Purification of patulin from Penicillium expansum culture: high-speed counter-current chromatography (HSCCC) versus preparative high-performance liquid chromatography (prep-HPLC)

Abstract

Patulin is a mycotoxin produced by species of Penicillium and Aspergillus and is toxic to a wide range of organisms, including humans and livestock. To produce large amount of pure patulin for research purposes, high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were applied to the purification of patulin. Apple juice was inoculated with P. expansum and containing 0.5 mg patulin per ml was used as a starting material for separation. For HSCCC, a biphasic solvent system consisted of ethyl acetate–hexane–pH 4 acetic acid (7.5:2.5:10, v/v/v) was used. For prep-HPLC, the separation was carried out in a C18 reversed-phase preparative column with a mobile phase containing acetonitrile–pH 4 acetic acid (5:95, v/v). Fractions containing patulin were collected and analysed by analytical HPLC and identified by congruent retention time and ultraviolet/visible (UV–VIS) spectrum of the standard. The structure of the purified patulin was confirmed by mass spectrometry and nuclear magnetic resonance. HSCCC produced 21.9 mg of patulin from 50 ml apple juice culture whereas the prep-HPLC yielded 18.1 mg. HSCCC also produced purer patulin than the prep-HPLC (98.6 versus 96.3%) and higher recovery (86.2 versus 71.3%). In addition, the HSCCC method is advantageous for its lower cost and a simpler procedure compared with the prep-HPLC. This one-step HSCCC method can potentially provide a simple, effective and environmentally friendly tool for obtaining gram-level pure patulin for toxicology, detoxification and many other patulin-related studies.

Keywords:

• patulin
• purification
• apple juice
• Penicillium expansum
• high-speed counter-current chromatography (HSCCC)
• preparative high-performance liquid chromatography (prep-HPLC)

Acknowledgements

The authors would like to thank Honghui Zhu, Guelph Food Research Centre, AAFC, for technical support in MS analysis, and Dr Jun Gu, University of Guelph, for technical support in nuclear magnetic resonance analysis.