Pretreatment procedure for selenium speciation in shellfish using high‐performance liquid chromatography–microwave‐assisted digestion–hydride generation‐atomic fluorescence spectrometry

A pretreatment procedure based on an enzymatic hydrolysis extraction followed by a two‐step clean‐up has been performed for selenium speciation in shellfish samples. Bivalve samples were extracted with protease XIV, lipase VII and protease VIII. By using a protease VIII–lipase VII mixture, quantitative recoveries were obtained for all the selenium species, except for selenocystine (59%). Owing to the complexity of the matrix, clean‐up procedures were required to remove interferents that affected the chromatographic separation. The extracts were first partitioned in dichloromethane and then passed through a column with aminopropylsilane. Speciation of selenocystine, selenomethionine, selenoethionine, selenite and selenate was obtained using a high‐performance liquid chromatography–microwave‐assisted digestion–hydride generation‐atomic fluorescence spectrometry coupling. The chromatographic system consisted of an anion exchange and a reversed‐phase column, both connected through a six‐port switching valve. On‐line microwave‐assisted digestion and hydride generation steps were performed prior to atomic fluorescence detection. The method was applied to clam and prawn samples collected from the southwest coast of Spain. Copyright­© 2002 John Wiley & Sons, Ltd.     

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     

Use of gel permeation chromatography for automatic and rapid extract clean-up for the determination of diarrhetic shellfish toxins (DSP) by liquid chromatography-mass spectrometry

Summary A new analytical technique was established to improve the performance of DSP toxin determination in different sample matrices. High performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algal cells and mussel tissue prior to the determination of DSP toxins by LC/MS. The proposed protocol can be performed totally automatically and enables fast and sensitive analysis of large sample numbers. The recovery of the entire method protocol (consisting of extraction, clean-up and LC/MS determination) was approximately 70% with good repeatability (standard deviation ranging from 1.9% to 5.0% in the concentration range analyzed).     

液相色谱-高分辨质谱测定贝类中的4种贝毒素及16种兽药残留

建立了贝类组织中米氏裸甲藻贝毒素(Gymnodimine,GYM)、螺环内酯毒素(Spirolides,SPX1)、大田软骨酸贝毒素(Okadaic acid,OA-C)、蛤毒素(Pectenotoxins,PTX2)4种腹泻性贝类毒素、氯霉素、氟甲砜霉素以及14种磺胺类药物的液相色谱-高分辨质谱分析方法。样品采用甲醇提取,正己烷去除脂肪,乙酸乙酯反萃取,ODS粉分散固相萃取净化,经Agilent ZORBAX SB-C18色谱柱(3.0 mm×100 mm,1.8μm)分离,高分辨质谱仪进行检测。结果表明,各化合物在一定的质量浓度范围内线性良好,相关系数(r)均大于0.99。GYM,SPX1,OA-C,PTX2 4种腹泻性贝类毒素的定量下限分别为0.5,0.1,2.0,0.5μg/kg,各化合物在低、中、高3个浓度加标水平下的回收率为70.1%~105.8%,相对标准偏差(RSD)为10.1%~14.8%。该方法具有简单、快速、灵敏等特点,能满足贝类产品中贝类毒素、抗生素的检测要求。     

高效液相色谱/四极杆-飞行时间质谱测定神经性贝毒

采用高效液相色谱/四极杆 飞行时间质谱(HPLC/Q TOFMS)联用技术对贝类样品中的短裸甲藻毒素 PbTx-2 进行了检测研究。样品经丙酮提取、C18小柱净化后,用Zorbax XDB C18色谱柱(2.1 mm i.d.×150 mm,3.5 μ m)进行分离,流动相为甲醇 水(体积比为85∶15)溶液(含0.5 mmol/L NH4Ac),流速0.20 mL/min 。电喷雾正离子模式,选择质子化PbTx 2分子离子 ［M+H］ +作为前体离子进行TOFMS扫描、测定。结果表明,样品的平均加标     

Saxiphilin蛋白分离纯化及其活性测定

Saxiphilin是一种能与麻痹性贝类毒素（PSPs）特异性结合的可溶性血清蛋白,利用超滤离心及凝胶层析从牛蛙血浆中分离纯化Saxiphilin活性蛋白使其达到一定纯度,通过酶联免疫检测技术对其活性进行研究.结果表明：分离纯化目标蛋白时,采用pH值为6.5的Tris-HCl缓冲液,使用超滤离心、凝胶层析时目标蛋白的纯化倍数分别为5.9和11.2,回收率为67.8%和50%.通过SDS-聚丙酰胺凝胶电泳验证其分子量为90 kDa,并通过毒素粗蛋白的结合曲线可得粗蛋白与PSP浓度比达到46.86时,目标蛋白结合PSP毒素的量达到饱和.将Saxiphilin蛋白应用于微量滴定板来检测PSPs,具有快速、简便、灵敏、特异、经济等优点.     

几种饵料微藻基因组DNA的提取及其特异性引物的设计

用改良CTAB法提取我国沿海东南部滩涂贝类常见的4种饵料微藻的基因组DNA,结果发现提取的DNA产率高、完整性好,认为是一种简便而高效的微藻基因组DNA提取方法.对其18S rRNA 基因(18S rDNA)进行克隆与测序,并利用序列比对软件 MEGA 5.0对各微藻的18S rDNA序列进行两两比对,设计出了能够用于快速区分此4对饵料微藻的特异性PCR引物(Cha.F/Cha.R、Iso.F/Iso.R、Pla.F/Pla.R及Nan.F/Nan.R). PCR扩增验证实验结果显示,4对引物均具有很强的特异性,无交叉扩增现象.扩增片段大小范围为100~200 bp,满足实时荧光定量PCR的实验要求,在检测滩涂贝类对此4种饵料微藻的摄食选择性研究方面具有重要意义.     

基于三维荧光的产麻痹性贝毒藻浓度监测研究

近年来,我国沿海赤潮发生的次数和面积持续增加,经济损失严重。根据赤潮的毒性特点,通常分为三类,分别为无毒赤潮、鱼毒性赤潮和有毒赤潮。其中有毒赤潮产生的毒素主要是麻痹性贝毒,其由于分布广,毒性强成为危害最大的生物毒素之一。根据麻痹性贝毒的摄入量不同,人类误食染毒的贝类后,身体各部位会出现刺痛或灼热的感觉,然后全身麻痹,严重者甚至在短时间内死亡。近年来,多地出现人类误食染毒的贝类后死亡的事件。麻痹性贝毒的摄入量主要取决于产麻痹性贝毒藻的浓度,因此,对产麻痹性贝毒藻浓度的监测就显着尤为重要。提出了用三维荧光光谱结合化学计量学方法建立产麻痹性贝毒藻定量分析模型。首先,利用F-4600荧光光度计采集微小亚历山大藻（Alexandrium minimum）、链状裸甲藻（Gymnodinium catenatum）和太平洋亚历山大藻（Alexandrium pacificum）三种典型的产麻痹性贝毒藻类三维荧光光谱数据,获取藻类样本的三维荧光光谱等高线图,并进行图谱分析;然后,利用不同激发波长下的发射光谱数据建立产麻痹性贝毒藻三维荧光光谱的串行表示模型,提取新的特征;最后,将新的特征数据分别作为粒子群优化最小二乘支持向量机算法（particle swarm optimization-least squares support vector machine, PSO-LSSVM）和偏最小二乘回归（partial least squares regression, PLSR）的输入,建立产麻痹性贝毒藻的定量分析模型。结果表明,运用粒子群优化最小二乘支持向量机算法建立的产麻痹性贝毒藻的定量分析模型普遍优于偏最小二乘回归算法。当激发波长选择460和530 nm,发射波长选择650～750 nm作为PSO-LSSVM的输入数据,建立的产麻痹性贝毒藻的定量分析模型效果最好,结果显示R_c=0.999 9,RMSEC=0.017 1,R_p=0.949 2,RMSEP=0.291 0。这体现出三维荧光光谱结合PSO-LSSVM定量分析模型可有效地监测活体产麻痹性贝毒藻的浓度数值,为产麻痹性贝毒藻浓度检测提供了一种在线检测的新方法。     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

阴/阳离子交换色谱-电感耦合等离子体质谱法分析鱼和贝类海产品砷的形态

采用阴(Hamilton PRP X100柱)阳(Dionex Ionpac CS- 10柱)离子交换色谱-电感耦合等离子体质谱联用技术,分别以pH 10.3的20 mmol/L NH4HCO3和pH 2.0的5 mmol/L吡啶溶液为流动相,建立了三价砷As(Ⅲ)、五价砷As(V)、一甲基砷酸MMA、二甲基砷酸DMA...