Development and validation of HPLC method for simultaneous quantitative determination of Azilsartan medoxomil potassium and Chlorthalidone in human plasma

Doctoral Thesis / Dissertation, 2014

75 Pages, Grade: 3



List of abbreviations


Acceptance criteria

Chapter – 1: Introduction
Section- 1: General introduction
1 Bioanalytical techniques
2 Types of chromatography
3 Sample preparation techniques
4 Method validation
5 Bioavailability and bioequivalence
6 References
Section- 2 Introduction of Active Pharmaceutical Ingredients (API) used for present work by various instrumental method development and validation
1 Introduction of Azilsartan medoxomil
2 Introduction of Chlorthalidone
3 References:

Chapter-2: Development and validation of RP-HPLC methods for quantitative determination of some bioactive molecules in human plasma
Section-1: Development and validation of HPLC method for simultaneous quantitative determination of Azilsartan medoxomil potassium and Chlorthalidone in human plasma by HPLC
1 Aim of present work
2 Experimental
3 Method development
4 Results and discussion
5 Conclusion


It is moment of gratification and pride to look back with a sense of contentment at the long traveled path, to be able to recapture some of the fine moments, to think of the infinite number of people, some who were with me from the beginning, some who joined me at different stages during this journey, whose kindness, love and blessings has brought me to this day. I wish to thank each of them from the bottom of my heart.

First and foremost, I am extremely thankful to the almighty god for making me capable of doing all that I had proposed, the work leading to my Ph. D. thesis submission is one of them.

At the outset, I would like to extol Prof. Hitendra Joshi for his continues guidance in my doctoral research endeavor during the yester years. As my supervisor, he constantly forced me to remain focused to achieve my goal. His observation and comments helped me to establish the overall direction of the research and to move forward expeditiously with investigation in depth. I thank him for providing me the opportunity to work with numerous local and global peers.

I am also thankful to whole departmental staff. I owe a great deal to Dr. P. H. Parsania, Professor and Head, Department of Chemistry and Dr. Anamik Shah, Professor, Department of Chemistry. I am especially thankful of Department of Chemistry and National Facility for Drug Discovery centre, Saurashtra University, Rajkot for providing instrumental facility. I am also thankful to all administrative staff of this department for their timely help.

From bottom of heart I especially thanks to my seniors Dr. Vijay ram, Dr. Renis Ghetiya, Dr Bhavesh dodiya, Dr. Govind Kher, Dr. Kapil Dubal and Dr. Gaurang Pandya for their selfless help, moral support and guidance during my Ph. D. work. I heartily express special thanks to my colleagues Vaishali, Jalpa, Nirav Nayan, Reema, Chirag and Pankaj for their kind help and support.

I offer my gratitude to my friends Hasmukh, Bharat, Vipul, Mitaa, Kapil, Batuk, Hetal, Jayesh, Pinakin, Satish, Kalpesh, Devang, Hardik, Nayan, Rajesh, Viral, Pankaj, Pratik, Chirag, Bhavin, Ashish, Denish, Mitesh, Mayank, Gaurav, Khushal, Jignesh and all the Ph. D. students for their fruitful discussion at various stages.

Who have given us everything that we possess in this life? The life itself is their gift to us, so I bow my head with utter respect to my beloved parents Smt. Savitaben and Shri Pravinbhai. I bow my head in utter humility and complete dedication from within my heart. I am very much grateful to my brother Narendra and for His love, affectionate and caring. I am also obliged to my younger cousin Ketan.

As with completion of this task, I find myself in difficult position on attempting to express my deep indebtedness to Samir Pandya, Amit Chapala, Dakshesh Patel, Kapil, Ritesh Bhatt, Kunal Gupte, Vijay Vekariya, Rushikesh Joshi, Narendra Garaniya, Jigisha.

Big thanks to the staff of Department of chemistry and also Mr. Harshad Joshi and Mrs. Namrata for their kind support and providing chemicals and glassware on time.

I also remember well wishers and all those persons who helped me directly or indirectly during my Ph.D.

Paras P. Vekariya


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Absolute bioavailability: It is the extent or fraction of drug absorbed upon extra vascular administration in comparison to the dosage size administered.

Absorption: It is the process of uptake of the compound from the site of administration into the systemic circulation. A prerequisite for absorption is that the drug should be in aqueous solution. The only relatively rare exception is absorption by pinocytosis.

Accuracy: The degree of closeness of the determined value to the nominal or known true values under prescribed conditions. This is sometimes termed trueness.

Analyte: A specific chemical moiety being measured, this can be intact drug, bio molecule or its derivative, metabolite, and/or a degradation product in a biological matrix.

Analytical run (or batch): A complete set of analytical and study samples with appropriate number of standards and QCs for their validation. Several runs (or batches) may be completed in one day, or one run (or batch) may take several days to complete.

Bioequivalence: It is a relative term that denotes that the drug substance in two or more identical dosage forms, reaches the systemic circulation at the same relative rate and to the same relative extent i.e. their plasma concentration-time profiles will be identical without significant statistical differences.

Biological matrix: A discrete material of biological origin that can be sampled ad processed in a reproducible manner. Examples are blood, serum, plasma, urine, faeces, saliva, sputum and various discrete tissues.

Blank (Control) plasma: A sample of human plasma to which no analytes have been added that is used to assess the specificity of the bioanalytical method.

Blood: It consists of cellular material (99% red blood cells, with white blood cells and platelets making up the remainder), water, amino acids, proteins, carbohydrates, lipids, hormones, vitamins, electrolytes, dissolved gases and cellular wastes. Each red blood cell is about 1/3 haemoglobin by volume. The primary blood gases are oxygen, carbon dioxide and nitrogen.

Blood, Plasma or Serum levels: It demonstrates the drug concentration in blood, plasma or serum upon administration of a dosage form through various routes of administration. Blood, plasma or serum-level curves are plots of drug concentration versus time on numeric or semi-log graph paper. These levels are obtained from blood samples by venopuncture in certain time intervals after administration of the drug product and chemical or microbiological analysis of the drug in the biological fluid.

Calibration standard: A biological matrix to which a known amount of analyte has been added or spiked. Calibration Standards are used to construct calibration curves from which the concentration of analyte in QCs and in unknown study sample are determined.

Drug: It is a chemical compound of synthetic, semi synthetic, natural or biological origin that interacts with human or animal cells. The interaction may be qualified, whereby these resulting actions are intended to prevent, to cure or to reduce ill effect in the human or animal body, or to detect disease-causing manifestations.

Drug product or dosage form: It is the pharmaceutical form containing the active ingredient and vehicle substance necessary in formulating a medicament of desired dosage, desired volume and desired application form, ready for administration.

Excretion: It is the final elimination of the drug from the body’s systemic circulation via the kidney into urine, via bile intestines and saliva into faeces, via sweat, via skin and via milk.

Lower Limit Of Quantification (LLOQ): The lowest amount of an analyte in a sample that can be quantitatively determined with suitable precession and accuracy.

Matrix effect: The direct or indirect alteration for biotransformation of endogenous and exogenous substance, which take place in the living cell.

Method: A comprehensive description of all procedure used in sample analysis.

Pharmaceutic equivalence: This term implies that two or more drug products are identical in strength, quality, purity, content uniformity and disintegration and dissolution characteristics, they may however differ in containing different excipients.

Pharmacokinetics: It deals with the changes of drug concentration in the drug product and changes of concentration of drug and/or its metabolite in the human or animal body following administration, i.e. the changes of drug concentration in the different body fluids and tissue in the dynamic system of liberation, adsorption, distribution, body storage, binding, metabolism, and excretion.

Plasma: It consists of about 92% water, with plasma protein as the most abundant solute. Plasma appearance is transparent with a faint straw colour. It is mainly composed of water, blood proteins (albumins, globulins, and fibrinogens), and inorganic electrolytes. It serves as transport medium for glucose, lipids, amino acids, hormones, metabolic end products, carbon dioxide and oxygen. Plasma is the largest single component of blood, making up about 55% of total blood volume.

Precession: the closeness of agreement (degree of scatter) between a series of measurement obtained from multiple sampling of the some homogeneous sample under the prescribed conditions.

Processed: The final extract (prior to instrumental analysis) of a sample that has been subjected to various manipulations (e.g., extraction, dilution, concentration).

Quantification range: The range of concentration, including ULOQ and LLOA, that can be reliably and reproducible quantified with accuracy and precession through the use of a concentration- response relationship.

Quality Control sample (QC): A spiked sample used to monitor the performance of a bioanalytical method and to assess the integrity and availability of the results of the unknown samples analysed in an individual batch.

Recovery: The extraction efficiency of an analytical process, reported as percentage of the known amount of the analyte carried the through the sample extraction and processing steps of the method.

Selectivity: The ability of the bioanalytical method to measure and differentiate th analytes in the presence of component that may be expected to be present. These could include metabolites, impurities, degradants, or matrix components.

Serum: It refers to blood plasma in which clotting factors have been removed.

Stability: The chemical stability of an analyte in a given matrix under specific conditions for given time intervals. Drug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system. Stability evaluation is done to show that the concentration of analyte at the time of analysis corresponds to the concentration of the analyte at the time of sampling.

Solution Stability: The stability test for the standard stock solution of analyte must be done at the same temperature (Room or refrigerated), container and solvent as that to be used for the study.

Bench top stability: It is the stability of the analyte in matrix at working temperature conditions over a short period covering the sample time, when all precaution are taken to avoid specifically known stability problems of the analyte.(e.g. light sensitivity).

Post preparative stability (Extracted sample stability): It is evaluated over the maximum time from completion of sample work-up to completion of data collection, with allowance also for potential delay in analysis due to equipment failure.

Freeze and thaw stability: This stability test is done to ensure that the sample remains stable after it is subjected to multiple freeze thaw cycle in the process of the study.

Long term stability : This is done to assess whether the analyte is stable in plasma matrix under the sample storage conditions for the time period required for the samples generated in a clinical study to the last date of analysis.

Standard/ calibration curve: The relationship between the experimental response value and the known analytical concentration.

Therapeutic equivalence: The term indicates that two more drug products that contain the same theoretically active ingredient elicit identical pharmacologic effects and can control the disease to the same extent.

Upper Limit Of Quantitation (ULOQ): The highest amount of an analyte in a sample that can be quantitatively determined with precession and accuracy.

Acceptance criteria of validation parameters performed for the drugs and their active metabolites

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[1] Guidance for Industry: Bioanalytical Method validation, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER), Centre for Veterinary Medicine (CVM), May (2001).

[2] R.N. Xu, L. Fan, M.J. Rieser, T.A. El-Shourbagy, J. Pharm. Biomed. Anal. 44 (2007) 342.

[3] V.P. Shah, K.K. Midha, W.A.J. Findlay, M.H. Hill, D.J. Hulse, J.I. McGilveray, G. Mckay, J.K. Miller, N.R. Patnaik, L.M. Powell, A. Tonelli, C.T. Viswanathan, A. Yacobi, Pharm. Research 17 (2000) 1551.

[4] B.K. Matuszewski, M.L. Constanzer, C.M. Chavez-Eng, Anal. Chem. 75 (2003) 3019.

[5] FDA Guidance for Industry: Bioavailability Studies for Orally Administered Drug-Products - General Considerations, US Department of Health and Human Services, Food and Drug Administration Centre for Drug Evaluation and Research (CDER), (2000).

[6] Guidance for Industry: ICH E6 Good Clinical Practice, U.S. Department of Health and Human Services, Food and Drug Administration, Centre for Drug Evaluation and Research (CDER), Centre for Biologics Evaluation and Research (CBER), April (1996).

[7] M.L. Rocci, Jr., V. Devanarayan, D.B. Haughey, P. Jardieu, AAPS Journal 9 (2007) E336.

Section 1. General introduction

1 Bioanalytical techniques

Bioanalytical chemistry is the qualitative and quantitative analysis of drug substances in biological fluids such as blood, plasma, serum, urine and tissues. It plays a significant role in the evaluation and interpretation of bioavailability, bioequivalence and pharmacokinetic data [1].

The process by which a specific bioanalytical method is developed, validated and used in routine sample analysis can be divided into following method

(1) Reference standard preparation.

(2) Bioanalytical method development and establishment of assay procedure (full method validation).

(3) Application of validated bioanalytical method to routine drug analysis and acceptance criteria for the analytical run and/or batch.

The main analytical segments that comprise bioanalytical methodology are method development, method validation and application in routine sample analysis.

A bioanalytical method is a set of all the procedures involved in collection, processing, storing and analysis of a biological matrix for an analyte [2]. Analytical methods employed for quantitative determination of drugs and their metabolites in biological fluids are the key determinants in generating reproducible and reliable data for sample analysis [3]. Method development involves evaluation and optimization of the various stages of sample preparation, chromatographic separation, detection and quantification. Initially, an extensive literature survey on the same or similar analyte is done followed by summarizing the main features of the work, which is of primary importance. Based on this information, the following selections could be made for bioanalytical technique:

(1) The choice of instrument that is suitable for the analysis of the analyte of interest. This includes the choice of the column associated with the instrument, the detector, the mobile phase in the high performance liquid chromatography (HPLC), and the choice of carrier gas in gas chromatography (GC).

(2) Choice of internal standard, which is best for the study. It must have similar chromatographic and ionization properties compared to the analyte.

(3) The choice of extraction procedure, which is quick and efficient and gives the highest possible recovery without interference at the elution time of the analyte of interest and has acceptable accuracy and precision which meets the intended study requirement.

Method performance is determined primarily by the quality of the procedure itself. The two factors that are most important in determining the quality of the method, are selective recovery and standardization (Analytical recovery of a method refers to whether the analytical method in question provides response for the entire amount of analyte that is contained in a sample). Recovery is usually defined as the percentage of the reference material that is measured, to that which is added to a blank. This should not be confused with the test of matrix effect in which recovery is defined as the response measured from the matrix (e.g. plasma) as a percentage of that measured from the pure solvent (e.g. water). Results of the experiment that compare matrix to pure solvent is referred to as relative recovery and true test of recovery is referred to as absolute recovery [4, 5].

Matrix effect, often described as matrix ionization effect or ion suppression effect, is a phenomenon observed when the signal of analyte can be either suppressed or enhanced due to the co-eluting components that originate from the sample matrix. When a rather long isocratic or gradient chromatographic program is used in the quantitative assay, matrix effect may be not present at the retention time for an analyte. However, in the case of high-throughput LC–MS/MS analysis, matrix effect is one of the major issues to be addressed in method development and validation, especially when analyte is not well separated from the LC-front. One problem brought by matrix suppression effect is reduced sensitivity when analyte signal is suppressed. Detailed studies on matrix effects revealed that the ion suppression or enhancement is frequently accompanied by significant deterioration of the precision of the analytical method as demonstrated by Matuszewski et al. [5]. The authors studied the precision (%CV) upon repetitive injection of post-extraction spiked plasma samples as a function of the analyte concentration for a single lot and for five different lots of plasma. While for the single plasma lot the precision is acceptable, it may not be acceptable when different plasma lots are taken into account. Generally, matrix effect impacts more on the low end of calibration curve than the mid range or high end. When discussing matrix effects, it is useful to discriminate between ion suppression (or enhancement) by the matrix at one hand, and different matrix effects exerted by different sample lots at the other hand. The difference in response between a neat solution sample and the post-extraction spiked sample is called the absolute matrix effect, while the difference in response between various lots of post-extraction spiked samples is called the relative matrix effect. If no counteraction is taken, an absolute matrix effect will primarily affect the accuracy of the method, while a relative matrix effect will primarily affect the precision of the method.

Another important issue in method development stage is the choice of internal versus external standardization. Internal standardization is common in bioanalytical methods especially with chromatographic procedures. The assumption for the use of internal standard is that the partition coefficient of the analyte and the internal standard are very similar [4]. For internal standardization, a structural or isotopic analogue of the analyte is added to the sample prior to sample pre-treatment and the ratio of the response of the analyte to that of the internal standard is plotted against the concentration [6]. Additionally, the tests performed at the stage of method development should be done with the same equipment that will actually be used for subsequent routine analysis. The differences found between individual instruments representing similar models from the same manufacturer is not surprising and should be accounted for [7].

Various sophisticated techniques have been developed to allow the rapid separation and quantification of trace component of complex mixtures in biological matrix. In order to extract the drug from biological matrix and make it available for analysis different extraction techniques are used like precipitation, liquid-liquid and solid-phase extraction. Several methods have been applied in the analysis of drug and their metabolite, such as radio immune assay(RIA), capillary electrophoresis (CE), gas chromatography (GC), GC-mass spectrometry (GC-MS), high performance liquid chromatography (HPLC) with UV, fluorescence, radioactivity and mass spectrometric detection (MS).

2 Types of chromatography

Chromatography can be classified by various ways (I) Interaction of solute to the stationary phase, (II) Chromatographic bed shape, ( III) Physical state of mobile phase and (V) Advanced chromatographic techniques[8].

2.1 Interaction of solute to stationary phase

2.1.1 Adsorption chromatography

Adsorption chromatography is perhaps one of the oldest types of chromatography around. It has a solid stationary phase and a liquid or gaseous mobile phase. Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent, the least soluble or best adsorbed ones travel more slowly. The result is a separation into bands containing different solutes. Liquid chromatography using a column containing silica gel or alumina is an example of adsorption chromatography (Refer Figure 1) [9, 10].

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Figure 1: Figure for adsorption phenomenon in chromatography

2.1.2 Partition chromatography

In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface of an inert solid. The mixture to be separated is carried by a gas or a liquid as the mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble component in the mobile phase reaching the end of the chromatography column first (Figure 2). Paper chromatography is an example of partition chromatography [11].

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Figure 2: Figure for partition phenomenon in chromatography

2.1.3 Ion exchange chromatography

Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase [12]. The coating is referred to as a resin, and has ions (either cat ions or anions, depending on the resin) covalently bonded to it and ions of the opposite charge are electrostatically bound to the surface [13]. When the mobile phase (always a liquid) is eluted through the resin the electrostatically bound ions are released as other ions, bonded preferentially (Figure 3) [14]. Domestic water softeners work on this principle.

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Figure 3: Figure for ion exchange phenomenon in chromatography

2.1.4 Molecular exclusion chromatography

Also known as gel permeation or gel filtration, it differs from other types of chromatography in that no equilibrium state is established between the solute and the stationary phase [15]. Instead, the mixture passes as a gas or a liquid through a porous gel. The pore size is designed to allow the large solute particles to pass through uninhibited. The small particles however permeate the gel and are slowed down so the smaller the particles, the longer it takes for them to get through the column [16]. Thus separation is according to particle size (Figure 4).

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Figure 4: Figure for molecular exchange phenomenon in chromatography


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Development and validation of HPLC method for simultaneous quantitative determination of Azilsartan medoxomil potassium and Chlorthalidone in human plasma
Saurashtra University  (Department of Chemistry)
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development, hplc, azilsartan, chlorthalidone
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Dr. Vijay Ram (Author)Hitendra S. Joshi (Author)Paras P. Vekariya Rajesh Ram (Author), 2014, Development and validation of HPLC method for simultaneous quantitative determination of Azilsartan medoxomil potassium and Chlorthalidone in human plasma, Munich, GRIN Verlag,


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