Estimation of aflatoxin-albumin adduct in serum sample by optimized HPLC-fluorescence

With consideration of lethal effects of aflatoxins specially B1 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. In this study, blood serum of three groups of rats as A) positive controls (treated with AFB1), B) negative controls (without treatment) and standard rats (treated with radiolabeled AFB1) 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. The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were 10, 13 and 12.5 mg/ml, respectively. Detection limit (20 pg/mg Alb) for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were 92% and 100% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was 10 ng/mg Alb and the reproducibility of the method after several repeat was very good. 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.