Synthesis and Application of Glibenclamide Imprinted Polymer for Solid Phase Extraction in Serum Samples Using Itaconic Acid as Functional Monomer

INTRODUCTION Molecular Imprinted Polymer (MIP) is a polymer that is made using molecular imprinting techniques with an affinity for the template molecule, its prepared by the existence of template as a print for the conformation of a complementary binding site of the template (Zheng et al., 2002; Yoshimi et al., 2013). The use of MIP in the Solid Phase Extraction (SPE) has high benefits because it produces selective extraction of analytes and eliminates sample matrices. It is able to produce the receptor binding site-like artificial memory on the shape and position of the functional groups of the template molecule (Rezaei et al., 2010; Khodadadian and Farhad, 2010). Molecular Imprinted Solid Phase Extraction (MISPE) is able to provide the stationary phase which selectively isolate the specific compound or its structural analog of a complex matrix (Qiao et al., 2006; Lulinski et al., 2014; Pichon, 2007; Yin et al., 2005). The selectivity of the MIP comes from synthetic procedure to prepare the MIP wherein a template molecule is linked by noncovalent or covalent bonding to a monomer with functional groups (Caro et al., 2006). Glibenclamide is a second-generation sulfonylurea drugs for the treatment of noninsulin dependant diabetes mellitus (NIDDM) diabetes type. Glibenclamide is capable to stimulate 1288 www.ansinet.com | Volume 15 | Issue 11 | 2015 | J. Applied Sci., 15 (11): 1288-1296, 2015 the release of insulin from pancreatic beta cells (Binz et al., 2012). As a drug that was used for long term treatment, drug monitoring was needed for glibenclamide. The concentration of glibenclamide was small in biological matrices so preparation of the sample with high accuracy and precision was important in this case. Hence, the development of molecular imprinted polymer for selective extraction of glibenclamide from biological matrices was needed. Up to now, a glibenclamide imprinted polymer for selective extraction of glibenclamide from biological samples never been reported with full study (Hasanah et al., 2014). Thus, this study evaluates the polymer performance for selective recognition of glibenclamide which are made from selection of ten common monomer used in imprinting technology. In addition, this study also aims to determines the best extraction condition for glibenclamide recognition in serum samples with high selectivity. MATERIALS AND METHODS Chemicals and apparatus: Itaconic Acid (ITA), ethylene glycol dimethacrylate (EGDMA), 2,2-azoisobutyronitrile (AIBN), tetrafluoroacetic acid (TFA) were provided by Sigma Aldrich. Methanol pro HPLC, chloroform pro analysis, acetonitrile pro HPLC, dimethylformamide (DMF) pro analysis, acetone and acetic acid pro analysis were purchased from JT Baker. Glibenclamide (GC) were provided by Hexpharm Pharmaceuticals Industry. Glipizide (GP) were purchased from Indonesia National Agency of Drug and Food Control. Gliclazide (GL) were provided by Dexa Medica Pharmaceuticals Industry. Blood were provided by Indonesian Red Cross after full examination. ChemDraw and Chem 3D Ultra 8.0.3 software (Cambridgesoft Corporation, USA), Gaussian 09 (Gaussian Inc., Wallingford, CT), Waterbath shaker (Memmert), HPLC (Waters 1525 binary pump HPLC pump with photodiode array 2998 and C18 column sunfire 4, 6, 150 mm), Fourier Transform Infra Red (FTIR) Shimadzu prestige-21, scanning electron microscope (Hitachi TM 3000), centrifugation (eppendorf centrifuge 5424R). This study has been carried out from January 2013 until December 2014 at Pharmacochemistry Research Group Bandung Institute of Technology and Research Center Laboratory Universitas Padjadjaran. Monomer template interaction studies using computational studies: The molecular models of the template molecules, functional monomer and their complexations were drawn using ChemDraw and Chem 3D Ultra 8.0.3 (Cambridgesoft Corporation, USA). The 3D structures were drawn and cartessian coordinates of stable conformers were generated to prepare input file for running Gaussian 09 (Gaussian Inc., Table 1: Composition of the produced polymers (MIP and NIP) Polymer Template (T) Monomer (M) Crosslinker (C) Ratio T:M:C MIP 1 GC ITA EDGMA 1:6:60 NIP 1 ITA EDGMA 0:6:60 MIP 2 GC ITA EDGMA 1:6:70 NIP 2 ITA EDGMA 0:6:70 Wallingford, CT) simulations. The Hartree-Fock level of theory in combination with the 6-31 G (d) basis set was used for geometry optimization to obtain structures with minimum energy. The possible modes of interaction between template molecules and functional monomers at molar ratio 1:1 were sampled by manually docking the functional monomer to each functional group of the template molecule in a systematic manner. The Gibbs free energy gains of the complexes were calculated using Eq. 1: ΔG = G template-monomer complex-|Gtemplate+Gmonomer| (1) where, ΔG is the change in Gibbs free energy on the formation of template-monomer complex, Gtemplate-monomer complex is the Gibbs free energy of template-monomer complex, Gtemplate is the Gibbs free energy of template and Gmonomer is the Gibbs free energy of monomer molecules. Synthesis of Molecular Imprinted Polymer (MIP): The Molecular Imprinting Polymer (MIPs) and Non Imprinted Polymers (NIPs) were prepared by bulk polymerization. The polymers were prepared as follows: GC as a template, ITA as functional monomer, followed by cross linker EGDMA and initiator AIBN (0.082 mmol) were dissolved in DMF (4.5 mL) in a thick-walled glass tube. The appropriate homogenous solutions were sonicated for 40 min. Then the mixtures were incubated in the waterbath 60C for 18 h. The final bulk rigid polymers were ground in a laboratory mortal pestle and wet-sieved with acetone to get particles below 100 μm diameter. The particles were extracted to remove the glibenclamide as a template using Soxhlet apparatus for 24 h in methanol: Acetic acid (9:1) and dried at 55°C for 3 h and stored at room temperature for further experiments. Quantitative removal of the template was ensured by monitoring the amount of template remaining in the extraction solvent by HPLC. The Non Imprinted Polymers (NIP) were prepared in a similar ways as used for the corresponding imprinted polymers except without template molecule during polymerization. Composition of the produced polymers can be seen in Table 1. Characterization: Infrared (IR) spectra from 4000-400 cmG were obtained on FTIR Shimadzu prestige-21. Morphology of the MIP and NIP were obtained by using SEM Hitachi TM 3000. 1289 www.ansinet.com | Volume 15 | Issue 11 | 2015 | J. Applied Sci., 15 (11): 1288-1296, 2015 Batch rebinding and isoterm adsorption studies: The rebinding batch-mode experiments were performed in methanol, chloroform, methanol pH 4, acetonitrile pH 4 and acetonitrile. Binding analysis was carried out by incubating 20 mg of polymer in 5 mL volume of the GC solution (5 μg mLG) on a vial and oscillated by a shaker 120 rpm for 3 h at room temperature. Then the mixtures were filtrated and an aliquot of solvent was used to analyze by HPLC. Amount of GC bound to the polymer was calculated by subtracting the amount determined after the experiment from starting amount of GC in standard solution. Isoterm Adsorption Studies were performed by incubating 10 mg of polymer in 1.5 mL volume of GC in several concentration (0.05, 0.1, 0.2, 0.5, 1 and 2 mmolar) on a vial for 24 h. The solution were then analyzed by HPLC. The isoterm adsorption graph were then made with the curve between bound and free GC. Optimation of Solid Phase Extraction (SPE) system: The 200 mg of MIP and NIP particles were dry packed in 3 mL Chromabond SPE cartridges using 20 mm porous PTFE frits. This further called MISPE and NI-SPE. Equilibration of the columns, loading and washing were performed using 1 mL aliquots of the corresponding solutions and elution of the retained analytes with different elution solvents. Full vacuum was applied between each step in order to dry the stationary phases. The collected fractions were analytes by HPLC and isocratic elution using a mixture of CH3CN: TFA 0.01% in water (60:40) with the flow-rate set at 1.2 mL per min. SPE separation of a mixture of structurally related compounds: After establishing the optimum condition of GC application on the MISPE, a mixture of structurally compounds were used to evaluate the selectivity of the MISPE produced. The compounds were from other sulfonylurea antidiabetic drugs which are GL and GP. Equilibration of the MISPE cartridge with 3 mL of methanol, loading with mixture of GL, GP and GC solution in methanol pH 4 and washing with methanol:water (5:95) were performed and elution of the retained analytes and structurally related compounds with 3×1 mL CH3CN: Methanol (1:1). The recovery percentage of the elution fraction was then calculated after analysis by using HPLC. Application of MIP for extraction of glibenclamide from serum samples: Blood serum samples were prepared by centrifugation the collected blood from Indonesian Red Cross 5000 rpm for 5 min at 14°C and careful collection of the clear top layer. The blood serum samples then were spiked with 0.5, 1, 2, 3, 4, 5 and 6 ppm of GC in methanol pH 4. The spiked serum then applied to MISPE and NI-SPE system. The SPE system was conditioned with methanol, washing with water:methanol 95:5 and elute with 3×1 mL CH3CN:Methanol 1:1. Full vacuum was applied between each step in order to dry the stationary phase. The elution fraction then analytes by HPLC and isocratic elution using a mixture of acetonitrile: TFA 0.01% in water (60:40) with the flow-rate set at 1.2 mL per min. The recovery percentage of the elution fraction was then calculated. RESULTS AND DISCUSSION Computational studies of monomer-template interaction: In order to optimize the preparation of the MIP, ten monomer were tested for the interaction with template by using Gaussian 09. The result of the studies can be seen in Table 2. Based on computational studies from Gibbs energy, complex between ITA-GC has a lower Gibbs energy