QC and Management Slides.pdf for 4th year

PHARMACEUTICAL QUALITY
MANAGEMENT
SLIDES
4TH PROF
Prepared By:
Ma'am Chritina Peter
Compiled By:
Muhammad Huzaifa

Index
1) QA, QC, GMP, QM
2) Validation And Its Types
3) GMP
4) Tablets
5) Capsules
6) Powders And Granules
7) Syrups
8) Viscosity Of Liquids
9) Emulsions and Suspensions
10) Suppositories
11) Parenterals
12) Biological Assays
Index
13) Alcohal Determination
14) Alkaloidal Drug Assay
15) Vaccines
16) Ointments
17) Alkalinity of Glass
18) QC Charts

Quality Assurance
GMP
Quality Control
Quality Management
1
WHAT IS QUALITY?
The word ‘QUALITY’ is originated from a Latin word-
“QUALITAS” means “general excellence” or a
distinctive feature’.
It is the best possible expected outcome of an effort
under given conditions and terms of skill, experience,
financial and available resources.
Customer Satisfaction
2
QUALITY IN THE HEALTHCARE FIELD
Targets (customers) of the healthcare team:
•The Patient
•The Community
The aim of the healthcare team:
•To provide quality patient care
•To provide quality health service to the community
3
QUALITY IN PHARMACEUTICAL INDUSTRY
The degree to which a set of inherent properties of a
product, system or process fulfills requirements
specifically for quality of drug substance and drug
product (ICH Q9).
The suitability of either a drug substance or drug product
for its intended use. This term includes such attributes as
identity, strength and purity (ICH Q6A).
4

QUALITY ASSURANCE
•It is the sum total of the organized
arrangements with the objective of ensuring
that products will be of the quality required
for their intended use
ISO 9000 defines as "part of quality
management focused on providing
confidence that quality requirements will be
fulfilled"
5
• According to WHO, quality assurance is a wide- ranging concept
covering all matters that individually or collectively influence the
quality of a product. With regard to pharmaceuticals, quality assurance
can be divided into major areas: development, quality control,
production, distribution, and inspections.
6
Aim:
oTo prevent defects with a focus on the process
used to make the product. It is a proactive
quality process.
oTo improve development and test processes
so that defects do not arise when the product is
being developed.
How:
Establish a good quality management system
and the assessment of its adequacy.
Periodic conformance through audits of the
operations of the system.
Benefit:
Prevention of quality problems through
planned and systematic activities including
documentation.
Responsibility:
Everyone on the team involved in developing
the product is responsible for quality
assurance.
As Tool: Managerial Tool
Orientation: Process oriented

The objectives of Quality Assurance are achieved when processes
have been defined which, when followed, will yield a product that
complies with its specification, and when the finished product:
a.Contains the correct ingredients in the correct proportions,
b.Has been correctly processed, according to the defined procedures,
c.Is of the purity required,
d.Is enclosed in its proper container, which bears the correct label (or
is otherwise suitably marked or identified), and
f.Is stored, distributed and subsequently handled so that its quality is
maintained throughout its designated or expected life.
Responsibilities of QA in
Pharmaceutical Industry
To Ensure:
•Raw materials used in the manufacturing are approved and
procured from approved vendor.
•All data are recorded as per cGMP and is reviewed for accuracy
and traceability.
•Procedures are in place for performing the activities, operating
and calibrating the equipment
•Quality is built up in the plant, process, product. That a Robust
Quality system is in place
•Trainings like induction, On job, Scheduled and after any changes
are conducted to respective individuals on time.
• To prepare and approve Quality Policy, Quality Objectives, Quality
Manual and Validation Master Plan.
• Periodic Monitoring of the Quality Objectives.
• Monitors all validation & stability activities are completed as per the
schedule.
• Ensures that all changes impacting the product and the established
systems are documented and reviewed to analyze the impact.
• Ensures that all deviations, OOS/OOT & Market complaints are logged,
investigated to identify the root cause so as to take CAPA to prevent
recurrence.
• Preparation of Annual product quality reports, trending of data,
determining product and process performance.
• To arrange and conduct the self inspection, identify gaps and take CAPA.
• Review of related batch manufacturing records and QC testing data Prior
to release of any batch.
Job Description of QA
1. Technology transfer
2. Validation
3. Documentation
4. Assuring quality of products
5. Quality improvement plans

1. Technology transfer
• Receipt of product design documents from research
centre
• Receipt of the trial and error data and its final
evaluation
• Distribution of documents received from research
centre
• Checking and approval of documents generated
based on research centre documents i.e. batch
manufacturing record
• Scale-up and validation of product
2. Validation
• Preparation of Validation Master plans for
facility/equipment/process Utility, Cleaning and all
the sections of the validation
• Approval of protocol for validation of facility/
equipment/product/ process/Utility
• Team member for execution of validation of
facility/equipment / product/ process
• Final approval of the facility/ equipment/product/
process/Utility validation
3. Documentation
• Written statement or proof. It is an essential part of Quality
Assurance and Quality Control system and is related to all
aspects of Good Manufacturing Practices (GMP). It is mainly
defines the specification for all materials, method
of manufacturing and control.
• Main objective
– To establish, monitor and record “Quality” for all aspects of Good
Manufacturing Practices (GMP) and other Quality System
pertaining
• Type of documents
– Standard operating procedures
– Protocols of tests
– Results
– Reports
“IF you have not documented it
you have not done it”
• Laboratory records
–Description and identification of sample received
–Description of method of testing
–Record of all data secured in the course of the test
–Record of test results and how they compare with
standards of identity, strength and quality
–Record of all deviations and modification of test
–Record of standardization of reference standards
–Record of calibration of equipment

• SOP: Standard operating procedures describe
in a detailed form the activities performed in
the laboratory
• Provide uniformity, consistency and reliability
in each of the activities performed in the
laboratory
• Reduce systematic errors
• Provide training and guidance for new staff
4. Assuring Quality of products
• CGMP training
• SOP compliance
• Audit of facility for compliance
• Line clearance
• In-process counter checks
• Critical sampling
• Record verification
• Release of batch for marketing
• Investigation of market complaints
5. Quality improvement plan
• Feedback received from the compliance team
• Customer complaint history
• Proposals for corrective and preventive actions
• Annual Products review
• Trend analysis of various quality parameters for
products, environment and water
• Review of the Deviations, Change Controls, Out of
Specifications and Failures
Good Manufacturing Practice
(GMP)
“Part of Quality Assurance which ensures that
products are consistently produced and
controlled to the quality standards appropriate to
their intended use and as required by their
Marketing Authorisation or Product
Specification”.
GMP is thus concerned with both production and
quality control

Basic Needs of GMP
a) All manufacturing processes should be clearly defined, and known
to be capable of achieving the desired ends.
b) All necessary facilities are provided, including:
1. Appropriately trained personnel;
2. adequate premises and space;
3. suitable equipment and services;
4. correct materials, containers and labels;
5. approved procedures (including cleaning procedures);
and
6. suitable storage and transport.
c) Procedures are written in instructional form, in clear
and unambiguous language, and are specifically
applicable to the facilities provided.
d) Operators are trained to carry out the procedures correctly.
e) Records are made during manufacture (including
packaging) which demonstrate that all the steps required
by the defined procedures were, in fact , taken and that the
quantity and quality produced were those expected.
f) Records of manufacture and distribution, which enable the
complete history of a batch to be traced, are retained in a
legible and accessible form.
g) A system is available to recall from sale or supply any
batch or product, should that become necessary.
Principles of GMP
• Design and construct the facilities and equipment properly.
• Follow written procedure and instruction.
• Documentation work
• Validation work.
• Check the facilities and equipment.
• Write step by step operating procedure and work on in
sanitation.
• Design , demonstrate, develop job competence.
• Protect against contamination
• Control component and product related to process.
• Conduct planned and periodic audit.
Why GMP is important
• A poorqualitymedicinemaycontaintoxic
substances that have been unintentionally added.
•A medicine that contains little or none of the
claimed ingredient will not have the intended
therapeutic effect.

Importance of GMP
• Ensure that the product are safe for human use
and dose.
• Prevent the toxicity
• Side effect due to variation in drug content.
• Prevent or control contamination.
Objective of GMP
• To understand where the regulation come from, who
has enforcement authority, and why you need to
comply.
• To understand the fundamental benefits, key part of
GMP.
• To minimize the risk involved in pharmaceutical
production that can’t be eliminated by testing the
final product.
• Poor chance for the patient to detect anything wrong.

QUALITY CONTROL - QC
•Part of GMP concerned with sampling, specification &
testing, documentation & release procedures which
ensure that the necessary & relevant tests are performed
& the product is released for use only after ascertaining
it’s quality
•ISO 9000 defines quality control as "A part of quality
management focused on fulfilling quality
requirements".
•QC is a set of activities (testing and analysis) for
ensuring quality in pharmaceuticals.
29
Scope of QC
Items concerned :
•Starting materials
•Packaging materials
•Bulk products
•Intermediate and finished products
•Environmental conditions
30
Aim:
To produce a defect free pharmaceutical, a
regulatory requirement to safeguard the health of
end user;
Objectives:
Use of latest techniques and technologies
31
How:
Development of Standard Testing and Analytical
Procedures.
(mostly by the Pharmacopeias editing and
compiling teams with researcher who developed
the drugs molecules and brand leader companies)
Responsibility:
A specific team that tests the product for defects.
As tool:QC is a corrective tool
Orientation: It is product oriented
32

HOW: BASIC REQUIREMENTS OF QUALITY
CONTROL
1.Adequate facilities and staff should be available for
sampling, inspecting and testing starting materials,
packaging materials, intermediate, bulk and finished
products.
2.Samples of starting materials, packaging materials, and of
intermediate products, bulk products, finished products
should only be taken by, and methods approved by the
person responsible for Quality Control.
3.Results of the inspection and testing of materials, and of
intermediate, bulk or finished products should be formally
assessed against specification by the person responsible for
Quality Control (Qualified Person - or a person designated
by (him/her) before products are released for sale or supply.
4.Product assessment should include a review
and evaluation of relevant manufacturing
(including packaging) documentation.
5.Sufficient reference samples of starting
materials and products should be retained (the
latter, where possible, in the final pack) to
permit future examination if necessary.
•Sampling of raw materials
•Initially quarantined
•Sample are taken and tested to ensure that the materials meets
strict purity guidelines
•Microbiological and chemical testing as written in Pharmacopeia
•In process check
•At intervals QC staff takes samples to check for contamination
and ensure that the composition is as expected
•E.g. 5 tablets (every 15 minutes)
•Final Product checking
•All results are compiled and recorded with name and batch
number on it
Responsibilities of QC in
Pharmaceutical Industry
• QC is responsible for the day-to-day control of quality
within the company.
• This department is responsible for analytical testing of
incoming raw materials and inspection of packaging
components, including labeling.
• They conduct in-process testing when required, perform
environmental monitoring, and inspect operations for
compliance.
• They also conduct the required tests on finished dosage
form.

• Improve the quality of production and reduction in the
production cost.
• Uniform the production and supply of standard quality goods
to consumers.
• Offers full return of the price paid by the consumers and
giving convenience and satisfaction to consumers.
• Reduces spoiled production and rejection from consumers and
dealers.
• Promote exports due to superior and standard quality
production.
• Reduces inspection cost.
• Make products popular in market.
The 4 Main responsibilities of quality control in pharmaceutical industry
include :
a)Efficacy
b)Safety
c)Quality
d)Compliance
Job Description of Quality Control
• Quality Control in the pharmaceutical industry is required for :
• Raw Materials and API: The techniques used include Raman
and IR spectroscopy, Assay( HPLC and Titration ), Physical tests.
• Packaging Components : The various packaging components
which are in contact with the drug are tested. The techniques
include appearance, color, text and monograph etc.
• Finished Products : The techniques include HPLC, Assay,
Dissolution, Content uniformity.
Job Description of Quality Control
Steps in quality control
a)Devising the control over raw materials:
– The quality of the finished products is determined mostly by the
quality of raw materials.
b)Fixing standards and specifications:
– In order to make any scheme of quality control successful, it is
necessary to predetermine standards and specifications.
c)Exercising control over production operations:
– In order to execute efficient practices, the technical expert of the
quality control department must investigate the operating methods.

d) Locating inspection points:
– When the points at which defects occur are wrongly located
or located with delay, it hinders quality control. Hence there
should first be inspection of raw material at vendors place,
then at company’s plant then at various stages during process.
e) Maintaining quality of equipment:
– The final quality of the products is conditioned by the quality
of the equipment and other devices used.
f) Maintaining records:
– The QC department is responsible for setting records related
to quality inspection and control and the number rejected
QA GMP QC
Relationships between GMP, QA
& QC
QA serves as a management tool while GMP and quality control are interrelated
aspect of quality management.
QA QC
•Quality of process
•All those planned actions
necessary to provide
adequate confidence that a
product will satisfy the
requirements for quality,
includes implementation of
cGMP, personnel control.
•Quality of product
•Operational laboratory
techniques and activities
used to fulfill the
requirements for that a
product will satisfy the
requirements quality
43
QA QC
•QA is company Base
•Improving the process
and prevents defects
•Receive Complaints
from Market
•QA is a managerial tool
•QC is Lab based
•Finding defects and
then fixing them
•Test and correct the
complaints
•QC is a corrective tool
44

What are its goals and on what does it focus?
QA aims to prevent
defects with a focus on
the process used to
make the product. It is a
proactive quality
process.
The goal of QA is to
improve development
and test processes so
that defects do not arise
when the product is
being developed.
QC aims to identify (and
correct) defects in the
finished product. Quality
control, therefore, is a
reactive process.
The goal of QC is to
identify defects after a
product is developed and
before it's released.
What and how does it work?
Prevention of quality
problems through planned
and systematic activities
including documentation.
Establish a good quality
management system and the
assessment of its adequacy.
Periodic conformance audits
of the operations of the
system.
The activities or techniques
used to achieve and maintain
the product quality, process
and service.
Finding & eliminating
sources of quality problems
through tools & equipment so
that customer's requirements
are continually met.
Whose responsibility is it and what is the example of it?
Everyone on the team
involved in developing
the product is responsible
for quality assurance.
Verification is an
example of QA.
Quality control is usually
the responsibility of a
specific team that tests
the product for defects.
Validation is an example
of QC.
“The aspect of management function which
determines and implements the “quality
policy”, i.e. the overall intention and direction
of an organization regarding quality, as
formally expressed and authorized by top
management”.
TOTAL QUALITY MANAGEMENT IN
PHARMACEUTICALS

49
The pharmaceutical industry is a vital segment of health care
system which is regulated heavily because; any mistake in
product design or production can be severe, even fatal.
The poor qualities of drug are not only a health hazard but
also a waste of money for both the government and the
individual customers.
So, the maintenance of the quality with continuous
improvement is very important for pharmaceutical industries.
From this concept, Total Quality Management (TQM) was
established.
The aim of TQM is “prevention of defects rather than
detection of defects”.
So TQM is very important for pharmaceutical industries to
produce the better product and ensure the maximum safety
of health care system and also protect waste of money for
both government and individual customers.
The basis of this approach is that the organizational units
should be working harmoniously to satisfy the customer.
50
Therefore, total quality management (TQM) means:
1. Satisfying customers first time, every time;
2. Enabling the employees to solve problems and eliminate
wastage;
3. A style of working, a culture more than a management
technique;
4. Philosophy of continuous improvement, never ending,
only achievable by/or through people.
51
Definition:
52
TQM has been defined as an integrated organizational effort
designed to improve quality at every level.
The process to produce a perfect product by a series of
measures requiring an organized effort by the entire company
to prevent or eliminate errors at every stage in production is
called Total Quality Management.

According to International Organization for Standards (ISO)
TQM is defined as, a management approach for an organization,
centred on quality, based on the participation of all its members
and aiming at long-term success through customer satisfaction
and benefits to all members of the organization and to the
society.
It uses strategy, data and effective communications to integrate
the quality discipline into the culture and activities of the
organization.
53
TQM Model:
Customer
Focus
Total
Participation
Planning
Process
Process
Improvement
Process
Management
TQM
MODEL
54
Basic elements of quality management
The basic elements of quality management are:
oan appropriate infrastructure or “quality
system”, encompassing the organizational
structure, procedures, processes and resources;
osystematic actions necessary to ensure adequate
confidence that a product (or service) will satisfy
given requirements for quality. The totality of
these actions is termed “quality assurance”.
55
Requirements of Quality Management
Sanitation & hygiene

The scope of sanitation and hygiene covers personnel, premises,
equipment and apparatus, production materials and containers, products for
cleaning and disinfection, and anything that could become a source of
contamination to the product.

Potential sources of contamination should be eliminated through an
integrated comprehensive program of sanitation and hygiene.
Qualification & validation

Identify what qualification and validation work is required to prove that
the critical aspects of their particular operation are controlled.

The key elements of a qualification and validation program of a company
should be clearly defined and documented in a validation master plan.

It is of critical importance that particular attention is paid to the validation
of analytical test methods, automated systems and cleaning procedures.

Complaints
All complaints and other information concerning potentially
defective products should be carefully reviewed according to
written procedures and the corrective action should be taken.
Product recalls

There should be a system to recall from the market, promptly
and effectively, products known or suspected to be defective.

The progress of the recall process should be monitored and
recorded. Records should include the disposition of the
product. A final report should be issued, including a
reconciliation between the delivered and recovered quantities
of the products.
Self-inspection and quality audits

To evaluate the manufacturer’s compliance with GMP in all aspects
of production and quality control.

Should be designed to detect any shortcomings in the
implementation of gmp and to recommend the necessary corrective
actions.

Should be performed routinely, and may be, in addition, performed
on special occasions, e.G. In the case of product recalls or repeated
rejections, or when an inspection by the health authorities is
announced.

The team responsible for self-inspection should consist of
personnel who can evaluate the implementation of gmp objectively.

All recommendations for corrective action should be implemented.

The procedure for self-inspection should be documented, and there
should be an effective follow-up program.
Personnel
The establishment and maintenance of a satisfactory system of
quality assurance rely upon people.

Sufficient legally qualified and professionally competent personnel
must be employed to carry out all the tasks for which the
manufacturer is responsible;
 Pharmacists
 Microbiologist
 Chemist / biochemist
 Biotechnologist

Individual responsibilities should be clearly defined and understood
by the persons concerned and recorded as written descriptions.

Personnel should be aware of the principles of gmp that affect them
and receive initial and continuing training, including hygiene
instructions, relevant to their needs.

All personnel should be motivated to support the establishment and
maintenance of high-quality standards.
Premises
Premises must be located, designed, constructed, adapted, and maintained
to suit the operations to be carried out.
 The layout and design of premises must aim to minimize the risk of errors and
permit effective cleaning and maintenance in order to avoid cross-
contamination, build-up of dust or dirt, and, in general, any adverse effect on the
quality of products.
 Premises should be designed to ensure the logical flow of materials and
personnel.
 Premises should be situated in an environment that, when considered together
with measures to protect the manufacturing process, presents minimum risk of
causing any contamination of materials or products.
 Premises used for the manufacture of finished products should be suitably
designed and constructed to facilitate good sanitation, carefully maintained,
electrical supply, lighting, temperature, humidity and ventilation should be
appropriate and such that they do not adversely affect, directly or indirectly,
either the pharmaceutical products during their manufacture and storage, or the
accurate functioning of equipment.
 Rest and refreshment rooms should be separate from manufacturing and control
areas.
 Facilities for changing and storing clothes and for washing and toilet purposes
should be easily accessible and appropriate for the number of users. Toilets
should not communicate directly with production or storage areas.

Equipment
Equipment must be located, designed, constructed, adapted, and
maintained to suit the operations to be carried out.
The layout and design of equipment must aim to minimize the risk
of errors and permit effective cleaning and maintenance in order to
avoid cross-contamination, build-up of dust or dirt, and, in general,
any adverse effect on the quality of products.
Equipment should be installed in such a way as to minimize any
risk of error or of contamination.
 Production equipment should be thoroughly cleaned on a
scheduled basis.
Laboratory equipment and instruments should be suited to the
testing procedures undertaken.
Washing, cleaning and drying equipment should be chosen and
used so as not to be a source of contamination.
Material
Both apis, excipients, regents etc. Should be quarantined
before use and when testified by the QC as are according to
the specs should then be used.
All materials and products should be stored under the
appropriate conditions established by the manufacturer and in
an orderly fashion to permit batch segregation and stock
rotation by a first-expire, first-out rule.
Water used in the manufacture of pharmaceutical products
should be suitable for its intended use.
Documentation
Good documentation is an essential part of the quality assurance
system and, as such, should exist for all aspects of GMP.
To define the specifications and procedures for all materials and
methods of manufacture and control;
To ensure that all personnel concerned with manufacture know
what to do and when to do it;
To ensure that authorized persons have all the information
necessary to decide whether or not to release a batch of a drug for
sale,
To ensure the existence of documented evidence, traceability, and
to provide records and an audit trail that will permit investigation.
To ensures the availability of the data needed for validation,
review and statistical analysis.
The design and use of documents depend upon the manufacturer.
Categories of TQM
64
 Total quality management ensures that every single
employee is working towards the improvement of work
culture, processes, services, systems and so on to ensure
long term success.
 Total Quality management can be divided into four
categories:
 Plan
 Do
 Check
 Act
 Also referred to as PDCA cycle.

Planning Phase
65
 Planning is the most crucial phase of total quality
management.
 In this phase employees have to come up with their problems
and queries which need to be addressed.
 They need to come up with the various challenges they face in
their day to day operations and also analyze the problem’s
root cause.
 Employees are required to do necessary research and collect
relevant data which would help them find solutions to all the
problems.
Doing Phase
66
 In the doing phase, employees develop a solution for the
problems defined in planning phase.
 Strategies are devised and implemented to overcome the
challenges faced by employees.
 The effectiveness of solutions and strategies is also
measured in this stage.
Checking Phase
67
 Checking phase is the stage where people actually do a
comparison analysis of before and after data to confirm the
effectiveness of the processes and measure the results.
Acting Phase
 In this phase employees document their results and prepare
themselves to address other problems.
Thanks
68

VALIDATION
According to FDA
• Validation is "Establishing documented evidence that provides a high
degree of assurance that a specific process will consistently produce a
product meeting its pre-determined specifications and quality
attributes.
• This is to maintain and assure a higher degree of quality of food and
drug products.
• The action of making or declaring something legally or officially
acceptable
According to WHO
• Validation is the documented act of proving that any procedure,
process, equipment, material, activity or system actually leads to the
expected result.
ISO definition
• Validation is the confirmation by examination and the provision of
objective evidence that the particular requirements for a specific
intended use are fulfilled.
• Validation can be defined as a procedure that demonstrates that a
process under standard conditions is capable of consistently
producing a product that meets the established product
specifications.
Need of validation
• Before introduction of a new method into routine use.
• Whenever the conditions change for which a method has been validated, e.g.,
instrument with different characteristics
• Whenever the method is changed, and the change is outside the original scope of
the method.
• Customer satisfaction
• Product liability
• Control production cost
• Safety
• Regulatory Requirement

Advantages of validation
• During the process the knowledge of process increases
• Assures the repeatability of the process
• Assures the fluency of production
• Assures that the product is continuously made according to the marketing
authorisation
• Decreases the risk of the manufacturing problems
• Decreases the expenses caused by the failures in production
• Decreases the risks of failing in GMP
• Decreases the expenses of the every day production even though the
validation itself will create expenses
Scope of validation
Validation requires an appropriate and sufficient infrastructure
including:
• organization, documentation, personnel and financial
• Involvement of management and quality assurance personnel
• Personnel with appropriate qualifications and experience
• Extensive preparation and planning before validation is performed
• A specific programme for validation activities in place
• Validation done in a structured way according to documentation
including procedures and protocols.
• Validation should be performed:
• for new premises, equipment, utilities and systems, and processes and
procedures;
• at periodic intervals;
• when major changes have been made.
• A written report on the outcome to be produced.
• Validation over a period of time, e.g.
• at least three consecutive batches (full production scale) to demonstrate
consistency. (Worst case situations should be considered.)
• Demonstrate suitability for new manufacturing formula or method
Major phases of validation
1. Prevalidation qualification ( phase 1)
2. Process validation (phase 2)
3. Validation maintainance (phase 3)
1. PREVALIDATION Q UALIFICATION PHASE:
This includes all activities relating to product research and
development , pilot batch studies, scale up studies, commercial scale
batches, establishing stability conditions and storage and analysis of in
process and finished dosage forms, equipment qualification,
installation qualification, master production document, operational
qualification and process capacity.

2. PROCESS VALIDATION (PHASE 2)
In this phase the limits of all critical process parameters are
established, verified and validated to ensure that the desired quality of
product can be achieved even under the worst condition.
3. VALIDATION M AINTAINANCE PHASE
This includes review of all documents related to process validation of
audit reports, to make sure that no changes, deviations, standard
operating procedures including change control procedures have been
followed . Hence there is no need for requalification and revalidation.
Documentation of Validation
• The validation activity cannot be completed without proper
documentation of each and every minute activity with utmost details.
Documentation of validation is generally different types such as:
A. Validation Master Plan(VMP)
B. Validation Protocol(VP)
C. Validation Reports(VR)
D. Standard Operating Procedure(SOP)
TYPES OF VALIDATION
The major types of Validation
I. PROCESS VALIDATION
II. CLEANING VALIDATION
III. EQUIPMENT VALIDATION
IV. VALIDATION OF ANALYTICAL METHODS
I. PROCESS VALIDATION
• As per FDA Nov.2008, The collection of data from the process design
stage throughout production, which establishes scientific evidence
that a process is capable of consistently delivering quality products.
• The Process Validation may be/types;
a) Prospective validation.
b) Retrospective validation.
c) Concurrent validation.
d) Revalidation.

a) Prospective validation.
o Carried out during the development stage
oapplies to new processes and new equipment, where studies are
conducted and evaluated, and the overall process/equipment system is
confirmed as validated before the commencement of routine
production.
b) Retrospective validation
• Retrospective validation involves the examination of past experience
of production on the assumption that composition, procedures, and
equipment remain unchanged
• applies to existing processes and equipment, and is based solely on
historical information. Unless sufficiently detailed past processing and
control records are available, retrospective validation studies are
unlikely to be either possible or acceptable.
c) Concurrent validation.
 carried out during normal production.
 The first three production-
scale batches must be monitored as
comprehensively as possible.
 applies to existing processes and equipment. It consists of studies
conducted during normal routine production and can only be
considered acceptable for processes which have a manufacturing and
test history indicating consistent quality production.
d) Revalidation
• Revalidation is needed to ensure that changes in the process and/or
in the process environment, whether intentional or unintentional, do
not adversely affect process characteristics and product quality.
• Revalidation may be divided into two broad categories:
• Revalidation after any change in the process, material or machine
• Periodic revalidation carried out at scheduled intervals.

II. CLEANING VALIDATION
• “A process of attaining and document in sufficient evidence to give
reasonable assurance, given the current state of Science and
Technology, that the cleaning process under consideration does, and /
or will do, what it purpose to do.”
Objective:
• To minimize cross contamination.
• To determine efficiency of cleaning process.
• To do troubleshooting in case problem identified in the cleaning
process and give suggestions to improve the process
• It mainly include 5 stages;
• Stage 1 :Prerequisites
• Determine the most appropriate cleaning procedure for the equipment.
• Evaluate equipment surface
• Equipment qualification
• Hazard evaluation
• Acceptance criteria
• Sampling techniques
• Analytical methods
• Cleaning procedure
• Stage 2 :
• Develop a cleaning validation protocol for the product and equipment being
cleaned
• Stage 3:
• Under taking the cleaning validation process
• Stage 4:
• Generate a cleaning validation report detailing the acceptability of cleaning
procedure for the product and equipment
• Stage 5:
• Maintaining the validated state
iii. EQUIPMENT VALIDATION/QUALIFICATION
• As per FDA, May 1987,‘
Action of proving that any equipment works
correctly and leads to the expected result is equipment qualification.
• It is not a single step activity but instead result from many activities.

Types
a. Design qualification (
DQ)
b. Installation qualification (
IQ)
c. Operational qualification (
OQ)
d. Performance qualification (
PQ)
a. Design qualification (DQ)
DQ defines the functional and operational specifications of the
instrument and details for the conscious decisions in the selection of
the supplier".
Points to be considered for inclusion in a DQ :
• Description of the intended use of the equipment
• Description of the intended environment
• Preliminary selection of the functional and performance
specifications (
technical, environmental, safety)
b. Installation Qualification (IQ)
• IQ establishes that the instrument is received as designed and
specified, that it is properly installed in the selected environment, and
that this environment is suitable for the operation and use of the
instrument.”
c. Operational Qualification (OQ)
• OQ is the process of demonstrating that an instrument will function
according to its operational specification in the selected environment
d. Performance Qualification (PQ)
• PQ is the process of demonstrating that an instrument consistently
performs according to a specification appropriate for its routine use

iv. VALIDATION OF ANALYTICAL METHODS
• The process established by laboratory studies, that the performance
characteristics of the method meet the requirements for the intended
analytical application.
Accuracy:
• The closeness of test results obtained by that method to the true
value. This accuracy should be established across its range.”
Precision:
• The degree of agreement among individual test results when the
method is applied repeatedly to multiple sampling of a homogenous
sample.
Detection limit:
Detection limit is a characteristic of limit tests. It is the lowest amount
of analyte in a sample that can be detected, but not necessarily
quantitated, under the stated experimental conditions.
Quantitation limit
The quantitation limit is a characteristic of quantitative assays for low
levels of compounds in sample matrices, such as impurities in bulk drug
substances and degradation products in finished pharmaceuticals. It is
the lowest amount of analyte in a sample that can be determined with
acceptable Precision and Accuracy under the stated experimental
conditions.

Linearity and range
The linearity of an analytical procedure is its ability to elicit test results
that are directly, or by a well-
defined mathematical transformation,
proportional to the concentration of analyte in samples within a given
range.
The range of an analytical procedure is the interval between the upper
and lower levels of analyte (
including these levels) that have been
demonstrated to be determined with a suitable level of precision,
accuracy, and linearity using the procedure as written
Robustness
The robustness of an analytical procedure is a measure of its capacity
to remain unaffected by small but deliberate variations in procedural
parameters listed in the procedure documentation and provides an
indication of its suitability during normal usage.
Specificity
The ICH documents define specificity as the ability to assess
unequivocally the analyte in the presence of components that may be
expected to be present, such as impurities, degradation products, and
matrix components.
Good Laboratory Practice (GLP)
A quality system concerned with the organizational
process and the conditions under which
laboratory studies are planned, performed,
monitored, recorded, archived and reported.
GLP covers the whole range of Laboratory Processes
History/why was GLP created?
GLP was instituted in US following cases of fraud generated by toxicology
labs in data submitted to the FDA by pharmaceutical companies.
Industrial Bio Test Lab (IBT) was the most notable case, where thousands of
safety tests for chemical manufacturers were falsely claimed to have been
performed or were so poor.
 First introduced in New Zealand in 1972.
 FDA promulgated the Good Laboratory Practice (GLP) Regulations, on
December 22, 1978. The regulations became effective June 1979.
 In 1981 Organization for Economic Co-operation & Development (OECD)
produced GLP principles which were accepted in all other OECD
Member Countries and are in practice.

Objectives of GLP
 To makes sure that the data submitted are a true reflection of the
results that are obtained during the study.
 To makes sure that not to indulge in any fraud activity by labs.
 To promote international acceptance of tests / Data thus
generated .
Purpose
 Ensure quality test data
 Ensure sound laboratory management
 Ensure robust conductance of laboratory testing
 Ensure accurate reporting of test findings
 Ensure safe archival of laboratory data
Main Focus of GLP
W hatever the industry targeted, GLP stresses the importance of the
following main points:
1. Resources:Organization, personnel, facilities and equipment;
2. Characterization:Test items and test systems;
3. Rules:Protocols, standard operating procedures (SOPs);
4. Results:Raw data, final report and archives;
5. Q uality Assurance:Independent monitoring of research processes.
1. Resources: Organization, personnel, facilities and
equipment;
 Services of sufficient relevant professional shall be available with
defined j
obs and responsibilities
 Sufficient number of rooms or areas assure the isolation of test
systems and the isolation of individual proj
ects involving
substances or organisms known to be or suspected of being
biohazardous with biohazard auto detect and control system
(where applicable).
 There should be storage rooms or areas as needed for supplies and
equipment.

 Archive facilities should be provided for the secure storage and
retrieval of study plans, raw data, final reports, samples of test
items and specimens. Archive design and archive conditions should
protect contents from untimely deterioration.
 Handling and disposal of wastes should be carried out in such a way
as not to j
eopardies the integrity of studies. This includes provision
for appropriate collection, storage and disposal facilities, and
decontamination and transportation procedures
 Apparatus, including validated computerized systems, used for the
generation, storage and retrieval of data, and for controlling
environmental factors relevant to the study.
 Apparatus used in a study should be periodically inspected, cleaned,
maintained, and calibrated according to Standard Operating Procedures.
 Apparatus and materials used in a study should not interfere adversely
with the test systems.
 Chemicals, reagents, and solutions should be labelled to indicate
identity(with concentration if appropriate), expiry date and specific
storage instructions. Information concerning source, preparation date
and stability should be available. The expiry date may be extended on
the basis of documented evaluation or analysis.
2. Characterization: Test items and test systems;
 Test item-product going to be
tested
 identity, impurity
profile, potency,
solubility, composition,
stability, chemical
nature.
 new formula or
modified
 previous product
formula
 Test system-to which animal is
going to be administered
Drug Product
Toxicity Testing Q C Testing and Analysis
Characterization
Results submitted to
regulatory agency
Release to the market
& mass produced
3. Rules:Protocols, standard operating procedures (SOPs);
1. Study Plan
Content of the Study Plan:
i. Dates
ii. Test Methods (Description of Materials and Test Methods)
iii. Issues (where applicable)
iv. Records.
v. A list of records to be retained
vi. Conduct of the Study.

4. Results: Raw data, final report and archives;
Content of the Final Report:
i. Identification of the Study, the Test Item and Reference Item
ii. Information Concerning the Sponsor and the Test Facility
iii. Dates
iv. Statement
v. Description of Materials and Test Methods
vi. Results
vii. Storage
5. Q uality Assurance:Independent monitoring of
research processes.
 The study plan, raw data, samples of test and reference items,
specimens and the final report of each study.
 Records of all inspections performed by the Q uality Assurance
Program, as well as master schedules.
 Records of qualifications, training, experience and j
ob descriptions
of personnel.
 Records and reports of the maintenance and calibration of
apparatus.
 Validation documentation for computerized systems.
GLP in Pharmaceutical Industry
 GLP from a Q uality System perspective
 GLP from a Regulatory perspective
GLP AT GLANCE
Product Development / Manufacturing
Non-clinical &
Environmental Clinical
Study;GLP
Data submitted to
Regulatory Agency
Release to market
Audit Inspections
Regulatory Body
Corporate Audit
Inter departmental Audit
Intra-Departmental Audit

QC Tests for Solid Dosage Forms
Quality Control Tests
Physical and chemical Tests
•Appearance, colour, identity, optical rotation, specific
gravity, pH, Solubility, viscosity, disintegration time,
hardness, friability, average weight variation, content
uniformity, impurities, assay, dissolution
Biological and microbiological tests
•Microbiological assay, tests for safety, potency, toxicity,
pyrogenicity, sterility, histamine, phenol coefficient,
antiseptic activity, preservative action
• Dosage Form
Physical form in which a drug is produced and dispensed
Classification
Route of AdministrationPhysical Form
• OralSolid
• TopicalLiquid
• ParenteralSemi Solid
• InhaledGas
• OphthalmicOther
• Otic
Solid Dosage Forms
•Cachets
•Capsules
•Powders
•Insufflations
•Dentrifices
•granules
•Lozenges
•Tablets
•Suppositories

Tablets
Is solid pharmaceutical dosage forms containing
drug substances and excipients and prepared
either by compression or molding methods
Advantages
• Production aspect
– Large scale production at lowest cost
– Easiest and cheapest to package and ship
– High stability
• User aspect (doctor, pharmacist, patient)
– Easy to handle
– Lightest and most compact
– Greatest dose precision & least content variability
– Coating can mark unpleasant tastes & improve pt.
acceptability
– Ease of Administration
Disadvantages
• Some drugs resist compression into dense
compacts
• Drugs with poor wetting, slow dissolution,
intermediate to large dosages may be difficult
or impossible to formulate and manufacture
as a tablet that provide adequate or full drug
bioavailability
• Bitter taste drugs, drugs with an objectionable
odor, or sensitive to oxygen or moisture may
require encapsulation or entrapment prior to
compression or the tablets may require
coating
Essential properties of tablets
• Accurate dosage of medicament, uniform in
weight, appearance and diameter
• Have the strength to withstand the rigors of
mechanical shocks encountered in its
production, packaging, shipping and
dispensing
• Release the medicinal agents in the body in a
predictable and reproducible manner
• Elegant product, acceptable size and shape
• Chemical and physical stabilities

The quantitative evaluation and assessment of a tablet’s
chemical, physical and bioavailability properties are
important in the design of tablets and to monitor product
quality.
These properties are important since chemical breakdown or
interactions between tablet components may alter the
physical tablet properties, and greatly affect the
bioavailability of the tablet system.
There are various standards that have been set in the various
pharmacopoeias regarding the quality of pharmaceutical
tablets.
10
QC Tests to be Performed
• Physical TestsChemical Tests
• General AppearanceContent Uniformity
• Thickness and DiameterAssay
• FriabilityDissolution
• Weight Variation
• Hardness
• Disintegration
• GENERAL APPEARANCE
The general appearance of a tablet, its identity and general
elegance is essential for:
• consumer acceptance,
• for control of lot-to-lot uniformity and
• tablet-to-tablet uniformity.
The control of general appearance involves the measurement
of:
• size, shape, color, presence or absence of odor, taste etc.
12

• ORGANOLEPTIC PROPERTIES
• Color is a vital means of identification for many pharmaceutical tablets
and is also usually important for consumer acceptance.
• The color of the product must be uniform within a single tablet, from tablet
to tablet and from lot to lot.
• Techniques used: Reflectance spectrophotometry, tristimulus colorimetric
measurements and micro reflectance photometer have been used to
measure color uniformity and gloss on a tablet surface .
• Odor may also be important for consumer acceptance of tablets and can
provide an indication of the quality of tablets as the presence of an odor in
a batch of tablets could indicate a stability problem, example such as the
characteristic odor of acetic acid in degrading aspirin tablets.
• Taste is also important for consumer acceptance of certain tablets (e.g.
chewable tablets) and many companies utilize taste panels to judge the
preference of different flavors and flavor levels in the development of a
product.
• Taste preference is however subjective and the control of taste in the
production of chewable tablets is usually based on the presence or absence
of a specified taste. 13
WEIGHT VARIATION TEST
•To ensure constant dose between individual tablets
•Traditionally dose variation is determined by
– Weight variation and
– Content Uniformity
WHY?
•If the drug forms the greater part of the tablet mass, any weight
variation reflects variations in the content of active ingredient.
•In case of potent drugs, the excipients form the greater part of the
tablet weight & so the correlation between tablet weight and
amount of active ingredient can be poor.
14
• If the weight of tablets is higher than the
recommended range then the assay or
content uniformity may also be high.
&
• If the weight of tablets is lower than the
recommended range then the assay or
content uniformity may also be low.
Method:
• Take 20 tablet and weighed individually.
• Calculate average weight and compare the
individual tablet weight to the average.
The tablet pass the U.S.P. test if not more than 2 tablets are
outside the percentage limit and if no tablet differs by
more than 2 times the percentage limit.

% error= (Avg wt-act wt/avg wt) x 100
USP LIMIT
Average weightPercent difference
• 130 mg or less±10 ±20
• More than 130 mg to 324 mg ± 7.5 ± 15
• More than 324 mg ± 5 ±10
BP LIMIT
Average weightPercent difference
• 80 mg or less ± 10 ±15
• More than 80 mg to 250 mg ± 7.5 ±12.5
• More than 250 mg ± 5 ±10
17
• Limits:
Upper limit: average weight + (Average weight * % error)
Lower Limit: average weight - (Average weight * % error)
Causes of weight variation
1. The size & distribution of the granules being compressed (presence of too large
or too fine granules).
• Granules size: For small size tablets if we use large size granules it will result in
improper die filling and improper die filling results in weight variation.
• For large size tablets if we use small size granules it may also result in weight
variation of tablet during tablet compression.
More Fine:
• More fine means our final product has less granules and more fine powders.
• If we have more fine in our formulation it may result in poor flow of product
from compression machine hoper to the feeder.
2. Poor flow (cause incomplete filling of the die).
3. Poor mixing. (Sometimes the lubricants &
glidants have not been well distributed).
4. When lower punches are of unequal lengths,
the fill of each die varies.
SIZE & SHAPE
It can be dimensionally described & controlled.
Use of: Vernier Caliper, Screw Gauge
•thickness can vary with no change in weight, due to
= difference in density of granules and
= pressure applied to the tablets
20

• Thickness range = 2-4 mm (depending on
diameter of tablet).
• Diameter range = 4-13 mm.
• Apply on tablets which are not sugar coated,
enteric or film coated.
• Deviation/Tablet thickness should be
controlled within a ± 5% variation of standard
value/stated diameter.
• For diameter exceeding 12.5 mm = ±3%
deviation.
HARDNESS: Breaking Force/Crushing Strength
•Def.: The force required to break a tablet along its
diameter by applying compression loading.
•Why hardness required? Tablets require a certain
amount of strength or hardness to:
1. Withstand mechanical shocks of handling in
manufacture, packaging and shipping.
2. Withstand reasonable abuse when in the hands of
the consumer.
3. The relationship of hardness to tablet
disintegration, dissolution and solubility .
22
Hardness Variation:
It depends on:
•Compression force
•Concentration and type of binding agent
•Method of tablet preparation
 If the tablet initially is TOO HARD, it may not disintegrate in the
requisite period of time.
 If it is TOO SOFT, it may not withstand the necessary multiple shocks
occurring during handling, shipping, and dispensing.
 If a tablet is too hard, first check the disintegration. If it is
in limits then the batch or lot is passed
23
Instrument: Monsanto, Erweka, Pharmatest, pfizar
In Erweka and pharmatest Constant speed (0.05—3.5 mm/sec)
Constant force(10-200 N/sec)
Test Description:
• A tablet is placed between two anvils, force is applied to the
anvils, & the crushing strength that just causes the tablet to break
is recorded
Unit: (in Kgf or N). 1 kgf= 9.8N= 1kp=1kg/cm2
Limits: Tablet hardness should be between: 5–10 kg/cm2
In case of Hypodermic and chewable tablets: 3 kg/cm2
Sustained or modified Release:10-20 kg/cm2
24

Sun Mar 24 09:24:32 2024
25
Stokes monsanto type
26
FRIABILITY (Attrition-resistance test)
• Def: It's the measure of a tablet's strength against friction and shocks
that can cause tablets to chip/powder or break.
•Why measure friability?
•Tablets that tend to powder & fragment when handled:
1.lack elegance & consumer acceptance,
2.Create excessively dirty processes in areas of manufacturing
as coating & packaging.
3. Can also add to a tablet's weight variation or content
uniformity
4. When tabs loose moisture on aging they may become more friable
Instrument used-------Roche friabilator by Pharmatest
•Drum/plastic chamber, Diameter 287…. Depth 38 mm
•It subjects a number of tablets to the combined effects of abrasion & shock by
utilizing a plastic chamber that revolves at 25 rpm, dropping the tablets with
each revolution. they fall 6 inches on each turn. friabilator, which is then
operated for 100 revolutions/4min (i.e., 25 rev/min).
Test Description:
•A pre-weighed tablet sample is placed in the friabilator, which is then
operated for 100 revolutions.
 For tablets whose unit weight is ≤ 650 mg------sample of 6500 mg/6.5 g
 For tablets whose unit weight is > 650 mg---- sample of 10 tablets
•The tablets are reweighed.
•Calculate %wt loss= (Initial weight-Final weight)/Initial Weight x 100
Limits == expressed as %w/w ------------- Less than 0.8 %
27
• Tablets are then dusted and reweighed.
• Conventional compressed tablets that lose less than 1.0% of
their weight are generally considered acceptable.
•
• •% friability = (W0 – Wf / W0) x %.
• W0 = initial weight, Wf = final weight.
Sun Mar 24 09:24:32 2024
322 PHT
28

DISINTEGRATION TEST:
Definition: The state in which any residue of the unit, except
the fragments of insoluble coating or capsule shell,
remaining on the screen of the test apparatus or adhering to
the lower surface of the discs, if used, is a soft mass having
no palpably firm core
- Does not mean complete dissolution
- Attempts to simulate in-vivo conditions.
29
Apparatus: Basket Rack Assembly
•6 open tubes, 7.75 ± 0.25 cm long, 2mm thick, and diameter
21.5 mm
•Tubes held vertically by 2 plastic plates (diameter 9cm) with 6
holes (diameter 24mm) equidistant from center and from one
another.
•Plates are held rigidly in position, 7.5cm apart by means of 3
metal bolts
•10 mesh (2mm opening) stain less steel wire cloth on the lower
end of tubes.
•Glass tubes and upper plastic plate connected to steel plate 9cm
diameter
Sun Mar 24 09:24:32 2024
32

• To the stainless steel plate, an 8cm (length) and
7mm(diameter) metal rod attached that is provided to
suspend the assembly from raising and lowering device.
• A cylindrical glass jar diameter 15cm, and height 20-
21cm
• Thermostat arrangement to maintain the fluid at 37 ± 2
°C. (Water Bath)
• Discs: Used when specified.. Perforated cylindrical
disc. 9.5 ± 0.15 mm thick and 20.7 ± 0.15 diameter.
Discs Purpose;
• to obtain solid to solid contact as in stomach.
• to prevent floating tablets from coming out of tubes.
Condition
•Temperature:37 ± 2 °C.
•Movement: Upward and downward
•On upward movement tablets should remain 2.5cm below the
surface of liquid and on downward movement tablets should
remain 2.5 cm above the bottom of fluid containing vessel
•Distance 5-6cm
•Speed… 28-32 cycles per minute
Uncoated tablets
•Place one tablet in each tube
•Add a disc to each tube
•Water as immersion fluid (unless specified)
•At the end of time specified in monograph lift the
basket, observe the tablets
Limit:
•All tablets should disintegrate
•If one or 2 fail to disintegrate, repeat with 12
more tablets. 16 out of 18 tablets should
disintegrate completely.
•USP------ less than 30 minutes
•BP------ less than 15 minutes

Plain Coated Tablets
•Place one tablet in each tube,
•Add disc to each tube.
•Simulated gastric fluid as immersion fluid
•After 30 minutes, lift the basket, observe the
tablets
Limit:
•If one or 2 fail to disintegrate, repeat with 12 more
tablets. 16 out of 18 tablets should disintegrate
completely.
•Film coated tablets ----- NMT 30 minutes
•Other---------------------- NMT 60 minutes
Enteric Coated Tablets
•Place one tablet in each tube,
•If soluble coating first immerse the assembly in water for 5
minutes at room temp.
•Operate without the discs with Simulated gastric fluid as
immersion fluid
•After (1 hour-USP; 2 hours-BP) , lift the basket, observe the
tablets
•No tablets should disintegrate
•Then Operate with the discs in Simulated intestinal fluid as
immersion fluid
•After (2 hour-USP; 1 hour-BP), lift the basket, observe the
tablets
•If one or 2 fail to disintegrate, repeat with 12 more tablets. 16
out of 18 tablets should disintegrate completely after 3rd hour.
Buccal and Sublingual tablets
•Same procedure as for uncoated tablets
•Only the use of discs is omitted
•Time is specified in the monographs----Buccal
Tablets ------ 4 hours
Sublingual Tablets ----2- 3 minutes
•Limits – same as for uncoated tabs.
•Effervescent - less than 5 min
•Soluble - less than 1-2 min
•Chewable ???
•Sustained or modified Release ???
•
ASSAY
• 1st step in assay – grinding of 20 tabs.
• Analysis of an aliquot – representing a certain
amount of drug, normally in single unit.
• Method of analysis – prescribed in
monograph.
• Results – as percentage of active drug in
tablet.
• Compared with limits in monograph.

CONTENT UNIFORMITY
• When ingredients of tablet granulation are
homogenous – tablet weigh test can be
considered as measure of drug content.
• Content uniformity test performed – when
individual monograph requires.
DOSE AND RATIO OF THE
API
DOSE AND RATIO OF THE
API
≥25mg or ≥25% <25mg or <25%
Uncoated WV CU
Film coated WV CU
Other coating CU CU
Method:
•30 tabs selected randomly .
•Out of which – 10 tabs crushed and assayed individually.
For 10 individual tabs:
•All tabs = 85 to 115% (±15) of the labeled drug content.
If;
•9 tabs = ±15 of the labeled drug content.
•1 tab but NOT more than one = falling out of ±15% range but
within ± 25%
Then; assay remaining 20 tabs.
The requirements are met if NOT more than 1/30 tabs results is
outside the limits of 85-115%
DISSOLUTION
• Solubility vs dissolution ???
• Def: process where a solute dissolves in a
solvent to form a solution.
• Is a chemical test
• Dissolution rate: amount of drug that goes
into solution per unit time under standardized
conditions of temp, solid-liq interface and
solvent composition

• Drugs having solubility – greater than 1% w/v,
have no problem.
• But drugs having lower solubility – dissolution
systems are designed.
• So test not used for all drugs hence specified
in monographs
• Dissolution ---------- Tool for predicting the
Bioavailibility ----- IVIVC
Two methods used for dissolution.
•Method I & method II
•Used method – prescribed in individual
monograph.
Method I (apparatus 1)
Basket type
Apparatus – consists of following:
•covered vessel – made of plastic , glass or other
inert transparent material.
•motor
•metallic drive shaft
•cylindrical basket
•constant temp. bath

Vessel
•immersed in suitable water bath.
•(Water bath – maintain temperature at 37 ± 0.5°C.)
•made of plastic , glass or other inert transparent
material
•cylindrical with slightly concave bottom.
•Height = 16 cm
•Internal diameter = 10 cm
•Capacity = 1000 ml
•Sides are flanged near top – to accept a fitted cover.
fitted cover
Cover has 4 ports – one of which is centered.
•Shaft of motor placed in centre port.
•One outer port for insertion of thermometer.
•1 port – for sample removal &
•1 port – for dissolution medium replacement.
Motor – fitted with speed regulating device.
•Speed limit of motor = 25 to 200 rpm
•Maintained – as mentioned in monograph,
within ±5%.
Shaft/rod
•Length = 30 cm
•Diameter = 6mm
•Can be raised or lowered to position the basket.
•Shaft is centred – so basket rotates smoothly.
Basket – 2 parts.
•Top – attached to shaft (solid metal having 2 mm
vent).
•Fitted to lower part by 3 clips – that allow removal of
lower part.
•Detachable part (lower part) – made of 40 mesh
stainless steel cloth (sieve opening = 420μm).
Mesh is in the form of cylinder.
•Height = 3.66 cm
•Diameter= 2.5cm

• Gold plated basket – recommended for dilute
acid media.
• Dissolution medium – as specified in individual
monograph. 500-900 ml
Procedure
• Take 900ml dissolution medium in vessel.
(Vessel – already immersed in constant temp. Bath.)
• Allow dissolution medium to – temp. 37 ± 0.5 °C.
• Place 1 tablet in the basket.
• Assemble apparatus & immerse basket in vessel.
-distance b/w basket & bottom of vessel = 2 ± 0.2cm.
• Rotate basket at rate – specified in individual
monograph.
• After time specified – withdraw samples for
analysis.
-Analysis method – specified in individual
monograph.
• Repeat test on 5 additional tablets.
Limits
• If 1 or 2 tablets fail – repeat test on 6
additional tablets.
• 10 out of 12 tabs – must meet the
requirements.

Apparatus 2
• Apparatus – same as apparatus 1.
Except:
• Paddle (formed from blade) – replaced with basket.
• Distance b/w paddle & bottom of vessel = 2.5 ± 0.2cm.
• Dosage unit is allowed to sink to the bottom of the vessel
– before rotation of paddle.
• Small wire helix may be attached or placed over dosage units.
-in order to prevent floating.
• Apparatus 1 is superior to 2 for non
disintegrating tablets and floating tablets
(helix as a sinker may be used for floating
tablets in case of apparatus 2)
• Inferior in case of tablets contains gums---may
clog to the mesh.
CAPSULES

Capsules
• Capsules are solid dosage forms in which the
medication contained within gelatin shells.
• Medication may be – a powder, a liquid or a
semisolid mass.
• Usually intended to be administered orally.
• Two types – soft gelatin & hard gelatin
capsules.
QC TESTS FOR CAPSULES
Tests for capsules:
• Uniformity of weight
• Disintegration test
• Assay of active ingredients
• Content uniformity test (same as for tablets)
• Dissolution test (same as for tablets)
UNIFORMITY OF WEIGHT
• This test applies to all types of capsules and it
is to be done on 20 capsules.
Two methods used:
Method A
• for capsules with dry content
Method B
• for capsules with liquid or paste
Method A (Hard Gelatin Capsules):
• Weigh an intact capsule.
• Open the capsule without losing any part of the
shell and remove the contents as completely as
possible.
• Weigh the shell.
• The weight of the contents is the difference
between the weight of whole capsule and empty
shell.
• Repeat the procedure with a further 19 capsules
selected at random.
• Determine the average weight.

Limits:
Not more than two of the individual weights
deviate from the average weight by more than
the percentage deviation shown in the table
below, and none deviates by more than twice
that percentage.
Method B (Soft Gelatin Capsules):
• Weigh an intact capsule
• Open it by cutting delicately.
• Remove the contents and wash the shell with a
solvent
• Wait for the solvent to evaporate(if ether is used—
wait until the odour of the ether is no longer
perceptible).
• Again weigh the shells.
• Difference between the weights is the weight of
contents
• Repeat with remaining 19 capsules
Limits:
• 18 out of 20 should have percent deviation of
±7.5% from the average weight and none
should be out of ±15%
Avg cap wt % dev % dev
±7.5% ±15%
DISINTEGRATION TEST
• Def: Disintegration is the state in which no
residue except fragments of capsule shell,
remains on the screen of the test apparatus
or adheres to the lower surface of the disc.
• The disintegration test determines whether
tablets or capsules disintegrate within a
prescribed time when placed in a liquid
medium under the prescribed experimental
conditions.

Method
Applies to hard and soft capsules.
• Introduce one capsule into each tube and suspend the
apparatus in a beaker containing 600 ml water @ 37oC.
• If hard capsules float on the surface of the water, the discs
may be added.
• Operate the apparatus for 30 minutes; remove the
assembly from the liquid.
The capsules pass the test if
• No residue remains on the screen of the apparatus OR,
• If a residue remains, it consists of fragments of shell OR,
• Is a soft mass with no palpable core.
• If the disc is used, any residue remaining on its lower
surface should only consist of fragments of shell.
ASSAY OF ACTIVE INGREDIENTS
• 1st step in assay – take contents of 20 capsules.
• Analysis of an aliquot – representing a certain
amount of drug, normally in single unit.
• Method of analysis – prescribed in monograph.
• Results – as percentage of active drug in tablet.
-Compared with limits in monograph.
QUALITYCONTROLTESTFOR
POWDERS&GRANULES
A pharmaceutical powders is a mixture of finely divided drug
and chemicals in dry form in a solid dosage form meant for
internal and external use and available in crystalline (or)
amorphous form.
CLASIFICATION OFPOWDERS
1.Bulk powder for internal use
2.Bulk powder for external use.
3.Simple and compound powder for internal use
4.Powder enclosed in sachets and capsules.
5.Compressed powders(tablets).

QUALITYCONTROLTESTFORPOWDERSANDGRANULES
1. Particle size and shape determination
2. Surface area
3. Flow properties
4. Moisture content
5. % fine
6. Granule strength and friability
7. Dissolution
8. Assay
6. Percentage fines
PARTICLE SIZE
Size affects the average weight of tablet, disintegration time,
weight variation, friability, hardness , flowability and drying rate.
The methods for determining size and shape are:
a. Sieving
b. Sedimentation rate
c. Microscopy (SEM)
d. Laser light Scattering
e. Light energy diffraction
f. Electronic sensing zone
Sieving method
 Sieving method is an ordinary and simple method.
It is widely used as a method for the particle size
analysis .
 Sieving method directly gives weight distribution.
Thus percent of coarse, moderate, fine powder is
estimated by this method.
 Most sieve analyses utilize a series, stack (layer)
of sieves which have the coarser mesh at the top
of the series and smallest mesh at the bottom.
 A sieve stack usually comprises 6-8 sieves.
 Powder is loaded on to the coarsest sieve of the
stack and then it is subjected to mechanical
vibration for specified time, eg 20 minutes.
 After this time, the powder retained on each sieve
is weighed .
 The particles are considered to be retained on the
sieve mesh with an aperture corresponding to the
sieve diameter.

 It is not commonly used for granules but generally used for
drug substances.
 If required particle size is measured and from this surface area
is measured
 Mostly used methods are gas adsorption method and air
permeability method.
 In gas adsorption, gas is adsorbed as monolayer on particles.
This is in term calculated and converted to surface area.
 In air permeability, the rate of air permeates a bed of powder
is used to calculate surface area of powder sample.
SURFACE AREA
 Specific surface area: surface area per unit weight
(Sw) or unit volume (Sv) can be estimated as
follows:
 Sv = surface area of particles
volume of particles.

The flow properties of powder determine by
following methods.
 Angle of Repose
 Compressibility Index and
 Hausner’s ratio
ANGLE OF REPOSE
 The angle of repose is a relatively simple technique for
estimating the flow properties of a powder.
 It can easily be determined by allowing a powder to flow
through a funnel and fall freely onto a surface.
 The height and diameter of the resulting cone are
measured and the angle of repose is calculated from this
equation:
tan q = h/r
θ = tan-1(h/r)
Where h = height of pile and r = radius of the base of the
pile

 The amount of moisture present in the granule is
called moisture content.
 Generally granules contain 2% moisture. It is
required for the binding of the powder or granules
during compression in die cavity.
 Percentage of moisture is calculated by using
moisture balance or IR balance. IR balance
consist of simple balance containing IR bulb
which produces heat inside the chamber.
 A small amount of sample is placed in IR balance and
weight is recorded (Initial reading) .
 IR balance is operated at specified time and
temperature. Sample is reweighed (Final reading)
Moisture content = [(Initial wt – Final wt )/ Initial wt] * 100
PERCENTAGE FINES
 % fines means amount of powder remained in the granule.
 It is necessary for the tablet compression because if we are using 100%
granules then it is difficult to maintain hardness and the weight of tablet
because granules have free space in the die cavity and after compression
tablet will crack due to air. Thus fine particles fill those spaces and and air bubbles
will notbe trapped
 % fines can be calculated by using sieve method.
 % fine should not be more than 15%.

Quality Control Test for Oral
Liquids
QC Tests for Syrups and Elixirs
• Syrups:
Concentrated, Aqueous Preparations of Sugar/
substitutes with or without adding flavouring and
medicinal agents
• Medicated Syrups
Contains Medicinal Agents
• Non medicated (Flavouring Syrups)
Cherry Chocolate, Strawberry
• ELIXIRS:
Clear sweetened, hydro alcoholic preparations generally
intended for oral use and are usually flavored to enhance
their palatability
• Medicated
• Non medicated (used as vehicles for medicated)
Aromatic , isoalcoholic elixir
• Water
• Light transmittance
• Visual inspection
• Odour and taste
• Determination of sucrose concentration
• Determination of alcohol concentration (for elixirs only)
• pH determination
• Weight per ml
• Labelling
• Assay of active content(s).
• Viscosity determination
• Uniformity of mass
• Uniformity of volume

LIGHT TRANSMITTANCE METER:-
In a light transmittance meter, sample is checked
for color by passing light through the sample.
The percent of light transmission is compared to
light transmission rates set for different grades.
When using one, you need to be sure:
• there are no fingerprints on the test bottle,
• the sample has no bubbles or cloudiness.
Any of these conditions may diminish the light
that is transmitted through the sample and
therefore lowers the grade of the sample.
VISUAL INSPECTION:- panel board
• for purity
• for appearance.
• Elegance
• No solid Material
ODOUR AND TASTE===Palatable
CLEAN AND PURIFIED VEHICLE (WATER):-
The water is filtered and purified at the plant to destroy
any micro-organisms and to remove particles from the
water. Quality control technicians test the water
frequently to ensure that it is clean and pure beforehand.
WEIGHT PER mL
Def: Weight expressed in grams, of a 1 millilitre of a
liquid when weighed in air at specified temperature.
Method:
• Weigh a clean and dried pycnometer
• Fill the pycnometer with sample. Remove any excess
and weigh.
• Subtract the total weight from weight of pycnometer
• Weight per ml --- divide the weight of
sample by volume of pycnometer
• Unit: g/ml
pH DETERMINATION
• Negative log of hydrogen ion concentration
pH = − log [H+]
• Hydroxyl ion concentration is explained as
pOH = − log [OH+]
• pH is the measurement of Acidity or basicity
of a solution

• pH Scale/Sorenson’s pH Scale
The scale on which the pH values are
computed
• pH Measurement
–pH Paper Method
• Simplest and cheapest
• Dip a piece of pH paper in sample. Paper
impregnated with chemicals and colour changes.
• Colour is compared with a chart supplied
• pH Meter Method
• For greater accuracy
• Consists of glass electrode connected to an
electronic meter that measures and displays the
pH.
Importance of pH
• Drug Solubility and Absorption
• pH can effect solubility of Drugs
• Weakly acidic drugs are more soluble in
alkaline solution---converted into salt form
(more soluble)--- if pH is lowered acidic drug
will precipitate.
• Weakly basic drugs are more soluble in acidic
solution--- if pH is raised, basic drug will
precipitated.

• Drug stability
The pH of the solution can affect the degradation
rate of the drug. Depending on the drug, a pH at
which the drug is most stable can vary
A Drug in the non-ionized form is more
permeable than in its ionized form.
• Irritation:
The pH of a pharmaceutical solution can not be
too acidic or too basic. The further away we get
from the physiological pH (7.4) the greater the
irritation
DETERMINATION OF SUCROSE CONCENTRATION:
• If the concentration of sucrose in the syrup is very high it
may crystallize the syrup and less sucrose concentrations
may favor the microbial growth.
• There is no specific method for the determination of sucrose
in syrup, we use HPLC and UV-spectroscopy for this
purpose.
DETERMINATION OF ALCOHOL CONCENTRATION
(FOR ELIXIRS ONLY):
Elixir usually contains 5 to 40% alcohol.
Distillation , Specific gravity.
Specific gravity from alcoholometric table
LABELLING
Every pharmaceutical preparation must comply with the labelling
requirements established under Good Manufacturing Practice
The label should include:
• the name of the pharmaceutical product;
• the name(s) of the active ingredients; INNs (International
Nonproprietary Names) should be used wherever possible;
• the amount of active ingredient in a suitable dose-volume;
• the name and concentration of any antimicrobial preservative and the
name of any other excipient;
• the batch (lot) number assigned by the manufacturer;
• the expiry date and, when required, the date of manufacture;
• any special storage conditions or handling precautions that may be
necessary;
• directions for use, warnings, and precautions that may be necessary;
• the name and address of the manufacturer or the person responsible for
placing the product on the market.

• Assay
–Specified in monograph
–Performed on liquids whose net volume is not more
than 300ml
–For viscous liquids known amount of water is added
and mixed with liquids and final volume is determined
–For non viscous liquids, pour the contents of each
container into calibrated volume measures and
determine the volume
• First 10 containers are studied for uniformity of volume. If
one is out of range. Repeat with 10 more.
• Not more than 1 out of the 20 containers should be out of
the given range
Uniformity of mass
• Liquid preparations for oral use that are
presented as single-dose preparations comply
with the following test. Weigh individually the
contents of 20 containers, emptied as
completely as possible, and determine the
average mass.
• Not more than 2 of the individual masses
deviate by more than 10% from the average
mass and none deviates by more than 20%.
Uniformity of mass of doses delivered by the
measuring device.
The measuring device provided with a multidose
liquid preparation for oral use complies with the
following test. Weigh individually 20 doses taken at
random from one or more multidose containers with
the measuring device provided and determine the
individual and average masses.
Not more than two of the individual masses deviate
by more than 10% from the average mass and none
deviates by more than 20%.

VISCOSITY OF LIQUIDS
DEF:
• The force required to move one plane surface past
another under specified conditions when the space
between them is filled by the liquid in question
• Viscosity is a property of liquids that is closely related
to the resistance to flow.
• The viscosity is due to friction between neighbouring
particles. A liquid’s viscosity depends on size and
shape of its particles and attractions between
particles.
Unit:
• The basic unit is the poise (according to USP)
• However, viscosities commonly encountered represent
fractions of the poise, so that the centipoise (1 poise =
100 centipoises) proves to be the more convenient
unit.
• Other units : Stroke/ centistroke
Temperature specification is important as
viscosity changes with temperature
• 1o rise in temperature decreases viscosity by
2%
• Should be held to within ± 0.1o
Determination of viscosity.
In Newtonian System, rate of shear is directly proportion
to shear stress. There fore, a single point instrument with a
single rate of shear will be sufficient, as the curve passes
through origin.

Non-Newtonian systems.
These systems do not follow Newton's law of flow and they
are liquid and solid heterogeous dispersions as colloidal
solutions, emulsion, liquid suspensions, ointments, creams and
many other similar products.
Their viscosity can not be determined by ordinary viscometer.
There fore, by using Rotational Viscometer, we have various
consistency curves showing THREE Classes of flow as:
I). Plastic- fluid will behave as a solid under static conditions.
II). Pseudo plastic - decreasing viscosity with an
increasing shear rate
III). Dilatants - Increasing viscosity with an increase in shear rate
In Non-Newtonian system, operation of instrument
at different rate of shear can only characterize the
system. The instrument is known as “Multipoint
instrument”.
Hence, all instruments can be used for Newtonian
system’s viscosity determination. Where as , only
multipoint instruments can be used for
determination of viscosity of Non-Newtonian
system i.e. operated at various rate of shear.
Measurement of viscosity
Rheometers
• A rheometer is a laboratory device used to
measure the way in which a liquid flows in
response to applied forces. It is used for those
fluids which cannot be defined by a single value of
viscosity and therefore require more parameters
to be set and measured than is the case for a
viscometer.
• For liquids with viscosities which vary with flow
conditions
• Brookfield rheometer

VISCOM ETER
• Used to measure viscosities which are under one flow
conditions
• either the fluid remains stationary and an object moves
through it, or the object is stationary and the fluid moves past
it.
• Type used Depends upon the individual monograph
• Capillary Tube Viscometer
• Falling sphere viscometers
• Falling Ball Viscometer
• Falling Piston Viscometer
• Oscillating Piston Viscometer
• Vibrational viscometers
• Rotational viscometers
– Electromagnetically Spinning Sphere Viscometer (EMS Viscometer)
– Stabinger viscometer
• Bubble viscometer
• Rectangular-Slit Viscometer
Capil
l
ary tube viscometer
• The usual method for measurement of
viscosity involves the determination of the
time required for a given volume of liquid to
flow through a capillary.
• Many capillary-tube viscosimeters have been
devised, but Ostwald and Ubbelohde
viscosimeters are among the most frequently
used.
Capillary tube viscometer
• Viscosity is measured by measuring the flow
rate of fluid flowing through the capillary tube
• Before each measurement, clean with
cleaning liquids like benzene followed by
acetone, then rinse with purified water and
dry
• Temperature of liquids must be controlled

• Ostw al
d viscometer (U shaped viscometer)
• Works on the basis of time of flow of liquid
between two points under action of force
of gravity
• U shaped… two bulbs
• Definite volume of liquid is placed in
Bulb C and sucked upto mark A.
• Allowed to fall from mark A to B
• Repeated with water
ɳ1= × × ɳ
• ɳ1= viscosity of liquid
• ɳ2= viscosity of water (reference)
• d1= density of liquid
• d2= density of water (reference)
• t1= time of flow of liquid
• t2= time of flow of water (reference)
η1 = Absolute viscosity,
η1/ η2 = Relative viscosity
• Ubbel
ohde viscometer
• A liquid is introduced
into the reservoir then
sucked through the
capillary and measuring
bulb. The liquid is
allowed to travel back
through the measuring
bulb and the time it
takes for the liquid to
pass through two
calibrated marks is a
measure for viscosity.
Rotating viscometer method
• The principle of the method is to measure the
force
acting on a rotor (torque) when it rotates at a
constant
angular velocity (rotational speed) in a liquid.
• Rotating viscometers are used for measuring the
viscosity of Newtonian (shear-independent viscosity)
or non-Newtonian liquids (shear dependent
viscosity or apparent viscosity).
• Different measuring systems are available for
given viscosity ranges as well as several rotational
speeds.

Apparatus
The following types of instruments are most common.
CON CEN TRIC CYLIN DER VISCOM ETERS (absolute
viscometers)
In the concentric cylinder viscometer (coaxial double
cylinder viscometer or simply coaxial cylinder
viscometer), the viscosity is determined by placing the
liquid in the gap between the inner cylinder and the
outer cylinder.
Viscosity measurement can be performed by rotating the
inner cylinder (Searle type viscometer) or the outer
cylinder (Couette type viscometer), as shown in Figures.
CON E-
PLATE VISCOM ETERS (absolute viscometers)
• In the cone-plate viscometer, the liquid is introduced
into the gap between a flat disc and a cone forming a
define angle.
• Viscosity measurement can be performed by rotating
the cone or the flat disc, as shown in Figures below. For
laminar flow, the viscosity (or apparent viscosity) h
expressed in Pascal-seconds is given by the following
formula: η = c. T/v, where, c = Instrument
constant.
T = Torque.
v = speed (r.p.m.)

SPIN DLE VISCOM ETERS
In the spindle viscometer, the viscosity is determined by
rotating a spindle (for example, cylinder- or disc-shaped,
as shown in Figures) immersed in the liquid.
Relative values of viscosity (or apparent viscosity) can be
directly calculated using conversion factors from the
scale
reading at a given rotational speed.
In a general way, the constant k of the apparatus
may be determined at various speeds of rotation
using a certified viscometer calibration liquid.
The viscosity ƞ then corresponds to the formula:
Method
• Measure the viscosity (or apparent viscosity) according to
the instructions for the operation of the rotating viscometer.
• The temperature for measuring the viscosity is indicated in
the monograph.
• For non-Newtonian systems, the monograph indicates the
type of viscometer to be used and if absolute viscometers
are used the angular velocity or the shear rate at which the
measurement is made.
• If it is impossible to obtain the indicated shear rate exactly,
use a shear rate slightly higher and a shear rate slightly
lower and interpolate.

With relative viscometers the shear rate is not the same
throughout the sample and therefore it cannot be defined.
• Under these conditions, the viscosity of non-Newtonian
liquids determined from the previous formula has a relative
character, which depends on the type of spindle and the
angular velocity as well as the dimensions of the sample
container (Ø = minimum 80 mm) and the depth of
immersion of the spindle.
• The values obtained are comparable only if the method is
carried out under experimental conditions that are rigorously
the same.
FALLIN G BALL VISCOM ETER
The determination of dynamic viscosity of Newtonian
liquids using a suitable falling ball viscometer is
performed at 20 ± 0.1 o C, unless otherwise prescribed in
the monograph.
The time required for a test ball to fall in the liquid to be
examined from one ring mark to the other is determined.
If no stricter limit is defined for the equipment used, the
result is valid only if 2 consecutive measures do not differ
by more than 1.5 per cent.
Apparatus
• The falling ball viscometer consists of: a glass tube enclosed
in a mantle, which allows precise control of temperature;
• six balls made of glass, nickel-iron or steel with different
densities and diameters.
• The tube has 2 ring marks which define the distance the ball
has to roll.
• Commercially available apparatus is supplied with tables
giving the constants, the density of the balls and the suitability
of the different balls for the expected range of viscosity.
Method
• Fill the clean, dry tube of the viscometer, previously brought to 20 ±
0.1 oC, with the liquid to be examined, avoiding bubbles.
• Add the ball suitable for the range of viscosity of the liquid so as to
obtain a falling time not less than 30 s.
• Close the tube and maintain the solution at 20 ± 0.1 oC for at
least 15 min.
• Let the ball run through the liquid between the 2 ring marks once
without measurement.
• Let it run again and measure with a stop-watch, the time required
for the ball to roll from the upper to the lower ring mark.
• Repeat the test run at least 3 times.

• Calculate the dynamic viscosity ƞ in millipascal seconds
using the formula:
k = constant, expressed in millimeter squared per second
squared,
ρ1 = density of the ball used, expressed in grams per cubic
centimeter,
ρ2 = density of the liquid to be examined, expressed in grams
per cubic centimeter.
t = falling time of the ball, in seconds.
Application in pharmacy
• Useful in formulations like syrups, suspensions,
ointments
• Important for flow from container
• Flow through needle
• The viscosity of a solution influences heat transfer
through it. For example, the cooling of hot syrup, the
evaporation of a viscous plant extract, all depend on
their viscosities. So in unit processes such as
evaporation, mixing, cooling, distillation etc., viscosity
is a parameter which influences heat flow and thus the
time required to carry out the process.
• Rheological behaviour can be studied

Quality Control Test for
Emulsion and Suspensions
Emulsion and Suspensions
• Emulsion:-
An emulsion is a biphasic liquid preparation containing two
immiscible liquids, one of which is dispersed as minute globules
into the other. Emulsifying Agents are used
O/W &W/O
• Suspension:-
Pharmaceutical suspensions are uniform dispersions of solid drug
particles in a vehicle in which the drug has minimum solubility.
The internal phase consisting of insoluble solid particles which is
maintained uniformly throughout the suspending medium with aid
of single or combination of suspending agents.
The external phase (suspending medium) is generally aqueous in
some instance, may be an organic or oily liquid for non oral
use.
Why suspensions
• Insoluble drugs
• Soluble drugs but instable
• Soluble drugs suspended in non aqueous
vehicle
• To mask the test
• Some materials are needed to be present in
GIT in a finely divided form, to increase the
surface area. E.G. Mg carbonate and Mg
trisilcate are used to adsorb some toxins
Quality Control Test for Emulsions:
• Type of Emulsion
• Dispersibility/Pourability
• pH
• Water content
• Stability
• Labelling
• Weight per ml
• Viscosity
• Assay

Type of Emulsion
• Type of emulsion is determined whether it w/o or o/w as follow. Dispersibility/
pourability:
• It is used to check the spreadability &
pourability of emulsion.
Proc
ess:
Determined by the continuous phase
• If continuous phase is oil soluble, emulsion is
diluted with oil & if continuous phase is water
soluble then emulsion is diluted with water.
• And effects of dispersibility on skin are
checked by spreading the emulsion on skin.
pH
• Stability of active drug
• Stability of Emulsifying systems
–Emulsion that have soaps --- have pH 8 or more
–Nonionic, cationic, acid stable anionic ---- pH less
than 7
• pH paper/pH meter
• For calibration of pH meter--- first use pH 7
then pH 4 or 9
Water c
ontent
• Determination by Karl Fischer titration method
(USP)
• The principle of Karl Fischer titration is based
on the ox
idation reac
tion between iodine
and sulphur diox
ide. Water reacts with iodine
and sulphur dioxide to form sulphur trioxide
and hydrogen iodide. An endpoint is reached
when all the water is consumed

Stability of Emulsion
• An emulsion is said to be stable when
–Absence of coalescence of internal phase (the merging
of two or more droplets, bubbles or particles into one)
–Maintenance of elegance—appearance, colour, odour
–Absence of creaming
• Creaming results in lack of uniformity of drug distribution
• Determination of phase separation
• Phase separation may be observed visually Or by
subjecting the emulsions to various stress
conditions like boiling, temperature variations
• (causes--density difference, particle size-----
viscosity)
• Recovered by gelling agents
Stability
• Determination of elec
trophoreticproperties:
Determination of electrophoretic properties
like zeta potential is useful for assessing
flocculation since electrical charges on particles
influence the rate of flocculation.(zeta potential
is the potential difference between the dispersion
medium and the stationary layer of fluid attached
to the dispersed particle)
Determination of partic
le siz
e and partic
le c
ount:
• It is performed by optical microscopy and Coulter
counter apparatus.
• - O/W emulsion having a fine particle size will
exhibit low resistance but if the particle size increase,
then it indicates a sign of oil droplet aggregation and
instability.

Quality Control Test for Suspensions:
• Degree of Sedimentation, Sedimentation volume
• Degree of flocculation
• Determination of particle size and particle count
• Determination of electrophoretic properties
• Stability of Suspension
• Electro kinetic method
• Particle size determnation - Micromeritic method
• Crystal growth in suspensions
• Labelling
• Weight per ml
• Physic
al appearanc
e
• pH determination
• Assay & c
ontent uniformity
• Surface tension
• Visc
osity
Sedimentation
The velocity of sedimentation is expressed by
Stoke’s law.
v =
d = the diameter of the particle in cm.
s = the density of the dispersed phase (particles).
o = the density of the dispersed medium.
g = the acceleration due to gravity
 = the viscosity of the dispersion medium in poise.
d2 (s

o) g
18
o
 One aspect of physical stability in pharmaceutical suspensions is concerned with
keeping the particles uniformly distributed throughout the dispersion.
 While it is seldom possible to prevent settling completely over a prolonged period of
time, it is necessary to consider the factors which influence the velocity of
sedimentation.
 Partic
le siz
e of any suspension is critical.
 Larger particles will settle faster at the bottom of the container.
 The particle size can be reduced by using mortar and pestle
But very fine particles will easily form hard cake at the bottom of the container.
Sedimentation, Stability problem
Theory of suspension: Brownian Movement
For particles having a diameter of about 2- 5 m
Brownian movement counteracts sedimentation to a
measurable extent at room temperature by keeping the
dispersed material in random motion.

•Thixotropic Suspension
•Thixotropy is defined as the isothermal slow reversible conversion of
gel to sol.
•Thixotropic substances on applying shear stress convert to sol(fluid)
and on standing they slowly turn to gel(semisolid).
•At rest the solution is sufficient viscous to prevent sedimentation and
thus aggregation or caking of the particles.
•When agitation is applied the viscosity is reduced and provide good
flow characteristic from the mouth of bottle.
• Determination of elec
trophoreticproperties:
Determination of electrophoretic properties
like zeta potential is useful for assessing
flocculation since electrical charges on particles
influence the rate of flocculation.(zeta potential
is the potential difference between the dispersion
medium and the stationary layer of fluid attached
to the dispersed particle)
• ELECTRO KI
NETI
C M ETHOD : The
determination of zeta potential of suspension is
helpful to find out the stability of suspension.
• Deflocculation of particles is obtained when the zeta
potential is higher than the critical value and the
repulsive forces supersede the attractive forces.
• The addition of a small amount of electrolyte reduces
the zeta potential. When this zeta potential goes
below the critical value, the attractive forces
supersede the repulsive forces and flocculation
occurs.
Stability of Suspension
• Physic
al stability.
• Two useful parameters for the evaluation of
suspensions are;
• A- sedimentation volume
B- degree of flocculation

Sedimentation volume
• The ratio of the final volume of sediment (Vu) to the original volume of
suspension (Vo) before settling
F=
Vu /
Vo
• Range of F-------- < 1 >
• normally F < 1
When F < 1    Vu < Vo
When F =1    Vu = Vo
The system is in flocculated equilibrium and show sedimention and no
clear supernatant on standing.
When F > 1  Vu > Vo
Sediment volume is greater than the original volume due to the network of
flocs formed in the suspension and so loose and fluffy sediment
Degree of flocculation (β) (minimum 1)
It is a very useful parameter for flocculation
Sedimentation behaviour of
floc
c
ulated and defloc
c
ulated
suspensions
  In flocculated suspension, formed flocks (loose
aggregates). flocculated suspensions sediment more
rapidly. The volume of final sediment is thus
relatively large and is easily redispersed by agitation.
 In deflocculated suspension, individual particles
are settling, so rate of sedimentation is slow which
prevents entrapping of liquid medium which makes it
difficult to re-disperse by agitation. This
phenomenon also called ‘cracking’ or ‘claying’.
• M I
CROM ERI
TI
C M ETHOD : The stability
of a suspension depends on the particle size of
the disperse phase. A change in particle size
distribution & crystal habit may be studied by
microscopy & counter coulter method.
• Dilute a sample with glycerol, water or liquid
paraffin (As in monograph)
• Mount on a slide and observe microscopically
• Observe 1000 particles diameter and take the
average and compare with the monograph

• Assay
–Specified in monograph
–Performed on liq
uids whose net volume is not more
than 3
0
0
ml
–For visc
ous liq
uids known amount of water is added
and mixed with liquids and final volume is determined
–For non visc
ous liquids, pour the contents of each
container into calibrated volume measures and
determine the volume
• First 10 containers are studied for uniformity of volume. If
one is out of range. Repeat with 10 more.
• Not more than 1 out of the 20 containers should be out of
the given range
Uniformity of mass
• Liquid preparations for oral use that are presented as single-
dose preparations comply with the following test. Weigh
individually the contents of 20 containers, emptied as
completely as possible, and determine the average mass. Not
more than 2 of the individual masses deviate by more than
10% from the average mass and none deviates by more than
20%.
Uniformity of mass of doses delivered by the
measuring devic
e.
• The measuring device provided with a multidose liquid
preparation for oral use complies with the following test.
Weigh individually 20 doses taken at random from one or
more multidose containers with the measuring device
provided and determine the individual and average masses.
Not more than two of the individual masses deviate by more
than 10% from the average mass and none deviates by more
than 20%.
Surfac
e tension
• Determined by Wilhelmy plate pull method and Ring
detachment method
• The Wilhelmy plate method involves measuring force
on a plate immersed vertically in the liquid. The
Wilhelmy plate is a rectangular, thin plate made of
glass or platinum which has good wetting properties on
contact with a liquid.
• The sample is attached to a very precise balance and
allowed to immerse in the liquid.
• The force acting on the immersed
sample is measured.

Ring detac
hment method:
Sample is poured in beaker and brought in contact to
platinum iridium ring, as ring should be light in weight to
avoid settlement.
For measurement of surface tension, the ring is pulled
away from the surface of liquid
The force required to detach the platinum ring from
surface is proportional to surface tension.
Method is applicable for liquid in which ring can be
dipped, as non-wet able and sticky liquids cannot give
true surface tension, procedure can be carried out on a
controlled temperature.
y = K . F
K = proportionality constant that depends on geometry of ring
F = surface tension force
Crystal growth in suspension:
• It is a common cause of deterioration of suspension.
Process:
Crystal growth is achieved by simulating the
temperature fluctuation under normal storage conditions.
But at greatly increased frequency as daily variation of
temperature has been reproduced by cycling time of 16
min.
Maintain temperature at 23-33 oC, alter temperature
from this range after every 16 minutes then crystal growth
Crystal growth depends upon the particles
concentration, the bulk particles solubility, the slope of
solubility curve, the temperature fluctuation range and
frequency of fluctuation
pH
• For determination of pH of suspension---- First
centrifuge or pH may differ upto 3 pH units
due to effect of dispersed phase
• pH paper/ pH meter
• For calibration of pH meter--- first use pH 7
then pH 4 or 9

Suppositories
 Suppositories are specially shaped solid
dosage form of medicament for insertion into
body cavities other than mouth.
 They may be used for both local and systemic
effect.
 These products are so formulated that after
insertion, they will either melt or dissolve in
the cavity fluids to release the medicament.
TYPES OF SUPPOSITORIES
 Rectal suppositories:
 Cylindrical
 32 mm in length: weight- 2-4 gm (adult), 1gm (Infants)
 Vaginal Suppositories (Pessaries)
 oviform or cone shaped
 These are larger than rectal suppositories (3 – 6 gm).
Generally 5 gms
 Urethral suppositories (Urethral bougies):
 2-4gm and 10-15 cm long for male and 6-7.5 cm long for
female, pencil shaped.
 Nasal suppositories/ bougies:
 Aural Suppositories / Bougies
Advantages of suppositories:
 Local effect:
 Hemorrhoids, antiseptic, antifungal
 Systemic effect.
 Absorbed into the blood Stream
 Mechanical Effect:
 increase bowl evacuation in constipation
 Suppositories are convenient mode of
administration for those drugs which irritate the
gastrointestinal tract, cause vomiting, are
destroyed by the hepatic circulation, or are
destroyed in the stomach by pH changes, enzymes
etc.

 The lower portion of the rectum affords a large absorption
surface area from which the soluble substances can absorb
and reach the systemic circulation.
 e.g. aminophylline used in asthmatic and chronic bronchitis.
morphine a powerful analgesic
ergotamine tartarate used to treat migraine
indomethacin and phenyl butaz
one analgesic and anti-
inflammatory actions.
 Systemic treatment by the rectal route is of particular value
for
 treating patients who are unconscious, mentally disturbed or
unable to tolerate oral medication because of vomiting or
pathological conditions of the alimentary tract.
 administering drugs, such as aminophylline, that cause gastric
irritation, and
 treating infants.
Quality control of suppositories:
 1- Appearance:
This includes odour, colour, surface condition and shape.
W hen cut longitudinally, and ex
amined with nak
ed eye, the
internal surfaces are uniform in appearance and bloom free
(white powdery deposit). Check
ed for fissuring, pitting, fat
blooming, ex
udation, sedimentation
 A change in the odor may also be indicative of a degradation
process
2- Weight Uniformity:
 If the weight is found to be too small, it is advisable to check
whether the mold is being well filled and whether there are ax
ial
cavities or air bubbles
 If the weight is found to be too high, checkthat scraping has been
carried out correctly, and also that the mix
ture is homogeneous.
 Lastly, the weight may decrease during aging when the
suppositories contain volatile substances, especially if the
pack
aging is not airtight.
 Method:
- W eigh 20 suppositories individually
- Calculate the average weight.
- calculate the % error for each suppository
Limit: Not more than 2 suppositories differ from the average weight by
more than 5%, and no suppository differs from the average weight
by more than 10%.

3-Disintegration Test
 Method 1 (for water-soluble, hydrodispersible and fat-based
suppositories):
 consists of a 60-mm long and 52 mm diameter cylinder of glass or
transparent plastic
 A metal device consisting of two perforated stainless steel discs, held
about 30 mm apart.
 These discs each have 39holes, 4mm in diameter, which are evenly
spaced in a concentric pattern.
 Once inserted into the cylinder, the metal device is attached to the rim of
the cylinder by means of three spring clips.
 Apparatus is placed in a beak
er with a minimum capacity of 4 litres filled
with water unless otherwise prescribed. The beak
er is fitted with a slow
stirrer and a support that holds the apparatus vertically 9
0 mm below the
surface of the water so that it can be inverted without emerging from the
water.
 Recommended procedure
 Unless otherwise described in the individual monograph, use
water maintained at a temperature of 36-37°
C as the
immersion fluid.
 The test req
uires three suppositories and the procedure is
applied to each of the suppositories.
 Place the sample on the lower disc of the metal device and
then insert it into the cylinder.
 Place the apparatus into the beak
er and invert it every 10
minutes without removing it from the liq
uid. Repeat the
operation with the remaining two suppositories. Record the
time req
uired for the disintegration of the suppositories.
 Limits: According to BP 30 minutes for fat based and 60
minutes for water soluble suppositories
 NA for USP
 Method 2 (alternative for fat-based suppositories):
 The apparatus consists of a flat-bottomed glass tube about
140mm long with an internal diameter of 15.5 mm
 and a two-part rod.
 The tube is closed with a removable plastic cover that has an
opening 5.2 mm in diameter. The rod has two parts: both
made of plastic, or the lower part made of plastic and the
upper of metal. The rod is 5 mm in diameter and widens at
the lower end to a diameter of 12mm.
 To the bottom of the lower end is fix
ed a metal needle 2mm
long and 1mm in diameter.
 The upper part of the rod has an adj
ustable sliding ring and a
weighted disc is attached to the top. The weight of the entire
rod should be 30 g ±0.1 g.

 Recommended procedure
 Unless otherwise described in the individual monograph, use water
maintained at a temperature of 36-37°
C as the immersion fluid. The test
req
uires three suppositories and the procedure is applied to each of the
suppositories.
 Place the glass tube containing 10 mL of water in the water-bath.
 Fixthe glass tubes vertically and immerse to a depth of at least 7 cm
below the surface but without touching the bottom of the water bath.
 Introduce a suppository, tip first, into the tube followed by the rod into the
glass tube until the metal needle touches the flat end of the suppository.
Put the cover on the tube. Note the time which elapses until the rod sink
s
down to the bottom of the glass tube and the markring reaches the upper
level of the plastic cover.
 Each of the three suppositories should melt within 30 minutes, unless
otherwise stated in the individual monograph.
4- Melting Range Test
 Time tak
en by an entire suppository to melt when immersed
in a constant temperature water bath at 37°
C.
 Also called macro-melting range.
 The release rate of the suppository is related to its melting
point;
it is therefore critical that this test be evaluated using a
non-destructive method.
 The melting point can be determined by placing a small-diameter
wire into the mold containing the suppository melt before the form
solidifies. The form is then immersed in water, held by the wire,
and the temperature of the water is raised slowly (about 1◦
C every
2–3 minutes) until the suppository slips off the wire;
this is the
melting point of the suppository.
 Melting test
1 . Heat a 200 mL beak
er of water to 37◦
C on a magnetic stirring unit
set at about 50 rpm.
2 Add a dosage unit to the water.
3 After 30 minutes, record your observations as yes, no or partially
melts on the scale provided.
NOTE: It may be necessary to add a weight to these dosage units to
pull them below the water surface.

 The ex
periment can also be done by using the BP
suppository Disintegration Apparatus.
 Procedure:
 The suppository is completely immersed in the constant
temperature water bath, and the time for the entire
suppository to melt or disperse in the surrounding water is
measured.
 The suppository is considered melted when:
 It is completely dissolved or
 Dispersed into its component part.
 Become soft “
change in shape”with formation of core which is not
resistant to pressure with glass rod.
5- Liquefaction time
 Also called softening time.
 Liq
uefaction testing provides information on the behaviour/
withstanding power of a suppository when subj
ected to a
max
imum temperature of 37◦
C (body temperature) and
pressure which will determine the release of the active
ingredients.
 The test commonly measures the time req
uired for a
suppository to liq
uefy under pressures similar to those found
in vivo in the presence of water at 37◦
C.
 In general, liq
uefaction should tak
e no longer than about 30
minutes.
Cellophane tube Method
 Apparatus consists of a glass cylinder (Condenser) with an ex
ternal
diameter of 50mm, narrowing down to 22mm at either end.
 The cylinder is fitted with two connections through which water that is
maintained at 37◦
C can circulate in such a manner that the lower half of
the cellophane collapse and the upper part gapes
 A 34–35cm length of cellulose dialyz
er tubing is moistened, opened and
placed in the cylinder.
 The cellophane tube is drawn out of either end of the cylinder and secured
with two elastic bands.
 W hen the appropriate temperature is reached, the suppository is placed in
the dialysis tubing
 As a result of water pressure the upper part of the tubing widens and the
lower half collapse ex
erting pressure on the suppository
 and the time to liq
uefaction is measured.

6-Breaking Test (hardness)
 Performed for measuring the fragility or
brittleness of a suppository
 Measured to assess their ability to withstand
the possible harsh conditions or shock
s during
normal handling,
 Hardness indicates the max
imum force which
the suppository can withstand during
production, pack
ing and handling.
 Double wall chamber in which suppository is
placed in an inner chamber. W ater is pumped
through the double wall of the chamber.
 the suppository is placed in the inner chamber
which hold a disc to which a rod is attached.
The other end of the rod is attached with
another disc on which weights are placed.
 Place the disc in the inner chamber.
 First 600 g weight is placed on the upper disc.
And left for one minute.
 If the suppository is not brok
en, add 200 g
weights after 1 minute till the suppository
crumbles.
 Range 1.8
-2 k
g.

 7- Assay
 8- Dissolution
 BP suppository disintegration apparatus
 USP dissolution apparatus

Quality Control Tests for
Parenteral
• Parenteral preparations are sterile preparations
intended for administration by injection,
infusion, or implantation into the human body
or animal body.
• Parenteral preparations must be sterile
• free of microorganisms
• To ensure sterility, parenterals are prepared
using
– aseptic techniques
– special clothing (gowns, masks, hair net, gloves)
– laminar flow hoods placed in special rooms
• Advantages and Disadvantages
Advantages
• Rapid onset of action
• Administrable to nonresponsive patients
• Patient convenience and comfort
• Administrable directly to site of action
• Better absorption

Parenteral Routes of Administration
1. Intra-articular
–joints
2. Intracisternal
-cisterna magna at the
base of the skull
3. Intra-arterial
–arteries
4. Intravenous
–veins
5. Intradermal
–skin
6. Intrasynovial
–joint fluid
7. Intrathecal
–spinal fluid
8. Intracardiac
–heart
9. Intramuscular
–muscles
10. Subcutaneous
–under the skin
GENERAL REQUIREMENTS OF PARENTERAL
PREPARATIONS
• Stability
• Sterility
• Free from Pyrogens
• Free from foreign particles
• Isotonicity
• Specific gravity
• Chemical purity
Official Types of Injections
1. Drug Injection
- Liquid preparations that are drugs substances or
solutions thereof.
Example: Insulin Injection, USP
2. Drug for Injection
- Dry solids that, upon the addition of suitable
vehicles, yield solutions conforming in all
respects to the requirement for Injections
Example: Cefamandole Sodium for
Injection

3. Drug Injectable Emulsion
- Liquid preparations of drug substances
dissolved or dispersed in a suitable
emulsion medium
Example: Lipofundin
4. Drug Injectable Suspension
- Liquid preparations of solids suspended in a
suitable liquid medium
Example: Methylprednisolone Acetate
Suspension
5. Drug Injectable for Suspension
- Dry solids that, upon the preparations conforming
in all respects to the requirements for Injectable
Suspensions
Example: Imipenem + Cilastatin for Injection
Suspension, (Tienem)
Factors affecting Stability
1- Environmental factors
- Temperature - Light
- Oxygen - Moisture
- Carbon dioxide
2- Drugs or excipients in the dosage form
- Particle size of drug
- pH of the vehicle
3- Microbial contamination .

Processing of parenteral
preparations
• 1. Cleaning of containers, closures & equipment’s: Thoroughly
cleaned with detergents, with tap water, distilled water finally rinsed
with water for injection. Rubber closures are washed with 0.5% sod.
Pyrophosphate in water.
• 2. Collection of materials: All raw material of preparation should
be collected from warehouse after accurate weighing. Water for
injection should be Pyrogen free.
• 3. Preparation of parenteral products: The parenteral
preparation must be prepared in aseptic conditions. The ingredients
are accurately weighed separately and dissolved in vehicle as per
method of preparation to be followed.
• 4. Filtration: The parenteral preparation must be filtered by
bacteria proof filter such as, filter candle, membrane filter.
• 5. Filling the preparation in final container: The filling operation
is carried out under strict aseptic precautions.
• 6. Sealing the container: Sealing should be done immediately after
filling in aseptic environment.
• 7. Sterilization: For thermo stable substances the
parenteral products are sterilized by autoclaving method at
different temp. & pressure.
• 10 lb. pressure (115.50C, or 2400F) for 30 minutes
• 15 lb. pressure (121.50C, or 2500F ) for 20 minutes
• 20 lb. pressure (126.50C, or 2600F) for 15 minutes
• Heat sensitive or moisture sensitive material are
sterilized by exposure to ethylene oxide or propylene oxide
gas .
• 8. Evaluation of the parenteral preparation: The
following tests are performed in order to maintain quality
control:
• 1. Sterility test 2. Clarity test 3. Leakers
test
• 4. Pyrogen test 5. Assay for active ingredients
• 9. Labeling & packaging .

Sterility Test
• It is a procedure carried out to detect and
confirm the absence of any viable form of
microbes in pharmacopeial preparation or
product.
OBJECTIVE OF STERILITY TESTING:
• For validation of sterilization process.
• To check presence of microorganisms in
preparation which are sterile.
• To prevent issue of contaminated product in
market.
Steps
1. Sampling
• The sample must be representative of the whole of the bulk material
& a lot of final containers.
Number Of Items In the Batch Minimum Number of Items
Recommended to be tested
• Not more than 100 containers
• More than 100 but not more than
500 containers
• More than 500 containers
• 10% or 4 container whichever is
greater
• 10 containers
• 2% or 20 containers (10
containers for LVP) whichever is
less
2. Selection of the quantity of the product to be used
• Selection of the quantity of the product to be used for sterility testing
depends mainly on the volume or weight in the container
Quantity of Each Container Minimum Quantity to be Used For Each
Culture Medium
• FOR LIQUIDS
• ≤ 1 ml
• > 1ml – 40 ml
• > 40ml and ≤ 100 ml
• >100ml
• The whole contents of the container.
• Half of the contents of the container
but not less than 1 ml.
• 20 ml
• 10 % of the contents of the container
but not less than 20 ml.
FOR SOLIDS
• Less than 50 mg
• 50 mg to < 300 mg
• 300 mg- 5g
• > 5g
• The whole of the contents of the
container
• Half of the contents of the container
but not less than 50mg
• 150 mg
• 500 mg
3. Method of sterility testing
• Membrane filtration method (METHOD 1):
• Membrane filtration is Appropriate for :
– Filterable aqueous preparations.
– Alcoholic preparations.
– Oily preparations.
– Preparations miscible with or soluble in aqueous or oily
solvents (solvents with no antimicrobial effect).
• All steps of this procedure are performed aseptically in a Class
100 Laminar Flow Hood.

• Direct Inoculation method ( Method 2):
• In this method suitable quantity of the
preparation to be examined is transferred
directly into an appropriate culture medium so
that the volume of the product is not more than
10% of the volume of the medium, unless
otherwise prescribed
• Incubate for not less than 14 days.
• It is a suitable method for samples with small
volumes.
• It is suitable for oily liquids , ointments and
creams.
• Culture medium is examined during and after the end
of incubation . The following observations are
possible :
– If there is no evidence of growth , pass the test for sterility.
– If there is evidence of growth , test is re-performed using
the same no. or volume of sample and medium as in the
original test.
– Now if there is no evidence of growth , pass the sterility
test for the sample. But if again there is evidence of
microbial growth , retesting is done with twice amount of
the sample and medium. If there is no evidence of growth ,
pass the sterility test and if there is evidence of microbial
growth , the batch is then rejected .
Leaker Test
• Ampule intends to provide a
hermetically sealed container for a single dose of a product.
• Leaker test is performed to detect incompletely sealed
ampules
• Leakers are detected by producing a negative pressure
within an incompletely sealed ampule, usually in a vacuum
chamber, while the ampule is entirely submerged in a
deeply coloured dye solution (usually 0.5 – 1 % methylene
blue). Subsequent atmospheric pressure causes the dye to
penetrate an opening into the ampule. It is visible after
washing the ampule from dye.
• Only a tiny drop of dye may penetrate a very small opening
at vacuum of 27 inches of hg or more. (15-30 minutes)

• Vials and bottles are not subjected to such test
because rubber closure is not rigid.
• Vials are tested for sealing efficacy
– Into few vials, air is injected through syringe and
these vials are submerged in water.
– Incompletely sealed vials will eject air into the
water in the form of bubbles.
Clarity Test
• Particulate matter is defined as unwanted mobile,
insoluble matter other than gas or air bubbles present in
the product.
• If the particle size of foreign matter is larger than the
size of RBC. It can block the blood vessel.
• Methods
Subjective Method (visual Method)
Quantitative Method (particle Count Methods)
• based on the
• Light obstruction/blockage (HIAC),
• Electrical resistance (Coulter Counter) methods
• Microscopic count method
Subjective Method (Visual Inspection)
• Simple method
• All containers are observed
• Apparatus
• A white and black matt panel held vertically
• An adjustable lampholder with shaded white light
source and with a light diffuser.
• The intensity of illumination at the viewing point
is maintained between 2000 lux and 3750 lux for
clear glass ampules. For coloured glass ampules
higher values are used

Quantitative Methods
• HIAC particle counters: based on the principle of light blockage
which allows an automatic determination of the size of particles and
the number of particles according to size.
General precautions
• Carried out in a laminar-flow cabinet.
• Very carefully wash the glassware and filtration equipment used,
with a warm detergent solution and rinse with abundant amounts of
water to remove all traces of detergent. Immediately before use,
rinse the equipment from top to bottom, outside and then inside,
with particle-free water.
• Take care not to introduce air bubbles into the preparation to be
examined, especially when fractions of the preparation are being
transferred to the container in which the determination is to be
carried out.
• Procedure (HIAC, Light obstruction)
• Mix the contents of the sample by slowly inverting the
container 20 times successively. If necessary, cautiously
remove the sealing closure. Clean the outer surfaces of
the container opening using a jet of particle-free water
and remove the closure, avoiding any contamination of
the contents. Eliminate gas bubbles by appropriate
measures such as allowing to stand for 2 min or
sonicating.
• For large-volume parenterals, single units are tested.
• For small-volume parenterals less than 25 ml in
volume, the contents of 10 or more units is combined in
a cleaned container to obtain a volume of not less than
25 ml;
• Light obscuration works by passing a dilute
stream of particles in a liquid between a light
source and a detector. In the HIAC liquid particle
counter, the light source is a laser diode, which
illuminates individual particles in the stream to
generate a shadow or blockage of light on the
detector. This blockage is called ‘obscuration’.
The detector measures the reduction in light
intensity and employing a calibration curve,
processes the signal to determine particle size.

• Procedure (Coulter Counter)
• Coulter Counter: is based on the principle of electrical
resistance produced by particles
• The sample solution is added to an electrolyte solution
which is drawn through a small orifice
• As particle passes through orifice, it displaces its own
volume of electrolyte
• Particle size detected by increase in electrical resistance
• Voltage pulses are proportional to the particle size

MICROSCOPIC PARTICLE COUNT TEST
• The microscope is equipped with an ocular
micrometer calibrated with an objective
micrometer, a mechanical stage capable of
holding the sample, two suitable illuminators
to provide illumination, and is adjusted to 100
± 10 magnifications.
BP Limits
Small Volume Injections Large Volume Injections
Method Method
Visual Control A (Coulter Counter) B (Light obstruction,
HIAC Counters)
Limits Limits
Practically free from visible
Particles
≥ 2 µm
Maximum 1000/ml
≥ 2 µm
Maximum 500/ml
≥ 5 µm
Maximum 100/ml
≥ 5 µm
Maximum 80/ml
USP Limits Pyrogen test
• Pyrogen = “Pyro” (Greek = Fire)
• + “gen” (Greek = beginning).
• Thus pyrogen are fever producing , metabolic
bi-products of microbial growth and death.
Pyrogens can be classified into two groups:
• endotoxins and
• non-endotoxin pyrogens (NEPs).

• Endotoxins are heat stable lipopolysaccharides
(LPS) present in bacterial cell walls (gram
negative), released only when bacteria die.
• Endotoxins are stable to at least 175oC; steam
sterilization ineffective. Can pass through 0.22
µm filter
Importance
• The sterility of a product does not imply that it is
free of pyrogens. Therefore, drugs that are
purported to be sterile must also be tested for
pyrogens to prevent febrile reactions in patients.
• Pyrogen contamination can occur during
production or the administration of
pharmaceuticals, biotherapeutics, and medical
devices, but the presence of pyrogens can also be
an inherent characteristic of the product, such as
adjuvants in vaccines or synthetic lipopeptides.
• In vivo Test
• Rabbits are used to perform this test
• 3 healthy adult rabbits of either sex, each
weighing not less than 1.5 kg are selected
• Do not use any rabbit
• having a temp < 38 or > 39.8 o C
• Showing temp variation > 0.2 o C between two
successive reading in the determination of initial temp.
• All syringes, needles and glasswares should
be pyrogen free (heating at 250 o C for 30
minutes).
• Method:
• Dissolve or dilute the sample with a pyrogen free saline
solution.
• Warm the sample to approx. 38.5o C temp before injection.
• The volume of injection should not be less than 0.5ml/kg &
not more than 10ml/kg of body weight.
• Withhold water during test.
• Record body temperature of Rabbits.
• 2 normal reading of temperature should be taken prior to
the test injection at an interval of half an hour & its mean is
calculated.
• The solution under test is injected through an ear vein .
• Record the temp of each rabbit in an interval of 30 minutes
for 3 hours.
• The difference between initial temp & maximum temp is
recorded- taken as response .

• Results
• If no rabbit shows an individual rise in temperature of
0.6 °C or more above its respective control temperature,
and if the sum of the 3 temperature rises does not
exceed 1.4 °C, the tested material meets the
requirements for the absence of pyrogens.
• If 1 or 2 rabbits show a temperature rise of 0.6 °C or
more, or if the sum of the temperature rises exceeds 1.4
°C, continue the test using 5 other rabbits. If not more
than 3 of the 8 rabbits show individual rises in
temperature of 0.6 °C or more, and if the sum of the 8
temperature rises does not exceed 3.7 °C, the tested
material meets the requirements for the absence of
pyrogens.
• In vitro Test (LAL Test)
• Limulus Amebocyte Lysate Test
• More sensitive and accurate than in vivo test
• Extract from the blood cells of horse shoe crab
(Limulus Polyphemus) contains an enzyme called
limulus amebocyte lysate which reacts with
pyrogens so that an assay mixture increases in
viscosity and opacity until an opaque gel is
formed
• LAL collected by bleeding healthy mature
specimen by heart puncture. Amebocytes are
carefully concentrated, washed and lysed by
osmotic effect.
Mechanism :
• Crab's amebocyte blood cells form Protinecious gel with endotoxin.
• Before the test, equipment is depyrogenated.
• During the Test performance, Avoid endotoxin contamination.
• Procedure :
– Equal Volume of LAL reagent and test solution (usually 0.1 ml of each)
are mixed in a depyrogenated test-tube .
– The test tube is then incubated at 37°C for 1 hour.
– After incubation tube is put out of the incubator , inverted at 180° and
observed for the result.
Result
• If gel-clot is formed in the tube , test is positive i.e endotoxins are
present in the test solution and the sample is rejected.
• If the test solution is turned opaque, it also indicates the presence of
pyrogens and so the sample is rejected.
• If test solution gives no “ gel – clot” formation with LAL reagent,
test is negative i.e there is no presence of endotoxins , and thus the
test solution is accepted.

• Assay
• An assay is an investigative (analytic) procedure in laboratory
medicine, pharmacology, environmental biology, and molecular
biology for qualitatively assessing or quantitatively measuring the
presence or amount or the functional activity of a target entity (the
analyte) which can be a drug or biochemical substance or organic
sample.
• Types of Assay:
• Chemical Assay
• Immunoassay
• Bioassay
• Chemical Assay
It is the study of the separation, identification, and quantification of the
chemical components of natural and artificial materials.
• Immunoassay
A technique that makes use of the binding between an antigen and its
homologous antibody in order to identify and quantify the specific
antigen or antibody in a sample.
Biological Assay OR Bioassay:
• Bioassay literal meaning
• Bio – living tissue
• Assay- assessment / measurement
• Bioassay: Assessment of a biological substance
• the term “bioassay” is a general name given to any experiment in
which the potency or preparation of a drug is measured by its effect
on living organism or tissue (Olaniyi and Ogunlana, 1998).
• Bioassay involves the use of live animals, plant, tissue or cells (in-
vitro) to determine the biological activity of a substances such as
hormones or drugs.
• Detection and measurement of the concentration of the substance in
a preparation using biological methods
• Estimation or determination of concentration or potency of a
physical, chemical or biological substance (agent) by means of
measuring and comparing the magnitude of the response of the test
with that of standard over a suitable biological system under standard
set of conditions

Synonymes
Comparison between Chemical &
Bioassay
Bioassay
• Less Precise
• More time consuming
• More expensive
• Active constituent & structure not
known.
• More sensitive
• More men power Required
• Difficult to handle
Chemical Assay
• More Precise
• Less time consuming
• Less expensive
• Active constituent & structure fully
established.
• Less sensitive
• Less men power required
• Easy to handle
Need of Bioassay
1. Chemical method is either
• Not available
• If available, too complex,
• Insensitive to low doses e.g. Histamine can be bioassayed in microgram conc.
2. If active principle of drug is not known
3. Unknown Chemical composition
4. Chemical composition of drug variable but has same
pharmacological action--- cardiac glycosides isolated from different
sources
5. Active principle cannot be isolated
6. Biological activity of drug cannot defined by a chemical assay e.g.
Cis and Trans form of methyl phenidate.
Principles of Bioassay
• All bioassays should be comparative against a standard drug
• Standard & new drug should be as far as possible identical to each other
• Activity assay should be the activity of interest
• The degree of pharmacological response produced should be reproducible
under identical conditions. e.g. Adrenaline.
• Method of comparison preferably (not essentially) test therapeutic
property of drug.
• Individual variations must be minimised.
• Active principle to be assayed should show the same measured response in
all animal species

Characteristics of a good assay method
Sensitivity
Specificity
Reproducibility
Precision
Accuracy
Stability – tissue has to stay “bioassay-fit
Bioassay can be performed on
• Intact Animal
In-vivo
(inside the living
body)
• Isolated tissues
• Specific cells
• Organisms
In-vitro
(in Glass)
• Cells or tissues from
human or animal donor.
Ex vivo
(outside of living
body)
Most common Bioassy:
WHOLE ANIMALS
• Nor Adrenaline – Spinal Cat
• Cardiac Glycosides – Guinea Pig
• Insulin – Mice
• Estrogens – Ovariectamised (OVX) Female Rat
MICRO ORGANISMS
• Vit B12 – Euglena gracilis
• Tetracycline - Bacillus pumilus (to detect tetracycline in milk
ISOLATED TISSUE
• Acetyl Choline – Frog Rectus Abdominus muscle
• Histamine – Guinea Pig ileum
• Adrenaline – Rat uterus
• Oxytocin – Rat uterus
DISPERSED CELLS
• Plasma LH estimation by stimulation of testosterone synthesis - on
isolated Leydig cells (also known as interstitial cells of Leydig)

Purpose of Bioassay
1. Compare test sample with standard substance to determine quantity of
test sample required to produce an equivalent biological response to
that of the standard substance.
2. Measuring pharmacological activity of new or chemically undefined
substance.
3. Test the method employed in measuring the response of living animals to
toxicity of chemical contaminant. e.g. Certain no. of individuals of
sensitive specie are exposed to specific conc. of contaminant for specific
period to examine toxic effects.
4. Determine conc. As well as potency of unknown substance.
5. Improving and maintaining standards of basic environmental conditions
affecting well-being of people e.g. pollutants released by particular
source.
6. To determine specificity of compounds to be used e.g. penicillin's are
effective against G+ve but not against G-ve
Advantages
• Bioassays are procedures that can determine the concentration of
purity or biological activity of a substance such as vitamin, hormone,
and plant growth factor.
• While measuring the effect on an organism, tissue cells, enzymes or
the receptor is preparing to be compared to a standard preparation.
• Bioassays may be qualitative or quantitative.
• Qualitative bioassays are used for assessing the physical effects of a substance
that may not be quantified, such as abnormal development or deformity.
• Quantitative bioassays involve estimation of the concentration or potency of a
substance by measurement of the biological response that it produces.
Quantitative bioassays are typically analyzed using the methods of
biostatistics.
• They not only help to determine the concentration but also the potency of
the sample.
• It is especially used to standardize drugs, vaccine, toxins or poisons,
disinfectants, antiseptics etc. as these are all used over biological system in
some or other form.
• These also help determine the specificity of a compound to be used ex:
testing sputum of infected patients helps to determine which antibiotic be
given for quick recovery
• Certain complex compounds like Vitamin B- 12 which can't be analyzed by
simple assay techniques can be effectively estimated by Bioassays.
• Sometimes the chemical composition of samples are different but have
same biological activity.
• For samples where no other methods of assays are available.
• Biological products like toxin, anti-toxin, sera can be conveniently assayed.
• Measure minute (Nano mole & Pico mole) quantities of active substances
can detect active substance without prior extraction or other treatment.
Disadvantages
• Key problem is variability in response
• Large number of animal to be used
• Expertise in experimental design, execution of assay & analysis of
data required
• Leads to expensive & time consuming
• Time related changes in sensitivity of test organ.
• Tachyphylactic responses of substance being assayed.

Standard Preparation for Bioassay
A selective representative sample of a substance for which it is to serve as a
basis of the measurement is called standard preparation.
• Uniform quality
• Stable
Type of standard preparation:
1) International standard and reference standard
• USP units(highly recognized-able and authorized standard)
2) British standard and reference standard
• Country want to have its own standard preparation, then used according to
its own law.
• For biological assay and tests, units are referred called GREAT BRITIAN.
• For specific biological activity small quantity of standard preparation are
used.
Classification of
Bioassay
There are two types of
bioassay:
a. Quantal
b. Graded
a. Quantal
• A quantal assay involves an "all or none response".
• For example:
• Insulin induced hypoglycemic convulsive reaction. The response is either +ve
or -ve, there is no intermediate response e.g.—either convulsion occurs or
doesn't occur;
• similarly the cardiac arrest caused by digitalis. In case of toxicity studies, the
animal receiving a dose of drug either dies or does not die. Also, no
intermediate response is possible
• Such study can be applied for:
• Comparison of LD50 and ED50
• Comparison of Threshold response
• Drugs producing quantal effect can be bioassayed by End-point
method
End- point method
• Here the threshold dose producing a positive effect is measured on
each animal and the comparison between the average results of two
groups of animals (one receiving standard and other the test) is done.
• e.g. bioassay of digitalis in cats.
The cat is anaesthetized with chloralose and its blood pressure is recorded.
The drug is slowly infused into the animal.
The moment the heart stops beating and blood pressure falls to zero, the
volume of fluid infused is noted down.
Two series of such experiments-one using standard digitalis and the other
using test preparation of digitalis is done.
potency is calculated as follows:
Conc. of Unknown = Threshold dose of the Standard X Conc. of the Std
Threshold dose of the Test

b. Graded Bioassay
Graded assays are based on the observation that there is a proportionate
increase in the observed response following an increase in the concentration
or dose.
The parameters employed in such bioassays are based on the nature of the
effect the substance is expected to produce.
For example: contraction of smooth muscle for assaying histamine or the
study of blood pressure response in case of adrenaline
Types of Graded Assay:
a) Bracketing /direct matching
b) Graphical
c) Multiple point assays
i. Three point assay
ii. Four point assay
iii. Six point assay
a) Bracketing or Direct Matching
1. A constant dose of the standard is bracketed by varying dose of test
sample until an exact matching between the response of std & that
of the sample is achieved
2. Strength of unknown/test drug can be found by simple
interpolation of bracketed response.
3. Initially, two responses of the standard are taken.
4. The doses are adjusted such that one is giving response of
approximately 20% and other 70% of the maximum.
5. The response of unknown which lies between the two responses of
standard dose is taken.
6. The panel is repeated by increasing or decreasing the dose(s) of
standard until all three equal responses are obtained.
7. The dose of test sample is kept constant.
8. At the end, a response of the double dose of the standard and test
which match each other are taken.
9. These should give equal responses. Concentration of the test
sample can be determined as follows:
Conc. of Unknown = Dose of the Standard X Conc. of Std.
Dose of the Test
bioassay of histamine on guinea pig ileum
Step No. 4
Step No. 8
Step No. 5
Step No. 6 & 7

b) Graphical method
• This method is based on the assumption of the dose-response relationship.
• Log-dose-response curve is plotted and the dose of standard producing the
same response as produced by the test sample is directly read from the
graph.
• In simpler design, 5-6 responses of the graded doses of the standard are
taken and then two equiactive responses of the test sample are taken.
• The height of contraction is measured and plotted against the log- dose.
• The characteristic of log-dose response curve is that it is linear in the
middle (20-80%).
• Thus, the comparison should be done within this range only. In other
words, the response of test sample must lie within this range
c) Multiple point assays
• are based on the dose-response relationship include 3 point, 4 point and 6 point
methods.
• In these 3 methods, the responses are repeated several times and the mean of
each is taken.
• Thus, chances of error are minimized in these methods.
• In 3 point assay method 2 doses of the standard and one dose of the test are
used.
• In 4 point method 2 doses of standard and 2 doses of the test are used.
• In 6 point method 3 doses of standard and 3 doses of the test are used.
• Similarly one can design 8 point method also.
3 point 4 point
Bioassay of histamine by multiple point method

3 point assay [2+1 dose assay]
• Fast & convenient
• e.g. Ach bioassay
• – Log dose response [LDR] curve plotted with varying conc of std Ach.
• – Select two std doses s1& s2 [ in 1:2 dose ratio] from linear part of
LDR
• – Choose a test dose T between S1 & S2
• Record 4 sets data [Latin square: Randomisation reduces error] as
follows
• s1 s2 t
• t s1 s2
• S2 t s1
•s1 s2 t
• Plot mean of S1, S2 and T against dose. Calculate – Log Potency ratio
[ M ] = [ (T –S1) / (S2-S1) ] X log d
where d = s2/s1
• Now calculate the conc of unknown as UK= s1/t X potency ratio X
conc of unknown
• Error is calculated as error= calculate- actual/actual X 100
• n1 = Lower Standard dose
• n2 = Higher Standard dose
• t = Test dose
• S1 = Response of n1
• S2 = Response of n2
• T = Response of test (t)
• Cs = Concentration of standard
Equation for 3 point method
4 point assay [2 +2 dose assay]
• Procedure [Eg Ach bioassay]
• Log dose response [LDR] curve plotted with varying conc. of std. Ach
• Select two std. doses s1& s2 from linear part of LDR
• Choose two test doses t1 & t2

• Record 4 data sets with Randomisation
s1 s2 t1 t2
s2 t1 t2 s1
t1 t2 s1 s2
t2 s1 s2 t1
• Plot mean of S1, S2 and T1, T2 against dose.
Calculate
• Log Potency ratio [M] = [ (T1 –S1 + T2 –S2) / (S2-S1 + T2-T1) ] X log d
• [d = dose ratio] d= s2/s1
• Error is calculated as error= calculate- actual/actual X 100
Equation for 4 point method
t1 = lower dose of test
t2 = higher dose of test
T1 = response of t1
T1 – response of t2
Bioassay of Some Important Drugs

Biological Assays: Definition
– Estimation or determination of concentration or potency of a
physical, chemical or biological substance (agent) by means of
measuring and comparing the magnitude of the response of the
test with that of standard over a suitable biological system
under standard set of conditions
– i.e. Observation of pharmacological effects on
• living tissues, or cells
• microorganisms
• animals
Indications for Bioassay
• Active principle of drug is unknown
• Active principle cannot be isolated, etc.
• Chemical method is either
– not available
– if available, too complex,
– insensitive to low doses e.g. Histamine can be bioassayed in
microgram conc.
• Unknown Chemical composition.
• Chemical composition of drug variable but has same
pharmacological action e.g. cardiac glycosides isolated
from different sources
DEMERITS
• Key problem is variability in response.
• Large No. of animal is to be used.
• Expertise in experimental design, execution of assay
and analysis of data required.
• Expensive and time consuming.
• Time related changes in sensitivity of test organ.
• Tachyphylactic responses of substance being assayed.

Principles of Bioassay
• Active principle to be assayed should show the same
measured response in all animal species
• The degree of pharmacological response produced
should be reproducible under identical conditions [Eg
Adrenaline shows same rise in BP in the same species
under identical conditions: wt, age, sex, breed etc]
• Activity assayed should be the activity of interest
• Individual variations must be minimised
TYPES OF BIOASSAY:
• Quantal Assays
• Show ‘All or None’ response in different animals.
• As the name indicates, the threshold dose of the
sample required to elicit a complete or a
particular pharmacological effect is determined
and compared with standard.E.g..
• Digitalis induced cardiac arrest in guinea pigs
• hypoglycaemic convulsions in mice by insulin.
• Graded Response Assays [mostly on tissues]
• In these assays, as the dose increases there is an equivalent
rise in response. The potency is estimated by comparing the
Test sample responses with the standard response curve.
• e.g contraction of smooth muscles for histamine assay and the
study of blood pressure responses in case of adrenaline.
Microbiological assay of antibiotics
• The potency (activity) of an antibiotic product is determined by
comparing the dose that inhibits the growth of a suitable
susceptible microorganism with the dose of an International
Biological Standard of that antibiotic that produces same degree
of inhibition.
• the determination or estimation of concentration or potency of
an antibiotic by means of measuring and comparing the area of
zone of inhibition or turbidity produced by test substance with
that of standard over a suitable microbe under standard
conditions.

• Microbial assay/Bioassay of antibiotics is done because of the
following reasons.
• Resistance: The use of antimicrobial is increasing day by day
and thus the chance of resistance to these microbes also
increases.
• Introduction of new strands of pathogen.
• Determination of concentration
• Multicomponents antibiotics
Methods for microbial assay
• Two general methods are used ;
– Cylindrical- plate or plate method
– Turbidity method
Cylindrical- plate or plate method (Petri dish method)
• Use Petri dishes / plates (20 * 100 mm) filled to a depth of 3-4
mm, unless otherwise indicated in the monograph, with a culture
medium.
• During the filling they should be placed on a flat, horizontal
surface so as to ensure that the layer of the medium will be of a
uniform thickness.
• Inoculate the medium with a susceptible bacterial suspension
previously prepared
• temperature of the molten agar medium must not exceed 48-50
°C at the time of inoculation (vegetative organisms).
• The inoculated plates or allowed to dry for 30 minutes at room
temperature or solidified by storage in a refrigerator.
• 4-8 Sterile cylinders of 10mm height and 6mm inside diameter
approximately made of glass, porcelain, aluminium or stainless
steel, are placed on the surface of the inoculated medium.
• Instead of cylinders, 5-8 mm holes can be bored in the medium
using sterile borer.
• Solutions of the reference material of known concentration and
corresponding dilutions of the test substance, presumed to be of
approximately the same concentration, are prepared in a sterile
buffer of a suitable pH value.
• The solutions are placed in cylinders or holes by means of
pippete which deliver a uniform amount of solution.
• Different solutions are arranged in an alternate manner.

• Then incubate at suitable temperature (32-35 or as mentioned)
for 16-18 hours unless specified.
• After incubation measure the zone of inhibition in mm.
• Compare the zone of inhibition of the standard and the test
antibiotic.
Turbidimetric Tube Method
• In this method, a uniform solution of an antibiotic is
made. The microbial culture is added to the fluid. The
biggest advantage of this method is that it requires a
relatively shorter incubation period. However, there is
also a big disadvantage. The presence of foreign
material that may be inhibitory to the growth of microbes
may influence the results of this assay. This method is
therefore appropriate when the samples are clear.

Turbidity method
• Principle:
• This method is based on inhibition of microbial
growth as indicated by measurement of the
turbidity (transmittance) of suspensions of a
suitable micro-organism in a fluid medium to
which have been added graded amount of test
compound.
• Changes in transmittance produced by the test
compound are compared with those produced by
reference material.

QC and Management Slides.pdf for 4th year

Bioassays of Insulin
• The cells in your body need sugar for energy.
However, sugar cannot go into most of your cells
directly. After you eat food and your blood sugar
level rises, cells in your pancreas (known as beta
cells) are signaled to release insulin into your
bloodstream. Insulin then attaches to and signals
cells to absorb sugar from the bloodstream.
Insulin is often described as a “key,” which
unlocks the cell to allow sugar to enter the cell
and be used for energy.
• Insulin contains 51 amino acids arranged in 2
chains (A and B) linked by disulfide bridges. The
A chain is composed of 21 amino acids and the B
chain is composed of 30 amino acids.
• Since insulin first isolation, In vivo animal bioassays
have been used to assess the potency of insulin. Insulin
manufacturers have been required to conduct in-vivo
assay of new insulin batches according to
pharmacopoeial methods.
• The potency of insulin injection (expressed in
International Units/ml) is determined by comparing its
hypoglycemic activity with that produced by the
Standard Insulin using one of the following methods:
• Rabbit blood sugar method:
• Mouse convulsion method:
• Rat diaphragm method
• Rat epididymal fat-pad method
• Radioimmunoassay

Rabbit blood sugar method
• Principle:
– The potency of a test sample is estimated by comparing the
hypoglycaemic effect of the sample with that of the std.
preparation of insulin.
• Standard preparation and unit:
– It is pure, dry and crystalline insulin.
– One unit contains 0.04167 mg (1mg contains 24 units)
– This unit is specified by NIMR (National Institute of
Medical Research) U.K.
• Preparation of standard solution
– Accurately weigh specified quantity of insulin and dissolve
it in a normal saline. Acidify it with HCl to pH 2.5-3.5. Add
0.1-0.25% phenol & 1.4% -1.8% glycerin as preservative.
Final volume should contain 40 units/ml. Store the solution
in a cold place (2-8°C) and use it within six months.
• Preparation of test sample solution:
– The solution of the test sample is prepared in the same
way as the standard solution described above.
• Dilution of standard and sample solutions
– These are freshly prepared by diluting with normal
saline solution so as to contain 1 unit/ml and 2
units/ml.
• Selection of rabbits
– All Rabbits should be healthy, weighing about 1800-
3000 gms.
– They should be maintained on uniform diet but are
fasted for 18 hrs before assay.
– Water is withdrawn during the experiment.
• Experimental Procedures:
– Animals are divided into 4 groups of 3 rabbits each. The rabbits are
then put into an animal holder. They should be handled with care to
avoid excitement.
• First part of the Test:
– A sample of blood is taken from the marginal ear vein of each rabbit
and the average concentration of blood glucose in mg/dl is determined
in each group. This concentration is called ‘Initial Blood Sugar Level’.
– The four groups of rabbits are then given sc. injections of insulin (0.3-
0.5ml).
– 1 unit/ml (Dilution. I) and 2 units/ml (Dilution. II)
– And after specified time, blood sugar level is again determined
12
Rabbits
1 (n=3)
(Stnd I)
2 (n=3)
(Stnd II)
3 (n=3)
(Test I)
4 (n=3)
(Test II)
• Second part of the test (Cross over Test)
• The same animals are used for the second part. The experiment can
be carried out after a day but not more than one week. Again they
are kept in fasting condition and initial blood sugar is determined.
• In this part of test ,the grouping is reversed
• This test is known as “ Twin cross-over test”.
• And after specified time, blood sugar level is again determined
• Mean percentage decrease of the first and second part is then
calculated after proper statistical calculations
12
Rabbits
1 (n=3)
(Test II)
2 (n=3)
(Test I)
3 (n=3)
(Stnd II)
4 (n=3)
(Stnd I)

Mouse convulsion method
• Albino mice are selected as they show convulsion
after s/c injection of insulin.
• Preparation of standard and sample solution:
– Solutions are prepared with sterile N/S solution so as
to contain 0.015 units/0.5ml and 0.030 units/0.5ml.
• Selection of mice
– Minimum 96 mice are selected , each having weight
lying in the range of 18-22gm. They should be
maintained at constant diet , however should be kept in
fasting condition 2 hrs before experiment.
• The mice are divided into four groups and the insulin preparations
are injected .
• After injecting the insulin , the mice are incubated at 29-35°C for 90
mints. Temperature is maintained thermostatically. The incubator
consists of six shelves , with transparent front.
• After 90 minutes, the mice are taken out from the incubator , placed
with their backside down position and observed for convulsion or
death. Then the convulsion to death ratio percentage is calculated.
• The convulsed mice may be recovered by 0.5ml dextrose solution
injection.
96 Mouse
1 (n=24)
(Stnd I)
2 (n=24)
(Stnd II)
3 (n=24)
(Test I)
4 (n=24)
(Test II)
• 3. Rat diaphragm method: In this method
increase in glycogen content of the muscle or
increase in glucose uptake by muscle in response
to insulin is taken as the index of potency of
insulin.
• 4. Rat epididymal fat-pad method: Here, the
ability of insulin to increase CO2 production by
the fat-pad is taken as the parameter for the
measurement of potency of the insulin
preparation.
• 5. Radioimmunoassay: It is the estimation of
the concentration of the substance in a unit
quantity of preparation using radiolabelled
antigens. A number of drugs are estimated now
days by radioimmunoassy methods because
these methods are highly specific and highly
sensitive.

BIOASSAY OF DIGITALIS
• Digitaloid group of drugs includes digitalis
• Dried leaves of Digitalis Purpurea
• Product contains Cardio active glycosides,
digitoxins ,gitoxins ,digitonin or saponine
• Bioassay of Digitalis
• It is mainly based on determinations of the
amount of test materials required to cause death
due to cardiac arrest in anaesthetized pigeons,
relative to the amount of a reference standard
preparation required to produce the same effect
• Principle
• Potency of the test sample is compared with that of the standard
preparation by determining the action on the cardiac muscles
• Standard preparation and units
• The standard preparation consists of dry powdered leaves of
digitalis purpurea. Supplied in ampules containing 2.5 g.
• ( 1 unit =76 mg) 1mg= 0.01316 Units
• Preparation of extracts
• Weigh contents of 1 container accurately and transferred to a 50 ml
glass stoppered container.
• 10 ml of Menstruum (Alcohol 4 parts, water 1 part) is added for
each gram of the powder.
• Closed and shacked for 24 ± 2 hours at 25 ± Co by mechanical
means.
• Contents is then centrifuged and transferred to a hard glass container
• Stored at 5Co - -5 Co and can be used for one month.
• Extract of the sample is prepared in the same manner
• Methods;
• Bioassay of digitalis is usually done using any
of the following method.
• Pigeon method
• Guinea pigs method

PIGEON METHOD
• Minimum 12 pigeons in two groups are used for testing
standard and sample.
• They should be of the same sex, breed, same weight free
from gross evidence of disease.
• The weight of the heaviest one should not exceed twice the
weight of the lightest one and the mean weight of the two
groups should not differ by more than 30 percent.
• Food but not water is withheld 16-20 hrs before the
experiment.
• The standard and test sample is diluted with normal saline
in such a way that the estimated fatal dose will be 15 ml/kg
body weight.
• They are lightly anaesthetized with ether.
• Alar vein is exposed and cannulated by means of a venous
cannula.
• The injection is continued at the dose of 1ml/kg through venous
cannula with a 5 minutes interval until the pigeon dies of cardiac
arrest.
• The amount of extract required to produce this effect is taken as
the lethal dose.
• If the average number of doses required to produce cardiac
arrest is less than 13 or more than nineteen or the average
number of doses of the two groups differ by more than four, the
test is repeated using suitably adjusted dilutions
• Potency= Mean lethal dose of standard/mean lethal dose of
sample.
GUINEA PIGS METHOD
• Selection of animals
• 12 guinea pigs are selected , each weighing
200-600 grams.
• These are divided into two groups.
• The weight difference between lightest and
heaviest should not be more than 100 gms and
the mean weight difference between the two
groups should not be more than 10%.
• Procedure
• The guinea pigs are anaesthetized with suitable
anesthetic agent , usually urethane.
• The jugular vein is traced by removing the skin
and I.V cannula is adjusted.
• Then the solutions are injected through the
cannula continuously until the heart arrest occurs.
(duration of injection may be between 20-40
minutes).
• The amount of the solution required to produce
this effect is called Lethal dose. The mean of
both, the standard and the sample solution is
calculated and compared.

BIO ASSAY OF VITAMIN D
• Antirachitic Vitamin
• Calciferol
• Vitamin D3 (cholcalciferol), D2 (ergocalciferol)
• 7-dehydrocholesterol is a precursor and is converted into Vit-D3
in the skin
• osteomalacia
Bioassay
• Performed by comparing the antirachitic activity of Vit-D3
sample with that of the standard
• Standard Preparation and Units:
• International standard established in 1949
• Contains activated crystalline 7-dehydrocholesterol.
• Supplied in bottles (1000 units/gram)
• Solution in vegetable oil (Cotton seed Oil). Dilution upto the
extent that on a single day the dose fed is not more then 0.2ml.
• Two selected dosage levels with ratio not less than 1.5 and not
more than 2.5.
•Method
• 40 young rats of either sex
• Preliminary period: from birth upto 30 days. Under the
researcher. Normal diet but limited in contents vitamin D.
• Weight of heaviest should not be greater by 10 gm than the
lightest one or between (44-60 gm) and should be free from any
abnormality (disease, injury).
• Depletion period: Fed for 19-25 days on rachitogenic diet
• Rickets is developed and is determined in each rat under light
anaesthesia by taking X-ray photograph of proximal ends of tibia
or distil end of ulna and radius

• Divided into four groups.
• The rats in two groups receive doses of X and nX of standard
• The other two groups the same amount of doses of X and nX of
the sample
• Each rat may receive its dose in whole or 8 divided doses/day
• 0.2 ml for 10-14 days
• 2 selected dosage levels ---- dilutions (1:1.5-2.5)
• After 10-14 days of treatment again examination of bones is
carried out by X-ray images
• The recovery is compared between sample and standard
40 rats
1 (n=10)
(Stnd X)
2 (n=10)
(Stnd nX)
3 (n=10)
(Test X)
4 (n=10)
(Test nX)
• Line test
• Remove the proximal end of tibia or distill end of radius and
clean it from tissues.
• Cut longitudinally. Rinse with purified water. Immerse
immediately in silver nitrate (1 in 50) for one minute
• Rinse with water again
• Expose the cut surface to daylight or untill the calcified area
develop clear defined stain without marked discolouration of the
uncalcified area

Alcohol Determination
Methods of alcohol determination
• Distillation method (Method I)
• Gas Chromatographic method (Method II)
Specific gravity from alcoholometric table
Distillation method
• Method I must be used for the determination of
alcohol, unless otherwise specified in individual
monograph.
• This method is useful for examining most fluid
extracts and tinctures, provided ;
– The capacity of the distilling flask is sufficient
(commonly two to four times the volume of the liquid
to be heated)
– Rate of distillation is such that clear distillates are
produced

• Problems and their solution
• Distillate should be clear. If cloudy, add talc or CaCO3
and filter, adjust temperature and determine the
alcohol content from the specific gravity.
• Treat the liquids that froth to a troublesome extent
during distillation by ;
– 1- Rendering them strongly acidic with H3PO4 , H2SO4 ,
tannic acid
– 2- Treating with a slight excess of calcium chloride solution
or with a small amount of paraffin or silicon oil before
starting the distillation.
• Loss of alcohol by evaporation should be minimized.
• Prevent bumping during distillation by adding porous
chips of insoluble material such as small pieces of
broken glass, glass beads, porous chips or silicon
carbide.
For liquids presumed to be containing less than 30%
v/v of alcohol
• Take 25ml sample in a suitable distilling apparatus and note
the temperature at which the volume was measured.
• Add equal volume of water and distill.
• Collect distillate 2ml less than original volume of the test
liquid (23ml) , adjust the temperature at which the original
test liquid was measured , add water to make 25ml and
mix.
• The distillate must be clear and not more than slightly
cloudy and does not contain more than traces of volatile
substances other than alcohol and water.
• Find specific gravity at 25 ° (as specified in pharmacopoeia
under the heading “specific gravity”) .
• From alcoholometric table find the %age of alcohol.
For liquids presumed to be containing more
than 30% v/v of alcohol
• Take 25ml sample in a suitable distilling apparatus and note the
temperature at which the volume was measured.
• Add 50ml of water and distill.
• Collect distillate 2ml less than the double amount of original
sample( 48ml ) , adjust the temperature at which the original test
liquid was measured , add water to make 50ml and mix.
• The distillate must be clear and not more than slightly cloudy and
does not contain more than traces of volatile substances other than
alcohol and water.
• Find specific gravity at 25 ° (as specified in pharmacopoeia under
the heading “specific gravity”).
• From alcoholometric table find the %age of alcohol .
• The proportion of alcohol is ½ of liquid so multiply it with 2 to get
exact % age of alcohol in sample

Special Treatments
Volatile acids and bases
• Render the preparations containing volatile bases
slightly acidic with dilute sulphuric acid before
distillation
• If volatile acids are present, render the
preparation slightly alkaline with sodium
hydroxide test solution
• Acidifying the bases or alkalinizing the acids
causes them to be converted to ionized form and
form salts. These salts then do not interfere with
ethanol evaporation in distillation process.
• Glycerin and iodine
• Glycerin: To the preparation containing
glycerin, add sufficient water so that after the
distillation, the residue contains not less than
50% of water.
• Iodine: Solution containing free iodine must
be treated with;
– 1- Powdered zinc (zinc iodide will be formed)
– 2- Decolorize with sufficient sodium thiosulfate
solution followed by few drops of sodium
hydroxide
• Other Volatile substances
• Spirits, elixirs, tinctures and similar preparations that contain
appreciable proportion of volatile materials other than alcohol
and water such as volatile oils, chloroform, ether, camphor, etc ,
they require special treatment as follow:
• For liquids presumed to be containing 50% alcohol or less --
Mix 25ml sample with equal volume of water in a separator
• Saturate with NaCl & add 25ml hexane and shake well to remove
any interfering volatile substances
• Draw off the separated lower layer into a second separator and
repeat the extraction twice with hexane (2, 25 ml portions)
• Extract the solution (combined solvent hexane ) thrice with 10ml
portions of saturated saline solution
• Combine all these portions and distil as usual
• Take volume having simple ratio to original specimen
• For liquids presumed to be containing more than
50% alcohol
• Adjust the conc. of alcohol to approximately 25%
v/v by diluting with water.
• Perform the same procedure as above beginning
with “saturate this mixture with sodium chloride”.
• If volatile substances are in small portion.
Shake distillate with about one-fifth its
volume of solvent hexane, or filter it through a thin
layer of talc

Gas-liquid Chromatographic Methods
• Now-a-days gas chromatography is widely used in
pharmaceutical analysis. This method is used for
certain specified samples containing alcohol. It gives
accurate results rapidly.
• Principle
• This technique is based on the partition of molecules
between the two phases i.e. mobile phase and
stationary phase.
• The mobile phase is gas, most often Helium (nitrogen).
• The stationary phase is usually liquid and may be;
– Polyethylene glycol for separation of polar compounds.
– Polymer of silicon for separation of non-polar compounds.
• Apparatus
• The apparatus consists of:
• Injector
• Detector
• Retention time (RT) data analyzer
• Procedure
– Two standard solutions of Ethanol are prepared
• Liquid containing more than 10 % alcohol
• For its detection two standards are used:
– Standard-I containing 5 % more alcohol (Ethanol) than the
sample.
– Standard-II containing 5 % less alcohol than the sample.
• Liquid containing less than or 10 % alcohol
• For its detection, the standard used is:
– Standard-I containing 1 % more alcohol than the sample.
– Standard-II containing 1 % less alcohol than the sample.
Standard Solutions
Samples presumed to
contain more than 10 %
alcohol
SS-II containing
5% Less alcohol
than presumed
SS-I containing 5%
more alcohol than
presumed
Samples presumed to
contain less than or equal to
10 % alcohol
SS-I containing 1%
more alcohol than
presumed
SS-II containing
1% Less alcohol
than presumed
• Procedure
• Take 25 ml of each standard and sample solution in a
container bearing stopper.
• Add 10 ml of internal reference solution of Acetone.
• Use flame ionization detector, Carbowax as stationary
phase, Helium as mobile phase and column of 2-4 mm
dimension.
• Set the flow rate at 30 ml/sec.
• Maintain the injector temperature at 210°C, detector
temperature at 150°C and column temperature at
120°C.
• Inject 2 µl for each solution five times and note the
retention time.
•

• Calculate the %age of alcohol from the following
formula:
• % alcohol = S1 ( Y – Z ) + S2 ( Z – X )
• Y – X
• S1 = % age of alcohol of standard-I.
• S2 = %age of alcohol of standard-II.
• X = Ratio of RT of the internal reference solution to the
RT of standard-I.
• Y = Ratio of RT of the internal reference solution to the
RT of standard-II.
• Z = Ratio of RT of the internal reference solution to the
RT of sample solution.
• Ratio = peak area of sample/peak area of acetone

ALKALOIDAL DRUG ASSAY
Alkaloids are the cyclic organic
compounds normally with basic chemical
properties, containing nitrogen in its
negative oxidation state and occurring
chiefly in many vascular plants and some
fungi.
❑ Alkaloids are slightly or very slightly soluble in water but soluble in
certain organic solvents immiscible with water such as chloroform, ether,
amyl alcohol and benzene or mixture of these.
❑ Salts of alkaloids however are usually practically insoluble in nearly all
the organic solvents. The process of assay by immiscible solvents,
generally known as the “shaking out” process is based on these
partitioning properties of alkaloids.
❑ It is carried out by treating the drug or a concentrated liquid extract of it
with a solvent immiscible with water, in the presence of an excess of alkali,
which liberates the alkaloid. The immiscible solvent from which it is
subsequently removed by means of an excess of dilute aqueous acid
dissolves the free alkaloid The acid solutions are then extracted with an
immiscible solvent in the presence of an excess of alkali, and immiscible
solvent evaporated to obtain the alkaloid, which is either weighed or
determined volumetrically.
❑ The amounts of alkaloids is calculated by multiplying the factor under
individual monograph with the volume of standard acid used.

PREPARATION OF DRUG FOR ASSAY
Grind the drug to be extracted to a powder of fineness designated. Care should be taken
to avoid the loss of water during the powdering of the drug.
WEIGHING FOR ASSAY
In weighing bulky, crude drugs for the assay, accuracy to within 10 mg for quantities of 5
gm and over is sufficient.
Portions of pilular (soft) extracts or ointments may be weighed on a tared piece of waxed
paper and transferred into the vessel containing the solvent. In transferring weighed
portions to a separator, thoroughly rinse the vessel in which the material to be assayed
was weighed and add the rinsing to the separator.
EXTRACTION OF DRUGS
❑Because of the structural diversity of alkaloids, there is no single
method of their extraction from natural raw materials. Most methods
exploit the property of most alkaloids to be soluble in organic solvents
but not in water, and the opposite tendency of their salts.
❑Most plants contain several alkaloids. Their mixture is extracted first,
and then individual alkaloids are separated.
❑Plants are thoroughly ground before extraction. Most alkaloids are
present in the raw plants in the form of salts of organic acids. The
extracted alkaloids may
❑remain salts or change into bases.
METHODS OF EXTRACTION
The alkaloid content of alkaloid bearing drugs is usually extracted by
one of the following methods;
Maceration
Percolation
Continuous extraction
MACERATION
❑ The term maceration comes from the Latin “macerare” which means “to
soak”.
❑ It is a process in which the properly comminuted drug is permitted to soak in
the solvent until the cellular structure is softened and penetrated by the
solvent, as well as nearly all the constituents are dissolved in the solvent. In
the maceration process, the drug to be extracted is generally placed in a
wide mouth container with a prescribed solvent, the vessel is stoppered
tightly, and contents are agitated repeatedly over a period usually ranging
between 2 to 14 days.

MACERATION
❑ Agitation permits the repeated flow of fresh menstrum over the entire surface
area of comminuted drug. An alternative to repeated shaking is to place the drug
in a porous cloth bag i.e., tied and suspended in the upper portion of menstrum,
much the same as a tea bag is suspended in water to make a cup of tea. As the
soluble constituent dissolve in the menstrum ,they tend to settle to the bottom
because of an increase in the specific gravity of liquid due to its added weight.
❑ The extractive is separated from the marc by expressing the bag of drug and
washing it with additional fresh menstrum, these washings being also added to
the extractive. If maceration is performed with loose drug, then marc may be
removed by straining or filtration.
Maceration Process for Organized and Unorganized Crude Drugs
Organized drugs have a defined cellular structure whereas unorganized
drugs are non-cellular. Bark and roots are examples of organized crude
drugs, while gum and resin are unorganized crude drugs. The processes of
maceration for organized and unorganized drugs are slightly different, as
shown in Table 1.
Steps Organized drugs Unorganized drugs
1. Drug + entire volume of menstrum Drug + four-fifths of menstrum (in most
cases)
2. Shake occasionally for 7 days Shake occasionally on days 2 to 7, as
specified
3. Strain liquid, press the marc Decant the liquid. Marc is not pressed
4. Mix the liquids, clarify by subsidence for, Filtrate
is not adjusted for volume
Filter the liquid and add remaining
menstrum through the filter.
PERCOLATION
Organized vegetable drug in a suitably powdered form. Uniform moistening of the
powdered vegetable drugs with menstruum for a period of 4 hours in a separable
vessel (Imbibition). Packed evenly into the percolator. A piece of filter paper is
placed on surface followed by a layer of clean sand so that top layers of drugs are
not disturbed.
Sufficient menstruum is poured over the drug slowly and evenly to saturate it,
keeping the tap at bottom open for passing of occluded gas to pass out. Sufficient
menstruum is also added to maintain a small layer above the drug and allowed to
stand for 24 hours.
After maceration, the outlet is opened, and solvent is percolated at a control rate
with continuous addition of fresh volume. 75% of the volume of the finished
product is collected. Marc is pressed and expressed liquid is added to the percolate
giving 80% to 90% of the final volume. Volume is adjusted with calculated quantities
of fresh menstruum. Evaporation and concentration to get finished products by
applying suitable techniques and apparatus.
CONTINUOUS EXTRACTION
❑ Finely ground crude drug is placed in a porous bag or
“thimble” made of strong filter paper, which is placed in
chamber E of the Soxhlet apparatus.
❑ The extracting solvent in flask A is heated, and its vapors
condense in condenser D.
❑ The condensed extractant drips into the thimble containing
the crude drug and extracts it by contact.
❑ This process is continuous and is carried out until a drop
of solvent from the siphon tube does not leave residue
when evaporated.
The advantage of this method is that large amounts of drug can be
extracted with a much smaller quantity of solvent. This affects tremendous
economy in terms of time, energy and consequently financial inputs. At
small scale, it is employed as a batch process only, but it becomes much
more economical and viable when converted into a continuous extraction
procedure on medium or large scale.

Purification:
The extraction of the alkaloid from the bulk of the crude alkaloid solution is
invariably carried out by shaking with an acid solution.
In usual practice, the use of HCl is restricted when chloroform remains as
the solvent as few alkaloidal hydrochlorides are distinctly soluble in the
chloroform. Dilute H2SO4 is always preferred over HCl for general use in the
extraction of alkaloids.
Acid solution is rendered alkaline with dilute NH4OH solution to liberate the
alkaloids which is then extracted with an organic solvent.
Solvent is removed under reduced pressure and the traces of moisture is
removed with anhydrous sodium sulphate.
DETERMINATION OF ALKALOIDS
❑ Evaporate the solution of the purified alkaloids in the immiscible solvent to
dryness on a steam bath or with a current of air.
❑ When the alkaloid residue is to be determined volumetrically. Soften it by the
addition of about 1 ml of neutralized alcohol or ether; add an accurately
measured volume of the standard acid.
❑ Equivalent to about one and one half to two times the volume estimated for
the quantity of alkaloid present. Warm the mixture gently to ensure the
complete solution of the alkaloid. If preferred. Dissolve the alkaloid residue
in chloroform.
❑ Add the standard acid of higher normality and remove the chloroform
completely by evaporation. Then add enough water to make the volume
of the mixture measure at least 25 ml. Titrate the excess of acid with
standard alkali, using the appropriate indicator. If the alkaloidal residue is
to be weighed, dry it at 105 ºC to a constant weight. If the solvent has
been chloroform remove last traces of the solvent by addition of 5 ml of
neutralized ether or alcohol followed by evaporation.
Assay of the SPECIFIC ALKALOID
given in the relevant
Official Monograph

QUALITY ASSUARANCE FOR
VACCINES
Amna Zarar Khan
Pharm-D
University of Peshawar
DEFINITION
•Immunization, is the process by which an
individual's immune system becomes
fortified against an infectious agent.
BIOLOGICAL PRODUCTS:
• According to Public Health;
• In the U.S., a biological product is defined as “a virus,
therapeutic serum, toxin, antitoxin, vaccine, blood, blood
component or derivative, allergenic product, or analogous
product, or arsphenamine, or derivative of arsphenamine (or
any other trivalent organic arsenic compound), applicable to
the prevention, treatment or cure of a disease or condition
of human beings.
VACCINES
• Vaccines are microbial preparations of killed or modified
microorganisms that can stimulate an immune response in the body
to prevent future infection with similar microorganisms.
• Vaccine is a biological preparation that consists of either a whole
organism or a part of it against which immunization has to be
achieved.
• Vaccines provide active immunity as they stimulate the immune
system of the recipient to produce T cells or antibodies that impede
the attachment of infectious agents and promote their destruction

HOW VACCINES WORK
• Vaccines contain weakened or inactive parts of a particular organism
(antigen) that triggers an immune response within the body.
• Newer vaccines contain the blueprint for producing antigens rather
than the antigen itself. Regardless of whether the vaccine is made up
of the antigen itself or the blueprint so that the body will produce the
antibody.
• This weakened version will not cause the disease in the person
receiving the vaccine, but it will prompt their immune system to
respond much as it would have on its first reaction to the actual
pathogen.
TYPES OF VACCINES
I. Live vaccines
II. Killed vaccines
III. Adjuvants
IV. Sub unit vaccines
V. Conjugated vaccines
PREPERATION OF VACCINES:
• Seed Lot System
• Production of Bacteria
• Blending
• Filling and Drying
A- SEED LOT SYSTEM
• The starting point for the production of all microbial vaccines is the
isolation of the appropriate infectious agent.
• Bacterial strains may need to be selected for high toxin yield or
production of abundant capsular polysaccharide; viral strains may
need to be selected for stable attenuation.
• Once a suitable strain is available, the practice is to grow, often from
a single viable unit, a substantial culture which is distributed in small
amounts in a large number of ampoules and then stored at -70°C or
below, or freeze- dried.
• This is the original seed lot. From this seed lot, one or more ampoules
are used to generate the working seed from which a limited number
of batches of vaccine are generated. Full history of the seed lot should
be known including the media composition.

B- PRODUCTION OF BACTERIA:
• To obtain specific components from the bacteria, generally
fermentation methods are used.
• Production of bacterial vaccine begins with resuscitation of bacterial
strains from the seed lot.
• Resuscitated bacteria are first cultivated through one or more
passages in pre-production media.
• Then, when the bacteria have multiplied sufficiently, they are used to
inoculate a batch of production medium.
• The medium in the fermenter should have an optimum pH as well as
it should be free of any (Transmissible Spongiform Encephalopathy)
TSE agents.
• At the end of the growth period the contents of the fermenter, which
are known as the harvest, are ready for the next stage in the
production of the vaccine.
PROCESSING F HARVEST:
• Killing – Live bacteria are killed by either Heat or by certain disinfectants
like Formalin, Thiomersal.
• Separation- Bacterial cells are separated from the culture fluid and soluble
products. Centrifugation, Ultrafiltration and Precipitation methods are
commonly used.
• Fractionation – Components are extracted from the bacterial cells or from
the medium in which they are grown in a purified from. Example: The
polysaccharide antigens of Neisseria meningitidis are usually separated
from the bacterial cells by treatment with hexadecyltrimethylammonium
bromide followed by extraction with calcium chloride and selective
precipitation with ethanol.
• The purity of an extracted material may be improved by resolubilization in
a suitable solvent and re-precipitation.
• After purification, a component may be freeze-dried, stored
indefinitely at low temperature and, as required, incorporated into a
vaccine in precisely weighed amount at the blending stage.
• Detoxification - Carried out by formaldehyde to obtain toxoids.
Detoxification may be performed either on the whole culture in the
fermenter or on the purified toxin after fractionation.
• Further Processing – These may include many physical and chemical
treatments to modify the product. Example: Polysaccharides may be
further fractionated to produce material of a narrow molecular size
specification. They may then be activated and conjugated to carrier
proteins to produce Glyco-conjugate vaccines.
• Adsorption: It’s the process of adsorbing the vaccine to the mineral
adjuvant. It helps in improving immunogenicity and decreasing
toxicity.
• Conjugation: The linking of a vaccine component that induces an
inadequate immune response, with a vaccine component that
induces a good immune response.

PRODUCTION OF VIRUSES:
There are 3 main ways;
1) Inoculation of virus into animals.
2) Inoculation of virus into embryonated eggs (Eggs should be from
disease free flocks.)
3) Tissue culture (Media composition should be known and it should
be free of TSE agents)
BLENDING
• Blending is the process in which the various components of a vaccine
are mixed to form a final bulk.
• When bacterial vaccines are blended, the active constituents usually
need to be greatly diluted and the vessel is first charged with the
diluents, usually containing a preservative.
• Thiomersal has been widely used in the past but is now being phased
out and replaced by phenoxy ethanol or alternatives.
FILLING AND DRYING:
• Bulk vaccine is distributed into single-dose ampoules or into
multi-dose vials as necessary.
• Vaccines that are filled as liquids are sealed and capped in
their containers, whereas vaccines that are provided as dried
preparations are freeze-dried before sealing.
QUALITY CONTROL OF VACCINES:
Mainly to provide assurances of both the probable efficacy and
safety of every batch of every product. 3 main ways:
1) In-process control
2) Final product control
3) Quality control tests

IN PROCESS CONTROL:
• In-process quality control is the control exercised over starting
materials and intermediates.
• The toxoid concentrates used in the preparation of the vaccines have
been much diluted and, as the volume of vaccine that can be
inoculated into the test animals (guinea-pigs) is limited, the tests are
relatively insensitive.
• In-process control, however, provides for tests on the undiluted
concentrates and thus increases the sensitivity of the method at least
100-fold.
FINAL PRODUCT CONTROL
1- ASSAY
Vaccines containing killed microorganisms or their products are
generally tested for potency in assays in which the amount of the
vaccine that is require to protect animals from a defined challenge dose
of the appropriate pathogen, or its product, is compared with the
amount of a standard vaccine that is required to provide the same
protection.
• The number of survivors in each group is used to calculate
the potency of the test vaccine relative to the potency of the
standard vaccine by the statistical method.
• The potency of the test vaccine may be expressed as a
percentage of the potency of the standard vaccine.
• Vaccines containing live microorganisms are generally tested
for potency by determining their content of viable particles.
• Example: In the case of BCG vaccine, dilutions of vaccine are
prepared in a medium which inhibits clumping of cells, and fixed
volumes are dropped on to solid media capable of supporting
mycobacterial growth.
• After a fortnight the colonies generated by the drops are counted and
the live count of the undiluted vaccine is calculated.

2- SAFETY TEST:
• Bacterial vaccines are regulated by relatively simple safety
tests. Those vaccines composed of killed bacteria or bacterial
products must be shown to be completely free from the
living microorganisms used in the production process.
• Those vaccines prepared from toxins, for example, diphtheria and
tetanus toxoids, require in addition, a test system capable of revealing
inadequately detoxified toxins.
• This can be done by inoculation of guinea- pigs, which are exquisitely
sensitive to both diphtheria and tetanus toxins.
• A test for sensitization of mice to the lethal effects of histamine is
used to detect active pertussis toxin in pertussis vaccines.
• With killed vaccines the potential hazards are those due to
incomplete virus inactivation and the consequent presence of
residual live virus in the preparation.
• With attenuated viral vaccines the potential hazards are those
associated with reversion of the virus during production to a degree
of virulence capable of causing disease in recipients.
QUALITY CONTROL TEST FOR VACCINES:
1) Staining test
2) sterility test
3) Inactivation test
4) Pyrogen test
5) Freedom from abnormal toxicity

1-STAINING TEST:
• Approximately 10 mL of the test sample is centrifuged in a
pointed centrifuge tube at approximately 2,000 × g for 30
minutes.
• The sediment or the bottom portion is spread on a slide
glass, dried and heat-fixed over a flame.
• The smear is then stained by the Gram’s method and, unless
otherwise specified, examined microscopically at an
approximately 1,000-fold magnification.
CRITERIA FOR JUDGEMENT:
•No bacteria shall be observed other than those
defined in the individual monographs.
2- STERILITY TEST
• It is a procedure carried out to detect and confirm the absence of any
viable form of microbes in pharmacopeial preparation or product.
• The broad basis of the test for sterility is that it examines samples of
the final product for the presence of microorganisms.
• Theoretically, the test for sterility should be applied to all products
that are designated as sterile.
• The test results can be valid only if all the products of a batch are
treated similarly.
1-Membrane filtration method (METHOD 1):
• Membrane filtration is Appropriate for :
• Filterable aqueous preparations.
• Alcoholic preparations.
• Oily preparations.
• Preparations miscible with or soluble in aqueous or oily solvents
(solvents with no antimicrobial effect).
• All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood.

2- DIRECT INNOCULATION METHOD
• In this method suitable quantity of the preparation to be examined is
transferred directly into an appropriate culture medium so that the
volume of the product is not more than 10% of the volume of the
medium, unless otherwise prescribed Incubate for not less than 14
days. It is a suitable method for samples with small volumes. It is
suitable for oily liquids , ointments and creams.
OBSERVATIONS:
• Culture medium is examined during and after the end of incubation .
The following observations are possible :
• If there is no evidence of growth , pass the test for sterility.
• If there is evidence of growth , test is re-performed using the same
no. or volume of sample and medium as in the original test.
• Now if there is no evidence of growth , pass the sterility test for the
sample.
• But if again there is evidence of microbial growth , retesting is done
with twice amount of the sample and medium.
• If there is no evidence of growth , pass the sterility test and if there is
evidence of microbial growth , the batch is then rejected
3- INACTIVATION TEST:
• Each purified bulk material shall be tested in mice for effective
inactivation of the virus before the addition of preservative and other
substances.
• The test should be performed with undiluted purified bulk material
injected intra-cerebrally into at least 20 mice, each weighing between
15 and 20 g. these mice shall be observed for 14 days.
• Any symptoms caused by the virus shall be confirmed by immuno-
florescence assay. At the end of the observation period, no cytopathic
effects should be observed.
Interpretation of results:
• If more than 1 mouse dies, the test is failed.
• If one mouse dies , repeat the test.
• If no mouse dies , the test is passed.

4- PYROGEN TEST:
•In vivo Test
• Rabbits are used to perform this test 3 healthy adult rabbits of either
sex, each weighing not less than 1.5 kg are selected.
• Do not use any rabbit having a temp < 38 or > 39.8 o C Showing temp
variation > 0.2 o C between two successive reading in the
determination of initial temp.
• All syringes, needles and glasswares should be pyrogen free (heating
at 250 o C for 30 minutes).
METHOD OF TEST
• Insert thermometer in rectum of each rabbit. Note the normal
readings. Two readings are taken .
• First reading is taken and then after 30 minutes , next reading is
taken.
• Calculate the mean value.
• Then test solution /vaccine is injected into ear veins of rabbits. Record
the temperature of each rabbit.
• Six readings of temperature are recorded each after the interval of 30
minutes.
• If no rabbit shows an individual rise in temperature of 0.6 °C or more
above its respective control temperature, and if the sum of the 3
temperature rises does not exceed 1.4 °C, the tested material meets
the requirements for the absence of pyrogens.
• If 1 or 2 rabbits show a temperature rise of 0.6 °C or more, or if the
sum of the temperature rises exceeds 1.4 °C, continue the test using 5
other rabbits. If not more than 3 of the 8 rabbits show individual rises
in temperature of 0.6 °C or more, and if the sum of the 8 temperature
rises does not exceed 3.7 °C, the tested material meets the
requirements for the absence of pyrogens.
IN VITRO TEST
LAL TEST
• In vitro Test (LAL Test) Limulus Amebocyte Lysate Test More sensitive
and accurate than in vivo test Extract from the blood cells of horse
shoe crab (Limulus Polyphemus) contains an enzyme called limulus
amebocyte lysate which reacts with pyrogens so that an assay
mixture increases in viscosity and opacity until an opaque gel is
formed LAL collected by bleeding healthy mature specimen by heart
puncture. Amebocytes are carefully concentrated, washed and lysed
by osmotic effect.

PROCEDURE:
• Equal Volume of LAL reagent and test solution (usually 0.1 ml of
each) are mixed in a depyrogenated test-tube .
• The test tube is then incubated at 37°C for 1 hour. After incubation
tube is put out of the incubator , inverted at 180° and observed for
the result.
RESULTS:
• If gel-clot is formed in the tube , test is positive i.e endotoxins are
present in the test solution and the sample is rejected.
• If the test solution is turned opaque, it also indicates the presence of
pyrogens and so the sample is rejected.
• If test solution gives no “ gel – clot” formation with LAL reagent, test
is negative i.e there is no presence of endotoxins , and thus the test
solution is accepted.
5-FREEDOM FROM ABNORMAL TOXICITY:
• This test, also commonly referred to as the general safety
test, innocuity test or test for freedom from abnormal
toxicity, was specifically designed to identify non-specific
toxicity and contamination from exogenous substances.

Evaluation of Ointments and
Creams
• Topicals
– that is applied to body surfaces such as the skin or mucous
membranes to treat ailments e.g. creams, foams, gels,
lotions and ointments
– Nasal decongestant
– Eye, Ear
Others:::::: Enteral, inhalation or parenteral
• Transdermal
– deliver the drug through the skin to the general circulation
for systemic effects. These systems follow absorption of
active ingredients through the transdermal barriers
especially through stratum corneum by various mechanisms
for absorption through the blood capillaries present in the
dermis or epidermis layers for systemic effects.
– drugs for birth control, hormone replacement therapy and
prevention of motion sickness. Antibiotic like
chloramphenicol
Ointments
• Greasy semisolid preparations for application to the skin
• Fatty preparation of anhydrous nature
– May be
• Medicated
– Medicament is either dissolved or dispersed in vehicle
• Non Medicated
– Used as vehicle for preparations of medicated ointment or
used for physical effects like as emollient, soothens the skin
• Properties of an ideal ointment
– Does not interfere with healing process
– None sensitizing Non irritating
– Elegant No dehydrating effect
– Neutral in reaction
– Stability Efficient
– Spreadability and extrudablity
Ointment Base
• It determines the use of the ointment.
• Base that penetrate into the skin and release the medication
is an ideal base for antiseptic ointment,
• while having ‘poor penetrating power’ is ideal for a protective
ointment against moisture, air, UV lights and other external
factors.
• Divided in to 4 types
• Oleaginous or Hydrocarbon Bases
• Absorption Bases
• Oil-in-water (Water-miscible/ water removable) Emulsion
Bases
• Water Soluble Bases

• Oleaginous or Hydrocarbon Bases
– composed of oleaginous compounds, low water content (Anhydrous),
hydrophobic, difficult to be spreaded and washed, stay for prolong
time on skin. Donot dry out quickly. Mainly used as protectants and
emollients.
Examples are white petrolatum and white ointment
• Absorption Bases (W/O)
– composed of oleaginous base and an emulsifying agent (Wool Fat),
low water content, difficult to spread and wash,
– Examples: Hydrophilic Petrolatum,, anhydrous lanolin, Aquabase, Cold
Creams
• Oil-in-water (Water-miscible/ water removable ) Emulsion Bases
– Composed of oleaginous base contains an O/W emulsifier ,
Hydrophilic, soothing effect due to high water content
– Examples are hydrophilic ointments, Dermabase, Velvachol and
vanishing cream
• Water Soluble Bases
– composed of polyethylene glycols, hydrophilic, greasless, easy to be
spreaded and washed, mainly used as drug carriers. Examples are PEG
Ointment and Polybase.
Creams
• Viscous semi solid emulsions intended for topical application
• Medicated or non medicated
• Types
– Water in oil or oily cream
• Contains W/O emulsifier e.g. wool fat, wool alcohols
• Example: Zinc Cream BP. Cold cream
– Oil in Water or Aqueous Cream
• Contains O/W emulsifier e.g. emulsifying wax, alkali salt or fatty acid
• Example: vanishing Cream, Hydrocartisone Cream
• Physical Appearance
• Particle Size Determination
• Weight Variation Test
• Viscosity Determination
• Potency / Assay of Active Ingredient
• Microbial Contamination
• Metal Particles in Opthalmic Ointment
• Sterility test of ophthalmic ointments
• Spreadability
• Skin irritation test
• Cracking (of creams ----
separation of oil and water)
• Development of granular and
lumpy appearance
• Marked change in viscosity
• Crystal growth
• Color change
The main
characteri
stics need
to be
checked
are
Physical Appearance
Factors
• Incompatibility
• Temperature
• Packaging
• Microbial contamination

• Dilute a suitable quantity of preparation with
equal volume of glycerol or liquid paraffin, as
specified
• Mount on a glass slide and examine under light
microscope
• Count the number of particles with daimeter above
or below than that specified in monograph
• Compare the percentage with official limits
Particle Size Determination Weight Variation Test/Minimum Fill
It is applied to those products in which labeled net weight is
not more than 150 g.
Procedure
Select 10 filled containers and remove the label (can alter
the weight) .
Clean and rinse from out side and weigh individually.
Remove the contents by cutting the containers and wash
with suitable solvent
Dry and again weigh each empty container together with its
corresponding part.
 Take the difference between the two weights as weight of
contents.
10 containers (Average should not be less than
the labelled amount)
For ≤ 60 g Net weight of None should be < 90 % of the
labelled amount
For > 60 g
and ≤ 150 g
Net weight of None should be < 95 % of the
labelled amount
If it fails repeat with 20 more (Average of 30 should not be less
than the labelled amount)
For ≤ 60 g Net weight of not more than 1 should be < 90 %
of the labelled amount
For > 60 g
and ≤ 150 g
Net weight of not more than 1 should be < 95 %
of the labelled amount
• Viscosity is determined using a method specified
in official monograph.
• Instruments used is called “rheometers” and
“viscometers”
• (brookfield viscometer)
Viscosity Determination

Assay of active
ingredients
should be
performed
according to
monograph
Percentage
contents
should be
within the
official
limits
Assay of Active Ingredients Avoidance of Microbial Contamination
Microorganisms may grow if no preservatives is
included specially in Aqueous Creams
If preservatives are present its efficacy may be reduced
due to incompatibility
It is better not to depend solely on preservatives to kill
microorganisms that are accidently introduced during
manufacturing, handling and storage
Aseptic conditions and suitable container should be
chosen
Metal Particle in Ophthalmic Ointments
• Extrude completely the contents of 10 containers separately
into flat bottom 60 mm Petri dishes
• Cover the petri dishes , heat at 85oC for 2 hours. Slight increase
in temperature can be done to ensure complete fluid state.
then cool slowly to solidify.
• Remove the cover and invert the petri dishes on the stage of
microscope individually very carefully.
• Adjust the magnification upto 30 times. In addition to usual
light source an illuminator can also be used
• Examine the entire bottom of Petri dish for metal particles.
Varying the intensity of the illuminating light allows such metal
particles to be recognized by their characteristic reflection of
light
• Count the metal particles of 50µm or larger in any dimension.
• The requirement is met :
• if total number of such particles in all 10 tubes
does not exceed 50 .
• Not more than 1 tube contains more than 8 such
particles.
• If the requirement is not met , repeat with 20
more tubes
• The test is passed if total number of particles of
50 µm or larger in 30 tubes is not more than 150.
• And not more than 3 containers contain more
than 8 such particles , individually.

STERILITY TESTING
• It is a procedure carried out to detect and confirm
the absence of any viable form of microbes in
pharmacopeial preparation or product.
OBJECTIVE OF STERILITY TESTING:
• For validation of sterilization process.
• To check presence of microorganisms in
preparation which are sterile.
• To prevent issue of contaminated product in
market.
Sterility test of ophthalmic ointments
Number Of Items In the Batch Minimum Number of Items Recommended to
be tested
• Not more than 200
containers
• 5 per cent or 2 containers whichever is the
greater
• 10 containers
• If the product is presented in the form of single-dose containers, apply the
scheme shown in preparations for parenteral use
Sampling
Number Of Items In the Batch Minimum Number of Items
Recommended to be tested
• Not more than 100 containers
• More than 100 but not more
than 500 containers
• 10% or 4 container whichever is
greater
• 10 containers
• 2% or 20 containers (10
containers for LVP) whichever is
less
Quantity to be Used
Minimum Quantity to be Used For Each Culture Medium
Use the contents of each container to provide not less
than 200 mg

Method of sterility testing
Membrane filtration method
• Diluted to 1 percent in isopropyl myristate R, by heating, if
necessary, to not more than 40-44 °C. filter it rapidly.
• Wash the membrane at least three times by filtering through
it each time about 100 ml of a suitable sterile solution such as
peptone (1 g/l) containing a suitable emulsifying agent for
example polysorbate 80.
Direct Inoculation method
• Prepare by diluting by emulsifying with the
chosen emulsifying agent in a suitable sterile
diluent such as peptone (1 g/l). Transfer the
diluted product to a medium not containing
an emulsifying agent
• Incubate for not less than 14 days.
• Culture medium is examined during and after the end of
incubation . The following observations are possible :
– If there is no evidence of growth , pass the test for sterility.
– If there is evidence of growth , test is re-performed using the
same no. or amount of sample and medium as in the original
test.
– Now if there is no evidence of growth , pass the sterility test for
the sample. But if again there is evidence of microbial growth ,
retesting is done with twice amount of the sample and
medium. If there is no evidence of growth , pass the sterility test
and if there is evidence of microbial growth , the batch is then
rejected .

Spreadability
• The term spreadability is used to express the ease of spreading on the
skin. For the measurement of the spreadability glass slide apparatus is
used. For this purpose two glass slides are used.
• The lower slide is kept fixed while the upper slide is kept moveable with a
pan attached to it. About 20gm of the dosage form is placed between the
two slides and the spreadability is measured in terms of time taken by the
two slides to slip off from the dosage form placed in between the two
slides under the action of a certain load placed in the pan attached to the
upper slide.
• Lesser the time taken to separate the two slide, better will be the
spreadability.
• 𝐒 = 𝐌×𝐋
𝐓
Where,
S= Spreadability in gm.cm/sec______M=Weight tied to the upper slide
L=Length of glass slides_______T=Time taken by the two slides to separate
Skin irritation test
• To evaluate any potential of the dosage form
for skin irritancy.
• About 9 cm2 area of the rabbit skin is shaved
to remove hair and then about 1g is applied on
the shaved area.
• Observed for 7 days for the development of
erythema (redness) and edema/swelling.

ALKALINITY OF
GLASS
•Glass
•Glass is an amorphous inorganic product formed by
fusion of chemicals.
•Glass is said to be a frozen liquid.
•General composition of Glass
•SiO2, Na2O, K2O, CaO, MgO
• Advantages of glass
• Impermeable to gases , odour and microorganisms.
• Transparent and elegance in appearance
• Because of its high M.P and heat resistance , it can be sterilized by dry or
moist heat.
• Mostly inert to all packaging materials.
• It can be fabricated to produce variety of shapes.
• Can be easily cleaned due to its smooth surface.
• Re-useable and re-cyclable.
• Disadvantages
• Heavy weight
• Susceptibility to mechanical breakage.
• Unable to withstand sudden temperature changes
• Leaching of alkali
• Potential hazards may be caused by any of its component , leached into
pharmaceutical components.
• What is alkalinity of glass?
• From pharmaceutical point of view , it can be defined as ;
• “ The release of alkaline contents of a glass into the pharmaceutical
product it contains “

• How Alkalinity of Glass occur?
• In all glasses, the sodium and potassium oxides are hygroscopic;
therefore, the surface of the glass absorbs moisture from the air.
• The absorbed moisture and exposure to carbon dioxide causes the
Na2O or NaOH and K2O or KOH to convert to sodium or potassium
carbonate.
• Both Na2CO3 and K2CO3 are also hygroscopic. In water, especially salt
water, the Na and K carbonates in unstable glass may leach out, leaving
only fragile, porous hydrated silica (SiO2) network. This causes the glass
to craze, crack, flake, and pit, and gives the surface of the glass a frosty
appearance.
• Types of glass:
• Glass containers suitable for packaging pharmaceuticals may be
classed in to four types. Type I, II and III are intended for
parenteral products
• Type O is intended for oral or topical use.
Type General Description Type of Test Limit
Size.ml ml of acid
I Highly resistant, borosilicate
glass
Powdered Glass All 1.0
II Highly resistant, Soda Lime
Glass
Water attack 100 or less 0.7
Over 100 0.2
III Moderately resistant Soda Lime
Glass
Powdered Glass All 8.5
O General Purpose/ Soda Lime
Glass
Powdered Glass All 15
Type I glass containers
(Borosilicate glass / Neutral
glass)
This is a type of glass container that contains 80% silica, 10% boric
oxide, small amount of sodium oxide and aluminium oxide. It is chemically
inert and possess high hydrolytic resistant due to the presence of boric
oxide. It has the lowest coefficient of expansion and so has high thermal
shock properties.
Uses of Type I glass containers
Type I glass is suitable as packaging material for most
preparations whether parenteral or non-parenteral.
Type II glass containers
(treated soda-lime glass)
This is a modified type of Type III glass container with a high
hydrolytic resistance resulting from suitable treatment of the inner surface
of a type III glass with sulfur. This is done to remove leachable oxides and
thus prevents blooming/weathering from bottles. Type II glass has lower
melting point when compared to Type I glass and so easier to mould.
Uses of Type II glass containers
They are suitable for most acidic and neutral aqueous
preparations whether parenteral or non-parenteral.

Type III glass containers
(Regular soda lime glass)
This is an untreated soda lime glass with average chemical
resistance. It contains 75% silica, 15% sodium oxide, 10% calcium oxide,
small amounts of aluminium oxide, magnesium oxide, and potassium oxide.
Aluminium oxide impacts chemical durability while magnesium oxide
reduces the temperature required during moulding.
Uses of Type III glass containers
They are used as packaging material for parenteral products
or powders for parenteral use ONLY WHERE there is suitable stability test
data indicating that Type III glass is satisfactory.
Type IV glass containers
(Type NP glass/General-
purpose soda lime glass)
This type of glass container has low hydrolytic resistance.
This type of glass containers are not used for products that need to be
autoclaved as it will increase erosion reaction rate of the glass container.
Uses of type IV glass containers
It is used to store topical products and oral dosage forms
Test for Alkalinity of Glass:
• Two types of tests are designed
• Powdered Glass Test
• Water attack test
• These tests are performed on new (not previously used) glass
containers.
• The degree of attack is determined by the amount of alkali released
from the glasses under the conditions specified.
• This quantity of alkali is very small so strict precautions is to be followed.
• Euipments of High quality and precision should be used and area should
be free from fumes and excessive dust
Apparatus used:
• An autoclave capable of maintaining a temperature of
121 ±0.5 °C, equipped with a thermometer, a pressure gauge, a
vent and a rack.
• hardened-steel mortar and pestle.
• Other Equipment required are sieves No. 20, 40 and 50 along
with the pan and cover, 250ml conical flask made of resistant
glass as specified , 2 lb hammer, and adequate volumetric flask

Reagents:
1) Special Distilled water:
The water used in these tests has a specific conductivity of 0.5 µ
mho to 1 µ mho. After double distillation , reject the first 10% or 15% of
the distillate, and retain the remaining 75%.
2) Methyl red solution:
Dissolve 24 mg of methyl red sodium in sufficient purified water to
make 100ml. Neutralize the solution with 0.02 N sodium hydroxide
Powdered Glass Test:
• Take 6 or more containers. Rinse with purified water and dry steam of
clean and dry air
• Crush into fragments.
• Take 3 samples of 100 grams
• Crush them further separately in special mortar with hammer.
• Nest the sieves and put the three samples combinely on the No. 20
sieve.
• Shake the sieves for a while and remove the glass powder from No. 20
and 40 sieves and repeat the crushing in special mortar on them
• After that Continue the shaking process for 10 minutes.
• Collect the glass powder (excess of 10 grams) from No. 50 sieve and
place in a close container and store in a desiccator
• Spread the sample on a gazzed paper.
• Pass a magnet to remove any iron particle
• Transfer sample to 250ml special conical flask.
• Wash the sample with six 30-ml portions of acetone
• Dry the flask along the sample for 20 minutes at 140 C. transfer to
desiccator and use within 48 hours
Procedure:
• Take 10 gram of the sample in special conical flask
• Add 50ml of the special distilled water to this flask.
• Cap all flasks with special beakers so that the bottom of the beaker fit
the tip of the flasks.
• Place both in autoclave, close the autoclave leaving the vent open and
heat.
• When steam appear from the vent. Close the vent and heat further with
1degree rise in temperature per minute upto 121°C

• Hold the temperature for 30 minutes at 121°C . then slowly cool the
autoclave and remove the flasks.
• After fast cooling, decant the water into a vessel, and wash the residual
powder with four 5 ml portions of special distilled water, adding the
decanted washings into the main portion
• Add 5 ml methyl red solution and titrate against 0.02 N Sulphuric acid.
Record the volume of the acid. The volume of acid should not exceed
from that indicated in monograph.
Water Attack test:
• Rinse 3 or more containers with special distilled water
• Fill each containers upto 90 % of their over flow capacity with special
distilled water
• The flasks are capped and placed in autoclave.
• The sample is heated for 1 hour instead of 30 minutes.
• Flasks are taken out from autoclave and with the help of graduated
cylinder, 100ml of water from each container is transferred to conical
flask.
• 5 drops of methyl red solution are added to the sample and titrated
against 0.02N H2SO4.The volume of H2SO4 is noted and compared
with limits.
LIMITS:
• O.7 ml of sulphuric acid for containers less than or
equal to 100 ml
• 0.2 ml for containers greater than 100 ml
• Test performed after some time of storage of the containers
• Same procedure as water attack test
• only following differences
• Solution of methyl red: 0.2 g of methyl red dissolved in 60ml of alcohol,
7.5 ml of 0.5 N sodium hydroxide and diluting upto 100 ml with special
distilled water.
• Cover the containers with aluminum foil washed thoroughly with
acetone, instead of glass beakers
• Cool at room temperature instead of rapid cooling after removal from
autoclave. Titrate with 0.01 N Sulphuric acid instead of 0.02N sulphuric
acid.
• Limits: 1.5 ml of sulphuric acid for containers less than or equal to 100
ml
• 0.5 ml for containers greater than 100 ml

Control Chart:
A statistical based tool used to determine whether a manufacturing of
dosage form in pharmaceutical industry is with in predetermined
specification range (quality) or not.
▪ Control charts, also known as Shewhart Charts or Process-behavior
Charts
▪ The purpose of the chart is to indicate deviating trends in order that
the system may be brought back into control.
▪ It contain a centerline representing the process average or expected
performance, as well as upper and lower control limits that set bounds
for an acceptable area of deviation from the centerline. Control limits
are not the same as specification limits.
▪ Control limits indicate the process capability. They are not an
indication of how a process should perform.
Q Chart is a tool used by pharma industry to gather and analyze data on
the variations in specs of the product, which occur during production
processes, in order to determine corrective and preventive measure, if
so needed to ensure the quality of the product.
Variations in the production process are of two types;
❑Common Cause Variation: Variation because of inbuilt fault in the
design of the system leading to numerous, ever-present differences in
the process is called common cause variation.
❑Special Cause Variation; Variation because of causes which
are not normally present in the process but caused by
employees or by unusual circumstances or events is called
special cause variation.
✓Thus, a stable process is a process that exhibits only
common variation, or variation resulting from inherent system
limitations.
✓A stable process is a basic requirement for process
improvement efforts.

Need for Q charts
a) It is a proven technique for improving productivity.
b) It is effective in defect prevention.
c) It prevents unnecessary process adjustment.
d) It provides diagnostic information.
e) It provides information about process capability.
Quality Control chart
“Out of control” Process
“In control” Process
Function of Q.C. Charts
The main purpose of using a control chart is to monitor, control,
and improve process performance over time by studying
variation and its source. There are several functions of a control
chart:
1. It provides statistical ease for detecting and monitoring
process variation over time.
2. It provides a tool for ongoing control of a process.
3. It differentiates special from common causes of variation in
order to be a guide for local or management action.
4. It helps to improve a process to perform consistently and
predictably to achieve higher quality, lower cost, and higher
effective capacity.
5. It serves as a common language for discussing process
performance.
Two categories of control charts:
❑ Variable Control Charts
❑ X bar-R chart
❑ X bar-S chart
❑ Attribute control charts
❑ np-chart
❑ p-chart
❑ c-chart
❑ u-chart
TYPES OF CONTROL CHARTS

X bar-R chart
It is a set of control chart for variables data (data that are both
quantitative and continuous in measurement, such as a measured
dimension or time).
The X-bar chart monitors the process location over time, based on the
average of a series of observations, called a subgroup.
The range chart monitors the variation between observations in the
subgroup over time.
X-bar-R charts are used when collected measurements are in groups
(subgroups) of between two and ten observations.
o X-bar and R charts consist of two charts, both with the same horizontal axis
denoting the sample number.
o The vertical axis on the top chart depicts the sample means (X-bar) for a series of
lots or subgroup samples.
o It has a centerline represented by X-bar bar, which is simply the overall process
average, as well as two horizontal lines, one above and one below the centerline,
known as the upper control limit or UCL and lower control limit or LCL, respectively.
o These lines are drawn at a distance of plus and minus three standard deviations
(that is, standard deviations of the sampling distribution of sample means) from the
process average.
X-bar chart Range (R) chart
X bar-S chart
The X-bar chart monitors the process location over time, based on the average
of a series of observations, called a subgroup.
The sigma (S) chart monitors the variation between observations in the
subgroup over time.
X bar-S charts are generally employed for plotting variability of sub-groups with
sizes greater than 10. X bar-S charts plot the process mean (the X bar chart) and
process standard deviation (the S chart) over time for variables within
subgroups.
Both the X bar-R and -S charts must be seen together to interpret the stability of
the process. The X bar-S chart must be examined first as the control limits of the
X bar chart are determined considering both the process spread and center

S-charts
X-bar chart
Attributes control charts
It is used when measurements are qualitative, for example,
accept / reject.
a. np-chart: It is a control chart for measurements which
are counted, such as number of parts defective per
batch per day per machine but more then 5 % of the
samples inspected means not rare.
b. p-chart: It is a control chart for
fraction nonconforming, i.e., for
percentage measurements, such
as percentage of parts defective
with reference to whole number
of papulation of samples.
The p-chart monitors the percent
of samples having the condition,
relative to either a fixed or
varying sample size, when each
sample can either have this
condition, or not have this
condition.
c. c-chart: It is a control chart for
number of defects or
nonconformities, i.e., for
measuring defects in units of
constant size, for example, number
of imperfections in tablets of one
batch of 50,000 tablets.

d. u-chart: It is a control chart for
number of nonconformities per
unit, i.e., for measuring defects in
units of varying size, for example,
number of imperfections in micro
encapsulates. u-chart is typically
used to analyze the number of
defects per inspection unit in
samples that contain arbitrary
numbers of units.
Advantages of Attribute Control Charts
❑ Easy to classify products as acceptable or unacceptable,
based on various quality criteria.
❑ Bypass the need for expensive, precise devices and time-
consuming measurement procedures.
❑ More easily understood by managers unfamiliar with quality
control procedures;
❑ Provide more persuasive (to management) evidence of quality
problems.
❑ Used for graphical representation of optimization result in drug
formulation.
Rules for detecting “OUT OF CONTROL” situations
A process is said to be in a state of STATISTICAL CONTROL if the values
are within the control limits and the pattern is random.
The process is called “out of control” when
2 or 3 points fall outside the warning limits (shift), OR
8 points in a row fall above or below the center line (shift), OR
6 points in a row are steadily increasing or decreasing (trend), OR
14 points in a row alternate up and down (two feed sources),
Implementation of these rules if show on the chart the lines for;
± 2 SE (warning limits) in addition to the control limits.

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