Cannabis Consumption in the University Population in Portugal: Determination of Biomarkers in Hair and Oral Fluid Samples Consumo de Cannabis na População Universitária em Portugal: Determinação de Biomarcadores em Amostras de Cabelo e Saliva

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A review of publications in hair testing: 2020-2023

Publication . Simão, Ana Y.; Antunes, Mónica; Pires, Bruno; Catarro, Gonçalo; Soares, Sofia; Matos, Francisco; Rosado, Tiago; Gallardo, Eugenia; Barroso, Mário

Introduction and Aims: Since its first use until now, the relevance of hair in the toxicological expertise has evolved, mainly due to its advantages over traditional samples. Despite the fact that it is one of the most used alternative specimens, hair still presents important challenges. The objective of this study was to perform a bibliographic review of scientific publications on toxicological applications of hair testing between the years 2020 and 2023. Materials & Methods: The Scopus database was used for literature search, for papers published from 2020 to 2023 (10th April), and only articles written in English were considered. The search strings for terms included in the title, abstract or keywords were “hair testing” and “hair samples”. The papers were independently reviewed by four of the authors to determine their relevance in the context of this study. Results & Discussion: Taking into consideration these criteria and search strings, 218 articles were found, from which 18 were excluded since animal hair specimens had been used. The papers were classified according to type (e.g. review, research paper), analytes, extraction and clean-up approaches. Each paper could be placed under more than one category, except review papers (32 articles). Concerning analytes, the highest number of papers involved drugs of abuse (175, from which 45 involving new psychoactive substances), drugs (44) and alcohol biomarkers (17). Other biomarkers included hormones, endocannabinoids, etc. (44 papers, 25 of which involved cortisol). Most studies involve protein precipitation and direct injection (91), and only 12 studies address the issues of cosmetic treatments or drug adulterants. Conclusions: Hair testing is still mainly directed to the determination of drugs of abuse, and new psychoactive substances are still seldom included. Additionally, other applications are becoming more relevant, for instance the use of hair to determine biomarkers in some diseases.

2023-06Other

Open access

Documenting human exposure to cannabinoids using oral fluid

Publication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João Miguel; Gallardo, Eugenia; Barroso, Mário

The importance of studying non-conventional biological matrices such as oral fluid (OF) is increasingly being recognized. This sample presents several advantages, mainly related to its collection procedure: it is non-invasive, easy to perform by non-medical personnel, can be performed under supervision to prevent adulteration, and provides low biohazard risk. OF samples are more likely to contain parent drugs, reflecting recent drug use – a major advantage of this matrix1. A fast and robust analytical methodology was developed in OF samples for the determination of tetrahydrocannabinol (THC), 11-hydroxy-tetrahydrocannabinol (THC-OH), 11-carboxytetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD) by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), aiming at documenting cannabis consumption. Briefly, 200-μL aliquots of OF were subjected to protein precipitation with a refrigerated methanol/acetonitrile mixture (80:20, v/v). After centrifugation, the extracts were evaporated to dryness, reconstituted in methanol, and 5-μL aliquots were injected into the UPLC-QTRAP-MS 6500+ (SCIEX®) system (in a 14-minute run). The analysis was carried out in MRM mode with two transitions for each compound and one transition for each internal standard. The method was validated according to the guidelines of ANSI/ASB 0362. Parameters such as ion suppression/enhancement, interferents, linearity, precision and accuracy, limits of detection and quantification, dilution integrity and stability were studied and showcased satisfactory results. The 2 ng/mL cut-off for THC3 was achieved, and the method was successfully applied to real samples (57.95- 898.28 ng/mL for THC; 0.17-4.09 ng/mL for THC-COOH; 1.26-44.57 ng/mL for CBN; 0.42-1007.86 ng/mL).

2023-12Conference object

Open access

Desenvolvimento e validação de um método analítico para a determinação de canabinóides em amostras de fluido oral por LC-MS/MS

Publication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, Mário

A canábis é a droga de abuso mais consumida na Europa, pelo que várias metodologias analíticas têm sido desenvolvidas para a quantificação dos seus componentes em matrizes biológicas, especialmente sangue e urina. No entanto, cada vez mais se está a reconhecer a importância do estudo de matrizes biológicas não-convencionais, nomeadamente cabelo e fluido oral (ou saliva) – os seus procedimentos de recolha não são invasivos e estas amostras fornecem informações complementares sobre o uso de drogas, permitindo o controlo da exposição recente (saliva) e de médio a longo prazo (cabelo). Desta forma, foi desenvolvida uma metodologia analítica para a determinação e quantificação de tetrahidrocanabinol (THC), 11-hidroxi-tetrahidrocanabinol (THC-OH), 11-carboxitetrahidrocanabinol (THC-COOH), canabinol (CBN) e canabidiol (CBD) em amostras de saliva por cromatografia líquida acoplada à espectrometria de massa em tandem (LC-MS/MS). Segundo as Mandatory Guidelines for Federal Workplace Drug Testing Programs, o cut-off de THC em amostras de saliva é 2 ng/mL, exigindo um procedimento de extração otimizado e a utilização de uma técnica cromatográfica sensível. Resumidamente, a 200 μL de saliva foi adicionado o padrão interno, e as amostras foram submetidas a precipitação proteica com uma mistura de metanol/acetonitrilo (80:20, v/v) refrigerada. Depois de centrifugados, os extratos foram evaporados até à secura, reconstituídos em metanol, e 5 μL foram injetados no equipamento UPLC-QTRAP-MS 6500+ (SCIEX®), numa corrida de 14 minutos. A análise foi efetuada em modo MRM com duas transições para cada composto e uma transição para cada padrão interno. Os métodos foram validados de acordo com as diretrizes da norma ANSI/ASB 036, tendo sido estudados os parâmetros supressão iónica, interferentes, linearidade, precisão e exatidão, carryover, limites de deteção e quantificação, efeito de diluição e estabilidade das amostras extraídas. Todos os estudos destes parâmetros apresentaram resultados satisfatórios, e o método foi aplicado com sucesso a amostras reais. O método desenvolvido mostrou-se fácil, rápido, eficaz e robusto, estando apto para ser utilizado na rotina do laboratório, e sendo uma ferramenta complementar útil para o estudo do consumo recente de canabinóides.

2023-10Conference object

Open access

Analysis of Cannabinoids in Biological Specimens: An Update

Publication . Antunes, Mónica; Barroso, Mário; Gallardo, Eugenia

Cannabinoids are still the most consumed drugs of abuse worldwide. Despite being considered less harmful to human health, particularly if compared with opiates or cocaine, cannabis consumption has important medico-legal and public health consequences. For this reason, the development and optimization of sensitive analytical methods that allow the determination of these compounds in different biological specimens is important, involving relevant efforts from laboratories. This paper will discuss cannabis consumption; toxicokinetics, the most detected compounds in biological samples; and characteristics of the latter. In addition, a comprehensive review of extraction methods and analytical tools available for cannabinoid detection in selected biological specimens will be reviewed. Important issues such as pitfalls and cut-off values will be considered.

2023Journal article

Open access

Detection and quantification of selected cannabinoids in oral fluid samples by protein precipitation and LC-MS/MS

Publication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, Mário

Cannabis is the most widely consumed illicit drug worldwide. As consumption rates increase, partially due to the decriminalization of its use for medicinal and recreational purposes, analytical methods for monitoring different cannabinoids in several biological matrices have been developed. Herein, a simple and fast extraction procedure to extract natural cannabinoids from oral fluid (OF) samples was developed and fully validated according to the ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Using only 0.2 mL of neat OF, the analytes [Δ9-tetrahidrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), cannabinol (CBN) and cannabidiol (CBD)] were extracted by protein precipitation with a mixture of methanol:acetonitrile (80:20, v/v); the extracts were centrifuged, evaporated to dryness and reconstituted in 100 µL of methanol. Analysis was performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The developed methodology produced linear results for all compounds, with working ranges of 0.1–50 ng/mL for THC, 0.5–50 ng/mL for THC-OH, CBN and CBD, and 0.05–1 ng/mL for THC-COOH. Ion suppression was observed for THC, CBN and CBD, which did not impair sensitivity considering the low limits of quantification (LOQs) and limits of detection (LODs) obtained (which varied between 0.05 and 0.5 ng/mL). The extraction procedure produced great recoveries, and the compounds were stable. No interferences were found, and the method proved to be extremely fast, selective, precise, and accurate for use in routine analysis. The method was successfully applied to authentic samples.

2024Journal article

Open access