SYNTHESIS OF NEW LEVOFLOXACIN SELECTIVE MEMBRANE SENSOR BASED ON MOLECULARLY IMPRINTED POLYMERS.

Two molecular imprinted polymer (MIP) membranes for Levofloxacin (LEV) were prepared based on PVC matrix. The imprinted polymers were prepared by polymerization of styrene(STY) as monomer, N,N methylene di acrylamide as a cross linker ,benzoyl peroxide (BPO) as an initiator and levofloxacin as a template. Di methyl adepate (DMA) and acetophenone (AOPH) were used as plasticizers , the molecular imprinted membranes and the non molecular imprinted membranes were prepared. The slopesanddetectionlimits of the liquid electrodes ranged from-21.96–-19.38mV/decade and 2×10 -4 M- 4×10 -4 M,and Its response time was around 1 minute, respectively. The liquid electrodes were packed with 0.1 M standard drug solution and its response were stable at pH ranges from 1.0 to 11.0 and with good selectivity for more than several type. The electrodes produced have been successfully applied in preparation of the pharmaceutical sample for the determination of the analyte without any time consuming pretreatment steps.


INTRODUCTION
The first documented in the molecular imprinting technique in 1970Nishide&Tsuchida (1976), molecular imprinting is a commonly emerging technique for producing polymers with different molecular properties for a given compound, its analogs, or an enantiomer(Whitcombeet al., 1998; Yan & Row, 2006).MIPs are the sensing elements of a molecular imprinting electrochemical sensor (MIECS) which are molecularly imprinted. MIECS has many characteristics include: high selectivity, simplest techniques, lower costs, low D.L, highly stable. The combination of both the template molecules and the cavities can also be easily achieved and is carefully applicable even with harmful chemical materials. Thus, MIECS was used for optical and electrochemical application (Tadi&Motghare, 2016; Al-Bayati&Abd, 2017; Al-Bayati& Al-Safi, 2018; Momeneh & Gholivand, 2018; D'Aurelioet al.,2020).Molecularly imprint polymers (MIPs) are prepared By combining template molecule(LEV) with functional monomers styrene (STY), a cross-linker N,Nmethylene di acrylamide (N,N MDAM) and initiator benzoyl peroxide (BPO) in appropriate solvent. Most of the time, Aprotic and unipolar solvents.So after polymerization, template molecule extraction reveals recognition cavities that complement the template molecule's shape, height, and chemical functionality, allowing the resulting polymers to selectively rebind the template molecule from a mixture of closely related compounds (Lavignacet al., 2004) LEV is a broad-spectrum racemic fluoroquinolone antimicrobial ( Figure 1).It is the ofloxacin 's active L-isomer, and its antibacterial efficacy is around two times that ofloxacin. and are widely used today in both human and veterinary applications Medicine as antibacterial agents of different diseases (Turielet al., 2007). Over the past few years,the research on antibiotic residues in the multiple environmental compartments has been of great importance due to the biodiversity and public health consequences (Turielet al., 2007).

MATERIALS AND METHODS
Levofloxacin was purchased from company of drug manufacturing and medical supplies (IRAQ-SID-Samara). generic levofloxacin tablets purchased from local pharmacies are; Levoneer 10 tablets 500 mg from (Pioneer-Iraq) levofloxacin 7 tablets 750 mg from (Ilflox-Turkey).Di methyl adepate (DMA) and acetophenone (AOPH) also metal salts, they were obtained by Sigma-Aldrich and used as provided. Styrene (99%), monomer N,Nmethylenediacrylamide (N,N-MDAM) (99%),and benzoyl peroxide (BPO)(78%) from Sigma-Aldrich is obtained. the chemical used was the largest purity reagent and was used when obtained without further purification.

Apparatus
A digital voltmeter (HANA pH211instrument Microprocessor pH meter) was used to perform potentiometric measurements. Digital pH meters (wissenschaftlich-Technische Werkstätten GmbH WTW pHmeter in lab pH720-Germany) were used for pH measurements. Electrode efficiency was investigated by calculating the potential of levofloxacin solutions at ambient temperature with a variety of concentrations from 5×10 -4 M to 10 -1 M. Every solution was stirred, and the reading potential was recorded at equilibrium. The calibration curves obtained by plotting the response against the levofloxacin concentration logarithmic functions.

Synthesis of the Imprinted Polymer (MIP)
For the preparation of levofloxacin molecularly imprinted polymer (LEV-MIP), 1 mmol (0.34 g) of levofloxacin was mixed with 2mmol (0.208 g) styrene as the monomer. After this, 10.6mmol (1.634 g) N, Nmehtylene di acrylamide was added to the solution as the cross linker, followed by (0.3g) benzoyl peroxide as the initiator. All these materials were subsequently dissolved in 5 mL mixture of methanol and chloroform, and the mixture was stirred for 5 minutes to obtain a homogenous solution. Afterwards, the gas N2 was passed through the ‫املستهلك‬ ‫ية‬ ‫ومحا‬ ‫لسىق‬ ‫ا‬ ‫لبحىث‬ ‫لعراقية‬ ‫ا‬ ‫اجمللة‬

Iraqi Journal of Market Research and Consumer Protection
solution for 30 minutes to remove oxygen from it, and the solution was placed in a water path at 65°C. When the reaction was complete, the molecularly imprinted polymer became hard, and, after the polymerization process, the polymer was dried and crushed to obtain it as particles. Finally, these particles was sonicated in CHCL3/ CH3OH / CH3COOH (7 : 2 : 1)( v/ v/v) to remove the template from the MIP. The particles size of LEV-MIP (125 μm).
The preparation of non-molecularly imprinted polymers was carried out using the same method, using the same compounds and under the same conditions as in LEV-MIPpreparation, but without levofloxacin. A PVC tube (1-2 cm long) was flattened and polished by placing it on a glass plate and soaking it with THF. Similar to the actual diameter of the PVC tubing, the membrane was then sliced and pasted onto the polished end. The other end of that was attached to an electrode of Ag-AgCl.

Scanning Electron Microscope (SEM)
The SEM is used to get an understanding of the pore 's membrane thickness, composition, and surface distribution. SEM analysis showed that molecular impressed polymer has a strongly ordered and normal pore structure in surface and cross-section which serves as interface sites. Several papers have shown that due to the shape and function of the porous structures, the amolecular impressed membrane of this type recognizes that the molecule of the template is quickly transported with good efficacy. The morphology of MIP and non-imprinted polymer NIP membranes before and after washing showed by electron microscope in (Figure2).

Construction of Ion-Selective Electrodes
Construction of Ion-Selective Electrodes As shown by Mahajanet al. (2007), electrode body building and immobilization have been achieved. The solution of Levofloxacin 0.1 M was filled as an internal solution in the glass tube. Membrane was preferred tobe immersed in a standard solution of 0.1M levofloxacin for at least three hours before measurements representing membrane electrode stipulations.

Preparation of Pharmaceutical Samples
The drug tablets were ground to powder by using pestle and mortar. Subsequently, a required weight of the powder was used to prepare 100 mL solutions. Here, a certain amount of powder was dissolved in methanol (CH3OH) and stirred by magnetic stirrer for 30 minutes to completely dissolve the powder. The solution was completed to 100 mL by methanol to prepare 1×10-3 M and 1×10-4 M levofloxacin solutions.

RESULTS AND DISCUSSION
MIP based liquid electrodes, their concentrations range and slopes response to Nernstian equation has been investigated. The membranes of MIP made of the monomer styrene with a PVC matrix using two plasticizers DMA and AOPH. The internal solution was used 0.1M aqueous standard solution of drug for all liquid electrodes. Experimental results of synthesis of molecularly imprinted (MIP) and non-imprinted polymers (NIP) based on monomer Styrene indicate that monomer can be used for the preparation of effective MIP for Levofloxacin. The plasticizer is an essential part of the sensing membrane which have important role as a solvent for the different components and determines the mobility of the analyte in membrane. Both of the plasticizers that are used, DMA and AOPH, are suitable for the fabrication of MIP-based LEV electrodes. (Table 1) show the parameters of the fabricated and tested electrodes, Two membranes of the different compositions were prepared using two different plasticizers Di methyl adepate (DMA)and acetophenone (AOPH). The results of electrode specification were obtained from the calibration curves that listed in (Table 1). The slopes of the electrodes ranged between -21.96 --19.38 mV/decade ( Figure 3). In generally the preparation electrodes have a short response time (about 60 second) mostly at high concentrations. Table(

Effect of pH on electrode response
The pH effect of the two electrode potential values for pH varied from 2.5 to 10 was studied and the pH effect was modified by adding drops of 0.1 N HCl and 0.1 M NaOH to aqueous drug solutions and then the potentials obtained for each value were recorded. With three concentrations of standard drug solutions 1×10 -2 , 1×10 -3 and 1×10 -4 M, the pH effect on the electrode potential has been reported The results obtained are shown in (Table 2) and the typical pattern of electrode potential versus pH for electrode M1 and M2 as seen in (Figure 4).

Response time and life time
Response time for both MIP.DMA and MIP.AOPH electrodes was obtained from dynamic potential reaction at range of concentration 5×10 -5 -1 ×10 -1 M by calculating the time needed to achieve 95 percent of the equilibrium potential. The findings show that the electrode reaction time was approximately 15 seconds for the solution of levofloxacin at a high concentration of 10 -1 M and approximately 46 seconds at a low concentration of 10 -5 M. The electrode lifetime was obtained by measuring the slope periodically from calibration curves for Lev. MIP. short life time was noticed for electrodes M1 and M2 (Table-1

Interference Studies
The separate Solution Method (SSM) has been used to test potentiometric sensor selectivity coefficients for different species. In the SSM, two separate solutions are used to evaluate the potential of a cell comprising an active electrode and a reference electrode: one containing drug ions, E1, and the other containing interference ion potential (E2), respectively. The coefficient of selectivity was determined using the following equation: Log K pot =(E 2 -E 1 )Z 1 F/2.303RT(1-Z 1 /Z 2 ) log a 1 .

Titration Methods (Titrimetry)
These methods relied on the identification of the end point of the titration. Such techniques have been using volumetric analysis between the concentrations and the reactants often make certain shifts slowly, resulting in a significant shift in the electrode reaction. Titration between the levofloxacin and ligand phosphomolybic acid (PMA). The findings for parameters RSD percent, RC percent and RE percent for all electrodes are shown in (Table 5)

Applications of pharmaceuticals
Ion sensitive electrodes based on molecularly imprinted polymers have been used for the determination of levofloxacin in pharmaceuticals. This ISE tests including: direct, standard addition, multiple standard and Gran plot. Levofloxacin preparation solutions at concentrations of 1×10 -3 and 1×10 -4 M.The RE percent, RC percent and RSDpercent were measured for Levofloxacin in pharmaceuticals. The results obtained represented in the (Table6 and 7).