Characterisation of Cannabis-Based Products Marketed for Medical and Non-Medical Use Purchased in Portugal.

Cannabis-based products have gained attention in recent years for their perceived therapeutic benefits (with cannabinoids such as THC and CBD) and widespread availability. However, these products often lack accurate labelling regarding their cannabinoid content. Our study, conducted with products available in Portugal, revealed significant discrepancies between label claims and actual cannabinoid compositions. A fully validated method was developed for the characterisation of different products acquired from pharmacies and street shops (beverages, herbal samples, oils, and cosmetic products) using high-performance liquid chromatography coupled with a diode array detector. Linearity ranged from 0.4 to 100 µg/mL (0.04-10 µg/mg) (THC, 8-THC, CBD, CBG, CBDA, CBGA), 0.1-100 µg/mL (0.01-10 µg/mg) (CBN), 0.4-250 µg/mL (0.04-25 µg/mg) (THCA-A), and 0.8-100 µg/mL (0.08-10 µg/mg) (CBCA). Among sampled beverages, none contained detectable cannabinoids, despite suggestive packaging. Similarly, oils often differed from the declared cannabinoid compositions, with some containing significantly higher CBD concentrations than labelled. These inconsistencies raise serious concerns regarding consumer safety and informed decision-making. Moreover, our findings underscore the need for stringent regulation and standardised testing protocols to ensure the accuracy and safety of cannabis-based products.

The Determination of Cannabinoids in Urine Samples Using Microextraction by Packed Sorbent and Gas Chromatography-Mass Spectrometry.

Cannabis is the most consumed illicit drug worldwide, and its legal status is a source of concern. This study proposes a rapid procedure for the simultaneous quantification of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabidiol (CBD), and cannabinol (CBN) in urine samples. Microextraction by packed sorbent (MEPS) was used to pre-concentrate the analytes, which were detected by gas chromatography-mass spectrometry. The procedure was previously optimized, and the final conditions were: conditioning with 50 µL methanol and 50 µL of water, sample load with two draw-eject cycles, and washing with 310 µL of 0.1% formic acid in water with 5% isopropanol; the elution was made with 35 µL of 0.1% ammonium hydroxide in methanol. This fast extraction procedure allowed quantification in the ranges of 1-400 ng/mL for THC and CBD, 5-400 ng/mL for CBN and 11-OH-THC, and 10-400 ng/mL for THC-COOH with coefficients of determination higher than 0.99. The limits of quantification and detection were between 1 and 10 ng/mL using 0.25 mL of sample. The extraction efficiencies varied between 26 and 85%. This analytical method is the first allowing the for determination of cannabinoids in urine samples using MEPS, a fast, simple, and low-cost alternative to conventional techniques.

Rapid analysis of cocaine and metabolites in urine using microextraction in packed sorbent and GC/MS.

Cocaine is still one of the most abused drugs worldwide and, as such, it is often screened for in driving-under-the-influence or workplace drug - testing scenarios. A large number of samples have usually to be processed in those situations, and this requires fast and simple extraction procedures for the detection and quantification of the drugs. The present work describes an ultrafast and fully validated procedure for the simultaneous detection and quantification of cocaine and its two main metabolites, ecgonine methyl ester and benzoylecgonine, in urine using microextraction by packed sorbent and GC-MS. A small sample volume (200 µL) was used, and a fast extraction procedure together with a microwave-assisted derivatization (800 W, 2 min) allowed the quantification of all analytes in a range of 25 to 1000 ng/mL (r 2 > 0.99). Inter-day precision revealed coefficients of variation (CVs) lower than 10% for all analytes at the tested concentration levels, with an accuracy within a ±7% interval, with the exception of EME's lowest calibrator (±17%). Intra-day CVs were lower than 15% at the studied concentration levels, with a mean relative error within a ±13% interval. Recoveries ranged from 14.5 to 37.2% (EME), 67.0 to 83.3% (cocaine), and 24.6 to 43.5% (BEG), allowing the limits of detection and quantification to be set at 25 ng/mL for all compounds. Graphical Abstract Schematized analysis of cocaine and metabolites in urine by MEPS- GC/MS.

α-Pyrrolidinohexanophenone (α-PHP) and α-Pyrrolidinoisohexanophenone (α-PiHP): A Review.

New Psychoactive Substances are currently a serious and growing problem affecting public health worldwide. By 2022, 1184 of these substances had been identified over a period of 16 years. Within these, α-pyrrolidinohexanophenone (α-PHP) and α-pyrrolidinoisohexanophenone (α-PiHP) have emerged, two synthetic cathinones from the pyrovalerone derivates subgroup that are positional isomers of each other. Alpha-PHP appeared on the Japanese illicit drug market in 2014 and, two years later, α-PiHP was identified for the first time in China. They were placed in schedule II on the list of Psychotropic Substances under International Control in 2020 and in March 2023, respectively. Both cathinones have no therapeutic potential for medical use and therefore are abused for recreational habits, which can lead to fatalities. The most frequent adverse effects reported are cardiac, psychiatric, and neurologic, and fatal intoxications have already been described. In Portugal, their consumption and consequent seizures are more prevalent on the archipelagos, which has been aggravating the health situation. In conclusion, these types of substances are a challenge for forensic toxicology since they are easily synthesized, modified, and placed on the market. Therefore, more studies to develop analytical methods to detect them and more comprehensive legislation should be applied. Thus, this review aimed to address the legislative, physicochemical, toxicological, and analytical aspects of both substances.

Development and Validation of a GC-MS-EI Method to Determine α-PHP in Blood: Application to Samples Collected during Medico-Legal Autopsies.

New psychoactive substances (NPSs) still represent an issue of great concern worldwide despite efforts made by national and international control systems to limit the spread of these substances. Alpha-pyrrolidinohexanophenone (α-PHP) is a fairly recent synthetic cathinone (the second largest group of monitored substances in Europe) with only a few published studies on the substance. Though there is a low incidence of NPS consumption in Portugal, a recent increase in apprehensions and detections in biological matrices of the substance was verified. An analytical methodology was developed and validated for determining and quantitating α-PHP in blood. Solid-phase extraction was employed for sample preparation (500 µL), which was further analyzed by gas chromatography--mass spectrometry-electron ionization in single-ion monitoring mode with cocaine-d3 as the internal standard. Method validation followed the guidelines of the American National Standards Institute/AAFS Standards Board (ANSI/ASB Standard 036). The procedure was linear between 10 and 1,000 ng/mL, with determination coefficients (r2) higher than 0.999. Carryover was not observed. A limit of detection of 5 ng/mL and a limit of quantitation of 10 ng/mL were achieved. Intraday and intermediate precision and bias assessment showed satisfactory results (coefficient of variation <17.7%; bias <11.6%), and extraction efficiency ranged from 98.5% to 103.3%. The stability of the substance was considered acceptable for at least 6 h at room temperature, 48 h in the autosampler and 21 days after five freeze/thaw cycles. The developed methodology was applied to 15 real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch of the National Institute of Legal Medicine and Forensic Sciences, Portugal, with drug concentrations ranging from 15 to 227 ng/mL. Available information for each case is also detailed in the present article.

Analysis of opiates in urine using microextraction by packed sorbent and gas Chromatography- Tandem mass spectrometry.

Opiates recreational consumption has always been a concern in society, public health, and in clinical toxicology analysis. The aim of this study was to develop and fully validate an analytical method, which was simple and rapid for the determination of tramadol, codeine, morphine, 6- acetylcodeine, 6-monoacetylmorphine and fentanyl using gas chromatography coupled to tandem mass spectrometry. The procedure includes the use of microextraction by packed sorbent for sample clean-up. A mixed mode sorbent was used, allowing the minimal use of solvents. The method was validated in urine samples, with the ability to detect and quantify all analytes with satisfactory linearity (in the range of 1 - 1000 ng/mL for all analytes, except for fentanyl (10-1000 ng/mL)). Extraction efficiency varied from 17 to 107%, which did not impair sensitivity, taking into account the low LLOQs obtained (1 ng/ mL for all analytes; and 10 ng/mL for fentanyl). The developed procedure proved to be fast, selective, and accurate for use in routine analysis, with a low volume of sample (250 µL).

A Novel Bioanalytical Method for the Determination of Opioids in Blood and Pericardial Fluid.

Opioids are the drugs most commonly detected in overdose deaths and the second most consumed worldwide. An analytical methodology has been optimized and fully validated for the determination of codeine, morphine, 6-acetylmorphine, 6-acetylcodeine, oxycodone, oxymorphone and fentanyl in whole blood and pericardial fluid. The internal standards used were codeine-d3, morphine-d3, 6-acetylmorphine-d3 and fentanyl-d5. Before solid-phase extraction, volumes of 250 µL of blood and pericardial fluid were subjected to a protein precipitation (with 750 µL of ice-cold acetonitrile) and a microwave-induced oximation was performed using a solution of 1% aqueous hydroxylamine hydrochloride in phosphate-buffered saline (1:2, v/v). Finally, the dried extracts were further derivatized with a solution of n-methyl-n-(trimethylsilyl) trifluoroacetamide + 5% trimethylchlorosilane under microwave irradiation. The chromatographic analysis was carried out using gas chromatography-mass spectrometry operating in electron impact and selected ion monitoring mode. For all analytes, the method was linear between 5 and 1,000 ng/mL with determination coefficients (r2) >0.99. Depending on the analyte and matrix, the limit of detection varies between 3 and 4 ng/mL. Intra- and intermediate precision (<20%) and bias (±20%) were acceptable for all analytes in both matrices. The stability of the substances in the studied matrices was guaranteed, at least, 24 h in the autosampler, 4 h at room temperature and 30 days after three freeze/thaw cycles. This methodology was applied to real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Determination of amphetamine-type stimulants in urine samples using microextraction by packed sorbent and gas chromatography-mass spectrometry.

The aim of this work was the development, optimization and full validation of a method applying microextraction by packed sorbent (MEPS) coupled to gas chromatography-mass spectrometry (GC-MS) to determine amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylmethamphetamine (MDMA), 3,4-methylenedioxy-N-methyl-α-ethylfenilethylamine (MBDB), and 3,4-methylenedioxy-N-ethylamphetamine (MDE) in urine samples. Using 200 µL of sample, the MEPS procedure was optimized concerning type of sorbent, sample dilution, number of strokes, activation of the ion exchange mechanism and composition of both washing and elution solvents. The method was fully validated according to the Food and Drug Administration and the Scientific Working Group of Forensic Toxicology guidelines for the validation of bioanalytical methods. The studied parameters included selectivity, calibration model and linearity, limits of detection and quantification, precision, accuracy, stability, dilution integrity and recoveries. Linearity was obtained in the range of 25-1000 ng/mL for MAMP, MBDB and MDE, 35-1000 ng/mL for AMP and MDMA, and 50-1000 ng/mL for MDA, with coefficients of determination (R2) >0.99 for all analytes. Both intra- and inter-day precision and accuracy were adequate, and coefficients of variation lower than 15% and mean relative errors (RE) within a range of ±15% of the theoretical concentrations were obtained for all compounds under study. Analyte recoveries ranged from 19 to 71%, allowing LLOQs ≤50 ng/mL.

Massive intoxication involving unusual high concentration of amitriptyline.

Amitriptyline is a tricyclic antidepressant widely used in the treatment of depression. Antidepressant drugs are among the most commonly encountered causes of self-poisoning, as illustrated by several published cases in the literature. This investigation reports a case of massive amitriptyline intoxication, involving a 44-year old female found dead in bed. The presence of this tricyclic antidepressant was revealed by a routine screening procedure. The concentration was calculated by gas chromatography/ electron ionization-mass spectrometry in the selected ion monitoring mode after solid-phase extraction using proadifen as internal standard and was in the post-mortem whole blood sample 85.9 mug/mL. This value was much higher than the reported toxic values ever found in the literature, and may therefore have caused the victim's death. Nortriptyline was also detected in the toxic concentration range, as well as therapeutic levels of diazepam and nor-diazepam. Taking into account both the available circumstantial information and toxicological results, it is very likely that death was caused by self-poisoning. Human & Experimental Toxicology (2007) 26, 667-670.

Salvia divinorum: toxicological aspects and analysis in human biological specimens.

The identification and quantitation of the main psychoactive component of Salvia divinorum (salvinorin A) in biological specimens are crucial in forensic and clinical toxicology. Despite all the efforts made, its uncontrolled abuse has increased quickly, exposing its users' health to serious risks both in the short and long term. The use of alternative biological matrices in toxicological analyzes can be advantageous as complementary postmortem samples, or in situations when neither blood nor urine can be collected; they may be useful tools in those determinations, providing important information about prior exposure. The aim of this article is to present a brief summary of legal aspects of Salvia divinorum and salvinorin A, including the methods used for the determination of the latter in biological matrices.

Determination of "new psychoactive substances" in postmortem matrices using microwave derivatization and gas chromatography-mass spectrometry.

Despite worldwide efforts aiming to ban the marketing and subsequent abuse of psychoactive substances such as synthetic cathinones and phenethylamines, there has been an alarming growth of both in recent years. Different compounds similar to those already existing are continuously appearing in the market in order to circumvent the legislation. An analytical methodology has been validated for qualitative and quantitative determinations of D-cathine (D-norpseudoehedrine), ephedrine, methcathinone, 1-(4-methoxyphenyl)-propan-2-amine (PMA), mephedrone, methedrone, 2,5-dimethoxy-4-methylamphetamine (DOM), 4-bromo-2,5-dimethoxyamphetamine (DOB), 2,5-dimethoxyphenethylamine (2C-H), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), 4-iodo-2,5-dimethoxyphenethylamine (2C-I), 2-[2,5-dimethoxy-4-(ethylthio)phenyl]ethanamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4) and 2-[2,5-dimethoxy-4-(propylthio)phenyl]ethanamine (2C-T-7), in low volumes of vitreous humor (100 µL), pericardial fluid (250 µL) and whole blood (250 µL), using deutered amphetamine, ephedrine and mephedrone as internal standards. The validation parameters included selectivity, linearity and limits of detection and quantification, intra- and interday precision and trueness, recovery and stability. The method included mixed-mode solid phase extraction, followed by microwave fast derivatization and analysis by gas chromatography-mass spectrometry operated in selected ion monitoring mode. The procedure was linear between 5 and 600 ng/mL, with determination coefficients higher than 0.99 for all analytes. Intra- and interday precision ranged from 0.1 to 13.6%, while accuracy variability was within 80-120% interval from the nominal concentration at all studied levels. The extraction efficiencies ranged from 76.6 to 112.8%. Stability was considered acceptable for all compounds in the studied matrices. The developed assay was applied to authentic samples of the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal.

Determination of furosemide in whole blood using SPE and GC-EI-MS.

A simple and rapid method was validated to determine furosemide in whole blood. The experimental work was performed so that all validation parameters are considered simultaneously in a one-day assay protocol. A solid-phase extraction procedure using BondElut-LRC Certify columns was used to extract this compound from blood samples, while ketoprofen was used as an internal standard. The extracts were analyzed by gas chromatography-electron ionization-mass spectrometry after on-column derivatization with trimethylanilinium hydroxide (0.2M in methanol). Calibration curves were prepared daily, between 0.10 and 5.00 microg/mL, and the correlation coefficients were above 0.9910. The calculated limits of detection and quantitation were 0.010 and 0.045 microg/mL, respectively. Control samples at low, medium, and high concentrations (0.30, 0.75, and 3.00 microg/mL) of furosemide of an independent source were measured in the same day. Precision and trueness, calculated in terms of relative standard deviation (%), were less than 15% for all concentration levels. The relative recoveries calculated for the three levels of the control samples were 104%, 89%, and 91%, respectively. In general, a sensitive, specific, and reliable procedure has been developed for the determination of furosemide in whole blood samples and was found suitable for the application in postmortem forensic toxicology routine analysis.

Hair analysis for delta(9)-THC, delta(9)-THC-COOH, CBN and CBD, by GC/MS-EI. Comparison with GC/MS-NCI for delta(9)-THC-COOH.

A sensitive analytical method was developed for quantitative analysis of delta(9)-tetrahydrocannabinol (delta(9)-THC), 11-nor-delta(9)-tetrahydrocannabinol-carboxylic acid (delta(9)-THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in human hair. The identification of delta(9)-THC-COOH in hair would document Cannabis use more effectively than the detection of parent drug (delta(9)-THC) which might have come from environmental exposure. Ketamine was added to hair samples as internal standard for CBN and CBD. Ketoprofen was added to hair samples as internal standard for the other compounds. Samples were hydrolyzed with beta-glucuronidase/arylsulfatase for 2h at 40 degrees C. After cooling, samples were extracted with a liquid-liquid extraction procedure (with chloroform/isopropyl alcohol, after alkalinization, and n-hexane/ethyl acetate, after acidification), which was developed in our laboratory. The extracts were analysed before and after derivatization with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH) using a Hewlett Packard gas chromatographer/mass spectrometer detector, in electron impact mode (GC/MS-EI). Derivatized delta(9)-THC-COOH was also analysed using a Hewlett Packard gas chromatographer/mass spectrometer detector, in negative ion chemical ionization mode (GC/MS-NCI) using methane as the reagent gas. Responses were linear ranging from 0.10 to 5.00 ng/mg hair for delta(9)-THC and CBN, 0.10-10.00 ng/mg hair for CBD, 0.01-5.00 ng/mg for delta(9)-THC-COOH (r(2)>0.99). The intra-assay precisions ranged from <0.01 to 12.40%. Extraction recoveries ranged from 80.9 to 104.0% for delta(9)-THC, 85.9-100.0% for delta(9)-THC-COOH, 76.7-95.8% for CBN and 71.0-94.0% for CBD. The analytical method was applied to 87 human hair samples, obtained from individuals who testified in court of having committed drug related crimes. Quantification of delta(9)-THC-COOH using GC/MS-NCI was found to be more convenient than GC/MS-EI. The latter may give rise to false negatives due to the detection limit.

A validated procedure for detection and quantitation of salvinorin a in pericardial fluid, vitreous humor, whole blood and plasma using solid phase extraction and gas chromatography-mass spectrometry.

The use of vitreous humor and pericardial fluid as alternative matrices to blood and plasma in the field of forensic toxicology is described to quantitate low levels of Salvinorin A using ethion as internal standard. The method was optimized and fully validated using international accepted guidelines. The developed methodology utilizes a solid phase extraction procedure coupled to gas chromatography mass spectrometry operated in the selected ion monitoring mode. The method was linear in the range of 5.0-100ng/mL with determination coefficients higher than 0.99 in 100µL of vitreous humor and in 250µL of each matrix pericardial fluid, whole blood and plasma. The limits of detection and quantitation were experimentally determined as 5.0ng/mL, intra-day precision, intermediate precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented mean efficiencies between 80 and 106% in the different biological specimens analyzed. According to the low volumes of samples used, and the low limits achieved using a single quadrupole mass spectrometer, which is available in most laboratories, we can conclude that the validated methodology is sensitive and simple and is suitable for the application in forensic toxicology laboratories for the routine analysis of Salvinorin A in both conventional and unconventional biological samples.

Illicit drugs in alternative biological specimens: a case report.

Postmortem tissues (e.g. liver, kidney) have been long used in forensic applications especially in those cases where blood is unavailable. The aim of this paper is to demonstrate the importance of the information provided to the forensic toxicologist at the time of carrying out the toxicological analysis, especially in cases where the samples commonly used in forensic toxicology are unavailable. This work describes the toxicological findings in a violent death resulting from a man who was hit by a train. Vitreous humor, liver and kidney were sent for toxicological analysis, once it was not possible to obtain blood and urine. The validated procedures used in the routine casework of Forensic Toxicology Laboratory of the Centre Branch of the National Institute of Legal Medicine, were applied in the analysis of liver, kidney and vitreous humor, using gas chromatography-mass spectrometry after solid-phase extraction and gas chromatography-flame ionization detector for the analysis of drugs of abuse and ethanol, respectively. Morphine, codeine, cocaine, benzoylecgonine and ecgonine methyl ester were found in the liver and in the kidney and no ethanol was found in the vitreous humor. The method validation included the study of specificity, selectivity, limits of detection, recovery and carryover. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, sometimes the choice of specimen is determined by the case under investigation. The forensic pathologist must be aware that relevant information must be provided so that the toxicological analysis can be conducted in accordance with case history, particularly when the only samples available for analysis are these "unconventional" specimens, since the interpretation of the obtained results is more difficult.