Pilot Study of the Estimation of Amphetamines Consumption in the Polish City of Poznan

Amphetamines are a term that includes both amphetamine and methamphetamine, both of which stimulate the central nervous system. Ecstasy is a group of synthetic substances chemically related to amphetamines but with some differences in their effects. The most common substance belonging to the ecstasy group is 3,4-methylenedioxymethamphetamine (MDMA), but other analogues are also sometimes found in ecstasy tablets (MDA, MDEA). Illicit amphetamine use is associated with a broader set of negative consequences, such as shortterm negative effects (restlessness, tremor, anxiety, dizziness), a ‘crash’ or coming-down after-effect (depression, sleeping difficulties, suicidal behavior), and also psychological and psychiatric effects of long-term use (psychosis, suicidal behavior, anxiety and violent behavior) [1].


Introduction
Amphetamines are a term that includes both amphetamine and methamphetamine, both of which stimulate the central nervous system. Ecstasy is a group of synthetic substances chemically related to amphetamines but with some differences in their effects. The most common substance belonging to the ecstasy group is 3,4-methylenedioxymethamphetamine (MDMA), but other analogues are also sometimes found in ecstasy tablets (MDA, MDEA). Illicit amphetamine use is associated with a broader set of negative consequences, such as shortterm negative effects (restlessness, tremor, anxiety, dizziness), a 'crash' or coming-down after-effect (depression, sleeping difficulties, suicidal behavior), and also psychological and psychiatric effects of long-term use (psychosis, suicidal behavior, anxiety and violent behavior) [1].
The United Nations Office on Drugs and Crime (UNODC 2011) reported that in 2009-2010 4.5% of the world's population aged 15-64 used cannabis, 1.3% used amphetamines, 0.6% Ecstasy-group stimulants and cocaine 0.5% [2]. According to the European Monitoring Centre for Drugs and Drug Addiction Annual Report 2011, in many European countries amphetamines or ecstasy are the second most commonly used illicit substances after cannabis. Significant methamphetamine use is restricted to the Czech Republic and Slovakia (our neighbors) but recent reports indicate an increase in amphetamine use in some countries in northern Europe [3]. Based on surveys conducted in Poland in 2008, cannabis and amphetamines are the most commonly used illicit drugs [4].
The official figures on the prevalence of drug abuse are currently derived from population surveys integrated with crime statistics, medical records, drug production and seizure rates. Such estimation is subjective and very expensive. In 2005, the Zuccato group estimated the level of consumption of cocaine based on the determination of cocaine and their metabolite in surface water and wastewater [5]. This methodology appeared to be much more relevant and objective. Nevertheless, the procedure needs to be standardized to make comparison of results in many countries more reliable, but the methodology gives an objective insight into the level and profile of illicit drugs consumed by local community in real time. Recently, many reports concerning the estimation of illicit drug abuse using 'sewage epidemiology' have been published in many European countries, such as Belgium [6][7][8], United Kingdom [9], Italy -Florence [10], Spain [11][12][13], Croatia [14], Switzerland [15] and also in Canada [16] and the United States of America [17][18][19].
According to surveys conducted in Poland amphetamine is a commonly used illicit drug, and this is why the group of amphetamine-like stimulants was chosen for analysis (amphetamine, methamphetamine, MDA, MDMA, MDEA) [4]. The aims of this study were to implement 'sewage epidemiology' to estimate the level of amphetamines consumed in Poland's fifth largest city, Poznan, describe the profile of amphetamines used and monitor the long-term trends in amphetamines use.

Wastewater treatment plant in poznan
The Wastewater Treatment Plant is located in north-east of Poznan, in the township of Kozieglowy. The facilities are located on the right bank of the Warta river and occupies an area of approximately 60 hectares. The central wastewater treatment plant has undergone several modifications to meet present demands from the year 1995 to 2009. The new and highly efficient biological section was constructed, which incorporates a system enabling the integrated biological removal of carbon, nitrogen and phosphorus. The capacity of the plant is one of the largest of such facilities of Poland. During the time when samples were collected the central plant served almost the whole city with the suburbs, about 687,000 people.

Sample collection and processing
Two wastewater samples (10 L each) were collected twice a week, on Monday and on Wednesday, from June 2009 to December 2010. All samples were collected at the same point before any chemical and physical treatment with the exception of sedimentation and the mean flow rate was 130,000 m 3 /day. The analyses of the samples were performed the same day just after collection.

Sample treatment and analysis
Samples were filtered on a glass microfiber filter GF/A 1.6 µm (Whatman, Kent, U.K.) prior to the extraction and were spiked with 15 ng of each internal standards and the pH was adjusted to 7.0 ± 0.4 with phosphate buffer (pH=7.0). Solid-phase extraction of analyzed substances was performed using a Bakerbond Narc-2 mixed mode cartridges, which were conditioned with methanol (2 mL) followed by deionized water (2 mL) followed by phosphate buffer (2 mL, 0.1 M, pH 7.0). Next the sample was passed through the cartridges under vacuum at a flow rate of 10 mL/min. When the sample eluted under the gravity, the column was washed with deionized water (2 mL) followed by hydrochloric acid (0.1 M, 0.5 mL) followed by methanol (0.5 mL). Vacuum was applied and the cartridges were dried for 20-30 min. The analytes were eluted into a vial with a mixture of chloroform: isopropanol: ammonium hydroxide (80:30:3, 2 mL). The eluates of two samples (each 10 L) were pooled and dried under a nitrogen stream.

Liquid chromatography-tandem mass spectrometry
Pooled and dried samples were redisolved in 200 µL of mobile phase, centrifuged and transferred into glass vials for instrumental analysis. 20 µL of the solution were injected in the LC-MS spectrometer (Agilent HPLC 1200 series, 6410B Triple Quad LC/MS System). Chromatographic separation was performed using capillary column (Agilent Zorbax XDBC18, 4.6×50 mm×1.8 µm) at a flow rate of 0.45 mL/min. The mobile phases were solutions: water with formic buffer (pH~3.2) and acetonitrile with the gradient from 10 to 70% of acetonitrile (6.5 min.). The capillary voltage was 4000 V and the temperature was 300°C, the auxiliary and collision gas was N2. The collision energy and tube lens were optimized for each analyte and standards separately. All selected analytes were analyzed in positive ionization mode (ESI+). Identification and quantification were performed using two characteristic transitions in multiple reaction monitoring (MRM) mode for the fragmentation products of the protonated or deprotonated pseudomolecular ions of each substance and each deutered analogue (Table 1). 8-point calibration curve was built at 4, 8, 12, 16, 20, 24, 28, 32 ng for amphetamine, methamphetamine, MDA and MDMA and the solutions were spiked with 30 ng of all internal standards. Validation was done according to Funk methodology, including testing homogeneity, linearity, homogeneity of variances (precision), outliers and securing the lower range limit [20,21]. The matrix effect was determined by analyzing 50 mL of wastewater samples spiked with internal standards. The recoveries for the whole process of sample preparation, filtration and extraction were set within the range 0.80-0.93. The detection limits (LOD) and quantification limits (LOQ) for the whole method were calculated by spiking wastewater samples with different amounts of the substances analogously like at the calibration curve. The results all of validation activities are shown in Table 2.

Back-calculation of community drug use
The first time back-calculation of illicit drug consumption was carried out was by Zuccato et al. [5,19,20]. Based on the determination of the major metabolite of cocaine (benzoylecgonine, BE) in wastewater and surface water, cocaine consumption in a few Italian cities was estimated. This approach has been developed for other illicit drugs. For each drug the substance, called drug target residues (DTRs), is specified. An ideal DTR is a major and exclusive excretion product, metabolite or unchanged parent drug, which is stable in wastewater. Amphetamines are excreted mainly as unchanged compounds, so the DTRs for them were parent drugs. The concentrations of these substances in wastewater samples were very low and therefore the dried residues of two untreated wastewater samples (each 10 L) after filtration and SPE extraction were pooled and joined together by redissolving in the mobile phase to perform HPLC-MS-MS analysis. The mean concentrations of DTR in ng/L of all samples collected in one month were multiplied by the monthly mean flow rate in the wastewater treatment plant (WWTP) to give the amount of DTR (grams) discharged per month. This value was then divided by the number of people served by the WWTP to estimate the grams of DTR excreted in wastewater per person per month and finally normalized to a value of grams per month per 1000 people. To estimate the consumption of the illicit drug a correction factor is applied, which takes into account the percentage of the parent drug excreted as the chosen DTR and the parent drug-to-DTR molar mass ratio. The correction factors for amphetamines were 3.3, 2.3 and 1.5 for amphetamine, methamphetamine and ecstasy respectively. As a result, the amount of illicit drugs consumed monthly by 1000 people was estimated.
It is also possible to estimate the number of doses consumed by local community (1000 people) per day or month by dividing the  consumption value by a single typical dose for each illicit drug. A typical oral dose for amphetamine and methamphetamine is 30 mg, but for MDMA (ecstasy) it is 100 mg. But it is recommended to focus on the loads of illicit drugs estimated for 1000 people per day or loads of DTR excreted per day per 1000 people and these results should be monitored for a period of time to give an objective insight into the level and profile of illicit drugs consumed by local community.

Statistical analysis
Graphical method and Shapiro Wilk test were used for testing the normal distribution of samples. To check whether the mean value of determinations in one month differs from mean in another the analysis of variance-one-way ANOVA-was performed (Matlab v. 7120635). The null hypothesis assumed the all monthly means of determinations are equal. It was interested not only if there are any differences among means but even more important which pairs of means differ significantly, that is why series of t-tests were performed. Because this procedure has a pitfall when you compare more than five variables (means) multiple comparison procedure was applied to compensate for multiple tests. It is easier to visualize the difference between monthly means using a graph which is presented in Figure 1. The procedure applied for the collection of samples and determination of illicit substances did not allow daily variations in the drugs consumed to be compared.

Results and Discussion
The results of determination of amphetamine, methamphetamine and ecstasy in wastewater samples are collected in Table 3. The normal distribution of variables was checked by graphical method and Shapiro-Wilks test (p>0.05). Based on the determination of DTR in wastewater samples population-standardized mean monthly loads of DTR were calculated. To check an increasing or decreasing tendency between months one way ANOVA and Multiple Comparison Test were applied. The results of ANOVA are presented in Table 4. Based on the F and p-values the null hypothesis was rejected. P-values are very below the cut-off level of 0.05% therefore it can be stated that     Processing of DTR extraction data allowed us to calculate the consumption rate (mg/month/1000 inhabitants) for the amphetamines group substances (in our case amphetamine, methamphetamine and ecstasy) as described in the section named "Materials and methods" (Figure 2). Because of the low limit of DTRs in wastewater, significantly more samples were subjected to analysis. Comparison of drug consumption with other reports [19,11,14] by other Western European scientists using a similar methodology shows significant differences in consumption amounts and the profiles of amphetamine-group substances ( Table 5). Generally speaking, consumption of amphetamines is one order of magnitude lower than in the countries compared. The mean drug consumption of amphetamine amounts to 0.33% (incase of London) to 2.9% (in case of Milan) of overall drug consumption. In case of ecstasy and methamphetamine, the percentage range is 5.30-13.25% (in case of Catalonia even 0.24%) and 5.1-9.67% respectively. Because drug consumption is standardized per day and per thousand people, these results seem to be comparable. An interesting fact is that cocaine was detected only once during the two-year study period. It is important to remember that these data were collected in different time periods (a few weeks monitoring was compared with a continuous two-year study) but in the authors' opinion, for general comparison purposes this approach is justified. The choice of city where our studies were carried out resulted from the author's place of residence and work, and also poll based data not indicating that Poznan is a city with special addiction problem. Therefore, it is possible that monitoring in other cities might give different results. The official Polish statistics do not offer any integrated data on the consumption of different types of illicit drugs in Poznan and other Polish cities. Official data are based on the number of people treated and hospitalized. Our calculations indicate that amphetamine consumption in Poznan (mean 0.26 mg/day/1000 inh.) is several times lower than in Western Europe (Table 5) and slight lower compared to London. Consumption of ecstasy is relatively high, but still makes up 13.25% at the most. An interesting fact is that the amphetamine to ecstasy consumption rate in case of Poznan is 0.55, while it is 2.7, 1.4, and 7.6 for Zagreb, Milan and Catalonia respectively. This indicates a different profile of consumption and a different 'liking for illicit drug' among Poznan's inhabitants. In London and Milan, the data show a higher level of methamphetamine consumption compared to Poznan. At this point it is important to take into consideration the sample collection and processing, which can be distinguished from other authors with regard to the amount of samples prepared and analyzed. Using a 20 L sample for analysis made this more complicated (because of the matrix effect) and more laborious, but it did make it possible to detect traces in wastewater. When interpreting the comparison of consumption in Poznan and other cites, it is important to note that this comparison concerns only those cities where the concentration of    amphetamine-type drugs was especially high. In other cites which were monitored, where the authors did not give figures for amphetamine consumption, it was under the detection or quantification limit using a 0.5 L sample. It is possible that in these cities the level of consumption is comparable with Poznan. It is therefore worth noting that the low level of consumption in Poznan compared with other cites may result from the selection of cities.
In our study an attempt was made not only to estimate the number of mg of consumed illicit drugs by local population but to indicate trends in consumption. To achieve this aim, the authors changed the way of collecting samples compared with others research (see Sample collection and processing). The data obtained allowed us to generate two-year profiles of consumption for amphetamine methamphetamine and ecstasy (Figure 2). The profile indicates a gentle decrease in the monthly consumption of amphetamine in Poznan from May 2010 to the end of the monitoring period. It is difficult to make a comparison with other works because there are no such long-term monitoring data and these data would need to be compared over the same period of time.

Conclusion
Wastewater analysis and monitoring of illicit drug content and calculation of consumption is a promising complementary tool for the assessment of trends in illicit drug abuse. Our research was the first of its kind carried out in Poland. The monitoring focused particularly on the amphetamine group. Consumption of amphetamine is lower than in other compared European countries, but consumption of ecstasy is relatively high. The profile of consumption of drug abuse substances is different in city of Poznan.