Chemicals and Materials

The reference standards (99% purity) of cocaine and BE were from MacFarlan-Smith Ltd (Edinburgh, UK), and LGC Promochem s.r.l. (Milan, Italy), respectively. The internal standard (IS), salbutamol-D 3 (99.1% D) was from CDN Isotopes (Quebec, Canada). Standards were dissolved in methanol at 1 mg/ml and subsequently diluted to 10 ng/μl. Purity of the solutions was checked before each analytical run by HPLC-MS-MS. All solutions were stored at -20°C in the dark. The cartridge used for solid phase extraction was a 3-ml disposable OASIS MCX (60 mg, Waters Corp., Milford, MA).

Sample collection

Composite water samples (pools of five 500-ml samples collected every 30 min) were collected on four different days from the River Po at Mezzano, Pavia (Figure 1). At this sampling site (average flow rate for the period, 743 m3sec-1) the basin's population equivalent is 5.4 × 106. The flow rates were kindly provided by the Ufficio Mareografico ed Idrografico del Po. Water samples were also taken from influent WW at four treatment plants (WWTPs) serving medium-size Italian cities (Cagliari, Latina, Cuneo, and Varese; location shown in Figure 1). Flow rates of the WWTPs were 1.0, 0.36, 0.22 and 0.46 m3sec-1, and population equivalents 270, 140, 45 and 110 × 103, respectively. For each plant, a 24-h, two-liter composite sample was obtained by pooling water collected every 20 min by an automatic sampling device. Water samples were stored at 4°C, and processed within 3 days, to minimize possible degradation.

Solid phase extraction

Cocaine and BE were measured by adapting our method for pharmaceuticals in river water [10]. Water samples (500 ml) were filtered on a glass micro-fiber filter and spiked with 10 ng of the IS. The pH was then adjusted to 2.0 with 37% HCl. Oasis MCX cartridges were conditioned before use by washing with 6 ml methanol, 3 ml MilliQ water and 3 ml water acidified to pH 2. Samples were then passed through the cartridges under vacuum, at a flow rate of 20 ml/min. Cartridges were vacuum-dried for 5 min and eluted with 2 ml methanol, and 2 ml 2% ammonia solution in methanol. The eluates were pooled and dried under an air stream.

Liquid chromatographic separation

Before analysis, samples were re-dissolved in 100 μL acetic acid 0.01% in water (pH 3.5), then centrifuged and transferred into glass vials. Aliquots of 10 μl were injected using an auto sampler. The HPLC system consisted of two Series 200 pumps and a Series 200 auto sampler (Perkin-Elmer, Norwalk, CT). A Luna C8 column 50 mm × 2 mm i.d., 3 μm particle size (Phenomenex, Torrance, CA, USA) was used for the chromatographic separation. The elution started with 100% of eluent A (formic acid 0.1% in water, pH 2) followed by a 10-min linear gradient to 100% of eluent B (acetonitrile), 2-min isocratic elution and a 2-min linear gradient to 100% of eluent A, which was maintained for 6 minutes to equilibrate the column. During the analysis the flow rate was 200 μl/min and the column was kept at room temperature.

Mass spectrometry (MS-MS)

An API 3000 triple quadrupole mass spectrometer (Applied Biosystems – Sciex, Thornhill, Ontario, Canada) was used for quantitative determinations. Analyses were done in ESI positive mode, with a spray voltage of 5.4 kV, orifice skimmer voltages that varied from 30 to 54 V and ring electrode voltages from 180 to 280 V. Data acquisition was performed with multiple reaction monitoring (MRM) of selected fragmentation products of the protonated pseudo-molecular ions (m/z 290 -> 105 and 290 -> 168 for BE, 304 -> 105 and 304 -> 182 for cocaine, 243 -> 151 and 243 -> 169 for the IS). Five-point calibration curves were generated for each compound by injecting 10 μl of 0.01% acetic acid solutions containing known amounts (0–1 ng/μl) of BE and cocaine, and the IS (0.1 ng/μl). Calibration curves run with each batch of samples showed excellent linearity (r2 > 0.998). Instrumental blanks (standard solution with IS only), showed no traces of interfering compounds. Procedural blanks and recoveries were performed using mineral water. Blanks showed no detectable cocaine and BE. Recoveries were >90% for both compounds. The limits of detection were respectively 0.06 and 0.12 ng/liter for BE and cocaine (calculated as the concentration giving a signal-to-noise ratio of 3 in recovery tests). The identity of cocaine and BE, and the absence of interfering compounds, were further confirmed by MS/MS qualitative analyses performed on a LCQ DecaXP Plus (Thermo Electron, Waltham, MA) ion trap mass spectrometer. In this case, chromatographic conditions were the same previously described, while the mass analysis was made by acquiring ESI-MS and MS/MS spectra, corresponding to the pseudo-molecular ions of BE and COC. The relative amounts of the fragment ions of the substances were in accordance (+/-20%) with those of reference standards.

Calculations and assumptions

Given that about half a cocaine dose is excreted in urine as BE, and only a small fraction as the unchanged drug, we used the concentrations of BE in WW or SW to estimate the amounts of cocaine consumed locally. Concentrations of cocaine were useful to verify that the BE/cocaine ratio was stable and in the expected range, thus giving confidence about their source being human consumption. If an unlikely accidental or intentional disposal of a significant amount of cocaine were to occur at any of these sites, the normal BE/cocaine ratio (see Results) would be transiently and markedly altered in favor of cocaine. BE loads (g/day) at each sampling site – calculated from the BE concentration in water (ng/liter) and water flow rate (m3/sec) – were used to estimate the loads of parent cocaine, multiplying by a factor of 2.33. This takes into account the BE/cocaine molar mass ratio (0.954) and the average molar fraction (45%) of a cocaine dose that is excreted as BE, according to different studies [14, 15]. Cocaine loads were then related to the local population equivalents (i.e. the number of people served by a WWTP or living in the river's catchment basin), using data from the Italian 14th General Population and Housing Census (2001) [16]. The estimated consumption (g per day per 1000 people) at each site was referred both to the general population and to young adults (15–34 y), since the latter group reportedly includes almost all consumers [2]. The data were also expressed as the number of doses per day per 1000 people, assuming 100 mg as an average dose [1] (the equivalent of four 25-mg "lines" of cocaine).