A novel bifunctional molecularly imprinted polymer for determination of Congo red in food

Abstract

A novel bifunctional molecularly imprinted polymer was synthesized in an aqueous system with Congo red as template, beta-cyclodextrin-maleic anhydride (β-CD-MA) and [2-(methacryloyloxy) ethyl] trimethylammonium chloride (DMC) as co-functional monomers and N,N-methylenebisacryl amide (MBA) as cross-linking agent, respectively. MIP was used as selective sorbent for the solid-phase extraction (SPE) of Congo red from food. And the elution was determined by HPLC. The mean recovery was 84.2–105.2%, the RSD ≤ 1.2%, the repeatability was 1.1–3.8%. The LOD and LOQ were 0.07 and 0.23 μg kg⁻¹, respectively. The evaluation of validation data verified that the MIP has high selectivity and recovery, and it can be applied for the selective extraction and determination of Congo red in food.

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1. Introduction

Molecularly imprinted polymers (MIPs) are a kind of synthetic material with artificially generated binding sites to recognize a target molecule in preference to other compounds with similar structures. Thanks to the obvious advantages of predictable specific recognition, chemical stability, and comparatively cheap and easy preparation,¹ they have been widely developed for a variety of applications including separation,² solid-phase extraction (SPE),³ sensors,⁴ enzyme inhibitors⁵ and antibodies.⁶

The non-covalent approach technique, used to produce MIPs, involves arranging functional monomers around a templating ligand. This ligand is a selected target substance and it should form a prepolymerization complex with the monomer by non-covalent interactions such as hydrogen bonding, ionic or hydrophobic interactions. Hence, the choice of functional monomer is very important to the property of MIPs. The most common functional monomers include methacrylic acid,⁷ vinyl pyridine,⁸ acrylamide,⁹ beta-cyclodextrins (β-CD),¹⁰ organic salt.¹¹ And two of them also could be chosen as co-functional monomers to improve the property of MIPs. The bifunctional molecularly imprinted polymers have different effort and higher selectivity.^12–14

Nowadays, the synthetic colorants are widely used in food and feed because of their low cost and high stability.¹⁵ However, many of the synthetic colorants are genotoxic or carcinogenic or both. The illegal dyes that detected in food samples so far are Sudan I to IV, Para Red, Rhodamine B, Orange II, Acid Red, Sudan Red 7B, Metanil Yellow, Auramine, Congo Red, Butter Yellow, Solvent Red I, Naphthol Yellow, Malachite Green, Leucomalachite Green, Ponceau 3R, Ponceau MX and Orange SS.¹⁶ In recent years, many MIPs were used to detect the synthetic colorants for the special property of MIPs, and got good results.^17–22 But the MIPs for the determination of Congo red were seldom mentioned in references. The Congo red is a synthetic colorant which contains disazo functional groups as shown in Fig. 1. In generally, it was used as indicator and cloth dyeing. However, some merchants added it to food to improve appearance and colour, and this is an illegal act.

Fig. 1 Structural formula.

In this work, beta-cyclodextrins-maleic anhydride (β-CD-MA) and [2-(methacryloyloxy) ethyl] trimethylammonium chloride (DMC) were chose as co-functional monomers, owing to their ability to form inclusion interaction and ionic binding effect with Congo red within the imprinting polymers. The MIPs then were applied to the determination of Congo red in food.

2. Experimental

2.1. Instruments and reagents

Congo red, naphthol green, carmine, sunset yellow, were supplied by Pure Crystal Shanghai Reagent Co. Ltd (Shanghai, China). Beta-cyclodextrin (β-CD) was from Houjin Chemical Co. Ltd (Suzhou, China). [2-(Methacryloyloxy) ethyl] trimethylammonium chloride (DMC), N,N-methylenebisacryl amide (MBA), maleic anhydride (MA) dimethylformamide (DMF) and ammonium persulfate (APS) were from Gaoyu Special Chemical Co. Ltd (Tianjin, China). Methanol, acetic acid, ammonia solution (28.0%) and ammonium acetate were obtained from Guangzhou Chemical Reagent Factory (Guangzhou, China). HPLC-grade methanol and water were purchased from Burdick & Jackson (USA) and Hangzhou Wahaha Group (Zhejiang, China), respectively. The pork sample was purchased from the police and other food samples were purchased from the convenience stores in Xi'an.

The solid phase extraction method (SPE) was developed using an ASE-12 solid phase extraction system from Automatic Science Instrument Co. Ltd (Tianjin China). The spectrums were obtained by Shimadzu UV-2550 spectrophotometer.

High-performance liquid chromatography analysis. Aliquots of 10.0 μL was analyzed on an Agilent 1100 HPLC system (USA), which is consisted with a pump operating at flow rate 1.0 mL min⁻¹, and photodiode-array UC detector with the effluent at 254 nm for all compounds. The separations were carried out on a Germil-pC18 column (150 mm × 4.6 mm, 5 μm) from Wu-Ben Biotechnology Co. Ltd (Xi'an China). The mobile phase was methanol : 20 mM ammonium acetate buffer solution (10 : 90, v/v), and filtered through a 0.45 μm membrane filter, prior to use.

2.2. Synthesis of beta-cyclodextrins-maleic anhydride (β-CD-MA)

According to the literature that had been published,²³ β-CD-MA has been synthesized. 5.68 g of β-CD (0.005 mol) was dissolved in 30.0 mL DMF, and 4.90 g of MA (0.05 mol) was added afterwards. The mixture solution was heated at 80 °C for 10 h under the vigorously stirring. After the reaction was completed, the mixture was allowed to cooling to room temperature, and then, 30.0 mL of trichloromethane was added. A white precipitate obtained was filtrated, and washed at least three times using large amount of acetone, finally, dried in a vacuum oven.

2.3. Synthesis of molecularly imprinted polymers

The method for preparing MIP was shown in Fig. 2. Some β-CD-MA and Congo red were dissolved in water, and stirred for 4 h at a constant temperature, and then DMC was added. After the process of 12 h self-assembly, APS as initiator and MBA as cross-linker were added to the mixture solution. Then, the solution was deoxygenated with nitrogen for 20 min. After that, the reactor was sealed with parafilm and then polymerized at a certain temperature for 24 h. The resulting particles were washed by methanol–ammonia solution in a Soxhlet extraction system until no template molecule could be detected by HPLC. Subsequently, the products were washed with methanol to remove residual ammonia solution and dried. Non-imprinted polymers (NIPs) were prepared with the same procedures as above described and only difference was that no tratarzine was added into the reaction mixture.

Fig. 2 Preparation procedure of molecularly imprinted polymer.

2.4. Preparation of SPE column

The column was packed by wet filling. The dry MIPs (100.0 mg) were added to methanol–water (8 : 2, v/v), stirred to be uniform, and then the suspension of mixed solution was filled into the cartridges at a flow rate of 1.0 mL min⁻¹. The SPE-cartridges filled with MIP were conditioned with 3.0 mL of methanol and 3.0 mL water prior to use.

2.5. Sample pretreatment

Pork, beef, jelly and hawthorn volumes got from the local market were pulverized. 10.0000 g samples were accurately weighed into 50 mL centrifuge tubes, and extracted with 30.0 mL distilled water on an ultrasonic oscillator for about 1 h. The mixture was filtered and the residue was extracted for two times with the same process, and the supernatant was mixed as loading solution. For the recovery test, 10.00 mL 50.0 μg mL⁻¹ Congo red standard was added to the pulverized samples. After overnight of rest, the mixtures were treated with the aforementioned process.

3. Results and discussion

3.1. Preparation of MIP

In order to obtain MIP with good imprinted efficiency, some factors have to be assessed. The inclusion reaction between β-CD-MA and Congo red was investigated by spectrophotometry. The different ratios of β-CD-MA and Congo red (1 : 2, 1 : 1, 2 : 1, 3 : 1) were dissolved in water, and reacted in different temperature (20, 40, 60 °C). The results were shown in Fig. 3 and 4. From the Fig. 3, the absorbance was increased after the inclusion reaction, and the ratio of β-CD-MA and Congo red at 2 : 1 was best. From the Fig. 4, they were prone to react at higher temperature. At last, the ratio of β-CD-MA and Congo red at 1 : 2 and 60 °C was chosen as the reaction condition.

Fig. 3 Absorbance at different ratios of β-CD and Congo red: (a) Congo red solution; (b) 1 : 2; (c) 1 : 1; (d) 2 : 1; (e) 3 : 1.

Fig. 4 Absorbance at different temperature: (a) 20 °C; (b) 40 °C; (c) 60 °C.

Several imprinted polymers were prepared using the functional monomers DMC at template : monomer : cross-linker ratios of 1 : 4 : 10, 1 : 4 : 20, 1 : 4 : 30, 1 : 2 : 20, 1 : 3 : 20. The MIPs (100 mg) were added into the aqueous solution of Congo red (20.0 mg L⁻¹, pH = 4.5), stirred 24 h at room temperature. Then the solution was detected by spectrophotometry, and the adsorbing capacity was shown in Fig. 5. From the Fig. 5, the adsorbing capacity of the MIP of 1 : 4 : 10 was highest.

Fig. 5 Adsorbing capacity at different ratios of template, monomer and cross-linker.

According to the results, the most effective polymers seemed to be those polymerized at 60 °C, with template : β-CD-MA : DMC : cross-linker ratios of 1 : 2 : 4 : 10.

3.2. Characteristics of molecular imprinting polymers

FTIR spectra of Congo red, Congo red-MIP, MIP and NIP are shown in Fig. 6. The absorbance band of COOH at 1724 cm⁻¹ and 953 cm⁻¹ in Congo red-MIP, MIP and NIP spectrum shows β-CD-MA had polymerized to MIP. And the absorption peak of 2950 cm⁻¹ was attributed to methyl groups. The characteristic peaks of benzene ring in the fingerprint region demonstrate that Congo red-MIP has been successfully synthesized.

Fig. 6 FTIR spectra of (a) Congo red; (b) Congo red-MIP; (c) MIP; (d) NIP.

3.3. The adsorption characteristic of MIP

The absorption characteristic was carried out in the Congo red concentration range of 0.1–6.0 mmol L⁻¹, shown in Fig. 7. The binding amount of Congo red on the imprinted polymer was much higher than that on the non-imprinted polymer, displaying the molecular imprinting effect.

Fig. 7 Adsorption isotherm and Langmuir model: (a) MIP; (b) NIP.

The Freundlich isotherm model and Langmuir isotherm model were used to account for the absorption characteristic of MIP.

where q_e (mg g⁻¹) was the amount of adsorbed analyte per unit of polymer mass at equilibrium, and C_e (mg L⁻¹) was the concentration of the analyte in solution at equilibrium, K_f and n were the two Freundlich constants, q_m was maximal amount of adsorbed analyte per unit of polymer mass, K was the Langmuir constant.

Table 1 summarizes the fitting coefficients of the MIP and NIP. The data showed that the absorption conformed to the Langmuir model. The maximal amount of adsorbed analyte of MIP and NIP calculated by Langmuir model were 270.3 and 144.9 mg g⁻¹, which illustrated that good accessibility to Congo red was higher in MIP than that in NIP, demonstrating the imprinting phenomenon.

Table 1 Adsoption model

Type	MIP	NIP	R^2
Freundlich model	log qe = 0.35 log Ce + 1.49	log qe = 0.52 log Ce + 0.37	0.8448
Langmuir model	1/qe = 0.093Ce + 0.0037	1/qe = 4.94Ce + 0.0069	0.9994

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3.4. Optimize MIP-SPE condition

To optimize the pH value of the loading solution, 2.0 mg L⁻¹ Congo red solution were adjusted to different pH values (pH = 4.0, 6.0, 8.0, 10.0) with phosphate buffer. 50.0 mL solutions were percolated through the MIPs-SPE. The recovery in this loading process was determined by HPLC analysis. When the pH of the loading solution was 4.0, 6.0, 8.0 and 10.0, the recovery was 96.9%, 79.2%, 47.6% and 37.3%, respectively. These observations illustrated that the MIPs in the acidic solution could generate higher adsorption than that in alkaline and neutral solutions.

The washing step was performed to minimise the non-specific interactions between polymeric matrices and template. In this study, 10.0 mL different ratios of methanol and water (9 : 1, 7 : 3, 5 : 5, 3 : 7, v/v) were used to wash the cartridge. The experimental results showed that less methanol could not remove all of the polar impurities from the columns while excess methanol could result in the loss of Congo red, and the ratio of methanol and water at 3 : 7 was the optimized condition.

5.0 mL different ratios of methanol : ammonia solutions (9 : 1, 7 : 3, 5 : 5, 3 : 7, v/v) were used to elute solution. The recoveries were 92.3%, 99.2%, 95.1%, 89.7%, separately. Based on these results, 5.0 mL mixed methanol : ammonia solutions of 7 : 3 was employed as elute solution.

3.5. Recovery test

Recovery determination can reflect the extraction efficiency and sample loss during sample preparation. Adding known amounts of Congo red standards in the samples before sample extraction could be used to assess the recovery of Congo red in real samples. Percentage recovery was determined from the amount of Congo red added compared with the amount found. The results of recovery, which were repeated three times of the tested samples, were summarized in Table 2. It was found that the recovery was 84.2–105.2%, the RSD was 0.26–1.2%, and this demonstrated that our sample preparation methodology and MIP-SPE was satisfactory.

Table 2 Recovery test of Congo red for some tested food samples (n = 3)

Food type	Mean Congo red level (μg kg^−1)	Amount of Congo red added (μg kg^−1)	Amount of Congo red found (μg kg^−1)	Recovery (%)	RSD (%)
Pork	13.3	50.0	65.9 ± 0.17	105.2	0.26
Beef	0	50.0	47.0 ± 0.25	94.1	0.53
Jelly	0	50.0	42.1 ± 0.51	84.2	1.2
Hawthorn volumes	0	50.0	45.4 ± 0.42	90.8	0.93

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3.6. Selectivity, limits of detection and quantification, and repeatability

Two sets of 20.0 mL mixed solution of Congo red, sunset yellow, carmine, naphthol green (1.0 mg L⁻¹, pH = 4.5) was treated by MIP-SPE and NIP-SPE, respectively. The recovery was shown in Fig. 8, and this demonstrated that MIP had better selectivity than NIP.

Fig. 8 The average recoveries extracted by MIP-SPE and NIP-SPE.

The limits of detection (LOD) and quantification (LOQ) of SPMIP-HPLC were determined with S/N equal to 3 and 10 respectively. It was found that the LOD and LOQ were 0.07 and 0.23 μg kg⁻¹ respectively. The LOD of the MIP was lower than other MIPs.^11,24

The repeatability of this method, expressed as the RSD (n = 3), was determined by three sets of standard sample. The resulting RSD were from 1.1 to 3.8%.

3.7. The determination of samples

In order to verify the reliability of the MIP-SPE, the determination of Congo red in food samples included pork, beef, jelly and hawthorn volumes were carried on. The results showed that mean Congo red level of the pork was 13.3 μg kg⁻¹, and it was not legal. The chromatogram was shown in Fig. 9. From Fig. 9, the signal of the sample with pretreatment is better than untreated sample, which verify the MIP-SPE can be applied for the selective extraction and determination of Congo red in food.

Fig. 9 Chromatograms: (a) Congo red solution; (b) pork sample untreated with MIP; (c) pork sample extracted by MIP; (d) spiked pork sample extracted by MIP.

4. Conclusions

In this work, a novel bifunctional MIPs using β-CD-MA and [2-(methacryloyloxy) ethyl] trimethylammonium chloride as co-functional monomer were synthesized and utilized as sorbents in SPE to extract Congo red in food. The evaluation of validation data verified that the bifunctional MIP has high selectivity and recovery, and the LOD of the MIP was lower than other MIPs. It can be applied for the selective extraction and determination of Congo red in food.

Acknowledgements

The authors gratefully acknowledge the financial support from Shaanxi Provincial Science and Development projects (2012k08-14).

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