The rs2601796 variant in ADCY9 gene is associated with severe asthma and less bronchodilator response.

Asthma is a respiratory disease caused by the interaction of genetic and environmental factors. The adenylyl cyclase type 9 (ADCY9) enzyme produces the cyclic-adenosinemonophosphate (cAMP), important mediator involved in bronchodilation and immunomodulatory response. The aim of this study was to investigate if rs2601796 and rs2532019 variants in the ADCY9 gene are associated with asthma and lung function. The study comprised 1,052 subjects. Logistic regressions were done using PLINK 1.9 adjusted by sex, age, BMI, smoke and principal components. Bronchodilator responsiveness was assessed using the percentage of difference in FEV1 before and after the bronchodilator use. The in silico analysis for gene expression was performed in the GTEx Portal. The variant rs2601796 (AA/AG genotype) was positively associated with asthma severity (OR: 1.60 IC95%: 1.08-2.39) and with obstruction in individuals with severe asthma (OR: 3.10, IC95%: 1.11-8.62). Individuals with severe asthma and the AA/AG genotype of rs2601796 had less responsiveness to bronchodilators and also a lower expression of ADCY9 in lung and whole blood. The variant rs2532019 (TT/GT genotype) also downregulated the ADCY9 gene expression, but no significant association with the studied phenotypes was found. Thus, the variant in ADCY9 was associated with worse asthma outcomes, including a lower response to bronchodilators, likely due to the impact on its gene expression rate. This variant may be useful in the future to assist in personalized management of patients with asthma.

LC-MS-MS-MS3 for the determination and quantification of ∆9-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 × 2.1 mm 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 min. 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 and 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% and 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.

Variants in interferon gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) genes that modulate inflammatory response are associated with periodontitis.

OBJECTIVE: Evaluate the association of genetic variants of the interferon gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) genes with periodontitis. METHODS: The study involved 117 individuals with periodontitis and 389 without periodontitis, all Brazilians, miscegenated. Individuals with periodontitis presented at least 4 teeth with ≥ 1 site with probing depth ≥ 4 mm; clinical attachment level ≥ 3 mm on the same site and bleeding upon stimulus. Genotyping was performed using the Infinium Multi-Ethnic AMR/AFR-8 Bead Chip focused on Hispanic and African American populations with approximately 2 million markers of the human genome. Multivariate logistic regression was performed to identify associations in additive, dominant and recessive models adjusted for covariates age, obesity, mouth breathing, flossing, asthma, and ancestry. RESULTS: In IFI16, the rs75985579-A is positively associated with periodontitis in the additive (Odds Ratio adjusted (ORadjusted) 2.65, 95% confidence interval (CI):1.25-5.60, p value: 0.007) and dominant models (ORadjusted 2.56, 95%CI:1.13-5.81, p value: 0.017). In AIM2, the rs76457189-G, is associated negatively with periodontitis in two genetic models evaluated, additive (ORadjusted 0.21, 95%CI:0.05-0.94, p value: 0.022) and dominant (ORadjusted 0.21, 95%CI:0.05-0.94, p value: 0.022). CONCLUSIONS: These results have shown that variants in the IFI16 and AIM2 genes are associated with periodontitis. Individuals with at least one A (adenine) allele of the rs75985579 (IFI16) are more than twice as likely to have periodontitis, while individuals with the G (guanine) allele of rs76457189 (AIM2) are less likely to be diagnosed with periodontitis, providing a negative association with periodontitis.

Structure Assignment of Seized Products Containing Cathinone Derivatives Using High Resolution Analytical Techniques.

The innovation of the new psychoactive substances (NPS) market requires the rapid identification of new substances that can be a risk to public health, in order to reduce the damage from their use. Twelve seized products suspected to contain illicit substances were analyzed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), gas chromatography coupled to mass spectrometry (GC-MS), and nuclear magnetic resonance spectroscopy (NMR). Synthetic cathinones (SCat) were found in all products, either as a single component or in mixtures. Infrared spectra of all products were consistent with the molecular structure of SCat, showing an intense absorption band at 1700-1674 cm-1, corresponding to the carbonyl stretching, a medium/strong peak at 1605-1580 cm-1, indicating stretching vibrations in the aromatic ring (C=C) and bands with relative low intensity at frequencies near 2700-2400 cm-1, corresponding to an amine salt. It was possible to identify a total of eight cathinone derivatives by GC-MS and NMR analysis: 4'-methyl-α-pyrrolidinohexanophenone (MPHP), α-pyrrolidinohexanophenone (α-PHP), 3-fluoromethcathinone (3-FMC), methedrone, methylone, buphedrone, N-ethylcathinone, and pentedrone. Among the adulterants found in these samples, caffeine was the most frequently detected substance, followed by ethylphenidate. These results highlight the prevalence of SCat in seized materials of the Portuguese market. Reference standards are usually required for confirmation, but when reference materials are not available, the combination of complementary techniques is fundamental for a rapid and an unequivocal identification of such substances.

Highly sensitive screening and analytical characterization of synthetic cannabinoids in nine different herbal mixtures.

The popularity of new psychoactive substances among drug users has become a public health concern worldwide. Among them, synthetic cannabinoids (SCs) represent the largest, most diversified and fastest growing group. Commonly known as 'synthetic marijuana' as an alternative to cannabis, these synthetic compounds are easily accessible via the internet and are sold as 'herbal incenses' under different brand names with no information about the chemical composition. In the present work, we aim to integrate gas chromatography-tandem mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) data as useful strategy for the identification and confirmation of synthetic cannabinoids present in nine seized herbal incenses. The analysis of all samples allowed the initial identification of 9 SCs, namely 5 napthoylindoles (JWH-018, JWH-073, JWH-122, JWH-210, MAM-2201), APINACA, XLR-11 and CP47,497-C8 and its enantiomer. JWH-018 was the most frequently detected synthetic compound (8 of 9 samples), while APINACA and XLR-11 were only identified in one herbal product. Other non-cannabinoid drugs, including oleamide, vitamin E and vitamin E acetate, have also been detected. Oleamide and vitamin E are two adulterants, frequently added to herbal products to mask the active ingredients or added as preservatives. However, to our knowledge, no analytical data about vitamin E acetate was reported in herbal products, being the first time that this compound is identified on this type of samples. The integration data obtained from the used analytical technologies proved to be useful, allowing the preliminary identification of the different SCs in the mixture. Furthermore, the examination of mass spectral fragment ions, as well as the results of both 1D and 2D NMR experiments, enabled the identification and confirmation of the molecular structure of SCs.

Prevalence of, and disability approaches to, temporomandibular joint trauma: A 17-year cross-sectional study.

BACKGROUND/AIM: Sociodemographic, cultural and environmental factors play significant roles in orofacial trauma. The main aim of the present work was to study the epidemiological data regarding temporomandibular trauma, in the orofacial trauma context. Secondly, follow-up data between injury and sequelae were assessed, in the medico-legal approach for this disability. MATERIAL AND METHODS: An observational, cross-sectional study, from 2000 to 2017, was performed, using the clinical reports of the National Institute of Legal Medicine and Forensic Sciences Institute of Portugal-Coimbra Central Branch. A descriptive analysis was performed. RESULTS: The injury group, 8.9% of the analysed population (2622 reports), included a majority of men (60.7%) with a mean age of 33.9 years. They were mainly students or retired (38.9%) and reported physical violence as the main external cause (56.4%) and temporomandibular joint (TMJ) trauma occurred with dental and facial trauma (84.2%). The sequela group, 2.9% of the analysed population, included a majority of men (65.6%) with a mean age of 40.1 years, focusing on a professional active population. They reported physical violence as the main external cause (46.3%) and TMJ (13.2%), soft tissues and teeth (12.8%) sequelae. CONCLUSIONS: Not all TMJ injuries corresponded to sequelae (63.4%). The sequela resulting from TMJ trauma encompass soft and hard tissues and TMJ sequelae, isolated or not. Physical violence was the primary external cause of TMJ injuries in both groups. Conversely, road accidents were the major external cause of isolated TMJ sequelae, highlighting the need to reinforce safety measures.

The synthetic cannabinoids phenomenon: from structure to toxicological properties. A review.

The word "cannabinoid" refers to every chemical substance, regardless of structure or origin, that joins the cannabinoid receptors of the body and brain and that have similar effects to those produced by the Cannabis plant and based on their source of production, cannabinoids can be classified into endocannabinoids, phytocannabinoids and synthetic cannabinoids. Synthetic cannabinoids represent the largest class of drugs detected through the EU Early Warning System with a total of 190 substances notified from 2008 to 2018 and about 280 have been reported worldwide to the United Nations Office on Drugs and Crime. Sprayed on natural herb mixtures with the aim to mimic the euphoria effect of cannabis and sold as "herbal smoking blends" or "herbal incense" under brand names like "Spice" or "K2", synthetic cannabinoids are available from websites for the combination with herbal materials or more recently, for the use in e-cigarettes. Currently labeled as "not for human consumption" to circumvent legislation, their legal status varies by country with many government institutions currently pushing for their control. However, due to the emergence of new substances, it requires a constant update of the list of controlled drugs. Little is known about how these substances work and their toxic effects in humans and the same product could vary not only in the amount and in the type of substance added. In the last years, synthetic cannabinoids have been associated with deaths and acute intoxications in Europe and, despite a range of new measures introduced in this area, continue to represent a challenge to current drug policy models. These synthetic substances are much more potent than natural cannabis, as well as displayed greater efficacy, acting as full agonists at the cannabinoid receptors. It is possible that, along with being highly potent, some may also have long half-lives, potentially leading to a prolonged psychoactive effect. The present work provides a review on existing literature about the development of synthetic cannabinoids as substances of abuse, current patterns of abuse and their legal status, chemical classification, and some pharmacological and toxicological properties.

MDMA Intoxication in a Potential Organ Donor with Cardiac Arrest.

Amphetamine and its derivatives' consumption is still an important public health issue, namely in terms of compounds variability and disposition to consumers. However, some of them, like 3,4-methylenedioxymethamphetamine (MDMA), still live in the illicit market, with continuous success. Nevertheless, there is always new information and data on MDMA intoxication, both in vivo and in postmortem context. The authors report an intoxication case with MDMA, in an 18-year-old male, considered a potential organ donor after a cardiac arrest. Whole blood samples were collected in vivo, at the emergency room (ER), and postmortem, at the National Institute of Legal Medicine and Forensic Sciences. After a general screening procedure, samples were extracted by solid phase extraction (OASIS® MCX), followed by gas chromatography-mass spectrometry analysis. The whole blood postmortem sample was positive for lidocaine (<500 ng/mL), compatible with the ER intervention, and positive for MDMA (2278 ng/mL) and methylenedioxyamphetamine (MDA) (49 ng/mL), while whole blood samples collected in vivo (during the maintenance of the individual under advanced life support), were positive for MDMA (504-1918 ng/mL) and MDA (20-89 ng/mL). Samples were negative for other substances, namely ethanol, other drugs of abuse and medicines. Results interpretation is pivotal to understand the behavior of the substance. Thus, in this case, MDMA postmortem behavior should be carefully evaluated, considering as possible influencers, in the specific context of the case, the time lapse between death verification, maintenance of the advanced life support and body manipulation for organ collection purposes. Also referred and discussed is the antemortem/postmortem ratio of MDMA obtained values, compared with literature references. There is no doubt that death was due to MDMA intoxication, but information from the analysis performed on the in vivo samples suggests that this type of sample should also be considered, in a complementary role, whenever possible.

Synthetic cathinones: an evolving class of new psychoactive substances.

Synthetic cathinones (SCat) are amphetamine-like psychostimulants that emerged onto drug markets as "legal" alternatives to illicit drugs such as ecstasy, cocaine, and amphetamines. Usually they are sold as "bath salts," "plant food," or "research chemicals," and rapidly gained popularity amongst drugs users due to their potency, low cost, and availability. In addition, internet drug sales have been replacing the old way of supplying drugs of abuse, contributing to their rapid spread. Despite the legislative efforts to control SCat, new derivatives continue to emerge on the recreational drugs market and their abuse still represents a serious public health issue. To date, about 150 SCat have been identified on the clandestine drugs market, which are one of the largest groups of new psychoactive substances (NPS) monitored by the United Nations Office on Drugs and Crime and the European Monitoring Center for Drugs and Drug Addiction. Similar to the classical stimulants, SCat affect the levels of catecholamines in the central nervous system, which results in their psychological, behavioral and toxic effects. Generally, the effects of SCat greatly differ from drug to drug and relatively little information is available about their pharmacology. The present work provides a review on the development of SCat as substances of abuse, current patterns of abuse and their legal status, chemical classification, known mechanisms of action, and their toxicological effects.

Epidemiology and forensic aspects of carbon monoxide intoxication in Portugal: A three years' analysis.

The presented manuscript describes the carbon monoxide (CO) related deaths in Portugal over a period of 3 years, based on autopsies carried out at the National Institute of Legal Medicine and Forensic Sciences, from January 2012 to December 2014. Three hundred and forty-seven forensic autopsy reports with carboxyhaemoglobin (COHb) analysis requests were analysed and subdivided into three main groups: (1) improbable CO intoxication; (2) possible CO intoxication; (3) highly probable CO intoxication. In group 1, COHb analysis was negative, and the death circumstances, as well as the post mortem findings, didn't corroborate an exposition to CO. In group 2, with COHb positive in 1/3 of the cases, the death circumstances corroborated an exposition to CO, but the post mortem findings weren't enough to confirm an exposition to this substance. In group 3, the results of COHb were positive, and both circumstances of death and post mortem findings corroborated an exposition to CO. The first group (113 cases) had no specific suspicion of a CO intoxication and, thus, the request of a COHb analysis had no particular basis, reflected in the low COHb achieved percentage (between 0 and 12). In the second group (164 cases), 29% of the cases were directly or indirectly related to CO exposure (between 0% and 94%). In the third group (70 cases), 56 deaths were due to CO intoxication and 14 due to burns after CO inhalation (between 18% and 91%). This study intended to do, not only a 3-year assessment of CO poisoning, but also to enhance the fact that circumstantial information, as well as a correct evaluation at the forensic autopsy data are crucial, and allow an enhanced diagnosis of possible intoxication, as well as a better guidance for the consequent toxicological analysis requests.

Quantification of GHB and GHB-GLUC in an 1,4-butanediol intoxication: A case report.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with known action at the neural level. Its psychoactive effects led to an illicit use context including recreational purposes, muscle building effects in bodybuilders and drug-facilitated crimes, specifically in sexual assaults. Besides the misuse of the main compound, there are precursors like Gammabutyrolactone (GBL) and 1,4-butanediol (1,4-BD), usually non controlled substances, becoming a much easier way to obtain the target-compound. The authors present the first reported intoxication case in Portugal with 1,4-Butanediol, including the quantification of GHB and GHB-GLUC in serum, by GC-MS/MS TQD. A suspicious liquid and a serum sample were sent by an hospital ER and analysed by GC-MS-single quadrupole and GC-MS/MS TQD, respectively. A methodology including protein precipitation and GC-MS/MS TQD analysis was used to detect and quantify GHB and GHB-GLUC in serum. Toxicological analysis revealed the presence of 1,4-Butanediol in the liquid and GHB [171 mg/L] and GHB-GLUC [13,7 mg/L] in serum. The victim reverted the coma with no neurological sequelae. This was the first detected case, in Portugal, with 1,4-Butanediol, suggesting that it is important to be aware that consumers have different options to obtain illicit compounds, such as GHB. On the other hand, GHB-GLUC was identified and quantified for the first time in a real case, due to intoxication. This case highlights the importance of analysing all samples for active compounds, precursors and metabolites that can lead to the main intoxication origin.

Analysis of fatal accidents with tractors in the Centre of Portugal: Ten years analysis.

Tractors have been described as one of the deadliest farming implements concerning agricultural activity. In Portugal, the scientific investigations about this problem are practically non-existent, with only statistical studies performed by entities related to road traffic safety, not in accordance to the study now performed, pinpointing the possibility of an underreporting of these accidents. This work aims to characterize the fatal tractor accidents in Portugal, autopsied at the Forensic Pathology Department of the Centre Branch of the National Institute of Legal Medicine and Forensic Sciences of Portugal, analysing several variables: gender, age, occupation, survival time, the victim position in the tractor, cause of death, toxicological and histological exams, year/month/day of the week, type of agricultural machine, existence of rollover protective structures (ROPS), type of accident, ground conditions, circumstantial information and geographic distribution of the accidents. All the autopsies between 2005 and 2014 were analysed. The victim profile corresponded to a man (89.5%), between 61 and 70 years old (33.3%), retired (43.9%), being the tractor driver (45.6%). In most of the cases, death occurred in less than 24h after the accident. These fatalities arose mainly in May and October. Rollover in sloping land was the most common type of accident, and cranioencephalic, thoracic and abdominal traumatic injuries were the main cause of death. In 16.2% of the cases, blood alcohol concentration was above the lower limit established in our road traffic law (<0.5g/L). There was lack information about the use of ROPS (95.9%), and even when existent, the protections were not used or were incorrectly used. This is the first national study involving the description of the forensic achievements in each autopsy related to tractor accidents, and the corresponding circumstances that contributed to the death. Many barriers remain about this matter, but the Portuguese government begin to design several laws to prevent this type of accidents. Future investigations should be performed to understand the real dimension of tractor accidents in Portugal, and to comprehend the efficiency of laws implemented by the government.

Postmortem in vitro ethanol production-It could be more common than we think!

The blood alcohol concentration (BAC) is the most frequent determination in a Forensic Toxicology Laboratory. Despite its apparent simplicity, the results interpretation can be complex and always have relevant social and legal implications, particularly in postmortem analysis. In the present report we describe the case of a 55-year-old male with an apparent natural death by myocardial infarction, whose initial BAC was 0.18g/L but, in repeated determinations prompted by discrepancies observed in the first two, it rapidly increased to 0.85g/L three days later, leading to the suspicion of in vitro ethanol production. A microbiological examination of the sample revealed the presence of the bacteria Escherichia coli and Enterococcus faecalis, and yeast Candida parapsilosis, known for their involvement in ethanol production. Although this is a case report and it is not meant to be generalizable, we discuss an existing large body of scientific literature showing the difficulties, limitations and some relevant medico-legal questions regarding BAC determinations in postmortem samples and their interpretation, particularly in the context of plausible in vitro ethanol production. The key conclusion is that evaluating a postmortem BAC is a complex and multifactorial process that always deserves a thorough analysis and a careful interpretation.

Adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in Brazilian children.

Asthma is a chronic inflammatory disease of the respiratory tract. This heterogeneous disease is caused by the interaction of interindividual genetic variability and environmental factors. The gene adenylyl cyclase type 9 (ADCY9) encodes a protein called adenylyl cyclase (AC), responsible for producing the second messenger cyclic AMP (cAMP). cAMP is produced by T regulatory cells and is involved in the down-regulation of T effector cells. Failures in cAMP production may be related to an imbalance in the regulatory immune response, leading to immune-mediated diseases, such as allergic disorders. The aim of this study was to investigate how polymorphisms in the ADCY9 are associated with asthma and allergic markers. The study comprised 1309 subjects from the SCAALA (Social Changes Asthma and Allergy in Latin America) program. Genotyping was accomplished using the Illumina 2.5 Human Omni bead chip. Logistic regression was used to assess the association between allergy markers and ADCY9 variation in PLINK 1.07 software with adjustments for sex, age, helminth infection and ancestry markers. The ADCY9 candidate gene was associated with different phenotypes, such as asthma, specific IgE, skin prick test, and cytokine production. Among 133 markers analyzed, 29 SNPs where associated with asthma and allergic markers in silico analysis revealed the functional impact of the 6 SNPs on ADCY9 expression. It can be concluded that polymorphisms in the ADCY9 gene are significantly associated with asthma and/or allergy markers. We believe that such polymorphisms may lead to increased expression of adenylyl cyclase with a consequent increase in immunoregulatory activity. Therefore, these SNPs may offer an impact on the occurrence of these conditions in admixture population from countries such as Brazil.

Pentobarbital in the context of possible suicides: Analysis of a Case.

Pentobarbital is a barbiturate, acting as a central nervous system depressant (CNS), being used for its anticonvulsant, sedative, hypnotic and anaesthetic properties. Barbiturates were replaced by benzodiazepines, leading to a decrease in poisoning cases with these compounds. However, pentobarbital is still used in many countries as an anaesthetic in veterinary medicine. Due to its properties, this compound is sought after by people who wish to commit suicide, acquiring it on the black market. The authors present an unusual fatal pentobarbital intoxication case, in a 37 years-old male salesperson, with no known connection with the veterinary field, being more difficult to obtain this compound. Toxicological results in cardiac blood revealed the presence of pentobarbital (111mg/L), ethanol (0.94g/L), diazepam (33ng/mL), nordiazepam (50ng/mL), oxazepam (3.3ng/mL), temazepam (5.3ng/mL), and metoclopramide. No illicit drugs were detected. Pentobarbital analysis in urine and gastric content was also positive, as well as its presence in the glass powder and in the bottle residue sent to the laboratory. In the present case, it was possible to conclude that the death was a suicide due to pentobarbital intoxication in association with other depressants of the CNS (benzodiazepines and ethanol). It is important to search pentobarbital in routine toxicological analyses, since it is one of the drugs most frequently mentioned by entities defending "painless death", advising the simultaneous use of metoclopramide for emesis avoidance.

Improved Analytical Approach Based on QuECHERS/UHPLC-PDA for Quantification of Fluoxetine, Clomipramine and their Active Metabolites in Human Urine Samples.

This paper reports, for the first time, the development of a modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) combined with a dispersive SPE (d-SPE) based clean-up procedure as a new and powerful strategy for the simultaneous and efficient isolation of two different antidepressants, fluoxetine and clomipramine, and their active metabolites in human urine samples. A univariate experimental design with four independent variables such as sample volume, extraction solvent, buffered salts and clean-up step, was performed and used to investigate the effect of process variables on the extraction efficiency. Good linearity was achieved at the studied concentration range (0.1-5.0 µg mL-1), with correlation coefficients (R2) higher than 0.9961. Low detection limits, ranging between 0.060 and 0.092 µg mL-1 were obtained for all analytes, whereas the lowest quantification limit was 0.1 µg mL-1, corresponding to the lowest concentration of the standard curve. The method also showed good results for accuracy, with values ranging from 91% to 105%. Intra- and inter-day precision, expressed as the relative standard deviation (RSD), were also satisfactory (<10%). Consistent recoveries of antidepressants ranging from 86% to 109% were observed when urine samples were fortified at three concentrations, namely 0.1, 2.5 and 5.0 µg mL-1 In order to evaluate the proposed method for clinical use, the QuEChERS/UHPLC-PDA method was applied to analysis of 12 urine samples from depressed patients.

Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels.

Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem interval.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960's. The possibility to use a multi-sample approach for GHB evaluation, including whole blood and hair, to better characterize a forensic toxicology case and evaluate a possible causal association with the death is an exciting up-to-date issue. In addition, its post-mortem behaviour, namely regarding degradation and metabolism, has been increasingly investigated as a putative biomarker for post-mortem interval (PMI) estimation. Thus, in order to contribute to clarification of this specific aspect, whole blood and hair post-mortem GHB levels were evaluated in 32 real cases with previous information on death and autopsy data. The results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, but not GHB levels in hair samples. No differences were encountered for the other parameters evaluated, including age, gender, cause of death and presence or absence of substances. This study brings new insights regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the context of legal medicine.

Inorganic mercury intoxication: A case report.

Mercury is a heavy metal with unique physico-chemical properties, and it is well distributed throughout the environment, being present in soil, water and air. This non-essential element is considered by the World Health Organization (WHO) as one of the ten most troublesome chemical to public health. Its toxicity spectrum depends on the chemical form in which it presents: elemental (metallic), organic or inorganic. The known intoxications are mainly occupational (mining, agriculture, incineration) or related to the use of dental amalgams or the consumption of contaminated fish and shellfish. Nowadays, acute exposures to toxic amounts of mercury are increasingly rare, especially those involving inorganic mercury compounds. The rate is even lower if we refer to intentional poisonings. Although there is a growing understanding of the toxicokinetics of mercury, there is still a lack of studies that support the emerging theories about its bioavailability in humans. In this manuscript we describe a rare case of an individual who committed suicide by ingesting mercuric oxide. The aim is to offer a medical contribution to the better understanding of the kinetics of this metal, making a discussion based on published literature and analyzing its distribution, metabolism, internal doses, target and reservoir organs. The whole case - clinical course of the victim and her fatal destiny, the ante- and post-mortem sample concentrations and the necropsy findings - illustrates a situation that meets specific features of acute poisoning by ingestion of inorganic mercury, thus constituting an important support towards a more realistic and a based on evidence understanding of mercury biodistribution in humans.

A fast and reliable method for GHB quantitation in whole blood by GC-MS/MS (TQD) for forensic purposes.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with a story of clinical use since the 1960s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market. A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC-MS/MS (TQD) in whole blood is presented. Using 100µL of whole blood, obtained results included a LOD and LLOQ of 0.1mg/L and a recovery of 86% in a working range between 0.1 and 100mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors' knowledge, the first GC-MS-MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.