1.

INTRODUCTION
[1]
1.1. INTRODUCTION TO ANALYTCAL CHEMISTRY

Analytical chemistry is a measurement science consisting of a set of

powerful ideas and methods that are useful in all fields of science and

medicine. Analytical chemistry is applied throughout industry, medicine,

and all the sciences for qualitative and quantitative analysis.

The scope of analytical chemistry is very broad and embraces a

wide range of chemical, instrumental techniques and procedures. Most

people apply some form of chemical analysis almost every day, such as

when smelling food to see if it as deteriorated or tasting substances to

determine if they are sweet or sour. These constitute simple analytical

processes, as compared with some of the more complex processes,

described in this volume that can be achieved with modern instruments.

It is not always necessary to apply advanced instrumental

procedures to carry out accurate analyses and there may be many times

when simple, rapid analysis may actually be more desirable than a

complicated and time consuming process. The objective and purpose of

the analysis has to be sensibly assessed before selecting an appropriate

procedure.

When a completely unknown sample is presented to analyst, the

requirement is usually to ascertain what substances are present in it. This

fundamental problem may sometimes be encountered in the modified

form of deciding what impurities are present in a given sample, or

1
perhaps of confirming that certain specified impurities are absent. The

solution lies within the province of qualitative analysis.

Having ascertained the nature of the constituents of a given

sample, then upon to determine how much of each component, or of

specified components, is present. Such determinations lie within the realm

of quantitative analysis, and a variety of techniques are available to

supply the required information.

Production and pharmacopoeial controls:

Once established as marketable products, all drug substances and

their formulated dosage forms are subjected to strict quality assurance

procedures. These encompass manufacturing methods, in-process

controls, release specifications, and finally check specifications that are

applicable throughout the shelf life of the product.

Sophisticated instrumental methods capable of automation to deal

with large numbers of samples linked to computerised data recording

systems for automatic calculation and recording of results.

The widespread availability of relatively cheap ultraviolet

spectrometers has brought a degree of precision into the characterization

and standardization of drugs, and such techniques are now widely used in

both the factory and independent control laboratories. Thin layer

chromatography, is sensitive, quick and easy to apply, and hence is also

in widespread use both for verification of identity, and in control tests to

limit the degree of contamination by related substances and

decomposition products.

2
 The move towards instrumental methods, particularly in the drive

to reduce dependence on biological assays and limit test requiring

animals, has led to the acceptance of a number of less widely used

methods.

The sensitivity of spectroflourimetry is also the reason for it being

chosen as the most suitable technique for the control of AI levels in

haemodialysis solutions.

Methods are the need to compensate for instrument and operator

variables, both within and between laboratories. Many of these techniques

require the use of reference compounds or internal standards of known

quality and composition.

Analytical instrumentation in small control laboratories, their

selectivity, sensitivity, and speed of operation ensure their continuous use

on an expanding scale.

Gas chromatography, capillary gas chromatography and high

performance liquid chromatography all have their place. But much use is

now made of such powerful coupled separative detector systems as gas

chromatography- mass spectroscopy (GC-MS) and liquid chromatography-

mass spectroscopy (LC-MS). These systems are extremely versatile but

very expensive, and much valuable work is still achieved with the less

sophisticated and less versatile through sensitive detection capabilities of

ultraviolet spectrophotometry, spectroflourimetry and polarography.

Applications:

3
 With increasing demands for pure water, better food control and

cleaner atmospheres, the analytical chemist has a greater and

greater role to play with in modern society.

 Manufacturing industries rely upon both qualitative and quantitative

chemical analysis to ensure their raw materials meet certain

specifications and to check the equality of the final product. Raw

materials are examined to ensure that there are no unusual

substance present which might upset the manufacturing process or

appear as a harmful impurity in the final product.

 The manufacturing product is subject to quality control to ensure

weather the components are present within a predetermined range

of composition and impurities does not exceed certain specified

limits.

 The development of new products (which may be mixtures rather

than pure materials) requires the services of the analytical chemist.

It will be necessary to ascertain the composition of the mixture

which shows that optimum characteristics for the materials has

been developed.

 Many industrial processes give rise to pollutants which can present

a health problem. Quantitative analysis of air, water, and sometimes

soil samples, must be carried out to determine the level of pollution,

and to establish safe limits for pollutants

4
Stages of analysis [2]

The stages of analysis should be carefully considered and assessed

in order to minimize errors and to maintain accuracy and reproducibility.

Sampling

Preparation of
Analytical sample

Dissolution of sample

Removal of interference

Sample measurement and

control of instrumental factors

Results

Presentation of data

Selecting the methods:

Several approaches can often be used to analyse a particular

sample. To choose the most appropriate, analyst must be familiar with the

practical details of the various techniques and the theoretical principles on

which they are based. They must also understand the conditions under

which each method is reliable consider possible interference which may

5
arise and find ways to circumvent and problems. Analyst will also be

concerned with accuracy and precision, time and costing. The most

accurate method for a certain determination may prove to be lengthy or it

may requires expensive reagents, and in the interests of economy it may

be necessary to choose a method which, although somewhat less exact,

yields results sufficient accuracy in a responsible time.

The main techniques employed in a quantitative analysis are

based on

 The quantitative performance of suitable chemical reactions and

either measuring the amount of reagent needed to complete the

reaction, or ascertaining the amount of reaction product obtained.

 Appropriate electrical measurements ( e.g.potentiometry )

 The measurement of certain spectroscopic properties (e.g.

absorption spectra).

 The characteristics moment of a substance through a defined

medium under controlled conditions. Sometimes two or more of

these techniques may be used in combination to achieve both

identification and quantification ( e.g. gas chromatography may be

linked to mass spectrometry)

Factors affecting the choice of analytical methods:

 The type of analysis required.

 Problems arising from the nature of the material to be investigated.

 Possible interference from components of the material other than

those of interest.

 The concentration range to be investigated.

6
  The accuracy required.

 The facilitates available, particularly the instrument.

 The time required to complete the analysis.

This will be particularly relevant when the results are required

quickly for the control of manufacturing process.

 It is always worth bearing in mind that single or exploratory analysis

may often be carried out which move cheaply and often more

quickly by a traditional titrimetric and gravimetric procedure in

which only limited sample preparation may be required.

 The cost of analysis does not only include the actual time the

equipment is used, but also a proportion of the maintenance costs

and the time the instruments may be standing idle.

 The method finally chosen for the required determination should be

a specific method. It should be capable of measuring the amount of

desired substance accurately, no matter what other substance may

be present.

 Improved selectivity is frequently achieved by carrying out the

analytical procedure under carefully controlled conditions.

Data handling:

 The analysis should be carried in duplication and preferably in

triplicate.

 All analytical results should be carefully recorded in a suitable note

book to provide a permanent record of the work.

 Many modern instruments are either computer operated or

interfaced with computers, so the results are not only displayed on a

7
 visual display unit but also presented graphically and or in tabular

form to serve as a detailed record.

 Calculations on the analytical results may be necessary present

them in the form required.

 Many instruments are now able to treat the read data and to relate

it to calibration charts action limits and statistical analysis.

 Any results are obtained are subject to a degree of uncertainty and

it is necessary to establish the magnitude of this uncertainty in

order that meaningful results can be presented. This is commonly

expressed in the terms of numerical difference between a given

experimental value and the mean value of all the experimental

results.

 The most important measures of precision are the standard

deviation on the variants.

 The difference between the most probable analytical results are the

true value for the sample is termed the systematic error in the

analysis, it indicates the accuracy of the analysis.

1.2 INTRODUCTION TO ABSORPTION SPECTROSCOPY[3]

Spectrophotometric techniques depend on the absorption or

emission of electromagnetic radiation of characteristic energy changes

within an atomic or molecular system.

Ultraviolet spectroscopy is concerned with the study of absorption of

UV radiation, which ranges from 200-400 nm. When radiation is passed

through a layer of a solution containing an absorbing substance, part of

8
radiation is absorbed. The intensity of the radiation emerging from the

solution is less than the intensity of radiation entering it. The wavelength

at which maximum absorption occurs is called max. It is independent of

concentration. The absorption of radiation is governed by the Beer-

Lamberts law.

Figure 1.1 UV Visible spectrophotometer

Lambert’s Law is defined as: the intensity of a beam of parallel

monochromatic radiation decreases exponentially as it passes through a

medium of homogeneous thickness (or) the absorbance is directly

proportional to the thickness (path length) of the solution.

Lambert investigated the relationship between I o and It for various

thickness of substance and found that the rate of decrease in the intensity

of light with the thickness, b, of the medium is proportional to the

intensity of incident light, and it is expressed as

A =
log10 Io
It
Beer’s law is defined as: the intensity of a beam of parallel

monochromatic radiation decreases exponentially with the number of

9
absorbing molecules (or) the absorbance is proportional to the

concentration of the solution.

Beer showed that a similar relationship exists between the absorbance

and the concentration, and it is expressed as

Log Io/It = k”c /2.303

A combination of the two laws yields the Beer-Lambert law

A = abc

Where

A = absorbance

a = absorptivity

b = path length

c = concentration

The name and value of ‘a’ depend on the units of concentration. When

‘C’ is in moles per liter, the constant is called molar absorptivity, and

has symbol ‘’. The equations therefore take the form

A = bc

Another form of the Beer-Lambert proportional constant is the

specific absorbance, which the absorbance of a specified concentration in

a cell of specified path length. The most common form in pharmaceutical

analysis is the A (1% 1cm), which is the absorbance of a 1g/100ml (1%

w/v) solution in a 1cm cell. Therefore, the Beer-Lambert equation

A = A1%1cmbc

Where, C is in g/100ml and b is in cm, the units of A (1% 1cm) are dl g-1 cm-
1
.

10
1.3 INTRODUCTION TO INFRARED SPECTROSCOPY

Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy)

is the measurement of the interaction of infrared radiation with matter by

absorption, emission, or reflection. It is

used to study and identify chemical substances or functional groups in

solid, liquid, or gaseous forms. The method or technique of infrared

spectroscopy is conducted with an instrument called an infrared

spectrometer (or spectrophotometer) which produces an infrared

spectrum. An IR spectrum can be visualized in a graph of infrared light

absorbance (or transmittance) on the vertical axis v/s frequency or

wavelength on the horizontal axis. Typical units of frequency used in IR

spectra are reciprocal centimetres (sometimes called wave numbers),

with the symbol cm−1. Units of IR wavelength are commonly given in

micrometres (formerly called "microns"), symbol μm, which are related to

wave numbers in a reciprocal way. A common laboratory instrument that

uses this technique is a Fourier transform infrared (FTIR) spectroscopy.

Infrared (IR) radiation refers broadly to that part of the

electromagnetic spectrum between the visible and microwave region. Of

greatest practical use to the organic chemist is the limited portion

between 4000 and 400cm -1. There has been sum interest in the near-IR

(14,290-4000cm-1) and the far-IR region (700-200cm-1)

11
 Figure 1.2 FTIR

Although the IR spectrum is characteristics of the entire molecule, it

is through that certain groups of atoms give rise to bands at or near the

same frequency regard less of the structure of the rest of the molecule. It

is the persistence of these characteristics band that permits the chemist

to obtain useful structural information by simple inspection and reference

to generalized charts of characteristics group frequencies.

1.4 INTRODUCTION TO DISSOLUTION TESTING

Definition: Material transfer from solid state to solution per unit time

from unit dose. The release of drug rate and extent was measured using

dissolution testing method, for confirming the absorption. Dissolution and

drug release are the terms used interchangeably. Dissolution is an

important CQA for modified release formulations.

12
 Evolution of drug Dissolution testing: In 1897 Noyes and Whitney

studied the dissolution for two compounds using glass cylinders. The

rationale for selection of benzoic acid and lead chloride were based on

poor solubility. The compounds were submerged in water containing glass

vessel, by maintaining constant temperature and revolution, to

understand the time required for completely dissolving the compounds,

which becomes the basis for Noyes-Whitney equation.

Dissolution requirements were emphasized by regulatory bodies and

pharmacopoeia.

In 1971, USP Apparatus 1 was adopted in monograph. USP

Apparatus 2 was introduced in 1985, USP Apparatus 3 and USP Apparatus

4 was introduced in 1995 for modified release formulation monograph.

There are seven dissolution test apparatus recommended by USP.

Capsule dissolution is a critical process in pharmaceutical sciences

that refers to the rate and extent to which the active pharmaceutical

ingredient (API) is released from a capsule dosage form and becomes

available for absorption in the body. This process plays a vital role in

determining the bioavailability, efficacy, and therapeutic performance of

oral medications. The dissolution behavior of a capsule is influenced by

several factors including the type of capsule shell (e.g., gelatin or

hydroxypropyl methylcellulose), the properties of the API, excipients used

in the formulation, and environmental conditions such as pH and

temperature.

13
 Figure 1.3 Dissolution test apparatus - USP II

[4,5]
1.5 VALIDATION OF ANALYTICAL PROCEDURES

Validation of analytical procedures is to provide some guidance and

recommendations on how to consider the various validation

characteristics for each analytical procedure. In some cases the overall

capabilities of a number of analytical procedures in combination may be

investigated in order to ensure the quality of the drug substance or drug

product. The parameters for method validation are summarised below:

1. LINEARITY:

Ability (within a specified range) to obtain test results which are

directly proportional to the concentration of analyte in the sample.

- Test across the range (at least 5 concentrations).

14
- Evaluate linearity by visual inspection of the plot and by statistical

techniques.

- Calculate corr. coefficient, y-intercept and slope.

2. SPECIFICITY:

Ability to assess unequivocally the analyte in the presence of

components which may be expected to be present (E.g. impurities,

degradants, matrix etc).

- Identification

- Purity tests

- Assay (Content/potency)

3. RANGE:

Interval between upper and lower concentration of the analyte in the

sample for which it has been demonstrated that the procedure has a

suitable level of precision, accuracy and linearity.

- Defined from linearity study

- Depends on the application of the method (assay, dissolution test,

content uniformity)

4. ACCURACY:

Expresses the closeness of agreement between the value which is

accepted either as a conventional true value or an accepted reference

value and the value found.

15
 Accuracy studies, for drug substance and drug product are performed

at 50%, 75% and 100% levels of label claim.

Drug substance

- Use of reference standard with known purity

- Comparison with independent, well-characterized procedure

- May be inferred once precision, linearity and specificity are Established

Drug product

- Spiking of placebo mixture

- Addition of analyte to ‘active’ material

- Comparison of results obtained with independent, well-characterized

procedure

- May be inferred once precision, linearity and specificity are established

Impurities

- Spiking of product samples

- Use of independent, well-characterized procedure

Recommended data

- Assessed by 9 determinations over a minimum of 3 concentration levels

covering the specified range

- To be reported as percent recovery

5. PRECISION:

Closeness of agreement between a series of measurements obtained

from multiple sampling of the same homogeneous sample.

- Repeatability

16
- Intermediate precision

- Reproducibility

PRECISION - REPEATABILITY

Precision under the same operating conditions over a short interval of

time.

- 9 determinations covering the specified range

- 6 determinations at 100% of the test concentration

PRECISION - INTERMEDIATE PRECISION

Expresses within laboratory variations.

- Depends on circumstances of usage of the methods

- Should include variations in days, columns

PRECISION - REPRODUCIBILITY

Precision between laboratories

- Dependent on usage of method

- Should include interlaboratory study

6. DETECTION LIMIT:

Lowest amount of an analyte in a sample which can be detected but

not necessarily quantitated.

- Based on visual evaluation

- Based on signal-to-noise ratio (3:1)

- Based on st.dev. (SD) of response and slope (DL=3.3xSD/S)

- Report results and method of choice

7. QUANTITATION LIMIT:

Lowest amount of an analyte in a sample which can be quantitatively

determined with a suitable precision and accuracy.

17
- Based on visual evaluation

- Based on signal-to-noise ratio (10:1)

- Based on st.dev. (SD) of response and slope (DL=10xSD/S)

- Report results and method of choice

8. ROBUSTNESS:

Measure of the capacity of a method to remain unaffected by small

variations in method parameters.

- To be considered during development

- To be used for establishment of system suitability criteria

- Include testing of stability of solutions

- To be tested by introducing small variations in method parameters

9. RUGGEDNESS:

The ruggedness of an analytical procedure is the degree of

reproducibility of test results obtained by analyzing the sample under

variety of normal test conditions E.g. different analysts, different

instruments, different laboratories, different days, different reagents etc.,

10. SYSTEM SUITABLITY PARAMETERS:

System suitability is done to assure validity of analytical procedure. It

is done to demonstrate that the analytical system is performing properly.

1.6 STATISTICAL VALIDATION

Linear regression

Once a linear relationship has been shown to have a high probability

by the value of the correlation coefficient ‘r’, then the best straight line

18
through the data points has to be estimated. This can often be done by

visual inspection of the calibration graph, but in many cases it is far more

sensible to evaluate the best straight line by linear regression (the

method of least squares).

The equation of straight line is y = bx + a

Where y, the dependent variable, is plotted as a result of changing

x, the independent variable.

To obtain the regression line ‘y on x’, the slope ‘b’ of the line and

the intercept ‘a’ on the y-axis are given by the following equation:

nΣx1 y 1 −Σx1 Σy1

b=
nΣx 2−( Σx1 )2
1

and a= ȳ− x̄

Where x̄ the mean of all is values of x1 and ȳ is the mean of all

values of y1 (Kamboj, 2003).

Correlation Coefficient

To establish whether there is a linear relationship between

two variables x1 and y1, Pearson’s correlation coefficient r is used

nΣx 1 y 1 −Σx 1 Σy 1
r=
{[ nΣx 2−( Σx 1 )2 ][ nΣy 2−( Σy 1 )2 ]}1/2
1 1

Where, n = Number of data points

The value of r must lie between +1 and –1; the nearer it is to

±1, the greater the probability that a definite linear relationship exists

between the variables x and y; values close to +1 indicate positive

correlation and values close to -1 indicate negative correlation. Values of

19
r that tend towards zero indicate that x and y are not linearly related

(They may be related in a non linear fashion.)

Note: A value of the correlation coefficient r of near +1 or –1 does

not confirm a linear relationship. Many non linear plots will give a high

positive value of the correlation coefficient. A scatter diagram should

always be plotted first to ensure the calibration curve is line.

Standard deviation

It is commonly used in statistics as a measure of precision and

is more meaningful than is the average deviation. It may be thought of as

a root-mean-square deviation of values form their average and is

expressed mathematically as;

√
i=n
∑ ( x i − x̄ )
i =1
S=
N −1 Where S is standard deviation

If N is large (50 or more), then of course, it is immaterial

whether the term in the denominator is N-1 or N.

S = Sum

x̄ = Mean or arithmetic average

x - { x̄ ¿ = deviation of a value from the mean

N = Number of observations.

Percentage relative standard deviation [% RSD]

 This is also known as coefficient of variation, v.

 It is defined as the standard deviation (S.D) expressed as the

percentage of mean.

20
 S.D
V or % RSD= ×100
x̄

 The variance is defined as S2 and is more important in

statistics than S itself. However, the latter is much more commonly used

with chemical data.

Standard error of mean (S.E.)

 Standard error of mean can be defined as the value obtained

by division of standard deviation by square root of number of

observations.

 It is mathematically expressed as

S. D.
S.E.=
√n n = number of observations

Drug Profile

21
2. DRUG PROFILE
[6]
2.1 NINTEDANIB ESYLATE:

Nintedanib Esylate is a small molecule kinase inhibitor used to treat

idiopathic pulmonary fibrosis (IPF) and some types of non-small cell lung

cancer (NSCLC). Is is also used for the treatment of chronic fibrosing

interstitial lung diseases (ILDs) with a progressive phenotype and used for

slowing the rate of decline in pulmonary function in patients with systemic

sclerosis (Scleroderma)-associated interstitial lung disease (SSc-ILD).

Chemical Structure :

2.1 NINTEDANIB ESYLATE

Molecular Formula : C33H39N5O7S

Molecular Weight : 649.76 g/mol

pKa : 2.2 and 7.6

Chemical Name : Methyl(3Z)-3-[({4-[N-methyl-2-(4-methylpiperazin-yl)

acetamido]phenyl}amino)(phenyl)methylidene]2-oxo-2,3-

dihydro-1H-indole-6-carboxylate ethanesulfonate

22
CAS number : 656247-18-6

Melting point : 244 °C (471 °F) to 251 °C (484 °F).

Description : a yellow, crystalline solid

Solubility : Methanol and Hydrochloric acid

Category : Tyrosine kinase inhibitor

PHARMACOKINETICS [7]

Only a small percentage of orally taken nintedanib is absorbed in

the gut, partially due to transport proteins (such as P-glycoprotein)

moving the substance back into the lumen.

Nintedanib is mainly inactivated by esterases that cleave the methyl

ester, resulting in the free carboxylic acid form, which is

then glucuronidated by uridine diphosphate- glucuronosyl

transferases and excreted mostly via the bile and faeces. No

relevant cytochrome P450 mediated metabolism has been observed.

ABSORPTION: Combined with a high first-pass effect, nintedanib reaches

a maximum plasma concentration approximately 2 to 4 hours after oral

administration as a soft gelatin capsule under fed conditions.

The absolute bioavailability of a 100 mg dose was 4.7%.

After food intake, nintedanib exposure increased by approximately

20% compared to administration under fasted conditions (90% CI: 95.3%

to 152.5%) and absorption was delayed (median t max fasted: 2.00 hours;

fed: 3.98 hours), irrespective of the food type.

23
DISTRIBUTION: It is widely distributed in the body, with a volume of

distribution of about 1050 L. It binds to plasma proteins, primarily

albumin.

METABOLISM: The prevalent metabolic reaction for nintedanib is

undergoes first pass metabolism in liver through hydrolytic ester

cleavage, resulting in the formation of the free acid moiety.

EXCRETION: Half-life (t1/2) is 10 to 15 hrs. The major route of excretion for

nintedanib is through the feces, with less than 1% of the drug-related

radioactivity eliminated in urine.

PHARMACOLOGY [8]

Nintedanib competitively inhibits both nonreceptor tyrosine

kinases (nRTKs) and receptor tyrosine kinases (RTKs). NRTK targets of

nintedanib include Lck, Lyn, and Src. RTK targets of nintedanib

include platelet-derived growth factor receptor (PDGFR) α and β; fibroblast

growth factor receptor (FGFR) 1, 2, and 3; vascular endothelial growth

factor receptor (VEGFR) 1, 2, and 3; and FLT3. Its use in IPF is predicated

on its inhibition of PDGFR, FGFR, and VEGFR, which increase fibroblast

proliferation, migration, and transformation.

Chronic Fibrosing Interstitial Lung Diseases with a Progressive

Phenotype

 Indicated for chronic fibrosing interstitial lung diseases (ILDs) with a

progressive phenotype

24
  Unclassifiable ILDs

 autoimmune ILDs,

 chronic hypersensitivity pneumonitis,

 sarcoidosis,

 myositis,

 Sjögren syndrome,

 coal workers pneumoconiosis, and

 idiopathic forms of interstitial pneumonias (eg, idiopathic

nonspecific interstitial pneumonia) are among the diseases that may

develop a progressive form of chronic fibrosing.

Systemic Sclerosis-associated Interstitial Lung Disease

Indicated to slow the rate of decline in pulmonary function in

patients with systemic sclerosis-associated interstitial lung disease (SSc-

ILD) 150 mg PO q12hr

Figure 2.2 Nintedanib Soft Gelatin Capsules150mg

CONTRAINDICATIONS: [9]

25
According to the FDA-approved labeling, there are no absolute

contraindications.

Warning and Precautions

 The use of nintedanib during pregnancy and breastfeeding is not

recommended.

 Moderate to severe hepatic impairment (Child-pugh class B and C) is

also a relative contraindication to nintedanib therapy. Nintedanib is

not well-studied in patients with severe hepatic impairment.

 Tobacco use is associated with reduced efficacy of nintedanib, as

well as worsening of preexisting disease, so patients should be

advised to stop smoking before initiating treatment.

 Caution should be exercised when prescribing nintedanib to women

older than 65 with low body mass index (BMI) or patients with

coronary artery disease, thromboembolic events, anticoagulation,

recent abdominal surgery, and a history of gastrointestinal

perforation.

 Hepatotoxicity

ADVERSE EFFECTS:-

Diarrhea, nausea, vomiting, abdominal pain, decreased appetite,

and weight loss may occur.

Other side-effects include:

 raise your blood pressure.

26
  unusual bruising/bleeding,

 cough/stuffy chest,

 foamy urine,

 swelling ankles/feet/hands,

 symptoms of liver problems (such as dark urine,

yellowing eyes/skin).

DOSAGE AND ADMINISTRATION:

Soft gelatin capsule

 100mg

 150mg

150 mg taken orally twice daily approximately 12 hours apart taken

after food

3. LITERATURE SURVEY

A review of literature was done to enumerate the methods available

for the analysis.

3.1 Keating G.M., 2015[10]

This review highlights the clinical utility of Nintedanib in managing

Idiopathic Pulmonary Fibrosis (IPF). The study noted the drug’s efficacy
in slowing disease progression, improving symptom control, and its
tolerability, confirming its therapeutic relevance in IPF treatment.

3.2 Hattimare K. et al., 2025[11]

The study presents a comprehensive review on UV method

validation and development. It summarizes various analytical

27
strategies, validation parameters, and regulatory expectations, serving
as a useful guide for method development in pharmaceutical analysis.

3.3 Chhippa Y, Saraswathy T.,2024 [12]

In their 2024 review, Chhippa and Saraswathy provided a

comprehensive overview of various analytical techniques—such as
HPLC, UV spectrophotometry, and LC-MS—used for the quantification of
Nintedanib in bulk and pharmaceutical dosage forms. The study also
focused on the identification and characterization of degradation
products under different stress conditions, following ICH guidelines.
Their work emphasizes the significance of method validation
parameters like accuracy, precision, linearity, and robustness in
ensuring reliable drug analysis, making it a valuable reference for
future analytical research on Nintedanib.

3.4 Parmar VK, Desai SB, Vaja T.,2014 [13]

The study presents validated RP-HPLC and UV

spectrophotometric methods for the estimation of pirfenidone in
pharmaceutical formulations. These analytical techniques offer simple,
accurate, and reproducible means for routine quality control. The RP-
HPLC method showed good linearity, precision, and sensitivity, while
the UV spectrophotometric method provided a cost-effective alternative
with satisfactory accuracy. This work contributes significantly to
pharmaceutical analysis by offering robust methods for the
quantification of pirfenidone, a drug used in the treatment of idiopathic
pulmonary fibrosis

3.5 Kim JS, Murray S ,et al.,2024 [14]

The study by Kim et al. (2024) presents a post hoc analysis

of the CleanUP-IPF study, comparing the efficacy and safety of
pirfenidone and nintedanib in treating idiopathic pulmonary fibrosis
(IPF). Both antifibrotic agents were found to slow disease progression,
with similar impacts on lung function decline. However, differences in
adverse event profiles and tolerability were observed, suggesting that

28
individual patient factors may guide treatment choice. This analysis
adds to the growing body of evidence supporting the comparable
effectiveness of these therapies, while emphasizing the need for
personalized management in IPF

3.6 Joshi D. et al., 2021[15]

An analytical method was developed and validated for the

estimation of Saxagliptin in gastric medium using a UV-visible
spectrophotometric technique. The study focused on assessing the
precision and accuracy of the method, confirming its applicability for
pharmaceutical analysis in gastric conditions. (Global Journal of
Pharmacy & Pharmaceutical Sciences)

3.7 Shrivastava A, Gupta V.,2011 [16]

The review provided various approaches used to determine

the limit of detection (LOD) and limit of quantitation (LOQ) in analytical
methods. Their study outlines the significance of these parameters in
ensuring the sensitivity and reliability of analytical techniques,
especially in pharmaceutical and chemical analysis. The authors discuss
statistical methods, such as the signal-to-noise ratio, standard deviation
of the response, and calibration curve approaches, emphasizing their
importance in method validation as per regulatory guidelines. This
review serves as a valuable reference for researchers aiming to
enhance method accuracy and sensitivity

3.8 Blessy M. et al., 2014[17]

This review focused on the development of forced degradation and

stability-indicating studies of drugs. It provides comprehensive insights
into stress condition evaluations and regulatory implications for drug
stability. (Journal of Pharmaceutical Analysis)

3.9 Haque MA .,2018 [18]

Haque MA (2018) contributed to this body of work by

developing and validating a UV spectrophotometric method specifically
for the estimation of Lamivudine in both bulk drug and tablet dosage

29
forms. The study established the method's reliability through key
validation parameters such as linearity, specificity, reproducibility, and
robustness. The simplicity and effectiveness of this method underscore
its utility in routine pharmaceutical analysis for the quality assurance of
Lamivudine-containing prodcts.

3.10 Raul S.K. et al., 2016[19]

A UV spectrophotometric method was developed and validated for

the estimation of Valsartan in both bulk and pharmaceutical dosage
forms. The study established key validation parameters such as the
limit of detection (LOD) and the limit of quantification (LOQ), which
confirmed the reliability of the method. (Asian Journal of Pharmaceutical
Analysis)

30
 4.AIM AND OBJECTIVES

4.1 AIM

Drug analysis plays an important role in the estimation of the purity

and quality of drugs, which are used in pharmaceutical formulations.

Standard analytical procedures for these drugs or formulations may

not be available, or the original methods are cumbersome, time

consuming. Hence, it is important to develop methods such that, they

are widely available and in common use in control laboratories.

Literature survey revealed that few methods have been reported

for the estimation of Nintedanib Esylate in capsule dosage forms.

The present work aims at developing an analytical technique using

UV spectroscopy, and the method would be of simple, precise and

accurate validated method for quantitative estimation of Nintedanib

Esylate in drug substance and capsule dosage form by spectrophotometry

method.

4.2 OBJECTIVES

Perform a comprehensive literature review on existing analytical methods

available for the estimation of Nintedanib Esylate

Procurement of drug and formulation

Identification of drug by FT-IR

Develop a UV spectrophotometric method for the estimation of Nintedanib

Esylate that is efficient, cost-effective, and suitable for routine quality

control analysis

31
Optimize analytical parameters such as wavelength of maximum

absorbance (λmax), solvent system, and sample preparation technique

Validate the developed method in accordance with ICH guidelines

Apply the validated method for the estimation of Nintedanib Esylate in

marketed capsule dosage form

Ensure the developed method is suitable for use in pharmaceutical quality

control laboratories

Develop dissolution method and optimize the conditions

Perform forced degradation studies

Calculation of statistical parameters

5. MATERIALS AND METHODS

5.1 Drug substance and Finished product

32
 Drug substance Nintedanib Esylate was received as gift sample from

Viora Innovations Private Limited and used for analytical method

development. The marketed finished product manufactured by

Glenmark Pharmaceuticals was procured from pharmacy.

5.2 Chemicals and Solvents used

 Distilled water

 Hydrogen Peroxide

 Hydrochloric acid (AR grade)

 Buffer solution

 Sodium hydroxide

5.3 Formulation used:

The commercially available marketed capsules NINTANIB 150mg was

procured from local pharmacy.

Each capsule contains Nintedanib Esylate 150mg.

5.4 Instruments used:

 Shimadzu Double Beam UV Visible Spectrophotometer, UV-1900i UV-

Visible Spectrophotometer with UV probe software.

 Electrolab Dissolution tester(inspire 8), Electrolab (india) Private

Limited, based in Mumbai, is the manufacturer.

 FTIR Spectrophotometer

33
  Weighing balance

 Sonicator

 pH meter

4.5. Identification of Drug substance Nintedanib Esylate:

The drug substance was evaluated for identification by using Fourier

Transformer Infrared spectroscopy.

5.5 UV Spectroscopic method development:

5.5.1. Selection of solvent and optimization

Solvent was selected by testing the solubility for Nintedanib. The

drug was dissolved in solvents such as distilled water, methanol,

acetonitrile, sodium hydroxide, phosphate buffer and hydrochloric acid.

Nintedanib was soluble and stable in methanol and hydrochloric acid. The

sample was scanned at different scanning speed and it was found that

there is no impact. Hence, the drug was dissolved in methanol and further

dilutions were made with hydrochloric acid solution. It was used for the

detection of wavelength and preparation of standard and working

concentration.

4.5.2. Preparation of standard stock solution

Pure raw material of Nintedanib Esylate (NTB) 100mg was weighed

and dissolved in 0.1M HCl to produce 1mg/1ml solution.

4.5.3. Selection of wavelength

34
 The wavelength for the estimation of NTB, a suitable standard

solution to contain 10µg/ml was prepared and scanned at medium speed

in the entire range from 200nm - 400nm, the lmax of NTB was found to be

285nm. Hence, this wavelength was selected for the estimation of NTB.

ANALYTICAL METHOD VALIDATION:

Validation is an important parameter to ascertain the developed

analytical procedure, which will give reproducible and reliable results that

are adequate for the intended purpose. Taking this into consideration,

validation was performed in terms of linearity, range, accuracy, precision,

LOD and LOQ. Further statistical validations like S.D, %R.S.D, and S.E are

shown in the Table .

4.5.4. Linearity and calibration

The linearity study was carried out for NTB in zero order spectrum at

the above said wavelength. The calibration curve was obtained by plotting

absorbance versus concentration of the standard solution.

PRECISION

Precision of the method was demonstrated by repeatability studies.

a) Intra-day precision: Intra-day precision was carried out by analyzing

the standard drug solutions in the linearity range for three times on the

same day and % R.S.D was calculated.

b) Inter-day precision: Inter-day precision was carried out by analyzing

the standard drug solutions in the linearity range for three times on

different days and the % R.S.D was calculated.

35
36
Quantification in capsule formulation - Nintedanib Soft gelatin

capsule 150mg:

1. Manual Extraction (Cutting & Squeezing)

Soft gelatin capsule was weighed. The capsule shell was carefully cut

using a sterile scalpel or fine scissors. The semi-solid contents was

squeezed into a clean, dry beaker or test tube, to this methanol was

added and sonicated to ensure complete dissolution, and transferred to a

100ml volumetric flask and the contents of the flask and made up to

volume. This solution was then filtered through Whattman filter paper (no

41). This is sample stock solution. Further dilutions were made from this

stock solution to get the required concentration. Absorbances of the

diluted sample solutions were measured for the method in the above said

wavelength.

ACCURACY

In order to ensure the accuracy of the proposed method, recovery studies

were carried out. To the preanalysed sample solution, a definite

concentration was added and then its recovery was studied. 5 ml of a

preanalysed formulation were taken in separate 10 ml volumetric flasks.

With these, known concentration of pure drug NTB at 50%, 75% and 100%

levels were added. The absorbances of the resulting solutions were

measured at their corresponding wavelengths and the percentage

recovery was then calculated.

37
Quantification in capsule formulation - Nintedanib Soft gelatin

capsule 150mg:

2. Solvent Extraction Method (Dissolution of the Shell)

The capsules were placed in a beaker containing a small volume of

methanol to dissolve the gelatin shell, gently stirred and warmed upto

60°C until the shell dissolves and releases the contents. The solution was

filtered through a Whatman filter paper to remove undissolved particles.

Dilute the filtrate with a 0.1M HCl and measure absorbance.

ACCURACY

Accuracy of the method was established by recovery studies by external

standard addition method. The known amount of standard was added at

three different levels to the preanalysed sample solution, each

determination was performed in replicate. The amount recovered and the

percentage recovered was calculated.

LOD and LOQ:

The LOD (Limit of Detection) and LOQ (Limit of Quantification) of the

method was determined from the linearity studies which has been done

six times and then it was calculated by using slope and standard

deviation.

DISSOLUTION METHOD DEVELOPMENT

Dissolution of NTB was performed using dissolution test apparatus

38
1. Dissolution parameters:

Apparatus type : USP Type I (Basket)

Basket speed : 100 rpm (standard)

Dissolution Medium : 0.1 N HCl

Media Volume : 900 mL

Temperature : 37.0 ± 0.5°C

Sampling time points : 10,20,30,40,50 and 60minutes

2. MEDIUM PREPARATION:

Preparation of 0.1M HCl:

8.5 mL of concentrated HCl (≈37%) was mixed in water and made up to

1L with distilled water.

3. SAMPLE PREPARATION:

One Nintedanib Esylate capsule was placed into each basket.

Carefully lowered baskets into the preheated dissolution medium.

4. SAMPLING:

At each time point, 10 mL of the dissolution sample was withdrawn using

a pipette.

Replaced the withdrawn volume with fresh, prewarmed dissolution

medium to maintain sink conditions.

5. ANALYSIS:

39
The sample solution was filtered and the sample dilution was optimized

and analyzed by using UV spectrophotometry, on validation.

UV Detection was done at 285nm based on λmax of NTB in selected

medium.

The % drug release at each time point was calculated.

FORCED DEGRADATION STUDIES

Forced degradation studies ( stress testing) are critical for understanding

a drug's stability and degradation pathways. These studies are required

under ICH Q1A(R2) guidelines and are used to support method

validation per ICH Q2(R2).

Oxidative degradation: To 1.5ml of the stock solution of NTB

(1000μg/ml), 1ml of 3%w/v of hydrogen peroxide added in 10ml of

volumetric flask by using methanol. The volumetric flask kept at room

temperature for 15min. For the blank, 1ml of the 3%w/v of hydrogen

peroxide was kept at normal condition for overnight in 10ml of

volumetric flask. Both the solutions were heated on boiling water

bath to remove excess of hydrogen peroxide. Finally, after 15mins

dilutions were made from the stock solution to achieve the

concentration (30μg/ml). The solution was then taken in a cuvette and

analyzed in UV spectrophotometer.

Hydrolysis under acidic condition: To 3ml of stock solution

(1000μg/ml) of NTB, 1ml of 3N HCl was added in 10ml volumetric flask

and the volume were made upto the mark with methanol. Kept at

normal condition for 60mins, 1ml of solution was pipette out from

40
this flask, and diluted with methanol the volume up to 10ml the

appropriate concentration (30μg/ml). This solution was taken in cuvette.

For the blank, 0.5ml solution of 3N HCl were diluted with methanol in 10ml

of volumetric flask.

Hydrolysis under alkaline condition: To 3ml of stock solution

(1000μg/ml) of Carvedilol, 1ml of 0.1N NaOH was added in 10ml

volumetric flask methanol. After 60mins, time interval, 1ml of solution was

pipette out from this flask, diluted with methanol in order to make the

volume up to 10ml (30μg/ml). This solution was taken in cuvette. For

the blank, 0.5ml solution of 0.1N NaOH were diluted with methanol in

10ml of volumetric flask.

41
6. RESULTS AND DISSCUSSION

The selected drug NINTEDANIB ESYLATE is currently available in the

form of capsules.

The present work deals with the estimation of NTB in pure drug and

pharmaceutical formulation by spectrophotometric method.

6.1. Identification by infra red spectroscopy:

The drug substance was identified by infra red spectroscopy and

spectrum is shown in Figure 6.1

42
Figure 6.1 FTIR Spectrum of Nintedanib Esylate

Wavenumber Observed Peak Functional Group Assignment/Type of

(cm−1 ) Vibrations
3396-3212 3396,3365,3333, Amide, N-H stretching
3277,3212 Indole (secondary amide and
indole NH)
1746-1715 1746,1715 Amide C=O stretching (amide
carbonyl)
1623-1572 1623,1598,1572 Amide, N-H bending (Amide-II)
Aromatic ring and C=C stretching
1260-1217 1260,1217 Esylate S=O asymmetric
stretching
824-910 824,853,882,910 Phenyl, Aromatic C-H out-of-
Indole ring plane bending

6.1. UV spectroscopic method:

NTB was evaluated for solubility in various solvents.

The drug was scanned in the range of 200-400 nm. It was found that the

drug shows good stability in methanol and hydrochloric acid. Hence, 0.1M

Hydrochloric acid was selected as the solvent. The lmax of NTB was found

to be 285 nm.

Figure 6.1: Selection of Wavelength for Nintedanib Esylate

The linearity of the standard NTB in 0.1M Hydrochloric acid was

shown in Figure 6.1

43
 Figure 6.2 Standard NTB in 0.1M Hydrochloric acid

Figure 6.1 Overlay spectrum of NTB in 0.1M Hydrochloric acid

Table 6.1 Absorbance of NTB at 285nm

Concentration in µg/ml Absorbance at 285nm

44
 0 0

5 0.107

10 0.271

15 0.413

20 0.531

25 0.641

30 0.755

35 0.906

40 1.074

Stock solution of NTB was prepared by using 0.1N Hydrochloric acid

and the absorbance was measured at above said wavelength. A plot of

absorbance versus concentration was done. From the calibration curve

the concentration range in which both the drug obey Beer’s law was found

out.

Calibration curve of the NINTEDANIB ESYLATE was shown in Figure 6.3

45
 Figure 6.2: Linearity graph of Nintedanib Esylate

The optical and regression characteristics of NTB was shown in Table-6.2

Table 6.2 Interpretaion of Nintedanib Esylate

Parameters Observed Value

Correlation Coefficient 0.9971

B-Intercept 0.004

Slope 0.026

Range: From the calibration curve it was found that DLX obeys

Beer’s Law limit in the range of 5-40µg/ml at 285nm.

Precision: The precision of the methods were performed by

Intra-day precision: Intra-day precision was carried out by analyzing the

standard drug solutions in the linearity range for three times on the same

day and % R.S.D was calculated, which are shown in Figure - 6.3 and

Table - 6.3.

46
 Figure 6.3 Intra-Day precision for Nintedanib Esylate

Table 6.3 Intra-Day precision for Nintedanib Esylate 285 nm (LQC

– 20 ppm)

LQC Abs Mean SD %RSD Abs SD %RSD Abs SD %RSD

(20
ppm)
1 0.557 0.552
2 0.558 0.551 0.0075 1.36 0.548 0.0033 0.60%
3 0.541 0.552
4 0.555 0.557
5 0.551 0.552
6 0.542 0.549
Mean 0.551 0.552

47
 Inter-day precision: Inter-day precision was carried out by analyzing

the standard drug solutions in the linearity range for three times on

different days and the % R.S.D was calculated, which are shown in Figure

6.4 and Table -6.4.

Figure 6.4 Inter-Day precision for Nintedanib Esylate

Table 6.4 Inter-Day precision for Nintedanib Esylate at 285

nm (LQC – 20 ppm)

48
LQC (20 Abs Day 1 Day 1 Day 1 Day 2 Day 2 Day 2 Day 3
ppm) Mean SD %RSD Mean SD %RSD Mean/SD/
%RSD

1 0.542 0.552
2 0.554 0.549
3 0.551 0.551 0.0052 0.94% 0.552 0.0091 1.65%
4 0.553 0.548
5 0.557 0.546
6 0.549 0.546

Day 3 0.5517 /
0.0043 /
0.78%

Quantification in capsule formulation - Nintedanib Soft gelatin

capsule 150mg:

1. Manual Extraction (Cutting & Squeezing)

NINDANIB 150 marketed brand was selected for the study and

analyzed by the proposed method. From the absorbance obtained from

the zero order spectrum, the amount of NTB was calculated by using the

above method is shown in Figure - 6.5 and 6.6. The results are tabulated

in Table - 6.5 for the above mentioned method.

Figure 6.5 Assay of commercial formulation (Method 1)

49
2. Solvent Extraction Method (Dissolution of the Shell)

Figure 6.6 Assay of commercial formulation (Method 2)

Table 6.6 Assay of commercial formulation

Accuracy: Accuracy of the method was established by recovery

studies by external standard addition method. The known amount of

standard was added at three different levels to the preanalysed sample

solution, each determination was performed in replicate. The amount

recovered and the percentage recovered was calculated. The values are

shown in Table – 6 .7.

Recovery studies in capsule formulation - Nintedanib Soft gelatin

capsule 150mg:

Manual Extraction (Cutting & Squeezing)

Table 6.5 Recovery study data of commercial formulation Method 1

50
Table 6.5 Recovery study data of commercial formulation Method 1

Table: Accuracy for Nintedanib Esylate:

Levels sample std Abs Mean SD % Amt. Amt. Amt. %

RSD found present added Recovered

50% 1 1 0.520 0.519 0.0011 0.2223 19.824 10 10 98.243

1 1 0.518

1 1 0.521

100% 1 2 0.784 0.784 0.005 0.6377 30.003 10 20 100.01

1 2 0.789

1 2 0.779

150% 1 3 1.045 1.04 0.0055 0.5353 39.85 10 30 99.5

1 3 1.034

1 3 1.041

51
 From the results obtained it was found that the above method shows best reproducible

results.

Recovery studies in capsule formulation - Nintedanib Soft gelatin

capsule 150mg: Solvent Extraction Method (Dissolution of the

Shell)

Figure : Absorbance spectra of Accuracy for Nintedanib

Table: Accuracy for Nintedanib Esylate:

Levels Sample Std Abs Mean SD % RSD Amt. Amt. Amt. %

found present added Recovered

50% 1 1 0.520 0.519 0.0011 0.2223 19.824 10 10 98.243

1 1 0.518

1 1 0.521

52
100% 1 2 0.784 0.784 0.005 0.6377 30.003 10 20 100.01

1 2 0.789

1 2 0.779

150% 1 3 1.045 1.04 0.0055 0.5353 39.85 10 30 99.5

1 3 1.034

1 3 1.041

LOD and LOQ

The LOD and LOQ of the method was determined from the linearity

studies which has been done six times and then it was calculated by using

slope and standard deviation. The LOD & LOQ of NINTEDANIB ESYLATE

was found to be 0.280 µg/ml & 0.848 µg/ml respectively.

LOD 0.280

LOQ 0.848

6.2 Dissolution method

The dissolution studies were carried out using 0.1M HCL as the medium of

solvent and the samples on capsules in the medium was withdrawn 10 ml

per sample size in the intervals of 10 minutes upto 40 minutes. The

withdrawn samples were diluted with suitable solvent and the aliquot’s

were analysted for % release using UV spetroscopy at 285nm. The

maxium % release was found at 20 minutes to be 180.44%.

53
Table : Absorbance of the Nintedanib Esylate at 285 nm using UV-

Spectroscopy

Time (mins) S1 S2 S3

10 0.174 0.185 0.178

20 0.541 0.533 0.493

30 0.685 0.663 0.674

40 0.757 0.776 0.741

50 0.989 0.993 0.952

60 1.025 1.043 1.038

Table : Absorbance of Nintedanib Esylate in 0.1M HCL

Table : Cumulative % drug release of the Nintedanib Esylate using

UV-Spectroscopy

Solution S1 S2 S3

1 170.52%

54
 2 180.44%

3 176.63%

4 173.74&

Figure : Dissolution graph of Nintedanib Esylate in 0.1M HCL

Absorbance (285nm)
1.2

0.8
Absorbance

0.6

0.4

0.2

0
10 20 30 40
Time (minutes)

6.3 Stability studies:

The stability testing for the Nintedanib Esylate capsules were performed

by employing three different degradation studies: Stress degradation study, Stress

55
degradation by hydrolysis under alkaline condition, Oxidative degradation. And the results

were recorded.

Figure Degradation of NTB in Hydrogen peroxide

Figure Degradation of NTB in Hydrochloric acid

56
Figure Degradation of NTB in Hydrogen peroxide in Sodium hydroxide

57
 7. SUMMARY AND CONCLUSION

For the estimation of NTB in pure durg and commercial formulations

of NTB in soft gelatin capsule dosage form few studies were published.

The present work on UV spectroscopic method development of NTB was

found to be simple accurate and sensitive when compared to the other

published methods.

The developed method was found to be simple and economical,

since 0.1M Hydrochloric acid was used as the solvent. The method is less

time consuming and no tedious steps are involved during the method

development.

The method is feasible at normal testing laboratories. The accuracy

of the developed method is evident from the analytical and statistical

validation.

Sample preparation is very easy. It does not suffer from any

interference due to excipients present in this pharmaceutical preparation

and can be used for routine analysis. The results obtained were in good

agreement with the labeled amount.

Hence this method is highly useful in the routine batch analysis of

NTB in soft gelatin capsules.

58
 Hence, it is concluded that the UV spectrophotometry method can

be effectively used for the estimation of NTB from pharmaceutical dosage

forms.

59
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