Synthesis and Characterization of Molecularly Imprinted Polymers for Metronidazole by using Allyl Chloride and Allyl Bromide as Monomers

 

Fadhel Ibrahem Aljabari ¹, Haider Abdulkareem Almashhadani^2,5*, Marowah H. Jehad³, Mustafa M. Kadhim^4,6

¹Department of Dentistry, Dijlah University College, Baghdad, Iraq.

²Department of Dentistry, Al-Rasheed University College, Baghdad, Iraq.

³Department of Dentistry, Al-Farabi University College, Baghdad, Iraq.

⁴College of Dentistry, Al- Farahidi University, Baghdad, Iraq.

⁵College of technical engineering, The Islamic University, Najaf, Iraq.

⁶Medical Laboratory Techniques Department, Al-Turath University College, Baghdad, Iraq.

 

ABSTRACT:

Metronidazole-MIPs were prepared by using (MDZ) as the template as well as allylchloride (AYC) or allylbromide (AYB) as monomer, used (TMPTA) tri-methylol propane tri-acrylate or ethylene glycol di-methyl acrylate (EGDMA)  as cross-linker and initiator used  (BP) benzyl peroxide. By using different plasticizers (di butyl Phthalate (DBPH), Nitrobenzene (NB), oleic acid (OA) and paraffin) for MDZ-MIP1 and (Di-butyl sebecate (DBS), Di-methyl acrylate (DMA), Tributylphosphate(TBP) and Tris(ethylhexyl phosphate (TEHP) ) for MDZ-MIP2. Membranes of MIPs were prepared in PVC matrix. The characterizations of each electrode were determined The Slope range from (55.083 - 43.711) mV/decade, Limit of Detection (8 X 10 ^-4- 2 X 10^-6) and Linearity range of electrodes MIPs from (1 X 10^-5 - 1 X 10 ^-1). Stable Signe of electrode pH from (2.5-9) and study the selectivity with additives of drugs synthesis (Glucose, Calcium stearate, sodium benzoate and benzoic acid) demonstrate strong selectivity.

 

 

 

INTRODUCTION: 

Chemically, metronidazole is (2-methyl-5-nitroimidazole-1-ethanol)¹. The biological activity of nitroimidazoles is dependent upon the nitro group reduction due to the formation of active intermediate species² that interact with DNA to produce biochemical damage. Prototype of the nitroimidazole class of antimicrobials been evaluated in the treatment of diverse anaerobic and gastrointestinal tract infections. Moreover, metronidazole has often been studied for antibacterial activity against gram-negative aerobes and some gram-positive bacteria, including Bacteroides fragilis that produces β-lactamases³.

 

 

In healthy humans, metronidazole is absorbed rapidly and completely from the gastrointestinal tract and is metabolized in the liver by an oxidative pathway⁴. Liver is the main site of metabolism by side chain oxidation and glucuronide conjugation. A major portion of the dose of the drug is excreted in urine, largely as metabolites⁵. Metronidazole is one of the most widely used antibacterial compounds in the treatment of some types of periodontal disease such as aggressive periodontitis.

 

Metronidazole is officially determined by titrimetric, potentiometry and HPLC methods. Indian Pharmacopoeia⁶ describes the non-aqueous titration method using perchloric acid as titrant and malachite green as indicator for the assay of metronidazole. British Pharmacopoeia^7,8 describes potentiometric and non-aqueous titration methods using perchloric acid as titrant. United States Pharmacopoeia⁹ describes HPLC and nonaqueous titration methods for the assay of metronidazole. Several methods have been reported for the determination of metronidazole, including flow injection^10-12 and polarography^13-15. Most of the spectrophotometric methods found in the literature for the determination of metronidazole in the visible region involve initial reduction by treatment with Zinc powder and HCl followed by the diazotization and coupling of the resulting amine^10,16-22.

 

Molecular Imprinting polymers (MIPs) is a technique for developing Receptors that are artificial for a given analyte with a predetermined specificity and selectivity that In various application areas, they can be used as ideal materials^23,24. Polymeric matrix obtained using the   imprinting technology, (MIPs), are robust molecular recognition components able to mimicking natural recognition entities such as biological receptors and antibodies that are used for distinguishing and the study of difficult samples such as environmental samples and biological fluids^25-27.The aim of MIT is the formation of a complex between an analyte (template) and a functional monomer. In the presence of a large excess of a cross-linking agent, a three-dimensional polymer network^28-30 is formed. After the polymerization process, the template is removed from the polymer, leaving specific recognition sites that are complementary in shape, scale, and chemical flexibility for the molecule prototype^31,32. Typically, molecular recognition phenomena are powered by intermolecular interactions such as the ionic interactions, the hydrogen bonds H-bond and dipoledipole between the functional groups of monomers and the template molecule in the matrix of polymer. The resulting polymer thus selectively recognizes and binds only the template molecules^33,34.

 

Figure 1. Concept of Molecularly imprinted polymers ³⁵.

 

MATERIALS AND METHODS:

Materials:

This work was conducted with an (Germany, WTW model), a pH meter (WTW model pH 720, Germany) expandable ion analyzer and a (Gallenkamp, USA) (SCE) saturated calomel electrode. All of the highest purity chemical reagents used are: allyl chloride (99%), tri-methylol propane tri-acrylate (99%) and benzoyl peroxide (78percent). Sigma Aldrich obtained plasticizers ((DBPH) di-butyl phthalate, nitrobenzene (NB), oleic acid (OA) and (PRF) Parrafin . Fluka was responsible for acquiring other chemicals and reagents.  Standard solution of 0.1M Metronidazole (MDZ) was prepared by dissolving 1.715g of standard metronidazole in methanol and diluting to 100mL, ultrasonicator equipment was prepared used to aid in drug dissolution, and several 100mL of standard solution range (10^-6-10^-1 M) prepared freshly. Interfering 0.1M solution for (Glucose, Calcium stearate, sodium benzoate and benzoic acid) were prepared and then series (10^-6-10^-1 M) were prepared. 0.1M stock solution of each of interfering; (1.8015)g of glucose, (1.2212)g of benzoic acid, (1.4411)g sodium benzoate and (6.0702)g of calcium stearate were prepared by dissolving in Distilled water 100mL standard solution ranged from (10^-6-10^-1 M) were freshly prepared.

 

Preparation of Metronidazole molecularly imprinted polymers:

The preparation of the Metronidazole molecularly imprinted polymers (MDZ-MIPs) 0.5mmol of Metronidazole (MDZ) was mixed with  3mmol of (allyl chloride (AYC) or allyl bromide (AYB)) as a monomer, In after that was added 15 mmol of the (Ethyleneglycol dimethylacrylate (EGDMA) or Trimethylolpropanetriacrylate (TMPTA) as Cross-linker, and then added the initiator (BPO) benzoyl peroxide 0.3mmol and 5mL of  CHCl₃ chloroform for obtaining the homogeneous solution, the mixture was stirred for 5 minutes. N2 gas passed for 30 min on the mixture to remove oxygen from the solution. After that, the solution was put at 70^oC for 6 hours in a water bath. When the reaction completed and the MDZ-MIP1or MDZ-MIP2 formed as very hard material leave 24 hours to dried and then crushed and the Metronidazole extracted from polymers by using soxhlet using (1:9) (CH₃COOH: CH₃OH). After ensuring the template is removal completely.   The polymer was dried at 45^oC for 48 hours. The MDZ-MIP1 and MDZ-MIP2 were then crushed and ground by a mortar, pestle and sieve to get 125-150μm was collected and then used as an active substance in the selective sensors membrane.

 

Preparation of electrodes:

0.02g of MDZ-MIPs polymers were mixed with different plasticizers used in this work such as DBPH, NB, O.A , PRF, DBS, DMA, TBP and TEHP. Then (0.2)g of PVC powder was added and dissolved in  (7mL) of Tetra Hydro Furan with shaking until obtaining A pure, viscous solution. Then mix the solutions and shake until the mixture is homogeneous .Pour the mixture into a glass ring moulds of diameter (30-35mm) and spread it on a glass plate and places a filter paper over the mouth of the mug. The solvent was then kept to evaporate at room temperature for at least 24hours. The thickness of the film obtained was different from one film to another, so the thickness was within (0.4-0.7mm). This membrane size was suitable for preparing the electrodes. The construction and assembly of the electrodes by taking a PVC tube (1-2 cm long) that has been plunged into THF solution from one of it is sides and positioned vertically on the prepared membrane, cut to fit the outer diameter of the  PVC tube .The other end of a glass container assembly was connected to plastic cover, Ag/AgCl through wire that was mounted. 3/4 glass tube filled with 0.1M Metronidazole solution. The electrodes were placed in a standard drug solution for three hours before use.  After good results were obtained when we used the prepared sensors depend on MDZ-MIPs, in determine Metronidazole in pure form.  Applied to estimate the Metronidazole drug in the pharmaceutical preparation whose found as tablets with 500mg. These ISEs measurements have been tested by different potentiometric methods. Preparation solutions of Metronidazole at concentrations 1×10^-3 and 1×10^-4 M then calculation’s the Erel.%, Rec.% and RSD% of Metronidazole in pharmaceuticals.

 

RESULT AND DISCUSSION:

Characterization of MDZ-MIPs:

Scanning Electron Microscopy (SEM) was used to Examination and analysis the surface and topography of MDZ-MIP1 and MDZ-MIP2 before and after Template removal as shown in figures (2a) for MDZ-MIP1 and figures (2b) for TMP-MIP2³⁶.

 

The molecular imprinted polymer of Metronidazole used after extraction of the template (Metronidazole) to constructed four membranes for each polymer MIP1 and MIP2 with different plasticizers such as di-butyl phthalate (DBPH), Nitrobenzene (NB), oleic acid (O.A) paraffin (PRF), Dibutyl sebecate (DBS), Dimethylacrylate (DMA), tributylphosphate (TBP) and trisnethyl hexyl phosphate(TEHP) With Matrix PVC. All membrane displays near the slopes of Nernst were acquired^37,38. The results are given in tables (1).

 

 

Figure 2. Scanning Electron Microscopy for MDZ- MIP1 and MDZ-MIP2, (a) before (b) after extraction the template.

 

 

 

Table 1. The parameters of MDZ-MIP1 and MDZ-MIP2 selective electrodes using different plasticizers.

 

Table 2. Selectivity Coefficients for (MDZ-MIP2+DBS) electrode at different concentrations of MDZ .

 

Selectivity coefficient Calculation:

For the selectivity coefficient calculation, a different solution approach was used and was determined by following the equation (1) ^{9, 39}.

 

Log Kpot = [(E_B − E_A)/(2.303RT/ZF)]+ (1 − z_A/z_B) logaA……….(1)

 

pH Effects:

The effect of changing the pH function with which the Metronidazole electrodes for MDZ-MIP1 depend on (DBPH, NB, O.A and PRF) as plasticizers and MDZ-MIP2 depend on (DBS, DMA, TBP and TEHP) as plasticizers operate was studied by determining the electrode response by measuring the potential of the Metronidazole electrode for three different concentrations 10^-4,10^-3,10^-2 M for ranges of pH from 10 -1.0, the pH values of the solutions were adjusted using a solution. Dilute hydrochloric acid or dilute sodium hydroxide solution. Different responses appear with the difference in the pH range of the solution, but there is a specific range of pH in which the response to a particular electrode stabilizes and represents the appropriate range within which potentiometric measurements can be made, followed by a decrease in response values with increasing pH values Solution.

 

Table 3. Effect pH ranges used for MDZ-MIP1 and MDZ-MIP2-selective electrodes.

 

Sample analysis:

Three techniques were used to determine sulfamethoxazole via direct, standard addition method (SAM) and multi-standard addition (MSA) method in pure form and pharmaceutical preparation. Equation (2)^{29, 30}   used to SAM.

 

C_U = C_S/ 10 ΔE/S [ 1+ (V_U/ V_S)] – (V_U/ V_S) ……….(2)

 

Where the volume and concentration of an unknown and standard solution respectively are VU, VS, CU and CS

 

Table 4. Determination of metronidazole pure samples by ion selective electrodes techniques based on PVC membranes for MDZ-MIP1.

 

 

Metronidazole estimation in pharmaceutical preparation:

After good results were obtained when we used the prepared sensors depending on MDZ-MIP1, in determining Metronidazole in pure form.  Applied to estimate the Metronidazole drug in the pharmaceutical preparation whose found as tablets with 500 mg. These ISEs measurements have been tested by different potentiometric methods. Preparation solutions of Trimethoprim at concentrations 1×10^-3 and 1×10^-4 M then calculation’s the Erel.%, Rec.% and RSD% of Metronidazole in pharmaceuticals. The results obtained are represented in tables (5)^40,41,42.

 

Table 5. Sample analysis of pharmaceuticals Metronidazole by using MIPs membrane electrode (MDZ-MIP1+NB)

 

CONCLUSION:

Metronidazole molecularly imprinted electrodes based on allyl chloride (AYC)  and allyl bromide (AYB) as a monomer,  the cross-linker tri-methylolpropanetri acrylate (TMPTA) and Etheleyene glycol di-methyl acrylate (EGDMA) was used  and (BP) benzoyl peroxide as (initiator)was constructed based on PVC matrix membrane. Excellent electrode parameters were obtained including Nernstain slopes, detection limit and pH. The prepared electrodes were used for Metronidazole determination in commercial drugs which gives recovery ranged from 95.7 to 104.7.

 

ACKNOWLEDGEMENT:

The authors are thankful to Middle-east Laboratories Company limited for pharmaceuticals industries Baghdad, Iraq for providing the gift Sample of Trimethoprim. I also thank everyone who supported me during my work on this research.

 

REFERENCES:

1.     Adegoke, O.A. and O.E. Umoh, A new approach to the spectrophotometric determination of metronidazole and tinidazole using p-dimethylaminobenzaldehyde. Acta Pharmaceutica, 2009. 59(4): p. 407-419. http://dx.doi.org/10.2478/v10007-009-0039-2

2.     Knox, R., R. Knight, and D. Edwards, Interaction of nitroimidazole drugs with DNA in vitro: structure-activity relationships. British journal of cancer, 1981. 44(5): p. 741-745.https://dx.doi.org/10.1038/bjc.1981.261

3.     Lamp, K.C., et al., Pharmacokinetics and pharmacodynamics of the nitroimidazole antimicrobials. Clinical pharmacokinetics, 1999. 36(5): p. 353-373.https://doi.org/10.2165/00003088-199936050-00004

4.     Loft, S., et al., Characterization of metronidazole metabolism by human liver microsomes. Biochemical pharmacology, 1991. 41(8): p. 1127-1134. https://doi.org/10.1016/0006-2952(91)90650-t

5.     Goodman, L.S., Goodman and Gilman's the pharmacological basis of therapeutics. Vol. 1549. 1996: McGraw-Hill New York.

6.     Manohara, Y., et al., Novel and rapid estimation of metronidazole in tablets. Der Pharma Chem, 2010. 2(3): p. 148-51.

7.     Jejurkar, L. and A. Jejurkar, HPLC method of detection for 5ASA in pure and in tablets. Journal of Pharmaceutical Science and Technology, 2012. 4: p. 792-797.

8.     Mansour, O., D. Nashed, and A.A. Sakur, Determination of clopidogrel bisulphate using drug selective membranes. Research Journal of Pharmacy and Technology, 2018. 11(5): p. 2017-2021. https://doi.org/10.5958/0974-360X.2018.00374.8

9.     Aljabari, F.I. and Y.K. Al-Bayati, Estimation of Trimethoprim by using a New Selective Electrodes dependent on Molecularly Imprinted Polymers. Egyptian Journal of Chemistry, 2021. 64(10): p. 6089-6096. https://dx.doi.org/10.21608/ejchem.2021.72564.3617

10.   Simões, S.S., et al., Flow injection determination of metronidazole through spectrophotometric measurement of the nitrite ion produced upon alkaline hydrolysis. Journal of the Brazilian Chemical Society, 2006. 17(3): p. 609-613. https://doi.org/10.1590/S0103-50532006000300028

11.   Yu, M., et al., In vitro/in vivo characterization of nanoemulsion formulation of metronidazole with improved skin targeting and anti-rosacea properties. European Journal of Pharmaceutics and Biopharmaceutics, 2014. 88(1): p. 92-103. https://doi.org/10.1016/j.ejpb.2014.03.019

12.   Palomeque, M., et al., Flow injection biamperometric determination of metronidazole with on-line photodegradation. Analytica chimica acta, 1999. 401(1-2): p. 229-236. http://dx.doi.org/10.1016/S0003-2670(99)00479-1

13.   Joshi, D. and A. Joshi, Polarographic determination of metronidazole and chloramphenicol. Journal of the Indian Chemical Society, 1997. 74(7).. http://dx.doi.org/10.5281/zenodo.5908860

14.   Sindhu, S.K., Y. Singh, and R. Singh, Potentiometric Determination of Heavy Metals Concentrations in Trace by Using Calixarene as Electroactive Materials. Asian Journal of Research in Chemistry, 2010. 3(1): p. 229-000.

15.   Sakur, A.A., et al., Determination of prasugrel hydrochloride in bulk and pharmaceutical formulation using new ion selective electrodes. Research Journal of Pharmacy and Technology, 2018. 11(2): p. 631-636. https://doi.org/10.5958/0974-360X.2018.00118.X

16.   Ijaz, A. and A. Raza, Spectrophotometric determination of metronidazole in pharmaceutical pure and dosage forms using p-benzoquinone. Journal of the Iranian Chemical Society, 2005. 2(3): p. 197-202. https://doi.org/10.1007/BF03245922

17.   Sastry, C., M. Aruna, and A.R.M. Rao, Spectrophotometric determination of some antiamoebic and anthelmintic drugs with metol and chromium (VI). Talanta, 1988. 35(1): p. 23-26. https://doi.org/10.1016/0039-9140(88)80006-7

18.   Dinesh, N.D., P. Nagaraja, and K.S. RANGAPPA, A sensitive spectrophotometric assay for tinidazole and metronidazole using a Pd-C and formic acid reduction system. Turkish Journal of Chemistry, 2004. 28(3): p. 335-344.

19.   Siddappa, K., et al., Spectrophotometric determination of metronidazole through Schiff's base system using vanillin and PDAB reagents in pharmaceutical preparations. Eclética Química, 2008. 33(4): p. 41-46. https://doi.org/10.1590/S0100-46702008000400005

20.   Abu Zuhri, A.Z., S.I. Al-khalil, and M.S. Suleiman, Electrochemical reduction of metronidazole and its determination in pharmaceutical dosage forms by DC polarography. Analytical Letters, 1986. 19(3-4): p. 453-459. https://doi.org/10.1080/00032718608064510

21.   El-Sayed, G., Polarographic determination of metronidazole in pharmaceutical formulations and urine. Microchemical journal, 1997. 55(1): p. 110-114. https://doi.org/10.1016/j.mset.2021.06.001

22.   Ahmed, B. and J. Onah, Colorimetric determination of chloramphenicol and metronidazole in pharmaceutical formulations after Schiff-base formation with vanillin and anisaldehyde. Global Journal of Pure and Applied Sciences, 2003. 9(3): p. 359-364. https://doi.org/10.4314/gjpas.v9i3.15940

23.   Rani, S. and G. Singh, Novel Polymeric Membrane Sensor for the Selective Determination of Citrazine. Asian Journal of Research in Chemistry, 2012. 5(10): p. 1210-1215.

24.   Maliwal, D., et al., Simultaneous spectrophotometric estimation of metronidazole and norfloxacin in combined tablet formulations using hydrotropy. Research Journal of Pharmacy and Technology, 2008. 1(4): p. 357-361.

25.   Guo, T.-Y., et al., Chitosan beads as molecularly imprinted polymer matrix for selective separation of proteins. Biomaterials, 2005. 26(28): p. 5737-5745. https://doi.org/10.1016/j.biomaterials.2005.02.017

26.   Whitcombe, M.J., C. Alexander, and E.N. Vulfson, Smart polymers for the food industry. Trends in Food Science & Technology, 1997. 8(5): p. 140-145.

27.   Al-Bayati, Y.K. and F.I. Aljabari, Synthesis of ibuprofen-molecularly imprinted polymers used as sensors to determine drug in pharmaceutical preparations. Asian Journal of Chemistry, 2016. 28(6): p. 1376.

28.   Al-Bayati, Y.K. and F.I. Aljabari, Mefenamic Acid Selective Membranes Sensor and Its Application to pharmaceutical Analysis. Baghdad Science Journal, 2016. 13(4).. https://doi.org/10.21123/bsj.2016.13.4.0829

29.   Al-bayati, Y.K. and F.I. Aljabari, Construction of New Ion Selective Electrodes for Determination Ibuprofen and Their Application in Pharmaceutical Samples. 2015, IJRPC.

30.   Ramström, O. and K. Mosbach, Synthesis and catalysis by molecularly imprinted materials. Current Opinion in Chemical Biology, 1999. 3(6): p. 759-764. https://doi.org/10.1016/s1367-5931(99)00037-x

31.   Thulluru, A., et al., Optimization of HPMC K100M and sodium alginate ratio in Metronidazole Floating Tablets for the Effective Eradication of Helicobacter pylori. Asian Journal of Pharmacy and Technology, 2019. 9(3): p. 195-203. http://dx.doi.org/10.5958/2231-5713.2019.00033.3

32.   Tiwari, G., et al., Simultaneous estimation of metronidazole and amoxicillin in synthetic mixture by ultraviolet spectroscopy. Asian Journal of Research in Chemistry, 2008. 1(2): p. 91-94.

33.   Samarth, N.B., et al., A historical perspective and the development of molecular imprinting polymer-A review. Chem. Int, 2015. 4: p. 202-210.

34.   Ramalakshmi, N. and B. Marichamy, Sensing of Lead and Copper Metal Ions by Substituted N-Methyl Piperazine Compound on Glassy Carbon Electrode. Asian Journal of Research in Chemistry, 2011. 4(12): p. 1920-1927.

35.   Ulmer-Scholle, D.S., et al., A color guide to the petrography of sandstones, siltstones, shales and associated rocks. Vol. 109. 2014: American Association of Petroleum Geologists Tulsa, OK, USA.

36.   Sivasankari, G., S. Boobalan, and D. Deepa, Dopamine sensor by Gold Nanoparticles Absorbed Redox behaving metal Complex. Asian Journal of Pharmacy and Technology, 2018. 8(2): p. 83-87. https://doi.org/10.5958/2231-5713.2018.00013.2

37.   AlMashhadani, H.A., Synthesis of a CoO-ZnO nanocomposite and its study as a corrosion protection coating for stainless steel in saline solution. International Journal of Corrosion and Scale Inhibition, 2021. 10(3): p. 1294-1306. http://dx.doi.org/10.17675/2305-6894-2021-10-3-26

38.   Almashhadani, H. and K. Alsaadie, Corrosion Protection of Carbon Steel in seawater by alumina nanoparticles with poly (acrylic acid) as charging agent. Moroccan Journal of Chemistry, 2018. 6(3): p. 6-3 (2018) 455-465. https://doi.org/10.48317/IMIST.PRSM/morjchem-v6i3.6214

39.   Sebaiy, M.M., et al., Rapid RP-HPLC method for simultaneous estimation of sparfloxacin, gatifloxacin, metronidazole and tinidazole. Asian Journal of Pharmaceutical Research, 2011. 1(4): p. 119-125.

40.   Almashhadani, H.A., et al., Corrosion inhibition behavior of expired diclofenac Sodium drug for Al 6061 alloy in aqueous media: Electrochemical, morphological, and theoretical investigations. Journal of Molecular Liquids, 2021. 343: p. 117656. https://doi.org/10.1016/j.molliq.2021.117656

41.   AlMashhadani, H.A. and K.A. saleh, Electro-polymerization of poly Eugenol on Ti and Ti alloy dental implant treatment by micro arc oxidation using as Anti-corrosion and Anti-microbial. Research Journal of Pharmacy and Technology, 2020. 13(10): p. 4687-4696. https://doi.org/10.5958/0974-360X.2020.00825.2

42.   Naji, A.M., I.Y. Mohammed, and S.A. AL-BAYATY, Mechanical and Thermal Degradation Kinetic Study of Basalt Filled Polyvinyl Chloride Composite Material. Egyptian Journal of Chemistry, 2021. 64(2): p. 893-901.https://dx.doi.org/10.21608/ejchem.2020.35343.2739

 

 

 

 

Accepted on 14.06.2022        © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(2):715-720.