Fluopyram standard stock solution (1000 mg/L, ethyl acetate) was configured using a solid standard of fluopyram. The fluopyram standard was weighed 10.0 mg (accurate to .0001 g) and added to 10.0 ml volumetric bottles, fixed with ethyl acetate and stored at −20°C. The standard working solutions (10.0 mg/L and 1.0 mg/L) diluted with ethyl acetate were placed in the refrigerator at 4.0°C until use.

The sample was weighed 5.00 g and deposited in centrifuge tube (50.0 ml). Added 10.0 ml of acetonitrile and mix the sample for 5 min using a high-speed homogenizer. Afterwards, sodium chloride was accurately weighed 5 g and added to the centrifuge tube and vortexed for 3 min, followed centrifuge the processed tubes for 5 min (5000 r/min). Then supernatant (2 ml) was added to 5.0 ml centrifuge tube containing purifying agent and anhydrous MgSO₄ and vortexed for 5 min and centrifuged at 5,000 r/min for 10 min. Removed 1 ml of supernatant from centrifuge tube and rotary evaporated at 40°C, then was re-dissolved with ethyl acetate and subsequently injected into the GC-MS/MS system. Sample preparation flow chart in Supplementary Figure S1.

The analysis of fluopyram was carried out on a Thermo Fisher gas chromatograph equipped with a TRACE 1300 gas chromatograph and a TSQ 9000 triple quadrupole mass spectrometer. The column was a Thermo Fisher TG-5SilMS weakly polarised capillary column (30 m × .25 mm×.25 μm). The programmed ramp-up was started at 120°C and held for 2 min, then increased to 300°C at a rate of 30°C min⁻¹ and held for 2 min, for a total run time of 10 min. The transmission line temperature and ion source temperature were both 280°C. The mass spectrometer sampled electron ionization (EI) for the quantitative analysis of fluopyram in SRM. Two transitions were used for quantification and qualification, with 144.9 > 95 (14 eV) and 144.9 > 75 (20 eV) for fluopyram.

The established method for the determination of fluopyram in fruits and vegetables was evaluated in terms of linearity, accuracy and precision according to the document SANTE/11312/2021 (European Commission, 2021). Linearity was evaluated by linear fitting of fluopyram measurements for all foodstuffs matrix at seven concentration levels. The analysis was considered accurate when R ² > .99 (Li et al., 2021).

Accuracy was verified by spiking experiments at three concentration levels. Fluopyram was added to blank matrix of nine fruits and vegetables for spiked recovery at levels of .01, .2, and 2 mg/kg in grapes, tomatoes, and peppers; and .01, .1, and 1 mg/kg in the remaining fruits and vegetables. The treatments were performed according to the extraction method in Section 2.2.2. The spiking experiment was repeated three times for each level and the relative standard deviation (RSD) was calculated to verify the precision.

Matrix effects (ME) can affect the reliability and sensitivity of analytical methods. The ME of fluopyram in each matrix was calculated using the following formula (Zaidon et al., 2019): M E = B − A A × 100 %

A and B represent the slope of the standard curve under ethyl acetate solution and blank matrix solution, respectively.

Market samples are collected and analysed in order to verify the reliability of the detection method in the application process. The samples are randomly selected and are representative. 128 samples were collected from the market in Sanya, Hainan Province, during June and July 2022. The market samples were analysed for residues according to the established detection method to count the residues of fluopyram in fruits and vegetables.

In addition to market samples, residue analysis was performed on field samples as an example of cowpea samples. The field trial was conducted in Sanya, Hainan Province, China (18°23′N, 109°9′E). Cowpeas were selected for the dissipation and terminal residue tests, with an area of 100 m² (20 m × 5 m). A buffer zone of 2 m width was divided between adjacent test areas. The field trials referred to NY/T 788-2018 (Guidelines for pesticide residue trials in crops) issued by China.

Dissipation: 41.7% fluopyram suspension was applied at 62.55 g a.i./ha (the maximum recommended dose). The dissipation experiment was applied when the cowpea fruits were halfway through growth to mature fruit. Cowpeas were collected in the upper, middle and lower layers of the cowpea plants, respectively. Cowpea samples were collected randomly for analysis at 0 (2 h), 1, 3, 5, 7, 10, and 14 days after application. All samples were labelled and stored at −20°C for further analysis. The dissipation of fluopyram in cowpea samples from field was delineated by a first-order kinetics model.

Terminal residues: The applied doses included a low dose of 62.55 g a.i./ha and a high dose of 93.825 g a.i./ha. Fluopyram was applied three or four times. The spraying interval of fluopyram was 7 days. Cowpea samples were collected at 5, 7, and 10 days after the last application. The picked cowpea were briefly processed and stored in a −20°C refrigerator.

In the analysis of pesticide residues, the selection of a suitable extraction solvent can be effective extraction of pesticides from the foodstuff matrix, the commonly used organic solvent extractants include acetonitrile, acetone and ethyl acetate (Dušek et al., 2020; Maragou et al., 2020; Tong et al., 2021). Three different extraction solvents were added to the nine foodstuff samples, and spiked recovery tests (.1 mg/kg) were conducted to screen the suitable extraction solvents. The results in Figure 1 showed that the highest average recovery among the nine fruits and vegetables was obtained from acetonitrile (93.91%), followed by acetone (91.12%), while ethyl acetate was the least effective extraction (85.39%). However, the more polar acetone may extract more impurities that would interfere with the analysis of the target compound (Zhou et al., 2021), and that ethyl acetate did not perform satisfactorily in grape (77.56%) and eggplant (76.66%), acetone and ethyl acetate were not suitable as extraction solvents for fluopyram. The extraction of acetonitrile was satisfactory. Therefore, the extraction study of acidic acetonitrile was not performed in this study. In several previous studies, acetonitrile was also used for the extraction of fipronil from meat products and acetamiprid from cowpea (Fu et al., 2020; Han et al., 2021). Therefore, acetonitrile was chosen as the extraction solvent for fluopyram for the further study.

The choice of sorbent for QuEChERS could vary depending on the analyte being used. Fruit and vegetable matrices are rich in pigments, water, polysaccharides and acids, a comparison of seven purification agents, including MWCNTs, MWCNTs-NH₂, MWCNTs-OH, PSA, GCB, C18, and ALO, was used to select suitable purification agents for the next step in the study. These adsorbents have been used extensively for the pretreatment of pesticide residues in foodstuffs (Gonzalez-Curbelo et al., 2013; Chen et al., 2014; Chen et al., 2019; Chen H. et al., 2021).

The sorbents were compared at the .1 mg/kg level of fluopyram additive, the results are shown in the Supplementary Table S1, where the extraction of the seven sorbents in different fruit and vegetable matrices varied considerably. The recovery was unsatisfactory in different food matrices when any adsorbent was used alone. For example, the highest recovery in banana was only 71.35%, and in grapes the highest recovery was only 80.90% except for C18 and MWCNTs-NH₂. However, in cucumber, which was a relatively simple matrix, the lowest recovery of all adsorbents was 85.20%, and all were in the range of 80%–110%.

As can be seen from Figure 2, the adsorption of GCB, MWCNTs and ALO was not satisfactory and the average recovery was below 80%, especially for banana, which were only 57.60%, 55.86% and 61.10% (Supplementary Table S1). The average recovery of PSA, C18, MWCNTs-NH₂ and MWCNTs-OH were all in the range of 80%–110%. PSA contains amino functional groups and is effective in adsorbing acids from fruit and vegetable matrices. C18 can produce relatively high adsorption of non-polar compounds through van der Waals forces (Wang et al., 2019; Liu et al., 2021). MWCNTs-NH₂ and MWCNTs-OH has a strong adsorption effect on interfering substances due to their large specific surface area (Pallavi et al., 2021). After a comprehensive evaluation, PSA, C18, MWCNTs-NH₂ and MWCNTs-OH were selected for combined adsorption in this study to determine the optimum adsorbent formulation.

PSA, C18, MWCNTs-OH, and MWCNTs-NH₂ were identified as adsorbents for optimization. Considering the similar functions of MWCNTs-NH₂ and MWCNTs-OH, both of which have good pigment removal, two combination formulations were designed, as shown in the Table 1. In previous studies, QuEChERS has mostly used 50 mg of sorbent (Fu et al., 2020; Zhou et al., 2021; Wang et al., 2022), so this study has added 50 mg of each sorbent.

To verify the reliability of two methods, three levels of spiked recovery tests were designed in nine food matrices, with the spiked levels containing MRL. The results of the comparison of the recoveries at the three fluopyram levels are shown in Table 2. Method 1 showed a range of 70.38%–108.31% with RSD of 1.17%–8.25% at the three spiked levels; Method 1 showed a range of 87.02%–101.42% with RSD of .81%–8.81%. The recoveries and RSD of both methods were in accordance with the SANTE guidelines to analyse the pesticides residues. However, Method 1 is not as satisfactory as Method 2 in grapes and bananas. The recoveries of Method 1 were 70.38% (.01 mg/kg) and 78.19% (.1 mg/kg) in grapes and 78.46% (.1 mg/kg) in bananas. Method 2 had the lowest recovery of 87.02% across all foodstuffs. What is clear is that Method 2 is a better method. This may be because MWCNTs-NH₂ contains more amino groups than MWCNTs-OH (Chen S. et al., 2021), which can additionally adsorb more acidic substances and ensure a more stable pretreatment method. It has been reported that the higher number of nitrogen atoms in MWCNT-NH₂ can form hydrogen bonds with the hydrogen atoms in other interfering substances and thus adsorb more interfering substances (Yu et al., 2020). Therefore, Method 2 was chosen as the final pre-treatment method in this study.

Based on the analytical method developed in this study, a total of 128 fruit and vegetable samples were tested for fluopyram residues in June and July 2022 in Sanya City, Hainan Province (Table 4), with a total of 19 positive samples. There were 10 cases of cucumber, 6 cases of cowpea and 3 cases of tomato. All samples did not exceed the limit according to GB 2763-2021, but one sample of tomato exceeded the EU MRL (.5 mg/kg).

The samples from the field trials were analysed using the established method and the dissipation of fluopyram on cowpea was in accordance with the first order kinetic model. As shown in Figure 4, the half-life of fluopyram in cowpea plants ranged from 3.07 to 3.95 days. The half-life values reported in this study were similar to those of mango under similar treatments. On mango fruit, the half-life of fluopyram was 4.3–5.4 days (Mohapatra et al., 2018). This indicated that the persistence of fluopyram was weak in cowpea. The half-life of fluopyram in watermelon was 6.48–6.60 days (Dong and Hu, 2014). The dissipation in cowpea is faster than that in watermelon. The main reasons for this may be caused by differences in climatic conditions and crop types.

Apart from this, the half-life of fluopyram in cowpea was 3.07 days in the upper layer and 3.03 and 3.95 days in the middle and lower layers, respectively (Figure 4). The dissipation rate of cowpea in the upper and middle layers was higher than that in the lower. Cowpeas in the upper and middle layers have a stronger tendency to grow and a faster metabolism. Thus, there was a higher growth dilution effect than the lower cowpeas. Pesticides are generally degraded gradually as plants grow, which is one of the significant pathways for pesticide dissipation. Similar results have been reported in previous studies that significant differences in pesticide dissipation rate between tender and old cowpeas (Wang et al., 2022). In addition, environmental factors such as sunlight, temperature and humidity also play a fundamental role in the dissipation (Farha et al., 2016). For example, pesticides on greenhouse crops commonly dissipate more slowly than in the open due to lack of light and confined space (Chen H. et al., 2021). Upper and middle cowpeas have more exposure to sunlight, which promoted faster dissipation of fluopyram.

As shown in Table 5, the terminal residues of fluopyram in cowpea ranged from .031–.596 mg/kg. The terminal residues of fluopyram in cowpea increased with the frequency of spraying and application rate. In terms of spatial distribution, fluopyram residues were higher in the lower cowpea than in the middle and upper layers, with the least residues in the upper layer. This was similar to the findings following the application of boscalid and pyraclostrobine in grape fields (Chen et al., 2019). In China and EU, the MRL for fluopyram in cowpea (with pods) are 1 mg/kg and 3 mg/kg. Residue levels at 5, 7, and 10 days after fluopyram application were below the MRLs (China and EU). This indicates that cowpea is relatively safe to consume for 5 days after the application of fluopyram at the recommended dose.