Development of a micro-solid-phase extraction molecularly imprinted polymer technique for synthetic cannabinoids assessment in urine followed by liquid chromatography–tandem mass spectrometry

Several molecularly imprinted polymers (MIPs) have been synthesized for the first time using various synthetic cannabinoids (JWH007, JWH015 and JWH098) as template molecules. Ethylene dimethacrylate (EDMA) was used as a functional monomer for all cases. Similarly, divinylbenzene (DVB) and 2,2′-azobisisobutyronitrile (AIBN) were used as cross-linker and initiator, respectively. The prepared MIPs have been fully characterized and evaluated as new selective adsorbents for micro-solid phase extraction (μ-SPE) of synthetic cannabinoids in urine. The developed MIP-μ-SPE devices consisted of a polypropylene (PP) porous membrane containing the adsorbent (novel porous membrane protected micro-solid phase extraction based on a cone-shaped device) for operating in batch mode, which allowed a fast and integrated extraction-cleanup procedure. High performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was used for quantifying the analytes after MIP-μ-SPE. The best performances were obtained for MIPs prepared from JWH015 as a template. Optimum loading conditions were found to be urine pH of 5.0 and adsorption time of 8.0 min under mechanical (orbital-horizontal) stirring at 100 rpm. The composition of the eluting solution consisted of 75:20:5 heptane/2-propanol/ammonium hydroxide. The elution was assisted by ultrasounds (37 kHz, 325 W) for 8.0 min. In addition, studies regarding selectivity have also been addressed for several drugs of abuse under optimized loading/adsorption conditions. Validation of the method showed good precision and analytical recovery by intra-day and inter-day assays (RSD values lower than 7 and 10% for intra-day and inter-day precision, and within the 83–100% range for intra-day and inter-day analytical recovery)

A Probable Fatal Case of Oleander (Nerium oleander) Poisoning on a Cattle Farm: A New Method of Detection and Quantification of the Oleandrin Toxin in Rumen

Oleander (Nerium oleander) is an ornamental plant common in tropical and sub-tropical regions that is becoming increasingly widespread, even in temperate regions. Oleander poisoning may occur in animals and humans. The main active components contained in the plant are cardiac glycosides belonging to the class of cardenolides that are toxic to many species, from human to insects. This work describes a case of oleander poisoning that occurred on a small cattle farm and resulted in the fatality of all six resident animals. Furthermore, the investigation of the poisonous agent is described, with particular focus on the characterization of the oleandrin toxin that was recovered from the forage and rumen contents. The innovation of this study is the first description of the detection and quantification of the oleandrin toxin by liquid chromatography-high resolution mass spectrometry (LC-HRMS) in rumen

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

High-throughput dispersive liquid/liquid microextraction (DLLME) method for the rapid determination of drugs of abuse, benzodiazepines and other psychotropic medications in blood samples by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and application to forensic cases

The process of dispersive liquid liquid microextraction (DLLME) was successfully applied for the simultaneous extraction and subsequent ultrahigh performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) determination of many different classes of drugs from whole blood samples. Main drugs of abuse (cocaine and metabolites, amphetamines and analogues, LSD, ketamine, opiates including buprenorphine, methadone and fentanyl and analogues), benzodiazepines, Z-compounds and other psychotropic drugs were effectively extracted in a single step and determined with satisfactory sensitivity, accuracy, repeatability and linearity. The matrix effect obtained was very low for all the analytes (maximum of 26% of ion suppression or 28% enhancement at low concentrations), demonstrating the effectiveness of sample purification. The limits of detection (LODs) varied from 0.05 to 2 ng/mL, limits of quantitation (LOQs) from 0.2 to 10 ng/mL. Accuracy and precision were satisfactory: %errors spanned from 0.1 to 15% for drugs of abuse and from 0.4 to 18%, at LOQs, for benzodiazepines and other psychotropic drugs. Interday %CV ranged from 2 to 15 % for drugs of abuse and from 0.2 to 18% (at low concentrations) for other drugs. The method was linear for all the studied analytes, giving regression coefficients (R2) always higher than 0.994. The developed method was successfully applied to the analysis of 50 blood samples from forensic cases, allowing determining the presence of different benzodiazepines in 13 cases, of drugs of abuse in 14 cases, and of other psychotropic drugs in four cases

A μ-SPE PROCEDURE FOR THE DETERMINATION OF CANNABINOIDS AND THEIR METABOLITES IN URINE BY LC–MS/MS

tIn this paper the development and validation of a method for the analysis of THC-COOH, THC, THC-OH,CBD and CBN in their total form in urine by LC–MS/MS is presented.Tandem hydrolysis, i.e. enzymatic and basic, has been found optimal for the simultaneous analysis of theselected analytes in urine: basic hydrolysis is more effective for the cleavage of THC-COOH glucuronidewhile enzymatic hydrolysis allows the cleavage of the conjugated cannabinoids possessing ether bonds(THC, THC-OH, CBD).The whole procedure requires a 2 h enzymatic hydrolysis using only 90 L of urine by -SPE extractiontechnique with C18tips. Clear advantages in terms of time and of enzyme reduction are obtained andthe cost of the analysis can be dramatically reduced. Satisfactory recovery values and matrix effect areobtained, and the chromatographic run, performed with a fused-core column, allowed the completeanalyte separation in only 3 min (total run 5.8 min) with a common HPLC system.Furthermore the whole procedure has been validated according to SWGTOX guidelines: LOQs arebetween 6 and 10 ppb, quite lower than the requested cut-off for urine testing; intermediate repro-ducibility of the selected analytes is below 10% and accuracy is between 85% and 113%, except for CBD,included only for semi-quantitative determination

A SNAPSHOT ON NPS IN ITALY: DISTRIBUTION OF DRUGS IN SEIZED MATERIALS ANALYSED IN AN ITALIAN FORENSIC LABORATORY IN THE PERIOD 2013-2015

The diffusion of New Psychoactive Substances (NPS) in the illicit drug market is a worldwide problem. The aim of the study is to describe the qualitative distribution of drugs of abuse in seized materials confiscated in the Italian territory over the last two years. Between 2013 and 2015 162 seizures of substances purchased through the Internet and confiscated by police authorities were analyzed: 35 seizures (22%) were crystals of 3-methylmethcathinone (3-MMC). Although 3-MMC is subject to the relevant legislation in Italy, it is not controlled in other countries such as the Netherlands, from which the shipments originated. 33 seizures (20%) were crystals of 4-methylethcathinone (4-MEC), 19 seizures (12%) were powders containing methylenedioxypyrovalerone (MDPV). N,N-diallyl-5-methoxytryptamine (5-MeO-DALT) was identified in 5 powders, whereas ethylphenidate in six and pyrrolidinophenones in fourteen seized powders: 6 α-PVP (alpha-pyrrolidinovalerophenone), 6 α-PHP (alpha-pyrrolidinohexiophenone) and 1 α-PVT (alpha-pyrrolidinopentiothiophenone). Other substances identified were cathinones such as pentedrone, methylone, buthylone, ethylone, methedrone, 3-CMC (3-chloromethcathinone), 3,4-dimethylmethcathinone (3,4-DMMC), flephedrone (4-fluoromethcathinone or 4-FMC), 2-FMC and 3-FMC (2- and 3-fluoromethcathinone), MPPP (4-methyl-alpha-pyrrolidinopropiophenone), bk-2C-B (2-amino-1-(4-bromo-2,5-dimethoxyphenyl)ethan-1-one). Other compounds were NM2AI (N-methyl-2-aminoindane), MPA (1-(thiophen-2-yl)-2-methylaminopropane), MTTA (mephtetramine), 4-APB and 6-APB (4- and 6- (2-aminopropyl)benzofuran), 2-fluoromethamphetamine, 1mCPP (1-meta-chlorophenylpiperazine) and diphenidine, detected for the first time in Europe. Only three seizures contained synthetic cannabinoids, consisting of herbal blends soaked in N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB48), or a mixture of 5-F-AKB48 and BB-22 (1-(cyclohexylmethyl)-8-quinolinyl ester-1H-indole-3-carboxylic acid). In some mixtures of drugs - such as granules - 4-MEC and pentedrone were detected, also with traces of diphenidine on one occasion. In other cases 5-MeO-DALT, ethylphenidate and caffeine were mixed together. In one batch, the mixture was flephedrone and methoxethamine, whereas in another one the sample contained methylone, ethylone, methedrone, 4-fluoroamphetamine, 5-MeO-DALT and 5MeO-MiPT (N-methyl-N-isopropyl-5-methoxytryptamine). In 9 seizures, tablets shipped together with NPS were also found to contain sildenafil. The analyses performed on these seizures showed the presence of a wide number of NPS within the Italian boundaries coming from abroad, therefore this study confirms the threat for the public health, especially when the content of NPS being sold is not reported on the label or misleading

LC-HRMS characterization of the skin pigmentation and sexualenhancers melanotan II and bremelanotide sold on the blackmarket of performance and image enhancing drugs

The spread of performance and image enhancing drugs (PIEDs) often requires foren-sic toxicology laboratories to identify unknown compounds without reference stan-dards. We characterized the PIEDs melanotan II and bremelanotide, not legallymarketed, in eight unknown samples confiscated by police together with anabolicsteroids, hormone modulators, sexual enhancers and stimulants, intended for theblack market of bodybuilders, using liquid chromatography–high resolution/highaccuracy Orbitrap mass spectrometry (LC-HRMS). The characterization was carriedout by the accurate mass measurements of MH+ionic species, the study of theirisotopic patterns and the associated relative isotopic abundance (RIA) values, as wellas the accurate mass measurements of collision-induced product ions obtained infragmentation experiments. LC-HRMS confirmed itself as a powerful analytical toolto elucidate the elemental composition and structural characteristics of unknowncompounds

High-throughput screening for new psychoactive substances (NPS) in whole blood by DLLME extraction and UHPLC-MS/MS analysis

The increasing number of new psychoactive substances (NPS) present in the illicit market render their identification in biological fluids/tissues of great concern for clinical and forensic toxicology. Analytical methods able to detect the huge number of substances that can be used are sought, considering also that many NPS are not detected by the standard immunoassays generally used for routine drug screening. The aim of this work was to develop a method for the screening of different classes of NPS (a total of 78 analytes including cathinones, synthetic cannabinoids, phenethylamines, piperazines, ketamine and analogues, benzofurans, tryptamines) from blood samples. The simultaneous extraction of analytes was performed by Dispersive Liquid/Liquid Microextraction DLLME, a very rapid, cheap and efficient extraction technique that employs microliters amounts of organic solvents. Analyses were performed by a target Ultrahigh Performance Liquid Chromatography tandem Mass Spectrometry (UHPLC-MS/MS) method in multiple reaction monitoring (MRM). The method allowed the detection of the studied analytes with limits of detection (LODs) ranging from 0.2 to 2ng/mL. The proposed DLLME method can be used as an alternative to classical liquid/liquid or solid-phase extraction techniques due to its rapidity, necessity to use only microliters amounts of organic solvents, cheapness, and to its ability to extract simultaneously a huge number of analytes also from different chemical classes. The method was then applied to 60 authentic real samples from forensic cases, demonstrating its suitability for the screening of a wide number of NPS