New psychoactive and classic substances in pooled urine samples collected at the Ultra Europe festival in Split, Croatia

Until 2019, the main event would take place at the city’s football stadium (Figure 1A, upper left corner), last three days, and then the festival would move on to the nearby Dalmatian islands for another couple of days.

Figure 1

Our first urine sampling in 2016 (16–18 July) was arranged with the organiser to take place inside the stadium, but one chemical toilet was (mis)placed on the main road connecting the city centre with the stadium (Figure 1A, location 1) and had no chemicals, while the other, which contained added chemical agents, was placed close to the stadium (Figure 1A, location 2). Each had a 265 L capacity and no rinse system to dilute urine. The assumption was that the toilets would mostly be used by the festival audience but also by those who did not enter the stadium at all, such as youth gathering around the stadium to listen to music. The musical part of the festival was scheduled to take place 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 toilets outside the stadium were available for use, and we collected one sample per toilet only once that day. On the second day, the organiser decided to start the programme earlier (1 pm) to compensate for the lost time. We took two samples from both locations, one at the start and the other at the end of the event. On the last day, samples were taken after the festival had ended, which makes eight collected samples in total, four from either toilet (Table 1).

In 2017, sampling from four toilets with added chemicals placed at three locations by the stadium (Figure 1B) was scheduled to take place once a day at 2 am (Table 1), but on the second and the third day one of the two toilets at location 3 was out of commission, which is why we collected only ten urine samples.

In 2018, the organiser let us enter the stadium. Twelve samples were taken from toilets with added chemicals at four locations: three from three toilets placed inside the stadium, five from five toilets located next to the stadium, and four samples from three toilets a bit further away from the stadium (Figure 1 C). All but the inside-the-stadium samples were taken once at 4 am (Table 1). At location 3 one sample was taken on the first day of the festival and two samples on the second day, while only one sample was taken at location 4 (on the second day), which was the farthest away from the stadium.

All urine samples were taken with syringes directly from the toilets and transferred to 100 mL containers (Figure 1A – lower left corner), delivered to the laboratory, and stored at -20 °C within 6 h. Overall, 30 samples were analysed (Table 1).

The analysis was done according to the laboratory protocol and the protocols described by Vaiano et al. (9, 10). Solid-phase extraction was performed using the Supelclean Envi Florisil and Supelclean LC-18 for reversed-phase extraction (Supelco, Sigma Aldrich, Taufkirchen, Germany). For liquid-liquid extraction we used TOXI Tubes A (Varian, Palo Alto, CA, USA). Drugs were qualitatively analysed with a Shimadzu GC/MS-QP2010 Ultra (Shimadzu, Kyoto, Japan) gas chromatograph with mass spectrometer using the full-scan mode (m/z range 40–600). The chromatographic column was InterCap 5MS/NP (5 % phenyl-95 % methyl polysiloxane, length 30 m, diameter 0.25 mm, film thickness 0.25 μm). The initial column temperature of 90 °C was held for 3 min, then increased to 270 °C at 15°C/min and held there for 5 min, and then increased to 320 °C at 15 °C/min and held there for 27 min. The total run time was 50.33 min. Ultra-pure grade helium was used as a carrier gas at the flow rate of about 1.5 mL/ min.

The obtained data were compared with the Wiley mass spectra library of designer drugs (DD2015), the SWGDRUG free database, and our own in-house library containing about 1000 compounds and metabolites (11, 12). Each sample was analysed twice.