characterization of the oxygen heterocyclic compounds
TRANSCRIPT
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Characterization of the Oxygen Heterocyclic
Compounds (Coumarins, Psoralens, and
Polymethoxylated Flavones)
in Food Products
Mariosimone Zoccali1, Adriana Arigò1, Marina Russo2,
Francesca Rigano3, Paola Dugo1,2,3 and Luigi Mondello1,2,3
1Dipartimento di Scienze Chimiche, Biologiche,
Farmaceutiche ed Ambientali, University of Messina, Italy2University Campus Bio-Medico of Rome, Italy3Chromaleont s.r.l. University of Messina, Italy
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Focus on the Oxygenated Heterocyclic Compounds
Coumarin
Furocoumarin or
psoralen Polymethoxyflavone
Beneficial effect on humans:
➢Anticancer
➢Antioxidant
➢Antibacterial
➢Anti-inflammatory
Harmful effect on humans:
➢Phototoxic
➢Inhibitor of intestinal and liver drug
metabolism
Isolation of pure
compounds
Development of high sensitive
analytical methods
secondary metabolites commonly found in all citrus plants
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Citrus essential oils
Food industry Cosmetic industry
Flavour and fragrance industry
Citrus bergamia Risso
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Opinion on Coumarin in Food products
The Panel considered the toxicity studies and the studies on the metabolism
of coumarin in humans with CYP2A6 polymorphism that have become
available since the last opinion of 2004, as well as clinical studies, and
concluded to maintain the TDI of 0.1 mg coumarin/kg
bw allocated in the 2004 opinion.
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Furocoumarins in Food products
Furocoumarins are a class of photoactive
compounds found in several plant species
and may be responsible for the observed
association between consumption of citrus
products and the risk of skin cancer.
When irradiated with UV light, furocoumarins can
undergo photoactivation, putting them into an excited and
highly reactive triplet state. In this state, certain
furocoumarins can form adducts with DNA, induce protein
denaturation, form cycloadducts with saturated fatty acids,
and react with ground state oxygen to form reactive
oxygen species that can cause cellular damage
Un
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EI-MS
APCI-MS
PDA/UV
LRI
Analytical techniques employed for the characterization of the oxygenated
heterocyclic compounds
Chromatography Detection/identification
TLC
NP-LC
RP-LC
LC×LC
Micro-LC
Nano-LC
SFC
Comprehensive two dimensional separation of oxygen
heterocyclic components of lemon oil
(P. Dugo, M. Ramírez Fernández, A. Cotroneo, G.
Dugo, L. Mondello. J. Chromatogr. Sci. 44, (2006) 561)
Normal phase (NP-LC)
Rev
erse
d p
has
e (R
P-L
C)
16
APCI-
MS
PDA/U
V
RF
EI-MS
7
9
10 11
1
25
3
4
6
7
8
910
11 The elution order in both dimensions represents a
key information for the identification of unknown
in the 2D plot
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Identification strategies
1 The UV spectrum easily allows identifying the chemical class (coumarin,
furocoumarin or polymethoxyflavones), as well as the position of substituents in
the heterocyclic structure
2 The interpretation of MS spectra can lead to a more reliable identification
3 Both UV and MS spectra can be included in UV and MS libraries to make the
identification process automatic and faster
4 Retention time information can be used complementarily to spectral data
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IUPAC NAME CAS
UV library
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Both UV and
retention time are
necessary to
confirm the
identification.
UV library: automatic identification
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MS library
Usually, only the molecular weight information is provided (impossibility to identify
isomers)
Interferences from the matrix or a matrix effect can significantly reduce the spectral
similarity (low identification reliability)
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MS library
The use of a MS library help the identification of species characterized by
identical UV spectrum
Phellopterin
Byakangelicin
Byakangelicol
mAU
222
195
270
314
200
254
289
200 250 300 350nm
221
268
195
313
254
201
222
269
196
314
202
254
288
287
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Remarks/Evaluations
In the case of molecules characterized by the same molecular weight, more
powerful MS instrumentation (high resolution, tandem MS systems) would be
necessary for structure elucidation.
One of the main purpose of the present research is the finding of less
expensive and easier solution to improve identification reliability of LC
methods
Linear retention index (LRI) approach
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More recent approaches
Identification tools in chromatography
Retention behaviour Spectral information
No universal
LRI system
EI-MS spectra
LC GC LC
API-MS and/or
UV spectra
HIGHLY RELIABLE
IDENTIFICATION
Stable LRI system
by Van Den Dool
and Kratz*
GC
POOR IDENTIFICATION POWER
*Van Den Dool and Kratz , J. Chrom. A, 11 (1963) 436-471
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Use of LRI as additional filter
• In gas chromatography LRI represents a system in which the retention times of
analytes are correlated to a reference standard mixture, making retention data
dependent only on the three terms interaction analyte-stationary phase-mobile phase
and indipendent from other chromatographic conditions (column dimension,
temperature program, mobile phase linear velocity)
• LRI are normally employed as an extra criterion of mass spectral library searching:
compounds with a high spectra matching but with a LRI value falling out from a
selected range are automatically excluded from the list of possible candidates
• In liquid chromatography retention data are strongly dependent from the mobile
phase composition, thus increasing the number of parameters to be considered.
• The building of an LRI database would be more meaningful in the LC-MS system
where retention data might be complementary to the identification capability of MS,
as in GC-MS.
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LRI in LC
A consistent literature refers about the employment of retention index during the
decades ’80s-90s, sometimes combined with UV spectral information for
identification purposes.
The influence of several experimental conditions, such as the stationary phase
chemistry or mobile phase composition, was evaluated, allowing to conclude that a
standardization of the LC conditions is necessary to create a usable database.
An example
Bogusz and Wu,
J. Anal. Toxicol. 1991,
15, 188-197.
«The elution conditions should be carefully standardized in
order to obtain reproducible results»
Bogusz et al.
J. Liq. Chromatogr. R.T.
1996, 19, 1291-1316.
In the last decades, the higher batch-to-batch reproducibility in LC columns and
instrumentation can lead to a more reliable and stable LRI system, also at
interlaboratory levels.
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Bogusz et al. J. Anal. Toxicol. 1988, 12, 325-329
Reference standard mixture “many
attempts”
Three homologue series have been reported in
literature:
alkan-2-ones from C3 to C23 (low UV absorption)
alkyl aryl ketones from acetophenone to
heptanophenone (long elution times)
1-nitroalkanes from nitromethane to 1-nitrooctane
(low availibility)
1-nitroalkanes
Analytes
Alkyl aryl ketones
LRI calculation
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LC-MS/MS approach as a novel unified tool
for the quali-quantitative characterization
of oxygen heterocyclic compounds
Comparison with the recent LC-PDA method
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Nexera-i Nexera X2 UHPLC LCMS-8060
▪ Validation of the LC-PDA
method (LoD, LoQ, repeatability, accuracy,
linearity)
▪ Creation of an UV-library
▪ Calculation of the LRI of each target
compounds
▪ Identification of oxygenated
heterocyclic compounds through
both UV library and LRI
▪ Quantitative determination of targets
compounds in the real samples by means of
calibration curves.
▪ Validation of the LC-MS/MS
method (LoD, LoQ, repeatability, accuracy,
linearity)
▪ Optimization of MRM transition
parameters for each target compouds
▪ Creation of MS and MS/MS-libraries
▪ Calculation of the LRI of each target
compounds
▪ Identification of oxygenated
heterocyclic compounds through
both MS and MS/MS libraries and LRI
▪ Realization of calibration curves
for each STD compounds
▪ Quantitative determination of targets
compounds in the real samples by means of
calibration curves
Project schedule (work-flow)
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Analysis conditionsColumn: Ascentis Express C18 (50 × 4.6 mm, 2.7 mm)
Solvent A: Water/Methanol/THF 85:10:5 v/v)
Solvent B: Methanol/THF 95:5 v/v)
Gradient:
Flow rate: 2 mL/min
Oven Temperature: 40° C
PDA parameters
Time constant: 0.48 sec
Sampling: 4.1667 Hz
Range: 190-370
MS parameters
Interface: APCI positive
MRM mode
Nebulizing Gas Flow: 3 L/min
Interface Temperature: 450° C
DL Temperature: 300° C
Heat Block Temperature: 300° C
Drying Gas Flow: 15 L/min
CID GAS: 270 kPa
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Inject the sample solution
Inject the reference homologue
series for LRI calculation
Identify the sample components by
OHCs library using UV and LRI filters
Quantify the sample by external
calibration
LC-PDA experimental work-flow
UHPLC-PDA (iseries)
instrumentation used in
this study
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Mix of all STDs (35 compounds among coumarins, psoralens and polymethoxyflavones)
1. Coumarin; 2. Meranzin hydrate; 3. Herniarin; 4. Byakangelicin; 5. 8-methoxypsoralen; 6. Psoralen; 7. Angelicin; 8.
Oxypeucedanin hydrate; 9. Citropten; 10. Isopimpinellin; 11. Meranzin; 12. Isomeranzin; 13. Heraclenin; 14.
Bergapten; 15. Sinensetin; 16. Isobergapten; 17. Byakangelicol; 18. Oxypeucedanin; 19. Nobiletin; 20. Tetra-O-
methylscutellarein; 21. Imperatorin; 22. Tangeretin; 23. Epoxyaurapten; 24. Phellopterin; 25. 5-O-demethylnobiletin;
26. Cnidilin; 27. Gardenin A; 28. Isoimperatorin; 29. Epoxybergamottin; 30. Gardenin B; 31. Cnidicin; 32. 8-
geranyloxypsoralen; 33. Aurapten; 34. Bergamottin; 35. 5-geranyloxy-7-methoxycoumarin.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min
0
5
10
15
20
mAU
315nm
1
2
3
4
5
6
7
8 9
10
11
12
13+14
15
16
1718
19
20
21
2223
24+25
26+27
2829
30
31
32
3334
35
PDA chromatogram of all the target analytes
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LC-PDA validation parameters
Compound Class Equation R2
Linearity
range
(mg/L)
CV%
LOD
(mg/L)
LOQ
(mg/L)
LOD
(mg/L)
LOQ
(mg/L)RE %* RE %*
Orange terpene
e.o.
Distilled lemon
e.o.
10 ppm
spiked
50 ppm
spiked
Psoralen
Isopimpinellin
Heraclenin
Cnidilin
Byakangelicin
Oxypeucedanin hydrate
Bergapten
Byakangelicol
Isoimperatorin
8-methoxypsoralen
Imperatorin
Phellopterin
Epoxybergamottin
8-geranyloxypsoralen
Angelicin
Isobergapten
Cnidicin
Bergamottin
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
y = 2824.8 x
y = 2903.5 x
y = 2820.2 x
y = 2628.0 x
y = 2207.3 x
y = 2902.0 x
y = 3970.9 x
y = 2394.5 x
y = 3115.6 x
y = 2623.8 x
y = 2195.9 x
y = 2546.4 x
y = 2309.6 x
y = 1011.4 x + 285.14
y = 2643.3x + 1026.7
y = 2974.7x + 1087.4
y = 1956.7x + 2219.6
y = 1956.7x + 2219.6
0.9996
0.9996
0.9996
0.9995
0.9997
0.9997
0.9997
0.9997
0.9997
0.9999
0.9999
0.9999
0.9998
0.9998
0.9994
0.995
0.995
0.9988
0.1-100
0.05-100
0.05-100
0.05-100
0.05-100
0.05-100
0.1-100
0.1-100
0.05-100
0.05-100
0.05-100
1-100
0.05-100
0.1-100
0.1-100
0.05-100
0.1-100
0.1-100
14.7
10.6
5.3
26.1
15.8
19.7
16.9
19.7
18.8
18.2
21.7
9.3
5.3
25.2
7.75
17.4
8.9
13.2
0.020
0.021
0.019
0.016
0.026
0.012
0.021
0.034
0.010
0.024
0.016
0.229
0.008
0.028
0.018
0.021
0.126
0.233
0.066
0.071
0.064
0.054
0.086
0.039
0.070
0.112
0.035
0.079
0.052
0.764
0.027
0.093
0.060
0.069
0.420
0.776
0.060
0.096
0.102
0.063
0.043
0.026
0.011
0.110
0.055
0.720
0.055
0.109
0.015
0.019
0.027
0.039
0.023
0.022
0.202
0.321
0.342
0.213
0.143
0.089
0.037
0.565
0.185
2.402
0.185
0.365
0.051
0.065
0.09
0.131
0.078
0.075
103.4
104.2
104.5
101.6
106.2
106.5
106.1
110.4
117.4
108.8
131.6
111.6
125.4
104.6
99.4
102.2
101.1
109.0
105.6
105.7
106.5
105.5
108.6
108.4
107.8
109
111.5
110.3
115.3
111.2
115.5
113.2
105.5
106.2
113.6
111.6
*10 ppm and 50 ppm spiked on distilled lemon Essential Oil. 10 Replicates
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Need of more sensitive and selective instrumentation
UV detectors are not a good choice for very complex samples in which a number of
coelutions occur.
Some interferences were sometimes observed also in the analysis of cold-pressed
essential oils, concluding that
«LC-MS would be the most reliable technique»
Frerot et al., J. Agric. Food Chem. 2004, 52, 6879
Particularly, «an approach based on HPLC-UV is not applicable to the
quantification of furocoumarins in complex fragrance products such as a fragrance
concentrate, or a fortiori consumer products».
Macmaster et al., J. Chromatogr. A, 2012, 1257, 34
UHPLC-MS/MS
(Nexera X2-LCMS8060)
instrumentation used in
this study
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Inject the sample solution
Inject the reference homologue
series for LRI calculation
Identify the sample components using
MS, MS/MS and LRI filters
Quantify the sample by external
calibration
LC-MS/MS experimental work-flow
N. Compound Class Optimized[M+H]+
MRMtransition
Q/q Ratio (%)
CE Q1Pre Bias
Q3Pre Bias
1 Coumarin C 146.80 Q 75.25
q 73.15
50 -7
-9
-25
-16
-17
-16
7 Auraptene C 299.00 Q 163.00
q 107.20
27 -15
-40
-11
-11
-10
-10
8 Herniarin C 177.00 Q 121.00
q 77.10
37 -21
-25
-30
-30
-28
-30
10 Citropten C 207.00 Q 192.05
q 162.90
61 -20
-15
-30
-28
-25
-29
14 5-geranyloxy-7-methoxy-coumarin C 329.00 Q 192.95
q 149.10
10 -20
-25
-21
-22
-21
-28
18 Tangeretin PMF 372.90 Q 342.95
q 211.20
8 -30
-34
-25
-25
-24
-24
19 Meranzin C 260.90 Q 188.95
q 131.10
81 -15
-29
-26
-28
-22
-16
20 Meranzin hydrate C 278.90 Q 189.15
q 261.20
95 -17
-7
-29
-30
-22
-30
22 Tetra-O-methylscutellarein PMF 343.00 Q 313.00
q 282.00
97 -30
-25
-24
-23
-22
-30
25 Epoxyaurapten C 315.20 Q 162.90
q 107.30
33 -16
-25
-20
-23
-13
-30
26 Sinensetin PMF 373.00 Q 343.00
q 312.15
57 -30
-21
-27
-27
-26
-25
27 Nobiletin PMF 402.90 Q 372.95
q 327.20
-34
-31
-30
-29
-29
-27
28 5-O-demethylnobiletin PMF 388.90 Q 358.95
q 341.15
34 -30
-27
-29
-27
-28
-27
29 Gardenin A PMF 418.90 Q 389.00
q 371.15
36 -32
-28
-28
-29
-30
-30
32 Isomeranzin C 260.90 Q 189.25
q 131.15
95 -17
-30
-30
-30
-30
-29
33 Gardenin B PMF 358.90 Q 328.95
q 311.20
28 -29
-25
-26
-26
-26
-25
The Q/q ratio
and/or the LRI
value can be
employed as
additional
identification
parameters
MRM
transitions
MRM
transitions
N. Compound Class Optimized[M+H]+
MRMtransition
Q/q Ratio (%)
CE Q1Pre Bias
Q3Pre Bias
2 Byakangelicin FC 317.20
334.70
Q 233.05
q 231.05
174.95
93 -19
-13
-31
-12
-16
-24
-26
-26
-28
3 Psoralen FC 186.90 Q 131.10
q 77.20
71 -21
-40
-30
-10
-30
-10
4 Isopimpinellin FC 246.90 Q 216.95
Q 232.10
49 -25
-18
-30
-17
-30
-19
5 Isoimperatorin FC 270.90 Q 203.00
q 147.15
47 -15
-31
-16
-30
-11
-30
6 Phellopterin FC 301.10 Q 233.15
q 218.10
34 -14
-30
-21
-30
-13
-29
9 8-methoxypsoralen FC 216.90 Q 202.00
q 173.80
67 -21
-25
-29
-30
-26
-30
11 Byakangelicol FC 316.90 Q 218.10
q 175.05
79 -29
-25
-30
-30
-30
-30
12 Cnidilin FC 301.00 Q 232.90
q 217.95
24 -15
-28
-30
-30
-30
-2713 8-geranyloxypsoralen FC 338.90 Q 202.95
q 95.10
77 -25
-25
-24
-27
-26
-29
15 Bergapten FC 217.20 Q 202.00
q 174.10
42 -19
-25
-25
-14
-25
-10
16 Bergamottin FC 338.90 Q 203.10
q 147.10
23 -14
-35
-23
-12
-24
-12
17 Imperatorin FC 270.90 Q 203.10
q 147.20
50 -15
-31
-29
-30
-22
-30
21 Oxypeucedanin FC 286.90 Q 203.05
q 59.10
29 -18
-38
-29
-13
-24
-12
23 Epoxybergamottin FC 355.20 Q 203.10
q 215.20
23 -18
-19
-26
-24
-26
-25
24 Angelicin FC 186.90 Q 131.10
Q 77.30
48 -25
-35
-30
-30
-30
-30
30 Heraclenin FC 286.90 Q 202.90
q 147.05
30 -17
-33
-28
-30
-26
-30
31 Oxypeucedanine hydrate FC 304.90 Q 202.90
q 147.15
29 -20
-32
-30
-30
-29
-30
34 Isobergapten FC 217.00 Q 201.90
q 174.20
41 -21
-26
-30
-26
-29
-29
35 Cnidicin FC 355.00 Q 219.05
q 172.95
-16
-32
-18
-14
-24
-10
The Q/q ratio and/or
the LRI value can
be employed as
additional
identification
parameters
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MS/MS library
Only 1 candidate
➢ An MS/MS library containing all the 35 compounds was built.
➢ In many cases it was able to provide only one candidate.
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MS/MS library
In the case of isomers, the
library can totally fail.
The Linear retention
index information play an
important role in the
identification.
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Need of the additional LRI
filter
The alkylarylketone mixture was used as
homologue series in both the instruments.
N. Compound LRILC-PDA LRILC-MS D
1
2
3
4
5
6
7
8
9
10
11
1213
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Byakangelicin
Psoralen
Isopimpinellin
Isoimperatorin
Phellopterin
Auraptene
Herniarin
8-methoxypsoralen
Citropten
Byakangelicol
Cnidilin
8-geranyloxy psoralen5-geranyloxy-7-methoxy-coumarin
Bergapten
Bergamottin
Imperatorin
Tangeretin
Meranzin
Meranzin hydrate
Oxypeucedanin
Tetra-O-methylscutellarein
Epoxybergamottin
Angelicin
Epoxyaurapten
Sinensetin
Nobiletin
5-O-demethylnobiletin
Gardenin A
Heraclenin
Oxypeucedanine hydrate
Isomeranzin
Gardenin B
Isobergapten
Cnidicin
827
844
886
1161
1108
1336
800
839
877
951
1131
1317
1363
912
1354
1084
1088
890
785
975
1031
1170
853
1096
938
1016
1102
1130
975
866
902
1174
939
1305
822
840
882
1156
1100
1336
798
834
871
948
1126
1314
1364
909
1356
1079
1085
882
780
972
1022
1161
853
1098
935
1015
1097
1128
973
861
898
1170
941
1301
5
4
4
5
8
0
2
5
6
3
5
3
1
3
2
5
3
8
5
3
9
9
0
2
3
1
5
2
3
5
6
4
2
4
LRI variability was minor
than 10 units (0.1 min) for
all the compounds
1 LRI=0.01 min
Average peak width: 0.1
min
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Combination of LRI and MS/MS libraries
Any mismatching between isomeric compounds (meranzin, isomeranzin) occurs
Un
iver
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Compound Class Equation R2
Linearity
range
(mg/L)
CV%
LOD
(mg/L)
LOQ
(mg/L)
LOD
(mg/L)
LOQ
(mg/L)RE %
Orange terpene
e.o.Distilled lemon e.o.
0.5 ppm
spiked
Psoralen
Isopimpinellin
Cnidilin
Byakangelicin
Oxypeucedanin hydrate
Bergapten
Byakangelicol
Isoimperatorin
8-methoxypsoralen
Imperatorin
Phellopterin
Epoxybergamottin
8-geranyloxypsoralen
Angelicin
Isobergapten
Bergamottin
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
y = 4878762 x
y = 49307077 x + 127574
y = 8172949 x
y = 1339307 x - 69973
y = 10461879 x - 255551
y = 15084 014 x
y = 695753x - 14379
y = 8555177 x
y = 8008825 x
y = 700559 x
y = 1279152 x
y = 1906971 x
y = 91092 x
y = 2716527 x
y = 7453506 x
y = 9868507 x
0.9998
0.9992
0.9994
0.9968
0.9985
0.9990
0.9994
0.9995
0.9995
0.9997
0.9995
0.9996
0.9997
0.9995
0.9998
0.9996
0.005-5
0.001-1
0.001-5
0.005-5
0.001-5
0.001-5
0.005-5
0.001-5
0.001-5
0.005-5
0.001-5
0.005-5
0.05-5
0.005-1
0.001-5
0.001-5
2.0
7.3
12.6
16.6
14.0
5.0
12.1
10.4
15.5
10.4
12.6
10.2
12.2
14.2
12.2
10.1
0.0010
0.0001
0.0003
0.0014
0.0002
0.0002
0.0013
0.0003
0.0003
0.0010
0.0002
0.0007
0.013
0.0021
0.0006
0.0004
0.0033
0.0005
0.0011
0.0043
0.0008
0.0008
0.0045
0.0008
0.0009
0.0035
0.0008
0.0025
0.043
0.0034
0.0007
0.0008
0.0016
0.0003
0.0011
0.0011
0.0015
0.0015
0.0020
0.0012
0.0105
0.0011
0.0012
0.0014
-
0.0025
0.0013
0.0019
0.005
0.0011
0.0036
0.0038
0.0049
0.0050
0.0067
0.0042
0.0352
0.0036
0.0385
0.0048
-
0.0082
0.0042
0.0066
90.0
84.3
78.4
98.6
85.1
103.0
86.3
73.9
76.3
77.4
88.6
77.6
81.6
79.8
83.3
79.1
LC-MS/MS validation parameters
On going data processing…
LOD & LOQ = 0.05 ppm spiked on orange terpenes Essential oil/distilled lemon Essential Oil. 10 Replicates
RE% = 0.5 ppm spiked on distilled lemon Essential Oil. 10 Replicates
Un
iver
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Comparison between PDA and MS/MS qualitative profiles
Bergamot
Bitter orange
Lemon
I.S.
Her
nia
rin
Cit
ropte
n
Mer
anzi
n
Isom
eran
zin
Ber
gap
ten
Sin
ense
tin
Nobil
etin
Tet
ra-O
-met
hyl
scute
llar
ein
Tan
ger
etin
Aura
pte
nB
ergam
ott
in
5-g
eran
ylo
xy-7
-m
ethox
yco
um
arin
5-O
-dem
ethyln
ob
ilet
in
Cit
ropte
n
Byak
angel
icol
Ox
ypeu
cedan
in
Her
acle
nin
Ber
gam
ott
in
5-g
eran
ylo
xy-7
-m
ethox
yco
um
arin
Mer
anzi
n
Ber
gap
ten
Nobil
etin
Tan
ger
etin
Imper
atori
n
Isoim
per
atori
n
Aura
pte
n
Cn
idic
in8-g
eran
ylo
xypso
rale
n
Ox
ypeu
cedan
inhydra
te
Mer
anzi
nhydra
te
Iso
mer
anzi
n
Mer
anzi
n
Ber
gap
ten
Nobil
etin
Ber
gam
ott
in5-g
eran
ylo
xy-7
-m
ethox
yco
um
arin
Tan
ger
etin
5-O
-dem
ethyln
ob
ilet
in
Epox
yber
gam
ott
in
Gar
den
inB
Cit
ropte
n
Gar
den
inA
Aura
pte
n
Tet
ra-O
-met
hyl
scute
llar
ein
Ox
ypeu
cedan
in
PDA (315 nm)
PDA (315 nm)MS/MS (MRM)
MS/MS (MRM)
PDA (315 nm)MS/MS (MRM)
Epox
yber
gam
ott
in
Un
iver
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Comparison between PDA and MS/MS quantitative profiles
Compounds ClassE.O. Lemon E.O. Bitter orange E.O. Bergamot
PDA MS/MS PDA MS/MS PDA MS/MS
Meranzin hydrate C 321.5 ± 4 361.9 ± 19
Meranzin C 8.3 ± 1 1437.3 ± 4 1464.4 ± 39 19.0 ± 1
Herniarin C < LOQ 24.2 ± 1 15.7 ± 1
Isomeranzin C 1146.4 ± 16 1605.3 ± 52 48.0 ± 2
5-geranyloxy-7-methoxycoumarin C 1641.2 ± 8 1685.0 ± 29 < LOQ 935.3 ± 5 956.7 ± 36
Citropten C 968.4 ± 2 898.8 ± 13 < LOQ 2062.5 ± 10 1766.4 ± 51
Gardenin A C 174.8 ± 1
5-O-demethylnobiletin PMF 236.3 ± 2 140.9 ± 2
Sinensetin PMF 32.7 ± 4 51.1 ± 3
Tangeretin PMF 85.0 ± 2 772.1 ± 0.2 662.1 ± 33 109.3 ± 6 94.2 ± 1
Nobiletin PMF 17.4 ± 0.5 1229.1 ± 5 1266.4 ± 66 120.9 ± 12
Tetra-O-methylscutellarein PMF 138.6 ± 0.2 159.6 ± 1 141.9 ± 1
Epoxybergamottin FC 688.6 ± 5 473.8 ± 21 < LOQ
Cnidicin FC 61.6 ± 2 68.5 ± 4
Bergamottin FC 2680.9 ± 12 2406.2 ± 122 103.7 ± 6 16852.2 ±
58
13724.2 ±
359
Bergapten FC < LOQ 389.7 ± 9 331.5 ± 5 2393.4 ± 8 1883.9 ± 56
Byakangelicol FC 961.9 ± 7 1024.8 ± 37
Heraclenin FC 250.4 ± 2 239.1 ± 15
Cnidilin FC 12.7 ± 1
Aurapten FC 5.2 ± 0.1 < LOQ 3.9 ± 0.2
Isobergapten FC 26.5 ± 0.2
Oxypeucedanin hydrate FC 62.8 ± 0.3
Isoimperatorin FC 80.6 ± 4 < LOQ
Imperatorin FC 22.5 ± 5
Oxypeucedanin FC 3767.6 ± 13 3681.4 ± 240 < LOQ
Phellopterin FC 126.9 ± 10 99.6 ± 8
8-geranyloxypsoralen FC 1278.7 ± 7 1082.6 ± 38
Tot of furocoumarins 9128 8786 1078 909 19246 15612
All 11737.6 11480 5984 6819 22430 18967
*content expressed as ppm, average of 3 replicates
Un
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Home-made “Limoncello” (lemon alcoholic beverage)
LC-MS/MS chromatograms
Cit
rop
ten
Ber
gam
ott
in
Ox
yo
peu
ced
anin
hyd
rate
Ox
yp
euce
dan
in
5-g
eran
ylo
xy-7
-met
ho
xyco
um
arin
0,5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0min
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
mV (x 10000000)B
yak
ang
elic
in
Ber
gap
ten
Byak
ang
elic
ol
Iso
imp
erat
ori
n
Phel
lopte
rin
8-g
eran
ylo
xyp
sora
len
Her
acle
nin
Nobil
etin
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 min0
0.4
0.8
1.2
mV (x 1000000)
Her
nia
rin
Mer
anzi
nIs
om
eran
zin
8-m
ethox
ypso
rale
n
Tet
ra-O
-met
hyl
scute
llar
ein
Imper
atori
nT
anger
etin
Epox
yber
gam
ott
in
Cnid
icin
Aura
pte
n
PDA315 nm
Un
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Bergamot alcoholic beverage
LC-MS/MS chromatograms
Ber
gam
ott
in
5-g
eran
ylo
xy-7
-met
ho
xyco
um
arin
Her
nia
rin
Pso
rale
n
Sin
ense
tin
Isoim
per
atori
n
Nobil
etin
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 min0
0.5
1.0
1.5
2.0
2.5
mV (x 100000)
Byak
angel
icin
Ox
ypeu
cedan
in h
ydra
te
Isopim
pin
elli
n
Tet
ra-O
-met
hyl
scu
tell
arei
n
Tan
ger
etin
5-O
-dem
ethyln
ob
ilet
in
Epox
yber
gam
ott
in
Au
rap
ten
8-g
eran
ylo
xyp
sora
len
PDA315 nm
Cit
rop
ten
Ber
gap
ten
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0min0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
mV (x 10000000)
Un
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Comparison between PDA and MS/MS quantitative profiles
Compounds Class“Limoncello” Bergamot alcoholic beverage
PDA MS/MS PDA MS/MS
Meranzin hydrate C
Meranzin C
Herniarin C 0.014 ± 0.002 0.011 ± 0.001
Isomeranzin C
5-geranyloxy-7-methoxycoumarin C 3.46 ± 0.06 2.88 ± 0.24 0.183 ± 0.022 0.185 ± 0.017
Citropten C 5.4 ± 0.21 3.98 ± 0.39 1.07 ± 0.01 0.94 ± 0.106
Gardenin A C
5-O-demethylnobiletin PMF
Sinensetin PMF 0.835 ± 0.018 0.97 ± 0.01
Tangeretin PMF 0.063 ± 0.0003 0.066 ± 0.001
Nobiletin PMF 1.53 ± 0.25 0.051 ± 0.006
Tetra-O-methylscutellarein PMF 0.265 ± 0.062 0.11 ± 0.001
Epoxybergamottin FC 0.559 ±0.064 0.401 ± 0.003 1.101 ± 0.12 1.263 ± 0.003
Cnidicin FC 0.140 ± 0.01
Bergamottin FC 4.96 ± 0.102 3.93 ± 0.67 3.67 ± 0.325 3.50 ± 0.25
Bergapten FC 0.161 ± 0.014 0.107 ± 0.007 9.05 ± 0.5 9.18 ± 0.64
Byakangelicin FC 4.44 ± 0.73 3.913 ± 0.3
Byakangelicol FC 1.49 ± 0.69 1.286 ± 0.3
Heraclenin FC 0.819 ± 0.109 0.794 ± 0.138
Cnidilin FC 0.01 ± 0.002
Aurapten FC 0.019 ± 0.002 0.001 ± 0.0002
Isobergapten FC
Oxypeucedanin hydrate FC 4.39 ± 0.73 3.69 ± 0.22 0.057 ± 0.001
Isoimperatorin FC 0.246 ± 0.013 0.157 ± 0.007
Imperatorin FC 0.013 ± 0.002
Isopimpinellin FC 0.003 ± 0.0004 0.001 ± 0.0001
Oxypeucedanin FC 1.96 ± 0.59 2.247 ± 1.420
Phellopterin FC 0.650 ± 0.031 0.804 ± 0.008
8-geranyloxypsoralen FC 5.267 ± 0.070 6.337 ± 0.389
Psoralen FC 0.010 ± 0.001
Tot of furocoumarins 24.9 ± 3.148 23.85 ± 3.47 13.8 ± 0.05 14.1 ± 0.89
All 35.6 ± 3.73 30.8 ± 4.13 16.0 ± 0.99 16.3 ± 1.04
Un
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Final remarks
➢ Advanced and innovative analytical techniques were developed for the isolation
and characterization of pure compounds from citrus essential oils.
➢ A novel identification approach based on the Linear Retention Index concept
was successfully employed for the identification of the oxygen heterocyclic
fraction, even at trace level (under the limit of identification of typical UV
detectors).
➢ A new LC-MS/MS method has been validated for the determination of the
oxygen heterocyclic fraction in food samples.
Un
iver
sity
of
Mes
sin
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Italy
Acknowledgments:
Shimadzu Corporation
Thank you for
your attention!
Palazzo dei Congressi, Riva del Garda, Italy May 13 - 18, 2018