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Development and Validation of a Novel Approach for the Analysis of Marine Biotoxins

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Development and Validation of a Novel Approach for the Analysis of Marine Biotoxins DEVELOPMENT AND VALIDATION OF A NOVEL APPROACH FOR THE ANALYSIS OF MARINE BIOTOXINS Bing Cheng Chai College of Engineering and Science Victoria University Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy June 2017 ABSTRACT Harmful algal blooms (HABs) which can produce a variety of marine biotoxins are a prevalent and growing risk to public safety. The aim of this research was to investigate, evaluate, develop and validate an analytical method for the detection and quantitation of five important groups of marine biotoxins in shellfish tissue. These groups included paralytic shellfish toxins (PST), amnesic shellfish toxins (AST), diarrheic shellfish toxins (DST), azaspiracids (AZA) and neurotoxic shellfish toxins (NST). A novel tandem liquid chromatographic (LC) approach using hydrophilic interaction chromatography (HILIC), aqueous normal phase (ANP), reversed phase (RP) chromatography, tandem mass spectrometry (MSMS) and fluorescence spectroscopic detection (FLD) was designed and tested. During method development of the tandem LC setup, it was found that HILIC and ANP columns were unsuitable for the PSTs because of the lack of chromatographic separation power, precluding them from being used with MSMS detection. In addition, sensitivity for the PSTs at regulatory limits could not be achieved with MSMS detection, which led to a RP-FLD combination. The technique of RP-MSMS was found to be suitable for the remaining four groups of biotoxins. The final method was a combination of two RP columns coupled with FLD and MSMS detectors, with a valve switching program and injection program. A novel sample preparation method was also developed for the extraction and clean-up of biotoxins from mussels. It was determined that Strata-X was a suitable sorbent for use in the clean-up of mussel extracts. A validation study was carried out on the developed method via analysis of certified reference materials for AZAs, DSTs and ASP, and naturally contaminated mussel material for PSTs. A major limitation to this research was the scarcity and the restrictions in obtaining and receiving biotoxin reference materials. In addition, no reference materials were available for brevetoxins. Therefore, spiking trials were conducted for brevetoxins and it was found that no recoveries could be observed, possibly due to irreversible binding to matrix components. Determination of measurement uncertainty was performed based on the validation data. The method was shown to be capable of meeting current regulatory needs with respect to specificity, analytical range, and limits of detection for PSTs and DSTs. Low recoveries were observed for AZAs and ASTs, which may be accounted for by the application of correction factors determined on a per batch basis. ii Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template DECLARATION OF ORIGINALITY I, Bing Cheng Chai, declare that the Ph.D. thesis entitled “Development and Validation of a novel approach for the analysis of Marine Biotoxins” is no more than 100,000 words in length including quotes and exclusive of tables, figures, appendices, bibliography, references and footnotes. This thesis contains no material that has been submitted previously, in whole or in part, for the award of any other academic degree or diploma. Except where otherwise indicated, this thesis is my own work. Signed:______________________________________________________________ Bing Cheng Chai Date:_________________________________________________________________ iii Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template Dedicated to my parents and to Eleanor Whatever your hand finds to do, do it with your might, for there is no work or thought or knowledge or wisdom in Sheol, to which you are going. — Ecclesiastes 9:10 ESV And whatever you do, in word or deed, do everything in the name of the Lord Jesus, giving thanks to God the Father through him. — Colossians 3:17 ESV iv Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my supervisors at VU, Dr. Rohani Paimin for initiating this project. Also, to Dr. Nicholas Milne and Professor Stephen Bigger for their help and support throughout the writing process. I would not have been able to complete this work without your valuable contributions. I would also like to thank those at the National Measurement Institute, Dr. Saman Buddhadasa, who provided the environment and resources without which this project would not be possible. Also to the NMI General Manager, James Roberts, and the Port Melbourne Branch Manager, Shyam Kumaran, as well as Section Manager, Timothy Stobaus, and Katherine Stockham, the Technical Development Coordinator. Their guidance and support throughout this project was crucial. Many thank also to Peter Anstis and Paul Armishaw for their help in helping me understand measurement uncertainty and their patience in walking me through the calculations. To James Pyke and Jim Tsiotinas from Agilent Technologies, for providing the hardware and technical expertise when I had questions about chromatography and tandem LC, which brought the tandem LC setup into fruition. To Allison Turnbull and Tom Madigan from the South Australia Research and Development Institution, for supplying the toxic shellfish material critical for the research. Finally, special thanks goes to Samantha Duong and Amanda Sheard, whose friendship have made these past years go by in the blink of an eye. And to my friends and housemates who have provided vital emotional support at the most stressful times, thank you sincerely. v Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template PRESENTATIONS ARISING FROM RESEARCH Chai B.C. (2012) “Developing a more sensitive and rapid method for the detection of Paralytic Shellfish Toxins”, Australian Institute of Food Science and Technology (AIFST) Young Members’ Presentation Evening, Deakin University, Burwood, VIC, Australia. Chai B.C., Paimin R., Stockham K. (2012) “Developing a Rapid Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) method for the detection of Paralytic Shellfish Toxins (PST)”, 20th Royal Australian Chemical Institute Research & Development Topics Conference, Deakin University, Geelong, VIC, Australia. Chai B.C (2013) “Development of a HILIC-MS/MS method for the determination of Paralytic Shellfish Toxins in mussel”, 24th Conference for Residue Chemists (CRC), Victoria University, Melbourne, VIC, Australia. Chai B.C., Paimin R., Buddhadasa S. (2013) “Marine Biotoxins” — Collaborative Research Partnerships Symposium (CRPS), Victoria University, Werribee, VIC, Australia. Chai B.C., Paimin R., Stockham K. (2013) “Characterisation of matrix components which suppress ionisation of Paralytic Shellfish Toxins for analysis with Hydrophilic Interaction Chromatography Tandem Mass Spectrometry”, 40th International Symposium on High Performance Liquid Phase Separations and Related Techniques Hobart 2013 (HPLC 2013), Hobart, TAS, Australia. Chai B.C. (2014) “The Trouble With Toxins”, 3 Minute Thesis (3MT) Competition, Victoria University, Footscray, VIC, Australia. Chai B.C., Milne N., Buddhadasa S., Bigger S., Pyke J.S. (2015) “Marine Biotoxins”, Collaborative Research Partnerships Symposium (CRPS), Victoria University, Werribee, VIC, Australia. vi Word Template by Friedman & Morgan 2014 Morgan & Friedman by Word Template TABLE OF CONTENTS 1 GENERAL INTRODUCTION .................................................................................. 1 1.1 HARMFUL ALGAL BLOOMS ...................................................................................... 1 1.1.1 Factors Affecting Biotoxin Production ............................................................ 4 1.1.2 Effects on Humans ........................................................................................... 6 1.1.3 Impact on Animals ........................................................................................... 7 1.1.4 Economic Impacts ........................................................................................... 8 1.2 CONTROL AND MONITORING OF HARMFUL ALGAL BLOOMS ................................. 10 1.2.1 Establishing regulatory limits ....................................................................... 11 1.2.2 Emerging Toxins ............................................................................................ 12 1.2.2.1 Cyclic Imines ..................................................................................................... 13 1.2.2.2 Yessotoxins and Pectenotoxins .......................................................................... 13 1.2.2.3 Palytoxin ............................................................................................................ 14 1.2.2.4 Tetrodotoxin ....................................................................................................... 14 1.2.2.5 Ciguatera Fish Poisoning ................................................................................... 14 1.3 SIGNIFICANCE AND AIM OF RESEARCH .................................................................. 15 1.4 THESIS ORGANISATION .......................................................................................... 16 2 LITERATURE REVIEW ......................................................................................... 17 2.1 INTRODUCTION .....................................................................................................
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