Monitoring carbamazepine in surface and waste

Monitoring carbamazepine in surface and waste waters by an immunoassay based on a monoclonal antibody Arnold Bahlmann • Michael G. Weller • Ulrich Panne • Rudolf J. Schneider*

BAM Federal Institute for Materials Research and Testing Richard-Willstätter-Str. 11, 12489 Berlin, Germany Tel.: +49 30 8104 1151 Fax: +49 30 8104 1157 E-mail: [email protected]

* corresponding author

Abstract. The pharmaceutical compound carbamazepine (CBZ) is an emerging pollutant in the aquatic environment and may potentially be used as a wastewater marker. In this work, an enzyme-linked immunosorbent assay (ELISA) for the detection of carbamazepine in surface and sewage waters has been developed. The heterogeneous immunoassay is based on a commercially available monoclonal antibody and a novel enzyme conjugate (tracer) that links the hapten via a hydrophilic peptide (triglycine) spacer to horseradish peroxidase. The assay achieves a limit of detection of 24 ng/L and a quantitation range of 0.05 - 50 µg/L. The analytical performance and figure of merits were compared to liquid chromatography- tandem mass spectrometry (LC-MS/MS) after solid-phase extraction (SPE). For nine Berlin surface water samples and one wastewater sample a close correlation of results was observed. A constant overestimation relative to the CBZ concentration of ca. 30 % by ELISA is probably caused by the presence of 10,11-epoxy-CBZ and 2-hydroxy-CBZ in the samples. The ELISA displayed crossreactivities for these compounds of 83 and 14 %, respectively. In a first screening of 27 surface water samples, CBZ was detected in every sample with concentrations between 0.05 and 3.2 µg/L. Since no sample clean-up is required the assay allowed for the determination of carbamazepine with high sensitivity at low costs and with much higher throughput than with conventional methods.

Keywords: Carbamazepine · ELISA · Antibody · Immunoassay · Surface Water · LC-MS/MS · Pharmaceuticals 1

Introduction The presence of pharmacologically active compounds and their metabolites in the aquatic environment has become a matter of concern, especially after a reconnaissance study on U.S. streams in the years 1999-2000 [1]. In Germany the first comprehensive reports on the presence of pharmaceuticals in surface water appeared in 1998 [2, 3]. In 1994, the lipid regulator clofibric acid had been identified in Berlin’s surface, ground and drinking water [4, 5]. Apart from their relevance to drinking water some of the compound classes detected, like hormones [6] and antibiotics, cause concern about their potential ecotoxicological effects [7-9]. One of the most frequently reported pharmaceuticals in surface water is carbamazepine [10]. This high-selling drug (61 tons prescribed in Germany in 2006 [11, 12]) is used as an anticonvulsant primarily in the treatment of epilepsy to control seizures. Other indications are attentiondeficit disorder, bipolar disorder and schizophrenia. A typical daily dose is 1,000 mg [12]. Like most other pharmaceuticals, carbamazepine and its metabolites enter the water cycle through human excretions. Furthermore, a considerable amount of unused medication is flushed down the toilet and contributes to wastewater concentrations of pharmaceuticals [13, 14]. Accordingly, CBZ has been found in the influent of wastewater treatment plants (WWTP) in concentrations up to 3.8 µg/L [15]. In common activated sludge treatment plants little to negligible amounts of carbamazepine are removed [16]. Several studies even reported an increase in CBZ concentration during the treatment process due to the cleavage of metabolic conjugates [17-20]. Hence, carbamazepine is introduced into rivers and streams by WWTPs discharge. A literature survey resulted in a mean carbamazepine concentration of 0.5 µg/L in German surface waters [10] or 0.45 µg/L as reported in [21]. In many studies the concentrations even exceeded the 1 µg/L level [2, 15, 20], reaching maximum concentrations of up to 7.1 µg/L [22]. After entering the environment CBZ has been shown to be very slowly degraded by sunlight with a half-life of ca. 100 days [13]. It shows a moderate [23] to low [24] adsorption to soil 2

and is therefore eventually introduced into groundwater after soil passage. This mainly happens through the input from surface waters [15], bank filtration and groundwater recharge [25]. Because of the little attenuation of CBZ during transport up to 900 ng/L have been detected in German groundwater [26]. Being so persistent and almost ubiquitously present in environmental water carbamazepine has been proposed as a marker for anthropogenic input to the aquatic environment [24,27-31]. Its aquatic toxicity is hitherto not concludingly assessed but the nonbiting midge Chironomus riparius was affected by carbamazepine which might also be the case for other aquatic insect populations [32]. When assessing environmental concentrations diurnal and seasonal variations have to be taken into account. Medication patterns, seasonal variations in the emergence of diseases and different flow regimes across the year may lead to temporal fluctuations of the pollutant’s occurrence in the environment [33-36]. Therefore profiles and time series of environmental concentrations have to be recorded. For this purpose a fast and inexpensive analytical method for the quantitation of carbamazepine in wastewater and surface waters is needed. At present the common analytical procedure for determining carbamazepine in aquatic samples is LC coupled with single or tandem MS. The limit of quantitation highly depends on the matrix and the sample preconcentration. LOQs between 1 and 50 ng/L are usually reported [20, 37]. Gas chromatography-mass spectrometry (GC-MS) has also been used to detect CBZ [2, 38] and usually reaches a sensitivity comparable to LCMS/MS. These methods are time-consuming, expensive and require costly instrumentation and dedicated personnel. Immunoassays have proven to be valuable tools for sensitive detection of various pollutants in water [39, 40]. Our objective was to establish an ELISA for the determination of carbamazepine in wastewater and surface water samples without prior enrichment. Several immunoassays using monoclonal antibodies and polyclonal sera produced against carbamazepine immunogens have been reported, using

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various formats [41-45]. By now these assays have exclusively been applied to biological samples with CBZ concentrations in the mg/L range.

Experimental Reagents and Materials Reagents The anti-carbamazepine monoclonal antibody (mouse IgG 1, clone B3212M, lot 5K32007) was purchased from BIODESIGN International (Meridian Life Science Inc., Saco, MN, USA). A polyclonal antibody against mouse IgG F(c) domain (from goat, lot 20185) was purchased from Acris Antibodies (Herford, Germany). Horseradish peroxidase (HRP), EIA grade, was obtained from Roche (Mannheim, Germany), Guardian™ (Peroxidase Conjugate Stabilizer/Diluent) was purchased from Thermo Scientific (Schwerte, Germany). 3,3’,5,5’-Tetramethylbenzidine (TMB), research grade, and Tween™ 20, pure, were purchased from Serva (Heidelberg, Germany). Buffer salts (sodium phosphate dibasic dihydrate, sodium phosphate monobasic dihydrate, potassium phosphate monobasic, potassium dihydrogen citrate, sodium chloride) were of Fluka "ultra” quality (Sigma-Aldrich, Taufkirchen, Germany). Potassium sorbate (purum), tetrabutylammonium borohydride (purum), hydrogen peroxide 30 % (Trace select®), ethylenediaminetetraacetic acid disodium salt (Na2EDTA, puriss), N,N-dimethylformamide (puriss.), N,N′dicyclohexylcarbodiimide (puriss), N,N'-disuccinimidyl carbonate (purum) and N-hydroxysuccinimide (purum) were also from Fluka. Tris(hydroxymethyl)aminomethane (TRIS, p.a.) was obtained from Merck (Darmstadt, Germany), Sulphuric acid 95 - 97 %, and hydrochloric acid 32 %, were of “Baker analyzed grade”, methanol was HPLC gradient grade (Mallinckrodt Baker, Griesheim, Germany). Ultrapure reagent water was obtained by running demineralized (by ion exchange) water through a Milli-Q® water purification system (Millipore Synthesis A 10, Schwalbach, Germany). Bovine serum albumine (BSA, 98 %), carbamazepine (CBZ, 4

99 %), dibenz[b,f]azepine-5-carbonyl chloride (95 %), 10,11-dihydro10,11-epoxycarbamazepine (EP-CBZ, 98 %), 10,11dihydrocarbamazepine (DiH-CBZ, 99 %), dibenz[b,f]azepine (iminostilbene, 97 %), oxcarbazepine (98 %), amitriptyline (98 %), protriptyline hydrochloride (99 %), imipramine hydrochloride (99 %), opipramol dihydrochloride (98 %), doxepine hydrochloride (98 %), glycylglycylglycine (triglycine, 99 %), phosphoric acid (85 %), tetrahydrofuran (puriss), diethylether (puriss) and N,N-dimethylacetamide (puriss) were purchased from Sigma-Aldrich (Taufkirchen, Germany). 10,11-Dihydro-10hydroxycarbamazepine (10OH-CBZ, 98 %), 10,11-dihydro-trans-10,11dihydroxycarbamazepine (DiOH-CBZ, 98 %), 2-hydroxycarbamazepine (2OH-CBZ, 98 %) and carbamazepine-d2 (98 %) were purchased from Toronto Research Chemicals (North York, Canada). Acridine (98 %) was purchased from ABCR (Karlsruhe, Germany). Sinapic acid was purchased from Protea Biosciences (Nîmes, France). All reagents were used as received except DiH-CBZ which was purified by HPLC as follows. A Gynkotek LC system consisting of a degasser, a M480 binary pump (Dionex, Germering, Germany), a Phen1 RP-18 analytical column (250  3 mm, 5 µm; Sepserv, Berlin, Germany), a column heater at 40 °C and a Gynkotek UVD 320S UV detector was employed. A mobile phase consisting of 61 % (v/v) methanol in water was used in isocratic mode at a flow rate of 0.4 mL/min. DiH-CBZ (3.2 g/L) was dissolved in 61 % (v/v) methanol in water and 80 µL of this nearly saturated solution was injected for each run. The retention times of CBZ and DiH-CBZ as observed by the UV signal were 7.8 and 8.6 minutes, respectively. Thus the fraction between 8.5 and 9.1 minutes was collected into a glass vial. 10 runs were conducted and the pooled fractions were reduced under nitrogen to about 500 µL. The supernatant was discarded and the white precipitate of DiH-CBZ was vacuum dried over silica gel for 36 hours. 2 mg of dry DiH-CBZ were obtained. Materials Transparent bottom microtiter plates with 96 flat-bottom wells possessing high binding capacity (MaxiSorpTM) were purchased from Nunc (Thermo 5

Scientific). PD-10 columns containing Sephadex G-25 were obtained from GE Healthcare (Munich, Germany). Grade 288 paper filters were from Sartorius (Göttingen, Germany). Strata™-X (200 mg, 6 mL) SPE cartridges were purchased from Phenomenex (Aschaffenburg, Germany). 0.22 µm cellulose acetate membrane syringe filters were obtained from Nalgene (Thermo Scientific). Polypropylene tubes (1.5 mL) were purchased from Eppendorf (Hamburg, Germany). Zeba™ Micro Desalt Spin Columns were from Pierce (Thermo Scientific).

Methods Hapten and enzyme conjugate synthesis 41 mg (0.22 mmol) triglycine, 54 mg sodium carbonate and 84 mg sodium hydrogen carbonate were dissolved in 8.4 mL water. 168 mg (0.66 mmol) dibenz[b,f]azepine-5-carbonyl chloride were dissolved in 11.2 mL dry tetrahydrofuran and immediately added to the triglycine solution. After stirring overnight at room temperature, the solvent was evaporated. During the evaporation process a white precipitate was formed. The cloudy suspension was transferred to a centrifuge tube and centrifuged at 3,000 rpm for 5 minutes. The supernatant was collected and acidified with 0.2 mL of 85 % phosphoric acid to a final pH of ca. 2. After extracting three times with diethyl ether (3 x 7 mL), the remaining ether was gently removed from the aqueous phase using a rotary evaporator. The white precipitate was collected, washed three times with 0.01 M hydrochloric acid and dried under vacuum over silica for 48 hours. Yield of N(dibenz[b,f]azepine-5-ylcarbonyl)glycylglycylglycine (CBZ-triglycine) was 17 mg (0.042 mmol; 19 % based on triglycine). 1.9 mg of CBZ-triglycine was dissolved in 50 µL of dry N,N-dimethylformamide (DMF) in a 1.5 mL microcentrifuge tube. A small spatula tip (ca. 1-2 mg) of N,N'-disuccinimidyl carbonate was added. 0.95 mg N-hydroxysuccinimide (NHS) was dissolved in 20 µL DMF and added. 1.11 mg N,N'dicyclohexylcarbodiimide was dissolved in 20 µL DMF and added. The activated ester was formed during shaking on an orbital shaker (700 rpm)

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at room temperature overnight. The precipitate of mainly dicyclohexyl urea was removed by centrifugation and discarded. 1.0 mg (23 nmol) of HRP was dissolved in 100 µL sodium hydrogen carbonate solution (0.13 M, pH 8.1) in a 1.5 mL micro centrifuge tube. An overall volume of 30 µL of activated ester was added subsequently in five intervals of 10 minutes (room temperature). After shaking for one more hour the solution was pipetted onto a PD-10 column conditioned with 25 mL of PBS (1 mM sodium phosphate, 0.015 M NaCl, pH 7.6). The same buffer was used for elution of the enzyme conjugate. Fractions were collected in a microtiter plate and their absorbance measured photometrically at 403 nm. Fractions of highest absorbance were pooled, mixed with an equal amount of HRP stabilizer (Guardian™), aliquoted and stored at 4 °C. The concentration of peroxidase was determined photometrically using a molar absorption coefficient of 102 cm-1 mM-1 at 403 nm [46]. Immunoassay procedure The CBZ ELISA is based on a commercial antibody and the novel enzyme conjugate described above. The direct competitive ELISA format using sequential saturation was adapted from Zettner et al. [47]. The entire ELISA procedure was performed at room temperature. Transparent highbinding microtiter plates were coated with polyclonal antibody against mouse IgG (1 mg/L, 200 µL per well) in PBS (10 mM sodium dihydrogen phosphate, 70 mM sodium hydrogen phosphate, 145 mM sodium chloride, pH 7.6). The plates were covered with Parafilm® to prevent evaporation. After overnight incubation on a Titramax 101 plate shaker (Heidolph, Schwabach, Germany) at 750 rpm, the plates were washed three times with PBS containing 0.05 % Tween™ 20 (PBS-T) using an automatic 96channel plate washer (ELx405 Select™, BioTek Instruments, Bad Friedrichshall, Germany). Monoclonal antibody against carbamazepine diluted in PBS (8.6 µg/L, 200 µL per well) was added and incubated for one hour. To establish the calibration curve a CBZ stock solution was prepared in methanol and then further diluted with ultrapure water to obtain calibrators. After the three-cycle washing step (PBS-T), 25 µL of 7

sample buffer (1 M TRIS, 1.5 M sodium chloride, 107 mM Na2EDTA , 1 % (w/v) BSA, pH 7.6) were pipetted into each well. Afterwards the calibration standards and the samples (175 µL per well) were added. This was followed by the addition of enzyme conjugate (118 pmol/L in PBS, 50 µL per well); the plates were shaken for 30 min, followed by a second threecycle washing step (PBS-T). Finally, substrate solution was added (200 µL per well) and incubated for 30 min. The TMB-based substrate (41 mM TMB, 8 mM tetrabutylammonium borohydride in N,N-dimethylacetamide) was prepared according to Frey et al. [48]. The actual substrate solution consists of 540 µL TMB solution in 21.5 mL substrate buffer (200 mM citric acid containing 3 mM hydrogen peroxide and 0.01 % sorbic acid potassium salt, pH 4.0) and was freshly prepared for each run. The enzyme reaction was stopped by addition of sulphuric acid (1 M, 100 µL per well). After mixing optical density was read on a microplate spectrophotometer (SpectraMax Plus384, Molecular Devices, Ismaning, Germany) controlled by SoftMax® Pro software (v 5.2, Molecular Devices). Optical density was measured at 450 nm and referenced to 650 nm. All samples and standards were determined in triplicate on each plate. The mean values were fitted to a four-parametric logistic equation (4PL) [49]. Usually 8 calibrators were used, including two ‘anchor’ concentrations at zero and 10 mg/L for a better convergence of the curve fitting. The remaining six calibrators were arranged in logarithmic intervals (factor of 3) around the average test midpoint of 0.13 µg/L. Calibrators and samples were evenly distributed over the plate to average possible systematic errors. Curve fitting was done using Softmax’ least square algorithm. The inverse of the square of the standard deviation was used as a weighting function for the calibrators [50]. Cross-reactivity determination The relative sensitivity of the immunoassay towards other compounds was determined by assaying an aqueous dilution series of the substances listed in Figure 1. Molar cross-reactivity was calculated as the ratio of molar concentrations at the inflection points (midpoints, parameter C in the 4PL ≈ 50 % inhibitory concentration (IC50)) of the corresponding 8

calibration curves and expressed in percentage relative to the midpoint for CBZ (Equation 1). CR 

C S tan dard * 100 (Eq. 1) CTest

CR describes the cross-reactivity in percent, CStandard is a parameter of the 4PL giving the CBZ concentration at the inflection point and CTest refers to the concentration of the cross-reacting compound at its inflection point. Mass-related cross-reactivity (“specific cross-reactivity”) was calculated after conversion of the respective molar C values into mass concentrations. Water sampling Water samples (1 liter) were collected close to the surface, stored in brown glass bottles at 4 °C and usually analyzed within one week. On two occasions nine surface water samples were taken at the river Spree and at two channels in Berlin, the Teltowkanal and the Britzer Verbindungskanal. One effluent grab sample from wastewater treatment plant Ruhleben, Berlin, was provided by the operating company, Berliner Wasserbetriebe. Further samples of 10 mL were taken from the Teltowkanal on the 1st of October 2008. Sample preparation and solid-phase extraction Water samples were filtered through (folded) paper filters and used without any further treatment for ELISA analysis. For LC analysis the samples were cleaned up and concentrated using solid-phase extraction. Each Strata™-X column was preconditioned with 5 mL of 50 : 50 acetone : ethyl acetate on an AutoTrace™ SPE workstation (Dionex). After rinsing the cartridges with 5 mL of methanol and 15 mL of water, 200 mL of sample (including 2.5 µg/L of internal standard (IS) CBZ-d2) was loaded onto the cartridge. The column was dried by passing a nitrogen (purity 5.0) stream for 20 minutes. Elution was performed with 9 mL of 50 : 50 acetone : ethyl acetate. The eluate was reduced under a gentle stream of nitrogen (SLS 02 Evaporator, SLS-Labor, Bad Münstereifel) and reconstituted in 10 mL of 30 : 70 MeOH : H2O. Each sample was filtered 9

through a 0.22 µm syringe filter before LC injection. The pH of each water sample was measured using an Inlab Expert Pro pH electrode (Mettler Toledo, Gießen, Deutschland). LC-MS/MS method The LC-MS/MS experiments were carried out with an Agilent 1100 LC system (Agilent Technologies, Waldbronn, Germany) consisting of degasser, binary pump, autosampler, and column heater. The column outlet was coupled to an API 4000 mass spectrometer from Applied Biosystems (Darmstadt, Germany). Ionisation was performed using a Turbo V™ ion source in ESI positive mode. Data acquisition and mass spectrometric evaluation was carried out using data analysis software Analyst™ 1.4.1 (Applied Biosystems). For the chromatographic separation a Purospher RP-C18 (250  3 mm; 5 µm) (VDS Optilab, Berlin, Germany) chromatographic column with guard column (10  3 mm) was used. The column oven was set to 40 °C, the flow rate was kept at 0.4 mL/min and 20 µL sample volume was injected. A binary gradient consisting of 5 mM ammonium acetate in water (A) and 100 % methanol (B) was used: started with 25 % B, isocratic for 3 min, linear increase to 95 % B within 12 min, kept at 95 % B for 7 min, return to the initial conditions 25 % B within 1 min, kept for 9 min. The mass spectrometer was used with the following parameters: collision gas: 41 kPa; curtain gas: 172 kPa; ion source gas #1: 345 kPa and ion source gas #2: 414 kPa; source temperature: 400 °C; entrance potential: 10 V; declustering potential: 60 V. The ion spray voltage was adjusted to 4.5 kV. For quantitation and identification of the analytes two transitions for each substance were analyzed in multiple reaction monitoring mode (MRM). MRM1 (CBZ): m/z 237→ 194 ; collision energy (CE): 25 V; cell exit potential (CXP): 15 V; MRM2 (CBZ): m/z 237→ 179; CE: 50 V; CXP: 12 V; MRM3 (CBZ-d2): m/z 239→ 196; CE: 25 V; CXP: 15 V; MRM4 (CBZd2): m/z 239→ 180; CE: 50 V; CXP: 12 V. MALDI-TOF-MS All MALDI-TOF mass spectra were acquired on a Bruker Reflex III MALDI mass spectrometer (Bruker-Daltonik, Bremen, Germany) operated with a 10

nitrogen laser and at 20 kV acceleration voltage. 10 µL of protein or enzyme conjugate sample was loaded onto a Zeba™ Micro Desalt Spin Column, centrifuged for 90 seconds at 10,000 rpm, eluted with 10 µL water and mixed with 50 µL of matrix. The sinapic acid matrix was freshly prepared as a 10 g/L aqueous solution that contained 50 % acetonitrile and 0.1 % trifluoroacetic acid. The sample target was precoated with a droplet of 0.5 µL of matrix solution and dried for 5 minutes. Then 0.5 µL of protein sample was added onto the same spot and dried for one hour. Data was processed using Origin™ 8.0 (OriginLab, Northampton, USA). The mass peaks were fitted with a Lorentzian function and the centres of the fitting curves were assigned to the protein and conjugate masses, respectively.

Results and Discussion Tracer characterization CBZ-triglycine (see Figure 1) was used as the precursor for the synthesis of the enzyme conjugate needed to perform the ELISA. It was obtained by attaching a triglycine linker to a CBZ precursor, dibenz[b,f]azepinecarbonyl chloride. The CBZ-triglycine reacted mainly with lysine residues present in horseradish peroxidase via the activated ester route producing an intermediary NHS ester. HRP carries 6 lysine residues, but only 2 of them are considered readily available for reaction under these mild conditions [51, 52]. The hapten : protein molar ratio in the conjugate can be controlled by varying the molar ratios of reactants. The aim was to introduce at least one equivalent of carbamazepine per mole of HRP. The masses of native HRP and the enzyme conjugate were determined by MALDI-TOF-MS as 44,343 ± 41 Da (average and standard deviation of n = 6 measurements) and 44,924 ± 60 Da (n = 8), respectively. This accounts for an average conjugation ratio of 1.5 ± 0.3 moles carbamazepine per mole HRP, provided that only hapten and nothing else did bind to the peroxidase during the synthesis. The conjugate was stored

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at 4 °C in Guardian™ stabilizer solution and showed no significant decrease in activity within 1 year.

Assay optimization Figure 2 reveals the ELISA’s dose response curve and its precision profile [53] after optimization using 32 calibrators and 3 replicates per calibrator to obtain a well-founded profile. For sample quantitation, regularly 8 calibrators were used. The midpoint of the calibration curves is mainly influenced by the concentrations of primary antibody and tracer [54]. The reciprocal value of the minimum midpoint can be considered as an estimate of the antibody’s affinity constant [55]. By sequential dilution of both components it is possible to reduce the test midpoint and therefore decrease the limit of detection. Figure 3 shows the relationship between the effective concentrations of antibody and tracer and the test midpoint. By diluting both antibody and tracer it was possible to reduce the test midpoint from 0.8 to 0.1 µg/L. Further dilutions only slightly influenced the midpoint because the affinity limit is reached [39], but strongly decreased the signal intensity resulting in lowered precision. We chose a final setting that permitted a signal intensity between 0.5 and 1.0 OD maintaining a low test midpoint, the average C value of 38 independent experiments being 0.13 ± 0.02 µg/L. Surface water and wastewater samples often vastly differ in composition. The pH value and the ionic composition of the sample’s matrix can affect the binding between antibody and antigen. We used a sample buffer adapted from Zeck et al. [56] with high buffer capacity containing high concentrations of salt and EDTA to counter the influence of matrix effects. Furthermore BSA was added to inactivate any contaminants that might cause a denaturation of proteins. As a side effect, the high salt concentration increased the ELISA signal to two-fold. It has been reported that EDTA decreases the activity of peroxidase by removing complexed calcium from its active site [57], but we did not observe that effect. Due to the high dilutions of the immunoreagents the ELISA requires only very small amounts of tracer and antibody making the method very cost12

effective. One milligram of both reagents is sufficient to run several thousand test plates, permitting the measurement of more than 100,000 samples.

Cross-reactivity of the antibody In order to assess the specificity of the monoclonal antibody used in this study, we tested several compounds for cross-reactivity. Care was taken that the tested compounds, especially CBZ metabolites, did not contain trace contaminations of CBZ. The manufacturers certified purities of at least 97 % for all substances tested. Still, we checked all compounds for contaminations with CBZ. Analysing aqueous solutions (0.5 mg/L) of each analyte using LC-MS/MS in MRM mode, we found CBZ to be present in DiH-CBZ, OxCBZ and EP-CBZ. The percentages of CBZ in these compounds were roughly estimated by single measurements applying two-point calibration to be 10 %, 0.5 % and 0.8 %, respectively. All other substances assayed did not contain any detectable amounts of CBZ. Accordingly, DiH-CBZ was purified by liquid chromatography before measuring its cross-reactivity. The purified DiH-CBZ contained a remaining impurity of 0.1 % CBZ. Figure 1 contains the molar cross-reactivities of the antibody. The list includes the various metabolites of carbamazepine and pharmaceutical compounds that are structurally similar to CBZ. More than 33 metabolites have been identified in urine [58]. Among them are EP-CBZ, which has the same pharmacological potency as the parent compound, the monohydroxylated compounds 2OH-CBZ and 3OH-CBZ and the dihydroxylated derivative DiOH-CBZ. The shares of a dose excreted in urine are ca. 80 % for DiOH-CBZ, 3 % EP-CBZ and 5 - 6 % of each monohydroxy compound [59] and 3 % unchanged CBZ [60]. Accordingly several metabolites were found to be present in sewage and surface water, mainly DiOH-CBZ, 10OH-CBZ, 3OH-CBZ, 2OH-CBZ, EP-CBZ and 9-hydroxymethyl-10-carbamoylacridan [61-63]. EP-CBZ and 2OH-CBZ showed substantial cross-reactivities of 83 % and 14 %, respectively.

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Cross-reactivity against EP-CBZ seems to be a common drawback with CBZ antibodies. Polyclonal antibodies raised in sheep [42] and rabbits [64] revealed cross-reactivities of 10 - 30 % and 20 %, respectively. 8 out of 13 tested automated clinical immunoassays displayed a significant positive bias in presence of EP-CBZ [65], one assay had a CR of 90 % [66]. In contrast, Walter et al. obtained a monoclonal antibody that showed only 8.4 % CR [67]. We also observed considerable cross-reactivities for several tricyclic antidepressants. Protriptyline, opipramol, amitriptyline, imipramine and doxepine showed cross-reactivities of 9.6 %, 7.4 %, 4.9 %, 4.6 % and 0.8 %, respectively. Only little is known about the presence of these compounds in the environment. Doxepine has been detected in German WWTP effluents and surface waters in levels up to 0.36 µg/L and 0.22 µg/L, respectively [68]. In several studies amitriptyline was found in WWTP effluents in rather low concentrations of up to 32 ng/L [69-71] and in Wales 21 ng/L were found in surface water [72]. Imipramine [73] and opipramol [70] have been studied but not been found in surface and wastewater. To our knowledge nothing is known about the presence of protriptyline in the aquatic system. Furthermore, it should be noted that there are several additional tricyclic pharmaceuticals, e.g. nortriptyline and loratadine that have not been included in this study. In summary, only doxepine has been reported in levels comparable to that of carbamazepine but due to its low cross-reactivity of 0.8 % it is not prone to interfere significantly. All other pharmaceuticals seem to be present in the environment in concentrations 1 - 2 orders of magnitude lower than carbamazepine showing cross-reactivities below 10 %, thus resulting in a maximum positive bias of 1 %. In conclusion, tricyclic antidepressants do not affect accuracy of the CBZ ELISA established, when comparable surface waters are measured. In addition, we determined the molar CR of DiH-CBZ and CBZ-triglycine as 98 % and 190 %, respectively. Both compounds are unlikely to occur in the environment. They have been found neither in the human metabolic system [58, 74] nor during studies on CBZ photodegradation [75], oxidation [76] and ozonation [77]. Furthermore, DiH-CBZ has been used 14

as internal standard for CBZ quantitation via LC-MS/MS in previous studies [2, 34, 63]. However, the cross-reactivity of these compounds gives insight in the nature of the binding between antigen and antibody. Obviously, in CBZ-triglycine the antibody recognizes the molecular bridge that is attached to the carboxamide moiety in CBZ. This has been observed before for a long-chain biotin [78] and TNT derivates [79]. Furthermore, to some extent, the antibody seems to be “blind” for variations in the 10,11-position. For the binding process no significant difference can be noted whether carbamazepine’s double bond at that position is hydrogenated or epoxidized. However, hydroxy and carbonyl groups at that position prevent the antibody from binding 10OH-CBZ, DiOH-CBZ and OxCBZ, which all showed cross-reactivites below 1 %. The cross-reactivities give opportunity for a series of other immunoassays. In single compound studies this system can be used for the detection of opipramol, protriptyline, amitriptyline, imipramine and doxepine. The detection limits for these substances would be in the range of 0.5 - 5 µg/L which should be sufficient for clinical purposes.

ELISA validation SPE-LC-MS/MS reference method An LC-MS/MS method was established as a reference method for validation of the ELISA method. A solid-phase extraction procedure for clean-up and pre-concentration of samples was adapted from Barron et al. [80]. The complete SPE-LC-MS/MS method was tested for the influence of the matrix on recoveries. Pure water and one surface water sample were both spiked with 1 µg/L carbamazepine. After addition of IS the samples were handled and measured as described above. The relative recoveries (in relation to IS) were 100 ± 2 % and 99 ± 6 % (mean and standard deviation of 2 LC-MS/MS runs) for pure water and surface water, respectively. Absolute recoveries determined without internal standard were 94 ± 4 % and 104 ± 20 %, respectively. Direct injection of surface water without SPE was also tested and the results only slightly differed from the ones obtained after solid-phase extraction (Table 1). It can be 15

concluded that the sample’s matrix interference is compensated by internal standardisation. Yet, for routine analysis, a direct injection is often not feasible because it requires increased maintenance of the LC system and frequent cleaning of the mass spectrometer’s ion source. Sensitivity, accuracy and precision of the ELISA The median limit of detection (LOD) was established at a signal-to-noise ratio (S/N) of 3 from a blank sample to be 0.024 µg/L (n = 38), the median limit of quantitation (LOQ, S/N of 10) was 0.085 µg/L. The precision of the method can be visualized by the precision profile (Figure 2). An allowable maximum relative error < 30 % is obtained within a quantitation range between 0.05 and 50 µg/L, i.e. over 3 orders of magnitude. It is noteworthy that the quantitation range is not limited to the quasi-linear range around the test midpoint. For high doses it extends far into the asymptotic range due to a high precision of the determination in this interval. This finding graphically manifests in an asymmetric precision profile that is not centred around the inflection point. Accuracy of CBZ ELISA results has been investigated by comparing the findings to LC-MS/MS as the reference method. 10 Samples (9 surface water grab samples and one WWTP effluent sample) were analysed. We found CBZ in all ten samples, concentrations ranging from 0.4 to 2.4 µg/L (Table 1). Consistently higher concentrations have been found by the ELISA method. Correlation analysis between ELISA and SPE-LC-MS/MS results (c(ELISA) = 1.29 c(LC) + 0.01, r2 = 0.996) more clearly shows the close correlation between the findings of both methods, the ELISA showing a constant overestimation of ca. 30 % relative to the CBZ concentration by the reference method. We believe that most of this overestimation is caused by the cross-reacting metabolites EP-CBZ (83 % CR) and 2OH-CBZ (14 % CR) which are known to occur in the environment in significant amounts [61-63]. The concentrations of these metabolites have not been determined in this study. Furthermore, a surface water sample was spiked with different concentrations of CBZ in the range of 0.5 to 5 µg/L and the concentrations were determined with both ELISA and LC-MS/MS (without SPE). The 16

ELISA overestimated CBZ concentrations by a nearly constant absolute value of ca. 0.6 µg/L compared to the reference method (Figure 4). This systematic absolute overestimation might be caused by the aforementioned presence of cross-reacting metabolites in the sample. Both methods determined the resulting concentrations (background level + spike) with high precision. The precision very much depends on the concentration range measured. For the data shown in Figure 4 the average inter-assay and intra-assay coefficients of variance (CV) were 3.6 % and 5.7 %, respectively. The linear regression line from spiked samples (Figure 4) shows a slope of 1.0 and a correlation coefficient of r2 = 0.997 in the range of 2.3 to 7.5 µg/L. This indicates that there is no matrix effect caused by interferents, like humic acids. These interferents are known to inhibit the formation of antigen-antibody-complexes by unspecifically binding to the antibodies or by adsorption/complexation of the analyte [39, 40, 78]. This kind of interference is usually non-linear and/or concentration dependent, causing slopes different from unity [81].

Monitoring CBZ in surface waters and wastewater by ELISA The data used for the accuracy study in Table 1 originates from environmental samples. The highest CBZ concentration of 3.2 µg/L was found in the Teltowkanal of Berlin at km 35.5, close to one of the two effluents of WWTP Waßmannsdorf (km 34.7 and km 32.2). Receiving the discharge of three adjacent sewage treatment plants the Teltowkanal can be described as a worst-case scenario with respect to surface water quality consisting of as much as 84 % of wastewater in dry periods [15]. In 2002, Heberer extensively studied the presence of CBZ in Berlin waters and found a peak concentration of 1.1 µg/L in the Teltowkanal [15]. In the river Spree and in one of its branches, Britzer Verbindungskanal, we determined CBZ concentrations between 0.5 and 0.6 µg/L by ELISA. In the effluent of WWTP Ruhleben we found 1.8 µg/L by SPE-LC-MS/MS and 2.3 µg/L by CBZ ELISA. Zühlke et al. had monitored pollutants in Berlin’s sewage treatment plants in 2002 [20]. They found a maximum concentration of 2.10 µg/L and a median concentration of 1.65 µg/L (n = 17

12) in June/July 2002 in WWTP Ruhleben effluent. In a study including WWTP Waßmannsdorf a maximum concentration of 5.0 µg/L and an average concentration of 1.63 µg/L in 1999 [15] were found, respectively. In addition, we collected 18 samples from the Teltowkanal between kilometer 28 and its tributary, the river Dahme, at kilometer 37.5. Figure 5 shows the longitudinal concentration profile we observed in the channel. Starting at km 37.5, the CBZ concentration was found to be around 50 ng/L. A strong increase in CBZ concentration with a peak concentration of 2.7 µg/L occurs at km 35.5. This can be related to WWTP Waßmannsdorf that is discharging its effluent into the channel at km 34.7. The increase is well noticeable 800 m before the WWTP’s discharge which can be explained by the low flow velocity of the Teltowkanal causing diffusive mixing upstream. A similar scenario was found on the 8th of August 2008 when the CBZ concentration at km 35.5 (upstream WWTP) was higher than at km 29.5 (downstream WWTP). It is noteworthy that the pH value of the stream seems to be influenced by the WWTP’s discharge, too. pH changes of this magnitude (0.5 units) do not affect the ELISA due to the sample buffer used.

Conclusions A CBZ ELISA method has been developed that allows determination of environmental CBZ concentrations in different kinds of water samples. Neither tedious and expensive solid-phase extraction nor sample clean-up was necessary to achieve a detection limit of 24 ng/L with less than 1 mL of sample volume required. Concentrations up to 50 µg/L can be determined with good precision without prior dilution. The carbamazepine monoclonal antibody used in this study proved to have sufficient affinity and selectivity to establish an ELISA suitable for screening and monitoring purposes in surface waters and wastewater. This inexpensive and fast technology for CBZ monitoring is beneficial since many studies, including ours, revealed a large variation of diurnal, seasonal and local

18

concentrations that could be accounted for by immunochemical screening with frequent and locally close-meshed samplings. Since there is still no decisive progress in “bottling the immune system” [82, 83] antibody availability is still the major challenge in establishing an immunochemical method for the desired analyte. Even with 300,000 – 500,000 commercially available antibodies, good antibodies for trace analytical applications are still very scarce – and according to our experience, many antibodies listed are of very limited quality. Only a few of them might be useful for environmental analysis – after careful selection, thorough optimization of the assays and in-depth validation.

Acknowledgements A. Bahlmann wants to thank BAM Bundesanstalt für Materialforschung und -prüfung for a grant within its PhD program. The authors thank F. Finn for assistance in sample collection and characterisation, S. Flemig, BAM, for MALDI-TOF-MS measurements and A. Lehmann, BAM, for helpful discussions.

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Table 1.

Carbamazepine concentrations present in 10 water samples determined with

ELISA and LC-MS/MS

Sampling site and codea

Date

km

Carbamazepine concentration [µg/L] LC-MS/MSb

SPE-LC-MS/MS ELISA

Teltowkanal 1a

7th Jul 08

35.5

0.69 ± 0.01

0.63 ± 0.01

0.86 ± 0.01

Teltowkanal 2a

7th Jul 08

29.5

2.4 ± 0.04

2.2 ± 0.1

3.0 ± 0.2

Teltowkanal 3a

7th Jul 08

27.7

1.9 ± 0.03

1.8 ± 0.1

2.3 ± 0.03

Teltowkanal 1b

11th Aug 08

35.5

2.4 ± 0.1

2.4 ± 0.04

3.2 ± 0.1

Teltowkanal 2b

11th Aug 08

29.5

1.8 ± 0.03

1.8 ± 0.03

2.3 ± 0.1

th

Teltowkanal 3b

11 Aug 08

27.7

0.66 ± 0.10

0.57 ± 0.01

0.75 ± 0.05

Spree 1a

7th Jul 08

26.0

0.45 ± 0.01

0.40 ± 0.01

0.55 ± 0.01

Spree 1b

11th Aug 08

26.0

0.52 ± 0.09

0.45 ± 0.01

0.59 ± 0.04

Britzer Verbindungskanal

7th Jul 08

31.3

0.47 ± 0.01

0.41 ± 0.01

0.54 ± 0.01

WWTP Berlin-Ruhleben,

04th Apr 08

1.8 ± 0.03

1.8 ± 0.1

2.3 ± 0.1

effluent a

site code: numbers depict different locations along the stream, letters codify for different sampling dates

b

no sample pretreatment

23

Figures

Chemical structure

Name, CAS number (quantity prescribed)

Chemical structure Name, CAS number (quantity prescribed) 10,11-Dihydrocarbamazepine (DiH-CBZ)

Carbamazepine (CBZ) 298-46-4 Anticonvulsant (61.2 t)

N H2N

O

HO

OH

3564-73-6

10,11-Dihydro-trans-10,11dihydroxycarbamazepine (DiOH-CBZ)

N

58955-93-4 Metabolite of CBZ

H2 N

N H2 N

O

O

10,11-Epoxycarbamazepine (EP-CBZ)

O


N H2 N

O

HO OH N H2 N

2-Hydroxycarbamazepine (2OH-CBZ)

10,11-Dihydro-10-hydroxycarbamazepine (10OH-CBZ)


O

N

58955-93-4 Metabolite of OxCBZ and CBZ

H2 N

O

Acridine

Iminostilbene 256-96-2 Metabolite of CBZ

N H


N

Imipramine

Protriptyline

NH

438-60-8 Antidepressant (prescription data not available)

N N

Amitriptyline

Doxepine

N

CH3

50-49-7 Antidepressant (0.3 t)

CH3

CH3

O

CH3


N

CH3


CH3

CH3 O

Oxcarbazepine (OxCBZ) 28721-07-5 Anticonvulsant (12.3 t)

N H2 N

N-(Dibenz[b,f]azepine-5ylcarbonyl)glycylglycylglycine (CBZ-triglycine)

N O

O

81879-94-9 Precursor for enzyme conjugate

HN

Opipramol

O NH

N


N

OH N

O HN

O OH

Fig. 1 Metabolites of carbamazepine and structurally similar compounds examined in this study including molar and specific (mass-related) cross-reactivities (CR) of the CBZ immunoassay (average and standard deviation of three experiments). Annual prescription quantities for Germany (2006) were calculated from the number of prescriptions [11] and the defined daily doses [12]

24

Fig. 2 Calibration curve of ELISA and precision profile (dotted line shows moving average of three adjacent points) using 3 replicates per calibrator

Fig. 3 Relationship between ELISA test midpoint and concentrations of tracer and antibody in the reaction mixture

25

Fig. 4 CBZ in a surface water sample after spiking with different concentrations of CBZ. Mean values and standard deviation measured with ELISA (3 plates) and LC-MS/MS (2 injections)

Fig. 5 Carbamazepine concentration and pH value in the transport channel Teltowkanal in southern Berlin between kilometers 28 and 38 on October 1, 2008. Effluents of WWTP Waßmannsdorf are discharged at km 32.2 and 34.7. ELISA was done in duplicate (average and threefold of standard deviation shown)

26