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.

Analysis of Citrinin in Cereals, Red Yeast Rice Dietary Supplement, and Animal Feed by Immunoaffinity Column Cleanup and LC with Fluorescence Detection.

The analysis of citrinin in various cereals (wheat, oats, maize, rice, and rye and multigrain breakfast cereal), red yeast rice (dietary supplement and traditional medicine), distillers dried grain with solubles, and barley (animal feed) was carried out using a citrinin immunoaffinity column (IAC) for sample cleanup before LC analysis with fluorescence detection (LC-fluorescence). To establish method performance characteristics, wheat was spiked with citrinin at levels of 10-200 µg/kg, whereas red yeast rice was spiked at levels of 100-3000 µg/kg. Methanol-water (75 + 25, v/v) was used for the extraction of cereals and animal feed, and extraction was with 100% methanol for red yeast rice. Cleanup used a commercial citrinin IAC, followed by LC-fluorescence (λex, 330 nm; λem, 500 nm). Recoveries ranged from 80 to 110%, with r from 0.7 to 4.3%. The LOQ for citrinin in both wheat and red yeast rice was 10 µg/kg, with an LOD of 3 µg/kg. Satisfactory performance was demonstrated in a proficiency testing exercise for a sample of maize contaminated with both citrinin and ochratoxin A. It was concluded that the commercial citrinin IAC was capable of providing an efficient and effective cleanup of complex food and feed matrixes to enable citrinin to be reliably determined with the specific LC-fluorescence system used.