SYNTHESIS, CHARACTERIZATION AND APPLICATIONS OF MOLECULARLY IMPRINTED POLYMER FOR ANALYSIS ENOXAPARIN IN BIOLOGICAL MATRIX

• Main Author: Juni Astuti, Engrid
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/81721
• Institution: Institut Teknologi Bandung

The clinical symptoms that often appear in Covid-19 are generally respiratory infections. Covid-19 can cause some patients to experience severe complications within a short time after infection, namely Adult Respiratory Distress Syndrome (ARDS) or Disseminated Intravascular Coagulation (DIC), sepsis followed by organ failure and death. In Covid-19 patients with severe symptoms, interleukin-6 (IL-6) levels were significantly higher than in patients with mild symptoms. Patients with Covid-19 can experience severe symptoms due to cytokine levels reaching or exceeding a certain threshold, known as a cytokine storm. Low molecular weight heparin (LMWH) has been reported to reduce the release of IL-6 in the body by inhibiting NF-KB expression. Based on recommendations from the World Health Organization (WHO), Covid-19 patients who are hospitalised are given a dose of LMWH, namely enoxaparin, at a dose of 40 mg subcutaneously once a day for adults. Giving heparin-type anticoagulants to Covid-19 patients has a risk of developing thrombocytopenia, especially those accompanied by impaired kidney function, so monitoring is needed regarding administering this type of heparin therapy. So far, enoxaparin measurements have been carried out using Size Exclusion HighPerformance Liquid Chromatography (HPLC), Biophen® heparin anti-Xa, cellulose-based photoacoustic sensors, and fluorescence sensors. Sample preparation in complex liquid biological matrices, such as urine, plasma, and oral fluids, usually requires lengthy sample preparation stages. Molecularly imprinted polymer (MIP) innovations have been widely used to speed up sample preparation. One of the challenges in making MIPs is that water-soluble macromolecular or biomacromolecular compounds limit the selection of functional monomers and cross-linkers. Therefore, developing a sample preparation method using MIP to analyse enoxaparin and monitor its quality, efficacy, and safety during use in biological matrices is necessary. MIP synthesis began in silico using the Gaussian ® Ab initio method and Density Functional Theory (DFT) to determine the functional monomer suitable for enoxaparin. The best functional monomer composition ratio with enoxaparin was determined using DFT and GFN2-xTB ® , and molecular dynamics simulations were carried out using Yasara®, which was proven by the Job's plot test. MIP synthesis using a precipitation polymerization method with the help of a microwave and stirrer. After the MIP is formed, the MIP will be characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscope (SEM), Thermal Gravimetry Analysis (TGA), Particle Size Analysis (PSA) and BET. The adsorption ability was calculated using an enoxaparin analysis method validated and developed to determine enoxaparin levels using a UV-VIS spectrophotometer and an HPLC ion pair. The final stage was to develop a method for enoxaparin sample preparation in a biological matrix, namely blood plasma, using MIP, which had been prepared and analyzed using a validated method. In silico results using Ab initio and DFT showed that itaconic acid was the functional monomer that interacted best with enoxaparin, indicated by the binding energy and Gibbs free energy values using Ab initio of ?45.6419 kcal/mol and ?30.1186 kcal/mol respectively, while using DFT are ?62.3199 kcal/mol and ?40.8691 kcal/mol respectively. Based on molecular dynamics simulations, the ratio of the enoxaparin complex to itaconic acid of 1:1 gives the best results in terms of root-mean-square deviation (RMSD), radius of gyration (Rg) and hydrogen bonds formed respectively at 6.6 Å, 8.2 Å and 4. These results are also supported by Jobs plot analysis. Polymers synthesized using the microwave precipitation polymerization (MIPM) method provide better morphological characterization results than those produced by stirring (MIPS), which is proven by testing the adsorption capacity and imprinting factor (IF). Method development and validation using a UV-VIS spectrophotometer were used to analyze enoxaparin during adsorption with MIP. Optimisation results show that enoxaparin can be read at a maximum wavelength of 231 nm using 0.01 N HCl solvent. Validation of the analytical method provides results that meet the acceptance requirements for parameters of linearity, detection limit, quantitation limit, accuracy and precision of 0.9999, 2.95 mg/ L, 9.89 mg/L, 96.71 % and 1.26 % in the measurement range of 25 –500 mg/L. Method validation was carried out using ion-pair HPLC to analyze the selectivity of MIP towards enoxaparin and heparin. Optimization of the HPLC system showed that the best separation was obtained with a C8 column with a length of 25 cm and a particle size of 5 µm, using a gradient elution system with mobile phase A, namely 300 mM NaCl plus ten mM tetra n-butyl ammonium hydroxide with mobile phase B, namely acetonitrile, a flow rate of 1 mL/minute with an injection volume of 100 µL, detection was carried out at a wavelength of 231 nm. Validation of the analytical method provides results that meet the acceptance requirements for specificity, linearity, detection limit, quantitation limit, accuracy and precision. The MIPM adsorption capacity value was 43.47 ± 0.40 mg/g, better than the MIPS adsorption capacity of 40.77 ± 0.75 mg/g. The IF values of MIPM and MIPS are 1.21 and 0.71. The results of adsorption kinetics testing show that all MIPs produced follow a pseudo-second-order kinetic model characterized by a correlation coefficient value close to one. The adsorption isotherm test results show that all MIPs follow the Freundlich isotherm adsorption model. The BET test results show that MIPM has the largest surface area, 20,186 m2/g. The selectivity test results showed that MIPM had better selectivity for enoxaparin than MIPS. The application results show that MIPM can extract enoxaparin from the blood plasma matrix compared to MIPS. MIPM with a sorbent amount of 20 mg can extract enoxaparin in blood plasma with a per cent recovery of 100.31 ± 0.21 %.