LC-MS/MS method for simultaneous determination of biomarkers of aflatoxin B1, fumonisin B1, ochratoxin A, deoxynivalenol and zearalenone in human and animal urines (Rapporti progetti di ricerca)

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  • LC-MS/MS method for simultaneous determination of biomarkers of aflatoxin B1, fumonisin B1, ochratoxin A, deoxynivalenol and zearalenone in human and animal urines (Rapporti progetti di ricerca) (literal)
Anno
  • 2013-01-01T00:00:00+01:00 (literal)
Alternative label
  • Solfrizzo M. and Gambacorta L. (2013)
    LC-MS/MS method for simultaneous determination of biomarkers of aflatoxin B1, fumonisin B1, ochratoxin A, deoxynivalenol and zearalenone in human and animal urines
    (literal)
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  • Solfrizzo M. and Gambacorta L. (literal)
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Titolo
  • LC-MS/MS method for simultaneous determination of biomarkers of aflatoxin B1, fumonisin B1, ochratoxin A, deoxynivalenol and zearalenone in human and animal urines (literal)
Abstract
  • This deliverable describes a new LC-MS/MS method for simultaneous determination of biomarkers of exposure to aflatoxin B1 (AFB1), deoxynivalenol (DON), zearalenone (ZEA), fumonisin B1 (FB1) and ochratoxin A (OTA) in human and animal urines at naturally occurring concentrations. The biomarkers selected for these mycotoxins were: the parent toxin for FB1 and OTA; a mixture of parent toxin and phase I metabolites for DON (DON and de-epoxydeoxynivalenol (DOM-1)) and ZEA (ZEA, alpha-zearalenol (?-ZOL) and beta-zearalenol (?-ZOL)); phase I metabolite for AFB1 (aflatoxin M1 (AFM1)). Urine samples were purified and concentrated by a double cleanup approach, using a multitoxin immunoaffinity column (Myco6in1) and a reversed-phase SPE Oasis HLB column. Separation of the biomarkers was performed by reversed-phase chromatography using a multi-step linear gradient of methanol/water containing 0.5% acetic acid as mobile phase. Detection and quantification of the biomarkers were performed by triple quadrupole mass spectrometry (LC-ESI-MS/MS). The clean-up conditions were optimised to obtain maximum analyte recovery and the sensitivity necessary to measure urinary biomarker concentrations deriving from natural exposure to the principal mycotoxins. Recovery experiments were performed at four spiking levels in the range of 0.03-12 ng/mL using matrix-matched calibration curves for quantification. Mean recoveries of the tested biomarkers ranged from 61 to 95% with relative standard deviations of 3-20%. The limits of quantification were 1.51, 1.50, 0.02, 0.12, 0.054, 0.029, 0.007 and 0.006 ng/mL for DON, DOM-1, AFM1, FB1, ?-ZOL, ?-ZOL, ZEA and OTA, respectively. A new multiple mycotoxin biomarker method based on a \"dilute-and-shoot\" approach was also developed for human urine. The limits of quantification for this method were 13, 33, 0.2, 3, 2, 2, 1 and 0.2 ng/mL for DON, DOM-1, AFM1, FB1, ?-ZOL, ?-ZOL, ZEA and OTA, respectively The performances of these two methods were checked in a mini interlaboratory study which also involved a third laboratory that used two different methods for determination of DON and FB1 biomarkers. The limits of quantification for these two methods were 0.5 and 0.02 ng/mL for DON and FB1, respectively. The results of the mini intercomparison study showed that blank sample was found to contain low levels of DON and DON-glucuronide (DON-Glu) therefore the results of spiked samples were corrected for the endogenous concentration of either free DON and DON derived from DON-Glu according to the use or not of the enzymatic pre-treatment. The results of each laboratory were evaluated for their repeatability and z-score. The overall results of the three laboratories, for each biomarker, were also evaluated for their reproducibility by means of coefficient of variation (CV). Acceptable values of repeatability were obtained by all laboratories for all biomarkers, at the two concentrations, (RSDr < 15%) with the exception of laboratory 1 for FB1 (RSDr 40% and 50%). Acceptable values of reproducibility results were obtained for 6 biomarkers (DON, DOM-1, AFM1, ?-ZOL, ?-ZOL, ZEA) at the two concentrations, since the CVs ranged from 5.3 to 33.3%. High values of reproducibility were obtained for FB1 (67.5 and 47.5%) and OTA (90.9 and 85.7%) at the two concentrations. The overall percentage of satisfactory z-scores (|z| < 2) for all biomarkers was 84% (64 results out of 76). In particular, the 3 laboratories scored individually a percentage of satisfactory z-scores (|z| < 2) of 78 % (Lab. 1), 67% (Lab. 2) and 97% (Lab. 3). (literal)
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