http://www.e-polymers.org e-Polymers 2013, no. 017 ISSN 1618-7229

Enantioselective analysis of naproxen using chiral molecular imprinting polymers based thin-layer

Fengyun Huangfu,1 Bing Wang,2* Juanjuan Shan,2 Zhiliang Zhang2

1 Textile Auxiliaries Company Ltd. of Tianjin Polytechnic University, Tianjin 300160 2 State Key Laboratory of Hollow Fiber Membrane Materials and Processes (Tianjin Polytechnic University), School of Environmental and Chemical Engineering, Tianjin Polytechnic University, Tianjin 300160, China; tel: 086- 022-8395-5113; fax: 086-022- 8395-5113; e-mail: [email protected]. cn

(Received: 31 January, 2012; published: 30 May, 2013)

Abstract: This paper describes a rational design and testing of molecularly imprinted polymers (MIPs) as chiral stationary phases of thin-layer chromatography (TLC) for enantiomeric purity of naproxen. Using D-naproxen as template, MIPs with particle size between 10~90 μm were prepared by precipitation polymerization in acetonitrile/methanol mixed solvent. The interactions between functional monomers and template were verified by UV absorption spectrometry. The morphology, particle size distribution and specific surface area of MIPs were also observed by scanning electron microscopy, particle size distribution meter and liquid nitrogen instrument, respectively. Binding capacities of MIPs had been studied by equilibrium binding assay. Preparation conditions of TLC and impact of acetic acid content on the separation of enantiomers were investigated. The results indicated that when acetic acid content was 4%, the racemates of templates were completely separated, and the chiral separation factor α was 1.58.

Introduction Naproxen(Npx, Fig.1), as a non-steroidal anti-inflammatory drug, is widely used to relieve pain and inflammation. This compound exists as enatiomers, in which the efficacy of D-naproxen (D-Npx) is 28 times larger than L-naproxen (L-Npx). And L- Npx causes some unwanted side effects. So it is meaningful to separate the naproxen enantiomers.

CH3 CH3 C COOH C COOH H H H3C H3C O O

L Npx D Npx

Fig. 1. The molecular structure of Npx enantiomers.

1 Molecular imprinting is a very useful method for the generation of based on molecular recognition, which has been applied in various fields such as those of separations, and [1-4]. The technique involves the polymerization of crosslinker and functional monomer in the presence of template. Then extraction of template leaves behind selective cavities which are complementary to the template in size, shape and chemical functionality. Therefore molecularly imprinted polymers (MIPs) are able to rebind the template with high affinity and specificity. As a result of its notable features, such as predictable elution order, high separation selectivity, good physical and chemical stability, etc., MIPs prepared by this technique could be used as a kind of novel chiral stationary phases in chromatographic chiral separations fields (HPLC, TLC, etc.) [5-8]. Compared with the HPLC, TLC possesses several advantages such as simplicity, rapidity, low cost and simultaneous detectability, and its combination with the molecular imprinting technique will provide simple, sensitive and rapid methods for analysis and characterization of enantiomers of optically active compounds. For this purpose, the first report on the employment of MIP as a TLC stationary phase was made by Kriz et al., involving the successful separation of enantiomers of amino acid derivatives [9], which proves the possibility of the MIPs as chiral stationary phases in TLC. And using this method, S. Roongnapa [10] and F. Rong [11] successfully completed the chiral resolution of the racemates of four kinds of adrenalines and three kinds of mandelic acids, respectively. Conventional MIPs have been synthesized by bulk polymerization. The copolymers then suffered grinding which is time-consuming. And the copolymers prepared have low capacity and poor site accessibility to template because the tedious grinding process of bulk polymerization may destroy some imprinting sites. On the contrary precipitation polymerization has overcome these shortcomings. Taking all these comments in mind, this paper describes MIPs as stationary phase of TLC to the determination of enantiomers of naproxen were synthesized using the method of precipitation polymerization. The preparation conditions of TLC and the impact of acetic acid content in the development system on the separation of enantiomers were systematically investigated. This method provides a rapid, sensitive and convenient way of analyzing and determining the enantiomers of chiral compounds.

Results and discussion In this study the selected imprinted molecule (Npx) and functional monomer (AM) were expected to form a stable supramolecular complex via the non-covalent self- assembly process, which would impact electronic energy levels in the imprinted molecular. The UV-vis spectrum of Npx and AM with different ratios is described in Fig. 2. As shown in Fig. 2, with the increasing concentration of functional monomer (AM), the absorption wavelength between 220 nm and 270 nm of D-Npx showed a redshift, and the absorption intensity decreased significantly. It indicated that some strong inter-molecular forces occurred between Npx and AM. On the basis of molecular structures of Naproxen (Npx with the methoxyl and carbonyl groups) and acrylamide (AA with the amide group), it could be deduced that hydrogen bonding might play a key role between them and a template-functional monomer complex formed via the molecular self-assembly process, in which the electron cloud distribution and density

2 of big pi (π) bond on naphthalene are transformed. This is the result of changes in the ultraviolet spectrum of Npx. So we deduced that the synthesis route and recognition mechanism of the imprinted polymer were as follows (Fig. 3)

3.0

2.5

2.0

1.5 Abs 1.0

0.5

0.0 200 250 300 350 400 nm

Fig. 2. UV–vis spectra of Npx samples with different molar ratios of Npx to AM.

CH3

CH3 C OH C O C OH H C AA H3C O H O H2N H3C O O O NH2

GDMA / DVB

CH3 C OH C O H O Adsorption H3C O O H2N H2N O NH2 Elution O NH2

Fig. 3. Synthesis route of the imprinted polymer and its recognition mechanism.

The adsorption isotherms drawn from the experimental data are shown in Fig. 4. As shown in Fig. 4, the adsorption of polymers (P1, P2) adhered to the rules of monolayer adsorption of the Langmuir model [12]. From the comparison of the three binding isotherms of P1, NP and P2, with increasing initial concentration of D-Npx, the adsorption capacities of the polymers (P1, P2) to D-Npx increased. However, the combination amount of D-Npx on P1 was always greater than those on NP and P2 obviously. Also, it was saturated in a high concentration range. Thus, P1 showed a better binding ability. During the experiments, the total pore volume and specific surface area of polymer 3 P1 were measured using multi-point BET method. The values are 0.1132cm /g and 2 99.64m /g respectively, which are higher than those of polymer P2 with a total pore volume of 0.0678cm3/g and a specific surface area 55.32m2/g, and higher than those of polymer NP with a total pore volume of 0.0171 cm3/g and a specific surface area

3 0.9457 m2/g. These results suggest that MIPs are porous materials, the pore volume and specific surface area of polymer P2 are smaller than those of polymer P1, the reason is that part of the imprinted holes were destroyed during the grinding process. While for polymer NP, because of the absence of imprinted cavities, its pore volume and specific surface area are relatively much less.

70 P1 60

-1 50 g P2

mol 40

Q/ 30 20 NP 10 0 0 1 2 3 4 5 -1 c/mmol L

Fig. 4. Adsorption (Q) of P1, P2 and NP to the D-Npx with different initial concentration.

Fig. 5. SEM images of P1 with different magnification. 30 100

80

20 ) %

) 60

%

on

( (

q q 40 10 Q 20

0 0 0 20 40 60 80 100120140160180200 diameter ( mm)

Fig. 6. Particle size distributions of P1.

Compared synthesis process of the three kinds of polymers, we can see that many specific structure holes with fixed three-dimensional shape and position of functional groups matching to D-Npx formed during the synthetically process in presence of

4 imprinted molecule. These imprinted holes with active binding site in them caused a good ability to bind the imprinted molecules on the MIPs.

Fig. 5 was the SEM of P1 particles with different magnification. It can be seen that MIP particles showed agglomerates of random irregular particles in the morphology. And as shown in Fig. 6, its particle size distribution was between the 10 ~ 90 μm, and the median particle size was about 36 μm. In this experiment, two kinds of binder were investigated for the preparation of the TLC plates. The tendency of the polymer to swell in acetonitrile and water made it difficult to manufacture the plates in these solvents unless a binder was found which was strong enough not to allow the coating forming cracks as it dried due to polymer shrinking. A small amount of ethanol was used to increase the wet ability of the particles. Plaster of Paris was found to be a suitable binder when water was used as solvent. When using methyl cellulose as the binder, the prepared TLC plates showed poor mechanical properties and emerged cracks after drying.

Tab. 1. Effect of the particle size of MIPs and binder amount on the physical properties of TLC.

Size distribution of Binder/MIPs Crack Mechanical TLC MIPs [µm] [w/w] formation stability Ⅰ 1:1 no good 15~38.5 Ⅱ 1:2 yes bad Ⅲ 1:1 no good 38.5~74 Ⅳ 1:2 no good Ⅴ 15~74 1:1 no good

Coatings were made with three different size distributions of MIP particles (as shown in Tab. 1). It was found that the coating had a greater tendency to form cracks when the smaller particles were used. Thus, a ratio of binder to MIP of 1: 2 (w/w) was used with the large MIP particles, but a ratio of 1: l had to be used with the small MIP particles. With different concentrations of acetic acid in acetonitrile as mobile phase, the enantiomeric resolution capacity of TLC plates I, III, IV, V were investigated. It was found that under the same conditions, TLC plates (III, IV) required shorter development time (4 ~ 5 min), but gave less effective separation. TLC plates (I) gave better separation values due to the increased time of migration (about 7 min), and the racemates was separated into isomers. However, the chromatographic separation effect of plate ( ) was worst, and there has been a serious tailing phenomenon and no clear separation of the chromatographic spots. This maybe because the size distribution of MIP particles is too wide, and the development speed of solvent in different regions of the chromatography plate is uneven, resulting in different interaction time between the drug molecules and stationary phase. Tab. 2 gives the retention and resolution data of the racemates of Npx on TLC Plate (I).

It can be seen from Tab. 2, the Rf values of D-Npx are less than those of L-Npx with various concentrations of acetic acid (0, 2, 4, 7or 10%) in acetonitrile, which shows that D-Npx is much more retained than L-Npx on the D-specific plate. This is

5 because many specific structure holes with fixed three dimensional shapes and positions of functional groups matching the D-Npx were formed during the synthesis process of MIP particles in the presence of D-Npx. These imprinted holes with active binding sites in them caused better ability to selective binding of D-Npx than L-Npx on the molecularly imprinted TLC plate due to the imprinting effect.

Tab. 2. Chromatographic data of Npx racemates separation on TLC Plate (I).

Acetic acid Template content in mobile Rf(D) Rf(L) α phase (%) 0 0.12 0.16 1.33 2 0.33 0.49 1.47 D-Npx 4 0.41 0.65 1.58 7 0.58 0.74 1.28 10 0.67 0.79 1.17

In addition, the importance of solvent comprised in the separation of products was examined by incorporating different concentrations of acetic acid in mobile phase system of acetonitrile. As shown in Table 2, modification of the mobile phase with acetic acid in the range of 2~10% resulted in the analytes being less retained. When acetic acid concentration was low, such as 0%, 2%, the separation factor α and Rf values were small, but with the increased concentration of acetic acid, both increased. When acetic acid content was 4%, the separation factor α reached a maximum of 1.58 and enabled better separation of the enantiomers. However, when the acid content continued to increase, such as 7%, 10%, the separation factor α reduced to 1.28 and 1.17, respectively. This is because addition of strong polarity of acetic acid enhanced the solubility of the substrate in the mobile phase and allowed the substrate to achieve a quick adsorption-desorption equilibrium on the TLC plate, and thus obtained better separation of the enantiomers. However, when acetic acid content was too high, the presence of hydrogen would weaken the interaction force between the template molecules and functional groups, resulting in the reduction of effective recognition sites, which led to lower separation factor.

Conclusions The employment of MIPs as stationary phases in TLC is a substantially useful method of determining optical isomers for enantiomeric purity. In this work, we successfully prepared the thin-layer chromatography plates coated with molecularly imprinted polymers particles synthesized by the method of precipitation polymerization, avoiding the tedious grinding process of bulk MIP and the loss of imprinting sites, and achieved the resolution of the naproxen racemates. This work may provide a potentially powerful tool for resolving chiral compounds and this is a useful method for quality control of optically active compounds.

Experimental part

Materials Naproxen (Npx) and D, L-Naproxen (99.5 %) were obtained from Zhejiang Charioteer Pharmaceutical Co., Ltd. Acrylamide (AM) was supplied by Tianjin Chemical Reagent

6 Research Institute. Ethylene glycol dimethacrylate (EGDMA) was purchased from Shanghai Haiqu Chemical Co., Ltd. All of the monomers were purified by vacuum distillation or recrystallization to remove inhibitor prior to use. Other chemicals were of analytical grade as noted.

UV-vis spectroscopic analysis A series of solutions were prepared with a fixed concentration of D-Npx (0.5 mmol/L) and various amounts of AM in methanol. The ratios of the molar concentration between D-Npx and AM among this set of solutions were 1 : 0, 1 : 1, 1 : 2, 1 : 3, 1 : 4, and 1 : 5. The solutions were shaken for about 3 h, and then placed for 10 min aiming at achieving sufficient interaction between D-Npx and AM. The changes of different absorption spectra of these solutions were obtained by the Ultraviolet-visible Spectrophotometer under a wavelength range of 200~400 nm.

Preparation of the MIP particles The template D-Npx 0.1151 g (0.5 mmol) and the functional monomer AM 0.1422 g (2 mmol) were dissolved in 100 ml 10 vol% methanol-acetonitrile solution with shaking for 4 h to form homogeneous solution, then the cross linker EGDMA 10 mmol and free radical initiator 2,2-azo-bis-isobutyronitrile (AIBN) 40 mg were added into the solution in turns. The solution was deoxygenated with nitrogen gas for 10 min, and then the reactor was sealed and placed in constant temperature bath oscillator. The polymerization was carried out at a constant temperature 60 0C and a rotational speed 300 r/min for 24 h. The resulting MIP particles (P1) were placed in a soxhlet extractor and washed with 10 vol% acetic acid-methanol solution until the template could no longer be detected in the elution. Then, the particles were washed with pure methanol to remove the residual acetic acid and dried to constant weight under vacuum at 60 0C. For verifying that rebinding to template was due to molecular recognition and not to non-specific binding, a control (the non-imprinted polymer particles, NP) were prepared by the same method, but in the absence of the template D-Npx. At the same time, the imprinted polymer (P2) was prepared by bulk polymerization with the dosage of solvent 10 ml, which needed grinding process before the template molecule being washed off. The remaining steps were the same as above.

Isothermal adsorption experiments The dry MIPs (50 mg) were placed in 25 mL conical flasks and mixed with 5 mL of a known concentration of D-Npx solution (0.2-5 mmol/L). The mixtures were oscillated with constant temperature bath at 28 0C for 10 h, and then the concentration of D- Npx in the solution was determined and binding amount (Q) was calculated according to the Equation (1).

Q = (c0 – ct) ×V / m (1) where, c0 is the initial concentration of D-Npx (mmol/L), ct is the equilibrium concentration of D-Npx (mmol/L), V and m are the volume of the substrate solution (5 mL) and the weight of dry polymer (50 mg) in the adsorption experiment, respectively.

7 BET analysis

The total pore volume and specific surface area of polymer P1 were measured using multi-point BET method by Omnisorp-100CX adsorption instrument. Polymers were degassed at 105 0C under nitrogen flow for 4 h. The total pore volume was calculated by Barrett-Joyner-Halenda (BJH) method, and the specific surface area was determined by BET equation.

Preparation of the TLC plates The synthesized MIP particles were sieved through 200 mesh, 400 mesh and 800 mesh sieve, respectively. Each imprinted polymer particles (1 g) and plaster of Paris (1g, CaSO4·1/2H2O) were gradually mixed with distilled water (15 ml) and a small amount of ethanol (0.2 ml) as wetting agent with a pestle and mortar. The slurry was carefully poured on standard glass microscope slides (76×26 mm), which then spread as a thin layer with a layer of thickness 0.25 mm. Then the plates were dried at room temperature for at least 24 h before use.

Chromatographic method The sample to be analyzed was dissolved in acetonitrile solution at a concentration of approximately 2 mg/ml and carefully applied as spots at 1 cm above the bottom edge of the plate using 1µl glass capillaries. For resolution studies, the chromatograms were developed with various concentrations of 0, 2, 4, 7 and 10 vol% acetic acid in acetonitrile solution. All separations were performed at room temperature. The detection of the substance on the TLC plate was carried out by spraying the detection reagent (alkaline potassium permanganate reagent). This detection reagent gave a pink background with yellow spots for the samples.

The Rf value of the substance is defined as the distance traveled by the compound divided by the distance traveled by the solvent. And the chiral separation factor α between two separated spots is defined as the ratio of the higher Rf value and the lower Rf value (imprinted enantiomer) for the two spots.

Acknowledgements The authors gratefully acknowledge the financial support of the Nature Science Foundation of Tianjin, China. Contract grant number: 10JCZDJC21900 and 13JCQNJC02600.

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