Selective Extraction of Metformin in Pharmaceutical Preparation via Synthesized MIP-SPE Technique

This work demonstrates the synthesis and storage of molecular-imprinted polymers (MIP) at room temperature using bulk polymerisation of Metformin (Met) characterized by high sensitivity, low cost, and high stability. To ensure an acceptable adsorption capacity, the research employed 0.8:4:20 mmol ratios of template, monomer, and cross-linking agents for the polymerization. A functional monomer, 2-acrylamido-2-methyl-1-propane sulphonic acid C 7 H 13 NO 4 S, was cross-linked with N,N -methylene bisacrylamide C 7 H 10 N 2 O 2 to form Met-MIP, which could be characterized using a UV-VIS spectrophotometer at 236 nm, FT-IR spectroscopy, and scanning electron microscopy. The elution process that was applied to the template Metformin from the Met-MIP created cavities that were caused by the porogenic mixture solution of methanol, chloroform, and acetic acid (70:20:10, respectively). In accordance with the Freundlich isotherm model, Met-MIP had a maximum adsorption capacity of 5.2998 µmol/g and a template to monomer ratio of 1:2. A solid-phase extraction syringe packed with molecular imprinted polymers was used for the selective separation and pre-concentration of Metformin from aqueous solutions and estimation of Metformin by MIP and HPLC instruments in multiple pharmaceutical drugs of Metformin from several sources. The comparison with standard analytical techniques by MIP and RP-HPLC showed no significant difference between the two methods.


Introduction
Metformin (Met) or Glucofage is an oral diabetes medication that helps control blood sugar levels and is used together with diet and exercise to improve blood sugar control in adults with type 2 diabetes mellitus. It is a component of drugs like metformin-alogliptin (Kazano) and Metformin-canagliflozin (Invokamet). In the last stages of type 2 diabetes, metformin can also be used with insulin. Metformin decreases hepatic glucose production, increases insulinmediated peripheral glucose uptake, and decreases intestinal glucose absorption. It is also used off-label to treat polycystic ovary syndrome (PCOS) [1,2]. Common adverse effects include diarrhea, nausea, and abdominal pain. It has a low risk of causing low blood sugar. High blood lactic acid levels are a concern if the medication is used in overly large doses or prescribed to patients with severe kidney problems. It is not recommended for those with significant liver disease [3]. Metformin is a biguanide antihyperglycemic agent that works by decreasing glucose production by the liver, increasing the insulin sensitivity of body tissues, and increasing GDF15 (growth differentiation factor, a protein coding gene) secretion, which reduces appetite and caloric intake [4]. Figure 1 shows the structure of metformin. In the beginning, the imprint molecule with the present monomers forms a complex in molecular imprinted polymers (MIP). The functional groups are maintained in situ following the polymerization cycle, as depicted in Figure 2, by a strongly cross-linking polymer structure [5]. In addition, the steric configuration of all these connections around a given substratum and the template is really an important characteristic for the formation of binding sites, providing additional shape, size, and flexibility to promote the selective identification followed by a high target affinity. As a result, the process of recognition in MIPs can be characterized in resemblance to enzyme-proven mechanisms-substratum-complex is formed in the (lock and key) model [6][7][8][9][10]. SPE and HPLC are used to prepare certain MIP applications [12][13][14][15][16]. The adsorption isotherm is provided by the solute concentration in the fluid phase at constant temperature. An isotherm is the relation between the concentrations of a solid and fluid, used to describe the states of a sorption process [17]. Solid phase extraction (SPE) is a technique created for quick, selective sample preparation and purification prior to chromatographic analysis (e.g. HPLC, GC, and TLC). In SPE, one or more analytes from a liquid sample are isolated by extracting, partitioning, and/or adsorbing onto a solid stationary phase, as shown in Figure 3 [18]. In this study, RP-HPLC was used because of its wide applicability and reproducibility. The preparation of MIP with recognition sites 2-acrylamido-2-methyl-1-propanesulphonic acid, C7H13NO4S as a monomer with cross-linker N,N-methylene bisacrylamide (C7H10N2O2) and benzoyl peroxide (BPO) as an initiator for the target molecule (Met). The effects of monomer dosages on the adsorption performance were studied. The adsorption behaviors of various functional monomers, cross-linking agents, and solvents were also investigated. SEM and FT-IR were used to characterize the prepared MIP. In addition, the effects of RP-HPLC, solid phase extraction, and initial Metformin concentration on adsorption capacity were studied.

Preparation and processing
Ultra -high-purity grade Chemicals was used for preparation process and bought from Sigma -Aldrich (St. Louis, MO, USA). The solutions of Clopidogrel 30, 60, 90,120,150 ppm were prepared by dissolving 0.0015, 0.003, 0.0045, 0.006, and 0.0075 gm of Clopidogrel in 50 ml of methanol, respectively, at pH 8 by using volumetric flasks.
The solutions were passed through the column at a flow rate of 70 rpm. The extraction column was cleaned twice by using 2 mL of methanol in order to remove the interference from the matrix and then separated from MIP. In general, 1mmol of Clopidogrel (CLO) was dissolved in 9 mL of porogen (methanol), then 4 mmol of acrylic acid (AA) was added, sonicated for 5 minutes at 35 MHz at room temperature. In addition, 15 mmol cross-linker (EGDMA) and 0.32 mg initiator (benzoyl peroxide) were added to the above solution. The solution was bubbled for 20 min with nitrogen and used as a pre-polymer solution, then, the rubber sealed the tube. Solution was left in a SAM PLES COLLECTION AND PREPARATION Ultra -high-purity grade Chemicals was used for preparation process and bought from Sigma -Aldrich (St. Louis, MO, USA). The solutions of Clopidogrel 30, 60, 90,120,150 ppm were prepared by dissolving 0.0015, 0.003, 0.0045, 0.006, and 0.0075 gm of Clopidogrel in 50 ml of methanol, respectively, at pH 8 by using volumetric flasks.
The solutions were passed through the column at a flow rate of 70 rpm. The extraction column was cleaned twice by using 2 mL of methanol in order to remove the interference from the matrix and then separated from MIP.
In general, 1mmol of Clopidogrel (CLO) was dissolved in 9 mL of porogen (methanol), then 4 mmol of acrylic acid (AA) was added, sonicated for 5 minutes at 35 MHz at room temperature. In addition, 15 mmol cross-linker (EGDMA) and 0.32 mg initiator (benzoyl peroxide) were added to the above solution. The solution was bubbled for 20 min with nitrogen and used as a pre-polymer solution, then, the rubber sealed the tube. Solution was left in a High purity grade chemicals were used for the preparation process: Met-MIP was prepared by dissolving Metformin.HCl (129.2 mg, 0.8 mmol) in methanol (4 mL), 2-acrylamido-2 methyl-1-propanesulphonic acid (829 mg, 4 mmol) dissolved in methanol (2 mL) with H2SO4 (4 drops, 1 mol/L) and added to the Metformin solution, then left for few seconds at room temperature, then cross-linker N,N-methylene bisacrylamide (3.0834 g, 20 mmol) dissolved in H2SO4 (12 drops, 1 mol/L), methanol (6 mL), and chloroform (2 mL), ), before adding a solution of benzoyl peroxide (300 mg) in chloroform, which is used as an initiator. The solution was then shacked, bubbled for 20 minutes with pure nitrogen gas to remove the dissolved oxygen from the monomer solution immediately. Following this, the tube was sealed with a rubber stopper, and the solution was left in a water bath at 60 ºC overnight. In this step, the polymerization process of Met-MIP (0.8:4:20) was completed. A white-colored polymer with a rigid structure and fine particles was formed. This was observed with the naked eye, and left to dry at room temperature overnight. The self-assembly (non-covalent) technique of bulk polymerization was used to synthesize Met-MIP. Soxhlet solid liquid phase extraction of the template has been done to remove it from the MIP using porogenic solvent v/v (acetic acid, chloroform, and methanol; 1:2:7 respectively), and has been successfully removed by repeated washing for 16-18 hours. The polymer was dried at room temperature, crushed with a mortar and sieved to a particle size of 125 μm. A plastic syringe (3 mL) of solid phase extraction vacuum (column) was used, and each syringe was packed with (100 mg) of Met-MIP and the flow rate (70 mL/min) of the Metformin standard solution. A series of Metformin.HCl standard solutions (0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, and 0.2 μmol/mL) were prepared by dissolving Metformin (11.6 mg) in methanol (100 mL) as a stock solution. The calibration curve between Metformin concentration and its absorption (A) was achieved at 236 nm using the UV-VIS spectrophotometer. The samples of pharmaceuticals were prepared by taking the average weight of powder of Metformin tablets, as shown in Table 1. This was done by dissolving in methanol (100 mL) before filtration through a cellulose filter paper of 0.07 µm in order to obtain the concentrations from the calibration curve 1.0×10 -4 , 1.4×10 -4 , and 1.6×10 -4 mmol/mL (0.1, 0.14 and 0.16 µmol/mL) of Metformin drugs (Metforal/Germany, Glucophage/Italy and Piophage/Iraqi, respectively), which have the lowest standard addition (SD) value. These were used with MIP in a solid phase extraction (SPE) column, MIP-SPE, which was prepared.

Results and discussion
For a good expressive example of the advantages of the use of impressed polymers in SPE in the quantification of Metformin, Figures 4, 5, and 6 were measured by UV-VIS Spectrophotometer. The residue that has less absorption was measured using a UV-VIS Spectrophotometer, which indicates a lower concentration in the final process.   It can be seen that the spectra for Metformin and MIP before and after Metformin removal have similar bands, which means that the elution process has almost no influence on the primary polymer network structure. The spectrum of the Met shows strong bands at 3392 and 3371 cm -1 for NH2 stretching. The absorptions at 3463 and 3431 cm -1 belong to the Met-MIP before elution, and 3433 cm -1 after elution. The absorptions at 3294, 3294, and 3288 cm -1 for N-H bands are explained in Table 2. The bands at 1625, 1639, and 1641 cm -1 are attributed to the C=N stretching. The C-H aliphatic bands in Metformin appeared at 2972 and 2939 cm -1 . The

A A B A
Met-MIP before elution was at 2975 cm -1 and after elution at 2972 and 2941 cm -1 . To improve that, Metformin was removed successfully, and the C=O group disappeared before eluting, which means there was interaction between C=O of the monomer and N-H of the template, and it appeared after removing Metformin, and the smallest peak of C=N indicates that the template was removed [19]. The FT-IR spectroscopy is used to indicate the composition of the molecular imprinted polymer of Met drug, as is observed by the above diagram and table showing the beam at 1681 cm -1 for the C=O compared with the FT-IR spectrum before and after drug removal. Metformin shows the appearance of a C=O stretching vibration band, which indicates that the Metformin drug was removed and the C=N band became the smallest. The RP-HPLC method (Liquid-Solid) has been used for quantitative estimation of Metformin.HCl in pharmaceutical dosage form. This was by a reverse phase chromatographic method utilizing HPLC column C8 (5 µm, 25 cm × 4.6 mm). Column chromatographic runs were performed at a flow rate of 1.2 mL/min with acetonitrile: phosphate buffer (65:35) as the mobile phase. The pH was adjusted to 5.5 with phosphoric acid and then filtered through a micro-membrane filter. The flow rate was 1.5 mL/min. The detection of eluent using the RP-HPLC-UV detector was at 235 nm. Figure 9 illustrates the chromatogram of the Metformin standard with a concentration of 0.1µmol/mL at two volumes; 25 and 10 µL. In Figures 10, 11 and 12, the solutions at a concentration of 0.1µmol/mL for pharmaceutical drugs [Metformin-Germany (Glucophage), Metformin-Italy (Metforal), Metformin-Iraqi (Piophage)] were prepared and 25 μL was injected three times and a chromatogram was recorded, respectively. The mean retention time for Metformin was 6 minutes. A chromatogram was recorded for each solution after filtration through a 0.2 μm membrane filter. When we measure the area of the peak according to the Metformin standard peak and calculate the concentration of these drugs after the isotherm process, the effect of the isotherm process before and after passing through the SPE column appears clear, as shown in Table 6. Figure 11: Metformin-Italy chromatogram at 25µL before and after isotherm process Figure 12: Chromatogram of Metformin-Iraqi at 25µL before and after isotherm process Scanning electron microscopy (SEM: Metformin), the morphological evaluation is critical to the appreciation of the morphological traits, cavity sizes, and surface configurations of MIPs both before and following the Metformin template removal. The morphology of the Met-MIPs was examined using SEM images. Figure 13 (A and B) displays the particle surface morphologies for Metformin-MIP before and after elution as well as the relative cavity calculation. Table 3 shows the surface morphologies of the particles before and after elution for Metformin-MIP and the relative cavity calculation.  From Figure 13 and Table 3, the 3D of cavities between the minimum mean area (13281.36 nm) and maximum mean area (21273.02 nm), it was noticed that the holes vary in diameter range between 13281.36 and 21273.02 nm, and most of the holes are large, which leads to the

A B
retention of large quantities of the drug, which is consistent with the high value of the capacity in isotherm.  Adsorption capacity and pre-concentration:A series of absorption achievements for different initial concentrations of Met-MIP ranging from 0.02 to 0.2 μmol/mL on adsorption capacity μmol/g was studied using the following equation [20]: The process of attaining a high local concentration at the sensor surface is referred to as preconcentration.*1 The concentrations from 0.02 to 0.08 μmol/mL consume volumes of 27-15 mL, while the concentrations between 0.1 and 0.2 μmol/mL consume 4-3 mL only when using Met-MIP (100 mg), as shown in Table 4.
Relation between initial concentration and capacity    * For n = 5 absorptions of drugs before isotherm process (passing through MIP column), * The true value is the absorption at 0.1,0.12 µmol/mL in calibration curve of Metformin. In Tables 5 and 6, the volumes that pass through the MIP column for pharmaceutical drugs consume milliliters more than standard due to interferences, additions, and active materials used in the manufacture of drugs. To compare the capacity of Metformin selectivity between two analytical techniques by RP HPLC and our method of MIP determination of Metformin in pharmaceutical drugs (Table 7).

Conclusion
The monomer 2-acrylamido-2-methyl-1-propane sulphonic acid (AMPS) C7H13NO4S and the crosslinker N,N-methylene bis-acrylamide C7H10N2O2 as Met-MIP were used to generate a novel bulk polymer. Numerous studies and experiments were conducted to achieve the selective molecular imprinted polymer. This was achieved by preparing and optimizing the required monomers and cross-linkers using suitable solvents, porogen solvents for template removal, and the optimal molar ratios of template (Metformin) to monomer to cross-linker. SEM reveals an irregular three-dimensional network structure of polymer before and after template removal; FT-IR, HPLC, and isotherm processing all improve the healthy work. Two slops are gained when studying the capacity of adsorption of Met-MIP, which follows the Freundlich isotherm model with scatter values (heterogeneous structure) and the ratio of template to monomer is 1:2. The maximum adsorption capacity of Met-MIP was 5.2998 µmol/g and when comparing the capacity between two analytical techniques by RP-HPLC and our method MIP by UV for Metformin drugs, there was no significant difference between the two methods.