Gas chromatography-mass spectrometry determination of nicotine and cotinine in urine: A study of the effect of passive smoking.

RATIONALE: Recent data suggest that passive smoking has a risk comparable to active smoking. Passive smoking is considered dangerous in children and is suspected as a cause of asthma. However, some reports are opposing such claims, indicating the need for solid results and large-scale studies. This scientific work aims to develop a method for the determination of nicotine (NCOT) and major nicotine's metabolite cotinine (COT) in urine samples, using gas chromatography-mass spectrometry (GC-MS). METHODS: Analysis was performed using a gas chromatograph Agilent Technologies 7890A with an MS 5975C inert XL, EI/CI MSD with Triple-Axis detector. For sample preparation, liquid-liquid extraction was applied after an optimization study with different extraction media. Eventually, 1 mL of dichloromethane was selected for the extraction of 0.5 mL of urine. Suitable chromatographic conditions were found for the rapid and accurate determination of NCOT and COT. Injection of 2 µL was performed using GC-MS, and selected ion monitoring (SIM) analysis was performed with the following ions (m/z): 162 (quantifier ion) and 84, 133, 161 qualifier ions for NCOT, and 176 (quantifier ion) and 98, 118, 119, 147 qualifier ions for COT. Nicotine-D4 (NCOT-D4) and cotinine-D3 (COT-D3) were used as internal standards with quantifier ions 101 and 166, respectively. The retention time (Rt) for NCOT was 7.557 min and 9.743 min for COT. RESULTS: The method was validated following international principles, assessing characteristics such as absolute recovery, carryover, linearity, specificity, selectivity, accuracy, precision, and stability. The method showed a linear dynamic range from 0.5 to 50 ng/mL, and the limits of detection and quantification were for both NCOT and COT 0.2 and 0.5 ng/mL, respectively. Validation results were found satisfactory. Finally, the method was applied to the analysis of 60 clinical pediatric samples obtained from Aristotle University's pediatric clinic to check for possible exposure to smoke. Concentration levels ranged between 0.5 and 16.2 ng/mL for NCOT and between 1.0 and 25.1 ng/mL for COT. CONCLUSIONS: A rapid, sensitive, accurate, and simple method was developed and used as a tool for the confirmation of passive smoking in children. It is the first method applied to the analysis of such samples belonging to nonsmokers of young age. The total runtime of the GC-MS analysis was short (20 min), and the pretreatment protocol was simple, giving the ability for analysis of a large number of samples on a daily routine basis.

Perceptions and experiences of female nurses when confronted with expressing a conscientious objection towards end-of-life care in Greece.

BACKGROUND: Conscientious objection in nursing has been a topic of much discussion in recent years. Healthcare providers' conscientious objection has been included in Greek legislation. However, little is known about the real experiences of nurses who want to apply conscientious objections in their practice. This study aimed to contribute to filling that gap. METHODS: This qualitative study was conducted with eighteen experienced female nurses. Data were collected through semi-structured in-depth qualitative interviews conducted with purposively selected nurses during the period from October 2019 to January 2020. Interviews were transcribed verbatim and analysed thematically. The ethical principles of anonymity, voluntary participation and confidentiality were considered. RESULTS: Eight major themes and seven subthemes emerged from the thematic data analysis. Oppressive behaviors in the workplace and subservient interactions between nurses and physicians, suboptimal communication and inadequate support of nurses, perceived ineffectiveness of nurses' conscientious objections, missing legal protection against job insecurity, provision of care labeled 'futile', nurses' false knowledge and perceptions on medical situations related to conscientious objections, nurses' fears of isolation bullying and negative gossip in the workplace and a trivial amount of nurses' involvement in medical decisions emerged as barriers to nurses raising conscientious objection. Furthermore, from data analysis, it emerged that some nurses had false knowledge and perceptions on medical situations related to conscientious objections, some nurses experienced mild uncertainty distress about their ethical concerns, nurses considered their remote contribution as participation that can give rise to conscientious objection, a collective conscientious objection raised by nurses might have increased chances of being effective, and upbringing, childhood experiences, education and religion are factors shaping the nurses' core values. CONCLUSION: A total of fifteen themes and subthemes emerged from this study. Most of the findings of this study were previously unknown or undervalued and might be helpful to inform nurses and nursing managers or leaders as well as healthcare policy makers. The results of this study might contribute to addressing the need for creating ethically sensitive health care services and ensuring nurses' moral integrity and high quality of patient care.

Detection and determination of C12-, C14-, C16-alkyldimethylamines in human blood using gas chromatography-mass spectrometry.

RATIONALE: N,N-Dimethyldodecylamine is produced from lauryl alcohol and dimethylamine. C12-C16 alkyldimethylamines are used as intermediates for the manufacture of amineoxides and quaternary amino compounds. In the present study a gas chromatography-mass spectrometry (GC/MS) method for the determination of C12-C16 alkyldimethylamines in blood was developed and validated. The reason for this study was the detection of the above compounds in the postmortem blood sample of a fatal suicide case. METHODS: Analysis of amines was performed using a gas chromatograph (Agilent Technologies 7890A) with an MS 5975C inrXL, EI/CI MSD with triple-axis detector in selected ion monitoring mode, after liquid-liquid extraction. Four different organic solvents (butyl acetate, ethyl acetate, n-hexane and n-heptane) were used for the optimization of the extraction procedure, resulting in ethyl acetate being the solvent of choice for the extraction procedure. A QuEChERS step was applied (20 mg of MgSO4 , 5 mg of NaCl) to 1 mL of blood and pH was adjusted at 12 (K2 CO3 buffer solution). After the addition of the extraction solvent, samples were vortexed, centrifuged and directly injected into the GC/MS system. RESULTS: In validation, the method was found to be selective and sensitive (limit of detection from 0.3 to 0.5 ng/mL, limit of quantitation from 10.0 to 20.0 ng/mL), whilst validation included recovery, stability, accuracy and precision (relative standard deviation). Validation results were found satisfactory: intra- and interday precision ranged from 0.4% to 2% and from 0.6% to 1.9% respectively, while intra- and interday accuracy ranged from 87% to 109% and from 86% to112.8%. C12-C16 alkyldimethylamines were detected in blood samples at a concentration of 8.39 µg/mL (C12), 3.01 µg/mL (C14) and 0.42 µg/mL (C16). CONCLUSIONS: A rapid, sensitive and reliable method was developed for the determination of C12-C16 alkyldimethylamines in postmortem blood, after optimization of the sample preparation procedure, and finally successfully applied to a real postmortem blood sample from a fatal case involving these compounds.

Liquid chromatography-mass spectrometry method for the determination of polyethylene terephthalate and polybutylene terephthalate cyclic oligomers in blood samples.

Food contact materials (FCM) polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) used extensively in food packaging may contain cyclic oligomers which may migrate into food and thus cause toxic effects on human health. A simple, fast, and sensitive ultra-high-performance liquid chromatography method quadrupole time-of-flight mass spectrometer was developed for the analysis of 7 cyclic oligomers in post-mortem blood samples. The targeted analytes were separated on a Waters BEH C18 (150 × 2.1 mm, 1.7 µm) analytical column by gradient elution. Calibration curves were constructed based on standard solutions and blood samples and Student's t-test was applied to evaluate the matrix effect. The LODs ranged from 1.7 to 16.7 µg mL-1, while the method accuracy was assessed by recovery experiments and resulting within the range 84.2-114.6%. Such an analytical method for the determination of PET and PBT cyclic oligomers in biological samples is reported for the first time. The developed methodology allows the determination of these oligomers in blood providing a useful analytical tool to assess the exposure and thus the potential hazard and health risks associated with these non-intentionally added substances (NIAS) from PET and PBT FCM through food consumption. The method was validated and successfully applied to the analysis of 34 post-mortem whole blood samples. Polyethylene terephthalate trimer was detected in four of them, for the first time in literature.

Efficacy and safety of anti-sclerostin antibodies in the treatment of osteoporosis: A meta-analysis and systematic review.

Osteoporosis is a chronic disease with an increasing prevalence. Anti-sclerostin antibodies are being investigated for the treatment of osteoporosis. The aim of this systematic review and meta-analysis is to evaluate the efficacy and safety of antis-sclerostin antibodies compared to placebo and conventional therapies (alendronate and teriparatide) in the treatment of osteoporosis. Randomized controlled trials were searched from PubMed, EMBASE and Cochrane Central Register of Controlled Trails (CENTRAL) from their inception up to June 2021 by using Medical Subject Headings terms "anti-sclerostin antibody", "romosozumab", "blosozumab", "AMG 785″, "LY2541546", and "osteoporosis". Two investigators independently screened eligible studies, assessed the risk of bias and extracted the data from each study. The I2 index was used to assess heterogeneity. Meta-analysis was conducted using the Review Manager Software (RevMan, Version 5.4). The GRADE approach was used to rate the quality of evidence for all the pooled outcomes. 8 RCTs with 12,416 patients met the inclusion criteria. Anti-sclerostin antibodies significantly increased lumbar spine, total hip and femoral neck bone mineral density compared to placebo, alendronate and teriparatide at both 6 and 12 mo. Adverse events were comparable between anti-sclerostin antibodies and other treatments, except for the incidence of injection-site reactions that was higher in the anti-sclerostin antibody groups. Anti-sclerostin antibodies represent a valid theurapeutic option in the treatment of osteoporosis. Further studies with longer duration and follow-up are needed to confirm the results of this meta-analysis.

Risk factors for fatal drowning in a Greek region: a retrospective case-control study.

BACKGROUND: Fatal drowning is one of the leading causes of unintentional injury mortality worldwide and a persistent public health concern in Greece. While several pathologic and sociodemographic contributing factors have been previously identified, these have not been extensively investigated in conjunction with the effects of psychoactive substances. METHODS: A retrospective case-control study of drowning deaths was conducted in the Greek regions of Northern Greece and Thessaly during a 10-year period. A regression model was constructed examining differences in detected substances, autopsy findings and sociodemographic characteristics between 240 victims of unintentional fatal submersion and 480 victims of other causes of sudden or violent death. RESULTS: The majority of victims were males (69.4%) and foreign nationality was associated with increased odds of drowning. Cardiomegaly and coronary bypass grafts were significantly more likely to have been recorded among drowning victims, while the frequency of other circulatory system disorders was also elevated. Several of these findings were potential arrhythmogenic substrates which could adversely interact with the diving reflex. Selective serotonin reuptake inhibitors (SSRIs) were the most commonly detected pharmacological group (9.0%), and along with tramadol, there was an increased likelihood of exposure to them. These drugs have been previously associated with QT prolongation and other adverse effects which may contribute to fatal outcomes in a seawater environment. In contrast, there was a decreased risk of exposure to dependence-inducing drugs and paracetamol. CONCLUSIONS: Male sex, older age, foreign nationality and cardiovascular disease predisposed individuals to an elevated risk of fatal submersion. SSRI antidepressants and tramadol may contribute to this outcome.

Evaluation of Cocaine Effect on Endogenous Metabolites of HepG2 Cells Using Targeted Metabolomics.

Cocaine toxicity has been a subject of study because cocaine is one of the most common and potent drugs of abuse. In the current study the effect of cocaine on human liver cancer cell line (HepG2) was assessed. Cocaine toxicity (IC50) on HepG2 cells was experimentally calculated using an XTT assay at 2.428 mM. The metabolic profile of HepG2 cells was further evaluated to investigate the cytotoxic activity of cocaine at 2 mM at three different time points. Cell medium and intracellular material samples were analyzed with a validated HILIC-MS/MS method for targeted metabolomics on an ACQUITY Amide column in gradient mode with detection on a triple quadrupole mass spectrometer in multiple reaction monitoring. About 106 hydrophilic metabolites from different metabolic pathways were monitored. Multivariate analysis clearly separated the studied groups (cocaine-treated and control samples) and revealed potential biomarkers in the extracellular and intracellular samples. A predominant effect of cocaine administration on alanine, aspartate, and glutamate metabolic pathway was observed. Moreover, taurine and hypotaurine metabolism were found to be affected in cocaine-treated cells. Targeted metabolomics managed to reveal metabolic changes upon cocaine administration, however deciphering the exact cocaine cytotoxic mechanism is still challenging.

Determination of illicit drugs and psychoactive pharmaceuticals in wastewater from the area of Thessaloniki (Greece) using LC-MS/MS: estimation of drug consumption.

This study presents the development of an analytical method for the simultaneous determination of multiclass illicit drugs (cocainoids, opiates, amphetamines, and cannabinoids) and psychoactive pharmaceuticals (anxiolytics, hypnotics, antipsychotics, antidepressants, and antiparkinsonian), in municipal wastewater. The analytical method was validated in terms of specificity, linearity, precision, and accuracy. The recoveries (%) for the majority of the analytes ranged between 70 and 120%, while the method showed good repeatability (2.4-29.2%). The limits of detection (LOD) of the method ranged between 0.8 and 9.4 ng L-1. The method was implemented on influent and effluent samples from Thessaloniki (N. Greece) wastewater treatment plant (WWTP), and it revealed the daily presence of benzoylecgonine (BEG) (84.0-202.2 ng L-1), methadone (12.3-17.5 ng L-1), 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) (80.3-171.9 ng L-1), morphine (144.2-264.3 ng L-1), and 6-monoacetylmorphine (6-MAM) (5.8-12.0 ng L-1) in the influent samples of WWTP. Clozapine (101.6-315.5 ng L-1), quetiapine (33.5-109.7 ng L-1), and fluoxetine (20.9-124.4 ng L-1) were pharmaceutical psychotics with the highest concentration in the influents. Back calculation estimated that the daily consumption of cocaine, heroin, cannabis, and methadone was 36-95, 86-164, 2300-5400, and 8-12 mg day-1 per 1000 inhabitants, respectively. The consumption was estimated between 7-16 and 15 mg day-1 per 1000 inhabitants for methyl diethanolamine (MDEA) and 3,4-methylenedioxymethamphetamine (MDMA), respectively.

Serum-Targeted HILIC-MS Metabolomics-Based Analysis in Infants with Ureteropelvic Junction Obstruction.

Ureteropelvic junction obstruction (UPJO) constitutes the predominant cause of obstructive nephropathy in both neonates and infants. Fundamental questions regarding UPJO's mechanism, assessment, and treatment still remain unanswered. The aim of the present study was to elucidate potential differences through serum metabolic profiling of surgical cases of infants with UPJO compared to both nonsurgical cases and healthy age-matched controls. Early diagnosis of renal dysfunction in this cohort based on highlighted biomarkers was the ultimate goal. Thus, serum samples were collected from 20 patients preoperatively, 19 patients with mild stenosis treated conservatively, and 17 healthy controls. All samples were subjected to targeted metabolomics analysis by hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC LC-MS/MS). Both univariate and multivariate statistical analyses were performed. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots showed that the studied groups differed significantly, with a panel of metabolites, including creatinine, tryptophan, choline, and aspartate, distinguishing patients who required surgery from those followed by systematical monitoring as well as from healthy controls, showing high performance as indicators of UPJO disease.

Lesbians' experiences and attitudes towards parenthood in Greece.

Same-sex parenthood is controversial subject. In this paper, we provide insights into the attitudes and experiences of self-reported lesbians regarding parenthood or the prospect of becoming a parent in the current Greek social and cultural context. In Greece, lesbians are not allowed access to in vitro fertilisation (IVF), while a solitary ('single') woman is allowed access for medical reasons. Fifty-nine (59) semi-structured in-depth interviews were conducted with women. What emerged from our data was a clear trend for participants to wish to have their own biological children based mostly on the belief that pregnancy would lead to a sense of self-completeness and/or fulfilment. Women also reported the negative impact of prejudice and social oppression on their reproductive autonomy. Interviewees reported that their reproductive choices were negatively influenced by their family and the wider socio-cultural environment. Even within a semi-permissive legal framework, impaired social acceptance of lesbian parenthood prohibits lesbians from becoming mothers. A major reason responsible for the positive attitude of most participants to shared biological motherhood was an altruistic attitude towards their partners.

ABCB1 and CYP2D6 polymorphisms and treatment response of psychotic patients in a naturalistic setting.

OBJECTIVES: The aim of our study was to examine the association between ABCB1 polymorphisms G2677T/A (rs2032582) and C3435T (rs1045642) and common CYP2D6 variants, with the response to antipsychotic treatment of psychotic patients, in a naturalistic setting, in Greece. METHODS: One hundred patients suffering from schizophrenia and other psychotic disorders were included in the study. Dosages were normalized to chlorpromazine equivalents. Response following 1 month of treatment was assessed as either a continuous variable, using the distribution of the corrected Positive and Negative Syndrome Scale percent change, or as a dichotomous variable defined as the number of patients scoring ≥30% from the corrected baseline Positive and Negative Syndrome Scale score. Genotyping was achieved with established polymerase chain reaction-restriction fragment length polymorphism methods. RESULTS: With response treated as a continuous variable, the homozygous recessive rs2032582 genotypes (TT) who were simultaneously carriers of a loss-of-function CYP2D6 allele (*4 or *5) responded significantly worse than the rest of the patients. Comparison of genotype frequencies revealed a statistically significant association of the above combination. No significant association between chlorpromazine equivalents and the tested genotypes was detected. CONCLUSION: We have detected a possible interaction between ABCB1 and CYP2D6 in affecting response of psychotic patients to drug treatment, in a naturalistic setting.

Fatal intoxication with antidepressants: a case with many culprits.

Serotonin-specific reuptake inhibitors (SSRIs) are generally considered safe drugs but fatal adverse effects do sometimes occur, often as a consequence of interactions with other serotonin active drugs. Polypharmacy is usually a problem that the elderly encounter, but it can also have dire consequences for young people, especially when an underlying heart condition is present. Thus, failure to diagnose heart disease and the use of contraindicated medications can be a lethal combination, irrespective of age. Here we present a case of a young adult suffering from bipolar disorder who used a combination of two SSRIs (citalopram and fluoxetine) and a monoamine oxidase inhibitor (MAO; moclobemide) with tragic consequences. The deceased also suffered from undiagnosed hypertrophic cardiomyopathy and was carrier of a genotype that may have predisposed him to increased sensitivity to SSRIs. The apparent difficulty in establishing the manner of death in this case is also discussed.

Amniotic Fluid and Maternal Serum Metabolic Signatures in the Second Trimester Associated with Preterm Delivery.

Preterm delivery (PTD) represents a major health problem that occurs in 1 in 10 births. The hypothesis of the present study was that the metabolic profile of different biological fluids, obtained from pregnant women during the second trimester of gestation, could allow useful correlations with pregnancy outcome. Holistic and targeted metabolomics approaches were applied for the complementary assessment of the metabolic content of prospectively collected amniotic fluid (AF) and paired maternal blood serum samples from 35 women who delivered preterm (between 29 weeks + 0 days and 36 weeks +5 days gestation) and 35 women delivered at term. The results revealed trends relating the metabolic content of the analyzed samples with preterm delivery. Untargeted and targeted profiling showed differentiations in certain key metabolites in the biological fluids of the two study groups. In AF, intermediate metabolites involved in energy metabolism (pyruvic acid, glutamic acid, and glutamine) were found to contribute to the classification of the two groups. In maternal serum, increased levels of lipids and alterations of key end-point metabolites were observed in cases of preterm delivery. Overall, the metabolic content of second-trimester AF and maternal blood serum shows potential for the identification of biomarkers related to fetal growth and preterm delivery.

Complex suicide involving pyrethroid ingestion (mosquito coils) and fatal self-wounding by sharp force.

"Complex suicide" is a term referring to the suicidal pattern in which more than one suicidal method is applied in the purpose of inducing death. The present paper aims to review complex suicide literature shortly and investigate an unusual planned complex suicide incident, the rarity of which is attributable to the combination of suicide methods as well as the type and quantity of substances applied to induce poisoning. A 33-year-old man with a history of psychotic depression was found dead in his bedroom lying within a large quantity of blood. He had already committed two previous suicide attempts and he was under treatment with antidepressants. The forensic examination revealed the use of the following successive suicide methods: benzodiazepine and alcohol intake, pyrethroid poisoning due to ingestion of mosquito coils, wrist cutting, and a fatal cut in the victims neck. Death occurred due to hemorrhagic shock. Furthermore, the authors extensively discuss the use of sharp force in suicide and the discrimination "tools" between suicide and homicide.

Infantile Respiratory Distress Syndrome and Its Probable Links With Parameters of the Maternal Patient History: A Forensic Case Report.

Respiratory distress syndrome (RDS) has a major contribution to neonatal mortality worldwide. Multiple factors associated with increased risk for RDS have been documented to effectively understand the emergence and progression of this disorder. A portion of these parameters has been broadly examined whereas the role of others, despite being clinically described, has not been fully evaluated. In this report, we analyze a forensic RDS case of a late preterm infant. Taking the maternal medical history into account, we focused on 2 not widely established risk factors, oligohydramnios and maternal age, discussing their possible pathophysiological relation to the development of RDS. Simultaneously, the fundamental role of the histopathological examination as a diagnostic tool resurfaces. Following a multidisciplinary approach derived from the collaboration of clinicians and researchers, the identification of factors that precipitate or contribute to this syndrome can be enhanced, leading to novel prognostic and therapeutic strategies against RDS.

Development and validation of a HILIC-MS/MS multitargeted method for metabolomics applications.

The paper reports the development of a multianalyte method and its application in metabolic profiling of biological fluids. The initial aim of the method was the quantification of metabolites existing in cell culture medium used in in-vitro fertilization (IVF) and in other biological fluids related to embryo growth. Since most of these analytes are polar primary metabolites a hydrophilic interaction liquid chromatography system was selected. The analytical system comprised Ultra-HPLC with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes. Mobile phase and gradient elution conditions were studied with the aim to achieve the highest coverage of metabolic space in a single injection namely the largest number of analytes that could be detected and quantified. The developed method provides absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, aminoacids, nucleotides, organic acids, and amines. Following validation, the method was applied for the metabolic profiling of hundreds of samples of spent culture medium originating from human IVF procedures and several hundreds of biological samples such as amniotic fluid, human urine and blood serum from pregnant women. The bioanalytical end-point was to provide assistance in the process of embryo transfer and improving IVF success rates but also to provide insight in complications related to the subsequent embryo growth during pregnancy.

Community Antibiotic Consumption in Cyprus for the Period 2015 to 2022.

BACKGROUND: Cyprus currently reports to ESAC-Net the total consumption of antimicrobials, without distinguishing between hospital and community-based antibiotic use. As a result, these data can only provide generalized insights into antimicrobial trends in the country. AIM: This study is a first attempt to retrospectively analyze community antibiotic consumption in Cyprus for the period of 2015 to 2022. MATERIAL AND METHODS: Data on community antimicrobial consumption between 2015 and 2022 were extracted from Pharmatrack's database. Orally administered dispensed antibiotics were categorized under the J01 group of the WHO Anatomical Therapeutic Chemical (ATC) classification and by the WHO's AWaRe classification of antibiotics. Antibiotic consumption was calculated in both packages consumed and per 1000 inhabitants, overall, by year of consumption and districts. RESULTS: During the period of 2015-2022, there was variability in the mean outpatient antibiotic consumption per 1000 inhabitants among the five districts in Cyprus. Community consumption increased by 38% throughout the study period. Additionally, a decrease of 3% in the consumption of WHO 'Access' antibiotics was observed, accompanied with a concurrent increase of 3% in the 'Watch' group. Specifically, in 2022 the WHO 'Access' group consumption in the Cypriot community was 48%, significantly lower than the WHO's goal of 60% and the EU's goal of 70% for 'Access' antibiotic consumption. CONCLUSIONS: Antibiotic consumption in the community of Cyprus between 2015 and 2022 demonstrated substantial variability among districts, with higher consumption in less populated areas. There was an increasing trend in community consumption over the years and a decreasing trend in the percentage of 'Access' antibiotics prescribed.

Metallothionein I/II Expression and Metal Ion Levels in Correlation with Amyloid Beta Deposits in the Aged Feline Brain.

Brain aging has been correlated with high metallothionein I-II (MT-I/II) expression, iron and zinc dyshomeostasis, and Aß deposition in humans and experimental animals. In the present study, iron and zinc accumulation, the expression of MT-I/II and Aß42, and their potential association with aging in the feline brain were assessed. Tissue sections from the temporal and frontal grey (GM) and white (WM) matter, hippocampus, thalamus, striatum, cerebellum, and dentate nucleus were examined histochemically for the presence of age-related histopathological lesions and iron deposits and distribution. We found, using a modified Perl's/DAB method, two types of iron plaques that showed age-dependent accumulation in the temporal GM and WM and the thalamus, along with the age-dependent increment in cerebellar-myelin-associated iron. We also demonstrated an age-dependent increase in MT-I/II immunoreactivity in the feline brain. In cats over 7 years old, Aß immunoreactivity was detected in vessel walls and neuronal somata; extracellular Aß deposits were also evident. Interestingly, Aß-positive astrocytes were also observed in certain cases. ICP-MS analysis of brain content regarding iron and zinc concentrations showed no statistically significant association with age, but a mild increase in iron with age was noticed, while zinc levels were found to be higher in the Mature and Senior groups. Our findings reinforce the suggestion that cats could serve as a dependable natural animal model for brain aging and neurodegeneration; thus, they should be further investigated on the basis of metal ion concentration changes and their effects on aging.

Analysis, Stability and Distribution of Pharmaceuticals and Drugs of Abuse over a Period of One Year in Formalin-Fixed Liver and Formalin Solutions.

The present study reports a thorough research on the stability of drugs of abuse and pharmaceuticals over a time period of 12 months. Fixed-liver tissues and formalin solutions where the tissues were preserved were analyzed using an ultra high performance liquid chromatography--tandem mass spectrometry method that has been developed and validated for this purpose. The method monitors 84 drugs in a 13-minute run. The concentrations of the drugs found were compared with their concentrations determined in the fresh liver tissues in a previous study. In the study, 14 cases with forensic interest were included with the main objective of the analysis and the study of the stability and the distribution of drugs of abuse and pharmaceuticals in the human liver and the formalin solution during preservation. The results showed that the number of detected compounds in the first month was significantly lower than the compounds found in fresh tissues. The effect of formalin was catalytic, and few substances could be detected. Specifically, out of the 86 positive detections of the monitored substances in the fresh tissues (in which 25 different substances were found), only 32 (37%) remained detectable 3 months after, 20 (23%) 6 months after and 15 (17%) 12 months after.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of 25 psychoactive drugs in cerumen and its application to real postmortem samples.

PURPOSE: In the present study, a method for the detection of 25 psychoactive substances in cerumen was developed and validated. This method targets opiates, cocaine, antidepressants, benzodiazepines, antipsychotics and antiparkinsons. METHODS: Analysis was performed on a SCIEX Triple Quad 6500+ system after liquid-liquid extraction. Methanol with 1% acetic acid was chosen as the extraction solvent. After the addition of the solvent, samples were vortexed, sonicated, centrifuged and directly injected into the liquid chromatography-tandem mass spectrometry system. RESULTS: The method was found to be selective and sensitive (limit of detection: 0.017 ng-0.33 ng/mg), the assay was linear for all analytes with linear regression coefficient ranging 0.9911-1.00. The values for intra-assay precision was between 4.34 and 14.6% and inter-assay precision between 5.81 and 17.7%, with accuracy within the acceptable criteria for all analytes. All analytes in cerumen specimens were stable for 48 h at 4 °C and 72 h at - 20 °C, whilst no significant matrix effect or carryover was observed. Applicability was proven by analyzing cerumen samples from 25 deceased with a history of drug abuse. All analytes were detected in real samples, thus confirming the sensitivity of the developed method. CONCLUSIONS: According to our knowledge, it is the first time that a method for the simultaneous detection of 25 psychoactive drugs in cerumen was developed, fully validated and finally applied to 25 postmortem samples.