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Download this complete Project material titled; Development And Validation Of Three New Spectrophotometric Methods For The Determination Of Lamivudine In Pure And Pharmaceutical Dosage Forms with abstract, chapters 1-5, references, and questionnaire. Preview Abstract or chapter one below

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ABSTRACT

In this study three rapid, simple, accurate, economical and reproducible spectrophotometric methods for the quantitative determination of lamivudine in pure form and tablet formulations were developed and validated. The first method is based on dissolution of standard lamivudine powder or extraction of the drug from tablet formulation using methanol as solvent. The resulting extract was filtered and scanned using Helios Zeta Model 164617 UV/Vis spectrophotometer; having a λmax of 273 nm. The second method is based on the formation of a coloured hydrazone by reacting the hydrazine group in 2,4-dinitrophenyl hydrazine with the carbonyl carbon in lamivudine under acidic condition (85 % H2SO4) for 10 minutes. The orange-red coloured hydrazone formed was allowed to stand for 2 hours for complete colour development, then scanned using spectrophotometer and was observed to have a λmax of 438 nm. The third method is based on the diazotization reaction of lamivudine using sodium nitrite and sulfamic acid under acidic condition (2 % H2SO4) for 5 minutes followed by coupling with paratoluidine reagent leading to the formation of yellow chromogen which was allowed to stand for 1 hour for complete colour development, then scanned using spectrophotometer and was observed to have a λmax of 282 nm. The proposed methods were used to prepare a calibration curves for lamivudine and also to assay sample of standard lamivudine powder and three different brands of lamivudine tablets and compared with International Pharmacopoeial method for assay of lamivudine. The three proposed methods obeyed Beer’s law within a concentration range of 2.5 to 15.0, 5.0 to 35.0, and 2.5
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to 12.5 μg/ml respectively. Correlation coefficients for the respective methods are 0.9997, 0.9988, and 0.9975. The precision (% coefficient of variation) and accuracy (% relative error) for the respective methods are 1.7, 1.8, 0.5 and 2.1, 4.0, 2.0 %. Percentage recoveries for the three methods obtained were 99.4, 98.9 and 99.6 % respectively. Limits of detection and limits of quantitation for the respective methods were 0.25, 1.3, 1.5 and 0.77, 3.8, 4.5 μg/ml. The percentage content of lamivudine in the standard powder and the three different tablet brands assayed in all the proposed methods were within the BP range of 97.5% to 102.0%. No statistically significant difference was observed between the percentage drug content of the proposed methods and International Pharmacopoeia method at P < 0.05. The proposed methods can be interchangeably used with the International Pharmacopoeia method for quantitative estimation of lamivudine in pure and tablet dosage forms.
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TABLE OF CONTENTS

Chapter 1 INTRODUCTION Page
1.1 General Introduction……………………………………………………………….1
1.2 Colour and Molecular Structure…………………………………………………….1
1.3 Lamivudine as Drug of Analysis…………………………………………………..2
1.4 Statement of Research Problem…………………………………………..…………3
1.5 Justification of the study……………………………………………………………5
1.6 Aims and Objectives…………………………………………….…………………..6
1.7 Research Hypothesis……………………………………………………………….6
Chapter 2 LITERATURE REVIEW
2.1 Lamivudine………………………………………………………………………….7
2.2 Properties of Lamivudine……………………………………….…………………..7
2.2.1 IR Spectra of Lamivudine…………………………………………………………..8
2.2.2 Synthesis of Lamivudine…………………………………….…………………….9
2.2.3 Mechanism of Lamivudine Antiviral action…………………….………………..11
2.2.4 Adverse effects of Lamivudine…………………………………………………..12
2.2.5 Pharmacokinetics of Lamivudine…………………………….……………………12
2.2.6 Precautions……………………………………………………….……………….13
2.2.7 Uses and Administration of Lamivudine………………………….……………..14
2.2.8 Interactions of Lamivudine……………………………………..………………..14
2.3 Absorption spectroscopy……………………………………………..…………….15
2.3.1 UV/VIS Spectroscopy……………………………………………………………..17
2.3.2 IR Spectroscopy………………………………………………………………….19
2.3.2.1 Number of Vibrational Modes…………………………………………………..20
2.3.2.2 Uses and Applications of IR Spectroscopy…………………………………….21
2.4 Schiff Bases and their Chemistry……………………………….………………….22
2.4.1 Biological importance of Schiff Bases…………………………………………….23
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2.4.2 Aryl Diazonium Salts………………………………………………………………….24
2.5 Reported UV Methods for Lamivudine Determination……………………………24
Chapter 3 MATERIALS AND METHODS
3.1 Materials…………………………………………………………..………………….31
3.1.1 Chemicals and Reagents………………………………………………..…………..31
3.1.2 Drug and Drug Reference Standard…………………………………………………..31
3.1.3 Equipment and Glassware………………………………………….………………32
3.1.4 Instrumentation……………………………………………………………………..32
3.2 Methods………………………………………………………….……………………33
3.2.1 Preparation of Solutions and Reagents……………………………………………..33
3.2.2 Identification and Assay of Lamivudine (Official methods)………….…………….34
3.2.3 Method 1: Extraction of Lamivudine with Methanol………………………………..35
3.2.3.1 Determination of λmax……………………………………..……………………….35
3.2.3.2 Preparation of Calibration curve for Method 1…………………………………..35
3.2.3.3 Validation of Method 1……………………………………………..……………..36
3.2.3.4 Assay of Lamivudine Tablets using Method 1…………………………………….38
3.2.4 Method 2: Condensation of Lamivudine with 2,4-DNPH…………………………39
3.2.4.1 Preparation of Calibration Curve for Method 2…………………………………….40
3.2.4.2 Validation of Method 2……………………………………………….………….40
3.2.4.3 Assay of Lamivudine Tablets using Method 2……………………………………41
3.2.5 Method 3: Diazotization of Lamivudine and Coupling with P-toluidine………….42
3.2.5.1 Preparation of Calibration Curve for Method 3……………………..……………43
3.2.5.2 Validation of Method 3…………………………………………………………..43
3.2.5.3 Assay of Lamivudine Tablets using Method 3……………………..…………..44
3.2.5.4 Statistical Analysis…………………………………………………….……….45
Chapter 4 RESULTS
4.1 Calibration Curves…………………………………………………..……………..46
4.2 Validation of Methods…………………………………………………..…………..50
4.3 Assay Results…………………………………………………………….…………..51
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4.4 FTIR Analysis Results……………………………………………………..………51
Chapter 5 DISCUSSION
5.1 Calibration Curves………………………………………………………………..58
5.2 Validation Parameters……………………………………………..……….…….58
5.3 Assay Results and Statistical Analysis………………………………….………..59
5.4 FTIR Analysis…………………………………………………………….………..60
5.4.1 Method 2…………………………………………………………………………60
5.4.2 Method 3…………………………………………………………………………..60
Chapter 6 CONCLUSIONS AND RECOMMENDATIONS
6.1 Conclusions………………………………………………………………….………62
6.2 Recommendations………………………………………………………………….62
REFERENCES…………………………………………………………………………63
APPENDICES
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CHAPTER ONE

INTRODUCTION
1.1 General Introduction
UV-visible spectrophotometric methods are the instrumental methods of choice which are commonly used in industrial and research laboratories because of their simplicity, accuracy, precision and low cost (Raza et al., 2003; 2005a, 2005b). The act of identifying materials based on their color was probably one of the earliest examples of qualitative molecular absorption spectrophotometry. Also, the first recognition that color intensity can be an indicator of concentration was probably the earliest application of employing molecular absorption spectroscopy for quantitative determination. The first measurements were made by using the human eye as the detector and undispersed sunlight or artificial light as the light source (Marczenko, 2000). Later it was found that the accuracy and the precision could be improved by isolating specific frequencies of light using optical filters. Further improvement of the measurement came with the use of prism and grating monochromators for wavelength isolation. Photoelectric detectors were soon developed, but were quickly replaced with phototubes and photomultiplier tubes. The development of solid state microelectronics has now made available a wide range of detector type which if coupled with the computers; provide highly sophisticated readout electronic systems (Marczenko, 2000).
1.2 Colour and molecular structure
Absorption spectrophotometry in the ultra-violet and visible regions is considered as one of the valued techniques for quantitative analysis. Visible light represents a very small part of the electromagnetic spectrum and is generally considered to extend from 380-780 nm. A
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solution or object appears coloured when it transmits or absorbs only part of the radiation in the visible spectrum. The optical characteristic of the substance is its absorption spectrum. There is a close relation between the colour of a substance and its electronic structure. A molecule exhibits absorption in the visible or ultraviolet range, when radiation causes an electronic transition, raising the molecule (ion) from the ground state to an exited state. The production or change of a colour is connected with deformation of the normal electronic structure of the molecule. Irradiation causes variations in the electronic energy of the molecules containing one or more chromophoric groups, i.e. atomic groupings with unsaturated linkages. Two or more chromophoric groups in the molecule often enhance one another’s effect, to deepen the colour by displacing the maximum absorption towards longer wavelengths (from the ultraviolet towards the red). This is called bathochromic shift. The displacement of the absorption maximum from the red towards the ultraviolet is known as a hypsochromic shift (Blaedel and Meloche, 2001). The colour of a molecule may be intensified by substituents called auxochromic groups. These groups may also affect bathochromic shifts. The colour determining factor in a number of molecules is the introduction of conjugation of double bonds by means of electron donor and electron acceptor groups. The quantitative applicability of the absorption method is based on the fact that the number of photons absorbed is directly proportional to the number or concentration of atoms, ions or molecules (Blaedel and Meloche, 2001).
1.3 Lamivudine as drug for analysis
Lamivudine is 4-Amino-1-((2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl) pyrimidin-2(1H)-one (BP, 2009) a synthetic nucleoside analogue with activity against the human
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immunodeficiency virus (HIV) and hepatitis B virus (HBV) (Sean, 2009). The molecule has two chiral centers and is manufactured as the pure 2R, cis(−)-enantiomer. The racemic mixture from which lamivudine originates has antiretroviral activity but is less potent and substantially more toxic than the pure (−)-enantiomer. Compared with the (+)-enantiomer, the phosphorylated (−)-enantiomer is more resistant to cleavage from nascent RNA/DNA duplexes by cellular 3′-5′ exonucleases, which may contribute to its greater potency (Skalski et al., 1993). Lamivudine is either formulated alone as a tablet formulation or in combination with zidovudine and it is also available as oral pediatric formulation. The method for assay of lamivudine is HPLC (BP, 2009 and USP, 2007). A few high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC) techniques have been suggested for analysis of the formulation (Shalini et al., 2009). HPLC is the most widely used technique for the estimation of lamivudine in human plasma, saliva, cerebrospinal fluid, and human blood cells, as well as for studying the drug metabolites in the urine (Basavaiah and Somashekar, 2009). The suggested HPTLC and HPLC methods for assay of lamivudine are expensive and need complex and sophisticated instrumentation. Lamivudine can also be determined by Reverse Phase-HPLC method with lesser runtime, but the aforementioned drawback still persists (Babu and Kumar, 2009).
1.4 Statement of Research Problem
The major problem that led to this research is the lack of easy access to the HPLC machine (used for BP and USP assay of lamivudine) in our environment and the technical hands to
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operate the equipment. One of the first methods for visible spectrophotometric determination of lamivudine was based on the colored condensation products of aromatic aldehydes, specifically para-dimethyl benzaldehyde (Baig et al., 2001). This method suffers the drawback of significant interference from the excipients; since the determination is carried out at much shorter wavelengths. There are also reports on the development of methods using chloramines T (Basavaiah and Somashekar, 2006), para dimethyl cinnameldehyde (Sriker et al., 2009), paradimethylaminobenzaldehyde and vanillin (Babu and Kumar, 2009), potassium permanganate (Sarma et al., 2002), N-bromosuccinamide (Sarma et al., 2002), potassium bromated and bromide mixture (Basavaiah and Somashekar, 2006), bromophenol blue and MBTH (Kumar et al., 2011), chloranillic acid and DDQ (Kenneth et al., 2011), ethanol, tetrahydrofuran and formic acid (Venkatesh et al., 2012) reagents for the estimation of lamivudine in pharmaceuticals. It is also reported that lamivudine can also be assayed by titrimetric methods based on diazocoupling, redox reaction using Folin-Ciocalteu reagent, and redox-complexation reaction using ferric chloride-orthophenanthroline (Appalaraju et al., 2002). However, some of the above mentioned UV spectrophotometric methods are reported to suffer from disadvantages like instability of the reagents, high cost of the chemicals, reduced sensitivity (Sarma et al., 2002).
Also a major challenge to treatment scale-up is the low availability of and delays in the delivery of ARVs. Presently, the Nigerian government is the main provider of antiretroviral services in the country (WHO, 2004), and there is a determined effort to further reduce the cost of ARVs to make it affordable to patients. With the local manufacture of generic ARVs,
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the need to monitor the quality of these drugs cannot be over emphasized as our contemporary drug market is frequently eroded with fake and substandard drugs. A study carried out by Abuga et al., (2003) three samples failed to meet the percentage active ingredient content required out of the thirty three samples selected. In view of this, there is need to provide a simple, accurate and sensitive analytical method by which such problem of monitoring the quality of these drugs can be addressed particularly lamivudine which occupy a strategic position in clinical practice.
1.5 Justification of the study
The cost and unavailability of the HPLC machine in this environment as well as the number of limitations associated with the reported UV spectrophotometric methods for lamivudine assay justifies the need for development of simpler methods that will take care of the limitations.
The presence of free –NH2 and -C=O groups in lamivudine can serve as a suitable site for coupling with a suitable reagent or formation of Schiff base and subsequent generation of coloured compound. The amino group can also be utilized in the formation of coloured diazo compounds. The intensity of the colour can serve as a means for the determination of the drug spectroscopically.
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1.6 Aims and Objectives
The aim and specific objectives of the study are:
1. Development of UV spectrophotometric methods for the determination of lamivudine, using diazotization and Schiff’s base formation.
2. Validation of the methods so developed.
3. Application of the developed methods in determination of lamivudine in pure form and in tablet dosage form.
1.7 Research Hypothesis
A simple, accurate, precise, quantitative and cost effective spectrophotometric method for the determination of lamivudine can be developed by extracting the drug with methanol, formation of a Schiff’s base via the carbonyl group or diazotization of the free primary amino group in the drug.
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