A fully automated and fast method using direct sample injection combined with fused-core column on-line SPE-HPLC for determination of ochratoxin A and citrinin in lager beers
Abstract
A new fast and sensitive method based on on-line solid-phase extraction on a fused-core precolumn coupled to liquid chromatography with fluorescence detection has been developed for ochratoxin A (OTA) and citrinin (CIT) determination in lager beer samples. Direct injection of 100 mu L filtered beer samples into an on-line SPE-HPLC system enabled fast and effective sample extraction including separation in less than 6 min.
Preconcentration of OTA and CIT from beer samples was performed on an Ascentis Express RP C18 guard column (5 x 4.6 mm), particle size 2.7 mu m, with a mobile phase of methanol/0.5 % aqueous acetic acid pH 2.8 (30:70, v/v) at a flow rate of 2.0 mL min(-1). The flow switch from extraction column to analytical column in back-flush mode was set at 2.0 min and the separation was performed on the fused-core column Ascentis Express Phenyl-Hexyl (100 x 4.6 mm), particle size 2.7 mu m, with a mobile phase acetonitrile/0.5 % aqueous acetic acid pH 2.8 in a gradient elution at a flow rate of 1.0 mL min(-1) and temperature of 50 A degrees C.
Fluorescence excitation/emission detection wavelengths were set at 335/497 nm. The accuracy of the method, defined as the mean recoveries of OTA and CIT from light and dark beer samples, was in the range 98.3-102.1 %.
The method showed high sensitivity owing to on-line preconcentration; LOQ values were found to be 10 and 20 ng L-1 for OTA and CIT, respectively. The found values of OTA and CIT in all tested light, dark and wheat beer samples were significantly below the maximum tolerable limits (3.0 mu g kg(-1) for OTA and 2000 mu g kg(-1) for CIT) set by the European Union.