Schweizerisches Zentrum für angewandte Ökotoxikologie Centre Suisse d écotoxicologie appliquée Eawag-EPFL State of the art in chromatographic analysis (LC MS/MS) of steroidal estrogens in surface water Status and Outlook Nadzeya Homazava contact: nadzeya.homazava@oekotoxzentrum.ch Bioanalytical options for the monitoring of steroidal estrogens in surface water Federal Insiture of Hydrology Koblenz (BfG), DE, 27.02.2013
Overview Steroidal estrogens and why it s so difficult to measure them (LC MS/MS) LOD, LOQ and EQS. What do we need to compare What can be done to improve current analytical methods What about analysis of waste water (effluents, influents) Conclusions
Steroidal estrogens: why it s so difficult to measure them Reason 1. Concentrations of steroidal estrogens in surface waters are very low (sub ng/l and pg/l range). Majority of the studies report E2 and EE2 concentrations in river water as n.d. or < LOQ Reason 2. Chemical structure and ionization. Estrone (E1) 17β Estradiol (E2) 17α Ethinylestradiol (EE2) - - - they all form negative ions during ionization in LC MS/MS BUT It s well known that negative electrospray ionization (ESI ) mode is less sensitive in LC MS/MS than positive mode
LOD, LOQ and EQS. Definitions and comparison Limit of detection (LOD) a smallest concentration that can be recognized as a chromatographic peak with a signal to noise ratio (S/N) = 3 Limit of quantification (LOQ) a smallest concentration that can be recognized as a chromatographic peak with a signal to noise ratio (S/N) = 10 LOQ = 3.3 x LOD Compliance monitoring for the WFD requires LOQ 0.3 EQS
Scientific literature search To present the state of art in the chromatographic analysis of steroidal estrogens: > 40 scientific publications included in the literature search 2 comprehensive literature overviews Time period: 2000 2012
Comparison of the LODs and EQS for fresh surface waters 100000 LOD (S/N = 3), Fresh Surface Water E1 E2 EE2 10000 pg/l 1000 EQS for E2 = 400 pg/l EPA EPA 1698 539 100 EQS for EE2 = 35 pg/l 10 0 2000 2003 2004 2005 2007 2009 2010 2011 2007 2010 Scientific publications (Year) 35% of the reviewed methods are able to detect E2 at EQS level Only 1 published method is able to detect EE2 at EQS level
LOQ and EQS Comparison 100000 LOQ (S/N = 10 or 3.3 x LOD), Fresh Surface Water E1 E2 EE2 10000 1000 pg/l 30% of EQS for E2 = 120 pg/l 100 10 30% of EQS for EE2 = 10.5 pg/l 0 2000 2003 2004 2005 2007 2009 2010 2011 2007 2010 Scientific publications (Year) At least 10x 100x decrease of current LOQs is needed to comply with the requirement of the WFD
What can be done to improve current analytical methods To decrease LOD/LOQ we need to: 1. Increase the signal 2. Reduce the noise How to increase the signal: 1. Increase the sensitivity of the instrument 2. Increase the concentration of the analyte in the sample 3. Increase amount of the sample in the instrument 4. Improve ionization efficiency How to reduce the noise: 1. Reduce the matrix interferences in the sample (clean up step after extraction) 2. Increase the sensitivity of the instrument
Increasing the signal of steroidal estrogens in LC MS/MS methods How to increase the signal: 1. Increase the sensitivity of the instrument new generation of instruments Critical point: Wait 5 10 15 or 20 years?
Increasing the signal of steroidal estrogens in LC MS/MS methods 2. Increase the concentration of the analyte in the sample increase enrichment factors during extraction typically, SPE extracts 1 L of water into 0.2 1 ml of extract = 1000x 5000x 3. Increase amount of the sample in the instrument large volume injection 100 µl injection vs. typical 5 10 µl 100000 E1 E2 EE2 10000 1000 SPE 1000x large volume injection 100 µl pg/l 100 SPE 20000 x 10 0 Critical points: These strategies lead to the increase of the noise level as well High volume of the sample is needed (> 2L) time consuming and impractical
Increasing the signal of steroidal estrogens in LC MS/MS methods 4. Improve ionization efficiency switch from negative to positive ionization Derivatization (e.g. with Dansyl chloride) EE2: negative ionization dansylated EE2: positive ionization Critical points: Additional step = more work Loss of analytes? Less specific detection Lien et al., Journal of Chromatography A, 1216 (2009) 956 966
Reducing the noise during the estrogens analysis Selective SPE (e.g. immunosorbents, molecular imprinted polymers MIP) Clean up of the extract after normal SPE (Florisil columns, size exclusion chromatography etc.) Clean up is especially relevant for waste water samples (high matrix content) Total ion chromatogram of raw and immunosorbent treated effluent extract Critical points: Fergusson, Anal. Chem. 2001, 73, 3890 3895 Clean up = additional step + custom made sorbents (MIP) Loss of analytes during clean up?
Comparison of LOD for river water and waste water 100000 10000 LOD, E2 river water effluent influent LOD, EE2 river water influent effluent pg/l 1000 100 10 0 E2 IS E 5x 25 x increase of LOD for the same method when analyzing waste water vs. river water Schlüsener et al., Rapid Commun. Mass Spectrom. 2005; 19: 3269 3278
Conclusions: In the ideal world or take home message So is it possible to reach the requirement LOQ 0.3 EQS with current analytical methods? Currently No, BUT: For E2: there are some methods published that are able to detect E2 at EQS level (LOD EQS ) For EE2: It is possible to assess if EE2 concentration in water is highly above EQS Outlook YES, only if: We take a large volume sample (> 2L) Clean up the sample after extraction Derivatize it to switch to the positive ionization Wait for a new generation of LC MS/MS instruments (5 15 years), and high end analytics become routine and affordable ( years?) until then Combine the advantages of the best bioanalytical tools with the lower LOQs with the best possible chemical analysis (provides compound specific information as opposed to bioanalytical tools)
Contact information and references Contact information: Dr. Nadzeya Homazava Oekotoxzentrum/Centre Ecotox Eawag/EPFL Überlandstrasse 133 Postfach 611 8600 Dübendorf Schweiz phone +41 58 765 50 60 nadzeya.homazava@oekotoxzentrum.ch References: 1. Görög, S., Advances in the analysis of steroid hormone drugs in pharmaceuticals and environmental samples (2004 2010). Journal of Pharmaceutical and Biomedical Analysis, 2011, 55, 728 743. 2. Tomšíková, H., et al., High sensitivity analysis of female steroid hormones in environmental samples. TrAC Trends in Analytical Chemistry, 2012, 34, 35 58. 3. Loos, R., Analytical methods relevant to the European Commission's 2012 proposal on Priority Substances under the Water Framework Directive. 2012.