Synthesis of Molecularly Imprinted Polymers for Selective Extraction Followed by Solid Phase Determination of Metformin in Pharmaceutical Preparation and in Human Serum

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Published Online First: October, 2023 https://dx.doi.org/10.21123/bsj.2023.8225P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal 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 1 .Fig. 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 4 , as depicted in Fig. 2, by a strongly crosslinking polymer structure.
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 5,6 , 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 mechanismssubstratum-Complex is formed in the (lock and key) model 7,8 .
Certain MIP applications were prepared in SPE 10,11 .
The concentration of the solute in the fluid phase at constant temperature provides the adsorption isotherm.An isotherm is the relation between the concentrations of a solid and fluid, used to describe states of sorption process 12 .
-Solid phase extraction (SPE) is a technique designed for rapid, selective sample preparation and purification prior to the chromatographic analysis (e.g.HPLC(high performance liquid chromatography) , GC( gas chromatography), TLC(thin layer chromatography)) 13,14 .In SPE, one or more analytes from a liquid sample are isolated by extracting, partitioning, and adsorbing onto a solid stationary phase, Fig. 3 15 .

Figure3. Illustrate the process of SPE.
In this work identify the MIP preparation was performed in conjunction with the recognition cite styrene C8H8 with crosslinking ethylene glycol dimethacrylate EGDMA C10H14O4, whereby benzoyl peroxide BPO functioned as the target molecule (Metformin) initiator.Subsequently, the impact of the monomer dosages on the adsorption performance was observed.The study also examined adsorption behaviors with diverse functional monomers, crosslinking agents, and solvents.SEM, FTIR was employed to characterise the primed MIPs.Furthermore, this research investigated the impact of solid phase extraction and initial Metformin concentration on adsorption capacity.

Preparation and Processing:
The Met-MIP preparation process used high-purity chemicals: Specifically, 0.8 mmol of Metformin (Metformin.HCl) 0.1325g was dissolved in 4 mL of methanol with stirring, 4 mmol of Styrene 0.4180g with 2 mL methanol was added, waiting for few seconds at room temperature.Subsequently, 20 mmol of crosslinker Ethylene glycol dimethacrylate EGDMA 3.9644g, 2mL of methanol, and 0.3 g of benzoyl peroxide were dissolved in chloroform to create an initiator.These were added to the mixture, which was shaken well in order to produce a clear solution.The solution was bubbled with Nitrogen gas for 20 minutes in order to eliminate the dissolved oxygen from the monomer solution.It was then sealed in the tube.
The solution was placed in a water bath, where it remained overnight at a temperature of 60 ºC.Thus, the Met-MIP polymerization process 0.8:4:20 was completed, leading to the formation of a white polymer with a fluff structure has been formed.This was left to dry overnight at room temperature.The Met-MIP was synthesized using the self-assembly (non-covalent) bulk polymerization method.
Soxhlet solid liquid extraction was performed to remove template (Metformin) from MIP.This relied on the use of a porogenic solvent consisting of 10 mL of acetic acid, 20 mL of chloroform, and 70 mL of methanol.The removal process was successful following repeated for 16-18 hours, following which the particles were rinsed in methanol and water in order to eliminate residual acetic acid.Subsequently, the polymer was dried at room temperature, before being crushed with a mortar and sieved to produce 125μm particles.A proposed successive mechanism for Met-MIP can be represented by the following scheme 1 and illustrate the structure of synthesized

Sample Preparation of (Metformin. HCl) (Glucophage)
The samples of pharmaceutical were prepared by taking the average weight of ten tablets of Metformin, they were crushed and grinded.Tablets containing 500 mg of Metformin were weighed 0.5264, 0.5598, 0.5567 g of Metformin drugs (Metforal/Germany, Glucophage/ Italy and Piophage / Iraq) dissolved in 100 mL of methanol solution, then filtered through cellulose filter paper 0.07µm in order to obtain the concentrations 1.0×10 -4 , 1.4×10 -4 and 1.6×10 -4 mmol/mL (0.1, 0.14, 0.16µmol/mL) (equivalent to 0.00166, 0.00232, 0.00265)g of active ingredients (Table 1), which have lowest standard deviation (SD) value and these concentrations were used with MIP in a solid phase extraction (SPE) column MIP-SPE which was prepared.

Accuracy of the work for extraction and determination of Metformin 1. FT-IR Spectrum of Molecularly Imprinted Polymers for (MET):
The functional groups present in a compound can be detected using FT-IR Fourier transmission infrared spectrometer, which comprises a significant chemical characterization process.The Metformin FT-IR spectra presents multiple functional groups, in addition to Met-MIP both prior to and following the Metformin template removal. 2 show the details of bands.The spectrum of Met shows strong bands at 3392 and 3326 cm −1 for N-H2 stretching, 3307, 3288 cm −1 for Met -MIP before elution while in 3294, 3217 cm −1 after elution become smallest.As it explain in Table 2, C=N stretching bands appear at 1625, 1639 cm −1 in both Met and MIP before extraction respectively but after extraction it be very smallest, C-H aliphatic bands in Metformin have been seen at 2972, 2939 cm −1 , in Met -MIP before elution at 2975 cm −1 and after elution at 2987and 2962 cm −1 .To improve that Metformin had been removed successfully in addition to N-H2, NH str.and C=N, there is C=C group of monomer in 1413 cm −1 which disappears after eluting that means there is an interaction between C=C of monomer and N-H of template(Met.),C=O takes two form ketone1730,1731 cm −1 and enole form in (3423,3436,3458), (3502, 3440) cm −1 respectively.(The bands values 16 ).

Scanning Electron Microscopy SEM for MIP-Met:
The morphological evaluation is critical to the appreciation of the morphological traits, cavity sizes, and surface configurations of MIPs both prior to and following the Metformin template removal.SEM images were used to analyze the morphology of the Met-MIP .Fig. 6 (A,B) and the results of calculated the dimentions of six cavities are cleared in Table 3 by using image j program.We notice that the holes vary in diameter range between (12.19921-20.76062)µm and most of the holes are large, which leads to the retention of large quantities of the drug and this is consistent with the high value of the capacity in isotherm.

UV-VIS Spectrophotometry
A column (10 mL solid phase extraction of plastic syringe (height=7cm, diameter=1.5cm)(was used and each syringe was packed with different weights 0.1 and 0.2g from Met-MIP.The resulting solutions ( standard solution, pharmaceutical drugs of Metformin and serum) was poured from the top of the column and the movement of the solution was 70 rpm by electric vacuum.
A series of standard solutions of Metformin.HCl 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2 μmol/mL was prepared by dissolving 0.0116g in methanol volumetric flask 100 mL as a stock solution, after passing the solution of Metformin in syringe packed with Met-MIP, the residue which has less absorption was measured by UV-VIS instrument at 236 nm 17 , that indicates to lower concentration at final process, for good expressive example of the advantages of the use of impressed polymers in SPE in the quantification of the Metformin.
A calibration curve between standard solutions of different concentrations of Metformin.HCl (0.02-0.2) µmol /mL and their absorbance are plotted in Fig. 7.

 Adsorption Capacity and Pre-concentration:
A series of adsorption achievement for different initial concentrations of Met-MIP ranging from 0.02 to 0.2 μmol/mL on adsorption capacity μmol/g was studied for 0.1and 0.2 g weight of MIP using the following equation Eq.The relation between initial concentration Ci (μmol/ml) and capacity Q (μmol/g): The relation between capacity Q (μmol/g) and Q/Cf (mL/g):   The relation of Q µmol/g and Q/Cf (mL/g): The volumes solution of the analytes was 3mL for all concentrations 0.02-0.2μmol/mL for 0.1g weight of Met-MIP, (Table 4), while the volumes for concentrations 0.02-0.2μmol/mL for 0.2g weight of Met-MIP were 14-3 mL (Table 5), therefore, a good capacity 2.4448 µmol/g has been achieved when using 0.2g weight of Met-MIP than 1.2320 µmol/g for Met-MIP using 0.1g, that mean a larger amount of interaction position available in 0.2 g so a promote linking sites has been done and the interaction taking place in more than site.* For n=5 absorptions of drugs before isotherm process.
Thetrue value is the absorption at 0.08 ,0.1µmol/ml in calibration curve for MIP.* For n=5 absorptions of drugs before isotherm process (passing through MIP column), * The true value is the absorption at 0.1,0.12μmol/mL in calibration curve of Metformin.
In Table 6 the volumes passing through MIPs column for pharmaceutical drugs consuming mils more than standard due to interferences and additions using in manufacture drugs.

-In Human Serum 1-Sample Collection
In total, 5 ml of blood was gathered and placed in serum separator tubes (SST).The clot activator SST contained a gel in the form of an inert thixotropic polymer 19,20 , which was located at the bottom, its purpose being to separate blood cells from serum through centrifugation.This was performed for each patient and healthy individual.Blood samples were allowed to stand for 5 minutes following centrifugation at ~ 2000 rpm.The serum was frozen at 20ºC , so that it could later be employed for the estimation of Metformin.

2-Procedure
This method uses one ml of each human serum.In other words, it requires serum from the control group (healthy individuals who do not take Metformin) and the patients' group (who took Metformin drug), both of which were diluted in 10 mL of deionized water.Subsequently, 1 mL of diluted serum was placed in a 10 ml volumetric flask, to which was added 2-3 drops of 1 N HCl solution, the purpose being to eliminate the viscosity of the serum 21,22 .Methanol was used to make the volume up to 10 ml.The solutions were then warmed in a water bath for 10-15 minutes at a temperature not exceeding 60 ºc in order to create a transparent solution.
Several series of solutions were created for each control and patient group.This was realized through the transferal of 1 ml to each eleven volumetric flask (10 mL) )we doubled the amount of serum to get the quantity needed for 11 volumetric flask) followed by the addition of constant volumes of standard Metformin (0.1 mL) from different concentrations 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2 µmol/mL to obtain 0, 0.0002, 0.0004, 0.0006, 0.0008, 0.001, 0.0012, 0.0014, 0.0016, 0.0018, 0.002 µmol/mL.Flask No.1 is the sample (serum).The findings were subjected to mathematical evaluation (M1V1=M2V2 for the standard addition method) (see Table 7).Furthermore, the absorption recorded for https://dx.doi.org/10.21123/bsj.2023.8225P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal each volumetric flask was gauged with the assistance of UV-Visible spectrophotometry, which focused on the control serum and then measured the patient serum at the maximum 236 nm absorption, the objective being to eradicate the majority of interferences.Subsequently, the resultant solution was scanned in the 190-300 nm range.Fig. 15 presents the calibration curve that was plotted between the concentrations and absorptions.Metformin in serum was statistically evaluated by considering the length of time the drug was in the body of the patient, the rate at which it was metabolized, and the medication dose.These variables differ between patients.In addition, it has an onset of action of about 1.5 hours, half-life in the circulation of about 1.5-4.9hours, and duration of action of 16-20 hours 23 Calibration curve between concentrations and absorptions.
Figure 10.Calibration curve between concentrations of Metformin in serum using standard addition method µmol/ml and its absorbance.

Discussion
This paper presents a comparison between two approaches to the drug Metformin.The T-Test statistical evaluation 24,25  It found t calculated < t tab at confidence level 95% therefor there is no significant difference between two approaches, So Null hypothesis will be accepted.

Conclusion
New and bulk polymers were created by using styrene and crosslinking ethylene glycol dimethacrylate EGDMA C10H14O4 as Met-MIP, different studies and experiments were used to reach for selective molecular imprinted polymer by prepare and optimize required monomers, crosslinker using suitable solvents , porogen solvent for template removal and the optimal molar ratios of Template (Metformin)to monomer to cross-linker.Irregular shapes three-dimension network structure of polymers can be seen by SEM before and after removal template, FTIR, all improve the healthy work.
One slope gain when studying the capacity of adsorption of Met-MIP with uniform values (homogeneous structure) in this study proves that the capacity increases with increasing the weight of the MIP.The maximum adsorption capacity was 1.2320, 2.4448 µmol/g for two studies of 0.1,0.2g of Met-MIP respectively.A standard addition method using to eliminate the interferences when detect the concentration of Metformin in human serum.T-Test statistical evaluation was designed to facilitate a comparison between the identification of Metformin once it passed through the Met-MIP syringe solid phase extraction process and the human serum at 236 nm and when it found that t calculated < t tab at confidence level 95% by UV for Metformin drug therefor there is no significant difference between two methods.So Null hypothesis will be accepted.

PublishedScheme 1 .
Scheme 1. Suggested mechanism of the synthesized of Met-MIP, using styrene as a functional monomer, EGDMA as cross-linker and the Structure of synthesized polymer after elution process.

Figure 6 .
Figure 6.A, B surface morphologies of the particles before and after elution for Met-MIP respectively, and three dimensions of cavities with their areas.

Figure 7 .
Figure 7. Calibration curve between concentration of Metformin standard µmol/mL and its absorbance.

Concentration of Metformin µmol /mL by standard addition method Absorbance
Published Online First: October, 2023 https://dx.doi.org/10.21123/bsj.2023.8225P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal through Met-MIP column was studied in pharmaceutical drugs solution and human serum.*To know the concentration of drug in human serum we must multiply this concentration 0.0209µmol /mL x 10 (Dilution coefficient).