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Article

Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection

by
Cong Ji
,
Xinyang Sun
,
Yong Fang
and
Peng Li
*
College of Food Science and Engineering, Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing University of Finance and Economics, Nanjing 210023, China
*
Author to whom correspondence should be addressed.
Foods 2024, 13(5), 640; https://doi.org/10.3390/foods13050640
Submission received: 11 January 2024 / Revised: 7 February 2024 / Accepted: 16 February 2024 / Published: 20 February 2024
(This article belongs to the Section Food Toxicology)
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Abstract

:
Aflatoxin B1 (AFB1) is a highly teratogenic and carcinogenic secondary metabolite produced by Aspergillus. It is commonly detected in agricultural products such as cereals, peanuts, corn, and feed. Grains have a complex composition. These complex components severely interfere with the effective extraction and separation of AFB1, and also cause problems such as matrix interference and instrument damage, thus posing a great challenge in the accurate analysis of AFB1. In this study, an aptamer affinity column for AFB1 analysis (AFB1-AAC) was prepared for the enrichment and purification of AFB1 from grain samples. AFB1-AAC with an AFB1-specific aptamer as the recognition element exhibited high affinity and specificity for AFB1. Grain samples were enriched and purified by AFB1-AAC, and subsequently analyzed by high performance liquid chromatography with post-column photochemical derivatization-fluorescence detection (HPLC-PCD-FLD). The average recoveries of AFB1 ranged from 88.7% to 99.1%, with relative standard deviations (RSDs) of 1.4–5.6% (n = 3) at the spiked levels of 5.0–20.0 μg kg−1. The limit of detection (LOD) for AFB1 (0.02 μg kg−1) was much below the maximum residue limits (MRLs) for AFB1. This novel method can be applied to the determination of AFB1 residues in peanut, corn, and rice.

Graphical Abstract

1. Introduction

Aflatoxins (AFs) are difuranocoumarin metabolites, which are mainly produced by food fungi Aspergillus, particularly Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius [1]. Among these AFs, AFB1 is recognized as the most toxic and prevalent, widely present in corn, rice, nuts, peanuts, and oil [2]. Annually, significant quantities of grains are contaminated with AFB1 due to extreme weather conditions or improper storage practices, particularly in hot and humid environments conducive to the growth of Aspergillus fungi [3,4]. As the International Agency for Research on Cancer (IARC) has classified AFB1 as a Group I human carcinogen, many countries and regions have set maximum residue limits (MRLs) for AFB1 in grains [5,6]. For example, the European Union (EU) has set the maximum level of AFB1 at 5.0 μg kg−1 in maize or rice, and 2.0 μg kg−1 in other cereals. In China, the mandated maximum level for AFB1 ranges from 5.0 to 20 μg kg−1 in different cereals [7]. The presence of AFB1 raises numerous health concerns in humans and livestock, as it exhibits mutagenic, teratogenic, carcinogenic, neurotoxic, and immunosuppressive properties [8]. Therefore, there is an urgent need for the development of robust analytical methods for detection of AFB1.
Thus far, analytical methods based on chromatography, spectroscopy, and immunoassay have been developed and used to determine AFB1. The chromatographic methods include high performance liquid chromatography (HPLC) [9], thin-layer chromatography (TLC) [10], and liquid chromatography-mass spectrometry (LC-MS) [11]. The spectroscopic methods include fluorescence spectrophotometry [12], infrared (IR) spectroscopy [13], and chromatin interacting protein-mass spectrometry (Chip-MS) [14]. In addition to these chromatographic and spectroscopic methods, immunoassays and biosensors have emerged as rapid and convenient tools for AFB1 detection, utilizing various recognition elements such as antibodies, enzymes, cells, and molecularly imprinted polymers [15]. However, while these rapid methods enable quick identification of AFB1, instrumental analytical techniques remain essential for accurate quantitative detection.
HPLC is one of the most powerful analytical instruments for mycotoxin analysis because of its advantageous features, including good usability, low cost, and high sensitivity. However, rigorous sample pre-treatment is required for AFB1 determination with HPLC. In particular, grain samples containing a large amount of complex matrices pose a great challenge for accurate detection, unless effective extraction and separation steps are performed. Therefore, for the determination of AFB1 in grain samples, pre-treatments such as extraction, purification, and concentration are required to achieve separation of interfering components and to effectively enrich traces of the analyte. The common pre-treatment techniques currently available include liquid-liquid extraction (LLE) [16], solid phase extraction (SPE) [17], supercritical fluid extraction (SFE) [18], immunoaffinity column (IAC) [19], and molecularly imprinted polymer (MIP) [20]. Among these, IAC based methods are widely utilized for the analysis of AFB1 in grain samples, as they enable the efficient and rapid separation and concentration of AFB1 from samples, while facilitating the removal of matrix interferences. For example, Shen et al. [21] determined aflatoxins in raw peanuts using IAC as sample clean-up method followed by normal-phase HPLC-fluorescence detector (HPLC-FLD) analysis. Aliakbarzadeh et al. [22] verified a standard method based on IAC cleanup and HPLC-FLD analysis for determination of aflatoxins in peanut kernels; the limit of detection (LOD) of the method was found to be 0.15 μg kg−1 for AFB1. IACs are usually prepared by binding AFB1-specific antibodies to an activated solid phase. As the sample extract passes through the column, AFB1 in the extract is bound by the antibody and adsorbed on the column. However, the use of antibodies as recognition elements has many disadvantages, including high production costs and susceptibility to denaturation during elution [23], underscoring the need for alternative technologies to IAC.
Aptamers are oligonucleotide compounds comprised of 20–100 nucleotides with a complex tertiary or quaternary structure [24]. Aptamers have been developed that can detect targets in the micromolar range, which is comparable to antibodies. High affinity aptamers are isolated by systematic evolution of ligands by exponential enrichment (SELEX), a process that relies on cycles of ligand selection and amplification of sequence libraries [25]. Xu et al. [26] determined the high-resolution structure of AFB1–AF26 DNA aptamer by solution NMR spectroscopy. Furthermore, based on the complex structure, they revealed the molecular mechanism of the aptamer’s discriminatory recognition of AFB1. To date, several aptamers have been applied as recognition elements for AFB1 binding. For example, Fan et al. [27] constructed an electrochemical aptamer sensor for the sensitive detection of AFB1, with a LOD as low as 4.8 fg mL−1. Li et al. [28] developed a colorimetric sensor using gold nanoparticles coupled with AFB1 and ochratoxin A (OTA) aptamers, the LOD of AFB1 was 0.07 ng mL−1. De Girolamo et al. [29] prepared an aptamer-based SPE column for OTA purification in wheat samples prior to HPLC-FLD analysis, with a LOD of 23 pg g−1. Zeng et al. [30] prepared Fe3O4@SiO2–NH2 material with a core–shell structure. Using this material, they developed an aptamer-based magnetic solid-phase extraction method for the pretreatment of AFB1 in bean sauce samples; the recoveries of AFB1 ranged from 80.19% to 113.92%. In comparison with antibodies, aptamers exhibit several outstanding features, including simple large-scale chemical production, easy chemical modification, low cost, and resistance to denaturation [31,32]. Thus, they are promising alternatives for various analytical applications, including AFB1 detection. However, the use of aptamers in enriching and purifying AFB1 from grains remains relatively underexplored.
In the present study, a novel AFB1-AAC was developed for the enrichment and purification of AFB1 in grains. After coupling with an amino-modified AFB1 aptamer, AAC immobilization was realized through a covalent reaction between isocyanate and amino groups. The operation procedures of AFB1-AAC were optimized, and its reproducibility and specificity were evaluated and compared with AFB1-IAC. The proposed method of AFB1-AAC enrichment and purification coupled with HPLC detection was validated by standard quality control materials (QCMs), and applied for the analysis of AFB1 in peanut, corn and rice samples.

2. Materials and Methods

2.1. Reagents and Chemicals

The sequence of a known AFB1-specific DNA aptamer was obtained from the literature [33]. The AFB1-specific aptamer sequence—5′-NH2C12-AAA AAA GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3′—was then synthesized by Sangon BioEngineering (Shanghai, China). CNBr-activated sepharose (particle size, 45–165 μm) was purchased from Chromsep Biotechnology (Qingdao, China). AFB1 standard (10 µg mL−1) was purchased from RomerLabs (Tulln, Austria). AFs mixture standard (AFB1, AFB2, AFG1 and AFG2, 1000 ng mL−1), OTA (10 µg mL−1), zearalenone (ZEN, 100 µg mL−1), and deoxynivalenol (DON, 100 µg mL−1) were all purchased from Tan-Mo Technology (Changzhou, China). QCMs of AFB1-contaminated corn and wheat were provided by Meizheng Bio-Tech (Rizhao, China). Methanol and acetonitrile were HPLC grade. All other chemicals were at least analytical grade and were used without further purification. Ultrapure water from a Millipore Milli-Q system was used throughout. The grain samples were collected from a local supermarket (Nanjing, China) and stored at 4 °C.

2.2. Instrumentation

HPLC analyses were conducted using an Agilent 1260 series liquid chromatography system equipped with a fluorescence detector (Agilent, Santa Clara, CA, USA). A photochemical reactor (Huaan Magnech Bio Tech, Beijing, China) was also used for enhanced photochemical derivatization (PCD) detection. Empty columns for the fabrication of AAC columns were procured from Biocomma (Shenzhen, China). Commercial IACs for aflatoxins and mycotoxins were obtained from Romer Labs (Washington, DC, USA). The C18 column (150 mm × 4.6 mm i.d., particle size 5 µm) for HPLC separation was sourced from Phenomenex (Guangzhou, China). The benchtop rotary evaporator for removing solvent was provided by Heidolph (Shanghai, China).

2.3. Preparation of AFB1-AAC

The aptamer was first immobilized onto the surface of CNBr-activated sepharose. Briefly, the aptamer was dissolved in reaction buffer (200 mM Na2HPO4, 5 mM MgCl2, pH 8.0), heated at 85 °C for 5 min, and subsequently left at room temperature for 30 min to activate the aptamer. Concurrently, 0.5 mL of CNBr-activated sepharose was pre-washed and activated. Subsequently, the activated aptamers with different amounts (0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, and 0.5 nmol) were coupled to the CNBr-activated sepharose at room temperature with overnight shaking. Following the coupling step, the above sepharose was washed once with 1 mL of Na2HPO4 (200 mM, pH 8.0) and then blocked to deactivate any remaining active sites using 0.5 mL of Tris-HCl (0.1 M, pH 8.0). Any remaining uncoupled aptamer was removed by washing alternately with 1 mL of acetic acid buffer (0.1 M acetic acid, 0.5 M NaCl, pH 4.0) and 1 mL of Tris-HCl buffer (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0) three times. After centrifugation, the supernatant was discarded. The obtained AFB1 aptamer-sepharose was resuspended in binding buffer (20 mM Tris-HCl, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, pH 7.4), and loaded onto an empty AC column. Lastly, the prepared AFB1-AAC was washed with 10 mL of binding buffer, and stored at 4 °C in 0.5 mL of binding buffer.

2.4. Operation of AFB1-AAC

Grain sample extraction procedure: grain samples (5 g) were first pulverized and sonicated with 20 mL methanol for 20 min. Subsequently, the resulting mixture was centrifuged at 6800× g for 10 min. The obtained supernatant was removed using a rotary evaporator and the pellet was then re-solubilized with 25 mL of loading buffer. The obtained sample extract was filtered using a 0.22 µm filter membrane for subsequent enrichment and purification.
Enrichment and purification procedure: 20 mL of the above sample extract was loaded onto the AFB1-AAC. Next, the AFB1-AAC was washed with 1 mL of loading buffer to remove nonspecifically adsorbed interfering substances. Finally, bound AFB1 was eluted with 1 mL of elution buffer (methanol-acetic acid elution buffer, 98:2, v/v). The collected eluent was subsequently injected into the HPLC-PCD-FLD system for analysis. All the solutions were driven through the AFB1-AAC by gravity flow (flow rate, approx. 1 mL min−1).
AFB1-AAC regeneration procedure: the AFB1-AAC was equilibrated with 10 mL binding buffer and stored at 4 °C for further use.

2.5. HPLC-PCD-FLD Analysis

The conditions for HPLC-PCD-FLD analysis were optimized as follows. Chromatographic separation was performed on a C18 column (150 mm × 4.6 mm i.d., particle size 5 µm) maintained at 30 °C. The separation procedure was performed using an isocratic eluent of methanol-water (50:50, v/v) as the mobile phase. To achieve optimal resolution, a flow rate of 1.0 mL min−1 was employed. The sample injection volume was set at 20 µL. The excitation and emission wavelengths of the fluorescence detector were 360 nm and 440 nm, respectively.

3. Results and Discussion

3.1. AFB1-AAC Working Principle

AFB1-specific aptamer was covalently linked to activated sepharose through a reaction between the -NH2 group of the amino modified aptamer and the -O-CN group of the CNBr-activated sepharose. The -NH2 group acts as a nucleophile, attacking the electrophilic carbon in the -O-CN group, leading to a stable amide bond formation. This reaction can easily occur at room temperature and is widely used in the field of biochemistry and molecular biology [34]. Detailed procedures for linking AFB1-specific aptamers with CNBr-activated sepharose, and the workflow for AFB1-AAC are illustrated in Figure 1.
The AFB1-AAC enrichment and purification procedure can be summarized in four steps: (1) column conditioning; (2) sample loading; (3) washing to remove interfering substances; and (4) elution of the target analyte. Prior to use, the AFB1-AAC is conditioned with 5 mL of loading buffer, equilibrating the column’s environment to optimize aptamer-AFB1 interaction. Following this, the AFB1-containing solution is introduced onto the column, where the aptamer’s unique three-dimensional structure and chemical properties facilitates selective binding of AFB1, distinguishing it from other molecules in the mixture. Subsequent washing removes non-specifically bound components, exploiting the differential binding affinities to ensure that only the target AFB1 remains adsorbed. Finally, the specifically bound AFB1 is released from the column using a carefully chosen elution reagent (methanol-acetic acid), which disrupts the aptamer-AFB1 interaction without damaging the integrity of either molecule. The eluted AFB1 is then collected for HPLC-PCD-FLD analysis.

3.2. Optimization of Operation Conditions of AFB1-AAC

The amount of immobilized aptamer plays a decisive role in AFB1 capture. Various AFB1-AACs were prepared using differing quantities of aptamers to explore how aptamer dosage influences the recovery of AFB1. As depicted in Figure 2a, when these columns were treated with a 20 mL solution containing 5 ng of AFB1, it was found that AFB1 recovery improved with an increase in the aptamer amount from 0 to 0.1 nmol. When the amount of aptamer exceeded 0.2 nmol, the recovery rate increased to between 98.1% and 106.2%, meeting the requirements for quantitative recovery. These results also indicated that 0.2 nmol of immobilized aptamer can effectively bind approximately 0.016 nmol of AFB1.
To further elucidate the relationship between aptamer and AFB1, a standard curve was generated, with the molar amount of aptamer as the X-axis and the molar amount of bound AFB1 as the Y-axis. As shown in Figure 2b, the standard curve exhibited a slope of 0.13 and a correlation coefficient (R2) of 0.984. These results indicated that the amount of immobilized aptamer significantly influenced the enrichment and purification of AFB1. Thus, the amount of aptamer should be at least eight times that of AFB1.
For grain sample analysis, the first step is extraction with solvent. In general, AFB1 is extracted from samples using organic solvents, such as pure methanol. However, organic solvents may impact the aptamer’s structure, potentially diminishing the affinity between the aptamer and its target [35]. Hence, organic components carried over from the extraction procedure may affect AFB1 capture on the AFB1-AAC. To address this issue, the maximum tolerance of the AFB1-AAC to methanol during the sample loading process was evaluated. First, the AFB1 standard solution was diluted with loading buffer containing varying concentrations of methanol (10%, 20%, 30%, 40%, 50%, 60%, and 70% (v/v)). Subsequently, an AFB1-AAC purification test was conducted. As shown in Figure 3a, AFB1 recovery decreased as the methanol concentration increased, indicating that the methanol concentration should be controlled below 10%. To ensure AFB1 retention on the column, the methanol in sample extract was evaporated using a rotavapor. Then, the sample was reconstituted with loading buffer to reduce the concentration of organic reagents and minimize the matrix interference.
The solution pH value has a significant impact on the affinity of an aptamer, as it directly affects the three-dimensional structure of the aptamer and its interactions with the target molecule [36]. If the pH value is too high or too low, it may lead to protonation or deprotonation reactions of the aptamer’s bases, altering their charge and consequently affecting the spatial structure of the aptamer. Such structural changes can weaken the binding affinity between the aptamer and the target molecule, or even completely inhibit their interaction [26]. Moreover, the structure and charge characteristics of the target molecule may also be affected by the pH value, further influencing the binding affinity of the aptamer [37]. Therefore, in the application of aptamers, researching and optimizing the impact of solution pH on aptamer affinity is crucial for achieving efficient and specific recognition. To address this issue, the pH tolerance of AFB1-AAC was evaluated by testing the AFB1 solutions with different pH (4.0–11.0). As depicted in Figure 3b, AFB1 recovery increased as the pH increased from pH 4.0 to pH 8.0, and then decreased drastically (from 104.1% to 9.7%) as the pH increased further from pH 9.0 to pH 11.0. Consequently, the optimal pH of the loading solution was set at pH 8.5 for this study.
In addition, the effect of flow rate on recovery was investigated at different flow rates in the range of 0.2−3 mL min−1. The average recovery for AFB1 exceeded 90% at each flow rate, showing that the variation in flow rate did not affect the recovery. Considering the convenience of use, the final design relied directly on natural gravity for operation, resulting in a solution flow rate of approximately 1 mL min−1.

3.3. Reusability of AFB1-AAC

The reusability of a column for sample pretreatment is a crucial factor in practical applications. The inability of IACs to be recycled is a major factor contributing to their high cost of utilization. The tolerance of aptamers to harsh conditions, including high salt and organic solvents, is known to be much higher than that of antibodies [38]. To evaluate the reusability of the prepared AFB1-AAC, it was tested over 30 cycles of loading, washing, and elution. As shown in Figure 4, during the initial 24 cycles, the average recovery of AFB1 ranged from 82.9% to 103.3%, and RSDs were within the range of 0.9–4.3%. However, the AFB1 recovery declined to 80% after the 24th cycle. These results demonstrate the favorable reproducibility of AFB1-AAC and its potential for cost-effective, repetitive usage.

3.4. Specificity of AFB1-AAC

In general, coexisting interfering substances in the solution may compete with the target analyte for active binding sites. To assess the specificity of AFB1-AAC, several AFB1 structural analogs—AFB2, AFG1, and AFG2—and several other toxins commonly found in grains—ZEN, DON, and OTA—were tested using the same procedure and conditions. As shown in Figure 5, the recoveries of AFG1, AFG2, ZEN, DON, and OTA were all very poor after AFB1-AAC extraction. However, the recovery of AFB2 was around 64.1%, suggesting that AFB1-AAC also possessed an affinity for AFB2. Nonetheless, due to the marked disparity in retention times between AFB2 and AFB1 on the HPLC-PCD-FLD chromatogram (7.13 min and 8.42 min, respectively), the specificity of AFB1 remained largely unaffected. Therefore, the presence of other mycotoxins in the grain does not affect the detection of AFB1 by AFB1-AAC. Besides, it is conceivable that with the selection of AFB1 aptamers with higher specificity, the possible for cross-reactivity could be further reduced.

3.5. Comparison of AFB1 Purification Using AFB1-IAC and AFB1-AAC

To evaluate the practical applicability of AFB1-AAC for the enrichment and purification of AFB1, it was compared with a commercial AFB1-IAC for the determination of AFB1 in peanuts. The resulting chromatograms are displayed in Figure 6. The results showed a significant intensification of the AFB1 peak, indicating that the target AFB1 was efficiently enriched using both the AFB1-AAC and the AFB1-IAC. In addition, both baselines were relatively clean. However, a meticulous comparison of the chromatograms of AFB1-IAC and AFB1-AAC revealed that there was less interference around the AFB1 peak by using AFB1-AAC. This suggested that AFB1-AAC may exhibit the greater tolerance to the complicated sample matrices, effectively removing interfering substances from sample extracts. Most importantly, there was no significant difference in the detected AFB1 between AFB1-AAC and AFB1-IAC extractions. Therefore, the AFB1-AAC can be confidently used as an alternative sample treatment method to AFB1-IAC.

3.6. Storage Stablility and Preparation Reproducibility of AFB1-AAC

The storage stability of the AFB1-AAC was assessed by evaluating its performance at three and six weeks post-preparation, with storage conditions maintained at 4 °C in a fridge. The recoveries of AFB1 consistently exceeded 90.0%, demonstrating the robust stability of the AFB1-AAC over time. Moreover, the preparation reproducibility of the AFB1-AACs was examined by testing the columns prepared within the same batch (n = 6) and across different batches (n = 6). The resulting RSDs for analyses of the same AFB1-contaminated sample were 3.4% for intra-batch and 4.6% for inter-batch comparisons, respectively. These low RSD values indicated that the AFB1-AACs were reliably reproducible, ensuring consistent performance in the detection of AFB1 in contaminated samples [39].

3.7. Method Validation and Application

Under optimized conditions of AFB1-IAC enrichment and purification, the LOD (calculated as a signal-to-noise ratio (S/N) of 3), and the limit of quantitation (calculated as a S/N of 10), were 0.02 µg kg−1 and 0.06 µg kg−1, respectively. Table 1 presents a comparison of the analytical performance obtained using the AFB1-AAC method with the previous works. The developed method for AFB1-AAC enrichment and purification in this study displayed good sensitivity and a high enrichment factor.
The developed method was validated by determination of AFB1 in QCMs. The analytical results are reported in Table 2. The good agreement between certified and determined values demonstrates the excellent accuracy of the proposed method. Based on the abovementioned findings, this successfully validated method was applied to the determination of AFB1 in peanut, corn and rice samples. The analytical results along with recoveries for the spiked samples are given in Table 3. The average recoveries of AFB1 in peanut, corn, and rice samples were 93.3–95.3%, 88.8–99.1%, and 88.7–95.0%, with RSDs of 1.4–3.0%, 1.8–5.6%, and 2.1–4.6%, respectively. These results demonstrate that the proposed AFB1-AAC enrichment and purification coupled with HPLC-PCD-FLD determination can be effectively used for the quantitative analysis of AFB1 in grain.
It is worth noting the construction of the AFB1-AAC offers significant benefits, including low raw material costs and an eco-friendly synthesis process. All the materials for the AFB1-AAC are readily available. According to the calculations based on quotations from the suppliers of raw materials, the manufacturing cost per AFB1-AAC would typically be less than USD $0.9, which is at least ten times lower than the IAC. Moreover, given its potential for reuse, the AFB1-AAC is economically acceptable and suitable for routine sample testing, which enhances its practicality in various applications.

4. Conclusions

In the present study, an AFB1-AAC for the enrichment and purification of AFB1 was prepared by covalently coupling an amino-modified AFB1-specific aptamer to CNBr-activated sepharose. The conditions for the use of this AFB1-AAC were optimized, and its reproducibility and specificity were investigated. The AFB1-AAC exhibited superior reproducibility, maintaining consistent performance over 24 repeated cycles. For spiked peanut, corn, and rice samples, the AFB1-AAC was able to remove matrix interferences and enable the quantitative analysis of AFB1. Comparison of AFB1-AAC with commercial AFB1-IAC also showed excellent results. AFB1-AAC exhibits the advantages of low cost, environmentally friendly attributes, and high reproducibility, and the proposed method can be used for the enrichment and separation of AFB1 in peanut, corn, and rice. Future efforts will focus on identifying AFB1 aptamers with superior performance and extending the method’s applicability to a broader range of food matrices.

Author Contributions

C.J.: Conceptualization, Methodology, Software. X.S.: Data curation, Writing- Original draft preparation. Y.F.: Funding acquisition. P.L.: Supervision, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32072317), the National Key Research and Development Program of China (2021YFD2100601), the Jiangsu Provincial Key Research and Development Project (Modern Agriculture) (BE2021370), the Natural Science Foundation of Jiangsu Province (BK20210672).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Conflicts of Interest

All authors declare that they have no conflict of interest.

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Foods 13 00640 g001
Figure 1. Schematic showing the production and use of AFB1-AAC. Top, conjugation of AFB1 and activated sepharose. Bottom, work flow for AFB1 purification on AFB1-AAC.
Figure 1. Schematic showing the production and use of AFB1-AAC. Top, conjugation of AFB1 and activated sepharose. Bottom, work flow for AFB1 purification on AFB1-AAC.
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Figure 2. Effect of the immobilized aptamer on AFB1 recovery. (a) AFB1 recovery versus immobilized aptamer. (b) Aptamer molarity in relation to AFB1 (n = 3).
Figure 2. Effect of the immobilized aptamer on AFB1 recovery. (a) AFB1 recovery versus immobilized aptamer. (b) Aptamer molarity in relation to AFB1 (n = 3).
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Figure 3. Effects of methanol concentration (a) and pH (b) of sample solution on AFB1 recovery (n = 3).
Figure 3. Effects of methanol concentration (a) and pH (b) of sample solution on AFB1 recovery (n = 3).
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Figure 4. Effect of repeated use of AFB1-AAC on AFB1 recovery (n = 3).
Figure 4. Effect of repeated use of AFB1-AAC on AFB1 recovery (n = 3).
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Figure 5. Recovery of various mycotoxins using the AFB1-AAC extraction procedure (n = 3).
Figure 5. Recovery of various mycotoxins using the AFB1-AAC extraction procedure (n = 3).
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Figure 6. Comparison of AFB1 purification using AFB1-IAC and AFB1-AAC. Chromatograms of AFB1 contaminated peanut treated by AFB1-AAC (1) and AFB1-IAC (2), AFB1 standard solution in methanol (3), peanut extract without purification (4).
Figure 6. Comparison of AFB1 purification using AFB1-IAC and AFB1-AAC. Chromatograms of AFB1 contaminated peanut treated by AFB1-AAC (1) and AFB1-IAC (2), AFB1 standard solution in methanol (3), peanut extract without purification (4).
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Table 1. Comparison of analytical performance of the proposed AFB1-AAC method with related previous methods.
Table 1. Comparison of analytical performance of the proposed AFB1-AAC method with related previous methods.
Pretreatment TechniqueEnrichment
Factor		Determination TechniqueLOD (µg kg−1)Reference
SiO2@Fe3O45HPLC-MS/MS0.04[40]
QuEChERS5LC-MS/MS0.03[41]
Immunoaffinity column12.5HPLC-PCD-FLD0.03[42]
HLB SPE cartridge14LC-ESI-MS/MS0.017[43]
AFB1-AAC20HPLC-PCD-FLD0.02This work
Table 2. Analytical results for determination of AFB1 in QCMs (mean ± SD, n = 3).
Table 2. Analytical results for determination of AFB1 in QCMs (mean ± SD, n = 3).
QCMsMatrixCertified (µg kg−1)Found (µg kg−1)
MRM0021-0 (A101903B)wheat0.0 ± 0.0ND
MRM0001-0 (A012001B)corn23.3 ± 3.521.3 ± 1.5
MRM0021-0 (A032303B)wheat33.2 ± 5.031.6 ± 1.4
Table 3. Recoveries of AFB1 in grain samples with different spiking levels.
Table 3. Recoveries of AFB1 in grain samples with different spiking levels.
No.Spiked (µg kg−1)Recovery (%)RSD (%, n = 3)
Peanut1593.32.6
21095.33.0
32094.21.4
Corn1599.11.8
21088.82.4
32091.45.6
Rice1594.33.7
21095.02.1
32088.74.6
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Ji, C.; Sun, X.; Fang, Y.; Li, P. Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection. Foods 2024, 13, 640. https://doi.org/10.3390/foods13050640

AMA Style

Ji C, Sun X, Fang Y, Li P. Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection. Foods. 2024; 13(5):640. https://doi.org/10.3390/foods13050640

Chicago/Turabian Style

Ji, Cong, Xinyang Sun, Yong Fang, and Peng Li. 2024. "Determination of Aflatoxin B1 in Grains by Aptamer Affinity Column Enrichment and Purification Coupled with High Performance Liquid Chromatography Detection" Foods 13, no. 5: 640. https://doi.org/10.3390/foods13050640

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