Novel sample-substrates for the determination of new psychoactive substances in oral fluid by desorption electrospray ionization-high resolution mass spectrometry

A reliable screening and non invasive method based on the use of microextraction by packed sorbent coupled with desorption electrospray ionization-high resolution mass spectrometry was developed and validated for the detection of new psychoactive substances in oral fluid. The role of different sample substrates in enhancing signal intensity and stability was evaluated by testing the performances of two polylactide-based materials, i.e. non-functionalized and functionalized with carbon nanoparticles, and a silica-based material compared to commercially available polytetrafluorethylene supports. The best results were achieved by using the nonfunctionalized polylactide substrates to efficiently ionize compounds in positive ionization mode, whereas the silica coating proved to be the best choice for operating in negative ionization mode. LLOQs in the low μg/L, a good precision with CV% always lower than 16% and RR% in the 83(±4)-120(±2)% range, proved the suitability of the developed method for the determination of the analytes in oral fluid. Finally, the method was applied for screening oral fluid samples for the presence of psychoactive substances during private parties, revealing mephedrone in only one sample out of 40 submitted to analysis

Determination of methadone and eight new psychoactive substances in hair samples by gas chromatography/mass spectrometry

Many new psychoactive substances (NPSs) with different chemical structures have emerged in the illicit drug market in the last decade. The present work was aimed at the development of a simple method in gas chromatography/mass spectrometry (MS) for the determination of NPS of different classes, the use of cannabinoids, and, at the same time, the evaluation of methadone therapy in hair matrix, within our routine analysis control for methadone treatment or from autopsy cases. The determination of synthetic cannabinoids and methadone therapy used an extraction method based on incubation in concentrated sodium hydroxide (NaOH) solution, providing a dissolution of the keratin matrix. The described method was applied on 15 authentic specimens from our cases: five showed the presence of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The described method can be useful not only in the forensic investigation of NPS-related addiction histories but also in epidemiological and retrospective studies on the spread of NPS among specific safety-sensitive social workers. The GC instrument was an Agilent 7820A (Agilent Technologies, Santa Clara, CA, USA), and the detection system was an Agilent 5977B single quadrupole MS operating in selective ion monitoring mode. Validation parameters such as limit of detections (LODs), limit of quantifications (LOQs), repeatability, accuracy, and linearity were satisfactory for its application on real specimens. LODs, LOQs, R%CV, standard deviation, and the mean concentration for the analyzed compounds are reported in Table 1b. Accuracy and repeatability were acceptable for all the analytes at their respective LOQs. Recovery experiments varied from 58.3% to 103.0%, thus allowing the application on authentic specimens. The described method can be useful not only in the forensic investigation of NPS-related addiction histories but also in epidemiological and retrospective studies on the spread of NPS among specific safety-sensitive social workers, such as drivers

“Toxicological findings: a retrospective overview of medico-legal investigations in Parma (Italy)”

The aim of this study was to collect all available data from 2009 to 2016 focusing on the epidemiological, clinical and pharmacological issues only related to acute intoxication fatalities in the Unit of Legal Medicine of the Department of Medicine and Surgery at the University of Parma. All death certificates and autopsy reports were retrieved from the archives and evaluated to identify cases in which only acute intoxication from xenobiotics could be defined as the cause of death, however statistical and descriptive analyses were applied to all the data. A more comprehensive analysis on all causes of death showed that out of 1005 total cases the most common is haemorrhagic shock/traumatic shock (36.5%), followed by cardiogenic shock with 27.4%; asphyxia ranks as the third cause of death (11.8%); concerning encephalic injuries, our data show 10.9% of cases, while acute intoxication by xenobiotics accounts for 5.7%. Data show that the majority of subjects are poly-abuser (75.4%); people not enrolled within a preventive treatment (59.4%) were more likely to commit suicide (28.1%), whereas only 6.2% in the sub-population in treatment (40.6%) committed suicide: therefore, data strongly suggest the evidence that joining a preventive programme can decrease the probability of extreme action. Access to a full case history may indeed save considerable time and expense in carrying out tests, but also valuable targeted samplings. The investigating officer should, therefore, submit as much information as possible about the case, as this may influence the type and extent of analysis undertaken, as well as the interpretation of analytical results

Planned and unplanned complex suicides: Casuistry of the Institute of Legal Medicine of Parma (Italy)

Complex suicide is performed using more harmful methods, simultaneously or consecutively. In these cases, the distinction between suicide and homicide represents a challenge for forensic pathologists. In literature, complex suicide is divided in two subgroups: â\u80\u9cplanned complex suicideâ\u80\u9d or â\u80\u9cunplanned complex suicideâ\u80\u9d depending from forensic features and often related to psychiatric variables. Aim of this study was to show the casuistry of complex suicide in Parma's Forensic service analyzing, for each case, the forensic medical problems (type and site of lesions on the body), and the supplementary data [Police's inspection report, toxicological analysis and psychiatric anamnesis (when available)], trying, through a multidisciplinary approach, to determine a possible correlation between the victim's mood and suicide's method chosen, whether planned or unplanned. Our results showed the importance of all the elements collected on the crime scene to distinguish suicide from homicide, and the correlation between bipolar disorder, borderline personality disorder and schizophrenia with unplanned complex suicide (because of the impulsiveness), and major depression disorder and anxiety disorder with planned complex suicide. Being able to understand the causes behind this extreme gesture may become important not only for forensic pathologists and judicial authority, but also, and above all, for the family as well

Cleaning up blood samples using a modified "QuEChERS" procedure for the determination of drugs of abuse and benzodiazepines by UPLC-MSMS

The "QuEChERS" (quick, easy, cheap, effective, rugged, and safe) dispersive SPE (dSPE) method is an emerging sample preparation technique that is becoming increasingly popular in the area of multi-residue pesticide analysis in food and agricultural products. A simplified QuEChERS extraction method followed by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed for the simultaneous determination of forensically relevant drugs of abuse (opiates including buprenorphine, methadone and fentanyl and analogues, cocaine and metabolites, amphetamines, LSD) and benzodiazepines and analogues (Z-drugs) in 1mL of human whole blood performing a sole extraction. The method was validated showing good repeatability, accuracy and linearity; LODs were 0.5ng/mL for all benzodiazepines tested while for drugs of abuse LODs varied from 0.05 to 2ng/mL. The method showed high throughput capabilities and was applied on various forensic cases for determination of pharmaceuticals and drugs of abuse

Determination of synthetic and natural cannabinoids in oral fluid by solid-phase microextraction coupled to gas chromatography/mass spectrometry: A pilot study

In 2008, several synthetic cannabinoids were detected in herbal smoking blends sold on websites and in the so-called “smart shops”. These compounds, as well as new psychoactive substances, flooded the market of illicit drugs and are sold at street level. Development and validation of rapid analytical methods for the detection and quantification of synthetic cannabinoids in biological matrices are essential for the investigation of pharmacokinetic, pharmacodynamic and toxicological properties. In this pilot study, the potential of direct immersion solid-phase microextraction coupledto GC/MS for the determination of synthetic and natural cannabinoids in oral fluid was proved. Validation proved the suitability of the developed method for the determination of the analytes at trace levels in oral fluid: linearity was assessed in the LOQ – 1000 ng/mL range, whereas a good precision was observed with CV below 20%. The method was then applied to more than 100 real oral fluid samples collected from a population of young students, showing a positivity rate of 3.8

Simplifying sample pretreatment: application of dried blood spot (DBS) method to blood samples, including postmortem, for UHPLC-MS/MS analysis of drugs of abuse

The complexity of biological matrices, such as blood, requires the development of suitably selective and reliable sample pretreatment procedures prior to their instrumental analysis. A method has been developed for the analysis of drugs of abuse and their metabolites from different chemical classes (opiates, methadone, fentanyl and analogues, cocaine, amphetamines and amphetamine-like substances, ketamine, LSD) in human blood using dried blood spot (DBS) and subsequent UHPLC-MS/MS analysis. DBS extraction required only 100μL of sample, added with the internal standards and then three droplets (30μL each) of this solution were spotted on the card, let dry for 1h, punched and extracted with methanol with 0.1% of formic acid. The supernatant was evaporated and the residue was then reconstituted in 100μL of water with 0.1% of formic acid and injected in the UHPLC-MS/MS system. The method was validated considering the following parameters: LOD and LOQ, linearity, precision, accuracy, matrix effect and dilution integrity. LODs were 0.05-1ng/mL and LOQs were 0.2-2ng/mL. The method showed satisfactory linearity for all substances, with determination coefficients always higher than 0.99. Intra and inter day precision, accuracy, matrix effect and dilution integrity were acceptable for all the studied substances. The addition of internal standards before DBS extraction and the deposition of a fixed volume of blood on the filter cards ensured the accurate quantification of the analytes. The validated method was then applied to authentic postmortem blood samples

Oral fluid analysis to monitor recent exposure to synthetic cannabinoids in a high-risk subpopulation

Among novel psychoactive substances, synthetic cannabinoid receptor agonists (SCRA) seem to have the widest diffusion in the population with no limitation to any particular demographic group. Information on drug consumption relies mostly on anonymized surveys and less on clinical or analytical data; notwithstanding, the huge efforts constantly made to enroll subjects to gather epidemiological data. In the present study, we collected 66 oral fluid samples from volunteers in a drug rehabilitation center in 2019 and early 2020. A liquid chromatography-mass spectrometry method previously developed and validated by our Unit was applied to detect SCRA (n = 10) in oral fluid. Results proved the presence of synthetic cannabinoids at a positivity rate of almost 20%, with detection frequency HU211(5/13) > UR144/JWH122 (3/13) > JWH019/JWH081/AM2201 (1/13). Concentrations were in the range < LOQ -0.36 ng/ml. Synthetic cannabinoids consumption had not been declared by any volunteer. This study enabled for the unprecedent detection of synthetic cannabinoids use in the territory of Parma (Italy) in a high-risk subpopulation. The identified SCRA proved the persistence into the market of the "first-generation" JWH family into the Italian territory and the marketing of relatively new ones (AM-2201). Public health consequences represented by NPS consumption are still scarce; therefore, further studies are needed to understand the real diffusion in the population

Hygrine and cuscohygrine as possible markers to distinguish coca chewing from cocaine abuse in workplace drug testing.

Cocaine abuse is widespread all over the world, and is performed generally by sniffing, injecting or smoking cocaine or crack. The distinction between the recreational use of cocaine from the practice of the so called \u2018\u2018coqueo\u2019\u2019 is still an issue in those countries where this habit is diffused and where it is not considered an addiction, by this reason is necessary to develop a method for to distinguish the coca chewers and cocaine abusers. The use of an unique marker to distinguish between cocaine abuse and chewing of coca leaves is of fundamental importance in those countries where this habit is diffused. Certain alkaloids of the leaves of Erythroxylum coca are lost during the process of extraction/purification of cocaine and it is not possible to find them neither in seizures of chlorhidrate of cocaine nor urine samples of cocaine abusers. These markers are the hygrine and cuscohygrine that are present in the leaves of E. coca. A fast GC/MS method involving a liquid:liquid extraction procedure with tertbutylmethylether (TBME) is proposed for the determination of some alkaloids in cocaine leaves, cocaine seizures and biological samples. All specimens were alkalinized to pH 9 with a carbonate/bicarbonate buffer and then extracted with TBME. The analysis was carry out by GC/MS with electron impact at 70 eV and in full scan mode. The results demonstrate that hygrine and cuscohygrine are not found neither in the urine of cocaine abusers nor in cocaine seizures. For this reason this compounds could be considered as markers of coca chewing. This developed method permits to distinguish coca chewing from cocaine abuse in workplace drug testing through the analysis of urine samples