Monitoring hexythiazox residues in vegetables using LC-MS/MS: Dissipation kinetics, terminal residues, risk assessment, and washing efficiency

2.1. Chemicals and Reagents

A certified reference material of hexythiazox (99.5% purity) was obtained from Chem Service Inc. (West Chester, PA, USA). HPLC-grade acetonitrile and MeOH, LC-MS grade formic acid, and ammonium formate were purchased from Fisher Scientific (Loughborough, UK). Anhydrous magnesium sulfate (MgSO₄) and sodium chloride (NaCl) were purchased from Chem-Lab NV (Zedelgem, Belgium). A ceramic homogenizer from Chrom Tech, Inc. (Copure^®, Apple Valley, MN, USA) was used. Cleanup sorbents include primary secondary amine (PSA) procured from Macherey-Nagel (Chromabond diamino, Düren, Germany) and Multi-Walled Carbon Nanotubes (MWCNTs) from Shilpa Enterprises (Shilpent, Maharashtra, India). The wettable powder (WP) formulation of hexythiazox (10% WP), used for field application, was a commercial product (Nissorun, Nippon Soda Co., Ltd., Tokyo, Japan), sourced locally. Deionized water was prepared via the Ultra-Clear™ purification unit (Evoqua Water Technologies LLC, Guenzburg, Germany)

2.2. Standard preparation

A stock standard solution of hexythiazox was prepared at a concentration of 1000 mg/L using acetonitrile as the solvent. A 100 mg/L mid-level standard solution was prepared by diluting the appropriate amount of the standard with acetonitrile. Subsequently, a working dilution of 10 mg/L was made. These working solutions were used for multiple purposes: spiking blank samples for recovery and precision studies and preparing calibration curves using solvent-based and matrix-spiked standards to evaluate linearity and matrix effects. Standard solutions were kept frozen at −20°C until required.

2.3. Field experiment

The field experiment occurred in February 2025 in the Khubash Governorate of the Najran Region, southern Saudi Arabia. The study consisted of six greenhouses, each measuring 10 × 40 m, with two greenhouses allocated for each of the three test crops: tomatoes, cucumbers, and sweet peppers. Each greenhouse was divided into five subplots, consisting of three treatment subplots and two buffer subplots. Plants were spaced at 0.5 × 0.5 m, and all crops were cultivated under controlled greenhouse conditions using a drip irrigation system. Throughout the experiment, environmental conditions fluctuated between 16 and 28°C, with relative humidity levels ranging from 70% to 85%.

Pesticide application was carried out during the fruiting stage of each crop using hexythiazox (10% WP) at two distinct dosages. These application rates were selected to represent both standard agricultural practice and a potential high-dose misuse scenario. The first dose, 50 g a.i./ha, reflects the label-recommended rate for hexythiazox (10% WP), while the second dose, 100 g a.i./ha, corresponds to double the recommended rate, consistent with approaches adopted in previous pesticide dissipation and risk assessment studies [12,18]. This dual-dose strategy ensures that the survey captures residue levels under both compliant use and worst-case conditions. A summary of dosage levels from related studies has been provided in Table S1. The appropriate volume of 10% WP was diluted to a total spray volume of 1000 L per hectare and was administered using a handheld knapsack sprayer. For the residue decline assessment, triplicate samples weighing 2-3 kg each were randomly gathered at 0 (2 h), 1, 3, 7, 10, and 14 days following treatment. Sampling was conducted after two or three applications at 14-day intervals to assess final residue levels. Post final pesticide treatment, samples were collected for laboratory testing on days 3, 7, and 14. Immediately after collection, samples were transferred under cold conditions, cut into 2-3 cm segments, and frozen at –20°C until analysis.

2.4. Removal of hexythiazox residues by washing

To assess the effectiveness of household washing in reducing hexythiazox residues, a washing experiment was conducted using samples collected 1 day after pesticide application at two treatment levels: T1 (50 g a.i./ha) and T2 (100 g a.i./ha). Random samples (approximately 6 kg) were collected from the greenhouse for each of the three crops: tomatoes, cucumbers, and sweet peppers. Washing was performed under simulated household conditions. Each sample (approximately 1 kg) was placed in a stainless-steel sieve and positioned 30 cm below the tap nozzle, where running tap water was applied at a consistent flow rate of 10 L/min. Its temperature was maintained at 20–22°C. Two washing durations were tested: 2 and 5 min. Each treatment was conducted in triplicate (n = 3) [26]. After washing, samples were drained on clean tissue paper and allowed to air-dry at room temperature to remove surface moisture. The tap water used for the washing treatments was sourced from the local municipal supply and was pre-tested using the validated LC-MS/MS method to ensure the absence of hexythiazox or any other detectable pesticide residues. All washing experiments were performed using the same water source under consistent conditions to maintain comparability of results. Residue levels in the washed samples were then quantified using the following analytical procedure.

2.5. Analytical procedure

To prepare the samples, frozen sub-samples were initially processed using a Stephan Universal UM5 blender (Stephan Machinery GmbH, Germany) to achieve uniform homogenization. 10 ± 0.1 g of the homogenate was weighed into a 50 mL centrifuge tube with a ceramic grinding aid. An aliquot of 10 mL of acetonitrile was introduced as the extraction medium.

The mixture was manually agitated for 2 min, after which 4 g of anhydrous magnesium sulfate (MgSO₄) and 1 g of sodium chloride (NaCl) were added to enhance phase separation. The contents were mixed for 30 s and centrifuged at 5000 rpm for 5 min. To perform sample cleanup, a 2 mL portion of the resulting supernatant was transferred to a clean tube containing 300 mg of MgSO₄, 50 mg of PSA, and 5 mg of MWCNTs. Following cleanup, the mixture was vortexed for 1 min and then subjected to a second centrifugation step at 5000 rpm for 5 min. The cleaned extract was subsequently filtered using a 0.22 µm nylon syringe filter and transferred into auto sampler vials for LC-MS/MS analysis.

2.6. LC-MS/MS

The application of LC-MS/MS, which offers high sensitivity and specificity for multi-residue detection, has become a central component in modern food safety and environmental monitoring studies [27]. Our method aligns with this trend, enabling reliable trace-level quantification consistent with current sustainability-focused analytical practices. Hexythiazox residues were analyzed using a Dionex Ultimate™ 3000 RS UHPLC system connected to a TSQ Altis triple quadrupole mass spectrometer (Thermo Fisher Scientific, Austin, TX, USA). The chromatographic separation was performed using a C18 reversed-phase column (100 mm × 2.1 mm, 2.6 µm particle size; Thermo Fisher) operated at a column temperature of 40°C.

The mobile phase consisted of eluent A (0.1% formic acid and 5 mM ammonium formate in water) and eluent B (MeOH containing 0.1% formic acid and 5 mM ammonium formate). The system was run in gradient mode with a constant flow rate of 0.3 mL/min. The injection volume was set at 2 µL. The gradient program began at 10% eluent B for 1 minute, followed by a ramp to 90% B over 3 min, held for 4 min, and then re-equilibrated to the initial conditions for 6 min.

Detection was done using the multiple reaction monitoring (MRM) mode under electrospray ionization (ESI) in positive polarity. The operating parameters included a spray voltage of 3500 V, a capillary temperature of 320°C, and a sheath gas setting of 35 arbitrary units. The auxiliary gas was set at 10 units, and the collision gas pressure was maintained at 1.5 mTorr. Quantification was accomplished using matrix-matched calibration standards. Data were acquired and processed with Trace Finder™ software version 4.1 (SP4) from Thermo Fisher Scientific.

2.7. Method validation

The analytical method was validated following the SANTE guidelines [28] to confirm the reliability of residue determination. The evaluated validation criteria included linearity, matrix influence, detection and quantification thresholds (limit of detection-LOD and limit of quantification-LOQ), measurement precision, and method accuracy (recovery). Linearity was tested by constructing matrix-based calibration standards at nine incremental concentration levels ranging from 0.001 to 0.5 mg/kg. Calibration graphs were generated by charting the peak areas of the analyte against the matching concentration levels.

Matrix effects were assessed by comparing the slope of calibration curves derived from post-extraction spiked matrix blanks with those from standards spiked into solvent at the same concentrations. The LOD and LOQ were defined using the lowest concentration spikes in matrix blanks, calculated using signal-to-noise ratios of 3 and 10, respectively. The LOQ was considered acceptable when recovery fell within 70–120%, and the relative standard deviation (RSD) did not exceed 20%. Method precision was determined through intra-day repeatability (RSD_r) and inter-day reproducibility (RSD_R). Six replicates (n = 6) were analyzed at the LOQ level across multiple sessions for precision assessment.

Recovery testing was conducted by spiking blank tomato, cucumber, and pepper samples at four concentration levels (0.002, 0.01, 0.1, and 1 mg/kg). Each level was analyzed in six replicates (n = 6). Accuracy was evaluated by comparing the measured residue levels to the known spiked concentrations to calculate recovery rates.

2.8. Calculations and statistical analysis

The dissipation of hexythiazox residues in tomato, cucumber, and pepper samples was modeled using a first-order kinetic approach, as recommended in EFSA guidance [29]. The change in residue concentration over time was expressed by the exponential decay function (Eq. 1). The dissipation half-life (t₁/₂) was derived using the corresponding rate equation Eq. (2). To determine the pre-harvest interval (PHI), Eq. (3) was employed, based on methodologies described by Abdallah et al. [30] and Hingmire et al. [31].

C_t represents the residue concentration at a given time (t), C₀ represents the initial concentration, k represents the dissipation rate constant, and MRL is the maximum permissible residue level established by European Union regulatory standards.

All statistical analyses were performed using Microsoft Excel (version 2024). Differences in residue levels between washing treatments (2 min vs. 5 min) and between application doses were evaluated using two-tailed Student’s t-tests. Statistical significance was set at p < 0.05.

3.1. MS/MS optimization

The collision energy and source parameters were carefully adjusted to enhance signal clarity (Figure 2). The analytical approach utilized MRM with three ion transitions, which were explicitly fine-tuned for quantitation and confirmation. A 0.5 mg/kg standard solution was infused into the mass spectrometer at a controlled rate of 5 µL/min via a Harvard pump. Heated ESI in positive mode (HESI⁺) was employed to ensure stable ion formation and consistent instrument response. For hexythiazox, the precursor ion [M+H]⁺ was identified at m/z 353.237 and selected for fragmentation analysis. A fragmentation profile (breakdown curve) was generated to determine optimal dissociation settings, ensuring sensitivity and efficiency. Three product ions were optimized at m/z values of 228.0, 168.0, and 270.917, with collision energies set at 15 V, 25 V, and 13 V, respectively. Among these, the ions at 228.0 and 168.0 m/z were prioritized for quantitation and confirmation due to their superior intensity and reproducibility. The RF lens voltage was set to 61.1 V, maximizing ion transmission and enhancing signal response (Figure 3).

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Figure 2.

MS/MS optimization parameters

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Figure 3.

Representative chromatograms of hexythiazox, showing quantifier and qualifier ions, in tomato (a), cucumber (b), and pepper (c) matrices spiked at 1 µg/kg

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3.2. Method validation

3.3. Dissipation of hexythiazox in tomato, cucumber, and pepper

The dissipation of hexythiazox residues was studied in tomato, cucumber, and pepper (Figure 4). For the low-dose, the initial deposits at 0 days were 0.248, 0.341, and 0.452 mg/kg, respectively. Residues decreased progressively over time across all commodities. At day 1, residue levels declined to 0.178, 0.157, and 0.352 mg/kg, respectively, indicating an early dissipation trend. At day 3, cucumber exhibited the most rapid dissipation, with residues decreasing to 0.069 mg/kg (79.77%), followed by tomato (0.113 mg/kg, 54.44%) and pepper (0.268 mg/kg, 40.71%). By day 7, cucumber residues had further declined to 0.011 mg/kg (96.77%), while tomato and pepper residues had retained 0.087 mg/kg (64.92%) and 0.176 mg/kg (61.06%), respectively. At day 10, cucumber residues were nearly depleted, with a remaining amount of 0.003 mg/kg (99.12%). In contrast, the residues of tomato and pepper were 0.033 mg/kg (86.69%) and 0.080 mg/kg (80.53%), respectively. By the 14^th day post-application, residue levels in cucumber samples had declined below the LOQ. In contrast, trace amounts persisted in tomato and pepper, measured at 0.004 mg/kg (representing 98.39% dissipation) and 0.017 mg/kg (96.24% dissipation), respectively.

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Figure 4.

Dissipation curves of hexythiazox (10% WP) in (a) tomatoes, (b) cucumbers, and (c) peppers.

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For the high-dose treatment (Figure 4), the initial deposits at day 0 were 0.381 mg/kg in tomatoes, 0.571 mg/kg in cucumbers, and 0.746 mg/kg in peppers. At day 1, residues declined to 0.265 mg/kg in tomatoes, 0.304 mg/kg in cucumbers, and 0.564 mg/kg in peppers, reflecting a similar dissipation pattern to the low dose but at slightly higher levels. By day 3, cucumber residues decreased to 0.141 mg/kg (75.31%), followed by tomato (0.185 mg/kg, 51.44%) and pepper (0.412 mg/kg, 44.77%). By day 7, cucumber residues had been reduced to 0.029 mg/kg (94.92%), while tomato and pepper residues retained 0.112 mg/kg (70.60%) and 0.255 mg/kg (65.82%), respectively. At day 10, cucumber residues decreased further to 0.008 mg/kg (98.60%), whereas tomato and pepper residues were 0.057 mg/kg (85.04%) and 0.134 mg/kg (82.04%), respectively. By day 14, cucumber residues were below the LOQ, while tomato and pepper exhibited minor remaining residues of 0.005 mg/kg (98.69%) and 0.053 mg/kg (92.90%), respectively.

Table 3 presents the kinetic parameters describing the dissipation of hexythiazox across tomato, cucumber, and pepper. The data exhibited a firm fit to the first-order kinetic model, as evidenced by high correlation coefficients (R²) ranging from 0.8975 to 0.9978. This confirms that the dissipation pattern in all three crops closely followed first-order behavior.

The initial residues (C₀) varied among commodities, with the highest deposition observed in pepper, followed by cucumber and tomato. These values are comparable to previous studies, where initial deposits ranged from 0.23 to 2.30 mg/kg, depending on crop type, pesticide formulation, and application rates [12,20]. Studies on hexythiazox in strawberries reported higher initial residues (3.64 mg/kg) due to the soft surface and increased pesticide retention [17].

The dissipation rate constant (K) was highest in cucumber, with values of 0.4614 day⁻¹ (low dose) and 0.4153 day⁻¹ (high dose), followed by tomato, with values of 0.2619 day⁻¹ and 0.2714 day⁻¹. In comparison, pepper had the lowest K values of 0.2123 days⁻¹ and 0.1788 days⁻¹, indicating slower degradation.

The calculated half-life (t_1/2) values reflected the dissipation trend, with cucumber showing the shortest persistence at 1.47 days (low dose) and 1.57 days (high dose). Tomato had t_1/2 values of 2.03 days (low dose) and 1.99 days (high dose), while pepper exhibited the most prolonged half-life, at 2.24 days (low dose) and 2.41 days (high dose), confirming its slower residue decline. These values align with previous research on hexythiazox, where strawberries showed a half-life of 2.0-3.59 days [18] and tea leaves exhibited faster dissipation with a half-life of 1.1-1.82 days [20], and brinjal (1.42-2.32 days) demonstrated faster dissipation than grapes (3.24-4.01 days) [22], which parallels the trend observed for cucumber and pepper in our study.

The dissipation behavior of hexythiazox in the tested matrices is influenced by its physicochemical properties, crop morphological characteristics, and growth dilution effect.

Hexythiazox exhibits low aqueous solubility, approximately 0.1 mg/L, a characteristic that significantly influences its environmental behavior and persistence in residues [10]. This limited solubility reduces its mobility and wash-off potential, allowing the compound to remain on plant surfaces for extended periods. Such persistence is especially notable in crops with waxy cuticles, like pepper, where Hexythiazox demonstrates a relatively long half-life of 2.24-2.41 days. Its log P value (2.67) [10] indicates moderate lipophilicity, suggesting it can bind to the cuticle layer, further slowing dissipation in crops with thick, waxy surfaces. This explains why pepper retained residues longer than tomato and cucumber. In contrast, with its smooth and permeable surface, cucumber exhibited the shortest half-life (1.47–1.57 days), indicating faster pesticide absorption and metabolic degradation. The low vapor pressure (1.33 × 10⁻³ mPa) [10] suggests negligible volatilization loss, indicating that dissipation is primarily driven by plant metabolism and environmental degradation rather than evaporation.

In addition to physicochemical interactions, plant morphology and growth patterns significantly impact the dissipation of pesticides. Cucumber is characterized by rapid vegetative growth, leading to a strong growth dilution effect, where pesticide residues decline due to degradation and increased biomass expansion over time [13,14]. This effect contributes to the shortest PHI (3.79-3.93 days) in cucumber, compared to tomato (4.01-5.51 days) and pepper (8.23-11.78 days). With an intermediate cuticle structure and moderate growth rate, Tomato exhibited a moderate dissipation rate. In contrast, pepper retains residues for the longest due to its slow growth rate, reduced metabolic breakdown, and thick cuticle, which restricts the penetration of pesticides. Our results are broadly consistent with those reported by Algethami et al. in Najran-grown eggplant [12]. They documented initial residues of 0.415 mg/kg (the recommended dose) and 0.698 mg/kg (double the recommended dose), with first-order dissipation and half-lives of 2.7 days (for the recommended dose) and 2.3 days (for the double recommended dose), respectively. In comparison, our study found initial residues in tomato and cucumber (0.286-0.511 mg/kg) and half-lives of 1.47-1.57 days in cucumber and 1.99-2.03 days in tomato. However, pepper exhibited slower dissipation with a half-life of 2.24-2.41 days, approaching that of eggplant. Notably, eggplant’s half-life aligns more closely with pepper due to similarly waxy cuticles that impede pesticide dissipation. Cucumber, in contrast, showed considerably faster degradation, likely due to its thin and smooth surface. These cross-crop comparisons highlight how crop surface morphology and growth patterns impact residue behavior, reinforcing the requirement for crop-specific dissipation data and tailored PHI recommendations. These findings suggest that growers should prioritize crops with faster dissipation rates for short PHI harvesting and adjust application schedules accordingly.

3.4. Effect of washing on reduction of hexythiazox residues

The effectiveness of washing treatments in reducing hexythiazox residues varied among the tested crops (Table 4). It was influenced by the pesticide’s physicochemical properties, crop surface morphology, application dose, and washing duration. Hexythiazox exhibits moderate hydrophobicity (Log P = 2.67) and low water solubility (0.1 mg/L), which typically limits its rinse-off potential, especially from crops with waxy or rough surfaces.

Despite this low water solubility, a significant reduction in residue was observed, particularly in tomatoes. A 2-minute wash reduced residues by 81.62% in T1 and 85.44% in T2, and a 5-minute wash achieved 84.22% and 86.76% removal, respectively. The lack of a significant difference between durations (P > 0.05) suggests that shorter washing times can reduce residues to below the MRL of 0.1 mg/kg. This aligns with Krol et al. [35] and Yang et al. [36], who reported that water solubility is not the sole factor in pesticide removal during washing. Instead, Randhawa et al. [37] demonstrated that pesticides such as chlorpyrifos can be effectively removed from crops like tomatoes due to their smooth, non-waxy surfaces, facilitating the easier physical dislodgement of residues.

In cucumbers, characterized by semi-rough epidermis with ridges, washing reduced residues by 66.74% (T1) and 68.55% (T2) after 2 min and up to 74.46% after 5 min. The increase was statistically significant (P < 0.05), although residues in T2 remained above the MRL of 0.05 mg/kg. This supports findings from Krol et al. [35], indicating that crops with textured surfaces may retain pesticides in surface crevices, thereby reducing the efficacy of washing. Pepper, which showed the highest initial residues (up to 0.564 ± 0.155 mg/kg in T2), retained the most pesticide after washing. A 2-minute wash reduced residues by 54.18% (T1) and 58.32% (T2), while a 5-minute wash reduced it to 65.17% in T2. Although reductions were statistically significant (P < 0.01), residues still exceeded the MRL (0.09 mg/kg), suggesting washing alone is insufficient. These findings support the conclusion by Amvrazi [38] that multiple variables, including pesticide-crop surface interactions and washing method, determine residue removal, rather than relying solely on solubility or K_ow values. Moreover, as hexythiazox is a contact pesticide that remains primarily on crop surfaces, it is more amenable to physical removal, especially in smooth-skinned produce. This is consistent with earlier works, which demonstrated that pesticide residues are generally more easily removed from the outer surfaces of fruits and vegetables. The findings confirm that washing is highly effective for tomatoes, moderately effective for cucumbers, and less effective for peppers at higher application rates. For peppers, additional treatments such as peeling, soaking in dilute salt solutions, or detergent-assisted washing may improve residue reduction [39-41].

3.5. Terminal residues

The terminal residue levels of hexythiazox in tomato, cucumber, and pepper declined progressively over the 14-day sampling period, with higher residue concentrations consistently observed under the high-dose treatment (T2, 100 g a.i./ha) compared to the recommended dose (T1, 50 g a.i./ha). Residue levels were influenced by both the number of applications and the interval post-treatment (Table 5). In tomatoes, residues under T1 decreased from 0.136 mg/kg on day 3 to 0.007 mg/kg by day 14, while T2 residues declined from 0.168 to 0.013 mg/kg over the same period. Cucumber residues followed a similar dissipation pattern, with concentrations under T1 and T2 dropping to below quantifiable levels by day 14. Pepper exhibited higher initial residues, particularly under T2, where levels reached up to 0.688 mg/kg at day 3 but declined to 0.061 mg/kg by day 14. Compared to the European Union MRLs of 0.1 mg/kg (for tomato), 0.05 mg/kg (for cucumber), and 0.09 mg/kg (for pepper), residues exceeded these thresholds at 3 and 7 days, especially under treatment 2 (T2). However, by 14 days post-treatment, residues in tomatoes and peppers had declined below their respective MRLs, while cucumbers were not evaluated beyond 7 days. Residue levels were notably higher with three applications, suggesting that extended PHIs may be necessary to achieve compliance with regulatory limits.

3.6. Risk assessment

The risk assessment of hexythiazox residues in tomatoes, cucumbers, and peppers was evaluated using the National Estimated Daily Intake (NEDI) and Health Quotient (HQ%) to determine chronic dietary exposure risks. In tomato, NEDI ranged from 1.88E-04–2.28E-04 mg/kg bw at 3 days, decreasing to 8.43E-05 – 1.55E-04 mg/kg bw at 7 days, and further reducing to 9.67E-06-1.24E-05 mg/kg bw at 14 days, while HQ% declined from 0.626–0.999% at 3 days, to 0.281–0.787% at 7 days, and 0.032–0.069% at 14 days. In cucumber, NEDI values ranged from 3.87E-05-4.89E-05 mg/kg bw at 3 days, decreasing to 9.26E-06-1.09E-05 mg/kg bw at 7 days, while HQ% followed a similar pattern, declining from 0.129-0.211% at 3 days to 0.031–0.054% at 7 days. In pepper, NEDI ranged from 1.06E-05 to 2.38E-05 mg/kg bw at 3 days, decreasing to 7.30E-06 to 1.05E-05 mg/kg bw at 7 days, and further decreasing to 1.24E-06-2.38E-06 mg/kg bw at 14 days. HQ% values declined from 0.035-0.065% at 3 days to 0.024-0.035% at 7 days, and finally 0.004-0.008% at 14 days. Across all crops and intervals, HQ% values remained well below 100%, indicating that the dietary exposure to hexythiazox residues did not pose an acute health risk. The steady decline in NEDI and HQ% over time highlights the progressive dissipation of residues and the reduced dietary risk associated with increasing waiting periods, reinforcing the importance of monitoring residue levels to ensure food safety compliance.