method development, validation and …
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METHOD DEVELOPMENT, VALIDATION AND
DETERMINATION OF PIOGLITAZONE HCL
WITH SUCRALOSE IN RATS SERUM BY USING
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
(HPLC/UV)
By
Lina Nasser Al –Tamimi
Under Supervision of
Prof. Tawfiq Arafat
Dr. Wael Abu Dayyih
A Thesis Submitted in Partial Fulfillment of the Requirements for
the Degree of
Master of Sciences
in Pharmaceutical Sciences
At
University of Petra
Faculty of Pharmacy and Medical Sciences
Amman-Jordan
June-2014
II
Method Development, Validation and Determination of Pioglitazone
HCl with Sucralose in Rats Serum by using High Performance Liquid
Chromatography
( HPLC/ UV)
By
Lina Nasser Al –Tamimi
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
June 2014
Major supervisor
Name signature
Prof. Tawfiq Arafat ................................
Co-Supervisor
Name signature
Dr. Wael Abu Dayyih ................................
Examination Committee
Name signature
Dr. Eyad Al Mallah .................................
Dr. Nidal Al Qinnah .................................
Prof. Maher Lutfi Saleem .................................
III
Acknowledgment
I would like to express my special appreciation and thanks to my major supervisor Prof.
Tawfiq Arafat and my co-supervisor Dr. Wael Abu Dayyih, who have been tremendous
mentors for me. I would like to thank them for encouraging my research and for allowing
me to grow as a research scientist. I would also like to thank my committee members, Dr.
Eyad Al Mallah and Dr. Nidal Al Qinnah for offering all supportive needs and serving as
my committee members. I also want to thank Prof. Maher Saleem (president of Middle
East University) for his brilliant comments and suggestions and for being an external
examiner at my committee. I would like to thank Enas Al Qasem and Jumana Al Qasem
for their help and support too.
A special thanks to my family. Words cannot express how grateful I am to my parents,
thank you dad (mercy upon you), your prayer for me was what sustained me thus far. I
would like also to thank my husband Eng. Sami Nazzal and my kids who had supported
me and donated me the strength that I needed every moment to keep on my way strongly.
I would like to thank my brothers, Eng. Bader Al Tamimi, Ghassan Al Tamimi, Eng.
Tamim Al Tamimi , Bilal Al Tamimi and Chef. Yazan Al Tamimi for their infinite
encouragement, my aunt who helped me to surpass any obstacle and keep going toward
the best. I would like to thank all my friends, the best friends who supported me and
encouraged me to achieve my goal, especially Dr. Hanan Al Hourani for her distinctive
support and amiable heart.
IV
ABSTRACT
Method Development , Validation and Determination of Pioglitazone HCl with
Sucralose in Rats Serum by using High Performance Liquid Chromatography
( HPLC/ UV)
By
Lina Nasser Al -Tamimi
University of Petra, 2014
Supervisor Co-supervisor
Prof. Tawfiq Arafat Dr. Wael Abu Dayyih
A new simple, rapid, sensitive and validated method for quantification of pioglitazone
HCl in the presence of sucralose has been carried out using High Performance Liquid
Chromatography –Ultra Violet ( HPLC-UV) spectroscopy in rats serum. Mobile phase
was consisted of (51.50%) acetonitrile and (48.50%) 0.025 mM ammonium acetate with
pH of 8 , column of separation was C8 at temperature of 40 C° using injection volume of
90 µl , mobile phase flow rate was 1 ml/min and samples run time was 10 min, the
signals were monitored and analyzed at λ= 269 nm and sildenafil citrate was used as
internal standard. Overall intra-day precision and accuracy were reasonable with CV %
values range (0.16-3.54) and accuracy % range ( 98.4-107.9), while inter-day precision
and accuracy showed accepted precision with CV% range ( 0.15- 4.13) and accuracy %
range (99.35-103.99). The coefficient of correlation was 0.9991 with reasonable
sensitivity and selectivity. Over all combination effect was considerable as the serum
concentrations of pioglitazone in presence of sucralose showed a significant decrease
during all time intervals of samples pooling after pioglitazone oral administration
according to statistical analysis results, while the difference between the area under the
curve of pioglitazone time profile in presence and absence of sucralose also illustrated a
significant combination effect referring to data statistical analysis results.
V
The statistical significance seen in the combination results could be justified by the
induction effect of sucralose over the CYP3A4 liver metabolic enzyme in rats by which
pioglitazone is extensively metabolized.
Further clinical research is warranted to investigate the extent of pioglitazone-sucralose
combination interaction in humans.
VI
TABLE OF CONTENTS
No.
Subject
Page
No.
I
Chapter One : Introduction
1
1 Introduction 2
1.1 Diabetes Mellitus 2
1.2 Type 1 Diabetes Mellitus 3
1.3 Type 2 Diabetes Mellitus 3
1.4 Gestational Diabetes Mellitus (GDM) 5
1.5 Oral Antidiabetics 5
1.5.1 Insulin Sensitizers 5
1.5.2 Insulin Secretagogues 6
1.5.3 Alpha Glucosidase Inhibitors 7
VII
1.5.4 Incretin Based Therapies 7
1.6 DM Treatment Protocols 8
1.6.1 DM Type 1 Therapy 8
1.6.2 DM Type 2 Therapy 9
1.6.2.1 DM Type 2 Monotherapy 9
1.6.2.2 DM Type 2 Combination Therapy 9
1.7 Thiazolidinediones 11
1.8 Pioglitazone HCl 11
1.8.1 Pioglitazone HCl Absorption 14
1.8.2 Pioglitazone HCl Pharmacokinetics 14
1.8.3 Pioglitazone HCl Distribution 14
1.8.4 Pioglitazone HCl Metabolism 15
VIII
1.8.5 Pioglitazone HCl Excretion and Elimination 16
1.8.6 Pioglitazone HCl Adverse Reactions 16
1.8.7 Drug-Drug Interaction 17
1.8.8 Drug Metabolism and Cytochrome P450 Enzymes 18
1.8.9 Pioglitazone – Drug Interaction 20
1.8.10 Determination of Pioglitazone HCl in Pharmaceutical Preparations and
Biological Fluids( Literature Survey)
22
1.9 Sucralose 24
1.9.1 Sucralose Manufacturing 25
1.9.2 Sucralose Brand Names 25
1.9.3 Sucralose Pharmacokinetics and Metabolism 26
1.9.4 Artificial Sweeteners – Drug Interactions 26
1.10 High Performance Liquid Chromatography (HPLC) Method 27
IX
1.10.1 HPLC Definition and Principle 27
1.10.2 Types of HPLC 29
1.10.2.1 Normal Phase HPLC 29
1.10.2.2 Reversed Phase HPLC 25
1.10.2.3 Methods of Detection 29
1.10.3 Internal Standard 31
1.10.4 HPLC Instrument Calibration 31
1.10.5 Bioanalytical HPLC Method Validation Parameters Definitions ( EMEA) 32
1.10.5.1 Precision 32
1.10.5.2 Accuracy 32
1.10.5.3 Linearity 33
1.10.5.4 Range 34
X
1.10.5.5 Ruggedness 34
1.10.5.6 Limit of Detection 35
1.10.5.7 Lower limit of Quantification 35
1.10.5.8 Selectivity 35
1.10.5.9 Sensitivity 36
1.10.5.10 Recovery 36
1.10.5.11 Stability 37
1.11 Aim of the Study 39
II Chapter Two : Experimental Part 40
2 Experimental Part 41
2.1
Reagents
41
2.2
Instrumentation
42
XI
2.3
Preclinical Study
44
2.4
Preparation of Stock Solutions and Working Solutions
46
2.4.1 Preparation of Pioglitazone HCl Oral Solution 46
2.4.2
Preparation of Sodium Hydroxide Solution
46
2.4.3
Preparation of Sucralose Oral Solution
47
2.4.4
Preparation of Pioglitazone HCl Stock Solution
47
2.4.5
Preparation of Pioglitazone HCl Serial Dilutions in Methanol
48
2.4.6
Preparation of Pioglitazone HCl Standard Solutions in Serum( QC
Solutions)
49
2.4.7
Preparation of Mobile Phase
50
2.4.8
Preparation of Buffer Solution
50
2.4.9
Preparation of Internal Standard Stock
50
XII
2.4.10
Solution Preparation of Internal Standard Working Solution
50
2.4.11
Sample Preparation ( Extraction Procedure)
51
2.4.12
Method Development (Chromatographic Conditions )
51
2.5
Method Validation
54
2.5.1
Inter-day Accuracy and Precision
54
2.5.2 Intra-day Accuracy and Precision 55
2.5.3
Selectivity and Sensitivity
56
2.5.4
Linearity
56
2.5.5
Recovery
57
2.5.6
Stability
57
2.6
Statistical Analysis
59
XIII
III
Chapter Three : Results and Discussion
62
3
Results and Discussion
63
3.1
Method Validation
63
3.1.1
Inter-day Precision and Accuracy
63
3.1.2
Intra-day Precision and Accuracy
69
3.1.3
Linearity
71
3.1.4
Selectivity and Sensitivity
79
3.1.5
Recovery
84
3.1.6
Stability
87
3.2
Sucralose - Pioglitazone HCl Combination Effect on Pioglitazone HCl
Serum Levels
99
XIV
IX
Chapter Four : Conclusion
117
4.1
Conclusion
118
4.2
Appendix : Chromatograms
119
4.3 References 133
4.5 Abstract in Arabic 151
LIST OF FIGURES
Figure
No.
Caption Page
No.
1.
Pioglitazone HCl Structural Formula
12
2.
Sucralose Structural Formula
25
3.
Calibration 1
72
4.
Calibration 2
73
XV
5.
Calibration 3
74
6.
Calibration 4
75
7.
Calibration 5
76
8.
Calibration 6
77
9.
Long Term Stability Day 30 Calibration Curve
97
10.
First Day Trials Calibration Curve
100
11.
First Day Trial Serum – Plasma Time Profile Curve
101
12.
Second Day Trial Serum – Plasma Time Profile Curve
103
13.
Third Day Trial Serum – Plasma Time Profile Curve
104
14.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 30 minutes of Drug Administration
106
15.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 1 hour of Drug Administration
107
XVI
16.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 2 hours of Drug Administration
108
17.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 3 hours of Drug Administration
109
18.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 4 hours of Drug Administration
110
19.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 6 hours of Drug Administration
111
20.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 8 hours of Drug Administration
112
21.
Dot Diagram with Error Bars for Mean Comparison of Pioglitazone HCl
Serum Concentration after 24 hours of Drug Administration
113
22.
Serum Concentration – Time Profile Graph ( 0-24) Hours of Oral
Administration of Drug,( PG = Pioglitazone HCl alone , PG Plus =
Pioglitazone HCl with Sucralose)
114
23.
Serum Blank Chromatogram with IS
119
24.
Serum Blank Chromatogram 1
120
25.
Serum Blank Chromatogram 2
121
26.
Serum Blank Chromatogram 3
122
XVII
27.
Serum Blank Chromatogram 4
123
28.
Serum Blank Chromatogram 5
124
29.
Serum Blank Chromatogram 6
125
30.
Piolitazone LLOQ Chromatogram ( Peak 1 for Pioglitazone HCl , Peak 2
for IS)
126
31.
Pioglitazone HCl QCL Chromatogram ( Peak 1 : Pioglitazone HCl, Peak 2
: IS)
127
32.
Pioglitazone HCl QCM Chromatogram ( Peak 1 : Pioglitazone HCl, Peak 2
: IS)
128
33.
Pioglitazone HCl QCH Chromatogram ( Peak 1 : Pioglitazone HCl, Peak 2
: IS)
129
34.
Pioglitazone HCl Zero Concentration with IS Chromatogram (Peak 2 :IS)
130
35.
Pioglitazone HCl Unknown Concentration Chromatogram after 30 minutes
Oral Administration ( Pioglitazone HCl : Peak 1 , IS : Peak 2 )
131
36.
Pioglitazone HCl Unknown Concentration Chromatogram after 3 hours of
Oral Administration ( Peak 1 : Pioglitazone HCl , Peak 2 : IS )
132
XVIII
LIST OF SCHEMES
Scheme
No.
Caption Page No.
1. PPARs Functions 10
2. HPLC Flow 28
3. UV Detector Detection Pathway 30
LIST OF TABLES
Table
No.
Caption Page No.
1. Pioglitazone HCl – Drugs Interactions 21
2. Sildenafil Citrate Information ( Internal Standard ) 42
3. Pioglitazone HCl Serial Dilutions in Methanol 48
4.
Pioglitazone HCl QC Standard Solutions in Serum
49
5.
Chromatographic and Detection Conditions
53
6.
Concentrations Used for Method Validation
54
7.
Inter- day Precision and Accuracy : Day 1
66
XIX
8. Inter- day Precision and Accuracy : Day 2 67
9.
Inter- day Precision and Accuracy : Day 3
68
10.
Intra- day Precision and Accuracy
70
11.
Linearity Calibration 1 Data 72
12.
Linearity Calibration 2 Data 73
13.
Linearity Calibration 3 Data
74
14.
Linearity Calibration 4 Data
75
15.
Linearity Calibration 5 Data
76
16.
Linearity Calibration 6 Data
77
17.
LLOQ Sensitivity Data
80
18.
QCL Sensitivity Data
81
19.
QCM Sensitivity Data
82
20.
QCH Sensitivity Data
83
21.
Pioglitazone HCl Recovery Data
85
XX
22.
Internal Standard Recovery Data
86
23.
Freeze and Thaw Stability Data
91
24.
Room Temperature Stability Data for Pioglitazone HCl in Serum
93
25.
Room Temperature Stability Data for Pioglitazone HCl in
Stock/Working Solution
94
26.
Room Temperature Stability Data for Internal Standard in
Stock/Working solution
94
27.
Long Term Stability Day 30 Calibration Data
97
28.
Long Term Stability Data of Pioglitazone in Serum
98
29.
Long Term Stability Data of Pioglitazone in Stock/Working Solution
98
30.
Long Term Stability Data of Internal Standard in Stock/Working
Solution
99
31. First Day Trials Calibration Curve Data
100
32. First Day Trial Serum Data 101
33. Second Day Trial Serum Data 103
XXI
34. Third Day Trial Serum Data 104
35. Serum Data Statistical Analysis Results 105
36. Serum Concentration – Time Profile Kinetic Parameters 115
ABBREVIATIONS
ADI Approved Daily Intake
AUC Area Under The Curve
BW By Weight
CL Clearance
Conc. Concentration
CPK Creatine Phosphokinase
CV Coefficient of Variation
XXII
CVD Cardiovascular Diseases
CYP Cytochrome P
D Day
DM Diabetes Mellitus
DPP-4 Dipeptidyl peptidase-4
EMEA European Medicines Agency
F Bioavailability
FDA Food and Drug Administration
GIT Gastrointestinal Tract
GLP-1 Glucagon-Like Peptide-1 Receptor Agonist
Gm Gram
HbA1c Hemoglobin A1c Subtype
XXIII
HCL Hydrochloric Acid
HDL High Density Lipoprotein
HPLC High Performance Liquid Chromatography
IS Internal Standard
IUPAC International Union of Pure and Applied
Chemistry
Kg Kilogram
KOH Potassium Hydroxide
L Liter
LC-MS Liquid Chromatography – Mass Spectroscopy
LLOQ Lower Limit Of Quantification
LOD Limit Of Detection
XXIV
LOQ Limit Of Quantification
MCP Mytochondrial Pyruvate Carrier
mM Milimolar
Mwt Molecular Weight
NaOH Sodium Hydroxide
PG Pioglitazone HCL
PG Plus Pioglitazone HCl Plus Sucralose
P-gp P Glycoproteins
PPARs Peroxisome Proliferator-Activated Receptors
QC Quality Control
QCH Quality Control High Concentration
QCL Quality Control Low Concentration
XXV
QCM Quality Control Medium Concentration
R² Regression Factor
RF Rate of Flow
S.C Subcutaneous
SD Standard Deviation
SOPs Standard Operating Procedures
St Standard Solution Concentration
TZDs Thiazolidinediones
RT Room Temperature
UKPDS United Kingdom Prospective Diabetes Study
USFDA United States Food and Drug Administration
UV Ultra Violet
XXVI
Vd Volume of Distribution
WHO World Health Organization
Wt Weight
1
CHAPTER ONE
INTRODUCTION
2
1. Introduction
1.1 Diabetes Mellitus
Diabetes mellitus is defined as a metabolic disorder of multiple etiologies,
characterized by hyperglycemia associated with alterations of carbohydrate, protein
and fat metabolism as a result of defect in insulin secretion and/or insulin activity
(Alberti and Zimmet, 1998).
The long term of diabetes mellitus consequences include: damage, dysfunction and
failure of various organs (Wolff, 1993; Turner et al,. 1998). Diabetes mellitus may
present with severe symptoms such as polyuria, thirst, weight loss and blurred vision
(Nathan, 1993). In severe forms, it may develop which may lead to coma and finally
death (Seshasai et al., 2011).
World Health Organization (WHO) has estimated that there are around 220
millions of diabetics around the world. Diabetes is responsible of 1.5 million of death
cases, the figure could be doubled till 2030. This disease is increasingly distributed in
the world which forces the health organizations to consider it as a critical warning that
needs a serious attention to avoid any possible threats in the future. World Health
Organization (WHO) has recognized and classified three forms of Diabetes mellitus:
type 1, type 2 and gestational diabetes (WHO 2010).
Diabetes is firstly classified as type 1 and type 2 by Sir Harold Pervival (Hary)
Himsworth and it was published in January 1936 (Himsworth, 2011).
The other subtypes of diabetes include: gestational diabetes and diabetes due to
any secondary specific cause.
3
1.2 Type 1 Diabetes Mellitus
Type 1 diabetes mellitus is primarily and specifically occur due to the loss of beta
cells at islets of Langerhans in pancreas which leads to insulin deficiency as a
consequence (Meier et al., 2005), but it can be also classified as idiopathic or
immune-mediated disease as the body immune system performs a defense mechanism
against beta cells causing its death (Rother, 2007).Type 1 diabetes can occur in both
children and adults which justifies the publically nomination of "juvenile diabetes”
because it is majorly founded in children.
Patient diagnosis with symptoms of hyperglycemia (polyuria, thirst and weight
loss) can be proceeded with a single random serum glucose test with result of 200
mg/dl or higher, without any need of measurement repetitions (Gavin et al., 1997).
It is considered as immune mediated disease in more than 90% of cases, and it is
mediated with unknown causes in less than 10% of cases (Kukreja et al., 2002).
The most serious and apparent symptoms are: rapid breathing, dryness of skin and
mouth, flushed face, fruity acetone like breath odor, nausea or vomiting, frequent
urination and stomach pain (Daneman, 2006).
It is effectively managed using insulin in order to control serum glucose levels due
to insufficient insulin levels in blood stream.
1.3 Type 2 Diabetes Mellitus
Type II diabetes mellitus is considered as a chronic endocrinal disease, which is
defined as a metabolic disorder (complex) characterized by hyperglycemia due to
inappropriate insulin secretion and action associated with insulin resistance in which
4
normal levels of insulin do not activate the glucose absorption signal (DeFronzo, 1999
; Scheen, 2007).
A high rate of glucose production by liver associated with hyperinsulinemia case
is the primary essential reason of fasting hyperglycemia incidence. After a meal,
impaired inhibition of hepatic glucose production by insulin and a decrease in the rate
of insulin-glucose uptake by muscles contribute equally to postprandial
hyperglycemia (Genuth et al., 2003).
Disease development usually occur due to genetic and environmental factors such
as high calorie diet and unhealthy lifestyle with mentionable low physical activity
(Hu et al., 2001 ; Tuomilehto et al., 2001).
Unfortunately, it can be unnoticed for years because of its invisible symptoms
which are non-existent and typically mild.
In United States, five classes of oral agents with different mechanism of action for
each, are currently available to improve glycemic control in patients with type 2
diabetes (Levetan, 2007).
A recent study that done by (UKPDS) has shown that type 2 diabetes mellitus is a
progressive disorder that can be treated initially with oral agent monotherapy but will
require an additional different oral agents, and usually in many patients, insulin
therapy will be needed to achieve targeted blood glucose levels (Miyazaki et al.,
2001; Genuth et al., 2003).
5
Glycemic control, regardless of the agent that is used will decrease the incidence
of several vascular complications such as: retinopathy, neuropathy, and nephropathy
(Patel et al., 2008).
1.4 Gestational Diabetes Mellitus
Gestational diabetes mellitus is strongly resembles Type 2 Diabetes Mellitus in
involving inadequate insulin secretion and response. Gestational diabetes is
characterized by high blood sugar levels during pregnancy in a woman without
previously diagnosed diabetes (Bellamy et al., 2009).
It is considered as a degree of glucose intolerance either with onset or during
pregnancy ( Buchanan and Xiang, 2005), treatable with appropriate medicines while
in 50% of women it may developed into Type 2 Diabetes Mellitus in future, if it is
neither treated nor controlled carefully (Langer et al., 2005).
1.5 Oral antidiabetics
Many oral antidiabetics are used either as a single or combination of two or more
antidiabetic according to diabetes type, blood glucose controlling factors. (Meltzer et
al., 1998 ; Krentz and Bailey, 2005). Healthy daytime blood sugar levels before meals
(fasting) is (80 - 120 mg/dl) and (100 - 140 mg/dl) at bedtime.
The most widely used antidiabetics are:
1.5.1 Insulin Sensitizers
This class of type 2 diabetes medications includes biguanides and thiazolidinediones.
6
Biguanides is a class of medication that is used for treatment of diabetes type 2.
Metformin is the only currently available biguanide which control the blood glucose
levels in type 2 diabetics by decreasing hepatic glucose output and increasing glucose
utilization in peripheral tissues including muscles and liver tissues via the activation
of AMP-protein kinase enzyme (Klip and Leiter, 1990 ; Bailey, 1992 and Miller et al.,
2013).
Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma
(PPARγ) agonists, this class of type 2 diabetes has its pharmacological effect through
the enhancement of insulin receptors sensitivity in muscle, fat and liver tissues
(Diamant and Heine, 2003 ; Nathan et al., 2006).
1.5.2 Insulin Secretagogues
This class of medications includes sulfonylureas and glinides drugs subclasses.
The first insulin secretagogues used were sulfonylureas, its hypoglycemic effect
was discovered in Montpellier in 1945. sulfonylureas stimulate insulin secretion by
pancreatic beta cells via sensitizing them to glucose blood levels.
Sulfonylureas bind to plasma membrane sulfonylurea receptor, activation of these
receptors inhibits (ATP-sensitive K+ channels) that yields in cellular depolarization,
as a result, voltage-dependant calcium channels will be opened which leads to insulin
secretion.
On the other hand, sulfonylureas can inhibit glucagon secretion and enhances
tissues insulin sensitivity.
7
Sulfonylureas second generation members are: glipizide, glibenclamide, gliclazide,
glibornuride and glimepiride, while the first generation drugs are: tolbutamide,
chlorpropamide and carbutamide.
Glinides are compounds of insulin secretion activation activity. Their
hypoglycemic effect is faster than sulfonylureas.
Glinides available drugs are: nateglinide and repaglinide, this antidiabetic class has
an antihyperglycemic action through blocking ATP-regulated K channels leading to
depolarization and Ca influx and finally, insulin release from pancreatic beta cells will
be achieved (Luna et al., 1999 ; Malaisse, 2003).
1.5.3. Alpha Glucosidase Inhibitors
This class of type 2 antidiabetics includes: acarbose and miglitol (Van de Laar et
al., 2005), which helps to control the normal blood glucose levels in type 2 diabetics
by inhibition of intestinal alpha-glucosidase enzyme and a weak effect on pancreatic
alpha-amylase causing a reduction of monosaccharides production and absorption at
the small intestine (Ye F et al., 2003).
1.5.4. Incretin Based Therapies
This type of diabetes type 2 therapy is majorly depends on the use of two recently
approved classes: glucagon-like peptide-1 receptor agonists (GLP-1) and dipeptidyl
peptidase-4 inhibitors (DPP-4) (Drucker, 2010).
Sitagliptin, saxagliptin, and sinagliptin (approved in 2011 and are not available
yet) are oral DPP-4 inhibitors while exenatide and liraglutide are injectable GLP-1
analogs.
8
Incretin based therapies mechanism of action could be described as a potentiation
of incretin receptor signaling, as inhibition of DPP-4 enzymes will lead to an increase
in incretin and GLP-1 levels, which enhances the release of insulin and decrease the
glycogen secretary levels.
1.6 DM Treatment Protocols
1.6.1 DM Type 1 Therapy
Treatment Guidelines: (Rodbard et al., 2007).
1. Controlling of blood glucose levels as follows:
Before meals = 70–120 mg/dL
2 hours post-meals = 160 mg/dL
Bed time = 70–120 mg/dL
2. Diet and physical activity.
3. Weight management.
4. Foot care.
Insulin is the first line of treatment, it is available as S.C (subcutaneous) injections
( Fonseca and Kulkarni, 2008). Insulin types are: rapid-acting insulin, intermediate
and long-acting insulin.
9
1.6.2 DM Type 2 Therapy
1.6.2.1 DM Type 2 Monotherapy
DM type 2 monotherapy line includes: metformin, thiazolidinediones (Tan, 2000),
secretagogues, dipeptidyl-peptidase 4 inhibitors and alpha-glucosidase inhibitors
(Bennett et al., 2011).
1.6.2.2 DM type 2 Combination Therapy
Combination Therapy of DM Type 2 includes the following: (Yki-Järvinen, 2001)
Secretagogue + metformin
Secretagogue + thiazolidinedione (Kipnes et al., 2001).
Secretagogue + alpha-glucosidase inhibitor
Thiazolidinedione + metformin (Einhorn et al., 2000).
Dipeptidyl-peptidase 4 inhibitor + metformin
Dipeptidyl-peptidase 4 inhibitor + thiazolidinedione
Secretagogue + metformin + thiazolidinedione
1.7 Thiazolidinediones:
A group of potent synthetic PPAR ligands, effectively used in treatment of DM
type 2 (Nathan, 2009). These ligands have their glucose lowering effects through
mediation of insulin sensitivity in skeletal muscles (Yki-Järvinen, 2004 ; Scheen,
10
2007) and facilitating glucose uptake by binding to the PPARγ receptors ( Divakaruni
et al., 2013).
The peroxisome-proliferator–activated receptors (PPARs) are a subfamily of 48-
member nuclear-receptor which regulates gene expression as response to ligand
binding (Nathan, 2002), three types of PPARs: PPARα, PPARβ and PPARγ have
been identified to date (Braissant et al., 1996).TZDs are potent synthetic PPAR
ligands, it as it is predominantly distributed and expressed mostly in adipose tissue but
is also found in pancreatic beta cells, vascular endothelium and macropages, as shown
in scheme 1(Radhika et al., 2012).
Scheme 1: PPARs Functions
11
TZDs group members are: pioglitazone, rosiglitazone, troglitazone and
rivoglitazone.To date, the only available drugs are pioglitazone and rosiglitazone.
In January 1997, the first thiazolidinedione; troglitazone, was approved for patients
with type 2 diabetes in the United States. (FDA 1999 ; Scheen, 2007).
In March 2000, troglitazone was subsequently withdrawn from the market because
of its hepatotoxicity side effect (Diamant and Heine, 2003). The two currently
available PPARγ agonists, rosiglitazone and pioglitazone were approved in the United
States in 1999 and still be used up to date.
Patients with type 2 diabetes have an increased bladder cancer risk of 40%
(Colmers et al., 2012 ; Chapman, 2013), thiazolidinediones, especially pioglitazone
can increase this risk, but recently, a systematic review was carried out to evaluate the
bladder cancer risk with type 2 diabetics taking thiazolidinediones, the result was with
limited evidence to support this claim concerning (Clomers et al., 2012 ; Wei et al.,
2013; Bosetti et al., 2013).
1.8 Pioglitazone
Pharmacological review: it is a compound that belongs to a group named
“thiazolidinediones” family, an oral antidiabetic agent that acts by decreasing insulin
resistance (Nissen, 2007). Pioglitazone has the same mechanism of action by which
all thiazolidinediones act inside the body.Its mechanism of action made it as one of
the most effective drugs that is used in the management of type 2 diabetes mellitus
(Grossman, 2001 ; Chilcott et al., 2001), pioglitazone was marketed at USA in 1999,
nowadays, it is marketed in more than 40 countries worldwide (Sohda et al., 2002).
12
Several pharmacological studies accentuated that pioglitazone can improve the
sensitivity to insulin in muscle and adipose tissue (Smith , 2001 ; Pavo et al., 2003)
which helps in hepatic gluconeogenesis inhibition (Tan, 2000) and improves glycemic
control by reducing circulating insulin levels, (Haffner et al., 1999 ; Aronoff et al.,
2000 ; Richter et al., 2006).
Pioglitazone monotherapy is an alternative to metformin monotherapy if
metformin cannot be used (either for intolerance or for contraindications), it is also
used as a combined therapy if monotherapy with metformin is insufficient to achieve
the required HbA1c blood glucose level target, or as a tripled therapy line with other
oral antidiabetics as a complementary treatment thus, pioglitazone HCl is still
considered as effective and useful antidiabetic drug with a efficient insulin-sensitizing
action. However, the therapeutic use of pioglitazone HCl is still under monitoring and
control because of conflicting safety issues and newer drugs availability such as DPP-
4 inhibitors, glucagon-like peptide-1 receptor agonists, and sodium glucose co-
transporter 2 inhibitors, even though, none of these new drugs affects or targets
insulin resistance.
Recent studies approved that insulin-resistant patients with increased waist
circumference, low HDL cholesterol level, fatty liver or with a high risk of CVD are
almost best treated with pioglitazone (Schernthaner et al., 2013).
Physical and chemical properties: pioglitazone: [ (±)-5-[ [4-[2- (5-ethyl-2-
pyridinyl) ethoxy] phenyl] methyl] -2, 4-] thiazolidinedione monohydrochloride
(Radhika et al., 2012), it belongs to a different chemical class with different
pharmacological action than the sulfonylureas, metformin, or the α-glucosidase
inhibitors (Smith, 2001).The structural formula is as shown in figure 1.
13
Figure 1: Pioglitazone HCl Structural Formula
Pioglitazone hydrochloride is a weak acid salt of pioglitazone, organic
compound; benzoids super class of phenol ethers (Picha and Zhu, 2007), odorless
white crystalline powder with molecular formula of C19H20N2O3S•.HCl and
molecular weight of 392.90 daltons (Takeda Canada, Inc. 2012).
Chemical properties: it is soluble in dimethylformamide and dimethyl sulfoxide,
Methanol, chloroform and acetonitrile (Brahmaiah and Raju, 2012), practically
insoluble in water and ether (Lawrence, 2001).
Preparations: Tablets: 15, 30 and 45 mg with brand names of: Actos®, Glustin
®
and Actost®,
while other brand names of combination are: Actoplus Met®
(containing metformin and pioglitazone HCl), Actoplus Met®
XR (containing
metformin and pioglitazone HCl), Duetact® (containing glimepiride and
pioglitazone HCl).
Stability and storage: stable under ordinary conditions, tablets should be kept at
room temperature, 15-30°C (59-86°F) (Takeda Canada, 2012).
14
1.8.1 Pioglitazone HCl Absorption
Following oral administration in fasting, pioglitazone HCl is first measurable in
serum within 30 minutes, with peak concentrations observed within 2 hours, its site of
absorption is stomach ,food slightly delays peak serum concentration time to (3 – 4)
hours, but does not alter the extent of absorption (Budde et al., 2003).
1.8.2 Pioglitazone HCl Pharmacokinetics
Serum concentrations of total pioglitazone HCl (pioglitazone HCl plus active
metabolites) remain elevated 24 hours after once daily dosing. Steady-state serum
concentrations of pioglitazone HCl are achieved within 7 days (Nathan, 2009).
At steady-state, two of the pharmacologically active metabolites of pioglitazone
HCl: M-III and M-IV can reach serum concentrations equal to or greater than
pioglitazone HCl in healthy and type 2 diabetic rats and human (Shah and Mudaliar,
2010 ; Aquilante et al., 2013), pioglitazone HCl involves approximately 30% to 50%
of peak serum concentrations and 20% to 25% of the total area under the serum
concentration-time curve (Eckland and Danhof,
2000).
Bioavailability of pioglitazone HCl in rats serum is about 50% (Umathe et al.,
2008) while clinical studies had indicated a higher bioavailability of pioglitazone HCl
in human serum about 80% (Tornio et al., 2008).
1.8.3 Pioglitazone HCl Distribution
The mean apparent volume of distribution (Vd/F) of Pioglitazone HCl following
single oral dose of administration is 0.63 ± 0.41 (mean ± SD) L/kg of body weight. It
is extensively protein bound (> 99%) in human serum, principally to serum albumin
15
and also binds to other serum proteins too, but, with lower affinity. Metabolites M-III
and M-IV are also extensively bound (> 98%) to serum albumin (Budde et al., 2003).
1.8.4 Pioglitazone HCl Metabolism
Pioglitazone HCl is extensively metabolized by hydroxylation and oxidation via
liver CYP450 enzymatic system (Jaakkola et al., 2006), in vitro data emphasized that
multiple CYP isoforms are involved in pioglitazone HCl metabolism, mostly:
CYP2C8 and CYP3A4 (Tornio et al., 2008 ; Holstein et al., 2012).
Pioglitazone HCl metabolites M-II & M-IV (hydroxy derivatives of pioglitazone
HCl) and M-III (keto derivative of pioglitazone HCl) are pharmacologically active in
animal models of type 2 diabetes ( Tanis et al., 1996 ; Scheen, 2007), M-III and M-IV
are the principal drug-related species found in human serum following multiple
dosing (Muschler et al., 2009).
P-glycoprotein (P-gp) is a trans-membrane efflux pump, which can extrude many
drugs from the cell. It was suggested that CYP3A4 function is complementary to P-gp
function through gastrointestinal tract, CYP3A4 is a prevalent CYP3A type that
mostly expressed in the gastrointestinal tract (Canaparo et al., 2007).
Interaction between enzymes that responsible for drug metabolism and active
transporters is a substantial concept of drug pharmacokinetics. In gastrointestinal tract
mucosa; P-glycoprotein and cytochrome P450 (CYP)3A may interact via three
mechanisms(Christians et al., 2005):
1- Drugs are continuously taken up and pumped out of the enterocytes by P-
glycoprotein which increases drugs metabolism probability.
16
2- P-glycoprotein keeps intracellular concentrations of drug within the linear range of
CYP3A metabolizing capacity.
3- P-glycoprotein transports drug metabolites formed in the mucosa back into lumen
mucosa.
1.8.5 Pioglitazone HCl Excretion and Elimination
Following oral administration, 15% to 30% of pioglitazone HCl dose is retrieved
in urine. Renal elimination of pioglitazone HCl is negligible as it is excreted primarily
in forms of metabolites and their conjugates into the bile followed by elimination in
feces.
The mean serum half-life of pioglitazone HCl and total pioglitazone HCl ranges
from 3 to 7 hours and 16 to 24 hours, respectively. Pioglitazone HCl has an apparent
clearance of CL/F = 5 - 7 L/hr (Radhakrishna et al., 2002).
1.8.6 Pioglitazone HCl Adverse Reactions
The most common include: weight gain and upper respiratory tract infection, but
the less common adverse reactions are: edema, headache, fatigue, hypoglycemia,
anemia, sinusitis and pharyngitis (Shah and Mudaliar, 2010), serious life threatening
adverse reactions are: CHF, dyspnea, hepatic failure and hepatitis (Padwal, 2008 ;
Takeda Canada, Inc. 2012).
17
1.8.7 Drug– Drug Interaction
Drug interactions are very important concept in clinical research and therapeutic
drug treatment. Drug interactions can lead to many serious side effects which forces
health authorities to: terminate drug use, refuse drug approval and withdraw it from
markets (Bjornsson et al., 2003). Therefore, clinicians, pharmaceutical industries and
regulatory authorities have increased and still the attention to drug-drug interactions.
There are a number of drugs interaction mechanisms: pharmacokinetic and
pharmacodynamic interactions. In pharmacokinetic drug interactions a drug affects
the absorption, distribution, metabolism, or excretion of another drug while in
pharmacodynamic drug interactions two drugs have additive or antagonistic
pharmacologic effects (Ho and Kim, 2005).
Pharmacokinetic Drug Interactions
Inhibition of absorption: where the drugs act as binding agents which impair the
bioavailability of other drugs which will result in a reduction in the therapeutic effect
of the desired drug, in some cases, the amount of these drugs that is absorbed from the
gut may be increased or decreased by drugs that increase stomach pH.
Enzyme inhibition: as most drugs are metabolized to either inactive or less active
metabolites by liver and intestine enzymes, inhibition of this metabolism can increase
the effect of the required drug which may result in drug toxicity. This is one of the
most commonly observed clinically important mechanisms of interactions (Tanaka,
1998).
A small number of drugs are administered to patients in inactive forms. These
drugs are known as prodrugs which require activation by body enzymes in order to
18
produce their effect. Inhibition of these prodrugs metabolism may reduce the amount
of active drug and decrease the desired therapeutic effect.
Enzyme induction: as some drug are called enzyme inducers, they are capable of
increasing the drug metabolizing enzymes activity which results in a decrease in the
therapeutic effect of other drugs.
Some drugs are biotransformed into toxic metabolites by metabolizing enzymes,
enzyme inducers can increase the formation of drug toxic metabolites and increase the
hepatotoxicity risk which may lead to other organs damage (Zhou et al., 2003).
Altered renal elimination: some drugs are actively secreted into the renal tubules as
a route of elimination, any drug that may alter the renal elimination of other drug
could lead to drug toxicity (Shitara Y et al., 2005).
Pharmacodynamic Drug Interactions
Additive effects: which occurs when two or more drugs with similar
pharmacodynamic effects are given concurrently, the additive effects may result in
excessive response and toxicity.
Antagonistic effects: when drugs with opposing pharmacodynamic effects are
administered concurrently, combination may lead to reduction in the response to one
or both drugs (Eschenhagen T, 2000).
1.8.8 Drug Metabolism and Cytochrome P450 Enzymes
Liver is the major site of drug metabolism. Metabolism could inactivate drugs,
while some drug metabolites are pharmacologically active more than the parent drug.
19
Drugs are usually metabolized by oxidation, reduction, hydrolysis, hydration,
conjugation or condensation in order to make the drug easier to be excreted. The
enzymes involved in metabolism are present in many tissues but mostly in the liver.
Drug metabolism rates are individually influenced by genetic factors, coexisting
disorders such as chronic liver disorders and advanced heart failure, and drug
interactions via induction or inhibition of metabolism (Tucker et al., 2001).
Generally, metabolism occurs in 2 phases. Phase I reactions involve formation of a
new functional group by oxidation, reduction or hydrolysis, these reactions are
considered as nonsynthetic. Phase II reactions involve conjugation with an
endogenous substance and these reactions are synthetic. Synthetic reactions produce
more polar metabolites that are readily to be excreted by the kidneys (urine) and the
liver (bile). (Xu and Kong, 2005)
First pass effect: an important concept of drugs metabolic pathways, it is
responsible for the reduction in total drug bioavailability which reflects the amount of
drug that delivered to the systemic circulation, where the drug is metabolized in the
GIT tissues and liver tissues before it reaches the systemic circulation.
Cytochrome P450 represents a family of isozymes that responsible for many drugs
biotransformation via oxidative reactions. These enzymes are heme-containing
membrane proteins, which are predominantly located in the smooth endoplasmic
reticulum of several tissues. It is majorly located in liver, but it could be also found in
kidneys, skin, gastrointestinal tract, and lungs where further non hepatic metabolism
may occur (Cupp and Tracy, 1998).
20
In humans upto 21 families, 20 subfamilies and 57 genes have been described
while CYP 1, 2 and 3 represent 70% of total hepatic CYPs content which are
responsible for 94% of drugs metabolism in liver (Rendic and Carlo, 1997).
1.8.9 Pioglitazone-Drug Interaction
In vitro data demonstrated that multiple CYP isoforms are involved in the
metabolism of pioglitazone HCl, involved cytochrome P450 isoforms are CYP2C8
mainly and CYP3A4 but with lesser degree (Takeda Canada, Inc. 2012), as a result,
any simultaneous intake of drug / food which can contribute in inhibition or activation
of these isoforms may lead to serious interactions with pioglitazone HCl resulting in
pathological complications which requires intensive patients care and critical drug
monitoring (Shah and Mudaliar, 2010).
Several studies have emphasize the pioglitazone – drug interaction through
CYP2C8 hepatic isoform, strong CYP2C8 inhibitors as gemfibrozil can increase
pioglitazone HCl plasma concentrations (Deng and Wang, 2005), while CYP2C8
inducers as rifampin may decrease pioglitazone HCl concentrations (Jaakkola et al.,
2006).
On the other hand, drugs that inhibit or activate CYP3A4 also contribute with
pioglitazone metabolic interaction (Radhika et al., 2012), itraconazole can inhibit
pioglitazone metabolism through CYP 3A4 inhibition which results in pioglitazone
serum levels elevation. Querciten ; a bioflavonoid that is used for treatment of some
heart and blood vessels diseases, has the same mechanism and interactive effect with
Pioglitazone (Jaakkola et al., 2005 ; Umathe et al., 2008), table 2 summarizes some
clinically approved pioglitazone – drugs interactions.
21
Table 1: Pioglitazone HCl – Drugs Interactions
Drug Mechanism Therapeutic Effect
Gemfibrozil Gemfibrozil is potent
CYP2C8 and CYP2C9
Inhibitor.
Increased AUC, enhanced
efficacy and increased
concentration-dependent
adverse effects
of pioglitazone HCl (Deng et al., 2005).
Itraconozole Itraconazole is a weak
inhibitor of CYP3A4 and
CYP2C9.
Enhanced the anti-diabetic
effect of the pioglitazone HCl.
(Jaakkola et al., 2005).
Rifampicin Rifampicin is an inducer of
CYP2C8.
Decreased serum
concentration of
pioglitazone HCl
leading to therapeutic failure.
Quercetin Quercetin is a potent
inhibitor of CYP3A4.
increased pioglitazone HCl bioavailability of
by 75% ( Umathe et al., 2008).
Ketoconazole Inhibitor of CYP3A4 and
CYP2C8.
Increased AUC of pioglitazone HCl
22
1.8.10 Determination of Pioglitazone HCl in Pharmaceutical Preparations and
Biological Fluids (Literature Survey)
Literature survey reveals many methods that were validated to be used for
determination of pioglitazone HCl in either pharmaceutical preparations or in
biological fluids for human and animals.
HPLC is one of the most frequently used analysis method for determination of
pioglitazone HCl, Ravikanth C. has improved a validated HPLC method for
bioanalysis of pioglitazone HCl in rats serum, the stationary phase was C18 column,
the mobile phase used consisted of Methanol and Ammonium acetate buffer (pH
adjusted to 5 with ortho-phosphoric acid) in ratio of 60:40, pioglitazone HCl and
internal standard were isolated from serum by liquid-liquid extraction. The organic
phase was separated and evaporated, and the remaining residue was reconstituted with
mobile phase before injected to the HPLC system, UV detector was operated at 269
nm for pioglitazone HCl serum concentrations determination (Ravikanth et al., 2011).
Another sensitive and rapid HPLC method was designed for determination of
pioglitazone HCl is serum by Kolte B. Rosiglitazone in Methanol was used as internal
standard, acetonitrile was used for extraction, and the final prepared sample was
injected into HPLC system for analysis. UV detector was set at 269 nm for the
determination of pioglitazone HCl concentrations in serum, HPLC system consisted
of C18 column and the mobile phase consisted of phosphate buffer (pH=3),
acetonitrile and methanol in a ratio of 70:25:5 (v/v/v) (Kolte et al., 2003), HPLC
technique was also used for determination of pioglitazone HCl in bulk and
pharmaceutical formulations, a validated experiment method was done by
Radhakrishna T., C18 column eluted with a mobile phase consisting of a mixture of
23
50% (v/v) acetonitrile and 10 mM potassium dihydrogen phosphate buffer, pH was
adjusted to 6 with 0.1 M KOH, wavelength was set at 225 nm (Radhakrishna et al.,
2002).
In addition, Sripalakit P., has developed an analytical method using high-
performance liquid chromatography (HPLC) with UV (269 nm) for the determination
of pioglitazone HCl in human serum. Rosiglitazone was used as an internal standard,
a reversed-phase chromatographic separation was obtained using C18 column and a
mobile phase of methanol and acetonitrile ,mixed with phosphate buffer 10 mM (pH=
2.6) in ratio of 40:12:48 (v/v/v) using a flow rate of 1.2 ml/min (Sripalakit et al.,
2006).
A validated HPLC –UV method for separation and quantification of pioglitazone
in plasma was developed, Zhong and Williams have used C18 separation column (250
mm × 4.6 mm i.d., 5 μm particle size) with mobile phase consisting of acetonitrile-
water (40:60, v/v) and containing 3 ml / L of acetic acid in mobile phase (pH=5.5).
Ultraviolet detector was operated at 269 nm (Zhang and Williams, 1996).
A different instrumentation, liquid chromatography tandem mass spectrometry
(LC-MS/MS) is also considered as a validated method that used for detection of
pioglitazone HCl in serum, Samanthula G. has carried this method procedure
successfully (Souri et al., 2008 ; Gananadhamu et al., 2012).
24
1.9 Sucralose
A synthetic organochlorine sweetener, it is considered as one of the most common
sweeteners and excipients in the world’s food industries (Broderick, 1992) and
pharmaceutical manufacturing (Blasé and Shah, 1993).
Physical and chemical properties: chemical formula: Dichloro-,1,6-dideoxy-β-D-
fructofuranosyl-4-chloro-4-deoxy α-D-galactopyranoside (Mann et al., 2000), the
chemical structure is shown in figure 2. Sucralose is a derivative of the halogenated
sucrose, mellow, with aromatic flavor and good stability, low in calories (around 3
calories per 1 gm) which justifies frequent usage of sucralose by diabetic patients and
people who follow low calories diet, in order to avoid sweetening of drinks or food
with table sugar which has high calories content. Sucralose is 600 times sweeter than
sucrose, good in sweet taste and mouth feel (Grotz and Munro, 2009).
It is a strongly stable sweetener with steady sweetness taste, these properties
permit safe usage of sucralose under high temperatures during manufacturing
processes of food or by home consumers, in addition, sucralose has a long term
stability of storage and even at low pH media (Binns, 2003). US FDA acceptable
daily intake (ADI) of sucralose is 5 mg/kg body weight/day.
25
Figure 2: Sucralose Chemical Structure.
1.9.1 Sucralose Manufacturing
Sucralose was artificially prepared and developed by British Professor Hough L in
1970 by chlorination, and at last deacetylation of sucrose (Luo et al., 2008).
1.9.2 Sucralose Brand Names
Splenda®,
which is widely distributed all over the world as either powder in sachets
or as tablets. Researchers analyzed Splenda®
and founded it as: 1.10% Sucralose,
1.08% glucose, 4.23% moisture and 93.59% maltodextrin.
Splenda® is only 1.10% sucralose, as a solitary sweetening agent (Schiffman and
Rother, 2013), FDA’s ADI of sucralose is 5 mg/ kg body weight/day which equals
about 454.5 mg/kg/day of Splenda®.
Another sucralose containing sweetener in markets named Tropicana Slim®, it
contains sorbitol 98.8% which is extracted from corn, and sucralose 1.2% as a
sweetness fortifying agent.
(http://www.amazon.com/Tropicana-Extracted-Sweetener-Calorie-Sticks).
26
Sucralose has not any teratogenicity, discontinuity or reproductive toxicity; it also
does not have any effect on blood sugar level and insulin secretion. Therefore, it is
freely used for obese patients, patients with cardiovascular diseases and diabetics.
(Goldsmith, 2000 ; Baird et al., 2000).
1.9.3 Sucralose PHarmacokinetics and Metabolism
Several studies stated that the majority of ingested sucralose is not absorbed from
gastrointestinal tract, 70–80% of ingested sucralose is excreted in feces and remainder
is excreted in urine (John et al., 2000).
The low absorption of sucralose from GIT is unpredictable as this sweetener is an
organochlorine molecule with good lipid solubility. Sucralose half-life has been
reported from 2 to 5 hours in most studies (Roberts et al., 2000) while serum peak
concentration is achieved 2 hours after oral dose, low bioavailability of sucralose
suggests that it is mostly extruded back into intestinal lumen during first-pass
metabolism in GIT (CYP-450) metabolism system ((Roberts et al., 2000 ; Schiffman
and Rother, 2013).
1.9.4 Artificial Sweeteners – Drug Interactions
Artificial sweeteners have historically been considered as inert compounds
without physiological complications. However, recent studies suggested that some of
these sweeteners have remarkable biological effects that may impact human health
(Rocha et al., 2008), on the other hand, there are significant gaps in our current
knowledge concerning pharmacokinetics of these sweeteners and their potential for
sweetener–drug interactions. Recently, previous studies have approved that sucralose
increases the expression of the P-gp intestinal transporter proteins and induces
27
CYP3A4 enzyme activity in intestine and liver (Schiffman and Rother, 2013 ; Abou-
Donia et al., 2008) at levels that have been associated with reduced bioavailability,
pharmacokinetic and pharmacodynamic parameters of drug, it may occur if the drug
is to be metabolized by CYP3A4 enzyme and concurrently taken with sucralose at
doses that are approved by the FDA, these findings justify the need of further studies
concerning potential drug interactions and monitoring of critical parameters during
concurrent intake of two possible interacting compounds, therefore, serious studies
concerning possible interaction are highly worthwhile(Grotz and Munro, 2009).
1.10 High Performance Liquid Chromatography (HPLC) Method
1.10.1 HPLC Definition and Principle
High performance liquid chromatography technique is used as separation and
quantification method to determine amount of specific compound that have been
dissolved in appropriate solution (Zhang et al., 2014).
HPLC system is mainly constructed by combining of three major components:
stationary phase, mobile phase and analyte (Kirkland et al., 1993).
Stationary phase is a matrix which is filled in column, it is usually made of inert
materials. Polarity of analyte determines the nature of stationary phase to be either
Normal Phase or Reverse Phase (Gloo and Johnson, 1977).
Mobile phase is liquid phase which is composed of solvent that carries sample to
be tested with its components through column stationary phase, composition of
mobile phase is basically depends on stationary phase composition and sample to be
tested too.
28
In HPLC, a pump use is a necessity to enhance mobile phase passage with sample
through column in a constant rate of flow to get a uniform separation of sample
components (Tang Y, 1996).
During analysis, where the sample solution is in contact with a second solid or
liquid phase that located in column, different solutes in sample solution will interact
with stationary phase, different interaction in column will occur which helps to
separate different components from each other.
Different factors should be properly selected and adjusted to obtain the most
suitable condition of separation and quantification, such as: type of column, column
length, column diameter, mobile phase, column temperature and flow rate (Bird,
1989), as shown in scheme 2.
Scheme 2: HPLC Flow
29
1.10.2 Types of HPLC
1.10.2.1 Normal Phase HPLC
In normal phase HPLC, column is filled with tiny silica particles and non-polar
solvent such as (hexane). A typical column has an internal diameter of 4.6 mm or less,
and a length of 150 - 250 mm.
Polar compounds in the mixture which will pass through the column will stick
stronger and longer to the polar silica than non-polar compounds. The non-polar
components will pass more quickly through the column (Kirkland et al., 1993).
1.10.2.2 Reversed Phase HPLC
In reversed phase HPLC, column is filled with silica which is modified to make it
non-polar by attaching long hydrocarbon chains to its surface with either 8 or 18
carbon atoms. A polar solvent is usually used such as mixture of water and alcohol.
A strong attraction between polar solvent and polar compounds in sample mixture
will occur, mixture will pass through column, non polar compounds will pass faster
while polar compounds will take longer passage time through column as it will bind
strongly with polar solvent. Reversed phase HPLC is the most commonly used form
of HPLC (Hearn et al., 1979).
1.10.2.3 Methods of Detection
There are several methods of detecting when compound passed through column. A
common and easy method is ultra-violet absorption.
30
Many organic compounds absorb UV light at various wavelengths. a beam of UV
light passing through a stream of liquid coming out of column, as UV detector is
located on opposite side of a stream, you can get a measurement of light amount that
was absorbed.
The amount of light absorbed is correlated to amount of specific compound that is
passing through a beam at that time, as shown in scheme 3.
Scheme 3: UV Detector Detection Pathway
The output will be recorded as a series of peaks, each one representing a
compound passing through the detector and absorbing UV light, peaks are used as a
way of measuring quantities of compounds present. Supposing that a particular
compound X, if a solution is injected containing a determined amount of X into
HPLC system, amount of X is related to peak that will be formed.
The area under the peak is proportional to amount of X, this area can be calculated
automatically by the computer linked to display (Lykkesfeldt, 2001).
31
1.10.3 Internal Standard (IS)
It is defined as a chemical substance that is added in constant amounts to samples
to be tested, blanks and calibration standards, the main purpose of internal standard is
for plotting ratio between signal that obtained from sample to signal that obtained
from internal standard in order to be used as a function of analytes unknown
concentrations calculations (Adibpour et al., 2013).
Both internal standard and analytes should perform a similar but distinguishable
signals and performance by instrument during analysis which facilitates analysis
technique and enhance the analysis procedure results (Goidin et al., 2001).
The internal standard must be available in pure form, stable and eluted after
sample components.
1.10.4 HPLC Instrument Calibration
Also named instrument qualification, it is defined as a procedure that carried to
ensure if instrument performance complies with method’s requirements specifications
resulting with reliable and valid obtained results (Bliesner, 2006).
To ensure that the instrument performs satisfactorily and gives accurate and
reproducible data, the instrument must be tested on site (Miller, 2005), within specific
time intervals and after repair, it should be inspected, maintained, calibrated and
cleaned periodically according to Standard Operating Procedures (SOPs) (Ermer,
2001; Shah et al., 1992).
32
1.10.5 Bioanalytical HPLC Method Validation Parameters Definitions (EMEA)
1.10.5.1 Precision
The precision of the analytical method describes closeness of repeated individual
measures of analyte. Precision is expressed as coefficient of variation CV% (Shabir,
2003).
Precision should be demonstrated for LLOQ, low, medium and high QC samples,
within a single run and between different runs, using the same runs and data as for the
demonstration of accuracy too (Taverniers et al., 2004).
Intra-day precision: five samples at least per each concentration of calibration
(LLOQ, QCL, QCM, and QCH) should be performed in a single run. Obtained
concentration results will be then evaluated.
Inter-day precision: LLOQ, QCL, QCM, and QCH samples should be run three
times separately on three different days, then results will be evaluated (Beavis, 1998).
Precision can be calculated as a coefficient of variation, CV% is a parameter with
high value in precision expression, for intra-day and inter-day precision: CV% should
not exceed 15% of QC values, except for LLOQ to be not more than 20%.
1.10.5.2 Accuracy:
The accuracy of an analytical method describes the closeness of determined value
obtained by method to nominal concentration of analyte (expressed in percentage).
Accuracy should be carried on samples spiked with determined analyte amounts as for
quality control samples (QC samples). QC samples should be spiked independently
33
from calibration standards, using separately prepared stock solutions ( Huber, 1999 ;
González et al., 1999).
QC samples should be analyzed, then compared with calibration curve nominal
values. Accuracy should be reported in percentage form of nominal value (Peduzzi et
al., 1995).
Accuracy % is calculated as follows:
Accuracy % = (calculated value / true value) * 100%
Intra- day accuracy: it is determined by analyzing a single run of minimum 5
samples per level at a minimum of 4 concentration levels covering calibration curve
range: LLOQ, 3 times of LLOQ: (QCL), 50% of calibration curve range: (QCM), and
75% of the upper calibration curve range: (QCH), mean concentration should be
within ±15% of the nominal values of QC samples, while for the LLOQ it should be
within ±20% of the nominal values.
Inter-day accuracy: LLOQ, QCL, QCM, and QCH are analyzed and evaluated on
three different days, mean concentration should be within ±15% of nominal values of
QC samples while for LLOQ it should be within ±20% of nominal values
1.10.5.3 Linearity
It is the ability to obtain test results which are directly proportional to
concentration of analyte in sample within a given range ( Huber, 1999).
A recommended protocol for establishing linearity of analytical method which
involves 6 calibration standards or more over concentration range of interest, with
34
repeating standard calibration for 3 or more runs in a random order, a direct
proportion between response and concentrations of analytes should be existed.
( Huber, 1999 ; Shah et al., 2000).
Regression factor (R²) is a value that is used to evaluate linearity of any calibration
curve data in order to evaluate closeness between predicted and target data, the closer
data reflects best linear relationship between values with R² closer to 1.
1.10.5.4 Range
Range of analytical method is the interval between upper and lower concentrations
including these concentrations of analyte in a sample with a suitable precision,
accuracy and linearity, range is derived from linearity studies and depends on
intended procedure application ( Huber, 1999).
1.10.5.5 Ruggedness
It is a measurement of method capacity to remain unaffected by small variations in
method parameters and supplies, it donates an indication of method reliability during
normal carrying (HÄsselbarth, 1998).
It can be also described as the ability to reproduce method in different laboratories
or under different conditions without any differences in obtained results (Vander et
al., 2001).Ruggedness test is usually performed for pharmaceutical preparations and
drug industries rather than other types of samples.
35
1.10.5.6 Limit of Detection
It is the lowest amount of analyte in a sample which can be detected but not
necessarily quantified as an exact value, but also represents the amount of analyte in
sample permitting the analyte identification qualitatively without accurate and precise
quantification (Taverniers et al., 2004 ; Armbruster and Pry et al., 2008).
1.10.5.7 Lower limit of Quantification
It is the lowest amount or concentration of analyte in a sample which can be
determined quantitatively with suitable precision and accuracy (Viswanathan et al.,
2007). Lower limit of quantification (LLOQ) is always higher than the limit of
detection (LOD) and is recommended for LLOQ as 3 times of LOD and not higher
than 5% of Cmax (Taverniers et al., 2004).
1.10.5.8 Selectivity
The analytical method should be able to differentiate between analyte(s) of interest
and IS from endogenous components in matrix or other components in sample.
Normally, absence of interfering components is accepted where the response is less
than 20% of lower limit of quantification for analyte and 5% for the internal standard.
It may also be necessary to investigate the extent of any interference caused by
metabolites of drug(s), interference from degradation products formed during sample
preparation, and interference from possible co-administered medications.Co-
medications normally used in the subject population studied which may potentially
interfere should be taken into account at method validation. (Miller and Miller, 1988 ;
Kazakevich and LoBrutto, 2007)
36
1.10.5.9 Sensitivity
Sensitivity can be defined as the ability to measure analyte concentrations
accurately in presence of interfering materials such as excipients and degradation
products that may be present in sample (Medina, 2003).
Sensitivity test is usually performed by analysis of 10 repetitionss of each quality
control level then evaluating the results according to the adopted validation
guidelines.
1.10.5.10 Recovery
A parameter which describes method ability to detect distinguishingly internal
standard and drug in presence of both at the sample to be tested, and obtaining a result
with less errors and more close to the nominated value.
It is an essential component of method validation as it is important to be aware of
problems and basis on which the results reporting depend (EMEA, 2012).
Recovery test should be carried out for analyte to be tested and internal standard, at
each level of QC samples, five different times analysis should be run at least.
37
1.10.5.11 Stability
Stability evaluation should be carried out to ensure that sample preparation,
analysis and storage conditions used, do not affect analyte concentration.
Analyte stability is usually studied using the matrix in low and high QC samples,
(LLOQ and close to the ULOQ) which analyzed immediately after preparation and
after the applied evaluation storage conditions. QC samples are analyzed against a
calibration curve, prepared freshly, and the obtained results of concentrations are
compared to nominal concentrations. Mean concentration at each QC used level
should be within ±15% of nominal concentration.
Essential stability tests that should be evaluated are as follow:
Stability of stock solution and working solutions of analyte and internal
standard.
Freeze and thaw stability of analyte in matrix from freezer storage conditions
to room temperature or sample processing temperature.
Short term stability of analyte in matrix at room temperature or sample
processing temperature.
Long term stability of the analyte in matrix stored in the freezer.
Regarding the freeze and thaw stability
QC samples are stored and frozen in freezer at a determined temperature then
thawed at room or processing temperature. After complete thawing, samples are
refrozen again under same conditions. At each cycle, samples should be frozen for at
least 12 hours before thawing. Number of cycles in the freeze-thaw stability should
equal or exceed that of freeze/thaw cycles of study samples.
38
Regarding long term stability of the analyte in matrix stored in the freezer
QC samples should be stored in freezer under the same storage conditions and at
least for same duration passed by study samples. For small molecules it is considered
acceptable to apply a bracketing approach, (in case stability has been proved for
instance at -70°C and -20°C, it is not necessary to investigate stability at temperatures
in between).
39
1.11 Aim of the Study
The aim of the present study is to develop a simple, valid and rapid
chromatographic method for quantifying pioglitazone HCl in rats serum under
feasible conditions to find out an appropriate method of analysis which comply
scientific research requirements.
Study the pharmacokinetic parameters of pioglitazone HCl in rats serum fed with
sucralose simultaneously in order to examine possibility of interaction between
pioglitazone HCl and sucralose in rats.
40
CHAPTER TWO
EXPERIMENTAL PART
41
2. Experimental Part
2.1 Reagents
All reagents that were used for analysis procedures and other experimental stages
are listed with details as follow:
Reagents List
Deionized water HPLC (TEDIA, B# 7732185), USA.
Rats serum (Pooled from animals).
Methanol HPLC gradient (FULLTIME, B# 6508ET30), USA.
Acetonitrile HPLC gradient (FULLTIME, B# 6308GT30), USA.
Ammonium acetate HPLC gradient (TEDIA, B# AR-OK17), USA.
Ammonia solution (MERCK, B# K41763423047), Germany.
Sodium Hydroxide pellets Purified 40Mwt (SDFCL, Fine – Chem
limited, B# G11Z/2911/1202/08), India.
Pioglitazone HydroChloride Raw material (JPM, B# 10713180), purity
˃99%, Jordan.
Splenda®
Sucralose containing sweetener (McNeil
Nutritionals, LLC. B# S6040543973-
071494), Indonesia.
Sildenafil Citrate (YASHICA pharmaceuticals, B#
LS/026/01/10). India.
Product information in table 2.
42
Table 2: Sildenafil Citrate Information (Internal Standard)
Wt 0.065 g
Assay % 100.905
Water content % 1.00%
Counter ion % 27.70%
Equivalent wt. 0.047 g
Volume prepared 100 ml
Sildenafil Citrate Chemical Structure
2.2 Instrumentation
A) HPLC
HPLC instrument parts and laboratory instruments with tools used in analysis
procedure are mentioned in the following list:
43
HPLC System Parts
Pump (S # L-2130 VWR-HITACHI, Japan).
Autosampler thermostat (S# L-2200 VWR-HITACHI, Japan).
Column oven (S# L-2300 VWR-HITACHI, Japan).
UV detector (S# L-2420 VWR-HITACHI, Japan)
Lab Instruments and Tools
Data system (Ez Chrome VWR-HITACHI, Japan)
Analytical balance (Sartorius, Germany.
Top loading balance (Sartorius, Germany)
Centrifuge (Eppendorf mini spin, Germany)
Vortex mixer (Heidolph, Germany)
Adjustable micropipettes
(20-200), ml
(Dragon, China)
pH meter (Bante instrument, model # PHS-3BW)
Sonicater (Decon FS 100, England)
Water bath (Hetotherm BWO)
44
2.3 Preclinical Study
The study protocol was approved by the Research Committee (October;
5/10/2013) at the Faculty of Pharmacy, University of Petra, Amman, Jordan.
Adult male Sprague Dawley laboratory rats were supplied at Petra University
animal house. Rats average weight was (0.230 kg ± 0.03). Rats were placed in air-
conditioned environment with temperature of (20-25 C°) and exposed to a
photoperiod cycle (12 hour light /12 hour dark) with humidity of 50% daily. Rats
were under fasting for 24 hours, and weighed directly before the experiment. All used
rats were in healthy conditions before and after experiment as rats were monitored for
one month post analysis.
Pioglitazone HCl and sucralose test solutions were freshly prepared directly in the
laboratory before rats feeding in order to avoid any possible decomposition of either
pioglitazone HCl or sucralose.
A group consisting of total 80 healthy rats was used for the experiment. Rats were
divided into groups of 8 rats, rats then were weighed and numbered orderly.
Pioglitazone HCl and sucralose oral doses were calculated according to each rat
weight then given orally according to ordered numbers using gastric gavage.
Trials analysis was performed among 3 days according to following arrangement:
At first day of trials: two groups of rats were used for analysis ; the first one has
received water at experiment zero time, then followed by pioglitazone HCl after 1
hour of water feeding, while the second group has received sucralose at zero time of
experiment followed by pioglitazone HCl after 1 hour of sucralose feeding.
45
Time intervals of blood samples pooling were: 0, 30 min, 1 hr, 2 hr, 3 hr, 4 hr and
6 hr.
At second day of trials: two groups have received water at zero time of
experiment followed by pioglitazone after one hour, while other two groups have
received sucralose at zero time followed by pioglitazone after 1 hour of suralose
feeding.
Time intervals of blood samples pooling were: 0, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6
hr, 8 hr and 24 hr.
At third day of trials: one groups have received water at zero time of experiment
followed by pioglitazone after one hour, while another three groups have received
sucralose at zero time followed by pioglitazone after 1 hour of suralose feeding.
Time intervals of blood samples pooling were: 0, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 6 hr
and 8 hr.
Tail tip of each rat was cut after weighing and numbering, approximately 200 µl of
blood was pooled into eppendorf tube at each time interval under the same numbering
order, after total time intervals of blood pooling is finished, samples were centrifuged
for 10 minutes (12000 rpm) in order to obtain pure serum that is needed for analysis
than frozen at -20 C°.
46
2.4 Preparation of Stock Solutions and Working Solutions
2.4.1 Preparation of Pioglitazone HCl Oral Solution:
10 mg/kg of pioglitazone HCl oral dose for rats was recommended at the
experiment.The current study was carried out with a reasonable dose of pioglitazone
for rodents (10 mg/kg). Other experiments with daily oral administration of
pioglitazone in rodents have been performed by using pioglitazone doses between 2.3
and 35 mg/kg, but with most studies 10 mg/kg.was used, it represents around 50 times
of 70 kg weighing patient who would receive a pioglitazone dose of 45-mg tablet.
(Sakai et al., 2002 ; Matsuura et al., 2004 ; Umathe et al., 2008 ; Janadri et al., 2009).
Although these doses seem disproportionate, but thiazolidinediones are eliminated
and cleared approximately 10 times faster by rats when compared with humans
because of higher CYP2C expression (Lamontagne et al., 2013).
As a result, using of 10 mg/kg dose is considered as effective dose with similar
acute therapeutic effects and corresponding bioactivities as the 45 mg / day human
daily dose of pioglitazone.
Freshly solution per day of trials was prepared : accurate weight of pioglitazone
HCl raw drug = (0.3 g) was prepared, dissolved in 0.0375 M NaOH solution to get
100 ml final solution volume.
Solution was heated to 35 C° using water bath, and sonicated for 5 minutes
sequentially till drug dissolved completely.
Pioglitazone HCl oral dose volume was calculated corresponding to each rat
weight according to the following formula:
47
Rat oral dose volume = rat weight (kg) x pioglitazone HCl experiment approved dose
(mg/kg) ÷ pioglitazone HCl solution concentration (mg/ml).
Rat oral dose = rat weight (kg) x 10 mg/kg ÷ 3 mg/ml
2.4.2 Preparation of Sodium Hydroxide Solution
0.15 g of sodium hydroxide pellets was weighed, then dissolved in distilled water
to obtain a 100 ml final solution volume of (0.0375 M).
2.4.3 Preparation of Sucralose Oral Solution
Splenda® contains 1.10% sucralose, as a solitary sweetening agent (Schiffman S. et
al., 2013), to reach the FDA’s ADI of 5 mg/kg/day of sucralose, adult human would
need to consume 454.5 mg/kg/day of Splenda®, corresponding to this calculations,
rats oral dose of sucralose was 11 mg/kg/day which equals 1000 mg /kg/day of
Splenda®. According to FDA approved daily dose, 11 mg/kg/day of sucralose is a
double dose, but it is approved in our experiment as an acute treatment in order to
reach the desired biological effects of sucralose in rats gastrointestinal system at the
required time.
Freshly prepared solution of sucralose was prepared daily, Splenda®
powder was
weighed (12.5 g), dissolved in distilled water to obtain 50 ml final solution volume of
250 mg/ml concentration of Splenda®.
Sucralose oral dose volume for each rat was calculated according to the following
formula:
Rat oral dose = rat weight (kg) x Splenda® experiment approved dose (mg/kg) ÷
Splenda® solution concentration (mg/ml).
48
Rat oral dose = rat weight (kg) x 1000 (mg/kg) ÷ 250 mg/ml
2.4.4 Preparation of Pioglitazone HCl Stock Solution
Daily prepared solutions were made by dissolving a weight of pioglitazone HCl
equals (25 mg) in 50 ml methanol to provide a solution concentration of 500 µg/ ml.
2.4.5 Preparation of Pioglitazone HCl Serial Dilutions in Methanol
Samples for standard calibration curve and method validation analysis were
prepared by taking different volumes from pioglitazone HCl stock solution, each
sample was added to sufficient volume of methanol HPLC gradient to reach 10 ml
final solution volume per sample with specific concentration per each in order to be
used for calibration and method validation analysis later, as shown in table 3 and 4.
Table 3: Pioglitazone HCl Serial Dilutions in Methanol
Serial solution (C) no.
Volume of
stock
solution
(ml)
Final
volume (ml) Final concentration (µg\ml)
C 1 0.25 10 12.5
C 2 0.75 10 37.5
C 3 2 10 100
C 4 3 10 150
C5 4 10 200
C 6 5 10 250
C7 (LLOQ) 0.25 10 12.5
C8 (QCL) 2 10 100
C9 (QCM) 4 10 200
C10 (QCH) 8 10 400
49
2.4.6 Preparation of Pioglitazone HCl Standard Solutions in Serum (QC
Solutions)
Serial dilutions of samples were prepared by taking an appropriate volume of each
concentration that is previously prepared in Methanol (as shown in table 3 and 4),
sufficient volume of serum was added to reach 5 ml final volume with corresponding
pioglitazone HCl concentration as detailed in table 4. Samples were vortexed, then
used for analysis procedure. All final standard solutions were prepared freshly and
directly before analysis. Concentrations: St1, St2, St3, St4, St5 and St6 were the
concentrations that used for calibrations, while concentrations of: St7 (LLOQ), St8
(QCL), St9 (QCM) and St10 (QCH) were the concentrations that used for method
validation analysis, as shown in table 4.
Table 4: Pioglitazone HCl QC Standard Solutions in Serum
Final standard
solution no.
Volume of
standard
(C) (ml)
Standard
solution
(C) conc.
(µg\ml)
Final
standar
d
volume
(ml)
Final concentration
(µg\ml)
St1 0.1 12.5 5 0.25
St2 0.1 37.5 5 0.75
St3 0.15 100 5 3
St4 0.167 150 5 5
St5 0.175 200 5 7
St6 0.32 250 5 16
St7 (LLOQ) 0.1 12.5 5 0.25
St8 (QCL) 0.1 100 5 2
St9 (QCM) 0.175 200 5 7
St10 (QCH) 0.16 400 5 12.8
50
2.4.7 Preparation of Mobile Phase
515 ml acetonitrile HPLC gradient was mixed with 485 ml buffer solution to get
the mobile phase with the defined ratios of the chromatographic conditions, as shown
in table 5.
2.4.8 Preparation of Buffer Solution
Amount of 1.927 g of ammonium acetate was weighed, then dissolved in 1L
distilled water, pH was adjusted to 8 by using 300 µl ammonia solution, pH=8 was
selected as pioglitazone HCl chromatographic peaks showed accepted resolution and
separation at this value.
2.4.9 Preparation of Internal Standard Stock Solution
Sildenafil citrate was used as internal standard as it has similar chemical
properities and solubility to pioglitazone HCl, it behaves quietly similar to
pioglitazone HCl during HPLC analysis, it elutes after pioglitazone at a different
retention time and sharp peaks with appropriate separation resolution. Blanks and
internal standard zero drug blanks explain and ensure sildenafil citrate suitability to be
used as IS. Sildenafil citrate stock solution was prepared freshly by weighing (65.04
mg) of sildenafil citrate which is equivalent to (46.97 mg), added into a volumetric
flask and dissolved in 100 ml methanol to provide a 469.7 µg /ml base equivalent.
2.4.10 Preparation of Internal Standard Working Solution
Internal standard solution was prepared by diluting 3 ml from stock solution with
sufficient volume of acetonitrile to obtain 100 ml final solution of(14.091) µg /ml
concentration. Sildenafil citrate information was listed in table 2.
51
2.4.11 Sample Preparation (Extraction Procedure)
Mixing of 100 µl of rats serum with 75.0 µl of IS working solution, then vortex for
30 seconds and centrifuged at 12000 rpm for 5 min. Supernatant was transferred into
rack, and 90 µl of supernatant was injected into HPLC unit.
2.4.12 Method Development (Chromatographic Conditions)
Separation column selection
Referring to previous studies that demonstrated reverse phase HPLC method for
detection of pioglitazone HCl in plasma and pharmaceutical preparations, C8 column
was selected for analysis, other studies suggested the use of C18 column and methods
were validated, C8 has the same characteristics of C18 but with difference in
hydrophobicity properties as C18 column is more hydrophobic than C8 due to higher
number of alkyl groups attached to silica, concerning separation technique: both have
same performance but with sharper peaks of separation chromatograms with C8,
which enhances analytes separation and results sharpness (Souri et al., 2008), this
finding suggested the proper selection of C8 column in our analysis procedure.
Mobile phase selection
Mobile phase selection was dependant on the following concepts:
1- Pioglitazone solvent dependant solubility.
2- Pioglitazone stability in mobile phase.
3- Pioglitazone pH dependant stability and solubility.
As detailed previously in literature survey heading, and referring to previous
studies that determined pioglitazone in serum, mobile phase was consisting of
52
solvents that has moderate to efficient pioglitazone HCl solubility, but with ratios
which enhances sample elution and avoid drug decomposition or complete
solubilization, which may interfere compounds separation and detection.
The most popularly used solvents for pioglitazone detection in plasma and serum
referring to literature surveys were: acetonitrile, methanol, water, ethyl acetate and
buffers with acidic pH.
Pioglitazone showed reasonable solubility with basic pH> 6 while many studies
claim that pioglitazone HCL could be decomposed under basic conditions with pH>8
with degradation percentage = 3.04 (Narsimha et al., 2012).
Other studies found that pH degradation was at pH=7-12 or less, with degradation
5.76% at 60min and 9.61% at 90min after drug solution preparation and under stress
conditions (Dubey, 2014), literature outcomes conclude the safe use of basic pH
values for pioglitazone HCl solutions if experimental procedure follows freshly
prepared drug solutions under normal conditions.
In our experiment, mobile phase was formulated with several solvents ratios and
pH values, it was founded to be (51.50%) acetonitrile and (48.50%) 0.025 mM
ammonium acetate, pH= 8 by which sharp peaks and reasonable resolution of
pioglitazone chromatograms were obtained.
53
UV Detector wave length selection
Referring to literature survey, several trials have been made to determine the most
precise UV wavelength at which pioglitazone can be detected, in our experiment it
was 269 nm.
Flow rate selection
1ml/min was founded as accepted rate of flow depending on peaks shapes
obtained, as it enhances mobile phase elution with uniform separation.
All chromatographic conditions were detailed in table 5.
Table 5: Chromatographic and Detection Conditions
Analytical technique HPLC
Detection method UV (269) nm
Analytical column ACE C8, 5 µm (250 x 4.6 mm i.d.)
Auto-sampler temperature 10 C°
Column temperature 40 C°
Mobile phase (51.50%) acetonitrile + (48.50%)
0.025 mM ammonium acetate
pH= 8
Injection volume 90 µl
Run time 10 min.
Flow rate 1 ml/min
Pioglitazone HCl retention time 5 min ±0.03
Internal standard sildenafil citrate
retention time
8 min ±0.01
54
2.5 Method Validation
EMEA guidelines of bioanalytical method validation were relied as validation
reference for method development.
2.5.1 Inter- day Accuracy and Precision
Inter-day accuracy and precision were determined by analyzing of 5 samples of
each QC concentration mentioned in table 6 on 3 different days with daily
randomization of samples before analysis. Evaluating of coefficient of variation CV%
was done to determine precision while accuracy% was used to determine accuracy
level.
Precision: CV% should not exceed 15% for QCL, QCM and QCH as acceptance
criteria, while it should be less than 20% for LLOQ according to EMEA guidelines.
Accuracy: mean of calculated concentrations should be within ±15% of nominated
concentrations for: QCL, QCM, and QCH, while it should be within ±20% of LLOQ
concentration.
Table 6: Concentrations Used for Method Validation
QC solution Final Concentration (µg\ml)
S1 (LLOQ) 0.25
QCL 2
QCM 7
QCH 12.8
55
2.5.2 Intra-day Accuracy and Precision
Intra–day accuracy and precision were determined by analyzing 5 samples of each
QC concentration mentioned in table 6. Evaluating of CV% values was done to
determine precision while accuracy% was employed to determine accuracy level.
Precision: CV% to be < 15% for QCL, QCM and QCH was adopted as acceptance
criteria, while it should be less than 20% for LLOQ according to EMEA guidelines.
Accuracy: mean of calculated concentrations should not exceed ±15% of
nominated concentrations of: QCL, QCM, and QCH, while it should be within ±20%
of LLOQ concentration.
Each set of samples were run simultaneously with a calibration curve samples in
order to calculate the concentrations per each, every calibration curve had a formula
which was used for concentrations calculation:
Y = a * X + b
Y= Area ratio
a = Slope of the plotted curve
X= Calculated concentration
b = Curve intercept
56
2.5.3 Selectivity and Sensitivity
Selectivity test was carried out by analysis of 6 different blanks of serum then
compared with LLOQ clarifying absence of any interfering peaks of components in
samples.
It is accepted if endogenous peak appeared in the chromatogram of blanks and
referred to either sample component or internal standard, but when response is less
than 20% of lower limit of quantification for analyte and 5% for internal standard
according to EMEA guidelines.
Sensitivity test was proceeded by analysis of 10 repetitionss of each quality
control level, mean calculated concentrations will be evaluated according to EMEA
guidelines as it should be within ± 20% for LLOQ, while it should be within ±15%
for other QC levels.
2.5.4 Linearity
Six calibration curves were designed for each group of quality control samples by
spiking of pioglitazone HCl and internal standard in serum to get standard
concentrations of (0.25, 0.75, 3, 5, 7, 16) µg/ml, samples then prepared for analysis by
extraction followed by injection for analysis.
Plotting of area ratios versus concentrations was done to perform calibration curve
for each group of samples.
R² values were calculated for each calibration in order to evaluate linearity and to
calculate concentrations at each level.
57
2.5.5 Recovery
Analyte recovery
QC samples per level were prepared in serum, and then treated to be analyzed with
compatible calibration. After obtaining calculated concentrations, mean concentration
was calculated, it must be within ± 20 for LLOQ and within ±15 for other QC
samples.
Internal standard recovery
Two groups of all QC samples with corresponding levels were prepared; the first
group was prepared with mobile phase while the other group was prepared with
serum.
After obtaining IS areas of each group per QC level, area ratios% were calculated
depending on its corresponding calibration as follows:
IS area in in serum / IS area mobile phase * 100%
2.5.6 Stability
1- Freeze and thaw stability
Three samples of (QCL and QCH) were prepared properly in serum with sufficient
final volume covering all test cycles analysis.
At zero time (FTS0): with corresponding calibration, samples were analyzed and
concentrations were calculated, then, samples were stored and frozen at -20C°.
After 12 hours (FTS1): samples were thawed at room temperature 25C° which was
the samples processing temperature during analysis stages. After complete thawing,
58
samples were analyzed again with corresponding calibration, and refrozen again for
24 hours.
After 24 hours (FTS2): samples were thawed and analyzed under same conditions.
After each cycle, concentrations were calculated referring to related calibration.
2- Room temperature stability
Room temperature stability of pioglitazone in serum and stock/working
solutions: 3 samples of (QCL and QCH) were prepared properly in serum. Another
group of QC samples were prepared in mobile phase for stock and working solution
stability, with sufficient volume depending on test requirements.
Samples were prepared and analyzed with freshly prepared calibration at zero time
and after 8 hours at room temperature of 28C°. Area ratios % per QCL and QCH were
calculated referring to related calibration.
Room temperature stability of internal standard in serum and stock/working
solutions: 3 samples of (QCL and QCH) were prepared properly in serum. Another
group of QC samples were prepared in mobile phase for stock and working solution
stability, with sufficient volume depending on test requirements.
Samples were prepared and analyzed with freshly prepared calibration at zero time
and after 8 hours at room temperature of 28C°.
In serum stability: area ratios% per QCL and QCH were calculated according to
freshly prepared combined calibration.
In working/stock solutions stability: area ratios% were calculated for each QC
sample properly.
59
3- Long term stability
Long term stability of pioglitazone in serum and working/stock solutions
3 samples of (QCL and QCH) were prepared properly in serum. Another group of
QC samples were prepared in mobile phase for stock and working solution stability,
with sufficient volume depending on test requirements.
Samples were prepared and analyzed with freshly prepared calibration at zero
time. Samples then were frozen for 30 days at temperature of -20 C°. After long term
freezing period was finished, samples were removed and thawed at room temperature.
In serum stability: concentrations per QCL and QCH were calculated according to
freshly prepared combined calibration.
In working/stock solutions stability: area ratios% were calculated for each QC
sample then evaluated.
3.2 Long term stability of internal standard in working/stock solutions: area
ratios% were calculated for each QC sample then evaluated.
2.6 Statistical Analysis
Statistic is a number that is derived from a specific data, such as mean or a
standard deviation. It is helpful when data are going to be examined to obtain a
relevant descriptive statistics.
Comparing statistics obtained from different sets of data can give an idea of
similarities or differences between sets of data.
60
Our experimental data was gathered then treated to be easily translated in to proper
computerized data structure, examining of data were carried to evaluate errors and
figures reasonability using T-test and Cohen’s d, statistical analysis tools were carried
by statistical science expert and performed using statistical package for social
sciences (SPSS) of (IBM version 20).
Confidence Interval: confidence interval determines an estimated range of values
which mostly includes unknown parameter, this estimated range is calculated from
given data, for example; a confidence interval for difference between two means
specifies a range of values where the difference between means could be found.
Confidence intervals are often calculated as 95% but producing 90%, 99% or
99.9% confidence intervals for unknown parameter is possible too.
Confidence interval width indicates a clear idea about how certain we are
concerning unknown parameter which helps to determine acceptance or rejection of
hypothesis. Cohen’s d: Cohen’s d is a measurement of effect size, which is usually
used in experiments to compare differences between two groups of data.
Cohen’s d = mean 1 – mean 2 / pooled SD
SD pooled = √ { (SD1)² + (SD2)² } ÷ 2
If Cohen’s d ˂ 0.3, effect size is considered as small, while value within (0.3-0.7)
is considered as medium effect, and exceeding ≥ 0.8 as large effect.
P value: when performing a hypothesis test in statistics, P-value helps to determine
the significance of experiment results. Hypothesis tests are used to test validity of a
claim concerning category, this claim which is on trial is called null hypothesis.
Alternative hypothesis is the one which should be approved if null hypothesis is
61
considered as untrue. The evidence is the data, and the statistics that go along with it.
All hypothesis tests use P-value to evaluate the strength of evidence.
P-value is a number between 0 and 1 and interpreted in the following way:
A small P-value (P ≤ 0.05) indicates strong evidence against null hypothesis, so you
reject it.
A large P-value (P > 0.05) indicates weak evidence against null hypothesis, so you
fail to reject it.
62
CHAPTER THREE
RESULTS AND DISCUSSION
63
3. Results and Discussion
3.1 Method Validation
3.1.1 Inter-day Precision and Accuracy
Inter-day Precision
At first day of validation: QCL showed the lowest coefficient of variation
(CV%) = 0.188 (less than 15%) which reflected acceptable precision while ST1
(LLOQ) showed the highest CV% = 1.77 which is also agreeable to be considered as
accepted (less than 20% for LLOQ) according to EMEA guidelines, both QCH and
QCM showed medium CV% values with reasonable precision equals 0.81 and 1.28
respectively, as shown in table 7.
First day validation calculated concentrations were obtained using corresponding
calibration that was run simultaneously with samples, as shown in figure 3 and table
11.
At second day of validation: QCL showed reasonable accepted precision with
CV% = 0.16, as it is less than 20 % for LLOQ, while QCH showed the least but also
reasonable precision with CV% value = 3.54 according to EMEA guidelines.
Concerning ST 1 (LLOQ) and QCM: both showed reasonable precision with CV% =
1.43 and 1.33 respectively as values were less than 20% for LLOQ and less than 15%
for QCM, as shown in table 8.
Second day validation calculated concentrations were obtained using
corresponding calibration that was run simultaneously with samples, as shown in
figure 5 and table 13.
64
At third day of validation: QCL showed the most valid precision value as it
obtained CV% value of 0.40 while QCH reflected the least precision over the third
day with accepted validity as it showed CV% value of 2.94, both QCM and
ST1(LLOQ) showed plausible precisions with CV% values less than 15% for QCM
and less than 20% for LLOQ to confirm reasonability for both, CV% values were 1.18
and 1.56 respectively, as shown in table 9.
Third day validation calculated concentrations were obtained using corresponding
calibration that was run simultaneously with samples, as shown in figure 7 and table
15.
Inter - day Accuracy
At first day of validation: QCH showed highly reasonable accuracy % = 105.6 %
and mean concentration within ± 15% of nominated value, QCL showed the least but
also accepted accuracy % = 99.45% with a mean concentration within± 15% of
nominated value indicating the passing of EMEA guidelines too.
Regarding QCM and ST1 (LLOQ), both levels showed approved accuracy as they
got accuracy% of 102.4 % and 102.8 % respectively with mean concentrations within
±15% of QCM and within ±20% of LLOQ.
First day overall accuracy % was 102.54 % which is approved according to EMEA
guidelines, as shown in table 7.
First day validation calculated concentrations were obtained using corresponding
calibration that was run simultaneously with samples, as shown in figure 3 and table
11.
65
At second day of validation: QCH showed highly reasonable accuracy % = 105.7
% while QCL showed the least but also accepted accuracy % = 101.2%.
Concerning ST1 (LLOQ) and QCM, both showed very plausible accuracy as they
got accuracy% of 102.4 % and 104.3% respectively.
Second day overall accuracy % was 103.4% with calculated concentrations mean
within permitted limits of EMEA, as shown in table 8.
Second day validation calculated concentrations were obtained using
corresponding calibration that was run simultaneously with samples, as shown in
figure 5 and table 13.
At the third day of validation: QCH showed a plausible accuracy % of 107.85
with mean concentration within ±15 % of nominated concentration, while
ST1(LLOQ) showed the least but also appreciable accuracy % = 98.4% and mean
concentration within ±20% of nominated concentration. In addition, QCL and QCM
showed very good accuracy as they got accuracy% of 102.2 % and 105.95%
respectively. Third day overall accuracy % = 103.6% considered as valid according to
EMEA guidelines, as shown in table 9.
Third day validation calculated concentrations were obtained using corresponding
calibration that was run simultaneously with samples, as shown in figure 7 and table
15.
66
Table 7: Inter- day Precision and Accuracy: Day 1
INTER- DAY ACCURACY AND PRECISION: DAY 1
Sample ID Pioglitazone
area 1
IS area
2
Area
ratio
Calculated
conc.
(µg/ml)
True
conc.
(µg/ml)
Mean SD CV% Error Accuracy
%
ST(LLOQ)
1 1
117581 477005 0.0246 0.254 0.25 0.26 0.01 1.77 0.01 102.8
ST(LLOQ)
1 2
118865 476363 0.0250 0.257 0.25
ST(LLOQ)
1 3
119333 476870 0.0250 0.258 0.25
ST(LLOQ)
1 4
121301 472719 0.0257 0.264 0.25
ST(LLOQ)
1 5
114968 470081 0.0245 0.252 0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 - 0.30)
QCL Day
1 1
951839 477832 0.1992 1.985 2.00 1.989 0.004 0.188 -0.01 99.45
QCL Day
1 2
950899 476217 0.1997 1.990 2.00
QCL Day
1 3
953577 476862 0.2000 1.993 2.00
QCL Day
1 4
947473 475692 0.1992 1.985 2.00
QCL Day
1 5
946498 473482 0.1999 1.992 2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QCM Day
1 1
3427503 472442 0.7255 7.205 7.00 7.16 0.09 1.28 0.16 102.3
QCM Day
1 2
3422696 472160 0.7249 7.200 7.00
QCM Day
1 3
3422376 471742 0.7255 7.205 7.00
QCM Day
1 4
3418429 470867 0.7260 7.210 7.00
QCM Day
1 5
3301435 468458 0.7047 7.000 7.00
QCM ±15% = ±1.05...... (5.95 - 8.050)
QCH Day
1 1
6297812 468234 1.3450 13.350 12.80 13.52 0.11 0.81 0.72 105.6
QCH Day
1 2
6381626 465840 1.3699 13.597 12.80
QCH Day
1 3
6250955 461026 1.3559 13.458 12.80
QCH Day
1 4
6184986 452207 1.3677 13.575 12.80
QCH Day
1 5
6172602 450621 1.3698 13.596 12.80
QCH ±15 %= ±1.92...... (10.88 – 14.74)
67
Table 8: Inter- day Precision and Accuracy: Day 2
INTER- DAY ACCURACY AND PRECISION: DAY 2
Sample ID Pioglitazone
area 1
IS area 2 Area
ratio
Calculated
conc.
(µg/ml)
True conc.
(µg/ml)
Mean SD CV
%
Error Accuracy
%
ST(LLOQ)
2 1
121255 486709 0.0249 0.254 0.25 0.26 0.004 1.43 0.01 102.40
ST(LLOQ)
2 2
122537 483712 0.0253 0.259 0.25
ST(LLOQ)
2 3
121916 489363 0.0249 0.254 0.25
ST(LLOQ)
2 4
125213 488775 0.0256 0.262 0.25
ST(LLOQ)
2 5
120435 486815 0.0247 0.253 0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 - 0.30)
QCL Day
2 1
971323 487586 0.1992 2.023 2.00 2.02 0.003 0.16 0.024 101.20
QCL Day
2 2
961354 482569 0.1992 2.023 2.00
QCL Day
2 3
960881 482985 0.1989 2.020 2.00
QCL Day
2 4
966005 483509 0.1998 2.029 2.00
QCL Day
2 5
965241 484606 0.1992 2.023 2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QCM Day
2 1
3504031 485146 0.7223
7.331
7.00 7.30 0.10 1.33 0.30 104.30
QCM Day
2 2
3444259 475134 0.7249
7.357
7.00
QCM Day
2 3
3478431 479926 0.7248
7.356
7.00
QCM Day
2 4
3507246 484583 0.7238 7.346
7.00
QCM Day
2 5
3460054 492446 0.7026 7.131
7.00
QCM ±15% = ±1.05...... (5.95 - 8.05)
QCH Day
2 1
6442610 489963 1.3149 13.344 12.80 13.53 0.48 3.54 0.73 105.70
QCH Day
2 2
6499104 458415 1.4177 14.388 12.8 0
QCH Day
2 3
6459534 493133 1.3099 13.293 12.80
QCH Day
2 4
6446871 491834 1.3108 13.302 12.80
QCH Day
2 5
6447860 490902 1.3135 13.33 12.80
QCH ±15% = ±1.92...... (10.88 – 14.74)
68
Table 9: Inter- day Precision and Accuracy: Day 3
INTER -DAY ACCURACY AND PRECISION: DAY 3
Sample ID Pioglitazone
area 1
IS area 2 Area ratio Calculated
conc.
(µg/ml)
True conc.
(µg/ml)
Mean SD CV
%
Error Accuracy
%
ST(LLOQ)
3 1
127844 476104 0.0269 0.247 0.25 0.25 0.004 1.56 -0.004 98.40
ST(LLOQ)
3 2
130431 478226 0.0273 0.251 0.25
ST(LLOQ)
3 3
122158 464357 0.0263 0.241 0.25
ST(LLOQ)
3 4
124118 461288 0.0269 0.247 0.25
ST(LLOQ)
3 5
122016 460724 0.0265 0.243 0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 - 0.30)
QCL Day
3 1
980839 478395 0.205 2.05 2.00 2.04 0.008 0.40 0.043 102.20
QCL Day
3 2
982727 479271 0.205 2.051 2.00
QCL Day
3 3
934037 459898 0.2031 2.031 2.00
QCL Day
3 4
940772 460934 0.2041 2.041 2.00
QCL Day
3 5
941802 460723 0.2044 2.044 2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QCM Day
3 1
3415499 462505 0.7385 7.451 7.00 7.42 0.088 1.18 0.42 105.95
QCM Day
3 2
3403102 458932 0.7415 7.482 7.00
QCM Day
3 3
3421587 463638 0.738 7.446 7.00
QCM Day
3 4
3351959 454486 0.7375 7.442 7.00
QCM Day
3 5
3301078 458575 0.7199 7.263 7.00
QCM ±15% = ±1.05...... (5.95 - 8.050)
QCH Day
3 1
6325417 461149 1.3717 13.862 12.80 13.81 0.405 2.94 1.01 107.90
QCH Day
3 2
6334111 452690 1.3992 14.141 12.8 0
QCH Day
3 3
6173948 445117 1.387 14.017 12.80
QCH Day
3 4
6213802 451902 1.375 13.896 12.80
QCH Day
3 5
4549269 350723 1.2971 13.107 12.80
QCH ±15% = ±1.92...... (10.88 – 14.74)
69
3.1.2 Intra- Day Precision and Accuracy
Intra-day Precision
A reasonable precision with CV% range of (0.15- 4.1) was achieved, all CV%
values were less than 15% for QC samples for QCL, QCM and QCH and less than
20% for LLOQ which confirms precision validity according to EMEA guidelines, as
shown in table 10.
Intra-day Accuracy
Accuracy% ranged between (99.35 - 103.99), while all calculated mean of
concentrations were within ±15% of QCM, QCH and QCL, and within ± 20% of
LLOQ, which indicates the validity of method accuracy according to EMEA
guidelines, as shown in table 10.
Intra- Day accuracy and precision calculated concentrations were obtained using
corresponding calibration that was run simultaneously with samples, as shown in
figure 3 and table 11.
70
Table 10: Intra- day Precision and Accuracy
INTRA- DAY ACCURACY AND PRECISION
Sample ID Pioglitazone
area 1
IS area
2
Area
ratio
Calculated
conc.
(µg/ml)
True conc.
(µg/ml)
Mean SD CV% Error Accuracy
%
ST(LLOQ)
1
120249 484105 0.0248 0.256 0.25 0.26 0.003 1.31 0.01 103.20
ST(LLOQ)
2
121541 481135 0.0253 0.26 0.25
ST(LLOQ)
3
120605 486736 0.0248 0.256 0.25
ST(LLOQ)
4
123770 486304 0.0255 0.262 0.25
ST(LLOQ)
5
119434 484381 0.0247 0.254 0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 - 0.30)
QCL
1
966488 485107 0.1992 1.986 2.00 1.99 0.003 0.15 -0.01 99.35
QCL
2
956768 480112 0.1993 1.986 2.00
QCL
3
956844 480365 0.1992 1.985 2.00
QCL
4
961728 481112 0.1999 1.992 2.00
QCL
5
960276 482162 0.1992 1.985 2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QCM
1
3491285 482558 0.7235 7.186 7.00 7.16 0.096 1.34 0.16 102.20
QCM
2
3430021 472448 0.726 7.211 7.00
QCM
3
3464505 477387 0.7257 7.208 7.00
QCM
4
3494237 482284 0.7245 7.196 7.00
QCM
5
3445729 489882 0.7034 6.986 7.00
QCM ±15% = ±1.05...... (5.95 - 8.05)
QCH
1
6428716 487289 1.3193 13.095 12.80 13.31 0.550 4.13 0.51 103.99
QCH
2
6484961 450279 1.4402 14.294 12.80
QCH
3
6445076 490655 1.3136 13.038 12.80
QCH
4
6433374 489376 1.3146 13.048 12.80
QCH
5
6435618 488427 1.3176 13.078 12.80
QCH ±15% = ±1.92...... (10.88 – 14.74)
71
3.1.3 Linearity
Calculating R² values for 6 calibration curves of QC samples to determine
closeness of R² value to 1 was performed, the closer value indicated highly reasonable
linearity while all R² values for 6 calibrations were with accepted fitness and
calculated concentrations of calibration levels was within ± 20% for LLOQ, and ±
15% for other QC levels.
Results indicated reasonable and valid method linearity, as shown in the following
tables and figures:
Linearity calibration 1 R² = 0.997383 See figure 3 and table 11
Linearity calibration 2 R² = 0.994147 See figure 4 and table 12
Linearity calibration 3 R² = 0.999127 See figure 5 and table 13.
Linearity calibration 4 R² = 0.996554 See figure 6 and table 14.
Linearity calibration 5 R² = 0.99955 (highest value with corresponding linearity), see
figure 7 and table 15.
Linearity calibration 6 R² = 0.996124 See figure 8 and table 16.
Six calibrations mean of linearity test was calculated. R² value = 0.9991 which
reflects valid linearity performance of method between plotted concentrations and
their responses.
72
Figure 3: Linearity Calibration 1
Table 11: Linearity Calibration 1 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 120560 296153 1490928 2587688 3378180 7666202
IS area 2 4766532 4689973 4708993 4733321 4793227 4887632
Area ratio 0.025293 0.065514 0.316613 0.546696 0.704782 1.568490
RF
0.1022
0.0842
0.1055
0.1093
0.1001
0.0980 Back calculated conc.
(µg/ml) 0.2606 0.6400 3.1500 5.4321 7.0000 15.5665
± 15% of St true value 0.0375 0.1125 0.45 0.75 1.05 2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.100824 x - 0.000986181 R²= 0.997383
0.0000000
0.2000000
0.4000000
0.6000000
0.8000000
1.0000000
1.2000000
1.4000000
1.6000000
1.8000000
0 2 4 6 8 10 12 14 16 18
Calibration 1
Conc.(µg/ml)
Are
a R
atio
73
Figure 4: Linearity Calibration 2
Table 12: Linearity Calibration 2 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml)
0.25
0.75
3
5
7
16
Pioglitazone area 1 126092 316816
1536496 2609269 3665146 7156039
IS area 2
4659633
4647634
4632825
4707404 5070670
4568116
Area ratio 0.027061
0.068167 0.331650 0.554290 0.722813 1.56652
RF
0.1012
0.0892
0.1054
0.1092
0.1101
0.0880
Back calculated conc.
(µg/ml) 0.2582 0.692728 3.2097 5.3670 7.0000 15.1755
± 15% of St true value 0.0375 0.1125 0.45 0.75 1.05
2.4
-15%
+15% 0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.103200 x + 0.000412951
R²=0.994147
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 2
Conc. (µg/ml)
Are
a R
atio
74
Figure 5: Linearity Calibration 3
Table 13: Linearity Calibration 3 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1
122771
305287
1509126
2618433
3275154
7431479
IS area 2
4815662
4816772
4890544
4993543
4748733
4742707
Area ratio 0.025494 0.063138 0.308580 0.524364 0.689690 1.566930
RF
0.1002
0.0896
0.1051
0.1082
0.1301
0.0955 Back calculated conc.
(µg/ml) 0.2603 0.6422 3.1328 5.3224 6.9999 15.9015
± 15% of St true value
0.0375 0.1125 0.45 0.75 1.05 2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.0985494 x - 0.000155154 R²= 0.999127
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 3
Conc.(µg/ml)
Are
a R
atio
75
Figure 6: Linearity Calibration 4
Table 14: Linearity Calibration 4 Data
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 4
Conc.(µg/ml)
Are
a R
atio
Standard solution no. St1 St2 St3 St4 St5 St6
Concentration(µg/ml)
0.25
0.75
3
5
7
16
Pioglitazone area 1 97842 293043 1477999 2550193 3326130 7357360
IS area 2
4728226
4718827
4747801
4721216
4725073
4730326
Area ratio 0.0206932 0.0621008 0.311302 0.540156 0.7039319 1.55536
RF
0.1016
0.0932
0.1021
0.1193
0.0987
0.0988
Back calculated conc.
(µg/ml) 0.2576 0.6662 3.1254 5.3838 6.9999 15.4021
± 15% of St true
value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15% 0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y= 0.101335 x - 0.00541304 R²=0.996554
76
Figure 7: Linearity Calibration 5
Table 15: Linearity Calibration 5 Data
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 5
Conc.(µg/ml)
Are
a R
atio
Standard solution no. St1 St2 St3 St4 St5 St6
Concentration(µg/ml)
0.25
0.75
3
5
7
16
Pioglitazone area 1
131669
315855
1461283
2133438
3268153
7318102
IS area 2 4683446
4716375
4659510
4732606
4709745
4593191
Area ratio 0.028114 0.066969 0.313613 0.450796 0.693913 1.593250
RF
0.1019
0.0841
0.1028
0.1048
0.09753
0.0979
Back calculated conc.
(µg/ml) 0.2593 0.6527 3.1498 4.5386 7.0000 16.10510
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.0987729 x + 0.00250227 R²=0.99955
77
Figure 8: Linearity Calibration 6
Table 16: Linearity Calibration 6 Data
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12 14 16 18
Calibration 6
Conc.(µg/ml)
Pe
ak A
rea
Rat
io
Standard solution no. St1 St2 St3 St4 St5 St6
Concentration(µg/ml)
0.25
0.75
3
5
7
16
Pioglitazone area 1 66663 263646 1360390 2396680 3146246 6958257
IS area 2
4729226
4728827
4719710
4720616
4724973
4729326
Area ratio 0.014096 0.055753 0.288236 0.507705 0.665876 1.471300
RF
0.1014
0.0832
0.1071
0.1093
0.09771
0.09788
Back calculated conc.
(µg/ml) 0.2556 0.6867 3.0923 5.3633 6.9999 15.3342
± 15% of St true
value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15% 0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.0966408 x - 0.0106092 R²=0.996124
78
The following calibration represents the overall linearity performance of analysis
method.
Y= 0.0972x + 0.0099 R² = 0.9991
79
3.1.4 Selectivity and Sensitivity
Selectivity: sample preparation for HPLC analysis by protein precipitation method
was a suitable procedure, six serum blanks chromatograms showed absence of any
endogenous compounds as no corresponding peaks were founded specially at the
expected retention time of both pioglitazone HCl and internal standard.
Evaluation of selectivity was done through the comparison of the 6 serum blanks
chromatograms with the chromatogram of ST1(LLOQ), HPLC detector did not detect
any sensible peaks over the retention time of analyte and IS, as shown in blanks
chromatograms and ST1(LLOQ) chromatogram (figures: 22-30).
Sensitivity: sensitivity tests was carried out through the analysis of 10 repetitions
of each QC samples level, the mean calculated concentrations were within EMEA
guidelines values as all mean concentrations were within ±15% of QCL, QCM and
QCH with CV% values less than 15%, while it was within ±20% of LLOQ with CV%
value less than 20% as shown in tables (17-20), sensitivity test was carried out in
combination of calibration 1 for day one of validation.
80
Table 17: LLOQ Sensitivity Data
Sample ID Pioglitazone
area 1
IS area 2 Area
ratio
Calculated
concentration
(µg/ml)
True
concentration
(µg/ml)
CV%
average
ST(1) (LLOQ)
System
Precision 1 105362 5014991 0.0210
0.218
0.25
9.810
ST(1) (LLOQ)
System
Precision 2
98865
4918648 0.0201
0.209
0.25
ST(1) (LLOQ)
System
Precision 3
97770
4840107 0.0202
0.210
0.25
ST(1) (LLOQ)
System
Precision 4 97016 4861460 0.0200 0.208
0.25
ST(1) (LLOQ)
System
Precision 5 101023 5001690 0.0202 0.210
0.25
ST(1) (LLOQ)
System
Precision 6 120421 4917461 0.0245 0.253
0.25
ST(1) (LLOQ)
System
Precision 7 119278 4861460 0.0245 0.253
0.25
ST(1) (LLOQ)
System
Precision 8 123473 4918648 0.0251 0.259
0.25
ST(1) (LLOQ)
System
Precision 9 120421 4917461 0.0245 0.253
0.25
ST(1) (LLOQ)
System
Precision 10 101287 4936063 0.0205 0.213
0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 - 0.30)
81
Table 18: QCL Sensitivity Data
Sample ID Pioglitazone
area 1
IS area 2 Area
ratio
Calculated
concentration
(µg/ml)
True
concentration
(µg/ml)
CV%
average
QCL System
Precision 1
952025 480712 0.1980 1.974 2.00
0.156
QCL System
Precision 2
928469 467743 0.1985 1.979 2.00
QCL System
Precision 3
961432 483118 0.1990 1.984 2.00
QCL System
Precision 4
924853 466708 0.1982 1.975 2.00
QCL System
Precision 5
949735 478938 0.1983 1.977 2.00
QCL System
Precision 6
926074 466174 0.1987 1.980 2.00
QCL System
Precision 7
979325 493793 0.1983 1.977 2.00
QCL System
Precision 8
923893 464912 0.1987 1.981 2.00
QCL System
Precision 9
970168 489682 0.1981 1.975 2.00
QCL System
Precision 10
928396 467821 0.1985 1.978 2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
82
Table 19: QCM Sensitivity Data
Sample ID Pioglitazone
area 1
IS area 2 Area
ratio
Calculated
concentration
(µg/ml)
True
concentration
(µg/ml)
CV%
average
QCM System
Precision 1
355760 493225 0.7213 7.164 7.00
0.097
QCM System
Precision 2
333112 461405 0.7220 7.170 7.00
QCM System
Precision 3
348471 483036 0.7214 7.165 7.00
QCM System
Precision 4
329734 457162 0.7213 7.163 7.00
QCM System
Precision 5
354823 491815 0.7215 7.165 7.00
QCM System
Precision 6
335025 464364 0.7215 7.166 7.00
QCM System
Precision 7
353060 489046 0.7219 7.170 7.00
QCM System
Precision 8
351195 486447 0.7220 7.170 7.00
QCM System
Precision 9
350355 484204 0.7236 7.186 7.00
QCM System
Precision 10
348780 483639 0.7212 7.162 7.00
QCM ± 15%= ±1.05...... (5.95 - 8.05)
83
Table 20: QCH Sensitivity Data
Sample ID Pioglitazone
area 1
IS area 2 Area
ratio
Calculated
concentration
(µg/ml)
True
concentration
(µg/ml)
CV%
average
QCH System
precision 1
638993 483710 1.3210 13.112 12.80
0.193
QCH System
Precision 2
638853 481211 1.3276 13.177 12.80
QCH System
Precision 3
638023 479911 1.3295 13.196 12.80
QCH System
Precision 4
640465 483803 1.3238 13.140 12.80
QCH System
Precision 5
641094 484469 1.3233 13.135 12.80
QCH System
Precision 6
641045 484439 1.3233 13.134 12.80
QCH System
Precision 7
641602 484834 1.3233 13.135 12.80
QCH System
Precision 8
641287 484118 1.3246 13.148 12.80
QCH System
Precision 9
640413 483040 1.3258 13.159 12.80
QCH System
Precision 10
641897 484962 1.3236 13.138 12.80
QCH ±15% = ±1.92...... (10.88 – 14.74)
84
3.1.5 Recovery
Analyte recovery (pioglitazone HCl)
As indicated in table 21, mean calculated concentration of pioglitazone per QC
level was within ±15% of QCL, QCM and QCH nominated values while it was within
± 20% of LLOQ nominated value which indicated accepted recovery of pioglitazone
in serum. Calibration used for calculation in figure 3 and table 11.
Figure 3: Linearity Calibration 1
Table 11: Linearity Calibration 1 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 120560 296153 1490928 2587688 3378180 7666202
IS area 2 4766532 4689973 4708993 4733321 4793227 4887632
Area ratio 0.025293 0.065514 0.316613 0.546696 0.704782 1.568490
RF
0.1022
0.0842
0.1055
0.1093
0.1001
0.0980
Back calculated conc.
(µg/ml) 0.2606 0.6400 3.1500 5.4321 7.0000 15.5665
± 15% of St true value
0.0375 0.1125 0.45 0.75 1.05 2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.100824 x – 0.000986181 R² = 0.997383
0.0000000
0.2000000
0.4000000
0.6000000
0.8000000
1.0000000
1.2000000
1.4000000
1.6000000
1.8000000
0 2 4 6 8 10 12 14 16 18
Calibration 1
Conc.(µg/ml)
Are
a R
atio
85
Table 21: Pioglitazone HCl Recovery Data
PIOGLITAZONE HCL RECOVERY DATA:
Sample ID Pioglitazone
area 1
IS area
2
Area
ratio
Calculated
conc.
(µg/ml)
True conc.
(µg/ml)
Mean SD CV
%
Error Accuracy
%
ST(LLOQ)
R1 121132 473512 0.0256 0.264
0.25
0.265
0.002
0.69
0.01
106
ST(LLOQ)
R2 122897 475679 0.0258 0.266
0.25
ST(LLOQ)
R3 117911 461679 0.0255 0.263
0.25
ST(LLOQ)
R4 119016 458502 0.0260 0.267
0.25
ST(LLOQ)
R5 117269 457995 0.0256 0.264
0.25
ST1 (LLOQ) ± 20% = ±0.05...... (0.20 – 0.30)
QCL
R1 953036 475684 0.2004 1.997
2.00
1.999
0.00
0.10
0.00
99.95
QCL
R2 957200 476657 0.2008 2.002
2.00
QCL
R 3 917393 457253 0.2006 2.000
2.00
QCL
R 4 918793 458523 0.2004 1.997
2.00
QCL
R5 919888 458289 0.2007 2.001
2.00
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QCM
R1 334098 460008 0.7263 7.213
7.00
7.212
0.01
0.17
0.21
103
QCM
R 2 332098 456309 0.7278 7.228
7.00
QCM
R3 334205 461134 0.7247 7.198
7.00
QCM
R4 328220 452269 0.7257 7.208
7.00
QCM
R5 334098 460008 0.7263 7.213
7.00
QCM ±15% = ±1.05...... (5.95 – 8.05)
QCH
R 1 622801 456138 1.3654 13.552
12.80
13.63
0.12
0.87
0.83
106.5
QCH
R2 625063 458545 1.3631 13.530
12.80
QCH
R 3 626550 450146 1.3919 13.815
12.80
QCH
R4 610466 442942 1.3782 13.679
12.80
QCH
R5
614502
449332
1.3676
13.574
QCH ±15%
= ±1.92
(10.88-
14.74)
12.80
86
Internal standard recovery
Table 22 illustrates acceptable recovery ratios of sildenafil citrate between mobile
phase and serum, QCL showed the highest recovery percentage which equals 97.89%,
while QCH indicated the least recovery of 93.54 %. Figure 3 and table 11 show
calibration that used for calculations.
Table 22: Internal Standard Recovery Data
INTERNAL STANDARD RECOVERY
Sample ID. Internal
standard
in mobile
phase
area 1
Internal
standard in
serum
area 2
Area ratio:
Area 2 /
Area 1
Recovery % Mean
recovery %
ST(LLOQ) 1 501499 473512 0.944193 94.42
94.48
ST(LLOQ) 2 491865 475679 0.967093 96.71
ST(LLOQ) 3 484011 461679 0.953861 95.39
ST(LLOQ) 4 486146 458502 0.943136 94.31
ST(LLOQ)5 500169 457995 0.91568 91.57
QCL 1 480712 475684 0.989541 98.95
97.89 QCL 2 467743 476657 1.019057 101.91
QCL 3 483118 457253 0.946462 94.65
QCL 4 466708 458523 0.982462 98.25
QCL 5 478938 458289 0.956886 95.69
QCM 1 493225 460008 0.932653 93.27
96.02 QCM 2 461405 456309 0.988955 98.90
QCM 3 483036 461134 0.954658 95.47
QCM 4 457162 452269 0.989297 98.93
QCM 5 491815 460008 0.935327 93.53
QCH 1 483710 456138 0.942999 94.30
93.54
QCH 2 481211 458545 0.952898 95.29
QCH 3 479911 450146 0.937978 93.80
QCH 4 483803 442942 0.915542 91.55
QCH 5 484469 449332 0.927473 92.75
87
3.1.6 Stability
Freeze and Thaw Stability
Data obtained after each cycle analysis were used for concentrations calculations
depending on a combined calibration, all results were reasonable and accepted
according to EMEA guidelines as the mean calculated concentrations were within
±15%, QCH showed higher stability percentage at cycle 1 and cycle 2 rather than
QCL as shown in table 23.
Calibrations used for calculation were as follows:
FTS 0: at 0 hours
Figure 3: Linearity Calibration 1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 1
Conc.(µg/ml)
Are
a R
atio
88
Table 11: Linearity Calibration 1 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 120560 296153 1490928 2587688 3378180 7666202
IS area 2 4766532 4689973 4708993 4733321 4793227 4887632
Area ratio 0.025293 0.065514 0.316613 0.546696 0.704782 1.568490
RF 0.1022
0.0842
0.1055
0.1093
0.1001
0.0980
Back calculated conc.
(µg/ml) 0.2606 0.6400 3.1500 5.4321 7.0000 15.5665
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.100824 x – 0.000986181 R²= 0.997383
FTS 1 at 12 hours
Figure 5: Linearity Calibration 3
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 3
Conc.(µg/ml)
Are
a R
atio
89
Table 13: Linearity Calibration 3 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1
122799
122799
1509126
2618433
3275154
7431479
IS area 2
4816772
4816772
4890544
4993543
4748733
4742707
Area ratio 0.025494 0.063138 0.308580 0.524364 0.689690 1.566930
RF 0.1002
0.0896
0.1051
0.1082
0.1301
0.0955
Back calculated conc.
(µg/ml) 0.2603 0.6422 3.1328 5.3224 6.9999 15.9015
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.0985494 x – 0.000155154 R²= 0.999127
FTS2: at 12+24 hours.
Figure 7: Linearity Calibration 5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 5
Conc.(µg/ml)
Are
a R
atio
90
Table 15: Linearity Calibration 5 Data
Standard solution no. St1 St2 St3 St4 St5 St6
Concentration(µg/ml)
0.25
0.75
3
5
7
16
Pioglitazone area 1 131669
315855
1461283
2133438
3268153
7318102
IS area 2 4683446
4716375
4659510
4732606
4709745
4593191
Area ratio 0.028114 0.066969 0.313613 0.450796 0.693913 1.593250
RF
0.1019
0.0841
0.1028
0.1048
0.09753
0.0979
Back calculated conc.
(µg/ml) 0.2593 0.6527 3.1498 4.5386 7.0000 16.10510
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.0987729 x + 0.00250227 R²=0.99955
91
Table 23: Freeze and Thaw Stability Data
QC Low (2 µg/ml)
Time Pioglitazone
area 1
IS area
2
Area
ratio
Measured
conc.
(µg/ml)
Mean Accuracy
%
Stability
%
FTS 0
0 hours
991662 4723889 0.209925 2.10
2.03
105
946089 4729311 0.200048 2.00 100
941299 4728722 0.199060 1.990 99.5
FTS 1
12 hours
924399 4719693 0.19586 1.989
1.99
99.95
98 923389 4714537 0.19586 1.988 99.90
924307 4719222 0.19586 1.989 99.95
FTS 2
12+24
hours
926216 4680693 0.19788 1.978
1.98 99.90
97.5 926046 4677711 0.19797 1.979 99.95
924940 4676612 0.19778 1.977 99.85
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QC High (12.8 µg/ml)
Time Pioglitazone
area 1
IS area
2
Area
ratio
Measured
conc.
(µg/ml)
Mean Accuracy
%
Stability
%
FTS
0 hours
5928455 4716660 1.256918 12.76
12.75
99.66
5926348 4718693 1.255930 12.75 99.58
5922855 4719621 1.254943 12.74 99.53
FTS 1
12 hours
5846681 4703345 1.243090 12.56
12.61
98.13
98.9 5842460 4688773 1.246053 12.59 98.36
5882194 4690913 1.253955 12.67 98.98
FTS 2
12+24
hours
5988118 4723299 1.267783 12.58
12.58
98.28
98.7
5974591 4719982 1.265808 12.56 98.13
5993074 4723527 1.268771 12.59 98.36
QCH ±15 %= ±1.92 (10.88-14.74)
Room Temperature Stability
Room temperature stability of pioglitazone in serum and stock/working solution
Data obtained at zero time and after 8 hours at room temperature of (28C° ± 1)
confirm accepted stability of pioglitazone HCl in both,
92
In serum stability: mean calculated concentrations were within ±15% for QCL and
QCH, which illustrates reasonable stability of both QC levels, QCH showed higher
stability at room temperature rather than QCL as shown in table 24.
In stock/working solutions: as shown in table 25, QCH showed higher stability of
99.10% while QCL represented lower but also accepted stability% of 92.39.
Both stability percentages represented a reasonable stability of pioglitazone in
stock and working solution.
Room temperature stability of internal standard in stock/ working solution
Data obtained at zero time and after 8 hours at room temperature of 28C°±1
confirms accepted stability of pioglitazone HCl, stability % of area ratios between
zero and after 8 hours were accepted as shown in table 26, QCH has obtained larger
level of stability while QCL obtained reasonable stability% value of 98.86. QCL and
QCH concentrations were calculated according to the following calibration:
Figure 3: Linearity Calibration 1
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration 1
Conc.(µg/ml)
Are
a R
atio
93
Table 11: Linearity Calibration 1 Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 120560 296153 1490928 2587688 3378180 7666202
IS area 2 4766532 4689973 4708993 4733321 4793227 4887632
Area ratio 0.025293 0.065514 0.316613 0.546696 0.704782 1.568490
RF 0.1022
0.0842
0.1055
0.1093
0.1001
0.0980
Back calculated conc.
(µg/ml) 0.2606 0.6400 3.1500 5.4321 7.0000 15.5665
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.100824 x – 0.000986181 R²= 0.997383
Table 24: Room Temperature Stability Data for Pioglitazone HCl in Serum
QC Low (2 µg/ml)
Time
(hour)
Pioglitazone
area 1
IS area
2
Area
ratio
Measured
concentration
(µg/ml)
Mean Accuracy% Stability
%
0 hour 1056494 4762522 0.221835 2.21 2.10 110.5
96.7 988363 4689873 0.210744 2.10 105
945077 4709793 0.200662 2.00 100
8 hours 1002921 4736301 0.211752 2.11 2.03 105.5
952528 4795127 0.198645 1.98 99
977852 4897732 0.199654 1.99 99.5
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QC High (12.8 µg/ml)
Time
(hour)
Pioglitazone
area 1
IS area
2
Area
ratio
Measured
concentration
(µg/ml)
Mean Accuracy% Stability%
0 hour 6124878 4753299 1.288553 12.79 12.79 99.9
99.5 6041041 4680913 1.290569 12.81 100.1
6071026 4718893 1.286536 12.77 99.8
8 hour 6068639 4739321 1.280487 12.71 12.73 99.3
6171882 4793527 1.287545 12.78 99.8
6248819 4887732 1.278470 12.69 99.1
QCH ±15% = ±1.92 (10.88-14.74)
94
Table 25: Room Temperature Stability Data for Pioglitazone HCl in
Stock/Working Solution
QC Low (2 µg/ml)+ QC High (12.8 µg/ml)
Sample
ID.
Pioglitazone
area at 0
hours
Pioglitazone
area at
8 hours
Area
time
8/area
time 0
Stability% Stability
average %
QCL1 1077494 969261 0.899551 89.96
92.39 QCL2 989343 879524 0.888998 88.90
QCL3 986037 969332 0.983059 98.31
QCH1 6194888 6118171 0.987616 98.76
99.10 QCH2
6081149 6065551 0.997435 99.74
QCH3 6016102 5941797 0.987649 98.76
Table 26: Room Temperature Stability Data for Internal Standard in
Stock/Working solution
QC Low (2 µg/ml)+ QC High (12.8 µg/ml)
Sample
ID.
IS area at 0
hours
IS area at
8 hours
Area time 8/
area time 0
Stability% Stability
average%
QCL1
4764523
4751442
0.997254
99.73
98.86
QCL2
4787873
4687883
0.979115
97.91
QCL3
4759792
4708790
0.989284
98.93
QCH1
4747302
4738321
0.998108
99.81
99.80
QCH2
4765027
4755167
0.997930 99.79
QCH3
4887562
4877732 0.997988 99.80
95
Long Term Stability
Long term stability of pioglitazone HCl in serum
Data was collected at zero and after 30 days, treated then concentrations were
calculated, results approve reasonable stability of piolgitazone in serum as mean
concentrations were within ±15% for QCL and QCH with accepted stability%, QCH
represented higher stability at long term test of 99.68% while QCL showed lower but
also reasonable stability % of 98.5 as shown in table 28.
Long term stability of pioglitazone HCl in stock/working solution
Data was collected at zero and after 30 days, prepared then analyzed,
concentrations were calculated and results approved reasonable stability of
pioglitazone, QCL showed higher stability percentage of 99.49 while QCH was with
lower but reasonable stability% of 98.12, as shown in table 29.
Long term stability of internal standard in stock/working solution
Area ratios of sildenafil citrate were collected at zero and after 30 days of freezing,
results approved reasonable stability of IS. Sildenafil stability was high at QCH and
QCL as shown in table 30.
Long term stability test was run using the following calibrations simultaneously:
96
Zero Time Calibration:
Figure 3: Long Term Stability Zero Time Calibration (Linearity Calibration 1)
Table 11: Long Term Stability Zero Time Calibration Data (Linearity
Calibration 1)
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 120560 296153 1490928 2587688 3378180 7666202
IS area 2 4766532 4689973 4708993 4733321 4793227 4887632
Area ratio 0.025293 0.065514 0.316613 0.546696 0.704782 1.568490
RF 0.1022
0.0842
0.1055
0.1093
0.1001
0.0980
Back calculated conc.
(µg/ml) 0.2606 0.6400 3.1500 5.4321 7.0000 15.5665
± 15% of St true value 0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y = 0.100824 x – 0.000986181 R²= 0.997383
0.0000000
0.2000000
0.4000000
0.6000000
0.8000000
1.0000000
1.2000000
1.4000000
1.6000000
1.8000000
0 2 4 6 8 10 12 14 16 18
Calibration 1
Conc.(µg/ml)
Are
a R
atio
97
Long Term Stability Day 30 Calibration:
Figure 9: Long Term Stability Day 30 Calibration Curve
Table 27: Long Term Stability Day 30 Calibration Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 112973 341691 1608632 2330399 3317295 7594075
IS area 2 4756542 4688983 4718793 4735351 4793017 4817622
Area ratio 0.023751 0.072871 0.340899 0.492128 0.69211 1.576312
RF
0.1021
0.0942
0.1035
0.1083
0.1221
0.0990
Back calculated conc.
(µg/ml) 0.249 0.745 3.453 4.980 7.000 15.931
± 15% of St true
value
0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4 y= 0.099000x-0.000900000 R²=0.998330
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Calibration at Day 30
Conc.(µg/ml)
Are
a R
atio
98
Table 28: Long Term Stability Data of Pioglitazone in Serum
QC Low (2 µg/ml)
Time
Pioglitazone
area 1
IS area 2 Area
ratio
Measured
concentration(
µg/ml)
Mean Accuracy% Stability
%
0 hour 933004 4710001 0.19809 2.01
1.99 98.72 98.50
916224 4700033 0.19494 1.98 97.25
909413 4684555 0.19413
1.97 97.74
30 days 935210 4721139 0.19809 1.97 1.96 100.50
920955 4719945 0.19512 1.95 98.91
926290 4723321 0.19611 1.95 98.50
QCL ± 15%= ±0.3...... (1.97 – 2.30)
QC High (12.8 µg/ml)
Time
Pioglitazone
area 1
IS area 2 Area
ratio
Measured
concentration
(µg/ml)
Mean Accuracy
%
Stability%
0 hour 5958805 4720444 1.26234 12.53 12.5 97.89
99.68 5934047 4719337 1.25739 12.48 97.51
5915547 4712044 1.25541 12.46 97.35
30 days 5745581 4679993 1.22769 12.41 12.46 96.95
5776574 4690110 1.23165 12.45 97.27
5788297 4673333 1.23858 12.52 97.81
QCH ±15% = ±1.92 (10.88-14.74)
Table 29: Long Term Stability Data of Pioglitazine in Stock/Working
Solution
QC Low (2 µg/ml)+ QC High (12.8 µg/ml)
Sample
ID.
Pioglitazone
area at 0
hours
Pioglitazone
area after
30 days
Area
time
30/area
time 0
Stability% Stability average
%
QCL1 935210 930001 0.99443 99.44 99.49
QCL2 920955 919330 0.998236 99.82
QCL3 926290 918997 0.992127 99.21
QCH1 5958805 5801177 0.973547 97.35 98.12
QCH2 5934047 5812229 0.979471 97.95
QCH3 5915547 5860225 0.990648 99.06
99
Table 30: Long Term Stability Data of Internal Standard in Stock/Working
Solution
QC Low (2 µg/ml)+ QC High (12.8 µg/ml)
Sample
ID.
IS area at 0
hours
IS area after
30 days
Area
time 30/
area
time 0
Stability% Stability
average %
QCL1 4783334 4599833 0.961637 96.16 97.32
QCL2 4779222 4701122 0.983658 98.36
QCL3 4765110 4643301 0.974437 97.44
QCH1 4775990 4599981 0.963147 96.31 97.65
QCH2 4783112 4689297 0.980386 98.03
QCH3 4756633 4690218 0.986037 98.60
3.2 Sucralose –Pioglitazone HCl Combination Effect on Pioglitazone HCl Serum
Levels
Rats serum levels of pioglitazone HCl were calculated through serum samples
analysis that obtained from rats groups per day of trials.
On each day of trials: serum samples of overall samples pooling time intervals
were prepared, then analyzed with combined calibration curve simultaneously.
A calibration was run for each samples group of analysis for samples
concentration calculations.
First day trial samples were analyzed and calculations were obtained using its
combined calibration, as shown in figures 10 and 11, tables 31 and 32.
100
Figure 10: First Day Trials Calibration Curve
Table 31: First Day Trials Calibration Curve Data
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 69194 211054 888665 1282666 2147128 4650648
IS area 2 3434439 3501072 3357176 3018770 3450005 3267527
Area ratio 0.020147 0.060283 0.2647061 0.424897 0.622355 1.423293
RF
0.1051
0.1042
0.1230
0.0993
0.1331
0.0992
Back calculated conc.
(µg/ml) 0.25 0.70 2.99 4.79 6.99 15.98
± 15% of St true
value
0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4
y =0.089190 x- 0.001972 R²=0.997962
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 2 4 6 8 10 12 14 16 18
Tria 1 Calibration
Are
a R
atio
Conc.(µg/ml)
101
Table 32: First Day Trial Serum Data
*NA: Not Available
Figure 11: First Day Trial Serum – Time Profile Curve
Second and third day of trials samples were analyzed at the third day of trials
using the same calibration of day 30 long term stability test, as shown in figures 9,12
and 13, table 27, 33 and 34.
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
0.00 2.00 4.00 6.00 8.00 Co
nce
ntr
atio
n (
µg
/ml)
Time (hr)
Day -1- trials
PG alone
PG+Sucralose
Day-1- trials
PG alone, N=1
PG+Sucralose, N=1
Serum concentration(µg/ml)
Group no. 30 min 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 24 hr
Group 1
(PG alone)
5.536
10.192 13.013 15.139 10.359 8.541 *NA *NA
Group 2
(PG+Sucralose)
5.726 10.316 11.987 12.982 12.931 9.054 *NA *NA
102
Data was obtained, and then a statistical analysis was carried out to evaluate it in
order to determine acceptance or rejection of intended proposal, as shown in table 35.
Figure 9: Second and Third Day Trials Calibration Curve (Long Term Stability
at Day 30)
Table 27: Second and Third Day Trials Calibration Curve Data(Long Term
Stability at day 30)
Sample ID St1 St2 St3 St4 St5 St6
Concentration(µg/ml) 0.25 0.75 3 5 7 16
Pioglitazone area 1 112973 341691 1608632 2330399 3317295 7594075
IS area 2 4756542 4688983 4718793 4735351 4793017 4817622
Area ratio 0.023751 0.072871 0.340899 0.492128 0.69211 1.576312
RF 0.1021
0.0942
0.1035
0.1083
0.1221
0.0990
Back calculated conc.
(µg/ml) 0.249 0.745 3.453 4.980 7.000 15.931
± 15% of St true
value
0.0375
0.1125
0.45
0.75
1.05
2.4
-15%
+15%
0.2125
0.2875
0.6375
0.8625
2.55
3.45
4.25
5.75
5.95
8.05
13.6
18.4 y= 0.099000x-0.000900000 R²=0.998330
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 2 4 6 8 10 12 14 16 18
Trials 2+3 Calibration
Conc.(µg/ml)
Are
a R
atio
103
Table 33: Second Day Trial Serum Data
Figure 12: Second Day Trial Serum –Time Profile Curve
Table 34: Third Day Trial Serum Data
0.000
5.000
10.000
15.000
20.000
0.00 5.00 10.00 15.00 20.00 25.00 30.00
Co
nce
ntr
atio
n a
vera
ge
(µg/
ml)
Time ( hr)
Day -2- trials
PG alone
PG+ Sucralose
Day-2- trials
PG alone, N=2
PG+Sucralose, N=3
Serum concentration(µg/ml)
Group no. 30 min 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 24 hr
Group 1
(PG alone)
5.591
10.294
13.143
15.290
13.493
8.626
5.858
0.556
Group 2
(PG alone)
5.656
10.605
13.484
15.352
13.585
8.989
6.070
0.596
Group 3
(PG+Sucralose)
5.736
10.666
12.241
13.130
12.928
8.434
5.045
0.303
Group 4
(PG+Sucralose)
5.783
10.419
12.107
13.112
12.050
8.282
4.949
0.242
104
Day-3- trials
PG alone, N=1
PG+Sucralose, N=3
Serum concentration(µg/ml)
Group no. 30 min 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 24 hr
Group 1
(PG alone)
5.681
10.671
13.256
15.281
14.095
8.807
5.689
*NA
Group 2
(PG+Sucralose)
5.882
10.544
12.325
12.923
11.713
8.707
5.010
*NA
Group 3
(PG+Sucralose)
5.690
10.581
12.144
13.026
12.826
8.367
5.005
*NA
Group 4
(PG+ Sucralose)
5.737
10.337
12.011
13.008
12.957
8.216
4.910
*NA
Figure 13: Third Day Trial Serum – Time Profile Curve
0.000
5.000
10.000
15.000
20.000
0.00 2.00 4.00 6.00 8.00 10.00
Co
nce
ntr
atio
n (
µg
/ml)
Time ( hr)
Day-3- trials
PG alone
PG+Sucralose
105
Table 35: Serum Data Statistical Analysis Results
[ Serum Concentrations Statistical Results for all Samples (Without Sucralose)](µg/ml)
Time 30 min 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 24 hr
N 4 4 4 4 4 4 3 2
Range (5.54-
5.60)
(10.19-
10.67)
(13.01-
13.48)
(15.14-
15.35)
(10.36-
14.10)
(8.54-
8.99)
(5.69-
6.07)
(0.56-
0.60)
Mean 5.62 10.44 13.22 15.27 12.88 8.74 5.87 0.58
Std. Error 0.03 0.12 0.10 0.05 0.85 0.10 0.11 0.02
Std.
Deviation 0.066 0.23 0.20 0.09 1.70 0.2 0.19 0.03
[ Serum Concentrations Statistical Results for all Samples (With Sucralose)](µg/ml)
Time 30 min 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 24 hr
N 6 6 6 6 6 6 5 2
Range (5.69-
5.88)
(10.32-
10.67)
(11.99-
12.33)
(12.92-
13.13)
(11.71-
12.96)
(8.22-
9.05)
(4.91-
5.05)
(0.24-
0.30)
Mean 5.76 10.48 12.14 13.03 12.57 8.51 4.98 0.27
Std. Error 0.03 0.06 0.05 0.03 0.22 0.13 0.02 0.03
Std.
Deviation 0.07 0.14 0.13 0.08 0.54 0.32 0.05 0.04
Concentrations Comparison [ Without Sucralose vs. With Sucralose ] (µg/ml)
P-value
(t-Test)
0.024 E-4 5.48 E-4 0.67 E-4 0.01 E-4 4.86 E-4 1.01 E-4 3.83 E-4
*NA
Means
Difference
-0.14 -0.04 1.09 2.24 0.32 0.23 0.89 0.30
Effect Size
[Cohen’s
d] -2.16 -0.19 6.45 26.43 0.25 0.87 6.34 8.32
percentage
change
between
means % -0.026 -0.004 0.08 0.15 0.024 0.03 0.15 0.53
* NA: Not Available
106
After statistical analysis, data was analyzed and assessed as follow: serum data
after 30 minutes of administration showed a strong significant result as P value was
less than 0.05, while a very small Cohen’s d value was gotten which indicated small
effect size of combination of sucralose on pioglitazone HCl in serum with reduction
percentage of mean concentrations between pioglitazone HCl alone and combined
with sucralose groups equals -2.5%, results represented a medium worthy
combination effect, as shown in figure 14 and table 35.
Figure 14: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 30 minutes of of drug administration.
107
Serum data at 1 hour showed a strong significant result as P value was less than
0.05 with a small Cohen’s d value which indicated small combination effect size of
sucralose on pioglitazone HCl in serum with reduction percentage of mean
concentrations between pioglitazone HCl alone and combined with sucralose groups
equals -0.35%, results showed the absence of any worthy combination effect.
See figure 15 and table 35.
Figure 15: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 1 hour of of drug administration.
108
Serum data at 2 hours showed also a very strong significant result as P value was
less than 0.05 while Cohen’s d value indicated large effect size of combination of
sucralose on pioglitazone HCl in serum, large effect size of Cohen’s d value indicates
the necessity of larger samples size to get more rational P value correlated with
founded large size effect. Reduction percentage of mean concentrations between
pioglitazone HCl alone and combined with sucralose groups equals 8.2%. Results
evaluation represented strong mentionable combination effect, as shown in figure 16
and table 35.
Figure 16: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 2 hours of of drug administration.
109
Serum data at 3 hours showed a significant result as P value was P < 0.05, Cohen’s
d value indicated large effect size of combination of sucralose on pioglitazone HCl in
serum, with a reduction percentage of mean concentrations between pioglitazone HCl
alone and combined with sucralose groups equals 14.64 % which indicated strong
effect of sucralose combination on pioglitazone serum levels, the greatest effect of
combination was obtained at 3 hours, as shown in figure 17 and table 35.
Figure 17: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 3 hours of of drug administration
110
Serum data at 4 hours showed a significant result as P value was less than 0.05
with a Cohen’s d value indication of moderate to small effect size of combination,
with a reduction percentage of mean concentrations between equals 2.44 %, P value
and Cohen’s d values correlation indicates the need of larger samples size to
rationalize between obtained values, as shown in figure 18 and table 35.
Figure 18: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 4 hours of of drug administration.
111
Serum data at 6 hours showed a significant result as P value was less than 0.05
with a Cohen’s d value indication of large to moderate effect size of combination in
serum, which emphasizes the need of larger samples size for obtaining more rational
P value. Reduction percentage of mean concentrations between solitary use of
pioglitazone and combination equals 2.63%, result represented combination effect to
be taken in consideration, as shown in figure 19 and table 35.
Figure 19: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 6 hours of of drug administration.
112
Serum data at 8 hours showed a significant result as P value was ˂ 0.05 with
Cohen’s d value of large effect size (larger sample size is needed).Reduction
percentage of mean concentrations between solitary and combined use equals 15.13%.
Data analysis indicated strong effect of combination, as shown in figure 20 and table
35.
Figure 20: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 8 hours of of drug administration.
113
Serum data at 24 hours showed a Cohen’s d value of large effect size of
combination, with percentage reduction of mean concentrations equals 52.69 %.
Cohen’s d value represented strong combination effect while P value was not
calculated as number of samples was too small, as shown in figure 21 and table 35.
Figure 21: Dot diagram with error bars for mean comparison of pioglitazone
HCl serum concentration (µg/ml) within 95% of confidence interval between PG
(Pioglitazone HCl alone) and PG plus (Pioglitazone HCl combined with
Sucralose) after 24 hours of of drug administration.
114
Cmax showed a significant serum level reduction with P-value = 0.008 E-4 which
indicates statistically significant decrease in pioglitazone Cmax serum concentration.
Cmax showed a value of 15.27 µg/ml with pioglitazone alone administration while it
was reduced to be 13.03 µg/ml when it is combined with sucralose. Effect size of
serum concentration differences was considered as strong due to large Cohen’s d
value, as shown in figure 22 and table 36.
Figure 22: Serum Concentration – Time Profile Graph (0-24) hours of Oral
Administration of Drug,(PG = Pioglitazone HCl alone, PG Plus = Pioglitazone
HCl with Sucralose).
0.000
2.000
4.000
6.000
8.000
10.000
12.000
14.000
16.000
18.000
20.000
0 5 10 15 20 25 30
Ser
um
con
cen
tra
tio
n (
µg
/ml
time (hr)
PG
PG Plus
115
AUC of pioglitazone in serum showed a strong significant P –value equals 0.001,
while difference between AUCs in presence and absence of sucralose was with
reduction of (12.07), as shown in table 36.
Table 36: Serum Concentration – Time Profile Kinetic Parameters
Statistical analysis results showed a statistically significant combination interaction
between pioglitazone and sucralose when both compounds are given concurrently, as
P values represented a strong combination effect, this combination interaction could
be justified according to sucralose induction effect on CYP 450 enzymes, specifically
3A4 subtype by which pioglitazone is extensively metabolized.
Pioglitazone is basically absorbed in stomach where CYP3A4 isoenzymes are
located profusely in parietal cells endoplasmic reticulum, this enzyme will exert its
metabolic biotransformational effect over pioglitazone once absorbed.
On the other hand and after pioglitazone absorption, drug will be metabolized via
another pathway as it will be extensively bounded to plasma proteins then distributed
through the circulatory system to reach the liver where most of metabolic reactions
occur.
Drug C max (µg/ml)
T max (hr)
T ½ (hr)
AUC (µg/ml*hr)
PG 15.27 ±0.03 3 3.6 130.59 ±2.10
PG Plus Sucralose 13.03 ± 0.1 3 3.6 118.52 ±2.90
P value 0.008 P values ˂ 0.05 0.001
116
Induction effect of sucralose over the CYP3A4 metabolic enzyme will also
activate the metabolism of pioglitazone in liver too, which results in pioglitazone
plasma/serum levels reduction and over production of pioglitazone active and inactive
metabolites.
As mentioned before that the active metabolites of pioglitazone have stronger
pharmacological effect than pioglitazone in most cases, thus precise detection and
quantification of pioglitazone active metabolites should be performed to investigate
the further clinical complications that could occur due to high levels of pioglitazone
active metabolites production.
In rats, CYP3A1, 3A2, 3A9, 3A18, 3A23 and 3A62 have been reported as CYP3A
forms. CYP3A23 was classified as identical to CYP3A1 by the analysis of its gene.
CYP3A62 form has been identified as a new rat CYP3A isoenzyme with
expression profile similar to human CYP3A4 and rat CYP3A9. CYP3A62 is a
predominant form in the intestinal tract, where CYP3A1 and -3A2 were found only in
liver (Matsubara et al., 2004).
As recent studies illustrated a distinctive binding affinity variance of pioglitazone
to reactive sites of CYP3A enzymes during metabolic reaction and which justifies its
variable bioavailability between humans and rats, this variance impresses a possible
perceptible clinical differences in pioglitazone human plasma levels when combined
with sucralose, which strongly recommends further clinical research in humans.
117
CHAPTER FOUR
CONCLUSION
118
4. Conclusion
A successful HPLC method was validated and developed to quantify pioglitazone
HCl in rats serum, the method was precise and accurate with rational linearity
performance and reasonable sensitivity and selectivity.
Concerning stability and recovery tests, all obtained results were reasonable and
accepted according to EMEA guidelines.
Combination effect of pioglitazone with sucralose over all time intervals of
pioglitazone serum profile was demonstrated as strong statistical effect according to
Cohen’s d and significant P values too.
Cmax showed a significant change between presence and absence of sucralose
while Tmax didn’t show any change, which suggests the possibility of interaction
between pioglitazone HCl and sucralose during combination.
Advanced clinical research on human volunteers to make more precise results
concerning pioglitazone HCl – sucralose combination interaction is suggested
through the detection and quantification of pioglitazone HCl and its active metabolites
as these metabolites are also pharmacologically active in human body of diabetic
patient ( Tanis et al., 1996 ; Scheen, 2007).
119
5. Appendix: Chromatograms
Figure 23: Serum Blank Chromatogram with IS
120
Figure 24: Serum Blank Chromatogram 1
121
Figure 25: Serum Blank Chromatogram 2
122
Figure 26: Serum Blank Chromatogram 3
123
Figure 27: Serum Blank Chromatogram 4
124
Figure 28: Serum Blank Chromatogram 5
125
Figure 29: Serum Blank Chromatogram 6
126
Figure 30: Piolitazone LLOQ Chromatogram (Peak 1 for Pioglitazone HCl, Peak
2 for IS)
127
Figure 31: Pioglitazone HCl QCL Chromatogram (Peak 1: Pioglitazone HCl,
Peak 2: IS)
128
Figure 32: Pioglitazone HCl QCM Chromatogram (Peak 1: Pioglitazone HCl,
Peak 2: IS)
129
Figure 33: Pioglitazone HCl QCH Chromatogram (Peak 1: Pioglitazone HCl,
Peak 2: IS)
130
Figure 34: Pioglitazone HCl Zero Concentration with IS Chromatogram (IS:
Peak 2)
131
Figure 35: Pioglitazone HCl Unknown Concentration Chromatogram after 30
minutes Oral Administration (Pioglitazone HCl: Peak 1, IS: Peak 2)
132
Figure 36: Pioglitazone HCl Unknown Concentration Chromatogram after 3
hours of Oral Administration (Peak 1: Pioglitazone HCl, Peak 2: IS)
133
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الملخص
والتيسبقاعطاؤهاالفئرانبيوغليتازونهيدروكلورايدفيمصلدراسةتحليليةذاتمصداقيةلمعايرةال
باستخدامجهازالاستشرابالمائيعاليالاداءوالطيفللضوئيللاشعةفوقالبنفسجيةالسكرالوز
اعداد
لينةناصرعبدالخالقالتميمي
المشرفالمشاركالمشرف
الدكتوروائلابوديةالاستاذالدكتورتوفيقعرفات
باستخدامجهازوزلسةلتحديدالبيوغليتازونبوجودالسكرااقدتمذلكباستخدامطريقةبسيطةوسريعةوحس
من%5..5ناتيرايلواسيتو%5..5)تماستخدامخليطمن.يالاداءوالطيفالكتليلالاستشرابالمائيعا
مايكروميترومعدل5بقطرحوالي.العمودالفاصلنوعكربون,(تاتيمحلولامونيوماسمليمولار0.0.5
.مايكروليتروتماستخدامالسيلدينافيلكمعيارداخلي00وحجمالحقنللعيناتكان(دقيقةلمللك.)تدفق
1..5.يعادلكيزللبيوغليتازونوحدهفيالمصلفقدكاناعلىتر,اعتماداعلىالنتيجةالتيتمالحصولعليها
علىعالبيوغليتازونليصبحاهذاالمستوىعنداقترانوجودالسكرالوزمبينماانخفض,ميكروغراملكلمل
".ملحوظاحصائياميكروغراملكلملوقداعتبرهذاالتاثير1.01.يساويتركيزللبيوغليتازونفيالمصل
لمستوياتالبيوغليتازونفيودقةقياساتالتحاليل%(5.معدلمعاملالتبايناقلمن)كانتدقةالقياساتعالية
تيتكفلصحةطريقةالتحليلاستناداالىالمعاييرالاوروبيةالمعتمدةلقبولضمنالمعاييرالمقبولةالالمصل
ازونفقدكانتمطابقةللشروطكونهاالمنحنياتالقياسيةللبيوغليتأما,طرقالتحليلالبيولوجيةللمحليلالحيوية
..0.0تزيدعن
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