Simultaneous high-performance liquid chromatographic determination of ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium

Abstract

A simultaneous reversed-phase HPLC determination of two major mycotoxins, ochratoxin A and citrinin, in soft cheese is proposed. Both mycotoxins are eluted on a C 18 RP support (25 × 4.6 mm I.D.) using an isocratic eluent consisting of methanol-water (70:30, v/v) containing tetrabutylammonium hydroxide (10 −3 M), acidified to pH 5.5 with HCl, and pumped at a flow-rate of 0.8 ml/min. Prior to detection, a butanolic solution of 5·10 −3 M terbium-5 · 10 −4 M trioctylphosphine oxide (TOPO)-2.5 · 10 −2 M triethylamine (TEA) was pumped in a postcolumn mode at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates ( λ ex = 331 nm/ λ em = 545 nm). The method is linear from 3.5·10 −6 to 2·10 −5 M for citrinin and from 1·10 −5 to 5·10 −5 M for ochratoxin A. The repeatability and reproducibility (R.S.D.) are 1.9 and 2.4% for citrinin ( c = 3.5·10 −6 M; n = 10), and 7.2 and 8.3% for ochratoxin A ( c = 1.0·10 −5 M; n = 10). The limits of detection, for a signal-to-background ratio of 3, are 2·10 −6 and 3·10 −6 M for citrinin and ochratoxin A, respectively. With the proposed method, ochratoxin A and citrin are easily determined in soft cheeses, with a significative increase in selectivity in comparison with direct fluorescence detection.