Rapid fabrication of molecularly imprinted polymer fibers for solid phase microextraction of bisphenol A

HU Mei，ZHANG Yijun，YANG Jinghua，，ZHOU Xiaomao，WEI Zhuqing，DING Xiaoqing，ZHANG Yuping*

（1.Institute of Applied Chemistry，Henan Institute of Science and Technology，Xinxiang 453003，China：2.Pesticide Research Institute，Hunan Agricultural University，Changsha 410128，China）

Bisphenol A （BPA）is an additive in epoxy resin and polycarbonate plastic，and is also a known environmental estrogen.It is widely used in the production of foodstuff containers， tableware，baby bottles，dental sealants，etc［1］.Since BPA is a highly lipid soluble substance，it can be easily infiltrated into foods or beverages，and then moved into the human bodies to form a pseudoestrogen.The pseudo-estrogen can cause endocrine disorder，harm human reproductive function，affect endocrine systems and cause adverse effects such as andocrie dyscrasia，precocity and even cancers［2，3］.Generally，the BPA exists in the environment and food at a low concentration.Usually，in order to detect the BPA effectively，a complex sample pre-preparation should be employed.The analytical methods must ensure individual quantitative determination of BPA so as to monitor the accurate concentration of trace BPA in foodstuff and beverage samples.Currently，mass spectrometry （MS）［4］，liquid chromatography-mass spectrometry （LC-MS）［5，6］and high performance liquid chromatography （HPLC）［7－9］are normally used for BPA identification and quantification.

Molecular imprinting is an emerging technology to synthesize the materials with high specific binding sites towards the target molecules.Molecularly imprinted polymers（MIPs）are a class of highly cross-linked polymers that can bind certain target compounds with high selectivity.The polymers are prepared in the presence of the target molecules as the template［10，11］.As a novel artificial affinity medium，MIPs have specific recognition and combining capacity towards the particular template molecules.The key of the technology is the preparation and choice of MIPs.Molecular recognition properties are attracting widespread interest，especially in solid-phase extraction （SPE）.In the last years，many successful applications of MIPs in solid phase extraction have been widely reported in literature.Vicente et al.［12］prepared the BPA-MIPs and packed the polymers in a column which was used for continuous on-column solid phase extraction（MISPE）of aqueous samples.The recoveries were in the range of 92%－101%；and the detection and quantification limits were 1.0 and 3.3 ng／mL，respectively.Baggiani et al.［13］synthesized the MIPs，superporous monolithic hydrogels，in an aqueous medium through a cryotropic gelation technique.The MIPs were used to selectively extract and preconcentrate BPA from river water and red wine samples in a reliable MISPE manner.Zhang et al.［14］successfully prepared BPA-MIPs by precipitation polymerization and used it as the SPE sorbent for the direct extraction of BPA from different biological and environmental samples（such as human serum，pig urine，tap water and shrimp）.The recoveries of the proposed method ranged from 94.03%to 105.3%，with the relative standard deviations（RSDs）lower than 7.9%.Undoubtedly，these methods had good selectivity and high sensitivity，while their pre-treatment processes were time-consuming （2－48 h）and needed large volume of organic solvents.The application of MIPs involving SPME is also a good choice.Jin et al.［15］prepared MIPs which were used for the selective determination of the model mixtures of olivetol，phenol and m-toluidine in lake water and wheat bran samples by SPME procedure.Chen et al.［16］adopted hollow-fiber membrane tube embedded with a MIP monolithic bar to micro-extract the triazine pesticides.

In this study，we have successfully prepared BPA-MIP fibers for the application in SPME based on our previous works［15－17］.The entire synthesis process needs only 7 min by microwave irradiation instead of a constant water bath for many hours.The molar ratios of template／monomer（BPA／α-methacrylic acid）were measured to confirm the most suitable synthesis conditions.Some important factors in the SPME procedures were investigated by evaluating the adsorption efficiency of BPA under different conditions.The selectivity and reproducibility of MIP-SPME were also studied.Finally，the proposed method was applied to the analysis of BPA in mineral water samples.

1 Experimental

1.1 Chemicals and instruments

Bisphenol A （99%），phenol（P，95%）and 4-phenylphenol（PP，99%）were purchased from AlfaAesarCo.， Ltd.， China. Dicyandiamide（DCD）was obtained from Aladdin Industrial Corporation. Ethylene dimethacrylate （EDMA，98%），α-methacrylic acid（MAA）and azodiisobutyronitrile（AIBN）were purchased from Beijing J＆ K Chemical Reagent Company.ACN （99.9%）was purchased from Tianjin Fuchen Chemical ReagentsFactory.Themonomerswerepurified through distillation to remove the inhibitors.The mineral water was purchased from local（Xinxiang，Henan Province）market randomly.Prior to the detection，the mineral water was filtrated and the dissolved carbon dioxide was removed by ultrasonication.Then it was kept in dark for next experiments.The stock solutions of BPA，P，PP and DCD （1.0 mg／L）were prepared by dissolving them into ethanol and kept in a refrigerator at 4℃，separately.The working solutions of the relevant substances were obtained by diluting appropriate amounts of the stock solutions with deionized water.

HPLC analysis was performed on an HP1100 pump system （Agilent Technologies，Inc.，Walbronn，Germany）.TWCL-D Magnetic Stirrer was from Henan Aibote Science and Technology Development Co.， Ltd， Zhengzhou， Henan.KQ-5200DE ultrasonic cleaner was from Kunshan ultrasonic instrument Co.，Ltd，Kunshan，Jiangsu.The irradiation experiments were carried out in a home microwave oven （Midy，China）with a microwave output power of 700 W and a frequency of 2450 Hz.Fourier transform infrared （FTIR）spectroscopic measurements were performed on TENSOR27infraredspectrometer （BRUKER，Germany）.The morphologies of different fibers were studied on a Quanta 200 scanning electronic microscope at an accelerating voltage of 15 kV（FEI， Hillsboro，Oregon，USA）.Fused-silica capillaries （inner diameter of 530 μm）were purchasedfrom Yongnian Photoconductive Fiber Factory （Hebei，China）.

1.2 Preparation of MIP fibers

Fused-silica capillaries about 2 m length was rinsed with 1 mol／L NaOH for 30 min and then with 1 mol／L HCl for 30 min，finally with water for 30 min.After being dried by passage of nitrogen gas，the pretreated capillaries were cut into 7-cm-long pieces.

BPA （0.25 mmol，0.057 g）was dissolved completely into a mixture containing 1 mmol（86 μL）MAA and 1.25 mL ACN to form a homogeneous solution for prearrangement，and ultrasonicated for about 10 min.Then 5 mmol EDMA （943 μL）and 10 mg AIBN （initiator）were added and the mixture solution was again degassed thoroughly by ultrasonication for about 10 min.Afterwards，the mixed solution was injected into the 7-cmlong capillary after degassing with N2stream for 15 min to remove the dissolved oxygen.The two ends of the capillary were sealed with rubbers.Then the capillaries were placed in a home microwave oven to react for 7 min in the microwave with an output power of 700 W and the frequency of 2450 Hz.

Compared to the previous work［18］，the great modification involved in this study was the elution procedure of the template.Instead of etching away the silica wall in 3 mol／L NH4HF2for 9－12 h，the entire monolithic bars in the capillaries were pushed out slowly and carefully.Then the MIP fibers were immersed in 20 mL methanol to elute the template molecules and unreacted compounds with a magnetic stirrer at an agitation of 400 r／min.The procedure was repeated for several times until no template was detected on HPLC.Finally the MIP fibers without silica wall were cut into 2-cm-long pieces and again packed into the 7-cm-long hollow capillaries with 1-cm-long exposed which was used as SPME fibers in the following steps.The greatest benefit of the elution method was to decrease the divulging of the template.Simultaneously，it led to a reduction in time and organic solvent consumptions.For comparison，the non-molecularly imprinted polymer（NIP）fibers were prepared in parallel without the addition of BPA.

1.3 MIP-SPME procedure

A volume of 5 mL of standard aqueous solution was added into a 10 mL vial which contained a 8 mm×3 mm Teflon stirring bar.The 1-cm-long exposure portion of the prepared MIP-SPME fiber was immersed into the standard solution for 30 min at a stirring speed of 400 r／min.Then the fiber was washed in ultrapure water for 2 min to remove the nonspecific interaction.Afterwards，the fiber was desorbed by 200 μL of desorption solvent in a 400 μL test tube for sonication for about 20 min.The obtained desorption solution was filtered by a 0.45 μm filter membrane.Finally，20 μL of the filtrate was injected for HPLC analysis.To avoid contamination，each segment of monolithic fiber was used for only once.

1.4 Sample preparation and chromatographic analysis

Mineral water was used for the evaluation of BPA MIP fibers.Prior to use，the mineral water was degassed by a ultrasonic cleaner for 2 h and then filtrated by suction through a 0.45 μm filter membrane for 3 times.Then the obtained mineral water was stored in a refrigerator at 4℃for the following use.

The chromatographic separation and detection of BPA were performed by an HP1100 Series pump system，equipped with an injector with a 20 μL quantitative tube （Rheodyne 7725i），a C18 column （250 mm×4.6 mm I.D.，5.0 μm），and a detector with a changeable ultraviolet-visible wavelength between 190 and 600 nm（Beijing Cailu Scientific Instrument Ltd.，China）.ACN／H2O（60 ∶40，v／v）was selected as the mobile phase with a flow rate of 0.7 mL／min at room temperature，the injection volume of 20 μL，and the detection wavelength of 218 nm.

2 Results and discussion

2.1 Optimization of preparation formula

In order to investigate the influence on the extraction efficiency，the preparation conditions were optimized carefully.When the molar ratios of template／monomer （BPA／MAA）were varied from 1 ∶1，1 ∶2，1 ∶4，1 ∶6 to 1 ∶8，and the other conditions were kept constant， namely， 0.25 mmol（0.0577 g）BPA，1.25 mL ACN，5 mmol（943 μL）EDMA，and 0.01 g AIBN，we selected the BPA extraction solvent at the concentration of 200 μg ／L to evaluate the preparation of BPA-MIP fibers.The extraction time of 30 min，desorption time by sonication of about 20 min，and stir speed at 400 r／min with room temperature were initially proposed.The extraction efficiencies of the prepared MIP fibers with the different molar ratios of template／monomer were measured through the peak area of the BPA from HPLC.The corresponding values were 67.04 （1∶1），80.99 （1∶2），119.02 （1∶4），98.78 （1∶6）and 47.38 （1∶8）mAU （Fig.1）.The results illustrated that when the molar ratio of template／monomer was 1 ∶4，the prepared MIP fibers possessed the highest adsorption efficiency.BPA and MAA are held together by hydrogen bonds.When the amount of MAA increases properly，the BPA imprinting molecules also increase.Then the cavities which could effectively imprinted the BPA molecules gradually increase，and the self-assembled function between MAA and BPA is more sufficient.Consequently，when themolar ratiobetween MAA and BPA was 1∶4，the adsorption of the MIP fibers attained its maximum performance.Hence，the best molar ratio of template／monomer （1 ∶4）was selected to prepare the MIP fibers throughout the experiments.

2.2 Optimization of extraction procedures

In order to facilitate the rapid mass-transfer for extraction and desorption of the template molecular from the prepared monolithic fibers，some effective parameters including extraction and desorption time，desorption solvent，the concentration of NaCl and pH value were optimized for the SPME procedure.Each standard extraction solution （5 mL）was placed in a 10 mL vial.A micro Teflon magnetic stirrer bar was introduced into the vial before sealing with a silicone-rubber septum cap.During the extraction process，the fiber was directly inserted into the sample solution with a length of 1.0 cm by continuously stirring at a constant speed of 400 r／min.Then the fibers were inserted into a 400 μL test tube and desorbed into a 200 μL of desorption solvent by sonication for about 20 min.

Fig.1 Optimization of the molar ratio of BPA and MAA during the polymerization

The evaluation of the extraction parameters under different conditions was carried out with HPLC peak area which manifested the analyte concentration.Since most of the environmental and biological samples are in aqueous solution and the real sample analysis is conducted in aqueous solution，water was chosen as the extraction solvent.During the whole optimized extraction procedures，200 μg／L BPA standard solution was selected as the extraction solution in the SPME procedures.The factors which can affect the extraction time including the selectivity distribution coefficient and diffusion coefficient of MIP fibers were studied.In this research，the extraction time was selected in the range of 10－70 min with the constant desorption procedure of ultrasonication for 20 min.At the beginning of the extraction process，the extraction efficiency increased rapidly during 10－30 min due to the large mass transfer driving force which was resulted by the difference of solute concentrations between the solid and liquid phases，and the equilibrium for BPA reached at 30 min.The desorption time （5－50 min）was also evaluated with a constant extraction of 30 min and the results demonstrated that the equilibrium for BPA reached at 20 min by ultra-sonication.Therefore，the extraction and desorption yields increased with time and arrived nearly equilibrium at 30 and 20 min，respectively.Extending the extraction or desorption times was no need for the results.Thus，the optimum extraction time of 30 min and the desorption time of 20 min were finally set in the following experiments to ensure high recovery and fast analysis speed.

Several organic solvents including methanol（MeOH），acetonitrile （ACN），MeOH／acetic acid（AA）（90 ∶10，v／v），ACN／AA （95 ∶5，v／v）and water were utilized as desorption solvents to investigate their desorption efficiency.The results indicated that the highest desorption efficiency was obtained with ACN／AA （95 ∶5，v／v）.Adding acetic acid into the desorption solvents could enhance the desorption efficiency.It might be due to that the acetic acid changed the pKavalue of the solution and weakened or destroyed the hydrogen bond between BPA and MIP fibers［19］.The addition of inorganic salt into the sample solution reduced the solubility of the polar organic compound because of the salt-out effect，and enhanced the distribution coefficient thereby improving the adsorption ability toward the analysis component of the MIP fibers.In order to investigate the extraction efficiency of BPA-MIP fibers，the salting-out effect was tested by adding NaCl at 0，50，100，150，200，250 g／L into the standard solution，respectively.When the mass concentration of NaCl was up to 100 g／L，the MIP fibers attained its maximum adsorption performance.When the amount of NaCl was continuously increased，the excessive NaCl would block some of the imprinted cavities and spoiled the imprinting effect.Hence，the mass concentration of NaCl of 100 g／L as selected throughout the experiments.MIP fibers could be damaged when the pH values were too high or too low due to the corrosive action.However，the hydrophobicity of the component changed when the pH value was adjusted，which affected its extraction efficiency.With other parameters kept constant，the pH value of the matrix was varied in the range of 3.0－12.0.The extraction capacity increased corresponding to the increase of pH in the range of 3.0－9.0，whilst the extraction capacity decreased corresponding to the increase of pH in the range of 9.0－12.0.This was probably because that most of the BPA analytes were in molecular state，which was beneficial for the matching with the functional groups of imprinting affinity binding pore when pH value was less than 9.0.Thus the optimum pH value was set at 9.0 throughout the experiments.

2.3 Characterization of the prepared fibers

Scanning electron microscope（SEM）was employed to observe the surface microscopic characteristics of the prepared fibers.The photographs of the morphological structure for the cross and side faces of the prepared fibers under the magnifications of 400 and 300 are shown in Fig.2a and 2b，respectively.MIP monolithic material was intact with a smooth surface after the monolithic polymer bar was removed from the capillary mold.The photographs under the magnification of 104for the MIP fibers in Fig.2c and NIPs in Fig.2d indicate that both fibers possessed a homogeneous and dense porous structure despite distinct differences.The photographs in Fig.2e and Fig.2f show that MIP possessed more pores and larger average pore diameters than NIP.Moreover，more irregular cavities were appeared in the resulted MIP monoliths through which the analytes could reach the inner part of the fibers.The pores of the polymers are usually formed by two methods.（1）Large pores are obtained by the presence of porogenic agent（organic solvent）.（2）Cavities are formed in the imprinting procedure to provide the specificity for their complementarity towards the template molecules［20］.Compared with the NIP fibers，more speckles and bigger cavities were formed in the MIP polymers after the BPA embedded in the framework was removed.The difference between NIP and MIP monoliths was probably attributed to the imprinting effect or the introduction of BPA in the polymerization.Thus，the prepared fiber was a porous cross-linked polymer.A large amount of smaller cavities existed in the large pores on the surface of the polymer.This porous structure provided a good venue and channel forsolute diffusion and massexchange.Therefore，on both the surface and inner part of the MIP fibers，the recognition sites could adsorb the template molecules more effectively［21－23］.

Fig.2 SEM images of MIP and NIP fibers

2.4 Reproducibility of the MIP fibers

The repeatability was investigated in terms of the relative standard deviation（RSD）of the recoveries for 400 μg ／L BPA using fibers prepared in different batches.The RSD of the recoveries using a single fiber（n＝5）under similar conditions was 4.3%.The RSD of fiber-to-fiber extraction efficiency was 7.6% in a single batch.The RSD of batch-to-batch extraction efficiency was 9.2%.To avoid contamination，each MIP-fiber was used only once because of the low cost and easy of preparation.

2.5 Extraction performance of the prepared MIP fibers

The equilibrium adsorption experiments of MIP and NIP fibers were combined with HPLC to determine trace BPA in a series of standard aqueous solutions at different mass concentration levels of 10－1000 μg／L.The extraction capabilities on MIP and NIP fibers are shown in Fig.3.It shows that the adsorption process was very quickly within the initial stage for the MIP fibers，and the adsorption equilibrium was achieved at 400 μg ／L.The adsorption capacities of both MIP and NIP fibers increased with the addition of the initial BPA concentrations.Under the optimal conditions，the maximum chromatographic peak areas of MIP and NIP fibers were 263.3 and 68.5，respectively.It indicated that the MIP fibers displayed much higher affinity （about 4 times）to BPA than NIP fibers.This capacity difference is probably caused by dissimilar extraction mechanisms.For MIP fibers，there are specific recognition cavities formed on them，so the analytes can be caught at the affinity sites via hydrogen bonds.

2.6 Selectivity of MIP and NIP SPME fibers

Fig.3 Adsorption curves of MIP and NIP fibers to BPA

The selectivity of the prepared BPA-MIP monolithic fibers were compared with BPA，its analogues P，PP and non-analogue DCD at the mass concentration of 400 μg／L.PP and P are structure similar compounds to BPA with phenol hydroxyl groups.DCD is a non-analogue substance with totally different molecular structure to BPA.Under the optimal conditions，the extraction amounts of BPA，P，PP and DCD with the MIP and NIP monolithic fibers were studied.As can be seen in Fig.4，the amount of BPA bound on BPA-MIP fibers was much larger than that of the other compounds.The extraction amount of MIP fibers were 3.5，1.8，2.8 and 1.0 times of that of NIPs，accordingly.Compared with the NIP fibers，the MIP fibers possessed better affinity to the template molecule BPA.It indicated that the MIP fibers might selectively extract the template molecule from the complex matrix effectively.

Fig.4 Comparative extraction effects for the target compounds with MIP and NIP fibers

2.7 Validation oftheMIP-SPME-HPLC method

In order to validate the method for the quantification of BPA in water samples，the linearity of the analytical method was estimated by analyzing a serial of BPA，P and PP mixed standard solutions of 10－400 μg／L under the optimized conditions.The calibrations with good linearity were obtained with chromatographic peak area（y）and mass concentration （x，μg／L）.The regression equations and limits of detection （LODs，S／N＝3）were calculated as follows：yBPA＝0.1596x＋7.679（r2＝0.9991，LOD＝0.22 μg／L）；yP＝0.0557x＋9.3921 （r2＝ 0.9635，LOD ＝ 1.07 μg／L）；yPP＝0.1613 x＋4.5056 （r2＝0.9878，LOD ＝0.48 μg／L）.To demonstrate the suitability and the potential application of the established method for sample pre-treatment，the mineral water was processed.No BPA was detected by HPLC after the sample was extracted by MIP fibers.In order to verify the feasibility of the approach，the sample was spiked with three levels of BPA，P and PP at 10，50 and 100 μg／L，respectively.The accuracy and precision of the method were evaluated by the detection of spiked samples in five replicates.Satisfactory accurate results were achieved for all the fortification levels tested with recoveries of 88.4%－102.8%，78.4%－91.6%and 84.9%－94.5%for BPA，P and PP，respectively.The corresponding RSDs were 2.7%－4.1%，5.9%－8.2% and 4.9%－6.2% （n＝5）.Some typical chromatograms are comparatively shown in Fig.5.It should be noted that the baseline of the mineral water sample became flatter and cleaner after pretreatment.Moreover，the peak areas of P，BPA and PP of the spiked samples （100 μg／L）pretreated by NIP and MIP fibers were obviously different.It resulted from the different enrichment abilities of the MIP and NIP fibers.For MIP fibers，the enrichment coefficients were determined experimentally from the ratio of the analytical peak areas with the same concentration after and before extraction.The peak areas of P，BPA and PP from the spiked mineral water after pretreatment by MIP fibers were 3.1，7.8 and 5.8 times accordingly than those of the standard BPA work solution with 100 μg／L.The high recoveries and enrichment coefficients indicated that the established MIP-SPME-HPLC method was successfully applied in the real sample analysis.

3 Conclusions

Fig.5 Comparative HPLC chromatograms in the application of SPME

With a capillary as the mold，the BPA MIP fibers were synthesized by microwave irradiated in situ polymerization.The MIP fibers could selectively recognize and effectively enrich the analyte from mineral water samples.Due to the simplicity and rapidity of the preparation in batch reproducibly，the obtained fibers can be used for disposable purpose to avoid the cross-contamination in different complex sample analysis.The method is sensitive，rapid，and has good repeatability for the determination of the trace BPA in real samples.

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