Determination of Benzimidazoles in Chicken and Egg Samples by Urea-Based Covalent Organic Polymer (UCOP) Pipette Tip–Solid-Phase Extraction (PT–SPE) and High-Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS)

Abstract A urea-based covalent organic polymer (UCOP), was synthesized using 1,4-phenylene diisocyanate (PPDI) and 4,4′,4″-(1,3,5-triazine-2,4,6-triyl) trianiline (TTTA) and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and nitrogen adsorption-desorption isotherm analysis. The prepared material was employed as an adsorbent for pipette tip–solid-phase extraction (PT–SPE) for benzimidazoles before high-performance liquid chromatography–tandem mass spectrometry analysis (HPLC–MS). The parameters affecting the isolation efficiency, including salt concentration, sample pH, mass of adsorbent, and types and volumes of eluent, were optimized. Using these conditions, PT–SPE provided good linearity from 0.1 to 10 μg kg−1 with low limits of detections (LODs) of 0.02 μg kg−1 together with good precision (relative standard deviations from 4.2 to 8.3%) and accuracy (recoveries from 76.2 to 84.8%). The results demonstrate that the urea-based covalent organic polymer has the potential for the enrichment of trace benzimidazoles.


Introduction
Benzimidazoles are broad-spectrum anthelmintics that have been widely used for the prevention and treatment of endoparasite infections in animals (Reuter et al. 2000).With the intensive breeding of livestock and poultry, frequent outbreaks of parasitic disease result in the massive use of benzimidazoles, including albendazole (ABZ), fenbendazole (FBZ), and oxfendazole (OFZ).However, their residues in the food may cause malformation, diarrhea, and anemia in humans.To avoid health problems, the establishment of sensitive methods to determine benzimidazole and their main metabolites at low levels in food is necessary.
Several methods have been published for the determination of benzimidazoles in environmental and food samples, including spectrophotometry (Soto et al. 2010), spectrofluorimetry (K€ uc ¸€ ukkolbas ¸ı, G€ und€ uz, and Kılıc ¸2008), capillary zone electrophoresis (Hu et al. 2012), and liquid chromatography with mass spectrometry (LC-MS) (Chen et al. 2011;Robert et al. 2015;Sun et al. 2016), ultraviolet (Santaladchaiyakit, Srijaranai, and Burakham 2014;Zhang, Huang, and Yuan 2015), or fluorescence detection (Batzias, Theodosiadou, and Delis 2004).Due to low concentrations of the drugs and the complex matrices, it is difficult to perform drug determination by direct LC.Thus, a separation and preconcentration step is required prior to analysis.
Several preparation methods including liquid-liquid extraction (LLE) (Whelan et al. 2010), solid-phase extraction (SPE) (Sun et al. 2016), solid-phase microextraction (Zhang, Huang, and Yuan 2015), and liquid-phase microextraction (LPME) (Santaladchaiyakit and Srijaranai 2013;Vichapong et al. 2015) have been used.However, traditional sample pretreatment methods are time-consuming as well as labor-intensive.In addition, LLE needs large volumes of organic solvents.To overcome these limitations, sample pretreatment has moved toward miniaturization.Pipette tip-solid-phase extraction (PT-SPE) offers short extraction periods, as well as reduced consumption of adsorbents and organic solvents (Seidi et al. 2019).The performance of PT-SPE depends upon the adsorbent; therefore, it is crucial to develop new materials with improved properties (Turo nov a, Kujovsk a Kr cmov a, and Svec 2021).
Covalent organic polymers (COPs) are endowed with high surface areas, exceptionally high thermal stability, tunable pore structures, and multifunctionality with excellent adsorption abilities.However, most synthetic methodologies need harsh conditions, such as high temperature (Zhong et al. 2017;Zheng et al. 2018), inert atmosphere (Liu et al. 2019), or a precious metal catalyst (Peng et al. 2019;Liao et al. 2018;Huang et al. 2022), which limit the applications.Therefore it is desirable to develop effective and mild synthetic methods for the preparation of COPs.A urea-based covalent organic polymer (UCOP) was synthesized by the polymerization of 1,4-phenylene diisocyanate and 1,3,5-tris(4-aminophenyl) benzene under mild conditions (Li et al. 2021) for the isolation of fluoroquinolones in food.
In this study, the urea-based covalent organic polymer was employed as the adsorbent for benzimidazoles and their metabolites from chicken and egg samples using pipette tip-solid-phase extraction.The pH, ionic strength, and eluent were optimized and the methodology was employed for the determination of benzimidazoles in chicken and eggs.
Scanning electron microscopy (SEM) images were obtained using a field-emission instrument (Quanta 650, FEI, Hillsboro, OR, USA) at 20 kV.Fourier transform infrared spectra (FT-IR) were recorded on a Perkin Elmer spectrometer as KBr pellets.Powder X-ray diffraction (PXRD) measurements were collected on an X'pert diffractometer (Malvern Panalytical, Netherlands) with Cu K a radiation.Nitrogen sorption studies were carried out using a Quadrasorb 2MP (Kantar, USA) specific surface and aperture analyzer.The thermogravimetric analysis (TGA) was conducted from 30 to 800 C in the air (Mettler Toledo TGA/DSC 1, Switzerland).
The MS triple quadrupole detector operated in positive electrospray ionization mode (ESI þ ) with a capillary voltage of 3 kV with multiple reaction monitoring (MRM).The gas temperature was 300 C, desolvation gas flow rate was 11 L min À1 , and nebulizer gas pressure was 30 psi.The specific MS/MS conditions and LC retention times for the analytes are shown in Table S1.

Sample collection and pretreatment
Chicken and eggs were purchased from a local supermarket (Nanchang, China).Briefly, the chicken was chopped and homogenized with a blender.The eggs were directly homogenized by the blender.Five grams of homogenized samples were spiked with three concentrations of mixed standard solutions, followed by vortex mixing for 1 min.The samples were subsequently treated with 10 mL acetonitrile and 5 g of anhydrous sodium sulfate and sonicated for 30 min.The mixture was centrifuged (10,000 r min À1 ) for 5 min at room temperature.The supernatant was collected and transferred to another centrifuge tube.The residual sample was extracted again with 10 mL acetonitrile.The supernatants were combined and concentrated to dryness by a rotary evaporator at 40 C. The residues were re-dissolved in 5.0 mL of 0.1 mol L À1 pH 7 phosphate buffer containing 2.5% (m/v) NaCl for further purification.

PT-SPE procedure
The homemade PT-SPE cartridge has been previously described (Yuan et al. 2020).The PT-SPE device was made using a pipette tip (200 mL) with 3.0 mg of urea-based covalent organic polymer as the adsorbent.Degreased cotton was placed at both ends of the pipette tip to avoid the loss of adsorbent.After the PT-SPE cartridge was preconditioned by methanol (2.0 mL) and deionized water (2.0 mL), 5.0 mL of phosphate buffer was loaded on the cartridge followed by deionized water (2.0 mL) as the washing solution and 1% acetic acid methanol (2.0 mL) as eluent.The effluent was concentrated to dryness under a gentle nitrogen flow to obtain the residue that was dissolved in 1.0 mL of 2:8 methanol-water and passed through a 0.22 lm organic membrane before the injection of 10.0 lL of extract into the LC-MS for analysis.

Characterization of adsorbent
Figure 2a shows the characteristic peaks for 4,4 0 ,4 00 -(1,3,5-triazine-2,4,6-triyl) trianiline were from 3000 to 3500 cm À1 (N-H).A peak at 1695 cm À1 in 1,4-phenylene diisocyanate was due to the C ¼ O stretching vibration.The peaks at 1663 cm À1 (C ¼ O) and 3325 cm À1 (N-H) indicate the formation of the urea moiety.The XRD pattern of the urea-based covalent organic polymer showed diffraction peaks at 25 and 41 (Figure 2b), which suggests that the material is amorphous.Thermogravimetric analysis (Figure S1) shows there was a larger mass loss before 100 C due to the removal of water, and further heating showed obvious weight loss up to 300 C, indicating weak thermal stability.
Scanning electron microscopy (Figure 2c) of the urea-based covalent organic polymer shows irregular small particles.From Figure 2d, the BET surface area was determined to be 67.5 m 2 g À1 .The total pore volume was evaluated to be 0.21 cm 3 g À1 by the Barrett-Joyner-Halenda (BJH) approach.In addition, the pore size distributions from 1.50 to 1.96 nm suggest the presence of microporous and mesoporous components in the urea-based covalent organic polymer (Figure S2) with an average pore diameter equal to 9.25 nm.These results demonstrate that the urea-based covalent organic polymer was a macroporous material.

Mass of sorbent
To appraise the influence of sorbent mass upon the extraction efficiency, masses of sorbent from 1.0 to 5.0 mg were added to the pipette tip.The recoveries of the benzimidazoles increased from 1.0 to 3.0 mg and reached a plateau.Therefore, 3.0 mg of the urea-based covalent organic polymer was deemed to be optimum.High recoveries of benzimidazoles were achieved by using a small quantity of sorbent due to the too many adsorption sites and the strong interactions with the benzimidazoles.

pH
The pH plays an important role in extraction efficiency as it influences the ionization of the analytes and the surface charge of the adsorbent.When the pH is <5, the imidazole ring moiety is protonated and benzimidazoles are present as water-soluble cations which result in poor adsorption.From pH 5 to 9, benzimidazoles are primarily in the zwitterionic form which is more hydrophobic, so high extraction efficiency is obtained.Lastly, at pH values exceeding 9, the urea groups of the adsorbent are unstable, which affects the interaction with the benzimidazoles.
In this study, the pH was varied from 3.0 to 11.0.Figure 3 shows the highest benzimidazole adsorption was observed at pH 7.0 due to strong hydrogen bonding and p-p non-covalent interactions between the analytes and urea-based covalent organic polymer.

Ionic strength
To investigate the influence of ionic strength upon the benzimidazoles, various concentrations of NaCl (0-15%, m/v) were incorporated into the pre-concentration process.Figure 4 shows that the highest response was obtained using 2.5% (m/v) NaCl which was employed in subsequent experiments.

Desorption conditions
Several eluents, including methanol, acetonitrile, 5% (v/v) ammonia-methanol, 1% (v/v) acetic acid-methanol, ethyl acetate, and acetone were investigated using the same conditions.The highest efficiency was achieved with 1% (v/v) acetic acid-methanol as the eluent (Figure 5) that was employed in subsequent measurements.Furthermore, the influence of the elution volume from 1.0 to 5.0 mL on the efficiency was also investigated.The results showed that all analytes were completely desorbed by 2.0 mL of 1% (v/v) acetic acid-methanol.

Reusability
To investigate the reusability of the procedure, each pipette tip was rinsed with 3 mL of 1% (v/v) acetic acid-methanol twice before each subsequent application After five recycling periods, there was no change in the analyte recoveries.The results demonstrate suitable reusability with no analyte carryover.

Method validation
The proposed method was evaluated in terms of linearity, limit of detection (LOD), limits of quantification (LOQ), accuracy, and precision under the optimized conditions.The linearity of the working curve was studied by spiking a series of standard solutions in chicken and egg samples from 0.1 to 10.0 lg kg À1 for all benzimidazoles.Good linear ranges from 0.1 to 10.0 lg kg À1 were obtained for all with correlation coefficients (R 2 ) from 0.9964 to 0.9986.The limits of detection and quantification were calculated using the baseline noise method at signal-to-noise ratios (S/N) of 3 and 10, respectively.The values for the benzimidazoles were from 0.02 to 0.1 lg kg À1 , respectively.The limits of quantification were lower than the maximum residue limits (MRLs) established by the European Union, indicating that the developed procedure was sufficiently sensitive for the determination of the benzimidazoles in food.
The precision was estimated by the intra-and inter-day relative standard deviation (RSD), which were obtained by analyses of six replicates in one day and on six contiguous days, respectively.The intra-day relative standard deviations were from 4.2 to 8.3%, and the inter-day assay values were between 4.9 and 9.0%, demonstrating satisfactory precision.
The accuracy was accessed through the spiking recovery experiments on chicken and egg samples at 0.5, 1.0, and 2.0 lg kg À1 .The recoveries of benzimidazoles in Table 1 ranged from 76.2 to 84.8% with standard deviations between 4.2 and 8.3%.The results demonstrate satisfactory recovery and precision for the determination of trace benzimidazoles in chicken and eggs.

Real samples analysis
The validated method was employed for the determination of benzimidazoles in 50 chicken and 60 egg samples.Albendazole and its major metabolites were detected in nine chicken samples and five eggs at levels from 1.8 to 228.2 lg kg À1 .Figure S3 shows no interfering peaks in the MRM chromatogram for a positive egg sample following PT-SPE, indicating the purification protocol was satisfactory.

Comparison with other methods
The developed method was compared with the literature for the determination of benzimidazoles (Table 2).The developed procedure had low limits of detection and high recoveries compared to most previous protocols.In addition, the reported approach used less adsorbent that was easily prepared to employ mild conditions.

Conclusions
A sensitive, economical, and miniaturized self-assembled PT-SPE coupled with LC-MS/MS detection was developed for the rapid determination of benzimidazoles and their metabolites in chicken and egg samples.A porous urea-based covalent organic polymer was prepared using mild conditions and employed for a novel PT-SPE cartridge with high adsorption capacity for benzimidazoles due to the large surface area and structure of the urea-based covalent organic polymer.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 1 .
Figure 1.Synthesis of the urea-based covalent organic polymer.

Table 1 .
Spiked recoveries of benzimidazoles in chicken and eggs.

Table 2 .
Comparison of the reported work to the literature for the determination of benzimidazoles in food.