Quantitative liquid chromatographic method using fluorescence detection for determining zearalenone and its metabolites in blood plasma and urine

Abstract

The liquid chromatographic (LC) method described, suitable for use with both blood plasma and urine, is applicable for determination of zearalenone and α-zearalenol at levels as low as 0.5 ng/mL plasma and 5 ng/mL urine. The sample is incubated overnight with β-glucuronidase to analyze for both conjugated and unconjugated forms of zearalenone. The next day, the sample is acidified with H3PO4, extracted with chloroform, and evaporated to dryness. The residue is dissolved in toluene and loaded onto a silica gel cartilage which is washed with toluene and eluted with toluene-acetone (88 + 12). The eluate is evaporated, and the residue is dissolved in chloroform, extracted with 0.18M NaOH, neutralized with H3PO4, and re-extracted with chloroform. The chloroform extract is evaporated, dissolved in mobile phase for LC, and injected onto a normal phase column under the following chromatographic conditions: mobile phase of water-saturated dichloromethane containing 2% 1-propanol, and fluorescence detector, excitation wavelength 236 nm, and 418 nm cut-off emission filter. Recoveries of zearalenone and its metabolites from blood plasma urine are 80-89% in the range 2.0-10 ng standard/mL plasma, and 81-90% in the range 10-30 ng standard/mL urine. This method was used to analyze blood and urine samples from a pig fed zearalenone-contaminated feed (5 mg/kg), corresponding to 80 μg/kg body weight. Zearalenone was rapidly metabolized to α-zearalenol, which appeared in the blood only 30 min after feeding. Almost all zearalenone and α-zearalenol was found conjugated with glucuronic acid in both blood plasma and urine.