Headspace solid phase microextraction and gas chromatography–quadrupole mass spectrometry methodology for analysis of volatile compounds of marine salt as potential origin biomarkers

Analytica Chimica Acta 635 (2009) 167–174

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Headspace solid phase microextraction and gas chromatography–quadrupole mass spectrometry methodology for analysis of volatile compounds of marine salt as potential origin biomarkers Isabel Silva, Sílvia M. Rocha ∗ , Manuel A. Coimbra Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e

i n f o

Article history: Received 6 October 2008 Received in revised form 24 December 2008 Accepted 5 January 2009 Available online 14 January 2009 Keywords: Marine salt Saltpans Volatiles Headspace solid phase microextraction combined with gas chromatography–quadrupole mass spectrometry Geographic origin chemical biomarkers

a b s t r a c t The establishment of geographic origin chemical biomarkers for the marine salt might represent an important improvement to its valorisation. Volatile compounds of marine salt, although never studied, are potential candidates. Thus, the purpose of this work was the development of a headspace solid phase microextraction (SPME) combined with gas chromatography–quadrupole mass spectrometry (HS-SPME/GC–qMS) methodology to study the volatile composition of marine salt. A 65 ␮m carbowax/divinylbenzene SPME coating fibre was used. Three SPME parameters were optimised: extraction temperature, sample quantity, and presentation mode. An extraction temperature of 60 ◦ C and 16 g of marine salt in a 120 mL glass vial were selected. The study of the effect of sample presentation mode showed that the analysis of an aqueous solution saturated with marine salt allowed higher extraction efficiency than the direct analysis of salt crystals. The dissolution of the salt in water and the consequent effect of salting-out promote the release of the volatile compounds to the headspace, enhancing the sensitivity of SPME for the marine salt volatiles. The optimised methodology was applied to real matrices of marine salt from different geographical origins (Portugal, France, and Cape Verde). The marine salt samples contain 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 three main sources: algae, surrounding bacterial community, and environment pollution. Since these volatile compounds can provide information about the geographic origin and saltpans environment, this study shows that they can be used as chemical biomarkers of marine salt. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The marine salt is a natural product that is obtained in the saltpans. Saltpans are man-made systems where the salt is produced by the evaporation of seawater due to a combined effect of wind blow and sunlight heat. In saltpans the seawater circulates by gravity, flowing through different ponds with increasing levels of salinity due to a continuous evaporation. Along the way, decantation of silt and algae occurs. The harvest of the salt is possible when the point of crystallisation is achieved (s(NaCl) = 35.92 g/100 g of aqueous solution at 25 ◦ C) and the salt crystals precipitate [1]. There is a typical environment associated to saltpans. Some species of grass (Dactylis glomerata L.), bushes (Sueda vera) and shrubs (Quercus ilex L.) have been already identified in the surroundings 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). 0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2009.01.011

algae (Spartina maritima, Euglena spp., Dinobryon spp., Eudorina spp., Scendesmus spp.) [2,3]. Nowadays, there is a growing concern for the protection and revalorisation of saltpans identity. This valorisation is intrinsically associated to the quality of the marine salt produced, which can be evaluated by its physico-chemical proprieties. Concerning the chemical characterisation of this natural product, the establishment of geographic origin chemical biomarkers for the marine salt might represent an important improvement for its valorisation. There are references about the presence of volatile compounds, such as halocarbons, aliphatic and aromatic hydrocarbons, and ketones, in seawater [4–6]. In coastal atmosphere, volatile compounds such as hydrocarbons, aldehydes, ketones, and norisoprenoids have been also identified [7]. In addition, the presence of carotenoids, that can give rise to volatile compounds (norisoprenoids), has been identified in some of the above-mentioned flora growing in the surroundings of saltpans (Q. ilex L., Euglena spp., Scendesmus spp.), as well as the emission of volatile compounds such 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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from seawater in coastal zones, can contain volatile compounds coming from these sources, present during all the crystallisation process. These volatile compounds may be considered as potential candidates for geographic origin chemical biomarkers of marine salt. Solid phase microextraction (SPME) is a rapid, easy, solventfree and sensitive sampling technique, evidenced by studies from a large number of products [15]. The methodology, that comprises SPME associated with GC–qMS, is able to identify and quantify volatile compounds, namely the compounds that occur in the headspace of different matrices. An advantage of SPME over the conventional solvent extraction methods is that the extracts do not have to be concentrated prior to analysis, preventing losses of low boiling point volatiles [16,17], and could allow their detection, which is usually impossible due to their co-elution with the solvent. Since, as far as we know, no information exists about the volatile composition of marine salt, the main purpose of this work was to develop a methodology for the analysis of marine salt volatiles based on the headspace solid phase microextraction combined with gas chromatography–quadrupole mass spectrometry (HS-SPME/GC–qMS). Three important SPME experimental parameters that can influence the extraction efficiency, namely extraction temperature, sample quantity, and presentation mode [18], were considered on this study. The optimised methodology was applied to real matrices of marine salt from different geographical origins (Portugal, France, and Cape Verde). 2. Experimental 2.1. Samples Marine salt produced in saltpans of Aveiro, Castro Marim, and Tavira, in Portugal, Guérande, in France, and Sal Island, in Cape Verde, were analysed. The European samples were supplied by the participants in project SAL – Sal do Atlântico- “Revalorisation of the Atlantic traditional saltpans identity. Recovery and promotion of the biological, economical, and cultural potential of the humid zones from the coast”, supported by the European Commission (INTERREG IIIB Programme). The salt from Cape Verde was obtained directly from a Sal Island producer. Salt from Aveiro saltpan (Peijota) was used for the optimisation of the methodology. Samples were stored in glass bottles until analysis. 2.2. HS-SPME methodology The SPME holder for manual sampling and the fibre used were purchased from Supelco (Aldrich, Bellefonte, PA, USA). The SPME device included a fused silica fibre coating partially cross-linked with 65 ␮m Carbowax/divinylbenzene (CW/DVB). CW/DVB coating combines the absorption properties of the liquid polymer with the adsorption properties of porous particles, which contains macro (>500 Å), meso (20–500 Å) and microporous (2–20 Å). The mutually synergetic effect of adsorption and absorption of the stationary phase promotes a high retention capacity and, consequently, a higher sensitivity than fibres based on absorption only. According to these properties, CW/DVB presents a wide range capacity of sorbing compounds with different physicochemical properties within a molecular weight ranging from 40 to 275. As there are no references about the volatile composition of marine salt, this fibre, presenting a wide range capacity of sorbing compounds with different physicochemical properties, was considered the most adequate choice for this first exploratory study. The SPME fibre was conditioned at 250 ◦ C for 30 min in the GC injector, according to the manufacturer’s recommendations. Blanks,

corresponding to the analysis of the coating fibre not submitted to any extraction procedure, were run between sets of three analyses. All measurements were made with, at least, three replicates, being each replicate the analysis of one different aliquot of marine salt. 2.2.1. Effect of extraction temperature and sample quantity Aqueous solutions saturated with 16 or 25 g of salt were analysed using extraction temperatures of 40, 50, or 60 ◦ C. Lower quantities of salt were tested without achieving adequate volatiles enrichment (data not shown). First, 16 g of marine salt were placed in a 120 mL glass vial containing a 30 mm stirring bar (500 rpm), and ultra-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 and an aluminium cap (Chromacol Ltd., Herts, UK), and placed in a thermostatted bath at 40.0 ± 0.1 ◦ C for 18 h (overnight). Although adequate volatiles enrichment could be achieved in 4 h (data not shown), the longer periods have been used due to laboratory schedule convenience. After this step, the SPME fibre was manually inserted into the sample vial headspace for 90 min. This procedure was repeated also for the assays with the extraction temperatures of 50 ± 0.1 ◦ C and 60 ± 0.1 ◦ C. The assays for these three extraction temperatures were also performed for 25 g of marine salt, where the undissolved salt in the bottom of the flask was visible during all experiments. 2.2.2. Effect of sample presentation mode The 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) of marine 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 mL glass vial containing a 30 mm stirring bar (500 rpm) and ultra-pure water was added until a volume of 40 mL was achieved (1/␤ ratio of 0.5). Both vials were capped with a PTFE septum and an aluminium cap (Chromacol Ltd., Herts, UK), and were placed in a thermostatted bath adjusted to 60.0 ± 0.1 ◦ C for 18 h (overnight). After this step, the SPME fibre was manually inserted into the sample vial headspace for 90 min. An aqueous saturated solution of p.a. NaCl (99.5%, Aldrich) was also analysed using the methodology described for Aq. This analysis was done in order to depict possible artefacts arising from the water used to prepare the solutions. 2.2.3. Analysis of volatile compounds of marine salts In 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 volume of 40 mL was achieved. The vial was capped with a PTFE septum and an aluminium cap (Chromacol Ltd., Herts, UK), and was placed in a thermostatted bath adjusted to 60.0 ± 0.1 ◦ C for 18 h (overnight). After this step, the SPME fibre was manually inserted into the sample vial headspace for 90 min. 2.3. GC–qMS analysis The SPME coating fibre containing the marine salt volatile compounds was manually introduced into the GC injection port at 250 ◦ C and kept for 5 min for desorption. The injection port was lined with a 0.75 mm I.D. splitless glass liner. The desorbed volatile compounds were separated in an Agilent Technologies 6890N Network gas chromatograph, equipped with a 30 m × 0.32 mm I.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 initial temperature of 35 ◦ C for 2 min, raised to 100 ◦ C at 4 ◦ C min−1 , than raised to 200 ◦ C at 2 ◦ C min−1 , and held there for 5 min. Helium carrier gas had a flow rate of 1.7 mL min−1 and the column head

Table 1 Sample 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

Identificationa

Compound

m/zb

Peak areac (×10−6 ) 16 g

25 g

40 ◦ C 16 22 23

6 8 9 12 20 33

43 47

5 10 17 26 28 29 39

Alcohols and phenols 3-Octanol 2-(1,1-Dimethylethyl)-phenol ?-(1,1-Dimethylethyl)-phenol Subtotal (GC peak area) Subtotal (%) Ketones 6-Methyl-2-heptanone 3-Octanone 2,2,6-Trimethylcyclohexanone ␣-Isophorone 6,10-Dimethyl-2-undecanone 6,10,14-Trimethyl-2-pentadecanone Subtotal (GC peak area) Subtotal (%) Pyrroles and amines Benzopyrrole Diphenylamine Subtotal (GC peak area) Subtotal (%) Terpenoids and norisoprenoids 1,8-Cineole 6-Methyl-5-heptene-2-one ␤-Cyclocitral ␣-Ionone ␤-Ionone trans-␤-Ionone-5,6-epoxide Dihydroactinidiolide Subtotal (GC peak area) Subtotal (%) Total

60 ◦ C

B, C B, C B, C

57, 71, 43, 85 57, 71, 85, 43 83, 97, 69, 55

3.90d – 34.87 36.17 47.55

– – (69) (72) (27)

B, C B, C B, C

59, 83, 55, 101 135, 107, 150, 91 135, 107, 150, 95

– 5.74 12.09 17.83 31.48

– (52) (44) (46) (57)

0.44d 8.83 22.60 31.58 16.41

– (6) (8) (8) (2)

A, B, C A, B, C B, C B, C B,C B, C

43, 58, 57, 71 57, 43, 99, 72 82, 69, 140, 56 82, 138, 54, 43 58, 43, 71, 85 43, 58, 71, 57

1.09 7.34d 0.67 0.41c – 7.83d 6.95 9.20

(27) – (6) – – – (69) (38)

0.79 6.58e 0.71 0.34 5.87 21.69 33.79 17.27

(32) (79) (18) (21) (17) (20) (27) (18)

B, C B, C

117, 90, 89, 63 169, 168, 167, 170

– – 0.00 0.00

A, B, C B, C, C B, C A, B, C A, B, C B, C B, C

108, 43, 154, 81 43, 69, 108, 93 137, 123, 109, 152 121, 93, 136, 91 177, 43, 57, 71 123, 43, 135, 124 111, 137, 180, 43

– 0.88d – 2.50 3.47 – 3.76e 8.77 11.77 69.71

– – – – – – – (47) (70) – (38) (73) (16)

8.40 7.30 87.18 102.87 53.43

– – 0.00 0.00 – – 1.70 6.23 8.56 2.65d 7.84 25.21 13.18 193.45

(20) (28) (8) (9) (2)

9.42 13.32 98.83 121.57 53.15

40 ◦ C

50 ◦ C

60 ◦ C

(14) (18) (12) (12) (12)

– – 26.90 26.90 52.18

– – (24) (24) (41)

3.70 2.77 52.33 58.80 37.70

(20) (15) (20) (20) (4)

0.41c 5.98e 17.82d 24.00 10.36

– (6) (3) (2) (10)

– 8.35e 16.16e 24.52 41.66

– (2) (2) (2) (10)

– 14.47 35.83 50.30 32.20

– (19) (22) (21) (6)

– 11.05e 33.95e 45.00 18.13

– (1) (3) (2) (0)

0.68 – 0.60 0.28 7.16 42.48 51.20 22.09

(9) – (17) (8) (18) (32) (28) (19)

1.23 – 0.85 0.31 – – 2.39 4.70

(42) – (6) (24) – – (26) (46)

1.09 – 1.48 0.46 4.06 10.94e 14.39 9.69

(31) – (23) (28) (19) (28) (36) (46)

0.90 – 0.83 0.43 5.76 46.15 54.06 22.83

(19) – (4) (23) (33) (11) (8) (2)

– – – –

– – 0.00 0.00

– – – –

– – – –

– 21.81c 21.81 9.84

– – – –

0.95d – 0.95 2.18

– – – –

– – (15) (7) (9) – (9) (9) (18)

– – 2.39 7.22 13.35 5.68e 9.68e 33.21 14.58

– – (8) (7) (2) (30) (16) (29) (32)

– 1.06d – 2.63 1.66 – 4.86e 7.88 14.62

– – – (13) (23) – (9) (19) (5)

229.25

53.84

– – 0.00 0.00 0.79e 0.23 1.83e 6.62 6.74 5.57 12.35 32.02 20.41 155.51

(117) (70) (21) (24) (37) (14) (14) (27) (13)

7.64 9.02 91.48 108.15 46.36

1.35d – 2.17 8.44 10.93 8.54 14.19 44.72 18.72

(27) (22) (22) (19) (28)

– – (18) (7) (9) (7) (70) (22) (15)

I. Silva et al. / Analytica Chimica Acta 635 (2009) 167–174

13 34 37

Hydrocarbons Pentadecane Heptadecane 8-Heptadecene Subtotal (GC peak area) Subtotal (%)

50 ◦ C

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 mass spectral 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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pressure was 12 psi. The mass spectrometer was operated in the electron impact mode (EI) at 70 eV scanning the range 33–300 m/z in a 3 scans s−1 , in a full scan acquisition mode. The identification of the chromatogram peaks was done comparing all mass spectra with the library data system of the GC–qMS equipment (Wiley 275). The spectra were also compared with spectra found in the literature. The identification of each volatile compound was confirmed by comparing its mass spectrum and retention time with those of the pure standard compounds, when available. Reproducibility was expressed as coefficient of variation (CV) in Tables 1–3. The GC peak areas were used as an indirect approach to estimate the relative content of each volatile compound.

3. Results and discussion 3.1. HS-SPME methodology 3.1.1. Evaluation of extraction temperature and sample quantity effects Sample preparation is one of the most critical steps in chromatographic analysis. The temperature used for extraction is one of the most important parameters for the evaluation of efficiency in SPME and other extraction methodologies [19]. The effect of temperature was studied by the analysis of saturated solutions of marine salt using three different temperatures (40, 50, and 60 ◦ C). Two sam-

Table 2 Sample presentation effect (solid salt and aqueous saturated salt solution) on the GC–qMS peak area (×10−6 ) and coefficient of variation (CV% in parentheses) of the volatile compounds found in marine salt. Aqueous saturated solution of NaCl (99.5%) use to control possible artefacts arising from the water. Peak number

Identificationa

Compound

m/zb

Peak areac (×10−6 ) Aqueous saturated solutionMarine salt of NaCl (p.a., 99.5%)

16 22 23 24

15 30

40 6 9 20 32 33 42

38 41 45 48 11

5 7 10 17 26 28 39 44

Hydrocarbons Pentadecane Heptadecane 8-Heptadecene Heptadecadiene Subtotal (Peak area) Subtotal (%) Alcohols 2-Ethyl-1-hexanol 2-Methyl-1-dodecanol Subtotal (Peak area) Subtotal (%) Aldehydes and ketones ␣-Hexylcinnamaldehyde 6-Methyl-2-heptanone 2,2,6-Trimethylcyclohexanone 6,10-Dimethyl-2-undecanone 2,6-di(t-Butyl)-4-hydroxi-4-methyl-2, 5-cyclohexadiene-1-one 6,10,14-trimethyl-2-pentadecanone Acetylethyltetramethyltetralin Subtotal (Peak area) Subtotal (%) Esters and thioethers Dihydromethyljasmonate Ethylphthalate Isobutylphthalate Butylphthalate 2,3,4-Trithiapentane Subtotal (Peak area) Subtotal (%) Terpenoids and norisoprenoids 1,8-Cineole p-Cimene 6-Methyl-5-hepten-2-one ␤-Cyclocitral ␣-Ionone ␤-Ionone Dihydroactinidiolide 4-Oxo-␤-ionone Subtotal (Peak area) Subtotal (%) Total

a

Solid salt (S)

Aqueous saturated solution (Aq)

B, C B, C B, C B, C

57, 71, 43, 85 57, 71, 85, 43 83, 97, 69, 55 67, 81, 95, 82

– – – – 0.00 0.00

– – – – – –

– – 81.86 – 81.86 19.58

– – (9) – (9) (29)

21.81 (44) 11.95 (25) 336.93 (26) 17.96 (19) 388.65 (24) 53.28 (44)

B, C B, C

57, 40, 41, 43 57, 43, 69, 71

– – 0.00 0.00

– – – –

– – 0.00 0.00

– – – –

1.42d 4.33d 5.74 0.59

(25) (43) (26) (32)

B, C A, B, C B, C B, C B, C

129, 115, 117, 216 43, 58, 57, 71 82, 140, 55, 69 58, 43, 71, 85 165, 180, 57, 137

– – – – –

– – – – –

6.45 0.26 0.26 1.90d 9.57

(33) (23) (6) (28) (53)

4.41 0.32 0.24 3.66 38.65

(32) (27) (45) (9) (114)

B, C B, C

43, 58, 71, 59 243, 258, 43, 244

– – 0.00 0.00

– – – –

35.77 3.76d 56.09 13.53

(33) (39) (5) (32)

42.25 3.24 92.66 11.05

(28) (69) (59) (37)

B, C B, C B, C B, C B, C

83, 156, 153, 82 149, 177, 150, 176 149, 223, 57, 150 149, 150, 223, 205 126, 45, 111, 79

– – 5.65 (92) 39.13 (20) – – – – 44.78 (19) 100.00 (0)

9.34 161.57 – 36.03 0.31e 227.79 44.39

(60) (134) – (45) – (85) (45)

7.68 (59) 10.10 (58) 179.39 (79) 108.11 (77) – – 305.29 (77) 32.38 (67)

A, B, C A, B, C B, C B, C A, B, C A, B, C B, C B, C

108, 43, 154, 81 119, 134, 91, 117 43, 69, 108, 93 137, 123, 109, 152 121, 93, 136, 91 177, 43, 57, 71 111, 109, 137, 243 163, 206, 121, 43

– – – – – – – – 0.00 0.00

– – – 2.90 6.51 18.04 58.69 4.13 90.27 22.50

– – – (6) (18) (50) (12) (12) (14) (45)

0.34e 1.54 0.51 – 5.76 12.65 – – 20.58 2.90

44.78

– – – – – – – – – –

456.01

– (24) (59) – (6) (6) – (4) (50)

811.01

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 mass spectral 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 two replicates. e The compound was detected only in one replicate.

Table 3 GC–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, and Tavira), France (Guérande) and Cape Verde (Sal Island). Peak number

Identificationa

Compound

Peak areac (×10−6 )

m/zb

Aveiro

Castro Marim

Tavira

Guérande

Sal Island

Hydrocarbons 1 4 14 16 18 21 22 23 24 25

3 40 6 8 9 20 33 42

38 41 45 48

2 7 10 19 26 28 31 35

Alcohols and phenols 2-Ethyl-1-hexanol BHT 2-(1,1-Dimethylethyl)-?-methylphenol o-Phenylphenol Subtotal (Peak area) Subtotal (%) Aldehydes and ketones 2-Ethylhexanal ␣-Hexylcinnamaldehyde 6-Methyl-2-heptanone 3-Octanone 2,2,6-Trimethylcyclohexanone 6,10-Dimethyl-2-undecanone 6,10,14-trimethyl-2-pentadecanone Acetylethyltetramethyltetralin Subtotal (Peak area) Subtotal (%) Esters Dihydromethyljasmonate Ethylphthalate Isobutylphthalate Butylphthalate Subtotal (Peak area) Subtotal (%) Terpenoids and norisoprenoids Limonene p-Cimene 6-Methyl-5-hepten-2-one ␣-Humulene ␣-Ionone ␤-Ionone Viridiflorol ␤-Eudesmol Subtotal (peak area) Subtotal (%) Total

B, C B, C B, C B, C B, C B, C B, C B, C B, C B, C

57, 43, 71, 85 57, 71, 43, 85 57, 71, 43, 85 57, 71, 43, 85 57, 71, 43, 85 57, 71, 85, 43 83, 97, 69, 55 67, 81, 95, 82

1.13e 3.64 7.30d 10.08 4.00 – 9.64 132.93 6.77 – 170.25 58.24

(71) (56) – (19) (75) – (23) (12) (51) – (18) (23)

– – – – – – – – – – 0.00 0.00

B, C A, B, C B, C B, C

57, 40, 41, 43 205, 220, 206, 145 149, 121, 164, 150 170, 169, 141, 115

1.24 36.04 5.56 – 42.84 13.88

(9) (121) (8) – (103) (96)

– 1.35 2.31d – 2.12 1.98

B, C B, C A, B, C A, B, C B, C B, C B, C B, C

72, 57, 41, 43 129, 115, 117, 216 43, 58, 57, 71 57, 43, 99, 72 82, 140, 55, 69 58, 43, 71, 85 43, 58, 71, 59 243, 258, 43, 244

– 1.87 0.31 14.72e 0.20 3.69 29.31 1.40 46.60 15.68

– (32) (14) (136) (38) (11) (18) (25) (24) (17)

– – – – – – – – 0.00 0.00

83, 156, 153, 82 149, 177, 150, 176 149, 223, 57, 150 149, 150, 223, 205

7.45 – – 15.05 22.50 7.72

(12) – – (26) (20) (27)

68, 93, 67, 79 119, 134, 91, 117 43, 69, 108, 93 93, 121, 88, 147 121, 93, 136, 91 177, 43, 57, 71 161, 109, 43, 107 59, 149, 164, 108

– – 0.66 – 3.55 8.93 – – 13.14 4.49

– – (44) – (7) (25) – – (14) (20)

B, C B, C B, C B, C

B, C A, B, C B, C A, B,C A, B, C A, B, C B, C B, C

295.32

6.29d 7.58 60.00 31.88 101.56 97.56

– – 0.43 – – – – – 0.43 0.46 104.11

– – – – – – – – – – – –

– (90) – – (45) (30)

– – – – – – – – – – 0.00 0.00

– tre 3.46e – 3.46 1.53

– – – – – – – – – – – –

– – – – – 28.71 – 29.98 – 13.82 72.51 53.23

– – – – – (12) – (29) – (23) (4) (4)

– – (25) – (25) (15)

– tr – – 0.00 0.00

– – – – – –

– – – – – – – – – –

– – – – – – – – 0.00 0.00

– – – – – – – – – –

– (83) (19) (17) (26) (0)

10.67‘ 22.24 89.89 54.24 173.49 98.10

– – (48) – – – – – (48) (74)

– 0.60e 0.64 – – – – – 1.04 0.88 176.84

– – – – – – – – – – 0.00 0.00 1.50e 1.32 – 1.82e 3.54 3.48

– – – – – – – – – – – –

(94) (27) – (44) (79) (66)

1.23 – 0.88 1.53e 5.63 – – – 8.76 6.46

(17) – (8) (28) (8) – – – (11) (15)

– – – – – – – – 0.00 0.00

– – – – – – – – – –

(2) (67) (37) (49) (47) (0)

– – 7.12 – 7.12 5.19

– – (30) – (30) (27)

8.99 15.33 28.29 16.55 69.16 76.44

(17) (17) (30) (11) (17) (24)

– (6) (56) – – – – – (57) (107)

– – tr – 10.16 12.40 19.32 6.02 47.90 35.12

– – – – (4) (13) (14) (37) (7) (4)

7.92e 14.18e 0.81 2.81e – 7.24e – – 22.25 20.08

(29) (27) (41) (0) – (37) – – (85) (84)

136.30

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15 27 36 46

Decane Dodecane Tetradecane Pentadecane Hexadecane n.i. (m/z 57, 71, 43, 85, 113) Heptadecane 8-Heptadecene Heptadecadiene n.i. (m/z 57, 71, 85, 43, 113) Subtotal (Peak area) Subtotal (%)

94.95

a

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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 mass spectral 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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ple quantities were also tested (16 and 25 g). The optimisation of these two parameters was done using saturated solutions, based on the principle that the addition of water to a matrix can increase the extraction efficiency [20]. The results of these analyses are presented in Table 1. For both sample quantities tested, the increase of extraction temperature from 40 to 50 ◦ C enabled the identification of a highest number of compounds. This fact must be related with the increment of the volatility of some compounds within the matrix [17]. For the analysis with 16 g of salt the increment was from 13 to 17 compounds identified, while for the analysis with 25 g of salt the increment was from 11 to 17 compounds identified (Table 1). For 50 and 60 ◦ C the number of compounds identified was almost the same. These compounds have been grouped in the following chemical families: hydrocarbons, alcohols, phenols, ketones, pyrroles, amines, terpenoids, and norisoprenoids. Analysing the GC peak areas of the compounds identified, it is possible to verify that, using 16 g of salt, the increase from 40 to 50 ◦ C increased the GC peak areas of ketones and alcohols and phenols, while the increase from 50 to 60 ◦ C increased the GC peak areas of ketones but decreased those of alcohols and phenols. Using 25 g of salt, the increase from 40 to 50 ◦ C increased the GC peak areas of hydrocarbons, alcohols and phenols and terpenoids and norisoprenoids, while the increase from 50 to 60 ◦ C increased the GC peak areas of hydrocarbons and ketones. For all the other chemical families the differences of the GC peak areas were not significant (Table 1). The extraction temperature has two opposing effects on the SPME technique, resulting the extraction efficiency from a compromise between the solubility and the volatility of the compounds [17,20]. Reproducibility, expressed as coefficients of variation (CV), ranged from 1% to 117%. The high values obtained may be explained by the fact that the marine salt is a natural heterogeneous product.

For each extraction temperature, no significant differences were observed between the total GC peak areas obtained for 16 and 25 g of salt (Table 1). For the conditions investigated, the extraction temperature seems to have a higher influence than the sample quantity used. The CV values for almost all compounds were higher when 25 g of salt were used. For 16 g of salt the increment of total GC peak area between 50 and 60 ◦ C was not significant (Table 1). However, although the GC peak areas for the majority of the compounds were 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 (2and ?-(1,1-dimethylethyl)-phenol) presented higher GC peak areas for 50 ◦ C. According to the previous results, it was established an extraction temperature of 60 ◦ C and a sample quantity of 16 g for the analysis of the volatile composition of marine salt by HSSPME/GC–qMS. 3.1.2. Evaluation of sample presentation effect Based 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 the analysis 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 chromatograms of 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 compounds found in marine salt. For S analysis, 17 compounds were identified, while the analysis of Aq results in 22 identified compounds (Table 2 and Fig. 1). These compounds have been grouped in the

Fig. 1. Typical HS-SPME/GC–qMS chromatograms of the volatile composition of solid marine salt (a) and of an aqueous saturated salt solution (b). a.u.: arbitrary units.

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following chemical families: hydrocarbons, alcohols, aldehydes, ketones, esters, thioethers, terpenoids, and norisoprenoids. With the exception of ␤-cyclocitral, the alcohols and terpenoids were identified only in Aq. For S, esters represented the chemical family with the higher contribution to the total GC peak area, while the aldehydes and ketones presented the higher number of compounds. For Aq, hydrocarbons presented the higher contribution to the total GC peak area, and aldehydes and ketones (as observed for S) presented the higher number of compounds. Reproducibility, expressed as CV values, ranged from 6% to 134% for S and from 6% to 114% for Aq. The total GC peak area of the identified compounds was 78% higher for Aq (Table 2 and Fig. 1) when compared to S, showing that the higher extraction efficiency was attainted for Aq mode. Thus, the analysis of a saturated solution enhances the sensitivity of SPME for the marine salt volatiles. A reason for this experimental behaviour can be explained by the effect of the salt dissolution in water plus the effect of salting-out. The salt dissolution in water promotes the release to the aqueous phase of compounds that could be adsorbed on the surface of the salt crystals and/or entrapped by them during crystallisation and crystals deposition. The saturated salt solution formed promotes the release of these volatile compounds to the headspace by the salting-out effect. Based on this principle, the addition of salt is routinely applied in the analysis of liquid matrices by SPME to increase the extraction efficiency. In the present study (based on the same principle) the salt itself was the object under study. In order to depict possible artefacts arising from the ultra-pure water used to prepare the solutions, an aqueous saturated solution of p.a. NaCl (99.5%) was analysed. Table 2 shows that just two compounds were detected in common to marine salt: ethylphthalate and isobutylphthalate. However, the data obtained allowed to infer that these compounds should also be considered marine salt volatile components: ethylphthalate showed higher GC areas in solid salt than in the other samples investigated, and aqueous saturated solution of marine salt exhibited the highest GC area for isobutylphthalate. According to the previous results, it was established that the sample presentation mode for the HS-SPME/GC–qMS methodology applied to the analysis of the volatile composition of marine salt (for a vial of 120 ml) should be 40 mL of an aqueous solution saturated with 16 g of marine salt. 3.2. Application – analysis of marine salts from different geographical origins Using the optimised conditions described in Section 3.1, it was analysed marine salts from different geographical origins: Portugal (Aveiro, Castro Marim, and Tavira), France (Guérande) and Cape Verde (Sal Island) (Table 3). The analysis of the volatile composition of these marine salts allowed the identification of compounds from different chemical families, including hydrocarbons, alcohols, phenols, aldehydes, ketones, esters, terpenoids, and norisoprenoids. The number and type of compounds varied according to the salt geographical origin. The marine salt from Aveiro presented the higher number of identified compounds (23) while Castro Marim salt presented the lowest (7). The families of compounds with a greater contribution to the total chromatographic area were the hydrocarbons, for the marine salt from Aveiro and Guérande, and the esters, for those from Castro Marim, Tavira, and Sal Island. For the majority of these salts, the chemical family of esters was the one that presented the highest number of compounds. For the salt from Aveiro, the chemical family with more identified compounds was the hydrocarbons, and for the marine salt from Guérande were the terpenoids and norisoprenoids. Reproducibility, expressed as CV values, ranged from 2% to 136%. Similar high reproducibility values

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were observed previously for the optimisation of the methodology. Among all compounds identified in marine salts from different geographical origins, it was possible to find some compounds in common to all or almost all of the analysed samples. The 2,6-bis(1,1-dimethylethyl)-4-methyl-phenol (BHT) and 6-methyl-5-hepten-2-one were present in all samples, and dihydromethyljasmonate, isobutyl phthalate, and butyl phthalate were not identified only in one of the five different salts analysed. The possible sources of some compounds found in the marine salts analysed are the following: - 6-Methyl-5-hepten-2-one, ␤-ionone, 2,2,6-trimethyl-cyclohexanone, ␣-ionone, and 6-methyl-2-heptanone have been identified in algae, aquatic plants, and bacteria [7,21–25]; - 8-Heptadecene has been identified in aquatic plants and in bacterial communities specific of hyper saline environments [21,26]; - ␣-Humulene, ␤-eudesmol, and viridiflorol are sesquiterpenoids that have been identified in plants and ␤-eudesmol also in aquatic fungi [27–30]; - Dihydromethyljasmonate has been related with the metabolites responsible for the protection mechanisms of plants [31,32]; - Some of the identified compounds probably come from environment pollution. The BHT is an antioxidant used in food stuffs, but also in paints and gasoline [33–35]; its presence could be related with the traffic of boats nearby the water supply of the saltpans. The 2-ethyl-1-hexanol, a compound that exists in nature, can be produced by bacteria and fungi while degrading plasticizing substances [36,37]. The ␣-hexylcinnamaldehyde is a compound labelled “Generally Recognized as Safe” (GRAS) food additive by the Food and Drug Administration [38], that may come from contaminated water that supplies the saltpans. The acetylethyltetramethyltetralin, a synthetic musk fragrance used in detergents, cosmetics, and perfumes, are present in the atmosphere. This ketone undergoes bioaccumulation and causes ecotoxicity in aquatic environments [39]. The o-phenylphenol, a compound used as fungicide in food and other products like wood and textiles [40], may also come from contaminated water that could supply the saltpans. The results presented in Table 3 and the possible sources of volatile compounds found in marine salt showed that the volatile composition of each marine salt can be related to its geographic origin and, consequently, to the environment of each saltpan. Thus, according to these results, the volatile compounds identified in the marine salt of the different geographical origins may have three main sources: (i) algae, (ii) surrounding bacterial community, and/or (iii) environment pollution. These compounds, coming from the saltpans environment, are present during all the crystallisation process of the marine salt. Along the crystallisation process, these compounds can be retained in the salt crystals. Therefore, there is a possibility that the volatile compounds can be used as chemical biomarkers of geographic origin for marine salt, since these biomarkers can provide information about the geographic origin as well as the saltpans environment. 4. Conclusions This paper is the first investigation on volatile compounds present in marine salt. In this work it was developed a HSSPME/GC–qMS methodology that allowed the detection and identification of volatile and semi-volatile compounds from marine salt. Optimisation of SPME parameters, including extraction temperature and sample quantity, showed a good compromise between efficiency and reproducibility for 60 ◦ C of extraction temperature

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using 16 g of salt. The study on the effect of sample presentation mode showed that the analysis of an aqueous solution saturated with marine salt allowed a higher efficiency of extraction than the direct analysis of salt crystals, by enhancing the sensitivity of SPME for the marine salt volatiles. The salt dissolution in water and the consequent effect of salting-out promote the release of volatile compounds to the headspace. Concerning the application of the developed methodology to samples of marine salt from different origins, it was possible to identify in their headspace ca. 40 volatile compounds from different chemical families, which included hydrocarbons, alcohols, phenols, aldehydes, ketones, esters, terpenoids, and norisoprenoids. These seem to arise from three main sources: (i) algae, (ii) surrounding bacterial community, and/or (iii) environment pollution. In the future, more studies should be done including salt samples from different harvests and more origins. Acknowledgements This work was financially supported by FCT, Research Unit QOPNA and by a PhD grant (SFRH/BD/31076/2006) to Isabel Silva. The authors tank project SAL – “Sal do Atlântico” (INTERREG IIIB) for providing the European marine salt samples, and Professor Filomena Martins and Mrs. Margarida Silva, from Universidade de Aveiro, for helpful discussions. References [1] I.B. Thompson, J. Hist. Geogr. 25 (1999) 216. [2] http://www.biorede.pt/, website supported by the University of Aveiro, 16 June, 2008. [3] http://www.valorizarariadeaveiro.com/, website supported by the University of Aveiro, 16 June, 2008. [4] C. Schall, K.G. Heumann, G.O. Kirst, Fresenius J. Anal. Chem. 359 (1997) 298. [5] C.M. Bravo-Linares, S.M. Mudge, R.H. Loyola-Sepulveda, Mar. Pollut. Bull. 54 (2007 1742). [6] E.D. Hudson, K. Okuda, P.A. Ariya, Anal. Bioanal. Chem. 388 (2007) 1275. [7] J.H. Sartin, C.J. Halsall, B. Davison, S. Owen, C.N. Hewitt, Anal. Chim. Acta 428 (2001) 61. [8] J.I. García-Plazaola, U. Artetxe, J.M. Becerril, Plant Sci. 143 (1999) 125. ˜ [9] S.M. Owen, J. Penuelas, Trends Plant Sci. 10 (2005) 420.

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