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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

mzoccali@unime.it

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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

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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

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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

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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

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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)

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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

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of

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Acknowledgments:

Shimadzu Corporation

Thank you for

your attention!

Palazzo dei Congressi, Riva del Garda, Italy May 13 - 18, 2018