for PHB and the calibration curve was linear (r2 > 0.99) for both analytes. The RSD% in within-run precision varied from 3.2% to 8.7% and between-run precision varied from 3.4% to 8.4%. Accuracy ranged from 85.4% to 105.0%. Normalized matrix factor (NMF%) ranged from 96.1% to 101.2% and the obtained recovery extraction was from 33.9% to 41.5%. The method was successfully applied to 42 plasma samples from suspected poisoning cases. All samples were positive for at least CBZ and/or PHB. The developed and validated method proved to be efficient, easy to execute, and implement, helping in the diagnosis of poisoning.

A novel strategy for the study on molecular mechanism of prostate injury induced by 4,4'-sulfonyldiphenol based on network toxicology analysis.

The study aimed to investigate the underlying molecular mechanisms of prostate injury induced by 4,4'-sulfonyldiphenol (BPS) exposure and propose a novel research strategy to systematically explore the molecular mechanisms of toxicant-induced adverse health effects. By utilizing the ChEMBL, STITCH, and GeneCards databases, a total of 208 potential targets associated with BPS exposure and prostate injury were identified. Through screening the potential target network in the STRING database and Cytoscape software, we determined 21 core targets including AKT1, EGFR, and MAPK3. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses conducted through the DAVID database illustrated that the potential targets of BPS in prostatic toxicity were primarily enriched in cancer signaling pathways and calcium signaling pathways. These findings suggest that BPS may actively participate in the occurrence and development of prostate inflammation, prostatic hyperplasia, prostate cancer, and other aspects of prostate injury by regulating prostate cancer cell apoptosis and proliferation, activating inflammatory signaling pathways, and modulating prostate adipocytes and fibroblasts. This research provides a theoretical basis for understanding the molecular mechanism of underlying BPS-induced prostatic toxicity and establishes a foundation for the prevention and treatment of prostatic diseases associated with exposure to plastic products containing BPS and certain BPS-overwhelmed environments.

Autopsy, histological and toxicological findings in deaths associated with new psychoactive substances (NPS): a study with 12 autopsies

Background“New psychoactive substances” (NPS), has been described as a “growing global epidemic”. The aim of our study was to document acute and chronic lesions that can be induced by NPS from autopsy cases for which complete toxicological analyses (including hair analysis) have been performed, in order to better define the role of NPS in the mechanisms of death. Material and methodsA retrospective study was performed at the Department of Forensic Medicine and Pathology, jointly with the Department of Pharmacology and Toxicology. All records for forensic autopsy case within a 6-year period, from January 2016 to January 2021, were reviewed for the diagnosis “death associated with NPS”. Results and discussionTwelve young male adults complied with the inclusion criteria. Lifestyles were dominated by drug addiction and homosexual context which could be anchored in a practice of ChemSex or even of “slamming”. In regard to autopsy findings, previous pathological state was dominated by cardiovascular and hepatic lesions possibly of toxic etiology. Causes and manners of death were, on the whole, quite heterogeneous; however, an accident as a manner of death was the most frequently observed. In terms of histological findings, the most notable lesions were: (1) cardiovascular (sequelae of myocardial infarction probably secondary to a vasospasm, accelerated atherosclerosis); (2) hepatic (steatohepatitis); (3) neurological (peri mortem convulsions); (4) renal (granular cylinders that may indicate rhabdomyolysis); (5) pulmonary (chronic instertitial inflammation); all of them may have had a toxic origin. In regard to toxicological findings, our case series was mostly subject to chronic consumption of synthetic cathinones, in particular 3-MMC; their involvement in the fatal determinism was retained in 7 cases. Tissue distribution was highlighted as increased in brain, explaining at least partially psychoactive effects of these NPS. Average cardio-femoral ratio of 3.78 suggested a cardiac tropism. A lethal concentration of mexedrone, measured at 640 ng/mL, was described for the first time. ConclusionWe provide autopsy data on NPS consumption habits in Ile-de-France, namely men who have sex with men in ChemSex. Macroscopic and microscopic lesions observed were cardiovascular, hepatic, neurological, renal and pulmonary. From a toxicological point of view, we collect additional data on toxic, even lethal concentrations of NPS. Concerning 3-MMC, calculated cardio-femoral ratio is positive; moreover, it is to our knowledge the first study that demonstrates its pharmacokinetic tissue distribution.

In Vitro Production of Gamma-Hydroxybutyrate in Antemortem Blood Under Various Storage Conditions.

Gamma-hydroxybutyrate (GHB) is an endogenous compound and central nervous system depressant drug used recreationally for its intoxicating effects. In a medico-legal context, the interpretation of blood GHB concentrations can be complicated by its endogenous nature and potential for formation during storage. In Canada, the per se limit for GHB in blood is 5 mg/L. Endogenous GHB concentrations in blood are typically well below 5 mg/L; however, there is a paucity of literature regarding the potential production of GHB in antemortem blood during storage. Changes in GHB concentrations were evaluated over the course of 306 days in preserved and unpreserved antemortem blood stored at 4°C and 21°C. Results were compared to 22 impaired driving cases in Ontario between 2019-2022 where GHB was detected in antemortem blood by toxicological analysis at the Centre of Forensic Sciences. Preservative was effective at minimizing GHB production (< 2.5 mg/L) regardless of storage temperature whereas significant in vitro production of GHB occurred in unpreserved antemortem blood. GHB production occurred rapidly in unpreserved blood stored at 21°C; a significant increase was detected after 5 days. The rate of GHB production in unpreserved blood stored at 4°C occurred more gradually but increased significantly by day 30 and reached a maximum concentration of 10 mg/L at 114 days. In unpreserved blood, GHB concentrations were significantly lower at 4°C compared to 21°C for the first 44 days; however, refrigeration had no significant effect then onward. Blood concentrations of GHB detected in the majority of impaired driving cases were markedly higher than the maximum concentration of 10 mg/L detected in the study; however, in 4 of the 22 cases, concentrations were below 10 mg/L. The results demonstrate that concentrations of GHB less than 10 mg/L in blood collected for the purposes of a drug impaired driving investigation should be carefully interpreted.

Production of the dinoflagellate Amphidoma languida in a large scale photobioreactor and structure elucidation of its main metabolite AZA-39

Shellfish contamination with azaspiracids (AZA) is a major and recurrent problem for the Irish shellfish industry. Amphidoma languida, a small thecate dinoflagellate of the family Amphidomataceae, is widely distributed in Irish coastal waters and is one of the identified source species of azaspiracids. Irish and North Sea strains of Am. languida have been found to produce as major metabolites AZA-38 and -39 whose structures have only been provisionally elucidated by mass spectrometry and their toxic potential is currently unknown. In order to provide pure AZA-38 and -39 for subsequent structural and toxicological analyses, we present the first successful large-scale culture of Am. languida. A 180 L in house prototype bioreactor was used for culture growth and harvesting in semi-continuous mode for two months. Two different runs of the photobiorector with different light and pH setting showed the highest toxin yield at higher light intensity and slightly higher pH. AZA-38 and -39 cell quota were measured throughout the complete growth cycle with AZA-39 cell quota increasing in proportion to AZA-38 at late stationary to senescence phase. Over two experiments a total of 700 L of culture was harvested yielding 0.45 mg of pure AZA-39. The structure of AZA-39 was elucidated through NMR data analyses, which led to a revision of the structure proposed previously by mass spectrometry. While the spirotetrahydrofuran/tetrahydrofuran of rings A and B has been confirmed by NMR for AZA-39, a methyl is still present in position C-14 and the carboxylic acid chain is different from the structure proposed initially.

Proteins from toad’s parotoid macroglands: do they play a role in gland functioning and chemical defence?

BackgroundParotoid gland secretion of bufonid toads is a rich source of toxic molecules that are used against predators, parasites and pathogens. Bufadienolides and biogenic amines are the principal compounds responsible for toxicity of parotoid secretion. Many toxicological and pharmacological analyses of parotoid secretions have been performed, but little is known about the processes related to poison production and secretion. Therefore, our aim was to investigate protein content in parotoids of the common toad, Bufo bufo, to understand the processes that regulate synthesis and excretion of toxins as well as functioning of parotoid macroglands.ResultsApplying a proteomic approach we identified 162 proteins in the extract from toad’s parotoids that were classified into 11 categories of biological functions. One-third (34.6%) of the identified molecules, including acyl-CoA-binding protein, actin, catalase, calmodulin, and enolases, were involved in cell metabolism. We found many proteins related to cell division and cell cycle regulation (12.0%; e.g. histone and tubulin), cell structure maintenance (8.4%; e.g. thymosin beta-4, tubulin), intra- and extracellular transport (8.4%), cell aging and apoptosis (7.3%; e.g. catalase and pyruvate kinase) as well as immune (7.0%; e.g. interleukin-24 and UV excision repair protein) and stress (6.3%; including heat shock proteins, peroxiredoxin-6 and superoxide dismutase) response. We also identified two proteins, phosphomevalonate kinase and isopentenyl-diphosphate delta-isomerase 1, that are involved in synthesis of cholesterol which is a precursor for bufadienolides biosynthesis. STRING protein-protein interaction network predicted for identified proteins showed that most proteins are related to metabolic processes, particularly glycolysis, stress response and DNA repair and replication. The results of GO enrichment and KEGG analyses are also consistent with these findings.ConclusionThis finding indicates that cholesterol may be synthesized in parotoids, and not only in the liver from which is then transferred through the bloodstream to the parotoid macroglands. Presence of proteins that regulate cell cycle, cell division, aging and apoptosis may indicate a high epithelial cell turnover in parotoids. Proteins protecting skin cells from DNA damage may help to minimize the harmful effects of UV radiation. Thus, our work extends our knowledge with new and important functions of parotoids, major glands involved in the bufonid chemical defence.

Extracts from the Leaf of Couroupita guianensis (Aubl.): Phytochemical, Toxicological Analysis and Evaluation of Antioxidant and Antimicrobial Activities against Oral Microorganisms.

The study of phytotherapy in dentistry holds great relevance because of the scarcity of research conducted on the treatment of oral pathologies, specifically, caries and periodontal disease. Therefore, this research aimed to analyze the chemical composition of extracts from Couroupita guianensis Aubl. leaves, evaluate their toxicity, and assess their antioxidant and antimicrobial properties against Staphylococcus aureus, Streptococcus mutans, and Candida albicans. Three extracts were prepared using assisted ultrasound and the Soxhlet apparatus, namely, Crude Ultrasound Extract (CUE), Crude Soxhlet Extract (CSE), and the Ethanol Soxhlet Extract (ESE). Flavonoids, tannins, and saponins were detected in the chemical analysis, while LC-DAD analysis revealed the presence of caffeic acid, sinapic acid, rutin, quercetin, luteolin, kaempferol, and apigenin in all extracts. GC-MS analysis identified stigmasterol and β-sitosterol in the CUE and CSE. The ESE showed higher antioxidant activity (2.98 ± 0.96 and 4.93 ± 0.90) determined by the DPPH• and ABTS•+ methods, respectively. In the toxicity evaluation, the CUE at 50 μg/mL and the ESE at 50 μg/mL stimulated the growth of Allium cepa roots, while all extracts inhibited root growth at 750 μg/mL. None of the extracts exhibited toxicity against Artemia salina. Antibacterial activity was observed in all extracts, particularly against the microorganisms S. aureus and S. mutans. However, no antifungal activity against C. albicans was detected. These results suggest that extracts of C. guianensis have therapeutic potential for controlling microorganisms in the oral microbiota.

Spectral and theoretical analysis of derivatives of 1,2,3,3-tetramethyl-3H-indolium iodide (TMI), a highly selective derivatization reagent of cyanide, and their utility for the analysis of cyanide concentrations in beverages.

We develop a specific derivatization gas chromatography-mass spectrometry (GC-MS) method for cyanide using 1,2,3,3-tetramethyl-3H-indium iodide as the derivatization reagent. The derivative compounds were synthesized and characterized using 1H nuclear magnetic resonance (NMR), 13C NMR, and Fourier transform infrared (FT-IR) spectroscopy. The high selectivity of this derivatization for cyanide is supported by calculations and activation energy comparisons. We applied this method to pure water, green tea, orange juice, coffee cafe au lait, and milk. Derivatization was performed by diluting 20μL of sample solution with 0.1M NaOH and adding 100μL of saturated borax solution and 100μL of 8mM TMI solution, each drink was completed in 5min at room temperature, and selected ion (m/z = 200) monitoring analysis was linear (R2 > 0.998) at 0.15 to 15μM, with detection limits of 4-11μM were shown. This method is expected to be widely used in forensic toxicology analysis and can be applied to beverages, which are forensically important field samples.