Application Note AN 113-01-C
Determination of Aflatoxins in Foods by OPLC
A
Aflatoxins are toxic, carcinogenic compounds produced by fungi that can contaminate food and foodstuffs. Optimum Performance Laminar ChromatographyTM bstract (OPLC) can provide an extremely sensitive, selective and robust methodology for their analysis in a broad range of foodstuffs including grains and fish. The analytical procedure involves three steps: extraction of the aflatoxins and related compound from the sample, separation and quantitation. OPLC is easy to perform and can analyze a number of samples (standards and blanks) in a single run. In contrast, HPLC requires that samples be run in series, requiring a longer time frame for an overall analytical protocol. This paper describes the analysis of aflatoxins in a variety of different foodstuffs and includes validation and robustness testing of the procedure.
Introduction
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There are three steps in an analysis for aflatoxins in foodstuffs, extraction of the compounds of interest and related compounds from the matrix, purification (typically via chromatography) and quantitation, typically by fluorescence detection. In recent years OPLC has become an extremely powerful tool for the chromatographer for separation and quantitation of complex mixtures. In this technique, the sample is applied to a sorbent bed and the separation is performed by an appropriate mobile phase under pressure (ca 50 bar). Quantitation can be performed either on line with a flow through detector or off line via densitometric evaluation with a TLC scanner. OPLC provides a number of advantages: it provides considerably better resolution than TLC and can separate a number of samples and standards in a single run (as opposed to HPLC which involves the sequential analysis of samples).
Experimental HTSorbTM
All separations were performed on columns having Silica Gel 60 as the stationary phase (BSLA 001) with an OPLC 50 system (Bionisis SA, Le Plessis Robinson, France). Quantitation was performed by an OPTON KM3 densitometer, λex = 365 nm, λem = 436 nm or a Shimadzu CS920 TLC/HPTLC scanner λex= 365 nm, λem= 380 nm. The sample pretreatment steps and solvents used to develop the separation are dependent on the sample type and are presented in the discussion of this note.
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Fish Samples1 Ground fish samples were mixed with methanol:water (7:3) for 20 min. The solution was then extracted with dichloromethane, and the extract was evaporated to dryness and re-dissolved in dichloromethane: acetone (9:1). The sample was applied to the stationary phase (50 µL) and separated using a solvent mixture of chloroform:ethyl acetate:tetrahydrofuran (10:15:1 v/v). The HTSorb column was evaluated by densitometry using the OPTON system. The densitograms of standards, a fish sample spiked with aflatoxins and an unspiked sample is presented in Figure 1. Fish samples were analyzed for aflatoxins to the 2 ng/g level by OPLC, which is the range required for government monitoring. These workers also described an HPLC method for aflatoxins in fish, which has similar performance characteristics, but requires an expensive cleanup step with an immunoaffinity column. In the OPLC method, recovery of the aflatoxins was in the 80-90% range.
Figure 1: OPLC Densitograms obtained from a) aflatoxin standards (1 ng), b) fish sample spiked with aflatoxins (1ng), c) blank fish sample [1=G2, 2=G1, 3=B2, 4=B1] .
Corn2 Otta, et al. have investigated the determination of aflatoxins in corn. Ground corn was added to a mixture of acetonitrile:water (9:1) and shaken. After 15-30 minutes, the liquid was decanted, filtered, evaporated to dryness and dissolved in 9:1 chloroform:acetone (100µL). The extract was applied to the OPLC column and separated with chloroform:ethyl acetate:tetrahydrofuran (10:15:1) for a period of 1157 seconds. When the extract was spiked with various aflatoxins, linear calibration relationships were obtained up to 15 ng.
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Discussion
Aflatoxins are toxic, carcinogenic compounds that are produced by fungi and are commonly found in foodstuffs such as grains, nuts and fish. In most instances, they are formed either before the grain is harvested or while the grains are in storage. Several aflatoxins have been identified and four are found in nature (B1, B2, G1, G2). Due to their toxicity, foodstuffs are monitored throughout the world via a variety of methods including HPLC, TLC, GC-MS, electrophoresis and ELISA. The acceptable level of aflatoxin B1 (which is the most toxic) is typically 1-5 ppb, depending on the jurisdiction and the acceptable level of all aflatoxins is 5-20 ppb.
Wheat3 Wheat samples were ground and extracted with acetonitrile:water (9:1). The solution was filtered and evaporated to dryness and the residue was dissolved in dichloromethane:acetone. The residue was separated with chloroform:ethyl acetate:tetrahydrofuran (10:15:1) and the densitogram was obtained via the OPTON densitometer. A typical densitogram from a spiked wheat sample is presented in Figure 2 and recovery studies were performed, the values are shown in Table 1 and the accepted range is between 85 and 115%. Figure 2 Densitogram obtained from a wheat sample spiked with 5 ng/g aflatoxins
ng/g
G2
G1
B2
B1
7
83.47
89.21
88.27
85.42
5
90.03
89.10
84.55
98.04
2
93.64
97.21
105.51
111.81
Table 1: Recovery of Aflatoxins from Wheat Samples
The RSD, Detection Limit and Quantitation Limit for the assay are shown in Table 2. The acceptance limit is 7 % for repeatability according to ICH guidelines but for food chemistry with complex biological matrices, 10% is acceptable. As we can see, OPLC clearly meets these guidelines. Compound
RSD (%)
Detection Limit
Quantitation Limit
G2
8.77
0.018
0.5
G1
8.53
0.10
0.5
B2
9.04
0.15
0.5
0.14
0.5
B1
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Table 2: Repeatability, Detection Limits and Quantitation Limits
A calibration curve for each aflatoxin was obtained, the curve for Aflatoxin G1 is presented as Figure 3
Robustness testing was performed on seven different parameters: the type of plate, the volume of each of the three components of the mobile phase, the external pressure, the mobile phase volume and the linear velocity of the mobile phase. It was determined that OPLC sorbent bed 5 µm (BSLA 001) performed significantly better than TLC grade silica (11 µm, BSLA 011). The mobile phase volume and the ratio of toluene to chloroform are important.
Conclusions OPLC can provide an extremely sensitive, and accurate and reproducible assay for the determination of aflatoxins in foods. An important benefit of the analysis is that many samples, blanks and standards can be analyzed on a simultaneous basis, therefore increasing the laboratory productivity. In contrast, most chromatographic techniques can only perform one analysis at a time. Figure 3 Calibration curve for Aflatoxin G1.
References 1) Papp, E., Bagocsi, B., H-Otta, K., Kovacsics-Acs, L., and Mincsovics, E. The Role of Overpressure Layer Chromatography among Chromatographic Methods for the Determination of Aflatoxins in Fish. J. Planar Chrom. 12 383387 (1999). 2) H.-Otta, K., Papp, E, Mincsovics, E. and. Zaray, G.H., Determination of Aflatoxins in Corn by Use of the Personal OPLC Basic System, J. Planar Chromatography, 11 370-373 (1998). 3) Papp, E., Farkas, A., H Otta, K., and Mincsovics, E.. Validation and Robustness Testing of an OPLC method for Determination of Aflatoxins in Wheat. J. Planar Chromatography 13, 328-332 (2000).
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