Selective Extraction and Low Level Quantification of the Mycotoxin Patulin from Organic Apple Puree

By: K. G. Espenschied, Olga Shimelis, Michael Ye, Jennifer Claus, Reporter US Volume 33.2

Introduction

Patulin (Figure 1) is a mycotoxin produced by several genera of mold including blue Penicillium species. The toxin was discovered and characterized in the 1940’s during routine general research conducted to locate and isolate medical antibiotics. Studies in animals exposed to patulin indicated toxicity with potential adverse immunological, genetic, and gastrointestinal health effects. As a result, patulin was deemed unsuitable for antibiotic therapy1-4.

Structure of Patulin

Figure 1. Structure of Patulin

Penicillium expansum on apple skin

Figure 2. Penicillium expansum on Apple Skin

One of the Penicillium molds, P.expansum (Figure 2) commonly infects apples used to produce juice and other apple products. The mold primarily affects damaged fruit and can propagate unobserved while apples are in pre-process storage2,5. Patulin, therefore, is often found in apple products such as juice or puree. Table 1 shows low levels of patulin observed in locally acquired commercial juice samples analyzed in-house prior to this study.

Table 1. Patulin Observed In-House in Commercial Juice Samples - µg/L (n=1)

Juice From Concentrate Juice From Fresh Apples
Kosher Infant Drink Box Commercial Organic
3.6 10.4 1.9 6.4 4.7


Because exposure to patulin may negatively impact human health, most developed countries regulate patulin concentrations in apple products6. In the United States, the Food and Drug Administration (USFDA) has set an action level of 50 µg/kg for patulin in apple products for all United States consumers.

European Union member states have a regulated limit of 50 µg/kg for patulin in apple juice products labeled for adults. In addition to limits set for adults, the EU has established a 10 µg/kg limit for patulin in apple products specifically labeled for consumption by infants and young children (EC no. 1881/2006.) The more stringent limit was established on these products as children have lower body weights and tend to consume greater amounts of apple juice than adults. They are at higher risk for patulin exposure than the general population6,7.

The goal of this study was to concentrate and analyze patulin spiked into apple puree at the EC no. 1881/2006 standard for infants and children under the associated sample recovery guidelines using SupelMIP® SPE and UHPLC with UV detection8. A major brand of commercially available organic apple puree marketed specifically for infant consumption was used for testing. Cleanup and analysis was conducted using two Supelco products: SupelMIP SPE - Patulin (Product No. 52776-U) and a newly developed Titan™ C18 10 cm x 2.1 mm I.D. UHPLC column packed with proprietary 1.9 µm monodisperse silica (Product No. 577124-U). SupelMIP SPE was chosen for the selective specificity characteristic inherent in Molecularly Imprinted Polymers (MIPs), that is their ability to select low levels of a targeted analyte to the exclusion of even highly similar compounds9. The Titan HPLC column was chosen because it enhances UHPLC analysis in terms of short analysis times at lower pressures than standard sub-2 µm UHPLC columns.

Experimental

Sample Preparation and Spike
Puree samples were prepared by weighing 10 g of sample into 50 mL conical centrifuge tubes. Pectinase (150 µL) was pipetted into the apple puree followed by 100 µL of 1 µg/mL patulin stock solution (1 µg/mL patulin in water containing 0.1% acetic acid). Deionized water (9.750 mL) was added to the mixture, which was subsequently vortexed for 1 minute. The puree solution was incubated for 120 minutes in a 40 °C water bath and the mixture was centrifuged at 4,500 rpm for 5 minutes. The resulting supernatant was filtered into 15 mL conical centrifuge tubes using 10 mL syringes with 33 mm tips with 0.22 µm PVDF syringe-tip filters.

SPE Procedure
The SPE procedure for patulin selection and cleanup using filtered apple puree supernatant is described in the conditions section of Figure 3. To ensure sufficient binding interaction between the analyte of interest and the SupelMIP polymer, flow rates were maintained at 1 drop / 2 seconds when loading the samples. Carefully controlling the sample loading flow rates during the SPE process is critical to good sample recovery. Drop rates for all other steps were maintained at 1 drop / second.

UHPLC analysis of Patulin and Apple puree matrix blank

Figure 3. UHPLC Analysis of (a) Patulin spiked at 10 µg/kg in Apple Puree after SPE cleanup, (b) Patulin standard at 10 µg/kg in 0.1% Acetic Acid (aq), and (c) Apple Puree Matrix Blank

Sample Preparation and Conditions

sample/matrix: (a) apple puree samples spiked to 10 µg/kg with patulin, prepared in the folling manner: 10 grams of apple puree spiked with 150 µL of pectinase and 100 µL of 1 µg/mL patulin in 0.1% acetic acid. Dilution with 9.750 mL of deionized water, followed by heating at 40 °C, centrifugation at 4,500 rpm for 5 minutes, and filtration produces the final sample. (b) patulin standard at 10 µg/kg in 0.1% acetic acid (aq), and (c) apple puree matrix blank
SPE cartridge: SupelMIP® SPE – Patulin, 100 mg/3 mL (Product No. 52776-U)
conditioning: 2 mL acetonitrile, then 1 mL water
sample addition: 4 mL patulin spiked apple puree, patulin standard, or apple puree blank
washing: 4 mL 1% acetic acid, 1 mL 1% sodium bicarbonate, 3 mL water (immediately)
drying: 15 seconds, 6” vacuum
washing: 500 µL diethyl ether
elution: 2 mL ethyl acetate with 10 µL glacial acetic acid to stabilize patulin
elate post-treatment: Evaporate under nitrogen stream at 40 °C. Reconstitute in 1 mL of 0.1% acetic acid.
column: Titan™ C18, 10 cm × 2.1 mm, 1.9 µm (Product No. 577124-U)
mobile phase: (A) 95:5 water:acetonitrile (B) 100% acetonitrile
flow rate: 0.3 – 0.4 mL/min
column temp.: 35 °C
detector: UV, 276 nm, bw 16
injection: 15 µL

Gradient

Time (min) A(%) B(%) mL/min
0 100 0 0.3
6 100 0 0.3
6.1 20 80 0.4
7.6 20 80 0.4
7.7 100 0 0.3
9 100 0 0.3


Table 2.
UHPLC Results of the Analysis of Patulin in Apple Puree

Sample Area Adev RSD Recovery RT (min) Symmetry
Solvent (n=3) 4.77 0.25 5.30% 89.8% 3.6 0.7
Apple (n=3) 3.70 0.10 2.70% 70.2% 3.6 0.8
Blank (n=1) ND

Results and Discussion

The duration of the SPE procedure was approximately 30 minutes. Approximately 20 minutes were required to evaporate the elution fraction to dryness. Concentrations were calculated using a calibration curve with points ranging from 5 to 35 µg/kg and an R2 value of 0.9988. The chromatography indicated no evidence of patulin in non-spiked matrix samples and patulin signal response in spiked samples showed no matrix interference (Figure 3). Sample recoveries and variability (% RSD) were well within ranges set forth in Commission Regulation EC No. 401/2006, (Table 2)8. The retention time for patulin was 3.6 minutes (Table 2). A column wash was included to mitigate matrix or patulin carry over, bringing the total analysis time to 9.0 minutes.

Conclusion

A method utilizing SupelMIP SPE- Patulin cartridges and a Titan C18 UHPLC column was developed to successfully demonstrate the extraction, detection, and quantification of patulin at low concentrations in organic apple puree. SupelMIP SPE-Patulin sample cleanup enables the selective concentration of very low levels of the targeted compound along with matrix interference removal. The subsequent analysis using the Titan C18 UHPLC column provides short analysis times with good analyte signal response and peak shape (Figure 3). Together, under the applied methodologies, these products were used to effectively extract and analyze patulin with good recoveries and low variation at the most stringent testing requirements currently specified for patulin in international regulations.

Legal Information

Supel and Titan are trademarks of Sigma-Aldrich Co. LLC
SupelMIP is a registered trademark of Sigma-Aldrich Co. LLC

Materials

     

 References

  1. Demain, A. L.; Fang, A. The natural functions of secondary metabolites. Adv. Biochem. Eng. Biotechnol. 2000, 69, 1-39.
  2. Welke, J. E; Hoeltz, M.; Dottori, H. A.; Noll, I. B. Effect of processing stages of apple juice on patulin levels. Food Control 2009, 20, 48-52.
  3. Puel, O.; Galtier, P.; Ozwald, I. P. Biosynthesis and Toxicological Effects of Patulin. Toxins 2010, 2, 613-631.
  4. FDA U.S. Food and Drug Administration Foodborne Illness and Contaminants http://www.fda.gov/food/foodborneillnesscontaminants/naturaltoxins/ucm212520.htm (accessed June 2014)
  5. Rosenberger, D. A.; Engle, C. A.; Meyer, F. W.; Watkins, C. B. Penicillium expansum invades apples through stems during controlled atmosphere storage. Plant Health Progress 2006, doi:10.1094/PHP-2006-1213-01-RS.
  6. FDA U.S. Food and Drug Administration CPG Sec.510.150 Apple Juice, Apple Juice Concentrates, and Apple Juice Products – Adulteration with Patulin Site. http://www.fda.gov/ICECI/ComplianceManuals/CompliancePolicyGuidanceManual/ucm074427.htm (accessed June 2014)
  7. Commission Regulation (EC) 1881/2006. Official Journal of European Union 2006, L364, 16-17.
  8. Commission Regulation (EC) 401/2006. Official Journal of European Union 2006, L 70/32.
  9. Osman, R.; Siam, N.; Anuar, N. M.; Subari, S.N.M. Application of Molecularly Imprinted Polymer Solid Phase Extraction (MISPE) in the Extraction of Caffeine from Coffee. The Open Conference Proceedings Journal 2013, 4, (Suppl-2, M25), 111-114.