State of the art in chromatographic analysis (LC MS/MS) of steroidal estrogens in surface water Status and Outlook

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1 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,

2 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

3 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

4 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

5 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:

6 Comparison of the LODs and EQS for fresh surface waters LOD (S/N = 3), Fresh Surface Water E1 E2 EE pg/l 1000 EQS for E2 = 400 pg/l EPA EPA EQS for EE2 = 35 pg/l 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

7 LOQ and EQS Comparison LOQ (S/N = 10 or 3.3 x LOD), Fresh Surface Water E1 E2 EE pg/l 30% of EQS for E2 = 120 pg/l % of EQS for EE2 = 10.5 pg/l Scientific publications (Year) At least 10x 100x decrease of current LOQs is needed to comply with the requirement of the WFD

8 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

9 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 or 20 years?

10 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 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 E1 E2 EE SPE 1000x large volume injection 100 µl pg/l 100 SPE 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

11 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)

12 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, Clean up = additional step + custom made sorbents (MIP) Loss of analytes during clean up?

13 Comparison of LOD for river water and waste water LOD, E2 river water effluent influent LOD, EE2 river water influent effluent pg/l 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:

14 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)

15 Contact information and references Contact information: Dr. Nadzeya Homazava Oekotoxzentrum/Centre Ecotox Eawag/EPFL Überlandstrasse 133 Postfach Dübendorf Schweiz phone References: 1. Görög, S., Advances in the analysis of steroid hormone drugs in pharmaceuticals and environmental samples ( ). Journal of Pharmaceutical and Biomedical Analysis, 2011, 55, Tomšíková, H., et al., High sensitivity analysis of female steroid hormones in environmental samples. TrAC Trends in Analytical Chemistry, 2012, 34, Loos, R., Analytical methods relevant to the European Commission's 2012 proposal on Priority Substances under the Water Framework Directive