Determination of the variability of both hydrophilic and lipophilic toxins in endemic wild bivalves and carnivorous gastropods from the Southern part of Chile

The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55–2505 μg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 μg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 μg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 μg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 μg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.     

Inter-species variability of okadaic acid group toxicity in relation to the content of fatty acids detected in different marine vectors

Okadaic acid group (OA-group) is a set of lipophilic toxins which are characterised by being produced by species associated with the genera Dinophysis and Prorocentrum. OA-group has been regularly detected in endemic shellfish species from the southern zone of Chile only through the mouse bioassay. The purpose of this work was to determine the variability of OA-group toxins in endemic aquatic organisms (bivalves, crabs, gastropods and fish) and to establish the relationship with the concentration of fatty acids (FAs) detected in the evaluated species. The toxicity of OA-group and the FA profiles were determined using LC-MS/MS and gas chromatography with flame-ionisation detection, respectively. In the study area, the dinoflagellate Dinophysis acuta was detected in densities ≈2000 cells ml^?1 with a toxicity ≈18.3 pg OA equiv cel^?1. The analysis identified OA and dinophysistoxin-1 in shellfish in a range of ≈90 to ≈225 μg OA eq kg^?1, where no toxins in fish were detected. A positive relationship between the FA level and the concentration of OA-group toxins in the digestive glands of bivalves and gastropods was established, noted for high levels of saturated FAs (C14:0 and C16:0). The toxic variability of OA-group toxins determined in the different species allowed us to establish that the consumption of these vectors, regulated by non-analytical methods, can be harmful when consumed by humans, thus suggesting that the sanitary regulations for the control of OA-group in Chile should be updated.     

海产品贝毒素大田软海绵酸ELISA及HPLC-MS/MS检测方法的研究

为了检测海产品贝毒大田软海绵酸,保障其食用安全,利用活泼酯法将小分子OA与载体蛋白偶联,免疫BALB/c小鼠,细胞融合技术建立分泌抗OA的杂交瘤细胞株,并对其各种特性进行分析;小鼠腹水法大量生产抗体,纯化后建立ELISA方法,同时建立OA的高效液相色谱-串联质谱(HPLC-MS)检测法,并对部分市售海产品进行了实际检测,ELISA方法标准曲线为y=－34.212x+83.49,相关系数为0.9784,线性范围0.4～25μg/L,灵敏度0.18μg/L;HPLC-MS/MS检测方法标准曲线y=193.07x－780.6(Q1/Q3：m/z 827.4～m/z 723.5)和y=83.021x－335.6(Q1/Q3：m/z 827.4～m/z 809.5),R2 均为0.9991,线性范围10～800μg/L,灵敏度小于2μg/L,平均RSD为4.34%。在检测的实际样品中两种样品ELISA呈阳性反应,其中一种经过了HPLC-MS/MS的验证。所建立的ELISA及HPLC-MS检测方法均可用于海产品腹泻性贝毒OA限量标准检测,为进出口海产品OA标准方法的建立提供实验基础。     

Lipophilic toxin profiles detected in farmed and benthic mussels populations from the most relevant production zones in Southern Chile

Lipophilic toxins associated with diarrhoeic toxins were found in Mytilus chilensis (Blue mussels) and Aulacomya ater (Ribbed mussels). These shellfish samples were collected from Chiloe Island, Southern Chile. The samples were tested by liquid chromatography–tandem mass spectrometry (LC-MS/MS). After the analysis, four toxins were found: DTX-1, DTX-3, YTX and PTX. All toxins were identified by comparing their HPLC retention times with those of analytical standards and confirmed by LC-MS/MS. Dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) toxins were the major components within the mussel extracts. Nevertheless, the percentages of these toxins differed depending on the area they were collected from and/or the sampling date. The levels detected in Butacheuques Island for okadaic acid (OA) was 267?±?3.5?µg OA?eq?kg^?1 (p?<?0.05) and for DTX-3 was 183.4?±?7.5?µg?kg^?1 in ribbed mussels. Pectenotoxin (PTX) and yessotoxin (YTX) were the toxins detected in minor proportions in the toxic profile of the bivalves. The maximum concentration of YTX detected in ribbed mussels was 85.2?±?2.8?µg?kg^?1 in Mechuque Island, whereas the PTX-2 level in ribbed mussels was 82.0?±?2.4?µg?kg^?1 in Cailin Island. Analogues of YTX and PTX-2 were not detected in any of the analysed mussels, which did not support the supposed presence of isomers of toxins as a result of the enzymatic metabolism of bivalves. This study found evidence proving co-occurrence of lipophilic toxins – like PTX and YTX – with diarrhoeic toxin in samples collected in Southern Chile, which is, to date, the more complex mix of lipophilic toxins ever found in mussels samples from Southern Chile.     

Approaches to the evaluation of matrix effects in the liquid chromatography-mass spectrometry (LC-MS) analysis of three regulated lipophilic toxin groups in mussel matrix (Mytilus edulis)

Liquid chromatography coupled to mass spectrometry (LC-MS) is seen as an integral part of methods of choice for the replacement of animal tests in the determination of lipophilic shellfish toxins. However, these techniques are prone to matrix effects that need to be considered when developing and validating methods. The analysis of shellfish is a challenging task due to the complexity of the shellfish matrix and the number of shellfish species encountered in monitoring laboratories. Therefore, it is crucial that the cause and the extent of matrix effects is fully understood in order to apply corrective measures to the analytical method and to develop efficient sample clean-up steps. This paper presents different approaches to evaluate matrix effects associated with the analysis of okadaic acid (OA), azaspiracid-1 (AZA1) and pectenotoxin-2 (PTX2) in cooked and raw mussel flesh. Post-extraction addition and standard addition experiments were carried out and analysed using various LC-MS methods. Gradient and isocratic elution were compared and ultra-performance liquid chromatography (UPLC), using C8 and C18 Acquity BEH columns, was evaluated for the extent of matrix effects. When matrix effects were observed, OA and PTX2 were always prone to signal enhancement and AZA1 to signal suppression. For all the toxins studied, matrix effects were dependent on chromatographic conditions. UPLC separation using a C8 column significantly reduced matrix effects compared to the other conditions assessed. Furthermore, sample dilution has proven to be an efficient way of reducing matrix effects associated with OA analysis.     

Analysis of paralytic shellfish toxins and their metabolites in shellfish from the North Yellow Sea of China

Samples of toxic scallop (Patinopecten yessoensis) and clam (Saxidomus purpuratus) collected on the northern coast of China from 2008 to 2009 were analysed. High-performance liquid chromatography with post-column oxidation and fluorescence detection was used to determine the profile of the main paralytic shellfish poisoning (PSP) toxins in these samples and their total toxicity. Hydrophilic interaction liquid ion chromatography with mass spectrometric detection confirmed the toxin profile and detected several metabolites in the shellfish. Results show that C1/2 toxins were the most dominant toxins in the scallop and clam samples. However, GTX1/4 and GTX2/3 were also present. M1 was the predominant metabolite in all the samples, but M3 and M5 were also identified, along with three previously unreported presumed metabolites, M6, M8 and M10. The results indicate that the biotransformation of toxins was species specific. It was concluded that the reductive enzyme in clams is more active than in scallops and that an enzyme in scallops is more apt to catalyse hydrolysis of both the sulfonate moiety at the N-sulfocabamoyl of C toxins and the 11-hydroxysulfate of C and GTX toxins to produce metabolites. This is the first report of new metabolites of PSP toxins in scallops and clams collected in China.     

An investigation of presence of 2- and 3-monochloropropanediol fatty acid esters in Canadian human milk samples

ABSTRACT

Occurrence of 2- and 3-monochloropropanediol fatty acid esters (MCPDEs) in 199 human milk samples collected from Canadian women was examined in this study. MCPDEs were determined via indirect analytical approach by using acidic hydrolysis/transesterification and derivatization with cyclohexanone using a fluorinated sulfonic acid resin as a catalyst followed by GC-MS analysis. Analyses were done by a stable isotope dilution assay (SIDA) using 3-monochloropropanediol-d₅ dipalmitate and 2-monochloropropanediol-d₅ distearate as internal standards. In all samples 2-MCPD esters were not detected, at LOD = 1 ng/g for 2-MCPD equivalent. 3-MCPD esters were not detected, at LOD = 2 ng/g for 3-MCPD equivalent, in 193 samples. Six samples had the apparent concentration of 3-MCPD equivalent in the range of 2.0–5.1 ng/g, which was likely caused by accidental contamination during processing of samples. For selected samples, these findings were confirmed by direct analysis via LC-MS/MS analysis based on SIDA for 3-MCPD dipalmitate and 2-MCPD distearate. To our best knowledge, this is the first study to analyze human milk samples for 2-MCPD esters.     

Triacylglycerols and their fatty acid composition in edible Mediterranean molluscs and crustacean

The triacylglycerol (TAG) composition of edible Mediterranean molluscs (Eledone moschata, Sepia officinalis, Todarodes sagittatus) and crustacean (Penaeus kerathurus) was studied using a combination of preparative RP–HPLC and GC/MS. In S. officinalis and T. sagittatus mantle TAG, the main fatty acids were C16:0 and C18:0 while in E. moschata they were C18:1ω-9, C16:0, C20:5ω-3 and C22:6ω-3. In P. kerathurus muscle and cephalothorax TAG, the main fatty acids were C16:0, C18:0, C18:1ω-9, C20:4ω-6, C20:5ω-3, C22:6ω-3 and C16:0, C18:1ω-9, C20:4ω-6, C20:5ω-3, C22:6ω-3, respectively.     

Colour properties,stability, and free radical scavenging activity of jambolan (Syzygium cumini) fruit anthocyanins in a beverage model system: Natural and copigmented anthocyanins

The colour and stability properties of jambolan anthocyanins, both natural and copigmented forms, were investigated in beverage model as well as their radical scavenging ability. Natural anthocyanins of jambolan revealed low colour intensity due to glycosylation structure of the anthocyanins as diglucoside. The intermolecular copigmentation of anthocyanins with sinapic acid, caffeic acid, ferulic acid, and rosemary polyphenolic extract could enhance the colour intensity, which was observed through spectrometric parameters, such as hyperchromic effect (ΔA_vis-max) and bathochromic shift (Δλ_vis-max). In addition of sinapic acid, caffeic acid, and rosemary polyphenolics also increased the stability of the anthocyanin colour during exposure to white fluorescent light and storage at refrigeration and room temperatures, whereas on high thermal treatments, this phenomenon was not observed. Furthermore, beverage model coloured with natural or copigmented anthocyanins revealed DPPH radical-scavenging activity. The AEAC values indicated that the beverage models with copigmented anthocyanins had higher scavenging activity than the beverage with natural anthocyanins.