Receptor binding assay for the detection of paralytic shellfish poisoning toxins: comparison to the mouse bioassay and applicability under regulatory use

The receptor-binding assay (RBA) method for the detection of paralytic shellfish poisoning (PSP) toxins was evaluated for its overall performance in comparison with the mouse bioassay (MBA). An initial study to evaluate the effects of filtering shellfish extracts prior to running the RBA indicated no significant difference between filtered and unfiltered extracts on the determined saxitoxin (STX) concentrations. Next, we tested the RBA assay on 295 naturally contaminated mussel tissue samples, ranging in concentrations from 320 µg STX equiv. kg^?1 to 13,000 µg STX equiv. kg^?1 by MBA. An overall trend was observed with the RBA giving higher results (256 µg STX equiv. kg^?1 on average) than the MBA; however, at low concentrations (< 500 µg STX equiv. kg^?1) the RBA results were marginally lower. A third study was conducted using spiked mussel tissue analysed by three independent laboratories, two of which performed the RBA and one the MBA. This multi-laboratory study again showed the RBA to give higher results than the MBA; however, it also revealed that STX determination was accurate by the RBA, unlike the MBA. To optimise the assay for efficient usage under regulatory practice, three suggestions have been made: the use of an initial screening plate to separate those samples that exceed the alert level; use of rapid PSP test kits in the field and in the laboratory for screening negative samples and for early detection of toxicity; and use of an alternate commercially available porcine membrane in place of the laboratory-prepared rat membrane homogenate. The large number of samples analysed and the diversity of the tests conducted in this study further support the RBA as an affordable rapid method for STX detection that is also free of the routine sacrifice of live animals.     

Contamination of shellfish from Shanghai seafood markets with paralytic shellfish poisoning and diarrhetic shellfish poisoning toxins determined by mouse bioassay and HPLC

This paper reports the results of investigations of shellfish toxin contamination of products obtained from Shanghai seafood markets. From May to October 2003, 66 samples were collected from several major seafood markets. Paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP) toxins in shellfish samples were monitored primarily by a mouse bioassay, then analysed by HPLC for the chemical contents of the toxins. According to the mouse bioassay, eight samples were detected to be contaminated by PSP toxins and seven samples were contaminated by DSP toxins. Subsequent HPLC analysis indicated that the concentrations of the PSP toxins ranged from 0.2 to 1.9 µg/100 g tissues and the main components were gonyautoxins 2/3 (GTX2/3). As for DSP, okadaic acid was detected in three samples, and its concentration ranged from 3.2 to 17.5 µg/100 g tissues. Beside okadaic acid, its analogues, dinophysistoxins (DTX1), were found in one sample. According to the results, gastropod (Neverita didyma) and scallop (Argopecten irradians) were more likely contaminated with PSP and DSP toxins, and most of the contaminated samples were collected from Tongchuan and Fuxi markets. In addition, the contaminated samples were always found in May, June and July. Therefore, consumers should be cautious about eating the potential toxic shellfish during this specific period.     

Assessment of the quantitative determination of paralytic shellfish poisoning toxins by pre-column derivatization and elimination of interfering compounds by solid-phase extraction

Monitoring programmes for paralytic shellfish poisoning toxins in bivalve molluscs still rely heavily on the use of mouse bioassays (MBA) for consumer protection. A high-performance liquid chromatography (HPLC) methodology (Lawrence method) was implemented in 1996 in the Portuguese monitoring programme as a complementary means of analysis. Comparison between MBA and HPLC was done at the time only by a qualitative approach due to the scarce number of positive samples tested. More quantitative data were obtained recently when studying toxin profiles in Moroccan shellfish, and the correlation found between these two methodologies is reported here for the first time. Two different matrices were studied: blue mussel and the giant cockle Acanthocardia tuberculatum. A good linear correlation was obtained for both matrices. However, a second-degree polynomial best fitted the data at both low and high extremes of toxicity. According to the HPLC quantitative results, 13% of false-negatives could be obtained by MBA due to an underestimation of toxicity near the limit of detection of the MBA. Difficulties on relying solely on HPLC for consumer protection have been aroused with uncommon matrices, such as imported clams or crustaceans, due to the presence of high concentrations of interfering compounds. The solid-phase extraction step of the Lawrence method was implemented to eliminate an unknown compound that could be mistaken for saxitoxin, and an 80% reduction of another common unknown compound eluting close to decarbamoylsaxitoxin. The implementation of the HPLC methodology achieved so far allows a high degree of consumer protection without the need to resource to animal sacrifice.     

草鱼鱼鳞胶原蛋白酸酶分步提取工艺研究

为实现鱼鳞胶原蛋白的高效提取,在优化脱钙、混酸法和酶法提取工艺基础上,对鱼鳞胶原蛋白的酸酶进行分步提取。研究发现,鱼鳞最佳脱钙工艺为料液比8:100(g/mL),盐酸浓度1.0 mol/L,反应时间1.0 h,反应温度20℃;混合酸法提取鱼鳞胶原蛋白的最佳条件为醋酸料液比1:12(g/mL),柠檬酸料液比1:10(g/mL),乳酸料液比1:12(g/mL),即混合酸料液比为1:34(g/mL),其中,0.8 mol/L柠檬酸、1 mol/L乳酸、0.8 mol/L醋酸的体积比为6:5:6,提取时间2 d,胶原蛋白的提取率为48.14%;最佳酶法提取条件为胃蛋白酶用量450 U/g,提取温度30℃,提取时间72 h,该条件下提取率为45.26%。酸酶耦合法优于单一方法或同种方法两次提取的效果,可实现酸溶性和酶溶性胶原蛋白的连续提取,先酸后酶法胶原蛋白的提取率达84.61%,SDS-PAGE凝胶电泳发现其为Ⅰ型胶原蛋。     

两种不同形式的固相萃取技术相结合测定复杂基质中的三聚氰胺

针对饲料、巧克力制品、肉制品等一些复杂基质,采用分散性固相萃取技术与固相萃取技术相结合的方法进行净化,然后由高效液相色谱技术对三聚氰胺的含量进行测定。结果表明：该方法能够有效去除复杂基质中的油脂、蛋白质等基体的干扰,降低三聚氰胺假阳性的概率;在0.5～8.0μg/mL 范围内线性良好,相关系数R＞ 0.9995,检出限为0.5mg/kg。     

一起雪卡毒素中毒事件中裸胸鳝毒性的研究

目的研究厦门一起引发食物中毒的裸胸鳝毒性及种类,以预防雪卡毒素中毒。方法对中毒事件进行流行病学调查,采集裸胸鳝样品共5份,采用小鼠生物法、雪卡毒素免疫膜检测试剂盒检测毒性大小,高效液相色谱-串联质谱法测定太平洋雪卡毒素(P-CTX-1),同时提取中毒鱼肉组织中的mtDNA,PCR技术扩增细胞色素b(Ctyb)后测序鉴定中毒鱼种。结果小鼠生物法试验检测5份样品,鱼肉毒性为无毒性(ND)～0.052 MU/g,内脏毒性为ND～0.23 MU/g;高效液相色谱-串联质谱法未检出P-CTX-1。5种鱼种经鉴定均为裸胸鳝属(Gymnothorax),其中中毒鱼种经基因测序为波纹裸胸鳝(Gymnothorax undulates)。结论引发该起食物中毒的是一种含有雪卡毒素的波纹裸胸鳝。     

Use of folk tests to detect ciguateric fish: a scientific evaluation of their effectiveness in Raivavae Island (Australes,French Polynesia)

Ciguatera fish poisoning is a seafood intoxication commonly afflicting island communities in the Pacific. These populations, which are strongly dependent on fish resources, have developed over centuries various strategies to decrease the risk of intoxication, including the use of folk tests to detect ciguateric fish. This study aims to evaluate the effectiveness of two folk tests commonly used in Raivavae Island (Australes, French Polynesia): the rigor mortis test (RMT) and the bleeding test (BT). A total of 107 fish were collected in Raivavae Lagoon, among which 80 were tested by five testers using the RMT versus 107 tested by four testers using BT. First, the performance between testers was compared. Second, the efficiency of these tests was compared with toxicity data obtained via the receptor binding assay (RBA) by assessing various parameter's values such as sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV). Comparisons of outcomes between folk tests and RBA analyses were considered: tests used separately or in a parallel versus the series approach by each tester. The overall efficiency of the RMT and BT tests was also evaluated when the judgments of all testers were “pooled”. The results demonstrate that efficiencies varied between testers with one showing the best scores in detecting toxic fish: 55% with RMT and 69.2% with BT. BT gave the best results in detecting toxic fish as compared with RMT, giving also better agreement between testers. If high NPV and Se values were to be privileged, the data also suggest that the best way to limit cases of intoxication would be to use RMT and BT tests in a parallel approach. The use of traditional knowledge and a good knowledge of risky versus healthy fishing areas may help reduce the risk of intoxication among communities where ciguatera fish poisoning is highly prevalent.     

Optimisation of the supercritical extraction of toxic elements in fish oil

This study aims to optimise the operating conditions for the supercritical fluid extraction (SFE) of toxic elements from fish oil. The SFE operating parameters of pressure, temperature, CO₂ flow rate and extraction time were optimised using a central composite design (CCD) of response surface methodology (RSM). High coefficients of determination (R²) (0.897–0.988) for the predicted response surface models confirmed a satisfactory adjustment of the polynomial regression models with the operation conditions. The results showed that the linear and quadratic terms of pressure and temperature were the most significant (p < 0.05) variables affecting the overall responses. The optimum conditions for the simultaneous elimination of toxic elements comprised a pressure of 61 MPa, a temperature of 39.8ºC, a CO₂ flow rate of 3.7 ml min^?1 and an extraction time of 4 h. These optimised SFE conditions were able to produce fish oil with the contents of lead, cadmium, arsenic and mercury reduced by up to 98.3%, 96.1%, 94.9% and 93.7%, respectively. The fish oil extracted under the optimised SFE operating conditions was of good quality in terms of its fatty acid constituents.     

石墨烯作为新型固相萃取填料结合UPLC-MS/MS测定黑鱼中13种磺胺类药残

建立了以石墨烯为固相萃取填料,结合高效液相色谱-电喷雾离子阱质谱联用技术测定黑鱼中磺胺类兽药残留的检测方法。采用甲醇作为提取溶剂,经石墨烯固相萃取柱净化富集,质谱采用电喷雾正离子源,多反应监测模式。方法的线性良好,相关系数大于0.998,检出限范围0.048⁰.288μg/kg,定量限为0.160⁰.960μg/kg,线性范围内的加标回收率为70.6%⁹3.8%。本方法灵敏、可靠、稳定,满足复杂水产品基质中磺胺类药残检测的需要。      

Solid-phase extraction (SPE)—a comparison of 16 materials for the purification of anthocyanins from aronia melanocarpa var Nero

There is an increasing interest in anthocyanins, not only as natural food colourants but also for pharmaceutical products due to their antioxidative potential. Common extraction procedures of anthocyanins from plant material are non-selective and yield pigment solutions with large amounts of by-products such as sugars, sugar alcohols, organic acids, amino acids and proteins. Some of these impurities may accelerate anthocyanin degradation or cause problems in further processing steps such as spray drying. In order to obtain a highly concentrated and purified anthocyanin pigment from black chokeberry (Aronia melanocarpa var Nero), juices and skin extracts were purified by solid-phase extraction (SPE). Sixteen solid-phase materials were tested on a laboratory scale, whereby the anthocyanin and sugar content of collected fractions were determined to represent elution profiles. Among these, reversed-phase silica gels and macroreticular non-ionic acrylic polymer adsorbents such as Serdolit PAD IV or Amberlite XAD-7 turned out to be most suitable. SPE was investigated with these materials in an enlarged scale, improving elution gradient and column purification. Amberlite XAD-7 was successfully applied in a 36-litre-scale separation. © 1998 Society of Chemical Industry.     

Differences in fatty acid composition of fish faeces as determined by two extraction methods

Chloroform-methanol, a supposedly efficient solvent system for lipid extraction, was applied to heterogeneous samples, respectively fish diets and fish faeces. Comparisons were made of the compositions of the fatty acids prepared from solvent-recovered lipids with those obtained by total saponification. These show that solvent extraction, followed by saponification prior to fatty acid methyl ester preparation, gives differences attributable to incomplete extraction. Direct sample saponification in ethanolic alkali is recommended if all fatty acids in the diet are of interest.     

Solid-phase extraction for selective determination of bisphenol A in drinks and fruits by dummy surface molecularly imprinted polymer with direct synthetic method

A reliable and selective method was developed for the determination of bisphenol A (BPA) in drinks and fruit using dummy surface molecularly imprinted polymer (DSMIP) as a solid-phase extraction (SPE)-enrichment and separation sorbent coupled with high-performance liquid chromatography (HPLC). Tetrabromobisphenol A (TBBPA), whose structure is similar to BPA, was selected as a dummy template molecule. DSMIP has a higher selectivity for BPA than surface non-imprinted polymer (SNIP) when used as sorbents for SPE. Potential factors affecting the extraction efficiency, including conditioning, sample loading, washing and elution, and the breakthrough volume were optimised. Under the optimum experimental conditions, the recoveries of BPA in drinks and fruit were in the range from 98% to 105% with relative standard deviations (RSDs) below 7%, and a limit of detection (LOD) of 3 ng ml^?1. The developed extraction protocol eliminated the effect of template leakage on quantitative analysis and could be applied to the trace determination of BPA in complicated functional samples.     

超声波提取-气相色谱法测定鱼类食品中的多氯联苯

采用超声波提取鱼肉样品,探索以下超声波条件对食品中多氯联苯提取效果的影响:溶剂用量、温度、超声功率、超声波提取时间.研究结果表明,最佳提取条件为:5g鱼肉样品用30mL正已烷提取,提取温度为35℃,超声波功率为90kHz,超声时间为120min.方法的回收率为40.7％～96.7％,平均为66.0％.当PCBs浓度在0.08μg/mL以下时,标准曲线的直线相关系数达到0.9966 (n=4).     

鱼体中两种挥发性有机残留物的提取与检测

目的 研究水蒸汽蒸馏方法提取鱼体中挥发酚和硝基苯的效果, 并研究鱼体中挥发酚和硝基苯残留量的测定方法。方法 用自制水蒸汽发生器蒸馏提取挥发酚和硝基苯, 并测定两种物质含量。结果 水蒸汽发生器蒸馏法泡沫产生少, 蒸馏过程稳定、容易控制, 不存在暴沸现象。鱼体中挥发酚测定平均回收率在85.5%以上, 硝基苯测定平均回收率为96.83%。硝基苯出峰时间为10.051 min, 分离度高, 检测时间短、消耗有机溶剂量少、试验简便, 完全能够满足检测分析的需要。 结论 水蒸汽蒸馏提取法效果好, 方法实用, 便于操作, 适用于处理后样品的分析测定, 已应用于鱼体中挥发酚和硝基苯残留量的测定。     

Dynamic microwave-assisted extraction coupled on-line with clean-up for determination of caffeine in tea

A rapid method based on dynamic microwave-assisted extraction (DMAE) coupled on-line with clean-up was developed for the determination of caffeine in tea samples. A TM₀₁₀ microwave resonance cavity was applied to concentrate the microwave energy. An extraction vessel was placed in microwave irradiation zone of the TM₀₁₀ microwave resonance cavity. A silica gel column connected with the extraction vessel was used to remove chlorophyll in tea. The extraction and clean-up procedures were carried out simultaneously in a single step. The DMAE parameters were optimized by the Box-Behnken design. The maximum extraction efficiency was achieved using 70 W of microwave power, 3.5 mL of extraction solvent and 1.0 mL min⁻¹ of extraction solvent flow rate. The limit of detection obtained is 0.012 mg g⁻¹. The RSDs of intra- and inter-day of eight kinds of tea samples are 2.7%-5.4% and 5.1%-7.3%, respectively. The recovery of caffeine in the tea samples is in the range of 88.2%-99.3%.The proposed method has selectivity and sensitivity and considerably less labor and time for determination of caffeine in tea compared with conventional extraction methods, such assoxhlet extractionand liquid-liquid extraction.     

固相萃取净化-高效液相色谱串联质谱法测定鱼肉制品中链霉素、双氢链霉素、卡那霉素的含量

本文建立了一种简单、快速测定鱼肉制品中链霉素、双氢链霉素、卡那霉素的固相萃取净化-液相色谱串联质谱的分析方法。样品经庚烷磺酸钠和磷酸盐缓冲溶液提取,C18固相萃取柱净化后,在ACQUITYUPLC BEH HILIC C18色谱柱上以乙腈和0.1%甲酸水溶液为流动相进行梯度洗脱分离,采用HPLC-MS/MS选择反应监测(SRM)模式测定进行定性、定量分析,质谱采集模式为电喷雾正离子监测模式。结果表明:在5.0~100μg/kg范围内,峰面积与浓度线性良好,相关系数均大于0.9992;方法的检出限(LOD)为2.5μg/kg,定量限(LOQ)为5.0μg/kg,检测结果的相对标准偏差为1.16%~7.35%(n=6),平均加标回收率达到77.4%~107.3%。该方法具有较高的重现性和选择性,适用于鱼肉制品中链霉素、双氢链霉素、卡那霉素的残留分析检测。     

Towards the standardisation of the neuroblastoma (neuro-2a) cell-based assay for ciguatoxin-like toxicity detection in fish: application to fish caught in the Canary Islands

The ouabain/veratridine-dependent neuroblastoma (neuro-2a) cell-based assay (CBA) was applied for the determination of the presence of ciguatoxin (CTX)-like compounds in ciguatera-suspected fish samples caught in the Canary Islands. In order to avoid matrix interferences the maximal concentration of wet weight fish tissue exposed to the neuro-2a cells was set at 20?mg tissue equivalent?(TE)?ml^?1 according to the sample preparation procedure applied. In the present study, the limit of quantification (LOQ) of CTX1B equivalents in fish extract was set at the limit of detection (LOD), being defined as the concentration of CTX1B equivalents inhibiting 20% cell viability (IC₂₀). The LOQ was estimated as 0.0096?ng CTX1B eq.?g?TE^?1 with 23–31% variability between experiments. These values were deemed sufficient even though quantification given at the IC₅₀ (the concentration of CTX1B equivalents inhibiting 50% cell viability) is more accurate with a variability of 17–19% between experiments. Among the 13 fish samples tested, four fish samples were toxic to the neuro-2a cells with estimations of the content in CTX1B?g^?1 of TE ranging from 0.058 (±0.012) to 6.23 (±0.713) ng CTX1B eq.?g?TE^?1. The high sensitivity and specificity of the assay for CTX1B confirmed its suitability as a screening tool of CTX-like compounds in fish extracts at levels that may cause ciguatera fish poisoning. Species identification of fish samples by DNA sequence analysis was conducted in order to confirm tentatively the identity of ciguatera risk species and it revealed some evidence of inadvertent misidentification. Results presented in this study are a contribution to the standardisation of the neuro-2a CBA and to the risk analysis for ciguatera in the Canary Islands.     

固相萃取-气相色谱法同时检测草莓中13种 农药残留

目的 建立一种固相萃取-毛细管柱气相色谱方法,可以同时检测草莓中13种农药残留量。方法 草莓样品匀浆后,经乙腈提取,NH₂固相萃取柱净化,采用HP-5毛细管气相色谱柱进行分离,GC-ECD 进行定性及定量分析。结果 13 种农药残留的色谱图分离效果良好,线性相关系数均大于0.996,方法检出限在0.01 mg.kg_-1~0.5 mg.kg_-1之间。添加回收试验表明,该方法平均回收率在70.5~114.5 % 之间,相对标准偏差在2.17~6.85 %之间。结论 该方法简单、快速、灵敏、净化效果好、回收率高,适合草莓中多种农药残留的检测和安全监控。通过对50份草莓样品进行检测,检出百菌清、乙草胺、毒死蜱、粉唑醇、醚菌酯、烯酰吗啉6种农药。     

分散固相萃取技术在动物源性食品兽药残留检测中的应用进展

高效的样品前处理是实现准确分析的前提条件。分散固相萃取(dispersive solid-phase extraction,DSPE)是一种通过将固体吸附剂分散在液体分析物中,从复杂基质中分离和净化不同的分析物的方法。这种方法具有高效、快速、选择性强、易操作等优点,已经被广泛应用于环境、食品、药物、生物等领域的样品前处理中。本文首先对DSPE做了概述,然后以DSPE的设计原理为切入点,阐述固体吸附剂的不同分散方式、固体吸附剂的种类和性质对样品前处理过程的影响。随着纳米科技的蓬勃发展,尤其是磁性纳米材料、分子印迹聚合物、碳质材料及金属有机框架化合物等新材料的发现为DSPE注入了新的活力。本文重点讨论了这几种新材料在动物源性食品兽药残留方面的应用情况,并对DSPE的未来发展方向进行了预测,以期为相关技术人员提供新的思路。