Development of a deep eutectic solvent-based matrix solid phase dispersion methodology for the determination of aflatoxins in crops

Accepted Manuscript Development of a deep eutectic solvent-based matrix solid phase dispersion methodology for the determination of aflatoxins in crops Xi Wu, Xinxin Zhang, Yanqiang Yang, Yurong Liu, Xiaoni Chen PII: DOI: Reference:

S0308-8146(19)30680-6 https://doi.org/10.1016/j.foodchem.2019.04.030 FOCH 24631

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

2 November 2018 14 March 2019 8 April 2019

Please cite this article as: Wu, X., Zhang, X., Yang, Y., Liu, Y., Chen, X., Development of a deep eutectic solventbased matrix solid phase dispersion methodology for the determination of aflatoxins in crops, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.04.030

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Development of a deep eutectic solvent-based matrix solid phase dispersion methodology for the determination of aflatoxins in crops Xi Wua*#, Xinxin Zhangb#, Yanqiang Yangb, Yurong Liua, Xiaoni Chena* a

Department of Chemistry, Changzhi University, Changzhi 046011, China

b

Changzhi Entry-Exit Inspection and Quarantine Bureau, Changzhi 046011, China

Corresponding address: Department of Chemistry, Changzhi University, Changzhi 046011, P. R. China Tel: +086-0355-2178113, Fax: +086-0355-2178113, E-mail: [email protected], [email protected]  Corresponding author. #

These authors contributed equally to this work.

1

Abstract A novel and sensitive deep eutectic solvent-based matrix solid phase dispersion (DES-MSPD) method for the determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in various crops was established using high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The DES-MSPD sample preparation procedure was optimized. Based on the optimal conditions, the intra-day and inter-day variability for AFs in all crop samples was less than 7.5%. Linearity was observed with R2 values (> 0.994). Using the present method, HPLC-FLD gave the limits of detection (LODs) of 0.03-0.10 μg/kg and the limits of quantification (LOQs) of 0.10-0.33 μg/kg for AFs. This work represents the first attempt of using DESs as a green extraction medium for the extraction of AFs in MSPD. Compared with conventional MSPD methods, the DES-MSPD procedure looks promising as a relatively simple and low cost process to build an assay that can be used for monitoring concentrations of AFs in crops.

Keywords: Aflatoxins; Matrix solid phase dispersion extraction; Deep eutectic solvents; Crops

2

1. Introduction Aflatoxins (AFs) are highly toxic and carcinogenic mycotoxins produced by the secondary metabolism of certain Aspergillus species. These species can grow on a variety of food stuffs under a wide range of environmental conditions (Ali, Hashim, Saad, Safan, Nakajima, & Yoshizawa, 2005). The most frequently detected aflatoxins are aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). AFs can contaminate nuts, cereals, oilseed crops, and their realted by-products under specific conditions (Bacaloni, Cavaliere, Cucci, Foglia, Samperi, & Lagana, 2008). Considering the serious threat to public health, the presence of AFs in food has become a worldwide concern. Sensitive and accurate determination of AFs is currently an important requirement to meet official regulations and relieve concerns of food safety. At present, many methods for the determination of AFs in food and related products have been investigated. Due to the presence of various interferents and the low concentrations of AFs (µg/kg), the determination of AFs was often very difficult in complex food samples. Thus, sample pretreatment has always been a chanllenging and important step in the detection of AFs. The most popular official methods of AF detection in complex samples rely on the use of immunoaffinity chromatography (IAC) followed by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and post-column derivatization (PCD) (Cano-Sancho, Ramos, Marin, & Sanchis, 2012; Xie, Chen, & Ying, 2016; Zollner, & Mayer-Helm, 2006). However, this method requires expensive disposable immunoaffinity columns 3

and the corresponding procedures are labor-intensive and time-consuming. Additionally, these methods use large amounts of solvents. Hence, there is a need for improvement in the analytical methods of the determination of AFs. For this reason, other sample pretreatment methods have been used to detect AFs. These include solid-phase extraction (SPE) (Campone, Piccinelli, Celano, Pagano, Russo, & Rastrelli, 2016; Khayoon, Saad, Salleh, Manaf, & Latiff, 2014), solid-phase microextraction (SPME) (Es’haghi, Sorayaei, Samadi, Masrournia, & Bakherad, 2011; Nonaka, Saito, Hanioka, Narimatsu, & Kataok, 2009), accelerated solvent extraction (ASE) (Li, Xie, Lu, Ping, Ma, Luan, & Wang, 2014; Sheibani, & Ghaziaskar, 2009), microwave-assisted extraction (MAE) (Chen, & Zhang, 2013), and matrix solid-phase dispersion (MSPD) (Rubert, Soler, & Manes, 2010). MSPD is an SPE-based extraction technology that has previously been applied to the pre-process of a variety of samples (Li, Li, Xu, Zhang, Wang, & Luo, 2017). The main advantage of this technology is its ability to achieve extraction and clean-up in a single step. Moreover, MSPD reduced the amount of solvent needed for sample extraction and simplifies the process of sample pretreatment. In general, functional dispersants play an important role in MSPD. Primary secondary amine (PSA) and octadecylsilane chemically bonded silica (C18) are the most popular functional dispersants, with several kinds of homemade dispersants also used in MSPD (Arabi, Ghaedi, & Ostovan, 2016; Fotouhi, Seidi, Shanehsaz, & Naseri, 2017). However, the types of functional dispersant used in MSPD are still relatively limited and expensive. The MSPD method lacks a degree of flexibility and 4

selectivity. Moreover, the elution solvents used in the classical MSPD method are usually toxic and not environmentally friendly (Iparraguirre, Rodil, Quintana, Bizkarguenaga, Prieto, Zuloaga, Cela, & Fernandez, 2014; Montes, Canosa, Lamas, Pineiro, Orriols, Cela, & Rodriguez, 2009). Furthermore, the optimization process of MSPD is also a very time-consuming and laborious task. Finding a simple, cost-effective, and environmentally friendly MSPD method remains a challenge. In recent years, green solvents have gained much attention in the field of analytical chemistry. The literature base on the use of these solvents, which include room temperature ionic liquids (RTILs) and deep eutectic solvents (DESs), has grown considerably (Espino, Fernandez, Gomez, & Silva, 2016). RTILs are a new type of reagent with the unique and interesting characteristics (Welton, 1999). Research indicates that RTILs have the characteristics of non-volatility, non-flammability, high ion density, good ionic conductivity, etc. Thus, RTILs have attracted significant attention in many fields of chemistry (Shiddiky, & Torriero, 2011; Bohm, Tietze, Heimer, Chen, & Imhof, 2014; Armstrong, He, & Liu, 1999; Gao, Jin, You, Ding, Zhang, Wang, Ren, Zhang, & Zhang, 2011). For example, the adoption of ILs as eluting solvents in MSPD with high affinity and extraction efficiency has been reported by Xu, Yang, Ye, Cao, Cao, Hu, & Peng (2016). In their study, ILs were used as elution solvents instead of common organic elution solvents. A highly effective miniaturized MSPD method coupled with a UPLC-UV instrument was developed for analysis of flavonoid glycosides in lime. However, some studies have suggested that most RTILs are toxic and have poor biodegradability (Romero, 5

Santos, Tojo, & Rodriguez, 2008; Plechkova, & Seddon, 2008). In addition, RTILs are relatively expensive. These drawbacks limit the widespresd application of RTILs. DESs are a new generation of RTILs that are also known as being green solvents (Abbott, Capper, Davies, Rasheed, & Tambyrajah, 2003). A DES usually consists of two or three types of cheap and safe components that are linked through hydrogen bonds. These components are defined as hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). At present, a number of DESs are prepared by simply mixing and heating organic halide salts (e.g. choline chloride or tetrabutylammonium chloride) as the HBA with urea, organic acids, alcohols, amines, or amides as the HBD (Zhang, Vigier, Royer, & Jerome, 2012). Since DESs have the advantages of low toxicity, biodegradability, affordability, non-volatility, and availability, they have become the subject of much research in the field of chemistry (Chen, Xie, Zhang, & Liu, 2013; Roehrer, Bezold, Garcia, & Minceva, 2016). Limited research has begun to discuss the application of DESs in sample pretreatments. For example, Khezeli, Daneshfar, & Sahraei (2016) proposed an ultrasonic-assisted liquid-liquid microextraction (LLME) method based on DES for the determination of ferulic, caffeic, and cinnamic acid from oil samples. The same team also extracted BTE (benzene, toluene, ethylbenzene) and seven polycyclic aromatic hydrocarbons (PAHs) from water samples using a method based on DESs referred to as emulsification LLME (Khezeli, Daneshfar, & Sahraei, 2015). Habibi, Ghanemi, Fallah-Mehrjardi, & Dadolahi-Sohrab, (2013) developed an efficient digestion method based on DES for the flame atomic absorption spectrometry 6

(FAAS) determination of metal elements in biological samples. Xu, Wang, Li, Lin, Zhang, & Zhou (2016) synthesized a new DES-grafted, silica-coated, magnetic microsphere that was used to extract trypsin. However, to the best of our knowledge, no MSPD method based on DESs has been reported for the determination of AFs in a complex food matrix. The purpose of the

present

study was

to

develop

a

simple,

sensitive,

low-cost,

and

environment-friendly method for the determination of AFs in millet, peanut, and heepseed samples. Additionally, the effects of various experimental parameters (e.g. types of DESs and dispersants used), as well as the mass ratio of sample:dispersant, were investigated for optimization.

2. Materials and methods 2.1. Reagents and samples Acetonitrile and methanol of HPLC grade were obtained from Kermel Chemical Reagents Development Center (Tianjin. China). Hexyl alcohol, dodecyl alcohol, hexanoic acid, and tetrabutylammonium chloride (TBAC) were obtained from Aladin Reagent (Shanghai, China). C18 and PSA were purchased from Varian Technologies China Co., Ltd. (Beijing, China). Diatomite and Al2O3 were purchased from Tianjin Caratton Industry Co., Ltd. (Tianjin, China). Silica gel was purchased from Qingdao Jiyida Silica Reagent Co., Ltd. (Qingdao, China). G1010 AflaTest P immunoaffinity columns were purchased from VICAM (Milford, MA, USA). Water used throughout the study was purified with an Ultrapure Water Purification System 7

(High Wycombe, UK). All standard solutions were stored in the dark at 4 C. Aflatoxin standards (98% purity) were purchased from the Clover Technology Group. Inc. (Beijing, China). A mixed stock solution containing AFB1, AFB2, AFG1, and AFG2, each at a concentration of 1.0 mg/mL, was prepared in methanol. A series of standard solutions were prepared by diluting the stock solution with methanol. Three types of AFs-positive crop samples and the corresponding negative samples, including millet, peanut, and heepseed samples, were acquired from the Changzhi Entry-Exit Inspection and Quarantine Bureau. The HPLC-FLD method was used for determination of AFs concentrations. The samples were classified as AFs-positive or AFs-negative according to whether AFs were present in samples. All samples were homogenized in a homogenizer and stored in a desiccator. All spiked samples were prepared by spiking the AF stock solutions into negative samples. After being dried at room temperature, the spiked samples were then stored in a desiccator.

2.2. Instrumental and analytical conditions In this work, the content of AFs were determined with HPLC-FLD. A Shimadzu LC-20AD HPLC system with an RF-10AXL fluorescence detector (FLD) and a CRB-6A post-column derivatization (PCD) was used to separate and detect AFs. The separation was performed on an Inertsil ODS-SP column (250 mm  4.6 mm  5µm). AFs were detected by FLD and the wavelengths for excitation and emission were 365 and 435 nm respectively. The mobile phase was composed of methanol (solvent 8

A) and ultrapure water (solvent B). Isocratic elution (A:B = 45:55, volume ratio) was used with a flow rate of 1.0 mL/min. The temperature of the column was maintained at 30 C. The injection volume was 20 L. Post-column derivatization was used to enhance responses of the AFs. The PCD reagent was prepared by dissolving 5 mg of pyridinium tribromide in 1 L of ultrapure water. This was delivered at a flow rate of 0.2 mL/min. The reaction coil was maintained at 70 C. All chromatographic data was reprocessed on LC solution software (Shimadzu, Japan). A Midea homogenizer (Foshan, China) was used in this study. 2.3. Deep eutectic solvents-matrix solid phase dispersion procedure The usual MSPD procedures (Blesa, Soriano, Molto, Marin, & Manes, 2003) can be summarized as follows: (1) the sample is mixed with a certain functional dispersant, and the mixture is grinded using a mortar and pestle; (2) the homogenized mixture is then packed in an SPE cartridge; (3) the SPE column is then eluted with a suitable solvent. The DES-MSPD procedures are illustrated in Fig. 1. An aliquot of 0.5 g dispersant and 1.0 g sample was weighed accurately into an agate mortar. Then, 200 L DES was added into the mixture. The ternary mixture was blended using a pestle until a visually homogeneous powder was obtained. This blend was then carefully transferred to a 6.0 mL polypropylene SPE cartridge with a frit at the bottom. A second frit was placed on top of the mixture by gentle compression with a glass rod. The mixture was eluted with 6.0 mL of acetonitrile, and the elution process was carried out using an SPE vacuum manifold. The eluent was collected into a 10.0 mL centrifuge tube and blown with N2 at 50 °C until the 9

volume of residue was constant. Finally, the residue was diluted with 1.0 mL of acetonitrile and filtered through nylon filters (0.22 m) prior to the HPLC analysis.

3. Results and discussion 3.1. Selection of the DESs In the DSE-MSPD method, the extraction efficiency can be influenced by the properties of the DESs. Considering the solubility of AFs, three types of TBAC-based DESs (TBAC-hexyl alcohol DES, TBAC-dodecyl alcohol DES, and TBAC-hexanoic acid DES) were chosen as alternative extraction mediums for our study. The sample used was 1.0 g spiked millet, and the concentrations of AFG2, AFG1, AFB2, and AFB1 in the spiked sample were 3.0, 10.0, 3.0, and 10.0 µg/kg respectively. Diatomite was used as a dispersant as it is very common in MSPD. The mass ratio of sample to diatomite is 1:1. After repeated attempts, the volume of DES used in this study was validated as 200 L per 1.0 g sample. The average recoveries of the AFs obtained with different DESs are shown in Fig. 2. The TBAC-hexyl alcohol DES clearly showed the best recoveries for all AFs, and the corresponding recoveries with the addition level were 97.87±2.85%, 97.67±3.28%, 98.07±1.10%, and 93.67±3.28% (n=3) for AFB1, AFB2, AFG1, and AFG2 respectively. In contrast, a blank experiment was also carried out without the addition of DES. The recoveries of all AFs obtained using the blank experiment were lower than those obtained using the DES-MSPD method (Figure 2). This result proved that DESs play a significant role in the proposed method. Thus, further studies were performed using 10

TBAC-hexyl alcohol DES as the extraction medium.

3.2. Real crop samples analysis 3.2.1. Selection of the dispersants The performance of the MSPD method is determined by differences in the physicochemical properties of the dispersants. The selection of dispersants has a significant effect on the extraction efficiency. In this study, silica gel (Al2O3) and diatomite were evaluated as dispersants, with 200 L TBAC-hexyl alcohol DES used as the extraction medium. The amount of each AF-positive crop sample (millet, peanut, and heepseed) was 1.0 g, and the mass ratio of sample to dispersants was 1:1. Corresponding experimental results are presented in Fig. 3. The results show that AFB1 was detected in all crop samples, while AFB2 was detected only in the peanut sample. These results indicate that changing the dispersants greatly affects the performance of the DES-MSPD method. This may be ascribed to the hydrophobic characteristics, particle sizes, or rigidity of the dispersants, as well as the nature of the sample. Evidently, diatomite was the best dispersant for the peanut and hempseed samples, and silica gel was the best dispersant for the millet sample.

3.2.2. Mass ratio between sample and dispersant Besides the types of DESs and dispersants used, another important parameter for the proposed method is the ratio between the sample and dispersant. According to the result of the previous step, diatomite was used as the dispersant 11

peanut

and heepseed samples, and silica gel was used as the dispersant for the millet sample. Four different sample : dispersant ratios (1:0.5, 1:1, 1:1.5, and 1:2) were tested. The effect of the sample-dispersant ratio on the extraction efficiency of analytes was significant (Figure 4). The results suggested that the extraction efficiency of AFB1 and AFB2 from the millet and peanut samples was highest when the sample-dispersant ratio is 1:1. For the heepseed sample, the extraction efficiency of AFB1 increased when the sample-dispersant ratio increased from 1:0.5 to 1:1.5. Moreover, the extraction efficiency decreased when the mass ratio increased to 1:2. Thus, the optimal sample-dispersant ratio for the millet and peanut samples was 1:1, and the optimal sample-dispersant ratio for the heepseed sample was 1:1.5.

3.3. Comparison with other method The conventional MSPD method which use C18 or PSA as sorbents has proven to be an effective method for investigation of AFs in peanut samples (Blesa, Soriano, Molto, Marin, & Manes, 2003). To evaluate the performance of the proposed method in the present study, the samples were also processed using the conventional MSPD method. The extraction effects using the PAS and C18 dispersants were investigated for all samples. The extracts obtained using both methods were analyzed by HPLC-FLD under the same conditions. The results were shown in Fig. 5. In this figure, the solid line represents the experimental signals obtained by the DES-MSPD method, whilst the dashed and dotted lines represent the experimental signals obtained by the conventional MSPD method, which used C18 and PAS as sorbents 12

respectively. The naturally contaminated-positive millet and heepseed samples only contained only AFB1, while the naturally contaminated-positive peanut sample contained AFB1 and AFB2 (Figure 5). It was evident that the intensity of the peak areas of the AFs obtained using DES-MSPD was significantly greater than that obtained using conventional MSPD. This can be seen, for example, in the millet sample, where the concentration of the AFB1 obtained using the proposed method was eight times that of the concentration obtained when using the conventional MSPD method with PSA as a dispersant. This suggests that the DES-MSPD method is much more effective than the conventional MSPD method. Compared to the dispersive liquid-liquid microextraction (DLLME) method (a common cleanup method for AFs analysis), the proposed method has many advantages (Somsubsin, Seebunrueng, Boonchiangma, & Srijaranai, 2018). Less solvent was required and fewer parameters needed to be optimized for the DES-MSPD method.

3.4. Method validation The feasibility of the proposed DES-MSPD method was established by studying limit of detection (LOD), limit of quantitation (LOQ), linearity range, and precision on non-infected crop samples (peanut, millet and hempseed) spiked with AFs. The main parameters of the developed analytical method are summarized in Table S1 (see supplementary material). LODs and LOQs were determined by decreasing the concentration of the analytes gradually until signals could still be 13

discovered at a signal : noise ratio of 3 (S/N =3) and 10 (S/N =10). The LODs of AFB2, AFB1, AFG2, and AFG1 ranged from 0.03 to 0.10 μg/kg, and the LOQs of AFB2, AFB1, AFG2, and AFG1 ranged from 0.10 to 0.33 μg/kg. Calibration curves of 4 kinds of AFs were constructed by plotting the HPLC peak areas of AFs in the spiked samples against their concentrations. The concentrations of AFB2 and AFG2 ranged from 0.03 to 30.0 g/kg, while the concentrations of AFB1 and AFG1 ranged from 0.10 to 100.0 g/kg. Excellent linearity was shown within the studied concentration range. The correlation coefficients were between 0.9994 and 0.9998, further highlighting this strong linearity. The precision was represented by the intra- and inter-day relative standard deviations (RSDs) of the results obtained through DES-MSPD. This was evaluated in terms of peak area of each AF at a fixed concentration. The intra-day RSDs were obtained by analyzing samples from three replicates on the same day. The inter-day RSDs were determined by analyzing the samples three times per day for three consecutive days. The intra- and inter-day RSDs for all AFs were in the range of 1.6-4.8% and 3.8-7.5% respectively. These results demonstrate that the precision of the developed method was satisfactory.

4. Conclusion In this study, a simple and effective analytical method based on the DES-MSPD procedure was developed for the determination of AFs in millet, peanut, and heepseed. This method used TBAC-hexyl alcohol DES as an adsorption medium, 14

with silica gel and diatomite used as dispersants. The main advantages of the DES-MSPD method are its affordability and environmental friendliness. This method also involves a simple pretreatment process and good applicability in analytical laboratories. Moreover, the proposed method is environmentally friendly because it only required DESs and a small amount of acetonitrile. This novel method could therefore be widely adopted in the field of food safety and control.

Acknowledgments This work is sponsored by the Fund for Shanxi Key Subjects Construction and it is supported by the Basic Research Project of Shanxi Province (2014011036_3).

Conflicts of interest There are no conflicts of interest to declare.

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chromatographic analysis of aflatoxins in rice samples. Talanta, 176, 172-177. Welton, T. (1999). Room-temperature ionic liquids. Solvents for synthesis and catalysis. Chemical Reviews, 99, 2071-2084. Xie, L., Chen, M., & Ying, Y. (2016). Development of methods for determination of aflatoxins. Critical Reviews in Food Science and Nutrition, 56, 2642-2664. Xu, J. J., Yang, R., Ye, L. H., Cao, J., Cao, W., Hu, S. S., & Peng, L. Q. (2016). Application of ionic liquids for elution of bioactive flavonoid glycosides from lime fruit by miniaturized matrix solid-phase dispersion. Food Chemistry, 2016, 204, 167-175. Xu, K. J., Wang, Y. Z., Li, Y. X., Lin, Y. X., Zhang, H. B., & Zhou, Y. G. (2016). A 20

novel poly(deep eutectic solvent)-based magnetic silica composite for solid-phase extraction of trypsin. Analytica Chimica Acta, 946, 64-72. Zhang, Q. H., Vigier, K. D. O., Royer, S., & Jerome, F. (2012). Deep eutectic solvents: syntheses, properties and applications. Chemical Society Reviews, 41, 7108-7146. Zollner, P., & Mayer-Helm, B. (2006). Trace mycotoxin analysis in complex biological and food matrices by liquid chromatography-atmospheric pressure ionisation mass spectrometry. Journal of Chromatography A, 1136, 123-169.

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Captions Figure 1 Schematic diagram of the proposed DES-MSPD method for the analysis of AFs in crop samples. Figure 2 Recoveries of AFG1, AFG2, AFB2, and AFB1 in spiked millet samples. Figure 3 The effect of dispersant type on the extraction yield of the AFs. Figure 4 The effect of mass ratio of sample:dispersant on the extraction yield of AFs. Figure

5

Chromatograms

of

AFs

extracted

from

three

naturally

contaminated-positive crop samples using different MSPD methods. The solid lines represent chromatograms obtained using the DES-MSPD method. The dashed and dotted lines represent chromatograms obtained using conventional MSPD with C18 and PSA sorbents. Table captions in supplementary material Table S1 Validation parameters of the proposed method

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Highlights 

A deep eutectic solvent-based matrix solid phase dispersion (DES-MSPD) method was first developed to analyze AFs in crop samples.



The DES was used as an adsorption medium and sample preparation can be simplified.



The DES-MSPD method was lower costs, environment-friendly, and good applicability in analytical laboratories.

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