Determination of avermectins residues in soybean, bean and maize using a QuEChERS-based method and ultra-high- performance liquid chromatography coupled to tandem mass spectrometry

Soybean, maize and bean are crops of great economic importance, but in the last years suffered with infestations of the caterpillar Helicoverpa armigera, being the main problem the resistance of this pest to most pesticides. Avermectin emamectin benzoate was recently released to control this pest. Other avermectins, like abamectin, doramectin, eprinomectin and ivermectin are used in large scale because they potent acaricidal, anthelmintic, and insecticidal activities. Thus, a simple and fast method for the determination of avermectins in these crops based on a quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction procedure and ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) analysis was developed and validated. For extraction, water followed by acetonitrile:isopropanol and a partition step with salts was stablished. With the clean-up step using activated EMR-Lipid, limits of detection of 1.2 μg kg-1 for abamectin, doramectin, emamectin benzoate and ivermectin, and of 2.4 μg kg-1 for eprinomectin were achieved. Accuracy and precision evaluated at low levels presented satisfactory results. The method was successfully applied in commercial samples and is a good alternative for routine analysis.


Introduction
Soybean (Glycine max) is among the most important crops in the world and their demand increases every year, as long as soybean may be applied for animal feed and biodiesel production to raw material for cosmetics [1]. Maize (Zea mays) also has its economic importance related to various forms of use, from animal feed to high technology industry. Common bean (Phaseolus vulgaris), an oleaginous crop such as soybean, is an important source of protein.
In 2013, the first attacks of Helicoverpa armigera were reported on soybean and bean crops [4]. This caterpillar affects several crops, such as soybean, bean, cotton, chickpea, tomato, eggplant, canola and sunflower. In general, this pest is controlled by the use of insecticides, especially carbamates, organochlorines, organophosphates and pyrethroids.
However, due to extensive use of these pesticides, the caterpillar developed resistance against them, causing great damages in the crops [5]. Thus, a phytosanitary emergency was declared in Brazil, and the use of emamectin benzoate was released on an emergency basis. From the class of avermectins, only the use of abamectin as a pesticide was initially allowed in soybean, bean and maize crops [6]. The need for using emamectin benzoate In recent years, ultra-high performance liquid chromatography coupled with mass spectrometry has been a preferred technique for the determination of pesticides and veterinary drugs in food samples [7,8]. However, the determination in complex matrices with high fat, sugar and protein content requires special attention in the sample preparation stage, in order to obtain extracts with low concentration of interferents and containing the analytes of interest [9].
As extraction techniques for determination of pesticide residues in cereals and oleaginous crops we can mention the QuEChERS method, which stands for quick, easy, cheap, effective, rugged and safe [10] and the matrix solid-phase dispersion (MSPD) [11].
Huang et al. [12] analyzed abamectin and ivermectin in olive, soybean, maize and peanut oils and in lard after liquid-liquid extraction (LLE). The extractive solvent used was nhexane, followed by addition of acetonitrile while maintaining vortexing. Macedo et al. [13] analyzed abamectin, doramectin and ivermectin in butter using LLE at high temperatures and a mixture of acetonitrile, ethyl acetate and water (90:4:6, v/v/v) as solvent.
López-Blanco et al. [14] used a modified QuEChERS method for the multiresidue analysis of abamectin and other pesticides in avocado and olive oil. Several sorbents were tested for the clean-up step. Du et al. [15] established a QuEChERS method, with a cleanup step using primary secondary amine (PSA), to analyze residues of avermectins, pyriproxyfen and diflubenzuron in mushrooms. Liu et al. [16] developed a QuEChERS method for multiresidue determination of pesticides, including abamectin, in grains, with water addition before extraction with acetonitrile. Wang et al. [17] analyzed abamectin, emamectin benzoate and other pesticides in soybean applying a modified QuEChERS acetate method. Thus, giving the importance of avermectins residue analysis in soybean, 3 of 13 bean and maize crops, the aim of this work was to develop a suitable method for determination of residues of the avermectins abamectin, doramectin, emamectin benzoate, eprinomectin and ivermectin in soybean, maize and bean crops using the same sample preparation step in order to simplify the execution in routine analyses. To the best of our knowledge, there are no methods available in the literature for the determination of avermectins residues in more than one cereals or legumes matrix.

Standards, Chemicals and Materials
Analytical standards of abamectin, doramectin, emamectin benzoate, eprinomectin and ivermectin with purities from 95.0 to 98.3% were purchased from Dr. Ehrenstorfer For method development, soybean, bean and maize blank samples were obtained from controlled experiments without use of pesticides. None of the evaluated pesticides were detected. Sample were processed for 1 min in a IKA A11 Basic (Staufen, Germany) analytical mill. 1 min, increasing for 2 min to reach 80% B and then increases to 100% B, remaining for 0.5 min, and returning to 50% B maintaining constant until the end of the analysis. The flow rate was 0.225 mL min -1 , with 10 μL of injection volume. The mass spectrometer was operated using selected reaction monitoring (SRM) mode. The electrospray ionization operated in positive mode (ESI+). The transition with the highest intensity was selected for quantification, and the transition with the second highest intensity was used for identification. The following conditions were used for the ESI source: desolvation and cone gas flow rate were set at 500 and 60 L N2 h -1 , respectively; the capillary voltage at 2.5 V; desolvation temperature at 350 °C and source temperature at 150 °C. The collision gas was argon used at 0.15 mL min -1 . Partition coefficients of avermectins and UHPLC parameters for the determination of their residues in soybean, bean and maize are presented in Table 1.

Optimized Sample Preparation Procedure
The proposed sample preparation method for determination of avermectin residues in soybean, bean and maize is based on the citrate QuEChERS procedure using 5 g of sample weighed in a 50-mL polypropylene (PP) tube followed by addition of 10 mL of water and homogenization for 1 min in ultra-turrax IKA T25 Digital (Staufen, Germany) at high speed to produce a slurry. Extraction was done with 10 mL of a mixture of acetonitrile:isopropanol 9:1 (v/v) vortexed for 1 min. Partition and salting-out effect were achieved by adding 2 g of MgSO4, 0.5 g of NaCl, 0.

Evaluation of the Different QuEChERS Procedures
The initial tests consisted in the evaluation of the three QuEChERS versions: original [19], citrate [20] and acetate [21] using amounts of sample, solvent and partition salts established in each version. Considering the low percentage of moisture, it was necessary to prepare a slurry using a ultra-turrax. Different ratio matrix:water (m/v) of 1:1; 1:1.

Validation Procedure
After evaluating the sample preparation procedure for determination of avermectin residues in soybean, bean and maize, the method was validated according to SANTE guidelines [22]. The following parameters were evaluated: selectivity, matrix effect,

Application in Real Samples
The developed and validated method was applied for the determination of residues of the avermectins under study in 18 soybean samples, 12 bean samples and 15 maize samples obtained from supermarkets from the Rio Grande do Sul State, Brazil. Each commercial sample of at least 1 kg was collected, milled and stored in a freezer at -20 °C. adducts. The sodium adducts are avoided due to its high stability and poor fragmentation response [24]. The column Acquity UPLC™ BEH C18 provided high resolution and good peak shape for the compounds under study. Still, the chromatographic column chosen is the same used in routine analysis in our laboratory.

Sample Preparation Evaluations
Soybean and bean are considered complex matrices due to the high amount of fats and proteins, besides having a low humidity, needing to add water for the extraction.
Maize presents a low percentage of water and a particular characteristic that is the presence of starch, which increases its complexity. The preparation of slurry was uniformized for the proportion 1:2 of sample:water which produced suitable consistence.
This proportion of sample:water was reported for low moisture matrices like maize [25], wheat and oat [26], and barley and wheat [27]. Lower protortions of water of 1:1 and 1:1.5 were not effective to have a homogeneous slurry.

Clean-up Procedure Evaluations
In the clean-up step, it was decided to evaluate d-SPE, without considering SPE, in order to keep the QuEChERS advantages. Figure 1 shows recovery results for five sorbents evaluated for soybean. Recoveries above 70% for most of the compounds were obtained.
Similar resuts were observed for bean and maize. The use of C18+PSA presented the lowest recoveries, with a mean recovery < 70% for abamectin (69%), ivermectin (63%) and doramectin (64%), while abamectin presented RSD > 20%. Regarding the EMR-Lipid and Florisil ® , the five compounds presented adequate recoveries and RSD. Results for Z-Sep+ were satisfactory, except for eprinomectin in which presented recovery of 67%. Silica presented recoveries > 70% for all compounds, however the RSD for abamectin was >20%. emamectin benzoate and a RSD of more than 30% for eprinomectin. Z-Sep+ was the only sorbent that presented recoveries between 70-120%. For maize, all sorbents evaluated presented satisfactory recoveries (70 to 120%). The RSD was satisfactory for all analytes, except for eprinomectin, when using C18 + PSA that presented RSD > 20%. As maize obtained recoveries between 70-120% for the five sorbents, the values obtained for soybean and bean were taken into account to select the most suitable sorbent. C18 + PSA was discarded because of the lowest recovery values in comparison to the other sorbents for both matrices. The claen-up that generated the best results for bean was Z-Sep+ and for soybean was EMR-Lipid. In general, EMR-Lipid presented lighter advantage in terms of recovery and RSD, as well the final extract presented less coextractives when evaluated by gas chromatography-mass spectrometry in full scan mode.
The non-activated EMR-Lipid was tested in the clean-up step to verify if it would have better results or similar to the activated one. As a result, the non-activated presented higher recoveries for abamectin, emamectin benzoate and ivermectin, compared to the activated EMR-Lipid. For eprinomectin the recovery was similar, but doramectin presented a much lower recovery. However, the RSD values for the five compounds increased (RSD >20%) with the use of non-activated EMR-Lipid. With activated EMR-Lipid the RSD were < 10% for all avermectins. Therefore, the activated EMR-Lipid was chosen for use in the clean-up step of the three matrices.

Application of the Method in Real Samples
From the analysis of 18 soybean samples, one sample presented abamectin at 21.1 μg kg -1 , that is two times higher than the MRL (10 μg kg -1 ) established in Brazil and USA.
Another three samples also presented residues of abamectin, however below the method LOQ. Figure 2 demonstrates the determination of abamectin residues in a soybean sample at concentration higher than the method LOQ and the MRL established in Brazil and USA.
For the 12 bean samples and 5 maize samples, no residues of the analyzed avermectins were detected. The absence of detectable residues may be due to the reduced number of samples analyzed. However, it is important to monitor these compounds in the crops under study and in other agricultural crops in order to guarantee the food safety and health of the population.

Conclusions
The results obtained with the modifications of the QuEChERS citrate method indicate good efficiency of the proposed method for the determination of avermectins residues in soybean, bean and maize samples. The addition of isopropanol to the acetonitrile improved the extraction efficiency and the clean-up step using EMR-Lipid permitted to obtain clean extracts, avoiding the need of frequent maintenance of the UHPLC-MS/MS system. The use of UHPLC-MS/MS with additives in the mobile phase and specific conditions allowed the determination with method limits of quantification below the MRL values. The validation indicated very good results of accuracy and precision, proving that the proposed method is reliable. The method was efficiently applied to real samples with positive results, including with abamectin in a soybean at concentration higher than the MRL established in Brazil and USA. We emphasize that there are no available studies reporting the development of a method for the determination of avermectins in more than one agricultural crop, highlighting the relevance of this work. The proposed modified QuEChERS citrate method for determination of avermectins in soybean, bean and maize using UHPLC-MS/MS was effective and is a good alternative for routine laboratories.  Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.