DEVELOPMENT OF VALIDATED ANALYTICAL AND BIOANALYTICAL METHODS FOR NEWER DRUG FORMULATIONS

Transcription

1 DEVELOPMENT OF VALIDATED ANALYTICAL AND BIOANALYTICAL METHODS FOR NEWER DRUG FORMULATIONS Thesis submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai for the award of the degree of DOCTOR OF PHILOSOPHY In the Faculty of Pharmacy Submitted by Susheel John Varghese, M. Pharm. Under the guidance of Dr. T. K. Ravi, M. Pharm., Ph.D., FAGE COLLEGE OF PHARMACY SRI RAMAKRISHNA INSTITUTE OF PARAMEDICAL SCIENCES COIMBATORE , TAMIL NADU, INDIA JUNE 2011

2 Certificate This is to certify that the Ph.D. dissertation entitled DEVELOPMENT OF VALIDATED ANALYTICAL AND BIOANALYTICAL METHODS FOR NEWER DRUG FORMULATIONS being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in PHARMACY was carried out by SUSHEEL JOHN VARGHESE, in the College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, under my direct supervision and guidance to my fullest satisfaction. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma. Dr. T.K. Ravi, M. Pharm., Ph.D., FAGE, Principal, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore , Tamil Nadu, India Place: Coimbatore Date:

3 Certificate This is to certify that the Ph.D. dissertation entitled DEVELOPMENT OF VALIDATED ANALYTICAL AND BIOANALYTICAL METHODS FOR NEWER DRUG FORMULATIONS being submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in PHARMACY was carried out by SUSHEEL JOHN VARGHESE, in the College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, under my co-guidance to my fullest satisfaction. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma. Mr. Francis Saleshier M., M. Pharm. Professor and Head, Department of Pharmaceutical Chemistry, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore , Tamil Nadu, India Place: Coimbatore Date:

4 Declaration This is to certify that the Ph.D. dissertation entitled DEVELOPMENT OF VALIDATED ANALYTICAL AND BIOANALYTICAL METHODS FOR NEWER DRUG FORMULATIONS submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai, for the award of degree of DOCTOR OF PHILOSOPHY in PHARMACY was carried out by me under the supervision of Dr. T. K. RAVI, M. Pharm., Ph.D., FAGE, in the College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or University for the award of any degree or diploma. Susheel John Varghese Place: Coimbatore Date:

5 CONTENTS Page LIST OF ABBREVIATIONS & SYMBOLS LIST OF APPENDICES LIST OF PUBLICATIONS i iv v CHAPTER 1: INTRODUCTION 1.1. What are cardiovascular diseases? Cardiovascular drugs Drug analysis Quantification Procedure Method Validation Statistics 12 CHAPTER 2: LITERATURE REVIEW 2.1. Amlodipine Aspirin Atorvastatin Ezetimibe Fenofibrate Hydrochlorothiazide Nebivolol Olmesartan medoxomil Ramipril Rosuvastatin Telmisartan Valsartan 43

6 CHAPTER 3: AIMS AND OBJECTIVES 46 CHAPTER 4: DRUG PROFILE 48 CHAPTER 5: INSTRUMENTS AND MATERIALS 54 CHAPTER 6: METHODOLOGY 6.1. High-performance liquid chromatography High-performance thin layer chromatography UV spectroscopy Liquid chromatography-mass spectrometry (bioanalysis) 61 CHAPTER 7: EXPERIMENTAL WORK Part1- Analytical method development 7.1. Amlodipine-hydrochlorothiazide-valsartan Atorvastatin-ezetimibe-fenofibrate Telmisartan-ramipril-hydrochlorothiazide Atorvastatin-ramipril-aspirin Rosuvastatin-ezetimibe Telmisartan-atorvastatin Rosuvastatin-fenofibrate Olmesartan medoxomil-amlodipine Telmisartan-amlodipine Nebivolol-amlodipine 95 Part 2- LC-MS bioanalytical method development Rosuvastatin-ezetimibe Telmisartan-atorvastatin 103

7 CHAPTER 8: RESULTS AND DISCUSSION Part1- Analytical method development 8.1. Amlodipine-hydrochlorothiazide-valsartan Atorvastatin-ezetimibe-fenofibrate Telmisartan-ramipril-hydrochlorothiazide Atorvastatin-ramipril-aspirin Rosuvastatin-ezetimibe Telmisartan-atorvastatin Rosuvastatin-fenofibrate Olmesartan medoxomil-amlodipine Telmisartan-amlodipine Nebivolol-amlodipine 180 Part 2- LC-MS bioanalytical method development Rosuvastatin-ezetimibe Telmisartan-atorvastatin 193 CHAPTER 9: CONCLUSION 198 REFERENCES APPENDICES

8 LIST OF ABBREVIATIONS & SYMBOLS % Percentage > Greater than µ Micro µg Microgram µg/ml Microgram per millilitre µl Microlitre µv Microvolt AML ANOVA API ASP ATO CV CVDs DAD ESI EZE FEN Fig. h H 0 HPTLC HYD ICH Amlodipine besylate Analysis of variance Active pharmaceutical ingredients Aspirin Atorvastatin Coefficient of variation Cardiovascular diseases Diode-array detector Electrospray ionization Ezetimibe Fenofibrate Figure Hour Null hypothesis High-performance thin layer chromatography Hydrochlorothiazide International conference on Harmonization i

9 IS LC LC-MS LC-MS/MS LLE LLOQ LOD LOQ m/z mau mg MHz min ml mm mm/s NEB ng nm NMR o C ODS OLM PC pg/ml QC Internal standard Liquid chromatography Liquid chromatography mass spectrometry Liquid chromatography/tandem mass spectrometry Liquid liquid extraction Lower limit of quantification Limit of detection Limit of quantification Mass-to-charge ratio Milli absorbance unit Milligram Megahertz Minute Millilitre Millimolar Millimetre per second Nebivolol Nanogram Nanometre Nuclear magnetic resonance Degree celsius Octadecylsilane Olmesartan medoxomil Personal computer Picograms per millilitre Quality control ii

10 R f ROS RP-HPLC RPL rpm RSD R t S/N s-aml SIM TLC TLM ULOQ USFDA UV v v/v VAL λ max Retention factor Rosuvastatin Reverse phase high-performance liquid chromatography Ramipril Revolutions per minute Relative standard deviation Retention time Signal-to-noise ratio s-amlodipine Selected ion monitoring Thin layer chromatography Telmisartan Upper limit of quantification United states food and drug administration Ultraviolet Volume Volume to volume Valsartan Wavelength of maximum absorbance iii

11 LIST OF APPENDICES Appendix 1 Column certificates Appendix 1.1 Luna C18 (4.60 mm 150 mm, 5 µ particle size), Phenomenex Appendix 1.2 Lichrospher RP18, (4 mm 250 mm, 5 µ particle size), Merck Appendix 1.3 Shim-pack C18 (6 mm 150 mm, 5 µ particle size), Shimadzu Appendix 1.4 Chromolith RP18 (6 mm 100 mm, 5 µ particle size), Merck Appendix 2 Instrument settings for LC-MS Appendix 3 Publications Appendix 3.1 Determination of rosuvastatin and ezetimibe in a combined tablet dosage form using high-performance column liquid chromatography and high-performance thin-layer chromatography Appendix 3.2 Simultaneous determination of ramipril, hydrochlorothiazide and telmisartan in tablet dosage form using high-performance liquid chromatography method Appendix 3.3 Quantitative simultaneous determination of amlodipine, valsartan and hydrochlorothiazide in exforge hct tablets using high-performance column liquid chromatography and highperformance thin-layer chromatography IV

12 LIST OF PUBLICATIONS Susheel John Varghese, Thengungal Kochupappy Ravi. Determination of rosuvastatin and ezetimibe in a combined tablet dosage form using highperformance column liquid chromatography and high-performance thin-layer chromatography. Journal of AOAC International. 2010; 93(4): Susheel John Varghese, Thengungal Kochupappy Ravi. Simultaneous determination of ramipril, hydrochlorothiazide and telmisartan in tablet dosage form using high-performance liquid chromatography method. Der Pharmacia Lettre. 2011; 3(2): Susheel John Varghese, Thengungal Kochupappy Ravi. Quantitative simultaneous determination of amlodipine, valsartan and hydrochlorothiazide in exforge hct tablets using high-performance column liquid chromatography and high-performance thin-layer chromatography. Journal of Liquid Chromatography & Related Technologies. 2011; 34(12): v

13 Introduction CHAPTER 1. INTRODUCTION Most countries face high and increasing rates of cardiovascular disease (CVDs). Cardiovascular diseases are the number one cause of death globally. The variety and scope of cardiovascular drugs have increased immensely in the past few decades and new drugs are being approved annually. The term cardiovascular drug refers to any medication that affects the heart, blood vessels or the circulatory system. These drugs can be used alone or in combination with each other in the treatment of a variety of disease states such as hypertension, acute coronary syndromes, congestive heart failure (CHF), arrhythmias and dyslipidemias (1). This thesis deals with the development of validated analytical and bioanalytical methods for the determination of newer cardio vascular drugs in combination form in formulations and biological fluids Cardiovascular diseases CVDs are caused by disorders of the heart and blood vessels and include coronary heart disease (heart attacks), cerebrovascular disease (stroke), raised blood pressure (hypertension), peripheral artery disease, rheumatic heart disease, congenital heart disease and heart failure (2). More people die annually from CVDs than from any other cause. An estimated 17.1 million people died from CVDs in 2004, representing 29 % of all global deaths. Among these deaths, an estimated 7.2 million were due to coronary heart disease and 5.7 million were due to stroke. Low- and middle-income countries are disproportionally affected: 82% of CVD deaths take place in low- and middleincome countries and occur almost equally in men and women. By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. These are projected to remain the single leading cause of death (3). Higher CVD rates will also 1

14 Introduction have an economic impact (4). As the number of medications that a patient requires increases, adherence and compliance to therapy are likely to decrease (5). Hence, to avoid these problems and to give better therapeutic effect, combination therapy has become more popular Cardiovascular drugs The classification of cardiovascular drugs is as follows (6): General: β-receptor blockers ("beta blockers"), calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, nitrates, antianginals, vasoconstrictors, vasodilators, peripheral activators. Affecting blood pressure (antihypertensive drugs): ACE inhibitors, angiotensin receptor blockers, α blockers, calcium channel blockers. Coagulation: Anticoagulants, heparin, antiplatelet drugs, fibrinolytics, antihemophilic factors, haemostatic drugs. Atherosclerosis/cholesterol inhibitors: Hypolipidaemic agents, statins Drug analysis The main aim of drug analysis is to contribute to the improvement of drug safety. Controlling and minimizing the side effects of drugs are the key issues in assuring the safety of drug therapy. Since side effects are inherent properties of the drug material, these cannot be influenced by drug analysis. At the same time, drug analysts play a predominant role in assuring the quality of bulk drug materials and drug formulations and this is also closely related to the safety issue. The three main attributes of drug quality are identity, strength and purity. The development in the field of chromatographic and spectroscopic methods in the last decades had led to changes in the philosophy, structure and requirements in the monographs of drug materials in the principal pharmacopoeias (7). 2

15 Introduction Analytical method development Analytical method development and validation play vital role in the discovery, development and manufacture of pharmaceuticals. The test methods that result from these processes are used by quality control laboratories to ensure the identity, purity, potency and performance of drug products. Pharmaceutical products formulated with more than one drug are referred to as combination products. These combination products can present challenges to the analyst responsible for the development and validation of analytical methods. The number of drugs introduced into the market is increasing every year. These drugs may be either new entities or partial structural modification of the existing ones. Very often, there is a time lag from the date of introduction of a drug into the market to the date of its inclusion in pharmacopoeias. This happens because of the possible uncertainties in the continuous and wider usage of these drugs, reports of new toxicities (resulting in their withdrawal from the market), development of patient resistance and introduction of better drugs by competitors. Under these conditions, standards and analytical procedures for these drugs may not be available in the pharmacopoeias. Hence, it becomes necessary to develop newer analytical methods for such drugs. This thesis will discuss the development and validation of analytical methods [spectrophotometric, high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC)] for drug products containing more than one active ingredient. 3

16 Introduction Basic criteria for new method development of drug analysis The drug or drug combination may not be official in any pharmacopoeias. A proper analytical procedure may not be available for the drug in the literature due to patent regulations. Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients. Analytical methods for the quantitation of the drug in biological fluids may not be available. Analytical methods for a drug in combination with other drugs may not be available. The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable Separation methods Chemical separations are achieved using chromatographic and, to a much lesser extent, electrophoretic methods. In chromatographic methods, separation is based on variation in the distribution of different compounds between two dissimilar phases-a stationary phase and a mobile phase. Further differentiation can be made between chromatographic procedures in which the individual components elute from the stationary phase and are monitored online (column chromatography) and procedures in which the components are measured in situ on the chromatographic stationary phase [e.g., thin-layer chromatography (TLC)]. Chromatographic methods utilizing a liquid as the mobile phase are termed liquid chromatography. Modern liquid chromatography performed in a highly automated format is termed (HPLC). 4

17 Introduction The primary goal of chromatographic separations is to separate the components (of interest) in the sample HPLC HPLC is the premier analytical technique in pharmaceutical analysis, which is predominantly used in the pharmaceutical industry for a large variety of samples. It is the method of choice for checking the purity of new drug candidates, monitoring changes or scale ups of synthetic procedures, evaluating new formulations and scrutinizing quality control of final drug products. All column chromatographic separations operate on the same principle: the distribution of solute between two dissimilar phases-a mobile phase and a stationary phase. In each procedure, sample is introduced into one end of the column and the mobile phase transports the sample components toward the other end of the column. In the absence of interaction with the stationary phase, all components would exit the other end of the column after a time, t 0, based on the column volume and mobile-phase flow rate and could be detected at the other end of the column using an on-line detector. If an interaction with the stationary phase occurs, the time taken for a solute to elute from the column would be increased by the time that the solute spends in association with the stationary phase (R t ). Isocratic vs gradient analysis Traditionally, most pharmaceutical assays are isocratic analysis employing the same mobile phase throughout the elution of the sample. Notable disadvantages of isocratic analysis are limited peak capacity (the maximum number of peaks that can be accommodated in the chromatogram), and problems with samples containing analytes of diverse polarities. In contrast, gradient analysis in which the strength of 5

18 Introduction the mobile phase is increased with time during sample elution is suited for complex samples and those containing analytes of wide polarities TLC TLC is a chromatographic technique used to separate mixtures of compounds. Modern TLC can be done in both the normal-phase and the reversed phase modes. The role of TLC in qualitative or semi quantitative analysis is large. TLC is a useful technique to complement HPLC during early-phase method development (8). A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantization. This method is referred to as HPTLC. Advantages The sample preparation is usually relatively simple. Samples may be directly compared, often as they are running. A range of detection procedures can be applied to the same plate. Solvents and other reagents are required in very small volumes (9) Ultraviolet spectroscopy UV spectroscopy is the most frequently employed technique in the analysis of various pharmaceuticals. This technique utilizes the measurement of intensity of electromagnetic radiation emitted or absorbed by the analyte. UV spectroscopy is usually applied to molecules in solution. The concentration of an analyte in solution can be determined by measuring the absorbance at some wavelength and applying the Beer-Lambert law. The following technique is used for multicomponent analysis of drugs. 6

19 Introduction Derivative spectrophotometry A conventional spectrum is a plot of absorbance against wavelength (A vs λ). An analyst regularly comes across the condition where the amount of one or more substances in the sample is known to have other absorbing substances which might affect the assay. The interference which is difficult to be quantified may arise in the analysis of formulations from manufacturing impurities, decomposition products and formulation excipients. Unwanted absorption from these sources is termed as irrelevant absorption and if not removed, imparts a systematic error to the assay of the drug in the sample. In order to solve this problem various methods like derivative spectroscopy are used. Derivative spectroscopic method involves the transformation of a normal spectrum to its 1 st, 2 nd or higher derivative spectrum. The transformations that occur in the derivative spectra are understood with reference to a Gaussian band which represents an ideal absorption band. The D 1 (1 st derivative) spectrum is a plot of da/dλ Vs λ. It is characterised by a cross- over point at the λ max of the absorption band. The D 2 (2 nd derivative) spectrum is a plot of d 2 A/dλ 2 Vs λ. It is characterised by a reversed band which corresponds to the λ max of the fundamental band and two satellite maxima (10) Bioanalysis Bioanalysis is the quantitative determination of drugs and their metabolites in biological fluids. This technique is used very early in the drug development process to provide support to drug discovery programs on the metabolic fate and pharmacokinetics of chemicals in living cells and in animals. Its use continues throughout the preclinical and clinical drug development phases, into post-marketing 7

20 Introduction support and may sometimes extend into clinical therapeutic drug monitoring. A reliable analytical method is achieved with the successful combination of efficient sample preparation, adequate chromatographic separation and a sensitive detection technique. Liquid chromatography (LC) coupled with detection by mass spectrometry (MS) is the preferred analytical technique for drug analysis Fundamental strategies for bioanalytical sample preparation It is a challenge to develop bioanalytical methods that selectively separate drugs and metabolites from endogenous materials in the sample matrix. Sample preparation is a technique used to clean up a sample before analysis and/or to concentrate a sample to improve its detection. When samples are biological fluids such as plasma, serum or urine, this technique is described as bioanalytical sample preparation. Many different sample preparation techniques are available in order to meet the desired objectives for an assay Major goals for sample preparation 1. To remove unwanted matrix components that can cause interferences upon analysis. 2. Concentrate an analyte to improve its limit of detection. 3. Exchange the analyte from an environment of aqueous solvent into a high percentage organic solvent suitable for injection into the chromatographic system Typical choices of sample preparation techniques useful in bioanalysis Dilution followed by injection Protein precipitation Filtration Protein removal by equilibrium dialysis or ultrafiltration Liquid-liquid extraction (LLE) 8

21 Introduction Solid-supported liquid-liquid extraction LLE LLE is a technique used to separate analytes from interferences in the sample matrix by partitioning the analytes between two immiscible liquids. Analytes distribute between these two liquid phases (aqueous and organic) and partition takes place preferentially into the organic phase when the analytes are unionized (uncharged) and have solubility in that organic solvent. Isolation of the organic phase, followed by evaporation and reconstitution in a mobile phase compatible solvent, yields a sample ready for injection. This technique provides efficient sample cleanup as well as sample enrichment. A major advantage of LLE is that it is widely applicable for many drug compounds and is a relatively inexpensive procedure. With proper selection of organic solvent and adjustment of sample ph, very clean extracts can be obtained with good selectivity for the target analytes. Inorganic salts are insoluble in the solvents commonly used for LLE and remain behind in the aqueous phase along with proteins and water soluble endogenous components. These interferences are excluded from the chromatographic system and a cleaner sample is prepared for analysis Liquid chromatography-mass spectrometry (LC-MS) The preferred analytical technique in the bioanalytical research environment is liquid LC-MS, used for qualitative and quantitative drug identification. LC-MS is preferred for its speed, sensitivity and specificity. LC is a powerful and universally accepted technique that offers chromatographic separation of individual analytes within liquid mixtures. These analytes are subjected to an ionization source and then are introduced into the mass spectrometer. The mass spectrometer separates these ions based on their mass-to-charge ratio (m/z) and then sends them to the detector. The 9

22 Introduction data generated are used to provide information about the molecular weight, structure, identity and quantity of specific components within the sample. A general scheme of this process is shown below. Sample Inlet Ionization Source Mass Analyzer Detector Data Analysis LC-MS Interface The LC-MS interface is the most important element of this system. It is the point at which the liquid from the LC (operated at atmospheric pressure) meets the mass spectrometer (operated in a vacuum) Electrospray Ionization (ESI) ESI generates ions directly from the solution phase into the gas phase. The ions are produced by applying a strong electric field to a very fine spray of the analyte in solution. The electric field charges the surface of the liquid and forms a spray of charged droplets. The charged droplets are attracted toward a capillary orifice where heated nitrogen drying gas shrinks the droplets and carries away the uncharged material. As the droplets shrink, ionized analytes escape the liquid phase through electrostatic forces and enter the gas phase, where they proceed into the low pressure region of the ion source and into the mass analyzer Mass Analyzers The ionization source and the mass analyzer are linked since the mass analyzer requires a charged particle for separation to occur. The mass analyzer contains electric or magnetic field, or combination of the two, which can manipulate the trajectory of the ion in a vacuum chamber. The most popular mass analyzer is the quadrupole. The quadrupole mass analyzer is available as a single quadrupole or is configured in tandem (called a triple quadrupole) to greatly enhance its capabilities. A quadrupole 10

23 Introduction mass filter typically consists of four cylindrical electrodes (rods) to which precise DC and RF voltages can be applied. A mass spectrometer with a single quadrupole is capable of either full scan acquisition or selected ion monitoring (SIM) detection. In the full scan mode, the instrument detects signals over a defined mass range during a short period of time. All the signals are detected until the full mass range is covered. A mass spectrum is generated in which ion intensity (abundance) is plotted versus m/z ratio. Scan mode is used for qualitative analysis when the analyte mass is not known. In the SIM mode, a single stage quadrupole is used as a mass filter and monitors only a specific m/z ratio, allowing only that m/z ratio to pass through the detector (11) Quantification Procedures Peak-height or peak-area measurements only provide a response in terms of detector signal. This response must be related to the concentration or mass of the compound of interest. To accomplish this, some type of calibration must be performed. They are, Normalised peak area method External standard calibration method Internal standard calibration method Method of standard addition (12) Method Validation Method validation is defined as the process of proving that an analytical method is acceptable for its intended use. Guidelines for method validation for new noncompendial test methods are provided by the international conference on harmonization (ICH) draft document, text on validation of analytical procedures (Q2A) (13). Typical analytical parameters used in validation include accuracy, 11

24 Introduction precision, limit of detection, limit of quantification, selectivity, specificity, reproducibility and stability. Bioanalytical method validation includes all procedures that demonstrate that a particular method used for quantitative measurement of analytes in a given biological matrix such as blood, plasma, serum or urine, is reliable and reproducible for the intended use. Guidelines for bioanalytical method are provided by the food and drug administration (FDA) (14) Statistics Statistical methods are necessary part of the development and testing of drug products. An important aspect of any method transfer or crossover study is determining whether the results are equivalent. Following statistical tools are used during this study (9, 12) Arithmetic Mean (Average) The average result, denoted as x, is calculated by summing the individual results and dividing this by the number (n) of individual values: x = x 1 + x 2 + x 3 n + x Standard Deviation It is one of the most common statistical terms employed in analytical chemistry. The standard deviation is a measure of how precise the average is, that is, how well the individual numbers agree with each other. It is a measure of a type of error called random error. It is calculated as follows: Standard deviation, S = ( X X ) n

25 Introduction Relative Standard Deviation Precision often is expressed by the standard deviation (SD) or relative standard deviation (RSD) of a data set. The square of the standard deviation is called the variance. A further measure of precision, known as the relative standard deviation (RSD), is given by RSD = s x This measure is often expressed as a percentage, known as the coefficient of variation (CV) or %RSD. CV S = 100 x Comparison of results (Determining equivalence) An important aspect of any method transfer or crossover study is determining whether the results are equivalent. There are two common methods for comparing results, Student s t-test Analysis of variance (ANOVA) Each of these tests has a specific purpose in analyzing data Student s t-test The t-test compares the mean results obtained from two experiments or determines if a sample mean is different from a standard value. It is also used to test the difference between the means of two sets of data Analysis of variance (ANOVA) The comparison of more than two means is done by using ANOVA. The variations between techniques may be calculated and their effects estimated by this technique. 13

26 Literature Review CHAPTER 2. LITERATURE REVIEW 2.1. Amlodipine Naidu et al. reported a stability indicating RP-HPLC method for simultaneous determination of amlodipine and benazepril hydrochloride from their combination drug product. The mobile phase consisted of phosphate buffer and acetonitrile in the proportion of 65:35 (v/v) with apparent ph adjusted to 7. UV detection was done at 240 nm using a photodiode array detector. The described method was linear over a range of 6-14 µg/ml for amlodipine and µg/ml for benazepril hydrochloride (15). Zarghi et al. reported a rapid, simple and sensitive HPLC method was developed for quantification of amlodipine in plasma. The assay enabled the measurement of amlodipine for therapeutic drug monitoring with a minimum detectable limit of 0.2 ng/ml. The method involved simple, one-step extraction procedure and analytical recovery was about 97%. The calibration curve was linear over the concentration range ng/ml (16). Nesrin et al. reported HPLC and HPTLC methods for the simultaneous estimation of amlodipine besylate and valsartan. Separation by HPLC was achieved using a C18 column and methanol-acetonitrile-water-0.05% (v/v) triethylamine in a ratio 40:20:30:10, v/v/v/v, as mobile phase, ph was adjusted to 3 with ortho-phosphoric acid. The HPTLC method used silica gel 60 F 254 plates; the optimized mobile phase was ethyl acetate-methanol-ammonium hydroxide (55:45:5, v/v/v) (17). Syed et al. reported RP-HPLC method for the simultaneous determination of olmesartan medoxomil and amlodipine in pharmaceutical tablet formulations. 14

27 Literature Review Analysis was carried out using reverse phase isocratic elution with a C18 column and a mobile phase consisting of 0.05 M ammonium acetate (ph 6.8) and acetonitrile in the ratio of 40:60, v/v. Detection of the analytes was achieved using UV detector at 239 nm. The retention times of olmesartan medoxomil and amlodipine were 2.50 and 3.94 minutes respectively (18). Sohan et al. reported a RP-HPLC method for the simultaneous estimation of amlodipine and metoprolol. Mobile phase used was 0.02 M phosphate buffer solution: acetonitrile (70:30, v/v, ph 3.0). The retention times for amlodipine and metoprolol were 2.57 and 4.49 min respectively. Amlodipine and metoprolol showed a linear response in the concentration range of µg/ml (19). Rajeswari et al. reported a RP-HPLC method for the simultaneous determination of atorvastatin and amlodipine in combined tablet dosage form. The mobile phase used was a mixture of acetonitrile and 0.03M phosphate buffer ph 2.9 (55:45, v/v). The detection of atorvastatin and amlodipine was carried out on dual absorbance detector at 240 nm and 362 nm respectively (20). Meyyanathan and Suresh reported a HPTLC method for the simultaneous analysis of amlodipine and benazepril. The method used zolpidem as an internal standard (IS). The mobile phase consisted of ethyl acetate-methanolammonia solution (8.5:2.0:1.0, v/v/v). Detection was performed densitometrically at a wavelength of 254 nm. The R f values of amlodipine, benazepril and zolpidem (IS) were 0.58, 0.50 and 0.78 respectively (21). 15

28 Literature Review Wankhede et al. reported two UV-spectrophotometry methods and a RP- HPLC method for the simultaneous estimation of amlodipine besylate, losartan potassium and hydrochlorothiazide in tablet dosage form. The first UV spectrophotometric method used the simultaneous equation method over the concentration range 5-25, and 5-25 µg/ml for amlodipine besylate, losartan potassium and hydrochlorothiazide respectively. The second UV method applied area under curve method over the concentration range of 5-25, 5-25 and µg/ml for amlodipine besylate, hydrochlorothiazide and losartan potassium respectively. RP-HPLC analysis was carried out using M phosphate buffer (ph 3.7)-acetonitrile (57:43, v/v) as the mobile phase (22). Bahrami and Mirzaeei reported a RP-HPLC method using fluorescence detection for analysis of amlodipine in human serum. Amlodipine was extracted from serum by ethyl acetate and involved precolumn derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl). The mobile phase used was sodium phosphate buffer (ph 2.5) containing 1 ml/l triethylamine and methanol at flow rate of 2.8 ml/min. Propranolol was used as IS. The standard curve was linear over the range ng/ml for amlodipine in human serum (23). Ramakrishna et al. reported a LC-MS/MS method for the assay of amlodipine in human plasma. Following LLE, the analytes were separated using an isocratic mobile phase on a reverse-phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M+H] + ions, m/z 409/238 for amlodipine and m/z 409/228 for the IS. The assay exhibited a 16

29 Literature Review linear dynamic range of 50 10,000 pg/ml for amlodipine in human plasma (24). Miroshnichenko et al. reported a LC-MS/MS method for the determination of amlodipine in blood plasma. The HPLC system was coupled to a MS detector via an electrospray ionization interface in the positive ion mode. The plasma samples were extracted with a diethylether-hexane (8:2, v/v) mixture by means of solid-phase extraction (25). Massaroti et al. reported a LC-MS/MS method for quantifying amlodipine in human plasma. Sample preparation was based on LLE using sodium hydroxide and a mixture of ethyl acetate-hexane (80:20, v/v). Chromatography was performed on a C18 analytical column and the retention times were 1.9 and 3 min for amlodipine and nimodipine (IS) respectively (26) Aspirin Anandakumar et al. reported a HPLC method for the separation of aspirin and clopidogrel bisulphate by C18 column and acetonitrile-methanol-20 mm phosphate buffer at ph 3 (50:7:43, v/v/v) as eluent at a flow rate of 1 ml/min. Detection was carried out at 240 nm. Linearity for aspirin and clopidogrel bisulphate were in the range of µg/ml for both the drugs (27). Londhe et al. reported a HPTLC method for the simultaneous quantification of aspirin, atorvastatin calcium and clopidogrel bisulphate in the bulk drug and in capsule dosage form. Mobile phase used was toluene-methanol-formic acid, 6.5:3.5:0.1 (v/v/v) as mobile phase. Densitometric evaluation of the separated zones was performed at 254 nm (28). 17

30 Literature Review Purushotam et al. reported a stability-indicating HPTLC method for the analysis of aspirin and clopidogrel bisulphate. The method employed TLC aluminum plates precoated with silica gel 60 F 254 as the stationary phase. The solvent system consisted of carbon tetrachloride-acetone (6:2.4, v/v). This system gave compact spots for both aspirin (R f value=0.13) and clopidogrel bisulphate (R f value=0.78) (29). Prakash et al. reported a RP-HPLC method for the simultaneous determination of aspirin and dipyridamole in pharmaceutical formulations. Chromatographic separation was achieved on a C18 column using a mobile phase system consisting of 0.1% ortho phosphoric acid and acetonitrile in the ratio of 75:25, v/v. The method was found to be linear over the concentration range of 4-80 µg/ml for dipyridamole and µg/ml for aspirin (30). Shah et al. reported a RP-HPLC method for the simultaneous determination of atorvastatin calcium and aspirin in capsule dosage forms. The mobile phase consisted of 0.02 M potassium dihydrogen phosphate: methanol (20:80, v/v) adjusted to ph 4 using ortho phosphoric acid was used. The flow rate was 1 ml/min and effluents were monitored at 240 nm. The linearity for atorvastatin calcium and aspirin were in the range of mg/ml and 5-25 mg/ml respectively (31). Altun et al. determined acetylsalicylic acid, caffeine and codeine phosphate simultaneously. Acetylsalicylic acid, caffeine and codeine phosphate were separated using a C8 column by isocratic elution with flow rate 1 ml/min. The mobile phase consisted of isopropyl alcohol-acetonitrile-water-ortho 18

31 Literature Review phosphoric acid in ratio of 125:125:250:0.5, v/v/v/v. The samples were detected at 215 nm using photo-diode array detector (32). Sultana et al. described a RP-HPLC method for simultaneous determination of clopidogrel and aspirin. The mobile phase consisted of methanol-water (80:20, v/v); the ph of the mobile phase was adjusted to 3.4 with orthophosphoric acid and pumped at a flow rate of 1 ml/min. Multivariate chromatographic calibration technique was subjected to HPLC for simultaneous quantitative analysis of binary mixtures of clopidogrel and aspirin. HPLC data based on the analyte peak areas were obtained at five wavelengths (225, 230, 235, 240 and 245 nm). The mathematical algorithm of multivariate chromatographic calibration technique was based on the use of the linear regression equations (33) Atorvastatin Jamshidi et al. reported a two-step isocratic chromatography method on silica gel 60F 254 HPTLC layer for the separation of atorvastatin from plasma. Densitometric quantitation was done at a wavelength of 280 nm. The calibration function of the analyte was linear in the range ng/band (34). Altuntas and Erk reported a HPLC method for determination of atorvastatin in bulk drug, tablets and human plasma. Liquid chromatography was performed on a C18 column and the mobile phase consisted of acetonitrile-methanolwater (45:45:10, v/v/v). The effluent was monitored on a UV detector at 240 nm. A calibration curve was constructed for atorvastatin in the range of µg/ml (35). 19

32 Literature Review Gholamreza et al. described a HPLC method for determination of atorvastatin in human serum. Following LLE, chromatographic separation was accomplished using C18 analytical column with a mobile phase consisting of sodium phosphate buffer (0.05 M, ph 4) and methanol (33:67, v/v). Atorvastatin and diclofenac sodium (IS) were detected by ultraviolet absorbance at 247 nm. The calibration curve was linear over a concentration range of ng/ml for atorvastatin in human serum (36). Shen et al. reported a RP-HPLC method with UV detection for the assay of atorvastatin in beagle dog plasma using Indomethacin as the IS. Atorvastatin was extracted by protein precipitation. The extracts were injected into a C8 column with wavelength set at 270 nm. The mobile phase consisted of acetonitrile-0.1 mol/l ammonium acetate buffer (ph 4) (65:35, v/v) at a flow rate of 1 ml/min. A linear relationship was obtained between the peak area ratios of atorvastatin to indomethacin in the concentration range of µg/ml (37). Alaa reported a validated stability-indicating HPLC method for determination of atorvastatin in bulk drug and tablet form. Atorvastatin was subjected to different stress conditions. Atorvastatin and its degradation products were analyzed on a C18 column using isocratic elution with acetonitrile 0.02 M sodium acetate, ph 4.2 (45:55, v/v) for 25 min. The samples were monitored with fluorescence detection at excitation wavelength of 282 nm and emission wavelength of 400 nm (38). Mohammadi et al. reported a simple, rapid, precise and accurate isocratic stability indicating RP-HPLC method for the simultaneous determination of 20

33 Literature Review atorvastatin and amlodipine in commercial tablets. Separation was achieved on a C18 column using a mobile phase consisting of acetonitrile M sodium dihydrogen phosphate buffer (ph 4.5) (55:45, v/v) at a flow rate of 1 ml/min and UV detection at 237 nm. The linearity was investigated in the range of 2 30 µg/ml for atorvastatin and 1 20 µg/ml for amlodipine (39). Shah et al. reported a chromatographic method for the simultaneous determination of atorvastatin calcium and amlodipine besylate in tablet dosage forms. A C18 column in isocratic mode, with mobile phase containing methanol-acetonitrile-50 mm potassium dihydrogen phosphate, ph 3.5(20:50:30, v/v/v) was used. The flow rate was 1 ml/min and eluent was monitored at 240 nm. The linearity for atorvastatin calcium and amlodipine besylate was in the range of µg/ml and µg/ml respectively (40). Bharat et al. reported a stability indicating RP-HPLC method for the simultaneous estimation of atorvastatin and amlodipine from their combination drug product. The separation was carried out on a C18 column using the mobile phase consisting of acetonitrile and 50 mm potassium dihdrogen phosphate buffer (60:40, v/v), apparent ph adjusted to 3 with 10% phosphoric acid solution. The flow rate was 1 ml/min and eluents were monitored at 254 nm. Atorvastatin and amlodipine and their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analyzed by proposed method (41). Erturk et al. described a HPLC method for the analysis of atorvastatin and its impurities in bulk drug and tablets. This method showed good resolution for atorvastatin, desfluoro-atorvastatin, diastereomer-atorvastatin, unknown 21

34 Literature Review impurities and formulation excipients of tablets. A gradient RP-HPLC method was used with UV detection. Good resolution was obtained using a C18 column with acetonitrile ammonium acetate buffer (ph 4)-tetrahydrofuran as mobile phase (42). Unnam and Chandrasekhar reported an isocratic RP-HPLC method for quantitative determination of atorvastatin and ezetimibe in pharmaceutical formulations. Atorvastatin and ezetimibe were determined using 0.01 M ammonium acetate buffer (ph 3)-acetonitrile (50:50, v/v) as mobile phase. The detection was done at 254 nm. The linearity of the method was studied over the concentration range of µg/ml for atorvastatin and µg/ml for ezetimibe (43). Gulam et al. described an isocratic stability indicating RP-HPLC method with UV detection at 247 nm for analysis of atorvastatin. Retention time of atorvastatin was found to be 4.02 min. A mobile phase consisting of 0.05 M sodium phosphate buffer (ph 4.1) and methanol (3:7, v/v) at flow rate of l ml/min was used in this study (44). Zahid et al. reported a stability-indicating HPLC method for the analysis of atorvastatin calcium in pharmaceutical dosage form. The mobile phase consisted of a mixture of methanol-acetonitrile-phosphate buffer solution, (45:45:10, v/v/v). Detection was carried out at 246 nm. The retention time of atorvastatin was 6.98 min. Atorvastatin Calcium was subjected to acid and alkali hydrolysis, oxidation, photochemical degradation and thermal degradation (45). 22

35 Literature Review Sandeep et al. described two methods for the simultaneous determination of atorvastatin calcium and ezetimibe in binary mixture. The first method was based on UV-spectrophotometric determination using simultaneous equation method. It involved absorbance measurement at nm and 246 nm in methanol. Linearity was obtained in the range of 5 25 µg/ml for both the drugs. The second method was based on HPLC separation of the two drugs in reverse phase mode using C18 column. Linearity was obtained in the concentration range of 8-22 µg/ml for both the drugs (46). Hiral et al. established a HPTLC method for simultaneous analysis of atorvastatin calcium, ramipril and aspirin in a capsule dosage form. Ultraviolet detection was performed at 210 nm. The R f values were 0.38, 0.06 and 0.86 for atorvastatin calcium, ramipril and aspirin, respectively. The linearity ranges were ng/band for atorvastatin calcium, ng/band for ramipril and ng/band for aspirin (47). Panchal and Suhagia established RP-HPLC and HPTLC methods for simultaneous determination of atorvastatin calcium and losartan potassium in tablet dosage forms. The mobile phase consisted of 0.05 M potassium dihydrogen phosphate buffer (ph 5.4)-acetonitrile, 45:55 (v/v). Quantification was achieved at 238 nm over the concentration range µg/ml, for each compound. For HPTLC method, methanol-carbon tetrachloride-ethyl acetateglacial acetic acid, 8:63.6:28:0.4 (v/v/v/v) was used as mobile phase. Quantification was achieved by UV detection at 238 nm over the concentration range of ng/band for each drug (48). 23

36 Literature Review Chaudri et al. reported a HPTLC method for the estimation of atorvastatin calcium and ezetimibe simultaneously in combined dosage forms. The mobile phase used was a mixture of chloroform-benzene-methanol-acetic acid (6:3:1:0.1, v/v/v/v). The detection of spots was carried out at 250 nm. The calibration curve was found to be linear from µg/band for atorvastatin calcium and µg/band for ezetimibe (49) Ezetimibe Li et al. reported a simple, reliable and sensitive LC MS/MS method for quantification of free and total ezetimibe in human plasma. The analyte and IS ( 13 C6-ezetimibe) were extracted by LLE with methyl tert-butyl ether. The assay exhibited linear ranges from ng/ml for free ezetimibe and ng/ml for total ezetimibe in human plasma (50). Sistla et al. reported a RP-HPLC method for assaying ezetimibe in pharmaceutical dosage forms. The assay involved an isocratic elution of ezetimibe on a C18 column using a mobile phase composition of water (ph 6.8, 0.05 % w/v 1-heptane sulfonic acid) and acetonitrile (30:70, v/v). The flow rate was 0.5 ml/min and the analyte was monitored at 232 nm. The method was found to be linear from µg/ml (51). Oliveira et al. reported a LC-MS-MS method for the determination of ezetimibe in human plasma. Ezetimibe and etoricoxib (IS) were extracted from the plasma by LLE and separated on a C18 analytical column with acetonitrile-water (85:15, v/v) as mobile phase. Detection was carried out by positive electrospray ionization in multiple reactions monitoring (MRM) mode (52). 24

37 Literature Review Oliveira et al. reported a validated RP-HPLC method for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical dosage forms. The mobile phase consisted of 0.03 M phosphate buffer, ph 4.5-acetonitrile (35:65, v/v) run at a flow rate of 0.6 ml/min. The detection was made using a photodiode array detector at 234 nm. Calibration graphs were linear in the concentration range of µg/ml (53). Chaudhari et al. reported a stability-indicating RP-HPLC method for simultaneous estimation of simvastatin and ezetimibe from their combination drug product. The method utilized mobile phase consisting of acetonitrile water methanol (60:25:15, v/v/v) with apparent ph adjusted to 4. The detection wavelength was 238 nm. The drugs and their combination drug products were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions and the stressed samples were analyzed by the proposed method. The method was linear over the range of 1 80 and 3 80 µg/ml for simvastatin and ezetimibe respectively (54). Saranjit et al. established a methodology where, ezetimibe was subjected to different ICH prescribed stress conditions. The drug was particularly labile under neutral and alkaline hydrolytic conditions. A stability-indicating HPLC method was developed for analysis of the drug in the presence of the degradation products. It involved a C8 column and mobile phase composed of ammonium acetate buffer (0.02 M, ph adjusted to 7 with ammonium hydroxide) and acetonitrile, in gradient mode (55). Rahul et al. reported a HPTLC method for analysis of simvastatin and ezetimibe. The method used hexane acetone 6:4 (v/v) as mobile phase. 25

38 Literature Review Densitometric analysis of both drugs was carried out in absorbance mode at 234 nm. This system gave compact bands for simvastatin (R f =0.39) and ezetimibe (R f =0.50). The drugs were linear in the concentration range 200 1,600 ng/band (56). Bharat et al. reported a stability-indicating RP-HPLC method for simultaneous estimation of atorvastatin and ezetimibe from their combination drug product. The proposed method utilized C18 column. The optimum mobile phase consisted of acetonitrile water methanol (45:40:15, v/v/v) with apparent ph adjusted to 4. The response was linear over the concentration range of 1 80 µg/ml for atorvastatin and ezetimibe (57). Imran et al. reported a rapid stability indicating ultraviolet spectroscopic method for the estimation of ezetimibe and carvedilol in pure form and in their respective formulations. The linearity range for ezetimibe and carvedilol at their respective wavelength of detection of 232 nm and 238 nm was found to be 2-50 µg/ml and 2-20 µg/ml respectively (58). Muhammad et al. reported a RP-HPLC method for the simultaneous determination of ezetimibe and simvastatin in pharmaceutical formulations. Chromatographic separation was performed on a C18 column at a wavelength of 240 nm using a mixture of 0.1M ammonium acetate buffer (ph 5) and acetonitrile in the ratio of (30:70, v/v) (59). 26

39 Literature Review 2.5. Fenofibrate Jain et al. reported a RP-HPLC method for the simultaneous estimation of atorvastatin calcium and fenofibrate in tablet formulation. The separation was achieved by C18 column using methanol-acetate buffer, ph 3.7 (82:18, v/v) as mobile phase, at a flow rate of 1.5 ml/min. Linearity for atorvastatin calcium and fenofibrate were in the range of 1-5 µg/ml and µg/ml respectively (60). El-Guindy et al. reported graphical spectrophotometric methods as first derivative of ratio spectra or first and second derivative spectrophotometry for determination of vinpocetine and fenofibrate respectively. HPLC method was developed using ODS column with mobile phase consisting of acetonitrilewater (80:20, v/v, ph 4) with UV detection at 287 nm for fenofibrate (61). Atul and Sanjay reported simple, rapid, selective and precise densitometric HPTLC method for simultaneous analysis of atorvastatin calcium and fenofibrate in pharmaceutical dosage forms in accordance with ICH guidelines. Polynomial regression data for the calibration plots showed that there was a good linear relationship between response and amount in the range ng/band for atorvastatin calcium and ng/band for fenofibrate (62). Gupta et al. reported a HPTLC method for estimation of fenofibrate in capsules. Aluminum plates precoated with Silica gel 60 F 254 was used as stationary phase and toluene: chloroform (7:3, v/v) was employed as mobile phase. Quantification was carried out at 296 nm (63). 27

40 Literature Review Rupali et al. reported a RP-HPLC method for simultaneous estimation of atorvastatin calcium and fenofibrate in tablet formulation. The separation was achieved by a C8 column with a mobile phase consisting of methanol-water, ph 3.2 (90:10, v/v) at a flow rate of 1mL/min. Detection was carried out at 260 nm. Retention times of atorvastatin calcium and fenofibrate was found to be 3.32 and 4.51 min respectively (64). Abe et al. reported a HPLC method for the determination of fenofibric acid and reduced fenofibric acid in the biological samples. After addition of the IS solution and 0.5 M HCl to the biological sample, fenofibric acid, reduced fenofibric acid and the IS were extracted with a mixed solvent of n-hexane and ethyl acetate (90:10, v/v) from the mixture. The acids were back-extracted from the organic phase with 0.1 M disodium hydrogen phosphate and then reextracted from the aqueous phase with a mixed solution of n-hexane and ethyl acetate (95:5, v/v) after addition of 0.5 M HCl. The organic phase was evaporated to dryness under the vacuum. The residue was dissolved in methanol and diluted with distilled water. An aliquot of the resulting solution was injected on the HPLC (65). Shirkhedkar and Surana reported a HPTLC method for simultaneous analysis of atorvastatin calcium and fenofibrate in pharmaceutical dosage forms. Detection and quantification were performed densitometrically at 258 nm. Polynomial regression data for the calibration plots showed there was a good linear relationship between response and amount in the range ng/band for atorvastatin calcium and ng/band for fenofibrate (66). 28

41 Literature Review 2.6. Hydrochlorothiazide Tian et al. reported a simple RP-HPLC method for simultaneous determination of valsartan and hydrochlorothiazide in tablets. Mobile phase containing methanol-acetonitrile-water-isopropylalcohol (22:18:68:2, v/v/v/v; ph was adjusted to 8 using triethylamine) was used. The flow rate was 1 ml/min and effluent was monitored at 270 nm. The linearity range for valsartan and hydrochlorothiazide were and µg/ml respectively (67). Alaa et al. reported two methods for the simultaneous determination of benazepril hydrochloride and hydrochlorothiazide in binary mixture. The first method was based on HPTLC separation of the two drugs followed by densitometric measurements of their spots at 238 and 275 nm for benazepril hydrochloride and hydrochlorothiazide, respectively. The second method was based on HPLC separation of the two drugs on reversed phase column using a mobile phase consisting of acetonitrile and water (35:65, v/v) and adjusted to ph 3.3 with acetic acid. Quantitation was achieved with UV detection at 240 nm (68). Shah et al. reported an HPTLC method for the simultaneous estimation of olmesartan medoxomil and hydrochlorothiazide in combined dosage forms. The mobile phase used was a mixture of acetonitrile-chloroform-glacial acetic acid (7:2:0.5, v/v/v). The detection of spots was carried out at 254 nm. The calibration curve was found to be linear between ng/band for olmesartan medoxomil and ng/band for hydrochlorothiazide (69). Girija et al. reported a HPTLC method for simultaneous determination of quinapril hydrochloride and hydrochlorothiazide in pharmaceutical 29

42 Literature Review formulations. The solvent system used was ethyl acetate-acetone-acetic acid (6.5:3:0.5, v/v/v). The detection wavelength was found to be 208 nm. The linearity was found to be in the range of and ng/band for quinapril hydrochloride and hydrochlorothiazide respectively (70). Bipin et al. reported a HPTLC method for analysis of candesartan cilexetil and hydrochlorothiazide in their tablet dosage forms. Acetone-chloroform-ethyl acetate-methanol 3:3:3:0.5, (v/v/v/v) was used as mobile phase. Detection was performed at 280 nm. The R f values were 0.27 for candesartan cilexetil and 0.45 for hydrochlorothiazide. Regression plots showed good linear relationships in the concentration ranges µg/band for hydrochlorothiazide and µg/band for candesartan cilexetil (71). Sathe and Bari reported a HPTLC method for separation and quantitative analysis of losartan potassium, atenolol and hydrochlorothiazide in bulk and in pharmaceutical formulations. The mobile phase consisted of toluene methanol triethylamine 6.5:4:0.5 (v/v/v). The bands were scanned densitometrically at 274 nm. The R f values of losartan potassium, atenolol and hydrochlorothiazide were 0.60, 0.43 and 0.29 respectively. Calibration plots were linear in the ranges of ng/band for losartan potassium and atenolol and ng/band for hydrochlorothiazide (72). Fei et al. reported a LC-MS/MS method for the determination of hydrochlorothiazide in human plasma. The analyte and irbesartan (IS) were precipitated and extracted from plasma using methanol. Analysis was performed on a C8 column with water and methanol (27:73, v/v) as the mobile phase. Linearity was obtained in the range of ng/ml in plasma (73). 30

43 Literature Review Eda et al. reported first-derivative UV spectrophotometry and HPLC methods to determine valsartan and hydrochlorothiazide simultaneously in combined pharmaceutical dosage forms. For derivative procedure, the calibration graphs were linear in the range of µg/ml for valsartan and µg/ml for hydrochlorothiazide. Mobile phase consisting of mixture of 0.02 M phosphate buffer (ph 3.2)-acetonitrile (55:45, v/v) was used to separate valsartan and hydrochlorothiazide (74) Nebivolol Patel et al. reported RP-HPLC and HPTLC methods for the estimation of nebivolol in tablet dosage form. The methods were validated in terms of linearity, accuracy and precision. The calibration curves were found to be linear over the concentration range of µg/ml for HPLC and ng/band for HPTLC (75). Pankaj et al. reported an isocratic stability indicating RP-HPLC method for the determination of nebivolol. The mobile phase consisted of acetonitrile ph 3.5 phosphate buffer (35:65, v/v) at a flow rate of 1 ml/min and detection was performed at 280 nm using a photodiode array detector. The drug was subjected to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was linear in the concentration range of µg/ml (76). Meyyanathan et al. reported a HPLC method for the simultaneous estimation of nebivolol and hydrochlorothiazide. Mobile phase consisted of acetonitrile- 50 mm ammonium acetate (adjusted to ph 3.5 using ortho-phosphoric acid) (70:30, v/v). Detection was carried out at 254 nm. The retention times of 31

44 Literature Review probenecid (IS), nebivolol and hydrochlorothiazide were 13.05, 3.32 and 4.25 min respectively (77). Khandelwal et al. reported a HPLC method for estimation of nebivolol in pharmaceutical dosage form. The method was carried out on a C18 column consisting of acetonitrile-0.3m potassium dihydrogen phosphate in ratio 50:50 (ph 3.2 adjusted with orthophosphoric acid), v/v as mobile phase at a flow rate of 1.2 ml/min. Detection was carried out at 278 nm. The retention time of nebivolol was 4.34 min (78). Kokil and Bhatia reported an ion-pair RP-HPLC method for the simultaneous estimation of nebivolol and valsartan in their capsule formulation. The chromatographic method was carried out on a C18 column with UV detection at 289 nm and flow rate of 1 ml/min. The mobile phase consisting of methanol-water (80:20, v/v) with addition of 0.1 percent 1-hexanesulfonic acid monohydrate sodium salt as an ion-pairing reagent was selected for the study (79). Tarte et al. reported a HPLC method for the simultaneous determination of nebivolol and hydrochlorothiazide in combined tablet dosage from. A C18 column with mobile phase, methanol-water (60:40, v/v) adjusted to ph 3.2 with ortho-phosphoric acid was used. The flow rate was 1 ml/min and the effluent was monitored at 281 nm. The retention times for nebivolol and hydrochlorothiazide were and min respectively. The linearity range was found to be 5-50 µg/ml for nebivolol and µg/ml for hydrochlorothiazide (80). 32

45 Literature Review Shirkhedkar et al. reported a stability-indicating HPTLC method for determination of nebivolol both in bulk drug and in formulation. The mobile phase system consisted of toluene-methanol-triethylamine (3.8:1.2:0.2, v/v/v). Densitometry analysis of nebivolol was carried out in the absorbance mode at 281 nm. The system gave compact spot for nebivolol at R f value of The linear regression analysis data for the calibration plots showed good relationship in the concentration range ng/band (81). Damle et al. reported a stability-indicating HPTLC method for analysis of nebivolol and hydrochlorothiazide. Separation was achieved with ethyl acetate-methanol-acetic acid 6.5:1:0.5 (v/v/v) as mobile phase. Detection and quantification were performed at 280 and 270 nm for nebivolol and hydrochlorothiazide respectively. The drugs got resolved with R f values of 0.46 and 0.78 for nebivolol and hydrochlorothiazide respectively (82). Mahesh et al. reported stability indicating HPTLC method for simultaneous analysis of amlodipine and nebivolol. The method employed TLC aluminum plates precoated with silica gel 60F 254 as the stationary phase. The solvent system consisted of ethyl acetate-methanol-dilute ammonia (8.5:1:1, v/v/v) (83). Ramakrishna et al. reported a LC-MS/MS method for the quantitation of nebivolol in human plasma. The method involved single step LLE with diethyl ether/dichloromethane (70:30, v/v). The analyte was chromatographed on a C18 reversed-phase chromatographic column with water-acetonitrile-formic acid (30:70:0.03, v/v/v) as the mobile phase. The chromatographic runtime 33

46 Literature Review was 2 min and the calibration curves were linear over the range 50 10,000 pg/ml (84) Olmesartan medoxomil Sagirili et al. reported a validated HPLC method for the simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablets. Chromatography was carried out on a cyano column with methanol 10 mm phosphoric acid containing 0.1% triethylamine (ph 2.5, 50:50, v/v) as the mobile phase. The UV detector was set at 260 nm. Valsartan was used as IS. A linear response was observed in the range of µg/ml for olmesartan medoxomil and µg/ml for hydrochlorothiazide, respectively (85). Sayali et al. reported a validated densitometric method for determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage forms. Separation of the drugs was carried out using chloroform-methanoltoluene (6:4:5, v/v/v) as mobile phase on precoated silica gel 60 F 254 plates. The calibration curve was linear in the concentration range ng/band for olmesartan medoxomil and ng/band for hydrochlorothiazide (86). Raveendra et al. reported a HPLC method for the assay of olmesartan medoxomil in tablet dosage form. HPLC analysis used C8 column and mobile phase consisted of buffer and acetonitrile (55:45, % v/v). The buffer was composed of 3 g of sodium perchlorate and 3 ml of triethylamine in 1000 ml of water and the ph of the solution was adjusted to 3 with orthophosphoric acid. The wavelength of the detection was 250 nm. The method was linear from µg/ml (87). 34

47 Literature Review Rote and Bari reported a UV method for simultaneous determination of olmesartan medoxomil and hydrochlorothiazide by absorption ratio spectrophotometric method. The method was based on measurements of absorbance at isoabsoptive point. The Beer's law obeyed in the range of µg/ml for both olmesartan medoxomil and hydrochlorothiazide (88). Sharma and Pancholi reported a RP-HPLC method for the determination of olmesartan medoxomil in the presence of its degradation products. Olmesartan medoxomil and all the degradation products were separated on a C18 column. The mobile phase was composed of methanol-acetonitrile-water (60:15:25, v/v/v, ph was adjusted to 3.5 by orthophosphoric acid). The method was linear over the concentration range of 1-18 µg/ml (89). Sultana et al. reported a HPLC method for analysis of serum samples and commercial tablet formulation containing hydrochlorothiazide, olmesartan medoxomil and irbesartan. Chromatographic separation was achieved using a C18 column and mobile phase consisted of acetonitrile-0.2% (v/v) acetic acid aqueous solution (50:50, v/v) at a flow rate of 1 ml/min. The ultraviolet detector was set at a wavelength of 260 nm. Hydrochlorothiazide, olmesartan medoxomil and irbesartan were eluted at 1.2, 3.8 and 4.4 min respectively. The method used protein precipitation with acetonitrile for the preparation of serum sample. The linear ranges for hydrochlorothiazide, olmesartan medoxomil and irbesartan were , and ng/ml respectively (90). Bahia et al. reported a HPTLC method for simultaneous quantitative analysis of olmesartan medoxomil and hydrochlorothiazide in the presence of 35

48 Literature Review olmesartan medoxomil degradation products. R f values of olmesartan medoxomil, its degradation products and hydrochlorothiazide were different when chloroform-methanol-formic acid, 8:1.5:0.5 (v/v/v) was used as mobile phase. Detection was performed at 260 nm and 272 nm for olmesartan medoxomil and hydrochlorothiazide respectively (91). Bahia et al. reported RP-HPLC and HPTLC densitometry methods as stability indicating assays for olmesartan medoxomil in presence of its acid or alkaline induced degradation products. Olmesartan medoxomil and its degradation products were analyzed at 257 nm. Mobile phase was composed of acetonitrile-methanol-water-glacial acetic acid (40:35:25:0.1, v/v/v/v) at flow rate 1 ml/min. For HPTLC method, chloroform-methanol-formic acid (8:1.5:0.5, v/v/v) was used as mobile phase. Densitometric evaluation of drug was carried out at 260 nm (92). Asmita et al. reported two methods for simultaneous determination of amlodipine besylate and olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on using n-butanolacetic acid-water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on a C18 column using acetonitrile-0.03 M ammonium acetate buffer (ph 3) in a ratio of 55:45, v/v as the mobile phase (93) Ramipril Kurade et al. reported a rapid HPLC method for the estimation of ramipril and telmisartan simultaneously in combined dosage form. A C18 column in isocratic mode with mobile phase containing a mixture of 0.01 M potassium 36

49 Literature Review dihydrogen phosphate buffer (adjusted to ph 3.4 using orthophosphoric acid)- methanol-acetonitrile (15:15:70, v/v/v) was used. Linearity for ramipril and telmisartan were found in the range of µg/ml and µg/ml respectively (94). Rao and Srinivas reported a simple, specific and accurate RP-HPLC method for the simultaneous determination of losartan potassium and ramipril in table dosage forms. A C18 column in isocratic mode with mobile phase, acetonitrile-methanol-10 mm tetra butyl ammonium hydrogen sulphate in water in the ratio of 30:30:40, v/v/v, was used. The linearity range for losartan potassium and ramipril were in the range of µg/ml and µg/ml respectively (95). Kiran et al. reported a HPLC method for quantitative simultaneous estimation of telmisartan and ramipril. The mobile phase consisted of buffer acetonitrile (55:45, v/v). The buffer used in mobile phase contained 0.1 M sodium perchlorate monohydrate in double distilled water; ph was adjusted to 3 with trifluoroacetic acid. The wavelength of detection was 215 nm (96). Bankey et al. reported a UV method for simultaneous determination of ramipril, hydrochlorothiazide and telmisartan in tablet formulation. The wavelengths selected for these drugs were 218 nm, 271 nm and 296 nm respectively using methanol as solvent. The linearity for these drugs at all the selected wavelengths were between µg/ml for ramipril, µg/ml for hydrochlorothiazide and 4-28 µg/ml for telmisartan (97). Lincy et al. reported RP-HPLC and spectroscopic methods for the analysis of combined dosage form of atorvastatin and ramipril. A wavelength of 215 nm 37

50 Literature Review was fixed for HPLC method. The linearity range was found to be µg/ml for atorvastatin and for ramipril for HPLC method. In UV method, absorbances of solutions were measured at 247 nm for atorvastatin and 208 nm for ramipril (98). Belal et al. reported a HPLC method for the simultaneous determination of ramipril and hydrochlorothiazide in their dosage forms. Acetonitrile-sodium perchlorate solution (0.1 M) adjusted to ph 2.5 with phosphoric acid (46:54 v/v), was used as the mobile phase, at a flow rate of 1.5 ml/min. Detection was done at 210 nm. Clobazam was used as IS. The method was also applied for the determination of ramipril in the presence of its degradation products. Linearity ranges for ramipril and hydrochlorothiazide were and µg/ml respectively (99). Baing et al. reported a HPLC method for simultaneous determination of losartan potassium, ramipril and hydrochlorothiazide. The mobile phase was M sodium perchlorate acetonitrile, 62:38 (v/v), containing 0.1% heptanesulphonic acid. The ph of the solution was adjusted to 2.85 with orthophosphoric acid. UV detection was performed at 215 nm. The method was found to be linear in the ranges µg/ml for losartan, µg/ml for ramipril and µg/ml for hydrochlorothiazide (100) Rosuvastatin Ravi et al. reported a simple, sensitive and specific LC MS/MS method for simultaneous determination of rosuvastatin and fenofibric acid using carbamazepine as IS. Linearity was established for the range of concentrations 38

51 Literature Review 1 50 ng/ml and µg/ml for rosuvastatin and fenofibric acid respectively (101). Tushar et al. reported a stability-indicating LC method for the determination of rosuvastatin in the presence of its degradation products. Degradation of the drug was done at various ph values. The drug was degraded under oxidative, photolytic and thermal stress conditions. The developed method was successfully applied for an accelerated stability study of the tablet formulation (102). Thammera et al. reported a HPLC method for the estimation of rosuvastatin. The assay procedure involved simple LLE of rosuvastatin and ketoprofen (IS) from plasma directly into acetonitrile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 C. The residue was reconstituted in the mobile phase and injected onto a C18 column. Mobile phase consisted of 0.05 M formic acid and acetonitrile (55:45, v/v). The detection of the analyte peak was achieved by using a UV detector set at 240 nm. Retention times of rosuvastatin and IS were 8.6 and 12.5 min respectively (103). Hull et al. reported an automated solid-phase extraction followed by LC MS/MS method for the quantification of rosuvastatin in human plasma. The standard curve range in human plasma was ng/ml (104). Ke et al. reported a LC MS/MS method for the quantification of rosuvastatin in human plasma. The chromatographic separation was performed on a C18 column with a mobile phase consisting of 2 % v/v formic acid-methanol (20:90, v/v) at a flow rate of 1 ml/min with a split of 200 µl to mass 39

52 Literature Review spectrometer. The retention times of rosuvastatin and IS were 2.3 and 3.4 min respectively. The assay exhibited a linear range of ng/ml (105). Gao et al. reported a LC MS/MS method for the determination of rosuvastatin in human plasma. The plasma samples were prepared using LLE with ethyl ether. Chromatographic separation was accomplished on a C18 column. The mobile phase consisted of methanol water (75:25, v/v, adjusted to ph 6 by aqueous ammonia). Detection of rosuvastatin and the hydrochlorothiazide (IS) was achieved by ESI MS/MS in the negative ion mode. The linear range of the method was from ng/ml (106). Dong-Hang et al. reported a LC MS/MS method for determining rosuvastatin in human plasma. The analyte and cilostazol (IS) were extracted by simple one-step LLE with ether. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 C. The chromatographic separation was performed on an C18 column with a mobile phase consisting of 0.2% (v/v) formic acid-methanol (30:70, v/v) at a flow rate of 0.2 ml/min. Linearity was found to be in the range of ng/ml (107). Vittal et al. reported a HPLC method for simultaneous determination of rosuvastatin and gemfibrozil in human plasma using celecoxib as IS. The assay procedure involved extraction of rosuvastatin, gemfibrozil and IS from plasma into acetonitrile. Following separation and evaporation of the organic layer, the residue was reconstituted in the mobile phase and injected onto a C18 column. The chromatographic run time was less than 20 min using flow gradient ( ml/min) with a mobile phase consisting of 0.01 M ammonium acetate-acetonitrile-methanol (50:40:10, v/v/v) and UV detection 40

53 Literature Review at 275 nm. Retention times of rosuvastatin, gemfibrozil and IS were 6.7, 13.9 and 16.4 min respectively. Linearity was found in the range of µg/ml and µg/ml for rosuvastatin and gemfibrozil respectively (108). Celebier and Altinoz reported a spectrophotometric method for the determination of rosuvastatin calcium in pharmaceutical preparations. The solutions of standard and pharmaceutical samples were prepared in methanol. A wavelength of 243 nm was chosen for measuring absorbances of rosuvastatin calcium. The linearity range of the method was 1 60 µg/ml (109) Telmisartan Tingting et al. reported a LC MS/MS method for the simultaneous determination of telmisartan and hydrochlorothiazide in human plasma. Sample preparation involved LLE with diethyl ether dichloromethane (60:40, v/v). The analytes and probenecid (IS) were separated on a C8 column using gradient elution with acetonitrile 10 mm ammonium acetate formic acid at a flow rate of 1.2 ml/min. For both analytes, the method was linear in the range ng/ml (110). Wankhede et al. reported a RP-HPLC method for simultaneous estimation of telmisartan and hydrochlorothiazide in tablet formulation. Mobile phase consisted of acetonitrile-0.05 M potassium dihydrogen phosphate, ph 3 (60:40, v/v). The flow rate was 1 ml/min and the eluent was monitored at 271 nm. The selected chromatographic conditions were found to separate telmisartan (R t = 5.19 min) and hydrochlorothiazide (R t = 2.97 min) (111). 41

54 Literature Review Lories et al. reported four sensitive methods for determination of telmisartan and hydrochlorothiazide in combined dosage forms without prior separation. The first method utilized first derivative spectophotometry ( 1 D) using a zerocrossing technique of measurement at and nm for telmisartan and hydrochlorothiazide respectively. The second method was first derivative of ratio spectrophotometry ( 1 DD) method where the amplitudes were measured at nm for telmisartan and nm for hydrochlorothiazide. The third method was based on TLC separation of the two drugs followed by the densitometric measurements of their spots at 295 and 225 nm for telmisartan and hydrochlorothiazide respectively. The fourth method was spectrofluorimetric determination of telmisartan, depending on measuring the native fluorescence of the drug in 1 M sodium hydroxide at excitation wavelength of 230 nm and emission wavelength of 365 nm (112). Pengfei et al. established a method for the determination of telmisartan in human plasma by LC-MS/MS. Telmisartan and diphenhydramine (IS), were extracted from plasma using diethyl ether dichloromethane (60:40, v/v), and separated on a C18 column using methanol 10 mm ammonium acetate (85:15, v/v) adjusted to ph 4.5 with formic acid as mobile phase. The assay was linear over the range ng/ml (113). Londhe et al. reported a stability-indicating HPLC method for quantitative analysis of telmisartan. The drug was separated from its degradation products on a C18 column with methanol-water 80:20 (v/v), ph 4 (adjusted by addition of orthophosphoric acid), as mobile phase at a flow rate of 1mL/min. 42

55 Literature Review Quantification was achieved by UV detection at 225 nm. Calibration plots were linear in the concentration range µg/ml (114) Valsartan Agnivesh et al. reported a stability-indicating HPTLC method for analysis of valsartan both in bulk drug and in formulations. The method used tolueneethyl acetate-methanol-formic acid 60:20:20:1 (v/v/v/v) as mobile phase. The system gave compact bands for valsartan (R f value=0.44). Densitometric analysis of valsartan was performed in absorbance mode at 250 nm. Linear regression analysis data for the calibration plots revealed good linearity in the working concentration range ng/band (115). Piao et al. established a method for determining valsartan concentration in human plasma samples using HPLC combined with UV detection. After protein precipitation using methanol, the analytes were separated on a C18 column using acetonitrile-15 mm potassium dihydrogen phosphate in water (ph 2; adjusted with phosphoric acid), 42:58, v/v as the mobile phase at a flow rate of 1.2 ml/min. Standard calibration curve was constructed in the concentration range of ng/ml (116). Kadam and Bari reported a HPTLC method for simultaneous analysis of valsartan and hydrochlorothiazide in tablet formulations. The plates were developed with chloroform ethyl acetate acetic acid, 5:5:0.2 (v/v/v), as mobile phase. UV detection was performed densitometrically at 248 nm. The retention factors of valsartan and hydrochlorothiazide were 0.27 and 0.56 respectively. The linear range was ng/band for valsartan and ng/band for hydrochlorothiazide (117). 43

56 Literature Review Shah et al. reported a HPTLC method for the simultaneous estimation of valsartan and hydrochlorothiazide in combined dosage forms. The mobile phase used was a mixture of chloroform-methanol-toluene-glacial acetic acid (6:2:1:0.1, v/v/v/v). The detection of spots was carried out at 260 nm. The calibration curve was found to be linear between ng/band for valsartan and ng/band for hydrochlorothiazide (118). Chitlange et al. reported a RP-HPLC method for simultaneous estimation of amlodipine besylate and valsartan on C18 Column using acetonitrilephosphate buffer (0.02M, ph 3.0), (56:44, v/v) as mobile phase at a flow rate of 1 ml/min and the detection wavelength was 234 nm. The retention times for amlodipine besylate and valsartan were found to be 3.07 and 6.20 min respectively (119). Afshin et al. reported a HPLC method using fluorescence detection for determination of valsartan in human plasma. The assay was based on protein precipitation using acetonitrile. The mobile phase consisted of 0.01 M disodium hydrogen phosphate buffer-acetonitrile (60:40, v/v) adjusted to ph 3.5 with diluted phosphoric acid. The excitation and emission wavelengths were 230 and 295 nm respectively. The calibration curve was linear over the concentration range ng/ml (120). Lakshmi and Lakshmi reported a HPLC method to determine valsartan and ramipril simultaneously. Chromatographic separation was achieved on a C18 column using a mixture of acetonitrile and water in the ratio 55:45 (v/v), ph adjusted to 3.6 with 88% orthophosphoric acid at a wavelength of 215 nm. 44

57 Literature Review Linearity of the method was found to be in the concentration range of µg/ml for valsartan and l µg/ml for ramipril (121). Sevgi and Serap reported a normal UV and second derivativespectrophotometric and HPLC methods for the determination of valsartan. For the first method, UV-spectrophotometry, standard solutions were measured at nm. For the second method, distances between two extreme values (peak-to-peak amplitudes), and nm were measured in the second order derivative-spectra of standard solutions. Calibration curves were constructed by plotting d 2 A/dλ 2 values against concentrations. The third method was based on HPLC using acetonitrile, phosphate buffer as a mobile phase and losartan as IS. Detection was carried out at 265 nm using a UV detector. (122). 45

58 Aims & Objectives CHAPTER 3. AIMS AND OBJECTIVES This thesis deals with the development of precise and accurate validated analytical and bioanalytical methods for the determination of newer cardio vascular drug combinations from formulations and biological fluids. The plan of work carried out for the present study has been divided into following sections. Part-1(Analytical method development) Development of high-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC) methods for the following combinations Amlodipine + Hydrochlorothiazide + Valsartan Atorvastatin + Ezetimibe + Fenofibrate Telmisartan + Ramipril + Hydrochlorothiazide Rosuvastatin + Ezetimibe Telmisartan + Atorvastatin Olmesartan medoxomil + Amlodipine Rosuvastatin + Fenofibrate Telmisartan + Amlodipine Nebivolol + s-amlodipine Development of high-performance thin layer chromatography (HPTLC) method for the following combination Atorvastatin + Ramipril + Aspirin 46

59 Aims & Objectives Development of UV spectroscopy methods (simultaneous equation method & derivative spectroscopy) for the combinations given below Rosuvastatin + Ezetimibe Telmisartan + Atorvastatin Olmesartan medoxomil + Amlodipine Part-2 (LC-MS bioanalytical method development) Development of sensitive methods based on liquid chromatography-mass spectrometry (LC-MS) for the following drug combinations from human plasma. Rosuvastatin + Ezetimibe Telmisartan + Atorvastatin 47

60 Drug Profile CHAPTER 4. DRUG PROFILE The profiles of the selected drugs are as follows ( ). Drug Amlodipine Hydrochlorothiazide Structure IUPAC name Molecular weight Solubility Category 3-Ethy l-5-methyl (±)-2-[(2-6-chloro-3,4-dihydro-2H-1, 2, 4- aminoethoxy)methyl]-4-(2- benzothiadiazine-7-sulfonamide chlorophenyl)-1,4-dihydro-6-methyl- 1,1-dioxide 3,5-pyridinedicarboxylate, monobenzenesulphonate Slightly soluble in water and Practically insoluble in water, sparingly soluble in ethanol chloroform and ether, soluble 1 in 200 of ethanol and 1 in 20 of acetone, freely soluble in dimethyl formamide and solutions of alkali hydroxides Calcium channel blocker used to treat Antihypertensive high blood pressure 48

61 Drug Profile Drug Valsartan Telmisartan Structure IUPAC name N-(1-oxopentyl)-N-[[2 '-(1Htetrazol-5-yl) [1,1 '-biphenyl]-4- yl]methyl]-l-valine 4'-[(1,4'-dimethyl-2'-propyl [2,6'-bi- 1H-benzimidazol]-1'-yl)methyl]- [1,1'-biphenyl]-2-carboxylic acid Molecular weight Solubility Category Soluble in ethanol and Practically insoluble in water; methanol; slightly soluble in sparingly soluble in strong acid; water soluble in strong base Nonpeptide, orally active and Non-peptide angiotensin II receptor specific angiotensin II receptor (type AT 1 ) antagonist blocker 49

62 Drug Profile Drug Ramipril Aspirin Structure IUPAC name (2S,3aS,6aS)-1[(S)-N-[(S)-1Carboxy-3- phenylpropyl] alanyl] octa hydrocyclopenta [b]pyrrole-2-carboxylic acid, 1-ethyl ester 2-(Acetyloxy)benzoic acid Molecular weight Solubility Category Soluble in methyl alcohol; sparingly soluble in water. ACE inhibitor used for treating high blood pressure, heart failure and for preventing kidney failure due to high blood pressure and diabetes Soluble 1 in 300 of water, 1 in 5 of ethanol, 1 in 17 of chloroform, and 1 in 10 to 15 of ether Nonsteroidal antiinflammatory drug (NSAID) effective in treating fever, pain and inflammation in the body. It also prevents blood clots (antithrombotic) 50

63 Drug Profile Drug Rosuvastatin Fenofibrate Structure IUPAC name bis[(e)-7-[4-(4-fluorophenyl)-6- isopropyl-2-[methyl (methylsulfonyl) amino] pyrimidin- 5-yl](3R,5S)-3,5-dihydroxyhept-6- enoic acid] calcium salt 2-[4-(4-chlorobenzoyl) phenoxy]-2-methyl-propanoic acid, 1-methylethyl ester Molecular weight Solubility Category Sparingly soluble in water and methanol, and slightly soluble in ethanol Help lower "bad" cholesterol and fats (such as LDL, triglycerides) and raise "good" cholesterol (HDL) in the blood. It belongs to a group of drugs known as statins. Lowering "bad" cholesterol and triglycerides and raising "good" cholesterol decreases the risk of heart disease. Insoluble in water, soluble in acetonitrile Help lower "bad" cholesterol and fats (such as LDL, triglycerides) and raise "good" cholesterol (HDL) in the blood. It works by reducing the amount of cholesterol made by the liver. Lowering "bad" cholesterol and triglycerides and raising "good" cholesterol decreases the risk of heart disease. 51

64 Drug Profile Drug Ezetimibe Atorvastatin Structure IUPAC name 1-(4-fluorophenyl)3(R)-[3-(4- fluorophenyl)-3(s)- hydroxypropyl]-4(s)-(4- hydroxyphenyl)-2-azetidinone [R-(R *,R * )]-2-(4-Fluorophenyl)- β,δ-dihydroxy 5-(1 methylethyl)-3 phenyl 4- [(phenylamino)carbonyl]-1hpyrrole 1 heptanoic acid Molecular weight Solubility Category Freely to very soluble in ethanol, methanol and acetone and practically insoluble in water Ezetimibe belongs to a class of lipid-lowering compounds that selectively inhibits the intestinal absorption of cholesterol. Very slightly soluble in distilled water, phosphate buffer (ph 7.4) and acetonitrile; slightly soluble in ethanol; freely soluble in methanol Synthetic lipid-lowering agent, inhibitor of 3-hydroxy-3- methylglutaryl-coenzyme A (HMG-CoA) reductase. 52

65 Drug Profile Drug Nebivolol Olmesartan medoxomil Structure IUPAC name α,α -[Iminobis(methylene)]bis[6 fluoro 3,4 dihydro 2H-1 benzopyran 2 methanol] 2,3-dihydroxy-2-butenyl 4-(1- hydroxy-1-methylethyl)-2- propyl-1-[p-(o-1h-tetrazol-5- ylphenyl)benzyl]imidazole-5- carboxylate, cyclic 2,3- carbonate Molecular weight Solubility Category Soluble in methanol, dimethyl sulfoxide and N, N-dimethyl formamide; Sparingly soluble in ethanol and propylene glycol Beta-blocker used to treat hypertension Practically insoluble in water and sparingly soluble in methanol Selective AT 1 subtype angiotensin II receptor antagonist 53

66 Instruments & Materials CHAPTER 5. INSTRUMENTS AND MATERIALS The experimental requirements for this thesis work including the analytical instruments, statistical software, reagents/chemicals, active pharmaceutical ingredients (API) and formulations used are discussed below Analytical Instruments HPLC Shimadzu Prominence UFLC (Shimadzu corporation, Kyoto, Japan) equipped with LC-20 AD pump, SPD-M20A diode array detector, DGU-20A 3 degasser, SIL-20 AC auto sampler and CTO-10ASVP column oven. Chromatograms were recorded and integrated on PC installed with LC solutions chromatographic software, version Shimadzu Liquid chromatograph (Shimadzu corporation, Kyoto, Japan) equipped with LC-10AT VP pump, SPD-M10A VP diode array detector and rheodyne 7725i injector with a 20 µl loop. Chromatograms were recorded and integrated on PC installed with CLASS-M10A chromatographic software, version Stationary phases used Luna C18 (4.60 mm 150 mm, 5 µ particle size) column, Phenomenex, USA. Chromolith RP-18 (6 mm 100 mm, 5 µ particle size) column, Merck, Germany. Lichrospher RP-18, (4 mm 250 mm, 5 µ particle size) column, Merck, Germany. Shim-pack C18 (6 mm 150 mm, 5 µ particle size) column, Shimadzu, Japan. 54

67 Instruments & Materials Mass spectrometer Mass spectrometric detection was performed using a Shimadzu LCMS-2010 EV quadrupole mass spectrometer interfaced with electrospray ionization (ESI) probe (Shimadzu corporation, Kyoto, Japan) HPTLC CAMAG Linomat V sample applicator and TLC scanner III controlled by wincats-planar chromatography manager, version: (CAMAG, Muttenz, Switzerland). Stationary phase used Merck TLC plates coated with silicagel 60F 254 on aluminum sheets were used as the stationary phase (Merck Chemicals Ltd., Darmstadt, Germany) UV visible spectrophotometer Jasco V-530 double-beam UV visible spectrophotometer with data processing software, spectra manager version (Jasco Corporation, Tokyo, Japan) Analytical balance Shimadzu electronic balance, BL-220H (Shimadzu Corporation, Kyoto, Japan) PH meter Elico LI 127 ph meter (Elico Ltd., Hyderabad, India) Sonicator LeelaSonic Ultrasonicator (Leela Electronics, Mumbai, India) Vacuum pump Gelman Sciences (Pall Pharmalab Filtration Pvt. Ltd., Mumbai, India) Centrifuge Remi Centrifuge (Remi Motors Ltd., Mumbai, India) 55

68 Instruments & Materials 3.2. Statistical software GraphPad InStat software version 3.05(GraphPad Software, Inc.) 3.3. Chemicals and reagents The details of the chemicals and reagents obtained from companies are as below. Chemicals Ethyl acetate (GR grade), dichloromethane (GR grade), formic acid (GR grade), diethyl ether (GR grade), toluene (GR grade), chloroform (GR grade) n-hexane (SQ grade), triethyl amine (SQ grade) Acetonitrile (HPLC grade), methanol (SQ grade) Ammonium formate (AR grade), tertbutyl methyl ether (HPLC grade), ammonium acetate (AR grade) Methanol (HPLC grade), glacial acetic acid, ammonia solution (25%) (AR grade), n-butyl acetate (LR grade) HPLC grade water Source Merck Specialities Pvt. Ltd., (Mumbai, India) Qualigens Fine Chemicals Pvt. Ltd., (Mumbai, India) Fischer Scientific Pvt. Ltd., (Mumbai, India) Himedia Laboratories Pvt. Ltd., (Mumbai, India) S.D. Fine-Chem Pvt. Ltd., (Mumbai, India) Prepared by use of a Millipore Milli-Q Academic water purifier (Bangalore, India) Active pharmaceutical Ingredients The details of API procured from pharmaceutical companies are as below. API Amlodipine besylate, s-amlodipine, atorvastatin, fenofibrate, ramipril, telmisartan, valsartan Hydrochlorothiazide Ezetimibe, aspirin Rosuvastatin Nebivolol Olmesartan medoxomil Source Aristo Pharmaceuticals Pvt. Ltd., (Mumbai, India) Medreich Pvt. Ltd., (Bangalore, India) Orchid chemicals & pharmaceuticals Ltd., (Chennai, India) Glenmark Pharmaceuticals Ltd., (Mumbai, India) Hetero Drug Ltd., (Hyderabad, India) Macleod s Pharmaceutical Pvt. Ltd., (Mumbai, India) 56

69 Instruments & Materials 3.5. Pharmaceutical formulations The details of pharmaceutical formulations used in the study are as follows. Formulation Teram-H tablets labeled to contain ramipril 5 mg, telmisartan 40 mg and hydrochlorothiazide 12.5 mg Fibator EZ tablets, labeled to contain atorvastatin 10 mg, ezetimibe 10 mg and fenofibrate 160 mg Tele Act ST 40 tablets, labeled to contain atorvastatin 10 mg and telmisartan 40 mg Rosuvas tablets labeled to contain rosuvastatin 10 mg and ezetimibe 10 mg Exforge HCT tablets labeled to contain amlodipine besyalte 5 mg, hydrochlorothiazide 12.5 mg and valsartan 160 mg Rosuvas F 10 tablets, labeled to contain rosuvastatin 10 mg and fenofibrate 160 mg Olmezest Am tablets labeled to contain amlodipine besylate 5 mg and olmesartan medoxomil 20 mg Ramitorva capsules labeled to contain ramipril 5 mg, atorvastatin 10 mg and aspirin 75 mg Nebicard-SM tablets labeled to contain s-amlodipine 2.5 mg and nebivolol 5 mg Tele Act-AM tablets labeled to contain amlodipine besylate 5 mg and telmisartan 40 mg Source Atoz Life Sciences, Pondicherry, India Sun Pharmaceutical Industries Ltd., Jammu & Kashmir, India Ranbaxy Laboratories Ltd., New Delhi, India Glenmark Pharmaceuticals Ltd., Mumbai, India Novartis Pharmaceuticals Ltd., Mumbai, India Ranbaxy Laboratories Ltd., New Delhi, India Sun Pharmaceuticals Ltd., Gujarat, India Zydus Cadila Pharmaceuticals Ltd., New Delhi, India Torrent Pharmaceuticals Ltd., Himachal Pradesh, India Ranbaxy Laboratories Ltd., New Delhi, India 57

70 Methodology CHAPTER 6. METHODOLOGY 6.1. High-performance liquid chromatography Selection of stationary phase-normal/reverse Selection of mobile phase-isocratic/gradient Sample/standard preparation Selection of detection wavelength Optimization of chromatographic conditions like organic phase composition, ph and ionic strength of buffer etc. Detection using PDA detector Method validation as per ICH guidelines in terms of parameters like, Linearity Precision Specificity LOD & LOQ Accuracy Stability Application of the method for formulation studies 58

71 Methodology 6.2. High-performance thin layer chromatography Selection of stationary phase-normal/reverse Selection and optimization of mobile phase t Sample/standard preparation Sample application Plate equilibration and chamber saturation Development and scanning of the plate Method validation as per ICH guidelines in terms of parameters like, Linearity Precision Specificity LOD &LOQ Accuracy Stability Application of the method for formulation studies 59

72 Methodology 6.3. UV spectroscopy Selection of solvents Sample/standard preparation Solving simultaneous equation Derivative spectroscopy Selection of wavelengths Determination of zero crossing points Recording of absorbances at the selected wavelengths, calculating absorptivities and solving the equation Recording of derivative absorbances of drugs at the selected zero crossing wavelengths Method validation as per ICH guidelines Application of the method for formulation studies 60

73 Methodology 6.4. Liquid chromatography-mass spectrometry (bioanalysis) Selection of stationary phase Selection and optimization of mobile phase t Selection of internal standard Optimization of extraction procedure for extracting analytes from plasma Recording of mass spectra of analytes to select the detection ions for analysis Optimization of mass spectrometer parameters like detector, interface, Q-array, RF and CDL voltages; CDL and heat block temperatures; nebulising gas flow rate etc. Bioanalytical method validation according to US Food and Drug Administration in terms of parameters like Linearity Precision Recovery Selectivity Accuracy Stability 61

74 Experimental Work CHAPTER 7. EXPERIMENTAL WORK Part1- Analytical method development 7.1. Amlodipine-hydrochlorothiazide-valsartan Preparation of mobile phase HPLC The chromatographic separation was performed using a mobile phase system consisting of 10 mm ammonium acetate buffer (ph 6.7) and methanol in solvent gradient elution for 20 min at a flow rate of 1mL/min (Table 7.1). Solvents were filtered through a nylon membrane filter (Rankem, New Delhi, India) of 0.45µm porosity HPTLC The mobile phase system consisted of a mixture of chloroform-glacial acetic acid-n-butyl acetate (8:4:2, v/v/v) Stock and standard solutions HPLC Preparation of the standard stock solution AML, HYD and VAL standard stock solution was prepared by transferring accurately 5 mg of AML, 12.5 mg of HYD and 160 mg of VAL reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of AML, 250 µg/ml of HYD and 3200 µg/ml of VAL Preparation of standard solutions From the above solution, standard solutions containing µg/ml of AML, µg/ml of HYD and µg/ml of VAL were prepared in water- 62

75 Experimental Work methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as response factor against concentration of each drug Preparation of sample solution Twenty tablets containing 5 mg of AML, 12.5 mg of HYD and 160 mg of VAL were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of AML, 12.5 mg of HYD and 160 mg of VAL were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with watermethanol (50:50, v/v) mixture to obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of AML, HYD and VAL (2.0, 3.0, 4.0, 5.0 and 6.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from , and µg/band for AML, HYD and VAL respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 320 nm with deuterium lamp and analyzed in triplicate. The calibration curves were constructed by plotting peak areas 63

76 Experimental Work versus concentrations for each drug. After separation of the drugs on the TLC plate, UV spectra of the individual drugs in solid state were recorded using the deuterium lamp of the TLC scanner Preparation of sample solution Preparation was similar to HPLC method. Suitable aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures Linearity Linearity was studied for the standards using standard drug solutions prepared as described above. Peak areas of drugs were plotted versus concentration and leastsquares analysis was performed Precision The precision of the methods was determined by repeatability (intraday precision) and intermediate precision (interday precision) and was expressed as RSD of a series of measurements. Intraday precision was evaluated by six replicate readings at three concentration levels within the linearity range. Interday precision was studied by comparing the results on 3 different days Accuracy To study the accuracy of the method, recovery studies were carried out by addition of standard drug solution to preanalyzed sample at three different levels: 80, 100 and 120%. The resulting solutions were then reanalyzed by the proposed method. 64

77 Experimental Work Detection and quantitation limits Standard solutions were prepared by sequential dilutions and injected at lower concentrations. LOD and LOQ were calculated using the S/N method. LOD was taken as the concentration of analyte at which S/N was 3. LOQ was taken as the concentration of analyte at which S/N was Specificity Specificity of the method was evaluated by studying the peak purity index values. Spectral purities of AML, HYD and VAL were evaluated using the UV spectra recorded by a diode array detector. In addition, a solution containing a mixture of the tablet excipients were prepared using the sample preparation procedure and injected onto the chromatograph Robustness Robustness was evaluated by studying the influence of small deliberate variations of the analytical parameters on the elution time, peak area or peak shape. The method should be robust enough with respect to all critical parameters so as to allow routine laboratory use Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.2 and 7.3 respectively. 65

78 Experimental Work Table 7.1. Gradient program Time (min) Methanol (%) Buffer Flow rate (ml/min) Table 7.2. HPLC conditions Parameter Fixed conditions Mobile phase 10 mm ammonium acetate buffer (ph 6.7) and methanol in solvent gradient elution for 20 min Stationary phase Phenomenex Luna C18 (4.60 mm 150 mm) column Column temperature 25 o C Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 238 nm 66

79 Experimental Work Table 7.3. HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Chloroform-glacial acetic acid-n-butyl acetate (8:4:2, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 320 nm 6 mm 30 min 8.5 cm mm 20 mm/s 67

80 Experimental Work 7.2. Atorvastatin-ezetimibe-fenofibrate Preparation of mobile phase HPLC The chromatographic separation was performed using a mobile phase system consisting of 0.1% formic acid and acetonitrile in solvent gradient elution for 25 min at a flow rate of 1.5 ml/min (Table 7.4). Solvents were filtered through a nylon membrane filter (Rankem, New Delhi, India) of 0.45µm porosity HPTLC The mobile phase consisted of a mixture of toluene-methanol-triethylamine (8:1.5:0.1, v/v/v) Stock and standard solutions HPLC Preparation of the standard stock solution ATO, EZE and FEN standard stock solution was prepared by transferring accurately 5 mg of ATO, 5 mg of EZE and 80 mg of FEN reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get a concentration of 100 µg/ml of ATO, EZE and 160 µg/ml of FEN Preparation of standard solutions From the solution, standard solutions containing µg/ml of ATO and EZE and 8-80 µg/ml of FEN were prepared in water-methanol (50:50, v/v) mixture. The peaks obtained were integrated, the peak areas were noted and respective calibration curves were plotted as response factor against concentration of each drug. 68

81 Experimental Work Preparation of sample solution Twenty tablets containing 10 mg of ATO, 10 mg of EZE and 160 mg of FEN, were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of ATO, 5 mg of EZE and 80 mg of FEN were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A further 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with watermethanol (50:50, v/v) mixture to obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of ATO, EZE and FEN (1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from , and µg/band for ATO, EZE and FEN respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 245 nm with deuterium lamp and analyzed in triplicate. The calibration curves were constructed by plotting peak areas versus concentrations for each drug. After separation of the drugs on the TLC plate, 69

82 Experimental Work UV spectra of the individual drugs in solid state were recorded using the deuterium lamp of the TLC scanner Preparation of sample solution Solution preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on precoated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.5 and 7.6 respectively. Table 7.4. Gradient program Time (min) Acetonitrile(%) Buffer Flow rate (ml/min)

83 Experimental Work Table 7.5. HPLC conditions Parameter Fixed conditions Mobile phase 0.1% formic acid and acetonitrile in solvent gradient elution for 25 min Stationary phase Shim-pack C18 column (6 mm 150) Injection volume 20 µl Flow rate 1.5 ml/min Detection wavelength 245 nm Table 7.6. HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Toluene-methanol-triethylamine (8:1.5:0.1, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 245 nm 6 mm 30 min 8.5 cm mm 20 mm/s 71

84 Experimental Work 7.3. Telmisartan-ramipril-hydrochlorothiazide Stock and standard solutions HPLC Preparation of the standard stock solution RPL, HYD and TLM standard stock solution was prepared by transferring accurately about 5mg of RPL, 12.5 mg of HYD and 40 mg of TLM reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of RPL, 250 µg/ml of HYD and 800 µg/ml of TLM Preparation of standard solutions An aliquot of 1 ml of above stock solution was transferred to a 10 ml volumetric flask and diluted with methanol, to obtain solution of concentrations 10, 25 and 80 µg/ml of RPL, HYD and TLM respectively. From this solution, standard solutions containing µg/ml of RPL, µg/ml of HYD and 4-24 µg/ml of TLM were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were noted and respective calibration curves were plotted as response factor against concentration of each drug Preparation of sample solution Twenty tablets containing 5 mg of RPL, 12.5 mg of HYD and 40 mg of TLM were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of RPL, 12.5 mg of HYD and 40 mg of TLM were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A 30 ml 72

85 Experimental Work portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. A 1 ml portion of the supernatant solution was transferred to a 10 ml volumetric flask and diluted to volume with methanol. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of RPL, HYD and TLM (2.0, 3.0, 4.0, 5.0 and 6.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from , and µg/band for RPL, HYD and TLM respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 210 nm. Calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Solution preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of 73

86 Experimental Work parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.7 and 7.8 respectively. Table 7.7. HPLC conditions Parameter Fixed conditions Mobile phase 0.1% phosphoric acid (ph adjusted to 2.5 with triethylamine)-acetonitrile, (58:42, v/v) Stationary phase Lichrospher RP-18, (4mm 250 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 210 nm Table 7.8. HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Ethylacetate-chloroform-methanol, 6.5:3:1.3 (v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 210 nm 6 mm 30 min 8.5 cm mm 20 mm/s 74

87 Experimental Work 7.4. Atorvastatin-ramipril-aspirin Stock and standard solutions Preparation of the standard stock solution RPL, ATO and ASP standard stock solution were prepared by transferring accurately about 5 mg of RPL, 10 mg of ATO and 75 mg of ASP reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of RPL, 200 µg/ml of ATO and 1500 µg/ml of ASP Preparation of standard solutions Standard solutions of RPL, ATO and ASP (4, 6, 8, 10, 12 and 14 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from , and 6-21 µg/band for RPL, ATO and ASP respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 210 nm with deuterium lamp and analyzed in triplicate. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Twenty tablets containing 5 mg of RPL, 10 mg of ATO and 75 mg of ASP were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of RPL, 10 mg of ATO and 75 mg of ASP were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min and aliquots of 75

88 Experimental Work supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness Chromatographic conditions HPTLC chromatographic conditions are summarized in table 7.9. Table 7.9. HPTLC conditions Parameter Fixed conditions Mobile phase Methanol-benzene-ethyl acetate-glacial acetic acid (0.36:2.5:4:0.04, v/v/v/v) Stationary phase Merck TLC plates coated with silica gel 60F 254 on aluminum sheets Detection wavelength 210 nm Band width 6 mm Chamber saturation time 30 min Developing distance of the plate 8.5 cm Slit dimension mm scanning speed 20 mm/s 76

89 Experimental Work 7.5. Rosuvastatin-ezetimibe Stock and standard solutions HPLC Preparation of the standard stock solution Standard stock solution containing each of ROS (100 µg/ml) and EZE (100 µg/ml) was made by dissolving the pure drugs in methanol Preparation of standard solutions From standard stock solution, standard solutions containing 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 µg/ml of both ROS and EZE were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as response factor against concentration of each drug Preparation of sample solution Twenty tablets were weighed and average weight was calculated. An amount of powder equivalent to 10 mg of both ROS and EZE were transferred to a 100 ml volumetric flask and 40 ml methanol was added. The flask was shaken for 10 min, followed by making up to volume with the same diluent to provide a solution containing 100 µg/ml of ROS and EZE. The solution was filtered and aliquots of the filtrate were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method (section ). 77

90 Experimental Work Preparation of standard solutions Standard solutions of ROS and EZE (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from µg/band. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning by camag TLC scanner III at 245 nm with deuterium lamp and analyzed in triplicate. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Solution preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range UV spectroscopy Preparation of the standard stock solution Standard stock solution containing 100 µg/ml of ROS and EZE were made separately by dissolving the pure drugs in methanol Preparation of standard solutions From standard stock solutions, standard solutions containing 5, 7, 9, 11, 13, 15, 17 and 19 µg/ml of both ROS and EZE were prepared in methanol. These solutions were scanned in the UV range against methanol as a blank in 1 cm quartz cuvettes Preparation of sample solution Twenty tablets were weighed and average weight was calculated. An amount of powder equivalent to 10 mg of both ROS and EZE were transferred to a 100 ml 78

91 Experimental Work volumetric flask and 40 ml methanol was added. The flask was shaken for 10 min, followed by making up to volume with the same diluent to provide a solution containing 100 µg/ml of ROS and EZE. The solution was filtered and aliquots of the filtrate were diluted with methanol to obtain concentrations in the linearity range and scanned in the UV range against methanol as blank Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.10 and 7.11 respectively. Table HPLC conditions Parameter Fixed conditions Mobile phase 0.1% v/v ortho-phosphoric acid solution (ph 3.5)-acetonitrile (63:37, v/v) Stationary phase Chromolith RP18 (6 mm 100 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 245 nm 79

92 Experimental Work Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions n-butyl acetate-chloroform-glacial acetic acid (1:8:1, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 245 nm 6 mm 30 min 8 cm mm 20 mm/s 80

93 Experimental Work 7.6. Telmisartan-atorvastatin Stock and standard solutions HPLC Preparation of the standard stock solution ATO and TLM standard stock solution was prepared by transferring accurately 5 mg of ATO and 20 mg of TLM reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of ATO and 400 µg/ml of TLM Preparation of standard solutions From the above solution, standard solutions containing µg/ml of ATO and µg/ml for TLM were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as peak areas against concentration of each drug Preparation of sample solution Twenty tablets containing 10 mg of ATO and 40 mg of TLM were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of ATO and 20 mg of TLM were transferred to a 50 ml volumetric flask, added 15 ml methanol and sonicated for a few minutes. A 35 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to 81

94 Experimental Work obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of ATO and TLM (2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from µg/band and µg/band for ATO and TLM respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 255 nm with deuterium lamp and analyzed in triplicate. The calibration curves were constructed by plotting peak areas versus concentrations for each drug. After separation of the drugs on the TLC plate, UV spectra of the individual drugs in solid state were recorded using the deuterium lamp of the TLC scanner Preparation of sample solution Preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range UV spectroscopy Preparation of the standard stock solution ATO and TLM standard stock solutions were prepared by transferring accurately 5 mg of ATO and 20 mg of TLM reference standards to separate 50 ml volumetric flasks. Twenty milliliters of methanol was added initially to solubilize the 82

95 Experimental Work drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of ATO and 400 µg/ml of TLM Preparation of standard solutions From standard stock solutions, standard solutions containing 3, 5, 7, 9 and 11 µg/ml of ATO and 12, 20, 28, 36 and 44 µg/ml of TLM were prepared in methanol. These solutions were scanned in the range of nm against methanol as a blank in 1 cm quartz cuvettes Preparation of sample solution Preparation was similar to HPLC method. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with methanol to obtain concentrations in the linearity range. The solutions were filtered and scanned in the range of nm against methanol as blank Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.12 and 7.13 respectively. 83

96 Experimental Work Table HPLC conditions Parameter Fixed conditions Mobile phase 10 mm ammonium acetate solution (adjusted to ph 4 with formic acid)- acetonitrile (30:70, v/v) Stationary phase Shim-pack C18 (6 mm 150 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 245 nm Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Ethyl acetate-chloroform-glacial acetic acid (8:3:0.5, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 255 nm 6 mm 30 min 8.5 cm mm 20 mm/s 84

97 Experimental Work 7.7. Rosuvastatin-fenofibrate Stock and standard solutions HPLC Preparation of the standard stock solution ROS and FEN standard stock solution was prepared by transferring 5 mg of ROS and 80 mg of FEN reference standards to a 50 ml volumetric flask. Twenty millilitres of acetonitrile was added initially to solubilize the drugs and the solution was diluted to volume with acetonitrile and mixed well to get 100 µg/ml of ROS and 1600 µg/ml of FEN Preparation of standard solutions From the above solution, standard solutions containing µg/ml of ROS and µg/ml of FEN were prepared in water-acetonitrile (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as peak areas against concentration of each drug Preparation of sample solution Twenty tablets containing 10 mg of ROS and 160 mg of FEN were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of ROS and 80 mg of FEN were transferred to a 50 ml volumetric flask, added 20 ml acetonitrile and sonicated for a few minutes. A 30 ml portion of acetonitrile was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-acetonitrile (50:50, v/v) mixture to 85

98 Experimental Work obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of ROS and FEN (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from and µg/band for ROS and FEN respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 240 nm. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.14 and 7.15 respectively. 86

99 Experimental Work Table HPLC conditions Parameter Fixed conditions Mobile phase 0.01% (v/v) formic acid solution (ph 3.9) acetonitrile (15:85, v/v) Stationary phase Luna C18 (4.60 mm 150 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 240 nm Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Chloroform methanol-toulene (4:3:6, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 240 nm 6 mm 30 min 8.5 cm mm 20 mm/s 87

100 Experimental Work 7.8. Olmesartan medoxomil-amlodipine Stock and standard solutions HPLC Preparation of the standard stock solution AML and OLM standard stock solution was prepared by transferring accurately 5 mg of AML and 20 mg of OLM reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of AML and 400 µg/ml of OLM Preparation of standard solutions From the above solution, standard solutions containing µg/ml of AML and 2-20 µg/ml of OLM were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as peak areas against concentration of each drug Preparation of sample solution Twenty tablets containing 5 mg of AML and 20 mg of OLM were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of AML and 20 mg of OLM were transferred to a 50 ml volumetric flask, added 20mL methanol and sonicated for a few minutes. A 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to 88

101 Experimental Work obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of AML and OLM (3.0, 4.0, 6.0, 8.0, 10.0 and 12.0µL) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from and µg/band for AML and OLM respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 238 nm. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range UV spectroscopy Preparation of the standard stock solution AML and OLM standard stock solutions were prepared by transferring accurately 5 mg of AML and 20 mg of OLM reference standards to separate 50 ml volumetric flasks. Twenty milliliters of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of AML and 400 µg/ml of OLM. 89

102 Experimental Work Preparation of standard solutions From standard stock solutions, standard solutions containing 7, 9, 11, 13 and 14 µg/ml of AML and 28, 36, 44, 52 and 56 µg/ml of OLM were prepared in methanol. These solutions were scanned in the range of nm against methanol as blank Preparation of sample solution Preparation was similar to HPLC method. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with methanol to obtain concentrations in the linearity range. The solutions were filtered and scanned in the range of nm against methanol as blank Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.16 and 7.17 respectively. Table HPLC conditions Parameter Fixed conditions Mobile phase 0.01% (v/v) formic acid solution (adjusted to ph 3.5 using triethylamine) methanol (35:65, v/v) Stationary phase Lichrospher RP-18, (4mm 250mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 238 nm 90

103 Experimental Work Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Ethyl acetate chloroform-glacial acetic acid (3:5:4, v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 238 nm 6 mm 30 min 8.5 cm mm 20 mm/s 91

104 Experimental Work 7.9. Telmisartan-amlodipine Stock and standard solutions HPLC Preparation of the standard stock solution AML and TLM standard stock solution was prepared by transferring accurately 5 mg of AML and 40 mg of TLM reference standards to a 50 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of AML and 800 µg/ml of TLM Preparation of standard solutions From the above solution, standard solutions containing µg/ml of AML and µg/ml of TLM were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as peak areas against concentration of each drug Preparation of sample solution Twenty tablets containing 5 mg of AML and 40 mg of TLM were weighed and average weight was calculated. An amount of powder equivalent to 5 mg of AML and 40 mg of TLM were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to 92

105 Experimental Work obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution Preparation was similar to that of the HPLC method Preparation of standard solutions Standard solutions of AML and TLM (2.0, 3.0, 4.0, 5.0 and 6.0 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from and µg/band for AML and TLM respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 254 nm. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.18 and 7.19 respectively. 93

106 Experimental Work Table HPLC conditions Parameter Fixed conditions Mobile phase 0.1% v/v formic acid (ph adjusted to 4 with triethylamine)-methanol(25:75, v/v) Stationary phase Lichrospher RP-18, (4 mm 250 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 238 nm Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate Slit dimension Scanning speed Fixed conditions Ethyl acetate-chloroform-glacial acetic acid, 3:5:4, (v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 254 nm 6 mm 30 min 8.5 cm mm 20 mm/s 94

107 Experimental Work Nebivolol-amlodipine Stock and standard solutions HPLC Preparation of the standard stock solution s-aml and NEB standard stock solution was prepared by transferring accurately 10 mg of s-aml and 20 mg of NEB reference standards to a 100 ml volumetric flask. Twenty millilitres of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of s-aml and 200 µg/ml of NEB Preparation of standard solutions From the above solution, standard solutions containing 2-10 µg/ml of s-aml and 4-20 µg/ml of NEB were prepared in water-methanol (50:50, v/v) mixture and analyzed in triplicate. The peaks obtained were integrated, the peak areas were calculated and respective calibration curves were plotted as peak areas against concentration of each drug Preparation of sample solution Twenty tablets containing 10 mg of s-aml and 20 mg of NEB were weighed and average weight was calculated. An amount of powder equivalent to 10 mg of s-aml and 20 mg of NEB were transferred to a 50 ml volumetric flask, added 20 ml methanol and sonicated for a few minutes. A 30 ml portion of methanol was then added and sonicated for 15 min to ensure complete extraction. This solution was centrifuged at 4000 rpm for 10 min. Aliquots of this solution were transferred to 10 ml volumetric flasks and diluted with water-methanol (50:50, v/v) mixture to 95

108 Experimental Work obtain concentrations in the linearity range. The solutions were filtered through a 0.45 µm membrane filter before injection into the column HPTLC method Preparation of the standard stock solution s-aml and NEB standard stock solution was prepared by transferring accurately 5 mg of s-aml and 10 mg of NEB reference standards to a 50 ml volumetric flask. Twenty milliliters of methanol was added initially to solubilize the drugs and the solution was diluted to volume with methanol and mixed well to get 100 µg/ml of s-aml and 200 µg/ml of NEB Preparation of standard solutions Standard solutions of s-aml and NEB (3.0, 4.0, 5.0, 6.0, 8.0, 10 and 12 µl) from standard stock solution were applied on precoated TLC plate to get concentrations ranging from and µg/band for s-aml and NEB respectively. The plate was developed in a developing chamber previously saturated with the mobile phase for 30 min. After development, the plate was air dried and standard zones were quantified by scanning at 268 nm. The calibration curves were constructed by plotting peak areas versus concentrations for each drug Preparation of sample solution Preparation was similar to that of the HPLC method. Aliquots of supernatant were applied on pre coated TLC plate to obtain concentrations in the linearity range Method validation The developed methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures in terms of parameters like linearity, precision, accuracy, detection and quantitation limits, specificity and robustness. 96

109 Experimental Work Chromatographic conditions HPLC and HPTLC chromatographic conditions are summarized in tables 7.20 and 7.21 respectively. Table HPLC conditions Parameter Fixed conditions Mobile phase 20 mm ammonium acetate (ph adjusted to 3.5 with formic acid)-acetonitrile, 55:45, v/v) Stationary phase Lichrospher RP-18, (4 mm 250 mm) column Injection volume 20 µl Flow rate 1 ml/min Detection wavelength 263 nm Table HPTLC conditions Parameter Mobile phase Stationary phase Detection wavelength Band width Chamber saturation time Developing distance of the plate slit dimension scanning speed Fixed conditions Ethyl acetate-chloroform-glacial acetic acid, 2:6:2, (v/v/v) Merck TLC plates coated with silica gel 60F 254 on aluminum sheets 268 nm 6 mm 30 min 8.5 cm mm 20 mm/s 97

110 Experimental Work Part 2- Bioanalytical method development Rosuvastatin-ezetimibe Instrumentation and operating conditions Liquid chromatography Liquid chromatography was carried out on a Shimadzu Prominence UFLC system. Luna C18 column (4.60 mm 150 mm, 5 µ particle size) was used as stationary phase. The chromatographic separation was performed using a mobile phase system consisting of 0.1 % (v/v) formic acid-methanol (20:80, v/v). The flow rate was 1mL/min and the column effluent was split so that 0.4 ml/min entered the mass spectrometer. Solvents were filtered through a nylon membrane filter (Rankem, New Delhi, India) of 0.45µm porosity. The column was maintained at 25 o C and the injection volume was 25 µl Mass spectrometry Mass spectrometric detection was performed using a Shimadzu LCMS-2010 EV quadrupole mass spectrometer interfaced with ESI probe. The ESI source was set at negative ionization mode. The [M-H] - ions at m/z 480, 408 and 557 were selected as detection ions for ROS, EZE and ATO(IS) respectively. These ions were decided by negative scanning from m/z Calibration of mass spectrometer was done by auto tuning. Tuning of mass spectrometer was performed with the help of auto tuning option in LC solutions software using tuning standard solution (polypropylene glycol). The main working parameters of the mass spectrometer are summarized in table Data acquisition and processing were accomplished with Shimadzu LC solutions chromatographic software. 98

111 Experimental Work Preparation of stock solutions, calibration standards and quality control (QC) samples Solutions (100 µg/ml) each of ROS, EZE and ATO were prepared separately in methanol. Working solutions for calibration and controls were prepared by appropriate dilution with the diluents, water-methanol (50:50, %v/v) mixture. Stock solution of ATO was also diluted with water-methanol (50:50, %v/v) to give an 800 ng/ml IS working solution. These solutions were then used to spike blank plasma to get a series of calibration standards containing 0.10, 0.25, 0.50, 2, 4, 8 and 10 ng/ml ROS and EZE. Low, medium and high concentration QC samples containing 0.25, 2 and 8 ng/ml of each analyte were prepared using the same procedure. The lower limit of quantification (LLOQ) and upper limit of quanification (ULOQ) were 0.1 and 10 ng/ml respectively Sample preparation Plasma samples stored in the freezer were allowed to thaw at room temperature and then subjected to LLE. To 250 µl of plasma in a glass-stoppered 10 ml tube was added 25 µl of IS working solution. After vortex mixing for about 30 seconds, 3 ml of extraction mixture, diethyl ether-dichloromethane (70:30, %v/v) was added. This solution was vortexed for about 2 minutes and centrifuged at 3000 rpm for 5 minutes. The organic layer was transferred to another clean glass tube and evaporated to dryness under a stream of nitrogen gas at room temperature. The residue was reconstituted in 200 µl of mobile phase and 25 µl injected into the LC-MS system Bioanalytical method validation Method validation was carried out according to US FDA bioanalytical method validation guidelines. 99

112 Experimental Work Selectivity To study selectivity, six lots of plasma extracts from different sources were prepared as in section and analyzed. In order to evaluate the matrix effect on the ionization of analytes, three QC samples (0.25, 2 and 8 ng/ml) and IS (20 ng/ml) were dried and reconstituted in mobile phase (neat standard). Plasma samples were processed as in section and the residues were reconstituted in 200 µl of mobile phase to contain 0.25, 2 and 8 ng/ml of ROS and EZE and 20 ng/ml of IS Linearity Calibration curves were constructed using six non-zero standards ranging from 0.1 to 10 ng/ml. A blank sample (a plasma sample processed without the IS) and a zero sample (a plasma sample processed with the IS) were also processed and analyzed. Calibration curves were acquired by plotting the peak area ratio of analyte (ROS or EZE): IS against the nominal concentrations of the calibration standards. The acceptance criterion for calibration curve was a correlation coefficient (r) of 0.99 or better Precision and accuracy Precision and accuracy were determined for both intra-day and inter-day runs. They were determined by replicate analyses of the QC samples of three concentrations. Intra-day precision was studied by replicate analysis of the samples on one day (n=5). Inter-day precision was determined by repeated analysis on five consecutive days. The acceptance criterion for precision is that CV should be less than 15% Recovery The extraction efficiencies of ROS, EZE and ATO by the LLE procedure were determined by comparing the responses of the analytes extracted from replicate QC 100

113 Experimental Work samples (n=5) with the response of analytes from non-extracted standard solutions at equivalent concentrations. Recoveries were determined for low, medium and high concentration QC samples (viz. 0.25, 2 and 8 ng/ml) Stability studies Freeze and thaw stability: Three aliquots of two concentration levels (low and high QC samples) were frozen and thawed unassisted at room temperature. When completely thawed, the samples were frozen again at the same temperature and thawed. This freeze-thaw cycle was repeated twice and after the third cycle, the samples were analyzed. Short-term temperature stability: Three aliquots of each of the low and high QC samples were kept at room temperature for a period that exceeded the routine preparation time of sample and analyzed. Long-term stability: Long-term stability was evaluated by storing three aliquots of each of the low and high QC samples in freezer for a period of around one week. Stock solution stability: The stability of ROS, EZE and IS were evaluated at room temperature for around 6 hours and stability was tested by comparing the instrument response with that of freshly prepared solutions. Post-Preparative Stability: This stability was determined for about hours to cover the anticipated run time for the analytical batch and to allow for delayed injection owing to unforeseen circumstances. The extracted QC samples (ready to inject) were kept in autosampler conditions and analyzed with fresh standards. 101

114 Experimental Work Table Mass spectrometer working parameters Parameter Value Detector voltage, KV 2 Interface voltage, KV 4.5 Q-array DC voltage, V +5 RF voltage, V +150 CDL voltage, V -10 CDL temperature, o C 250 Heat Block temperature, o C 200 Nebulising gas flow, L/min 1.5 Mode of analysis Negative 102

115 Experimental work Atorvastatin-telmisartan Instrumentation and operating conditions Liquid chromatography Liquid chromatography was carried out on a Shimadzu Prominence UFLC system. Luna C18 (4.60 mm 150 mm, 5 µ particle size) column was used as stationary phase. The chromatographic separation was performed using a mobile phase system consisting of 10 mm ammonium acetate-methanol (20:80, v/v) adjusted to ph 4 after mixing with formic acid. The flow rate was 1mL/min and the column effluent was split so that 0.4 ml/min entered the mass spectrometer. Solvents were filtered through a nylon membrane filter (Rankem, New Delhi, India) of 0.45µm porosity. The column was maintained at 25 o C and the injection volume was 25 µl Mass spectrometry Mass spectrometric detection was performed using a Shimadzu LCMS-2010 EV quadrupole mass spectrometer interfaced with ESI probe. The ESI source was set at positive ionization mode. The [M+H] + ions at m/z 482, 515 and 559 were selected as detection ions for ROS (IS), TLM and ATO respectively. These ions were decided by positive scanning from m/z The main working parameters of the mass spectrometer are summarized in table Data acquisition and processing were accomplished with Shimadzu LC solutions chromatographic software Preparation of stock solutions, calibration standards and quality control (QC) samples Solutions (100 µg/ml) of ATO, TLM and ROS were prepared separately in methanol. Working solutions for calibration and controls were prepared by appropriate dilution with the diluents, water-methanol (50:50, v/v) mixture. Stock 103

116 Experimental work solution of ROS was also diluted with water-methanol (50:50, % v/v) mixture to give a 1200 ng/ml working solution. These solutions were then used to spike blank plasma to give a series of calibration standards ranging from 1-35 ng/ml ATO and TLM. Low, medium and high concentration QC samples containing 2, 15 and 30 ng/ml of each analyte were prepared using the same procedure. Their LLOQ and ULOQ were 1 and 35 ng/ml respectively Sample preparation Plasma samples stored in the freezer were allowed to thaw at room temperature and then subjected to LLE. To 250 µl plasma containing analytes in a glass-stoppered 10 ml tube, added 25 µl of IS working solution. After vortex mixing for about 1 min, 3 ml of extraction mixture, ethyl acetate-dichloromethane (80:20, %v/v) was added. This solution was vortexed for about 5 minutes and centrifuged at 3000 rpm for 5 minutes. The organic layer was transferred to another clean glass tube and evaporated to dryness under a stream of nitrogen gas at room temperature. The residue was reconstituted in 200 µl of mobile phase and 25 µl injected into the LC-MS system Bioanalytical method validation Method validation was carried out according to US FDA bioanalytical method validation guidelines Selectivity To study selectivity, six lots of plasma extracts from different sources were prepared as in section and analyzed. In order to evaluate the matrix effect on the ionization of analytes, three QC samples (2, 15 and 30 ng/ml) and IS (30 ng/ml) were dried and reconstituted in mobile phase (neat standard). Plasma samples were 104

117 Experimental work processed as in section and the residues were reconstituted in 200 µl of mobile phase to contain 2, 15 and 30 ng/ml of ATO and TLM and 30 ng/ml of IS Linearity Calibration curves were constructed using six non-zero standards ranging from 1 to 35 ng/ml. A blank sample (a plasma sample processed without the IS) and a zero sample (a plasma sample processed with the IS) were also processed and analyzed. Calibration curves were acquired by plotting the peak area ratio of analyte (ATO or TLM): IS against the nominal concentrations of the calibration standards Precision and accuracy Precision and accuracy were determined for both intra-day and inter-day runs. They were determined by replicate analyses of the QC samples of three concentrations. Intra-day precision was studied by replicate analysis of the samples on one day (n=5). Inter-day precision was determined by repeated analysis on five consecutive days. The acceptance criterion for precision is that CV should be less than 15% Recovery The extraction efficiencies of the analytes by the LLE procedure were determined by comparing the responses of the analytes extracted from replicate QC samples (n=5) with the response of analytes from non-extracted standard solutions at equivalent concentrations. Recoveries were determined at low, medium and high concentration QC samples (viz. 2, 15 and 30 ng/ml) Stability studies Freeze and thaw stability: Three aliquots of two concentration levels (low and high QC samples) were frozen and thawed unassisted at room temperature. When completely thawed, the samples were frozen again at the same temperature and 105

118 Experimental work thawed. This freeze-thaw cycle was repeated twice and after the third cycle, the samples were analyzed. Short-term temperature stability: Three aliquots of each of the low and high QC samples were kept at room temperature for a period that exceeded the routine preparation time of sample and analyzed. Long-term stability: Long-term stability was evaluated by storing three aliquots of each of the low and high QC samples in freezer for a period of around one week. Stock solution stability: The stability of ATO, TLM and IS were evaluated at room temperature for around 6 hours and stability was tested by comparing the instrument response with that of freshly prepared solutions. Post-Preparative Stability: This stability was determined for about hours to cover the anticipated run time for the analytical batch and to allow for delayed injection owing to unforeseen circumstances. The extracted QC samples (ready to inject) were kept in autosampler conditions and analyzed with fresh standards. Table Mass spectrometer working parameters Parameter Value Detector voltage, KV 1.7 Interface voltage, KV 4.5 Q-array DC voltage, V +5 RF voltage, V +150 CDL voltage, V -20 CDL temperature, o C 250 Heat Block temperature, o C 200 Nebulising gas flow, L/min 1.5 Mode of analysis Positive 106

119 CHAPTER 8. RESULTS AND DISCUSSION Part1- Analytical method development 8.1. Amlodipine-hydrochlorothiazide-valsartan Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the best resolution and peak shape. Mobile phase selection was based on peak parameters (symmetry, theoretical plates and capacity factor), run time, ease of preparation and cost. Initially, a mobile phase system consisting of 0.1 % (v/v) formic acid and methanol in different proportions was tried. With this system, the peak characteristics were not good and hence the aqueous phase was substituted with ammonium acetate buffer. Although good peak shapes were achieved with this system, VAL and HYD peaks were not resolved. Trials with different strengths and ph s of buffer also did not give good results. Hence, several proportions of ammonium acetate buffer-methanol and flow gradients were evaluated in order to achieve optimum separation of analytes in short time of analysis. With the developed gradient system, symmetrical peaks with good separation (retention times, HYD=2.0 min, VAL=7.5 min and AML=10.2 min) were obtained on a Phenomenex Luna C18 (4.60 mm 150 mm, 5 µ particle size) column at a flow rate of 1 ml/min. To select the absorption wavelength for detection, UV spectra of the three drugs were acquired and overlaid. It was found that the best response for all the drugs was obtained at 238 nm (fig. 8.1). The chromatograms of standards and formulation are shown in fig

120 HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of AML, HYD and VAL. Good separation of the drugs (R f values of HYD = 0.18, AML= 0.40 and VAL = 0.75) with good symmetrical peaks were obtained by using the mobile phase, chloroform-glacial acetic acid-n-butyl acetate (8:4:2, v/v/v). The separated spots of the three drugs were scanned at 320 nm (fig. 8.9). Densitograms obtained from the analysis of drugs using the proposed method are shown in fig Method Validation HPLC Linearity To assess linearity, standard calibration curves for AML, VAL and HYD were constructed by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of , and µg/ml for AML, HYD and VAL respectively, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations (x). Table 8.1 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies indicate good repeatability and low interday variability (table 8.1). 108

121 Accuracy The recovery study results ranged from 98 to 101% for AML, HYD and VAL showing the accuracy of the method (Table 8.2) Detection and quantitation limits The LOD values for AML, HYD and VAL were 0.004, and µg/ml respectively and their LOQ values were found to be 0.03, 0.08 and µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for AML, HYD and VAL were found to be close to 1) Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition, ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C were found to be stable for around 1and 3 days respectively. 109

122 System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of AML, HYD and VAL and peak areas of the spots was investigated. Good linearity was observed for in the concentration range of , and µg/band for AML, HYD and VAL respectively, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.1 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies indicate good repeatability and low interday variability (table 8.1) Accuracy The recovery study results ranged from 98 to 100% for AML, HYD and VAL showing the accuracy of the method (table 8.2). Low RSD values indicate that the method is also precise. 110

123 Detection and quantitation limits The LOD values for AML, HYD and VAL were found to be 0.09, 0.03 and 3.2 µg/band respectively and their LOQ values were 0.2, 0.06 and 6.4 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of AML, HYD and VAL were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for AML [correlation r(s,m) = and r(m,e) = ], HYD [correlation r(s,m) = and r(m,e) = ] and VAL [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times, etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C were found to be stable for around 2 and 4 days respectively. 111

124 Application of the method The proposed methods were used for the assay of commercially available tablets containing AML, HYD and VAL. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of AML, HYD and VAL in samples is presented in table Statistical Analysis The developed analytical methods were compared using statistical analysis using GraphPad InStat software version 3.05(GraphPad Software, Inc.). The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table 8.5. This result shows that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 112

125 Table 8.1. Summary of validation parameters for the proposed methods HPLC HPTLC Parameters AML VAL HYD AML VAL HYD Linearity, (µg/ml or µg/band) Linear regression equation a Intercept Slope Correlation coefficient LOD, (µg/ml or µg/band) LOQ, (µg/ml or µg/band) Precision(RSD) Intraday(n=3), % Interday (n=3), % Repeatability of injection (n=10), % a y=mx+c Table 8.2. Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC AML HYD VAL a Average of six determinations 113

126 Table 8.3. System suitability studies Method Drug Resolution Theoretical Plate# Tailing Factor Capacity Factor AML HPLC HYD VAL Table 8.4. Analysis of formulation Drug,labeled Amount found, amount, mg/tablet Amount found, % a R.S.D., % a mg/tablet HPLC HPTLC HPLC HPTLC HPLC HPTLC AML, HYD, VAL, a Average of six determinations Table 8.5. Statistical comparison of the results obtained by the proposed methods Methods AML HYD VAL HPLC vs HPTLC t c = t c = t c = P value= * P value=0.6029* P value=0.7321* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 114

127 Figure 8.1. Overlain UV spectra of AML, HYD and VAL HPLC chromatograms of standards Figure 8.2. AML-0.5 µg/ml, HYD-1.25 µg/ml, VAL-16 µg/ml

128 Figure 8.3. AML-1 µg/ml, HYD-2.5 µg/ml, VAL-32 µg/ml Figure 8.4. AML-2 µg/ml, HYD-5 µg/ml, VAL-64 µg/ml Figure 8.5. AML-3 µg/ml, HYD-7.5 µg/ml, VAL-96 µg/ml

129 Figure 8.6. AML-4 µg/ml, HYD-9 µg/ml, VAL-128 µg/ml Figure 8.7. AML-5 µg/ml, HYD-11.5 µg/ml, VAL-160 µg/ml HPLC chromatogram of formulation Figure 8.8. AML-4 µg/ml, HYD-9 µg/ml, VAL-128 µg/ml

130 Figure 8.9. Overlain UV spectra of AML, HYD and VAL on pre-coated TLC plate HPTLC chromatograms of standards Figure AML- 0.2 µg/band, HYD-0.5 µg/band, VAL-6.4 µg/band Figure AML-0.3 µg/band, HYD-0.75 µg/band, VAL-9.6 µg/band

131 Figure AML-0.4 µg/band, HYD-1 µg/band, VAL-12.8 µg/band Figure AML-0.5 µg/band, HYD-1.25 µg/band, VAL-16 µg/band HPTLC chromatogram of formulation Figure AML-0.6 µg/band, HYD-1.5 µg/band, VAL-19.2 µg/band Figure AML-0.3 µg/band, HYD-1.25 µg/band, VAL-16 µg/band

132 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of AML (0.5-5 µg/ml) Concentration (µg/ml) Peak Area Figure Calibration graph of HYD ( µg/ml)

133 Concentration (µg/ml) Peak Area Figure Calibration graph of VAL ( µg/ml) HPTLC calibration graphs Concentration (µg/band) Peak Area Figure Calibration graph of AML ( µg/band)

134 Concentration (µg/band) Peak Area Figure Calibration graph of HYD ( µg/band) Concentration (µg/band) Peak Area Figure Calibration graph of VAL ( µg/band)

135 8.2. Atorvastatin-ezetimibe-fenofibrate Method Development and Optimization HPLC Chromatographic conditions were optimized to achieve the best resolution and peak shape. The chromatography parameters were initially evaluated using a mobile phase system consisting of different proportions of 0.1 % (v/v) formic acid and acetonitrile. With different proportions of 0.1 % (v/v) formic acid and acetonitrile in different ratios like 50:50, v/v and 40:60, v/v, ATO and EZE peaks got resolved but FEN peak did not elute even after 30 min. At higher ph s, ATO and EZE peak shapes were not good. Hence, several proportions of 0.1% formic acid-acetonitrile and different flow gradients were evaluated in order to achieve optimum separation of analytes in short time of analysis. With the developed gradient system, symmetrical peaks with good separation (retention times, EZE=4.5 min, ATO=5.5 min and FEN=15.6 min) were obtained on a Shim-pack C18 (6 mm 150 mm, 5 µ particle size) column at a flow rate of 1.5 ml/min. The optimum wavelength for detection and quantification was 245 nm, at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of ATO, EZE and FEN. Good separation of the drugs (R f values of ATO = 0.10, EZE=0.20 and FEN = 0.80) with symmetrical peaks was obtained by using the mobile phase, toluene-methanol-triethylamine (8:1.5:0.1, v/v/v). 115

136 The separated spots of the three drugs were scanned at 245 nm (fig. 8.30). Chromatograms obtained from the analysis of drugs using the proposed method are shown in fig Method Validation HPLC Linearity To assess linearity, standard calibration curves for ATO, EZE and FEN were obtained by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of µg/ml for ATO and EZE and 8-80 µg/ml of FEN, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations (x). Table 8.6 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.6 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 97 to 102% for ATO, EZE and FEN, showing the accuracy of the method (Table 8.7) Detection and quantitation limits The LOD values for ATO, EZE and FEN were found to be 0.08, 0.07 and 0.25 µg/ml respectively and their LOQ values were 0.5, 0.5 and 0.75 µg/ml respectively. 116

137 Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for ATO, EZE and FEN were found to be close to 1) Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition and ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of ATO, EZE and FEN and peak areas of the spots was investigated. Good linearity was observed in the concentration 117

138 range of , and µg/band for ATO, EZE and FEN respectively, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.6 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.6, indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 97 to 102% for ATO, EZE and FEN, showing the accuracy of the method (Table 8.7). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for ATO, EZE and FEN were found to be 0.06, 0.08 and 0.09 µg/band respectively, and their LOQ values were 0.09, 0.1 and 0.15 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of ATO, EZE and FEN were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for ATO [correlation r(s,m) = and r(m,e) = ], EZE [correlation r(s,m) = and r(m,e) = ] and FEN [correlation r(s,m) = and r(m,e) = 118

139 0.9999]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times, etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 4 days, respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing ATO, EZE and FEN. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of ATO, EZE and FEN in samples is presented in table Statistical Analysis The developed HPLC and HPTLC methods were compared using statistical analysis. The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table This result shows 119

140 that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. Table 8.6. Summary of validation parameters for the proposed methods HPLC HPTLC Parameters ATO EZE FEN ATO EZE FEN Linearity (µg/ml or µg/band) Linear regression equation a Intercept Slope Correlation coefficient LOD (µg/ml or µg/band) LOQ (µg/ml or µg/band) Precision(RSD) Intraday(n=3), % Interday (n=3), % Repeatability of injection (n=10), % a y=mx+c Table 8.7. Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC ATO EZE FEN a Average of six determinations 120

141 Table 8.8. System suitability studies Method Drug Resolution Theoretical Plate # Tailing Factor Capacity Factor HPLC EZE ATO FEN Table 8.9. Analysis of formulation Drug, labeled Amount found, amount, mg/tablet Amount found, % a R.S.D., % a mg/tablet HPLC HPTLC HPLC HPTLC HPLC HPTLC ATO, EZE, FEN, a Average of six determinations Table Statistical comparison of the results obtained by the proposed methods Methods ATO EZE FEN HPLC vs HPTLC t c = P value= * t c = P value= * t c = P value=0.5923* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 121

142 Figure Overlain UV spectra of ATO, EZE and FEN HPLC chromatograms of standards Figure ATO-0.5 µg/ml, EZE-0.5 µg/ml, FEN-8 µg/ml

143 Figure ATO-1 µg/ml, EZE-1 µg/ml, FEN-16 µg/ml Figure ATO-2 µg/ml, EZE-2 µg/ml, FEN-32 µg/ml Figure ATO-3 µg/ml, EZE-3 µg/ml, FEN-48 µg/ml

144 Figure ATO-4 µg/ml, EZE-4 µg/ml, FEN-64 µg/ml Figure ATO-5 µg/ml, EZE-5 µg/ml, FEN-80 µg/ml HPLC chromatogram of formulation Figure ATO-3 µg/ml, EZE-3 µg/ml, FEN-48 µg/ml

145 Figure Overlain UV spectra of ATO, EZE and FEN on pre-coated TLC plate HPTLC chromatograms of standards Figure ATO- 0.1 µg/band, EZE-0.1 µg/band, FEN-1.6 µg/band Figure ATO-0.15 µg/band, EZE-0.15µg/band, FEN-2.4 µg/band

146 Figure ATO- 0.2 µg/band, EZE-0.2 µg/band, FEN-3.2 µg/band Figure ATO-0.25 µg/band, EZE-0.25µg/band, FEN-4 µg/band Figure ATO- 0.3 µg/band, EZE-0.3 µg/band, FEN-4.8 µg/band Figure ATO-0.35 µg/band, EZE-0.35µg/band, FEN-5.6 µg/band

147 HPTLC chromatogram of formulation Figure ATO µg/band, EZE-0.25 µg/band, FEN-4 µg/band

148 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of ATO (0.5-5 µg/ml) Concentration Peak (µg/ml) Area Figure Calibration graph of EZE (0.5-5 µg/ml)

149 Concentration Peak (µg/ml) Area Figure Calibration graph of FEN (8-80 µg/ml) HPTLC calibration graphs Concentration (µg/band) Peak Area Figure Calibration graph of ATO ( µg/band)

150 Concentration (µg/band) Peak Area Figure Calibration graph of EZE ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of FEN ( µg/band)

151 8.3. Telmisartan-ramipril-hydrochlorothiazide Method Development and Optimization HPLC Chromatographic conditions were optimized to achieve the best resolution and peak shapes. Preliminary trials using different compositions of mobile phases consisting of acetonitrile and orthophosphoric acid solutions gave poor peak shape and high tailing factors. Symmetrical peaks with good separation (retention time, HYD=2.9 min, RPL=10.5 min and TLM=12.9 min) were obtained on a RP18 column with mobile phase consisting of 0.1% (v/v) phosphoric acid (ph adjusted to 2.5 with triethylamine) and acetonitrile (58:42, v/v) at a flow rate of 1 ml/min. The optimum wavelength for detection and quantification was 210 nm at which good detector response was obtained for the drugs, fig The chromatograms of standards and formulation are shown in fig HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of RPL, HYD and TLM. Good separation of the drugs (R f values of RPL = 0.18, TLM =0.45 and HYD = 0.60) with symmetrical peaks was obtained by using the mobile phase, ethyl acetate-chloroform-methanol, 6.5:3:1.3 (v/v/v). The separated spots of the three drugs were scanned at 210 nm (fig. 8.51). Densitograms obtained from the analysis of drugs using the proposed method are shown in fig

152 Method Validation HPLC Linearity The relationship between the concentration of RPL, HYD and TLM and peak areas was investigated. Good linearity was observed in the concentration range of 0.5-4, and 4-32 µg/ml for RPL, HYD and TLM respectively, fig The regression equations for the drugs were found by plotting peak areas (y) versus concentrations (x). Table 8.11 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for precision studies are presented in table Accuracy The recovery study results ranged from 98 to 103% for RPL, HYD and TLM, showing the accuracy of the method (Table 8.12). Low RSD values indicate that the method is precise Detection and quantitation limits The LOD values for RPL, HYD and TLM were found to be 0.05, 0.02 and 0.03 µg/ml respectively, and their LOQ values were 0.5, 0.15 and 0.20 µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to 123

153 more than one component (diode array detector peak purity index values for RPL, HYD and TLM were found to be close to 1) Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition and ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 5 days, respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of RPL, HYD and TLM and peak areas of the spots was investigated. Good linearity was observed for in the concentration range of , and µg/band for RPL, HYD and TLM respectively, fig The regression equations for the drugs were found by 124

154 plotting peak areas (y) versus the concentration (x). Table 8.11 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.11, indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 103% for RPL, HYD and TLM, showing the accuracy of the method (Table 8.12). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for RPL, HYD and TLM were found to be 0.09, 0.04 and 0.25 µg/band respectively, and their LOQ values were 0.2, 0.09 and 0.45 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of RPL, HYD and TLM were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for RPL [correlation r(s,m) = and r(m,e) = ], HYD [correlation r(s,m) = and r(m,e) = ] and TLM [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs. 125

155 Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 1½ and 5 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing RPL, HYD and TLM. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of RPL, HYD and TLM in samples are presented in table Statistical Analysis The developed HPLC and HPTLC methods were compared using statistical analysis. The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table This result shows that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 126

156 Table Summary of validation parameters for the proposed methods HPLC HPTLC Parameters RPL HYD TLM RPL HYD TLM Linearity (µg/ml or µg/band) Linear regression equation a Intercept Slope Correlation coefficient LOD (µg/ml or µg/band) LOQ (µg/ml or µg/band) Precision(RSD) Intraday(n=3), % Interday (n=3), % Repeatability of injection (n=10), % a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC RPL TLM HYD a Average of six determinations 127

157 Table System suitability studies Method Drug Resolution Theoretical Plate # Tailing Factor Capacity Factor HYD HPLC RPL TLM Table Analysis of formulation Drug, labeled amount, mg/tablet Amount found, Amount found, % a R.S.D., % a mg/tablet HPLC HPTLC HPLC HPTLC HPLC HPTLC RPL, HYD, TLM, a Average of six determinations Table Statistical comparison of the results obtained by the proposed methods Methods RPL TLM HYD HPLC vs HPTLC t c = P value= * t c = P value=0.7380* t c = P value=0.5691* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 128

158 Figure Overlain UV spectra of RPL, HYD and TLM HPLC chromatograms of standards Figure RPL-0.5 µg/ml, HYD-1.25 µg/ml, TLM-4 µg/ml Figure RPL-1 µg/ml, HYD-2.5 µg/ml, TLM-8 µg/ml

159 Figure RPL-2 µg/ml, HYD-5 µg/ml, TLM-16 µg/ml Figure RPL-3 µg/ml, HYD-7.5 µg/ml, TLM-24 µg/ml Figure RPL-4 µg/ml, HYD-10 µg/ml, TLM-32 µg/ml

160 HPLC chromatogram of formulation Figure RPL-1 µg/ml, HYD-2.5 µg/ml, TLM-8 µg/ml Figure Overlain UV spectra of RPL, HYD and TLM on pre-coated TLC plate

161 HPTLC chromatograms of standards Figure RPL- 0.2 µg/band, HYD-0.5 µg/band, TLM-1.6 µg/band Figure RPL-0.3 µg/band, HYD-0.75µg/band, TLM-2.4 µg/band Figure RPL- 0.4 µg/band, HYD-1 µg/band, TLM-3.2 µg/band Figure RPL-0.5 µg/band, HYD-1.25µg/band, TLM-4 µg/band

162 HPTLC chromatogram of formulation Figure RPL- 0.6 µg/band, HYD-1.5 µg/band, TLM-4.8 µg/band Figure RPL-0.2 µg/band, HYD-0.5 µg/band, TLM-1.6 µg/band HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of RPL (0.5-4 µg/ml)

163 Concentration Peak (µg/ml) Area Figure Calibration graph of HYD ( µg/ml) Concentration Peak (µg/ml) Area Figure Calibration graph of TLM (4-32 µg/ml)

164 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of RPL ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of HYD ( µg/band)

165 Concentration Peak (µg/band) Area Figure Calibration graph of TLM ( µg/band)

166 8.4. Atorvastatin-ramipril-aspirin Method Development and Optimization Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of RPL, ATO and ASP. Good separation of the drugs (R f values of RPL = 0.28, ATO=0.48 and ASP = 0.68) with good symmetrical peaks was obtained by using the mobile phase, methanol-benzene-ethyl acetate-glacial acetic acid, 0.36:2.5:4:0.04 (v/v/v/v). The separated spots of the three drugs were scanned at 210 nm (fig. 8.64). Chromatograms obtained from the analysis of drugs using the proposed method are shown in fig Method Validation Linearity The relationship between the concentration of RPL, ATO and ASP and peak areas of the spots was investigated. Good linearity was observed in the concentration range of , and 6-21 µg/band for RPL, ATO and ASP respectively, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.16 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.16 indicate good repeatability and low interday variability. 129

167 Accuracy The recovery study results ranged from % for RPL, ATO and ASP showing the accuracy of the method (Table 8.17). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for RPL, ATO and ASP were found to be 0.06, 0.05 and µg/band respectively and their LOQ values were 0.1, 0.07 and µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of RPL, ATO and ASP were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for RPL [correlation r(s,m) = and r(m,e) = ], ATO [correlation r(s,m) = and r(m,e) = ] and ASP [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times, etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged 130

168 solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C were found to be stable for around 1 and 2 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing RPL, ATO and ASP. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of RPL, ATO and ASP in samples is presented in table Table Summary of validation parameters for the proposed method Parameters RPL ATO ASP Linearity, µg/band Linear regression equation a Intercept Slope Correlation coefficient LOD, µg/band LOQ, µg/band Precision Intraday(n=6), % Interday (n=6), % Repeatability of injection (n=10), % a y=mx+c 131

169 Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a RPL ATO ASP a Average of six determinations Table Analysis of the formulation Drug, labeled Amount found, Amount found, amount, mg/tablet mg/tablet % a R.S.D., % a RPL, ATO, ASP, a Average of six determinations 132

170 Figure Overlain UV spectra of RPL, ATO and ASP on pre-coated TLC plate HPTLC chromatograms of standards Figure RPL-0.4 µg/band, ATO-0.8 µg/band, ASP-6 µg/band Figure RPL-0.6 µg/band, ATO-1.2 µg/band, ASP-9 µg/band

171 Figure RPL-0.8 µg/band, ATO-1.6 µ g/band, ASP-12 µg/band Figure RPL-1 µg/band, ATO-2 µg/band, ASP-15 µg/ /band Figure RPL-1.2 µg/band, ATO-2.4 µ g/band, ASP-18 µg/band Figure RPL-1.4 µg/band, ATO-2.8 µg/band, ASP-21 µg/band

172 HPTLC chromatogram of formulation Figure RPL-0.6 µg/band, ATO-1.2 µg/band, ASP-9 µg/band HPTLC calibration graphs Concentration (µ µg/band) Peak Area Figure Calibration graph of RPL ( µg/band)

173 Concentration Peak Area (µg/band) Figure Calibration graph of ATO ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of ASP (6-21 µg/band)

174 8.5. Rosuvastatin-ezetimibe Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the best resolution and peak shape. Preliminary trials using different compositions of mobile phases consisting of acetonitrile and ortho-phosphoric acid solutions gave poor peak shape and higher tailing factors. Symmetrical peaks with good separation (retention time of ROS=4.8 min and EZE=10.9 min) was obtained using C18 Chromolith column and mobile phase consisting of 0.1% v/v orthophosphoric acid solution ph 3.5 (adjusted with triethyl amine solution)-acetonitrile (63:37, v/v), at a flow rate of 1 ml/min. The optimum wavelength for detection and quantification was 245 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in pharmaceutical formulations HPTLC Experimental conditions such as mobile phase and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of ROS and EZE by HPTLC method. Good separation of both drugs (R f values of ROS=0.30 and EZE=0.58) with minimum tailing was obtained by using the mobile phase consisting of n-butyl acetate-chloroform-glacial acetic acid (1:8:1, v/v/v). The separated spots of the three drugs were scanned at 245 nm (fig.8.88).typical densitograms obtained from the analysis of drugs using the proposed method are shown in fig

175 UV spectroscopy Two simple, accurate and precise UV methods were developed for the simultaneous determination of ROS and EZE in combined dosage form. First method employs formation and solving of simultaneous equation (Vierodt s method). Second method depends on second derivative UV-spectrometry with zero-crossing measurement technique. Methanol was selected as the solvent because it provided the highest solubility and UV absorbance without interference from sample matrix for both the methods Technique of simultaneous equations (Vierodt s method) Absorbances of the solutions were measured at wavelengths, 234 and 244 nm for EZE and ROS respectively. Zero order absorption spectra of ROS, EZE and formulation are shown in fig Quantitative determination of these drugs was carried out by solving the simultaneous equations, C X =A 2 a Y1 -A 1 a Y2 /a X2 a Y1 -a X1 a Y2 (1), C Y =A 1 a X2 -A 2 a X1 /a X2 a Y1 -a X1 a Y2 (2), where, A 1 and A 2 are absorbances of the mixture at 234 and 244 nm respectively, a X1 and a X2 are absorptivities of x at 234 and 244 nm respectively, a Y1 and a Y2 are absorptivities of y at 234 and 244 nm respectively, C X is the concentration of EZE and C Y is the concentration of ROS Derivative UV- spectrophotometry The derivative procedure was performed to check whether sample matrices of the investigated formulation would interfere with the drugs spectra. Zero-crossing second-order spectrophotometry permits a more selective identification and determination of the two drugs in a mixture. The second order derivative spectra of drugs gave sharper and better-defined peaks when compared with the original UV absorption and other order derivative spectrum. The selection of the optimum wavelength is based on the fact that the absolute value of the total derivative spectrum 134

176 at the selected wavelength has the best linear response to the analyte concentration. It is not affected by the concentration of any other component and gives a near-zero intercept on the ordinate axis of the calibration curve. Therefore, nm (zerocrossing wavelength of EZE) and nm (zero-crossing wavelength of ROS) were chosen as optimum working wavelengths for the simultaneous determination of ROS and EZE in a binary mixture respectively. Second derivative spectra of standard drugs in methanol are shown in fig Method Validation HPLC Linearity To assess linearity, standard calibration curves for ROS and EZE were constructed by plotting concentrations versus their peak areas. The curves showed good linearity over a concentration range of µg/ml for both ROS and EZE, fig The regression equations for drugs were found by plotting peak areas (y) versus the concentrations (x). Table 8.19 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.19 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 99 to 101 for both ROS and EZE, showing the accuracy of the method (Table 8.20). 135

177 Detection and quantitation limits The LOD values for ROS and EZE were 0.03 and 0.09 µg/ml respectively and their LOQ values were found to be 0.09 and 0.5 µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for both ROS and EZE were found to be Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition, ph values of the aqueous phase of the mobile phase and flow rate. During these investigations, it was found that there was not much change in the retention times, area and symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing the solutions at ambient temperature and 2-5 o C and tested at intervals of 12, 24, 36 and 48 h etc. The responses for the aged solution were evaluated using a freshly prepared standard solution. The solutions stored under ambient and 2-5 o C were found to be stable for around 1½ and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table

178 HPTLC Linearity Good linearity was observed for both ROS and EZE in the concentration range of µg/band, fig The regression equations for drugs were found by plotting peak areas (y) versus the concentrations (x). Table 8.19 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate precision (interday precision) and was expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.19; indicating good repeatability and low inter day variability Accuracy The recovery study results ranged from 98 to 101 for both ROS and EZE, showing the accuracy of the method (Table 8.20). Low % RSD values indicate that the method is precise Detection and quantitation limits The LOD values for ROS and EZE were found to be 0.04 and 0.07 µg/band respectively, and their LOQ values were found to be 0.07 and 0.1 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of both ROS and EZE were assessed by comparing their respective spectra at peak start, peak apex and peak end positions of the spots. Good correlation among spectra acquired at start(s), apex(m) and end(e) of the peaks indicate the peak purity for both ROS {correlation r(s,m)=0.9999, and 137

179 r(m,e)=0.9998} and EZE {correlation r(s,m)= and r(m,e)=0.9999}. It can be concluded that no impurities or degradation products migrated with the peaks obtained from solutions of drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times, etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing the solutions at ambient temperature and 2-5 o C and tested at intervals of 12, 24, 36 and 48 h etc. The responses for the aged solution were evaluated using a freshly prepared standard solution. The solutions stored under ambient and 2-5 o C were found to be stable for around 1½ and 4 days respectively UV spectroscopy Linearity For the Vierodt s method, absorbances of the solutions were measured at wavelengths, 234 and 244 nm for EZE and ROS respectively. Calibration curves were constructed by plotting absorbance values against concentrations, fig For derivative spectroscopy method, calibration curves were constructed by plotting d 2 A/dλ 2 values against concentrations, fig Good linearity was observed for both ROS and EZE in the concentration range of 5-19 µg/ml. Tables 8.23 and 8.24 summarize the linearity range and linear regression equation for both methods. 138

180 Precision The precision of the method was determined by repeatability (intraday) and intermediate precision (interday precision) and was expressed as the RSD of the results. The values obtained for precision studies are presented in tables 8.23 and 8.24, indicating good repeatability and low inter day variability Accuracy The recovery study results ranged from 97 to 100 for both ROS and EZE showing the accuracy of the method (table 8.25). Low % RSD values indicate that the method is precise Stability Studies The stability of solutions was evaluated by storing the solutions at ambient temperature and 2-5 o C and tested at intervals of 12, 24, 36 and 48 h etc. The responses for the aged solution were evaluated using a freshly prepared standard solution. The solutions stored under ambient and 2-5 o C were found to be stable for around 1½ and 4 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing ROS and EZE. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of drugs in samples are presented in table 8.22 and Statistical Analysis The results of the accuracy experiment were compared statistically by oneway ANOVA followed by Tukey-Kramer multiple comparison test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between the four methods. The results indicated that there was no significant 139

181 difference between the methods compared, tables 8.27 and Hence, the methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters ROS EZE ROS EZE Linearity, 0.5 to to to to 0.9 µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intra day (n=3) Inter day (n=3) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC ROS EZE a Average of six determinations 140

182 Table System suitability studies Method Drug Resolution Theoretical Plate# Tailing Factor Capacity Factor ROS HPLC EZE Table 8.22 Analysis of formulation Drug, labeled amount, mg/tablet Amount found, Amount found, % a R.S.D., % a mg/tablet HPLC HPTLC HPLC HPTLC HPLC HPTLC ROS, EZE, a Average of six determinations Results of UV-Spectroscopic analysis Table Summary of validation parameters for simultaneous equation method Parameters At wavelength, 234 nm At wavelength, 244 nm ROS EZE ROS EZE Linearity, µg/ml Linear regression equation Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) a y=mx+c 141

183 Table Summary of validation parameters for derivative method At wavelength, nm At wavelength, nm Parameters ROS EZE Linearity, µg/ml Linear regression equation Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a Simultaneous Derivative Simultaneous Derivative ROS EZE a Average of six determinations Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, Simultaneous Derivative Simultaneous Derivative Simultaneous Derivative mg/tablet ROS, EZE, a Average of six determinations 142

184 Table 8.27: Statistical analysis of ROS by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between the four methods. H 0 hypothesis is accepted (P>0.05) ns-not significant Table 8.28: Statistical analysis of EZE by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between the four methods H 0 hypothesis is accepted (P>0.05) ns-not significant 143

185 Figure Overlain UV spectra of ROS and EZE HPLC chromatograms of standards Figure ROS-0.5 µg/ml, EZE-0.5 µg/ml

186 Figure ROS-1µg/mL, EZE-1 µg/ml Figure ROS-2 µg/ml, EZE-2 µg/ml Figure ROS-3 µg/ml, EZE-3 µg/ml

187 Figure ROS-4 µg/ml, EZE-4 µg/ml Figure ROS-5 µg/ml, EZE-5 µg/ml Figure ROS-6 µg/ml, EZE-6 µg/ml

188 Figure ROS-7 µg/ml, EZE-7 µg/ml Figure ROS-8 µg/ml, EZE-8 µg/ml Figure ROS-9 µg/ml, EZE-9 µg/ml

189 Figure ROS-10 µg/ml, EZE-10 µg/ml HPLC chromatogram of formulation Figure ROS-3 µg/ml, EZE-3 µg/ml

190 Figure Overlain UV spectra of ROS and EZE on pre-coated TLC plate HPTLC chromatograms of standards Figure ROS- 0.1 µg/band, EZE-0.1µg/band Figure ROS- 0.2 µg/band, EZE-0.2 µg/band

191 Figure ROS- 0.3 µg/band, EZE-0.3 µg/band Figure ROS- 0.4 µg/band, EZE-0.4 µg/band Figure ROS- 0.5 µg/band, EZE-0.5 µg/band Figure ROS- 0.6 µg/band, EZE-0.6 µg/band

192 Figure ROS- 0.7 µg/band, EZE-0.7 µg/band Figure ROS- 0.8 µg/band, EZE-0.8 µg/band HPTLC chromatogram of formulation Figure ROS- 0.9 µg/band, EZE-0.9 µg/band Figure ROS- 0.2 µg/band, EZE-0.2 µg/band

193 UV spectra Figure Zero order absorption overlain spectra of ROS (5-19 µg/ml) Figure Zero order absorption overlain spectra of EZE (5-19 µg/ml)

194 Figure Zero order absorption spectrum of formulation (ROS-9 µg/ml, EZE-9 µg/ml) Figure Second derivative overlain spectra of ROS (5-19 µg/ml)

195 Figure Second derivative overlain spectra of EZE (5-19 µg/ml) Figure Second derivative spectrum of formulation (ROS-9 µg/ml, EZE-9 µg/ml)

196 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure ROS ( µg/ml) Concentration Peak (µg/ml) Area Figure EZE ( µg/ml)

197 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure ROS ( µg/band) Concentration Peak (µg/band) Area Figure EZE ( µg/band)

198 UV calibration graphs Zero order Concentration Absorbance (µg/ml) Figure ROS at 234 nm (5-19 µg/ml) Concentration Absorbance (µg/ml) Figure ROS at 244 nm (5-19 µg/ml)

199 Concentration Absorbance (µg/ml) Figure EZE at 234 nm (5-19 µg/ml) Concentration Absorbance (µg/ml) Figure EZE at 244 nm (5-19 µg/ml)

200 Derivative spectroscopy Concentration II derivative (µg/ml) absorbance Figure ROS at nm (5-19 µg/ml) Concentration II derivative (µg/ml) absorbance Figure EZE at nm (5-19 µg/ml)

201 8.6. Telmisartan-atorvastatin Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the good resolution and peak shape. Several proportions of ammonium acetate buffer- acetonitrile were evaluated in order to achieve optimum separation of drugs in short time of analysis. With the developed mobile phase system symmetrical peaks with good separation (retention times, ATO=4.8 min and TLM=6.6 min) were obtained on a Shim-pack C18 column at a flow rate of 1 ml/min. The fixed wavelength for detection and quantification was 245 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in the pharmaceutical formulation HPTLC Samples were applied to the plates as bands 6 mm wide. The plates were developed in a CAMAG twin trough chamber previously saturated for 30 min with the mobile phase consisting of ethyl acetate-chloroform-glacial acetic acid (8:3:0.5, v/v/v) to a distance of 8.5 cm. The separated spots of the drugs were scanned at 255 nm (fig ). Densitograms obtained from the analysis of drugs using the proposed method are shown in fig UV spectroscopy Two simple, accurate and precise UV methods were developed for the simultaneous determination of ATO and TLM in combined dosage form. First method employs formation and solving of simultaneous equation (Vierodt s method). Second 144

202 method depends on second derivative UV-spectrometry with zero-crossing measurement technique. Methanol was selected as the solvent because it provided the highest solubility and UV absorbance Technique of simultaneous equations (Vierodt s method) Absorbances of the solutions were measured at wavelengths, 247 and 296 nm forato and TLM respectively. Zero order absorption spectra of ATO, TLM and formulation are shown in fig Quantitative determination of these drugs was carried out by solving the simultaneous equations, C X =A 2 a Y1 - A 1 a Y2 /a X2 a Y1 -a X1 a Y2 (1), C Y =A 1 a X2 -A 2 a X1 / a X2 a Y1 -a X1 a Y2 (2), where, A 1 and A 2 are absorbances of the mixture at 247 and 296 nm respectively, a X1 and a X2 are absorptivities of x at 247 and 296 nm respectively, a Y1 and a Y2 are absorptivities of y at 247 and 296 nm respectively, C X is the concentration of ATO and C Y is the concentration of TLM Derivative UV- spectrophotometry Wavelengths, 265 nm (zero-crossing wavelength of TLM) and 275 nm (zerocrossing wavelength of ATO) were chosen for the simultaneous determination of ATO and TLM in a binary mixture respectively. Second derivative spectra of standard drugs in methanol are shown in fig Method Validation HPLC Linearity To assess linearity, standard calibration curves for ATO and TLM were constructed by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of µg/ml for ATO and µg/ml for TLM, fig The regression equations for the drugs were obtained by 145

203 plotting peak areas (y) versus concentrations (x). Table 8.29 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.29 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 97 to 102% for ATO and TLM, table Detection and quantitation limits The LOD values for ATO and TLM were and µg/ml respectively and their LOQ values were found to be 0.05 and 0.07 µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for ATO and TLM were found to be close to 1) Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition, ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations, it was found that there was not much change in the retention times, area or symmetry of the peaks. 146

204 Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 1and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of ATO and TLM and peak areas of the spots was studied. Good linearity was observed in the concentration range of µg/band and µg/band for ATO and TLM, fig The regression equations for the drugs were found by plotting peak areas in AU (y) versus the concentration (x) in µg/band. Table 8.29 summarizes the linearity ranges and linear regression equations for the drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies presented in table 8.29, indicate good repeatability and low interday variability. 147

205 Accuracy The recovery study results ranged from 97 to 100% for ATO and TLM, showing the accuracy of the method (table 8.30). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for ATO and TLM were found to be 0.07 and 0.08 µg/band respectively, and their LOQ values were 0.2 and 0.3 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of ATO and TLM were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for ATO [correlation r(s,m) = and r(m,e) = ] and TLM [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions 148

206 stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 5 days respectively UV spectroscopy Linearity For the Vierodt s method, absorbances of the solutions were measured at wavelengths, 247 and 296 nm for ATO and TLM respectively. Calibration curves were constructed by plotting absorbance values against concentrations, fig For derivative spectroscopy method, calibration curves were constructed by plotting d 2 A/dλ 2 values against concentrations, fig Good linearity was observed for ATO and TLM in the concentration range of 3-11 and µg/ml respectively. Tables 8.33 and 8.34 summarize the linearity range and linear regression equation for both methods Precision The precision of the method was determined by repeatability (intraday) and intermediate precision (interday precision) and was expressed as the RSD of the results. The values obtained for precision studies are presented in tables 4.33 and 4.34, indicating good repeatability and low inter day variability Accuracy The recovery study results ranged from 97 to 101 for both ATO and TLM, showing the accuracy of the method (table 8.35) Stability Studies The stability of solutions was evaluated by storing the solutions at ambient temperature and 2-5 o C and tested at intervals of 12, 24, 36 and 48 h etc. The responses for the aged solution were evaluated using a freshly prepared standard 149

207 solution. The solutions stored under ambient and 2-5 o C were found to be stable for around 1½ and 4 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing ATO and TLM. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of drugs in samples are presented in tables 8.32 and Statistical Analysis The results of the accuracy experiment were compared statistically by oneway ANOVA followed by Tukey-Kramer multiple comparison test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between the four methods. The results indicated that there was no significant difference between the methods compared, tables 8.37 and Hence the methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 150

208 Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters ATO TLM ATO TLM Linearity, µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intraday (n=6) Inter day (n=6) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC ATO TLM a Average of six determinations 151

209 Table System suitability studies Method Drug Resolution Theoretical Plate# Tailing Factor Capacity Factor HPLC ATO TLM Table Analysis of formulation Drug, labeled Amount found, amount, mg/tablet Amount found, % a R.S.D., % a mg/tablet HPLC HPTLC HPLC HPTLC HPLC HPTLC ATO, TLM, a Average of six determinations Results of UV-Spectroscopic analysis Table Summary of validation parameters for simultaneous equation method Parameters At wavelength, 247 nm At wavelength, 296 nm ATO TLM ATO TLM Linearity, µg/ml Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) a y=mx+c 152

210 Table Summary of validation parameters for derivative method At wavelength, 265 nm At wavelength, 274 nm Parameters ATO TLM Linearity, µg/ml Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a Simultaneous Derivative Simultaneous Derivative ATO TLM a Average of six determinations Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, mg/tablet Simultaneous Derivative Simultaneous Derivative Simultaneous Derivative ATO, TLM, a Average of six determinations 153

211 Table Statistical analysis of ATO by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns* HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between four methods; H 0 hypothesis is accepted (P>0.05). *not significant Table Statistical analysis of TLM by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns* HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between four methods; H 0 hypothesis is accepted (P>0.05) *not significant 154

212 Figure Overlain UV spectra of ATO and TLM HPLC chromatograms of standards Figure ATO-0.05 µg/ml, TLM-0.2 µg/ml Figure ATO-1 µg/ml, TLM-4 µg/ml

213 Figure ATO-5 µg/ml, TLM-20 µg/ml Figure ATO-10 µg/ml, TLM-40 µg/ml Figure ATO-15 µg/ml, TLM-60 µg/ml

214 Figure ATO-20 µg/ml, TLM-80 µg/ml HPLC chromatogram of formulation Figure ATO-1 µg/ml, TLM-4 µg/ml

215 Figure Overlain UV spectra of ATO and TLM on pre-coated TLC plate HPTLC chromatograms of standards Figure ATO- 0.2 µg/band, TLM-0.8 µg/band Figure ATO- 0.3 µg/band, TLM-1.2 µg/band

216 Figure ATO- 0.4 µg/band, TLM-1.6 µg/band Figure ATO- 0.5 µg/band, TLM-2 µg/band Figure ATO-0.6 µg/band, TLM-2.4 µg/band Figure ATO- 0.7 µg/band, TLM-2.8 µg/band HPTLC chromatogram of formulation Figure ATO-0.8 µg/band, TLM-3.2 µg/band Figure ATO-0.3 µg/band, TLM-1.2 µg/band

217 UV spectra Figure Zero order absorption overlain spectra of ATO (3-11µg/mL) Figure Zero order absorption overlain spectra of TLM (12-44 µg/ml)

218 Figure Zero order absorption spectrum of formulation (ATO-5 µg/ml, TLM-20 µg/ml) Figure Second derivative overlain spectra of ATO (3-11 µg/ml)

219 Figure Second derivative overlain spectra of TLM (12-44 µg/ml) Figure Second derivative spectrum of formulation (ATO-5 µg/ml, TLM-20 µg/ml)

220 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of ATO ( µg/ml) Concentration Peak (µg/ml) Area Figure Calibration graph of TLM ( µg/ml)

221 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of ATO ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of TLM ( µg/band)

222 UV calibration graphs Zero-order Concentration Abs. (µg/ml) Figure ATO at 247 nm (3-11 µg/ml) Concentration Abs. (µg/ml) Figure ATO at 296 nm (3-11 µg/ml)

223 Concentration Abs. (µg/ml) Figure TLM at 247 nm (12-44 µg/ml) Concentration Abs. (µg/ml) Figure TLM at 296 nm (12-44 µg/ml)

224 Derivative spectroscopy Concentration II derivative (µg/ml) absorbance Figure ATO at 265 nm (3-11 µg/ml) Concentration II derivative (µg/ml) absorbance Figure TLM at 274 nm (12-44 µg/ml)

225 8.7. Rosuvastatin-fenofibrate Method Development and Optimization HPLC The chromatographic conditions were optimized to achieve the good resolution and peak shape. Several proportions of formic acid and acetonitrile were evaluated in order to achieve optimum separation of analytes in minimum analysis time. With the developed mobile phase system, symmetrical peaks with good separation (retention times, ROS=1.9 min and FEN=5.4 min) were obtained on a Phenomenex Luna C18 (4.60 mm 150 mm, 5 µ particle size) column at a flow rate of 1 ml/min. The fixed wavelength for detection and quantification was 240 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in the pharmaceutical formulation HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of ROS and FEN by HPTLC. Good separation of the drugs (R f values of ROS = 0.30 and FEN = 0.80) with good symmetrical peaks was obtained by using the mobile phase, chloroform methanol-toulene (4:3:6, v/v/v). The separated spots of the drugs were scanned at 240 nm (fig ). Densitograms obtained from the analysis of drugs are shown in fig

226 Method Validation HPLC Linearity To assess linearity, standard calibration curves for ROS and FEN were constructed by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of µg/ml for ROS and µg/ml for FEN, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations (x). Table 8.39 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.39 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for ROS and FEN, showing the accuracy of the method (Table 8.40) Detection and quantitation limits The LOD values for ROS and FEN were and µg/ml respectively, and their LOQ values were found to be 0.1 and 0.01µg/mL respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for ROS and FEN were found to be 1). 156

227 Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition, ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 1and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of ROS and FEN and peak areas of the spots was investigated. Good linearity was observed for in the concentration range of µg/band for ROS and µg/band for FEN, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.39 summarizes the linearity ranges and linear regression equations for the drugs. 157

228 Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.39, indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for ROS and FEN, showing the accuracy of the method (table 8.40). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for ROS and FEN were found to be 0.03 and 0.04 µg/band respectively, and their LOQ values were 0.05 and 0.06 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of ROS and FEN were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for ROS [correlation r(s,m) = and r(m,e) = ] and FEN [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks. 158

229 Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 4 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing ROS and FEN. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of drugs in samples are presented in table Statistical Analysis The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table This result shows that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 159

230 Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters ROS FEN ROS FEN Linearity, µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intraday (n=6) Inter day (n=6) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC ROS FEN a Average of six determinations 160

231 Table System suitability studies Method Drug Resolution Theoretical Plate# Tailing Factor Capacity Factor ROS HPLC FEN Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, HPLC HPTLC HPLC HPTLC HPLC HPTLC mg/tablet ROS, FEN, a Average of six determinations Table Statistical comparison of the results obtained by the proposed methods Methods ROS FEN HPLC vs HPTLC t c = P value= * t c = P value=0.6585* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 161

232 Figure Overlain UV spectra of ROS and FEN HPLC chromatograms of standards Figure ROS-0.1 µg/ml, FEN-1.6 µg/ml

233 Figure ROS-0.5 µg/ml, FEN-8 µg/ml Figure ROS-1 µg/ml, FEN-16 µg/ml Figure ROS-2 µg/ml, FEN-32 µg/ml

234 Figure ROS-4 µg/ml, FEN-64 µg/ml Figure ROS-6 µg/ml, FEN-96 µg/ml Figure ROS-8 µg/ml, FEN-128 µg/ml

235 Figure ROS-10 µg/ml, FEN-160 µg/ml HPLC chromatogram of formulation Figure ROS-4 µg/ml, FEN-64 µg/ml

236 Figure Overlain UV spectra of ROS and FEN on pre-coated TLC plate HPTLC chromatograms of standards Figure ROS µg/band, FEN-0.8 µg/band Figure ROS- 0.1 µg/band, FEN-1.6 µg/band

237 Figure ROS µg/band, FEN-2.4 µg/band Figure ROS- 0.2 µg/band, FEN-3.2 µg/band Figure ROS µg/band, FEN-4 µg/band Figure ROS- 0.3 µg/band, FEN-4.8 µg/band HPTLC chromatogram of formulation Figure ROS µg/band, FEN-2.4 µg/band

238 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of ROS ( µg/ml) Concentration Peak (µg/ml) Area Figure Calibration graph of FEN ( µg/ml)

239 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of ROS ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of FEN ( µg/band)

240 8.8. Olmesartan medoxomil-amlodipine Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the good resolution and peak shape. Several proportions of formic acid, ph adjusted to 3.5 and methanol were evaluated in order to achieve optimum separation of analytes in minimum analysis time. With the developed mobile phase system symmetrical peaks with good separation (retention times, OLM=7.1 min, AML=10.5 min) were obtained on a Lichrospher C18 column at a flow rate of 1 ml/min. The fixed wavelength for detection and quantification was 238 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in the pharmaceutical formulation HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of AML and OLM by HPTLC. Good separation of the drugs (R f values of AML = 0.47, OLM = 0.71) with symmetrical peaks was obtained by using the mobile phase, ethyl acetate chloroform-glacial acetic acid (3:5:4, v/v/v). The separated spots of the drugs were scanned at 238 nm (fig ). Densitograms obtained from the analysis of drugs are shown in fig

241 UV spectroscopy Two simple accurate and precise UV methods were developed for the simultaneous determination of AML and OLM in combined dosage form. First method employs formation and solving of simultaneous equation (Vierodt s method). Second method depends on second derivative UV-spectrometry with zero-crossing measurement technique. Methanol was selected as the solvent because it provided the highest solubility and UV absorbance Technique of simultaneous equations (Vierodt s method) Absorbances of the solutions were measured at wavelengths, 238 and 256 nm for AML and OLM respectively. Zero order absorption spectra of AML, OLM and formulation are shown in fig Quantitative determination of these drugs was carried out by solving the simultaneous equations, C X =A 2 a Y1 - A 1 a Y2 /a X2 a Y1 -a X1 a Y2 (1), C Y =A 1 a X2 -A 2 a X1 / a X2 a Y1 -a X1 a Y2 (2), where, A 1 and A 2 are absorbances of the mixture at 238 and 256 nm, respectively, a X1 and a X2 are absorptivities of x at 238 and 256 nm, respectively, a Y1 and a Y2 are absorptivities of y at 238 and 256 nm respectively, C X is the concentration of AML and C Y is the concentration of OLM Derivative UV- spectrophotometry Wavelengths, 233 nm (zero-crossing wavelength of OLM) and nm (zero-crossing wavelength of AML) were chosen for the simultaneous determination of AML and OLM in binary mixture respectively. Second derivative spectra of standard drugs in methanol are shown in fig

242 Method Validation HPLC Linearity To assess linearity, standard calibration curves for AML and OLM were constructed by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of µg/ml for AML and 2-20 µg/ml for OLM, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations (x). Table 8.44 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.44 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for AML and OLM, showing the accuracy of the method (table 8.45) Detection and quantitation limits The LOD values for AML and OLM were 0.1 and µg/ml respectively, and their LOQ values were found to be 0.5 and 0.05µg/mL respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for AML and OLM were found to be close to 1). 164

243 Robustness The robustness of the proposed method was evaluated by making slight modifications in the organic composition, ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations, it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 1and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of AML and OLM and peak areas of the spots were investigated. Good linearity was observed for in the concentration range of µg/band for AML and µg/band for OLM, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentrations (x). Table 8.44 summarizes the linearity ranges and linear regression equations for the drugs. 165

244 Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.44, indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for AML and OLM, showing the accuracy of the method (table 8.45). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for AML and OLM were 0.09 and 0.06 µg/band respectively, and their LOQ values were found to be 0.2 and 0.09 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of AML and OLM were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for AML [correlation r(s,m) = and r(m,e) = ] and OLM [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks. 166

245 Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 4 days respectively UV spectroscopy Linearity For the Vierodt s method, absorbances of the solutions were measured at wavelengths, 238 and 256 nm for AML and OLM respectively. Calibration curves were constructed by plotting absorbance values against concentrations, fig For derivative spectroscopy method, calibration curves were constructed by plotting d 2 A/dλ 2 values against concentrations, fig Linearity was observed for AML and OLM in the concentration range of 7-14 and µg/ml respectively. Tables 8.48 and 8.49 summarize the linearity range and linear regression equation for both methods Precision The precision of the method was determined by repeatability (intraday) and intermediate precision (interday precision) and was expressed as the RSD of the results. The values obtained for precision studies are presented in tables 8.48 and 8.49, indicating good repeatability and low inter day variability Accuracy The recovery study results ranged from 97 to 100 for both AML and OLM, showing the accuracy of the method (table 8.50). 167

246 Stability Studies The stability of solutions was evaluated by storing the solutions at ambient temperature and 2-5 o C and tested at intervals of 12, 24, 36 and 48 h etc. The responses for the aged solution were evaluated using a freshly prepared standard solution. The solutions stored under ambient and 2-5 o C were found to be stable for around 1½ and 4 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing AML and OLM. Six replicate determinations were performed on accurately weighed tablets. Results of the formulation analysis are presented in tables 8.47 and Statistical Analysis The results of the accuracy experiment were compared statistically by oneway ANOVA followed by Tukey-Kramer multiple comparison test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between the four methods. The results indicated that there was no significant difference between the methods compared, tables 8.52 and Hence, the methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 168

247 Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters AML OLM AML OLM Linearity, µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intraday (n=6) Inter day (n=6) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC AML OLM a Average of six determinations 169

248 Table System suitability studies Method Drug Resolution Theoretical Plate # Tailing Factor Capacity Factor OLM HPLC AML Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, HPLC HPTLC HPLC HPTLC HPLC HPTLC mg/tablet AML, OLM, a Average of six determinations Results of UV-Spectroscopic analysis Table Summary of validation parameters for simultaneous equation method Parameters At wavelength, 238 nm At wavelength, 256 nm AML OLM AML OLM Linearity, µg/ml Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) a y=mx+c 170

249 Table Summary of validation parameters for derivative method At wavelength, nm At wavelength, 233 nm Parameters AML OLM Linearity, µg/ml Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) Precision (% R.S.D) Intra day (n=6) Inter day (n=6) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a Simultaneous Derivative Simultaneous Derivative AML OLM a Average of six determinations Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, mg/tablet Simultaneous Derivative Simultaneous Derivative Simultaneous Derivative AML, OLM, a Average of six determinations 171

250 Table 8.52: Statistical analysis of AML by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between four methods. H 0 hypothesis is accepted (P>0.05) ns-not significant Table 8.53: Statistical analysis of OLM by one-way ANOVA (Tukey Kramer multiple comparison test) Comparison q P value HPLC vs HPTLC P>0.05 ns HPLC vs UV (simultaneous) P>0.05 ns HPLC vs UV (derivative) P>0.05 ns HPTLC vs UV (simultaneous) P>0.05 ns HPTLC vs UV (derivative) P>0.05 ns UV (simultaneous) vs UV (derivative) P>0.05 ns H 0 hypothesis: no statistical significant difference exists between four methods. H 0 hypothesis is accepted (P>0.05). ns-not significant 172

251 Figure Overlain UV spectra of AML and OLM HPLC chromatograms of standards Figure AML-0.5 µg/ml, OLM-2 µg/ml

252 Figure AML-1 µg/ml, OLM-4 µg/ml Figure AML-2 µg/ml, OLM-8 µg/ml Figure AML-3 µg/ml, OLM-12 µg/ml

253 Figure AML-5 µg/ml, OLM-20 µg/ml HPLC chromatogram of formulation Figure AML-3 µg/ml, OLM-12 µg/ml

254 Figure Overlain UV spectra of AML and OLM on pre-coated TLC plate HPTLC chromatograms of standards Figure AML- 0.3 µg/band, OLM-1.2 µg/band Figure AML- 0.4 µg/band, OLM-1.6 µg/band

255 Figure AML- 0.6 µg/band, OLM-2.4 µg/band Figure AML- 0.8 µg/band, OLM-3.2 µg/band Figure AML- 1 µg/band, OLM-4 µg/band Figure AML- 1.2 µg/band, OLM-4.8µg/band HPTLC chromatogram of formulation Figure AML- 0.6 µg/band, OLM-2.4 µg/band

256 Figure Zero order absorption overlain spectra of AML (7-14 µg/ml) Figure Zero order absorption overlain spectra of OLM (28-56 µg/ml)

257 Figure Zero order absorption spectrum of formulation (AML-5 µg/ml, OLM-20 µg/ml) Figure Second derivative overlain spectra of AML (7-14 µg/ml)

258 Figure Second derivative overlain spectra of OLM (28-56 µg/ml) Figure Second derivative spectrum of formulation (AML-5 µg/ml, OLM-20 µg/ml)

259 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of AML (0.5-5 µg/ml) Concentration Peak (µg/m Area Figure Calibration graph of OLM (2-20 µg/ml)

260 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of AML ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of OLM ( µg/band)

261 UV calibration graphs Zero-order Concentration Abs. (µg/ml) Figure AML at 238 nm (7-14 µg/ml) Concentration Abs. (µg/ml) Figure AML at 256 nm (7-14 µg/ml)

262 Concentration Abs. (µg/ml) Figure OLM at 238 nm (28-56 µg/ml) Concentration Abs. (µg/ml) Figure OLM at 256 nm (28-56 µg/ml)

263 Derivative spectroscopy Concentration II derivative (µg/ml) absorbance Figure AML at nm (7-14 µg/ml) Concentration II derivative (µg/ml) absorbance Figure OLM at 233 nm (28-56 µg/ml)

264 8.9. Telmisartan-amlodipine Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the good resolution and peak shape. Several proportions of formic acid (ph adjusted to 4 with triethylamine) and methanol were evaluated in order to achieve optimum separation of analytes in minimum analysis time. With the developed mobile phase system symmetrical peaks with good separation (retention times, AML=2.0 min, TLM=6.7 min) were obtained at a flow rate of 1 ml/min. The fixed wavelength for detection and quantification was 238 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in the pharmaceutical formulation HPTLC Experimental conditions, such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of AML and TLM. Good separation of the drugs (R f values of TLM = 0.28, AML = 0.48) with symmetrical peaks was obtained by using the mobile phase, ethyl acetate-chloroform-glacial acetic acid, 3:5:4, (v/v/v). The separated spots of the three drugs were scanned at 254 nm (fig ). Densitograms obtained from the analysis of drugs using the proposed method are shown in fig

265 Method Validation HPLC Linearity To assess linearity, standard calibration curves for AML and TLM were constructed by plotting concentrations versus peak areas. The curves showed linearity over the concentration range of µg/ml for AML and µg/ml for TLM, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations in (x). Table 8.54 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.54 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for AML and TLM, showing the accuracy of the method (Table 8.55) Detection and quantitation limits The LOD values for AML and TLM were 0.01 and µg/ml respectively and their LOQ values were found to be 0.1 and 0.08 µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for AML and TLM were found to be close to 1). 174

266 Robustness The robustness of the proposed method was evaluated by modifications in the organic composition and ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations, it was found that there was not much change in the retention times, area or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 1and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor, capacity factor and RSD of the peak areas were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of AML and TLM and peak areas of the spots was investigated. Linearity was observed in the concentration range of µg/band for AML and µg/band for TLM, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.54 summarizes the linearity ranges and linear regression equations for the drugs. 175

267 Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies, presented in table 8.54, indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 100% for AML and TLM, showing the accuracy of the method (Table 8.55). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for AML and TLM were 0.09 and 0.2 µg/band respectively and their LOQ values were found to be 0.3 and 0.45 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of AML and TLM were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for AML [correlation r(s,m) = and r(m,e) = ] and TLM [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks. 176

268 Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C were found to be stable for around 2 and 3 days, respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table Application of the method The proposed methods were used for the assay of commercially available tablets containing AML and TLM. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of drugs in samples are presented in table Statistical Analysis The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table This result shows that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 177

269 Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters AML TLM AML TLM Linearity, µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intraday (n=6) Inter day (n=6) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC AML TLM a Average of six determinations 178

270 Table System suitability studies Method Drug Resolution Theoretical Plate# Tailing Factor Capacity Factor HPLC AML TLM Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, HPLC HPTLC HPLC HPTLC HPLC HPTLC mg/tablet AML, TLM, a Average of six determinations Table Statistical comparison of the results obtained by the proposed methods Methods AML TLM HPLC vs HPTLC t c = P value= * t c = P value=0.6737* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 179

271 Figure Overlain UV spectra of AML and TLM HPLC chromatograms of standards Figure AML-0.1 µg/ml, TLM-0.8 µg/ml Figure AML-0.5 µg/ml, TLM-4 µg/ml

272 Figure AML-1 µg/ml, TLM-8 µg/ml Figure AML-2µg/mL, TLM-16µg/mL Figure AML-3 µg/ml, TLM-24 µg/ml

273 Figure AML-4 µg/ml, TLM-32 µg/ml Figure AML-5 µg/ml, TLM-40 µg/ml Figure AML-6 µg/ml, TLM-48 µg/ml

274 Figure AML-7 µg/ml, TLM-56 µg/ml Figure AML-8 µg/ml, TLM-64 µg/ml HPLC chromatogram of formulation Figure AML-5 µg/ml, TLM-40 µg/ml

275 Figure Overlain UV spectra of AML and TLM on pre-coated TLC plate HPTLC chromatograms of standards Figure AML- 0.2 µg/band, TLM-1.6 µg/band Figure AML- 0.3 µg/band, TLM-2.4 µg/band

276 Figure AML-0.4 µg/band, TLM-3.2 µg/band Figure AML-0.5 µg/band, TLM-4 µg/band HPTLC chromatogram of formulation Figure AML- 0.6 µg/band, TLM-4.8 µg/band Figure AML- 0.3 µg/band, TLM-2.4 µg/band

277 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of AML (0.1-8 µg/ml) Concentration Peak (µg/ml) Area Figure Calibration graph of TLM ( µg/ml)

278 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of AML ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of TLM ( µg/band)

279 8.10. Nebivolol-amlodipine Method Development and Optimization HPLC For the RP-HPLC method, chromatographic conditions were optimized to achieve the good resolution and peak shape. Several proportions of formic acid and methanol were evaluated in order to achieve optimum separation of analytes in minimum analysis time. With the developed mobile phase system, symmetrical peaks with good separation (retention times, s-aml =6.6 min, NEB=8.6 min) were obtained at a flow rate of 1 ml/min. The fixed wavelength for detection and quantification was 263 nm at which good detector response for the drugs was obtained, fig The chromatograms of standards and formulation are shown in fig There was no interference from the diluents or excipients present in the pharmaceutical formulation HPTLC Experimental conditions such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible results for simultaneous determination of s-aml and NEB. Good separation of the drugs (R f values of s-aml = 0.28, NEB = 0.50) with good symmetrical peaks was obtained by using the mobile phase, ethyl acetate-chloroform-glacial acetic acid, 2:6:2, (v/v/v). The separated spots of the three drugs were scanned at 268 nm (fig ). Densitograms obtained from the analysis of drugs using the proposed method are shown in fig

280 Method Validation HPLC Linearity To assess linearity, standard calibration curves for AML and NEB were constructed by plotting concentrations versus peak areas. The curves showed good linearity over the concentration range of 2-10 µg/ml for s-aml and 4-20 µg/ml for NEB, fig The regression equations for the drugs were obtained by plotting peak areas (y) versus concentrations (x). Table 8.59 summarizes the linearity range and linear regression equation for both drugs Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and is expressed as the RSD of the results. The values obtained for precision studies are presented in table 8.59 and indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 101% for s-aml and NEB, showing the accuracy of the method (Table 8.60) Detection and quantitation limits The LOD values for s-aml and NEB were 0.02 and 0.2 µg/ml respectively, and their LOQ values were found to be and 0.4 µg/ml respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The chromatographic peak of each drug was not attributable to more than one component (diode array detector peak purity index values for s-aml and NEB were found to be 1). 181

281 Robustness The robustness of the proposed method was evaluated by modifications in the organic composition, ph value of the aqueous phase of the mobile phase and the flow rate. During these investigations, it was found that there was not much change in the retention times, area, or symmetry of the peaks Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C, and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C were found to be stable for around 1and 4 days respectively System Suitability Studies A system suitability test of the chromatographic system was performed before each validation run. Five replicate injections of standard preparation were made and resolution, theoretical plate number, tailing factor and capacity factor were determined. The results of the system suitability studies are shown in table HPTLC Linearity The relationship between the concentration of s-aml and NEB and peak areas of the spots was investigated. Good linearity was observed for in the concentration range of µg/band for s-aml and µg/band for NEB, fig The regression equations for the drugs were found by plotting peak areas (y) versus the concentration (x). Table 8.59 summarizes the linearity ranges and linear regression equations for the drugs. 182

282 Precision The precision of the method was determined by repeatability (intraday) and intermediate (interday) precision and was expressed as the RSD of the results. The values obtained for the precision studies presented in table 8.59 indicate good repeatability and low interday variability Accuracy The recovery study results ranged from 98 to 101% for s-aml and NEB, showing the accuracy of the method (Table 8.60). Low RSD values indicate that the method is also precise Detection and quantitation limits The LOD values for s-aml and NEB were 0.12 and 0.2 µg/band respectively, and their LOQ values were found to be 0.2 and 0.3 µg/band respectively Specificity The specificity of the developed method was evaluated by studying the peak purity index values. The peak purities of s-aml and NEB were assessed by comparing their respective in situ spectra at peak start (s), peak apex (m) and peak end (e) positions of the spots. Good correlation among the spectra indicate the peak purity for s-aml [correlation r(s,m) = and r(m,e) = ] and NEB [correlation r(s,m) = and r(m,e) = ]. Hence, it could be concluded that no impurities or degradation products migrated with the spots obtained from solutions of the drugs Robustness Robustness studies were done by making slight alterations in the detection wavelength, mobile phase composition and influence of different conditions (different chamber saturation/plate equilibration times etc.). It was found that there was not much change in the R f values, area or symmetry of peaks. 183

283 Stability Studies The stability of solutions was evaluated by storing them at ambient temperature and 2 5 o C and testing at regular intervals. The responses for the aged solutions were evaluated using a freshly prepared standard solution. The solutions stored at ambient temperature and 2 5 o C was found to be stable for around 2 and 4 days respectively Application of the method The proposed methods were used for the assay of commercially available tablets containing s-aml and NEB. Six replicate determinations were performed on accurately weighed tablets. Experimental values obtained for determination of drugs in samples are presented in table Statistical Analysis The results of the accuracy experiment were compared statistically by Student s t-test and P>0.05 was considered to test the hypothesis that no statistical significant difference exists between two methods. The values of t-test obtained at 95% confidence level did not exceed the theoretical table value, indicating no significant difference between the methods compared, table This result shows that the two methods are equivalent for the quantitative simultaneous determination of the drugs in formulations. 184

284 Table Summary of validation parameters for proposed methods HPLC method HPTLC method Parameters s-aml NEB s-aml NEB Linearity, µg/ml or µg/band Linear regression equation a Intercept (c) Slope(m) Correlation coefficient (r) LOD, µg/ml or µg/band LOQ, µg/ml or µg/band Precision (% R.S.D) Intraday (n=6) Inter day (n=6) Repeatability of injection (n=10) a y=mx+c Table Accuracy data (analyte recovery) Drug Level,% Recovery, (%) a R.S.D., (%) a HPLC HPTLC HPLC HPTLC s-aml NEB a Average of six determinations 185

285 Table System suitability studies Method Drug Resolution Theoretical Plates # Tailing Factor Capacity Factor HPLC s-aml NEB Table Analysis of formulation Drug, Amount found, labeled mg/tablet Amount found, % a R.S.D., % a amount, HPLC HPTLC HPLC HPTLC HPLC HPTLC mg/tablet s-aml, NEB, a Average of six determinations Table Statistical comparison of the results obtained by the proposed methods Methods s-aml NEB HPLC vs HPTLC t c = P value= * t c = P value=0.3989* t c = calculated t values; t t = table t values (t t = for n=6). H 0 hypothesis: no statistical significant difference exists between two methods. t c < t t : H 0 hypothesis is accepted (P>0.05) *The two-tailed P value was considered not significant 186

286 Figure Overlain UV spectra of s-aml and NEB HPLC chromatograms of standards Figure s-aml-2 µg/ml, NEB-4 µg/ml

287 Figure s-aml-3 µg/ml, NEB-6 µg/ml Figure s-aml-4 µg/ml, NEB-8 µg/ml Figure s-aml-5 µg/ml, NEB-10 µg/ml

288 Figure s-aml-6 µg/ml, NEB-12 µg/ml Figure s-aml-7 µg/ml, NEB-14 µg/ml Figure s-aml-8 µg/ml, NEB-16 µg/ml

289 Figure s-aml-9 µg/ml, NEB-18 µg/ml Figure s-aml-10 µg/ml, NEB-20 µg/ml HPLC chromatogram of formulation Figure s-aml- 4 µg/ml, NEB-8 µg/ml

290 Figure Overlain UV spectra of s-aml and NEB on pre-coated TLC plate HPTLC chromatograms of standards Figure s-aml- 0.3 µg/band, NEB-0.6 µg/band Figure s-aml- 0.4 µg/band, NEB-0.8 µg/band

291 Figure s-aml- 0.6 µg/band, NEB-1.2 µg/band Figure s-aml- 0.8 µg/band, NEB-1.6 µg/band Figure s-aml- 1 µg/band, NEB-2 µg/band Figure s-aml- 1.2 µg/band, NEB-2.4 µg/band HPTLC chromatogram of formulation Figure s-aml- 0.6 µg/band, NEB-1.2 µg/band

292 HPLC calibration graphs Concentration Peak (µg/ml) Area Figure Calibration graph of s-aml (2-10 µg/ml) Figure Calibration graph of NEB (4-20 µg/ml) Concentration Peak (µg/ml) Area

293 HPTLC calibration graphs Concentration Peak (µg/band) Area Figure Calibration graph of s-aml ( µg/band) Concentration Peak (µg/band) Area Figure Calibration graph of NEB ( µg/band)

294 Part 2- Bioanalytical method development Rosuvastatin-ezetimibe Chromatography The chromatographic conditions, especially the composition of the mobile phase and strength of buffer were optimized to achieve good resolution and symmetric peak shapes for the analytes and IS, as well as short run time. It was found that a mobile phase system consisting of 0.1 % (v/v) formic acid-methanol (20:80, v/v) gave good results and hence was selected. Formic acid was added to improve the peak characteristics. The percentage of formic acid and proportion of organic phase was optimized to maintain this peak shape and also to give the best ionization in mass spectrometer. The retention times of ROS, EZE and ATO under these conditions were found to be 2.7, 3.4 and 3.8 min respectively Mass spectrometry LC-MS method employing ESI in the negative mode was used for simultaneous determination of ROS and EZE since it provides better limit of detection for trace analysis. In order to determine the concentration of the analytes in plasma with high sensitivity, ESI interface parameters as given in table 7.22 were optimized and fixed. Electrospray in positive mode was less sensitive for EZE than the negative mode. Negative ESI mass spectra of ROS, EZE and ATO (IS) are shown in fig The [M-H] - ions at m/z 408, 480 and 557 were selected as detection ions for EZE, ROS and ATO respectively. The chromatograms are given in fig

295 Selection of internal standard It is necessary to use an IS to get good accuracy in bioanalytical studies. Accordingly, drugs like simvastatin, telmisartan, olmesartan, atorvastatin etc. were tried to find out the most suitable one. ATO was selected because of its similarity in retention and ionization with the analytes and less interference at [M-H] - ions m/z 408 and Sample preparation LLE was used as the sample preparation technique. LLE is helpful in producing a spectroscopically clean sample and avoiding the introduction of nonvolatile materials into the column and MS system. Different organic solvents like n-hexane, diethyl ether, dichloromethane, chloroform, tert-butyl methyl ether, ethyl acetate and their mixtures in different combinations and ratios were evaluated. From these trials, a mixture of diethyl ether-dichloromethane (70:30, v/v) was selected because it gave high extraction efficiency Method validation Selectivity and matrix effect The selectivity of the method was studied by analyzing blank human plasma from different sources and blank plasma spiked only with internal standard. It was found that there was no significant interference in the blank plasma from endogenous substances at the retention times of the analytes (Fig ). It was also found that there was no interference from the internal standard. The matrix effects i.e. the possibility of ionization suppression or enhancement for the analytes were examined. The coefficient of variation (CV, %) of mean peak areas of analytes at any given concentration were small (< 10%), indicating little or no difference in ionization efficiency and consistent recovery of analytes from plasma. 188

296 Linearity The calibration curves were constructed for both ROS and EZE using six nonzero standards ranging from ng/ml. A blank sample (matrix sample processed without IS) and a zero sample (matrix sample processed with IS) were also prepared and analyzed. The correlation coefficient values for ROS and EZE were found to be The calibration curve of ROS had the regression equation of y= x , where y was the peak area ratio of ROS to IS, and x was the concentration of ROS, fig The calibration curve of EZE had the regression equation of y= x , where y was the peak area ratio of EZE to IS, and x was the concentration of EZE, fig Table 8.64 summarizes the calibration curve results. For both analytes, lower limit of quantification (LLOQ) was found to be 0.1 ng/ml Precision and accuracy Precision and accuracy were determined by replicate analyses of QC of three concentrations of plasma samples. The accuracy and precision data for the LC-MS analysis of blank human plasma spiked with ROS and EZE are presented in tables 8.65 and Recovery The extraction efficiency was determined by analyzing the quality control samples. The average absolute recoveries for ROS and EZE at three QC levels are shown in table The recoveries of the analytes were high and also the extent of recovery was consistent, precise and reproducible. 189

297 Stability studies Table 8.68 summarizes the freeze and thaw stability, short-term stability, longterm stability and post-preparative stability data of the analytes. All the results showed that there was no stability related issues during the analyses. The stability of working solutions was tested at room temperature for 6 h and it was found that these solutions were stable for 6 h. Table Regression parameters Parameter ROS EZE Correlation coefficient Slope Intercept Table Intra-day precision and accuracy Analyte Nominal concentration, ng/ml Mean concentration found, ng/ml Intra-day precision, RSD (%) a Accuracy b (%) ROS EZE a n=5, b Accuracy is given by the % deviation of mean concentration from nominal concentration 190

298 Table Inter-day precision and accuracy Analyte Nominal concentration, ng/ml Mean concentration found, ng/ml Inter-day precision, RSD (%) a Accuracy b (%) ROS EZE a n=5, b Accuracy is given by the % deviation of mean concentration from nominal concentration Table Absolute recoveries of ROS and EZE from human plasma Analyte Concentration, ng/ml Recovery (%); %RSD, n=5) ROS (4.6639) (5.222) (4.4742) EZE (4.009) (4.7736) (3.7640) 191

299 Table Stability data for ROS and EZE in plasma Conditions Recovery a (%) ROS EZE Three freeze-thaw cycles Short-term stability for 24 h in plasma in room temperature Auto sampler stability for 24 h Long-term stability for one week Stock solution stability for 6h a Recovery is expressed as the response of ROS and EZE stored in plasma following stability conditions compared to the response of the analytes freshly spiked in plasma 192

300 Figure ESI-MS negative ion full-scan spectrum of ROS Figure ESI-MS negative ion full-scan spectrum of EZE Figure ESI-MS negative ion full-scan spectrum of ATO

301 Figure Chromatograms for blank plasma (drug and IS free) at m/z (A) 408, (B) 480 and (C) 557

302 Figure Chromatograms at LLOQ level for EZE-0.1 ng/ml, ROS-0.1 ng/ml and ATO-20 ng/ml

303 Figure Chromatograms for EZE-0.25 ng/ml, ROS-0.25 ng/ml and ATO-20 ng/ml

304 Figure Chromatograms for EZE-2 ng/ml, ROS-2 ng/ml and ATO-20 ng/ml

305 Figure Chromatograms for EZE-4 ng/ml, ROS-4 ng/ml and ATO-20 ng/ml

306 Figure Chromatograms for EZE-8 ng/ml, ROS-8 ng/ml and ATO-20 ng/ml

307 Figure Chromatograms for EZE-10 ng/ml, ROS-10 ng/ml and ATO- 20 ng/ml

308 Calibration graphs Concentration Peak (ng/ml) Area Ratio Figure Calibration graph of ROS ( ng/ml) Concentration Peak (ng/ml) Area Ratio Figure Calibration graph of EZE ( ng/ml)

309 8.12. Atorvastatin-telmisartan Chromatography The chromatographic conditions like the composition of the mobile phase and strength of buffer were optimized to achieve good resolution and symmetric peak shapes for the analytes and IS, as well as short run time. It was found that a mobile phase system consisting of 10 mm ammonium acetate, adjusted to ph 4 after mixing with formic acid-methanol (20:80, v/v) gave good results and hence was selected. The substitution of 10 mm ammonium acetate for water in the mobile phase reduced the matrix effects. The retention times of ROS, ATO and TLM under these conditions were found to be 2.6, 3.7 and 4.6 min respectively Mass spectrometry LC-MS method employing ESI in the positive mode was used for simultaneous determination of ATO and TLM, since it provides better limit of detection for trace analysis. In order to determine the concentration of the analytes in plasma with high sensitivity, ESI interface parameters as given in table 7.23 were optimized and fixed. Positive ESI mass spectra of ATO, TLM and ROS are shown in fig The [M+H] + ions at m/z 482, 515 and 559 were selected as detection ions for ROS, TLM and ATO respectively. The chromatograms are shown in fig Selection of internal standard It is necessary to use an IS to get good accuracy in bioanalytical studies. Accordingly, drugs like simvastatin, rosuvastatin, olmesartan, etc. were tried to find out the most suitable one. Rosuvastatin was selected because of its similarity in 193

310 retention and ionization with the analytes and less interference at [M+H] + ions m/z 515 and Sample preparation LLE was used as the sample preparation technique in the present work. LLE is helpful in producing a spectroscopically clean sample and avoiding the introduction of non-volatile materials into the column and MS system. Different organic solvents like ethyl acetate, n-hexane, diethyl ether, dichloromethane, chloroform, tert-butyl methyl ether and their mixtures in different combinations and ratios were evaluated. From these trials, a mixture of ethyl acetate-dichloromethane (80:20, v/v) was selected because it gave high extraction efficiency Method validation Selectivity and matrix effect The selectivity of the method was studied by analyzing blank human plasma from different sources and blank plasma spiked only with IS. It was found that there was no significant interference in the blank plasma from endogenous substances at the retention times of the analytes (Fig.8.262). It was also found that there was no interference from the internal standard. The matrix effects i.e. the possibility of ionization suppression or enhancement for the analytes were examined. The coefficient of variation (CV, %) of mean peak areas of analytes at any given concentration were small (< 10%), indicating little or no difference in ionization efficiency and consistent recovery of analytes from plasma Linearity The calibration curves were constructed for both ATO and TLM using six non-zero standards ranging from 1-35 ng/ml. A blank sample (matrix sample 194

311 processed without IS) and a zero sample (matrix sample processed with IS) were also prepared and analyzed. The correlation coefficient values for ATO and TLM were found to be and respectively. The calibration curve of ATO had the regression equation of y=0.0173x , where y was the peak area ratio of ATO to IS, and x was the concentration of ATO, fig The calibration curve of TLM had the regression equation of y=0.1232x , where y was the peak area ratio of TLM to IS, and x was the concentration of TLM, fig Table 8.69 summarizes the calibration curve results. For both analytes, lower limit of quantification (LLOQ) was found to be 1 ng/ml Precision and accuracy Precision and accuracy were determined by replicate analyses of QC of three concentrations of plasma samples. The accuracy and precision data for the LC-MS analysis of blank human plasma spiked with ATO and TLM are presented in tables 8.70 and Recovery The extraction efficiency was determined by analyzing the quality control samples. The average absolute recoveries for ATO and TLM at three QC levels are shown in table The recoveries of the analytes were high and also the extent of recovery was consistent, precise and reproducible Stability studies Table 8.73 summarizes the freeze and thaw stability, short-term stability, long-term stability and post-preparative stability data of the analytes. All the results showed that there was no stability related issues during the analyses. The stability of working solutions was tested at room temperature for 6 h and it was found that these solutions were stable for 6 h. 195

312 Table Regression parameters Parameter ATO TLM Correlation coefficient Slope Intercept Table Intra-day precision and accuracy Analyte ATO TLM Nominal concentration, ng/ml Mean concentration found, ng/ml Intra-day precision, RSD (%) a Accuracy b (%) a n=5, b Accuracy is given by the % deviation of mean concentration from nominal concentration Table Inter-day precision and accuracy Analyte ATO TLM Nominal concentration, ng/ml Mean concentration found, ng/ml Inter-day precision, RSD (%) a Accuracy b (%) a n=5, b Accuracy is given by the % deviation of mean concentration from nominal concentration 196

313 Table Absolute recoveries of ATO and TLM from human plasma Analyte Concentration, ng/ml Recovery (%); %RSD, n=5) (4.0925) ATO (3.5444) (2.8905) (2.5595) TLM (3.9087) (2.5550) Table Stability data for ATO and TLM in plasma Conditions Recovery a (%) ATO TLM Three freeze-thaw cycles Short-term stability for 24 h in plasma in room temperature Auto sampler stability for 24 h Long-term stability for one week Stock solution stability for 6h a Recovery is expressed as the response of ATO and TLM stored in plasma following stability conditions compared to the response of the analytes freshly spiked in plasma 197

314 Figure ESI-MS positive ion full-scan spectrum of ROS Figure ESI-MS positive ion full-scan spectrum of ATO Figure ESI-MS positive ion full-scan spectrum of TLM

315 Figure Chromatograms for blank plasma (drug and I.S. free) at m/z (A) 482, (B) 515 and (C) 559

316 Figure Chromatograms at LLOQ level for ATO-1 ng/ml, TLM-1 ng/ml and ROS-30 ng/ml

317 Figure Chromatograms for ATO-2 ng/ml, TLM-2 ng/ml and ROS-30 ng/ml

318 Figure Chromatograms for ATO-5 ng/ml, TLM-5 ng/ml and ROS- 30 ng/ml

319 Figure Chromatograms for ATO-15 ng/ml, TLM-15 ng/ml and ROS-30 ng/ml

320 Figure Chromatograms for ATO-20 ng/ml, TLM-25 ng/ml and ROS-30 ng/ml

321 Figure Chromatograms for ATO-30 ng/ml, TLM-30 ng/ml and ROS-30 ng/ml

322 Figure Chromatograms for ATO-35 ng/ml, TLM-35 ng/ml and ROS-30 ng/ml

323 Calibration graphs Concentration Peak (ng/ml) Area Ratio Figure Calibration graph of ATO (1-35 ng/ml) Concentration Peak (ng/ml) Area Ratio Figure Calibration graph of TLM (1-35 ng/ml)

324 Conclusion CHAPTER 9. CONCLUSION An attempt has been made to develop validated analytical and bioanalytical methods for the determination of newer cardio vascular drugs in combination form from formulations and biological fluids. The analytical and bioanalytical methods were validated according to International Conference on Harmonization guidelines for validation of analytical procedures and US Food and Drug Administration bioanalytical method validation guidelines respectively. A comparison of analytical and bioanalytical methods developed in this thesis are shown in tables Part-1(Analytical method development) Amlodipine-hydrochlorothiazide-valsartan For the combination containing amlodipine, hydrochlorothiazide and valsartan, precise and accurate, HPLC and HPTLC methods were developed and validated. The developed methods were selective and specific for the drugs as there was no interference from the tablet excipients. The HPTLC method was found to be more sensitive than the HPLC method. The formulation analyzed by the methods showed adequate quality and drug contents in concordance with the label claim. The developed methods found to be suitable in quantifying these cardiovascular drugs might be employed for quality control analysis, as well as in further studies in other matrices like plasma. A RP-HPLC method (126) was reported for the simultaneous determination of amlodipine besylate, hydrochlorothiazide and valsartan in bulk drugs and pharmaceutical dosage forms. The developed RP-HPLC method has the following advantages when compared to the reported method. The buffer used in the present methodology is LC-MS compatible and hence can be adopted for LC-MS analysis. The reported methodology used higher 198

325 Conclusion buffer strength (50 mm phosphate) which may sometimes damage the columns or reduce its life, but the developed HPLC method utilized buffer of nominal strength (10 mm ammonium acetate). The mobile phase system developed for the HPLC method is simpler and cost effective. Atorvastatin-ezetimibe-fenofibrate Simultaneous analysis of atorvastatin, ezetimibe and fenofibrate in their pharmaceutical formulation has been successfully achieved by the application of the developed RP-HPLC and HPTLC methods. The HPTLC method was found to be more sensitive than the HPLC method. The developed methods are accurate and precise and hence can be applied for quality control evaluation of drugs in formulations and other matrices. The developed RP-HPLC and HPTLC methods are more sensitive and use economic mobile phase system when compared to the methods (127) reported. Telmisartan-ramipril-hydrochlorothiazide For the combination consisting of telmisartan, ramipril and hydrochlorothiazide, precise and accurate RP-HPLC and HPTLC methods were developed and validated. Analytical results of samples were in accordance with those of standard solution in the same concentrations. The drug peaks were well resolved with the use of mobile phase systems in case of both the methods. The HPTLC method was found to be more sensitive than the HPLC method. No methods are reported for the analysis of this combination. Hence, the developed methods can be applied for the simultaneous determination of these components in their pharmaceutical formulations and biological fluids. 199

326 Conclusion Atorvastatin-ramipril-aspirin An optimized HPTLC method employing UV detection has been developed and validated for the simultaneous determination of atorvastatin, ramipril and aspirin in combined dosage form. The linearity, precision, accuracy, reproducibility and selectivity of this methodology have been established. The developed method showed no interference with the formulation excipients and there was good resolution between the drugs. A HPTLC method (47) has been reported for the simultaneous determination of atorvastatin, ramipril and aspirin in dosage forms. The developed method has the advantage that all the drug peaks are well resolved (R f values, ramipril=0.28, atorvastatin=0.48 and aspirin=0.68) when compared to the reported method. For the reported method, the R f value of ramipril was found to be 0.06 which shows that the developed spot is very close to the application position, which may affect accurate quantification of the drug. The proposed HPTLC method is more sensitive in terms of the linearity range when compared to the HPLC method (128) reported for the analysis of these drugs in combination. Rosuvastatin-ezetimibe RP-HPLC, HPTLC and UV spectroscopy (simultaneous equation & derivative) methods have been developed and validated for the determination of rosuvastatin and ezetimibe in tablet formulations. The separation methods like HPLC and HPTLC methods have several advantages compared with UV spectrophotometry methods such as lower detection and quantification limits, small sample volumes and specificity. Among the chromatographic methods, HPTLC method was found to be more sensitive than the HPLC method. Specificity tests were successfully performed 200

327 Conclusion for HPLC and HPTLC methods. The developed chromatography methods are rapid, specific and reliable, whereas the UV methods are more cost effective and simple. The current methods were found to be more sensitive than the UV spectrophotometric method (129) reported for the simultaneous determination of the drugs. Telmisartan-atorvastatin For the simultaneous determination of telmisartan and atorvastatin in tablet dosage form RP-HPLC, HPTLC and UV spectroscopy methods (simultaneous equation & derivative) have been developed and validated. The chromatographic methods were found to be sensitive than the UV methods. Among the chromatographic methods, HPLC method was found to be more sensitive than the HPTLC method. Because there is no pharmacopoeial method for the analysis of the drugs, developed methods can be used for routine quality control of formulations containing these drugs. A RP-HPLC method (130) has been reported for the simultaneous determination of the drugs. The proposed HPLC method has the following advantages when compared to the reported method. The mobile phase used in the reported method is phosphate buffer which is not compatible with the LC-MS system, whereas ammonium acetate buffer used in the developed HPLC method is compatible with LC-MS. Hence, the developed HPLC method can be applied to LC-MS bioanalytical studies. The proposed method was found to be more sensitive in terms of linearity range. 201

328 Conclusion Rosuvastatin-fenofibrate For the simultaneous analysis of rosuvastatin and fenofibrate, precise and accurate, HPLC and HPTLC methods were developed and validated. The developed methods showed no interference from the formulation excipients. HPTLC method was found to be more sensitive than the HPLC method. The formulation analyzed by the methods showed adequate quality and drug contents in concordance with the label claim. The developed methodologies can be applied for the analysis of these drugs in biological fluids. The developed HPLC method is more sensitive when compared to the reported method (131). Olmesartan medoxomil-amlodipine RP-HPLC, HPTLC and UV spectroscopy (simultaneous equation & derivative) methods have been developed and validated for the simultaneous determination of olmesartan medoxomil and amlodipine in tablet formulations. The method was validated for accuracy, precision, linearity and stability. The chromatographic methods were found to be more sensitive than the UV methods. Among the chromatographic methods, HPTLC method was more sensitive. The developed HPLC method is more economic and sensitive when compared to the reported method (132). Telmisartan-amlodipine Rapid and reliable isocratic RP-HPLC and HPTLC methods for simultaneous determination of telmisartan and amlodipine have been developed and validated. These chromatographic methods fulfilled all the requirements to be identified as a reliable and feasible method, including accuracy, linearity and precision data. The HPLC method was found to be sensitive than the HPTLC method. In the case of 202

329 Conclusion HPLC method, a run time of around 7.5 min allows the analysis of a large number of samples in a short period of time. System suitability tests were done for the HPLC method and the results complied with the limits. The proposed HPLC method was found to be more economic when compared with the reported methods (133, 134) because the current methodology used less costly methanol in place of acetonitrile in the mobile phase system. The developed HPTLC method used more economic mobile phase system when compared to the reported HPTLC method (135) Nebivolol-amlodipine HPLC and HPTLC methods have been developed for the simultaneous determination of nebivolol and amlodipine. The method was validated for parameters like accuracy, precision, linearity and stability. The methods used simple mobile phase composition. Among the developed methods, HPLC method was found to be more sensitive. Because there is no pharmacopoeial method for simultaneous analysis of the drugs, the developed methods can be recommended for quality control of these drug contents in pharmaceutical preparations. The proposed HPLC method it is more sensitive and economic when compared to the reported method (136). The developed HPTLC method used simpler and more economic mobile phase when compared to the reported method (137). Statistical analysis The proposed analytical methods were compared using statistical analysis by applying student s t-test and one-way ANOVA followed by Tukey-Kramer multiple comparison tests. The results ensured that there was no significant difference between 203

330 Conclusion the developed methods for the combinations studied and hence can be applied for the routine analysis of these drugs in formulations. Part-2 (LC-MS bioanalytical method development) Sensitive and reliable LC-ESI-MS method for the simultaneous determination of rosuvastatin and ezetimibe & atorvastatin and telmisartan at subnanogram levels in human plasma has been developed and validated. Acceptable precision and accuracy were obtained within the calibration range. In addition, the present methods utilized a single step simple liquid-liquid extraction methodology for extraction of analytes from plasma samples. The proposed methods have advantages like good extraction efficiency, resolution and short analysis time (approximately 5 min). As no bioanalytical LC-MS methods are reported for the simultaneous analysis of these drugs, the developed bioanalytical methods can be used for pharmacokinetic, bioavailability or bioequivalent studies in human plasma. The analytical methods developed in the thesis were validated according to ICH/US FDA guidelines and also statistically evaluated; hence these methods can be used to ensure quality of newer selected cardiovascular drug combinations and to monitor their drug levels in biological fluids. 204

331 Conclusion 205 Table Summary of analytical method development (chromatographic methods) HPLC HPTLC Combination Drugs Linearity, Recovery,% a Amount Linearity, Recovery,% a Amount µg/ml found, % µg/band found, % AML AML + HYD + VAL HYD VAL ATO ATO + EZE + FEN EZE FEN RPL + HYD + TLM RPL HYD TLM RPL RPL + ATO + ASP ATO ASP ROS + EZE ROS EZE ATO + TLM ATO TLM ROS + FEN ROS FEN AML + OLM AML OLM AML + TLM AML TLM s-aml + NEB s-aml NEB a Recovery values at 100 % level (Average of six determinations)

332 Conclusion Combination ROS + EZE ATO + TLM AML + OLM Table Summary of analytical method development (UV-spectroscopy methods) Drugs Simultaneous Equation Method Derivative Spectroscopy Method Linearity, Recovery, % a Amount Linearity, Recovery, % a Amount µg/ml found, % µg/ml found, % ROS EZE ATO TLM AML OLM a Recovery values at 100 % level (Average of six determinations) 206 Table Summary of LC-MS bioanalytical method development Combination Drugs Linearity, ng/ml Recovery,% at ROS + EZE ROS ng/ml b 2 ng/ml c 8 ng/ml d 0.25 ng/ml b EZE ng/ml c 8 ng/ml d ATO + TLM ATO ng/ml b 15 ng/ml c 30 ng/ml d 2 ng/ml b TLM ng/ml c 30 ng/ml d b Low concentration QC sample, c Medium concentration QC sample, d High concentration QC sample

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351 References determination of amlodipine and telmisartan in pharmaceutical dosage form. J Appl Chem Res. 2010; 12: Aniruddha RC, Swati CJ, Kumbhar SV, Vinayak JK, et al. Simultaneous HPTLC estimation of telmisartan and amlodipine besylate in tablet dosage form. Arch Appl Sci Res. 2010; 2(3): Sudhakar M, Venkateshwara RJ, Devika GS, Ramesh PR. A validated RP-HPLC method for simultaneous estimation of nebivolol hydrochloride and s-amlodipine in tablet dosage forms. Int J Chem Pharm Sci. 2010; 1(2): AnilKumar S, Bhavesh P, Rakshit P. Simultaneous estimation of nebivolol hydrochloride and s-amlodipine by high-performance thin layer chromatography. Int J Pharm Bio Sci. 2010; 1(4):

352 Column Certificates Appendix 1.1

353 Column Certificates Appendix 1.2

354 Column Certificates Appendix 1.3

355 Column Certificates Appendix 1.4

356 Column Certificates Appendix 1.4

357 LC-MS Instrument Settings Appendix 2

358 LC-MS Instrument Settings Appendix 2

359 Publications Appendix 3.1

360 Publications Appendix 3.1

361 Publications Appendix 3.1

362 Publications Appendix 3.1

363 Publications Appendix 3.1

364 Publications Appendix 3.1

365 Publications Appendix 3.2

366 Publications Appendix 3.2

367 Publications Appendix 3.2

368 Publications Appendix 3.2

369 Publications Appendix 3.2

370 Publications Appendix 3.2

371 Publications Appendix 3.2

372 Publications Appendix 3.2

373 Publications Appendix 3.3

374 Publications Appendix 3.3

375 Publications Appendix 3.3

376 Publications Appendix 3.3

377 Publications Appendix 3.3

378 Publications Appendix 3.3

379 Publications Appendix 3.3

380 Publications Appendix 3.3

381 Publications Appendix 3.3

382 Publications Appendix 3.3

383 Publications Appendix 3.3

384 Publications Appendix 3.3

385 Publications Appendix 3.3

386 Publications Appendix 3.3