1. Measurement of Brevetoxin Levels in Ocean Water Erik Sahlin Chem 4101 Fall 2011 Image - http://www.scripps.edu/newsandviews/e_20010226/hi-brevetoxin.gif Molecular Mass 895.08 amu

2. Why High Levels of Brevetoxin is a Problem? Harmful to Marine Life [7] Brevetoxin (neurotoxin) binds to the gills on fish Reaches the protein structure of cell membranes Eventually enters food cycle and contaminates most marine life in area High concentrations can kill marine life Harmful to Humans [7] Causes Neurotoxic Shellfish Poisoning (NSP) – Human must consume shellfish – Leads to tingling sensations, muscle pain, nausea, and headaches May lead to respiratory problems

3. Hypothesis There is a higher concentration of Brevetoxin in the ocean water near the algal blooms than in the surrounding ocean water. Image - http://www.shenzhen-standard.com/wp-content/uploads/2010/08/red-tide.jpg Ocean water samples in the open ocean that are far away from algal blooms Ocean water samples in the open ocean that are close to algal blooms Ocean water samples near the shoreline that are far away from algal blooms Ocean water samples that are near shoreline that are close to algal blooms Samples Controls and Standards Standards of Brevetoxin can be purchased from EMD4Biosciences

4. Possible Techniques [10] SeparationAdvantagesDisadvantages Micellular Electrokinetic Chromatography High column efficiency, small sample volumes Temperature must be closely regulated, moderate reproducibility Capillary Zone Electrophoresis Fast retention time, small sample volumes Low LOD, low quantitative precision

5. Possible Detectors [10] DetectorAdvantageDisadvantage Mass SpectrometryDetect several components, good sensitivity Expensive, moderate levels of sensitivity UV-Visible Absorption Easy to use, high sensitivity, fast retention time Difficult to measure low concentrations, complex matrix will make it hard to detect the analyte

6. Why Choose High Performance Liquid Chromatography? Advantages Easy separation of similar compounds Can detect small sample volumes Very common and widely used technique High resolution High selectivity and sensitivity Disadvantages Longer retention time to increase resolution

7. Sample HPLC Preparation [11] Sample Spiked to Enriched Filter (100-1000ng) Dry Sample (60min) Add Acetone (10mL) and Vortex (20s) Sonicate Sample(20min) Evaporate off Impurities(100µL) Vortex and Add Solvent(200 µL)

8. High Performance Liquid Chromatography Parameters Column temperature 32 ⁰ C Flow rate 150-200µL/min Injection volume 50µL Solvents used 90:10 ration of 1mM ammonium acetate and water LOD 10pg/m 3 LOQ 500 ng C18 Aqua Column (3µm, 125Å, 75mm x 2 mm) [8] C18 Guard Column (3µm, 125Å, 30mm x 2 mm) [8]

9. Why Use Fluorescence? Advantages Cheaper instrument than mass spectrometry High sensitivity High selectivity Fast retention times Image http://www.chemistry.adelaide.edu.au/external/soc-rel/content/mol-fluo.htm

10. JASCO Fluorescence Detector for HPLC FP-2020 [5] Excitation at 354nm Emission at 410nm S/N greater than 350:1 16µL flow cell High Sensitivity – LOD 40fg http://www.jasco.co.uk/fluorescence.asp

11. Conclusions The sample size is small and it is affordable Several ocean samples need to be gathered to compare against the standard Brevetoxins Reverse Phase Liquid Chromatography could be used to separate Brevetoxins from an ocean water sample and detected by fluorescence

12. References 1.Cohen, Jonathan H. Tester, Patricia A. Forward Jr., Richard B. Oxford Journals: Journal of Plankton Research. Sublethal Effects of the Toxic Dinoflagellate Karenia Brevis on Marine Copepod Behavior. January 12th, 2007. http://plankt.oxfordjournals.org/content/29/3/301.full 2.Errera, Reagan M. Campbell, Lisa. “Osmotic stress triggers toxin production by the dinoflagellate Karenia brevis.” May 23 rd, 2011. http://www.pnas.org/content/early/2011/06/08/1104247108.abstract 3.Hua, Yousheng. Cole, Richard B. Electrospray Ionization Tandem Mass Spectrometry for Structural Elucidation of Protonated Brevetoxins in Red Tide Algae. http://pubs.acs.org/doi/full/10.1021/ac990433o. 4.Hua, Yousheng. Cole, Richard B. "High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry for the Determination of Brevetoxins in “Red Tide” Algae." http://pubs.acs.org/doi/pdf/10.1021/ac00107a010. 5.Jasco UK. “JASCO Fluorescence detector for HPLC FP-2020.”. Jasco UK. 2011. http://www.jasco.co.uk/fluorescence.asp 6.Mattley, Yvette D. Garcia-Rubio, Luis H. Multiwavelength Spectroscopy for the Detection, Identification and Quantification of Cells. Nov. 5th, 2000. http://www.marine.usf.edu/sapd/spiep00ym.pdf 7.National Oceanic and Atmospheric Administration. “Brevetoxin and Florida Red Tides.” National Oceanic and Atmospheric Administration. 2004. http:// www.nmfs.noaa.gov/pr/pdfs/health/brevetoxin.pdf 8.Phenomenex. "C18 "Aqua"column [3 μm, 125 A, 75 × 200 mm, (Phenomenex #003-4311-B0) and C18 guard column (Phenomenex #AJO4287)" Phenomenex-DNV. Torrance, CA. 2011. http://www.phenomenex.com/Products/Part/00A-4311- B0 9.Shea, Damian. "Analysis of Brevetoxins by Micellar Electrokinetic Capillary Chromatography and Laser-induced Fluorescence Detection. Wiley Online Library. Wiley Online Library, 14 Apr. 2005. 10.Skoog, Douglas A. Holler, F. James. Crouch, Stanley R. “Principles of Instrumental Analysis.” Brooks/Cole Cengage Learning. Belmont, CA. 6 th edition, 2007. 11.Yung Sung Cheng. McDonald, Jacob D. "Concentration and Particle Size of Airborne Toxic Algae (Brevetoxin) Derived from Ocean Red Tide Events." National Institutes of Health. May 15, 2005. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652738/