CHAPTER 1

INTRODUCTION
1.1 Imidazole
Imidazole is a heterocyclic molecule with five members that has two nitrogen atoms
arranged non-adjacent within its ring. Due to this structure, Imidazole and its derivatives
have special chemical and biological properties, making it an essential aspect of medicinal
chemistry. Imidazole and its derivatives were first discovered in the 19th century and have
since become important pharmacophores, being utilized extensively in the creation of
medicinal medicines for a range of illnesses (Sahoo, Biswal et al. 2012).

Figure 1.1: Structure of imidazole

1.2 Chemical Properties of Imidazole
The unique molecular characteristics of imidazole, a five-membered heterocyclic
molecule with two nitrogen atoms at positions 1 and 3, are essential to its adaptability in
both chemistry and biology. The imidazole ring's atom configuration confers special
properties on it, including amphoteric behavior, aromaticity, and hydrogen bonding capacity
(Tolomeu and Fraga 2023).
Imidazole has two nitrogen atoms, which give it both basic and acidic properties: N1
(Pyrrole-like Nitrogen): This nitrogen atom is weakly basic because it is a component of a
resonance structure and has a lone pair of electrons that are difficult to protonate. N3, or
pyridine-like nitrogen, is more basic and proton-accepting because it possesses a lone pair
that is not included in the delocalized π-electrons of the aromatic ring (Kumar, Mary et al.
2020).
Imidazole can take part in proton-transfer reactions because of its amphoteric
character, which make it capable of having acidic and basic character. This characteristic is
essential to its biological function in proteins and enzymes, especially in histidine residues.
Imidazole's ring's delocalized π-electrons make it an aromatic chemical. The six π-electrons
that move across the ring two from the lone pair on the N1 nitrogen and four from the
carbon-carbon double bonds are what give the ring its aromatic character. This characteristic
affects the reactivity of imidazole and provides it with chemical stability (Mesu, Visser et al.
2005).
Imidazole is a highly effective compound due to its two nitrogen atoms, N1 and N3.
Its hydrogen bonding capabilities are crucial in biological systems, particularly in enzyme
catalysis and protein-ligand interactions. Its pKa values are 7.0 and 14.5, making it soluble in
water and polar solvents. It is a nucleophilic compound, participating in nucleophilic
substitution reactions and coordinating with metal ions like zinc, copper, and iron. Imidazole
derivatives are valuable as ligands in organometallic chemistry. Its reactivity allows it to
undergo electrophilic substitution reactions and alkylation and acylation, resulting in various
derivatives with different pharmacological properties (Al-Otaibi, Almuqrin et al. 2020).
1.3 Pharmacological Activities and Mechanism of Action
Imidazole medications target a broad spectrum of disorders and are very adaptable
in their therapeutic applications. Antifungal, antibacterial, anticancer, anti-inflammatory,
and antiparasitic chemicals are only a few of the pharmacologically active substances that
rely on the imidazole ring structure as its fundamental scaffold. Imidazole-based
medications' principal pharmacological actions are as follows:
1.3.1 Antifungal Activity
Most well-known for its strong antifungal qualities, imidazole derivatives are
frequently used to treat fungal infections of the skin, nails, and internal organs.
Ketoconazole, miconazole, and clotrimazole are a few examples. The mechanism of action of
these antifungal drugs involves the inhibition of lanosterol 14α-demethylase, an enzyme
that is vital for the manufacture of ergosterol, which is a crucial element of fungal cell
membranes. The fungal cell membrane's integrity is compromised by the imidazole
medicines' disruption of ergosterol formation, which increases permeability, allows
intracellular contents to seep out, and ultimately results in cell death (Serdaliyeva,
Nurgozhin et al. 2022).
1.3.2 Antibacterial Activity
Additionally, some imidazole compounds have antibacterial properties, especially
against anaerobic microorganisms. One of the most often prescribed imidazole medications
for the treatment of bacterial infections is metronidazole. Metronidazole, a derivative of
nitroimidazole, works against anaerobic bacteria by entering their cells and going through a
reduction process. Bacterial cell death results from the lowered drug's generation of harmful
radicals that harm bacterial DNA and cellular proteins. It is commonly used to treat
anaerobic infections, bacterial vaginosis, and other illnesses caused by Clostridium difficile
(Balakrishnan, Jarzecki et al. 2012).
1.3.3 Anticancer Activity
A few imidazole derivatives are being researched for potential application in cancer
treatment since they have shown anticancer activity. The mode of action Some imidazole
medications function by blocking cytochrome P450 enzymes that are necessary for the
manufacture of steroids, which may have an impact on the growth of cancer cells. In
addition, these substances can accomplish their goals by inhibiting angiogenesis—the
formation of new blood vessels that are crucial for tumor growth—or by inducing
programmed cell destruction, often known as apoptosis, in cells in tumors. For example, the
anticancer potential of benzimidazole derivatives has been investigated (Borgers 1980).
1.3.4 Anti-inflammatory and Analgesic Activity
Aside from their demonstrated analgesic and anti-inflammatory qualities, imidazole
derivatives may also be used as therapies for rheumatoid, the mode of action The ability of
imidazole medications to prevent the synthesis of pro-inflammatory cytokines inhibit the
function of enzymes like cyclooxygenase (COX), or lower reactive oxygen species (ROS) levels
is usually what causes their anti-inflammatory effects. Certain imidazole derivatives can
relieve pain and edema by modifying immune responses and decreasing inflammation
(Thomason 2013).
1.3.5 Antiviral Activity
Research on the antiviral capabilities of imidazole derivatives has focused on their
ability to combat viruses including the human immunodeficiency virus (HIV). The mode of
action by focusing on viral enzymes or proteins necessary for the virus's life cycle, these
substances can prevent viral reproduction. For instance, it has been demonstrated that
specific imidazole derivatives inhibit HCV protease, hence blocking the processing of viral
proteins necessary for viral reproduction (Gougoula, Cole et al. 2020).
1.4 Therapeutic Applications of Imidazole-Based Drugs
Modern medicine relies heavily on imidazole-based medications because of their
many therapeutic applications. One important pharmacophore that is used in the creation of
medications that target a variety of illnesses and ailments is the imidazole ring. Common
medications used to treat fungal infections, bacterial infections, protozoan infections,
anticancer therapy, anti-inflammatory and analgesic therapy, anti-ulcer therapy, antiviral
therapy, antihistaminic therapy, CNS manipulation, and sedative effects include
ketoconazole, Clotrimazole, Miconazole, and Econazole (Arenas, Batra et al. 2021).
The lanosterol 14α-demethylase enzyme is inhibited by antifungal medications such
as ketoconazole, clotrimazole, miconazole, and econazole, which results in cell death.
Metronidazole is one antibacterial medication that breaks down bacterial DNA, which kills
the organism. Anticancer medications such as imidazolium salts and benzimidazole
derivatives block tumor growth and important enzymes that are necessary for cancer cells to
survive. Benzimidazole derivatives, which are anti-inflammatory and analgesic medications,
work by blocking pro-inflammatory cytokines and reactive oxygen species to decrease
inflammation. Cimetidine, an antiviral medication, blocks the H2 receptors on the parietal
cells of the stomach, allowing ulcers to heal (Zhang, Peng et al. 2014).

1.5 Current Trends and Future Prospects

Enhancing pharmacokinetics (absorption, distribution, metabolism, and excretion)
and pharmacodynamics (interaction with biological targets) is the goal of the continuous
creation of innovative imidazole derivatives. Imidazole analogs with improved
characteristics, like increased selectivity for particular enzymes or receptors, better
bioavailability, and decreased toxicity, are being created by researchers. Structure-activity
relationship (SAR) research advancements are making it possible to create molecules with
fewer adverse effects and greater potency. The investigation of imidazole derivatives with
numerous targets is gaining momentum. These drugs can operate on various biological
pathways, offering a more comprehensive approach to treating diseases such as
neurological disorders and cancer (Alghamdi, Suliman et al. 2021).
Because they can block enzymes essential to cancer cell growth and survival,
imidazole derivatives have demonstrated promise as anticancer treatments. Targeting
several elements of cancer progression like tumor growth, metastasis, and angiogenesis,
new imidazole-based medicines are being created. The goal is to create imidazole-based
inhibitors of proteins (such as kinases, matrix metalloproteinases, and cytochrome P450
enzymes) that are implicated in the growth of cancer cells. With imidazole medications
created to specifically block cancer-specific pathways while limiting harm to healthy cells,
the future appears bright for tailored cancer therapy. A further improvement in therapeutic
outcomes could come from the combination of imidazole-based medications with currently
available cancer treatments, such as immunotherapy or chemotherapy (Hachuła, Polasz et
al. 2017).
Imidazole derivative research for antiviral action has accelerated significantly with
the emergence of new viral illnesses (such as COVID-19, hepatitis, and HIV). Researchers are
looking at whether imidazole derivatives can block viral enzymes that are essential for viral
replication, such as proteases and polymerases. Imidazole-based antivirals will probably find
their way into the treatment of viral infections in the future, as concerns about drug
resistance to conventional antivirals develop. Their application in stopping viral alterations
and combating drug-resistant virus strains is also possible (Gaba and Mohan 2016).
To combat chronic inflammatory illnesses, researchers are looking at imidazole
derivatives for their potential as immunomodulatory and anti-inflammatory drugs that
target the synthesis of cytokines and the activation of immune cells. These drugs could
revolutionize treatments for autoimmune and inflammatory diseases with fewer side effects
compared to traditional immunosuppressive drugs. They are also being explored for treating
neurological disorders like Alzheimer's, epilepsy, Parkinson's, and anxiety. Green chemistry
practices are being adopted to reduce the environmental impact and manufacturing costs of
imidazole-based drugs. Nanotechnology and drug delivery systems are being integrated to
improve drug delivery, particularly for poorly soluble or unstable compounds. Personalized
medicine is also being explored, with pharmacogenomics being used to study patients'
metabolism and response to imidazole-based drugs. Genetic testing could help identify
patients who will respond best to specific imidazole drugs, optimizing treatment and
reducing adverse effects (Hachuła, Polasz et al. 2017).
1.6 Esomeprazole (EMeprazole) Drugs
Esomeprazole, often referred to as EMeprazole, is a proton pump inhibitor (PPI) that
is mostly used to treat peptic ulcers, GERD, and disorders like Zollinger-Ellison syndrome that
cause the overproduction of stomach acid (Figure 1.2). The omeprazole S-enantiomer is
what increases the medication's ability to prevent the generation of stomach acid (Spencer
and Faulds 2000).

Figure 1.2: Structure of Esomeprazole

1.7 Chemical properties of Esomeprazole
Esomeprazole, a compound in the benzimidazole class, has a benzimidazole ring
linked to a pyridine ring via a sulfoxide group, contributing to its chirality. As a racemic
combination of two enantiomers (the R- and S-forms), omeprazole has an S-enantiomer
known as esomeprazole. More acid suppression has been demonstrated by the S-
enantiomer (esomeprazole), which has a superior pharmacokinetic profile and better
bioavailability than the R-enantiomer (Jayabharathi, Thanikachalam et al. 2011).
1.7.1 Absorption
Because esomeprazole is acid-labile, or unstable in acid, enteric-coated versions of
the drug are usually used to avoid stomach breakdown. It is taken up in the intestines and
transformed by the stomach's Bioavailability: Esomeprazole is more bioavailable than
omeprazole, with a bioavailability of around 64% following a single oral dose of 20 mg and
rising to 89% following several doses (Babar, Munawar et al. 2019).
 1.7.2 Metabolism
The cytochrome P450 system (CYP2C19 and CYP3A4) is principally responsible for
the liver's breakdown of esomeprazole. The S-enantiomer exhibits less interindividual
variability in metabolism than the R-enantiomer. While esomeprazole is stable in basic
circumstances, it breaks down rapidly in acidic ones. To maintain its stability until it enters
the more alkaline environment of the intestine for absorption, it is therefore packaged in
enteric-coated granules or tablets (Naveed and Qamar 2014).
1.8 Pharmacological Activities and Mechanism of Action
Proton pump inhibitors (PPIs) like esomeprazole are used to treat a range of
gastrointestinal problems because of their strong acid-suppressive properties. By inhibiting
the gastric parietal cells' H+/K+ ATPase enzyme, it lessens the production of stomach
acid. Beyond merely suppressing acid production, its pharmacological actions also aid in
ulcer repair and the alleviation of GERD symptoms. The main pharmacological effect of
esomeprazole is to decrease the production of gastric acid by blocking the stomach lining's
proton pumps. Both baseline and induced acid output are significantly decreased as a result.
This acid suppression relieves heartburn and acid reflux symptoms, protects the lining of the
esophagus from harm, and promotes the healing of gastric and duodenal ulcers (Colucci,
Fornai et al. 2009).
Esomeprazole helps erosive esophagitis, a condition where long-term exposure to
acid causes inflammation or ulceration of the esophageal lining, to heal by reducing the
amount of gastric acid in the stomach. For people with GERD, this is crucial. Compared to
alternative medications, clinical studies have demonstrated that esomeprazole aids in a
faster and more thorough healing process, particularly in cases of severe esophagitis.
Treatment and prevention of peptic ulcers, especially those caused by NSAID use or
H. pylori infections, are common uses for the medicine esomeprazole. It not only
keeps the pH of the stomach more neutral, which inhibits the production of new ulcers, but
it also aids in the healing of existing ulcers by reducing the acidic environment that hinders
healing. Esomeprazole is used as part of triple therapy, which also includes antibiotics, to get
rid of H. pylori, a bacterium that causes peptic ulcers and stomach cancer (Sharma, Verma et
al. 2015).
The medications that treat the infection work better when esomeprazole lowers
stomach acidity. The stomach lining can be harmed by long-term NSAID use, which can
result in bleeding ulcers. Since esomeprazole lowers stomach acid and increases mucosal
protection, it is frequently recommended prophylactically to patients taking long-term
NSAIDs to avoid these problems. The proton pump in the parietal cells of the stomach is
responsible for the last stage of gastric acid generation; esomeprazole is a prodrug that
selectively inhibits this pump. It gets activated in an acidic environment, focusing on the
stomach's acidic environment. This selective activation minimizes systemic effects and
provides a long-lasting effect, typically reducing acid production for up to 24 hours with a
single dose. Esomeprazole is useful for the management of chronic disorders such as GERD
since it decreases acid output, both basal and provoked (Curkovic, Stupnisek-Lisac et al.
2009).
Pharmacological Activities: Esomeprazole significantly reduces gastric acid secretion,
promotes healing of ulcers, treats GERD and erosive esophagitis, prevents NSAID-induced
ulcers, and helps eradicate H. pylori infections. Mechanism of Action: By permanently
blocking the parietal cell H+/K+ ATPase (proton pump), esomeprazole lowers acid
production in the stomach. An potent and extensively used proton pump inhibitor, it
selectively activates within acidic environments and has a sustained activity (Tonini, Vigneri
et al. 2001).
1.9 Therapeutic Applications of esomeprazole
A severe kind of GERD known as erosive esophagitis occurs when the esophagus
becomes inflamed and damaged as a result of continuous exposure to stomach acid.
Esomeprazole reduces acid exposure and protects the mucosa, which makes it a very
successful treatment for erosive esophagitis. In comparison to other PPIs, clinical trials have
demonstrated faster and more thorough recovery. It is possible to cure peptic ulcers (also
called gastric ulcers or duodenal ulcers) with esomeprazole. These open sores develop on
the inside of the stomach or duodenum. Esomeprazole aids in the healing of existing ulcers
and inhibits the production of new ones by lowering the production of stomach acid. It
works very well to treat Helicobacter pylori infections and ulcers brought on by NSAID use
(Fan, Xie et al. 1986).
Helicobacter pylori (H. pylori) is a bacterium that has been linked to gastric cancer.
To eradicate H. pylori entirely, a patient undergoing triple therapy must take antibiotics in
addition to esomeprazole. Esomeprazole improves the efficiency of antibiotics by lessening
stomach acidity, which makes the environment less conducive to bacterial longevity. Gastric
cancer has been connected to the bacteria Helicobacter pylori (H. pylori), which is a major
cause of peptic ulcers. Triple therapy involves the use of antibiotics and esomeprazole to
completely remove H. pylori. Esomeprazole improves the efficiency of antibiotics by
lessening stomach acidity, which makes the environment less conducive to bacterial
longevity (Giuliano, Bizzocchi et al. 2019).
A rare disorder known as Zollinger-Ellison syndrome is typified by tumors called
gastronomes that cause the stomach to create excessive amounts of acid. One of the best
medications for controlling acid hypersecretion in Zollinger-Ellison syndrome is
esomeprazole, which helps to regulate acid levels and guard against patients with dyspepsia,
or chronic indigestion, which can result in symptoms including nausea, bloating, and
stomach discomfort, are frequently prescribed esomeprazole. Esomeprazole relieves these
symptoms and improves gastric comfort and digestion by reducing stomach acid levels
(Molina, Tárraga et al. 2012).
Long-term acid exposure alters the esophageal lining in Barrett's esophagus, a
disorder that raises the risk of esophageal cancer. People with Barrett's esophagus take
esomeprazole to lessen acid reflux, delay or stop further damage to the lining of the
esophagus, and lower the risk of developing esophageal cancer. Critically sick patients
are at risk of getting stress-related ulcers due to extended illness or surgery. Internal
bleeding
may result from these sores. In hospital settings, esomeprazole is used as a component of
stress ulcer prophylaxis to lower the risk of gastrointestinal bleeding and ulcer formation in
high-risk patients (Chen, Zhang et al. 2006).
Esomeprazole is a highly effective and versatile proton pump inhibitor used to
manage a variety of gastrointestinal conditions, including GERD, erosive esophagitis,
bacterial infection, and Zollingar-Ellison syndrome. Its ability to suppress gastric acid
production makes it essential for both treatment and prevention of acid-related disorders,
offering relief and promoting healing in patients with chronic and acute acid-related
conditions (Kucuk, Yurdakul et al. 2022).
1.10 Current Trends and Future Prospects
Esomeprazole, an anti-inflammatory medication, is being used in personalized
medicine due to its efficacy and potential side effects. Genetic variations in CYP2C19, an
enzyme responsible for metabolizing esomeprazole, have been found to impact its efficacy.
Personalized dosing is being explored to optimize therapeutic outcomes and minimize side
effects. New formulations like dual delayed-release esomeprazole are emerging to provide
better control of acid suppression. Combination therapies with Potassium-Competitive Acid
Blockers (PCABs) are also being explored to achieve faster onset of action. Esomeprazole is
also being used in multidrug-resistant H. pylori infections, where it enhances antibiotic
efficacy through its acid-suppressing action. Esomeprazole is available over-the-counter in
many countries, making it accessible for self-treatment of heartburn and indigestion.
However, patient education and responsible use of PPIs are crucial to avoid long-term
overuse or inappropriate self-medication (Srinivasan, Sawant et al. 2007).
Future research into nanotechnology could improve the delivery and absorption of
esomeprazole, particularly in targeted delivery systems. This could improve bioavailability,
ensure precise release in the stomach, and reduce side effects. Esomeprazole's anti-
inflammatory and antioxidant properties may be beneficial in treating non-gastrointestinal
conditions like neuroinflammation, cardiovascular disease, and certain cancers. Clinical trials
may expand its therapeutic applications into oncology or neurology. As new proton pump
inhibitors and non-PPI acid suppressors are developed, esomeprazole's role may evolve, but
it remains a key player in acid suppression. Concerns about long-term use of PPIs, including
esomeprazole, have led to heightened scrutiny regarding potential side effects. Future
efforts may focus on developing safer formulations with fewer long-term risks and
establishing clearer guidelines for appropriate PPI use, particularly in elderly patients or
those with pre-existing conditions. Longer-acting PPIs could be dosed once a week, providing
more convenience for patients while maintaining effective acid control (Jayabharathi,
Thanikachalam et al. 2011).
 CHAPTER 2
REVIEW OF LITERATURE
Dargent, Martin et al. (2000) studied that cervical cancer is common among young
women who are trying to avoid having children. In order to keep the uterus's reproductive
abilities intact, the authors developed and executed radical trachelectomy, a surgical
technique for patients with superficial invasive lesions.
In a radical trachelectomy, the surgeon uses both transvaginal and laparoscopic techniques
to remove lymph nodes from the pelvis. Of the 56 patients scheduled for this operation from
April 1987 to December 1996, only 47 actually had it done. Medical and obstetric history,
surgical procedure details and problems, pathologic findings, postoperative obstetric
outcomes, and cancer recurrences were all examined in a retrospective manner in these
patients' charts. Laparoscopic and vaginal portions of the treatment averaged 62 and 67
minutes, respectively. Seven problems occurred after the operation (drainage of pelvic
collection) and one occurred during the procedure (cystotomy). In five cases, the pathologic
tumor classification was pIA1 according to the International Union Against Cancer (UICC)
system; in thirteen cases, it was pIA2 according to the FIGO system; in twenty-five cases, it
was pIB according to the FIGO system; in one case, it was pT2a according to the FIGO
system; and in three cases, it was pT2b according to the FIGO system. Average follow-up
duration was 52 months. One patient passed away due to the disease's progression, and two
recurrences (one lateropelvic and one distant) were noted, accounting for 4% of the total.
Thirteen healthy infants were born following radical trachelectomy, even though a quarter
of pregnancies ended in miscarriage. Radical trachelectomy does not seem to raise the
recurrence rate in young patients with early invasive cervical cancer. Some individuals may
be able to conceive and have healthy babies, although there is a relative risk of infertility and
late miscarriage. Therefore, it appears to be a reasonable option to conduct this operation in
certain instances, as long as the surgeon is well trained and every patient is informed.
Schuster and Visvesvara (2004) studied that different species of amoeba can infect
humans and cause them to contract deadly parasitic diseases. The fast progression of the
disease and diagnostic delays cause most patients to die from the infection, even though
amphotericin B can kill N. fowleri. Combinations of antimicrobial drugs, such as azoles and
pentamidine, are commonly used to treat infections caused by Acanthamoeba and
Balamuthia. This helps to overcome drug resistance and maximize the effectiveness of the
treatment. Nevertheless, difficulties arise from the fact that drug sensitivity varies
throughout species and strains, and relapse might occur due to dormant cysts. In order to
lower the high mortality rates linked with these illnesses, the study stresses the need of
improved diagnosis tools and treatment approaches.
Basit (2005) studied that there has been a lot of buzz in recent years about directing
medicine delivery devices toward the colonic area of the GI tract. The desire for improved
treatment of colon-specific illnesses as some gastrointestinal disorders such as IBS and
cancer has motivated scientific research in this field. Many studies are concentrating on the
colon as a way for medications to enter the bloodstream. There are a number of different
delivery systems and strategies that have been suggested for colonie targeting. Their
operation is typically dependent on the use of microorganisms, transit duration, pressure, or
pH in the gastrointestinal tract. There are now commercially available coated systems that
take advantage of the pH differential in the intestines and prodrugs that depend on colonie
bacteria for release. There are obvious limitations to both methods. Lots of systems are still
in the research and development phase, and some of them are either too costly, too
complicated, or don't have the site-specificity that would be ideal. Given their usefulness
and ability to capitalize on the colon's most unique characteristic—its rich microflora—
universal polysaccharide systems stand out as the most promising.
Aulin, Gällstedt et al. (2010) explained that details the process of making
carboxymethylated micro fibrillated cellulose (MFC) films by dispersing-casting aqueous
dispersions onto base papers and then coating their surfaces. Researchers tested MFC films'
oxygen permeability over a range of RH values. At a relative humidity of 0%, the oxygen
permeability of MFC films was comparable to that of traditional synthetic films, such as
ethylene vinyl alcohol, and significantly lower than that of films made from plasticized
starch, whey protein, and arabinoxylan. Presumably as a result of water molecules
plasticizing and expanding the carboxymethylated nanofibers, the oxygen permeability
increased dramatically with higher relative humidity. By the help of different electron
microscopy such as FE-SEM, the researchers examined how the nano fibrillation/dispersion
degree affected the films' microstructure and optical characteristics. Nanofibers ranging in
thickness from 5 to 10 nm were observed to be randomly assembled in the MFC films,
according to FE-SEM micrographs; however, bigger aggregates were also observed. Coating
different base papers with MFC significantly decreased their air permeability. Electron
scanning microscope (E-SEM) images taken in an environmental setting showed that the
MFC layer improved oil barrier characteristics by decreasing sheet porosity, a phenomenon
caused by the dense structure created by the nanofibers.
Artsimovitch, Seddon et al. (2012) explained that for the treatment of Clostridium
difficile infection, fidaxomicin has just been approved. It blocks the action of bacterial RNA
polymerase on transcription. Because transcription is a multistep process, researchers
performed studies to determine which step was stopped by adding fidaxomicin at various
points in the commencement of transcription. To further understand the stage blocked, DNA
foot printing tests were also carried out. To prevent initiation, fidaxomicin must be
introduced prior to the development of the "open promoter complex," a state in which RNA
synthesis has not yet started but the template DNA strands have parted. The early
separation of DNA strands, which is necessary for RNA production, is prevented by
fidaxomicin binding. In contrast to streptolydigin and other elongation inhibitors, as well as
myxopyronin and the rifamycins, which impede transcription initiation, this compound's
mechanism is unique, according to these investigations.
Bratzler, Dellinger et al. (2013) stated that organizations such as the IDSA, SIS, and
SHEA that deal with infectious diseases. The ASHP Therapeutic Guidelines on Antimicrobial
Prophylaxis in Surgery, along with the guidelines from IDSA and SIS, have been revised and
updated in this study. Based on the best available clinical evidence and new developments,
the guidelines aim to standardize the use of antimicrobial medicines to prevent surgical site
infections (SSIs) in a way that is reasonable, safe, and effective. There are three distinct types
of prophylaxis, all of which aim to reduce the likelihood of infection: primary, secondary, and
elimination. Preventing an initial infection is known as primary prophylaxis. The goal of
secondary prophylaxis is to keep an infection from coming back or becoming worse if it
already exists. A colonized organism can be "eradicated" if all attempts to keep it from
spreading are unsuccessful. Primary perioperative prophylaxis is the main emphasis of these
guidelines.
Desai, Maheta et al. (2014) stated that arylamides 3a-l were synthesized using both
conventional and microwave methods. The synthesis of quinoline-based imidazole
derivatives using solvent-free microwave thermolysis is found to be an eco-friendly, fast,
convenient, and high-yielding alternative to traditional reaction in a solution phase. Different
microorganisms were used to test the antimicrobial activity of the newly synthesized
compounds. The in vitro antibacterial activity of all the bio-active compounds that have been
produced is evaluated using a bioassay, specifically serial broth dilution. Three, four, five,
and jolly are the most effective of these chemicals against various types of microbes. Mass
spectrum data, 1H NMR, and infrared spectra have all been used to characterize the
molecules. Statistical research has shown that there is a strong correlation between these
chemicals.
Gu, Yan et al. (2016) studied that the low catalytic effectiveness of molecularly imprinted
polymer (MIP) is a common drawback of using it as an electrochemical sensor. In this paper,
we offer a different strategy that integrates mimetic enzyme technology with the idea of
MIP. Melamine acted as both a functional monomer of metronidazole imprinted polymer
(MIP) and a component of mimetic enzyme, allowing for the effective electrochemical
synthesis of a polymer with nitro reductase-like activity. The catalytic redox-active core was
inserted into the imprinted cavities during the imprinting process. Consequently, the
imprinted polymer demonstrated improved electrocatalytic activity and selectivity due to its
dual nature as a catalyst and recognition site. This biomimetic sensor that was imprinted
with metronidazole was thoroughly tested for its sensing capabilities. The findings showed
that metronidazole exhibited a linear response. Furthermore, we tested the suggested
sensor's ability to identify metronidazole in an injection solution, and the findings suggested
that it could be useful in the real world.
Herbinger, Alberer et al. (2016) explained that The purpose of this research was to
examine the range of infectious diseases (IDs) brought into the country from other regions
between 1999 and 2014 among individuals who sought medical attention at the University
of Munich in Germany after visiting the subtropics. The study looked at comprehensive
datasets containing different travelers across Germany, Africa, America and other sick
emigrants. Fever (29% of cases), skin disorder (22% of cases), and diarrhea (38% of cases)
were the most often evaluated symptoms. Intestinal infections caused by Blastocystis,
Giardia, Campylobacter, Shigella, and Salmonella were the most common types of infections
identified. Some other common parasites included dengue (257 cases), malaria (160 cases),
and cutaneous larva migrants (379 cases). The result shows that German immigrants and
travelers use a wide variety of imported IDs, which differ significantly depending on factor.
Bouchemal, Bories et al. (2017) explained that The most prevalent non-viral STD
globally is caused by the human protozoa parasite. It is known that T. vaginalis can cause
vaginitis. For males, it can spread to the prostate and urethra. There have been reports of
dysuria and discharge in men who carry T. vaginalis, although most men do not experience
any symptoms. Up to half of women may not experience any symptoms at all, and in the
other half, the disease can be quite severe and leave lasting effects. A foul-smelling, purulent
discharge that causes localized pain and irritation is a hallmark symptom. In most cases,
infections of the reproductive tract following surgery are limited to the lower urogenital
region and are thought to be caused by T. vaginalis. Different serious diseases can also be
caused by this parasite. Furthermore, there is evidence that both men and women are more
likely to contract HIV. Worldwide, the incidence of Trichomonas vaginalis is higher than the
combined incidence of chlamydia and gonorrhea, according to the most recent estimates.
Recent developments in the diagnosis of Trichomonas vaginalis and methods for its
treatment and prevention have been the subject of this critical review, which brings the
current level of knowledge up to speed. In specifically, the article reviews and discusses
recent research on the effectiveness of formulations when applied topically.
Balarak, Mostafapour et al. (2017) studied that the adsorption of the antibiotic
amoxicillin (AMO) on activated carbon made from Azolla filiculoides (ACAF) was examined in
the batch adsorption studies. Critical variables impacting this investigation include ACAF
dosage, starting AMO dose, and reaction time. At an initial AMO concentration of 100 mg/L,
the testing findings showed that the elimination percentage of AMO increased from 49% to
90% as the ACAF amount increased from 0.15 to 0.60 g/L. Raising the starting dose of AMO
reduced its removal efficiency.
 Agarwal (2017) explained that metronidazole is an antibiotic prodrug, and this study
shows how to immobilize it onto cellulose nanofibrils in a new and environmentally
acceptable way using chemistry. The ester-containing prodrug in water-based suspension is
covalently attached in two stages. Nuclear polarization-enhanced dynamic nuclear magnetic
resonance (NMR) and Raman spectroscopy are among the cutting-edge analytical methods
that have verified the successful attachment. The ester functional group of the prodrug is
enzymatically cleavable, which allows for regulated release of the antibiotic. Making use of
cellulose nanoparticles that are already on the market, this novel approach shows great
promise for in situ drug delivery devices. The article discusses the use of Raman
spectroscopy to study nanocelluloses and nanocellulose composites, and it demonstrates
how different Raman techniques can yield valuable insights. Finding cellulose nanoparticles,
estimating cellulose crystallinity, studying CNC dispersion in polymers, and evaluating
CNC/matrix interactions were among the most significant applications. Additionally, it was
shown that molecular-level information about cellulose orientation may be retrieved using
polarized Raman spectroscopy, and this was done using a bleached fiber. Because of this
capability, research into the structure of composites containing nanocelluloses can be
conducted. The use of Raman spectroscopy to measure the stress or strain imparted to
nanocelluloses from the surrounding polymer matrix was lastly demonstrated in the context
of nanocellulose composites.
(Rajendran, Anwar et al. 2017) stated that The objective of this research was to find
out if the current medications for primary amoebic meningoencephalitis caused by
Naegleria fowleri are more effective when conjugated with silver nanoparticles. Using UV-
visible spectrophotometry, the production of silver nanoparticle conjugates of amphotericin
B, nystatin, and fluconazole was confirmed. Atomic force microscopy put their size
somewhere between twenty and one hundred nanometers. Incubation of N. fowleri with
medicines alone, silver nanoparticles alone, or medications conjugated with silver
nanoparticles allowed us to determine the amoebicidal effects. Comparing the antiamoebic
effects of Fluconazole and Nystatin at micromolar concentrations to those of Amphotericin B
and silver nanoparticles conjugated together, the results showed that Fluconazole was not
affected.
Our research provided the first evidence that antiamoebic medication efficacy against
Neisseria fowleri can be improved by conjugating them with silver nanoparticles. We
anticipate that by tweaking current medications to boost their antiamoebic activities, we can
improve the treatment of brain-eating amoebae infection owing to N. fowleri. This is
especially promising given the rarity of the condition and the hurdles in generating new
drugs.
Rebiere, Guinot et al. (2017) explained that addressing the problem of counterfeit
pharmaceuticals has emerged as a top concern for many groups concerned about the impact
on public health. To help determine the risks to patients from chemically characterized
fabricated samples, analytical laboratories conduct analyses. There is a plethora of methods
available for determining specifics such the formulation's crystalline structure, the existence
of an active ingredient or contaminants, and the inorganic and organic composition. This
review begins by outlining each of these methods separately, and then it proposes a strategy
for integrating them. Scientific literature examples (weight loss aids, erectile dysfunction
treatments, and malaria prevention items) are used to demonstrate this process. The analyst
can determine whether a sample is fabricated, whether it complies with pharmaceutical
quality standards, and, finally, whether it is safe for patients by combining analytical
techniques.
Anwar, Rajendran et al. (2018) explained that a uncommon but deadly condition is
infection of the central nervous system (CNS) by free-living amoebae like Acanthamoeba
species and Naegleria fowleri. The development of new drugs with the ability to penetrate
the central nervous system (CNS) and cure infections caused by these amoebae is a
significant issue. Since clinically licensed medications are known to effectively penetrate the
blood-brain barrier and influence eukaryotic cell targets, it is reasonable to test them against
CNS illnesses for their possible antiamoebic benefits. In amoebicidal, cysticidal, and host-cell
cytotoxicity experiments, both drugs alone and drug attached silver nanoparticles were
evaluated. Drugs were used as capping agents in the synthesis of nanoparticles, which
involved the sodium borohydride reduction of silver nitrate. Nanoconjugates that were
conjugated with drugs were studied using atomic force microscopy (AFM), (UV-vis) and FTIR
spectroscopy, and other imaging techniques. When tested against A. castellanii and N.
fowleri, diazepam, phenobarbitone, and phenytoin conjugated to AgNPs had stronger
amoebicidal effects than the medicines alone in an in vitro moebicidal assay. In addition,
there were strong cysticidal effects observed with both the medicines and drug attached
AgNPs. The antiacanthamoebic activity of drugs was increased through their coupling with
silver nanoparticles. Interestingly, both the medicines alone and their nanoconjugates
considerably decreased amoeba-mediated host-cell cytotoxicity. It appears viable to
evaluate these medications against brain-eating amoebae since they are being used to treat
central nervous system (CNS) problems. The clinically available medications that were
examined here show promise for future in vivo investigations against brain-eating amoebae,
which is a significant improvement over the current pharmacological landscape.
McDonald, Gerding et al. (2018) stated that a traditional Chinese drug called
triptolide (TP) has shown promise in treating autoimmune illnesses and exhibiting anti-
cancer effects in various human tumor cell lines. this study delves into the anticancer effects
of TP and sheds light on the potential molecular mechanism at work. Varieties of TP were
administered to SW114 and K562 cells at concentrations ranging upto fifty. There was a
dose-dependent inhibition of tumor cell growth by TP, according to the results. In order to
delve deeper into its workings, an enzyme-linked immunosorbent assay (ELISA) was used to
assess prostaglandin E2 (PGE2) and nitric oxide (NO). Results demonstrated that TP
significantly reduced NO and PGE2 synthesis. In line with these findings, real-time RT-PCR
and Western blotting demonstrated an increase in the expression of cyclooxygenase-2 (COX-
2) and inducible NO synthase (iNOS) at the protein expression level. These findings
presented evidence that TP's anticancer processes may involve iNOS activity and the
suppression of the inflammatory factor COX-2.
Gonzales, Dans et al. (2019) explained that this work studies comparing
metronidazole, tinidazole, and combination treatments for amoebic colitis were the most
effective. Compared to metronidazole, tinidazole may have fewer side effects and be more
successful in minimizing clinical failures, according to the review. When compared to
metronidazole alone, combination therapy seemed to decrease parasitological failures;
nevertheless, no particular combination was determined to be the most beneficial.
Especially in countries with limited resources, the review stressed the need of bigger trials
with better designs and more precise diagnostic tools to evaluate treatment results.
Shedoeva, Leavesley et al. (2019) stated that skin heals itself, a process known as
cutaneous wound healing. Hemostasis, inflammation, proliferation, and remodeling are the
four stages that are commonly recognized as cutaneous wound healing processes. In
humans, keratinocytes reepithelialize, or quickly produce a functioning epidermis, to close
the wound and restore tissue homeostasis. The migration and proliferation of dermal
fibroblasts into the wound bed produces "granulation tissue" that is abundant in
extracellular matrix proteins and helps to foster the development of new blood vessels. The
goal of the long-term remodeling process is to restore the wounded tissue to its pre-injury
condition. Skin diseases such as nonhealing or chronic ulceration can occur when any step of
the wound healing cascade is dysregulated, which in turn slows healing. Half of the world's
pharmaceuticals come from indigenous and traditional medicine systems, which rely heavily
on natural ingredients and their derivatives. In light of the ongoing significance of traditional
medicine, we conducted a comprehensive literature review on the topic of cutaneous
wound treatment using medicinal plants and products derived from plants. For 36 different
medicinal plant species, we detail their bioactivities, active components, clinical applications,
formulations, preparation methods, and therapeutic value. Centella asiatica, Curcuma longa,
and Paeonia suffruticosa are three species that stand out; they are widely utilized as wound
healing items across many civilizations and ethnic groups. Traditional techniques can
nevertheless teach us a lot, judging by their sustained popularity and application. There may
be undiscovered combinations, adjunct compounds, and undescribed reagents among the
vast array of natural products and their derivatives that could find a home in the modern
therapeutic toolbox.
Rice, Troth et al. (2020) studied that serious brain disorders caused by pathogenic
free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and a number of
Acanthamoeba species, can cause fatalities in excess of 90% in certain cases. Misdiagnosis
and treatments that only partially work are two of the many limitations that contribute to
these alarmingly high death rates. To alleviate these concerning death rates, there is a
medical need for novel medications that are both fast-acting and extremely powerful. In this
study, we detail the pharmacological development of novel compounds with anti-amoebic
activity. Furthermore, the Medicines for Malaria Ventures (MMV) Pandemic Response Box
was screened using the Cell Titer-Glo 2.0 high-throughput screening methodologies to
identify potential novel active chemical scaffolds. At 10 and 1 µM, the library was first tested
as a single-point assay. We discovered 12 hits against B. mandrillaris, 29 against N. fowleri,
and 14 against A. castellanii ranging from nanomolar to low micromolar potency based on
these findings, which were confirmed by conducting quantitative dose response
experiments. Additional information was provided regarding eleven new compounds that
showed activity against B. mandrillaris, twenty-two against N. fowleri, and nine against A.
castellanii. These structures show how useful phenotypic screening is for drug development
in the treatment of diseases caused by free-living amoebas, and they also provide a
foundation for medicinal chemistry research.
Akbar, Hussain et al. (2023) detailed the possible anti-amoebic medication
possibilities against Balamuthia mandrillaris and Naegleria fowleri based on Nano
formulations of azoles and 5-nitroimidazoles. The study discovered that by combining
fluconazole, itraconazole, and metronidazole into Nano formulations, the amoebicidal and
cytocidal effects were much stronger than when the medications were used alone. Nano
formulations also showed little cytotoxicity and reduced amoeba-mediated host-cell
damage. These new Nano formulations show promise as potential candidates for the
creation of more efficient treatments for illnesses caused by brain-eating amoebae,
according to the results.
Velásquez-Torres, Trujillo-Ferrara et al. (2023) explained that riluzole's (a
benzothiazole derivative) anti-amoebic activity against Entamoeba histolytica. The study
showed that riluzole drastically decreased the vitality of amoebas in vitro, leading to
abnormalities in the parasites' nuclei and cell membranes' ultrastructure. An upsurge in
reactive oxygen species and nitric oxide generation occurred with the drug-induced cell
death that resembled apoptosis. Among the candidates for the treatment of amoebiasis,
riluzole stands out due to its stronger affinity for amoebic antioxidant enzymes compared to
metronidazole, as shown in in silico docking experiments. To verify its effectiveness,
additional in vivo trials are suggested.
 CHAPTER 3
Research Methodology
Based out of Faisalabad, Pakistan, the Axis Pharmaceutical Industry is where this
inquiry is occurring. The individuals were administered pharmaceutical medicine active
components in both the stationary and mobile phases, and every method used in this
investigation adhered to scientific standards. Because imidazole is so unique in its use—just
one tablet—the stationary phase was selected. Developed and validated analytical processes
make use of the following equipment, which is summarized below. It was determined and
measured that sampling and other components satisfied the standards needed to carry out
the current activity while undertaking practical work and keeping all conditions towards a
perfect and exact output.
3.1 Chemicals
Here is a comprehensive list of every single substance, reagents, and materials that
were utilized in the testing process, along with the suppliers from whom they were
procured. For storage instructions, see the drug's MSDS (material safety data sheet). For this
research, we rely for use with AR and HPLC-certified reagents and solvents.
 Dibasic Potassium Hydrogen Phosphate
 Acetonitrile (HPLC Grade)
 Phosphoric Acid
 Acetic Acid
 Sodium Acetate Trihydrate
3.2 Equipment & Glassware
Table 3.1: Capacity of the various types of glassware
Glassware Capacity/Grade
Bulb Pipette-I waki Pyrex 2, 5 & 10mL
Beaker-I waki Pyrex 50, 100,250 & 1000mL
Pyrex measuring cylinder 100mL,250mL,1000mL
Test tube 15mL
Syring Filter 0.45µm
Whatsman-Filter Paper 42 grade
Volumetric Flask-I waki Pyrex 50, 100 & 500mL
 3.3 Analytical Apparatus
All of the equipment that was utilized in developing and validating the methods complies
with 21 CFR standards and has the necessary certifications and calibrations. Here are the
instruments, accompanied by their model and model names:
 HPLC (High Performance Liquid Chromatography)
 Ultrasonic Batch Analytical Weighing Balance
 Dissolution Apparatus
 Moisture Analyzer
 Magnetic stirrer
 pH meter
 Bath sonicator
Following is documentation of the instruments used and their qualification status:
Table 3.2: The Analytical Equipment: A Catalog
Instruments Manufacturer Qualification/Calibration Model
HPLC Shimadzu Calibrated & Qualified LC-20AC
Dissolution Apparatus Dawn Externally Calibrated DIS-010
Analytical Column Shimadzu Calibrated C18, (4.6 mm × 25cm), 5µm
Moisture Assessor Sartorius Calibrated MA-150
Weighing Balance Sartorius Externally calibrated BSA224S – CW
Bath Sonicator -- Calibrated HC-19U-0001
Magnetic Stirrer -- Calibrated MS300-HS
pH Meter HANNA Calibrated HI 2210

Figure 3.1: High liquid pressure chromatography’s Auto-sampler

Figure 3.2: Dissolving device

Figure 3.3: Imidazole

3.4 Analytical Method Development

3.4.1 Solubility
Both water and methanol have a weak solubility for imidazole.
3.4.2 Choosing the detection wavelength
Prior to formulating a plan to evaluate pharmacological ingredient toxins caused by
processing imidazole with a focus on high sensitivity and low noise, choosing the right
wavelength is essential. The UV absorption spectra of each analyte were examined, paving
the way for more studies. Significant ultraviolet (UV) absorption at 230 nanometers was
observed for imidazole and related foreign chemicals (Ramasamy 2015).
3.4.3 Preparation for the mobile phase
Resolution efficiency of various mobile phase was tested, including H2O-C2H3N, H2O
and CH3OH, Sodium Phosphate buffer with C2H3N, and Sodium Phosphate buffer-CH3OH, all
of which had different arrangements and separations. Because it was able to separate the
test compounds better than the alternatives, a mobility state consisting of Sodium
Phosphate buffer, CH3OH, and C2H3N was selected for further testing. Additional studies
were conducted to improve the determination by experimenting with different ratios of
Na3PO4 buffer, CH3OH, and C2H3N. The successful separation was achieved using a sodium
phosphate buffer-acetonitrile mixture in thirty to seventy volume/volume and a sodium
phosphate buffer-methanol-acetonitrile mixture seventy two to thirty to three volume by
volume ratio as mobile phases A and B, respectively (Serrano-Andrés, Fülscher et al. 1996).
3.4.4 Choosing a Diluents
The effect of various solvents that dilute the solution, such as ethanol, distilled
water, methanol, acetonitrile, acetone, and dimethylformamide, was investigated. As a
result of its high luminosity, great reproducibility, and low blank measurement rates,
methanol was determined to be the best diluting solvent (Ahmad, Khalid et al. 2018).
3.5 Assaying
3.5.1 Preparation of Standard Solutions
Using a volumetric flask of 100 mL, add 50 milligrams of the weighed Imidazole
working standard. Top out the flask with diluents after adding 30 mL of sonicating the active
ingredient, and then mix well. Fill a 50 mL volumetric flask with injection-use diluents until it
is full, then pour 5 mL of the diluted solution to it (Jayabharathi, Thanikachalam et al. 2012).
Suggested dilution: 50 mg/100 mL x 5 mL/50 mL.

3.5.2 Preparation of Sample solution

Make fine powder by crushing at least ten tablets by a pestle and mortar. A 100 mL
volumetric flask transferred 100 mg of the sample, or 10 mg of Imidazole, after weighing it.
Complete dissolution is achieved by sonicating for 15 minutes after 90 milliliters of Diluent
have been added. Once it has cooled, add diluent to make the volume the right amount and
keep sonicating for another 15 minutes. Use a centrifuge set to 4000 rpm for five minutes.
Fill a 10-milliliter volumetric flask with diluent after transferring 5-milliliters of the
supernatant to it using a pipette. After ensuring thorough mixing, inject the mixture after
passing it through a 0.45 µm membrane (Wójcik, Kwiendacz et al. 2010).
Proposed dilution: Sample (100 mg/100 mL × 5 mL/10 mL portions)
3.5.3 Chromatographic Conditions
Table 3.3: Equipment Requirements for Chromatography
Apparatus Conditions
Column C18, (4.6 mm × 25 cm), 5 µm
UV-Vis Detector 230 nm
Rate of Flow 1.0 mL/min
Column Temperature Ambient
Injection Volume 20 µL
Auto-Sampler Temp. 2 – 8 oC
3.5.4 Procedure
Insert a syringe filter with a particle size of 0.45μ and record the volumes of both the
standard and sample solutions discretely. Determine the imidazole concentration as a
percentage by comparing the standard and sample solutions' reactions to the main peaks.
A appropriate system was determined by meeting the following criteria:
3.5.5 Determinants of Approval
 Just around 2% is the tailing
factor
 Standard deviation relative
to the mean is under 2%.

3.5.6 Calculation
Avg. Area of the sample Dilution of the Standard Avg. Weight of Tablet
% Assay= × × ×% Purity
Avg. Area of Standard Dilution of Sample Label Claim ×1.34
3.6 Dissolution
3.6.1 Preparation of dissolving media
So as to assign a way for analytical dissolution testing of imidazole tablets, we
exposed them to a range of dissolving media, such as H2O and acidic to neutral buffers.
Sodium ethanoate trihydrate (40.8 g) and ethanoic acid (23.1 mL) should be
dissolved in water last, and then add enough water (H2O) to make the volume 6000 milli
Liter. Use acetic acid to bring the pH down to 4.5 (Saini, Kaur et al. 2011).
3.6.2 Factors affecting solubility
The following conditions must be met: a solution media of 900 mL of acetate buffer
(pH 4.5), an apparatus with a frequency of 50 rpm, a temperature of 37 ± 0.5°C, and
a duration of 15 minutes.
3.6.3 Procedure
Get the temperature set to 37 ± 0.5°C and add 900 mL of Dissolving medium
to each of the six vessels. Put a single tablet into each of the six containers. Run the
machine for the allotted time. Gather around 25 milliliters of the sample solution and quickly
strain it (Wang, Lee et al. 2006).
3.6.4 Sample Solution Preparation
Using a filter paper sheet or spinning the mixture at 4000 rpm for 10 minutes, strain
off approximately 25 milliliters of sample when the timer goes off. The absorbance can be
determined from the translucent supernatant (Hossain, Thomas et al. 2018).
3.6.5 Standard Solution Preparation
Transferring 25 milligrams from an imidazole working standard was done
using a volumetric flask measuring fifty milliliters. The dosage was properly measured.
Add 30 mL of dissolving liquid to dissolve the active component by stirring or sonication. A
50 mL volumetric flask was pipetted with 1 mL of the solution stated before. After that,
reach the mark by adding more dissolving medium. A solution is injected.
Suggested dilution: Tablet weight 25 mg / 50 mL× 1mL/50mL
3.6.6 Calculations
Area of Sample Dilution of standard 1
% Dissolution= × × × % Purity
Avg. Area of standard Dilution of sample Factor (1.34)
3.7 Accreditation of Analytical Methods
Following the guidelines set down by the ICH, the following parameters were utilized to
validate this method.
 Systemic Appropriateness
 Particularity
 Preciseness
 Precision (Rotation)
 Repetition
 Consistency
 Resilience
 Limit for Detecting Low Molecular Density
 Limit of Quantitation (LOQ)
3.7.1 Systemic Appropriateness
One way to make sure the technique worked was to examine duplicate
injections of the reference solution. Inject working/reference standard five times at 100%
target concentration using the authorized analytical process, as described in the section
above. Calculate the standard deviation, tailing factor and number of theoretical plates
(Rajarajan, Thanikachalam et al. 2018).
3.7.1.1 Acceptance Criteria
This system satisfies all requirements set out by the US Pharmacopeia:
 Just around 2% is the tailing factor and Standard deviation relative to the mean is
under 2%.
 3.7.2 Particularity
To further understand the specificity of the approach, experiments were carried out
on forced deterioration. To prove that additional excipients do not hinder the API's activity,
the specificity test was conducted. The API's peak response area was tested for interference
by injecting three replicates of the sample, standard solution, blank (mobile phase), and
placebo. Content percentages and relative standard deviations (% RSD) were computed
(Richmond, Faguy et al. 1998).
3.7.2.1 Preparation of Placebo Sample Solution
All the excipients were mixed together to make the placebo in a lab.
These subjects were chosen from the master formulation of imidazole tablets while
the plant was operating in Faisalabad, Pakistan, by Axis Pharmaceuticals Industry.
The ingredients used to make the control sample are as follows:
 Microcrystalline cellulose 102
 Colloidal silicon dioxide
 Magnesium stearate
 Croscarmellose sodium
 Mannitol
 Hydroxy propylmethyl cellulose
 Sodium starch glycolate
 Purified water
Three sections of each solution category were gathered to make the control group:
blank, sample, and standard. Steps for modifying the dilution ratio were adhered to as
outlined in the Sample Preparation section of the technique development document
(Siddekha, Nizam et al. 2011).
3.7.2.2 Acceptance Criteria
The blank sample shouldn't show a peak reaction at the given retention
time. Ideally, the placebo sample shouldn't show a peak response at the given retention
time.
3.7.3 Accuracy
We repeated the aforementioned procedure twice to get VPZ samples with varying
concentrations. Utilizing the corresponding models of regression, the concentrations were
ascertained. Standard deviations and mean recovery percentages were subsequently
calculated. The accuracy experiment included three separate sample concentrations: 90%,
100%, and 110%. The percentage recovery, percentage variation, and 95% confidence
interval were calculated for each concentration after three injections were made in
duplicate (Devine, Cronin et al. 2006).
3.7.3.1 Preparation of 90% Sample concentration
Get ten pills and crush them into a powder. Transfer 180 milligrams of
powder to a 100 milliliter volumetric flask after precisely weighing it. To make sure the 90
mL of diluent dissolves completely, sonicate it for 15 minutes after adding it. After the
solution has cooled, add the diluent until the flask is full, then stir or sonicate for another 15
minutes. Spin the mixture in a centrifuge at 4000 rpm for 5 minutes. Add diluent to a 20 mL
volumetric flask after transferring 5 mL of the supernatant using a pipette. Blend the mixture
completely and pass it through a 0.45 µm membrane filter. This solution has been filtered
and is then injected.
Proposed Dilution: Weight taken/100mL x 5mL/20mL
3.7.3.2 100% Sample concentration Preparation
Whip up a powder by crushing ten pills. Divide the powder into 200
mg portions and pour 100 milliliters of it into a volumetric flask. To make sure the 90
mL of diluent dissolves completely, sonicate it for 15 minutes after adding it. When
using diluent, wait for the solution to cool before filling the flaskad equate. Beat or
sonicate the ingredients for another fifteen minutes. Spin the mixture in a centrifuge
at 4000 rpm for 5 minutes. Fill a 20-milliliter volumetric flask with diluent after
pipetting 5 milliliters of the supernatant into the flask. Make sure the solution is well
combined before passing it through a 0.45 µm membrane filter. This solution has
been filtered and is now prepared for injection (Thomas, Hossain et al. 2018).
Proposed Dilution: Weight taken/100mL*5mL/20mL
3.7.3.3 Preparation of 110% Sample concentration
Make a fine powder by grinding ten tablets. Measure out 220 milligrams of
powder and pour it into a 100 milliliter volumetric flask. Complete dissolution is achieved by
sonicating for 15 minutes after 90 milliliters of Diluent have been added. Fill the flask to the
required level with Diluent when the solution has cooled. Beat or sonicate the ingredients for
another fifteen minutes. Spin the mixture in a centrifuge at 4000 rpm for 5 minutes. Before
filling a 20 mL container with diluent, pipette five milliliters of the residue into the flask.
Thoroughly combine all ingredients. Then, pass the mixture through a membrane filter that
has a 0.45 µm aperture. Injectable preparations include the filtered mixture (Carver,
Tregenna-Piggott et al. 2003).
Proposed Dilution: Weight taken/100mL•5mL/20mL
3.7.3.4 Determinants of Approval
 Sample Recovery Rate: 98.0 to 102.0 percent
 The 95% confidence interval is greater than or equal to ± 1.0%.
 More than 2000 theoretical plates
3.7.4 Spiking
The imidazole sample (0.5 mg mL−1) was contaminated with ten contaminants
at a concentration of 0.1% in order to assess the scheme's adaptability. Before they
were employed, all of the solutions were passed through 0.22 μm membrane filters
(Li and Kobayashi 2016).
3.7.4.1 Spiked Sample Preparation (125%):
Combine 200 milligrams of powder with 100 milliliters of volumetric
flask, then add 25 milligrams of imidazole. Sonicate it for five minutes to create the
control group. Dilute the solution to the required volume when it has cooled. For fifteen
minutes, vigorously stir or sonicate it. Put it in a centrifuge and spin it at 4000 rpm for 5
minutes. Following the pipetting of 5 mL of the supernatant into a 20 mL volumetric
flask, the volume was modified with diluent. Injectable solutions should be thoroughly
mixed before passing them through a 0.45 µm filter (Mostafa and Bazzi 2011).
Suggested Dilution: Weight taken/100mL x 5mL/20mL
3.7.4.2 Acceptance Criteria
• The standard sample NMT can vary by around 2.0%.
3.7.5 Accuracy
3.7.5.1 Consistency
On the same day, six distinct combinations of system suitability
solutions were injected using the same apparatus to guarantee uniformity. The
percentage of relative standard deviation (RSD) was calculated for the response
regions of each analyte peak. Six injections of the material at 100% concentration
were used to test repeatability (Cao, Gu et al. 2003).
3.7.5.2 Intermediate Precision
Not only that, but the intermediate accuracy was double-checked by a
large number of analyzers on different days using different instruments in different
labs. Results for the detected impurity contents' percentage RSD values were
calculated (Eseola and Obi-Egbedi 2010).
3.7.5.3 Acceptance Criteria
 %RSD NMT 2.0%
3.7.6 Linearity
A sufficient analytical method size will provide a confidence range of test
results that are directly proportional to the concentration of the analyte in the
sample. To guarantee linearity, at least five separate doses were prepared, and three
times were each level replicated (Ahmad, Alam et al. 2018).
3.7.6.1 Standard Stock Solution Preparation
Put 50 milligrams of imidazole (the experimental reference) into a 100
milliliter capacity flask. Incorporate 30 mL of diluent, followed by adjusting the
volume to the desired amount. Use sonication to thoroughly mix the active
ingredient by shaking the mixture firmly. With the help of a pipette, transfer 4, 5, 6,
8, and 10 mL of the diluted solution to an individual 50 mL volumetric flask. Then,
inject the diluted mixture after diluting it with the diluent to the necessary volume
for the number of serial dilutions (Prenesti and Berto 2002).
3.7.6.2 Standard solution preparation
 For 80% Concentration
To achieve the desired volume, additional diluent was added to the 50 mL
volumetric flask after 4.0 mL of the standard stock solution had been transferred to
it.
Imidazole reached a final concentration of 0.008 mg/mL, while the reference
solution reached a concentration of 0.08 mg/mL.

 For 90% Concentration

In order to get the desired amount of volume, more diluent was added to the
same 50 mL volumetric flask after 4.5 mL of the standard stock solution had been
transferred. Imidazole reached a final concentration of 0.009 mg/mL, while the
reference solution reached a concentration of 0.09 mg/mL (Kagami, Sahara et al. 2016).
 For 100% concentration
The necessary quantity of diluent was added to a 50 mL volumetric flask that
was previously full with five milliliters of the standard stock solution. The reference
solution had an imidazole final concentration of 0.001 mg/mL, while the
experimental solution had a concentration of 0.01 mg/mL.
 For 110% Concentration
Once the volumetric flask was half full with the standard stock solution (5.5
mL), it was filled up to the necessary level with more diluent. Both the standard
solution and the imidazole solution reached ultimate concentrations of 0.011 mg/mL
and 0.0011 mg/mL, respectively.
 For 120% Concentration
The 50 mL volumetric flask was filled to the proper level with diluent after 6.0
mL of the standard stock solution was transferred to it. After all processes were
complete, the concentrations of imidazole and the reference solution were 0.0012 and 0.012
mg/mL, respectively (Polat and Yurdakul 2014).
3.7.6.3 Determinants for acceptance
• The squared correlation coefficient (r2) is more than 0.999.
3.7.7 Range
 Per ICH guidelines, the suggested analytical procedure was tested for concentrations
range, reliability, responsiveness, consistency, exactness, and stabilization. A range of
concentrations (80%, 90%, 100%, 110%, and 120% by weight) was determined by creating a
sample solution and testing the analytical procedure (Asher and Murtaugh 1988).
3.7.7.1 Preparation of Sample Stock Solution
Make a fine powder by crushing ten pills. Using a 100 mL volumetric flask,
combine 200 mg of powder with 90 mL of diluent. Five minutes of sonication should be
enough to dissolve the ingredients completely. Give it a 15-minute sonication or stir to mix
when it has cooled, and then add more diluent to get the desired consistency. Run it through
a centrifuge at 4000 rpm for five minutes. Transfer sections of the supernatant into separate
20 mL volumetric flasks by pipetting 4 upto 6mililitres with gradually increasing 0.5millilitres
concentration upto 6, respectively. Dilute the solution at the proper volume using diluent to
provide the necessary serial dilutions. To filter it, use a 0.45 pm membrane. Then, inject this
(Trivedi, Branton et al. 2018).
3.7.7.2 Preparation of Sample Solutions
The majority (80%) To get a concentration of 0.12 mg/mL, add 1.6 mL
of the stock solution to a 50 mL flask and measure the volume. A whopping 90%
Determine the volume of the sample stock solution (1.8 mL) and add it to a 50 mL
flask; the concentration should be 0.135 mg/mL. Hundred percent. Determine the
volume of the stock solution and add 2.0 mL to a 50 mL flask to produce a
concentration of 0.15 mg/mL. A hundred One percent 0.165 mg/mL: Measure out
2.2 mL of the sample stock solution and pour it into a 50 mL flask. A hundred
Determine the volume at a concentration of 0.18 mg/mL by adding stock solution.
This will yield twenty percent (Akçay, Bayrak et al. 2012).
3.7.7.3 Determinants for Approval:
 Relative Standard Deviations (RSD) NMT 2.0%
 % Variation NMT 2.0%

3.7.8 Longevity and Resilience

How discriminating or measurable outcomes were affected by small but significant
modifications to specific analytical limits was one way to test robustness (Peral and Gallego
1997).
Table 3.4: Determinants of Stability
Parameters Attribute Variations
Solution preparation Stability of stock Extended stability for up to four
Stability of sample hours
Mobile phase / Diluent Concentration, pH, and ± 10% less than the quantity or
stability of the mobile phase half a unit Maintains stability for
at least four hours.
Chromatography Flow rate Column temperature up to 40 degrees Celsius, or the
inverse, in milliliters per minute
3.7.9 Limit of Detection (LOD)
In order to find the LOD, the following is used:
3.3×σ
Limit of Detection =
S
Where,
σ =¿ Standard deviation of regression line / y-intercept
S = Slope of the calibration curve
 3.7.10 Limit of Quantification (LOQ)
To find the LOQ, we utilize the following:
10×σ
Limit of Quantification=
S
Where,
σ =¿ Standard deviation of regression line / y-intercept
S = Slope of the calibration curve
 CHAPTER 4
RESULTS AND DISCUSSION
Creating a gastro retentive dose form that effectively delivers medication to the
stomach is a challenging task. In order to accomplish the needed gastro retention, several
methods were used, the most promising of which was the floating drug delivery system. The
efficacy of medicine absorption from the upper stomach is enhanced by these mechanisms.
Maximizing sensitivity, minimizing duration, and optimizing separation efficiency were our
primary objectives throughout the method's development. According to their unique
structures, all three medications exhibit polar and ionic characteristics (Mulloev, Majidov et
al. 2022).
After the biological solution for the sample had been combined with the VPZ
standard solution in defined proportions, it was moved to the measurement cell.
Next, the manufactured CuO/CPE was calibrated to measure VPZ concentration
voltammetrically. Using 230 nm UV detection, the HPLC method yielded significantly
different results from the average recoveries calculated above. High recoveries and
sufficient relative standard deviation values demonstrated that the suggested
analytical technique was appropriate for VPZ analysis in both pure forms and
biological samples with or without phenylephrine. Present strategy showed respectable
average recoveries and sensitivity when compared to the published HPLC methodology. It
also benefited from speedy and economic analysis (Jayabharathi, Thanikachalam et al. 2012).
This study aims to develop methodologies for imidazole validation testing,
with a particular emphasis on the evaluation of Amibazole tablets. The medicinal
advantages of amibazole pills have made them popular in the pharmaceutical
business and among patients. In my line of work, I develop and implement reliable
analytical procedures to precisely measure the amount of active substances in a
tablet. To guarantee the procedure's precision, accuracy, and reliability, it must
undergo rigorous testing, data analysis, and optimization. Several comprehensive
investigations were conducted to evaluate the created method's performance and
ensure its validity. Linearity, accuracy, precision, specificity, and adaptability are
some of the parameters that are evaluated in this technique (Zhong, Xu et al. 2016).
4.1 High Performance Liquid Chromatography (HPLC)
Using an HPLC method, the quantitative determination of imidazole in tablet dosage
form was carried out. Use of high-performance liquid chromatography (HPLC) for drug
confirmation and quantitative analysis. Concurrent analysis of imidazole in pharmaceutical
dosage form. There are two analytical procedures that have been described in the literature
for determining the amount of imidazole in tablets: infrared spectroscopy and ultraviolet
spectrophotometer. Imidazole couldn't be monitored by any of these at the same time
because of how selective they are. The goal of developing and validating analytical methods
is to use what is known about scientific procedures and quality control to the process of drug
analysis using HPLC (High Performance Liquid Chromatography) (Mol, Aruldhas et al. 2019).
4.2 Analytical Method Development
The process of developing the approach began after a literature review on imidazole
was completed. Thus, the basic column's stationary phase was initially selected as a
50:50 mixture of acetonitrile and phosphate buffer at a pH of 6.0.
When this analytical method was first developed, separation could be achieved with
a flow rate of 1.0 ml/min and a column temperature between 2 and 8°C. Using a C18
Welchrom HPLC column as the stationary phase, keeping the column temperature at
 30-35°C, and changing the detector wavelength to 230 nm, the desired practical
results were finally achieved. The column dimensions are 4.6 mm × 25 cm and the
particle size is 5µm (Marboutin, Desbois et al. 2009).
4.3 Validation of the technique
4.3.1 Systemic appropriateness
In order to ensure that the system is running smoothly, its appropriateness is
assessed either before or during the analysis. To ensure the chromatographic system is
suitable for the analysis, it must undergo system suitability tests to ensure the resolution and
repeatability are adequate. To ensure the procedure's accuracy, five standard working
injections were performed at the target concentration of 100%. The tailing aspect, %
relevant deviation from the mean, and number of theoretic plates were all reported
(Benzon, Varghese et al. 2015).
Table 4.1: Imidazole Working Standard Solution Systemic appropriateness
Observational Findings
Working / Reference Standard Solution
Actual Dilution Sample Response Std. % RSD Tailing No. of
Weight Conc. ID Area Average Dev. Factor theoretical
Plate
50.2 g 0.05020 STD=01 1199732 1199296.00 947.15 0.08 1.668 4425
mg/ mL STD=02 1200743 % 1.667 4455
STD=03 1198875 1.662 4487
STD=04 1198736 1.653 4512
STD=05 1198394 1.646 4568
All three of the calculated numbers—Imidazole 1.659 for the Tailing Factor,
Imidazole 0.08% for the mean Results percent RSD, and Imidazole 4489.4 for the
theoretical Plates—are within the range of acceptable values. Consequently, all
parameters were considered acceptable.

4.3.2 Particularity
Analysis of sample spiked with all potential contaminants demonstrates the absence
of mild interruption during the heights of interests in maintenance periods of time, a
phenomenon called particularity. To ensure that the analytical approach could reliably
signal stability, stress tests were conducted. There are three measurements taken of each of
the standard, stock and control solution. Determine extent to which the API's peak response
region is affected by interference by comparing the percentages of RSD and content
computed (Lagutschenkov, Lorenz et al. 2011).
 Figure 4.1: Blank solution chromatogram
Figure 4.2: Sample solution chromatogram

Figure 4.3: Control solution chromatogram

Table 4.2: Imidazole standard solution suitability reports
 50.2 g 0.05020 STD = 01 1199732 1199783.33 953.06 0.08 %
mg/mL STD = 02 1200743
STD = 03 1198875
Actual Dilution Sample ID Response % Recovery
Weight Conc. Area Average
Table 4.3: Imidazole sample solution suitability values
Sample Solution
200.1 g 0.50025 SPL = 01 1582084 1581406.67 98.99 %
mg/mL SPL = 02 1581261
SPL = 03 1580875
Table 4.4: Imidazole control solution suitability values
Control Solution
200.5 g 0.50125 Control=01 unanswered unanswered ----
mg/mL Control=02 unanswered
Control=03 unanswered
Table 4.5: Imidazole blank solution suitability values
Blank solution
---- ---- blank=01 unanswered Unanswered -----
blank=02 unanswered
blank=03 unanswered
The results shown in the list and the chromatograms showing the Imidazole
outcome clearly illustrate that at the appropriate RT of the major peak periods, neither the
Blank Sample (Mobile Phase) nor the Control sample exhibited any detectable reaction.
There was a 98.99 percent concentration of imidazole. Because it satisfies the stated
standards, the parameter is considered validated (Benzon, Varghese et al. 2015).
4.3.3 Precision
This process was repeated twice so that different quantities of pure VPZ
could be determined. The standard deviation and mean recovery percentage were
computed once the concentrations were obtained using the respective coefficients
of regression.
Ninety percent, one hundred percent, and eleven hundred percent of the samples were
obtained to encompass the test range. Injecting three replicates of each concentration
allowed us to calculate recovery, variation, and an interval of confidence equal to 95%
(Douhal, Amat-Guerri et al. 1994).
Table 4.6: Findings on the Precision of Imidazole sample concentration
Working Standard Concentration (100%) (Imidazole)
Actual Weight Dilution Conc. Sample ID Response Average Std. Dev %RSD
Area
50.2 g 0.05020 mg/mL STD = 01 1199732 1199783.33 953.06 0.08 %
STD = 02 1200743
STD = 03 1198875
The table of observations revealed a percent RSD of 0.08 and a standard
variation of 953.06 for the effective standards concentration for imidazole (100%)
Table 4.7: Predictability of Imidazole Concentration in Samples
Predictability of Sample Concentration (Imidazole)
Target Actual Dilution Sample ID Area Percent Average Std. 95% CI
Conc. Weight Conc. Recovery Dev.
 90.0% 180.2 mg 0.45050 90%= 01 1461610 101.60 101.69 % 0.14 ± 0.35
mg/mL 90%= 02 1465298 101.85
90%= 03 1461923 101.62
100.0% 200.1 mg 0.50025 100%= 01 1582084 99.04 98.99 % 0.04 ± 0.10
mg/mL 100%= 02 1581261 98.98
100%= 03 1580875 98.96
110.0% 250.3 mg 0.50750 110%= 01 1770512 100.67 100.52 % 0.38 ± 0.95
mg/mL 110%= 02 1760326 100.09
110%= 03 1773077 100.81
Based on the outcomes, the value is proven because the imidazole sample level was
found to be 101.69% ± 0.35 (90%), 109.99% ± 0.04 (100%), and 100.52% ± 0.95 (11%). Actual
weight of the sample concentrations were 180.2 mg, 200.1 mg, and 250.3 mg, respectively
(Khan, Alam et al. 2019).
4.3.4 Spikings
A rough calculation was done to determine the nominal content of the
pharmaceutical dosage form. The standard addition method was also employed to
investigate the effects of adding different concentrations of only standards medications to
the pharmaceutical formulation (Sen, Mallick et al. 2016).
Table 4.8: The striking findings of the imidazole working standard concentration
Working Standard Concentration (100%) (Imidazole)
Actual Weight Dilution Conc. Sample ID Response Average Std. Dev %RSD
Area
50.2 g 0.05020 mg/mL STD = 01 1199732 1199783.33 953.06 0.08 %
STD = 02 1200743
STD = 03 1198875

Table 4.9: Spiking outcomes of spiked imidazole sample solution concentration

Spiked sample solution
Actual Weight Dilution Conc. Sample ID Response % Recovery %Variation
Area Average
62.2 g 0.06220 mg/mL SPK = 01 2017152 2021290.33 101.26 % 1.26 %
SPK = 02 2027688
SPK = 03 2019031
There is an overall RSD of 0.08 and a percentage variation of 1.26 for the samples
spiked with imidazole. The sample and standard percentage variations must not be more
than ± 2.0% in order to meet the spiking approval standards. This means it satisfies the
acceptance criteria that were set out. Therefore, the parameter is considered validated
(Andreev, Budantseva et al. 2009).

4.3.5 Accuracy
4.3.5.1 Repetitiveness
There were six separate injections of a 100% sample concentration,
and the standard deviation of those injections was determined as a percentage.
Table 4.10: The Reliability and Accuracy of Imidazole Sample Concentrations
Imidazole Sample Concentration Accuracy (Reliability)
Target Dilution Conc. Sample Response Std. Dev. % RSD
Conc. ID Area Average
 200.1% 0.50025 SPL01 1582084 1576621.50 7562.24 0.48 %
mg/mL SPL02 1581261
SPL03 1580875
SPL04 1562944
SPL05 1580102
SPL06 1572463
By utilizing a goal concentration of 100%, the results for percent relativistic and
variation for the sample are 0.09, which meets the predetermined acceptance conditions. As
a result, the parameter was deemed valid. Typical accuracy level The intermediate
correctness was also checked by many analysts using different instruments in different labs
on different days (Adesokan, Chaban et al. 2007).
Table 4.11: Accuracy of the Imidazole Working Standard Concentration: An
Intermediate-Level Assessment
Concentration of Imidazole Working Standard with Intermediate Accuracy (100%)
Analyst Actual Dilution Conc.
Sample Response Std. Dev. % RSD Difference
Weight ID Area Average
Analyst A 200.1 mg 0.50025 STD01 1582084 1576621.50 7562.24 0.48 % 0.03 %
mg/mL STD02 1581261
STD03 1580875
STD04 1562944
STD05 1580102
STD06 1572463
Analyst B 200.5 mg 0.50125 mg/mLSTD01 1563093 1571953.83 8059.35 0.51 %
STD02 1584279
STD03 1575077
STD04 1564272
STD05 1576035
The study's experimental design allowed for investigation of intermediate
precision. The findings are displayed in table 4.9 up there. Results for the standard
deviation and percent RSD were computed using a 100% imidazole solution as the
working standard (Reddy, Cho et al. 1996).
4.3.6 Linearity
Imidazole was dissolved in a stock solution (as described in section 3.6.6). In order to
create the calibration plot, five different concentration solutions were prepared: 80%, 90%,
100%, 110%, and 120%. To test the linear relationship of the analytical technique , the
standard solution of stock was diluted to create a working standard. The Y-axis and the
X-axis represent the peak area and the concentration values, respectively. To assess the
linearity study, the following criterion was employed. Present a statistically insignificant
intercept. One way to check if your data is linear is with an analysis of variances (ANOVA).
That the regression was probably not an accident is demonstrated by this (Giuliano, Bizzocchi
et al. 2019).
Table 4.12: Impact of Imidazole on The linear approach
Voloprazan Operational Grade Concentration (100%) Accuracy (Intermediate)
Sr. Dilution Conc. Response Std. Dev. % RSD Intercept Slope r2
No. Area Average
1 80.0 % 977404 977134.00 909.57 0.09 27503.8 23752193.33 1.000
(0.04 mg/mL) 976120
977878
 2 90.0 % 1088890 1098963.33 8726.05 0.79
(0.045 mg/mL 1103800
1104200
3 100.0 % 1212032 1210682.00 1637.83 0.14
(0.05 mg/mL 1208860
1211154
4 110.0 % 1335295 1336735.00 1247.22 0.09
(0.055 mg/mL 1337474
1337436
5 120.0 % 1455824 1452053.00 7716.63 0.53
(0.06 mg/mL 1443176

Examining five sets of varying concentrations between 0.04 and 0.06 revealed vonoprazan's
stability. Vonoprazan achieved a highly significant correlation coefficient (r2 value, 1.000) in
the 0.04–0.06 range. Accordingly, the values satisfied the established acceptance criteria,
indicating that the approach was deemed valid (Molina, Tárraga et al. 2012).

Figure 4.4: Graphical representation of Imidazole

Linearity Graph
1550000

1450000
f(x) = 23752193.4 x + 27503.7959999999
1350000 R² = 0.999750591991912

1250000
Area

1150000

1050000

950000
0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065
Concetration

4.3.7 Range
The exactness, precision, and predictability indicated by the set procedure as it
pertains to an analytical method's range is defined as the range between the higher and
lower results. Imidazole range was determined by preparing a variety of
concentrations in five distinct ranges. Based on what is stated in section 3.6 (Al-Otaibi,
Almuqrin et al. 2020).
Table 4.13: Concentration Range of Imidazole Used as a Standard
Concentration of Imidazole for Working Standards (100%)
Actual Dilution Conc. Sample Response Std. Dev. %RSD
Weight ID Area Average
50.2 mg 0.05020 mg/mL STD 01 1199732 1199783.33 953.06 0.08 %
STD 02 1200743
STD03 1198875
Table 4.14: Range of Sample concentration of Imidazole
Range of Sample Concentration (Imidazole)
Actual DilutionCon SampleID Response %RSD % %Variatio
Weight c. Area Average Content n
161.8 mg 0.40450 80% = 01 1319071 1315296.33 0.25 % 81.46 % 1.82 %
(80%) mg/mL 80% = 02 1313478
80% = 03 1313340
180.2 mg 0.45050 90% = 01 1461610 1462943.67 0.14 % 91.52 % 1.69 %
(90%) mg/mL 90% = 02 1465298
90% = 03 1461923
200.1 mg 0.50025 100% = 01 1582084 1581406.67 0.04 % 98.99 % -1.01 %
(100.0 %) mg/mL 100% = 02 1581261
100% = 03 1580875
220.3 mg 0.55075 110% = 01 1770512 1767971.67 0.38 % 110.58 % 0.52 %
(110%) mg/mL 110% = 02 1760326
110% = 03 1773077
240.8 mg 0.60200 120% = 01 1951913 1958600.00 0.31 % 122.26 % 1.88 %
(120%) mg/mL 120% = 02 1959898
120% = 03 1963989
The variation of Imidazole concentrations in the sample was determined by
fabricating five unique concentrations: 80%, 90%, 100%, 11%, and 120%. Each outcome is
within the permissible range. All concentrations' percentage variation values are within the
specified acceptable range. As a result, the value is meant to be confirmed (Chen, Zhang et
al. 2006).
4.3.8 Robustness
The robustness of specific surveillance techniques fluctuates in normal
operating settings. One way to determine whether an analytical technique is suitable
for routine usage is to look at its robustness, which is the extent to which it can resist
purposeful, minor modifications to any of the parameters listed in the technique's
protocol. Here, we'll use a 100% Imidazole standard concentration and a 100%
sample concentration that we created according to the steps in section 3.6.2. (Kucuk,
Yurdakul et al. 2022).
Table 4.15: Criteria for durability with respect to changes in imidazole
Factors Properties Variance
Solution Preparation Standard solution durability Extended durability for up to
four hours
Sample solution durability Extended durability for up to
four hours
Mobile Phase / Diluent Mobile phase durability Extended durability for up to
four hours
pH +/- half a unit
Concentration Part in a reduced amount of ± 1
Chromatography Column Temperature Controlled to a temperature of
40oC or the opposite
Rate of flow 0.5% per minute
Using slightly different buffers, temperatures, and other factors from the
method settings, an experiment was conducted to ascertain the percent drug content.
Table 4.15 shows that the analytical results were unaffected by changes in rate of flow
and wavelengths, proving that the existing approach is robust.
 Table 4.16: Consistent Imidazole Solution Consistency for All Varieties
Repeatability of a Standard Solution
Sample ID Variation Time Retention Time Area Average
RT Average
STD = 01 Initial 3.63 3.63 1207073 1207581.00
STD = 02 3.63 1208089
STD = 01 After 2 hours 3.66 3.66 1202388 1201892.50
STD = 02 3.66 1201397
STD = 01 After 4 hours 3.64 3.64 1203652 1203465.00
STD = 02 3.64 1203278
The new HPLC method is durable, as no substantial change was found in the
resultant durability parameters of the imidazole standard solution (Srinivasan, Sawant et al.
2007).
Table 4.17: Ratio of Imidazole via the Mobile Phase
Mobile Phase Concentration
Sample ID Variation (Conc.) Retention Time Area Average
RT Averag
STD = 01 Initial 3.63 3.63 1207073 1207581.00
STD = 02 3.63 1208089
STD = 01 + 10.0 % 3.33 3.33 1100708 1100506.50
STD = 02 3.33 1100305
STD = 01 - 10.0 % 4.05 4.05 1301547 1301556.00
STD = 02 4.05 1301565
The devised HPLC method is robust, as indicated by the substantial outcomes
in the the bands (no response) for the mobile phase concentration of imidazole.
Table 4.18: Temperature of the imidazole column
Mobile Phase Concentration
Retention Time
Sample ID Variation (Conc.) Area Average
RT Average
STD = 01 3.63 1207073
Initial 3.63 1207581.00
STD = 02 3.63 1208089
STD = 01 3.61 1206273
40oC 3.60 1205975.50
STD = 02 3.60 1205678
Table shows that altering the column temperatures for imidazole does not
significantly alter the chromatogram findings. This proves the established HPLC
process is reliable.
Table 4.19: Rapidity of imidazole flow
Column Flow Rate
Sample ID Variation (Flow) Retention Time Area Average
RT Average
STD = 01 Initial 3.63 3.63 1207073 1207581.00
STD = 02 3.63 1208089
STD = 01 + 0.5 mL/min 2.66 3.33 879285 876823.00
STD = 02 2.66 874361
STD = 01 - 0.5 mL/min 5.89 5.89 1958060 1958224.50
STD = 02 5.89 1958389
As the table illustrates, there is discernible difference in the chromatogram's results
following a little adjustment of the column's Imidazole temperature. This proving the
 robustness of the established HPLC approach shown in table 4.18 (Boghaei, Askarizadeh et
al. 2008).
Table 4.20: Imidazole viability in a mobile state
Mobile phase stability
Sample ID Variation (Time) Retention Time Area Average
RT Average
M. Ph. = 01 Initial --- --- Unanswered ---
M. Ph. = 02 --- Unanswered

M. Ph. = 01 After 2 hours --- --- Unanswered ---

M. Ph. = 02 --- Unanswered

M. Ph. = 01 After 4 hours --- --- Unanswered ---

M. Ph. = 02 --- Unanswered

The devised HPLC method is robust, as shown by the result values, It showed
that changing the mobile phase makeup and flow rate had a major impact, but
changing the other parameters had nothing to do with it. In light of this, the verified
parameter (Jayabharathi, Thanikachalam et al. 2012).

Table 4.21: Ensuring the Durability of Imidazole Sample Solutions

Sample Solution Durability
Sr. Sample ID Variation (Time) Retention Time Area Average
RT Average
1. SPL = 01 Initial 3.64 3.64 1708219 1709837.50
SPL = 02 3.64 1711056
2. SPL = 01 After 2 hours 3.65 3.65 1710077 1708730.50
SPL = 02 3.65 1707384
3. SPL = 01 After 4 hours 3.63 3.64 1703493 1702701.00
SPL = 02 3.63 1701909
There was no discernible change in the sample solution stability values of
imidazole, proving the robustness of the devised HPLC technique.
Table 4.22: Preservative Resistance in Imidazole Sample Solutions
Mobile Phase pH
Sample ID Variation (Time) Retention Time Area Average
RT Average
STD = 01 Initial 3.63 3.63 1207073 1207581.00
STD = 02 3.63 1208089
STD = 01 + 0.5 unit 4.28 4.28 1191341 1192421.50
STD = 02 4.28 1193502
STD = 01 - 0.5 unit 3.58 3.29 1213615 1208594.00
STD = 02 2.99 1203573
No substantial variation was found in the result values of mobile phase pH of
imidazole, indicating that the HPLC method devised is robust. The devised HPLC method is
robust, as shown by the result values: chromatograms changed significantly when the flow
rate and mobile phase composition were changed, but no other parameters changed
noticeably. Consequently, the validated parameter (Ricciardi, Romanchick et al. 1983).
4.3.9 Limit of Detection (LOD)
Assessing the quantitative testing and pollutant limit of detection is standard
procedure. "The detection limit of a character analytical technique is the lowest amount of
analyze in a sample that can be detected but not necessarily quantities as an exact
number,"statedICHQ2".
The formula was used to calculate the LOD value,
3.3 ×σ
Limit of Detection=
S
The determined limit of detection for Imidazole is 0.00014 µg/ mL.
4.3.10 Limit of Quantitation (LOQ)
LOQ, also called the quantitation limit (QL) on occasion, is the minimal
detectable concentration of an analyte that can be reliably quantified using a certain
technique. An honest and reliable way, where truthfulness and correctness are
paramount and can be confirmed (Thirumurugan and Anitha 2018).
The following formula was used to obtain the LOQ value:
10 × σ
Limit of Detection =
S
The concentration of imidazole that may be measured is 0.00042 µg/mL.