Molecularly Imprinted Polymer for Solid Phase Extraction of Patulin mycotoxin Delphine Derrien, Mélissa Mulet, Florent Alix, Céline Pérollier, Olivier Lépine, Kaynoush Naraghi, Sami Bayoudh*

POLYINTELL- Voie de l’innovation Pharmaparc II 27100 Val de Reuil , FRANCE. www.polyintell.com [email protected] Introduction Patulin is a mycotoxin produced by a variety of molds, particularly Aspergillus We have developed a new class of solid intelligent polymers based on and Penicillium . It is commonly found in rotting apples, and the amount of molecularly imprinted polymers and this phase has proved to be a powerful patulin in apple products is generally viewed as a measure of the quality of technique for clean-up and preconcentration of Patulin. the apples used in production. O OH Studies showed that Patulin is genotoxic. Several countries have instituted Patulin patulin restrictions in apple products. The World Health Organization, the FDA and the European Union recommend a maximum concentration of 50 µg/kg in O apple juice and 25µg/kg in apple puree and 10µg/kg in babyfood. O Principle of AFFINIMIP ® Application of AFFINIMIP ® SPE MIP is a process that creates a three-dimensional network that has a « memory » of the shape and functional group positions of the template molecule.

Loading Washing Elution

This is a process where functional and cross-linking monomers are copolymerized in the presence of the target analyte (the imprint molecule). The functional monomers form a complex with the imprint molecule, and following Treated sample copolymerization, their functional groups are held in position by the highly crosslinked polymeric structure. Subsequently thanks to the removal of the imprint molecule, it reveals binding sites that are complementary in size and shape to the analyte. Extraction from Apple Juice using AFFINIMIP ® Patulin

800

Step Solvent 70 70 700 Before treatment Patulin Spiked at 40µg/kg 4mL of apple juice 60 60 600 Loading (after dilution by 2 with Water-2% After AFFINIMIP ® Patulin 50 50 Spiked at 10µg/kg acetic acid) 500 40 40

30 30 400 1mL 1% NaHCO mAU mAU 3 Concentration Spiked at 0µg/kg mAU 2mL Water Recoveries RSD 20 20 300 of Patulin Washing % % Dry 10 seconds (µg/kg) 10 10

200 1mL Diethyl Ether Patulin 0 0 10 (n=9) 97.9 11 Elution 2mL Ethyl Acetate 100 -10 -10

40 (n=41) 90.6 11 -20 -20 0 HMF Analysis by LC-UV 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Minutes 9 10 11 12 13 14 15 16 17 18 19 Column : Atlantis T3 column 150 X 2.1mm id (3µm) Minutes Mobile Phase : ACN/Water 5/95 (v/v) Apple juice spiked at 46ng/mL UV: 276nm, F : 0.2mL/min Extraction from Apple Puree using AFFINIMIP® Patulin

Patulin UV 6000 -276nm- UV 6000 -276nm UV 6000 -276nm Step Solvent C07 C10 C15 140 140 Before treatment 50 Spiked at 25µg/kg50 5mL of apple puree 120 120 40 40 Loading (after treatment with pectinase After AFFINIMIP ® Patulin 30 30 100 100 Spiked at 10µg/kg enzyme) 100

20 20 8080 80 mAU 4mL Water-1% acetic acid mAU mAU Spiked at 0µg/kg 10 10

6060 60 mAU 4mL Water Concentration mAU Washing Recoveries RSD 0 0 Dry 10 seconds 4040 40 of Patulin

% % -10 -10

500µL Diethyl Ether 20 20 (µg/kg) 20

Patulin -20 -20 Elution 2mL Ethyl Acetate 00 0 10 (n=9) 77.4 8.1

-30 -30 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Minutes

Analysis by LC-UV 25 (n=8) 90.9 11 -40 -40

15 16 17 18 19 20 21 22 23 24 25 Column : Atlantis T3 column 150 X 2.1mm id (3µm) Apple puree spiked at 40µg/kg Minutes Mobile Phase : gradient ACN/Water 2 different apple purees UV: 276nm, F : 0.2mL/min spiked at 10 and 25µg/kg Conclusion The detection of patulin at trace level is a real interest to determine the quality of apples used in apple juice or puree. A new AFFINIMIP ® SPE has been developed for extraction of patulin from apple juice and apple puree and more than 75 % recovery were obtained with an efficient clean up (suitable chromatograms with a limited furfural major interference have been obtained) . Other related AFFINIMIP ® SPE

AFFINIMIP ® Zearalenone: SPE from cereal extract AFFINIMIP ® Ochratoxin A : SPE from cereal extract

Perfect clean-up method with AFFINIMIP ® Zearalenone Perfect clean-up method with AFFINIMIP ® Ochratoxin A

9

140 8

120 7 With AFFINIMIP ® ZON With AFFINIMIP ® OTA

100 6

80 5 OTA

No pre-treatment 4 No pre-treatment 60 ZON

40 3

2 20

1 0

0

2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

Chromatograms obtained after a purification with Chromatograms obtained after a purification with AFFINIMIP ® ZON (80µg/kg) of a cereal sample AFFINIMIP ® OTA (2.5µg/kg) of a cereal sample

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