198 Sutlović D, et al.

D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204

Original article DOI: 10.2478/aiht-2021-72-3509

New psychoactive and classic substances in pooled urine

Davorka Sutlović1,2, Sendi Kuret1, and Marija Definis3,4

[Received in November 2020; Similarity Check in November 2020; Accepted in September 2021]

We believe that analysing pooled urine samples for recreational drugs used at mass events can provide useful information
about trends in drug use. An opportunity arose with the Ultra Europe music festival, which is attended by more than
150,000 people from over 150 countries every year. We analysed 30 pooled urine samples collected from portable chemical
toilets located at or close to the Ultra Europe music festival venue in Split, Croatia in 2016–2018 to detect the presence
of classic and new psychoactive substances (NPS). Four urine samples collected in 2016 were from a toilet without added
chemicals (otherwise used to kill the smell) while the remaining samples were collected from toilets with added
chemicals. Samples were qualitatively analysed with gas chromatography-mass spectrometry (GC/MS) using the full-
scan mode. Data were compared with the Wiley mass spectral library of designer drugs and our in-house library containing
about 1000 compounds and metabolites. We identified forty-six different substances and metabolites, 26 of which were
classic substances/metabolites, mostly from the stimulants group, while 20 were NPS. In the NPS group, most of them
were phenethylamines and cathinones. The variety of substances was the highest on the first day of the festival regardless
of the year, but 2018 showed a significant drop compared to the previous two years. The results of our study revealed
a stable trend of classic drug consumption, while NPS trend changed from one year to another.
KEY WORDS: amphetamines; cathinones; music festival; portable chemical toilet; recreational drugs

The term new psychoactive substances (NPS), which confirm the presence of both classical drugs and NPS
according to the United Nations Office on Drugs and Crime, (5, 6). The latter two studies point to the utility of urine
denotes “substances of abuse, either in a pure form or a sample analysis to evaluate NPS consumption. Similar tests
preparation, that are not controlled by the 1961 Single have been performed on pooled urine samples taken at
Convention on Narcotic Drugs or the 1971 Convention on music festivals in Belgium and the UK (7), while an analysis
Psychotropic Substances, but which may pose a public in Poland, carried out on wastewater samples, also showed
health threat” (1). On the market they are referred to as a rise in popularity of NPS there (8).
“legal highs”, “bath salts”, or “research chemicals” and are Ultra Europe held in Split, Croatia each summer since
known to affect mental health, behaviour, and perception 2013 has been the largest electronic music festival in
(2). They are analogues or chemical derivatives of Europe. According to organizers and the media, it has the
controlled drugs designed to achieve similar effects to those audience of about 150,000 people from more than 150
of illegal ones but still have not been listed as such (2, 3). countries every year and takes pride in being “…one of the
Their use is most common among young recreational users key weeks in the continent’s dance music calendar”,
and is constantly increasing (3, 4). Presently, many countries
according to its official website. As in other countries, this
are experimenting with a number of control measures to
festival has been connected with increasing availability,
address this issue, which is why any information on the
possession, and distribution of illegal drugs. As we did not
trends of their use is welcome.
have any official data about NPS use at the festival, we
According to online forums, people consume NPS at
asked the Croatian Ministry of Internal Affairs for more
electronic music festivals to enhance their musical
experience. There are reports of an increasing number of detail. They reported back that the police seized 1100 pieces
acute intoxications at music festivals in the Netherlands (4) of ecstasy, 73 pieces and 92.5 g of MDMA, 13 pieces and
and nightclubs, bars, and disco clubs in central London, 79.8 g of amphetamine, 253.4 g of marijuana, 12.5 g of
hashish, 34.1 g of cocaine, and 1.7 g of ketamine in 2016
alone. In 2017, the police seized 1097 pieces of ecstasy,
Corresponding author: Davorka Sutlović, University of Split,
Department of Health Studies, Ruđera Boškovića 35, 21000 Split, Croatia, 39.27 g of MDMA, 105.93 g of amphetamine, 342.19 g of
E-mail: [email protected] marijuana, 0.85 g of hashish, 13.31 g of cocaine, three pieces
Sutlović D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204 199
of LSD, and 0.72 g of heroin. The report from 2018 referred stadium (Figure 1A, location 2). Each had a 265 L capacity
only to seizure of a large amount of marijuana, 49 kg. and no rinse system to dilute urine. The assumption was
Considering that NPS consumption seems to increase that the toilets would mostly be used by the festival audience
worldwide and that no NPS were reported by the police, but also by those who did not enter the stadium at all, such
we decided to identify the trends at the Split Ultra, hoping as youth gathering around the stadium to listen to music.
to get more accurate information about what was actually The musical part of the festival was scheduled to take place
being consumed. from 5 pm to 5 am every day, and the toilets were emptied
and cleansed at 7 every morning. However, the first day
was cancelled over stormy weather, which is why only the
MATERIALS AND METHODS toilets outside the stadium were available for use, and we
collected one sample per toilet only once that day. On the
Sample collection second day, the organiser decided to start the programme
earlier (1 pm) to compensate for the lost time. We took two
Until 2019, the main event would take place at the city’s samples from both locations, one at the start and the other
football stadium (Figure 1A, upper left corner), last three at the end of the event. On the last day, samples were taken
days, and then the festival would move on to the nearby after the festival had ended, which makes eight collected
Dalmatian islands for another couple of days. samples in total, four from either toilet (Table 1).
Our first urine sampling in 2016 (16–18 July) was In 2017, sampling from four toilets with added
arranged with the organiser to take place inside the stadium, chemicals placed at three locations by the stadium (Figure
but one chemical toilet was (mis)placed on the main road 1B) was scheduled to take place once a day at 2 am (Table
connecting the city centre with the stadium (Figure 1A, 1), but on the second and the third day one of the two toilets
location 1) and had no chemicals, while the other, which at location 3 was out of commission, which is why we
contained added chemical agents, was placed close to the collected only ten urine samples.

Figure 1 Location of the sampled chemical toilets during the Split Ultra music festival in 2016 (A), 2017 (B), and 2018 (C)
200 Sutlović D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204

In 2018, the organiser let us enter the stadium. Twelve phenyl-95 % methyl polysiloxane, length 30 m, diameter
samples were taken from toilets with added chemicals at 0.25  mm, film thickness 0.25  μm). The initial column
four locations: three from three toilets placed inside the temperature of 90 °C was held for 3 min, then increased to
stadium, five from five toilets located next to the stadium, 270  °C at 15°C/min and held there for 5  min, and then
and four samples from three toilets a bit further away from increased to 320 °C at 15 °C/min and held there for 27 min.
the stadium (Figure 1 C). All but the inside-the-stadium The total run time was 50.33 min. Ultra-pure grade helium
samples were taken once at 4 am (Table 1). At location 3 was used as a carrier gas at the flow rate of about 1.5 mL/
one sample was taken on the first day of the festival and min.
two samples on the second day, while only one sample was The obtained data were compared with the Wiley mass
taken at location 4 (on the second day), which was the spectra library of designer drugs (DD2015), the SWGDRUG
farthest away from the stadium. free database, and our own in-house library containing about
All urine samples were taken with syringes directly 1000 compounds and metabolites (11, 12). Each sample
from the toilets and transferred to 100 mL containers (Figure was analysed twice.
1A – lower left corner), delivered to the laboratory, and
stored at -20  °C within 6  h. Overall, 30 samples were
analysed (Table 1). RESULTS
Methods Table 2 shows the findings for 18 samples collected in
The analysis was done according to the laboratory 2016 and 2017. Nineteen substances were identified as NPS
protocol and the protocols described by Vaiano et al. (9, and 25 as classic substances. Most (11 classic and 7 NPS)
10). Solid-phase extraction was performed using the were found in sample 1 from 2016, which was taken from
Supelclean Envi Florisil and Supelclean LC-18 for reversed- the toilet with added chemicals on the first night when the
phase extraction (Supelco, Sigma Aldrich, Taufkirchen, festival was not held. Between 4 and 18 substances were
Germany). For liquid-liquid extraction we used TOXI Tubes found in other urine samples. In two samples from 2016,
A (Varian, Palo Alto, CA, USA). Drugs were qualitatively taken from the toilet with added chemicals, NPS were not
analysed with a Shimadzu GC/MS-QP2010 Ultra found (Figure 2). In 2017, 19 classic and 13 NPS were
(Shimadzu, Kyoto, Japan) gas chromatograph with mass identified, most of which in sample 1 (9 classic and 7 NPS)
spectrometer using the full-scan mode (m/z range 40–600). and 4 (7 classic and 8 NPS) (Figure 2). In addition, all 2016
The chromatographic column was InterCap 5MS/NP (5 % and in 2017 samples showed the presence of caffeine,

Table 1 Location and time of urine sampling at the Split Ultra festival by day
Day 2 Day 3
Day 1
2016 Saturday/Sunday Sunday/Monday
Friday/Saturday
(13 pm – 5 am) (17 pm – 5 am)
Collection time 1 am 2 pm 6 am 6 am
Location 1 sample 1a sample 2a sample 3a sample 4a
Location 2 sample 1 sample 2 sample 3 sample 4
Day 1 Day 2 Day 3
2017 Friday/Saturday Saturday/Sunday Sunday/Monday
(5 pm – 5 am) (5 pm – 5 am) (5 pm – 5 am)
Collection time 2 am 2 am 2 am
Location 1 sample 1 sample 5 sample 8
Location 2 sample 2 sample 6 sample 9
sample 3
Location 3 sample 7 sample 10
sample 4
Day 1 Day 2
2018
Friday/Saturday Saturday/Sunday
Collection time 4 am 4 am
Location 1 samples 1, 2, 3 -
Location 2 samples 4, 5 samples 7, 8, 9
Location 3 sample 6 samples 10, 11
Location 4 - sample 12
a
– toilet from location 1 without added chemicals
Sutlović D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204 201

Table 2 Compounds detected in 18 pooled urine samples at the Split Ultra music festival in 2016 and 2017 by festival days
2016 2017
Group Substances Substance class Day Day
1 2 3 1 2 3
Nicotine Stimulant + + + + + +
Cotinine Stimulant + + + + + +
Theobromine Stimulant + + + + + +
Caffeine Stimulant + + + + + +
Amphetamine Stimulant + +
MDEA (3,4-methylenedioxy-N-ethylamphetamine) Stimulant + +
HMMA (4-hydroxy-3-methoxy-methamphetamine) Stimulant +
MDMA (3,4-methylenedioxy-methamphetamine) Stimulant + + + + +
MDDMA (3,4-methylenedioxy-N,
Stimulant + +
N-dimethylamphetamine)
3,4-MDA (3,4-methylenedioxyamphetamine) Stimulant + + + +
pMMA (para-methoxyamphetamine) Stimulant +
Propylhexedrine Stimulant + + + +
Classic
(25) Cocaine Stimulant + +
Ecgonine methyl estere Stimulant + + +
Cocaethylene Stimulant +
Butabarbital Hypnotic + +
Pentobarbital Hypnotic + + +
D9-THC (delta-9-tetrahydrocannabinol) Hallucinogen + +
Methadone Opiate + + +
Oxymorphone Opioid analgesic +
Tramadol Opioid analgesic + +
Phenacetine Analgesic +
Benzocaine Local anesthetic +
Paracetamol Antipyretic + + + + + +
Nonsteroidal anti-
Ibuprofen + + + + + +
inflammatory drug
Bk-MPA (β-keto-methiopropamine) Cathinone + +
3,4-dimethylmethcathinone Cathinone + + + +
3,4-methylenedioxyphenyl-2-propanone Cathinone +
Methedrone Cathinone +
Mephedrone Cathinone + +
Methylone Cathinone +
Buphedrone Cathinone +
Phenetylamine Phenethylamine + +
3-fluoromethamphetamine Phenethylamine + +
NPS 4-fluoromethamphetamine Phenethylamine + + +
(19) Bk-MBDB (β-keto-N-
methylbenzodioxolylbutanamine) Phenethylamine + +
metabolite 3-OH-4-MeO
N-ethyl-N-hexylamphetamine Phenethylamine + + +
N-hexyl, N-methylbezylamine Phenethylamine + +
Ketamine Anesthetic +
1-benzylpiperazine Piperazine + +
5 MeO DMT (5-methoxy-N,N-dimethyltryptamine) Tryptamine + +
Psilocin Tryptamine alkaloid +
N-ethyl-1,2-diphenylethylamine Hallucinogen +
Salvinorin A Hallucinogen +
202 Sutlović D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204

Figure 2 Number of classic and NPS drugs identified in urine samples during the Split Ultra music festival from 2016 to 2018 by
sampling site (chemical toilet). 1a to 4a – samples taken from the toilet without added chemicals. NPS – new psychoactive substances

theobromine, paracetamol, ibuprofen, nicotine, and its samples showed the presence of caffeine, theobromine,
metabolite cotinine (Table 2). nicotine, and its metabolite cotinine. Cocaine was identified
In the 12 samples collected in 2018, we identified 16 in six and MDMA in three urine samples. Ten of the 12
substances, 14 of which were classic and 2 NPS (from the samples had ibuprofen (Table 3). The largest signal area
cathinone and phenethylamine groups) (Table 3), most of
(dominating over all other substances) was for MDMA in
which in sample 1 (10 and 2, respectively) taken from one
sample 12 (taken on the second day of the festival from the
of the toilets placed inside the stadium on the first day. In
other urine samples 5 to 12 substances and metabolites were farthest toilet) and for cocaine in sample 7 (from the toilet
found. Seven samples had no NPS (Table 3 and Figure 2). located in the stadium ring). A higher cocaine signal was
Most of the classic substances and metabolites were from also found in all samples collected from the stadium toilet.
the stimulants group. As in the previous two years, all In addition we identified methcathinone for the first time.

Table 3 Compounds detected in 12 pooled urine samples at the Split Ultra music festival in 2018 by festival days
Day
Substance 1 2
Group Substances
class Sample
1 2 3 4 5 6 7 8 9 10 11 12
Nicotine Stimulant + + + + + + + + + + + +
Cotinine Stimulant + + + + + + + + + + + +
Theobromine Stimulant + + + + + + + + + + + +
Caffeine Stimulant + + + + + + + + + + + +
HMMA (4-hydroxy-3-methoxy-
Stimulant + + +
methamphetamine)
MDMA (3,4-methylenedioxy-
Stimulant + + +
methamphetamine)
MDDMA (3,4-Methylenedioxy-
Stimulant +
Classic N,N-dimethylamphetamine)
(14) 3,4-MDA
Stimulant +
(3,4-methylenedioxyamphetamine)
Cocaine Stimulant + + + + + +
Ecgonine methyl ester Stimulant + + + + +
Cocaethylene Stimulant +
Local
Mepivacaine + +
anaesthetic
Paracetamol Antipyretic +
Nonsteroidal
Ibuprofen anti-inflammatory + + + + + + + + + +
drug
NPS Methcathinone Cathinone + + + + +
(2) 4-fluorometh-amphetamine Phenethy1amine +
Sutlović D, et al. New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia
Arh Hig Rada Toksikol 2021;72:198-204 203

Figure 3 Number of classic and NPS drugs identified in urine samples during the Split Ultra music festival from 2016 to 2018 by
festival day. NPS – new psychoactive substances

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Nove psihoaktivne tvari u uzorcima urina prikupljenima tijekom Ultra Europe festivala u Splitu

Analiza uzoraka urina prikupljenih iz prijenosnih kemijskih zahoda korisna je u otkrivanju droga za rekreaciju koje su
se koristile tijekom Ultra Europe glazbenoga festivala. Zbog prisutnosti više od 150 000 ljudi iz više od 150 svjetskih
zemalja, moguće je dobiti više informacija o svjetskom trendu NPS-a. Analiza uzoraka urina koji su se prikupljali od
2016. do 2018. tijekom glazbenoga festivala Ultra Europe u Splitu provedena je radi otkrivanja prisutnosti novih
psihoaktivnih tvari (NPS). Analizirano je trideset uzoraka urina prikupljenih iz prijenosnih kemijskih zahoda smještenih
na mjestu festivala ili blizu njega. U 2016. godini prikupljeno je osam uzoraka urina iz dvaju zahoda koji su bili smješteni
na dvama različitim mjestima, i to jedan s dodanim kemikalijama, a drugi bez njih. U 2017. godini prikupljeno je deset
uzoraka urina iz triju zahoda na trima mjestima, svi iz zahoda s dodanim kemikalijama. U 2018. godini prikupljeno je
dvanaest uzoraka urina iz devet prijenosnih kemijskih zahoda na četirima mjestima, svi s dodatkom kemikalija. Uzorci
su kvalitativno analizirani tehnikom plinske kromatografije-spektrometrije masa (GC/MS) korištenjem načina ukupnog
skeniranja. Podatci su uspoređeni s bazom spektara masa Wiley (DD2015), kao i s vlastitom bibliotekom koja sadržava
oko 1000 spojeva i metabolita. U analiziranim uzorcima pronađeno je 46 različitih tvari i metabolita, od kojih 26 klasičnih
tvari/metabolita, uglavnom iz skupine stimulansa, te 20 tvari iz skupine novih psihoaktivnih tvari (NPS). U skupini NPS-a
najviše ih je bilo iz skupina fenetilamina i katinona. U svakoj promatranoj godini, prvoga festivalskog dana otkriveno je
više tvari nego u ostalim danima. Suprotno 2016. i 2017. godini, u 2018. godini broj otkrivenih tvari bio je znatno manji.
Rezultati našeg istraživanja pokazali su stabilnost konzumacije klasičnih droga, ali su se trendovi NPS-a mijenjali.

KLJUČNE RIJEČI: amfetamini; droga za rekreaciju; glazbeni festival; katinoni; prijenosni kemijski zahod