Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 76 No. 1 pp. 137ñ145, 2019 ISSN 0001-6837 DOI: 10.32383/appdr/94615 Polish Pharmaceutical Society COCAINE CONSUMPTION IN THE CITY OF MURCIA (SOUTHEAST OF SPAIN) ESTIMATED BY WASTEWATER ANALYSIS: APPLYING AN ACCURATE AND VALID TOOL TO OBTAIN OBJECTIVE DATA FOR DRUG ABUSE LUCÕA FERN¡NDEZ-L”PEZ2, JAVIER NAVARRO-ZARAGOZA1*, FRANCISCO CARAVACA3, MARÕA FALC”N2 and AURELIO LUNA2 1Department of Pharmacology, University of Murcia, Spain 2Health and Social Sciences Department, University of Murcia, Spain 3Department of Social Work and Social Services, University Pablo de Olavide, Spain Abstract: Cocaine consumption is one of the most relevant public health issues in America and Europe. There are different methods to estimate cocaine consumption, with different levels of accuracy. Although surveys are one of the most employed methods, it usually underestimates cocaine consumption. Recent studies have vali- dated the use of wastewater analysis as the method that would provide more accurate results, as it is based on the analysis of residues of illicit drugs and their metabolites that are excreted by human flow through waste- water treatment plants. This study aims to estimate cocaine consumption in the southeast of Spain by waste- water analysis. Samples were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC- MS/MS). According to this analysis, cocaine consumption has been estimated at 410.6 milligrams per day and per 1000 inhabitants, a higher result than that obtained at other European cities of similar size. Highest con- sumption occurs during the weekend, which is consistent with cocaine being categorized as a recreational drug. The results of this analysis show that cocaine use is increasing public health and economic problem. Finally, wastewater analysis offers different options to estimate its use in this region of Spain. Keywords: wastewater, cocaine, mass spectrometry, drug consumption, benzoylecgonine, cocaethylene Abbreviations (in alphabetical order):BOD: Biochemical Oxygen Demand, DPNSD: Government Delegation for the National Plan on Drug, EMCDDA: European Monitoring Centre for Drugs and Drug Addiction, ED: Emergency Department, EDADES: Household Survey on Alcohol and Drugs in Spain, FWHM: Full Width Half Maximum, GC/MS: Gas Chromatography-Mass Spectrometry, LC/MS: Liquid Chromatography-Mass Spectrometry, LOQ: Limit of Quantification, Q: Flow rate, RSD: Relative Standard Deviation, S/N: Signal-to- noise, SRM: Selective Reaction Monitoring, UNODC: United Nations Office on Drugs and Crime, UPLC- qTOF-MS/MS: Ultra-performance liquid chromatography quadrupole time of flight mass spectrometry The illicit drugs consumption is increasing consumed cocaine so in the present year. According worldwide, mainly in America and Europe, which to previous studies, United Kingdom is the country leads to significant health and social behavior issues with the highest number of cocaine-related emer- that affect 27 million users (1-2). Consumption of gency episodes, followed by Spain (7). Both coun- heroin, cocaine and other drugs causes around 0.2 tries share a high consumption rate among young million deaths per year (183.000 drug-related deaths adults (aged 15ñ34) (5, 8, 9). were reported in 2012 (3)). Cocaine consumption in Spain is a critical Although cocaine, one of the most potent and issue. According to different surveys (10), the num- addictive illicit drugs, has been consumed for over a ber of adult population that have consumed cocaine century, its consumption seems to be increasing (4- at least once in their life has constantly increased 6). Cocaine is now the most commonly consumed since 1995 (11). Cocaine is currently the drug that illicit stimulant drug in Europe (4, 6). More than generates more visits (45.1%) to the Emergency 14.5 million Europeans have consumed cocaine at Department (ED) (5). Moreover, although mortality least once, and it has been estimated that 3.5 have after psychoactive substances use and abuse has * Corresponding author: e-mail: [email protected] 137 138 LUCÕA FERN¡NDEZ-L”PEZ et al. decreased in Spain, the proportion of deaths where separation and determination using liquid (LC) or cocaine or its metabolites are involved continues gas chromatography (GC), coupled with mass spec- increasing (11). trometry (LC-MS or GC-MS). Gas chromatography- For many years, surveys have been the only mass spectrometry (GC-MS) and liquid chromato- affordable analytic method to measure drug con- graphy-mass spectrometry (LC-MS) are the tech- sumption. This system has different inconveniences, niques of choice for quantitative analysis. To have a including underestimation of consumption (12). The tandem mass offers better results than single-quadru- treatment of the sample let to obtain acceptable pole instruments, in terms of sensitivity and selecti- results for some drugs of abuse, but in contrast, vity. In contrast, LCñMS/MS has been the technique these surveys do not permit to obtain trustful results most referenced for polar and semi-polar organic for illicit drugs with less consumption, such as compounds analysis until currently (24). cocaine. One reason is statistical, another important The selection of appropriate DTRs is also reason is the fear of some minors to answer this kind essential to obtain reliable estimates of drug con- of surveys, although are anonymous (12). Another sumption. For this reason, the new good practices analytic method, wastewater-based drug epidemiol- protocol for WBE studies specifies that the sub- ogy (WBDE), has been increasingly employed as an stances should fulfill the following requirements: 1) alternative approach to socio-epidemiological sur- being excreted in consistent amounts in urine; 2) veys. This method consists analyzing drugs and being detectable in urban wastewater; 3) being sta- their metabolites or drug biomarkers in wastewater ble in wastewater and 4) have human excretion as samples from a target community (13). their unique source (25). Different methods have been developed in Since the proposal of this method, several stud- order to diagnose substances consumption by ies have been carried out in different countries (26- WBDE. Solid phase and headspace solid-phase 27). However, in Spain it has only been developed methods allow extracting residues and metabolites in cities of other regions (Santiago, Barcelona, of illicit drugs from hair and biological fluids like CastellÛn, Valencia) and one town from Murcia urine, sweat, blood or saliva. However, previous (Molina de Segura) but it has not become the pre- analyses of biological fluids have allowed to meas- ferred method to measure drug consumption, uring drug consumption by individuals, not by a although as said above, cocaine is a predominant community (1, 14-15). Residues of illicit drugs that drug of abuse very difficult to measure. Surveys are excreted by humans flow into and through suggest that the southeast of Spain is one of the wastewater treatment plants. These residues can regions where drugs consumption is higher. For produce a serious potential effect on humans, being these reasons, the aim of this study is to determine persistent (such as cocaine and its metabolite ben- the concentrations of cocaine and its metabolites zoylecgonine) in the environment (16). The metabo- in samples of wastewater from a water treatment lites maintain the same action of the original com- plant in the city of Murcia, in the Region of Mur- pound, thus generating toxicological effects on non- cia, Spain, in order to back-calculate cocaine con- target microorganisms (17). sumption. The proposed analysis of illicit drugs in waste- water is a reliable source of data to estimate sub- EXPERIMENTAL stances consumption in the population (18-19). This idea was proposed by Daughton in 2001 and applied Materials and chemicals by him and Zuccato (4, 20-22). Daughton hypothe- Cocaethylene-d3 and benzoylecgonine-d3 were sized that in a similar manner to medicinal com- purchased from Sigma-Aldrich (Steinheim, Germa- pounds, residues of illicit drugs may also be detected ny) as 0.1 mg/mL solutions in acetonitrile or in the aquatic environment as a result of human use. methanol. Diluted standard solutions were prepared As a consequence, it is possible to determine the in methanol HPLC (Sigma-Aldrich) until 10 µg/mL amount of drug used by a population by measuring as required. Ultrapure water was obtained from a the concentration of drugs residues in wastewater (1, Milli-Q Gradient A-10 system (Millipore, Bedford, 23). There are different procedures used to perform Massachusetts, USA). Ammonium acetate, acetic the determination of organic compounds in aquatic acid, formic acid and tetrabutylammonium bromide environmental matrices, i.e. solid-phase extraction (TBA, 99% purity) were purchased also at Sigma- (SPE), solid-phase microextraction (SPME), stir bar Aldrich. Cartridges (6-mL disposable Oasis MCX) sorptive extraction (SBSE) or liquid-liquid extrac- for solid-phase extraction (SPE) were acquired from tion (LLE). Besides, they are remarkable methods of Waters (Milford, Massachusetts, USA). Cocaine consumption in the city of Murcia (southeast of Spain) estimated by... 139 Wastewater sampling ESI interface operating in positive ion mode. The Raw influent wastewater was collected at the UPLC separation was performed using an Acquity inlet (before the primary clarifier) of the main UPLC BEH C18 1.7 µm particle size analytical col- wastewater treatment plant located in the province umn 50 mm ◊ 2.1 mm (Waters) at a flow rate of 0.3 of Murcia. The flow rate of this plant is around mL/min. Ther acquisition was performed in 108.000 m3/day and it covers 445.000 inhabitants. Selected Reaction Monitoring (SRM) mode. The We decided to collect wastewater samples from the mobile phases used were A= H2O with 0.01% central area of the city, which consists of a popula- HCOOH and B = MeOH. The percentage of organ- tion of approximately 170.000 inhabitants and it ic modifier (B) was changed linearly as follows: 0 comprises a 70% of total flow rate according to data min, 10%; 14 min, 90%; 7 min, 90%; 9.0 min, 90%; from the wastewater treatment plant (Table 1).