SIMULTANEOUS HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC DETERMINATION OFOCHRATOXIN-A AND CITRININ IN CHEESE BY TIME-RESOLVED LUMINESCENCE USING TERBIUM

A simultaneous reversed-phase HPLC determination of two major mycotoxi ns, ochratoxin A and citrinin, in soft cheese is proposed. Both mycoto xins are eluted on a C-18 RP support (25 x 4.6 mm I.D.) using an isocr atic eluent consisting of methanol-water (70:30, v/v) containing tetra butylammonium 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 s olution 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 mo de at a flow-rate of 0.2 ml/min to perform time-resolved luminescence (TRL) detection of the corresponding terbium chelates (lambda(ex) = 33 1 nm/lambda(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 ochra toxin 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 och ratoxin 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, och ratoxin A and citrinin are easily determined in soft cheeses, with a s ignificative increase in selectivity in comparison with direct fluores cence detection.