Study on optimization of solid-phase microextraction and gas chromatography-mass spectrometry analysis for the volatile fraction of pastures

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1 Romanian Biotechnological Letters Copyright 2011 University of Bucharest Vol. 16, No.6, 2011, Supplement Printed in Romania. All rights reserved ORIGINAL PAPER Study on optimization of solid-phase microextraction and gas chromatography-mass spectrometry analysis for the volatile fraction of pastures Received for publication, October 10, 2011 Accepted, November 22, 2011 AMALIA MITELUŢ 1, ALINA CULEŢU 1,2(*), MONA POPA 1, PETRU NICULIŢĂ 1 1 University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bd. Mărăsti, no.59, Bucharest, Romania, phone: National Institute of Research & Development for Food Bioresources IBA Bucharest, Str. Dinu Vintilă, no.1-6, Bucharest, Romania, phone: (*) Corresponding author, alinaculetu@yahoo.com Abstract In this study, identification of volatile compounds from pasture collected from Bucegi area (Romania) was performed using solid-phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS) technique. Optimum conditions of SPME analysis of the headspace volatile compounds of pastures in sealed bottles, using a carboxen/polydimethylsiloxane fiber were developed. Also, GC-MS operating parameters were varied in order to find a better profile of the volatile compounds. Accordingly, there have been investigated different sample processing methods in order to improve the analytical procedure. Keywords: pasture, solid-phase microextraction, gas chromatography-mass spectrometry, volatile compounds Introduction Analysis of volatile fraction from pastures contributes to improving the traceability of food products (milk and dairy products, meat). Among volatile compounds from plants, terpenes are of interest, because they can be used as biochemical markers helping to characterize products from different geographical areas [1-5]. They are lipophilic aliphatic compounds (usually contain one or more C=C double bonds) involved as secondary metabolites of plants. They are thoroughly distributed in the plant kingdom, especially in dicotyledons [6]. Gas chromatography (GC) coupled with mass spectrometry (MS) is used as analytical method for volatile compounds identification. Solid-phase microextraction (SPME) is used for extraction of volatile compounds; this technique enables the isolation and concentration of compounds of interest on a silica fiber coated with an appropriate stationary phase. It integrates sampling, extraction, concentration and sample introduction to GC [7]. The principle of headspace SPME is an equilibrium partitioning of analytes among the coating of solid phase or fiber, sample and headspace. SPME was developed by Pawliszyn [8] and has been applied to a wide variety of volatile compounds from different food products [9-14]. The aim of this study was to identify volatile compounds, especially terpenes, from a type of sub-alpine pasture, using SPME under different operating conditions, optimizing these conditions in order to obtain the best chromatographic profile. 113

2 AMALIA MITELUŢ, ALINA CULEŢU, MONA POPA, PETRU NICULIŢĂ Materials and Methods Plant materials The plant material used in the present study was collected from a sub-alpine pasture (Research Base Blana Bucegi, Romania) in August 2010, being sealed in plastic bags and preserved by freezing at 20 C until analysis. Equipment A gas-chromatograph (GC 7890 A) coupled with a mass spectrometer (MS 5975 C with Triple-Axis Detector, Agilent Technologies, Santa Clara, CA, USA) was used to analyze the pasture headspace components. The injection port temperature was 230 C; the injection mode split less with fiber and an HP 5 capillary column (30 m long, 0.25 mm i.d., 0.25 m film thickness). The GC oven temperature initial was maintained at 50 C for 10 min, and then increased up to 200 C by 10 C/min, followed by 15 C/min to 260 C. The carrier gas was helium with a flow rate of 1 ml/min. The mass-spectrometer detector was operating under electron impact mode (70eV). The MS temperatures adopted were: source 230 C, quadrupole 150 C; the acquisition range m/z. The samples were injected through an automatic auto-sampler CombiPAL (CTC Analytics) and the whole analytical procedure was controlled with the program ChemStation (Agilent Technologies, Santa Clara, CA, USA). The SPME fiber used to collect volatile fraction was carboxen / polydimethylsiloxane (CAR/PDMS) 85 m (StableFlex Supelco), a mixed fiber containing a porous activated carbon support (Carboxen) which increase retention capacity, being better for trace level of analytes [15]. According to the manufacturer recommendation, the fiber was conditioned at 300 C for 1 h in GC injector before analysis. A single fiber was used for this study. Chromatograms were recorded in the scan mode. For finding the peaks and their area, auto-integrate option from ChemStation software was used. Volatile compounds identification was done by comparing the mass spectra with those stored in the National Institute of Standards and Technology library (NIST Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library version 2.0, USA). Sample preparations The sample was brought to ambient temperature and 2 g cut up roughly with scissors. There were tested two variants: direct extraction in pasture in a 20 ml vial, respectively, extraction with an organic solvent (5 ml methylene chloride, Sigma Aldrich). In this case, the extraction was performed on the shaker for 24 h. The vials were sealed with an aluminum cap provided with a pierceable septum. The vial sample was allowed to equilibrate, before the SPME fiber is inserted at 80 C for 15 min, and then the fiber was exposed to the headspace for 10 min, performing the extraction of volatiles from pasture. Blanks with fiber after GC-MS sample analysis were performed in order to indicate the absence of any compound in the fiber or column. Results and Discussions Development of the method In the present study, in order to optimize the analytical technique, different factors were considered, such as: sample preparation, sample heating temperature, vial penetration, helium flow rate. 114 Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

3 Study on optimization of solid-phase microextraction and gas chromatography-mass spectrometry analysis for the volatile fraction of pastures Different sample preparations were tested; direct extraction with SPME fiber provided a higher number of compounds identified, 22 compounds compared to 4 in case of solvent extraction. These compounds were: 2-carene, caryophyllene, coumarin and apiol. The effect of sample-heating temperatures (80 C, 90 C, respectively 100 C) for extraction of volatile compounds from pasture was tested. At higher temperatures the number of volatile compounds identified is less, the adsorption being affected. Fig.1 shows that sample-heating with 80 C allows identification of more terpenes compounds, except for - ionone and nonanal where the abundance is lower compare to higher temperature. 5.E+08 4.E+08 3.E+08 Area 2.E+08 1.E+08 0.E+00 o-cymene a-pinene Ocimene g-terpinen Terpinolene Caryophyllene b-ionone Nonanal b-cyclocitral 800C 0 C 900C 0 C 1000C 0 C Fig.1. Comparison among some volatile compounds peak areas detected using different sampling temperature. Direct extraction with CAR/PDMS fiber; vial penetration 26 mm; equilibrium time 15 min; extraction time 10 min; desorption temperature 230 C; desorption time 3 min. Vial penetration which determines how far the fiber extends into the vial was tested also. At 22 mm depth of the fiber in the vial were identified 14 compounds, while in the case of 26 mm, the number was higher (22 compounds). Fig.2 shows that the abundance of volatile compounds presented is higher in the case of 26 mm vial penetration. 5.E+08 4.E+08 3.E+08 Area 2.E+08 1.E+08 0.E+00 o-cymene a-pinene Ocimene g-terpinen Terpinolene Caryophyllene b-ionone Nonanal b-cyclocitral 22 mm 26 mm Fig.2. Comparison among some volatile compounds peak areas detected using two different values for vial penetration. Direct extraction with CAR/PDMS fiber; equilibrium time 15 min; extraction time 10 min; desorption temperature 230 C; desorption time 3 min. Helium flow rate through GC column was tested at 0.8 ml/min and 1 ml/min. As it can be noticed from Fig.3, a flow rate of 1 ml/min allowed detecting a higher number of terpenes and also a higher abundance compared to a flow of 0.8 ml/min. Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011) 115

4 AMALIA MITELUŢ, ALINA CULEŢU, MONA POPA, PETRU NICULIŢĂ 5.E+08 Area m 4.E+08 3.E+08 2.E+08 1.E+08 0.E+00 o-cymene a-pinene Ocimene g-terpinen Terpinolene Caryophyllene b-ionone Nonanal b-cyclocitral 1 ml/min 0.8 ml/min Fig.3. Comparison among terpene areas detected with different helium flow rate. Direct extraction with CAR/PDMS fiber; vial penetration 26 mm; equilibrium time 15 min; extraction time 10 min; desorption temperature 230 C; desorption time 3 min. For a good peak separation, there were used two temperature steps in the program rate of the GC oven. An equilibrium step of 15 min during which the sample was preheated was needed, so that the equilibrium was reached the moment the fiber was introduced into the headspace. A sampling time of 10 min was optimum for most of the components of interest. Volatile fractions of pasture sample Fig.4 shows an SPME-GC/MS chromatogram obtained from the pasture sample studied. The headspace sampling technique allowed the detection in sample of 22 different volatile compounds, which were grouped according to chemical classes in terpenes, aldehydes, ketones, alcohols and hydrocarbons. Table 1 summarizes the compounds identified with their retention time on the HP 5 capillary column Fig.4. SPME-GC/MS chromatogram obtained from a sub-alpine pasture sample (peaks are numbered as in Table1). Table 1. Compounds identified in the volatile fraction of sub-alpine pasture sample. No. Compound 1,2 Retention time (min) Area 1 Fenchol, exo (T) o-cymene (T) Pinene (T) Terpinene (T) ,5-octadien-2-one (K) Terpinolene (T) Nonanal (Ad) cis--mentha-2,8-dien-1-ol (Al) Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

5 Study on optimization of solid-phase microextraction and gas chromatography-mass spectrometry analysis for the volatile fraction of pastures 9 Benzyl carbinol (Al) Ocimene (T) ,6-dimethyl-1,3,5,7-octatetraene (H) Dodecane (H) Decanal (Ad) Cyclocitral (T) Decenal (Ad) Tridecane (H) Caryophyllene (T) Ionone (T) Apiol (H) Octadecanal (Ad) ,7,11,15-tetramethyl-2-hexadecen-1-ol (Al) Pentadecanone- 6,10,14-trimethyl (K) Compounds are listed in order of elution from the HP 5 column. Identification made by comparison of mass spectra with the respective data of NIST library in total ion current (TIC). 2 In bracket is set the chemical class of the compound identified: T-terpene, K-ketone, Ad-aldehyde, Al-alcohol, H-hydrocarbon. In the chromatogram obtained from the sub-alpine pasture analyzed, 68.18% from all the compounds identified is represented by terpenes (with terpinolene and o-cymene as main constituents), followed by aldehydes and hydrocarbons. So, the predominated compounds are terpenes, which can be used as biochemical markers for traceability purposes. Fig.5 shows an example for the fragmentation pattern of -terpinene in pasture sample analyzed and that of the NIST library. The comparison of the two fragmentation pattern allowed the compounds identification. Fig.5. Mass fragmentation pattern of -terpinene in the pasture sample analyzed (lower graphic) and of library reference (upper graphic). Conclusions A headspace solid-phase microextraction method in combination with gas chromatography mass spectrometry has been used for extraction and identification of Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011) 117

6 AMALIA MITELUŢ, ALINA CULEŢU, MONA POPA, PETRU NICULIŢĂ components from the volatile fraction of a sub-alpine pasture from Bucegi area. The effects of various conditions were studied in order to optimize the technique. From the chromatographic profile obtained for the sample analyzed, 22 compounds belonging to different chemical classes were identified. Among these, terpenes were the most abundant; thus, these can be used as biochemical markers for traceability purposes. Acknowledgements The authors thank for the financial support from National Research Program PN II within the project no /2008. References 1. C. VIALLON, I. VERDIER-METZ, C. DENOYER, P. PRADEL, J.B. COULON, J.L. BERDAGUE, Desorbed terpenes and sesquiterpenes from forages and cheeses. J. Dairy Res., 66, (1999). 2. S. CARPINO, S. MALLIA, S. LA TERRA, C. MELILLI, G. LICITRA, T.E. ACREE, D.M. BARBANO, P.J. VAN SOEST, Composition and Aroma Compounds of Ragusano Cheese: Native Pasture and Total Mixed Rations. J. Dairy Res., 87, (2004). 3. G. ZEPPA, M. GIORDANO, M. BERTOLINO, V. GERBI, Application of artificial neural network on mono- and sesquiterpenes compounds determined by headspace solid-phase microextraction gas chromatography mass spectrometry for the Piedmont ricotta cheese traceability. J. Chromatogr. A, 1071, (2005). 4. E. ABILLEIRA, M. DE RENOBALES, A.I. NAJERA, M. VIRTO, J.C. RUIZ DE GORDOA, F.J. PEREZ- ELORTONDO, M. ALBISU, L.J.R BARRON, An accurate quantitative method for the analysis of terpenes in milk fat by headspace solid-phase microextraction coupled to gas chromatography mass spectrometry. Food Chem., 120, (2010). 5. M. POVOLO, G. CONTARINI, M. MELE, P. SECCHIARI, Study on the Influence of Pasture on Volatile Fraction of Ewes Dairy Products by Solid-Phase Microextraction and Gas Chromatography-Mass Spectrometry. J. Dairy Res., 90, (2007). 6. J. DEGENHARDT, T.G. KOLLNER, J. GERSHENZON, Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry, 70, (2009). 7. C.L ARTHUR, J. PAWLISZYN, Solid phase microextraction with thermal desorption using fused silica optical fibers. Anal. Chem., 62, (1990). 8. J. PAWLISZYN, Solid phase microextraction. Theory and practice. Wiley-VCH, New York, [M. KOVACEVIC, M. KAC, Solid-phase microextraction of hop volatiles: Potential use for determination and verification of hop varieties. J. Chromatogr. A, 918, (2001). 10. A.J. MATICH, D.D. ROWAN, N.H. BANKS, Solid Phase Microextraction for Quantitative Headspace Sampling of Apple Volatiles. Anal. Chem., 68, (1996). 11. E. IBANEZ, S. LOPEZ-SEBASTIAN, E. RAMOS, J. TABERA, G. REGLERO, Analysis of volatile fruit components by headspace solid-phase microextraction. Food Chem., 63, (1998). 12. G FITZGERALD, K.J. JAMES, K. MACNAMARA, M.A. STACK, Characterization of whiskeys using solid-phase microextraction with gas chromatography mass spectrometry. J. Chromatogr. A, 896, (2000). 13. D. BERTELLI, G. PAPOTTI, M. LOLLI, A.G. SABATINI, M. PLESSI, Development of an HS-SPME GC method to determine the methyl anthranilate in citrus honeys. Food Chem., 108, (2008). 14. J. IGLESIAS, I. MEDINA, Solid-phase microextraction for the determination of volatile compounds associated to oxidation of fish muscle. J. Chromatogr. A, 1192, 9-16 (2008). 15. SUPELCO CombiPAL SPME Option Manual. Supplement to the CombiPAL System Users Manual. 118 Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)