1. M. Kainz 1, G. Mitterer 1, M. Freudenschuss 2, G. Häubl 3, R. Krska 3 1 ROMER LABS Division Holding GmbH, Technopark 1, 3430 Tulln, Austria 2 Biopure Referenzsubstanzen GmbH, Technopark 1, 3430 Tulln, Austria 3 Christian Doppler Laboratory for Mycotoxin Research, Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Str. 20, A-3430 Tulln, Austria 13 C 24 -T-2 Toxin O Stable 13 C Isotope Labeled Mycotoxin Calibrants* The mass spectrometric detection of mycotoxins in analytical chemistry is a very powerful tool. Nowadays, so called LC-MS/MS based multi-mycotoxin analysis methods allows the determination of more than 100 toxins in single run*. However, interferences from matrix components lead to a different ionization of the analyte in a sample in comparison of pure standard calibrants and hence results in different signal intensity. This so called “matrix effect” limits the methods using a mass spectrometer as detector for liquid chromatography. “Matrix effects” can be overcome by adding an internal standard (IS) to the sample, which behaves similar to the analyte and therefore can both correct recovery losses during the sample preparation process and ion suppression effects in the MS source. Stable isotope-labeled analogs of natural mycotoxins provide the best IS for these toxins. However, it should be noted that deuterated compounds still run the risk of H/D exchange in protic solvents and retention time shifts relative to the natural toxin. Moreover, partially labeled toxins frequently contain considerable amounts of “lighter” isomers, leading to mass peaks that interfere with natural toxin isotopes. Therefore, fully 13 C- substituted compounds can be regarded as the best standard for quantification by LC–MS/MS based methods. *Sulyok M, Krska R, Schuhmacher R, Food Addit Contam. 24, 1184-95 (2007) We thank the Christian Doppler Society for their financial support Correspondence should be addressed to: Georg Mitterer ROMER LABS Division Holding GmbH, Technopark 1 3430 Tulln, Austria email: georg.mitterer@romerlabs.comgeorg.mitterer@romerlabs.com www.romerlabs.com Full scan MS spectra of ( 13 C 15 )-DON (Biopure) showing the isotopic distribution (blue line). Overlayed is the isotopic pattern of natural occuring DON (red line). The use of fully 13 C isotope labeled mycotoxin internal standards can correct fluctuations that may occur during extraction, clean-up, separation and ionization of the sample. Therefore, isotope labeled mycotoxins are well suited for performing a fast and accurate LC-MS analysis of mycotoxins. Fully 13 C- substituted compounds can be regarded as the best internal standard for quantification of analytes by MS based methods. Determination of the suitability of the labeled DON IS using stable isotope dilution mass spectrometry*: 2.) Comparison of recovery functions for a blank maize extract spiked with DON (0-1000 µg/kg, 7 levels, 3 per level) and 250 µg/kg IS.. 1.) Measured DON concentrations of matrix reference materials without cleanup. Results were evaluated with external (yellow colums) and internal calibration (blue colums). Certified values are shown with horizontal lines The extracts were purified with Mycosep® 225 (RomerLabs) clean-up columns before measured with HPLC-MS Recovery without IS: 76% Full scan MS spectra of sodium adducts of ( 13 C 24 )-T-2 toxin showing the isotopic distribution (blue line). Overlayed is the isotopic pattern of sodium adducts of the natural occuring T-2 toxin (red line). Determination of the suitability of the labeled T-2 toxin as IS using stable isotope dilution mass spectrometry*: The comparison of the recovery functions shows an overestimation of approx 40%, if no IS was used (ion enhancement). Additionally blank maize was also spiked with T-2 toxin (0 - 1000 µg/kg, 8 levels, 3 per level) and IS (250 µg/kg). Maize is one of the cereals which creates the highes matrix interferences. Also here an overestimation (+50%) was observed, if no IS was used. Conclusion *Häubl G, Berthiller F, Krska R, Schuhmacher R., Anal Bioanal Chem. 384, 692-6 (2006).,Häubl G, Berthiller F, Rechthaler J, Jaunecker G, Binder EM, Krska R, Schuhmacher R., Food Addit Contam. 23, 1187-93 (2006). *Häubl G, Berthiller F, Hametner C, Rechthaler J, Jaunecker G, Freudenschuss M, Krska R, Schuhmacher R., Anal Bioanal Chem. 389, 931-940 (2007) 13 C 15 -Deoxynivalenol (DON) The Use of Fully Stable Isotope Labeled Mycotoxins as Internal Standards for Mycotoxin Analysis with LC-MS/MS Component Conc. (μg/mL) Solvent Unit of Issue Cat. No. U-[ 13 C 15 ]-Deoxynivalenol 25acetonitrile1.2 mL002005 U-[ 13 C 17 ]-3-Ac-DON 25acetonitrile1.2 mLILM006 U-[ 13 C 17 ]-Aflatoxin B1 0.5acetonitrile1.2 mLILM010 U-[ 13 C 17 ]-Aflatoxin B2 0.5acetonitrile1.2 mLILM011 U-[ 13 C 17 ]-Aflatoxin G1 0.5acetonitrile1.2 mLILM012 U-[ 13 C 17 ]-Aflatoxin G2 0.5acetonitrile1.2 mLILM013 U-[ 13 C 17 ]-Mycophenolic acid 0.5acetonitrile1.2 mLILM014 U-[ 13 C 18 ]-Zearalenone 25acetonitrile1.2 mLILM009 U-[ 13 C 20 ]-Ochratoxin A 10acetonitrile1.2 mLILM007 U-[ 13 C 22 ]-HT-2 Toxin 25acetonitrile1.2 mLILM008 U-[ 13 C 24 ]-T-2 Toxin 25acetonitrile1.2 mL002044 U-[ 13 C 34 ]-Fumonisin B1 25 acetonitrile/ water 1.2 mLILM003 U-[ 13 C 34 ]-Fumonisin B2 10 acetonitrile/ water 1.2 mLILM004 U-[ 13 C 34 ]-Fumonisin B3 10 acetonitrile/ water 1.2 mLILM005 U-[ 13 C 15 ]-Nivalenol25acetonitrile1.2 mL ILM-019- 1.2ML U-[ 13 C 7 ] -Patulin25acetonitrile1.2 mL ILM-015- 1.2ML U-[ 13 C 22 ]-Roquefortine C25acetonitrile1.2 mL ILM-016- 1.2ML U-[ 13 C 18 ]-Sterigmatocystin25acetonitrile1.2 mL ILM-017- 1.2ML * Available at www.romerlabs.com