INTRODUCTION TO QUALITY BY DESIGN (QbD):

It was not too long ago that pharmaceutical and biopharmaceutical organizations only
recognized and employed Quality by Design (QbD) guidelines to manufacturing processes.
However, there has been a clear downstream trend to adopt QbD principles earlier in product
research and development efforts. A catalyst to facilitate continuous improvement, not only
to processes and product quality, but also productivity, QbD is now evolving as a means to
facilitate continuous improvement of the drug development process.
Quality by Design is a concept which was first described by Joseph M. Juran. Quality by
design (QbD) deals with getting to market reliably and knowing enough about the
limitations and risks associated with formulation, production and analytical methods in order
to establish appropriate mitigation and contingency plans. Quality by design (QbD)
principles have been used to advance product and process quality in every industry and most
recently been adopted by the U.S. Food and Drug Administration (FDA) (USFDA 2004,
ICH Quality Guideline Q8 (R2) 2009, ICH Quality Guideline Q2 (R1) 2005) as a vehicle for
how the drugs are discovered, developed, and commercially manufactured. Since first
initiated by the U.S. Food and Drug Administration (FDA) in its "Pharmaceutical cGMPs
for the twenty first century" , Quality by Design (QbD) has become an important concept
for the pharmaceutical industry that is further defined in the International Conference on
Harmonization (ICH) guidance on pharmaceutical development as "a systematic approach
to development that begins with predefined objectives and emphasizes product and
process understanding and process control, based on sound science and quality risk
management". The overarching goal of QbD is to embed quality into pharmaceutical
products to ultimately protect patient safety. ICH Q10 describes a holistic and integrated
quality management system applicable to the QbD environment. The scientific
understanding gained during the method development process can be used to devise method
control elements and to manage the risks identified. (2)
Quality by Design (QBD) is a philosophy that refines the level of knowledge associated with
a product that uses process understanding to deliver a product with the desired critical
quality attributes. Quality by design (QbD) became the answer to assist the industry as well

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as regulatory agencies to move toward a more scientific, risk-based, holistic and proactive
approach to pharmaceutical development.
One may define QbD for analytical methods as the compilation and evaluation of knowledge
from the method design stage throughout the methods lifecycle of use, that establish that a
method, when executed appropriately and consistently, delivers quality data.
From these definitions, it can be seen that there are a number of key factors that are
important in a Quality by design (QbD) /lifecycle approach. These include:

The importance of having predefined objectives

The need to understand the method, or being able to explain the method performance

as a function of the method input variables

The need to ensure that controls on method inputs are designed such that the method

will deliver quality data consistently in all intended environments in which it is used
The need to evaluate method performance from the method design stage throughout
its lifecycle of use.

Advantages

Easier method transfer.

Regulatory flexibility.
Highly robust methods.
Fewer or no methods specific out-of-specification situations when used in routine.(3)

Analytical methods play a key role in assuring that drug substances and drug products
conform to their specifications. Demonstrating that these methods consistently perform
appropriately for their intended purpose can be both challenging and resource intensive. By
using a pro-active, quality-by-design (QbD) approach, it is possible to design better
methods, understand the strengths, weaknesses and capabilities of those method and to
perform validation exercises in a scientifically rational way that will maximize success and
minimize overall resource consumption.
The application of Quality by design (QbD) principles to analytical method development is
focused on the concept of building quality into the method during development, as opposed
to testing methods after development for quality. QbD methodology is now actively
promoted and encouraged by regulatory pharmaceutical agencies both in manufacturing and
analytical development. From a QbD perspective, development should be carried out in a

Page | 2

systematic manner, beginning with predefined objectives, emphasizing understanding and

control and based on sound science and quality risk management. It has been emphasized
that analytical procedures are also processes, and that QbD could and should be
implemented for the development of analytical procedures, as they are non-negligible
components of the global pharmaceutical-product process.[19]
Traditional analytical method development involves typically selecting suitable method
parameters based on experience and knowledge and then check to ensure that we can
achieve the desired results by applying analytical method validation characteristics. Thus we
can ensure that the method can quantify at the levels required, that the results are always the
same and that they give the true value, etc. This approach does not result in a full
understanding of how the parameters of the method can affect the results. Applying Quality
by design (QbD) would involve moving these studies which provide an understanding of the
method to the beginning of the method development process instead of performing at the
end of method validation. It also includes an assessment of method variability compared
with the specification limits, which is one of the most important method attributes to test
when deciding whether the method is fit for its purpose. This means that the method
parameters would be chosen on the basis of these experiments and would be within a design
space of the method. The use of automation in these experiments would be desirable to
reduce the effort involved and analytical chemists would benefit from a good understanding
of the necessary statistics. The comparison of the traditional and quality by design approach
is shown in the Table no: 1.
Potential benefits in adopting the concept of QbD to analytical methods:

Facilitation of continuous improvement and technological innovation through more

advanced regulatory approaches to change management.

More effective use of industry and regulatory resources.
Enhanced method robustness and ruggedness throughout the method lifecycle.
Fewer investigations regarding the method performance.
More robust method knowledge transfer as a result of enhanced analytical method
understanding as well as improved knowledge management.

QUALITY RISK MANAGEMENT:

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Quality risk management is a systematic process for the assessment, control, communication
and review of risks to the quality of the drug (medicinal) product. It can be applied both
proactively and retrospectively. It is an important part of science-based decision making
which is essential for quality management of pharmaceutical manufacturing.
Principles of Quality risk Management:
The quality risk management system should ensure that:
-

The evaluation of the risk to quality is based on scientific knowledge, experience

with the process and ultimately links to the protection of the patient.
The level of effort, formality and documentation of the quality risk management
process is commensurate with the level of risk.

Figure 1: The scheme of a Quality Risk Management process as proposed by ICH Q9

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Risk assessment tools employed during design space evaluation help to identify where
variability in a factor, or failure in a part of system, represent a potential risk to the ability of
the method to deliver the design intent. Examples of tools utilized for risk assessment
include but are not limited to Fishbone diagram/Ishikawa diagram, Failure Mode Effect
Analysis (FMEA) and Prioritization Matrices.
Successful application of risk assessment includes robustness studies for high risk factors,
including the design of experiment (DOE) for assessing multidimensional combination and
interactions of the high risk factors.
In analytical drug development studies the application of quality risk management plays a
crucial role to help identify and analyze the risk against the given risk criteria.

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COMPARISON TABLE:
Comparison Of Traditional And Quality By Design Approaches To A Pharmaceutical
Process
S.
Aspect
Traditional approaches
QbD approaches
No

1.

2.

3.

4.

5.

Method
Developmen
t

Method
Validation

Method
Transfer

Method
Operation

Lifecycle
management

Material, product, and process

attributes to be measured are
based on standard guidance
(e.g., ICH Q6).Selection of
techniques
and
method
conditions using empirical
and univariate approaches.
One-time exercise based on
required
validation
characteristics in ICH Q2 and
pharmaceutical guidelines.

One time exercise using

limited numbers of operators
or
instruments
that
demonstrates the receiving
site can run the method.

Relies on general system

suitability checks to indicate
how well the method is
operating. Occasional method
failures and investigations
into method performance as
out-of-specification results.
Introduction
of
method
changes or improvements is
constrained by the costs of
regulatory actions. Methods
only changed if major issues
are identified (reactionary).

Material attributes to be measured

are based on structural risk
assessment. Method conditions are
selected based on a systematic and
multivariate strategy that is aligned
with the end-users (manufacturing
sites,
quality
control
testing
laboratories, etc.) preferences.
Starts with end in mind (i.e.,
predefined analytical target profile,
based on the intended use of the
method). Considers all variables that
have a potential to impact the
method performance criteria and
uses risk assessment tools to identify
potentially critical and critical
variables for study.
Considered as a change control
process where variables likely to
change as a consequence of
operating the method at the receiving
site are risk assessed, if appropriate,
included in a measurement systems
analysis study to assess conformance
to the analytical target profile.
Robust methods with system
suitability tests specifically derived
from a formal assessment of the risk
of method factor variation on the
method performance criteria.
Changes are made with reference to
the
accumulated
knowledge
generated
during
method
development,
validation,
and
technology transfer. Changes are
made without the need for the prior
regulatory approval, including the
introduction of more appropriate
technology as long as the analytical
Page | 6

target profile is met.

INTRODUCTION TO CHROMATOGRAPHY:
At the beginning of the twentieth century, the Russian botanist Mikhail Tswett invented and
named chromatography. He separated plant pigments by passing solution mixtures through a
glass column packed with fine particles of calcium carbonate. The separation of those
pigments appeared as colored bands on the column. Tswett named his separation method for
the two Greek words chroma and graphein, which mean color and to write,
respectively (Skoog et al., 1998).
Chromatographic separation techniques are multistage separation methods in which the
components of a sample are distributed between two phases, of which one is stationary
phase and the other is mobile. The stationary phase may be a solid or liquid supported o a
solid or a gel. The stationary phase may be packed in a column, spread as a layer, distributed
as a film, or applied by other techniques. The mobile phase may be a gaseous or liquid or a
supercritical fluid.

CHROMATOGRAPHY

Gas
chromatography

Gas-liquid

Gas-solid

Liquid
chromatography

Ion exchange

Supercritical fluid
chromatography

Exclusion

Partition
Chromatography

Liquid solid
Chromatography

Paper
Chromatography

Thin layer
Chromatography

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I)

GAS CHROMATOGRAPHY:

Gas chromatography (GC) is a separation technique in which the mobile phase is a gas. Gas
chromatography is always carried out in a column, which is typically "packed" or
"capillary". It is based on partition equilibrium of analyte between a solid stationary phase
(often a liquid silicone-based material) and a mobile gas (most often helium). The stationary
phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid
matrix inside a larger metal tube (a packed column).
II)

LIQUID CHROMATOGRAPHY:

Liquid Chromatography (LC) synonymous with high-pressure liquid chromatography and

high performance liquid chromatography (HPLC) is a separation technique based on a solid
stationary phase and a liquid mobile phase. It is further classified based upon:
1. MODES OF SEPARATION: (Beckett AH, 2007).There are two different modes of
separation in HPLC.
1.1 Normal phase mode: The stationary phase is polar and the mobile phase is nonpolar in
nature. In this technique, nonpolar compounds travel faster and are eluted first. This is
because of the lower affinity between the nonpolar compounds and the stationary phase.
Polar compounds are retained for longer times because of their higher affinity with the
stationary phase. These compounds, therefore take more times to elute. Normal phase mode
of separation is therefore, not generally used for pharmaceutical applications because most
of the drug molecules are polar in nature and hence take longer time to elute. The attractive
forces exits are dipole-dipole interaction & hydrogen bonding interactions.
1.2 Reversed phase: The stationary phase is nonpolar hydrophobic packing with octyl or
octadecyl functional group bonded to silica gel and the mobile phase is polar solvent. An
aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as
ionization control, ion suppression, ion pairing and complexation) to control retention and
selectivity. The polar compound gets eluted first in this mode and nonpolar compounds are
retained for longer time. As most of the drugs are polar in nature, they are not retained for
longer times and hence elute faster. The different columns used are octadecylsilane (ODS) or
Page | 8

C18, C8, C4, etc. the attractive forces which exist are mainly nonspecific hydrophobic
interactions.
2. TYPES BASED ON SEPARATION PRINCIPLES: (David Watson G, 2005)
2.1 Adsorption chromatography: In adsorption chromatography, the mobile phase
containing the dissolved solutes passes over the surface of the stationary phase. Retention of
the component and their consequent separation depends on the ability of the atoms on the
surface to remove the solutes from the mobile phase and adsorb them temporarily by means
of electrostatic forces. Usually silica or alumna is utilized as the adsorbent with relatively
non-polar solvents such as hexane as the mobile phase in normal phase adsorption whereas
in reversed phase adsorption non polymer beds with relative polar solvents such as water,
acetonitrile and methanol are used as mobile phase.
2.2 Partition chromatography: In partition chromatography an inert solid material such as
silica gel or diatomaceous earth serves to support a thin layer of liquid which is the effective
stationary phase. As the mobile phase containing the solutes passes in close proximity to this
liquid phase, retention and separation occurs due to the solubility of the analytes in the two
fluids as determined by their partition coefficients.
The method suffers from disadvantage due to some solubility of stationary phase in the
mobile phase. Hence precautions must be taken to limit dissolution of stationary phase.
2.3 Ion exchange chromatography: In ion exchange chromatography, the stationary phase
contains ionic groups like NR3+ or SO3- , which interact with the ionic groups of the sample
molecules. This is suitable for the separation of charged molecules only. Changing the pH
and salt concentration can modulate the retention. Ion pair chromatography may be used for
the separation of ionic compounds and this method can also substitute for ion exchange
chromatography.
2.4 Affinity chromatography: Affinity chromatography uses highly specific biochemical
interactions for separation. The stationary phase contains specific groups of molecules which
can adsorb the sample if certain steric and charge related conditions are satisfied. This

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technique can be used to isolate proteins, enzymes as well as antibodies from complex
mixtures.
2.5 Size exclusion chromatography: Size exclusion chromatography separates molecules
according to their molecular mass. Largest molecules are eluted first and the smallest
molecules last. This mode can be further subdivided into gel permeation chromatography
(with organic solvents) and gel filtration chromatography (with aqueous solvents).
4. ACCORDING TO THE TECHNIQUE (methods of holding the stationary phase)
4.1 Planar or Plane Chromatography
In this type of chromatography the stationary phase is used in the form of layer. Plane
chromatography is further classified into:

a)

Thin Layer Chromatography (TLC): The stationary phase in the form of fine
powder is spread on glass or plastic or aluminum sheets. Compared to paper, it has
the advantage of faster runs, better separations, and the choice between different

adsorbents.
b) Paper Chromatography: A specific type of papers is used as stationary phase in the
form of sheets. It is a technique that involves placing a small dot or line of sample
solution onto a strip of chromatography paper. The paper is placed in a jar containing
a shallow layer of solvent and sealed. As the solvent rises through the paper, it meets
the sample mixture, which starts to travel up the paper with the solvent. This paper is
made of cellulose, a polar substance, and the compounds within the mixture travel
farther if they are non-polar. More polar substances bond with the cellulose paper
more quickly, and therefore do not travel as far.
4.2 Columnar or Column Chromatography
The stationary phase is held in to a tube made of glass or metal. Silanized chromatographic
siliceous earth is used as a solid support for reverse-phase partition chromatography.
5. ACCORDING TO PURPOSE OF USE (www.chemagilent.com)

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5.1 Analytical The objective of an analytical HPLC run is the qualitative and quantitative
determination of a compound. Sample goes from detector into waste.
5.2 Preparative The objective of a preparative HPLC is isolation and purification of a
valuable product Sample goes from detector into fraction collector.
III) SUPERCRITICAL FLUID CHROMATOGRAPHY:
Supercritical fluid chromatography is a separation technique in which the mobile phase is a
fluid above and relatively close to its critical temperature and pressure.
INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
High performance Liquid Chromatography (HPLC) is the most common analytical
separation tool and is used in many aspects of drug manufacture and research.
HPLC is particularly suitable for the separation of compounds having one or more of the
following characteristics: a) high polarity b) high molecular weight c) thermal instability d)
tendency to ionize in solution. [www.chemistry.sjsu.edu]

Figure 1: HPLC instrument

There are six basic components of an HPLC:
a. Solvent system

Page | 11

b.
c.
d.
e.
f.

Pumping system
Injection system
Column (Separating system)
Detecting system
Data collecting system

a. Solvent system:
Mobile phase (solvent) reservoirs for analytical separations is that the solvent reservoir
should be ideally about 2 litres capacity, and is usually constructed from glass. Before
solvent is used, however it should be degassed. Degassing is required to remove dissolved
gases (in particular oxygen), which if not removed will produce degassing in the detector
which will produce either base-line drift or continuous spikes on the chromatographic trace.
Before solvent is used it should also be filtered to remove any particulate matter which
would cause wear to the pumping system. [24]
b. Pumping system:
The development of suitable pumping systems has been one of the main factors in the
growth of modern liquid chromatography. Two types of elution are required, namely: (i)
isocratic, in which the solvent composition does not change during an analysis (ii) gradient
elution, during which continuous changes in composition are made at a controlled rate
during the analysis.
There are two basic types of pump in common use: constant pressure and constant volume
pumps. Constant pressure pumps are of two main types:
(i)
(ii)

coil pumps, in which inert gas at high pressure drives eluent out of a narrow tube, and
air-driven pressure intensifiers (amplifiers) in which moderate air pressure drives a
large area piston connected rigidly to a much smaller area piston bearing on the eluent.
Consequently constant pressure pumps have been superseded by constant volume pumps
in virtually all modern HPLC systems.
Constant volume pump: They are of two types:

(i)
(ii)

Constant displacement (syringe-type pumps) and

Reciprocating pumps - the latter being the most universally employed.
c. Sample injection system:

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For maximum efficiency on a chromatographic column, the sample should be introduced

ideally in an infinitely narrow band. Valve-loop injectors are the most widely used sample
introduction system and are designed to operate at pressures in excess of 3.5x10 7N m-2,
without the use of septa. The solvent flow is bypassed into the column and an external loop
is filled with sample, which is then introduced into the column by switching a valve.
Auto samplers are currently employed in most industrial analytical laboratories for rapid
unattended analysis of samples. They can be used as stand-alone LC automation units or can
become part of a remote controlled system. The sample injection valve is usually
pneumatically activated, and provides excellent injector reproducibility. Since these systems
can be operated 24 hours a day (with the aid of a time switch), the savings in time and
manpower soon outweigh the cost of their installation. [25]
d. Column (Separating system)
The heart of the system is the column where separation occurs. They are commonly filled
with a stationary phase with a particle size of 5 - 10m.
Types of columns in HPLC:
1

Analytical(internal diameter 1.0 -4.6-mm; lengths 15 250 mm)

Preparative ( internal diameter > 4.6 mm; lengths 50 250 mm)

Capillary (internal diameter 0.1 -1.0 mm; various lengths)

Nano (internal diameter < 0.1 mm or sometimes stated as < 100 m)

Materials of construction for the tubing:
1

Stainless steel (the most popular; gives high pressure capabilities)

Glass (mostly for biomolecules)

PEEK- polymer (biocompatible and chemically inert to most solvents)

The column is usually made up of heavy glass or stainless steel tubing to withstand high
pressure. The columns are usually 10-30 cm long .Columns with an internal diameter of 5
mm gives good results because of compromise between efficiency, sample capacity, and the
amount of packing and solvent required.

Page | 13

Column packing (Stationary phase): The packing used in modern HPLC consists of small,
rigid particles having a narrow particle size distribution.
There are three main types of column packing in HPLC.
1. Porous, polymeric beds: Porous, polymeric beds based on styrene divinyl benzene copolymers used doe ion exchange and size exclusion chromatography.
2. Porous layer beds: Consisting of a thin shell (1-3 m) of silica or modified silica on a
spherical inert core (e.g. Glass).
3. Totally Porous silica particles (diameter <10 m): These packing have widely been used
for analytical HPLC in recent years. Particles of diameter >20 m are usually dry packed.
While particles of diameter <20 m are slurry packed in which particles are suspended on a
suitable solvent and the slurry so obtained is driven into the column under pressure.
e. Detecting system:
The function of the detector in HPLC is to monitor the mobile phase as it merges from the
column. Detection of the eluting components is important, and this can be either selective or
universal, depending upon the detector used. The response of the detector to each
component is displayed on a chart recorder or computer screen and is known as a
chromatogram.
An ideal detector should have the following properties:
Low drift and noise level (particularly crucial in trace analysis)
High sensitivity and fast response
Wide linear dynamic range (this simplifies quantitation).
Low dead volume (minimal peak broadening)
Insensitivity to changes in type of solvent, flow rate, and temperature
Operational simplicity, reliability and non-destructive.
Detectors are usually of two types:

Page | 14

Bulk property detectors: It compares overall changes in a physical property of the

mobile phase with and without an eluting solute. e.g. refractive index, dielectric

constant or density.
Solute property detectors: It responds to a physical property of the solute which is
not exhibited by the pure mobile phase. E.g. UV absorbance, Fluorescence and
Electrochemical detectors. Such detectors are about 1000 times more sensitive giving
a detection signal for a few nanograms of sample.

The commonly employed detectors in the HPLC analysis study include:

Spectroscopic Detection: Ultraviolet (UV) Absorption:
An ultraviolet light beam is directed through a flow cell and a sensor measures the light
passing through the cell. If a compound elutes from the column that absorbs this light
energy, it will change the amount of light energy falling on the sensor. The resulting change
in this electrical signal is amplified and directed to a recorder or data system. A UV
spectrum is sometimes also obtained which may aid in the identification of a compound or
series of compounds.
Photo Diode Array Detector:
The PDA Photodiode Array Detector is an optical detector capable of measuring the
absorbance spectrum from 190 nm to 800 nm. A deuterium lamp optimizes the UV range
(190 nm to 380 nm) and a tungsten lamp optimizes the visible range (380 nm to 800 nm). In
this detector light from an UV source passes through the flow cell into a polychromator
which disperses the beam so that full spectrum falls on the array of diodes. Each diode
detects light at a discrete wavelength and the array provides an almost instantaneous
absorption spectrum of the solute in the eluate. Monochromatic variable wavelength
detectors monitor eluting components of the sample at a single wavelength (ideally, the
wavelength of maximum absorbance), whereas photodiode array (PDA) detectors scan a
range of wavelengths every few milliseconds and continually generate spectral information.
PDA detectors provide three-dimensional information that allows an accurate assessment of
peak identity, purity, and quantitation in a single run.

Page | 15

Figure 2: Diode Array Detector

Vis
Lamp

UV
Lamp

Achromatic
Lens
Detector Diode Array
Flow Cell
Homium
Filter
Optical
Slit

Grating

f. Data collecting system:

Signals from the detector may be collected on chart recorders or electronic integrators that
vary in complexity and in their ability to process, store, and reprocess chromatographic data.
The data storage capacity of these devices is usually limited.

Analytical Method Development under Quality by Design (QbD)

paradigm

Page | 16

Quality by Design (QbD) when used in analytical laboratory during the development
process is all about building quality into the methods used by focusing on the goal of
reducing variation and producing consistent results through formalized experimentation.
A Stepwise Strategy to Quality by Design Method Development
FDA encourages a systematic science and risk based approach in pharmaceutical
development, including analytical development.

Target measurement
Define
(ATP)
Method
Intent
Select a technique

Risk assessment
Method
Design experiment to
Understanding
define method
Control strategy
Lifecycle
Manage risks for longManagement
term performance

Figure: Stepwise strategy of QbD method development

I)

Method Intent:

Page | 17

Fundamental to any method development is being clear about the method intent i.e., the
criteria that must be met. It includes establishing the method performance requirements,
developing a method that will meet these requirements and then performing appropriate
studies to understand the critical method variables that must be controlled to assure that the
method is robust and rugged. It is an iterative process which is repeated as required in
accordance with the lifecycle phase of the product.
Analytical Target Profile (ATP):
Utilizing the QbD approach, the first step is to define the intended purpose of the method.
This has been called the Analytical Target Profile (ATP), which is similar to the Quality
Target Product Profile (QTPP) for pharmaceutical process. This ATP will be the set of
criteria that defines what will be measured, in which matrix, over what concentration range,
and the required performance criteria of the method, together with the specifications. To
build the ATP, it is necessary to define the characteristics that will be indicators of method
performance. Typically these characteristics will be subset to those defined in ICH Q2, such
as accuracy, precision. It is this ATP that would drive the design and development of
appropriate analytical methods and it is proposed that the ATP could also form the basis of a
regulatory submission.
The establishment of an ATP is in complete alignment with ICH Q10, that is, the application
of a holistic quality-management system. Although additional information is gained when
using ATP combined with a QbD approach, the ATP can also facilitate the establishment of
some of the recommendations of the ICH Q10 when used with a traditional approach. The
application of the ATP provides confidence that future method changes and improvements
may be introduces with full knowledge of their likely effect on the product.
Critical Quality Attributes:
The first step is to define the CQAs of the analytical method that have to be included into
the ATP. These CQAs are the responses to be measured to judge the quality of the developed
analytical methods. CQAs are defined as a physical, chemical, biological, or
microbiological property or characteristic that should be within an appropriate limit, range,
or distribution to ensure the desired product quality. For separative analytical methods (e.g.
chromatography), the CQAs can be related to the method selectivity (e.g. resolution

Page | 18

criteria). Additionally CQAs can be the run time of the analysis, the precision of the
analytical method, the dosing range of the analytical method, etc.
Method Selection:
The method which can meet the defined method objectives and goal is selected utilizing the
principles of Quality Risk Management (QRM). Various risk assessment tools can be used in
selecting an analytical method like the fishbone analysis/Ishikawa diagram, Failure Effects
and Mode analysis (FMEA), Hazard Analysis and Critical Control Point (HACCP) etc. The
appropriate method for the drug is chosen from the possible methods available in the
literature. Using this information as input, a risk assessment can begin. One of the most
common ways to perform a structured risk assessment is to use a Fishbone diagram, also
known as an Ishikawa or cause-and-effect diagram, to identify potential factors that may
influence the method performance. Fishbone diagrams segregate risks into different
categories,

such

as

those

associated

with

instrumentation,

materials,

methods,

measurements, laboratory climate, and human factors. After risks have been assessed, they
are typically grouped into three categories: high-risk factors that should be tightly
controlled, potential noise factors, and factors that can be probed experimentally to
determine acceptable ranges. Typical high risk factors that can be fixed at the time of
method development include data analysis methods and sample preparation methods. The
third category of risks identified from the Fishbone analysis contains instrumental
parameters that can be prioritized and probed using the Design of Experiments approach
(DoE).
Method Understanding:
Adequate method understanding can be achieved from risk assessment and focus on the
concept of design space applied to analytical methods.
Risk assessment:
With the method goal in hand, method scouting can commence, typically with the use of
detailed flowcharts and decision trees to aid the analyst in deciding among the options.
Although it is not possible to capture all knowledge about a technique in such a format, it is
possible to capture many important points frequently encountered during method
development for many of the techniques of interest, such as HPLC. The risk assessment

Page | 19

involves the identification of risks in a structured fashion, followed by ruggedness and

robustness testing. As previously noted, ICH Q8 and Q9 require a risk-based approach as a
key component of QbD (Quality by Design). To implement this guidance for analytical
methods, structured methodologies for risk assessment can be used. These include tools such
as Fishbone diagrams which help identify potential risks to the method parameters, and
changes that might occur between laboratories, analysts, instruments, reagents, and the
period of time in which the method is performed.
Finally, it should be noted that if the final method is selected against well-chosen desirable
method attributes, it is likely that the selected method will be reliable. This consideration
also factors into the risk assessment for a method, because it can limit the amount of work
necessary to demonstrate ruggedness and robustness and the number of controls that must be
fixed.
Design of Experiments (DOE):
A key component of the development of analytical procedure using QbD is what has been
called the Design Space (DS). The analytical Design Space is finally a multivariate domain
of input factors ensuring that critically chosen responses are included within the predefined
limits with an acceptable level of probability.
The experiments are typically performed on parametric variables using Design of
Experiments to ensure that maximum understanding is gained while minimizing the total
number of experiments. Analytical methods need to be robust so that they can be used
routinely without problems and can be easily transferred for use in another laboratory if
necessary.
Control strategy:
ICH guidelines Q8, Q9, Q10 and Q11 and the reflections around their concrete
implementation reinforced the need for improvement in Control Strategy. The parent
guideline on Pharmaceutical Development ICH Q8 (R2) introduced the concept of Control
Strategy and its link with the control of product and process critical attributes. This guideline
explains that the process control strategies that provide process adjustment capabilities to
ensure control of all the critical attributes should be described. The ICH Q10 guideline goes

Page | 20

further in the principles of this concept, defining the Control Strategy as a planned set of
controls, derived from current product and process understanding that ensures process
performance and product quality.
A control strategy is designed to ensure consistent product quality. The control strategy is an
important QbD feature of an analytical method, because it assures that the method is
performing as intended on a routine basis. Using the appropriate risk assessment tools, the
critical factors and their acceptable ranges (from the risk assessment or experimental work)
are explicitly defined in the method and considered when implementing a method control
strategy. If the risk assessment activities indicate that the overall understanding of method
performance can be improved, and the risk to obtaining reliable data is high and difficult to
manage, a more appropriate method may be needed. If the risks are low and well managed,
then the method control strategy can be defined, which generally consists of appropriate
system suitability criteria to manage risk and ensure the method delivers the desirable
method attributes. An appropriate system suitability test may be the only control element
needed to ensure performance of the selected method. System suitability tests are an integral
part of chromatographic methods and are used to ensure the performance of the analytical
system. Additionally, system suitability tests verify that the resolution and reproducibility of
the chromatographic system are adequate for the analysis. The risk assessment can help
identify a specific control strategy.
Validation and Post Method Development Considerations:
Method validation is a key activity that traditionally occurs after method development. The
validation exercise is typically a separate activity, removed from development, which occurs
only once. Once a QbD analytical method is developed and assessed for risk, resulting in
appropriate definition and control of method parameters, formal method validation can
commence. Although validation still follows ICH Q2 guidance, a proper method
development and risk assessment process for the methods makes validation a formality.
Because the method has been thoroughly developed and evaluated, issues with the method
are unlikely to be discovered during validation.

Page | 21

Because of the large amount of knowledge accumulated during analytical QbD method
development, there is a general need for repositories that can maintain this knowledge for
future use. Throughout the lifecycle of the product, this repository can be maintained and
updated by the R&D and QC organizations. This repository allows for potential changes to
the method to be considered in light of method scouting and knowledge, and the previous
risk assessment.
Product lifecycle Management and Continual Improvement:
Throughout the product lifecycle, companies have opportunities to evaluate innovative
approaches to improve product quality. (ICH Q10)Process performance can be monitored to
ensure that it is working as anticipated to deliver product quality attributes as predicted by
the deign space.
Applying the principles of QbD to analytical method development, and establishing a
control strategy are merely starting points for the real challenge: managing and updating
methods as the process

evolves to assure they perform as intended throughout the

development and manufacturing lifecycles.

Page | 22

Figure 1: Components of application of Quality by Design (QbD) to Analytical Methods

METHOD VALIDATION:
Method Validation: Validation of an analytical procedure is the process used to confirm
that the analytical procedure employed for a specific test is suitable for its intended use.
According to ICH Q2 (R1), the typical validation characteristics which are to be considered:
1. Specificity
2. Accuracy
3. Precision
Repeatability
Intermediate Precision
4. Detection limit
5. Quantitation limit
6. Linearity
7. Range
8. Robustness
9. System suitability
Type of analytical

Identificatio

Testing for impurities

Assay

Page | 23

Limit

Dissolution
Content/potenc
y

Intermediate Precision

Specificity

Detection Limit

Quantitation Limit

Linearity

Range

procedure
Characteristics

Accuracy

Repeatability

Quantitatio
n

Precision

- signifies that this characteristic is not normally evaluated

+ signifies that this characteristic is normally evaluated
1. Specificity
Specificity is the ability to assess unequivocally the analyte in the presence of components
which may be expected to be present. Typically these might include impurities, degradants,
matrix, etc.
2. Accuracy
Page | 24

The accuracy of an analytical procedure 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.
Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3
concentration levels covering the specified range (e.g., 3 concentrations /3 replicates each of
the total analytical procedure).
3. Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of
scatter) between a series of measurements obtained from multiple sampling of the same
homogeneous sample under the prescribed conditions. Precision may be considered at three
levels: repeatability, intermediate precision and reproducibility.
The precision of an analytical procedure is usually expressed as the variance, standard
deviation or coefficient of variation of a series of measurements.
Repeatability
Repeatability expresses the precision under the same operating conditions over a short
interval of time. Repeatability is also termed intra-assay precision.
A minimum of 9 determinations covering the specified range for the procedure (e.g., 3
concentrations/3 replicates each); or b) A minimum of 6 determinations at 100% of the test
concentration.

Intermediate precision
Intermediate precision expresses within-laboratories variations: different days, different
analysts, different equipment, etc.
Reproducibility

Page | 25

Reproducibility expresses the precision between laboratories (collaborative studies, usually

applied to standardization of methodology). Reproducibility is assessed by means of an
inter-laboratory trial. Reproducibility should be considered in case of the standardization of
an analytical procedure, for instance, for inclusion of procedures in pharmacopoeias. To
validate this characteristic, similar studies need to be performed at different laboratories
using the same homogeneous sample lot and the same experimental design.
4. Detection Limit (LOD)
It is the lowest amount of analyte in a sample that can be detected but not necessarily
quantitated under the stated experimental conditions.
Visual evaluation may be used for non-instrumental and instrumental methods. In this case,
the DL is determined by the analysis of a series of samples with known concentrations and
establishing the minimum level at which the analyte can be reliably detected. For
instrumental procedures that exhibit background noise, it is common to compare measured
signals from samples with known low concentrations of analyte with those of the blank
samples. The minimum concentration at which the analyte can reliably be detected is
established using an acceptable signal - to - noise ratio of 2 : 1 or 3 : 1.
5. Quantitation Limit (LOQ)
The quantitation limit of an individual analytical procedure is the lowest amount of analyte
in a sample which can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays for low levels of compounds in
sample matrices, and is used particularly for the determination of impurities and/or
degradation products.
6. Linearity
The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the
sample.

Page | 26

To determine linearity, prepare a stock solution of high concentration. Linearity is then

demonstrated directly by dilution of the standard stock solution. Linearity is best evaluated
by visual inspection of a plot of the signals as a function of analyte concentration. At least
five concentration levels should be used. Under normal circumstances, linearity is
acceptable with a coefficient of determination (r2) of 0.997. Data from regression line itself
may be helpful to provide mathematical estimate of degree of linearity. The slope, residual
sum of squares, and y intercept should also be reported.
7. Range
The range of an analytical procedure is the interval between the upper and lower
concentration (amounts) of analyte in the sample (including these concentrations) for which
it has been demonstrated that the analytical procedure has a suitable level of precision,
accuracy and linearity. The following minimum specified ranges should be considered:
For the assay of a drug substance or a finished (drug) product: normally from 80 to 120
percent of the test concentration; For content uniformity, covering a minimum of 70 to 130
percent of the test concentration, unless a wider more appropriate range, based on the nature
of the dosage form (e.g., metered dose inhalers), is justified;
For dissolution testing: +/-20 % over the specified range.
8. Robustness
The robustness of an analytical procedure 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. In the 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,
Flow rate.
9. System Suitability:
System suitability tests are an integral part of gas and liquid chromatographic methods.
These tests are used to verify that the chromatographic system is adequate for the intended

Page | 27

analysis. The tests are based on the concept that the equipment, electronics, analytical
operations, and samples analyzed constitute an integral system that can be evaluated as such.
Parameter

Recommendation

Capacity Factor (k)

k > 2.0

Repeatability

RSD < 1 % for N > 5 is desirable.

Resolution (Rs)

Rs of > 2

Tailing Factor (T)

T of 2

Theoretical Plates (N)

In general should be > 2000.

Plate number or number of theoretical plates (N)

This is a measure of the sharpness of the peaks and therefore the efficiency of the Column.
This can be calculated in various ways, for example the USP uses the peak width at the base
and the BP at half the height. Higher the plate number, more efficient is the column.

Fig 11: Theoretical plates

Wh = peak width at 1/2 peak height

Wb = peak width at base

t = retention time of peak

Peak Resolution (R)

Page | 28

This is not only a measure of the separation between two peaks, but also the efficiency of the
column. It is expressed as the ratio of the distance between the two peaks.

Fig 12: Peak Resolution

Separation Factor (relative retention)
This describes the relative position of two adjacent peaks. Ideally, it is calculated using the
capacity factor because the peaks' separation depends on the components' interaction with
the stationary phase.

Fig 13: Separation Factor

Symmetry Factor (AS):
The symmetry factor (also known as the tailing factor) of a peak is calculated by:
AS = W0.05/2f

Page | 29

Where W0.05 is the width of the peak at 5% height and f is the distance from the peak
maximum to the leading edge of the peak, the distance being measured at a point 5% of the
peak height from the baseline.

Fig 14: Symmetry Factor

Capacity Factor (k): Capacity factor, k, is defined as the ratio of the number of molecules
of solute in the stationary phase to the number of molecules of the same in the mobile phase.

.
Fig 15: Capacity Factor
Where, tR = retention volume at the apex of the peak (solute) and
t0 = void volume of the system.

SYSTEM SUITABILITY ALLOWABLE VARIATIONS

Page | 30

pH of Mobile Phase (HPLC):The pH of the aqueous buffer used in the preparation of the
mobile phase can be adjusted to within 0.2 units of the value or range specified.
Concentration of Salts in Buffer (HPLC): The concentration of the salts used in the
preparation of the aqueous buffer employed in the mobile phase can be adjusted to within
10% if the permitted pH variation (see above) is met.
Ratio of Components in Mobile Phase (HPLC): The following adjustment limits apply to
minor components of the mobile phase (specified at 50% or less). The amounts of these
components can be adjusted by 30% relative. However, the change in any component
cannot exceed 10% absolute (i.e., in relation to the total mobile phase).
Wavelength of UV-Visible Detector (HPLC): Deviations from the wavelengths specified
in the procedure are not permitted. The procedure specified by the detector manufacturer, or
another validated procedure, is used to verify that error in the detector wavelength is, at
most, 3 nm.
Column length (GC, HPLC): Can be adjusted by as much as 70%.
Column Inner diameter (HPLC): Can be adjusted if the linear velocity is kept constant.
Particle Size (HPLC): The particle size can be reduced by as much as 50%, but cannot be
increased.
Flow Rate (HPLC): When column dimensions have been modified, the flow rate can be
adjusted using:

Where F1 is the flow rate indicated in the monograph, in mL/min; F2 is the adjusted flow
rate, in mL/min; l1 is the length of the column indicated in the monograph; l 2 is the length of
the column used; d1 is the column inner diameter indicated in the monograph; and d 2 is the
internal diameter of the column used. Additionally, the flow rate can be adjusted by 50%.

Page | 31

Injection Volume (HPLC): The injection volume can be reduced as far as is consistent with
accepted precision and detection limits; no increase is permitted.
Column Temperature (HPLC): The column temperature can be adjusted by as much as
10 C. Column thermo stating is recommended to improve control and reproducibility of
retention time.

DRUG PROFILE

Page | 32

Generic Name

Cefuroxime sodium

Brand Name

Zinacef

Chemical Structure

IUPAC Name

Sodium salt of (6R, 7R)-3-carbamoyloxymethyl-7[Z-2-methoxyimino-2-(fur-2-yl)acetamido]ceph-3em-4-carboxylate

Molecular Formula

C16H15N4NO8S Na

Molecular Weight

446.40

Description

It is a white to slightly yellowish powder.

pH value

pH of freshly constituted solutions usually ranges

from 6 to 8.5.

Melting Point

240C -245C

pKa

2.5 (water), 5.1 (DMF)

Categories

Anti-Bacterial Agents , Cephalosporin

Solubility

Soluble in DMSO, Methanol and Water.

Stability

Stable under ordinary conditions.

Incompatibilities

Cefuroxime sodium is reported to be clinically

incompatible with amino glycosides.

Decomposition Products

During thermal decomposition, it may be possible

to generate irritating vapors and/or toxic fumes of
carbon oxides (COx), nitrogen oxides (NOx),

Page | 33

sodium oxides (Na2O), and sulfur oxides (SOx).

medicaldrugstudy.info/cefuroxime-drug-study

MECHANISM OF ACTION:
Cefuroxime is a second-generation bactericidal cephalosporin antibiotic which is resistant to
most -lactamases and is active against a wide range of Gram-positive and Gram-negative
organisms. It is indicated for the treatment of infections that inhibits cell-wall synthesis,
promoting osmotic instability; usually bactericidal before the infecting organism has been
identified or when caused by sensitive bacteria.
PHARMACOKINETICS:
Peak levels of Cefuroxime are achieved within 30 to 45 minutes after intramuscular
administration. The serum half-life after either intramuscular or intravenous injection is
approximately 70 min. In the first weeks of life the serum half-life of Cefuroxime can be 3 5 times that in the adult. Cefuroxime is not metabolized and is excreted by glomerular
filtration and tubular secretion. Serum levels of Cefuroxime are reduced by dialysis.
Cefuroxime passes the blood-brain barrier when the meninges are inflamed.
ADVERSE REACTIONS:
Cardiovascular
GI

track:

Hematologic:

system:
pseudomembranous

phlebitis
colitis,

nausea,

and
anorexia,

thrombophlebitis
vomiting,

diarrhea

transient neutropenia, eosinophilia, hemolytic anemia, thrombocytopenia

Skin: maculopapular and erythematous rashes, urticaria, pain, induration, sterile abscesses,
temperature elevation, tissue sloughing at intramuscular injection site, hypersensitivity
reactions, serum sickness, anaphylaxis.
INDICATIONS AND USAGE:
Cefuroxime for Injection is indicated for the treatment of patients with infections caused by
susceptible strains of the designated organisms in the following diseases:

Page | 34

a) Lower Respiratory Tract Infections, including pneumonia, caused by Streptococcus

pneumoniae, Haemophilus influenzae (including ampicillin-resistant strains), Klebsiella
spp.,

Staphylococcus aureus (penicillinase- and non-penicillinase-producing

strains), Streptococcus pyogenes, and Escherichia coli.

b) Urinary Tract Infections caused by Escherichia coli and Klebsiella spp.
c) Skin and Skin-Structure Infections caused by Staphylococcus aureus (penicillinaseand

non-penicillinase-producing

strains), Streptococcus

pyogenes,Escherichia

coli, Klebsiella spp., and Enterobacter spp.

DOSAGE AND ADMINISTRATION:
Adults
Many infections respond to 750mg three times daily by IM or IV injection. For more severe
infections the dose should be increased to 1.5g three times daily IV. The frequency of
administration may be increased to 6-hourly if necessary, giving total daily doses of 3 to 6g.
Infants and Children
30 - 100mg/kg/day given as 3 or 4 divided doses. A dose of 60mg/kg/day is appropriate for
most infections.
Neonates
30 - 100mg/kg/day given as 2 or 3 divided doses.
AVAILABLE FORMS:
Cefuroxime sodium Infusion 750 mg, 1.5 g premixed, frozen solution
Cefuroxime sodium for Injection 750 mg, 1.5g.
DRUG INFORMATION:
Name

: Cefuroxime sodium

Formulation

: Powder for Injection

Strength

: 1.5g

Route of administration

: Intravenous (IV)

Literature Review

1.

R. Kitzes Cohen et al. developed an HPLC method for the determination of

Cefuroxime in human plasma. The method uses solid phase extraction technique (SPE)

Page | 35

and has acceptable sensitivity, precision and accuracy. The limit of quantification in
plasma cells is 0.1g/mL. Calibration curves were linear within 0.1-20 g/mL, with mean
correlation coefficient of 0.9982. Mean inter-day precision and accuracy were 7.8% and
6.4% respectively. The method was applied to determine Cefuroxime levels in patients
receiving Cefuroxime, 3 times a day.

2.

A. B. Devkhile et al. described a simple, accurate, precise and cost effective UV-Vis

Spectrophotometric method for the estimation of Cefuroxime sodium, a second

generation cephalosporin antibiotic in dry powder injection and drug substances. The
solvent used throughout the experiment was distilled water. The max or the absorption
maxima of the drug was found at 275 nm. Beers law obeyed in the range of 2.0-16.0
g/ml. The developed method was successfully validated with respect to linearity,
accuracy and precision. The sample concentrations are measured on weight basis
throughout the experiment in UV-Vis Spectrophotometric method. The method was
validated and shown linear in the mentioned concentrations. The correlation coefficient
for Cefuroxime sodium was 0.9999. The recovery values for Cefuroxime sodium ranged
from 100.0-100.3. The relative standard deviation of six replicates of assay was less than
2 %. The percent relative standard deviation of inter-day precision ranged 1.0-1.4 % and
intra-day precision 1.2-1.6 % of Cefuroxime sodium. The limit of detection and limit of
quantification of Cefuroxime sodium was 0.11g/mL and 0.39g/mL. The developed
method was cross checked with high performance liquid chromatography for six replicate
assays as per USP 32, the mean assay was 100.3% and %RSD was 0.35%. The
degradation study of Cefuroxime sodium was studied for the purpose of stability
indicating method. Hence proposed method was precise, accurate and cost effective. This
method can be applicable for the quantitative determination of the titled drug with respect
to assay from their new commercial formulation of injection in quality control
laboratories.

3.

H.R.N. Salgado et al. described the development and evaluation of a HPLC and UV

spectrophotometric methods to quantify Cefuroxime sodium in injectables. HPLC

analysis were carried out using a C18 Water 054275 column and a mobile phase

Page | 36

composed of methanol and water (70:30), with a flow rate of 0.8 mL/min and UV
detection at 280 nm. For the spectrophotometric analysis, water was used as solvent and
the wavelength of 280 nm was selected for the detection. Both methods were found to
quantify Cefuroxime sodium in injectables accurately. Therefore HPLC and UV methods
presented the most reliable results for the analyses of injectables.

4.

Ingrid Nilsson Ehle et al. described a procedure for determining Cefuroxime

concentrations in serum by using high performance liquid chromatography. The drug is

extracted from serum with dimethylformamide and separated from other substances in the
extract by reverse phase chromatography. The ultraviolet (280nm) absorption of the
effluent was monitored and quantitated based on the height of the Cefuroxime peak. Inter
and Intra- assay ambiguity was less than 3.1 and 4.3% respectively. Serum concentrations
as low as 1.0mg/L cold be accurately measured. No interference from other drugs and
antibiotics was found. A biological half life of 52min in serum was observed after
intravenous injection of the drug into a human volunteer.

5.

F.M. Patel et al. described a spectrophotometeric method for the simultaneous

estimation of Cefuroxime sodium and Sulbactam sodium injection. Cefuroxime is a 2ndgeneration cephalosporin and Sulbactam is a -Lactamase inhibitor. The combination
formulation is used for the treatment of lower respiratory tract infection. Two new,
simple, accurate and precise UV spectrophotometric methods have been developed and
validated for the simultaneous determination of Cefuroxime Sodium (CEF) and
Sulbactam Sodium (SUL) in their combined dosage forms. First method is based on
simultaneous estimation of Cefuroxime at 279nm and Sulbactam at 259 nm, while other
Qabsorption Ratio method using two wavelengths, 259nm (max of SUL) and 272nm
(Isoabsorptive point). 0.01 N NaOH was the solvent used in all methods. Cefuroxime
Sodium showed linearity in the range of 8-32g/mL and Sulbactam sodium showed
linearity in the range of 4-16g/mL in all the methods. All methods were validated
statistically and recovery studies were carried out. All methods were found to be accurate,
precise and reproducible. These methods were applied to the assay of the drugs in
marketed formulation, which were found in the range of 98.0% to 100.0% of the labelled

Page | 37

value for both Cefuroxime and Sulbactam. Hence, the methods herein described can be
successfully applied in quality control of combined pharmaceutical dosage forms.

6.

P Santhosh Kumar et al. described a rapid and reproducible High Performance

Liquid Chromatographic method for the estimation of Cefuroxime axetil CFA in its pure
form as well as in pharmaceutical dosage forms. Chromatography was carried out on an
ODS C18column (150 x 4.6 mm x 5 m length), using a mixture of methanol and 0.01M
potassium dihydrogen orthophosphate buffer (pH-2.00.05) (60:40 v/v) as the mobile
phase at a flow rate of 0.8 mL/min and the detection was done at 248 nm was developed
and fully validated for the determination of CFA. The retention time of the drug was
3.693 min. The method produced linear responses in the concentration range of 0.45 to 80
g/mL of CFA. Developed HPLC method was sensitive with LOD= 0.26 gmL1 and
LOQ= 0.58 gmL1. The method was successfully validated in accordance to ICH
guidelines and was found to be reproducible for analysis of the drug in parental
preparations.

7.

Corina-Ciresica Mondea et al. described a simple, rapid and

sensitive LC/MS/MS method for quantification of Cefuroxime sodium in

cattle plasma involving development and validation study. The analyte
was extracted from plasma samples using protein precipitation with a
mixture of perchloric acid 12% in acetonitrile. Separation was achieved
using a Zorbax SB-C18 column using a mobile phase containing
methanol: ammonium acetate 1mM in water (14:86 v/v). The detection
was realized in MS-MRM mode using an Ion Trap mass spectrometer
with electrospray negative ionization. The linearity domain was
established between 63 - 6120 ng/mL. Accuracy and precision were
less than -4.9% and 9.3% for intra-day assays and - 6.9% and 9.3% for
inter-day assays, respectively. The recovery ranged between 95 and
105%. The method is very simple and rapid and was used for analysis
of Cefuroxime sodium in cattle plasma for pharmacokinetics and drug
residue analysis.

Page | 38

8.

Devesh A. Bhatt et al. described the development of a comprehensive science and

risk based HPLC method and subsequent validation for the analysis of zidovudine active
pharmaceutical ingredient (API) using a quality by design approach. An efficient
experimental design based on systematic scouting of all three key components of the RP
HPLC method (column, pH and mobile phase) is presented. The described method was
linear. (r(2)=0.9998). The precision, ruggedness and robustness values were also within
the prescribed limits (<1% for system precision and <2% for other parameters).
Chromatographic peak purity results indicated the absence of coeluting peaks with the
main peak of zidovudine.The proposed method can be used for routine analysis of
zidovudine in quality control laboratories.

9.

Cleber A. Schmidt et al. described the development and validation of a simple,

sensitive, accurate and reproducible agar diffusion method to quantify CFU sodium in
injectable formulations. The assay is based on inhibitory effect of CFU upon the strain
Staphylococcus aureus ATCC 6538P used as test organism. The results were treated
statistically by analysis of variance and was found to be linear (r = 0.9998) in the selected
range of 8.0-32.0g/mL; precise [repeatability: RSD = 1.56%; intermediate precision:
between-day RSD= 1.27%; between analyst RSD= 1.13%] and accurate (101.58%). The
bioassay specificity was studied by evaluation of degraded sample at 50C with analysis
at 0,24 and 48h in parallel with pharmacopoeial liquid chromatography method for CFU.

10.

Kanakapura Basavaiah et al. described the development of an integrated

multivariate approach to develop and quantify the constituent concentrations of

pioglitazone hydrochloride (PGZ) drug in its pure and formulated forms. To facilitate
studies investigating the determination of PGZ in bulk drug and its pharmaceutical
formulations, a rapid UPLC method was developed and validated for the determination of
PGZ accompanied by its degradation studies in different stress conditions. The method
fulfilled validation criteria and was shown to be sensitive, with limits of detection (LOD)
and quantitation (LOQ) of 0.01 and 0.05 g mL-1 respectively. The percentage relative
standard deviation for robustness and ruggedness were observed within the range of 0.1

Page | 39

and 1.74. The calibration graph was linear in the range of 0.05300 g mL-1. The
applicability of the method was shown by analysis of formulated drug samples and spiked
human urine. The proposed method can be used for routine analysis in quality control
laboratories for its bulk and formulated product and this is the first reported UPLC
method for the assay of PGZ.

11.

Pavagada Jaganathamurthi Ramesh et al. presented a QbD approach to

method development and validation on nateglinide, an anti-diabetic drug. To facilitate

studies investigating the determination of Nateglinide (NTG) in bulk drug and its
pharmaceutical formulations, development and validation of a rapid UPLC method used
for determining NTG. The validated limit of quantitation (LOQ) of 0.06 g mL -1 and limit
of detection (LOD) of 0.02 g mL -1 are low enough to allow determination of low
concentrations of the drug. NTG showed no degradation at different stress conditions.
The percentage relative standard deviation for robustness and ruggedness were observed
within the range of 0.1 and 1.74. The calibration was linear in the range of 0.06250 g
mL-1. The proposed method was compared with a pharmacopoeial reference method and
found to give equivalent result. The proposed method can be used for routine analysis in
quality control laboratories for its bulk and formulated product and this is the first
reported UPLC method for the assay determination of Nateglinide.

12.

Kathryn Ellen Monks presented the investigation and evaluation of

reversed-phase high pressure liquid chromatography (HPLC) method development

strategies in accordance with Quality by Design (QbD) principles for a number of
practical applications of pharmaceutical interest. Workflow schemes were established
targeting the current needs of the pharmaceutical industry to develop robust, reliable,
durable and flexible HPLC methods with limited investment of resources, time and
money. A fundamental tool employed throughout such schemes is chromatography
modeling software which is shown to greatly aid the understanding and visualization of
chromatographic behavior in silico and therefore significantly reduces the experimental
burden typically associated with attaining good robust methods.

Page | 40

AIM AND PLAN OF WORK

Aim and Objective:

The aim of the analytical method is to identify and quantify the main compound while
meeting the method performance criteria based on regulatory requirements, such as
specificity, linearity, accuracy, precision, sensitivity, robustness and ruggedness.
The primary objective of this study was to implement Quality by Design (QbD) approach to
develop and validate a Reverse Phase High Performance Liquid Chromatography (RPHPLC) method that could identify the drug and to establish an in-depth understanding of the
method and build in the quality during the method development to ensure optimum method
performance over the lifetime of the product.
The objectives of this work are as follows:
1. To develop accurate, precise and robust method for identification of critical attributes
by QbD approach of the parenteral drug by RP-HPLC assay method.
2. To establish a validated test method as per ICH guidelines for the determination of
assay of the parenteral drug by RP-HPLC assay method.
PLAN OF PRESENT WORK:
The present work is planned to develop and validate an effective RP-HPLC method for the
estimation of using Quality by Design (QbD) principles for the estimation of Cefuroxime
sodium for injection dosage form.

Understand the method requirements and develop an Analytical Target Profile (ATP)
Select, develop and validate an analytical method that meets the ATP
Determine any critical analytical method parameters
Understand how these critical parameters will affect the result and develop a strategy
to control them.
Page | 41

Monitor method performance and continually improve.

MATERIALS AND METHODS

RP_HPLC ASSAY METHOD:
DRUG SAMPLES:
S.No

Material

1.

Cefuroxime sodium API

2.

Cefuroxime sodium for Injection

Manufactured By
Orchid Chemicals and
Pharmaceutical LTD.
Hospira Healthcare Pvt. Ltd.

REAGENTS AND CHEMICALS:

S.No

Reagent

Grade

Make

Acetonitrile

HPLC

Rankem

Methanol

HPLC

Rankem

Sodium acetate

HPLC

Sigma Aldrich

Milli Q water

HPLC

Millipore

Ortho phosphoric acid

HPLC

Rankem

Acetic acid

HPLC

Rankem

Sodium hydroxide

HPLC

Rankem

Note: All the materials used were within the expiry date and stored at recommended
storage conditions.

Page | 42

INSTRUMENTS:
S.No

Component

Make

Model

HPLC

Waters

Waters Aquity H class

Micro Balance

Mettler Toledo

MX5

Analytical balance

Sartorius

MSA225P-100-DA

pH meter

Fisher scientific

XL15

Sonicator

Auro bio medical

UT020

Vortex

Ika vortex genius3

v-03

Water bath

Equitron

BHJ-12-S

Empower software connected to the HPLC system.

STEPWISE STRATEGY OF HPLC METHOD DEVELOPMENT:

Drug development using the principles of QbD emphasizes a systematic approach, starting
with predefined objectives, and applying scientific understanding and risk management
Page | 43

approaches. This emphasis underlies the QbD principles and approaches to analytical
method development. Liquid chromatography, a long preferred technology for analysis by
drug companies; builds and enables the development of robust methods when used for a
QbD approach to chromatographic method development.
1. ANALYTICAL TARGET PROFILE:
The first step is to define the intended purpose of the analytical method. This has been called
the Analytical Target Profile (ATP) which is similar to the Quality Target Product Profile
(QTPP) for pharmaceutical process. The following table defines the criteria to be met for the
proposed method.
Table: 1
Analytical Target Profile (RP-HPLC technique)
S.N
o

Parameters

Target Profile

1.

Retention time

Min 10% variation from 10 repetitive injections

2.

Capacity factor

Min 2

3.

Tailing factor

Min 2

4.

Theoretical Plates

Min 2000

5.

RSD* (Precision)

Not more than 2%

6.

Selectivity

Method should be selective

7.

Accuracy

Method should be accurate to analyze the drug

*RSD = relative standard deviation

Having defined the ATP, the principles of QbD can be used during method development and
evaluation to ensure that an appropriate analytical method is selected and designed to meet
its intended performance requirements.

Page | 44

2. CRITICAL QUALITY ATTRIBUTES:

The critical quality attributes affecting the HPLC assay method are identified utilizing the
principles of Quality Risk Management presenting a risk management tool accounting for
the potential risk factors from the selected method and a risk assessment matrix which
categorize the risk to improve the method understanding and help achieve the analytical
target profile (ATP).
Method Selection:
According to the literature survey, there are few publications for the estimation of the drug
Cefuroxime sodium which include:

High performance liquid chromatography

Ultraviolet Spectroscopy
Direct titration
Microbiological assay method

Apart from the expense of the considerable disadvantages in terms of cost, space, materials,
analyst

skill,

operability

from

the

other

methods

High

performance

liquid

chromatography (HPLC) has been selected for the estimation of the drug Cefuroxime
sodium to carry out the present work because of the advantages which only it provides and
not replaceable by other equipments.
Risk Assessment:
Fishbone diagram, also known as an Ishikawa or cause-and-effect diagram, is used to
perform a structured risk assessment to identify potential factors that may influence the
method performance.

Environment
Temperature
(Column temp,
Room temp)

Machine
Pump
pressure

Man
Weighing

Visual
observation

Sample &
Page | 45

Response
time

Humidity

Diluent

Column

Flow rate

Mobile phase

Retention time

Detectors
Pumps
Glassware

Wavelength
Column
Efficiency

Run time
Solubility
Elution
Method

Standard
preparation

Stationary
Phase

Peak area &

Peak height
Response factor
Injection volume
Resolution

Measurement

Materials
Figure 2: Fishbone diagram of HPLC method

The above Fishbone diagram of the selected method HPLC segregates the risks into
different categories, such as those associated with instrumentation, materials, methods,
measurements, laboratory climate, and human factors. After risks have been assessed, they
are typically grouped into three categories: high-risk factors that should be tightly
controlled, potential noise factors, and factors that can be probed experimentally to
determine acceptable ranges. Typical high risk factors that can be fixed at the time of
method development include data analysis methods and sample preparation methods. The
third category of risks identified from the Fishbone analysis contains instrumental
parameters that can be prioritized and probed using the Design of Experiments approach
(DoE).

Initial Risk Assessment matrix:

S.No

Factors affecting ATP

Assay

Solubility

High

Detector wavelength

Low

Peak area and

Peak height

Page | 46

Column

High

Standard and Sample concentration

Medium

Mobile phase

High

Mobile phase composition

High

Flow rate

Low

Run time

Low

Column temperature

Medium

10

Injection volume

Low

11

Elution mode

Medium

Relative risk ranking:

Low no further investigation is needed
Medium further investigation may be needed
High further investigation is needed
From the above risk assessment matrix Solubility, Column, Mobile phase, Mobile phase
composition, Mobile phase pH, Flow rate, and Column temperature are identified as the
high risk factors directly or indirectly affect the accuracy, precision and robustness of the
method.
The identified, assessed risk factors are focused on processes to mitigate or avoid risk to an
acceptable level. So for each risk factor a mitigation plan is presented which has been
applied and that quality risk is reduced to a specified (acceptable) level.
1) Solubility: As per the literature review Cefuroxime sodium being a polar molecule is
freely soluble in water, soluble in methanol and DMSO (dimethyl sulphoxide). Hence
water was selected as the diluent. And the reverse phase HPLC is appropriate to select for
the estimation of the drug where the stationary phase is non-polar and mobile phase is
polar.
Risk: The risk associated with solubility which accounts for the effect to assay includes:
Page | 47

Use of non-calibrated glassware (volumetric flask, pipettes, measuring cylinder).

Use of contaminated water.
Insufficient knowledge regarding the physicochemical properties of the drug.
Use of non-calibrated weighing machine.
Low solubility may be lead to bad peak shapes.
Mitigation plan: The high risk concerned with solubility can be reduced by:

Following SOP for weighing of the drug substance in the calibrated weighing balance.
Using HPLC grade Milli Q Water as the diluent.
Using Class A glassware as accuracy is critical.
- Sufficient study of the physicochemical properties, nature of the drug molecule, pKa
-

which enhance the solubility and account for the correct choosing of the diluents.
Visual examination for the complete solubility of the drug in the diluents for good peak
shapes.

2) Detector wavelength: Selection of detector is based on Chemical nature of analyte,

potential interferences, Limit of detection required, Availability and/or cost of detector. If
the baseline noise continues under zero-flow conditions the problem may be related to a
defective detector lamp or an environmental problem. However, the majority of
reversed-phase and normal-phase HPLC method development in the pharmaceutical
industry is carried out with UV detection. But Photo-Diode Array (PDA) has been used
to select the detector wavelength as the PDA UV-Vis spectrophotometer has a multichannel detector, controlled by a microprocessor, which can collect spectral data for
many wavelengths simultaneously, while a conventional UV-Vis spectrophotometer has
a single channel detector which can collect data for a selected wavelength. The PDA UVVis spectrophotometer is much faster and gives more reproducible results than a
conventional UV-Vis. spectrophotometer because the latter has moving parts, such as a
grating or prism, which are operated by a stepper motor for selecting a specific

wavelength. These moving parts can affect the reproducibility.

Risk:
Using the of the detector lamp which is not calibrated.
Air bubbles in the detector.
Mitigation plan:

Check the purge valves to remove air bubbles in the detector.

Detector lamp should be checked for calibration.

Page | 48

3) Column: As per the literature review the efficient column for the estimation of
Cefuroxime sodium is L15 (Hexylsilane C6 chemically bonded to a totally porous silica
particle- 3-10m in diameter).
Risk: The risk associated for the selection of the column:
- High column back pressure: backpressures generally increases as particulate matter
accumulates on the inlet frit of the column and may lead to band distortion of the peaks
-

in the chromatogram.
Clogging of the column inlet and outlet frits from sample/mobile phase: Injection of

sample/mobile phase without filtering.

pH of the mobile phase- mobile phase pH also has an effect on the column stability, and
this is indirectly related to the peak efficiency and the retention of the analyte. The
operating pH range is 2.0-8.0 as the column cannot withstand above or below this range

as the siloxane linkages are cleaved below pH 2.0 and above pH 8.0 silica may dissolve.
Age of the column
Packing material (type of stationary phase)
- Particle diameter 5 based on the chemistry of the analyte.
- Improper column washing and conditioning with the mobile phase prior to the starting of
the analysis.
Mitigation plan: The high risk associated with column to the assay method can be
mitigated:
-

The high column back pressure and clogging of inlet and outlet frits with particulate
matter in the mobile phase can be prevented by filtering the sample and mobile phase

prior to injection to the column. Hence the life of the column is maintained.
The column should be properly washed and conditioned with the respective mobile

phase for efficient analysis.

The use of very old columns is not recommended.
pH of the mobile phase for Cefuroxime sodium was selected based on pKa value of the
drug in water which is suitable for the proper column functioning, identification and

quantifying the drug.

Use of guard column.
4) Standard and Sample preparation: The standard and sample concentration imparts on
the detection system, affects the accuracy and precision of the assay method.
Risk: The concentration of both standard and sample has a direct impact on the assay. The
other factors include:
Page | 49

Improper weighing of the drug (concentration mg/mL) as per monograph.

Use of non-calibrated weighing balance, glassware.
Incorrect dilution factor.
Improper skill and training on the weighing of the drug substance.
Use of contaminated diluent.
Use of unfiltered sample for the analysis.
Calculation identified as per on as in basis, anhydrous basis, on dried basis.
Expired date of the standard.

Mitigation plan: The risk concerned with the standard and sample preparation of the drug
can be mitigated by following the measures.
-

The calibration of the weighing balance and the glassware should be strictly considered

for accurate and precise measurement.

The standard and sample should be sonicated and filtered prior to the injection into the

system as to prevent the clogging of the column which affects the accurate detection.
The calculation identified is as per on as in basis for the assay as mentioned.
The valid date of the standard should be checked without fail.
The Milli Q water of HPLC grade should be used as diluent.
The analyst should be properly trained to follow the SOP of weighing the drug

substances.
The use of correct dilution factor for preparing the right concentration.
Use of Class A glassware for the standard and sample preparation.

5) Mobile phase: The mobile phase is composed of a buffer and a polar organic solvent.
The components are retained due to hydrophobic interactions with the non-polar
stationary phase and are eluted in order of increasing hydrophobicity (decreasing
polarity).
Risk: The considerable factors for the high risk in choosing the correct mobile phase
includes:
- pH of the mobile phase: It is one of the important factors allowing simultaneous change
-

in the retention time and selectivity of the components.

Solubility of the analyte in mobile phase.
Use of appropriate buffer: In order to develop rugged HPLC methods, knowledge of

using the right buffer is very important.

Inaccurate concentration of the salt component in the preparation of the buffer.
Non-degassing of the mobile phase.
Use of expired organic solvents i.e., 6 months from the date of opening of the organic
component container.

Page | 50

Improper mixing of the mobile phase containing both the aqueous component and

organic component.
Inaccurate adjustment of pH of mobile phase as known.

Mitigation plan:
-

Selection of pH of mobile phase from the monograph and accurate adjustment of pH of

the buffer prior to the addition of the organic component.

Correct pH leads to good peak shape.
Appropriate selection of the buffer with strength not exceeding 0.2M varying about 10-

20% as to avoid injection load on the column and peak tailing.

Degassing and proper mixing of the mobile phase containing both the organic and

aqueous phase.
Checking for the valid date of the organic solvents used.
Compatibility of the analyte and chemically bonded phase with the mobile phase.
Use of calibrated pH meter.
Covering the mobile phase after its preparation to prevent the evaporation of the organic

component from the final volume of the mobile phase.

Maintaining the concentration of the salt content in the buffer i.e., about 30
millimoles/litre as increasing the buffer concentration can reduce the tailing factor.

6) Mobile phase composition: The mobile phase composition affects the system
specificity, selectivity and efficiency.
Risk:
- Improper calculation of the exact composition of the mobile phase required.
- Preparation of the buffer without checking the pH.
- Adding the organic component without adjusting the buffer pH.
- Improper making up of the mobile phase with the exact volume.
- Using the buffer with undissolved salts and improper sonication.
Mitigation plan:
-

Proper adjustment of the buffer pH.

Dissolving the salts completely and sonicating the buffer after its preparation.
Using design of experiments can help develop the preferable mobile phase composition
required for the study.

7) Flow rate: The flow rate impacts system pressure, chromatographic quality and analysis
time.
Risk:

Page | 51

Flow rate may affect the peak elution, the retention time is decreased.
Reproducibility is affected.

Mitigation plan:
-

To sustain the reproducibility the flow rate needs to be optimized by using experimental

approach.
The flow rate needs to be maintained constant throughout the analysis.

8) Run time: The run time of the drug is fixed as the appropriate chromatographic
conditions like the column temperature, mobile phase, pH and all the contributing
factors are optimized. There is no potential risk to the assay of the HPLC assay method.
9) Column temperature: The column temperature depends on the viscosity of the solvent
used for the analysis. It may then impact the retention time and column performance.
Hence, the column temperature can be fixed based on the type of the solvent (diluent)
used for the analysis.
Risk:
- External temperature fluctuations may affect the column temperature affecting the peak
-

shape.
Increase in column temperature will decrease the actual retention time.
Elevation in column temperature may decrease the viscosity of the solvent.

Mitigation plan:
-

To improve the peak shape and detect ability using highly aqueous component i.e.,

water.
Maintaining the external room temperature constant.
Set the column temperature before the analysis starts.

10) Injection volume: The injection volume generally for the HPLC estimation is 10-20L.
It mainly impacts the peak symmetry. So the volume of injection is to checked out for
attaining good peak shape.
Risk:
- Strong injection of the solvent may cause bad peak shape.
- Peak fronting may result from sample mass overload more sample than can effectively
-

partition results in some sample preceding the rest of the peak.

Peak tailing from overload of silanols with basic compounds may be seen as peak
tailing.

Page | 52

Mitigation plan:
The injection volume of the standard, sample solution and the solvent needs to be kept
constant throughout the analysis to maintain good peak shape. Hence to reduce peak tailing
and peak fronting.
11) Elution mode: The elution mode includes the gradient type and the isocratic type. It is
selected based on the chemistry of the analyte. In isocratic separation equilibrium
condition in the column is maintained and actual velocity of components moving
through the column is constant. Gradient system generally increases the separation
power.
Risk:
-

In isocratic elution, the separation power is low.

Peak capacity is low and longer the component is retained on the column, the wider is
the resultant peak.

Mitigation plan:
-

Based on the chemistry of the analyte the elution mode is selected.

METHOD DEVELOPMENT AND OPTIMIZATION OF CHROMATOGRAPHIC

CONDITIONS
The risk assessment study described all the potential risk factors involved in the
development of the HPLC assay method.
The assay method development utilizing the principles of QbD, optimization of the
chromatographic conditions are done in order to mitigate the risk of the critical quality
attributes using both one-factor at a time (OFAT) approach, where each factor is varied
without changing any of the other method parameters and experimental design approach,

Page | 53

where factors are investigated simultaneously, the advantage being significant saving in the
time required for the study.

Method development and optimization includes the following parameters:

1. Solubility
2. Selection of detector wavelength
3. Selection of column
4. Selection of column temperature
5. Selection of concentration
6. Optimization of buffer/mobile phase pH
7. Optimization of mobile phase composition
8. Selection of flow rate
9. Solution stability
10. Method Robustness
High risk instrumental parameters (optimization of mobile phase pH, mobile phase
composition and selection of flow rate) are assessed experimentally using statistical
Design of Experiments (DoE) methodology. These parameters are the critical process
parameters (CPPs). From the DoE results and their interpretation, the design space (DS)
of the analytical method will be obtained.
1. SOLUBILITY:
According to the literature review the drug Cefuroxime sodium is found to be freely soluble
in water (500mg/mL), soluble in methanol and DMSO (dimethyl sulphoxide).
Selection of Chromatographic Method:
Selection of chromatographic conditions depends upon the characteristic nature of the
sample, its molecular weight and solubility. The nature of Cefuroxime sodium is polar,
hence it can be dissolved in polar mobile phase. Perhaps, reverse phase chromatography was
used. Hence polar mobile phase and non polar stationary phase was selected.
2. SELECTION OF DETECTOR WAVELENGTH:

Page | 54

The sensitivity of the HPLC depends on the selection of detection wavelength. An ideal
wavelength is one that gives good response for the drugs to be detected. Known
concentration of standard solution in the suitable diluent was injected into the
chromatographic system with photodiode array detector and the spectrum was collected.
Selection of wavelength was done based on the higher response of the compound.
3. SELECTION OF COLUMN
Selection of column was done by using different columns like Waters Spherisorb C6,
150x4.6 mm, 5, YMC Pack ODS C8, 150x4.6 mm, 5 and Princeton Sphere C18, 150x4.6
mm, 5 to achieve best separation of analyte peak.
4. SELECTION OF COLUMN TEMPERATURE:
Always it is preferable to optimize the chromatographic conditions with column temperature
as ambient. The column temperature depends on the viscosity of the solvent used for the
analysis. It may then impact the retention time and column performance. Hence, the column
temperature can be fixed based on the type of the solvent (diluent) used for the analysis.

5. SELECTION OF CONCENTRATION:
The different concentrations like 50ppm and 100ppm of standard and sample were prepared
and injected into the chromatographic system and the system suitability was observed.
EXPERIMENTAL APPROACH (DoE)
High-risk instrumental parameters can also be assessed experimentally using Design of
Experiments (DoE) methoodology.
a) Selection of the factors:
The first step of testing is to decide which factors are going to be studied. The number of
factors selected determines how much information is gathered about the way in which
Page | 55

changes in method parameters affect the results, but with increasing numbers of factors the
time required to complete the investigation also increases.
The systematic scouting of the three factors of the RP-HPLC method is presented:
6. Optimization of buffer/mobile phase pH.
7. Optimization of mobile phase composition.
8. Selection of flow rate.
b) Selection of the factor levels:
The amount of variation is referred to as the factor level, typically limits around a nominal
value are investigated and the magnitude of these limits has to be defined. When using a
QbD approach to method development the aim is to understand the effect of changing a
method parameter.

Levels
S.No

Factors

1.

Flow rate

2.

pH of the mobile phase

3.

Mobile phase composition

Low

High

1.0 mL/min

2.0 mL/min

3.0

4.0

100:10

70:40

c) Selection of method responses:

The factors and factor levels define the way in which the method (and thus HPLC system)
will be set up for the experiments. This needs to take into account quantitative aspects of the
method such as an assay result, e.g., %w/w for main components or impurities, but also
chromatographic criteria such as resolution, relative standard deviation (RSD), tailing of a
major peak and theoretical plates. These are referred to as the method responses.
d) Design of Experiments:
Design of experiments provide an effective, efficient approach to evaluate simultaneously
the effect of factors and their interactions, and to model and to predict the relationship
between these factors and the CQAs or responses.

Page | 56

The experimental design selected for the evaluation of the factors is Two level Full
Factorial Design which maintains the experiments as low as possible. The study involves the
number of experiments for the 3 factors and 2 levels is 8 (23).
e) Execution of experiments:
To obtain the design space (DS) of analytical methods, the method parameters are set up
correctly for each experiment using suitable test solutions and sequence of injections. A
random order is advised when using experimental design but this may involve changeover
between the mobile phase components and columns. The order of experiments requires a
careful planning.
f) Interpretation of results:
The two-level factorial design has been selected to interpret the results using MINITAB
software.
9. SOLUTION STABILITY
Bench Top Stability of Test Preparation And Standard Preparation:
A study to establish the Cefuroxime in test preparation and standard preparation on bench
top was calculated at initial, 1 day, and 2 days. The assay of Cefuroxime sodium test
preparation and standard preparation were estimated against freshly prepared standard each
time. The difference in % assay of standard and test preparations from initial to 1 day and 2
days was calculated and observed that standard and test were not stable on bench top for a
period of 1 day.

Refrigerator Stability of Test Preparation and Standard Preparation:

A study to establish stability of Cefuroxime in test preparation and standard preparation in

refrigerator was conducted over a period of 5 days. The assay of Cefuroxime sodium test
preparation and standard preparation was estimated at initial, 1 day, 2 days, and 5 days
against freshly prepares standard each time. The difference in % assay of standard and test
preparation from initial to 1 day, 2 days and 5 days was calculated and found that standard
and test preparation were stable in refrigerator for a period of 1 days.

Bench Top Stability of Mobile Phase:

Page | 57

A study to establish bench top stability of mobile phase at initial, 1 day and 2 days was
conducted. The assay of Cefuroxime in Cefuroxime sodium for injection was estimated
using the same lot of mobile phase each time. The system suitability parameters were
evaluated as per the test method and found to be within the limits. The difference in % assay
from initial to 1 day and 2 days was found to be within the acceptable limits.
From the above study, it was established that the mobile phase for Assay analysis of
Cefuroxime sodium for injection is stable for a period of 2 days on bench top.

Refrigerator Stability of Mobile Phase:

A study to establish Refrigerator stability of mobile phase was conducted over a period of 5
days. The Assay of Cefuroxime in Cefuroxime sodium for injection was estimated using the
same lot of mobile phase each time. The system suitability parameters were evaluated as per
the test method and found to be within the limits. The difference in % assay from initial to 5
days was found to be within the limits.
From the above study, it was established that the mobile phase for Assay analysis of
Cefuroxime sodium for injections is stable for a period of 5 days in refrigerator.

10. ROBUSTNESS:
Effect of Variation in Mobile Phase Composition:
A study to establish the effect of variation in mobile phase composition was conducted. Two
mobile phases were prepared having 90% and 110% of the method organic phase
composition. Standard solution prepared as per test method was injected into HPLC system.
The system suitability parameters were evaluated with both the mobile phases as per test
method and found to be within the limits. From the above study, it was established that the
allowable variation in organic phase composition in mobile phase is from 90% to 110% of
the method organic phase composition.

Effect of Variation in Flow Rate:

Page | 58

A study was conducted to determine the effect of variation in flow rate. Standard preparation
prepared as per test method was injected into the HPLC system with flow rate 1.8 mL/min
and 2.2 mL/min.
The system suitability parameters were evaluated as per the test method with both the flow
rates and found to be within the limits. From the above study, it was established that the
allowable variation in flow rate is from 1.8 mL/min to 2.2 mL/min.

Effect of Variation in Column Temperature:

A study was conducted to determine the effect of variation in column temperature. Standard
preparation prepared as per the test method was injected into the HPLC system at 20 C and
at 30 C column temperatures. The system suitability parameters were evaluated as per the
test method with both the column temperatures and found to be within the limits. From the
above study, it was established that the allowable variation in column temperature is from
20 C to 30 C.

Filter validation:

A study to establish the suitability of filters was conducted using two different filters
namely, 0.45m HNN filter (Mfg. by: Advanced Micro devices) and 0.45m HVF filter
(Mfg. by: Advanced Micro devices). Test preparations prepared in triplicate were
centrifuged and filtered through different filters, were assayed against unfiltered standard.
The differences in % Assay values between centrifuged and filtered samples were found to
be within limits. The above study indicates that both the filters are suitable for filtration.

Page | 59

Rationale for Method development and Optimization:

The emergence of the use of Quality by Design (QbD) principles in pharmaceutical
manufacturing has led to the application of QbD to analytical methods. This in turn has
highlighted the importance of robustness during method development as the design space
concept of QbD translates into knowledge about the effect of each method parameter on the
final analytical result. For many analytical chemists this means that the robustness of a
method may now be considered during method development.
Ideally robustness should be investigated as part of method development because a method
is not complete without an evaluation of its reliability in routine use, but unfortunately it is
often deferred, or completely overlooked, because of the time-consuming nature of the

Page | 60

study. When the method is being validated, a robustness study may then be performed. In the
ICH validation guidelines [1], robustness is not included in the tabular summary of required
characteristics which should be tested during validation, which could lead to the mistaken
belief that a study of robustness is not required. However, in the section of the guidelines
relating to robustness, it is expected that the evaluation of robustness should be considered
during the development phase and depends on the type of procedure under study. It should
show the reliability of an analysis with respect to deliberate variations in method
parameters. So not only is robustness good practice in terms of developing fit-for-purpose
analytical methods, it is also a regulatory requirement. It is interesting to note that
robustness is included as a requirement for validation in the summary table provided in the
draft FDA guidelines [2].
FINAL RISK ASSESSMENT:
By carrying out the above studies and optimizing the assay method development parameters
using the OFAT (one-factor at a time) approach and experimental design (DoE) approach,
the risk is found to be reduced by following the mitigation plan. All the risks contributing to
the critical quality attributes are ranked low by optimizing the chromatographic conditions
and showing the design space of the selected parameters.

FINAL METHOD PARAMETERS:

Page | 61

PARAMETERS

OPTIMIZED RESULT

Mode of separation

Reverse phase

Mode of elution

Isocratic

Instrument

Waters HPLC

Column

Waters Spherisorb C6,

SYSTEM

Phenomenex Sphereclone C6-

PARAMETERS

150 x 4.6mm, 5
Column temperature

Ambient

Flow rate

2mL/min

Injection volume

10L

Run time

6 min

Detector

PDA

Wavelength

254 nm

SAMPLE PARAMETERS:
DILUENT: HPLC grade water (milli Q water).
MOBILE PHASE:
-

Preparation of 0.1M sodium acetate buffer:

Weigh and transfer about 1.36g of sodium actetate into a 100 mL volumetric flask,

dissolve it in milli Q water and dilute to volume with milli Q water and mix.
Preparation of 0.1N acetic acid:
Pipette 5.8 mL of acetic acid into a 1000 mL volumetric flask, dilute to volume with

milli Q water and mix.

Preparation of pH 3.4 Acetate buffer:
Transfer 50 mL of 0.1M sodium acetate solution into a 1000 mL volumetric flask,

dilute to volume with 0.1N acetic acid solution and mix.

Preparation of Mobile Phase:
1. Mix pH 3.4 acetate buffer and acetonitrile in the ratio of 100:10 (v/v)
respectively.
2. Filter through 0.45m membrane filter.

Page | 62

Note: Do not use the mobile phase preparations beyond 2 days on bench top or 5 days in
refrigerator.
PREPARATION OF STANDARD SOLUTION:
Weigh accurately and transfer 54 mg of Cefuroxime sodium working standard into a 50 mL
volumetric flask, add about 30 mL of diluents, sonicate to dissolve the material completely,
dilute to volume with diluents and mix.
Immediately pipette 5.0 mL of the above solution and into a 100 mL volumetric flask, dilute
to volume with diluents and mix.
Filter about 2 mL with HNN/HVF filter.
Note: Use freshly prepared standard solution on bench top and do not use beyond 1 day in
refrigerator.
ASSAY PREPARATION:
Weigh accurately and transfer 54 mg of sample into a 50 mL volumetric flask, add about 30
mL of diluents, sonicate to dissolve the material completely, dilute to volume with diluents
and mix.
Immediately pipette 5.0 mL of the above solution and into a 100 mL volumetric flask, dilute
to volume with diluents and mix.
Filter about 2 mL with HNN/HVF filter.
Calculation:
Assay of Cefuroxime
(g/mg, on anhydrous basis)

AT x WS x 5 x 50 x 100 x P x 100
AS x 50 x 100 x WT x 5 x (100-W)

Where,
AT
AS
WS
WT
P

=
=
=
=
=

Peak area of Cefuroxime from Test preparation

Peak area of Cefuroxime from standard preparation
Weight of Cefuroxime sodium working standard taken, in mg
Weight of sample taken in mg
Potency of Cefuroxime sodium working standard in g/mg (on as in basis, as

Page | 63

Cefuroxime)
= Water content of the sample in %w/w

CONTROL STRATEGY:
As a result of development and robustness studies, the overall method understanding of
method performance under various conditions can be improved and an analytical method
performance control strategy along with appropriate system suitability criteria can be
defined to manage risk and ensure the method delivers the desirable method attributes. If the
risk is high and hard to manage, it is an opportunity to go back to the database described in
performing the experimental design to find a more appropriate method and to go through the
procedure to ensure method robustness and ruggedness.

RESULTS:
1. SOLUBILITY:
The drug Cefuroxime sodium being freely soluble in water (500mg/2.5mL), hence the same
(water) is chosen to be the diluents for developing the assay method.

2.

SELECTION OF DETECTOR WAVELENGTH:

S.N
o
1

Wavelength

254 nm

Response

2905930

Page | 64

3. SELECTION OF COLUMN:
Column Description

Observation

Remarks

Princeton Spher C18,

150x4.6 mm, 5

Theoretical plates found to be less

than the specification limit

Not Satisfactory

YMC- Pack ODS C8,

150x4.6 mm, 5

Peak was asymmetrical and fronting

was observed

Not Satisfactory

Waters Spherisorb C6,

150x4.6 mm, 5

Peak was found to be symmetrical,

with tailing factor 1.2

Satisfactory

4. OPTIMIZATION OF COLUMN TEMPERATURE:

As the solvent used in the mobile phase is chosen as water which is less viscous. Hence the
column is run at ambient temperature.

5. OPTIMIZATION OF CONCENTRATION:
The optimum concentration 50ppm was found satisfying the system suitability parameters theoretical plates, relative standard deviation (RSD %) and tailing factor.

System Suitability
parameters
The column efficiency
Cefuroxime peak

for

Observed
value

Acceptance
criteria

3349

NLT: 1300

Page | 65

USP
tailing
factor
Cefuroxime peak

for

%RSD for peak area of

Cefuroxime peak from five
replicate injections of standard

Solvent/diluen

1.1

NMT: 2.0

0.5

NMT: 2.0

Preparatio

Peak response

% Assay

n No

( R)

451389

97.4

452268

97.5

452677

97.4

453200

98.6

453543

97.5

RSD (%)

Water

0.5

Acceptance criteria:
The % RSD should be NMT 2.0.

EXPERIMENTAL DESIGN (DOE):

Two-level Full factorial design in MINITAB software is used for obtaining design space.
The three parameters which are to be evaluated under the experimental design space study:
6. Optimization of mobile phase/buffer pH
7. Optimization of mobile phase composition
8. Selection of flow rate

Contour plots of the method responses (RT, Tf and N):

Page | 66

RT Retention time, Tf Tailing factor, N Theoretical plates

The contour plots show the feasible region (white area) i.e. the design space for the
optimization of the selected high risk method parameters of the selected method responses
(RT, Tf and N). The grey area is not feasible for satisfying the method criteria defined in
Analytical Target Profile.
Table: Creation of the design experiments:
S.N
o

Flow
rate

Mobile
phase
pH

Mobile
phase
compositio
n

1.0

4.0

70:40

1.0

3.0

100:10

1.0

3.0

70:40

2.0

3.0

100:10

2.0

4.0

70:40

2.0

4.0

100:10

1.0

4.0

100:10

2.0

3.0

70:40

Retentio
n time
(RT)

Tailing
factor
(Tf)

Theoretica
l plates
(N)

Page | 67

(1) Chromatogram with Flow rate 1.0 mL/min, pH 4.0 and 70:40 (Buffer: ACN)

(2) Chromatogram with Flow rate 1.0 mL/min, pH 3.0 and 100:10 (Buffer: ACN)

Page | 68

(3) Chromatogram with Flow rate 1.0 mL/min, pH 3.0 and 70:40 (Buffer: ACN)

(4) Chromatogram with Flow rate 2.0 mL/min, pH 3.0 and 100:10 (Buffer: ACN)

Page | 69

(5) Chromatogram with Flow rate 2.0 mL/min, pH 4.0 and 70:40 (Buffer: ACN)

(6) Chromatogram with Flow rate 2.0mL/min, pH 4.0 and 100:10 (Buffer: ACN)

Page | 70

(7) Chromatogram with Flow rate 1.0mL/min, pH 4.0 and 100:10 (Buffer: ACN)

(8) Chromatogram with Flow rate 2.0mL/min, pH 3.0 and 70:40 (Buffer: ACN)

Page | 71

1. Contour plot for Flow rate and pH of Mobile phase

Contour Plot of RT, Tf, N

4.0

RT
2.355
4.699

pH mobile phase

3.8

Tf
1
1.5
N
2500
4000

3.6

Hold Values
composition of mobile phase

3.4

3.2

3.0
1.0

70

Feasible
region
1.2

1.4
1.6
flow rate

1.8

2.0

In the above contour plot the design space (white area) shows the range of pH of mobile
phase and flow rate where the criteria for the three response variables (RT, T f and N) are
satisfied.
The optimized condition can be obtained when the method parameters are operated in the
range:
pH of the mobile phase/buffer: 3 - 4
Flow rate: 1.6 2 mL/min

2. Contour plot for Flow rate and composition of mobile phase

Page | 72

Contour Plot of RT, Tf, N

composition of mobile phase

100

RT
2.355
4.504

95

Tf
1
1.2

Feasible
region

90

N
2500
3500

85

Hold Values
pH mobile phase 3.5

80
75
70
1.0

1.2

1.4
1.6
flow rate

1.8

2.0

In the above contour plot the design space (white area) shows the range of mobile phase
composition and flow rate where the criteria for the three response variables (RT, T f and N)
are satisfied.
The optimized condition can be obtained when the method parameters are operated in the
range:
Mobile phase composition: 70 100%
of organic phase
Flow rate: 1.4 2 mL/min

3. Contour plot for Flow rate and pH of mobile phase

Page | 73

Contour Plot of RT, Tf, N

composition of mobile phase

100

RT
2.255
5.204

95

Tf
1
1.2

90

N
2000
4500

85

Hold Values
flow rate 1

80
75
70
3.0

3.2

3.4
3.6
pH mobile phase

3.8

4.0

In the above contour plot the design space (white area) shows the range of pH of mobile
phase and composition of mobile phase where the criteria for the three response variables
(RT, Tf and N) are satisfied.
The optimized condition can be obtained when the method parameters are operated in the
range:
Mobile phase composition: 80 100 % of organic phase
pH of mobile phase: 3.0 3.5 mL/min

9. SOLUTION STABILITY:
Bench Top Stability of Test Preparation And Standard Preparation:
Page | 74

%Assay of
standard
preparatio
n

%Differenc
e from
initial

Initial

94.1*

1
2

Time in
days

%Assay of test
preparation

%Difference from
initial

Test 1

Test - 2

Test 1

Test - 2

NA

99.9

100.2

NA

NA

84.8

9.3

91.1

91.2

8.8

9.0

75.7

18.4

81.3

81.0

18.6

19.2

* Potency of working standard as Cefuroxime sodium

Acceptance criteria:
Difference in % Assay of standard and test from initial should be NMT 3.0.

Refrigerator Stability of Test Preparation and Standard Preparation:

%Assay of
%Assay of test
%Differenc
Time in standard
preparation
e
from
days
preparatio
initial
Test 1
Test - 2
n

%Difference from
initial
Test 1

Test - 2

Initial

94.1*

NA

99.9

100.2

NA

NA

93.0

1.1

99.5

99.5

0.4

0.7

90.2

3.9

96.4

96.5

3.5

3.7

89.3

4.8

95.8

95.9

4.1

4.3

* Potency of working standard as Cefuroxime sodium

Acceptance criteria:
Difference in % Assay of standard and test from initial should be NMT 3.0.

Bench Top Stability of Mobile Phase:

Page | 75

Observed values

System Suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak

for

%RSD for peak area of

Cefuroxime peak from five
replicate injections
of
standard

Acceptance
criteria

Initial

1 day

2 day

2974

3265

3519

NLT: 1300

1.1

1.1

1.1

NMT: 2.0

0.2

0.3

0.2

NMT: 2.0

Time in days

% Assay

% difference w.r.t. Initial

Initial

100.1

NA

100.4

0.3

99.7

0.4

Acceptance criteria:
1) The % Assay result should not differ from initial value by more than 3.0.
2) All system suitability parameters shall meet the requirements as per the test method.

Page | 76

Refrigerator Stability of Mobile Phase:

System Suitability parameters

Observed value

Acceptance
criteria

Initial

5 day

2974

4335

NLT: 1300

USP tailing factor for Cefuroxime

peak

1.1

1.1

NMT: 2.0

%RSD for peak area of

Cefuroxime peak from five
replicate injections of standard

0.2

1.8

NMT: 2.0

The Column Efficiency

Cefuroxime peak

for

Time in days

%Assay

%Difference w.r.t. initial

Initial

100.1

NA

5 day

99.6

0.5

Acceptance criteria:
1) The % Assay result should not differ from initial value by more than 3.0.
2) All system suitability parameters shall meet the requirements as per the test method.

10. ROBUSTNESS:
Effect of Variation in Mobile Phase Composition:

Page | 77

Acceptance
criteria

Observed value

System suitability
parameters

90%

100%

110%

The Column Efficiency

for Cefuroxime peak

3357

33495

2995

NLT: 1300

USP tailing factor for

Cefuroxime peak

1.1

1.1

1.1

NMT: 2.0

%RSD for peak areas of

Cefuroxime peak five
replicate injection of
standard

0.2

0.1

0.1

NMT: 2.0

Acceptance criteria:
All system suitability parameters shall comply as per the test method.

Effect of Variation in Flow Rate:

Observed value

System suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak

for

%RSD for peak areas of

Cefuroxime peak from five
replicate
injection
of
standard

Acceptanc
e criteria

1.8mL/min

2.0mL/min

2.2mL/mi
n

3560

3349

2870

NLT: 1300

1.1

1.1

1.1

NMT: 2.0

0.1

0.1

0.1

NMT: 2.0

Acceptance criteria:
All system suitability parameters shall comply as per the test method.

Page | 78

Effect of Variation in Column Temperature:

System Suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak

Acceptance
criteria

Observed value

for

%RSD for peak areas of

Cefuroxime peak from five
replicate injections
of
standard

20C

25C

30C

2934

3349

3114

NLT: 1300

1.1

1.1

1.1

NMT: 2.0

0.1

0.1

0.1

NMT: 2.0

Acceptance criteria:
All system suitability parameters shall comply as per the test method.

Filter validation:

Filter description
Manufacturers Name
Lot No.
Size

Filters
HNN

HVF

Advanced Microdevices

Advanced Microdevices

NB169695

420/720/1

0.45m

0.45m

Page | 79

%Assay

Difference

Centrifuged

100.2

100.4

99.5

NA

NA

NA

Sample filtered through

HNN filter

100.1

100.8

100.4

0.1

0.4

0.9

Sample filtered through

HVF filter

100.1

100.7

100.3

0.1

0.3

0.8

NA Not Applicable
Acceptance criteria:
The difference of %assay result from centrifuged sample to filtered samples should be not
more than 3.0.

METHOD VALIDATION:

Page | 80

1) SYSTEM PRECISION AND SYSTEM SUITABILITY:

The standard solution, prepared by using Cefuroxime sodium working standard as per test
method was injected 10 times into the HPLC system. The system suitability parameters were
evaluated and found to be within the limits. The RSD for peak areas of ten replicate
injections was found to be 0.2% for Cefuroxime and 0.1% for internal standard.

Table 1A: System Suitability

System Suitability
parameters
The column efficiency
Cefuroxime peak

for

USP
tailing
factor
Cefuroxime peak

for

%RSD for peak area of

Cefuroxime peak from five
replicate injections of standard

Observed
value

Acceptance
criteria

3349

NLT: 1300

1.1

NMT: 2.0

0.1

NMT: 2.0

Table 1B: System Precision

Page | 81

Injections

Cefuroxime sodium peak

area

01

451413

02

451134

03

450542

04

452111

05

451472

06

450922

07

452000

08

452238

09

453137

10

453157

Average

451813

%RSD

0.2

Acceptance criteria

The relative standard

deviation should be not more
than 1.0%

Chromatogram of System Precision

2) SPECIFICITY:
Page | 82

i) Interference from degradation products:

A study was conducted to demonstrate the effective separation of degradants from
Cefuroxime in Cefuroxime sodium for injection. Separate portions Drug product were
exposed to following stress conditions to induce degradation.
a) Acid stress: Stressed with 0.1N HCl solution by keeping on water bath at 60C for
5minutes.
b) Base stress: Stressed with 0.1N NaOH solution by keeping on bench top for 30
seconds.
c) Peroxide stress: Oxidized with 1% H2O2 solution by keeping on water bath at 40C
for 10 minutes.
d) Heat stress: Exposed to heat at 100C for about 13hrs and 50 minutes.
e) Humidity stress: Exposed to humidity at 25%C/90% RH for about 596 hours and 10
minutes.
f) Water stress: Stressed with purified water on water-bath at 60C for 10minutes.
g) Photolytic stress:
Visible stress: Exposed to Visible light for 596 Lux hours and 10 minutes.
UV stress: Exposed to UV light for 596 Lux hours and 10 minutes.
Stressed samples were injected into the HPLC system with diode array detector by following
test method conditions. All degradants peaks were resolved from Cefuroxime sodium peak
in the chromatograms of all samples.
The chromatograms of the stressed samples were evaluated for peak purity of Cefuroxime
using Empower software. For all forced degradation samples, the purity angle for
Cefuroxime peak is less than purity threshold. Cefuroxime peak does not have any flag in
purity results table. This indicated that there is no interference from degradants in
quantitating the Cefuroxime in Cefuroxime sodium for injection. Thus, this method is
considered to be Stability indicating.
Page | 83

Table 2: Peak Purity Results of Forced Degradation Studies

Drug product
Stress condition

%
Degradation

Purity
angle

Purity
threshold

Purity
flag

Stressed with 0.1 N HCl

solution for about 5 minutes on
water bath at 60C.

24.53

0.207

1.086

No

Stressed with 0.1N NaOH

solution for 30 seconds on
bench top

14.08

0.136

1.065

No

Oxidized with 1% H2O2

solution on water bath at 40C
for 10 minutes

11.22

0.125

1.083

No

Refluxed with purified water

for about 10 minutes on water
bath at 60C

21.57

0.141

1.119

No

Exposed to heat at 100C for

13 hours and 50 minutes

Nil

0.050

1.049

No

Exposed to humidity at
25C/90% RH for 596 hours
and 10 minutes

Nil

0.054

1.049

No

Exposed to Visible light for

596 Lux hours and 10 minutes

Nil

0.069

1.080

No

Exposed to UV light for 596

Lux hours and 10 minutes

Nil

0.084

1.106

No

Acceptance criteria:
1) Purity angle should be less than purity threshold.
2) Cefuroxime sodium peak should not have any Flag in purity results table. (Waters
Empower).

Page | 84

Chromatogram and Purity plot of Acid Stressed Drug Product

Chromatogram and Purity plot of Base Stressed Drug Product

Page | 85

Chromatogram and Purity plot of Oxidation Stressed Drug Product

Chromatogram and Purity plot of Heat Stressed Drug Product

Page | 86

Chromatogram and Purity plot of Humidity Stressed Drug Product

Chromatogram and Purity plot of Water Stressed Drug Product

Page | 87

Chromatogram and Purity plot of Visible Light Stressed Drug Product

Chromatogram and Purity plot of Ultraviolet Light Stressed Drug Product

Page | 88

ii)

Interference From Impurities:

A study was conducted to demonstrate the effective separation of all known impurities of
Cefuroxime sodium from Cefuroxime peak. All impurities are spiked in assay test
preparation and injected in to the chromatographic system and evaluated for peak purity
of Cefuroxime and found that all impurities are well separated. Thus, this method is
considered to be Stability indicating.

Chromatogram and Purity plot of Drug Product spiked with Known Impurities
3) ACCURACY:
Accuracy studies were evaluated by determining the percentage recovery of different
levels (80%-120%) of standard solution under same condition. The results of accuracy
determination were shown in Table 16 & 17. Recoveries of Cefuroxime sodium laid
between 100.07% and 102.13%; this demonstrated that the method was accurate within
the desired range.

Page | 89

Spike
level
(%)

80

100

120

Weight taken Weight added Weight found

as Cefuroxime as Cefuroxime as Cefuroxime
sodium(mg)
(mg)
(mg)

%
Recovery

43.20

40.60

40.90

100.73

43.08

40.49

41.1

101.50

42.99

40.41

40.83

101.03

54.32

51.06

51.1

100.07

54.16

50.91

51.23

100.62

54.23

50.97

51.09

100.23

64.83

60.94

61.89

101.55

64.92

61.02

62.32

102.13

64.79

60.90

61.55

101.06

Cumulative % RSD

%
RSD

0.38

0.27

0.53

0.66

Acceptance criteria:
1) The recovery of the drug product should lie within 97% to 103%.
2) The % RSD should be NMT 2.0.

Chromatogram 50%

Page | 90

Chromatogram 100%

Chromatogram 120%
Linearity of Test Method:
The linearity of the test method is determined by plotting the graph for the average mg
found and the response of the different percentage levels (80%, 100% and 120%) obtained
from the accuracy data.

Page | 91

900000
800000
700000
600000
500000

Area 400000
300000
200000
100000
0
0

20

40

60

Amount found (mg)

Acceptance criteria:
The correlation coefficient should be NLT 0.999.
4) LINEARITY OF DETECTOR RESPONSE:
Linearity of detector response was established by plotting a graph to concentration versus
average area and determining the correlation coefficient. A series of solutions of Cefuroxime
sodium standard, were prepared in the concentration range of 11.262g/mL to 81.905g/mL
of Cefuroxime corresponding to about 25% to 150% of target concentration and analyzed as
per test method. A graph was plotted to concentration in g/mL on X-axis versus response
on Y-axis. The detector response was found to be linear with a correlation coefficient of
0.999.

Page | 92

Table 4: Linearity of Detector Response

S. No.

Concentration (g/mL)

01

11.262

02

15.357

03

20.476

04

25.595

05

30.714

06

40.952

07

51.190

08

61.428

09

71.667

10

81.905

Peak area

Acceptance criteria:
The correlation co-efficient should be not less than 0.999
Linearity graph:

Page | 93

5) PRECISION OF TEST METHOD:

Repeatability: The precision of test method was evaluated by assaying six samples of
Cefuroxime sodium for injection for the test preparation. The average assay and the relative
standard deviation of assay were calculated and found to be within the acceptance criteria.
Table 4D: Precision of Test Method: Assay preparation: 1.5g/vial (IV)

Sample No.

%Assay

99.0

99.5

99.8

98.9

99.2

99.6

Average

99.3

%RSD

0.4

Acceptance criteria:
1) The average of six assays should be not less than 97.0% and not more than 103.0%.
2) The relative standard deviation for six determinations should be not more than 2.0%.

Page | 94

Chromatogram of Precision of Test method

6) RANGE
The range of an analytical procedure is the interval between the upper and lower
concentrations of analyte in the sample to be determined in which it has been demonstrated
that the analytical procedure has a suitable level of precision, accuracy and linearity.
Result: The method was linear in the range of
7) RUGGEDNESS:
i) System To System Variability:
System to system variability was conducted on two HPLC systems by using the same
column by assaying six different test preparations of Cefuroxime sodium for injection Test
preparation (1.5g/vial IV) under similar conditions. The system suitability parameters were
evaluated as per the test method on both the systems and found to be within limits.
The average assay for the two systems and relative standard deviation were found to be
within acceptance criteria. Comparison of the results obtained on two systems shows that the
assay method is rugged for system to system variability.

HPLC system

HPLC system 1

HPLC system 2
Page | 95

HPLC system ID No.

CQCE075

CQCE154

Acceptance criteria:
1) The average of six assays should be not less than 97.0% and not more than 103.0%.
2) The relative standard deviation for six determinations should be not more than 2.0%.

Chromatogram of System 1

Chromatogram of System 2

Page | 96

ii) Column To Column Variability:

Column to column variability study was conducted on two columns by using the same
system by assaying six different test preparations of Cefuroxime sodium for injection
test preparation 1 (1.5g/vial IV). The system suitability parameters were evaluated as per
the test method on both the columns and found to be within limits.
The average assay for the two columns and relative standard deviation were found to be
within acceptance criteria. Comparison of the results obtained on two columns shows
that the assay method is rugged for column to column variability.
HPLC Column

Column 1

Column 2

Column ID No.

LCC368

LCC791

Acceptance criteria:
1) The average of six assays should be not less than 97.0% and not more than 103.0%.
2) The relative standard deviation for six determinations should be not more than 2.0%.

Chromatogram of Column 1

Page | 97

Chromatogram of Column 2
iii)

Column to Column Variability on Equivalent Column:

Column equivalency was studied on Phenomenex Sphereclone, C6 150x4.6mm, 5m by
assaying six different test preparations of Cefuroxime sodium for injection test preparation
(1.5g/vial IV). The system suitability parameters were evaluated as per the test method on
both the columns (Waters Spherisorb 150x4.6mm, 5m, C6 and Phenomenex Sphereclone,
150x4.6mm, 5m, C6) and found to be within limits.
The average assay and relative standard deviation for the two columns were found to be
within acceptance criteria. Comparison of the results obtained on two columns shows that
Phenomenex Sphereclone, C6 150x4.6mm, 5m is also suitable for assay of Cefuroxime in
Cefuroxime for injection.
HPLC Column

Column 1

Column 2

Column ID No.

LCC368

LCC813

Acceptance criteria:
1) The average of six assays should be not less than 97.0% and not more than 103.0%.
2) The relative standard deviation for six determinations should be not more than 2.0%.

Page | 98

iv)

Analyst to Analyst Variability:

Analyst to analyst variability study was conducted by two analysts by assaying six different
test preparations of Cefuroxime sodium for injection Test preparation (1.5g/vial IV).
The average assay obtained by both the analysts and relative standard deviations of assay are
found to be within acceptance criteria.
The system suitability parameters were evaluated as per the test method by both the analysts
and found to be within limits. Comparison of the results obtained by both the analysts
shows that the assay method is meeting intermediate precision acceptance criteria.
Analyst I

Analyst II

Chromatogram for Analyst 1

Page | 99

Chromatogram for Analyst 2

Acceptance criteria:
1) The average of six assays should be not less than 97.0% and not more than 103.0%.
2) The relative standard deviation for six determinations should be not more than 2.0%.

Acceptance
criteria

Analyst

System

Column

3210

4211

3499

3850

3210

3042

NLT 1300

USP tailing factor of Cefuroxime

peak

1.1

1.2

1.1

1.1

1.1

1.1

NMT 2.0

% RSD for Cefuroxime peak

area of five replicate standard
injections

0.1

0.6

0.4

0.1

0.1

0.6

NMT 2.0

System suitability parameters

The Column Efficiency of

Cefuroxime peak

Table 21: Assay data for Analyst, System and Column variability
S. No.

Assay % as Cefuroxime
Analyst 1

Analyst 2

System 1

System 2

Column 1

Column 2

99.0

100.1

99.4

102.8

99.0

100.5

99.5

100.0

99.6

102.4

99.5

100.7

99.8

100.7

99.1

102.3

99.8

100.2

98.9

99.9

99.4

102.3

98.9

100.4

99.2

100.5

99.2

101.9

99.2

100.5

99.6

100.5

99.4

102.3

99.6

100.4

Average

99.3

100.3

99.4

102.3

99.3

100.5

% RSD

0.4

0.3

0.2

0.3

0.4

0.2

Page | 100

METHOD VALIDATION SUMMARY:

S.No

1.

2.

Parameter
System
suitability
and System
Precision

Specificity

Experiment
System
suitability

Observation
a) 0.1

a) RSD NMT 2.0%

b) 1.1

b) Tailing factor NMT 2.0

c) 3349

c) Column efficiency NLT 1300

System
Precision

0.2

Impurities

No interference
from impurities

All known impurities are well

separated from the active.

Degradation
products

No interference
from degradation
products

All degradation products are

well separated from the active.

100.07% -102.13%
3.

4.

Accuracy

Linearity

% Recovery
Correlation
coefficient(r)

Cumulative RSD is
0.66
0.999
0.4

5.

Precision

Acceptance criteria

Repeatability
99.3

RSD NMT 1.0%

Recovery of Cefuroxime should

lie within 97% to 103%
RSD NMT 2.0%
Minimum acceptable 0.999
a) RSD of % assay results
should NMT 2.0%
b) Assay of Cefuroxime should
be NLT 97.0 and NMT 103%
Limit derived from accuracy,
linearity and precision studies

6.

Range

S.No

Parameter

Experimen
t

7.

Ruggednes

System

to

Observation

Acceptance criteria

0.4

a) RSD of % assay results should

Page | 101

System
99.3
Column
Column

to

0.2

100.5
s

Column to
equivalent
column
Analyst
Analyst

to

NMT 2.0%
b) Assay of Cefuroxime should
be NLT 97.0 and NMT 103%
a) RSD of % assay results should
NMT 2.0%
b) Assay of Cefuroxime should
be NLT 97.0 and NMT 103%
a) RSD of % assay results should
NMT 2.0%
b) Assay of Cefuroxime should
be NLT 97.0 and NMT 103%

0.3

100.3

a) RSD of % assay results should

NMT 2.0%
b) Assay of Cefuroxime should
be NLT 97.0 and NMT 103%

SUMMARY

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In the present study, Stability indicating Assay method by Reverse Phase HPLC method for
the estimation of Cefuroxime sodium in powder for injection by using Quality by Design
(QbD) and Quality Risk Management (QRM) principles has been developed and optimized.
A simple isocratic HPLC method was developed with Waters Spherisorb C 6 column of
length 150mm, internal diameter 4.6mm and 5 column. pH 6.0 Acetate buffer, Acetonitrile
(100:10) was used as Mobile phase and HPLC grade water was used as Diluent with a Flow
rate of 2.0 mL/min. 50ppm concentration of Standard and Sample preparations were used
with ambient Column temperature and with a Detector wavelength of 254 nm.
The developed method was optimized by Design of Experiments approach using the
parameters like selection of detector wavelength, selection of column, optimization of buffer
pH and mobile phase composition, selection of flow rate, column temperature, and
optimization of standard and sample concentration.

The specificity of the method was demonstrated by analysing the sample with different
stressed condition and injected into the HPLC system with diode array detector. All
degradants peaks were resolved from Cefuroxime peak in the chromatograms of all samples.
The chromatograms of the stressed samples were evaluated for peak purity using Empower
software. For all forced degradation samples, the purity angle for Cefuroxime peak is less
than purity threshold. Cefuroxime peak does not have any Flag in purity results table. This
indicates that there is no interference from degradants in quantitating Cefuroxime. Thus this
method was proved to be Stability Indicating.
The accuracy of the method was determined by performing the recovery experiment at 3
levels (80%-120%). The % recovery obtained between 100.07% and 102.13% proved that
the method was accurate. The drug content in injection was quantified using the proposed
analytical method.
The calibration plot of peak area against concentration was linear in the range investigated
(11.262 81.905g/mL). The low values of RSD showed that method is precise. The linear
regression data for the calibration plot are indicative of a good linear relationship between

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peak area and concentration over a wide range. The method was found to be linear (r 2>
0.999), precise (% RSD <2), accurate and selective.
Robustness of the proposed method was ascertained by deliberately changing the flow rate,
column temperature and mobile phase composition. There was no significant change in the
system suitability factors of Cefuroxime Sodium when the organic composition, flow rate
and column temperature were changed. The low values of the % RSD indicated the
robustness of the method.
Ruggedness of the method was studied by Analyst to analyst, System to system, Column to
column variation. The results of tailing factor, theoretical plates and % RSD of the peak
areas of five replicates were found to be within limit to indicate the method precise. From
the solution stability studies it was proved that test preparations and standard preparations
were stable for 2 day in refrigerator. Mobile phase was found to be stable on the bench top
for two days and five days when kept in refrigerator.
Thus all the validated parameters were found within the acceptance criteria. The validated
method is specific, linear, precise, accurate, robust and rugged for determination based on
the knowledge of method obtained through the method development and the detailed
analytical method performance control strategy can be defined to manage the risk. This
approach has been successfully to develop HPLC method for Cefuroxime sodium.

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