Detecting Endocrine Disrupting Compounds in Wastewater with GC-MS

1. Detecting Endocrine Disrupting Compounds in Wastewater with GC-MS Abbey Kopan Chem 4101, Fall 2008

2. Introduction Endocrine disrupting compounds (EDCs) such as bisphenol A (BPA) have been of particular interest in recent years due to the deleterious effects they have been suspected of having on humans and wildlife, even at concentrations of ng/L or μg/L.1 The effects of low concentrations of EDCs in wastewater on fathead minnows has been extensively studied, and they were found to have significant reproductive effects on the fish.2 http://fwp.mt.gov/fieldguide/mediaDisplay.aspx? id=4568&elcode=AFCJB32020

3. The effects of EDCs were more prominent when the compounds were present in complex mixtures such as wastewater compared with EDCs in Milli-Q water.3 This leads to the hypothesis that chlorinated derivatives of BPA, produced during disinfection of wastewater with sodium hypochlorite (bleach), may have increased estrogenic potency.4 NaOCl NaOCl

4. Experimental Methodology Gas chromatography-mass spectrometry -Predictable electron ionization fragmenting -SIM provides low LOD, low matrix interference due to selectivity, and a high number of scans per second -Quantification of analytes by comparison to calibration curve from standards Sample Preparation Sample injection port Electron Multiplier Diode Electron Impact Ionization He Quadrupole MSD (SIM) Capillary Column Oven Data System/ Display Transfer line

5. Why GC-MS? • GC • Separates compounds at different retention times to make detection and analysis of mixtures less complicated • Uses less hazardous solvents than LC, since mobile phase is gas, not liquid MS • Very selective, so avoids co-elution of similar compounds • Gives extremely specific structural information • Effective electrode design for these analytes is complicated, and electrochemistry does not give quantitative analysis • UV-visible and fluorescence detection do not give structural information • IR detection limits are too high to detect trace amounts The combination of these two methods allows for great ease and precision in identifying compounds.

6. Sampling5 Preconcentration (SPE of 500mL sample, pH 3, eluted in 6 mL diethyl ether/methanol, 9:1) Spike with BPA, 2.5 μg/L Wastewater collection Filter 0.2 μm Centrifuge Injection into GC-MS Derivatization • Derivatization of the concentrated samples using a silylation agent will provide better separation of analytes because of higher volatility and less interaction with the stationary phase of the column. • 30 μl 1:1 v/v of ethyl acetate and BSTFA/TMCS • (N,O-bis(trimethylsilyl)trifluoroacetamide and trimethylchloro silane) • React at 60 ºC for 30 min

7. GC-MS Intstrumentation5 • Agilent 6890 GC with quadrupole mass filter • 7683 automatic liquid sampler injection • 5976 network mass selective detector (MSD) • HPCHEM chromatography software • GC separates compounds using a capillary column, depending on the interaction of the analytes with the stationary phase of the column. Analytes will come out of the GC at different times and then are ionized and detected by the mass spectrometer. • MS ionizes analytes into fragments and detects them using their mass to charge ratio (m/z). • Destructive detection http://www.chem.agilent.com/en-us/products/ instruments/gc/6890ngc/pages/default.aspx

8. Instrument Parameters5 • Carrier gas: High purity He (99.999%); 1 mL/min • Injection port: 250 ºC, splitless injection mode • Injection volume: 1 μl • Column: Zebron ZB-5; 30m x 0.25mm; 0.25 μm film thickness • Low polarity • 5%-Phenyl 95%-Dimethylpolysiloxane • Oven temperature program • 120 ºC, 1 min • Ramp to 230 ºC, 15 ºC/min • Ramp to 260 ºC, 30 ºC/min • Hold at 260 ºC for 8 min • Transfer line from GC to MS: 270 ºC • EI ion source: 250 ºC; 70 eV • Mass Analyzer: Quadrupole • MSD operation mode: Selected ion-monitoring (SIM)

9. GC-MS Method Characteristics5 *Compounds detected in their derivatized form **Cl-35/Cl-37 isotopes Precision: <5% RSD

10. Results: MS Spectra5 • Selectivity: Well resolved peaks, appear to be no interference of peaks. Peaks identified by retention time as well as mass spectrum.

11. Conclusions • Pre-concentration, using solid-phase extraction, and derivatization of analytes increase sensitivity. • Environmental concentrations of the analytes can be detected using this method. • Sensitive, precise, and selective method for detecting BPA and its chlorinated derivatives.

12. References • Maffini, Maricel V., et al. “Endocrine disruptors and reproductive health: The case of bisphenol A.” Molecular and Cellular Endocrinology. 2006. 179-186. • Sohoni, P., et al. “Reproductive effects of long-term exposure to bisphenol a in the fathead minnow (Pimephales promelas).” Environmental Science & Technology 35. 2001. 2917-2925. • Kuruto-Niwa, Ryoko, et al. “Identification of estrogenic activity of chlorinated bisphenol A using a GFP expression system.” Environmental Toxicology and Pharmacology 12. 2002. 27-35. • Filby, Amy L., et al. “Health Impacts of Estrogens in the Environment, Considering Complex Mixture Effects.” Environmental Health Perspectives 115. 2007. 1704-1710. • Ballesteros, Oscar, et al. “Sensitive gas chromatographic-mass spectrometric method for the determination of phthalate esters, alkylphenols, bisphenol A and their chlorinated derivatives in wastewater samples.” Journal of Chromatography A, 1121. 2006. 154-162.