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Journal of Chromatography A, 1216 (2009) 3731–3739

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

ssessment of specific migration to aqueous simulants of a new active foodackaging containing essential oils by means of an automatic multipleynamic hollow fibre liquid phase microextraction system�

esús Salafranca, Davinson Pezo, Cristina Nerín ∗

epartment of Analytical Chemistry, Aragon Institute of Engineering Research I3A, CPS-University of Zaragoza, Torres Quevedo Building,aría de Luna St. 3, E-50018 Zaragoza, Spain

r t i c l e i n f o

rticle history:eceived 11 November 2008eceived in revised form 19 January 2009ccepted 3 March 2009vailable online 11 March 2009

a b s t r a c t

The determination of specific migration in the three aqueous food simulants (water, 3% acetic acid and10% ethanol) from experimental active packaging polypropylene-based films containing natural essentialoils as active agents has been carried out for the first time by a two-phase hollow fibre liquid phasemicroextraction (HFLPME). Due to the high number of variables involved, an experimental design hasbeen applied. High throughput, with six samples running simultaneously in a highly automated system

eywords:ollow fibrequeous food simulantsigration

ctive packaging

working in dynamic extraction mode, has been achieved. The main analytical characteristics are detectionlimits as low as 0.01 �g kg−1, linearity higher than 0.99 for almost 5 magnitude orders, average precisionbelow 16% as RSD and concentration factors ranging from 4 to 189. Migration of 43 compounds includingterpenes, alkanes, plastic additives and degradation compounds is reported. According to the resultsobtained and European legislation, the packaging prototypes tested could be safely marketed.

actorial designolypropylene

. Introduction

Active food packaging is a promising technology with increasingpplications due to the advantages over traditional packaging sys-ems. Their main objective is to extend the shelf-life or to maintainr to improve the condition of packaged food by deliberately incor-orating components that would release (those called emitters) orbsorb substances (for absorbers) into or from the packaged foodr its surrounding environment [1–4].

According to the European regulation on active food packag-ng materials [5], they should not change the composition nor therganoleptic properties of food, e.g. to avoid frauds or to masknpleasant odours in foods which are past their sell-by date, andonsequently with a high risk for the health of consumers. Besides,nd pending the adoption of additional rules in a specific measuren active packaging, incorporated active agents shall be authorized

nd used in accordance with the relevant provisions applicable toood. Consequently, all the substances incorporated from the pack-ging to the foodstuffs must comply with the Directive on fooddditives [6].

� Presented at the 12th Conference on Instrumental Analysis, Barcelona, Spain,1–23 October 2008.∗ Corresponding author. Tel.: +34 976 761 873; fax: +34 976 762 388.

E-mail address: cnerin@unizar.es (C. Nerín).

021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2009.03.001

© 2009 Elsevier B.V. All rights reserved.

Directive 89/107/EEC makes a classification of food additivesas colours, preservatives, sweeteners, flavour enhancers,. . . as afunction of the intended effect when incorporated to the food.There is also specific regulation for each additive group. Morespecifically, essential oils and most of their components, as thoseused in this work incorporated in polypropylene films as activeagents—perceived as safer, healthier and much less toxic thansynthetic ones for consumers [7]—are accepted and have beenapproved as flavouring substances by a Decision [8] of the EuropeanCommission.

Plastic materials in contact with food are regulated as well by theDirective 2002/72/EC [9] and its amendments, which establish theglobal and specific migration limits for a wide series of compounds,commonly known as “positive list”. As essential oils are acceptedand used as food additives, they do not appear in such list, althoughthey consist of a complex mixture of chemicals.

However, the launching into the market of any new active mate-rial requires that migration tests must be carried out to certify thatthese materials fulfil the legislation and guarantee the health ofconsumers. Migration tests [10] consist of the exposure of the plas-tic sample in contact with several established liquid simulants [11]

which mimic the behaviour of food. Distilled water, 3% (w/v) aceticacid, 10% (v/v) ethanol and olive oil were selected as aqueous, acidicor fatty food simulants respectively. Alternatively, isooctane or 95%(v/v) ethanol can be used instead of olive oil as fatty simulants. Afterexposure of the plastic samples to simulants for a determined time

3732 J. Salafranca et al. / J. Chromatogr. A 1216 (2009) 3731–3739

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nd temperature, the released substances—migrants—are analyzedy appropriate analytical methods and their concentration is com-ared with the established values. Thus, the plastic is consideredafe and can be marketed if all the found values are below theirpecific migration limits.

As the migration values are usually at trace or ultratrace lev-ls, very sensitive analytical techniques and concentration stepsuring sample treatment are required. In the particular case oferpenes, which are the main components of essential oils, mostf methods make use of gas chromatography for the final deter-ination due to their volatile and semivolatile characteristics.ell consolidated techniques such as liquid–liquid extraction with

ichloromethane and diethyl ether/pentane mixtures have beensed for the determination of linalool, nerol, �-terpineol, cit-onellol and geraniol in hydroalcoholic samples [12]. Solid-phasextraction (SPE) with granular activated carbon cartridges [13] orolid-phase microextraction (SPME) [14–16] has also been reported.ome applications of innovative techniques such as single dropicroextraction (SDME) [17], headspace [18] or stir bar sorp-

ive extraction (SBSE) [19] have been developed with promisingesults.

In a previous study recently published [20] liquid phaseicroextraction assisted by semipermeable membranes (more

pecifically hollow fibres, HFLPME), proved to be a powerful tech-ique when applied to the determination of migrants from activeaterials. Membrane extraction combines preconcentration, sepa-

ation and clean-up steps in only one operation [21–24] with highersatility, selectivity, cheapness and easiness for both miniaturiza-ion and automatization.

Taking the cited work [20] as starting point, several importantovel aspects are considered in this study, which can be expressed

n the following objectives:

(a) To extend the goals of the HFLPME procedure recently achievedin water to the other two aqueous simulants (3% w/v aceticacid and 10% v/v ethanol), for the first time in full dynamic andnearly automated extraction mode. Thus, the applicability of

this extraction technique is extended to acidic and hydroalco-holic solutions, which are the food simulants used for a broadrange of foodstuffs.

b) To get a more accurate knowledge of the real migration sit-uation. To reach this objective, and based on the previous

iple dynamic HFLPME set-up.

migration results, the number of compounds considered foroptimization purposes will be notoriously increased.

(c) To study in depth the most relevant variables influencing extrac-tion, as well as their possible interrelation or cross-interactions,by applying chemometrics to the experimental design opti-mization data.

(d) To minimize the migration values detected in the previouswork, thus obtaining active films for a wide spectrum of applica-tions in the market. With regard to this objective, considerablechanges will be considered, including modifications in theformulations of active compounds, substitution of the basepolymer, and production of active films with potential antiox-idant [25] and antimicrobial activity [26] at industrial levelinstead of laboratory scale, thus evaluating real materials.

Numerous details about the optimization, the analytical char-acteristics and the migration values found in the three aqueoussimulants are shown and discussed.

2. Experimental

2.1. Apparatus

Experimental set-up for performing membrane microextrac-tions (see Fig. 1 for a schematic diagram) consisted of anAladdin AL-8000 programmable multisyringe pump from WorldPrecision Instruments Ltd. (Stevenage, Hertfordshire, UK); a mul-tipoint magnetic stirrer ACS-06 from Science Basic SolutionsS.L. (Barcelona, Spain) set at 300 rpm, with 8 mm × 3 mm pivotring stir bars from VWR International (Barcelona, Spain), and animmersion thermostat Tectron 200 from P. Selecta (Barcelona,Spain) with a poly(methylmethacrylate) water bath. Syringe pumpwas fitted with 100 �L capacity microsyringes 701 RN fromHamilton (Bonaduz, Switzerland), with 28-gauge, point style#3 needles (5 cm for solvent feeding to the membrane, 20 cmfor microsyringe–membrane connections), narrow enough to fitdirectly into the membrane without tear.

Since the previous work [20] demonstrated that withdraw wasbetter than direct pumping by minimizing the risk of looses, sol-vent flows during extraction from the 2 mL vial to the syringe barrel.Once 50 �L of toluene is drawn in, pump stops and bended needlesare removed from solvent vials. Then, 30 �L of air are sucked in

atogr. A 1216 (2009) 3731–3739 3733

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Table 1Experimental design: evaluated variables, tested values and optimum values foundfor the three simulants.

Variable Extreme values(low–high)

Optimum values

Simulant A Simulant B Simulant C

Extraction time (min) 5–20 20 20 20

J. Salafranca et al. / J. Chrom

min to allow all the toluene entering in the syringe body, long nee-les are disconnected from fibres and extract is flushed to 200 �Licrovials, which after closing with a crimp cap are ready to be

nalyzed by GC.Specific migration tests at 40 ± 0.1 ◦C for 10 days were done into

Binder ATP Line KB incubator (Tuttlingen, Germany).Qualitative analyses were performed by gas chromatography–

ass spectrometry (GC–MS) by using a Hewlett-Packard (Palo Alto,A) 6890 Series GC System coupled to a HP 5973 detector. Capil-

ary column was a HP-5 (60 m × 0.25 mm, 0.25 �m film thickness).njection (1 �L) was carried out in splitless mode (270 ◦C, split valvelosed for 0.25 min). Data were collected and processed with MSDhemstation D.03.00.611. Carrier gas was helium (C-50 quality)t a constant flow rate of 1.0 mL min−1 from Carburos MetálicosZaragoza, Spain). Temperature program was as follows: initialemp. 50 ◦C held for 2 min, then raised at 6.4 ◦C min−1 up to 95 ◦C,t 1.6 ◦C min−1 up to 110 ◦C and at 10 ◦C min−1 up to 250 ◦C held formin. Electron impact was selected as ionization mode (mass range

rom 40 to 400 amu), with a solvent delay of 9.1 min. Compoundsere identified by matching their mass spectra vs. NIST05 (USational Institute of Standards and Technology, Gaithersburg, MD,SA) and Hewlett-Packard Wiley275 commercial libraries (purity

hreshold >85%). All the analyses were carried out in triplicate.Quantitative analyses were carried out with a Trace GC Ultra

rom Thermo Electron Corp. (Madrid, Spain) equipped with an AS000 autosampler and flame ionization detector (FID). Capillaryolumn was a Supelco (Madrid, Spain) SLB-5ms (30 m × 0.25 mm,.25 �m film thickness). Injection (1 �L) was carried out in split-

ess mode (270 ◦C, split valve closed for 0.25 min). Carrier gas waselium at a constant flow rate of 1.0 mL min−1. For FID, hydrogen,itrogen and synthetic air, all of them C-50 quality from Carburosetálicos, were also used. Temperature program was as follows:

nitial temp. 50 ◦C held for 2 min, then raised at 10 ◦C min−1 up to5 ◦C, at 2 ◦C min−1 up to 110 ◦C and at 10 ◦C min−1 up to 250 ◦C heldor 5 min. Detector temperature was set at 280 ◦C.

.2. Reagents and materials

�-Pinene 98% [CAS 80-56-8], camphene ≥95% [79-92-5], ben-aldehyde ≥99% [100-52-7], �-phellandrene ≥95% [4221-98-1],-terpinene ≥95% [99-86-5], �-terpinene 97% [99-85-4], p-cymene9% [99-87-6], 1,8-cineole 99% [470-82-6], �-terpinolene 97% [586-2-9], linalool ≥97% [78-70-6], camphor 96% [76-22-2], estragole8% [140-67-0], borneol 98% [464-43-7], trans-cinnamaldehyde9% [14371-10-9], verbenone 94% [1196-01-6], bornyl acetate97% [76-49-3], thymol ≥99.5% [89-83-8], carvacrol ≥97% [499-5-2], cinnamyl alcohol 98% [104-54-1], eugenol 99% [97-53-0],-humulene ≥90% [116-04-1], �-humulene ≥98.0% [6753-98-6],-pentadecane ≥99% [629-62-9], n-hexadecane ≥99% [544-76-], n-heptadecane 99% [629-78-7], n-octadecane 99% [593-45-3],-eicosane 99% [112-95-8], benzyl benzoate 99% [120-51-4],iisobutyl phthalate 99% [84-69-5], tri-n-butyl citrate ≥97% [77-4-1] and tributyl 2-acetyl citrate ≥98% [77-90-7] were fromigma–Aldrich (Madrid, Spain).

Ethyl acetate ≥99.5% [141-78-6], toluene ≥99.9% [108-88-3],cetic acid glacial ≥99.8%, ethanol absolute ≥99.9% [64-17-5] andodium chloride ≥99.5% [7647-14-5] all of them reagent grade, ACS,SO quality were from Scharlab (Barcelona, Spain). Ultrapure wateras obtained from a Millipore Milli-Q system (Billerica, MA, USA).

thyl acetate was chosen as solvent for stock solutions of about000 mg kg−1 containing all the compounds. Appropriate dilutions

ere made with ultrapure water, being all the solutions gravimet-

ically controlled for improved accuracy.Hollow fibres Accurel® PP 150/330 (polypropylene, hydropho-

ic, 0.2 �m nominal pore size, 150 �m wall thickness, 600 �m inneriameter), were purchased from Membrana GmbH (Wuppertal,

Extraction temperature (◦C) 25–50 50 50 50Salt concentration (% w/w) 0–10 5a 5a 0

a Not significant; central value selected according to experimental design.

Germany), and they were used without any pretreatment. Carry-over was minimized by using each fibre only one time [27] andby performing regular blank analysis to confirm the absence ofcontamination.

The active polymer prototypes were prepared and supplied byArtibal (Sabinánigo, Spain). The polypropylene films were 30 �mthickness and contained the following natural essential oil extractsincorporated into the formulations: ginger (Zingiber officinalis, CAS8007-08-7), oregano (Origanum vulgare, 8007-11-2), cinnamon(Cinnamomum zeylanicum, 8015-91-6) and a fortified fraction ofcinnamon. Polypropylene film of 30 �m was also used as blanksample.

2.3. Migration studies

In this study, all the aqueous simulants were selected: water(simulant A, “SimA” hereafter); 3% (w/v) acetic acid (simulant B,“SimB”), and 10% (v/v) ethanol (simulant C, “SimC”), adequate topredict the behaviour of the active films against most of food-stuffs, except those of fatty nature, for which olive oil, isooctaneor 95% aqueous ethanol could be used as simulant. The mostusual testing conditions (10 days at 40 ◦C, area-to-volume ratio6 dm2 per dm3) were chosen according to Directive 97/48/CE [28].The active polypropylene film samples were cut in 3 cm × 4 cmrectangles, which were completely immersed in 20 mL of the cor-responding aqueous simulants contained in glass vials. After beingclosed with PTFE/butyl crimp caps, the vials were stored in a ther-mostatic oven. After the exposure time, the vials were opened,the active film was removed and the simulant was subjected toHFLPME. Six replicates were carried out for each experiment.

2.4. Experimental design optimization procedure

The software package Modde 6.0 from Umetrics AB (Umeå,Sweden) was used for planning and evaluating the results of theexperimental design, due to the high number of potential variablesinvolved in HFLPME. The most studied ones [21,29] are the natureof hollow fibre, acceptor solution, shaking degree, ionic strength,acceptor/donor volume ratio, pH and extraction time.

Following the guidelines of a previous work [20] ionic strength,extraction time and extraction temperature were selected. OnlyNaCl was considered as salt because of the very low acid–basebehaviour of the selected compounds and, especially, to avoidpH modifications. Experimental design consisted of a cubic, face-centred model, with 14 experiments plus 4 repetitions in thecentre. Despite all the compounds were individually evaluated,as will be commented later, the optimization criterion was thesum of the chromatographic peak areas of the 20 most represen-tative compounds selected (�-pinene, camphene, benzaldehyde,

�-phellandrene, �-terpinene, p-cymene, 1,8-cineole, terpinolene,linalool, camphor, borneol, trans-cinnamaldehyde, bornyl acetate,thymol, carvacrol, cinnamyl alcohol, eugenol, �-humulene, benzylbenzoate and tributyl acetyl citrate). Table 1 shows the variablesevaluated, their experimental domain as well as their optimum val-

3734 J. Salafranca et al. / J. Chromatogr. A 1216 (2009) 3731–3739

Fig. 2. GC–MS chromatogram from a cinnamon migration test from simulant B (3% acetic acid, upper trace) and its corresponding simulant B blank (lower trace), bothextracted by dynamic HFLPME.

Fig. 3. Significant factors for individual compounds according to the experimental design optimization for simulant A (water). Error bars corresponding to n = 4, 95% confidencelevel.

J. Salafranca et al. / J. Chromatogr. A 1216 (2009) 3731–3739 3735

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ig. 4. Significant factors for individual compounds according to the experimental donfidence level.

es for each simulant. In all the experiments, the extraction volumeas 50 �L of toluene, which withdraw speed depended inversely on

xtraction time. The procedure was identical for the three studiedimulants.

. Results and discussion

.1. Qualitative analysis

In order to get the maximum information, essential oil extracts,ctive packagings and food simulants have been analyzed fromhe qualitative point of view. Despite the high potential of bothIST05 and Wiley275 spectral libraries, some compounds couldot be correctly identified due to the complexity of the essentialil extracts on which active agents are based. Taking into accountrecent review [30] and to get accurate and unequivocal identifi-

ation, chromatographic data were correlated with retention indexatabases [31–33]. Thus, only those peaks whose spectral match inoth commercial libraries was higher than 85% and agreed in all thehree databases have been finally here reported.

Taking into account both legislative and consumer safetyiewpoints, the most relevant data are by far those derivedrom migration studies. Consequently, only the description ofhe results from food simulants is reported. The most abun-ant compounds found have been a wide range of terpenes,resent in the original essential oil extracts, n-alkanes fromhe polypropylene matrix, specific compounds from active for-

ulation such as ethyl citrate, benzyl benzoate or isopropylyristate, and even a degradation compound from phenolic

ntioxidants present in the polypropylene: 7,9-di-tert-butyl-1-xaspiro[4,5]-deca-6,9-dien-2,8-dione, already described in theiterature [34]. As an example, Fig. 2 shows a GC–MS chromatogramf a migration test from a cinnamon sample to SimB, where alank sample (lower trace) of a polypropylene film subjected to

optimization for simulant B (3% acetic acid). Error bars corresponding to n = 4, 95%

migration and extracted by HFLPME is included for comparisonpurposes.

3.2. Experimental design optimization procedure

In the previous work [20], 11 representative compounds fromthe natural essential oils used were initially selected for opti-mization purposes. Nevertheless, results from migration testsdemonstrated that up to 32 compounds were able to migrateto SimA. Taking into account these results, 9 additional chemi-cals found in such tests (benzaldehyde, �-phellandrene, p-cymene,terpinolene, borneol, bornyl acetate, cinnamyl alcohol, benzylbenzoate and tributyl acetyl citrate, considered as the most rep-resentative ones) have been included in this study to achievemore robust results from the experimental design, by evaluatingnow 20 different compounds. Standard solutions containing about500 �g kg−1 of each compound in each of the three simulants wereprepared. Aliquots of 20 mL were extracted by HFLPME accordingto the experimental design conditions. Toluene extracts were ana-lyzed by GC–FID, individual peak areas were calculated and foroptimization purposes, the sum of all of them was used.

Fig. 3 shows the individual behaviour of each compound in SimA,where the significant variables are plotted together with confidenceintervals. Considered factors have been the individual variables(extraction time, temperature and salt), as well as their quadraticand crossed by pairs ones. The central zero represents the averagearea of all the experiments of the design. Bars express the changein the response when the factors vary from the low to the highlevel. Thus, positive bars show the increase noticed in area when

rising the variable value (e.g. when sampling from 25 to 50 ◦C),whereas negative bars indicate that the most favourable conditionsoccur when using their lowest values. For clarity purposes, only thesignificant coefficients are represented here, since the confidenceinterval does not cross zero.

3736 J. Salafranca et al. / J. Chromatogr. A 1216 (2009) 3731–3739

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ig. 5. Significant factors for individual compounds according to the experimentalonfidence level.

As can be seen, extraction time demonstrated to be the mostnfluential variable. In 11 out of 20 studied compounds, a signifi-ant increase of area was observed when using 20 min instead of, whereas in the other 9 compounds no noticeable effect was pro-uced. This fact is still more marked in SimB (Fig. 4), where the

xtraction of up to 16 compounds was significantly improved whensing the highest extraction time. Despite the trend is the same inimC (extraction performance of 14 compounds is directly corre-ated with time, as can be seen in Fig. 5), an anomalous behaviourppeared in �-humulene, for which the best result was obtained

able 2nalytical characteristics of the developed automatic HFLPME procedure for the aqueous

ompound Detection limit (�g kg−1) Accuracy (�g kg−1) calculated

SimA SimB SimC SimA SimB

amphene 0.05 0.04 0.03 55.9 [56.2] 48.1 [55.8]enzaldehyde 0.02 0.02 0.04 54.4 [57.7] 52.7 [57.3]-Cymene 0.04 0.21 0.02 45.4 [45.8] 50.3 [45.5],8-Cineole 0.01 0.02 0.01 51.3 [56.2] 62.8 [54.9]-Terpinene 0.09 0.19 0.04 53.5 [48.0] 42.9 [47.7]inalool 0.01 0.02 0.01 45.1 [50.2] 44.7 [49.9]amphor 0.01 0.01 0.01 51.0 [57.7] 51.9 [57.3]stragole 0.02 0.02 0.02 56.7 [53.7] 49.8 [53.4]erbenone 0.02 0.01 0.03 50.1 [56.2] 52.9 [55.8]

rans-Cinnamaldehyde 0.01 0.01 0.02 61.5 [58.6] 79.0 [70.2]ornyl acetate 0.01 0.02 0.02 55.9 [52.8] 34.1 [30.4]hymol 0.01 0.01 0.01 56.5 [51.7] 47.1 [51.4]arvacrol 0.01 0.01 0.01 58.5 [62.5] 59.5 [62.1]ugenol 0.01 0.01 0.02 67.3 [63.0] 66.0 [62.6]-Humulene 0.16 0.01 0.06 55.6 [50.2] 54.0 [49.9]-Humulene 0.09 0.01 0.04 46.5 [54.0] 50.1 [53.6],4-Ditertbutylphenol 0.01 0.01 0.01 43.0 [48.4] 53.3 [48.1]enzyl benzoate 0.01 0.01 0.01 50.9 [46.7] 49.8 [46.4]iisobutylphthalate 0.01 0.01 0.02 51.8 [53.1] 55.9 [52.7]ributyl citrate 0.02 0.02 0.02 47.0 [43.4] 46.3 [43.1]

n optimization for simulant C (10% ethanol). Error bars corresponding to n = 4, 95%

when sampling 5 min. These results could be expected, since thetransfer of compounds through the membrane to toluene is moreefficient when increasing extraction time. Besides, as the finaltoluene volume is the same in all cases (50 �L), the flow rate isalso lower at high extraction times.

When considering temperature extraction, a moderate improve-ment in extraction performance was observed in SimA (4 casespositive influence vs. 2 negative) when increasing temperature,but this effect is much more notorious in SimB (12 cases positive,none negative) and SimC (15 positive vs. 3 negative for thymol, �-

simulants A (water), B (3% acetic acid) and C (10% ethanol).

[real] Precision (RSD, %); N = 6, 95% Concentration factor

SimC SimA SimB SimC SimA SimB SimC

58.6 [63.2] 10.5 17.8 16.4 8 11 956.3 [64.9] 16.3 13.3 12.2 51 67 8951.8 [51.6] 6.3 13.7 8.9 14 4 1862.3 [62.2] 7.1 17.2 9.9 76 50 7758.6 [54.0] 8.7 11.9 14.8 8 14 1456.3 [56.5] 8.8 12.2 9.5 99 69 8958.6 [64.9] 11.0 16.6 8.2 106 96 9659.7 [60.5] 15.2 17.9 9.8 60 45 6865.0 [63.2] 12.9 17.0 12.5 88 126 52

125.9 [121.0] 6.4 14.5 6.3 106 118 8152.3 [59.5] 12.2 15.4 11.9 93 74 7256.3 [58.2] 13.8 11.3 7.2 124 112 10068.7 [70.4] 12.6 14.0 8.3 136 126 10470.5 [70.9] 13.0 8.4 10.3 111 120 8551.4 [56.5] 20.7 9.1 11.8 77 69 4661.6 [60.7] 15.1 14.9 14.0 97 82 6249.0 [54.5] 15.5 11.4 10.7 189 126 12445.9 [52.5] 8.3 9.3 14.6 184 122 12254.7 [59.7] 10.1 11.0 14.4 101 134 12249.9 [48.8] 12.9 14.7 12.0 188 123 118

J.Salafrancaet

al./J.Chromatogr.A

1216(2009)

3731–37393737

Table 3Concentration of migrants (expressed as �g kg−1, N = 6, 95% confidence level) in simulants after migration tests by HFLPME.

Compound {retention index} Active packaging with ginger Active packaging with oregano Active packaging with cinnamon Active packaging with fortified cinnamon

Simulant A Simulant B Simulant C Simulant A Simulant B Simulant C Simulant A Simulant B Simulant C Simulant A Simulant B Simulant C

Camphene {939} 13.2 ± 2.2 7.7 ± 2.0 7.2 ± 0.8Benzaldehyde {961} 23.5 ± 1.4 <QL 36.5 ± 2.1p-Cymene {1022} <QL 11.6 ± 1.4 40.3 ± 10.3 5.3 ± 1.2 <QL 2.5 ± 0.41,8-Cineole {1033} 54.5 ± 8.2 59.8 ± 12.8 45.2 ± 6.5 22.2 ± 2.4 19.6 ± 3.8 13.6 ± 2.0 8.3 ± 0.3 <QL 2.2 ± 0.4�-Terpinene {1062} 11.1 ± 0.8 11.5 ± 1.7 16.3 ± 4.3 2.5 ± 0.7Linalool {1098} <QL 17.4 ± 1.1 11.7 ± 0.8 39.8 ± 5.4 31.4 ± 4.3 7.5 ± 0.7Chrysanthenonea {1125} 17.1 ± 1.6 15.6 ± 1.0 10.0 ± 0.7 8.2 ± 0.2 12.8 ± 0.6 8.4 ± 0.6 10.0 ± 0.6 11.1 ± 1.4 8.6 ± 0.4 12.3 ± 0.4 9.0 ± 0.7Camphor {1143} <QL 8.1 ± 0.4 10.0 ± 1.5 21.0 ± 2.4 <QLIsoborneol {1156} 13.1 ± 0.8Borneol {1165} 15.1 ± 1.4 23.6 ± 2.2 22.8 ± 2.2 19.5 ± 1.31-Terpinen-4-ola {1178} 18.7 ± 1.6 29.3 ± 2.5 9.8 ± 0.4 4.9 ± 0.2Myrtenala {1167} 26.6 ± 1.8�-Terpineol {1189} 12.6 ± 0.1 12.1 ± 1.3 14.1 ± 0.5 24.1 ± 1.3 7.0 ± 0.6Estragole {1195} 13.6 ± 2.9Verbenone {1204} 74.9 ± 7.7 24.4 ± 2.3 57.8 ± 5.5 84.8 ± 10.3 17.0 ± 0.8 40.0 ± 2.5 76.3 ± 2.6 22.8 ± 2.4 47.2 ± 2.5 74.9 ± 7.6 21.1 ± 2.0 43.3 ± 2.5Carvotanacetonea {1246} 20.0 ± 1.6Carvonea {1246} 22.3 ± 1.8 7.0 ± 0.1trans-Cinnamaldehyde {1266} 93.0 ± 3.5 111.9 ± 4.8 947.8 ± 93.7 947.3 ± 149.3Bornyl acetate {1285} 15.7 ± 0.2 <QLSafrola {1285} 14.2 ± 0.8 54.3 ± 2.8 16.0 ± 1.0Thymol {1290} 13.3 ± 1.5 72.6 ± 6.0 72.6 ± 8.2 65.5 ± 2.5p-Thymola {1296} 18.7 ± 0.9 25.2 ± 3.1 17.0 ± 2.3 19.4 ± 1.5Carvacrol {1298} 1241.5 ± 108.5 990.1 ± 116.4 876.5 ± 59.2Eugenol {1356} <QL 2330.5 ± 321.9 1754.5 ± 332.1 2112.2 ± 356.1 90.7 ± 11.1 51.0 ± 3.8 85.6 ± 11.2�-Humulene {1419} 1.7 ± 0.2Cinnamyl acetatea {1443} 11.6 ± 0.4 11.2 ± 0.3 11.7 ± 1.4�-Humulene {1455} <QLar-Curcumenea {1483} 8.6 ± 0.1 7.1 ± 0.2n-Pentadecane {1500} 39.8 ± 6.1 12.9 ± 0.7 10.2 ± 0.9 8.6 ± 0.3 12.2 ± 0.2 9.6 ± 0.9 46.2 ± 2.8 8.2 ± 1.5 11.8 ± 0.3 10.7 ± 1.12,4-Ditertbutylphenol {1512} 9.4 ± 1.6 <QL <QL 3.6 ± 0.6 <QL <QL 16.8 ± 3.0 20.9 ± 4.6 <QL 2.9 ± 0.4 15.0 ± 1.5 <QL�-Bisabolenea {1513} 8.6 ± 0.3Eugenol acetatea {1524} 78.0 ± 9.5 80.8 ± 13.8 47.3 ± 6.4 8.8 ± 0.7Elemola {1549} 15.5 ± 0.7 20.7 ± 3.0trans-Neridola {1564} 15.4 ± 1.5n-Hexadecane {1600} 30.8 ± 5.3 34.4 ± 4.0 27.7 ± 4.4 24.2 ± 2.8 39.4 ± 4.9 32.1 ± 4.2 17.0 ± 1.5 36.7 ± 4.8 37.0 ± 5.0 21.1 ± 2.0 41.6 ± 5.6 40.0 ± 5.8Ethyl citratea {1627} 340.9 ± 52.7 779.4 ± 98.8 362.8 ± 48.1 313.4 ± 56.0 810.2 ± 68.7 249.3 ± 11.9 280.6 ± 44.3 1079.4 ± 109.1 260.8 ± 44.0 373.0 ± 51.7 921.5 ± 95.2 7.3 ± 2.0n-Heptadecane {1700} 16.2 ± 1.3 19.2 ± 2.0 16.0 ± 2.2 10.7 ± 0.3 18.3 ± 1.6 10.6 ± 0.8 19.3 ± 1.7 12.8 ± 1.5 11.1 ± 0.6 18.2 ± 1.5 17.6 ± 2.2Benzyl benzoate {1762} <QL <QL 10.3 ± 1.7n-Octadecane {1800} 17.4 ± 2.3 24.8 ± 3.0 19.7 ± 2.5 16.3 ± 1.7 25.4 ± 1.5 18.3 ± 2.2 14.2 ± 1.3 28.0 ± 2.4 21.4 ± 2.5 14.8 ± 1.3 28.1 ± 3.3 24.8 ± 2.6Isopropyl myristatea {1829} 29.6 ± 3.5 33.8 ± 4.3 32.6 ± 3.8 18.7 ± 2.1 30.9 ± 2.4 23.9 ± 2.4 16.5 ± 1.6 33.9 ± 3.0 26.0 ± 4.1 17.8 ± 1.6 34.9 ± 3.8 29.4 ± 3.1Diisobutylphthalate {1868} <QL <QL <QL <QL <QL <QL <QL <QL <QL <QL <QL <QL7,9-Di-tert-butyl-1-

oxaspiro[4,5]-deca-6,9-dien-2,8-dionea

{1929}

22.5 ± 2.8 17.4 ± 1.0 14.9 ± 1.2 19.5 ± 2.1 16.5 ± 1.1 11.5 ± 0.6 12.0 ± 0.9 15.0 ± 0.9 10.4 ± 0.9 12.4 ± 2.9 15.2 ± 1.2 5.3 ± 0.2

n-Eicosane {2000} 8.9 ± 0.3 16.2 ± 1.0 11.6 ± 0.5 11.0 ± 0.6 15.5 ± 0.4 11.2 ± 0.3 9.6 ± 0.4 12.6 ± 1.9 10.0 ± 1.0 9.4 ± 0.4 15.6 ± 0.5 11.4 ± 1.5

a Quantitated as linalool since no standards were available.

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umulene and benzyl benzoate), being the main effect a higherass transfer ratio, still improved by stirring. Temperature was

lso the most important variable to be taken into account whenvaluating the effect of crossed variables. In this way, the fac-or temperature × time is very marked in SimA (10 cases of 20how positive influence), whereas for simulant B is negative onlyn one compound (�-humulene, which in fact proved to be the

ost anomalous one), and only in 3 cases positive for SimC, forhich the quadratic term temperature × temperature was positive

or �-humulene and benzyl benzoate.Considering the effect of ionic strength, modified by the addi-

ion of NaCl to avoid an undesired modification of pH values whichould strongly modify the nature of simulant B, this variable wasot significant for SimA and SimB. Thus, 5% was selected as centralalue of the experimental design, despite a negative trend was ineneral observed. For SimA, extraction of 8 compounds was madeifficult by the addition of salt, 5 for SimB and up to 13 for SimC,hereas only 1, 2 and 0 cases, respectively, were improved for each

imulant. With respect to crossed variables, in SimA salt × salt wasositive for benzyl benzoate, whereas temperature × salt was pos-

tive in SimB for �-humulene and negative in SimC for �-pinene.onsequently, very different effects depending on the particularompounds have been observed, which have been already reported,uch as extraction enhancement [35], no noticeable changes [36] orven a performance decrease [37].

Despite this detailed study compound-by-compound, an overallptimum extraction was pursued. Thus, the sum of all the areas waselected for fixing the best possible operating conditions, as shownn Table 1. Most of the compounds showed a similar behaviour, tak-ng into account their terpenic nature and close chemical structure,

hereas �-humulene, benzyl benzoate and benzaldehyde provedo be the more anomalous ones.

.3. Analytical characteristics of the automatic multiple HFLPMEethod

In all the simulants, the followed procedure was exactly theame. Table 2 shows the main analytical characteristics of the opti-ized procedure for SimA, SimB, and SimC. Besides given data,

inear ranges were also calculated with at least 6 calibration points,ach one in sextuplicate. In general, similar both detection anduantitation limits were found in the three simulants. Limits ofuantitation as low as 0.02 �g kg−1 for benzyl benzoate in SimA,.03 �g kg−1 for carvacrol, �-humulene and 2,4-ditertbutylphenol

n SimB, and 0.04 �g kg−1 for the same compounds in SimC werebserved, whereas the worst cases corresponded to �-humulene inimA and SimC (0.54 and 0.19 �g kg−1, respectively) and p-cymenen SimB (0.7 �g kg−1). With respect to upper linearity limits, theest performance was obtained for carvacrol and eugenol in all theimulants (from 1490.8 to 1627.5 �g kg−1), reaching almost 5 ordersf magnitude; on the other hand, p-cymene (45.8 and 94.6 �g kg−1

n SimA and SimC, respectively), and �-humulene (49.9 �g kg−1 inimB) showed the lowest linear range, around 3 orders of mag-itude. Calibration graphs fitted adequately to straight lines, withinimum R2 of 0.9851 for �-humulene in SimC (0.9895 in SimB)

r 0.9878 for camphor in SimA, and up to 0.9999 for camphene,-cymene, �-humulene (SimB) or p-cymene and 1,8-cineole inimC. Accuracy was measured at intermediate concentration level,esulting relative errors from −0.5% (camphene) to −13.9% (�-umulene) in SimA; from −4.2% (carvacrol) to +14.4% (1,8-cineole)

n SimB, or from +0.2% (1,8-cineole) to −13.3% (benzaldehyde) in

imC, with typical values below ±6%. Reproducibility—expresseds %RSD—was adequate for these concentration levels, since con-idering the whole extraction process, values ranging from 6.3%p-cymene in SimA and trans-cinnamaldehyde in SimC) up to a

oderate 20.7% for the anomalous �-humulene in SimA were

. A 1216 (2009) 3731–3739

found. In the last columns the values of calculated concentrationfactors [38] are shown, being poor for camphene (8, 11 and 9,respectively in SimA, SimB and SimC), p-cymene (14, 4 and 18)and �-terpinene (8, 14 and 14). On the other hand, the best perfor-mance was obtained for 2,4-ditertbutylphenol (189, 126 and 124),benzyl benzoate (184, 122 and 122) and diisobutyl phthalate (101,134 and 122). Butyl citrate reached a value of 188 in SimA, and ver-benone and carvacrol equalled the value of 2,4-ditertbutylphenolin SimB. The maximum theoretical value, taking into account theaqueous-to-organic phase ratio was about 400. In general, no sig-nificant differences among simulants occurred, despite individualresults for certain compounds were observed.

3.4. Results of migration tests

One of the main objectives of this work was to obtain directlymarketable active food packaging films. In the previous work [20]the migration values for some individual compounds were quitehigh for 3 of the 4 active plastic prototypes tested. In consequence,several modifications were considered to get profitable products.On the one hand, the concentration of active agents in the origi-nal formulation was reduced to one fourth, in an attempt to reducethe migration of some compounds: eugenol, camphor, benzalde-hyde and benzyl alcohol. On the other hand, polyethylene used asbase polymer has been replaced by polypropylene, also widely usedin food contact applications [39] with lower diffusion properties[40], and consequently its expected behaviour and performance vs.migration tests could be more favourable than in the first study.Additionally, the active films have been successfully manufacturedat industrial level instead of at laboratory scale. So, the resultsobtained will reflect accurately the behaviour of these real pack-aging films.

Table 3 shows the results obtained when the migration sam-ples were analyzed. Where no data appears, compounds are belowthe detection limit or simply not present into the compositionof the essential oil extracts. It can be pointed out that up to 37compounds migrated from ginger-based films to the different sim-ulants, followed by 24 from cinnamon, 23 from oregano and 19 fromfortified cinnamon-based ones. From a qualitative point of view,the behaviour of the three simulants was quite different depend-ing on the particular film. While similar performance in oreganoand fortified cinnamon-based films was observed, significant dif-ferences were noticed with ginger films, where 28 compounds weredetected in SimB vs. 25 in SimA and only 19 in SimC. Just the oppo-site was noticed in cinnamon-based films, where 24 compoundsmigrated to SimC, followed by 20 to SimB and 16 to SimA. Thehighest concentrations of migrants also depended on the simulantand the active film. Camphene, myrtenal, verbenone, carvotanace-tone, carvone, bornyl acetate, carvacrol, eugenol, ar-curcumene,�-bisabolene and trans-neridol always showed higher migrationvalues in SimA. Camphor, isoborneol, �-terpineol, estragole, safrol,p-thymol, �-humulene, eugenol acetate, ethyl citrate, isopropylmyristate and alkanes migrated preferably to SimB. Finally, cin-namyl acetate and elemol showed highest affinity by SimC in all thecases. The rest of compounds did not follow specific guidelines. Tak-ing into account overall results, SimB could be considered a priorias the most problematic one, due to both high number of migrantsand their concentrations. So, it should be recommended that thepriority use of these active films was for aqueous (pH > 4.5) andalcoholic foodstuffs instead of those of acidic nature.

In general, the migration values found are very low and no prob-

lems are expected. More in detail, most of the detected terpenesappear in legislation [41] as flavouring substances without restric-tions. Small risk could be expected only for estragole, since it wasinitially classified as “substance the use of which in certain Mem-ber States is subject to restrictive or prohibitive measures” and

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J. Salafranca et al. / J. Chrom

substance to be given priority evaluation” whereas a subsequentmendment of legislation [8] concluded that estragole was geno-oxic, and consequently should be deleted from the register. In anyase, its acute toxicity (LD50 1.23 g kg−1 oral dose in rats) can be clas-ified as moderate. Anyway, these substances are always presentn the ginger, cinnamon, oregano or rosemary used as spices inood.

From the point of view of packaging materials, and taking intoccount the European Legislation for plastics in contact with food-tuffs, only two migrating compounds are subjected to restrictionsn Directive 2002/72/CE and 1st amendment (13/02/2003) [9] whenhey are synthetically produced and included as chemical additivesnto the plastic material. Thus, while the only limitation for cam-hor is that its concentration must be in compliance with note 9

n annex VI (risk of deterioration of organoleptic characteristicsf food), eugenol has a restriction of 0.02 mg kg−1. So, one of theain goals of this work has been achieved, since introduced mod-

fications made the new oregano-based films safe. The reductionn concentration of active agents (one fourth of the original one)ave a very significant decrease in migration of eugenol of 12.4nd up to 25.7 times for cinnamon and fortified cinnamon-basedlms.

Despite restriction appears for 4-tertbutylphenol (0.05 mg kg−1)o reference is made to 2,4-ditertbutylphenol present in somextracts, but no risk is expected since its concentration is lowerhan 0.021 mg kg−1 in the worst case. Finally, some restrictions arepplied to several phthalates, such as dibutyl phthalate (0.3 mg kg−1

imulant), but the only detected one (diisobutyl phthalate) is alwayselow the quantitation limit (0.04–0.06 �g kg−1). In consequence,he studied active films are, according to specific migration val-es here reported, safe for consumers. Thus, they could be directlyommercialized for food contact applications in the European mar-et.

. Conclusion

A fully automated multiple dynamic HFLPME procedure haseen optimized and successfully applied to the evaluation of migra-ion values from active food packaging containing essential oils tohe three established aqueous simulants. Analytical characteristicsave been calculated, with detection limits as low as 0.01 �g kg−1,ery high linearity for almost 5 orders of magnitude, typical RSDelow 13% and concentration factors near to 200 for several com-ounds. Active polypropylene film prototypes including in theiromposition ginger and oregano at industrial level can be directlyaunched to the market without any problem, since they accomplish

ith the present European legislation as food contact materials andhe active agents used as food additives.

cknowledgements

This research has been financed by the Government of AragónConsolidated Research Group T-10); the projects AGL-07545 by

inistry of Science and Technology, Interreg IIIA-2005-326-C (Nat-ralpack) from and FEDER funds.

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. A 1216 (2009) 3731–3739 3739

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