ELECTROCHEMICAL PREPARATION OF A MOLECULAR IMPRINTED POLYMER ELECTRODE FOR ESTEMATION OF ASPIRIN USING TWO DIFFERENT FUNCTIONAL MONOMERS.

For aspirin estimated, a molecularly imprinted polymer MIP-ASP electrodes were generated by electro-polymerization process, the electrodes were prepared by combining the template (aspirin) with (vinyl acetate (VA), 1-vinylimidizole (VIZ) as a functional monomer and N, N-methylene bisacrylamide (MBAA) as crosslinkers using benzoyl peroxide (BPO) as an initiator. The efficiency of the membrane electrodes was analyzed by differential pulse voltammetry (DPV). Four electrodes were synthesized using two different plasticizers, di-butyl sebacate (DBS), di-octyl phthalate (DOP) in PVC matrix. Scanning electron microscopy (SEM) was used to describe the generated MIP, studying the electrodes properties, the slope, detection limit, and life time and linearity range. The effect of PH and interferes on the efficiency of the MIP electrode was investigated. The study has shown that the molecularly imprinted electrodes have high sensitivity and responsiveness to aspirin. The DPV value was linearly dependence on the aspirin concentration and a linear curve was obtained within the range of (1×10−1-5×10-4) M of aspirin with correlation coefficients are about (0.9974, 0.9966, 0.9938 and 0.9961) with slops value of (-21.41, -17.67, -17.47 and -18.67) and the detection limit for all electrodes ranging from (7.5×10-5-1×10-4) M. The molecularly imprinted electrode exhibited a good response with highly reproducible and no effect on interferes frequently available in pharmaceutical formulations. The approach employed is easy and fast. Also ASP membranes get a limited time of response, excellent mechanical stability, removable and are easy to construct. The results indicate that the strongest two peaks are missing at ~1691 cm -1 and ~1755 cm -1 for carbonyl group stretching C=O and C=C aromatic stretching respectively, while the characteristically peak appears at ~1627 cm -1 due to interaction between the ASP-template and the VIZ-monomer, However when the ASP-template removed after washing noticed that the carbonyl group at ~1658 cm -1 and the hydroxyl group at ~3300 cm -1 are missing, that also indicated the template are completely extracted in the soxhlet extraction stage from MIP 2.


INTRODUCTION
An advance in molecularly imprinted polymers (MIPs) is a useful method to a preparatory work of the polymeric materials with active sites. (Lata et al., 2015). MIPs are polymers that are manufactured to create cavities with attraction to a selected "template" molecule within the polymer chains. They have been prepared by complexing the target (template) with the FM, whether by covalently or non-covalently bonding, accompanied by polymerization with large quantities crosslink's to establish a highly cross linked polymeric network. When the target-molecule is extracted from of the polymeric matrix , different recognizing locations are identified which are comparable to the target in terms of size, shape and function  Once the template have been extracted, the active site representing the spatial configuration of a target molecules will be obtained as well as the electrochemical response has been received because when MIP is associated with a particular template the electrosignal was recorded depending on the active site, as well as the targeted molecule concentrations can be estimated (Crapnell et  As in present study, four of the MIPs have been manufactured as recognizing materials using [Vinylacetate (VA), 1-Vinylimidizol (VIZ)] as functional monomer, N, N-methylene bisacrylamide (MBAA) is a crosslinkers also benzoyl peroxide (BPO) as an initiators using methanol as porogen solvent. The efficiency of the MIPs was tested using rebinding equilibrium assays. The highest efficiency of the MIP was selected as the identification substance in PVC membrane for the estimation of aspirin (ASP) in pharmacological samples.
Aspirin (ASP) is widely used in pharmaceutical formulations as an analgesic and antipyretic agent for relieving headaches, fever, muscle pains and inflammation severe arthritis. so the wide use of ASP had led to the therapeutic intoxication due to overdose , which can be found in individuals with chronic inflammatory diseases and routinely take ASP

MATERIALS AND METHODS
The standard of aspirin was gained from of the government drug manufacturer (IRAQ-SDI-Samara). Aspirin tablets (100 mg) Chewable tablet, acetylsalicylic acid ((SDI)-IRAQ and Memphis/Bayer) were purchased from local pharmacies.Plasticizers: (DBS) (97.0% purity) and (DOP) (99.5% purity), were purchased from Sigma Aldrich. Other chemicals and reagents materials were obtained from Fluka, BDH and Sigma Aldrich.

Molecular imprinting polymer synthesis
The first aspirin molecularly imprinted polymer (ASP-MIP 1 ) prepared by mixed 0.5 mmol (0.0901 g) from aspirin then mixed with 3 mmol (0.26 g) vinyl acetate (VA) as the monomer, after that added 15 mmol (2.313 g) N,N-methylene bis-acrylamide (MBAA) to the solution as the cross-linker, followed that added (0.32 g) benzoyl peroxide as the initiator. All these materials were dissolved in 5±mL methanol (CH 3 OH) except the initiator have dissolved in 3 Ml chloroform. While the second aspirin molecularly imprinted polymer (ASP-MIP 2 ) were achieved by mixed 0.4 mmol (0.0721 g) from aspirin as the template, 2.4 mmol (0.226 g) 1vinylimidizol (VIZ) as the monomer, 12 mmol (1.85 g) N,N-methylenbisacrylamide (MBAA) act as crosslinkers also (0.3 g) benzoyl peroxide as the initiator which dissolved in 5mL of methanol (CH 3 OH). For obtaining a homogeneous solution, the mixture was stirred for 5 minutes. N 2 e f 3 i e he i e e e ge f he i f e h he i i e h he he reaction completes the molecularly imprinted polymer became hardened, after the polymerization process the polymer was drying and crashed to obtain a polymer particle. These particles were sonicated in acetonitrile/ CH 3 COOH (18:2 v/v) to remove the template from MIP. The polymer was dried for (42-75) hours at room temperature, crushed and ground the polymers by mortar and pestle and sieve to ge 125 μ ic e ize ( i g 125µ e h ie e); f e d ied c e e e h been used in the membrane of the selective sensors as an active substance. To fabricate of electrode, The PVC tubing (1-2 cm long) was placed that on a glass slide and soaking it with THF. Similar to an average thickness of the PVC tubing, the membranes also was cut and pasted onto the end tube other end of that was connected to an electrode of Ag-AgCl.

Preparation of pharmaceutical samples.
The mortar has used to create the powder of the pharmaceutical tablets. There were two different types of ASP-tablets used it to estimate the molarity concentration of ASP-drugs. An appropriate weight of powder was then taken to prepare of (1×10 -3 and 1×10 -4 ) M from the pharmaceutical sample solutions. Suitable quantity of methanol has been used to dissolve pharmaceutical samples and to complete volumes of up to 100 mL.

Scanning Electron Microscope (SEM)
A morphological characteristics of the MIPs before and after template removal membranes was evaluated by scanning the electron microscope using Tokyo / Japan-JSM-6390 A, in order to show the differences between both the SEM image of both the MIPs before and after the template obtained in proportion to the size and surface morphology of a polymeric particles. SEM analysis indicates that molecular imprinted polymer in surface and in crosssection, had a highly ordered and regular pore structure which serves as the sites of interaction, in (Figure 2a

RESULTS AND DISCUSSION
Four MIP membranes are prepared using two different monomers VA and VIZ with PVC matrix and two different plasticizers DBS and DOP, the ions selective polymer membrane is one of the most crucial components of ISEs. It isolates the internal reference solution from the external analytical sample solution. Polymeric membranes must have selectivity for different analyte ions, un-porous, insoluble in water and mechanically stable, based on the natural of the membrane material used. Other critical components of an ISE assembly are the internally and externally reference electrodes. Collection consisting of both reference electrodes and also the polymeric membrane is referred to as cell the characteristic of these membranes were studied, including: slope, detection limit, linearity, life time and the response to the Nernstian equation were investigated; the results in (

Infrared spectroscopy (FTIR-Analysis)
The FTIR is a commonly utilized method of substance characterisation. The spectral generated from of the analysis provides the specific samples their identities. The peak frequency of absorption relate to signature vibration of binds between all the atoms that create up the product. The FTIR spectrum is therefore a substance characteristics and allows accurate recognition.The listed in (Table, 2& 3) showed a band that before/after removal ASP-template from MIP 1 & MIP 2 respectively. Table (2). The FT-IR spectra for ASP-VA polymer before/after removal clop-template.
From this table can be seen (before ASP removal ) that the characteristically peak at ~1733 cm -1 is attributable to vibrational mode of -COOR and the vibration version of amide N-C=O is allocated at ~1654 cm -1 these notes indicated to fact that the interaction occurred between the template (ASP) and the monomer (VA), while after ASP removed the detected peak characteristics are missing in carboxylic spectra at ~1654 cm -1 , carbonyl group at ~1720 cm -1 and also missing the absorption band of C=C at ~1521 cm -1 . The results indicated that the height contrast of imprinted polymer FT-IR spectra before and after template removal proves that ASP-template has been fully extracted from MIP 1 in the extraction stage by soxhlet.  The results indicate that the strongest two peaks are missing at ~1691 cm -1 and ~1755 cm -1 for carbonyl group stretching C=O and C=C aromatic stretching respectively, while the characteristically peak appears at ~1627 cm -1 due to interaction between the ASP-template and the VIZ-monomer, However when the ASP-template removed after washing noticed that the carbonyl group at ~1658 cm -1 and the hydroxyl group at ~3300 cm -1 are missing, that also indicated the template are completely extracted in the soxhlet extraction stage from MIP 2.

Effect of pH on ASP-electrodes
The pH dependency of the electrode sensor membrane was measured at a pH range of 1× (10 -4 , 10 -3 and 10 -2 ) M aspirin concentrations (Figure 3). PH modifications were made with (HCl or NaOH) solutions. The results in (Table, 4), indicate that the potentials slightly change and remain constant from ~pH (3.0 to 9.0). Therefore, this range can be considered as the pH of working electrode senses. The behaviors of this membrane can be explained as follow: a) the pKa of aspirin is about 3.0, i.e. at acidic pH, observed that the potential will be relatively high at this range; this might be because the membrane can responds to H + activity. b) at higher pH 9.0, it becomes increasingly dissociated for this explanation we have noticed a decline in potential.

Selectivity of ASP-electrodes potentiometric
The influence of interferences on the electrods-response behavior is usually described as the selectivity coefficients. Nernst response, or when the ions concerned is different in charge. As seen from results in (Table 5 and 6) the selectivity coefficients achieved for all prepared electrode sensors, that most of these compounds did not interfere with the response of the electrode sensors membrane.

Analysis of commercial tablets
In order to illustrate the technical use of the electrochemical sensor, two tablets for (ASP) were analyzed by MIP electrodes. The solutions was obtained by dissolving the specific weight of commercial tablets in methanol solvent and diluted so that the tablet concentration range lies within calibration plot values. The DPVs value then was recorded under precisely the same conditions. The suggested technique was used to measure the concentration of all selected drugs in two types of pharmaceutical products. In order to verify the electro-chemical detection, we have compartmented the data results for parameters RSD%, RC% and Erel% with both detection methods including: direct potentiometric, standard addition method (SAM), multi standard addition method (MSA), by using ISE, as well as titration method and optimal chromatographic conditions. The results was indicated in (Table, 7&8) Table (7). Recovery results and standard deviation of ASP-drugs obtained through the use of (MIP1+DBS).

Adsorption Isotherm
Adsorption isotherm is useful in understanding the adsorption mechanism of the adsorption template on a polymer surface. The collected data from the adsorption isotherm equilibrium were studied to illustrate the isotherm model Langmuir or Freundlich this was accomplished by plotting the ability of binding (Q) against free drug concentration, Q is determined according to the equation below: Adsorption isotherm produced after preparation of different concentrations of standard solution at room temperature is shown in (Figure,3). Experimental results for classifying experiments have been included in the (Table, 9).

CONCLUSION
In this research, four electrodes were prepared based on MIP method using two monomer (VA, VIZ) and two different plasticizer (DBS, DOP), as it was observed that the interaction between template and the monomer was non-covently, therefore the ASP-drug was extracted easily to form selective cavity for estimation commercial ASP and excellent results obtained at lowest costs and with high accuracy.