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Authors: Chatterjee, Sanghamitra
Issue Date: 2010
Abstract: The power of the electrochemical techniques to probe deep insights into the mechanistic pathways of compounds of biological significance permits an elucidation of the redox mechanism of various physiologically important compounds. Electrochemistry at carbon based electrodes has emerged over the last few years, significantly changing the scope and sensitivity of electroanalytical methods. Pyrolytic graphite electrode is the most widely used in electroanalysis as it incorporates almost all the features of a good electrode material like broad potential window, low background current, chemical inertness and suitability for various sensing and detecting applications. Modified electrodes obtained by the fabrication of the surface of the electrode with nanomaterials has been gaining significance in the last few decades as it imparts functionality distinct from the base electrode. In an attempt to increase the sensitivity, selectivity and to improve the detection limit of electrochemical sensors, application of the modified electrodes has been envisaged. In view of the importance of electrochemical determination of biologically important compounds, drugs and doping agents, efforts have been made in the present thesis to investigate these compounds at bare and nanomaterial modified pyrolytic graphite electrode. For simplicity and clarity, the results of the determination are presented in the dissertation as follows: The first chapter of the thesis is "General Introduction" which presents a compendious review of the pertinent work and highlights the importance of electrochemical studies in biological systems along with its application in diverse areas. The salient features of the various techniques employed for present investigations have also been described. The chapter also highlights the merits of carbon based electrodes along with the advantages imparted by nanomaterial modified electrodes. The second chapter of the thesis deals with the electrochemical oxidation of 2', 3'- dideoxyadenosine at pyrolytic graphite electrode. The compound is a synthetic nucleoside analogue of deoxyadenosine in which the 3' hydroxyl group on the ribose moiety is replaced by a hydrogen atom. It exhibits antiviral and antibactertial activities with a principal side effect of nephrotoxicity. It is an inhibitor of HIV replication, acting as a chain-terminator of viral DNA by binding to reverse transcriptase. The ultimate goal of investigating the oxidation properties of purines and their nucleosides at solid electrodes is to elucidate the redox behavior of nucleic acids. Voltammetric, coulometric, spectral studies and product analysis indicate that the oxidation of 2', 3'- dideoxyadenosine occurs in an EC reaction involving 6e , 6H process at pH 7.2 to give allantoin, C-C dimer and dideoxyribose as the major products and a C-O-O-C linked dimer as a minor iii product. Tentative mechanisms for the formation of the products have also been suggested. A comparison of peak potential value of 2', 3'-dideoxyadenosine with adenosine and 2'- deoxyadensoine indicated that the difference is insignificant which has further been supported by the calculations of difference of energies of lowest unoccupied and highest occupied molecular orbitals. Steroids including adrenocortical steroids and anabolic androgenic steroids affect the nervous system, cause euphoria, alleviate pain and improve athlete's ability to concentrate in performance of endurance and power events and are thus considered as doping agents by World Anti-Doping Agency and International Olympic Committee. It was therefore mandatoryto develop an effective method which aims at the determination of some steroids like dexamethasone and results obtained are presented in the third chapter of the thesis. It deals with dexamethasone, which is considered as a doping agent as it is frequently abused by athletes in sports such as cycling and horse racing to improve the performance. It is a potent glucocorticoid which acts as an anti inflammatory and immunosuppressant. It is used for the treatment of various cancers, viral infections, respiratory diseases, liver disorders, gastrointestinal diseases, skin disorders and nervous system abnormalities. The studies have been carried out at fullerene - C6o - modified edge plane pyrolytic graphite electrode which enhances the sensitivity of electrochemical determinations as compared to bare electrode. Calibration plot having good linearity with a correlation coefficient 0.9983 is obtained in the concentration range of 0.05 - 100 uM and the sensitivity of the method has been found to be 0.685 uA uM"1. The detection limit is estimated to be 5.5 x 10"8 M. The practical analytical utility of the method is illustrated by quantitative determination of dexamethasone in several commercially available pharmaceutical formulations and human blood plasma of patients being treated with dexamethasone. HPLC method was used to compare the results obtained for the quantitative estimation of dexamethasone in biological fluids. The fourth chapter of the thesis describes the simultaneous determination of purine nucleosides (adenosine and inosine) and corticosteroid isomers (testosterone and epitestosterone) at single-wall carbon nanotubes modified edge plane pyrolytic graphite electrode which have been dealt in two different sections of this chapter. The first section presents the voltammetric oxidation of adenosine and inosine simultaneously at pH 7.2 using square wave voltammetry. Adenosine known as 6-amino-9-P-D-ribofuranosyl-9-H-purine is an endogenous purine nucleoside occurring in all cells of the human system. It is a potent anti-inflammatory and antiarrhythmic agent which is important in the control of coronary and cerebral blood flow. Adenosine is converted to inosine in iv biological systems by hydrolytic deamination and the enzyme adenosine deaminase is involved in purine metabolism. The conversion is site specific and takes place in the mRNA of the cell and specifically enhances deep sleep and slow wave activity during sleep. The increase in the plasma adenosine and inosine concentration affects the metabolic pathway occurring in the human system which led to the need of simultaneous determination of these nucleosides. The modified electrode exhibits remarkable electrocatalytic properties towards adenosine and inosine oxidation with a peak potential of- 1229 mV and 1348 mV respectively. Linear calibration curves are obtained over the concentration range 0.5 uM - 1.0 mM in adenosine and 10 uM - 1.0 mM in inosine with sensitivity of 1.0 uA uM and 1.9 uA uM for adenosine and inosine respectively. The proposed method was also used to estimate these compounds in human blood plasma and urine samples and the method was validated using HPLC. Testosterone, a potent member of the naturally occurring androgens and epitestosterone, the inactive 17a- epimer of testosterone are found in the ratio of about 1:1 in the normal human urine. The urinary testosterone/epitestosterone ratio > 6 is an indication of exogenous use of testosterone enhancing compounds and is taken as the hallmark of drug abuse. Since the concentration and ratio of testosterone and epitestosterone are the key indicators to determine the abusing of testosterone, the simultaneous detection of them with high selectivity is of great significance. The reduction of the two isomers occurred in a pH dependent, 2e, 2H+ process and well-defined voltammetric peaks were observed. Under the optimum experimental conditions, linear calibration curves are obtained within the concentration range 5 - 1000 nM for both the steroids with the limit of detection 2.8 * 10"9 Mand 4.1 x 10"9 Mfor testosterone and epitestosterone respectively. The developed protocol is successfully implemented for the analysis of both the compounds in the urine samples of normal subjects as well as in patients undergoing treatment with testosterone. The results are compiled in the second section of chapter 4. CH3 OH CH3 OH Adenosine Inosine Testosterone Epitestosterone The last chapter of the thesis has been subdivided in two parts. The first part deals with the development of a sensitive voltammetric sensor for determination of synthetic corticosteroid triamcinolone, abused for doping. This compound was selected due to the fact that on July 3, 2008, Olympic team member Morgan Hamm of Columbus, an athlete in the sport of gymnastics received an official warning from the United States Anti-Doping Agency after testing positive for triamcinolone acetonide after the second day of the US Nationals on May 24, 2008. It is a longacting corticosteroid with reported efficacy when given by intravitreal or subtenon injection as a treatment for diabetic macular edema, uveitis, retinal vein occlusion and age-related macular degeneration. It is for this reason that the analysis of triamcinolone is of great significance in pharmaceutical research and clinical chemistry. A comparison of the voltammetric behavior between single-wall carbon nanotubes modified edge plane pyrolytic graphite electrode and fullerene - C6o - modified edge plane pyrolytic graphite electrode indicated that single-wall carbon nanotubes serves as a better modifier. The square wave voltammetric response of the electrode to triamcinolone is linear in the range 0.1 - 25 nM with a detection limit and sensitivity of 8.9 x 10"11 M and 2.06 uA nM" respectively. The method was applied for the determination of triamcinolone in several commercially available pharmaceuticals and real urine samples obtained from patients undergoing pharmacological treatment with triamcinolone. A comparison of the observed results with HPLC analysis indicated a good agreement. The product obtained after reduction of triamcinolone was also characterized using !H NMR and GC-MS and the site of reduction is found to be carbonyl group at position 20. Paracetamol or acetaminophen, an analgesic medicament similar to acetylsalicylic acid lacking anticoagulatory properties and gastric irritation which is widely used as an active ingredient in pharmaceutical preparations has been discussed in the second part of this chapter. The voltammetric oxidation of paracetamol on single-wall carbon nanotubes modified edge plane pyrolytic graphite electrode was explored in phosphate buffer solution by using square wave voltammetry. Cyclic and square wave voltammetry studies indicated the oxidation of paracetamol at the electrode surface through a two electron reversible step and fundamentally controlled by adsorption. A comparison of the electrocatalytic activity of multi-wall carbon nanotubes and single-wall carbon nanotubes has also been made on the electrooxidation of the drug. The sensitivity at single-wall carbon nanotubes modified edge plane pyrolytic graphite electrode is ~ 2 ^ times more than that at multi-wall carbon nanotubes modified edge plane pyrolytic graphite electrode. Paracetamol gave a sensitive oxidation peak at ~ 187 mV at pH 7.2 which was used to vi quantitate the drug in the range 5-1000 nM with a detection limit of 2.9 x 10" M at single-wall carbon nanotubes modified edge plane pyrolytic graphite electrode. The interfering effect of physiologically common interferents on the current response of paracetamol has been reported. The procedure was successfully applied for the assay of paracetamol in pharmaceutical formulations. The applicability of the developed method to determine the drug in human urine samples obtained after 4 h of administration of paracetamol is illustrated
Other Identifiers: Ph.D
Appears in Collections:DOCTORAL THESES (chemistry)

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