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Detailed profiles of 7-O-acyl esters in plankton and shellfish from the Portuguese coast
Journal of Chromatography A, 1128 (2006) 181–188
Detailed profiles of 7-O-acyl esters in plankton and shellfish from the Portuguese coast Paulo Vale ∗ Instituto Nacional de Investiga¸ca˜ o Agr´aria e das Pescas – IPIMAR, Av. Bras´ılia, 1449-006 Lisboa, Portugal Received 30 December 2005; received in revised form 5 June 2006; accepted 21 June 2006 Available online 24 July 2006
Abstract In bivalve mollusks from the Portuguese coast contaminated by diarrhetic shellfish poisoning (DSP), most of the parent toxins, okadaic acid (OA) or dinophysistoxin-2 (DTX2), are found esterified, and toxicity assessment is only performed after an alkaline hydrolysis step to recover the parent molecules in their free form. The presence of 7-O-acyl esters with fatty acids (FAs) has already been confirmed previously in Mytilus galloprovincialis and Donax trunculus samples. This paper reports the presence of acyl esters in a wider range of estuarine and offshore bivalve species found by direct analysis in LC–MS. The total of acyl esters found in each species represented the percentages commonly found by hydrolysis in those species in previous years, justifying the majority of the esters commonly found in shellfish. This implies that any diol esters remaining after digestion of toxic microalgae would represent only a minor contribution to the ester’s contents. Esters with C14:0, C16:0, C16:1, C20:5 and C22:6 FAs were the most abundant, followed by esters with C18:0, C18:1, C18:2, C18:3 and C18:4. This is the first report of OA and DTX2 esters with odd FAs: C15:0, C17:0, C17:1, and probably a branched FA: iso-C16:0. Esters with iso-C16:0 where found in high percentages particularly in two species of estuarine clams, where they represented 13–34% of total esters found. Esters were also found in plankton, predominantly with C16:0. Total esters in plankton were not higher than 10%, not enough to justify per se the high levels found in bivalves. © 2006 Elsevier B.V. All rights reserved. Keywords: 7-O-acyl esters; Fatty acids; Dinophysistoxin-3; Dinophysis spp.; Diarrhetic shellfish poisoning; Bivalves; LC–MS
1. Introduction Episodes of contamination of bivalve mollusks with diarrhetic shellfish poisoning (DSP) toxins are recurrent phenomenons at the Portuguese coast. Two types of marine microalgae are responsible for these events: Dinophysis acuminata, which produces only okadaic acid (OA), and D. acuta, which produces simultaneously OA and dinophysistoxin-2 (DTX2) at a constant ratio of 60%:40% [1]. However, in shellfish it has been found that both OA and DTX2 are commonly esterified to a high degree in all commercial bivalve species, with the exception of Mytilus galloprovincialis and Donax trunculus [2,3]. In Japanese scallops it was discovered that these esterified toxins were mainly composed of a mixture of 7-Oacylderivatives of DTX1 (Fig. 1), ranging from tetradecanoic acid (C14:0) to docosahexaenoic acid (C22:63) and desig-
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nated as dinophysistoxin-3 (DTX3) [4]. DTX3 was not found in marine microalgae and so it was presumed that they originated in the bivalve by acylation [5]. Direct evidence of this biotransformation by shellfish was obtained by artificially feeding the scallops with the microalgae Dinophysis fortii collected from the sea [6]. It was later demonstrated in Irish mussels that also OA and DTX2 might be acylated to produce DTX3 [7]. Due to their high molecular weight and lipophilicity, acyl esters cannot be easily detected by derivatization with the fluorescent reagent 9-anthryildiazomethane (ADAM) [8], however a hot alkaline hydrolysis reaction with sodium hydroxide releases fatty acids (FAs) from the parent toxins and can give an indirect estimation of their abundance [9]. Their direct detection by LC–LC after ADAM labeling has been proposed however requires a more complicated LC system and standards [10]. A recent multi-toxin LC–MS method, allowing simultaneous detection of several lipophylic marine toxins, can separate these acyl derivatives [11]. This method has previously been used to confirm the presence of OA and DTX2 7-O-acyl esters with C14:0, C16:1, C16:0, C18:1 and C18:0 in samples of M. gal-
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Fig. 1. Structure of the 7-O ester of DSP with palmitic acid. For OA, R1 = CH3 and R2 = H; for DTX2, R1 = H and R2 = CH3 ; for DTX1, R1 = R2 = CH3 .
into an autosampler vial through a 0.45 m nylon disposable syringe filter. A plankton sample was collected in October 2005 inside Ria de Aveiro and filtered on 20-m membranes after passing by a cut-off 90-m mesh. Cells were disrupted in a test tube with 5 mL methanol with a probe set at 100 W for 4 min (Vibra-Cell, Sonics and Materials, Connecticut, USA). The methanol was extracted with dichloromethane and further treated as bivalve samples above. 2.3. LC–MS
loprovincialis and D. trunculus collected from the Portuguese coast [12]. A modified version of this method was employed here to confirm the presence of acyl esters in a larger number of shellfish species. 2. Experimental 2.1. Reagents and standards Dichloromethane and hexane were of analytical grade from Riedel-de-Haen (Honeywell, Germany). Acetonitrile and methanol were LC grade from Lab-Scan (Dublin, Ireland). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). A certified reference standard solution (25.3 g OA/ml) was obtained form the National Research Council of Canada (Halifax, Canada). Retention time for DTX2 was obtained from contaminated material, and molar response was assumed to be identical to OA on a peak area basis. Molar response for all acyl esters was assumed to be identical to OA on a peak area basis. 2.2. Sample collection and preparation D. trunculus specimens were collected off Lisbon coast, Portugal, in June 2005. Spisula solida was collected off Aveiro coast in October 2005. Remaining bivalve species (Mytilus edulis, blue mussel; Cerastoderma edule, common cockle; Solen marginatus, razor clam; Ruditapes decussates and Venerupis senegalensis, clams) and green crab (Carcinus maeanas) samples were collected inside a costal lagoon, Ria de Aveiro, during September–October 2005. Bivalves and crabs were dissected, the digestive glands (DG) removed and homogenized on a Polytron PT3100 (Kinematica, Littau, Switzerland) mixer. A 1-g DG aliquot was extracted once with 5 ml methanol by vortex mixing and centrifuged at 3000 rpm for 10 min. A 2-ml aliquot of the supernatant was transferred to 10 ml glass test tubes, 1 ml of water was added and the lipidic components extracted with dichloromethane (2 × 2 ml). The combined dichloromethane layers were dried with anhydrous sodium sulphate, centrifuged for 1 min, transferred to small glass test tubes and dried at 38 ◦ C under reduced pressure on a RapidVap (Labconco, USA). A 120-mg silica-gel cartridge (Waters, Sep-Pak Light, n◦ 23537) was conditioned with 6 ml hexane. The dried residue was dissolved in 1 ml dichloromethane and applied on the cartridge, followed by washing with 10 ml dichloromethane. Toxins were eluted with 4 ml dichloromethane/methanol (4:1, v/v). The eluate was vacuum dried, dissolved in 500 l methanol and filtered
Analyses were performed on a LC–MS system from HewlettPackard 1100-Series, consisting of an in-line degasser, a quaternary pump, an autosampler, a column oven and the 1946A single-quadrupole mass detector. Separation of toxins was carried out isocratically on a 50 mm × 2 mm column packed with 3 m Hypersil-BDS-C8 (ThermoQuest, Cheshire, England), protected by a 10 mm × 2 mm guard cartridge packed with the same material, as described in [11]. However, the mobile phase and detection mode where different from [11] to improve the efficiency of negative mode ionization in this MS system. The mobile phase consisted of an 83:17 acetonitrile/water (v/v) solution, both containing 0.05% acetic acid. The flow rate was 200 l/min, the injection volume was 5-L and column temperature was maintained at 30 ◦ C. The LC flow was introduced into the ESI interface without any splitting. The spray capillary voltage on the ESI interface was maintained at 4.0 kV and the nebulizer pressure at 25 psig. High-purity nitrogen, obtained through an N2 -Generator (Dominick-Hunter, Durham, England), was used as a drying gas at 10 l/min. and heated to 350 ◦ C. The fragmentor was kept at 180 V. Full-scan mass spectra in the range 960–1160, were acquired in negative ion with gain set at 2. Selected ion monitoring (SIM) was performed in negative ion with gain set at 1. For SIM analysis, the [M-H]− ions from the range of esters obtainable by esterification with thirteen of fatty acids commonly found in bivalves were chosen based on literature survey of fatty acid profiles [13–16]. For analysis of OA, DTX2 and diol esters, separation was performed with the same mobile phase but with 55:45, acetonitrile/water (v/v) during 13 min, and the following [M-H]− ions were screened: 803.5, 913.5, 927.5, 941.5. 3. Results Despite the absence of standards, the retention time (RT) for 7-O-acyl esters was determined by comparison with bivalves contaminated with OA alone or simultaneously with OA and DTX2. In the first case, only one type of 7-O-acyl ester would be expected with each naturally occurring fatty acid, while in the second case, two types of esters would be expected, one with OA and another with DTX2, respectively. Fig. 2 exemplifies profiles of 7-O-acyl esters with OA in a sample of the clam Donax spp. where OA was the only parent toxin found after alkaline hydrolysis in routine analysis. Fig. 3 exemplifies profiles of 7-O-acyl esters when the parent toxins found where OA and DTX2 in a sample of the clam Ruditapes decussatus. In Table 1 are listed the
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Fig. 2. LC–MS chromatograms of 7-O esters of OA with several fatty acids from the clam Donax trunculus, harvested in June 2005 off Lisbon. Eluting conditions for free OA and DTX2 were different from remaining acyl derivatives. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
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Fig. 3. LC–MS chromatograms of 7-O esters of OA and DTX2 with several fatty acids from the clam Ruditapes decusstus, harvested in October 2005 at Ria de Aveiro. Eluting conditions for free OA and DTX2 were different from remaining acyl derivatives. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
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Fig. 4. LC–MS chromatograms of 7-O esters of OA and DTX2 with several fatty acids from: (a) the razor clam Solen marginatus; (b) plankton concentrate; (c) the crab Carcinus maenas. Samples were collected at Ria de Aveiro during September/October 2005. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
Fig. 5. Mass spectra extracted from a Ruditapes decusstus sample of an: (a) isomer of C16:0-DTX2; (b) C16:0-DTX2 (superposed with C18:0–OA, arrow); (c) C20:5-DTX2 and C22:6-DTX2.
retention times obtained under the experimental conditions used. Similar retention times were obtained for the putative OA-esters in Donax spp., and the corresponding OA-esters in Ruditapes spp. The retention times increased with the decreasing degree of saturation. In samples with double contamination, the relative retention time (RRT) of DTX2 esters over OA esters was generally comprised between 1.08/1.09. Under the same eluting conditions, DTX2 and OA co-eluted, but under different eluting conditions their RRT was 1.131 (Table 1). The solid phase
1 In this column the RRT of DTX2/OA increases with the percentage of water used in the separation.
extraction used was effective in producing clean chromatograms, without many interfering peaks. Most of the interfering peaks were attributable to 13 C isotopes of lower molecular weight acyl esters by match of RT, and were marked with an (*) in Figs. 2 and 3. An unexpected finding in the chromatograms was the observation of a prominent pair of peaks eluting before the pair of peaks of C16:0 esters, and tentatively attributed to iso-C16:0OA and iso-C16:0-DTX2 (Fig. 3). The RRT obtained for the iso-DTX2-ester over iso-OA-ester was 1.09, similar to the RRT obtained for most of the other esters. Not all bivalve species tested presented such a great abundance of these uncommon esters: the clam R. decussatus and the clam V. senegalensis
23.3 16 9.8 25.8 21.9 11.6 15.1 26.7 12.7 4.1 0.5 4.6 2.1 0.9 0 0.9 0.6 0.6 0 0.6 Harvest dates: A = 19 September 2005; B = 10 October 2005; C = 27 June 2005; D = 13 October 2005. The harvests A and B were performed inside Ria de Aveiro, C off Lisbon coast and D off Aveiro coast.
21.9 6.5 19.1 25.6 20.7 7.1 33.8 40.9 28.7 5.6 12.8 18.4 24.1 5.3 14.8 20.1 17.7 10.9 19 29.9 16.4 10 20.4 30.4 19.9 7.5 21.8 29.3 14.7 7.6 20.4 28.0 28.9 9.5 1.5 11.0 21.1 9.1 1.9 11.0 31.6 8.8 2.2 11.0 24.8 8.6 2.7 11.3 26.5 5.5 8.3 13.8 25.2 4.8 9.4 14.2 29.4 5.1 6.7 11.8 22.6 4.7 5.9 10.6 7.4 1.7 0.9 2.6 21.4 2.4 2.1 4.5 17.2 3.6 2.4 6.0
9.2 3.2 1.9 5.1
7.7 8.9 5.3 11.5 9.8 10.9 4.2 6.5 2.6 0.0 2.8 4.5 7.4 7.4 10.5 27.2 16 4.9 11.3 3.7 9.6 15.1 11.9 3.7 4.2 7.3 0.0 1.1 3.7 10.0 7.3 6.2 33.2 13.3
OA OA
9.0 24.2 2.7 14.9 0.5 27.0 1.0 0.4 2.2 1.8 2.1 4.8 2.7 4.6 2.0 53.9 19.7 89.8 0.6 0.4 0.6 0.0 5.3 0.0 0.5 0.0 0.0 0.0 0.7 0.0 0.2 1.9 5.9 1.3
DTX2 OA
91.8 0.6 0.1 0.5 0.0 5.2 0.0 0.5 0.0 0.0 0.0 0.7 0.0 0.1 0.5 5.8 1.2 3.4 13.4 3.0 5.4 19.1 12.4 0.8 2.7 2.7 0.8 1.2 3.2 1.6 12.8 4.4 27.4 8.6
DTX2 OA
2.8 15.2 3.0 7.1 33.8 18.1 1.2 2.9 2.2 0.8 1.0 3.2 2.4 13.3 3.4 35.7 10.3 4.1 12.4 2.2 5.5 12.8 24.5 0.8 2.6 5.0 1.8 2.5 2.9 3.7 9.5 9.9 40.6 8.4
DTX2 OA
5.9 10.7 2.7 4.2 14.8 25.5 0.8 1.8 3.4 2.3 2.2 3.6 5.7 9.2 7.0 41.9 7.8 3.9 13.6 5.3 7.6 19.0 21.4 0.9 4.7 2.4 1.0 1.7 4.0 2.0 9.0 3.6 37 11.6
DTX2 OA
5.0 12.0 4.9 6.9 20.4 21.8 1.2 3.9 2.2 0.8 1.5 4.8 2.8 10.3 1.6 36.6 11.7 4.9 10.5 3.1 5.5 21.8 24.3 1.2 3.2 2.2 1.4 2.4 3.2 2.6 5.4 8.5 37.4 8.7
DTX2 OA
6.9 10.4 2.9 4.7 20.4 27.0 1.4 3.3 1.6 1.3 1.7 4.3 3.9 5.0 5.1 41.3 9 9.5 4.9 3.1 9.8 1.5 26.5 1.7 4.7 3.5 1.1 2.1 6.4 3.1 7.7 14.5 34.5 16.2
DTX2 OA
9.9 4.7 3.5 8.3 1.9 32.9 1.8 3.8 2.4 1.2 2.0 7.5 4.5 9.4 6.1 42.1 15.8 6.0 3.8 3.1 7.5 2.2 30.8 1.8 3.9 2.9 1.6 3.2 6.0 3.3 5.9 18.0 37.9 13.5
DTX2 OA DTX2 OA DTX2
DTX2
OA
DTX2 OA OA
5.7 4.1 3.5 5.9 2.7 35.4 2.0 3.1 2.2 2.0 2.9 7.3 5.4 6.3 11.4 44.9 13.2
Spisula solida
D B
Ruditapes decussatus
A B
Solen marginatus
A B
B
Cerastoderma edule
A
Mytilus galloprovincialis
A
Acyl moiety
Table 2 Distribution of OA and DTX2 between unesterified forms and acyl ester forms (expressed in molar percentage)
presented an abundance similar to that of C16:0 esters, while in mussels, cockles, razor clams, and other clams, these esters were found in lower percentages over the C16:0 esters (Fig. 4a, Table 2). The screening for diol esters, either in shellfish or in plankton samples, did not produce any conclusive results, because also a pair of peaks would be expectable and was not found. Analysis in scan mode allowed the confirmation of peak identity by mass spectra. In negative ionization mode, only the molecular ion was obtained, similar to what is obtained in this MS system with the OA standard. Most of the esters identified in SIM mode were confirmed by mass spectra and some of these are exemplified in Fig. 5. Both esters with the C16:0 isomer presented mass spectra undistinguishable from C16:0-OA or C16:0-DTX2. Table 2 lists the relative abundance of acyl esters in a pair of samples from five bivalve species harvested in the same lagunar area three weeks apart, and also data on a plankton sample and two offshore clam species. The relative abundance of esters seems to be partially dependent on the species–specific profile of fatty acids due to the constancy observed from one sampling date to the next sampling. For example, while in R. decussatus and V. senegalensis the abundance of C14:0 esters was higher than the abundance of C16:1 esters in both sampling dates, in S. marginatus the opposite was found at both sampling dates. In all lagunar shellfish the most abundant ester was the C16:0. The presence of esters with eicosapentaenoic acid or EPA (C20:5) and docosahexaenoic acid or DHA (C22:6) was also prominent in all bivalves, while esters with eighteen carbons in chain length were generally the least abundant. In the offshore Donax spp. the profile was dominated only by three esters: C14:0, C16:1 and C16:0. Also in Donax spp., and in one of the mussel samples, the total percentage of polyunsaturated fatty acids (PUFA) was lower than in remaining species.
10.7 6.3 2.0 10.0 8.3 24.1 1.2 2.3 5.0 1.1 2.0 5.8 2.8 10.3 8.1 33.2 15.8
1.13 1.08 1.09 1.08 1.09 1.08 1.08 1.08 1.08 1.07 1.10 1.10 1.09 1.08 1.11
6.3 9.6 2.8 9.7 9.4 24.0 1.0 1.0 3.5 1.4 1.9 7.4 3.5 11.4 7.0 37.1 17.1
3.04 8.70 10.28 9.28 11.41 13.29 11.54 15.88 5.86 7.45 10.19 14.27 20.98 6.79 8.03
4.4 3.9 1.4 7.4 6.7 35.1 1.0 2.7 4.3 2.3 2.8 4.7 3.4 6.4 13.6 42.4 12.1
2.68 8.06 9.46 8.61 10.46 12.36 10.64 14.65 5.44 6.97 9.28 13.03 19.29 6.30 7.24
2.3 9.8 2.8 6.4 5.9 36.5 0.8 1.1 2.7 3.2 3.1 7.2 4.5 5.8 7.8 50.8 13.6
2.71 7.99 9.84 8.61 10.52 12.22 10.64 14.60 5.49 6.92 9.26 13.05 19.20 6.27 7.35
70.0 2.0 0.4 6.0 0.9 10.3 0.5 0.8 0.6 0.3 0.5 1.2 0.5 1.0 5.0 12.8 7.2
803.5 1013.7 1027.7 1039.7 1041.7 1041.7 1053.7 1055.7 1061.7 1063.7 1065.7 1067.7 1069.7 1087.7 1113.7
40.5 6.7 1.6 9.6 1.9 22.5 0.9 0.7 1.2 0.6 1.5 3.8 2.6 2.3 3.6 31.8 13.4
RRT (DTX2/OA)
34.3 3.5 1.0 6.9 2.1 25.4 0.8 0.6 1.2 1.0 1.3 2.4 1.5 2.7 15.2 30.4 9.3
RT DTX2
14.3 10.0 2.0 8.0 2.4 36.5 0.8 0.8 1.9 1.4 2.5 4.3 3.8 3.5 7.9 50.3 12.3
RT OA
Free C14:0 C15:0 C16:1 iso C16:0 C16:0 C17:1 C17:0 C18:4 C18:3 C18:2 C18:1 C18:0 C20:5 C22:6 Saturated Monoinsaturated PUFA Odd iso Bacterial
RT OA
Donax trunculus
Ion mass
C
Venerupis senegalensis
Plantkon
Donax spp.
B
Free toxina C14:0 C15:0 C16:1 iso C16:0 C16:0 C17:1 C17:0 C18:4 C18:3 C18:2 C18:1 C18:0 C20:5 C22:6
Species
Venerupis senegalensis
Fatty acid
B
Table 1 Retention times (min) obtained in a sample contaminated only with OA, and comparison with retention times obtained in a sample contaminated with OA and DTX2 (Relative retention times (RRT) of DTX2 esters in relation to OA esters are also presented. a Eluting conditions: ACN/H2 O 55:45)
DTX2
P. Vale / J. Chromatogr. A 1128 (2006) 181–188
A
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P. Vale / J. Chromatogr. A 1128 (2006) 181–188
Fig. 6. Relationship between the percentage of OA and DTX2 acyl esters with odd FA and iso FA in five shellfish species harvested at Aveiro lagoon in September and October 2005.
In all bivalve samples low levels of some odd FA were found (C15:0, C17:0, C17:1). Either OA or DTX2 were found esterified with these FA. In some species the total amount of FA was greater than others and bared some relationship with what was found with the iso-C16:0 FA (Fig. 6). Mussels presented the lowest percentages of both odd and iso FA, while both clam species presented high levels of odd and iso FA. Razor clams presented high levels of odd FA but low levels of iso FA, while cockles presented an intermediate profile. In a plankton sample from Aveiro lagoon the presence of C16:0-OA and C16:0-DTX2 was clearly found (Fig. 4b), as well as traces of a few other esters (Table 2). The percentage of esters in relation to free OA or free DTX2 was equal or less than 10% of total of toxins found. In the guts contents of the green crab (Carcinus maenas) the presence of C16:0 esters and C22:6 could be demonstrated (Fig. 4c). Other esters may have been present at concentrations below the detection limit of the analytical method. 4. Discussion The increase in retention time with the decreasing degree of saturation has been reported for liquid chromatography of fatty acids [17] or of 7-O-acyl derivatives of DSP toxins [7,12]. Similar elution profiles were obtained previously with this column after acetonitrile gradient separation and detection with a triple quadrupole mass spectrometer [12]. However, the same mobile phase modificators and ionization mode could not be used with the Hewlett-Packard mass spectrometer. Instead, the mobile phase modificator and ionization conditions that have been used previously for obtaining the negative ion of OA proved to work well for the acyl esters [2]. The presence of 7-O-acyl esters with C14:0, C16:1, C16:0, C18:1 and C18:0 had been reported previously in Portuguese shellfish in samples of M. galloprovincialis and D. trunculus. This is the first report for the presence of esters with acyl
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chains ranging from C14 to C22 in a larger variety of bivalve species. The relative abundance of acyl chains in these esters is similar to that demonstrated for the first time in Japanese shellfish where the fatty acids obtained after hydrolysis of DTX3 were dominated by C14:0, C16:0, C20:5 and C22:6 chains [4]. The profile of fatty acids in bivalves is strongly dependent on seasonal variations in plankton and on important physiological processes such as reproduction [13–16]. The esters found in the present report were composed of fatty acids commonly found in bivalves from the northwest Iberian coast [13–16]. The presence of DSP esters with isomers of C16:0 has never been reported before. Three known fatty acids with a C15 or a C13 methyl-branched chain could explain the results found: 3methyl-pentadecanoate, 14-methyl-pentadecanoate or 4, 8, 12trimethyl-tridecanoate. The presence of trace levels of iso-C16 has been reported in oysters and scallops from the NW Iberian coast [13,14], but in general little attention has been paid in analyzing this fatty acid in shellfish from this region [15]. Also the presence of DSP acyl esters with odd FA has never been reported before. The presence of iso- and anteiso-branched chain fatty acids and unbranched 15:0 and 17:0 has been used as biomarkers to determine the presence of bacteria [18,19], and might reflect here the relative importance of a detritivourous diet in shellfish. Free fatty acids were also screened in bivalves by LC–MS with a methodology similar to the one used here, and the presence of a fatty acid eluting before C16:0 was observed. The average RRT of C16:0 over this isomer was 1.16, while the average RRT observed for OA-C16:0 over OA-isoC16:0 was 1.17, and for the DTX2-C16:0 over DTX2-isoC16:0 was 1.16 (Vale, unpublished data). It has been reported that acyl esters are produced in shellfish and are absent in plankton [5,6]. In the present study, low levels of the C16:0 acyl esters were found in plankton. However, their relative low levels do not account for the abundance of acyl esters found in bivalves. In Dinophysis, diol esters, with esterification at C1 instead of C7, have been found [20]. The percentages of free toxins in relation to total toxin levels found in Mytilus and Donax, and the lower percentages found in remaining species, are in accordance with typical ratios found after alkaline hydrolysis [3,21]. This suggests the acyl esters detected accounted for the large majority of the esters present in bivalves. This would imply diol esters originating from Dinophysis spp. would have been converted onto OA or DTX2 so quickly that would contribute to a minor contamination of shellfish tissues. In the benthic microalge Prorocentrum lima it was shown these esters are destroyed by algal enzymes after cell rupture [22]. However, the tentative in the present research of finding the diol ester already reported in Dinophysis spp. was not successful [20]. Acyl esters were also present in the green crab. Their low concentrations did not allow confirmation of more derivatives than those reported here. Further concentration of sample extract would not have improved the detection limit, as the use of the bivalve matrix clean-up method was inadequate to sufficiently reduce matrix interference.
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Acknowledgements Program “Safety, surveillance and quality of bivalve molluscs” (QCAIII/med.4/MARE Programme) supported this work. Gratitude is due to Mike Quilliam for helpful discussions and hints on LC–MS analysis. References [1] P. Vale, M. Cerejo, M.G., Vilarinho, in: Proceedings of 5th International Conference on Molluscan Shellfish Safety, Galway, Ireland, 14–18 June 2004, in press. [2] P. Vale, M.A.M. Sampayo, Toxicon 40 (2002) 33. [3] P. Vale, Toxicon 44 (2004) 123. [4] T. Yasumoto, M. Murata, Y. Oshima, M. Sano, G.K. Matsumoto, J. Clardy, Tetrahedron 41 (1985) 1019. [5] J.S. Lee, T. Igarashi, S. Fraga, E. Dahl, P. Hovgaard, T. Yasumoto, J. Appl. Phycol. 1 (1989) 147. [6] T. Suzuki, H. Ota, M. Yamasaki, Toxicon 37 (1999) 187. [7] J.C. Marr, T. Hu, S. Pleasance, M.A. Quilliam, J.L.C. Wright, Toxicon 30 (1992) 1621. [8] J.S. Lee, T. Yanagi, R. Kenma, T. Yasumoto, Agric. Biol. Chem. 51 (1987) 877. [9] T. Yasumoto, M. Murata, J. Lee, K. Torigoe, in: S. Natori, K. Hashimoto, T. Ueno (Eds.), Mycotoxins and Phycotoxins, 88, Elsevier, Amsterdam, 1989, p. 375.
[10] K. Akasaka, H. Ohrui, H. Meguro, T. Yasumoto, Anal. Sci. 12 (1996) 557. [11] M.A. Quilliam, P. Hess, C. Dell’Aversano, in: W.J. de Koe, R.A. Samson, H.P. van Egmond, J. Gilbert, M. Sabino (Eds.), Mycotoxins and Phycotoxins in Perspective at the Turn of the Millenium, WJ de Koe, Wageningen, The Netherlands, 2001, p. 383. [12] M.A. Quilliam, P. Vale, M.A.M. Sampayo, in: A. Villalba, B. Reguera, J.R. Romalde, R. Beiras (Eds.), Molluscan Shellfish Safety, Conseller´ıa de Pesca e Asuntos Mar´ıtimos da Xunta de Galicia and IOC of UNESCO, 2003, p. 67. [13] A.J. Pazos, C. Ruiz, O. Garc´ıa-Mart´ın, M. Abad, J.L. S´anchez, Comp. Biochem. Physiol. 114B (1996) 171. [14] A.J. Pazos, G. Rom´an, C.P. Acosta, J.L. S´anchez, M. Abad, Comp. Biochem. Physiol. 117B (1997) 393. [15] M.J. Fern´andez-Reir´ız, U. Labarta, M. Albentosa, A.P. Camacho, Comp. Biochem. Physiol. 117B (1999) 309. [16] L. Freites, U. Labarta, M.J. Fern´andez-Reir´ız, Exper. Mar. Biol. Ecol. 268 (2002) 185. [17] T. Suzuki, J. Chromatogr. A 677 (1994) 301. [18] G.J. Perry, J.K. Volkman, R.B. Johns, H.J. Bavor Jr., Geochim. Cosmochim. Acta 43 (1979) 1715. [19] S.M. Budge, C.C. Parrish, C.H. Mackenzie, Mar. Chem. 76 (2001) 285. [20] T. Suzuki, V. Beuzenberg, L. Mackenzie, M.A. Quilliam, Rapi. Commun. Mass Spectrom. 18 (2004) 1131. [21] P. Vale, M.A.M. Sampayo, Toxicon 40 (2002) 989. [22] M.A. Quilliam, N.W. Ross, in: A.P. Snyder (Ed.), Biochemical and biotechnological applications of electrospray ionization mass spectrometry., American Chemical Society, Washington, DC, 1996, p. 351.
Detailed profiles of 7-O-acyl esters in plankton and shellfish from the Portuguese coast Paulo Vale ∗ Instituto Nacional de Investiga¸ca˜ o Agr´aria e das Pescas – IPIMAR, Av. Bras´ılia, 1449-006 Lisboa, Portugal Received 30 December 2005; received in revised form 5 June 2006; accepted 21 June 2006 Available online 24 July 2006
Abstract In bivalve mollusks from the Portuguese coast contaminated by diarrhetic shellfish poisoning (DSP), most of the parent toxins, okadaic acid (OA) or dinophysistoxin-2 (DTX2), are found esterified, and toxicity assessment is only performed after an alkaline hydrolysis step to recover the parent molecules in their free form. The presence of 7-O-acyl esters with fatty acids (FAs) has already been confirmed previously in Mytilus galloprovincialis and Donax trunculus samples. This paper reports the presence of acyl esters in a wider range of estuarine and offshore bivalve species found by direct analysis in LC–MS. The total of acyl esters found in each species represented the percentages commonly found by hydrolysis in those species in previous years, justifying the majority of the esters commonly found in shellfish. This implies that any diol esters remaining after digestion of toxic microalgae would represent only a minor contribution to the ester’s contents. Esters with C14:0, C16:0, C16:1, C20:5 and C22:6 FAs were the most abundant, followed by esters with C18:0, C18:1, C18:2, C18:3 and C18:4. This is the first report of OA and DTX2 esters with odd FAs: C15:0, C17:0, C17:1, and probably a branched FA: iso-C16:0. Esters with iso-C16:0 where found in high percentages particularly in two species of estuarine clams, where they represented 13–34% of total esters found. Esters were also found in plankton, predominantly with C16:0. Total esters in plankton were not higher than 10%, not enough to justify per se the high levels found in bivalves. © 2006 Elsevier B.V. All rights reserved. Keywords: 7-O-acyl esters; Fatty acids; Dinophysistoxin-3; Dinophysis spp.; Diarrhetic shellfish poisoning; Bivalves; LC–MS
1. Introduction Episodes of contamination of bivalve mollusks with diarrhetic shellfish poisoning (DSP) toxins are recurrent phenomenons at the Portuguese coast. Two types of marine microalgae are responsible for these events: Dinophysis acuminata, which produces only okadaic acid (OA), and D. acuta, which produces simultaneously OA and dinophysistoxin-2 (DTX2) at a constant ratio of 60%:40% [1]. However, in shellfish it has been found that both OA and DTX2 are commonly esterified to a high degree in all commercial bivalve species, with the exception of Mytilus galloprovincialis and Donax trunculus [2,3]. In Japanese scallops it was discovered that these esterified toxins were mainly composed of a mixture of 7-Oacylderivatives of DTX1 (Fig. 1), ranging from tetradecanoic acid (C14:0) to docosahexaenoic acid (C22:63) and desig-
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nated as dinophysistoxin-3 (DTX3) [4]. DTX3 was not found in marine microalgae and so it was presumed that they originated in the bivalve by acylation [5]. Direct evidence of this biotransformation by shellfish was obtained by artificially feeding the scallops with the microalgae Dinophysis fortii collected from the sea [6]. It was later demonstrated in Irish mussels that also OA and DTX2 might be acylated to produce DTX3 [7]. Due to their high molecular weight and lipophilicity, acyl esters cannot be easily detected by derivatization with the fluorescent reagent 9-anthryildiazomethane (ADAM) [8], however a hot alkaline hydrolysis reaction with sodium hydroxide releases fatty acids (FAs) from the parent toxins and can give an indirect estimation of their abundance [9]. Their direct detection by LC–LC after ADAM labeling has been proposed however requires a more complicated LC system and standards [10]. A recent multi-toxin LC–MS method, allowing simultaneous detection of several lipophylic marine toxins, can separate these acyl derivatives [11]. This method has previously been used to confirm the presence of OA and DTX2 7-O-acyl esters with C14:0, C16:1, C16:0, C18:1 and C18:0 in samples of M. gal-
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Fig. 1. Structure of the 7-O ester of DSP with palmitic acid. For OA, R1 = CH3 and R2 = H; for DTX2, R1 = H and R2 = CH3 ; for DTX1, R1 = R2 = CH3 .
into an autosampler vial through a 0.45 m nylon disposable syringe filter. A plankton sample was collected in October 2005 inside Ria de Aveiro and filtered on 20-m membranes after passing by a cut-off 90-m mesh. Cells were disrupted in a test tube with 5 mL methanol with a probe set at 100 W for 4 min (Vibra-Cell, Sonics and Materials, Connecticut, USA). The methanol was extracted with dichloromethane and further treated as bivalve samples above. 2.3. LC–MS
loprovincialis and D. trunculus collected from the Portuguese coast [12]. A modified version of this method was employed here to confirm the presence of acyl esters in a larger number of shellfish species. 2. Experimental 2.1. Reagents and standards Dichloromethane and hexane were of analytical grade from Riedel-de-Haen (Honeywell, Germany). Acetonitrile and methanol were LC grade from Lab-Scan (Dublin, Ireland). Water was purified using a Milli-Q system (Millipore, Bedford, MA, USA). A certified reference standard solution (25.3 g OA/ml) was obtained form the National Research Council of Canada (Halifax, Canada). Retention time for DTX2 was obtained from contaminated material, and molar response was assumed to be identical to OA on a peak area basis. Molar response for all acyl esters was assumed to be identical to OA on a peak area basis. 2.2. Sample collection and preparation D. trunculus specimens were collected off Lisbon coast, Portugal, in June 2005. Spisula solida was collected off Aveiro coast in October 2005. Remaining bivalve species (Mytilus edulis, blue mussel; Cerastoderma edule, common cockle; Solen marginatus, razor clam; Ruditapes decussates and Venerupis senegalensis, clams) and green crab (Carcinus maeanas) samples were collected inside a costal lagoon, Ria de Aveiro, during September–October 2005. Bivalves and crabs were dissected, the digestive glands (DG) removed and homogenized on a Polytron PT3100 (Kinematica, Littau, Switzerland) mixer. A 1-g DG aliquot was extracted once with 5 ml methanol by vortex mixing and centrifuged at 3000 rpm for 10 min. A 2-ml aliquot of the supernatant was transferred to 10 ml glass test tubes, 1 ml of water was added and the lipidic components extracted with dichloromethane (2 × 2 ml). The combined dichloromethane layers were dried with anhydrous sodium sulphate, centrifuged for 1 min, transferred to small glass test tubes and dried at 38 ◦ C under reduced pressure on a RapidVap (Labconco, USA). A 120-mg silica-gel cartridge (Waters, Sep-Pak Light, n◦ 23537) was conditioned with 6 ml hexane. The dried residue was dissolved in 1 ml dichloromethane and applied on the cartridge, followed by washing with 10 ml dichloromethane. Toxins were eluted with 4 ml dichloromethane/methanol (4:1, v/v). The eluate was vacuum dried, dissolved in 500 l methanol and filtered
Analyses were performed on a LC–MS system from HewlettPackard 1100-Series, consisting of an in-line degasser, a quaternary pump, an autosampler, a column oven and the 1946A single-quadrupole mass detector. Separation of toxins was carried out isocratically on a 50 mm × 2 mm column packed with 3 m Hypersil-BDS-C8 (ThermoQuest, Cheshire, England), protected by a 10 mm × 2 mm guard cartridge packed with the same material, as described in [11]. However, the mobile phase and detection mode where different from [11] to improve the efficiency of negative mode ionization in this MS system. The mobile phase consisted of an 83:17 acetonitrile/water (v/v) solution, both containing 0.05% acetic acid. The flow rate was 200 l/min, the injection volume was 5-L and column temperature was maintained at 30 ◦ C. The LC flow was introduced into the ESI interface without any splitting. The spray capillary voltage on the ESI interface was maintained at 4.0 kV and the nebulizer pressure at 25 psig. High-purity nitrogen, obtained through an N2 -Generator (Dominick-Hunter, Durham, England), was used as a drying gas at 10 l/min. and heated to 350 ◦ C. The fragmentor was kept at 180 V. Full-scan mass spectra in the range 960–1160, were acquired in negative ion with gain set at 2. Selected ion monitoring (SIM) was performed in negative ion with gain set at 1. For SIM analysis, the [M-H]− ions from the range of esters obtainable by esterification with thirteen of fatty acids commonly found in bivalves were chosen based on literature survey of fatty acid profiles [13–16]. For analysis of OA, DTX2 and diol esters, separation was performed with the same mobile phase but with 55:45, acetonitrile/water (v/v) during 13 min, and the following [M-H]− ions were screened: 803.5, 913.5, 927.5, 941.5. 3. Results Despite the absence of standards, the retention time (RT) for 7-O-acyl esters was determined by comparison with bivalves contaminated with OA alone or simultaneously with OA and DTX2. In the first case, only one type of 7-O-acyl ester would be expected with each naturally occurring fatty acid, while in the second case, two types of esters would be expected, one with OA and another with DTX2, respectively. Fig. 2 exemplifies profiles of 7-O-acyl esters with OA in a sample of the clam Donax spp. where OA was the only parent toxin found after alkaline hydrolysis in routine analysis. Fig. 3 exemplifies profiles of 7-O-acyl esters when the parent toxins found where OA and DTX2 in a sample of the clam Ruditapes decussatus. In Table 1 are listed the
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Fig. 2. LC–MS chromatograms of 7-O esters of OA with several fatty acids from the clam Donax trunculus, harvested in June 2005 off Lisbon. Eluting conditions for free OA and DTX2 were different from remaining acyl derivatives. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
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Fig. 3. LC–MS chromatograms of 7-O esters of OA and DTX2 with several fatty acids from the clam Ruditapes decusstus, harvested in October 2005 at Ria de Aveiro. Eluting conditions for free OA and DTX2 were different from remaining acyl derivatives. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
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185
Fig. 4. LC–MS chromatograms of 7-O esters of OA and DTX2 with several fatty acids from: (a) the razor clam Solen marginatus; (b) plankton concentrate; (c) the crab Carcinus maenas. Samples were collected at Ria de Aveiro during September/October 2005. (*) Denotes interfering signals from 13 C isotope peaks of lower molecular weight esters.
Fig. 5. Mass spectra extracted from a Ruditapes decusstus sample of an: (a) isomer of C16:0-DTX2; (b) C16:0-DTX2 (superposed with C18:0–OA, arrow); (c) C20:5-DTX2 and C22:6-DTX2.
retention times obtained under the experimental conditions used. Similar retention times were obtained for the putative OA-esters in Donax spp., and the corresponding OA-esters in Ruditapes spp. The retention times increased with the decreasing degree of saturation. In samples with double contamination, the relative retention time (RRT) of DTX2 esters over OA esters was generally comprised between 1.08/1.09. Under the same eluting conditions, DTX2 and OA co-eluted, but under different eluting conditions their RRT was 1.131 (Table 1). The solid phase
1 In this column the RRT of DTX2/OA increases with the percentage of water used in the separation.
extraction used was effective in producing clean chromatograms, without many interfering peaks. Most of the interfering peaks were attributable to 13 C isotopes of lower molecular weight acyl esters by match of RT, and were marked with an (*) in Figs. 2 and 3. An unexpected finding in the chromatograms was the observation of a prominent pair of peaks eluting before the pair of peaks of C16:0 esters, and tentatively attributed to iso-C16:0OA and iso-C16:0-DTX2 (Fig. 3). The RRT obtained for the iso-DTX2-ester over iso-OA-ester was 1.09, similar to the RRT obtained for most of the other esters. Not all bivalve species tested presented such a great abundance of these uncommon esters: the clam R. decussatus and the clam V. senegalensis
23.3 16 9.8 25.8 21.9 11.6 15.1 26.7 12.7 4.1 0.5 4.6 2.1 0.9 0 0.9 0.6 0.6 0 0.6 Harvest dates: A = 19 September 2005; B = 10 October 2005; C = 27 June 2005; D = 13 October 2005. The harvests A and B were performed inside Ria de Aveiro, C off Lisbon coast and D off Aveiro coast.
21.9 6.5 19.1 25.6 20.7 7.1 33.8 40.9 28.7 5.6 12.8 18.4 24.1 5.3 14.8 20.1 17.7 10.9 19 29.9 16.4 10 20.4 30.4 19.9 7.5 21.8 29.3 14.7 7.6 20.4 28.0 28.9 9.5 1.5 11.0 21.1 9.1 1.9 11.0 31.6 8.8 2.2 11.0 24.8 8.6 2.7 11.3 26.5 5.5 8.3 13.8 25.2 4.8 9.4 14.2 29.4 5.1 6.7 11.8 22.6 4.7 5.9 10.6 7.4 1.7 0.9 2.6 21.4 2.4 2.1 4.5 17.2 3.6 2.4 6.0
9.2 3.2 1.9 5.1
7.7 8.9 5.3 11.5 9.8 10.9 4.2 6.5 2.6 0.0 2.8 4.5 7.4 7.4 10.5 27.2 16 4.9 11.3 3.7 9.6 15.1 11.9 3.7 4.2 7.3 0.0 1.1 3.7 10.0 7.3 6.2 33.2 13.3
OA OA
9.0 24.2 2.7 14.9 0.5 27.0 1.0 0.4 2.2 1.8 2.1 4.8 2.7 4.6 2.0 53.9 19.7 89.8 0.6 0.4 0.6 0.0 5.3 0.0 0.5 0.0 0.0 0.0 0.7 0.0 0.2 1.9 5.9 1.3
DTX2 OA
91.8 0.6 0.1 0.5 0.0 5.2 0.0 0.5 0.0 0.0 0.0 0.7 0.0 0.1 0.5 5.8 1.2 3.4 13.4 3.0 5.4 19.1 12.4 0.8 2.7 2.7 0.8 1.2 3.2 1.6 12.8 4.4 27.4 8.6
DTX2 OA
2.8 15.2 3.0 7.1 33.8 18.1 1.2 2.9 2.2 0.8 1.0 3.2 2.4 13.3 3.4 35.7 10.3 4.1 12.4 2.2 5.5 12.8 24.5 0.8 2.6 5.0 1.8 2.5 2.9 3.7 9.5 9.9 40.6 8.4
DTX2 OA
5.9 10.7 2.7 4.2 14.8 25.5 0.8 1.8 3.4 2.3 2.2 3.6 5.7 9.2 7.0 41.9 7.8 3.9 13.6 5.3 7.6 19.0 21.4 0.9 4.7 2.4 1.0 1.7 4.0 2.0 9.0 3.6 37 11.6
DTX2 OA
5.0 12.0 4.9 6.9 20.4 21.8 1.2 3.9 2.2 0.8 1.5 4.8 2.8 10.3 1.6 36.6 11.7 4.9 10.5 3.1 5.5 21.8 24.3 1.2 3.2 2.2 1.4 2.4 3.2 2.6 5.4 8.5 37.4 8.7
DTX2 OA
6.9 10.4 2.9 4.7 20.4 27.0 1.4 3.3 1.6 1.3 1.7 4.3 3.9 5.0 5.1 41.3 9 9.5 4.9 3.1 9.8 1.5 26.5 1.7 4.7 3.5 1.1 2.1 6.4 3.1 7.7 14.5 34.5 16.2
DTX2 OA
9.9 4.7 3.5 8.3 1.9 32.9 1.8 3.8 2.4 1.2 2.0 7.5 4.5 9.4 6.1 42.1 15.8 6.0 3.8 3.1 7.5 2.2 30.8 1.8 3.9 2.9 1.6 3.2 6.0 3.3 5.9 18.0 37.9 13.5
DTX2 OA DTX2 OA DTX2
DTX2
OA
DTX2 OA OA
5.7 4.1 3.5 5.9 2.7 35.4 2.0 3.1 2.2 2.0 2.9 7.3 5.4 6.3 11.4 44.9 13.2
Spisula solida
D B
Ruditapes decussatus
A B
Solen marginatus
A B
B
Cerastoderma edule
A
Mytilus galloprovincialis
A
Acyl moiety
Table 2 Distribution of OA and DTX2 between unesterified forms and acyl ester forms (expressed in molar percentage)
presented an abundance similar to that of C16:0 esters, while in mussels, cockles, razor clams, and other clams, these esters were found in lower percentages over the C16:0 esters (Fig. 4a, Table 2). The screening for diol esters, either in shellfish or in plankton samples, did not produce any conclusive results, because also a pair of peaks would be expectable and was not found. Analysis in scan mode allowed the confirmation of peak identity by mass spectra. In negative ionization mode, only the molecular ion was obtained, similar to what is obtained in this MS system with the OA standard. Most of the esters identified in SIM mode were confirmed by mass spectra and some of these are exemplified in Fig. 5. Both esters with the C16:0 isomer presented mass spectra undistinguishable from C16:0-OA or C16:0-DTX2. Table 2 lists the relative abundance of acyl esters in a pair of samples from five bivalve species harvested in the same lagunar area three weeks apart, and also data on a plankton sample and two offshore clam species. The relative abundance of esters seems to be partially dependent on the species–specific profile of fatty acids due to the constancy observed from one sampling date to the next sampling. For example, while in R. decussatus and V. senegalensis the abundance of C14:0 esters was higher than the abundance of C16:1 esters in both sampling dates, in S. marginatus the opposite was found at both sampling dates. In all lagunar shellfish the most abundant ester was the C16:0. The presence of esters with eicosapentaenoic acid or EPA (C20:5) and docosahexaenoic acid or DHA (C22:6) was also prominent in all bivalves, while esters with eighteen carbons in chain length were generally the least abundant. In the offshore Donax spp. the profile was dominated only by three esters: C14:0, C16:1 and C16:0. Also in Donax spp., and in one of the mussel samples, the total percentage of polyunsaturated fatty acids (PUFA) was lower than in remaining species.
10.7 6.3 2.0 10.0 8.3 24.1 1.2 2.3 5.0 1.1 2.0 5.8 2.8 10.3 8.1 33.2 15.8
1.13 1.08 1.09 1.08 1.09 1.08 1.08 1.08 1.08 1.07 1.10 1.10 1.09 1.08 1.11
6.3 9.6 2.8 9.7 9.4 24.0 1.0 1.0 3.5 1.4 1.9 7.4 3.5 11.4 7.0 37.1 17.1
3.04 8.70 10.28 9.28 11.41 13.29 11.54 15.88 5.86 7.45 10.19 14.27 20.98 6.79 8.03
4.4 3.9 1.4 7.4 6.7 35.1 1.0 2.7 4.3 2.3 2.8 4.7 3.4 6.4 13.6 42.4 12.1
2.68 8.06 9.46 8.61 10.46 12.36 10.64 14.65 5.44 6.97 9.28 13.03 19.29 6.30 7.24
2.3 9.8 2.8 6.4 5.9 36.5 0.8 1.1 2.7 3.2 3.1 7.2 4.5 5.8 7.8 50.8 13.6
2.71 7.99 9.84 8.61 10.52 12.22 10.64 14.60 5.49 6.92 9.26 13.05 19.20 6.27 7.35
70.0 2.0 0.4 6.0 0.9 10.3 0.5 0.8 0.6 0.3 0.5 1.2 0.5 1.0 5.0 12.8 7.2
803.5 1013.7 1027.7 1039.7 1041.7 1041.7 1053.7 1055.7 1061.7 1063.7 1065.7 1067.7 1069.7 1087.7 1113.7
40.5 6.7 1.6 9.6 1.9 22.5 0.9 0.7 1.2 0.6 1.5 3.8 2.6 2.3 3.6 31.8 13.4
RRT (DTX2/OA)
34.3 3.5 1.0 6.9 2.1 25.4 0.8 0.6 1.2 1.0 1.3 2.4 1.5 2.7 15.2 30.4 9.3
RT DTX2
14.3 10.0 2.0 8.0 2.4 36.5 0.8 0.8 1.9 1.4 2.5 4.3 3.8 3.5 7.9 50.3 12.3
RT OA
Free C14:0 C15:0 C16:1 iso C16:0 C16:0 C17:1 C17:0 C18:4 C18:3 C18:2 C18:1 C18:0 C20:5 C22:6 Saturated Monoinsaturated PUFA Odd iso Bacterial
RT OA
Donax trunculus
Ion mass
C
Venerupis senegalensis
Plantkon
Donax spp.
B
Free toxina C14:0 C15:0 C16:1 iso C16:0 C16:0 C17:1 C17:0 C18:4 C18:3 C18:2 C18:1 C18:0 C20:5 C22:6
Species
Venerupis senegalensis
Fatty acid
B
Table 1 Retention times (min) obtained in a sample contaminated only with OA, and comparison with retention times obtained in a sample contaminated with OA and DTX2 (Relative retention times (RRT) of DTX2 esters in relation to OA esters are also presented. a Eluting conditions: ACN/H2 O 55:45)
DTX2
P. Vale / J. Chromatogr. A 1128 (2006) 181–188
A
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P. Vale / J. Chromatogr. A 1128 (2006) 181–188
Fig. 6. Relationship between the percentage of OA and DTX2 acyl esters with odd FA and iso FA in five shellfish species harvested at Aveiro lagoon in September and October 2005.
In all bivalve samples low levels of some odd FA were found (C15:0, C17:0, C17:1). Either OA or DTX2 were found esterified with these FA. In some species the total amount of FA was greater than others and bared some relationship with what was found with the iso-C16:0 FA (Fig. 6). Mussels presented the lowest percentages of both odd and iso FA, while both clam species presented high levels of odd and iso FA. Razor clams presented high levels of odd FA but low levels of iso FA, while cockles presented an intermediate profile. In a plankton sample from Aveiro lagoon the presence of C16:0-OA and C16:0-DTX2 was clearly found (Fig. 4b), as well as traces of a few other esters (Table 2). The percentage of esters in relation to free OA or free DTX2 was equal or less than 10% of total of toxins found. In the guts contents of the green crab (Carcinus maenas) the presence of C16:0 esters and C22:6 could be demonstrated (Fig. 4c). Other esters may have been present at concentrations below the detection limit of the analytical method. 4. Discussion The increase in retention time with the decreasing degree of saturation has been reported for liquid chromatography of fatty acids [17] or of 7-O-acyl derivatives of DSP toxins [7,12]. Similar elution profiles were obtained previously with this column after acetonitrile gradient separation and detection with a triple quadrupole mass spectrometer [12]. However, the same mobile phase modificators and ionization mode could not be used with the Hewlett-Packard mass spectrometer. Instead, the mobile phase modificator and ionization conditions that have been used previously for obtaining the negative ion of OA proved to work well for the acyl esters [2]. The presence of 7-O-acyl esters with C14:0, C16:1, C16:0, C18:1 and C18:0 had been reported previously in Portuguese shellfish in samples of M. galloprovincialis and D. trunculus. This is the first report for the presence of esters with acyl
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chains ranging from C14 to C22 in a larger variety of bivalve species. The relative abundance of acyl chains in these esters is similar to that demonstrated for the first time in Japanese shellfish where the fatty acids obtained after hydrolysis of DTX3 were dominated by C14:0, C16:0, C20:5 and C22:6 chains [4]. The profile of fatty acids in bivalves is strongly dependent on seasonal variations in plankton and on important physiological processes such as reproduction [13–16]. The esters found in the present report were composed of fatty acids commonly found in bivalves from the northwest Iberian coast [13–16]. The presence of DSP esters with isomers of C16:0 has never been reported before. Three known fatty acids with a C15 or a C13 methyl-branched chain could explain the results found: 3methyl-pentadecanoate, 14-methyl-pentadecanoate or 4, 8, 12trimethyl-tridecanoate. The presence of trace levels of iso-C16 has been reported in oysters and scallops from the NW Iberian coast [13,14], but in general little attention has been paid in analyzing this fatty acid in shellfish from this region [15]. Also the presence of DSP acyl esters with odd FA has never been reported before. The presence of iso- and anteiso-branched chain fatty acids and unbranched 15:0 and 17:0 has been used as biomarkers to determine the presence of bacteria [18,19], and might reflect here the relative importance of a detritivourous diet in shellfish. Free fatty acids were also screened in bivalves by LC–MS with a methodology similar to the one used here, and the presence of a fatty acid eluting before C16:0 was observed. The average RRT of C16:0 over this isomer was 1.16, while the average RRT observed for OA-C16:0 over OA-isoC16:0 was 1.17, and for the DTX2-C16:0 over DTX2-isoC16:0 was 1.16 (Vale, unpublished data). It has been reported that acyl esters are produced in shellfish and are absent in plankton [5,6]. In the present study, low levels of the C16:0 acyl esters were found in plankton. However, their relative low levels do not account for the abundance of acyl esters found in bivalves. In Dinophysis, diol esters, with esterification at C1 instead of C7, have been found [20]. The percentages of free toxins in relation to total toxin levels found in Mytilus and Donax, and the lower percentages found in remaining species, are in accordance with typical ratios found after alkaline hydrolysis [3,21]. This suggests the acyl esters detected accounted for the large majority of the esters present in bivalves. This would imply diol esters originating from Dinophysis spp. would have been converted onto OA or DTX2 so quickly that would contribute to a minor contamination of shellfish tissues. In the benthic microalge Prorocentrum lima it was shown these esters are destroyed by algal enzymes after cell rupture [22]. However, the tentative in the present research of finding the diol ester already reported in Dinophysis spp. was not successful [20]. Acyl esters were also present in the green crab. Their low concentrations did not allow confirmation of more derivatives than those reported here. Further concentration of sample extract would not have improved the detection limit, as the use of the bivalve matrix clean-up method was inadequate to sufficiently reduce matrix interference.
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