Rapid and sensitive detection of citrinin production during fungal fermentation using high-performance liquid chromatography

Abstract

A rapid and sensitive assay was developed for the detection of the mycotoxin citrinin by reversed-phase chromatography. Citrinin was eluted from a radical-compression C18 column with a retention time of 3.86 min (flow-rate of 2.5 ml/min) with acetonitrile-water-acetic acid (40:59:1) containing tetrabutylammonium phosphate (0.0025 M) [corrected]. Comparative analysis revealed fluorescence detection to be 100 times more sensitive than detection by conventional ultraviolet absorbance. The fluorescence excitation and emission maxima of citrinin were 330 and 500 nm, respectively. The assay was linear over the concentration range between 0.01-100 micrograms/ml. Recovery experiments conducted by addition of citrinin to fermentation samples, revealed the assay quantitation efficiency to be 91-102%. Assay utility was demonstrated by using an Aspergillus niveus culture, propagated in complex liquid medium. Citrinin production was detected as early as 20 h following inoculation and increased dramatically when the culture entered the stationary phase of growth, analogous to other secondary metabolites. Unlike previously reported methods, this procedure has the advantage of enabling the direct quantitative analysis of citrinin in crude microbial fermentations without sample extraction.