1.

Introduction 0

1. INTRODUCTION
 1. Introduction

1. INTRODUCTION
1.1. INTRODUCTION OF ANALYTICAL METHOD 1-4
The science and art of figuring out the elements or compounds that make up a material's
composition is known as analytical chemistry. This area of chemistry is practiced in
numerous laboratories in a wide variety of ways and deals with theoretical and practical
aspects of science. Methods of analysis are frequently created, enhanced, verified, jointly
studied, and used. Gaining knowledge of the qualitative and quantitative composition of
substances and chemical species—that is, learning exactly what a substance is composed
of and how much of it—is crucial for analytical chemistry. How much is present is the key
question in quantitative analysis. This criterion serves as the foundation for the research
in this thesis. Pharmaceutical analysis deals with both medicines (drugs and their
formulations) and their precursors, or the raw materials that determine the purity and
quality of the medication. The purity and quality of the pure substance in the drug and its
formulations are tested to establish the authenticity of the drug before determining its
quality. Quality matters in every good or service, but it is especially crucial in medicine
because it involves saving lives. Contrary to common consumer goods, drugs cannot be of
"second quality." A concept known as "quality control" aims to produce a flawless product
through a series of steps meant to prevent and get rid of mistakes made at various
production stages.
1.2. Need of Analytical Development

The discovery, development, and production of pharmaceuticals depend heavily on the

development and validation of analytical methods. To guarantee the identity, purity,
potency, and performance of drug products, quality control laboratories use the official test
methods that come from the development of analytical methods. ii. To guarantee that quality
and safety standards are being followed. As a result, the United States, Europe, Japan, and
other nations have released compendia, or pharmacopoeias, that outline the official test
procedures for many drugs that are currently on the market.
The analytical approach addresses quality standards that are assigned to products to have
desired medication efficacy. It is assumed that drugs or medicines that meet these
standards are having the desired effects on use when a sample from any batch is analyzed
for these standards. Quality control is a concept that aims to create a flawless product

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through a series of steps meant to stop and get rid of mistakes at various stages of
production. One or more types of control actions are used to determine whether to release
or reject a product. There has been rapid advancement in the field of pharmaceutical
analysis as a result of the pharmaceutical industry's recent rapid growth, which has led to
the introduction of numerous pharmaceutical formulations into the healthcare system. It is
crucial to develop analytical methods for newly introduced pharmaceutical formulations
because they may not be officially recognized by any pharmacopoeias and thus lack
analytical methods for quantification. Various analytical techniques are employed to verify
the medication's quality standards. In evaluating the chemical quality standards of medicine,
modern analytical techniques are crucial. As a result, the establishment of drug standards
and their ongoing monitoring require analytical techniques. Chromatography is the method
of analysis that is most frequently used to evaluate the quality of drugs.

1.3. Relevance of Analytical Methods 5-7

Drug development and follow-up activities heavily rely on analytical methods that serve as
a gauge of the quality of the drugs. It guarantees that a drug product complies with the
required standards, is stable, and will maintain the claimed level of quality for the duration
of its shelf life. These methods must be written in a format that allows them to be repeated
over time and from laboratory to laboratory, which is to say that they must be validated.
They must also be selective and sensitive to monitor the known and unknown impurities.
1.4. Important of Analytical methods

Pharmaceutical analysis is crucial for testing raw materials, conducting quality checks
during production, and examining finished goods. To ascertain the identity, potency,
quality, and purity of drug samples, pharmaceutical analysis is used.
In analytical chemistry, it is of prime importance to gain information about the
qualitative and quantitative compositions of substances and chemical species, that
is, to find out what a substance is composed of and exactly how much. In general
terms, pharmaceutical analysis comprises of those procedures
necessary to determine the “identity, strength, quality and purity” of drugs.
In all stages of pharmaceutical development, analytical methods are necessary to characterize
the drug substances and drug product composition. Early phase techniques must

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accommodate modifications in synthetic routes and dosage form and clarify the structures
and concentrations of impurities. Later phases have the development of quick and reliable
methods for release and stability evaluation as their objectives.
Numerous straightforward and instrumental analytical techniques can be used for analysis,
but spectroscopy and chromatography are the techniques most frequently employed for
quality control. The majority of quantitative analyses call for measuring specific
components while they are mixed with the sample matrix or other substances, so isolating
or separating the components is necessary before the analysis. Chromatographic methods
are used in these situations for quantitative analysis. Quantitative measurements are made
using spectroscopic or titration techniques when matrix interference is not seen.
Method development must include method validation. It is the process of proving that
analytical techniques are appropriate for the purposes for which they were designed and that
they support the purity, potency, identity, and quality of drug substances and drug products.
Method validation is the process of demonstrating that an analytical method is suitable for
the goal for which it was developed.

1.5. High Performance Liquid Chromatography8 -10

In 1903, Mikhail Tswett, a Russian botanist, separated plant pigments on chalk (CaCO 3)
packed in glass columns and coined the term "chromatography," which means "colour
writing." Midway through the 1970s, high pressure liquid chromatography was
developed. With the advent of column packing materials and the added convenience of
online detectors, it quickly advanced. In the late 1970s, improved separation between
chemically similar compounds was made possible by new techniques, such as reverse
phase liquid chromatography. By the 1980s, chemical compound separation was
frequently accomplished using HPLC. Automation and computers improved HPLC's
convenience.
The physical separation method known as liquid chromatography (LC) is carried out in the
liquid phase. Analyte is compelled by intense pressure to pass through a column. Then, it
is distributed between the mobile phase (a flowing liquid) and a stationary phase to be
separated into its component parts (sorbents packed inside a column). Normal phase,
reversed phase, ion exchange chromatography, and size exclusion chromatography (also
known as gel permeation and gel filtration chromatography) are the four principal

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separation modes of HPLC.

Figure no. 1.1 Diagram of HPLC

1.5.1 Critical Steps for HPLC Method Development
Information on a sample, define separation goals
↓
Need for special HPLC procedure, sample pretreatment, etc.?
↓
Choose detector and detector settings
↓
Choose LC method; preliminary run; estimate best separation conditions
↓
Optimize separation conditions
↓

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Check for problems or requirement for special procedure

↓
Recover purified material, Quantitative calibration, Qualitative method
↓
Validate method for release to routine laboratory

1.5.2 Selecting an HPLC Method and Initial Conditions

Because HPLC method development involves a lot of trial and error, it is impossible to
provide a precise recipe.
Following is an explanation of the recommended method for selecting the first
separation's experimental conditions. Which chromatographic method is most promising
for this specific sample, given knowledge of sample composition and separation
objectives? Although HPLC is assumed to have been chosen, other options should also be
taken into account.
Identifying the solubility of the sample components is the first step in developing an HPLC
method. Selecting the best HPLC mode will depend on understanding the nature of the
analyte. Reversed-phase should be tried first when choosing an appropriate
chromatography method for organic compounds, and if that doesn't work, normal-phase
should be taken into account.

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Figure 1.2: Phase Selection Process

1.5.3 Some of the advantages are:

ŽSpeed (analysis can be accomplished in 20 minutes or less)

ŽGreater sensitivity (various detectors can be employed)

ŽPrecise and reproducible

ŽCalculations are done by integrator itself

Reusable columns (expensive columns but can be used for many analysis)

ŽIdeal for the substances of low volatility

ŽEasy sample recovery, handling and maintenance

Instrumentation tends itself to automation and quantitation (less time and less labor)
ŽSuitable for preparative liquid chromatography on a much larger scale
In HPLC, the analyst has a wide choice of chromatographic separation methodologies

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from normal to reverse phase and a whole range of mobile phases using isocratic or
gradient elution techniques
1.5.4 Different Modes of Separation

1.5.4.1 Normal-Phase Chromatography (NPC)8-9

The traditional separation method known as NPC relies on the analyte's adsorption and
desorption onto a polar stationary phase (typically silica or alumina). Because of their lower
affinity for the stationary phase in this method, nonpolar compounds move more quickly and
are eluted first. Due to their greater affinity for the stationary phase, polar compounds are
retained for a longer period of time. Because most drug molecules are polar in nature and
therefore take longer to elute, normal phase mode of separation is therefore not typically
used for pharmaceutical applications.
1.5.4.2 Reversed-Phase Chromatography (RPC)

For analytical and preparative separations of compounds of interest in chemical, biological,

pharmaceutical, food, and biomedical sciences, reversed phase mode is the most widely used
mode. In this mode, a polar solvent serves as the mobile phase, and the stationary phase is a
nonpolar hydrophobic packing with an octyl or octadecyl functional group bonded to silica
gel. With an aqueous mobile phase, retention and selectivity can be controlled by secondary
solute chemical equilibrium processes like ionization control, ion suppression, ion pairing,
and complexation. In this mode, nonpolar compounds are retained for a longer period of
time while the polar compound is eluted first. Due to the polar nature of the majority of
pharmaceuticals and drugs, they elute quickly because they are not retained for long periods
of time. The different columns used are octadecylsilane (ODS) or C18, C8, C4 etc., (in the
order of increasing polarity of the stationary phase).

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Fig 1.3: Principle pattern of a HPLC instrument

The core of HPLC is a set of columns. The most common material used to build liquid
chromatographic columns is smooth bore stainless steel tubing. made from polymer tubes
like PEEK and glass tubes with thick walls on occasion. By removing sample components
that bind permanently to the stationary phase as well as particulate matter and other
contaminants from solvents, guard columns are used before analytical columns to extend the
life of the latter. The length of analytical columns varies from 5 to 25 cm, and their inside
diameter is typically 3 to 5 mm. The most typical packing particle size is 3 to 5 µm.
Pellicular and porous particles are the two primary types of column packing used in
LC. Spherical, nonporous beads made of glass or polymers are used in pellicular
packing. On the surface of these beads, a thin layer of silica, alumina, synthetic
polystyrene-divinylbenzene resin, or an ion-exchange resin was deposited. The
common porous particle packing of LC is made up of silica, alumina, synthet ic
polystyrene-divinylbenzene resin, or an ion-exchange resin. Surface functionalization
of silica is used to prepare columns for bonded phase chromatography. Fully
hydrolyzed silica has silanol groups on its surface, which are chemically reactive. The
most practical bonded phase coatings are siloxanes, which are created when
organochlorosilanes react with the hydrolyzed surface.

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Fig no. 1.4 Reaction of silanol group with organochlorosilane leads to formation of
siloxanes

For Example, where R is an alkyl group or substituted alkyl group like C8, C18
Different types of detectors used in HPLC are absorbance detectors, fluorescence detectors,
electrochemical detectors, refractive index detectors, conductivity d e t e c t o r s ,
photo ionization detectors etc.

1.6 Method Development and Design of Separation Method10-13

If one is aware of the characteristics of the sample, such as its molecular weight, polarity,
ionic character, and solubility parameter, one can develop methods for analyzing drugs in
single or multi component dosage forms. The lack of a precise recipe for HPLC, however, is
due to the extensive amount of trial and error that goes into developing new methods.
Choosing what type of column to try with what kind of mobile phase is typically the most
challenging problem. When the compounds have many polar groups and are hydrophilic in
nature, one typically starts with reversed phase chromatography.
Gradient elution method can be used to estimate the organic phase concentration needed
for the mobile phase. Gradient reversed phase chromatography is the best place to start
when working with aqueous sample mixtures. Gradients can begin with 5–10% organic
phase in the mobile phase and can increase the concentration of the organic phase
(acetonitrile or methanol) to 100% within 30–45 minutes. The initial mobile phase's
composition and gradient's slope can then be changed to optimize separation in
accordance with the preliminary run's chromatogram. Based on the location and mobile
phase composition at which the target compounds were eluted, the initial mobile phase
composition can be estimated.
Drug molecules can elute differently depending on the polarity of the mobile phase. A
mobile phase's polarity determines how strongly it will elute; the stronger the polarity, the

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stronger the elution. If they are present in undissociated form, ionic samples - whether acidic
or basic - can be separated. The right pH choice can prevent the dissociation of ionic
samples. It is necessary to choose the mobile phase's pH so as to prevent ionization of the
compounds. The concentration of the organic phase in the mobile phase can decrease in
steps of 5% if the retention times are too short. An increase in the concentration of the
organic phase is required if the retention times are too long.
Good analytical results in UV detection can only be attained with careful wavelength
selection. Understanding the UV spectra of the person included in the sample is necessary for
this. Prior to developing an HPLC method, the UV spectra of analyte standards can be
measured.
Another crucial factor is the molar absorbance at the detection wavelength. If no peaks
are seen in the chromatograms, it's possible that there isn't enough sample to make a
detection. For UV active compounds at around 220 nm, a volume injection of 20 L from a
solution of 1 mg/mL concentration typically yields good signals. Even though the
compounds have higher max, they strongly absorb at shorter wavelengths. Compounds
need not always be detected at their maximum absorbance. For precise quantitation, it is
advantageous to avoid detection at the sloppy part of the UV spectrum. The analysis of
the peak shapes can aid in the development of new methods when acceptable peaks are
found on the chromatogram. The separation of ionic samples may be impacted by the
addition of peak modifiers to the mobile phase. As an illustration, adding trace amounts of
the peak-modifier triethylamine to the mobile phase can affect the retention of the basic
compounds. Similar to this, acetic acid or other small amounts of acids can be used with
compounds that are acidic. This may result in beneficial selectivity changes. When tailing
or fronting is seen, it indicates that the solutes and mobile phase are not completely
compatible. In the majority of cases, the pH is not chosen properly, which results in partial
dissociation or protonation. Ion-pair chromatography can be used when the peak shape is not
improved by a lower (1-2) or higher (8-9) pH. Anionic ion-pair molecules at lower pH can be used
for basic compounds, while cationic ion-pair molecules at higher pH can be used for acidic
compounds. Ion-pair chromatography is the preferred technique for amphoteric solutes or a blend of
acidic and basic compounds.
Bad peak shapes can also be brought on by the sample's low solubility in the mobile
phase. To prevent compound precipitation in the column or injector, it is always advisable
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to prepare the sample solution using the same solvents as the mobile phase.
Only after a decent chromatogram has been obtained can optimization be started. When all
the compounds are detected by more or less symmetrical peaks on the chromatogram, the
chromatogram is considered reasonable. Within the scope of the investigated changes, the
position of the peaks can be predicted by a sight change in the composition of the mobile
phase. A chromatogram is said to be optimized when all of the peaks are symmetrical and
well separated in a short amount of time. By using a more effective column (column with a
higher theoretical plate number, N), which can be accomplished by using a column of
smaller particle size, or a longer column, the peak resolution can be increased. However,
these elements will lengthen the analysis period. While flow rate has little bearing on
resolution, it significantly affects analysis time. Although theoretical predictions of the
interactions between the mobile phase and stationary phase with a particular set of sample
components are not always accurate, they do aid in limiting the options for method
development. Until a satisfactory separation is achieved, the separation scientist must
typically conduct a number of trial-and-error experiments with various mobile phase
compositions.

Fig 1.5: A HPLC chromatogram

The parameters that are affected by the changes in chromatographic conditions are:

1. Resolution (RS).
2. Capacity factor (k‟).
3. Selectivity (α).
4. Plate number (N).
5. Asymmetry factor (T).

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1.6.1 Quantitative Analysis in HPLC14

Two methods are generally used for quantitative analysis. They are the external standard
method, the internal standard method and the standard addition method.
1. External standard method
Utilizing one standard solution or a maximum of three standard solutions is part of the
external standard method. Direct comparisons can be made between the sample's peak
area or height and the reference standard, as well as the slope of the calibration curve
using standards with known concentrations of the target compounds.
2. Internal standard method
The addition of an internal standard to a quantitation method is a common way to account
for various analytical flaws. In this method, the loss of the target compounds during sample
pretreatment steps is made up for by adding a known compound with a fixed concentration
to the known quantity of samples to produce distinct peaks in the chromatograms. Any loss
of the interest component will also result in the equivalent loss of internal standard. The
structural similarity of the compounds of interest and the internal standard are obviously
necessary for this method to be accurate.
Prior to the sample preparation process, the internal standard should be added to the
sample and homogenized with it. The concentration of a sample component in the initial
sample is calculated using the response factor. The internal standard (AISTD) and sample
component (Ax) peak areas obtained by injecting the same volume are compared to
determine the response factor (RF).

1.7 ANALYTIC METHOD DEVELOPMENT AND VALIDATION15-22

In order to improve the nature of drugs, Ted Byers and Bud Loftus, two Food and Drug
Administration (FDA) officials, first proposed the idea of approval in the 1970s. The
primary approval activities initially focused on the production cycles for these products,
but they quickly spread to related cycles such as ecological control, media fill, equipment
sterilization, and clean water production. The purpose of the approval is to ensure that
quality is built into the framework at every stage, rather than just being tested at the end,
as such approval exercises will frequently recall preparation for creation material and
working procedures, training of those involved, and checking of the framework while in
production and have become an important part of flow great manufacturing practices

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(CGMPs).

Figure 1.6: Method Validation Parameters as per USP and ICH

1.7.1 Observed as:
 When methods are properly developed, they can be readily validated.
 Validation does not make a method better or more efficient.
 A validated method does not necessarily imply that it meets all criteria of a properly
developed method.
 Validation acceptance criteria should be based on method development experience.
1.7.2 Method Validation is required for the following reasons:
1. A new method is been developed.
2. Revision of established method.
3. When established methods are used in different laboratories and different analysts etc.
4. Comparison of methods.
5. When quality control indicates method changes.
1.7.3 Advantages of analytical method validation:
 The biggest advantage of method validation is that it builds a degree of
confidence, not only for the developer but also to the user.
 Although the validation exercise may appear costly and time consuming, it
results inexpensive, eliminates frustrating repetitions and leads to better time
management in the end.
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 Minor changes in the conditions such as reagent supplier or grade, analytical setup
are unavoidable due to obvious reasons but the method validation absorbs the
shock of such conditions and pays for more than invested on the process.

Guidelines from the following sources provide a framework for performing validation.
 United States Pharmacopoeia (USP)
 International conference on harmonization (ICH)
 Food and drug administration (FDA)
 Validation according to ICH Guidelines
1.7.4 Typical validation parameters are:
i) Accuracy
ii) Precision (Repeatability, Intermediate precision and Reproducibility)
iii) Linearity
iv) Range
v) Specificity
vi) Robustness
vii) System suitability testing
viii) Limit of detection (LOD) and Limit of quantitation (LOQ)
A) Accuracy:
Definition: It 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.
This is sometimes termed trueness.
The degree to which test results obtained using an analytical method are accurate in
relation to the true value Recovery studies determined the method's accuracy. According
to the ICH document on validation methodology, accuracy should be evaluated using at
least nine determinations made at a minimum of three concentration levels that fall within
the prescribed range.
The percentage of recovery from the assay of the known additional amount of analyte in
the sample or the difference between the mean and the recognized true value should be
used to describe accuracy.
B) Precision:
Definition: It expresses the closeness of agreement between a series of measurements
obtained from multiple sampling of the same homogeneous sample under the prescribed

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conditions.
Precision may be considered at three levels: repeatability, intermediate precision and
reproducibility.
C) Repeatability: It conveys the precision over a brief period of time while operating under
the same conditions. Precision within an assay is another name for repeatability. At least six
replications must be measured at the test target concentration or at least nine replications must
cover the entire specified range in order to test for repeatability.
D) Intermediate precision: It describes variations within labs, such as various days, various
analysts, various equipment, and so forth. When the development phase of a method is complete,
the aim of intermediate precision validation is to confirm that the method will produce the same
result in the same laboratory.
E) Reproducibility: It conveys the level of accuracy between laboratories. Reproducibility's
goal is to confirm that a method will produce the same outcomes in various labs. Aliquots from
homogeneous lots are examined in various laboratories by various analysts to gauge the
reproducibility of an analytical method.
F) Linearity:

Definition: Linearity of an analytical procedure is its ability (within a given range) to

obtain test results that are directly proportional to the concentration of analyte in the
sample.
Using the suggested method, it can be demonstrated directly on the drug substance (by
diluting a standard stock solution) and/or separately weighing synthetic mixtures of the drug
product's constituent parts.
G) Range:
Definition: Range of an analytical procedure is the interval from the upper to the lower
concentration (amounts) of analyte in the sample for which it has been demonstrated that
the analytical procedure has a suitable level of precision, accuracy and linearity. For the
assay of a drug substance or a finished (drug) product: normally from 80 to 120 percent
of the test concentration should be tested/checked for range.
H ) Specificity:
Definition: It is the ability to assess unequivocally the analyte in the presence of
components, which may be expected to be present. Typically, these might include

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impurities, degradants, matrix, etc.

I) Robustness:
Definition: It is a measure of its capacity to remain unaffected by small, but deliberate
variations in method parameters and provides an indication of its reliability during normal
usage.
J) System suitability testing:
Definition: The tests, based on the concept that the equipment, electronics, analytical
operations and samples to be analyzed constitute an integral system that c a n be
evaluated as such. System suitability test parameters to be established for a
particular procedure depend on the type of procedure being validated. System
suitability testing is an integral part of procedures.

K) Limit of detection and Limit of quantitation:

The detection limit of an individual analytical procedure is the lowest amount of analyte
in a sample, which can be detected but not necessarily quantitated as an exact value. The
quantitation limit of an individual analytical procedure is the lowest concentration of
analyte in a sample, which can be quantitatively determined with a suitable level of
precision and accuracy.
Several approaches for determining are possible, depending on whether the procedure is
a non- instrumental or instrumental.
 Based on visual evaluation
 Based on signal-to-noise
 Based on the standard deviation of the response and the slope.

The LOD and LOQ were estimated from the set of 5 calibration curves used
to determine method linearity. Limit of detection and Limit of quantitation can be
calculated by the following equation.
L) LOD = 3.3 (σ/S), LOQ = 10 (σ/S)
Where,
σ = Standard deviation of y-intercepts of regression lines
S =Slope of the calibration curve
M) Data Elements Required for Assay Validation
There are various analytical methods for the examination of pharmaceutical materials. Not

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all of the aforementioned traits will always need to be taken into account. According to W HO,
analytical methods can be broadly categorized as follows.
Class A: tests intended to determine the identity of drug substances in bulk or a specific
ingredient in a finished dosage form.
Class B: Techniques intended to identify and measure impurities in a finished dosage form or a
bulk drug substance.
Class C: Methods used to quantify the concentration of a major ingredient or a bulk drug
substance in a finished dosage form.
Class D: A technique for evaluating the properties of finished dosage forms, such as content
uniformity and dissolution profiles.

Table 1.1 : Validation parameters that should be considered for different types of
analytical procedures

Class B

Quantitative Limit tests

Parameters Class A tests Class C Class D

Accuracy * * *

Precision * * *

Robustness * * * * *

Linearity & Range * * *

Selectivity * * * * *

LOD * *

LOQ *

1.8 Introduction of Drugs:23

Under the trade name Koselugo, selumetinib is a drug used to treat neurofibromatosis type I
(NF-1), a genetic nervous system disorder that results in tumors growing on nerves, in
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children two years of age and older. It is consumed orally twice daily on an empty stomach.
Headaches, pain in the stomach and other gastrointestinal issues, fatigue, pain in the
muscles, as well as dry skin and other skin issues are typical side effects. In the United
States, selumetinib was given the go-ahead for medical use in April 2020. The Food and
Drug Administration (FDA) in the United States regards it as a first-in-class drug.

Neurofibromas are treated with selumetinib in people with type I neurofibromatosis (NF-1).
This is a rare, developing condition brought on by a mistake or mutation in the gene that
codes for the neurofibromin 1 protein. A diagnosis of NF-1 is typically made in the early
years of life and is thought to affect one in every 3,000 newborns. It is characterized by
alterations in skin pigmentation, impairments of the nervous and skeletal systems, and a
lifetime risk of developing benign and malignant tumors. It is specifically approved for
children with symptomatic, incurable plexiform neurofibromas (PN), which are tumors of
the nerve sheaths (the covering around nerve fibers) and can develop anywhere in the body,
including the face, extremities, regions around the spine, and deep within the body where
they may affect organs. Children born with NF-1 develop one or more PNs in between 30%
and 50% of cases.

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