Simplified Synthesis and Stability Assessment of Aflatoxin B1-Lysine and Aflatoxin G1-Lysine

Abstract

Aflatoxins B1 (AFB1) and G1 (AFG1) are carcinogenic mycotoxins that contaminate crops such as maize and groundnuts worldwide. The broadly accepted method to assess chronic human aflatoxin exposure is by quantifying the amount of aflatoxin adducted to human serum albumin. This has been reported using ELISA, HPLC, or LC-MS/MS to measure the amount of AFB1-lysine released after proteolysis of serum albumin. LC-MS/MS is the most accurate method but requires both isotopically labelled and unlabelled AFB1-lysine standards, which are not commercially available. In this work, we report a simplified synthetic route to produce unlabelled, deuterated and 13C6 15N2 labelled aflatoxin B1-lysine and for the first-time aflatoxin G1-lysine. Additionally, we report on the stability of these compounds during storage. This simplified synthetic approach will make the production of these important standards more feasible for laboratories performing aflatoxin exposure studies.

Optimization of Aflatoxin B1-Lysine Analysis for Public Health Exposure Studies

“…AFB 1 -epoxide undergoes rapid hydrolysis in an aqueous environment to form the AFB 1 -dihydrodiol, which is in equilibrium with AFB 1 -dialdehyde [ 12 ]. The initial AFB 1 solution in dichloromethane (DCM) was clear, however following the addition of DMDO, drying and reconstitution in 0.1 M sodium bicarbonate (pH 8.1), the solution was an intense yellow colour, in line with previous observations that indicated the presence of the dialdehyde [ 19 ]. To maximize the amount of AFB 1 adducts on the reference material (RM) serum, while ensuring that the adducts formed were similar to those formed in vivo it was necessary to determine the theoretical maximum molar ratio of AFB 1 to HSA.…”

“…Blank serum (H4522; from human plasma, USA origin, sterile-filtered; Millipore Sigma Burlington, MA, USA), sodium bicarbonate and Oxone ® was purchased from Sigma-Aldrich (Oakville, ON, Canada). Labelled AFB 1 -Lys adduct was prepared according to Renaud et al, (2022) [ 19 ]. In that work, which minimized the production of unwanted reaction by-products, quantitative NMR was used to demonstrate that the previously reported molar attenuation coefficient (ε 400 30,866/M cm) [ 30 ] used for the generation of AFB 1 -Lys standards is valid.…”

“…The sample was incubated at room temperature for 4 h. 100 μL of 0.1 M sodium bicarbonate (pH 8.1) was added to the solution and the vial was placed at 45 °C until all the DCM had evaporated. The presence of the AFB 1 -dialdehyde could be observed by an intense yellow colour in the solution [ 19 ]. 900 μL of sterile-filtered human serum was added to AFB 1 -dialdehyde solution and vortexed for 15 s. The serum solution was then incubated with gentle shaking for 16 h overnight at room temperature.…”

“…There is an additional barrier to more effective use of biomarker studies namely the lack of alternatives to shipping blood samples over long distances and storage in −80 °C freezers. Recently, progress was made towards addressing the first gap though the publication of a simplified method to synthesize the standards required to quantify AFB 1 -Lys by LC-MS/MS [ 19 ].…”

Assessing the Impacts of Preanalytical Field Sampling Challenges on the Reliability of Serum Aflatoxin B1-Lysine Measurements by Use of LC-MS/MS

“…Studies in which serum samples were subjected to ambient room and transportation temperatures over timeframes of 4-5 days found that albumin concentrations were stable [17] or increased slightly (2.2%) [18]. It is important to state that the apparent stability of albumin-bound AFB1-lys in serum is not commutable to the storage of free AFB1-lys that would be used as a calibrator, and that stability can vary widely depending on the solvation conditions [19], nor should prolonged storage at room temperature or higher be substituted for the proven robust stability of low temperature (−80 • C) storage [20] when available.…”