Portable magnetic adsorbent self-assembly coupled with effervescence-assisted dispersive liquid-liquid microextraction for rapid detection of pyrethroid residues in Chrysanthemum morifolium Ramat

Portable magnetic adsorbent self-assembly coupled with effervescence-assisted dispersive liquid-liquid microextraction for rapid detection of pyrethroid residues in Chrysanthemum morifolium Ramat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Portable magnetic adsorbent self-assembly coupled with effervescence-assisted dispersive liquid-liquid microextraction for rapid detection of pyrethroid residues in Chrysanthemum morifolium Ramat Gaotian Li, Menglei Wang, Zheyuan Xu, Hongqing Wang, Guofang Shen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8582392/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract An innovative magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method was developed for rapid pyrethroid detection in Chrysanthemum morifolium Ramat ( C. morifolium ). Effervescent tablets (CO 2 /H + donors, Fe 3 O 4 , carboxylated multi-walled carbon nanotubes) were prepared via one-pot synthesis. During extraction, the tablet was introduced into C. morifolium powder that had been moistened with acidified acetonitrile as the extracting agent. By integrating effervescence, salting-out, and magnetic aggregation, this method simplified the sample preparation steps, eliminated vortexing/centrifugation, and reduced total time to approximately 3 min. Coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS), it showed excellent linearity (R 2 > 0.998), sensitivity (limits of quantifications: 1.5-4.1 μg·kg -1 ), accuracy (recovery: 73.9-108.7 %), and precision (relative standard deviations < 5.7%). The application of MEALLE to commercial C. morifolium validated its robustness and versatility. Overall, this approach offers a time-effectiveness, equipment-independent solution for pesticide residue analysis, serving as a valuable tool for ensuring the safety of processed floral products. Physical sciences/Chemistry Earth and environmental sciences/Environmental sciences magnetic effervescence-assisted liquid-liquid extraction carboxylated multi-walled carbon nanotubes Chrysanthemum morifolium Ramat pesticide residues pyrethroid GC-MS/MS Figures Figure 1 Figure 2 Figure 3 Highlights 1. An innovative magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method was developed for ultrafast pyrethroid extraction from C. morifolium . 2. By eliminating vortexing, centrifugation, and complex steps; enables high‑throughput, equipment‑free analysis. 3. The recovery rates of detected 12 pyrethroid residues were 73.9%- 108.7%. 4. MEALLE was simpler, cheaper, and less equipment‑dependent than QuEChERS and similar methods. 1. Introduction Chrysanthemum morifolium Ramat ( C. morifolium , belonging to the Asteraceae family and commonly known as Hangbaiju) is a traditional “drug-homologous food” plant widely cultivated in Zhejiang province, China. Owing to its anti-inflammatory, antioxidant, and antipyretic properties, C. morifolium has found widespread application in beverages, seasonings, and herbal teas, driving continuous expansion in local cultivation and breeding practices (Huang et al., 2024 ; Liu et al., 2024 ). Nevertheless, this agricultural intensification has coincided with escalating pest pressures, particularly from aphids, leafminers, and caterpillars, which threaten crop yields and quality (Hernández-Valencia et al., 2024 ). To reduce crop losses, pyrethroids are frequently employed, raising concerns about potential pesticide residues in processed chrysanthemum products (Tewari, 2024 ). Given that pyrethroids are prone to bioaccumulation in humans and may induce neurotoxicity, endocrine disruption and other adverse health effects (Saito, Hara, & Tanemura, 2017 ; Wang et al., 2017 ), the establishment of efficient analytical methods for detecting pyrethroids in C. morifolium is crucial for guaranteeing product safety and consumer health (Ortiz et al., 2024 ; Ueyama et al., 2022 ; Xue et al., 2019 ). Effervescence-assisted extraction techniques has gained significant attention as an effective method for its ability to quickly and efficiently isolate target analytes from complex matrices in recent years (Legesse, Megersa, & Chandravanshi, 2025 ; Li et al., 2025 ). The carbonation process generated by acid-carbonate reactions in effervescent tablets creates a dynamic microenvironment that enhances mass transfer and extraction efficiency, while concurrently reducing reliance on organic solvents and minimizing labor-intensive sample preparation steps (Legesse et al., 2025 ). This technology has demonstrated broad applicability in pesticide extraction from diverse agricultural products, including fruits, tea beverages, and herbal extracts, underscoring its potential for pesticide residue analysis (Fu et al., 2025 ; Piao et al., 2020 ). Notably, the integration of effervescence extraction with magnetic separation technology remains underexplored, particularly in the context of pesticide residue analysis for flower tea, especially chrysanthemum tea. Magnetic adsorbent-based extraction techniques offer distinct advantages in terms of rapid separation, high selectivity, and reusability (Pajewska-Szmyt et al., 2020 ). By combining magnetic separation with effervescence-assisted extraction, this magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method aims to further streamline the sample preparation workflows. The magnetic nanoparticles enable efficient adsorbent-matrix separation through rapid magnetic collection, obviating the need for time-consuming vortexing and centrifugation steps. This innovation not only shortens the overall processing time but also reduces manual labor and the risks of sample loss or contamination, rendering the method particularly suitable for high-throughput pesticide residue analysis (Huang et al., 2024 ; Li, Qiu, & Qian, 2020 ; Zhang et al., 2019 ). To enable the rapid quantification of pyrethroid residues in C. morifolium , we developed a portable analytical approach combining MEALLE with gas chromatography-tandem mass spectrometry (GC-MS/MS). This involved creating a streamlined protocol that utilizes effervescence, salting-out phase separation, and magnetic aggregation via Fe 3 O 4 /MWCNTs-COOH. A functional composite tablet—comprising a CO 2 donor, H + donor, and magnetic nanoparticles—was designed for direct addition to hydrated C. morifolium powder, with acidified acetonitrile serving as the extraction solvent. The CO 2 and H + donors released salts upon dissolution, driving salting-out separation to enrich pyrethroids into the acetonitrile phase. Simultaneously, the gas bubbles generated by the CO 2 -H + reaction enhanced extraction efficiency, accelerated phase separation, and promoted the magnetic aggregation of pesticide residues. By eliminating the need for mechanical separation (vortexing/centrifugation), this approach significantly streamlines the workflow while improving extraction speed and reducing sample handling risks. The analytical workflow was rigorously optimized and validated for key performance parameters, including linearity, sensitivity, accuracy, and precision. Furthermore, its practical utility was further evaluated through the analysis of commercial C. morifolium samples, demonstrating its potential as a robust tool for ensuring the safety of processed floral products and supporting quality control in the chrysanthemum industry. 2. Materials and methods 2.1. Chemicals and reagents Nanomaterials: Magnetic Fe 3 O 4 nanoparticles (100 nm) and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH; 10–20 nm of outer diameter and 10–30 µm of length) were obtained from XFNANO (XFNANO, Nanjing, China). Pesticide Standards: High-purity (≥ 98%) mixed standard stock solutions (100 µg·mL − 1 ) containing twelve pyrethroid pesticides—tetramethrin, bifenthrin, fenpropathrin, lambda-cyhalothrin, acrinathrin, biopermethrin, cyfluthrin, cypermethrin, flucythrinate, fenvalerate, fluvalinate, and deltamethrin—were procured from the Agricultural Environmental Protection Institution (Tianjin, China). These stock solutions were formulated in methanol and maintained at -20 ℃ for storage. Calibration curves were constructed through serial dilution, utilizing either solvent-aligned or matrix-aligned reference standards. Another reagents: analytical grade sodium carbonate (Na 2 CO 3 ), sodium chloride (NaCl), dihydrogen phosphate (NaH 2 PO 4 ), ammonium sulfate ((NH 4 ) 2 SO 4 ), and magnesium sulfate (MgSO 4 ) were obtained from Aladdin (Shanghai, China). LC-MS-grade acetonitrile and formic acid (FA) were supplied by Thermo (Waltham, MA, USA). Milli-Q purified ultrapure water was obtained through a dedicated purification system (Millipore, Billerica, MA, USA). Octadecylsilane (C18), graphitized carbon black (GCB), and primary secondary amine (PSA) were provided by Agilent (Santa Clara, CA, USA). 2.2. Effervescent tablets preparation process Effervescent tablets were fabricated using an optimized one-pot grinding-and-pressing technique (refer to Fig. 1 A). Briefly, Na 2 CO 3 (61 mg), NaH 2 PO 4 (139 mg), Fe 3 O 4 nanoparticles (6 mg), MWCNTs-COOH (2 mg) were homogeneously ground in a mortar, then compressed into tablets using a powder tablet press (SPECAC, Operton, Kent, UK). Each tablet measured approximately 7 mm in diameter, 2 mm in thickness, and weighed 208 mg. This straightforward and economical fabrication procedure, which utilizes easily accessible and inexpensive materials along with high-speed production, makes the tablets extremely suitable for routine analytical applications. To prevent cross-contamination, and taking into account their ease of operation, wide availability, and low cost of tablet manufacturing, these effervescent tablets were designed for single use only. 2.3. Sample collection and pretreatment A total of forty samples of C. morifolium were gathered from various commercial retail stores throughout Zhejiang Province, China. To preserve their integrity before analysis, all the floral samples were promptly stored at 4℃ in a dark environment. For sample preparation, 10 g of a well-homogenized C. morifolium sample was combined with 100 mL of ultrapure water to form a uniform suspension. For spiked samples, pyrethroids were introduced by pipetting a precise volume of the standard stock solution to the suspension, achieving final concentrations of 0.05 mg·kg − 1 for optimization of the analytical procedure and 0.01, 0.05, and 0.10 mg·kg − 1 for validating method. The spiked suspensions were thoroughly vortexed and equilibrated for 30 min to guarantee the even distribution of pesticides within the matrix. These resultant samples were then processed through the following extraction process. 2.4 Magnetic effervescence-assisted liquid-liquid extraction procedure The MEALLE procedure for extracting pyrethroid residues from C. morifolium samples is illustrated in Fig. 1 B. Precisely, a 1.0 grams of homogenized C. morifolium was accurately weighed and placed into a 10 mL polytetrafluoroethylene (PTFE) tube. Subsequently, 800 µL of acetonitrile and 30 µL of FA were introduced into the tube. The mixture was thoroughly vortexed to ensure uniform dispersion. A specially prepared magnetic effervescence tablet was then added to the vortexed mixture. Effervescent precursors (Na 2 CO 3 as the CO 2 donor and NaH 2 PO 4 as the H + donor) generated CO 2 gas via acid-base reaction, enhancing extraction process. Simultaneously, the reaction released salts (Na 3 PO 4 and Na 2 HPO 4 ) that functioned as salting-out agents, effectively driving the partitioning of the acetonitrile phase from the aqueous matrix. The magnetic nanoparticles within the tablet interacted with the pesticide residues, forming magnetic aggregates. After a 3-minute reaction period, the acetonitrile-rich organic phase migrated to the upper portion. An external magnet was utilized to quickly immobilize the magnetic aggregates, facilitating the clean isolation of the supernatant. Subsequently, 500 µL of the supernatant was carefully collected via 0.22 µm organic membrane filtration before undergoing GC-MS/MS analysis. 2.5. Pesticide analyses by GC–MS/MS For the GC-MS/MS analysis, an Agilent 8890 GC system was employed, which was integrated with a 7963A autosampler and connected to an Agilent 7000E triple quadrupole (GC/TQ) mass spectrometry system. Separation was achieved using an HP-5MS UI fused-silica capillary column (30 m × 0.25 mm inner diameter, 0.25 µm film thickness, Agilent Technologies). A carrier gas of high-purity helium (99.999%) flowed at a rate of 1.0 mL·min − 1 . Sample injection was carried out in splitless mode with a 1 µL injection volume. The oven temperature profile consisted of maintaining 80 ℃ for 1.5 min, then increasing at a rate of 40 ℃·min − 1 to 170 ℃, followed by further heating to 300℃ at 10 ℃·min − 1 and holding for 3.25 min, yielding a total analytical time of 20 min. In multiple reaction monitoring (MRM) mode, both the transfer line and electron ionization (EI) source temperatures were maintained at 280 ℃. Ultra-high purity nitrogen (99.9999%) was utilized as the collision gas, flowing at 1.5 mL·min − 1 . A solvent delay of 3.5 min was implemented. The characteristic precursor ions, corresponding product ions, and respective collision energies for all target analytes are specified in Supplementary Table S1 . 2.6. Method verification and quality assurance Validation of the analytical approach was conducted per the standardized protocol defined in the European Commission’s official technical guideline (SANTE 11312/2021) (European Commission, 2021 ). Comprehensive evaluation of method performance was conducted using matrix-free C. morifolium samples, assessing critical analytical parameters including linearity, matrix effects, detection capabilities, accuracy, precision, and analyte recovery. Method linearity was established through construction of six-point calibration curves using matrix-matched standards. Matrix effects were quantified by calculating the slope ratios of matrix-matched calibration curves relative to those prepared with solvent-based standards. The limits of detections (LODs) were set as three times the signal-to-noise ratio (S/N) of the quantitative ion, while limits of quantifications (LOQs) were set at ten times the S/N ratio. Analytical accuracy and precision were systematically evaluated through replicate recovery assays (n = 3) conducted at three distinct fortification levels (0.01, 0.05, and 0.10 mg·kg − 1 ), with each concentration level analyzed in sextuplicate. To ensure data integrity and identify potential procedural contamination, strategically positioned solvent blanks were incorporated at random intervals within the analytical sequence. This comprehensive validation approach guarantees the reliability and robustness of the developed analytical methodology for pyrethroid residue analysis in botanical matrices. 2.7. Statistical analysis All the data were handled utilizing IBM SPSS Statistics 20.0 and Excel 2019. Prior to one-way ANOVA, homogeneity of variance was assessed, followed by least significant difference (LSD) multiple comparison tests to assess the differences among groups. 3. Results and discussion 3.1. Preliminary effectiveness evaluation Owing to their strong affinity for polycyclic aromatic compounds, large specific surface area, favorable π-π stacking interactions, and excellent porosity, MWCNTs were identified as promising adsorbents (Musarurwa et al., 2019). To enhance their dispersibility in sample matrices, functionalized MWCNTs including hydroxylated, aminated, and carboxylated variants were evaluated. Based on our prior research, MWCNTs-COOH demonstrated superior purification performance in C. morifolium matrices (Ruan et al., 2025), making it the optimal choice as the adsorbent. The selection of acetonitrile as the extraction medium was based on its superior aqueous solubility, reduced viscosity, and effective binding with moderately non-polar pesticide compounds such as pyrethroids. Moreover, it shows notable proficiency in driving salting-out mediated phase separation, which was an essential characteristic for the MEALLE methodology. To verify the effectiveness of the proposed MEALLE method, two critical aspects were specifically evaluated. The initial focus was on confirming the in-situ formation of a magnetically responsive Fe 3 O 4 /MWCNTs-COOH composite through self-assembly of bare magnetic Fe 3 O 4 nanoparticles with MWCNTs-COOH during the effervescence reaction, and assessing its capability for rapid magnetic separation using an external magnet. The secondary objective was to determine whether this composite could function as an effective dispersant within the effervescent sample solution to achieve target analyte purification. Experimental results demonstrated successful in-situ composite formation (Fig. S1) with 61.15 emu·g − 1 saturation magnetization (Fig. S2), enabling rapid and efficient solid-liquid separation within 10 seconds under an applied magnetic field. The purification efficacy of the self-assembled magnetic adsorbent is further validated in the subsequent extraction condition. 3.2. Extraction condition optimization To attain the best extraction efficiency in the MEALLE process, key operational parameters influencing the method’s performance were thoroughly examined. These parameters consist of the quantity of acetonitrile (the extraction solvent), the content of FA in the solvent, the molar ratio of anhydrous Na 2 CO 3 to NaH 2 PO 4 (CO 2 donor to H + donor), and the mass ratio of Fe 3 O 4 to MWCNTs-COOH (magnetic nanoparticles to adsorbent). For this optimization, C. morifolium samples were fortified with 12 pyrethroid pesticides at 0.02 mg·kg − 1 . Optimal extraction parameters were identified by systematically analyzing and comparing the recovery efficiencies and chromatographic responses of the target compounds. 3.2.1. Optimization of acetonitrile volume The optimization of extraction solvent (acetonitrile) volume aimed to balance high detection sensitivity with clear salt-induced phase separation. Variations in acetonitrile volume directly affected the thickness of the upper organic phase: insufficient volume led to overly thin upper layers that were difficult to collect, while excessive volume diluted analytes in the upper phase. To address this, peak areas of analytes (rather than recovery rates, due to variable dilution across acetonitrile volumes) were compared across 500–1000 µL. The peak areas peaked at 500 µL but declined with further volume increases as depicted in Fig. 2A. However, volumes < 800 µL produced insufficient upper organic phases for practical collection (specialized sample vials need to be matched). Thus, 800 µL was determined as the optimal parameter, prioritizing ease of collection while maintaining acceptable detection sensitivity (slightly lower peak areas than 500 µL but ensuring operational feasibility). 3.2.2. Effect of formic acid content Pyrethroid pesticides are generally stable under acidic conditions, and FA-acidified acetonitrile enhances their extraction efficiency (Fu et al., 2022; Ruan et al., 2023). Additionally, FA promotes effervescence by facilitating CO 2 generation through reactions between CO 2 donor and H + donor (Fu et al., 2025). To evaluate its impact, varying FA volumes (10–50 µL) were tested in C. morifolium samples extraction. As shown in Fig. 2B, all FA-added groups exhibited higher recovery rates than the FA-free control. Recovery rates of most pesticides significantly increased as FA volume rose from 10 µL to 30 µL, after which further increases (up to 50 µL) led to plateaued recoveries. This indicated that 30 µL of FA saturated the effervescence reaction, maximizing CO 2 generation and extraction efficiency. Thus, 30 µL of FA was chosen as the optimum. 3.2.3. Optimization of adsorbent (MWCNTs-COOH) mass Adsorbent quantity is critical: insufficient mass provides inadequate active sites, leading to matrix interference, while excess mass may cause target analyte loss (Ma et al., 2022; Parsayi Arvand, Moghimi, & Abniki, 2023). The effect of MWCNTs-COOH mass (0.5-3.0 mg) on extraction recovery was evaluated. Recovery rates improved with increasing mass from 0.5 mg to 2.0 mg but plateaued at 3.0 mg as illustrated in Fig. 2C. Thus, considering reagent conservation, 2.0 mg was determined as the ideal adsorbent dosage, balancing efficiency and practicality. 3.2.4. Influence of Fe 3 O 4 to MWCNTs-COOH mass ratio on magnetic adsorbent performance The Fe₃O₄/MWCNTs-COOH mass ratio significantly affects both nanostructure assembly characteristics and magnetic decantation efficiency (Li et al., 2025). With MWCNTs-COOH mass fixed, ratios of 2:1, 3:1, 4:1, and 5:1 (Fe 3 O 4 : MWCNTs-COOH) were systematically examined in this experiment. As illustrated in Fig. 2D, recovery rates remained stable across ratios, as MWCNTs-COOH was held constant. From a magnetic separation perspective, formulations exhibiting a 3:1 or higher ratio demonstrated optimal phase separation kinetics. Conversely, the 2:1 composition exhibited suboptimal separation behavior, with residual adsorbent material observable at the magnet interface even after prolonged exposure. This phenomenon suggests incomplete magnetic clustering of the Fe 3 O 4 component with the MWCNTs-COOH, resulting in inadequate magnetic dipole interactions. The observed phase separation deficiency would necessitate supplementary centrifugation protocols to achieve acceptable purification standards. Based on these findings, the 3:1 mass ratio was strategically selected as the optimal operational parameter. This composition successfully balances robust magnetic responsiveness with rapid solid-liquid separation dynamics, offering superior performance for magnetic nanocomposite-based extraction systems without compromising process efficiency. 3.2.5. Molar ratio of CO 2 donor to H + donor The proportion of the carbonate source (Na 2 CO 3 ) to the acid source (NaH 2 PO 4 ) dictates both the vigor and the thoroughness of the effervescence generated, which affects analyte dispersion via bubble-induced mixing (Jia et al., 2020). The molar ratio of Na 2 CO 3 to NaH 2 PO 4 regulates effervescence intensity and completeness, which affect analyte dispersion via bubble-induced mixing. Four ratios (1:1, 1:1.5, 1:2, and 1:3) were evaluated to ensure the smooth progress of the effervescent reaction and achieve an effective extraction efficiency of the target analytes. Notably, employing a 1:3 ratio resulted in an overly intense and uncontrolled foaming that exceeded the capacity of the centrifuge tube, consequently halting the subsequent analytical steps. Among the remaining ratios, a 1:2 ratio yielded the highest recoveries for all 12 pyrethroids (Fig. 2E) and the shortest reaction time. Thus, a 1:2 molar ratio (Na 2 CO 3 : NaH 2 PO 4 ) was optimal for extraction efficiency. 3.2.6. Type of salts Conventional salting-out agents (e.g., NaCl, MgSO 4 , (NH 4 ) 2 SO 4 ) were compared with the effervescent byproducts Na 3 PO 4 and Na 2 HPO 4 . The latter achieved comparable salting-out effects. To test if traditional salts could enhance extraction, NaCl, MgSO 4 , and (NH 4 ) 2 SO 4 were added to the effervescent tablets. Results showed negligible or no significant impact on pyrethroid recoveries from C. morifolium samples (Fig. 2F). Thus, additional salts were omitted from the final effervescent tablet formulation for the subsequent extraction protocol. Notably, the optimal sample mass was not further optimized due to its interdependence with other parameters (e.g., acetonitrile volume, adsorbent mass, and magnetic nanoparticle quantity). For future applications requiring larger sample quantities, proportional adjustments of all codependent parameters will ensure consistent extraction efficiency without compromising method robustness. 3.3. GC-MS/MS condition optimization The refined protocols for GC-MS/MS were devised utilizing a proprietary library detailing characteristic compound transitions, underpinned by the pivotal role of MS parameters in attaining superior analytical sensitivity (Chen et al., 2021). The initial phase involved the comprehensive screening of all feasible ion transitions for the targeted pesticides. This was accomplished by injecting a 1.00 mg·L − 1 mixed standard solution, with the objective of pinpointing the most analytically advantageous transitions. Given the complex matrix of chrysanthemum, selection of transitions prioritized selectivity over sensitivity to minimize potential matrix interferences. This selection was validated by comparing the signal response in the presence of the same concentration of target analytes in a prepared matrix extract. Upon establishing the optimal set of diagnostic transitions, a meticulous optimization of their respective collision-induced dissociation (CID) energies was undertaken. This involved systematically injecting the calibration standard solution and subjecting it to a wide range of collision energies (from 0 V to 65 V), with incremental adjustments made in 5 V steps to identify the energy yielding the most intense and selective product ions. To ensure both sensitivity and efficient data acquisition, the selected transitions were distributed across 12 MRM windows, with appropriate dwell times assigned to each transition. Additionally, since most pyrethroid pesticides exhibit chiral isomerism and other structural variations, the agricultural applications and commercial standards often consist of mixtures of multiple isomers. Consequently, during qualitative ion scanning and quantitative ion integration using triple quadrupole mass spectrometry, manual screening and integration steps were incorporated to ensure that all isomeric forms could be summed and included in the calculations. This approach guarantees the accuracy of both qualitative identification and quantitative analysis. Acetonitrile was chosen as the extraction medium owing to its favorable characteristics for straightforward sample extraction and subsequent purification processes, allowing for its direct introduction into the GC system. However, due to its relatively high boiling point (~ 81 ℃), acetonitrile is not commonly used in GC analysis, as its instantaneous condensation on the inner walls of the GC column can lead to reduced column efficiency, peak tailing, and peak broadening. To mitigate these adverse effects, the initial operating temperature was increased to 80 ℃, which represents a departure from the more typical setpoint range of 60 to 70 ℃. Furthermore, the temperatures of the EI source were optimized by testing a series of values: the temperature was varied in 10 ℃ increments from 250 to 320 ℃. Concurrently, the transfer line temperature was similarly adjusted from 270 to 320 ℃. Ultimately, both were set to 280 ℃ based on performance evaluation. The quantitative ions, qualitative ions, retention times, collision energies, and dwell times for all target compounds are summarized in Supplementary Table S1. 3.4. Method validation The newly developed analytical method’s quantitative validation parameters are listed in Table 1. Figure 3A shows a typical chromatographic profile of an unspiked C. morifolium sample, while Fig. 3B and Fig. 3C illustrate the corresponding chromatographic patterns of matrix-fortified samples at a target concentration of 0.1 mg·kg − 1 . Matrix effects, a critical parameter in method validation, were carefully evaluated. Notably, the MEALLE method incorporates magnetic adsorbent-based sample purification and magnetic cluster formation, which introduces unique challenges for matrix effect assessment. Standard solutions, devoid of solid residues, fail to replicate the magnetic particle aggregation behavior observed in authentic sample matrices. Consequently, the evaluation of matrix effects solely based on standard solutions lacks reliability. To address this, matrix-matched calibration was intentionally employed to more accurately mirror the actual extraction environment. Calibration curves constructed using matrix-matched standards demonstrated outstanding linearity for all 12 pyrethroid analytes under optimized conditions, with R 2 exceeding 0.998. LODs and LOQs were established by progressively diluting the mixed standard intermediate solutions with blank matrix extracts, using the lowest concentration within the validated linear dynamic range as the reference point. Considering an 8-fold dilution factor during C. morifolium sample pretreatment, the final LODs and LOQs for the analytes are shown in Table 1. Notably, the calculated LOQs of the target pesticides were exceptionally low (1.5–4.1 µg·kg − 1 ), bellowing the maximum residue limits (MRLs) specified in GB 2763 − 2021 (The State Health Commission et al., 2021). These results demonstrate the method’s exceptional sensitivity for trace-level pesticide detection in C. morifolium samples. Accuracy was verified for the method by means of spike-recovery tests at three different concentration levels (low: 0.01 mg·kg − 1 , medium: 0.02 mg·kg − 1 , high: 0.10 mg·kg − 1 ), enabling comprehensive evaluation of recovery performance across the analytical concentration spectrum. Precision was further validated by evaluating both recovery percentages and their RSDs. As shown in Table 2, recoveries for the 12 pyrethroid pesticides at these three fortified levels ranged from 73.9% to 108.7%, with intra- and inter-day RSDs not exceeding 5.7%. The method demonstrates satisfactory performance for pesticide residue analysis in C. morifolium , as the obtained recovery rates and RSDs align with the acceptability criteria outlined in the European Union (EU) guidelines on food safety (Food Safety, 2009), which specify that recovery values between 70–120% and relative standard deviations below 10% represent acceptable analytical precision and trueness. 3.5. Application to commercial C. morifolium samples To validate the method’s suitability for routine use, 40 commercial C. morifolium samples gathered from local farms or markets were analyzed. Significantly, none of the targeted pyrethroids were found in these 40 samples. This might be attributed to China’s increasingly stringent regulatory controls, which have significantly minimized or nearly eliminated pesticide residues in agricultural products. Ten samples were randomly chosen and spiked with pyrethroids at 0.1 mg·kg − 1 , and then analyzed by the verified method to further assess method suitability for commercial samples. Results confirmed accurate detection of all 12 pyrethroids in fortified samples, with retention times and signal intensities matching those of standard solutions. Therefore, the robustness and reliability of the method for analyzing pesticide residues in commercial C. morifolium matrices were confirmed. 3.6. Advantages of the MEALLE method This study presents an innovative approach, the MEALLE method, specifically designed for the efficient detection of pyrethroids in C. morifolium . This method employs a simplified one-pot preparation technique to produce effervescent tablets, combining Na 2 CO 3 , NaH 2 PO 4 , bare magnetic Fe 3 O 4 nanoparticles, and the functionalized adsorbent MWCNTs-COOH, which are then directly compressed into tablets. During the effervescent reaction, the adsorbent simultaneously self-assembles with the bare Fe 3 O 4 nanoparticles to form in-situ magnetic composites and interacts thoroughly with the aqueous sample, ensuring effective purification of the target analytes. The residual magnetic Fe 3 O 4 nanoparticles exhibit strong adhesion to solid particulates, forming magnetic aggregates that facilitate rapid phase separation upon application of an external magnetic field, thereby obviating the necessity for centrifugation and streamlining the extraction process. The MEALLE method uniquely integrates three key mechanisms: effervescence-driven extraction, salting-out phase separation, and magnetic nanoparticle aggregation, all within a self-activating system that removes the requirement for mechanical agitation. This integration circumvents labor-intensive (vortex mixing and centrifugation) and environmentally burdensome steps (only 800µL of acetonitrile), thereby reducing operational complexity and costs. A critical advantage lies in the concurrent execution of effervescence-driven extraction and salting-out phase separation, which eradicates the demand for high-energy input devices (e.g., vortex mixers) while maintaining extraction efficiency. Moreover, the magnetic nanoparticles’ adherence to solid residues, paired with their rapid isolation from the supernatant via magnetic response, removes the time-consuming centrifugation typically necessary after sample homogenization. Comparative evaluation against established methods (Table 3) highlights MEALLE’s superiority in simplicity, speed, and analytical efficiency. The protocol requires a streamlined three-step procedure and about 3 minutes of sample preparation, markedly less than conventional techniques such as SPE (19 steps, ≥ 45 minutes), QuEChERS (10 steps, ≥ 42 minutes), and GCT-DES-EVA-DLLME (9 steps, ≥ 35 minutes). By eliminating vortexing and centrifugation through integrated mechanisms, MEALLE optimizes workflow practicality for routine analysis. Analytical performance, assessed via GC-MS/MS, demonstrates exceptional sensitivity, with LODs spanning from 0.5 to 1.4µg·kg − 1 . These values are either comparable to or surpass those of advanced alternatives, including MEASO-LLE (0.7–3.5µg·kg − 1 ) and MEA-SHS-HLLME (6.7–32.9µg·kg − 1 ). Collectively, the MEALLE method provides a rapid, reliable, and user-centric solution for pyrethroid residue analysis in C. morifolium , aligning with the stringent requirements of safety monitoring for floral agricultural products. 4. Conclusion In this research, a brand-new MEALLE method was devised and effectively utilized for accurately quantifying pyrethroid residues in C. morifolium . Through the composition of effervescence-driven extraction, salting-out phase separation, and in-situ magnetic modified adsorbent aggregation, this method considerably streamlines the sample preparation workflows and gets rid of labor-consuming steps like vortexing and centrifugation. These enhancements have cut down the overall processing time to around 3 minutes, which improves its practicality for regular analysis. The optimized MEALLE method demonstrated exceptional analytical performance, including outstanding linearity, accuracy, precision, and sensitivity (LOQs: 1.5–4.1 µg·kg − 1 ). Its successful application to authentic C. morifolium matrices, including spiked samples, underscored its robustness and versatility in real-world scenarios. Compared to conventional techniques (e.g., QuEChERS), the MEALLE method excels in simplicity, cost-effectiveness, and adaptability to high-throughput analysis. By streamlining extraction processes and leveraging magnetic separation, it addresses key limitations of traditional methods, such as lengthy procedures and equipment dependence, while maintaining high sensitivity. Overall, this work provides a potent analytical instrument for monitoring of pyrethroid residues in flower-based agricultural products, contributing to the development of efficient and reliable quality control protocols in the chrysanthemum industry. Declarations Author Contribution Gaotian Li: Methodology, Writing-original draft preparation. Menglei Wang: Methodology, Data curation, Software. Zheyuan Xu: Visualization, Investigation. Hongqing Wang: Software, Validation. Guofang Shen: Software, Validation. Zhi Yang: Supervision. Hailong Xiao: Supervision. Jinhua Yan: Supervision. Xuming Ji: Conceptualization. Jiazhao Ruan: Conceptualization, Visualization, Writing- review and editing. Acknowledgements This work was supported by the National Natural Science Foundation of China (82274406 and 82474385), the Natural Science Foundation of Zhejiang Province (LZ24H270001), the Science and Technology Program of Zhejiang Province (2025C02178), the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (CI2023C009LH), and the Zhejiang Medical Products Administration Science and Technology Plan (2025035). References Chen, R., Xue, X., Wang, G., & Wang, J. (2021). Determination and dietary intake risk assessment of 14 pesticide residues in apples of China. Food Chemistry , 351, 129266. https://doi.org/10.1016/j.foodchem.2021.129266 . Dai, Z., Liang, S., Zhang, C., Sun, H., Zhou, L., Luo, F., & Chen, Z. (2024). Detection of 13 pyrethroid pesticides in jasmine ( Jasminum sp. ) by modified QuEChERS method and gas chromatography-tandem mass spectrometry. 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Amphiphilic block copolymer-grafted magnetic multi-walled carbon nanotubes as QuEChERS adsorbent for simultaneous determination of mycotoxins and pesticides in grains via liquid chromatography tandem mass spectrometry. Microchimica Acta , 187 (12). https://doi.org/10.1007/s00604-020-04632-w . Li, W., Xu, Y., Wang, M., Wang, Y., Wei, J., & Chen, D. (2025). Effervescence-assisted salting-out liquid-liquid extraction for rapid and convenient analysis of pyrethroid pesticide residues. Talanta, 287 . https://doi.org/10.1016/j.talanta.2025.127704 . Li, Z., Deng, B., Chen, J., Feng, R., Wang, S., Li, S.,.. . Hua, L. (2025). One-pot synthesis of magnetic adsorbent with integrated pH regulation for convenient and rapid determination of antidepressant in biofluids. Microchemical Journal , 209 . https://doi.org/10.1016/j.microc.2025.112834 . Liu, Y., Lu, C., Zhou, J., Zhou, F., Gui, A., Chu, H., & Shao, Q. (2024). Chrysanthemum morifolium as a traditional herb: A review of historical development, classification, phytochemistry, pharmacology and application. Journal of Ethnopharmacology , 330 , 118198. https://doi.org/10.1016/j.jep.2024.118198 . Ma, W., Yang, B., Li, J., & Li, X. (2022). Amino-functional metal–organic framework as a general applicable adsorbent for simultaneous enrichment of nine neonicotinoids. Chemical Engineering Journal , 434 , 134629. https://doi.org/10.1016/j.cej.2022.134629 . Musarurwa, H., Chimuka, L., Pakade, V. E., & Tavengwa, N. T. (2019). Recent developments and applications of QuEChERS based techniques on food samples during pesticide analysis. Journal of Food Composition and Analysis , 84 , 103314. https://doi.org/10.1016/j.jfca.2019.103314 . Nemati, M., Farajzadeh, M. A., Mogaddam, M. R. A., Mohebbi, A., Azimi, A. R., Fattahi, N., & Tuzen, M. (2022). Development of a gas–controlled deep eutectic solvent–based evaporation–assisted dispersive liquid–liquid microextraction approach for the extraction of pyrethroid pesticides from fruit juices. Microchemical Journal , 175 , 107196. https://doi.org/10.1016/j.microc.2022.107196 . Ortiz, D. M. D., Park, J., Lee, H., & Park, K. (2024). Integrated assessment for the estrogenic effects of pyrethroid compounds: defining the molecular initiating events and key events for the adverse outcome pathway. Toxics , 12 (3). https://doi.org/10.3390/toxics12030218 . Pajewska-Szmyt, M., Biniewska, E., Buszewski, B., & Gadzala-Kopciuch, R. (2020). Synthesis of magnetic molecularly imprinted polymer sorbents for isolation of parabens from breast milk. Materials , 13 (19). https://doi.org/10.3390/ma13194328 . Parsayi Arvand, M., Moghimi, A., & Abniki, M. (2023). Extraction of alprazolam in biological samples using the dispersive solid-phase method with nanographene oxide grafted with α-pyridylamine. IET Nanobiotechnology , 17 (2), 69–79. https://doi.org/10.1049/nbt2.12105 . Piao, H., Jiang, Y., Qin, Z., Tao, S., Ma, P., Sun, Y.,.. . Song, D. (2020). Development of a novel acidic task-specific ionic liquid-based effervescence-assisted microextraction method for determination of triazine herbicides in tea beverage. Talanta , 208 . https://doi.org/10.1016/j.talanta.2019.120414 . Qiao, Y., Qiao, J., Cao, J., Cheng, F., Cheng, Y., Chang, M.,.. . Feng, C. (2023). Magnetic effervescence-assisted switchable solvent dispersive liquid-liquid microextraction for the determination of pyrethroids in edible fungi. Journal of Food Composition and Analysis , 122 , 105473. https://doi.org/10.1016/j.jfca.2023.105473 . Ruan, J., Li, G., Lu, X., Wang, D., Yang, Z., Wang, S., & Ji, X. (2023). Monitoring residue levels of multiple types pesticides in chrysanthemum ( Chrysanthemum morifolium Ramat) and its residue pattern in diet consumption. Journal of Food Composition and Analysis , 121 , 105403. https://doi.org/10.1016/j.jfca.2023.105403 . Ruan, J., Li, G., Wang, Y., Li, A., Wang, J., Niu, H.,.. . Ji, X. (2025). Development of a modified QuEChERS-UPLC-MS/MS method based on multi-walled carbon nanotubes for 27 pesticide residues followed by determination of the residue levels and dietary intake risk assessment in Chrysanthemum morifolium Ramat. Food Chemistry: X , 31 , 103154. https://doi.org/10.1016/j.fochx.2025.103154 . Saito, H., Hara, K., & Tanemura, K. (2017). Prenatal and postnatal exposure to low levels of permethrin exerts reproductive effects in male mice. Reproductive Toxicology , 74 , 108–115. https://doi.org/10.1016/j.reprotox.2017.08.022 . Tewari, A. (2024). Respiratory system: Highly exposed yet under-reported organ in pyrethrin and pyrethroid toxicity. Toxicology and Industrial Health , 40 (11), 622–635. https://doi.org/10.1177/07482337241273808 . The State Health Commission, the Ministry of Agriculture and Rural Affairs, & the State Administration of Market Regulation. (2021). Standard for maximum residue limits of pesticides in food. GB 2763 – 2021. http://2763.foodmate.net . Ueyama, J., Ito, Y., Hamada, R., Oya, N., Kato, S., Matsuki, T.,.. . Kamijima, M. (2022). Simultaneous quantification of pyrethroid metabolites in urine of non-toilet-trained children in Japan. Environmental Health and Preventive Medicine , 27 . https://doi.org/10.1265/ehpm.21-00037 . Wang, X., He, B., Kong, B., Wei, L., Wang, R., Zhou, C.,.. . Fu, Z. (2017). β-Cypermethrin and its metabolite 3-phenoxybenzoic acid exhibit immunotoxicity in murine macrophages. Acta Biochimica Et Biophysica Sinica , 49 (12), 1083–1091. https://doi.org/10.1093/abbs/gmx111 . Xue, J., Zhang, D., Wu, X., Pan, D., & Hua, R. (2019). In-tube ultrasound assisted dispersive solid-liquid microextraction based on self-assembly and solidification of an alkanol-based floating organic droplet for determination of pyrethroid insecticides in chrysanthemum. Chromatographia , 82 (3), 695–704. https://doi.org/10.1007/s10337-018-3678-y . Zhang, M., Yang, J., Geng, X., Li, Y., Zha, Z., Cui, S., & Yang, J. (2019). Magnetic adsorbent based on mesoporous silica nanoparticles for magnetic solid phase extraction of pyrethroid pesticides in water samples. Journal of Chromatography A , 1598 , 20–29. https://doi.org/10.1016/j.chroma.2019.03.048 . Zhang, W., Jiang, Q., Xiu, F., Bao, W., Hu, B., & Xu, D. (2025). Residue analysis and degradation studies of two chiral pyrethroids in cabbage by ultra performance convergence chromatography. Microchemical Journal , 212 , 113174. https://doi.org/10.1016/j.microc.2025.113174 . Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files npjScienceofFoodSupplementary.docx Appendix A. Supplementary data Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 May, 2026 Reviews received at journal 03 May, 2026 Reviews received at journal 25 Apr, 2026 Reviews received at journal 14 Apr, 2026 Reviewers agreed at journal 12 Apr, 2026 Reviewers agreed at journal 10 Apr, 2026 Reviewers agreed at journal 01 Apr, 2026 Reviewers invited by journal 13 Mar, 2026 Editor assigned by journal 17 Jan, 2026 Submission checks completed at journal 17 Jan, 2026 First submitted to journal 12 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8582392","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":605844627,"identity":"6ab70ddd-1f92-4123-b5e8-522fe54a2b82","order_by":0,"name":"Gaotian Li","email":"","orcid":"","institution":"Zhejiang Chinese Medical University","correspondingAuthor":false,"prefix":"","firstName":"Gaotian","middleName":"","lastName":"Li","suffix":""},{"id":605844628,"identity":"bbc2fefa-b7b7-4301-9c9c-c1e974471510","order_by":1,"name":"Menglei Wang","email":"","orcid":"","institution":"Zhejiang Chinese Medical 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sample.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/0aba81a0d40633eec7eaf746.png"},{"id":104735378,"identity":"6fde6122-96fe-4338-b894-96576428f0b0","added_by":"auto","created_at":"2026-03-16 15:16:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5474981,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of key experimental parameters: (A) acetonitrile volume, (B) formic acid content, (C) MWCNTs-COOH mass, (D) Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e to MWCNTs-COOH mass ratio, (E) Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e to NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e molar ratio, (F) the type of added salt.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/520693f0989ec5e90bcd1ce7.png"},{"id":104735380,"identity":"e062a3fa-51c6-43e1-9ab9-d2b32f119a04","added_by":"auto","created_at":"2026-03-16 15:16:09","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12628309,"visible":true,"origin":"","legend":"\u003cp\u003eTotal ion chromatograms (A and B) and extracted quantitative ion chromatograms (C) of the 12 pyrethroid pesticides: A. blank \u003cem\u003eC. morifolium\u003c/em\u003e sample; B. blank sample spiked with target analytes at 0.10 mg·kg\u003csup\u003e-1\u003c/sup\u003e; C. extracted quantitative ion chromatograms.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/d06a6dd620c8885c42d930d0.png"},{"id":104783381,"identity":"dbab73f5-442c-46d3-ae82-1df970d8b53a","added_by":"auto","created_at":"2026-03-17 07:58:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":75947059,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/e9ec3d26-bacc-4b26-a74f-7d73f53c907c.pdf"},{"id":104735379,"identity":"10b9544a-0f3e-4599-b439-22b61f27d504","added_by":"auto","created_at":"2026-03-16 15:16:08","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7690407,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAppendix A. Supplementary data\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"npjScienceofFoodSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/940f769b649bac5085a06705.docx"},{"id":104735377,"identity":"2d429b7d-78eb-471b-b336-4e0c96da9563","added_by":"auto","created_at":"2026-03-16 15:16:08","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":32849,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8582392/v1/4eb4a6a8d0914289c3428ca4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Portable magnetic adsorbent self-assembly coupled with effervescence-assisted dispersive liquid-liquid microextraction for rapid detection of pyrethroid residues in Chrysanthemum morifolium Ramat","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. An innovative magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method was developed for ultrafast pyrethroid extraction from \u003cem\u003eC. morifolium\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e2. By eliminating vortexing, centrifugation, and complex steps; enables high‑throughput, equipment‑free analysis.\u003c/p\u003e\n\u003cp\u003e3. The recovery rates of detected 12 pyrethroid residues were 73.9%- 108.7%.\u003c/p\u003e\n\u003cp\u003e4. MEALLE was simpler, cheaper, and less equipment‑dependent than QuEChERS and similar methods.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e Ramat (\u003cem\u003eC. morifolium\u003c/em\u003e, belonging to the Asteraceae family and commonly known as Hangbaiju) is a traditional \u0026ldquo;drug-homologous food\u0026rdquo; plant widely cultivated in Zhejiang province, China. Owing to its anti-inflammatory, antioxidant, and antipyretic properties, \u003cem\u003eC. morifolium\u003c/em\u003e has found widespread application in beverages, seasonings, and herbal teas, driving continuous expansion in local cultivation and breeding practices (Huang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Nevertheless, this agricultural intensification has coincided with escalating pest pressures, particularly from aphids, leafminers, and caterpillars, which threaten crop yields and quality (Hern\u0026aacute;ndez-Valencia et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To reduce crop losses, pyrethroids are frequently employed, raising concerns about potential pesticide residues in processed chrysanthemum products (Tewari, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Given that pyrethroids are prone to bioaccumulation in humans and may induce neurotoxicity, endocrine disruption and other adverse health effects (Saito, Hara, \u0026amp; Tanemura, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), the establishment of efficient analytical methods for detecting pyrethroids in \u003cem\u003eC. morifolium\u003c/em\u003e is crucial for guaranteeing product safety and consumer health (Ortiz et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Ueyama et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xue et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEffervescence-assisted extraction techniques has gained significant attention as an effective method for its ability to quickly and efficiently isolate target analytes from complex matrices in recent years (Legesse, Megersa, \u0026amp; Chandravanshi, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The carbonation process generated by acid-carbonate reactions in effervescent tablets creates a dynamic microenvironment that enhances mass transfer and extraction efficiency, while concurrently reducing reliance on organic solvents and minimizing labor-intensive sample preparation steps (Legesse et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). This technology has demonstrated broad applicability in pesticide extraction from diverse agricultural products, including fruits, tea beverages, and herbal extracts, underscoring its potential for pesticide residue analysis (Fu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Piao et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Notably, the integration of effervescence extraction with magnetic separation technology remains underexplored, particularly in the context of pesticide residue analysis for flower tea, especially chrysanthemum tea.\u003c/p\u003e \u003cp\u003eMagnetic adsorbent-based extraction techniques offer distinct advantages in terms of rapid separation, high selectivity, and reusability (Pajewska-Szmyt et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). By combining magnetic separation with effervescence-assisted extraction, this magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method aims to further streamline the sample preparation workflows. The magnetic nanoparticles enable efficient adsorbent-matrix separation through rapid magnetic collection, obviating the need for time-consuming vortexing and centrifugation steps. This innovation not only shortens the overall processing time but also reduces manual labor and the risks of sample loss or contamination, rendering the method particularly suitable for high-throughput pesticide residue analysis (Huang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li, Qiu, \u0026amp; Qian, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo enable the rapid quantification of pyrethroid residues in \u003cem\u003eC. morifolium\u003c/em\u003e, we developed a portable analytical approach combining MEALLE with gas chromatography-tandem mass spectrometry (GC-MS/MS). This involved creating a streamlined protocol that utilizes effervescence, salting-out phase separation, and magnetic aggregation via Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MWCNTs-COOH. A functional composite tablet\u0026mdash;comprising a CO\u003csub\u003e2\u003c/sub\u003e donor, H\u003csup\u003e+\u003c/sup\u003e donor, and magnetic nanoparticles\u0026mdash;was designed for direct addition to hydrated \u003cem\u003eC. morifolium\u003c/em\u003e powder, with acidified acetonitrile serving as the extraction solvent. The CO\u003csub\u003e2\u003c/sub\u003e and H\u003csup\u003e+\u003c/sup\u003e donors released salts upon dissolution, driving salting-out separation to enrich pyrethroids into the acetonitrile phase. Simultaneously, the gas bubbles generated by the CO\u003csub\u003e2\u003c/sub\u003e-H\u003csup\u003e+\u003c/sup\u003e reaction enhanced extraction efficiency, accelerated phase separation, and promoted the magnetic aggregation of pesticide residues. By eliminating the need for mechanical separation (vortexing/centrifugation), this approach significantly streamlines the workflow while improving extraction speed and reducing sample handling risks. The analytical workflow was rigorously optimized and validated for key performance parameters, including linearity, sensitivity, accuracy, and precision. Furthermore, its practical utility was further evaluated through the analysis of commercial \u003cem\u003eC. morifolium\u003c/em\u003e samples, demonstrating its potential as a robust tool for ensuring the safety of processed floral products and supporting quality control in the chrysanthemum industry.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e \u003cp\u003eNanomaterials: Magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles (100 nm) and carboxylated multi-walled carbon nanotubes (MWCNTs-COOH; 10\u0026ndash;20 nm of outer diameter and 10\u0026ndash;30 \u0026micro;m of length) were obtained from XFNANO (XFNANO, Nanjing, China).\u003c/p\u003e \u003cp\u003ePesticide Standards: High-purity (\u0026ge;\u0026thinsp;98%) mixed standard stock solutions (100 \u0026micro;g\u0026middot;mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) containing twelve pyrethroid pesticides\u0026mdash;tetramethrin, bifenthrin, fenpropathrin, lambda-cyhalothrin, acrinathrin, biopermethrin, cyfluthrin, cypermethrin, flucythrinate, fenvalerate, fluvalinate, and deltamethrin\u0026mdash;were procured from the Agricultural Environmental Protection Institution (Tianjin, China). These stock solutions were formulated in methanol and maintained at -20 ℃ for storage. Calibration curves were constructed through serial dilution, utilizing either solvent-aligned or matrix-aligned reference standards.\u003c/p\u003e \u003cp\u003eAnother reagents: analytical grade sodium carbonate (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e), sodium chloride (NaCl), dihydrogen phosphate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), ammonium sulfate ((NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e), and magnesium sulfate (MgSO\u003csub\u003e4\u003c/sub\u003e) were obtained from Aladdin (Shanghai, China). LC-MS-grade acetonitrile and formic acid (FA) were supplied by Thermo (Waltham, MA, USA). Milli-Q purified ultrapure water was obtained through a dedicated purification system (Millipore, Billerica, MA, USA). Octadecylsilane (C18), graphitized carbon black (GCB), and primary secondary amine (PSA) were provided by Agilent (Santa Clara, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Effervescent tablets preparation process\u003c/h2\u003e \u003cp\u003eEffervescent tablets were fabricated using an optimized one-pot grinding-and-pressing technique (refer to Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Briefly, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (61 mg), NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (139 mg), Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles (6 mg), MWCNTs-COOH (2 mg) were homogeneously ground in a mortar, then compressed into tablets using a powder tablet press (SPECAC, Operton, Kent, UK). Each tablet measured approximately 7 mm in diameter, 2 mm in thickness, and weighed 208 mg. This straightforward and economical fabrication procedure, which utilizes easily accessible and inexpensive materials along with high-speed production, makes the tablets extremely suitable for routine analytical applications. To prevent cross-contamination, and taking into account their ease of operation, wide availability, and low cost of tablet manufacturing, these effervescent tablets were designed for single use only.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sample collection and pretreatment\u003c/h2\u003e \u003cp\u003eA total of forty samples of \u003cem\u003eC. morifolium\u003c/em\u003e were gathered from various commercial retail stores throughout Zhejiang Province, China. To preserve their integrity before analysis, all the floral samples were promptly stored at 4℃ in a dark environment.\u003c/p\u003e \u003cp\u003eFor sample preparation, 10 g of a well-homogenized \u003cem\u003eC. morifolium\u003c/em\u003e sample was combined with 100 mL of ultrapure water to form a uniform suspension. For spiked samples, pyrethroids were introduced by pipetting a precise volume of the standard stock solution to the suspension, achieving final concentrations of 0.05 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for optimization of the analytical procedure and 0.01, 0.05, and 0.10 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for validating method. The spiked suspensions were thoroughly vortexed and equilibrated for 30 min to guarantee the even distribution of pesticides within the matrix. These resultant samples were then processed through the following extraction process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Magnetic effervescence-assisted liquid-liquid extraction procedure\u003c/h2\u003e \u003cp\u003eThe MEALLE procedure for extracting pyrethroid residues from \u003cem\u003eC. morifolium\u003c/em\u003e samples is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB. Precisely, a 1.0 grams of homogenized \u003cem\u003eC. morifolium\u003c/em\u003e was accurately weighed and placed into a 10 mL polytetrafluoroethylene (PTFE) tube. Subsequently, 800 \u0026micro;L of acetonitrile and 30 \u0026micro;L of FA were introduced into the tube. The mixture was thoroughly vortexed to ensure uniform dispersion. A specially prepared magnetic effervescence tablet was then added to the vortexed mixture. Effervescent precursors (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e as the CO\u003csub\u003e2\u003c/sub\u003e donor and NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e as the H\u003csup\u003e+\u003c/sup\u003e donor) generated CO\u003csub\u003e2\u003c/sub\u003e gas via acid-base reaction, enhancing extraction process. Simultaneously, the reaction released salts (Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e) that functioned as salting-out agents, effectively driving the partitioning of the acetonitrile phase from the aqueous matrix. The magnetic nanoparticles within the tablet interacted with the pesticide residues, forming magnetic aggregates. After a 3-minute reaction period, the acetonitrile-rich organic phase migrated to the upper portion. An external magnet was utilized to quickly immobilize the magnetic aggregates, facilitating the clean isolation of the supernatant. Subsequently, 500 \u0026micro;L of the supernatant was carefully collected via 0.22 \u0026micro;m organic membrane filtration before undergoing GC-MS/MS analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Pesticide analyses by GC\u0026ndash;MS/MS\u003c/h2\u003e \u003cp\u003eFor the GC-MS/MS analysis, an Agilent 8890 GC system was employed, which was integrated with a 7963A autosampler and connected to an Agilent 7000E triple quadrupole (GC/TQ) mass spectrometry system. Separation was achieved using an HP-5MS UI fused-silica capillary column (30 m \u0026times; 0.25 mm inner diameter, 0.25 \u0026micro;m film thickness, Agilent Technologies). A carrier gas of high-purity helium (99.999%) flowed at a rate of 1.0 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Sample injection was carried out in splitless mode with a 1 \u0026micro;L injection volume. The oven temperature profile consisted of maintaining 80 ℃ for 1.5 min, then increasing at a rate of 40 ℃\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 170 ℃, followed by further heating to 300℃ at 10 ℃\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and holding for 3.25 min, yielding a total analytical time of 20 min. In multiple reaction monitoring (MRM) mode, both the transfer line and electron ionization (EI) source temperatures were maintained at 280 ℃. Ultra-high purity nitrogen (99.9999%) was utilized as the collision gas, flowing at 1.5 mL\u0026middot;min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A solvent delay of 3.5 min was implemented. The characteristic precursor ions, corresponding product ions, and respective collision energies for all target analytes are specified in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Method verification and quality assurance\u003c/h2\u003e \u003cp\u003eValidation of the analytical approach was conducted per the standardized protocol defined in the European Commission\u0026rsquo;s official technical guideline (SANTE 11312/2021) (European Commission, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Comprehensive evaluation of method performance was conducted using matrix-free \u003cem\u003eC. morifolium\u003c/em\u003e samples, assessing critical analytical parameters including linearity, matrix effects, detection capabilities, accuracy, precision, and analyte recovery. Method linearity was established through construction of six-point calibration curves using matrix-matched standards. Matrix effects were quantified by calculating the slope ratios of matrix-matched calibration curves relative to those prepared with solvent-based standards. The limits of detections (LODs) were set as three times the signal-to-noise ratio (S/N) of the quantitative ion, while limits of quantifications (LOQs) were set at ten times the S/N ratio. Analytical accuracy and precision were systematically evaluated through replicate recovery assays (n\u0026thinsp;=\u0026thinsp;3) conducted at three distinct fortification levels (0.01, 0.05, and 0.10 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), with each concentration level analyzed in sextuplicate. To ensure data integrity and identify potential procedural contamination, strategically positioned solvent blanks were incorporated at random intervals within the analytical sequence. This comprehensive validation approach guarantees the reliability and robustness of the developed analytical methodology for pyrethroid residue analysis in botanical matrices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were handled utilizing IBM SPSS Statistics 20.0 and Excel 2019. Prior to one-way ANOVA, homogeneity of variance was assessed, followed by least significant difference (LSD) multiple comparison tests to assess the differences among groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003e3.1. Preliminary effectiveness evaluation\u003c/h2\u003e\n \u003cp\u003eOwing to their strong affinity for polycyclic aromatic compounds, large specific surface area, favorable \u0026pi;-\u0026pi; stacking interactions, and excellent porosity, MWCNTs were identified as promising adsorbents (Musarurwa et al., 2019). To enhance their dispersibility in sample matrices, functionalized MWCNTs including hydroxylated, aminated, and carboxylated variants were evaluated. Based on our prior research, MWCNTs-COOH demonstrated superior purification performance in \u003cem\u003eC. morifolium\u003c/em\u003e matrices (Ruan et al., 2025), making it the optimal choice as the adsorbent. The selection of acetonitrile as the extraction medium was based on its superior aqueous solubility, reduced viscosity, and effective binding with moderately non-polar pesticide compounds such as pyrethroids. Moreover, it shows notable proficiency in driving salting-out mediated phase separation, which was an essential characteristic for the MEALLE methodology.\u003c/p\u003e\n \u003cp\u003eTo verify the effectiveness of the proposed MEALLE method, two critical aspects were specifically evaluated. The initial focus was on confirming the \u003cem\u003ein-situ\u003c/em\u003e formation of a magnetically responsive Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e/MWCNTs-COOH composite through self-assembly of bare magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles with MWCNTs-COOH during the effervescence reaction, and assessing its capability for rapid magnetic separation using an external magnet. The secondary objective was to determine whether this composite could function as an effective dispersant within the effervescent sample solution to achieve target analyte purification. Experimental results demonstrated successful \u003cem\u003ein-situ\u003c/em\u003e composite formation (Fig. S1) with 61.15 emu\u0026middot;g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e saturation magnetization (Fig. S2), enabling rapid and efficient solid-liquid separation within 10 seconds under an applied magnetic field. The purification efficacy of the self-assembled magnetic adsorbent is further validated in the subsequent extraction condition.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003e3.2. Extraction condition optimization\u003c/h2\u003e\n \u003cp\u003eTo attain the best extraction efficiency in the MEALLE process, key operational parameters influencing the method\u0026rsquo;s performance were thoroughly examined. These parameters consist of the quantity of acetonitrile (the extraction solvent), the content of FA in the solvent, the molar ratio of anhydrous Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e to NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (CO\u003csub\u003e2\u003c/sub\u003e donor to H\u003csup\u003e+\u003c/sup\u003e donor), and the mass ratio of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e to MWCNTs-COOH (magnetic nanoparticles to adsorbent). For this optimization, \u003cem\u003eC. morifolium\u003c/em\u003e samples were fortified with 12 pyrethroid pesticides at 0.02 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Optimal extraction parameters were identified by systematically analyzing and comparing the recovery efficiencies and chromatographic responses of the target compounds.\u003c/p\u003e\n \u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003e3.2.1. Optimization of acetonitrile volume\u003c/h2\u003e\n \u003cp\u003eThe optimization of extraction solvent (acetonitrile) volume aimed to balance high detection sensitivity with clear salt-induced phase separation. Variations in acetonitrile volume directly affected the thickness of the upper organic phase: insufficient volume led to overly thin upper layers that were difficult to collect, while excessive volume diluted analytes in the upper phase. To address this, peak areas of analytes (rather than recovery rates, due to variable dilution across acetonitrile volumes) were compared across 500\u0026ndash;1000 \u0026micro;L. The peak areas peaked at 500 \u0026micro;L but declined with further volume increases as depicted in Fig.\u0026nbsp;2A. However, volumes\u0026thinsp;\u0026lt;\u0026thinsp;800 \u0026micro;L produced insufficient upper organic phases for practical collection (specialized sample vials need to be matched). Thus, 800 \u0026micro;L was determined as the optimal parameter, prioritizing ease of collection while maintaining acceptable detection sensitivity (slightly lower peak areas than 500 \u0026micro;L but ensuring operational feasibility).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec14\"\u003e\n \u003ch2\u003e3.2.2. Effect of formic acid content\u003c/h2\u003e\n \u003cp\u003ePyrethroid pesticides are generally stable under acidic conditions, and FA-acidified acetonitrile enhances their extraction efficiency (Fu et al., 2022; Ruan et al., 2023). Additionally, FA promotes effervescence by facilitating CO\u003csub\u003e2\u003c/sub\u003e generation through reactions between CO\u003csub\u003e2\u003c/sub\u003e donor and H\u003csup\u003e+\u003c/sup\u003e donor (Fu et al., 2025). To evaluate its impact, varying FA volumes (10\u0026ndash;50 \u0026micro;L) were tested in \u003cem\u003eC. morifolium\u003c/em\u003e samples extraction.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig.\u0026nbsp;2B, all FA-added groups exhibited higher recovery rates than the FA-free control. Recovery rates of most pesticides significantly increased as FA volume rose from 10 \u0026micro;L to 30 \u0026micro;L, after which further increases (up to 50 \u0026micro;L) led to plateaued recoveries. This indicated that 30 \u0026micro;L of FA saturated the effervescence reaction, maximizing CO\u003csub\u003e2\u003c/sub\u003e generation and extraction efficiency. Thus, 30 \u0026micro;L of FA was chosen as the optimum.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec15\"\u003e\n \u003ch2\u003e3.2.3. Optimization of adsorbent (MWCNTs-COOH) mass\u003c/h2\u003e\n \u003cp\u003eAdsorbent quantity is critical: insufficient mass provides inadequate active sites, leading to matrix interference, while excess mass may cause target analyte loss (Ma et al., 2022; Parsayi Arvand, Moghimi, \u0026amp; Abniki, 2023). The effect of MWCNTs-COOH mass (0.5-3.0 mg) on extraction recovery was evaluated. Recovery rates improved with increasing mass from 0.5 mg to 2.0 mg but plateaued at 3.0 mg as illustrated in Fig.\u0026nbsp;2C. Thus, considering reagent conservation, 2.0 mg was determined as the ideal adsorbent dosage, balancing efficiency and practicality.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec16\"\u003e\n \u003ch2\u003e3.2.4. Influence of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e to MWCNTs-COOH mass ratio on magnetic adsorbent performance\u003c/h2\u003e\n \u003cp\u003eThe Fe₃O₄/MWCNTs-COOH mass ratio significantly affects both nanostructure assembly characteristics and magnetic decantation efficiency (Li et al., 2025). With MWCNTs-COOH mass fixed, ratios of 2:1, 3:1, 4:1, and 5:1 (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: MWCNTs-COOH) were systematically examined in this experiment. As illustrated in Fig.\u0026nbsp;2D, recovery rates remained stable across ratios, as MWCNTs-COOH was held constant. From a magnetic separation perspective, formulations exhibiting a 3:1 or higher ratio demonstrated optimal phase separation kinetics. Conversely, the 2:1 composition exhibited suboptimal separation behavior, with residual adsorbent material observable at the magnet interface even after prolonged exposure. This phenomenon suggests incomplete magnetic clustering of the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e component with the MWCNTs-COOH, resulting in inadequate magnetic dipole interactions. The observed phase separation deficiency would necessitate supplementary centrifugation protocols to achieve acceptable purification standards.\u003c/p\u003e\n \u003cp\u003eBased on these findings, the 3:1 mass ratio was strategically selected as the optimal operational parameter. This composition successfully balances robust magnetic responsiveness with rapid solid-liquid separation dynamics, offering superior performance for magnetic nanocomposite-based extraction systems without compromising process efficiency.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003e3.2.5. Molar ratio of CO\u003csub\u003e2\u003c/sub\u003e donor to H\u003csup\u003e+\u003c/sup\u003e donor\u003c/h2\u003e\n \u003cp\u003eThe proportion of the carbonate source (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e) to the acid source (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) dictates both the vigor and the thoroughness of the effervescence generated, which affects analyte dispersion via bubble-induced mixing (Jia et al., 2020). The molar ratio of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e to NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e regulates effervescence intensity and completeness, which affect analyte dispersion via bubble-induced mixing. Four ratios (1:1, 1:1.5, 1:2, and 1:3) were evaluated to ensure the smooth progress of the effervescent reaction and achieve an effective extraction efficiency of the target analytes. Notably, employing a 1:3 ratio resulted in an overly intense and uncontrolled foaming that exceeded the capacity of the centrifuge tube, consequently halting the subsequent analytical steps. Among the remaining ratios, a 1:2 ratio yielded the highest recoveries for all 12 pyrethroids (Fig.\u0026nbsp;2E) and the shortest reaction time. Thus, a 1:2 molar ratio (Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e: NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) was optimal for extraction efficiency.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.2.6. Type of salts\u003c/h2\u003e\n \u003cp\u003eConventional salting-out agents (e.g., NaCl, MgSO\u003csub\u003e4\u003c/sub\u003e, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) were compared with the effervescent byproducts Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. The latter achieved comparable salting-out effects. To test if traditional salts could enhance extraction, NaCl, MgSO\u003csub\u003e4\u003c/sub\u003e, and (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were added to the effervescent tablets. Results showed negligible or no significant impact on pyrethroid recoveries from \u003cem\u003eC. morifolium\u003c/em\u003e samples (Fig.\u0026nbsp;2F). Thus, additional salts were omitted from the final effervescent tablet formulation for the subsequent extraction protocol.\u003c/p\u003e\n \u003cp\u003eNotably, the optimal sample mass was not further optimized due to its interdependence with other parameters (e.g., acetonitrile volume, adsorbent mass, and magnetic nanoparticle quantity). For future applications requiring larger sample quantities, proportional adjustments of all codependent parameters will ensure consistent extraction efficiency without compromising method robustness.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.3. GC-MS/MS condition optimization\u003c/h2\u003e\n \u003cp\u003eThe refined protocols for GC-MS/MS were devised utilizing a proprietary library detailing characteristic compound transitions, underpinned by the pivotal role of MS parameters in attaining superior analytical sensitivity (Chen et al., 2021). The initial phase involved the comprehensive screening of all feasible ion transitions for the targeted pesticides. This was accomplished by injecting a 1.00 mg\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e mixed standard solution, with the objective of pinpointing the most analytically advantageous transitions. Given the complex matrix of chrysanthemum, selection of transitions prioritized selectivity over sensitivity to minimize potential matrix interferences. This selection was validated by comparing the signal response in the presence of the same concentration of target analytes in a prepared matrix extract. Upon establishing the optimal set of diagnostic transitions, a meticulous optimization of their respective collision-induced dissociation (CID) energies was undertaken. This involved systematically injecting the calibration standard solution and subjecting it to a wide range of collision energies (from 0 V to 65 V), with incremental adjustments made in 5 V steps to identify the energy yielding the most intense and selective product ions. To ensure both sensitivity and efficient data acquisition, the selected transitions were distributed across 12 MRM windows, with appropriate dwell times assigned to each transition.\u003c/p\u003e\n \u003cp\u003eAdditionally, since most pyrethroid pesticides exhibit chiral isomerism and other structural variations, the agricultural applications and commercial standards often consist of mixtures of multiple isomers. Consequently, during qualitative ion scanning and quantitative ion integration using triple quadrupole mass spectrometry, manual screening and integration steps were incorporated to ensure that all isomeric forms could be summed and included in the calculations. This approach guarantees the accuracy of both qualitative identification and quantitative analysis.\u003c/p\u003e\n \u003cp\u003eAcetonitrile was chosen as the extraction medium owing to its favorable characteristics for straightforward sample extraction and subsequent purification processes, allowing for its direct introduction into the GC system. However, due to its relatively high boiling point (~\u0026thinsp;81 ℃), acetonitrile is not commonly used in GC analysis, as its instantaneous condensation on the inner walls of the GC column can lead to reduced column efficiency, peak tailing, and peak broadening. To mitigate these adverse effects, the initial operating temperature was increased to 80 ℃, which represents a departure from the more typical setpoint range of 60 to 70 ℃. Furthermore, the temperatures of the EI source were optimized by testing a series of values: the temperature was varied in 10 ℃ increments from 250 to 320 ℃. Concurrently, the transfer line temperature was similarly adjusted from 270 to 320 ℃. Ultimately, both were set to 280 ℃ based on performance evaluation. The quantitative ions, qualitative ions, retention times, collision energies, and dwell times for all target compounds are summarized in Supplementary Table S1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.4. Method validation\u003c/h2\u003e\n \u003cp\u003eThe newly developed analytical method\u0026rsquo;s quantitative validation parameters are listed in Table 1. Figure 3A shows a typical chromatographic profile of an unspiked \u003cem\u003eC. morifolium\u003c/em\u003e sample, while Fig. 3B and Fig. 3C illustrate the corresponding chromatographic patterns of matrix-fortified samples at a target concentration of 0.1 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Matrix effects, a critical parameter in method validation, were carefully evaluated. Notably, the MEALLE method incorporates magnetic adsorbent-based sample purification and magnetic cluster formation, which introduces unique challenges for matrix effect assessment.\u003c/p\u003e\n \u003cdiv\u003eStandard solutions, devoid of solid residues, fail to replicate the magnetic particle aggregation behavior observed in authentic sample matrices. Consequently, the evaluation of matrix effects solely based on standard solutions lacks reliability. To address this, matrix-matched calibration was intentionally employed to more accurately mirror the actual extraction environment. Calibration curves constructed using matrix-matched standards demonstrated outstanding linearity for all 12 pyrethroid analytes under optimized conditions, with R\u003csup\u003e2\u003c/sup\u003e exceeding 0.998.\u003c/div\u003e\n \u003cp\u003eLODs and LOQs were established by progressively diluting the mixed standard intermediate solutions with blank matrix extracts, using the lowest concentration within the validated linear dynamic range as the reference point. Considering an 8-fold dilution factor during \u003cem\u003eC. morifolium\u003c/em\u003e sample pretreatment, the final LODs and LOQs for the analytes are shown in Table 1. Notably, the calculated LOQs of the target pesticides were exceptionally low (1.5\u0026ndash;4.1 \u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), bellowing the maximum residue limits (MRLs) specified in GB 2763\u0026thinsp;\u0026minus;\u0026thinsp;2021 (The State Health Commission et al., 2021). These results demonstrate the method\u0026rsquo;s exceptional sensitivity for trace-level pesticide detection in \u003cem\u003eC. morifolium\u003c/em\u003e samples.\u003c/p\u003e\n \u003cp\u003eAccuracy was verified for the method by means of spike-recovery tests at three different concentration levels (low: 0.01 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, medium: 0.02 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, high: 0.10 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), enabling comprehensive evaluation of recovery performance across the analytical concentration spectrum. Precision was further validated by evaluating both recovery percentages and their RSDs. As shown in Table 2, recoveries for the 12 pyrethroid pesticides at these three fortified levels ranged from 73.9% to 108.7%, with intra- and inter-day RSDs not exceeding 5.7%. The method demonstrates satisfactory performance for pesticide residue analysis in \u003cem\u003eC. morifolium\u003c/em\u003e, as the obtained recovery rates and RSDs align with the acceptability criteria outlined in the European Union (EU) guidelines on food safety (Food Safety, 2009), which specify that recovery values between 70\u0026ndash;120% and relative standard deviations below 10% represent acceptable analytical precision and trueness.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e3.5. Application to commercial C. morifolium samples\u003c/h2\u003e\n \u003cp\u003eTo validate the method\u0026rsquo;s suitability for routine use, 40 commercial \u003cem\u003eC. morifolium\u003c/em\u003e samples gathered from local farms or markets were analyzed. Significantly, none of the targeted pyrethroids were found in these 40 samples. This might be attributed to China\u0026rsquo;s increasingly stringent regulatory controls, which have significantly minimized or nearly eliminated pesticide residues in agricultural products. Ten samples were randomly chosen and spiked with pyrethroids at 0.1 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and then analyzed by the verified method to further assess method suitability for commercial samples. Results confirmed accurate detection of all 12 pyrethroids in fortified samples, with retention times and signal intensities matching those of standard solutions. Therefore, the robustness and reliability of the method for analyzing pesticide residues in commercial \u003cem\u003eC. morifolium\u003c/em\u003e matrices were confirmed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\"\u003e\n \u003ch2\u003e3.6. Advantages of the MEALLE method\u003c/h2\u003e\n \u003cp\u003eThis study presents an innovative approach, the MEALLE method, specifically designed for the efficient detection of pyrethroids in \u003cem\u003eC. morifolium\u003c/em\u003e. This method employs a simplified one-pot preparation technique to produce effervescent tablets, combining Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, bare magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles, and the functionalized adsorbent MWCNTs-COOH, which are then directly compressed into tablets. During the effervescent reaction, the adsorbent simultaneously self-assembles with the bare Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles to form \u003cem\u003ein-situ\u003c/em\u003e magnetic composites and interacts thoroughly with the aqueous sample, ensuring effective purification of the target analytes. The residual magnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles exhibit strong adhesion to solid particulates, forming magnetic aggregates that facilitate rapid phase separation upon application of an external magnetic field, thereby obviating the necessity for centrifugation and streamlining the extraction process.\u003c/p\u003e\n \u003cp\u003eThe MEALLE method uniquely integrates three key mechanisms: effervescence-driven extraction, salting-out phase separation, and magnetic nanoparticle aggregation, all within a self-activating system that removes the requirement for mechanical agitation. This integration circumvents labor-intensive (vortex mixing and centrifugation) and environmentally burdensome steps (only 800\u0026micro;L of acetonitrile), thereby reducing operational complexity and costs. A critical advantage lies in the concurrent execution of effervescence-driven extraction and salting-out phase separation, which eradicates the demand for high-energy input devices (e.g., vortex mixers) while maintaining extraction efficiency. Moreover, the magnetic nanoparticles\u0026rsquo; adherence to solid residues, paired with their rapid isolation from the supernatant via magnetic response, removes the time-consuming centrifugation typically necessary after sample homogenization.\u003c/p\u003e\n \u003cp\u003eComparative evaluation against established methods (Table 3) highlights MEALLE\u0026rsquo;s superiority in simplicity, speed, and analytical efficiency. The protocol requires a streamlined three-step procedure and about 3 minutes of sample preparation, markedly less than conventional techniques such as SPE (19 steps, \u0026ge; 45 minutes), QuEChERS (10 steps, \u0026ge; 42 minutes), and GCT-DES-EVA-DLLME (9 steps, \u0026ge; 35 minutes). By eliminating vortexing and centrifugation through integrated mechanisms, MEALLE optimizes workflow practicality for routine analysis.\u003c/p\u003e\n \u003cp\u003eAnalytical performance, assessed via GC-MS/MS, demonstrates exceptional sensitivity, with LODs spanning from 0.5 to 1.4\u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These values are either comparable to or surpass those of advanced alternatives, including MEASO-LLE (0.7\u0026ndash;3.5\u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and MEA-SHS-HLLME (6.7\u0026ndash;32.9\u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Collectively, the MEALLE method provides a rapid, reliable, and user-centric solution for pyrethroid residue analysis in \u003cem\u003eC. morifolium\u003c/em\u003e, aligning with the stringent requirements of safety monitoring for floral agricultural products.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this research, a brand-new MEALLE method was devised and effectively utilized for accurately quantifying pyrethroid residues in \u003cem\u003eC. morifolium\u003c/em\u003e. Through the composition of effervescence-driven extraction, salting-out phase separation, and \u003cem\u003ein-situ\u003c/em\u003e magnetic modified adsorbent aggregation, this method considerably streamlines the sample preparation workflows and gets rid of labor-consuming steps like vortexing and centrifugation. These enhancements have cut down the overall processing time to around 3 minutes, which improves its practicality for regular analysis. The optimized MEALLE method demonstrated exceptional analytical performance, including outstanding linearity, accuracy, precision, and sensitivity (LOQs: 1.5\u0026ndash;4.1 \u0026micro;g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Its successful application to authentic \u003cem\u003eC. morifolium\u003c/em\u003e matrices, including spiked samples, underscored its robustness and versatility in real-world scenarios. Compared to conventional techniques (e.g., QuEChERS), the MEALLE method excels in simplicity, cost-effectiveness, and adaptability to high-throughput analysis. By streamlining extraction processes and leveraging magnetic separation, it addresses key limitations of traditional methods, such as lengthy procedures and equipment dependence, while maintaining high sensitivity. Overall, this work provides a potent analytical instrument for monitoring of pyrethroid residues in flower-based agricultural products, contributing to the development of efficient and reliable quality control protocols in the chrysanthemum industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGaotian Li: Methodology, Writing-original draft preparation. Menglei Wang: Methodology, Data curation, Software. Zheyuan Xu: Visualization, Investigation. Hongqing Wang: Software, Validation. Guofang Shen: Software, Validation. Zhi Yang: Supervision. Hailong Xiao: Supervision. Jinhua Yan: Supervision. Xuming Ji: Conceptualization. Jiazhao Ruan: Conceptualization, Visualization, Writing- review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (82274406 and 82474385), the Natural Science Foundation of Zhejiang Province (LZ24H270001), the Science and Technology Program of Zhejiang Province (2025C02178), the Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences (CI2023C009LH), and the Zhejiang Medical Products Administration Science and Technology Plan (2025035).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen, R., Xue, X., Wang, G., \u0026amp; Wang, J. (2021). 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Residue analysis and degradation studies of two chiral pyrethroids in cabbage by ultra performance convergence chromatography. \u003cem\u003eMicrochemical Journal\u003c/em\u003e, \u003cem\u003e212\u003c/em\u003e, 113174. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microc.2025.113174\u003c/span\u003e\u003cspan address=\"10.1016/j.microc.2025.113174\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"magnetic effervescence-assisted liquid-liquid extraction, carboxylated multi-walled carbon nanotubes, Chrysanthemum morifolium Ramat, pesticide residues, pyrethroid, GC-MS/MS","lastPublishedDoi":"10.21203/rs.3.rs-8582392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8582392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn innovative magnetic effervescence-assisted liquid-liquid extraction (MEALLE) method was developed for rapid pyrethroid detection in \u003cem\u003eChrysanthemum morifolium\u003c/em\u003e Ramat (\u003cem\u003eC. morifolium\u003c/em\u003e). Effervescent tablets (CO\u003csub\u003e2\u003c/sub\u003e/H\u003csup\u003e+\u003c/sup\u003e donors, Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e, carboxylated multi-walled carbon nanotubes) were prepared via one-pot synthesis. During extraction, the tablet was introduced into \u003cem\u003eC. morifolium\u003c/em\u003e powder that had been moistened with acidified acetonitrile as the extracting agent. By integrating effervescence, salting-out, and magnetic aggregation, this method simplified the sample preparation steps, eliminated vortexing/centrifugation, and reduced total time to approximately 3 min. Coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS), it showed excellent linearity (R\u003csup\u003e2\u003c/sup\u003e \u0026gt; 0.998), sensitivity (limits of quantifications: 1.5-4.1 μg·kg\u003csup\u003e-1\u003c/sup\u003e), accuracy (recovery: 73.9-108.7 %), and precision (relative standard deviations \u0026lt; 5.7%). The application of MEALLE to commercial \u003cem\u003eC. morifolium\u003c/em\u003e validated its robustness and versatility. Overall, this approach offers a time-effectiveness, equipment-independent solution for pesticide residue analysis, serving as a valuable tool for ensuring the safety of processed floral products.\u003c/p\u003e","manuscriptTitle":"Portable magnetic adsorbent self-assembly coupled with effervescence-assisted dispersive liquid-liquid microextraction for rapid detection of pyrethroid residues in Chrysanthemum morifolium Ramat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-16 15:16:03","doi":"10.21203/rs.3.rs-8582392/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-03T16:29:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T05:31:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-25T09:26:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-15T03:18:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154481402688037815409370436216398171461","date":"2026-04-12T10:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282932864814196364175132124919048196274","date":"2026-04-11T00:54:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255307635654875100466498684778291304933","date":"2026-04-01T04:59:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T15:23:23+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-17T05:03:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-17T05:02:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2026-01-12T13:28:21+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23984213-13e6-4b3c-abba-3a6b315a057c","owner":[],"postedDate":"March 16th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-03T16:29:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T05:31:00+00:00","index":49,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64481120,"name":"Physical sciences/Chemistry"},{"id":64481121,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2026-05-17T04:08:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-16 15:16:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8582392","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8582392","identity":"rs-8582392","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}