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.

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.

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.

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.

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.

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.

Determination of fentanyl and norfentanyl in cerumen in the setting of postmortem investigation.

Cerumen is an emerging alternative biological matrix in the field of forensic toxicology. An ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry [UHPLC-MS/MS] method for the determination of fentanyl and norfentanyl in cerumen was developed and applied in a mixed drug toxicity fatal case. The method was found to be selective and sensitive (LOQ: 0.05 ng/mg for fentanyl and 0.02 ng/mg for norfentanyl), while validation included recovery, carryover, short-term stability, matrix effect, accuracy, and precision (RSD%). Accuracy ranged from 83.1% to 103.5%, while intra- and inter-day precision ranged from 8.6% to 13.1% and from 8.3% to 15.8%, respectively. Matrix effect experiments showed that matrix did not significantly affect signal intensity (82.3%-96.8%). Short-term stability concerning sample extracts was found satisfactory. Fentanyl and norfentanyl were detected in cerumen at a concentration of 1.17 and 0.36 ng/mg respectively. The findings in cerumen corroborate the cause of death and suggest that cerumen is a potential specimen for detecting drugs of abuse in forensic cases.

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.

Efficacy and safety of Mydriatic Microdrops for Retinopathy Of Prematurity Screening (MyMiROPS): study protocol for a non-inferiority crossover randomized controlled trial.

BACKGROUND: Retinopathy of prematurity (ROP) eye examination screening presupposes adequate mydriasis for an informative fundoscopy of preterm infants at risk, on a weekly basis. Systemic absorption of the instilled mydriatic regimens has been associated with various adverse events in this fragile population. This report aims to present the fully developed protocol of a full-scale trial for testing the hypothesis that the reduced mydriatic drop volume achieves adequate mydriasis while minimizing systemic adverse events. METHODS: A non-inferiority crossover randomized controlled trial will be performed to study the efficacy and safety of combined phenylephrine 1.67% and tropicamide 0.33% microdrops compared with standard drops in a total of 93 preterm infants requiring ROP screening. Primary outcome will be the pupil diameter at 45 (T45) min after instillation. Pupil diameter at T90 and T120 will constitute secondary efficacy endpoints. Mixed-effects linear regression models will be developed, and the 95% confidence interval approach will be used for assessing non-inferiority. Whole blood samples will be analyzed using hydrophilic liquid chromatography-tandem mass spectrometry method (HILIC-MS/MS), for gathering pharmacokinetic (PK) data on the instilled phenylephrine, at nine specific time points within 3 h from mydriasis. Pooled PK data will be used due to ethical restrictions on having a full PK profile per infant. Heart rate, oxygen saturation, blood pressure measurements, and 48-h adverse events will also be recorded. DISCUSSION: This protocol is designed for a study powered to assess non-inferiority of microdrops compared with standard dilating drops. If our hypothesis is confirmed, microdrops may become a useful tool in ROP screening. TRIAL REGISTRATION: ClinicalTrials.gov NCT05043077 . Registered on 2 September 2021.

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.

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.

Liquid chromatography tandem mass spectrometry for the determination of nine insecticides and fungicides in human postmortem blood and urine.

Pesticide poisoning is a common occurrence due to their widespread use, easy access and high toxicity even in small concentrations. The most common poisoning fatalities have been observed due to exposure to organophosphates, carbamates and neonicotinoids, thus development of a method for the rapid determination of these compounds in blood and urine is of great importance for clinical and toxicology laboratories. A simple, fast and reliable method was developed for the determination of 9 pesticides in blood and urine using HPLC-MS/MS instrumentation. In order to find the most suitable sample pretreatment technique, three different sample preparation procedures: SPE, protein precipitation and QuEChERS were compared. The final optimized analytical method was fully validated with the values of parameters such as calibration linearity, accuracy, precision, recovery, matrix effect and stability being acceptable. The method proved reliable, accurate, robust and sensitive and was successfully applied for the quantitation of pesticides in three postmortem cases of pesticides poisoning.

Investigation of patterns and dynamics of substance users: A long-term toxicology study on a sample of the Greek population between 2005 and 2019.

The purpose of the present study is to investigate dynamics underlying drug abuse and identify statistical correlations/patterns of forensic findings and sociodemographic factors in a population of illicit substance users. The following long-term studies were conducted: (a) a retrospective autopsy cohort study on autopsy incidents with available toxicological screening results (N = 482) in investigation of any possible forensic associations of exposure to illicit drugs; and (b) a cross-sectional study on a sample of arrested drug law offenders with available toxicological screening results (N = 195) as well as cluster analysis in order to possibly identify user profiles. Although outside the scope of the present study, ethanol was generally considered to be the main substance of abuse, as more than half of the premature deaths reported tested positive on the ethanol toxicology screen. Cannabis and opioid use was associated with unintentional causes of death, while an association of deliberate self-harm was noted with opioid and benzodiazepine use. Both cannabis and opioid use correlated with significantly younger ages (more than a decade) of premature death. Most frequently, an onset of substance use was reported in the early 20s with cannabis use. Although 65.3% of the subjects were diagnosed as dependent and unable to eliminate substance use on their own, only 7.7% of the subjects in the users' population had ever accessed appropriate support through rehabilitation programs.

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.

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.

A UHPLC-MS-MS Method for the Determination of 84 Drugs of Abuse and Pharmaceuticals in Blood.

The analysis of blood samples for forensic or clinical intoxication cases is a daily routine in an analytical laboratory. The list of 'suspect' drugs of abuse and pharmaceuticals that should be ideally screened is large, so multi-targeted methods for comprehensive detection and quantification are a useful tool in the hands of a toxicologist. In this study, the development of an ultra-high performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method is described for the detection and quantification of 84 drugs and pharmaceuticals in postmortem blood. The target compounds comprise pharmaceutical drugs (antipsychotics, antidepressants, etc.), some of the most important groups of drugs of abuse: opiates, cocaine, cannabinoids, amphetamines, benzodiazepines and new psychoactive substances. Sample pretreatment was studied applying a modified Mini-QuEChERS single step, and the best results were obtained after adding a mixture of 20 mg MgSO4, 5 mg K2CO3 and 5 mg NaCl together with 600 µL of cold acetonitrile in 200 µL of sample. After centrifugation, the supernatant was collected for direct injection. LC-MS analysis took place on a C18 column with a gradient elution over 17 min. The method was found to be selective and sensitive, offering limits of detection ranging from 0.01 to 9.07 ng/mL. Validation included evaluation of limit of quantification, recovery, carryover, matrix effect, accuracy and precision of the method. The method performed satisfactorily in relation to established bioanalytical criteria and was therefore applied to the analysis of blood obtained postmortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.

GC-MS analysis of underivatised new psychoactive substances in whole blood and urine.

Herein a method was develop and validated for the detection and quantification of five new psychoactive substances (NPS) belonging to three categories: synthetic cathinones (mephedrone, 3,4-MDPV), opioids (AH-7921) and cannabinoids (JWH-018, AM-2201) by EI GC-MS. Target analytes were quantified in whole blood; in urine the same compounds plus methylone were detected. Liquid-liquid extraction by MTBE - butyl acetate (1:1, v/v) in blood and butyl acetate in urine was applied for the recovery of analytes, while no derivatization was necessary for their sensitive detection and quantification. The method showed good linearity for all analytes within a concentration range from 0.25 to 2 µg/mL for mephedrone, from 0.02 to 0.16 µg/mL for 3,4-MDPV and AH-7921 and from 0.005 to 0.04 µg/mL for JWH-018 and AM-2201. LOD ranged from 0.002 µg/mL (JWH-018 and AM-2201 in blood and urine), to 0.08 µg/mL (mephedrone in urine). LOQ in blood ranged from 0.005 µg/mL for JWH-018 and AM-2201 to 0.25 µg/mL for mephedrone. Accuracy was within acceptable limits with % bias ranging from +20% to -17.98% for intra-assay study and from +18.87% to -11.16% for inter-assay study. Precision was found to be between 2.60% and 17.17% (CV%) for intra-assay study and from 6.03% to 13.72% (CV%) for inter-assay study. An intra laboratory comparison provided proof of the method robustness. The developed method can be used for the reliable and fast quantification of five NPS in blood and the detection of six NPS in urine within the practice of a clinical or forensic toxicology laboratory.

Development of a UHPLC-MS/MS method for the determination of 84 pharmaceuticals and drugs of abuse in human liver.

Analysis of post-mortem liver for toxicological reasons is a considerable option when blood is unavailable. The development of analytical methods for tissue specimens is not as straightforward as for biological fluids as tissue presents challenges to the analytical chemist. The present study reports the development of a UHPLC-MS/MS method for the detection and quantification of 84 drugs and pharmaceuticals in human liver. The selected target drugs include pharmaceutical drugs and drugs of abuse. Sample preparation was studied using QuEChERS and different ratios of solvent volume and sample mass. Best results were attained by homogenizing 1 g of liver with acetonitrile K2CO3 buffer (pH = 10), QuEChERS salts MgSO4/ NaCl (1st purification step) and PSA/ 150 mg MgSO4 (2nd purification step). The extracted sample was analysed on UHPLC-MS/MS in multiple reaction monitoring mode (MRM) on a reversed-phase (Acquity BEH C18) column. Elution was accomplished by gradient program of mobile phase A: water, 0.1% formic acid and B: methanol, 0.1% formic acid that lasted 17 min. The method was specific, without interferences from the complex matrix. Sensitivity was satisfactory with limit of detection (LOD) ranging from 0.01 ng/g to 4.94 ng/g. Validation study was based on the guidelines of international bodies and included evaluation of recovery, carry-over, matrix effect, accuracy, stability, and precision of the method. The method performed satisfactory in relation to established bioanalytical criteria and was therefore applied to the analysis of liver tissue obtained post-mortem from chronic drug abusers, offering unambiguous identification and quantitative determination of drugs in postmortem blood.