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Quantitation of aflatoxins in walnut kernels by highperformance liquid chromatography with fluorescence detection Bulent Kabak

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Faculty of Engineering, Department of Food Engineering, Hitit University, Corum, Turkey Accepted author version posted online: 28 May 2014.Published online: 25 Jun 2014.

Click for updates To cite this article: Bulent Kabak (2014) Quantitation of aflatoxins in walnut kernels by high-performance liquid chromatography with fluorescence detection, Food Additives & Contaminants: Part B: Surveillance, 7:4, 288-294, DOI: 10.1080/19393210.2014.928372 To link to this article: http://dx.doi.org/10.1080/19393210.2014.928372

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Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 4, 288–294, http://dx.doi.org/10.1080/19393210.2014.928372

Quantitation of aflatoxins in walnut kernels by high-performance liquid chromatography with fluorescence detection Bulent Kabak* Faculty of Engineering, Department of Food Engineering, Hitit University, Corum, Turkey

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(Received 27 March 2014; accepted 22 May 2014) A total of 85 walnut samples collected between October 2012 and April 2013 in different provinces of Turkey were analysed for the presence of aflatoxins (AFs). The method involved methanol–water extraction, clean-up with immunoaffinity columns and a sensitive high-performance liquid chromatography coupled with fluorescence detection after postcolumn derivatisation. The method was validated for selectivity, linearity, trueness, precision, limit of detection and limit of quantification (LOQ), which met the performance criteria as set by EC regulation No. 401/2006. LOQs were 0.07, 0.04, 0.09 and 0.05 µg kg–1 for AFB1, AFB2, AFG1 and AFG2, respectively. AFs were present in 9.4% of walnut samples (8/85) at total AFs levels ranging from 0.09 to 15.4 µg kg–1. Only one of eight walnut samples exceeded the European Union limit of 2 and 4 µg kg–1 for AFB1 and total AFs, respectively. Keywords: aflatoxins; walnuts; occurrence; Turkey; HPLC-FLD

Introduction The genus Juglans (family Juglandaceae) consists of approximately 20 species and is widely distributed throughout the world. The best-known member of this genus is walnut (Juglans regia L.), native to the mountain ranges of Central Asia and Southwest Asia (Georgia, Armenia, Azerbaijan, Iran and Turkey). Walnut tree is now cultivated in the United States, South America (Chile, Argentina), Western and Southern Europe, Australia, New Zealand, South Africa and Japan (FAO 2004). Of the over 3.3 million metric tonnes of walnut, with shell produced annually in the world, Turkey is the fourth major producer, with a production of 194,298 metric tonnes, followed by China (1,700,000 tonnes), Iran (450,000 tonnes) and the United States (425,820 tonnes) (FAO 2012). While China and United States are leading exporters, Iran and Turkey consume the bulk of their walnut production domestically. The major importers of walnut are Germany, Spain, the United Kingdom, Canada and Japan (FAO 2004). Walnuts, like other tree nuts, are susceptible to fungal infection, occurring during the loosening of hulls from shells which usually takes 10–28 days, and subsequently mycotoxin formation (Singh & Shukla 2008). The Aspergillus section Flavi is of particular interest, since three members of this section, Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, produce aflatoxins (AFs). AFs are considered to be potent carcinogens, mutagens, teratogens and immunosuppressants to human and animals (Eaton & Gallagher 1994). Among *Email: [email protected] © 2014 Taylor & Francis

the several structurally related AFs, only four, AFB1, AFB2, AFG1 and AFG2, are naturally found in feed and foodstuffs. AFB1 is the most potent genotoxic and carcinogenic AFs and amongst the most commonly found in agricultural products (Sweeney & Dobson 1998). Furthermore, the International Agency for Research on Cancer (IARC) classifies AFB1 and mixtures of AFs as a Group 1 human carcinogen (IARC 1993), with a role in the aetiology of liver cancer, notably among subjects who are carriers of hepatitis B virus surface antigens (IARC 2002), and proposes no safe dose. To protect consumers from the harmful effects of certain contaminants, regulations have been established by the national and international organisations. The European Commission (2010) established maximum limits (MLs) of 2 µg kg–1 for AFB1 and 4 µg kg–1 for the sum of AFB1, AFB2, AFG1 and AFG2 in walnut kernels and processed products thereof, intended for direct human consumption or use as ingredient in foodstuffs. Republic of Turkey Ministry of Food, Agriculture and Livestock adopted the European Union (EU) limits in foodstuffs. A number of survey and monitoring programmes have been carried out to determine the AF contamination in main crops of Turkey, including figs, dried fruits, chilli peppers, peanuts and tree nuts such as hazelnuts and pistachios. However, little information exists regarding the presence of AFs in walnuts. The aim of this study is therefore to obtain representative data on the occurrence and levels of AFs in walnuts consumed in Turkey, using a sensitive high-performance liquid

Food Additives & Contaminants: Part B chromatography-fluorescence detector method with post-column derivatisation.

(HPLC-FLD)

this stock solution, a series of working standards (0.5–15 ng ml–1 for AFB1 and AFG1 and 0.15–4.5 ng ml–1 for AFB2 and AFG2) were prepared freshly in LC mobile phase consisting of water–acetonitrile–methanol (6/2/3, v/v/v) and renewed every two weeks.

In this survey, 85 samples of walnuts (35 walnuts with shells and 50 walnuts without shells) were analysed. Samples were collected randomly from retail shops, local markets and bazaar in different province of Turkey including Corum, İstanbul, Adana, Karaman, Gaziantep, Ankara, Antakya and Yozgat from October 2012 to April 2013. According to EC 401/2006, the aggregate sample of dried figs, groundnuts and nuts at retail stage might be less than 1 kg (European Commission 2006). Walnuts without shells were taken either in their original packages (500 g) or picked at random from the same batch. For walnuts with shells, 1 kg was sampled from the same batch. Samples were transported to the laboratory in an insulated container. All samples were ground and mixed thoroughly with Waring blender to ensure homogeneity and stored in plastic containers in a refrigerator until analysis. Then, a 25 g subsample was collected randomly before analysis.

Apparatus A Waring blender with a 1-l container, operating at high speed (Waring Products Co., Torrington, Connecticut, USA), and a vacuum manifold (Agilent Technologies, Santa Clara, CA, USA), which is capable of processing 20 samples simultaneously, were used. The HPLC system consisted of a Shimadzu (Tokyo, Japan) liquid chromatographic apparatus coupled to a fluorescence detector (Shimadzu RF20AXL) equipped with a LC-20AD pump, a SIL-20AHT autosampler, a DGU-20A3 online degasser and a CBM20Alite system controller. Shimadzu LC solution software was used for data acquisition and processing. The HPLC column used was a reversed-phase Inertsil® ODS-3 (5 μm, 250 × 4.6 mm i.d.) supplied by GL Sciences Inc. (Tokyo, Japan). Post-column derivatisation was carried out with electrochemically generated bromine in a Kobra® cell (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340 × 0.5 mm i.d. politetrafluoroethylene to enhance the fluorescence intensity of AFB1 and AFG1.

Materials and methods Samples

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Chemicals and reagents Methanol and acetonitrile used for sample preparation and mobile phase were both HPLC grade and were supplied by VWR International (Leuven, Belgium). Sodium chloride (NaCl) and potassium chloride (KCl) were also purchased from VWR International. Potassium dihydrogen phosphate (KH2PO4), disodium hydrogen phosphate (Na2HPO4) and sodium hydroxide (NaOH) were supplied by SigmaAldrich (St. Louis, MO, USA). Nitric acid and potassium bromide were obtained from Merck (Darmstadt, Germany). Phosphate-buffered saline solution was prepared by dissolving 8 g NaCl, 1.16 g Na2HPO4, 0.2 g KH2PO4 and 0.2 g KCl in 1000 ml of ultra-pure water. The pH of the solution was adjusted to 7.4 with 0.1 N NaOH. Ultra-pure water was produced on Direct-Q3 purification system, from Millipore (Molsheim, France). Immunoaffinity columns (IACs), AflaTest® were purchased from Vicam (Watertown, MA, USA). GF/A glass microfibre filters (125 mm) were from Whatman International (Kent, UK).

Standard solutions The AF mixture (AFB1 and AFG1, 1 μg ml–1; AFB2 and AFG2, 0.3 μg ml–1) reference standard solution in methanol (Aflatoxin Mix kit, catalogue no. 46304-U) was provided from Supelco® (Bellefonte, PA, USA). From this standard solution, stock solutions of AFs (AFB1 and AFG1, 0.1 μg ml–1; AFB2 and AFG2, 0.03 μg ml–1) were prepared in methanol and stored in amber glass vials at –18°C. From

Sample preparation The 25 g finely ground representative subsamples were extracted with 125 ml methanol–water (80:20, v/v) and 5 g NaCl using a Waring blender at high speed for 2 min. The sample extract was filtered using a pre-folded filter paper. An aliquot of 10 ml of the filtrate was diluted with 50 ml water, shaken vigorously and filtered once more through a glass microfibre filter. Then, a 60 ml of diluted filtrate was passed through an AflaTest® IAC attached onto a vacuum manifold at a speed of 2–3 ml min–1. The column was washed twice with 10 ml of ultra-pure water, and then air was passed through a syringe at least three times. Subsequently, the AFs bound to the specific antibody were eluted by passing twice with 0.5 ml of methanol through the IAC at a flow rate of one to two drops per second and collected in HPLC vials. The eluate was evaporated until dryness at 45°C under N2 stream, and the residue was reconstituted in 1 ml of water–acetonitrile–methanol (6/2/3, v/v/v). The samples were stored at 4–8°C until HPLC-FLD analysis. HPLC-FLD analysis The chromatographic separation of AFs was carried out in the isocratic mode using a mixture of potassium bromide (120 mg l–1)–nitric acid (350 μl l–1) and water– acetonitrile–methanol (6/2/3, v/v/v) at a flow rate of 1 ml

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B. Kabak

min–1. The column temperature was set at 35°C. The injection volume into HPLC system for both standard and sample was 100 μl. The fluorescence detector was set to excitation and emission wavelengths of 360 and 440 nm, respectively. Each chromatographic run was 20 min, and retention times recorded were around 15.2, 12.7, 11.3 and 9.6 min for AFB1, AFB2, AFG1 and AFG2, respectively. The method was validated in-house for the quantification of AFB1, AFB2, AFG1 and AFG2 in finely ground walnuts in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), trueness and precision. The selectivity of the method was tested by analysing blank and spiked walnut samples at levels of 2 μg kg–1 for AFB1 and AFG1 and 0.6 μg kg–1 for AFB2 and AFG2. The linear range of the method was estimated by injecting six calibration solutions ranging from 0.5 to 15 μg l–1 for AFB1 and AFG1 and 0.15 to 4.5 μg l–1 for AFB2 and AFG2 and plotting respective peak areas against the concentrations. The linearity was determined by linear regression analysis and expressed as coefficient of determination (R2). LODs and LOQs were determined as the minimum concentration of analyte in the spiked blank samples as described in ISO11843 part 2, with a signalto-noise (S/N) ratio of 3 and 10, respectively. The trueness of the analytical method was evaluated by recovery experiments. To determine the recoveries of AFs, blank samples of finely ground walnut were spiked with AFs at two different levels of 2 and 5 μg kg– for AFB1 and AFG1 and 0.6 and 1.5 μg kg–1 for AFB2 and AFG2. The spiked materials were then analysed according to the method protocol and analytes were quantified. The measured concentration was determined using the obtained calibration curves, and the recovery value was calculated by: % recovery ¼ 100  measured concentration for spiked sample=actual ðaddedÞ concentration (1)

The precision of the method was determined in terms of intra-day and inter-day repeatability expressed as % relative standard deviation (RSD) associated with the recovery experiment on the same day and on three consecutive days at the respective spiking levels.

Results and discussion Method validation HPLC-FLD conditions selected for analysis of AFs ensure a satisfactory selectivity, as the four analyte peaks were well separated and no interfering peaks in the retention times of analytes were observed. The selectivity of the method was assured by the use of IAC for clean-up and a very selective fluorescence detector. The retention times of AFB1, AFB2, AFG1 and AFG2 were 15.2, 12.7, 11.3 and 9.6 min, respectively. The calibration level, linear regression equation and coefficient of determination (R2) for target analytes are given in Table 1. The calibration curves for all the analytes were linear in the range of 0.5–15 μg kg–1 for AFB1 and AFG1 and 0.15–4.5 μg kg–1 for AFB2 and AFG2, with R2 for the six-point calibration curves not lower than 0.99 for all analytes. The LODs and LOQs of the method, summary of recovery and precision data are shown in Table 2. Based on the S/N ratios of 3 and 10, LODs ranged from 0.01 to 0.03 μg kg–1 for AFs, while LOQs of global method were established between 0.04 and 0.09 μg kg–1, respectively. Table 1. Analyte AFB1 AFB2 AFG1 AFG2

Linearity data for aflatoxins (AFs). Range (μg l–1) 0.5–15 0.15–4.5 0.5–15 0.15–4.5

Equations y y y y

= = = =

2.812x 1.472x 3.286x 2.798x

+ + + +

0.0752 0.0027 0.0704 0.0224

R2 0.9998 0.9999 0.9998 0.9998

Note: R2, coefficient of determination.

Table 2. Trueness, precision, recoveries, limit of detection and limit of quantification. Analyte AFB1 AFB2 AFG1 AFG2

Spiking level (μg kg–1)

Recovery (%)

Intra-day repeatabilitya (RSDr) (%)

Inter-day repeatabilityb (RSD) (%)

0.66 × Horwitz value (%)

LOD (μg kg–1)

LOQ (μg kg–1)

2 5 0.6 1.5 2 5 0.6 1.5

95.7 96.4 93.6 93.8 95.4 94.6 91.3 91.9

4.1 3.4 3.6 2.9 3.0 1.9 5.2 4.9

2.9 3.7 5.2 2.8 5.2 4.8 7.6 5.2

26.9 23.4 32.2 28.1 26.9 23.4 32.2 28.1

0.02

0.07

0.01

0.04

0.03

0.09

0.01

0.05

Notes: aIntra-day repeatability (RSDr) was estimated by the analysis of six replicate samples at two concentration levels of each toxin on the same day (n = 6). b Inter-day repeatability was estimated by the analysis of six replicate samples at two concentration levels of each toxin on the three consecutive days (n = 18).

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Food Additives & Contaminants: Part B Thus, the method is suitable for AF determination, since the calculated LOD and LOQ values were much smaller than the enforcement limits as set by the European Commission (2010). All recovery values, ranging between 91.3% and 96.4%, are in agreement with the requirements of the Commission Regulation (EC) No 401/2006 (European Commission 2006) laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. It is stated that recovery rate of 50– 120% at the concentration range of

Quantitation of aflatoxins in walnut kernels by high-performance liquid chromatography with fluorescence detection.

A total of 85 walnut samples collected between October 2012 and April 2013 in different provinces of Turkey were analysed for the presence of aflatoxi...
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