METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS

ESTIMATION OF LOBEGLITAZONE SULPHATE AND GLIMEPIRIDE BY HPLC

METHOD

A THESIS SUBMITTED
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE DEGREE OF
MASTER OF PHARMACY
IN
PHARMACEUTICAL QUALITY ASSURANCE

TO
PARUL UNIVERSITY

BY
SHIKHA MISHRA
(2208422130007)

Supervised by
Mrs. MANISHA JADAV
M.PHARM
Assistant Professor
Co-Supervised by
Mrs. Minal Salunkhe
M.PHARM
Assistant Professor

SCHOOL OF PHARMACY
FACULTY OF PHARMACY
PARUL UNIVERSITY, VADODARA
MAY, 2024

I
 / 05 / 2024

CERTIFICATE

It is certified that the work contained in the thesis titled “ METHOD DEVELOPMENT AND
VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF LOBEGLITAZONE SULPHATE
AND GLIMEPIRIDE BY HPLC METHOD,” BY “SHIKHA MISHRA (2208422130007)” has been
carried out under my/our supervision and that this work has not been submitted elsewhere for a degree.

Signature of Supervisor Signature of Co-Supervisor

Mrs. Manisha Jadav Mrs.Minal Salunkhe

School of Pharmacy School of Pharmacy
Faculty of Pharmacy Faculty of Pharmacy
Parul University. Parul University.

Signature of HOD Signature of Principal

Dr. Shital V. Patel Dr. Lalit Lata Jha

School of Pharmacy School of Pharmacy
Faculty of Pharmacy Faculty of Pharmacy
Parul University. Parul University.

Seal of Institute

DECLARATION OF ORIGINALITY

II
I hereby declare that this work is my original work and further confirm that:

 This work has been composed by me with the assistance of my Supervisor.

 I have clearly referenced in accordance with the university requirements, in both the text
and the bibliography or references, all sources (either from a printed source, internet or any
other source) used in the work.

 All data and findings in the work have not been falsified or embellished.

 This work has not been previously or concurrently used either for other courses or within
other exam processes as an exam work.

 This work has not been published.

I understand that any false claim in respect of this work will result in disciplinary action in
accordance with university regulations.

I confirm and agree that my work may be electronically checked for plagiarism by the use of
plagiarism detection software and stored on a third party’s server for eventual future comparison.

Signature of Student with Date:

Name of Student: SHIKHA MISHRA
Enrolment No.: 2208422130007
Name of Institute: School of Pharmacy

THESIS APPROVAL

Student: SHIKHA MISHRA Enrolment No.: 2208422130007

III
In partial fulfilment of the requirements for the degree of: MASTER OF PHARMACY in
QUALITY ASSURANCE. Thesis Title: METHOD DEVELOPMENT AND VALIDATION
FOR THE SIMULTANEOUS ESTIMATION OF LOBEGLITAZONE SULPHATE AND
GLIMEPIRIDE BY HPLC METHOD.

As research supervisor for the above student, I hereby certify that I have read the student’s defended
thesis, titled above and have also received the plagiarism report which was found to be within the
prescribed limit. Hence, I recommended the thesis to the Faculty of Pharmacy, Parul University for
the partial fulfilment of Degree in Master of Pharmacy.

___________________ ________________

Name of Supervisor Signature of Supervisor Date

___________________ ________________

Name of Co-Supervisor Signature of Co-Supervisor Date

The undersigned hereby certify that they recommend the thesis to the Faculty of Pharmacy, Parul
University for the partial fulfilment of the Degree in Master of Pharmacy.

________________ ___________________ ________________

Name of Examiner Signature of Examiner Date

________________ ___________________ ________________

Name of Examiner Signature of Examiner Date

PLAGIARISM REPORT

IV
V
DEDICATED TO GOD & MY
FAMILY

VI
 ACKNOWLEDGEMENT

A little ‘thank you’ that you will say to someone for a ‘little favour’ shown to you is a key to
unlock the doors that hide unseen ‘greater favours’.

First and foremost, praises and thanks to the God, the almighty, for his showers of blessings
throughout my research work to complete the research work successfully.
I would like to extend my warmest thanks and deepest gratitude to my supervisor, mentor, and
guide, Mrs. Manisha Jadav, Assistant Professor at the School of Pharmacy, Parul University.
Her consistent support, invaluable guidance, and expertise have been essential throughout my
research and the entire thesis process. Her dynamism, vision, sincerity, and motivation have
profoundly inspired me. She has taught me the methodology for conducting and presenting
research as clearly as possible. It has been a great privilege and honor to work and study under
her guidance, and I am extremely grateful for all she has offered me during the preparation of
this thesis.
I would also like to thank my co-supervisor, Mrs. Minal Salunkhe, Assistant Professor at the
School of Pharmacy, Parul University, for her guidance, help, and support.. I am also deeply
grateful to Dr. Shital V. Patel, Head of the Department of Quality Assurance at the School of
Pharmacy, for her guidance in completing this thesis.

I wish to express my sincere and heartfelt gratitude to our respected Principal, Dr. Lalit Lata
Jha of the School of Pharmacy, for providing me with an excellent platform, kind cooperation,
and valuable suggestions that guided my project work in the right direction.

I am sincerely grateful to Dr. L.D. Patel, PG guide of the Faculty of Pharmacy, for his valuable
suggestions, ideas, profound knowledge, and insightful remarks.

I would like to express my gratitude to the non-teaching staff of the School of Pharmacy,
especially Mr. Dinesh Patel (Store Incharge), Mr. Brijesh dhave (Lab Assistant), and Mrs.
Arpita Pandit (Librarian), for providing all the necessary requirements and facilities for my
project work.
I would like to extend my deepest gratitude to Akums Drugs and Pharmaceuticals Ltd,
located in Haridwar, Uttarakhand, India, for generously providing me with a gift sample of
Lobeglitazone sulfate for my project work.

VII
I extend my sincere gratitude to the Centre for Research and Development at Parul
University for providing the necessary facilities that enabled me to conduct my research work
and complete my tasks on time. I would also like to thank Mr. Tejas Bhatt for his consistent
support and dedication.
No words can adequately express my profound love and gratitude to my parents for their
unwavering love, prayers, care, and sacrifices in educating and preparing me for the future. I
am also deeply thankful to my brothers and sister for their invaluable support. Additionally, I
extend my heartfelt thanks to my friends for their keen interest and support, which greatly
contributed to the successful completion of this thesis.

I am grateful to all those who have directly or indirectly offered their support towards the
completion of this thesis.

Thankful I ever remain………...

SHIKHA MISHRA

VIII
 TABLE OF CONTENTS

Sr.no Particulars Page no.

1. Title Page I
2. Certificate II
3. Declaration of Originality III
4. Thesis Approval IV
5. Plagiarism Report V
6. Dedication VI
7. Acknowledgement VII
8. Table of Contents IX
9. List of Tables XI
10. List of Figures XII
11. List of Abbreviations XIII
12. Abstract XVII
Chapter 1 INTRODUCTION
1-13
1.1 Introduction to Diabetes 1
1.1.1 Categorization of diabetes 1
mellitus
1.1.2 Pathophysiology 2
1.1.3 Cause of diabetes 8
1.1.4 Some common sign and 9
symptoms
1.1.5 Diagnosis of diabetes 10
mellitus
1.1.6 Treatment of diabetes 11
mellitus
1.1.7 Prevention 14
1.1.8 Treatment of diabetes 15
mellitus
1.2 High performance liquid 15-35
chromatography
1.2.1 Introduction of Hplc 15
1.2.2 Principle of Hplc 16
1.2.3 Instrumentation 17
1.2.4 The Factor which influence 30

IX
 the Hplc performance

1.2.5 Parameter used in Hplc 31

1.2.6 Advantages and 32
disadvantages of Hplc
1.2.7 Application of Hplc 33
1.3 Method development on Hplc 36-43
1.3.1 Method development steps 36
1.3.2 Component of method 42
development
1.4 Drug profile 43-46
1.4.1 Drug profile of 44
lobeglitazone sulfate
1.4.2 Drug profile of Glimepiride 44
1.4.3 Marketed formulation 45

Chapter 2 LITERATURE REVIEW AND PSAR 47-53

SUMMARY
2.1 Review of literature 47

2.1.1 Patent search for 54

glimepiride
2.1.2 Patent search for 57
lobeglitazone sulfate
Chapter 3 AIM, OBJECTIVES AND RATIONALE 59

Chapter 4 MATERIALS & METHODS 60-61

4.1 List of Materials 60

4.2 List of Equipment 61

Chapter 5 RESULTS AND DISCUSSION 62-71

5.1 Method validation 62

5.2 Result 63

5.2.1 Solubility 63

5.2.2 Study of IR identification 63

5.2.3 Selection of wavelength 65

5.2 HPLC Method Development and 66
validation
5.2.1 Linearity and range 66

X
 5.2.2 System suitability test 68

5.2.3 Repeatability 69

5.2.4 Precision 69

5.2.5 Accuracy 70

5.2.6 LOD and LOQ 72

5.2.7 Robustness 72

Chapter 6 SUMMARY AND CONCLUSION 72-73

6.1 Summary 72

6.2 Conclusion 73

75-84
Chapter 7 REFERENCE
85
Appendix A Comment Sheet
Appendix B Proof of Paper Acceptance / Publication and of 86
Poster/Oral presentation and Any special Activity

List of Tables
Table Title Page
No. No.

1.1 Treatment of diabetes mellitus 15

1.2 The overall study of technique 39

1.3 Table of different detectors & type of compounds 39
detected by them
1.4 List of Marketed Formulation 45

1.5 Patent Search Analysis Report Summary 54-58

1.6 List of Materials 60

1.7 List of Equipment 61
1.8 Solubility 63
1.9 Linearity and range 67

1.10 System suitability parameters 68

1.11 Repeatability data 69

XI
 1.12 Intraday and Interday precision data 70

1.13 Accuracy data of lobeglitazone sulfate 71

1.14 LOD and LOQ 72

1.15 Robustness data 72

1.16 Summary of optimization chromatographic condition 72

1.16 Summary of validation parameters of HPLC 72-73

List of Figures

Figure Title Page

No. No.
1.1 Pathophysiology of T2DM – ominous octet. 2
1.2 Targets of treatment for T2DM [TZDs – 4
Thiazolidinediones, DPP – 4i – Dipeptidyl peptide – 4
inhibitor, GLP-1RA – Glucagon like peptide – 1
receptor agonist, SGLT-2i - Sodium–Glucose co-
transporter 2 inhibitor] sodium–glucose co-transporter
2 inhibitor].

1.3 Schematic Mechanism of action of Peroxisome 5

Proliferator Activated Receptor (PPAR) agonists

1.4 Schematic mechanism of α – glucosidase inhibitor 5

to lower the blood glucose level.

1.5 Pathophysiology of Type I and Type II diabetes. 7

Abbreviations: Aβ- Amyloid- β, GSK-3β-glycogen
synthase kinase 3β, LTP- long term potentiation, P-
Phosphate

1.6 flow diagram of Hplc 17

1.7 component of Hplc 18

XII
 1.7 Manual injection 20
1.8 Guard column 22
1.9 Typical Hplc column. 22
1.8 Evaporative light scattering detectors (ELSDS 23
1.9 UV detector 24
1.10 Fluorescence detectors 25
1.11 Electrochemical detectors 25
1.12 Mass spectrometry 26
1.13 SEC separation 29
1.14 Steps involved in Hplc method development 36
1.15 Typical chromatogram of lobeglitazone sulfate and 64
glimepiride
1.16 FTIR of lobeglitazone sulfate 64
1.17 65
FTIR of Glimepiride
1.18 66
UV spectra of lobeglitazone sulphate
1.19 UV spectra of glimepiride 66
1.20 calibration Curve of lobeglitazone sulfate 68
1.21 68
calibration Curve of Glimepiride

List of Abbreviations
Abbreviation Expanded form
DM Diabetes Mellitus
PPARγ Peroxisome proliferator activated receptor gamma
DPP Dipeptidyl peptidase
ADA American Diabetes Association
DNA Deoxyribonucleic acid
FDA Food and Drug Administration
HDL High density Lipoprotein
HbA1C Glycated Haemoglobin
TZD Thiazolidinediones
TG Triglyceride
FPG Fasting Plasma Glucose
COPD Chronic Obstructive Pulmonary Disease
T2DM Type 2 Diabetes mellitus
GLP-1RA Glucagon like peptide – 1 receptor agonist
SGLT-2i Sodium–Glucose co-transporter 2 inhibitor
DPP -4i Dipeptidyl peptide – 4 inhibitor
Pcos polycystic ovary syndrome
XIII
IDDM Insulin-dependent diabetes mellitus
NIDDM Non-insulin-dependent diabetes mellitus
GDM Gestational diabetes mellitus
MODY maturity onset diabetes of the young
PAH polyaromatic hydrocarbons
UV Ultraviolet
Vis Visible
SEC Size exclusion chromatography
RS Resolution
GC Gas chromatography
TNT trinitrotoluene
EMR Electromagnetic radiation
HPLC High Performance Liquid Chromatography
ELSDS Evaporative light scattering detectors
LC Liquid chromatography
DMSO Dimethyl sulfoxide
FTIR Fourier-transform infrared radiation
IR Infrared radiation
°C Degree Centigrade
ml/min Millilitre per minute (s)
µg/ml Micrograms per millilitre (s)
ppm Parts per million
µL Microliter (s)
mg Microgram (s)
nm Nanometer (s)
LOD Limit of detection
LOQ Limit of Quantification
% RSD Percentage relative standard deviation
Min Minute (s)
ICH International Conference for Harmonisation
ng Nanogram (s)
cm Centimetre (s)
Conc. Concentration
Fig. Figure
API Active Pharmaceutical Ingredient

XIV
 “METHOD DEVELOPMENT AND VALIDATION FOR THE
SIMULTANEOUS ESTIMATION OF LOBEGLITAZONE SULPHATE AND
GLIMEPIRIDE BY HPLC METHOD.”

BY

Ms. Shikha Mishra

UNDER THE GUIDANCE OF

Mrs. Manisha Jadav

AT

School of Pharmacy, Parul University

ABSTRACT

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-

HPLC) method was developed for the simultaneous estimation of lobeglitazone sulfate and
glimepiride in bulk and tablet dosage forms. The method involved selecting various
chromatographic parameters. Specifically, a new method was developed using a 250 mm × 4.6
mm (HSS) reverse-phase C18 column with 5 µm particle size (Shim-pack C18, 250 × 4.6 mm;
5 µm). The mobile phase consisted of 40 volumes of phosphate buffer (pH 3) and 60 volumes
of acetonitrile, with methanol as the diluent, running as an isocratic elution. The flow rate was
set at 1.0 mL/min, and UV detection was performed at 254 nm. The injection volume was 2
µL, and the total runtime was 10 minutes. The recoveries for lobeglitazone sulfate were found
to be in the range of 101% to 100.6%, while those for glimepiride were in the range of 101.5%
to 100%. The method’s accuracy is evident from these results. The inter-day and intra-day
precision of the new method were both below the maximum allowable limit (RSD% ≤ 2.0), as
per International Council on Harmonisation (Q2 R1) guidelines. The method exhibited a linear
response, with correlation coefficient (r2) values of 0.9972% for pioglitazone and 0.998 for
glimepiride. The validation parameters, such as accuracy, precision, linearity, specificity,
stability in the analytical solution, and robustness, met the acceptance criteria. Hence, this

XV
method can be effectively used for the simultaneous estimation of lobeglitazone sulfate and
glimepiride in both bulk and pharmaceutical dosage forms.

KEYWORDS: Reverse Phase High Performance Liquid Chromatography, Lobeglitazone

sulphate, Glimepiride, Method Development, Analytical Method Validation.

XVI
CHAPTER 1
INTRODUCTION
CHAPTER 1 INTRODUCTION

1.1 INTRODUCTION TO DIABETIES MELLITUS

Diabetes is the most prevalent disease. The presence of sugar in the urine of diabetics was first demonstrated
by Dobson in 1755. It is a group of metabolic disorders characterized by high blood sugar levels. Diabetes
mellitus (DM), often referred to as "sugar," is the most common endocrine disorder. It typically occurs due
to a deficiency or absence of insulin, or, less commonly, an impairment of insulin activity (insulin
resistance).[2] Insulin and glucagon are both hormones secreted by the pancreas. Insulin is produced by the
beta (ß) cells, while glucagon is produced by the alpha (α) cells, both located in the islets of Langerhans.
Insulin decreases blood glucose levels by promoting glycogenesis and transporting glucose into muscles,
the liver, and adipose tissue. Neural tissue and erythrocytes do not require insulin for glucose utilization.
In contrast, alpha (α) cells play an important role in regulating blood glucose levels by producing glucagon,
which increases blood glucose levels by accelerating glycogenolysis. [3,4].

The various complications associated with diabetes mellitus (DM) include nephropathy, neuropathy,
cardiovascular and renal complications, retinopathy, foot-related disorders, and more. There are two types
of DM: Type 1 and Type 2. Type 1 DM is an autoimmune disorder that affects pancreatic cells, reducing
or impairing insulin production. Type 2 DM results from the impairment of pancreatic beta cells, which
hinders the individual's ability to use insulin effectively.[5].

The primary conventional drug classes for treating hyperglycemia include: Sulfonylureas (Stimulate insulin
release from pancreatic islets), Biguanides (glucose Decrease hepatic production), Peroxisome proliferator-
activated receptor-γ (PPARγ) agonists (Enhance insulin action.) α-Glucosidase inhibitors (Inhibit glucose
[6]
absorption in the gut). These drugs can be used as monotherapy or in combination with other
hypoglycemic agents. However, they have notable drawbacks, including severe hypoglycemia, weight gain,
and reduced therapeutic efficacy due to improper or ineffective dosage regimens, low potency, side effects
influenced by drug metabolism, and issues with target specificity, solubility, and permeability. [7]

1.1.1 Categorization of diabetes mellitus.

The initial widely accepted classification of diabetes mellitus was published in 1980 [8], and it was
subsequently revised in 1985[9].

1. Diabetes mellitus:

Diabetes mellitus comprises a group of metabolic diseases characterized by abnormal blood sugar levels.
Blood glucose, derived from the food you consume, serves as the primary energy source. The hormone
insulin, produced by the pancreas, facilitates the entry of glucose from food into your cells for energy
utilization. Normal human blood glucose levels typically range between 70 to 90 mg per 100 ml.

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CHAPTER 1 INTRODUCTION

Hyperglycemia occurs when there is an excess of sugar concentration beyond the normal range. Conversely,
hypoglycaemia develops when blood sugar levels drop below the normal range. [10]

2. Diabetes insipidus:

Diabetes insipidus is a relatively rare disorder that differs from diabetes mellitus in that it does not affect
blood glucose levels. However, like diabetes mellitus, it also results in increased urination. It is
characterized by acquired polyuria and polydipsia. [11]

1.1.2 Pathophysiology:

Type 2 diabetes mellitus is characterized by insulin resistance, reduced insulin production, and eventual
failure of pancreatic beta cells. This leads to a situation where glucose is not efficiently converted into
[12]
glycogen and there is increased breakdown of fat, resulting in hyperglycemia. Patients with type 1
diabetes are typically young and may not notice symptoms when they first develop. Sometimes, these
symptoms can be attributed to environmental factors and viral infections, which damage pancreatic beta
cells, leading to insulin deficiency. Prolonged insulin deficiency in this type can potentially impact long-
term cognitive function, including learning and memory, and impair insulin secretion. [13, 14]

Fig. no: 1.1 Pathophysiology of T2DM – The Ominous Octet

The pathophysiology of Type 2 diabetes mellitus (T2DM) involves eight major mechanisms, often
referred to as the "ominous octet":[48]

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CHAPTER 1 INTRODUCTION

1. Decreased Insulin Secretion by the pancreas.

2. Increased Insulin Resistance in muscle and liver cells.

3. Increased Hepatic Glucose Production.

4. Neurotransmitter Dysfunction leading to appetite dysregulation.

5. Increased Glucagon Secretion by pancreatic alpha cells.

6. Enhanced Lipolysis leading to increased free fatty acids.

7. Increased Glucose Reabsorption by the kidneys.

8. Decreased Incretin Effect, reducing insulin release post-meal.

Gestational diabetes occurs due to hormonal changes during pregnancy. The placenta produces hormones
that make cells less sensitive to the effects of insulin, leading to increased blood sugar levels. [49, 50]

Gestational diabetes occurs due to hormonal changes during pregnancy. The placenta produces
hormones that make cells less sensitive to the effects of insulin [49, 50]. Genetic mutations can lead to
various forms of diabetes mellitus. For example, monogenic diabetes is caused by mutations in a single
gene.

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CHAPTER 1 INTRODUCTION

Fig. no: 1.2 “Targets of treatment for T2DM [TZDs – Thiazolidinediones, DPP – 4i – Dipeptidyl
peptide – 4 inhibitor, GLP-1RA – Glucagon like peptide – 1 receptor agonist, SGLT-2i - Sodium–
Glucose co-transporter 2 inhibitor] sodium–glucose co-transporter 2”

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CHAPTER 1 INTRODUCTION

Fig no: 1.3 “Schematic Mechanism of action of Peroxisome Proliferator Activated Receptor (PPAR)
agonists”

Several factors increase the risk of developing diabetes[51. The dominant factors are detailed below:

Fig.1.4 “Schematic mechanism of α – glucosidase inhibitor to lower the blood glucose level.”

In the case of Type 1 diabetes mellitus (T1DM), the risk of developing diabetes increases for
[52]
children or teenagers if a parent or sibling has the condition. . In the case of Type 2 diabetes

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 CHAPTER 1 INTRODUCTION

mellitus (T2DM), the risk increases due to several factors, including being overweight, dietary habits,
age over 45 years, a family history of diabetes, physical inactivity, having pre-diabetes or gestational
diabetes, and high cholesterol or triglyceride levels.[53, 55].

• The risk of gestational diabetes increases if a person is overweight, over the age of 25, had
gestational diabetes during a previous pregnancy, gave birth to a baby weighing more than 9
pounds, has a family history of Type 2 diabetes mellitus (T2DM), or has polycystic ovary
syndrome (PCOS). [56]

Diabetes is associated with numerous complications due to high blood sugar damaging organs and tissues
throughout the body. The longer the body is exposed to high blood sugar levels, the greater the risk of
developing additional complications. These complications can be: Microvascular: Nephropathy,
Retinopathy, Vision loss; Macrovascular: Heart diseases, Heart attack, Stroke; Other complications
include: Neuropathy, Infections and sores that don’t heal, Bacterial and fungal infections, Depression,
Dementia [54]

The old terms "insulin-dependent diabetes mellitus (IDDM)" and "non-insulin-dependent diabetes
mellitus (NIDDM)," proposed by WHO in 1980 and 1985, have been replaced. The new classification
system identifies four types of diabetes mellitus: Type 1 Diabetes Mellitus (IDDM); Type 2 Diabetes
Mellitus (NIDDM); Other Specific Types; Gestational Diabetes.

These categories were reflected in the subsequent International Nomenclature of Diseases (IND) in 1991
and the tenth revision of the International Classification of Diseases (ICD-10) in 1992. [15].The classification
of diabetes mellitus is described as follows: “Type 1 Diabetes Mellitus (IDDM); Type 2 Diabetes Mellitus
(NIDDM); Other Specific Types; Gestational Diabetes”

1. Type 1 Diabetes Mellitus (Insulin-Dependent Diabetes Mellitus, Type 1 IDDM):

Type 1 diabetes mellitus, also known as insulin-dependent diabetes mellitus (IDDM), primarily affects children and
young adults, with a sudden onset that can be life-threatening. [16] It is an autoimmune disease characterized by the
destruction of beta cells in the pancreas, which are responsible for producing insulin .[17]This type of diabetes mellitus
is also referred to as autoimmune diabetes and was previously known as juvenile-onset or ketosis-prone diabetes.
Individuals with type 1 diabetes may also have other autoimmune disorders such as Graves' disease, Hashimoto's
thyroiditis, and Addison's disease. [18]
Type 1 is usually characterized by the presence of anti–glutamic acid decarboxylase, islet cell or insulin
[19]
antibodies which identify the autoimmune processes which leads to beta-cell destruction . Type 1
diabetes (due to the destruction of b-cell which is usually leading to absolute insulin deficiency) (American

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CHAPTER 1 INTRODUCTION

diabetes association, 2014). The rate of destruction of beta cell is quite variable; it can be occur rapidly in
some individuals and slow in others [20]. There is a severe deficiency or absence of insulin secretion due to
destruction of ß-islets cells of the pancreas. Treatment with injections of insulin is required [16]

2. Non-insulin dependent diabetes mellitus(type2 NIDDM)

Type 2 diabetes mellitus is also known as adult-onset diabetes. The progressive insulin secretary defect on
[20]
the background of insulin resistance (American diabetes association, 2014) . People with this type of
diabetes frequently are resistant to the action of insulin [21].

The long-term complications in blood vessels, kidneys, eyes and nerves occur in both types and are the
[24]
major causes of morbidity and death from diabetes . The causes are multifunctional and predisposing
factor includes: obesity, sedentary lifestyle, increasing age (affecting middle aged and older people), genetic
factor (ross and Wilson 2010), such patients are at increased risk of developing macro vascular and micro
vascular complications [22, 23].

It occurs when the our body resistance or secreted the low amount of insulin thus increases the sugar level
in your blood , because insulin manage the blood sugar level .in this condition of blood glucose level may
rises up to 6-20 time of the normal range and altered the state develop or person loss of function [29].

Fig no 1.5: Pathophysiology of Type I and Type II diabetes. Abbreviations: Aβ- Amyloid- β, GSK-3β-
glycogen synthase kinase 3β, LTP- long term potentiation, P- Phosphate

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CHAPTER 1 INTRODUCTION

3. Gestational diabetes mellitus

The glucose intolerance occurring for the first time or diagnosed during pregnancy is referred to as
[25]
gestational diabetes mellitus (GDM) . Women who develop type1 diabetes mellitus during pregnancy
and women with undiagnosed asymptomatic type 2 diabetes mellitus that is discovered during pregnancy
are classified with gestational diabetes mellitus (GDM) [26]. Gestational diabetes mellitus (GDM) (diabetes
[27]
diagnosed during pregnancy that is not clearly over diabetes) . The gestational diabetes mellitus may
develops during pregnancy and may disappear after delivery; in the longer term, children born to mothers
with GDM are at greater risk of obesity and type 2 diabetes in later life, a phenomenon attributed to the
effects of intrauterine exposure to hyperglycaemia.

It is high blood sugar during the pregnancy. It causes due to insulin-blocking hormone producing by the
placenta .get help from your doctor and take simple steps to manage your blood sugar. After baby is born,
gestational diabetes usually goes away .gestational diabetes makes you more likely to develop type 2
diabetes.[30] different condition in which your kidney excreted more fluid from your body

4. Other specific type (monogenic types)

The most common form of monogenic types of diabetes is developed with mutations on chromosome 12 in
a hepatic transcription factor referred to as hepatocyte nuclear factor (hnf)-1a.they also referred to as genetic
defects of beta cells. These forms of diabetes are frequently characterized by onset of hyperglycemia at an
early age (generally before age of 25 years). They are also referred to as maturity onset diabetes of the young
(MODY)[27] or maturity-onset diabetes in youth or with defects of insulin action; persons with diseases of
the exocrine pancreas, such as pancreatitis or cystic fibrosis; persons with dysfunction associated with other
endocrinopathies (e.g. Acromegaly); and persons with pancreatic dysfunction caused by drugs, chemicals or
infections[26]. Some drugs also used in the combination with the treatment of HIV/ aids or after organ
transplantation. Genetic abnormalities that result in the inability to convert proinsulin to insulin have been
identified in a few families, and such traits are inherited in an autosomal dominant pattern. They comprise
less than 10% of DM cases [28]

1.1.3 Cause of diabetes:

Causes of diabetes mellitus disturbances or abnormality in glucoreceptor of ß cell so that they respond to
higher glucose concentration or relative ß cell deficiency. In either way, insulin secretion is impaired; may
[34]
progress to ß cell failure . The theory of principal in micro vascular disease leading to neural hypoxia,
and the direct effects of hyperglycaemia on neuronal metabolism [34].

 Reduced sensitivity of peripheral tissues to insulin: reduction in number of insulin receptors, ‘down
regulation’ of insulin receptors. Many hypersensitive and hyperinsulinemia, but normal glycaemic; and have

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CHAPTER 1 INTRODUCTION

associated dyslipidaemia, hyperuricemia, abdominal obesity. Thus there is relative insulin resistance,
particularly at the level of liver, muscle and fat. Hyperinsulinemia has been implicated in causing angiopathy
[33]

 Excess of hyperglycaemia hormone (glucagon) etc/obesity; causes relative insulin deficiency –the ß cells lag
behind. Two theories have demonstrated abnormalities in nitric oxide metabolism, resulting in altered
perineural blood flow and nerve damage [34].
 Other rare forms of diabetes mellitus are those due to specific genetic defects (type 3) like “maturity onset
diabetes of young” (MODY) other endocrine disorders, pancreatectomy and gestational diabetes mellitus
(GDM). [33].
 Due to imbalance of specific receptor can cause diabetes mellitus. Some specific receptors are glucagon-like
peptide-1(glp-1) receptor, peroxisomes proliferator activated (γ) receptor (PPARγ), beta3 (ß3) ardent-
receptor some enzymes like α glycosidase, dipeptidyl peptidase IV enzyme etc [33].
 Current research on diabetic neuropathy is focused on oxidative stress, advanced glycation-end products,
protein kinase c and the polyol pathway [34].
 Different causes are associated with each type of diabetes
 Type 1 diabetes:
In this type of diabetes generally occurs due to the abnormal immune system and destroyed insulin
producing beta cell in the pancreas, so insulin does not secreted.

 Type 2 diabetes:
Type 2 DM are the occurs combination of genetic and life style factors .being overweight or obese
increase your risk too .so that the insulin does not maintain your blood sugar level.

 Gestational diabetes:
This type of diabetes occurs during the pregnancy due to the some hormonal changes. The placenta
produce a different type of hormone during pregnancy an affect the insulin .this can cause high blood
sugar during the pregnancy [31]

1.1.4 Some common sign and symptoms

In Diabetes Mellitus, cells fails to metabolized glucose in the normal manner, effectively become starved
[25]
. The long term effect of diabetes mellitus which includes progressive development of the specific
complications of retinopathy with potential blindness, nephropathy that may lead to renal failure, and
neuropathy with risk of foot ulcer, Charcot joint and features of autonomic dysfunctions and sexual
dysfunction [32] people with diabetes are at increases risk of diseases.

Other, various symptoms are observed due to

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CHAPTER 1 INTRODUCTION

i. Gluconeogenesis from amino acids and body protein, causing muscle wasting, tissue breakdown and
further increases the blood glucose level.
ii. Catabolism of body fat, releasing some of its energy and excess production of ketone bodies [25]

1.1.5 Diagnosis of diabetes mellitus

• The diagnosis of patients with diabetes or pre diabetes some test are needed to performed ,like oral glucose
tolerance testing, hba1c testing etc.
• A high risk factor of diabetes mellitus is following such as obesity, hypertension, and family history diabetes
[26]
.The 1997 American diabetes association (ADA) recommendation for diagnosis of d m factor. Focus on
[41]
fasting plasma glucose (FPG). While who is focus on the ogtt .diabetes mellitus is diagnosed by any
following type of test [42]
• The diagnosis of diabetes in an asymptomatic subject should never be made on the basis of a single abnormal
blood glucose value. If a diagnosis of diabetes is made, the clinician must feel confident that the diagnosis is
fully established since the consequences for the individual are considerable and lifelong [35].
• The diagnosis of diabetes mellitus include, urine sugar, blood sugar, glucose tolerance test, renal threshold
of glucose, diminished glucose tolerance, increased glucose tolerance, renal glycosuria, extended glucose
tolerance curve, cortisone stressed glucose tolerance test, intravenous glucose tolerance test, oral glucose
tolerance test.

1-fasting plasma glucose level: it should be 8 hour fasting before taking this test. Condition of DM more
than 126mg/dl.
2-plasma glucose: more than or equal to 200mg/dl two hours after a 75 gram oral glucose load as in a Ogtt.
3-symptoms of high blood sugar and casual plasma glucose: it is greater than or equal to 200 mg/dl.
4-glycated haemoglobin (hba1c): it is greater than or equal to 48 mmol/mol [43]
1.1.6 Treatment of diabetes mellitus

The treatment is to overcome the precipitating cause and to give high doses of regular insulin. The insulin
requirement comes back to normal once the condition has been controlled the aims of management of
diabetes mellitus can be achieved by:

1. To restore the disturbed metabolism of the diabetic as nearly to normal as is consistent with comfort and
safety.

2. To prevent or delay progression of the short and long term hazards of the disease.

3. To provide the patient with knowledge, motivation and means to undertake this own enlightened care.

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A. Types of therapy involved in diabetes mellitus

1. Stem cell therapy: researchers have shown that monocytes/ macrophages may be main players which
contribute to these chronic inflammations and insulin resistance in T2dm patients [36]. Stem cell educator
[37]
therapy, a novel technology, is designed to control or reverse immune dysfunctions . The procedure
includes: The process involves collecting blood from patients, which circulates through a closed-loop system.
Lymphocytes are purified from the whole blood and then co-cultured with adherent cord blood-derived
multipotent stem cells (CB-SCS) in vitro. Subsequently, the educated lymphocytes, but not the CB-SCS, are
administered back into the patient's circulation. [37].
2. Antioxidant therapy: involves the use of various antioxidants, including vitamins, supplements, plant-
derived active substances, and drugs with antioxidant effects, for treating oxidative stress in patients with
Type 2 diabetes mellitus (T2DM). Vitamins C and E, as well as β-carotene, are considered ideal supplements
for combating oxidative stress and its associated complications. [38] Antioxidants play a significant role in
reducing the risk of developing diabetes and its complications.
3. Anti-inflammatory treatment: it is essential, as research indicates that inflammation plays a crucial role in
the pathogenesis of Type 2 diabetes mellitus (T2DM) and its complications. [38, 39] In T2DM, inflammation
is particularly evident in adipose tissue, pancreatic islets, the liver, the vasculature, and circulating
leukocytes. This inflammation involves altered levels of specific cytokines and chemokines, changes in the
number and activation state of various leukocyte populations, increased apoptosis, and tissue fibrosis.[40,
19]
Immunomodulatory drugs are administered to address these inflammatory processes.

B. Dietary management

Dietary management plays a central role in the overall treatment of diabetes mellitus. It involves carefully
planning meals to regulate blood sugar levels and prevent complications. Key aspects of dietary
management include:

Carbohydrate Control: Monitoring carbohydrate intake and choosing complex carbohydrates with a low
glycemic index to prevent spikes in blood sugar levels.

Portion Control: Controlling portion sizes to manage calorie intake and maintain a healthy weight.

Balanced Diet: Emphasizing a balanced diet rich in fruits, vegetables, lean proteins, and whole grains to
provide essential nutrients and promote overall health.

Fibre Intake: Including high-fiber foods such as fruits, vegetables, legumes, and whole grains to improve
blood sugar control and support digestive health

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Limiting Sugary Foods: Avoiding or limiting foods and beverages high in added sugars to prevent rapid
increases in blood sugar levels.

Monitoring Blood Sugar: Regularly monitoring blood sugar levels and adjusting dietary choices
accordingly.

Hydration: Ensuring adequate hydration by drinking plenty of water and avoiding sugary beverages.

Meal Timing: Spreading food intake evenly throughout the day and avoiding long periods of fasting to
help stabilize blood sugar levels.

Individualized Approach: Tailoring dietary recommendations to the individual's nutritional needs,

preferences, and lifestyle factors.

Collaboration with Healthcare Team: Working closely with healthcare professionals, including dietitians
or nutritionists, to develop and implement a personalized dietary plan.

C. Newer insulin delivery devices

Advancements in insulin delivery technology have led to the development of innovative devices that offer
improved convenience, precision, and ease of use for individuals with diabetes mellitus. Some of the
newer insulin delivery devices include:

Insulin Pens: These devices resemble writing pens and allow for convenient and discreet insulin
administration. They come in disposable or reusable forms and offer features such as dose memory, dose
adjustment, and half-unit dosing for precise insulin delivery.

Insulin Pumps: Insulin pumps are small, wearable devices that deliver insulin continuously throughout the
day, mimicking the function of a healthy pancreas. They offer customizable basal and bolus insulin
delivery, enabling tight glucose control while minimizing the need for multiple daily injections.

Patch Pumps: Patch pumps are tubeless insulin delivery devices that adhere directly to the skin, providing
continuous insulin infusion without the need for tubing. They offer flexibility and discretion, allowing for
comfortable insulin delivery during daily activities.

Closed-Loop Systems: Also known as artificial pancreas systems, closed-loop systems combine insulin
pumps with continuous glucose monitoring (CGM) devices and algorithms that automatically adjust insulin

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delivery based on real-time glucose levels. These systems offer improved glycemic control and reduce the
risk of hypoglycemia by providing personalized insulin dosing.

Smart Insulin Pens: Smart insulin pens integrate Bluetooth connectivity and smartphone apps to track
insulin doses, monitor blood glucose levels, and provide personalized insulin recommendations. They offer
enhanced data management and connectivity for improved diabetes management.

Inhaled Insulin: Inhaled insulin devices deliver insulin powder directly to the lungs, where it is absorbed
into the bloodstream. These devices offer an alternative to injectable insulin for individuals who prefer non-
invasive insulin delivery.

Implantable Insulin Pumps: Implantable insulin pumps are surgically placed devices that deliver insulin
directly into the peritoneal cavity. They offer continuous insulin infusion with reduced risk of site infections
and skin irritation compared to external insulin pumps.

These newer insulin delivery devices provide individuals with diabetes mellitus with a range of options to
customize their insulin therapy and improve their quality of life. It is important for healthcare providers to
stay updated on these advancements and work collaboratively with patients to select the most appropriate
device based on their individual needs and preferences.

D. Oral hypoglycaemic or antidiabetic agents

Oral hypoglycemic or antidiabetic agents are medications used to manage blood sugar levels in individuals
with diabetes mellitus. These agents are taken orally and work through various mechanisms to lower blood
glucose levels and improve insulin sensitivity. Some common classes of oral hypoglycemic agents include:

Biguanides: Examples include metformin, which works by reducing glucose production in the liver and
improving insulin sensitivity in peripheral tissues.

Sulfonylureas: Examples include glyburide, glipizide, and glimepiride, which stimulate insulin release
from pancreatic beta cells and help lower blood sugar levels.

Meglitinides: Examples include repaglinide and nateglinide, which stimulate insulin secretion from
pancreatic beta cells in a glucose-dependent manner, helping to control postprandial blood glucose levels.

Thiazolidinediones (TZDs): Examples include pioglitazone and rosiglitazone, which improve insulin
sensitivity in peripheral tissues and reduce insulin resistance.

Alpha-glucosidase inhibitors: Examples include acarbose and miglitol, which delay the digestion and
absorption of carbohydrates in the small intestine, resulting in lower postprandial blood glucose levels.

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Dipeptidyl peptidase-4 (DPP-4) inhibitors: Examples include sitagliptin, saxagliptin, and linagliptin,
which increase insulin secretion and decrease glucagon secretion in a glucose-dependent manner, helping
to lower blood sugar levels. [32]

Sodium-glucose co-transporter 2 (SGLT2) inhibitors: Examples include empagliflozin, dapagliflozin,

and canagliflozin, which inhibit glucose reabsorption in the kidneys, leading to increased urinary glucose
excretion and lower blood sugar levels.

These oral hypoglycemic agents are often used alone or in combination with each other or with insulin
therapy to achieve optimal blood glucose control in individuals with diabetes mellitus. The selection of a
specific agent or combination of agents depends on various factors, including the individual's medical
history, comorbidities, medication tolerance, and treatment goals. It is essential for healthcare providers to
tailor treatment regimens to meet the unique needs of each patient and to regularly monitor blood glucose
levels to assess treatment efficacy and safety.

1.1.7 Prevention:

Prevention strategies are crucial in reducing the risk of developing diabetes mellitus and its associated
complications. These strategies include:

Healthy Lifestyle: Adopting a healthy lifestyle that includes regular physical activity, a balanced diet
rich in fruits, vegetables, whole grains, and lean proteins, and maintaining a healthy weight can help
prevent or delay the onset of Type 2 diabetes mellitus. [44]

Regular Exercise: Engaging in regular physical activity, such as brisk walking, jogging, swimming, or
cycling, can help improve insulin sensitivity, lower blood sugar levels, and reduce the risk of developing
diabetes.

Weight Management: Maintaining a healthy weight through a combination of regular exercise and a
balanced diet can reduce the risk of obesity-related insulin resistance and Type 2 diabetes mellitus.

Healthy Eating Habits: Adopting healthy eating habits, such as consuming smaller portion sizes,
limiting intake of sugary and high-fat foods, and focusing on nutrient-dense foods, can help prevent
excessive weight gain and insulin resistance.

Regular Screening: Undergoing regular health screenings for diabetes mellitus and related risk factors,
such as high blood pressure, high cholesterol, and family history of diabetes, can help detect the condition
early and prevent complications.

Stress Management: Managing stress through relaxation techniques, such as deep breathing, meditation,
yoga, or mindfulness, can help reduce cortisol levels and improve insulin sensitivity.

Avoiding Smoking and Excessive Alcohol Consumption: Quitting smoking and limiting alcohol
consumption can reduce the risk of developing Type 2 diabetes mellitus and its associated complications.

Education and Awareness: Educating individuals about the risk factors for diabetes mellitus, the
importance of early detection, and the benefits of adopting a healthy lifestyle can empower them to make
informed decisions about their health and take preventive measures.

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By implementing these preventive strategies, individuals can reduce their risk of developing diabetes
mellitus and improve their overall health and well-being. [45, 46, 47]

Table no: 1.1 Treatment of diabetes mellitus

Types of drug Working Example(s)
Alpha-glucosidase Slow your body’s breakdown of Acarbose (precose) and miglitol
Inhibitors sugars and starchy foods (glyset)
Dpp-4 inhibitors Improve your blood sugar without Linagliptin (tradjenta), saxagliptin
making it drop too low (onglyza), and sitagliptin (januvia)
Glucagon-like To help the produces sufficient Dulaglutide (trulicity), exenatide
Peptides Amount of insulin by beta cell of (byetta),
Pancreas And liraglutide (victoza)
Meglitinide Stimulate your pancreas to release Nateglinide (starlix) and
More insulin which is control the repaglinide
blood (prandin)
Glucose level in blood
Sglt2 inhibitors Release more glucose into the urine Canagliflozin (invokana) and
dapagliflozin (farxiga)
Sulfonylureas Stimulate your pancreas to release Glyburide(diabeta,Glynase),
More insulin glipizide(glucotrol),And
glimepiride (amaryl)
Thiazolidinediones Increase the amount of insulin those Pioglitazone (actos) and
Reduces the blood glucose at the rosiglitazone (avandia)
Normal range

1.2 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

1.2.1 INTRODUCTION OF HPLC
High Performance Liquid Chromatography (HPLC) originated from classical column chromatography and
stands as one of the foremost analytical tools in modern chemistry. [71]
Also referred to as high-pressure liquid chromatography, HPLC is a form of column chromatography
extensively employed in biochemistry and analysis to separate, identify, and quantify active chemicals. It
serves as a widely favored analytical method for isolating, characterizing, and quantifying individual
components within a mixture, showcasing its status as a sophisticated column liquid chromatography

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CHAPTER 1 INTRODUCTION

technology. [57] In traditional column chromatography, the solvent flows through the column due to gravity.
However, in HPLC, the solvent is propelled under high pressures, reaching up to 400 atmospheres. This
high pressure enables the separation of the sample into its various constituents based on differences in their
relative affinities. Typically, an HPLC system consists of a column containing packing material (stationary
phase), a pump responsible for driving the mobile phase(s) through the column, and a detector that measures
[58]
the retention times of the molecules. The retention time in HPLC is influenced by interactions among
the stationary phase, the molecules under analysis, and the solvents used. Samples for analysis are
introduced in small quantities into the mobile phase stream, where they undergo specific chemical or
physical interactions with the stationary phase. These interactions slow down the movement of the
molecules, leading to differences in retention times. [59] The degree of retardation is determined by both the
characteristics of the analyte and the composition of both the stationary and mobile phases. Retention time
[60]
refers to the duration it takes for a particular analyte to elute. Common solvents in HPLC include any
combination of water or organic liquids that are miscible. Gradient elution, a technique used to alter the
composition of the mobile phase during analysis, helps separate analyte mixtures based on their affinity for
the current mobile phase. The choice of solvents, additives, and gradients is influenced by the nature of the
stationary phase and the analyte being analyzed. [61]
The technique of HPLC possesses the following features: [72]
- High resolution
- Employment of small-diameter stainless steel or glass columns
- Rapid analysis capabilities
- Utilization of relatively higher mobile phase pressures
- Controlled flow rate of the mobile phase
1.2.2 Principle of Hplc
HPLC is a separation technique that involves injecting a small volume of liquid sample into a tube packed
with tiny particles, typically 3 to 5 microns in diameter, known as the stationary phase. The individual
components of the sample are then carried down the packed tube, or column, by a liquid, called the mobile
phase, which is forced through the column by high pressure from a pump.

As the components travel through the column, they interact with the packing material, undergoing various
chemical and/or physical interactions that separate them from one another. These separated components
are detected at the exit of the column by a flow-through device known as a detector, which measures their
quantity.

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In principle, both LC (liquid chromatography) and HPLC work similarly, but HPLC offers superior speed,
efficiency, sensitivity, and ease of operation. While HPLC is primarily used for analytical purposes,
traditional liquid chromatography still finds applications in preparative techniques.

Figure Figure no : 1.6 flow diagram of Hplc

As shown in the schematic diagram in the figure above, Hplc instrumentation includes a solvent reservoir,
pump, injector, column, detector, and integrator or acquisition and display system. The heart of the system
is the column where separation occurs.

1.2.3 INSTRUMENTATION
Solvent reservoir
The solvent reservoir contains the mobile phase, which typically consists of a mixture of polar and non-
polar liquid components. The concentrations of these components vary based on the composition of the
sample being analyzed. High-grade solvents are used in HPLC, and the type and composition of the mobile
phase have a significant impact on the separation of components.
Different types of HPLC utilize different solvents. In normal-phase HPLC, the solvent is typically non-
polar, while in reverse-phase HPLC, the solvent is typically a mixture of water and a polar organic solvent.
It is crucial to use high-purity solvents and inorganic salts when preparing the mobile phase to ensure
accurate and reliable results. [73].
The most common solvent reservoirs are as simple as glass bottles with tubing connecting them to the pump
inlet.
Degassers typically consist of the following components:
- Vacuum pumping system
- Distillation system
- Devices for heating and stirring
- System for sparging, where dissolved gases are removed from the solution by introducing fine bubbles of
an inert gas that is not soluble in the mobile phase
- Often, these systems also include a means of filtering dust and particulate matter from the solvents to
prevent damage to pumping or injection systems or clogging of the column.
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CHAPTER 1 INTRODUCTION

Figure no: 1.7 component of Hplc

Degassers and filters are not always integral components of the HPLC system. Elution with a single solvent
or a solvent mixture of constant composition is termed isocratic elution. On the other hand, gradient elution
involves using two or more solvent systems with significantly different polarities, and the composition of
these solvents is varied during the separation process.
Pump
A pump in HPLC can be likened to the human heart, which continuously circulates blood throughout the
body. However, unlike the human heart, which can tolerate changes in blood pressure within a certain
range, the HPLC pump must deliver a constant flow of mobile phase at a consistent pressure and flow rate.
Any deviations in these parameters can result in errors in the analytical results.
Put simply, the HPLC pump must be robust yet capable of providing reproducible flow characteristics run
after run. The operational pressure limits vary widely depending on the specific analysis requirements. In
typical analytical operations, the pressure may range between 2000 to 5000 psi, but in ultra-high-
performance liquid chromatography (uHPLC) applications, operating pressures can reach as high as 15000
to 18000 psi.Every HPLC system includes at least one pump to drive the mobile phase through the column,
which typically has compact packing. This results in an increase in pressure at the injector, which can reach
up to 20000 kPa (200 bars) depending on factors such as the flow rate of the mobile phase, its viscosity,
and the particle size of the stationary phase.
HPLC pumps are designed to maintain a stable flow rate, minimizing pulsations even when the composition
of the mobile phase varies. Typically, these pumps contain two pistons in series, working in opposition to
ensure uninterrupted flow. [74].
A pump in HPLC serves to aspirate the mobile phase from the solvent reservoir and propel it through the
system's column and detector. Depending on various factors such as column dimensions, particle size of
the stationary phase, flow rate, and mobile phase composition, operating pressures of up to 42000 kPa
(about 6000 psi) can be generated.

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The primary function of the pump is to drive a liquid, known as the mobile phase, through the liquid
chromatography system at a specific flow rate, typically expressed in milliliters per minute (ml/min).
Normal flow rates in HPLC typically range from 1 to 2 ml/min. Typical pumps can achieve pressures in
the range of 6000 to 9000 psi (400 to 600 bar).
During chromatographic experiments, a pump can deliver either a constant mobile phase composition
(isocratic) or a changing mobile phase composition (gradient). Variations in the flow rates of the mobile
phase can impact the elution time of sample components, leading to errors. Pumps are designed to provide
a consistent flow of the mobile phase to the column under a steady pressure.
An ideal pump should possess the following desirable characteristics:
- Solvent compatibility and resistance to corrosion
- Consistent flow delivery independent of back pressure
- Ease of replacement of worn-out parts
- Low dead volume to minimize issues during solvent changeover
Three commonly used types of pumps in HPLC are syringe-type pumps, constant pressure pumps, and
reciprocating piston pumps.
Constant Pressure Pumps: These pumps ensure a consistent continuous flow rate through the column by utilizing
pressure from a gas cylinder. A valving arrangement allows for rapid refill of the solvent chamber. However, a low-
pressure gas source is required to generate high liquid pressures.
Syringe Type Pumps: These pumps are suitable for small-bore columns. They deliver a constant flow rate to the
column using a motorized screw arrangement. The solvent delivery rate is adjusted by varying the voltage on the
motor. Syringe pumps provide pulseless flow independent of column back pressure and changes in viscosity.
However, they have limitations such as limited solvent capacity and restrictions on gradient operation.
Reciprocating Piston Pumps: These pumps deliver solvent(s) through the reciprocating motion of a piston in a
hydraulic chamber. During the backstroke, the solvent is drawn in, while it is delivered to the column during the
forward stroke. Flow rates can be adjusted by modifying piston displacement in each stroke. Dual and triple head
pistons feature identical piston chamber units that operate at 1800 or 1200 phase differences. Reciprocating pump
systems offer smooth solvent delivery since one pump is in the filling cycle while the other is in the delivery cycle.
They can achieve high-pressure output at a constant flow rate, and gradient operation is feasible. However, pulse
dampening is necessary to further eliminate pressure pulses..
Sample injector
The injector in an HPLC system can either be a single injection setup or an automated injection system. Its
primary function is to deliver the liquid sample within a volume range of 0.1 to 100 ml with high
reproducibility and under high pressure (up to 4000 psi). It must be capable of withstanding the high
pressures within the liquid system.

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An autosampler is an automated version of the injector, suitable for scenarios where there are numerous
samples to analyze or manual injection is impractical. Injectors ensure a constant volume injection of
samples into the mobile phase stream. The inertness and reproducibility of the injection process are crucial
for maintaining a high level of accuracy in the analysis. [75].
In HPLC, the injection of a precise volume of sample onto the head of the column must be performed
rapidly to minimize disturbance to the dynamic regime of the mobile phase, ensuring stable flow from the
column to the detector. This is achieved using a special high-pressure valve, either manual or motorized,
with multiple flow paths, situated just before the column. This valve must be a precision component capable
of withstanding pressures greater than 30,000 kPa. The valve functions in two positions:
In the load position, only communication between the pump and the column is established. The sample,
contained in a solution, is introduced at atmospheric pressure into a small tubular curved section called a
loop. Each loop has a predefined volume and is either integrated into the rotor of the valve or connected to
the outside of the valve's casing.
In the inject position, the sample contained in the loop is introduced into the flow of the mobile phase by a
60-degree rotation of a part of the valve, connecting the sample loop to the mobile phase circulation. Highly
reproducible injections are achieved only if the loop has been completely filled with the sample.
Injectors serve to accurately introduce the required sample volume into the HPLC system. Sample injection
into the moving mobile phase stream in HPLC differs significantly from injection into a gas stream in gas
chromatography, as precise injection against high back pressure is required.In such a situation it is not
possible to simply inject using a syringe alone. The type of injector is:
1. Manual injection, facilitated by Rheodyne or Valco injectors:
The injection process in HPLC involves a specially designed 6-port rotary injection valve. The sample is
introduced at atmospheric pressure using a syringe into a constant volume loop. In the load position, the
loop is not in the path of the mobile phase.
When rotated to the inject position, the sample in the loop is pushed by the mobile phase stream into the
column for analysis. It is crucial to allow some samples to flow into waste from the loop to ensure there are
no air bubbles present and that any residue from the previously used sample is completely washed out,
preventing memory effects. This ensures the accuracy and reliability of the analysis.

Figure no: 1.8 Manual injection

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CHAPTER 1 INTRODUCTION

1. Automatic injection:
Automatic injection enhances laboratory productivity and minimizes human errors. Modern HPLC systems
are equipped with an auto-injector and auto-sampler, controlled by software programs that manage the filling
of the loop and sample delivery to the column. The computer also orchestrates the sequence of sample
injections from vials stored in numbered positions on the auto-sampler.

To ensure consistent results and prolong the service life of the automated system, certain precautions should
be taken. It's essential to prime the injector with solvents compatible with those used previously and to
conduct needle washes between samples to prevent carry-over between injections.

Before initiating and concluding an analysis, ensure that the tubing is thoroughly washed of any buffers or
previously used solvents. Additionally, ensure accurate vial numbering on the auto-sampler rack and
correctly list the sequence on the computer to maintain the integrity of the analytical process. [76].
Columns
Columns in chromatography are typically constructed from polished stainless steel, ranging from 50 to 300
mm in length, with an internal diameter between 2 and 5 mm. They are commonly packed with a stationary
phase consisting of particles sized between 3 and 10 μm. Columns with internal diameters smaller than 2
mm are often termed microbore columns.
Maintaining a constant temperature of both the mobile phase and the column throughout an analysis is
ideal. Regarded as the "heart of the chromatograph," the column's stationary phase separates the
components of interest from the sample using various physical and chemical parameters.
The small particles within the column contribute to the high back pressure experienced at normal flow rates.
Consequently, the pump must exert significant force to propel the mobile phase through the column,
resulting in elevated pressure within the chromatograph. [77].
It is crucial to maintain the column properly according to the supplier's instructions to ensure reproducible
separation efficiency from one run to another. Various types of columns include:
1. Guard columns:
A guard column is installed prior to the analytical column to extend the lifespan of the latter by
eliminating particulate matter, contaminants from solvents, and sample components that irreversibly bind
to the stationary phase. It acts to saturate the mobile phase with the stationary phase, thereby reducing
solvent losses from the analytical column. The composition of the packing material in the guard column is
akin to that of the analytical column, though the particle size is typically larger. When the guard column
becomes contaminated, it is either repacked or replaced with a new one.

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CHAPTER 1 INTRODUCTION

Figure no : 1.9 Guard column

2. Analytical columns:
The liquid-chromatographic column serves as the core of high-performance liquid chromatography
(HPLC). These columns typically range in length from 10 to 30 cm, with straight configurations being
common. Additional length requirements are met by coupling two or more columns together. The inside
diameter of liquid columns typically falls within the range of 4 to 10 mm, and the most prevalent particle
size of packing material is 5 or 10 μm. One of the most widely used column configurations is a 25 cm
length column with a 4.6 mm inside diameter, packed with 5 μm particles. Columns of this specification
typically boast 40,000 to 60,000 plates per meter, indicating their efficiency in separating components.

Figure no: 1.10 Typical Hplc column.

HPLC columns are primarily constructed from smooth-bore stainless steel. However, they are also
occasionally crafted from heavy-walled glass tubing or polymer tubing, such as polyether ether ketone
(PEEK).Temperature control of the column is essential for certain applications to ensure consistent and
reproducible chromatograms. While some columns may be operated at room temperature, maintaining a
constant column temperature often yields better results. There are three main methods for controlling
column temperature: using an oven, a heater block, or a water bath. Modern instruments may require higher
temperatures for certain applications, although there may be limitations to how high the temperature can be
raised. [78]
Detector
The HPLC detector, positioned at the column's end, identifies analytes as they elute from the
chromatographic column. Commonly utilized detectors include evaporative light scattering, UV-
spectroscopy, fluorescence, mass spectrometric, and electrochemical detectors. The detector observes and
detects individual molecules eluting from the column, enabling quantitative analysis of sample components

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CHAPTER 1 INTRODUCTION

by measuring their amounts. Its output is transmitted to a recorder or computer, generating the liquid
chromatogram, which graphically represents the detector response. A detector offers specific responses for
separated components and ensures necessary sensitivity, remaining unaffected by changes in mobile phase
composition. It monitors the mobile phase as it emerges from the column. [79].
The ideal characteristics of an HPLC detector include:
 Adequate sensitivity tailored to the specific task.
 Good stability and reproducibility of measurements.
 Wide linear dynamic range of response to accommodate varying analyte concentrations.
 Short response time that remains independent of flow rate changes.
 Insensitivity to alterations in solvent composition, flow rate, and temperature.
 Cell design that prevents remixing of separated bands.
 High reliability and user-friendly operation.
 Non-destructive for the sample, preserving its integrity throughout the analysis.
Type of detector
1. Evaporative Light Scattering Detectors (ELSDs):
Evaporative light scattering detectors (ELSDs) are commonly used in high-performance liquid chromatography
(HPLC) for their ability to detect compounds that have low or no UV absorption. These detectors operate by
nebulizing the eluent from the column and then evaporating the solvent to produce fine particles. These particles
scatter light, and the intensity of the scattered light is measured to detect analytes. ELSDs offer several advantages,
including sensitivity, wide linear dynamic range, and insensitivity to changes in solvent composition. They are
particularly useful for analyzing compounds that lack UV absorption or are present in low concentrations.

Fig no: 1.11 Evaporative light scattering detectors (ELSDS)

2. Refractive Index Detectors:
Refractive index detectors are commonly used in high-performance liquid chromatography (HPLC) to
detect analytes based on changes in the refractive index of the eluent as it passes through the detector cell.
These detectors work by measuring the difference in refractive index between the eluent and the reference
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CHAPTER 1 INTRODUCTION

solvent. When analytes elute from the column, they cause a change in refractive index, which is then
detected and recorded.
Refractive index detectors offer several advantages, including high sensitivity, simplicity, and compatibility
with a wide range of solvents. They are particularly useful for detecting analytes that lack UV absorption
or are present in low concentrations. However, they may have limited selectivity compared to other
detectors and can be affected by changes in temperature and solvent composition.
3. U.V detectors
UV detectors, short for ultraviolet detectors, are widely utilized in high-performance liquid chromatography (HPLC)
for detecting analytes based on their absorption of ultraviolet light. These detectors operate by passing the eluent
from the column through a flow cell where it is exposed to UV light at a specific wavelength. Analytes that absorb
UV light will exhibit a decrease in UV transmission, which is then measured and recorded by the detector.
UV detectors offer several advantages, including high sensitivity, specificity, and compatibility with a wide range of
analytes. They are particularly useful for detecting compounds that contain chromophores, which absorb UV light.
However, UV detectors may not be suitable for analytes that lack UV absorption or are present in low concentrations.
Additionally, they can be affected by changes in solvent composition and baseline drift. Based on electronic
transitions within molecules.

Figure no: 1.12 UV detector

4. Fluorescence detectors
Fluorescence detectors are commonly employed in high-performance liquid chromatography (HPLC) to detect
analytes based on their fluorescent properties. These detectors operate by irradiating the eluent from the column with
a specific wavelength of light, typically ultraviolet (UV) or visible light. When analytes containing fluorescent
molecules elute from the column, they absorb the incident light energy and re-emit it at longer wavelengths, resulting
in fluorescence emission. The emitted fluorescence is then measured and recorded by the detector.
Fluorescence detectors offer several advantages, including high sensitivity and selectivity, as well as the ability to
detect analytes at low concentrations. They are particularly useful for analyzing compounds that exhibit natural
fluorescence or have been derivatized to enhance their fluorescent properties. However, fluorescence detectors may

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CHAPTER 1 INTRODUCTION

require additional sample preparation steps, such as derivatization, and can be sensitive to changes in experimental
conditions such as pH and solvent composition.

Fig no: 1.13 Fluorescence detectors

5. Electrochemical Detectors:
Electrochemical detectors are commonly employed in high-performance liquid chromatography (HPLC) for the
detection of analytes based on their electrochemical properties. These detectors operate by converting the chemical
changes occurring at the surface of an electrode into an electrical signal, which is then measured and recorded.
Electrochemical detectors offer several advantages, including high sensitivity, selectivity, and compatibility with a
wide range of analytes. They are particularly useful for detecting compounds that undergo redox reactions or possess
electroactive functional groups. Additionally, electrochemical detectors can provide quantitative information about
analytes, making them valuable tools in HPLC analysis. However, they may require specialized electrodes and
experimental conditions, and can be sensitive to interference from other compounds present in the sample.

Fig no: 1.14 Electrochemical detector

6. Mass Spectrometry Detector:
Mass spectrometry (MS) detectors are widely utilized in high-performance liquid chromatography (HPLC)
for the detection and identification of analytes based on their mass-to-charge ratios. These detectors operate
by ionizing analyte molecules eluting from the column and then separating and detecting ions based on
their mass-to-charge ratios.
In HPLC-MS systems, the eluent from the column is directed into the mass spectrometer, where analyte
molecules are ionized by techniques such as electrospray ionization (ESI) or atmospheric pressure chemical
ionization (APCI). The ions are then separated based on their mass-to-charge ratios using techniques such
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CHAPTER 1 INTRODUCTION

as quadrupole, time-of-flight (TOF), or ion trap mass analyzers. Finally, the ions are detected and recorded,
providing information about the molecular composition and structure of the analytes.
MS detectors offer several advantages, including high sensitivity, selectivity, and the ability to provide
detailed structural information about analytes. They are particularly useful for analyzing complex mixtures,
identifying unknown compounds, and quantifying trace levels of analytes. However, MS detectors can be
expensive and require specialized training and expertise for operation and data analysis. Additionally, they
may not be suitable for all analytes and can be sensitive to experimental conditions such as solvent
composition and ionization efficiency.

Fig no: 1.15 Mass spectrometry

7. Infrared (IR) Detectors:
Infrared detectors are utilized in high-performance liquid chromatography (HPLC) for the detection of analytes
based on their absorption of infrared radiation. These detectors operate by measuring the changes in infrared
radiation caused by the presence of analytes in the eluent from the chromatographic column.
In HPLC-IR systems, the eluent from the column is directed into the IR detector, where it passes through an
infrared beam. Analyte molecules in the eluent absorb specific wavelengths of infrared radiation, leading to
changes in the intensity of the transmitted light. These changes are then measured and recorded, providing
information about the presence and concentration of analytes in the sample.
IR detectors offer several advantages, including high sensitivity and selectivity, as well as the ability to detect a
wide range of analytes. They are particularly useful for analyzing compounds that exhibit characteristic
absorption bands in the infrared region of the electromagnetic spectrum. However, IR detectors may require
specialized sample preparation techniques and can be sensitive to interference from other compounds present in
the sample. Additionally, they may not be suitable for all analytes and experimental conditions.
 Instrument filter or FTIR.
 Similar cell with variable volumes (v, 1.5 ~ 10 μL and b, 0.2 ~ 1.0 mm).
 Limitations include unsuitability for certain solvents and requiring special optics.
 FTIR enables spectrum recording of flowing systems, akin to the diode array system.
 Water and alcohols can significantly interfere with solute detection.
 Limit of detection (LoD) is 100 mg.

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CHAPTER 1 INTRODUCTION

Data collection devices (computer)

Signals from the detector can be captured using chart recorders or electronic integrators, which differ in
complexity and their capacity to process, store, and reprocess chromatographic data. The computer, often
referred to as the data system, plays a central role in this process. It not only oversees all the components
of the HPLC instrument but also takes the signal from the detector to ascertain the elution time (retention
time) of sample components for qualitative analysis and to determine the sample amount for quantitative
analysis. Additionally, the computer integrates the detector's response to each component, presenting it in
a chromatogram format that is straightforward to interpret. [80].
Sample preparation for Hplc
Liquid samples can be directly analyzed after suitable cleanup to remove any particulate matter or through
extraction to eliminate matrix interferences. For example, in the analysis of polyaromatic hydrocarbons
(PAH) in wastewater, an initial extraction with CH2Cl2 concentrates the analytes and isolates them from
matrix interferents.
Solid samples typically need to be dissolved in a suitable solvent, or the analytes must be brought into
solution through extraction. For instance, in an HPLC analysis of active ingredients and degradation
products in a pharmaceutical tablet, the process often begins by extracting the powdered tablet with a
portion of the mobile phase.
Gaseous samples are collected by passing them through a trap containing an appropriate solvent. For
instance, organic isocyanates in industrial atmospheres can be quantified by bubbling the air through a
solution of 1-(2-methoxyphenyl)piperazine in toluene. This method stabilizes the isocyanates against
degradation before HPLC analysis and forms a derivative that can be monitored via UV absorption.. [81] The
sample is easily fractionated and purified. [62]
1.2.3 Classificatiion of Hplc
A. Based on modes of chromatography [63,65]
1. Normal–phase chromatography:
Normal-phase chromatography, an early type of HPLC, is also known as NP-HPLC. In this method,
analytes are separated based on their interaction with a polar stationary surface, typically silica. This relies
on the analyte's ability to form polar interactions, like hydrogen bonding or dipole-dipole interactions, with
the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase, such as chloroform or octane,
and is effective for separating analytes soluble in non-polar solvents. The analyte binds to and is retained
by the polar stationary phase.[82].
In normal-phase chromatography, the mobile phase is nonpolar, while the stationary phase is polar.
Consequently, the polar analyte is retained by the stationary phase.[66] The enhanced polarity of solute
molecules enhances their adsorption capacity, leading to a longer elution time. This chromatography
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CHAPTER 1 INTRODUCTION

employs a stationary phase composed of chemically modified silica, such as cyanopropyl [67]. For instance,
a standard column typically features an internal diameter of approximately 4.6 mm and a length ranging
from 150 to 250 mm. Polar compounds within the mixture will adhere to the polar silica within the column
for a longer duration compared to non-polar compounds. Consequently, non-polar compounds will pass
through the column more rapidly. [68]
Hydrophilic interaction chromatography:
Hydrophilic interaction chromatography (HILIC) is a type of liquid chromatography that utilizes a
hydrophilic stationary phase and a hydrophobic mobile phase. In this technique, analytes interact with both
the water-rich stationary phase and the organic-rich mobile phase, leading to a separation based on their
varying degrees of hydrophilicity. HILIC is particularly useful for the separation of polar and hydrophilic
compounds, which may be challenging to separate using traditional reversed-phase chromatography.
2. Reversed-phase chromatography (RPC):
Reversed-phase chromatography (RPC) is a widely used technique in liquid chromatography where the
stationary phase is nonpolar and the mobile phase is polar. [83] In RPC, the analytes interact more strongly
with the nonpolar stationary phase than with the polar mobile phase. This leads to the separation of analytes
based on their varying degrees of hydrophobicity. [69] RPC is particularly effective for separating nonpolar
and hydrophobic compounds, making it a versatile technique in analytical chemistry.[70].
A. Based on the principle of separation
Affinity chromatography, adsorption chromatography, size exclusion chromatography, ion-exchange
chromatography, chiral phase chromatography.[64]
Ion exchange chromatography:
Ion exchange chromatography (IEC) is a technique used to separate ions based on their interactions with
charged groups attached to a solid support. In this method, a stationary phase containing charged functional
groups is packed into a column. When a sample containing ions of interest is passed through the column,
the ions interact with the charged groups on the stationary phase.
Depending on their charge and affinity for the stationary phase, ions are either retained or eluted from the
column at different rates. Cations will bind to negatively charged groups (anion exchange chromatography),
while anions will bind to positively charged groups (cation exchange chromatography). By adjusting the
pH and ionic strength of the mobile phase, as well as the type and density of charged groups on the
stationary phase, specific ions can be selectively retained or separated.
IEC is widely used in biochemistry, molecular biology, and protein purification to separate and purify
charged molecules such as proteins, peptides, nucleic acids, and other biomolecules based on their net
charge and affinity for the stationary phase.

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CHAPTER 1 INTRODUCTION

Size exclusion chromatography (SEC):

Size exclusion chromatography (SEC), also known as gel filtration chromatography or gel permeation
chromatography, is a technique used to separate molecules based on their size and molecular weight. In
SEC, a porous stationary phase is packed into a column, consisting of porous beads with specific pore sizes.
When a sample containing molecules of different sizes is passed through the column, smaller molecules
can enter the pores of the stationary phase and take longer to elute, while larger molecules cannot enter the
pores and elute more quickly. As a result, larger molecules are eluted first, followed by smaller molecules.
SEC is often used for the analysis and purification of biomolecules such as proteins, nucleic acids,
polysaccharides, and synthetic polymers. It can separate molecules ranging from small ions and metabolites
to large proteins and polymers, making it a versatile technique in biochemical and pharmaceutical research.
Additionally, SEC is valuable for determining the molecular weight distribution of polymers and the
oligomeric state of proteins.[85]
Size exclusion chromatography (SEC) can be classified into two categories based on the nature of the
columns and their packing:
1. Gel filtration chromatography: In gel filtration chromatography, the stationary phase consists of porous
beads made of cross-linked polymers such as agarose or dextran. These beads have a range of pore sizes,
allowing molecules to enter the pores based on their size. Smaller molecules are excluded from the pores
and take longer to elute, while larger molecules pass through the column more quickly.
2. Gel permeation chromatography: Gel permeation chromatography (GPC), also known as gel
permeation chromatography, employs porous beads made of synthetic polymers such as styrene-
divinylbenzene (SDVB) as the stationary phase. Similar to gel filtration chromatography, GPC separates
molecules based on their size, with smaller molecules being excluded from the pores and larger molecules
eluting more quickly. However, GPC is often used for higher-resolution separations and is more suitable
for analyzing synthetic polymers and other large molecules.
It is classified into two categories based on the nature of the columns and their packing

Figure no: 1.16 SEC separation

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CHAPTER 1 INTRODUCTION

Affinity chromatography
Affinity chromatography involves the covalent bonding of a specialized reagent called an affinity ligand
to a solid support. Common affinity ligands include antibodies, enzyme inhibitors, cofactors, or other
molecules that selectively bind to target molecules in the sample. The stationary phase comprises a
support medium (e.g., cellulose beads) with the ligand bound covalently, exposing reactive groups
essential for binding. As the protein mixture passes through the column, proteins with binding sites for
the immobilized ligand attach to the stationary phase, while other proteins elute in the column's void
volume.
During column passage, only molecules that specifically bind to the affinity ligand are retained, while
non-binding molecules flow through with the mobile phase. After removing undesired molecules, the
retained analyte is eluted by altering mobile-phase conditions. Subsequently, the bonded analyte need
separation from the stationary phase using a solvent with good separation capacity. Affinity
chromatography is predominantly useful for biomolecule separation, such as proteins. [86]
1.2.4 THE FACTORS WHICH INFLUENCE THE HPLC PERFORMANCE

 Mobile Phase Composition: The choice and composition of the mobile phase, including the solvent
type, pH, and buffer concentration, significantly influence chromatographic separation and peak
resolution.
 Stationary Phase Properties: The characteristics of the stationary phase, such as particle size, pore
size, surface chemistry, and packing material, affect the interaction between analytes and the
column, thus impacting separation efficiency.
 Column Temperature: Controlling the temperature of the column can influence retention times,
selectivity, and resolution. Temperature variations can also affect the viscosity of the mobile phase
and analyte diffusion rates.
 Flow Rate: The rate at which the mobile phase is pumped through the column affects peak shape,
resolution, and analysis time. Higher flow rates may lead to broader peaks but shorter analysis times,
while lower flow rates offer improved resolution but longer analysis times.
 Detector Sensitivity and Specificity: The sensitivity and specificity of the detector used in HPLC
impact the detection limits, accuracy, and precision of the analysis. Different detectors may be more
suitable for specific analytes or applications.
 Injection Volume and Technique: The volume of sample injected onto the column and the
injection technique (e.g., manual or automatic) influence peak shape, resolution, and detection
sensitivity.

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CHAPTER 1 INTRODUCTION

 Column Conditioning and Equilibration: Proper conditioning and equilibration of the column
before analysis are essential to achieve reproducible results. This includes removing air bubbles,
stabilizing column temperature, and ensuring consistent mobile phase flow.
 pH and Ionic Strength of Mobile Phase: pH and ionic strength adjustments of the mobile phase
can alter analyte retention and selectivity by affecting ionization states and interactions with the
stationary phase.
 Sample Preparation: The quality of sample preparation, including extraction, filtration, and
derivatization techniques, can impact chromatographic performance by minimizing matrix effects
and sample impurities.
 Instrument Calibration and Maintenance: Regular calibration and maintenance of HPLC
instruments, including pumps, detectors, and autosamplers, are crucial to ensure accurate and
reproducible results.
 These factors collectively influence the performance of HPLC and must be carefully optimized to
achieve reliable chromatographic separations and quantitative analyses

1.2.5 PARAMETER USED IN HPLC

 In High-Performance Liquid Chromatography (HPLC), several parameters are crucial for
optimizing separation efficiency, peak resolution, and detection sensitivity. These parameters
include:
 Retention Time (RT): The time taken by an analyte to travel through the column from injection to
detection. Retention time is influenced by the affinity of the analyte for the stationary phase and the
mobile phase composition.
 Peak Width: The width of a chromatographic peak at its base, often measured as the full width at
half maximum (FWHM). Narrower peaks indicate better resolution and separation efficiency.
 Peak Area: The integrated area under a chromatographic peak, which is proportional to the amount
of analyte present in the sample.
 Peak Height: The maximum intensity or height of a chromatographic peak, which is also
proportional to the analyte concentration.
 Resolution (Rs): The separation between two adjacent peaks, often expressed as the ratio of the
difference in retention times to the average peak width. Higher resolution indicates better separation
of analytes.
 Selectivity (α): The relative separation between two closely eluting peaks, calculated as the ratio of
their retention factors (k values). Greater selectivity ensures discrimination between similar
compounds.

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CHAPTER 1 INTRODUCTION

 Retention Factor (k): The ratio of the retention time of an analyte to the retention time of an
unretained compound (typically the void volume). Retention factor indicates the degree of
interaction between the analyte and the stationary phase.
 Efficiency (N): The theoretical plates or number of theoretical stages of separation in the column,
which determines the chromatographic resolution. Higher efficiency leads to sharper peaks and
better separation.
 Mobile Phase Composition: The composition and ratio of solvents used in the mobile phase, which
affects analyte retention, selectivity, and peak shape.
 Column Temperature: The temperature at which the chromatographic separation is performed,
which influences analyte retention, solubility, and column efficiency.
 Flow Rate: The rate at which the mobile phase is pumped through the column, which affects
analysis time, resolution, and peak shape.
 Detector Sensitivity: The ability of the detector to detect and quantify analytes accurately, which
depends on factors such as detector type, wavelength, and signal-to-noise ratio.
 Optimizing these parameters according to the specific requirements of the analyte and the analytical
method is essential for achieving robust and reliable HPLC analyses.

1.2.6 Advantage and disadvantages of Hplc

High-Performance Liquid Chromatography (HPLC) offers several advantages and disadvantages, making it a
versatile technique for various analytical applications:

Advantages:

 High Sensitivity: HPLC can detect and quantify analytes at trace levels, making it suitable for
applications requiring high sensitivity.
 Wide Applicability: It can separate and analyze a wide range of compounds, including polar and
non-polar substances, small molecules, proteins, peptides, and nucleic acids.
 High Resolution: HPLC can achieve excellent resolution between closely related compounds,
enabling accurate identification and quantification.
 Automation: Modern HPLC systems are highly automated, allowing for rapid and reproducible
analyses with minimal manual intervention.
 Quantitative Analysis: HPLC is widely used for quantitative analysis due to its ability to generate
accurate and precise concentration measurements.
 Sample Flexibility: HPLC can analyze various sample types, including liquids, gases, and solids,
with minimal sample preparation.
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CHAPTER 1 INTRODUCTION

 Versatility: It offers various separation modes (e.g., normal phase, reversed phase, ion exchange)
and detection methods (e.g., UV, fluorescence, MS), making it adaptable to different analytical
needs.

Disadvantages:

 Cost: HPLC instrumentation and consumables can be expensive, especially for high-end systems
with advanced features.
 Complexity: HPLC method development and optimization can be time-consuming and require
expertise in chromatography principles and instrumentation.
 Solvent Usage: HPLC often requires large volumes of organic solvents, which can be costly and
environmentally unfriendly.
 Column Maintenance: Columns need regular cleaning and replacement to maintain performance,
adding to operational costs.
 Sensitivity to Operating Conditions: Small changes in temperature, flow rate, or mobile phase
composition can affect chromatographic results, requiring careful control of experimental
parameters.
 Limited Robustness: HPLC methods may lack robustness when applied to complex matrices or
samples with high variability, requiring frequent method validation and optimization.
 Limited Throughput: Although modern HPLC systems offer rapid analysis times, they may have
limited throughput compared to other analytical techniques like mass spectrometry.
 Despite these limitations, HPLC remains one of the most widely used techniques in analytical
chemistry, offering unparalleled versatility and performance for a wide range of applications.

1.2.7 Applications of high-performance liquid chromatography (Hplc)

High-Performance Liquid Chromatography (HPLC) finds extensive applications across various industries
and scientific fields due to its versatility and reliability. Some common applications include:

 Pharmaceutical Analysis: HPLC is widely used in pharmaceutical industries for drug discovery,
development, and quality control. It helps in the analysis of drug compounds, impurities,
degradation products, and formulation stability studies.
 Environmental Monitoring: HPLC is employed for the analysis of environmental samples, such
as water, soil, and air, to detect and quantify pollutants, pesticides, herbicides, and other
contaminants.

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CHAPTER 1 INTRODUCTION

 Food and Beverage Analysis: HPLC is utilized for the analysis of food and beverage products to
determine the presence of additives, preservatives, vitamins, antioxidants, pesticides, mycotoxins,
and other compounds.
 Clinical Diagnostics: HPLC plays a crucial role in clinical laboratories for the analysis of
biomarkers, hormones, vitamins, drugs, metabolites, and other compounds in biological samples
like blood, urine, serum, and plasma.
 Forensic Science: HPLC is used in forensic laboratories for the analysis of drugs of abuse, toxins,
poisons, explosives, and other compounds in criminal investigations and toxicological
examinations.
 Chemical Research: HPLC is a valuable tool in chemical research for the purification, isolation,
and characterization of organic compounds, natural products, synthetic polymers, and catalysts.
 Biochemical Analysis: HPLC is employed in biochemistry and biotechnology for the analysis of
proteins, peptides, nucleic acids, carbohydrates, lipids, and other biomolecules in research and
diagnostic applications.
 Phytochemical Analysis: HPLC is utilized in the analysis of plant extracts and herbal medicines
to identify and quantify bioactive compounds, such as flavonoids, alkaloids, terpenoids, and
phenolic compounds.
 Quality Control: HPLC is used for quality control and assurance purposes in various industries,
including pharmaceuticals, food and beverages, cosmetics, chemicals, and environmental
monitoring.
 Petroleum and Petrochemical Analysis: HPLC is employed in the analysis of petroleum products,
lubricants, and petrochemicals to determine the composition, purity, and quality of hydrocarbons
and additives.
 These are just a few examples of the diverse applications of HPLC across different sectors. Its
flexibility, sensitivity, and ability to separate and quantify a wide range of compounds make it an
indispensable tool in analytical chemistry and research.

Other application of HPLC includes

 Pharmacokinetics Studies: HPLC is used to study the absorption, distribution, metabolism, and
excretion (ADME) of drugs in biological systems, helping in the determination of drug
concentrations in plasma, tissues, and other biological matrices over time.

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CHAPTER 1 INTRODUCTION

 Biopharmaceutical Analysis: HPLC is employed for the analysis of biopharmaceuticals, such as

monoclonal antibodies, peptides, proteins, and nucleic acids, in terms of purity, identity, structure,
and stability.
 Metabolomics: HPLC is utilized in metabolomics studies to analyze and identify small molecule
metabolites present in biological samples, providing insights into metabolic pathways, biomarkers,
and disease mechanisms.
 Chiral Separation: HPLC is used for the separation of enantiomers (chiral compounds) into their
individual optical isomers, which is crucial in pharmaceuticals, agrochemicals, and flavor and
fragrance industries.
 Polymer Analysis: HPLC is employed in polymer chemistry for the analysis of polymer molecular
weight, composition, branching, and end-group functionality, aiding in polymer characterization
and quality control.
 Natural Product Analysis: HPLC is utilized in the analysis of natural products, such as herbal
extracts, essential oils, and botanicals, to identify and quantify bioactive compounds, antioxidants,
and phytochemicals.
 Toxicology and Drug Screening: HPLC is used in toxicology laboratories for the analysis of drugs,
metabolites, and toxins in forensic and clinical toxicology, drug screening, and antidoping testing.
 Bioavailability and Bioequivalence Studies: HPLC is employed in pharmaceutical research to
assess the bioavailability and bioequivalence of generic drug products compared to their reference
formulations.
 Quality Assurance in Manufacturing: HPLC is utilized in manufacturing industries for quality
assurance and control of raw materials, intermediates, and finished products, ensuring compliance
with regulatory standards and specifications.
 Proteomics and Peptidomics: HPLC is employed in proteomics and peptidomics research for the
separation and analysis of proteins, peptides, and amino acids, facilitating protein identification,
quantification, and characterization.
 These applications demonstrate the versatility and significance of HPLC across various scientific
disciplines and industries, contributing to advancements in research, development, and quality
control.

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CHAPTER 1 INTRODUCTION

1.3 METHOD DEVELOPMENT ON HPLC

Figure no: 1.17 steps involved in Hplc method development

1.3.1 Method development involves the following steps:
1. Understanding the physicochemical properties of the drug molecule.

2. Selection of chromatographic conditions.

3. Developing the approach of analysis. Sample preparations

4. Method optimization

5. Method validation [87]

1. Understanding the physicochemical properties of the drug molecule

The development of a method heavily relies on understanding the physicochemical characteristics of a

therapeutic molecule. Before initiating this process, it's crucial to evaluate essential physical properties of the
drug, such as solubility, polarity, pKa, and pH. Among these properties, polarity holds particular significance.
It assists analysts in determining the composition of solvents and mobile phases. The solubility of a molecule
can be inferred from its polarity. For example, polar solvents like water and nonpolar solvents like benzene
remain separate due to their differing polarities, adhering to the principle of "like dissolves like." This principle
suggests that substances with similar polarities dissolve more effectively in each other. Consequently, the
solubility of the analyte influences the selection of diluents. [89] The pH value is a fundamental indicator used
to assess the acidity or basicity of a substance. Choosing the right pH for an ionizable analyte is critical for
achieving symmetrical and well-defined peaks in HPLC. It represents the negative logarithm, base 10, of the
hydrogen ion concentration. [90]
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CHAPTER 1 INTRODUCTION

pH = -log10 [H3O+]

Selecting the appropriate pH for an ionizable analyte is crucial as it often results in the generation of
symmetrical and well-defined peaks in HPLC. These sharp, symmetrical peaks are vital in quantitative
analysis as they contribute to achieving low detection limits, minimal relative standard deviations
[91]
between injections, and consistent retention times. .

2. Selection of chromatographic conditions

In the initial stages of method development, a predetermined set of starting conditions, including the
choice of detector, column, and mobile phase, is selected to generate the initial "scouting" chromatograms
for the sample. These chromatograms usually involve reversed-phase separations on a C18 column with
UV detection. At this juncture, a critical decision must be made regarding whether to adopt an isocratic
or a gradient method. [92]

Selection of column

The selection of the stationary phase or column is pivotal in method development. Without a reliable,
high-performance column, establishing a robust and reproducible procedure can be challenging. Columns
need to exhibit stability and reproducibility to tackle issues related to inconsistent sample retention during
method development. Typically, a C8 or C18 column, made from carefully purified, low-acidic silica and
designed for separating basic compounds, is widely applicable and highly recommended for various
sample types. [93].

Key factors to consider include column diameters, the quality of the silica substrate, and the properties
of the bonded stationary phase. Silica-based packing is predominantly preferred in modern HPLC
columns because of its versatile physical properties. The three main components of an HPLC column are
the hardware, matrix, and stationary phase. [94]

Silica, polymers, alumina, and zirconium are among the matrices utilized to support the stationary phase
in HPLC columns. Silica is the most prevalent matrix for HPLC columns due to its strength, ease of
derivatization, consistent sphere sizes, and resistance to compression under pressure. Additionally, silica
exhibits chemical stability to most organic solvents and low pH solutions. However, one drawback of
silica as a solid support is its dissolution above pH 7. [95].

Selection of chromatographic mode

Chromatographic modes are determined by the molecular weight and polarity of the analyte. In all case
studies, the primary focus will be on reversed-phase chromatography (RPC), the most commonly used
technique for small organic compounds. RPC is frequently employed for separating ionizable substances,

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CHAPTER 1 INTRODUCTION

such as acids and bases, utilizing buffered mobile phases to prevent analyte ionization. Alternatively, ion-
pairing reagents may be used in RPC. [96]

Buffer selection

Several buffers, such as potassium phosphate, sodium phosphate, and acetate, underwent testing to
evaluate their compatibility with the system and their overall chromatographic performance.

General consideration for buffer selection

When selecting a buffer for HPLC applications, several considerations come into play to ensure optimal
performance and compatibility with the system:

1. pH Range: Choose a buffer with a suitable pH range that matches the analyte's ionization state and
stability. Consider the pKa of the analyte and select a buffer within a pH range that provides adequate
ionization.

2. Ionic Strength: The buffer's ionic strength should be compatible with the separation conditions and the
detector used in the HPLC system. Excessive ionic strength can lead to baseline drift and affect peak
resolution.

3. Chemical Compatibility: Ensure that the buffer is chemically compatible with the mobile phase,
stationary phase, and detector components to prevent corrosion, degradation, or contamination.

4. Buffer Capacity: Select a buffer with sufficient buffering capacity to maintain the desired pH throughout
the chromatographic run, especially in gradient elution methods where pH changes occur.

5. UV Absorbance: Consider the UV absorbance of the buffer to minimize interference with UV

detection, especially in UV-sensitive applications.

6. Stability: Choose a buffer that is stable under the chromatographic conditions, including temperature,
pressure, and mobile phase composition, to avoid changes in pH or degradation during the analysis.

7. Cost and Availability: Evaluate the cost-effectiveness and availability of the buffer, considering factors
such as procurement, storage, and disposal.

By considering these factors, analysts can effectively select buffers that ensure reliable and reproducible
HPLC separations while minimizing potential issues and optimizing performance.[97].

Buffer concentration
A buffer concentration between 10 to 50 mM is typically suitable for small molecules in HPLC
applications. It's important to note that a buffer should generally not exceed 50% organic material, which
is contingent upon the buffer type and its concentration. While phosphoric acid and its sodium or
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CHAPTER 1 INTRODUCTION

potassium salts are widely utilized buffer systems for reversed-phase HPLC, sulfonate buffers can be
preferred over phosphonate buffers when analyzing organophosphate chemicals. [98].

Selection of mobile phase

The mobile phase plays a pivotal role in determining resolution, selectivity, and efficiency in RP-HPLC.
The composition of the mobile phase, or the solvent strength, is particularly crucial in separation.
Commonly employed solvents in RP-HPLC include acetonitrile (ACN), methanol (MeOH), and
tetrahydrofuran (THF), each with UV cut-offs of 190 nm, 205 nm, and 212 nm, respectively. These
solvents are highly miscible with water. During method development, an acetonitrile-water mixture is
often the preferred initial choice for the mobile phase. [99]
Sr. Mode Solvent type Type of compound used
No used
1 Reversed- Water/ buffer, Neutral or non-ionized compound
phase can, methanol which can be dissolved in water.
2 Ion-pair Water/ buffer, Ionic or ionizable compound
can, methanol
3 Normal Organic A mixture of isomer & compound not
phase solvent soluble in organic / water mixture

4 Ion exchange Water/ Inorganic ions, protein, nucleic acid,

buffer organic acid.
5 Size exclusion Water, High molecular weight compound
chloroform

Table 1.2: The overall study of technique [100].

Sr. No Detector Type of compound can be detected

1 UV-visible & Compounds with chromophores, such as

photodiode array aromatic rings or multiple alternating double
bonds.
2 Fluorescence detector Fluorescent compounds, usually with fused
rings or highly conjugated planer system
3 Conductivity detector Charged compounds, such as inorganic ions
and organic acid.

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CHAPTER 1 INTRODUCTION

4 Electrochemical For easily oxidized compounds like

detector quinines or amines
5 Refractive index detector Compounds that do not show
& evaporative light characteristics usable by the other
Scattering detectors, eg. Polymers, saccharides.
Detector.

Table 1.3: “Table of different detectors & type of compounds detected by them “[102].

Selection of detectors

The detector is a critical component of HPLC, and selecting the appropriate one depends on several
factors, including the chemical composition of the samples, potential interferences, required detection
limits, availability, and cost. Commonly available detectors in LC include UV detectors, fluorescence
detectors, electrochemical detectors, refractive index (RI) detectors, and mass spectrometry (MS)
detectors. The selection of a detector is influenced by the characteristics of the sample and the analytical
objectives.[101]

3. Developing the approach of analysis

The initial phase of developing an analytical method for RP-HPLC involves selecting various
chromatographic parameters, including the mobile phase, column, mobile phase flow rate, and mobile
phase pH. These parameters are chosen through iterative testing, and their effectiveness is subsequently
assessed against system suitability criteria. Common system suitability parameters include a retention
time exceeding 5 minutes, a theoretical plate count greater than 2000, a tailing factor below 2, a resolution
above 5, and a percent relative standard deviation (% RSD) of analyte peak areas in standard
chromatograms not exceeding 2.0%. When simultaneous estimation of two components is necessary, the
detection wavelength typically aligns with an isosbestic point. Moreover, the practicality of the laboratory
setup is evaluated to ensure feasibility. Once the proposed method for simultaneous estimation is
established, the subsequent step involves analyzing the marketed formulation. This entails diluting it to
fall within the concentration range of linearity as determined during method development. [103]

Sample preparation

Sample preparation stands as a pivotal stage in method development, requiring meticulous scrutiny from
the analyst. For instance, when confronted with insoluble components within the sample, the analyst must
evaluate the necessity of centrifugation (including determining optimal rpm and duration), agitation,

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CHAPTER 1 INTRODUCTION

and/or filtration. The aim is to ensure that sample filtration does not influence the analytical outcome by
causing adsorption and/or extraction of leachables. The effectiveness of syringe filters in removing
contaminants/insoluble components without introducing undesirable artifacts (such as extractables) into
the filtrate determines their efficiency. The sample preparation protocol should be clearly delineated in
the pertinent analytical method applied to real in-process samples or dosage forms intended for
subsequent HPLC analysis. [104]

In the analytical technique, it's crucial to specify the manufacturer, type of filter, and pore size of the
filter media used in sample preparation. The primary objective of sample preparation is to generate a
processed sample that yields superior analytical outcomes compared to the raw sample. This prepared
sample should be an aliquot that is reasonably free from interferences, compatible with the HPLC
procedure, and safe for the column. Hence, the goal of sample preparation is to provide a sample aliquot
that is reasonably free from interferences, compatible with the column, and suitable for the intended
HPLC procedure. This ensures that the sample solvent dissolves in the mobile phase without adversely
affecting sample retention or resolution. Sample preparation encompasses the entire process from sample
collection to sample injection onto the HPLC column.[105].

4. Method optimization

Identifying the method's "weaknesses" and improving its performance through experimental design
entails evaluating its efficacy across various settings, instrument configurations, and sample types.
Primarily, the focus in optimizing HPLC method development has been on fine-tuning HPLC conditions.
This involves careful consideration of both the mobile phase and stationary phase compositions.
Typically, optimizing mobile phase parameters takes precedence over stationary phase parameters due
to its relative simplicity and practicality. [107]

To streamline the optimization process and reduce the number of trial chromatograms, it's crucial to
concentrate on parameters that have a substantial effect on selectivity. In liquid chromatography (LC)
optimization, key control variables include factors related to the mobile phase, such as acidity, solvent
composition, gradient profile, flow rate, temperature, sample size, injection volume, and the type of
diluent solvent. Focusing on these variables allows for efficient optimization and enhancement of
chromatographic performance. [108]

After achieving adequate selectivity, the next step involves finding the optimal balance between
resolution and analysis time. Parameters like column dimensions, particle size of the column packing
material, and flow rate become crucial at this stage. Adjusting these parameters allows for fine-tuning of
chromatographic performance without compromising the capacity factor or selectivity. [109]

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CHAPTER 1 INTRODUCTION

5. Method validation

Validation is the process of systematically evaluating and providing objective evidence that a particular
analytical method meets the defined requirements for its intended application. It serves as a means of
assessing method performance and demonstrating its suitability for specific conditions. Essentially,
validation helps in understanding the capabilities of a method, especially in detecting substances at low
concentrations.

Analytical methods need to undergo validation or revalidation before being implemented into routine
use, especially when there are changes in the conditions for which the method was initially validated or
when modifications are made to the method itself. [111]

1.3.2 Components of method validation

Method validation typically involves the following components:

1. Accuracy: This assesses the closeness of the measured value to the true value of the analyte. Accuracy
can be determined through recovery studies, where known amounts of analyte are added to samples, and
the difference between the added and recovered amounts is measured. [113,114]

2. Precision: Precision evaluates the degree of repeatability or reproducibility of results obtained under
similar conditions. It includes both the within-run (repeatability) and between-run (reproducibility)
variations. Precision can be assessed using replicate injections of samples.[116,117,118]

3. Specificity: Specificity measures the ability of the method to differentiate the analyte from other
components in the sample matrix. It ensures that the measured signal is solely due to the analyte of interest
and not influenced by interfering substances. [124]

4. Limit of Detection (LOD) and Limit of Quantification (LOQ): LOD is the lowest concentration of
an analyte that can be reliably detected but not necessarily quantified, while LOQ is the lowest
concentration that can be quantitatively determined with acceptable precision and accuracy. [121,122]

LoD = 3.3 × s /SD and

LoQ = 10 × s /SD

5. Linearity: Linearity evaluates the relationship between the analyte concentration and the detector
response. It determines whether the method produces results that are directly proportional to the analyte
concentration over a specified range. [119,120]]

6. Range: The range of the method defines the concentrations over which it has been validated to provide

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CHAPTER 1 INTRODUCTION

accurate and precise results. It encompasses the lowest to highest concentrations that fall within the
method's linearity, accuracy, and precision criteria. [126].

7. Robustness: Robustness assesses the method's ability to remain unaffected by small variations in
experimental parameters, such as changes in mobile phase composition, column temperature, or flow rate.
It demonstrates the method's reliability under different conditions.

These components collectively ensure that the analytical method is reliable, accurate, and suitable for its
intended purpose. [125]
1.4 Drug profile [127,128]
1.4.1 Drug profile of Lobeglitazone sulphate

Name of drug Lobeglitazone sulphate

Category Antidiabetic drug

Agonist indication Type 2 diabetes mellitus

Structure

IUPAC 5-[(4-[2-([6-(4-methoxyphenoxy)pyrimidin-4-yl]-
methylamino) ethoxy] phenyl)methyl]-1,3-thiazolidine-
2,4-dione.

Class of drug Thiazolidinedione

Chemical formula C24H26N4O9S2

Dose 0.5mg once a daily

Elimination half-life 7.8–9.8 hours

Trade names Duvie (Chong kun dang corporation)

Molecular weight 578.611

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CHAPTER 1 INTRODUCTION

Storage Dry , dark and at 0 - 4°C for short term (days to weeks) or
-20° C for long term (months to years)

Solubility Soluble in DMSO

Route of administration Oral

Melting point Found in results and discussion

1.4.2 Drug profile of Glimepiride [129]

Name of Drug GLIMPERIDE

Category Antidiabetic drug

Agonist indication Type 2 Diabetes mellitus

Structure

IUPAC 3-Ethyl-4-methyl-N-[2-(4-{[(trans-4-
methylcyclohexyl)carbamoyl] sulfamoyl} phenyl)ethyl]-2-oxo-
2,5-dihydro-1H-pyrrole-1-carboxamide

Class of drug Sulfonylurea

Chemical Formula C24H34N4O5S

DOSE 1 to 2 mg once a day

Elimination half-life 5-8 hrs

Trade names Amaryl, others

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CHAPTER 1 INTRODUCTION

Molecular weight 490.6 g/ mol

Melting point 207 to 209°C (405 °F)

Storage Store at room temperature

Solubility Soluble in DMSO

Route of Oral
administration

1.4.3 Marketed Formulation

Table no: 1.4 List of Marketed Formulation

Sr No. Drug name Brand Contents Manufacturer

name

LOBG®-G1 LOBG® lobeglitazone Glenmark pharmaceutical

1.
-G1 sulfate 0.5mg ltd. Mumbai (India )
and
HEALTHON(
glimepiride1
GLENMARK)
mg

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CHAPTER 2

LITERATURE REVIEW
CHAPTER 2 LITERATURE REVIEW

2.1 LITERATURE REVIEW

KARTHIK A et.al (2008) [130]: A simple, fast, and precise reverse phase, isocratic HPLC
method was developed for the separation and quantification of pioglitazone and glimepiride in
bulk drug and pharmaceutical dosage form. The quantification was carried out using Inertsil
ODS (250 × 4.6 mm, 5µ) column and mobile phase comprised of acetonitrile and ammonium
acetate (pH 4.5; 20mM) in proportion of 60:40 (v/v). The flow rate was 1.0 ml/min and the
effluent was monitored at 230 nm. The retention time of pioglitazone and glimepiride were
7.0±0.1 and 10.2±0.1 min respectively. The method was validated in terms of linearity,
precision, accuracy, and specificity, limit of detection and limit of quantitation. Linearity of
pioglitazone and glimepiride were in the range of 2.0 to 200.0µg/ml and 0.5-50µg/ml
respectively. The percentage recoveries of both the drugs were 99.85% and 102.06% for
pioglitazone and glimepiride respectively from the tablet formulation. The proposed method is
suitable for simultaneous determination of pioglitazone and glimepiride in pharmaceutical
dosage form and bulk drug.

Nahed M El-Enany et.al (2012)[131]: A simple reversed phase high performance liquid
chromatographic (RP-HPLC) method was developed and validated for the simultaneous
determination of Rosiglitazone (ROS) and Glimepiride (GLM) in combined dosage forms and
human plasma. The separation was achieved using a 150 mm × 4.6 mm i.d., 5 μm particle size
Symmetry® C18 column. Mobile phase containing a mixture of acetonitrile and 0.02 M
phosphate buffer of pH 5 (60: 40, V/V) was pumped at a flow rate of 1 mL/min. UV detection
was performed at 235 nm using nicardipine as an internal standard. The method was validated
for accuracy, precision, specificity, linearity, and sensitivity. The developed and validated
method was successfully used for quantitative analysis of Avandaryl™ tablets. The
chromatographic analysis time was approximately 7 min per sample with complete resolution
of ROS (tR = 3.7 min.), GLM (tR = 4.66 min.), and nicardipine (tR, 6.37 min). Validation
studies was performed according to ICH Guidelines revealed that the proposed method is
specific, rapid, reliable and reproducible. The calibration plots were linear over the
concentration ranges 0.10-25 μg/mL and 0.125-12.5 μg/mL with LOD of 0.04 μg/mL for both
compounds and limits of quantification 0.13 and 0.11 μg/mL for ROS and GLM respectively.

Nalini Shastri et.al (2014) [132]: There are many analytical methods available for estimation of
glimepiride in biological samples and pharmaceutical preparations. To our knowledge, there is
no specific reverse-phase high-performance liquid chromatography (RP-HPLC) method for

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CHAPTER 2 LITERATURE REVIEW

estimation of glimepiride and its dissolution study in self-nanoemulsifying powder (SNEP)

formulation. Methods: A simple method was carried out on a 5-μm particle octadesyl silane
(ODS) column (250 × 4.6 mm) with acetonitrile: 0.2 M phosphate buffer (pH = 7.4) 40:60 v/v
as a mobile phase at a flow rate of 1 mL/min, and quantification was achieved at 228 nm using
PDA detector.

Mohammed M. Amin et.al (2017) [133] : A new isocratic HPLC method is optimized and
validated for simultaneous determination of Vildagliptin (VLD), Pioglitazone Hydrochloride
(PIO) and Glimepiride (GLIM) in Bulk and tablets (Gliptus® and Amaglust® tablets). The
chromatographic separation was achieved on a reversed-phase analytical column
[Hypersilgold® C18 (10µm, 150 x 4.6 mm) column] at ambient temperature. The separation
was achieved by applying an isocratic elution system using acetonitrile and 0.05M potassium
dihydrogen phosphate buffer, adjusted by orthophosphoric acid to a pH of 3.5 with a ratio of
(45:55 v/v) respectively, at a flow rate of 1.5 ml/min. The UV detection was performed at 200
nm, the drugs calibration curves exhibited linear concentration ranges of 5–75, 3–45 and 1–8
µg/ml for Vildagliptin, Pioglitazone and Glimepiride respectively with correlation coefficients
not less than 0.9996.

K. Poonam et.al (2019)[134]: In the present work, a rapid, accurate and precise RP-HPLC
method for the estimation of Pioglitazone and Glimepiride in tablets dosage form [500/15/2mg]
by selecting the various chromatographic parameters. A new method was developed using
250mm x 4.6 mm, reverse phase C 18 column, 5µm (XBridge C18, 250 X 4.6 mm; 5µ) with
mobile phase of 40 volumes of potassium dihydrogen phosphate (Phosphate buffer pH 6.8) and
60 volumes of Methanol as mobile phase and Methanol as diluent run as isocratic elution. Flow
rate was 1.0 mL-1 with UV detection at 257nm and the injection volume was set at 20µL with
20 minutes of runtime. The method was validated by using various validation parameters like
accuracy, precision, linearity, specificity and stability in analytical solution and robustness. All
the validation parameters were found to be well within the acceptance criteria. Hence the
method can be used for routine estimation of Pioglitazone and Glimepiride tablets [500/15/2
mg].

Tejaswini Kande, et.al (2019)[135]: Glimepiride and Pioglitazone in combination are available
as tablet dosage forms in the ratio of 2:15. A simple, reproducible and efficient
spectrophotometric method has been developed for the simultaneous estimation of Glimepiride
and Pioglitazone in bulk and tablet dosage forms. The sampling wavelengths selected are 227

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CHAPTER 2 LITERATURE REVIEW

nm and 235 nm, Absorption Ratio Method, the sampling wavelengths selected are 251 nm (iso-
absorptivity wavelength) and 235 nm.

Mahmoud M. Sebaiy et.al (2019)[136]: An ISOCRATIC RP-HPLC method has been

developed for rapid and simultaneous separation and estimation of three antidiabetic drugs,
metformin, gliclazide and glimepiride in tablet dosage forms within 6 minutes. Separation was
carried out on a Thermo Scientific® BDS Hypersil C8 column (5µm, 2.50 x 4.60 mm) using a
mobile phase of MeOH : 0.025M KH2 PO4 adjusted to pH 3.20 using ortho - phosphoric acid
(70: 30, v/v) at ambient temperature. The flow rate was 1 mL/min and UV detection was set at
235 nm. The retention time of metformin, gliclazide and glimepiride was noted to be 3.06, 4.33
and 6.00 minutes respectively, indicating a very short analysis time rather than other reported
methods. Also, limits of detection were reported to be 0.05, 1.21 and 0.11 µg/mL for
metformin, gliclazide and glimepiride, respectively, showing a high degree of the method
sensitivity. The method was then validated according to ICH guidelines where it was found to
be accurate, reproducible and robust. Finally, the method was compared statistically with
reference methods indicating that there is no significant difference between them in respect of
precision and accuracy.

Mr. Shoheb S. Shaikh et.al (2021) [137]: A stability-indicating UV spectroscopic and high-
performance liquid chromatography (RP-HPLC) method is developed for the quantification of
Pioglitazone, Glimepiride & Metformin Hydrochloride drug substances. UV spectroscopic
method was developed and validated, the wavelength selected for simultaneous estimation
were 226nm for Pioglitazone, 229nm for Glimepiride and 232nm for metformin hydrochloride.
The isosbestic point found for the analysis was 229nm. Selected mobile phase was a
combination of methanol and water with a ratio of 70% Methanol and 30 % HPLC water with
the flow rate of 0.85ml/min. The analyte was analysed on the C18 HPLC column having the
pore size of 5 microns at room temperature. The method is validated according to ICH
guidelines, the retention time of about 4.0min for metformin, 5.5min for Pioglitazone and
6.8min for Glimepiride was observed. The linearity range with regression co-efficient for
Pioglitazone, Glimepiride & Metformin Hydrochloride is 3-15 μg/mL,0.4-1.2 μg/mL and 100-
500 μg/mL and 0.9998, 0.9991, 0.9991 respectively.

Pamuru Raja, J. C. Thejaswini et.al (2012)[138] :

A simple, specific, accurate stability
indicating RP-HPLC method was developed for simultaneous determination of metformin,
pioglitazone and glimepiride in pure and tablets form using C18 (250 × 4.6 mm, 5µ) Column

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CHAPTER 2 LITERATURE REVIEW

and a mobile phase composing of methanol and phosphate buffer (pH 3) in proportion of 80:20
(v/v). The flow rate was 1.0 ml/min and the effluent were monitored at 230 nm. The retention
time of metformin, pioglitazone and glimepiride were 2.3 ±0.1, 3.8±0.2 and 6.2±0.2 min
respectively. Drugs were subjected to acidic, alkali, neutral hydrolysis, oxidation, photolytic
and UV degradation. The degradation studies indicated the susceptibility of drugs to various
degradations. The method was statistically validated for accuracy, precision, linearity and
forced degradation. Quantitative and recovery studies of the dosage form were also carried out
and the % RSD was found to be less than 1. The developed method is simple, rapid and accurate
and hence can be used for routine quality control analysis.

[139]
FREDDY H. HAVALDAR et.al (2010) : A simple, specific, accurate and economical
gradient reversed phase liquid chromatographic (RP-HPLC) method was developed and
subsequently validated for the determination of glimepiride, rosiglitazone and pioglitazone
hydrochloride. Separation was achieved with a nucleodur C18 column having 250x4.6 mm i.d.
with 5 µm particle size and water HPLC grade adjusted to pH 3.0 using diluted orthophosphoric
acid and acetonitrile (80:20v/v) with gradient program as eluent at a constant flow rate of 0.8
ml per min. UV detection was performed at 215 nm. The retention time of glimepiride,
rosiglitazone and pioglitazone hydrochloride were about 17.9 min, 6.31 min and 8.24 min
respectively. This method is simple, rapid and selective and can be used for routine analysis of
antidiabetic drugs in pharmaceutical preparation. The proposed method was validated and
successfully used for estimation of glimepiride, rosiglitazone and pioglitazone hydrochloride
in the pharmaceutical dosage form.

Vinod Kumar K et.al (2011) [140]: The present work deals with the development of a reliable
method for the simultaneous estimation of Pioglitazone and Glimepiride in a combined tablet
dosage form by using RP – HPLC method and Spectrophotometric method. Both methods were
validated and compared for sensitivity and linearity. The RPHPLC method utilized Gliburide
(Glibenclamide) as an internal standard and the mobile phase composition was Methanol and
water which gave a retention time of 4.34 min and 5.19 min for Glimepiride and Pioglitazone
respectively. The linearity range was between 1 -15μg/ ml and 3 - 45μg/ml for glimepiride and
pioglitazone respectively. The accuracy of the method was found to be in the range of 98 – 102
%. The precision studies were carried out on three concentrations in three replicates and the %
RSD was found to be less than 2%. The method proved specificity for both drugs. The UV
spectrophotometric method utilized simultaneous equation for the estimation of the drugs. The
linearity range was between 3- 24 μg/ml for glimepiride and 4 – 32 μg/ml for pioglitazone. The

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CHAPTER 2 LITERATURE REVIEW

recovery was in the range of 103 – 110 %. The precision studies were carried out using three
concentrations in three replicates and the %RSD was found to be less than 2 %. Both these
methods have been successively applied to pharmaceutical dosage formulation and were
validated according to ICH guidelines

N.Ramathilagam et.al (2014) [141]: A simple, accurate, precise and linear Isocratic RP-HPLC
has been developed and subsequently simultaneous for the determination of glimepiride,
pioglitazone Hcl and metformin HCl in pharmaceutical dosage form. Kromosil C18 (150mm
X4.6 mm) 5µ with flow rate of 1ml/min by using JASCO PU-1580 and UV/VIS JASCO UV-
1570 at 217nm. The separation was carried out using a mobile phase consisting of the mixture
of methanol and 25mM phosphate buffer (pH-2.0) in the ratio of 50:50. The retention time for
glimepiride, metformin HCl and pioglitazone HCl were found to be 2.24, 3.76 and 10.20 min.
the correlation coefficient was found to be 0.999 for GLI, 0.9993 for PIO and 0.9997 for MET.
The mean percentage recovery was found to be 98.58 (GLI), 98.30 (PIO) and 98.87 (MET).
The percentage the accuracy of the drugs were found to be near to 100% representing the
accuracy of the method. The proposed method was also validated and applied for the analysis
of the drugs on tablet formulations.[141]

Deepti Jain et.al (2008) [142] : A simple, precise, rapid, and reproducible reversed-phase high-
performance liquid chromatography method is developed for the simultaneous estimation of
metformin hydrochloride (MET), pioglitazone hydrochloride (PIO), and glimepiride (GLP)
present in multicomponent dosage forms. Chromatography is carried out isocratically at 25°C
± 0.5°C on an Inertsil-ODS-3 (C-18) Column (250 × 4.60 mm, 5 µm) with a mobile phase
composed of methanol–phosphate buffer (pH 4.3) in the ratio of 75:25 v/v at a flow rate of 1
mL/min. Detection is carried out using a UV-PDA detector at 258 nm. Parameters such as
linearity, precision, accuracy, recovery, specificity, and ruggedness are studied as reported in
the International Conference on Harmonization guidelines. The retention times for MET, PIO,
and GLP are 2.66 + 0.5 min, 7.12 + 0.5 min, and 10.17 + 0.5 min, respectively. The linearity
range and percentage recoveries for MET, PIO, and GLP are 10–5000, 10–150, and 1–10
µg/mL and 100.4%, 100.06%, and 100.2%, respectively. The correlation coefficients for all
components are close to 1. The relative standard deviations for three replicate measurements
in three concentrations of samples in tablets are always less than 2%.

Saurabh Chaudhari et.al (2022) [143]: As per requisition of current regulatory requirements, a
simple, rapid, and sensitive method by 33 factorial QbD approach was established and

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CHAPTER 2 LITERATURE REVIEW

validated for Pioglitazone (PGZ) Glimepiride (GPR) by RP-HPLC. Method: A simple RP-
HPLC method has been developed and validated with different parameters such as linearity,
precision, repeatability, LOD, LOQ, accuracy as per International Conference for
Harmonisation guidelines (Q2R1). Statistical data analysis was done for data obtained from
different aliquots Runs on Agilent Tech. Gradient System with Auto injector, UV (DAD) &
Gradient Detector. Results: Equipped with Reverse Phase (Agilent) C18 column (4.6 mm ×
100 mm; 2.5µm), a 20µl injection loop and UV730D Absorbance detector at 231nm
wavelength and running chemstation 10.1 software and drugs along with degradants were
separated via Methanol: (0.1% OPA) Water (70:30) of pH 3.2 as mobile phase setting flow rate
0.7 ml/min at ambient temperature. The developed method was found linear over the
concentration range of 15-75 μg/ml for PGZ and 2-10 μg/ml for GPR, while detection and
quantitation limit was found to be 1.39 μg/ml and 0.28 μg/ml as LOD and 3.85 μg/ml and 0.77
μg/ml respectively for PGZ and GPR .

Ravi Sharma et.al (2011) [144]: A simple reverse phase liquid Chromatographic method has
been developed and subsequently validated for simultaneous determination of Pioglitazone and
Glimepiride in combination. The separation was carried out using a mobile phase of phosphate
buffer (pH-4.5): Acetonitrile (45:55) v/v and using methanol as diluent. The column used was
Inertsil ODS (250 mm x 4.6 mm i.d., 5μm) with flow rate of 1 ml/min using UV detection at
225 nm. The described method was linear over a concentration range of 5-50µg/ml and 5-25
µg/ml for the assay of Pioglitazone and Glimepiride respectively. The retention times of
Pioglitazone and Glimepiride were found to be4.6 and 7.7min respectively. Results of analysis
were validated statistically and by recovery studies. The results of the study showed that the
proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the
routine determination of Pioglitazone and Glimepiride bulk drug and in its pharmaceutical
dosage form.

[145]
Shoheb S Shaikh et.al (2020) : A stability-indicating UV spectroscopic and high-
performance liquid chromatography (RP-HPLC) method is developed for the quantification of
Pioglitazone, Glimepiride & Metformin Hydrochloride drug substances. UV spectroscopic
method was developed and validated, the wavelength selected for simultaneous estimation
were 226nm for pioglitazone, 229nm for glimepiride and 232nm for metformin hydrochloride.
The isosbestic point found for the analysis was 229nm. Selected mobile phase was a
combination of methanol and water with a ratio of 70% Methanol and 30 % HPLC water with
the flow rate of 0.85ml/min. The analyte was analysed on the C18 HPLC column having the

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CHAPTER 2 LITERATURE REVIEW

pore size of 5 microns at room temperature. The method is validated according to ICH
guidelines, the retention time of about 4.0min for metformin, 5.5min for Pioglitazone and
6.8min for Glimepiride was observed. The linearity range with regression co-efficient for
Pioglitazone, Glimepiride & Metformin Hydrochloride is 3-15 µg/mL,0.4-1.2 µg/mL and 100-
500 µg/mL and 0.9998, 0.9991, 0.9991 respectively.

Piyusha D. Gulhane et.al (2023) [146]: A simple, rapid, reliable, precise, accurate, sensitive
and selective analytical method for the estimation of lobeglitazone. In human plasma and using
as an internal standard (IS). Lobeglitazone is a novel thiazolidinedione (TZDs) based
peroxisome proliferator-activated receptor (PPAR) agonist, used for the management of type-
2 diabetes. After mixing the A, dissolved in acetonitrile, with a plasma sample containing
lobeglitazone, the method was developed using acetonitrile-methanol-water (6:3:1, v/v) 10 μL
of supernatant was injected into the HPLC system. The method showed good linearity
subsequently, serial dilutions of five different concentrations ranging between 3.12–50 ppm
were made, ultra-sonicated and then analysed as per the chromatographic condition in section
5.x. for Plasma (r 2 ≥ 0.9996). The mean percent extraction recovery of lobeglitazone was 90.8
% for plasma. Freshly prepared stock solution of lobeglitazone (100 ppm) was analysed were
tested and evaluated. The intra-day precision of plasma ranged from 0.233, 0.290, 1% (RSD),
respectively, and the inter-day precision of plasma ranged from 1.5 to 0.115 and 0.99, 1%,
respectively. This method is simple, sensitive, and applicable for the pharmacokinetic study of
lobeglitazone in human plasma. Most of the plasma concentrations of lobeglitazone were below
the LOQ because the lobeglitazone is extensively metabolized. The method was developed
using acetonitrile-methanol-water (6:3:1, v/v). The peaks obtained for the drug of interest by
the present method was symmetrical in nature with acceptable tailing factor and from the
plasma endogenous proteins by Protein precipitation Extraction. The retention time of
lobeglitazone was shorter and proves that the method is rapid.

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CHAPTER 2 LITERATURE REVIEW

2.1.1 Patent search for Glimepiride

Table 1.5 Patent Search Analysis Report Summary

Sr Patent Title of Name of Date of Summary

No. application patent inventor filling
number/patent
Number with
Country
1. EP1928421A2 Formulation Delsams 2006- The invention relates,
s containing Marta 05-30 in general, to new
glimepiride Tarruella formulations and
and/or its dosage units
salts containing
glimepiride of
defined particle size
and/or salts thereof
that are useful for the
therapeutic treatment
(including
prophylactic
treatment) of
mammals, including
humans, without the
need for micronizing
any excipients
together with the
glimepiride that
advantageously saves
time, energy and
resources and a
process for making
the same. In
particular, the
invention can be

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CHAPTER 2 LITERATURE REVIEW

useful for the

treatment of diabetes
2 1WO200610369 A novel Venkat 2005- The present invention
0A1 process for asubra 05-18 discloses a novel
preparation manian process for
of Radhak purification of trans-
substantiall rishnan 4-methyl
y pure Tarur; cyclohexylamine
glimepiride HC1 and 4-[2-(3-
Ethyl-4-methyl-2-
carbonyl pyrrolidine
amido) ethyl]
benzene sulfonamide
used in the synthesis
of 3-Ethyl-2,5-
dihydro-4-methyl-N-
[2-[4-[(trans-4-
methyl
cyclohexyl)amino]ca
rbonyl]amino]sulfon
yl]phenyl]ethyl]-2-
oxo-1H-pyrrole-1-
carboxamide(I),
popularly known as
Glimepiride. The
present invention
also discloses a novel
purification of
Glimepiride Form I
(I), having the
undesired cis isomer
below 0.15%.
Glimepiride (I) is

SCHOOL OF PHARMACY PAGE 55

CHAPTER 2 LITERATURE REVIEW

useful in the
treatment of diabetes
mellitus.

3. US-7282517-B2 Method of RADL 2003/02/ A method of

manufacturi STANIS 21 manufacturing
ng LAV glimepiride of
glimepiride (CZ) formula I wherein
and the JARRA trans-4-
respective H methylcyclohexylam
intermediate KAMA ine pivalate of
L (CZ) formula VII is
reacted, either
directly or after
conversion to trans-4
methylcyclohexylam
ine or to its another
salt, with an alkyl [4-
(2-{[(3-ethyl-4-
methyl-2-oxo-2,5-
dihydro-1H-pyrrol-1
yl)carbonyl]amino)et
hyl) phenyl]-sulfonyl
carbamate of general
formula IV wherein
R is a C1-C5 alkyl,
giving glimepiride of
formula I, trans-4-
Methylcyclohexylam
ine pivalate of
formula VII

SCHOOL OF PHARMACY PAGE 56

CHAPTER 2 LITERATURE REVIEW

2.1.2 Patent search for lobeglitazone sulfate

Sr Patent application Title of Name of Date of Summary

No. number/patent patent inventor filling
Number with
Country
1 WO2017073897A1 Pharma Eun Ji 2016-08- The present invention
ceutical Shin 24 provides a
compos pharmaceutical
ition composition comprising
compris metformin or a
ing pharmaceutically
metfor acceptable salt thereof
min and and lobeglitazone or a
lobeglit pharmaceutically
azone acceptable salt thereof
and a pharmaceutical
formulation comprising
the same. The
pharmaceutical
composition can exhibit a
synergistic effect on
glycemic control and can
block the potential of side
effects, and the
pharmaceutical
formulation can be
provided in the form of a
combination formulation
that is able to be
administered once a day
to thus improve
medication compliance.

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CHAPTER 2 LITERATURE REVIEW

2. WO2016072748A1 Pharma Shin Jung 2015-11- The present invention

ceutical Park 04 relates to pharmaceutical
compos compositions for oral
itions administration
compris comprising
ing lobeglitazone. The
lobeglit pharmaceutical
azone compositions of the
for oral present invention have
adminis excellent dissolution rate
tration and express prompt
pharmacological effects
by comprising cellulose
derivatives.

SCHOOL OF PHARMACY PAGE 58

CHAPTER 3

AIM, OBJECTIVE AND

RATIONALE
CHAPTER 3 AIM, OBJECTIVE AND RATIONAL

AIM

The development and validation of an analytical method for the simultaneous estimation of
Lobeglitazone Sulfate and Glimepiride in both bulk and tablet dosage forms.

OBJECTIVE

To develop a novel, specific, precise, accurate, and cost-effective analytical method for
simultaneously detecting Lobeglitazone Sulfate and Glimepiride in combination, and
subsequently validate the method using HPLC.

To perform all the validation parameters according to the ICH guidelines.

RATIONALE

The combination of Lobeglitazone Sulfate and Glimepiride is utilized in managing Type 2

Diabetes Mellitus.

A simultaneous estimation method for Lobeglitazone Sulfate and Glimepiride is currently

unavailable.

Moreover, there is a lack of scientific evidence available to support the development and
validation of a method for Lobeglitazone Sulfate and Glimepiride using HPLC.

Hence, it is valuable to undertake the development and validation of an HPLC method for
quantifying Lobeglitazone Sulfate and Glimepiride in combination for the treatment of
Diabetes Mellitus.

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CHAPTER 4

MATERIALS AND
METHODS
CHAPTER 4 MATERIALS AND METHODS

4.1 Materials
Table no: 1.6 List of Materials

Sr.
Material Name Category Supplier
No.
1 Lobeglitazone API Akums Drugs and
sulphate Pharmaceutical Ltd.
2 Glimepiride API Akums Drugs and
Pharmaceutical Ltd.

3 Methanol AR Solvent Avantors Performance Materials

grade India Pvt Ltd

4 HPLC grade solvent Avantors Performance Materials

water India Pvt Ltd
solvent
5 Hplc grade Avantors Performance Materials
Acetonitrile India Pvt Ltd
6 HPLC grade solvent Avantors Performance Materials
methanol India Pvt Ltd

7 0.5 µm membrane ------- PALL Life Sciences

filter
8 Phosphate buffer solvent

9 Potassium ------ Laboratory Aatur Rasayan

dihydrogen
orthophosphate

10 Trimethylamine ------

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CHAPTER 4 MATERIALS AND METHODS

4.2 Equipment

Table no: 1.7 List of Equipment

Sr. Equipment Use Company

No.
1 UV Spectrophotometer Method shimadzu
development
and
validation

2 HPLC Method Shimadzu(I series)

development
and
validation

3 Filtration Assembly Filtration PCI analytics

4 Sonicator Sonication Navyug

4.3 MATERIALS AND METHODS

Materials and Reagents:
Lobeglitazone sulfate and Glimepiride were obtained as a gift sample from Akums Drugs and
Pharmaceuticals Ltd. located in Haridwar, Uttarakhand, India. Lobeglitazone sulfate and
Glimepiride tablet formulation in combined dosage form was procured from the local drug
store. Analytical grade Phosphate buffer, Potassium dihydrogen orthophosphate,
trimethylamine, orthophosphoric acid, Methanol, Acetonitrile and Water were used as solvent.
The solvents were of HPLC grade obtained from Avantor Performance Materials India Pvt.
Ltd.

Instrumentation
Spectrophotometric method development was performed on Shimadzu model (UV 1900i) used
for selection of detection wavelength. Infrared spectroscopy study of standard drug was carried
out on Bruker Optics. The analysis was performed by using HPLC system, specifically the
Shimadzu (i-series) HPLC model containing the LC-2050 pump and equipped with Photo
Diode Array detector, automatic sample injector and column thermostat.

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CHAPTER 4 MATERIALS AND METHODS

Preparation of buffer

Buffer was prepared by dissolving 0.68g of Potassium dihydrogen orthophosphate in 500mL

of water and add 1 ml of trimethylamine adjusts the pH 3.0 using 1% o-phosphoric acid solution
then filtered followed by the degassing of the solution.

Preparation of Mobile phase

HPLC grade Acetonitrile and Buffer solution was taken in the ratio 60:40 (v/v). The mobile
phase was filtered with 0.45 µ membrane filter and was sonicated for 20 minutes.

Preparation of standard stock solution.

Accurately weighed 2.5 mg of lobeglitazone sulfate and 5 mg of glimepiride was taken and
transferred it to a 100 ml volumetric flask. The drug was dissolved in methanol to obtain a
solution with a concentration of 50 μg/ml and 100 μg/ml respectively. Above prepared solution
of lobeglitazone sulfate and glimepiride were used as standard.

Preparation of working standard stock solution

1 ml of Lobeglitazone sulfate and glimepiride working standards were accurately weighed and
transferred from 50 ppm and 100pm respectively. Solution into a 10 ml volumetric flask and
dissolved in Methanol and made up to the volume with the same solvent to produce 10 μg/ml
of Lobeglitazone sulfate and glimepiride.

Sample solution preparation

Twenty tablets were weighed, made into fine powder in a mortar with pestle and average weight
was taken. Accurately weighed powder equivalent to average weight of each tablet were taken
in a 100 ml volumetric flask and methanol was added and sonicated to mix uniformly. The
final volume was adjusted with mobile phase to get the sample solution. (100 μg/ml) .

METHOD DEVELOPMENT
Various chromatographic conditions were explored to enhance the resolution and efficiency of
the chromatographic system. Parameters such as mobile phase composition, detection
wavelength, column type, column temperature, and mobile phase pH were carefully optimized.
According to the literature.

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CHAPTER 4 MATERIALS AND METHODS

Lobeglitazone sulfate and Glimepiride show high solubility in methanol and Dimethyl
sulfoxide (DMSO). The solubility of these compounds was assessed using various dilutions of
methanol and acetonitrile, resulting in the choice of methanol as the solvent for this study.
Based on UV-Visible spectrophotometric results, a detection wavelength of 247 nm for
Lobeglitazone sulfate and 226 nm for Glimepiride was chosen due to their maximum
absorbance at these wavelengths. Additionally, 230 nm was selected as the common
wavelength for the simultaneous estimation of both drugs, as they elute in the same mobile
phase at maximum absorbance. Furthermore, a chromatogram was observed at 254 nm using a
PDA detector

CHROMATOGRAPHIC CONDITION

Table 1.8: Optimized Chromatographic conditions of Lobeglitazone sulfate and

Glimepiride.

Parameters Method

Stationary phase (column) shim-pack 5µm C18 column

Mobile Phase Acetonitrile: Phosphate Buffer pH

3.0 (70:30%v/v)

Flow rate (ml/min) 1.0ml/min

Run time (minutes) 30.0

Column temperature (°C) 40ºC

Volume of injection loop (uL) 2uL

The typical chromatogram obtained from final HPLC conditions are depicted in Figure1.8 .

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CHAPTER 4 MATERIALS AND METHODS

Figure 1.18: Typical chromatogram of Lobeglitazone sulfate and Glimepiride by optimized

Method validation

As per ICH guidelines, the method validation parameters checked were system suitability,
specificity, precision, accuracy, linearity, and robustness, limit of detection and limit of
quantification.

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CHAPTER 5
RESULT AND
DISCUSSION
CHAPTER 5 RESULT AND DISCUSSION

5.1 METHOD VALIDATION

The proposed method was validated as per ICH guidelines Q2 (R1).

Linearity and range

The linearity of detector response was determined by constructing a graph correlating the concentration
versus the area of Lobeglitazone Sulfate and Glimepiride standards, and subsequently calculating the
correlation coefficient. Solutions containing Lobeglitazone Sulfate ranging from approximately 10 to 50
µg/ml and Glimepiride ranging from 20 to 100 µg/ml were prepared, and injections of these solutions were
made into the HPLC system to assess their respective target concentrations.

System Suitability

Standard solutions of Lobeglitazone Sulfate and Glimepiride were prepared following the procedure and
injected into the HPLC system six times. The system suitability parameters were assessed using standard
chromatograms, calculating the % Relative Standard Deviation (% RSD) of retention times, tailing factor,
theoretical plates, and peak areas from five replicate injections.

Precision: The system precision of the test method was evaluated by injecting triplicate determinations of
the test sample against a qualified reference standard (n=3). The % Relative Standard Deviation (% RSD)
was calculated for both interday and intraday precision. The % RSD should not exceed 2% to ensure
acceptable precision.

Accuracy:

Accuracy was assessed using tablet samples with known concentrations of the drugs at 50%, 100%, and
150% of the expected levels. Each concentration was injected six times, and the assay was conducted
according to the developed method. The % recovery and the calculated amount present or recovery were
determined from these experiments. The results of the recovery study are presented in Table 1.15

Limit of Detection (LOD)

LOD of lobeglitazone sulfate was determined by the following equation.

LOD =3.3×σ/S
Where, σ= Standard deviation of Y-intercepts
S= Slope
Limit of Quantification (LOQ)

LOQ of lobeglitazone sulfate was determined by the following equation.

LOQ = 10xσ/S

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CHAPTER 5 RESULT AND DISCUSSION

Where, S= slope
σ= Standard deviation of Y-intercepts15

Robustness

To determine the robustness of the method, experimental conditions such as the composition of the mobile
phase, pH of the mobile phase, and flow rate of the mobile phase were altered and the chromatographic
characteristics were evaluated. The altered conditions are detailed in Table 1.17

5.2 RESULT

5.2.1 SOLUBILITY

Lobeglitazone sulfate exhibits solubility in methanol and DMSO, with limited solubility in acetone. On the
other hand, Glimepiride demonstrates very slight solubility in methanol, solubility in DMSO, and
insolubility in acetonitrile. Both compounds share solubility in methanol.

Table 1.9: Results of solubility for lobeglitazone sulfate and Glimepiride

CHARACTERISTICS LOBEGLITAZONE GLIMEPIRIDE

SULPHATE

Appearance Solid white powder White powder

Melting Range 104 to108°C 201 to 205°C

Solubility Methanol Very soluble Methanol Very slightly

soluble
DMSO Very soluble DMSO Soluble

Acetone Sparingly Acetonitrile Insoluble

soluble

5.2.2 IR IDENTIFICATION

FOURIER TRANSFORM INFRARED (FTIR) STUDY

Lobeglitazone sulfate: The FTIR absorption spectrum of sample obtained for lobeglitazone sulfate. By the
interpretation of the spectra Peak at 1646 cm-1attribution to C=H stretching vibration, peak at 3000 cm-1 was assigned

SCHOOL OF PHARMACY PAGE 63

CHAPTER 5 RESULT AND DISCUSSION

to C-S stretching, peak at 1214 cm-1was assigned C-N, Stretching and peak at 1152 cm-1was assigned S=0 Stretching
which conform the structure of lobeglitazone sulfate.

Fig no: 1.19 FTIR of lobeglitazone sulfate

Sr no. Stretching Report wavenumber Observed wavenumber

1 C=O 1600-1850 cm-1 1646 cm-1

2 C-H 2850-3300 cm-1 3000 cm-1

3 C-N 1000-1400 cm-1 1214 cm-1

4 S=O 1150-1380 cm-1 1152 cm-1

Glimepiride: By the interpretation of the spectra Peak at 3368 cm-1 attribution to N-H stretching vibration, peak at
1671 cm-1was assigned to C=O stretching, peak at 1540 cm-1was assigned C=C Stretching, peak at 3006 cm-1 was
assigned C-H, peak at 1273 cm-1 was assigned S=O, peak at 1152 cm-1 was assigned C-N Stretching which conform
the structure of glimepiride

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CHAPTER 5 RESULT AND DISCUSSION

Fig no: 1.20 FTIR of Glimepiride

Functional group Reported Observed wavenumber

wavenumber

N-H 3300-3500 cm-1 3368 cm-1

C=O 1650-1700 cm-1 1671 cm-1

C=C 1400-1600 cm-1 1540 cm-1

C-H 3000-3100 cm-1 3006 cm-1

S=O 1100-1300 cm-1 1273 cm-1

C-N 1000-1200 cm-1 1152 cm-1

5.2.3 SELECTION OF WAVELENGTH (λ max)

Lobeglitazone sulfate: For the selection of analytical wavelength range for method 10 µg/ml lobeglitazone sulfate
was scanned in the spectrum mode from 200 nm to 400 nm against methanol as blank. From the above scan
selected wavelength maxima was 247 nm.

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CHAPTER 5 RESULT AND DISCUSSION

Fig no: 1.21 UV spectra of lobeglitazone sulphate

Glimepiride: For the selection of analytical wavelength range for method 10 µg/ml metformin was scanned in the
spectrum mode from 200 nm to 400 nm against methanol as blank. From the above scan selected wavelength
maxima was 226 nm.

Fig no: 1.22 UV spectra of glimepiride

5.3 VALIDATION OF HPLC

5.3.1 Linearity and range

A strong linear correlation between concentration and peak areas was observed over a concentration range of 10-50
µg/ml for Lobeglitazone Sulfate and 20-100 µg/ml for Glimepiride. The correlation coefficients obtained were 0.9972
for Lobeglitazone Sulfate and 0.9988 for Glimepiride, both exceeding 0.999, indicating excellent linearity.

Table 1.11: Results of Linearity parameter for lobeglitazone sulfate and Glimepiride

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CHAPTER 5 RESULT AND DISCUSSION

Average
Conc.
µg/ml Area Area Area (n=3) SD %RSD

Lobeglitazone sulfate

10 170537 172564 170537 171212.67 1170.29 0.68

20
305265 305285 305295 305281.66 15.27 0.005

30 422643 422694 422695 422677.33 29.73 0.007

40 578090 578095 579094 578426.33 578.22 0.099

50 680909 680906 682914 681576.33 1158.45 0.169

Average
Conc.
µg/ml Area Area Area (n=3) SD %RSD

GLIMEPIRIDE

20 70816 71840 71949 71535 625.05 0.87

40 261193 262039 261949 261727 464.64 0.17

60 456970 459502 456939 457803.66 1470.88 0.32

80 688623 683461 687192 686425.33 2665.03 0.38

100 910317 916434 916462 914404.33 3539.76 0.38

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CHAPTER 5 RESULT AND DISCUSSION

Lobeglitazone sulphate
800000 y = 12939x + 43673
700000 R² = 0.9972
600000
PEAK AREA 500000
400000
300000
200000
100000
0
0 10 20 30 40 50 60
CONC. (µg/ml)

Figure no: 1.20 Calibration Curve of lobeglitazone sulfate

Glimepiride
1000000
y = 10552x - 154752
800000 R² = 0.9988
PEAK AREA

600000

400000

200000

0
0 20 40 60 80 100 120
CONC. (µg/ml)

Figure no: 1.21 Calibration Curve of Glimepiride

5.3.2 System suitability test

Five replicate injections of a single standard solution were performed on an HPLC system to assess parameters
including the number of theoretical plates, peak tailing, and retention time. The results obtained are presented in
Table 1.12.
Table: 1.12Results System Suitability for lobeglitazone sulfate and Glimepiride.

Parameters Proposed Method Standard

Value
Glimepiride Lobeglitazone

Theoretical 8992±1.923538 10670±40693.2 Should be >

plates (N) 2000

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CHAPTER 5 RESULT AND DISCUSSION

Tailing 1.179±0.002739 1.161±0.003347 T ˂ 1.5

Factor (T)

Height 3445 1139 --

Area 31826 12596 --

5.3.3 Repeatability

Six replicate injections at the same concentration were analyzed on the same day, and the % Relative Standard
Deviation (% RSD) was calculated, as illustrated in Table 1.13.

Table: 1.13 Result Repeatability for lobeglitazone sulfate and Glimepiride.

Lobeglitazone sulfate Glimepiride

Conc. Area RT Conc. Area RT

µg/ml µg/ml
(n=6) (min) (min)

30 425011 4.231 60 226970 7.884

30 424990 4.23 60 226834 7.874

30 423968 4.23 60 226970 7.891

30 425477 4.232 60 226984 7.882

30 424953 4.231 60 226735 7.891

30 425789 4.23 60 226974 7.892

Mean 425031.3 4.230 Mean 226911.2 7.885

SD 618.3225 0.0008 SD 103.1163 0.0070

% RSD 0.145477 0.019 % RSD 0.045443 0.089

5.3.4 Precision: The % Relative Standard Deviation (% R.S.D.) for Lobeglitazone Sulfate and Glimepiride assay
during method precision ranged from 0.936% to 0.782% and 0.46% to 0.24%, respectively. These results demonstrate
excellent precision of the method.

Table: 1.14 Results of Method Precision parameter for Lobeglitazone sulfate and Glimepiride

LOBEGLITAZONE SULFATE

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CHAPTER 5 RESULT AND DISCUSSION

INTRADAY PRECISION INTERDAY PRECISION

Conc. Peak area ± SD Conc.

µg/ml (n=3) %RSD µg/ml peak area ± SD %RSD

10 171029±599.54 0.35 10 176660.3±1654.94 0.936

30 424064.333±2366.34 0.558 30 423845.7±1013.97 0.239

50 681852.3±1031.65 0.151 50 678720.7±5311.28 0.782

GLIMEPIRIDE

INTRADAY PRECISION INTERDAY PRECISION

Conc. Peak area ± SD Conc.

µg/ml (n=3) %RSD µg/ml Peak area ± SD %RSD

20 71535 ± 625.0528 0.873 20 716495±3296.166 0.46

456606.667
60 ±461.061095 0.100 60 458570.3±1534.935 0.33

914856±
100 6891.52748 0.753 100 913933±2268.539 0.24

5.3.5 Accuracy

The percent recovery of Lobeglitazone Sulfate ranged from 101.5% to 100.6%, while for Glimepiride samples, it
ranged from 101.5% to 100.5%. These findings indicate the good accuracy of the method. Detailed results are
provided in Table 1.15.

Table no : 1.15 Results of accuracy of the HPLC method developed for Lobeglitazone sulfate and Glimepiride

Sample Standard Area Amount Mean

Spiked %
% recovere (n=3)±SD
Level (µg/ml) (µg/ml) amount (n=3) recovery
d

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CHAPTER 5 RESULT AND DISCUSSION

Lobeglitazone sulfate

20 10
426359 29.58 98.59
50% 20 10 30 99.29±0.6
431206 29.95 99.84
38
20 10
429654 29.83 99.44
20 20
548569 39.02 97.55
100% 20 20 40 98.38±0.8
557564 39.72 99.29
76
20 20 552506
39.33 98.31
20 15
677896 49.02 98.03
150% 20 15 50 685241 98.81±0.6
49.58 99.17
76
20 15
679203 49.12 98.23

Sample Standard Spiked Area Amount % Mean

%
Level (µg/ml) (µg/ml) amount (n=3) recovered recovery (n=3)±SD

GLIMEPIRIDE

40 20 483436 60.48 100.80

100.96±0.1
50% 40 20 60 485237 60.65 101.08
45
40 20 484740 60.60 101.01

40 40 699534 80.96 101.20

99.75±1.25
80
3
100% 40 40 681023 79.21 99.01

40 40 681405 79.24 99.05

40 60 917534 101.62 101.62

100.25±1.1
150% 40 60 100 895620 99.54 99.54
78
40 60 896405 99.62 99.62

5.3.6 Limit of Detection (LOD) and Limit of Quantification (LOQ)

The Limit of Detection (LOD) for Lobeglitazone Sulfate was determined to be 1.320719 µg/ml, while for
Glimepiride, it was found to be 4.402395 µg/ml. Additionally, the Limit of Quantification (LOQ) was calculated as
18.41433 µg/ml for Lobeglitazone Sulfate and 61.38109 µg/ml for Glimepiride.

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CHAPTER 5 RESULT AND DISCUSSION

Table no: 1.16 LOD&LOQ for lobeglitazone sulfate and Glimepiride.

Parameter LOD LOQ

Lobeglitazone 1.320µg/ml 4.402µg/ml

sulfate

Glimepiride 1.841µg/ml 61.381µg/ml

5.3.7 Robustness:

To assess the robustness of the method, variations were introduced in experimental conditions such as the
composition and pH of the mobile phase, as well as the flow rate. Subsequent evaluation of chromatographic
characteristics revealed no significant changes under altered conditions.

Table no : 1.17 Results Robustness of parameter for Lobeglitazone sulfate and Glimepiride

Sr.no. Parameters Conditions RT(min) Area

Lobeglitazone sulfate

0.9 3.754 1279703

1. Flow rate (ml/min)
1.1 3.953 1274959

Mobile phase 65.35.00 3.659 1059383

2.
(70:30) 75.25.00 3.832 1495950

35 3.823 1099438
3. Temperature (°C)
45 3.823 1649599

Sr.no. Parameters Conditions RT Area

(min)
Glimepiride
1. Flow rate 0.9 7.139 145914

1.1 7.017 159495

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CHAPTER 5 RESULT AND DISCUSSION

2. Mobile phase 65.35.00 7.294 129495

(70:30)
75.25.00 7.425 169494

3. Temperature 35 6.985 105954

(°C) 45 7.659 201040

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CHAPTER 6
SUMMARY AND
CONCLUSION
CHAPTER 6 SUMMARY AND CONCLUSION

6.1 summary

Table no: 1.18 Summary of Optimized Chromatographic conditions of Lobeglitazone

sulphate and Glimepiride

Parameters Method

Stationary phase C18 column

Mobile phase Acetonitrile: phosphate buffer pH 3.0 (70:30 v/v)

Flow rate (ml/min) 1.0 ml/min

Run time 10 min

Column temperature 40ºC

Volume of injection (ml) 2 uL

Detection wavelength (nm) 254 nm

Retention time (min) Lobeglitazone sulfate: 7.831±0.019209 min

Glimepiride: 6.089 min±0.013737 min

Table no: 1.19 Summary of validation parameters by HPLC method which results indicate the
validity of the method

Sr.no Parameters Result

Lobeglitazone
Glimepiride
sulfate

1. Linearity and Range 10-50(µg/ml) 20-100 (µg/ml)

2. Precision:

Intraday: Intraday:

10(µg/ml) 171029±599.54 20 (µg/ml) 71535 ± 625.0528

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CHAPTER 6 SUMMARY AND CONCLUSION

30(µg/ml) 60 (µg/ml) 456606.667

424064.333±2366.34 ±461.061095

50 (µg/ml) 100 (µg/ml) 914856±

681852.3±1031.65 6891.52748

Intermediate: Intermediate:

10 (µg/ml) 176660.3±1654.94 20(µg/ml) 176660.3±1654.94

30 (µg/ml) 423845.7±1013.97 60 (µg/ml) 423845.7±1013.97

50 (µg/ml) 678720.7±5311.28 100 (µg/ml) 678720.7±5311.28

3 Repeatability: 425031.3±618.3225 Repeatability:

30 µg/ml 60µg/ml 226911.2 ±103.1163

4. Accuracy:

50% 99.29 ±0.638 100.96±0.145

100% 98.38 ± 0.876 99.75±1.253

150% 98.81 ± 0.676 100.25±1.178

5. Limit of detection 1.320µg/ml 4.402µg/ml

7. Limit of 18.414µg/ml 61.381µg/ml

Quantification

8. Robustness Robust Robust

6.2 Conclusion

The method underwent validation for accuracy, repeatability, and precision. Lobeglitazone Sulfate and
Glimepiride displayed a strong linear relationship within their respective concentration ranges of 10-
50μg/mL and 20-100μg/mL, with correlation coefficients of 0.9972 and 0.9988, respectively. Precision
assessment demonstrated the method's reproducibility. Assay experiments verified the presence of
Lobeglitazone Sulfate and Glimepiride in the tablet dosage form without interference from excipients.
Overall, a reversed-phase high-performance liquid chromatographic method, designed to be simple,
rapid, reliable, robust, and optimized, was successfully developed and validated in accordance with the

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CHAPTER 6 SUMMARY AND CONCLUSION

International Conference on Harmonization guidelines for the estimation of Lobeglitazone Sulfate and
Glimepiride.

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CHAPTER 7
REFERENCE
CHAPTER 7 REFERENCES

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 APPENDIX A

COMMENT SHEET

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 APPENDIX B

POSTER PRESENTATION CERTIFICATES

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