Using ultrasonic liquid extraction for estrogens analysis in

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Using ultrasonic liquid extraction for estrogens analysis in sludge by HPLC with fluorescence detection

Using ultrasonic liquid extraction for estrogens analysis in sludge by HPLC with fluorescence detection Vitória Lourosa, Diana Limab, Jorge Leitãoc, Valdemar Estevesb, Helena Nadaisa a Department of Environment and Planning, CESAM, University of Aveiro, Aveiro b Department of Chemistry, CESAM, University of Aveiro, Aveiro c Centre for Biological and Chemical Engineering, IBB, Instituto Superior Técnico, Lisbon Introduction Recently, a group of organic pollutants called endocrine-disrupting chemicals (EDCs) has attracted some attention due to their impact in the environment and living beings. Natural estrogens estrone (E 1), 17β-estradiol (E 2) and the synthetic estrogen 17α-ethinylestradiol (EE 2) are reported as the EDCs with higher disrupting potency, even at trace levels. Wastewater Treatment Plants (WWTPs) are a significant point source of EDCs released into the environment. In the literature is reported that E 2 is readily oxidized to E 1 during the sewage treatment process. Due the high sorption potential of estrogens, in WWTPs, large proportion of removed estrogens are adsorbed onto sludge. Therefore, it is crucial to develop a selective, reliable and affordable analytic method for the simultaneous determination of E 1 and EE 2 in fresh digested sludge from WWTP. Table 1 / E 1 and EE 2 analytical curves and recovery results. Analyte Linear Correlation Linearity Limit of detection range coefficient sludge E 1 EE 2 (μg L-1) (r) (%) 0. 99800 97. 164 67. 5 1. 2 102. 9 0. 9 b 10 -500 0. 99994 99. 451 7. 0 0. 1 104 4 b sludge of 5 min, 30 min and 1 h. b Mean value standard deviation (n = 9). Our study Literature Standards spiked onto freezedried sludge [1, 2] overestimation of recovery rates of this compounds and low LOD Standards spiked onto fresh sludge Longer consumption times degradation of estrogens during cleanup steps [1] Spike Addition of 100 μg E 1 and 1 μg EE 2 with contact times of 5 min, 30 min and 1 h Fig 2 / Comparison between the methodology applied in this study and in literature. Solid phase Storage (-80 ºC) Mass estrogen (µg) (a) Centrifugation (4000 rpm, 10 min) 120 100 Freeze-dried Storage (4 ºC) (μg g-1) 100 -1000 Fresh digested sludge samples (50 m. L) Filtration (Pore size 0. 2 μm) (μg L-1) a Values obtained for a mass spike of 100 μg E 1 and 1 μg EE 2 with contact times of spiked estrogen with fresh Experimental procedure The treatment of digested sludge sample is illustrated in Fig 1. Liquid phase Extraction recovery a Fresh sludge Solid phase of 80 60 40 20 0 E 1 in liquid phase E 1 in solid phase (b) 3 extraction Shaken, 1 min ULE*, 1 h Centrifugation (4000 rpm), 10 min Filtration (Pore size 0. 2 μm) Analysis by HPLC-FLD Fig 1 / Schematic diagram for analysis of hormones in liquid and solid phases of digested sludge. *Ultrasonic liquid extraction (ULE) was carried out with 9 m. L of methanol, 4. 5 m. L of methanol and 4. 5 m. L of acetone per gram of dried sludge. Results The extraction recovery rates were determined by spiking a known amount of E 1 and EE 2 into fresh digested sludge with contact times of 5 min, 30 min and 1 h. This parameter was calculated by the estrogen mass measured in fresh sludge after spiking with estrogen divided by total mass spiked of each compound in sample. The individual mass measured of E 1 and EE 2 was determined by adding the masses quantified in liquid and in solid phases. Results indicated good recovery efficiencies of the overall method, which ranged from 102. 99% to 104. 33% for E 1 and EE 2, respectively, indicating good accuracy; also good precision were observed with relative standard deviation <4% (Table 1). A comparison between the methodology applied in this study and in literature is presented in Fig 2. The different adsorption values obtained for E 1 and EE 2 (Fig 3) may be associated with the higher sorption potential of EE 2. At contact times of 5 min, 30 min and 1 h, no differences were observed of mass E 1 and EE 2 in liquid phase and solid phase. Mass estrogen (µg) Abstract A method for the simultaneous determination of estrone (E 1) and 17α-ethinylestradiol (EE 2) in fresh digested sludge samples was developed, based on ultrasonic liquid extraction (ULE) and high-performance liquid chromatography with fluorescence detector (HPLCFLD). The developed method is fast, cheap and easy-to-use. During the pretreatment procedure the standards were spiked onto the fresh sludge and extracted using three-steps extraction procedure with methanol and acetone as extraction solvents. Good calibration linearity, recovery and precision, for E 1 and EE 2, in real digested sludge samples were observed. The recoveries of E 1 and EE 2 in digested sludge were 102. 9 0. 9% and 104 4%, respectively. Limits of detection (LOD) for E 1 and EE 2 in fresh sludge were detected up to 67. 5 µg L− 1 and 7. 0 µg L− 1, respectively. In solid phase of sludge LODs of 1. 2 µg g− 1 for E 1 and 0. 1 µg g− 1 for EE 2 were achieved. 1. 2 More than 78. 7% of spiked E 1 and 90. 7% of spiked EE 2 were adsorbed onto sludge phase after 5 min of contact time 1. 0 0. 8 0. 6 0. 4 0. 2 0. 0 EE 2 in liquid phase before spiked 0 min EE 2 in solid phase 5 min 30 min 1 h Fig 3 / Mass profiles of E 1 (a) and EE 2 (b) using the spike of 100 μg E 1 and 1 μg EE 2 in fresh digested sludge under anaerobic condition. Conclusion Comparing with the other methods reported in the literature, ULE-HPLC-FLD presents the following advantages: high extraction recoveries, rapid and easy-touse analysis. Overall, the method suggested in this study shows good efficiency for determination of E 1 and EE 2 in fresh sludge matrix. References [1] Ternes, T. A. , Andersen, H. , Gilberg, D. , Bonerz, M. (2002) Determination of Estrogens in Sludge and Sediments by Liquid Extraction and GC/MS/MS, Anal. Chem. , Vol. 74, pp. 3498– 3504. [2] Nie, Y. , Qiang, Z. , Zhang, H. , Adams, C. (2009) Determination of endocrinedisrupting chemicals in the liquid and solid phases of activated sludge by solid phase extraction and gas chromatography-mass spectrometry, J. Chromatogr. A, Vol. 1216. Acknowledgements Thanks are due for the financial support to CESAM (UID/AMB/50017 - POCI-01 -0145 -FEDER-007638), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT 2020 Partnership Agreement and Compete 2020. Vitoria Louros and Diana Lima would like to thank for their doctoral (SFRH/BD/112907/2015) and post-doctoral (SFRH/BPD/80315/2011) grants, respectively.