Analysis of Aflatoxins, Fumonisins, Deoxynivalenol, Ochratoxin A, Zearalenone, HT-2, and T-2 Toxins in Animal Feed by LC-MS/MS Using Cleanup with a Multi-Antibody Immunoaffinity Column.

BACKGROUND: Regulations limiting aflatoxin levels in animal feed and guidance values for maximum levels for fumonisins (FB1 and FB2), deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZON), HT-2, and T-2 toxins are in place both to protect animal health and to minimize potential transfer to animal products for human consumption. A multi-mycotoxin method which can handle complex feed matrices such as distillers dried grains with solubles (DDGS) is essential for analysis and accurate quantification without the need to revert to separately analyze individual mycotoxins. OBJECTIVE: The objective of this study is to generate single laboratory validation data for a method employing a multi-antibody immunoaffinity column (IAC) capable of providing cleanup for eleven mycotoxins, followed by LC-MS/MS quantification without the need for isotopic labelled and matrix-matched standards. The applicability of method is to be demonstrated for corn feed, pig feed, and DDGS by fortification and naturally occurring mycotoxins covering the range of regulated limits. METHODS: Feed sample (1 kg) ground by milling to approximately 1-2 mm particle size and sub-sample (5 g) extracted with acetonitrile-water-formic acid, passing through a multi-mycotoxin IAC, washing, and eluting prior to LC-MS/MS analysis monitoring selected ion transitions. RESULTS: Recoveries were in the range 74 to 117% (excluding five outliers) for aflatoxins, FB1, FB2, DON, OTA, ZON, HT-2, and T2- toxins spiked into three commercial animal feed matrixes (n = 84) and within-day RSDs averaged 1.7 to 10.3% (n = 99). CONCLUSION: Single laboratory validation of a multi-antibody IAC method coupled with LC-MS/MS has shown the method to be suitable for accurate quantification of eleven regulated mycotoxins in DDGS, pig feed, and poultry feed. HIGHLIGHTS: IAC method capable of accurately quantifying eleven regulated mycotoxins in complex feed matrices.

Assessment of Citrinin in Spices and Infant Cereals Using Immunoaffinity Column Clean-Up with HPLC-Fluorescence Detection.

Historically, the analysis of citrinin has mainly been performed on cereals such as red yeast rice; however, in recent years, more complex and abnormal commodities such as spices and infant foods are becoming more widely assessed. The aim of this study was to develop and validate clean-up methods for spices and cereal-based infant foods using a citrinin immunoaffinity column before HPLC analysis with fluorescence detection. Each method developed was validated with a representative matrix, spiked at various citrinin concentrations, based around European Union (EU) regulations set for ochratoxin A (OTA), with recoveries >80% and % RSD < 9% in all cases. The limit of detection (LOD) and the limit of quantification (LOQ) were established at 1 and 3 µg/kg for spices and 0.1 and 0.25 µg/kg for infant cereals, respectively. These methods were then tested across a variety of spices and infant food products to establish efficacy with high recoveries >75% and % RSD < 5% across all matrices assessed. Therefore, these methods proved suitable for providing effective clean-up of spices and infant cereals, enabling reliable quantification of citrinin detected. Samples such as nutmeg and infant multigrain porridge had higher levels of citrinin contamination than anticipated, indicating that citrinin could be a concern for public health. This highlighted the need for close monitoring of citrinin contamination in these commodities, which may become regulated in the future.

Determination of Aflatoxin M1 in Liquid Milk, Cheese, and Selected Milk Proteins by Automated Online Immunoaffinity Cleanup with Liquid Chromatography‒Fluorescence Detection.

BACKGROUND: Aflatoxins are secondary metabolites produced by a number of species of Aspergillus fungi. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 and is found in the milk of cows fed with feed spoilt by Aspergillus species. AFM1 is carcinogenic, especially in the liver and kidneys, and mutagenic, and is also an immunosuppressant in humans. OBJECTIVE: A high-throughput method for the quantitative analysis of AFM1 that is applicable to liquid milk, cheese, milk protein concentrate (MPC), whey protein concentrate (WPC), whey protein isolate (WPI), and whey powder (WP) was developed and validated. METHOD: AFM1 in cheese, milk, and protein products is extracted using 1% acetic acid in acetonitrile with citrate salts. The AFM1 in the resulting extract is concentrated using RIDA®CREST/IMMUNOPREP® ONLINE cartridges followed by quantification by HPLC‒fluorescence. RESULTS: The method was shown to be accurate for WP, WPC, WPI, MPC, liquid milk, and cheese, with acceptable recovery (81-112%) from spiked samples. Acceptable precision for WP, WPC, WPI, MPC, liquid milk, and cheese was confirmed, with repeatabilities of 4-12% RSD and intermediate precisions of 5-13% RSD. Method detection limit and ruggedness experiments further demonstrated the suitability of this method for routine compliance testing. An international proficiency scheme (FAPAS) cheese sample showed that this method gave results that were comparable with those from other methods. CONCLUSIONS: A method for high-throughput, routine testing of AFM1 is described. The method was subjected to single-laboratory validation and was found to be accurate, precise, and fit-for-purpose. HIGHLIGHTS: An automated online immunoaffinity cleanup HPLC‒fluorescence method for milk proteins, cheese, and milk was developed and single-laboratory validated. It allows for high-throughput analysis of AFM1 and can be used for the analysis of AFM1 in whey protein products.