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Validation and Determination of Candesartan with Different Juices in Rat Plasma

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Validation and Determination of Candesartan with Different Juices in Rat Plasma 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 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 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 ------------------------ II 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 Chromatography–Mass 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), 5µ, 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% <10%). The accuracy for candesartan over all the three days of validation and all the four tested target concentration was within the accepted criteria. The standard curves for candesartan matched the requirements, linear relation (R2) ranged between (0.996 to 1). According to the result obtained, the Cmax for candesartan alone was (964.692 ng/ml), there was no significant effect (P>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<0.05). candesartan plasma level was lowered to the half when combined with pomegranate, and almost at the same level when combined with both of orange and liquorice. III 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. IV 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. V 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 ARB’s 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 VI 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 VII 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 VIII List of Figures Figure Caption Page No. No. 1. Renin-Angiotensin-Aldosterone System 9 2. Candesartan Chemical Structure 11 3. The plot of calibration curve levels against their analytical response, in 84 day one validation for candesartan 4. The plot of calibration curve levels against their analytical response, in 85 day two validation for candesartan 5. The plot of calibration curve levels against their analytical response, in 86 day three validation for candesartan 6. The plot of linearity of calibration curve levels for candesartan 89 quantification against their analytical response and regression linear equation 7.
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