Environ. Sci. Technol. 2006, 40, 7321-7328
Simultaneous Determination of Psychoactive Drugs and Their Metabolites in Aqueous Matrices by Liquid Chromatography Mass Spectrometry† DANIELA HUMMEL, DIRK LO ¨ FFLER, GUIDO FINK, AND THOMAS A. TERNES* Federal Institute of Hydrology (BFG), D-56068 Koblenz, Am Mainzer Tor 1, Germany
A multi-residue method was developed that allows for the simultaneous determination of psychoactive compounds such as opioids, tranquilizers, antiepileptics (primidone, carbamazepine plus two metabolites), the cocaine metabolite benzoylecgonine, the antidepressant doxepin, as well as the calcium channel blocker verapamil in raw and treated wastewater, surface water, groundwater, and drinking water. After solid-phase extraction with Oasis HLB at neutral pH, the analytes were detected by LC electrospray tandem MS in the positive ion mode. With a few exceptions relative recoveries of the analytes exceeded 70%. The limits of quantification were in the low ng/L range. Matrix effects were compensated by using appropriate deuterated or 13C-15N-labeled surrogate standards. For raw and treated wastewater, concentration factors were lowered to reduce matrix effects. Most analytes (15 of 20) were found in raw and treated wastewater as well as in surface water, and hence, are presumably ubiquitously present in the environment. Antiepileptics, the opium alkaloids morphine and codeine, dihydrocodeine, the two tranquilizers oxazepam and temazepam, the opioid tramadol, doxepin, and verapamil were detected in STP discharges and German rivers at concentrations up to the µg/L range. In drinking water, only carbamazepine, its metabolite 10,11-dihydroxy10,11-dihydrocarbamazepine, and primidone were present at concentrations up to 0.020 µg/L.
1. Introduction Approximately 3000 different pharmaceutical ingredients are registered in the EU today, including psychoactive drugs, painkillers, antibiotics, antidiabetics, betablockers, contraceptives, lipid regulators, antidepressants, antineoplastics, tranquilizers, and anti-impotence drugs. For most addictive drugs, including opioids, prescription and sale are strongly regulated and controlled. Despite all regulations there is an extensive consumption of illicit drugs in most developed countries. As these compounds are frequently metabolized in the body, a combination of unchanged drug and metabolites is excreted. These pharmaceuticals are discharged from private households and from hospitals, and they eventually reach †
This article is part of the Emerging Contaminants Special Issue. * Corresponding author phone: +49 261 1306 5560; fax: +49 261 1306 5363; e-mail:
[email protected]. 10.1021/es061740w CCC: $33.50 Published on Web 11/29/2006
2006 American Chemical Society
municipal sewage treatment plants (STPs). However, most drugs are only partly removed during sewage treatment and are present in receiving waters. In some cases those compounds were even detected in groundwater and drinking water (1-6). Systematic studies with regard to their appearance and fate in STPs are already known for several medicinal classes such as antibiotics, estrogens, antiphlogistics, betablockers, antiepileptics, lipid regulators, and iodinated contrast media (3, 7-10). Miao and Metcalfe (4) investigated the occurrence of carbamazepine metabolites in Canadian STP effluents and found that the 10,11-dihydroxy-10,11dihydro-carbamazepine exhibited three times higher concentrations than carbamazepine itself. So far, there have been no published comprehensive studies on the environmental occurrence of opioids and tranquilizers, although individual compounds have already been detected. The tranquilizer diazepam has been found in 8 of 20 German STP effluents at concentrations up to 0.040 µg/L (5), while it was not detected in a Spanish STP (11). Codeine, hydrocodone and dihydrocodeine were present in STP effluents in an overall German monitoring study at concentrations up to 0.9 µg/L. Zuccato et al. (12) quantified cocaine and its metabolite benzoylecgonine in the river Po at concentrations of 0.0012 µg/L and 0.025 µg/L, respectively. To our knowledge, psychoactive drugs such as morphine, tramadol, methadone, temazepam, nordiazepam, doxepin, and the calcium channel blocker verapamil have not been investigated in environmental samples. Analytical methods have been developed for the determination of psychoactive drugs and metabolites in biological matrices such as urine, blood plasma, or hair. Using GC/ion trap MS, Pirnay et al. (13) detected 22 benzodiazepines in blood and urine, and Hadidi (14) detected tramadol in hair. An analytical method has been described by Dams et al. (15) for the determination of opioids in blood and urine with LC-diode array-fluorescence detection. Nevertheless, the most recent technique for these psychoactive compounds is LC-MS including LC tandem MS. Screening methods with LC-MS for psychoactive drugs and metabolites including tranquilizers, methadone, and morphine are reported by several authors (16, 17). LC APCI tandem MS has been used for the analysis of opioids and cocaine in hair (18) and urine (19). The electrospray interface was also frequently applied, for instance for the analysis of morphine and its glucuronides (20, 21) or for the detection of hydrocodone in blood plasma (22). Liang et al. (23) were even able to determine the R- and S-enantiomers of methadone simultaneously by LC tandem MS using a chiral LC column. The objective of this paper was to develop a multi-residue method for the simultaneous determination of various psychoactive drugs in the aqueous matrices with LC tandem MS. Opioids, antiepileptics, tranquilizers, the antidepressant doxepin, and cocaine metabolite benzoylecgonine were selected as analytes. Additionally, the calcium channel blocker verapamil was included due to the toxicological concerns, as it is known to inhibit the cellular export of xenobiotics at micromolar concentrations (24). The analytical method was applied to native samples such as wastewater, surface water, and drinking water in order to determine its strength and limitations as well as to produce first results about the environmental relevance of the selected drugs.
2. Experimental 2.1. Reference Compounds. The reference compounds (see Table 1; analytical grade >98% purity) were obtained from VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Substances, CAS Numbers, Chemical Structures, Application Quantity Prescribed in Germany 2004 () number of Prescribed Daily Doses × Defined Daily Dose) (32, 33), and log KOW, pKa (37)
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the following suppliers: codeine, dihydrocodeine (Th.Geyer, Renningen, Germany), bromazepam, diazepam, nordiazepam, oxazepam, temazepam, carbamazepine, doxepin, primidone, hydrocodone, oxycodone, methadone, verapamil (Sigma, Deisenhofen, Germany), morphine (Cambridge Isotopes Lab., Saarbru ¨ cken, Germany), benzoylecgonine (LGC Promochem, Wesel, Germany), tramadol (Fluka, Buchs, Switzerland), 10,11-dihydrocarbamazepine (Alltech, U.S.), and 10,11-dihydroxy-10,11-dihydrocarbamazepine (µ-Mol, Luckenwalde, Germany). Medazepam was kindly provided by Altana AG, Oranienburg, Germany. The surrogate standards (analytical grade >98% purity) were obtained from the following suppliers: benzoylecgonine-d8 (LGC Promochem, Wesel, Germany), codeine-d6, diazepam-d5, methadone-d9, morphine-d6, nordiazepam-d5 (Cambridge Isotopes Lab., Saarbru ¨cken, Germany), oxazepam-d5 (Sigma, Deisenhofen, Germany), 13C-15N-carbamazepine (Campro Scientific, Berlin, Germany). A standard solution of all target analytes, and a standard solution of all surrogate standards, were separately prepared at a concentration of 10 ng/µL in methanol. Both were stored in the dark at 4 °C. The information about which surrogate standard was used for quantitation of each analyte is provided in Table 4. 2.2. Sampling of Wastewater and Surface Water. In March 2005 and November 2005, grab samples of the influent (after mechanical treatment) and of the effluent were taken from 12 German STPs (see Table S2, Supporting Information for technical data). The STPs investigated consisted of a preliminary clarification, an activated sludge system (nitrification, denitrification), phosphate removal and a final clarification. At the same days, grab samples were taken from the rivers Rhine (Koblenz, km 590.3), Saar (Fremersdorf, km 46.1), Mosel (Koblenz, km 2.0) and Lahn (Lahnstein, km 38.0). Furthermore, grab samples were taken from small German creeks from the Hessian area: Schwarzbach (Trebur), Landgraben (Trebur), Wickerbach (Flo¨rsheim), Rodau (Mu ¨ hlheim), Bieber (Mu ¨ hlheim), Erlenbach (Bad Vilbel) and Eschbach (Harheim). All these rivers and streams are used as receiving waters for STPs, but the sampling points are not directly located in the discharge of a STP. The samples were taken in glass bottles and were immediately cooled down to 4 °C. The storage time before sample extraction did not exceed 24 h. 2.3. Sample Preparation and Extraction. All water samples were filtered through glass fiber filters (GF 6, < 1 µm, diameter 55 mm from Schleicher and Schuell, Dassel, Germany). For SPE of water samples, 100 mL of raw wastewater, 200 mL of treated wastewater, and 1 L of surface water, groundwater and drinking water were spiked with 200 ng of each surrogate standard. Hence, surrogate concentrations were 2 µg/L and 1 µg/L for raw and treated wastewater, respectively, and 0.20 µg/L for surface water, groundwater and drinking water. The Oasis HLB cartridges (200 mg, 30 µm, Waters, Milfort, U.S.) were conditioned with 1 × 2 mL n-heptane, followed by 1 × 2 mL acetone, 3 × 2 mL methanol and 4 × 2 mL water (pH 7). The water samples were then passed through the pre-conditioned SPE-cartridges at a flow rate of approximately 20 mL/min. Subsequently, the solid-phase material was dried completely with a commercially available drying device (GrenzahlWyhlen, Germany) by a continuous nitrogen stream for 1 h. Then the analytes were eluted four times with 2 mL acetone (total 8 mL). The extracts were evaporated to approximately 50 µL by a gentle nitrogen stream and were finally made up to a volume of approximately 500 µL with a 1:9 (v/v) solution of methanol in milli-Q-water. 2.4. LC-MS Analysis. Chromatographic separation was achieved at ambient temperature using a Synergi Polar-RP 80 Å column (150 × 3 mm, 4 µm) (Phenomenex, Aschaffenburg, Germany). The samples extracts (25 µL) were injected
into the LC system (Agilent 1100 with degasser, quaternary pump and autosampler, Agilent Technologies, Waldbronn, Germany) using acetonitrile (A) and 10 mM ammonium formiate in water, adjusted to pH 4 with formic acid (B), as a mobile phase. The following binary gradient was used: Start of the run with 10% A kept isocratic for 5 min, linearily increased to 80% A within 13 min, return to the initial conditions 10% A within 2 min which were kept for the last 10 min. During the analysis the flow rate was kept constant at 0.5 mL/min. The tandem mass spectrometer (API 4000 with turbo/electrospray ionization, collision gas: nitrogen; Applied Biosystems, Foster City, CA) was operated in positive ion mode using multiple reaction monitoring (MRM) for all measurements. Parameters adjusted were collision gas, 41 kPa; curtain gas, 172 kPa; ion source gas 1 and ion source gas 2, both 275 kPa; source temperature, 450 °C; entrance potential, 10 V. The ionspray voltage was adjusted to 5 kV. Two MRM transitions for each substance were monitored for identification and quantification of the analytes. Parameters such as declustering potential, collision energy, and cell exit potential were optimized in the auto-tuning routine of the Analyst 1.4 software. The retention times, the selected ion masses, and the optimized parameters are listed in Table 2.
3. Method validation 3.1. Determination of Recoveries. Individual recoveries for the entire method were determined by spiking samples of groundwater, surface water, drinking water, as well as raw and treated wastewater with analytes and surrogate standards. The absolute recoveries were determined in comparison to a non-enriched standard solution. For calculation of the relative recoveries, the absolute recoveries were corrected with the recovery of the spiked surrogate standards. All statistical errors are given as 95% confidence intervals. 3.2. Determination of the Calibration and the Quantitation Limits. Calibration curves with 15 calibration points ranging from 0.0002 to 12 µg/L were prepared by spiking groundwater. The groundwater used was not influenced by anthropogenic contamination, and hence, no residues of the analytes were detectable. The linearity range was between 0.0002 and 0.20 µg/L. A quadratic fitting (y ) ax2 + bx + c) was used from 0.20 to 12 µg/L. In all cases the correlation coefficients were higher than 0.997. Peak areas of the chromatograms were integrated and the ratios of the analyte/internal surrogate standards were calculated for each analyte. Resulting analyte concentrations were plotted versus the respective ratios of analyte and surrogate standard peak areas. The limit of quantification (LOQ) was defined as the second lowest calibration point in the linear regression as long as the calculated signal/noise ratio of the analytes in the native samples extracts was at least 10. For STP influents and effluents the LOQs were determined by multiplying the LOQ achieved for groundwater by the factor of 10 and 5, respectively, considering the enriched sample volume. Still the signal/noise ratio of the analytes in the extracts of STP influent and effluent has to be at least 10. However, mostly it was much higher and the empirical factors 5 and 10 guarantee that also for highly polluted samples a precise quantification was possible. Blank groundwater samples were spiked with the surrogate standards only and were included in each series of the analysis. 3.3. Matrix Effects. Ion suppression and ion enhancement during electrospray ionization is known to be a major reason for incorrect quantitative results, especially for complex matrices such as wastewater. In order to investigate the influence of matrix components during electrospray ionization, aliquots of the final sample extracts were spiked with the analytes to determine recoveries excluding any losses caused by SPE and further sample preparation. VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Precursor, Product Ions, and Retention Times in LC/MS/MS Detectiona substances
retention time in min
precursor ion in m/z (M+H)+
product ion 1 (P1) in m/z
product ion 2 (P2) in m/z
DP in V
CE(P1/P2) in eV
CXP (P1/P2) in V
opioids, cocaine-metabolite benzoylecgonine codeine dihydrocodeine hydrocodone methadone morphine oxycodone tramadol
11.3 9.6 8.1 11.7 18.3 4.0 11.2 13.2
289.9 300.0 302.1 300.0 310.0 285.9 315.9 263.5
168.0 215.0 128.0 198.9 105.0 201.0 256.0 58.0
104.9 164.9 200.9 128.0 76.9 152.0 212.0
61 71 71 91 51 86 56 46
27/41 37/53 85/39 41/77 37/75 35/77 35/59 45
12/8 16/12 12/16 6/10 6/6 6/14 8/14 4
tranquilizers bromazepam diazepam medazepam nordiazepam oxazepam temazepam-1 temazepam-2
13.4 17.9 17.9 16.6 14.3 16.8 16.8
317.9 284.9 272.9 270.9 286.9 300.9 302.9
182.0 193.0 91.0 208.0 240.9 254.8 256.9
209.1 221.9 207.0 140.0 103.9
86 76 71 71 61 56 56
45/37 45/34 43/39 39/41 47/81 31 31
16/18 14/20 8/6 6/12 8/6 18 6
antiepileptics carbamazepine DH-CBZ DHH-CBZ primidone
15.7 15.8 12.2 11.5
236.9 238.9 270.9 218.9
193.9 196.0 252.9 162.1
179.1 180.0 236.0 90.9
71 66 41 46
27/49 31/55 13/19 19/39
16/12 14/14 16/6 14/8
tricyclic antidepressant doxepin
17.1
280.0
107.0
90.9
46
37/59
10/6
calcium channel blocker verapamil
19.2
455.1
165.2
303.2
86
41/35
4/10
surrogate standards benzoylecgonine-d8 carbamazepine-13C-15N codeine-d6 diazepam-d5 methadone-d9 morphine-d6 nordiazepam-d5 oxazepam-d5
10.9 15.2 11.8 17.6 18.3 4.2 16.4 14.3
298.0 239.0 306.0 290.0 319.0 292.0 275.9 291.9
171.0 191.9 165.0 198.0 105.0 152.0 164.9 235.9
109.9
66 61 91 91 61 61 86 81
29/43 29 57/89 45/31 41/77 81/59 43/43 31/49
14/8 12 12/12 14/8 8/6 12/10 12/18 20/8
a
151.9 261.9 77.0 152.9 213.1 109.0
DP ) Declustering potential, CE ) Collision energy, CXP ) Collision cell exit potential
4. Results and Discussion 4.1. Recoveries and Limits of Quantification. The limits of quantification (LOQ) ranged between 0.010 and 0.050 µg/L for raw wastewater, 0.005-0.025 µg/L for STP effluent, and 0.001-0.010 µg/L for surface water, groundwater and drinking water (Table 4). 4.1.1. Wastewater (Influent, Effluent). In general, the selected opioids, tranquilizers, the metabolite benzoylecgonine, the antiepileptics plus metabolites, doxepin, and verapamil can be analyzed with an acceptable accuracy in raw and treated wastewater considering the 95 confidence intervals of less than 25%. A LC-MS chromatogram is shown in Figure S1 of the Supporting Information. The relative recoveries mainly exceeded 75% (Table 3). Although low absolute recoveries for a few compounds down to 18% (diazepam) exhibited major matrix effects, reliable data was achieved due to the effective compensation using selected deuterated surrogate standards (see Table S1, Supporting Information). The relative recovery of diazepam was 98 ( 9%. However, the surrogate standard noradiazepam-d5 is not appropriate for the determination of medazepam in STP influent, and of verapamil in the STP effluent indicated by an elevated relative recovery of 162 ( 30%, and a low relative recovery of 49%, although both are quantitatively recovered in the remaining matrices. Therefore, it is crucial to determine individual relative recoveries in complex matrices and if these are too low (130%), the standard addition method should be applied, as for medazepam in STP influent and for verapamil in STP effluent. Optimally, there should 7324
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be one stable isotope labeled (13C, 15N-labeled or deuterated) surrogate for each analyte that elutes from the chromatographic column at the same time as the native compound, and therefore, is equally affected by the sample matrix. However, at the beginning of the current study, no more isotope labeled compounds from commercial suppliers could be identified. 4.1.2. Surface Water, Drinking Water and Groundwater. The analytical method enables the analysis of all analytes in surface water, groundwater, and drinking water down to 0.001 µg/L with a sufficient accuracy (Table 3). Relative recoveries are comparable to those obtained in treated wastewater and were generally greater than 75%. Since, for most compounds, the differences between absolute and relative recoveries are low or negligible, matrix effects do not constitute a major problem for surface water and groundwater. Nevertheless, in drinking water, very low absolute recoveries down to 21 ( 3% were obtained for morphine, benzoylecgonine, and bromazepam. This might have been caused by chlorine residues in drinking water which are known to oxidize compounds containing phenolic hydroxy groups or amino groups selectively (25). The surrogate standard morphine-d6 and benzoylecgonine-d8 efficiently compensated for these losses. However, benzoylecgonine-d8 was inappropriate as surrogate standard for doxepin and primidone in drinking water, and hence its use as surrogate standard would lead to an over-compensation. 4.2. Matrix Effects During Ionization: Ion Suppression, Ion Enhancement. During ionization of LC-MS detection,
TABLE 3. Recoveries of Opioids, Benzoylecgonine, Tranquilizers, Doxepin, and Verapamil in Different Aqueous Matrices with 95% Confidence Intervals after Subtracting the Original Contamination (Raw and Treated Wastewater Samples Spiked with 2 µg/L and 1 µg/L, Respectively; and Rhine Water, Groundwater, and Drinking Water Are Spiked to 0.20 µg/L, n ) 4)a STP influent
STP effluent
Rhine water
drinking water
groundwater (n ) 1)
recovery in %
absoluteb recovery
relativec recovery
absoluteb recovery
relativec recovery
absoluteb recovery
relativec recovery
absoluteb recovery
relativec recovery
opioids, cocaine-metabolite benzoylecgonined codeinef dihydrocodeinef hydrocodonef methadone h morphinei oxycodonef tramadolj
40 ( 5 48 ( 2 36 ( 1 49 ( 1 44 ( 2 29 ( 1 50 ( 5 20 ( 1
65 ( 5 80 ( 5 60 ( 2 92 ( 6 123 ( 6 102 ( 14 85 ( 13 121 ( 27
42 ( 2 64 ( 6 70 ( 5 71 ( 3 77 ( 2 43 ( 8 69 ( 10 49 ( 15
77 ( 9 77 ( 8 85 ( 12 85 ( 7 119 ( 6 90 ( 12 84 ( 20 66 ( 20
70 ( 5 76 ( 5 92 ( 5 81 ( 10 83 ( 7 86 ( 8 86 ( 6 74 ( 9
95 ( 12 74 ( 3 89 ( 4 78 ( 7 98 ( 3 115 ( 14 84 ( 9 74 ( 18
43 ( 14 70 ( 7 81 ( 10 69 ( 7 87 ( 19 25 ( 18 53 ( 15 80 ( 16
105 ( 10 79 ( 10 91 ( 6 78 ( 7 96 ( 6 119 ( 12 60 ( 24 77 ( 15
71 90 88 83 92 83 85 94
89 120 118 111 118 125 114 87
tranquilizers bromazepami diazepamg medazepamj nordiazepamj oxazepamk temazepamj
19 ( 2 18 ( 1 40 ( 10 18 ( 3 42 ( 5 19 ( 7
56 ( 9 98 ( 9 162 ( 30 101 ( 14 86 ( 5 105 ( 14
39 ( 7 61 ( 2 74 ( 18 69 ( 2 61 ( 7 57 ( 4
81 ( 13 105 ( 6 99 ( 21 93 ( 4 109 ( 8 77 ( 8
66 ( 14 84 ( 5 82 ( 5 93 ( 3 93 ( 9 78 ( 4
88 ( 14 21 ( 3 95 ( 14 88 ( 8 81 ( 11 76 ( 10 92 ( 14 95 ( 19 102 ( 7 102 ( 10 78 ( 17 89 ( 16
77 ( 22 100 ( 9 73 ( 4 91 ( 7 101 ( 9 85 ( 9
87 102 98 123 96 99
131 108 92 114 92 92
antiepileptics carbamazepinee DH-Carbamazepinee DHH-Carbamazepinee primidoned
36 ( 4 25 ( 2 59 ( 5 40 ( 2
111 ( 12 77 ( 4 -118 ( 10
36 ( 10 29 ( 2 40 ( 11 44 ( 8
79 ( 20 63 ( 7 88 ( 25 80 ( 6
76 ( 7 66 ( 5 94 ( 13 63 ( 9
101 ( 7 87 ( 3 124 ( 12 86 ( 20
90 ( 12 105 ( 7 85 ( 9 99 ( 7 99 ( 4 115 ( 16 64 ( 9 (146 ( 16)l
98 92 103 71
112 105 117 89
tricyclic antidepressant doxepind
40 ( 1
107 ( 8
50 ( 6
92 ( 22
71 ( 5
97 ( 19
73 ( 17 (156 ( 35)l
75
95
calcium channel blocker verapamilj
31 (1
116 ( 19
36 ( 3
49 ( 4
95 ( 4
95 ( 15
93 ( 10
47
44
89 ( 9
absoluteb relativec recovery recovery
DHH- Carbamazepine ):10,11-Dihydroxy-10,11-dihydrocarbamazepine, DH- Carbamazepine: 10,11-dihydrocarbamazepine. b Calculated in comparison to a non-enriched standard solution. cCalculated using the recovery of the spiked surrogate standard d Benzoylecgonine-d8. e Carbamazepine-13C-15N. f Codeine-d . g Diazepam-d . h Methadone-d . i Morphine-d . j Nordiazepam-d . k Oxazepam-d l Benzoylecgonine-d is 6 5 9 6 5 5 8 inappropriate as surrogate standard, since it might react with chlorine residues in drinking water. a
matrix effects can cause deviations of the absolute recoveries up to nearly 100%. Matrix effects in LC-MS occur when molecules coeluting with the compounds of interest alter their ionization efficiency in the interface, leading to ion suppression or ion enhancement (26). Annesley (27) pointed out that ion suppression might result from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn, affects the amount of charged ions in the gas-phase that ultimately reaches the detector. The detailed mechanism of these processes is still unknown, but it occurs in complex matrices (e.g. wastewater). The extent of matrix effects for the analytes varies with the specific composition of the sample (28). However, there are several common options to obtain correct quantitative results of samples with complex matrices: (1) using standard addition as an alternative quantitation method, (2) using appropriate internal/surrogate standards which are coeluting with the analytes (preferably deuterated, 15N- or 13C-labeled compounds) to compensate ion suppression and ion enhancement; (3) including effective cleanup steps to remove interfering matrix components, and hence, minimize matrix effects; (4) to reduce the injected matrix quantity by a smaller concentration factor or a smaller injection volume, and (5) the use of an alternative ionization technique (e.g. APCI) or another source design where matrix effects are less significant. However, currently all ionization techniques in LC-MS may be affected by matrix effects, since different ionization mechanisms may not simply influence the extent but also the nature of the matrix effects (29). In the current analytical method, (a) seven deuterated and one 13C-15N-labeled surrogate standards were used, and (b) the concentration factors were varied for the different
matrices. For raw and treated wastewater, a concentration factor of 200 and 400 (0.1 L/0.2 L reduced to 500 µL) was applied, respectively, whereas for surface water, groundwater, and drinking water, a concentration factor of 2000 (1 L was reduced to 500 µL) was used. Furthermore, the sample injection volume was optimized to be 25 µL, being a compromise between sensitivity and the occurrence of matrix effects. Matrix effects can be determined following the protocols by Chin et al. (30) or Mallet et al. (31). In the current paper, an alternative approach was used similar to Miao and Metcalfe (4). The extent of ion suppression was determined by subtracting the absolute recoveries of the spiked (200 ng) final sample extracts from the nonspiked final sample extracts from STP influent, STP effluent, and Rhine water (Figure 1). In Rhine water, ion suppression was of minor relevance since it did not exceed 20%. A higher level of ion suppression was detected in the municipal STP effluent up to maximum of 45% for carbamazepine. However, for most analytes it varied between 10 and 25%. In STP influents, matrix effects were as high as 90%, and thus, the use of appropriate isotope labeled surrogate standards is absolutely essential. 4.3. Occurrence of Psychoactive Compounds and Verapamil in Environmental Samples. 4.3.1. Opioids. The plant alkaloids, morphine and codeine, were detected in all influents of the 12 German municipal STPs, monitored in this study, up to concentrations of 0.82 µg/L and 0.54 µg/L, respectively (Table 4). The removal of these compounds in STPs seems to be quite substantial, since the effluent concentrations of both were reduced by about a factor of two for codeine and a factor of about eight for morphine. However, a more detailed and comprehensive study is needed to confirm those conclusions and to investigate the efficiencies of different technologies. Further opioids, such as VOL. 40, NO. 23, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Ion suppression for the analytes in water from the river Rhine as well as influent and effluent of a municipal STP.
TABLE 4. Occurrence of Opioids, Benzoylecgonine, Tranquilizers, Doxepin, and Verapamil in Wastewater (Influent and Effluent) and Surface Water Samples, LOQ, Number of STPs and Rivers/streams Sampled, Median, and Maximum in µg/La
g
a LOQ ) limit of quantification. b Confirmed by standard addition. c Benzoylecgonine-d . 8 methadone-d9. h morphine-d6. i nordiazepam-d5. j Oxazepam-d5.
hydrocodone, oxycodone, and dihydrocodeine could be detected in raw wastewater, but only dihydrocodeine was ubiquitously present with a median above the LOQ (influent: 0.10 µg/l; effluent: 0.06 µg/L). Several analytes (tramadol, benzoylecgonine, 10,11-dihydrocarbamazepine, verapamil) were integrated into the analytical method at a later stage, and thus were only monitored in one municipal STP. Tramadol was found in both the influent and the effluent at concentrations of 1.5 µg/L and 0.61 µg/L respectively at this municipal STP serving population equivalents of 320 000. Benzoylecgonine, the main metabolite of cocaine, was found in the STP influent and effluent at concentrations of 0.078 µg/L and 0.049 µg/L as well as in all three investigated rivers Rhine, Mosel, and Lahn at concentrations of up to 0.003 µg/L (Figure 2). In the majority of the monitored German 7326
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d
carbamazepine-13C-15N. e codeine-d6. f diazepam-d5.
surface waters, codeine, dihydrocodeine, hydrocodone, morphine, and tramadol were found within the low ng/L range, with a maximum concentrations of 0.094 µg/L for codeine (Table 5). Methadone was not found in any sample, at concentrations above the LOQ of 0.005 µg L in surface water, 0.025 µg/L in effluent and 0.050 µg/L in influent. 4.3.2. Tranquilizers. The two hydroxylated tranquilizers, oxazepam and temazepam (see Figure S2, Supporting Information), were present in similar concentrations in STP influents and effluents up to 0.86 µg/L, as well as in German rivers and streams with a maximum concentrations of 0.40 µg/L (oxazepam) in the stream Erlenbach (Figure 2). Their removal in municipal STPs seems to be limited. The other tranquilizers medazepam and nordiazepam were not detected in surface waters, nor was bromazepam found above
FIGURE 2. The occurrence of psychoactive drugs in rivers and streams in Southwest Germany (n.a.: not analyzed; LOQ: limit of quantification). LOQ. Obviously, only the two hydroxylated tranquilizers, oxazepam and temazepam, are present in the aquatic environment at higher concentrations. 4.3.3. Other Pharmaceuticals such as Antiepileptics, Verapamil, and Doxepin. Maximum concentrations up to 3.7 µg/L were detected for the antiepileptic carbamazepine and its metabolite 10,11-dihydro-10,11-dihydroxycarbamazepine with an obviously negligible removal percentage in STPs as already described (2, 5). Also Miao and Metcalfe (4) pointed out that the two metabolites of carbamazepine have been found in Canadian STPs discharges with maximum concentrations of 1.3 µg/L. Verapamil, similarly to tramadol only monitored in one STP, exhibited relative high concentrations with 3.1 µg/L (influent) and 0.51 µg/L (effluent). Even doxepin seems to be ubiquitously present in the environment with maximum concentration up to 0.92 µg/L in the STP influents, 0.36 µg/L in the STP effluents, and up to 0.22 µg/L (Rodau) in German rivers and streams (see Figure 2). These first results of the occurrence of psychoactive drugs in wastewater and surface water samples clearly showed that the selected compounds are introduced via municipal STPs into rivers and streams. However, for a detailed discussion, the database of these compounds needs to be enlarged. Furthermore, for opiates, tranquilizers, doxepin, and verapamil, the environmental impacts are not known and hence an appropriate environmental risk assessment is currently not possible. 4.4. Exposure of Drinking Water. Drinking water samples were taken from three different waterworks in Germany using water resources which are known to be contaminated by pharmaceuticals due to a significant influence of treated wastewater. The processes used in these waterworks did contain bank filtration, flocculation, disinfection, acidification, but not ozonation, activated carbon filtration, nanofiltration, or reverse osmosis. In the drinking water of all three waterworks the two antiepileptics, carbamazepine and primidone, were detected at concentrations up to 0.020 µg/L and 0.016 µg/L, respectively. Furthermore, the metabolite 10,11-dihydroxy-10,11-dihydro-carbamazepine were found at concentrations up to 0.013 µg/L (LOQ ) 0.005 µg/L). All
other analytes were not detected at concentrations above the LOQ of 0.002-0.010 µg/L.
Acknowledgments This study was part of the EU Reclaim Water project (018309) which was financially supported by grants obtained from the EU Commission in the sixth Framework. We thank Maria Ramil, BfG for the internal review of the manuscript and the Hessische Landesanstalt fu ¨ r Umwelt and Geologie (HLUG) for providing river and wastewater samples.
Supporting Information Available Further information on the technical data of the sewage treatment plants sampled and the metabolization of several tranquilizers are shown in the Supporting Information. This material is available free of charge via the Internet at http:// pubs.acs.org.
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Received for review July 21, 2006. Revised manuscript received September 15, 2006. Accepted September 25, 2006. ES061740W
