DEVELOPMENT OF AN ANALYTICAL METHOD FOR β-METHYLAMINO-L-ALANINE, A CYANOBACTERIAL METABOLITE AND POTENTIAL ENVIRONMENTAL TOXIN AND SELECTIVE EXTRACTION PROTOCOL AND STRUCTURE OF FORMAMIDES OF β-METHYLAMINO-L-ALANINE (BMAA) FROM CYCAS THOUARSII A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI'I AT M NOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY MAY 2014 by Yoshiaki Miyasaka Thesis Committee: Thomas K. Hemscheidt, Chairperson Phillip Williams Joseph T. Jarrett ii We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Chemistry THESIS COMMITTEE _________________________ _________________________ _________________________ iii Acknowledgement I would like to thank the members of my thesis committee for their time, helpful comments and suggestions. First, I would like to show my greatest appreciation to my advisor, Professor Hemscheidt, for his continuous and generous support during my years in a graduate program. Additionally, I would like to express my gratitude to Professor Williams and Professor Jarrett for their support when needed. I have been also supported by all faculty members and graduate students at the chemistry department. I would like to show my deep gratitude to them wholeheartedly. In addition, I would like to thank my collaborators in the Bidigare laboratory. (Professor Bidigare, Stephanie Christensen and Daniel Elsey). My work could not have been accomplished without their continuous support. I would also like to thank the University of Hawaii for financial support in the form of teaching assistantship. Being a TA was great learning experience along with my research experience. Finally, I would like to express my gratitude to my family and friends for their moral support and warm encouragements. iv Abstract Part I: The development of a qualitative/quantitative analysis of β-Methylamino-L-alanine (BMAA), including a sample preparation protocol and SPE protocol is described. A common interfering metabolite was isolated as the FMOC-derivative. Its structure was determined by spectroscopic methods and confirmed by chemical synthesis. Part II: The qualitative analysis of the BMAA content in samples from cycad leaf and oyster are described. Three different extraction protocols have been developed to categorize the form of BMAA present in either cycad leaf or oyster muscle. The low molecular mass conjugate of β-Methylamino-L-alanine (BMAA) in Cycas thouarsii has been identified as a mixture of regioisomeric formamides. The structures were elucidated on the basis of spectroscopic data and confirmed by chemical synthesis. v Table of Contents Acknowledgements....................................................................................................... iii Abstract......................................................................................................................... iv List of Tables................................................................................................................. viii List of Figures............................................................................................................... ix List of Schemes............................................................................................................. xi List of Abbreviations..................................................................................................... xii Part I: Development of an Analytical Method for β-Methylamino-L-alanine, a Cyanobacterial Metabolite and Potential Environmental Toxin 1.1 Introduction............................................................................................................. 2 1.2 Approaches to BMAA analysis, pros/cons.............................................................. 5 1.2.1 Direct method........................................................................................... 5 1.2.2 Indirect method........................................................................................ 6 1.3 Detections for BMAA............................................................................................. 8 1.3.1 UV/FD detection...................................................................................... 8 1.3.2 MS/MS detection..................................................................................... 9 1.4 False positive detection of BMAA due to co-elution of similar compounds.......... 11 1.5 The isolation and structure elucidation of the contaminant peak............................ 12 1.6 Development of a DNFB based pre-column derivatizing method.......................... 16 1.6.1 MRM method development..................................................................... 16 1.6.2 Isomers of BMAA: Possible interferences............................................... 18 1.7 Qualitative Analysis of BMAA............................................................................... 20 1.8 Quantitative Analysis.............................................................................................. 24 vi 1.8.1 Internal standard....................................................................................... 24 1.8.2 Calibration Standards............................................................................... 28 1.8.3 Calibration Curves................................................................................... 28 1.8.4 Detection limit/Sensitivity....................................................................... 32 1.9 Matrix effect............................................................................................................ 34 1.10 SPE cartridge cleaning/concentration, Dowex-50 strong cation resin.................. 36 1.11 Sample and LC/MS sample preparation protocol................................................. 39 1.11.1 Large Scale Extraction........................................................................... 40 1.11 Summary............................................................................................................... 42 1.12 Experimental section............................................................................................. 43 1.13 Reference............................................................................................................... 52 vii Part II: Selective Extraction Protocol and Structure of Formamides of β-Methylamino- L-alanine (BMAA) from Cycas thouarsii 2.1 Introduction........................................................................................................... 56 2.2 Free BMAA vs. Protein associated BMAA in Cycad leaf.................................... 58 2.3 Isolation of the formamide of BMAA from Cycas thouarsii............................... 63 2.4 Elucidation of the formamide of BMAA.............................................................. 65 2.5 mono-DNB deivatives of the BMAA formamides............................................... 71 2.6 The absolute configuration................................................................................... 75 2.7 Several notes......................................................................................................... 77 2.8 Conclusion............................................................................................................ 78 2.9 Experimental section............................................................................................ 79 2.10 References........................................................................................................... 85 viii List of Tables Table Page 1.1 List of isobaric molecules of BMAA and observed fragmentations in order of decreasing ion intensity............................................................... 18 1.2 Summary of calibration standards............................................................ 28 1.3 Summary of three calibration curves, A, B and C.................................... 29 1.4 Summary of curve A with separate ranges................................................ 30 1.5 Summary of Matrix-adapted curves......................................................... 35 1.6 Recovery from SPE protocol.................................................................... 38 1.7 Recovery after entire sample preparation................................................. 40 2.1 500 MHz (1H) and 125 MHz (13C) NMR data for the two observable rotamers of 1a and for 1b in CD3OD........................................................ 70 ix List of Figures Table Page 1.1 Structure of BMAA.................................................................................... 3 1.2 Section of the UPLC/FD chromatogram of hydrolyzed Spirulina. (UPLC/FD Method A)................................................................................ 9 1.3 Mass spectrum of AQC-tagged co-eluting peak with fragmentation at 10 eV................................................................................................................ 12 1.4 Structure of the compound co-eluting with BMAA................................... 15 1.5 Derivatizing reaction of BMAA and DNFB.............................................. 16 1.6 Fragmentation of bis-DNB-BMAA at 10 eV.............................................. 17 1.7 Chromatogram of standard isomer mix (Top) and hydrolyzed Spirulina (Bottom) by LCMS/MS Method A............................................................ 21 1.8 LC Chromatogram