Liquid chromatography–tandem mass spectrometry method for the detection of marine lipophilic toxins under alkaline conditions

A new LC–MS/MS method for the separation and detection of the most prominent marine lipophilic toxin groups comprising okadaic acid, dinophysistoxins, yessotoxins, azaspiracids, pectenotoxins, spirolides and some okadaic acid fatty acid esters has been developed. With this method 28 different marine lipophilic biotoxins can be analysed in a single run. Separation was achieved with an acetonitrile/water gradient containing ammonium hydroxide (pH 11). All toxins were stable under these basic conditions. Compared to chromatography using an acidic gradient, the limit of detection (LODs) for okadaic acid, yessotoxin, gymnodimine and 13-desmethyl spirolide C were improved two- to three-fold, mainly due to better peak shapes. The azaspiracids and pectenotoxins-2 showed comparable LODs under acidic and basic conditions. A major advantage of the developed method is that toxins can be clustered in retention time windows separated for positively and negatively ionized molecular ions. Therefore, there is no need for rapid polarity switching or two separate runs for one sample. The new method showed good repeatability and reproducibility and is an important step in the development of alternatives to the animal tests currently in use for shellfish toxin analysis.     

Results of a European interlaboratory method validation study for the quantitative determination of lipophilic marine biotoxins in raw and cooked shellfish based on high-performance liquid chromatography–tandem mass spectrometry. Part I: collaborative study

A European interlaboratory collaborative study was conducted to validate a method for the quantitative determination of lipophilic marine biotoxins based on high-performance liquid chromatography–tandem mass spectrometry. During this study, the diarrhetic shellfish poisoning toxins okadaic acid, dinophysis toxin1 and 2 including their esters, the azaspiracids 1-3, pectenotoxin2, and the yessotoxins were investigated at concentration levels near the limit of quantification and near the legal limit. Naturally contaminated blue mussels, both raw and cooked and spiked extracts of clams and oysters were studied and results were obtained for 16 test samples from 16 laboratories representing eight different countries. This article summarizes the study outcome concerning validation key parameters like specificity, linearity, limit of detection, accuracy/recovery, and precision. Further, influences of cooking of mussels before homogenization or hydrolysis on method robustness have been evaluated.     

Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography–mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300–500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 μg/L (parts per billion) for the positive ions, 1.6–5.1 μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.     

Selective enrichment and quantification of okadaic acid in shellfish using an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry assay

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean‐up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 μg/kg, respectively. Fifty‐three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.     

Derivatization of azaspiracid biotoxins for analysis by liquid chromatography with fluorescence detection

Azaspiracids (AZAs) are an important group of regulated lipophilic biotoxins that cause shellfish poisoning. Currently, the only widely available analytical method for quantitation of AZAs is liquid chromatography-mass spectrometry (LC-MS). Alternative methods for AZA analysis are needed for detailed characterization work required in the preparation of certified reference materials (CRMs) and by laboratories not equipped with LC-MS. Chemical derivatization of the amine and carboxyl groups on AZAs was investigated for the purpose of facilitating analysis by LC with fluorescence detection (FLD). Experiments towards chemical modification of AZA1 at the amine achieved only limited success. Derivatization of the carboxyl group, on the other hand, proved successful using the 9-anthryldiazomethane (ADAM) method previously applied to the okadaic acid (OA) group toxins. Extraction and clean-up methods were investigated for shellfish tissue samples and a post-reaction solid phase extraction procedure was developed for the AZA ADAM derivatives. Chromatographic separations were developed for the LC-FLD analysis of derivatized AZAs alone or in the presence of other derivatized toxins. This new analytical method for analysis of AZAs enabled verification of AZA1-3 concentrations in recently certified reference materials. The method demonstrated good linearity, repeatability and accuracy showing its potential as an alternative to LC-MS for measurement of AZAs.     

Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry

Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the analysis of the 13 toxins that are stated in European Union legislation. However, to date over 200 lipophilic marine toxins have been described in the literature. To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100 000) for marine lipophilic toxins has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin (PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were obtained from Norway, Ireland, United Kingdom, Portugal and Italy. Each sample extract was analyzed with and without collision induced dissociation fragmentation. Based on their mass and specific fragmentation pattern, 85 different toxins were identified comprising 33 OA, 26 YTX, 18 AZA and 8 PTX group toxins. A major complication of full scan HRMS is the huge amount of data generated (file size), which restricts the possibility of a fast search. A software program called metAlign was used to reduce the orbitrap MS data files. The 200-fold reduced data files were screened using an additional software tool for metAlign: ‘Search_LCMS’. A search library was constructed for the 85 identified toxins. The library contains information about compound name, accurate mass, mass deviation (<5 ppm), retention time (min) and retention time deviation (<0.2 min). An important feature is that the library can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested by analyzing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. A toxin profile was determined for each of the contaminated samples. No toxins were found in the blank sample, which is in line with the results obtained for this sample in the routine monitoring program (rat bioassay and tandem LC–MS).     

Development of a novel immunobiosensor method for the rapid detection of okadaic acid contamination in shellfish extracts

The mouse bioassay is the methodology that is most widely used to detect okadaic acid (OA) in shellfish samples. This is one of the best-known toxins, and it belongs to the family of marine biotoxins referred to as the diarrhetic shellfish poisons (DSP). Due to animal welfare concerns, alternative methods of toxin detection are being sought. A rapid and specific biosensor immunoassay method was developed and validated for the detection of OA. An optical sensor instrument based on the surface plasmon resonance (SPR) phenomenon was utilised. A polyclonal antibody to OA was raised against OA–bovine thyroglobulin conjugate and OA–N-hydroxy succinimide ester was immobilised onto an amine sensor chip surface. The assay parameters selected for the analysis of the samples were: antibody dilution, 1/750; ratio of antibody to standard, 1:1; volume of sample injected, 25 μl min⁻¹; flow rate, 25 μl min⁻¹. An assay action limit of 126 ng g⁻¹ was established by analysing of 20 shellfish samples spiked with OA at the critical concentration of 160 ng g⁻¹, which is the action limit established by the European Union (EU). At this concentration of OA, the assay delivered coefficient of variations (CVs) of <10%. The chip surface developed was shown to be highly stable, allowing more than 50 analyses per channel. When the concentrations of OA determined with the biosensor method were compared with the values obtained by LC–MS in contaminated shellfish samples, the correlation between the two analytical methods was found to be highly satisfactory (r ² = 0.991). Figure Biacore     

Freeze-drying for the stabilisation of shellfish toxins in mussel tissue (<Emphasis Type="Italic">Mytilus edulis</Emphasis>) reference materials

Two samples of mussels (Mytilus edulis) were collected from the southwest of Ireland. One sample contained domoic acid, the other sample contained okadaic acid, dinophysistoxin-2 and azaspiracid-1, -2 and -3. Wet and freeze-dried reference materials were prepared from each of the two samples to test for differences in homogeneity, stability and extractability of the analytes in either condition. Wet materials were homogenised, aliquoted and hermetically sealed under argon and subsequently frozen at −80 °C. Dry materials were similarly homogenised but frozen in flat cakes prior to freeze-drying. After grinding, sieving and further homogenisation, the resulting powder was aliquoted and hermetically sealed. Domoic acid materials were characterised using HPLC–UV, while LC–MS was used for the determination of lipophilic toxins. The extractabilities of all phycotoxins studied were comparable for wet and freeze-dried materials once a sonication step had been carried out for reconstitution of the freeze-dried materials prior to extraction. Homogeneity was assessed through replicate analysis of the phycotoxins (n = 10), and was found to be similar for wet and freeze-dried materials, for both hydrophilic and lipophilic toxins. Water contents were determined for both wet and freeze-dried materials, and particle size was determined for the freeze-dried materials. Stability was evaluated isochronously over eight months at four temperatures (−20, +4, +20 and +40 °C). The freeze-dried material containing domoic acid was stable over the whole duration at all temperatures, while in the wet material domoic acid degraded to some extent at all temperatures except −20 °C. In freeze-dried and wet materials containing lipophilic toxins, okadaic acid, dinophysistoxin-2, azaspiracid-1 and azaspiracid-2 were stable over the whole duration at all conditions, while concentrations of azaspiracid-3 changed significantly in both materials at some storage temperatures. Figure Aliquots of freeze-dried and wet mussel tissue reference materials containing the various shellfish toxins examined in the study     

Feasibility of gamma irradiation as a stabilisation technique in the preparation of tissue reference materials for a range of shellfish toxins

The effect of γ-irradiation on concentrations of hydrophilic and lipophilic phycotoxins has been investigated by use of HPLC–UV and LC–MS. Pure toxins in organic solvents and toxins in mussel (Mytilus edulis) tissues were irradiated at three different doses. In solution all toxin concentrations were reduced to some extent. Most severe decreases were observed for domoic acid and yessotoxin, for which the smallest dose of irradiation led to almost complete destruction. For pectenotoxin-2 the decrease in concentration was less severe but still continuous with increasing dose. Azaspiracid-1 and okadaic acid were the least affected in solution. In shellfish tissue the decrease in toxin concentrations was much reduced compared with the effect in solution. After irradiation at the highest dose reductions in concentrations were between ca. 5 and 20% for the lipophilic toxins and there was no statistical difference between control and irradiated samples for azaspiracids in tissue. Irradiation of shellfish tissues contaminated with domoic acid led to a more continuous decrease in the amount of the toxin with increasing dose. The effect of irradiation on the viability of microbial activity in shellfish tissues was assessed by using total viable counting techniques. Microbial activity depended on the type of shellfish and on the pretreatment of the shellfish tissues (with or without heat treatment). As far as we are aware this is the first investigation of the effectiveness of irradiation as a technique for stabilising tissue reference materials for determination of phycotoxins. Our results suggest that this technique is not effective for materials containing domoic acid. It does, however, merit further investigation as a stabilisation procedure for preparation of shellfish tissue materials for some lipophilic toxins, in particular azaspiracids. Chemical structures of the toxins investigated in the study     

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.     

Sensitive method for the determination of lipophilic marine biotoxins in extracts of mussels and processed shellfish by high-performance liquid chromatography–tandem mass spectrometry based on enrichment by solid-phase extraction

A solid-phase extraction (SPE) method for the enrichment and clean-up of lipophilic marine biotoxins from extracts of different species of bivalve molluscs and processed shellfish products was developed. Okadaic acid (OA), pectenotoxin2 (PTX2), azaspiracid1 (AZA1) and yessotoxin (YTX) were determined by LC–MS/MS in hydrolyzed and non-hydrolyzed extracts. Applying a concentration factor of 10 the limit of quantification for the four toxins was determined to be 1 μg/kg. An organized in-house ring trial proved transferability of the method protocol and satisfactory results for all four toxins with a relative standard deviation (RSD) of 5–12%. The precision of the whole method including LC–MS detection was determined by processing seven independent extractions analyzed in independent sequences. RSD ranged between 12% and 24%. This SPE method was tested within a concentration range corresponding to the range of the current European Union regulatory limits (up to 160 μg/kg for the OA group), but it would also be applicable to a lower μg/kg range which is important in view of a possible decrease of regulatory limits as proposed by a working group of the European Food Safety Authority. The potential of SPE as a cleaning tool to cope with matrix effects in LC–MS/MS was studied and compared to liquid–liquid portioning.     

Mass spectrometric analysis of the marine lipophilic biotoxins pectenotoxin-2 and okadaic acid by four different types of mass spectrometers

The performances of four different mass spectrometers [triple-quadrupole (TQ), time-of-flight (ToF), quadrupole ToF (Q-ToF) and ion trap (IT)] for the detection of the marine lipophilic toxins pectenotoxin-2 (PTX2) and okadaic acid (OA) were investigated. The spectral data obtained with the different mass spectrometric analyzers were used to propose fragmentation schemes for PTX2 in the positive electrospray mode and for OA in the negative electrospray mode. TQ data were used to obtain product ions, while ToF and Q-ToF-MS produced accurate mass data of the precursor ion and product ions, respectively. IT data provided a better understanding of the fragmentation pathways using MS(n) experiments. With respect to analytical performance, all four mass analyzers showed a good linearity (R(2) > 0.97) and repeatability (CV < 20%). Detection limits (LoDs) (S/N = 3) were the lowest on triple-quad MS: 12.2 and 2.9 pg on-column for PTX2 and OA, respectively.     

Discovery of new analogs of the marine biotoxin azaspiracid in blue mussels (Mytilus edulis) by ultra-performance liquid chromatography/tandem mass spectrometry

Azaspiracids (AZAs) are a group of lipophilic marine biotoxins that were first discovered in blue mussels harvested in 1995 in Killary Harbour on the west coast of Ireland. At least eight people fell ill after the consumption of contaminated mussels and developed symptoms of nausea, stomach cramps, vomiting and severe diarrhoea. Until now, eleven different analogs of these toxins have been described, with a twelfth one theoretically postulated. This paper describes the detection and identification of twenty new analogs of azaspiracid, including dihydroxy-AZAs and carboxy-AZAs, using state-of-the-art techniques including ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS). Blue mussels (Mytilus edulis) from a toxic event of the northwest coast of Ireland in 2005 were extracted and analysed using LC/MS. The mass spectra obtained from different instruments enabled identification of previously unknown analogs of azaspiracid with additional hydroxyl and carboxyl substituents. Mass fragmentation patterns of the dihydroxy-AZAs indicated the positions of these substituents to be at the C3 and C23 position. The previously theoretically postulated AZA12 was also observed in this study. Product ion spectra showed the presence of a unique fragment ion at m/z 408 for all C23-hydroxylated analogs. This fragmentation competes with the fragmentation leading to m/z 362, a fragment ion that has shown to be present in all AZAs. The novel analogs have not been seen in plankton or water samples and are believed to be metabolites of AZAs formed in mussels. All the new AZA analogs were present at low concentrations in the shellfish and it is probably safe to assume that they do not pose a risk for the shellfish consumer.     

Inter-laboratory validation of the fluorescent protein phosphatase inhibition assay to determine diarrhetic shellfish toxins: intercomparison with liquid chromatography and mouse bioassay

Toxic episodes of diarrhetic shellfish toxins (DSP) in shellfish harvesting areas have serious economic and public health implications, where fluorescent protein phosphatase inhibition assay (FPPIA) may be a highly useful tool for monitoring purposes. This paper presents results from the first inter-laboratory study to validate the assay. Three laboratories participated in the design and development of the inter-laboratory work. Standard solutions and spiked samples of the main toxin, okadaic acid, were used at the beginning of the validation exercise to avoid cross-inhibition of other toxins that would otherwise deteriorate the quantitative significance of the data. HPLC with fluorimetric detection of okadaic acid was also submitted to inter-laboratory validation to be subsequently used as a quantitative reference method. FPPIA results from spiked samples were free of systematic bias in any laboratory and determinations repeated over 3 days showed that the classic “repeatability” was the main within-laboratory source of variability (15-26% R.S.D. depending on the sample).After the inter-laboratory validation of both HPLC and FPPIA methods, 83 samples of mussel hepatopancreas collected during a toxic DSP episode were analyzed over 9 weeks. Toxic levels determined with FPPIA were in line with mouse bioassay results, highlighting the lack of false negative results of the FPPIA test: 98.7% of samples whose concentration of okadaic acid equivalents was over 0.8 μg/g hep., provided positive bioassay results within 24 h of observation time. The reliability and the quantitativeness of the FPPIA method in naturally contaminated samples was demonstrated by intercomparison with mouse bioassay and HPLC.     

超高效液相色谱-串联质谱法同时测定血浆与尿液中12种脂溶性贝类毒素

生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持。目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题。该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法。实验对提取试剂以及流动相的选择进行了优化,采用乙腈对尿液和血浆样品进行提取。采用Phenomenex Kinetex C18色谱柱(50 mm×3 mm, 2.6 μm)进行分离,以0.05%(v/v)氨水水溶液、90%(v/v)乙腈水溶液为流动相,以流速0.40 mL/min梯度洗脱时,12种目标化合物分离效果最好。串联质谱的离子源为电喷雾离子(ESI)源,采用多反应监测(MRM)模式检测。12种目标物的基质效应均在0.8~1.1之间,表明该前处理方法的基质干扰低,采用外标法可对化合物进行准确定量。12种贝类毒素的线性范围为0.03~36.25 μg/L,相关系数均大于0.995。尿液检测的方法定量限为0.23~0.63 μg/L,血浆检测的方法定量限为0.31~0.84 μg/L。3个加标水平的回收率为72.7%~124.1%,日内精密度为2.1%~20.0%,日间精密度为2.1%~15.3%。利用该方法检测健康人尿液和血浆样本,以及经腹腔注射12种贝类毒素的小鼠尿液和血液样本。20份健康人样本中未检出目标物,20份小鼠样本中12种贝类毒素均有检出。该方法操作简便,样品取样量少,方法灵敏高,适用于血浆和尿液中脂溶性贝类毒素的快速检测。     

Strategies for the elimination of matrix effects in the liquid chromatography tandem mass spectrometry analysis of the lipophilic toxins okadaic acid and azaspiracid-1 in molluscan shellfish

Considerable efforts are being made worldwide to replace in vivo assays with instrumental methods of analysis for the monitoring of marine biotoxins in shellfish. Analysis of these compounds by the preferred technique of liquid chromatography tandem mass spectrometry (LC-MS/MS) is challenged by matrix effects associated with the shellfish tissues. In methods validation, assessment of matrix interferences is imperative to ensure the validity and accuracy of results being produced. Matrix interferences for the analysis of okadaic acid (OA) and azaspiracid 1 (AZA1) were assessed using acidic methods on electrospray triple stage quadrupole (TSQ) and hybrid quadrupole time of flight (QToF) instruments by the use of matrix matched standards for different tissue types. Using an acidic method no matrix interference and suppression was observed on the TSQ for OA and AZA1 respectively, whilst the opposite was observed on the QToF; matrix enhancement for OA and no matrix interference for AZA1. The suppression of AZAs on the TSQ was found to be due to interfering compounds being carried over from previous injections. The degree of suppression is very much dependant on the tissue type ranging from 15 to 70%. Several strategies were evaluated to eliminate these interferences, including the partitioning of the extract with hexane, optimisation of the chromatographic method and the use of on-line SPE. Hexane clean up did not have any impact on matrix effects. The use of an alkaline method and a modified acidic method eliminated matrix suppression for AZA1 on the TSQ instrument while an on-line SPE method proved to be effective for matrix enhancement of OA on the QToF.     

Development of a High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Lipophilic Toxins in Marine Shellfishes and Edible Safety Evaluation

At present, edible marine shellfishes are often contaminated by a combination of different kinds of marine lipophilic toxins. In this study, several common lipophilic shellfish toxins (LSTs) in marine shellfishes were simultaneously detected by liquid chromatography-tandem mass spectrometry, and the safety risk of commercial marine shellfishes was evaluated based on the materiome of LSTs. Under the optimal conditions, the developed method displayed satisfactory recovery values (63.2%–88.8%), precision (relative standard deviations ≤ 14.5%), and sensitivity (limit of detection in the range of 0.54–2.69 ng g^?1) for all analytes. Among the 105 commercially available shellfish samples, 42.86% of the samples had at least one kind of toxins. The highest average content was 47.60 μg kg^?1 of DTX1, which was the most serious contaminant in marine shellfish samples. Total Exposure Risk Index (∑ERI) was calculated based on Total Daily Intake (TDI) and Acute Reference Dose (ARfD) of each toxin to evaluate the safety risk of commercial marine shellfishes. The results indicated that the risk of toxin poisoning was 19.05% in the commercial available marine shellfishes, and the scallops (Chlamys farreri) have the highest poisoning risk among different shellfishes used in this study. In summary, a new method based on the combined contamination of LSTs was successfully developed for the risk assessment of commercial marine shellfishes. The proposed method is stricter than that in the relevant rules of European Food Safety Authority (EFSA) and can benefit to protect shellfish consumers from poisoning risk.     

Determination of lipophilic toxins by LC/MS/MS: single-laboratory validation

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.     

超高效液相色谱-串联质谱法测定海水悬浮颗粒物中的8种典型脂溶性藻毒素

海水悬浮颗粒物对海洋环境中污染物的迁移转化有着重要的影响,在海水悬浮颗粒物上富集的脂溶性藻毒素会严重的毒害海洋滤食性生物。本研究建立了海水悬浮颗粒物中8种典型脂溶性藻毒素同步测定的超高效液相色谱-串联质谱( UPLC-MS/MS)分析方法。海水悬浮颗粒物样品经甲醇超声辅助提取后,以5 mmol/L 乙酸铵水溶液和乙腈为流动相,经1.7微米C18色谱柱分离,采用电喷雾串联质谱( ESI-MS/MS)多反应监测( MRM )模式检测,外标法定量。结果表明,在最佳实验条件下,8种目标物在5 min内分离良好,加标回收率在83.8%~110.4%之间,方法具有良好的精密度(相对标准偏差( RSD)≤14.1%)和灵敏度(检出限介于2.9~103 pg/g之间),在线性范围内,相关系数(R2)均大于0.996,能满足海水悬浮颗粒物中8种典型脂溶性藻毒素同步检测的要求。采用本方法初步分析了青岛沿岸海域海水悬浮颗粒物中的脂溶性藻毒素,其中PTX2被检出,含量最高可达790 pg/g(干重)。     

Immunosensor for the Determination of Okadaic Acid Based on Screen-Printed Electrode

A disposable immunosensor for okadaic acid (OA), using a screen-printed electrode (SPE), was developed and characterised. Detection of the product, p-aminophenol, resulting from the reaction catalysed by alkaline phosphatase (AP), was carried out using an amperometric three-electrode system poised at a voltage of + 300 mV versus Ag/AgCl. Alkaline phosphatase was used as a label for the antigen, OA, and two kinds of alkaline phosphatase preparation were studied for the conjugation of okadaic acid. The calibration curve for okadaic acid obtained from the conjugate created from low-activity AP, 969 units/mg, was unsatisfactory in terms of sensitivity, but a high-activity conjugate delivered the required sensitivity and limit of detection. Studies on the stability of the sensor with α-OA antibody and OA-AP conjugate showed that the current response decreased drastically after one day. Stabilisation strategies have been formulated to overcome this problem. The calibration curve obtained with the high activity conjugate was linear up to 40 ng/ml of okadaic acid with a minimum concentration of analyte detected of 5 ng/ml and a detection limit of 2 ng/ml.