Szent István University COMPREHENSIVE SELENIUM SPECIATION OF A SELENIUM ACCUMULATOR PLANT, CARDAMINE VIOLIFOLIA Doctoral (Ph.D.) thesis of ESZTER BORBÁLA BOTH Budapest 2020 Doctoral School/Program Name: Doctoral School of Food Sciences Field: Food Science Head: Prof. Livia Simonné Sarkadi, DSc Professor SZIU, Faculty of Food Science Institute of Food Quality, Safety and Nutrition Department of Food Chemistry and Nutrition Supervisors: Mihály Dernovics, habil. PhD Senior researcher Centre for Agricultural Research Agricultural Institute, Martonvásár Department of Plant Physiology Zsuzsanna Jókainé Szatura, PhD Associate professor SZIU, Faculty of Food Science Institute of Food Quality, Safety and Nutrition Department of Applied Chemistry ……………………………………. …………………………….......... ………………………………….. Signature of Head of Doctoral School Signatures of Supervisors TABLE OF CONTENTS LIST OF ABBREVIATONS ..................................................................................... 5 1. INTRODUCTION .................................................................................................. 9 2. OBJECTIVES ....................................................................................................... 11 3. REVIEW OF LITERATURE ............................................................................. 12 3.1. SELENIUM IN PLANTS .................................................................................................................... 12 3.1.1. Relation of selenium and plants .......................................................................................... 12 3.1.2. Seleniferous regions worldwide ........................................................................................... 15 3.1.3. Biochemistry of selenium assimilation in plants ................................................................ 20 3.1.4. Selenium species identified in the Brassicaceae family ..................................................... 25 3.1.5. Selenium metabolism and the plant microbiome ............................................................... 29 3.1.6. Cultivation methods for selenium metabolism studies ...................................................... 32 3.2. ANALYTICAL APPROACHES IN SELENIUM SPECIATION ................................................................ 35 3.2.1. Direct methods in selenium speciation ............................................................................... 35 3.2.2. Orthogonal LC-MS techniques ........................................................................................... 39 3.2.3. Detection and basics of structural identification of selenocompounds by LC-ESI-MS and MS/MS ..................................................................................................................................... 43 3.3. QUANTIFICATION WITHOUT AUTHENTIC STANDARDS ................................................................. 48 3.3.1. Background ........................................................................................................................... 48 3.3.2. Making uniform analytes: general aspects ......................................................................... 49 3.3.2.1. Making uniform analytes for LC-ultraviolet and fluorescence detections through sample preparation and/or methodology ................................................................................. 49 3.3.2.2. Making uniform analytes through LC-ESI-QQQ methodology ............................... 50 3.3.3. Solutions with LC-ICP-MS; Part 1 ..................................................................................... 50 3.3.4. Possibilities in LC-ICP-MS Part 2: isotope dilution .......................................................... 52 4. EXPERIMENTAL ............................................................................................... 55 4.1. INVESTIGATION OF SELENOMETABOLITES IN CARDAMINE VIOLIFOLIA ...................................... 55 4.1.1. Reagents and samples .......................................................................................................... 55 4.1.2. Selenium distribution in C. violifolia .................................................................................. 56 4.1.2.1. Total selenium and sulphur determination ................................................................. 56 4.1.2.2. Sequential extraction procedure .................................................................................. 56 4.1.3. Identification of selenocompounds in C. violifolia ............................................................. 57 4.1.3.1. Extractions ..................................................................................................................... 57 4.1.3.2. LC-ICP-MS set-ups (SAX, SCX and IP-RP) .............................................................. 58 4.1.3.3. LC-ESI-QTOF-MS set-up ............................................................................................ 58 4.1.3.4. LC-Unispray-QTOF-MS set-up ................................................................................... 59 4.1.4. Synthesis of selenolanthionine ............................................................................................. 60 4.1.4.1. Chemical synthesis ........................................................................................................ 60 4.1.4.2. Chromatographic purification – analytical scale ....................................................... 60 4.1.4.3. Chromatographic purification – semi-preparative scale ........................................... 60 4.1.5. Quantification procedures ................................................................................................... 61 4.1.5.1. Quantification of selenolanthionine standard ............................................................. 61 4.1.5.2. Quantification of lanthionine and selenolanthionine with LC-ESI-QQQ-MS ......... 61 4.1.5.3. Quantification of selenolanthionine with isotope dilution analysis (IDA) ................ 61 4.2. COMPARISON OF SELENIUM METABOLISM IN C. VIOLIFOLIA AND C. PRATENSIS ........................ 63 4.2.1. Reagents and biological materials ....................................................................................... 63 4.2.2. Cultivation of plants ............................................................................................................. 63 4.2.3. Elemental analysis ................................................................................................................ 66 4.2.4. Selenium distribution and speciation analysis using X-ray microprobe analysis ........... 66 4.2.5. Physiological parameters: chlorophyll fluorescence, antioxidant capacity analysis and total phenolic content ..................................................................................................................... 67 4.2.6. Strong cation exchange (SCX) – ICP MS analysis............................................................. 67 4.2.7. Statistical analysis................................................................................................................. 68 5. RESULTS AND DISCUSSION .......................................................................... 69 5.1. SELENIUM METABOLITES IN CARDAMINE VIOLIFOLIA ................................................................. 69 5.1.1. Total selenium, sulphur contents and selenium distribution ............................................ 69 5.1.2. Chromatographic characterisations ................................................................................... 70 5.1.3. Identification of the major selenocompound by HPLC-ESI-QTOF-MS and -MS/MS .. 72 5.1.4. Analytical and semi-preparative chromatographic purification of synthesised selenolanthionine ............................................................................................................................ 75 5.1.5. Confirmation of selenolanthionine by chemical synthesis ................................................ 79 5.1.6. Identification of minor selenocompounds in C. violifolia .................................................. 81 5.1.6.1. Chromatographic characterisation ............................................................................. 81 5.1.6.2. Screening for selenium species in the LC-Unispray-QTOF-MS acquisitions .......... 82 5.1.6.3. Structural elucidation of novel selenocompounds: N-glycosylated selenolanthionines ...................................................................................................................... 84 5.1.7. Quantification of selenolanthionine in C. violifolia samples ............................................. 89 5.1.7.1. Quantification of lanthionine and selenolanthionine with HILIC-QQQ-MS .......... 89 5.1.7.2. Quantification of selenolanthionine with isotope dilution analysis (IDA) ................ 90 5.2. SELENIUM METABOLISM STUDIES OF C. VIOLIFOLIA IN COMPARISON WITH C. PRATENSIS ....... 91 5.2.1. Selenium tolerance and accumulation ................................................................................ 91 5.2.2. Chlorophyll fluorescence, antioxidant capacity and total phenolic content .................... 95 5.2.3. Selenium localisation and speciation based on X-ray microprobe analysis..................... 96 5.2.4. Investigation of the presence of selenocystine by SCX-ICP-MS in Cardamine samples .......................................................................................................................................................