The mutagenic and deleterious mold fungus product patulin can formulate during incorrect cider-making processes of fruits such as apple. In apple juice, the permitted daily exposure for patulin was stated as 0.05 µg/mL by the scientific committee for foodstuffs of the European Commission.
The specified determination routines must be carried out with HPLC to adhere to official guidelines. In this article, a quick protocol for the quantification of patulin from apple juice with AZURA® HPLC plus system and a Eurospher II column is outlined.
The lactone patulin is part of the chemical group of polyketides. It is soluble in acidic water and numerous organic solvents such as methanol. Different species of fungi, for example byssochlamys, aspergillus, and penicillium, which grow on rotting parts of fruits such as blueberries, plums, apples, cherries, pears, and strawberries can produce the mycotoxin patulin.
If affected fruits are used for the cider-making process, juices can be contaminated with patulin. Patulin cannot be destroyed by pasteurization or thermal denaturation due to its thermal stability. In addition to its antibiotic qualities, patulin is seen as a possible carcinogen but the toxicity of patulin is mostly because of its affinity to sulfhydryl groups, which result in inhibition of enzymes.
Major acute toxicity findings are inclusive of gastrointestinal problems, pulmonary congestion, neurotoxicity, and edema. In order to protect customers from patulin in juices, the FDA and the European Commission (EU) recommend a maximum daily ingestion concentration of 0.4 µg/kg body weight for humans according to 93/5/EWG and 0.05 µg/mL in apple juice per day.
Additionally, the EU has set a cap of 10 µg/kg in baby food and 25 µg/kg in solid apple products (2003/598/EG).
The extremely quick and robust technique with AZURA HPLC plus for the identification of patulin in juices such as apple juice allows the validation of the concentration according to the regulation of the FDA and EU, and at lower concentrations. The minimum concentration, which was calculated using a 50 µL injection, was 0.05 µg/mL apple juice with a signal to noise ratio (S/N) of 216.4.
The short retention times of patulin and hydroxymethylfurfural (HMF) allow high efficiency for the analysis. The low noise value of 50 µAU permits a detection of patulin at the limit of quantification (LOQ) with a concentration of 0.003 µg/mL. As can be observed in Fig. 2 A, a good separation from all matrix peaks (Rs >1.5) comfortably assures the quality of the measurements of the concentration of 0.25 µg/mL patulin and HMF.
Figure 1: Patulin contamination in apple juice
Figure 2: Chromatogram of 50 μL injections from apple juice concentrate spiked with 0.25 μg/ mL patulin and HMF (A) and an concentration curve of patulin with the same injetion volume (B). Concentrations of 1.00 μg/mL, 0.50 μg/mL, 0.40 μg/mL, 0.25 μg/ mL, 0.10 μg/mL and 0.05 μg/mL patulin (n=3) for the concentration curve
Materials and Methods
To develop a quick and simple isocratic technique, an AZURA HPLC plus system which can cool the autosampler rack to restore the quality of the food samples, plus a DAD detector with booster flow cell to monitor impurities at different wavelengths were utilized. The eluent was a mixture of 10 % acetonitrile in water.
Within seven minutes, the separation was realized using a phenyl end-capped Eurospher II 100-5 Phenyl column at an ambient temperature of 40 °C. So, 10 mL of juice was combined with 600 µL pectinase enzyme and incubated at 37 °C for three hours before being centrifuged at 3500 U/min for ten minutes.
The liquid was then centrifuged again under the same conditions after removing the centrifugate. The apple juice, which is now concentrated, is prepared for HPLC analysis.
The concentrated juice was combined with a standard solution of patulin and HMF to attain a concentration of 0.25 µg/mL for the measurements in Fig. 2 A. A concentration curve from 0.05 µg/mL to 1 µg/mL of patulin was measured utilizing a standard of patulin in water (Fig. 2 B) to evaluate the concentration in apple juice.
A quick and efficient HPLC technique was realised with AZURA HPLC using an isocratic method with a retention time of just seven minutes. The cooling possibility of the autosampler enhances the lifespan of sensitive food samples and a column thermostat allows a continuous separation atmosphere above room temperature, which is vital for the high robustness of the technique.
The identification of patulin from apple juice can be realized down to the very low concentration of 0.003 µg/mL using these benefits. According to 2003/598/EG of the EU, this is 16 times lower than the recommended maximum daily ingestion concentration for humans.
- 2003/598/EG, Amtsblatt der Europäischen Union zur Prävention und Reduzierung der Patulinkonzentration in Apfelsaft und Apfelsaftzutaten in anderen Getränken, 2003, L203/54. https://eur-lex.europa.eu/legal-content/DE/TXT/PDF/?uri=CELEX:32003H0598&from=EN
- G. C. Llewellyn, J. A. McCay, R. D. Brown, D. L. Musgrove, L. F. Butterworth, A. E. Munson, Jr. K. L. White, Immunological evaluation of the mycotoxin patulin in female B6C3F1 mice. Food Chem. Toxicol. 1998, 36, 1107–1111.  O. Puel, P. Galtier, I. P. Oswald, Biosynthesis and Toxicological Effects of Patulin, Toxins, 2010, 2 (4): 613–631.
Additional Materials and Methods
Table A1. Method parameters
Table A2. Instrument set up
||AZURA P6.1L, LPG
||AZURA AS 6.1L
||AZURA DAD 6.1L
||LuightGuide 50 mm
||AZURA CT 2.1
||KNAUER Vertex Plus column, Eurospher II 100-5 Phenyl, 150 x 4.6 mm
||ClarityChrom 7.4.2 – Workstation, autosampler control included
ClarityChrom 7.4.2 – PDA extension
KNAUER is an owner-managed middle-sized company with more than 120 employees situated in Berlin, Germany. Since 1962, KNAUER has been producing and developing high performance liquid chromatography (HPLC) systems.
The HPLC technique is used to separate, identify and quantify substances in a mixture. It can be used for many applications, including the analysis of toxics in drinking water, the detection of drugs in blood as well as for the purification or quality inspection of pharmaceutical and chemical products.
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