Quantitative Analysis of Natural and Synthetic Estrogens in Surface and Final Effluent Waters at Low ppq Levels Using UPLC-MS/MS

Estrogens are routinely used either as contraceptive medicines or in hormone replacement therapy and they can enter aquatic environments via the discharge of final effluent waters. Estrogens are believed to have a negative effect on aquatic environments by disrupting the hormonal systems of fish. For the European Union, EU Directive 2013/39/EU includes 15 additional priority substances to the water framework Directive 2000/60/EC. In this update, 17α-ethinylestradiol and 17β-estradiol were not included in the priority substance list, but instead added to a watch list in order to gather further data regarding the  presence of these compounds in aquatic environments, and the risks they may pose.

This application note summarizes a method for the analysis of estrone, 17α-ethinylestradiol and 17β-estradiol in surface and final effluent waters. To achieve the challenging EU LLOQ parts per quadrillion (ppq) levels required for these compounds, a combination of off-line solid phase extraction (SPE) clean-up and pre-concentration, combined with a large volume injection and triple quadrupole mass spectrometry were utilized.

Benefits

Application benefits include:

• High sensitivity to achieve EU target LLOQs.
• Baseline separation and good peak shapes for target analytes.
• Suitable linearity and repeatability data in surface water and final effluent.
• Acceptable SPE spiked recoveries and repeatability.
• Quantitation of detected residues in final effluent using standard addition in the absence of isotopically labeled internal standard.

Estrogens are routinely used either as contraceptive medicines or in hormone replacement therapy and they can enter aquatic environments via the discharge of final effluent waters.¹ Estrogens are believed to have  a negative effect on aquatic environments by disrupting the hormonal systems of fish.¹ For the European Union, EU Directive 2013/39/EU² includes 15 additional priority substances to the water framework Directive 2000/60/EC.³ In this update, 17α-ethinylestradiol and 17β-estradiol were  not included in the priority substance list, but instead added to a watch list² in order to gather further data regarding the presence of these compounds in aquatic environments, and the risks they may pose. 

This application note summarizes a method for the analysis of estrone, 17α-ethinylestradiol and 17β-estradiol in surface and final effluent waters. To achieve the challenging EU LLOQ parts per quadrillion (ppq) levels⁴ required for these compounds, a combination of off-line solid phase extraction (SPE) clean-up and pre-concentration, combined with a large volume injection and triple quadrupole mass spectrometry were utilized.

Sample Description

Extracted samples were prepared and supplied by Scottish Water. Spiked surface water and final effluent samples were first filtered, extracted, and concentrated using an off-line solid phase extraction (SPE) method^5-6 (Figure 1). After evaporation and reconstitution in LCMS grade water, the samples were then analyzed by UPLC-MS/MS using a large volume injection (100 µL).

Method optimization was accomplished by evaluating various columns, mobile phase compositions, gradients, and MS transitions. The conditions detailed in the Experimental section provided the best overall performance of those tested. Baseline separation of the target analytes was achieved, and a example of the chromatography from a 50 ng/L solvent standard is shown in Figure 2. Due to the hydrophobic nature of the analytes, they elute in the high organic part of the gradient. As a result, it is challenging to separate the analytes away from matrix components in the final effluent used for this analysis, as shown in the RADAR scan (full scan m/z 100 to 1000) in Figure 3. To minimize any contamination of the MS source, the integrated fluidics system on the  Xevo TQ-XS was used to divert the chromatographic region to the MS system for analysis, and unwanted regions to waste. 

Satisfactory linearity was achieved for all compounds in matrix matched (spiked post extraction) bracketed calibration curves, over the appropriate ranges. In surface water, a range of 10 to 320 ng/L for 17α-ethinylestradiol, and 62.5 to 2000 ng/L for 17β-estradiol and Estrone provided good linearity (R² >0.998, residuals <15%). An example of the calibration and associated residuals is shown for all three compounds in Figure 4. For final effluent, the matrix matched calibration curves ranged from 120 to 2000 ng/L for all three compounds, also giving acceptable linearity (R²>0.997, residuals <10%). The robustness of the method was assessed using spiked water samples (n=8 for each matrix type) where %RSD values below 6% were obtained.

To evaluate the method performance in surface water, matrix samples were pre-spiked at  suitable pg/L (ppq) levels prior to extraction, and prepared in accordance with the methodology detailed in Figure 1. An example of the chromatography and sensitivity observed for a sample pre-spiked at low ppq level in surface water, before clean-up and concentration, is shown in Figure 5. 

Matrix effects were determined by quantifying post spiked surface water samples against a solvent calibration curve. For 17β-estradiol and estrone, matrix effects were calculated at ≤-22% (suppression), 17α-ethinylestradiol ≤16% (enhancement). Final effluent, even after the SPE clean-up remained a complex sample, as shown in the RADAR scan in Figure 3, this resulted in significant matrix suppression ≤-72% for all compounds. However even with this significant suppression, low levels of each compound are still detectable.

The recovery and repeatability data for the analytes, spiked at two levels in triplicate in surface water are detailed in Table 2. 

The method showed high sensitivity, achieving the target European (2015/495/EU)⁴ LLOQ (PtP s/n=10) levels for each compound in the surface water matrix. Low level concentrations of all compounds were detected in the final effluent matrix. A standard addition method was used to quantify the analytes present, where 17α-ethinylestradiol was measured at 16.9 pg/L, as shown in Figure 6.

This method highlights the analysis of low ppq levels of synthetic estrogens in surface and final effluent waters. Utilizing Oasis SPE and Sep-Pak SPE sample preparation technologies, the optimized extraction method was found to provide the required concentration and clean-up, giving acceptable recoveries and repeatability in spiked surface water samples. The use of a large volume injection in combination with the ACQUITY UPLC H-Class and Xevo TQ-XS, allowed for the challenging detection requirements of this analysis to be achieved in surface water. The use of standard addition allowed for accurate quantitation of trace residues in final effluent samples.

1. Schwindt A, Winkelman D, Keteles L, et al. An Environmental Oestrogen Disrupts Fish Population Dynamics through Direct and Transgenerational Effects on Survival and Fecundity. Journal of Applied Ecology. 2014.
2. Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 Amending Directive 2000/60/EC and 2008/105/EC in Respect of Priority Substances in the Field of Water Policy Text with EEA Relevance. August 2013.
3. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 Establishing a Framework for Community Action in the Field of Water Policy. October 2000.
4. Commission Implementing Decision (EU) 2015/495 of 20 March 2015 Establishing a Watch List of Substances for Union-Wide Monitoring in the Field of Water Policy Pursuant to Directive 2008/105/EC of the European Parliament and of the Council (Notified under Document C(2015) 1756) Text with EEA Relevance. March 2015.
5. Ross E, Boag A, Todd H, Gatward N. Analysis of Natural and Synthetic Estrogens at Sub-PPT Levels in Surface Water and Crude Influent Water Utilizing the ACQUITY UPLC System with 2D LC Technology and Xevo TQ-S. Waters Technology Brief No. 720005626en. March 2016.
6. Chang H. et al. Occurrence of Androgens and Progestogens in Wastewater Treatment Plants and Receiving River Waters: Comparison to Estrogens. Waters Research. 45(2):732–740. 2011.