Trace Determination of Macrolide and Sulfonamide Antimicrobials, a

Jul 10, 2004 - Trace Determination of Macrolide and Sulfonamide Antimicrobials, a Human Sulfonamide Metabolite, and Trimethoprim in Wastewater Using ...
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Anal. Chem. 2004, 76, 4756-4764

Trace Determination of Macrolide and Sulfonamide Antimicrobials, a Human Sulfonamide Metabolite, and Trimethoprim in Wastewater Using Liquid Chromatography Coupled to Electrospray Tandem Mass Spectrometry Anke Go 1 bel,* Christa S. McArdell, Marc J.-F. Suter, and Walter Giger

Swiss Federal Institute for Environmental Science and Technology (EAWAG), CH-8600 Du¨bendorf, Switzerland

An analytical method has been developed and validated for the simultaneous trace determination of four macrolide antibiotics, six sulfonamides, the human metabolite N4acetylsulfamethoxazole, and trimethoprim in wastewater. The method was validated for tertiary, secondary, ands unlike in previously published methodssalso for primary effluents of municipal wastewater treatment plants. This wide range of application is necessary to thoroughly investigate the occurrence and fate of chemicals in wastewater treatment. Wastewater samples were enriched by solid-phase extraction, followed by reversed-phase liquid chromatography coupled to tandem mass spectrometry using positive electrospray ionization. Recoveries from all sample matrixes were generally above 80%, and the combined measurement uncertainty varied between 2 and 18%. Concentrations measured in tertiary effluents ranged between 10 ng/L for roxithromycin and 423 ng/L for sulfamethoxazole. Corresponding levels in primary effluents varied from 22 to 1450 ng/L, respectively. Trace amounts of these emerging contaminants reach ambient waters, since all analytes were not fully eliminated during conventional activated sludge treatment followed by sand filtration. In the case of sulfamethoxazole, the amount present as human metabolite N4-acetylsulfamethoxazole had to be taken into account in order to correctly assess the fate of sulfamethoxazole in wastewater treatment. Since 1997, interest in the occurrence and behavior of pharmaceuticals in the aquatic environment has significantly increased.1-7 One motivation for this attention is the fact that these chemicals are designed to trigger specific biological effects and, * Corresponding author. E-mail: [email protected]. Fax: +41 1 823 5311. Voice: +41 1 823 5371. (1) Stan, H. J.; Heberer, T. Analusis Mag. 1997, 27, 20-23. (2) Ternes, T. A. Water Res. 1998, 32, 3245-3260. (3) Halling-Sorensen, B.; Nors Nielson, S.; Lanzky, P. F.; Ingerslev, F.; Holten Lu ¨ tzenhoft, H. C.; Jorgensen, S. E. Chemosphere 1998, 36, 357-393. (4) Daughton, C. D.; Ternes, T. A. Environ. Health Perspect. 1999, 107, 907938. (5) Ku ¨ mmerer, K. Chemosphere 2001, 45, 957-969. (6) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Buxton, H. T. Environ. Sci. Technol. 2002, 36, 1202-1211. (7) Heberer, T. Toxicol. Lett. 2002, 131, 5-17.

4756 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

hence, pose a potential threat for the aquatic environment. In the case of antimicrobial agents (including both naturally and synthetically derived compounds), the possible maintenance and spread of bacterial resistance is a major point of concern. In 1997, the human consumption of antibacterials in Switzerland exceeded 30 tons per annum, (t/a).8-10 In addition ∼55 t/a of antimicrobials were used in veterinary medicine. Macrolides (4.3 t/a), sulfonamides (5.1 t/a), and fluoroquinolones (3.9 t/a) represent the most important groups in human medicine, next to β-lactams (17.5 t/a). The latter include penicillins and cephalosporins and seem to be hydrolyzed shortly after excretion. In industrialized countries, most human use antimicrobials and other pharmaceuticals reach the aquatic environment, unchanged or transformed, mainly via discharge of effluents from municipal wastewater treatment plants (WWTPs). The residual concentrations of these bioactive compounds in the treated effluents depend on their removal during wastewater treatment. They can potentially pose a hazard for aquatic organisms if the removal is incomplete. In addition, exposure via sewage sludge disposal on land could represent a hazard for soil organisms. Detailed knowledge of the behavior of antimicrobials in wastewater treatment and the aquatic environment will help to achieve a reliable basis for environmental risk assessment (e.g., by providing measured environmental concentrations, MECs). MECs can be used in environmental risk assessment studies since they provide accurate indications of actual concentrations present in environmental systems. Investigations on the occurrence and fate of antimicrobial agents in various wastewater treatment steps can be exploited in order to evaluate wastewater treatment technologies with respect to elimination of specific contaminants. Reducing the release of residual pharmaceuticals into the aquatic environment would presumably decrease any potential environmental risks. By monitoring receiving surface waters as well as wastewater treatment plants, locations of particular concern can be identified and mitigated specifically. (8) Annual Report; Swiss Importers of Antibiotics (TSA): Berne, Switzerland, 1998. (9) Pharmaceuticals Sold in Switzerland; Swiss Market Statistics, 1999. (10) Antibiotics Used in Veterinary Medicine; Swiss Federal Office for Agriculture (BLW): Berne, Switzerland, 2001. 10.1021/ac0496603 CCC: $27.50

© 2004 American Chemical Society Published on Web 07/10/2004

To reach the aims stated above, selective and sensitive analytical methods for many different sample matrixes are essential. Until now, published methods for antimicrobial agents have focused on wastewater treatment plant effluents and surface waters,11-17 with the exception of fluoroquinolones, which were studied in detail by Golet et al.18-21 Analytical methods for wastewater matrixes other than final effluents including sludge extracts, however, are lacking. Another important aspect that has not yet been sufficiently addressed is the presence of human metabolites of antibacterials in wastewaters. Sulfamethoxazole, for example, is metabolized in the human body and ∼50% of the administered dose is excreted as the inactive human metabolite N4-acetylsulfamethoxazole and only 10% as the unchanged compound.22 The retransformation of N4-acetylsulfamethazine to the active sulfamethazine during the storage of manure has already been shown by Berger et al., suggesting a similar cleavage of N4acetylated sulfonamides, for instance in wastewater treatment.23 Observed elimination rates may be biased, if the possible retransformation to the active pharmaceutical is not considered. To the best of our knowledge, only one study included N4-acetylsulfamethoxazole in the analysis of surface water and WWTP effluentssindicating concentrations of up to 2200 ng/L in WWTP effluents.24 Unfortunately, the state of treatment has not been reported. This clearly shows the importance of considering the main human metabolite of sulfonamides when assessing the occurrence and fate of sulfamethoxazole in wastewater treatment. In this article, we present a reliable analytical method for the trace determination of the most important macrolide and sulfonamide antibiotics in the various aqueous compartments of a WWTP, including primary effluent. In addition, the human metabolite N4-acetylsulfamethoxazole and trimethoprimsfrequently used as a synergist to sulfamethoxazoleswere measured. Table 1 lists the selected macrolides and sulfonamides; their respective chemical structures are given in Charts 1 and 2. Using solid-phase extraction combined with liquid chromatography tandem mass spectrometry (positive electrospray ionization), concentrations down to the low-nanogram per liter range can be determined. The presented method is feasible to study the occurrence and fate of (11) Hirsch, R.; Ternes, T. A.; Haberer, K.; Mehlich, A.; Ballwanz, F.; Kratz, K.L. J. Chromatogr., A 1998, 815, 213-223. (12) Sacher, F.; Lange, F. T.; Brauch, H.-J.; Blankenhorn, I. J. Chromatogr., A 2001, 938, 199-210. (13) Lindsey, M. E.; Meyer, M.; Thurman, E. M. Anal. Chem. 2001, 73, 46404646. (14) Hartig, C.; Storm, T.; Jekel, M. J. Chromatogr., A 1999, 854, 163-173. (15) McArdell, C. S.; Molnar, E.; Suter, M. J.-F.; Giger, W. Environ. Sci. Technol. 2003, 37, 5479-5486. (16) Giger, W.; Alder, A. C.; Golet, E. M.; Kohler, H.-P. E.; McArdell, C. S.; Molnar, E.; Siegrist, H. R.; Suter, M. J.-F. Chimia 2003, 57, 485-491. (17) Vanderford, B. J.; Pearson, R. A.; Rexing, D. J.; Snyder, S. Anal. Chem. 2003, 75, 6265-6274. (18) Golet, E. M.; Alder, A. C.; Hartmann, A.; Ternes, T. A.; Giger, W. Anal. Chem. 2001, 73, 3632-3638. (19) Golet, E. M.; Strehler, A.; Alder, A. C.; Giger, W. Anal. Chem. 2002, 74, 5455-5462. (20) Golet, E. M.; Alder, A. C.; Giger, W. Environ. Sci. Technol. 2002, 36, 36453651. (21) Golet, E. M.; Xifra, I.; Siegrist, H. R.; Alder, A. C.; Giger, W. Environ. Sci. Technol. 2003, 37, 3243-3249. (22) Vree, T. B.; Hekster, Y. A. Pharmacokinetics of sulfonamides revisited; Karger: Basel, 1985. (23) Berger, K.; Petersen, B.; Buening-Pfaue, H. Arch. Lebensmittelhyg. 1986, 37, 85-108. (24) Hilton, M. J.; Thomas, K. V. J. Chromatogr., A 2003, 1015, 129-141.

Table 1. Investigated Compounds name

acronym

sulfadiazine sulfathiazole sulfamethazine sulfapyridine sulfamethoxazole N4-acetylsulfametoxazole trimethoprim azithromycin erythromycin clarithromycin roxithromycin tylosina josamycina sulfamerazinea

SDZ STZ SMZ SPY SMX N4AcSMX TRI AZI ERY CLA ROX TYL JOS SMR

a

CAS 68-35-9 72-14-0 57-68-1 144-83-2 723-46-6 738-70-5 83905-01-5 114-07-8 81103-11-9 80214-83-1 1401-69-0 16846-24-5 127-79-7

pKa 1/pKa 2a 6.428/2.129 7.228/2.129 7.428/2.329 8.428/2.630 5.728/1.829 5.028 7.231 8.726/9.526 8.827 8.926 9.227 7.126 7.028/2.229

Used as internal standards.

the selected compounds in all compartments of a wastewater treatment plant as well as for environmental monitoring studies. Preliminary results on the occurrence of macrolides and sulfonamides in Swiss wastewater treatment plants are presented. EXPERIMENTAL SECTION Chemicals and Reagents. HPLC-grade methanol, acetonitrile, and water are purchased from Scharlau (Barcelona, Spain). Analytical ethyl acetate, ammonia solution, 25% sulfuric acid, sodium chloride, sodium hydroxide, ammonium acetate, and formic acid were obtained from Merck (Darmstadt, Germany). Sulfamethazine, sulfamethoxazole, sulfadiazine, and roxithromycin were purchased from Sigma-Aldrich (Buchs, Switzerland). Sulfathiazole, sulfapyridine, trimethoprim, tylosin, josamycin, and erythromycin were obtained from Fluka Chemicals (Buchs, Switzerland), and sulfamerazine was from Riedel-de Hae¨n (Seelze, Germany). Sulfamethazine-phenyl-13C6 was purchased from Cambridge Isotope Laboratories (Andover, MA), and sulfamethoxazoled4, sulfadiazine-d4, sulfathiazole-d4, and N4-acetylsulfamethoxazoled5 were purchased from Toronto Research Chemicals (North York, ON, Canada). Clarithromycin was kindly supplied by Abbott (Wiesbaden, Germany) and azithromycin by Pfizer (Zurich, Switzerland). Azithromycin is also commercially available from Sigma-Aldrich (Buchs, Switzerland). Standard solutions for erythromycin-H2O were prepared from erythromycin as described by McArdell et al.15 The acidic solution was readjusted to pH 6 after 4 h using 1 M NaOH to ensure stability during storage. N4Acetylsulfamethoxazole was synthesized by acetylation with acetic acid anhydrate according to Neumann with a yield of 70%.25 Identity and purity was confirmed by LC/UV, LC/MS/MS, and H NMR analysis. Internal Standards. Deuterated sulfonamide standards were commercially available in most cases. Erythromycin-13C2 was tested as an internal standard for the macrolides but proved to be unsuitable due to the significant natural contribution to M + 2 from unlabeled erythromycin (11.6%). Similar observations were described by Vanderford and co-workers for erythromycin-13C1.17 The absence in water samples of all internal standards used was confirmed by enriching a representative samples from each matrix, to which no surrogate or instrumental standard was added. No (25) Neumann, J., Ph.D. Thesis in Pharmacy, Free University of Berlin, 1989.

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Chart 1. Chemical Structures of Macrolide Antibiotics

Chart 2. Chemical Structures of Sulfonamide Antibiotics and Trimethoprim

peaks could be detected at the retention times of the used internal standards. Surrogate standards were added prior to enrichment to assess possible losses during the analytical procedure. Instrumental standards were added to the final extracts prior to measurement. The following substances were used as surrogate standards: sulfamethazine-phenyl-13C6 (13C6SMZ) for SMZ, TRI, and SPY, sulfamethoxazole-d4 (d4SMX) for SMX, sulfadiazine-d4 (d4SDZ) for SDZ, sulfathiazole-d4 (d4STZ) for STZ, N4-acetylsulfamethoxazole-d5 (d5N4AcSMX) for N4AcSMX, and tylosin (TYL) for ERY-H2O, AZI, CLA, and ROX. As instrumental standards sulfamerazine (SMR) was used for all sulfonamides and TRI, and 4758

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josamycin (JOS) for all macrolides. While the surrogate standards were used for quantification, the instrumental standards were used to check the instrument performance during measurement. Its peak area was monitored over the whole measurement series in order to detect problems with, for example, instrument sensitivity or injection volume. If the area of the instrumental standard decreases significantly (signal reduction of >20% within same matrix), the series was stopped and the instrument cleaned. Sample Collection and Preparation. Flow-proportional composite samples of the primary effluent (1°EFFL) after mechanical treatment, the secondary effluent (2°EFFL) after biological treat-

ment, and the tertiary effluent (3°EFFL) after sand filtration were collected. The samples were transferred into amber glass bottles and filtered as soon as possible but no later than 6 h after sampling through 0.45-µm cellulose nitrate filters (Schleicher & Schuell). The filtered samples were directly extracted or kept at -20 °C in half-filled amber glass bottles in horizontal position until extraction. The sample volumes were 250 mL for 2°EFFL and 3°EFFL samples and 50 mL for 1°EFFL samples. The latter were diluted with 150 mL of water prior to extraction. After addition of 1 g of sodium chloride, the pH was adjusted to 4 with sulfuric acid, and the surrogate standard (50-100 ng) was added. Solid-phase extraction was performed on 6-mL Oasis HLB sorbent cartridges (200 mg; Waters, Bergen op Zoom, The Netherlands) using a 12fold vacuum extraction box (J.T. Baker, Phillipsburg, NY). The sorbent material is a copolymer of two monomers, N-vinylpyrrolidone and divinylbenzol. The cartridges were preconditioned with 2 × 1.5 mL of methanol-ethyl acetate (1:1), 2 × 1.5 mL of methanol containing 1% (v/v) ammonia, and 2 × 1.5 mL of water adjusted to pH 4 with H2SO4. The wastewater samples were percolated through the cartridges at a flow rate of less than 5 mL/min. After percolation, the cartridges were washed with 1.5 mL of water-methanol (95:5) and the eluent was discarded. Subsequently, the cartridges were dried completely in a nitrogen flow for 1 h. The analytes were then eluted with 2 × 1.5 mL of methanol-ethyl acetate (1:1) and 2 × 1.5 mL of methanol containing 1% ammonia into 10-mL graduated glass vessels. Eluates were reduced to ∼50 µL by a gentle flow of nitrogen at room temperature. After the addition of the instrumental standard (100 ng), the sample volume was adjusted to 0.5 mL with water. Final extracts were stored in amber glass vials at -15 °C until analysis. Liquid Chromatography. HPLC analyses were performed using a Rheos 2000 pump equipped with a solvent degasser (Flux Instruments AG), a HTS Pal autosampler (CTC Analytics, Zwingen, Switzerland), and a Jones chromatography column oven, model 7956 (Omnilab AG, Mettmenstetten, Switzerland). Sample aliquots of 20 µL were injected. Two analytical columns were tested for separation. Initially, a 125 × 2 mm Nucleosil 100-5 C18HD end-capped column (Macherey-Nagel, Dueren, Germany) equipped with a 8 × 2 mm precolumn containing the same sorbent material was used (column 1). Gradient elution was performed with water adjusted to pH 4.6 by acetic acid and acetonitrile, both containing 10 mM ammonium acetate. Later, a 150 × 2 mm YMC Pro C18, 120 Å, 3 µm (Stagroma, Reinach, Switzerland) column equipped with a 10 × 2 mm precolumn containing the same sorbent was applied (column 2) was used. Optimal separation was achieved using column 2 maintained at 30 °C and with a flow rate of 0.15 mL/min. Solvent A was water acidified with 1% (v/v) formic acid, resulting in a pH of 2.1, and solvent B was methanol acidified with 1% (v/v) formic acid. The run (0.15 mL/min) started at 10% B for 5 min, followed by a 5-min linear gradient to 15% B, a 5-min linear gradient to 40% B, and another 5-min linear gradient to 45% B and was terminated by a 10-min linear gradient to 70% B. Afterward, the eluent was brought to 100% B in 2.5 min and the column washed at a flow rate of 0.25 mL/min for 10 min. Initial conditions were reestablished in 2.5 min, and the column was equilibrated for 10 min at a flow rate of 0.25 mL/min prior to the next analysis. The total time per analysis was 55 min. Table 2

Table 2. Precursor Ions, Selected Fragment Ions, and Retention Times of the Measured Compounds analyte

precursor ion (m/z)

product ions (m/z)

retention time (min)

SDZ d4SDZa STZ d4STZa SMZ 13C SMZa 6 SPY SMX d4SMXa N4AcSMX d5N4AcSMXa TRI AZI ERY-H2O CLA ROX TYLa SMRa JOSa

251.06 255.08 256.02 260.05 279.09 285.09 250.07 254.06 258.08 296.07 301.10 291.15 375.26 716.46 748.49 837.53 916.53 265.08 828.48

156.01; 108.04 160.01; 112.04 156.01; 108.04 160.01; 112.04 124.09; 186.03 124.09; 186.03 156.01; 184.09 156.01; 108.04 160.01; 112.04 134.06; 198.02 139.06; 203.02 123.07; 275.14 591.40; 158.12 540.33; 558.34 158.12; 590.37 158.12; 679.41 174.11; 772.45 156.01; 172.02 108.91; 174.11

10.3 10.0 12.7 12.4 17.6 17.6 12.6 20.4 20.3 24.1 24.0 17.1 21.1 30.1 31.5 31.6 29.1 14.9 31.1

a

Used as internal standards.

gives the retention times of the individual analytes. To prevent sensitivity losses of the mass spectrometer, the eluate of the first 8 min and of the last 20 min of the chromatographic run were bypassed and discarded. Tandem Mass Spectrometry. A triple quadrupole mass spectrometer, TSQ Quantum Discovery (Thermo Finnigan, San Jose, CA), equipped with electrospray ionization was used for detection. Analyses were performed in the positive mode, with a spray voltage of 3500 V and an ion-transfer capillary temperature of 350 °C. Nitrogen was used as sheath gas (40 bar) and as auxiliary gas (10 bar), and argon as collision gas (1.5 mTorr). Both mass analyzers were set to unit resolution. Usually, the protonated molecular ion ([M + H]+) of the compounds was selected as precursor ion except for azithromycin, for which the doubly charged molecular ion ([M + 2H]2+) was chosen as precursor ion because of its greater abundance under the given conditions. Detection was performed in multiple reaction monitoring mode using the two most intense and specific fragment ions. Table 2 lists the monitored transitions for the individual analytes. The detection of the compounds was divided in time windows during the course of the chromatographic run with a dwell time of 100 ms. Figure 1 shows a chromatogram of a 1°EFFL sample. In the case of SDZ, STZ, and SMZ, which were not present in the sample, the peaks obtained from the measurement of a 1°EFFL sample spiked with 25 ng prior to sample preparation are included (dashed lines). Method Validation. For the method validation, flow-proportional composite samples from the respective effluents of a WWTP (Kloten-Opfikon) were taken. Breakthroughs were determined by extracting spiked wastewater samples (duplicate analyses) using two stacked cartridges. A breakthrough on the first cartridge triggered an enrichment on the consecutive cartridge, which was then eluted separately. For the 1°EFFL a 250-mL sample with a spiked analyte concentration of 2000 ng/L was used, and for the 3°EFFL a 500-mL sample with a spiked analyte concentration of 5000 ng/L was extracted. Complete elution of the cartridges was Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

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Figure 1. Total ion chromatogram (sum of two transitions) of an extract from a primary wastewater effluent (1°EFFL). a The peaks shown for SDZ, STZ, and SMZ correspond to a 1°EFFL sample spiked with 25 ng of these compounds since they were not present in the unspiked sample.

verified by eluting cartridges of spiked samples for a second time with 1.5 mL of acetone as a stronger solvent. The acetone extract was then treated as a separate sample. Instrumental limits of detection (LODs) and limits of quantification (LOQs) were calculated on the basis of standard deviation of the repeated measurement (n ) 10) of a standard mixture (100 pg on column). The LOD is defined as 3 times and the LOQ as 10 times the standard deviation. If the resulting value for the LOQ was below the linear range, the lower limit of the linear range was set as LOQ. Sample-based LOD and LOQ were defined as concentrations in a sample matrix resulting in peak areas with signal-to-noise ratios (S/N) of 3 and 10, respectively. Since samples typically contained analytes in higher amounts, the concentration corresponding to the defined S/N was determined by downscaling, using the measured concentration and the assigned S/N of the peaksassuming a linear correlation through zero. Instrumental precision of the measurement was assessed using an average of 10 independent injections of 100 pg on column of a standard mixture. The precision of the entire method was determined using four replicates of each matrix investigated, spiked with 50 ng of analyte prior to extraction. It is indicated by the relative standard deviation of the measured concentrations of native plus spiked analyte. For recovery studies over the entire procedure, wastewater samples (duplicate analyses) were spiked prior to extraction with surrogate standard and with 25 and 50 ng of analytes, respectively. The calculated amount of antibacterials minus the 4760 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

amount already present before spiking was then divided by the spiked concentration. Identification and Quantification. For each substance, two transitions of the precursor ion were monitored. Together with the retention times, they were used to ensure correct peak assignment and to evaluate peak purity. For instrumental and surrogate standards, peak purity was tested using the area ratio of the two product ions monitored. Their individual ratio was calculated as well as the mean ratio of all samples and its relative standard deviation. The ratio in one sample was compared to the mean ratio of all the samples measured in one series. The variance had to be within the range of twice the standard deviation of the mean sample ratio. The peak purity of the analytes was tested by calculating a concentration (as described below) for both product ions measured. The respective surrogate product ion was used. If no surrogate product ion resulting from the same fragmentation reaction can be used, i.e., if no isotope labeled surrogate standard is available, the sum of both product ions of the compound assigned as surrogate standard was used for quantification to simplify the procedure. The relative average deviation of the calculated concentrations from the two product ions had to be less than 10%. Peaks not fulfilling the requirements for peak purity were not used for quantification. Quantification was performed using the ratio of the peak areas of the analytes and of the surrogate standard. The sum of the two monitored product ions was used. An external calibration

curve, plotting ratio against concentration, was obtained by diluting standards in HPLC water. A standard curve was acquired at the beginning, at the end, and also in the middle of a measurement series. At least five concentration points in the appropriate concentration range were used for quantification. Concentrations in the samples were calculated by comparing the peak area ratios of the analytes and their assigned surrogate standards in the SPE extracts, to the corresponding ratios in the standard solutions. These results were corrected with the corresponding recovery rates obtained in the same matrix and sample batch to provide accurate amounts. For routine determination, duplicate analyses of all samples were performed. Procedural blanks, consisting of deionized water, were analyzed with each set of 12 extractions as a control for laboratory contamination. Additional instrumental blanks using deionized water were checked with each calibration curve in order to uncover potential analytical interferences. RESULTS AND DISCUSSION Method Development. The crucial parameters for enrichment, separation, and detection of the analytes were identified and optimized. The pH of the sample proved to be the most influential variable during sample extraction. A critical impact on the retention of the analytes on the cartridge material was observed, especially for sulfonamides caused by their amino groups. Our enrichment tests between pH 2 and 6 revealed, as expected, highest recoveries at pH 4 for the sulfonamides, while the recovery of the macrolides and trimethoprim showed no strong pH dependence. This behavior can be explained by the charge state of the sulfonamides at the particular pH values (Table 1).26-31 With a compound specific pKa of 5-8 for the sulfonamino groups (pKa 1) and a pKa of 2-2.5 for the arylamin (pKa 2), the sulfonamides are positively charged at pH 2 and negatively at a pH above 5. The interaction with the cartridge material is strongest for analytes in uncharged forms occurring at a pH of ∼4 in the case of the sulfonamides. The dilution of the 1°EFFL samples prior to enrichment additionally increased signal intensity provided by the mass spectrometer for the sulfonamidessin most cases by a factor of 2. For the macrolides and trimethoprim, no significant improvement was observed. While N4-acetylsulfamethoxazole is stable during sample preparation, erythromycin present in the samples is completely transformed to erythromycin-H2O at pH 4. This is in agreement with the reported instability of erythromycin under acidic conditions resulting in the formation of the inactive erythromycin-H2O.27 Erythromycin was therefore assessed as the main environmental metabolite, erythromycin-H2O. Tandem mass spectrometric conditions were optimized for each analyte and internal standard through automated tuning procedures implemented in the instrument software. Scheme 1 (26) McFarland, J. W.; Berger, C. M.; Froshauer, S. A.; Hayashi, S. F.; Hecker, S. J.; Jaynes, B. H.; Jefson, M. R.; Kamicker, B. J.; Lipinski, C. A.; Lundy, K. M.; Reese, C. P.; Vu, C. B. J. Med. Chem. 1997, 40, 1340-1346. (27) Bryskier, A. J.; Butzler, J.-P.; Neu, H. C.; Tulkens, P. M. Macrolides; Arnette Blackwell: Paris, 1993. (28) Vree, T. B.; Hekster, Y. A. Clinical pharmacokinetics of sulfonamides and their metabolites; Karger: Basel, 1987. (29) Lin, C.-E.; Chang, C.-C.; Lin, W.-C. J. Chromatogr., A 1997, 768, 105-112. (30) Petz, M. Habilitationsschrift in Chemie; Westfa¨lische Wilhelms-Universita¨t, Mu ¨ nster, 1986. (31) Neuman, M. Antibiotika-Kompendium; Verlag Hans Huber: Bern, 1981.

Scheme 1. Breakdown Curves of N4-Acetylsulfamethoxazole and Proposed Product Ion Structures (Absolute Intensity 3.56 × 105, Collision Pressure 1.5 mTorr)

shows the breakdown curves for N4AcSMX and its four most intense fragments as a function of the collision energy. As expected, the collision energy, which gives the most intense signal, increases for the formation of smaller fragments. Tentative product ion structures are given also. These structures have not been reported previously but are in agreement with transitions known to be typical for sulfonamides.32, 33 During LC/MS/MS measurement, matrix compounds can be deposited on the instrument’s sample interface, especially on the ion-transfer capillary, and can thus significantly reduce instrument sensitivity. The higher the sample volume the more matrix will be introduced into the mass spectrometer within one run. On the other hand, a high enrichment factor is desirable to achieve the low limits of detection, which are necessary for the environmental analysis of antimicrobials. The sample volume was optimized by using a variable splitting device prior to the electrospray interface. For this experiment, higher sample volumes were chosen. Therefore, 200 mL of 1°EFFL and 1000 mL of 2°EFFL and 3° EFFL, respectively, were enriched and measured in one series. The eluent flow and split ratio were varied, so that the instrument remained sensitive enough for the measurement of up to 30 samples of each matrix. The sample volume used was then adjusted according to the split ratio. Subsequent samples were measured without the additional splitting device that may pose (32) Volmer, D. Rapid Commun. Mass Spectrom. 1996, 10, 1615-1620. (33) Haller, M. Y.; Mu ¨ ller, S. R.; McArdell, C. S.; Alder, A. C.; Suter, M. J.-F. J. Chromatogr., A 2002, 952, 111-120.

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Table 3. Linear Ranges and Limits of Quantification sample based LOQa (ng/L)

a

primary effluent

analyte

linear range (pg on column)

average

range

SDZ STZ SMZ SPY SMX N4AcSMX TRI AZI ERY-H2O CLA ROX

20-30000 20-30000 10-30000 50-30000 10-30000 100-30000 5-2000 10-4000 5-4000 10-6000 10-4000

68 214 42 96 62 212 21 7 19 4 3

60-77 194-236 35-48 51-150 35-104 155-288 15-27 4.6-9.9 4.4-13 2.3-5.7 1.2-6.3

secondary effluent average range 11 21 9 14 12 23 6 3 5 2 1

6-32 15-30 7-12 8-31 8-17 17-29 4-9 2.0-3.4 3.5-8 1.3-2.9 0.3-2.0

tertiary effluent average

range

7 16 9 9 11 22 4 2 6 2 1

5-11 10-22 4-17 6-19 6-15 16-28 3-7 1.6-2.6 3.4-9.5 1.3-3.1 0.4-1.4

Concentration estimated from measured samples for a signal-to-noise ratio of 10.

as a source for errors (for example, plugging of the capillary). The given sample volumes therefore represent a compromise between method sensitivity and routine analysis. Two columns were tested for the separation of the selected antibacterial agents. In both cases, a reversed-phase end-capped C18 column was chosensone belonging to the older (column 1) and one belonging to the newer (column 2) generation of silica gels. On column 1, azithromycin produces a peak with substantial tailing. To our knowledge, azithromycin has not been included in analytical methods for environmental samples so far, likely also due to analytical difficulties like this. The observed tailing on column 1 is probably due to the interaction of the two basic functional groups with residual silanol groups and metal impurities of the column material. In the case of the other macrolides, which contain one amino group less than azithromycin in the lactone moiety, this interaction was sufficiently suppressed by the addition of ammonium acetate. On column 2, belonging to the new generation of silica gels, however, good separation was achieved with almost symmetrical peaks for all analytes. In addition, the use of ammonium acetate in the eluent was no longer necessary. This significantly increased the sensitivity of the method for sulfonamides, which tend to form ammonium adducts during ionization. In the case of some 1° EFFL samples, the extracts needed to be diluted up to five times in order to obtain good peak shapes for azithromycin, which seems to form complexes with matrix compounds. For most analytes, the assumed loss of sensitivity due to dilution is partly compensated by the simultaneous reduction of ion suppression, since signal intensities observed are reduced to a lesser extent. Method Validation. The developed method was validated for primary effluents after mechanical treatment, secondary effluents after biological treatment, and tertiary effluents after sand filtration. For breakthrough studies, samples representing unnaturally high concentrations and high loads of sample matrix were enriched on two stacked cartridges. No quantifiable amounts of the analytes could be detected on the second cartridge for both sample matrixes (1°EFFL and 3°EFFL). When testing for complete elution, no quantifiable amounts of analytes could be measured in the acetone eluates of already eluted cartridges. Thus, the 4762

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analytes are quantitatively enriched by one cartridge and exhaustively eluted by the procedure described above. For the standard curves, good linearity was observed with correlation factors typically above 0.99. The linear range of the measurement varied with the analyte due to differences of the ionization efficiencies (Table 3). The instrumental LOQ ranges between 16 and 100 pg of analyte on column. In the case of the sample-based LOQ and LOD, the range and the average of the resulting values for each matrix from different samples are given in Table 3. Since the LOD and LOQ in an individual sample can be higher or lower than the average LOD and LOQ, all concentrations resulting from peaks with S/N greater than or equal to 3 and 10, respectively, are considered valid results. The instrumental precision of the method was addressed for various aspects, and the following relative standard deviations were obtained: the retention time ranged between 0.06 and 0.35% and the peak area between 1.3 and 9.2%. The peak area ratios of analyte versus surrogate standard varied to a lesser extent in most cases (between 1.3 and 7%), since the surrogate standard compensates for analytical variability. The precision of the entire method (reproducibility) is indicated by the standard deviation of multiple analyses and ranged between 0.5 and 15%. Detailed results are shown in Table 4. Accuracies of the method were determined by recovery studies over the entire procedure (Table 4). The resulting recoveries obtained in all matrixes investigated were generally above 80%, with the exception of TRI where they ranged between 30 and 47%. For TRI, this was caused by the use of a nonideal surrogate standard (13C6SMZ), but none better suited could be found. Recoveries, and thereby LODs and LOQs, of the analytes vary between samples, mainly due to varying matrix effects, if no isotopically labeled surrogate standard is available. Correct quantification can still be ensured if recovery studies are performed in each matrix and sample batch, as was the case within the work presented here. The combined measurement uncertainty was quantified using data from the method validation as described in example A4 of the EURACHEM/CITAG Guide Quantifying Uncertainty in Analytical Measurement.34 Therefore, all uncertainty sources were identified and quantified. The main contributions result from the repeatability of the measurement, calculated from

Table 4. Method Precisions, Accuracies, and Combined Measurement Uncertainties precision relative SDa (%), nb ) 4

accuracy recovery ( SDa (%), nb ) 4

combined measurement uncertainty (%)

analyte

primary effluent

secondary effluent

tertiary effluent

primary effluent

secondary effluent

tertiary effluent

primary effluent

secondary effluent

tertiary effluent

SDZ STZ SMZ SPY SMX N4AcSMX TRI AZI ERY-H2O CLA ROX

3.4 0.7 1.1 4.3 3.0 0.7 8.5 2.4 5.6 5.9 6.6

0.5 5.2 1.6 4.5 0.4 2.4 2.6 5.7 4.1 3.0 5.9

1.0 2.0 2.1 2.2 1.4 4.2 12 11 8.9 15 15

102 ( 1.8 101 ( 1.8 98 ( 0.9 108 ( 3.6 101 ( 3.3 91 ( 3.8 47 ( 2.8 83 ( 6.5 91 ( 1.0 89 ( 7.5 100 ( 4.9

95 ( 1.8 102 ( 2.1 98 ( 3.3 101 ( 4.6 105 ( 10 100 ( 6.7 30 ( 12 85 ( 4.4 82 ( 1.0 81 ( 8.3 108 ( 1.8

98 ( 4.8 103 ( 3.1 98 ( 5.8 106 ( 0.9 105 ( 6.0 93 ( 6.1 35 ( 10 86 ( 10 86 ( 7.9 78 ( 6.4 124 ( 2.6

3.1 2.6 2.4 6.1 5.2 3.8 10 9.4 4.8 6.5 8.6

2.7 3.3 3.9 3.8 3.6 4.4 8.0 11 3.8 7.4 8.7

4.0 5.7 4.9 13 3.4 5.7 13 16 15 12 14

a

SD, standard deviation. b n, number of samples.

Table 5. Sulfonamide and Macrolide Concentrations Measured in Two Municipal Wastewater Treatment Plants in Switzerland sample concn ( ADa (ng/L), nb ) 2 WWTP

1c

WWTP 2c

analyte

primary effluent

secondary effluent

tertiary effluent

primary effluent

secondary effluent

tertiary effluent

SDZ STZ SMZ SPY SMX N4AcSMX TRI AZI ERY-H2O CLA ROX

ndd nd nd 72 ( 1.6 343 ( 5.8 518 ( 13 168 ( 6.1 86 ( 8.4 67 ( 3.6 234 ( 30 22 ( 0.5

nqd nd nd 82 ( 0.6 344 ( 0.1 86 ( 3.3 170 ( 6.3 110 ( 8.5 96 ( 1.1 374 ( 8.4 11 ( 0.2

nq nd nd 88 ( 0.6 352 ( 0.9 82 ( 0.4 81 ( 0.9 85 ( 3.3 75 ( 1.7 329 ( 10 10 ( 0.2

nd nd 39 ( 1.0 135 ( 1.1 641 ( 4.0 943 ( 2.9 110 ( 3.2 224 ( 22 44 ( 3.9 160 ( 9.4 30 ( 0.9

nd nd 18 ( 0.1 63 ( 1.3 352 ( 11 nq 86 ( 0.6 129 ( 5.9 54 ( 0.6 188 ( 0.5 21 ( 1.1

nd nd 19 ( 0.6 85 ( 2.6 352 ( 2.9 71 ( 1.0 68 ( 8.0 255 ( 13 55 ( 2.4 220 ( 7.6 23 ( 1.4

a Concentration measured in filtered 72-h flow proportional composite sample. Mean and average deviation (AD) of duplicate determination. Number of measurements. c WWTP 1, Kloten-Opfikon (canton Zurich); WWTP 2, Altenrhein (canton St. Gall). d nd, not detected, signal-to-noise below 3; nq, not quantifiable, signal-to-noise below 10.

b

duplicate sample analysis, and its accuracy, represented by recovery studies. The relative values of all uncertainty sources are finally combined using statistical methods. The values for the combined measurement uncertainty vary between 2 and 18% with the analyte and the matrix investigated (Table 4). Wastewater Applications. The developed method was successfully applied to the analyses of wastewater samples from two urban wastewater treatment plants in Switzerland: WWTP KlotenOpfikon, located near the international airport of Zurich (WWTP1), and WWTP Altenrhein, located in the canton St. Gall close to the border with Austria (WWTP2). In both cases, mechanically treated wastewater (primary effluent) passes through conventional activated sludge treatment, followed by secondary settling (secondary effluent). After biological treatment, both treatment plants use sand filtration as a tertiary treatment step (tertiary effluent). Table 5 shows the results obtained from duplicate analyses of 72-h flowproportional composite samples of the primary, secondary, and (34) http://www.measurementuncertainty.org/mu/guide/index.html, Quantifying uncertainty in analytical measurement/prep. by the EURACHEM Working Group on Uncertainty in Chemical Measurement (ISBN 0 948926 15 5).

tertiary effluents. Samples were taken in February 2003. With 54 100 and 40 000 inhabitant equivalents, the two investigated treatment plants are of similar size and have comparable volumes of wastewater inflow. This is also reflected in the similar concentration ranges found at each plant, with the exception of azithromycin. The latter appears to be more frequently used in the catchment area of WWTP2. Sulfamethoxazole and clarithromycin were found to be the most commonly used sulfonamides and macrolides, respectively. Sulfadiazineswhich is very rarely applied in human medicinescould not be quantified in any of the samples, nor could sulfathiazoleswhich is almost exclusively used in veterinary medicine. Sulfamethazinesanother sulfonamide used mainly for veterinary applicationsswas only present in one of the treatment plants (WWTP2). N4-Acetylsulfamethoxazole is typically present in high amounts in the primary effluents, but only small amounts can be found in the tertiary effluents. If the amount of sulfamethoxazole present as acetyl metabolite is neglected, the elimination of sulfamethoxazole will be underestimated. Concentrations of the analytes in both tertiary effluent range between 19 and 352 ng/L. This clearly shows that the compounds investigated Analytical Chemistry, Vol. 76, No. 16, August 15, 2004

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are not eliminated completely and reach receiving surface waters. Compared to results obtained in Germany,14,35 the concentrations found are in the same range but generally lower. CONCLUSIONS Solid-phase extraction Oasis HLB cartridges coupled with reversed-phase liquid chromatography and tandem mass spectrometry were successfully applied for the determination of selected sulfonamide and macrolide antibiotics, in addition to trimethoprim and the human sulfonamide metabolite N4-acetylsulfamethoxazole, in municipal wastewater. As a result of this method’s applicability to wastewater samples spanning the whole treatment process (including primary effluent samples), it can be used to investigate the fate of these compounds through the various steps of wastewater treatment. The resulting information can be used to evaluate the performance of wastewater treatment procedures and to highlight options for the optimization of WWTPs with the aim of minimizing the input of antibiotics into ambient receiving waters. Additionally, by including N4-acetylsulfamethoxazolesthe main human metabolite of sulfamethoxazolesthe fate of the most commonly used sulfonamide in human medicine can be investigated more thoroughly. The presented method provides the necessary basis for a comprehensive study on antibacterials in wastewater treatment (35) Hirsch, R.; Ternes, T. A.; Haberer, K.; Kratz, K.-L. Sci. Total Environ. 1999, 225, 109-118. (36) Go ¨bel, A., Ph.D. Thesis, ETH Zurich, in preparation. (37) http://www.eu-poseidon.com. (38) http://www.nrp49.ch/pages/.

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including alternative wastewater technologies such as biofilter technology and membrane filtration.36 Our ongoing studies are also aimed at achieving complete mass balances of antimicrobials in wastewater treatment plants, including sewage sludge treatment steps. Preliminary results show that the method can easily be adapted for the analyses of sewage sludge extracts. Applications to drinking water, ambient waters, and hospital wastewaters also seem to be possible judging from first measurements without major changes in the procedure. With this method, we therefore present a powerful tool to fully assess the fate and occurrence of macrolides and sulfonamides throughout their main pathways to and within the aquatic environment. ACKNOWLEDGMENT Abbott GmbH (Wiesbaden, Germany) is acknowledged for supplying clarithromycin and Pfizer A.G. (Zu¨rich, Switzerland) for supplying azithromycin. Partial financial support came from the EU project POSEIDON (EVK1-CT-2000-00047)37 and the EAWAG project on human-use antibiotics (HUMABRA) within the framework of the National Research Program on antibiotic resistance funded by the Swiss National Science Foundation.38 We thank Eva Molnar, Norriel Nipales, and Rene Scho¨nenberger for their technical assistance and advice. For helpful comments on the manuscript, we acknowledge our colleagues Alfredo Alder, Michael Dodd, Stephan Mu¨ller, and Krispin Stoob. Received for review March 3, 2004. Accepted May 12, 2004. AC0496603