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

Abstract

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.

Aflatoxin Contamination, Its Impact and Management Strategies: An Updated Review

“…A study demonstrated the use of a high-performance liquid chromatography-mass spectrometry (LC–MS) technique to analyze the aflatoxin concentration in baby food in the range 0.003–0.008 μg/L [ 90 ]. In milk samples, high performance liquid chromatography (HPLC) coupled with mass spectroscopy or fluorescence detection is reported as a reliable technique for estimating aflatoxin concentrations [ 91 ].…”

Developments in mycotoxin analysis: an update for 2020-2021

“…Meanwhile, Wood et al (2020) developed a sophisticated method for determining AFM 1 in liquid milk, cheese, and other selected milk-based proteins. The technique used automated online immunoaffinity clean-up with HPLC-FLD.…”

“…In developing this high-throughput approach for the quantitative analysis of AFM 1 , Wood and colleagues extracted AFM 1 in protein products using 1% ethanoic https://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2021.2752 -Sunday, March 06, 2022 7:43:18 PM -IP Address: 44.224.250.200 acid diluted in acetonitrile with citrate salts. The AFM 1 in the resulting extract was subsequently concentrated and purified by utilising RIDA® CREST/IMMUNOPREP® cartridges (R-Biopharm, Pfungstadt, Germany) (Wood et al, 2020). This method was found accurate and precise, with 81-112% recoveries and 4-12% repeatability.…”

Selection and Characterization of DNA Aptamers for Constructing Aptamer-AuNPs Colorimetric Method for Detection of AFM1

“…There are many detection methods for AFM1. Although instrumental methods, such as gas chromatography and high-performance liquid chromatography [ 8 , 9 ], have the advantages of high sensitivity and strong stability, the complex equipment and tedious sample preparation limit their application in the rapid monitoring of AFM1 in the field [ 10 ].…”