Corso di dottorato di ricerca in Scienze e Biotecnologie Agrarie in convenzione con Fondazione Edmund Mach Ciclo XXX Titolo della tesi "Tracciabilità dell'origine del latte alpino mediante lo studio del profilo alcaloidico naturale" "Origin traceability of alpine milk using natural alkaloid profiles" Dottorando Supervisore Tiziana Nardin Edi Piasentier, Professor Co-supervisore Roberto Larcher, Dr. Anno 2018 Responsabile del procedimento: dott. Edi Piasentier – [email protected] Compilatore del procedimento: Tiziana Nardin – [email protected] via Palladio 8, 33100 Udine (UD), Italia - t +39 0432 556306 - +39 0432 556214 - www-uniud.it CF80014550307 P.IVA 01071600306 ABI 02008 CAB 12310 CIN R c/c 000040469443 2/2 Contents Tables ................................................................................................................................................... 5 Figures .................................................................................................................................................. 5 Abbreviations ....................................................................................................................................... 6 Acknowledgements .............................................................................................................................. 8 ABSTRACT ......................................................................................................................................... 9 1. Introduction .................................................................................................................................... 11 1.1. Food authenticity and fraud ..................................................................................................... 11 1.1.1. Milk characterisation and traceability .............................................................................. 12 1.1.2. Alpine milk ....................................................................................................................... 13 1.1.3. Alkaloids in alpine herbs .................................................................................................. 14 1.1.4. Alks in milk ...................................................................................................................... 15 1.2. Alks ......................................................................................................................................... 17 1.2.1. Biosynthesis of alkaloids .................................................................................................. 17 1.2.2. Classification .................................................................................................................... 18 1.2.3. Indole alks ......................................................................................................................... 20 1.2.4. Pyrrole, pyrrolidine, pyridine and piperidine alks ............................................................ 22 1.2.5. Pyz alks ............................................................................................................................. 24 1.2.6. Quinoline, isoquinoline, isoquinolone and benzylisoquinoline alks ................................ 25 1.2.7. Quinolizidine alks ............................................................................................................. 27 1.2.8. Steroidal and glycosteroidal alks ...................................................................................... 29 1.2.9. Terpenoid alks .................................................................................................................. 30 1.2.10. Tropane alks .................................................................................................................... 31 1.3. Liquid chromatography and mass spectrometry ..................................................................... 34 1.3.1. Average mass, nominal mass and monoisotopic mass ..................................................... 35 1.3.2. Electrospray ionisation ..................................................................................................... 36 1.3.3. Ion suppression and data quality ....................................................................................... 37 1.3.4. Quadrupole analyser ......................................................................................................... 38 1.4. High Resolution Mass Spectrometry ....................................................................................... 39 1.4.1. Orbitrap ............................................................................................................................. 41 3 1.4.2. Hybrid instrument: Q-Orbitrap ......................................................................................... 42 1.4.3. Resolving power and resolution ....................................................................................... 44 1.5. References ............................................................................................................................... 46 2. Objectives....................................................................................................................................... 61 3. Experimental section and results .................................................................................................... 62 3.1. Method development ............................................................................................................... 62 3.1.1. Targeted and untargeted profiling of alkaloids in herbal extracts using online solid-phase extraction and high-resolution mass spectrometry (Q-Orbitrap). ............................................... 62 3.2. Alkaloid profiles of herbal and alpine plants .......................................................................... 98 3.2.1. Alkaloid profiling of herbal drugs using high resolution mass spectrometry .................. 99 3.2.2. Targeted and untargeted profiling of alkaloids in Italian alpine herbs using high resolution mass spectrometry and their use for distinguishing the herbage grazed by dairy cows .................................................................................................................................................. 139 3.3. Alkaloid profiles of alpine milk samples .............................................................................. 175 3.3.1 Alkaloid profiles for dairy product traceability using high resolution mass spectrometry .................................................................................................................................................. 176 4. Conclusions to the thesis .......................................................................................................... 208 5. Other scientific contributions ....................................................................................................... 209 4 Tables 1.1. Alkaloid classification, biosynthetic origin and chemical structure ………………………...…20 Figures 1.1. Biosynthesis of isoprenoid alkaloids (Huang et al., 2016)…………………………………….21 1.2. Biosynthesis of Camalexin a non-isoprenoid alkaloids (adapted from Moldrup et al. 2013a)...22 1.3. Pyridine alkaloid biosynthesis in Nicotiana tabacum (Hӓkkinen et al., 2007)………………...23 1.4. Common necine bases of pyrrolizidine alkaloids (Fu et al., 2002)………………………….24 1.5. HSS catalyzes the first specific step of the biosynthesis of the necine base moiety common to all pyrrolizidine alkaloids. Exemplified with senecionine N-oxide (adapted from Reimann et al., 2004)………………………………………………………………………………………………..25 1.6. Biosynthesis of quinoline alkaloids (Diaz et al., 2015)………………………………………..26 1.7. Biosynthesis of isoquinoline alkaloids (Diaz et al., 2015)……………………………………..27 1.8. Biosynthetic pathway of quinolizidine alkaloids (Bunsupa et al., 2012)………………..……..28 1.9. Biosynthesis of steroidal alkaloids from cholesterol (Ohyama et al., 2013)…………………..30 1.10. Proposed biosynthetic pathway for the production of Daphniphyllum alkaloids (Cherney and Baran, 2011)……………………………………………………………………………...…………31 1.11. The first steps of tropane biosynthesis leading to the formation of the N-methyl-Δ1- pyrrolidinium cation (Jirschitzka et al., 2013)……………………………………………………....32 1.12. The mid- and late-biosynthetic reactions in tropane alkaloid production (Jirschitzka et al., 2013).………………………………………………………………………………………….…….33 1.13. Representation of the three steps in electrospray ionization (De Hoffmann and Stroobant, 2007).……………………………………………………………………………………………..…36 1.14. A cut-away mod-el of the orbitrap mass analyzer (Scigelova and Makarov, 2006)……………………………………………………………………………………..……….…41 1.15. Hybrid Q-Orbitrap instrument (adapted from www.planetorbitrap.com)...................................................................................................................42 5 Abbreviations ACD: acridone ACN: acetonitrile AGC: automatic gain control AIF: all ion fragmentation ALK: alkaloid BIQ: Benzylisoquinoline BZP: benzophenantridine CID: collision-induced dissociation dd-MS/MS: data-dependent MS/MS EIC: extracted ion chromatogram ESI: electrospray ionization FA: formic acid FID: flame ionization detector FSA: food standard agency GC: gas chromatography HCD: higher-energy collisional dissociation HQOMS: hybrid quadrupole orbitrap mass spectrometer HRMS: high resolution mass spectrometry IND: indole IQL: isoquinolone IQN: isoquinoline IRMS: isotope ratio mass spectrometry IT: