Development of a liquid–chromatography tandem mass spectrometry and ultra-high-performance liquid chromatography high-resolution mass spectrometry method for the quantitative determination of zearalenone and its major metabolites in chicken and pig plasma

A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC–MS/MS) and high-resolution Orbitrap® mass spectrometry ((U)HPLC–HR–MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 μm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases.Both mass spectrometers were operated in the negative h-ESI mode.The method was in-house validated for all analytes: matrix-matched calibration graphs were prepared and good linearity (r ≥ 0.99) was achieved over the concentration range tested (0.2–200 ng mL−1). Limits of quantification (LOQ) in plasma were between 0.2 and 5 ng mL−1 for all compounds. Limits of detection in plasma ranged from 0.004 to 0.070 ng mL−1. The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values (within-day precision: RSDmax = 2(1–0.5logConc) x 2/3; between-day precision: RSDmax = 2(1–0.5logConc)). The accuracy fell within −50% to +20% (concentrations <1 ng mL−1), −30% to +10% (concentrations between 1 and 10 ng mL−1) or −20% to +10% (concentrations >10 ng mL−1) of the theoretical concentration.The method has been successfully used for the quantitative determination of ZEN in plasma samples from broiler chickens and pigs. α-ZEL and β-ZEL were the only metabolites that could be detected, but the concentrations were around the LOQ levels. The intact ZEN-glucuronide conjugate could be detected using the (U)HPLC–HR–MS instrument. A good correlation (r2 = 0.9979) was observed between the results for ZEN obtained with the LC–MS/MS and (U)HPLC–HR–MS instruments. The results prove the usefulness of the developed method for application in the field of toxicokinetic analysis and for exposure assessment of mycotoxins.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlights► Qualitative + quantitative analyses of zearalenone with LC–MS/MS and (U)HPLC–HR–MS. ► Development of a generic, rapid and cheap sample preparation procedure. ► Chromatograhy: baseline separation of metabolites with same precursor ion, run-time: 10 min. ► Method validation: linearity, accuracy, precision, LOQ, LOD, specificity, recovery and matrix effects. ► Investigation of toxicokinetics of zearalenone in chickens.