validation and determination of candesartan with … issa… · by using high performance liquid...

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



(HPLC/MS/MS).

By


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



(HPLC/MS/MS).

By


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


day two validation for candesartan

85


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


freshly spiked calibration standard, showing candesartan bench

stability

93


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


98



candesartan with licorice juice and solitary drug use , each data point


99

13. Rat plasma profile showing the changes in mean serum candesartan 99

X


candesartan with pomegranate juice and solitary drug use , each data

point represents the mean ± SEM (n=3)


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


interval) serum candesartan drug concentration after one hour of drug

administration between single and combined drug use

103


interval) serum candesartan drug concentration after 2 hours of drug


104




105




106




107




108




109




110




111

25. Diagram with error bars comparing the mean (with its 95% confidence 112

XI






113




114




115




116




117




118




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



73



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


validation

76

16. Intra-day precision and accuracy data for QC high samples of


validation

76


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



validation

78



validation

78


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



validation

80



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


and intercept for day two for candesartan

85

30. Standard calibration curve of day three validation, intraday accuracy

data for candesartan

85


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



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



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



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


Kingdom Plantae


(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


Kingdom Plantae


(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


high samples was (93.68%-108.88%), (94.46%-109.59%), (90.30%-99.81%),


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



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



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


Theo.

Conc.

AUC

Drug


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


Theo. Conc. AUC

Drug


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


Theo.

Conc.

AUC Drug AUC IS Ratios Measured

Conc.


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


candesartan based on the standard calibration curve of day two validation.

Theo.

Conc

.

AUC

Drug


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


Candesartan based on the standard calibration curve of day two validation.

Theo.

Conc.

AUC

Drug


Conc.


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



Theo.

Conc.

AUC

Drug


Conc.


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



Theo.

Conc.


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


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



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



Theo.

Conc.

AUC

Drug


Conc.


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



Theo.

Conc.


Conc.


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 accuracy data, shows an accuracy range of (90.20-109.05).

83


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


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


and intercept for day two for candesartan.

Correlation (R) Slope R² intercept

0.9978 0.0036 0.9956 +0.0268


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


Validation of Day Two

86


Measured conc.= (ratio/0.00366)-(+0.0355)

Function is Y = 0.00366X + 0.0355 (R= 0.9987 )

p<0.05


and intercept for day three for candesartan

Correlation (R) Slope R² intercept

0.9987 0.00366 0.9974 +0.0355


three validation for candesartan.

0.00

1.00

2.00

3.00

4.00

0 200 400 600 800 1000 1200

Rat

io


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.


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


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)




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


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


Function is Y = 0.00326X + 0.0265 (R= 0.9977 )




0.9977 0.00326 0.9954 +0.145

93


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


Function is Y = 0.00281X + 0.0369 (R= 0.9984 )




0.9984 0.00281 0.9968 +0.0369

95


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).


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


concentration with time after drug administration, comparing candesartan with

liqourice juice and solitary drug use, each data point represents the mean ± SEM

(n=6).


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)


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


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 (

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ml)


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

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of statistical significance (possibly because of very small sample size), table 11 and

figure 16.


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


figure 17.

0

100

200

300

400

500

600

700

800

900

1000


Pla

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interval) serum candesartan drug concentration after 2 hours of drug administration


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


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con

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afte

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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


Pla

sma

con

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(n

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l)

afte

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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


Pla

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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


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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

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with liqourice was decreased by 84.459 ng/ml compared to its isolated administration.


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


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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


Pla

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with liqourice was lowered by 18.85 ng/ml compared to its isolated administration.


use was evaluated as a moderate effect (Cohen’s d=0.38). This 5.6% reduction in



figure 25.

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100

150

200

250

300

350

400

450

500


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with liqourice was increased by 3.787 ng/ml compared to its isolated administration.


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


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113




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


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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

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115





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

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1000


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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

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700


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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

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300

350

400

450

500


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with liqourice was lowered by 84.509 ng/ml compared to its isolated administration.


use was evaluated as a moderate effect (Cohen’s d=1.398). This 30.99% reduction in


of statistical significance, table 11 and figure 32.

0

50

100

150

200

250

300

350

400


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0

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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

122

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


high samples was (98.70%-109.52%), (93.90%-109.07%), (92.90%-97.80%),


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


high samples was (93.68%-108.88%), (94.46%-109.59%), (90.30%-99.81%),


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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158

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

٪ 0.2٪ 50 ٪ ايثاي ، 50 )ت استخذا خيظ . جاص االستششاب اائي عاي االداء اطيف اىتي

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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validation and determination of candesartan with … issa… · by using high performance liquid...

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