OPTIMIZATION AND VALIDATION OF HPLC-FLD METHOD FOR DETERMINATION OF AFLATOXIN B1 AND PRODUCTION OF THIS MYCOTOXIN BY ASPERGILLUS SECTION FLAVI ISOLATES

Abstract

A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was evaluated for the monitoring of aflatoxin B₁ (AFB₁) production by Aspergillus section Flavi isolates, which were found in the commodities of Slovak origin (barley and tomatoes). Rapid validation of the method was performed and the method was applied for the determination of the mycotoxin. Based on the calculated validation characteristics, the method showed high sensitivity and reproducibility. The LOD and LOQ determined were 0.03 and 0.10 µg.mL^-1 which is fully acceptable for the determination of the AFB₁ in isolates from plant commodities. The calibration graph was linear in the concentration range of 0.01– 10.0 µg.mL^-1. The optimized method was used for the analysis of the mycotoxin in three Aspergillus section Flavi isolates. The submerse cultivation was performed at 25±1°C in yeast extract sucrose liquid medium and material was collected on the 7th, 14th and 21st day of cultivation.  Simple sample extraction was carried out with use of ethyl acetate, and measurement was done with use of the mobile phase consisting of water and methanol by HPLC-FLD. Production of the AFB₁ by isolates was ranging from <LOD (0.03 µg. mL^-1) to 49.38 µg.mL^-1 with the highest concentration on the 14th day of cultivation. The maximum level of AFB₁ was achieved by the Isolate 1 (isolated from barley, Kolíňany, 2018).