Synthesis and characterization of meropenem imprinted polymer

Meropenem imprinted polymer (M-MIP) has been successfully synthesized. It is prepared by using meropenem (MERP) as template and acrylamide as functional monomer. For comparison, non-imprinted polymer (NM-MIP) was synthesized in the absence of MERP. The synthesized polymers were then characterized by infrared spectroscopy (IR) and thermogravimetry analysis (TGA). Extraction of template from polymer was conducted using soxhletation method and methanol-acetate acid (85:15) as the leaching solvent.. The prepared M-MIP showed higher adsorption capacity than the NM-MIP. Adsorption capacity of MIP and NIP were 4.8942 mg/g and 1.0078 mg/g respectively.


Introduction
Meropenem, (4R,5S,6S)-3-[[(3S,5S)-5-dimethylcarbamoyl pyrrolidin-3-yl]-thio]-6-[(1R)hydroxyethyl]-4-methyl-7-oxo-1 azabicyclo[3,2,0] hept-2-ene-2-carboxylic acid, is a new parenteral carbapenem antibiotic with a very broad spectrum of antibacterial activity against the majority of grampositive and gram-negative pathogens [1]. Meropenem showed excellent efficacy in clinical studies involving seriously ill patients with intraabdominal, central nervous system, lower respiratory tract, skin and soft tissue, urinarytract, and febrile neutropenic infections [2]. Meropenem demonstrated to be unstable in aqueous solution when submitted to thermal and alkaline treatment. Because of the instability of meropenem, special care must be taken to avoid exposure of the drug to the degradation conditions during the handling and storage of the pharmaceutical preparation [3]. In this study, we intend to synthesis sorbent for solid phase extraxtion of meropenem by molecularly imprinted polymer (MIP). MIP is a technique to design artificial receptors with a predetermined selectivity and specificity for a given analyte, which can be used as ideal materials in various application fields. Molecularly Imprinted Polymer (MIP), the polymeric matrices obtained using the imprinting technology, are robust molecular recognition elements able to mimic natural recognition entities, such as antibodies and biological receptors, useful to separate and analyze complicated samples such as biological fluids and environmental samples. MIP synthesized by a noncovalent imprinting approach, which exhibit more selective recognition sites and higher adsorption capabilities for specific analytes or groups of structurally related species in contrast to the conventional sorbents. Molecular imprinting is a process where the target molecule acts as a template around which interacting and cross-linking monomers are arranged and copolymerized to form a castlike shell (Fig. 2). Initially, the monomers form a complex with the template through covalent or noncovalent interactions. After polymerization and removal of the template, binding sites are exposed that are complementary to the template in size, shape, and position of the functional groups, which are held in place by the cross-linked structure. In essence, a molecular memory is imprinted in the polymer, which is now capable of rebinding or adsorbing the template selectively [4] Figure 2. General principle of molecular imprinting. A molecular template (T) is mixed with functional monomers (M) and a cross-linker (CL) resulting in the formation of a self-assembled complex (1). The polymerization of the resulting system produces a rigid structure bearing imprinted sites (2). Finally removal of the template liberates cavities that can specifically recognize and bind the target molecule (3).

Material and instrumentation
Meropenem powder for injection was obtained commercially and claimed to contain 500 mg and 1000 mg as anhydrous base. Meropenem reference standard, acrylamide was bought from Sigma Aldrich. Acetonitrile for chromatography, potassium dihydrogenphosphate p.a. and ortho-phosphoric acid analytical grade were obtained from Merck. Dimethylsulfoxide (DMSO), ethylenglygol methacrylate (EGDMA) were also obtained from Merck. Ultrapure water pyrogen free (water for injection) was obtained from PT Ikafarmindo and was used to prepare all solutions for HPLC All solutions were prepared daily. Infrared absorption spectra for polymer was obtained using Fourier Transform Infrared (FT-IR) Prestige21 (Shimadzu), evaluation of leached polymer is observed by HPLC (Agilen).

Synthesis of MIP
To prepare the M-MIP, 1 mmol of MERP and the appropriate amount of functional monomer (Table-1) were dissolved in 20 ml of DMSO. To this mixture then cross-linker (EGDMA) and initiator benzoyl peroxide (BPO) were added. There were sonicated for 5 min. The solution was degassed with a stream of pure nitrogen for about 10 min. The vessel was caped under nitrogen flow and transferred to oven. After that, it was heated at temperature maximum of 60˚C during 4 h. The polymeric particles were dried at the temperature of 60˚C and rinsed with acetone. A Non-imprinted polymer (NM-MIP) as control polymer was also prepared using an identical procedure without adding MERP. Table 1 shows the recepies of the polymers synthesized in this study.

Extraction of MERP from polymer
The template was removed by a soxhlet extractor using (methanol: acetic acid = 85:15) for 24 h. The leached polymer particles were sieved to obtain particles sizes between 60 and 80 mesh. Characterization was conducted using Infrared Spectrophotometer for functional group analysis and TGA for thermal analysis.

Synthesis of M-MIP and NM-MIP
The composition of M-MIP and NM-MIP was as follows:

Characterization of M-MIP
The infrared spectra of M-MIP showed in fig. 4. The important absorption bands observed was at 1152 cm -1 which correspond to C-N stretching bond in β lactam, and also 1724 cm -1 which corresponds to the stretching of C=O bond in β lactam [6]

Extraction of template from polymer
After polymerization completed, extraction of the template would be the next step to make M-MIP. Suitable solvent must be found to leach the meropenem (template) well. The complete leaching process was observed using UV spectrophotometer. As shown in fig 5 (a), the peak of meropenem was found at λ = 309 nm. As we can see, the peaks was in different level of absorbance according to the different concentration of meropenem in the proper solvent of extraction (methanol-acetic acid) = (85:15). From the red and blue line, which is the spectra of leached polymer, in Fig. 5 (b) we can see that was no peak found at the λ = 309 nm. It indicated that no meropenem found. As the control, there is peak of black line in fig. 4b that gave certain absorbance from meropenem standard added

Thermal analysis
In order to study thermal stabilities of M-MIP NM-MIP and after-leached M-MIP, the thermogravimetric analysis (TGA) was employed. Figure 6 below showed the resulting thermogram The leached polymer showed only 10% weight loss at the temperature around 100-200˚C. It showed the loss of porogenic solvent involved in the synthesis process, DMSO. There was different treatment in leaching the polymer. Polymer was leached in the presence of methanol and acetic acid, the volatile solvent. DMSO evaporated quickly together with methanol-acetic acid. As the information, the melting point of meropenem is at 150-153 ˚C, as seen in line blue (C) and line black (A), on that area, there were different percentage of weight loss. Blue line had more weight loss compared to the black line. It might be the loss of meropenem (blue line). The selectivity for meropenem was 4.8563. These could be ascribed to the hydrogen-bond interaction between the meropenemand functional monomers in the specific recognition sites of the imprinted polymers. These results demonstrated that the selective recognition was built on the complementarities of functional groups, size, and shape between the analytes and recognition sites [7].

Conclusion
Molecularly imprinted polymer for meropenem has been successfully synthesized and was characterized by its IR spectrum, TGA curve and the imprinting factor calculation.