Cyromazine imprinted polymers for selective stir bar sorptive extraction of melamine in animal feed and milk samples.

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DOI: 10.1039/C5AN00325C

Cyromazine imprinted polymer for selective stir bar sorptive extraction of melamine in animal feed and milk samples Wenying Fan, Mingqi Gao, Man He, Beibei Chen, Bin Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P R China

ABSTRACT: In this work, a molecularly imprinted polymers (MIPs) coated stir bar was prepared by using a self-designed polytetrafluoroethylene (PTFE) mold and in situ polymerization, with cyromazine as the dummy template for target melamine. The prepared MIP coated stir bar presented uniform and porous surface as well as good chemical stability and selectivity for melamine. Based on it, a method of MIP coated stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography-ultraviolet

detection

(HPLC-UV)

was

developed

for

the

quantification of melamine in food samples. The significant factors affecting the extraction efficiency of melamine by MIP-SBSE, such as extraction solvent and time, stirring rate, desorption solvent and time, were investigated thoroughly. Under the optimal conditions, the analytical performance of this method was evaluated. The detection limit of the developed method was 0.54 µg L-1 for melamine with an enrichment factor of 42-fold and the relative standard deviation (RSD) of 6.1% (c = 5 µg L-1, n = 7), and the linear range was 2-200 µg L-1. The established method was applied for the determination of melamine in a variety of real samples including cat food, dog food, chicken feed A, chicken feed B and milk powder, and the recoveries for melamine in the spiked samples were in the range of 76.2-98.2%, 80.0-85.5%, 89.5-113%, 85.0-95.5% and 65.0-111%, respectively. The proposed method presented good specific recognition ability and matrix interference resistance, and was demonstrated to be effective and sensitive for the analysis of melamine in animal food and milk samples.

*

Corresponding author, tel: 86-27-68752162; fax: 86-27-68754067, email: [email protected] (B. Hu)


Published on 30 March 2015. Downloaded by Imperial College London Library on 06/04/2015 21:44:39.

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DOI: 10.1039/C5AN00325C

KEYWORDS: molecularly imprinted polymers; stir bar sorptive extraction; melamine; dummy template; polytetrafluoroethylene mold

1. Introduction Melamine (1,3,4-triazine-2,4,6-triamine, MEL) is a typical triazine compound widely used in the production of melamine resins, which are used in the manufacture of plastics, laminates, glues, flame retardants and so on.1-3 Melamine has low oral acute toxicity but their excessive exposure to animals would cause renal stones and kidney stones.4,5 In recent years, melamine with high nitrogen content (66.7%) has been found to be used illicitly as food additive to artificially increase the apparent protein levels of dairy products, which are generally quantified by Kjeldahl method based on the measurement of total nitrogen content.6 Subsequently, melamine monitoring has been becoming a focus of international concern,7 and the Codex Alimentarius Commission has stipulated that the safety limit standard of melamine in baby formulas is 1 mg kg-1, 2.5 mg kg-1 in other food or animal feed. Therefore, reliable and sensitive methods are needed to determine melamine residues in food, particularly in dairy products for children, which is of biological, clinical and food industrial importance. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS),8,9 HPLC-diode array detection (DAD)10,11 and gas chromatography (GC)-MS12 have been widely employed for the analysis of melamine in different samples. Owing to the complexity of sample matrix and the low levels of melamine, sample pretreatment techniques are crucial for the separation and enrichment of melamine prior to instrument detection. Nowadays, solid phase extraction (SPE) is a widely used technique for preconcentration and clean-up in the analysis of melamine.13-18 Molecularly imprinted polymers (MIPs) are stable polymers with selective molecular recognition abilities, provided by the template used during their synthesis.19 So far, MIPs have been widely used as molecular recognition media in SPE-based methodologies for melamine



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analysis. Yang et al.20 prepared melamine imprinted polymers through bulk polymerization method. After polymerization, the polymers were ground with a mortar and pestle, and sieved. The obtained imprinted polymers (37 µm and 74 µm) showed high affinity to melamine and was successfully applied as special SPE sorbent for selective extraction of melamine from dairy products. Other similar preparation methods21,22 of MIP-SPE for selective extraction of melamine in complex samples also have been proposed in recent years. MIP microspheres with melamine as template were prepared by precipitation polymerization23,24. The MIPs synthesized by precipitation polymerization with melamine as template showed better separation and enrichment characteristics for melamine analysis compared with that prepared by bulk polymerization. Cheng et al.18 prepared a highly selective molecularly imprinted layer-coated silica gel (MIP@SiO2) for melamine by surface molecular imprinting technique. Characterization and performance tests of the obtained products revealed that MIP@SiO2 not only exhibited excellent selectivity to the target molecule melamine compared with non-imprinted polymer layer-coated silica gel (NIP@SiO2), but also displayed superior adsorption capacity to bulk MIP due to the molecular recognition sites on the surface of silica gel. A well documented drawback of MIPs is the residual template leaching or bleeding that may occur even after extensive washing.25 The special recognition sites of MIP are based on the combination of templates and functional monomers. With the addition of cross-linking agent, initiator and porogen, a three-dimensional polymer network is formed after polymerization. The removal of templates generates the specific recognition sites complementary in shape, size and chemical functionality to the template molecules. The incomplete removal of template will influence the method accuracy by using target analyte as template. To prevent the bleeding problem, the application of an analogue of the target analyte as template is a good alternative, with which the bleeding of the template does not interfere in the quantification of the target analyte. The dummy MIP with specific recognition sites to dummy template also has the ability to recognize target analyte which has similar properties to dummy template. Cyromazine was reported to be an ideal dummy template for melamine because its chemical structure is very similar to



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that of melamine and it is more soluble than melamine in polar solvent (e.g. methanol, acetonitrile).13,22,26 Traditional sample preparation techniques such as liquid-liquid extraction and SPE consume large amounts of organic solvent and often require complex time-consuming multi-step procedures which can lead to low accuracy, contamination and losses of analytes. Modern trends in analytical chemistry are toward the simplification, miniaturization and low-consumption of solvent/sample. Stir bar sorptive extraction (SBSE) is a novel environmentally friendly microextraction technique which was developed from solid phase microextraction (SPME) in 1999.27 Due to its advantages of simplicity, rapidity, strong ability of sample clean-up, high extraction efficiency, SBSE has been successfully applied in environmental, food and biological samples for pharmaceutical analysis. The selectivity and extraction efficiency of SBSE is mainly determined by the stir bar coatings. An ideal stir bar coating should be capable of enriching the target molecules with high concentration factors, whilst leaving other interfering substances in the sample matrix. The use of MIPs for stir bar coatings can improve the selective extraction capability for target analytes and decrease the interference of sample matrix. Nowadays, MIPs have also been successfully applied in SBSE as a special coating. MIP coated stir bars were first reported by Zhu and coworkers.28 They applied the phase-inversion imprinting technique to prepare the MIP coated stir bar with nylon-6 polymer solution. Compared with the conventional coating (non MIP coating) stir bars, MIP coated stir bars showed better sensitivity and higher enrichment capability. In the phase inversion imprinting system, the MIPs were casted from the polymer solutions of the template molecule rather than polymerized from monomers. However, lack of suitable polymer with high strength nature and desired functionality restricted wide application of this technique. Li’s group29-31 developed a synthetic method for linking the MIP to the stir bar by chemical bonding, which was realized through silylation of the substrate surface and then multiple co-polymerization reactions. The MIP coated stir bars were successfully applied to the determination of β2-agonists in animal-derived food samples, triazine herbicides in agricultural products, triazole fungicides in soil and sulfa drugs in food samples,



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respectively. By using the same MIP synthetic technique, the MIP-based SBSE methods were developed for the analysis of 2-aminothiazoline-4-carboxylic acid in forensic urine32 and sulfonylurea herbicide in water and soil,33 respectively. The MIP coating chemically bonded to the glass bar was homogeneous and porous and showed good mechanical and chemical stability. Gomez-Caballero et al.34 designed a chiral MIP-SBSE device for selectively extracting (S)-citalopram from a racemic mixture in an aqueous media. The prepared MIP coated stir bar was proved to be significant for enantiospecific sample pre-concentration and subsequent analysis without the need for any chiral chromatographic separation. To prevent the template bleeding problem, Sheng et al.35 and Zhan et al.36 prepared dummy MIP coated stir bars for bisphenol A (BPA) analysis by using 3,3’,5,5’-tetrabromobisphenol A (TBBPA) as dummy template molecule. Using dummy MIP instead of MIP avoided inaccurate determination of BPA due to the leakage of BPA remaining in MIP. In this work, an MIP coated stir bar was prepared by chemically bonding the MIP to the glass bar and an in situ polymerization reaction, with cyromazine as dummy template for the target melamine, avoiding the template bleeding problem. Based on it, a method of SBSE-HPLC-UV was developed for the analysis of melamine in animal food and milk samples, which presented good specific recognition ability and matrix interference resistance.

2. Experimental 2.1 Instrumentation An Agilent 1100 HPLC (Agilent Technologies, Waldbronn, Germany), equipped with a degassing device, a quaternary pump, a 100 µL sample loop, and an ultraviolet detector, was employed for the analysis of melamine and its metabolites. A Hypersil CN column (150 mm × 4.6 mm, 5 µm, Elite, Dalian, China) was employed for the analysis of melamine and the selectivity study of MIP coated stir bar. A mixture of methanol and 10 mmol L-1 ammonium acetate/acetic acid (pH 3) at a volume ratio of 35:65 was employed for the analysis of target melamine with the mobile phase at a flow rate of 0.7 mL min-1. Detection was performed with a variable wavelength UV



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detector at 234 nm. For selectivity experiment, the mobile phase for the separation of melamine, ammeline and cyanuric acid was a mixture of methanol and 5 mmol L-1 NaH2PO4·2H2O (pH 5) at a volume ratio of 35:65 and the flow rate was set as 1 mL min-1. Detection was performed with a variable wavelength UV-visible detector at 220 nm. The injection volume was 50 µL through the whole HPLC-UV analysis. The 85-2A constant temperature magnetic stirrer (Ronghua Instrument Factory, China) and SY 1200-T ultrasonic processor (Shengyuan Instrument Factory, China) were used for SBSE procedures. Mettler Toledo 320-S pH meter (Mettler Toledo Instruments Co. Ltd., China) was used to adjust the pH value of mobile phase. HH-6 Digits Show Thermostat Water Bath Tank (Changzhou Aohua Instrument Co., Ltd., China) was used for the polymerization reaction. Quanta 200 scanning electron microscope (FEI Nanoport, Japan), iS10 Fourier transform infrared spectrometer (Varian Medical Systems, America) and Setsys 16 TG/DAT/DSC thermal analyzer (Setaram Technologies, France) were employed for the characterization of home-made SBSE coating.

2.2 Standard solutions and reagents Melamine was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Cyromazine and ammeline were obtained from Adamas Reagent, Ltd. Cyanuric acid was obtained from Aladdin Reagent Database Inc. (Shanghai, China). The structure, pKa and logP of cyromazine, melamine and its metabolites are all listed in Table 1. Each standard solution of analyte was prepared in methanol at 100 mg L-1. Working standard solutions were prepared by diluting the standard solution with methanol to the required concentration. All standard stock solutions were kept in refrigerator at 4 oC away from light. Methacrylic acid (MAA) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Azodiisobutyronitrile (AIBN) was obtained from Shanghai No. 4 Reagent & H. V. Chemical Co., Ltd. (Shanghai, China). Ethylene glycol dimethyl acrylate (EGDMA) was obtained from Aladdin Reagent Database Inc. (Shanghai, China). γ-(Methacryloxypropyl)trimethoxysilane (KH-570) was obtained from WD



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Silicone Co., Ltd. (Wuhan, China). Acetonitrile (CH3CN), methanol (CH3OH), ethanol (CH3CH2OH), acetic acid (CH3COOH), acetone (CH3COCH3), sodium hydroxide (NaOH), hydrochloric acid (HCl) were all analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). High purity deionized water was purified by the Milli-Q water purification system (18.2 MΩ·cm, Millipore, Molsheim, France). The capillary glass bars (1.0 mm I.D., 0.10 mm wall thickness) were purchased from Apparatus Factory of West China University of Medical Sciences (Sichuan, China).

2.3 Sample preparation Milk powder, chicken feeds A and B with different ingredients, dog food and cat food were all purchased from local supermarket (Wuhan, China). The procedure of sample preparation was referred to Yang’s work10. The samples were ground into powder and then dried under the infrared lamp. 2.0 g sample powder was spiked with 15 mL methanol, mixed by vortex for 1 min. After ultrasonication for 30 min, the mixture was centrifuged for 10 min at a 7000 rpm. The supernatant was transferred to another tube. The powder was extracted again under the same conditions. The two supernatants were combined together and diluted with methanol to 40 mL for SBSE procedure. For recovery test, the standard solution of melamine was spiked into the sample powder, followed by the same procedure as described above.

2.4 Preparation of MIP coated stir bar A PTFE mold was designed as a vessel for polymerization reaction. The PTFE mold was composed of three parts, bottom cap, body and top cap. There is a hole in the centre of the top cap. The body size was adjustable to control the thickness and length of MIP coating. The selected body size of PTFE mold was 2.5 mm I.D., 2 cm length. With the O.D. of 1.1 mm for the employed bare stir bar, the thickness of obtained MIP coating was calculated to be about 700 µm. The structure of PTFE mold was shown in Figure 1. A capillary glass bar (1mm I.D. and 0.1 mm wall thickness) was cut into 60 mm



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long and sealed by alcohol flame at one end. Then the other end was sealed, and a spherical bubbled end (1.5-2.0 mm I.D.) was obtained because the air inside the glass bar was heated and expanded. The bare bars were cleaned with pure water and dichloromethane. Then they were treated with 1 mol L-1 NaOH for 8 h and 1 mol L-1 HCl for 2 h, respectively. The pretreated bars were washed several times to remove the residual moisture and dried with a stream of nitrogen. In order to immobilize the organic polymers on the bare bar to obtain extraction phase with low bleeding, good repeatability and long lifetime, KH-570 was chosen as a bridge agent to chemically combine the polymer coating and glass substrate. One end of KH-570 could react with hydroxyl group exposed on the glass surface through open-ring bonded reaction, while the double bond of KH-570 could participate in the co-polymerization of the MIP coating. Thus, the prepared bars were soaked into the mixture of KH-570 and acetone at the volume ratio of 1:3 for 1.5 h. Then the bars were taken out, washed with methanol and dried with a stream of nitrogen for further use. The pre-polymer solution for MIPs preparation was prepared in a vial by dissolving 8.3 mg cyromazine and 0.034 mL MAA in 1.5 mL acetonitrile. The mixture was completely dissolved by ultrasonication and then shaked on a shaking bed for 10 h at room temperature. Then 0.226 mL EGDMA and 6 mg AIBN were added into the pre-polymer solution. Subsequently the reaction solution was injected into a PTFE mold (2.5 mm I.D.) with a pretreated glass bar vertically placed in the center of the mold. The system was sealed and deoxygenized with a stream of nitrogen for 5 min. The polymerization was then maintained at 60 oC for 24 h. After polymerization, the MIP coated stir bar was pulled out and aged at 60 oC for 8 h. Then the end without spherical bulb of MIP coated bar was cut off and an iron wire (17 mm length) was inserted into the glass bar. The open side was sealed with alcohol flame and another spherical bubbled end was formed. A dumbbell shaped stir bar was obtained by sintering the two ends of glass capillary into bulbs. The preparation sketch of MIP coated sitr bar with PTFE mold are also shown in Figure 1. The template molecules were removed from the coating by soaking the obtained



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MIP coated stir bar in 2 mL of 10% (v/v) ammonia in methanol under ultrasonication for 1 h. The washing procedure was repeated several times until the concentration of template cyromazine was under the detection limit of HPLC-UV (20 ng mL-1). Then the MIP coated stir bar was cleaned by 10% (v/v) acetic acid in methanol and pure methanol before SBSE procedure. The non-imprinted polymer (NIP) coated stir bar was also prepared following the same procedure except for the addition of cyromazine.

2.5 SBSE procedures The extraction procedure was carried out in a 25 mL vial containing 10 mL sample solution with a MIP coated stir bar. The vial was sealed by vial cap and placed on the 85-2A constant temperature magnetic stir. After extraction for 180 min with a stirring rate of 600 rpm, the stir bar was taken out from the sample solution, washed with methanol and then gently put into a desorption vial. The stir bar was desorbed in 400 µL desorption solution of 8% (v/v) ammonia in methanol by ultrasonication for 20 min. Then the desorption solution was volatilized to dryness and diluted with methanol to 80 µL for subsequent HPLC-UV analysis. The MIP coated stir bar could be regenerated by ultrasonicating in 2 mL of 10% (v/v) acetic acid in methanol for 15 min and 2 mL methanol for 5 min in turn after each SBSE procedure.

3. Results and discussion 3.1 The optimization of MIP coating preparation For MIP coating preparation, cyromazine, acetonitrile, MAA, EGDMA, AIBN and KH-570 were selected as dummy template, porogen, functional monomer, cross-linking agent, initiator and bridging agent, respectively. The preparation scheme is illustrated in Figure S1. The volume of porogen directly impacted the morphology and mechanical property of MIP coatings. The effect of the volume of acetonitrile (0.9, 1.1, 1.3, 1.5, 1.7 and 1.9 mL) was investigated on the preparation of MIP coatings. It was found that the



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MIP coatings polymerized with 1.5 mL acetonitrile possessed best mechanical property and uniform surface morphology. MIP coatings polymerized with acetonitrile volume less than 1.5 mL were easy to fall off during SBSE procedure and the lifetime of MIP coated stir bar was relatively short. MIP coatings polymerized with acetonitrile volume more than 1.5 mL exhibited hard and crack surface. Finally, 1.5 mL acetonitrile was selected in polymerization reaction to form MIP coated stir bar. To improve the binding sites and recognition capacity, the molar ratio of MAA, EGDMA and cyromazine was also optimized by orthogonal test, and the results were presented in Figure S2. As can be seen, the MIP coated stir bars prepared by MAA, EGDMA and cyromazine with a molar ratio about 1:8:24 presented the best extraction efficiency to melamine. To remove the template quickly and efficiency, ultrasonication was adopted. The effect of acetic acid/methanol (1/9, v/v) and ammonia/methanol (1/9, v/v) was investigated on the elution of cyromazine template by breaking the hydrogen bonds between cyromazine template and functional monomer. Ammonia/methanol (1/9, v/v) presented better elution effect than acetic acid/methanol (1/9, v/v). By using 2 mL ammonia/methanol (1/9, v/v) under ultrasonication for 1 h for the elution of cyromazine template, the washing times were investigated, and it was found that the concentration of cyromazine could be decreased to below 20 ng mL-1 (the detection limit of HPLC-UV) after washing 5 times. Finally, the cyromazine template was eluted by 2 mL ammonia/methanol (1/9, v/v) under ultrasonication for 1 h for 5 times.

3.2 Characterization of the MIP coated stir bar Figure 2 shows the SEM images of MIP coating before and after template elution, and NIP coating at different magnification. As could be seen, MIP coating before elution presented a homogeneous dense surface with no obvious pore structure. The MIP coating after elution presented more pore structure than MIP coating before elution. This structure could promote the mass transfer of target analytes between MIP coated stir bar and sample solution. The NIP coating also presented pore structure, but



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the pore size was relatively smaller than that of MIP coating after template elution. The FT-IR spectra of MIP coating before and after template elution and NIP coating are shown in Figure 3. In the FT-IR spectra of NIP coating, several absorption peaks are observed, which are attributed to the stretching vibration peak of O-H bonds (3441 cm-1), C=O bonds (1729 cm-1), C=C (1635 cm-1), C-O bonds (1260 cm-1), C-O-C bonds (1151 cm-1), and bending vibration peak of O-H bonds (1455 cm-1), C-H bonds (1384 cm-1), respectively. These characteristic absorption peaks of NIP coating proved that the in situ polymerization reaction was successful. Compared with the FT-IR spectra of NIP coating, the only difference of the MIP coating before elution is the appearance of peak at 1578 cm-1, which is the stretching vibration peak of C=N bond attributed by cyromazine template. The appearance of C=N bond proved that a complex was successfully formed by the cyromazine template and functional monomer via non-covalent interaction. The FT-IR spectra of MIP coating after elution are similar to that of NIP coating, due to the removal of cyromazine template by template elution. The solvent resistance property of the MIP stir bar coating was investigated by using chloroform, toluene, n-hexane, ethanol, acetone, acetonitrile, methanol, water, acetic acid/methanol and ammonia/methanol, respectively. The prepared MIP coated stir bars were soaked into the above solvent respectively for 24 h, then taken out and cleaned with water. The treated MIP coated stir bars all kept the uniform integrity and their extraction performance was further investigated. The obtained results showed no obvious difference between the treated and untreated MIP coated stir bar on the extraction efficiency for melamine, indicating that the self-prepared MIP coated stir bars possessed good solvent resistance property.

3.3 Extraction selectivity of the MIP coated stir bar To study the recognition characteristics of the MIP coated stir bar, melamine and its metabolites (cyanuric acid, ammeline) of melamine were extracted with MIP coated stir bar and NIP coated stir bar. The chromatograms obtained by HPLC-UV were shown in Figure S3. As we can see, the MIP coated stir bar imprinted by cyromazine



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template presented good selectivity for melamine while there was no definite difference on extraction efficiency of melamine and its two metabolites by NIP coated stir bar. The selectivity factors of MIP coated stir bars for melamine, cyanuric acid and ammeline were 8.9, 0.69 and 2.2, respectively, which was calculated by the extraction efficiency ratio of MIP coated stir bar to NIP coated stir bar on the SBSE of melamine. This difference of extraction selectivity was probably caused by different extraction mechanism of MIP and NIP coated stir bar. For MIP coated stir bar, specific adsorption of melamine on the created imprinted sites contributes to higher extraction efficiency. Though the molecular structures of cyanuric acid and ammeline are familiar to melamine, the interaction between hydroxyl groups and carboxyl groups is relatively weaker than that between amino groups and carboxyl groups. Besides, the matching pore size and recognition sites of MIP coating also can improve the extraction efficiency and selectivity of melamine by MIP-SBSE. Thus, the MIP coated stir bar presented better selectivity to melamine than its analogues. For NIP coated stir bar, the non-specific adsorption is dominant.

3.4 Preparation reproducibility and lifetime of the MIP coated stir bar The preparation reproducibility of MIP coated stir bar was evaluated, and an acceptable reproducibility for the preparation of MIP coated stir bar was obtained, with RSDs of 5.0% for bar-to-bar (n = 7, c = 100 µg L-1) and 8.9% for batch-to-batch (n = 7, c = 100 µg L-1), respectively. The lifetime of the self-prepared MIP coated stir bar was investigated by repeating the extraction, desorption and regeneration process. It was found that the extraction performance for melamine decreased when the MIP coated stir bar was used more than 13 times because the MIP coating wore out during the extraction procedure. Thus, the MIP coated stir bar could be reused for 13 times.

3.5 Optimization of SBSE parameters To obtain high extraction efficiency for melamine by MIP coated stir bars, the factors influencing SBSE, such as sample solution, stirring rate, extraction time,



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desorption solution and desorption time were studied in detail. The recognition of MIP coating to melamine is based on hydrogen bonds, which means the polarity of sample solution has a significant impact on extraction performance for melamine. The influence of six solutions, including water, methanol, methanol/water, acetonitrile, methanol/acetonitrile and ethanol, on extraction of melamine by MIP coated stir bar and NIP coated stir bar was shown in Figure S4. And the selectivity factors of melamine on MIP coated stir bars obtained in different solutions, are shown in Table 2. As can be seen, the extraction selectivity of MIP coated stir bars for melamine in methanol and methanol/acetonitrile are the best among these six solutions. Considering that acetonitrile is more toxic and expensive than methanol, we finally chose methanol as sample solvent. The effect of stirring rate on the extraction efficiency of melamine was studied within the range of 200-700 rpm. The experimental results in Figure S5 indicated that the extraction efficiency of melamine increased with the increase of stirring rate from 200 to 600 rpm and then leveled off with further increase of stirring rate to 700 rpm. Considering the higher stirring rate might cause damage to the stir bar coating during the SBSE procedure, stirring rate of 600 rpm was selected in the following work. The effect of extraction time on the extraction efficiency of melamine was studied with the extraction time varying from 30 to 300 min. It is found in Figure S6 that the extraction efficiency of melamine increased with increasing the extraction time from 30 to 180 min, and the kept almost constant with further increase of the extraction time to 300 min. The long extraction equilibrium time was due to the thick MIP coating. Finally extraction time of 180 min was adopted in this work. Liquid desorption (LD) was employed in this study to desorb melamine from the MIP coated stir bar. Four solvents (acetonitrile, methanol, 10% acetic acid/methanol, 10% ammonia/methanol) were investigated as desorption solvents to break the hydrogen bonds between melamine and MAA, and the results are shown in Figure 4. As can be seen, 10% ammonia/methanol exhibited the best desorption for melamine. As the selective extraction is based on the interaction between amino groups of melamine and carboxyl groups of MAA, alkali aqueous would destroy the interaction



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between melamine and MAA. Therefore, the mixture of ammonia and methanol was adopted for desorption, and the content of ammonia in methanol was optimized in the range of 0-10%. It is found that melamine signal reached the maximum when the content of ammonia in methanol was higher than 6% (Figure S7). Considering that the desorption solution would be volatilized to dry and dissolved with methanol to 80 µL for HPLC-UV detection, and high concentration of ammonia decreased the speed of volatility, we finally selected 8% ammonia/methanol as desorption solution. After desorption process, the MIP coated stir bar was regenerated by ultrasonication in 10% acetic acid/methanol for 5 min before next use to remove melamine residues, counteract ammonia residues and active the recognition sites of the stir bar coating. The effect of desorption time within 5-30 min on the desorption efficiency was investigated, and the results are shown in Figure S8. As can be seen, the desorption efficiency for melamine increased with the increase of desorption time from 5 to 15 min, and then kept almost constant with further increase of desorption time to 30 min. Thus, desorption time of 20 min was adopted in this work.

3.6 Analytical performance Under the optimal conditions of SBSE, the analytical performance of the proposed method of SBSE-HPLC-UV with MIP coated stir bar for melamine analysis was evaluated and the results are shown in Table 3. The proposed MIP-SBSE-HPLC-UV procedure provided a linear range of 2-200 µg L-1 for melamine with R2 of 0.9962. The EF (theoretical EF was 125), calculated as the ratio of slope of the calibration curve obtained with and without SBSE, was 42 for melamine. The extraction efficiency (EE) of proposed method, calculated as the ratio of real EF and theoretical EF, was 34% for melamine. The LODs, calculated as the concentration of melamine that produced a signal-to-noise ratio (S/N) of 3 obtained by UV detection, was about 0.54 µg L-1. The RSDs (n=7) for melamine at 5 µg L-1 was 6.1%. Table 4 is the comparison of analytical performance obtained by this method and other SPE-based methods for the determination of melamine. The LODs of our proposed method is lower than those reported in References10,20 which involved no



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DOI: 10.1039/C5AN00325C

enrichment, and is comparable to some research works13,37. Cyromazine was used as the dummy template to prevent the template bleeding problem in the present method and References13,22,26. It should be noted that the MIP coating in this work was chemically bonded to the glass bar by in situ polymerization reaction, avoiding crushing, grinding and sieving applied in some works10,13,20,22,26. The lifetime of MIP coating is comparable to a series of reported information10,26,37 and much shorter than that in Reference17, in which suspension polymerization was applied to improve the stability and the lifetime of the prepared molecularly imprinted microspheres. The combination of MIPs with SBSE provided a sample pretreatment technique with high selectivity and enrichment for melamine as well as easy operation. It can be expected that the combination of the prepared MIP-SBSE with HPLC-MS will be a more accurate and efficient method for melamine analysis.

3.7 Sample analysis Under the optimized conditions, the developed method of SBSE-HPLC-UV with MIP coated stir bar was applied for the determination of melamine in cat food, dog food, chicken feed A, chicken feed B and milk powder. Table 5 lists the analytical results of melamine in above five real-world samples. As can be seen, no target melamine was detected in these five samples. To verify the accuracy of proposed method, recovery test were carried out and the analytical results for spiked samples are also listed in Table 5. The obtained recoveries are in the range of 76.2-98.2%, 80.0-85.5%, 89.5-113%, 85.0-95.5% and 65.0-111% for the spiked cat food, dog food, chicken feed (small particle), chicken feed (large particle) and milk powder, respectively. The HPLC-UV chromatograms for five real-world samples with/without MIP coated SBSE are shown in Figure 5, which demonstrated a good specific recognition ability and matrix interference resistance for the proposed method in melamine analysis.

4 Conclusions



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DOI: 10.1039/C5AN00325C

An MIP coated stir bar was prepared by in situ polymerization with a PTFE mold. The prepared MIP coated stir bar presented homogenous and porous surface, controllable coating thickness, good mechanical, chemical stability, as well as high extraction selectivity for strong polar compound of melamine. The use of cyromazine as dummy template effectively overcame the problem of template bleeding and ensured the quantification accuracy for target melamine. Although the prepared MIP coated stir bar can be used for 13 times, the prepared MIP coated stir bar is featured with easy preparation, low cost, and good preparation reproducibility. Based on it, a method of MIP-SBSE-HPLC-UV for melamine analysis was proposed for the determination of melamine in real-world samples including cat food, dog food, chicken feeds and milk powder. The proposed method presented good specific recognition ability and matrix interference resistance, and was effective and sensitive for melamine analysis.

Acknowledgments Financial support from the National Nature Science Foundation of China (20775057), the Science Fund for Creative Research Groups of NSFC (Nos. 20621502, 20921062) and the State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. KF2010-04) are gratefully acknowledged.



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DOI: 10.1039/C5AN00325C

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WHO.

Available:

http://www.who.int/foodsafety/fs_management/

Exec_Summary_melamine.pdf (accessed December 2, 2008). 8. M. S. Filigenzi, E. R. Tor, R. H. Poppenga, L. A. Aston, B. Puschner, Rapid Commun. Mass Spectrom., 2007, 21, 4027-4032. 9. K. Xia, J. Atkins, C. Foster, K. Armbrust, J. Agric. Food Chem., 2010, 58, 5945-5949. 10. H. H. Yang, W. H. Zhou, X. C. Guo, F. R. Chen, H. Q. Zhao, L. M. Lin, X. R. Wang, Talanta, 2009, 80, 821-825. 11. H. W. Sun, L. X. Wang, L. F. Ai, S. X. Liang, H. Wu, Food Control, 2010, 21, 686-691. 12. Y. L. Wong, C. S. Mok, Anal. Methods, 2013, 5, 2305-2314. 13. L. M. He, Y. J. Su, X. H. Shen, Y. Q. Zheng, H. B. Guo, Z. L. Zeng, J. Sep. Sci., 2009, 32, 3310-3318. 14. L. Meng, G. J. Shen, X. L. Hou, L. L. Wang, Chromatographia, 2009, 70, 991-994. 15. I. L. Tsai, S. W. Sun, H. W. Liao, S. C. Lin, C. H. Kuo, J. Chromatogr. A, 2009, 1216, 8296-8303.



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DOI: 10.1039/C5AN00325C

16. Z. H. Zhang, M. L. Zhang, L. J. Luo, X. Yang, Y. F. Hu, H. B. Zhang, S. Z. Yao, J. Sep. Sci., 2010, 33, 2854-2861. 17. B. Wang, Y. Z. Wang, H. Yang, J. Q. Wang, A. P. Deng, Microchim. Acta, 2011, 174, 191-199. 18. W. J. Cheng, Z. J. Liu, Y. Wang, Talanta, 2013, 116, 396-402. 19. G. Vlatakis, L. I. Andersson, R. Müller, K. Mosbach, Nature, 1993, 361, 645-647. 20. H. H. Yang, W. H. Zhou, X. C. Guo, F. R. Chen, H. Q. Zhao, L. M. Lin, X. R. Wang, Talanta, 2009, 80, 821-825. 21. M. Curcio, F. Puoci, G. Cirillo, F. Iemma, U. G. Spizzirri, N. Picci, J. Agric. Food Chem., 2010, 58, 11883-11887. 22. H. Y. Yan, X. L. Cheng, N. Sun, T. Y. Cai, R. J. Wu, K. Han, J. Chromatogr. B, 2012, 908, 137-142. 23. Z. C. Zhang, Z. Q. Cheng, C. F. Zhang, H. Y. Wang, J. F. Li, J. Appl. Polym. Sci., 2012, 123, 962-967. 24. N. A. Yusof, S. K. Ab. Rahman, M. Z. Hussein, N. A. Ibrahim, Polymers, 2013, 5, 1215-1228. 25. C. Crescenzi, S. Bayoudh, P. A. G. Cormack, T. Klein, K. Ensing, Anal. Chem., 2001, 73, 2171-2177. 26. L. M. He, Y. J. Su, Y. Q. Zheng, X. H. Huang, L. Wu, Y. H. Liu, Z. L. Zeng, Z. L. Chen, J. Chromatogr. A, 2009, 1216, 6196-6203. 27. E. Baltussen, P. Sandra, F. David, C. Cramers, J. Microcolumn Sep., 1999, 11, 737-747. 28. X. L. Zhu, J. B. Cai, J. Yang, Q. D. Su, Y. Gao, J. Chromatogr. A, 2006, 1131, 37-44. 29. Z. G. Xu, Y. F. Hu, Y. L. Hu, G. K. Li, J. Chromatogr. A, 2010, 1217, 3612-3618. 30. Y. L. Hu, J. W. Li, Y. F. Hu, G. K. Li, Talanta, 2010, 82, 464-470. 31. Y. L. Hu, J. W. Li, G. K. Li, J. Sep. Sci., 2011, 34, 1190-1197. 32. I. Petrikovics, J. C. C. Yu, D. E. Thompson, P. Jayanna, B. A. Logue, J. Nasr, R. K. Bhandari, S. I. Baskin, G. Rockwood, J. Chromatogr. B, 2012, 891, 81-84. 33. L. Q. Yang, X. M. Zhao, J. Zhou, Anal. Chim. Acta, 2010, 670, 72-77.



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34. A. Gomez-Caballero, A. Guerreiro, K. Karim, S. Piletsky, M. A. Goicolea, R. J. Barrio, Biosens. Bioelectron., 2011, 28, 25-32. 35. N. Sheng, F. D. Wei, W. Zhan, Z. Cai, S. H. Du, X. M. Zhou, F. Li, Q. Hu, J. Sep. Sci., 2011, 35, 707-712. 36. W. Zhan, F. D. Wei, G. H. Xu, Z. Cai, S. H. Du, X. M. Zhou, F. Li, Q. Hu, J. Sep. Sci., 2012, 35, 1036-1043. 37. H. B. Zhang, Z. H. Zhang, Y. F. Hu, X. Yang, S. Z. Yao, J. Agric. Food Chem., 2011, 59, 1063-1071.



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DOI: 10.1039/C5AN00325C

Figure captions Figure 1. The preparation sketch of MIP coated sitr bar with PTFE mold. Figure 2. SEM images of MIP coating before/after elution and NIP coating at different magnification. (a, MIP coating before elution, 1200×; b, MIP coating after elution, 1200×; c, NIP coating, 1200×; d, MIP coating before elution, 5000×; e, MIP coating after elution, 5000×; f, NIP coating, 5000×) Figure 3. FT-IR spectra of MIP coating after elution (a), MIP coating before elution (b) and NIP coating (c). Figure 4. Effect of desorption solvent on the desorption of melamine by MIP coated stir bar. Conditions: melamine, 100 µg L-1; sample solvent, methanol; stirring rate, 600 rpm; extraction time, 90 min; desorption time, 15 min. Figure 5. HPLC-UV chromatograms of five real-world samples (A. cat food; B. dog food; C. chicken feed of small particle; D. chicken feed of large particle; E. milk powder) with direct injection (a), MIP-SBSE (b), MIP-SBSE of 50 µg L-1 melamine spiked (c).



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Page 21 of 32



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. The preparation sketch of MIP coated sitr bar with PTFE mold.


DOI: 10.1039/C5AN00325C

Analyst



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b

c

d

e

f

Figure 2. SEM images of MIP coating before/after elution and NIP coating at different magnification. (a, MIP coating before elution, 1200×; b, MIP coating after elution, 1200×; c, NIP coating, 1200×; d, MIP coating before elution, 5000×; e, MIP coating after elution, 5000×; f, NIP coating, 5000×)


DOI: 10.1039/C5AN00325C

Page 23 of 32


1384.44 1260.23 1154.47

a

1258.73 1155.17

1578.01 1455.70

1634.17

b

1731.37 1384.12

3000

2500

2000

1500

1000

500

-1

Wavenumbers(cm )

Figure 3. FT-IR spectra of MIP coating after elution (a), MIP coating before elution (b) and NIP coating (c).


1455.58

1384.16

1729.95

3500

1260.84 1151.77

3441.48

1729.39

1635.00

c

3447.73

100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10

3445.24


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

T%

DOI: 10.1039/C5AN00325C

Analyst


DOI: 10.1039/C5AN00325C

800 700 600 500 400 300 200 100 0 10%

aceti c

acid /m

meth anol etha nol

10% a

mm o nia/m

aceto nitrile

etha n

ol

Figure 4. Effect of desorption solvent on the desorption of melamine by MIP coated stir bar. Conditions: melamine, 100 µg L-1; sample solvent, methanol; stirring rate, 600 rpm; extraction time, 90 min; desorption time, 15 min.


Peak Area


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Page 25 of 32


DOI: 10.1039/C5AN00325C

700

700

melamine

melamine

600

600

500

400

mAu

b 300

400

b

300

200

200

a

a

100

100

0

0 0

1

2

3

4

5

6

0

Time/min

1

2

3

4

5

6

Time/min

(A)

(B) melamine

700

700

melamine 600

600

c 500

c

500

400

400

b

b

300

300

200

200

a

a 100

100

0

0 0

1

2

3

4

5

6

0

Time/min

1

2

3

4

5

6

Time/min

(B)

(D)

700

melamine 600

c

500

400

b

300

200

a

100

0 0

1

2

3

4

5

6

Time/min

(E) Figure 5. HPLC-UV chromatograms of five real-world samples (A. cat food; B. dog food; C. chicken feed of small particle; D. chicken feed of large particle; E. milk powder) with direct injection (a), MIP-SBSE (b), MIP-SBSE of 50 µg L-1 melamine spiked (c).


mAu

c

c

500

mAu


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DOI: 10.1039/C5AN00325C

Table 1. The structures and properties of cyromazine, melamine and its metabolites Compound

Structure

pKa (25 oC)

logP (25 oC)

5.39±0.10 Melamine

-1.370±0.187 (Basic)

5.44±0.10 Cyromazine

-0.040±0.395 (Basic)

6.20±0.70 (Acidic) -1.281±0.350

Ammeline

3.96±0.20 (Basic)

5.21±0.20 -1.178±0.350

Cyanuric acid

(Acidic)



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Page 27 of 32


DOI: 10.1039/C5AN00325C

Table 2. Selectivity factors of MIP coated stir bars for melamine in different extraction solvent Extraction

Water

Methanol

Methanol/water

Acetonitrile

solvent Selectivity

Methanol/

Ethanol

acetonitrile 1.8

4.0

2.4

1.5

3.8

2.3

factor

The selectivity factor was calculated by the extraction efficiency ratio of MIP coated stir bar to NIP coated stir bar.



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Analyst


DOI: 10.1039/C5AN00325C

Table 3. Analytical performance of the proposed MIP-SBSE-HPLC-UV method for melamine analysis Analyte

Linear range (µg L-1)

R2

LOD (µg L-1)

EF

EE

RSD (c=5 µg L-1, n=7)

melamine

2-200

0.9962

0.54

42

34

6.1%



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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Analyst

Table 4. Comparison of LODs for the analysis of melamine by different methods LOD Dummy

Preparation

Operation

Method

Extraction Lifetime

template

method

Detection

(µg

time (min)

Recovery Sample

efficiency (%)

enrichment

Reference

91.6-102

No

[10]

86.3-98.6

Yes

[13]

(%)

L-1) bulk MIP-SPE

No

50

7

HPLC-DAD

60

-

65

-

HPLC-UV

0.1

70-85

dairy products

polymerization bulk MIP-SPE

Yes

environmental

polymerization

water

surface imprinting CIP-SPE

No

70

-

HPLC-UV

-

-

dairy products

87-92

Yes

[16]

-

60

HPLC-UV

-

89.8-100.6

milk sample

76.26-105.05

No

[17]

64

-

HPLC-DAD

20

-

dairy products

91.6–102.8

No

[20]

-

-

HPLC-UV

-

bovine milk

86.0-96.2

No

[22]

technique suspension MIM-SPE

No polymerization bulk

MIP-SPE

No polymerization bulk

MIP-MSPD

Yes polymerization

0.05 µg g-1


Published on 30 March 2015. Downloaded by Imperial College London Library on 06/04/2015

Page 29 of 32

Page 30 of 32

bulk MIP-SPE

Yes

60

10

HPLC-UV

-

-

15 (online)

20

HPLC-UV

0.3

-

feed and milk samples

83.4-103

No

[26]

-

Yes

[37]

56.0-113

yes

polymerization surface imprinting MIP-SPE

No

animal feed material and

technique

milk powder cat food, dog food,

In situ MIP-SBSE

Yes

This 200

13

HPLC-UV

0.54

34

chicken feed and milk

polymerization

work powder

CIP: complex-imprinted polymer; MIM: molecularly imprinted microsphere; MSPD: matrix solid-phase dispersion


Published on 30 March 2015. Downloaded by Imperial College London Library on 06/04/2015

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DOI: 10.1039/C5AN00325C

Table 5. Analytical results of melamine in cat food, dog food, chicken feed (small particle), chicken feed (large particle) and milk powder by proposed method Sample

Added (µg g-1)

Found (µg g-1)

Recovery (%)

0

N.D.

—

0.2

0.15±0.02

76.2

1.0

0.98±0.07

98.2

2.0

1.96±0.11

98.2

0

N.D.

—

Cat food

0.2

0.16±0.01

80.0

Dog food 1.0

0.81±0.04

81.0

2.0

1.71±0.15

85.5

0

N.D.

—

Chicken feed (small

0.2

0.23±0.03

113

particle)

1.0

0.98±0.06

97.9

2.0

1.79±0.10

89.5

0

N.D.

—

Chicken feed (large

0.2

0.17±0.01

85.0

particle)

1.0

0.91±0.11

91.5

2.0

1.91±0.07

95.5

0

N.D.

—

0.2

0.13±0.02

65.0

1.0

1.11±0.09

111

2.0

2.05±0.12

103

Milk powder

N.D.: not detected



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241x183mm (150 x 150 DPI)


DOI: 10.1039/C5AN00325C

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