Chapter 1 Introduction

1.1 INTRODUCTION[1-3]

1.1.1 Analytical chemistry

The science of analytical chemistry looks for constantly better ways to

determine the chemical makeup of both natural and manmade materials. The
full picture (composition) of a substance at the chemical scale is known as its
chemical composition, and it includes geometric aspects like species
distributions and molecular morphologies, as well as one-dimensional
parameters like percent composition and species identity Analyzing samples
effectively and efficiently involves knowledge of:

1. The chemistry that can occur in a sample.

2. Techniques for handling samples and conducting analyses for several
issues (the tools-of-the trade).
3. The method's accuracy and precision.
4. Accurate data analysis and documentation.

The following are the key steps in an analytical process:

The processes required to ascertain the "identification, strength, quality, and

purity" of such chemicals are included in the pharmaceutical analysis. It also
covers the study of starting materials and intermediates used in the production
of pharmaceuticals.

1.1.2 Classical methods

 Quantitative analysis
 Qualitative analysis

TYPES OF ANALYTICAL CHEMISTRY QUALITATIVE ANALYSIS

The qualitative inorganic Establishing the existence of a specific element or

inorganic compound in a sample is the aim analysis.

Establishing the existence of a specific functional group or organic molecule in

a sample is the goal of qualitative organic analysis.

1) QUANTITATIVE ANALYSIS
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Qualitative analysis

The analysis deals with the identification of elements, ions or compounds

presents in sample. The goal of quantitative analysis is to determine the
concentration of a specific element (or compound) in a sample.

Methods for identifying analyses

1. Physical properties include mass, color, refractive index, and thermal

conductivity
2. Using Electromagnetic Radiation (Spectroscopy)Absorption,
Emission, and Scattering.
3. Using electric charge, mass spectrometry, and electrochemistry.

Fig [Link] in analytical cycle

1.1.3 Ultraviolet Absorption Spectroscopy

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Spectrophotometry is typically chosen, particularly by small-scale enterprises,

as the equipment is less expensive and has fewer maintenance issues. The
analysis approach is based on evaluating the monochromatic light's absorption
by colorless substances in the near ultraviolet path of the spectrum (200-
380nm). The foundation for the photometric analysis techniques is According
to the Beer-Lambert law, Beer's solution's absorbance is inversely proportional
to its analyte concentration. The spectrophotometer's basic mode of operation
for the UV region involves light of a specific wavelength passing through a
cell filled with solvent before hitting a photoelectric cell, which converts the
radiant energy into electrical energy that can be measured by a galvanometer.

1.1.4 Spectroscopy.[4-6]

1.1.5 UV visible spectroscopy

Ultraviolet (UV) spectroscopy is a physical technique of the optical

spectroscopy that uses light in the visible, ultraviolet, and near infrared ranges,
the molecular absorption is studied in the wavelength region of 190 to 800 nm
of Electromagnetic spectrum, Ultraviolet region from 190nm - 400nm and
visible region from 400nm - 800 nm 1. When monochromatic radiation passes
through a homogeneous solution in a cell, the intensity of the emitted radiation
depends upon the thickness (b) and the concentration (C) of the solution. Io is
the intensity of the incident radiation and I is the intensity of the transmitted
radiation2.
1.1.6 Principle of UV-Vis Spectroscopy:

A molecule or ion will exhibit absorption in the visible or ultraviolet region

when radiation causes an electronic transition within its structure. Thus, the
absorption of light by a sample in the ultraviolet or visible region is
accompanied by a change in the electronic state of the molecules in the
sample. The energy supplied by the light will promote electrons from their
ground state orbital to higher energy, excited state orbital or anti-bonding
orbital. Potentially, three types of ground state orbitals may be involved.

1.1.7 Lambert –Beer law :

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Lambert’s law:

It states that, When a monochromatic light passes through a transparent

medium, layer of equal thickness of that homogeneous absorbing medium
absorbs equal proportions of incident radiation.7

Beer’s law:
The Beer’s law states that “The fraction of the monochromatic radiant energy
absorbed on passing through a solution is directly proportional to the
concentration of the absorber.” It is a relationship between the light absorptive
capacity and the concentration of the absorber in solution.

Ultraviolet –visible (UV-V) is spectrophotometry is a technique used to

measure light absorbance across the ultraviolet and visible range of the
electromagnetic spectrum. When incident light strikes matter it can either be
absorbed, reflected, or transmitted.

1.1.8 Instrumentation:

UV spectroscopy is the absorption or reflectance spectroscopy of the

ultraviolet and adjacent visible regions of the electromagnetic spectrum. It is
also known as UV-visible spectrophotometry (UV-Vis or UV/Vis). Because of
its low cost and ease of implementation, this methodology is widely used in a
wide range of applied and fundamental applications. The only requirement is
that the sample absorb in the UVVis range, indicating that it is a chromophore.
Absorption spectroscopy supplements fluorescence spectroscopy. Aside from
the wavelength, the parameters of interest are absorbance (A), transmittance
(%T), and reflectance (%R), as well as their variations over time.7

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Fig No.2. Instrumentation of UV spectrophotometer

Components of UV-VIS Spectrophotometer are as follows:8

1. Light Source

2. Monochromator

3. Sample & reference cells

4. Detector

5. Recorder

1. Light Source:

The light source used must provide consistent & stable light

a) Hydrogen & deuterium lamps

b) Tungsten filaments lamp

c) Xenon arc lamps.

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2. Monochromator :

It separates polychroma- tic light into single spectral line. A monochromator is

an optical device that is used to select a narrow band of a wavelength of light.

a) Slit

b) Mirror

c) Lens

d) Prism

e) Grafting
3. Sample & reference cells :

The cuvette are generally made up of quartz & borosilicate. One beam pass
through sample solution & second beam pass through reference solution. The
cuvette are generally transparent.

4. Detector :

The Detector is responsible for detection of radiation. The intensity of

radiation from reference cell is stronger than beam of sample cell.

a) Photovoltaic cell

b) Photo tubes

5. Recorder :

The recorder detect & record the data of the experiment. It also stores the data
in computer when it connected to computer.

1.1.9. SOURCES:

A continuous source, or one that produces radiation at a variety of

wavelengths, is necessary for UV-Vis Spectroscopy. Assorted UV radiation
sources include the following:

1) Hydrogen lamp: Hydrogen lamps are reliable, steady, and continuously

emit radiation between 160 and 380 nm. It consists of hydrogen gas at high
pressure, which causes an electrical discharge. The excited hydrogen
molecules produce radiation.

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2) Deuterium lamp: A gas discharge lamp called a deuterium lamp is

frequently employed as a UV source. It emits radiation in the 160–450nm
range. It costs more than a hydrogen lamp.

3) Tungsten lamp: The most typical light source utilized in

spectrophotometers is the tungsten lamp. With a wavelength range of roughly
330 to 900 nm, it comprises of a tungsten filament encased in a glass envelope
and is utilized for the visible spectrum.

4) Xenon discharge lamp: A xenon lamp is a discharge light source that

contains xenon gas inside a bulb. Radiation from xenon ranges from 250 to
600 nm.

1. The chemistry that can occur in a sample.

2. Techniques for handling samples and conducting analyses for several
issues (the tools-of-the trade).
3. The method's accuracy and precision
4. Accurate data analysis and documentation

1.2 High- performance liquid Chromatography (HPLC) [9-12]

High-Performance Liquid Chromatography (HPLC) was developed in the late

1960s and early 1970s. Today it is widely applied for separations and
purifications in a variety of areas including pharmaceuticals, biotechnology,
environmental, polymer and food industries. HPLC has over the past decade
become the method of choice for the analysis of a wide variety of compounds.
Its main advantage over GC is that the analytes do not have to be volatile, so
macromolecules are suitable for HPLC analysis. HPLC is accomplished by
injection of a small amount of liquid sample into a moving stream of liquid
(called the mobile phase) that passes through a column packed with particles
of the stationary phase. The separation of a mixture into its components
depends on different degrees of retention of each component in the column.
The extent to which a component is retained in the column is determined by its
partitioning between the liquid mobile phase and the stationary phase. In
HPLC this partitioning is affected by the relative solute/stationary phase and
solute/mobile phase interactions. Thus, unlike GC, changes in mobile phase

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composition can have an enormous impact on your separation. Since the

compounds have different mobilities, they exit the column at different times;
i.e., they have different retention times, tR. The retention time is the time
between injection and detection. Thus, HPLC is most often used when one is
performing a target compound analysis, where one has a good idea of the
compounds present in a mixture so reference standards can be used for
determining retention times .Pumps are used in HPLC to move pressurised
liquid solvent and the sample mixture into a column that is packed with solid
adsorbent material. Each sample component will interact differently, which
results in different flow rates for each component and, ultimately, leads to the
separation of column components. Adsorption is a component of the mass
exchange process that makes up chromatography. Pumps are used in HPLC to
pressurized a fluid and a sample blend through an adsorbent-filled section,
causing the specimen segments to separate. Adsorbent, the section’s dynamic
segment, is often a granular substance comprised of solid particles with sizes
ranging from 2 m to 50 m, such as silica and polymers. The different degrees
of connectivity between the segments of the example mixture/blend and the
retentive particles isolate them from one another. The mobile phase’, which is
the pressured fluid, is typically a mixture of solvents (such as water,
acetonitrile, and/or methanol). Its structure and temperature have a significant
impact on the connections between the sample segments and the adsorbent,
which is how the partition process works. Because HPLC operates at
fundamentally higher pressures (50 bar to 350 bar) than conventional ("low
weight") liquid chromatography, it can be distinguished from the latter.
Conventional liquid chromatography frequently relies on gravity to move the
portable stage through the segment.

1.2.1 Principle of Separation in HPLC.[12-14]

High-Performance Liquid Chromatography (HPLC) is a separation technique

utilizing differences in the distribution of compounds to two phases, called
stationary phase and mobile phase. The stationary phase designates a thin layer
created on the surface of fine particles and the mobile phase designates the
liquid flowing over the particles. Under a certain dynamic condition, each

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component in a sample has different distribution equilibrium depending on

solubility in the phases and/or molecular size. As a result, the components
move at different speeds over the stationary phase and are thereby separated
from each other. This is the principle behind HPLC. only when are in the
mobile phase. Compounds that tend to be distributed in the mobile phase,
therefore, migrate faster through the column while compounds that tend to be
distributed in the stationary phase migrate slower. In this way, each component
is separated on the column and sequentially elutes from the outlet. Each
compound eluting from the column is detected by a detector connected to the
outlet of the column. When the separation process is monitored by the recorder
starting at the time the sample is injected, a graph is obtained. This graph is
called a chromatogram. The time required for a compound to elute (called
retention time) and the relationship between compound concentration
(amount) and peak area depend on the characteristics of the compound.
Retention time is therefore used as an index for qualitative determination and
peak surface area (or height) as an index for quantitative determination.

1.2.2 METHOD DEVELOPMENT

The goal of the HPLC-method is to try & separate, quantify the main active
drug, any reaction impurities, all available synthetic inter-mediates and any
degradantes Analytical method development and validation are critical steps in
the discovery, development, and manufacturing of pharmaceuticals. These
techniques are used to ensure the identity, purity, potency, and performance of
pharmaceutical products. When developing methods, there are numerous
factors to consider. In the case of UV detection, they first gather information
about the analyte's physiochemical properties (pKa, log P, solubility) and
determine which mode of detection would be suitable for analysis. The
majority of the analytical development effort is spent validating an HPLC–
method for indicating stability. The purpose of the HPLC method is to separate
and quantify the main active drug, any reaction impurities, all available
synthetic intermediates, and any degradants.15

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Steps in Analytical Method Development:

1. The assay may be challenging to carry out using the analytical method
for biological fluids.
2. The formulation contains a large number of excipients that may cause
interference. Because of the patient, access to the full body of literature
regarding the drug's analytical techniques is not possible
3. The current analytical procedures may not be appropriate due to their
requirement for expensive reagents and solvents. It may also involve
challenging extraction and separation techniques that are inappropriate.
4. The medication or drug combination is not included in any
pharmacopeia. Validation of analytical procedure is required by law
and must be carried out.
5. The guidelines for the validation of the analytical method have been
established by the ICH guidelines [Q2 (R1)].

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Fig. No: 3 Instrumentation of HPLC

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[Link] Steps involved in HPLC Method Development

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The various parameters that include to be optimized during method

development,

1. Mode of separation
2. Selection of stationary phase
3. Selection of mobile phase
4. Selection of detector

Selection of Chromatographic mode

Chromatographic modes are dictated by the analyte's polarity and molecular

weight. Reversed-phase chromatography (RPC) takes precedence in case
studies, especially for small organic compounds. RPC is extensively utilized
for separating ionizable substances, such as acids and bases, employing ion-
pairing reagents or buffered mobile phases to prevent analyteionization.

Optimization of Mobile phase

1) Buffer Selection

Various buffers, including acetate, sodium phosphate, and potassium

phosphate, were evaluated based on overall chromatographic performance and
system suitability criteria. Through a series of experiments, potassium
dihydrogen phosphate emerged as the most suitable buffer for successful
separation of all peaks. Test concentrations of 0.02 M, 0.05 M, and 0.1 M were
examined. Interestingly, altering the buffer concentration did not significantly
impact the elution pattern and resolution, although the 0.05 M concentration
enhanced the sensitivity of the technique without substantial changes in the
separation characteristics.

2) Effect of pH

For ionizable analytes, determining the appropriate mobile-phase pH is crucial,

guided by the analyte's pKa . This ensures that the target analyte is either in a
neutral or ionized form. The ability to adjust the pH of the mobile phase is a
powerful tool in the chromatographer's toolkit. This capability allows
simultaneous modifications to retention and selectivity, providing a strategic

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means to optimize separation conditions, particularly for critical pairs of

components in the sample. pH adjustment plays a vital role in tailoring
chromatographic conditions to achieve desired separation outcomes.

Selection of detectors

Detector is a very important part of HPLC. Selection of detector depends on

the chemical nature of analyses, potential interference, limit of detection
required, availability and/or cost of detector. UV visible detector is versatile,
dual wavelength absorbance detector for HPLC. This detector offers the high
sensitivity required for routine UV-based applications to low-level impurity
identification and quantitative analysis. Photodiode Array (PDA). Detector
offers advanced optical detection for Waters analytical HPLC, preparative
HPLC, or LC/MS system solutions. Its integrated software and optics
innovations deliver high chromatographic and spectral sensitivity. Refractive
index chromatographic and spectral sensitivity, stability and reproducibility,
which make this detector the ideal solution for analysis of components with
limited or no UV absorption. Multi-wavelength Fluorescence Detector offers
high sensitivity and selectivity fluorescence detection for quantitating low
concentrations of target compounds. 16

1.3 Method Validation

The creation and validation of analytical methods are essential for maintaining
the standards of goods with high economic and market value, for moral
grounds, and in light of global competitiveness. The protocols for approving,
authenticating, and registering pharmaceutical items have been put to
standards by various international regulatory agencies. Some of the
organizations governing the quality standards are 17

 United States of Food and Drug Administration (USFDA)

 Good Laboratory Practice (GLP) regulations
 World Health Organization (WHO)
 The Pharmaceutical Inspection Cooperation Scheme’s (PIC/S)
 The International Conference of Harmonization (ICH).

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Different regulatory validation parameters

Different regulatory bodies like ICH, FDA, GMP, USP provides validation
parameters

 USP validation parameters

 FDA validation parameter
 GMP validation parameters
 ICH validation parameters

USP Parameters of method validation

1) Specificity

2) Linearity

3) Precision

4) Accuracy

5) Limit of detection

6) Limit of quantitation

7) Range

8) Robustness

9) Ruggedness
FDA validation parameters:

1) Specificity

2) Linearity

3) Precision

4) Accuracy

5) Ruggedness

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GMP validation parameters

 Accuracy
 Specificity
 Sensitivity
 Reproducibility

ICH validation parameters

1) Specificity

2) Linearity

3) Precision

4) Accuracy

5) Limit of detection

6) Limit of quantitation

7) Range

8) Robustness

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Analytical Major Minor Minor Major

task qualitative quantitative qualitative quantitative
analysis analysis analysis
Analysis

Specificiy Yes Yes Yes Yes

Linearity No Yes No Yes

Range No Yes No Yes

Accuracy No No Yes Yes

Precision No Yes No Yes

Intermedete No Yes No Yes

Precision

Limit of No No Yes No
detection

Limit of No Yes No No
quantificati
on

Table: 1 ICH characteristics and guidelines

1.3.1 Validation of an analytical method: [18-19]

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Method validation parameters as per ICH guidelines are summarized below.

Method validation is an integral part of the method development; it is the

process of demonstrating that analytical procedures are suitable for their
intended use and that they support the identity, quality, purity, and potency of
the drug substances and drug products. Simply, method validation is the
process of proving that an analytical method is acceptable for its intended
purpose.

Method Validation, however, is generally a one-time process performed after

the method has been developed to demonstrate that the method is scientifically
sound and that it serves the intended analytical purpose.

All the variables of the method should be considered, including sampling

procedure. sample preparation, chromatographic separation, and detection and
data evaluation. For chromatographic methods used in analytical applications
there is more consistency in validation practice with key analytical parameters
including:

 Specificity /Selectivity
 System suitability
 Precision

Repeatability

Intermediate precision

a) Interday

b) Intraday

Reproducibility

 Accuracy
 Linearity
 Range
 Limit of Detection
 Limit of Quantitation
 Robustness

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Specificity / Selectivity:

The terms selectivity and specificity are often used interchangeably.

According to ICH, the term specific generally refers to a method that produces
a response for a single analyte only while the term selective refers to a method
which provides responses for a number of chemical entities that may or may
not be distinguished from each other. If the response is distinguished from all
other responses, the method is said to be selective. Since there are very few
methods that respond to only one analyte, the term selectivity is usually more
appropriate. The analyte should have no interference from other extraneous
components and be resolved well from them. A representative chromatogram
or profile should be generated and submitted to show that the extraneous peaks
either by addition of known compounds or samples from stress testing are
baseline resolved from the parent analyte.

 Selectivity of the analytical method is defined as the degree to which

method can quantify the analyte in the presence of interferents.
 The other components which may include impurities, degradants,
matrix etc.
 The term specific generally refers to a method that produces a response
for a single analyte only, while the term selective refers to a method
that provides responses for a number of chemical entities that may or
may not be distinguished from each other.
 The forced degradation studies are carried out to challenge this method.
 During forced degradation studies, the sample is subjected to the
stressed conditions of light, heat, humidity, acid / base hydrolysis and
oxidation.
 The selectivity of chromatographic methods may be assessed by
examination of peak homogenecity or peak purity.
 Peak purity shows that there is no co-elution of any sample component

Precision:

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Precision of an analytical procedure expresses the closeness of agreement

between a series of measurements obtained from multiple sampling of the
same homogeneous sample under the prescribed conditions of Repeatability,
Intermediate precision, Reproducibility.26

To ensure precision of method for major analytes, RSD should be 2 %. For

low level impurities, RSD of 5-10 % is usually acceptable.

Intermediate precision
Involve in the estimation of variations in analysis when method is used within
a laboratory, as on different days (Inter-day). Intermediate precision was
studied by injecting 3 concentration of standard in triplicates on 3 different
days. The method was passed the test for intermediate precision, as the % RSD
≤ 1.5%.

Accuracy

The International Conference On Harmonization (ICH) defines the accuracy of

and analytical procedure as the closeness of agreement between the values that
are accepted either as conventional true values or an accepted reference value
and the value found. The accuracy of an analytical method may be defined as
the closeness of the test results obtained by the method to the true value
Accuracy is usually reported as percent recovery by the assay (using the
proposed analytical procedure) of known added amount of analyte in the
sample or as the difference between the mean and the accepted true value
together with the confidence intervals. The range for the accuracy limit should
be within the linear range. Typical accuracy of the recovery of the drug
substance is expected to be about 99 -101%. Typical accuracy of the recovery
of the drug product is expected to be about 98 - 102%. Values of accuracy of
recovery data beyond this range need to be investigated as appropriate. The
accuracy of an analytical method may be determined by spiked placebo
(product matrix) recovery method and standard addition method.

Linearity

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Linearity of an analytical method is its ability to elicit test results that are
directly proportional to the analyte concentration in samples within a given
range. Linearity usually expressed in terms of the variance around the slope of
regression line calculated according to an established mathematical
relationship from test results obtained by the analysis of samples with varying
concentrations of analyte. The linear range of detectability that obeys Beer’s
law is dependent on the compound analyzed and the detector used. The
working sample concentration and samples tested for accuracy should be in the
linear range.

Range:

Range is one of the valid parameters. The range is the range within which the
concentration of the API must be. This gives an overview of the upper and
lower limits of API concentration. Between this period, the API can show
good performance. The range should be set to a range that can show linearity.
accuracy and precision at an acceptable level. A linear result of a suitable
range is usually extracted, which must be favorable to the method. The range
must be set so that it does not affect the result of linearity, accuracy and
precision. Even at the extreme level it should fit. Areas to follow include: 1)
Typically, the assay range for the final drug product is 80 to 120 percent of the
label claim.

Limit of detection (LOD):

Lowest quantity of an analyte which may be detected by the

chromatographical separation however it is not necessary that this quantity
will quantify as a precise value. A blank resolution is injected and peak to peak
quantitative noise relation we have to calculate from blank chromatograms.
Then, calculate the concentration at the signal to quantitative noise relation is
concerning 3:1. 27

LOD can be expressed as

LOD = 3.3 σ/S

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σ = Standard deviation of the response

S = Slope of calibration curve

Limit of Quantification .[28-31]

The limit of quantification (LOQ) is defined as the lowest concentration of an

analyte in a sample that can be determined with acceptable precision and
accuracy under the stated operational conditions of the method. The ICH has
recommended a signal: noise ratio 10:1. LOQ may also be calculated based on
the standard deviation of the response (SD) and the slope of the calibration
curve at levels approximating the LOQ according to the formulae

LOQ = 10 σ/S

Where, σ = Standard deviation of the response

S = Slope of calibration curve

Robustness

The robustness of an analytical method is a measure of its capacity to remain

unaffected by small but deliberate variation in method parameters and
provides an indication of its reliability during normal usage. The evaluation of
robustness should be that a series of system suitability parameters (e.g.,
resolution test) is established to ensure that the validity of the analytical
procedure is maintained whenever used. In case of liquid chromatography,
examples of typical variations are influence of variations of pH in a mobile
phase, influence of variations in mobile phase composition, different columns
(different lots and/or suppliers), temperature etc. In the case of gas-
chromatography examples of typical variations are different columns (different
lots or suppliers), temperature, flow rate etc.32

Ruggedness

A different analyst, using a different batch of chemicals, tried the method

graph. There was not much variation in results. These studies showed that the
method is rugged.

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1.4 Stability- indicating assay method

A Stability-indicating assay method can be defined as Validated quantitative

analytical method that can detect the change with time in the chemical,
physical or microbiological properties of the drug substance and drug products
are specific so that the content of active ingredients and degradation products
can be accurately measured without interference the advent of International
Conference on Harmonisation (ICH) guidelines, the requirement of
establishment of stability indicating assay method (SIAM) has become more
clearly mandated. The guidelines explicitly require conduct of forced
decomposition studies under a variety of conditions, like pH, light, oxidation,
dry heat, etc. and separation of drug from degradation products. Stability
testing of drug substance requires an accurate analytical method that
quantitates active pharmaceutical ingredients (API) without interference from
degradation. products, process impurities and other potential impurities.
"Generally forced degradation/stress testing is used to generate the samples for
stability indicating assay methods, Forced degradation/stress testing is defined
as the stability testing of drug substance and drug product under conditions
exceeding those used for accelerated stability testing. Degradation can be
achieved by exposing the drug, for extended period of time, to extremes of pH
(HCl or NaOH solutions of different strengths), at elevated temperature, to
hydrogen peroxide at room temperature, to UV light, and to dry heat (in an
oven) to achieve degradation to an extent of 5-20%.33

1.4.1 Development of validated SIAMS 34-35

The practical steps involved in the development of SIAMs are discussed

below.

Step 1: critical study of the drug structure:

The structure, by study of the functional groups and other key components.
There are definite functional group categories, like amides, esters, lactams,
lactones, etc. that undergo hydrolysis, others like thiols, thioethers, etc,

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undergo oxidation. For information on degradation chemistry of like drugs,

one can look into the treatises like Analytical Profiles of Drug Substances and
the monographs provided by Connors et al., the internet search engines.

Step II: collection of information on physicochemical properties:

It is generally important to know various physicochemical parameters

Insolubility, absorptivity and wavelength maximum of the drug in question.
The knowledge of pKa is important as most of the pH-related changes in
retention occur at pH values within 1.5 units of the pKa value. The ionization
value also helps in selecting the pH of the buffer to be used in the mobile
phase. The availability of the solubility data in aqueous, organic and
commonly used UHPLC solvents and their combinations can thus prove to be
very useful in the selection of the sample solvent and the mobile phase.

Step III: Stress (Forced Decomposition) Studies:

 Hydrolytic degradation
 Oxidative degradation:
 Thermal degradation:
 Photolytic degradation:

[Link] degradation

Hydrolysis is one of the most common degradation chemical reactions over

wide range of pH. Hydrolysis is a solubility process in which drug reacts with
water to yield breakdown products of different chemical compositions. Water
either as a solvent or as moisture in the air comes in contact with
pharmaceutical dosage forms is responsible for degradation most of the drugs.
Hydrolytic study under acidic and basic condition involves breakdown of
ionisable functional groups present in the molecule. Alternatively if total
degradation is seen after subjecting the drugs to initial condition, acid/alkali
strength can be decreased with decrease in reaction temperature. In general
temperature and pH are the major determinant in stability of the drug prone to
hydrolytic decomposition.

2. Oxidative degradation:

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Many drug substances undergo autoxidation i.e., oxidation under normal

storage condition and involving ground state elemental oxygen. Autoxidation
is a free radical reaction that requires free radical initiator to begin the chain
reaction. Hydrogen peroxide, metal ions, or trace level of impurities in a drug
substance act as initiators for autoxidation. Hydrogen peroxide is very
common oxidant to produce oxidative degradation products which may arise
as minor impurities during long term stability studies. It can be used in the
concentration range of 3-30% at a temperature not exceeding 40 °C for 2-8
days. The oxidative stress testing can be carried out initially with 3% H2O2.

3. Thermal degradation

In general, rate of a reaction increase with increase in temperature. Hence, the

drugs are susceptible to degradation at higher temperature Effect of
temperature on thermal degradation of a substance is studied through. Thermal
degradation study is carried out at 40°C to 80°C. The most widely accepted
temperature is 70°C at low and high humidity for 1-2months. High
temperature (80°C) may not produce predictive degradation pathway. The use
of high- temperatures in predictive degradation studies assumes that the drug
molecule will follow the same pathway of decomposition at all temperatures.

4. Photolytic degradation

Exposure of drug molecules may produce photolytic degraded products. The

rate of photo degradation depends upon the intensity of incident light and
quantity of light absorbed by the drug molecule. Photolytic degradation is
carried out by exposing the drug substance or drug product to a combination of
visible and UV light. The most commonly accepted wavelength of light is in
the range of 300-800 nm to cause the photolytic degradation. The photolytic
degradation can occur through non oxidative or oxidative photolytic reaction.
The non- oxidative photolytic reaction include isomerization, dimerization,
cyclization, rearrangements, decarboxylation and hemolytic cleavage of X-C
hetero bonds, N-alkyl bond(de alkylation and deamination), SO2-C bonds etc.
and while oxidative photolytic reaction occur through either singlet
oxygen(10%) or triplet oxygen mechanism. The singlet oxygen reacts with the

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Chapter 1 Introduction

unsaturated bonds, such as alkenes, dienes, poly nuclear aromatic hydrocarbon

to form photo oxidative degradation products whereas triplet oxygen react
with free radical of the drug molecule, which than react with a triplet oxygen
molecule to form peroxide. Hence, light can also act as a catalyst to oxidation
reactions.

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