One pot green synthesis of m-aminophenol–urea–glyoxal resin as pipette tip solid-phase extraction adsorbent for simultaneous determination of four plant hormones in watermelon

Plant hormones (PHs) are a type of pesticide that can potentially affect human health. Therefore, their quantitative detection is particularly important. In this study, a green and economic method for the simultaneous extraction and determination of four PHs, namely thidiazuron, forchlorfenuron, 1-naphthylacetic acid, and 2-naphthoxyacetic acid.

Journal of Chromatography A 1623 (2020) 461214 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma One pot green synthesis of m-aminophenol–urea–glyoxal resin as pipette tip solid-phase extraction adsorbent for simultaneous determination of four plant hormones in watermelon juice Yanke Lu a, Pengfei Li a, Chunliu Yang a,∗, Yehong Han b, Hongyuan Yan a,b,∗ a Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Public Health, Hebei University, Baoding 071002, China Key Laboratory of Analytical Science and Technology of Hebei Province, College of pharmacy, Hebei University, Baoding 071002, China b a r t i c l e i n f o Article history: Received 16 March 2020 Revised May 2020 Accepted May 2020 Available online May 2020 Keywords: One pot fabrication Resin adsorbent Pipette tip solid-phase extraction Plant hormones Watermelon juice a b s t r a c t Plant hormones (PHs) are a type of pesticide that can potentially affect human health Therefore, their quantitative detection is particularly important In this study, a green and economic method for the simultaneous extraction and determination of four PHs, namely thidiazuron, forchlorfenuron, 1-naphthylacetic acid, and 2-naphthoxyacetic acid, in watermelon juice was developed by using maminophenol–urea–glyoxal resin as the adsorbent for pipette tip solid phase extraction (PT-SPE) coupled with liquid chromatography The resin was synthesized via a simple (one pot hydrothermal synthesis) and green (ethanol as the solvent and glyoxal as crosslinking agent) process The synthesized resin possesses multiple functional groups (hydroxyl, amino, and imino, among others), high adsorption capacity, larger specific surface area than the urea–glyoxal resin and m-aminophenol–glyoxal resin, and can be regenerated easily The PT-SPE device is simple, cheap, and easy to obtain, and the adsorbent dosage is only 5.0 mg The proposed method has a wide linear detection range, high recovery, good precision, and high sensitivity, and satisfies the measurement requirements for detecting trace levels of PHs in fruits and vegetables © 2020 Elsevier B.V All rights reserved Introduction Sample preparation, as a key step in modern analysis, plays an important role in the entire analysis process Currently, the development of new, economical, fast, and high-throughput sample pretreatment methods is still an urgent issue in the analytical field Sample pretreatment technologies include solid-phase extraction (SPE) [1], solid-phase microextraction [2,3], liquid–liquid extraction [4], matrix solid-phase dispersion [5], dispersive liquid–liquid microextraction [6], pipette tip SPE (PT-SPE) [7] Among them, SPE and methods derived therefrom are the most widely used technologies, especially the PT-SPE method have received widespread attention from researchers because of its advantages of low cost, small dead volume, longer flow path, and low consumption of the adsorbent and organic solvent [8] However, SPE and related methods are dependent on the adsorption performance of the adsor- ∗ Corresponding authors at: Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, College of Public Health, Hebei University, Baoding 071002, China E-mail addresses: yangchunliu@hbu.edu.cn (C Yang), yanhy@hbu.edu.cn (H Yan) https://doi.org/10.1016/j.chroma.2020.461214 0021-9673/© 2020 Elsevier B.V All rights reserved bent; therefore it is necessary to explore new adsorbents with excellent adsorption and purification abilities Phenolic resin, as a commercial synthetic resin, is widely used in a variety of fields, including in adsorbent owing to its low cost, easy availability, and simple preparation procedure [9,10] Phenol– formaldehyde resins are conventionally utilized, but formaldehyde, used as the crosslinker, is carcinogenic Therefore, researchers have attempted to find alternative crosslinking agents, such as glutaraldehyde, glyoxylic acid, hexamethylenetetramine, and glyoxal [11–15] Although the toxic side effects of the aforementioned adsorbents to the human body are relatively low, the functional groups of the phenolic resin are still limited as only phenolic hydroxyl groups are present in these resins, which in turn affects the adsorption ability Therefore, the development of new multifunctional phenolic resin adsorbents has become a hot research topic Plant hormones (PHs) are widely used in modern agriculture to increase crop yields [16–23] Thidiazuron (TDZ), forchlorfenuron (CPPU), 1-naphthylacetic acid (NAA), and 2-naphthoxyacetic acid (BNOA), as PHs, regulate watermelon growth, and have been widely used in different periods [24,25] Currently, watermelon juice has become one of the most popular fruit drinks in the sum- Y Lu, P Li and C Yang et al / Journal of Chromatography A 1623 (2020) 461214 mer, but long-term consumption of watermelon juice containing high-dose residues of PHs can cause a series of diseases [26,27] Therefore, many countries and organizations, such as the European Union, United States, Australia, and China have been established maximum residue limits for PHs [28–31] However, it is difficult to quantitatively analyze PHs in watermelon juice because of the complicated nature of the sample matrix and trace levels of PHs Therefore, establishing an accurate and efficient analytical method for the simultaneous determination of PHs in watermelon juice is desired In this work, aminophenol–urea–glyoxal resin (MAPUGR) with multiple functional groups is synthesized by using aminophenol and urea as bifunctional monomers, low toxicity glyoxal as the crosslinking agent to replace carcinogenic formaldehyde, and polyethylene glycol 60 0 (PEG 60 0) as a porogen Aminophenol– glyoxal resin (MAPGR) and urea–glyoxal resin (UGR) are also synthesized, and the adsorption capacity of the three adsorbents is compared A small PT-SPE cartridge is assembled by using a pipette tip, degreased cotton, and rubber suction bulb Finally, a method based on MAPUGR−PT-SPE coupled with high performance liquid chromatography (HPLC) for detecting these four PHs in watermelon juice is established by utilizing the optimized extraction parameters Fig Assembly and operation process of MAPUGR−PT-SPE Experimental section 2.1 Reagents and instruments The details are displayed in the Supplementary material 2.2 Synthesis of MAPUGR, MAPGR, and UGR Synthesis of MAPUGR: Aminophenol (30 mmol), urea (10 mmol), PEG 60 0 (0.015 mmol), and anhydrous ethanol (50 mL) were added to a flask (100 mL) and stirred until thoroughly mixed Glyoxal (30 mmol) was added, and pH of the solvent was adjusted to 9.0 with NaOH solution, the resulting solution was stirred at 50 °C for h After heating at 75 °C for 24 h, the solid product was washed with methanol and water, and then dried at 40 °C to obtain MAPUGR Synthesis of MAPGR: Aminophenol (40 mmol), PEG 60 0 (0.015 mmol), and anhydrous ethanol (50 mL) were added to a flask (100 mL) and stirred until thoroughly mixed Glyoxal (30 mmol) was added and the pH of the system was adjusted to 9.0 with NaOH solution; the resulting solution was stirred at 50 °C for h After heating at 75 °C for 24 h, the resulting solid product was washed with methanol and water, and then dried at 40 °C to obtain MAPGR Synthesis of UGR: Urea (40 mmol), PEG 60 0 (0.015 mmol), and anhydrous ethanol (50 mL) were added to a flask (100 mL) and stirred until thoroughly mixed Glyoxal (30 mmol) was added, the pH of the system was adjusted to acidic with HCl solution, the resulting solution was stirred at 50 °C for h After heating at 75 °C for 24 h, the obtained solid product was washed with methanol and water, and then dried at 40 °C to obtain UGR 2.3 Preparation of watermelon juice Watermelon samples were randomly purchased from the local supermarkets in Baoding China First, the watermelons were homogenized with a homogenizer after peeling The supernatant was obtained after centrifugation at 10 0 rpm for 10 to remove the solid residues, and the supernatant was stored frozen in a refrigerator Finally, the samples were passed through a 0.45 μm filter for further work 2.4 Procedure for MAPUGR−PT-SPE The PT-SPE was assembled from two pipette tips, degreased cotton and the adsorbent, as shown in Fig First, degreased cotton was inserted into the exit of the 200 μL pipette tip to avoid loss of the adsorbent Thereafter 5.0 mg of MAPUGR was added to the 200 μL pipette tip The top of the adsorbent was then covered with degreased cotton A 1.0 mL pipette tip that was cut off was connected to the 200 μL pipette tip filled with adsorbent The final height of the adsorbent in the PT-SPE was approximately 10 mm After activating MAPUGR with 2.0 mL of methanol and water, watermelon juice (1.0 mL) at pH 3.0 was loaded onto the cartridge, washed with water (1.0 mL), and eluted with methanol (1.0 mL) The tip of a rubber suction bulb was tightly inserted into the top of the pipette tip The pressure above the sample solution was controlled by squeezing the rubber suction bulb through a clamp fixed on an iron stand to control the flow rate The eluate was collected, dried under nitrogen, and then re-dissolved in 1.0 mL of the mobile phase for HPLC analysis 2.5 HPLC conditions Chromatographic separation was performed on an LC–20A high performance liquid chromatograph equipped with an LC–20AT solvent delivery unit and an SPD−20A ultraviolet detector (Shimadzu, Kyoto, Japan) A Labsolution workstation was used for data acquisition (Shimadzu, Kyoto, Japan) An Eclipse XDB–C18 column (4.6 × 150 mm, mm) was purchased from Agilent Tech Co., Ltd (California, United States) The wavelength of the detection was set at 224 nm The mobile phase comprised water-acetonitrile (72:28, v/v, containing 0.1% TFA) at a flow rate of 1.0 mL min−1 The column temperature was maintained at 40 °C, and the injection volume was 20 μL 2.6 Statistical analysis The confidence interval is represented by an error bar Because comparisons among three or more sample means were to be performed, one-way analyses of variance and multiple comparison Student−Newman−Keuls (SNK-q) were selected to analyze the sig- Y Lu, P Li and C Yang et al / Journal of Chromatography A 1623 (2020) 461214 Fig Schematic illustration of MAPUGR synthesis nificant differences Mean values were considered to have a significant difference when the significance test value (P) < 0.05 Results and discussion 3.1 Synthesis of MAPUGR, MAPGR, and UGR The purpose of the MAPUGR adsorbent synthesized herein was to extract and isolate four PHs in watermelon juice An attempt was made to synthesize the resin using m-aminophenol and urea with glyoxal to meet the requirements for pretreatment of the complex sample The synthesis process and reaction principle of the adsorbent are shown in Fig The methylolated intermediate was first formed from m-aminophenol, urea and glyoxal in ethanol After increasing the temperature, the intermediate polycondensed and solidified, and PEG 60 0 was also uniformly fixed in the solid product After washing the product with methanol and water, the unreacted monomers, crosslinker, and porogen in the solid product were removed, and the spatial arrangement of the MAPUGR structure was maintained by vacuum drying The hydrophilic groups of m-aminophenol and urea were retained in the product, which provided numerous adsorption sites and enhanced the hydrophilicity of the sorbent Glyoxal containing two aldehyde groups provides adequate reaction sites, and was used as a crosslinking agent in the synthesis of MAPUGR PEG 60 0, as a porogen and structure-directing agent, plays an important role in the formation of the three-dimensional porous structure of MAPUGR Anhydrous ethanol, as the reaction solvent in this experiment, does not exert the toxic effects of organic solvents on the human body and the environment, and thus enables green synthesis of the polymer 3.2 Characteristics of MAPUGR, MAPGR, and UGR Fig 3A shows the scanning electron microscope (SEM) image of MAPGR, which revealed that the product formed by the reaction of m-aminophenol with glyoxal had a non-uniform particle size with close adhesion of the components The average particle size of the individual microspheres was 3−5 μm, and the particle size of the adhesion structure was 5−30 μm The SEM image of UGR in Fig 3B shows a sheet-like adhesion structure with a particle size of 2–30 μm The SEM image of MAPUGR in Fig 3C shows the overall three-dimensional porous structure formed by the aggregation of multiple microspheres The microspheres/microspheres junction may be producted by the reaction of glyoxal with urea The size of the individual microspheres is 1–3 μm, but the overall particle size is 30–100 μm The particle size of MAPUGR satisfies the application range of SPE, and the solid layer formed by the aggregation of the microspheres in the three-dimensional network structure ensures that MAPUGR is in sufficient contact with the sample solution when loaded into the PT-SPE device The functional groups of MAPUGR, MAPGR and UGR were confirmed by Fourier-transform infrared spectrometry (FT–IR) Broad adsorption peaks were found at 3342 and 3232 cm−1 due to the N–H and O–H telescopic vibrations, and the peak of the C–H stretching vibration was appeared at 2973 cm−1 Furthermore, the stretching vibration of the C=O group of urea was observed at 1689 cm−1 , and the peaks associated with the C=C group in the maminophenol benzene ring skeleton were observed at 1612, 1493, and 1460 cm−1 The peaks at 1304, 1203, 1161, and 1105 cm−1 were attributed to C–O and C–N tensile vibrations Finally, the signals at 809 and 766 cm−1 were attributed to the out-of-plane bending vibration of C–H in the aromatic ring, respectively These results indicate that the amino and phenolic hydroxyl groups in maminophenol, the carbonyl group in urea, and the hydroxyl group from the reaction of urea and glyoxal were successfully introduced into MAPUGR The surface area and pore characteristics are listed in Table S1 The average pore diameters of MAPUGR, MAPGR, and UGR were ˚ respectively The surface area of MA96.42, 131.54, and 176.86 A, PUGR is 2.02 and 1.67 times that of MAPGR and UGR, respectively The adhesion of MAPGR results in a small specific surface area However, during the synthesis of MAPUGR, the addition of urea effectively prevented the adhesion of MAPGR; therefore, the synthesized MAPUGR had a high specific surface area 3.3 Evaluation of adsorption behavior The adsorption capacity of MAPGR, UGR and MAPUGR for the four PHs was evaluated by adding 2.0 mL of solution (30.0 μg mL−1 of the analytes) to a 10 mL centrifuge tube containing 3.0 mg of adsorbent As shown in Fig 4A, MAPUGR had a higher adsorption capacity for the four PHs than MAPGR and UGR (P < 0.05) This further confirmed that the addition of urea during the preparation procedure enhanced the adsorptive interactions with the analytes and increased the surface area, which improved the adsorption capacity of MAPUGR The experimental steps for evaluating the adsorption kinetics are presented in the Supplementary material As shown in Fig S1, the adsorption capacity of MAPUGR for TDZ, CPPU and BNOA could exceed 45% of the adsorption capacity at adsorption equilibrium, and the adsorption capacity of NAA could exceed 33% of the adsorption capacity at adsorption equilibrium within Therefore, the mass trans- Y Lu, P Li and C Yang et al / Journal of Chromatography A 1623 (2020) 461214 Fig SEM of MAPGR (A), UGR (B), and MAPUGR (C) fer rate of MAPUGR satisfies the requirements for extraction of the four PHs 3.4 Optimization of the MAPUGR−PT-SPE process The molecular forms of the compounds may be affected by the sample pH, which has an effect on the recovery of the four PHs When the sample pH is less than the pKa, the analytes are predominantly in the molecular or protonated form, which facilitates adsorption of the analytes by the adsorbent The pKa values of TDZ, CPPU, NAA, and BNOA were 8.86, 8.40, 4.26, and 4.75, respectively, and pH of the watermelon juice was 5.63; thus, the sample pH was adjusted in the range from 2.0 to 7.0 with M HCl and M NaOH Fig 4B shows that pH had a strong influence on the adsorption of NAA and BNOA (P < 0.05), but had little effect on TDZ and CPPU (P > 0.05) Therefore, the sample pH was optimized to 3.0 The loading volumes (1.0, 1.5, 2.0, 2.5, and 3.0 mL) of the samples were studied Fig 4C shows that the loss rate of the analytes gradually increased (P < 0.05) with increasing loading volume, which indicates that limited adsorption could be achieved with 5.0 mg of the adsorbent, and dynamic adsorption equilibrium was not reached Considering the extraction speed and efficiency, a loading volume of 1.0 mL was used for further work The washing solvent can effectively remove interfering substances Thus, 1.0 mL of water, methanol–water (1:9, v/v), acetonitrile–water (1:9, v/v), acetone–water (1:9, v/v), water–hydrochloric acid (pH 3), and water–acetic acid (pH 3) were studied as the washing agent As shown in Fig 4D, there was almost no loss of the analytes when water–hydrochloric acid (pH 3) or water was used as the washing agent (P > 0.05) Considering the purification effect and cost, 1.0 mL of water was used for the subsequent work Six eluents were studied for elution of the analytes from MAPUGR; the results presented in Fig 4E show that the recovery of four PHs was the highest when methanol was used as eluent (P < 0.05) The effect of different eluent volumes (0.20, 0.40, 0.60, 0.80, 1.0, and 1.2 mL) on the analyte recovery was also investigated As shown in Fig 4F, the optimal recovery was achieved when the eluent volume was 1.0 mL (P > 0.05) 3.5 Comparison with commercial adsorbents A spiked recovery experiment was used to evaluate the extraction efficiency of MAPUGR and seven commercial adsorbents (C18 , HLB, MCX, PSA, SCX, silica gel, and MAX) for the four PHs in watermelon samples The procedure for MAPUGR–PT-SPE was performed as described in the Experimental Section, and the extraction conditions for HLB, MCX, C18 , PSA, SCX, silica, and MAX are described in previous studies [32–35]; the results are presented in Fig and Fig S2 MAPUGR had higher recoveries for the four PHs than the commercial adsorbents (P < 0.05) MCX provided excellent recovery (>80%) because it is a cation exchange and reverse- phase dual-retention mode adsorbent, whereas the anionic adsorbents (MAX, PSA) presented low recoveries for the PHs, indicating that the cation exchange and reverse-phase dual-retention play an important role in the adsorption process In addition, the recovery of SCX, dominated by π –π bonds, was low (