Investigation of food products containing garlic or onion for a false positive sulphite response by LC-MS/MS.

In the US, sulphites must be declared on the label if they are present in concentrations greater than 10 mg/kg (determined as) SO2 because an allergic-like response has been reported in a small subset of the population upon consumption of sulphite-containing products. The most widely used method for sulphite determination, the optimised Monier-Williams (OMW), produces false positive results with vegetables from the Allium (garlic) and Brassica (cabbage) genera due to extraction conditions that are thought to cause endogenous sulphur compounds to release SO2. Recently, an LC-MS/MS method was developed for sulphites but has only been tested with samples that are 100% Allium or Brassica. Since regulatory samples may contain these vegetables as ingredients, additional investigations were necessary to determine the potential extent of false positives. Four blank matrices, chips, phyllo shells, hummus, and quinoa were spiked with various concentrations of onion and garlic powders. The sulphite concentrations were determined using an LC-MS/MS method. The matrix is extracted with a buffered formaldehyde solution, converting free and reversibly bound sulphite to the stable formaldehyde adduct, hydroxymethylsulfonate (HMS). It was determined that even at concentrations up to 8% garlic powder or 2% onion powder, the measured sulphite concentration was below the 10 mg/kg SO2 labelling threshold. Commercial dried garlic powders were evaluated to determine the variation in responses that might be encountered in future regulatory samples. Recovery studies were conducted to determine if these methods would detect added sulphite. The ability to eliminate false positives due to these ingredients will result in a greater reliability in the accurate determination of added sulphite to ensure compliance with labelling requirements.

Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit.

The cyanobacterium Aphanizomenon flos-aquae (AFA), from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA) dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 µg/g microcystin (MC) for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP) regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 µg/g. The objectives of this study were (1) identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2) use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA), based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51) distinct AFA-BGA products had MC ≥0.25 µg/g (the detection limit of the kit), 10 products had MC concentrations between 0.5 and 1.0 µg/g, and 4 products exceeded the limit (1.1-2.8 µg/g). LC-MS/MS confirmed PPIA results ≥0.5 µg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 µg/g MC, most were at or below 1.0 µg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC contamination found.

Surface plasmon resonance biosensor for detection of feline calicivirus, a surrogate for norovirus.

The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 10(4)TCID50FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.