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Chapter 1

The document provides an introduction to analytical chemistry and pharmaceutical analysis. It discusses how analytical chemistry is used to determine the elemental makeup, purity, and quality of pharmaceuticals and chemicals. Various analytical techniques are described, including chromatography, electrophoresis, mass spectroscopy, and specific methods like high performance liquid chromatography. The importance of quality control in pharmaceutical production and ensuring safety, effectiveness, and maintenance of product qualities is also highlighted.

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0% found this document useful (0 votes)
26 views24 pages

Chapter 1

The document provides an introduction to analytical chemistry and pharmaceutical analysis. It discusses how analytical chemistry is used to determine the elemental makeup, purity, and quality of pharmaceuticals and chemicals. Various analytical techniques are described, including chromatography, electrophoresis, mass spectroscopy, and specific methods like high performance liquid chromatography. The importance of quality control in pharmaceutical production and ensuring safety, effectiveness, and maintenance of product qualities is also highlighted.

Uploaded by

UpadhayayAnkur
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Chapter-1 INTRODUCTION

CHAPTER-1. INTRODUCTION

1.1 INTRODUCTION

Analytical chemistry is the study and practice of analyzing substances to determine


their elemental make-up. Purity, safety, and quality assessments of pharmaceuticals
and chemicals are the focus of pharmaceutical analysis, a branch of science. It has
tests for determining the composition, stability, quality, and purity of novel chemicals.
Methods for disentangling, identifying, and quantifying constituents in a material
sample are also a part of this process. Finding safe and effective drugs relies heavily
on quality assurance. For pharmaceutical R&D, quality assurance, and
standardization, it provides extremely specific and sensitive analytical procedures.
They're crucial for determining bioavailability and clinical response in
pharmacokinetics and medication metabolism investigations. Even for tiny material
samples, modern physical methods of examination are exceedingly sensitive. It can be
implemented quickly and is easily automatable. As a consequence, it plays a
significant role in the creation of novel goods as well as the formulation, manufacture,
and monitoring of medical procedures and medications. Pharmaceutical analysis
involves both quantitative and qualitative techniques, and it applies to all
pharmaceuticals and pharmaceutical substances, from bulk pharmaceuticals to
finished dosage forms. Thus, in modern medicine, a diagnostic tool is the examination
of chemical components in the human body, which may change during the illness
state. If a doctor expresses concerns about a drug's quality, it's the chemist's job to
address such concerns. This can be accomplished in a number of ways, including
consulting with the manufacturer of the drug, conducting in-house analysis of the
preparation using appropriate clinical laboratory equipment, submitting the sample to
a private laboratory for examination, or some combination of these. However, the
analyst is still responsible for resolving issues with medication quality. 'Quality' in the
context of pharmaceuticals refers to the aggregate of all variables that ensure the
drug's efficacy, safety, and dependability.

Significance of quality control:

The pharmaceutical business remains an integral part of the health care cycle, doing
critical research and producing medications that sustain and restore human life.
Standards for quality, safety, and effectiveness are quite high for today's
pharmaceuticals intended for human consumption. Adequate procedures for product
quality control are necessary for the assessment of safety, effectiveness, and the
ongoing maintenance of these qualities in practice. Quantitative analysis may be used
to identify species in a sample based on their chemical makeup. One or more of these
species or analytes may be detected using a quantitative analysis to determine their
abundances.

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1.1.1 Analytical Techniques

A pharmaceutical product's efficacy and safety can only be ensured by analytical


quality monitoring. Every step of a drug's life cycle, from production through patient
usage, has to be monitored for purity. The goal is more likely to be reached if the
applicable requirements are grounded on validated technology that can show the
quality connection between the substance under inspection and that which was
initially subject to pharmaceutical, toxicology, and pharmacological evaluation.

Estimation methods for various formulation components include the ones listed
below.

Optical methods

Some of the optical methods are

• Spectroscopy with X-rays


• Infrared spectroscopy
• Infrared spectroscopy
• Atomic absorption spectroscopy
• Photos of a flame
• NMR spectroscopy, or nuclear magnetic resonance
• Nephlo-turbidimetry
• Spectroscopy of the spin of electron

1.1.2 Methods of Electroanalysis:

Electro analytical techniques include the following:

• Amperometry
• Voltametry
• Potentiometry
• Conductometry

Methods of separation/chromatography

Several chromatographic techniques include

• Chromatography of Gas-Liquid
• Chromatography of Gases and Solids
• Chromatography of a liquid sample
• Chromatography, Liquid-Solid
• Chromatography on a thin layer
• Chromatography on paper
• Chromatography with gel permeation

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The choice of the most efficient method for a particular analysis is one of the most
important decisions an analyst must make. He needs to be familiar with the theoretical
foundations, real-world applications, and the circumstances under which each
approach is dependable in order to do this. He must also be aware of any potential
interferences that may occur and be able to devise solutions to avoid them.

1.1.3 There are two main types of instrumental separation methods.

1. Chromatography

2. Electrophoresis

3. Mass spectroscopy

Chromatography

Chromatography is a method for separating substances from a mixture based on the


relative ease with which each component can travel through or along a stationary
phase in the presence of a mobile phase. The sample is positioned on the periphery of
a solid or liquid stationary phase. The sample is swept along the length of the column
as the stationary phase flows over the mobile phase. Components that are heavily
"

adsorbed to the stationary phase move more slowly than those that are not over the
course of the stationary phase. The Greek roots of the word chromatography-chromos
and graphy-mean, roughly, ''writing in colour.'' It was Tswett who first used a
chromatography column made of adsorbent powder and rinsed with a liquid solvent to
separate colour bands of plant pigments, hence the words ''adsorbent powder'' and
''mobile phase.'' This is transmitted down the tube's length in a stationary solid or
liquid phase.

1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Researchers in the late 1960s and early 1970s built on the theoretical foundations laid
by earlier chromatographic methods, most notably column chromatography, to create
the high performance liquid chromatography technique (HPLC). Similar to traditional
column chromatography, this method employs a number of different separation
mechanisms. These methods include things like adsorption, partitioning (including
reverse phase partition), ion exchange, and gel permeation. In high-pressure liquid
chromatography (HPLC), the mobile phase is pushed through a less porous, more
densely packed column. Improved resolution of the compounds being separated,
shorter separation periods, and better accuracy, precision, and sensitivity in
quantifying the substances being separated are the primary benefits of HPLC in
comparison to traditional (gravity feed) column chromatography.

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Another enhancement compared to column chromatography is the choice of detection


technique. The level of automation and sensitivity of these techniques is
unprecedented.

 Solvent reservoir : glass / stainless steel, 1 liter


 Sonication or sparging with helium to elimate outgassing.
 Particals in column <50µ m, 500 psi pressure
 Pumps:
1. Mechanical = constant flow
2. Pneumatic = constant pressure
 Gas displacement =highly compressed gas
 Amplifier type = compressed gas at low pressure impinge on
large end
3. Reciprocating piston type = sapphire pluger
 Gradient analysis : particles sepration,
 Gradient controller : synchronization of pump = mobile phase mixture
 Solvent conditioning chamber : for silica gel column which dissolve below pH
2 and above pH 7 , small column 5-10 cm packed with silica gel after the
pump and before the injector , saturation of mobile phase
 Injection device ; septum injection ( self sealing rubber , Teflon disk )
 Precolumn / guard column : for thin layer of liquid coated on solid support ,
tapping particulate matter , 2-10 cm.
 Analytical column : 2-25 cm , 2-4.6 mm diameter , partical size > 10 µm, 6000
psi
 Packing material :
1. Porous
2. Pellicular : glass bead , 37-50 m
3. Totally porous : microparticulate, 3.5,10,20

1.2.1 TYPES of HPLC

1. Partition chromatography.
2. Normal–phase chromatography.
3. Displacement chromatography.
4. Reversed-phase chromatography (RPC)
5. Size-exclusion chromatography.
6. Ion-exchange chromatography.
7. Bio-affinity chromatography.
8. Aqueous normal-phase chromatography

1.2.2 Basic principle of HPLC

High performance liquid chromatography (HPLC) separates chemicals by distribution


using a stationary phase and a mobile phase. Small particles are what make up the

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stationary phase, while the liquid that moves over them constitutes the mobile phase.
The solubility of components in the phases and the molecular size of the sample
inform the distribution equilibrium under a specific dynamic solution.

As a result, during the static phase, the different parts move at different speeds,
creating physical separation between them. Spherical solid particles are jammed into a
tube made of stainless steel (or resin) to form the column. Liquid pumping keeps
mobile phase flowing steadily into the column's intake. The column's inlet is placed
close to the sample injector. The column is first loaded with mobile phase, and when
the sample's constituent parts move through it, they switch back and forth between the
stationary phase and the mobile phase.

Compounds that migrate quicker across the column are only those that are in the
mobile phase, whereas those that are more often found in the stationary phase travel
through the column more slowly. In this arrangement, the components are separated
on the column before eluting in a specified sequence from the output. A detector
affixed to the column's exit picks up every chemical that elutes from the column. The
instant the sample is entered, the separation process is recorded and graphed. This
diagram is known as a chromatogram. Both the retention time (or elution time) and
the ratio of compound concentration to peak area are affected by the compound's
specific properties.

Selectivity of HPLC

Due to a number of benefits, the majority of pharmaceuticals may be analyzed using


the HPLC technique.

 The analysis may be completed quickly (in 20 minutes or less).


 heightened sensitivity (using an array of detectors).
 More clarity (a plethora of stationary phase options).
 Reliable columns (a lot of options for the stationary phase).
 Low volatility materials should be used.
 Simple sample handling, maintenance, and recovery.
 Each module's many features may be easily programmed.
 Time-programmable operating sequence, such as starting the pump and
detector light to get a stable baseline and an equilibrated column before the
workday starts.
 Retention times are reliably repeatable.

1.2.3 Different Modes of Separation of HPLC

 Normal phase mode


 Reverse phase mode
 Reverse phase ion pair chromatography
 Ion exchange chromatography
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 Affinity chromatography
 Size exclusion chromatography (gel permeation and gel filtration
chromatography)

1.3 Instrumentation for HPLC

 a supply of mobile phase with variable flow and pressure that can feed the
column. To purge the solvents of dissolved air and other gases, a degasser is
required.
 A mobile phase pump that supplies the column with mobile phase. There can't be
any pulsations in the pumping system. Low-cost chromatography requires a
pump with a pressure range of at least 100 atm (1500 psi). However, a better
upper limit might be 400atm (600 psi). The generation of a modest flow rate of
0.5 to 2 ml per minute was all that was required for many analytical columns.
 A sampling valve or loop at the top of the separation column is used to inject the
sample into the moving mobile phase.
 A guard column or inline filter placed at the separation column's inlet can catch
any particles too tiny to pass through the main column's filtration system.
 The HPLC separation packing required to achieve the separation is housed in the
separation column. Silica for adsorption chromatography, bonded phases for
liquid-liquid chromatography, ion-exchange functional groups attached to
stationary support for ion-exchange chromatography, gels with a specific porosity
for exclusion chromatography, and so on are all examples of suitable media for
these separation techniques.
 The typical length of a column is between 10 and 30 centimeters, but shorter,
faster columns are just 3 to 8 centimeters in length and have an interior diameter
of 4 to 5 millimeters. Preparative chromatography typically uses particles with a
diameter of 3 to 5 nm, but can use particles as large as 10 nm.
 The bare-bones set of instruments is rounded out by a detector coupled to a data-
handling device.

1.3.1 The various detectors are

1. UV visible photometers
2. Refractive index detector
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3. Flourimetric detector
4. Conductivity detector
5. Amperometric detector
6. PDA

Modern high-performance liquid chromatography (HPLC) often employs detector


electronic integrators and computer integrators to measure peak regions. These
gadgets can detect peaks automatically and output the corresponding regions as
numbers. Peak width (using a triangle model of the peak) and the number of
theoretical plates (using peak area and height values) can be determined.

There is currently no universal detector available for HPLC that has sufficient
sensitivity. Therefore, deciding on a detector is task-specific.

1.4 Buffers in Reversed-Phase Liquid Chromatography:

Understanding the basic effect of pH on retention of ionic analytes and considering


some of the characteristics of the buffer alternatives can help you make a reasonable
and acceptable choice when selecting a buffered aqueous mobile phase for reversed-
phase liquid chromatography (RPC).The solubility or compatibility constraints of the
detection methods may call for a rethinking of this choice. For consistent and error-
free results, it's also important to follow best practices for buffer preparation. A
change in the mobile phase's pH of only 1.5 pH units may have a considerable effect
on retention if the analytes PKa is sensitive to changes in pH. The importance of
mobile phase pH regulation in ensuring reliable separations of ionic compounds for
functional groups is obvious. The retention of an analyte that is not ionic will not be
directly affected by the pH of the mobile phase.

1.4.1 Chromatographic parameters:

Resolution: Resolution Rs of adjacent bands is the standard by which


chromatographers evaluate separation quality.

where tw1 and tw2 are located at the point where the tangents meet the baseline. The
tangent lines for a Gaussian peak that is symmetrical in shape are drawn at a distance
of 0.60 times the height of the peak.

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Capacity factor: It's a metric for determining how effectively the column retains
sample molecules during isocratic separation. Solvent makeup, separation age, and
separation temperature all have a role.

K=tr-t0/t0
where, tr = Band separation time

t0 = column dead volume

Column efficiency: Theoretical plates number is the term for this concept. A peak's
band widening is quantified. Lower band spread results in a greater number of
theoretical plates. It's a sign of a well-performing column and overall system health. N
values may be used to characterise the performance of a column.

Where,

N = Number of theoretical plates

Ve = elution volume, retention time or retention distance (mL, sec, or cm)h = peak
height

wb = width of the peak at the base line (ml, sec, or cm)

Peak Asymmetry and Peak tailing:

Peak Asymmetry:

Peak with poor symmetry can result in

• Measurement errors in plate count and resolution


• Accurate measurements
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• Poorer detail and invisible low-level bands in the peak tail


• Poor predictability of retention

As = peak asymmetry factor

b = The 10 percent of the peak's height corresponding to the distance from its midway
to its leading edge.

a = distance from the peak's front to its center (described as a proportion of the peak's
total height)

Peak tailing:

Where,

T = tailing factor (measured at 5% of peak height)


b = distance the point from at peak midpoint to the tailing edge
a = distance from the leading edge of the peak to the midpoint
Increased peak asymmetry value, AS > 1.5 the sign that the column should be
changed

Selectivity:
It measures relative retention of two components. Selectivity is the function of
chromatographic surface ( column), melting point and temperature.
α= K2/K1 = V2-V0/V1-V0

1.5 DIAGRAM OF HPLC

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Figure 1.1 : High performance liquid chromatography

1.6 Quantitation methods in HPLC

Measuring the heights of peaks or the areas of peaks only gives you a response in
terms of the detector signal.

The answer should depend on how much of the target component is present or how
much mass there is to account too. This may be achieved by means of a calibration
procedure.

The four primary techniques for quantization are

a. Normalized peak area method.

b. External standard method.

c. Internal standard method.

d. Method of standard addition.

a) Normalized peak area method

The proportion of the overall area that each individual peak takes up is known as the
normalised peak area. In order to determine the relative quantities of trace impurities
or degradation products in a purified material, it is common practice to calculate the
response factor for each component.

b) External Standard method

Using a calibration plot or response factors, the unknown is computed visually or


numerically, and the standard is injected together with the unknown in this technique.
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Each standard has its own response factor (RF), which may be calculated as follows.

RF = Standard area (peak height) / Standard Concentration

To run most samples in HPLC without substantial preparation, the external standard
technique can be utilised.

c) Internal Standard method

The analyte is separated from the internal standard, which is a distinct component.
The internal reference point should be selected such that it behaves similarly to the
example item. Samples that need extensive processing or cleaning before analysis are
a prime candidate for the use of an internal standard.

d) Method of standard addition

Calibration plots for quantitative analysis may be generated using the standard
addition approach. It is more often used in trace analysis. In order for the standard
addition method to work, the response before spiking additional analytes must be
sufficiently large to establish an acceptable signal-to-noise ratio (10).(Vogel’s 2003).

1.7 Limitations of HPLC

High-performance liquid chromatography (HPLC) is widely used in chemistry and


biology to separate and identify compounds in complex mixtures. HPLC has its limits,
just like any other analytical technique. Some of HPLC's major drawbacks will be
highlighted, along with strategies for addressing them.

1. Sample size: The size of the sample used is a major drawback of high-
performance liquid chromatography. Analysis typically requires just a few
microliters of sample, which might be problematic for scarce or costly
materials. However, sample size may be increased by the use of sample
concentrators or through an increase in the detector's sensitivity.
2. Resolution: High-performance liquid chromatography (HPLC) separates
compounds based on their physical and chemical properties. High-
performance liquid chromatography is widely used in chemistry and biology
for chemical separation and identification (HPLC). Gas chromatography (GC),
mass spectrometry (MS), and our very own GC-UV solution are all tools we
advocate for use in such circumstances.
3. Detection limit: Another drawback is that HPLC is not as sensitive as other
analytical methods like GC or UV, hence its detection limit is less.
4. Column lifetime: HPLC columns are consumables that will need to be
changed at some point. Columns have different lifespans depending on their
types, the sample matrix, and the circumstances of the sample. Cleaning and
storing columns properly may prolong their useful lives, but doing so is
laborious and time-consuming.

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5. Cost: The high-cost method known as high-performance liquid


chromatography (HPLC) requires specialised columns and equipment.
Columns and other consumables might also build up in price over time.
Because of this, researchers working with low finances may find HPLC less
accessible.
6. When analyzing a combination, high-performance liquid chromatography
(HPLC) can be used to isolate and learn more about specific compounds. It is
a subset of liquid chromatography, a method for separating substances in a
mixture based on their affinities to a solid or gel stationary phase and a liquid
mobile phase (such as a liquid or gas). High-performance liquid
chromatography (HPLC) involves injecting a sample into a column containing
a stationary phase and then using a mobile phase, often a liquid, to elute (push)
the components of the sample out of the column. As the sample's constituents
elute from the column, they are detected and measured according to their
interactions with the stationary phase.

1.8 VALIDATION

Quality assurance relies heavily on the procedure of validation. A process or piece of


equipment is said to be validated if ''established documented evidence'' shows that it
reliably produces a result that consistently meets its specific requirements and quality
features.

Definition

Validation is ''established recorded proof that offers a high degree of confidence that a
certain method will consistently generate a product of predefined specifications and
quality characteristics,'' according to the US Food and Drug Administration.

The European Union Good Manufacturing Practice (EUGMP) defines validation as


''the action of showing in line with the concept of Good manufacturing practice
(GMP), that any material activity or system really led to intended outcome.''

Validation is defined by Australian Good Manufacturing Practice as ''the action of


proving that any material, process, procedure, system, equipment, or mechanism used
in manufacture or control can and will be reliable and achieve the desired and
intended result.''

Why Validate?

• Rapid and reliable up


• Robust process
• Reduction in rejections/rework/recalls
• Reduce testing

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• Rapid introduction
• To increase the productivity
• To target and reduce the number of controls
• To reduce product cost

1.8.1 Importance of validation

1. Since a statistically small sample size is used in most quality control


procedures, there is no guarantee that the product will always meet standards.
2. In order to provide the user a high degree of confidence that the same quality
is incorporated into each and every end product, batch after batch, the validation
process should guarantee that a system or product is sufficient and reliable in
achieving the given criteria or characteristics.
3. In order to compare trends or resolve concerns about outcomes from cGMP to
cGLP, retrospective validation is helpful.
4. For taking necessary measures in case of noncompliance.

1.8.2 Objectives of validation

The main goal of validation is to provide the foundation for documented


manufacturing and process control processes that are meant to ensure the claimed or
represented identity, quality, and purity of the medicinal products.

1. Assurance of quality.

2. Government regulation.

1.8.3 Types of validation


The following are common areas of a pharmaceutical process that need to be checked.

1. Verification of Equipment
2. Validation of Process
3. Confirmation of Cleaning
4. Verification of Analytical Methods
5. Checking the utilities and certifying the facility

1.9 ANALYTICAL METHOD VALIDATION

The process of assessing whether or not a certain analytical technique may be utilized
for its intended purpose is known as method validation. Only comprehensive
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Chapter-1 INTRODUCTION

analytical testing can provide the assurance that a pharmaceutical product is pure,
stable, safe, and effective. The quality control process is not complete without
validating the analytical methods used.

 Validation activities must be carried out in compliance with the validation


procedure. Procedures and criteria for acceptance of all qualities should be
included in the protocol. The validation report should include the outcomes.
 When procedures deviate from the pharmacopoeia, an explanation should be
supplied. Data like comparisons to the pharmacopoeial or other approaches
should be included in the argument if possible.
 A comprehensive overview of the standard test technique that includes enough
information for a suitably skilled analyst to carry out the analysis reliably is
required.
 The chromatographic condition (for chromatographic tests), required reagents,
reference standards, equations for computing results, and suitability tests
should all be included in the description.

Table 1.1 : Acceptance criteria of validation parameter for HPLC

S.no Parameter Acceptance criteria


1 Accuracy % Recovery 98-102%
% RSD of recovery concentration must be
<2
2 Precision RSD<2%
3 Range Concentration where data can be reliably
detected (80-120%)
4 Specificity No interference
5 Linearity Correlation coefficient – NLT 0.999
6 Detection limit S/N >2 or 3
7 Quantitation limit S/N>10
8 Ruggedness Should meet all system suitability
parameters
9 Robustness RSD <2%

Alterations to one technique during drug development could need revisions to a


different, preexisting analytical technique. Figure illustrates how these changes may
need new rounds of validation or transfer processes.

1.9.1 Method development

Method development is ongoing and occurs in tandem with the maturation of the
pharmaceutical product. With respect to time, money, and efficiency, the concept of
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Chapter-1 INTRODUCTION

phase-appropriate method development is crucial. Methodological goals and


purposes have to be aligned with the current stage of drug development. Methods may
center on active pharmaceutical ingredient (API) behaviour throughout the first stages
of drug development. They need to be able to back up investigations on the stability
of prototype products, as well as pre-clinical safety assessments and pre-formulation
research. With better understanding of APIs and finished drugs, analytical
methodologies may be improved and extended throughout the drug development
process. The procedures must be reliable and straightforward while yet conforming to
legal requirements.

Figure 1.2 : Analytical method development validation

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Chapter-1 INTRODUCTION

Figure 1.3 : Analytical method development validation

When developing a new technique, it is common practice to conduct preliminary


''scouting'' trials to determine the method's boundaries before moving on to the more
rigorous validation phase. Studies of forced deterioration are one possible kind of
stability indicator technique development. The most common enemies of API are
acids, bases, oxidizers, heat, and light. This gives us the opportunity to understand the
fundamental mechanisms behind degradation and assess the method's ability to
identify and measure degradation byproducts. The developed medicinal product may
be subjected to heat and light once a stability-indicating procedure has been designed
to determine if the API will deteriorate in the presence of the excipients used in the
formulation.

With the help of more trials, the standards for system suitability that will be applied to
upcoming analytical sample sets may be better specified. System suitability tests are a
set of routine inspections that assess the overall functioning of the instrument,
software, reagents, and analysts.3. The ultimate system suitability parameters may be
determined by assessments of method robustness carried out under statistical
experiment design. The objective is to determine which factors are most important
when evaluating a method's or system's potential utility.

1.9.2 Elements of Validation

To demonstrate the reliability of the measurement or characterization, it is necessary


to validate an analytical method as part of the regulatory submission process.
Analytical methods need to go through validation to prove they are measuring the
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Chapter-1 INTRODUCTION

right thing in the right range for the right samples. By doing so, the analyst may learn
about the method's characteristics and determine its capabilities. In the endnotes, you
will find a collection of useful references and strategies for validating your methods.

The laboratory should have in place a documented standard operating procedure


(SOP) that details how to carry out the technique validation process. The laboratory
equipment must be properly qualified and calibrated, and it must be run in accordance
with standard operating procedures. Before carrying out validation trials, a well-
developed and documented test procedure and an authorised protocol should be in
place. The protocol is a set of instructions for carrying out the testing of the method's
performance parameters, including the means by which those parameters will be
evaluated and the standards by which their results will be judged. In the end,
validation investigations need API or drug product samples, placebos, and reference
standards.

1.10 PARAMETERS USED FOR ASSAY VALIDATION

The following criteria are used to carry out the assay procedure validations..

SPECIFICITY:

Specificity is the ability to make a clear evaluation of an analyte in the presence of


other possible variables, such as contaminants, degradation products, matrix
components, and so on. When one analytical method is lacking in specificity, it may
be made up for by using a combination of techniques.

Specificity can be used for identification test, purity test, assays ,etc.

According to the ICH guidelines, a representative chromatogram should be shown to


indicate the level of specificity (selectivity), and peaks should be properly labelled if a
chromatographic method is utilized. It may be feasible to prove that the
chromatographic peaks seen for a given analyte are not due to a mixture of substances
by performing a peak purity test.

PRECISION:

Definition

The degree to which multiple measurements made from the same homogenous
material under controlled conditions agree with one another is considered a measure
of an analytical technique's accuracy. The precision of an analytical method is often
evaluated by using the variance, standard deviation, or co-efficient of variation of a
series measurement.

For reliable estimations of the relative standard deviation, the precision of an


analytical method must be evaluated using a statistically significant sample (also
known as the coefficient of variation).

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System precision

By comparing the peak responses of six replicate injections of the same standard
solution performed using the recommended method, the system's precision may be
determined. After calculating the RSD percentage, we know that it must be less than
or equal to 2%.

Method precision

The peak response of six duplicate injections of six distinct weighed sample solutions
prepared according to the suggested technique is measured to determine the method's
accuracy. The %RSD is determined, and it must be less than or equal to 2%.

Determination

Assaying sufficient replicates of a consistent sample allows for the calculation of


statistically reliable estimations of the standard deviation or relative standard
deviation, which in turn gives insight into the analytical method's accuracy.

ICH Requirements

According to ICH guidelines, nine determinations should be used to evaluate


repeatability (i.e., using six determinations at 100% of the test concentration or using
three concentrations and duplicates of each concentration).

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ACCURACY

Definition - An analytical procedure's precision is defined by how well the results


match up with what is generally acknowledged as the real value or a standard point of
reference.

Determination

Analytical methodologies used to estimate the concentration of a medication in a


produced product's formulation can have their efficacy confirmed by applying the
method to synthetic mixes of the drug product's constituent parts to which a known
amount of analyte has been added. It may be allowed to use substitutes if it is not
possible to obtain all of the product components. Methods that have been established
as reliable include the incorporation of a standard concentration of the analyte into the
therapeutic product and comparing the results to those obtained using a second,
similarly characterised method. Samples and analytical data for method validation
should be submitted at 80%, 100%, and 120% of the label claim for the drug
substance and drug product, respectively. Multiple samples are tested at each planned
investigational level. . Test method 14 provides analytical precision (or the standard
deviation of the replicates). The accuracy of the test may be calculated from the
average of the replicates as a percentage of the label claim.

ICH Requirements
The ICH guidelines suggest testing accuracy with nine determinations across three
concentration levels that span the intended range (i.e., three concentration and three
duplicates of each concentration).

LINEARITY

Definition

Analytical methods are said to be linear within a specific range if they reliably yield
test results that are proportional to the concentration (quantity) of analyte in the
sample.

.Determination

At least five concentrations are often used to determine whether or not an analytical
technique is linear. A plot of signals vs. analyte concentration is visually inspected to
make the first determination. Appropriate statistical methods are employed to
establish test results if a linear relationship is suspected (i.e., by computing the
regression line using the least squares approach).

LIMIT OF DETECTION (LOD)

Definition

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Chapter-1 INTRODUCTION

Lower Detection Limit (LOD) is the minimum substance concentration that a given
analytical technique can identify (but not necessarily measure). Below or above LOD
merely means that the sample is not at that level.

The LOD is often represented as the analyte sample's concentration (e.g., in


percentages, parts per million, etc.).

Determination

Non-instrumental methods frequently involve testing samples with a known


concentration of the analyte to identify the detection limit, or the lowest concentration
at which the analyte can be reliably recognised.

ICH Requirements

Methods for comparing the signal strength of samples containing known and blank
concentrations of analyte are detailed in the ICH. Figure out the smallest analyte
concentration at which a positive result is still feasible.

Calculations of LOD for instrument sensitivity

LOD(mg/L)=3×Noise/Signal×Lowest concentration of the linearity samples

Typically, signal-to-noise ratios of 2:1 or 3:1 are deemed acceptable.

LIMIT OF QUANTITATION (LOQ)

Definition

The limit of quantification (LOQ) refers to the concentration of an analyte at which


the proposed method can provide a quantitative estimate with the required degree of
precision, accuracy, and dependability. Finding the lowest concentration for which
acceptable precision and accuracy may be maintained requires analysing a series of
samples containing progressively decreasing levels of the chemical.

Determination

Analyzing samples of known analyte concentration is commonly used to identify the


lowest concentration at which the analyte may be detected with acceptable accuracy
and precision, and this serves as the quantization limit of a non-instrumental
technique.

ICH Requirements

Methods for comparing signals measured from samples with a known low
concentration of analytes to those measured from blank samples are provided in the
ICH papers.

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Chapter-1 INTRODUCTION

To verify these quantitation limits, it is necessary to conduct inspections on a


sufficient number of samples that were either created or are known to be very close to
the limit.

Calculations of LOQ values for instrument sensitivity:

LOQ(mg/L)=10×Noise/Signal×Lowest concentration of the linearity samples

RANGE

Definition

It has been shown that analytical procedures are sufficiently specific, accurate, and
linear throughout a range of analyte concentrations in a sample. The range represents
the space between the sample's greatest and lowest analyte concentrations.

Determination

Analytical precision, accuracy, and linearity are tested using samples with both low
and high concentrations of the analyte to guarantee they are within the method's
acceptable range.

ROBUSTNESS

Definition

Analytical procedures may be judged by their robustness based on how well they hold
up under typical conditions, which can be affected by modest but deliberate
modifications to the parameters of the analysis.

Determination

By carrying out the assay while deliberately varying some parameters (such as the
flow rate by 10%, the mobile phase ratio by ±2, the pH of the mobile phase through
±0.2, the wave length detection by ±5 nm, and the temperature by ±1 to 50 degrees),
the robustness of the method is evaluated to ensure that the results are unaffected by
the changes in the parameters listed above.

RUGGEDNESS

Definition

Analytical robustness is defined as the degree to which results from repeated analyses
of the same samples remain consistent when performed in different laboratories by
different analysts using different instruments using different lots of reagents using
different elapsed assay times using different temperatures using different days, etc.

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Chapter-1 INTRODUCTION

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Chapter-1 INTRODUCTION

Determination

To determine the resilience of the analytical technique, aliquots from homogenous


batches are subjected to numerous analyses in several laboratories under potentially
different operational and environmental circumstances but still within the assay's set
parameters. The assay factors influence the degree of reproducibility of results.
Against determine how resilient the analytical technique is, this repeatability may be
compared to the test's accuracy under typical circumstances.

SAMPLE SOLUTION STABILITY

After proper preparation in line with the test method, the solution stability of the drug
substance or drug product can be evaluated. It is common practise for laboratories to
use auto samples for overnight runs, during which the sample will remain in solution
for several hours. Particular care must be taken with drugs that can be broken down
by water or light or that stick to glass.

SYSTEM SUITABILITY SPECIFICATION AND TESTS

A well-behaved chromatographic system is the first step towards collecting accurate


and precise HPLC data. The parameters of the system suitability standards and testing
help in this direction.

It consists of following factors:

1. Capacity factor

2. Precision\Injection repeatability

3. Relative retention

4. Resolution

5. Tailing factor

6. Theoretical plate number

1. Capacity factor (K’)

K’ = ( tR-tO/tf )

The capacity factor, a gauge of where the peak of interest falls in respect to the empty
volume, determines how long the non-retained components take to extract.

2. Precision/Injection repeatability (RSD)

The performance of the HPLC (including the pumping, column, and ambient
conditions) is reflected in the injection accuracy, which is recorded as RSD (relative
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Chapter-1 INTRODUCTION

standard deviation) when the samples are analysed. Note that this does not account for
differences in sample preparation or manufacturing.

3. Relative retention (α)

α = K’1/K’2

The distance between two peaks may be quantified by analysing their relative
retention. As long as the resolution (Rs) is specified, this is not a required option.

4. Resolution (Rs)

Rs = (tR2-tR1)/(1/2)(tw1+tw2)

The degree of separation between two peaks is quantified in units of a parameter


called Rs. Peaks need to be evenly spaced for reliable quantification. This value is
extremely useful if the possibility of inference peaks (s) is a problem.

Tailing factor

T = Wx/2f

Increases in peak tailing reduce the accuracy of quantitation because the integrator has
a harder time identifying when the peak terminates and, thus, calculating the area
beneath the peak . The analyst has provided integrator variables to provide precise
"

determination of the region around the peak of interest. Integrator accuracy suffers if
it is uncertain when an upslope or downslope starts.

Theoretical plate number (N)

N = 16(tR/tw) 2 = L/H

A chromatograph's peak detection time is expressed as its theoretical plate number. N


is always the same in a chromatogram with the same number of peaks and the same
experimental procedures.

H- Height equivalent of a theoretical plate.

L- Length of column.

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