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A new HPLC method developed for the analysis ofoxygen heterocyclic compounds in Citrus essential oilsMarina Russo a , Germana Torre a , Caterina Carnovale a , Ivana Bonaccorsi a , Luigi Mondelloa b & Paola Dugo a ba Dipartimento Farmaco-chimico, University of Messina, Messina, Italyb University Campus Bio-Medico, Rome, Italy

Available online: 20 Mar 2012

To cite this article: Marina Russo, Germana Torre, Caterina Carnovale, Ivana Bonaccorsi, Luigi Mondello & Paola Dugo (2012):A new HPLC method developed for the analysis of oxygen heterocyclic compounds in Citrus essential oils, Journal of EssentialOil Research, 24:2, 119-129

To link to this article: http://dx.doi.org/10.1080/10412905.2012.659523

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A new HPLC method developed for the analysis of oxygen heterocyclic compounds in Citrusessential oils

Marina Russoa, Germana Torrea, Caterina Carnovalea, Ivana Bonaccorsia*, Luigi Mondelloa,b and Paola Dugoa,b

aDipartimento Farmaco-chimico, University of Messina, Messina, Italy; bUniversity Campus Bio-Medico, Rome, Italy

(Received 7 November 11; final form 25 November 2011)

In 2009, a temporary crisis in the world production of acetonitrile caused the abnormal increase of the price of thissubstance, commonly used as a solvent and in high-pressure liquid chromatography (HPLC) separation as a mobilephase. For this reason, a new HPLC method was developed to analyze the oxygen heterocyclic components of Citrusoils, substituting acetonitrile with methanol. The new method described in this article was validated, new componentswere isolated and characterized by nuclear magnetic resonance (NMR), and others were synthesized, to obtain purestandards, not commercially available. The results are evaluated in comparison with methods previously developed.Seven genuine cold-pressed Citrus essential oils – lemon (C. limon (L.) Burm.), Key lime (C. aurantifolia (Christm.)Swing.) type B, bergamot (C. bergamia), grapefruit (C. paradisi Macf.), bitter orange (C. aurantium L.), sweetorange (C. sinensis (L.) Osbeck) and mandarin (C. deliciosa Ten.) – were analyzed under identical conditions. In con-clusion, it was observed that the replacement of acetonitrile with methanol in the mobile phase and the increase ofthe column temperature (from 30 to 40°C) led to a better separation in terms of resolution and total analysis time.Limit of detection (LOD) and limit of quantification (LOQ) were comparable with methods previously developedwith acetonitrile in the mobile phase.

Keywords: Citrus oils; Coumarins; psoralens; polymethoxyflavones; HPLC method development; synthesis

Introduction

Citrus essential oils and their derived products are widelyused in food, cosmetics, flavor and fragrance industry,for the preparation of soft drinks, alcoholic beverages,ice creams, cookies, candies, air freshness, pharmaceuti-cal preparations, cleaning products as well as colognes,fine perfumes and other cosmetic products (1,2).

Cold-pressed Citrus essential oils consist of a vola-tile fraction, which represents 85–99% of the oil, and anon-volatile fraction 1–15%, which mainly containsoxygen heterocyclic compounds, especially coumarins,psoralens and polymethoxyflavones (3). These compo-nents play an important role in the characterization ofcold-pressed Citrus oils, since the composition of thisfraction is characteristic of each oil. Moreover, many ofthe pharmacological and toxicological activities pos-sessed by Citrus oils have been demonstrated to berelated to these components (4).

Because of their non-volatile nature, the mostsuitable analytical approach for oxygen heterocycliccompounds is high-pressure liquid chromatography(HPLC), both in normal (NP) and in reverse phase (RP)(5–8). The first chromatographic separations were carriedout on silica columns (NP), and different methods weredeveloped and optimized for each Citrus oil (6). Later,with the improvement of reversed phase HPLC columns

and the introduction of reduced particle size as a packingmaterial, new methods were developed on conventionaland fast RP-HPLC (7) for several Citrus oils.

Because of their common chromophore the optimaldetection of oxygen heterocyclic compounds is ultravi-olet (UV) at 315 nm. Thus UV and photodiode array(PDA) detectors are the most employed for these classof components. The successful introduction of modernHPLC systems coupled to mass spectrometry (HPLC-MS) allowed the identification of Citrus non-volatilefraction new minor components (8–10).

Recently, thanks to the more innovative chromato-graphic techniques now available, optimal separationswere achieved using comprehensive liquid chromatog-raphy (LC; LC × LC) (11).

A few years ago, a fast and efficient RP-HPLC-photodiode array detector (DAD) method for theanalysis of oxygen heterocyclic compounds in Citrusessential oils was proposed by Dugo et al. (12). In thisarticle, a C18 HPLC column packed with partially por-ous particles of 2.7 μm and, a linear gradient usingmixtures of water/acetonitrile/tetrahyrofuran (85:10:5)and of acetonitrile/methanol/tetrahydrofuran (65:30:5)as a mobile phase was used. This method allowed abetter separation of all the oxygen heterocycliccompounds of the Citrus essential oils, compared with

*Corresponding author. Email: ivabonaccorsi@unime.it

The Journal of Essential Oil ResearchVol. 24, No. 2, April 2012, 119–129

ISSN 1041-2905 print/ISSN 2163-8152 online� 2012 Taylor & Francishttp://dx.doi.org/10.1080/10412905.2012.659523http://www.tandfonline.com

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the methods previously employed. Hence, the use ofdifferent eluents did not appear to be necessary in orderto improve the separation of the compounds of interest.Shortly after, because of a temporary crisis in the pro-duction of acetonitrile in the year 2009, it became nec-essary to replace acetonitrile with another solvent thathad the same characteristics of viscosity and polarity.Methanol, which shows similar solvent strength andeluotropic values to acetonitrile, seemed a suitablechoice. Furthermore, the cost of methanol is less thanhalf that of acetonitrile.

The present article reports the work developed toset up a new analytical method that uses methanolinstead of acetonitrile in the mobile phase, keeping thegradient program and column previously used (12)unchanged. All the isolated compounds have been iden-tified by nuclear magnetic resonance (NMR) spectros-copy, and the most relevant data are reported under theExperimental section.

Experimental

Samples

This research was carried out on seven genuine cold-pressed Citrus essential oils: lemon (C. limon (L.)Burm.), Key lime (C. aurantifolia (Christm.) Swing.)type B, bergamot (C. bergamia), grapefruit (C. paradisiMacf.), bitter orange (C. aurantium L.), sweet orange(C. sinensis (L.) Osbeck) and mandarin (C. deliciosaTen.). Samples were analyzed without any pretreatment:c. 100 mg of oil was accurately weighed and diluted in6 mL of ethanol (lemon, lime and bergamot oils) or 5mL of ethanol (grapefruit, bitter orange, sweet orangeand mandarin oils). Before HPLC analysis, 50 μL ofinternal standard coumarin (64.8 mg in 50 mL of etha-nol) was added to 1 mL of the solution of the oil. Eachoil was analyzed in triplicate.

Reagents and materials

Tetrahydrofuran (THF) and cyclohexane were pur-chased from Carlo Erba Reagenti (Milan, Italy), water,methanol, n-hexane, dichloromethane (DCM), ethylacetate and ethanol were purchased from Sigma-Aldrich(Milan, Italy). All solvents were HPLC-grade. Phloro-glucinol, 3,3-dimethylallyl-bromide, ethylpropiolate,potassium carbonate anhydrous and iodomethane werepurchased from Fluka (Milan, Italy); granular zinc, 3-chloroperoxybenxoic acid, geranyl bromide and zincchloride P98% were purchased from Sigma-Aldrich.Coumarin was purchased from Fluka; umbelliferon,herniarin and isobergapten were purchased from Extra-synthese (Genay, France). Byakangelicol, psoralen,byakangelicin, cnidilin, 8-geranyloxy-psoralen andphellopterin were purchased from Herboreal Ltd (Edin-

burgh, UK). Bergapten and 8-methoxy-psoralen werepurchased from Sigma-Aldrich.

Isopimpinellin, bergamottin, 5-isopentenyloxy-7-methoxy-coumarin and 5-geranyloxy-7-methoxy-couma-rin were previously isolated in our laboratory (12,13).Meranzin and osthol were obtained by medium-pressureliquid chromatography (MPLC) separation from grape-fruit oil residue; tangeretin, nobiletin and heptameth-oxyflavone were obtained by MPLC and preparativeHPLC separation from sweet orange oil residue.

Cnidicin and imperatorin were synthesized startingfrom 8-methoxy-psoralen; isoimpertorin, oxypeucedaninand oxypeucedanin hydrate were synthesized startingfrom bergapten; epoxybergamottin was synthesizedstarting from bergamottin; aurapten and epoxyauraptenwere synthesized starting from umbelliferon; citroptenwas synthesized starting from phloroglucinol (Figure 1).

Isolation of meranzin and osthol

Non-volatile residue (13 g) obtained by evaporatinggrapefruit essential oil on a water bath for 6 hours wassubjected to MPLC on a silica gel column (5 g) using,as eluent, a mixture of n-hexane/ethyl acetate (from 9:1to 5:5); the fractions were monitored by thin layerchromatography (TLC), (cyclohexane/ethyl acetate 6:4)and then collected into six main groups (A, B, C, D, E,F). Each fraction was re-separated by MPLC on a silicagel column (1 g) using, as eluent, a mixture of n-hex-ane/ethyl acetate (from 8:2 to 6:4) and the fractionswere monitored by TLC. From D and E fractions, mer-anzin (45 mg) and osthol (15 mg) were isolated, whosestructures were confirmed by 1H and 13C NMR data.

Isolation of tangeretin, nobiletin and heptamethoxy-flavone

Non-volatile residue (12g) obtained by evaporatingmandarin essential oil on a water bath for 5 hours wassubjected to MPLC on a silica gel column (5 g) using,as eluent, a mixture of DCM/ethyl acetate (from 8:2 to5:5); the fractions were monitored by TLC (DCM/ethylacetate 6:4) and then collected into five main groups(A, B, C, D, E). Each fraction was separated by pre-parative RP-HPLC using a Discovery® HS C18 col-umn (25 cm × 10 mm, 5 μm) (Supelco, Bellefonte, PA)under isocratic mode using water/methanol/THF(55:40:5) as the mobile phase. The collected fractionswere evaporated by a Genevac EZ-2 evaporator toobtain compounds tangeretin (20 mg), nobiletin (20mg) and heptamethoxyflavone (10 mg), whose struc-tures were confirmed by 1H and 13C NMR data.

Synthesis of isoimperatorin (21) Oxypeucedanin (16)and Oxypeucedanin hydrate (6)

Isoimperatorin (21) and oxypeucedanin (16) were pre-pared according to literature procedure (14), slightly

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modified. In particular, BBr3 (7 mL, 1 M in DCM, 7mmol) was slowly added to a solution of bergapten(0.125 g, 0.7 mmol) in DCM (10 mL) and then thesolution was stirred under nitrogen at room tempera-ture. After 2 hours, the mixture was added slowly to asolution of saturated sodium bicarbonate (35 mL)obtaining a grey solid; after 30 minutes, the productwas recovered by filtration, washed with cold waterand ethyl acetate, and dried under a high vacuum,yielding 83% of bergaptol (0.120 g, 0.6 mmol).

To a solution of bergaptol (0.120 g, 0.6 mmol) andpotassium carbonate (0.100 g) in acetone (10 mL), 3,3-dimethylallyl bromide (160 μL, 1.4 mmol) was addeddropwise and the mixture was heated under reflux for 1hour. Aqueous chloridric acid (5% w/v) was added andthe solution was extracted with DCM (2 × 6 mL); thecombined organic layers were washed with water andbrine, dried (Na2SO4), then the solvent was evaporatedunder reduced pressure to afford a pale yellow oil,which was purified by column chromatography elutingwith ethyl acetate/cyclohexane (1/4). The resultingtranslucent pale yellow oil was crystallized while cool-ing with n-hexane to recover isoimperatorin (21) aswhite crystals in 73% yield (0.110 g, 0.43 mmol).

The synthesis of oxypeucedanin (16) was per-formed by applying the procedure used for the epox-yaurapten preparation. Starting from 0.250 g (0.92mmol) of isoimperatorin (21), oxypeucedanin wasobtained in 86% yield (0.228 g, 0.80 mmol).

To a solution of oxypeucedanin (16) (60 mg, 0.2mmol) in 1,4-dioxane (5 mL) diluted sulfuric acid (5μL) was added dropwise. The reaction mixture was stir-red at room temperature for 1 hour and then a saturatedaqueous sodium bicarbonate solution (10 mL) wasadded. The mixture was extracted with ethyl acetate (3× 10 mL) and the combined organic layers were driedon sodium sulfate and evaporated under reduced pres-sure. The residue was purified by column chromatogra-phy using ethyl acetate/cyclohexane 7/3 as eluent. Thefractions of interest were evaporated to give oxypeu-cedanin hydrate (6), which was re-crystallized from n-hexane to afford colorless needles in 53% yield (34mg, 0.11 mmol).

Synthesis of imperatorin (24)

To a solution of 8-methoxy-psoralen (0.250 g, 1.15mmol) in dry DCM (10 mL), BBr3 (10 mL, 10 mmol,1 M in DCM) was slowly added and the mixture wasstirred under nitrogen at room temperature. After 2hours, the mixture was poured slowly into a solution ofsaturated sodium bicarbonate (50 mL). Subsequentlythe product was recovered by filtration, washed withcold water and ether, and dried under a high vacuum,yielding the xanthotoxol as an white solid in 83% yield(0.190 g, 1.0 mmol).

To a solution of xanthotoxol (0.190 g, 1.0 mmol)and potassium carbonate (0.140 g) in acetone (15 mL),3,3-dimethylallyl bromide (250 μL, 2.2 mmol) was

Figure 1. Scheme of the synthesis of coumarins and furocoumarins. For compound identification, see Table 1.

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added dropwise. The mixture was heated under refluxfor 1 hour. Aqueous chloridric acid (5% w/v) wasadded and the solution was extracted with DCM (2 ×10 mL). The organic layers were dried (Na2SO4) andthe solvent evaporated under reduced pressure to afforda yellow oil, which was crystallized from hexane,recovering by filtration the formed colorless needles ofimperatorin in 82% yield (0.180 g, 0.9 mmol).

Synthesis of cnidicin (33)

The xanthotoxol (0.387 g, 1.92 mmol), obtained usingthe procedure outlined above, solubilized in glacial ace-tic acid, was oxidized with a chromium trioxide 15%solution (0.120 g in 150 mL acetic acid 80%, 1.2 mL).After 2 hours, the mixture was first heated in an oilbath, until boiling, and then cooled in an ice bath,allowing the formation of corresponding quinone (14).

Quinone (0.250 g, 1.7 mmol) and 1.5 g of zinc dustwere suspended in acetone/water (15/50 mL) at roomtemperature and 5 mL of concentrated HCl was addeddropwise. After 5 hours, the remaining zinc dust wasfiltered off, and the filtrate was concentrated underreduced pressure to half its volume and kept at 4°Covernight. The 5,8-dihydroxypsoralen was collected byvacuum filtration (0.160 g, 0.7 mmol, 62% yield). To asolution of 5,8-dihydroxypsoralen (0.100 g, 0.45 mmol)and potassium carbonate (2.5 g) in anhydrous acetone(50 mL), 3,3-dimethylallyl bromide (0.09 g, 0.6 mmol)was added. The mixture was heated at reflux undernitrogen for 3 hours and then was poured into coldwater (100 mL) and acidified with HCl. After 24 hours,the product was extracted with DCM (3 × 50 mL), theorganic layers were combined and the solvent wasremoved under reduced pressure to form a residue,which was purified by column chromatography using7/3 ethyl acetate/cyclohexane as eluent. The fractionswere concentrated and recrystallized from hexane togive cnidicin (33) as a yellow solid in 64% yield (0.08g, 0.2 mmol).

Synthesis of citropten (7)

Citropten (7) was prepared according to a literatureprocedure (14), with a little modification. To a solutionof anhydrous phloroglucinol (1.18 g; 0.12 mmol) in1,4-dioxane dry (25 mL), ethyl propiolate (1.18 g; 0.12mmol) and freshly dried zinc chloride (1.08 g) wereadded. After stirring under reflux for 24 hours, the mix-ture was poured into HCl (5%, 10 mL), concentratedunder reduced pressure to half its volume, and kept at4°C overnight. The precipitate, 5,7-dihydroxycoumarin,was collected by vacuum filtration, with 68% yield (1g) m.p. 280°C (Lit1 m.p. 280°C).

To a solution of 5,7-dihydroxycoumarin (1.0 g; 5.6mmol) and potassium carbonate (5 g) in dry acetone

(25 mL), iodomethane was added to (2 mL). Afterbeing heated under reflux for 6 hours, the mixture wasacidified with HCl, and kept at 4°C overnight. Theresulting precipitate citropten (7) (1 g, 4.8 mmol) wascollected by vacuum filtration and recrystallized fromethanol to give 7 as white crystals in 73% yield.

Synthesis of epoxyaurapten (23) and aurapten (35)

To a stirring solution of 7-hydroxycoumarin (0.200 g,1.2 mmol) in anhydrous acetone (20 mL), geranyl-bro-mide dropwise (0.23 mL, 1.2 mmol) and potassium car-bonate (0.350 g) were added. After 24 hours, theorganic layer was removed under reduced pressure andthe residue was extracted with ethyl acetate. Theorganic layers were concentrated and recrystallizedfrom hexane to give aurapten (35) as a colorless whitesolid in 70% yield (0.250 g, 0.8 mmol).

To a solution of aurapten (35) (0.200 g, 0.7mmol) in DCM (20 mL), 3-chloroperoxybenzoic acid(0.320 g, 1.8 mmol) was added and the mixture wasstirred at �10°C, in an ice–methanol bath, for 2.5hours and then at room temperature for 1 hour. Thesolvent was removed under reduced pressure and thecolorless oil was purified by column chromatographyeluting with ethyl acetate/n-hexane (3/7). The frac-tions of interest were combined and the solventremoved under reduced pressure. The residue wasrecrystallized from n-hexane to obtain as white crys-tals epoxyaurapten (21) in 76% yield (0.160 g, 0.5mmol).

Synthesis of epoxybergamottin (29)

For the synthesis of epoxybergamottin, see reference(12).

Physico-chemical data of coumarins and psoralens

(Isoimperatorin, 21) 4-(3-Methylbut-2-enyloxy)-7H-furo(3,2-g)chromen-7-one: m.p. 108–109°C (lit m.p.108–109°C (14) for 1H NMR and 13C data, see refer-ence (12). (Oxypeucedanin, 16) 4-((3,3-dimethyloxi-ran-2-yl)methoxy)-7H-furo(3,2-g)chromen-7-one: paleyellow crystal from n-hexane; m.p. 141–142°C (lit. m.p. 141–142°C (14). For 1H NMR and 13C data, see ref-erence (12). (Oxypeucedanin hydrate, 6) 4-(2,3-dihy-droxy-3-methylbutoxy)-7H-furo(3,2g)chromen-7-one: m.p. 129–131°C (lit. m.p. 129–132°C (15, 16)); 1H NMR(300 MHz, CDCl3) δ:1.35 (s, 3H); 1.40 (s, 3H); 4.05(t, 1H, J=3.7 Hz); 4.50 (dd, 1H, J=7.7 and 9.8 Hz),4.78 (dd, 1H, J=3.1 and 9.8 Hz); 6.35 (d, 1H, J=9.8Hz); 7.05 (d, 1H, J=2.4 Hz); 7.22 (s, 1H); 7.75 (d, 1H,J=2.4 Hz); 8.32 (d, 1H, J=9.8 Hz); 13C NMR (300MHz, CDCl3) δ: 25.1; 25.4; 71.1; 74.7; 76.5; 93.5;105.0; 106.7; 111.9; 113.5; 139.7; 144.6; 145.1; 148.9;157.7; 160.8. (Imperatorin, 24) 9-(3-methylbut-2-enyl-oxy)-7H-furo(3,2-g)chromen-7-one: m.p. 99–100°C (lit.

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97.5–99°C (17, 18)) 1H NMR (300 MHz, CDCl3) δ:1.90 (s, 3H); 1.92 (s,3H); 5.18 (d, 2H, J=7.3Hz); 5.78(t, 1H, J=7.3); 6.55 (d, 1H, J=9.3 Hz); 6.99 (d, 1H,J=2.4 Hz), 7.49 (s,1H); 7.90 (d, 1H, J=2.4 Hz); 8.01(d, 1H, J=9.3 Hz); 13C NMR (300 MHz, CDCl3)δ:18.0; 25.7; 69.9; 106.6; 113.2; 114.2; 116.2; 119.7;125.7; 131.3; 139.2; 143.3; 143.5; 146.3; 148.3; 160.2;(Cnidicin, 33) 4,9-Bis(3-methylbut-2-enyloxy)-7H-furo(3,2-g)chromen-7-one: m.p. 53–57°C (lit. m.p. 76°C(17) for 1H NMR and 13C data, see reference (12).(Citropten, 7) 5,7-Dimethoxy-2H-chromen-2-one: whitecrystals from ethanol; m.p. 143–144°C; for 1H NMRand 13C data, see references (12,19). (Aurapten, 35)(E)-7-(3,7-dimethylocta-2,6-dienyloxy)-2H-chromen-2-one: colorless white solid from n-hexane; m.p. 62–64°C (lit. 66–67°C (19, 20)) 1H NMR (300 MHz, CDCl3)δ: 1.58 (s, 3H); 1.64 (s, 3H); 1.73 (s, 3H); 2.08 (m,

2H); 2.08 (m, 2H); 4.57 (d, 2H, J=6.5 Hz); 5.08 (m,1H); 5.45 (t,1H, J=6.5 Hz), 6.20 (d, 1H, J=9.5 Hz);6.80 (dd, 1H, J=2.5 and 8.3 Hz); 6.82 (d, 1H, J=2.5Hz); 7.35 (d, 1H, J=8.3 Hz); 7.60 (d, 1H, J=9.5 Hz).13C NMR (300 MHz, CDCl3) δ:17.6; 18.9; 25.4; 26.3;41.9; 65.4; 107.4; 111.2; 114.7; 114.9; 122.4; 122.6;127.4; 132.5; 137.8; 141.1; 151.0; 161.4; 161.6. (Epox-yaurapten, 23) (E)-7-(5-(3,3-dimethyloxiran-2-yl)-3-methylpent-2-enyloxy)-2H-chromen-2-one: white crys-tals from hexane; m.p. 72–74°C, 1H NMR (300 MHz,CDCl3) δ:1.30 (s, 3H); 1.35 (s,3H); 1.70(m, 2H); 1.80(s,1H); 2.20 (m, 2H); 2.80 (t, 1H, J=6.2); 4.60 (d, 2H,J=6.6 Hz); 5.45 (t, 1H, J=6.6 Hz); 6.25 (d, 1H, J=9.5Hz); 6.80 (d, 1H, J=2. Hz); 6.82 (dd,1H, J=8.1,2.6Hz); 7.30 (d, 1H, J=8.1); 7.62 (d, 1H, J=9.5Hz);13C NMR (300 MHz, CDCl3) δ:18.2; 23.3; 23.7; 28.2;37.4; 59.8; 65.4; 68.0; 107.6; 111.0; 114.2; 114.7;

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Figure 2. Reverse phase high-pressure liquid chromatography (RP-HPLC) chromatograms of a separation on a C18 AscentisExpress coloumn of a standard mix of coumarins, psoralens and polymethoxyflavones obtained with two different methods: withacetonitrile (A) and with methanol (B). For peak identification, see Table 1.

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120.4; 127.2; 140.0; 151.0; 161.2; 161.7. (Bergamot-tin, 37) 4-((E)-3,7-Dimethylocta-2,6-dienyloxy)-7H-furo(3,2-g)chromen-7-one: white solid from hexane, m.p.55–56°C, for 1H NMR and 13C NMR data, see refer-ence (11). (5-Geranyloxy-7-OMe-coumarin, 38) 5-((E)-3,7-Dimethylocta-2,6-dienyloxy))-7-methoxy-2H-chromen-2-one: white solid from hexane, m.p. 86–87°C, for 1H NMR and 13C NMR data, see reference (11).(5-Isopentenyloxy-7-methoxy-coumarin, 30) 5-(3-Methylbut-2-enyloxy)-7-methoxy-2H-chromen-2-one:Colorless needles from hexane, m.p. 88–91°C, for 1HNMR and 13C NMR data, see reference (12). (Epoxy-bergamottin, 29)4-((E)-3-Methyl-5-(3,3-dimethyloxiran-2-yl)pent-2-enyl-oxy)-7H-furo(3,2-g)chromen-7-one: Colorless crystalsfrom n-hexane, m.p. 69–70°C, for 1H NMR and 13CNMR data, see reference (12).

RP-HPLC/PDA separation of Citrus essential oils

Instrumentation

HPLC separation was carried out on a Shimadzu systemequipped with two LC 10 AD Vp pumps, an SPD-M10Avp UV detector, a SCL-10-Avp controller, a CTO-20AC column oven thermostated at 40°C and a DGU-14A degasser. NMR spectra (1H NMR recorded at 300MHz, 13C NMR recorded at 75 MHz) were obtainedwith Varian instruments and are reported in ppm relativeto tetramethylsilane.

Method

LC analyses were carried out on a Ascentis ExpressC18, 150 × 4.6 mm I.D.. with a particle size of 2.7 μm(Supelco, Bellefonte, PA). The injection volume was 2μL, the mobile phase consisted of water/methanol/THF(85:10:5) (solvent A) and methanol/THF (95:5) (solventB), and the step-wise gradient profile was as follows:0–5 min, 0% B; 5–25 min, 0–40% B; 25–45 min, 40–90% B; 45–55 min, 90% B; 55 min, 0% B; 60 min,0% B. Flow-rate was 1.0 mL/min, data were acquiredusing a PDA detector in the range 190–370 nm and thechromatograms were extracted at 315 nm. The timeconstant was 0.64 seconds and sample frequency1.5625 Hz. Data acquisition was performed by Shima-dzu LCsolution software ver. 3.3.

Method validation

Calibration curves for coumarins, psoralens and poly-methoxyflavones as reference materials were con-structed in order to quantify the oxygen heterocycliccompounds content of Citrus essential oils. Five con-centration levels of the standards, in the 100–0.1 mg/Lconcentration range, were prepared by diluting a stocksolution of 1000 mg/L, using ethanol as solvent, and

analyzed five times by RP-HPLC under the same chro-matographic conditions optimized for the oil samples.Before injection, 50 μL of internal standard (I.S.) cou-marin (64.8 mg/ 50 mL) were added to 1 mL of eachstandard solution. Correction factors were then calcu-lated using the formula reported by Dugo et al. (12).Limit of detection (LOD) and limit of quantification(LOQ) values, following the EURACHEM guidelines,were also calculated (21). The detection limit (yD) andthe quantitation limit (yQ) were expressed for each ana-lyte as signals based on the mean value (yb) and thestandard deviation (sb) of the blank signal as follows:

yD ¼ yb þ 2tsb yQ ¼ yb þ 10sb

METHOD WITH ACETONITRILE

METHOD WITH METHANOL

RS14-15 4.84

min 23.0 25.0 27.0

14

16

18.0 20.0 22.0 min

14

16

RS14-15 3.08

A

14.5 16.5 18.5 min

9

11

10

C

19.5 21.5 23.5 min

9

10

11

19.0 21.0 23.0 min

11 8

RS8-11 5.39

7

14.5 16.5 18.5 min

11 8

7

RS8-11 1.99

B

Figure 3. Extracted chromatogram of (A) lemon essential oilfor a critical pair byakangelicol (14)–oxypeucedanin (15)analyzed with the two methods, (B) lime essential oil for acritical pair isopimpinellin (8)–bergapten (11) analyzed withthe two methods, (C) bitter orange essential oil for theinversion of elution order of the pair isomeranzin (10)–bergapten (11) with the new method.

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where t is a constant of the t-Student distribution(one-tailed) dependent on the confidence level anddegrees of freedom (df). The recovery, the instrumentalintraday repeatability and the interday repeatabilitywere calculated using the same methods reported byDugo et al. (12).

Results and discussion

The RP-HPLC method developed in this study providedan excellent baseline separation of all the oxygen hetero-cyclic compounds contained in Citrus essential oils. Fig-ure 2 shows the comparison of two RP-HPLCchromatograms where the same standard mixture of oxy-gen heterocyclic compounds is separated using two dif-

ferent mobile phases. Figure 2A shows the separationobtained using the method previously developed (11),which involved the presence of acetonitrile in mobilephase and the column thermostated at 30°C. The secondchromatogram (Figure 2B) shows the separationachieved by replacing acetonitrile with methanol and thecolumn thermostated at 40°C. The mixture of standardscontains all the oxygen heterocyclic components presentin the Citrus oils investigated. The method used toachieve the separation shown in Figure 2B can beapplied to investigate all the Citrus essential oils.

Using the mobile phase containing methanol and acolumn oven thermostated at 30°C, the total analysistime increases of about 5 minutes, and the backpressuresignificantly increases. All these problems were solved

Table 1. Values of correction factor (CF)±standard deviation, of R2, of limit of detection (LOD) and limit of quantification(LOQ), of λmax (nm) of coumarins, psoralens and polymethoxyflavones.

Compounds Source CF R2 LOD LOQ λmax

CoumarinsMeranzin hydrate (1) Grapefruit oil 0.84a±0.144 0.028a 0.038a 248,258,325Herniarin (2) Commercial 0.55±0.020 1.000 0.018 0.024 325Citropten (7) Synthesis 0.66±0.047 0.997 0.021 0.029 248,256,330Meranzin (9) Grapefruit oil 0.84±0.144 0.999 0.028 0.038 248,258,325Isomeranzin (10) Grapefruit oil 0.84a±0.144 0.028a 0.038a 248,258,325Epoxyaurapten (23) Synthesis 1.14±0.104 0.999 0.038 0.053 325Osthol (27) Grapefruit oil 0.70±0.069 1.000 0.022 0.031 259,3255-Isopentenyloxy-7-methoxy-coumarin (30) Lemon oil 1.05±0.078 0.996 0.021 0.028 249,257,330Aurapten (35) Synthesis 0.86±0.098 0.998 0.018 0.025 3255-Geranyloxy-7-methoxy-coumarin (38) Lemon oil 1.05±0.419 0.998 0.036 0.050 248,257,329

PsoralensByakangelicin (3) Commercial 2.24±0.209 1.000 0.045 0.062 269,3158-Methoxy-psoralen (4) Commercial 0.85±0.156 0.999 0.028 0.038 249,304Psoralen (5) Commercial 1.03±0.089 1.000 0.030 0.041 297Oxypeucedanin hydrate (6) Synthesis 1.06±0.031 0.998 0.037 0.051 251,261,313Isopimpinellin (8) Lime oil 0.86±0.069 1.000 0.018 0.024 242,249,269,315Bergapten (11) Commercial 0.56±0.039 0.996 0.018 0.025 250,269,315Isobergapten (12) Commercial 0.76± 0.050 1.000 0.025 0.035 251,269,315Byakangelicol (14) Commercial 1.06±0.111 0.999 0.022 0.030 242,249,269,315Oxypeucedanin (16) Lemon oil 0.81±0.060 0.996 0.016 0.022 251,311Epoxybergamottin hydrate (20) Grapefruit oil 1.72b±0.178 0.057b 0.079b 251,312Isoimperatorin (21) Synthesis 0.82±0.052 0.997 0.016 0.022 252,312Imperatorin (24) Synthesis 1.06±0.148 0.999 0.034 0.047 250,303Cnidilin (25) Commercial 1.03±0.116 0.998 0.035 0.048 269,314Phellopterin (26) Commercial 0.92±0.093 1.000 0.020 0.027 270,315Epoxybergamottin (29) Synthesis 1.72±0.178 0.999 0.057 0.079 251,312Cnidicin (33) Synthesis 1.21±0.068 0.997 0.043 0.060 270,3148-Geranyloxy-psoralen (34) Commercial 1.40±0.233 0.999 0.049 0.068 249,303Bergamottin (37) Lemon oil 1.11±0.154 0.998 0.039 0.054 251,312

PolimethoxyflavonesSinensetin (13) Mandarin oil 0.65c±0.038 0.021 0.029 271,328Hexamethoxyflavone (15) Sweet orange oil 0.86d±0.055 0.029 0.041 251,270,337Nobiletin (17) Sweet orange oil 0.86±0.055 1.000 0.029 0.041 251,270,337Tetra-O-methyl-scutellarein (18) Sweet orange oil 0.65c±0.038 0.021 0.029 271,328Heptamethoxyflavone (19) Sweet orange oil 1.58±0.195 0.998 0.031 0.043 255,346Tangeretin (22) Sweet orange oil 0.65±0.038 0.996 0.021 0.029 271,3285-Demethyl-tangeretin (31) Mandarin oil 0.65c±0.038 0.021 0.029 271,3285-Demethyl-nobiletin (32) Mandarin oil 0.86d±0.055 0.029 0.041 251,270,337

Notes: aCF, LOD and LOQ of meranzin; bCF, LOD and LOQ of epoxybergamottin; cCF, LOD and LOQ of tangeretin; dCF, LOD and LOQ ofnobiletin.

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by using a column oven thermostated at 40°C (Fig-ure 2B).

From the comparison of the two chromatograms inFigure 2, only phellopterin and osthol are completelycoeluted. However, these two components are neverpresent in the same oil simultaneously, thus the lack ofseparation does not compromise the application of thismethod to Citrus oils. The separation of all the other

components is improved by the use of methanol insteadof acetonitrile. In particular, the use of methanol for theseparation of oxygen heterocyclic compounds of Citrusoils led to an increase of baseline resolution of criticalpairs of psoralens such as byakangelicol/oxypeuceda-nin, both present in lemon oil and in lime oils, and asisopimpinellin/bergapten, both present in lime oil.

Figure 3 shows the enlargements of critical pairscompared for the two methods. The resolution valuesfor the pair byacangelicol/oxypeucedanin increase from3.08 to 4.84 when methanol is used instead of acetoni-trile. More evident is the improvement of separation forthe pair isopimpinellin/oxypeucedanin, which changesfrom 1.99 to 5.39 with the new method. Using metha-nol, the elution order of meranzin/isopimpinellin, nobi-letin/oxypeucedanin and bergapten/isomeranzin isinverted. The components of the first two pairs arenever present simultaneously in Citrus oils, whereas inFigure 3C the case of bergapten/isomeranzin presentsimultaneously in bitter orange and grapefruit oils isshown. These components present inverted elutionorder if eluted with methanol instead of acetonitrile.

Table 1 shows the values of correction factors (CF)obtained from calibration curves of each standard, thevalues of LOD and LOQ, and the values of maximumUV absorption of coumarins, psoralens and polymeth-oxyflavones. For some oxygen heterocyclic compounds,namely epoxybergamottin hydrate, isomeranzin, meran-

Figure 4. Correlation between the position of the substituenton the aromatic ring of psoralens and the increase ofcorrection factor (CF).

5.0 10.0 15.0 20.0 25. 30.0 35. 40.0 min

315nm,4nm (1.00)2

3

11 34

16

14 8 36

33 21 24 30 Lime

I.S.

6

737 38

25 28

3 34 16 14 33 21 30 6 7 37 38

24 28 26 Lemon

2

11

Bergamot

7 37

38 13 18

Bitter orange 1

10 11 17

29 27

19

9 22

20

1 10

11

27

19

9

22 20 17

35

37

29 23

Grapefruit

13 18

22

19 17 Mandarin

31 32

Sweet orange 13

18 22 19 17

15

Figure 5. Reverse phase high-pressure liquid chromatography (RP-HPLC) chromatograms of seven Citrus essential oils obtainedby means of a methanol-based method. For peak identification, see Table 2.

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Table2.

Con

centratio

n±SD

inmg/Lof

oxyg

enheterocyclic

compo

unds

intheanalyzed

Citrus

essentialoils.

No

Com

poun

dsLem

onLim

eBergamot

Grapefruit

Bitter

orange

Sweetorange

Mandarin

1Meranzinhy

drate

––

–85

±4.5

171±

6.1

––

2Herniarin

–38

80±4

5.8

67±3

.2–

––

–3

Byakang

elicin

23±2

.5–

––

––

–6

Oxy

peucedanin

hydrate

11±0

.816

90±2

0.3

––

––

–7

Citrop

ten

726±

6.9

1095

0±92

.822

32±2

6.3

––

––

8Isop

impinellin

–73

00±4

6.9

––

––

–9

Meranzin

––

–62

68±8

6.0

5479

±46.1

––

10Isom

eranzin

––

–13

87±9

.220

74±1

4.8

––

11Bergapten

–30

00±3

1.1

2474

±28.4

75±3

.636

6±8.4

––

13Sinensetin

––

103±

2.8

––

97±6

.132

±2.3

14Byakang

elicol

708±

5.2

920±

9.9

––

––

–15

Hexam

etho

xyflavon

e–

––

––

109±

3.7

–16

Oxy

peucedanin

1372

±30

1060

0±85

.1–

––

––

17Nob

iletin

––

–81

±5.0

399±

5.7

590±

14.2

1504

±27.2

18Tetra-O

-methy

l-scutellarein

––

195±

3.6

––

246±

12.0

102±

5.8

19Heptametho

xyflavon

e–

––

133±

9.8

144±

2.5

1202

±31.2

592±

20.7

20Epo

xybergam

ottin

hydrate

––

–31

2±12

.421

8±6.3

––

21Isoimperatorin

33±3

.688

±5.9

––

––

–22

Tangeretin

––

–15

4±9.5

700±

13.9

518±

16.0

3450

±70.5

23Epo

xyaurapten

––

–40

74±5

1.9

––

–24

Imperatorin

34±2

.939

0±10.3

––

––

–25

Cnidilin

a–

249±

7.6

––

––

–26

Phello

pterin

118±

5.6

––

––

––

27Ostho

l–

––

1179

±16.2

1108

±21.7

––

285-(Isopent-2′-eniloxy

)-8-(2′,3′-epox

y)-isopentenylox

y-psoralen

n.a.⁄

n.a.⁄

––

––

–29

Epo

xybergam

ottin

––

–80

98±8

7.5

2001

±54.1

––

305-Isop

enteny

loxy

-7-m

etho

xy-cou

marin

151±

6.2

2790

±15.0

––

––

–31

5-Dem

ethy

l-tang

eretin

––

––

––

375±

13.4

325-Dem

ethy

l-no

biletin

––

––

––

63±2

.933

Cnidicin

73±2

.125

0±6.2

––

––

–34

8-Geranylox

y-psoralen

806±

8.6

4470

.3±2

8.65

––

––

–35

Aurapten

––

–33

56±4

1.1

––

–36

5-Geranylox

y-8-metho

xy-psoralen

–n.a.⁄

––

––

–37

Bergamottin

2925

±12.3

3640

0.6±

150.9

1960

5±73

.239

4±7.4

––

–38

5-Geranylox

y-7-metho

xy-cou

marin

1754

±10.5

4314

0.9±

166.7

1065

±7.5

––

––

Total

8699

1258

6825

741

2185

112

664

2404

6121

Notes:⁄ n.a.,standard

notavalaiblein

sufficientam

ount

forquantitativecalculation.

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zin hydrate, sinensetin, hexametoxyflavone, 5-demeth-yl-nobiletin, 5-demethyl-tangeretin and tetra-O-methyl-scutellarein, CF values determined for epoxybergamot-tin, meranzin, nobiletin and tangeretin were adoptedthe, as indicated by the notes in the table, since refer-ence materials were not available. For epoxybergamot-tin hydrate the CF of epoxybergamottin was chosen,and for isomeranzin and meranzin hydrate the CF ofmeranzin was chosen, since they present high structuralsimilarity. For hexamethoxyflavone and 5-demethyl-nobiletin the CF of nobiletin was chosen, since all ofthem are hexamethoxyflavones, and for 5-demethyl-tan-geretin and tetra-O-methyl-scutellarein the CF of tan-geretin was chosen, for the similarity of their structure.

The CF values reported in Table 1 are influencedby the molecular structure of the compounds of inter-est. In particular, the CF values increase with thedimension of the substituent on the aromatic ring ofcoumarins and psoralens. Furthermore, with the samesubstituent on position 8 of the aromatic ring of thepsoralens, CF value increases with respect to positionnumber 5, as happens for the bergapten (CF: 0.56) and8-methoxy-psoralen (CF: 0.85), and bergamottin (CF:1.11) and 8-geranyloxy-psoralen (CF: 1.40), as seenfrom Figure 4. Comparing the CF values obtained bythe new method with methanol with those calculatedusing a mobile phase containing acetonitrile (12), noremarkable differences were observed for most of thecompounds. The only exceptions are the higher CF val-ues of byakangelicol, oxypeucedanin, bergamottin and8-geranyloxy-psoralen, determined when acetonitrile ispresent in the mobile phase.

The values in Table 1 highlight excellent linearityfor all the analytes, confirmed by the correlation coeffi-cient R2, ranging from 0.996 to 1.000. In addition,LOD and LOQ values determined for this method aresimilar to those determined with the previously reportedone (12). These show excellent sensitivity of themethod, which could be applied to the analysis of oxy-gen heterocyclic compounds in trace levels in food-stuffs. Values determined on standard solutions forintraday repeatability and coefficient of variation (CV)lower than 8% demonstrated good precision at the con-centration level tested. Recovery values ranged from85% to 97%, demonstrating good accuracy (30).

The newly validated method was then applied tothe analysis of lemon, lime, bergamot, grapefruit, bitterorange, mandarin and sweet orange oils. Figure 5shows a comparison of the HPLC chromatogramsobtained for these Citrus oils. The quantitative analysis,performed by means of calibration curves, allowed thedetermination of the values reported in Table 2, wherethe concentration is expressed as mg/L±standard devia-tion of the coumarins, psoralens and polymethoxyflav-ones. All the results obtained for the seven oils

analyzed are in good agreement with those reported inthe literature (4, 11, 23–26).

Conclusions

The new RP-HPLC method developed allowed anexcellent baseline separation of almost all the oxygenheterocyclic compounds present in Citrus essential oils,and resolution of critical pairs of psoralens was clearlyimproved by the use of methanol in the mobile phase.This method allows us to analyze, under identicalexperimental conditions, all the major Citrus oils.

The method was validated in terms of detectionlimit, quantitation limit, linearity and precision.

References1. E. Colombo, C. Ghizzoni and D. Cagni, Citrus oils in

food and beverages: Uses and analyses. In: Citrus TheGenus Citrus. Edits., A. Dugo and Di Giacomo, pp. 539–56, Taylor & Francis, London (2002).

2. F. Buccellato, Citrus oils in perfumery and cosmeticproducts. In: Citrus. The Genus Citrus. Edits., G. Dugoand A. Di Giacomo, pp. 557–67, Taylor & Francis, Lon-don and New York (2002).

3. A. Di Giacomo and B. Mincione, Gli oli essenzialiagrumari in Italia. Laruffa Editore, Reggio Calabria(1994).

4. P. Dugo and M. Russo, The oxygen heterocyclic com-ponents of Citrus essential oils. In: Citrus Oils: Composi-tion, Advanced Analytical Techniques, Contaminants,Biological Activity. Edits., G. Dugo and L. Mondello, pp.405–43, Taylor & Francis, London and New York(2010).

5. D. McHale and J.B. Sheridan, The oxygen heterocycliccompounds of citrus peel oils. J. Essent. Oil. Res., 1,139–149 (1989).

6. P. Dugo, L. Mondello, I. Stagno d’Alcontres, A. Cavazzaand G. Dugo, Oxygen heterocyclic compounds of citrusessential oils. Perf. Flav., 22, 25–30 (1994).

7. Bonaccorsi., H.M. McNair, L.A. Brunner, P. Dugo andG. Dugo, Fast HPLC for the analysis of oxygen heterocy-clic compounds of citrus essential oils. J. Agric. FoodChem., 47, 4237–4239 (1999).

8. P. Dugo, L. Mondello, L. Dugo, R. Stancanelli and G.Dugo, LC-MS for the identification of oxygen heterocycliccompounds in citrus essential oils. J. Pharm. Biomed.Anal., 24, 147–154 (2000).

9. P. Dugo, I. Bonaccorsi, C. Ragonese, M. Russo, P. Dona-to, L. Santi and L. Mondello, Analytical characterizationof mandarin (Citrus deliciosa Ten.). Flav. Fragr. J., 26,34–46 (2010).

10. G. Dugo, I. Bonaccorsi, D. Sciarrone, L. Schipilliti, M.Russo, A. Cotroneo, P. Dugo, L. Mondello and V. Raymo,2011. Characterization of cold-pressed and processed ber-gamot oils by GC-FID, GC-MS, enantio-GC, HPLC, GC-C-IRMS. J. Essent. Oil Res. (2011), sent.

11. P. Dugo, MD. Fernandez, A. Cotroneo, G. Dugo and L.Mondello, Optimization of a comprehensive two-dimensional normal-phase and reversed-phase liquidchromatography system. J. Chr. Sci., 44, 561–565(2006).

128 M. Russo et al.

Dow

nloa

ded

by [

Ivan

a B

onac

cors

i] a

t 08:

00 2

0 M

arch

201

2

12. P. Dugo, A. Piperno, R. Romeo, M. Cambria, M. Russo, C.Carnovale and L. Mondello, Determination of oxygen het-erocyclic components in citrus products by HPLC with UVdetection. J. Agric. Food Chem., 57, 6543–6551 (2009).

13. P. Dugo, L. Mondello, G. Lamonica and G. Dugo, Char-acterization of cold-pressed key and Persian lime oils bygas chromatography, gas chromatography/mass spectros-copy, high-performance liquid chromatography and phys-icochemical indices. J. Agric. Food Chem., 45,3608–3616 (1997).

14. E.C. Row, S.A. Brown, A.V. Stachulski and M.S. Len-nard, Design, Synthesis and evaluation of furanocouma-rin monomers as inhibitors of CYP3A4. Org. Biomol.Chem., 4, 1604–1610 (2006).

15. H. Wulff, H. Raurer, T. During, C. Hanselmann, K. Ruff,A. Wrisch, S. Grissmer and W. Hansel, Alkoxypsoralens,novel nonpeptide blockers of Shaker-type K+ channels:Synthesis and photoreactivity. J. Med. Chem., 41,4542–4549 (1998).

16. W.L. Stanley and S.H. Vannier, Psoralens and substitutedcoumarins from expressed oil of lime. Phytochemistry, 6,585–596 (1967).

17. T. Masuda, M. Takasugi and M. Anetai, Psoralen andother linear furanocoumarins as phytoalexins in GlehniaLittoralis. Phytochemistry, 47, 13–16 (1998).

18. K. Young-Kyoon, S.K. Young and R. Shi Young, Anti-proliferative effect of furocoumarins from the root ofAngelica dahurica on cultured human tumor cell lines.Phytoter. Res., 21, 288–290 (2007).

19. E. Fillion, A.M. Dumas, B.A. Kuropatwa, N.R. Malhotraand T.C. Sitler, Yb(OTf)3 catalyzed reactions of5-alkylidene Meldrum’s acids with phenols: One-potassembly of 3, 4-dihydrocoumarins, 4-chromanones, Cou-marins and chromones. J. Org. Chem., 71, 409–412(2006).

20. J. Reisch, K. Szendrei, E. Minker and I. Novak, Notizüber das vorkommen von aurapten in Dictamnus albus.Planta Med., 15, 320 (1967).

21. The Fitness for Purpose of Analytical methods: A Labo-ratory Guide to Method Validation and Related Topics.EURACHEM Guide, Prague (1998).

22. G.C. Hokanson, A life cycle approach to the validationof analytical methods during pharmaceutical productdevelopment, Part I. The initial validation process.Pharm. Technol., 18, 118–130 (1994).

23. P. Dugo, L. Mondello, L. Dugo, R. Stancanelli and G.Dugo, LC-MS for identification of oxygen heterocycliccompounds in citrus essential oils. J. Pharm. Biomed.Anal., 24, 147–154 (2000).

24. E. Frérot and E. Decorzant, Quantification of total fur-ocoumarins in Citrus oils by HPLC coupled with UV,fluorescence, and mass detection. J. Agric. Food Chem.,52, 6879–6889 (2004).

25. P. Dugo, I. Bonaccorsi, C. Ragonese, M. Russo, P. Dona-to, L. Santi and L. Mondello, Analytical characterizationof mandarin (Citrus deliciosa Ten.) essential oil. FlavourFragr J., 26, 34–46 (2010).

26. J.A. del Río, P. Gómez, A.G. Baidez, M.C. Arcas, J.M.Botía and A. Ortuño, Changes in the levels of polymeth-oxyflavones and flavones as part of the defense mecha-nism of Citrus sinensis (Cv. Valencia Late) fruits against.Phytophthora citrophthora. J. Agric. Food Chem., 52,1913–1917 (2004).

27. P. Dugo, C. Ragonese, M. Russo, D. Sciarrone, L. Santi,A. Cotroneo and L. Mondello, Sicilian lemon oil: Com-position of volatile and oxygen heterocyclic fractions andenantiomeric distribution of volatile components. J. Sep.Sci., 33, 3374–3385 (2010).

28. E. Gorgus, C. Lohr, N. Raquet, S. Guth and D. Schrenk,Limettin and furocoumarins in beverages containing cit-rus juices or extracts. Food Chem. Toxicol., 48, 93–98(2010).

29. L. Schipilliti, P.Q. Tranchida, D. Sciarrone, M. Russo, P.Dugo, G. Dugo and L. Mondello, Genuineness assess-ment of mandarin essential oils employing gas chroma-tography-combustion-isotope ratio MS (GC-C-IRMS). J.Sep. Sci., 33, 617–625 (2010).

30. A.M. Ortuño, M.C. Arcas, O. Benavente-García and J.A.del Río, Evolution of polymethoxyflavones during devel-opment of tangelo Nova fruits. Food Chem., 66, 217–220(1999).

31. G. Dugo, I. Bonaccorsi, D. Sciarrone, R. Costa, P. Dugo,L. Mondello, L. Santi and H.A. Fakhry, Characterizationof oils from the fruits, leaves and flowers of the bitterorange tree. J. Essent. Oil Res., 23, 45–59 (2011).

32. Bonaccorsi., P. Dugo, L. Mondello, D. Sciarrone, G.Dugo and L. Haro-Guzman, Analytical characterizationof industrial essential oils from fruits and leaves of C.aurantifolia Tan. and C. latifolia Swing. J. Essent. OilRes., 23, 68–79 (2011).

33. B. Weber, B. Hartmann, D. Stöckigt, K. Schreiber, M.Roloff, H. Bertram and C. Schmidt, Liquid chromatogra-phy/mass spectrometry and liquid chromatography/nuclear magnetic resonance as a complementary nanalyt-ical techniques for unambiguous identification of poly-methoxylated flavones in residue from moleculardistillation of oreange peel oils (Citrus sinensis). J. Agric.Food Chem., 54, 274–278 (2006).

34. P. Dugo and D. McHale, The oxygen heterocycliccompounds of citrus essential oils. In: Citrus. The GenusCitrus. Edits., G. Dugo and A Di Giacomo, pp. 355–90,Taylor and Francis, London (2000).

35. W. Feger, H. Brandauer, P. Gabris and H. Ziegler, Non-volatiles of commercial lime and grapefruit oils separatedby high-speed countercurrent chromatography. J. Agric.Food Chem., 54, 2242–2252 (2006).

36. F. de Villiers, B. Tiempont, F. David and P. Sandra,Comprehensive two-dimensional liquid chromatographyapplying two parallel columns in the second dimension.J. Chromatogr. A., 1178, 33–42 (2008).

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