Analytical and Bioanalytical Chemistry

, Volume 394, Issue 5, pp 1361–1373 | Cite as

Analysis of glutathione adducts of patulin by means of liquid chromatography (HPLC) with biochemical detection (BCD) and electrospray ionization tandem mass spectrometry (ESI-MS/MS)

  • Nils Helge Schebb
  • Helene Faber
  • Ronald Maul
  • Ferry Heus
  • Jeroen Kool
  • Hubertus Irth
  • Uwe KarstEmail author
Original Paper


A novel method for the identification of glutathione/electrophile adducts that are inhibiting glutathione-S-transferase (GST) activity was developed and applied for the analysis of the mycotoxin patulin. The method is based on high-performance liquid chromatography (HPLC) coupled to a continuous-flow enzyme reactor serving as biochemical detector (BCD) in parallel to electrospray mass spectrometric detection (ESI-MS). This HPLC-BCD technique combines a separation step and the detection of the inhibition and is therefore ideally suited for the analysis of the activity of single patulin/glutathione adducts within a complex mixture of adducts. Two out of at least 15 detected patulin–glutathione adducts showed strong GST inhibition. In ESI-MS, the inhibitory active adducts were characterized by [M + H]+ ions with m/z 462.1138 and m/z 741.2011, respectively. They could be identified as a dihydropyranone adduct containing one molecule glutathione and a ketohexanoic acid bearing two glutathione molecules.

Graphical Abstract



HPLC-BCD Patulin–glutathione adducts Glutathione-S-transferase inhibition ESI(+)-MS/MS Fragmentation reactions 



We thank Dr. Heinrich Luftmann (University of Münster, Germany) for helpful discussions. The “Studienstiftung des Deutschen Volkes” (Bonn, Germany) is gratefully acknowledged for financial support in form of a Ph.D. scholarship for Nils Helge Schebb.

Supplementary material

216_2009_2765_MOESM1_ESM.pdf (71 kb)
ESM I (PDF 71.1 kb)


  1. 1.
    Rychlik M, Schieberle P (1999) J Agric Food Chem 47:3749–3755CrossRefGoogle Scholar
  2. 2.
    Tangni EK, Theys R, Mignolet E, Maudoux M, Michelet JY, Larondelle Y (2003) Food Addit Contam 20:482–489CrossRefGoogle Scholar
  3. 3.
    Drusch S, Ragab W (2003) J Food Prot 66:1514–1527Google Scholar
  4. 4.
    WHO (1995) World Health Organ Tech Rep Ser 859:1–54Google Scholar
  5. 5.
    Alves I, Oliveira NG, Laires A, Rodrigues AS, Rueff J (2000) Mutagenesis 15:229–234CrossRefGoogle Scholar
  6. 6.
    McKinley ER, Carlton WW, Boon GD (1982) Food Chem Toxicol 20:289–300CrossRefGoogle Scholar
  7. 7.
    Mahfoud R, Maresca M, Garmy N, Fantini J (2002) Toxicol Appl Pharmacol 181:209–218CrossRefGoogle Scholar
  8. 8.
    Riley RT, Showker JL (1991) Toxicol Appl Pharmacol 109:108–126CrossRefGoogle Scholar
  9. 9.
    Thust R, Kneist S, Mendel J (1982) Mut Res Let 103:91–97CrossRefGoogle Scholar
  10. 10.
    Pfeiffer E, Gross K, Metzler M (1998) Carcinogenesis 19:1313–1318CrossRefGoogle Scholar
  11. 11.
    Liu B-H, Yu F-Y, Wu T-S, Li S-Y, Su M-C, Wang M-C, Shih S-M (2003) Toxicol Appl Pharmacol 191:255–263CrossRefGoogle Scholar
  12. 12.
    Schumacher D, Metzler M, Lehmann L (2005) Arch Toxicol 79:110–121CrossRefGoogle Scholar
  13. 13.
    Schumacher DM, Muller C, Metzler M, Lehmann L (2006) Toxicol Lett 166:268–275CrossRefGoogle Scholar
  14. 14.
    Dickens F, Jones HEH (1961) Brit J Cancer 15:85–100Google Scholar
  15. 15.
    Barhoumi R, Burghardt RC (1996) Fundam Appl Toxicol 30:290–297CrossRefGoogle Scholar
  16. 16.
    Pfeiffer E, Diwald TT, Metzler M (2005) Mol Nutr Food Res 49:329–336CrossRefGoogle Scholar
  17. 17.
    Luft P, Oostingh GJ, Gruijthuijsen Y, Horejs-Hoeck J, Lehmann I, Duschl A (2008) Environ Toxicol 23:84–95CrossRefGoogle Scholar
  18. 18.
    Rychlik M, Kircher F, Schusdziarra V, Lippl F (2004) Food Chem Toxicol 42:729–735CrossRefGoogle Scholar
  19. 19.
    Larsson P, Tjalve H (1992) Cancer Res 52:1267–1277Google Scholar
  20. 20.
    Lind RC, Gandolfi AJ, Hall PM (1992) Anesthesiology 77:721–727CrossRefGoogle Scholar
  21. 21.
    Fliege R, Metzler M (2000) Chem Res Toxicol 13:373–381CrossRefGoogle Scholar
  22. 22.
    Rychlik M (2005) Nutrition 29:61–68Google Scholar
  23. 23.
    Rychlik M (2003) Food Addit Contam 20:829–837CrossRefGoogle Scholar
  24. 24.
    Lindroth S, von Wright A (1990) J Environ Pathol Toxicol Oncol 10:254–259Google Scholar
  25. 25.
    Lindroth S, von Wright A (1978) Appl Environ Microbiol 35:1003–1007Google Scholar
  26. 26.
    van Bladeren PJ, van Ommen B (1991) Pharmacol Ther 51:35–46CrossRefGoogle Scholar
  27. 27.
    Schebb NH, Heus F, Saenger T, Karst U, Irth H, Kool J (2008) Anal Chem 80:6764–6772CrossRefGoogle Scholar
  28. 28.
    de Jong CF, Derks RJ, Bruyneel B, Niessen W, Irth H (2006) J Chromatogr A 1112:303–310CrossRefGoogle Scholar
  29. 29.
    van Elswijk DA, Diefenbach O, van der Berg S, Irth H, Tjaden UR, van der Greef J (2003) J Chromatogr A 1020:45–58CrossRefGoogle Scholar
  30. 30.
    Kool J, van Liempd SM, Ramautar R, Schenk T, Meerman JH, Irth H, Commandeur JN, Vermeulen NP (2005) J Biomol Screen 10:427–436CrossRefGoogle Scholar
  31. 31.
    Kool J, Eggink M, van Rossum H, van Liempd SM, van Elswijk DA, Irth H, Commandeur JN, Meerman JH, Vermeulen NP (2007) J Biomol Screen 12:396–405CrossRefGoogle Scholar
  32. 32.
    Lake BG (1987) In: Snell K, Mullock B (eds) Biochemical toxicology: a practical approach. IRL Press, Oxford, pp 183–215Google Scholar
  33. 33.
    Bradford MM (1976) Anal Biochem 72:248–254CrossRefGoogle Scholar
  34. 34.
    Baillie TA, Davis MR (1993) Biol Mass Spectrom 22:319–325CrossRefGoogle Scholar
  35. 35.
    Wen B, Ma L, Nelson SD, Zhu M (2008) Anal Chem 80:1788–1799CrossRefGoogle Scholar
  36. 36.
    Haroldsen PE, Reilly MH, Hughes H, Gaskell SJ, Porter CJ (1988) Biomed Environ Mass Spectrom 15:615–621CrossRefGoogle Scholar
  37. 37.
    Maul R, Schebb NH, Kulling SE (2008) Anal Bioanal Chem 391:239–250CrossRefGoogle Scholar
  38. 38.
    van Elswijk DA, Irth H (2003) Phytochem Rev 1:427–439CrossRefGoogle Scholar
  39. 39.
    van Ommen B, Ploemen JH, Bogaards JJ, Monks TJ, Gau SS, van Bladeren PJ (1991) Biochem J 276(Pt 3):661–666Google Scholar
  40. 40.
    Fliege R, Metzler M (1999) Chemico-Biological Int 123:85–103CrossRefGoogle Scholar
  41. 41.
    Ashoor SH, Chu FS (1973) Food Cosmet Toxicol 11:995–1000Google Scholar
  42. 42.
    Ashoor SH, Chu FS (1973) Food Cosmet Toxicol 11:617–624CrossRefGoogle Scholar
  43. 43.
    Arafat W, Kern D, Dirheimer G (1985) Chem Biol Interact 56:333–349CrossRefGoogle Scholar
  44. 44.
    Askelof P, Guthenberg C, Jakobson I, Mannervik B (1975) Biochemical J 147:513–522Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Nils Helge Schebb
    • 1
  • Helene Faber
    • 1
  • Ronald Maul
    • 2
  • Ferry Heus
    • 3
  • Jeroen Kool
    • 3
  • Hubertus Irth
    • 3
  • Uwe Karst
    • 1
    Email author
  1. 1.Institut für Anorganische und Analytische ChemieWestfälische Wilhelms-Universität MünsterMünsterGermany
  2. 2.Center for Cardiovascular Research (CCR), Institut für PharmakologieCharité-Universitätsmedizin BerlinBerlinGermany
  3. 3.Department of Chemistry and Pharmaceutical Sciences, Section Analytical Chemistry & Applied SpectroscopyVrije Universiteit AmsterdamAmsterdamThe Netherlands

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