Determination of Aflatoxins in Peanut Oil by One-Step Solid-Phase Extraction with Metal-Organic Framework MIL-101 (Cr) Filled Syringe Filter and HPLC-MS/MS

Abstract A simple, rapid and sensitive method for the determination of aflatoxins in peanut oil was developed using MIL-101(Cr) material filled syringe filter for one-step solid-phase extraction (SPE) with high-performance liquid chromatography—tandem mass spectrometry (HPLC-MS/MS) detection. The MIL-101(Cr) sorbent was characterized and its mass, the loading solution, and the eluent were optimized; under these conditions, a matrix matching internal standard method was developed. The linear range was from 0.1 to 20 ng/mL with correlation coefficient (r) exceeding 0.9998. The recoveries were between 94.7% and 105.9% with relative standard deviations from 2.02% 6.88%. The recoveries of inter-day and intra-day measurements were from 93.0% to 98.5% and 97.6% to 100.7%. The precision values for inter-day and intra-day analyses were from 6.09% to 7.68% and 2.56% to 4.30%, respectively. The pretreatment efficiency of the developed method was superior to literature procedures and used to determine aflatoxins in 126 peanut oil samples from local markets. 38 samples had positive results and 1 sample exceeded the allowable limit. Hence, the developed protocol was suitable for the determination of aflatoxins in peanut oil.


Introduction
Aflatoxins (AFs) are toxic secondary metabolites of Aspergillus species (mainly A. flavus and parasiticus) that exhibit immunotoxic, hepatotoxic, carcinogenic, and teratogenic effects in humans (Vijayanandraj et al. 2014;Acaroz 2019).Exposure to aflatoxins may occur by consumption of cereals, milk, and spices.Fungal contamination is an important problem in agricultural products resulting in economic and food safety concerns (Xue et al. 2019;Shabeer et al. 2022).Aflatoxin production is affected by environmental conditions including humidity and high temperature.
AFB 1 has been classified by the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) to be a class I carcinogen (IARC. 1993).There are lower carcinogenic properties for AFG 1 .Cancer cannot be caused directly by AFB 2 and AFG 2 , but it is reported that AFB 2 may be bio-transformed into AFB 1 in vivo.Therefore, there is some carcinogenicity in AFB 2 (Li, Zhang, and Zhang 2009).
Peanut oil is an important part of the human diet (Indelicato et al. 2017;Liu et al. 2018).; Peanuts and peanut products can easily infected by aflatoxins.If the peanut raw materials are not properly controlled in production, processing, transportation, and storage, then they are highly susceptible to fungal infections, resulting in the production of aflatoxins (Silva, Garcia, and Paiva-Martins 2010;Saini, Rani, and Kumari 2014) that may endanger human health (Manafi et al. 2015).Aflatoxins are difficult to eliminate by conventional methods because of the high temperature resistance and stability.However, ultraviolet light (Liu et al. 2011a;2011b;2012), microwave radiation, and photocatalysis (Magzoub et al. 2019) may destroy aflatoxins.Genetic engineering and antagonistic bacteria have been shown to inhibit the growth of toxin producing strains and aflatoxin production (Chen, Kong, and Liang 2019).
However, the primary preventive measure is to formulate standards.Strict standards for AFs have been specially formulated.GB 2761-2017 National Standard for Food Safety Limits of Mycotoxins in Food of China stipulates that the limit of AFB 1 in peanut oil and corn oil is 20 mg/kg and 10 mg/kg in other vegetable oils.There are no limits on the other aflatoxins.
EC Decree No. 1525/98 issued by the European Union stipulates that the limit of AFB 1 in food is 2 mg/kg, and the total mass of AFB 1 þ AFB 2 þ AFG 1 þ AFG 2 cannot exceed 15 mg/kg.Japan stipulates that AFB 1 shall not be presented in food, and the total mass of AFB 1 þ AFB 2 þ AFG 1 þ AFG 2 is below 15 mg/kg.Herein, it is of significance to develop a rapid, efficient, sensitive, and accurate method to monitor aflatoxins in peanut oil.
Current methods for aflatoxins include thin-layer chromatography (Vijayanandraj et al. 2014), enzyme-linked immunosorbent assay (Liu et al. 2016;Acaroz 2019), high performance liquid chromatography with fluorescence detection (Somsubsin et al. 2018;Kim et al. 2020), and liquid chromatography with mass spectrometry detection (Romero-S anchez et al. 2022;Wang et al. 2022).GB 5009.22-2016National Food Safety Standard Determination of Aflatoxins B and G in Food includes the above procedures.Limitations of these methods include cumbersome operation, poor reproducibility, low selectivity, and low accuracy (Yang et al. 2022a).
The primary method for the determination of aflatoxin in recent years is highperformance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) because of its high sensitivity and good selectivity.For sample pretreatment, the immunoaffinity column is the primary procedure, but this approach is expensive and time-consuming.An effective pretreatment procedure is required to remove the fat from the oil samples.Extraction by a packed sorbent (EPs) is a miniature solid-phase extraction (SPE) separation that seals the adsorbent in the syringe that provides high permeability, favorable enrichment, and good recoveries.This approach has been employed for the analysis of a variety of environmental and biological samples (Jiang et al. 2018).
Here a simple, rapid, efficient, and low-cost method is reported to determine aflatoxins in peanut oil.A MIL-101(Cr) sorbent was prepared and characterized for solidphase extraction.The extraction, rinsing, and elution were rapidly completed in the filter head.The determination of AFB 1 , AFB 2 , AFG 1 , and AFG 2 in peanut oil was performed with HPLC-MS/MS detection.Peanut oil from China was analyzed to demonstrate the applicability of the proposed method for the aflatoxin analytes.

Instruments and conditions
A high-performance liquid chromatograph (LC-30A, Shimadzu, Japan) was used for analysis.The analytes were separated by a Chromcore C18 column (inner diameter 2.1 mm Â 150 mm, particle size 1.8 lm) (Nanochrom, Suzhou) at a flow rate of 0.15 mL/min.The column temperature was 40 C. The mobile phase was composed of aqueous 0.1% formic acid (A) and acetonitrile (B).The gradient elution was 10.0% B in 2.0 min, 10.0% to 95.0% B in 3.0 min, 95.0%B in 3.0 min, 95.0% to 10.0% B in 0.01 min, and 10.0% B in 1.99 min.The injection volume was 3 lL.
The theoretical number of plates for the analytes exceeded 50,000, indicating rapid and efficient separation.The separation degree of four analytes were determined to be higher than 1.5, meeting the experimental requirements.The tailing factors for the analytes were between 0.98 and 0.99, demonstrating that the analyte peaks were symmetrical with no tailing.Example chromatograms are shown in Figure 1.
A triple quadrupole mass spectrometer (4500 QTrap, AB Sciex, USA) equipped with electrospray ionization was employed in the positive ion mode with multiple reaction monitoring (MRM).The optimized conditions were ion spray voltage, 4500 V; source temperature, 450 C; collision gas, medium, nebulizing gas, 50 psi; heater gas, 55 psi; and curtain gas, 35 psi.As shown in Table S1 (supplementary material), the MRM parameters included the measured ion, precursor ions, product ions, delocalization potential and collision energy.
A D8 Advance instrument (XRD, Bruker, Germany) was used to collect the X-ray diffraction patterns.A Zeiss Gemini 300 device (Germany) was used to record the scanning electron microscopic (SEM) images.

Preparation of MIL-101 (Cr)
MIL-101 (Cr) was synthesized by a hydrothermal reaction (Yang et al. 2022b).1.64 g terephthalic acid and 4 g Cr (NO 3 ) 3 9 H 2 O were treated with 70 mL of water and 0.125 mL of 40% HF and thoroughly mixed by 30 min of sonication.The mixture was sealed and heated at 220 C for 8 h.The product was collected using a 250 mesh stainless steel screen, centrifuged, and the supernatant was removed.The precipitate was washed 3 times with deionized water and dried under vacuum at 70 C for 24 h to obtain a green powder product.

Construction of solid phase extraction device
40 mg of MIL-101(Cr) were treated with 10 mL isopropyl alcohol and sonicated to prepare a 4 mg/mL dispersion.A total of 1.5 mL of the dispersion was introduced into a needle nylon filter with a 0.22 mm filter membrane.MIL-101(Cr) crystal was collected in the filter to prepare the MIL-101(Cr) solid phase extraction device.Sample preparation About 0.2 g of peanut oil were treated with the mixed internal standard solution and 2.0 mL of n-hexane, vortex mixed, placed in a syringe, and introduced into the MIL-101(Cr) solid phase extraction device at a flow rate of 1.0 mL/min.2.0 mL of n-hexane were used to remove the residual matrix components.3.0 mL of acetone were employed to elute the analytes.The eluent was evaporated to dryness at 40 C under nitrogen.The residue was dissolved in 0.2 mL of 20% acetonitrile water and passed through a 0.22 lm nylon membrane filter before HPLC-MS/MS analysis.

Characterization of MIL-101(Cr)
The X-ray diffraction pattern and the scanning electron microscope (SEM) characterization of MIL-101(Cr) in Figure 2 show agglomeration with good crystal structure and relatively uniform particle size, comparable to the literature (Yang et al. 2022b).Six diffraction peaks were present at 2h values of 1. 68 , 2.73 , 3.22 , 3.92 , 5.14 , and 8.99 , consistent with the standard results.

Optimization of HPLC-MS/MS conditions
The target compounds were determined by HPLC-MS/MS.Flow injection was used to inject the standards and samples into the mass spectrometer.The parent ion was scanned in positive and negative ion mode.The results showed that the response of the parent ion was stronger and the fragmentation was better in the positive mode because of the protonated [M þ H] þ ions of all analytes.The response and separation of the analytes were affected by the liquid chromatography conditions, including the mobile phase.The use of methanol water produced high viscosity, weak elution ability, delayed retention times, and peak broadening.Reduced background and higher response were obtained employing acetonitrile water.
Formic acid was added to the aqueous phase to produce an M þ 1 ion, significantly improving the ionization efficiency and the peak shape.The results showed that the 0.1% formic acid water-acetonitrile system provided good sensitivity with symmetrical peaks (Figure 1).Hence, aqueous 0.1% formic acid-acetonitrile solution was selected to be the mobile phase.

Optimization of extraction conditions
The mass of adsorbent, loading solution, and eluent solvent were varied to optimize the SPE conditions by monitoring the analyte recoveries.Peanut oil samples spiked with 1.0 lg/kg AFB 1 , AFB 2 , AFG 1 and AFG 2 were employed to optimize these parameters.

Mass of sorbent
The unsaturated metal sites of MIL-101 (Cr) may attract aflatoxins through hydrogen bonding.Several masses of MIL-101(Cr) were employed for the solid-phase extraction of AFB 1 , AFB 2 , AFG 1 and AFG 2 in peanut oil. Figure 3A shows that the extraction efficiency was improved when the mass of adsorbent was increased from 2 to 6 mg.However, higher masses of MIL-101(Cr) did not improve the extraction efficiency.Hence, 6 mg of sorbent were employed in the subsequent measurements.

Sample dilution conditions
Due to the high viscosity of peanut oil, the samples were diluted with solvent.Here, ethyl acetate, n-hexane, and dichloromethane were investigated as shown in Figure 3B.The highest extraction efficiency was obtained using n-hexane, with all analyte recoveries exceeding 80%.The higher polarities of ethyl acetate and dichloromethane caused some of the aflatoxins to be eluted from the MIL-101(Cr), resulting in low extraction efficiency.Therefore, n-hexane was selected to be the loading solvent.

Elution solvents
Methanol, acetonitrile, and acetone were investigated as elution solvents as shown in Figure 3C.Acetone was selected as the elution solvent due to its rapid concentration period.The volume of acetone was optimized as shown in Figure 3D.3.0 mL of acetone were sufficient to completely elute the aflatoxins.

Reusability of MIL-101 (Cr) filled syringe filter
To evaluate the reusability of MIL-101 (Cr) filled syringe filter, the device was rinsed three times with 3 mL of acetone after each adsorption-desorption cycle.The results showed insignificant changes in the adsorption efficiency.The recoveries were from 89.5% to 102.3% with a relative standard deviation of 2.5%-5.0%.Herein, MIL-101 (Cr) filled syringe filter was sufficiently stable for practical analysis.

Method validation
The developed procedure was validated in accordance with the European Union's SANTE 11813/2021 and China GB/T 27404-2008 on the bases of limits of detection, linear dynamic range, and matrix effects, as shown in Table 1.The accuracy and precision obtained for four concentration levels are summarized in Table 2.
The analyses were performed by matrix matching and the internal standard method for 0.1 (LOQ), 1.0, 2.0, and 5.0 lg/kg of AFB 1 , AFB 2 , AFG 1 and AFG 2 , respectively.These values were all within the linear range from 0.1 to 20 ng/mL with correlation coefficients (r) exceeding 0.9998.The limits of detection were evaluated on the bases of 3 and 10 times of signal-to-noise ratio, providing values of 0.03 and 0.1 lg/kg, respectively.The relative standard deviation at the limits of quantitation was between 2.02% and 4.56%.
Although countries have different aflatoxin limits, these parameters are within the linear range of the developed method, suggesting its application for practical analysis.

Matrix effects
Matrix effects were characterized by comparing the slopes of the calibration curve for peanut oil samples and solvent.The matrix effects were insignificant and within the acceptable range from 85% to 115%.(Yang et al. 2022a)  Table 1 shows that the external standard matrix effects of AFB 1 , AFB 2 , AFG 1 , and AFG 2 were 1.83, 1.91, 1.93, and 2.03, respectively, showing significant enhancement.Hence, matrix matching with external standard calibration was used to reduce the matrix effects.
Table 1 also shows that the internal standard matrix effects of AFB 2 and AFG 1 were 1.23 and 1.21, respectively, both greater than 1.15, showing strong enhancement.Conversely, the value for AFG 2 was 0.83, below 0.85, showing weak matrix inhibition; and the value for AFB 1 was 1.07, between 0.85 and 1.15, showing no influence.Herein, matrix matching internal standard method was used to improve the accuracy.

Analysis of peanut oil
In order to characterize the repeatability of the method, three concentration levels (1.0, 2, and 5 lg/kg) of spiked peanut oil were analyzed in six replicates.The recoveries for the targets were between 94.7% and 105.9% with relative standard deviations between 2.41% and 6.88%.About 1.0 lg/kg of AFB 1 , AFB 2 , AFG 1 , and AFG 2 were added to peanut oil to evaluate the inter-day and intra-day repeatability.The recoveries of inter-day and intra-day measurements were from 93.0% to 98.5% and 97.6% to 100.7%, respectively.The precision values for inter-day and intra-day analysis were from 6.09% to 7.68% and 2.56% to 4.30%, respectively.Therefore, the validated method met the monitoring requirements for aflatoxins in peanut oil.
The developed solid phase extraction procedure with HPLC-MS/MS detection was used to determine aflatoxins in 126 peanut oil samples.Blanks and 1.0 lg/kg spiked sample were employed as quality control standards.The analyses were performed with matrix matching and internal standard calibration.The results are summarized in Table S2 (supplementary material).AFB 1 was detected in 38 samples, with a detection rate of 30.1%.The determined values were between 0.175 and 27.6 lg/kg.One sample exceeded the GB 2761-2017 standard limit of 20 lg/kg, resulting in an unsatisfactory rate of 0.793%.The spike recoveries of AFB 1 , AFB 2 , AFG 1 , and AFG 2 in the samples were between 91.6% and 101.2%, and the relative standard deviations were less than 5.9%, indicating that the developed procedure was suitable for the determination of aflatoxins in peanut oil.
Comparison of this approach with the literature A comparison of the developed method with the standard GB 5009.22-2016procedure and the literature is provided in Table 3.The reported analytical figures of merit were satisfactory for practical applications and comparable with the previous studies.The literature procedures included extraction, centrifugation, loading, washing and elution, resulting in cumbersome procedures.In addition, the costs of these methods are relatively high.The reported procedure involved one-step extraction and cleanup of aflatoxins with packed needle filter solid phase extraction device based upon MIL-101 (Cr).The sorbent may be easily prepared in large quantities to reduce the cost.The pretreatment time of this method was only 40 min, much less than the other approaches.In addition, the volume of solvent was significantly lower.In summary, the reported method has significant advantages for the determination of alfatoxins in peanut oil.

Conclusion
A MIL-101(Cr) material was prepared by a hydrothermal reaction and characterized to prepare a simple, efficient and low-cost solid-phase extraction device with HPLC-MS/MS for the determination of aflatoxins in peanut oil.Compared with standard procedures with multiple extraction and cleanup steps, the developed protocol offers simple operation, reduced sorbent consumption, low cost, and high throughput.The wide linear range, high sensitivity, good recovery, and favorable precision meet the requirements of the European Union's SANTE 11813/2021 and the People's Republic of China GB/T 27404-2008 reference procedures.The validated procedure was employed to determine aflatoxins in 126 peanut oil samples.In addition, these results show that the proposed method was fast with low detection limits and good precision for the determination of aflatoxins in peanut oil, providing a new approach to characterize the safety of this foodstuff.

Disclosure statement
No conflicts of interest are reported by the authors.

Figure 3 .
Figure 3. Optimization of the (A) mass of sorbent, (B) loading solvent, (C) eluent, and (D) volume of eluent for the determination of aflatoxins.

Table 1 .
Analytical figures of merit for aflatoxins in peanut oil by MIL-101(Cr) based SPE-FSF coupled with high-performance liquid chromatography-tandem mass spectrometry.

Table 2 .
Recovery and precision of aflatoxins spiked in peanut oil (n ¼ 6).

Table 3 .
Comparison of the reported method with literature procedures.