A new method for rapid and quantitative detection of the Bacillus cereus emetic toxin cereulide in food products by liquid chromatography-tandem mass spectrometry analysis

The Bacillus cereus emetic toxin cereulide causes foodborne intoxication, which may occasionally result in severe disease, and even death. To differentially diagnose the emetic-type of foodborne disease caused by B. cereus and assess the safety of commercial food, we developed a rapid method to quantitate cereulide. This method was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for the extraction of cereulide from food using a normal-phase silica gel cartridge. The limits of detection and quantification were 0.1 and 0.5 ng of cereulide ml⁻¹, respectively. Spiked cereulide was reproducibly recovered with over 67% efficiency from nine diverse foods implicated in cereulide food poisoning. The recovery rate, reproducibility, and intermediate precision for this single laboratory validation using boiled rice were 87.1%, 4.4%, and 7.0%, respectively. Further, we detected a wide range of cereulide concentrations in leftover food and vomitus samples from two emetic foodborne outbreaks. LC-MS/MS analysis correlated closely with those acquired using the HEp-2 cell assay, and quantitated cereulide from 10 food samples at least five times faster than the bioassay. This new method will provide clinicians with an improved tool for more rapidly and quantitatively determining the presence of cereulide in food and diagnosing food poisoning caused by cereulide.     

郴州市一起灰花纹鹅膏中毒事件调查与分析

目的 调查2020年6月湖南省郴州市发生的一起毒蘑菇中毒事件,分析事件发生的原因,为制定防控措施提供依据。方法 对中毒原因开展流行病学调查,采集可疑毒蘑菇样品并进行形态学鉴定,同时应用液质联用法测定蘑菇样品和患者血液中的蘑菇毒素。结果 本次毒蘑菇中毒事件发病7人,死亡1人;患者均进食了自行采摘的野生蘑菇,潜伏期14～23 h,早期临床表现为呕吐、腹痛、腹泻等胃肠道症状,进而出现进行性肝损害,严重者因急性肝衰竭而死亡;患者血液生化检测丙氨酸氨基转移酶、门冬氨酸氨基转移酶、乳酸脱氢酶异常升高;采集现场剩余可疑毒蘑菇样品,经外观形态学鉴定和蘑菇毒素检测,综合鉴定为灰花纹鹅膏。结论 本次事件是因误采误食灰花纹鹅膏引起的中毒事件,建议加大毒蘑菇中毒宣传力度,建立蘑菇毒素快速筛查技术,完善食源性疾病监测网络,以减少毒蘑菇中毒和死亡的发生。     

甘肃省2004—2011年食物中毒事件流行特征分析

目的:分析2004—2011年甘肃省突发并网络报告的食物中毒事件,为预防食物中毒事件发生提供相关依据。方法:对2004—2011年甘肃省网络报告的食物中毒事件进行描述性流行病学分析。结果:2004—2011年甘肃省共报告食物中毒事件73起,中毒2 311例、死亡53例,病死率为2.3%。事件发生时间主要集中在4—10月,其中9月中毒人数最多。致病因素中微生物性食物中毒为30起、植物性和动物性13起、化学性为25起;致死人数较多的因素依次为农药/鼠药(30例)、毒蘑菇(7例)、肉毒毒素(4例)、亚硝酸盐(4例)和其它(8例)。结论:报告的食物中毒事件中,微生物所导致食物中毒仍是较大的食品安全问题;而农药/鼠药中毒和蘑菇中毒是导致食物中毒事件中的主要死亡原因。农村是食物中毒的防范重点。     

Azaspiracid shellfish poisoning: unusual toxin dynamics in shellfish and the increased risk of acute human intoxications

A number of recent acute human intoxications in Europe from the consumption of Irish mussels have been attributed to the presence of a new class of toxins named azaspiracids. The study demonstrates that azaspiracids behave differently from other polyether toxins, and this accounts for most false-negative results in the mouse bioassay employed by regulatory agencies to detect azaspiracids. Typically, polyether toxins are concentrated in the digestive glands of shellfish, but this is not always the situation with azaspiracids. Liquid chromatography-mass spectrometry (LC-MS), especially multiple tandem MS methods, have been applied to demonstrate that azaspiracid (AZA1) and its methyl- and demethyl- analogues, AZA2 and AZA3 respectively, are distributed throughout shellfish tissues. Using conventional mouse bioassay protocols, only 0-40% of the total azaspiracid content of shellfish was used in the assay, which could directly account for false-negative results. It was also observed that the toxin profiles differed significantly in various mussel tissues with AZA1 as the predominant toxin in the digestive glands and AZA3 predominant in the remaining tissues.     

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.     

Detection of the hepatotoxic microcystins in 36 kinds of cyanobacteria Spirulina food products in China

Gel filtration chromatography, ultra-filtration, and solid-phase extraction silica gel clean-up were evaluated for their ability to remove microcystins selectively from extracts of cyanobacteria Spirulina samples after using the reversed-phase octadecylsilyl ODS cartridge for subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The reversed-phase ODS cartridge/silica gel combination were effective and the optimal wash and elution conditions were: H(2)O (wash), 20% methanol in water (wash), and 90% methanol in water (elution) for the reversed-phase ODS cartridge, followed by 80% methanol in water elution in the silica gel cartridge. The presence of microcystins in 36 kinds of cyanobacteria Spirulina health food samples obtained from various retail outlets in China were detected by LC-MS/MS, and 34 samples (94%) contained microcystins ranging from 2 to 163 ng g(-1) (mean = 14 +/- 27 ng g(-1)), which were significantly lower than microcystins present in blue green alga products previously reported. MC-RR - which contains two molecules of arginine (R) - (in 94.4% samples) was the predominant microcystin, followed by MC-LR - where L is leucine - (30.6%) and MC-YR - where Y is tyrose - (27.8%). The possible potential health risks from chronic exposure to microcystins from contaminated cyanobacteria Spirulina health food should not be ignored, even if the toxin concentrations were low. The method presented herein is proposed to detect microcystins present in commercial cyanobacteria Spirulina samples.     

酶联免疫法与液相色谱-串联质谱法检测猪肉中沙丁胺醇

运用酶联免疫法（ELISA）与液相色谱-串联质谱法（LC-MS/MS）对猪肉中沙丁胺醇残留进行测定,并比较了二者在灵敏度、准确性和重现性等方面的差异。结果显示,ELISA法和LC-MS/MS法在猪肉样品中的检测限可达到0.5 μg/kg和0.25 μg/kg,分别向猪肉样品中添加3个质量浓度水平（1.0 μg/kg、2.0 μg/kg、4.0 μg/kg）的沙丁胺醇时,回收率分别为83.7%～90.9%和86.6%～93.5%,变异系数（CV）分别为5.4%～10.3%和6.0%～8.3%。用酶联免疫法实际样品进行检测,筛选出2个阳性样品,经液相色谱-串联质谱法确证亦为阳性,测定结果一致。研究表明,酶联免疫法重复性较好、准确度较高,适用于进行猪肉样品中沙丁胺醇的快速筛选,液相色谱-串联质谱法适用于阳性样品的确证和精确定量。     

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml^–1; R² ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g^–1; R² ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml^–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.     

Simultaneous determination of aconitine analogues in Aconitum plants and foods that caused food poisoning by liquid chromatography with tandem mass spectrometry

A simple method for the simultaneous determination of four aconitine analogues (AC; aconitine, HA; hypaconitine, MA; mesaconitine, JA; jesaconitine) in Aconitum plants (Aconitum subcuneatum NAKAI) and a food that caused food poisoning was developed, using liquid chromatography tandem mass spectrometry (LC/MS/MS). Aconitine analogues were extracted with 1 mmol/L HCl and then cleaned up with an Oasis HLB cartridge. The LC separation was performed with an octadecylated silica column (Develosil ODS-HG-5, 2.0 mm i.d. x 50 mm) at a flow rate of 0.2 mL/min, using A solution (5 mmol/L ammonium acetate dissolved in 0.1% acetic acid) and B solution (acetonitrile-THF=1 : 3), 90%A (0 min)-->60%A (15 min)-->const. (2 min). Mass spectral acquisition was performed in the positive mode and the analogues were targeted using multiple reaction monitoring (MRM) with electrospray ionization (ESI). The recoveries of aconitine analogues were 93-99% from Aconitum plants. The detection limits of AC, HA, MA and JA were 0.4, 0.4, 0.3 and 0.5 ng/g, respectively. The aconitine analogues, except JA, were detected in food that caused food poisoning at the level of 2.6-29.7 microg/g. These results indicate that the developed method is suitable for the determination of aconitine analogues in Aconitum plants and foods that cause food poisoning.     

Detection of the hepatotoxic microcystins in 36 kinds of cyanobacteria Spirulina food products in China

Gel filtration chromatography, ultra-filtration, and solid-phase extraction silica gel clean-up were evaluated for their ability to remove microcystins selectively from extracts of cyanobacteria Spirulina samples after using the reversed-phase octadecylsilyl ODS cartridge for subsequent analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The reversed-phase ODS cartridge/silica gel combination were effective and the optimal wash and elution conditions were: H₂O (wash), 20% methanol in water (wash), and 90% methanol in water (elution) for the reversed-phase ODS cartridge, followed by 80% methanol in water elution in the silica gel cartridge. The presence of microcystins in 36 kinds of cyanobacteria Spirulina health food samples obtained from various retail outlets in China were detected by LC-MS/MS, and 34 samples (94%) contained microcystins ranging from 2 to 163 ng g^?1 (mean?=?14?±?27 ng g^?1), which were significantly lower than microcystins present in blue green alga products previously reported. MC-RR?–?which contains two molecules of arginine (R)?–?(in 94.4% samples) was the predominant microcystin, followed by MC-LR?–?where L is leucine?–?(30.6%) and MC-YR?–?where Y is tyrose?–?(27.8%). The possible potential health risks from chronic exposure to microcystins from contaminated cyanobacteria Spirulina health food should not be ignored, even if the toxin concentrations were low. The method presented herein is proposed to detect microcystins present in commercial cyanobacteria Spirulina samples.     

Caribbean ciguatoxin profile in raw and cooked fish implicated in ciguatera

A cooked meal remnant and uncooked portion of a Caribbean barracuda suspected in ciguatera fish poisoning were examined for the presence of ciguatoxins (CTX). Samples were analysed using a tiered method of CTX analysis consisting of in vitro cell (N2a) assay to assess composite toxicity and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for structural confirmation. Meal remnant and uncooked fish extracts were cytotoxic by N2a cell assay and Caribbean ciguatoxin congener C-CTX-1 was structurally confirmed. Sample extracts were fractionated by LC and fractions analysed by the cell assay. The cytotoxicity profiles of cooked meal remnant and uncooked fish were similar. Cytotoxicity-guided LC-MS/MS analyses identified several CTX congeners contributing to the composite toxicity of the samples. C-CTX-1 was a major contributor, supporting its utility as a biomarker of Caribbean ciguatoxic fish.     

Occurrence of eight bisphenol analogues in indoor dust from the United States and several asian countries: implications for human exposure

Bisphenol A has been reported to be a ubiquitous contaminant in indoor dust, and human exposure to this compound is well documented. Information on the occurrence of and human exposure to other bisphenol analogues is limited. In this study, eight bisphenol analogues, namely 2,2-bis(4-hydroxyphenyl)propane (BPA), 4,4'-(hexafluoroisopropylidene)diphenol (BPAF), 4,4'-(1-phenylethylidene)bisphenol (BPAP), 2,2-bis(4-hydroxyphenyl)butane (BPB), 4,4'-dihydroxydiphenylmethane (BPF), 4,4'-(1,4-phenylenediisopropylidene)bisphenol (BPP), 4,4'- sulfonyldiphenol (BPS), and 4,4'-cyclohexylidenebisphenol (BPZ), were determined in indoor dust samples (n = 156) collected from the United States (U.S.), China, Japan, and Korea. Samples were extracted by solid-liquid extraction, purified by automated solid phase extraction methods, and determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total concentrations of bisphenols (∑BPs; sum of eight bisphenols) in dust were in the range of 0.026-111 μg/g (geometric mean: 2.29 μg/g). BPA, BPS, and BPF were the three major bisphenols, accounting for >98% of the total concentrations. Other bisphenol analogues were rare or not detected, with the exception of BPAF, which was found in 76% of the 41 samples collected in Korea (geometric mean: 0.0039 μg/g). The indoor dust samples from Korea contained the highest concentrations of both individual and total bisphenols. BPA concentrations in dust were compared among three microenvironments (house, office, and laboratory). The estimated median daily intake (EDI) of ∑BPs through dust ingestion in the U.S., China, Japan, and Korea was 12.6, 4.61, 15.8, and 18.6 ng/kg body weight (bw)/day, respectively, for toddlers and 1.72, 0.78, 2.65, and 3.13 ng/kg bw/day, respectively, for adults. This is the first report on the occurrence of bisphenols, other than BPA, in indoor dust.     

Tetrodotoxin poisoning due to smooth-backed blowfish Lagocephalus inermis and toxicity of L. inermis caught off the Kyushu coast, Japan

Food poisoning due to ingestion of a puffer fish occurred in Nagasaki Prefecture, Japan, in October 2008, causing neurotoxic symptoms similar to those of tetrodotoxin (TTX) poisoning. In the present study, we identified the species, toxicity, and toxins using the remaining samples of the causative puffer fish. The puffer fish was identified as smooth-backed blowfish Lagocephalus inermis by nucleotide sequence analysis of the 16S rRNA and cytochrome b gene fragments of muscle mitochondrial DNA. The residual liver sample showed toxicity as high as 1,230 mouse unit (MU)/g by bioassay and TTX was detected by liquid chromatography/mass spectrometry analysis. We therefore concluded that the food poisoning was due to TTX caused by consumption of the toxic liver of L. inermis. This is the first report that the liver of L. inermis caught in Japanese waters is strongly toxic, with levels exceeding 1,000 MU/g. In this context, we re-examined the toxicity of L. inermis collected off the coast of Japan. Of 13 specimens assayed, 12 were toxic, although the toxicity varied markedly among individuals and tissues. Because the intestine and ovary of L. inermis have been considered non-toxic, it is particularly noteworthy that these organs were determined to be toxic, with a maximum toxicity of 43.6 MU/g and 10.0 MU/g, respectively. Furthermore, kidney, gallbladder, and spleen, whose toxicity has been unknown, were frequently found to be weakly toxic with levels ranging from 10 to 99 MU/g. Therefore, further study is needed to re-examine the toxicity of smooth-backed blowfish L. inermis in the coastal waters of Japan.     

Mycotoxins in infant/toddler foods and breakfast cereals in the US retail market

The objective of this study was to conduct a mycotoxin survey of commercial infant/toddler foods (cereals and teething biscuits) and breakfast cereals in the United States. A total of 215 retail samples were collected from three geographical locations and analysed for aflatoxins, fumonisins, deoxynivalenol, HT-2 toxin, ochratoxin A, T-2 toxin, and zearalenone using a stable isotope dilution liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. One or more mycotoxins were found in 69% (101/147) of the infant/toddler foods and 50% (34/68) of breakfast cereals. Mycotoxin co-occurrence was observed in 12% of infant/toddler foods and 32% of breakfast cereals. However, the concentrations of detected mycotoxins were lower than the current FDA action and guidance levels. Aflatoxins and HT-2 toxin were not detected in any of the samples, while deoxynivalenol was the most frequently detected mycotoxin. Rice-based cereals appeared to be less susceptible to mycotoxin contamination than other cereal types.     

Diarrhetic shellfish poisoning toxin esters in Danish blue mussels and surf clams

Until recently, little focus was given to the presence of diarrhetic shellfish poisoning (DSP) toxin esters in seafood products. However, during the last few years, the occurrence of a high percentage of esters of the total amount of DSP toxins present in some seafood products has been observed. Samples of Danish surf clams (Spisola spp.) and blue mussels (Mytilus edulis) from 1999-2004 were analysed by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the presence of DSP toxin esters. The samples contained only okadaic acid and esters of okadaic acid. The level of total okadaic acid equivalents ranged from 224 to 2516 µg kg^-1 in surf clams. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 83 to 98%, mean 95%. The level of total okadaic acid equivalents ranged from 43 to 1631 µg kg^-1 in blue mussels. The percentage of okadaic acid esters of the total okadaic acid equivalents ranged from 21 to 86%, mean 59%. The probability of a high percentage of okadaic acid esters seems to increase with higher amounts of total okadaic acid equivalents in the bivalves. The large prevalence of DSP toxin esters are of particular importance because of the increased use of chemical methods instead of mouse bioassay for the detection of DSP toxicity.     

测定肉串源性成分的液相色谱-串联质谱与聚合酶链式反应方法对比研究

采用液相色谱-串联质谱（liquid chromatography-tandem mass spectrometry,LC-MS/MS）与聚合酶链式反应（polymerase chain reaction,PCR）方法分别对北京市场中的200 个烤肉串样品进行源性成分分析。其中PCR方法按照出入境标准进行操作,LC-MS/MS方法为自建方法,原理是基于测定物种的特异性多肽链进行物种鉴别。结果表明：2 种检测方法所得到的结果一致,肉串总体掺假比例为21%,其中羊肉串掺假比例为28%,牛肉串掺假比例为14%,主要掺假物种为猪和鸭;通过大批量样品实验对比,LC-MS/MS方法的准确性更高,实验操作也更为简便、耗时短、成本低,可用于日常食品的肉源性成分检测。     

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang,China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS ⁿ ). The toxicity of nassariid N. semiplicatus showed an ‘M’-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g^?1 of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.     

Toxin composition and toxicity dynamics of marine gastropod Nassarius spp. collected from Lianyungang, China

Consumption of nassariid gastropods often leads to poisoning incidents in some coastal provinces in China. To elucidate the pattern of toxicity dynamics and origin of toxins, samples of gastropod Nassarius spp. were collected from late May to early August 2007 from Lianyungang, Jiangsu province, where the poisoning incidents have been frequently reported. Toxicity was first screened with the mouse bioassay method, and tetrodotoxin and its analogues (TTXs) were analysed with high-performance liquid chromatography coupled with an ion-trap mass spectrometer (HPLC-MS(n)). The toxicity of nassariid N. semiplicatus showed an 'M'-shaped pattern of fluctuation during the sampling season. Two peaks of toxicity appeared in late May and late July. The maximum toxicity was recorded on 24 May, with the value of 846 mouse unit (MU) g(-1) of tissue (wet weight). TTX and its analogues trideoxyTTX, 4-epiTTX, anhydroTTX and oxoTTX were detected in the nassariid samples. TrideoxyTTX but not TTX was the major toxin in all the samples. No paralytic shellfish poison (PSP) was detected in the sample with the maximum toxicity by HPLC-FLD analysis. Variation of TTX content in the tissue of nassariid gastropods correlates well with the dynamics of toxicity. It is suggested that TTXs are the major toxins corresponding to the toxicity of the nassariids, and May and July are the high-risk seasons for consumption of nassariids, which is critical for the management of poisoning incidents.     

Identification of tetrodotoxin in a marine gastropod (Nassarius glans) responsible for human morbidity and mortality in Taiwan

The toxicity of the gastropod Nassarius glans was investigated. This gastropod was implicated in an incident of food paralytic poisoning on Tungsa Island, Taiwan, in April 2004. Six victims consumed both digestive glands and muscle. These tissues contained high concentrations of toxin; their highest toxicity scores were 2,048 and 2,992 MU/g, respectively, based on the tetrodotoxin (TTX) bioassay. The toxin was purified from these gastropods and analyzed by high-performance liquid chromatography, which revealed TTX and related compounds 4-epi TTX and anhydro-TTX; paralytic shellfish poisons were not found. The urine and blood samples from patients were cleansed using a C18 Sep-Pak cartridge column and 3,000 molecular weight cutoff Ultrafree microcentrifuge filters, and the eluate was filtered and analyzed by liquid chromatography and mass spectrometry. The detection limit for TTX was 1 ng/ml. The standard curves were linear in the range 30 to 600 ng/ml for urine and 1 to 30 ng/ml for blood. TTX was detected in all urine samples but in only three of four blood samples tested. Thus, the causative agent of gastropod food poisoning was identified as TTX.     

Monitoring phytoplankton and marine biotoxins in production waters of the Netherlands: results after one decade

Shellfish products may be contaminated with marine biotoxins which, after consumption, may lead to human illness. The Netherlands has a regular monitoring programme for marine biotoxins and the possible toxic phytoplankton in shellfish production waters. The aim of the current study was to evaluate the presence of potential toxic phytoplankton species and marine biotoxins in Dutch production waters over the last decade, and to analyse the relationship between toxin levels and abundance of possible causative phytoplankton species. The results of the monitoring programme of the period 1999-2009 were used. The presence of Alexandrium spp. were negligible, but Pseudo-nitzschia spp. and phytoplankton causing diarrhetic shellfish poisoning (DSP toxin-producing phytoplankton) were present in nearly all three main production areas and years. The main DSP toxin-producing species was Dinophysis acuminata followed by D. rotundata and Prorocentrum lima. Toxins causing paralytic shellfish poisoning (PSP) and amnesic shellfish poisoning (ASP) were present in only a few individual shellfish samples, all at low levels. At the end of 2002, an episode of DSP toxicity was recorded, based on the rat bioassay results. Of the samples that were chemically analysed for DSP toxins in 2007 and 2008, about half of the samples in 2007 contained these toxins, although levels were low and no positive results were obtained using the rat bioassay. There was a slight positive correlation between concentrations of DSP toxin-producing phytoplankton and levels of DSP toxins in 2007. Increased DSP toxin levels were found up to 5 weeks after the peak in DSP toxin-producing phytoplankton. This positive, but weak, relationship needs to be confirmed in future research using more samples and chemical methods to quantify the presence of DSP toxins. If this relationship is further substantiated and quantified, it could be used within the current monitoring programme in the Netherlands to predict the risk areas regarding DSP toxicity in shellfish.