100 200 300 400 500 600 700 800 m/z0.0

2.5

Inten.(x100,000)

335

235 403317 363 487 555199 731111 617 867773691 81783

Detection of biophenols from virgin olive oil in French-fried potatoes by high-performance liquid chromatography

tandem electrospray ionization mass spectrometry (HPLC-ESI/MS)

Detection of biophenols from virgin olive oil in French-fried potatoes by high-performance liquid chromatography

tandem electrospray ionization mass spectrometry (HPLC-ESI/MS)

criol Centro Ricerche per l’Industria Olearia

Università degli Studi di Napoli Federico II

Dipartimento di Scienza degli Alimenti

Savarese M.Savarese M.§§, Parisini C., Parisini C.§§, De Marco E., De Marco E.§§, Sacchi R., Sacchi R.§,§, §§CRIOL-Centro Ricerche per l’Industria Olearia, Industria Olearia Biagio Mataluni, Montesarchio (BN), Italy CRIOL-Centro Ricerche per l’Industria Olearia, Industria Olearia Biagio Mataluni, Montesarchio (BN), Italy Department of Food Science, University of Naples Federico II, Department of Food Science, University of Naples Federico II, Portici (Na), Italy; Portici (Na), Italy;

e-mail: e-mail: criol@mataluni.comcriol@mataluni.com; sacchi@unina.it

INTRODUCTIONINTRODUCTION

Deep frying is an important cooking technique employed in domestic, collective Deep frying is an important cooking technique employed in domestic, collective and industrial food preparation. During this process oil used as cooking medium and industrial food preparation. During this process oil used as cooking medium is submitted to high temperatures (180°C) in presence of oxygen and moisture. is submitted to high temperatures (180°C) in presence of oxygen and moisture.

These conditions promote a series of reactions determining deep modification in These conditions promote a series of reactions determining deep modification in the frying oil as well as in the food being fried (the frying oil as well as in the food being fried (Figure 1Figure 1). ).

Liquid Chromatography tandem MassLiquid Chromatography tandem Mass SpectrometrySpectrometry (LC-MS) (LC-MS) with Electrospraywith Electrospray Ionization (ESI) represents a very powerful Ionization (ESI) represents a very powerful tool for the analysis of natural phenolic tool for the analysis of natural phenolic extracts since the mass spectrometer is a extracts since the mass spectrometer is a universal detector, able to achieve very universal detector, able to achieve very high sensitivity and to provide data on high sensitivity and to provide data on molecular weights as well as structural molecular weights as well as structural information. information.

Components already present in the frying medium or generated during frying Components already present in the frying medium or generated during frying may migrate from the oil into the food product, which in its turn may release may migrate from the oil into the food product, which in its turn may release substances into the frying medium. substances into the frying medium.

Extra virgin olive oilExtra virgin olive oil (EVOO) contains (EVOO) contains different phenolic compounds acting as different phenolic compounds acting as antioxidants. These molecules may be antioxidants. These molecules may be partially transferred from oil to food during partially transferred from oil to food during frying and may act as antioxidants also in frying and may act as antioxidants also in fried food, extending the shelf-life and fried food, extending the shelf-life and improving the nutritional quality of the improving the nutritional quality of the products. products.

OBJECTIVEOBJECTIVE

Aim of the present study was the application of ESI-MS to evaluate the Aim of the present study was the application of ESI-MS to evaluate the possible migration of antioxidant compounds from extra virgin olive oil to possible migration of antioxidant compounds from extra virgin olive oil to the fried food during a deep-frying process. the fried food during a deep-frying process.

MATERIALS AND METHODSMATERIALS AND METHODS

Frying trialsFrying trials

An electrical deep fat fryer (Tefal, Milan), operating at a temperature of 180 °C, was An electrical deep fat fryer (Tefal, Milan), operating at a temperature of 180 °C, was used for frying frozen pre-fried French fries (Orogel, Cesena, Italy). The trial was used for frying frozen pre-fried French fries (Orogel, Cesena, Italy). The trial was carried out with an extra virgin olive oil, purchased in a local market, and with a refined carried out with an extra virgin olive oil, purchased in a local market, and with a refined olive oil, as control. The fryer was filled with 1 l of oil and 12 frying cycles were carried olive oil, as control. The fryer was filled with 1 l of oil and 12 frying cycles were carried out with 30 min interval between each other. During each cycle a French fries sample out with 30 min interval between each other. During each cycle a French fries sample (100 g) was fried. (100 g) was fried.

Both oil samples (50 ml) and French fried potato sample were collected every hour of Both oil samples (50 ml) and French fried potato sample were collected every hour of frying and stored at -20 °C until analysis. frying and stored at -20 °C until analysis.

The presence of these compounds, which proposed structures are shown in The presence of these compounds, which proposed structures are shown in Figure 3Figure 3, suggests that further investigation is required in order to verify , suggests that further investigation is required in order to verify their properties in terms of antioxidant power and nutritional impact. their properties in terms of antioxidant power and nutritional impact. Since oxidation regards only the elenolic group, these important Since oxidation regards only the elenolic group, these important properties may be preserved as they are due to the phenolic moiety. properties may be preserved as they are due to the phenolic moiety.

RESULTSRESULTS

Many different biophenols were identified in the frying oil samples, several Many different biophenols were identified in the frying oil samples, several of which were also revealed in fried potatoes. of which were also revealed in fried potatoes.

Identification (Identification (Figure 2Figure 2) of phenolic compounds was based on:) of phenolic compounds was based on:

search for pseudomolecular [M-H]search for pseudomolecular [M-H]-- ions, using extracted ion mass ions, using extracted ion mass chromatogramschromatograms

interpretation of collision-induced dissociation (CID) fragments. The interpretation of collision-induced dissociation (CID) fragments. The structural information resulting from the deprotonated molecular ion was structural information resulting from the deprotonated molecular ion was confirmed by modifying some MS parameters (interface voltage and Q-confirmed by modifying some MS parameters (interface voltage and Q-Array voltage) in order to obtain a moderate dissociation-fragmentation Array voltage) in order to obtain a moderate dissociation-fragmentation induced by the collisions in the ionic transport region.induced by the collisions in the ionic transport region.

comparison of retention times and mass spectra with those of standards, comparison of retention times and mass spectra with those of standards, when available, or with those reported in literature [3; 4]. when available, or with those reported in literature [3; 4].

Extraction of phenolic compounds Extraction of phenolic compounds from French fried potatoes.from French fried potatoes.

Extraction of phenolic compounds Extraction of phenolic compounds from French fried potatoes was from French fried potatoes was carried out following the procedure carried out following the procedure described by Brenes et al. [1], with described by Brenes et al. [1], with some modifications. some modifications.

Extraction of phenolic compounds Extraction of phenolic compounds from oilsfrom oils

Extraction of EVOO phenolic Extraction of EVOO phenolic compounds was carried out compounds was carried out according to the method described according to the method described by Sacchi et al. [2], to which some by Sacchi et al. [2], to which some modifications have been applied. modifications have been applied. Liquid chromatography tandem Liquid chromatography tandem electrospray electrospray ionization mass spectrometryionization mass spectrometry

The LC system consisted of two Shimadzu The LC system consisted of two Shimadzu LC-10AD VP pumps (Shimadzu, Milan, LC-10AD VP pumps (Shimadzu, Milan, Italy). Italy).

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

(x1,000,000)TIC

1122 33 44 55

66 77

88

991010

1111

1212

1313 1414

1515

1616

17171818

19192020

21212222

2323

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

(x10,000,000)TIC

1122 33

7799

1212

1414 1616

1717

21212222

2323

Figure 2. Figure 2. Total Ion Current (TIC) ESI-MS chomatograms of EVOO and French fried potatoes phenolic exctracts. Total Ion Current (TIC) ESI-MS chomatograms of EVOO and French fried potatoes phenolic exctracts.

Peaks IdentificationPeaks Identification: : 1.1. Hydroxytyrosol; ; 2.2. Tyrosol; ; 3.3. Decarboxymethyl elenolic acid oxidized; 4.4. Not Identified.; 5.5. Elenolic acid oxidized; 6.6. Elenolic acid; 7.7. Elenolic acid oxidized; 8.8. Dialdehydic form of decarboxymethyl oleuropein aglycon; 9.9. Oxidation product of of decarboxymethyl oleuropein aglycon; 10.10. Aldehydic form of oleuropein aglycon; 11.11. Aldehydic form of ligtroside aglycon; 12.12. Oxidation product of decarboxymethyl ligstroside aglycon; 13.13. H Hydroxytyrosol derivative; 14.14. Aldehydic form of oleuropein aglycon; 15.15. Aldehydic form of oleuropein aglycon; 16.16. Oxidation product of oleuropein aglycon; 17.17. Luteolin; 18.18. Aldehydic form of oleuropein aglycon; 19.19. Aldehydic form of oleuropein aglycon; 20. 20. Aldehydic form of oleuropein ligstroside; 21.21. Oxidation product of oleuropein ligstroside; 22. 22. Apigenin; 23. 23. Methoxyluteolin.

PeaPeak k

no. no.

[M-[M-H]H]--

((m/z)m/z)

1h frying1h frying 3h frying3h frying 6h frying6h frying

EVOEVOOO

FrencFrench h

friesfriesEVOOEVOO French French

friesfries EVOOEVOO French French friesfries

1 153153 - - - 2 137137 - - - 3 199199 -5 257257 - - - -6 241241 - - 7 257257 - 8 319319 - - - -9 335335 - -

10 377377 - - - - - -11 361361 - - - - -12 319319 14 377377 - - -15 377377 - - -16 393393 - -17 285285 18 377377 - - 19 377377 - - - 20 361361 - - -21 377377 22 269269 23 299299

The mass spectrometry system consisted of a The mass spectrometry system consisted of a mass spectrometer Shimadzu LCMS-2010EV mass spectrometer Shimadzu LCMS-2010EV (Shimadzu).(Shimadzu).

•interface voltageinterface voltage: 4 kV; : 4 kV; •nebulizer gas flownebulizer gas flow: 1.5 l min: 1.5 l min-1-1;;•block heater temperatureblock heater temperature: 250 °C; : 250 °C; •curved desolvation linecurved desolvation line (CDL) (CDL) temperaturetemperature and and voltagevoltage:: 300 °C and -5 V; 300 °C and -5 V;•Q-Array voltageQ-Array voltage: 0 V DC and 150 V RF;: 0 V DC and 150 V RF;•detector voltagedetector voltage: 1.5 kV;: 1.5 kV;•scan rangescan range: m/z 60-900.: m/z 60-900.

EXTRA VIRGIN EXTRA VIRGIN OLIVE OILOLIVE OIL

FRIED FRIED POTATOESPOTATOES

ReferencesReferences[1] Brenes M., Rejano L., Garcia P., Sanchez H.A., Garrido A. [1] Brenes M., Rejano L., Garcia P., Sanchez H.A., Garrido A. J. Agric. Food Chem.J. Agric. Food Chem., 1995, 43, 2702-2706. , 1995, 43, 2702-2706. [2] Sacchi R., Paduano A., Fiore F., Della Medaglia D., Ambrosino M.L., Medina I. [2] Sacchi R., Paduano A., Fiore F., Della Medaglia D., Ambrosino M.L., Medina I. J. Agric. Food Chem.J. Agric. Food Chem., , 2002, 50, 2830-2835.2002, 50, 2830-2835.[3] Rovellini P., Cortesi N. [3] Rovellini P., Cortesi N. Riv. Ital. Sost. Grasse, Riv. Ital. Sost. Grasse, 2002, 2002, 6969, 1-14., 1-14.[4] R[4] Ríos J.J., Gil M.J., Gutiérrez-Rosales F. íos J.J., Gil M.J., Gutiérrez-Rosales F. J. Chromatogr. AJ. Chromatogr. A, 2005, , 2005, 10931093, 167-176. , 167-176. [5] Kalogeropoulos N., Chiou A., Mylona A., Ioannou M.S., Andrikopoulos N.K. [5] Kalogeropoulos N., Chiou A., Mylona A., Ioannou M.S., Andrikopoulos N.K. Food Chem.Food Chem., 2007, , 2007, 100100, , 509-517. 509-517. [6] Brenes M., Garcia A., Dobarganes M.C., Velasco J., Romero C. J. Agric. Food Chem., 2002, 50, 5962-[6] Brenes M., Garcia A., Dobarganes M.C., Velasco J., Romero C. J. Agric. Food Chem., 2002, 50, 5962-5967. 5967.

AcknowledgementsAcknowledgementsThis work was supported by Italian Ministry of University and Research (MIUR) and by This work was supported by Italian Ministry of University and Research (MIUR) and by Industria Olearia Biagio Mataluni s.r.l. within the project “Controllo Qualità ed Industria Olearia Biagio Mataluni s.r.l. within the project “Controllo Qualità ed Innovazione Tecnologica nell’Industria olearia” (DM 593- 08/08/2000, Prot. MIUR 1866 – Innovazione Tecnologica nell’Industria olearia” (DM 593- 08/08/2000, Prot. MIUR 1866 – 18/02/2002). 18/02/2002).

The frying process proved to determine a partial loss of all antioxidants in The frying process proved to determine a partial loss of all antioxidants in the fried oils paralleled by their well as their enrichment in the fried food the fried oils paralleled by their well as their enrichment in the fried food ((Table 1Table 1). ).

Oxidation forms of elenolic acid and of secoiridoid derivatives were Oxidation forms of elenolic acid and of secoiridoid derivatives were observed in both frying oils and fried potatoes (observed in both frying oils and fried potatoes (Figure 2Figure 2; ; Table 1Table 1). ).

FOODFOOD

AERATIONAERATION

ABSORPTIONABSORPTION

VAPORIZATIONVAPORIZATION

steam

HYDROLYSISHYDROLYSIS

free fatty acids

diglycerides

monoglycerides

glycerine

SOLUBILIZATIONSOLUBILIZATION

oxygen

coloured compounds

food lipids

OXIDATIONOXIDATION

hydroperoxides

(conjugated dienes)

DEHYDRATIONDEHYDRATIONFISSION

FISSIONFREE RADICALS

FREE RADICALS

alcohols

aldehydes

ketones dimers

trimers

epoxides

alcohols

hydrocarbonsHEATING

HEATINGdimers

cyclic compounds

FOODFOOD

AERATIONAERATION

ABSORPTIONABSORPTION

VAPORIZATIONVAPORIZATION

steam

HYDROLYSISHYDROLYSIS

free fatty acids

diglycerides

monoglycerides

glycerine

SOLUBILIZATIONSOLUBILIZATION

oxygen

coloured compounds

food lipids

OXIDATIONOXIDATION

hydroperoxides

(conjugated dienes)

DEHYDRATIONDEHYDRATIONFISSION

FISSIONFREE RADICALS

FREE RADICALS

alcohols

aldehydes

ketones dimers

trimers

epoxides

alcohols

hydrocarbonsHEATING

HEATINGdimers

cyclic compounds

O

CH3

HOOC

O OOH

OH

[A]

[B]

[C]

[D]O

CH3

HOOC

O OOH

OH

[A]

[B]

[C]

[D]

[M-A]- (m/z 199); [M-A-B-C]- (m/z 111); [M-D]- (m/z 235)

100 200 300 400 500 600 700 800 m/z0.0

0.5

1.0

1.5

Inten.(x1,000,000)

319

249

387455 661199155 745409 535 81359199 317

887

O

CH3

HOOC

O OOH

[A]

[B]

[C]O

CH3

HOOC

O OOH

[A]

[B]

[C]

[M-A]- (m/z 199); [M-A-B]- (m/z 155); [M-C]- (m/z 249)

100 200 300 400 500 600 700 800 m/z0.0

1.0

2.0

3.0

Inten.(x100,000)

377

333249 445

513285 461 597 777665 733113 875181

[A]

[B]O

CH3

HOOC

O OOHCOOMe

[A]

[B]O

CH3

HOOC

O OOHCOOMe[M-A]- (m/z 333); [M-B]- (m/z 249)

100 200 300 400 500 600 700 800 m/z0.0

0.5

1.0

1.5Inten.(x1,000,000)

285

361 409221 545260 596471 643 776175 834101897

100 200 300 400 500 600 700 800 m/z0.0

2.5

5.0

7.5Inten.(x100,000)

269

377349291 455 521223 599563 660113

888167 721 813

O

O

OH

OH

OH

OH

O

O

OH

OH

OH

Figure 1.Figure 1. Changes occurring during deep-fat frying. Changes occurring during deep-fat frying.

Table 1.Table 1. Presence/absence of identified phenolic Presence/absence of identified phenolic

compounds in EVOO and French fried potatoes during compounds in EVOO and French fried potatoes during deep-frying (deep-frying (- undetected undetected detected) detected) Peak 9Peak 9

Peak Peak

1212

Peak Peak

2121

Peak Peak

1717 Peak Peak

2222

Figure 4.Figure 4. Full scan MS spectra of luteolin (peak 17) andFull scan MS spectra of luteolin (peak 17) and apigenin (peak 22) detected in EVOO and in fried potatoes after 6h of apigenin (peak 22) detected in EVOO and in fried potatoes after 6h of

deep-frying.deep-frying.

Figure 3.Figure 3. Full scan MS spectra of oxidation form of decarboxymethyl Full scan MS spectra of oxidation form of decarboxymethyl oleuropein aglycon (peak 9), oxidation form of decarboxymethyl oleuropein aglycon (peak 9), oxidation form of decarboxymethyl ligstroside (peak 12) and oxidationligstroside (peak 12) and oxidation form of ligstroside aglycon (peak 21) form of ligstroside aglycon (peak 21) detected in EVOO and in fried potatoes after deep-frying.detected in EVOO and in fried potatoes after deep-frying.

Tyrosol and hydroxytyrosol were revealed only in fried potatoes, confirming Tyrosol and hydroxytyrosol were revealed only in fried potatoes, confirming the previously reported hydrolysis and diffusion mechanisms and the previously reported hydrolysis and diffusion mechanisms and consequent partition between oil-water phases [2, 5, 6]. consequent partition between oil-water phases [2, 5, 6].

Flavonoids (luteolin, apigenin, methoxyluteolin) proved to be particularly Flavonoids (luteolin, apigenin, methoxyluteolin) proved to be particularly resistant to frying process (resistant to frying process (Figure 4Figure 4), being present in EVOO even after 6h ), being present in EVOO even after 6h of deep-frying, as well as in fried potatoes. of deep-frying, as well as in fried potatoes.

ESI or APCIFrom LC

ShutterSourcewindow

Drain

Block heater

CDL

Q-Array

Rotarypump

SkimmerOctapole

Turbo molecularpump 1

Focus lens

Entrance lens

Pre-rod

Quadrupole rod

Turbo molecularpump 2

Detector

ESI or APCIFrom LC

ShutterSourcewindow

Drain

Block heater

CDL

Q-Array

Rotarypump

SkimmerOctapole

Turbo molecularpump 1

Focus lens

Entrance lens

Pre-rod

Quadrupole rod

Turbo molecularpump 2

Detector

Although phenolic compounds give peaks of lower intensity in negative than in positive Although phenolic compounds give peaks of lower intensity in negative than in positive ion mode, in the present study analyses were performed in negative mode because ion mode, in the present study analyses were performed in negative mode because cleaner spectra were obtained. cleaner spectra were obtained.

•ColumnColumn: Discovery HS C18 column (5: Discovery HS C18 column (5m, 150 mm x m, 150 mm x 2.1 mm i.d., Supelco, USA),2.1 mm i.d., Supelco, USA),

•Flow rateFlow rate: 0.35 ml min: 0.35 ml min-1-1, ,

•Solvent system: Solvent system: solvent A (water + formic acid solvent A (water + formic acid 0.25%), solvent B (methanol + formic acid 0.25%), 0.25%), solvent B (methanol + formic acid 0.25%), with a step gradient from 5 to 55% B (45 min).with a step gradient from 5 to 55% B (45 min).

•Injected volumeInjected volume: : 20 20 l of methanol extractl of methanol extract

Mixing[30mL CH3OH/H2O (80/20) + 400 ppm sodium metabisulphite]

Mixing[30mL CH3OH/H2O (80/20) + 400 ppm sodium metabisulphite]

Centrifugation[5 min; 4000 rpm ]

Centrifugation[5 min; 4000 rpm ]

FiltrationFiltration

X 4

Hydroalcoholicextract

EXTRA VIRGIN OLIVE OIL[10 ml]

EXTRA VIRGIN OLIVE OIL[10 ml]

Evaporation organic solvent[under vacuum ]

Evaporation organic solvent[under vacuum ]

Washing[hexane; 15 mL X 3]

Washing[hexane; 15 mL X 3]

Extraction[ethyl acetate; 20 mL X 5]

Extraction[ethyl acetate; 20 mL X 5]

Evaporation organic solvent[under vacuum ]

Evaporation organic solvent[under vacuum ]

BIOPHENOLS EXCTRACT[2 ml methanol]

BIOPHENOLS EXCTRACT[2 ml methanol]

FRENCH FRIES POTATOES[10 g]

FRENCH FRIES POTATOES[10 g]

Mixing[10mL hexane]Mixing

[10mL hexane]

Extraction[CH3OH/H2O (60/40); 7mL X 3]

Extraction[CH3OH/H2O (60/40); 7mL X 3]

Washing[hexane 20 mL am centrifugation 10 min; 3500 rpm]

Washing[hexane 20 mL am centrifugation 10 min; 3500 rpm]

Hydroalcoholicextract

Evaporation organic solvent[under vacuum ]

Evaporation organic solvent[under vacuum ]

BIOPHENOLS EXCTRACT[2 ml methanol]

BIOPHENOLS EXCTRACT[2 ml methanol]

HPLC- ESI - MSANALYSIS

HPLC- ESI - MSANALYSIS