Research ArticleReceived: 8 October 2013 Revised: 16 January 2014 Accepted article published: 27 February 2014 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6628

Volatile profiles of flavedo, pulp and seedsin Poncirus trifoliata fruitsFabrizio Papa,a FilippoMaggi,b* Kevin Cianfaglione,c Gianni Sagratini,b

Giovanni Capriolib and Sauro Vittorib

Abstract

BACKGROUND: Poncirus trifoliata, also known as trifoliate orange, is a tree native to China and Korea and widely used all overthe world as a rootstock breeding material. In this study the differences among the volatile profiles of flavedo, pulp and seedsfrom two cultivars (var. trifoliata and var.monstrosa) grown in Italy (Marche, Abruzzo and Sicily) were determined. Headspacesolid phase microextraction and hydrodistillation techniques were used in combination with GC/FID and GC/MS to obtain thevolatile profiles of the samples.

RESULTS: Both techniques permitted the differentiation of fruit parts based on the main volatile components: the flavedo wascharacterized by monoterpene hydrocarbons such as limonene and myrcene, the seeds were characterized by sesquiterpenehydrocarbons such as (E)-caryophyllene and germacrene D, while the pulp showed an intermediate composition. The maindifferences in volatile profiles obtained by the two techniques were analyzed by chemometric techniques such as principalcomponent analysis.

CONCLUSION: The studydidnot highlight significant differences in volatiles between the two cultivars of trifoliate orange,whilefew differences in the number of volatiles in the fruit parts were revealed by the two techniques.© 2014 Society of Chemical Industry

Keywords: Poncirus trifoliata; Rutaceae; fruit; cultivars; headspace solid phase microextraction; hydrodistillation

INTRODUCTIONPoncirus trifoliata, also known as trifoliate orange or Korean bitterorange, is a member of the Rutaceae family, closely related tothe genus Citrus. The status rank of this species has undergonemany changes and some authors still call it Citrus trifoliata L.(http://www.theplantlist.org/). In certain countries the plant isused as an important rootstock breeding material with a goodinfluence on resistance against hard weather conditions andparasites.1

Being native to China and Korea, whence it was later introducedinto Japan, India and Australia, trifoliate orange is nowadays culti-vated all over theworld.2 In Italy, this species is cultivated3 and notconsidered as invasive. The P. trifoliata rootstock induces cold har-diness and dwarfing shape on the grafted tree; the cultivar mon-strosa gives a greater dwarfing effect.4

The plant is a large shrub or tree that can reach 8 m in height,with 3–5 cm thorns on the shoots, deciduous leaves with threeor rarely five leaflets, and green/yellow fruits, 3–4 cm in diameter,resemblinga small orangeandemitting amangoodour.5 The fruitsare bitter andused fresh inmarmalade;whendried andpowdered,they can be used as a condiment.1

Besides its uses as a rootstock, the plant is also employed ingardens to make decorations and barrier hedges owing to itsdensity and thorns.6 For this purpose, two cultivars are used:the nominate one (i.e. P. trifoliata var. trifoliata) and the so-called‘flying dragon’, ‘twisted’ or ‘contorta’ cultivar (i.e. P. trifoliatavar. monstrosa), which is recognizable by its contorted stems

and used for its dwarfing effect on grafting. The plant wasused for centuries in Italy, especially as a rootstock, in gardensand hedges.Trifoliate orange has been commonly used in Korean traditional

medicine for treating gastrointestinal disorders and as a rem-edy for allergic inflammation.7 Other recorded uses include thetreatment of hernia and sinusitis.6 The plant continues to occupyan important place in traditional oriental medicine and is usu-ally prescribed as an over-the-counter drug for patients sufferingfrom several diseases.7–16 Alkaloids,17 coumarins,11 limonoids,15

flavonoids,18 proanthocyanidins,19 sterols,20 triterpenoids21 andsulfur-containing compounds5 have been isolated from fruits,seeds, bark and roots of the plant.No exhaustive investigations have been made on the volatile

compounds released from plant organs of trifoliate orange. Thefirst study, dating back to 1966 and performed in California, USA,

∗ Correspondence to: FilippoMaggi, School of Pharmacy,University ofCamerino,Via Sant’Agostino 1, I-62032 Camerino, Italy. E-mail: filippo.maggi@unicam.it

a School of Science and Technology, Chemistry Division, University of Camerino,Via Sant’Agostino 1, I-62032 Camerino, Italy

b School of Pharmacy, University of Camerino, Via Sant’Agostino 1, I-62032Camerino, Italy

c School of Biosciences and Veterinary Medicine, University of Camerino, ViaPontoni 5, I-62032 Camerino, Italy

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www.soci.org F Papa et al.

reported limonene, myrcene and �-terpinene as the main volatilecomponents of the rind, linalool of the pulp, and �-terpinene,nonanol and myrcene of the leaves.22 In German samples, Hein-rich et al.23 identified 19 volatile components in the rind, amongwhich limonene, myrcene and �-phellandrene were the mostabundant, and 15 compounds in the endocarp, among which�-caryophyllene was by far the predominant one. Recently, thevolatile oil isolated from seeds gathered in Korea was found tobe characterized by sesquiterpenes such as viridiflorol (17.34%),spathulenol (14.21%), �-humulene (12.32%), �-cadinol (7.24%)and T-muurolol (6.23%) and showed inhibition against foodbornepathogens.24

In the present study we determined the differences amongthe volatile profiles of the flavedo (exocarp), pulp (endocarp)and seeds from two cultivars of trifoliate orange cultivated inthree Italian regions (Marche, Abruzzo and Sicily), using two dif-ferent approaches, i.e. traditional hydrodistillation (HD) (using aClevenger apparatus) and headspace solid phase microextrac-tion (HS-SPME), both coupled with gas chromatography/flameionization detection (GC/FID) and gas chromatography/massspectrometry (GC/MS). In addition, our study might be useful toapproach problems of rootstock identification and to providechromatographic fingerprints for use in the determination ofhybrids from crosses.

EXPERIMENTALPlant materialFully yellow fruits of P. trifoliata were collected in autumn 2012(end of November to beginning of December) at ripening fromthree Italian locations where the plant is cultivated in gardens.Samples of var. trifoliata were collected in Macerata (Marche, cen-tral Italy, 43∘ 17′ 59′′ N, 13∘ 27′ 13′′ E; Gino Cartechini privatecollection) and Sulmona (Abruzzo, central Italy, 42∘ 02′ 54′′ N,13∘ 55′ 39′′ E; Kevin Cianfaglione private collection). Samples ofvar. monstrosa (‘flying dragon’) were collected in Milazzo (Sicily,southern Italy, 38∘ 13′ 34′′ N, 15∘ 14′ 17′′ E; Vivai Torre Natalecollection). Voucher specimens were deposited in the HerbariumCamerinensis (included in the online edition of Index Herbari-orum (http://sweetgum.nybg.org/ih/) of School of Environmen-tal Sciences, University of Camerino, Italy) under the accessioncodes CAME 26176 (var. trifoliata from Sulmona), CAME 26177(var. trifoliata from Macerata) and CAME 26178 (var. monstrosafromMilazzo). Flavedo (exocarp), pulp (endocarp) and seeds weremanually separated in the laboratory, then washed, cut intosmall pieces and stored at −20 ∘C until sample preparation andchemical analyses.

HydrodistillationFlavedo, pulp and seeds (64–907 g) were cut into small piecesthat were crushed and then hydrodistilled in a Clevenger-typeapparatus using 1–3 L of deionized water for 3 h until no morevolatile oil was obtained. For seeds and pulp, n-hexane was usedas the collector solvent that was then removed under a nitrogenflow. Collected oils were dried using anhydrous sodium sulfateand stored in sealed vials protected from the light at −20 ∘CbeforeGC/FID andGC/MS analyses. The oil yield (1–3% for flavedo,0.1–0.2% for pulp, 0.02–0.04% for seeds) was estimated on a dryweight basis by calculationof thewater content prior to distillationby leaving plant material in an oven at 105 ∘C for 8 h according tothe standard procedure.25

Headspace solid phasemicroextractionFresh flavedowas homogenizedwith 10mLof liquid nitrogen for 5min in amortar and pestle to reduce it to a powder, while pulp andseeds were simply reduced to a powder with a mortar and pestle.Then 10 mg of flavedo, 100 mg of seeds and 200 mg of pulp werehermetically sealed in separate 4 mL vials with a polypropylenehole cap and a polytetrafluoroethylene/silicone septum (Supelco,Bellefonte, Palo Alto, CA, USA) and equilibrated in a thermostaticbath (RCT basic IKAMAG® safety control, IKA®-Werke GmbH &Co. KG, Staufen, Germany) using a contact thermometer (ETS-D5,IKA®-WerkeGmbH&Co. KG). Polydimethylsiloxane (PDMS, 100 μm,1 cm long) fibre and a manual SPME holder were purchased fromSupelco. The SPMEdevicewas inserted into the sealed vial byman-ually penetrating the septum and the fibre was exposed to theheadspace 1 cm above the plant material. Experimental condi-tions, after appropriate optimization, were set as follows: extrac-tion temperature, 30 ∘C; extraction time, 20min; desorption time, 3min; injector temperature, 250 ∘C (in splitlessmode). For eachplantpart investigated, SPME analysis was conducted in triplicate. Aftersampling, the SPME device was immediately inserted into the GCinjector and the fibre was thermally desorbed. Before analysis, thefibre was conditioned in the injector of the GC system accordingto the instructions provided by the manufacturer.

GC/FID and GC/MS analysesGC/FID analysis of volatile components was carried out usingan Agilent 4890D instrument (Agilent Technologies, Palo Alto,CA, USA) coupled to a flame ionization detector (FID) andequipped with an injector liner for SPME (Sigma, Milan, Italy).Compounds were separated on an HP-5 capillary column (5%phenylmethylpolysiloxane, 30 m, 0.32 mm i.d., 0.1 μm filmthickness; J&W Scientific, Folsom, CA, USA) using the follow-ing temperature programme: 5 min at 60 ∘C, subsequently 4 ∘Cmin−1 up to 220 ∘C, then 11 ∘C min−1 up to 280 ∘C, held for 15min; injector and detector temperatures, 280 ∘C (250 ∘C for SPME);carrier gas, helium at a flow rate of 1.9 mLmin−1; injection volume(for volatile oils), 1 μL; split ratio, 1:34 (splitless mode for SPME). Amixture of aliphatic hydrocarbons (C8 –C30) (Sigma) in n-hexanewas directly injected into the GC injector or loaded onto the SPMEfibre and injected using the above temperature programme inorder to calculate the retention indices of each extracted com-pound using both HD and HS-SPME. All GC/FID analyses wereconducted in triplicate. Data were collected using HP3398A GCChemStation software (Rev. A.01.01, Hewlett Packard, Palo Alto,CA, USA). Quantification of volatile components was performedaccording toMaggi et al.26 by FID peak area internal normalizationby calculating response factors (RFs) for the main chemical classesof compounds.GC/MS analysis was performed using an Agilent 6890N gas chro-

matograph (GC) coupled to a 5973N mass spectrometer (AgilentTechnologies) and equipped with an inlet liner for SPME (Sigma).Separation of volatiles was accomplished on an HP-5MS capillarycolumn (5% phenylmethylpolysiloxane, 30 m, 0.25 mm i.d., 0.1 μmfilm thickness; J&W Scientific). The GC was programmed at 60 ∘Cfor 5 min, then ramped at 4 ∘C min−1 up to 220 ∘C, then 11 ∘Cmin−1 up to 280 ∘C, held for 15 min, finally 11 ∘C min−1 up to 300∘C, held for 5 min; carrier gas, helium at a flow rate of 1 mL min−1;injector and transfer line temperatures, 280 ∘C (250 ∘C for SPME);injection volume (for volatile oils), 2 μL; split ratio, 1:50 (splitlessmode for SPME); scan time, 75 min; acquisition mass range, m/z29–400. All mass spectra were acquired in electron impact (EI)

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mode (single quadrupole) with an ionization voltage of 70 eV.Data were analyzed using MSD ChemStation software (VersionG1701DAD.01.00, Agilent, Palo Alto, CA, USA). Whenever possible,volatile compounds were identified by co-injection with authenticstandards. Otherwise, peak assignment was carried out accordingto the recommendations of the International Organization of theFlavor Industry (IOFI, http://www.iofi.org/),27 i.e. by the interactivecombination of chromatographic linear retention indices (RIs) con-sistent with those reported in the literature for apolar stationaryphases28–30 and MS data consisting of computer matching withWILEY275, NIST 08, ADAMS and FFNSC 2 libraries. In addition, ahome-made library based on analyses of reference oils and com-mercially available standards was used as well.

Principal component analysisTo reveal the relationship among the different samples and culti-vars of trifoliate orange based on headspace and volatile oil com-positions and to identify the main constituents influencing thevariability, two compositional datamatrices of nineheadspace andessential oil samples (three from flavedo, three from pulp, threefrom seeds) were analyzed by principal component analysis (PCA)using STATISTICA 7.1 (StatSoft Italia srl, 2005, www.statsoft.it). Atotal of 81 variables × 9 samples = 729 data from the headspacematrix and 146 variables × 9 samples = 1314 data from thehydrodistillate matrix were selected to be included in the multi-variate analysis. Prior to the PCA, the variables were standardizedfor a normalized PCA: missing data were substituted by 0.01%for this purpose. Eigenvalues were calculated using a covariancematrix among 81 and 146 chemical compounds respectively asinput, and a two-dimensional PCA biplot including both plantparts and compounds was generated.

RESULTSHeadspace analysisIn this work we investigated the volatile compounds of trifoliateorange by SPME, since this technique constitutes an analyticalmethod giving a realistic perspective regarding the odour offruits. All HS-SPME analyses were performed using a PDMS fibreat 30 ∘C with an extraction time of 20 min. The PDMS fibre wasused because of its good extraction efficiency and reproducibilityamong the various types of fibre previously screened in ourlaboratory. The repeatability of the method was determined byperforming three replicate analyses for each fruit part. Relativestandarddeviation (RSD) valueswerebelow20% in all cases,whichis satisfactory.The headspace volatiles of flavedo, pulp and seeds from the two

cultivars of P. trifoliata are reported in Table 1. A total of 81 volatilecompoundswere identified in all samples (74 in flavedo, 51 in pulp,27 in seeds), accounting for 92.5–99.3%of the total volatiles.Manyof them were detected at scant levels (traces or below 1%).The majority of compounds detected in the outer coloured part

of fruits (flavedo) were monoterpene hydrocarbons, represent-ing 80.7–88.6% of the total volatiles. The main representatives ofthis fraction were limonene (43.6–55.1%), myrcene (18.2–22.0%),�-pinene (2.9–7.0%) and (E)-�-ocimene (2.2–5.5%). Among theother families of volatile compounds, only sesquiterpene hydro-carbons and, to a minor extent, esters were present at noteworthylevels (5.6–7.2 and trace–8.1% respectively). Themain representa-tives of these groups were (E)-caryophyllene (3.3–4.8%) and ethylhexanoate (3.2% in fruits fromMacerata) respectively. Notably, no

differences were revealed by SPME in the flavedo headspace com-ponents of var. trifoliata and var.monstrosa.The odour of fruit pulp was given mostly by similar

levels of monoterpene and sesquiterpene hydrocarbons(33.2–43.4 and 38.7–45.0% respectively), with limonene(6.9–26.8%), (E)-�-ocimene (1.5–25.9%), myrcene (4.1–9.7%) and(E)-caryophyllene (31.6–38.4%) as the predominant compounds.Noteworthy here was the abundance of esters (2.1–16.3%) suchas butyl butanoate (5.1–6.3%), ethyl octanoate (trace–3.5%) andbutyl hexanoate (0.2–2.3%). Esters, having low odour thresholds,are significant aroma constituents of many fruits and plants. Alsoin this case, no differences in volatile patterns were detectedbetween var. trifoliata and var. monstrosa. Among the former,the sample from Sulmona exhibited a higher level of limonene(26.8%) and lower levels of (E)-�-ocimene (1.5%) and esters (2.1%)with respect to the sample from Macerata (6.9, 25.9 and 16.3%respectively). We assume that the different climatic conditionsin which the plants are growing could affect quantitatively thevolatile composition of the fruits.The headspace of seeds was represented mainly by

sesquiterpene hydrocarbons (63.2–89.9%), with germacreneD (29.3–37.6%), �-elemene (9.2–13.9%) and (E)-caryophyllene(8.1–12.3%) as the major components. Other compounds occur-ring in appreciable levels were elemane-type compounds suchas �-elemene (4.1–7.8%) and �-elemene (3.6–6.6%) resultingfrom Cope rearrangements of germacranes.31 The remainingseed volatiles comprised mainly monoterpene hydrocarbons(2.7–27.7%), with myrcene (1.7–22.9%) and limonene (0.5–3.4%)as the predominant compounds. There were no differencesbetween the volatiles of the two cultivars, but one of the samplesof var. trifoliata (Sulmona) showed higher levels of monoter-pene hydrocarbons, mainly myrcene (22.9%), and lower levelsof sesquiterpene hydrocarbons with respect to the other site ofcultivation (Macerata).

Composition of hydrodistillatesThe composition of volatile oils hydrodistilled from flavedo, pulpand seeds of the two cultivars of P. trifoliata are reported in Table 2.A total of 146 compounds were identified in all samples (78 inflavedo, 88 in pulp, 89 in seeds), accounting for 86.9–98.0% ofthe total compositions. As in the headspace, many compoundswere detected at scant levels (traces or below 1%). The oil yield ofsamples from var.monstrosa (3% for flavedo, 0.2% for pulp, 0.04%for seeds) growing in southern Italy (Sicily) was significantly higherthan that of var. trifoliata from central Italy (Marche and Abruzzo)(1.1–1.2% for flavedo, 0.1% for pulp, 0.02–0.03% for seeds).The fruit flavedo was characterized mainly by monoterpene

hydrocarbons (75.6–82.1%), with limonene (41.3–54.1%) andmyrcene (18.2–23.2%) as the major volatile components, consis-tent with those of bitter orange.32 Other compounds occurring atnoteworthy levels were �-pinene (2.2–4.8%), �-phellandrene(2.7–4.7%) and (E)-�-ocimene (0.7–4.2%). The remainingflavedo oil was given mainly by oxygenated monoterpenes(3.5–6.4%), esters (0.4–5.6%) and aromatics (1.5–3.0%), withlinalool (0.7–1.7%), trans-p-mentha-1(7),8-dien-2-ol (0.7–2.6%),butyl butanoate (0.7–3.4%) and p-cymene (0.7–2.9%) as themost representative. The occurrence of sesquiterpene hydrocar-bons (0.2–1.4%), oxygenated sesquiterpenes (trace–0.7%) andaliphatic alcohols (0.5–0.9%) was negligible. As in the flavedoheadspace, there were no significant differences in volatile oilcomposition between var. trifoliata and var.monstrosa.

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www.soci.org F Papa et al.

Table

1.Headspacevo

latiles

inflavedo

,pulpan

dseed

sof

Ponciru

strifoliata

var.trifo

liata

andvar.mon

strosa

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

1Hexan

al80

280

180

0trf

tr0.2

tr0.4

0.4

trStd

2Bu

tylacetate

815

811

811

0.2

0.6

MS,RI

3Ethy

lisovalerate

855

858

856

0.1

0.2

MS,RI

4(2Z)-Hexen

ol86

686

786

5tr

trMS,RI

5n-Hexan

ol86

987

087

0tr

trtr

MS,RI

6�-Thu

jene

932

930

930

trtr

trMS,RI

7�-Pinen

e94

093

993

90.8

1.5

1.4

0.2

0.7

0.5

1.1

0.1

trStd

8Cam

phe

ne95

595

495

4tr

0.1

trStd

9Isob

utylbutan

oate

959

958

trtr

MS,RI

10Sabinen

e97

997

597

90.5

tr0.7

tr0.6

0.1

trtr

MS,RI

11�-Pinen

e98

297

998

22.9

7.0

3.6

0.7

3.8

1.7

0.6

0.6

trStd

12Myrcene

992

990

992

22.0

19.3

18.2

4.9

9.7

4.0

1.8

22.9

1.Std

13Bu

tylb

utan

oate

995

994

993

2.3

tr6.3

5.1

MS,RI

14�-Phe

lland

rene

1003

1002

1003

1.4

1.6

4.8

trtr

0.4

Std

15Ethy

lhexan

oate

1005

998

1005

3.2

0.8

0.5

1.7

MS,RI

16�-3-Caren

e10

1310

1110

131.2

trStd

17Hexylacetate

1013

1009

1013

0.9

0.5

0.2

MS,RI

18�-Terpinen

e10

1810

1710

18tr

0.4

MS,RI

19p-Cy

men

e10

2410

2410

24tr

tr0.3

1.4

0.2

0.3

0.2

Std

211,8-Cineo

le10

3010

3110

30tr

0.3

trStd

20Limon

ene

1032

1030

1032

43.6

54.6

55.0

6.9

26.8

12.2

3.1

3.4

0.5

Std

22(Z)-�-O

cimen

e10

4010

3710

402.1

tr0.8

tr0.6

MS,RI

23Bu

tyl2-m

ethy

lbutan

oate

1045

1045

trtr

0.5

0.7

MS,RI

24Bu

tyl3-m

ethy

lbutan

oate

1052

1055

trtr

MS,RI

25(E)-�-O

cimen

e10

5310

5010

505.5

2.2

3.8

25.9

1.5

12.7

0.2

0.4

0.4

MS,RI

26�-Terpinen

e10

5810

5910

590.5

1.1

0.5

0.6

0.3

1.0

tr0.1

trStd

27Pe

ntylisob

utan

oate

1059

1055

1057

0.4

0.3

1.7

0.5

1.1

MS,RI

28Isop

entylb

utan

oate

1060

1058

1060

0.7

0.8

trtr

MS,RI

29cis-Sabinen

ehy

drate

1069

1070

1068

0.2

1.1

1.0

MS,RI

30n-Octan

ol10

7010

6810

70tr

0.8

0.3

0.4

Std

31Terpinolen

e10

8810

8810

890.3

0.3

0.4

0.2

trtr

Std

32tran

s-Sabinen

ehy

drate

1098

1098

1099

0.3

0.3

0.3

MS,RI

33Lina

lool

1100

1096

1100

0.2

0.5

0.1

0.7

1.9

0.7

0.5

Std

34tran

s-p-Men

tha-2,8-dien

-1-ol

1121

1122

1123

trtr

0.3

MS,RI

wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric (2014)

Volatile profiles of flavedo, pulp and seeds of trifoliate orange www.soci.org

Table

1.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

35(3E,5E

)-2,6-Dim

ethy

l-1,3,5,7-

octatetraene

1135

1134

0.1

trtr

0.4

tr0.2

MS,RI

36Hexan

oicacid,3-hyd

roxy-,ethy

lester

1138

1136

trMS,RI

37cis-p-Men

tha-2,8-dien

-1-ol

1140

1137

1138

trMS,RI

38tran

s-Limon

eneoxide

1144

1142

1143

trtr

tr0.6

MS,RI

392,7-Dim

ethy

l-2,6-octad

ien-4-ol

1150

1149

trtr

MS,RI

40Isob

utylhe

xano

ate

1154

1154

1148

trtr

trMS,RI

412,6-Dim

ethy

l-1,5,7-octatrien

-3-ol

1161

tr0.4

trMS

42Terpinen

-4-ol

1178

1177

1179

trtr

trtr

0.4

0.5

Std

43�-Terpineo

l11

8811

8811

89tr

0.1

trtr

0.1

tr0.1

trStd

44Bu

tylh

exan

oate

1189

1188

1188

0.5

0.4

2.3

0.2

2.1

MS,RI

45n-Hexylbutan

oate

1194

1192

1194

trtr

trMS,RI

46Ethy

loctan

oate

1199

1197

1199

0.8

tr0.4

3.4

tr3.5

MS,RI

47Decan

al12

0712

0112

07tr

trtr

trtr

0.2

0.6

0.2

0.3

MS,RI

48(Z,Z)-2,6-Dim

ethy

l-3,5,7-

octatriene

-2-ol

1208

1208

trtr

MS,RI

49Octan

olacetate

1213

1213

1213

trtr

tr0.2

0.4

0.3

MS,RI

50Citron

ellol

1229

1225

1229

trtr

Std

51Hexyl2-methy

lbutan

oate

1236

1236

trMS,RI

52(E)-2-Hexen

oicacid,butylester

1244

1243

trtr

tr0.1

tr0.2

MS,RI

53Carvo

ne12

5112

4312

53tr

trStd

54Isop

entylh

exan

oate

1252

1250

trtr

trtr

MS,RI

552-Methy

lbutylhe

xano

ate

1256

1246

trtr

trMS,RI

561-Decan

ol12

7212

6912

72tr

trtr

MS,RI

57Indo

le12

9512

9112

95tr

trtr

MS,RI

58n-Tridecan

e13

0013

0013

00tr

trtr

trtr

trtr

0.1

0.1

Std

59�-Elemen

e13

3613

380.3

trtr

0.4

0.4

0.1

6.6

3.6

6.5

MS,RI

60�-Cub

eben

e13

5413

4813

54tr

trtr

0.2

0.2

0.2

Std

61Citron

ellolacetate

1357

1352

1357

trtr

tr0.1

0.1

0.2

Std

62Nerylacetate

1368

1361

1368

trtr

trtr

trtr

Std

63�-Cop

aene

1379

1376

1379

trtr

0.3

0.3

0.3

Std

64�-Bou

rbon

ene

1392

1388

1388

trtr

tr0.4

trMS,RI

65�-Elemen

e13

9413

9013

990.4

0.1

tr0.8

0.8

0.5

7.5

4.1

7.8

MS,RI

66Bu

tyloctan

oate

1395

1392

trtr

0.1

0.1

MS,RI

67Ethy

ldecan

oate

1399

1395

1399

trtr

trtr

tr0.2

MS,RI

J Sci Food Agric (2014) © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa

www.soci.org F Papa et al.

Table

1.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

687-epi-S

esqu

ithujen

e14

0814

0514

08tr

trtr

MS,RI

69(E)-Caryo

phy

llene

1419

1419

1419

3.3

4.8

4.1

31.6

37.3

38.4

10.0

8.1

12.3

Std

70�-Ylang

ene

1420

1420

0.6

0.1

0.2

trtr

0.3

MS,RI

71�-Cop

aene

1432

1432

trtr

trMS,RI

72�-Elemen

e14

3614

36tr

trtr

1.6

1.6

0.3

13.7

9.2

13.9

MS,RI

73�-Hum

ulen

e14

5514

5414

550.4

0.7

0.4

0.3

0.8

0.8

1.4

1.6

1.8

Std

74(E)-�-Farne

sene

1460

1456

1460

0.2

0.3

0.2

1.9

2.2

2.3

1.9

0.7

2.1

Std

75Germacrene

D14

8514

8514

851.4

0.5

0.2

1.3

1.0

0.2

36.0

29.3

37.6

MS,RI

76Bicyclog

ermacrene

1502

1500

1502

0.3

tr0.1

0.1

0.1

3.0

2.1

2.5

MS,RI

77Germacrene

A15

0915

0915

09tr

trtr

0.3

0.3

0.3

0.2

0.3

MS,RI

78�-Cad

inen

e15

2315

2315

230.4

0.2

0.3

MS,RI

79Germacrene

B15

6215

6115

620.2

trtr

0.4

0.4

tr3.5

2.6

3.5

MS,RI

80Caryo

phy

llene

oxide

1583

1583

1583

trtr

tr0.2

0.2

0.1

Std

81n-Hep

tade

cane

1700

1700

1700

trtr

trStd

Totalide

ntified

(%)

97.4

98.4

99.3

97.0

96.0

94.3

93.9

92.5

93.1

Group

edcompou

nds(%

)Mon

oterpen

ehy

drocarbon

s80

.788

.188

.640

.143

.433

.27.0

27.7

2.7

Oxyge

natedmon

oterpen

es1.0

2.6

1.7

1.2

3.4

1.6

0.1

0.9

0.1

Sesquiterpen

ehy

drocarbon

s7.2

6.8

5.6

38.7

45.0

43.1

85.6

63.2

89.9

Oxyge

natedsesquiterpen

estr

trtr

0.2

0.2

0.1

Esters

8.1

tr3.0

16.3

2.1

16.3

Others

0.3

0.9

0.5

0.6

0.8

0.8

0.7

0.7

0.4

aCom

pon

entsarelistedin

orde

rofthe

irelutionfrom

anHP-5M

Scolumn.

bLine

arretentioninde

xon

HP-5M

Scolumn,experim

entally

determ

ined

usingho

molog

ousseries

ofC8–C30

alkane

s.cLine

arretentionindicesfrom

Ada

ms2

8an

dNIST08

.29

dPe

rcen

tage

values

aremeans

ofthreede

term

inations

with

RSDbelow

20%

inallcases.

eIden

tificatio

nmetho

ds:M

S,based

oncomparison

with

WILEY,A

DAMS,FFNSC

2an

dNIST08

MSda

tabases;RI,based

oncomparison

oflin

earreten

tionindiceswith

thosereportedinAda

ms,28

NIST08

29

andFFNSC

2;30

Std,based

oncomparison

with

authen

ticcompou

nds.

fTrace(m

eanvaluebelow

0.1%

).

wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric (2014)

Volatile profiles of flavedo, pulp and seeds of trifoliate orange www.soci.org

Table

2.Com

position

ofhy

drod

istillatesfrom

flavedo

,pulpan

dseed

sof

Ponciru

strifoliata

var.trifo

liata

andvar.mon

strosa

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

1Hexan

al79

980

180

01.8

1.7

0.9

Std

2Bu

tylacetate

812

811

812

trf

trMS,RI

3(E)-2-Hexen

al85

085

585

4tr

trMS,RI

4Hexan

ol86

587

086

70.2

0.1

0.1

trMS,RI

5Isop

entylalcoh

ol,acetate

875

878

trMS,RI

6�-Thu

jene

930

930

930

trtr

MS,RI

7�-Pinen

e93

793

993

90.8

1.7

1.6

0.2

trtr

Std

8�-Fen

chen

e95

095

295

1tr

trMS,RI

9Cam

phe

ne95

195

495

4tr

Std

10Isob

utylbutan

oate

959

958

trtr

0.2

MS,RI

11Bo

isde

Rose

oxide

974

972

0.2

1.3

MS,RI

12Sabinen

e97

697

597

90.3

0.5

0.4

trMS,RI

13�-Pinen

e97

897

998

22.2

4.8

3.0

tr0.8

0.2

trtr

Std

141-Octen

-3-ol

981

979

981

trtr

Std

15Myrcene

992

990

992

23.2

18.2

18.7

1.1

3.6

2.5

0.2

1.6

1.3

Std

16Bu

tylb

utan

oate

997

994

993

3.4

0.7

0.9

0.2

0.6

trMS,RI

17Hexan

oicacid,ethylester

1000

999

0.9

0.2

trMS,RI

18�-Phe

lland

rene

1004

1002

1002

2.7

0.9

4.7

tr0.3

0.3

Std

19�-3-Caren

e10

0810

1110

13tr

trStd

20Deh

ydro-cis-lina

looloxide

1009

1008

1007

0.1

0.2

0.1

MS,RI

21Hexylacetate

1016

1009

1013

0.4

0.2

0.2

MS,RI

22�-Terpinen

e10

1810

1710

180.2

0.3

0.9

tr0.1

trStd

23p-Cy

men

e10

2410

2410

250.7

2.9

1.1

tr1.7

0.3

trtr

Std

24Limon

ene

1033

1030

1032

41.3

54.1

45.3

1.1

9.6

3.5

tr0.6

0.4

Std

25(Z)-�-O

cimen

e10

4210

3710

400.1

0.2

0.3

0.2

0.6

trtr

0.10

MS,RI

26Be

nzen

eacetalde

hyde

1046

1042

1046

0.1

0.1

MS,RI

27Bu

tyl2-m

ethy

lbutan

oate

1046

1045

0.2

0.1

0.1

0.1

0.2

trMS,RI

28(E)-�-O

cimen

e10

5210

5010

504.2

0.7

3.9

7.4

1.5

4.8

0.3

0.1

0.4

MS,RI

29�-Terpinen

e10

5810

5910

590.5

0.4

0.6

0.2

trtr

Std

30Isop

entylb

utyrate

1059

1058

1061

0.2

MS,RI

312-Methy

lbutylbutyrate

1060

1058

0.2

MS,RI

32n-Octan

ol10

7110

6810

700.1

0.2

trMS,RI

33cis-Lina

looloxide

1075

1072

1073

tr0.1

0.1

0.8

trtr

trMS,RI

34Terpinolen

e10

8910

8810

890.2

0.3

0.5

0.1

0.8

trtr

Std

35tran

s-Lina

looloxide

1089

1086

1091

0.2

trMS,RI

362-Non

anon

e10

9310

9010

910.1

MS,RI

J Sci Food Agric (2014) © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa

www.soci.org F Papa et al.

Table

2.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

37Lina

lool

1099

1096

1100

0.7

0.3

1.7

19.2

5.8

26.5

1.3

1.5

1.3

Std

38cis-Ve

rben

ylacetate

1101

1282

0.6

MS,RI

39n-Non

anal

1104

1100

1.2

trtr

MS,RI

403,7-Dim

ethy

l-1,5,7-octatrien

-3-ol

1104

1105

0.2

0.4

0.3

MS,RI

41cis-Ro

seoxide

1111

1108

trtr

MS,RI

42p-Men

tha-1,3,8-triene

1111

1110

1111

tr0.1

MS,RI

43endo

-Fen

chol

1113

1116

1112

trtr

MS,RI

44tran

s-Ro

seoxide

1121

1125

1127

0.2

MS,RI

45tran

s-p-Men

tha-2,8-dien

-1-ol

1121

1122

0.2

MS,RI

46cis-p-Men

th-2-en-1-ol

1122

1121

1121

0.3

MS,RI

47Myrceno

l11

2211

2211

230.5

0.8

MS,RI

48(E,E)-2,6-Dim

ethy

l-1,3,5,7-octatetraen

e11

3211

340.5

0.7

0.3

MS,RI

49cis-p-Men

tha-2,8-dien

-1-ol

1137

1137

0.1

trtr

0.2

trMS,RI

50tran

s-Pino

carveo

l11

3711

3911

370.2

0.2

0.3

tr0.1

MS,RI

51tran

s-p-Men

th-2-en-1-ol

1141

1140

1140

0.7

trMS,RI

52cis-�-Terpineo

l11

4711

4411

45tr

MS,RI

53Ipsdieno

l11

5011

4511

50tr

tr0.5

0.2

-MS,RI

542,6-Dim

ethy

l-5,7-octad

ien-2-ol

1155

1150

0.2

0.5

MS,RI

55Neroloxide

1157

1158

1155

trMS,RI

56Bo

rneo

l11

6711

6911

680.3

trStd

57tran

s-�-Terpineo

l11

6911

630.2

trtr

0.5

trMS,RI

58Terpinen

-4-ol

1178

1177

1179

trtr

tr0.2

1.2

0.5

0.5

trStd

59tran

s-Isocarveol

1187

1185

0.3

trMS,RI

60tran

s-p-Men

tha-1(7),8-dien-2-ol

1187

1189

0.7

1.6

2.6

0.5

trMS,RI

61�-Terpineo

l11

9011

8811

890.8

0.3

0.4

1.8

1.3

5.3

0.2

0.6

0.4

Std

62Bu

tylh

exan

oate

1192

1188

1189

tr1.1

0.2

0.4

trMS,RI

63Ethy

loctan

oate

1198

1197

1199

1.7

0.2

1.0

tr0.2

MS,RI

64n-Decan

al12

0512

0111

981.0

0.2

0.8

MS,RI

65Octan

olacetate

1213

1213

1213

0.2

trtr

0.4

trMS,RI

66p-Men

th-1-en-9-al

1217

1217

0.2

0.3

0.2

0.1

trMS,RI

67tran

s-Carveol

1219

1216

1219

trtr

trtr

0.1

MS,RI

68Citron

ellol

1230

1225

1229

0.5

tr0.3

trStd

69(Z)-Nerol

1230

1229

1228

0.4

0.7

0.3

0.7

0.5

1.0

MS,RI

70Bu

tyl(2E

)-2-he

xeno

ate

1244

1243

trtr

tr0.2

trMS,RI

71Carvo

ne12

4512

4312

530.2

0.4

Std

wileyonlinelibrary.com/jsfa © 2014 Society of Chemical Industry J Sci Food Agric (2014)

Volatile profiles of flavedo, pulp and seeds of trifoliate orange www.soci.org

Table

2.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

72Geran

iol

1257

1252

1256

0.1

tr0.2

4.6

1.3

5.5

0.6

0.4

0.5

Std

73Isog

eran

iol

1272

1273

0.1

0.1

MS,RI

74Ph

elland

ral

1274

1273

1273

0.2

tr0.6

0.4

MS,RI

75n-Decan

ol12

7612

6912

79tr

tr0.7

2.0

0.1

1.3

0.3

0.7

MS,RI

76n-Tridecan

e13

0013

0013

00tr

trtr

trStd

77p-Viny

lgua

iacol

1313

1309

1313

0.1

0.1

MS,RI

78(2E,4E

)-Decad

iena

l13

1613

1613

090.3

0.3

0.6

0.1

MS,RI

79�-Elemen

e13

4013

38tr

0.3

0.5

tr4.4

0.2

5.0

MS,RI

80�-Cub

eben

e13

5213

4813

50tr

trStd

81tran

s-Cad

ina-1,4-dien

e15

3515

3415

35tr

MS,RI

82Citron

ellolacetate

1356

1352

1357

0.1

tr0.1

0.2

0.3

trMS,RI

83�-Ylang

ene

1366

1375

1367

tr0.2

MS,RI

84Nerylacetate

1367

1361

1368

0.2

tr0.4

0.4

0.2

MS,RI

85�-Cop

aene

1377

1376

trtr

trMS,RI

86�-Bou

rbon

ene

1382

1388

trMS,RI

87�-Elemen

e13

8913

9013

990.2

tr0.8

0.9

0.2

5.4

3.4

6.1

MS,RI

88�-Cub

eben

e13

8813

8813

88tr

MS,RI

89Bu

tyloctan

oate

1393

1392

tr0.9

0.3

trMS,RI

90Ethy

ldecan

oate

1396

1395

1399

tr0.3

0.2

MS,RI

91�-cis-Berga

moten

e14

0914

120.3

0.2

0.6

MS,RI

92Eu

geno

lmethy

lether

1409

1405

0.1

0.1

MS,RI

93(Z)-Caryo

phy

llene

1411

1408

1411

0.1

trMS,RI

94(E)-Caryo

phy

llene

1422

1419

1422

33.3

32.9

22.1

7.8

8.4

6.8

Std

95�-Cop

aene

1431

1432

0.2

0.2

0.2

MS,RI

96�-Elemen

e14

3614

360.7

1.0

0.2

6.8

5.2

7.0

MS,RI

97�-Gua

iene

1441

1439

1440

tr0.1

trMS,RI

986,9-Gua

iadien

e14

4714

440.1

0.1

0.1

MS,RI

99�-Hum

ulen

e14

5714

5414

580.4

0.2

0.3

2.7

2.6

1.7

2.0

2.4

2.8

Std

100

(E)-�-Farne

sene

1459

1456

1460

tr0.7

0.4

Std

101

Dod

ecan

ol14

7514

7014

740.3

MS,RI

102

Germacrene

D14

8414

8514

911.4

1.2

0.2

29.7

21.0

32.1

MS,RI

103

Erem

ophilene

1489

1489

1489

0.2

0.3

0.1

MS,RI

104

�-Selinen

e14

9314

9214

940.3

0.3

0.2

MS,RI

105

Isoe

ugen

olmethy

lether

1495

1495

1.3

0.4

MS,RI

106

Bicyclog

ermacrene

1498

1500

1500

0.5

0.2

0.4

0.2

0.3

2.4

2.2

2.5

MS,RI

107

Germacrene

A15

0715

0915

070.5

1.0

0.5

MS,RI

J Sci Food Agric (2014) © 2014 Society of Chemical Industry wileyonlinelibrary.com/jsfa

www.soci.org F Papa et al.

Table

2.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

108

�-Cad

inen

e15

0915

130.5

0.5

0.4

MS,RI

109

(E,E)-�-Farne

sene

1510

1505

1510

0.1

tr0.8

0.4

0.2

0.2

trtr

MS,RI

110

�-Cad

inen

e15

2315

230.3

0.31

1.1

2.0

0.7

MS,RI

111

�-Sesqu

iphe

lland

rene

1525

1522

1525

trMS,RI

112

�-Cad

inen

e15

4115

380.14

0.5

0.5

0.2

MS,RI

113

Selin

a-3,7(11

)-dien

e15

4515

460.3

0.3

TrMS,RI

114

Germacrene

B15

6115

6115

621.9

2.6

0.5

15.2

11.3

15.7

MS,RI

115

Elem

icin

1562

1557

1561

0.2

0.2

trMS,RI

116

(E)-Nerolidol

1564

1563

1565

0.3

1.3

0.3

0.1

0.2

trMS,RI

117

Spathu

leno

l15

8015

7815

78tr

0.2

trMS,RI

118

Caryo

phy

llene

oxide

1585

1583

1583

0.2

tr0.1

0.8

2.1

0.2

0.6

0.8

0.5

Std

119

Globulol

1591

1590

1590

0.2

0.3

0.1

MS,RI

120

Epiglobulol

1595

1589

tr0.2

0.2

0.1

0.8

0.7

0.3

MS,RI

121

Dod

ecan

oicacid,ethylester

1602

1597

0.1

tr0.6

0.3

0.4

MS,RI

122

Rosifolio

l16

0416

000.2

0.2

trMS,RI

123

Hum

ulen

eep

oxideII

1612

1608

1611

0.1

0.1

0.2

MS,RI

124

June

nol

1622

1619

1625

1.2

1.3

0.5

MS,RI

125

Selin

a-6-en

-4-ol

1629

1624

0.4

0.4

0.1

MS,RI

126

epi-�

-Muu

rolol

1643

1642

1.2

1.7

0.6

MS,RI

127

�-M

uurolol

1648

1646

1648

0.3

0.3

0.4

MS,RI

128

�-Cad

inol

1654

1654

2.6

3.4

1.3

MS,RI

129

Isoe

lemicin

1669

1568

1670

0.3

trtr

1.2

0.8

3.8

MS,RI

130

(E)-Asarone

1678

1676

1678

0.8

0.1

0.5

0.3

0.3

0.1

MS,RI

131

Eude

sm-7(11)-en-4-ol

1700

1699

1699

0.5

0.1

0.5

0.3

0.1

1.1

1.1

0.4

MS,RI

132

(2Z,6E)-Farne

sol

1724

1723

trMS,RI

133

(2E,6E

)-Farnesylacetate

1844

1846

1845

0.1

trtr

MS,RI

134

2-Hep

tade

cano

ne19

0219

010.1

0.1

0.1

MS,RI

135

Methy

lhexad

ecan

oate

1926

1921

1926

0.2

MS,RI

136

Hexad

ecan

oicacid

1962

1960

1962

0.2

0.4

1.5

4.3

4.8

MS,RI

137

Ethy

lpalmita

te19

9519

960.14

MS,RI

138

(Z)-9-Octad

ecen

al19

9519

950.3

0.6

0.7

MS,RI

139

Hexad

ecan

oicacid,ethylester

1995

1993

1997

tr0.2

0.1

MS,RI

140

Lino

leicacid,m

ethy

lester

2096

2085

2092

0.2

tr0.1

1.8

MS,RI

141

Methy

l-(9Z

,12Z

,15Z

)-oc

tade

catrieno

ate

2110

2108

0.7

MS,RI

142

Lino

leicacid

2140

2133

2140

1.5

Std

143

Lino

leicacid,ethylester

2161

2162

trtr

trtr

0.3

0.5

4.1

MS,RI

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Volatile profiles of flavedo, pulp and seeds of trifoliate orange www.soci.org

Table

2.Co

ntinued

Flaved

o(%

)dPu

lp(%

)dSe

eds(%

)d

RIlit.c

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

Trifo

liata

Mon

strosa

No.

Com

pon

enta

RIb

Ada

ms

NIST08

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

Macerata

Sulm

ona

Milazzo

IDe

144

Ethy

lstearate

2196

2195

0.4

tr0.2

trtr

MS,RI

145

n-Tricosan

e23

0023

0023

00tr

0.1

0.2

trtr

trStd

146

n-Tetracosan

e24

0024

0024

000.50

trtr

tr0.6

trStd

Totalide

ntified

(%)

91.9

94.3

93.2

93.0

86.9

93.3

95.3

93.0

98.0

Oilyield(%

)1.1

1.2

3.0

0.1

0.1

0.2

0.02

0.03

0.04

Group

edcompou

nds(%

)Mon

oterpen

ehy

drocarbon

s75

.682

.179

.910

.116

.613

.00.6

2.3

2.3

Oxyge

natedmon

oterpen

es3.5

4.5

6.4

27.3

12.8

40.9

2.2

4.2

2.3

Sesquiterpen

ehy

drocarbon

s1.4

0.2

1.0

42.2

43.2

25.8

78.0

59.7

81.3

Oxyge

natedsesquiterpen

es0.7

tr0.3

1.5

2.6

0.4

8.7

10.5

4.5

Alip

hatic

alcoho

ls0.5

0.9

0.6

2.7

4.8

3.4

1.4

0.8

0.8

Esters

5.6

0.4

2.0

6.0

1.9

4.0

1.1

8.5

0.1

Aromatics

2.2

3.0

1.5

2.3

2.7

4.7

0.6

0.8

0.4

Others

2.3

3.2

1.5

1.1

2.3

1.1

2.8

6.2

6.4

aCom

pon

entsarelistedin

orde

rofthe

irelutionfrom

anHP-5M

Scolumn.

bLine

arretentioninde

xon

HP-5M

Scolumn,experim

entally

determ

ined

usingho

molog

ousseries

ofC8–C30

alkane

s.cLine

arretentionindicesfrom

Ada

ms2

8an

dNIST08

.29

dPe

rcen

tage

values

aremeans

ofthreede

term

inations

with

RSDbelow

20%

inallcases.

eIden

tificatio

nmetho

ds:M

S,based

oncomparison

with

WILEY,A

DAMS,FFNSC

2an

dNIST08

MSda

tabases;RI,based

oncomparison

oflin

earreten

tionindiceswith

thosereportedinAda

ms,28

NIST08

29

andFFNSC

2;30

Std,based

oncomparison

with

authen

ticcompou

nds.

fTrace(m

eanvaluebelow

0.1%

).

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www.soci.org F Papa et al.

(a) (b)

(c) (d)

Figure 1. (a) PCA score plot for main variation in headspace volatiles among trifoliate orange samples. (b) PCA loading plot for headspace constituentsexplaining 56.50% of variation on horizontal axis (PC 1) and 24.25% on vertical axis (PC 2). (c) PCA score plot for main variation in essential oil componentsamong trifoliate orange samples. (d) PCA loading plot for essential oil constituents explaining 52.24% of variation on horizontal axis (PC 1) and 27.55% onvertical axis (PC 2).

The pulp hydrodistillate showed sesquiterpene hydrocarbons(25.8–43.2%) and oxygenatedmonoterpenes (12.8–40.9%) as theprevalent chemical classes, with (E)-caryophyllene (22.1–33.3%)and linalool (5.8–26.5%) as the predominant volatile compounds.The remaining oil was given mainly by monoterpene hydro-carbons (10.1–13.0%) and esters (4.0–6.0%), with limonene(1.1–9.6%), (E)-�-ocimene (1.5–7.4%), myrcene (1.1–2.5%), butylhexanoate (0.2–1.1%) and ethyl octanoate (0.2–1.7%) as themostrepresentative. A slight difference, more quantitative than qualita-tive, was shown by var. monstrosa, which contained higher levelsof oxygenated monoterpenes and lower levels of sesquiterpenehydrocarbons.The oil hydrodistilled from seeds showed a volatile pattern sim-

ilar to that of the headspace, being dominated by sesquiterpenehydrocarbons (59.7–81.3%) such as germacrene D (21.0–32.1%),germacrene B (11.3–15.7%) and (E)-caryophyllene (6.8–8.4%).Also in this case, elemane-type compounds such as �-elemene

(3.4–6.1%), �-elemene (5.2–7.0%) and �-elemene (0.2–5.0%)weredetected in appreciable amounts. The remaining oil was mainlyconstituted by oxygenated sesquiterpenes (4.5–10.5%) and esters(0.1–8.5%), with �-cadinol (1.3–3.4%), epi-�-muurolol (0.6–1.7%)and ethyl andmethyl esters of linoleic acid (0.5–4.1 and 0.1–1.8%respectively) as the most representative. As for the headspace,there were no significant differences in volatile oil compositionbetween the two cultivars, but one sample of var. trifoliata (Sul-mona) showed a higher level of esters (8.5 vs 1.1%) and a lowerlevel of sesquiterpene hydrocarbons (59.7 vs 78.0%) with respectto the sample from the other collection site (Macerata).

Multivariate analysisMultivariate analysis of headspace and volatile oil constituentsshowed that all trifoliate orange fruit parts are well separated fromeach other. The 2D graphical representations of PCA are shownin Fig. 1 and represent 80.75 and 79.79% respectively of the total

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Volatile profiles of flavedo, pulp and seeds of trifoliate orange www.soci.org

0

10

20

30

40

50

60

70

80

90%

HS-SPME

Flavedo Pulp

HS-SPMEHD HS-SPME HD HD

Seeds

MH MO SH SO EST ALC ARO

Figure 2. Percentages of main chemical groups extracted by HS-SPME andHD:MH,monoterpene hydrocarbons;MO, oxygenated sesquiterpenes; SH,sesquiterpene hydrocarbons; SO, oxygenated sesquiterpenes; EST, esters;ALC, aliphatic alcohols; ARO, aromatics. Data refer to Poncirus trifoliata var.trifoliata samples from Macerata and are means of three determinationswith RSD below 20% in all cases.

variance in the data set. In both cases the variability of data wasgenerated mostly by the content of limonene in the first PC andby sesquiterpenes such as germacrene D and (E)-caryophyllene inthe second PC. Samples on the upper left-hand side of the PCAscore plots (blue colour, Figs 1(a) and 1(c)) were those coming fromthe flavedo (peel); they were positively correlated with compo-nents in the same position of the loading plots (Figs 1(b) and 1(d))that mostly contributed to the total variance, such as limoneneand, to a lesser extent, myrcene. Samples from seeds were on thelower right-hand side of the score plots (yellow colour, Figs 1(a)and 1(c)), being more influenced by the content of sesquiterpenehydrocarbons such as germacrene D and (E)-caryophyllene. Sam-ples on the central part of the headspace score plot (pink colour,Fig. 1(a)) and on the right-hand side of the volatile oil score plot(pink colour, Fig. 1(c)) were those coming from the fruit pulp,whichshowed an intermediate composition between flavedo and seeds.Notably, the headspace of the pulp was a little more character-ized bymonoterpenes with respect to the hydrodistillate. Interest-ingly, no significant differences in both headspace and hydrodistil-late compositions between var. trifoliata and var. monstrosa wereobserved, since the samples of the latter (i.e. those with abbrevia-tions Mi) were strongly correlated with the samples of the former(Figs 1(a) and 1(c)).

DISCUSSIONLimited comparison can be done with previous work conductedon volatile compounds of flavedo and pulp of P. trifoliata owingto different analytical conditions employed. In the work of Scoraet al.,22 only 25 and 28 compounds were identified in the rindand pulp respectively. Limonene (40.7%), myrcene (20.4%) and�-terpinene (13.5%) were themajor volatile compounds of the for-mer, while linalool (13.0%) was the major compound of the latter.Notably, the authors did not detect sesquiterpene compounds. Onthe other hand, Heinrich et al.23 identified 19 volatile componentsin the rind, among which limonene, myrcene and �-phellandrenewere the most abundant, and 15 compounds in the pulp, with

�-caryophyllene as the major compound. Our analysis showeda higher number of detected peaks (78 in flavedo, 88 in pulp),thanks to the modern tools available for identification (i.e. inter-active combination of RI and MS matching with those of four dif-ferent libraries), and significant differences in the chemical pro-file of the pulp. Finally, the chemical profile of seeds was signif-icantly different with respect to that of Korean samples.24 In thelatter, only 36 compounds were detected (compared with 89 inour study), with oxygenated sesquiterpenes such as veridiflorol(17.34%), spathulenol (14.21%) and �-cadinol (7.24%) giving themajor contribution. In the Italian samples, this groupwas little rep-resented (4.5–8.5%). From our study, we can conclude that geo-graphical factors influence the volatile composition of pulp andseeds more than that of flavedo.As reported in Tables 1 and 2, HDpermitted us to obtain a higher

number of volatiles (146 vs 81) with respect to SPME. This canbe due to the high selectivity of PDMS fibre towards low-boilingpoint compounds and to the hydrolytic and oxidative processesoccurring with HD, leading to the formation of artefact products.As shown in Fig. 2, the behaviour of the main chemical groups

extracted by HS-SPME andHD fromdifferent fruit parts of trifoliateorange (var. trifoliata, sample from Macerata) was fairly constantfor flavedo and seeds, while some differences were noted for pulp.Monoterpene and sesquiterpene hydrocarbons were detected atsimilar levels in both headspaces and hydrodistillates (80.7 and75.6% and 85.6 and 78.0% respectively) of flavedo and seeds. Adifferent situation was observed for pulp, where HD extractedmore oxygenated compounds (monoterpenes, e.g. linalool andgeraniol, sesquiterpenes and aliphatics) with respect to SPME. Onthe other hand, esters, being quite unstable compounds, showedgreat variation between the two extraction techniques: a signif-icant decrease in their content was observed in the hydrodis-tillates of flavedo and pulp compared with the correspondingheadspaces (5.6 vs 8.1% and 6.2 vs 16.3% respectively). By com-parison of the two extraction methods, it is evident that monoter-pene hydrocarbons were extracted in slightly higher quantities bySPME with respect to HD (81.7, 41.3 and 7.1% vs 79.1, 37.4 and2.8% for flavedo, pulp and seeds respectively), while oxygenatedcompounds (i.e. monoterpenoids, sesquiterpenoids and aliphat-ics), some of them being formed during hydrolytic and oxidativeprocesses, were extracted more efficiently by HD.

CONCLUSIONSThe study conducted gave an insight on the volatile fractionof trifoliate orange fruit. Eighty-one and 146 different aromaticcompounds were detected in different fruit parts using HS-SPMEand HD, respectively. HD permitted us to extract more oxy-genated compounds such as monoterpenoids, sesquiterpenoidsand aliphaticswith respect to SPME.On theother hand,with SPME,more monoterpene hydrocarbons and esters were obtained. Bothtechniques permitted the differentiation of trifoliate orange fruitparts based on the main volatile components, showing similarchromatographic behaviour: the flavedo was characterized bymonoterpenes, the seeds were characterized by sesquiterpenes,while the pulp was intermediate, being rich in both. This groupingwas confirmed by multivariate analysis. Giving a realistic perspec-tive of fruit odour, SPME could be considered an analyticalmethodfor successful application in the analysis of trifoliate orange aroma,since it has the advantage of being a non-time-consuming, wastematerial- and solvent-free and non-artefact-producing technique.Finally, this study did not highlight significant differences in

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www.soci.org F Papa et al.

volatiles between the twocultivars ofP. trifoliatagrown in Italy. Fur-ther research will be focused on the detection of other secondarymetabolites suitable for use as marker compounds of these culti-vars.

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