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Romanian Journal of Food Science

EDITURA ACADEMIEI ROMNE


http://www.ear.ro

Official Journal of the Romanian Association


of Food Professionals
http://www.asiar.ro

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 UVVIS 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]pyran2(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): 65 69

Food Chemistry

Monica CATAN, Luminia CATAN, Gabriela LILIOS et al.

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).

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.

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 following: 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 purification 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.

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 external 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 concentration in injected sample, function peak area obtains

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

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.

Concentration of patuulin
(g/L)

250

y = 0,0020x + 1,7689

200

R = 1,0000

150
100
50
0
0

20000

40000

60000

80000

100000

120000

Peak area

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

Food Chemistry

Determination of patulin in apple juice

Food Chemistry

Monica CATAN, Luminia CATAN, Gabriela LILIOS et al.

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.

not detected; it was artificially contaminated with


patulin, using patulin standard solution for calibration 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.

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.

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.

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 samples). In one of the apple juice samples patulin was

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.

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).

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

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).

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

60
Patulin content ( g/L)

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.

50

Davis N.D. & Dinier N.L. 1978. Micotoxyn. In Food and


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

40
30
20
10
0
I

II

III

IV

VI

VII

VIII

IX

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 Bucharest 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 performance liquid chromatography was achieved.

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

Food Chemistry

Determination of patulin in apple juice