1. Forensic Toxicology Use Only Analysis of ETG, ETS using the Thermo Scientific Exactive Mass Spectrometer Kent Johnson Fortes lab, Portland Oregon

2. Forensic Toxicology Use Only Goal Development of an easy to use, interference free, less than 4 min analysis method for ETG and ETS in urine LOQ, 100ng/mL or better Method validation using QC’s and real samples

3. Exactive Benchtop LC-MS Resolution 100,000 at 1 scan per second 10,000 at 10 scans per second Mass accuracy Sub ppm Sensitivity 500 fg Buspirone with S/N >10:1 Dynamic range >10,000 within a spectrum Scan speed Up to 10 scans per second Mass range m/z 50 - 4000 Polarity switching One positive and one negative scan < 1 second (25K Resolution)

4. Ethinyl-Estradiol at Different Mass Resolutions Resolution: 10k, 30k, 50k, 100k 279.12279.14279.16279.18279.20 m/z 0 10 20 30 40 50 60 70 80 90 100 Relative Abundance Ethinyl-Estradiol, 279.17434 Butyl-Phthalate, 279.15909 (ubiquitous background ion)

5. Forensic Toxicology Use Only ETG and ETS in Urine

6. Forensic Toxicology Use Only Standards, QC and sample preparation ETG and ETS + 25uL int. std (IS) mix@2000ng/mL 100 uL urine + 900uL of mobile phase A Sample preparation method Inject 20uL Vortex Note: IS are ETG-D5 and ETS-D5 50 -100 x dilution with mobile phase recommended

7. Forensic Toxicology Use Only LC/MS Method LC method – Thermo Hypersil Gold 50 x 2.5 mm, 5u – Mobile Phase A: 5mM Dihexylammonium acetate in LC DI H2O – Mobile Phase B: 5mM Dihexylammonium acetate in CAN – Mobile Phase C: ACN/IPA/Acetone = 45/45/10 v/v/v – Column Temp: 50 Deg C Exactive MS method – HESI source – Full scan MS acquisition @ 100,000 Resolution – External mass calibration – Extract chromatogram for drugs of interest (m/z)@ better than 5ppm mass accuracy – Confirm the identity using mass accuracy and RT – Quantitate using peak area in the chromatogram

8. Forensic Toxicology Use Only ETG-ETS LC gradient Time (min)%A%B%CFlow (mL/min) 095500.8 0.195500.8 3.0643600.8 3.01010000.8 3.5010000.8 3.51001000.8 4.0001000.8 4.0195500.8 5.095500.8

9. 100 ng/mL EtG standard in urine TIC Spectrum 100- 250 amu Peak width@ ½ ht 0.0029 amu XIC: ETG XIC: ETG-D5 ETG IS ETG Very High Specificity

10. 100 ng/mL EtS standard in urine TIC Spectrum 100- 250 m/z Peak width@ ½ ht 0.0027amu XIC: ETS XIC: ETS-D5 ETS IS ETS Very High Specificity

11. Forensic Toxicology Use Only Ion Suppression Test - Water vs Urine 250 ng/mL std Urine Water No Ion Suppression Peak Areas Similar Analyte/IS ratio similar Urine Water EtG-D5 EtS-D5 EtG-D5 EtS-D5 EtG EtS EtG EtS Analyte IS

12. Forensic Toxicology Use Only Calibration curves and LOQ ETS 25- 20,000 ng/mL ETG 100- 20,000 ng/mL 25 ng/mL standards ETGETS ETG-D5ETS-D5 50 ng/mL standards ETGETS ETG-D5ETS-D5

13. Forensic Toxicology Use Only 100 ng/mL, 20 uL standards in urine ETG ETS ETG-D5 ETS-D5

14. Forensic Toxicology Use Only Urine – Negative control ETG ETS ETG < LOD ETS present ETG-D5 ETS-D5

15. Forensic Toxicology Use Only QC 375 ng/mL - ETG ETG ETS present ETG-D5ETS-D5 ETG=317 ng/mL, ETS present

16. Forensic Toxicology Use Only QC 1250 ng/mL - ETG ETG ETS not expected found 190 ng/mL ETG-D5 ETS-D5 ETG=1164 ng/mL, ETS=190 ng/mL

17. Forensic Toxicology Use Only Urine sample ETG < LOD ETS< LOD ETG-D5 ETS-D5 ETG and ETS < LOD

18. Forensic Toxicology Use Only Urine sample ETG ETS ETG-D5 ETS-D5 ETG=312247, ETS=262149 ng/mL

19. Forensic Toxicology Use Only Urine sample ETG=68116, ETS=17789 ng/mL ETG ETS ETG-D5 ETS-D5

20. Forensic Toxicology Use Only ETG ETS Urine sample ETG=14397, ETS=1176 ng/mL ETG-D5 ETS-D5

21. Forensic Toxicology Use Only ETGETS Urine sample Passes lie detector test for alcohol but drinks 1 bottle of Nyquil every night ETG=459.9 ETS=160.8 ng/mL ETG-D5 ETS-D5

22. Forensic Toxicology Use Only ETG ETS Urine sample ETG=864.9 ETS=688.3 ng/mL ETG-D5ETS-D5

23. Forensic Toxicology Use Only ETGETS Urine sample ETG=559.2 ETS=616.4 ng/mL ETG-D5ETS-D5

24. Forensic Toxicology Use Only ETG ETS Urine sample ETG<LOQ, ETS< LOQ (~51.7 ng/mL) ETG-D5ETS-D5

25. Forensic Toxicology Use Only Conclusions Assay based on Exactive ultra high resolution MS coupled to LC system is – Well suited for accurate analysis of ETG and ETS – Analysis time of 4 min for ETG and ETS – ETG LOQ = 100ng/mL, LOD = 25ng/mL – ETS LOQ = 25ng/mL, LOD = 10ng/mL Exactive with a resolution of 100,000 offers >/= SRM selectivity (SRM data comparison not shown) Very easy for method development and routine operation Can be multiplexed for even higher throughput

26. Forensic Toxicology Use Only Acknowledgements Kent Johnson, Fortes Lab Portland, Oregon Marta Kozak, Thermo Fisher Scientific Perkin Elmer (Steroid kit) LipoMed (EtS/EtS D5) Cerilliant (EtG/EtG D5) Thermo Scientific; Instrument, Technical, and application support TCI - Dihexylammonium acetate