This article was downloaded by: [University of Arizona] On: 15 September 2013, At: 14:20 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxins in selected Thai commodities Natthasit Tansakul
a b
c
b
b
, Sasithorn Limsuwan , Josef Böhm , Manfred Hollmann &
Ebrahim Razzazi-Fazeli
d
a
Department of Pharmacology, Faculty of Veterinary Medicine , Kasetsart University , Bangkok , Thailand b
Institute of Animal Nutrition , University of Veterinary Medicine Vienna , Vienna , Austria
c
Molecular Phytopathology and Mycotoxin Research, Department of Crop Sciences , Göttingen University , Göttingen , Germany d
VetCore Facility for Research, VetOMICS , University of Veterinary Medicine Vienna , Vienna , Austria Accepted author version posted online: 12 Jun 2013.Published online: 16 Jul 2013.
To cite this article: Natthasit Tansakul , Sasithorn Limsuwan , Josef Böhm , Manfred Hollmann & Ebrahim Razzazi-Fazeli , Food Additives & Contaminants: Part B (2013): Aflatoxins in selected Thai commodities, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.812148 To link to this article: http://dx.doi.org/10.1080/19393210.2013.812148
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2013 http://dx.doi.org/10.1080/19393210.2013.812148
Aflatoxins in selected Thai commodities Natthasit Tansakula,b*, Sasithorn Limsuwanc, Josef Böhmb, Manfred Hollmannb and Ebrahim Razzazi-Fazelid a Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand; bInstitute of Animal Nutrition, University of Veterinary Medicine Vienna, Vienna, Austria; cMolecular Phytopathology and Mycotoxin Research, Department of Crop Sciences, Göttingen University, Göttingen, Germany; dVetCore Facility for Research, VetOMICS, University of Veterinary Medicine Vienna, Vienna, Austria
Downloaded by [University of Arizona] at 14:20 15 September 2013
(Received 1 January 2013; final version received 3 June 2013) Aflatoxin (AF) B1, B2, G1 and G2 were determined in 120 samples of selected Thai commodities including unpolished rice, unpolished glutinous rice, chilli powder, whole dried chilli pods and raw peanut. The mean concentrations of the total AFs for analysed samples were 0.16, 25.43, 14.18, 6.62 and 1.43 µg kg−1 with positive incidences of 4%, 20%, 97%, 37% and 30%, respectively. Quantitative analysis was performed using HPLC equipped with post-column derivatisation and fluorescence detection. Sample clean-up was carried out using immunoaffinity columns for selective enrichment of AFs. The method was validated by using certified reference material, which showed recoveries over 85%. The limit of detections (LODs) and limit of quantifications (LOQs) were in a range between 0.01–0.11 µg kg−1 and 0.03–0.38 µg kg−1, respectively. The results clearly demonstrated that AFs were detectable in different matrices. Chilli powder was found to have the highest level of AFs contamination followed by chilli pods, peanut and rice, respectively. However, among the selected commodities, unpolished rice contained only trace levels of AFB1 and AFB2. With regard to the fact that AFs are a natural contaminant in commodities, this report calls to attention the regular monitoring and effective control of food commodities to prevent health hazards. Keywords: aflatoxins; rice; chilli; peanut; HPLC
Introduction Unavoidable mycotoxin contamination in food commodities is of global concern as they are one of the most harmful fungal metabolites to human and animal health. Aflatoxins are a group of toxic metabolites produced mainly by the fungi Aspergillus flavus and Aspergillus parasiticus growing on a variety of agricultural products during environmentally favourable conditions, particularly in the tropical zone. The four major aflatoxin analogues are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). AFB1 is classified as carcinogen class 1A (International Agency for Research on Cancer 1993). The EU food law has set maximum limits of 2 µg kg−1 for AFB1 and 4 µg kg−1 for total AFs in peanut and cereals. Maximum levels in spices are 5 µg kg−1 for AFB1 and 10 µg kg−1 for the sum of AFs (European Commission 2010). Rice (Oryza sativa L.), a tropical grain, is one of the staple food crops in the world. Rice and rice products are also used as feed ingredient in animal production. World rice production was forecasted to be about 476 million tons (on a milled basis) in 2011–2012 and Thailand is one of the major rice exporters in the world. It was estimated that the mean unpolished rice consumption by the Thai population was 25.60 g/person/day. The occurrence of mycotoxins in rice has been reviewed by Tanaka et al. *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
(2007). Aflatoxins were found worldwide in rice samples in the Philippines (Sales & Yoshizawa 2005), China (Liu et al. 2006), Vietnam (Nguyen et al. 2007), India (Reddy et al. 2009), Sweden (Fredlund et al. 2009), Austria (Reiter et al. 2010), Germany (Reinhold & Reinhardt 2011) and Canada (Bansal et al. 2011). In addition, glutinous rice, known as sticky rice, is a kind of rice mostly consumed as a main dish and also preferred to serve as a desert or sweet between meals in some regions of Asia. However, only few studies are available on aflatoxin contamination in unpolished rice and unpolished glutinous rice. Chilli, Capsicum genus of plants, is ranked second after black pepper among the world spices (Iqbal et al. 2010). Aspergillus spp. was found to be a predominant fungal infection in chilli (Reddy et al. 2001). AFs contamination in several kinds of spices has been reported elsewhere (Cho et al. 2008; Reinhold & Reinhardt 2011). Among monitoring of spices in the Irish market, chilli powder was found to be contaminated with AFs (range 0.35–27.50 µg kg−1) in higher concentrations than others (O’Riordan & Wilkinson 2008). In other former surveys, AFs have also been detected in the chillies in Portugal (Martins et al. 2001), India (Reddy et al. 2001), Pakistan (Iqbal et al. 2010) and Spain (Santos et al. 2010). Although most countries in the world have no specific legal limits on AFs for spices, the current study provides
Downloaded by [University of Arizona] at 14:20 15 September 2013
2
N. Tansakul et al.
data on AFs contamination in chilli with regard to the EU guidelines for AFs level in spices. High incidences and levels of AFs contamination in peanut has widely been reported for decades, that is, in Japan (Tabata et al. 1993), the Philippines (Ali et al. 1999), Malaysia (Sulaiman et al. 2007) and more recently in Sudan (Elzupir et al. 2011). During 1967–1987, there were reports on peanut contaminated with AFs in Thailand. Since then, a limited number of scientific reports on the incidence and levels of AFs in peanut in Thailand have been recorded (Waenlor & Wiwanitkit 2003). Countries in tropical zones suffer from AFs contamination in peanut as a cause of liver cancer (Liu & Wu 2010). Therefore, surveillance programmes on AFs in peanut are a necessity. Thailand, located in a tropical zone with suitable conditions for AFs biosynthesis, is one of the major agricultural food exporters in the world. Thus far, there are few reports on AFs in rice, chilli and peanut from Thailand (Waenlor & Wiwanitkit 2003; Tanaka et al. 2007). The present study aims to investigate the contamination of AFs content in selected Thai commodities using a selective and sensitive method.
Materials and methods Samples In total, 120 samples including 25 unpolished rice, 10 unpolished glutinous rice, 30 chilli powder, 30 whole dried chilli pods and 25 raw peanut samples were randomly collected from either retail fresh markets or modern trade department stores in the central region of Thailand. Sample sizes ranged between 0.5 and 1 kg. After mixing well, each sample was divided into 100 g subsamples, from which portions were taken to collect a final 100 g sample. The pooled 100 g sample was finely ground and 10 g was taken for analysis. Unpolished rice and unpolished glutinous rice originated from Thailand (without mixing of white milled rice), but the original source of chilli and peanut were not declared. All samples were ground and sieved at 1 mm diameter, except for peanut. Thereafter, they were labelled and put into polyethylene bags and placed at –20°C pending analysis.
Chemical and reagents A mixed aflatoxin standard was purchased from Supelco™ (Bellefonte, PA, USA), containing 1.026, 0.297, 1.054 and 0.302 µg ml−1 AFB1, AFB2, AFG1 and AFG2, respectively. HPLC grade solvents (acetonitrile and methanol) were purchased from Fisher Scientific (Essex, UK). Purified water was produced by a UPW2 system (F&L, Vienna, Austria). Sample clean-up was performed using AflaClean™ immunoaffinity columns
(IACs) (LC Tech™, Dorffen, Germany). The chilli powder certified reference material (CRM, reference number T04132) was purchased from FAPAS™ (Food Analysis Performance Assessment Scheme, The Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, UK). HPLC conditions The HPLC system consisted of a pump (LC9A, Shimadzu, Tokyo, Japan) connected to an autosampler equipped with a 100-µL injection loop (AS-2000™, Merck-Hitachi, Tokyo, Japan). A guard column (20 × 4 mm) was placed before the analytical column (250 × 4 mm), both filled with Lichrospher 100_RP 18E 5 µm (Bischoff chromatography Leonberg, Germany). Analysis was run at a flow rate of 1 ml min−1 by an isocratic mobile phase using a mixture of water/methanol/acetonitrile (62:22:16, v/v/v) where 119 mg/L of potassium bromide (KBr) and 350 µl/L 4 M HNO3 were added. The analytical column oven (Jetstream 2 plus, TECHLAB, Erkerode, Germany) temperature was set at 50°C. Detection was carried out by a fluorescence detector (Waters 474, MA, USA) coupled with a Kobracell (R-Biopharm Rhône LTD, Glasgow, UK) to achieve signal enhancement. Excitation and emission wavelengths were 365 and 440 nm, respectively. Chromatograms were displayed with a Stratos™ LC software (Polymer Laboratories, Version 4.5, Shropshire, UK). Sample preparation Briefly, 30 ml of acetonitrile/water (60:40, v/v) was added to 10 g sample, using a 100 ml Schott-Duran™ glass container. After stirring well with a magnetic bar for 30 min, the matrix was filtrated through a paper filter and transferred into a polypropylene test tube. For cleanup, IACs were allowed to stand at room temperature for 30 min prior to starting the clean-up protocol. Meanwhile, 3 ml of the extracted solution was diluted with 30 ml PBS except for chilli, where a PBS/Tween-20 (96:4, v/v) solution was applied. The diluted samples were flushed through an IAC column with a flow rate of one drop per second. After washing two times with 30 ml PBS and 10 ml of distilled water, respectively, the sample was eluted two times with 1 ml methanol. Each eluate was put into a glass test tube and allowed to dry under a gentle nitrogen stream. The sample was reconstituted with a 1-ml mobile phase and put in an autosampler vial.
Analytical procedure Standard curves were constructed daily by duplicate injection of five concentrations in a range of 0.5–15 ng ml−1 using mixed AFB1, AFB2, AFG1 and AFG2 solutions.
Food Additives & Contaminants: Part B Linear regression provided correlation coefficients (R2) above 0.997 for all curves. The limit of detection (LOD) and limit of quantification (LOQ) were determined by taking the signal-to-noise ratios of 3:1 and 10:1, respectively, using spiked mixed standard AFB1, AFB2, AFG1 and AFG2 concentrations of 10.2, 2.9, 10.5 and 3.0 ng ml−1, into blank matrices (n = 3). Repeatability and reproducibility were obtained by three replicates of each fortified matrices in three different days. To control the performance method, certified material reference of chilli (AFB1 and AFB2) was applied.
Downloaded by [University of Arizona] at 14:20 15 September 2013
Results and discussion Validation of the method Table 1 summarises the validation results of AFs analysis in different matrices. High recoveries at the spike levels for AFs contents, particularly aflatoxin B series in all matrices were noted. O’Riordan and Wilkinson (2009) found that using a mixture of methanol/water (80/20; v/v) as an extraction solvent for AFs in chilli may provide better recoveries. Low LOD and LOQ values indicated high sensitivity. According to the specifications of the certified reference material, mean level of AFs in the chilli CRM should be 8.84–22.73 µg kg−1 with a standard deviation of 3.52 for AFB1 and 0.42–1.07 µg kg−1 with a standard deviation of 0.16 for AFB2. The method provided z-scores < 2, which is satisfactory. According to the EU food regulation (European Commission 2006), recoveries in the concentration range of 1–10 µg kg−1 are acceptable when between 70 and 110% and RSD should be less than 20%. The applied
Table 1.
Validated values of AFs in the different matrices.
Intra-day (n = 3)
Inter-day (n = 3)
0.03 0.05 0.03 0.13
106.8 94.2 103.9 71.5
4.4 3.8 2.8 5.3
4.9 3.5 5.1 1.3
0.04 0.11 0.05 0.38
84.5 71.8 76.6 41.6
4.5 6.0 6.4 7.4
1.3 0.4 3.3 3.5
0.04 0.11
92.9 93.6
2.9 7.5
1.6 2.2
0.04 0.03 0.03 0.19
87.3 91.7 81.2 71.9
2.1 3.2 4.1 3.4
2.4 4.1 5.9 2.7
LOQ LOD Matrix (µg kg−1) (µg kg−1) Rice AFB1 0.01 AFB2 0.01 AFG1 0.01 AFG2 0.04 Chilli AFB1 0.01 AFB2 0.03 AFG1 0.01 AFG2 0.11 Chilli (CRM) AFB1 0.01 AFB2 0.03 Peanut AFB1 0.01 AFB2 0.01 AFG1 0.01 AFG2 0.06
RSD (%)
Mean recovery (%) (n = 3)
3
method in this study showed satisfactory results, except in the case of AFG2 in chilli. Occurrence of aflatoxins in rice samples In Table 2, the occurrence of AFs in unpolished Thai rice is presented, which clearly shows the low incidence of AFs in Thai rice. This is in agreement with previous reports, where only low content of AFs was detected in a few rice samples (Sales & Yoshizawa 2005; Reiter et al. 2010). As reported by Fredlund et al. (2009), AFB1 was detected in 71% basmati rice samples and in 20% jasmine rice samples. In this case, it was also detected in high fibre content in jasmine rice (unpolished Thai rice) sold at the Swedish market. The authors reported AF levels between 0.1 and 50.7 µg kg−1. In 2010, AFs were found in more than 70% of the 22 analysed basmati rice samples in Germany with a mean concentration of 1.53 µg kg−1 (Reinhold & Reinhardt 2011). In Canada, two years study of AFs contamination in rice reported mean values of 0.34–0.39 µg kg−1 in AFB1 (with a maximum level of 7.14 µg kg−1). These authors also reported AFs contamination in black and red rice exported from Thailand (Bansal et al. 2011). In the current study, only trace amounts of AFB1 and AFB2 in one sample unpolished rice was noted. Higher concentrations were found in unpolished glutinous rice than unpolished rice. In contrast, no AFG1 and AFG2 were detected in both unpolished rice and glutinous rice samples. This is in contrast to a former report of Jankhaikhot (2005) who found only AFG1 in unpolished rice of Thailand. It may indicate the vast diversity of aflatoxin-producing fungal species and environmental conditions for AFs production in Thailand (Erhlich et al. 2007). Due to processing and rice composition, unpolished rice is more prone to be contaminated with AFs than polished rice (Jankhaikhot 2005). Nevertheless, AFs concentrations in unpolished rice samples did not exceed EU regulations, except for one sample of unpolished glutinous rice. Occurrence of aflatoxins in chilli samples In Table 3, contamination in chilli powder and whole dried chilli pods is shown. Out of 30 chilli powder and chilli pods samples, 29 and 11, respectively, were contaminated with AFs. Among positive chilli tested samples in this study, there were 16 samples of chilli powder and 1 sample of chilli pods contaminated with AFs higher than the acceptable limit of 5/10 μg kg−1 for AFB1/sum AFs as prescribed by the EU (European Commission 2010). This finding confirms previous studies of incidences with high levels of AFs in chilli powder samples, like 93 µg kg−1 (Paterson 2007), 27.50 µg kg−1 (O’Riordan & Wilkinson 2008) and 32.20 µg kg−1
4
N. Tansakul et al.
Table 2. Occurrence of AFs in unpolished rice and unpolished glutinous rice. Sample (unpolished)
Averagea (µg kg−1)
Positive (n)
Rice (n = 25) AFB1 1 AFB2 1 AFG1 0 AFG2 0 Glutinous rice (n = 10) AFB1 2 AFB2 1 AFG1 0 AFG2 0
Mina (µg kg−1)
0.11 0.05
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxins in selected Thai commodities Natthasit Tansakul
a b
c
b
b
, Sasithorn Limsuwan , Josef Böhm , Manfred Hollmann &
Ebrahim Razzazi-Fazeli
d
a
Department of Pharmacology, Faculty of Veterinary Medicine , Kasetsart University , Bangkok , Thailand b
Institute of Animal Nutrition , University of Veterinary Medicine Vienna , Vienna , Austria
c
Molecular Phytopathology and Mycotoxin Research, Department of Crop Sciences , Göttingen University , Göttingen , Germany d
VetCore Facility for Research, VetOMICS , University of Veterinary Medicine Vienna , Vienna , Austria Accepted author version posted online: 12 Jun 2013.Published online: 16 Jul 2013.
To cite this article: Natthasit Tansakul , Sasithorn Limsuwan , Josef Böhm , Manfred Hollmann & Ebrahim Razzazi-Fazeli , Food Additives & Contaminants: Part B (2013): Aflatoxins in selected Thai commodities, Food Additives & Contaminants: Part B: Surveillance, DOI: 10.1080/19393210.2013.812148 To link to this article: http://dx.doi.org/10.1080/19393210.2013.812148
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2013 http://dx.doi.org/10.1080/19393210.2013.812148
Aflatoxins in selected Thai commodities Natthasit Tansakula,b*, Sasithorn Limsuwanc, Josef Böhmb, Manfred Hollmannb and Ebrahim Razzazi-Fazelid a Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand; bInstitute of Animal Nutrition, University of Veterinary Medicine Vienna, Vienna, Austria; cMolecular Phytopathology and Mycotoxin Research, Department of Crop Sciences, Göttingen University, Göttingen, Germany; dVetCore Facility for Research, VetOMICS, University of Veterinary Medicine Vienna, Vienna, Austria
Downloaded by [University of Arizona] at 14:20 15 September 2013
(Received 1 January 2013; final version received 3 June 2013) Aflatoxin (AF) B1, B2, G1 and G2 were determined in 120 samples of selected Thai commodities including unpolished rice, unpolished glutinous rice, chilli powder, whole dried chilli pods and raw peanut. The mean concentrations of the total AFs for analysed samples were 0.16, 25.43, 14.18, 6.62 and 1.43 µg kg−1 with positive incidences of 4%, 20%, 97%, 37% and 30%, respectively. Quantitative analysis was performed using HPLC equipped with post-column derivatisation and fluorescence detection. Sample clean-up was carried out using immunoaffinity columns for selective enrichment of AFs. The method was validated by using certified reference material, which showed recoveries over 85%. The limit of detections (LODs) and limit of quantifications (LOQs) were in a range between 0.01–0.11 µg kg−1 and 0.03–0.38 µg kg−1, respectively. The results clearly demonstrated that AFs were detectable in different matrices. Chilli powder was found to have the highest level of AFs contamination followed by chilli pods, peanut and rice, respectively. However, among the selected commodities, unpolished rice contained only trace levels of AFB1 and AFB2. With regard to the fact that AFs are a natural contaminant in commodities, this report calls to attention the regular monitoring and effective control of food commodities to prevent health hazards. Keywords: aflatoxins; rice; chilli; peanut; HPLC
Introduction Unavoidable mycotoxin contamination in food commodities is of global concern as they are one of the most harmful fungal metabolites to human and animal health. Aflatoxins are a group of toxic metabolites produced mainly by the fungi Aspergillus flavus and Aspergillus parasiticus growing on a variety of agricultural products during environmentally favourable conditions, particularly in the tropical zone. The four major aflatoxin analogues are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). AFB1 is classified as carcinogen class 1A (International Agency for Research on Cancer 1993). The EU food law has set maximum limits of 2 µg kg−1 for AFB1 and 4 µg kg−1 for total AFs in peanut and cereals. Maximum levels in spices are 5 µg kg−1 for AFB1 and 10 µg kg−1 for the sum of AFs (European Commission 2010). Rice (Oryza sativa L.), a tropical grain, is one of the staple food crops in the world. Rice and rice products are also used as feed ingredient in animal production. World rice production was forecasted to be about 476 million tons (on a milled basis) in 2011–2012 and Thailand is one of the major rice exporters in the world. It was estimated that the mean unpolished rice consumption by the Thai population was 25.60 g/person/day. The occurrence of mycotoxins in rice has been reviewed by Tanaka et al. *Corresponding author. Email: [email protected] © 2013 Taylor & Francis
(2007). Aflatoxins were found worldwide in rice samples in the Philippines (Sales & Yoshizawa 2005), China (Liu et al. 2006), Vietnam (Nguyen et al. 2007), India (Reddy et al. 2009), Sweden (Fredlund et al. 2009), Austria (Reiter et al. 2010), Germany (Reinhold & Reinhardt 2011) and Canada (Bansal et al. 2011). In addition, glutinous rice, known as sticky rice, is a kind of rice mostly consumed as a main dish and also preferred to serve as a desert or sweet between meals in some regions of Asia. However, only few studies are available on aflatoxin contamination in unpolished rice and unpolished glutinous rice. Chilli, Capsicum genus of plants, is ranked second after black pepper among the world spices (Iqbal et al. 2010). Aspergillus spp. was found to be a predominant fungal infection in chilli (Reddy et al. 2001). AFs contamination in several kinds of spices has been reported elsewhere (Cho et al. 2008; Reinhold & Reinhardt 2011). Among monitoring of spices in the Irish market, chilli powder was found to be contaminated with AFs (range 0.35–27.50 µg kg−1) in higher concentrations than others (O’Riordan & Wilkinson 2008). In other former surveys, AFs have also been detected in the chillies in Portugal (Martins et al. 2001), India (Reddy et al. 2001), Pakistan (Iqbal et al. 2010) and Spain (Santos et al. 2010). Although most countries in the world have no specific legal limits on AFs for spices, the current study provides
Downloaded by [University of Arizona] at 14:20 15 September 2013
2
N. Tansakul et al.
data on AFs contamination in chilli with regard to the EU guidelines for AFs level in spices. High incidences and levels of AFs contamination in peanut has widely been reported for decades, that is, in Japan (Tabata et al. 1993), the Philippines (Ali et al. 1999), Malaysia (Sulaiman et al. 2007) and more recently in Sudan (Elzupir et al. 2011). During 1967–1987, there were reports on peanut contaminated with AFs in Thailand. Since then, a limited number of scientific reports on the incidence and levels of AFs in peanut in Thailand have been recorded (Waenlor & Wiwanitkit 2003). Countries in tropical zones suffer from AFs contamination in peanut as a cause of liver cancer (Liu & Wu 2010). Therefore, surveillance programmes on AFs in peanut are a necessity. Thailand, located in a tropical zone with suitable conditions for AFs biosynthesis, is one of the major agricultural food exporters in the world. Thus far, there are few reports on AFs in rice, chilli and peanut from Thailand (Waenlor & Wiwanitkit 2003; Tanaka et al. 2007). The present study aims to investigate the contamination of AFs content in selected Thai commodities using a selective and sensitive method.
Materials and methods Samples In total, 120 samples including 25 unpolished rice, 10 unpolished glutinous rice, 30 chilli powder, 30 whole dried chilli pods and 25 raw peanut samples were randomly collected from either retail fresh markets or modern trade department stores in the central region of Thailand. Sample sizes ranged between 0.5 and 1 kg. After mixing well, each sample was divided into 100 g subsamples, from which portions were taken to collect a final 100 g sample. The pooled 100 g sample was finely ground and 10 g was taken for analysis. Unpolished rice and unpolished glutinous rice originated from Thailand (without mixing of white milled rice), but the original source of chilli and peanut were not declared. All samples were ground and sieved at 1 mm diameter, except for peanut. Thereafter, they were labelled and put into polyethylene bags and placed at –20°C pending analysis.
Chemical and reagents A mixed aflatoxin standard was purchased from Supelco™ (Bellefonte, PA, USA), containing 1.026, 0.297, 1.054 and 0.302 µg ml−1 AFB1, AFB2, AFG1 and AFG2, respectively. HPLC grade solvents (acetonitrile and methanol) were purchased from Fisher Scientific (Essex, UK). Purified water was produced by a UPW2 system (F&L, Vienna, Austria). Sample clean-up was performed using AflaClean™ immunoaffinity columns
(IACs) (LC Tech™, Dorffen, Germany). The chilli powder certified reference material (CRM, reference number T04132) was purchased from FAPAS™ (Food Analysis Performance Assessment Scheme, The Food and Environment Research Agency, Sand Hutton, York YO41 1LZ, UK). HPLC conditions The HPLC system consisted of a pump (LC9A, Shimadzu, Tokyo, Japan) connected to an autosampler equipped with a 100-µL injection loop (AS-2000™, Merck-Hitachi, Tokyo, Japan). A guard column (20 × 4 mm) was placed before the analytical column (250 × 4 mm), both filled with Lichrospher 100_RP 18E 5 µm (Bischoff chromatography Leonberg, Germany). Analysis was run at a flow rate of 1 ml min−1 by an isocratic mobile phase using a mixture of water/methanol/acetonitrile (62:22:16, v/v/v) where 119 mg/L of potassium bromide (KBr) and 350 µl/L 4 M HNO3 were added. The analytical column oven (Jetstream 2 plus, TECHLAB, Erkerode, Germany) temperature was set at 50°C. Detection was carried out by a fluorescence detector (Waters 474, MA, USA) coupled with a Kobracell (R-Biopharm Rhône LTD, Glasgow, UK) to achieve signal enhancement. Excitation and emission wavelengths were 365 and 440 nm, respectively. Chromatograms were displayed with a Stratos™ LC software (Polymer Laboratories, Version 4.5, Shropshire, UK). Sample preparation Briefly, 30 ml of acetonitrile/water (60:40, v/v) was added to 10 g sample, using a 100 ml Schott-Duran™ glass container. After stirring well with a magnetic bar for 30 min, the matrix was filtrated through a paper filter and transferred into a polypropylene test tube. For cleanup, IACs were allowed to stand at room temperature for 30 min prior to starting the clean-up protocol. Meanwhile, 3 ml of the extracted solution was diluted with 30 ml PBS except for chilli, where a PBS/Tween-20 (96:4, v/v) solution was applied. The diluted samples were flushed through an IAC column with a flow rate of one drop per second. After washing two times with 30 ml PBS and 10 ml of distilled water, respectively, the sample was eluted two times with 1 ml methanol. Each eluate was put into a glass test tube and allowed to dry under a gentle nitrogen stream. The sample was reconstituted with a 1-ml mobile phase and put in an autosampler vial.
Analytical procedure Standard curves were constructed daily by duplicate injection of five concentrations in a range of 0.5–15 ng ml−1 using mixed AFB1, AFB2, AFG1 and AFG2 solutions.
Food Additives & Contaminants: Part B Linear regression provided correlation coefficients (R2) above 0.997 for all curves. The limit of detection (LOD) and limit of quantification (LOQ) were determined by taking the signal-to-noise ratios of 3:1 and 10:1, respectively, using spiked mixed standard AFB1, AFB2, AFG1 and AFG2 concentrations of 10.2, 2.9, 10.5 and 3.0 ng ml−1, into blank matrices (n = 3). Repeatability and reproducibility were obtained by three replicates of each fortified matrices in three different days. To control the performance method, certified material reference of chilli (AFB1 and AFB2) was applied.
Downloaded by [University of Arizona] at 14:20 15 September 2013
Results and discussion Validation of the method Table 1 summarises the validation results of AFs analysis in different matrices. High recoveries at the spike levels for AFs contents, particularly aflatoxin B series in all matrices were noted. O’Riordan and Wilkinson (2009) found that using a mixture of methanol/water (80/20; v/v) as an extraction solvent for AFs in chilli may provide better recoveries. Low LOD and LOQ values indicated high sensitivity. According to the specifications of the certified reference material, mean level of AFs in the chilli CRM should be 8.84–22.73 µg kg−1 with a standard deviation of 3.52 for AFB1 and 0.42–1.07 µg kg−1 with a standard deviation of 0.16 for AFB2. The method provided z-scores < 2, which is satisfactory. According to the EU food regulation (European Commission 2006), recoveries in the concentration range of 1–10 µg kg−1 are acceptable when between 70 and 110% and RSD should be less than 20%. The applied
Table 1.
Validated values of AFs in the different matrices.
Intra-day (n = 3)
Inter-day (n = 3)
0.03 0.05 0.03 0.13
106.8 94.2 103.9 71.5
4.4 3.8 2.8 5.3
4.9 3.5 5.1 1.3
0.04 0.11 0.05 0.38
84.5 71.8 76.6 41.6
4.5 6.0 6.4 7.4
1.3 0.4 3.3 3.5
0.04 0.11
92.9 93.6
2.9 7.5
1.6 2.2
0.04 0.03 0.03 0.19
87.3 91.7 81.2 71.9
2.1 3.2 4.1 3.4
2.4 4.1 5.9 2.7
LOQ LOD Matrix (µg kg−1) (µg kg−1) Rice AFB1 0.01 AFB2 0.01 AFG1 0.01 AFG2 0.04 Chilli AFB1 0.01 AFB2 0.03 AFG1 0.01 AFG2 0.11 Chilli (CRM) AFB1 0.01 AFB2 0.03 Peanut AFB1 0.01 AFB2 0.01 AFG1 0.01 AFG2 0.06
RSD (%)
Mean recovery (%) (n = 3)
3
method in this study showed satisfactory results, except in the case of AFG2 in chilli. Occurrence of aflatoxins in rice samples In Table 2, the occurrence of AFs in unpolished Thai rice is presented, which clearly shows the low incidence of AFs in Thai rice. This is in agreement with previous reports, where only low content of AFs was detected in a few rice samples (Sales & Yoshizawa 2005; Reiter et al. 2010). As reported by Fredlund et al. (2009), AFB1 was detected in 71% basmati rice samples and in 20% jasmine rice samples. In this case, it was also detected in high fibre content in jasmine rice (unpolished Thai rice) sold at the Swedish market. The authors reported AF levels between 0.1 and 50.7 µg kg−1. In 2010, AFs were found in more than 70% of the 22 analysed basmati rice samples in Germany with a mean concentration of 1.53 µg kg−1 (Reinhold & Reinhardt 2011). In Canada, two years study of AFs contamination in rice reported mean values of 0.34–0.39 µg kg−1 in AFB1 (with a maximum level of 7.14 µg kg−1). These authors also reported AFs contamination in black and red rice exported from Thailand (Bansal et al. 2011). In the current study, only trace amounts of AFB1 and AFB2 in one sample unpolished rice was noted. Higher concentrations were found in unpolished glutinous rice than unpolished rice. In contrast, no AFG1 and AFG2 were detected in both unpolished rice and glutinous rice samples. This is in contrast to a former report of Jankhaikhot (2005) who found only AFG1 in unpolished rice of Thailand. It may indicate the vast diversity of aflatoxin-producing fungal species and environmental conditions for AFs production in Thailand (Erhlich et al. 2007). Due to processing and rice composition, unpolished rice is more prone to be contaminated with AFs than polished rice (Jankhaikhot 2005). Nevertheless, AFs concentrations in unpolished rice samples did not exceed EU regulations, except for one sample of unpolished glutinous rice. Occurrence of aflatoxins in chilli samples In Table 3, contamination in chilli powder and whole dried chilli pods is shown. Out of 30 chilli powder and chilli pods samples, 29 and 11, respectively, were contaminated with AFs. Among positive chilli tested samples in this study, there were 16 samples of chilli powder and 1 sample of chilli pods contaminated with AFs higher than the acceptable limit of 5/10 μg kg−1 for AFB1/sum AFs as prescribed by the EU (European Commission 2010). This finding confirms previous studies of incidences with high levels of AFs in chilli powder samples, like 93 µg kg−1 (Paterson 2007), 27.50 µg kg−1 (O’Riordan & Wilkinson 2008) and 32.20 µg kg−1
4
N. Tansakul et al.
Table 2. Occurrence of AFs in unpolished rice and unpolished glutinous rice. Sample (unpolished)
Averagea (µg kg−1)
Positive (n)
Rice (n = 25) AFB1 1 AFB2 1 AFG1 0 AFG2 0 Glutinous rice (n = 10) AFB1 2 AFB2 1 AFG1 0 AFG2 0
Mina (µg kg−1)
0.11 0.05
