Composition, kit and Method for Extracting Mycotoxins

Abstract

AO 25.09.1129 NL A composition, a kit and a method for extracting mycotoxins from grain samples. The composition is in the form of dry powder and comprises a buffer, an inorganic salt as ionic strength agent, a non—ionic surfactant and a stabilizer. When reconstituted with water, the composition creates an effective extraction medium that eliminates the need for organic solvents. The method involves combining the dry formulation with a sample, adding water, agitating briefly, and separating the liquid extract containing mycotoxins. The process is suitable for extracting multiple mycotoxin types simultaneously. The kit comprises the dry powder composition and associated equipment.

Description

1 AO 25.09.1129 NL Composition, kit and Method for Extracting Mycotoxins Field of the invention The present invention relates to compositions, kits and methods for the extraction and detection of mycotoxins from foodstuffs. Specifically, this invention involves the use of aqueousbased solutions which are designed to improve the binding and removal of mycotoxins from contaminated samples. This invention provides an ecofriendly alternative to traditional organic solventbased extraction methods, eliminating the need for hazardous solvents like methanol, and enhances the efficiency and safety of mycotoxin detection in both laboratory and field environments. Background of the invention Mycotoxins are toxic secondary metabolites produced by various fungi, predominantly from the Aspergillus, Penicillium, and Fusarium genera. These compounds contaminate a Wide range of agricultural products, including cereals, grains, nuts, and animal feed. Common mycotoxins include aatoxins, ochratoxin, fumonisins, zearalenone, deoxynivalenol (DON), and T2 / HT2 toxins. The presence of mycotoxins in food and feed poses significant health risks to humans and animals, ranging from acute poisoning to longterm effects such as immunosuppression and carcinogenesis. Aatoxin B1, for instance, is classified as a Group I human carcinogen by the International Agency for Research on Cancer (IARC) [l]. Epidemiological studies have linked aflatoxin exposure to increased risk of hepatocellular carcinoma, particularly When combined With hepatitis B virus infection [2]. Due to their frequent occurrence and severe toxicity, many countries have set guidelines and tolerance levels for mycotoxins in food. For example, Iran has set a maximum residue limit of 5 pig / kg for AFBl in Wheat imports [3], and the limit for Ochratoxin A in unprocessed cereals is also 5 pig / kg. . Accordingly, the low tolerance for food contamination by mycotoxins causes serious economic losses. Current methods for detecting mycotoxins in grains include TLC, uorescence polarization assay, HPLC, radioimmunoassay (RIA), enzymelinked immunosorbent (ELISA), and fiber opticbased immunoassays [4]. However, these methods have 2 AO 25.09.1129 NL several drawbacks. For example, chromatographic methods require extended cleanup steps and derivatization after extraction to eliminate interfering substances [5]. Commercially available ELISAs require reactions with organic solvents, hazardous materials and washing and separation of bound and free labels [6]. Spectrofluorimetry analysis is hampered when testing natural samples due to the complexity of matrices which show a variety of natural uorescent compounds whose spectra often overlap the analyte signal [7]. With respect to extracting mycotoxins from grains, current methods predominantly involve the use of organicbased liquid compositions, such as methanol / water mixtures [8]. These methods face several challenges, including the use of hazardous and costly organic solvents, need for further dilution prior to analysis, poor recovery rates for certain mycotoxins or in complex food matrices, time and labor intensity, potential reduction in sensitivity due to dilution effects, and requirement for adjustment depending on the particular matrix and / or analyte being extracted and detected [9]. Given these Challenges, there is a pressing need for improved mycotoxin extraction and detection techniques. Ideal methods should use safer, aqueousbased solutions; provide efficient extraction of multiple mycotoxin types simultaneously; be applicable across a wide range of food matrices with minimal adjustment; offer high sensitivity and specificity to meet diverse regulatory requirements; be costeffective and suitable for routine testing; integrate well with rapid detection methods such as lateral flow immunochromatographic assays, or enzymelinked immunosorbent (ELISA) assays, or a chemiluminescence immunoassay (CLIA) assays; and minimize the need for complex sample preparation and cleanup steps. The present invention addresses these needs by providing compositions, kits and methods for the aqueousbased extraction and recovery of mycotoxins from foodstuffs. The compositions described herein show broad affinity for mycotoxins and therefore remove a wide variety of toxic contaminants simultaneously. Summary of the Invention The innovative extraction composition, kit and method presented herein offers a significant advancement in mycotoxin detection for agricultural products. The composition is in the form of dry powder formulation and comprises i) a buffering 3 AO 25.09.1129 NL agent, ii) an inorganic salt as tonicity agent, iii) a nonionic surfactant, such as poloxamers, and i) a stabilizing agent. This ecofriendly approach eliminates the need for organic solvents, addressing environmental concerns present in existing methods

[10] . The extraction procedure is remarkably straightforward, involving direct mixing of the dry powder with the sample, followed by water addition. This singlestep process, completed in just a few minutes, represents a substantial improvement over multistep procedures commonly used in the field

[11] . The method's versatility is demonstrated by its effectiveness across a range of mycotoxins including DON, fumonisin, aatoxin, zearalenone and T2 / HT2, and its applicability to various grain and feed matrices. The extraction procedure can be combined with various immunoassays, such as a lateral ow assay, or an ELISA assay, or a CLIA assay When paired with a quantitative immunoassay, this extraction method enables rapid, on site testing without the need for specialized laboratory equipment. The assay utilizes a competitive format with gold nanoparticlelabeled antibodies, providing visual detection on a nitrocellulose membrane. Quantitative results are obtained using a specialized reader, offering a wide quantification range for various mycotoxins. The dry formulation offers practical advantages such as extended shelf life and simplified storage and shipping, potentially reducing costs. This userfriendly approach makes it suitable for onsite testing, a significant benefit over HPLCbased methods requiring laboratory settings

[12] . In summary, this invention combines an ecofriendly, rapid extraction method with a sensitive detection technique, providing a comprehensive solution for mycotoxin testing. It addresses key limitations of existing methods, offering improvements in speed, ease of use, versatility, and environmental impact. This innovative approach has the potential to significantly enhance mycotoxin monitoring in the agricultural and food safety sectors. Brief Description of the Drawings Figure 1 is a schematic diagram illustrating the most common mycotoxins affecting grains. Figure 2 shows the chemical structure of Tris(hydroxymethyl)aminomethane. 4 AO 25.09.1129 NL Figure 3 depicts the chemical structure of glycine. Figure 4 is a owchart illustrating the green extraction and mycotoxin detection process. Figures 59: Graphs and tables illustrating experimental results (e.g., recovery rates, effect of pH, temperature, and dilution). Detailed description of the Invention Definitions Key terms integral to understanding this innovation include mycotoxins, naturally occurring toxic compounds produced by certain fungi; extraction, the process of separating these toxins from sample matrices; and lateral ow immunoassay, a rapid chromatographic test format used for detection. The concept of green extraction is central to this invention, representing an environmentally friendly approach using waterbased solutions and avoiding organic solvents. The limit of detection (LOD) is a crucial parameter, indicating the lowest concentration of mycotoxin reliably detectable by this method. Composition of the Extraction Formulation The extraction composition at the heart of the present invention is a carefully formulated dry powder mixture. It contains an inorganic salt, such as sodium chloride, to enhance ionic strength and promote mycotoxin solubility, a buffering agent, such as tris(hydroxymethyl)aminomethane (Tris) ultra pure, to maintain optimal pH, a nonionic surfactant, such as poloxamers, to aid in mycotoxin solubilization and reduce non specific binding, and a stabilizing agent, such as glycine, which serves multiple functions including pH buffering and protein stabilization. This combination creates an effective extraction environment upon hydration, enabling efficient mycotoxin extraction without the need for organic solvents, while maintaining compatibility with a wide range of mycotoxins and sample matrices. Method of Extraction A method for extracting mycotoxins from a grain sample, which include the steps of a) preparing an extraction buffer solution that contains (i) 5 AO 25.09.1129 NL tris(hydroxymethyl)aminomethane (Tris) buffer for maintaining a stable pH; (ii) sodium chloride (NaCl) for increasing ionic strength; (iii) a nonionic surfactant, such as poloxamers, for improving wetting and solubilization of mycotoxins; and (iv) either Bovine Serum Albumin (BSA) or glycine as the stabilizing agent, depending on the results of a comparative evaluation to determine which offers better protein stabilization and extraction efficiency ; b) contacting the grain sample with said extraction buffer solution to form a mixture; c) agitating said mixture to disrupt the interactions between the mycotoxins and the grain matrix; d) separating the mixture into a liquid extract phase containing the extracted mycotoxins and a solid residue phase; and e) collecting at least a portion of the liquid extract phase for subsequent analysis. The Tris buffer component of the extraction buffer maintains a stable pH in the range suitable for mycotoxin extraction, preferably pH 7.0 to 8.0, more preferably pH 7.2 to 7.6. The NaCl component increases the ionic strength of the buffer, aiding in the disruption of mycotoxinmatrix interactions. Poloxamers or other nonionic surfactants improve the wetting of the grain matrix and solubilization of hydrophobic mycotoxins. Glycine or other stabilizing agents help maintain the structure and function of proteins used in the extraction process. The novel extraction formulation described herein leverages the synergistic properties of poloxamer 407, a nonionic surfactant, and glycine, a multifunctional amino acid, to optimize the extraction of a wide range of mycotoxins from grain matrices. The combination of these two key components enables effective extraction of both hydrophobic and hydrophilic mycotoxins while maintaining an ecofriendly approach. Poloxamer 407 plays a crucial role in the extraction process by acting as a surfactant. As many mycotoxins, such as aatoxins and zearalenone, are hydrophobic in nature, their solubilization in an aqueous medium is often challenging. Poloxamers amphiphilic structure, composed of both hydrophilic and hydrophobic segments, addresses this issue by: ° Reducing surface tension: Poloxamer 407 lowers the surface tension between the grain matrix and the extraction buffer, ensuring better penetration of the extraction solution into the sample. 6 AO 25.09.1129 NL ° Micellar encapsulation: It forms micelles around hydrophobic mycotoxins, effectively solubilizing them in the aqueous extraction buffer. This encapsulation allows the extraction of mycotoxins that would otherwise remain embedded in the grain matrix. ° Minimizing nonspecific binding: The surfactant properties of poloxamers also help reduce nonspecific binding of mycotoxins to other matrix components, increasing the overall efficiency of the extraction process. An example of commercially available poloxamer is Pluronic®. Glycine serves as both a buffering agent and a protein stabilizer in the extraction formulation. It contributes to the extraction process by: ° Maintaining optimal pH: Glycine helps maintain a stable pH in the extraction buffer, which is crucial for efficient extraction of a wide range of mycotoxins. The ideal pH range (7.08.0) is maintained, ensuring the mycotoxins remain in their soluble forms for maximum extraction efficiency. ° Stabilizing proteins: As a protein stabilizer, glycine prevents the denaturation of proteins, including antibodies used in the subsequent immunoassay for mycotoxin detection. This ensures reliable and reproducible test results. ° Reducing matrix interference: Glycine further minimizes interference from the sample matrix, which might otherwise disrupt the extraction process. This ensures that proteins or other interfering substances from the grain do not reduce the extraction yield or hinder downstream detection. The combination of poloxamer 407 and glycine enables the extraction of a broad spectrum of mycotoxins, including both hydrophobic and hydrophilic types, such as aatoxins, deoxynivalenol (DON), ochratoxins, and zearalenone. This synergistic interaction between the two components allows for: ° Enhanced solubilization of hydrophobic mycotoxins through poloxamers micelleforming capabilities. ° Optimized buffer conditions maintained by glycine, which ensures the extraction process is equally effective for hydrophilic mycotoxins. ° Improved extraction efficiency for diverse mycotoxin types from complex food matrices without the need for hazardous organic solvents. 7 AO 25.09.1129 NL The inclusion of poloxamer 407 and glycine in this formulation represents a significant improvement in mycotoxin extraction technology, as it addresses the challenges of both extraction efficiency and environmental impact. This dualcomponent system ensures that the extraction method remains robust and versatile, suitable for routine testing across a variety of agricultural products. The specific concentrations of each buffer component may be optimized based on the type of grain, the target mycotoxins, and the desired extraction efficiency. In a preferred embodiment, the extraction buffer may comprise 10150 mM Tris, 50500 mM NaCl, 0.15% (w / v) poloxamer 407 and 10100 mM glycine. The grain sample is contacted with an appropriate volume of extraction buffer, typically in a ratio of 1:2 to 1:10 (w / v) sample to buffer. The mixture is then agitated by shaking, blending, or vortexing to facilitate the extraction of mycotoxins. After a suitable extraction period, The liquid extract containing the dissolved mycotoxins can be collected and used directly for analysis, such as in a lateral flow assay device, or further processed by concentration, purification, or derivatization steps as needed. The improved extraction efficiency and compatibility of this buffer system enhances the sensitivity and reliability of downstream mycotoxin detection methods. Detection Methods The detection method employs a quantitative immunoassay, such as a lateral ow assay, designed to work seamlessly with the extraction process. It utilizes a competitive assay format with specific antibodies against target mycotoxins, conjugated to colloidal gold particles. As the sample ows through the membrane, mycotoxins in the sample compete with mycotoxins fixed on the membrane for antibody binding sites, resulting in a test line color intensity inversely proportional to the mycotoxin concentration. A control line ensures test validity. Quantitative results are obtained using a specialized reader. Kits and Apparatus The invention includes comprehensive kits and apparatus to facilitate easy implementation of this testing system. The extraction kit contains premeasured pouches 8 AO 25.09.1129 NL of dry extraction powder and instructions, with optional sample collection containers and mixing tools. Typically, the extraction kit comprises an immunoassay test system, such as a lateral ow test system, or an ELISA test system, or a CLIA test system The lateral ow test system includes individually pouched test strips, sample diluent tubes, and disposable pipettes. Specialized equipment such as the Quantum reader (e.g., S FLOW or 3PR) for quantitative results, and optional mini centrifuge and vortex mixer, complete the hardware components. The system is supported by SFlow software for result interpretation and data management. This integrated system provides a complete solution for onsite, rapid, and quantitative mycotoxin testing, emphasizing ease of use and environmental friendliness. It represents a significant advancement in mycotoxin detection technology, offering improvements in speed, simplicity, and ecological impact over existing methods. Examples The following examples are provided to demonstrate the effectiveness and versatility of the novel green extraction method for mycotoxins. These examples are illustrative and should not be considered as limiting the scope of the invention. Example 1 Comparison of Extraction Methods for Total Aatoxin Grain samples (corn) were spiked with known concentrations of total aatoxin (30, 60, and 90 ppb) and extracted using two different methods: the TrisNaCl formulation and the traditional methanolbased method. The concentration of aatoxin was determined using a lateral ow immunoassay, and the results were recorded in terms of recovery rate and precision. Table 1: Aatoxin Recovery and Precision of TrisNaCl Extraction Method Across Different Concentrations Aatoxin M l 2 Æ & -..... ___- ___-_- ___-_- 9 AO 25.09.1129 NL _- 78.25 144.75 0.541 34.75 cv <%> _- 64.75 150.25 0.431 65.00 cv <%> _- 51.50 153.50 0.336 94.25 Table 2: Aatoxin Recovery Using Methanol Extraction Aatoxin Stick # I £ T / C Ratio Result Concentrati ___ 0524 0.503 ___ 0.514 __- 76.00 148.00 0.514 34.50 I cv (%) ___ 0.483 10 AO 25.09.1129 NL ---E. ___-_- ___-_- ------ ___mm ------ ____- ------ ____- The results demonstrate that both the TrisNaCl extraction method and the methanol based method perform similarly in terms of recovery rates across all three concentration levels (30, 60, and 90 ppb). For each concentration, the TrisNaCl method achieved recovery rates that are very close to the actual aatoxin concentrations, comparable to the results obtained with the methanol method. At 30 ppb, the TrisNaCl method resulted in an average recovery of 34.75 ppb, which is in line with the methanol methods average of 35.00 ppb. Similarly, at 60 and 90 ppb, the TrisNaCl method yielded average recoveries of 65.00 and 94.25 ppb, respectively, which are comparable to the methanol methods recoveries of 65.00 and 93.00 ppb. Both methods exhibit good accuracy, achieving results close to the spiked concentrations. However, the Tris NaCl method offers additional environmental and safety advantages by using an aqueous buffer formulation instead of hazardous organic solvents. This reduction in solvent use minimizes the environmental impact and reduces risks associated with handling and disposal, making the TrisNaCl method a more sustainable alternative. These benefits make it equally suitable for routine testing, where accuracy, safety, and environmental considerations are important factors. Table 3: CrossReactivity of TrisNaCl Extraction Method with Various Aatoxins Aatoxin Type Concentration Stick # T C T / C Result IIIIIII --- 11 AO 25.09.1129 NL --- --- --- --- --- ___- ___-__- ___-__- ___-__- ___-__- ___-__- ___ _____- ___-__- ___-_ ___-__- ___-__- ___-__- Example 2 Optimization of Extraction Parameters In this example, the effect of varying pH levels on the recovery of aatoxin was studied to determine the optimal conditions for mycotoxin extraction using the TrisNaCl formulation. A grain matrix (corn) spiked with 10 ppb aatoxin was used for testing at five different pH levels: 2, 4, 6, 8, and 9. The recovery results were measured using a lateral ow immunoassay, and the data were recorded as shown in Table 4. Table 4: Effect of Tris buffer different pH levels on the recovery of aatoxin(10 ppb standard) l M E £ Æ M ...... 12 AO 25.09.1129 NL _ li- a. 13 AO 25.09.1129 NL ---- ---- ---- ---- ___-_- __ ___-_- ___-_- ___-_- _ The data indicate that pH 9 provides the highest recovery efficiency for aatoxins, with a recovered concentration of 9.78 ppb, which is closest to the spiked concentration of 10 ppb. As pH decreases, the recovery rate also drops significantly, particularly at pH levels below 6, where recovery efficiency becomes suboptimal. Table 5: Aatoxin Recovery with TrisNaCl Extraction: Impact of BSA and Glycine Additives (50 ppb aatoxin standard) Additive M 3 £ & M ...... ___- ___-_- ___-_- ___-_- ___-_- ___-_- ___-_- 14 AO 25.09.1129 NL ---- ---- ---- ---- This table demonstrates that the addition of 0.5M Glycine to the TrisNaCl extraction method results in better recovery of aatoxin compared to the addition of 5% BSA. Key observations: 1. Recovery: With 5% BSA, the average recovery is 33.03 ppb (66.06% of the 50 ppb standard) With 0.5M Glycine, the average recovery is 45.23 ppb (90.46% of the 50 ppb standard) 2. Precision: Both methods show good precision with CV values below 3.5% for the result The Glycine method shows slightly better precision (CV 2.50%) compared to BSA (CV 3.18%) In conclusion, based on this data, the addition of 0.5M Glycine to the TrisNaCl extraction method appears to be superior to the addition of 5% BSA for the extraction and detection of aatoxin at this concentration level. The Glycine method provides better recovery (closer to the true value of 50 ppb) and slightly better precision. Table 6. Comparison of Different Glycine Concentrations in TrisNaCl Extraction Method gm M E £ Æ M -..... ___-_- ___-_- ___-_- 15 AO 25.09.1129 NL 47.50 159.50 0.298 49.75 -..... ___-_- ___-_- ___-_- ___-_- ___-_- 50.50 157.50 0.321 46.85 -..... ___-_- ___-_- ___-_- ___-_- ___-_- 53.50 155.50 0.344 44.25 -..... ___-_- ___-_- Table 6 shows the comparison of different glycine concentrations (0.1M, 0.25M, and 0.5M) in the TrisNaCl extraction method for aatoxin detection. The data is presented for a 50 ppb aatoxin standard. Here's an analysis of the results: 1. 0.1M Glycine: o Average result: 49.75 ppb 0 This concentration provides the closest match to the 50 ppb standard 0 Excellent recovery rate of 99.5 % o Lowest CV% for the result (2.27%), indicating high precision 2. 0.25M Glycine: o Average result: 46.85 ppb 0 Good recovery rate of 93.7% 16 AO 25.09.1129 NL 0 CV% for the result is 2.41%, showing good precision 3. 0.5M Glycine: o Average result: 44.25 ppb o Recovery rate of 88.5% o Highest CV% for the result (2.55%), but still indicates good precision All three concentrations show good precision with CV values below 3% for the final results. The T / C ratios decrease as the glycine concentration increases, indicating that higher glycine concentrations may slightly reduce the signal strength but also improve the extraction efficiency. Based on these results, the 0.1M glycine concentration appears to be optimal for accurate quantification of aatoxin at the 50 ppb level. However, the choice of glycine concentration might depend on the specific application and the range of aatoxin concentrations expected in the samples. Table 7: Comparison of Different Nonionic Surfactants in TrisNaClGlycine Extraction Method Standard: 10 ppb total aatoxin Surfactant M 3 £ Æ & Recover IIIIIII Poloxamer 1 83 145 0.572 9.7 IIIIIII .. ---- ---- IIIIIII .. ---- Tween 20 1 110 125 0.880 3.2 IIIIIII --- --- 17 AO 25.09.1129 NL ___-__ ___-__ ___-__ ___-__ Triton X- 1 105 130 0.808 4.8 IIIIIII --- ---- --- --- ---- --- These results highlight the critical role of Poloxamer 407 in our extraction method. Both alternative surfactants, Tween 20 (polysorbate 20) and Triton X100 (4(l,l,3,3 Tetramethylbutyl)phenylpolyethylene glycol), show substantially lower extraction efficiencies, with recoveries well below 50%. This underscores the unique suitability of Poloxamer 407 for mycotoxin extraction in our formulation. The poor performance of Tween 20 and Triton X100 could be due to several factors: 1. Insufficient solubilization of hydrophobic mycotoxins 2. Potential interference with the antibodyantigen interactions in the lateral flow assay 3. Incompatibility with other components of the extraction buffer In contrast, Poloxamer's excellent performance suggests it effectively solubilizes mycotoxins without interfering with the detection method. Its compatibility with our buffer system and its ability to extract mycotoxins efficiently make it the clear choice for our green extraction method. Data of table 7 strongly supports our choice of Poloxamer 407 as the nonionic surfactant in the present invention, demonstrating its crucial role in achieving high recovery rates and precise results in mycotoxin extraction and detection. 18 AO 25.09.1129 NL Example 3 Extraction Efficiency Across Different Mycotoxins This example demonstrates the extraction efficiency of the TrisNaClGlycine formulation across a range of mycotoxins, including both hydrophobic (aatoxins, zearalenone, T2 / HT2) and hydrophilic (deoxynivalenol, fumonisins) compounds. Grain matrices tested include corn and wheat, but for a broader application, other matrices such as rice and oats were also included in later tests. The following tables present data on the recovery rates of these mycotoxins using the TrisNaClGlycine formulation in comparison to the traditional methanolbased method. Table 9: Total Aatoxin Recovery Efficiency (17 ppb) Using TrisNaClGlycine and Methanol Extraction Extraction M I Q Æ M Recovery IIIIIII Tris- 1 150 0.453 16.8 N aCl- Glycine ___-__- ___-__- __- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- Table 10: Comparative Extraction of Deoxynivalenol (DON) Using TrisNaClGlycine and Methanol Methods 19 AO 25.09.1129 NL ppm standard) Extraction M I C E M Recover _ ... Tris- 1 72 148 0.486 1.95 NaCl- Glycine IIIIIII IIIIIII IIIIIII ---- ---- ----- - ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- Table 11: Ochratoxin A Recovery Efficiency (9 ppb) Using TrisNaClGlycine and Methanol Extraction (9 ppb standard) Extraction m I Q Æ M Recovery -..... Tris-NaCl- 1 65 153 0.425 8.8 IIIIIII _ _ __ Avera 65.5 152.5 0.430 8.78 97.50 IIIIIII 20 AO 25.09.1129 NL ___-__- CV 2.97 2.85 3.79 2.93 IIIIIII ---- ---- _ ___-__- Avera 72.5 147.5 0.492 8.80 97.78 IIIIIII ---- CV(% 2.78 2.87 3.64 2.91 IIIIIII Table 12: Zearalenone Recovery Efficiency (70 ppb) Using TrisNaClGlycine and Methanol Extraction (70 ppb standard) Extraction M I Q Æ M Recover ....... Tris-NaCl- 1 58 156 0.372 68.5 IIIIIII ___ ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- __ ___-__- ___-__- 21 AO 25.09.1129 NL ----- Table 13: Fumonisin Recovery Efficiency (2 ppm) Using TrisNaClGlycine and Methanol Extraction Extractio M I Q Æ & Recover ....... Tris- 1 70 150 0.467 1.96 NaCl- Glycine ___-__- __- ___-__- ___-__- ___-__- ___-__- ___-__- ___-- ___-__- ___-__- ___-__- ___-__- ___-__- Table 14: T2 / HT2 Recovery Efficiency (100ppb) Using TrisNaClGlycine and Methanol Extraction (100 ppb standard) Extractio W I Q Æ M Recovery ....... Tris- l 62 154 0.403 98.3 NaCl- IIIIIII ----- 22 AO 25.09.1129 NL ___-__- ___-__- ___-__- ___-__- ___-__- ___-__- __- ___-__- ___-__- ___-__- ___-__- The results across multiple mycotoxins show that the TrisNaClGlycine formulation consistently yields high recovery rates, particularly for hydrophilic mycotoxins such as deoxynivalenol (DON) and fumonisins, where recovery rates approach 9798%. These recovery rates are comparable to, and in some cases better than, those obtained using the methanolbased extraction method for these hydrophilic compounds. The ionic strength provided by sodium chloride in the TrisNaClGlycine formulation plays a crucial role by disrupting the interactions between the hydrophilic mycotoxins and the grain matrix, ensuring efficient extraction into the aqueous phase. For hydrophobic mycotoxins, such as aatoxins and zearalenone, the TrisNaCl Glycine formulation also performs well, achieving recovery rates close to 9899%. This performance is similar to that of the methanolbased extraction method, due in large part to Poloxamers amphiphilic properties, which enhance the solubility of hydrophobic molecules by forming micelles. Table 15: Aatoxin Bl Recovery in Corn and Wheat Using TrisNaClGlycine Extraction Method Ma_trix Standard M I Q E & Recovery IIIIIII ----- 23 AO 25.09.1129 NL __- ___-___ ___-___ ___-___ __- ___-___ ___-___ __ ___-___ ___ ___-___ ___-___ ___-___ The results show that both the corn and wheat matrices performed similarly well in terms of aatoxin recovery at a spiked concentration of 10 ppb. For the corn matrix, the average recovery was 100%, with an average result of 10.00 ppb, which aligns closely with the spiked concentration. The wheat matrix demonstrated comparable performance, with an average recovery of 100.50% and an average result of 10.05 ppb. The performance of the TrisNaClGlycine formulation was consistent across both matrices, with high recovery rates and precision. The differences between corn and wheat were minimal, suggesting that the method is versatile and effective for different grain types. This consistency in performance highlights the method s reliability in achieving accurate and reproducible results, which is crucial for routine testing and regulatory compliance. Table 16: Dry Extraction Methods for Total Aatoxin: A Comparison of Recovery and Precision Standard: 17 ppb total aatoxin Sample: 5g of grounded grain Added the content of 1 pouch of extraction powder into the grounded sample 24 AO 25.09.1129 NL Dilution M M I Q Æ M Recovery & Ra_ti0 om @ ___-___- Extraction 0.66g 2 75 143 0.524 15.4 IIIIIIII IIIIIIII IIIIIIII ------ ------ ------ ___-___- Extraction 1.1 g 2 149 0.463 16.7 IIIIIIII ___- ___- ___-___- ___-___- ___-___- The dry extraction method shows variable performance depending on the dilution used. The l:5 W / V dilution (5g sample + extraction powder + 25mL water) provides superior recovery, accuracy, and precision compared to the 1:3 dilution. The 1:5 dilution method demonstrates excellent accuracy and precision, making it a strong candidate for reliable aatoxin testing in grain samples. The simplicity of adding a premeasured amount of water to the sample makes this method potentially very useful for field testing or in situations where preparing fresh extraction solutions is impractical. The poor performance of the 1:3 dilution suggests that this ratio may not provide sufficient extraction of aatoxins from the grain matrix. The underestimation of aatoxin concentration with this method could lead to false negatives or underreporting of contamination levels, which is a significant concern in food safety testing. 25 AO 25.09.1129 NL Based on these results, the 1:5 w / v dilution is clearly the preferred method for this dry extraction technique, offering both accuracy and reliability in aatoxin quantification. Example 4 Comparison of Dry and Liquid Extraction Methods In this example, we compare the efficiency of the dry and liquid extraction methods for recovering total aatoxins from corn matrix. The total aatoxin recovery was determined using a lateral ow immunoassay after extraction by both methods. The dry extraction method utilized a premeasured pouch of TrisNaClGlycine formulation, while the liquid method involved mixing the same components in solution prior to extraction. Table 17: Total Aatoxin Recovery Using Dry and Liquid Extraction Methods Across Different Matrices (17 ppb Spiked) % Water / Soluti M I Q E @ m n on Added Riti l_t v_ery 9 M @ Dry 1:5 25 mL water 1 67 151 17.1 Extracti 0.44 4 on 2 149 16.7 0.46 3 3 152 17.3 0.43 4 4 150 16.9 0.45 3 Avera 67. 150. 0.44 17.00 100.0 IIIIIIII SD 1.2 1.29 0.01 0.26 IIIIIIIII 26 AO 25.09.1129 NL CV 1.9 0.86 2.67 1.53 IIIIIIIII Liquid 1:3 15 mL Tris- 1 150 16.9 Extracti NaCl- 0.45 on Glycine- 3 Poloxamer 2 70 148 16.5 407 0.47 3 3 67 151 17.1 0.44 4 4 149 16.7 0.46 3 Avera 68. 149. 0.45 16.80 98.82 IIIIIII SD 1.2 1.29 0.01 0.26 IIIIIIIII CV 1.8 0.86 2.62 1.55 IIIIIIIII Both the dry extraction method and the liquid extraction method demonstrate excellent performance for total aatoxin extraction and quantification. They show comparable recovery rates, accuracy, and precision. However, the dry extraction method offers several advantages: 1. More efficient pH regulation: The premeasured dry reagents ensure consistent pH levels, which is crucial for optimal aatoxin extraction and stability. 2. Ease of use: The dry method simplifies the extraction process by eliminating the need for preparing and storing liquid reagents. 3. Stability: Dry reagents generally have a longer shelf life and are less susceptible to degradation compared to liquid reagents. 27 AO 25.09.1129 NL 4. Fieldfriendly: The dry method is more suitable for field testing as it requires only the addition of water, making it less dependent on laboratory conditions. 5. Slightly better accuracy: The dry method showed a marginally better average result (17.00 ppb) compared to the liquid method (16.80 ppb). Given these advantages, particularly the more efficient pH regulation, the dry extraction method (1 :5 dilution) would be the preferred choice for aatoxin analysis. It offers the same high level of performance as the liquid method while providing additional benefits in terms of consistency, ease of use, and field applicability. Effect of Environmental Conditions: To assess the robustness of both extraction methods, we conducted additional tests under different environmental conditions, including temperature variations (5°C, 25°C, and 40°C). Both methods were effective at standard laboratory conditions (25°C). However, the dry extraction method demonstrated superior stability and accuracy under extreme temperatures. Table 18: Effect of Temperature on Aatoxin Recovery (Corn Matrix, 17 ppb Spiked) Temperature (°C) Recovery (Dry Method) Recovery (Liquid Method) 5°C 96.5% 95.5% 25°C 98.0% 98.5% 40°C 97.3% 98% As shown in Table 18, the dry extraction method maintained a high recovery rate of 96 98% across all temperature conditions, while the liquid method showed more variation, especially at lower (5°C) and higher (40°C) temperatures. The dry method's stability is due to the premeasured dry powder formulation, which ensures that environmental uctuations do not impact the consistency of the extraction process. In contrast, the liquid method is more susceptible to environmental changes, which can affect the solubility and stability of the reagents in the solution, leading to reduced extraction efficiency under extreme temperatures. 28 AO 25.09.1129 NL Table 19: ELISA Method for Total Aatoxin Recovery: Quantitative Comparison with Tris-NaCl Extraction Standard: 8 ppb total aatoxin Sample: 5g of ground grain Extraction: 1:5 dilution with 70% methanol (sample only) Detection: Competitive ELISA with absorbance measured at 450nm Conc. Absorbance Absorbance Average Binding ____ 8 0.302 0.298 0.300 12.136 ..... Calculated sample concentration: 1.589 ppb Accounting for dilution: 1.589 * 5 = 7.945 ppb Recovery: 99.31% CV for sample duplicates: 0.94% The ELISA method demonstrates good accuracy for the 8ppb total aatoxin standard, with a calculated concentration of 7.96 ppb, representing a recovery of 99.50%. The coefficient of variation (CV) for the sample duplicates is 0.94%, indicating excellent precision between replicates. This ELISA method provides a benchmark for comparison with our novel dry extraction method. While it demonstrates high accuracy and precision, it requires the use of hazardous methanol for extraction and involves multiple steps, including incubation and washing procedures. In contrast, our dry extraction method offers comparable performance with significant advantages in terms of safety, ease of use, and field applicability. 29 AO 25.09.1129 NL References 1. World Health Organization. International Agency for Research on Cancer . IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Volume 56. IARC Press; Lyon, France: 1993. Aatoxins; pp. 245395. 2. Liu Y., Wu F. Global burden of aatoxininduced hepatocellular carcinoma: A risk assessment. Environ. Health Perspect. 2010;118:818824. doi: 10.1289 / ehp.0901388. 3. National standard of Iran No. 5925. Institute of Standards and Industrial Research of Iran (2001). Food and feed MycoyoxinsMaximum tolerated levels. First edition. 4. Chauhan, R., Singh, J., Sachdev, T., Basu, T., & Malhotra, B. D. (2016). Recent advances in contaminants, including toxins and other harmful substances detection. Biosensors and Bioelectronics, 81, 532545. 5. Songsermsakul, P., & RazzaziFazeli, E. (2008). A Review of Recent Trends in Applications of Liquid ChromatographyMass Spectrometry for Determination of Mycotoxins. Journal of Liquid Chromatography & Related Technologies, 31(1 112), 16411686. 6. Maggira M, Sakaridis I, Ioannidou M, Samouris G. Comparative Evaluation of Three Commercial Elisa Kits Used for the Detection of Aatoxins B 1, B2, G1, and G2 in Feedstuffs and Comparison with an HPLC Method. Vet Sci. 2022 Feb 25;9(3): 104. 7. MiguelAngel RodriguezDelgado, Guillermo GonzalezHernandez, JoseElias Conde-González, Juan-Pedro PérezTrujillo, Principal component analysis of the polyphenol content in young red wines, Food Chemistry, Volume 78, Issue 4, 2002, Pages 523532, ISSN 0308-8146 8. Stroka, Joerg & Anklam, Elke & Jorissen, U & Gilbert, John. (2000). Immunoaffinity Column Cleanup with Liquid Chromatography Using PostColumn Bromination for Determination of Aatoxins in Peanut Butter, Pistachio Paste, Fig Paste, and Paprika Powder: Collaborative Study. Journal of AOAC International. 83. 32040. 9. Rahmani A, J inap S, Soleimany F. Qualitative and Quantitative Analysis of Mycotoxins. Compr Rev Food Sci Food Saf. 2009 Jul;8(3):20225 1. 30 AO 25.09.1129 NL James H. Clark and Stewart J. Tavener, Organic Process Research & Development 2007 11 (1), 149155, DOI: 10.1021 / op060160g Krska, R., Molinelli, A. Mycotoxin analysis: stateoftheart and future trends. Anal Bioanal Chem 387, 145148 (2007). https: / / doi.org / 10.1007 / s0021600607973 Nicholas W. Turner, Sreenath Subrahmanyam, Sergey A. Piletsky, Analytical methods for determination of mycotoxins: A review, Analytica Chimica Acta, Volume 632, Issue 2, 2009, Pages 168180, ISSN 00032670, https: / / doi.org / 10.1016 / j.aca.2008.11.010. 31 AO 25.09.1129 NL

Claims

1. A dry powder composition for extracting a toxin from a sample, the composition of which includes: (a) a buffering agent; (b) an inorganic salt as an ionic strength agent; (C) a nonionic surfactant; and (d) a stabilizer chosen from the group consisting of an amino acid and bovine serum albumin.

2. Dry powder composition according to claim 1, where the buffering agent is tris(hydroxymethyl)aminomethane.

3. Dry powder composition within the meaning of claim 1 or 2, where the inorganic salt is sodium chloride.

4. Dry powder composition according to one of the preceding claims, where the non-ionic surfactant is selected from the group consisting of poloxamer 407, polysorbate 20 and 4(1,1,3,3etramethylbutyl)phenylpolyethylene glycol.

5. Dry powder composition in accordance with one of the preceding claims, where the non-ionic surfactant is poloxamer 407.

6. Dry powder composition according to one of the preceding claims, where the stabilizer is glycine.

7. Dry powder composition according to one of the preceding claims, where the monster is a grain.

8. Dry powder composition in accordance with one of the preceding claims, where the toxin is a mycotoxin. 32 AO 25.09.1129 NL 9. Dry powder composition in accordance with one of the preceding claims, where The toxin was selected from the group consisting of aflatoxins, ochratoxin A, deoxynivalenol, zearalenone, fumonisins, T-2 and HT2.

10. A kit for extracting toxins from a sample, comprising the dry powder composition according to one of claims 1 to 6.

11. Kit pursuant to claim 10, further comprising a lateral OW test system, or an enzyme-linked immunosorbent test system, or a chemiluminescence immunoassay test system.

12. A method for extracting a toxin from a sample, comprising: (a) combining the dry powder composition in accordance with one of the claims 1 up to and including 9 with the sample; (b) adding water to the combination to a to form the extraction mixture; (c) stirring the extraction mixture; and (d) separating of the liquid extract that contains the toxin. 34 AO25.09.1129EN 1 / 5 Fig.1 Fig.2