Metabolism of JWH-015, JWH-098, JWH-251, and JWH-307 in silico and in vitro: a pilot study for the detection of unknown synthetic cannabinoids metabolites.

This pilot study was performed to study the main metabolic reactions of four synthetic cannabinoids: JWH-015, JWH-098, JWH-251, and JWH-307 in order to setup a screening method for the detection of main metabolites in biological fluids. In silico prediction of main metabolic reactions was performed using MetaSite(™) software. To evaluate the agreement between software prediction and experimental reactions, we performed in vitro experiments on the same JWHs using rat liver slices. The obtained samples were analyzed by liquid chromatography-quadrupole time-of-flight and the identification of metabolites was executed using Mass-MetaSite(™) software that automatically assigned the metabolite structures to the peaks detected based on their accurate masses and fragmentation. A comparison between the experimental findings and the in silico metabolism prediction using MetaSite(™) software showed a good accordance between experimental and in silico data. Thus, the use of in silico metabolism prediction might represent a useful tool for the forensic and clinical toxicologist to identify possible main biomarkers for synthetic cannabinoids in biological fluids, especially urine, following their administration.

Cannabinoids determination in oral fluid by SPME-GC/MS and UHPLC-MS/MS and its application on suspected drivers.

The confirmation of Δ9-tetrahydrocannabinol (THC) in oral fluid (OF) is an important issue for assessing Driving Under the Influence of Drugs (DUID). The aim of this research was to develop a highly sensitive method with minimal sample pre-treatment suitable for the analysis of small OF volumes (100 µL) for the confirmation of cannabinoids in DUID cases. Two methods were compared for the confirmation of THC in residual OF samples, obtained from a preliminary on-site screening with commercial devices. An ultra high performance LC-MS (UHPLC-MS/MS) method and an SPME-GC/MS method were hence developed. 100 µL of the residual mixture OF/preservative buffer or neat OF was simply added to 10 µL of THC-D3 (1 µg/mL) and submitted to the two different analyses: A - direct injection of 10 µL in UHPLC-MS/MS in positive electrospray ionisation (ESI) mode and B - sampling for 30 min with SPME (100 µm polydimethylsiloxane or PDMS fibre) and direct injection by desorption of the fibre in the GC injection port. The lowest limit of detection (LLOD) of THC was 2 ng/mL in UHPLC-MS/MS and 0.5 ng/mL in SPME-GC/MS. In addition, cannabidiol (CBD) and cannabinol (CBN) could be detected in GC/MS equipment at 2 ng/mL, whilst in UHPLC-MS/MS the LLOD was 20 ng/mL. Both methods were applied to 70 samples coming from roadside tests. By SPME-GC/MS analysis, THC was confirmed in 42 samples, whilst CBD was detected in 21 of them, along with CBN in 14 samples. THC concentrations ranged from traces below the lowest limit of quantification or LLOQ (2 ng/mL) up to 690 ng/mL.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

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.

UHPLC-ESI-MS/MS method for direct analysis of drugs of abuse in oral fluid for DUID assessment.

An ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method for the direct analysis in oral fluid (OF) of several abused drugs and metabolites in a single chromatographic run was set up and validated. Amphetamine, methamphetamine, morphine, O-6-monoacetylmorphine, cocaine, codeine, methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine, methylenedioxyamphetamine, methadone, benzoylecgonine (BEG), Δ9-tetrahydrocannabinol (THC), ketamine, and cocaethylene were determined in a single chromatographic run with no sample pretreatment, after addition of the respective deuterated internal standards. The method was designed to perform a confirmation analysis on the residual OF samples after the preliminary on-site screening test, and it was applied on preservative buffers from different devices (Mavand Rapidstat, Concateno DDS, and Greiner Bio-One) or on neat OF samples. The method was suitable to be applied to the small amounts of sample available for the confirmatory analysis after the preliminary on-site screening or on undiluted OF samples. Limits of detection varied from 5 (morphine) to 0.2 ng/mL (methamphetamine, MDMA, BEG, and cocaethylene). The method was linear for all the substances involved, giving quadratic correlation coefficients of >0.99 in all the different preservative buffers checked. In addition, repeatability and accuracy were satisfactory for the majority of the substances, except for a few cases. The developed method was subsequently applied to 466 residual samples from on-site screening performed by police officers. Of these samples, 74 showed the presence of cocaine and metabolites; THC was detected in 49 samples. Two samples showed codeine and morphine while MDMA was detected in 11 samples and ketamine in four samples.

A gas chromatography/mass spectrometry method for the determination of sildenafil, vardenafil and tadalafil and their metabolites in human urine.

Sildenafil (SDF), vardenafil (VDF) and tadalafil (TDF) are phosphodiesterase type 5 enzyme inhibitors (PDE5Is), used in the treatment of erectile disorders and to improve breathing efficiency in pulmonary hypertension. The increasing incidence of their use among young athletes has drawn the attention of the anti-doping authorities to the possible abuse of PDE5Is by athletes due to their pharmacological activities. This paper describes a method for the determination in urine of PDE5Is and their metabolites by gas chromatography/mass spectrometry (GC/MS) after liquid/liquid extraction of the analytes from urine and derivatisation to obtain trimethylsilyl derivatives. The metabolic profile was studied on real samples collected from subjects taking PDE5Is (Viagra, Levitra or Cialis); the main urinary metabolites were identified and their MS fragmentation characterized. The sample pre-treatment and GC/MS conditions for the detection of the metabolites have been optimised. A method for their preliminary screening and subsequent confirmation is described that takes into account the general requirements of a routine doping analysis to be used for the screening of large numbers of samples. The main metabolites identified can be included in a general purpose screening method and all the metabolites in a more specific confirmation method. The method developed has been applied for the screening of PDE5Is in 5000 urine samples. Based on the obtained results, the proposed method appears to be of practical use in analytical and forensic toxicology, including doping analysis.

Validation of a Bioanalytical Method for the Determination of Synthetic and Natural Cannabinoids (New Psychoactive Substances) in Oral Fluid Samples by Means of HPLC-MS/MS.

New psychoactive substances (NPS) represent an important focus nowadays and are continually produced with minimal structural modifications in order to circumvent the law and increase the difficulty of identifying them. Moreover, since there are a high number of different compounds, it is arduous to develop analytical screening and/or confirmation methods that allow the identification and quantification of these compounds. The aim of this work is to develop and validate a bioanalytical method for detecting new synthetic drugs in biological samples, specifically oral fluid, using high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS) with minimal sample pretreatment. Oral fluid samples were simply centrifuged and denaturized with different rapid procedures before injection into the LC-MS/MS system. Calibration curves covered a linear concentration range from LOQ to 100 ng/mL. Validation parameters such as linearity, precision, accuracy, selectivity, matrix effect and thermal stability were evaluated and showed satisfactory results, in accordance with US Food & Drug Administration guidelines. The inter-day analytical bias and imprecision at two levels of quality control (QC) were within ±15% for most compounds. This method was able to identify and calculate the concentration of 10 NPS validated in this biological sample, even in the presence of matrix effect.

A case report positive for synthetic cannabinoids: are cardiovascular effects related to their protracted use?

The use of synthetic cannabinoids is being increasingly recognised worldwide, but the chemical compositions and physiological effects of these drugs are poorly characterised and are continually changing. New substances are constantly being added to the content of synthetic cannabinoids and they are rarely identified on toxicological screening tests. Due to their structures synthetic cannabinoids and their effects have been compared to the psychoactive compound, Δ-9-tetrahydrocannabinol (THC), found in marijuana. On the molecular level, they are potent cannabinoid receptor agonists that also may have affinity for other types of receptors such as those on platelets. Reported symptoms of toxicity include anxiety, agitation, paranoia, hallucinations, tachycardia, hypertension, excessive sweating, nausea, and vomiting. They can also be linked to serious adverse cardiovascular events and can affect the cardiovascular system causing hypotension and bradycardia, myocardial infarction, atrial fibrillation, prolonged QTc, and Mobitz type II atrioventricular block, as well as interfere with the aggregation of platelets. We present a case report of a cardiac tamponade with toxicological findings positive for synthetic cannabinoids. This case highlights the importance of testing routinely for novel psychoactive compounds, and recognising their potential to cause life-threatening conditions.

Screening methods for rapid determination of new psychoactive substances (NPS) in conventional and non-conventional biological matrices.

In the last years, a global awareness has arisen from the reported harmful effects and public health risks associated with the consumption of new psychoactive substances (NPSs). Improving efforts in the detection and identification of these substances have emerged as a global analytical challenge involving the large range of NPSs' chemical structures and the variety of conventional and non-conventional biological matrices. Indeed, detection capabilities and screening tools impact many fields and settings, including seized products analysis, workplace and roadside drug controls, emergency rooms, drug addiction treatment clinics, post-mortem and criminal caseworks, law enforcement and health interventions. Colorimetric, immunochemical and chromatographic-mass spectrometry techniques have been investigated and developed for the rapid identification of NPSs. Considering the continuous emergence of new substances, this review offers a panoramic view on the current status of analytical approaches for the rapid screening of NPSs, including, when available, data on conventional and non-conventional biological matrices. Although some of the presented methods are sound and promising, their applications are still limited, thus proving the importance of further investigations. New screening and sensitive targeted methods for NPS and their metabolites should be developed in different types of biological matrices, where concentration of substances and matrix effects can be significantly different.

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.

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 coupled to 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%.

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 non-functionalized 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.

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: "planned complex suicide" or "unplanned complex suicide" 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.

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.

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.

UHPLC-MS/MS and UHPLC-HRMS identification of zolpidem and zopiclone main urinary metabolites and method development for their toxicological determination.

Zolpidem and zopiclone (Z-compounds) are non-benzodiazepine hypnotics of new generation that can be used in drug-facilitated sexual assault (DFSA). Their determination in biological fluids, mainly urine, is of primary importance; nevertheless, although they are excreted almost entirely as metabolites, available methods deal mainly with the determination of the unmetabolized drug. This paper describes a method for the determination in urine of Z-compounds and their metabolites by ultra-high-pressure liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) and UHPLC coupled with high resolution/high accuracy Orbitrap® mass spectrometry (UHPLC-HRMS). The metabolic profile was studied on real samples collected from subjects in therapy with zolpidem or zopiclone; the main urinary metabolites were identified and their MS behaviour studied by MS/MS and HRMS. Two carboxy- and three hydroxy- metabolites, that could be also detected by gas chromatography/mass spectrometry (GC-MS) as trimethylsylyl derivatives, have been identified for zolpidem. Also, at least one dihydroxilated metabolite was detected. As for zopiclone, the two main metabolites detected were N-demethyl and N-oxide zopiclone. For both substances, the unmetabolized compounds were excreted in low amounts in urine. In consideration of these data, a UHPLC-MS/MS method for the determination of Z-compounds and their main metabolites after isotopic dilution with deuterated analogues of zolpidem and zopiclone and direct injection of urine samples was set up. The proposed UHPLC-MS/MS method appears to be practically applicable for the analysis of urine samples in analytical and forensic toxicology cases, as well as in cases of suspected DFSA.

Screening for new psychoactive substances in hair by ultrahigh performance liquid chromatography-electrospray ionization tandem mass spectrometry.

In the latest years, many new psychoactive substances (NPS) from several drug classes have appeared in the illicit drug market. Their rapid, sensitive and specific identification in biological fluids is hence of great concern for clinical and forensic toxicologists. Here is described a multi-analyte method for the determination of NPS, pertaining to different chemical classes (synthetic cannabinoids, synthetic cathinones, ketamine, piperazines and amphetamine-type substances-ATS) in human hair using ultrahigh performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in electrospray ionization mode. We focused on a sample preparation able to extract the different classes of NPS. About 30mg of hair was decontaminated and incubated overnight under sonication in different conditions depending on the type of analytes to be extracted: (a) with 300µL of HCOOH 0.1% for cathinones, piperazines and ATS; (b) with 300µL of MeOH for synthetic cannabinoids. Ten microliter of the extracts were then injected in UHPLC-ESI-MS/MS in MRM mode. The LODs varied from 2pg/mg to 20pg/mg. The method was linear in the range from the LOQ to 500pg/mg and showed acceptable precision (%RSD<15) and accuracy (%E<15) for all the analytes. The method was finally applied on 50 samples from real forensic cases (driving license re-granting, postmortem toxicological analyses, workplace drug testing). In three samples we detected synthetic cannabinoids, in four samples cathinones or ephedrines, in two samples ketamine.

Screening for exogenous androgen anabolic steroids in human hair by liquid chromatography/orbitrap-high resolution mass spectrometry.

A method for the screening of various anabolic steroids and their esters in human hair, based on liquid-chromatography-high resolution mass spectrometry using an Exactive benchtop Orbitrap mass spectrometer, has been set up and validated. This method involved methanolic incubation of 30 mg of hair and analysis of the relevant extract in HPLC using a C18 column. The mass detector, with nominal resolving power of 100,000, operated in full scan mode in APCI under positive ionization mode. Analytes were identified by exact mass, correspondence of isotopic cluster and retention times. The limits of detection obtained varied from 10 to 50 pg mg(-1), and limits of quantitation were 0.5 ng mg(-1) for all compounds. The method was linear for all analytes in the ranges from the LOQ to 6 ng mg(-1), giving correlation coefficients >0.99 for all analytes. Also accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Specificity was assessed by analysing ten blank samples and fifteen samples from polidrug abusers. This method was applied to a real-life case, resulting in the identification of testosterone undecanoate in the hair of a suspect. The analyte identity was confirmed by the analysis of its in-source fragmentation and comparison to a certified standard. Thanks to the scan acquisition, this method also enables retrospective re-analysis of the acquired datafile in case a further analyte needs to be screened.

Ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry screening method for direct analysis of designer drugs, "spice" and stimulants in oral fluid.

An ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) screening method for the direct analysis in oral fluid (OF) of 24 drugs, including new synthetic cannabinoids and so-called "smart" designer drugs, in a single chromatographic run was set up. Benzylpiperazine, methylone, 5,6-methylenedioxy-2-aminoindane (MDAI), fenproporex, 4-fluoroamphetamine (4-FA), 4-methyl-N-ethylcathinone (4-MEC), 4-methylamphetamine (4-MA), methylbenzodioxolylbutanamine (MBDB), mephedrone, methylthioamphetamine (MTA), methylenedioxypyrovalerone (MDPV), mefenorex, nabilone, furfenorex, clobenzorex, JWH-200, AM 694, JWH-250, JWH-073, JWH-018, JWH-019, JWH-122, HU 210 and CP 47497 were determined in a chromatographic run of 9 min only with no sample pre-treatment, after addition of ISs and dilution in mobile phase A. This method is designed to be applied to 250 µL of OF sample, anyway is suitable to be used on smaller volumes (till 100 µL). LODs vary from 1ng/mL to 20 ng/mL. No interfering peaks were observed due to similar analytes, common therapeutic drugs or endogenous compounds. Matrix effect, although present especially for mephedrone, is acceptable, allowing the detection of the compounds at the LODs described. The developed method was applied on 400 real OF samples from on-site tests performed by police officers.

Evaluation of four oral fluid devices (DDS®, Drugtest 5000®, Drugwipe 5+® and RapidSTAT®) for on-site monitoring drugged driving in comparison with UHPLC-MS/MS analysis.

New Italian legislation on driving under the influence of drugs considers oral fluid (OF) as a possible alternative drug testing matrix. On this basis, the present research was carried out to evaluate the applicability of four commercial on-site OF drug screening devices, namely DDS(®), Drugtest 5000(®), Drugwipe 5+(®) and RapidSTAT(®), in a real operative context. Preliminarily trained police officers tested randomly stopped drivers with two different kits side-by-side during roadside patrols. A central laboratory confirmed on-site kits' results by UHPLC-MS/MS analysis of the saliva specimen remaining after the screening analysis. 1025 drivers were submitted to the OF tests: 11.6% were positive for cocaine and metabolites, 11.1% for THC, 6% for amphetamines and amphetamine-type designer drugs and 2.3% for ketamine. The sensitivities of the kits were 81% (RapidSTAT(®)), 82% (DDS(®)), 90% (Drugwipe 5+(®)) and 97% (Drugtest 5000(®)) for cocaine and 38% (DDS(®)), 47% (Drugwipe 5+(®)), 72% (RapidSTAT(®)) and 92% (Drugtest 5000(®)) for THC. Drugtest 5000 was the only kit showing an acceptable sensitivity for on-site application. Only Drugtest 5000(®) and RapidSTAT(®) could be evaluated for amphetamines and methamphetamines: Drugtest 5000(®) showed a sensitivity of 100% in the case of amphetamines and 86% for methamphetamines, while RapidSTAT(®) 90% and 76% respectively. Nowadays, ketamine is not included in the target analytes of any on-site devices, but it was systematically included in the UHPLC-MS/MS confirmatory analysis. To ensure adequate reliability, MS confirmation of on-site OF screening tests is anyway always necessary, due to the presence of a significant number of false positive results even when using the commercial kit with the best performance.