Broekaert, Nathan

Croubels, Siska

De Mil, Thomas

De Saeger, Sarah

Devreese, Mathias

Fraeyman, Sophie

Osselaere, Ann

Ghent University Academic Bibliography

Centrifuge chicken blood sample to collect plasma 250 µl chicken plasma + 750 µl ACN + C13DON (IS) Centrifuge 10’ at 8517g and 4°C Isolate supernatant and dry at 70 °C under N2 Reconstitute in 200 µl UPLC water and vortex 15” Filter (0.22 µm) and analyse Determination of the toxicokinetic parameters and degree of in vivo hydrolysis of 3ADON and 15ADON in broiler chickens. For this, an LC-MS/MS method needed to be developed and validated for the above mentioned compounds in chicken plasma. For sample clean-up, three methods were tested, a liquid-liquid extraction, a solid-phase extraction and a deproteinization by means of organic solvents. In the following steps of the development, critical factors (e.g. precipitation solvent, temperatures, etc ..) were examined by linear regression with the use of Plackett-Burman designs in order to determine their statistical significance.   Development and validation of an LC-MS/MS method for the determination of DON, 3ADON and 15ADON in chicken plasma N. Broekaert*1, M. Devreese1, T. De Mil1, A. Osselaere1, S. Fraeyman1, S. De Saeger2 and S. Croubels1 1Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke; 2Department of Bio-analysis, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium, *e-mail: nathan.broekaert@ugent.be  INTRODUCTION The research towards the toxicity and occurrence of deoxynivalenol (and mycotoxins in general) has made significant progress. As the information and insight on native mycotoxins increases, the focus of this research domain tends to shift towards the gathering of occurrence and toxicity data on masked mycotoxins. These are mycotoxin conjugates or derivatives that are undetectable by conventional methods due to changes in their structural formation and physicochemical properties. For this study we focused on two important and frequently occurring masked forms of deoxynivalenol, 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON), both precursors in the fungal biosynthesis of deoxynivalenol (DON). Conversion of these masked mycotoxins back to their native form by in vivo hydrolysis cannot be excluded. Consequently, this would imply an underestimation of the degree of contamination upon analysis. Deepoxy-deoxynivalenol (DOM-1) is the main in vivo metabolite, with highly reduced toxicity due to the removal of the epoxide function.  OBJECTIVES METHOD DEVELOPMENT AND VALIDATION Linearity R² & GOF LOQ (ng/ml) LOD (ng/ml) Accuracy Within-day precision Between-day precision DON 0.9975 13.2% 1  0.04 -5.4% 28.7% 17.6% 3ADON 0.9989 4.9% 1  0.13 -28.4% 22.6% 41.2% 15ADON 0.9986 3.1% 2 0.70 2.0% 6.0% 19.2% DOM-1 0.9987 7.2% 2 0.51 5.5% 14.3% 32.8% MS/MS instrument: TSQ Quantum Ultra triple quadrupole mass spectrometer  HPLC instrument: Surveyor autosampler Plus and MS Pump Plus  Column: Hypersil Gold (50 mm x 2.1 mm i.d., d.p.: 1.9 mm) MFa: UPLC water + 0.1% acetic acid MFb: UPLC grade methanol + 0.1 % acetic acid Column temperature: 60°C Injection volume: 10 µl Flow rate: 0.5 ml/min Run Time: 12 minutes Detection: MRM-mode With thanks to Ghent University for the funding (BOF-project) Presented at the 35th MYCOTOXIN WORKSHOP  Ghent, Belgium 22 – 24 May 2013 www.mytox.be 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 0 min 1 1,1 5 5,1 8 8,1 9,1 9,2 12 MF B MF A CONCLUSION Linearity: three calibration curves (3 x n=7) in plasma up to 200 ng/ml LOQ:  lowest concentration (n=6) with acceptable results for accuracy and precision LOD: calculated from S/N values from the LOQ samples (n=6) Precision: determined at three levels (LOQ, 50 and 100 ng/ml), values presented in table are for LOQ, values are listed as relative standard deviation, within day (n=6), between day (n=3x3) Accuracy: determined at three levels (LOQ, 50 and 100 ng/ml), values presented in table are for LOQ (n=6) Specificity: results not shown, tested by injection of blank sample, no interfering peaks Carry-over: results not shown, tested by injecting a 50/50 (v/v) mixture of both mobile phases after a 200 ng/ml spiked plasma sample, none of the compounds showed carry-over • Solid-phase extraction as well as liquid-liquid extraction gave comparable results to a simple deproteinization by means of acetonitrile.  • The latter method was further developed and validated. • C13DON was well suited as internal standard for all the compounds included in the LC-MS/MS method. • The method will be used to determine toxicokinetic parameters (AUC, Cmax, tmax, bioavailability, …) of the compounds in broiler chickens. Matrix effect: results not shown, ion suppression was observed in plasma, demonstrating the need of matrix matched calibration curves  

Development and validation of an LC-MS/MS method for the determination of DON, 3A-DON and 15A-DON in chicken plasma

https://biblio.ugent.be/publication/4240902/file/4240912

Development and validation of an LC-MS/MS method for the determination of DON, 3A-DON and 15A-DON in chicken plasma

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Ghent University Academic Bibliography