Similarities and differences of the volatile profiles of six spicesexplored by Proton Transfer Reaction Mass Spectrometry

Silvis, I. C. J., Luning, P. A., Klose, N., Jansen, M., & van Ruth, S. M. (2019). Similarities and differences of thevolatile profiles of six spices explored by Proton Transfer Reaction Mass Spectrometry. Food Chemistry, 271,318-327. https://doi.org/10.1016/j.foodchem.2018.07.021

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Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

Similarities and differences of the volatile profiles of six spices explored byProton Transfer Reaction Mass Spectrometry

I.C.J. Silvisa,b, P.A. Luningb, N. Klosea,b, M. Jansenc, S.M. van Rutha,b,⁎

a RIKILT Wageningen University and Research, P.O. Box 230, 6700 AE Wageningen, The Netherlandsb Food Quality and Design Group, Wageningen University and Research, P.O. Box 17 / bode 30, 6700 AA Wageningen, The Netherlandsc Intertaste, Weverseinde 343, 3297 LJ Puttershoek, The Netherlands

A R T I C L E I N F O

Chemical compounds studied in this article:2-Butanone (PubChem CID: 6569)Acetic acid (PubChem CID: 176)Cinnamaldehyde (PubChem CID: 637511)Estragole (PubChem CID: 8815)P-cymene (PubChem CID: 7463)Methanol (PubChem CID: 887)Safranal (PubChem CID: 61041)

Keywords:AromaFingerprintsNon-destructivePTR-TOFMSVolatile compounds

A B S T R A C T

Aroma properties of spices are related to the volatile organic compounds (VOCs) present, which can providedistinct analytical signatures. The aim of the study was to examine similarity and diversity of VOC profiles of sixcommon market spices (black/white pepper, chili paprika, cinnamon, nutmeg and saffron). The key volatileswere identified by PTR-TOFMS. Twelve samples per spice were subjected to PTR-Quadrupole MS (PTR-QMS)and Principal Component Analysis to compare the groups and examine diversity. With PTR-TOFMS, 101 volatilecompounds were identified as total sum across all samples by mass and comparing them with literature data.Some spices comprised key character aroma compounds, e.g. cinnamaldehyde in cinnamon. For others, VOCgroups, such as terpenes, acids and aldehydes topped the list. The PTR-QMS in combination with variablesselection resulted in distinct PCA patterns for each spice. Variation within the spice groups was observed, butvaried with the kind of spice. The results are valuable for future authentication studies.

1. Introduction

Spices are globally used as flavouring ingredients (Sasikumar,Swetha, Parvathy, & Sheeja, 2016). They are also increasingly used inhealthcare products, cosmetics and as preservatives. Over the lastdecade, artificial flavours became less popular and the interest in naturalcompounds increased. Furthermore, spices and herbs can be a possibleintervention for reduced salt intake in human diets (Kilcast & Angus,2007). For these reasons the global spice market is growing steadily at anannual rate of 5% and is expected to exceed $10 billion by 2020 (CBI:Trends: Spices and Herbs in Europe, 2016). Especially the trade of spicesand herbs produced in developing countries has grown significantly inrecent years, whereas the growth in production in the EU is limited.Consequently, prices of many spices and herbs have increased stronglyand prices will likely not drop. However, the centre for the promotion ofimports from developing countries (CBI) emphasized that the Europeanspices and herbs market becomes stricter on food safety and quality re-quirements due to multiple adulteration incidents. When spices are nottraceable throughout the supply chain or buyers have undeclared, un-authorised, or excessively high levels of extraneous material, the spices

are rejected. Also the food industry is becoming stricter in applying foodfraud monitoring (Silvis, van Ruth, van der Fels-Klerx, & Luning, 2017).These developments in spice trade entail therefore increasing demandson quality control and assurance of authenticity.

Nowadays, quality control of spices is embedded in theInternational Standard Organization (ISO) and European SpiceAssociation (ESA) standards (ESA, 2015). The standards involve thedetermination of volatile oil and spectroscopy of key markers, such assafranal in saffron (“ISO 3632-1 Spices – Saffron (Crocus sativus L.)–Part 1: Specification,” 2011, “ISO 3632-2 Spices – Saffron (Crocus sa-tivus L.) Part 2: Test methods,” 2010). Aroma compounds that arepresent in the volatile (or essential) oil the spices cause the typicalflavour and smell. These aroma compounds are commonly determinedby gas chromatography. The techniques have been successfully appliedfor differentiation of pepper varieties (piper nigrum) (Jirovetz,Buchbauer, Ngassoum, & Geissler, 2002) and chili (capsicum products)(Rodr Guez-Burruezo et al., 2010). Another technique applied is thesensor array based technique ‘e-nose’, which has been used for theanalysis of the volatile fraction of saffron (Carmona et al., 2006). Someother studies identified key-compounds such as safranal in saffron and

https://doi.org/10.1016/j.foodchem.2018.07.021Received 6 March 2018; Received in revised form 29 June 2018; Accepted 2 July 2018

⁎ Corresponding author at: RIKILT Wageningen University and Research, P.O. Box 230, 6700 AE Wageningen, The Netherlands.E-mail addresses: isabelle.silvis@wur.nl (I.C.J. Silvis), pieternel.luning@wur.nl (P.A. Luning), saskia.vanruth@wur.nl (S.M. van Ruth).

Food Chemistry 271 (2019) 318–327

Available online 21 July 20180308-8146/ © 2018 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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the effect of aging on saffron quality by direct mass spectrometryanalysis of the volatile compounds (Nenadis, Heenan, Tsimidou, & VanRuth, 2016).

In view of authentication and quality control (QC) in this fastmoving industry, rapid, non-destructive methods are promising for fu-ture QC applications. A non-destructive technique used for volatileorganic compound (VOC) analysis is Proton-Transfer Reaction MassSpectrometry (PTR-MS). It is a soft chemical ionisation mass spectro-metry based on proton transfer from a protonated reagent, most com-monly H3O+. Compounds with higher proton affinity than H2O reactwith H3O+ and accept a proton. PTR-MS is among the methods thathave been used most extensively for on-line analysis of volatile organiccompounds (Ellis & Mayhew, 2014). The non-destructive technique,PTR-quadrupole MS (PTR-QMS), allows measurement of nominalmasses. It is a rapid method and has demonstrated its robustness overthe past 20 years (Lindinger & Jordan, 1998). However, the mass re-solution is not as high as it is for the newer PTR-Time of Flight MS (PTR-TOFMS). This instrument allows the analysis of a wider range of VOCsthan PTR-QMS and it provides information on the compound identity.However, due to its complexity it is also more challenging to maintaindatabases over time and the analyses are less cost-efficient.

Moreover, recent mass spectrometry studies identified and assessedcomposition of VOCs combined with chemometrics, i.e. the analysis ofmultivariate data. Chemometrics shows benefits in analysis of spec-trometric data, applied in targeted as well as non-targeted techniques toidentify authenticity of geographical origin, or the contamination/fraudof spices (Reinholds, Bartkevics, Silvis, van Ruth, & Esslinger, 2015). Afew authentication studies applied chemometrics, such as a study on thedifferentiation of chocolates by the geographical origin of the cocoabeans applied (Acierno, Yener, Alewijn, Biasioli, & van Ruth, 2016) andthe analysis of dairy authenticity (Kamal & Karoui, 2015). The numberof profiling studies with spices is limited, but the application of un-targeted techniques, combined with chemometrics, is promising. It isexpected that adulterations affect the genuine volatile profile. There-fore a fingerprint and the identification of main compounds will lead toa better understanding of the possibilities of PTRMS in authenticitydetermination and subsequently the detection of fraud.

The aim of the present study is to examine the similarity and di-versity in the profiles of VOCs of six common spices, i.e. black pepper(BP), chili paprika (blend of Capsicum annuum) (CH), cinnamon (CN),nutmeg (NM), saffron (SF) and white pepper (WP). Sets of samples ofeach spice were analysed by PTR-QMS. Furthermore, a selection ofrepresentative samples of each spice has been analysed by PTR-TOFMSto identify VOCs tentatively. The combination of both PTR-MS analysisand tentative compound identification will support in building a char-acterisation database that can be used for spice authenticity research inthe future.

2. Materials and methods

2.1. Sampling design

Six types of spices were selected for analysis, i.e. Black Pepper, ChiliPaprika, Cinnamon, Nutmeg, Saffron, and White pepper. Per spice type,12 packages from different brands were purchased in retail outlets inthe Netherlands. In total, 72 samples were collected. There was no in-formation available about how these spices were processed. After pur-chase, the spices were stored in the dark at ambient temperature. Allsamples were analysed with PTR-QMS, and for each spice type 3 out ofthe 12 packages were used for tentative compound identification byPTR-TOFMS.

2.2. Sample preparation

The exact sample size was determined based on preliminary ex-periments. Sample size varied with the type of spice due to the

sensitivity and potential saturation of the instruments. Details arespecified below for each spice.

2.2.1. SaffronSaffron stigmas were powdered with a pestle and mortar. The pestle

and mortar were cooled with liquid nitrogen before the saffron wasadded to the mortar. An amount of 35mg of ground saffron wasweighed into a 250mL flask.

2.2.2. Other spicesThe other spices were collected in powdered form, and did not re-

quire a grinding step. The sample material (CN 250mg; BP, CH, WP100mg; NM 40mg) was put in a 250mL flask, the flask was closed andplaced in a water bath at 25 °C for 30min to equilibrate the sample’sheadspace. Preliminary experiments showed that 30min were sufficientfor equilibration (Nenadis et al., 2016). The same sample preparationapproach was used for both PTR-TOFMS and PTR-QMS.

2.3. Ptr-QMS

After equilibration the sample’s headspace was analysed with aHigh Sensitivity-PTR-QMS (Ionicon Analytik GmbH., InnsbruckAustria). The sample flasks were connected to the HS-PTR-MS inletflow, which was heated to 60 °C, via Teflon (0.25 mm) tubing.Headspace air was sampled at a flow rate of 48mL/min. The masseswere analysed in a quadrupole mass spectrometer and detected as ioncounts per second (cps) by using a secondary electron multiplier (SEM).Mass ion intensities were converted to volume mixing ratio (ppbv)values, according to Lindinger, Hansel and Jordan (Lindinger, Hansel, &Jordan, n.d.). Sample measurements were performed in 5 cycles, re-sulting in an analysis time of∼3.0min. The mean of cycle 2, 3, and 4were used in further analysis. Cycle 1 and 5 were excluded to ensure theconsistent measurement of the VOC concentration and to minimize theeffect that connecting and disconnecting the flask can have on themeasurement. Before each sample measurement, a blank flask wasanalysed, for which the first and the last cycle were removed. The va-lues obtained for the blank were subtracted from the samples andcorrected for transmission. A full scan was performed with a mass rangebetween m/z 20 to 160 since preliminary experiments did not show anypeaks after mass 160. All spice samples were analysed in triplicate.

2.4. Ptr-TOFMS

The PTR-TOFMS conditions were as follows: drift tube voltage465 V, drift tube temperature 110 °C, drift tube pressure of 2.30mbarthat causes an E/N value of 130 Townsend (1Td= 10–17 cm2 V−1

s−1). Samples were analysed at a flow rate of 60mL/min with a mass/charge ratio between m/z 20 and 373. The Time of Flight covers abroader mass range than the quadrupole, because of its higher sensi-tivity. De mass resolution (m/Δm) was at least 3600. The headspace airin the flask was directly transferred in a heated line (60 °C). After 10 s,the flask was connected to the equipment. After 47 s, the valve wasmanually changed from valve 1 (sample inlet) to valve 4 (filtered air)with help of the software. Each sample was measured for 60 s. The PTR-TOFMS generates 1 spectrum per second. All spice samples were ana-lysed in duplicate. The higher resolution of the PTR-TOFMS providesdetermination of sum formula and tentative identification of masspeaks.

2.5. Data treatment and statistical methods

2.5.1. Data treatment PTR-QMSPTR-QMS resulted in nominal masses with associated concentration

in parts per billion by volume (ppbv) intensity. Concentrations of re-plicates were averaged for each sample and standard deviations cal-culated. In order to check reliability of the data coefficients of variance

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(percentage CV=mean/standard deviation * 100%) were calculatedfor sample means and for replicates. The three replicate measurementsper spice were subjected to Levene’s test to check the homogeneity ofvariance for the masses. Based on these results ANOVA was appliedwith post-hoc test Tamhane’s T2 (p < 0.05) to all the masses, and themasses that were significantly different were determined (XL-STAT,Addinsoft, Germany). In order to explore differences and similaritiesbetween spices, the data were subjected to Principal ComponentAnalysis (Pirouette 4.5, Infometrix, Bothell WA) and different datatreatments (autoscale. log10) were applied.

2.5.2. Data treatment PTR-TOFMSThe intensity of ions resulting from the TOF mass analyser is in

counts per second (CPS). The 60 acquired spectra were averaged andused for further data processing. The masses of the ions in the spectrumwere compared with the ptr-wid database (Holzinger, 2015). A sumformula is connected to each mass. Additionally, an elemental compo-sition calculator (TODO, United States) combined with consultingpreviously conducted research on volatile analysis in spices and in PTR-

TOFMS studies also other compounds were identified, which were notmentioned in spice literature before. For compound identification basedon measured masses, a tolerance of 10 mDa (millidalton) was accepted.It was decided to leave out the masses that have not been identified asthere was particular interest in type of molecule and flavour char-acteristics. This characterisation is not possible with unidentifiedcompounds. The masses that have not been identified in spice studies orPTR studies have been removed from the list.

3. Results and discussion

3.1. VOC patterns of the six spices measured by PTR-QMS

All samples were subjected to PTR-QMS analysis. Fig. 1 shows themean VOC fingerprints of the six spices, i.e. the average of the massspectra of the twelve samples of each of the spices. It appears that theconcentration range of the volatile compounds differs enormously forthe various spices. For instance, saffron shows relatively low con-centrations, whereas nutmeg presents very high values (> 10 ppmv).

Fig. 1. Mean PTR-QMS volatile fingerprints of the six spices.

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This is partly due to the volatile oil content of the spices. Nutmeg yields5–15 per cent of volatile oil (Periasamy, Karim, Gibrelibanos,Gebremedhin, & Gilani, 2016). This is much higher than for saffron,which has a volatile oil content of about 0.8 per cent (VelascoNegueruela, 2011). However, the air/spice partition coefficients andthus the matrix affect the headspace concentrations of the volatiles aswell.

3.2. VOC identification of the six spices by PTR-TOFMS

Three samples of each of the six spices were subjected to PTR-TOFMS. Table 1 shows the number of key compounds found in litera-ture and the number of tentatively identified compounds determined byPTR-TOFMS in the current study. In total, 101 compounds were ten-tatively identified, most of them in chili paprika. Table 2 shows themasses that were tentatively identified, their sum formulas, and inwhich spices the masses were detected and in which range. In total, 600mass peaks were extracted in the m/z range of 11.000–373.000.

3.3. Qualitative similarities in VOCs of the six spices – PTR-TOFMS

The unique character of a spice is affected by the presence of keycharacter compounds (i.e. contains the particular smell), the composi-tion, and the character and concentrations of other aroma compoundscan determine the kind of aroma perceived (Kamadia, Yoon, Schilling,& Marshall, 2006). As shown in Table 2, many of the identified com-pounds have pleasant aromas such as fruity and spicy. On the contrary,it is mostly the group of acids carrying unpleasant and rancid odours.

The major VOC class in the current study (Table 2) is the class ofterpenoids, which are widely produced by plants (Ercioglu, MuratVelioglu, & Hakki Boyaci, 2018). In the current study, the predominantcompounds present in the top 10 of three or more spices include ses-quiterpenes, monoterpenes, p-cymene, acetic acid, methanol and bu-tanone. Monoterpenes and sesquiterpenes are the main constituents ofessential oil in plants. p-Cymene is related to monoterpenes anddominant in essential oils too (“PubChem Compound Database;CID=7463,” 2017). The compounds acetic acid and methanol are de-tected by PTR-TOFMS in all spices and are mentioned as main com-pounds in several studies (e.g., Acierno et al., 2016; Liu, Koot, Hettinga,de Jong, & van Ruth, 2018). Acetic acid is a compound found in theessential oil of black pepper, which is a compound commonly found inplants (Ravindran & Kallupurackal, 2011). 2-Butanone has been foundin fruits and vegetables in trace amounts. The compound 2-butanonehas a sharp, sweet, butterscotch odour(Ferreira & López, 2013).

3.4. Qualitative diversity in VOCs of the six spices by PTR-TOFMS

The top 10 compounds in highest concentration of individual spicescovers also compounds that are not present in the top 10 of other spices.Those are listed with a hashtag (#) in the sections below with an ex-planation of their characteristics. Some of the compounds are keyaroma compounds. Examples are safranal in saffron and cinnamalde-hyde in cinnamon. Other compounds mentioned, have not been iden-tified in previous spice studies before. Those compounds are describedwith general characteristics.

3.4.1. Black pepperCompared to the other spices, black pepper contains the least dis-

tinctive adundant compounds, namely Estragole (#8), isoprene (#9)and ethylbenzene (#10). Estragole (#8) is a phenylpropene and com-ponent with an anise flavour found in anise, but also in bay and pine oil(Ozguven, 2011). Subsequently, isoprene (#9) is found in plants, and1,3-dimethylbenzene (#10) a sweet smelling aromatic compound(“CID=7929,” 2017) present in a chili study (Gahungu, Ruganintwali,Eric, Zhang, & Mukunzi, 2011) and found in cinnamon.

3.4.2. Chili paprikaChili paprika contains 5 compounds in its top 10 that are not

abundant in the other spices, namely propanoic acid (#4), acetaldehyde(#6) methylbutanal (#8), ethyl acetate (#9) and propanal (#10).Propanoic acid (#4) has an unpleasant odour and is used as an anti-fungal agent in food (“PubChem Compound Database; CID=1032,”2017). Acetaldehyde (#6) is produced by plants, present in bread,coffee and ripe fruit and found in pepper (Jirovetz et al., 2002). Bu-tyrolactone (#7) was found in a chili study before, and in saffron stu-dies as well (Ko et al., 2014; Urbani, Blasi, Chiesi, Maurizi, &Cossignani, 2015) with a caramellic note. Methylbutanal (#8) is aconstituent of black currant, isolated from coffee, tea, peppermint oiland soy, banana, orange juice and several other food and beverageproducts (“CID=6971249,” 2017). Ethyl acetate (#9) has a char-acteristic sweet smell and has been found in many food products(“CID=8857,” 2017). Propanal (#10) is known for its trigeminalsensation (Ko et al., 2014).

3.4.3. CinnamonCinnamaldehyde (#2) is the key aroma compound of cinnamon (Li,

Kong, & Wu, 2013). It is a flavonoid responsible for the typical cin-namon odour. The essential oil content of cinnamon contains about50% cinnamaldehyde (Senanayake, Lee, & Wills, 1978). Benzaldehyde(#6) is present in a relatively high concentration in cinnamon(> 500,000 cps) compared to the other spices, and it is known for itsalmond like flavour (“CID=240,” 2017). The following compoundshave not been found in previous cinnamon studies before, namely tri-methylbenzene (#7) with a strong petroleum odour (“CID=7247,”2017) occuring naturally in coal tar and petroleum, and 1,3-di-methylbenzene (#8) a sweet smelling aromatic compound(“CID=7929,” 2017), present in crude oil and gasonline, found in achili study (Gahungu et al., 2011). Finally curcumene, belonging to thesesquiterpenes (#10) (“CID=92139,” 2017).

3.4.4. NutmegSafrole (#5) is a phenylpropene in nutmeg (miscellaneous com-

pound), typically extracted in small amounts in a variety of plants andhigh in sassafras species (“CID=5144,” 2017). For nutmeg, the con-centration of safrole is higher than in the other spices. Acetone (#6) isnot specific for spices but is widely present in nature. Pyrrole is also notspecific for spices, but N-methylpyrrole (#7) is mentioned in a chilistudy (Ko et al., 2014). Benzene propanol or cinnamyl alcohol (#9) is

Table 1Overview of tentatively identified compounds in six spices.

Spice Number ofcompounds foundin literature

References Number of compoundsdetermined by PTR-TOFMS

Black pepper 34 [1] 22Chili paprika 131 [2–4] 64Cinnamon 26 [5–6] 24Nutmeg 14 [7–8] 10Saffron 58 [9–11] 38White pepper 14 [12–13] 12

BP [1]: (Jirovetz et al., 2002); CH [2–4]: (Ko et al., 2014), (Gahungu et al.,2011), (Bogusz Junior, Henrique Março, et al., 2015); CN [5–6]: (Li et al.,2013), (Senanayake et al., 1978); NM [7–8]: (Chandrasekharan, 1994),(Krishnamoorthy & Rema, 2011); SF [9–11]: (Urbani et al., 2015), (Carmonaet al., 2006), (Jiang, Kulsing, Nolvachai, & Marriot, 2015); WP [12–13]: (Liu,Zeng, Wang, Wu, & Tan, 2013), (Plessi, Bertelli, & Miglietta, 2002)

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Table 2Masses of VOCs with their sum formula and tentative identifications in six spices from PTR-TOF-MS analysis (3 left hand columns). The colour indications aredependent on concentration and the rankingsa for their presence in the various spices. The 6 right hand column highlights the spices in which the particularcompound is present based on information from previous reports.

(continued on next page)

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naturally occurring, present in cinnamon (Senanayake et al., 1978).Myristicin (#10) is an abundant and characteristic phenylpropene inthe essential oil of nutmeg (Chandrasekharan, 1994; Krishnamoorthy &Rema, 2011).

3.4.5. SaffronSafranal (#2), is the key aroma compound of saffron (Carmona

et al., 2006). Lanierone (#4) is a compound, which is also specific forsaffron. It is a pheromone constituent (Guijarro-Díez, Castro-Puyana,Crego, & Marina, 2017). B-cyclocitral (#5) is a compound in saffron,which is also present in other plant materials (“beta-Cyclocitral |C10H16O - PubChem,” n.d.). Furanone (#7) is also a key aroma com-pound, usually present in high concentration in saffron (Urbani et al.,2015). Likewise, linalool (#8) is a naturally occurring terpene alcoholfound in many flowers and spice plants and has a pleasant floral scent(“Linalool | C10H18O - PubChem,” n.d.). It is a heterocyclic compound.Butyrolactone (#9) is found in petals of saffron (Urbani et al., 2015),but also present in chili paprika. Isophorone (#10), another typicalvolatile of saffron, has a characteristic peppermint smell (Carmonaet al., 2006; Jiang, Kulsing, Nolvachai, & Marriott, 2015; Urbani et al.,2015; ISOPHORONE | C9H14O - PubChem, n.d.).

3.4.6. White pepperWhite pepper contains 2 compounds in the top 10 that are not

present in the other spices, namely isoprene (#8) and estragole (#10).Methylpyrrole (#4) is a pyrrole with CH3-group connected to N and is aMaillard product with nutty odour (“CID=7304,” 2017) and found innutmeg. Tri-metylbenzene (#5) is an aromatic compound. Many plantsproduce isoprene (#8). Estragole (methyl-chavicol) (#10) has an aniseflavour. White pepper differs from black pepper in intensity of a few

compounds namely skatole and cresol, known off odours (Jagella &Grosch, 1999).

3.5. Quantitative diversity in VOCs of the six spices by PTR-QMS

The masses that show significant differences between spice groupsin PTR-QMS analyses are presented in Table 3. The results from thetentative identifications from Table 2 are linked to the results of thePTR-QMS. The data of all samples of the masses showing significantdifferences were subsequently subjected to exploratory statistical ana-lysis, namely PCA (Fig. 2).

Chili paprika has 10 masses that are significantly higher in intensityincluding acetic acid (m/z 61), butanone (73), pentenone (85), butanoicacid (89), pentadione (1 0 1), ethyl propionate (1 0 3), heptenal (1 1 3),guaiacol (1 2 5) and butyrolactone (87), and a non-identified compound(m/z 63), that could be a water cluster of acetaldehyde. Eight of thesetentatively identified compounds have been found in chili studies be-fore, except from guaiacol (Bogusz Junior, Março, et al., 2015; Gahunguet al., 2011; Ko et al., 2014). The quantities of the other 7 compounds/masses is responsible for the separation of the chili paprika group inFig. 2, the PCA plot of the six spices. PCA is an explorative analysis,which allows fast comparison of the full set of the spice volatile com-pounds profiles. Saffron is the other spice showing a very distinct pat-tern from black/white pepper, cinnamon, and nutmeg.

Saffron contains 28 compounds, which are significantly lower inintensity compared to the intensity of the other spices. On the otherhand, there are two abundant masses, namely mass 85 and 151, whichwere tentatively identified as safranal and furanone by PTR-TOFMS(Table 2). Special care must be taken with the tentative identification,as mass 85 and 151 represent different compounds. Mass 85 also relates

Table 2 (continued)

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to pentenone and mass 151 involves piperonal, carvone, and cuminylalcohol as well. Mass 85 is significantly higher for both chili paprikaand saffron.

Black pepper is the only spice that does not comprise masses thatdiffer significantly from the others. For white pepper it is mass 89, i.e.butanoic acid and/or ethyl acetate, and mass 123, which is a terpenefragment. Both compounds have not been found in white pepper before,but butanoic acid has been found in black pepper.

The four discriminating compounds that are higher in intensity incinnamon are styrene (1 0 5), benzaldehyde (1 0 6), Ethyl 2-methylbu-tanoate (1 3 1), and cinnamaldehyde (1 3 3). Cinnamaldehyde is a keycompound in cinnamon, which is giving cinnamon its characteristicodour. Styrene and benzaldehyde are known compounds in cinnamon.Nutmeg has only one single compound that is significantly higher inintensity, namely mass 155.

Saffron is the least terpenic spice, when evaluating the concentra-tions of terpene related compounds. The terpenes (C10H16), (C10H18O),and terpene related compounds mentioned previously do not showsignificant differences for a specific spice, but are high in black/ whitepepper, cinnamon and nutmeg. This may explain why these terpenicspices nutmeg (NM, grey), white pepper (WP, red) black pepper (BP,green), and cinnamon (CN, orange) present less distinctive clusters inFig. 2. Variation within the spice groups was observed, with some

showing more variation than others, e.g. chili paprika more than whitepepper. The spice samples were commercially sourced and vary inprices and qualities. The origin and processing conditions of the spiceswere unknown. Some spice samples in the group (n= 12) show andistinct outlier pattern that could affect the clustering of the spicegroup.

4. Conclusions

The volatile composition of spices includes mainly terpenes, alde-hydes and esters. Similarities in black/white pepper, chili paprika,cinnamon, nutmeg, and saffron were attributed to acetic acid, me-thanol, terpenes and p-cymene. PTR-MS detected compounds in thespices that have not been found in spice studies before. The diversityamong spice groups is caused by specific spice markers, such as cin-namaldehyde in cinnamon and safranal in saffron. The compoundidentification analysis of the PTR-TOFMS aids in the data analysis ofPTR-QMS. The most distinct spices are saffron and chili paprika. Eventhough all the spices share plant related VOC groups, the combinationof both PTR-MS techniques and literature research on volatile analysisof spices, resulted in a comprehensive mass characterisation in spicesthat can be used for PTR authentication analysis in the future.

Table 2 (continued)

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>aTop ranking for the 10 highest intensities per spice. The highest intensity has a number 1 and the lowest intensity a number 10. The colour is representative for theintensity in counts per second, divided into five sections; ; ; , ; .bThe masses in the grey boxes are those that can be distinguished with the TOFMS due to the higher resolution that the QMS.cBP: black pepper, CH: chili paprika, CN: cinnamon, NM: nutmeg, SF: saffron, WP: white pepper [14]: (Yener et al., 2014); [15]: (Ionicon Analytik GmbH, 2008).

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Table 3VOCs and their mean concentrations for the six spices determined by PTR-QMS [ppbv]. Different letters in a row indicate significant differences(Tamhane’s T2 test, P < 0.05). VOCs showing a significant difference for (a) particular spice(s) are highlighted. Dark grey boxes represent a significanthigher concentration than the rest. A light grey box represents a significant lower concentration. Masses that do not show significant differences, andthose with mean concentrations of 1 ppbv or less for all spices are not shown.

cAn N.I. (not identified) not is given to masses that are not tentatively identified.

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5. Chemical compounds

CID=240. (2017). In PubChem Compound Database.CID= 5144. (2017). In PubChem Compound Database.CID= 6971249. (2017). In PubChem Compound Database.CID= 7247. (2017). In National Center for Biotechnology

Information.CID= 7304. (2017). In PubChem Compound Database.CID= 7929. (2017). In PubChem Compound Database.CID= 8857. (2017). In PubChem Compound Database.CID= 92139. (2017). In PubChem Compound Database.

Acknowledgement

This research was executed in the framework of the EU-projectSPICED (Grant Agreement: 312631) with the financial support from the7th Framework Programme of the European Union, EuropeanCommission - Directorate-General Enterprise & Industry. This publica-tion reflects the views only of the authors, and the EuropeanCommission cannot be held responsible for any use which may be madeof the information contained therein. Furthermore, authors acknowl-edge co-financing of the project through the ‘Kennisbasis’ fundingprogramme by the Ministry of Economic Affairs of the Netherlands andfinancial support of the PhD project by Intertaste, the Netherlands.

References

Acierno, V., Yener, S., Alewijn, M., Biasioli, F., & van Ruth, S. (2016). Factors contributingto the variation in the volatile composition of chocolate: Botanical and geographicalorigins of the cocoa beans, and brand-related formulation and processing. FoodResearch International, 84, 86–95. https://doi.org/10.1016/j.foodres.2016.03.022.

beta-Cyclocitral | C10H16O - PubChem. (n.d.). Retrieved November 22, 2017, fromhttps://pubchem.ncbi.nlm.nih.gov/compound/9895.

Bogusz Junior, S., Henrique Março, P., ıcia Valderrama, P., Cardoso Damasceno, F.,Silvana Aranda, M., audia Alcaraz Zini, C., & Teixeira Godoy, H. (2015). Analysis ofvolatile compounds in Capsicum spp. by headspace solid-phase microextraction andGC-TOFMS. Analytical Methods, 7, 521–529. https://doi.org/10.1039/c4ay01455c.

Bogusz Junior, S., Março, P. H., Valderrama, P., Damasceno, F. C., Aranda, M. S., Zini, C.A., ... Godoy, H. T. (2015). Analysis of volatile compounds in Capsicum spp. by

headspace solid-phase microextraction and GC × GC-TOFMS. Analytical Methods,7(2), 521–529. https://doi.org/10.1039/C4AY01455C.

Carmona, M., Martínez, J., Zalacain, A., Rodríguez-Méndez, L., José, De Saja, A., &Alonso, G. L. (2006). Analysis of saffron volatile fraction by TD–GC–MS and e-nose.European Food Research and Technology, 223, 96–101. https://doi.org/10.1007/s00217-005-0144-5.

Carmona, M., Martínez, J., Zalacain, A., Rodríguez-Méndez, M. L., de Saja, J. A., &Alonso, G. L. (2006). Analysis of saffron volatile fraction by TD–GC–MS and e-nose.European Food Research and Technology, 223(1), 96–101. https://doi.org/10.1007/s00217-005-0144-5.

CBI: Trends: Spices and Herbs in Europe. (2016). The Hague. Retrieved from https://www.cbi.eu/sites/default/files/market_information/researches/trends-europe-spices-herbs-2016_0.pdf.

Chandrasekharan, C. (1994). Nutmeg and derivatives. Rome Retrieved from http://www.fao.org/docrep/019/v4084e/v4084e.pdf.

Ellis, A. M., Andrew, M., & Mayhew, C. A. (2014). Proton transfer reaction mass spectro-metry: Principles and applications. Wiley.

Ercioglu, E., Murat Velioglu, H., & Hakki Boyaci, I. (2018). Determination of terpenoidcontents of aromatic plants using NIRS. Talanta, 178, 716–721. https://doi.org/10.1016/J.TALANTA.2017.10.017.

ESA. (2015). European Spice Association Quality Minima Document, rev 5(December).Ferreira, V., López, R., 2013. Flavour science : Proceedings from XIII Weurman Flavour

Research Symposium. Elsevier Science. Retrieved from https://books.google.nl/books?id=eUITAAAAQBAJ&dq=butterscotch+2-butanone&hl=nl&source=gbs_navlinks_s.

Gahungu, A., Ruganintwali, E., Eric, K., Zhang, X., & Mukunzi, D. (2011). VolatileCompounds and Capsaicinoid Content of Fresh Hot Peppers (Capsicum Chinense)Scotch Bonnet Variety at Red Stage. Advance Journal of Food Science andTechnology, 3.

Guijarro-Díez, M., Castro-Puyana, M., Crego, A. L., & Marina, M. L. (2017). Detection ofsaffron adulteration with gardenia extracts through the determination of geniposideby liquid chromatography–mass spectrometry. Journal of Food Composition andAnalysis, 55, 30–37. https://doi.org/10.1016/j.jfca.2016.11.004.

Holzinger, R. (2015). PTRwid: A new widget tool for processing PTR-TOF-MS data.Atmospheric Measurement Techniques, 8(9), 3903–3922. https://doi.org/10.5194/amt-8-3903-2015.

Ionicon Analytik GmbH (2008). Substances - Proton Affinities and Fragmentation List.IONICON Analytik GmbH.

ISO 3632-1 Spices – Saffron (Crocus sativus L.)– Part 1: Specification. (2011). ISO.ISO 3632–2 Spices – Saffron (Crocus sativus L.) Part 2: Test methods. (2010).ISOPHORONE | C9H14O - PubChem. (n.d.). Retrieved November 22, 2017, from https://

pubchem.ncbi.nlm.nih.gov/compound/isophorone.Jagella, T., Grosch, W. (1999). Flavour and off-flavour compounds of black and white

pepper (Piper nigrum L.). European Food Research and Technology, 209(1), 27–31.https://doi.org/10.1007/s002170050451.

Jiang, M., Kulsing, C., Nolvachai, Y., & Marriot, P. J. (2015). Improved Saffron Analysis inComprehensive Two Dimensional Gas Chromatography with Secondary Retention Indexand Accurate Mass. Huazhong.

Jiang, M., Kulsing, C., Nolvachai, Y., & Marriott, P. J. (2015). Two-dimensional retentionindices improve component identification in comprehensive two-dimensional gaschromatography of saffron. Analytical Chemistry, 87(11), 5753–5761. https://doi.org/10.1021/acs.analchem.5b00953.

Jirovetz, L., Buchbauer, G., Ngassoum, M. B., & Geissler, M. (2002). Aroma compoundanalysis of Piper nigrum and Piper guineense essential oils from Cameroon usingsolid-phase microextraction–gas chromatography, solid-phase microextraction–gaschromatography–mass spectrometry and olfactometry. Journal of Chromatography A,976(1), 265–275. https://doi.org/10.1016/S0021-9673(02)00376-X.

Kamadia, V. V., Yoon, Y., Schilling, M. W., & Marshall, D. L. (2006). Relationships be-tween Odorant Concentration and Aroma Intensity. Journal of Food Science, 71(3),S193–S197. https://doi.org/10.1111/j.1365-2621.2006.tb15640.x.

Kamal, M., Karoui, R. (2015, November 1). Analytical methods coupled with chemo-metric tools for determining the authenticity and detecting the adulteration of dairyproducts: A review. Trends in Food Science and Technology. Elsevier. https://doi.org/10.1016/j.tifs.2015.07.007.

Kilcast, D., Angus, F. (2007). Reducing salt in foods. Igarss 2014. Woodhead PublishingLtd. https://doi.org/10.1007/s13398-014-0173-7.2.

Ko, A.-Y., Musfiqur Rahman, M., Abd El-Aty, A. M., Jang, J., Choi, J.-H., Mamun, M. I. R.,& Shim, J.-H. (2014). Identification of volatile organic compounds generated fromhealthy and infected powdered chili using solvent-free solid injection coupled withGC/MS: Application to adulteration. Food Chemistry, 156, 326–332. https://doi.org/10.1016/j.foodchem.2014.02.001.

Krishnamoorthy, B., Rema, J. (2011) Nutmeg and mace. In: The handbook of herbs andspices. pp. 238–248.

Li, Y., Kong, D., & Wu, H. (2013). Analysis and evaluation of essential oil components ofcinnamon barks using GC–MS and FTIR spectroscopy. Industrial Crops and Products,41, 269–278. https://doi.org/https://doi.org/10.1016/j.indcrop.2012.04.056.

Linalool | C10H18O - PubChem. (n.d.). Retrieved November 22, 2017, from https://pubchem.ncbi.nlm.nih.gov/compound/6549.

Lindinger, W., Hansel, A., Jordan, A. (n.d.). Proton-transfer-reaction mass spectrometry(PTR–MS): on-line monitoring of volatile organic compounds at pptv levels.Retrieved from http://pubs.rsc.org/en/content/articlepdf/1998/CS/A827347Z.

Lindinger, W., & Jordan, A. (1998). Proton-transfer-reaction mass spectrometry(PTR–MS): On-line monitoring of volatile organic compounds at pptv levels. ChemicalSociety Reviews, 27(5), 347. https://doi.org/10.1039/a827347z.

Liu, H., Zeng, F. K., Wang, Q. H., Wu, H. S., & Tan, L. H. (2013). Studies on the chemicaland flavor qualities of white pepper (Piper nigrum L.) derived from five new

Factor 1 (45.6%)

Factor 3 (7.0%)

Factor 2 (7.7%)

Fig. 2. Principal component analysis of the PTR-QMS data of the 6 differentspices. Pre-processing: mean centering and log 10 transformation after variableselection based on ANOVA. the colours represent BP: red, CH: orange, CN: blue,NM: grey SF: pink, WP: green. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

I.C.J. Silvis et al. Food Chemistry 271 (2019) 318–327

326

genotypes. European Food Research and Technology, 237(2), 245–251. https://doi.org/10.1007/s00217-013-1986-x.

Liu, N., Koot, A., Hettinga, K., de Jong, J., & van Ruth, S. M. (2018). Portraying andtracing the impact of different production systems on the volatile organic compoundcomposition of milk by PTR-(Quad)MS and PTR-(ToF)MS. Food Chemistry, 239,201–207. https://doi.org/10.1016/j.foodchem.2017.06.099.

Nenadis, N., Heenan, S., Tsimidou, M. Z., & Van Ruth, S. (2016). Applicability of PTR-MSin the quality control of saffron. Food Chemistry, 196, 961–967. https://doi.org/10.1016/j.foodchem.2015.10.032.

Ozguven, M. (2011). Aniseed. In: Handbook of herbs and spices. pp. 39–51.Periasamy, G., Karim, A., Gibrelibanos, M., Gebremedhin, G., Gilani, A.-H. (2016).

Chapter 69 – Nutmeg (Myristica fragrans Houtt.) Oils. In: Essential Oils in FoodPreservation, Flavor and Safety (pp. 607–616). https://doi.org/10.1016/B978-0-12-416641-7.00069-9.

Plessi, M., Bertelli, D., & Miglietta, F. (2002). Effect of Microwaves on VolatileCompounds in White and Black Pepper. LWT - Food Science and Technology, 35(3),260–264. https://doi.org/10.1006/fstl.2001.0853.

PubChem Compound Database; CID=1032. (2017). In: PubChem Compound Database.PubChem Compound Database; CID=7463. (2017). In: PubChem Compound Database.

National Center for Biotechnology Information. Retrieved from https://pubchem.ncbi.nlm.nih.gov/compound/7463.

Ravindran, P., Kallupurackal, J. (2011). Black pepper. In: Handbook of herbs and spices(pp. 62–110).

Reinholds, I., Bartkevics, V., Silvis, I. C. J., van Ruth, S. M., & Esslinger, S. (2015).Analytical techniques combined with chemometrics for authentication and determi-nation of contaminants in condiments: A review. Journal of Food Composition and

Analysis, 44, 56–72. https://doi.org/10.1016/j.jfca.2015.05.004.Rodr Guez-Burruezo, A. N., Kollmannsberger, H., Carmen, M., Lez-Mas, G., Nitz, S., &

Nuez, F. (2010). HS-SPME Comparative Analysis of Genotypic Diversity in theVolatile Fraction and Aroma-Contributing Compounds of Capsicum Fruits from theannuum-chinense-frutescens Complex. J. Agric. Food Chem, 58, 4388–4400. https://doi.org/10.1021/jf903931t.

Sasikumar, B., Swetha, V. P., Parvathy, V. A., & Sheeja, T. E. (2016). 22 – Advances inAdulteration and Authenticity Testing of Herbs and Spices. In Advances in FoodAuthenticity Testing, 585–624. https://doi.org/10.1016/B978-0-08-100220-9.00022-9.

Senanayake, U. M., Lee, T. H., & Wills, R. B. H. (1978). Volatile Constituents of Cinnamon(Cinnamomum zeylanicum) Oils. Journal of Agricultural and Food Chemistry, 26(4),822–824. https://doi.org/10.1021/jf60218a031.

Silvis, I. C. J., van Ruth, S. M., van der Fels-Klerx, H. J., & Luning, P. A. (2017).Assessment of food fraud vulnerability in the spices chain: An explorative study. FoodControl, 81, 80–87. https://doi.org/10.1016/j.foodcont.2017.05.019.

Urbani, E., Blasi, F., Chiesi, C., Maurizi, A., & Cossignani, L. (2015). Characterization ofVolatile Fraction of Saffron from Central Italy (Cascia, Umbria). International Journalof Food PropertiesOnline) Journal. International Journal of Food Properties, 18(10),1094–2912. https://doi.org/10.1080/10942912.2014.968787.

Velasco Negueruela, A. (2011). Saffron. In: Handbook of herbs and spices (pp. 276–286).Yener, S., Romano, A., Cappellin, L., Märk, T. D., Del Pulgar, J. S., Gasperi, F., ... Biasioli,

F. (2014). PTR-ToF-MS characterisation of roasted coffees (C. arabica) from differentgeographic origins. Journal of Mass Spectrometry, 49(9), 929–935. https://doi.org/10.1002/jms.3455.

I.C.J. Silvis et al. Food Chemistry 271 (2019) 318–327

327