Introduction
In Europe, approximately 96 million adults, aged from 15-64 years, have used recreational drugs at some point in their lives.1 The use of alcohol and drugs can cause serious health issues, both with short-term and long-term risk.Reference Bolier, Voorham, Monshouwer, Van Hasselt and Bellis2 Furthermore, substance abuse is also a concern for the broader community as it leads to an increased risk of traffic accidents, risky sexual behavior, and substance-induced violence.Reference Bolier, Voorham, Monshouwer, Van Hasselt and Bellis2 Unfortunately, using drugs seems to have become more and more accepted, and the production and spread of new synthetic drugs has expanded significantly. However, there is limited knowledge about these new substances. They pose a considerable challenge due to their rapid evolution and they are hard to be controlled by international drug laws.1,Reference Hoegberg, Christiansen and Soe3
Many epidemiological studies have demonstrated a connection between a party scene and substance use.Reference Bolier, Voorham, Monshouwer, Van Hasselt and Bellis2 A party scene is related to freedom, escaping one’s responsibilities and obligations, and taking a break from everyday life in an increasingly more complex society. This creates an environment to experiment with substances that are designed to intensify feelings of pleasure and relaxation.Reference Hoegberg, Christiansen and Soe3–Reference Gjerde, Gjersing and Baz-Lomba5 In addition, studies pointed out that drug use is largely concentrated among young adults (aged 15 to 34 years).1 Major music festivals, with visitors from around the globe, are locations where recreational drugs are frequently found and new drugs, such as new psychoactive substances (NPS), will be explored.Reference Hoegberg, Christiansen and Soe3–Reference Gjerde, Gjersing and Baz-Lomba5
The aim of this study was to obtain objective data on drug use in Flanders, Belgium by analyzing urine samples at a major festival during the summer of 2019.
Methods
Study Design
An observational cross-sectional study, using pooled urine collected at a music festival with approximately 70,000 attendees per day, was performed. Samples were analyzed in the toxicology laboratory of the University Hospitals of Leuven (Leuven, Belgium). The festival organization gave permission to perform and publish the results anonymously.
Sample Collection
Samples were collected during three consecutive days at 2:00pm, 5:00pm, 8:00pm, and 11:00pm at four different festival locations. Three locations were situated on the festival site, all at different music stages. The fourth location was at a public camping site. Forty-eight urine samples were collected but only 43 samples were analyzed due to limited collection volume at specific times and at specific collection points (no activities at that time at that particular place). Pumps installed on collecting tubes of men’s urinals were used to tap urine into 100ml urine samples. This urine was stored at 4°C and later analyzed.
Urine Analysis
Determination of Ethanol Concentrations using Gas Chromatography with a Flame Ionization Detector (GC-FID)—For this, 200µL of homogenized urine sample, calibration solution, or blank was added to 300µL sterile water and 500µL internal standard solution. The analysis was performed using a Trace 1300 Gas Chromatograph outfitted with a flame ionization detector (Thermo Fisher Scientific; Merelbeke, Belgium). Following a 10-minute incubation at 80°C, a headspace injection was performed onto an RTX-Bac Plus 1 column (Restek Gmbh; Bad Homburg, Germany) with an isothermic program of six minutes at 40°C and He2 flow of 2mL/minute FID was set at 250°C using the following gas ratio:
Air Flow: 350mL/minute; Make-Up Gas: 40mL/minute; H2 Flow: 35mL/minute.
Data acquisition was processed using Chromeleon 7.2 software (Thermo Fisher Scientific; Merelbeke, Belgium; 2015). A linear calibration curve for methanol/ethanol was obtained by running the seven calibration solutions up to 4g/L.
Quantitative Determination of Drugs of Abuse using Multiple Reaction Monitoring (MRM) Analysis on Triple Quadrupole Linear Ion Trap (QTRAP) 5500 Spectrometer—For this, 13 different kinds of recreational drugs were analyzed. Samples were prepared by homogenizing all quality controls prior to preparing a 1:10 dilution with an internal standard solution containing benzoylecgonine-D8, 3,4-methylenedioxymethamphetamine (MDMA)-D5, amphetamine-D6, 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH)-D9, and halazepam. All urinary samples were homogenized before centrifugation. After centrifugation, samples were diluted to a 1:10 ratio with an internal standard solution. The high-performance liquid chromatography (HPLC) system was a Nexera XR LC-20AD Liquid Chromatograph (Shimadzu; Wemmel, Belgium).
Chromatographic separation was performed with a Kinetex C18 analytical column (100x3.0mm, 2.6mm; Phenomenex; Utrecht, The Netherlands). Mobile Phase A consisted of 10mM ammonium formate, 0.1% formic acid in SR water. Mobile Phase B consisted of 0.1% formic acid in acetonitrile. Mobile Phase gradient was programmed as follows: two percent Mobile B at the start linearly increased to 35% at a total flow rate of 0.5mL/minute. At minute 10, the flow rate was increased to 0.6mL/minute whilst continuing to increase Mobile B from 35% to 100% until minute 15. Mobile B at 100% was maintained until minute 19 before returning to initial conditions at minute 19. The mass spectrometer used was a QTRAP 5500 triple quadrupole mass spectrometer (AB Sciex LLC; Framingham, Massachusetts USA). The instrument was operated in electrospray using tandem positive and negative mode (electrospray ionization [ESI]+ and ESI-). All data were recorded using the scheduled MRM mode. Data acquisition was performed using Analyst 1.6.2 software (AB Sciex LLC; Framingham, Massachusetts USA; 2013) and processed using MultiQuant 3.0.2 software (AB Sciex LLC; Framingham, Massachusetts USA; 2015).
Qualitative Determination using Tandem Mass Spectrometry (MSMS) Analysis on Quadrupole Time-of-Flight (QTOF) 5600 Spectrometer—All samples were screened for classic drugs as well as NPS using QTOF. Samples were prepared as described above for analysis using QTRAP.
The HPLC system was a Nexera X2 LC-30AD Liquid Chromatograph (Shimadzu; Wemmel, Belgium). Chromatographic separation was performed as described above. The mass spectrometer used was a TripleTOF 5600 QTOF mass spectrometer (AB Sciex LLC; Framingham, Massachusetts USA). The instrument was operated in electron spray using positive and negative mode (ESI+ and ESI-). All data were recorded using the high-sensitivity time-of-flight information dependent acquisition (TOF-IDA) MSMS mode, combining a mass spectrometry survey scan with a TOF-MSMS IDA scan. Reference spectra to confirm the identity of each recorded analyte were obtained using diluted reference materials. Data acquisition was performed using Analyst TF 1.7.1 software (AB Sciex LLC; Framingham, Massachusetts USA; 2015) and processed using the PiekView 2.2 software (AB Sciex LLC; Framingham, Massachusetts USA; 2014) using a database containing more than 1,600 compounds.
Results
Quantitative Analysis
Ethanol—The findings are presented in Table 1. The median ethanol level of all samples was 0.88g/L. In the early afternoon, it was 0.49g/L. During the night, an increase to a level of 0.96g/L at 8:00pm and 11:00pm was observed.
Table 1. Ethanol Concentrations (g/L)
Note: The lower limit of detection was 0.05 g/L. ‘L’ stands for low urine volumes.
Drugs of Abuse—Data are presented in Table 2 (supplementary material; available online only). No samples contained sufficient cocaine to rise above the lower limit of detection for quantitative analysis using QTRAP. In contrast, qualitative analysis using QTOF demonstrated the presence of cocaine in every sample (Table 3; supplementary material; available online only). Both MDMA and 3,4-methyleendioxyamphetamine (MDA) were detected in every sample, mostly in quite high concentrations; MDA can be present as a parent compound, but also as a metabolite of MDMA, and demonstrated a similar evolution as MDMA. In 35% (15/43) of all samples, amphetamines were detected, most frequently at one specific collection point.
All samples contained ketamine above the limit of quantification, even in quite high concentrations. The highest concentration was found on Sunday at the festival site. Norketamine is the active metabolite of ketamine and was detected in similar quantities.
Also, THC-COOH, the main metabolite of cannabis, was abundantly detected. Much higher concentrations were observed at the camping site in comparison with any festival site. In contrast, the glucuronide of THC-COOH was never found above the limit of detection.
Qualitative Analysis
Cocaine was present in every sample (43/43) on qualitative analysis. Furthermore, different opiates were detected: codeine in 44% (19/43), dextromethorphan in 16% (7/43), dihydrocodeine in five percent (2/43), and tramadol in 49% (21/43). Ninety-five percent (41/43) of all samples were positive for paracetamol, 60% (26/43) for naproxen, 86% (37/43) for cetirizine, and 19% (3/43) for diphenhydramine. All samples were positive for caffeine, quinine, and nicotine. In addition, flecainide in 37% (16/43) and amlodipine in nine percent (4/43) were detected. Finally, in search for NPS, 2/3/4-fluoromethamphetamine (FMA) was found in 37% (16/43); FMA gave a stronger signal on QTOF compared to classic amphetamines, suggesting that the concentration of FMA was of a higher order of magnitude. Twenty-one percent (9/43) of all samples were also positive for 3/4-methylmethcathinone (MMC). All positive samples were collected on Saturday. Differentiation between the structural isomers was not possible with the used analytical technique.
Discussion
Ethanol
Music festivals are known to be a high-risk setting for alcohol consumption. Only six samples contained an ethanol concentration under 0.5g/L, 27 samples between 0.5 and 1.0g/L, and 10 samples above 1.0g/L. Overall, ethanol concentrations gradually rose throughout the day. In addition, ethanol concentrations were slightly higher at the festival site compared with the camping site. Ethanol is normally analyzed in blood or plasma. A careful interpretation of the results is needed as urine alcohol concentration are 1.3-times higher than blood alcohol concentration.Reference Jones6 However, it is clear that alcohol consumption is prominent at festivals. In comparison with other festivals, the levels are comparable.Reference Harris, Everhart, Medelson and Jones7
Drugs of Abuse
The European Drug Report (European Monitoring Centre for Drugs and Drug Addiction [EMCDDA]; Lisbon, Portugal) described an increasing role of cocaine.1 Cocaine could only be detected with qualitative analysis. First, detection of cocaine is possible up to a maximum of 12 hours following intake, whereas benzoylecgonine can be detected up to 72 hours.Reference Hamilton, Wallace and Shimek8 Benzoylecgonine has a half-life of 4.5 to 6.0 hours, as cocaine has 0.5 to 2.5 hours.Reference Puet, Claussen, Hild, Heltsley and Schwope9 All samples confirmed a benzoylecgonine concentration above the lower limit. Second, simultaneous consumption of cocaine and alcohol alters the biotransformation of cocaine. In this situation, 17% of cocaine is excreted as coca-ethylene in urine, resulting in a decrease of urinary benzoylecgonine and cocaine excretion.Reference Harris, Everhart, Medelson and Jones7 However, coca-ethylene poses a greater risk of cardiotoxicity and central nervous system toxicity. In 28 of 43 samples, coca-ethylene exceeded 1.0ng/mL (limit of quantitation). At the camping site, coca-ethylene concentrations were rather low, but were much higher at the festival site, suggesting that people at the festival site were more likely to be under the influence of alcohol and cocaine.
In contrast, MDMA and amphetamine did not demonstrate a clear pattern. However, these results may indicate that less people were consuming amphetamines in comparison to MDMA.Reference Gjersing, Bretteville-Jensen, Furuhaugen and Gjerde10 The evolution of MDA and MDMA concentrations throughout the day were quite similar; MDA was most likely detected as a metabolite of MDMA. In contrast with the data from the EMCDDA, no methamphetamine was detected.
Ketamine was prominently present. Ketamine is also described as an adulterant of various drugs, including MDMA.1 These results may support the observation that ketamine is becoming more popular as a party drug amongst a younger population. Not surprisingly, THC was prominently detected. Concentrations rose during evenings and were predominantly found at the camping site. One could postulate that cannabis was used for its relaxation properties at the camping site, whilst the more exciting drugs were used at the festival sites.
The most popular NPS are cannabinoid receptor agonists followed by synthetic cathinones.1 Searching for NPS, FMA and MMC were detected. Both could be used as a substitute for MDMA and amphetamines. Morphine screening was negative, indicating that heroine was hardly used at the festival.
Legal Medications and Adulterants
Various prescribed and over-the-counter medications were detected, including antihistamines. As the festival took place in the summer, tree and grass pollen cause allergic rhinitis. Diphenhydramine (eight samples), also an anti-histaminic, can potentially induce delirium and hallucinations.
Adulterants are intentionally added substances to an illicit drug, but increase perceived quantity and reduce purity. It can be either pharmacological active or inactive substances. Mostly, these are rather harmless agents such as sugars, caffeine, and paracetamol.Reference Cole, Jones, McVeigh, Kickman and Syed11 Findings on qualitative analysis, every sample was positive for caffeine, quinine, and nicotine, and also paracetamol was present in almost every sample. However, a remarkable finding was the presence of flecainide and amlodipine. Both can be used as adulterants of cocaine as they mimic or counteract the pharmacological effect of cocaine. Amlodipine is clinically used for cocaine dependence to prevent headaches and decrease systemic blood pressure.Reference Malcolm, Liao and Michel12 Flecainide is a Class I anti-arrhythmic agent with similar pharmacological properties as cocaine. Both act on the cardiac sodium channel.Reference Wood, Dargan and Hoffman13 Their use as prescription drug is less likely in a young festival population. It is needless to state that combinations of these substances could lead to dangerous clinical manifestations.
Limitations
It is important to understand the possibilities and limitations of the analytic techniques used. First, QTRAP is used to detect overdoses in a clinical setting, and the dilute and shoot preparation may be less-sensitive for pooled urine samples. Therefore, the quantitative analysis was a better instrument to detect an evolution of drug use over time, but not for absolute quantification. Both quantitative (QTRAP) and qualitative detection (QTOF) of various drugs could have differed depending on location and time of collection. However, the authors strived to obtain more insight in the current drug scene, not to deliver a complete summary of all drugs used during the festival.
Another important limitation is that urine, to be processed, cannot be mixed with feces, currently making male urinals the only option for sampling and data being limited to only a male population. Evidence is available that recreational drugs are more frequently used by males.Reference Hoegberg, Christiansen and Soe3
The urine flow rate was different amongst different sampling sites due to a variation in number of urinals connected to a collection point as well as the amount of people using the urinals over a given time period. At the camping site and one festival stage, there was a steady constant flow. At two other collection points, there was a clear fluctuation over time. Urine samples from low-flow sites could represent individual use or a “lucky catch” and not representative for a larger population. However, these low-rate sampling sites were still valuable in the search for different classical drugs and NPS.
Unwanted contamination could have been possible: for example, dilution by pouring liquids other than urine or concentration by pouring in a compound.Reference Jones6 One out of four sampling sites had roofing and there were some rain showers. The presence of metabolites demonstrated that drugs were consumed and metabolized rather than poured into the urinals.
Some substances were not detectable by the current techniques.
Conclusion
Insight in “classical” recreational drug use as well as information about the emergence of new drugs and trends is extremely valuable. Music festivals, a high-risk setting, can provide abundant information in this regard. Through analyses of pooled urine samples, the authors provided objective data on illicit drug use in Belgium. This study confirmed wide-spread use of several classical drugs and was able to detect two NPS during a major international music festival in the summer of 2019. Furthermore, the authors aimed to reach out a well-structured sampling method for drug analysis of pooled urine.Reference Bijlsma, Celma and Castiglioni4,Reference Gjerde, Gjersing and Baz-Lomba5,Reference Gjersing, Bretteville-Jensen, Furuhaugen and Gjerde10
From a preventive perspective, a collaboration was established between different actors, namely health care professionals, law enforcement members, and festival organizers. The authors hope to stimulate others to carry out equal projects in order to create a more global mapping and to detect arising trends on which prevention strategies can rely on.
Conflicts of interest/funding
All authors confirm that no conflict of interest has taken place. No funding has been received.
Supplementary Materials
To view supplementary material for this article, please visit https://doi.org/10.1017/S1049023X22001285.
