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- LC-MS/MS-MS3 for determination and quantification of ∆9-tetrahydrocannabinol and metabolites in blood samplesPublication . Proença, Paula; Martinho, Beatriz; Teixeira, Helena; Monteiro, Carla; Simões, Susana; Franco, João; Corte Real, F.Due to the high prevalence of cannabinoids in forensic toxicology, it is crucial to have an efficient method that allows the use of a small sample amount and that requires a minimal sample preparation, for the determination and quantification of low concentrations. A simple, highly selective and high throughput liquid chromatography tandem mass spectrometry method (LC-MS/MS-MS3) was developed for the determination and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-THC (THC-OH) and 11-nor-∆9-THC-9-carboxylic acid (THC-COOH) in blood samples. Chromatographic analysis was preceded by a protein precipitation of 0.1 mL of blood samples with acetonitrile, then THC, THC-OH, THC-COOH and deuterated internal standards were separated on an Acquity UPLC® HSS T3 (100 mm x 2.1 mm i.d., 1.8 mm) reversed-phase column using a gradient elution with 2 mM aqueous ammonium formate (0.1% formic acid) and methanol, at a flow rate of 0.4 mL/min, and with a run time of 10 min. For MS/MS-MS3 analysis, a SCIEX QTRAP® 6500+ linear ion trap triple quadrupole mass spectrometer was used via electrospray ionization (ESI), operated in multiple reaction mode (MRM) and linear ion trap mode (MS3). The method was validated in accordance with international accepted criteria and guidelines. The method was selective and linear between 0.5-100 ng/mL (r2>0.995). The lower limits of quantification (LLOQ) corresponded to the lowest concentrations used for the calibration curves. The coefficients of variation obtained for accuracy and precision were less than 15%. The mean recoveries were between 88.0-101.4% for the studied concentration levels (1 ng/mL, 5 ng/mL and 50 ng/mL). No significant interfering compounds, matrix effects or carryover were observed. The validated method provides a sensitive, efficient and robust procedure for the quantitation of cannabinoids in blood using LC–MS/MS-MS3 and a sample volume of 0.1 mL. This work is also a proof of concept for using LC-MS3 technique to determine drugs in biological samples.
- Spatio‐temporal assessment of illicit drug use at large scale: evidence from 7 years of international wastewater monitoringPublication . González‐Mariño, Iria; Baz‐Lomba, Jose Antonio; Alygizakis, Nikiforos A.; Andrés‐Costa, Maria Jesus; Bade, Richard; Bannwarth, Anne; Barron, Leon P.; Been, Frederic; Benaglia, Lisa; Berset, Jean‐Daniel; Bijlsma, Lubertus; Bodík, Igor; Brenner, Asher; Brock, Andreas L.; Burgard, Daniel A.; Castrignanò, Erika; Celma, Alberto; Christophoridis, Christophoros E.; Covaci, Adrian; Delémont, Olivier; Voogt, Pim; Devault, Damien A.; Dias, Mário; Emke, Erik; Esseiva, Pierre; Fatta‐Kassinos, Despo; Fedorova, Ganna; Fytianos, Konstantinos; Gerber, Cobus; Grabic, Roman; Gracia‐Lor, Emma; Grüner, Stefan; Gunnar, Teemu; Hapeshi, Evroula; Heath, Ester; Helm, Björn; Hernández, Félix; Kankaanpaa, Aino; Karolak, Sara; Kasprzyk‐Hordern, Barbara; Krizman‐Matasic, Ivona; Lai, Foon Yin; Lechowicz, Wojciech; Lopes, Alvaro; López de Alda, Miren; López‐García, Ester; Löve, Arndís S. C.; Mastroianni, Nicola; McEneff, Gillian L.; Montes, Rosa; Munro, Kelly; Nefau, Thomas; Oberacher, Herbert; O'Brien, Jake W.; Oertel, Reinhard; Olafsdottir, Kristin; Picó, Yolanda; Plósz, Benedek G.; Polesel, Fabio; Postigo, Cristina; Quintana, José Benito; Ramin, Pedram; Reid, Malcolm J.; Rice, Jack; Rodil, Rosario; Salgueiro‐González, Noelia; Schubert, Sara; Senta, Ivan; Simões, Susana; Sremacki, Maja M.; Styszko, Katarzyna; Terzic, Senka; Thomaidis, Nikolaos S.; Thomas, Kevin V.; Tscharke, Ben J.; Udrisard, Robin; Nuijs, Alexander L. N.; Yargeau, Viviane; Zuccato, Ettore; Castiglioni, Sara; Ort, ChristophBackground and aims Wastewater‐based epidemiology is an additional indicator of drug use that is gaining reliability to complement the current established panel of indicators. The aims of this study were to: (i) assess spatial and temporal trends of population‐normalized mass loads of benzoylecgonine, amphetamine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in raw wastewater over 7 years (2011–17); (ii) address overall drug use by estimating the average number of combined doses consumed per day in each city; and (iii) compare these with existing prevalence and seizure data. Design Analysis of daily raw wastewater composite samples collected over 1 week per year from 2011 to 2017. Setting and Participants Catchment areas of 143 wastewater treatment plants in 120 cities in 37 countries. Measurements Parent substances (amphetamine, methamphetamine and MDMA) and the metabolites of cocaine (benzoylecgonine) and of Δ9‐tetrahydrocannabinol (11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol) were measured in wastewater using liquid chromatography–tandem mass spectrometry. Daily mass loads (mg/day) were normalized to catchment population (mg/1000 people/day) and converted to the number of combined doses consumed per day. Spatial differences were assessed world‐wide, and temporal trends were discerned at European level by comparing 2011–13 drug loads versus 2014–17 loads. Findings Benzoylecgonine was the stimulant metabolite detected at higher loads in southern and western Europe, and amphetamine, MDMA and methamphetamine in East and North–Central Europe. In other continents, methamphetamine showed the highest levels in the United States and Australia and benzoylecgonine in South America. During the reporting period, benzoylecgonine loads increased in general across Europe, amphetamine and methamphetamine levels fluctuated and MDMA underwent an intermittent upsurge. Conclusions The analysis of wastewater to quantify drug loads provides near real‐time drug use estimates that globally correspond to prevalence and seizure data.
- UPLC MS/MS Method for the Analysis of Cardiovascular Medications in Putrefied Specimens obtained from an Exhumation AutopsyPublication . Simões, Susana; Castañera, Antonio; Dias, Mário
- Multi-year inter-laboratory exercises for the analysis of illicit drugs and metabolites in wastewater:Development of a quality control systemPublication . van Nuijs, Alexander L.N.; Lai, Foon Yin; Been, Frederic; Andres-Costa, Maria Jesus; Barron, Leon; Baz-Lomba, Jose Antonio; Berset, Jean-Daniel; Benaglia, Lisa; Bijlsma, Lubertus; Burgard, Dan; Castiglioni, Sara; Christophoridis, Christophoros; Covaci, Adrian; de Voogt, Pim; Emke, Erik; Fatta-Kassinos, Despo; Fick, Jerker; Hernandez, Felix; Gerber, Cobus; González-Mariño, Iria; Grabic, Roman; Gunnar, Teemu; Kannan, Kurunthachalam; Karolak, Sara; Kasprzyk-Hordern, Barbara; Kokot, Zenon; Krizman-Matasic, Ivona; Li, Angela; Li, Xiqing; Löve, Arndís S.C.; Lopez de Alda, Miren; McCall, Ann-Kathrin; Meyer, Markus R.; Oberacher, Herbert; O'Brien, Jake; Quintana, Jose Benito; Reid, Malcolm; Schneider, Serge; Simões, Susana; Thomaidis, Nikolaos S.; Thomas, Kevin; Yargeau, Viviane; Ort, ChristophThis study presents the development of a worldwide inter-laboratory testing scheme for the analysis of seven illicit drug residues in different matrices (standard solutions, tap- and wastewater). By repeating this exercise for six years with participation of 37 laboratories from 25 countries, the testing scheme was substantially improved based on experiences gained across the years (e.g. matrix type, sample conditions, spiking levels). From the exercises, (pre-)analytical issues (e.g. pH adjustment, filtration) were revealed for some analytes which resulted in formulation of best- practice protocols, both for inter-laboratory setup and analytical procedures. The results illustrate the effectiveness of the inter-laboratory testing scheme in assessing laboratory performance in the framework of illicit drug analysis in wastewater. The exercise proved that measurements of laboratories were of high quality (> 80% satisfactory results for 6 out of 7 analytes) and that analytical follow-up is important to assist laboratories in improving robustness of wastewater-based epidemiology results.
- Selection criteria for weighting factor in linear regressionPublication . Costa, Pedro; Simões, Susana; Castañera, Antonio; Franco, João; Teixeira, Helena
- Screening of recently seized new psychoactive substances in urine of potential consumers and urban wastewaterPublication . Celma, Alberto; Bijlsma, Lubertus; Sancho, Juan V.; Hernandez, Felix; Dias, Mário; Simões, Susana; Salgueiro, Noelia; Castiglioni, Sara; Zuccato, EttoreNewPsychoactive Substances (NPS) areconstantly being developed as legalsubstitution of traditional drugs of abuse.Although the traditional drugs maintain popular, new drugs are regularly introduced, changing the drug market ceaselessly. Monitoring NPS and its consumption is challengingas these compoundsare normally missed in routine drug analysis. Users do often not exactly know what they consume andinformation available islimitedof what is being sold. Furthermore, very little information exists onmetabolism of these newly introduced NPS. This highlights the needof applying a strategic workflow making use of modernanalytical techniques to face this novel public health safety challenge.The workflowpresented in this work consists of three stages: i)the creation of a databaseincluding approximately 200 NPS. These NPS wererecently reported to the National Early Warning Systems (EWS) ofSpain andItaly and the EWS of the European Monitoring Center for Drugsand Drug Addiction (EMCDDA). ii) The collection of urine samples of individuals suspected of drug consumption, pooled urine samples from festivals and urban wastewater samples. iii) The screening of NPS using ultra-high performance liquid chromatography coupled to high resolution massspectrometry with a hybrid quadrupole time-of-flight mass analyzer. Although the database is under constant development,including NPS reported to be present in wastewaters around Europe, new compounds appearing in the market, as well as metabolites reported in the literature, the workflow applied allowed the detection of several unchanged NPS in urine and pooled urine samples. In several cases, NPSwere also detected in wastewater samples, which indicatesthat consumptionof some of these compounds is elevated. The absence of reference standards for several NPS found, as well as for some NPS metabolites, made it unfeasible the full confirmation of some compounds in the urine/wastewater samples analyzed. However, accurate-mass full-spectrum data provided by HRMS allowed their tentative identification with high degree of reliability. The subsequent acquisition of reference standards, when available, will allow the unequivocal confirmation of their identity.
- Desenvolvimento e validação de um método analítico para a determinação de canabinóides em amostras de fluido oral por LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, MárioA 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.
- Documenting human exposure to cannabinoids using oral fluidPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João Miguel; Gallardo, Eugenia; Barroso, MárioThe 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).
- Mass Spectrometry and Workflow in Forensic ToxicologyPublication . Castañera, Antonio; Simões, Susana
- Detection and quantification of selected cannabinoids in oral fluid samples by protein precipitation and LC-MS/MSPublication . Antunes, Mónica; Simões, Susana; Fonseca, Suzana; Franco, João; Gallardo, Eugenia; Barroso, MárioCannabis 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.