1. An improved qualitative method for detecting and isolating shark-repelling semiochemicals Eric M. Stroud Michael M. Herrmann Dr. Samuel Gruber AES July 09, 2005

2. Introduction Analytical method Analytical method Observe decay process Observe decay process Field testing Field testing

3. HPLC-UV/Vis Solvents: Methanol + 0.1% acetic acid Water + 0.1% acetic acid

4. HPLC-UV/Vis Ternary HPLC Pump Gradient control: 0-10 minutes: 100% methanol/acetic acid 10-12 minutes: Linear gradient to 100% water/acetic acid 12-20 minutes: 100% water/acetic acid 20-22 minutes: Linear gradient to methanol/acetic acid 22-40 minutes: 100% methanol/acetic acid

5. HPLC-UV/Vis Injector with 50uL loop

6. HPLC-UV/Vis Guard column

7. HPLC-UV/Vis Column heater: Maintain temperature at 25C Column: Waters Novapak C18 RP 3.9x150 mm

8. HPLC-UV/Vis UV / Vis Detectors Monitor: 240nm-340nm - semios 440nm – secondary amines 570nm – primary amines

9. HPLC-UV/Vis Integrator or PC

10. Why ??? November 2004 Field Test November 2004 Field Test Visual/odor was OK Visual/odor was OK Delivery method same Delivery method same Same species Same species What happened? What happened?

11. Good Batch – Early Eluting

12. ~7 minutes ~6 minutes ~5 minutes

13. Bad Batch – Early Eluting

14. 5.2 5.9 5.8 5.4

15. Good Batch – Late Eluting

17. Bad Batch – Late Eluting

19. GWS Head – Early Eluting

22. GWS Head – Late Eluting

25. GWS Field Trials 6 C. perezii, 2 C. acronotus 6 C. perezii, 2 C. acronotus 500mL GWS, 2 minute span 500mL GWS, 2 minute span 4 C. perezii, 2 C. acronotus gone 4 C. perezii, 2 C. acronotus gone 2 C. perezii disinterested for 10 mins with constant attractant 2 C. perezii disinterested for 10 mins with constant attractant

27. A2, good batch, 1 yr A13N, 1 yr SQ1, 1 yr

29. GWS Head Tiger shark carcass Nurse shark carcass Lemon shark carcass, 2yr. C. limbatus carcass GWS carcass Lemon shark carcass, 1yr. Southern ray GWS Tail

31. 0 days

32. 7 days

33. 0 days 7 days 21 days

34. 0 days 7 days 21 days 40 days

35. Secondary Amines @ 440nm

36. Primary Amines @ 570nm

38. Conclusions HPLC is suitable for degradation studies HPLC is suitable for degradation studies GWS head currently has highest yield of target compounds GWS head currently has highest yield of target compounds Storage procedures / shelf life with extracts Storage procedures / shelf life with extracts