Development, validation and application of a gas chromatography method for the determination of dillapiole from Piper aduncum essential oil in skin permeation samples.

The essential oil extracted from the leaves of Piper aduncum has antifungal, insecticidal and antibacterial activity. Studies with its main compound, dillapiole (DIL) revealed antibacterial and anti-inflammatory potential. Despite all this bioactivity, there is no updated report on the development and validation of analytical and bioanalytical methodology to quantify DIL in skin samples. A selective, precise, accurate and adequate method for the determination of DIL in solutions, porcine ear skin samples and receptor fluid was developed and validated by headspace extraction-gas chromatography with flame ionization detection (HS-GC-FID). HS-GC-FID was applied to determine DIL in Franz cell permeation and retention studies using porcine ear skin samples. In the HS-GC-FID method, matrix-related interferences were not observed at the peak of the DIL retention time. The results showed a high recovery (>97%) after the extraction procedure, allowing the quantification of DIL in complex matrices. In vitro permeation/retention for DIL showed cumulative amounts permeated in the order: receptor fluid (21.98 ± 1.19 µg/cm2 ) > epidermis (15.40 ± 1.20 µg/cm2 ) > dermis (9.52 ± 1.13 µg/cm2 ). HS-GC-FID was successfully validated and the results point to DIL transdermal permeation and to the potential to develop pharmaceutical formulations for skin delivery to treat inflammation or infections.

Antifungal Properties of Essential Oils Derived from the Genus Cymbopogon: A Systematic Review.

Essential oils (EOs) are products of secondary metabolism with recognized organoleptic characteristics and biological properties. Recently, there has been a growing demand for EOs in the national and international market, mainly due to the recognition of their use as complementary medicine practices, and the increased use in the industries of pharmaceutics, cosmetics, well-being, veterinary and agroecology, boosting the productive sector. In this context, EOs from grasses of the Cymbopogon (Poaceae) are promising sources of bioactive compounds, due to their recognized biological properties, such as anti-inflammatory, antibacterial, antifungal, antidiabetic, repellent, and larvicide. Thus, the present study aims to carry out a review of the scientific literature of the main works related to the evaluation of the antifungal action of essential oils extracted from plants of the Cymbopogon genus, compiling the species that showed the best results and relating them to their main chemical constituents. This review covers the following species: C. citratus, C. flexuosus, C. winterianus, C. martinii, C. nardus, C. giganteus, C. schoenanthus, C. khasans, and C. proximus. Among them, C. citratus was the most assessed, being associated with the vast majority of studies (61.9 %), and it was also the species that showed the best results in terms of MIC.

Where should I start? A scoping review about the publications on clove cigarettes.

INTRODUCTION: The general lack of knowledge about the composition of clove cigarettes and the large number of terms that can be used to define different types of cigarettes that contain cloves makes it difficult to find scientific articles focused on the subject. We reviewed the publications that cited clove cigarettes to assess their relevance. METHODS: We searched publications on Scopus, ScienceDirect, PubMed, and Portal CAPES during the first semester of 2021, without date restrictions. Through a quality assessment, the studies found were assorted in ten different categories. RESULTS: Indonesian clove cigarettes-also known as kretek-are a mixture of tobacco and cloves rolled in a cigarette and sprayed with oils, plant extract, and food flavorings, in an unknown quantity and composition, different from each brand, which is usually kept in secret. Due to the relatively low prevalence of use in the general population, most publications on tobacco products tend to ignore clove cigarettes or place them in an existing category. Clove cigarettes can be hand-rolled, machine-made, filtered, unfiltered, and each form can be named differently. The interchangeable use of each term, sometimes grouping conventional cigarettes in the mix, can lead the researcher to consider a publication that should be excluded. CONCLUSION: The existing regulations on flavored cigarettes, although well-intentioned, are still somewhat vague and broad, leaving possible loopholes that can be exploited by the tobacco industries. Fully understanding the precise effects caused by clove cigarettes can be an important tool in future discussions about tobacco control.

DoE-assisted development and validation of a stability-indicating HPLC-DAD method for simultaneous determination of five cannabinoids in Cannabis sativa L. based on analytical quality by design (AQbD) concept.

INTRODUCTION: Medical uses of Cannabis sativa L. have gained interest in recent decades, which highlights the need for defining appropriate quality specifications for Cannabis-based products. However, the complexity of plant matrices and structural similarity between cannabinoids make analytical development a challenging task. Thus, the application of analytical quality by design (AQbD)-driven approaches can favour the development of fit-for-purpose methods. OBJECTIVES: To develop a high-performance liquid chromatography diode array detector (HPLC-DAD) method for simultaneous quantification of cannabidiol, Δ9 -tetrahydrocannabinol, cannabidiolic acid, tetrahydrocannabinolic acid, and cannabinol in C. sativa by applying an AQbD-driven approach. MATERIALS AND METHODS: Critical method attributes (CMA) were established following the analytical target profile. Critical method variables (CMV) were categorised based on risk assessment and literature review. Selected CMV regarding sample preparation and chromatographic conditions were optimised using response surface methodology (RSM). The working point was estimated by multiple response optimisation using Deringer's desirability function. The validity of the optimal conditions was confirmed experimentally. Method validation was performed according to ANVISA and ICH guidelines. Relative response factors (RRFs) were also determined. RESULTS AND DISCUSSION: Baseline resolution of 12 major cannabinoids was achieved in a 35 min chromatographic analysis. All experimental responses obtained during confirmatory analyses were within the prediction intervals (PI95% ). Method's selectivity, linearity (10-100 µg/mL), precision, bias, extraction recovery, and ruggedness were satisfactorily demonstrated. CONCLUSIONS: The application of an AQbD-driven approach allowed for a better understanding of the effects of the ensemble of CMV on the analyte's behaviour, enabling the definition of appropriate conditions to ensure consistent achievement of the intended method's performance.

Dried matrix spots: an evolving trend in the toxicological field.

Dried matrix spot (DMS) is a sampling technique, primarily used to analyze blood to diagnose metabolic diseases in newborns. As this technique has several advantages, DMS has started to be employed for other purposes using other biological matrices and increasingly in toxicology over the last decade. The aim of this work was to review the analytical methods using DMS which can be applied to drugs of abuse and which have been published since 2010. Three different databases were searched, using dried, spots, and drugs of abuse as the descriptors and using a snowball search. After applying the exclusion criteria, 39 papers remained. The most common publications were related to the use of blood, which corresponded to 77% of the papers, followed by urine and oral fluid, which corresponded to 13 and 10% of the papers, respectively. The selected studies covered different illicit drugs, sample sizes of 5 to 250 µL and spot sizes ranging from 3 to 18 mm in diameter. This review also examined the extraction techniques and the methods employed to analyze various biological matrices and drugs of abuse, mostly by liquid-extraction and liquid chromatography-tandem mass spectrometry. The benefits of DMS include: a simple sample pretreatment, better stability than liquid matrices, a simple extraction procedure, lower costs, and environmental benefits. DMS appears to be a promising technique in the field of toxicology and provides new perspectives for use in forensic laboratories.

[Toxicology of suicide cases in the state of Rio Grande do Sul, Brazil, 2017 to 2019Perfil toxicológico de los casos de suicidio en Rio Grande do Sul (Brasil), 2017-2019]. / Perfil toxicológico dos suicídios no Rio Grande do Sul, Brasil, 2017 a 2019.

OBJECTIVE: To describe the toxicology of suicide cases recorded in the state of Rio Grande do Sul, Brazil, from 2017 to 2019. METHOD: The present descriptive, cross-sectional study examined all the medico-legal reports and police records related to suicide deaths in the state. Multiple correspondence analyses were performed along with independent logistic regression models having ethanol, anxiolytic and antidepressant drugs, illicit drugs, and non-medical substances as dependent variables. RESULTS: Ethanol was investigated in 2 978 samples, with positive results in 28.5%. The odds of a positive ethanol finding were 0.5 time higher (95%CI: 1.1; 2.2) for suicides occurring at night, 1.0 (95%CI: 1.4; 2.9) time higher for suicides occurring on weekends, and 0.9 (95%CI: 1.3; 2.7) time higher in individuals with a prior criminal record. Investigation of psychotropic drugs (2 900 samples) was positive in 30.4% samples. Anxiolytics were the most common medication detected, with 1.5 (95%CI: 1.6; 4.1) time higher odds of occurrence in women and 0.8 time higher odds (95%CI: 1.2; 2.7) for suicides occurring in the fall-winter. The odds of detecting illicit drugs (n = 338) were 4.1 times higher (95%CI: 1.9; 14.4) in the regions of Pelotas (south of the state) vs. Passo Fundo (north), and 1.2 (95%CI: 1.3; 3.6) time higher in cases with positive ethanol results, without significant difference between adolescents and adults. CONCLUSIONS: Despite the lack of evidence on causality, the present results support a link between suicide and several psychoactive drugs. Medico-legal experts should be guided regarding the need to perform toxicological tests in all suicide cases.

OBJETIVO: Describir el perfil toxicológico de todas las víctimas de suicidio en Rio Grande do Sul desde el 2017 hasta el 2019. MÉTODOS: En este estudio descriptivo y transversal se consultaron todos los informes periciales y policiales sobre las muertes por suicidio en el estado. Se realizaron análisis de correspondencia múltiple y se crearon modelos independientes de regresión logística, con empleo de etanol, productos ansiolíticos y antidepresivos, sustancias ilícitas y agentes tóxicos no medicamentosos como variables dependientes. RESULTADOS: Se realizaron 2 978 exámenes de alcoholemia, con resultado positivo en un 28,5%. La probabilidad de obtener resultados positivos para alcoholemia aumentó 0,5 (IC95%: 1,1-2,2) en casos de suicidio durante la noche, 1,0 (IC95%: 1,4-2,9) en casos de suicidio en los fines de semana y 0,9 (IC95%: 1,3-2,7) cuando había antecedentes penales. En la investigación de productos psicotrópicos (2 900 muestras) se detectó algún medicamento en un 30,4%. Los ansiolíticos fueron la clase detectada con más frecuencia, con un aumento de la probabilidad de 1,5 (IC95%: 1,6-4,1) en las mujeres y de 0,8 (IC95%: 1,2-2,7) en casos de suicidio durante el otoño y el invierno. El aumento de la probabilidad de detección de sustancias ilícitas (n = 338) fue de 4,1 (IC95%: 1,9-14,4) en la macrorregión de Pelotas en comparación con la de Passo Fundo y de 1,2 (IC95%: 1,3-3,6) en personas con resultados positivos en la prueba de detección de etanol, sin que hubiera ninguna diferencia significativa entre adolescentes y adultos. CONCLUSIONES: Aun sin haberse comprobado la causalidad, los resultados muestran que existe un vínculo entre el suicidio y diversos productos psicoactivos. Es preciso orientar a los médicos legistas con respecto a la necesidad de realizar exámenes toxicológicos en todos los casos de suicidio.

Clinical and Toxicological Profile of NBOMes: A Systematic Review.

BACKGROUND: NBOMes are a new class of potent hallucinogens widely present in illicit drugs. Little is known about this class of drugs, regarding its detection and clinical manifestations of intoxication. OBJECTIVE: This study aims to enhance care involving NBOMes by reviewing the literature on their clinical manifestations and laboratorydetection. METHODS: A systematic review was performed on the clinical manifestations and laboratory tests of NBOMEs ingestion. Embase, Pubmed, PsycINFO, and Cochrane databases were employed in this analysis. RESULTS: Forty-five articles met the inclusion criteria out of the 2814 nonduplicated studies on the theme. Seventy case reports of intoxication were found in the analyzed articles (64.3% were men and 11.4% were women, mean age of 22.5). The technique most employed for NBOMes identification was chromatography of blood, urine, and oral fluids. Moreover, the studies identified 13 chemical structures differentfrom the NBOMes on their toxicological analyses.According to these studies, most of these drugs were ingested orally-nasal use was the second preferred administration route, followed by intravenous administration. CONCLUSION: Better identification of the clinicalmanifestations and laboratory profile of NBOMes is crucial to the recognition of intoxication as well as to its effective treatment.

Citrus essential oils inhalation by mice: Behavioral testing, GCMS plasma analysis, corticosterone, and melatonin levels evaluation.

The use of orange essential oils (EOs) as a complementary treatment is very common in Brazilian popular culture. The levels of melatonin (MEL) and corticosterone (CORT) hormones were investigated simultaneously, by the Luminex™ immunoassay system in mice plasma, after Citrus aurantium and Citrus sinensis EOs inhalation for 30 min. The plasma was analyzed by headspace through gas chromatography coupled to mass spectrometry for investigation of the EO components. Mice were submitted to behavioral testing to research anxiolytic-like, sedative, and antidepressant-like effects. The inhalation of atmosphere obtained from vaporization of 10% solution of this Citrus EO separately did not affect MEL or CORT plasma levels; that is, the MEL and CORT levels did not present variation in function of the EO in the schedule used. On the other hand, the imipramine positive control used altered the level of MEL as expected. The EO constituents were detected in plasma at different ratios that is present in inhaled EO. Behavioral tests showed that the inhalation of 10% C. sinensis EO presents an anxiolytic-like and sedative effect. Thus, C. sinensis EO can be a valuable tool for treatment of the anxiety disturbs, apparently without interference with MEL and CORT physiological levels.

Method validation and determination of lisdexamfetamine and amphetamine in oral fluid, plasma and urine by LC-MS/MS.

Lisdexamfetamine (LDX) is a long-acting prodrug stimulant indicated for the treatment of attention-deficit/hyperactivity disorder and binge-eating disorder symptoms. In vivo hydrolysis of LDX amide bond releases the therapeutically active d-amphetamine (d-AMPH). Since toxicological tests in biological samples can detect AMPH from the use of some legal medications, efficient methods are needed in order to correctly interpret the results. The aim of this study was to develop and validate an LC-MS/MS method for the simultaneous quantification of LDX and its main biotransformation product AMPH in human oral fluid, plasma and urine. Calibration curve range for both analytes was 1-128 ng/mL in oral fluid and plasma and 4-256 ng/mL in urine, being the lowest concentration the limit of quantification. Accuracy of the determined values of the target analytes for the five control levels ranged from 94.8 to 111.7% for oral fluid, from 91.3 to 100.2% for plasma and from 94.8 to 109.8% for urine. Imprecision for the five control levels did not exceeded 12.8% for oral fluid, 16.2% for plasma and 17.1% for urine. The method developed for the three matrices was validated and was also successfully applied to assess real samples, showing for the first time the detection of LDX in oral fluid.

Pharmacokinetics study of mazindol in plasma, oral fluid, and urine of volunteers.

PURPOSE: There are no pharmacokinetics studies in oral fluid reported in the literature, as well as there are no data on correlation of drug levels in plasma, urine, and oral fluid in order to propose alternative matrices to monitor the use of mazindol by drivers. The present work aimed to study, preliminarily, mazindol's pharmacokinetics in plasma and oral fluid, as well as investigate the correlation of drug levels in urine, plasma, and oral fluid. METHOD: Blood, urine, and oral fluid samples from seven healthy male volunteers were collected at 0, 1, 2, 4, 5, 6, 8, 10, and 24 h after administration of tablets of 2 mg mazindol and analyzed by a previously validated method by LC-MS with liquid-liquid extraction. Levels of the drug found were higher in plasma when compared with oral fluid and higher in urine in relation to plasma. The study of the mazindol's pharmacokinetics showed that the most suitable model to describe the variation of the concentration over time is the compartment open model with absorption and elimination following the first-order kinetics, and confirming literature data, drug is metabolized, being the major metabolite detected, but not quantified. CONCLUSION: It was not found a good correlation between the concentrations of mazindol in urine and plasma, but between plasma and oral fluid, there was a good correlation, suggesting this as an alternative matrix to plasma. However, studies involving more subjects are needed.

Seized cannabis seeds cultivated in greenhouse: A chemical study by gas chromatography-mass spectrometry and chemometric analysis.

Cannabis sativa L. is cultivated in most regions of the world. In 2013, the Brazilian Federal Police (BFP) reported 220 tons of marijuana seized and about 800,000 cannabis plants eradicated. Efforts to eradicate cannabis production may have contributed to the development of a new form of international drug trafficking in Brazil: the sending of cannabis seeds in small amounts to urban centers by logistics postal. This new and increasing panorama of cannabis trafficking in Brazil, encouraged the chemical study of cannabis seeds cultivated in greenhouses by gas-chromatography coupled with mass spectrometry (GC-MS) associated with exploratory and discriminant analysis. Fifty cannabis seeds of different varieties and brands, seized by the BFP were cultivated under predefined conditions for a period of 4.5 weeks, 5.5 weeks, 7.5 weeks, 10 weeks and 12 weeks. Aerial parts were analyzed and cannabigerol, cannabinol, cannabidiol, cannabichromene Δ9-tetrahydrocannabinol (THC) and other terpenoids were detected. The chromatographic chemical profiles of the samples were significantly different, probably due to different variety, light exposition and age. THC content increased with the age of the plant, however, for other cannabinoids, this correlation was not observed. The chromatograms were plotted in a matrix with 50 rows (samples) and 3886 columns (abundance in a retention time) and submitted to PCA, HCA and PLS-DA after pretreatment (normalization, first derivative and autoscale). The PCA and HCA showed age separation between samples however it was not possible to verify the separation by varieties and brands. The PLS-DA classification provides a satisfactory prediction of plant age.

Determination of mazindol in human oral fluid by high performance liquid chromatography-electrospray ionization mass spectrometry.

Brazil is one of the countries most affected by abuse of stimulant medications by professional drivers, especially fenproporex, amfepramone and mazindol. Even though their sale is banned, they can be found in illegal markets, such as those located on the country's borders. The use of oral fluid to monitor drug levels has many advantages over plasma and urine because it is noninvasive, easier to collect and more difficult to adulterate. The aim of this study was to develop and validate a sensitive and specific method to quantify mazindol in human oral fluid by liquid chromatography-mass spectrometry (LC-MS). The LC system consisted of an LC-MS system operated in selected ion monitoring mode. The mobile phase was composed of water at pH 4.0, acetonitrile and methanol (60:15:25 v/v/v) at a flow rate of 1.0 mL/min and propranolol was used as internal standard. Total running time was 10 min. The lower limit of quantification was 0.2 ng/mL and the method exhibited good linearity within the 0.2-20 ng/mL range (r = 0.9987). A rapid, specific, sensitive, linear, precise and accurate method was developed for determination of mazindol in human oral fluid according to European Medicines Agency guidelines, and is suitable for monitoring mazindol levels in oral fluid of professional drivers.

Fenproporex and amphetamine pharmacokinetics in oral fluid after controlled oral administration of fenproporex.

BACKGROUND: Fenproporex hydrochloride (FEN) is an anorectic drug used in the treatment of obesity, and its major metabolite is amphetamine (AMP), another central nervous system stimulant. The concentration versus time profile of FEN and its metabolite AMP has been described in classic biological matrices such as plasma and urine; however, there are no reports of such data in oral fluid. OBJECTIVE: The aim of this study is to describe the pharmacokinetics of FEN and AMP in oral fluid after intake of FEN. METHODS: Twenty-five milligrams of FEN (1 capsule of Desobesi-m) was orally administered to 6 male volunteers, and oral fluid samples were collected with a Quantisal device during 24.00 hours after drug ingestion. These samples were submitted to solid-phase microextraction before analysis by gas chromatography-mass spectrometry in the selected-ion-monitoring mode, using deuterium-labeled AMP as internal standard. RESULTS: After FEN administration, both analytes could be detected in oral fluid of all volunteers with an initial detection time varying from 0.50 to 1.00 hour. FEN peak concentrations occurred between 1.00 and 1.50 hours after administration and were between 70.7 and 227.5 µg/L. For AMP, peak concentration occurred between 1.50 and 4.00 hours, reaching 33.0-150.9 µg/L. CONCLUSION: The authors observed that oral administration of FEN resulted in significant amounts of FEN and AMP in oral fluid, showing that oral fluid could be a biological matrix suitable for pharmacokinetic studies for both analytes. Using a compartmental approach, FEN data were best fitted by 1-compartment model with first-order input and output, whereas AMP followed a 2-compartment model with first-order input and output.

Uncovering cloves: characterization of volatile compounds present in clove cigarettes.

Indonesian clove cigarettes-called "kretek" due to the crackling sound that can be heard when the product burns-are tobacco products containing clove and the "saus", a mixture of essential oils and plant extracts whose ingredients are mostly kept in secret. It is important to determine which ingredients those are to properly assess the effects that clove cigarettes can cause. An organoleptic, qualitative and quantitative analysis was made in 9 different brands of clove cigarettes obtained in Brazil. Nicotine, eugenol, menthol, and ß-caryophyllene were quantified through gas chromatography coupled to mass spectrometry. The samples presented 20 different compounds, and all samples had a different combination of the compounds. Nicotine concentrations were generally higher than eugenol, and lower than nicotine concentration in a conventional cigarette. One sample had menthol even though the cigarette pack did not inform that it was a menthol product. There were traces of 2 unusual substances. Clindamycin is an antibiotic that can be used to treat bacterial infections in respiratory airways, and octodrine is an amphetaminic stimulant used in nutritional supplements, considered as a substance of doping by the World Anti-Doping Association. The presence of both substances was not tested using certified reference materials, but its possible presence raises concern about the compounds in kretek cigarettes. There should be more studies about the contents of clove cigarettes, to improve antitobacco legislations and regulations. This way it would be possible to properly inform the risks of smoking clove cigarettes and to diminish the number of tobacco users throughout the world.

Medical cannabis regulation: an overview of models around the world with emphasis on the Brazilian scenario.

Cannabis sativa has accompanied humankind since ancient times, permeating the most diverse aspects of its existence, among which the search for health promotion and well-being stands out. Nevertheless, during the twentieth century, a series of restrictions and controls have been adopted internationally to prevent the abusive use of this species. Despite that, there has been an increased demand for the medical use of cannabis and its derivatives in the last few decades, especially among patients with debilitating conditions for which the existing therapeutic alternatives are limited. Accordingly, several countries have adopted regulatory strategies to allow access to cannabis-based products. This study aimed to overview the existing regulatory frameworks for medical cannabis around the world, focusing on the current Brazilian scenario. In addition to supply and access regulation aspects, some quality-related issues regarding cannabis-based pharmaceutical products were addressed, with emphasis on risks to patients. The literature research was performed between October 2020 and March 2021. According to the retrieved information, by the time the data collection was completed, thirty-six countries had already implemented regulatory frameworks regarding medical cannabis, and sixteen countries had models under development or in the process of implementation. The characteristics of the assessed regulatory strategies vary considerably from country to country, reflecting sociocultural, historical, and political aspects. Among the key aspects that differed between the assessed models, one can highlight the type of cannabis products that are made available and the technical requirements applied to them, as well as the possible access mechanisms. Different supply regulation strategies were also observed regarding cannabis cultivation, production licensing, and distribution mechanisms. In Brazil, an evolution of the regulatory framework has been noticeable since 2015, even though pending points are still to be addressed, among which are the species' cultivation and the access to it for scientific research purposes. Constructing a regulatory model which provides access to good quality cannabis-based medicines that may meet the patient's needs is still a challenge in the coming years, requiring the engagement of various stakeholders, including regulators, members of the academic community, prescribing professionals, and patients.

Piper aduncum Essential Oil Rich in Dillapiole: Development of Hydrogel-Thickened Nanoemulsion and Nanostructured Lipid Carrier Intended for Skin Delivery.

The essential oil extracted from the leaves of Piper aduncum, an aromatic plant from the Amazon region, is rich in dillapiole and presents anti-inflammatory activity. In this study, nanoemulsions (NE) and nanostructured lipid carriers (NLC), which are biocompatible nanostructured systems of a lipid nature, were prepared by high-pressure homogenization for the yet unexplored skin delivery of dillapiole. The addition of hydroxyethylcellulose produced hydrogel-thickened NE or NLC in view to improving the viscosity and skin adherence of the nanoformulations. Formulations were characterized with respect to dillapiole content, droplet size, polydispersity index, zeta potential, morphology, rheological behavior, bioadhesion, skin permeation profile, and in vitro irritancy (HET-CAM). The formulations developed presented spherical, homogeneous nanometric particle size (around 130 nm), narrow polydispersity index (<0.3), and negative zeta potential (around −40 mV). Dillapiole content was slightly lower in NLC compared to NE since the production process involves heating. The hydrogels containing nanocarriers showed pseudoplastic behavior with bioadhesive characteristics. The developed formulations exhibited a controlled release profile, dillapiole delivery up to the dermis, the layer of interest for anti-inflammatory potential, and low irritant potential in the chorioallantoic membrane (HET-CAM). Both hydrogels-thickened NE and NLC seemed to be promising formulations for skin delivery of Piper aduncum essential oil.

Fragmentation of Cannabinoids by Flow Injection Analysis Tandem Mass Spectrometry (FIA-MS/MS).

BACKGROUND: The analysis of plant material from Cannabis sativa L. has long been targeted on its main psychologically active metabolite, Δ9-tetrahydrocannabinol (THC). In addition to the diverse plant composition and medicinal interest in several cannabinoids, these compounds may also be related to the different characteristics of samples sold illegally. Currently, it is indisputable that other cannabinoids should also be considered in cannabis assays. Mass spectrometry has been used to identify and characterize substances in the most different scenarios, and knowing the analyte fragmentation profile is essential for characterizing samples of diverse origin. OBJECTIVE: In this work, flow injection analysis-tandem mass spectrometry with electrospray ionization (FIA-ESI-MS/MS) in positive and negative modes was used to evaluate the fragmentation profiles of eight cannabinoids commonly found in cannabis samples: THC, tetrahydrocannabinolic acid, Δ8-tetrahydrocannabinol, cannabidiol, cannabidiolic acid, cannabigerol, cannabigerolic acid and cannabinol. METHODS: By exploring the fragmentation data from mass spectrometry, the samples were classified using a chemometric model of partial least squares discriminant analysis (PLS-DA). RESULTS: When ESI in negative mode is used with adequate collision energies, it is possible to identify differences in the fragmentation of isomers. Based on that, chemometric tools were employed to classify different samples. The PLS-DA applied to FIA-ESI-MS/MS data yielded satisfactory classification. CONCLUSION: Thus, the results presented can be applied as a preliminary tool in the analysis of unknown samples, guiding more accurate investigations in terms of chemical composition. HIGHLIGHTS: This study of the cannabinoid fragmentation pattern by flow injection MS showed that cannabinoids can be distinguished by their fragmentation spectra after negative electrospray ionization. Multivariate data analysis (PLS-DA) allowed classification of different cannabis samples.

Forensic analysis of anabolic steroids tablets composition using attenuated total reflection Fourier transform infrared microspectroscopy (µATR-FTIR) mapping.

The use of falsified and unregistered drugs is a worldwide public health problem. Because these global market products usually do not follow the Good Manufacturing Practices required by health legislation, its composition may be completely different from the original or may contain relevant concentrations of impurities and toxic contaminants. Since anabolic steroids are among the main irregular therapeutic classes seized in Brazil, here we propose a new methodology for analyzing these products, in tablets form, using Attenuated Total Reflection Fourier Transform Infrared Microspectroscopy (µATR-FTIR) mapping. Spectra were acquired from solid tablets by attenuated total reflection, through point mapping methodology. In data processing, a characteristic absorption band for each Active Pharmaceutical Ingredient (API) was integrated and plotted to create its distribution map. This technique was applied in an unprecedented way for the forensic analysis of anabolic steroids and proved to be effective in distinguishing falsified products based on the detection of their APIs. It was possible to detect APIs in 26 out of 30 samples, five of which were classified as falsified only through µATR-FTIR analysis. We were able to create distribution maps of the detected substances associating the microspectroscopic results with characteristic band integration method, which can be used to detect substances and to study samples' homogeneity. We concluded that this methodology is promising for the analysis of anabolic steroid tablets, and can be used in a complementary way with techniques already consolidated in forensic laboratory routine for a better classification of questioned samples between authentic and falsified ones.

Study of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) extraction FROM dried oral fluid spots (DOFS) and LC-MS/MS detection.

BACKGROUND: Oral fluid is a widely studied matrix able to isolate the primary Cannabis constituent THC, facilitating its detection via mass spectrometry, and in most cases link these findings to recent drug use. As an alternative to liquid oral fluid, dried oral fluid spots (DOFS) is a simple and a low-cost sampling technique. It has shown improved stability compared to liquid samples, allowing for the possibility to preserve the specimens under various temperature and humidity conditions. The sampling strategy is straightforward and involves the application of a small quantity of oral fluid aliquot to a paper substrate that is set to air dry allowing for on-site collection at a large-scale demand. The goal of this study is to study THC and CBD extraction from DOFS, applying a previous established protocol for a LC-MS/MS qualitative method validation. Although other drugs of abuse have been included in DOFS methods, this is the first method validation including cannabinoids. An alternative oral fluid extraction method (WAX-S tips) is demonstrated to improve the recovery of the analytes. METHODS: A pool of blank oral fluid was used to prepare THC and CBD spiked DOFS samples for method validation and application. Spiked oral fluid was used to demonstrate WAX-S tips THC and CBD extraction. All samples were analyzed on a LC-MS/MS instrument. RESULTS: The qualitative method validation for THC and CBD confirmation in DOFS included method selectivity, matrix effects (< 20%), recovery (average of 25%), process efficiency (average of 21%), LOD (2 ng/mL for THC and 4 ng/mL for CBD), absence of carryover, and DOFS stability (70% in 35 days) as figures of merit. The method application in blindly prepared samples demonstrated the method capability to identify THC and CBD. WAX-S tips extraction showed an average of 91% recovery of THC and CBD from liquid oral fluid. CONCLUSIONS: THC and CBD extraction from DOFS showed low recoveries. However, the LC-MS/MS qualitative confirmation of THC and CBD in DOFS could improve cannabinoids screening in oral fluid, as it shows adequate LOD and stability over time. This method has potential for assisting the screening of drivers under possible drug influence by facilitating sample transportation and temporary storage in dried spot form. Additional research is suggested for WAX-S tips extraction and quantitative method validation.

A systematic review of quantitative analysis of cannabinoids in oral fluid.

Cannabis sativa L. is a substance widely used around the world for recreational and medicinal purposes. Oral fluid has been investigated as an alternative biological matrix for demonstrating the illegal use of cannabis, particularly in situations where its recent use needs to be identified. In the last two decades, many methods have been developed to detect and quantify cannabinoids in oral fluid, especially for Δ9 -tetrahydrocannabinol, the primary psychoactive substance of cannabis. However, some aspects must be considered in the use of these techniques, such as cannabinoids recoveries or extraction efficiency from different oral fluid collection devices/containers. Pharmacokinetic studies have shown that the presence of minor cannabinoids and metabolites in the analysis of oral fluid may be valuable in interpreting tests, which indicates the need to improve the sensitivity of detecting low concentrations. The aim of this review is to summarize and to describe the methodologies for the quantitative analysis of cannabinoids in oral fluid that have previously been investigated. A systematic search for articles was performed of four different databases, using the descriptor "cannabinoids and oral fluid". Forty-seven studies that examined quantitative methods were identified. The analytical data described in these articles, including oral fluid collection, sample preparation, cannabinoids recovery and extraction efficiency, detection instruments, and quantification limits, were analyzed. The discussion of these particular features of cannabinoid analysis in oral fluid could help to improve or to develop methods for use in Forensic Toxicology.