Determination of domoic acid toxins in shellfish by biosense ASP ELISA--a direct competitive enzyme-linked immunosorbent assay: collaborative study

A collaborative study was conducted on the Biosense amnesic shellfish poisoning (ASP) enzyme-linked immunosorbent assay (ELISA) for the determination of domoic acid (DA) toxins in shellfish in order to obtain interlaboratory validation data for the method. In addition, a method comparison study was performed to evaluate the ASP ELISA as an alternative to the current liquid chromatography (LC) reference method for DA determination. The study material comprised 16 shellfish samples, including blue mussels, Pacific oysters, and king scallops, spiked with contaminated mussel homogenates to contain 0.1-20 mg DA/kg shellfish flesh. The shellfish samples were extracted with 50% aqueous methanol, and the supernatants were directly analyzed. Sixteen participating laboratories in 10 countries reported data from the ASP ELISA, and 4 of these laboratories also reported data from instrumental LC analysis. The participating laboratories achieved interlaboratory precision estimates for the 8 Youden paired shellfish samples in the range of 10-20% for RSD(r) (mean 14.8 +/- 4%), and 13-29% for RSDR (mean 22.7 +/- 6%). The precision estimates for the ELISA data did not show a strong dependence on the DA concentration in the study samples, and the overall precision achieved was within the acceptable range of the Horwitz guideline with HorRat values ranging from 1.1 to 2.4 (mean HorRat 1.7 +/- 0.5). The analysis of shellfish samples spiked with certified reference material (CRM)-ASP-MUS-b gave recoveries in the range of 88-122%, with an average recovery of 104 +/- 10%. The estimate on method accuracy was supported by a correlation slope of 1.015 (R2 = 0.992) for the determined versus the expected DA values. Furthermore, the correlation of the ASP ELISA results with those for the instrumental LC analyses of the same sample extracts gave a correlation slope of 1.29 (R2 = 0.984). This indicates some overestimation of DA levels in shellfish by the ELISA, but it is also a result of apparent low recoveries for the LC methods. This interlaboratory study demonstrates that the ASP ELISA is suitable for the routine determination and monitoring of DA toxins in shellfish, and that it offers a rapid and cost-effective methodology with high sample throughput.     

High-throughput analysis of amnesic shellfish poisoning toxins in shellfish by ultra-performance rapid resolution LC-MS/MS

The application of ultra-performance rapid resolution LC on a 1.8 microm particle-size column coupled with tandem MS (RRLC-MS/MS) is described for the analysis of amnesic shellfish poisoning (ASP) toxins in shellfish. Complete resolution among domoic acid (DA) and the isomers was achieved in less than 3 min. The method was intralaboratory validated for direct analysis of crude extracts without further cleanup. It showed LODs ranging from 0.05 to 0.09 mg/kg and a working range that complied with the current regulatory level for DA of 20 mg/kg, and with the level of 4.5 mg/kg recently proposed by the European Food Safety Authority. Confirmatory capabilities were demonstrated according to the Commission Decision 2002/657/EC criteria. The results obtained by RRLC-MS/MS agreed with those provided by the reference LC-UV method, both intralaboratory for the analysis of blind samples (R2 = 0.9751) and interlaboratory through participation in the proficiency test for ASP toxins during 2009 (z-score = -0.962 and 0.177 for low- and high-contaminated samples, respectively). RRLC-MS/MS provided fast analysis and additional confirmatory capabilities for direct analysis of crude extracts while the performance and reliability of the results were maintained, even in very complex matrixes.     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.     

New fluorimetric method of liquid chromatography for the determination of the neurotoxin domoic acid in seafood and marine phytoplankton

Domoic acid (DA) is a neurotoxic amino acid that is responsible for the human toxic syndrome, amnesic shellfish poisoning (ASP). A new rapid, sensitive liquid chromatographic (LC) method has been developed for the determination of DA in various marine samples. DA in marine biological materials was derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and analysed using isocratic reversed-phase LC with fluorimetric detection. The calibration, based on standard DA solutions, was linear in the range 0.04-2 microg/ml (r2=0.998) and the detection limit (3:1, signal/noise) was better than 1 ng/ml. Using the certified reference material (MUS-1B), recoveries of DA from shellfish tissue were >95% (n=5). When a strong anion exchange SPE cartridge was used for sample clean-up the detection limit was 6 ng DA/g mussel tissue. Good reproducibility was achieved with RSD values ranging from 3% for 8 microg DA/g (n=5), to 5% for 0.04 microg DA/g (n=5). This new method was successfully applied to the determination of DA in naturally contaminated shellfish and in marine phytoplankton cultures of Pseudonitzschia sp.     

石墨烯-管尖固相萃取-液相色谱-串联质谱法测定贝类中10种脂溶性贝类毒素

以石墨烯为吸附剂，制作了石墨烯-管尖固相萃取装置，结合液相色谱-串联质谱，建立了一种同时测定贝类中10种脂溶性贝类毒素的方法。实验对提取剂、石墨烯的用量、淋洗剂的种类和用量、洗脱剂的种类和用量等实验参数进行了详细优化。在最优的实验条件下，10种脂溶性贝类毒素在各自相应浓度范围内线性良好，相关系数均大于0.99，方法检出限（LOD）和定量限（LOQ）分别在0.1~1.1 μg/kg和0.3~3.2 μg/kg之间；对阴性牡蛎样品进行3个水平的加标回收实验，10种脂溶性贝类毒素的回收率在72.0%~101.2%之间，相对标准偏差小于15%。结果表明，该方法灵敏度高，操作简单高效，适用于贝类水产品中脂溶性贝类毒素的检测分析。     

Determination of brevetoxins in shellfish by LC/MS/MS: single-laboratory validation

A single-laboratory validation is reported for an LC/MS/MS quantification of six brevetoxins in four matrixes (Greenshell mussel, eastern oyster, hard clam, and Pacific oyster). Recovery and precision data were collected from seven analytical batches using shellfish flesh at 0.05 mg/kg. Method recoveries and within-laboratory reproducibility ranged from 73 to 112%, with an RSD between 14 and 18% for brevetoxin-3, brevetoxin B5, brevetoxin B2, and S-desoxy brevetoxin B2. The recovery and within-laboratory reproducibility for brevetoxin-2 was 61%, with an RSD of 27%. Brevetoxin B1 gave an RSD of 12%, but no reference material was available and this toxin was only recorded in a hard clam sample naturally contaminated with brevetoxins. One naturally contaminated sample of each shellfish matrix, with brevetoxin levels ranging from 0.012 to 9.9 mg/kg, was tested in multiple batches, and the RSDs were similar to those for fortified samples at 0.05 mg/kg. Comparisons with limited data for the neurotoxic shellfish poisoning mouse bioassay for four naturally contaminated shellfish samples showed that the regulatory action limit of 0.8 mg/kg is conservative with respect to the bioassay regulatory limit of 20 mouse units/100 g.     

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.     

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.     

Development of a pressurised liquid extraction and liquid chromatography with electrospray ionization-tandem mass spectrometry method for the determination of domoic acid in shellfish

Amnesic shellfish poisoning (ASP) is a potentially lethal human toxic syndrome which is caused by domoic acid (DA) that originates in marine phytoplankton belonging to the Pseudonitzschia genus. A confirmatory and sensitive procedure has been developed and validated for the determination of DA in shellfish. The proposed method includes pressurised liquid extraction (PLE) with methanol/acetone (9:1), florisil clean-up purification inside the PLE extraction cell and detection by liquid chromatography (LC) coupled to electrospray ionization in positive mode tandem mass spectrometry (ESI-MS-MS). Comparison of ionization sources (ESI, atmospheric pressure ionization (APCI) atmospheric pressure photoionization (APPI) and combined APCI/APPI) were carried out in order to improve the analytical signal. The main parameters affecting the performance of the different ionization sources and PLE parameters were previously optimised using statistical design of experiments (DOE). Linear calibrations were obtained using mussel tissue extracts 0.05-5 microg DA/ml (R2>0.999). The limits of detection (LOD) and quantitation (LOQ) of the method were 0.2 and 0.5 microg/g respectively and recoveries ranged from 81 to 95%. This method was successfully applied to determine DA levels in 46 shellfish samples collected from Valencian (Spain) supermarkets, showing high sample throughput.     

液相色谱-串联质谱法同时测定贝类中大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素

建立了同时测定贝类中大田软海绵酸(okadaic acid, OA)及其衍生物鳍藻毒素(dinophysistoxin-1, DTX-1)、蛤毒素(pectenotoxin-2, PTX-2)和虾夷扇贝毒素(yessotoxin, YTX)的液相色谱-串联质谱分析方法。样品经甲醇提取,固相萃取柱净化,C18色谱柱分离,经含甲酸和甲酸铵的乙腈-水溶液为流动相梯度洗脱,选择反应监测(SRM)模式检测,正、负离子切换扫描,基质标准校正,外标法定量。结果表明,OA、DTX-1和YTX的线性范围为2.0～200.0 μg/L,定量限(以信噪比(S/N)≥10计)为1.0 μg/kg; PTX-2的线性范围为1.0～100.0 μg/L,定量限为0.5 μg/kg;几种化合物的添加平均回收率为83.1%～105.7%,相对标准偏差(RSD)为3.16%～9.29%。成功应用本法对黄海灵山湾海域采集的贝类样品进行了分析,发现部分样品中含有大田软海绵酸、鳍藻毒素、蛤毒素和虾夷扇贝毒素。     

Proficiency testing of eight French laboratories in using the AOAC mouse bioassay for paralytic shellfish poisoning: interlaboratory collaborative study

In an interlaboratory study, 8 French laboratories were tested for their proficiency in using the AOAC mouse bioassay for paralytic shellfish poisoning (PSP). Each laboratory received 1 saxitoxin (STX) standard solution, 1 STX acidified water solution for determination of the titer, 1 noncontaminated shellfish sample, 1 naturally contaminated shellfish sample, and 2 shellfish samples spiked, respectively, at low (152.8 microg STX/100 g meat) and moderate (334.7 microg STX/100 g meat) levels. All samples were analyzed in duplicate. Mean recoveries were 35.1% for the low level and 46.6% for the moderate level. Relative standard deviations (RSD) for within-laboratory variations (repeatability) ranged from 5.4 to 9.8%; RSD for between-laboratory variations (reproducibility) varied from 7.8 to 39.6%, depending on STX level. On the basis of overall performance, all 8 participating laboratories were proficient in their use of the AOAC mouse bioassay.     

Liquid chromatographic method to determine narasin in feedingstuffs and premixtures: development, validation, and interlaboratory study

A reversed-phase liquid chromatography (LC) method for narasin in feedingstuffs and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was methanol-K2HPO4 solution (9 + 1, v/v). Narasin was detected at 600 nm after post-column derivatization with dimethylamino-benzaldehyde. Recovery was >90%. The repeatability (RSDr) in feed (20-140 mg/kg) ranged between 1.2 and 10.5%; the within-laboratory reproducibility (RSD(R)) ranged between 2.2 and 4.9%. The limit of determination was <20 mg/kg. Other feed additives did not interfere in the assay. The method showed ruggedness against changes in the composition of extraction solvent, eluent, and conditions for post-column reactions. In an interlaboratory study, 5 broiler feeds (4 positive, 1 blank) and 1 premixture were analyzed by 13 laboratories. The RSDr of the feedingstuffs (20-120 mg/kg) varied between 2.17 and 7.57%. The HORRAT ranged between 0.77 and 0.88, with recoveries between 82 and 104%. One laboratory detected small signals in the blank sample, calculated as 0.6 and 2.8 mg/kg. For the premixture, acceptable results for reproducibility could only be obtained after modification of the method: the RSDr was 4.42% and the HORRAT was 1.56 (12 laboratories).     

Improved high-performance liquid chromatographic method for the determination of domoic acid and analogues in shellfish: effect of pH

Domoic acid (DA) is a naturally-occurring amino acid that causes a form of human intoxication called amnesic shellfish poisoning (ASP) following the consumption of shellfish. A rapid and sensitive HPLC-UV method has been developed for analysis of DA and analogues in shellfish without the need for SPE clean-up. Isocratic chromatographic separation of DA and its isomers from shellfish matrix interferences and from the prevalent amino acid, tryptophan, was achieved by careful control of the mobile phase pH. The optimised pH was found to be 2.5 when using a Luna(2) C₁₈ column. Sample extraction was verified with control extracts from shellfish spiked at 5.0 and 10.0 g/g of DA and with certified reference material. The average extraction efficiency was 98.5%. The calibration, based on mussel tissue spiked with DA standard, was linear in the range 0.05–5.0 g/ml (r=0.9999) and the detection limit (signal:noise 3:1) was better than 25 ng/ml. The DA assay achieved good precision; %RSD=1.63 (intra-day, n=6) and %RSD=3.7 (inter-day, n=8). This method was successfully applied to a variety of shellfish species, allowing the rapid screening of a large number of samples per day (20–30), without the need for SPE clean-up. Quantitative data were obtained for shellfish samples containing domoic acid in the concentration range 0.25–330 g/g. Using the same chromatographic conditions, LC-MS³ was used to determine DA and its isomers, isodomoic acid D and epi-domoic acid, in scallop tissues.     

Extension of the validation of AOAC Official Method 2005.06 for dc-GTX2,3: interlaboratory study

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.     

Liquid chromatography post-column oxidation (PCOX) method for the determination of paralytic shellfish toxins in mussels, clams, oysters, and scallops: collaborative study

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official Method 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.     

Gliadin as a measure of gluten in foods containing wheat, rye, and barley-enzyme immunoassay method based on a specific monoclonal antibody to the potentially celiac toxic amino acid prolamin sequences: collaborative study

The Working Group on Prolamin Analysis and Toxicity (WGPAT) organized a collaborative study to confirm whether the two R5 antibody-based ELISA test kits are able to detect gliadin in the lower mg/kg (ppm) level. Twenty laboratories investigated 12 blind-coded samples, spiked and naturally contaminated, to show the possibility of determining traces of gliadin in heat-treated or nonheat-treated foods by ELISA. It was shown that very small amounts of gliadin (below 100 ppm) could be detected by ELISA with a reproducibility RSD(R) (37%) and a repeatability RSD, (27%) common for ELISA under these conditions. The recovery of gliadin from the spiked samples was between 84 and 109%, based on the results of all laboratories, including those with poor performance. No false positives were found by the method (P < or =0.05), but one negative sample was contaminated during the bakery process. It is recommended that the method be accepted by AOAC as Official First Action.     

Integrated enzyme-linked immunosorbent assay screening system for amnesic, neurotoxic, diarrhetic, and paralytic shellfish poisoning toxins found in New Zealand

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.     

Determination of petroleum contamination in shellfish using solid phase micro-extraction with gas chromatography-mass spectrometry

Headspace solid phase micro-extraction (HS-SPME) has been applied as a sampling technique for the determination of petroleum contamination in shellfish using capillary gas chromatography-mass spectrometry (GC-MS). A poly(dimethylsiloxane) fused silica fibre (100 microm thickness) was found to be satisfactory for the extraction of a range of aliphatic hydrocarbons (HCs) from homogenised shellfish tissues. The SPME conditions, including temperature, salt content, extraction time and desorption temperature, were optimised for a range of aliphatic HCs (C9-C20). A methyl silicone column GC (12 m x 0.20 mm, 0.33 microm layer thickness) was used with a temperature programme from 40 to 260 degrees C and the HCs were determined within a mass range of m/ z=50-550 in electron impact mode. Calibration range was from 10 to 5000 ng/g with linear correlation coefficients ( r(2)) of 0.982 for nonane to 0.997 for octadecane. Detection limits for aliphatic HCs, spiked into shellfish (mussel) tissues, varied from 3.6 ng/g (tetradecane) to 51 ng/g (eicosane) and relative standard deviation (% RSD) values ranged from 1.4% (hexadecane) to 24.3%(eicosane).     

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。