Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.      

麻痹性贝毒产毒藻株的三维荧光光谱识别技术研究

利用三维荧光光谱技术,研究了以微小亚历山大藻 （台湾株）（AMSY）、塔玛亚历山大藻（大亚湾株）（ATDY）、链状裸甲藻（防城港株）（GCFC）、塔玛亚历山大藻（香港株）（ATHK）、链状亚历山大藻（南海株）（ACSY）5株4种产麻痹性贝毒(PSP)微藻以及21株不产PSP藻在不同温度条件下培养微藻的各生长期产毒能力与三维荧光的关系。研究结果表明,在不同温度条件下,产毒藻类单位藻细胞产毒量会发生显著变化,低温可促进麻痹性贝毒的产生。通过Db7小波分解,选取Ca3尺度分量的联合荧光谱作为特征谱并利用Fisher判别发现,产PSP藻与不产PSP藻荧光差异主要集中在λ_ex为400~425和450~545nm;λ_em为715~750 nm的波段,利用判别函数建立判别式,实现了对产麻痹性贝类毒素藻类的识别测定。对产PSP毒素藻类的判别率达到93.7%,不产PSP毒素藻类判别率达到93.3% ,综合判别率为93.5%。该研究可实现对水体中活体产PSP微藻的快速识别,为进一步开发产毒微藻识别仪提供理论基础。     

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H₂O₂/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.     

11种贝螺肉类27种微量元素的分析测试

采用微波消解法处理贝肉类样品,并用ICP-AES法测定11种贝肉类中的27种微量元素Ca,Mg,K,Na,P,S,Zn,Mn,Fe,Cu,Cr,Co,Se,Sn,Ni,Al,Ti,V,Mo,Ba,Ge,Si,Cd,I,Pb,As和Hg,结果表明：ICP-AES法在测定多种微量元素的仪器方法中显示巨大的优势,11种贝肉类中含有20多种以上微量元素,淡水养殖的贝肉类比海水养殖的钙含量要高得多,有害金属元素主要存在着As和Pb的污染概率。     

Shellfish waste-derived mesoporous chitosan for impressive removal of arsenic(V) from aqueous solutions: A combined experimental and computational approach

Novel shellfish waste-derived chitosan (CS) has been developed to adsorb As(V) from simulated wastewater under evaluating adsorption process parameters. The coexistence of some competing ions, like SiO₃^2-, Cl^-, NO₃^– and PO₄^3- as well as the regeneration capacity of the spent adsorbent, was explored. The experimental data were modeled using several kinetics and isotherm models to understand the mechanism related to the uptake process. As(V) uptake was relatively rapid and highly dependent on pH. The Avrami-fractional-order expression supported data best, while the Liu equation described well isotherm data at pH 5.0. The maximum uptake capability (Liu) was 12.32 mg/g, and the highest removal performance (99 %) was obtained at optimum pH 5.0. Molecular dynamics simulations were performed to more clearly illuminate the atomic-level interactions between arsenic species and CS surface in both acidic and basic mediums. After four adsorption–desorption cycles, CS exhibited more than 90 % As(V) removal efficiency. The results of this study indicates that low cost shellfish derived chitosan is promising for efficient removal of As(V) from water body and can be used to remove other pollutants from watewater.     

液相色谱-串联质谱检测贝类组织中5种脂溶性贝毒素

建立了液相色谱-串联质谱分析贝类组织中米氏裸甲藻(GYM)贝毒素、螺环内酯毒素(SPX1)、大田软骨酸(OA)贝毒素、蛤毒素(PTX2)、原多甲藻酸(AZA1)贝毒素的方法.用甲醇-水(4: 1, V/V)溶液对贝类组织中GYM, SPX1, OA, PTX2和AZA1进行提取,MAX阴离子交换柱净化后,采用液相色谱分离,除OA以负离子选择反应监测外,GYM, SPX1, PTX2和AZA1以电喷雾离子源正离子选择反应监测模式进行质谱分析.5种脂溶性贝毒素GYM, SPX1, OA, PTX2和AZA1在各自相应浓度范围内线性良好,相关系数＞0.99.扇贝闭壳肌空白样品添加5种贝毒素的提取率均为78.6%～94.4%(n=6); 精密度(RSD)为6.8%～14.9%.贝类组织中5种贝毒素GYM, SPX1, OA, PTX2和AZA1的检出限分别为0.10, 0.21, 2.00, 0.32和0.04 μg/kg.     

Supramolecular solvents in solid sample microextractions: Application to the determination of residues of oxolinic acid and flumequine in fish and shellfish

Supramolecular solvents are here proposed firstly as extractants in solid sample microextractions. The approach was evaluated by extracting flumequine (FLU) and oxolinic acid (OXO), two widely used veterinary medicines, from fish and shellfish muscle using a supramolecular solvent made up of decanoic acid (DeA) reverse micelles. The antibiotics were extracted in a single step (∼15 min), at room temperature, using 400 μL of solvent. After centrifugation, an aliquot of the extract was directly analyzed by liquid chromatography and fluorescence, without the need of clean-up or solvent evaporation. Contrary to the previously reported methods, both OXO and FLU were quantitatively extracted from fish and shellfish, independently of sample composition. The high extraction efficiencies observed for these antibiotics were a consequence of their amphiphilic character which resulted in the formation of DeA-OXO and DeA-FLU mixed aggregates. The quality parameters of this quantitative method including sensitivity, linearity, selectivity, repeatability, trueness, ruggedness, stability, decision limit and detection capability were evaluated according to the 2002/657/EC Commission Decision. Quantitation limits in the different samples analyzed (salmon, sea trout, sea bass, gilt-head bream, megrim and prawns) ranged between 6.5 and 22 μg kg⁻¹ for OXO and, 5 and 15 μg kg⁻¹ for FLU. These limits were far below the current maximum residue limits (MRLs) set by the European Union (EU) (i.e. 100 and 600 μg kg⁻¹, for OXO and FLU, respectively). The trueness of the method was determined by analyzing a Certified Reference Material (CMR, BCR^®-725) consisting of a lyophilised salmon tissue material. Recoveries for fortified samples (50–100 μg kg⁻¹ of OXO and 50–600 μg kg⁻¹ of FLU) and their relative standard deviations were in the intervals 99–102% and 0.2–5%, respectively. The repeatability, expressed as relative standard deviation, was 3.6% for OXO and 2.3% for FLU ([OXO] = [FLU] = 200 μg kg⁻¹ and n = 11).     

钯－抗坏血酸基体改进剂平台石墨炉原子吸收光谱法测定贻贝标样中的砷

本实验建立了钯－抗坏血酸基体改进剂平台石墨炉原子吸收光谱法测定贻贝中砷的方法。研究了基体改进剂氯化钯、抗坏血酸用量对测定的影响，比较了氯化钯－抗坏血酸、氯化钯、硝酸镍三种基体改进剂及平台石墨管、普通石墨管在测定贻贝中砷的灵敏度。方法的相对标准偏差为３．２％，回收率在９７．８％—１０７．４％之间，检出限为１２．０ｎｇ／ｍＬ。     

LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea

Phycotoxins produced by various species of toxigenic microalgae occurring in the plankton are a global threat to the security of seafood resources and the health of humans and coastal marine ecosystems. This has necessitated the development and application of advanced methods in liquid chromatography coupled to mass spectrometry (LC-MS) for monitoring of these compounds, particularly in plankton and shellfish. Most such chemical analyses are conducted in land-based laboratories on stored samples, and thus much information on the near real-time biogeographical distribution and dynamics of phycotoxins in the plankton is unavailable. To resolve this problem, we conducted ship-board analysis of a broad spectrum of phycotoxins collected directly from the water column on an oceanographic cruise along the North Sea coast of Scotland, Norway, and Denmark. We equipped the ship with a triple-quadrupole linear ion-trap hybrid LC-MS-MS system for detection and quantitative analysis of toxins, such as domoic acid, gymnodimine, spirolides, dinophysistoxins, okadaic acid, pectenotoxins, yessotoxins, and azaspiracids (AZAs). We focused particular attention on the detection of AZAs, a group of potent nitrogenous polyether toxins, because the culprit species associated with the occurrence of these toxins in shellfish has been controversial. Marine toxins were analyzed directly from size-fractionated plankton net tows (20 μm mesh size) and Niskin bottle samples from discrete depths, after rapid methanolic extraction but without any further clean-up. Almost all expected phycotoxins were detected in North Sea plankton samples, with domoic acid and 20-methylspirolide G being most abundant. Although AZA was the least abundant of these toxins, the high sensitivity of the LC-MS-MS enabled detailed quantification, indicating that the highest amounts of AZA-1 were present in the southern Skagerrak in the 3–20 μm size-fraction. The direct on-board toxin measurements enabled isolation of plankton from stations with high AZA-1 levels and from the most suspicious size-fraction, i.e. most likely to contain the AZA-producer. A large number (>100) of crude cultures were established by serial dilution and later screened for the presence of AZAs after several weeks growth. From one crude culture containing AZA, a small dinoflagellate was subsequently isolated and brought into pure culture. We have thus proved that even sophisticated mass spectrometers can be operated in ship laboratories without any limitation caused by vibrations of the ship’s engine or by wave movement during heavy seas at wind forces up to nine Beaufort. On-board LC–MS–MS is a valuable method for near real-time analysis of phycotoxins in plankton for studies on bloom dynamics and the fate of toxins in the food web, and for characterization and isolation of putatively toxigenic organisms.     

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.