Identification of a New Antidepressant and its Glucuronide

(9). Lamotrigine undergoes hepatic metabolism by the cytochrome P450 system. .... column of 150 mm ×4.6 mm and 5 μm particle size (Zorbax Eclipse XD...
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Anal. Chem. 2010, 82, 8161–8168

Identification of a New Antidepressant and its Glucuronide Metabolite in Water Samples Using Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry Imma Ferrer* and E. Michael Thurman Center for Environmental Mass Spectrometry, Department of Civil, Environmental and Architectural Engineering, University of Colorado, Boulder, Colorado 80309 This paper reports the first detections of an antidepressant, lamotrigine, and its major metabolite (2-N-glucuronide), in environmental water samples using a new chlorine mass-filter technique with accurate mass and high resolution. A quantitative method is described using solid phase extraction (SPE) followed by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS) for the simultaneous analysis of both compounds in aqueous samples, including drinking water, groundwater, surface water, and wastewater collected from sewage treatment plants. The recoveries of the analytes ranged from 75 to 99%, depending on the type of water extracted. The method detection limits were 1 and 5 ng/L for lamotrigine and its metabolite, respectively. The method was validated with more than a hundred aqueous samples analyzed and lamotrigine and its 2-N-glucuronide metabolite were mostly detected in both wastewater and surface water impacted sites at mean concentrations of 488 and 209 ng/L, respectively. Lamotrigine was detected in 94% of all the wastewater samples analyzed. Two detections for lamotrigine occurred in drinking water. To our knowledge, this is the first report of water samples containing lamotrigine, a relatively new drug used for the treatment of epilepsy and type I bipolar syndrome. It is also the first report of a glucuronide of an antidepressant surviving wastewater treatment plant operations and becoming a ground and surface water contaminant. Pharmaceutically active compounds, including drugs and their active metabolites, are an important, if not dominant, water-quality issue both scientifically1 and to the lay public.2,3 Pharmaceuticals are important because of their possible impact on humans,4,5 * To whom correspondence should be addressed. E-mail: imma.ferrer@ colorado.edu. (1) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Environ. Sci. Technol. 2002, 36, 1202–1211. (2) Pharmaceuticals found in Drinking Water. Associated Press, March 9, 2008. (3) Donn, J. Tons of Released Drugs Taint U.S. Water. U.S. News and World Report, 2009. (4) Stackelberg, P. E.; Furlong, E. T.; Meyer, M. T.; Zaugg, S. D.; Henderson, A. K.; Reissman, D. B. Sci. Total Environ. 2004, 329, 99–113. (5) Schultz, M. M.; Furlong, E. T. Anal. Chem. 2008, 80, 1756–1762. 10.1021/ac1014645  2010 American Chemical Society Published on Web 09/03/2010

wildlife, and fish.6 This is especially true for the psychoactive medications, such as antidepressants, which have targets in the brain that control mood stabilization.7,8 Lamotrigine, also known as Lamictal (6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine), is an important drug for the treatment of epilepsy, which is the second most common central nervous system disease after stroke. Lamotrigine has been available as an anticonvulsant for more than 15 years9 and has been widely prescribed along with carbamazepine. It was approved by the FDA in 1994 for seizures, and it is relatively new compared to carbamazepine, which was approved in 1974. Its efficacy, together with its acceptable safety profile, has made lamotrigine a common choice as an antiepileptic drug for a wide range of seizure disorders in children and adults. Lamotrigine is a first choice for several disorders including trigeminal neuralgia and as a psychotropic agent.9 Approximately 10 years ago, lamotrigine along with carbamazepine was introduced in clinical psychiatry for the treatment of schizophrenia because of its mood stabilizing properties. Since June 2003, it is also used for the treatment of bipolar disorder, which is a serious disease afflicting over 1% of adults in the United States. Finally, lamotrigine may be combined with other drugs for the treatment of alcohol withdraw.9 Lamotrigine undergoes hepatic metabolism by the cytochrome P450 system.7,8 Two metabolites of lamotrigine have been identified from human urine. The main metabolic pathway of lamotrigine is conjugation with glucuronide.7,8 A lesser metabolite is the formation of 2-N-methyl-lamotrigine. The glucuronide metabolite is a major pathway for excretion through the kidneys. It is less toxic than the parent compound but can undergo simple hydrolysis back to the parent. The metabolite does show some activity similar to the parent. Environmental field studies have not been reported for lamotrigine, to our knowledge, and that of an extensive literature search. However, a detailed study of carbamazepine and its metabolites by Miao an Metcalfe10,11 do mention that lamotrigine is used in conjunction with carbamazepine. The two papers by (6) Vajda, A. M.; Barber, L. B.; Gray, J. L.; Lopez, E. M.; Woodling, J. D.; Norris, D. O. Environ. Sci. Technol. 2008, 42, 3407–3414. (7) Ohman, I.; Beck, O.; Vitols, S.; Tomson, T. Epilepsia 2008, 49, 1075–1080. (8) Saracino, M. A.; Bugamelli, F.; Conti, M.; Amore, M.; Raggi, M. A. J. Sep. Sci. 2007, 30, 2249–2255. (9) Lamictal Prescribing Information; GlaxoSmithKline: Brentford, Middlesex, UK, 2007. (10) Miao, X. S.; Metcalfe, C. D. Anal. Chem. 2003, 75, 3731–3738.

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Table 1. Elemental Composition, Protonated Molecules, And Chemical Structures of Lamotrigine, Its 2-N-Glucuronide and Lamotrigine Labeled Standard

Miao and Metcalfe10,11 on the distribution of carbamazepine are useful and insightful into the importance of lamotrigine, which is used nearly as frequently as carbamazepine. Nonetheless there are no monitoring data, occurrence data, or fate data available on lamotrigine or its metabolites in water samples. This is in spite of more than 500 papers dealing with pharmaceutical occurrence in surface and wastewaters, based on several recent reviews.12,13 Our aim was to develop and apply a sensitive, specific, and reproducible analytical technique for the simultaneous determination of lamotrigine and its major metabolite (Table 1) in various aqueous environmental samples using solid phase extraction (SPE), followed by analysis with liquid chromatography/quadrupole timeof-flight mass spectrometry with accurate mass (LC/Q-TOF-MS). EXPERIMENTAL SECTION Chemicals and Reagents. Lamotrigine and its 2-N-glucuronide were purchased from Sigma-Aldrich (St. Louis, MO), and from Carbosynth (Compton, UK), respectively. Lamotrigine-13C3d3 labeled standard was purchased from Toronto Research Chemicals (North York, Ontario, Canada). HPLC grade acetonitrile and methanol were obtained from Burdick and Jackson (Muskegon, MI). Formic acid was obtained from Sigma-Aldrich (St. Louis, MO). A Milli-Q-Plus ultrapure water system from Millipore (Milford, MA) was used throughout the study to obtain the HPLC-grade water used during the analyses. (11) Miao, X. S.; Yang, J. J.; Metcalfe, C. D. Environ. Sci. Technol. 2005, 39, 7469–7475. (12) Calisto, V.; Esteves, V. I. Chemosphere 2009, 77, 1257–1274. (13) Corcoran, J.; Winter, M. J.; Tyler, C. R. Crit. Rev. Toxicol. 2010, 40, 287– 304.

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Individual pharmaceutical stock solutions (1000 µg/mL) were prepared in pure methanol and stored at -18 °C. From these solutions, working standard solutions were prepared by dilution with acetonitrile and water. Sample Collection. All samples were collected in baked, glass, 1 -L, amber bottles with Teflon lined caps to ensure sample integrity. The bottle head space was kept to a minimum by filling the bottles to the top. The bottles were rinsed in the field three times with sample and filled to the top on the fourth sampling. Disposable gloves were used by the sampler to prevent any personal care products from contaminating the sample bottles. All water samples were stored on ice before analysis, and sample extraction was completed within 7-days for all samples. No additives were placed in the samples to prevent contamination and sorptive removal and to leave any residual chlorine in drinking water to mimic the use of tap water that is commonly stored in pipes and houses before consumption. All samples were kept refrigerated at e4 °C from the time of collection until extraction by placing all samples in an appropriate ice-chest filled with blue ice packets. The field pH and specific conductance were recorded for the samples. Samples of drinking water, groundwater, surface water, and wastewater were collected from ten locations including locations in Alabama, Colorado, Kansas, Nebraska, Ohio, New York, Minnesota, Texas, and Utah. The wastewater samples were collected at the outfall of the treatment plant and not in the treatment plant itself; thus, these samples represent the impact to the source water and eventually to drinking water. Source water river samples were collected following standard U.S. Geological Survey (USGS) protocol14 and were taken by integrating sampling

across the river, when possible. If this were not available, then a grab sample was taken. Grab samples were taken from the rapid area of the stream or river where the majority of flow was occurring. Groundwater samples were collected from wells. The major issue in groundwater sampling is that the flow from the well is sufficient that at least three pore volumes have been passed before the sample is taken. Sample Extraction. An off-line SPE was used for the preconcentration of the water samples. All the extraction experiments were performed using an automated sample preparation with extraction columns system (GX-271 ASPEC, Gilson, Middleton, WI) fitted with a 25 mL syringe pump for dispensing the water samples through the SPE cartridges. Water samples were extracted with Oasis HLB cartridges (500 mg, 6 mL) obtained from Waters (Milford, MA). The cartridges were conditioned with 4 mL of methanol followed by 6 mL of HPLC-grade water at a flow rate of 1 mL/min. The water samples (200 mL) were loaded at a flow rate of 10 mL/min. Elution of the analytes from the cartridge was carried out with 5 mL of methanol. The solvent was evaporated to 0.5 mL with a stream of nitrogen at a temperature of 45 °C in a water bath using a Turbovap concentration workstation (Caliper Life Sciences, Mountain View, CA). The samples were transferred to vials and analyzed by LC/Q-TOFMS. LC/Q-TOF-MS Analyses. The separation of the analytes was carried out using an HPLC system (consisting of vacuum degasser, thermostatted autosampler, column compartment and a binary pump) (Agilent Series 1200, Agilent Technologies, Santa Clara, CA) equipped with a reversed phase C8 analytical column of 150 mm ×4.6 mm and 5 µm particle size (Zorbax Eclipse XDBC8). Column temperature was maintained at 25 °C. The injected sample volume was 50 µL. Mobile phases A and B were acetonitrile and water with 0.1% formic acid, respectively. The optimized chromatographic method held the initial mobile phase composition (10% A) constant for 5 min, followed by a linear gradient to 100% A after 30 min. The flow-rate used was 0.6 mL/min. A 10 min postrun time was used after each analysis. This HPLC system was connected to an ultra high definition quadrupole time-of-flight mass spectrometer model 6540 Agilent (Agilent Technologies, Santa Clara, CA) equipped with electrospray Jet Stream Technology, operating in positive ion mode, using the following operation parameters: capillary voltage: 4000 V; nebulizer pressure: 45 psig; drying gas: 10 L/min; gas temperature: 325 °C; sheath gas flow: 11 L/min; sheath gas temperature: 350 C; nozzle voltage: 1000 V, fragmentor voltage: 190 V; skimmer voltage: 45 V; octopole RF: 750 V. LC/MS accurate mass spectra were recorded across the range 50-1000 m/z at 4 GHz. The data recorded was processed with MassHunter software. Accurate mass measurements of each peak from the total ion chromatograms were obtained by means of an automated calibrant delivery system using a low flow of a calibrating solution (calibrant solution A, Agilent Technologies), which contains the internal reference masses (purine (C5H4N4 at m/z 121.0509 and HP-921 (hexakis(1H,1H,3H-tetrafluoro-pentoxy)phosphazene) (C18H18O6N3P3F24) at m/z 922.0098. The instrument worked providing a typical mass resolving power of 40 000 ± 500 (m/z 1522).

Method Validation. Recovery experiments with spiked samples were performed to determine precision and accuracy of the method. Drinking water, groundwater and surface water free of lamotrigine and its metabolite were spiked at 100 ng/L and extracted by SPE and analyzed by LC/TOF-MS. Peak areas for the extracts were compared to peak areas corresponding to a pure standard prepared in DI water, and recovery values were obtained. Since a sample of wastewater free of lamotrigine could not be obtained, the percent recoveries of the analytes spiked into wastewater samples were calculated as the measured spiked concentration minus the concentration in the original sample. The value obtained was then divided by the spiked concentration and multiplied by 100. The method detection limits were defined as the lowest concentration of the analytes that yielded an ion signalto-noise ratio of 3:1. HPLC-grade water, drinking water, groundwater, surface water, and wastewater were used as sample matrices to investigate matrix effects. To avoid losses due to extraction, the extracts from each of the matrices were spiked with the standard solution after the extraction procedure and then analyzed using LC/TOF-MS. The signal suppression was calculated by comparing the areas obtained in reagent water and the areas obtained in each sample matrix. Peak areas, regression parameters, and concentrations were obtained by using the quantification software of Agilent Mass Hunter. Aliquots of standard solutions of analytes were added to water samples at seven different concentrations to obtain the standard calibration curves, all of which went through the SPE system and treated like samples. To ensure accuracy, a calibration curve was developed for each type of matrix sample. An aliquot of 100 µL of surrogate labeled standard, lamotrigine-13C3-d3, was added to each calibration sample and to each environmental sample. The internal standard was used to account for recovery losses during SPE and any suppression from the matrix of the samples. RESULTS AND DISCUSSION Initial Discovery of Lamotrigine in Water Samples. The initial identification of lamotrigine in water samples was accomplished using a mass-defect filter that looks for chlorinated analytes in the extract of a wastewater sample after LC/TOF-MS analysis in MS-only mode. The mass defect filter essentially looks at the accurate mass of the monoisotopic mass of an analyte and the A+2 isotopic mass. Both the intensity and the accurate mass are used to detect chlorinated compounds using the mass defect filter. In the case of lamotrigine, the mass defect filter detected a peak at 13.7 min with a mass of m/z 256.0153 and an A+2 isotope with a mass of m/z 258.0122 and an intensity of 66% (see Figure 1S, Supporting Information (SI)). The mass defect filter showed that the A+2 peak had a relative isotopic mass defect of -0.0030 u, indicating a chlorinated compound with two chlorine atoms.15,16 The second step after the mass defect filter was to determine the molecular formula of the unknown chlorinated compound. The (14) National Field Manual for the Collection of Water-Quality Data; U.S. Geological Survey: Reston, VA, 2008; http://water.usgs.gov/owq/FieldManual. (15) Thurman, E. M.; Ferrer, I.; Zweigenbaum, J. A. Anal. Chem. 2006, 78, 6703–6708. (16) Thurman, E. M.; Ferrer, I.; Zweigenbaum, J. A.; Garcia-Reyes, J. F.; Woodman, M.; Fernandez-Alba, A. R. J. Chromatogr., A 2005, 1082, 71– 80.

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Figure 1. LC/Q-TOF-MS analysis of a surface water sample showing the MS-MS spectrum of peak at 13.7 min.

best fit for the ion formula was C9H8Cl2N5 with a match of 99 out of 100 based on MassHunter Software, which evaluates accurate mass of the A ion, the isotope matching for intensity, and spacing or accurate mass of the isotopes. The neutral formula, C9H7Cl2N5, was then run through the Merck Index database for a formula match and gave lamotrigine as its only formula. When the formula was put through a much larger database, ChemSpider, the match was for 65 compounds; however, there were only 13 patented structures and only 1 compound was listed in Wikipedia-available article and that was lamotrigine. A quick read showed that this compound is the number three most used bipolar medication in the U.S. at this time; thus, it was given the most likelihood of a correct identification. Next we performed an MS-MS experiment on the unknown peak using LC/Q-TOF-MS, which gave the complex and rich spectrum shown in Figure 1. The major fragment ions were identified by using the isotope cluster information and the formula generator in the software, which generated the elemental compositions shown also in Figure 1. After purchase of the lamotrigine standard, an LC/Q-TOF-MS analysis was performed and the retention time and accurate masses of the protonated molecule and all its fragment ions were verified against the initial batch of experiments carried out previously. Identification of 2-N-Glucuronide Metabolite. A second peak of much less intensity and earlier retention time (9.9 min) had been detected in the 256 m/z extracted ion chromatograms of several wastewater samples containing lamotrigine (Figure 2a). The spectrum of this peak revealed a much larger ion at 432.0472 m/z, thus an MS-MS experiment was carried out to confirm that the 256 ion formed indeed from the 432 ion, as shown in Figure 2a. A literature search for the empirical formula C15H15Cl2N5O6 (at 432.0472 m/z) revealed that this was a potential glucuronide metabolite of lamotrigine.7,8 The finding was verified by analyzing a pure standard of 2-N-glucuronide lamotrigine. Both retention times and accurate masses matched perfectly with the experimental peak observed at 9.9 min. A second MS-MS experiment was performed to fragment the ion at m/z 256, hence simulating 8164

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a pseudo MS3 experiment, and a spectrum that matched that of lamotrigine was obtained, thus totally confirming the identification of the 2-N-glucuronide metabolite (see Figure 2b). Proposed Fragmentation Pathways. Figure 3 shows the detailed fragmentation pathway for lamotrigine. Several major fragments containing one or two chlorine atoms were obtained by MS-MS. This molecule undergoes a complex fragmentation pathway through several losses occurring mainly in the triazine ring and further rearrangement of the chemical moiety. Accurate mass measurements were essential to propose exact chemical structures to the main fragments obtained by MS-MS. Two fragment ions were odd electron ions and the rest were all even electron ions. On the other hand, the 2-N-glucuronide metabolite showed the classic fragmentation pathway of a glucuronide to give rise to the 256.0151 m/z ion, after cleavage of the glucuronic moiety with an exact neutral mass of 176.0321. In general, the mass accuracies averaged 0.3 millidaltons (less than 2 ppm mass accuracy) for all the ions measured, including the isotope mass accuracies. Table 1S in the SI summarizes the main fragment ions and accurate mass errors obtained in real samples compared to calculated values. The accurate mass analysis of the protonated molecule together with that of additional characteristic fragment ion(s) (including characteristic isotopic signals and retention times) enables the unambiguous identification and confirmation of the two analytes at low concentration levels in MS-MS mode. This fits the requirements of the EU according to the identification point system, including resolving power for all product ions of greater than 20,000 at fwhm.17 Extraction Efficiency and Matrix Effects. The extraction of lamotrigine and its glucuronide metabolite from water samples was investigated using Oasis HLB cartridges. The mean overall recoveries and analytical precision from different types of water are shown in Table 2. The recoveries ranged from 75 to 99% in the aqueous samples, with standard deviations of 5-10%. The best recoveries were obtained in drinking water and groundwater, and (17) Commission Directive 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results, Official Journal L221/8.

Figure 2. (a) LC/Q-TOF-MS analysis of a wastewater sample showing the MS-MS spectrum of peak at 9.9 min and (b) pseudo MS3 for ions at m/z 432 and 256, respectively.

the lowest recoveries were in wastewater samples due to the coextraction of matrix interferences and matrix suppression as commented below. These results show that both analytes can be efficiently extracted and isolated from environmental water samples and further analyzed by LC-MS techniques. Electrospray ionization is usually susceptible to matrix-related signal suppression, which is believed to result from the competition between the analyte ions and matrix components for access to the droplet surface in gas phase.18 Therefore the presence of coextracted matrix components may affect analyte quantitation by LC/Q-TOF-MS. For this reason several types of water samples (drinking water, groundwater, surface water and wastewater) were chosen to investigate matrix effects. Figure 2S in SI shows the effect of different matrices on the ion intensity of lamotrigine and its metabolite. Groundwater did not show ion suppression for lamotrigine, but more severe suppression occurred with surface water and wastewater, as seen in this Figure. The unexpected matrix suppression for drinking water can be attributable to the presence of adjuvants or treatment substances such as alum and/ or organic coagulants. On the other hand, the 2-N-glucuronide metabolite of lamotrigine did not show significant suppression in any type of water. This compound has an early retention time (of about 10 min) eluting ahead of all the natural organic matter (18) Pascoe, R.; Foley, J. P.; Gusev, A. Anal. Chem. 2001, 73, 6014–6023.

interferents, therefore does not suffer a significant suppression similar to the parent compound, which comes in the middle of the humic signal in the chromatogram. The use of isotopically labeled standards is preferred in a quantitative mass spectrometric method and a labeled standard was used for this work when analyzing environmental water samples. Analytical Performance. To evaluate the usefulness of LC/ Q-TOF-MS for quantitative analyses of lamotrigine and its glucuronide in aqueous matrices, the analytical performance of the proposed method was studied and validated in terms of linearity, limits of detection and interday precision of the technique. Quantitation of the sample extracts was accomplished using a calibration curve based on matrix-matched standards: blank sample extracts from different sources were spiked with the standard solution at different concentrations ranging from 10 to 5000 ng/L in order to have a wide range of concentrations, extracted and analyzed, and they were used as external calibration. The internal standard was spiked at 80 ng/L in all the working standard solutions and used as a surrogate. Calibration charts were found to be linear between these concentrations. Values for the coefficient of determination, R2, were >0.99 for the two analytes. Quantitation was carried out in MS-only mode using the peak area from the extracted ion chromatograms (EIC) of the base Analytical Chemistry, Vol. 82, No. 19, October 1, 2010

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Figure 3. Fragmentation pathway for lamotrigine. Table 2. Recoveries (in %) and Standard Deviations (RSD) of Lamotrigine and Its Glucuronide Metabolite from Drinking Water, Groundwater, Surface Water and Wastewatera analyte lamotrigine lamotrigine 2-N-glucuronide

drinking water

groundwater

surface water

wastewater

91(5) 99(7)

95(6) 98(8)

82(7) 93(7)

75(10) 77(8)

a Recoveries are the average of 5 determinations at a concentration of 100 ng/L.

peak ion (in bold in Table 1S in SI), using a mass window of 0.1 Da, and the peak area from the EIC of the labeled lamotrigine standard. The analytical performance of the methodology for water samples is summarized in a Table (SI Table S2). Method detection limits for lamotrigine and its metabolite were 1 and 5 ng/L, respectively. Method quantitation limits were 10 and 50 ng/L, respectively. The method limits of detection (LODs) were estimated from the injection of spiked wastewater samples with low concentration levels giving a signal-to-noise ratio of 3 for the 8166

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quantifier ion. The first concentration level of the calibration curve corresponded to the method LOQ for each compound. The interday precision and repeatability of the method were also evaluated on extracted water samples at different concentration levels. The RSD (n ) 5) values for intraday analyses were in the range 1-3% and the RSD for interday (n ) 5) values were between 3 and 8%, showing good reproducibility of the methodology. Identification of Lamotrigine and Its Glucuronide in Environmental Water Samples. A variety of water samples, including wastewater, groundwater, surface water, and drinking water were analyzed for lamotrigine and its major metabolite. A total of 118 water samples were analyzed from 2008 to 2010 with the methodology described in this paper. Figure 3S in SI illustrates the chromatogram for an effluent sample from a location near Estes Park (Colorado), showing the presence of lamotrigine in the extracted ion chromatogram. In general, and for all the samples analyzed, the presence of lamotrigine and its main metabolite was confirmed by extracting the 256 m/z ion and by accurate mass of the corresponding protonated molecules for both compounds followed by MS-MS experiments with accurate mass at high resolving power (greater than 25 000 at fwhm). Table 3 summarizes the median concentrations and percentage of detec-

Table 3. Analysis of Representative Wastewater, Groundwater, Surface Water and Drinking Water from Different Locations in the U.S. Showing Concentrations for Lamotrigine and its 2-N-Glucuronide samples

lamotrigine

lamotrigine 2-N-glucuronide

wastewater (34 samples) mean concentration (ng/L) 488 median concentration (ng/L) 443 percentage detections (%) 94

209