Preparation and evaluation of novel surface molecularly imprinted polymers by sol–gel process for online solid-phase extraction coupled with high performance liquid chromatography to detect trace patulin in fruit derived products

Abstract

A new-type of surface molecularly imprinted polymers (MIPs) for the selective recognition of trace contaminant mycotoxin patulin (PAT) was prepared using oxindole as the dummy template by means of sol–gel polymerization on activated silica beads. Synthesis conditions were optimized by changing some factors to obtain MIPs with controllable adsorption capacity, selectivity and hardness. The prepared MIPs were characterized using Fourier transform infrared spectrometry and scanning electron microscopy, and its adsorption properties were evaluated by static and dynamic adsorption tests. These proved that the prepared MIPs showed excellent affinity, high selectivity adsorption and fast kinetics towards PAT. Then the imprinted material was employed as an online solid-phase extraction (SPE) sorbent for the separation and concentration of PAT in food samples, which was subsequently detected by high performance liquid chromatography (HPLC). The parameters of online MIPs based SPE-HPLC including the pH of loading sample, the loading flow rate and eluting time were optimized in detail. The factor of enrichment and the limit of detection (S/N = 3) of the established MIPs-SPE-HPLC method were 125 and 0.5 μg L⁻¹, respectively. The linear range (r² > 0.990) was 2–40 μg L⁻¹, and the precision of peak area (relative standard deviation, RSD) of nine consecutive enrichments for 2.0 μg L⁻¹ PAT detection was 7.80%. PAT in apple juice, pear juice, haw juice and haw flakes was determined at three spiked levels with recoveries ranging from 60.13 to 97.60%, suggesting the established MIPs-SPE-HPLC method is promising for the accurate quantification of PAT at trace levels in fruit derived samples.

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Introduction

Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, PAT) is a naturally occurring toxic and carcinogenic secondary metabolite produced by a variety of species, particularly Aspergillus, Brysochylamys and Penicillium. It is commonly found in many kinds of food in relatively high amounts, such as in apple, pear and haw.^1–3 PAT has been proved as a nerve poison. The poisoning symptoms include acute symptoms such as agitation, convulsions, edema, ulceration, intestinal inflammation, vomiting, and chronic symptoms of genotoxicity, immunotoxicity, and neurotoxicity while the mechanisms of these poisoning symptoms on humans are not yet clear.^4,5 Due to its harmful effects, a standard limit of PAT has been stipulated and recommended by many organizations. The Food and Agriculture Organization (FAO) has recommended the Hazard Analysis and Critical Control Points (HACCP) system to guarantee the control of PAT in food. The World Health Organization has recommended a maximum tolerable daily intake for PAT should be less than 0.4 μg kg⁻¹ of body weight.⁶ Association of the Industry of Juices and Nectars has realized PAT contamination of food as a serious problem in many countries and recommended that residual PAT level of juice for human consumption should not exceed 50 μg kg⁻¹, the maximum limit of PAT in the solid apple produces is 25 μg kg⁻¹ and the residues of PAT in apple products for infants and young children shall not exceed 10 μg kg⁻¹.^7,8

Up to now, several analytical methods have been reported for detecting and analyzing PAT in fruit products, such as thin-layer chromatography, gas chromatography-mass spectrometry (GC/MS),^9–12 high performance liquid chromatography (HPLC),^13,14 liquid chromatography-mass spectrometry (LC-MS),^15–19 ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)^20,21 and micellar electrokinetic capillary chromatography.^22,23 Among the methods mentioned above, HPLC has been widely applied for the determination of PAT due to relatively cheap instrument, good repeatability, high quantitative precision of the HPLC method.²⁴ However, HPLC with UV detector has poor resolution, which could not discriminate PAT at trace level from other interfering substances, especially 5-hydroxymethyl-furaldehyde (HMF) (Fig. 1) in complex matrix.¹⁵ Therefore, it is necessary to establish an efficient sample pretreatment for the HPLC analysis of PAT in food products.

Fig. 1 Molecular structures of patulin, oxindole and 5-hydroxymethyl-furaldehyde.

Solid-phase extraction (SPE) was the most widely used and well-established sample pretreatment technique, and some SPE methods using traditional sorbents for extraction of PAT from juice have been reported.^25–30 However, these traditional SPE is usually nonspecific for the target. Additional time and pretreatment steps are required to remove the matrix interference.³¹ Molecularly imprinted polymers (MIPs), as a kind of functional material with specific absorption towards target, displays good target selectivity, enrichment capability and chemical stability, which makes MIPs as an ideal adsorbent materials for SPE.³² In recent years, the MIPs based SPE have been developed and used for the matrix purification of mycotoxins, such as ochratoxin A,^33–36 deoxynivalenol,³⁷ moniliformin³⁸ and zearalenone.³⁹

However, the MIPs synthesized using traditional methods exhibit low mass transfer and poor site accessibility to the analytes. The kinetics of the sorption/desorption process is unfavorable. To overcome the drawbacks above, materials with large surface area and high porosity are used as support matrices for the preparation of MIPs by surface imprinting polymerization.^40,41

Due to high toxicity and expense of PAT, PAT-MIPs were generally prepared using oxindole, 6-hydroxynicotinic, and 3-hydroxyphtalic anhydride as dummy template.^42–47 Zhao et al. prepared new MIPs on silica beads using the radical “grafting from” polymerization method for selective extraction of minor contaminant mycotoxin of PAT.⁴⁸ Khorrami et al. described novel MIPs for PAT using oxindole as a dummy template. The MIPs was prepared in a non-covalent approach with methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross-linker via free radical polymerization.⁴²

In this study, a new-type of MIPs was prepared via sol–gel surface imprinting method on the surface of activated silica gel, using oxindole as dummy template (Fig. 1), 3-aminopropyltriethoxysilane as the functional monomer and tetraethoxysilicane as the crosslinker. The adsorption capacity and the selectivity specific of the prepared MIPs were evaluated. The prepared MIPs was applied as efficient and selective separation materials of SPE, coupled with HPLC to establish an online MIPs based SEP-HPLC method for the determination of PAT in apple juice, pear juice, haw juice, haw flakes. The factors affecting preconcentration and separation of the analytes were also discussed in detail.

Experimental

Materials and reagents

Template PAT (99.9%) and 5-hydroxymethyl-furaldehyde (99.9%) were obtained from Tianjin Bichenglan Biological Technology Co., Ltd. Dummy template oxindole (98%) was purchased from Sigma-Aldrich Chemicals (Madrid, Spain). Silica gel (80–120 mesh) obtained from Qingdao Ocean Chemical Co., Ltd. (Qingdao, China) was used as support material to prepare the surface-imprinted functionalized polymer. 3-Aminopropyltriethoxysilane (APTES) and tetraethoxysilicane (TEOS) (Hubei Wuhan University Silicone New Material Co., Ltd., Wuhan, China) were used as functional monomer and crosslinker. Acetonitrile, methanol, acetic acid and other chemicals used in this experiment were purchased from Concord Co., Ltd. (Tianjin, China) and at least of analytical grade. Water for experiment was purified in a Milli-Q ultrapure water system (Billerica, USA). Apple juice, pear juice, haw juice, haw flakes were purchased from a local market. Standard solutions of PAT were prepared in acetonitrile and stored at −20 °C.

Instrumentation and analytical conditions

HPLC system consisted of LC-10ATVP pump and SPD-20A ultraviolet detector (Shimadzu, Kyoto, Japan). All separations were achieved on a C₁₈ reverse-phase column (250 mm × 4.6 mm, 5 μm, Thermo) at a mobile flow rate of 1.0 mL min⁻¹ at 30 °C. The mobile-phase of HPLC was aqueous solution containing 0.1% (v : v) acetic acid containing of 10% (v : v) acetonitrile. Class-VP software from Shimadzu was used to acquire and process spectral and chromatographic data. A model FIA-3100 flow injection system (Beijing Titan Instruments Co., Ltd.) was applied for online SPE preconcentration. Tygon pump tubes were used for delivering the sample solution. Small bore (0.5 mm i.d) PTFE tubes were adapted for all collections, which were kept at the shortest possible length to minimize the dead volume. Fourier transform infrared (FT-IR) spectra (4000–400 cm⁻¹) in KBr were recorded using a Nicolet 380 (Thermo Electron Corporation, USA).

Activation of silica gel carrier

The silica gel surface was activated by refluxing 8.0 g of silica gel (80–120 mesh) with 60 mL of 33% (v : v) methane sulfonic acid aqueous solution under magnetic stirring for 8 h in a 250 mL three-necked flask. Then, the activated silica gel carrier were filtered, repeatedly washed with DDW to neutral pH and dried under vacuum at 70 °C for 8 h.

Molecularly imprinted sol–gel polymers synthesis

Dummy temple oxindole (133.15 mg) was dissolved in methanol (5 mL) at 35 °C in a 25 mL glass round-bottom flask and 936 μL of APTES was then added. After magnetically stirred for 15 min, 0.1 g of activated silica gel was added and the mixture was magnetically stirred for 50 min. Subsequently, 1115 μL of TEOS and 1000 μL of 0.1 mol L⁻¹ NH₃·H₂O solution were added to the prepolymer solution in turn. The copolymerization was carried out at 60 °C for 12 h in a water bath.

The polymer obtained was filtrated, flushed with methanol to remove unreacted oxindole and aged in a vacuum oven at 70 °C for 10 h. Then the resultant was washed with a mixture of methanol and 1.0 mol L⁻¹ HCl (v : v, 1 : 1) under magnetic stirring for 4 h at room temperature. After filtering, the polymers were washed with methanol, sodium hydroxide solution (0.1 mol L⁻¹) and doubly deionized water (DDW) in turn and the eluent were adjusted to pH = 7. Then the imprinted material were extracted with a mixture of methanol and acetic acid (v : v, 9 : 1) for 25 h using the Soxhlet technique. After they were washed and dried in vacuum oven at 60 °C, the MIPs particles were stored at room temperature until use.

For comparison, non-imprinted polymers (NIPs) were prepared and treated in an identical method, but without the addition of template during polymerization process.

Static and dynamic adsorption test

In order to validate the binding property of the MIPs, the static and dynamic adsorption tests were employed in this study. The solvent used during static and dynamic adsorption tests was acetate buffer solution (0.02 mol L⁻¹, pH = 4). The static adsorption test was conducted as following procedure: 5 mg of MIPs or NIPs was encased in a 3 mL centrifuge tube and 1 mL of the PAT of solution (concentration varying from 2 to 50 mg L⁻¹) was added. The tube was mechanically shaken for 2.5 h at room temperature and then separated centrifugally (5000 rpm) for 15 min. At last, the concentration of PAT after adsorption in the tube was measured by HPLC. The adsorption capacity (Q, mg g⁻¹) of the MIPs or NIPs for PAT was calculated according to the equation:
where C_i and C_f is the initial and the equilibrium concentration of PAT solution, respectively, and V is the volume of solution (mL), w is the weight of the MIPs or NIPs (g).

Dynamic adsorption tests was examined through shaking the mixture of PAT solution (20 mg L⁻¹) and MIPs (50 mg) for different time (10–180 min) at room temperature.

Competitive adsorption

To validate the selectivity of the MIPs, the most confusing interfering substance HMF was chosen as the competitor of PAT in the selective recognition studies. 5 mg MIPs were added to 1 mL of mixed solution containing PAT and HMF (20 mg L⁻¹). After magnetically stirred for 3 h at room temperature, the supernatant were measured and determined at 276.0 nm by HPLC after centrifugation (5000 rpm, 15 min). The NIPs and the activated silica gel were tested in the identical way.

The selective specificity of these materials (MIPs, NIPs and activated silica gel) could be generally estimated by the distribution coefficient (K_d), selectivity coefficient (K), and relative selectivity coefficient (K′) acquired from these competitive binding experiments. K_d, K, and K′ were calculated using the following equations:

where C_i and C_f represents the initial and final concentration of analytes, respectively.

Procedures of online MIPs-SPE-HPLC

A cylindrically shaped microcolumn (1.5 cm × 4 mm i.d.) packed with 50 mg MIPs as sorbent was used to evaluate the application of the MIPs for online MIPs-SPE-HPLC determination of trace PAT in food. The 20 μL injection loop was replaced by the prepared SPE microcolumn. The SPE microcolumn was washed with DDW before loading samples. The process of the online SPE preconcentration coupled with HPLC for determination of PAT was as follows: first, 50 mL of the PAT solution was injected into the SPE microcolumn at a flow rate of 2.0 mL min⁻¹ while the HPLC injector valve was on the load position, so that the PAT was adsorbed onto the MIPs in the SPE microcolumn and the unwanted solution was sent to the waste. Second, the analytes adsorbed in the SPE microcolumn were eluted in the back-flush mode by the HPLC mobile phase at a flow rate of 1.0 mL min⁻¹ into the chromatographic separation column for 2.0 min by switching the HPLC value from “load” to “inject”. As such, the sample band in the microcolumn was compressed into a narrow band before entering the analytical column and the band broaden effect was reduced. Third, the HPLC valve was turned to the load position for the next sample solution concentration while the analytes were separated in the chromatographic separation column. The corresponding chromatograms were recorded and stored in the computer. Areas at 276.0 nm were calculated and used for data evaluation.

Analysis of PAT in the spiked sample

Apple juice, pear juice, haw juice and haw flakes samples were obtained from a local market and spiking and recovery studies were used to evaluate the application capability of the developed method.

For juice samples, 15 mL of sample was accurately measured into a beaker, and spiked with PAT at three levels (20, 40, 60 μg L⁻¹). After overnight of rest in the dark, ethyl acetate (50 mL) was added. After magnetically stirred for 5 min and ultrasonic extracted for 5 min, the supernatant were collected after quiescence. The above process was then repeated twice, and the supernatants were combined. Then the supernatant was dried using rotary evaporator at 35 °C. Finally, the residue was dissolved with 1 mL acetonitrile and diluted to the scale of a 50 mL brown volumetric flask with acetate buffer solution (pH = 4) for online SPE enriching.

For the solid samples, the haw flakes was crushed firstly by high-speed mixer and then went through 80 mesh sieve to obtain uniform particles of haw flakes powder samples. 10 g of sample was accurately measured into a beaker and spiked with PAT at three levels (20, 40, 60 μg kg⁻¹). After overnight of rest in the dark, 15 mL DDW was added, the mixture was homogenized and ethyl acetate (50 mL) was added. After magnetically stirred for 5 min and ultrasonically extracted for 5 min, the supernatant was filtered using a Buchner funnel. The following process was the same with the juice samples.

Results and discussion

Synthesis method and recognition mechanism of the MIPs

In this study silica gel was selected as the support material, which contains siloxane groups (Si–O–Si) in the bulk and silanol groups (Si–OH) on its surface. The silanol groups are responsible for chemical modifications that may occur on the silica surface. In fact, commercial silica gel has less active Si–OH on the surface that cannot meet the requirement of the chemical modification. Thus, methanesulfonic acid (33%) was chosen as silica activator to increase the amount of silanol groups on account of its strong catalytic action, high boiling point, and its ability to be used repeatedly.

The possible preparation process of the MIPs and recognition mechanism of the MIPs were shown in Fig. 2. Under the condition of solvent, dummy template oxindole combined with functional monomer in the form of noncovalent hydrogen bond to form a complex. In the role of catalyst, hydrolysis silane groups of the complex highly crosslinked with tetraethoxysilane silicon on the surface of activated silicon gel. After elution process, cavities that matched the template in space structure and binding sites were obtained in the polymers.

Fig. 2 The preparation process of the molecularly imprinted sol–gel polymers.

Optimization of the MIPs preparation conditions

To realize the ideal selectivity of the MIPs, some key variables during the synthesis process of the MIPs were optimized, such as reaction solvent, the choice of the dummy template molecule, functional monomer, crosslinking agent, molar ratio of the reagents, catalyst and elution conditions.

The reaction solvent is an important factor which affects the formation of hydrogen. To prepare the MIPs, methanol with low polarity was selected as the proper reaction solvent due to its weak effect on the hydrogen bond and good ability of solubilizing the template molecules, functional monomer, and crosslinking agent.

Due to the presence of –NH₂ groups in the structure, APTES was selected as the monomer combining with the template to form noncovalent bonding. Prior to adding crosslinker TEOS, the activated silica gel was added as the support to achieve surface imprinting. After TEOS was added, the MIPs was synthesized by copolymerization in the role of catalyst. Generally, the molar ratios of the involved components influence the adsorption performance and imprinting effect of MIPs toward the template molecule. Changing the ratio of the dummy temple, the functional monomer APTES and the crosslinker TEOS (1 : 2 : 2, 1 : 2 : 4, 1 : 2 : 6, 1 : 4 : 5, 1 : 4 : 12, 1 : 3 : 4) caused a dramatically alteration in the adsorption capacity. When the molar ratio of oxindole/APTES/TEOS was 1 : 4 : 5, the polymers had the highest adsorption capacity for template. NH₃·H₂O and acetic acid were often used as catalyst during the synthesis of sol–gel MIPs, so a series of concentration and dosage of catalyst were tested. NH₃·H₂O (0.1 mol L⁻¹, 1 mL) was chosen as the optimal catalyst. The resulting imprinting material was washed repeatedly with a mixture of HCl (1.0 mol L⁻¹) and methanol to remove template in the polymers. The residual templates in the product were extracted with CH₃OH in a Soxhlet extractor. After removal of the temple molecules, the specific imprinted polymers will be maintained with tailormade cavities containing functional binding sites for the analyte.

SEM and FT-IR spectra characterization of the MIPs

SEM was employed to observe morphologies of the activated silica gel (a) and synthesized polymers (NIPs (b) and MIPs (c)). As shown in Fig. 3, the MIPs and NIPs possessed pore structure while activated silica gel had a relatively smooth surface, suggesting that synthesized polymers had been successfully coated on the surface of the silica gel. Compared with NIPs, the pore structure of MIPs was more obvious, which may result in different adsorption ability and dynamics.

Fig. 3 SEM of silica gel (A), NIPs (B) and MIPs (C).

To further ascertain occurrence of the intending synthetic reaction, FT-IR spectra of the activated silica gel (a), NIPs (b) and MIPs (c) were compared (Fig. 4). As shown in Fig. 4, a range of characteristic peaks of the Si–O–Si group (1101 cm⁻¹), Si–O–H group (974 cm⁻¹) and Si–O vibrations (805 cm⁻¹ and 470 cm⁻¹) were observed (curve a). The absorbance peak of the N–H around 2940 cm⁻¹, the C–H group around 1559 cm⁻¹, and the CH₂–N peak at 1422 cm⁻¹ of NIPs and MIPs were appeared after polymerization (curve b and c), indicating that the formation of chemical bond between the functionalized silica gel and the APTES which were not simply physically mixed. Furthermore, the FT-IR spectra of MIPs and NIPs had similar locations and appearances of the major bonds, indicating that the template molecules were mainly removed from the cavities of imprinted polymers after the elution process.

Fig. 4 FT-IR spectra of the silica gel (A), NIPs (B) and MIPs (C).

Evaluation of adsorption properties

In order to compare the recognition ability of MIPs and NIPs towards PAT, the static equilibrium adsorption experiments were measured by varying the initial concentrations of PAT from 2 mg L⁻¹ to 50 mg L⁻¹. As shown in Fig. 5, the adsorptions of MIPs and NIPs towards PAT increased with the increment of the initial concentration. The equilibrium adsorptive capacity of the MIPs were larger than that of NIPs at each concentration of PAT, and the adsorption capacity of MIPs (2.81 mg g⁻¹) was approximately five times larger than that of NIPs (0.546 mg g⁻¹) at the concentration of 40 mg g⁻¹. These results proved that the MIPs owned more cavities and binding sites for PAT rebinding than NIPs, which had strong absorption effect towards PAT.

Fig. 5 Adsorption isotherms of MIPs and NIPs towards PAT.

The dynamic adsorption test was carried out at different times from 10 to 180 min at the concentration of 20 mg L⁻¹, and the results were shown in Fig. 6. This high affinity of MIPs was demonstrated by dynamic uptake plots in which the MIPs always adsorbed larger amount of template than that of NIPs. More than 90.6% of binding was completed within 30 min, the rebinding rate slowed down after 30 min. The MIPs quickly reached adsorption equilibrium within 30 min and it resulted in a faster adsorption rate when the initial concentration of PAT was lower than 20 mg L⁻¹ PAT. The results indicated that the MIPs held fast adsorption rate, which was mainly due to the effect of surface imprinting. This is an obvious advantage of the silica gel surface imprinting polymer materials to be used as sorbent in the online SPE to detect PAT residues in food. This means that the surface imprinted polymers facilitate diffusion of PAT to the binding sites.

Fig. 6 Dynamic uptake plots of MIPs and NIPs towards PAT.

Specific adsorption experiments of the MIPs, NIPs and the activated silica gel was evaluated with PAT and its structurally related compound (HMF) in acetate buffer solution at pH = 4. The molecular selectivity of the polymers was valued by the static adsorption distribution coefficient (K_d), selectivity coefficient (K), and relative selectivity coefficient (K′). As shown in Table 1, the adsorption capacity of NIPs and activated silica gel towards any of the target analytes were relatively low, which because there were non-specific adsorption and no imprinted sites formed in the NIPs and activated silica gel. The MIPs offered higher selectivity towards PAT and the selectivity coefficient of MIPs was about 6.6 times than that of NIPs, which were attributed to the specific binding sites with suitable shape and functional groups in MIPs.

Table 1 The selectivity of MIPs, NIPs and activated silica gel for two relative structure matters

Parameters	Analytes	MIPs	NIPs	Activated silica gel
Loading capacity (mg g^−1)	PAT	1.657	0.978	0.1
	HMF	0.406	0.529	0.359
Kd	PAT	502.4	25.47	1.06
	HMF	98.02	32.56	21.88
K	PAT	5.13	0.78	0.048
K′	HMF	6.58

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The online MIPs-SPE-HPLC parameter optimization

In order to achieve good detection results of trace PAT in food using online SPE-HPLC method, various paraments, such as the pH of loading solution, the sample loading flow rates and eluting time were evaluated and optimized.

In general, the pH value of the sample solution is vital for the online SPE enrichment. In this study, we selected 50 mL of PAT acetate buffer solution at 2.0 μg L⁻¹ as sample solution at a loading flow rate of 1.5 mL min⁻¹ with the pH changing from 2.5 to 8.0. The maximum peak of PAT was obtained when pH was 4.0. Thus pH 4.0 was used as optimal pH in further experiments. Sample flow rate, another important factor affecting the detection result of online SPE-HPLC, was optimized using 50 mL of pre-concentration solution (2.0 μg L⁻¹) with different loading flow rate (1.5–3.5 mL min⁻¹). The result indicated that different flow rate had little effect on the chromatographic peak area of PAT. Considering the column pressure and column efficiency, 1.5 mL min⁻¹ was applied for the further studies. For simplicity, the HPLC mobile phase was used to elute the adsorbed PAT from the MIPs based SPE column. The time required for desorption of the adsorbed PAT when the HPLC injector valve was in the “inject” position was evaluated in order to determine when the HPLC injector valve should turn to the load position for the next online solid-phase extraction during the HPLC separation of the analytes. As the desorption time increased, the chromatographic peak area of PAT increased rapidly from 0 to 2.0 min, and then leveled off when elution time exceeded 2.0 min. Accordingly, 2.0 min was selected as the optimal desorption time.

The application and evaluation of online MIPs-SPE-HPLC

In order to validate the performance of the established MIPs-SPE-HPLC method for the sensitive and selective determination of PAT, the analysis parameters such as enrichment factor, linearity, limit of detection, and repeatability were evaluated under optimal experimental conditions. The enrichment factor was 125 compared with the direct injection of a 10 μL standard solution. The linear range of the calibration graph was 2–40 μg L⁻¹ and the limit of detection (S/N = 3) for PAT was 0.5 μg L⁻¹. The relative standard deviation of nine replicate extractions for 2.0 μg L⁻¹ PAT detection was below 7.80%.

To demonstrate the applicability and reliability of the developed MIPs-SPE-HPLC method, food samples (apple juice, pear juice, haw juice and haw flakes) obtained from a local market were determined using spiking and recovery studies. The chromatograms of four samples were given in Fig. 7–10. Those samples were determined free of PAT. As was shown in Table 2, the recoveries of PAT with three spiked levels (20 μg L⁻¹ kg⁻¹, 40 μg L⁻¹ kg⁻¹, and 60 μg L⁻¹ kg⁻¹) were ranging from 60.13–97.6%. These results demonstrated the applicability and reliability of the developed method for determination of PAT in the real samples.

Fig. 7 Chromatogram of apple juice sample (a) and spiked with 20 μg L⁻¹ PAT (b).

Fig. 8 Chromatogram of pear juice sample (a) and spiked with 20 μg L⁻¹ PAT (b).

Fig. 9 Chromatogram of haw juice sample (a) and spiked with 20 μg L⁻¹ PAT (b).

Fig. 10 Chromatogram of haw flakes sample (a) and spiked with 20 μg kg⁻¹ PAT (b).

Table 2 Results of MIPs based SPE-HPLC measurements of PAT content in real samples (n = 3)

Samples	Spiked (μg kg^−1/μg L^−1)	Recovery (%)	RSD (%)
Apple juice	20	83.50	3.50
	40	97.20	4.50
	60	97.60	5.40
Pear juice	20	94.20	5.00
	40	92.96	3.80
	60	92.37	4.90
Haw juice	20	63.12	7.30
	40	75.75	5.40
	60	63.86	7.50
Haw flakes	20	60.13	6.50
	40	70.75	5.80
	60	63.42	7.80

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Conclusions

We have successfully prepared a surface imprinted functionalized silica gel sorbent with an excellent selective adsorption to PAT. The sorbent have been successfully applied to online SPE coupled with HPLC to determine the spiked apple juice, pear juice, haw juice and haw flakes with the acceptable recovery for PAT, suggesting the established MIPs based SPE-HPLC method is promising for the accurate quantification of PAT at trace levels in fruit derived samples.

Acknowledgements

This work was supported by the Ministry of Science and Technology of China (project No. 2012AA101602) and Bei-jing Municipal Commission of Science and Technology, China (project No. Z151100001215002) and the PhD Training Foundation of Tianjin University of Science and Technology (project No. 201401 and No. 201501).

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Footnote

† These authors contributed equally to this work.

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