Aflatoxins and ochratoxin A in Brazilian paprika

Food Control 20 (2009) 1099–1102

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Aflatoxins and ochratoxin A in Brazilian paprika Luzia Shundo *, Adriana P. de Almeida, Janete Alaburda, Leda C.A. Lamardo, Sandra A. Navas, Valter Ruvieri, Myrna Sabino Instituto Adolfo Lutz, Divisão de Bromatologia e Química, Serviço de Química Aplicada, Seção de Química Biológica, Av. Dr. Arnaldo, 355, 01246-902 São Paulo, SP, Brazil

a r t i c l e

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Article history: Received 22 October 2008 Received in revised form 19 February 2009 Accepted 24 February 2009

Keywords: Paprika powder Aflatoxins Ochratoxin A

a b s t r a c t Seventy paprika samples collected in the city of São Paulo, Brazil, from January to April 2006 were analysed for aflatoxins and ochratoxin A (OTA) using an immunoaffinity column clean-up and HPLC-FLD. For aflatoxins, the limit of quantification (LOQ) were 0.23, 0.23, 0.45 and 0.45 lg/kg for AFB1, AFB2, AFG1 and AFG2, respectively. For OTA the LOQ was 0.80 lg/kg. Aflatoxins were found in 82.9% of samples and AFB1 was detected in 61.4% at levels ranging from 0.5 to 7.3 lg/kg with mean concentration of 3.4 lg/kg. OTA was found in 85.7% at levels ranging from 0.24 to 97.2 lg/kg with mean concentration of 7.0 lg/kg. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Aflatoxins are produced by certain species of Aspergillus where of the Aspergillus flavus and Aspergillus parasiticus are the most economically important. Aflatoxin (AFB1) is the most toxic compound of their group (Eaton, Ramsdell, & Neal, 1994). It is hepatotoxic and carcinogenic in humans. OTA is produced by some species of fungi including Aspergillus species and Penicillium verrucosum. It is nephrotoxic and hepatotoxic and it is likely to have carcinogenic potential in humans (IARC, 1993). Both groups of mycotoxins can occur jointly in a range of food commodities, including spices. Mycotoxins present in food products and animal feeds are an important problem concerning food and feed safety and significant economic losses are associated with their impact on human and animal health. Paprika is a spice normally coming from red pepper (Capsicum annum), obtained by grinding pods from dried fruits. Aflatoxins and OTA may contaminate red pepper spice at any stage of production, from preharvest to drying and storage. They plays an important role in national economy of several producing, exporting and importing countries. In 2004, the Hungarian Government banned ground paprika products after finding samples contaminated with aflatoxins above the regulatory limits of 10 lg/kg for total aflatoxins and 5 lg/kg for AFB1 (EC, 2006). Paprika has entered European Union via Spain, imported overseas from developing countries, including Brazil (FSIS, 2005). Following a notification from Hungary reporting levels of

* Corresponding author. Tel.: +55 1130682922; fax: +55 1130625363. E-mail addresses: [email protected] (L. Shundo), [email protected] (M. Sabino). 0956-7135/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2009.02.008

naturally occurring aflatoxin above the EC limits in paprika, UK’s Food Standard Agency (FSA) suggested to the European Commission a survey over popular spices including OTA analysis in paprika, in order to establish a future EC limit for this mycotoxin in this spice, since there are currently no legal limit for OTA in spices. In this regard, the present study aims to investigate contamination of aflatoxins and OTA in Brazilian paprika, using analytical methods evaluated in our laboratory to verify their efficacy for these analytes. 2. Materials and methods 2.1. Paprika samples Seventy paprika samples were purchased from local supermarkets and free markets in the city of São Paulo/Brazil, from January to April 2006. It was selected six most consumed trademarks available in supermarkets collected at least seven different lots and 28 samples purchased in free markets. Packs were from 200 g to the more frequent 50 g originated from various regions of country, mainly in the Southeast and Northeast regions of Brazil. Each sample was mixed and sub-sampled prior to analysis. 2.2. Standard and reagents All chemicals were analytical reagent grade, excepting methanol and acetonitrile (HPLC grade). They were purchased from Merck (Darmstadt, Germany). All solutions were prepared with deionized water. Standards of AFB1, AFB2, AFG1, AFG2 and OTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aflatoxins standard

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before LC analysis. The same procedure was applied for standard solutions used to calibrate the LC detector response (see Fig. 1a). 2.4.1.3. LC-FLD conditions. The mobile phase consisted in acetonitrile–methanol–water (20:20:60, V/V) solution at a flow-rate of 1 mL/min. The fluorescence detector was set to an excitation wavelength of 360 nm and an emission wavelength of 440 nm. Sample volumes of 20 lL were injected in triplicate. The linearity

200000

Derivatized aflatoxins standard Derivatized paprika sample 150000

uV

stock solutions were prepared in benzene-acetonitrile (98:2, V/V). Working solutions were prepared by diluting stock solution to 1.25 lg/kg for each aflatoxins. Concentration was checked by spectrophotometry at 350 nm according to AOAC (1997, chap. 49). OTA stock solution was prepared with toluene–acetic acid (99:1, V/V). Working solution were prepared by diluting stock solution to 1.66 lg/kg. Concentration was determined according to (AOAC, 1997, chap. 49) by spectrophotometry at 333 nm using e = 5440 L/(mol cm). A series of working standard solution was prepared by evaporation of known volumes from the stock solution under N2 stream, followed by dissolution in LC mobile phase. They were used to calibrate LC detector response and in recovery tests. The immunoaffinity clean-up columns specific for Aflatoxins (AflaStarTM) were obtained from Romer Labs. Diagnostic GmbH (Austria). For OTA (OchraprepR) it was obtained from R-Biopharm Rhone Ltd. (Scotland). 2.3. LC-FLD determination

100000

The detection of aflatoxins and OTA was carried out using a GBC system (GBC, Dandenong, Victoria, Australia) equipped with a HPLC pump (LC 1110) and a fluorescence detector (model LC 1255). Liquid chromatography separation was performed on a reversed phase LiChrosorb C18 (250  4 mm Merck, 5 lm, Darmstadt, Germany).

50000

0 0

5

2.4. Methodology

Time (min)

10

15

(a) 200000

OTA Standard Paprika sample 150000

uV

2.4.1. Aflatoxins 2.4.1.1. Sample extraction and immunoaffinity clean-up. The samples (25 g) were put into a 250 mL erlenmeyer flask with 100 mL of methanol–water (60:40, V/V) and the mixtures were shaken mechanically for 1 hour. The extracts were filtered through qualitative filter paper and 8 mL of the extracts were diluted in 16 mL PBS in order to reduce methanol content to about 20%. The diluted extracts were passed through an immunoaffinity column according to the manufacturer’s recommendation. The column was washed with 2  10 mL of deionized water (20 mL) to remove extraneous non-specific material. The aflatoxins bound to the antibody were released by elution with 2.0 mL methanol. The eluate was reduced to dryness under N2.

100000

50000

0 0

2.4.1.2. Precolumn derivatization. The trifluoroacetic acid (TFA) derivatization solution was prepared according to AOAC (1997, chap. 49). The residue from sample extracts was dissolved in 200 lL acetonitrile and added 700 lL derivatization solution. The vial was closed and heated for 8.5 min at 65 °C in water bath. This solution was filtered through 0.45 lm (Durapore Membrane Filter)

5

10

15

Time (min)

(b) 120000

Derivatized OTA standard Derivatized paprika sample

Table 1 Recovery of AFB1, AFB2, AFG1, AFG2 and OTA added to paprika powder samples. Mycotoxins

Quantity added (lg/kg)

Mean recoverya (%)

Relative standard deviations (RSD)a (%)

Aflatoxin B1

2.0 5.0

97.8 91.9

8.0 5.9

Aflatoxin B2

2.0 5.0

97.3 91.8

8.2 7.2

Aflatoxin G1

2.0 5.0

94.4 85.5

5.5 4.8

Aflatoxin G2

2.0 5.0

89.5 75.6

24.8 17.3

2.0 10.0

108.0 92.5

4.6 7.8

OTA a

Means of three determinations at 2.0, 5.0 and 10.0 lg/kg.

uV

90000

60000

30000

0 0

5

10

15

Time (min)

(c) Fig. 1. HPLC chromatograms – (a) standard solution containing 4 ng/mL of aflatoxins and paprika sample naturally contaminated with 2 lg/kg of aflatoxins, (b) standard solution containing 10 ng/mL of OTA and paprika sample naturally contaminated with 18.7 lg/kg of OTA and (c) derivatized OTA standard and derivatized paprika sample.

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determined ranged from 0.5 to 32 ng/mL of aflatoxins using eight standard solutions. 2.4.1.4. Recovery experiments. Recovery experiments were performed by spiking aflatoxin-free paprika samples with known amounts of Aflatoxins (2 and 5 lg/kg). Spiking was carried out in triplicate, besides a double analysis of blank. Aflatoxins concentrations were determined by LC analysis. 2.4.2. Ochratoxin A 2.4.2.1. Sample extraction and immunoaffinity clean-up. The samples (5 g) were extracted with 100 mL methanol–NaHCO3 1% (50:50, V/ V); the mixture was shaken vigorously for 3 min in a blender and filtered through qualitative paper filter; 20 mL of this extract were diluted in 30 mL of PBS. The diluted extract was passed through an immunoaffinity column. The column was washed with 2  10 mL PBS. OTA was eluted under gravity with 2 mL of methanol–acetic acid mixture (49:1, V/V). The eluate was reduced to dryness under N2 and the residue was dissolved in 300 lL mobile phase. This solution was filtered through 0.45 lm (Durapore Membrane Filter) before LC analysis (see Fig. 1b). 2.4.2.2. LC-FLD conditions. The mobile phase used was acetic acid aqueous solution 3.33%–acetonitrile–methanol (30:35:35, V/V) at a flow-rate of 0.8 mL/min. The fluorescence detector was set to an excitation wavelength of 332 nm and an emission wavelength of 476 nm. Sample volumes of 20 lL were injected in triplicate. The linearity was determined in the range of 2–256 ng/mL of OTA using eight standard solutions. The confirmation of OTA identity (see Fig. 1c) was performed by methyl ester formation according to AOAC (1997, chap. 49). 2.4.2.3. Recovery experiments. Recovery experiments were performed on OTA-free paprika samples, spiked with OTA levels of 2.0 and 10.0 lg/kg. Spiking was carried out in triplicate, besides a double analysis of blank. OTA concentration was determined by LC analysis. The limit of detection (LOD) was determined by repeated analysis of a blank matrix, giving a result equivalent to mean blank response plus three standard deviations. The LOQ was defined as the minimum level at which the analyte can be quantified with acceptable accuracy and precision (Garfield, Klesta, & Hirsh, 2000). 3. Results and discussion 3.1. Analytical 3.1.1. Aflatoxins The calibration curve obtained by least-squares linear regression was linear at 0.5–32 ng/mL with correlation coefficients of 0.9996, 0.9995, 0.9994 and 0.9993 for AFB1, AFB2, AFG1 and AFG2

respectively. The results of recovery of aflatoxins were summarized in Table 1. The LOD was established in 0.09, 0.09, 0.14 and 0.14 lg/kg and LOQ was 0.23, 0.23, 0.45 and 0.45 lg/kg for AFB1, AFB2, AFG1 and AFG2 respectively. These results complied with those values described in previous work about aflatoxins in spices (Fazekas, Tar, & Kovács 2005). 3.1.2. Ochratoxin A OTA calibration curve was linear at 2–256 ng/mL with correlation coefficient of 0.9999. The results of recovery of OTA were summarized in Table 1. The LOD was established in 0.24 lg/kg and LOQ was 0.80 lg/kg. Czerwiecki and Wilczynska (2005) found 0.02 and 0.06 lg/kg for LOD and LOQ for OTA analysis in paprika, respectively, with recovery ranging from 61% to 82%. 3.2. Incidence 3.2.1. Aflatoxins From 70 paprika samples analysed, 58 (82.9%) were contaminated with aflatoxins (Table 2). Forty three (61.4%) were contaminated only by AFB1, at levels ranging from 0.5 lg/kg to 7.3 lg/kg; 15 (21.5%) were contaminated by total aflatoxins with levels ranging from 1.7 lg/kg to 7.0 lg/kg. The average concentration was 3.4 lg/kg and 3.7 lg/kg for AFB1 and total aflatoxins, respectively. Thirteen (18.6%) samples analysed exceeded European Communities’ legal limits for AFB1 (5 lg/kg) and none for total aflatoxins (10 lg/kg). No sample exceeded the Brazilian legal limits (30.0 lg/kg for AFB1 + AFG1) (Brasil & Decretos, 1977). In a study over paprika, Zinedine et al. (2006) found high levels of contamination (100%) in red paprika commercialized in Morocco. The average concentration was 2.88 lg/kg for AFB1, similar to that found in our work and 5.23 lg/kg for total aflatoxins. In Spain, Hernández Hierro, Garcia-Villanova, Rodríguez Torrero, and Toruño Fonseca (2008) found AFB1 in 90% of red paprika with average concentration of 1.1 lg/kg. Tabata et al. (1993) reported low levels of concentration with 42% of positive samples. In Portugal, Martins, Martins, and Bernardo (2001) found AFB1 in 43% of samples, ranging from 1 to 20 lg/kg. Moreover, other authors described presence of aflatoxins in red pepper (Erdogan, 2004; Fufa & Urga, 1996; Vrabcheva, 2000). In a study developed in Hungary, Fazekas et al. (2005) found AFB1 in 26% of samples. According to those studies, aflatoxins were not found in home-made ground red pepper. They observed that aflatoxins are not produced under the climatic conditions prevailing in Hungary. Aflatoxins contamination of ground red pepper collected from commercial outlets was probably due to the mixing of aflatoxin-containing imported red pepper batches to batches of red pepper produced in Hungary. 3.2.2. Ochratoxin A OTA was found in 60 (85.7%) of 70 samples analysed at levels ranging from 0.2 to 97.2 lg/kg, with average concentration of

Table 2 Occurrence of aflatoxins and OTA in 70 paprika powder samples. Mycotoxins

Ochratoxin A Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 a b c d

Positive (n)

60 58 9 15 —

Range (lg/kg)

LODa–97.3 LODb–7.3 LODb–0.5 LODc–1.6
LOD for OTA = 0.24 lg/kg. LOD for AFB1 and AFB2 = 0.09 lg/kg. LOD for AFG1 and AFG2 = 0.14 lg/kg. Average = average contamination on positive samples.

Averaged (lg/kg)

7.0 3.4 1.0 0.4 —

Frequency distribution of samples (lg/kg) ND

[LOD–5.0]

[5.0–10.0]

P10.0

10 12 61 55 70

31 45 09 15 —

20 13 — — —

9 — — — —

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7.0 lg/kg. The results of OTA analysis of 70 paprika samples are presented in Table 2. Many researches revealed high contamination by OTA in different kinds of foods, although some studies address OTA contamination in spices, such as red peppers, black pepper and white pepper (El-Kady, El-Maraghy, & Eman Mostafa, 1995; Fazekas et al., 2005; Patel, Hazel, Winterton, & Mortby, 1996; Thirumala-Devi et al., 2001). According to Almela et al. (2007), despite the interest of the topic, information about the content of OTA in spices, specially in paprika, is scarce, with few known studies, or else published in journals of limited diffusion. OTA incidence and concentration levels observed in this work for paprika samples (85.7%) were higher than those described by others authors (Almela et al., 2007; Hernández Hierro et al. 2008). Hernández Hierro et al. (2008) found OTA in 67% of paprika samples, with a mean of 11.9 lg/kg. Almela et al. (2007), in a study of OTA in red paprika commercialized in Spain, found very low levels of OTA (mean of 3–4 lg/kg) in paprika cropped and produced in Brazil. The large extent of aflatoxins and OTA contamination in paprika samples, in Brazil, could be explained by tropical and subtropical climate, under which pepper is grown. High temperature and humidity offer a favorable environment for mould growth and mycotoxin development. Although there is not specific European Union regulation concerning OTA in spices in general or paprika in particular, maximum limits between 10 and 20 lg/kg can be assumed considering OTA provisional tolerable daily intake (Almela et al., 2007; JECFA, 2001). Only very few countries have regulations for OTA in food and feed products. In Brazil there is not limit for OTA in this product. Considering the high toxic potency of OTA, as well as the continuous exposure of the human population to this toxin, the present work may contribute to establish OTA limits for paprika samples. The consumption of paprika in Brazil is very low in comparison with European countries, specially Hungary and the Balcans. Most local paprika products have been designated for export, therefore, controlling aflatoxins and OTA levels in paprika produced in our country is important in order to guarantee a contamination-free product, at the same time decreasing the health risks and improving the quality of these products. References Almela, L., Rabe, V., Sánchez, B., Torrella, F., López-Pérez, J. P., Gabaldón, J. A., et al. (2007). Ochratoxin A in red paprika: Relationship with the origin of the raw material. Food Microbiology, 24(4), 319–327.

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