3 2 Farmaceutiche ed Ambientali, 1 Francesca Mariosimone Ch University Dipartimento Characterization Oxygen of the Heterocyclic romaleont s.r.l. Compounds ( Compounds Rigano Campus Campus Polymethoxylated Zoccali di Scienze Chimiche, Biologiche, 3 , University Paola in Food Products in Food Bio 1 , Coumarins Adriana Adriana - Medico of Rome, Dugo of Messina, University 1,2,3 Arigò and and Luigi [email protected] Flavones) Flavones) , 1 Psoralens , Marina of Messina, Italy Italy Mondello Russo , and 2 Italy , 1,2,3

University of Messina, Italy ➢ ➢ ➢ ➢ Beneficial effect on humans: Isolation Isolation pure of Anti Antibacterial Antioxidant Anticancer compounds secondary - Coumarin inflammatory Focus on the Oxygenated Heterocyclic Compounds metabolites Furocoumarin or commonly psoralen metabolism ➢ ➢ effect Harmful on humans: Inhibitor of intestinal Inhibitor intestinal and liver of drug Phototoxic found Development of high sensitive ofsensitive Development high analytical analytical methods in all Polymethoxyflavone citrus plants

University of Messina, Italy Food Food industry Citrus essential Citrus essential oils Flavour and fragrance industry Cosmetic industry Citrus Citrus Risso bergamia

University of Messina, Italy The The Panel considered the toxicity studies and the studies on the metabolism of of available since the last opinion coumarin concluded concluded to maintain the TDI of in humans Opinion on bw allocated in the 2004 with CYP2A6 with CYP2A6 polymorphism that have become Coumarin of of 2004 in , as well as clinical studies, and Food 0.1 0.1 mg opinion. products coumarin /kg

University of Messina, Italy products association and compounds Furocoumarins may oxygen and denaturation furocoumarins highly undergo When and be react between found species irradiated the responsible reactive photoactivation are with Furocoumarins risk , form in a can that consumption of several class ground triplet skin form cycloadducts with can for of cancer cause , adducts the plant putting UV state photoactive state observed in of . cellular species . Food light, oxygen citrus with with them In products DNA, saturated damage this furocoumarins into to an induce form state, excited fatty reactive protein certain acids, and can

University of Messina, Italy Analytical Analytical techniques employed for the characterization of the Reversed phase (RP-LC) 7 2 1 9 Normal phase (NP 10 3 4 5 6 7 8 PDA/U Chromatography 1 EI APCI 9 11 MS RF - V 10 MS Micro Nano LC RP - NP TLC SFC 11 - LC) × - - heterocyclic compounds LC LC - LC - 6 LC LC Dugo, ( heterocyclic Comprehensive P . The elution order in both dimensions represents a a represents dimensions both in order The elution key information for the identification of unknown unknown of identificationthe for keyinformation Dugo, L . Mondello M components . Ramírez two Detection/identification . J . dimensional in the 2D plot 2D the in Chromatogr of Fluorescence APCI Fernández, PDA/UV lemon EI LRI - MS - MS oil separation . Sci A . 44 . Cotroneo, , oxygen ( 2006 of oxygen ) 561 ated G ) .

University of Messina, Italy 4 3 2 1 The Retention identification Both The the furocoumarin heterocyclic interpretation UV UV and spectrum time process or MS information structure polymethoxyflavones), of spectra easily MS automatic Identification strategies spectra can can allows be be and can included used identifying faster lead complementarily as to well in a more UV as the and the reliable chemical position MS to libraries spectral identification class of substituents data to (coumarin, make the in

University of Messina, Italy IUPAC NAME UV UV library CAS

University of Messina, Italy UV UV library: automatic identification identification. confirm the necessary to retention Both and UV time are

University of Messina, Italy   similarity (low identification reliability) identification (low similarity spectral the reduce can significantly effect matrixa or matrix the from Interferences isomers) identify to (impossibility provided is information weight molecular the only Usually, MS library

University of Messina, Italy mAU 200 195 196 195 201 202 200 221 222 222 The use of a MS library help the identification of species characterized by characterized species of theidentification help MS library a of The use 250 254 254 254 268 269 270

300 287 288 289

313 314 314 350 nm Byakangelicol Phellopterin Byakangelicin identical UV spectrum identical MS library

University of Messina, Italy   methods expensive One necessary powerful In the of case the MS for Linear retention approach (LRI) retention Linear index and of main structure instrumentation molecules easier purpose elucidation solution Remarks/Evaluations characterized of (high the to . resolution, improve present by the research identification tandem same molecular is MS the systems) reliability finding weight, would of of more less LC be

University of Messina, Italy *Van Den Dool and Kratz , J. Chrom. A, 11 (1963) 436 11 A, (1963) Chrom.J. , Kratz and Den Dool *Van No universal universal No LRI systemLRI LC Retention behaviour Retention Identification tools in chromatography in tools Identification Stable LRI LRI systemStable by Van Den Dool VanDenDool by More More recent approaches POOR IDENTIFICATION POWERIDENTIFICATION POOR and Kratz*and HIGHLY RELIABLE RELIABLE HIGHLY IDENTIFICATION GC EI - MS spectra MS - 471 GC Spectral information Spectral API UV spectra - MS and/or and/or MS LC

University of Messina, Italy as where • phase • selected compounds • temperature and dependent analytes • The In LRI In in liquid GC gas indipendent building are composition, retention range - chromatography MS are normally only chromatography with program, . correlated are of on data a an automatically from the employed thus high LRI might mobile three Use of LRI as additional filter increasing spectra to database other be LRI a terms phase as retention complementary reference excluded an chromatographic represents matching would interaction the extra linear number data criterion be standard from velocity) but a are more system analyte to of with the strongly the parameters of meaningful list conditions mixture, a mass identification in - stationary of LRI which dependent possible spectral value making to in the (column be phase the falling candidates retention considered library capability from LC retention - mobile - MS out dimension, the searching times of . mobile system from phase MS data of a : ,

University of Messina, Italy interlaboratory instrumentation In standardization chemistry The identification decades A order order to obtain reproducible results» «The elution conditions should be carefully standardized in 188 15, Anal. J. Bogusz Anexample consistent the influence last - and and 197. Toxicol ’ or 80 decades, Wu s mobile purposes literature - 90 levels of of , . 1991, 1991, . can s, the several . the phase sometimes LC lead . refers higher conditions experimental to composition, about a batch more combined LRI in LC is the - necessary to reliable - employment conditions, was batch with evaluated, reproducibility to and create UV stable of such 1996, 1291 19, 1996, Liq. J. Bogusz allowing retention a spectral usable as LRI Chromatogr et al. al. et in the database to LC system, index information stationary - conclude 1316. columns . R.T. during . also that phase and the for at a

University of Messina, Italy (low 1 heptanophenone alkyl alkan literature Three - nitroalkanes Reference standard mixture “many availibility - 2 - homologue ones aryl : from Bogusz Bogusz al. et Anal. J. Toxicol. 1988, 12, 325 ketones from ) LRI calculation (long C attempts” 3 series nitromethane to elution C from 23 have (low times) acetophenone been UV to absorption) 1 reported - nitrooctane - 329 to in Analytes 1 - nitroalkanes Alkyl aryl ketonesaryl Alkyl

University of Messina, Italy Comparison with the recent the recent Comparison with LC LC for - MS/MS the of oxygen heterocyclic compounds quali approach as a novel unified approach as tool - quantitative quantitative characterization - PDA method

University of Messina, Italy compounds in thereal samples by means of ▪ both UV libraryand LRI heterocyclic compounds through ▪ compounds ▪ ▪ method ▪ calibrationcurves. Quantitative determination Identification Calculationof the linearity) Validation of the Creation of an ( L o D , L Nexera o of oxygenated Q UV , , repeatability, accuracy, LC LRI - library - - i PDA PDA of each target Project Project schedule of targets ▪ for each STD compounds ▪ both MSand MS/MSlibraries and LRI heterocyclic compounds through ▪ compounds ▪ ▪ parameters for each target ▪ method ▪ compounds inthe real samples by means of (work Nexera X2 X2 UHPLC Nexera LCMS calibrationcurves Quantitative determination Realization of calibrationcurves Identification Calculationof the Optimizationof linearity) Validation of theLC Creation of ( - L flow o D , L ) MS o of oxygenated Q , , repeatability, accuracy, MRM and LRI - MS/MS MS/MS of each target compouds transition of targets - libraries - 8060

University of Messina, Italy CIDGAS: Drying Heat DLTemperature: Interface Temperature: Nebulizing Interface MS Range: Sampling Time PDA Oven Flow rate: Gradient Solvent Solvent Column parameters Block constant parameters Temperature: Gas Gas Flow: B: A: : : : : Gas Flow: Temperature: MRM modeMRM APCI positive 190 4.1667 2 Methanol Water/ Ascentis mL : - 0.48 sec 370 / min Methanol Hz 40 Express Express C18 (50 /THF 95:5 ° 270 15 L/ 300 450 3 L/ C 300 /THF 85:10:5 ° ° min kPa min C C ° C v/v ) × Analysis Analysis 4.6 4.6 mm, 2.7 v/v ) conditions m m)

University of Messina, Italy OHCs library library OHCs Identify Identify components by sample the Inject solution Inject sample the using UV and LRI filters LRI using UVand LC - PDA experimental PDA work Quantify sample Quantify the by Inject Inject the series series for LRI calculation reference homologue reference calibration instrumentation used in used instrumentation UHPLC - PDA (iseries) external external this study this - flow

University of Messina, Italy geranyloxypsoralen 26 methylscutellarein Bergapten Oxypeucedanin 1 Mix 10 20 15 . 0 5 . Coumarin 0.0 mAU Cnidilin of 315nm all ; STDs 15 1.0 ; ; 1 2 27 . 2 . ( Sinensetin Meranzin . hydrate 35 3 ; Gardenin ; 21 compounds 4 33 2.0 5 . PDA PDA . 6 Imperatorin ; Aurapten 7 8 10 ; 9 hydrate 9 . 16 A Citropten 11 . 3.0 ; chromatogram among Isobergapten 12 28 ; 13+14 ; 34 ; 3 . . 22 Isoimperatorin . Herniarin Bergamottin 15 coumarins ; . 4.0 16 Tangeretin 17 10 . ; Isopimpinellin 18 17 ; 4 5.0 . , . psoralens ; Byakangelicol Byakangelicin ; ; 35 23 19 29 . . 20 5 . of of Epoxyaurapten - geranyloxy Epoxybergamottin 6.0 ; and all 11 21 22 . polymethoxyflavones ; ; the target 23 Meranzin 5 18 7.0 . - 8 7 . 24+25 - - 26+27 Oxypeucedanin methoxypsoralen methoxycoumarin ; 28 24 29 30 . ; ; 8.0 Phellopterin 30 12 . . analytes Gardenin Isomeranzin 31 ) 32 9.0 ; ; ; . 19 6 33 25 . . Psoralen B . Nobiletin 34 5 ; 10.0 ; 35 - 31 O 13 - . demethylnobiletin . ; Cnidicin Heraclenin 7 ; . 11.0 Angelicin 20 . Tetra ; 32 ; . min - ; 14 O 8 8 - - ; . .

University of Messina, Italy Oxypeucedanin 8 - 8 Epoxybergamottin geranyloxypsoralen - methoxypsoralen Isoimperatorin Isopimpinellin Byakangelicol Byakangelicin Isobergapten Bergamottin Phellopterin Imperatorin Compound Heraclenin *10 Bergapten Angelicin Cnidicin Psoralen Cnidilin ppm hydrate and and ppm 50 Class FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC y = 1011.4 x y= 1011.4x + 285.14 y = 1956.7x y= 1956.7x + 2219.6 y= 1956.7x + 2219.6 y= 2974.7x + 1087.4 y= 2643.3x + 1026.7 LC y= 2309.6x y= 2546.4x y= 2195.9x y= 2623.8x y= 3115.6x y= 2394.5x y= 3970.9x y= 2902.0x y= 2207.3x y= 2628.0x y= 2820.2x y= 2903.5x y= 2824.8x Equation spiked spiked on - PDA validation 0.9988 0.9994 0.9998 0.9998 0.9999 0.9999 0.9999 0.9997 0.9997 0.9997 0.9997 0.9997 0.9995 0.9996 0.9996 0.9996 0.995 0.995 distilled lemon R 2 Linearity 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.1 0.1 0.1 0.1 0.1 0.1 0.1 (mg/L) 1 range - 100 ------100 100 100 100 100 100 100 parameters 100 100 100 100 100 100 100 100 100 100 CV% 13.2 17.4 7.75 25.2 21.7 18.2 18.8 19.7 16.9 19.7 15.8 26.1 10.6 14.7 8.9 5.3 9.3 5.3 Essential Oil. 10 Replicates (mg/L) Orange terpeneOrange 0.233 0.126 0.021 0.018 0.028 0.008 0.229 0.016 0.024 0.010 0.034 0.021 0.012 0.026 0.016 0.019 0.021 0.020 LOD e.o. (mg/L) 0.776 0.420 0.069 0.060 0.093 0.027 0.764 0.052 0.079 0.035 0.112 0.070 0.039 0.086 0.054 0.064 0.071 0.066 LOQ (mg/L) 0.022 0.023 0.039 0.027 0.019 0.015 0.109 0.055 0.720 0.055 0.110 0.011 0.026 0.043 0.063 0.102 0.096 0.060 Distilled lemon Distilled LOD e.o. (mg/L) 0.075 0.078 0.131 0.065 0.051 0.365 0.185 2.402 0.185 0.565 0.037 0.089 0.143 0.213 0.342 0.321 0.202 LOQ 0.09 10 RE RE spiked 109.0 101.1 102.2 104.6 125.4 111.6 131.6 108.8 117.4 110.4 106.1 106.5 106.2 101.6 104.5 104.2 103.4 99.4 ppm % * 50 ppm RE % RE spiked 111.6 113.6 106.2 105.5 113.2 115.5 111.2 115.3 110.3 111.5 107.8 108.4 108.6 105.5 106.5 105.7 105.6 University of109 Messina, Italy * this study this in used instrumentation X2 (Nexera UHPLC concentrate, quantification Particularly, concluding essential oils, that Somewere sometimes the interferences observed also in cold analysis of coelutions occur. good UVvery choice detectors not a for complexsamplesinwhich are a number of - MS/MS «LC - Need of more sensitive and selective instrumentation LCMS8060) LCMS8060) or - MS would be the most reliable the would reliable be MSmosttechnique» «an of a fortiori furocoumarins approach consumer based in products» complex Macmaster et al., J. Chromatogr. A, 2012, 1257, 34 1257, 2012, A, Chromatogr. J. al., et Macmaster on Frerot et al., J. Agric. Food Chem. 2004, 6879 Chem. 52, 2004, Food Agric. J. al., et Frerot HPLC . fragrance - UV products is not such applicable as - a pressed fragrance to the

University of Messina, Italy Inject solution Inject sample the Identify Identify components sample the MS,and LRIfilters MS/MS LC - MS/MS MS/MS experimental work Quantify sample Quantify the by Inject Inject the series series for LRI calculation reference homologue reference calibration using external external - flow

University of Messina, Italy MRM N. Compound Class Optimized MRM Q/q CE Q1 Q3 [M+H]+ transition Ratio (%) Pre Bias Pre Bias transitions 1 Coumarin C 146.80 Q 75.25 50 -7 -25 -17 q 73.15 -9 -16 -16 7 Auraptene C 299.00 Q 163.00 27 -15 -11 -10 q 107.20 -40 -11 -10 8 Herniarin C 177.00 Q 121.00 37 -21 -30 -28 q 77.10 -25 -30 -30 10 Citropten C 207.00 Q 192.05 61 -20 -30 -25 q 162.90 -15 -28 -29 14 5-geranyloxy-7-methoxy-coumarin C 329.00 Q 192.95 10 -20 -21 -21 q 149.10 -25 -22 -28 18 Tangeretin PMF 372.90 Q 342.95 8 -30 -25 -24 q 211.20 -34 -25 -24 19 Meranzin C 260.90 Q 188.95 81 -15 -26 -22 q 131.10 -29 -28 -16 20 Meranzin hydrate C 278.90 Q 189.15 95 -17 -29 -22 q 261.20 -7 -30 -30 22 Tetra-O-methylscutellarein PMF 343.00 Q 313.00 97 -30 -24 -22 The Q/q ratio q 282.00 -25 -23 -30 and/or the LRI 25 Epoxyaurapten C 315.20 Q 162.90 33 -16 -20 -13 value can be q 107.30 -25 -23 -30 26 Sinensetin PMF 373.00 Q 343.00 57 -30 -27 -26 employed as q 312.15 -21 -27 -25 additional 27 Nobiletin PMF 402.90 Q 372.95 -34 -30 -29 identification q 327.20 -31 -29 -27 parameters 28 5-O-demethylnobiletin PMF 388.90 Q 358.95 34 -30 -29 -28 q 341.15 -27 -27 -27 29 Gardenin A PMF 418.90 Q 389.00 36 -32 -28 -30 q 371.15 -28 -29 -30 32 Isomeranzin C 260.90 Q 189.25 95 -17 -30 -30 q 131.15 -30 -30 -29 33 Gardenin B PMF 358.90 Q 328.95 28 -29 -26 -26 q 311.20 -25 -26 -25 N. Compound Class Optimized MRM Q/q CE Q1 Q3 MRM [M+H]+ transition Ratio (%) Pre Bias Pre Bias 2 Byakangelicin FC 317.20 Q 233.05 93 -19 -12 -26 transitions q 231.05 -13 -16 -26 334.70 174.95 -31 -24 -28 3 Psoralen FC 186.90 Q 131.10 71 -21 -30 -30 q 77.20 -40 -10 -10 4 Isopimpinellin FC 246.90 Q 216.95 49 -25 -30 -30 Q 232.10 -18 -17 -19 5 Isoimperatorin FC 270.90 Q 203.00 47 -15 -16 -11 q 147.15 -31 -30 -30 6 Phellopterin FC 301.10 Q 233.15 34 -14 -21 -13 q 218.10 -30 -30 -29 9 8-methoxypsoralen FC 216.90 Q 202.00 67 -21 -29 -26 q 173.80 -25 -30 -30 11 Byakangelicol FC 316.90 Q 218.10 79 -29 -30 -30 The Q/q ratio and/or q 175.05 -25 -30 -30 12 Cnidilin FC 301.00 Q 232.90 24 -15 -30 -30 the LRI value can q 217.95 -28 -30 -27 be employed as 13 8-geranyloxypsoralen FC 338.90 Q 202.95 77 -25 -24 -26 q 95.10 -25 -27 -29 additional 15 Bergapten FC 217.20 Q 202.00 42 -19 -25 -25 identification q 174.10 -25 -14 -10 16 Bergamottin FC 338.90 Q 203.10 23 -14 -23 -24 parameters q 147.10 -35 -12 -12 17 Imperatorin FC 270.90 Q 203.10 50 -15 -29 -22 q 147.20 -31 -30 -30 21 Oxypeucedanin FC 286.90 Q 203.05 29 -18 -29 -24 q 59.10 -38 -13 -12 23 Epoxybergamottin FC 355.20 Q 203.10 23 -18 -26 -26 q 215.20 -19 -24 -25 24 Angelicin FC 186.90 Q 131.10 48 -25 -30 -30 Q 77.30 -35 -30 -30 30 Heraclenin FC 286.90 Q 202.90 30 -17 -28 -26 q 147.05 -33 -30 -30 31 Oxypeucedanine hydrate FC 304.90 Q 202.90 29 -20 -30 -29 q 147.15 -32 -30 -30 34 Isobergapten FC 217.00 Q 201.90 41 -21 -30 -29 q 174.20 -26 -26 -29 35 Cnidicin FC 355.00 Q 219.05 -16 -18 -24 q 172.95 -32 -14 -10 ➢ ➢ In many cases it was able to provide only In many toprovide only cases one candidate. it was able An MS/MScontaining all library compounds 35wasbuilt. the MS/MS MS/MS library Only 1 candidate 1 Only

University of Messina, Italy MS/MS MS/MS library identification important index The library In the information case can Linear totally of role . isomers, retention fail in play . the the an

University of Messina, Italy homologue series in both the instruments. the both in series homologue as used was mixture alkylarylketone The Need of the additional LRI min Average 1 all than LRI LRI= the 10 variability compounds 0 units . 01 peak filter min ( 0 width . was 1 min) minor : 0 for . 1 2 1 N. 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 Psoralen Byakangelicin Compound Cnidicin Isobergapten Gardenin Isomeranzin Oxypeucedanine Heraclenin Gardenin 5 Nobiletin Sinensetin Epoxyaurapten Angelicin Epoxybergamottin Tetra Oxypeucedanin Meranzin Meranzin Tangeretin Imperatorin Bergamottin Bergapten 5 8 Cnidilin Byakangelicol Citropten 8 Herniarin Auraptene Phellopterin Isoimperatorin Isopimpinellin - - - - O geranyloxy geranyloxy methoxypsoralen - demethylnobiletin - O - methylscutellarein B A hydrate - psoralen 7 - methoxy hydrate - coumarin LRI 1305 1174 1130 1102 1016 1096 1170 1031 1088 1084 1354 1363 1317 1131 1336 1108 1161 886 844 827 939 902 866 975 938 853 975 785 890 912 951 877 839 800 LC - PDA LRI 1301 1170 1128 1097 1015 1098 1161 1022 1085 1079 1356 1364 1314 1126 1336 1100 1156 941 898 861 973 935 853 972 780 882 909 948 871 834 798 882 840 82 LC 2 - MS D 4 2 4 6 5 3 2 5 1 3 2 0 9 9 3 5 8 3 5 2 3 1 3 5 3 6 5 2 0 8 5 4 4 5

University of Messina, Italy Any Any mismatching between Combination of LRI and MS/MS libraries isomeric compounds ( meranzin , isomeranzin ) occurs

University of Messina, Italy RE% = RE% LOQ LOD = & Oxypeucedanin 8 - 8 Epoxybergamottin geranyloxypsoralen - methoxypsoralen Isoimperatorin Isopimpinellin Byakangelicol Byakangelicin Isobergapten Bergamottin Phellopterin Imperatorin Compound Bergapten Angelicin Psoralen Cnidilin 0.5 0.5 ppm spiked on on spiked ppm hydrate 0. 0 5 Class FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC ppm spiked on on spiked ppm y = 49307077 x y= 49307077x + 127574 y = 10461879 x y= 10461879x y = 1339307 x y= 1339307x y = 695753x y= 695753x distilled lemon distilled y= 15084014x y= 1906971x y= 1279152x y= 8008825x y= 8555177x y= 8172949x y= 4878762x y = 9868507 x y= 9868507x y= 7453506x y= 2716527x y= 700559x y= 91092x Equation LC orange - - MS/MS MS/MS parameters validation - - 14379 255551 69973 Essential Oil. 10 10 Oil. Essential terpenes 0.9996 0.9998 0.9995 0.9997 0.9996 0.9995 0.9997 0.9995 0.9995 0.9994 0.9990 0.9985 0.9968 0.9992 0.9998 0.9994 R 2 Essential Linearity 0.001 0.001 0.005 0.005 0.001 0.005 0.001 0.001 0.005 0.001 0.001 0.005 0.001 0.001 0.005 (mg/L) 0.05 range ------5 Replicates 5 5 1 5 5 5 5 5 5 5 5 5 5 1 5 oil / distilled CV% 10.1 12.2 14.2 12.2 10.2 12.6 10.4 15.5 10.4 12.1 14.0 16.6 12.6 5.0 7.3 2.0 (mg/L) 0.0004 0.0006 0.0021 0.0007 0.0002 0.0010 0.0003 0.0003 0.0013 0.0002 0.0002 0.0014 0.0003 0.0001 0.0010 Orange terpeneOrange 0.013 LOD lemon e.o. (mg/L) 0.0008 0.0007 0.0034 0.0025 0.0008 0.0035 0.0009 0.0008 0.0045 0.0008 0.0008 0.0043 0.0011 0.0005 0.0033 0.043 LOQ Essential Oil. 10 Replicates 10 Oil. Essential On On data going processing… Distilled lemon e.o. lemon Distilled (mg/L) 0.0019 0.0013 0.0025 0.0014 0.0012 0.0011 0.0105 0.0012 0.0020 0.0015 0.0015 0.0011 0.0011 0.0003 0.0016 LOD - (mg/L) 0.0066 0.0042 0.0082 0.0048 0.0385 0.0036 0.0352 0.0042 0.0067 0.0050 0.0049 0.0038 0.0036 0.0011 0.005 LOQ - 0.5 ppm spiked RE RE 103.0 79.1 83.3 79.8 81.6 77.6 88.6 77.4 76.3 73.9 86.3 85.1 98.6 78.4 84.3 90.0

University of Messina, Italy % I.S.

Meranzin hydrate Herniarin

Oxypeucedanin Citropten hydrate Citropten Citropten Meranzin Meranzin profiles qualitative PDA MS/MS and between Comparison Meranzin Bergapten Heraclenin Isomeranzin Bergapten Bergapten Isomeranzin Sinensetin Byakangelicol Oxypeucedanin Oxypeucedanin

Nobiletin Nobiletin Nobiletin Tetra-O-methyl Tetra-O-methyl scutellarein scutellarein Imperatorin Tangeretin Tangeretin Tangeretin 5-O-demethylnobiletin 5-O-demethylnobiletin Epoxybergamottin Isoimperatorin Epoxybergamottin Gardenin A Gardenin B Cnidicin 8-geranyloxypsoralen Aurapten Aurapten Aurapten Bergamottin Bergamottin Bergamottin 5-geranyloxy-7- 5-geranyloxy-7- methoxycoumarin 5-geranyloxy-7- methoxycoumarin methoxycoumarin PDA (315 nm) MS/MS MS/MS (MRM) PDA (315 nm) MS/MS (MRM) PDA (315 nm) MS/MS MS/MS (MRM) Bergamot Bitter Bitter orange Lemon

University of Messina, Italy * Tangeretin Sinensetin 5 Gardenin Citropten 5 Isomeranzin Herniarin Meranzin Meranzin All Tot of 8 Phellopterin Oxypeucedanin Imperatorin Isoimperatorin Oxypeucedanin Isobergapten Aurapten Cnidilin Heraclenin Byakangelicol Bergapten Bergamottin Cnidicin Epoxybergamottin Tetra Nobiletin - - - content geranyloxypsoralen O geranyloxy - demethylnobiletin - O furocoumarins - methylscutellarein A hydrate expressed Compounds - 7 - methoxycoumarin hydrate Comparison between PDA and MS/MS quantitative profiles quantitative PDA MS/MS and between Comparison as ppm , average Class PMF PMF PMF PMF PMF FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC C C C C C C C of of 3 3767.6 2680.9 1278.7 126.9 1641.2 250.4 961.9 968.4 61.6 11737.6 9128 PDA replicates ± ± ± ± ± ± ± ± ± E.O. E.O. Lemon 10 2 13 2 7 12 2 7 8 3681.4 2406.2 1082.6 1024.8 1685.0 239.1 898.8 62.8 26.5 17.4 5.2 99.6 22.5 80.6 12.7 68.5 85.0 MS/MS < LOQ < LOQ 8.3 11480 8786 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.1 ± ± ± 0.3 0.2 0.5 1 8 240 5 4 1 15 122 4 2 13 38 37 29 1146.4 772.1 1229.1 1437.3 321.5 389.7 688.6 5984 1078 PDA E.O. Bitter E.O. Bitter ± ± ± ± ± ± ± 0.2 4 9 5 16 5 4 1266.4 1605.3 1464.4 138.6 orange 361.9 473.8 662.1 331.5 103.7 236.3 174.8 MS/MS < LOQ < LOQ < LOQ < LOQ < LOQ 6819 909 ± ± ± ± ± ± ± ± ± ± ± 0.2 19 21 33 5 6 66 2 1 52 39 2062.5 2393.4 16852.2 159.6 109.3 935.3 32.7 24.2 22430 19246 PDA 58 E.O. E.O. Bergamot ± ± ± ± ± ± ± 4 1 1 6 10 5 ± 8 1883.9 1766.4 120.9 956.7 13724.2 141.9 140.9 3.9 94.2 51.1 48.0 15.7 19.0 MS/MS < LOQ 18967 15612 359 ± ± ± ± ± ± ± ± 0.2 ± ± ± ± 12 1 3 36 2 1 1 56 1 2 51 ±

University of Messina, Italy 1.0 2.0 1.5 2.5 1.2 3.5 0.5 3.0 0.4 0.8 0 0 mV 1000000(x mV mV 1000 (x mV 0.5 0,5 1.0 1.0

Herniarin 000 1.5 1.5 0 ) 8-methoxypsoralen Byakangelicin ) Home 2.0 2.0 Oxyopeucedanin hydrate Meranzin 2.5 2.5 Citropten

Isomeranzin - made “Limoncello” made “Limoncello” (lemon alcoholic beverage) Heraclenin 3.0 3.0 Bergapten 3.5 3.5 LC - 4.0 4.0

Byakangelicol MS/MS chromatograms 4.5 4.5 Oxypeucedanin 5.0 5.0 5.5 Nobiletin 5.5 Tetra-O-methyl scutellarein 6.0 6.0

6.5 Imperatorin Tangeretin 6.5 Phellopterin 7.0 7.0 Isoimperatorin 7.5 Epoxybergamottin 7.5 8.0 8.0 8.5 Cnidicin 8.5 8-geranyloxypsoralen 9.0 9.0 Aurapten 9.5 9.5 Bergamottin 10.0 10.0 5-geranyloxy-7-methoxycoumarin 315 nm PDA min min

University of Messina, Italy 2.0 2.5 0.5 1.0 1.5 2.0 0.5 2.5 3.0 3.5 1.0 1.5 4.0 mV 100000)(x mV 0 mV 1000000(x mV 0 0.5 0.5 1.0 1.0 Herniarin 1.5 1.5 0 Byakangelicin ) Psoralen 2.0 2.0 Oxypeucedanin hydrate Citropten 2.5 Isopimpinellin 2.5 Bergapten 3.0 3.0 Bergamot alcoholic alcoholic Bergamot beverage 3.5 3.5 Sinensetin LC - 4.0 4.0 MS/MS MS/MS chromatograms 4.5 4.5 5.0 5.0 Nobiletin 5.5 Tetra-O-methyl 5.5 scutellarein 6.0 6.0 6.5 Tangeretin 6.5 5-O-demethylnobiletin 7.0 7.0 Isoimperatorin Epoxybergamottin 7.5 7.5 8.0 8.0 8.5 8-geranyloxypsoralen 8.5 9.0 Aurapten 9.0

9.5 Bergamottin 9.5 10.0

10.0 5-geranyloxy-7-methoxycoumarin 315 nm PDA min min

University of Messina, Italy All Tot of Psoralen 8 Phellopterin Oxypeucedanin Isopimpinellin Imperatorin Isoimperatorin Oxypeucedanin Isobergapten Aurapten Cnidilin Heraclenin Byakangelicol Byakangelicin Bergapten Bergamottin Cnidicin Epoxybergamottin Tetra Nobiletin Tangeretin Sinensetin 5 Gardenin Citropten 5 Isomeranzin Herniarin Meranzin Meranzin - - - geranyloxypsoralen O geranyloxy - demethylnobiletin - O furocoumarins - methylscutellarein A hydrate Compounds - 7 - methoxycoumarin hydrate Comparison between PDA and MS/MS quantitative profiles quantitative PDA MS/MS and between Comparison Class PMF PMF PMF PMF PMF FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC FC C C C C C C C 5.267 0.650 0.246 0.819 0.161 0.265 0.559 24.9 4.96 35.6 1.96 4.39 1.49 4.44 1.53 3.46 5 .4 PDA ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3.148 0.102 0.21 0.064 3.73 0.070 0.031 0.59 0.013 0.73 0.109 0.69 0.73 0.014 0.062 0.25 0.06 “Limoncello” 0.003 0.063 6.337 0.804 2.247 0.013 0.157 0.019 0.794 0.107 0.014 0.401 23.85 0.01 0.140 30.8 3 1.286 3 3.93 3.98 2.88 .69 .913 MS/MS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.0004 0.002 ± ± 0.0003 4.13 0.389 0.008 1.420 0.002 0.007 0.22 0.002 0.138 0.007 0.67 0.39 0.24 0.002 0.003 3.47 0.01 0.3 0.3 0.835 0.183 3.67 1.101 Bergamot Bergamot 16.0 13.8 1.07 9.05 PDA ± ± ± ± ± ± ± ± 0.325 0.99 0.05 0.018 0.01 0.022 0.5 0.12 alcoholic 0.001 0.001 0.010 0.057 1.263 0.051 0.066 0.185 0.011 beverage 0.11 0.94 16.3 14.1 9 3 0.97 .18 .50 MS/MS ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.0001 0.0002 0.001 0.106 1.04 0.89 0.001 0.001 0.64 0.25 0.003 0.006 0.001 0.01 0.017 0.001

University of Messina, Italy ➢ ➢ ➢ oxygen A detectors) fraction, was A and Advanced novel new characterization successfully heterocyclic LC identification even . and - MS/MS innovative at trace employed fraction of method approach pure level analytical Final Final remarks compounds in for ha (under food s the based been techniques samples identification the on validated from limit the . citrus Linear of were for identification of essential developed the Retention the determination oxygen oils for . of Index the heterocyclic typical isolation concept of UV the

University of Messina, Italy Shimadzu Acknowledgments: Corporation Palazzodei Congressi, Rivadel Garda,Italy 13May your your attention! Thank you Thankyou for - 18,2018

University of Messina, Italy