headspace solid-phase microextraction combined with mass ...20solid-phase... · j. sep. sci. 2012,...

2282 J. Sep. Sci. 2012, 35, 2282–2296

Joao GoncalvesJose FigueiraFatima RodriguesJose S. Camara

CQM/UMa—Centro de Quımicada Madeira, Centro de CienciasExactas e da Engenharia,Universidade da Madeira,Campus Universitario daPenteada, Funchal, Portugal

Received March 6, 2012Revised April 16, 2012Accepted May 15, 2012

Research Article

Headspace solid-phase microextractioncombined with mass spectrometry as apowerful analytical tool for profiling theterpenoid metabolomic pattern ofhop-essential oil derived from Saaz variety

Hop (Humulus lupulus L., Cannabaceae family) is prized for its essential oil contents, used inbeer production and, more recently, in biological and pharmacological applications. In thiswork, a method involving headspace solid-phase microextraction and gas chromatography–mass spectrometry was developed and optimized to establish the terpenoid (monoterpenesand sesquiterpenes) metabolomic pattern of hop-essential oil derived from Saaz varietyas a mean to explore this matrix as a powerful biological source for newer, more se-lective, biodegradable and naturally produced antimicrobial and antioxidant compounds.Different parameters affecting terpenoid metabolites extraction by headspace solid-phasemicroextraction were considered and optimized: type of fiber coatings, extraction tem-perature, extraction time, ionic strength, and sample agitation. In the optimized method,analytes were extracted for 30 min at 40�C in the sample headspace with a 50/30 �m di-vinylbenzene/carboxen/polydimethylsiloxane coating fiber. The methodology allowed theidentification of a total of 27 terpenoid metabolites, representing 92.5% of the total Saazhop-essential oil volatile terpenoid composition. The headspace composition was domi-nated by monoterpenes (56.1%, 13 compounds), sesquiterpenes (34.9%, 10), oxygenatedmonoterpenes (1.41%, 3), and hemiterpenes (0.04%, 1) some of which can probably con-tribute to the hop of Saaz variety aroma. Mass spectrometry analysis revealed that the mainmetabolites are the monoterpene �-myrcene (53.0 ± 1.1% of the total volatile fraction), andthe cyclic sesquiterpenes, �-humulene (16.6 ± 0.8%), and �-caryophyllene (14.7 ± 0.4%),which together represent about 80% of the total volatile fraction from the hop-essential oil.These findings suggest that this matrix can be explored as a powerful biosource of terpenoidmetabolites.

Keywords: Essential oil / GC-qMS / Hop Saaz variety / HS-SPME / TerpenoidmetabolitesDOI 10.1002/jssc.201200244

1 Introduction

It is well known that plant-derived natural products are exten-sively used as biologically active compounds. From these, theessential oils, which represent a small fraction of a plant’scomposition, and some of their constituents are used notonly in pharmaceutical products for their therapeutic activi-ties but also in agriculture, as food preservers and additivesfor human or animal use, in cosmetics and perfumes, andother industrial fields [1, 2]. Particular emphasis has been

Correspondence: Professor Jose S. Camara, Centro de CienciasExactas e da Engenharia, Universidade da Madeira, Campus Uni-versitario da Penteada, 9000–390 Funchal, PortugalE-mail: [email protected]: +351-291705149

Abbreviations: HS-SPME, headspace solid phase microex-traction; RI, retention index

placed on their antibacterial, antifungal, and insecticidal ac-tivities [3]. In many cases, they serve as (i) plant defensemechanisms against predation by microorganisms, insects,and herbivores; (ii) metal transporting agents; (iii) agents ofsymbiosis between microbes and plants, nematodes, insects,and higher animals; (iv) sexual hormones; and (v) differenti-ation effectors [4,5]. Levels of secondary metabolites in plantsare both environmentally induced as well as genetically con-trolled [6].

Among the thousands of metabolites produced by plants,terpenoids represent, by far, the largest and the most diverseclass of secondary metabolites, followed by alkaloids and phe-nolic compounds [7]. They have received special attentionby the scientific community, due to their useful and widerange of biological and pharmacological activities [2,8]. Somemonoterpenes, such as �-pinene, cineole, eugenol, limonene,terpinolene, citronellol, citronellal, camphor, and thymol, arecommon constituents of a number of essential oils described

C© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

J. Sep. Sci. 2012, 35, 2282–2296 Other Techniques 2283

Figure 1. Biosynthetic pathways of terpenoid metabolites [2].

in the literature, as presenting mosquito repellent activity [9].Among sesquiterpenes, �-caryophyllene is most cited as astrong repellent against Aedes aegypti [10]. Although repel-lent properties of several essential oil regularly appear to beassociated with the presence of monoterpenes and sesquiter-penes [9, 11], other authors have found that farnesol has awide spectrum of desirable biological properties includingantitumor [12, 13], antioxidant [14], antifungal, and antibac-terial effects [15]. Moreover, farnesol has been demonstratedto selectively inhibit monoamine oxidase B of rat brain [16],a possible role for farnesol in prevention of Parkinson dis-ease [3]. Some others isoprenoids show antiviral (e.g. saponinand glycyrrhizin) [17], antihyperglycemic (e.g. stevioside) [18],anti-inflammatory (e.g. linalool) [19], and antiparasitic (e.g.artemisinin) [20] activities. The biosynthesis of the terpenoidcompounds in the essential oil uses the same building blocks

as required for the isoprenyl side chains of the hop resins(Fig. 1).

Essential oils represent a small fraction of the composi-tion of plants but confer the characteristics for which aromaticplants are used in the pharmaceutical, food, cosmetic, and fra-grance industries [21]. Are complex mixtures containing froma few dozen to several hundred volatile organic compoundsproduced as secondary metabolites in plants: they are consti-tuted by hydrocarbons (monoterpenes and sesquiterpenes)and oxygenated compounds (alcohols, esters, ethers, alde-hydes, ketones, lactones, phenols, and phenol ethers) [3].Their composition may vary considerably between aromaticplant species and varieties, and within the same variety fromdifferent geographic areas [22]. Frequently, both hydrocar-bons and oxygenated compounds are responsible for the dis-tinctive characteristic odors and flavors of plants.


2284 J. Goncalves et al. J. Sep. Sci. 2012, 35, 2282–2296

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Over centuries, hop (Humulus lupulus L.) was used pri-marily as an essential ingredient in the manufacturing ofbeer since its components add the typical bitter taste and con-tribute to the attractive aroma of the final beverage. Essentialoil of hop comprises two major fractions: the first belongs tothe group of hydrocarbons of which terpene hydrocarbons ac-count for about 70% [25]. The remaining 30% are compoundscontaining oxygen (oxygenated fraction that is generally morearomatic and less volatile) such as esters, aldehydes, ketones,acids, and alcohols [3]. Different hop varieties produce differ-ent essential oils that can have widely distinct taste, odor, andaroma, depending on their chemical nature (Table 1 ).

Geographical location, climate, and agronomical factorsalso affect the oil composition, potentially creating differentprofiles for hop samples with the same genetic material [3].

In recent years, essential oil has received much attentionas potentially useful bioactive compounds against insects. Al-though effective, the constant application of pesticides to con-trol insects, can disrupt the natural biological control systemsand has led to outbreaks of insect species, which sometimesresulted in the widespread development of resistance, hadundesirable effects on nontarget organisms, and fostered en-vironmental and human health concerns. These problemshave highlighted the need for the development of new strate-gies for selective and specific pest control. Furthermore, thedifferent activities of aromatic plants essential oils, such as an-timicrobial, antiviral, and anticarcinogenic activities, explaintheir broad use in phytotherapy [26]. Particularly, the antimi-crobial activity has formed the basis of many applications,including raw and processed food preservation, pharmaceu-tical, alternative medicine, and natural therapies. Accordingto Bozin et al. [26], this aspect assumes a unique relevancedue to an increased resistance of some bacterial strains to themost common antibiotics and antimicrobial agents for foodpreservation.

A range of extraction and concentration methods havebeen developed for the analysis of essential oil, which in-clude steam distillation [27] or extraction with a conventionalsolvent [28], supercritical fluid CO2 extraction [29], columnchromatography [30], and stir bar sorptive extraction [31].Nevertheless, these techniques present certain nonnegligi-ble drawbacks such as the use of high volumes of solvent, thetime required, and the use of expensive devices with a lim-ited lifetime that may entail carry-over or cross-contaminationproblems. Consequently, in order to overcome these draw-backs, solid-phase microextraction (SPME) has emerged asan efficient extraction-preconcentration method and a reliablealternative to traditional sample preparation techniques, dueto important features such as simplicity, low cost, selectivity,and sensitivity when combined with appropriate detectionmodes [32–37]. This method, developed by Pawliszyn andco-workers [38, 39], eliminates the use of organic solvents,and substantially shortens the time of analysis. SPME canintegrate sampling, extraction, concentration, and sample in-troduction into a single uninterrupted process, resulting inhigh sample throughput and also be used as a solvent-freesample preparation method with gas chromatography (GC)

mass spectrometry (MS) analysis, which has been success-fully applied for profiling the metabolomic pattern of fruits[40–44], and analysis of environmental [45], food [40, 42, 44],forensic [46], and pharmaceutical samples [47] and also as apowerful technique for extraction of urinary potential cancerbiomarkers [48, 49].

In the present communication, we report on usingSPME, in headspace mode (HS-SPME), coupled to GC-qMS(quadrupole first stage mass spectrometry) as a powerfulmethodology to investigate the metabolomic pattern of ter-penoid composition in hop-essential oil derived from Saazvariety as a mean to explore, in a near future, these matrixas a powerful biological source of antimicrobial (antibacte-rial and antifungal) and antioxidant agents, constituting anenvironmentally friendly alternative as potential substitutesfor synthetic compounds. Important SPME experimental pa-rameters that may affect extraction efficiency, namely, na-ture of fiber coating, extraction temperature, extraction time,ionic strength, and sample agitation, were considered on thisstudy. The optimized conditions were applied to the char-acterization of the terpernoid metabolites in hop-essentialoil from Saaz variety. The method is simple, requires smallamounts of sample, and was expected to provide global ter-penoid metabolomic signature of hop-essential oil while of-fering a significant time reduction when compared to othermethods commonly used.

2 Materials and methods

2.1 Reagents and materials

The SPME holder for manual sampling and the fibers usedwere purchased from Supelco (Bellefonte, PA, USA). Am-ber silanized glass vials (4.0 mL) were obtained from AgilentTechnologies (Palo Alto, CA, USA). According to manufac-turer’s recommendation, the fibers were first conditioned inthe GC injection port to remove fiber contaminants. Priorto extraction, the fiber was, daily, inserted in the hot injec-tion port for 6 min. A blank test was performed to checkpossible carry-over. The Kovat’s retention index (RI) wascalculated through injection of a series of C8–C20 straight-chain n-alkanes (concentration of 40 mg/L in n-hexane) pur-chased from Fluka (Buchs, Switzerland). Sodium chloride, ofanalytical grade, was purchased from Panreac Quimica SA(Barcelona, Spain).

2.2 Samples

Five hop-essential oil samples, obtained by supercritical CO2

extraction, were kindly provided by Empresa de Cervejasda Madeira (ECM), Madeira Island, Portugal. Samples weretransported under refrigeration (ca. 2–5�C) to the laboratoryand stored at −20�C until analysis. All samples were analyzedin triplicate.



2.3 HS-SPME extraction conditions

Right before analysis, samples were thawed at 20�C for 10min and then were subjected to HS-SPME. Extraction wascarried out using 0.5 g of hop-essential oil into a 4-mLglass HS vial. The samples were equilibrated during theincubation time (10 min in all assays) in a temperature-controlled six-vial agitator tray at the appropriate temper-ature and time (selected according to the optimization de-sign). Subsequently, the SPME fiber was manually insertedinto the sealed vial through the septum and the fiber wasexposed to the sample HS for a specific extraction time andextraction temperature. Following the extraction process, thefiber was retracted prior to remove from the sample vial andimmediately inserted into the GC-qMS injector for thermaldesorption of metabolites at 250�C for 6 min in splitlessmode. All measurements were made with, at least, threereplicates.

2.4 Optimization of SPME parameters

The effectiveness of analyte preconcentration using theSPME technique depends on several experimental parame-ters, from which the fiber coating, extraction time, and extrac-tion temperature are the most significant. For this reason, theextent to which each of these variables affects the efficiency ofSPME procedure was examined by application of univariateoptimization design.

2.4.1 Selection of the fiber coating

In the preliminary selection, all commercially avail-able silica SPME fibers, varying in polarity, thicknessof the stationary phase, and coated with the follow-ing polymers: polydimethylsiloxane (PDMS, 100 �m),PDMS/divinylbenzene (PDMS/DVB, 65 �m), DVB/carboxenon PDMS (DBV/CAR/PDMS; StableFlex, 50/30 �m),CAR/PDMS (CAR/PDMS, 75 �m), polyacrylate (PA,85 �m), and polyethyleneglycol (PEG, 60 �m) were testedin order to select the best polymer to extract the terpenoidmetabolites. In this step, all the fibers were exposed tothe sample HS under the following conditions: 10 min ofequilibrium time, 30 min of extraction time, and 40�C forextraction temperature (conditions arbitrarily establishedby the authors in the choice-of-fiber step). Fibers werethermally conditioned in accordance with the manufacture’srecommendations before first use. Before the first dailyanalysis, and in order to guarantee the absence of peaks inthe run blanks and the good quality of the SPME extractionand chromatographic procedures, each of the fibers wasreconditioned at 250�C for 15 min, following the manu-facturer’s recommendations. All the fibers were tested intriplicate and the results presented represent the mean valuesobtained.

2.4.2 Effect of extraction time and temperature

Extraction time and temperature are two of the most im-portant parameters affecting the volatility of analytes. There-fore, these two parameters were optimized. The proceduredescribed in Section 2.3 was employed to evaluate the ex-traction time and temperature. The HS-SPME extraction ofthe hop-essential oil samples (CO2 supercritical extract) wasdone using fiber exposure times between 15 and 60 min us-ing DVB/CAR/PDMS fiber at 40�C. In order to optimize theextraction temperature, up to three consecutive extractionswere carried out at each of the following temperatures: roomtemperature (24�C), 30, 40, and 50�C using DVB/CAR/PDMSfiber for 30 min.

2.5 GC-qMS conditions

The SPME-coating fibers containing the adsorbed terpenoidmetabolites extracted from the hop-essential oil were manu-ally introduced into the GC injection port at 250�C and keptfor 6 min for desorption. The split/splitless injector, operat-ing in the splitless mode, was equipped with an inlet liner forSPME (internal diameter 0.75 mm i.d., Supelco, Barcelone,Spain). The desorbed terpenoid metabolites were separatedin an Agilent Technologies 6890N Network GC equippedwith a BP-20 fused silica capillary column (30 m × 0.25 mmi.d. × 0.25 �m film thickness) supplied by SGE (Darmstadt,Germany) connected to an Agilent 5973N quadrupole massselective detector. Helium (Air Liquid, Portugal) was used ascarrier gas at 1.1 mL/min constant flow (column head pres-sure: 12 psi). The injections were performed in the splitlessmode (5 min). The GC oven temperature was programmed asfollows: 40�C for 1 min, 1.7�C/min ramp until 180�C (1 min)then to 220�C at 30�C/min and held isothermally at 250�Cfor a further 1 min. For the MS system, the temperatures ofthe transfer line, quadrupole, and ionization source were 250,180, and 230 ³C, respectively; electron impact mass spectrawere recorded at 70 eV and the ionization current was about30 �A. Data acquisitions were performed in scanning mode(mass range m/z 30–300; 6 scans per second). The GC peakarea of each compound was obtained from the ion extractionchromatogram by selecting target ions for each one. Repro-ducibility was expressed as relative standard deviation (RSD).Signal acquisition and data processing were performed usingthe HP Chemstation (Agilent Technologies).

Terpenoid metabolites identification was based on (i)comparison of the GC retention time and mass spectra, withthose, when available, of the pure standard compounds; (ii)comparison between the MS for each putative compoundwith those of the data system library (NIST, 2005 software,Mass Spectral Search Program V.2.0d; NIST 2005, Washing-ton, DC, USA); and (iii) Kovat’s RI determined according tothe Van den Dool and Kratz [50]. For the determination of theRI, a C8–C20 n-alkanes series was used, and the values werecompared, with available values reported in the literature for



similar chromatographic columns. All Identity SpectrumMach factor above 850 resulting from the NIST Identity Spec-trum Search algorithm (NIST MS Search 2.0) was determinedto be acceptable for positive identification.

Monoterpenes and specially sesquiterpenes are notori-ously difficult to resolve and identify because they have thesame molecular formulae and therefore interact with columnstationary phases in the same manner and exhibit very similarmass spectra.

3 Results and discussion

The influence of the main parameters that can affect the HS-SPME process from HS, i.e. fiber coating, extraction temper-ature, extraction time, ionic strength, and sample agitation,was evaluated. HS-SPME mode was used instead of directsampling mode because, for volatile analytes, in the formermode the equilibrium times are shorter compared to directextraction. The HS mode also protects the fiber from ad-verse effects caused by nonvolatile, high molecular weightsubstances present in the sample matrix. Temperature hasa significant effect on the extraction kinetics, since it deter-mines the vapor pressure of the analytes, and for that theirinfluence in the extraction process was also investigated. Inthe optimized method, analytes were absorbed for 30 min at40�C in the sample HS with a 50/30 �m DVB/CAR/PDMSfiber. The best conditions obtained for HS-SPME/GC-qMSmethodology was chosen based on intensity response (GCpeak area), number of identified compounds, and RSD (RSD,%). After the optimization step, the terpenoid metabolomicsprofile of the hop-essential oil derived from Saaz variety wasestablished.

3.1 Optimization of HS-SPME parameters

The optimization of the different parameters involved in HS-SPME was performed choosing the conditions that allowedobtaining the maximum response in terms of analyte peakarea.

3.1.1 Fiber-coating selection

The selection of a suitable fiber coating is an important step inSPME optimization. The sensitivity of the SPME extractiontechnique depends greatly on the value of the distributionconstant of analytes partitioned between the sample and fiber-coating material. For this reason, six different types of SPMEfibers were evaluated in this study, in order to assess that thecoating having highest affinity toward terpenoid metabolites.The comparison of the SPME fiber performance was basedon extraction efficiency, estimated by total peak area, numberof isolated compounds from the extract, and reproducibility.Table 2 reports the results of the relative extraction efficiencyof the six SPME fibers with respect to their capacity to ex-tract the terpenoid metabolites of the Saaz hop-essential oil.

Each fiber was exposed to the HS under the same conditionsof equilibrium time (10 min), extraction time (30 min), andtemperature (40�C), and although the extraction conditionswere the same, the differences in the areas obtained revealedthe behavior of each type of coating used for each fiber tested(Table 2). The results of this screening showed that the high-est extraction sensitivity was obtained with the CAR-relatedstationary phase. Although the means of the total areas ob-tained for the PDMS, DVB/CAR/PDMS, and CAR/PDMSfibers did not present significant statistical differences (Tukeyat P < 0.05), the fiber DVB/CAR/PDMS was chosen, since itpresented the best extraction efficiency for a highest numberof terpenoid metabolites (Table 2). The good performancesobtained with fibers containing PDMS coating were partiallyexpected since PDMS is a lypophilic coating, so with a higheraffinity than the partially polar PA and PEG, for nonpolarmolecules such as terpenoid metabolites.

Conversely, the lowest sorption capacity expressed aschromatographic areas (P < 0.05) were in general obtainedwith the PA fiber under the same experimental conditions.

DVB/CAR/PDMS coating (molecular weight rangingfrom 35 to 300) combines the absorption properties of theliquid polymer with the adsorption properties of porous par-ticles, which contains macro- (>500 A), meso- (20–500 A), andmicroporous (2–20 A), and has bipolar properties. The mu-tually synergetic effect of adsorption and absorption of thestationary phase explains its high retention capacity. Basedon the data evaluation completed within this particular opti-mization experiment, DVB/CAR/PDMS fiber was chosen tobe used for all further optimization steps and hop-essentialoil analysis experiments, without adding salt and without ag-itation of the sample. Using the DVB/CAR/PDMS 50/30 �mfiber, the addition of salt and the agitation of the sample led toa decrease of chromatographic peak areas (P < 0.05) for someanalytes. Similar results were obtained by Laura Campo et al.[51] in the quantification of 13 priority polycyclic aromatichydrocarbons in human urine by HS-SPME GC and isotopedilution MS.

3.1.2 Extraction time and temperature

Extraction time and temperature are very important experi-mental factors to define the optimum extraction conditionsfrom the HS. Since time affects the mass transfer of the an-alytes onto the fiber, optimum time is required for the fiberto reach equilibrium with HS. To study the effects of extrac-tion time, Saaz hop-essential oil samples were extracted forpredetermined extraction times ranging from 15 to 60 min at40�C. The results are shown in Fig. 2A. A typical extractiontime profile consists of an initial rapid portioning followed bya slower prolonged uptake and finally a steady-state equilib-rium between the fiber and the vapor phase of the analyte. Ascan be observed, over 30 min, no significant increase in the re-sponse was observed. Moreover, 30 min showed excellent re-producibility (RSD = 1.0%) when compared with 45 (RSD =8.4%) and 60 min (RSD = 6.8%).


J. Sep. Sci. 2012, 35, 2282–2296 Other Techniques 2289Ta

ble

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Figure 2. Influence of the extraction time on the extraction ef-ficiency of terpenoid metabolites by HS-SPME (fiber 50/30 �mDVB/CAR/PDMS, extraction temperature of 40�C), expressed as(A) total peak area; and (B) profile of major terpenoid metabolites(a.u. arbitrary units).

The extraction time profile of the major terpenoidmetabolites in essential oil from hop of Saaz variety is repre-sented in Fig. 2B. For some analytes, higher chromatographicresponses were observed for longer sampling time. The peakarea for �-myrcene decreased with time, while �-humuleneand �-caryophyllene reach the steady-state equilibrium at 30and 45 min, respectively. Considering the results for the 27identified terpenoid metabolites, an extraction time of 30 minwas chosen as a good compromise between obtaining an op-timized chromatographic signal and a reasonable analysistime. In addition, the lower extraction time can extend thelifetime of the SPME fiber.

The extraction temperature presents several effects onextraction efficiency. The temperature increases diffusion co-efficients and Henry’s constants while the time required toreach equilibrium decreases [54]. To evaluate the effect of tem-perature on SPME extraction efficiency, different extractiontemperatures (24, 30, 40, and 50�C) were investigated. Theresults concerning total GC-qMS peak area as a function oftemperature are illustrated in Fig. 3A. It can be observed thatthe extracted amount increases with the increase of the ex-traction temperature. Increase in extraction temperature willimproved the mobility of volatile compounds through liquid

Figure 3. Effect of the extraction temperature on the extractionefficiency of terpenoid metabolites from Saaz hop-essential oilby HS-SPME (fiber, 50/30 �m DVB/CAR/PDMS, extraction time, 30min; (A) total peak area of the terpenoid fraction; and (B) profileof the major terpenoid metabolites (a.u. arbitrary units).

and gas phases leading to an increase in extraction amounts.However, increasing temperature over 30�C, no significantincrease in the total response was observed. In addition, inwhat concerns the GC-qMS response (based on peak areas)as a function of temperature (Fig. 3A), a high reproducibilitywas obtained at 40�C (RSD = 2.9%) in comparison to 30�C(RSD = 11.4%) and 50�C (RSD = 16.4%), respectively; there-fore, 40�C was selected as extraction temperature for furtherstudies.

The absorption kinetics at different temperature of themost abundant terpenoid metabolites absorption found inhop-essential oil is shown in Fig. 3B. As well for extractiontime, and taking into account the three major compounds(�-myrcene, �-humulene, and �-caryophyllene), it can be ob-served that an extraction temperature of 30�C afforded thehighest extraction sorption for �-myrcene, in contrast to �-humulene and �-caryophyllene that extraction efficiency washighest when the HS-SPME extraction was performed at50�C. From these findings, an absorption temperature of 40�Cwas chosen in order to maximize the analytical response ofall compounds.

The conditions selected as optimal for the establishmentof the terpenoid metabolomics pattern from hop-essential oil



Figure 4. (A) A typical GC-qMS chromatogram of volatile fraction of the Saaz hop-essential oil isolated by HS-SPME (extraction conditions:DVB/CAR/PDMS fiber at 40�C during 30 min; for GC-qMS conditions see Section 2.5, for details on peaks identities see Table 2); and (B)pattern of the Saaz hop-essential oil terpenoid fraction obtained by HS-SPMEDVB/CAR/PDMS/GC-qMS methodology.

of Saaz variety were 50/30 mm DVB/CAR/PDMS fiber at40�C for 30 min.

3.1.3 Determination of terpenoid metabolites in

hop-essential oil derived from Saaz variety by

HS-SPMEDVB/CAR/PDMS/GC-qMS

The optimized HS-SPME/GC-qMS methodological condi-tions were used for profiling the terpenoid metabolomicspattern of the hop-essential oil from Saaz variety. A char-acteristic GC-qMS profile of Saaz hop-essential oil obtainedwith a DVB/CAR/PDMS fiber using the experimental opti-mized conditions is shown in Fig. 4A.

A total of 27 terpenoid metabolites (Table 2 and Fig. 4B)were identified in the HS of the essential oil from the Saazhop variety. Among these, 60.7% were monoterpene hydro-carbons, 37.7% were sesquiterpenes hydrocarbons, and it

also contained 1.52% oxygenated monoterpenes and 0.04%hemiterpenes. Table 2 shows the identified terpenoid metabo-lites, their RI values listed in order of elution on a BP-20capillary column, and the relative composition. The chemicalstructures of the terpenoid metabolites in the essential oil ofhop from Saaz variety are summarized in Fig. 5.

The major constituents in the hop-essential oil from Saazvariety were the monoterpene (5) �-myrcene (53.0 ± 1.1%of the total volatile fraction), and the cyclic sesquiterpenes(16) �-humulene (16.6 ± 0.8%) and (17) �-caryophyllene(14.7 ± 0.4%), which together account for 84.3 ± 1.5% of thevolatile essential oil. Metabolites found at low content include(4) �-pinene (1.8%), (19) methyl geranate (1.4%), (10) �-cis-o-cymene (0.7%), (22) (+)-�-cadinene (0.7%), and (6) limonene(0.5%). The results are according to Nance and Setzer [52],who reported that the main constituents of hop-essential oilderived from Saaz variety are �-myrcene, �-humulene, and�-caryophyllene.



Figure 5. Chemical structures of terpenoid metabolites determined in hop-essential oil from Saaz variety.

These compounds are interesting from the standpointof pharmacological, because of their antimicrobial activity[55] as well as to their anti-inflammatory effects [56]. On theother hand, limonene (6) and �-pinene (4) have been reportedas antitumor [57] and antimicrobial [55] agents, respectively.Biological activities and odor description of some plant sec-ondary metabolites found in hop-essential oil derived fromSaaz variety are summarized in Table 3.

The odor threshold of �-myrcene (5) in water has beendetermined to range between 13 and 36 ppb [69], and so isexpected to exert a large impact on the odor profile of the es-sential oils. This has been supported in studies using GC-O[3]. It has odor descriptors of resinous, herbaceous, balsamic,and geranium-like [3]. �-Myrcene (5) and essential oils con-taining this monoterpene have been widely used as scentingagents in cosmetics, soaps, detergents, and as flavoring addi-tives in food and beverages. Lorenzetti et al. [70] reported that�-myrcene is a peripheral analgesic substance and the active

ingredient in lemongrass (Cymbopogon citratus) tea. This po-tion is widely used in folk medicine to treat gastrointenstinaldisturbances and as a sedative and antipyretic [3]. Farnesenewas found to be present in some cultivars but not in others.It was not found in the oils of Saaz hops. Dehydration of�-terpineol gives limonene (6), the major hydrocarbon of cit-rus oils but also present in hop-essential oil. Limonene (6) isprobably the precursor of the bicyclic monoterpenes such as�- (2) and �-pinene (4) and camphene (3). Limonene (6) canalso disproportionate into o-cymene (11) [3].

4 Concluding remarks

A HS-SPMEDVB/CAR/PDMS/GC-qMS methodology was devel-oped that allowed to profiling the terpenoid metabolomic pat-tern of hop-essential oil from Saaz variety.



Ta

ble

3.

So

me

sele

cted

exam

ple

so

fth

eb

iolo

gic

alac

tivi

tyan

do

do

rd

escr

ipti

on

of

terp

enic

met

abo

lites

det

erm

ined

ines

sen

tial

oil

fro

mS

aaz

vari

ety

[53,

58–6

8]

Terp

ene

clas

sM

etab

olite

Odor

desc

riptio

nBi

olog

ical

activ

ityRe

fere

nces

Hem

iterp

ene

Isop

rene

Pene

tratin

gpe

trole

um-li

keod

orTh

erm

otol

eran

ce.

[58]

Tole

ranc

eof

ozon

ean

dot

herr

eact

ive

oxyg

ensp

ecie

s."S

afet

yva

lve"

toge

trid

ofun

wan

ted

met

abol

ites.

Mon

oter

pene

�-P

inen

eTe

rpen

ic,h

arsh

-pin

e-lik

e,w

eak

herb

alRe

pels

the

spru

cebe

etle

Dend

roct

onus

rufip

enni

sat

high

conc

entra

tions

,but

inte

rmed

iate

conc

entra

tions

elic

iten

tryan

dga

llery

cons

truct

ion.

[58]

Elic

itsol

fact

ory

rece

ptor

neur

ons

ofth

ew

eevi

lPis

sode

sno

tatu

s.En

hanc

esat

tract

ion

byTh

anas

imus

dubi

us,P

laty

som

acy

lindr

ica,

and

Corti

ceus

para

llelu

sto

the

pher

omon

esof

thei

rIps

prey

.M

onot

erpe

neCa

mph

ene

Cam

phor

aceo

us-o

ilyHa

vebe

enre

porte

dto

poss

ess

biol

ogic

alac

tivity

agai

nstG

ram

-pos

itive

and

Gram

-neg

ativ

eba

cter

ia.

[59]

Mon

oter

pene

�-P

inen

eRe

sino

us-p

iney

High

/med

ium

antim

icro

bial

effe

cts

agai

nstb

oth

Gram

-pos

itive

bact

eria

Stap

hylo

cocc

usau

reus

,Ent

eroc

occu

sfa

ecal

is,a

nda

high

activ

ityag

ains

tthe

Gram

-neg

ativ

eba

cter

iaEs

cher

ichi

aco

lian

dPr

oteu

svu

lgar

isas

wel

las

am

ediu

mon

eag

ains

tCan

dida

albi

cans

.

[55]

Mon

oter

pene

�-M

yrce

neRe

sino

us,h

erba

ceou

s,ba

lsam

ic,g

eran

ium

-like

1Hi

ghan

timic

robi

alac

tiviti

eson

lyag

ains

tEsc

heric

hia

coli

and

Prot

eus

vulg

aris

.[5

5]Ex

hibi

tgoo

dre

pelle

ntac

tivity

.M

onot

erpe

neLi

mon

ene

Mild

lyci

trus,

free

from

,cam

phor

aceo

usLo

tus

japo

nicu

spl

ants

infe

sted

with

two-

spot

ted

spid

erm

ites

(Tet

rany

chus

urtic

ae).

[58]

Sele

ctio

nof

the

ovip

ositi

onsi

teby

pred

ator

yho

verfl

ies

relie

son

the

perc

eptio

nof

avo

latil

ebl

end

com

pose

dof

prey

pher

omon

ean

dty

pica

lpla

ntgr

een

leaf

vola

tiles

.El

icits

olfa

ctor

yre

cept

orne

uron

sof

the

wee

vilP

isso

des

nota

tus.

Invo

lved

inth

ese

lect

ive

herb

ivor

yon

the

coni

ferP

inus

carib

aea

byth

ele

afcu

tting

antA

ttala

evig

ata.

Lim

onen

eha

sbe

ende

scrib

edin

the

liter

atur

eas

chem

opre

vent

ive

and

ther

apeu

ticag

ent

agai

nstm

any

tum

orce

lls.

Mon

oter

pene

�-P

hella

ndre

neHe

rbac

eous

,min

tyba

ckgr

ound

Have

been

repo

rted

due

toef

fect

inin

hibi

tion

the

path

ogen

Botry

tisci

nere

a.[5

8]M

onot

erpe

ne�

-tran

s-Oc

imen

eFr

esh-

terp

enic

,wea

kci

trus-

herb

alEx

posu

reof

Arab

idop

sis

thal

iana

toth

em

onot

erpe

neca

uses

incr

ease

dab

unda

nce

ofse

vera

lge

netra

nscr

ipts

and

incr

ease

dpl

antr

esis

tanc

eag

ains

tthe

path

ogen

Botry

tisci

nere

a.[5

8]

Gene

sof

the

octa

deca

noid

path

way

and

gene

skn

own

tore

spon

dto

octa

deca

noid

sar

eam

ong

the

mos

tpre

vale

ntw

ithin

the

stre

ss-g

ene

cate

gory

upre

gula

ted

inAr

abid

opsi

s.Th

e�

-oci

men

esy

ntha

seis

indu

ced

inLo

tus

japo

nicu

spl

ants

infe

sted

with

two-

spot

ted

spid

erm

ites

(Tet

rany

chus

urtic

ae).

Mon

oter

pene

�-T

erpi

nene

Herb

aceo

us,c

itrus

Have

been

repo

rted

topo

sses

sa

pote

ntre

pelle

ntac

tivity

.[6

0]M

onot

erpe

neo-

Cym

ene

n.i.a)

Reve

algo

odan

tibac

teria

lact

ivity

and

stro

ngan

tifun

galp

rope

rties

.[6

1]M

onot

erpe

ne�

-Ter

pino

lene

Plea

sant

,sw

eet-p

iney

,som

ewha

ttur

pent

ine

Exhi

biti

nsec

trep

elle

ntac

tivity

.[6

2]Pr

esen

tahi

gher

rela

tions

hip

with

the

antic

ance

ract

ivity

.



Ta

ble

3.

Co

nti

nu

ed

Terp

ene

clas

sM

etab

olite

Odor

desc

riptio

nBi

olog

ical

activ

ityRe

fere

nces

Sesq

uite

rpen

eYl

ange

neFr

uity

Ylan

gene

has

been

iden

tified

aska

irom

one

attra

ctan

tsfo

rmal

eM

edfly

.[6

3]Se

squi

terp

ene

�-C

ubeb

ene

Mild

wax

y,w

oody

�-C

ubeb

ene

has

been

iden

tified

aska

irom

one

attra

ctan

tsfo

relm

bark

beet

les

(Cur

culio

nida

e:Sc

olyt

inae

:Sco

lytu

ssp

p.).

[64]

Sesq

uite

rpen

e�

-Hum

ulen

eSo

ft-w

oody

,rem

indi

ngto

fresh

earth

,bal

sam

icPr

oduc

edin

high

amou

nts

inre

spon

seto

sim

ulta

neou

she

rbiv

ory

byth

epi

erci

ng–s

ucki

ngin

sect

wes

tern

flow

erth

rips

Fran

klin

iella

occi

dent

alis

and

the

chew

ing

herb

ivor

eHe

lioth

isvi

resc

ens.

[55,

58]

Prod

uced

bya

reco

mbi

nant

inse

ct-in

duce

dge

ne(A

lCar

S)w

ithhi

ghse

quen

cesi

mila

rity

toth

eflo

rally

expr

esse

d(E

)-�-c

aryo

phyl

lene

synt

hase

.M

ediu

man

timic

robi

alef

fect

sag

ains

tthe

Gram

-pos

itive

bact

eria

Ente

roco

ccus

faec

alis

and

Gram

-neg

ativ

eba

cter

iaEs

cher

ichi

aco

li,Ps

eudo

mon

asae

rugi

nosa

,and

Salm

onel

lasp

.Re

veal

anim

porta

ntan

ti-in

flam

mat

ory

prop

erty

.Se

squi

terp

ene

�-C

aryo

phyl

lene

Woo

dy,s

picy

Elic

itsel

ectro

ante

nnog

ram

resp

onse

s.[5

5,58

]In

volv

edin

inse

ctho

stlo

catio

n.In

volv

edin

the

sele

ctiv

ehe

rbiv

ory

onth

eco

nife

rPin

usca

ribae

aby

the

leaf

cutti

ngan

tAtta

laev

igat

a.Be

low

grou

ndsi

gnal

emitt

edby

inse

ctda

mag

edm

aize

root

s.In

duce

dby

apl

antp

atho

gen

and

perc

eive

dby

itsve

ctor

inse

ct,t

heph

loem

-feed

ing

psyl

lidCa

cops

ylla

pict

a.Bi

otra

nsfo

rmed

bypl

ant-h

oste

dba

cter

ia.

Rele

ased

byAr

abid

opsi

sup

onin

sect

feed

ing.

Stro

ngre

pelle

ntac

tivity

agai

nstA

.aeg

ypti.

High

antim

icro

bial

activ

ities

agai

nstt

heGr

am-n

egat

ive-

bact

eria

Pseu

dom

onas

aeru

gino

sa,

Prot

eus

vulg

aris

and

Salm

onel

lasp

.as

wel

las

med

ium

effe

cts

agai

nstt

heGr

am-p

ositi

veba

cter

iaEn

tero

cocc

usfa

ecal

isan

dth

eGr

am-n

egat

ive

bact

eria

Esch

eric

hia

coli.

Exhi

bita

nim

porta

ntan

ti-in

flam

mat

ory

prop

erty

.Se

squi

terp

ene

�-M

uuro

lene

Oily

,Her

bace

ous

Exhi

bita

ntifu

ngal

and

antib

acte

riala

ctiv

ity.

[53]

Oxy-

mon

oter

pene

Met

hylg

eran

ate

Flow

er,g

reen

,fru

itM

ethy

lger

anat

eha

sbe

enid

entifi

edas

pher

omon

epr

oduc

edby

Chlo

roch

roa

sayi

mal

es.

[65]

Sesq

uite

rpen

e�

-Sel

inen

eW

eak

spic

y,ba

lsam

ic,m

ildEx

hibi

tant

ifung

alan

dan

tibac

teria

lact

ivity

.[5

3]Se

squi

terp

ene

�-M

uuro

lene

Woo

dyPo

sses

san

tifun

gala

ctiv

ityag

ains

tCla

dosp

oriu

mcu

cum

erin

um.

[66]

Sesq

uite

rpen

e(+

)-�-C

adin

ene

Herb

aceo

usHi

ghan

timic

robi

alac

tiviti

esag

ains

tStre

ptoc

occu

spn

eum

onia

e.[6

7](+

)-�-c

adin

ene

isan

early

enzy

mat

icin

term

edia

tein

the

bios

ynth

esis

ofth

ese

squi

terp

enoi

dph

ytoa

lexi

nsby

upla

ndco

tton

Sesq

uite

rpen

eCu

bene

neW

eak

woo

dy,h

erba

l,sp

icy

�-C

uben

ene

has

been

iden

tified

aska

irom

one

attra

ctan

tsfo

relm

bark

beet

les

(Cur

culio

nida

e:Sc

olyt

inae

:Sco

lytu

ssp

p.).

[68]

Mon

oter

pene

3-Ca

rene

Citru

sfru

it,or

ange

peel

Exhi

bitr

epel

lent

activ

ityag

ains

tAno

phel

esga

mbi

ae.

[62]

Oxy-

mon

oter

pene

cis-

Gera

niol

Flor

al-r

ose,

gera

nium

Reve

alan

tibac

teria

lact

ivity

ata

broa

dsp

ectru

m.

[62]

Exhi

biti

nsec

trep

elle

ntac

tivity

.

a)N

oin

form

atio

n.



The importance of terpenoids is not limited to theiraromatic properties; they are also associated to insect re-pellent activity and to desirable properties in the humanhealth.

Concerning the application of the developed methodol-ogy to the Saaz hop-essential oil, it was possible to identifyin their HS 27 terpenoids, which include 13 monoterpenes,ten sequiterpenes, three oxygenated monoterpenes, and onehemiterpene. According to GC-qMS analysis, the terpenoidfraction of the Saaz hop-essential oil is dominated by themonoterpene �-myrcene, and the cyclic sesquiterpenes �-humulene, and �-caryophyllene, which together account for84% of the volatile hop-essential oil. Considering the widerange of biological and pharmacological activities associatedto terpenoid metabolites, and taking into account the highestcontent of these metabolites in hop-essential oil derived fromSaaz variety, we can estimate that this matrix can be used as apowerful and valuable natural biosource of terpenoid metabo-lites. In brief, our findings suggested that the essential oil de-rived from hop Saaz variety and its effective constituents canbe explored as a powerful biological source for newer, moreselective, biodegradable and naturally produced antimicrobialand antioxidant compounds, as an environmentally friendlyalternative to synthetic chemicals to control some bacterialstrains, fungs, and insects.

Future works will include studies on biological activity ofthe hop-essential oil through the evaluation of their antibac-terial, antifungal, and antioxidant activity.

The authors acknowledge the Empresa de Cervejas daMadeira (ECM) for the supply of CO2 supercritical Saazhop-essential oil samples and Portuguese Foundation for Sci-ence and Technology (FCT) through the MS Portuguese Net-works (REDE/1508/REM/2005) and Pluriannual base funding(QUI-Madeira-674).

The authors have declared no conflict of interest.

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