Académique Documents
Professionnel Documents
Culture Documents
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
Page | 1
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:
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
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
Page | 3
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:
-
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.
Page | 4
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.
Page | 5
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
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
Page | 7
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:
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
Page | 9
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)
Page | 10
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]
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)
Page | 12
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
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.
Page | 15
Vis
Lamp
UV
Lamp
Achromatic
Lens
Detector Diode Array
Flow Cell
Homium
Filter
Optical
Slit
Grating
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
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
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
Page | 22
Identificatio
Assay
Page | 23
Limit
Dissolution
Content/potenc
y
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
procedure
Characteristics
Accuracy
Repeatability
Quantitatio
n
Precision
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
Page | 26
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
k > 2.0
Repeatability
Resolution (Rs)
Rs of > 2
T of 2
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.
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 15: Capacity Factor
Where, tR = retention volume at the apex of the peak (solute) and
t0 = void volume of the system.
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
Molecular Formula
C16H15N4NO8S Na
Molecular Weight
446.40
Description
pH value
Melting Point
240C -245C
pKa
Categories
Solubility
Stability
Incompatibilities
Decomposition Products
Page | 33
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
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
non-penicillinase-producing
strains), Streptococcus
pyogenes,Escherichia
: Cefuroxime sodium
Formulation
Strength
: 1.5g
Route of administration
: Intravenous (IV)
Literature Review
1.
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
3.
H.R.N. Salgado et al. described the development and evaluation of a HPLC and UV
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.
5.
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.
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.
Page | 38
8.
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.
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.
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.
12.
Page | 40
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
Material
1.
2.
Manufactured By
Orchid Chemicals and
Pharmaceutical LTD.
Hospira Healthcare Pvt. Ltd.
Reagent
Grade
Make
Acetonitrile
HPLC
Rankem
Methanol
HPLC
Rankem
Sodium acetate
HPLC
Sigma Aldrich
Milli Q water
HPLC
Millipore
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
Micro Balance
Mettler Toledo
MX5
Analytical balance
Sartorius
MSA225P-100-DA
pH meter
Fisher scientific
XL15
Sonicator
UT020
Vortex
v-03
Water bath
Equitron
BHJ-12-S
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
2.
Capacity factor
Min 2
3.
Tailing factor
Min 2
4.
Theoretical Plates
Min 2000
5.
RSD* (Precision)
6.
Selectivity
7.
Accuracy
Page | 44
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
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).
Assay
Solubility
High
Detector wavelength
Low
Page | 46
Column
High
Medium
Mobile phase
High
High
Flow rate
Low
Run time
Low
Column temperature
Medium
10
Injection volume
Low
11
Elution mode
Medium
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.
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
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
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
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
-
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:
-
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
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:
-
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
-
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:
-
Mitigation plan:
-
Page | 53
where factors are investigated simultaneously, the advantage being significant saving in the
time required for the study.
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.
3.
Low
High
1.0 mL/min
2.0 mL/min
3.0
4.0
100:10
70:40
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.
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.
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.
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.
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
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.
Page | 61
PARAMETERS
OPTIMIZED RESULT
Mode of separation
Reverse phase
Mode of elution
Isocratic
Instrument
Waters HPLC
Column
SYSTEM
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:
-
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
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
=
=
=
=
=
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.
Wavelength
254 nm
Response
2905930
Page | 64
3. SELECTION OF COLUMN:
Column Description
Observation
Remarks
Not Satisfactory
Not Satisfactory
Satisfactory
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
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.
Page | 66
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
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
Page | 72
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
Page | 73
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
%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
Page | 75
Observed values
System Suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak
for
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
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
Observed value
Acceptance
criteria
Initial
5 day
2974
4335
NLT: 1300
1.1
1.1
NMT: 2.0
0.2
1.8
NMT: 2.0
for
Time in days
%Assay
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%
3357
33495
2995
NLT: 1300
1.1
1.1
1.1
NMT: 2.0
0.2
0.1
0.1
NMT: 2.0
Acceptance criteria:
All system suitability parameters shall comply as per the test method.
System suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak
for
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
System Suitability
parameters
The Column Efficiency for
Cefuroxime peak
USP tailing factor
Cefuroxime peak
Acceptance
criteria
Observed value
for
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
100.1
100.8
100.4
0.1
0.4
0.9
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
for
USP
tailing
factor
Cefuroxime peak
for
Observed
value
Acceptance
criteria
3349
NLT: 1300
1.1
NMT: 2.0
0.1
NMT: 2.0
Page | 81
Injections
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
%
Degradation
Purity
angle
Purity
threshold
Purity
flag
24.53
0.207
1.086
No
14.08
0.136
1.065
No
11.22
0.125
1.083
No
21.57
0.141
1.119
No
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
Nil
0.069
1.080
No
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
Page | 85
Page | 87
ii)
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
%
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
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
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
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
HPLC system
HPLC system 1
HPLC system 2
Page | 95
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
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 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 II
Page | 99
Acceptance
criteria
Analyst
System
Column
3210
4211
3499
3850
3210
3042
NLT 1300
1.1
1.2
1.1
1.1
1.1
1.1
NMT 2.0
0.1
0.6
0.4
0.1
0.1
0.6
NMT 2.0
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
S.No
1.
2.
Parameter
System
suitability
and System
Precision
Specificity
Experiment
System
suitability
Observation
a) 0.1
b) 1.1
c) 3349
System
Precision
0.2
Impurities
No interference
from impurities
Degradation
products
No interference
from degradation
products
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
6.
Range
S.No
Parameter
Experimen
t
7.
Ruggednes
System
to
Observation
Acceptance criteria
0.4
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
SUMMARY
Page | 102
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
Page | 103
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.
Page | 104