Determination Of New Psychoactive Substances Research Articles

Expanding the use of cork as biosorbent through the determination of new psychoactive substances in postmortem blood samples

This study aimed to investigate the applicability of cork in a solid phase-based extraction method coupled to a 96-deepwell plate for LC-MS/MS determination of new psychoactive substances and other drugs of abuse in blood. Nowadays, there is a concern in developing new analytical methods that follows the principles of “green analytical chemistry”. These include the miniaturization of methods, utilization of minimal sample and solvent volumes, development of greener solvents and sorbents, among others. Regarding sorbents, natural compounds such as cork can be a great choice to be employed as extraction phase due to its large availability and low cost. Nevertheless, these new materials can help overcome analytical challenges related to postmortem analysis. Regarding sample preparation, blood samples were primarily submitted to protein precipitation by mixing 200 μL of blood, 250 μL of water, 250 μL of zinc sulfate and 50 μL of NaOH, followed by agitation and centrifugation. An aliquot of 500 μL of the supernatant was added to a 96-deepwell plate containing 30 mg of cork powder with particle size ≤ 250 μm. Extraction was performed by agitation for 15 minutes in an orbital shaker at 300 rpm, with consecutive removal of the aqueous phase. Desorption was performed by the addition of 1150 μL of acetonitrile with further agitation at 300 rpm for 2 minutes. The organic layers were recovered and dried under nitrogen stream, reconstituted in 30 μL of methanol and 2 μL was injected in the analytical system. Sample preparation parameters were optimized through univariate and multivariate strategies with the software Statistica 8.0 (Statsoft, Tulsa, USA). A Nexera UFLC system coupled to a LCMS-8045 triple quadrupole mass spectrometer (Shimadzu, Japan) was used for the analysis. Separation was performed on an Acquity UPLC® BEH C18 (1.7 μm, 2.1 × 50 mm) column (Waters, USA) using water (A) and acetonitrile (B) both containing 0.1% formic acid as mobile phases. Total run time was 7.5 minutes. The method was validated according to ANSI/ASB Standard 036 for the following parameters: lower limit of quantification (LLOQ), linearity, carryover, selectivity, precision, bias and dilution integrity. To verify the applicability of the method, three real postmortem blood samples were analyzed. The optimization of extraction parameters was carried out for the following items: choice of cork conformation (powder or sheet), cork particle size, cork mass, extraction time, desorption time, solvent type and solvent volume. The best parameters values are described in “Methods” session. LLOQs ranged between 0.5 and 5 ng/mL. Calibration curves were linear through the ranges of 0.5–85 ng/mL for ethylone, MDA, MDMA, N-ethylpentylone, 25B-NBOMe, 25C-NBOMe, 25I-NBOMe, fentanyl, pentylone and LSD; 1–85 ng/mL for cathinone, mephedrone and PCP; and 5–100 ng/mL for 25-NBOH, with determination coefficient (r2) > 0.99 and absence of carryover effects. Bias, within-run and between-run precision and dilution integrity results were within the acceptable values. The three samples were positive for the presence of MDA and MDMA with concentrations ranging between 23.11–26.59 ng/mL and 79.21–198.13 ng/mL, respectively. Sample preparation optimization was found to be relevant for the achievement of better analytical responses. Additionally, the use of the 96-deepwell plate allows simultaneous processing of multiple samples, with further possibility of automation. This study demonstrate that cork can be employed as a biosorbent for the determination of NPS and other drugs of abuse in postmortem blood through proper optimization, validation and application of the methodology. Therefore, it can be an alternative extraction phase to be considered in method development for toxicological purposes.

Determination of new psychoactive substances and other drugs in postmortem blood and urine by UHPLC-MS/MS: method validation and analysis of forensic samples.

This study aimed to validate a modified QuEChERS method followed by ultra-high performance liquid chromatography-tandem mass spectrometry to determine 79 new psychoactive substances (NPS) and other drugs in blood and urine. Prescription drugs (n = 23), synthetic cathinones (n = 13), phenethylamines (n = 11); synthetic cannabinoids (n = 8), amphetamines (n = 7) and other psychoactive substances (n = 17) were included in the method. 500 µL of biological fluid was extracted with 2mL of water/ACN (1:1), 500mg of anhydrous MgSO4/NaOAc (4:1) added, followed by a supernatant cleanup with 25mg of primary secondary amine and 75mg of anhydrous MgSO4. Quantification was done using matrix-matched calibration curves and deuterated internal standards. The method was satisfactorily validated for blood and urine at limit of quantifications ranging from 0.4 to 16ng/mL, and applied to the analysis of 54 blood (38 postmortem and 16 antemortem) and 16 antemortem urine samples from 68 forensic cases. All urine samples and 59.3% of the blood samples were positive for at least one analyte. Twenty-two analytes were detected in at least one biological sample, including the synthetic cathinones ethylone (222ng/mL, antemortem blood), eutylone (246 and 446ng/mL, urine), and N-ethylpentylone (597 and 7.3ng/mL, postmortem and antemortem blood, respectively). The validated method was shown to be suitable for the analysis of blood and urine forensic samples and an important tool to collect information on emerging drug threats and understanding the impact of NPS and other drugs in poisoning cases.

Determination of Salt Forms of New Psychoactive Substances by Ion Chromatography

Objective To establish an ion chromatography method for the salt form determination of new psychoactive substances （NPS）. Methods The method of conducting qualitative and quantitative analysis of six types of organic acid ions （acetate ion, tartrate ion, maleate ion, oxalate ion, fumarate ion, citrate ion） and five types of inorganic anions （fluoride ion, chloride ion, nitrate ion, sulfate ion, phosphate ion） in NPS sample by ion chromatography was developed. The salt forms of 222 seized NPS samples （103 samples with synthetic cannabinoids, 81 samples with cathinones, 44 samples with phenylethylamines, 12 samples with tryptamines, 7 samples with phencyclidines, 6 samples with piperazines, 2 samples with aminoindenes, 26 samples with fentanyls and 43 samples with other types of NPS） were analyzed by this method. Results Each anion had good linearity in the corresponding linear range, the correlation coefficients （r） were greater than 0.999, the limits of detection were 0.01-0.05 mg/L, and the limits of quantitative were 0.1-0.5 mg/L. Except that 5F-BEPIRAPIM was hydrochloride, the salt forms of the other 102 synthetic cannabinoids were all base. The salt form of 81 cathinone samples, 44 phenylethylamine samples, 7 phencyclidine samples and 2 aminoindene samples were all hydrochloride. The salt forms of tryptamine samples included base, hydrochloride, fumarate and oxalate. The salt forms of piperazine samples included base and hydrochloride. The salt forms of fentanyl samples and samples of other types included base, hydrochloride and citrate. Conclusion Ion chromatography is a simple, accurate and efficient method for determining the salt form of NPS samples, which makes the qualitative and quantitative conclusions of NPS more scientific and rigorous.

Recent bionalytical methods for the determination of new psychoactive substances in biological specimens.

One of the problems associated with the consumption of new psychoactive substances is that in most scenarios of acute toxicity the possibility of quick clinical action may be impaired because many screening methods are not responsive to them, and laboratories are not able to keep pace with the appearance of new substances. For these reasons, developing and validating new analytical methods is mandatory in order to efficiently face those problems, allowing laboratories to be one step ahead. The goal of this work is to perform a critical review regarding bionalytical methods that can be used for the determination of new psychoactive substances (phenylethylamines, cathinones, synthetic cannabinoids, opioids, benzodiazepines, etc), particularly concerning sample preparation techniques and associated analytical methods.

Sample preparation strategies for the determination of psychoactive substances in biological fluids

This review focuses on the existing analytical procedures for the determination of new psychoactive substances (NPS) in biological fluids by chromatographic methods. Direct analysis of samples is scarcely employed and most proposed methodologies include a sample pre-treatment in order to remove matrix interferents and, in some cases, pre-concentrate extracts. Current extraction methods for NPS determination in plasma/serum, urine, and oral fluids have been widely discussed, such as liquid-liquid, solid-phase, and micro extraction approaches, highlighting the advantages and drawbacks of the proposed extraction methodologies. Regarding microextraction approaches, techniques like microextraction by packed sorbent, solid-phase microextraction, miniaturized solid phase extraction, and dispersive liquid-liquid extraction have been proposed for NPS determination in biological fluids with reliable analytical results.

Determination of New Psychoactive Substances in Whole Blood Using Microwave Fast Derivatization and Gas Chromatography/Mass Spectrometry.

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250μL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Simultaneous determination of new psychoactive substances and illicit drugs in sewage: Potential of micro-liquid chromatography tandem mass spectrometry in wastewater-based epidemiology

Wastewater-based epidemiology (WBE) can give valuable light on the extent and actual use of new psychoactive substances (NPS). In this work, a fully validated methodology for the simultaneous determination of illicit drugs and NPS in wastewater by solid-phase extraction followed by UHPLC-MS/MS has been developed. The small sample volume (5 mL) required for analysis is of high interest, especially when performing large sampling campaigns involving many locations of different geographical origin, as it has been performed in the past. The method was applied to wastewater samples from different European locations and permitted the simultaneous monitoring of conventional drugs and NPS. Cocaine, amphetamine, MDMA, methamphetamine and ketamine were found in all wastewater samples, and several NPS (dipentylone, butylone, mephedrone, methedrone and methylone) were observed in some of the samples monitored. It is noteworthy that dipentylone was detected in wastewater for the very first time. Furthermore, a detailed comparison of micro liquid chromatography (μLC) and UHPLC, both coupled to tandem mass spectrometry, in terms of sensitivity and reproducibility has been made for the first time in the application field of WBE. An average increase factor of 14 (mass normalized data) was observed in sensitivity for μLC-MS/MS. The overall method performance was also compared (un-normalized data), and an average increase sensitivity factor of 4.5 was observed for μLC-MS/MS. However, large deviations in retention time (up to 0.4 min) affected the reproducibility and robustness of the μLC-MS/MS method when it was applied to wastewater analysis. Although in this work μLC-MS/MS was strongly influenced by the amount of matrix loaded in the separation device, its enhanced sensitivity and promotion of green chemistry (faster analysis time and less solvent consumption) allow to expect improved future applications, especially when analytes are present at very low concentrations.

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.

Low-temperature plasma-probe mass spectrometry based method for determination of new psychoactive substances in oral fluid.

Owing to the widespread abuse of new psychoactive substances (NPSs), developing a rapid, easily operable method to detect NPSs in oral fluid is of high priority. Their ease of collection and non-invasive nature make oral fluid samples suitable for on-site tests and forensic cases. Herein we report a rapid and sensitive method to screen and quantitate 11 new NPSs in oral fluid. Low-temperature plasma-probe mass spectrometry (LTP-MS) was applied and, to improve the signal intensity, thermally assisted desorption was employed. Tandem mass spectrometry was performed to exclude false positive signals and to decrease noise at the m/z values of interest. Linearity was studied using matrix-matched calibration curves; all the analytes exhibited good linearity with R2 varying from 0.9907 to 0.9981. The estimated limits of detection (LODs) were in the range of 3.0-15.2ng/mL, which are comparable to those of immunoassay; relative standard deviations (RSDs) are no greater than 23% at the studied concentration levels. The proposed LTP-MS-based method was promising in forensic and on-site applications to curb the abuse of NPSs.

Early detection of emerging street drugs by near infrared spectroscopy and chemometrics

Near-infrared spectroscopy (NIRs) is spreading as the tool of choice for fast and non-destructive analysis and detection of different compounds in complex matrices. This paper investigated the feasibility of using near infrared (NIR) spectroscopy coupled to chemometrics calibration to detect new psychoactive substances in street samples. The capabilities of this approach in forensic chemistry were assessed in the determination of new molecules appeared in the illicit market and often claimed to contain “non-illegal” compounds, although exhibiting important psychoactive effects. The study focused on synthetic molecules belonging to the classes of synthetic cannabinoids and phenethylamines. The approach was validated comparing results with officials methods and has been successfully applied for “in site” determination of illicit drugs in confiscated real samples, in cooperation with the Scientific Investigation Department (Carabinieri-RIS) of Rome. The achieved results allow to consider NIR spectroscopy analysis followed by chemometrics as a fast, cost-effective and useful tool for the preliminary determination of new psychoactive substances in forensic science.

Screening for synthetic cannabinoids in hair by using LC-QTOF MS: A new and powerful approach to study the penetration of these new psychoactive substances in the population

The current analytical technology for the determination of New Psychoactive Substances in biological samples is still largely inadequate, because the immunoassays are unsuitable for the detection of most of these compounds and the use of traditional gas chromatography-mass spectrometry techniques is hampered by the lack of chromatographic standards and mass fragmentation patterns. Taking advantage of the molecular recognition capability of high-resolution mass spectrometry, the present work aimed to apply liquid chromatography-quadrupole-time of flight mass spectrometry for the rapid identification of New Psychoactive Substances in the hair, a peculiar tissue which "keeps memory" of the recent history of drug intake of the subject. All the samples were screened for the presence of 50 different New Psychoactive Substances (synthetic cannabinoids, cathinones and phenethylamines), substances that had been reported officially by the National Early Warning System in the period 2009-2011. Among the 435 samples analyzed, 8 were found "positive" for the following compounds: JWH-018, JWH-073, JWH-081, JWH-250, JWH-122, in a broad range of concentrations (0.010-1.28 ng/mg). Results strongly support the use of hair analysis to monitor the diffusion of new psychoactive drugs in the community.