SELECTIVE EXTRACTION OF GLIMEPIRIDE IN PHARMACEUTICAL PREPARATION AND IN HUMAN SERUM VIA SYNTHESIZED MIP-SPE TECHNIQUE

This paper demonstrates that the synthesizing and storage of molecular-imprinted polymers (MIP) at room temperature using bulk polymerisation of Glimepiride (Glim.) is characterized by high sensitivity, reduced costs, increased stability, and extended life. The research used 1:15:20 mmol ratios of template, monomer and cross-linking agents for the polymerisation in order to ensure an appropriate adsorption capacity. Benzoyl peroxide BPO was employed as the initiator for the functional monomer Allyl chloride C 3 H 5 Cl , cross-linked with Ethylene glycol dimethacrylate EGDMA C 10 H 14 O 4 , thereby creating MIP for Glimepiride (Glim-MIP) that could be characterised with UV-Visible Spectrophotometry at 274.5nm, for pharmaceutical drugs. Fourier-transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM) was used for the human serum. The elution process that was applied to the template (Glim.) from the Glim-MIP created cavities that were caused by the porogenic mixture solvents that were created from (acetic acid, methanol) 1:9 respectively, successfully removed by repeated washing for 20 hours, the polymer was dried at room temperature. The maximum adsorption capacity was 11.7797 µmol/g using (0.1) g weight of Glim-MIP. which adhered to the Langmuir isotherm model. A solid-phase extraction (SPE) syringe packed with molecular imprinted polymers (MIPs) was employed to selectively separate and pre-concentrate the Glimepiride in multiple pharmaceutical drugs from several sources. The human serum was based on the use of deionized water to dilute the serum, followed by heating of the serum with methanol. Subsequently, few drops of 1N hydrochloric acid were applied to detect Glimepiride at UV region 274.5 nm by applying the standard addition method.

It was found to be practically insoluble in water, slightly soluble in dichloromethane and very slightly soluble in methanol.It was soluble in DMSO (>10 mg/ml) and ethanol (<1 mg/ml).In acidic and neutral aqueous solutions glimepiride exhibits very poor solubility at 37 o C (<0.004 mg/ml) (Kari et al., 2023).
A molecular imprinting polymer (MIP) creates a multifaceted monomer (Samarth et al, 2015).A highly cross-linked polymer structure was used to secure functional groups in situ.Moreover, the steric patterns of these connections and the template are significant for the formation of binding sites that supply the shape, size, and flexibility required to encourage selective identification, in addition to elevated target correspondence.Consequently, the process can be deemed to be comparable to enzyme-proven mechanisms or substrata.Hence, the complex was created in a manner akin to a lock/key model    In this work identify the MIP preparation was performed in conjunction with the recognition site Allyl chloride C3H5Cl with crosslinking Ethylene glycol dimethacrylate EGDMA C10H14O4, whereby benzoyl peroxide BPO functioned as the target molecule (Glimepiride) initiator.Subsequently, the impact of monomer dosage on adsorption performance was observed.This study also examined the adsorption behavior of diverse functional monomers, cross-linking agents, and solvents.SEM, FTIR was employed to characterise the primed MIPs.Furthermore, this study investigated the impact of solid phase extraction and initial Glimepiride concentration on the adsorption capacity.

MATERIALS
Glimepiride standard as template from Samarra/Iraq was provided , Allyl chloride as monomer, EGDMA as cross-linker and Benzoyl peroxide as initiator were purchased from Sigma Aldrich (St. Louis, MO, USA, www.sigma-aldrich.com), Methanol, Nitrogen gas (99.99) supplied by Al-Watan factory (Al-Nahda street/ Baghdad/Iraq), Chloroform and Acetic acid were purchased from Merck (Darmstadt, Germany), Glimepiride/UK and Glypride/ julphar Emirate as pharmaceutical drugs of glimepiride purchased from pharmacy.

METHODOLOGY
With the recognition sites of monomer allylchloride C3H5Cl, crosslinking Ethylene glycol dimethacrylate EGDMA C10H14O4 with benzoyl peroxide BPO as initiator was synthesized for the target molecule Glimepiride: 1. Glim-MIP was prepared by dissolving 1mmol of Glimepiride 0.4906g in 5-6 drops of 1N HCl after that methanol was added.The resultant solution was stirred and warmed for 10-20 seconds to obtain a transparent solution.2. A 15 mmol of allylchloride 1.1480g with 2 ml methanol was added.3. The mixture (step1 and 2) was allowed to stand for a few seconds at room temperature.4. A cross-linker 20 mmol Ethylene glycol dimethacrylate EGDMA 3.9644g with 2ml methanol and 0.3 g benzoyl peroxide dissolved in chloroform as an initiator were added to the above solution.5.The ratio 1:15:20 Glim-MIP was completed, the solution was shaken and bubbled for 20 min with pure nitrogen gas to remove the dissolved oxygen from the monomer solution immediately.6.The tube was sealed with a rubber stopper.The solution was left overnight in a water bath at 60 ºC for 72 hours.A white color polymer with a fluff structure was formed, Figure 4. 7. Soxhlet solid liquid phase extraction for the template was performed to remove the template glimepiride from MIP using a porogenic solvent (acetic acid, methanol) 1:9 respectively, successfully removed by repeated washing for 20 hours, 8.The MIP was dried at room temperature, after that it was crushed with mortar, sieved to particle size 125μm.9.A plastic syringe (10 ml) of solid phase extraction vacuum (column) was used, and each syringe packed with 0.1 g of Glim-MIP.The preparation of imprinting polymer in the laboratory: firstly combined template, monomer, cross linker and initiator, mix well in shaker, bubbled with nitrogen gas, the polymer become solid MIP after placed in a water bath, drying and grinding, Soxhlet solid liquid extraction to separate the template, crushed and sieved the MIP to required a suitable particle size( using 125μm), packed in a cartridge to prepare a column for isotherm process, finely store MIPs in suitable containers.
A solutions (standard solution, pharmaceutical drugs of glimepiride and serum) was poured from the top of the column and the movement of the solution was by electric vacuum at 70 rpm.A series of standard solutions of Glimepiride (0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2) μmol/ml was prepared by dissolving 0.0589g Glim. in 1-2 drops of 1N HCl to create a buffer solution, after which methanol was added.The resultant solution was stirred and heated for approximately 15-20 seconds methanol volumetric flask 100 ml as a stock solution.A calibration curve was constructed between concentration of Glim.and its absorbance A, This was achieved using at (274.5 nm by UV-VIS instrument).

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It can be seen that the spectra for Glim-MIP before and after removal Glimepiride have approximately similar bands, that mean elution process has a slight impact on the architecture structure Table 2.

Table (2):
The structures of the main three compositions of Glim-MIP and the bands indicate MIP before &after removal template

SEM of molecularly imprinted polymers for (Glim.):
Figure 10 (A,B) shows the surface morphologies of the particles before and after elution for Glim-MIP and table 3 shows the measurements of 5 selected cavities   From Figure 10 and Table 3 the 3D of Cavities between min = 7395.47nm(7.3954µm) to max = 14655.9nm(14.6559µm ) we notice that the holes vary in diameter range between (7395.47-14655.9)nmand most of the holes are deep, which leads to the retention of large quantities of the drug and this is consistent with the high value of the capacity in isotherm.Adsorption capacity and pre-concentration for Glim-MIP: A series of absorption achievement for different initial concentrations of Glim-MIP ranging from 0.04 to 1.2 μmol/ml on adsorption capacity μmol/g was studied using the following equation (Al-Janabi, 2017;

Ci-initial concentration , Cf -final concentration (after passing through column packed with Glim-MIP)
Pre-concentration refers to the process of obtaining a high local concentration at the sensor surface, the concentrations from (0.06-1.2)μmol/ml consume (3)ml range of volumes while at concentration 0.04μmol/ml consume (4)ml, when using 0.1g weight of Glim-MIP, Table 4.

Table (4):
The optimal synthesis conditions for the molecularly imprinted polymer for Glimepiride developed in this study in 0.1 g of MIP Relation between initial concentration Ci (μmol/ml) and capacityQ (μmol/g):  Yasar & Konukoglu, 2020), 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 kept at 20ºC so that it could later be employed for the estimation of Glimepiride.

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 Glimepiride) and the patient group (who take Glimepiride 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 (Constable et al., 2019).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.

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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 Glimepiride (0.1 ml) from different concentrations ( 0, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.2) µmol/ml to obtain (0, 0.0004, 0.0006, 0.0008, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.012) 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 6).Furthermore, the absorption recorded for 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 274.5 nm absorption, the objective being to eradicate the majority of interferences.Subsequently, the resultant solution was scanned in the 200-350 nm range.Fig. 13 presents the calibration curve that was plotted between the concentrations and absorptions.

Table (6):
Results of standard addition for the estimation of Glim in human serum.
Glimepiride 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, Glimepiride is reported to undergo hepatic metabolism, the elimination half-life of glimepiride is approximately 5 -8 hours (Li et al., 2016) Calibration curve between concentrations and absorptions.When y= 0.4887 that mean the absorbance of Glimepiride in this sample of serum is 0.4887 It found that the absorption 0.4887 are nearest to the absorption 0.3314 which has concentration 0.20 μmol /ml in calibration curve (Figure 12) and substituting for y= 0.4887 the concentration is 0. 209µmol /ml.That mean the concentration of Metformin in this sample of serum is 0.2949 µmol /ml by ratio and proportion.so, a comparison for absorption of this concentration after passing through Glim-MIP column has been studied in pharmaceutical drugs solution and human serum.*To know the concentration of drug in human serum we must multiply this concentration 0.2949µmol /ml x 10(Dilution coefficient).

DISCUSSION
This paper presents a comparison between two approaches to the drug Glimepiride The T-Test statistical evaluation (Deckard, 2016;Haaland & Thomas, 1988), was designed to facilitate a comparison between the identification of Glimepiride once it had passed through the Glim-MIP syringe solid phase extraction process and the human serum at 274.5 nm: //=  − /((√ / + /))

(
Figure (4):The preparation of imprinting polymer in the laboratory: firstly combined template, monomer, cross linker and initiator, mix well in shaker, bubbled with nitrogen gas, the polymer become solid MIP after placed in a water bath, drying and grinding, Soxhlet solid liquid extraction to separate the template, crushed and sieved the MIP to required a suitable particle size( using 125μm), packed in a cartridge to prepare a column for isotherm process, finely store MIPs in suitable containers.

(
2024) 16(1): 70-87 Iraqi Journal of Market Research and Consumer Protection ‫المستهلك‬ ‫وحماية‬ ‫السوق‬ ‫لبحوث‬ ‫العراقية‬ ‫المجلة‬ least 30 minutes, the solution was filtered to get rid of undissolved materials, the residue was washed with methanol and completed the volume to 100ml with methanol.Table (1): Pharmaceutical drugs prepared for treating with Glim-MIP polymer work for extraction and determination of Glimepiride UV-VIS Spectrophotometry A calibration curve between concentrations of standard Glimepiride (0.04-1.2) µmol /ml and their absorbance was plotted figure 5.

Figure ( 10 )
Figure (10): A,B FTIR spectrum of Glim-MIP before and after extraction (after removal the template Glimepiride.
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Figure ( 11 )
Figure (11): A, B surface morphologies of the particles before and after elution for Glim-MIP respectively, and three dimensions of cavities with their areas.

Figure ( 14 ):
Figure (14):Calibration curve between concentrations of Glimepiride in serum using standard addition method µmol/ml and its absorbance.

Table ( 3
): Calculated mean, angle , lengths of some cavities (selected six of them) and their areas.

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Results of maximum capacity in µmol/g for Glim-MIP using 0.1g weight of MIP 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(Schrapp