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  • I

    Validation and Determination of Candesartan with Different Juices in Rat Plasma

    by using High Performance Liquid Chromatography/Mass Spectrometry

    (HPLC/MS/MS).

    By

    Ahmed Issam Al-Kawaz

    Supervisor:

    Prof. Tawfiq Arafat

    Co-Supervisor:

    Dr. Wael Abu Dayyih

    A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

    Master of Science in Pharmaceutical Sciences at

    University of Petra

    Faculty of Pharmacy and Medical Sciences

    Amman-Jordan

    January 2014

  • II

    Validation and Determination of Candesartan with Different Juices in Rat Plasma

    by using High Performance Liquid Chromatography/Mass Spectrometry

    (HPLC/MS/MS).

    By

    Ahmed Issam Al-Kawaz

    A Thesis Submitted in Partial Fulfillment of the Requirements for the

    Degree of Master of Science in Pharmaceutical Science at

    University of Petra

    Faculty of Pharmacy and Medical Sciences

    Amman-Jordan

    January 2014

    Supervisor: Signature

    Prof. Tawfiq Arafat -----------------------

    Co-supervisor:

    Dr. Wael Abu Dayyih -----------------------

    Examination committee:

    1. Prof. Zuhair Muhi-Eldeen -----------------------

    2. Dr. Eyad Al-Mallah -----------------------

    3. Dr. Naseer Hashim Ahmed ------------------------

  • III

    ABSTRACT

    Validation and Determination of Candesartan with Different Juices in Rat Plasma

    by using High Performance Liquid Chromatography/Mass Spectrometry

    (HPLC/MS/MS).

    By

    Ahmed Issam Al-Kawaz

    University of Petra, 2014

    Supervisor Co-supervisor

    Prof. Tawfiq Arafat Dr. Wael Abu Dayyih

    A new validated simple, rapid and sensitive method for determination of candesartan in

    the presence of each juice has been applied by using High Performance Liquid

    ChromatographyMass Spectrometry (HPLC/MS). The mobile phase was composed of

    (methanol, of 0.2% FA in water) was used as a mobile phase, ACE 5 C18 Column (50 X

    2.1 mm), 5, and a flow rate of 1.0 ml/min were used, the autosampler injection volume

    was 5 microliters, and Irbesartan was used as internal standard, The precision of

    predicted measurements for candesartan was high (mean CV% 0.05) of orange juice on candesartan Cmax

    (1253.163ng/ml). and for Licorice, the Cmax was (818.2868 ng/ml) which is also

    considered as a non- significant effect (P>0.05). on the other hand pomegranate shows to

    decrease the Cmax of candesartan (475.9673 ng/ml) which is a significant effect

    (P

  • IV

    ACKNOWLEDGEMENTS

    In The Name of Allah, the Most Gracious, the Most Merciful

    Foremost, I would like to express my special appreciation and thanks to my advisor Prof.

    Tawfiq Arafat and my co-advisor Dr. Wael Abu Dayyih, you have been a tremendous

    mentor for me. I would like to thank you for your patience, motivation, enthusiasm, and

    immense knowledge. your guidance helped me in my research, allowing me to grow as a

    research scientist. Your advice on both research as well as on my career have been

    priceless. your influence on my life will continue through my entire life, as one of my

    role models.

    I would also like to thank my committee members, Prof. Zuhair Muhi-Eldeen, Dr. Eyad

    Al-Mallah and Dr. Naseer Hashim Ahmed for serving as my committee members even at

    hardship. I also want to thank you for letting my defense be an enjoyable moment, and

    for your brilliant comments and suggestions, thanks to you.

    To my family, Words cannot express how grateful I am to my mother Dr.Intisar, my

    father Dr.Issam my brothers Anas and Zaid who spent sleepless nights with me and was

    always my support in the moments when there was no one to answer my queries, Your

    prayer for me was what sustained me thus far.

    My sincere thanks to Prof. Tawfiq Alhussainy for his support and continuous advices

    My sincere thanks also goes to Dr Nidal Qinna, for his support, advices and his help by

    allowing using his laboratory, and to Mohammad Albayed, for his great technical

    assistance in animal handling.

    Special thanks to Hamza Al-Hurob from JCPR who have been very helpful and

    supportive many thanks for the staff of JCPR who also helped me in this work.

    For my dear friends, Marwa nasir, Mustafa nawzad, Noor Taqi, Rawnaq Jalal, Hagop,

    Raghda Tobchi and Yaser Ahmed, I would like to specially thanking you for the help you

    gave me, and your continuous spiritual support until the finish of this research.

  • V

    I would especially like to thank my dear friends, Teeba Emad, Rafal Ammar, Ahmed

    Basim, Rafif Raad at University of Petra. All of you have been there to support me in

    writing, and incented me to strive towards my goal.

    Special thanks to my M.S.c friends (Abdullah Nabil, Nada Ali, Ibrahem Adil, Nibras

    Jamal, Noor Maan, Ragheed Adil, Mujtaba, Zena Hilal and Zainab Al Obaidy).

    Also I want to thank all my friends who have been very supportive. (Taha alKhanchi,

    Maashar, Usama Mezil, Bashar Younis, Ban Thiab, Shahad Faisal, Ahmed Al-Azzawi,

    Vegen, Mohannad Kattan, Hesham, Khalid Al-Haidari, Riham Nasir, Mais Jamal)

    In the end, my thanks go to for my beloved brothers, my extended family, especially my

    aunt Anaam. And I would love to dedicate this work, to my Father Dr.Issam and my

    mother Dr,Intisar whom I know their prayers and love still protecting and guiding me.

  • VI

    Table of Contents

    No. Subject Page

    No.

    Chapter One: Introduction 1

    1 Introduction 2

    1.1 Hypertension 2

    1.1.1 Hypertension Classification 3

    1.1.2 Management 4

    1.2 Anti Hypertensive Drugs 4

    1.2.1 The Angiotensin II Receptor Blockers (ARBs) 5

    1.2.2 ARBs Drug Interaction 7

    1.2.3 Side Effects of ARBs 7

    1.2.4 Action of Angiotensin II 8

    1.3 Candesartan 11

    1.3.1 Identification 12

    1.3.2 Mechanism of Action 13

    1.3.3 Indication and Clinical Use 14

    1.3.4 Contraindication 15

    1.3.5 Side Effects 16

    1.3.6 Drug Interaction 16

    1.3.7 Pharmacokinetic Data 17

    1.3.8 Toxicity 20

    1.4.0 Fruit Juices 20

    1.4.1 Liqurice 21

    1.4.2 Pomegranate 24

    1.4.3 Orange 26

    1.5.0 Chromatography 27

    1.6.0 High Performance Liquid Chromatography (HPLC) 28

    1.6.1 Advantages of HPLC 31

    1.6.2 HPLC Detectors 32

    1.7.0 Beverages Drug Interaction 36

    1.7.1 Drug-Drug Interaction 37

    1.7.1.1 Types of Drug Interaction Mechanisms 37

    1.7.1.2 The Cytochrome P-450 (CYP450) Enzyme System 38

  • VII

    1.7.1.3 The Transporters of Intestinal 39

    1.8 Pre-Clinical Studies 40

    1.9. Method Validation 40

    1.9.1 Precision 41

    1.9.2 Accuracy 41

    1.9.3 Linearity 42

    1.9.4 Range 42

    1.9.5 Ruggedness 42

    1.9.6 Limit of Detection 43

    1.9.7 Limit of Quantitation 43

    1.9.8 Selectivity 43

    1.9.9 Specificity 43

    1.9.10 Stability 43

    1.10. Internal Standard 44

    1.11. Previous Analytical Studies and Literature Survey 46

    1.12. Objective of This Study 55

    Chapter Two: Experimental Part 56

    2 Experimental Part 57

    2.1 Reagents 57

    2.2 Instrumentation 58

    2.3 Animals 58

    2.4 Preparation of Stock Solutions 59

    2.4.1 Preparation of Candesartan Solution to be Given to the Rats 59

    2.4.2 Preparation of Stock Solution of Candesartan 60

    2.4.3 Preparation of Stock Solution for Internal Standard 60

    2.4.4 Preparation of 2 ng/ml Irbesartan IS in methanol (precipitating agent) 60

    2.4.5 Preparation of Working Solution for Candesartan 60

    2.4.5 Preparation of Candesartan STD Serial Dilution and Spiked Plasma 60

    2.4.6 Preparation of Candesartan QC Serial Dilution and Spiked Plasma 60

    2.5 Preparation of Orange, Licorice and Pomegranate Juices 62

    2.6 Method of Sample Preparation 62

    2.7 Validation 63

    2.7.1 Accuracy and Precision 63

    2.7.2 Specificity and Selectivity 63

    2.7.3 Sensitivity 63

  • VIII

    2.7.4 Linearity 63

    2.7.5 Stability 64

    2.8 Chromatographic Conditions 64

    2.9 Irbesartan as Internal Standard 64

    2.10 Statistical Analysis 65

    Chapter Three: Results and Discussion 68

    3 Results 69

    3.1 Validation 69

    3.1.1 Precision 69

    3.1.2 Accuracy 70

    3.1.3 Measurement Error 71

    3.1.4 Linearity 82

    3.1.5 Stability 89

    3.1.6 Specificity and Sensitivity 95

    3.2 The Modifying Effect of Combining Fruit Juices with Candesartan 96

    3.2.1 Effect of Combination on Candesartan 96

    3.3 Discussion 120

    4 Chapter Four: Conclusion 129

    4.1 Conclusion 130

    4.2 References 132

    4.3 Appendix: Chromatograms 158

    4.4 Abstract (in Arabic) 163

  • IX

    List of Figures

    Figure

    No.