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Improved Separation of Fame Isomers Using Comprehensive Two-Dimensional Gas Chromatography. Application to Broccoli Samples

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Improved Separation of Fame Isomers Using Comprehensive Two-Dimensional Gas Chromatography. Application to Broccoli Samples
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  Improved Separation of Fame Isomers Using ComprehensiveTwo-Dimensional Gas Chromatography. Applicationto Broccoli Samples Pilar Manzano  &  Esther Arnáiz  &  Juan Carlos Diego  & Laura Toribio  &  María Jesús Nozal  &  José Luis Bernal  & José Bernal Received: 19 September 2011 /Accepted: 3 November 2011 /Published online: 19 November 2011 # Springer Science+Business Media, LLC 2011 Abstract  A new method has been developed to separate 47fatty acid methyl esters (FAMEs), including the linoleic andlinolenic acid group of isomers, using comprehensive two-dimensional gas chromatography (GC×GC) coupled to acapillary flow technology-based modulator and flameionization detector (FID). In addition a modification of theinverted phase, column combination was introduced, usinga high polar column (BPX-70) in the first dimension and asemi-polar column (ZB-35) in the second. Moreover, theinfluence of the length of the second dimension column onFAME isomer separation was studied. When comparedwith previously published methods devoted to FAMEanalysis, separation was improved, both in terms of analysistime, and with regard to the number of FAMEs separatedand identified in the same chromatographic run. Finally, theGC×GC-FID method proposed was successfully employedto identify FAMEs in supercritical fluid extracts from theleaves of three different broccoli cultivars (Naxos, Nubia,and Viola). Keywords  GC×GC.Capillary flow modulation.FAME.Broccoli Introduction The scientific interest in fatty acids (FAs) is clear when weconsider that more than 9,000 studies have been publishedin the last 5 years with FAs in the title (source: ISI Web of Knowledge data base, Copyright 2011 Thomson Reuters).This interest may principally be due to their influence onhuman health, not only in terms of metabolism as an energystore, or for their participation in biological membranes and protein acylation (Jones et al. 1992; Benatti et al. 2004), but  also because they may positively or negatively influence anumber of diseases (hypertension, cancer, etc.) (Benattiet al. 2004; Von Schacky 2003). For example, artificial trans-fat intake increases the risk of heart disease (Tan2011; Sun et al. 2007). The technique traditionally used to analyze FAs isone-dimensional gas chromatography (GC), usuallycoupled to flame ionization (FID) or mass spectrometry(MS) detection. In GC, FA detection is usually facilitated byconverting these compounds into their methyl ester derivatives(fattyacidmethylesters(FAMEs))andsometimes,intotheirownsterylesterscompounds(fattyacidssterylesters(FASEs)) (Harynuk et al. 2006; Bicalho et al. 2008; Hejazi et  al. 2009; Hauff and Vetter  2009; Toribio et al. 2011). However, the geometric isomers of unsaturated fatty acids incomplex mixtures are difficult to determine by one-dimensional GC. Nevertheless, with the development of newer columns and detectors, it has now become possible toanalyze a large number of fatty acids employing one-dimensional GC (Bicalho et al. 2008; Hejazi et al. 2009; Hauff and Vetter  2009; Toribio et al. 2011; Hejazi et al. 2011; Ragonese et al. 2009; Delmonte et al. 2011; Ando and Sasaki 2011; Gu et al. 2011; Janssen et al. 2009), including isomers such as the linoleic acid group of isomers (Bicalho et al. 2008; Ragonese et al. 2009; Delmonte et al. 2011) or the fatty acid isomer mixture of linolenic acid (Hejazi et al.2009; Hejazi et al. 2011; Ragonese et al. 2009; Delmonte et  al. 2011). P. Manzano :  E. Arnáiz :  J. C. Diego : L. Toribio :  M. J. Nozal : J. L. Bernal : J. Bernal ( * )I.U. CINQUIMA, Analytical Chemistry Group,University of Valladolid,Valladolid, Spaine-mail: jose.bernal@qa.uva.esFood Anal. Methods (2012) 5:920  –  927DOI 10.1007/s12161-011-9330-1  In recent years, comprehensive two-dimensional gaschromatography (GC×GC) has also emerged as a powerfulmultidimensional separation technique to analyze FAMEs.This approach has the advantage that the whole sample isseparated on two different GC columns connected in series(Herrero et al. 2009; Janssen et al. 2009). Accordingly, it is a technique that is suitable to characterize complex samplessuch as food (Herrero et al. 2009), resolving some of thelimitations in FAME analysis by significantly increasing the power of separation when compared with conventional(one-dimensional) GC.In GC×GC, the selection of the columns for bothdimensions is very important. The most commoncolumn combinations used, based on a differing degreeof orthogonality, are nonpolar   –   polar (conventional) and polar   –  nonpolar (inverted phase) (Gu et al. 2011; Gu et al.2010; Tranchida et al. 2010; Pyl et al. 2011; Manzano et al. 2011). Some such combinations have been tested previouslyin a research setting (Manzano et al. 2011) an in theexisting literature, one study was identified where avariation of this inverted phase setting was employed todefine FAMEs in milk and beef fat, which consisted of ahighly polar column in the first dimension and a semi- polar column in the second (Villegas et al. 2010). Sincethe results presented in this publication were promising,we decided to study this approach to see if it improved theresults we could obtain.Accordingly, we set out to achieve the best FAMEseparation possible using a GC×GC-FID systemequipped with a capillary flow technology (CFT) modulator and the high polar   –  semi-polar column combination. Different second dimension column lengths and chromatographicconditions have been tested in an attempt to minimizethe analysis time without losing any resolution whenanalyzing a large number of FAMEs, including that associated to the linoleic and linolenic acid isomersgroups. In terms of the analysis time and the number of FAMEs separated, the results obtained with the proposedmethod were compared with other protocols proposed in theliterature that used conventional GC (Harynuk et al. 2006;Bicalho et al. 2008; Hejazi et al. 2009; Hauff and Vetter  2009; Toribio et al. 2011; Hejazi et al. 2011; Ragonese et al. 2009; Delmonte et al. 2011; Ando and Sasaki 2011; Sánchez-Ávila et al. 2009), or that relied on GC×GCsystems equipped with thermal (Pyl et al. 2011; Villegaset al. 2010; Tranchida et al. 2008, 2010; Gardner et al. 2011; Beckstrom et al. 2011; Cordero et al. 2010) or  capillary flow modulators (Gu et al. 2011; Gu et al. 2010; Manzano et al. 2011). Finally, the GC×GC method developed was successfully applied to analyze FAMEsisolated by supercritical fluid extraction (SFE) of broccolileaves from three different cultivars (Naxos, Nubia, andViola). Materials and Methods Standard SolutionsStandard solutions of fatty acid methyl esters indichloromethane (reference 47885-u, S37, mix of 37FAMEs; reference 47792, linolenic acid methyl ester isomer mix; reference 47791, linoleic acid methyl ester isomer mix (Gu et al. 2010; Manzano et al. 2011)) and two kits of 10 saturated (ME10-1KT) and 14 unsaturated(ME14-1KT) individual FAME standards were purchasedfrom Supelco (Bellefonte, PA, USA).In this work, FAs were designated using the formula  Cx :  y (nz;catb) (Manzano et al. 2011). Where  “  x ”  is the number of carbon atoms in the fatty acid chain (the methyl alcohol part not included),  “  y ”  the number of double bonds, and  “  z  ” the location of the first double bond beginning at the methylterminal group,  “ a ”  and  “ b ”  were the conventional positions of the double bonds with  cis  ( “ c ” ) or   trans  ( “ t  ” )stereoisomerism, which were omitted from the formulawhen all the fatty acid double bonds were of the  cis -type.Standard stock solutions were prepared in dichloro-methane at a concentration of 1,000 mg/L, as the srcinalstandard mixtures were prepared in this solvent (Labscan,Dublin, Ireland). These solutions were diluted daily withdichloromethane to produce a set of working standards. Allstandards and stock solutions were kept in the dark at +4 °Cand they were stable at least for 1 month.SamplesLeaves from broccoli grown at CEBAS-CSIC (Murcia)were freeze dried as described previously (López-Berenguer et al. 2009). Briefly, broccoli seeds were pre-hydrated withaerated and de-ionized water for 12 h and germinated in anincubator for 2 days in vermiculite at 28 °C. The seeds werethen transferred to a controlled-environment chamber on a16 h light   –  8 h dark cycle at air temperatures of 25 and 20 °C, respectively, and with a relative humidity (RH) of 60%(day) and 80% (night). The environmental temperature andRH were strictly controlled throughout the experiment. After 5 days, the seedlings were placed in 15-L containers andthey were supplied with a modified Hoagland nutrient solution (López-Berenguer et al. 2006). This solution was prepared and replaced each week. After 15 days, the plantswere transplanted to perlite containers (one plant per container) irrigated with modified Hoagland nutrient solu-tion, and these containers were located in a controlled-environment greenhouse where the plants were grown untiltheywereharvested11weeksaftertransplanting.Thebroccolileaves used in this work belonged to the Nubia, Viola, and Naxos varieties. All the samples were lyophilized to preservethem until they were analyzed in triplicate. Food Anal. Methods (2012) 5:920  –  927 921  Supercritical Fluid ExtractionThe use of supercritical fluid extraction with carbon dioxide(CO 2 ) is being proposed as an alternative to the use of organic solvents (Toribio et al. 2011) since it is non-toxicand non-explosive. Hence, the solvent can be easilyremoved from the extracts by simple expansion without leaving any residue, and the selectivity of extraction can becontrolled by changes in pressure and temperature.The supercritical fluid extractor used to obtain the broccoliextracts was a home-built modular system (Arnáiz et al. 2011;Bernal et al. 2008) equipped with two intelligent preparative pumps (model PU-1586 from Jasco Corporation, Tokyo,Japan), one of which was used to supply the organicmodifier and the other to propel the CO 2 . The pump-headof the latter was maintained at 0 °C using a thermostatic bath(Frigomix U from B. Braun, Melsugen, Germany) and the pressure was controlled with a backpressure regulator (BP-1580-81, Jasco Corporation, Tokyo, Japan). The SFE procedure previously optimized by our research group toisolate FAMEs from broccoli leaves (Manzano et al. 2011)was employed here. Briefly, samples of broccoli leaves(0.3 g) were placed into the extraction cell (2 mL; Jasco,Tokyo, Japan) and introduced into an oven (CO-2056, Jasco,Tokyo, Japan) equipped with two (V3, V4) 7000 Rheodynevalves (Cotati, CA) and two (V1, V2) NV-5272 NOVASwiss valves (Cesson, France). After static extraction(10 min), extracts were obtained at 65 °C and 250 bar, witha flow rate of 3 mL/min and a dynamic extraction time of 60 min, and they were collected into 20 mL glass vials witha pierced-cap previously filled with methanol (1 mL) using aGilson233XLsampling-injector(Villiers-le-Bel,France).The233XLdevicehadacollectionneedlethatwasprogrammedtomove down to the bottom of the vial. It must be pointed out that CO 2  modified with methanol (15%) was used to extract the polar lipids from the broccoli leaves. Finally, the broccolileaf extracts were diluted to 20 mL with methanol.GC×GC ColumnsThree different column combinations were tested in this work (Table 1), each using the same column in the first dimension(BPX-70, SGE Analytical Science, Victoria, Australia) andan intermediate polarity column of different lengths (2, 5,and 10 m) in the second dimension (ZB-35, Phenomenex,Torrance, CA, USA). These column sets were abbreviated asIS-2, IS-5, and IS-10, respectively (IS = inverted set; 2, 5,and 10 = length of the second dimension column).GC×GC-FIDFirstly, and prior to analyzing the fatty acid content inthe broccoli SFE extracts by GC×GC-FID, fatty acidswere converted into their corresponding methyl estersusing Morrison ’ s method (Morrison and Smith 1964).Briefly, 2 mL of the extracts were hydrolyzed for 20 minat 90 °C in sealed tubes with 2 mL of KOH (1 M) inmethanol. Subsequently, 3 mL of BF 3  (14%) in methanolwas added to the mixture, which was heated again at 90 °C for 20 min. Finally, 2 mL of hexane was added andthe mixture was stirred for 15 s. The hexane layer waswithdrawn, partitioned twice against water and analyzed by GC×GC-FID.GC×GC experiments were performed on an Agilent 7890A GC apparatus (Agilent Technologies, CA, USA),equipped with an auto-sampler ALS 7683B injector that injected 1  μ  L at 250 °C in a pulsed split-less mode(Agilent Technologies, CA, USA). The injection pulse pressure was set at 31 psi until 0.75 min, the purgeflow to split vent worked at 50 mL/min at 0.75 min,meanwhile the septum purge flow and the gas saver flow were set at 3 and 15 mL/min (after 2 min),respectively. The GC×GC system was equipped with acapillary flow modulator (Agilent G3486A CFT Modulator)and a FID detector (operated at 260 °C and with a200 Hz data acquisition frequency). The air, nitrogen(make-up), and hydrogen flow (Carburos Metálicos;Barcelona, Spain) was 450, 20, and 25 mL/min for FID, respectively. The GC×GC parameters selected for each column set are shown in Table 1.It was necessary to use two different programs totransform and visualize the data. The Agilent ChemStationwas used to control the system and to acquire the data(version B.04.01, Agilent Technologies, CA, USA).Meanwhile, GC Image software was used to construct GC×GC contour plots from the modulated signals(version R1.9, Zoex Corp, Texas, USA). Results and Discussion Optimization of the GC×GC ConditionsThe chromatographic separation in GC×GC systems isinfluenced by four parameters: carrier gas flow in bothdimensions, modulation time and oven temperature (Guet al. 2010; Manzano et al. 2011). Hence, we studied how these four parameters affected the new column combina-tion employed here.In order to choose the GC×GC conditions that  provided the best FAME separation, several tests werecarried out with the distinct column sets and using thedifferent standard FAME mixtures to optimize the procedure. The conditions we reported previously weretaken as the starting point for each column set (Manzano et al.2011). The samples (1  μ  L) were injected at 250 °C in 922 Food Anal. Methods (2012) 5:920  –  927   pulsed split-less mode and the FID was operated at 260 °C, with a data rate of 200 Hz/0.001 min. Theseconditions were maintained constant because they did not significantly affect FAME separation. The initial oventemperature was programmed from 120 to 230 °C at 2 °C/min, and then to 260 °C at 20 °C/min (10 min),although it was found that with this program, it was not  possible to identify FAMEs with less than ten carbonatoms, so it was optimized to resolve this problem. Thestarting values for the modulation period and the first and second dimension carrier gas flows, were 1.40 sand 0.5 and 12 mL/min, respectively.The optimization of each column set was carried out  by varying only one of the most important parameters(oven temperature program, carrier gas flows in bothdimensions, and the modulation time), while the othersremained constant. Thus, the criterion employed toachieve the optimal conditions of this separation wasto find the best separation of the 47 standard FAMEs, primarily looking for the best separation and resolution between peaks of the two isomer mixtures (the linoleicand linolenic acids groups) in the shortest analysistime possible. The optimal values for each of the parameters studied were established using the threecolumn sets (Table 1) and there were no significant differences in terms of oven temperature, and the optimalvalues for the other parameters studied only differedslightly (e.g., 1D carrier gas flow and modulation time).By contrast, there was a much larger variation for the 2Dcarrier gas flow.It must be pointed out that the reproducibility of theretention times ( n =6) obtained was very good, with astandard deviation in the 1D less than 0.02 min and astandard deviation in the 2D less than 0.0002 min for theFAME analysis.Comparative Studies of the Column Sets Comparison Between Sets IS-2, IS-5, and IS-10 The influence of the second dimension column length onFAME separation was studied, which varied from 2 to10 m. After examining the results obtained, the elutionorder was identical in the three column sets, as would beexpected, with the non-saturated forms being eluted after the saturated ones. Regarding the geometric isomers, the trans  isomers were eluted first, followed by the  cis  –  trans and finally the  cis  isomers. When the FAMEs had the samenumber of carbons and unsaturated bonds, they eluted in adecreasing order according to their respective  “ n ”  values.It was noted that the retention time of each FAMEincreased when the 2D column length increased from 5 to10 m ( ∼ 3 min), whereas there was no important differenceswhen the column was extended from 2 to 5 m. Moreover,the peak shape and symmetry of the FAMEs improved withshorter 2D columns and the analysis time was faster.However, under these conditions the resolution betweenseveral FAME peaks was worse, and some pairs of peakswere not correctly identified when using the shortest 2 msecond dimension columns: C18:1(n9) and C18:1(n12);C18:2(n6;c9t12) and C18:2(n6;t9c12); C18:3(n3;c9t12c15);and C18:3(n3;t9c12c15). Hence, although a decrease in thelength of the second dimension column improved FAME peak shape and symmetry, as well as reducing the analysistime, the lack of resolution between some groups of FAMEs in this set up favored the use of the IS-5 set to Table 1  Optimized values of the most important GC×GC parametersGC×GC parameter Studied range Optimal valueIS-2 IS-5 IS-10First dimension column BPX-70 (30 m×0.25 mm×0.25  μ  m)BPX-70 (30 m×0.25 mm×0.25  μ  m)BPX-70 (30 m×0.25 mm×0.25  μ  m)Second dimension column ZB-35 (2 m×0.25 mm×0.25  μ  m)ZB-35 (5 m×0.25 mm×0.25  μ  m)ZB-35 (10 m×0.25 mm×0.25  μ  m)Oven temperature program 60 to 160 °C at 10 °C/min 60 to 150 °C at 10 °C/min 60 to 160 °C at 10 °C/min160 to 230 °C at 3 °C/min 150 to 230 °C at 3 °C/min 160 to 230 °C at 3 °C/min230 to 260 °C (5 min) at 20 °C/min230 to 260 °C (5 min) at 20 °C/min230 to 260 °C (6 min) at 15 °C/minFirst dimension carrier gasflow rate (mL/min)0.4  –  1.0 0.5 0.5 0.6Second dimension carrier gasflow rate (mL/min)10  –  25 15 20 22Modulation time (s) 1.40  –  2.00 1.50 1.70 1.70Food Anal. Methods (2012) 5:920  –  927 923  analyze broccoli leaves samples (The 3D chromatogramsobtained with the conditions showed in when using set IS-5are presented in Fig. 1). Under the conditions chosen, theseparation of the 47 FAMEs was achieved in less than39 min with this set up, including that of the linolenic andlinoleic group of isomers. The separation of these isomer groups is better seen in a close-up of the 3D chromatogramsof the area where these isomers eluted (Fig. 2). Comparison Between Set IS-5 and Previously Published  Methods The existing scientific literature regarding the separationand identification of FAMEs using GC and GC×GCequipped with thermal or CFT modulators was comparedwith the results obtained here. Despite the fact that one-dimensional GC separation of several FAME isomers issometimes faster than the method proposed (Harynuk et al.2006; Hauff and Vetter  2009), especially when using ionic liquid columns (Ragonese et al. 2009; Delmonte et al.2011; Ando and Sasaki 2011; Gu et al. 2011), the number  of FAMEs analyzed was smaller. Indeed, there are very few publications where linoleic and linolenic acids isomer mixtures were studied together (Bicalho et al. 2008;Ragonese et al. 2009; Delmonte et al. 2011; Sánchez-Ávila et al. 2009) or separately (Harynuk et al. 2006; Hauff and Vetter  2009), and only in our previous study (Manzano et al. 2011) were both groups of isomers simultaneouslyanalyzed with a large number of FAMEs.For example, an ionic liquid column length (12 to100 m) was seen to influence the separation of the twogroups of isomers, linoleic, and linolenic acids, amongother C18:1 isomers (10 FAMEs) over a time range between 6 and 70 min (Ragonese et al. 2009). However,the retention times and resolution of the eight linolenic acidisomers diminished in function of the column length, asevident with the 12 m column. Although the analysis timewas shorter in this case (6 min), the peaks were not separated at the baseline and some of them seemed tocoelute (Ragonese et al. 2009). In a separate study bothgroups of isomers were analyzed on the same type of columns in less time (Delmonte et al. 2011), and althoughonly FAMEs with carbon chain lengths between C14 andC22 were studied, they were not completely separated.Finally, the separation of all these isomers together withother FAMEs was improved by using cyano-basedcolumns, but these requiring much longer chromatographicruns lasting more than 60 min (Sánchez-Ávila et al. 2009).When compared to the existing GC×GC methods, longer (Gu et al. 2010; Villegas et al. 2010; Manzano et al. 2011) or shorter (Gu et al. 2011; Gardner et al. 2011) analysis times were used and in some cases, more FAMEs werestudied and including different groups of isomers. Using aGC×GC-FID with a cryogenic modulator, the suitability of conventional and inverted phase column sets to determineFAMEs was evaluated (Villegas et al. 2010). The resultswere better for the inverted phase column combinations,especially for the high polar   –  semi-polar arrangement,similar to that used here. However, the C18:1 isomer groupwas the main focal point of the study and thus, neither thelinolenic nor the linoleic acids groups of isomers werestudied, and longer analysis times were used. We recentlystudied the suitability of several column combinations(polar   –  nonpolar, nonpolar   –   polar) to determine a largenumber of FAMEs by GC×GC-FID with a CFT modulator,also assessing the separation of linoleic and linolenic acidisomers (Manzano et al. 2011). The polar   –  nonpolar arrange-ment (inverted phase) provided the best results of the columncombinations tested, although they required longer analysistimes ( ∼ 10 min longer), although fewer FAMEs were Fig. 1  GC×GC-FID 3D chromatogram of the standard mixture of 47 FAMEs employing column set IS-5. Column and chromatographicconditions described in GC×GC-FID section and Table 1924 Food Anal. Methods (2012) 5:920  –  927
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