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Romanian Journal of Food Science

Official Journal of the Romanian Association of Food Professionals

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Romanian Journal of Food Science 2011, 1(1): 65 69 65

Determination of patulin in apple juice Monica CATAN 1,*, Luminia CATAN 1, Gabriela LILIOS 2, Mioara NEGOI 1, Enua IORGA 1, Nastasia BELC 1 and Alina BALEA 1 1 National Institute of Research & Development for Food Bioresources IBA Bucharest, 6 Dinu Vintila St., Sector 2, 021102

Bucharest, Romania 2 Ovidius University of Constanta, 124 Mamaia St., 900527 Constanta, Romania Received 1 November 2010; received in revised form 19 January 2011; accepted 29 January 2011

Abstract

In this paper results of performed researches for patulin determination from apple juice, through high performance liquid chromatography are presented. Patulin is extracted in acetonitrile, and for cleanup of the obtained extract C.U. Patulin columns (MycoSep228) were used. The obtained solution is evaporated to dryness under nitrogen and redissolved. Patulin is separated on chromatographic column C18, 1504 mm, 5 m (high performance liquid chromatograph, Thermo Finnigan), eluted in mobile phase (acetonitrile/water) and detected on 276 nm, using an UV-VIS DIODE ARRAY detector. A study for validation method for patulin determination from apples juice, by high performance liquid chromatography was done. In concentration range 6.25400 g/L, the average recovery is 90.50%. The limit of detection (LOD) is 2.91 g/L, and the limit of quantification (LOQ) is 9.71 g/L. The contamination degree of apple juice from commerce, using high performance liquid chromatography, was evaluated. The level of patulin concentrations in apple juice samples analyzed is under 50 g/L, the maximum allowed limit by the legislation in force: Commission Regulation of EC No 1881/2006.

Keywords: mycotoxin, apple juice, high performance liquid chromatography.

1. Introduction

Patulin is a mycotoxin produced by some species of Penicillium, Aspergillus and Byssochylamys, moulds which can grow on various foods. Its chemical name is 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one and it is a heterocyclic unsaturated lactone, 154 molecular weight, stable in acid, but unstable in alkaline medium, with the structure presented in Figure 1. Patulin is a colorless compound, crystalline, with melting point of 110C. UV maximum absorption is at 276 nm (Davis and Dinier, 1978). *Corresponding author: Fax: +40 21 210.91.28 E-mail address: mona.catana@bioresurse.ro

Patulin is stable, especially in processed products from apples: apple juice, apple concentrate juice, apple nectar, fruit nectar (which has as ingredient the apple juice also), apple piuree, apples in syrup, etc. Wine and vinegar do not contain patulin, because it does not survive the alcoholic fermentation (Moake et al., 2005).

Figure 1. Structure formula of patulin.

Patulin occurs mainly in moldy fruit. In some

cases, indoor mold development is due to insect or

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Romanian Journal of Food Science 2011, 1(1): 6569 66

other invasions attack healthy tissue, leading to patulin in fruit, which from the outside appears to be unaffected. However, patulin can occur in fruits hit, after controlled atmosphere storage and exposure to ambient conditions, with or without damage to the flesh. Washing fruit and removing moldy tissue immediately before pressing will not remove patulin present in fruit, because its running in apparently healthy tissue (Order no. 163/2006).

In a study undertaken to assess the patulin contamination of apple juice, Ggmen and Acar (1998) analyzed 246 samples of commercial apple juice. Of these, 46% had a patulin content higher than the maximum permitted by legislation in force (50 g/L).

Although spores of most molds are capable of producing patulin present in the fruit while they are still in the tree, the fruit will not develop until after harvest. Moulds can grow and produce patulin in fruit before the harvest, when the fruits are affected by illness or attacked by insects. Fruit condition at harvest, how the fruit is handled after (especially during storage), as far as storage conditions, inhibit the growth of mold, and will determine the possibility of contamination with patulin of juice and other fruit products prepared from fresh and stored fruits (Order no. 163/2006). 2. Materials and methods

For determination of patulin content, samples of clear apple juice from the market were analyzed.

The determination of patulin from apple juice was performed by high performance liquid chromatography (Dombrink-Kurtzman, 2001). Steps of method for patulin determination are the follow-ing: preparation of test sample, patulin extraction, extracts purification, extract evaporation to dryness, residue re-dissolution, and HPLC detection of patulin. In figure 2 C.U. Patulin (Romer Labs Application Brief, 2003) columns, used for purifica-tion are presented.

HPLC perform conditions (high performance liquid chromatograph, Thermo Finnigan) are: Chromatographic column C18, 150 4 mm, 5

m (Figure 2): Mobile phase: acetonitrile: water = 5:95 (v/v); Mobile phase volume: 1 mL/minute; UV-VIS ,DIODE ARRAY detector, = 276 nm; Injection volume: 25 L; Temperature: 25C.

For calibration curve, patulin standard in crystalline form (Fluka) was utilized. It was dissolved in ethyl acetate (gradient grade for HPLC). This solution was re-dissolved in ethanol (c 99.7%, v/v). Working solution of patulin was prepared in ultra-pure water with pH = 4. This solution was utilized for calibration curve.

Figure 2. C.U. Patulin columns.

3. Results and discussion

For patulin determination from apple juice by high performance liquid chromatography, a calibration curve with 7 patulin standard levels (with each three repetitions), in concentrations range 0.00625 g/mL 0.400 g/mL was realized. The calibration curve is presented in Figure 3.

The linearity of response, expressed in terms of regression coefficient (R2), shown a value of 0.999912, implying a good suitability of the HPLC method.

Peak identification was based on retention time (tR), spectral information and fortification technique (spiking). Peak quantification was based on the ex-ternal standard method, using calibration curve (software ChromQuest 4.2 Thermo Finnigan).

For linearity domain establishment of patulin determination method from apple juice, by high performance liquid chromatography, apple juice samples in what was not detected patulin, were artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/mL (Figure 4).

Thus, there were obtained apples juice samples, with the following patulin concentrations: 10 g/L, 15 g/L, 25 g/L, 50 g/L 60 g/L, 80 g/L, 100 g/L, 125 g/L, 200 g/L and 400 g/L. These samples were analyzed for patulin concentration determination.

By graphic representation of patulin concen-tration in injected sample, function peak area obtains

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Determination of patulin in apple juice

Romanian Journal of Food Science 2011, 1(1): 6569 67

a right line with ecuation y = 0.0020x + 1.7689, and correlation coefficient, R2 = 1.000.

According to the obtained results, linearity domain of patulin determination method from apples juice, by high performance chromatography, is 5218 ppb.

For recovery establishment, in case of patulin determination method from apples juice, by high performance chromatography (Figure 5), apples juice samples in what was not detected patulin, were artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/mL.

In concentration range 6.25400 g/L, the average recovery had the following values: 96.64%, for patulin concentrations < 25 g/L;

90.31%, in patulin concentrations range 2550 g/L;

88.40%, for patulin concentrations > 50 g/L. In concentration range 6.25400 g/L, the

average recovery is 90.50%. For repeatability estimation, parallel apple juice

samples with patulin concentration 25 g/L, 50 g/L and 100 g/L (apple juice in what was not detected patulin, it was artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/ml) were analyzed and the following statistical indicators were calculated: relative standard deviation value RSD(r), for

determined patulin concentration; extended uncertainty; limit repeatability.

Figure 3. Patulin calibration curve.

y = 0,0020x + 1,7689R2 = 1,0000

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0 20000 40000 60000 80000 100000 120000

Peak area

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Figure 4. Linearity domain of method for patulin determination in apple juice,

by high performance liquid chromatography.

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Romanian Journal of Food Science 2011, 1(1): 6569 68

Figure 5. Chromatogram with patulin peak (spiked patulin in apple juice).

In case of 10 parallel samples of apples juice with

patulin concentration 25 g/L (apples juice in what was not detected patulin, it was artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/ml), relative standard deviation RSD(r), for determined concentration is 3.18%, and extended uncertainty is 4.20 g/L. Confidence interval is 24.994.20 g/L. Limit repeatability is: 2.22 g/L.

In case of 10 parallel samples of apple juice with patulin concentration 50 g/L (apple juice in what was not detected patulin, it was artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/mL), relative standard deviation RSD(r), for determined concentration is 1.84%, and extended uncertainty is 4.53 g/L. Confidence interval is 49.20 4.53 g/L. Limit repeatability is: 2.53 g/L.

In case of 8 parallel samples of apple juice with patulin concentration 100 g/L (apples juice in what was not detected patulin, it was artificially contaminated with patulin, using patulin standard solution for calibration, with patulin concentration 1 g/ml), relative standard deviation RSD(r), for determined concentration is 1.31%, and extended uncertainty is 5.36 g/L. Confidence interval is 100.365.36 g/L. Limit repeatability is: 3.67 g/L.

For intralaboratory reproducibility estimation, 8 apple juice samples with patulin concentration 50 g/L were performed by three analysts (analyst A 3 samples, analyst B 2 samples, analyst C 3 sam-ples). In one of the apple juice samples patulin was

not detected; it was artificially contaminated with patulin, using patulin standard solution for calibra-tion to obtain a patulin concentration of 1 g/mL. Than the following parameters were calculated: the relative standard deviation value RSD(R), for

determined patulin concentration; the extended uncertainty; the limit reproducibility.

In case of 8 samples of apple juice with patulin concentration 50 g/L, analyzed by three analysts (analyst A 3 samples, analyst B 2 samples, analyst C 3 samples), in the same laboratory, with the same equipment, relative standard deviation RSD(R) is 4.75% and extended uncertainty is 4.53 g/L.

Confidence interval is 49.77 4.53 g/L. Limit reproducibility is 6.62 g/L.

Limit of detection (LOD) is defined as being the concentration of which signalto-noise ratio is higher than 3 (S/N > 3). Limit of detection is the value which fixes inferior limit of work domain.

Limit of quantification (LOQ) is defined as being the lowest concentration of analyst, which can be determined with acceptable precision, under conditions of analysis method, at a signalto-noise ratio higher than 10 (S/N > 10).

In case of proposed method for patulin determination by high performance liquid chromatography, limit of detection (LOD) is 2.91 g/L, and limit of quantification (LOQ) is 9.71 g/L.

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Romanian Journal of Food Science 2011, 1(1): 6569 69

The proposed method for patulin determination by high performance liquid chromatography is selective. The patulin adequate peak is separated by baseline and by other compounds peaks. Resolution (DAB) is min. 2.7.

Using this method there were analysed clear apple juice samples, from commerce, in order to determine patulin content (Figure 6).

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I II III IV V VI VII VIII IX X

Sample Figure 6. Patulin content of apple juice.

Over 80% of the analyzed samples had not

patulin, and within rest of them patulin concentration was under 50 g/L, maximum allowed limit by the legislation in force (Commission Regulation (EC) No 1881/2006). In Figure 8 it is presented the patulin content of apple juice samples, in which patulin was detected (16.85 27.52 g/L). 4. Conclusions

The method for patulin determination from apple juice by high performance liquid chromatography was developed in the National Institute of Research & Development for Food Bioresources-IBA Bucha-rest laboratories.

Patulin is separated on chromatographic column C18, 150 4 mm, 5 m (high performance liquid chromatograph Thermo Finnigan), eluted in mobile phase and detected on 276 nm, using an UV-VIS DIODE ARRAY detector.

An internal study for validation of method for patulin determination from apple juice by high per-formance liquid chromatography was achieved.

In concentration range 6.25400 g/L, the average recovery is 90.50%. Limit of detection (LOD) is 2.91g/L, and limit of quantification (LOQ) is 9.71 g/L.

Patulin concentrations of the analyzed apple juice samples are under 50 g/L, and the maximum allowed limit by the legislation in force (Commission Regulation (EC) No 1881/2006). References Davis N.D. & Dinier N.L. 1978. Micotoxyn. In Food and

Beverage Mycology, Beuchat L.R. (Ed.) AVI, Westport, CT, 397444.

Dombrink-Kurtzman M.A. 2001. Comparison of cleanup procedures for analysis of patulin in apple juice by high-performance liquid chromatography with photodiode array detection, Mycotoxin Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, USA.

Ggmen V. & Acar J. 1998. Incidence of patulin in apple juice concentrates produced in Turkey. J. Chromatography A. 815: 99-102.

Moake M., Padilla-Zakour O. & Worobo R. 2005. Comprehensive Review of Patulin Control Methods in Foods. Comprehensive Reviews in Food Science and Food Safety. 1.

* * * Romer Labs Application Brief. 2003. Rapid quantization of patulin in various juices by HPLC UV. Applicable for: clear and cloudy apple juice, hawthorn juice.

* * * Commission Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs.

* * * Order no. 163/2006 for approval of Norme concerning prevention and reduction of patulin contamination in apple juice and apple juice used as ingredient for other drinks.

Abbreviations HPLC high-performance liquid chromatography LOD limit of detection LOQ limit of quantification

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2011-no1-09-Catana-p652011-no1-09-Catana-p66-69