Concentration of Human Hormones in Drinking Water Using Solid Phase Extraction and Analysis by High Performance Liquid Chromatography

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1 Concentration of Human Hormones in Drinking Water Using Solid Phase Extraction and Analysis by High Performance Liquid Chromatography Carl Fisher, Monika Verma, and Pranathi Perati; Thermo Fisher Scientific, Sunnyvale, CA, USA

2 Overview A method was developed to automate the capture of human hormones from drinking water onto solvent extraction cartridges and then eluting them using a solid phase extraction system. The resultant was subsequently quantified with HPLC. Introduction The presence of hormones in drinking water is a human health concern, although safe exposure limits have yet to be established due to the need for further studies to determine the impact of long-term and synergistic exposure. 1 Estriol, estrone, estradiol, ethynylestradiol, equilin, androstenedione, and testosterone (Figure 1) are routinely monitored as part of the U.S. Environmental Protection Agency (EPA) Unregulated Contaminant Monitoring program. 2 FIGURE 1. Sex hormones routinely monitored in drinking water. The naturally occurring human estrogens and androgens are highlighted. Estriol, estrone, and estradiol are estrogens that control female traits, while testosterone is an androgen controlling the development of male characteristics. These hormones are naturally present in humans and are derived from androstenedione during steroidogenesis. Various forms of estrogen are prescribed as a hormonal contraceptive device and to counter hormone deficiencies. Ethynylestradiol is an orally bioactive synthetic estrogen used as an oral contraceptive, while equilin is an equine-derived estrogen used for hormone replacement therapy. Testosterone is prescribed for hormone replacement therapy in males, while androstenedione is a performance enhancing anabolic steroid. 2 Concentration of Human Hormones in Drinking Water Using Solid Phase Extraction and Analysis by High Performance Liquid Chromatography

3 Methods Sample Preparation Drinking water was fortified with 1 mg/l hormones and mixed by vortexing. Instrumentation Thermo Scientific Dionex AutoTrace 280 Solid-Phase Extraction instrument Thermo Scientific Dionex UltiMate 3000 RSLC system: DGP-3600M Dual-Gradient Micro Pump WPS-3000TRS Rapid Separation Wellplate Sampler, Thermostatted TCC-3000RS Rapid Separation Thermostatted Column Compartment DAD-3000RS Rapid Separation Diode Array Detector (Without Flow Cell) Thermo Scientific Dionex AS-AP Autosampler Data Analysis Thermo Scientific Dionex Chromeleon Chromatography Data System (CDS) software. Sample Concentration and Elution Prior to loading, Dionex SolEx SPE HRPHS cartridges were conditioned by rinsing with methanol, then water, and then drying with nitrogen gas. Twenty ml of sample was loaded onto cartridges, followed by a rinse with water and elution with five ml acetonitrile. This results in a four fold concentration. Two µl from each eluent sample were then separated by HPLC and the hormones quantified using standard curves. FIGURE 2. Dionex AutoTrace 280 SPE instrument; a Dionex SolEx cartridge and the fluid connections are shown. Thermo Scientific Poster Note PN71117_HPLC_2014_E_05/14S 3

4 Results Chromatographic Separation An absorbance of 214 nm was chosen for run analysis due to the approximately equivalent signal response obtained for all hormones at this wavelength (Figure 3). Only testosterone and androstenedione, structurally very similar molecules, showed substantial absorbance at 240 and 254 nm. All of the peaks had close to baseline or better resolutions (R s (EP)), ranging from 1.4 to 2.9. FIGURE 3. Hormone standards at various wavelengths mau A -5 B C Minutes Column: Thermo Scientific Acclaim RSLC 120 C18 column, mm Mobile phase: Water/Acetonitrile (CH 3 CN) Gradient : CH 3 CN, 10 54% (0 4 min) 54% (4 12 min) 100% (12 16 min) 10% (16 20 min) Flow Rate: 0.20 ml/min Inj. Volume: 2 µl Column Temp.: 20 C Detection: UV, A: 214 nm, B: 240 nm, C:254 nm Sample: Standard mix, 50 mg/l each Peaks: R s (EP) 1. Estriol Estradiol Testosterone Ethynylestradiol Equilin Estrone Androstenedione -- Chromatographic Limits of Detection (LOD), Limits of Quantification (LOQ), and Linearity As can be seen in Table 1, the LOD and LOQ ranged from to 0.4 mg/l and to 1.3 mg/l, respectively. TABLE 1. LOD and LOQ for hormones at 214 nm. 4 To determine linearity, triplicate injections of standards ranging from 0.05 to 10 mg/l were run. The coefficient of determination (r 2 ) was >0.999, indicating good fit of the standard curve to the data. 4 Concentration of Human Hormones in Drinking Water Using Solid Phase Extraction and Analysis by High Performance Liquid Chromatography

5 Recovery To determine the accuracy of the method, three aliquots of drinking water fortified with 1 mg/l of seven hormones were extracted in parallel and analyzed using HPLC. Figure 4 shows a comparison of the chromatograms for the fortified water prior to cartridge loading, a cartridge elution, and an injection of acetonitrile (used as the eluent for SPE) to show the background signal. As can be seen in Table 2, recovery of hormones from drinking water was approximately 100%, ranging from 90 to 124% with an RSD from 1.2 to 4.4. FIGURE. 4. Chromatograms of hormones eluted from a Dionex SolEx SPE HRPHS cartridge (A), fortified drinking water prior to loading (B), and a blank injection (C) mau 3 7 A B C Minutes Column: Dionex Acclaim RSLC 120 C18 column, mm Mobile phase: Water/Acetonitrile (CH 3 CN) Gradient : CH 3 CN, 10 54% (0 4 min) 54% (4 12 min) 100% (12 16 min) 10% (16 20 min) Flow Rate: 0.20 ml/min Inj. Volume: 2 µl Column Temp.: 20 C Detection: UV, 214 nm Sample: A. Cartridge elution B. Drinking water + 1 mg/l hormones C. Acetonitrile (eluent blank) Peaks: 1. Estriol 2. Estradiol 3. Testosterone 4. Ethynylestradiol 5. Equilin 6. Estrone 7. Androstenedione TABLE 2. Percent recovery of hormones added to a drinking water sample from three Dionex SolEx SPE HRPHS cartridges. 4 Thermo Scientific Poster Note PN71117_HPLC_2014_E_05/14S 5

6 Conclusion The Dionex AutoTrace 280 SPE instrument greatly facilitates the analysis of aqueous samples that contain low-analyte concentrations by automating the process of concentrating them onto cartridges or discs containing absorbents with specific retention properties. Baseline resolution of all of the hormones analyzed was demonstrated using the Dionex SolEx SPE HRPHS cartridges for sample extraction, followed by analysis on the Dionex UltiMate 3000 RSLC system using the Acclaim 120 C18 analytical column. Good recoveries from drinking water, ranging from 90% for androstenedione to 124% for ethynylestradiol, were shown. The recoveries achieved verify the suitability of these cartridges as a means to concentrate human hormones for subsequent quantification as per EPA Method 539. References 1. World Health Organization (WHO), United Nations Environment Programme, Inter- Organization Programme for the Sound Management of Chemicals. State of the Science of Endocrine Disrupting Chemicals 2012: Summary for Decision-Makers. Bergman, A.; Heindel, J.J.; Jobling, S.; Kidd, K.A.; Zoeller, R.T. WHO Document Number WHO/HSE/PHE/IHE/ U.S. Environmental Protection Agency (EPA). Basic Information about the Unregulated Contaminant Monitoring Rule 3 (UCMR3). (accessed April 15, 2014). 3. Smith, G.A.; Zaffiro, A.D.; Zimmerman, M.L.; Munch, D.J. Method 539: Determination of Hormones in Drinking Water by Solid Phase Extraction (SPE) and Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI- MS/MS). Document No. 815-B U.S. Environmental Protection Agency, Cincinnati, OH, Thermo Fisher Scientific Technical Note 148: Automated Extraction and Determination of Human Hormones in Drinking Water Using Solid-Phase Extraction and HPLC with UV Detection. Thermo Scientific P/N TN70898_E, Sunnyvale, CA, [Online] SP-HPLC-Hormones-Water-TN70898-E.pdf (accessed April 15, 2014). 6 Concentration of Human Hormones in Drinking Water Using Solid Phase Extraction and Analysis by High Performance Liquid Chromatography

7 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. Africa Australia Austria Belgium Brazil Canada China (free call domestic) Denmark Europe-Other Finland France Germany India Italy Japan Korea Latin America Middle East Netherlands New Zealand Norway Thermo Fisher Scientific, Sunnyvale, CA USA is ISO 9001:2008 Certified. Russia/CIS Singapore Sweden Switzerland Taiwan UK/Ireland USA PN71117_E 05/14S

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