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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
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.
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 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
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.
Caption Page
No.
1. Renin-Angiotensin-Aldosterone System 9
2. Candesartan Chemical Structure 11
3. The plot of calibration curve levels against their analytical response, in
day one validation for candesartan
84
4. The plot of calibration curve levels against their analytical response, in
day two validation for candesartan
85
5. The plot of calibration curve levels against their analytical response, in
day three validation for candesartan
86
6. The plot of linearity of calibration curve levels for candesartan
quantification against their analytical response and regression linear
equation
89
7. The plot of QC samples against a calibration curve, obtained from
freshly spiked calibration standard, showing candesartan stability
autosampler procedure at 4°C
91
8. The plot of QC samples against a calibration curve, obtained from
freshly spiked calibration standard, showing candesartan bench
stability
93
9. The plot of QC samples against a calibration curve, obtained from
freshly spiked calibration standard, showing candesartan freeze and
thaw stability
95
10. Rat Plasma Profile Showing the Changes in Mean Serum Candesartan
Concentration with time after Drug Administration, each data point
represents the mean ± SEM (n=8)
98
11. Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, comparing
candesartan with grapefruit juice and solitary drug use, each data point
represents the mean ± SEM (n=3)
98
12. Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, comparing
candesartan with licorice juice and solitary drug use , each data point
represents the mean ± SEM (n=6)
99
13. Rat plasma profile showing the changes in mean serum candesartan 99
X
concentration with time after drug administration, comparing
candesartan with pomegranate juice and solitary drug use , each data
point represents the mean ± SEM (n=3)
14. Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration comparing combined
and solitary drug use
100
15. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of
drug administration between single and combined drug use
102
16. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug
administration between single and combined drug use
103
17. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug
administration between single and combined drug use
104
18. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug
administration between single and combined drug use
105
19. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 4 hours of drug
administration between single and combined drug use
106
20. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug
administration between single and combined drug use
107
21. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of
drug administration between single and combined drug use
108
22. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug
administration between single and combined drug use
109
23. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug
administration between single and combined drug use
110
24. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug
administration between single and combined drug use
111
25. Diagram with error bars comparing the mean (with its 95% confidence 112
XI
interval) serum candesartan drug concentration after 4 hours of drug
administration between single and combined drug use
26. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug
administration between single and combined drug use
113
27. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of
drug administration between single and combined drug use
114
28. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug
administration between single and combined drug use
115
29. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug
administration between single and combined drug use
116
30. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug
administration between single and combined drug use
117
31. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 4 hours of drug
administration between single and combined drug use
118
32. Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug
administration between single and combined drug use
119
33. Candesartan blank chromatogram
34. Candesartan zero chromatogram
35. Candesartan LLOQ chromatogram
36. Candesartan QC Mid chromatogram
XII
List of Tables
Table
No.
Caption Page
No.
1. Definitions and classification of blood pressure levels 3
2. Interaction of angiotensin II receptor blockers with other drugs 8
3. Physical and chemical properties of candesartan 17
4. Liqourice identification 21
5. Pomegranate identification 24
6. Orange identification 26
7. Spiked plasma samples 61
8. QC Spiked plasma samples 62
9. Summary table of Chromatographic Conditions and Mass Spectrometric
Conditions
64
10. The mean measurement error and accuracy for candesartan validation
experiment on the first day at 4 selected target concentrations (day 1)
72
11. The mean measurement error and accuracy for candesartan validation
experiment on the first day at 4 selected target concentrations (day 2)
73
12. The mean measurement error and accuracy for candesartan validation
experiment on the first day at 4 selected target concentrations (day 3)
74
13. Intra-day precision and accuracy data for LLOQ samples of candesartan
based on the standard calibration curve of day one validation
75
14. Intra-day precision and accuracy data for QC low samples of
candesartan based on the standard calibration curve of day one
validation
75
15. Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day one
validation
76
16. Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day one
validation
76
17. Intra-day precision and accuracy data for LLOQ samples of candesartan
based on the standard calibration curve of day two validation
77
18. Intra-day precision and accuracy data for QC low samples of 77
XIII
candesartan based on the standard calibration curve of day two
validation
19. Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day two
validation
78
20. Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day two
validation
78
21. Intra-day precision and accuracy data for LLOQ samples of candesartan
based on the standard calibration curve of day three validation
79
22. Intra-day precision and accuracy data for QC low samples of
candesartan based on the standard calibration curve of day three
validation
79
23. Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day three
validation
80
24. Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day three
validation
80
25. Inter day accuracy and precision for the quality control samples of
candesartan in the three days of validation
81
26. Standard calibration curve of day one validation, intraday accuracy data
for candesartan
83
27. Raw data of the standard curve with regards to correlation, slope, R2,
and intercept for day one for candesartan
83
28. Standard calibration curve of day two validation, intraday accuracy data
for candesartan
84
29. Raw data of the standard curve with regards to correlation, slope, R2,
and intercept for day two for candesartan
85
30. Standard calibration curve of day three validation, intraday accuracy
data for candesartan
85
31. Raw data of the standard curve with regards to correlation, slope, R2,
and intercept for day three for candesartan
86
32. Linearity and linear working range of six standard curves of candesartan
data based on the measured concentration
87
33. Linearity and linear working range of candesartan data based on 87
XIV
normalized concentration derived from standard calibration curves
34. Linearity and linear working range of six standard curves of candesartan
data based on the calculated area ratio
88
35. Raw data of six standard curves with regards to correlation, slope, R2,
and intercept for candesartan
88
36. Candesartan QC low Samples stability autosampler procedure at 4°C 89
37. Candesartan QC high Samples stability autosampler procedure at 4°C 90
38. Calibration curve for QC samples showing Candesartan stability
autosampler procedure at 4°C
90
39. Raw data of six standard curves with regards to correlation, slope, R2,
and intercept for candesartan
90
40. Candesartan QC low Samples stability after preparation at room
temperature
91
41. Candesartan QC high Samples stability after preparation at room
temperature
92
42. Calibration curve for QC samples showing bench stability for
candesartan
92
43. Raw data of six standard curves with regards to correlation, slope, R2,
and intercept for candesartan
92
44. The accuracy of three standard curves of candesartan showing freeze
thaw stability of QC low
93
45. The accuracy of three standard curves of candesartan showing freeze
thaw stability of QC high
94
46. Calibration curve for QC samples showing freeze and thaw stability for
candesartan
94
47. Raw data of six standard curves with regards to correlation, slope, R2,
and intercept for candesartan
94
48. Pharmacokinetic data of candesartan 97
49. Comparing the mean serum candesartan drug concentration at selected
time intervals after administration between single and combined drug
use
100
XV
Abbreviations
11b-HSD 11β-Hydroxysteroid dehydrogenase
ACE Angiotensin Converting Enzyme
AHH Aryl hydrocarbon hydroxylase
AT II Angiotensin II
API Atmospheric pressure ionization
ARBs Angiotensin Receptor Blocker
AUC Area under the curve
BC Before Christ
BP Blood pressure
C.V% Coefficient of variation
CAD Collision Gas
CE Collision energy
CEP Collision impotence potential
CL Clearance
C max Maximum concentration
Conc. Concentration
CUR Curtain Gas
CVD Cardiovascular Disease
CXP Collision cell exiting potential
CYP Cytochrome P
Dbp Diastolic blood pressure
DMSO Dimethyl sulfoxide
ECG Electro cardiogram
EMA European Medicines Agency
EP Entrance potential
FA Formic acid
FDA Food and drug administration
XVI
GC Gas chromatography
GS1 Ion Source Gas 1
GS2 Ion Source Gas 2
HCTZ Hydrochlorothiazide
HDL High density lipoprotein
HMGCoA 3-hydroxy-3-methylglutaryl-coenzyme A
HPLC High performance liquid chromatography
ICH International conference of harmonization
IS Internal standard
ISV Ion spray voltage
IV Intravenous
JPM Jordanian pharmaceutical manufacturing
K Da Kilo Dalton
LC Liquid chromatography
LC-MS Liquid chromatography–mass spectrometry
LFO Licorice flavonoid oil
LLOQ Low limit of quantification
MRM Multiple reaction monitoring
MS Mass spectrometry
NEB Nebulizer gas
NMR Nuclear magnetic resonance
NSAIDS Non-steroidal anti-inflammatory
P Probability
PC Paper chromatography
PDA Photodiode array
QC Quality Control
R Correlation
r.p.m. Run per minute
R2 Determination coefficient
RSD Relative standard deviation
XVII
Sbp Systolic blood pressure
SD Standard deviation
SE Standard error
STD Standard
T Temperature
T max Time needed to reach maximum concentration
TLC Thin layer chromatography
tR Retention time
ULOQ Lower limit of quantification
USFDA United States food and drug administration
USP United state pharmacopeia
UV Ultra violet
Vd Volume of distribution
1
CHAPTER ONE
INTRODUCTION
2
1. Introduction
1.1. Hypertension
Hypertension is a global health problem, (Lopez A, et al.. 2006), it is defined as either
a sustained systolic blood pressure (sbp) of greater than 140mm Hg or a sustained
diastolic blood pressure (dbp) of greater than 90mm Hg.Hypertension results from
increased peripheral vascular smooth muscle tone, (Human Hypertension advance
online publication, 2010), which leads to increased arteriolar resistance and reduced
capacitance of the venous system. In most cases, the cause of the increased vascular
tone is unknown. Elevated blood pressure is an extremely common disorder affecting
approximately 15% of the population of the united state (60million people). Although
many of these individuals have no symptoms, chronic hypertension—either systolic
of diastolic—can lead to cerebrovascular accidents (strokes), congestive heart failure,
myocardial infarction, and renal damage. The incidence of morbidity and mortality
significantly decreases when hypertension is diagnosed early and is properly
treated(American Heart Association Heart And Stroke Statistics update, 2005).In
recognition of the progressive nature of hypertension, the sixth report of the joint
national committee classifies hypertension into categories for the purpose of treatment
management.( Seventh report of the joint National committee on Detection,
Evaluation and Treatment of High Blood Pressure2003), The diagnosis of
hypertension is made when the average of 2 or more diastolic BP measurements on at
least 2 subsequent visits is ≥90 mm Hg or when the average of multiple systolic BP
readings on 2 or more subsequent visits is consistently ≥140 mm Hg. Isolated systolic
hypertension is defined as systolic BP ≥140 mm Hg and diastolic BP <90 mm
Hg.(Table 1) Individuals with high normal BP tend to maintain pressures that are
above average for the general population and are at greater risk for development of
3
definite hypertension and cardiovascular events than the general population. With the
use of these definitions, it is estimated that 43 million people in the United States have
hypertension or are taking antihypertensive medication, which is ≈24% of the adult
population.
Table 1. Definitions and Classification of Blood Pressure Levels.
Category Systolic, mm Hg Diastolic, mm Hg
Optimal <120 And <80
Normal <130 And <85
High normal 130–139 Or 85–89
Hypertension
Stage 1 (mild) 140–159 Or 90–99
Subgroup: borderline 140–149 Or 90–94
Stage 2 (moderate) 160–179 Or 100–109
Stage 3 (severe) ≥180 Or ≥110
Isolated systolic hypertension ≥140 And <90
Subgroup: borderline 140–149 And <90
*From JNC (Seventh report of the joint National committee on Detection, Evaluation
and Treatment of High Blood Pressure2003... (JNC-7) JAMA 289: 2560).
1.1.1 Hypertension Classification
Hypertension is classified in to two types primary (essential) hypertension and
secondary hypertension; Essential hypertension is a heterogeneous disorder, with
different patients having different causal factors that lead to high BP about 90–95% of
cases are categorized as "primary hypertension" which means high blood pressure
with no obvious underlying medical cause.(Career OA, et al.., 2000), The remaining
5–10% of cases (secondary hypertension) are caused by other conditions that affect
the kidneys, arteries, heart or endocrine system. (Edward Onusko, 2003)
4
1.1.2 Management
Lifestyle modifications
The first line of treatment for hypertension is lifestyle modification (changes), as with
prevention the life style modification includes dietary changes, physical exercise, and
weight loss. These have all been shown to significantly reduce blood pressure in
people with hypertension. If hypertension is high enough,, lifestyle modification in
conjunction with medication is recommended (Huang N, et al.. 2008).
Medications
To certain extent, lifestyle modification is not enough to control a patient's blood
pressure, which leaves them at risk of coronary heart disease, stroke and renal failure.
(National Heart Foundation of Australia, 2008).
―Antihypertensive drugs‖ refers to multiple classes of medications, used for treating
hypertension, the choice of antihypertensive drug should be based on the patient's age
and the presence of associated clinical conditions. ( Wright JM, Musini VM (July
2009).
1.2. Antihypertensive Drugs:
Hypertensive patients who takes antihypertensive drugs are advised to follow a
sodium restricted diet, antihypertensive drugs is classified in to 7 main catigories,
these are:
1. Diuretics.
2. Beta adrenergic blockers.
3. Calcium channel blockers.
5
4. Angiotensin converting enzyme inhibitors.
5. Angiotensin receptor antagonist.
6. Sympatholytics and adrenergic blockers.
7.Alpha blockers.
Combination therapy
Approximately 60% of patients with elevated blood pressure will not achieve their
blood pressure targets with monotherapy. Most patients will require a combination of
two or more drugs to achieve adequate blood pressure control. There are several
effective combinations, In the accomplish trial an ACE inhibitor and calcium channel
blocker combination reduced cardiovascular events more than a combination of the
ACE inhibitor with a diuretic.( Kjeldsen SE, et al.. 2008).
1.2.1 The Angiotensin II Receptor Blockers (ARBs)
The Angiotensin II Receptor Blockers (ARBs) were developed to overcome several of
the deficiencies of ACE inhibitors. The Angiotensin II receptor blockers (ARBs) are
used by patients with kidney disease, and heart failure. But it is mainly used for
treatment of hypertension, ( Steven G. Terra, 2003), (Hypertension is defined as either
a sustained systolic blood pressure (sbp) of greater than 140mm Hg or a sustained
diastolic blood pressure (dbp) of greater than 90mm Hg.) The Angiotensin II receptor
blockers (ARBs) are alternatives to the ACE inhibitors which are relatively
nonspecific enzyme and has substrates other than angiotensin, and so, inhibition of
ACE may result in accumulation of these substrates. The Angiotensin II receptor
6
blockers (ARBs) drugs block the AT1 receptors more specifically .(Joel M. Neutel, et
al.., 2007), Losartan is the prototypic ARBs; currently there are 6 additional ARBs,
candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan and valsartan, all
of the ARBs types are have similar clinical profiles with different pharmacokinetic
profiles( Dhiren K Patel, et al..,2013), The Angiotensin II produces a number of
effects that eventually leads to an increase in blood pressure and affects other
organs(heart and kidney) these effects include the activation of the sympathetic
nervous system, Constriction of blood vessels, increased salt and water retention,
,stimulation of blood vessel and heart fibrosis (stiffening).( Steven G. Terra, 2003),
Angiotensin receptor blockers prevent Angiotensin II from binding to its receptor and
thus reduce the effects of Angiotensin II. Most of the angiotensin receptor blockers
are available either alone or in combination with an additional medication called
hydrochlorothiazide (HCTZ), a diuretic that is very effective in lowering blood
pressure. The blood pressure-lowering effects of angiotensin receptor blockers are
made more effective by the addition of HCTZ. Pharmacologicaly, the effect of The
Angiotensin II receptor blockers (ARBs) are similar to those of ACE inhibitors in
that they produce arteriolar and venous dilation and block aldosterone secretion, thus
lowering blood pressure and decreasing salt and water retention.( Steven A. Atlas,
2007). ARBs do not increase bradykinin levels, ARBs decrease the nephrotoxicity of
diabetes making them an attractive therapy in hypertensive diabetics.( Earshad
Md,2012) Their adverse effects are similar to those of ACE inhibitors, although the
risks of cough and angioedema are significantly decreased.(Takeshi Morimoto MD
MPH, 2004) ARBs are also fetotoxic.( P Rachael James, 2004) Angiotensin II
receptor antagonists work by antagonizing the activation of angiotensin
receptors(Richard Finkel et al. 2009).
7
1.2.2 ARBs Drug Interactions
Concomitant use of ARBs with certain drugs affects both drugs and result in
fluctuations of their effectiveness for example lithium when taken with ARB's leads
lithium toxicity due to increased renal reabsorption of lithium. Another example is
Indomethacin which was reported to cause a decreased in the effectiveness of
losartan. (Hutchison TA, Shahan, 2001; Böhler S, 2005), table 2 shows Interactions of
angiotensin II receptor blockers with other drugs.
Table 2 Interactions of Angiotensin II Receptor Blockers with other Drugs
Precipitant drug Object drug CYP450 substrate Effect
Cimetidine Losartan −− ↑
Fluconazole Losartan 3A4, 2C9 ↑
Indomethacin Losartan − ↓
Phenobarbital Losartan − ↓loartan
↓ active metabolite
Rifampin Losartan 3A4, 2C9 ↓ losartan
Telmisartan Digoxin − ↑ digoxin
(Olin BR, 2002; Burnier M. 2001; Rodgers JE,2001).
1.2.3 The Side Effects of ARBs Include:
Dizziness, lightheadedness, or faintness upon rising, This side effect may be strongest
after the first dose, especially if you have been taking a diuretic (water pill).
Physical problems, Diarrhea, muscle cramps or weakness, back or leg pain,
insomnia, irregular heartbeat, upper respiratory infection.
8
Confusion and Abnormalities in blood chemistry laboratory tests.
Cough, (less than with ACE inhibitors), table 3 listed the side effects from angiotensin
II receptor blockers, (Sankyo Pharma Inc (US), 2002; Olin BR, 2002).
1.2.4 Actions of Angiotensin II
the rennin-angiotensin system plays a key role in the regulation of fluid and
electrolyte balance and arterial blood pressure. Excessive activity of the rennin-
angiotensin system can result in hypertension and disorders of fluid and electrolyte
homeostasis.
When rennin is released into the blood, it acts upon a circulating
substrate, angiotensinogen, that undergoes proteolytic cleavage to form the
decapeptide angiotensin I. Vascular endothelium, particularly in the lungs, has an
enzyme, angiotensin converting enzyme (ACE), that cleaves off two amino acids to
form the octapeptide, angiotensin II (AII), although many other tissues in the body
(heart, brain, vascular) also can form AII.( Fyhrquist, F. et al.. 1995)
9
Figure 1: The renin-angiotensin-aldosterone pathway
As seen in Figure 1, The renin-angiotensin-aldosterone pathway is regulated by the
mechanisms that stimulate rennin release and by natriuretic peptides released by the
heart. These natriuretic peptides acts as an important counter-regulatory system.( Jia
L. Zhuo, et al..,2011).
Angiotensin II is an octapeptide hormone, it was named on the basis of its first main
biological function that is the ability to act as a vasoactive agonist to induce
contraction of blood vessels.(Mark B. Taubman, 2003), Angiotensin II exerts
important actions at vascular smooth muscle, adrenal cortex, kidney, heart, and brain,
and so Ang II has been shown to play important roles in mediating hypertension, heart
failure, cardiac remodeling, diabetes, and the proliferative and inflammatory
responses to arterial injury through these actions(Crackower, M. A. et al.. 2002).
Angiotensin II is a very potent pressor agent, large portion of pressor response is due
to direct contraction of vascular—especialy arteriolar—smooth muscle. In addition
Angiotensin II can also increase blood pressure through actions on brain and
10
autonomic nervous system. (Phillips MI, et al.., 2002).Angiotensin II also interact
with autonomic ganglia, increases the release of epinephrine and norepinephrine from
the adrenal medulla, and facilitates sympathetic transmission by an action at
adrenergic nerve terminals. (Campos AH, et al.., 2003).The latter effect involves both
increased release and reduced reuptake of norepinephrine. Angiotensin II also has a
less important direct positive inotropic action on the heart.
Numerous studies examining the cellular effects of Ang II. Showed that signals
induced by Ang II in vascular smooth muscle cells (SMCs) and cardiomyocytes are
associated with contraction, it was found that Ang II activated phospholipase C,
resulting in the production of inositol trisphosphate (IP3) and diacylglycerol, which in
turn responsible for the mobilization of [Ca2_] I and the activation of protein kinase C
(PKC), respectively. ( Ruiz-Ortega M, et al.., 2001).
These findings have spawned the development of several classes of pharmacological
agents designed at inhibiting the synthesis of Ang II, eg, angiotensin II– converting
enzyme inhibitors or blocking its action as with angiotensin II receptor antagonists,
(Lonn, E. M et al.. 1994).
11
1.3. Candesrtan
Chemically, Candesartan is 2-ethoxy-3-[[4-[2-(2H-tetrazol-5 yl) phenyl] phenyl]
methyl] benzimidazole-4-carboxylic acid. Figure 2, (IUPHAR database; Kubo, K. et
al.. 1993).
Figure 2: candesartan chemical structure
12
1.3.1 Identification
Table 3: Physical and Chemical properties of candesartan(astrazenka inc. 2013)
Identification
Name Candesartan
Accession Number DB00796 (APRD00420)
CAS number 139481-59-7
Weight Average: 440.454
Monoisotopic: 440.159688536
Chemical Formula C24H20N6O3
Solubility Practically insoluble in water
Half life 5-10 hours
Protein binding >99%
Melting point 157-160 C
logP 4.02
logP 5.05
Logs -4.8
pKa (strongest acidic) 2.97
pKa (strongest basic) 1.71
physiological charge -1
hydrogen acceptor count 7
hydrogen donor count 2
rotatable bond count 7
Refractivity 134.92
Polarizability 45.35
* * available online at (ChemAxon, ALOGPS).
From table 3 candesartan chemical formula is C24H20N6O3
With Clearance: 0.37 mL/min/kg. and Volume of distribution: 0.13 L/kg.
Bioavailability: 15% (candesartan cilexetil), it is highly protein bonded
Candesartan cilexetil is a white to off-white powder with a molecular weight of
610.67. It is practically insoluble in water and sparingly soluble in methanol. With
melting point of 157-160 C Candesartan cilexetil is a racemic mixture containing one
chiral center at the cyclohexyloxycarbonyloxy ethyl ester group.
13
Candesartan is administered as candesartan celextil, commonly known as (±) -1-
[[(cyclohexyloxy)carbonyl]oxy]ethyl 2-ethoxy-1-[[2` -(1H-tetrazol -5- yl)[1,1`-
biphenyl]-4-yl]methyl]-1h-benzimidazole-7-carboxylate 1, which has better
availability than candesartan, the prodrug is rapidly and completely hydrolyzed to
candesartan during absorption by the gastrointestinal tract.
Candesartan belongs to the class of ARBs and binds to angiotensin ІІ receptor
type1 selectively and competitively, thus preventing action of angiotensin ІІ
and decreasing the blood pressure levels.( Ferreirós N, et al., 2007).
Candesartan is Metabolized by
CYP2C9 *1, CYP2C9 *2, and CYP2C9 *3 to O-Deethylated candesartan
UDP-glucuronosyltransferase 1-3 to Candesartan N2-glucuronide.
Prostaglandin G/H synthase 1 to Candesartan O-glucuronide.( Hanatani T, et
al. , 2001).
1.3.2 Mechanism of action
Candesartan blocks the vasoconstrictor and aldosterone-secreting effects of
angiotensin II by selectively blocking the binding of angiotensin II to the
AT1 receptor in many tissues including vascular smooth muscle and the adrenal
gland. and results in an overall decrease in blood pressure. ( Detroja C, et al.. 2011).
Candesartan does not bind to or block other hormone receptors or ion channels known
to be important in cardiovascular regulation, and so its action is, independent of the
pathways for angiotensin II synthesis. ( Easthope SE, et al.. 2002).
14
Candesartan is 10000 times more selective for AT1 than AT2, but AT2 is not known
to be associated with cardiovascular homeostasis(Kirk, J. K.et al.. 1999). Inhibition of
aldosterone secretion may increase sodium and water excretion while decreasing
potassium excretion. (Gasanin E, et al.. 2013).
candesartan does not inhibit ACE (kininase II), and so it does not affect the
bradykinin levels. Blockade of the angiotensin II receptor inhibits the negative
regulatory feedback of angiotensin II on renin secretion, but the resulting increased
plasma renin activity and angiotensin II circulating levels do not overcome the effect
of candesartan on blood pressure. (Husain A, Azim, et al.. 2011).
1.3.3 Indication and Clinical use
Candesartan is indicated for:
Hypertension
Candesartan cilexetil is indicated for the treatment of hypertension in adults and
children 1 to < 17 years of age. It may be used alone or in combination with other
antihypertensive agents. . (Karlson BW, et al.. 2009).
It may be used as a first line agent to treat uncomplicated hypertension, isolated
systolic hypertension and left ventricular hypertrophy., candesartan is also used for
patient with diabetic nephropathy, it slows the progression of nephropathy by
reducing albuminuria.( Hovind P, 2001; Hovind P, 2004).
Heart Failure
Candesartan cilexetil is indicated for the treatment of heart failure (NYHA class II-
IV) in adults with left ventricular systolic dysfunction (ejection fraction ≤ 40%),
15
myocardial infarction and coronary artery disease in those intolerant of ACE
inhibitors. (Malmqvist K, 2000) to reduce cardiovascular death and to reduce heart
failure hospitalizations. Candesartan cilexetil also has an added effect on these
outcomes when used with an ACE inhibitor (Fei Yu a, et al.. 2008; Ross A,
Papademetriou V, et al.. 2004).
The antihypertensive effect of candesartan cilexetil has been documented in doses up
to 16mg/day in European studies, receptor blockade by candesartan is more potent
than that by valsartan, irbesartan or losartan in vitro and it has been shown than
candesartan binds more tightly to and dissociates more slowly from the AT1 receptor
than those other ARBs. (US Food and Drug Administration, 2006; Karlson BW, et
al.. 2009).
1.3.4 Contraindication
Candesartan is contraindicated in patients who are hypersensitive to candesartan. Do
not co-administer aliskiren with candesartan in patients with diabetes.
Candesartan is FDA pregnancy category D Use of candesartan is not recommended
during the second and third trimesters of pregnancy, reports state that using
candesartan reduces renal function and increase fetal and neonatal morbidity.( Ouaret,
S et al.. 2004).
Breast feeding mother, because no information is available on the use of candesartan
during breastfeeding, an alternate drug may be recommended.
Severe hepatic impairment and/or cholestasis.( Gleiter, et al. 2004).
16
1.3.6 Side effects
Candesartan may cause side effects:
Dizziness or lightheadedness may occur as your body adjusts to the medication,
Headache, back pain, sore throat.
But Some side effects can be serious, swelling of the face, throat, tongue, lips, eyes,
hands, feet, ankles, or lower legs. Hoarseness, difficulty breathing or swallowing,
decreased urination. (See, S., & Stirling, A. L. et al.. 2000; Granger, C. B.,et al..
2003; Easthope, S. E et al.. 2002)
1.3.7 Candesartan Drug interaction:
Candesartan is not significantly metabolized by cytochrom p 450 systems so any drug
metabolized by this system will be not affected by candesartan. (Jonkman JH, et al..,
1997).
Clinical pharmacokinetic studies on hydrochlorothiazide, warfarin, digoxin, oral
contraceptives, glibenclamide, nifedipine and enalapril. Shows No clinical significant
pharmacokinetic interactions with candesartan, on the other hand some of chronically
used drug do interact with candesartan, table 4 listed drugs reported to intweract with
candesartan.( Easthope, S. E, et al. 2002).
As with ACE inhibitors, concomitant use of candesartan and NSAIDs may lead to an
increased risk of worsening of renal function,this include acute renal failure, and an
increase in serum potassium. (Jain, S. et al.. 2005).
17
Table 4 The mainly drugs candesartan have been reported to interact with.
Drug Interaction
Amiloride Increased risk of hyperkalemia
Drospirenone Increased risk of hyperkalemia
Lithium The ARB increases serum levels of lithium
Potassium Increased risk of hyperkalemia
Spironolactone Increased risk of hyperkalemia
Tobramycin Increased risk of nephrotoxicity
Trandolapril The angiotensin II receptor blocker, Candesartan, may increase
the adverse effects of Trandolapril.
Treprostinil Additive hypotensive effect. Monitor antihypertensive therapy
during concomitant use.
Triamterene Increased risk of hyperkalemia
(Andersson OK, et al.. 1998)
1.3.9 Pharmacokinetic data
Candesartan inhibits the effects of angiotensin II infusion in a dose-dependent
manner. After 1 week of once daily dosing with 8 mg of candesartan cilexetil, the
pressor effect was inhibited by approximately 90% at peak with approximately 50%
inhibition persisting for 24 hours. (See, S., & Stirling, A. L. et al.. 2000).
Plasma concentrations of angiotensin I and angiotensin II, and plasma renin activity
(PRA), increased in a dose-dependent manner after single and repeated administration
of candesartan cilexetil to healthy subjects, hypertensive, and heart failure patients.
ACE activity was not altered in healthy subjects after repeated candesartan cilexetil
administration. The once-daily administration of up to 16 mg of candesartan cilexetil
to healthy subjects did not influence plasma aldosterone concentrations, but a
decrease in the plasma concentration of aldosterone was observed when 32 mg of
candesartan cilexetil was administered to hypertensive patients. In spite of the effect
of candesartan cilexetil on aldosterone secretion, very little effect on
18
serum potassium was observed. (AstraZeneca LP, Wilmington, DE 19850, 2013,
©AstraZeneca 2010, 2013).
Absorption
After administration of the candesartan cilexetil (prodrug), candesartan cilexetil is
rapidly and completely biaoactivated by ester hydrolysis at the ester link to form the
active candesartan during absorption from the gastrointestinal (GI) tract McClellan
and Goa, 1998. Oral administration of candesartan shows low bioavailability,
approximately 15% in humans, due to its low water (pKa 6.0) solubility (Vijaykumar
et al., 2009) and efflux by drug resistance pumps in the gastrointestinal tract, limiting
the oral absorption (Zhang et al., 2012; Lee et al., 2009; Kamiyama et al., 2010; Zhou
et al., 2009). High fat content diet shows no affect on the bioavailability of
candesartan. (Joseph I. Boullata et al.., 2010)
Distribution
The volume of distribution of candesartan is 0.13 L/kg. Candesartan is highly bound
to plasma proteins (>99%) and does not penetrate red blood cells. In rats, it has been
demonstrated that candesartan crosses the blood-brain barrier poorly. It has also been
demonstrated in rats that candesartan passes across the placental barrier and is
distributed in the fetus. (Takara K, Kakumoto, et al.. 2002).
Metabolism
The prodrug candesartan cilexetil undergoes rapid and complete ester hydrolysis in
the intestinal wall to form the active drug, candesartan. ( Shantanu Bandyopadhyay,
2013), 75% of candesartan eliminated unchanged in the urine and, by the biliary
route, in the feces. Hepatic metabolism of candesartan (25%) occurs by O-
19
deethylation via cytochrome P450 2C9 to form an inactive metabolite. ( Fei Yu a, et
al.2008; Shantanu Bandyopadhyay, et al.. 2013), Candesartan undergoes N-
glucuronidation in the tetrazole ring by uridine diphosphate glucuronosyltransferase
1A3 (UGT1A3). O-glucuronidation may also occur. (Drug Metab. Dispos. (2010)
12:2302-2308; Takara K, Kakumoto, et al.. 2002).
Excretion:
Route of Elimination, when candesartan is administered orally, it is mainly excreted
unchanged (75%) in urine and feces (via bile). Renal 33%, faecal 67%.( Detroja C, et
al.. 2011).
Candesartan cilexetil is rapidly and completely bioactivated by ester hydrolysis
during absorption from the gastrointestinal tract to candesartan, Candesartan is mainly
excreted unchanged in urine and feces (via bile). It undergoes minor hepatic
metabolism by O-deethylation to an inactive metabolite. The elimination half-life of
candesartan is approximately 9 hours. Following single and repeated candesartan
administration, the pharmacokinetics of are linear for oral doses up to 32 mg of
candesartan cilexetil. (Detroja C, et al.. 2011)
As previously mentioned the absolute bioavailability of candesartan was estimated to
be 15%. After tablet ingestion, the peak serum concentration (Cmax) is reached after
3 to 4 hours. Food with a high fat content does not affect the bioavailability of
candesartan after candesartan cilexetil administration. (Hübner, R., et al.. 1997).
20
1.3.10 Toxicity
The Lethality was not observed when Candesartan were given as a single oral dose of
up to 2000 mg/kg , alone or in combination with 1000 mg/kg of hydrochlorothiazide
for both rats and dogs subjects. In mice given single oral doses of the primary
metabolite, candesartan, the minimum lethal dose was between 1001 mg/kg and
2000 mg/kg. (McClellan, K. J., & Goa, K. L. (1998).
1.4. Juices
Among all fruit juices, previously published data shows that grape fruit juice (GFJ)
have an interaction with many types of drugs. (GFJ) can alters the metabolism of the
medication by the body. Many reports have documented that drug interactions with
GFJ that occur through inhibition of CYP3A enzymes. other documented data
hypotheses that compounds present in grapefruit juice act as an inhibitor of the P-gp
activity which leads to the disposition of drugs that are P-gp substrates such as
talinolol.(Schwarz UI, et al.. 2005).
21
1.4.1 Liquorice
Table 5 liquorice scientific classification
Scientific classification
Kingdom Plantae
(unranked) Angiosperms
(unranked) Eudicots
(unranked) Rosids
Order: Fabales
Family: Fabaceae
Subfamily: Faboideae
Tribe: Galegeae
Genus: Glycyrrhiza
Species: G. glabra
Binomial name
Glycyrrhiza glabra
Synonyms
Glycyrrhiza glandulifera
Liquorices (Glycyrrhiza glabra) (table 5) is a tall shrub of the Leguminosae Family,
(Olukoga and Donaldson, 1998), There are about 14 species known like var.Typical,
var.glandulifera, var.violacea and var.lepidota. liquorice is one of the most popular
herb, it is widely used in both medicinal area and industrial area, the liqourice
originates from the warm regions of the word, it was firstly used by the pre-date
ancient civilization of Babylonian, Egyptian and Chinese cultures(Wang, Ma, Fu,
Lee, & Wang, 2004), (Fenwick et al.., 1990), Liqourice harvesting occurs in the
autumn of its third or fourth year of growth.
Liqurice root is the most part used. When harvested, the roots are dogged up, washed,
boiled, sorted and finally dried. The dried roots are crushed boiled to make the
extract. (Carmines et al., 2005).
Scribonius Largus (I century a.d.) indicated that liquorice was a valid remedy for
problems of the arteries (Scribonius, edition 1983). According to Ibn Al Baithar, Ibn
22
Sina (X century) stated that it is beneficial in palpitations (Von Sontheimer, 1842).
Hildegard von Bingen (1098–1179), advised the use of liquorice (which she called
liquiricium): together with fennel and honey, it could be useful for de cordis dolore
(with great likelihood: angina pain) (Hildegard von Bingen, edition 1903).
Chemically, liquorice roots contain several triterpenes, such as glycyrrhizin and
glycyrrhetic acid, together with variety of flavones,
isoflavones, chalcones. The constituent content varys on the base of species and
region of growth. (Leung and Foster, 1996).
Glycerhizien glycoside is the main active ingrideint of liquorice, it is a sweet-tasting
constituent. Making it 50 times sweeter than sugar, (Acharya,
Dasarathy, Tandon, Joshi, & Tandon, 1993).
Glycerhizien is hydrolysed by the intestinal bacteria and then absorbed
into blood only in the form of glycyrrhetic acid. (Ploeger, Mensinga, Sips,
Meulenbelt, & DeJongh, 2000).
The yellow color of liqourice root is related to liquiritin, isoliquiritin a Xavonoids
derivative. (Northern Echo, 2008).
The effect of glycyrrhiza uralensis, showed induction effect on CYP450 isozymes.
Efficacy and safety profiles of a drug may be affected when it administered
concomitantly with liqourice (Tang et al.., 2009). and 7-ethoxycumarin O-deethylase
(ECOD, 2.8 and 2.5 fold) were also shown to be increased (Asl and Hosseinzadah
2008). The metabolic rate of the drug, given concomitantly with liqourice, in the liver
microsomes was significantly higher in the herb pretreated rats. The pharmacokinetic
23
profile of the drug was significantly modified in the rats with the herbal pretreatment.
Elimination half-lives were shortened, and total clearances were increased, with the
pretreatment of glycyrrhiza uralensis. (Tang et al.., 2009).
As previously mentioned, liquorice has made its way and brought the attention in the
medical area due to its wide benefits, the medical use include:
Antimicrobial, Anti-virus. (Harada, 2005), Anti-atherostatic., Anti-hyperlipidemic.,
Hepatoprotective, Hepatitis treatment. (Orlent et al.., 2006; Sato et al.., 1996), Anti-
allergic, Anti-inflammatory. (Cho et al.., 2010), Anti-ulcer activities, Antioxidant
effects. (Cheel et al.., 2010; Visavadiya & Narasimhacharya, 2006), Tonic
expectorant, and in the immune system alterations.
and recently published report state that liquorice found to inhibit the replication of the
SARS-associated viruses. (Okimasu et al.., 1983), (Huang, 1993), (Akamatsu et al..,
1991), (Anon, 2005), (Hattori et al.., 1989; Hirabayashi et al.., 1991; Pliasunova et
al.., 1992), (Schulz et al.., 1998), (Nagai et al.., 1992), (Wang et al.., 2000), (Hikino,
1985), (Cinatl et al.., 2003), (Hattori et al..,1989), However, the quality and efficacy
of liqourice Differs according to the growing conidition, part of plant Used and also
to the area in which it was planted in (Demizu et al.. 1988; Hatano et al.., 1988;Okada
et al.., 1989). As with other herbs, liquorice use may precipitate some side effects that
must be taken in consideration (Eurekalert press, 2009).
In vitro study proves that liquorice can inhibit the functions of P-gp and CYP-
dependent monooxygenase. (Wang et al.., 1994), (Takeda et al.., 1979), (Huang et
al.., 2008), (Yoshida, Koizumi, Adachi, & Kawakami, 2006), (Paolini, Pozzetti,
Sapone,& Cantelli-Forti, 1998).
24
1.4.2 Pomegranate:
Table 6 pomegranate identification
Scientific classification
Kingdom Plantae
(unranked) Angiosperms
(unranked) Eudicots
(unranked) Rosids
Order: Myrtales
Family: Lythraceae
Genus: Punica
Species: P. granatum
Binomial name
Punica granatum L.
Synonyms
Punica malus
As seen in table 6 Pomegranate (punica granatum) from the family ―Lythraceae‖.
Pomegranate fruit made its way into the news recently due to its huge reported
benefits.( Arpita Basu, et al..2009).
The plant Grown on shrub-like trees with orange flowers and glossy leaves from
October to December. Its original native is Persian, and is cultivated in North Africa,
Asia and especially in the Middle East.( Sarkhosh et al.., 2006).
pomegranate are consumed fresh or transformed into fresh juices, beverages, jellies
and flavoring and coloring agents. (Oukabli et al.., 2004).
Historically; Pomegranates feature prominently in Islam, Judaism, Christianity,
Buddhism and Zoroastrianism.( Bashar Saad, et al.. 2011).
Pomegranate fruit is available around the year but freshly harvested between
September to January. (California Rare Fruit Growers. Crfg.org. 2012; LaRue, et al..
1980).
25
Recent studies proved that Pomegranate show to inhibit CYP3A in the body, thus it
will alter the pharmacokinatics of any drug metabolized by this enzyme.( Hidaka
M, et al.. 2005).
Chemically, pomegranate is a rich source of beneficial compounds. It contains a
potent antioxidant of the polyphenolic Xavonoid class, which includes tannins and
anthocyanins, (De Nigris et al.., 2005; Elfalleh et al.., 2011). Comparing with other
beverages pomegranate juice has showed to have more antioxidants than blueberries,
and cranberries. Another impressive antioxidant that only founded in pomegranate is
punicalagins which have many functions in lowering cholesterol level, blood pressure
and increase the speed at which heart blockages (atherosclerosis) melt away. (Aviram
M, et al.. 2000; Aviram M, et al.. 2004)
As previously mention Pomegranate juice has reduce blood pressure, by reducing
systolic blood pressure and inhibits serum angiotensin converting enzyme(Aviram M,
et al.. 2000). Also to note, pomegranate juice contains phytochemical compounds that
stimulate serotonin and estrogen receptors, thus improve symptoms of depression in
animals experiment, (Mori-Okamoto J, et al.. 2004).
26
1.4.3 Orange:
Table 7 Orange Identification
Scientific classification
Kingdom Plantae
(unranked) Angiosperms
(unranked) Eudicots
(unranked) Rosids
Order: Sapindales
Family: Rutaceae
Genus: Citrus
Species: G. glabra
Binomial name
Citrus × sinensis
Orange juice is probably the best known and most widespread fruit juice all over the
world. It has high content in vitamin C natural antioxidants, such as flavonoids,
phenylpropanoids, hesperidin. it is cholesrtol free, fat free and also it is free from any
added sugars (naturally sweet) (Gianni Galaverna, et al. 2008).with a typical pH of
around 3.5. (Walton B. Sinclair, et al.. 1945).
Orange originally come from a Different cultivars (e.g. Valencia, Hamlin, ruby red
and Mandarin) depending on the cultivars; Orange juice varies between orange to
yellow color with one expiations for ruby red orange that came in reddish-orange.(
Staff of the Citrus Experiment Station, College of Natural and Agricultural Sciences
(1910-2011). 2011).
According to recent report orange juice, acts as an inhibitor of organic anion
transporter proteins (OATPs) based on the report finding that the bioavailability of the
antihistaminic drug fexofenadine in humans was reduced with Orange juice intake,
(Dresser et al.. 2002).
27
Other report suggest that orange juice can inhibit intestinal cytochrome P450 (CYP)
isozyme 3A4 and P-glycoprotein
It was recently shown that vitamin C protects endothelial cells and LDL from either
intra- or extracellular oxidant stress and also may reduce the risk of atherosclerosis
(Nancy Preising Aptekmann, et al.. 2010).
Following are facts about a few of the important nutrients that make orange juice one
of the most naturally healthy beverages around:
Vitamin C is the most important antioxidants and it supports the immune system. As
an antioxidant, orange juice can neutralize free radicals. (Silvia Isabel Rech Franke,
Temenouga Nikolova Guecheva, João Antonio Pêgas Henriques, Daniel Prá. Orange
Juice and Cancer Chemoprevention. Nutrition and Cancer, 2013)
Thiamin which is associated with the conversion of food into energy and the.
Potassium, which is related to muscle function and helps to protect from strokes.
Folate (Folic Acid) is essential for red blood cells.
Calcium (in fortified orange juice) is essential for strong bones.
Vitamin B6 helps the body process energy from the food we eat and is needed for the
production of new cells.
1.5.Chromatography
It is the term used to describe the teqnique of separation compounds by distributed
between two phases, one of which is static and so called ''stationary phase'' and the
other is pass throw carrying it ''mobile phase'' (Ettre, et al., 2001).
28
1.5.1 Types of chromatography
Paper chromatography (PC).
Gas chromatography (GS).
Thin layer chromatography (TLC).
High performance liquid chromatography (HPLC). (Grob et al.., 2004; Skoog, DA et
al.., 2007).
1.6.High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC) is an chromatographic technique
used to separate a mixture of components on the bases of the time each component
needed to pass through a stationary phase when carried through it by a suitable mobile
phase; nowadays HPLC is used extensively in pharmaceutical industry with the
purpose to obtain qualitative, quantitative data about the composition of drugs or to
purify each individual component of the mixture. (Gopu C et al., 2008).
Mikhail Tswestt, a Russian botanist is the first scientist used chromatography to
separate plant pigments. (Tswett, M. S. 1906)
HPLC system mainly depends on three components
The stationary phase, The mobile phase and The analyte. (Liu Y. eatal,2006).
The stationary phase usually placed in the column which is usually made of inert
materials (stainless steel), depending on the polarity of sample; stationary phase is
either 'normal phase' or 'reserved phase'. ( William T. Cooper,2006; Akul
Mehta,2013).
29
The mobile phase is the liquid phase solvent that contain the analyte to be tested, it
flows continually through the stationary phase, the composition of the mobile phase
depends on the composition of both analyte and stationary phase. (Molnár etal,2013)
HPLC operation based on the bases of a pump that pumps pressurized liquid "mobile
phase" and analyt sample through a column filled with sorbent, then the sample
components will separated from each other due to their degree of interaction with the
used sorbent.
The HPLC instrument typically includes:
mobile phase reservoir (stors the mobile phase to be used).
Degresser.
Pump(which enable the flow of mobile phase through the system).
Injector (introduce the sample in to the system).
Column compartment (control the temperature of the column).
Detector(detects each component in separated mixture after it has eluted from
the column).
Data processor(converts the data from the detector into meaningful results).
Waste reservoir(collects the liquid waste).(Huber, U.,2006; Nägele, E,2002;
Rathore, A.S., 2003; Rosentreter U., 2004).
1.6.1 Types of HPLC
Classification of HPLC is based on the nature of the stationary phase and the
separation process.
A. Adsorption chromatography (liquid-solid): is one of the oldest types of
chromatography. Mobile phase is adsorbed onto the surface of a stationary solid
30
phase, the separation is based on equilibration between the mobile and stationary
phase of different solutes, adsorbent material such as silica gel or any other silica
based packing may be used. (B. S. Baharin etal, 1998).
B.partition Chromatography
This form of chromatography is based on a thin film formed on the surface of a solid
support by a liquid stationary phase. Solute equilibrates between the mobile phase and
the stationary liquid.
C. Ion-exchange chromatography: In this type of chromatography, the mechanism
is to use the stationary phase which is ionized (with opposite charge to the sample) at
the surface to covalently attach anions or cations onto it, where as The mobile phase is
an aqueous buffer Solute ions of the opposite charge in the mobile liquid phase are
attracted to the resin by electrostatic forces. The stronger sample charge, the
attachment to the stationary surface will be stronger, so longer time is needed to elute.
Figure 6 (Tatjana Weiss, 2005; Gjerde, 2000).
D. molecular exclusion chromatography :aka gel filtration chromatography
The technique is used for compounds with high molecular weight like proteins and
polymers.
The mechanism of Separation is sieving not partitioning so larger molecules is to be
eluted rapidly where as smaller molecules will be trapped inside the poruose packing
material to be eluted later.
Stationary phase porous silica or polymer particles(polystyrene, polyacrylamide)
(510mm) (Monika et al.. 2011).
31
E. Affinity Chromatography
This is the most selective type of chromatography employed. It utilizes the specific
interaction between one kind of solute molecule and a second molecule that is
immobilized on a stationary phase. For example, the immobilized molecule may be an
antibody to some specific protein. When solute containing a mixture of proteins are
passed by this molecule, only the specific protein is reacted to this antibody, binding it
to the stationary phase. This protein is later extracted by changing the ionic strength
or pH.
1.6.2 Advantages of HPLC over other chromatographic techniques:
Higher sensitivity e technique.
Can be applied for various types of samples.
Time effective (speed of analysis).
Automated with Greater Reproducibility.
Accurate.
Higher resolution (Snyder L. Et al.., 2011; Daniel J. Cziczo, 2004).
Disadvantages of HPLC
Cost.
Complexity.
Low sensitivity for some compounds.
Irreversibly adsorbed compounds not detected.
Coelution difficult to detect ( Daniel J. Cziczo, 2004; Monika et al.. 2011).
32
1.6.3 HPLC detectors
Routes of detection that are commonly employed include visible radiation methods
(measuring light scattering or refractive index), and absorbance methods (UV or
fluorescence spectroscopy and photodiode array (PDA) detection (Snyder 2011,
Meyer 2013).
These methods can be extremely useful for detecting certain classes of compounds
that either absorb UV or fluoresce. Indeed PDA detectors have been linked to HPLC
prior to MS detectors (Vorst et al.., 2005, Hanhineva et al.., 2008).
Mass detector is just one of many varieties of detector that can be linked to the HPLC
or UHPLC. For full structural elucidation, it is a necessary to employ MS and/or
NMR spectroscopy detectors. The three principal components found in all varieties of
MS are:
An ion source, which can convert gas phase sample molecules into ions.
A mass analyzer, which sorts the ions by their masses by applying electromagnetic
fields
A detector, which measures the value of an indicator quantity and thus provides data
for calculating the abundances of each ion present (Niessen 1998).
The gathering of MS to basic chromatography will introduce us to highly sensitive
analytical techniques.
Back in the middle of the last century the coupling of MS to gas chromatography
(MS-GS) was achieved. This was the first step in the way of introduce the till the
33
1980's, coupling of MS with LC (LC-MS) was almost impossible due to the
incompatibility of MS ion sources at that time with a continuous mobile phase.
At the 1980's, fenn an American chemistry scientist develop another MS ion source
''electrospray ion source''.
Nowadays , wide range of clinical applications of LC-MS, and this is because LC-MS
handle a wide range of single and complex mixture with high specificity.
Mass Spectrometry Instrumentation
MS operate by charging analyte molecules to a ionized state, with subsequent analysis
of the ions that are produced during the ionization process, according to their mass to
charge ratio(m/z).( snyder L. Et al.., 2011).
The following are different Ion Sources used in MS-LC
Electrospray Ionisation Source ESI:
This type of ion source are used with moderaitly polar.
ESI is a ―soft‖ ionization source, which means little energy is imparted to
the analyte, and little fragmentation occurs.
Atmospheric Pressure Chemical Ionisation Source
This type are used for neutraly or non-polar molecules that are thermally stable and
not well ionized by ESI. For APCI; multiple charging is not a feature and singly-
charged ions dominate.
34
Atmospheric Pressure Photo-ionisation
APPI uses photons energy to excite and ionise molecules, to minimise concurrent
ionization of solvents and ion source gases. (Cappiello, A. et al.., 2000).
Mass Analysers
Quadrupole Analysers
The quadrupole analyser consists of a set of four parallel
metal rods. Quadrupole analysers, comes single or triple quadrupole Configuration.
Time-of-flight Analysers
The time-of-flight (TOF) analyser ionize the molecules by high voltage. The velocity
of the ions,and the time required to travel down a flight tube to reach the
detector depends on their m/z values. The TOF analyser is usfull for small molecules.
Ion Trap Analysers
Ion trap analysers use three hyperbolic electrodes to trap ions using static and radio
frequency voltages.23the ejiction of Ions depends on m/z values to create a mass
spectrum.
Hybrid Analysers
mass spectrometers that use combinations of different
mass analysers. Two configurations
that are particularly useful for LC-MS. The
third quadrupole of a triple quadrupole MS can be replaced by
35
a TOF analyser to produce a hybrid quadrupole time-of-flight
(QTOF) mass spectrometer. (Chernushevich IV, 2001; Ens W, Standing KG, 2005).
Performance Monitoring and Practical Considerations
Highly specific MS does not require any chromatograic separation, the injection of
sample in to the ion source will measure analytes in a complex matrix typically in
less than two minutes but as with other analytical producers, LC-MS requires a
number of specific condition and protocols must be taken while handling each
instrument or sample.
System protocols are held to detect any error other than normal performance.
System protocols may include:
sensitivity assurance; by checking the retention time and peak shape of internal
standards.
Checking the ion optics and ion sources on regular bases.
Checking the internal standard response of each sample within a batch is useful way
to detect any default with individual samples.
Evaluation of solvents, chemicals used, tubes, bottles is necessary to minimize any
interference that may occur.( Bob Morrison, 2011).
36
1.7. Beverages – drug interaction :
In life Medicines are used to treat health problems. Nevertheless, it must be taken
accurately to ensure their efficacy and safety. Diet and beverages can sometimes have
a significant impact on drugs. Recently, drug interactions with fruit juices(beverages)
have received considerable attention from basic scientists, physicians, industry and
drug regulatory agencies. This interaction can affects the activity of a drug, such
effect are either increased or decreased, or even a newer effect that neither produces
on its own. Interaction occure Because a lot of juices shown to inhibit cytochrome P-
450 enzymes and P-glycoprotein transporters in the intestine and liver. The interaction
can affects the activity of a drug, Because a lot of juices shown to inhibit or induce
cytochrome P-450 enzymes or modulate intestinal drug absorption via the P-
glycoprotien mediated efflux and organic anion-transporting polypeptide(OATP)
mediated uptake transport systems the intestine and liver. (Alvarez-Gonzalez, et al..
2011).,and so are considered to be responsible for alterations in drug bioavailability,
and pharmacokinetic and pharmacodynamic changes when drugs are ingested
concurrently with it .( Hugo Vanden Bossche, et al.. 1995).however. It is well known
that risk factors for cardiovascular disease increase with advancing age, while hepatic
metabolic activity decreases in elderly individuals.( Rabia Bushra, et al.. 2010). It is,
therefore, possible that the combination of different juices with cardiovascular
medications may pose a health risk, especially in elderly patients. A number of studies
have shown interactions of friut juice with cardiovascular drugs such as calcium-
channel blockers, angiotensin II receptor antagonists, beta-blockers, and statins. Such
interactions are likely to change the pharmacokinetics and pharmacodynamics of
these drugs, consequently causing undesirable health effects. Therefore, health care
professionals and the public need to be advised of the potential risks associated with
37
the concomitant use of fruit juices and interacting medications, especially
cardiovascular drugs and Agents with a narrow therapeutic index.( Gareth E Lim, et
al. . 2003).
Bioavailability is a backbone pharmacokinetic indicator that Cross bond with the
clinical effect of most drugs. So a pharmacokinetic study must be conducted in order
to evaluate the effect of the beverages used on the drug. interactions associated with
treatment failure result from a reduced bioavailability in the fed state. This occurs due
to physiological response to intake, for example, gastric acid secretion, may reduce or
increase the bioavailability of certain drugs.( Rabia Bushra, et al... 2011; Ayo JA, et
al.. 2005).
1.7.1. Drug-Drug interaction:
The pharmacologic or clinical response to the administration of a drug combination
different from that anticipated from the known effects of the two agents when given
alone.(Tatro DS et al.. 1992).
1.7.1.1 Types of Drug Interaction Mechanisms
Pharmacokinetic Interactions
What the body does with the drug and how does a given drug alters the availability
(absorption, distribution, metabolism or excretion) of another drug.
Usually (but not always) mediated by cytochrome P450 (Ruiz-Garcia A, et al.. 2008.;
Scott R. Penzak, et al.. 2010).
38
Pharmacodynamic Interactions
This happens when One drug modulates the pharmacologic effect of another, these
modulates could be additive, synergistic, or antagonistic and Is caused by the
competition at receptor sites, or action of the interacting drugs on the same
physiological system. There is no change in the plasma concentrations of interacting
drugs in the pharmacodynamic interactions (Lees P, et al.. 2004).
Pharmaceutical Interactions
Type of interactions occurs prior to systemic administration of drugs, either during
synthesis or in the finished pharmaceutical product (Mellor and Jayasinghe 2011).
1.7.1.2 The Cytochrome P-450 (CYP450) Enzyme System
Cytochrome P450 (CYP) enzymes are a superfamily of mono-oxygenases that are
found in all kingdoms of life, it performs its metabolic function by oxidizing,
hydrolyzing or reducing the chemicals. This enables another group of enzymes i.e. the
conjugation enzymes, to attach polar groups to the parent molecule or the primary
metabolite to make the metabolites water-soluble so they can be excreted in the urine
The CYP450 system is important because it is involved in most clinically relevant
metabolic drug interactions (Ortiz de Montellano, P. 2005).
about 55 human isoforms of CYP450 have been discovered. The known clinically
relevant cytochromes are (CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C19 and
CYP2E1) but CYP1, CYP2 and CYP3 are the most important in drug metabolism.
(Dresser GK, et al.. 2000).
39
1.7.1.3 The Transporters of Intestinal Tract
Most drug products are administered orally. Influx transporters facilitate drug
absorption, whereas efflux transporters prevent the drug absorption (Scherrmann,
2009). The absorption of oral drug in the intestine is an important factor to determine
the drug bioavailability. There are many intestinal transporters expressed on the small
intestine, and the transporters can be classified into two major families, SLC family
and ABC family.
The ABC (ATP-binding cassette) transporters include the (P-gp (P-glycoprotein,
MDR1, ABCB1), MRP2 (multidrug resistance-associated protein 2,ABCC2),BCRP
(breast cancer resistance protein, ABCG2). P-gp inhibitors act as high avidity
substrates (e.g. verapamil,quinidine) or block its function by binding to it (e.g.
sulfhydryl-substituted purine) (Fo¨ ger, 2009). These two key factors (poor water
solubility and P-gp efflux pumps) are well known for incomplete absorption of orally
administered drugs and thus limit their bioavailability (Streubel et al., 2006; Dahan
and Amidon, 2009).
And the SLC transporters include the OCTs (organic cation transporter, SLC22A),
OCTNs (novel organic cation transporter, SLC22A), OATPs (organic anion-
transporting polypeptide, SLCO).
These transporters along with other enzymes and secreation in the liver and intestine
influence the absorbiton and/or metabolism of drugs.( Chan, L., et al. 2004).
40
1.7.1.4 Pre-Clinical Studies
Is the term used to describe the tests conducted to a new drug or a new medical device
using animal models, to see if it is really works and if it is safe to be tested on humans
(Edward D. Zanders, 2011).
Goals of Pre-Clinical Testing of Drugs and Biological
• To identify the pharmacologic properties of a pharmaceutical molecule
• To establish a safe initial dose level of the first human exposure
• To understand the toxicological profile of a pharmaceutical molecule (Karen and
Edward 2009).
Preclinical study Provide an imaginiation for the predicted clinical mechanism of
action and efficacy, guide schedule and dose escalation schemes, provide information
for selection of test species, aid in start dose selection, selection of investigations
biomarkers, justify pharmaceutical combinations, understand pharmacodynamic
properties (Karen and Edward 2009).
Preclinical safety testing should consider: selection of the relevant animal species,
age, physiological state, the manner of delivery (including dose, route of
administration, and treatment regimen) and stability of the test material under the
conditions of use (Brennan et al.., 2004).
1.9. Method validation
Validation of an analytical method is the conformation by examination to assure that
the performance characteristics of the method meet the requirements for the intended
analytical application and is capable of giving reproducible and reliable result, when
41
used by different individuals using the same equipment in the same or different
laboratories.( ISO/IEC, 2005; USP 36).
Validation Analytical Tools Include:
Precision, Accuracy, Linearity, Range, Ruggedness, Limit of detection, Limit of
quantitation, Selectivity and stability .(USP 36, ICH) Evaluation of stability should
be carried out to ensure that every step taken during sample preparation, sample
analysis and storage conditions used.(USP 36 , ICH guidelines, S. Seno et al.. 1997).
1.9.1 Precision
The precision of an analytical method is the degree of agreement among individual
test results obtained when the method is applied to multiple sampling of a
homogenous sample (USP 36 , ICH guidelines).
Precision is a measure of the reproducibility of the whole analytical method
(including sampling, sample preparation and analysis) under normal operating
circumstances.
Precision is determined by using the method to assay a sample for a sufficient number
of times to obtain statistically valid results (ie between 6 - 1 0). The precision is then
expressed as the relative standard deviation %RSD = SDmean X 100 %21, (USP 36).
1.9.2 Accuracy
Accuracy is a measure of the closeness of test results obtained by a method to the true
value. Accuracy indicates the deviation between the mean value found and the true
value ( USP 36 , ICH guidelines, JCGM 200:2008)
42
It is determined by applying the method to samples to which known amounts of
analyte have been added. These should be analyzed against standard and blank
solutions to ensure that no interference exists. Percentage of the analyte is recovered
by the assay (USP 36).
1.9.3 Linearity
Linearity of an analytical procedure as its ability (within a given range) to obtain test
results that are directly proportional to the concentration (amount) of analyte in the
sample (USP 36 , ICH guidelines).
Linearity is determined by calculating the regression line using a mathematical
treatment of the results (i.e. least mean squares) vs analyte concentration (USP 36).
1.9.4 Range
The range of the method is the interval between the upper and lower levels of an
analyte that have been determined with acceptable precision, accuracy and linearity,
(USP 36).
It is determined on either a linear or nonlinear response curve (where more than one
range is involved), and is normally expressed in the same units as the test results (USP
36 , ICH guidelines).
1.9.5 Ruggedness
Ruggedness is the degree of reproducibility of results obtained by the analysis of the
same sample under different test conditions (different analysts, laboratories,
instruments, reagents,days….etc).( USP 36 , ICH guidelines, Y. Vander Heyden,
2006).
43
1.9.6 Limit of Detection
This is the lowest concentration of a sample that can be detected, but not necessarily
quantitated, under the stated experimental conditions. The limit of detection is
important for impurity testing and the assay of drugs containing low active ingredient
level and placebo (USP 36, Lawson GM, et al.. 1994).
1.9.7 Limit of Quantitation
This is the lowest concentration of analyte in a sample that can be determined with
acceptable precision and accuracy ( Bansal and DeStefano 2007; USP 36 , ICH
guidelines), lt is quoted as the concentration yielding a signal-to-noise ratio of 10:1
and is confirmed by analyzing a number of samples near this value (USP 36).
1.9.8 Selectivity
Selectivity is the ability to measure accurately and specifically the analyte in the
presence of components that may be expected to be present in the sample matrix (USP
36; Kazakevich and Lobrutto 2007).
1.9.9 Specificity
Specificity for an assay ensures that the measured signal comes from the substance of
interest, and that there is no interference coming from excipients and/or degradation
products and/or impurities (USP 36 ; ICH guidelines; Kazakevich and Lobrutto 2007).
1.9.10 Stability
Stability of the analyte in the studied matrix is evaluated using low and high QC
samples (blank matrix spiked with analyte at a concentration of a maximum of 3 times
the LLOQ and close to the ULOQ) which are analysed immediately after preparation
44
and after the applied storage condition that are to be evaluated. The QC samples are
analysed against a calibration curve, obtained from freshly spiked calibration
standards, and the obtained concentration are compared to the nominal concentrations.
The mean concentration at each level should be within ±15% of the nominal
concentration. Stability should be ensured for every step in the analytical method,
meaning that the conditions applied to the stability tests, should be similar to those
used for the actual study samples. (USP 36; ICH guidelines; Armbruster DA et al..
1994).
The following stability tests should be evaluated:
Stability of the stock solution and working solutions of the analyte and internal
standard.
Short term stability of the analyte in matrix at room temperature or sample
processing temperature.
Freeze and thaw stability of the analyte in the matrix from freezer storage
conditions to room temperature or sample processing temperature.
Long term stability of the analyte in matrix stored in the freezer.( Armbruster
DA et al.. 1994).
1.10. Internal standard
An internal standard is a chemical substance that is added in a constant amount to
samples, the blank and calibration standards in the analysis. then the internal standrad
be used for calibration by plotting the ratio of the analyte signal to the internal
standard signal as a function of the analyte concentration of the standards. This is
done to correct for the loss of analyte during sample preparation or sample inlet. The
internal standard used needs to provide a signal that is similar to the analyte signal in
45
most ways but sufficiently different( because it is similler but not identical to the
chemical sample being tested so that the two signals are readily distinguishable by the
instrument (Guomin Shan, 2011).
In chromatography, internal standards are used to determine the concentration of other
analytes by calculating response factor
The internal standard selected should be again with certien chractarastic similar
to the analyte and have a similar retention time and similar derivitization. It
must be stable and must not interfere with the sample components.( Skoog, Douglas
A. 1998).
Accourding to ICH and USP guidelines, Ideal Internal standard must be with in
these criteria:
Never found in sample
Well-resolved (stable), The chromatographic system needs to be able to
independently measure the size of the analyte and IS peaks.
Ideally is eluted after the analyte, If the IS is eluted after the analyte, it can
offer additional information on the quality of the separation.
Stable, At a minimum, the IS must be sufficiently stable so that it does not
degrade during the sample preparation and chromatographic analysis
processes.
Available in pure form, in other word any impurities present will not be
coeluted with the analyte or otherwise interfere with the process.
Compatible with detector response, Adequate detector response certainly is
necessary. However, the response does not have to be the same as the analyte
46
in terms such as response/gram or detection wavelength, but it does need to be
easily detectable.
Structure similar to analyte, It often to select IS with a chemical relationship
close to the analyte of interest. This really is not necessary — if benzene has
all the other characteristics needed, it might be an adequate IS for a drug
analysis method. However, because the IS needs to have similar extraction
characteristics, retention times, stability, and detector response as the analyte,
it is highly likely that it will be a compound of similar structure. Most internal
standards are existing compounds with close structural relationships to the
analyte.
1.11. Previous study and letreture survey
Detection and determination candesartan cilexetil using RP-HPLC. in a simple, less
tedious, more economic, less time consuming method was obtained. Paracetamol used
as an internal standard. HPLC condition was achieved using C18 Intersil column (256
x 4.6 id) with an isocratic mobile phase composed of selected acetonitrile 40%:
methanol 60% with pH 6.0 and a flow rate of 1.0 mL/min with UV detection was
performed at 228 nm. The retention time of candesartan and internal standard was
1.96 and 3.33 min respectively.( Manisha P Puranik, et al.. 2012).
Georges Vauquelin et al.. Studied The interaction between (candesartan, irbisartan,
EXP3174) and the human angiotensin II type 1 (AT1) receptor in CHO-K1 cells by
incubating the cells with antagonist, followed by an exposure to angiotensin II and
measurement of the resulting inositol phosphate accumulation. In conclusion, the
findings provide further studies for insurmountable and surmountable AT1
antagonists.(Georges Vauquelin, et al.. 2001).
47
Patrick M. L. Vanderheyden,et al. studied whether insurmountable and surmountable
AT1 receptor antagonists The AT1 antagonists (candesartan, EXP3174 or losartan)
bind to a syntopic binding site in a competitively or by an allosteric mechanism.
Whilst there is recent evidence that both types of antagonists are competitive with AT,
it is proposed that an allosteric interaction between the AT1 antagonist EXP3174 and
AT may be responsible for its insurmountable behavior.( Patrick M. L.
Vanderheyden,et al.. 2000).
Validation and determination of Candesartan cilexetil and Hydrochlorthiazide in
pharmaceutical dosage forms was developed. using Hypersil ODS-C18 column (250
× 4.6 mm, 5 µm) with UV detection at 270 nm. Isocratic elution with a mobile phase
consisting of 10 mM (pH 3.37) Tetra butyl ammonium hydrogen sulphate: methanol
(15:85, V/V), at a flow rate 1.0 mL min-1 were used. Linearity was observed in the
concentration range 0.625-62.5 µg/mL for Hydrochlorthiazide and 0.8-80 µg/mL for
Candesartan cilexetil respectively. The LOD was found to be 0.1385 and 0.1892
µg/mL for Hydrochlorthiazide and Candesartan cilexetil respectively where as the
LOQ was found to be 0.4394 and 0.6187 µg/mL for Hydrochlorthiazide and
Candesartan cilexetil respectively. The mean analytical recovery in determination of
Candesartan cilexetil and Hydrochlorthiazide tablets was 99.31-100.08%
Hydrochlorthiazide and 99.58-100.39% for Candesartan cilexetil respectively.
(Mathrusri Annapurna M., et al.. 2012).
The developed HPLC technique which is precise, specific, accurate and stability
indicating to separate the candesartan and it’s impurities.The separations were
achieved by gradient elution using Acetonitrile: Buffer (80:20 v/v)[ Buffer pH- 3
48
(Buffer Preparation - 4.0 mL Ortho-Phosphoric Acid was mixed in 1000 mL of water
and pH -3 adjusted with Triethylamine )] as the mobile phase. The injection volume
was 20 μL. The working concentration was 100 μg/mL and mobile phase flow rate
was 1 mL/min with column oven temperature 30°C. The detection was carried out at
225 nm.(Sachin Bhagwate et al.. 2013).
Promprom W et al. ,investigated the effects of pomegranate (Punica granatum
L., Punicaceae) seed extract on uterine contractility. beta-sitosterol found to be the
main constituent of the extract (16%) and its effects were also
investigated. Pomegranate seed extract and beta-sitosterol increased spontaneous
contractions in a concentration-dependent manner with a maximum effect at 250
mg/100 mL and 1 mg/100 mL, respectively. And concluded that pomegranate seed
extract is a potent stimulator of phasic activity in rat uterus. due to nonestrogenic
effects of beta-sitosterol acting to inhibit K channels and SERCA and thereby
increasing contraction via calcium entry on L-type calcium channels and MLCK.
Fuhrman, B. et al. invistegated the possible mechanisms by which Pomegranate juice
reduces cholesterol accumulation in macrophages. J774.A1 macrophages were
preincubated with Pomegranate juice followed by analysis of cholesterol influx
[evaluated as LDL or as oxidized LDL (Ox-LDL) cellular degradation], cholesterol
efflux and cholesterol biosynthesis. Preincubation of macrophages with Pomegranate
juice resulted in a significant reduction (P<.01) in Ox-LDL degradation by 40%. On
the contrary, Pomegranate juice had no effect on macrophage degradation of native
LDL or on macrophage cholesterol efflux. Macrophage cholesterol biosynthesis was
inhibited by 50% (P<.01) after cell incubation with Pomegranate juice concluded that
Pomegranate juice mediated suppression of Ox-LDL degradation and of cholesterol
49
biosynthesis in macrophages can lead to reduced cellular cholesterol accumulation
and foam cell formation.
According to recent report, Two methods are created for the simultaneous
determination of candesartan cilexetil 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 270 nm. The separation was carried out
on Merck HPTLC aluminium sheets of silica gel 60 F 254 using chloroform:
methanol (80:20, v/v) as mobile phase. Linear regression analysis data used for the
regression line were in the range of 0.05–0.70 and 0.05–0.50µg. band for Candesartan
and Hydrochlorothiazide, respectively. The second method was based on difference
and derivative-difference spectrophotometry with a zero-crossing measurement
technique. Linear calibration graphs of absorbance difference values at 292 nm and
338 nm were obtained versus concentration in the range 20-100 mg.L for Candesartan
and Hydrochlorothiazide. Also linear regression equations of second derivative
difference values at 296 nm for CAN and first derivative difference values at 299 nm
for Hydrochlorothiazide versus concentration in the ranges 10–100 and 5– 70 mg.L
for Candesartan and Hydrochlorothiazide, respectively, were obtained. The two
methods were validated according to ICH guidelines and applied on bulk powder and
pharmaceutical formulation.(Youssef, RM, et al.. 2010).
validation and determination of candesartan in human plasma using irbesartan as the
internal standard (IS) was invented and proved to be linear, accurate, and precise over
the range of 2–200 ng/mL.. The analyte and IS were separated by a gradient program
with a mobile phase consisting of 0.1% formic acid (containing 2 mM ammonium
acetate) and methanol at a flow rate of 0.30 mL/min. Detection was performed on a
50
triple quadrupole tandem mass spectrometer via electrospray ionization in the positive
ion mode.( Hong-gang Lou, et al.. 2012).
KK Pradhan, et al.. develops A simple, specific, accurate and stability-indicating UV-
Spectrophotometric method for the estimation of candesartan cilexitil, using a
Shimadzu, model 1700 spectrophotometer and a mobile phase composed of methanol
90% : water 10% at wave length (λmax ) 254 nm. Linearity was established for
candesartan in the range of 10-90 μg/ml. The percentage recovery of was in the range
of 99.76-100.79%.( KK Pradhan, et al.., 2011).
Accourding to S. S. Qutab , et al.. 2007, A simple, sensitive, and inexpensive HPLC
method has been developed for simultaneous determination of hydro-chlorothiazide
and candesartan cilexetil in pharmaceutical formulations. separation were achieved on
a Phenyl-2 column with a 25:75:0.2 mixture of 0.02 M potassium dihydrogen
phosphate, methanol, and triethyl-amine, final pH 6.0 ± 0.1, as mobile phase.
Detection was at 271 nm. Response was a linear.
V. A. Eagling et al.. Reported that Grapefruit juice components inhibit CYP3A4-
mediated saquinavir metabolism and also modulate, to a extent, P-gp mediated
saquinavir transport in Caco-2 cell monolayers. The in vivo effects of grapefruit juice
coadministration result in an inhibition and down regulation on CYP3A4 and only to
a minor extent on modulation of P-gp function. The results shows that 6¾,7¾-
dihydroxybergamottin and bergamottin inhibited the metabolism of saquinavir.( V. A.
Eagling et al.. 2001).
Tuija H. Nieminen,et al.. 2010 conclude that dietary consumption of grapefruit
products may increase the concentrations and effects of oxycodone in clinical use.
Grapefruit juice increased the mean area under the oxycodone concentration–time
51
curve (AUC0) by 1.7-fold (p < 0.001), the peak plasma concentration by 1.5-fold (p <
0.001) and the half-life of oxycodone by 1.2-fold (p < 0.001) as compared to the
water. Grapefruit juice inhibited the CYP3A4-mediated first-pass metabolism of
oxycodone, decreased the formation of noroxycodone and noroxymorphone and
increased that of oxymorphone.( Tuija H. Nieminen,et al.. 2010).
Liqurice Extract supplementation can improve the physical quality of fresh meat, the
result obtained when a Fifty-four-month-old Tan male sheep were randomly allocated
among five dietary groups with Liqurice extarct supplementation at levels of 0 mg/kg,
1000 mg/kg, 2000 mg/kg, 3000 mg/kg and 4000 mg/kg feed. The results showed that
supplementation with Liqurice extract decreased (P < 0.05) temperature, drip loss,
metmyoglobin (MetMb) concentration and percentage, whereas it increased (P < 0.05)
myoglobin (Mb) concentration. As aging progressed after postmortem, temperature,
drip loss and Mb concentration decreased (P < 0.05), but MetMb concentration and
percentage increased (P < 0.05). (Yuwei Zhang, et al.. 2013).
The effect of oral administration of a water freeze-dried extract of liquorice showed to
suppress the adrenal–pituitary axis, accompanied by stimulation of renin production
from the kidney in a dose dependent manner, in rats on the plasma concentration of
cortisol, adrenocorticotrophic hormone (ACTH), aldosterone, renin, sodium (Na) and
potassium (K). The results indicated that treatment induced dose-dependent and
mostly significant decreases in the concentration of cortisol, ACTH, aldosterone and
K. There were concomitant dose-dependent increases in the concentrations of renin
and Na. (A.A. Al-Qarawi, et al.. 2002).
Concomitant take of Liquorice and Cyclosporine (CsA), an immunosuppressant, leads
to significantly reduced the oral bioavailability of CsA, this due Mechanism studies
52
revealed that glycyrrhetic acid (GA), the major metabolite of liquorice, significantly
activated the functions of P-gp and CYP3A4through activating P-gp and CYP3A4,
The results showes that Liquorice significantly decreased the peak blood
concentration and the areas under the curves of CsA in rats.(Yu-Chi Hou,et al.. 2012).
Muneaki Hidaka report that pomegranate juice components impairs the function of
enteric but not hepatic CYP3A. And a components of pomegranate inhibits the human
CYP3A-mediated metabolism of carbamazepine. The ability of pomegranate to
inhibit the carbamazepine 10,11-epoxidase activity of CYP3A was examined using
human liver microsomes, and pomegranate juice was shown to be a potent inhibitor of
human CYP3A. The inhibition potency of pomegranate juice was similar to that of
grapefruit juice. In addition, the in vivo interaction between pomegranate juice and
carbamazepine pharmacokinetics using rats. In comparison with water, and the area
under the concentration-time curve (AUC) of carbamazepine was approximately 1.5-
fold higher when pomegranate juice (2 ml) was orally injected.( Muneaki Hidaka, et
al.. 2005).
D. Adukondalu,et al.. investigate the effect of pomegranate juice pre-treatment on the
transport of carbamazepine across the rat intestine. Result showed that there was a
significant (p<0.05) difference in the transport of carbamazepine from the intestinal
sacs of pretreated with pomegranate juice and control. It seems that pomegranate juice
might have induced CYP3A4 enzymes and hence drug is extensively metabolized.( D.
Adukondalu,et al.. 2010).
Published data report that orange juice increases the bioavailability of pravastatin
administered orally. Oatp1 and oatp2 may be related to increases of pharmacokinetics
of pravastatin by orange juice. The pharmacokinetics of pravastatin (100 mg/kg p.o.)
53
were assessed with water, orange juice, and carbohydrates (12.5 ml/kg over 30 min)
and with acetic acid (0.1 M, pH 3.44). Orange juice significantly increased the area
under the curve (0–150 min) of pravastatin in rats. Results shows that Orange juice
had no effects on the pharmacokinetic parameters of intravenously administered
pravastatin in rats. Carbohydrates and acetic acid with pH and concentration
equivalent to those of orange juice also resulted in no statistically significant
differences in pravastatin pharmacokinetic parameters in rats. Orange juice
significantly increased oatp1 and oatp2 mRNA and protein in the intestine of rats.
Orange juice significantly increased the area under the curve (0–240 min) of
pravastatin in healthy volunteers.( Yu Koitabashi, et al.. 2006).
Orange juice found to interfere with the gastrointestinal absorption of
atenolol. Orange juice decreased the mean peak plasma concentration (C max) of
atenolol by 49% (P<0.01), and the mean area under the plasma atenolol
concentration–time curve (AUC0-33 h) by 40% (range 25–55%, P<0.01). The amount
of atenolol excreted into urine was decreased by 38% (range 17–60%, P<0.01.( J. J.
Lilja, et al.. 2005).
Concomment use of Orange juice and celiprolol leads to inhibition of celiprolol
intestional absorption, this inhibition reffered to Hesperidin, an ingrident of orange
juice, The pharmacokinetic interaction between celiprolol and orange juice was
characterized through in vivo experiments with rats. Celiprolol 5 mg/kg was injected
into the rat duodenum together with 5 ml/kg of neutralized orange juice. Plasma
celiprolol concentrations were measured by liquid chromatography-electrospray
ionization-mass spectrometry (LC-ESI-MS). Concomitant administration of orange
juice with celiprolol significantly decreased the area under the plasma concentration-
54
time curve (AUC) by 74% and 75%, respectively, compared with control.( Uesawa Y,
et al.. 2008).
55
1.12. Objective of This Study
1. Develop a sensitive and simple chromatographic method for quantifying
candesartan in rat plasma.
2. Study the efficacy and pharmacokinetic parameters for the candesartan as an
antihypertensive drug in animals pre-fed with different juices to Examine the possible
effect of these juices (liqourice, orange and pomegranate) on candesartan.
3. Enhance the use of rational drug therapy provided with examples of different juice
drug interactions that may have a deleterious effect on health.
56
CHAPTER TWO
EXPERIMENTAL PART
57
2. Experimental Part
2.1 Reagents
Deionized Water, Nanopure (Fisher Scientific, B# 1207702).
Rats Plasma, (harvested from Animals of UOP animal house).
Methanol advanced gradient grade (Fisher scientific, B# 1155904).
Acetonitrile advanced gradient grade (Fisher scientific, B# 1156250).
Formic acid advanced gradient grade (GPR Rectapur, B# 07L210512).
Dimethyl Sulfoxide DMSO (Tedia, B# DR0469-001)
Sodium hydroxide powder (GPR , B# B0057050)
Candesartan raw material (JPM B# WS/07/272)
Irbesartan raw material
Freshely prepared liquorice juice
Freshly squeezed pomegranate juice
Freshly squeezed orange juice
58
2.2 Instrumentation
An API Mass spectrometer was used and composed of the following:
On-line vacuum Degasser (Agilent 1200),
Solvent delivery systems pump (Agilent 1200).
Autosampler (Agilent 1200).
Thermostat column compartment (Agilent 1200).
API 3000 Mass Spectrometer,
ACE 5, C18 (50 x 2.1 mm), 5µm
Computer System, Windows XP, SP3, Data Management Software 1.5.2
Bath Sonicator Crest model-175T (Ultra Sonics CORP.)
Sartorius balance BP 2215
Sartorius pH meter (Professional meter PP-25)
Centrifuge (eppendorf 5417C)
2.3 Animals
All animal experiments were performed in compliance with FELASA guidelines
(Federation of European Laboratory Animal Science Association) and the study
protocol was approved by the Research Committee (No. 5, January 30/2013) at the
Faculty of Pharmacy, University of Petra, Amman, Jordan. Adult male Sprague
Dawley laboratory rats were supplied by the animal house of Petra University. The
Average weight of rats was approximately 220.0g, and they were in healthy condition.
59
They were placed in air-conditioned environment (20-25°C) and exposed to a
photoperiod cycle (12hours light/ 12 hours dark) daily.
After preparation of candesartan solution, the rats received a certain amount of
candesartan solution; this amount is to be calculated according to their weights the
solution was given by oral cavage.
The rats were divided into 20 groups, every group contain an average of six rats, eight
groups received candesartan only, six groups received candesartan with liqourice,
other three groups received candesartan with orange while the last three groups
received candesartan with pomegranate.
Fruit juices were given to the rats (liqourice, orange, and pomegranate) as multiple
doses, before the administration of the drug, by oral cavage.
Each rat had been weighed then we cut the tip of the tail and took a few drops of
blood in eppendoorf tube and then the rat received the drug solution orally, and after
giving the dose of drug to the rat we took a few drops of blood in eppendorf tubes
after 30 min and 1, 2, 3, 4 and 6 hours of administration.
Eppendorf tubes were centrifuged for 10 minutes to get the plasma required for the
analytical process.
2.4 Preparation of Stock Solutions
2.4.1 Preparation of Candesartan Solution to be given to the rats
0.022 g of candesartan raw material dissolved in 3 ml of DMSO then the volume were
completed to 100ml with distilled water.
60
Preparation of Stock Solutions:
2.4.2 Preparation of stock solution of Candesartan:
Weight equivalent to 10.00 mg of candesartan working standard was dissolved it in
10 ml of Methanol to get concentration 1000 µg/ml stock solution of candesartan.
2.4.3 Preparation of stock solution of Irbesartan Internal Standard:
Weight equivalent to 10.0 mg of irbesartan working standard was dissolved it in 10
ml of ACN to get concentration of 1000.0 µg/ml stock solution of irbesartan.
Preparation of working solutions:
2.4.4 Preparation of working solution of irbesartan I.S:
we took 200 µl from irbesartan stock solution (1000.0 µg/ml) and dilute it to 100 ml
of ACN which was considered to be I.S working solution (B-IS) that contains 2.0
µg/ml of irbesartan.
2.4.5 Preparation of working solution for candesartan:
200.0 μl from 1.0 mg/ml stock solution was added to 10.0 ml of 1:1 water/methanol in
volumetric flask to obtain 20.0 μg/ml working solution.
2.4.6 Preparation of Candesartan serial spiking samples in plasma:
Samples of standard curve in plasma were prepared by spiking 100.0 μl from serial
solution into 10.0 ml of plasma, using seven concentrations, not including zero to
obtain STD concentrations of: 10, 25, 75, 250, 400, 600, and 1000 ng /ml for
candesartan in plasma, Table 8. Each concentration of the plasma sample was divided
to 25 μl in 1.5 ml eppendorf tube and kept at (-30˚C), standard samples were given
daily together with the quality control samples.
61
Table 8 Serial Spiked Plasma Samples
Serial solution of Candesartan from working solution
of 1000 µg/ml
Plasma spiking solution
Solution
No:
Working
Solution
Conc.
(µg/ml)
Stock
Conc.
(µg/ml)
Volume
taken
from
stock
(µl)
Total
Volum
e (ml)
Cal ID Volume
taken
from
w.s (µl)
Total
Volume
(ml)
Final
concentration
(ng/ml)
1 0.4 1000 4 10 S1 25 1 10
2 1.0 1000 10 10 S2 25 1 25
3 3.0 1000 30 10 S3 25 1 75
4 10.0 1000 100 10 S4 25 1 250
5 16.0 1000 160 10 S5 25 1 400
6 24.0 1000 240 10 S6 25 1 600
7 40.0 1000 400 10 S7 25 1 1000
2.4.7 Preparation of Candesartan Quality Control Samples in plasma:
Samples of QC in plasma were prepared by spiking 100.0 μl from serial solution into
10.0 ml of plasma to obtain QC concentrations of: 30, 50 and 800 ng /ml for
candesartan in plasma. Table 9.
Each concentration of the plasma sample was divided to 25 μl in 1.5 ml eppendorf
tube and kept at (-30˚C), standard samples were given daily together with the quality
control samples.
62
Table 9 QC Spiked plasma samples
Serial solution of Candesartan from working
solution of 1000 µg/ml Plasma spiking solution
Solution
No:
Working
Solution
Conc.
(µg/ml)
Stock
Conc.
(µg/ml)
Volume
taken
from
stock
(µl)
Total
Volu
me
(ml)
Cal ID
Volume
taken
from
w.s (µl)
Total
Volume
(ml)
Final
concentration
(ng/ml)
8 1.2 1000 12 10 QcL 25 1 30
9 20.0 1000 200 10 QcM 25 1 500
10 32.0 1000 320 10 QcH 25 1 800
2.5 Preparation of fresh juices:
All of juices was freshly squeezed at the day of experiment.
2.6 Method of Sample preparation:
The procedures described were applied for subject samples, calibrator and quality
control samples. In order to perform the sample extraction, the following
experimental procedure was followed:
appropriate number of disposable Eppendorf tubes were placed in a rack. The tubes
are properly labeled.
We Pipette 50.0 µl aliquots of each test sample (blank, zero, standards, QCL, QCM,
QCH or Rat samples) into the appropriate tubes.
Then we Added 150.0 µl of Internal Standard (2.0 µg/ml Irbesartan)
Vortex each sample vigorously for 1.0 min.
63
Centrifuge at 14000 rpm for 15 minutes.
Validation
2.7.1 Accuracy and Precision
Within-batch accuracy and precision evaluations were determined by analysis of 6
replicates quality control samples from each level. The between-batch precision and
accuracy was determined by analyzing three sets of within-batch quality
controlsequence in three separate batches.
The quality control samples were randomized daily, processes and analyzed in
position either a) immediately following the standard curve,b) in the middle of batch
or c) at the end of the batch. The acceptance criteriafor withinandbetween –batch
precision and accuracy were 20% for LLOQ and 15 % for the otherconcentrations.
2.7.2 Specificity
Specificity is the ability of an analytical method to differentiate and quantify
theanalytes in presence of other components in the sample. The specificity of the
methodwas evaluated by screening six different lots of blank plasma. These lots were
analyzedas blank and zero samples then compared with LLOQ to confirm lack of
endogenouspeaks.
2.7.3 Linearity
Rats plasma samples were spiked with Candesartan to prepare calibrators, these
samples were extracted and assayed. Each calibration curve was completed by
plotting the ratio versus nominal concentration values.
64
2.8 Chromatographic Conditions:
Table 10: Summery Table of Chromatographic Conditions and Mass
Spectrometric Conditions
Column
Oven Temp
Autosampler
Temp
Autosampler Injection
Volume Pump Flow Rate HPLC
Conditions 30˚C 4˚C 5 µl 1.0 ml/min
B (%)
0.2%
F.A
A (%)
Methanol
Flow
Rate
(µl/min)
Total
Time(min) Step
50.0 50.0 1000 0.00 0
50.0 50.0 1000 0.01 1
0.0 100.0 1000 0.02 2
0.0 100.0 1000 0.70 3
50.0 50.0 1000 0.71 4
50.0 50.0 1000 2.00 5
Mobile phase
Gradient Elution
Chromatography
ACE 5 C18 Column (50 X 2.1 mm), 5µ Column type
Irbesartan (I.S) Candesartan Expected Retention
times(minutes) 1.0 1.4
CXP CE EP DP FP Dwell Q3 Mass Q1
Mass Analytes
MRM Detection
Conditions 22 19 10 81
70 150
263.200 441.20
0 Candesartan
22 8 10 26 70
150 207.300 429.45
3
Irbesartan
(IS)
NEB TEM IS CAD CUR MS Conditions
5 400 5500 6 10
2.9 Irbiartan as internal standard
Irbesartan as internal standard is used to determine the concentration of candesartan
by calculating response factor. irbesartan is similar to candesartan and have a similar
retention time. It is stable and does not interfere with the sample components.
65
2.10 Statistical Analysis
Data were translated into a computerized database structure. The database was
examined for errors using range and logical data cleaning methods, and
inconsistencies were remedied. Statistical analyses were done using SPSS version 20
computer software (Statistical Package for Social Sciences).
The 95% prediction interval in a linear regression model is a statistical procedure to
anticipate or predict the expected range of possible correct values of the mean
predicted concentration with 95% confidence.
The statistical significance of difference in mean of a normally distributed variable,
like drug concentration between 2 groups was assessed using the independent samples
Student’s t-test. The statistical significance of mean calculated errors between
predicted and target concentration was assessed by paired t-test.
Difference between 2 means is a measure of effect size presented in its original units
of measurements. It equals the mean of a quantitative outcome variable in a test group
minus that of a comparison group. Its usefulness is limited for comparison with other
contexts of similar units of measurements and magnitude of mean. The difference
between 2 means as a measure of effect is affected by the units of measurement for
the variable and the amount of variability (SD). Therefore such a measure is not
useful to compare the effect size across different type of variables or different studies.
Cohen’s d is a standardized measure of effect size for difference between 2 means,
which can be compared across different variables and studies, since it has no unit of
66
measurement. Cohen’s d = (mean1-mean2) / Pooled SD of the 2 groups. Cohen’s d <
0.3 small effect, 0.3-0.7 (medium effect), while 0.8 and higher is a large effect.
PS
xxdsCohen
21'
)2(
)1()1(
21
2
22
2
11
nn
SnSnS p
A multiple linear regression model was used to study the net and independent effect
of a set of explanatory variables, like ―Day of validation‖, ―Type of drug assessed:
Candesartan drug compared to Candesartan with fruit juice‖ and ―Target (standard)
concentration‖ on a quantitative outcome (dependent) variable like measurement
error. The linear regression model (both simple and multiple) provides the following
parameters:
P (model): In order to generalize the results obtained, the model should be statistically
significant.
Unstandardized partial regression coefficient: Measures the amount of change
expected in the dependent variable for each unit increase in the independent variable
after adjusting for other explanatory variables included in the model.
P for regression coefficient: reflects the statistical significance of the calculated partial
regression coefficient of each explanatory variable included in the model.
R² (Determination coefficient): measures the overall performance of the model since
it reflects the amount of variation in the dependent variable explained by the model.
Where Sp is the pooled standard deviation
N is the sample size
is the sample mean
67
The closer its value to 100% the better the model fit. It is also used to measure
linearity or strength of dose response relationship in validation of experiments.
68
CHAPTER THREE
RESULTS and DISCUSSION
69
3. Results
3.1 Validation
A full method validation were performed accourding to ICH and EMA guidelines for
any analytical method to demonstrate the reliability of a particular method for the
determination of an analyte concentration in a specific biological matrix.
3.1.1 Precision
At the first day of validation, the variability of errors (precision) in predicted
concentration ranged between as low as 2.645% observed with the High target
concentration of 800 ng/ml to a maximum coefficient of variation of (CV%) of
5.963% at the mid target concentration, table 11. The precision for low and mid
concentrations of target was 5.778%, 5.963% respectively, table 15 and 16.
At the second day of validation, the variability of errors (precision) in predicted
concentration ranged between as low as 1.839% observed with the mid target
concentration of 500 ng/ml to a maximum coefficient of variation of (CV%) of
5.677% at the low QC target concentration of 30 ng/ml, table 12. The precision for
low and high concentrations of target was 5.677%, 1.916% respectively, table 20 and
21.
At the third day of validation, the precision of predicted concentration ranged between
as low as 2.833% observed with the High QC concentration of target of 800 ng/ml to
a maximum coefficient of variation of (CV %) of 5.030% at the QC low target
concentration of 30 ng/ml, table 13. The precision for LLOQ and mid concentration
of target was 4.816%, 3.681% respectively, table 22 and 28
70
The precision (CV %) is not exceed 20% for LLOQ, and 15% for the other
concentrations which prove the closeness of the measurements.
3.1.2 Accuracy
At the first day validation, the accuracy of mean predicted value compared to target
concentration ranged between a minimum of 96.103% at the QC Low concentration
of target 30 ng/ml to a maximum accuracy of 103.319% at the QC high concentration
for target 800 ng/ml. The overall all average accuracy at the first day was 100.55 %,
table 11. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC high
samples was (97.01%-107.13%), (90.89%-102.56%), (106.42%-93.34%), (107.14%-
99.37%) respectively, table 14 to 17.
At the second day of validation, the accuracy of mean predicted value compared to
target concentration ranged between a minimum of 94.482% at the high concentration
of target 800 ng/ml to a maximum accuracy of 105.302% at the LLOQ target
concentration of 10 ng/ml. The overall all average accuracy at the second day was
98.78%, table 12. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC
high samples was (98.70%-109.52%), (93.90%-109.07%), (92.90%-97.80%),
(91.87%-96.91%) respectively, table 18 to 21.
At the third day validation, the accuracy of mean predicted value compared to target
concentration ranged between a minimum of 95.691% at the Mid concentration of
target 500 ng/ml to a maximum accuracy of 101.529% at the LLOQ target
concentration of 10 ng/ml. The overall all average accuracy at the third day was
99.78%, table 13. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC
high samples was (93.68%-108.88%), (94.46%-109.59%), (90.30%-99.81%),
(96.38%-105.07%) respectively, table 22 to 26.
71
Comparing with the accepted criteria which is 85-115% for all concentration except
for LLOQ which is 80-120%, the accuracy obtained is within the required criteria in
terms of accuracy.
3.1.3 Measurement error
The mean measurement error at the first day assessment ranged between 1.169 ng/ml
lower than the target concentration at the QC low concentration 30ng/ml of target to
26.549ng/ml higher than the target concentration) at the High concentration of target
500 ng/ml. The overall all mean measurement error at the first day was an
overestimate of 7.74 ng/ml, table 11.
The mean measurement error at the second day of assessment ranged between 44.145
ng/ml (lower than the target concentration) at the high concentration 800ng/ml of
target to 0.530 ng/ml higher than the target concentration at the LLOQ concentration
of target of 10 ng/ml. The overall all mean measurement error at the second day was
an underestimate of 17.91 ng/ml, table 12.
The mean measurement error at the third day of assessment ranged between 21.545
ng/ml lower than the target concentration at the mid concentration 500ng/ml of target
to 2.033 ng/ml higher than the target concentration at the high concentration of target
of 800 ng/ml. The overall all mean measurement error at the third day was an
underestimate of 6.07 ng/ml, table 13.
Looking at all the 3 days of validation one would conclude an overall mean
measurement error of 10.3 ng/ml (underestimate on average) for the validation
experiments of candesartan.
72
Table 11: The mean measurement error and accuracy for candesartan validation
experiment on the first day at 6 selected target concentrations (day 1).
First day of
validation
Target
(standard)
conc. (ng/ml)
(n=6)
Calculated
(predicted)conc.
(ng/ml)
(n=6)
Measurement
error (ng/ml)
Accuracy%
LLOQ
Mean 10 10.227 +0.227 102.270
SD 0.385 0.385 3.848
SE 0.157 0.157 1.577
CV% 3.763 3.763
Range (10 to 10) (9.701-10.713) (-.299-0.713) (97.01-
107.13)
Low
Mean 30 28.831 -1.169 96.103
SD 1.666 1.666 5.552
SE 0.680 0.680 2.27
CV% 5.778 5.778
Range (30 to 30) (27.266-30.769) (-2.744-0.769) (90.89-
102.56)
Mid
Mean 500 503.034 +3.034 100.607
SD 29.996 29.996 5.999
SE 12.24 12.24 2.449
CV% 5.963 5.963
Range (500 to 500) (466.683-532.078) (-33.317-32.078) (93.34-
106.42)
High
Mean 800 826.549 26.549 103.319
SD 21.866 21.866 2.733
SE 8.928 8.928 1.115
CV% 2.645 2.645
Range (800 to 800) (794.983-857.122) (-5.117-57.122) (99.37-
107.14)
Overall day1
Mean 7.724 100.568
73
Table 12: The mean measurement error and accuracy for candesartan validation
experiment on the first day at 4 selected target concentrations (day 2).
second day of
validation
Target
(standard)
conc.
(ng/ml)
(n=6)
Calculated
(predicted)
conc. (ng/ml)
(n=6)
Measurement
error (ng/ml)
Accuracy%
LLOQ
Mean 10 10.530 0.530 105.302
SD 0.421 0.421 4.205
SE 0.171 0.171 1.717
CV% 3.993 3.993
Range (10 to 10) (9.870-10.952) (-0.130-0.952) (98.700-
109.52)
Low
Mean 30 30.257 0.257 100.856
SD 1.718 1.718 5.726
SE 0.701 0.701 2.338
CV% 5.677 5.677
Range (30 to 30) (28.171-
32.722)
(-1.89-2.722) (93.90-
109.07)
Mid
Mean 500 473.277 -26.723 94.655
SD 8.702 8.702 1.740
SE 3.553 3.553 0.710
CV% 1.839 1.839
Range (500 to
500)
(464.492-
489.005)
(-35.508--10.995) (92.90-
97.80)
High
Mean 800 755.855 -44.145 94.482
SD 14.484 14.484 1.810
SE 5.914 5.914 0.739
CV% 1.916 1.916
Range (800 to
800)
(734.964-
775.249)
(-65.036- -24.751) (91.87-
96.91)
Overall day-2
Mean 17.910 98.825
74
Table 13: The mean measurement error and accuracy for candesartan validation
experiment on the first day at 4 selected target concentrations (day 3).
Third day of
validation Target
(standard)
conc.
(ng/ml)
(n=6)
Calculated
(predicted)
conc. (ng/ml)
(n=6)
Measurement
error (ng/ml)
Accuracy%
LLOQ
Mean 10 10.195 0.195 101.952
SD 0.491 0.491 4.910
SE 0.086 0.086 2.004
CV% 4.816 4.816
Range (10 to 10) (9.368-10.888) (0.632-0.888) (108.880-93.680)
Low
Mean 30 30.310 0.310 101.032
SD 1.540 1.540 5.132
SE 0.630 0.630 2.095
CV% 5.080 5.080
Range (30 to 30) (28.339-32.878) (-1.661-2.878) (94.460-109.590)
Mid
Mean 500 478.455 -21.545 95.691
SD 17.611 17.611 3.522
SE 7.191 7.191 1.438
CV% 3.681 3.681
Range (500 to 500) (451.491-
499.045)
(-48.509- -0.995) (90.30-99.81)
High
Mean 800 802.033 2.033 100.254
SD 22.724 22.724 2.840
SE 9.278 9.278 1.159
CV% 2.833 2.833
Range (800 to 800) (771.060-
840.571)
(-28.94-40.571) (96.38-105.07)
Overall day-3
Mean 6.0195 99.732
75
Table 14: Intra-day precision and accuracy data for LLOQ samples of
candesartan based on the standard calibration curve of day one validation.
Theo.
Conc.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision
%
Accuracy
%
10
QC
Low
3717 57830 0.064 10.713 3.763 107.13
3139 48985 0.064 10.647 106.47
3702 59394 0.062 10.056 100.56
3501 56022 0.062 10.113 101.13
3051 48768 0.063 10.132 101.32
3126 51014 0.061 9.701 97.01
Mean 3372.667 53668.833 0.063 10.227 102.270
STD 304.108 4661.220 0.001 0.385 3.848
CV% 9.017 8.685 1.814 3.763 3.763
Table 15: Intra-day precision and accuracy data for QC low samples of
candesartan based on the standard calibration curve of day one validation.
Theo.
Conc.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision
%
Accuracy
%
30ng/ml
QC Low
6062 53322 0.114 27.402 5.778 91.34
5807 51164 0.113 27.342 91.14
6389 56403 0.113 27.266 90.89
6385 51638 0.124 30.769 102.56
6018 49110 0.123 30.397 101.32
6474 53593 0.121 29.809 99.36
Mean 6189.167 52538.333 0.118 28.831 96.103
STD 264.928 2494.593 0.005 1.666 5.552
CV% 4.281 4.748 4.181 5.778 5.778
76
Table 16: Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day one validation.
Theo. Conc. AUC
Drug
AUC IS Ratios Measured
Conc.
Precision% Accuracy%
500.00ng/ml
QC Mid
86373 54194 1.594 527.377 5.963 105.48
83242 55682 1.495 493.988 98.80
91336 57287 1.594 527.567 105.51
84721 52697 1.608 532.078 106.42
69809 48973 1.425 470.512 94.10
79351 56114 1.414 466.683 93.34
Mean 82472.000 54157.833 1.522 503.034 100.607
STD 7342.090 2998.640 0.089 29.996 5.999
CV% 8.903 5.537 5.835 5.963 5.963
Table 17: Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day one validation.
Theo.
Conc.
AUC Drug AUC IS Ratios Measured
Conc.
Precision% Accuracy%
800.00
ng/ml
QC
High
138870 55575 2.499 833.078 2.645 104.13
129247 54169 2.386 794.983 99.37
137488 53498 2.570 857.122 107.14
134797 55155 2.444 814.572 101.82
140655 57277 2.456 818.531 102.32
125689 49832 2.522 841.007 105.13
Mean 134457.667 54251.000 2.479 826.549 103.319
STD 5851.739 2523.020 0.065 21.866 2.733
CV% 4.352 4.651 2.611 2.645 2.645
77
Table 18: Intra-day precision and accuracy data for LLOQ samples of
candesartan based on the standard calibration curve of day two validation.
Theo.
Conc
.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision
%
Accuracy%
10
QC
Low
3589 54526 0.066 10.838 3.993 108.38
3466 52636 0.066 10.846 108.46
3304 51704 0.064 10.309 103.09
3536 55152 0.064 10.366 103.66
3430 51787 0.066 10.952 109.52
3209 51478 0.062 9.870 98.70
Mean 3422.33
3
52880.50
0
0.065 10.530 105.302
STD 142.953 1579.498 0.002 0.421 4.205
CV% 4.177 2.987 2.343 3.993 3.993
Table 19: Intra-day precision and accuracy data for QC low samples of
Candesartan based on the standard calibration curve of day two validation.
Theo.
Conc.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision% Accuracy%
30
ng/ml
QC
Low
6775 50882 0.133 29.560 5.677 98.53
7556 53323 0.142 31.941 106.47
6912 51639 0.134 29.757 99.19
6927 52266 0.133 29.389 97.96
7051 48792 0.145 32.722 109.07
6776 52876 0.128 28.171 93.90
Mean 6999.500 51629.667 0.136 30.257 100.856
STD 291.757 1639.335 0.006 1.718 5.726
CV% 4.168 3.175 4.552 5.677 5.677
78
Table 20: Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day two validation.
Theo.
Conc.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision% Accuracy%
500.0
0
ng/ml
QC
Mid
77054 44698 1.724 471.878 1.839 94.38
82644 48691 1.697 464.492 92.90
88531 49584 1.785 489.005 97.80
84010 48412 1.735 475.061 95.01
83989 48614 1.728 472.934 94.59
90475 53102 1.704 466.292 93.26
Mean 84450.50
0
48850.16
7
1.729 473.277 94.655
STD 4718.554 2688.129 0.031 8.702 1.740
CV% 5.587 5.503 1.810 1.839 1.839
Table 21: Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day two validation.
Theo.
Conc.
AUC Drug AUC IS Ratios Measured
Conc.
Precision
%
Accurac
y%
800.00
ng/ml
QC
High
Mean
144966 51499 2.815 775.249 1.916 96.91
148565 54976 2.702 743.945 92.99
144407 52626 2.744 755.539 94.44
131192 47342 2.771 763.083 95.39
134489 48578 2.769 762.347 95.29
125368 46954 2.670 734.964 91.87
138164.500 50329.167 2.745 755.855 94.482
STD 9157.238 3213.052 0.052 14.484 1.810
CV% 6.628 6.384 1.898 1.916 1.916
79
Table 22: Intra-day precision and accuracy data for LLOQ samples of
candesartan based on the standard calibration curve of day three validation.
Theo.
Conc
.
AUC
Drug
AUC IS Ratios Measure
d Conc.
Precision
%
Accura
%
10
QC
Low
3759 49891 0.075 10.888 4.816 108.88
3583 49509 0.072 10.076 100.76
3641 49925 0.073 10.230 102.30
3836 52316 0.073 10.336 103.36
3716 50837 0.073 10.273 102.73
3637 52123 0.070 9.368 93.68
Mean 3695.33
3
50766.83
3
0.073 10.195 101.952
STD 92.996 1208.465 0.002 0.491 4.910
CV% 2.517 2.380 2.470 4.816 4.816
Table 23: Intra-day precision and accuracy data for QC low samples of
candesartan based on the standard calibration curve of day three validation.
Theo.
Conc
.
AUC
Drug
AUC IS Ratios Measure
d Conc.
Precision
%
Accura
%
30
ng/m
l
QC
Low
8033 51565 0.156 32.878 5.080 109.59
7342 50607 0.145 29.949 99.83
7458 50743 0.147 30.470 101.57
6694 48096 0.139 28.339 94.46
6617 46299 0.143 29.356 97.85
8311 55992 0.148 30.865 102.88
Mean 7409.16
7
50550.33
3
0.146 30.310 101.032
STD 685.326 3309.961 0.006 1.540 5.132
CV% 9.250 6.548 3.846 5.080 5.080
80
Table 24: Intra-day precision and accuracy data for QC mid samples of
candesartan based on the standard calibration curve of day three validation.
Theo.
Conc.
AUC
Drug
AUC IS Ratios Measured
Conc.
Precision% Accuracy%
500.00
ng/ml
QC
Mid
86062 51009 1.687 451.491 3.681 90.30
84685 48614 1.742 466.471 93.29
84219 46859 1.797 481.583 96.32
88387 49540 1.784 477.998 95.60
89640 48632 1.843 494.143 98.83
96158 51666 1.861 499.045 99.81
Mean 88191.833 49386.667 1.786 478.455 95.691
STD 4431.987 1755.362 0.064 17.611 3.522
CV% 5.025 3.554 3.608 3.681 3.681
Table 25: Intra-day precision and accuracy data for QC high samples of
candesartan based on the standard calibration curve of day three validation.
Theo.
Conc.
AUC Drug AUC IS Ratios Measured
Conc.
Precision% Accuracy%
800.00
ng/ml
QC
High
153531 52127 2.945 795.406 2.833 99.43
161763 52004 3.111 840.571 105.07
143703 48028 2.992 808.173 101.02
147175 50021 2.942 794.577 99.32
156006 52510 2.971 802.411 100.30
147578 51668 2.856 771.060 96.38
Mean 151626.000 51059.667 2.970 802.033 100.254
STD 6701.400 1718.973 0.083 22.724 2.840
CV% 4.420 3.367 2.800 2.833 2.833
Table represents inter day accuracy and precision for the quality control samples of
candesartan in three days of validation. All of the obtained accuracy and precision
data are within the required range.
81
Table 26: Inter day accuracy and precision for the quality control samples of
Candesartan in the three days of validation.
Mea
sure
d C
on
cen
trati
on
10 30 500 800
Day
One
Day
Two
Day
Three
Day
One
Day
Two
Day
Three
Day
One
Day
Two
Day
Three
Day
One
Day
Two
Day
Three
10.713 10.838 10.888 27.402 29.560 32.878 527.377 471.878 451.491 833.078 775.249 795.406
10.647 10.846 10.076 27.342 31.941 29.949 493.988 464.492 466.471 794.983 743.945 840.571
10.056 10.309 10.230 27.266 29.757 30.470 527.567 489.005 481.583 857.122 755.539 808.173
10.113 10.366 10.336 30.769 29.389 28.339 532.078 475.061 477.998 814.572 763.083 794.577
10.132 10.952 10.273 30.397 32.722 29.356 470.512 472.934 494.143 818.531 762.347 802.411
9.701 9.870 9.368 29.809 28.171 30.865 466.683 466.292 499.045 841.007 734.964 771.060
Mea
n
10.317 29.799 484.922 794.812
ST
D
0.437 1.696 23.591 35.549
CV
%
4.232 5.693 4.865 4.473
Acc
ura
cy %
103.174 99.330 96.984 99.352
82
3.1.4 Linearity
Linearity is determined by calculating the regression line using a mathematical
treatment of the results (i.e. least mean squares) vs. analyte concentration (Araujo
2009). The determination coefficient (R²) measures the amount of variation in the
response (dependent) variable explained by changes in the explanatory (independent
variable). A value of 1 for R² indicates a perfect linear relation between target
concentration and predicted concentration. The closer the value of R² to 1 the stronger
is the linear relation. A strong regression indicates a strong dose-response relationship
between predictor and outcome, which in turn supports a stronger validity for
predicted concentration of the drug.
The linear regression equation was used for calculating the predicted drug
concentration at the start of each validation experiment, using one unique target
concentration for getting the ―D area/ IS area‖ at each of the 3 days of validation for
each drug.
The R² was a perfect dose-response relationship for candesartan at 1st day of
validation. The remaining 2nd and 3rd day for candesartan validation showed an
almost perfect linear relation with an R² of 0.996. All the linear regression models
were statistically significant, figure 3 to 5.
Day 1 validation: table 27, which represents the standard calibration curve and intra-
day accuracy data, shows an accuracy range of (90.98-106.41). Represents the
standard calibration curve and intra-day accuracy data, shows an accuracy range of
(90.98-106.41).
Day 2 validation: table 28, which represents the standard calibration curve and intra-
day accuracy data, shows an accuracy range of (90.20-109.05).
83
Day 3 validation: table 31, which represents the standard calibration curve and intra-
day accuracy data, shows an accuracy range of (94.95-107.56).
Since the accepted criteria according to USFDA are 85%-115% except for the LLOQ
is 80%-120%, the results of three days of validation passed the required criteria in
terms of accuracy.
Table 27: Standard Calibration Curve of Day One Validation, intraday accuracy
data for candesartan.
Theoretical
conc.ng/ml
Drug
Area
IS
Area
Ratio Measured
Conc.
Accuracy%
10.00 3073 51659 0.059 9.098 90.98
25.00 5765 55295 0.104 24.217 96.87
75.00 14499 56771 0.255 75.269 100.36
250.00 42754 53723 0.796 257.830 103.13
400.00 80846 59901 1.350 444.913 111.23
600.00 102389 53257 1.923 638.432 106.41
1000.00 152936 56078 2.727 910.240 91.02
Ratio= drug area/IS area
Measured conc.= (ratio/0.00296)-(+ 0.0326)
Function is Y = 0.00296X + 0.0326 (R= 0.9962 )
P<0.05
Table 28: Raw data of the standard curve with regards to correlation, slope, R²,
and intercept for day one for candesartan.
Correlation (R) Slope R² Intercept
0.9962 0.00296 0.9924 +0.0326
84
Figure 3: The plot of calibration curve levels against their analytical response, in day
one validation for candesartan.
Table 29: Standard calibration curve of day two validation, intraday accuracy
data for Candesartan.
Theoretical
conc.
ng/ml
Drug
Area
IS
Area
Ratio Measured
Conc.
Accuracy%
10.00 3503 56813 0.062 9.682 96.82
25.00 6062 56165 0.108 22.551 90.20
75.00 16519 55345 0.298 75.531 100.71
250.00 52422 52043 1.007 272.624 109.05
400.00 81808 52448 1.560 426.250 106.56
600.00 118126 52358 2.256 619.878 103.31
1000.00 165841 49008 3.384 933.484 93.35
Ratio= drug area/IS area
Measured conc.= (ratio/0.0036)-(+ 0.0268 )
Function is Y = 0.0036X + 0.0268 (R= 0.9978 )
p<0.05
0.00
0.50
1.001.50
2.00
2.50
3.00
3.50
0 200 400 600 800 1000 1200
Rat
io
Measured Conc. ng/ml
85
Table 30: Raw data of the standard curve with regards to correlation, slope, R²,
and intercept for day two for candesartan.
Correlation (R) Slope R² intercept
0.9978 0.0036 0.9956 +0.0268
Figure 4: The plot of calibration curve levels against their analytical response, in day
two validation for candesartan.
Table 31: Standard calibration curve of day three validation, intraday accuracy
data for Candesartan.
Theoritical conc. Drug Area IS Area Ratio Measured Conc. Accuracy%
(ng/ml)
10.00 3435 48185 0.071 9.776 97.76
25.00 5407 44192 0.122 23.737 94.95
75.00 15731 48172 0.327 79.560 106.08
250.00 44708 47429 0.943 247.963 99.19
400.00 71748 48571 1.477 394.079 98.52
600.00 118419 49416 2.396 645.347 107.56
1000.00 178697 50397 3.546 959.538 95.95
0.000
1.000
2.000
3.000
4.000
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
Rat
io
Measured Conc. ng/ml
Validation of Day Two
86
Ratio= drug area/IS area
Measured conc.= (ratio/0.00366)-(+0.0355)
Function is Y = 0.00366X + 0.0355 (R= 0.9987 )
p<0.05
Table 32: Raw data of the standard curve with regards to correlation, slope, R²,
and intercept for day three for candesartan
Correlation (R) Slope R² intercept
0.9987 0.00366 0.9974 +0.0355
Figure 5: The plot of calibration curve levels against their analytical response, in day
three validation for candesartan.
0.00
1.00
2.00
3.00
4.00
0 200 400 600 800 1000 1200
Rat
io
Measured Conc. ng/ml
Validation of Day Three
87
Table 33: Linearity and linear working range of six standard curves of
Candesartan data based on the measured concentration.
Concentration for each Standard Point
Curve # 10.00 25.00 75.00 250.00 400.00 600.00 1000.00
1 9.098 24.217 75.269 257.830 444.913 638.432 910.240
2 9.682 22.551 75.531 272.624 426.250 619.878 933.484
3 9.776 23.737 79.560 247.963 394.079 645.347 959.538
4 9.495 25.251 75.088 241.489 427.222 635.725 945.729
5 9.949 25.009 68.976 253.792 426.317 642.156 933.800
6 9.704 23.590 76.920 249.280 431.006 624.428 945.071
Mean 9.617 24.059 75.224 253.830 424.965 634.328 937.977
STD 0.294 0.995 3.488 10.739 16.715 10.083 16.627
CV% 3.055 4.134 4.637 4.231 3.933 1.590 1.773
Accuracy
%
96.173 96.237 100.299 101.532 106.241 105.721 93.798
Min 9.098 22.551 68.976 241.489 394.079 619.878 910.240
Max 9.949 25.251 79.560 272.624 444.913 645.347 959.538
Table 34: Linearity and linear working range of candesartan data based on
normalized concentration derived from standard calibration curves.
Calibration
Curve #
Normalized Concentration
10.00 25.00 75.00 250.00 400.00 600.00 1000.00
1 0.910 0.969 1.004 1.031 1.112 1.064 0.910
2 0.968 0.902 1.007 1.090 1.066 1.033 0.933
3 0.978 0.949 1.061 0.992 0.985 1.076 0.960
4 0.950 1.010 1.001 0.966 1.068 1.060 0.946
5 0.995 1.000 0.920 1.015 1.066 1.070 0.934
6 0.970 0.944 1.026 0.997 1.078 1.041 0.945
Mean 0.962 0.962 1.003 1.015 1.062 1.057 0.938
STD 0.029 0.040 0.047 0.043 0.042 0.017 0.017
CV% 3.055 4.134 4.637 4.231 3.933 1.590 1.773
Min 0.910 0.902 0.920 0.966 0.985 1.033 0.910
Max 0.995 1.010 1.061 1.090 1.112 1.076 0.960
88
Table 35: Linearity and linear working range of six standard curves of
candesartan data based on the calculated area ratio.
Calibration
Curve #
AUC Ratio for Standard Point
10.00 25.00 75.00 250.00 400.00 600.00 1000.00
1 0.059 0.104 0.255 0.796 1.350 1.923 2.727
2 0.062 0.108 0.298 1.007 1.560 2.256 3.384
3 0.071 0.122 0.327 0.943 1.477 2.396 3.546
4 0.068 0.121 0.288 0.844 1.464 2.161 3.197
5 0.059 0.108 0.251 0.854 1.417 2.120 3.071
6 0.064 0.103 0.253 0.738 1.248 1.792 2.693
Mean 0.064 0.111 0.279 0.864 1.419 2.108 3.103
STD 0.005 0.008 0.031 0.098 0.109 0.220 0.345
CV% 7.814 7.585 11.005 11.330 7.668 10.444 11.109
Min 0.059 0.103 0.251 0.738 1.248 1.792 2.693
Max 0.071 0.122 0.327 1.007 1.560 2.396 3.546
Table 36: Raw data of six standard curves with regards to correlation, slope, R²,
and intercept for candesartan.
Correlation (R) Slope R² Intercept
0.9970 0.003147 0.994041 +0.074384
89
Figure 6: The plot of linearity of calibration curve levels for candesartan
quantification against their analytical response and regression linear equation.
3.1.5 Stability
From the table’s data, we find the autosampler stability test is passed according to the
ICH accepted range where the accuracy % doesn’t exceed 15%. Table 37 and 38
shows data for short term stability indicated by two QC concentrations (low, high) for
candesartan after preparation procedure (auto-sampler stability), T=4 C °.
Table 37: candesartan QC low samples stability autosampler procedure at 4 C °.
QC Low ( 30 ng/ml)
Time AUC
Drug
AUC IS Ratios Measured
Conc.
Mean
Measured
Accuracy% Stability
0.00
Hour
7032 50172 0.140 30.945 30.663 103.15 100.00
6893 51216 0.135 29.281 97.60
7501 52499 0.143 31.763 105.88
24.00
Hours
7373 52124 0.141 31.335 30.985 104.45 101.05
7039 50656 0.139 30.587 101.96
7448 53033 0.140 31.032 103.44
y = 0.003147x + 0.074384R² = 0.994041
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
Linearity for six calibration curves
90
Table 38: candesartan QC high samples stability autosampler procedure at 4 C°.
QC High (800.00)ng/ml
Time AUC
Drug
AUC
IS
Ratios Measured
Conc.
Mean
Measured
Accuracy
%
Stability
0.00
Hour
132859 49836 2.666 786.750 812.733 98.34 100.00
133477 48306 2.763 815.833 101.98
146874 51913 2.829 835.615 104.45
24.00
Hours
120410 44924 2.680 791.043 786.073 98.88 96.72
131163 49505 2.649 781.827 97.73
126637 47586 2.661 785.348 98.17
Table 39: Calibration curve for QC samples showing 4 C° stability for
candesartan.
Theoritical
conc.
ng/ml
Drug
Area
IS
Area
Ratio Measured
Conc.
Accuracy
10.00 3491 50980 0.068 9.495 94.95
25.00 5945 49079 0.121 25.251 101.00
75.00 14595 50734 0.288 75.088 100.12
250.00 41611 49316 0.844 241.489 96.60
400.00 75282 51407 1.464 427.222 106.81
600.00 113250 52400 2.161 635.725 105.95
1000.00 170741 53403 3.197 945.729 94.57
Ratio= drug area/IS area, Measured conc.= (ratio/0.00334)-(+0.0367)
Function is Y = 0.00334X + 0.0367 (R= 0.9982)
Table 40: Raw data of six standard curves with regards to correlation, slope, R²,
and intercept for candesartan.
Correlation (R) Slope R² Intercept
0.9982 0.00334 0.9964 +0.0367
91
Figure 7: The plot of QC samples against a calibration curve, obtained from freshly
spiked calibration standard, showing Candesartan 4 C° temperature
Regarding short term stability at room temperature or processing temperature, freshly
prepared 0 hour two QC’s concentrations were taken as a reference upon calculating
stability of candesartan at room temperature. All the results are within the accepted
criteria which are in the range 85%-115%, as shown in table 41 and 42.
Stability %= mean of measured concentration at 0 hr/mean of measured concentration
at 8 hr*100%.
Table 41: Candesartan QC low samples stability at (RT C°)
QC Low ( 30 ng/ml )
Time AUC
Drug
AUC IS Ratios Measured
Conc.
Mean
Measured
Accuracy% Stability
0.00
Hour
7032 50172 0.140 30.945 30.663 103.15 100.00
6893 51216 0.135 29.281 97.60
7501 52499 0.143 31.763 105.88
24.00
Hour
s
6666 52222 0.128 31.031 30.527 103.44 99.56
6062 47687 0.127 30.870 102.90
6528 52968 0.123 29.680 98.93
0.000
0.500
1.000
1.500
2.000
2.500
3.000
3.500
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
92
Table 42: Candesartan QC high samples stability at (RT C°) temperature
QC High ( 800 ng/ml )
Time AUC
Drug
AUC
IS
Ratios Measured
Conc.
Mean
Measured
Accuracy% Stability
0.00
Hour
132859 49836 2.666 786.750 812.733 98.34 100.00
133477 48306 2.763 815.833 101.98
146874 51913 2.829 835.615 104.45
24.00
Hours
130755 49836 2.624 796.483 794.742 99.56 97.79
129501 50030 2.588 785.688 98.21
139444 52783 2.642 802.054 100.26
Table 43: Calibration curve for QC samples showing candesartan stability
autosampler procedure at RT°C.
Theoritical conc.
ng/ml
Drug Area IS Area Ratio Measured Conc. Accuracy
10.00 3026 51374 0.059 9.949 99.49
25.00 5304 49101 0.108 25.009 100.04
75.00 12325 49027 0.251 68.976 91.97
250.00 42709 50008 0.854 253.792 101.52
400.00 74130 52329 1.417 426.317 106.58
600.00 103724 48916 2.120 642.156 107.03
1000.00 153889 50103 3.071 933.800 93.38
Ratio= drug area/IS area, Measured conc.= (ratio/0.00326)-(+0.0265)
Function is Y = 0.00326X + 0.0265 (R= 0.9977 )
Table 44: Raw data of six standard curves with regards to correlation, slope, R²,
and intercept for candesartan.
Correlation (R) Slope R² Intercept
0.9977 0.00326 0.9954 +0.145
93
Figure 8: The plot of QC samples against a calibration curve, obtained from freshly
spiked calibration standard, showing candesartan stability autosampler procedure at
Room temp.
Regarding the freeze and thaw stability: the QC samples are stored and frozen in the
freezer at the intended temperature and thereafter thawed at room or processing
temperature. After complete thawing, samples are refrozen again applying the same
conditions. At each cycle, samples should be frozen for at least 12 hours before they
are thawed. The accuracy for QC low and high after 3 cycles is within the accepted
range which is 85-115%, table 45 and 46.
Table 45: The accuracy of three standard curves of candesartan showing freeze
and thaw stability of QC low.
QC Low (30 ng/ml)
Time AUC
Drug
AUC
IS
Ratios Measured
Conc.
Mean
Measured
Accuracy% Stability
0.00
Hour
7032 50172 0.140 30.945 30.663 103.15 100.00
6893 51216 0.135 29.281 97.60
7501 52499 0.143 31.763 105.88
3
Cycles
6236 49732 0.125 31.475 30.596 104.92 99.78
5653 47407 0.119 29.281 97.60
6002 48347 0.124 31.031 103.44
0.000
1.000
2.000
3.000
4.000
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
94
Table 46: The accuracy of three standard curves of candesartan showing freeze
and thaw stability of QC high.
QC High (800ng/ml)
Time AUC
Drug
AUC
IS
Ratios Measured
Conc.
Mean
Measured
Accuracy% Stability
0.00
Hour
132859 49836 2.666 786.750 812.733 98.34 100.00
133477 48306 2.763 815.833 101.98
146874 51913 2.829 835.615 104.45
3
Cycles
115084 49559 2.322 813.097 797.785 101.64 98.16
112334 50308 2.233 781.362 97.67
111960 49057 2.282 798.897 99.86
Table 47: Calibration curve for QC samples showing freeze and thaw stability
for candesartan.
Theoritical
conc.
ng/ml
Drug
Area
IS
Area
Ratio Measured Conc. Accuracy%
10.00 2967 46208 0.064 9.704 97.04
25.00 4888 47348 0.103 23.590 94.36
75.00 13471 53221 0.253 76.920 102.56
250.00 37842 51308 0.738 249.280 99.71
400.00 62981 50454 1.248 431.006 107.75
600.00 88918 49622 1.792 624.428 104.07
1000.00 131982 49008 2.693 945.071 94.51
Ratio= drug area/IS area, Measured conc.= (ratio/0.00281)-(+0.0369)
Function is Y = 0.00281X + 0.0369 (R= 0.9984 )
Table 48: Raw data of six standard curves with regards to correlation, slope, R²,
and intercept for candesartan.
Correlation (R) Slope R² Intercept
0.9984 0.00281 0.9968 +0.0369
95
Figure 9: The plot of QC samples against a calibration curve, obtained from freshly
spiked calibration standard, showing candesartan freeze and thaw stability.
3.1.6 Sensitivity
The protein direct precipitation procedure was specified and sensitive for candesartan,
where both blank and zero samples that examined from six deferent lots of plasma
were attained the required clean chromatogram for specific method.
3.2 The modifying effect of combining fruit juices with candesartan
The serum concentration of candesartan with or without fruit juices was evaluated on
rats on a sample size of 8 for drug alone and another sample size of 6 when
candesartan is combined with liqourice and another sample size of 3 when
candesartan is combined with orange and another sample size of 3 when candesartan
is combined with pomegranate. The measurements were repeated at 6 time intervals
following drug administration to a maximum of 6 hours.
0.000
0.500
1.000
1.500
2.000
2.500
3.000
0.00 200.00 400.00 600.00 800.00 1000.00 1200.00
96
3.2.1 Effect of combination on candesartan
As shown in figures 11 to 13, when candesartan was administered alone its serum
level reached its maximum (964.692 ng/ml) after half an hour and then gradually
declines to reach a minimum concentration of (272.679 ng/ml) after 6 hours from the
administration of candesartan.
The same drug when administered combined with orange makes slightly increase in
serum concentration levels... It reaches its maximum serum concentration after half an
hour (1253.163 ng/ml) and then gradually declines to reach a minimum concentration
after 6 hours (253.149 ng/ml).
And when candesartan administered was combined with liquorice makes slightly
decrease in serum concentration levels. It reaches its maximum serum concentration
after half an hour (818.2868 ng/ml) and then gradually declines to reach a minimum
concentration after 6 hours (276.4665 ng/ml).
But the combination of candesartan and pomegranate shows a decrease in serum
concentration approximately the half the concentrations when candesartan is given
alone. At the first half an hour of administration it reaches its maximum serum
concentration (475.9673 ng/ml) and then gradually declines to reach a minimum
concentration of (188.1737 ng/ml) at the end of follow up period (6 hours).
97
Table 49: Pharmacokinetic data of candesartan
Drug Cmax (ng/ml) Tmax
(hr)
AUC (ng/ml*hr)
Candesartan 964.692±374.2553 0.5 2864.291±409.3088
Candesartan with orange juice 1253.163±136.9737# 0.5 3156.797±68.5176#
Candesartan with liqourice juice 818.2868±347.0212# 0.5 2551.162±587.6517#
Candesartan with pomegranate
juice
475.6973±139.5053# 0.5 1561.537±259.2953*
*P<0.05 (significant), #P>0.05 (insignificant)
The difference between Cmax of candesartan alone and candesartan with orange is
insignificant (P>0.05). Also for candesartan with liquorice, Cmax difference between
candesartan alone and candesartan with liquorice were insignificant, the difference in
Cmax between candesartan and candesartan with pomegranate is insignificant too
(P>0.05).
For the AUC, the difference between candesartan alone and candesartan with orange
is insignificant (P>0.05). Also for candesartan with liquorice, Cmax difference
between candesartan alone and candesartan with liquorice were insignificant
(P>0.05). But the difference between candesartan alone and candesartan with
pomegranate is significant (P<0.05).
98
Figure 10: Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, each data point represents the mean
± SEM (n=8).
Figure 11: Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, comparing candesartan with orange
juice and solitary drug use, each data point represents the mean ± SEM (n=3).
0
200
400
600
800
1000
1200
0 1 2 3 4 5 6 7
Dru
g p
lasm
a co
nce
ntr
atio
n (
ng/
ml)
Time after administartion (hour)
Candesartan
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5 6 7
Dru
g p
lasm
a co
nce
ntr
atio
n (
ng/
ml)
Time after administration (hour)
Cadesartan with orange
Candesartan
99
Figure 12: Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, comparing candesartan with
liqourice juice and solitary drug use, each data point represents the mean ± SEM
(n=6).
Figure 13: Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration, comparing candesartan with
pomegranate juice and solitary drug use, each data point represents the mean ± SEM
(n=3).
0
200
400
600
800
1000
1200
0 1 2 3 4 5 6 7
Dru
g p
lasm
a co
nce
ntr
atio
n (
ng/
ml)
Time after administration (hour)
Candesartan with licorice
Canesartan
0
200
400
600
800
1000
1200
0 1 2 3 4 5 6 7
Dru
g p
lasm
a co
nce
ntr
atio
n (
ng/
ml)
Time after adminsitration (hour)
Candesatran with pomagranate
Candesartan
100
Figure 14: Rat plasma profile showing the changes in mean serum candesartan
concentration with time after drug administration comparing combined and solitary
drug use.
Table 50: Comparing the mean serum candesartan drug concentration at
selected time intervals after administration between single and combined drug
use.
Plasma concentration (ng/ml)
Drug assessed
candesartan
30 min 1 hour 2 hours 3 hours 4 hours 6 hours
candesartan (n=8)
Mean 964.692 785.724 593.097 429.240 331.759 272.679
SD 377.7636 238.8949 90.2467 71.11296 45.06003 68.71602
SD.Error 133.5596 84.46212 31.90703 25.14223 15.93113 24.29478
Range (581.313-
1586.134)
(545.327-
1087.437)
(445.422-
701.449)
(350.796-
560.130)
(279.878-
404.249)
(175.348-
386.792)
candesartan with orange (n=3)
Mean 1253.163 893.117 626.629 469.421 340.785 253.149
SD 136.9735 66.632 93.98632 45.44197 25.83187 39.82667
SD Error 80.57262 39.19541 55.28607 26.73057 15.19522 23.42745
Range (1129.185-
1400.204)
(822.397-
954.723)
(568.667-
735.069)
(437.781-
521.492)
(325.397-
370.608)
(214.593-
294.135)
candesartan with liqourice (n=6)
0
200
400
600
800
1000
1200
1400
0 1 2 3 4 5 6 7
Dru
g p
lasm
a co
nce
ntr
atio
n (
ng/
ml)
Time after administration (hour)
Candesartan
Candesartan with orange
Candesartan with licorice
Candesartan with pomagranate
101
Mean 818.2868 669.094 508.638 385.7355 312.9038 276.4665
SD 351.8374 211.5204 102.9133 117.1978 52.08732 85.78684
SD Error 143.637 86.35286 42.01419 47.8458 21.26456 35.02233
Range (438.328-
1222.506)
(437.274-
949.166)
(359.936-
641.635)
(285-
606.909)
(260.1-
382.4)
(175.3-
389.9)
candesartan with pomegranate (n=3)
Mean 475.97 340.53 273.12 246.20 240.45 188.17
SD 139.51 71.38 21.50 47.99 36.87 50.76
SE 80.54 41.21 12.42 27.71 21.29 29.31
Range 315.02-
562.21)
(284.49-
420.89)
(248.31-
284.72)
(190.96-
277.60)
(200.21-
272.62)
(134.50-
235.41)
Effect of combination of candesartan with orange compared to solitary drug effect
Difference between
2 means
288.471 107.393 33.532 40.181 9.026 -19.53
Cohen's d 1.0153 0.8634 0.3639 0.6733 0.2457 0.3477
Percent change
compared to
solitary
29.90% 13.67% 5.65% 9.36% 2.72% -7.16%
P (t-test) 0.2409 0.4755 0.5998 0.3938 0.7557 0.6601
Effect of combination of candesartan with liqourice compared to solitary drug effect
Difference between 2
means
-146.4052 -116.63 -84.459 -43.5045 -18.8552 3.7875
Cohen's d 0.4010 0.5169 0.8730 0.4488 0.3871 0.0487
Percent change
compared to solitary
-15.18% -14.84% -14.%24 -10.14% -5.68% 1.39%
P (t-test) 0.4745 0.3620 0.1283 0.4040 0.4820 0.9283
Effect of combination of candesartan with pomegranate compared to solitary drug effect
Difference between 2
means
-488.722 -445.194 -319.977 -183.04 -91.309 -84.509
Cohen's d 1.7163 2.5251 4.8777 3.0173 2.2178 1.3989
Percent change
compared to solitary
-0.50.66% -56.66% -53.95% -42.64% -27.52% -30.99%
P (t-test) 0.0624 0.0131 0.0002 0.0029 0.0125 0.0876
102
The mean serum Candesartan concentration after half an hour when used in
combination with orange juice was increased by 288.471 ng/ml compared to its
isolated administration. This 29.90% increase in serum candesartan concentration,
however failed to reach the level of statistical significance. The effect of this
combination on serum candesartan level compared to its single drug use was
evaluated as a small effect (Cohen’s d=1.0153), table 11 and figure 15.
Figure 15: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of drug
administration between single and combined drug use.
The mean serum candesartan concentration after one hour when used in combination
with orange was increased by 107.393 ng/ml compared to its isolated administration.
The effect of drug combination on increasing serum candesartan level compared to its
single drug use was evaluated as a small effect (Cohen’s d=0.8634). This 13.67%
increase in serum concentration after using combination, also failed to reach the level
0
200
400
600
800
1000
1200
1400
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
h
alf
an h
ou
r
103
of statistical significance (possibly because of very small sample size), table 11 and
figure 16.
Figure 16: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug administration
between single and combined drug use.
The mean serum candesartan concentration after two hours when used in combination
with orange was increased by 33.532 ng/ml compared to its isolated administration.
The effect of drug combination on increasing serum candesartan level compared to its
single drug use was evaluated as a small effect (Cohen’s d=0.363). This 5.65%
increase in serum concentration after using combination, also failed to reach the level
of statistical significance (possibly because of very small sample size), table 11 and
figure 17.
0
100
200
300
400
500
600
700
800
900
1000
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
o
ne
ho
ur
104
Figure 17: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after three hours when used in
combination with orange juice was increased by 40.181 ng/ml compared to its
isolated administration. The effect of drug combination on increasing serum
candesartan level compared to its single drug use was evaluated as a moderate effect
(Cohen’s d=0.67). This 9.36% increase in serum concentration after using
combination, also failed to reach the level of statistical significance, table 11 and
figure 18.
500
520
540
560
580
600
620
640
660
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l)
afte
r tw
o h
ou
rs
105
Figure 18: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after four hours when used in combination
with orange juice was increased by 9.02 ng/ml compared to its isolated
administration. The effect of drug combination on elevating serum candesartan level
compared to its single drug use was evaluated as a small effect (Cohen’s d=0.24).
This 2.27% reduction in serum concentration after using combination, also failed to
reach the level of statistical significance (may be due to very small sample size), table
11 and figure 19.
360
380
400
420
440
460
480
500
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l)
afte
r th
ree
ho
urs
106
Figure 19: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 4 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after six hours when used in combination
with orange juice was decreased by -19.53 ng/ml compared to its isolated
administration. The effect of drug combination on lowering serum candesartan level
compared to its single drug use was evaluated as a moderate effect (Cohen’s d=-
0.3477). This 7.16% decrease in serum concentration after using combination, also
failed to reach the level of statistical significance (may be due to very small sample
size), table 11 and figure 20.
0
50
100
150
200
250
300
350
400
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
fo
ur
ho
urs
107
Figure 20: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after half an hour when used in
combination with liqourice was decreased by 146.405 ng/ml compared to its isolated
administration. This 15.18% decrease in serum candesartan concentration after using
combination, also failed to reach the level of significant. The effect of this
combination on serum candesartan level compared to its single drug use was
evaluated as a moderate (Cohen’s d=0.4010), table 11 and figure 21.
0
50
100
150
200
250
300
350
400
Candesartan Candesartan with orange
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
si
x h
ou
rs
108
Figure 21: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of drug
administration between single and combined drug use.
The mean serum candesartan concentration after one hour when used in combination
with liqourice was decreased by 116.63 ng/ml compared to its isolated administration.
The effect of this combination on serum candesartan level compared to its single drug
use was evaluated as a moderate effect (Cohen’s d=0.51), This 14.84% decrease in
serum concentration after using combination, also failed to reach the level of
statistical significance (may be due to very small sample size), table 11 and figure 22.
0
200
400
600
800
1000
1200
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
h
alf
an h
ou
r
109
Figure 22: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug administration
between single and combined drug use.
The mean serum candesartan concentration after two hours when used in combination
with liqourice was decreased by 84.459 ng/ml compared to its isolated administration.
The effect of this combination on serum candesartan level compared to its single drug
use was evaluated as a moderate effect (Cohen’s d=0.873), This 14.24% decrease in
serum candesartan concentration after using combination, also failed to reach the level
of statistical significance, table 11 and figure 23.
0
100
200
300
400
500
600
700
800
900
1000
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
o
ne
ho
ur
110
Figure 23: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after three hours when used in
combination with liqourice was decreased by 43.50ng/ml compared to its isolated
administration. The effect of this combination on serum candesartan level compared
to its single drug use was evaluated as a moderate effect (Cohen’s d=0.4488). This
10.14% decrease in serum candesartan concentration after using combination, failed
to reach the level of statistical significance (possibly because of very small sample
size), table 11 and figure 24.
0
100
200
300
400
500
600
700
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
tw
o h
ou
rs
111
Figure 24: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after four hours when used in combination
with liqourice was lowered by 18.85 ng/ml compared to its isolated administration.
The effect of this combination on serum candesartan level compared to its single drug
use was evaluated as a moderate effect (Cohen’s d=0.38). This 5.6% reduction in
serum candesartan concentration after using combination, also failed to reach the level
of statistical significance (possibly because of very small sample size), table 11 and
figure 25.
0
50
100
150
200
250
300
350
400
450
500
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
th
ree
ho
urs
112
Figure 25: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 4 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after six hours when used in combination
with liqourice was increased by 3.787 ng/ml compared to its isolated administration.
The effect of this combination on serum candesartan level compared to its single drug
use was evaluated as a weak effect (Cohen’s d=0.048). This 1.39% reduction in serum
candesartan concentration after using combination, also failed to reach the level of
statistical significance (possibly because of very small sample size), table 11 and
figure 26.
260
270
280
290
300
310
320
330
340
350
360
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
fo
ur
ho
urs
113
Figure 26: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after half an hour when used in
combination with pomegranate was lowered by 488.722 ng/ml compared to its
isolated administration. The effect of this combination on serum candesartan level
compared to its single drug use was evaluated as a large effect (Cohen’s d=1.716), yet
This 50% reduction in serum candesartan concentration after using combination, also
failed to reach the level of statistical significance, table 11 and figure 27.
0
50
100
150
200
250
300
350
Candesartan Candesartan with licorice
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
si
x h
ou
rs
114
Figure 27: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after half an hour of drug
administration between single and combined drug use.
The mean serum candesartan concentration after one hour when used in combination
with pomegranate was decreased by 445.149 ng/ml compared to its isolated
administration. This 56.66% decrease in serum candesartan concentration is
significant (P<0.05). The effect of this combination on serum candesartan level
compared to its single drug use was evaluated as a highly effective (Cohen’s
d=2.525), table 11 and figure 28.
0
200
400
600
800
1000
1200
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
h
alf
an h
ou
r
115
Figure 28: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after one hour of drug administration
between single and combined drug use.
The mean serum candesartan concentration after two hours when used in combination
with pomegranate was lowered by 319.977 ng/ml compared to its isolated
administration. This 53.95% decrease in serum candesartan concentration is
significant (P<0.05). The effect of this combination on serum candesartan level
compared to its single drug use was evaluated as a strong effect (Cohen’s d=4.877),
table 11 and figure 29.
0
100
200
300
400
500
600
700
800
900
1000
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
o
ne
ho
ur
116
Figure 29: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 2 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after three hours when used in
combination with pomegranate was decreased by 183.04 ng/ml compared to its
isolated administration. The effect of this combination on serum candesartan level
compared to its single drug use was evaluated as a strong effect (Cohen’s d=3.01).
This 42.64% decrease in serum candesartan concentration after using combination is
significant (P<0.05), table 11 and figure 30.
0
100
200
300
400
500
600
700
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
tw
o h
ou
rs
117
Figure 30: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 3 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after four hours when used in combination
with pomegranate was lowered by 91.30 ng/ml compared to its isolated
administration. The effect of this combination on serum candesartan level compared
to its single drug use was evaluated as a strong effect (Cohen’s d=2.217). This
27.52% reduction in serum candesartan concentration after using combination, is
significant (P<0.05), table 11 and figure 31.
0
50
100
150
200
250
300
350
400
450
500
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
th
ree
ho
urs
118
Figure 31: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 4 hours of drug administration
between single and combined drug use.
The mean serum candesartan concentration after six hours when used in combination
with liqourice was lowered by 84.509 ng/ml compared to its isolated administration.
The effect of this combination on serum candesartan level compared to its single drug
use was evaluated as a moderate effect (Cohen’s d=1.398). This 30.99% reduction in
serum candesartan concentration after using combination, also failed to reach the level
of statistical significance, table 11 and figure 32.
0
50
100
150
200
250
300
350
400
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
fo
ur
ho
urs
119
Figure 32: Diagram with error bars comparing the mean (with its 95% confidence
interval) serum candesartan drug concentration after 6 hours of drug administration
between single and combined drug use.
0
50
100
150
200
250
300
350
Candesartan Candesartan with pomagranate
Pla
sma
con
cen
trat
ion
(n
g/m
l) a
fter
si
x h
ou
rs
120
3.3 Discussion
Candesartan is an Angiotensin receptor blocker (ARBs) drug that binds to angiotensin
II receptor type1 selectively and competitively and thus blocks the vasoconstrictor
and aldosterone-secreting effects of angiotensin II in many tissues including vascular
smooth muscle and the adrenal gland. and results in an overall decrease in blood
pressure, it is a drug used chronically. In the recent years, an increase in beverage
intake was noticed , drug interactions with beverages have received considerable
attention. The interaction can affects the activity of a drug, Because a lot of juices
shown to inhibit or induce liver enzymes or modulate intestinal drug absorption via
the P-g pmediated efflux and OATP-mediated uptake transport systems the intestine
and liver. It has been well documented that the components of grapefruit, such as
bergamottin and (R)-6′,7′-dihydroxybergamottin, demonstrate potent inhibition of
CYP 450 system (Guo et al., 2000; Paine et al., 2004).
pomegranate has been widely consumed in many countries including the middle east
region. Pomegranate is a rich source of several chemicals such as pectin, tannins,
flavonoids, and anthocyanins. However, debaited data are available on whether the
component(s) of pomegranate inhibits or induce the metabolism and/or absorption of
drugs. Liquorices (Glycyrrhiza glabra),was firstly used by the pre-date ancient
civilization of Babylonian, Egyptian and Chinese cultures(Wang, Ma, Fu, Lee, &
Wang, 2004). Chemically, liquorice roots contain several triterpenes, such as
glycyrrhizin and glycyrrhetic acid, together with variety of flavones, Glycerhizien
glycoside is the main active ingrideint of liquorice, it is a sweet-tasting constituent.
Making it 50 times sweeter than sugar, (Acharya, et al., 1993).
121
The effect of glycyrrhiza uralensis, showed induction effect on CYP450 isozymes.
Efficacy and safety profiles of a drug may be affected when it administered
concomitantly with liqourice (Tang et al.., 2009). and 7-ethoxycumarin O-deethylase
(ECOD, 2.8 and 2.5 fold) were also shown to be increased (Asl and Hosseinzadah
2008).
Orange juice is probably the best known and most widespread fruit juice all over the
world. It has high content of flavonoids, phenylpropanoids, hesperidin. it is cholesrtol
free, fat free(Gianni Galaverna, et al. 2008), according to recent report orange juice,
acts as an inhibitor of organic anion transporter proteins (OATPs) based on the report
finding that the bioavailability of the antihistaminic drug fexofenadine in humans was
reduced with Orange juice intake, (Dresser et al.. 2002).
The aim of the current study was to develop a new validated simple chromatographic
method for quantifying candesartan and to study the effect of pomegranate, orange
and liquorice juices on candesartan serum concentration in a pre fed rats.
According to the data presented by in this study, the manner in which candesartan
concentration level was altered is caused by the component(s) of pomegranate.
Therefore, it is of interest to determine the identity of the chemical(s) in pomegranate
juice that exhibits the stated result. to enable health care professionals to avoid
beverage-drug interactions. Furthermore, identifying situations in which the inhibition
or induction of the liver and/or intestinal enzymes and/or transporters may be of
therapeutic benefit.
In this study, we demonstrated the influence of pomegranate, liquorice, and orange
juice on the pharmacokinetics of candesartan in rats, in comparison with water. the
AUC of candesartan decreased, (approximately 1 fold) upon adminstration to
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pomegranate juice.(table 49) The AUC ratio and Cmax of candesartan with water
were not significantly altered by administration of either liquorice or orange juice.
As shown in figures 10 to 13, when candesartan was administered alone its serum
level reached its maximum (964.692 ng/ml) after half an hour and then gradually
declines to reach a minimum concentration of (272.679 ng/ml) after 6 hours from the
administration of candesartan.
And when candesartan was administered to an orange pre-fed rat groups, slightly
increase in serum concentration levels were resulted, Candesartan reaches its
maximum serum concentration after half an hour (1253.163 ng/ml) and then gradually
declines to reach a minimum concentration after 6 hours (253.149 ng/ml).
But when candesartan was given to a liquorice pre-fed rat groups, a slightly decrease
in serum concentration levels were obsereved. Candesartan reaches its maximum
serum concentration after half an hour (818.287 ng/ml) and then gradually declines to
reach a minimum concentration after 6 hours (276.467 ng/ml).
Never the less, when candesartan was administered to a pomegranate pre-fed rat
groups, a significant decrease in serum concentration were resulted, at the first half an
hour of administration candesartan reaches its maximum serum concentration
(475.967 ng/ml) and then gradually declines to reach a minimum concentration of
(188.174 ng/ml) at the end of follow up period (6 hours). Regarding pomegranate
juice uptake, the metabolic effect is not necessary to be only via the hepatic enzymes,
and since candesartan in mainly excreted unchanged, we considered according to our
result that pomegranate juice might as well affect the intestinal metabolic system
which resulted in beverage-drug interactions. The mechanism of beverage-drug
interactions in the intestine consists of several systems (Lilja et al., 2003). mainly
123
divided into metabolism and absorption. The P-glycoprotein is believed to play an
important role in the efflux of drugs, which results in poor absorption of these drugs.
Therefore, pomegranate juice might be an inducer of P-glycoprotein in the intestine
and reduce the absorption of candesartan. Also it has recently been reported that
pomegranate juice affects intestinal uptake transporters as well as P-glycoprotein
(Dresser and Bailey, 2003; Lilja et al.., 2003). Thus, we conclude that the decrease in
the AUC of candesartan by pomegranate juice could be due to the induction of enteric
p-glycoprotien activity and/or due alteration in the intestinal uptake transporters
system. However, this hypothesis needs to be explored in future studies.
In addition the difference in T max between rats and humans, candesartan maximum
concentration where reached after 30min (½ hr) in rats compared to 3-4 hr’s in
human. The overall rate of biotransformation of candesartan in rats is markedly
different from that in humans; However, the major metabolic pathways of the drug are
almost similar in both rats and humans (Lertratanangkoon and Horning, 1982).,
However, it is difficult to extrapolate our results, which were obtained in rats, to
humans. Quantitative evaluation of pomegranate-drug interaction in humans needs to
be verified by studies in humans. Therefore, further investigations in humans are
necessary to develop our findings.
Validation
A full method validation according to ICH and EMA guidelines were performed for
our analytical method to demonstrate the reliability of a our method for the
determination of candesartan concentration in a specific rat plama.
124
In the current study, HPLC/MS/MS method is used to separate, identify and
determine the concentration candesartan in plasma. moreover that this method is very
fast, reproducible and easy to operate (Pharmacopoeial Forum, 2004). The developed
method was validated to meet the requirements for a global regulatory filing. The
validation parameters such as precision, linearity, specificity, accuracy, limit of
quantitation were carried out in accordance with ICH and US Pharmacopoeia
guidelines.
Linearity
The linearity of candesartan response is evaluated from the range of 10–1000 ng/mL
and showed a good correlation coefficient (r2) of more than 0.997. To validate
linearity, the standard curve of candesartan was constructed by plotting concentration
(ng/mL) versus area response (mAU) which is shown figuare3 to 5. The linear
regression and slope were calculated and are shown in Tables 28, 30, 32 and 36.
4.2.3. Precision
The precision of an analytical procedure expresses the closeness of the agreement
(degree of scatter) between a series of measurements obtained from the multiple
samples of the same homogeneous sample under the prescribed conditions.
Repeatability is a measure of the precision under the same operating conditions over a
short interval of time and it is also known as intra assay precision. A minimum six
determinations at 100% of the standard concentration were tested to find out the
average, standard deviation and related standard deviation, and all the calculated
125
parameters were well within the prescribed limit. Intra-day precision and intermediate
precision were done for ensuring the robustness of the method. The standard deviation
(SD) of both the tests was well within the desirable limit. which is clearly indicated
that the developed method is robust. Intraday and intermediate precision results are
shown in Table 26.
Accuracy
The accuracy of an analytical procedure is the closeness of agreement between the
values that are accepted either as conventional true values or an accepted reference
value. Accuracy is usually reported as percent recovery by an assay using the
proposed analytical procedure of known amount of analyte added to the sample. The
ICH also recommended assessing a minimum of three determinations over a
minimum of three concentration levels covering the specified range. The common
method of determining accuracy is to apply the analytical procedure to the drug
substance and to be quantitated against the reference standard of known purity.
At the first day validation, the accuracy of mean predicted value compared to target
concentration ranged between a minimum of 96.103% at the QC Low concentration
of target 30 ng/ml to a maximum accuracy of 103.319% at the QC high concentration
for target 800 ng/ml. The overall all average accuracy at the first day was 100.55 %,
table 11. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC high
samples was (97.01%-107.13%), (90.89%-102.56%), (106.42%-93.34%), (107.14%-
99.37%) respectively, table 14 to 17.
At the second day of validation, the accuracy of mean predicted value compared to
target concentration ranged between a minimum of 94.482% at the high concentration
of target 800 ng/ml to a maximum accuracy of 105.302% at the LLOQ target
126
concentration of 10 ng/ml. The overall all average accuracy at the second day was
98.78%, table 12. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC
high samples was (98.70%-109.52%), (93.90%-109.07%), (92.90%-97.80%),
(91.87%-96.91%) respectively, table 18 to 21.
At the third day validation, the accuracy of mean predicted value compared to target
concentration ranged between a minimum of 95.691% at the Mid concentration of
target 500 ng/ml to a maximum accuracy of 101.529% at the LLOQ target
concentration of 10 ng/ml. The overall all average accuracy at the third day was
99.78%, table 13. Accuracy range for six replicates of LLOQ, QC low, QC mid, QC
high samples was (93.68%-108.88%), (94.46%-109.59%), (90.30%-99.81%),
(96.38%-105.07%) respectively, table 22 to 26.
Comparing with the accepted criteria which is 85-115% for all concentration except
for LLOQ which is 80-120%, the accuracy obtained is within the required criteria in
terms of accuracy.
Measurement error
The mean measurement error at the first day assessment ranged between 1.169 ng/ml
lower than the target concentration at the QC low concentration 30ng/ml of target to
26.549ng/ml higher than the target concentration) at the High concentration of target
500 ng/ml. The overall all mean measurement error at the first day was an
overestimate of 7.74 ng/ml, table 11.
The mean measurement error at the second day of assessment ranged between 44.145
ng/ml (lower than the target concentration) at the high concentration 800ng/ml of
127
target to 0.530 ng/ml higher than the target concentration at the LLOQ concentration
of target of 10 ng/ml. The overall all mean measurement error at the second day was
an underestimate of 17.91 ng/ml, table 12.
The mean measurement error at the third day of assessment ranged between 21.545
ng/ml lower than the target concentration at the mid concentration 500ng/ml of target
to 2.033 ng/ml higher than the target concentration at the high concentration of target
of 800 ng/ml. The overall all mean measurement error at the third day was an
underestimate of 6.07 ng/ml, table 13.
Looking at all the 3 days of validation one would conclude an overall mean
measurement error of 10.3 ng/ml (underestimate on average) for the validation
experiments of candesartan.
Stability
From the table’s data, we find the autosampler stability test is passed according to the
ICH accepted range where the accuracy % doesn’t exceed 15%. Table 27 and 28
shows data for short term stability indicated by two QC concentrations (low, high) for
candesartan after preparation procedure (auto-sampler stability), T=4°C.
Regarding short term stability at room temperature or processing temperature, freshly
prepared 0 hour two QC’s concentrations were taken as a reference upon calculating
stability of candesartan at room temperature. All the results are within the accepted
criteria which are in the range 85%-115%, as shown in table 31 and 32.
Regarding the freeze and thaw stability: the QC samples are stored and frozen in the
freezer at the intended temperature and thereafter thawed at room or processing
temperature. After complete thawing, samples are refrozen again applying the same
128
conditions. At each cycle, samples should be frozen for at least 12 hours before they
are thawed. The accuracy for QC low and high after 3 cycles is within the accepted
range which is 85-115%, table 45 and 46.
Sensitivity
The protein direct precipitation procedure was specified and sensitive for candesartan,
where both blank and zero samples that examined from six deferent lots of plasma
were attained the required clean chromatogram for specific method.
129
CHAPTER FOUR
CONCLUSION
130
4.1 Conclusion
A new simple, rapid and sensitive method for validation and determination of
candesartan in the presence of each juices(pomegranate, liquorice and orange) has
been done by using High Performance Liquid Chromatography–Mass Spectrometry
(HPLC-MS/MS). Plasma candesartan level was affected by the administration of
pomegranate to a greater extent than that with orange or Liquorice. The reduction in
plasma candesartan level when in pomegranate pre-fed group was reduced to the half
comparing to candesartan alone drug use. Orange or liquorice consumption almost
have no effect on plasma level of candesartan.
The difference between Cmax (single administration vs. combination with orange),
(single administration vs. combination with liquorices) is insignificant, and the
difference in AUC is insignificant as well (P>0.05). The difference between Cmax
(single administration vs. combination with pomegranate) is insignificant too, never
the less the difference in AUC is significant (P<0.05).
we think according to our result that pomegranate juice might affect the intestinal
metabolic system which resulted in beverage-drug interactions. Thus, the decrease in
the AUC of candesartan by pomegranate juice could be due to the induction of enteric
p-glycoprotien activity and/or due alteration in the intestinal uptake transporters
system.
An interesting observation was the difference in T max between rats and humans,
candesartan maximum concentration where reached after 30min (½ hr) in rats
compared to 3-4 hr’s in human. However, it is difficult to extrapolate our results,
which were obtained in rats, to humans. Quantitative evaluation of pomegranate-drug
131
interaction in humans needs to be verified by studies in humans. Therefore, further
investigations in humans are necessary to develop our findings.
Our recomondation in the time being is not to take candesartan with pomegranate
juice due to its potentail interaction.
132
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APPENDIX
CHROMATOGRAMS
159
Figure 40: Candesartan blank chromatogram.
Figure 41: Candesartan zero chromatogram.
160
Figure 42: Candesartan LLOQ chromatogram.
Figure 43: Candesartan QC Mid chromatogram.
161
الملخص
التي سبق اعطبئهب بعض عصبئر الفئراندراسه تحليليه راث مصذاقيت لمعبيرة الكبنذيسبرتبن في بالزمب
جهبز االستشراة المبئي عبلي االداء والطيف الكتليالفىاكهه ببستخذام
اعذاد
احمذ عصبم خضير الكىاز
المشرف المشرف المشبرك
تىفيق عرفبث الذكتىر وائل ابى ديه ستبر الذكتىراال
تذذيذ اىاذيساستا بجد عصائش افاو باستخذا يلذ ت ره باستخذا طشيم بسيط سشيع دساس
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دليم، / 1.0 ايىشيتش، عذي اتذفك وا 5 بمطش 18، اعد افاص ع سي (دض افسيه في ايا
. داخيوعياس ايىشيتش، وا يستخذ ايشبيساستا 5دج اذم عيات وا
، يى (/غشا ا964.692 )فما تيج اتي ت اذصي عيا، ا اعى تشويض ىاذيساستا دذ وا
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475.9673)ل تشويض اىاذيساستا فمذ اشا اا (/ اغشا 818.2868)تأثيش اضخ
%(. 10 ال اذيساستاعذي عا اتباي ه) وات دل امياسات عاي . ز اضياد فعا (/اغشا
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