Un

iver

sity

of

Mes

sin

a, It

aly

Francesco Cacciola1, Luigi Mondello1,2,3

1University of Messina, Italy2Chromaleont s.r.l., c/o University of Messina, Italy

3University Campus Bio-Medico of Rome, Italy

Lipidomic analysis by using one- and

multidimensional chromatography coupled to mass

spectrometry

Un

iver

sity

of

Mes

sin

a, It

aly

Lipidomics

Lipidomics is a branch of metabolomics and aims to study all the lipids within a

living system or in complex biological samples

Lipid profiles can be related to very common illnesses such as cancer, diabetes,

and cardiovascular disease

BIOLOGICAL SAMPLES

ADVANCED ANALYTICAL TOOLS

HUMAN HEALTH STATUS

LC(xLC)-MS

LC-(x)-GC)-MS

GC(xGC)-MS

DATABASE

Informative featuresDisease

markers

Un

iver

sity

of

Mes

sin

a, It

aly

Structural components of cellular membranes (phosphoglycerides, sphingolipids

and steroids)

Cell signaling (e.g. phospholipase C and phospholipase A2 in modulating

immunological responses)

Endocrine actions (e.g. steroid hormones)

Biological function of lipids

Energy storage (triacylglycerols in adipocytes)

Un

iver

sity

of

Mes

sin

a, It

aly

More widespread approaches:

Shotgun

LC/GC-MS

Lipidomics approaches

Advantages:

- Fast

Disadvantages:

- Ion suppression

- Need of MS/MS and/or HRMS

Advantages:

- Pre-separation of lipidic species and/or

classes, which support MS identification

- Reduced ion-suppression effect

Disadvantages:

- Need of chromatographic optimization

0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0 16,0 min

0,00

0,25

0,50

0,75

1,00

1,25

1,50

(x100,000,000)

1:TIC(+)

400 500 600 700 800 900 1000 1100 1200 m/z

0

2500000

5000000

7500000

10000000

Inten.

857

601

575

368523 831 941

Un

iver

sity

of

Mes

sin

a, It

aly

Lipidomics approaches

FOLCH EXTRACTION

(CHCl3 / MeOH 2:1)

LC-MS analysis

BIOLOGICAL SAMPLES

COLD SAPONIFICATION

(KOH/EtOH 2N)

TRANS-ESTERIFICATION

(MeOH/TMSH)

GC(xGC)-FID/MS analysis

DERIVATIZATION

(BSTFA (1% TMCS) + Pyridin)

GC(xGC)-FID/MS analysis

Tocopherols

Sterols, dehydration

and oxydation products

Squalene

C1

2:0

Monoalkylglycerols

C3

0:0

Fatty

alcohols

C3

5:0

Hydrocarbons

C1

3:0

C1

8:0

C1

6:0

Fatty acids

GCxGC plasma sample: unsaponifiable lipid fraction

Un

iver

sity

of

Mes

sin

a, It

aly

LC-MS-Based Lipidomics

• (Q)TOF

• QqQ

• IT-TOF

• Q

BIOLOGICAL

SAMPLES

EXTRACTION

LC-MS

ANALYSIS

Separation of Lipid

Species

• Lipid identification

• Quantification

• RPLC

• NARPLC

• Silver ion-LC

Ionization Detection

• ESI

• APCI

• APPI

Separation of Lipid

Classes

• NPLC

• HILIC

T. Cajka, O. Fiehn, Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry,

Trends in Analytical Chemistry 61 (2014) 192–206.

DATA

PROCESSING

APCI

Un

iver

sity

of

Mes

sin

a, It

aly

1) Development of a fast liquid chromatography method to quickly and reliable

characterize the lipidic fraction of biological samples

2) Development of LC×LC-qMS for a complex food sample

Case studies

LC-MS

APCI

ESI

Un

iver

sity

of

Mes

sin

a, It

aly

The most commonly used API techniques for lipid characterization are:

Atmospheric pressure chemical ionization (APCI)

Electrospray ionization (ESI)

LC-MS analysis

Polar lipids Nonpolar lipids

[M±H]+/-

[M+X]+/-

(X= Li+, Na+, NH4+, HCOO-etc.)

[M±H]+/-

Abundant fragments

COMPLEMENTARY

TECHNIQUES

Spectra of a phosphatidylcholine (PC 16:0/18:2) extracted from plasma sample

500 600 700 800 m/z0

5000000

10000000

15000000

Inten.

575

758

717744

[M+H]+

[M+Na]+

[M+H]+

500 600 700 800 m/z0

5000000

10000000

15000000

Inten.

758

780

[M+2H-CH3]+

[M+H-41]+

[DAG]+ESI APCI

DIAGNOSTIC FRAGMENTS

Un

iver

sity

of

Mes

sin

a, It

aly

LC-MS analysis

Lipid extracts of different plasma samples were subjected to LC-MS analysis

The global lipid profiling method was based on ultrahigh-performance liquid

chromatography combined with a quadrupole MS analyzer through both ESI and

APCI interfaces operated in both positive and negative ionization mode

Significant advantages of UHPLC over conventional HPLC techniques:

use of columns with smaller particle sizes

more rapid analysis time

improved chromatographic resolution

Column: Titan C18, 100 x 2.1 mm I.D. with particle size of 1.9 µm (Supelco,

Bellefonte, PA)

Particles of uniform size

Lower pressure drop

Higher column permeabilityMobile Phase Velocity (mm/sec)

1 2 3 4 5

35.00

30.00

25.00

20.00

15.00

10.00

5.00

dp = 1.9 µm

dp = 3 µm

dp = 5 µm

dp = 10 µm

Monolithic or

Fused-core (2.7 µm)

Un

iver

sity

of

Mes

sin

a, It

aly

LC-APCI-MS analysis

Column: Titan C18 (100 x 2.1 mm, 1.9 µm).

Mobile phase: H2O with 20 mM HCOONH4 (solv.A) and IPA/ACN/H2O (60:36:4, v/v) with 0.1% HCOOH

(solv.B).

Flow rate: 0.4 mL/min. Injection volume: 10 µL. Oven temperature: 40 °C.

Gradient profile: : 0 min, 80% B, 6 min, 100% B (hold for 16 min). Starting conditions were achieved in

0.01 min and the column was re-equilibrated for 3 min, resulting in a total run time of 25 min.

MS conditions: APCI positive (+) and negative (-) mode. Full scan mode: mass spectral range, m/z 350-1250

(+) and m/z 150-1250 (-); event time, 0.2 s. SIM mode: fatty acid molecular ions were selected in negative

mode, cholesterol internal standard (TAG) were selected in positive ionisation mode. APCI parameters:

nebulizing gas (N2) flow, 3 L/min; drying gas (N2) flow, 15 L/min; interface temperature, 450 °C; DL

temperature, 250 °C; heat block temperature, 200 °C.

Experimental conditions

2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

0.00

0.25

0.50

0.75

1.00

1.25

1.50

(x100.000.000)

1:TIC(+)

LPLs

FFAsCholesterol

PLs

SLs

DAGs

TAGs

CEs

Un

iver

sity

of

Mes

sin

a, It

aly

ESI vs APCI

Inten.

Characteristic spectra of phosphatidylcholines

ESI spectra APCI spectra

400 500 600 700 800 900 m/z

0

25

50

75

100Inten.

599

782741768 804

[M+H]+

[M+Na]+

[M+H-CH3+H]+

[M+H-41]+

[DAG]+

Scan (+)

400 500 600 700 800 900 m/z

0

25

50

75

100826

894

962

[M+HCOO]-

Scan (-)

400 500 600 700 800 900 m/z

0

25

50

75

100

Inten.

782

804 [M+Na]+

[M+H]+

Scan (+)

400 500 600 700 800 900 m/z

0

25

50

75

100Inten.

766709

391

439

[M-CH3]-

[M-RCHCO-choline]-

[M-RCHCO-choline]-

Scan (-)

[M+H-73]-

SIM (-)

200 250 300 m/z

0

25

50

75

100

Inten.

303

255277

200 250 300 m/z

0

25

50

75

100

Inten.

303

255

SIM (-)

329

722

[M-H-N(CH3)3]-

(PC-16:0/20:4)

Un

iver

sity

of

Mes

sin

a, It

aly

Characteristic spectra of sphingomyelins

ESI vs APCI

400 500 600 700 m/z

0

25

50

75

100

Inten.

747

775737470419 507 585 663

Scan (-)

[M+HCOO]-

400 500 600 700 m/z

0

25

50

75

100

Inten.

725

703544 741

Scan (+)

[M+H]+

[M+Na]+

Scan (-)

0

25

50

75

100

[M+H-73]-

[M-CH3]-

[M+HCOO]-

400 500 600 700 m/z

Inten.

582

687630

613 747

[M+H-121]-

643

[M-H-N(CH3)3]-

Scan (+)

0

25

50

75

100

400 500 600 700 m/z

Inten.

520

626703

[M+H]+

689

[M+H-CH3+H]+

(SM-34:1)ESI spectra APCI spectra

[DAG]+

Un

iver

sity

of

Mes

sin

a, It

aly

ESI vs APCICharacteristic spectra of triacylglycerols of #scan 2036, min 11. 96. PN = 46

TIC (+)

400 500 600 700 800 900 m/z

0

25

50

75

100

Inten.

601 857

575

549883

831

603

523

577

805[C48:1+H]+

[C50:2+H]+

[C52:3+H]+

[C54:4+H]+

[C30:0]+

[C32:1]+

[C34:2]+

[C34:1]+

[C36:3]+

[C36:2]+

[C36:1]+

PARENT IONSDAUGHTER IONS

APCI

Only 7 TAGs

were

confirmed

TIC (+)

400 500 600 700 800 900 m/z

0

25

50

75

100

Inten.

874a

879b

905b

900a

853b

848a

822a

827b

[C52:3+X]+

[C48:1+X]+

[C50:2+X]+

[C54:4+X]+

PARENT IONS

30 different TAGs

could be tentatively

identified

ESI

X= a[M+NH4]+ ; b[M+Na]+

77% false positive

C52:3

C48:1

C50:2

C54:4

C20:4C18:0C16:0

C20:3C18:0C16:1

C20:3C18:1C16:0

C20:2C18:1C16:1

C20:2C18:1C16:1

C20:2C18:2C16:0

C20:1C18:3C16:0

C20:1C18:2C16:1

C18:0C18:2C18:2

C18:0C18:1C18:3

C18:1C18:1C18:2

C20:0C18:3C14:0

C20:1C16:1C16:1

C20:1C18:2C14:0

C20:2C16:0C16:1

C20:2C18:1C14:0

C20:3C16:0C16:0

C20:3C18:0C14:0

C18:0C18:2C16:1

C18:0C18:3C16:0

C18:1C18:2C16:0

C18:1C18:1C16:1

C18:2C16:0C16:0

C18:1C16:0C16:1

C18:1C18:1C14:0

C18:0C16:1C16:1

C18:2C18:0C14:0

C20:1C14:0C14:0

C18:1C16:0C14:0

C18:0C16:1C14:0

Un

iver

sity

of

Mes

sin

a, It

aly

ESI vs APCI

Characteristic spectra of cholesteryl esters

Scan (+)

[M+H-FA]+

400 500 600 700 m/z

0

25

50

75

100

Inten.

369

Scan (+)

400 500 600 700 m/z

Inten.

671369

666[M+NH4]

+

[M+Na]+

[M+H-FA]+

0

25

50

75

100

Scan (-)

200 250 300 350 m/z

0

25

50

75

100

Inten.

279

325loss of fatty acid from

cholesteryl ester

[C18:2+HCOO]-

STRUCTURAL INFORMATION

OF THE MOLECULAR SPECIES

(Chol-C18:2)

Scan (-)

no information

ESI spectra APCI spectra

Un

iver

sity

of

Mes

sin

a, It

aly

IT-TOF-ESI-MS/MS vs APCI-qMS

Lyso-phosphocholine PC-18:2

250 500 750 1000 1250 m/z

0.0

2.5

5.0

Inten. (x 1.000.000)

564.3303

504.3091

[M+HCOO]- TIC (-)

[M-CH3]-

200 250 300 m/z

0.0

0.5

1.0

1.5 279

Inten. (x 100.000)

[C18:2-H]-SIM (-)

CONFIRMED BY SIM (-)MS/MS Precursor ion from 504.3391

250 500 750 1000 1250 m/z

00

0.5

1.0279.2345

506.3180 739.1628

Inten. (x 1.000.000)

[C18:2-H]- TIC (-)

MS/MS Precursor ion from 564.3303

250 500 750 1000 1250 m/z

0.0

1.0

2.0

504.3087

279.2333

566.3403

Inten. (x 1.000.000)

[M-CH3]-

[C18:2-H]-

TIC (-) 250 500 750 1000 m/z

00

5.0

10.0

415

504

447

[M-104]-

[M-CH3]-

[M+H-73]-

Inten. (x 1.000.000)

279

TIC (-)

ESI MS/MS spectra

APCI-MS spectra

Un

iver

sity

of

Mes

sin

a, It

aly

POP

POS

PPO

SPO+PSO

MPoO

PPoO

POO

SOO

PLO

PPoL

POL

OOO

PoOO

PLL

OOL

OPoLOLL

POAr

ArOO

DB 0 1 2 3 4 5 6

TRIACYLGLYCEROLS

CP

CS

CM

CO

CPo

CL

CEt

CLn CAr CDh

MOP

DB (FA)

0 1 2 3 4 6

CHOLESTERYL ESTERS

M C14:0

P C16:0

Po C16:1w7

S C18:0

O C18:1w9

L C18:2w6

Ln C18:3w3

Et C20:3w6

Ar C20:4w6

Dh C22:6w3

C Cholesteryl

PPP

PPS

32 peaks identified10 CE:CN 14-22, DB 0-6

22 TAG:PN 44-50, DB 0-6

Ag+×RP-APCI(+)-MS analysis of plasma lipids

Future research will be focused on the development of an LC×LC method using

RPLC×HILIC but employing an APCI-qMS to improve the level of information,

without using a tandem MS

132.01 198.02 min

0.0

0

3

.00

6

.00

9

.00

12

.00

1

5.0

0 min

2D

RP

-LC

1D HILIC

18:0/18:1

18:1/20:2

18:1/18:1

18:0/20:4

16:0/18:120:2/20:4

16:0/20:3

16:0/18:2

16:0/20:4

18:1/20:5

16:1/20:4

147,5

150,0

152,5

155,0

157,5

min

0

2500000

5000000

7500000

10000000

12500000

15000000

17500000

20000000

22500000

25000000

27500000

30000000

32500000

35000000

TIC

(+) 1

62.5

165.0

167.5

170.0

172.5

min

35000000 0

2500000

5000000

7500000

10000000

12500000

15000000

17500000

20000000

22500000

25000000

27500000

30000000

32500000

TIC

(+)

14

5-1

60 m

in 2

D-L

C c

ut

16

0-1

75 m

in 2

D-L

C c

ut

18:1/20:5

20:2/20:4

18:0/20:4

18:0/18:1

18:1/20:2

18:1/18:1

16:0/18:1

16:0/20:3

16:0/20:4

16:0/18:2

16:1/20:4

HILIC×RP-LC contour plot for PC molecular species in plasma

HILIC×RP-LC contour plot for PC molecular

species in plasmaAg+×RP-APCI(+)-MS contour plot for TAGs

and CEs in plasma

5.0

Un

iver

sity

of

Mes

sin

a, It

aly

RPLC-APCI MS Analysis of Menhaden Fish Oil TAGs

30 PN 52

20.0 25.0 30.0 35.0 40.0 45.0

0.0

1.0

2.0

3.0

4.0

min

(TIC)

Inten. (x1,000,000)

Column: Ascentis Express C18, 150 × 4.6 mm i.d., 2.7 mm

Mobile phase, A: ACN; Solvent B: IPA; Injection volume: 5 L

Gradient: 0 min 0% B, 50 min 70% B, (hold for 5 min); 56 min, 0% B

Flow rate: 1 mL/min; DP at 70% IPA concentration: 21.0MPa

Detection: APCI-MS

1 column

Un

iver

sity

of

Mes

sin

a, It

aly

Column: 4 serially coupled Ascentis Express C18, total staionary phase lenght: 600 mm

Mobile phase, A: ACN; Solvent B: IPA; Injection volume: 5 L

Gradient: 0 min 0% B, 200 min 70% B, (hold for 20 min); 224 min, 0% B

Flow rate: 1 mL/min; DP at 70% IPA concentration: 89.5 MPa

Detection: APCI-MS

75.0 100.0 125.0 150.0 175.0min

Inten. (x1,000,000)

(TIC)

30

34

3638

40 42 4446+47

48

50

52

0.0

0.5

1.0

1.5

2.0

2.5

RPLC-APCI MS Analysis of Menhaden Fish Oil TAGs

4 columns

Un

iver

sity

of

Mes

sin

a, It

aly

68–125 min Enlargement of the TIC Chromatogram

Due the high

content of

omega-3 PUFAs,

the oil represent a

viable source of

dietary lipids.Eicosapentaenoic acid

20:5(v3)

70 80 90 100 110 120

0.0

1.0

2.0

3.0

4.0

Inten. (x1,000,000)

(TIC)

min

1

2 3

10

4 5

6 7+

8+

9 11

12

13

+1

4 15

+1

6+

17

18 19

20

2122

23

24

25

26

27

+2

8+

29

30

31

32

+3

334

35

36

37

+3

839

40

41

+4

2+

43

44

+4

5+

46

47

48

49

+5

051

52 5

3 54

55

56

57

EpEpEp

(DB=15, PN=30)

DhDhDh

(DB=18, PN=30)

Docosahexaenoic acid

22:6(v3)

Un

iver

sity

of

Mes

sin

a, It

aly

130 140 150 160 170 180

0.0

1.0

2.0

3.0

4.0

Inten. (x1,000,000)

(TIC)

min

58

+5

9+

60

61

+6

26

3+

64

+6

56

7+

68

66

69

+7

0+

71

72+

73+

74 7

57

677

78

+7

98

0+

81

83

82

84

+8

58

6+

87

+8

889

90

+9

192

93

94

95

96

97

98

+9

9 101

100

102

103

104

105

106

107108

109

11

0111

11

2

11

411

3

11

511

611

7+

11

811

9+

12

01

21

+1

22

123

12

4+

12

5126

12

7+

12

8

129 1

30

131

132

133

134

135

136

137

[PN values (30–52), DB values (0–18)]

One C18 column (150 mm) Four C18 columns (600 mm)

137 TAGs identified

125-188 min Enlargement of the TIC chromatogram

No unambiguous TAG identification

Un

iver

sity

of

Mes

sin

a, It

aly

1D

Column: Nucleosil ion-exchange loaded with silver ions (250 mm × 4.0 mm, 5 m)

Mobile phase: (A) 1.0 % BCN in Hex; (B) 5.0 % BCN in Hex

Linear gradient: 0 min 0% B; 100 min 100% B

Flow rate: 0.8 mL/min

Inj. vol.: 20 L

Sample: menhaden oil (40 mg in 10 mL Hex)

Off-line Ag+-LC×RPLC-APCI MS

2D

Column: Ascentis Express C18 Fused-core (150 x 4.6 mm)

Mobile phase: (A) ACN; (B) IPA

Linear gradient: 0 min 0% B; 50 min 70% B

Flow rate: 1.0 mL/min

Inj. vol.: 10 L

Detection: qMS (APCI+)

The combination of Ag+- and RP-separations provides information

that cannot be achieved by any of these when applied on its own

Un

iver

sity

of

Mes

sin

a, It

aly

10 20 30 40 50 60 70 80 90min

0

25

50

75

100

125

150

175

200

225mV

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

DB0 18

The 16 fractions were evaporated to dryness under a nitrogen stream

and re-dissolved in 1.0 mL acetone

2D

RPLC

1D

Ag+LC

Fraz.4

Po

C1

6:4

M

Po

PoH

+L

C1

6:2

M+

C1

6:2

C1

6:2

PO

tPoM

+P

oH

ME

pP

oM

+P

C1

6:4

P

Dh

MM

Ep

PP

d+

Ep

PoP

d+

Ep

MP

d

Ln

PoP

o+

OP

oH

ArP

oM

+ O

tPP

oE

pP

Po+

Ep

OM

Ep

PM

+P

PC

16

:4D

hP

MD

hP

Pd

OO

tPE

pO

PE

pP

PD

hP

P

ArO

P+

C21

:5P

P

Ep

SP

Dh

SP

Dh

SS

20 25 30 35 40 45 min

0

500000

1000000

1500000

TIC(+)

Fraz.4

Po

C1

6:4

M

Po

PoH

+L

C1

6:2

M+

C1

6:2

C1

6:2

PO

tPoM

+P

oH

ME

pP

oM

+P

C1

6:4

P

Dh

MM

Ep

PP

d+

Ep

PoP

d+

Ep

MP

d

Ln

PoP

o+

OP

oH

ArP

oM

+ O

tPP

oE

pP

Po+

Ep

OM

Ep

PM

+P

PC

16

:4D

hP

MD

hP

Pd

OO

tPE

pO

PE

pP

PD

hP

P

ArO

P+

C21

:5P

P

Ep

SP

Dh

SP

Dh

SS

20 25 30 35 40 45 min

0

500000

1000000

1500000

TIC(+)

Un

iver

sity

of

Mes

sin

a, It

aly

Ag+0

5

10

15

20

25

30

35

40

45

50

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

RP

min

0 18

DB

PN

28

54

Total number of identified TAGs: 273

Off-line Ag+LC×RPLC-APCI MS

Un

iver

sity

of

Mes

sin

a, It

aly

1D

Column: cation-exchange, 150 x 1.0 mm i.d., 5 mm dp (silvered in lab)

Mobile phase: 1% BCN in n-hexane (solvent A); 5 % BCN in n-hexane (gradient)

Flow rate: 20 L/min

Inj. vol.: 1 L

2D

Column: Ascentis Express C18 150 x 2.1 mm i.d.,

Mobile phase: A) isopropanol; B) acetonitrile (gradient)

Flow rate: 0.6 mL/min

Detection: qMS (APCI+)

Modulation time: 11 min

On-line Stop-flow Ag+LC×RPLC-APCI MS

Un

iver

sity

of

Mes

sin

a, It

aly

RP

-LC

Ag+-LC

DB 18110 20 30 40 50 60 min

1

2

3

4

5

67

8

9

10

11 12

13 14

151617 18

0

25

50

75

100

125

150

mV

Ag+LC-ELSD5

2

DB 18

PN

38 1

On-line Stop-flow Ag+LC×RPLC-APCI MS

Un

iver

sity

of

Mes

sin

a, It

alyPN

40

42

44

46 4

8 50

38

3

0

DB 1 18

Ag+

RP 5252

min

min

On-line Stop-flow Ag+LC×RPLC-APCI MS

Un

iver

sity

of

Mes

sin

a, It

aly

Serially coupled columns (4 C18):

• Flow rate: 1 mL/min

• Analysis time: 200 min

• Peak capacity: 210

• TAGs identified: 153

On-line Stop-flow Ag+-LC×RP-LC:

• 1st dimension flow rate: 20 mL/min

• 2nd dimension flow rate: 600 mL/min

• Analysis time: 600 min

• Peak capacity: 1265

• TAGs identified: 180

Method Performances for TAG Analysis in Menhaden Oil

600 mm

2.7 m

Off-line Ag+-LC×RP-LC:

• 1st dimension flow rate: 0.8 mL/min

• 2nd dimension flow rate: 1.0 mL/min

• Analysis time: 900 min

• Peak capacity: 2160

• TAGs identified: 253

Un

iver

sity

of

Mes

sin

a, It

aly

CONCLUSIONS

•Using a mono-dimensional LC-APCI-qMS we have identified 90 lipid species

• APCI provides informative spectra, with a protonated molecular ion and fragmentations,

which allow structural elucidation

• LC-APCI-qMS can be a valid and simple technique which can provide almost similar

information compared to more sophisticated ones (HRMS, QqQ MS, etc) for lipidomics

studies

• APCI-MS can often be used as an alternative to ESI MS/MS for analysis of lipids

• Powerful LC approaches e.g. UHPLC and comprehensive LC are valuable tools to

elucidate lipidic profile of complex food samples

•Comprehensive LC, due to the orthogonality of the stationary phases employed viz.

Ag+×RP-LC can be successfully employed for unravelling of complex lipidic samples

Un

iver

sity

of

Mes

sin

a, It

aly

…and you for your kind attention

I wish to thanks: