Objective: With consideration of lethal effects of aflatoxins specially B۱ on human health. Estimation of aflatoxin-albumin adduct, as an important marker of aflatoxin exposure, seems essential. The aim of this study is optimization of
HPLC-fluorescence method for measurement of this important marker in blood serum.
Materials and Methods: In this study, blood serum of three groups of rats as A) positive controls (treated with AFB۱), B) negative controls (without treatment) and standard rats (treated with radiolabeled AFB۱) were used. After albumin isolation using ammunium sulphate and acetic acid, purity of albumin was tested by SDS-PAGE electrophoresis and albumin concentration was quantified by bradford method. Then albumin was hydrolysed by pronase and aflatoxin bound to albumin was released as aflatoxin-lysine. Pronase was precipitated and albumin was digested by aceton in cold, the volume of supernatant was reduced by freeze-drier and injected into HPLC system. Aflatoxin was quantified in comparison to standard rats samples.
Results: The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were ۱۰, ۱۳ and ۱۲.۵ mg/ml, respectively. Detection limit (۲۰ pg/mg Alb) for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were ۹۲% and ۱۰۰% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was ۱۰ ng/mg Alb and the reproducibility of the method after several repeat was very good.
Conclusion: In this study, for AF-Alb adduct quantification by HPLC method, mobile phase, percentage of solvents and run time were changed and the affinity chromatography before HPLC, was deleted. Therefor HPLC- fluorescence which is a precise and specific method, and since it is fast, highly reproducible and cost effective, also with improvement made, could easily be used for the quantification of this important marker in serum.
