Characterization of Volatile Organic Compounds from Peel of Citrus medica L. by Headspace Trap (HS-Trap)Sampling TechniqueCoupled with GCMS.

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1 Page9330 Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: Journal home page: INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH Characterization of Volatile Organic Compounds from Peel of Citrus medica L. by Headspace Trap (HS-Trap)Sampling TechniqueCoupled with GCMS. Umesh Talekar* 1, Dr.D.N.Vora 1, Dr. E.R. Agharia 1, Dr.Shridhar Gawade 2, Dr. Anil Gopala 2, Dr. Yogesh Satpute 1 Department of Chemistry, Mithibai College (Vile Parle), Mumbai, 2 PerkinElmer (India) Pvt. Ltd., Thane ARTICLE INFO Article history Received 09/09/2013 Available online 03/12/2013 Keywords Volatile organic components (VOCs), Citrus medica L., GC-MS, Headspace Trap (HS-Trap), Monoterpene, d- limonene, Sabinene and Myrcene. ABSTRACT The volatile organic components obtained from fruit peel of Citrus medica L. using Headspace Trap (HS-Trap)sampling techniquewere characterized by gas chromatographymass spectrometry (GC-MS).The results shows that peak responses of44 components were obtained by using HS-Trap sampling technique coupled with GC-MS, predominantly containsmonoterpene group accounting for 98.46%,of which, d-limonene was the most abundant (85.93%), followed by sabinene (6.85%), myrcene (3.46%) while out of them 24 possesses specific aroma. The Headspace Trap (HS-Trap) sampling technique used in present study is advantageous over the conventional extraction technique. Technique is useful to avoid analyte losses, contamination. The other advantage of this technique is minimum sample requirement, capable to detect trace level component and minimum time require for extraction/sampling. The HS-Trap technique can be used as quick screening tool for authenticity determination and routine quality check of various citrus essential oil. The present study may found helpful for further work and may add as a valuable source of essential oil in flavor and fragrance industry. Corresponding author Umesh Talekar Department of Chemistry, Mithibai College (Vile Parle), Mobile No I.D. - umesh28t@rediffmail.com Please cite this article in press as Umesh Talekar et al. Characterization of Volatile Organic Compounds from Peel of Citrus medica L. by Headspace Trap (HS-Trap) Sampling TechniqueCoupled with GCMS. Indo American Journal of Pharm Research.2013:3(11). Copy right 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page9331 Introduction Citrus is the most widely produced fruit, as a group of several species and it is grown in more than 140 countries. According to 2009 data from the Food and Agriculture Organization of the United Nations (FAO); China, Brazil, USA, India, Mexico and Spain are the world s leading citrus fruit producing countries [1]. The citrus fruits are marketed as fresh or processed juice. The second important product of citrus fruit is the essential oil extracted mainly from the peel of various citrus fruits such as orange, lemon, lime, grapefruit and tangerine[1,2]. The volatile organic components(voc) isolated from plant parts are most commonly referred as Essential Oil.Essential oils are complex mixtures of volatile compounds produced by living organisms. Essential oil, also defined as essence, volatile oil, etheric oil or aetheroleum, is a complex mixture of volatile constituent s biosynthesized by living organisms and isolated by physical means (solvent extraction,distillation and pressing ) from a whole plant or plant part (leaf, flower and peel) of known taxonomic origin[3]. Citrus essential oils are greatly utilized as the flavorings in the food, beverage industries such as confectionary, cookies, desserts and fragrance materials are used in the perfumery, toiletries, fine chemicals, cosmetic products and aroma therapy[3-8]. Exploring essential oil seemed to be an alternative way to evaluate the economical values of citrus fruit due to the special roles it played in food, flavor and fragrance industries. The fruit selected in present work,citrus medica L. is known in local region. This fruit is known to be old, locally grown and used for its medicinal properties. Literature survey [9-15] reveals that, extensive research has been carried out on commercially important varieties of citrus fruitfrom all over world and India butthere is no reported study available on information of volatile organic component present in peel of selected fruit from Indian cultivar Citrus medica L.Therefore thefirst objective of this studywas to characterize the volatile organic component present in peel ofcitrusmedica L. fruit and explore the possibility of alternative source for essential oil industry. Literature survey also reveals that cold pressed, distillation is most preferable and commonly used extraction technique for commercial production and for compositional analysis of essential oil from citrus peel [9-15]. The new emerging technique has been tried by scientist to evaluate volatile organic components profile of plant material and to verify advantages of these techniques over conventional technique. The extraction technique like supercritical fluid extraction [16-19], headspace solid phase micro extraction (HS-SPME)[12,13,20]technique were used for extraction and analysis of volatile organic components present in citrus peel. The earlier reported study, on different types of adsorbents used for extraction of flavor, fragrance related compound and their analysis results suggest the use of Headspace Trap extraction/sampling technique in flavor and fragrance research [21]. In present work headspace trap (HS-Trap) technique was utilizedfor extractionof organic volatile componentsand characterization by gas chromatography- mass spectrometry (GCMS). Therefore another objective of this study was to evaluate the potential use of present technique in flavor and fragrance analysis.in HS-Trap technique, sample is placed in headspace vial and sealed with septa and cap. The headspace vial was equilibrated in automated, temperature controlled oven of instrument to extract the volatile organic components. The extracted volatile analytes were transferred on to adsorbent trap. After this pre-concentration step, volatile analytes were desorbed at high temperature and refocused on to gas chromatography-mass spectrometry (GC-MS) for further separation and analysis. Materials & Method Plant Material The leaves and fresh,mature fruits of selected plant were obtained from Harmadiya (Taluka-Kodinar, Dist-Junagarh, Gujrat). The plant material was identified and voucher specimen (No. BSI/WC/Tech/2012) was deposited at the Herbarium of Botanical Survey of India (BSI), Pune. Extraction of volatile organic components- The 500mg of fruit peel was chopped into small pieces and transferred to 22 ml headspace vial. The vial was sealed immediately with silicone/ptfe septa and aluminum cap. The vial was placed in TurboMatrix HS-40 Trap instrument which was connected through transfer line to Clarus 600 GC and Clarus 600C quadrupole mass spectrometer (PerkinElmer, USA). Instrumentation Clarus 600 GC (PerkinElmer, USA), Clarus 600 C Mass spectrometer (PerkinElmer, USA), TurboMatrix HS-40 Trap (PerkinElmer, USA) Analysis of volatile organic components Gas Chromatography analysis- The analysis and separation of extracted components were carried out on Elite-624 capillary column(60m x 0.32mm, 1.8um film thickness, PerkinElmer) using Clarus 600 GCPerkinElmer, USA).The flow rate of helium carrier gas was set at 2.0 ml/min with split flow 10ml/min.The oven temperature was held at 40 C for 10 min then programmed at rate of 8 C/min to 220 C and held isothermally for 15 min. The split/ splitless injector temperature was set at 230 C throughout analysis. Gas Chromatography-Mass Spectrometry analysis The single quadrupole mass spectrometer Clarus 600 C (PerkinElmer) was used for analysis and identification. The mass spectrometer was operated in electron impact ionization (EI) mode at a voltage of 70 ev. The ion source temperature, transfer line temperature was setat 200 o C, 230 o C respectively. The mass range was set from 30 to 500 m/z for scan time 0.2 sec, inter scan delay 0.1 sec.the peak areas were integrated with TurboMass softwareand volatile components wereidentified by comparing their mass spectra of each peak with the mass spectra of MS libraries: NIST 2008, WILEY 9.0 th Ed, FFNSC 1.3 (Flavor and Fragrance Library of Natural and Synthetic Chemicals).

3 Page9332 TurboMatrix HS-40 Trap: Oven Temperature : 80 C Needle Temperature : 160 C Transfer line Temperature : 170 C Trap Low Temperature : 40 C Trap High Temperature : 280 C Dry Purge (Helium) : 2 min Trap Hold Time : 10 min Desorb Time : 0.5 min Thermostatting Time : 30 min Pressurization Time : 1 min Decay Time : 1 min Outlet Split : Off Trap (Adsorbent used) : Mixed bed of graphitized carbon black & carbon molecular sieve RESULTS AND DISCUSSION Total 44 components were obtained and out of them, 43 components were identified.the monoterpene fraction was predominant; accounting for more than 98.43%, where d-limonene (85.93%) was the most abundant component, followed by sabinene (6.85%), β- myrcene (3.46%) and α-pinene (1.18%). Out 44 components 9 alcohols were identified viz. isobutanol, n-butanol, isopentyl alcohol, n-pentyl alcohol, prenyl alcohol, n-hexanol, cis-p-mentha-2,8-dien-1ol, Carveol, terpinen-4-ol. The two aldehyde components were identified viz. hexanal and prenal. The seven esters were identified viz. ethyl acetate, ethyl propionate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, citronellyl acetate and neryl acetate. The five sesquiterpene components were identified viz. elemene, α- bergamotene, aromadendrene, β-caryophyllene and β-bisabolene. The two ketone components were identified viz. carvone and 2- undecanone. The other component likemethyl-t-butyl ether, limonene oxide, 2-methylfuran, trichloromethane and (3E,5E)-2,6- Dimethyl-1,3,5,7-octatetraene were also detected and identified.tic chromatogram of peel sample presented in Figure 1. Figure 1: TIC chromatogram peel sample analysed in Headspace Trap mode; 1. methyl-t-butyl ether,2. 2-Methylfuran,3.Ethyl acetate, 4.Trichloromethane, 5.Isobutanol, 6.n-Butanol, 7.Ethyl propionate, 8.Acetoin, 9.Isopentyl alcohol, 10. Pentyl alcohol, 11.Ethyl butanoate, 12.Prenyl alcohol, 13. Hexanal, 14.Prenal, 15.n-Hexanol, 16. α-thujene, 17.Unknown, 18.α-Pinene, 19.Camphene, 20.Sabinene, 21.β-Myrcene, 22.Ethyl hexanoate, 23.α-Phellandrene, 24.α-Terpinene, 25. d-limonene, 26.β-Phellandrene, 27. γ- Terpinene, 28.α-Terpinolene, 29. Cis-p-Mentha-2,8-dien-1-ol, 30.trans-Limonene oxide, 31.Carveol, 32.Ethyl octanoate, 33.Terpinen- 4-ol, 34. α-fenchene, 35. 2, 6-Dimethyl-1,3,5,7-octatetraene,E,E-, 36. Carvone, 37.2-Undecanone, 38.Elemene, 39.Citronellyl acetate, 40.Neryl acetate,41.α -Bergamotene, 42.Aromoadendrene,43.β-Caryophyllene, 44. β-bisabolene. The component name, retention time and percent areaof components obtained by HS-Trap sampling technique and GC-MS aresummarized in followingtable 1.

4 Page9333 Table 1. Components obtained by HS-Trap sampling technique and GC-MS Sr. No. Component Name CAS No. Rt (min) % Area 1 Methyl-t-butyl ether Methylfuran Ethyl acetate Trichloromethane Isobutanol n-butanol Tr 7 Ethyl propionate Acetoin Isopentyl alcohol n-pentyl alcohol Ethyl Butanoate Prenyl alcohol Hexanal Prenal n-hexanol α-thujene Unknown α-pinene Camphene Sabinene β-myrcene Ethyl hexanoate α-phellandrene α-terpinene d-limonene β-phellandrene γ-terpinene α-terpinolene cis-p-mentha-2,8-dien-1-ol trans-limonene oxide Carveol Ethyl octanoate Terpinen-4-ol α-fenchene (3E,5E)-2,6-Dimethyl-1,3,5,7-octatetraene Carvone Tr 37 2-Undecanone Tr 38 Elemene Citronellyl acetate Neryl acetate α-bergamotene Aromadendrene β-caryophyllen β-bisabolene Tr = Trace compound whose percent are obtained is less than In current study, we have found some of the similar monoterpene component (α-thujene, α-pinene, sabinene, β-myrcene, α- terpinene, d-limonene, γ-terpinene, α-terpinolene, terpinene-4-ol, β-bisabolene, and α-bergamotene) compared with reported study carried out on peel composition of Citrus latifolia Tanaka using hydrodistillation and supercritical fluid extraction [17]. The

5 Page9334 component profile obtained from peel slurry of Japnese citrus fruit by using SFC extraction technique, matches with some of the component obtained in current study (α-pinene, β-myrcene, α-terpinene, d-limonene, terpinen-4-ol,γ-terpinene, camphene, α-elemene, β-caryophyllene) [19]. Similar observation is found in case of profile obtained from sweet lime (Citrus limetta Risso) by steam distillation technique(α-pinene, camphene, sabinene, β-myrcene, α-terpinene, d-limonene, carveol, neryl acetate, α-elemene, β- caryophyllene [15]. The comparative studies carried out for determination of chemical composition of Citrus sinesis cv. Valencia by HS-SPME technique and study carried out on Hallabong, lemon, orange, grapefruit shows some of the similar component, as obtained current study (α-pinene, sabinene, myrcene, α-phellandrene, d-limonene, γ-terpinene, terpinolene, α-thujene, limonene oxide, elemene, β-aromadendrene, Bergamotene ) [12]. The work carried out on Citrus nobilis Lour. Var deliciosa swingle using clevenger type hydrodsitillation extraction for volatile composition shows some of the similar monoterpene component obtained in current work (α-thujene, α-pinene, Sabinene, Myrcene,d-Limonene, γ-terpinene, Terpinolene, Terpinen-4-ol,Limonene oxide,β-elemene, β- caryophyllene[23]. The component profile obtained on Citrus reticulalta Blanco (Ponkan) by cold pressed oil technique is also comparable for some of the monoterpene component (α-pinene, camphene, sabinene, myrcene, α-phellandrene, β-phellandrene,dlimonene,α-terpinene, γ-terpinene, terpinolene, terpinen-4-ol, β-elemene) [11]. The other study carried out on Satsuma mandarian by using steam distillation technique also shows some of similar monoterpene component, obtained in current study (α-thujene, α-pinene, sabinene, β-myrcene, d-limonene, α-phellandrene, α-terpinene, β phellandrene, γ-terpinene, α-terpinolene, limonene oxide, α- Fenchene,carveol, β-elemene) [14]. Overall major components constituting monoterpene hydrocarbons were comparable with earlier reported data. The specific difference in some of constituent may be due to variety differences [1]. Literature survey reveals that 24 component obtained in current study,possess characteristic aroma property.the list of component and odour description is given in Table.2 Monoterpene Table 2: The component with characteristic odour description α-pinene Piney [10,19], Pinetree like [23] Monoterpene Camphene Camphor [19],Herbal [10] Monoterpene Sabinene Woody [10] Monoterpene β -Myrcene Lemon, Grapefruit [19], Geranium leaflike [23], Monoterpene sour,pungent, metallic [10] α-phellandrene Herbal,Chalky [10] Monoterpene α-terpinene Herbal, green [10] Monoterpene d-limonene lemon like, orange like, minty[19,23]pungent lemonlike Monoterpene [10] γ-terpinene Sweet,Citrusy [19]; Gasoline like [10] Monoterpene α-terpinolene Green [19], fresh sweet [10] Monoterpene trans-limonene oxide Fresh[10] Oxidized monoterpene Sesquiterpene δ-elemene Floral,Sweet, fruity,citrusy [10] Sesquiterpene β-caryophyllene Green, sweet [27] Sesquiterpene Alcohol Isopentyl alcohol Malty [25] Aliphatic alcohol Carveol Caraway [19] Monoterpene Alcohol Terpinen-4-ol Metallic, musty, green [24], Green [10,19],Earthy,green[10] Monoterpene Alcohol Aldehyde Hexanal Green [19, 24-26] Aliphatic aldehyde

6 Page9335 Ketone Carvone Mint like[23], Caraway-like [19,24], Green[10] Monoterpene Ketone Ester Ethyl acetate Fruity, solvent like [25,26] Aliphatic Ester Ethyl propionate Fruity [25] Aliphatic ester Ethyl Butanoate Fruit apple [19, 24-26] Aliphatic ester Ethyl Hexanoate Fruity, Orange [24-26] Aliphatic ester Ethyl Octanoate Spicy, Floral, Fruity [26] Aliphatic ester Citronellyl acetate Coriander like [10] Monoterpene Ester Neryl acetate Sour [10] Monoterpene Ester CONCLUSION In the present study, we have characterized the volatile organic components of Citrus medica L. by using GC-MS coupled with Headspace Trap technique.this Headspace Trap technique allows fast, automated extraction/sampling and analysis. This techniquecan be useful to avoid analyte losses, contamination and while other advantages of this technique are minimum sample requirement and detection of trace level component. This HS-Trap sampling technique can be used as quick screening tool for authenticity determination and routine quality check of various citrus essential oil. The experimental results show that techniquecan be helpful to obtain real aroma profiles of aroma with use of different adsorbent.the analytical resultsobtained in this work shows that 24 component out of 44, possessflavor and fragrance properties.thecomponent profileobtained in this study may found helpful for further studyand may add as a valuable source of essential oil in flavor and fragrance industry. The HS-Trap technique can be further evaluated with the use of different types of adsorbent material for characterization of aroma profile present in different citrus fruits. ACKNOWLEDGEMENTS We would like to thank, PerkinElmer (India) Pvt. Ltd. for providing support to carry out this research project and also express our appreciation to Dr. Shridhar Gawade(PerkinElmer),Dr. Yogesh Satpute (PerkinElmer) and Dr. Anil Gopala (PerkinElmer) for providing technical support. REFERENCES:- 1. Liu Y, Heying E, Tanumihardjo A; History, Global Distribution and Nutritional Importance of Citrus Fruits: Comprehensive reviews. Food Science and Food Safety.2012, 11, Ladaniya MS; Citrus fruit: Biology, Technology and Evaluation. San Diego, CA: Academic Press, 2008, Baser KHC, Demirci F; Chemistry of Essential Oils, In: Berger RG (eds). Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Berlin: Springer Verlag, 2007, Perez-Cacho PR, Rouseff RL; Citrus Flavour, In: Fresh squeezed orange juice odor, A Review. Crit Rev Food Sci Nutr, 48, 2008, Rouseff RL, Perez-Cacho PR; Citrus Flavours, In: Berger RG (eds), Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability. Berlin: Springer-Verlag, Colombo E, Ghizzoni C, Cagni D; Citrus Oils in Food and Beverages: uses and analyses In: Dugo G, Giacomo A (eds). Citrus: The Genus Citrus. London, New York:Taylor and Francis, 2002, Buccellato F, Citrus Oils in Perfumery and Cosmetic Products. In: Dugo G,Giacomo A (eds), Citrus-The Genus Citrus. London, New York: Taylor and Francis, 2002, Giacomo A, Giacomo G, Essential oil production. In: Dugo G, Giacomo A (eds), Citrus-The Genus Citrus, London, New York: Taylor and Francis, 2002, Haro-Guzman L, Production of distilled peel oils, In: Dugo G, Giacomo A (eds), Citrus-The Genus Citrus, London, New York: Taylor and Francis, 2002, Sawamura M, Tu NTM, Onishi Y, Ogawa E, Choi HS. Characteristic odor components of citrus reticulata blanco (ponkan) cold-pressed oil.2004, 68(8), Azar PA, Nekoel M., Larijani K, Bahraminasab S. Chemical composition of the Essential oils of Citrus sinensis cv. Valencia and a quantitative structure retention, Relationship study for the prediction of retention indices by multiple linear regressions.j.serb. Chem. Soc.,2011, 76 (0), Yoo ZW, Kim NS, Lee DS. Comparative analyses of the flavors from Hallabong (Citrus sphaerocarpa) with Lemon, Orange and Grapefruit by SPTE and HS-SPME combined with GC-MS.Bull. Korean Chem. Soc., 2004, 25 (2),

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