Paper_zuehlke_1.doc

LC-MS in environmental trace analyses – a challenge for reliable data monitoring
Sebastian Zuehlke
Introduction

The fate of pharmaceutical residues, personal care products and endocrine disrupting compounds has be en recognized as an emerging issue in environmental research [Heberer, 2002; Kümmerer, 2001; Daughton & Ternes 1999]. Thus, reliable data on occurrence and fate of such trace organic compounds need to be monitored comprehensively. This is a great task for the procedure of determination especially in case of substances with high potential of environmental or human risk in very low concentrations. Waste water treatment plants (WWTPs) receive a large spectrum of organic contaminants which are in part eliminated during treatment [Heberer, 2002, Daughton & Ternes 1999]. Nevertheless, several persistent organic chemicals such as some persistent pharmaceuticals or endocrine disrupting compounds are not completely removed during conventional wastewater treatment and discharges of municipal sewage effluents are therefore considered as a main source of environmental pollution [Heberer, 2002; Kümmerer, 2001]. Thus, they have been detected up to the µg/L level in municipal sewage [Heberer, 2002; Kümmerer, 2001; Ternes et al. 2001]. Due to dilution, degradation and sorption also lower concentrations can be expected and have already been detected in treated sewage, surface and ground water. Besides the original compounds metabolites can be detected in the aquatic environment. Thus, among others, degradation products of pharmaceuticals and industrial chemicals that have widely been used in Germany were found in the aquatic environment (Daughton & Ternes 1999; Today’s instrumental analytical methods apply gas chromatography (GC) or liquid chromatography (LC) [Lopez de Alda et al. 2003; Kolpin et al. 2002; Ternes et al. 2001] in combination with detection by mass spectrometry (MS). Especially for polar compounds, LC-MS is the method of choice for detection [Lopez de Alda et al. 2003; Ternes et al. 2001]. In comparison to GC analysis run times and sample preparation time is reduced due to the fact that no derivatization prior analysis with LC is necessary. LC with single mass spectrometric analysis offers good sensitivity but in the case of very complex matrices, such as sewage, insufficient selectivity often impairs the unequivocal identification of the analytes. LC with tandem mass spectrometric detection (LC-MS/MS) promises both high sensitivity and much better selectivity for the unambiguous identification and quantification of environmental contaminants at trace-level concentrations. Recently published methods for the determination of pharmaceuticals in environmental samples use electrospray ionization (ESI) or atmospheric Sample preparation and extraction

Water samples should be fortified with of suitable internal reference standards (IS) prior the
first sample preparation step. Sample extraction is often based on solid phase extraction (SPE)
or liquid-liquid-extraction (LLE). Nowadays SPE-materials promises high selectivity at high capacity. The best SPE extraction methods using automated extraction systems, enabling simultaneous conditioning of the cartridges with solvents and water, percolating of the samples through the SPE-material at defined flow rates and after drying eluting of the cartridges. Additional washing steps with solvent mixtures help to eliminate many compounds that might disturb the follow ing mass spectrometric analysis. Special clean-up procedure may be suitable for matrix containing samples. Additional procedures were found to be necessary especially for sewage samples. Thus, size exclusion chromatography or simple silica gel LC-MS Analysis

The LC measurements were generally performed using binary LC pumps, equipped with auto
samplers and degassing units. Separation is mostly carried out at room temperature (or up to 35°C) using RP18 or RP8 columns with water, methanol and acetonitrile as solvents. Modifiers like formic or acetic acid and ammonium acetate were generally used to enhance the resolution of the liquid chromatographic separation. The addition of buffers (ammonium acetate or ammonium hydroxide in varying concentrations) to the mobile phase may cause a dramatic decrease of the responses of the analytes due to lower ionization ratios and is therefore often omitted if low limits of detection (LOD) are required. The coupling of the LC-system to the mass spectrometer is the main factor of LC-MS. Atmospheric pressure ionization (API) is used in mainly two different techniques. Electrospray ionization (ESI) causes the ionization in the liquid phase and atmospheric pressure chemical ionization (APCI) in the gaseous phase. Fig. 1. General scheme of an electrospray ionization source (ESI) In general, ESI provides lower limits of detection than APCI but in case of complex matrices, such as sewage, false negative results due to matrix ionization suppression effects might be obtained [Christian et al. 2003; Zuehlke et al. 2004b & 2005]. APCI could in such cases act as a suitable tool for the sensitive and precise quantification even if appropriate surrogates for matrix compensation are not available or additional clean-up steps shall be omitted. Tandem mass spectrometric methods are mainly based on selected reaction monitoring (SRM). For all of the analytes, the precursor ions were chosen ever in the positive or the negative mode, resulting in the corresponding pr otonated/deprotonated molecular ions. After recording the individual product ion spectra suitable SRM transition ions were selected for multiple reaction monitoring (MRM) analysis. Characteristic precursor and product ion masses as well as the individual collision energy voltages representing a high selective method of detection. Data acquisition can be carried out using different retention time windows. Dwell times for SRM were set to a few milliseconds, enabling simultaneous Method recoveries, accuracy, and limits of quantification (LOQs)
The determination of LOQs should be based on the signal-to-noise (S/N) ratio of the analyte
peak and the baseline near to this peak obtained by analyses of field samples. LOQ is
characterized as the concentration with a minimum S/N ratio of 10 (S/N ratio of 3 for the LOD). Analyte recoveries should be determined by adding known amounts of the target compounds to previously analyzed samples. Non-contaminated samples of all sample types (e.g. soil, sewage) should be spiked at individual concentrations in the expected environmental concentration. Even at different types of environmental samples the recoveries (corrected by the internal standards) should be always in the range between 70 and 120% . Application of LC-MSMS for the determination of estrogenic steroids in wastewater

Over the last decades, several experiments have raised some concern that effluents from
municipal WWTPs might be responsible for endocrine disrupting effects in the aquatic
environment. The presence of endocrine disrupting compounds (EDCs) in river water such as estrogenic steroids originating from discharges of municipal sewage effluents may cause such effects that could result in a reproduction disorder in aquatic biota [Routledge et al. 1998; Desbrow et al. 1998]. Estrogenic steroids such as the synthetic steroid hormone 17a - ethinylestradiol (EE2) prescribed as oral contraceptive for birth control or estrogen-substitution therapies and the natural hormone 17ß-estradiol (E2) and its main metabolite estrone (E1) are among the most potent EDCs causing effects in aquatic organisms even at trace-level concentrations. Steroid hormones released into the aquatic system were identified as having the highest endocrine disrupting potential [Thorpe et al. 2003]. Snyder et al. [2001] estimated that E2 and EE2 concentrations represented between 88 and 99.5% of the total estrogen equivalents in sewage effluents and surface waters. In vivo investigations have also shown that exposure of fishes down to 1 ng/L E2 or 0.1 ng/L EE2 provoke feminization in some species [Routledge et al. 1998; Purdom et al. 1994]. Based on calculations of daily excretion by humans, amounts of wastewater, and dilution factors estrogen are expected to occur at concentrations at the low ng/l level in sewage effluents and in the receiving waters. Unequivocal identification and quantitation of such concentrations requires sensitive and selective techniques such as capillary gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS or even LC-MS/MS). GC analysis is more time consuming as suitable derivatization procedures are required for the analysis of the estrogenic compounds. LC with single MS can lead to sufficient sensitivity for the analysis of estrogens in environmental samples but only when a very specific cleanup step based on immunoaffinity extraction is applied. LC with tandem mass spectrometric detection (LC-MS/MS) promises both high sensitivity and much better selectivity for the unambiguous identification and quantification of environmental contaminants at trace-level concentrations even in complex matrices. 24h composite samples of the influent and effluent of a sewage treatment plant were collected and analyzed via SPE, silica gel clean up, LC separation and tandem mass spectrometric detection. In the influent of the investigated sewage treatment plant all the analyzed estrogens were detected at the ng/L-level (Table 1). Figure 2 shows a chromatogram of an analyzed raw sewage sample. The only sporadically in raw waste water measurable 17α-estradiol was never detected in treated sewage or surface and ground water. From the measured values, an elimination efficiency of 93 % for estrone (E1) and estradiol (E2) and a removal rate of 81 % for Ethinylestradiol (EE2) could be calculated for this specific WWTP. Table 1. Limits of quantification (LOQs), mean concentrations, and removal of Estrone (E1), 17ß-Estradiol (E2) and 17a-Ethinylestradiol (EE2) in a municipal sewage treatment plant; n = 18; from [Zuehlke et al. 2005] sewage influent
sewage effluent
estradiol
ethinylestradiol
Fig. 2. LC-MS/MS selected reaction monitoring (SRM) chromatograms for all analytes and the surrogates in a WWTP influent sample; quantifier and qualifier traces are shown; Refere nces

Christian, T.; Schneider, R.L., Färber, H.A.; Skutlarek, D.; Meyer, M.T.; Goldbach, H.E. Acta hydrochim. Hydrobiol. 2003 , 1, 36-44.
Daughton, C.G.; Ternes, T.A. Environ. Health Perspect ., 1999, 107 Suppl 6, 907-938.
Desbrow, C., E. J. Routledge, C. G. Brighty, J. P. Sumpter, M. Waldock, Environ. Sci. Technol. 1998, 32, 1549-
Heberer, Th. Toxicology Letters 2002, 131, 5-17.
Kolpin, D.W., E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D. Zaugg, L.B. Barber, H.T. Buxton, Environ. Sci. Technol. 2002, 36, 1202-1211.
Kümmerer, K. Chemosphere, 2001 , 45, 957-969.
Lopez de Alda, M.; Diaz-Cruz, S.; Petrovic, M.; Barcelo, D. J. Chrom. A, 2003, 1000, 503-526.
Purdom, C. E., P. A. Hardimann, V. J. Bye, N. C. Eno, C. R. Tyler, J. P. Sumpter, J. P. Chem. Ecol. 199 4, 8,
Reddersen, K.; Heberer, T.; Duennbier, U.; Chemosphere 2002, 49, 539-544.
Routledge, E. J., D. Sheahan, C. Desbrow, C. G. Brighty, M. Waldock, J. P. Sumpter, Environ. Sci. Technol. 1998, 32, 1559-1565.
Snyder, S.A., D.L. Villeneuve, E.M. Sny der, J.P. Giesy, Environ. Sci. Technol. 2001, 35, 3620 -3625.
Ternes, T.; Bonerz, M.; Schmidt, T. J. Chrom. A; 2001, 938, 175 -185.
Ternes, T.; Bonerz, M.; Schmidt, T. J. Chrom. A; 2001, 938, 175 -185.
Ternes, T.A., M. Stumpf, J. Mueller, K. Haberer, R.-D. Wilken, M. Servos, Sci. Total Environ. 1999, 225, 81-90
Thorpe, K.L., R.I. Cummings, Th.H. Hutchinson, M. Scholze, G. Brighty, J.P. Sumpter, Ch.R. Tyler, Environ. Sci. Technol. (in press) ASAP Article, Web Release Date: February 2003 , 12.
Zuehlke, S.; Duennb ier, U.; Heberer, Th. Anal. Chem. 2004b, 76, 6548 -6554.
Zuehlke, S.; Duennbier, U.; Heberer, Th. J. chromatogr. A 2004a , 1050, 201-209.
Zuehlke, S.; Duennbier, U.; Heberer, Th. Journal of separation science 2005 , 28, 52-58.

Source: http://www.infu.tu-dortmund.de/pdfs/paper/nyr_zuehlke.pdf