Targeted and untargeted analysis of secondary fungal metabolites by liquid chromatography-mass spectrometry Svetlana V. Malysheva Promoter: Prof. Dr. Sarah De Saeger Co-promoters: Prof. Dr. Irina Yu. Goryacheva Dr. José Diana Di Mavungu 2013 THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHARMACEUTICAL SCIENCES PROEFSCHRIFT VOORGELEGD TOT HET BEKOMEN VAN DE GRAAD VAN DOCTOR IN DE FARMACEUTISCHE WETENSCHAPPEN Targeted and untargeted analysis of secondary fungal metabolites by liquid chromatography-mass spectrometry Svetlana V. Malysheva Promoter: Prof. Dr. Sarah De Saeger Co-promoters: Prof. Dr. Irina Yu. Goryacheva Dr. José Diana Di Mavungu 2013 THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHARMACEUTICAL SCIENCES PROEFSCHRIFT VOORGELEGD TOT HET BEKOMEN VAN DE GRAAD VAN DOCTOR IN DE FARMACEUTISCHE WETENSCHAPPEN PhD thesis Title: Targeted and untargeted analysis of secondary fungal metabolites by liquid chromatography – mass spectrometry Author: Svetlana V. Malysheva Year: 2013 Pages: 338 ISBN: 978-94-6197-128-9 Printer: University Press, Zelzate, Belgium Refer to this thesis as follows: Malysheva SV (2013). Targeted and untargeted analysis of secondary fungal metabolites by liquid chromatography – mass spectrometry. Thesis submitted in fulfilment of the requirements of the degree of Doctor (Ph.D.) in Pharmaceutical Sciences. Faculty of Pharmaceutical Sciences, Ghent University Promoter: Prof. Sarah De Saeger (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Co-promoters: Prof. Irina Yu. Goryacheva (Chemistry Institute, Saratov State University, Russia) Dr. José Diana Di Mavungu (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Members of the reading committee: Prof. Ann Van Schepdael (Faculty of Pharmaceutical Sciences, KU Leuven, Belgium) Prof. Bart De Spiegeleer (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Prof. Patrice Chiap (Institute of Pathology, Academic Hospital of Liège, Belgium) Dr. Christof Van Poucke (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Prof. Sarah De Saeger (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Members of the examination committee: Dr. Els Daeseleire (ILVO, Melle, Belgium) Prof. Irina Yu. Goryacheva (Chemistry Institute, Saratov State University, Russia) Prof. Serge Van Calenbergh (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Prof. Em. Carlos Van Peteghem (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Dean: Prof. Stefaan De Smedt (Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium) Rector: Prof. Paul Van Cauwenberghe (Ghent University, Ghent, Belgium) The author and promoters give the permission to make this thesis available for consultation and allow the copying of any part of the manuscript for personal use. Any other use is subjected to the restrictions of author’s rights, in particular in relation to the obligation of explicit mention of the source when any results are taken from the thesis. Ghent, 10 June 2013 Author, Promoters, M.Chem. S. V. Malysheva Prof. Dr. S. De Saeger Prof. Dr. I. Yu. Goryacheva Dr. J. Diana Di Mavungu ACKNOWLEDGEMENTS Acknowledgements I would like to express appreciation and respect to my promoters, Prof. Dr. Sarah De Saeger, Prof. Dr. Irina Yu. Goryacheva and Dr. José Diana Di Mavungu, for the professional supervision and fruitful suggestions. I wish to thank my (ex-)colleagues for the good working atmosphere. Financial support from Research Foundation-Flanders (FWO-Vlaanderen) (research project G.0034.07) and Bijzonder Onderzoeksfonds (BOF) (Ghent University; grant code 01DI2611) are greatly acknowledged. Svetlana V. Malysheva 11 TABLE OF CONTENTS Table of contents ACKNOWLEDGEMENTS 9 TABLE OF CONTENTS 13 GLOSSARY 21 AIM OF THE STUDY 27 CHAPTER 1. INTRODUCTION TO SECONDARY FUNGAL METABOLITES 31 1.1. General information 33 1.2. Classification of secondary fungal metabolites 34 1.2.1. Classification challenges 34 1.2.2. Classification based on biosynthetic origin 34 1.2.3. Classification based on structure 37 1.2.3.1. Terpenoids 37 1.2.3.2. Polyketides 39 1.2.3.3. Non-ribosomal peptides 40 1.2.3.4. Alkaloids 41 1.2.4. Classification based on bioactivity 43 1.2.4.1. Anti-microbial activity 43 1.2.4.2. Mycotoxins 44 1.3. Occurrence 48 1.3.1. Feed 48 1.3.2. Food 50 1.3.3. Indoor environment 53 1.3.4. Biological material 54 1.4. References 56 CHAPTER 2. LIQUID CHROMATOGRAPHY - MASS SPECTROMETRY 71 2.1. General information 73 2.2. Targeted LC-MS analysis: strategy and methods 78 2.3. Untargeted LC-MS analysis: strategy and methods 82 2.4. References 87 CHAPTER 3. UNTARGETED SCREENING OF SECONDARY FUNGAL METABOLITES IN CRUDE EXTRACTS AND SAMPLES FROM MOULDY INDOOR ENVIRONMENTS BY TIME-OF-FLIGHT MASS SPECTROMETRY 97 3.1. Introduction 99 3.2. Experimental 100 3.2.1. Chemicals and material 100 3.2.2. Samples from mouldy buildings 101 3.2.3. Inoculation 101 3.2.4. Sample preparation 102 3.2.5. LC-MS conditions 102 15 Table of contents 3.2.6. Data treatment 103 3.3. Results and discussion 104 3.3.1. Mobile phase selection and optimization of MS parameters 104 3.3.2. Evaluation of experimental mass error in QTOF-MS analysis 107 3.3.3. Screening of fungal cultures 108 3.3.4. Screening of samples from mouldy environments 113 3.4. Conclusions 114 3.5. Acknowledgements 116 3.6. References 116 CHAPTER 4. METABOLIC PROFILING IN ASPERGILLUS FLAVUS: IDENTIFICATION AND FRAGMENTATION STUDY OF THE CLUSTER 27 POLYKETIDE SYNTHASE METABOLITES BY HIGH RESOLUTION AND MULTIPLE STAGE MASS SPECTROMETRY 121 4.1. Introduction 123 4.2. Experimental 124 4.2.1. Chemicals and material 124 4.2.2. Strains and growth conditions 125 4.2.3. LC-Orbitrap MS conditions 126 4.2.4. LC-ion trap MS conditions 127 4.2.5. Sample preparation 127 4.2.6. Statistical data treatment 128 4.3. Results and discussion 128 4.3.1. Development and evaluation of the LC-Orbitrap-MS method 128 4.3.1.1. Optimization of chromatographic conditions 130 4.3.1.2. Evaluation of experimental mass error in Orbitrap-MS analysis 131 4.3.1.3. Optimization of extraction procedure 132 4.3.2. Metabolic profiling and preliminary metabolite assignment 135 4.3.3. Fragmentation pathway and metabolite identification 138 4.3.4. Production of asparasone A derivatives in selected fungal cultures 147 4.4. Conclusions 149 4.5. Acknowledgements 149 4.6. References 150 CHAPTER 5. IONIZATION STRATEGY FOR PATULIN DETERMINATION BY LC-MS/MS: USEFULNESS IN MULTI-MYCOTOXIN ANALYSIS 155 5.1. Introduction 157 5.2. Experimental 158 5.2.1. Chemicals and material 158 5.2.2. MS conditions 159 16 Table of contents 5.2.2.1. Ionization efficiency of patulin and multi-mycotoxin analysis 159 5.2.2.2. Fragmentation behaviour 160 5.2.2.3. Accurate mass measurement 162 5.2.3. LC conditions 162 5.2.4. Clean-up of transdermal samples 163 5.2.5. Optimization of MS parameters by experimental design 163 5.2.6. Evaluation of the multi-analyte LC-ESI+-MS/MS method 165 5.2.7. Linear regression for the evaluation of analyte stability 165 5.3. Results and discussion 166 5.3.1. Optimization of patulin MS signal 166 5.3.1.1. Optimization of MS parameters by experimental design 168 5.3.1.1.1. Screening experiment 168 5.3.1.1.2. Response surface modelling 170 5.3.2. Confirmation of the methanol adduct of patulin (m/z 187) 172 5.3.3. Applicability in multi-mycotoxin LC-MS/MS analysis 177 5.3.4. Mycotoxin stability and injection solvent selection 178 5.3.5. Clean-up procedure and evaluation of the method 178 5.4. Conclusions 182 5.5. Acknowledgements 183 5.6. References 183 CHAPTER 6. MASS SPECTROMETRIC APPROACHES FOR TARGETED AND UNTARGETED ANALYSIS OF ERGOT ALKALOIDS 187 6.1. Background 189 6.2. LC-MS/MS determination of six major ergot alkaloids and their corresponding epimers in cereals and cereal products 192 6.2.1. Introduction 192 6.2.2. Experimental 193 6.2.2.1. Standards 193 6.2.2.2. Chemicals and material 194 6.2.2.3. Samples 195 6.2.2.4. Sample preparation 196 6.2.2.4.1. Cereals and cereal products 196 6.2.2.4.2. Corn and grass silages 196 6.2.2.5. Evaluation of extraction and clean-up recovery 196 6.2.2.6. Optimization of method parameters using experimental design 197 6.2.2.7. LC-MS/MS analysis 200 6.2.2.8. Method validation 201 6.2.2.9. Method robustness 203 6.2.3. Results and discussion 205 17 Table of contents 6.2.3.1. Optimization of the analytical method 205 6.2.3.1.1. MS/MS detection 205 6.2.3.1.1.1. Preliminary experiments 205 6.2.3.1.1.2. Optimization of MS parameters applying experimental design 205 6.2.3.1.2. LC-MS/MS analysis 213 6.2.3.1.3. Extraction and clean-up 215 6.2.3.2. Method validation 220 6.2.3.3. Robustness study 222 6.2.4. Application of the developed LC-MS/MS method to a European survey of cereals and cereal products 223 6.2.5. Conclusions 228 6.2.6. Acknowledgements 228 6.3. A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals 229 6.3.1. Introduction 229 6.3.2. Experimental 230 6.3.2.1. Standards 230 6.3.2.2. Chemicals and material 231 6.3.2.3. Spiking 231 6.3.2.4. Sample preparation 232 6.3.2.4.1. Liquid – liquid extraction (LLE) 232 6.3.2.4.2. SPE using PSA 232 6.3.2.4.3. SPE using strong cation exchange (SCX) columns 232 6.3.2.4.4.