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Analytica Chimica Acta 635 (2009) 167–174

Contents lists available at ScienceDirect

Analytica Chimica Acta

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

eadspace solid phase microextraction and gas chromatography–quadrupoleass spectrometry methodology for analysis of volatile compounds of marine

alt as potential origin biomarkers

sabel Silva, Sílvia M. Rocha ∗, Manuel A. Coimbraepartamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

r t i c l e i n f o

rticle history:eceived 6 October 2008eceived in revised form 24 December 2008ccepted 5 January 2009vailable online 14 January 2009

eywords:arine salt

altpansolatileseadspace solid phase microextractionombined with gas

a b s t r a c t

The establishment of geographic origin chemical biomarkers for the marine salt might represent animportant improvement to its valorisation. Volatile compounds of marine salt, although never stud-ied, are potential candidates. Thus, the purpose of this work was the development of a headspacesolid phase microextraction (SPME) combined with gas chromatography–quadrupole mass spectrom-etry (HS-SPME/GC–qMS) methodology to study the volatile composition of marine salt. A 65 �mcarbowax/divinylbenzene SPME coating fibre was used. Three SPME parameters were optimised: extrac-tion temperature, sample quantity, and presentation mode. An extraction temperature of 60 ◦C and 16 gof marine salt in a 120 mL glass vial were selected. The study of the effect of sample presentation modeshowed that the analysis of an aqueous solution saturated with marine salt allowed higher extraction effi-ciency than the direct analysis of salt crystals. The dissolution of the salt in water and the consequent effectof salting-out promote the release of the volatile compounds to the headspace, enhancing the sensitivity

hromatography–quadrupole masspectrometryeographic origin chemical biomarkers

of SPME for the marine salt volatiles. The optimised methodology was applied to real matrices of marinesalt from different geographical origins (Portugal, France, and Cape Verde). The marine salt samples con-tain ca. 40 volatile compounds, distributed by the chemical groups of hydrocarbons, alcohols, phenols,aldehydes, ketones, esters, terpenoids, and norisoprenoids. These compounds seem to arise from threemain sources: algae, surrounding bacterial community, and environment pollution. Since these volatilecompounds can provide information about the geographic origin and saltpans environment, this study

sed as
shows that they can be u
. Introduction

The marine salt is a natural product that is obtained in thealtpans. Saltpans are man-made systems where the salt is pro-uced by the evaporation of seawater due to a combined effect ofind blow and sunlight heat. In saltpans the seawater circulates by

ravity, flowing through different ponds with increasing levels ofalinity due to a continuous evaporation. Along the way, decanta-ion of silt and algae occurs. The harvest of the salt is possiblehen the point of crystallisation is achieved (s(NaCl) = 35.92 g/100 g

f aqueous solution at 25 ◦C) and the salt crystals precipitate1].

There is a typical environment associated to saltpans. Somepecies of grass (Dactylis glomerata L.), bushes (Sueda vera) andhrubs (Quercus ilex L.) have been already identified in the sur-oundings of saltpans, as well as aquatic plants (Zostera noltii) and

∗ Corresponding author. Tel.: +351 234401508; fax: +351 234370084.E-mail address: [email protected] (S.M. Rocha).

003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2009.01.011

chemical biomarkers of marine salt.© 2009 Elsevier B.V. All rights reserved.

algae (Spartina maritima, Euglena spp., Dinobryon spp., Eudorinaspp., Scendesmus spp.) [2,3].

Nowadays, there is a growing concern for the protection andrevalorisation of saltpans identity. This valorisation is intrinsicallyassociated to the quality of the marine salt produced, which canbe evaluated by its physico-chemical proprieties. Concerning thechemical characterisation of this natural product, the establishmentof geographic origin chemical biomarkers for the marine salt mightrepresent an important improvement for its valorisation.

There are references about the presence of volatile com-pounds, such as halocarbons, aliphatic and aromatic hydrocarbons,and ketones, in seawater [4–6]. In coastal atmosphere, volatilecompounds such as hydrocarbons, aldehydes, ketones, and noriso-prenoids have been also identified [7]. In addition, the presenceof carotenoids, that can give rise to volatile compounds (noriso-
prenoids), has been identified in some of the above-mentioned floragrowing in the surroundings of saltpans (Q. ilex L., Euglena spp.,Scendesmus spp.), as well as the emission of volatile compoundssuch as monoterpens from Q. ilex L. and Dinobryon spp. [8–14].Thus, it is possible that marine salt, as a natural product obtained
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dx.doi.org/10.1016/j.aca.2009.01.011

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68 I. Silva et al. / Analytica Ch

rom seawater in coastal zones, can contain volatile compoundsoming from these sources, present during all the crystallisationrocess. These volatile compounds may be considered as potentialandidates for geographic origin chemical biomarkers of marinealt.

Solid phase microextraction (SPME) is a rapid, easy, solvent-ree and sensitive sampling technique, evidenced by studies from aarge number of products [15]. The methodology, that comprisesPME associated with GC–qMS, is able to identify and quantifyolatile compounds, namely the compounds that occur in theeadspace of different matrices. An advantage of SPME over theonventional solvent extraction methods is that the extracts doot have to be concentrated prior to analysis, preventing lossesf low boiling point volatiles [16,17], and could allow their detec-ion, which is usually impossible due to their co-elution with theolvent.

Since, as far as we know, no information exists about theolatile composition of marine salt, the main purpose of this workas to develop a methodology for the analysis of marine salt

olatiles based on the headspace solid phase microextraction com-ined with gas chromatography–quadrupole mass spectrometryHS-SPME/GC–qMS). Three important SPME experimental parame-ers that can influence the extraction efficiency, namely extractionemperature, sample quantity, and presentation mode [18], wereonsidered on this study. The optimised methodology was appliedo real matrices of marine salt from different geographical originsPortugal, France, and Cape Verde).

. Experimental

.1. Samples

Marine salt produced in saltpans of Aveiro, Castro Marim, andavira, in Portugal, Guérande, in France, and Sal Island, in Capeerde, were analysed. The European samples were supplied by thearticipants in project SAL – Sal do Atlântico- “Revalorisation ofhe Atlantic traditional saltpans identity. Recovery and promotionf the biological, economical, and cultural potential of the humidones from the coast”, supported by the European CommissionINTERREG IIIB Programme). The salt from Cape Verde was obtainedirectly from a Sal Island producer. Salt from Aveiro saltpan (Pei-

ota) was used for the optimisation of the methodology. Samplesere stored in glass bottles until analysis.

.2. HS-SPME methodology

The SPME holder for manual sampling and the fibre used wereurchased from Supelco (Aldrich, Bellefonte, PA, USA). The SPMEevice included a fused silica fibre coating partially cross-linkedith 65 �m Carbowax/divinylbenzene (CW/DVB). CW/DVB coat-

ng combines the absorption properties of the liquid polymerith the adsorption properties of porous particles, which con-

ains macro (>500 Å), meso (20–500 Å) and microporous (2–20 Å).he mutually synergetic effect of adsorption and absorption ofhe stationary phase promotes a high retention capacity and,onsequently, a higher sensitivity than fibres based on absorp-ion only. According to these properties, CW/DVB presents a wideange capacity of sorbing compounds with different physico-hemical properties within a molecular weight ranging from 40o 275.

As there are no references about the volatile composition of
arine salt, this fibre, presenting a wide range capacity of sorbing
ompounds with different physicochemical properties, was con-idered the most adequate choice for this first exploratory study.he SPME fibre was conditioned at 250 ◦C for 30 min in the GCnjector, according to the manufacturer’s recommendations. Blanks,

Acta 635 (2009) 167–174

corresponding to the analysis of the coating fibre not submitted toany extraction procedure, were run between sets of three analy-ses. All measurements were made with, at least, three replicates,being each replicate the analysis of one different aliquot of marinesalt.

2.2.1. Effect of extraction temperature and sample quantityAqueous solutions saturated with 16 or 25 g of salt were analysed

using extraction temperatures of 40, 50, or 60 ◦C. Lower quantitiesof salt were tested without achieving adequate volatiles enrich-ment (data not shown). First, 16 g of marine salt were placed ina 120 mL glass vial containing a 30 mm stirring bar (500 rpm), andultra-pure water was added to complete a solution volume of 40 mL(1/� ratio of 0.5). The vial was capped with a PTFE septum andan aluminium cap (Chromacol Ltd., Herts, UK), and placed in athermostatted bath at 40.0 ± 0.1 ◦C for 18 h (overnight). Althoughadequate volatiles enrichment could be achieved in 4 h (data notshown), the longer periods have been used due to laboratory sched-ule convenience. After this step, the SPME fibre was manuallyinserted into the sample vial headspace for 90 min. This procedurewas repeated also for the assays with the extraction temperaturesof 50 ± 0.1 ◦C and 60 ± 0.1 ◦C. The assays for these three extractiontemperatures were also performed for 25 g of marine salt, wherethe undissolved salt in the bottom of the flask was visible during allexperiments.

2.2.2. Effect of sample presentation modeThe marine salt was analysed as a solid (S) and as an aqueous

saturated salt solution (Aq). For the analysis of S, 35 g (≈40 ml) ofmarine salt were placed in a 120 mL glass vial (1/� ratio of 0.5).For the analysis of Aq, 16 g of marine salt were placed in a 120 mLglass vial containing a 30 mm stirring bar (500 rpm) and ultra-purewater was added until a volume of 40 mL was achieved (1/� ratio of0.5). Both vials were capped with a PTFE septum and an aluminiumcap (Chromacol Ltd., Herts, UK), and were placed in a thermostat-ted bath adjusted to 60.0 ± 0.1 ◦C for 18 h (overnight). After thisstep, the SPME fibre was manually inserted into the sample vialheadspace for 90 min. An aqueous saturated solution of p.a. NaCl(99.5%, Aldrich) was also analysed using the methodology describedfor Aq. This analysis was done in order to depict possible artefactsarising from the water used to prepare the solutions.

2.2.3. Analysis of volatile compounds of marine saltsIn a 120 mL vial containing a 30 mm stirring bar (500 rpm) were

introduced 16 g of marine salt and ultra-pure water until a volumeof 40 mL was achieved. The vial was capped with a PTFE septum andan aluminium cap (Chromacol Ltd., Herts, UK), and was placed ina thermostatted bath adjusted to 60.0 ± 0.1 ◦C for 18 h (overnight).After this step, the SPME fibre was manually inserted into the sam-ple vial headspace for 90 min.

2.3. GC–qMS analysis

The SPME coating fibre containing the marine salt volatile com-pounds was manually introduced into the GC injection port at250 ◦C and kept for 5 min for desorption. The injection port waslined with a 0.75 mm I.D. splitless glass liner. The desorbed volatilecompounds were separated in an Agilent Technologies 6890NNetwork gas chromatograph, equipped with a 30 m × 0.32 mmI.D., 0.25 �m film thickness DB-FFAP fused silica capillary column(J&W Scientific, Folsom, CA, USA), connected to an Agilent 5973
quadrupole mass selective detector. Splitless injections were used(5 min). The GC oven temperature program was set at an initialtemperature of 35 ◦C for 2 min, raised to 100 ◦C at 4 ◦C min−1, thanraised to 200 ◦C at 2 ◦C min−1, and held there for 5 min. Heliumcarrier gas had a flow rate of 1.7 mL min−1 and the column head

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Table 1Sample quantity and extraction temperature effect on the GC–qMS peak area (×10−6) and coefficient of variation (CV% in parentheses) of the volatile compounds found in aqueous saturated marine salt solutions.

Peak number Compound Identificationa m/zb Peak areac (×10−6)

16 g 25 g

40 ◦C 50 ◦C 60 ◦C 40 ◦C 50 ◦C 60 ◦C

Hydrocarbons16 Pentadecane B, C 57, 71, 43, 85 3.90d – 8.40 (20) 9.42 (14) – – 3.70 (20) 7.64 (27)22 Heptadecane B, C 57, 71, 85, 43 – – 7.30 (28) 13.32 (18) – – 2.77 (15) 9.02 (22)23 8-Heptadecene B, C 83, 97, 69, 55 34.87 (69) 87.18 (8) 98.83 (12) 26.90 (24) 52.33 (20) 91.48 (22)

Subtotal (GC peak area) 36.17 (72) 102.87 (9) 121.57 (12) 26.90 (24) 58.80 (20) 108.15 (19)Subtotal (%) 47.55 (27) 53.43 (2) 53.15 (12) 52.18 (41) 37.70 (4) 46.36 (28)

Alcohols and phenols13 3-Octanol B, C 59, 83, 55, 101 – – 0.44d – 0.41c – – – – – – –34 2-(1,1-Dimethylethyl)-phenol B, C 135, 107, 150, 91 5.74 (52) 8.83 (6) 5.98e (6) 8.35e (2) 14.47 (19) 11.05e (1)37 ?-(1,1-Dimethylethyl)-phenol B, C 135, 107, 150, 95 12.09 (44) 22.60 (8) 17.82d (3) 16.16e (2) 35.83 (22) 33.95e (3)


Ketones6 6-Methyl-2-heptanone A, B, C 43, 58, 57, 71 1.09 (27) 0.79 (32) 0.68 (9) 1.23 (42) 1.09 (31) 0.90 (19)8 3-Octanone A, B, C 57, 43, 99, 72 7.34d – 6.58e (79) – – – – – – – –9 2,2,6-Trimethylcyclohexanone B, C 82, 69, 140, 56 0.67 (6) 0.71 (18) 0.60 (17) 0.85 (6) 1.48 (23) 0.83 (4)12 �-Isophorone B, C 82, 138, 54, 43 0.41c – 0.34 (21) 0.28 (8) 0.31 (24) 0.46 (28) 0.43 (23)20 6,10-Dimethyl-2-undecanone B,C 58, 43, 71, 85 – – 5.87 (17) 7.16 (18) – – 4.06 (19) 5.76 (33)33 6,10,14-Trimethyl-2-pentadecanone B, C 43, 58, 71, 57 7.83d – 21.69 (20) 42.48 (32) – – 10.94e (28) 46.15 (11)


Pyrroles and amines43 Benzopyrrole B, C 117, 90, 89, 63 – – – – – – 0.95d – – – – –47 Diphenylamine B, C 169, 168, 167, 170 – – – – 21.81c – – – – – – –

Subtotal (GC peak area) 0.00 – 0.00 – 21.81 – 0.95 – 0.00 – 0.00 –Subtotal (%) 0.00 – 0.00 – 9.84 – 2.18 – 0.00 – 0.00 –

Terpenoids and norisoprenoids5 1,8-Cineole A, B, C 108, 43, 154, 81 – – – – – – – – 0.79e (117) 1.35d –10 6-Methyl-5-heptene-2-one B, C, C 43, 69, 108, 93 0.88d – – – – – 1.06d – 0.23 (70) – –17 �-Cyclocitral B, C 137, 123, 109, 152 – – 1.70 (15) 2.39 (8) – – 1.83e (21) 2.17 (18)26 �-Ionone A, B, C 121, 93, 136, 91 2.50 (47) 6.23 (7) 7.22 (7) 2.63 (13) 6.62 (24) 8.44 (7)28 �-Ionone A, B, C 177, 43, 57, 71 3.47 (70) 8.56 (9) 13.35 (2) 1.66 (23) 6.74 (37) 10.93 (9)29 trans-�-Ionone-5,6-epoxide B, C 123, 43, 135, 124 – – 2.65d – 5.68e (30) – – 5.57 (14) 8.54 (7)39 Dihydroactinidiolide B, C 111, 137, 180, 43 3.76e (38) 7.84 (9) 9.68e (16) 4.86e (9) 12.35 (14) 14.19 (70)


Total 69.71 193.45 229.25 53.84 155.51 236.93

a The reliability of the identification or structural proposal is indicated by the following: A – mass spectrum and retention time consistent with those of an authentic standard; B – structural proposals given on the basis of massspectral data (Wiley 275); C – mass spectrum consistent with spectra found in literature.

b Ordered by decreasing intensity, being the peak base the fragment on the left side.c Mean of three replicates, numbers in parentheses correspond to the coefficient of variation (%).d The compound was detected only in one replicate.e The compound was detected only in two replicates.

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ressure was 12 psi. The mass spectrometer was operated in thelectron impact mode (EI) at 70 eV scanning the range 33–300 m/zn a 3 scans s−1, in a full scan acquisition mode. The identificationf the chromatogram peaks was done comparing all mass spectraith the library data system of the GC–qMS equipment (Wiley 275).

he spectra were also compared with spectra found in the litera-ure. The identification of each volatile compound was confirmedy comparing its mass spectrum and retention time with those of
he pure standard compounds, when available. Reproducibility wasxpressed as coefficient of variation (CV) in Tables 1–3. The GC peakreas were used as an indirect approach to estimate the relativeontent of each volatile compound.
able 2ample presentation effect (solid salt and aqueous saturated salt solution) on the GC–qMompounds found in marine salt. Aqueous saturated solution of NaCl (99.5%) use to contr

eak number Compound Identificationa m/zb

Hydrocarbons16 Pentadecane B, C 57, 71, 43, 822 Heptadecane B, C 57, 71, 85, 423 8-Heptadecene B, C 83, 97, 69, 524 Heptadecadiene B, C 67, 81, 95, 8

Subtotal (Peak area)Subtotal (%)

Alcohols15 2-Ethyl-1-hexanol B, C 57, 40, 41, 430 2-Methyl-1-dodecanol B, C 57, 43, 69, 7


Aldehydes and ketones40 �-Hexylcinnamaldehyde B, C 129, 115, 116 6-Methyl-2-heptanone A, B, C 43, 58, 57, 79 2,2,6-Trimethylcyclohexanone B, C 82, 140, 5520 6,10-Dimethyl-2-undecanone B, C 58, 43, 71, 832 2,6-di(t-Butyl)-4-hydroxi-4-methyl-2,

5-cyclohexadiene-1-oneB, C 165, 180, 57

33 6,10,14-trimethyl-2-pentadecanone B, C 43, 58, 71, 542 Acetylethyltetramethyltetralin B, C 243, 258, 4


Esters and thioethers38 Dihydromethyljasmonate B, C 83, 156, 15341 Ethylphthalate B, C 149, 177, 1545 Isobutylphthalate B, C 149, 223, 548 Butylphthalate B, C 149, 150, 211 2,3,4-Trithiapentane B, C 126, 45, 111


Terpenoids and norisoprenoids5 1,8-Cineole A, B, C 108, 43, 1547 p-Cimene A, B, C 119, 134, 9110 6-Methyl-5-hepten-2-one B, C 43, 69, 10817 �-Cyclocitral B, C 137, 123, 1026 �-Ionone A, B, C 121, 93, 13628 �-Ionone A, B, C 177, 43, 57,39 Dihydroactinidiolide B, C 111, 109, 1344 4-Oxo-�-ionone B, C 163, 206, 1


Total

a The reliability of the identification or structural proposal is indicated by the followitandard; B – structural proposals given on the basis of mass spectral data (Wiley 275); C

b Ordered by decreasing intensity, being the peak base the fragment on the left side.c Mean of three replicates, numbers in parentheses correspond to the coefficient of vard The compound was detected only in two replicates.e The compound was detected only in one replicate.

Acta 635 (2009) 167–174

3. Results and discussion

3.1. HS-SPME methodology

3.1.1. Evaluation of extraction temperature and sample quantityeffects

Sample preparation is one of the most critical steps in chromato-graphic analysis. The temperature used for extraction is one of the
most important parameters for the evaluation of efficiency in SPMEand other extraction methodologies [19]. The effect of temperaturewas studied by the analysis of saturated solutions of marine saltusing three different temperatures (40, 50, and 60 ◦C). Two sam-
S peak area (×10−6) and coefficient of variation (CV% in parentheses) of the volatileol possible artefacts arising from the water.

Peak areac (×10−6)

Aqueous saturated solutionof NaCl (p.a., 99.5%)

Marine salt

Solid salt (S) Aqueous saturatedsolution (Aq)

5 – – – – 21.81 (44)3 – – – – 11.95 (25)5 – – 81.86 (9) 336.93 (26)2 – – – – 17.96 (19)

0.00 – 81.86 (9) 388.65 (24)0.00 – 19.58 (29) 53.28 (44)

3 – – – – 1.42d (25)1 – – – – 4.33d (43)

0.00 – 0.00 – 5.74 (26)0.00 – 0.00 – 0.59 (32)

7, 216 – – 6.45 (33) 4.41 (32)1 – – 0.26 (23) 0.32 (27)

, 69 – – 0.26 (6) 0.24 (45)5 – – 1.90d (28) 3.66 (9), 137 – – 9.57 (53) 38.65 (114)

9 – – 35.77 (33) 42.25 (28)3, 244 – – 3.76d (39) 3.24 (69)

0.00 – 56.09 (5) 92.66 (59)0.00 – 13.53 (32) 11.05 (37)

, 82 – – 9.34 (60) 7.68 (59)0, 176 5.65 (92) 161.57 (134) 10.10 (58)

7, 150 39.13 (20) – – 179.39 (79)23, 205 – – 36.03 (45) 108.11 (77), 79 – – 0.31e – – –

44.78 (19) 227.79 (85) 305.29 (77)100.00 (0) 44.39 (45) 32.38 (67)

, 81 – – – – 0.34e –, 117 – – – – 1.54 (24)

, 93 – – – – 0.51 (59)9, 152 – – 2.90 (6) – –, 91 – – 6.51 (18) 5.76 (6)71 – – 18.04 (50) 12.65 (6)7, 243 – – 58.69 (12) – -21, 43 – – 4.13 (12) – –

0.00 – 90.27 (14) 20.58 (4)0.00 – 22.50 (45) 2.90 (50)

44.78 456.01 811.01

ng: A – mass spectrum and retention time consistent with those of an authentic– mass spectrum consistent with spectra found in literature.

iation (%).

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Table 3GC–qMS peak area (×10−6) and coefficient of variation (CV% in parentheses) of the volatile compounds found in marine salts from different geographic origins (aqueous saturated solution): Portugal (Aveiro, Castro Marim, andTavira), France (Guérande) and Cape Verde (Sal Island).

Peak number Compound Identificationa m/zb Peak areac (×10−6)

Aveiro Castro Marim Tavira Guérande Sal Island

Hydrocarbons1 Decane B, C 57, 43, 71, 85 1.13e (71) – – – – – – – –4 Dodecane B, C 57, 71, 43, 85 3.64 (56) – – – – – – – –14 Tetradecane B, C 57, 71, 43, 85 7.30d – – – – – – – – –16 Pentadecane B, C 57, 71, 43, 85 10.08 (19) – – – – – – – –18 Hexadecane B, C 57, 71, 43, 85 4.00 (75) – – – – – – – –21 n.i. (m/z 57, 71, 43, 85, 113) B, C – – – – – – 28.71 (12) – –22 Heptadecane B, C 57, 71, 85, 43 9.64 (23) – – – – – – – –23 8-Heptadecene B, C 83, 97, 69, 55 132.93 (12) – – – – 29.98 (29) – –24 Heptadecadiene B, C 67, 81, 95, 82 6.77 (51) – – – – – – – –25 n.i. (m/z 57, 71, 85, 43, 113) B, C – – – – – – 13.82 (23) – –

Subtotal (Peak area) 170.25 (18) 0.00 – 0.00 – 72.51 (4) 0.00 –Subtotal (%) 58.24 (23) 0.00 – 0.00 – 53.23 (4) 0.00 –

Alcohols and phenols15 2-Ethyl-1-hexanol B, C 57, 40, 41, 43 1.24 (9) – – – – – – 1.50e (94)27 BHT A, B, C 205, 220, 206, 145 36.04 (121) 1.35 (90) tre – tr – 1.32 (27)36 2-(1,1-Dimethylethyl)-?-methylphenol B, C 149, 121, 164, 150 5.56 (8) 2.31d – 3.46e (25) – – – –46 o-Phenylphenol B, C 170, 169, 141, 115 – – – – – – – – 1.82e (44)

Subtotal (Peak area) 42.84 (103) 2.12 (45) 3.46 (25) 0.00 – 3.54 (79)Subtotal (%) 13.88 (96) 1.98 (30) 1.53 (15) 0.00 – 3.48 (66)

Aldehydes and ketones3 2-Ethylhexanal B, C 72, 57, 41, 43 – – – – – – 1.23 (17) – –40 �-Hexylcinnamaldehyde B, C 129, 115, 117, 216 1.87 (32) – – – – – – – –6 6-Methyl-2-heptanone A, B, C 43, 58, 57, 71 0.31 (14) – – – – 0.88 (8) – –8 3-Octanone A, B, C 57, 43, 99, 72 14.72e (136) – – – – 1.53e (28) – –9 2,2,6-Trimethylcyclohexanone B, C 82, 140, 55, 69 0.20 (38) – – – – 5.63 (8) – –20 6,10-Dimethyl-2-undecanone B, C 58, 43, 71, 85 3.69 (11) – – – – – – – –33 6,10,14-trimethyl-2-pentadecanone B, C 43, 58, 71, 59 29.31 (18) – – – – – – – –42 Acetylethyltetramethyltetralin B, C 243, 258, 43, 244 1.40 (25) – – – – – – – –

Subtotal (Peak area) 46.60 (24) 0.00 – 0.00 – 8.76 (11) 0.00 –Subtotal (%) 15.68 (17) 0.00 – 0.00 – 6.46 (15) 0.00 –

Esters38 Dihydromethyljasmonate B, C 83, 156, 153, 82 7.45 (12) 6.29d – 10.67‘ (2) – – 8.99 (17)41 Ethylphthalate B, C 149, 177, 150, 176 – – 7.58 (83) 22.24 (67) – – 15.33 (17)45 Isobutylphthalate B, C 149, 223, 57, 150 – – 60.00 (19) 89.89 (37) 7.12 (30) 28.29 (30)48 Butylphthalate B, C 149, 150, 223, 205 15.05 (26) 31.88 (17) 54.24 (49) – – 16.55 (11)

Subtotal (Peak area) 22.50 (20) 101.56 (26) 173.49 (47) 7.12 (30) 69.16 (17)Subtotal (%) 7.72 (27) 97.56 (0) 98.10 (0) 5.19 (27) 76.44 (24)

Terpenoids and norisoprenoids2 Limonene B, C 68, 93, 67, 79 – – – – – – – – 7.92e (29)7 p-Cimene A, B, C 119, 134, 91, 117 – – – – 0.60e (6) – – 14.18e (27)10 6-Methyl-5-hepten-2-one B, C 43, 69, 108, 93 0.66 (44) 0.43 (48) 0.64 (56) tr – 0.81 (41)19 �-Humulene A, B,C 93, 121, 88, 147 – – – – – – – – 2.81e (0)26 �-Ionone A, B, C 121, 93, 136, 91 3.55 (7) – – – – 10.16 (4) – –28 �-Ionone A, B, C 177, 43, 57, 71 8.93 (25) – – – – 12.40 (13) 7.24e (37)31 Viridiflorol B, C 161, 109, 43, 107 – – – – – – 19.32 (14) – –35 �-Eudesmol B, C 59, 149, 164, 108 – – – – – – 6.02 (37) – –

Subtotal (peak area) 13.14 (14) 0.43 (48) 1.04 (57) 47.90 (7) 22.25 (85)Subtotal (%) 4.49 (20) 0.46 (74) 0.88 (107) 35.12 (4) 20.08 (84)

Total 295.32 104.11 176.84 136.30 94.95

a The reliability of the identification or structural proposal is indicated by the following: A – mass spectrum and retention time consistent with those of an authentic standard; B – structural proposals given on the basis of massspectral data (Wiley 275); C – mass spectrum consistent with spectra found in literature.

b Ordered by decreasing intensity, being the peak base the fragment on the left side.c Mean of three replicates, numbers in parentheses correspond to the coefficient of variation (%).d The compound was detected only in one replicate.e The compound was detected only in two replicates.

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le quantities were also tested (16 and 25 g). The optimisation ofhese two parameters was done using saturated solutions, basedn the principle that the addition of water to a matrix can increasehe extraction efficiency [20]. The results of these analyses are pre-ented in Table 1.

For both sample quantities tested, the increase of extractionemperature from 40 to 50 ◦C enabled the identification of a highestumber of compounds. This fact must be related with the incre-ent of the volatility of some compounds within the matrix [17].

or the analysis with 16 g of salt the increment was from 13 to 17ompounds identified, while for the analysis with 25 g of salt thencrement was from 11 to 17 compounds identified (Table 1). For0 and 60 ◦C the number of compounds identified was almost theame. These compounds have been grouped in the following chem-cal families: hydrocarbons, alcohols, phenols, ketones, pyrroles,mines, terpenoids, and norisoprenoids.

Analysing the GC peak areas of the compounds identified, it isossible to verify that, using 16 g of salt, the increase from 40 to0 ◦C increased the GC peak areas of ketones and alcohols and phe-ols, while the increase from 50 to 60 ◦C increased the GC peakreas of ketones but decreased those of alcohols and phenols. Using5 g of salt, the increase from 40 to 50 ◦C increased the GC peakreas of hydrocarbons, alcohols and phenols and terpenoids andorisoprenoids, while the increase from 50 to 60 ◦C increased theC peak areas of hydrocarbons and ketones. For all the other chemi-al families the differences of the GC peak areas were not significantTable 1). The extraction temperature has two opposing effects on
he SPME technique, resulting the extraction efficiency from a com-romise between the solubility and the volatility of the compounds17,20]. Reproducibility, expressed as coefficients of variation (CV),anged from 1% to 117%. The high values obtained may be explainedy the fact that the marine salt is a natural heterogeneous product.
Fig. 1. Typical HS-SPME/GC–qMS chromatograms of the volatile composition of solid m

Acta 635 (2009) 167–174

For each extraction temperature, no significant differences wereobserved between the total GC peak areas obtained for 16 and 25 gof salt (Table 1). For the conditions investigated, the extraction tem-perature seems to have a higher influence than the sample quantityused. The CV values for almost all compounds were higher when25 g of salt were used. For 16 g of salt the increment of total GCpeak area between 50 and 60 ◦C was not significant (Table 1). How-ever, although the GC peak areas for the majority of the compoundswere not significantly different, four compounds (heptadecane,6,10,14-trimethyl-2-pentadecanone, �-cyclocitral, and �-ionone)presented higher GC peak areas for 60 ◦C and two compounds (2-and ?-(1,1-dimethylethyl)-phenol) presented higher GC peak areasfor 50 ◦C.

According to the previous results, it was established an extrac-tion temperature of 60 ◦C and a sample quantity of 16 g forthe analysis of the volatile composition of marine salt by HS-SPME/GC–qMS.

3.1.2. Evaluation of sample presentation effectBased on the bibliography [20], it was expected to achieve

higher extraction efficiency by addition of water to the marine salt(aqueous solution) than by the direct analysis of the solid crystals.However, in order to confirm the application of this principle to theanalysis of the salt, the marine salt was analysed as solid crystals(S) and as aqueous saturated solution (Aq).

Fig. 1 shows the typical HS-SPME/GC–qMS chromatogramsof solid marine salt and of aqueous saturated salt solution, and
Table 2 shows the comparison of the two sample presentations(S and Aq) on the GC–qMS peak area of the volatile compoundsfound in marine salt. For S analysis, 17 compounds were identi-fied, while the analysis of Aq results in 22 identified compounds(Table 2 and Fig. 1). These compounds have been grouped in the
arine salt (a) and of an aqueous saturated salt solution (b). a.u.: arbitrary units.

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ollowing chemical families: hydrocarbons, alcohols, aldehydes,etones, esters, thioethers, terpenoids, and norisoprenoids. Withhe exception of �-cyclocitral, the alcohols and terpenoids weredentified only in Aq.

For S, esters represented the chemical family with the higherontribution to the total GC peak area, while the aldehydes andetones presented the higher number of compounds. For Aq, hydro-arbons presented the higher contribution to the total GC peak area,nd aldehydes and ketones (as observed for S) presented the higherumber of compounds. Reproducibility, expressed as CV values,anged from 6% to 134% for S and from 6% to 114% for Aq.

The total GC peak area of the identified compounds was 78%igher for Aq (Table 2 and Fig. 1) when compared to S, showing thathe higher extraction efficiency was attainted for Aq mode. Thus, thenalysis of a saturated solution enhances the sensitivity of SPME forhe marine salt volatiles. A reason for this experimental behaviouran be explained by the effect of the salt dissolution in water plushe effect of salting-out. The salt dissolution in water promotes theelease to the aqueous phase of compounds that could be adsorbedn the surface of the salt crystals and/or entrapped by them duringrystallisation and crystals deposition. The saturated salt solutionormed promotes the release of these volatile compounds to theeadspace by the salting-out effect. Based on this principle, theddition of salt is routinely applied in the analysis of liquid matricesy SPME to increase the extraction efficiency. In the present studybased on the same principle) the salt itself was the object undertudy.

In order to depict possible artefacts arising from the ultra-pureater used to prepare the solutions, an aqueous saturated solu-

ion of p.a. NaCl (99.5%) was analysed. Table 2 shows that just twoompounds were detected in common to marine salt: ethylphtha-ate and isobutylphthalate. However, the data obtained allowedo infer that these compounds should also be considered marinealt volatile components: ethylphthalate showed higher GC areasn solid salt than in the other samples investigated, and aqueousaturated solution of marine salt exhibited the highest GC area forsobutylphthalate.

According to the previous results, it was established that theample presentation mode for the HS-SPME/GC–qMS methodologypplied to the analysis of the volatile composition of marine salt (forvial of 120 ml) should be 40 mL of an aqueous solution saturatedith 16 g of marine salt.

.2. Application – analysis of marine salts from differenteographical origins

Using the optimised conditions described in Section 3.1, it wasnalysed marine salts from different geographical origins: Portu-al (Aveiro, Castro Marim, and Tavira), France (Guérande) and Capeerde (Sal Island) (Table 3). The analysis of the volatile compositionf these marine salts allowed the identification of compounds fromifferent chemical families, including hydrocarbons, alcohols, phe-ols, aldehydes, ketones, esters, terpenoids, and norisoprenoids.he number and type of compounds varied according to the salteographical origin. The marine salt from Aveiro presented theigher number of identified compounds (23) while Castro Marimalt presented the lowest (7). The families of compounds with areater contribution to the total chromatographic area were theydrocarbons, for the marine salt from Aveiro and Guérande, andhe esters, for those from Castro Marim, Tavira, and Sal Island. Forhe majority of these salts, the chemical family of esters was the one
hat presented the highest number of compounds. For the salt fromveiro, the chemical family with more identified compounds washe hydrocarbons, and for the marine salt from Guérande were theerpenoids and norisoprenoids. Reproducibility, expressed as CValues, ranged from 2% to 136%. Similar high reproducibility values
Acta 635 (2009) 167–174 173

were observed previously for the optimisation of the methodol-ogy.

Among all compounds identified in marine salts from dif-ferent geographical origins, it was possible to find somecompounds in common to all or almost all of the analysed sam-ples. The 2,6-bis(1,1-dimethylethyl)-4-methyl-phenol (BHT) and6-methyl-5-hepten-2-one were present in all samples, and dihy-dromethyljasmonate, isobutyl phthalate, and butyl phthalate werenot identified only in one of the five different salts analysed.

The possible sources of some compounds found in the marinesalts analysed are the following:

- 6-Methyl-5-hepten-2-one, �-ionone, 2,2,6-trimethyl-cyclohe-xanone, �-ionone, and 6-methyl-2-heptanone have been iden-tified in algae, aquatic plants, and bacteria [7,21–25];

- 8-Heptadecene has been identified in aquatic plants and in bac-terial communities specific of hyper saline environments [21,26];

- �-Humulene, �-eudesmol, and viridiflorol are sesquiterpenoidsthat have been identified in plants and �-eudesmol also in aquaticfungi [27–30];

- Dihydromethyljasmonate has been related with the metabolitesresponsible for the protection mechanisms of plants [31,32];

- Some of the identified compounds probably come from environ-ment pollution. The BHT is an antioxidant used in food stuffs,but also in paints and gasoline [33–35]; its presence could berelated with the traffic of boats nearby the water supply ofthe saltpans. The 2-ethyl-1-hexanol, a compound that exists innature, can be produced by bacteria and fungi while degrad-ing plasticizing substances [36,37]. The �-hexylcinnamaldehydeis a compound labelled “Generally Recognized as Safe” (GRAS)food additive by the Food and Drug Administration [38], thatmay come from contaminated water that supplies the saltpans.The acetylethyltetramethyltetralin, a synthetic musk fragranceused in detergents, cosmetics, and perfumes, are present in theatmosphere. This ketone undergoes bioaccumulation and causesecotoxicity in aquatic environments [39]. The o-phenylphenol, acompound used as fungicide in food and other products like woodand textiles [40], may also come from contaminated water thatcould supply the saltpans.

The results presented in Table 3 and the possible sources ofvolatile compounds found in marine salt showed that the volatilecomposition of each marine salt can be related to its geographicorigin and, consequently, to the environment of each saltpan. Thus,according to these results, the volatile compounds identified inthe marine salt of the different geographical origins may havethree main sources: (i) algae, (ii) surrounding bacterial community,and/or (iii) environment pollution. These compounds, coming fromthe saltpans environment, are present during all the crystallisationprocess of the marine salt. Along the crystallisation process, thesecompounds can be retained in the salt crystals. Therefore, thereis a possibility that the volatile compounds can be used as chem-ical biomarkers of geographic origin for marine salt, since thesebiomarkers can provide information about the geographic origin aswell as the saltpans environment.

4. Conclusions

This paper is the first investigation on volatile compoundspresent in marine salt. In this work it was developed a HS-
SPME/GC–qMS methodology that allowed the detection andidentification of volatile and semi-volatile compounds from marinesalt. Optimisation of SPME parameters, including extraction tem-perature and sample quantity, showed a good compromise betweenefficiency and reproducibility for 60 ◦C of extraction temperature

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sing 16 g of salt. The study on the effect of sample presentationode showed that the analysis of an aqueous solution saturatedith marine salt allowed a higher efficiency of extraction than

he direct analysis of salt crystals, by enhancing the sensitivity ofPME for the marine salt volatiles. The salt dissolution in water andhe consequent effect of salting-out promote the release of volatileompounds to the headspace.

Concerning the application of the developed methodology toamples of marine salt from different origins, it was possible todentify in their headspace ca. 40 volatile compounds from differenthemical families, which included hydrocarbons, alcohols, phenols,ldehydes, ketones, esters, terpenoids, and norisoprenoids. Theseeem to arise from three main sources: (i) algae, (ii) surround-ng bacterial community, and/or (iii) environment pollution. In theuture, more studies should be done including salt samples fromifferent harvests and more origins.

cknowledgements

This work was financially supported by FCT, Research UnitOPNA and by a PhD grant (SFRH/BD/31076/2006) to Isabel Silva.he authors tank project SAL – “Sal do Atlântico” (INTERREG IIIB) forroviding the European marine salt samples, and Professor Filom-na Martins and Mrs. Margarida Silva, from Universidade de Aveiro,or helpful discussions.

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http://www.biorede.pt/

http://www.valorizarariadeaveiro.com/

http://www.epa.gov/hpv/pubs/summaries/cinna/c12912tp.pdf/

http://www.epa.gov/hpv/pubs/summaries/cinna/c12912tp.pdf/

Headspace solid phase microextraction and gas chromatography–quadrupole mass spectrometry...

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