DEFINITIONS & SCOPE

Quality control
Quality control is a process employed to ensure a certain level of quality in a product or service.
It can be defined as:
“A system for ensuring the maintenance of proper standards in manufactured goods, especially by periodic
random inspection of the product”.
OR
“Quality control refers to a procedure or a set of steps taken during the manufacturing of a product or service
to ensure that it meets requirements and that the product/service is reproducible”.
Quality Assurance
Quality assurance is a wide ranging concept that covers all matters that individually or collectively influence
the quality of a drug. It starts from purchase to post market surveillance.
QA = QC + GMP’s
Pharmaceutical QC:
“It is mainly concerned with the analytical measurement processes and focuses on technical aspects of testing
of drugs at all stages.”

Quality Assurance Quality Control

Definition QA is a set of activities for ensuring QC is a set of activities for ensuring
quality in the processes by which quality in products. The activities focus
products are developed. on identifying defects in the actual
products produced.
Focus on QA aims to prevent defects with a QC aims to identify (and correct) defects
focus on the process used to make the in the finished product. Quality control,
product. It is a proactive quality therefore, is a reactive process.
process.
Goal The goal of QA is to improve The goal of QC is to identify defects
development and test processes so that after a product is developed and before
defects do not arise when the product it's released.
is being developed.
How Establish a good quality management Finding & eliminating sources of quality
system and the assessment of its problems through tools & equipment so
adequacy. Periodic conformance audits that customer's requirements are
of the operations of the system. continually met.
What Prevention of quality problems The activities or techniques used to
through planned and systematic achieve and maintain the product
activities including documentation. quality, process and service.
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Responsibility Everyone on the team involved in Quality control is usually the

developing the product is responsible responsibility of a specific team that
for quality assurance. tests the product for defects.
Example Verification is an example of QA Validation/Software Testing is an
example of QC
Statistical Statistical Tools & Techniques can be When statistical tools & techniques are
Techniques applied in both QA & QC. When they applied to finished products (process
are applied to processes (process outputs), they are called as Statistical
inputs & operational parameters), they Quality Control (SQC) & comes under
are called Statistical Process Control QC.
(SPC); & it becomes the part of QA.
As a tool QA is a managerial tool QC is a corrective tool
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Standard
Something established as a measure or model to which other similar things/materials should conforms.
Further two types:
a. Reference Standard
b. Working Standard
a. Reference Standard
Drug substance of highest purity which is reasonably attainable, specifically prepared by independent
synthesis or by further purification of existing production material and shown to be authentic by extensive
set of analytical tests.
b. Working Standard
Drug substance of established quality and purity as shown by comparison to the reference standard material
and used for routine quality.
Assay (Standardization)
The word assay comes from the French word essai, which means "trial".
It is type of analysis for the determination of amount of particular constituent in a mixture or biological and
pharmacological potency of a drug. It also refers to the measurement of active ingredient in a dosage form. For
example, an assay may be done of a vaccine to determine its potency.
OR
Determination of activity, potency and strength of substances either on absolute basis or in comparison with
that of standard.
1. Biological Assay:
Bioassays are typically conducted to measure the effects of a substance on a living organism and are
essential in the development of new drugs. Both are procedures by which the potency (pharmacology) or
the nature of a substance is estimated by studying its effects on living matter.
2. Chemical Assay:
It deals with the study of the chemical composition of substances. More broadly, it may be considered the
corpus of all techniques whereby any exact chemical information is obtained.
It has further two branches:
i. Qualitative assay:
Qualitative assay is the determination of those elements and compounds that are present in a sample
of unknown material.
ii. Quantitative assay:
Quantitative assay is the determination of the amount by weight of each element or compound
present.
Test
A technical operation that consists of determination of one or more characteristics or performance of a given
product, material, equipment, organism, process or service according to a specified procedure. Result of test is
normally recorded in a document called Test Certificate or Test Report.
Validation
The FDA defines validation as:
Establishing documented evidence which provides a high degree of assurance that a specific process or
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device will consistently produce data or a product that meets its predetermined specifications and quality
attributes.
OR
The documented act of demonstrating that any procedure, process and activity will consistently lead to the
expected results.
OR
Validation of any process is a scientific demonstration by appropriate tests that the process actually
accomplishes the intended effect under specified operating conditions.
It includes the qualification of systems and equipments. For example validation of Sterilization process.
Types of Validation
i. Prospective Validation
ii. Retrospective Validation
iii. Concurrent Validation
i. Prospective Validation
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Prospective validation is conducted before the distribution of either a new product or a product made under
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a modified production process. It is a preplanned scientific approach and includes:

 The Initial Stages Of Formulation Development
  Process Development
 Setting Of A Process Specifications
 Sampling Plans
 Designing Of Batch Records
 Defining Raw Material Specifications
 Completion Of Pilot Runs
 Transfer Of Technology From Scale Up Batches To Commercial Size Batches
 Listing Major Process Equipment
 Environmental control
ii. Retrospective Validation
Retrospective validation is conducted for a product already being marketed and is based on extensive data
accumulated over several slots. Retrospective validation may be used by the older products which were not
validated at the time that they were first marketed and which are now to be validated to conform to the
requirements of FDA.
iii. Concurrent Validation
It is the process in which current production batches are used to monitor processing parameters. It gives
assurance of the present batch being studied and offers limited assurance regarding consistency of quality
from batch to batch.
Accuracy
The accuracy of a measurement system is the degree of closeness of measurements of a quantity to its actual
(true) value.
Precision
The precision of a measurement system, also called reproducibility or repeatability, is the degree to which
repeated measurements under unchanged conditions show the same results.
Accuracy Precision
Definition:
“The ability of a measurement to match “The ability of a measurement to be
the actual value of the quantity being consistently reproduced.”
measured.” OR
OR “The number of significant digits to which
The accuracy of an analytical method is a value has been reliably measured.”
the extent to which test results generated
by the method and the true value agree.
Example:
If the temperature outside is 34.0 F and a If on several tests the temperature sensor
temperature sensor also reads 34.0 F, matches the actual temperature while the
then the sensor is accurate. actual temperature held constant, then the
sensor is precise.
By second definition: the no. 4.1215 is
more precise than the no. 4.12.
Accuracy is how close to true/actual Precision is how consistent your results are
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measurement are. for the same phenomena over several

measurements.
Single factor or measurement Multiple measurements or factors are
needed
Sensitivity
Capacity of test procedure or an instrument to record small variations in concentrations is called sensitivity.
Selectivity
Ability of method to measure accurately and specifically the analyte of interest even in the presence of
matrix andother components like impurities in the sample is called selectivity.
Linearity
Ability of method to produce test results that are directly proportional to concentration of analyte is called
linearity.
The linearity of a method gives the characteristic trend of parameters such as absorbance, peak
height, peak area or response ratio as a function of concentration of the component to be measured.
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Range
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Lowest and highest level of analyte that a method can determine with reasonable accuracy and precision is
called its range.
The range is normally expressed in the same units as the test results e.g percentage, parts per million
obtained by the analytical method.
Limit of Detection (LOD)
Lowest concentration of analyte in a sample that a method can detect but doesn’t necessarily quantify under
stated experimental conditions.
It simply indicates that analyte in the sample is below or above certain level.
Limit of Quantitation (LOQ)
LOQ is the lowest concentration of a substance in a sample that can be estimated quantitatively with acceptable
precision and accuracy.
Ruggedness
It is the degree of reproducibility of obtained by analyzing same sample under variety of normal test conditions,
different analysts, instruments, reagents and days.
Robustness
It is the measurement of capacity of a method to remain unaffected by small but considerable variations in
procedure.
Process Re-Validation
It is required when there is a change in any of the critical process parameters, formulation, primary packaging
components, raw material fabricators or major equipment. Failure to meet product or process specifications in
sequential batches would also require process re-validation.
Qualification
Qualification is a process of assurance that the specific system, premises or equipment are able to achieve the
predetermined acceptance criteria to confirm the attributes what it aims to do.
Calibration
The comparison of the measurement system or device of unknown accuracy to another measurement system or
device with a known accuracy to detect, correlate, report or eliminate any variation is called calibration.
Calibration is done to ensure that measuring equipment used in a manufacturing process or analytical
procedure gives measurements that are correct within established limits. For example calibration of a pH meter.
 Things are qualified. (For example equipments, systems etc).
 Processes and procedures are validated. (For example sterilization process).
 Calibration is one of the condition/step for qualification whereas Qualification is one of the
condition/step for validation.
Clean areas & rooms
Area with defined environment control of particulate and microbial contamination constructed and used in such
a way as to reduce the introduction, generation and retention of contamination within the area.
It is divided into 3 classes:
1. Class 100 area
Area not exceded from 100 particles/cubic feet of 0.5µ size
2. Class 1000 area
Area not exceded from 1000 particles/cubic feet of 0.5µ size
3. Class 100,000 area
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Area not exceded from 100,000 particles/cubic feet of 0.5µ size

Test
“A technical operation that consists of determination of one or more characteristics or performance of a
given product, material, equipment, organism, process or service according to a specified procedure.”
In Process Quality Control (IPQC)
In process quality control is a process of monitoring critical variables of manufacturing process to ensure
a quality of the final product. In-process manufacturing controls are established and documented by quality
control and production personnel to ensure that quality of the product is within the acceptable standard
range.
Statistical Quality Control (SQC)
“The monitoring of quality by application of statistical methods in all stages of production.”
Or
“The application of statistical methods to quality control.”
 It refers to characteristics of product from both quantitative and qualitative point of view to meet
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established standards.
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 By using charts and collecting few frequent samples, we can detect the change in process that may affect
the quality.
 These charts are:-
o Control charts for variables
o Control charts for attributes
Dissolution Test:
“Dissolution testing is required for all solid oral Pharmacopoeial dosage forms in which absorption of the drug
is necessary for the product to exert the desired therapeutic effect. Exceptions are for tablets meeting a
requirement for completeness of solution or for rapid (10 to 15 minutes) disintegration for soluble or
radiolabeled drugs.”
Laminar Flow Hood:
Laminar flow is unidirectional air moving at a steady velocity along parallel lines. Laminar flow cabinets may
or may not be biological safety cabinets.
OR
The laminar flow hood provides an aseptic work area while allowing the contaminant of infectious splashes
or by many microbiological procedures.
HEPA Filter:
It is designed to remove particles, including microorganisms, from the air. HEPA filters are effective at trapping
particulates & infectious agents but not at capturing volatile chemicals or gas. Only certain classes of biological
safety cabinets that are exhausted to the outside can be used when working with small amounts of volatile
chemicals.
Pyrogen Testing:
1. Rabbit Test:
This test involves measurement of rise in body temperature of rabbits following the IV injection of a
sterile solution of a substance to be examined. It is designed for products that can be tolerated by test
rabbit in a dose not exceeding 10ml per kg injected with in a period of not more than 10 minutes.
2. Lal’s Test:
Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe
crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a
membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used
for the detection and quantification of bacterial endotoxins.
Sterility:
Sterility or freedom from the presence of viable microorganisms, in a strict, uncompromising requirement of
an injectable dosage form.
Rheology:
Rheology is the study of the flow of matter, primarily in the liquid state, but also as 'soft solids' or solids under
conditions in which they respond with plastic flow rather than deforming elastically in response to an applied
force.
It applies to substances which have a complex microstructuring like muds, sludges, suspensions, polymers and
other glass formers (e.g., silicates), as well as many foods and additives, bodily fluids (e.g., blood) and other
biological materials or other materials which belong to the class of soft matter.
Pyrogens:
Pyrogen consists of two words:
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o Pyro-pyrexia (fever or rise in body temp.)

o Gen-producing or generation.
“Pyrogen is simply fever producing agent.”
“Pryrogens are substances that cause febrile reactions when sufficient amount enters in circulatory
system.”
Bacterial endotoxin is the most significant pyrogen because of its potency and ubiquity.
Friability:
“Friability (the condition of being friable) is the ability of a solid substance.”
A friable substance is any substance that can be reduced to fibers or finer particles by the action of a
small pressure or friction.
Stability Studies:
Stability studies are a critical part of the drug development process and are essential for drug product
marketing approval.
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Stability studies are conducted at all phases of the drug development cycle for different purposes with the
ultimate goal of having a stable product on the market. During development, stability studies are conducted to
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support the formulation development and safety and efficacy claims of investigational new drugs.
Difference Between Iodometry And Iodimetry:
Iodometry Iodimetry
1. When an analyte that is an oxidizing agent 1. When an analyte that is a reducing agent is
is added to excess iodide to produce titrated directly with a standard iodine solution,
iodine, and the iodine produced is the method is called "iodimetry"
determined by titration with sodium
thiosulfate, the method is called
"iodometry".
2. Iodometry a species is titrated with an 2. Iodimetry, a species is directly titrated with an
iodide solution and then the released iodine solution.
iodine is titrated with thiosulphate.
3. Iodometry is an indirect method. 3. Iodimetry is a direct method.
4. Iodometry can be used to quantify 4. Iodimetry can be used to quantify reducing
oxidizing agents agents.

Total Quality Management (TQM)

“The continuous process of reducing or eliminating errors in manufacturing, streamlining supply chain
management, improving the customer experience and ensuring that employees are up-to-speed with their
training. Total quality management aims to hold all parties involved in the production process as accountable
for the overall quality of the final product or service.’’
Weight Variation
This test is performed to check that the tablet contains the proper amount of drug and to ensure that the dose is
in safe therapeutic window.
Uniformity Of Content
Uniformity of Content is a pharmaceutical analysis technique for the quality control of capsules or tablets.
 Multiple capsules or tablets are selected at random and a suitable analytical method is applied to assay
the individual content of the active ingredient in each capsule or tablet.
 It is a test to ensure homogenous distribution of active ingredient in a dosage form.
 It involves weighting, crushing, making dilutions and analyzing by using a recommended analysis
method.
Quality Assurance System (QAS)
Formal Structures or techniques to make sure products and services consistently meet the standard required by
the customer; quality systems may be validated either within your organization, or by external auditors or by
both.
The basic steps followed to implement system for an organization are:
 Develop the system
 Document it
 Inform, instruct and train the staff to use it.
Features Of Quality Assurance System
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 Pharmaceutical products are designed and developed in a way that takes account of the
requirements of GMP and other associated codes such as those of good laboratory practice (GLP)
and good clinical practice (GCP).
 All necessary controls on starting materials, intermediate products, and bulk products and other in-
process controls, calibrations and validations are carried out.
 The finished products is correctly processed and checked according to the defined procedures.
 Satisfactory arrangements exist to ensure, that the pharmaceutical products are stored by the
manufacturer, distributed and subsequently handled.
 There is a procedure for self-inspection.
 Deviation are reported, investigated and recorded
 There is a system for approving changes that may have an impact on product quality.
 Regular evaluations of the quality of pharmaceutical products should be conducted.
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Standard Operating Procedures (SOPs):
"A Standard Operating Procedure is a document which describes the regularly recurring operations relevant to
the quality of the investigation. The purpose of a SOP is to carry out the operations correctly and always in the
same manner. A SOP should be available at the place where the work is done".
A SOP is a compulsory instruction. If deviations from this instruction are allowed, the conditions for these
should be documented.
Types of SOPs:
 Fundamental SOPs: These give instructions how to make SOPs of the other categories.
 Methodic SOPs: These describe a complete testing system or method of investigation.
 SOPs for safety precautions.
 SOPs for operating instruments, apparatus and other equipment.
 SOPs for analytical methods.
 SOPs for the preparation of reagents.
 SOPs for receiving and registration of samples.
 SOPs for Quality Assurance.
 SOPs for archiving and how to deal with complaints.
Quality Management (QM)
“The act of overseeing all activities and tasks needed to maintain a desired level of excellence. This includes
creating and implementing quality planning and assurance, as well as quality control and quality
improvement.’’
Total Quality Management (TQM)
“The continuous process of reducing or eliminating errors in manufacturing, streamlining supply chain
management, improving the customer experience and ensuring that employees are up-to-speed with their
training. Total quality management aims to hold all parties involved in the production process as accountable
for the overall quality of the final product or service.’’
Quality Management System (QMS)
“A system by which an organization aims to reduce and eventually eliminate nonconformance to
specifications, standards, and customer expectations in the most cost effective and efficient manner.”
Elements Of Quality Management System
 Organizational structure
 Responsibilities
 Processes
 Resources
 Data management
 Customer satisfaction
 Continuous improvement
 Product quality
Good Manufacturing practices (GMP’s)
Good manufacturing practices are the sets of the principles, regulations, codes (law or official standard),
guidelines and procedures and part of quality assurance system which must be followed by the
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manufacturers to ensure that the products that are consistently produce are of quality standard and
appropriate for their intented use and cover the manufacturing and testing of pharmaceutical dosage form
and active pharmaceutical ingredients, diagnostics, foods, various other pharmaceutical products and
medical devices.
Current Good Manufacturing practices (cGMP’s)
cGMP’s are used for the technology that is up to date.
GMP Principles:
Good manufacturing practice guidelines provides guidance for manufacturing, testing, and quality
assurance in order to ensure that drug product is safe for human consumption. Many countries have
legislatsed that pharmaceutical and medical device manufacturer must follow GMP procedures, and have
created their own GMP guidelines that correspond with their legislation.
All guidelines follow a few basic principles:
 Hygiene: Pharmaceutical manufacturing facility must maintain a clean and hygienic manufacturing area.
 Controlled environmental conditions in order to prevent cross contamination of drug product from other
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drug or extraneous particulate matter which may render the drug product unsafe for human consumption.
 Manufacturing processes are clearly defined and controlled. All critical processes are validated to ensure
consistency and compliance with specifications.
 Manufacturing processes are controlled, and any changes to the process are evaluated. Changes that have
an impact on the quality of the drug are validated as necessary.
 Instructions and procedures are written in clear and unambiguous language.
 Operators are trained to carry out and document procedures.
 Records are made, manually or by instruments, during manufacture that demonstrate that all the steps
required by the defined procedures and instructions were in fact taken and that the quantity and quality of
the drug was as expected. Deviations are investigated and documented.
 Records of manufacture that enable the complete history of a batch to be traced are retained in a
comprehensible and accessible form.
 The distribution of the drugs minimizes any risk to their quality.
 A system is available for recalling any batch of drug from sale or supply.
 Complaints about marketed drugs are examined, the causes of quality defects are investigated, and
appropriate measures are taken with respect to the defective drugs and to prevent recurrence.
Endotoxin
Endotoxins are part of the outer membrane of the cell wall of Gram-negative bacteria. Endotoxin is invariably
associated with Gram-negative bacteria whether the organisms are pathogenic or not. Although the term
"endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is properly
reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-
negative pathogens such as Escherichia coli,Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus
influenzae,Bordetella pertussis and Vibrio cholerae.

Difference b/w Content Uniformity and Assay:

Uniformity of Content Assay
Uniformity of Content is An assay is an investigative
a pharmaceutical analysis technique for (analytical) procedure for
the quality control of capsules or tablets. qualitatively assessing or
Multiple capsules or tablets are selected quantitatively measuring the
at random and a suitable analytical presence or amount or the
method is applied to assay the individual functional activity of a target entity
content of the active ingredient in each which can be a drug or biochemical
capsule or tablet. substance.”

It is a test to ensure homogenous It is the determination of the

distribution of active ingredient in a content of a specific component
dosage form. with no evaluation of other
components.

It involves weighting, crushing, making An assay may be biological or

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dilutions and analyzing by using a chemical.

recommended analysis method.
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 QUALITY CONTROL OF SOLID DOSAGE FORM

Dosage Forms:
Completed forms of the pharmaceutical preparation in which prescribed doses of medication are included.
They are designed to:
 Resist action by gastric fluids.
 Prevent Vomiting and Nausea
 Reduce or alleviate the undesirable taste and smell associated with oral administration.
 Achieve a high concentration of drug at target site.
 Produce a delayed or long-acting drug effect.
Solid Dosage Forms:
Solid dosage forms include
 Tablets
 Capsules
 Granules
 Powders
Tablets:
Tablets are solid dosage forms containing one or more active ingredients. They are unit dosage form. They are
obtained by single or multiple compression and may be coated or uncoated. They are usually intended for oral
applications but sometime they also have some alternative applications such as implants, tablets for injection,
irrigation or external use, vaginal tablets etc.
Capsules:
Capsules are solid dosage forms with hard or soft gelatin shells. They are of various shapes and sizes; contain
a single dose of one or more ingredients. They are intended for oral administration.
Powders:
A powder is intimate mixture of dry, finely divided drugs and/or chemicals that may be intended for internal
or external use.
Granules:
Granule is generic term used for small particle or grain, A granule is formed when small particles gathered
into longer, permanent aggregate in which the original particles can still be identified.
2.1. GENERAL APPEARANCE
Different parameters of tablet appearance are
 Size and Shape
 Color
 Odor
 Surface Texture
1. Size and Shape:
Purpose:
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To ensure the trouble free packing improve elegance and to control the weight of tablet.
Instruments:
 The size of tablet is directly related to thickness and diameter of tablets, that depends on the size
of die and punches, amount of fill material and force /pressure of compression.
 The thickness and diameter of the tablet may be measured manually (by micrometer screw gauge
and venire calipers) or by automatic equipment.
Procedure:
10 tablets are taken randomly to measure the thickness and diameter by placing them vertically and
horizontally in the Jaws of instruments.
For uncoated tablets
 The diameter should be 4mm-14mm
 The thickness should be 2mm-4mm
 Tablets having the diameter less than 12.5mm have variation of ±5%
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 Tablets having the diameter more than 12.5mm have variation of ±3%
Specification:
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±5% of stated to tablet hardness and can be used as initial control during production.
 Advantages:
 Thickness of tablets is directly related to tablet hardness and can be used as initial control parameter.
 Limits of thickness and diameter of tablet depend on tablet weight to ensure trouble free packaging
(when tablets are thicker than normal then packaging process is disturbed).
 The shape of tablet should be as provided by monograph i.e. round, circular, double convex etc and
for shaped tablets slotted punches must be run at slower speeds.
Application:
 Ensure the trouble free packaging.
 Used as control parameter.
2. Color:
 Color specification is helpful for the identification of specific product during manufacturing process.
 There should be equal distribution of color as uniform distribution of color increases the ethical appeal.
 Sometime during manufacturing the problem of mottling (unequal and uneven distribution of color)
occur that shows poor quality product.
3. Odour:
 Presence or absence of odor in a batch of tablet is also tested.
 Some drugs produce characteristic odor e.g. multivitamins and it is helpful for the detection of
material.
 While in some drugs presence of odour show the unstable drug or degradation e.g. aspirin has vinegar
like odour if degraded.
4. Surface texture:
 The surface of tablet should be smooth and there must be no chips, cracks, contamination from external
substances, capping and sticking.
2.2. PHYSICAL TESTS
Official Tests:
1. Disintegration
2. Weight Variation
3. Friability
4. Hardness
Non-Official Tests:
1. Thickness & Diameter
2.1.1 OFFICIAL TESTS:
1. DISINTEGRATION TEST (USP):
The state in which any residue of the unit, except fragment of insoluble coating or capsule shell, remaining
on the screen of the test apparatus or adhering to the lower surface of the disk, if used, is a soft mass having
no palpably firm core.
Theories of disintegration:
Several mechanisms of tablet disintegration has been proposed. Some of them are given below:
1. Evalution of gas:
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If the gas is evolved by a chemical reaction, when the tablet comes into contact with water, then
the tablet will disintegrate. This is the basis for manufacture of effervescent tablets.
Example of such a reaction is sodium bicarbonate with citric and tartaric acid, which yields carbon
dioxide.
2. Heat of wetting:
The heat produced when a tablet is immersed in water causes the entrapped air in the tablet to
expand and exert sufficient pressure to disintegrate the tablet.
3. Effect of water absorption :
The water absorbed by the tablet initiate disintegration, but this depends upon the solubility of the
drug and other ingredients present.
4. Swelling:
The grains of the disintegrant, particularly of starches, swell in the present of water and exert
pressure on the granules to force them apart.
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5. Porosity of tablets:
It has been shown that penetration of water into a tablet is proportional to its mean pore diameter or
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porosity. The porosity and permeability of tablets decrease as the tableting pressure increased, and as
the porosity decrease, the disintegration time increases.
 Apparatus-A Apparatus-B
For tablets and capsules less than 18 mm long For tablets and capsules more than 18 mm long
Tubes: Tubes:
 6 open ended tubes  3 open ended tubes
 77.5 ± 2.5 mm long  77.5 ± 2.5 mm long
 21.5 mm internal diameter  33.5 ± 0.5 mm internal diameter
 2 mm wall thickness  2 mm wall thickness
Plates: Plates:
 2 superimposable plates  2 superimposable plates
 90 mm diameter’  97 mm diameter’
 6 mm thick  9 mm thick
 6 holes  3 holes
Discs: Discs:
 Cylindrical discs  Cylindrical discs
 20.7 ± 0.15 mm diameter  31.4 ± 0.13 mm diameter
 9.5 ± 0.15 mm thick  15.3 ± 0.15 mm thick
 Transparent plastic material  Transparent plastic material
 Density 1.18-1.20  Density 1.18-1.20
 Weighing 3.0 ± 0.2g  Weighing 3.0 ± 0.2g
 Each disc with 5 holes & 4 groves  Each disc with 7 holes & without groves
 Diameter of perforation is 2.0 mm  Diameter of perforation is 3.15 ± 0.1mm

Purpose:
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This test is performed to determine that whether the tablets or capsules disintegrate within the
prescribed time when placed in a liquid medium under the specified experimental conditions
Apparatus:
Types of apparatus
Two types of apparatus are used:
Apparatus-A:
Most commonly used for tablets which are less than 18 mm long
Apparatus-B:
Most commonly used for tablets which are more than 18 mm long
Apparatus construction
1. Circular basket rack assembly
2. Suitable vessel for immersion fluid (1 liter beaker)
3. Thermostatic arrangement for maintaining the temperature at 37+2 oC
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4. A device for rising and lowering of basket rack in immersion fluid at constant frequency of 28-32
cycles/min through a distance of 50-60 mm.
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 Basket-rack assembly:
 The basket-rack assembly consists of 6 open-ended transparent tubes and a rack for holding these tubes
in vertical direction.
 Each tube is 77.5 ± 2.5 mm long and having an inside diameter of 21.5 mm and a wall 1 to 2.8 mm thick
 The tubes are held in a vertical position by two plastic plates which are circular in shape and made up
of transparent material having six holes of a diameter that allow the tube to be inserted.
 Attached to under surface of the lower plate is a woven stainless steel wire cloth, which has a plane
square weave with 1.8 to 2.2 mm mesh apertures and with a diameter of 0.63±0.03 mm.
 The parts of the apparatus are assembled and rigidly held by means of three bolts passing through the 2
plastic plates.
 A suitable means is provided to suspend the basket-rack assembly from the raising and lowering device
using a point on its axis.
Liquids used in disintegration:
 Water
 Simulated gastric fluid (PH = 1.2)
 Simulated intestinal fluid (PH = 7.5 )
Procedure:
Place 1 tablet in each of the six tubes of basket and operate the apparatus using water maintained at 37±20.
At the end of time limit, lift the basket from the fluid and observe the tablets, if all of the tablets have
disintegrated, test is clear, if 1-2 tablets fail to disintegrate, repeat the apparatus at 12 additional tablets.
Acceptance criteria:
All the 6 tablets or capsules must be disintegrated. If 1 or 2 dosage unit/s fails to disintegrate than repeat the
test with additional 12 tablets. The requirements of the test are meet if not less than 16 of the 18 dosage units
tested are disintegrated.
Acceptance/rejection criteria:
i. Uncoated tablets:
 If one or two of 6 tablets fails to disintegrate completely, repeat the test on 12 additional tablets, not
less than 16 of total 18 tablets disintegrate completely.
o According to USP disintegration time must be less than 30min.
o According to BP disintegration time must be less than 15min.
ii. Plain coated tablets (film/sugar coated):
Prescribed time and acceptance/rejection criteria are the same as to the uncoated tablets.
iii. Enteric coated tablets:
 One tablet in each tube.
 Operate the apparatus using simulated gastric fluid till 1-2 hours.
 Lift the basket from fluid and observed the tablets.
 There should be no evidence of disintegration, cracking and softening.
 Operate the apparatus using simulated intestinal fluid for one hour or the time specified in the
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monograph.
 Acceptance/rejection criteria are same as of the uncoated tablets.
iv. Buccal tablets:
 One tablet in each tube.
 Operate the apparatus using water or specified medium, if prescribed.
 After 4 hours all the tablets should have disintegrated.
 Acceptance/rejection criteria are same as of the uncoated tablets.
v. Sublingual tablets:
 Prescribed medium and A/R criteria is same.
 Omit the use of disc.
 Observe the tablets for time specified in monograph.
15

 For the most of the sublingual tablets time limit is within 2min.
vi. Hard gelatin capsules:
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 Apply the test of uncoated tablets.

  Attach the removable wire cloth, which has a plain square weave with 1.8 to 2.2 mm mesh
apertures and with a wire diameter of 0.60 – 0.655mm, as described under basket-rack assembly,
to the surface of the upper plate of basket-rack assembly.
 Observe the capsules within the time limit specified in the individual monograph.
 All the capsule have disintegrate except the capsule shell.
vii. Soft gelatin capsules:
Proceed as describer under hard gelatin capsules.
2. WEIGHT VARIATION (UNIFORMITY OF WEIGHT)
i. For Tablets:
 Weigh 20 tablets individually and calculate average weight.
 Now calculate the percentage deviation by following formula.

𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 − 𝑰𝒏𝒅𝒊𝒗𝒊𝒅𝒖𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕

𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝒅𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 = × 𝟏𝟎𝟎
𝐀𝐯𝐞𝐫𝐚𝐠𝐞 𝐰𝐞𝐢𝐠𝐡𝐭
Acceptance criteria:
Not more than 2 of the individual masses deviate from the average mass by more than the percentage
deviation given in the following table.
According to USP
Average weight of tablet(mg) %age deviation
130 mg or less ±10%
130-324 mg ±7.5%
More than 324 mg ±5%

According to BP
Average weight %age deviation
80 mg or less ±10%
More than 80 to less than 250 mg ±7.5%
More than 250 mg ±5%
ii. For Capsules BP:
 Weigh an intact capsule.
 Open the capsule without losing any part of the shell and remove the contents as completely as
possible.
 For soft shell capsules, wash the shell with a suitable solvent and allow to stand until the odour of
the solvent is no longer perceptible.
 Weigh the shell. The mass of the contents is the difference between the weighings.
 Repeat the procedure with another 19 capsules.
Acceptance criteria:
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Not more than 2 of the individual masses deviate from the average mass by more than the
percentage deviation given in the following table.
Average weight %age deviation
Less than 300 mg ±10%
More than 300 mg ±7.5%
iii. For Powders:
 Remove any paper label from outside then dry and wash it from outside.
 Open the container and weigh the container and its contents.
 Empty the container as completely as possible by gentle tapping; rinse it if necessary with
water or Alcohol.
 Dry it in oven.
 Allow to cool in desiccators and weigh the mass of contents i.e. the difference between the
weighing.
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 Repeat the procedure with another 19 containers.

Acceptance Criteria:
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Average weight %age deviation

More than 40mg ±10%
 Importance:
 This test is important in terms of
 Safety of dosage unit ̴ proper weight ̴proper dose
3. FRIABILITY:
It is the tendency of tablets to powder, chip, or fragment and this can affect the elegance appearance,
consumer acceptance of the tablet, and also add to tablet’s weight variation or content uniformity problems.
OR
It is the resistance of the tablets against the mechanical shock during packaging, handling and transportation
and helpful for checking the lamination and capping
Significance
 Check breakability
 Check drug loss
 Check capping and hardness
Apparatus:
An instrument called friabilator consisting of drum is used to evaluate the ability of the tablet to withstand
abrasion in packaging, handling, and shipping.
 Internal diameter 283-291mm
 Depth 36-40mm
 Made of transparent synthetic polymer with internal surface polished.
 A curved projection with an inside radius b/w 75.5-85.5mm that extent from middle of the drum to outer
wall from where tablets are tumbled.
 Drum is rotated at 25± 1rpm.
 Thus at turn, tablets rolls or slide, and fall onto the drum wall or onto each other.
Acceptance criteria:
 Not more than 1% USP
 Not more than 0.8% BP
Procedure:
 For tablets weighing upto 0.65g, take a sample of 20 tablets.
 For tablets weighing more than 0.65g, take a sample of 10 tablets.
 Place the tablet on a sieve no. 1000 and remove any loose dust with the aid of air pressure or a soft
brush.
 Accurately weigh the tablet samples and place the tablet in the drum.
 Rotate the drum 100 times (25 rpm for 4 minutes) and remove the tablets.
 Remove any loose dust from the tablets as before. If no tablets are cracked, split or broken, weigh the
tablets to the nearest mg.
 If the results are doubtful, repeat he test 3 times, take the average, average should be maximum to 1%.
Formula:
𝑾𝒐
𝑭 = 𝟏𝟎𝟎 (𝟏 − )
𝑾𝟏
 100 = Revolutions
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 Wo = Weight before test

 W1 = Weight after test
Disadvantages
 Affect the elegance of tablets.
 Affect the consumer acceptance of tablets.
4. HARDNESS (BP) OR BREAKING FORCE (USP)
It is the resistance of tablet against applied force till it break.
OR
It is the load required to crush the tablet when placed on its edge.
Importance
Check the breakage during
 Manufacture
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 Packaging
 Handling
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 Storage
 Why do we measure hardness?
 To determine the need for pressure adjustments on the tableting machine.
 To determine the disintegration time.
 To determine elasticity.
Factors Affecting the Hardness:
 Compression of the tablet and compressive force.
 Amount of binder. (More binder a more hardness)
 Method of granulation in preparing the tablet (wet method gives more hardness than direct method,
Slugging method gives the best hardness).
Types of hardness testers:
i. Manual or mechanical
 Strong-Cobb tester
 Monsanto Hardness tester
 Eureka Hardness tester
 Pfizer Hardness tester
ii. Motor driven
 Heberlein Schleuniger Hardness tester
 Erweka Hardness tester
 Casburt Hardness tester
Procedure:
i. Stoker-Monsanto Hardness Tester (manual):
 It is a small potable hardness tester, manufactured by Monsanto chemicals.
 Select 6 tablets randomly according to USP
 Place the tablets, diametrically, between the moving and fixed jaw one by one.
 Adjust the reading of indicator scale to zero.
 Gradually increase the force applied to the edge of tablet by moving the screw knob forward until
the tablet breaks.
 The reading is noted from the scale which indicates the pressure required in kg to break the tablet.
ii. Eureka Hardness Tester (mechanical):
 In this instrument the breaking force is applied by a beam fastened to one end to a pivot.
 The motor moves a weight along the beam at a constant speed and increase the force against the
tablet.
 When the tablet breaks, a micro switch is activated that stop the motor.
 An indicator is fastened to the weight shows the breaking strength on a scale in kg.
 The average hardness acceptable for compressed tablet ranges between 5-10 kg/cm2.
 German mode instrument has scale in Newton and 1N=9.8kg.
Acceptance criteria:
5-10 kg/cm2 according to USP
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i. According to USP:
Minimum of 6 tablets should be tested
ii. According to BP:
Carryout the measurement on each tested tablet.
2.1.2 NON-OFFICIAL TESTS:
1. THICKNESS AND DIAMETER:
Devices:
Micrometer Screw gauge, Vernier calipers or automatic equipments
Significance:
Proper packaging of solid dosage form, i.e. in blister, strip, bulk or bottle packagings.
Factors Affecting the thickness & diameter:
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Following factors are there:

 Tablet compression or force
 Amount of material in punch or die
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 Depth and diameter of die

 Procedure
 Select 10 tablets from the batch randomly and measure the thickness and diameter by the devices.
 All the tablets must follow the acceptance criteria.
Allowed variation:
Allowed range = ±5%
Acceptance criteria
Thickness = 2-4mm
Diameter = 4-14mm

2.3. CHEMICAL TESTS

1. CONTENT UNIFORMITY TEST (EP)
This test is used to confirm that every unit should contains same amount of drug or active ingredients with
little variation in the batch.
Principle:
The test of the uniformity of the content of single dose preparations is based on the assay of individual
contents of active substance(s) of a number of dosage units to determine whether the individual contents are
within the limits set with reference to the average content of the sample.
Method:
 Using a suitable analytical method, determine the individual contents of active substance(s) of 10
dosage units taken at random.
 Now apply the criteria of Test A, Test B or Test C as specified in the monograph for any specified
dosage form.
Test A:
 For tablets, powders for parenteral use, ophthalmic inserts, suspension for injection.
 The preparation complies with the test if each individual content is between 85-115% of the
Average content.
 The preparation fails to comply with the test if more than 1 individual content is outside 85-115%
of the average content or if 1 individual content is outside 75-125% of the average content.
 If 1 individual content is outside the limit of 85-115% but within the limit of 75-125%, determine
the individual content of another 20 dosage units taken at random.
 The preparation complies with the test if more than 1 individual contents of the 30 units is outside
85-115% of the average content and none is outside the limit of 75-125% of the average content.
Test B:
 For capsules, powders other than parenteral use, granules, suppositories, pessaries.
 Complies with test if not more than 1 individual content is outside 85-115% of the average content
and none is outside the limit of 75-125% of the average content.
 Fails to comply with test if more than 3 individual contents are outside 85-115% of the
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average content or if 1 or more individual contents are outside the limit of 75-125% of the
average content.
 If 2 or 3 individual are outside the limit of 85-115% than take 20 more units and perform as
in Test A.
Test C:
 For transdermal patches.
 Complies with test if the average content of the 10 dosage units is between the limit of 90-
110% of the content stated on the label and if the individual content of each dosage unit is
between the limit of 75-125% of the average content.
2. DISSOLUTION TEST (USP)
It is the amount of drug that is dissolved or goes into the solution per unit time under the specified
conditions.
19

Test Specification
Dissolution medium:
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Test is begin with aqueous media of pH 1.2-6.8. Simple water is usually not recommended due to the ionic
strength and pH can be vary. Different type of dissolution medium are as follow:
 i. Water or medium pH less than 6.8 with addition of purified pepsin (Intestinal environment)
ii. Medium with pH 6.8 or greater or pancreatic medium can be added (protease activity)
iii. 0.1N HCl with pH 1.2
iv. Acetate buffer with pH 4.5
v. Phosphate buffer with pH 6.8-7.5
vi. Delayed release dosage form pH 6.8
Temperature:
Temperature is maintained according to body temperature i.e. 37 ± 2 oC.
Speed of the apparatus:
50-100 rpm
Sink condition:
Concentration that yield saturation solubility of drug substances at least three time the weight dose of the
drug substance dissolved in the volume of the medium used for dissolution.
Example
If the dissolution of 100mg strength tablet is being performed in 900ml of the medium. A saturation
solubility is greater than 0.33mg/ml in the medium is required to maintain sink condition.
Reference Standard USP
Non-disintegrated
USP Chlorpheniramine Maleate Extended-Release Tablets (Apparatus III)
USP Salicylic Acid Tablets (Apparatus I & II)
Disintegrated
USP Prednisone Tablets (Apparatus I & II)
Dissolution Test
 In 1970’s dissolution test for dissolution introduce first time.
 In 1970’s USP recommended 7 apparatus for dissolution test
 In 1975’s BP recommended 4 apparatus for dissolution test
Dissolution Apparatus
According to USP 7 types of apparatus are used which are as follow:
Apparatus I (Rotating Basket Apparatus)
Apparatus II (Rotating Paddle Apparatus)
Apparatus III (Reciprocating Cylinder Apparatus)
Apparatus IV (Flow Through Cell Apparatus)
Apparatus V (Paddle Over Disc Apparatus)
Apparatus VI (Rotating Cylinder Apparatus)
Apparatus VII (Reciprocating Holder Apparatus)

Apparatus I (Rotating Basket Apparatus)

Assembly:
Vessel
May be covered (to retard evaporation), made of glass or other inert, transparent material of 100ml capacity
containing 900ml of dissolution medium. It is of cylindrical shape with hemispherical ends.
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Motor or Shaft
A metallic drive shaft for rotation
Cylindrical basket
Place dosage form on it having 40 mesh.
Temperature regulator or water bath
Maintain the temperature of medium at 37 ± 0.5 oC
Fitted cover
Use to retard the evaporation of dissolution medium
Working
 The vessel is partially immersed in a suitable water bath of any convenient size or heated by a
suitable device such as a heating jacket.
 The water bath or heating device permits holding the temperature inside the vessel at 37 ± 0.5
during the test and keeping the bath fluid in constant, smooth motion.
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 No part of the assembly, including the environment in which the assembly is placed, contributes
significant motion, agitation, or vibration beyond that due to the smoothly rotating stirring element.
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 An apparatus that permits observation of the specimen and stirring element during the test is preferable.
The vessel is cylindrical, with a hemispherical bottom and with one of the following dimensions and
 capacities: for a nominal capacity of 1 L, the height is 160 mm to 210 mm and its inside diameter is 98
mm to 106 mm; for a nominal capacity of 2 L, the height is 280 mm to 300 mm and its inside diameter
is 98 mm to 106 mm; and for a nominal capacity of 4 L, the height is 280 mm to 300 mm and its inside
diameter is 145 mm to 155 mm.
 The shaft is positioned so that its axis is not more than 2 mm at any point from the vertical axis of the
vessel and rotates smoothly and without significant wobble that could affect the results.
 A speed-regulating device is used that allows the shaft rotation speed to be selected and maintained at
the specified rate given in the individual monograph, within ±4%.
 Shaft and basket components of the stirring element are fabricated of stainless steel or other inert
material.
 A basket having a gold coating of about 0.0001 inch (2.5 µm) thick may be used.
 A dosage unit is placed in a dry basket at the beginning of each test.
 The distance between the inside bottom of the vessel and the bottom of the basket is maintained at 25 ±
2 mm during the test.
Uses:
 Generally preferred for capsules
 Dosage form that tend to dissolved slowly

Apparatus II (Rotating Paddle Apparatus)

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 Use the assembly from Apparatus I, except that a paddle formed from a blade and a shaft is used
as the stirring element.
 The shaft is positioned so that its axis is not more than 2 mm from the vertical axis of the vessel
at any point and rotates smoothly without significant wobble that could affect the results.
 The paddle blade and shaft may be coated with a suitable coating so as to make them inert.
 The dosage unit is allowed to sink to the bottom of the vessel before rotation of the blade is started.
 A small, loose piece of nonreactive material, such as not more than a few turns of wire helix, may
be attached to dosage units that would otherwise float.
 Rotation of blade is 50rpm
21
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Apparatus III (Reciprocating Cylinder Apparatus)
Assembly
Glass vessels
Cylindrical and flat-bottomed
Glass reciprocating cylinders
Inert fittings (stainless steel type 316 or other suitable material)
Mesh Screens
They are made of suitable nonsorbing and nonreactive material and that are designed to fit the tops and
bottoms of the reciprocating cylinders
Motor and drive assembly
To reciprocate the cylinders vertically inside the vessels and, if desired, index the reciprocating cylinders
horizontally to a different row of vessels.
Working:
 The vessels are partially immersed in a suitable water bath of any convenient size that permits holding
the temperature at 37 ± 0.5 during the test.
 Other parts of working are same as in Apparatus I but instead of rpm, dpm (dips per minute) are used.
5dpm = 50rpm
Uses
 Chewable tablets
 Conventional dosage form
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22
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Apparatus IV (Flow Through Cell Apparatus)
Assembly:
 Reservoir and a pump for the Dissolution Medium
 A flow-through cell
 A water bath that maintains the Dissolution Medium at 37 ± 0.5.
 Use the specified cell size as given in the individual monograph.
Working:
Pump
 The pump forces the Dissolution Medium upwards through the flow-through cell.
 The pump has a delivery range between 240 and 960 mL per hour, with standard flow rates of 4, 8, and
16 mL per minute.
Flow rate
 It must deliver a constant flow (±5% of the nominal flow rate); the flow profile is sinusoidal with a
pulsation of 120 ± 10 pulses per minute.
Flow through cell & material
 The flow-through cell, of transparent and inert material, is mounted vertically with a filter system
(specified in the individual monograph) that prevents escape of undissolved particles from the top of
the cell; standard cell diameters are 12 and 22.6 mm; the bottom cone is usually filled with small glass
beads of about 1-mm diameter with one bead of about 5 mm positioned at the apex to protect the fluid
entry tube; and a tablet holder is available for positioning of special dosage forms, for example, inlay
tablets.
Temperature maintenance
 The cell is immersed in a water bath, and the temperature is maintained at 37 ± 0.5 oC.

P. QUALITY CONTROL

Apparatus V (Paddle Over Disc Apparatus)

 Modified form of Apparatus II
 It is use to reduce the dead space (space between paddle and dosage unit)
 Use for individual monograph specifications
Working:
 Place the dosage form on disc (place cellular membrane for transdermal patches)
 Adjust the space 25 ± 2 mm between paddle and disc.
 Volume of medium used is 1000 ml.
 Maintain the temperature at 32 ± 0.5 oC.
Uses:
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 Transdermal patches
 Sustained release dosage form
 Modified or extended release dosage form
Page
 Apparatus VI (Rotating Cylinder Apparatus)
 Modified form of Apparatus I
 Basket is replaced with stainless steel cylinder.
 Transdermal patch is attached with cellular membrane which is than attached with cylinder.
 Maintain the temperature at 32 ± 0.5 oC.
Uses:
 Transdermal patches
Apparatus VII (Reciprocating Holder Apparatus)
 A set of volumetrically calibrated or tared solution container made up of glass or any other material
 A holder to hold the dosage unit
 Reciprocating frequency is about 30 cycles per minute.
 Motor and shaft drive assembly.
Uses:
 Transdermal patches
 Extented release drug products
Number
Stage Acceptance Criteria
Tested
S1 6 Each unit is not less than Q + 5%.
Average of 12 units (S1 + S2) is equal to or greater than Q, and no unit is less
S2 6
than Q 15%.
Average of 24 units (S1 + S2 + S3) is equal tour greater than Q, not more than 2
S3 12
units are less than Q 15%, and no unit is less than Q 25%.
Applications:
 To evaluate the formulation effect on the oral absorption of poorly water soluble drugs using a dissolution
system.
 To provide the criteria in-vitro drug release information for both the quality control purpose, i.e., to assess
batch to batch consistency of solid oral dosage forms such as tablets, and drug development, i.e., to
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predict in vivo drug release profile.

Difference between Absorbance and Dissolution:

Absorbance Dissolution
It is a movement of drug into the blood stream It is a situation, a tablet is ingested and pass
through the esophagus to the stomach
Absorption is primary focus in drug’s development The rate of dissolution is the key target for
and medicinal chemistry since the drug much be controlling drug duration in vive.
absorbed before any effect can take place.
No equation for the measurement of absorption. Noye’s Whitney equation is used for measuring
the rate of dissolution.
24

For the absorption the drug much be soluble in Dissolution of the drug in the equeous medium
water. depends upon the pH of medium
Page
3. ASSAY DETERMINATION:
Definition
 The determination of activity, potency, strength, etc of a substance, either on absolute basis or in
comparison with that of standard preparation.
 Qualitative or quantitative analysis of a substance, especially of the drug, to determine its components.
Specification of assay determination:
1. Temperature range = 15 and 250c.
2. Carried out in diffuse light.
3. Percentage limit is ± 15%
Procedure:
 Take 20 tablets and weighed individually.
 Calculate the average weight and crush them.
 Take the powder containing the stated amount of API and perform the assay according to the
monograph.
 Prepared the sample and standard.
 Measure the absorbance of the sample and standard at specified wave length.
 Deduce the result by comparison method.
Formulas:
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
%𝒂𝒈𝒆 𝒑𝒖𝒓𝒊𝒕𝒚 = × 𝟏𝟎𝟎
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
%𝒂𝒈𝒆 𝒑𝒖𝒓𝒊𝒕𝒚 = × 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅/ 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒔𝒂𝒎𝒑𝒍𝒆
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅
𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒘𝒆𝒊𝒈𝒉𝒕
× 𝒑𝒐𝒕𝒆𝒏𝒄𝒚 𝒐𝒇 𝒔𝒕𝒂𝒏𝒅𝒂𝒓𝒅 × × 𝟏𝟎𝟎
𝑺𝒕𝒂𝒕𝒆𝒅 𝒂𝒎𝒐𝒖𝒏𝒕
Qualities of standard:
1. It is a representative, selected sample of any given substance
2. Used to determine the potency and relative efficacy.
3. It should be uniform in quality and pure as possible.
Difference between Chemical and Biological Assay
Chemical Assay Biological Assay
Less time consuming More time consuming
Economical Expensive
More precise and accurate Less precise and accurate
Determine the amount of specific Measure the actual biological activity of a
compound or structure moiety present in a given sample
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given sample.
Less chance of error More chance of error
Less reliable More reliable
25
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Appar Name Assembly Critical parameters Dosage form
atus tested
1 Reciprocating Covered glass vessel, Rotating speed Capsules, Chewable
basket a motor and metallic 100rpm±4% tablets, Controlled
drive shaft, release dosage form
and a cylinder,
2 Rotating Vessel, Rotating speed 50rpm Tablets, suspension
paddle paddle form of shaft and
blade, stirring element
3 Reciprocating Flat bottom glass vessel, Dip rate 3dpm±5% Chewable tablets,
cylinder a set of glass Conventional dosage
reciprocating cylinders, form
a device ;control rate
4 Flow through A reservoir, Medium flow rate 4- Powder, Granules,
cell a pump, 16ml/min±5% Implants, Poor
flow through cell, water soluble
bath Drugs,Micropaticles,
5 Paddle over Same as apparatus 2, but Time specified in Transdermal
disk stainless steel disk is individual monograph fromulation
fitted at bottom
6 Rotating Same as apparatus 1, Time specified in Transdermal
cylinder except basket and shaft individual monograph fromulation
replaced with stainless
steel cylinder.
7 Reciprocating A motor and drive Reciprocating Transdermal
holder assembly, frequency 30 cycle formulation,
a set of solution glass /min Controlled release
containers,
a set of suitable holders.

Apparatus Dosage Unit Dosage Form Tested Advantages Disadvantages

Placement
1 Dry basket Immediate, extended pH can be changed, Limited volume,
and delayed release automatic degassing, dead zone,
dosage form. disintegration
dissolution interaction
2 Vessel Immediate, extended, Easy use, pH change Limited volume,
and delayed release possible, automated sticking, floating,
dosage forms. sinker require.
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3 Cylinder Bead type Easy pH change, Small volume i.e.

formulations. hydrodynamics can be 250ml, limited data,
directly influenced little experience.
using dip rate.
4 Cell Having limited Easy to change media Duration necessary,
solubility. pH, sink conditions high media volume.
can be maintained. Labor, intensive.
5 Disk Transdermal patches. Apparatus 2 can be Disk restrict patch
used by adding disk. size.
6 Cylinder Transdermal patches. Higher release rate as Slow release rate as
compare to App 7. compare to App 5.
7 Holder Transdermal parches No significant motion,
26

and non- agitation, vibration

disintegrating oral require.
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dosage forms.
 27
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 QUALITY CONTROL OF LIQUID DOSAGE FORM
“Liquid preparations for oral use are usually solutions, emulsions or suspensions containing one or more
active ingredients in a suitable vehicle; they may in some cases consist simply of a liquid active ingredient
used as such”. (WHO)
 Syrups
 Suspensions
 Emulsions
 Elixirs

3.1. GENERAL TESTS FOR ORAL LIQUID DOSAGE FORM

1. Physical appearance
2. PH determination
3. Assay & content uniformity
4. Surface tension & Applications
5. Viscosity & applications

1. PHYSICAL APPEARANCE
 It should be physically & chemically homogenous.
 Free from particle & microbial growth.
 Free from cloudiness and precipitation
 No phase separation present.
 No crystal growth occur in dosage form
 Free from color variation.

2. pH DETERMINATION
pH maintenance is very important for stability of active ingredient.
Method of pH determination:
i. pH paper
ii. pH Meter
i. pH paper:
pH paper normally dips into liquid dosage form and change in color is examined.

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ii. pH meter:
 First of all pH meter is calibrated with buffers of known pH i.e. of pH 4 & pH 10.
 Then dip the electrode of meter into dosage form and pH of solution appear on screen of
instrument.
Special consideration:
 pH of formulation is determined at each step of manufacturing.
 Syrup: pH determined by just dipping electrode into syrup.
28

 Suspension: first centrifuge it so that solid separate out and now dip electrode into it.
 Emulsion: first shake it then determine the pH.
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 Limit:
± 0.05 pH variation can be occur due to more than 1 time use of calibration curve.

3. VISCOSITY
Viscosity is a measure of the resistance of a fluid to deformation under shear stress.
Units: Pa·s = kg/(s·m)
Pharmaceutical Importance:
 Predict flow properties of material
 How to handle the material
 Predict the pourability of oral dosage form
 Predict the Stability of dosage form
Viscosity in different dosage form:
Syrups:
They having a specific value of viscosity if is change due to degradation of formulation then by measuring
viscosity we can claim its formulation.
Suspensions:
Due to flocculation the viscosity can be change.
Emulsion:
Due to coalescence the viscosity can be change.
Method of determination of viscosity:
Four basic methods are below but other instruments are also present but they are modification of these four
instruments:
i. Ostwald viscometer
ii. Falling sphere viscometer
iii. Cup and bob viscometer
iv. Cone & plate viscometer
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i. Ostwald’s viscometer:
Assembly:
Capillary viscometer it is U shape tube with two bulbs and two marks.
Principle: (flow of liquid due to gravity)
 When a liquid flows by gravity, the time required for the liquid to pass between two marks,
upper mark and lower mark, through a vertical capillary tube is determined.
 The time of flow of liquid under test is compared with the time required for a liquid of known
viscosity.
 Viscosity of unknown liquid η, can be determined using the equation,
𝒑 𝒕
η1= 𝒑𝟏𝒕𝟏 η2
𝟏 𝟐
p1=density of unknown liquid (sample), p2=density of known liquid (standard)
t1=time of unknown liquid t2=time of known liquid
29

η1=viscosity of unknown liquid η2= viscosity of known sample

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 ii. Falling sphere viscometer:
Assembly:
 Cylindrical transparent tube having graduated section near the middle of its length
 A steel ball that is allowed to fall through the tube.
Principle:
Tube is filled with liquid whose viscosity is to be determined and the ball is allowed to fall. The
velocity of falling ball is measured and viscosity is calculated using stoke’s law:
η = d2 (ρ2-ρ1)g/18V
ρ2 = Density of sphere ρ1 = density of liquid
g = gravitational acceleration V= settling velocity
d2g/18=K
So equation can become as
η = K(ρ2-ρ1)/V

iii. Cup and Bob viscometer:

Assembly
 Cup (rotate out side the sample)
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 Bob (rotate by holding the sample)

 Spring
 External jacket having insulating material
Principle
 Cup and bob viscometers works by defining the exact volume of sample which is to be
sheared within a test cell, the torque required to achieve a certain rotational speed is measured
and plotted.
 There are two classical geometries in “cup and bob” viscometers known as either the
“Cuvetles” or “Searle” systems-distinguished by whether the cup or bob rotates.
 Cup rotates with fixed angular velocity force transmitted to sample causing it to deform then
by fluid it is transferred to bob, this force is torque that can be determined by torsion spring
 We get the data which give the value about the shear stress and shear rate to find the fluid
30

viscosity.
Viscosity= shear stress/shear rate
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 iv. Cone and plate viscometer:
Mostly used for viscous fluids and semisolids (creams, ointments).
Assembly:
 Moving cone
 Fixed plate
Principle:
 This works by sandwiching a small amount of fluid in between a rotating cone and fixed plate as
shown.
 Cone and plate viscometers use a cone of very shallow angle in bare contact to a modest degree of
precision and deconvulation of a flow curve; a graph of shear stress (torque) against shear rate
(angular velocity) yields the viscosity in a straight forward manner.

4. SURFACE TENSION:
“It is defined as force per unit length which must be applied to surface so as to counter balance the net
inward pull”.
OR
“The force required to break the intermolecular attraction”.
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Units:
Dynes/cm---------------------------------------in CGS system
N/m----------------------------------------------in SI
Methods for measurement:
Choice of particular method often depends on whether surface or interfacial tension is to be determined
the accuracy and convenience desired size of sample available and whether the effect of time on surface
tension is to be studied.
Four methods are used for determination of surface tension:
i. Capillary rise Method:
Capillary tube is placed in liquid the surface of capillary the liquid will raise inside the capillary
tube and meniscus is formed which may be concave or convex due to two type of liquid
a) Wetting liquid (concave)
b) Non-wetting liquid (convex)
31

a) Wetting liquid:
Liquid e.g. water rises in capillary and then form the concave meniscus which form due to wetting
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ability of liquid to glass and from diagram we calculate “h” and “r”.
b) Non-wetting liquid:
 Liquid e.g. mercury don’t wet the capillary and form the convex meniscus at lower end of
capillary and we note the “h” this method is not use.
Apply formula
𝜸=1/2(drhg)
r = radius of capillary h = height of liquid
d = density of liquid g = gravitational constant.

ii. Ring detachment method:

It is used to measure both the surface tension (placed on surface) & interfacial tension (dip in sample)
Assembly:
A tensiometer (ring and balance attached) is used which consists of:
 A hanging platinum iridium ring of defined geometry
 A micro-balance,
Principle:
 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.
𝜸 = 𝑲. 𝑭
K = proportionality constant that depends on geometry of ring
F = surface tension force

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iii. Wilhelmy plate method:

Similar to ring method; instead of ring it uses a thin plate.
Assembly:
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A tensiometer (plate and balance is attached) is used which consists of

 A thin plate of mica, glass or platinum
 A suitable balance
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Mica is a group of minerals, can with stand high temperature and inert.
 Principle:
 Thin plate is place vertically so that its lower edge nearly touches the surface of liquid
 The plate is cleaned thoroughly and attached to a scale or balance via a thin metal wire.
 The force on the plate due to wetting is measured with microbalance and used to calculate the
surface tension by following formula.
𝑭
𝜸=
𝟐. 𝑳
L = is the wetted parameter (constant)

iv. Drop weight method:

Assembly:
A. Capillary tube (attached to liquid reservoir whose surface tension is to be measured).
B. Liquid drops into a tarred weighing bottle, placed in bottom (C).
C. Bottom (tensiometer or scale)
D. Water bath (whole apparatus is immersed in this constant temperature water bath)
Principle:
 Take a drop of liquid whose viscosity is to be measured on the top of capillary tube A.
 Slight vacuum is applied until a droplet from capillary tube A reaches full size and drops off by
itself.
 This process is repeated 5 times and average weight & volume of single drop is calculated.
 Surface tension is calculated by following formula.
𝒎𝒈
𝜸 = ( 𝒓 )𝑭
mg = weight of liquid droplet
r = capillary radius
F=correlation factor.

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5. ASSAY & CONTENT UNIFORMITY:

This test is used to confirm that every unit should contains same amount of drug or active ingredients
with little variation in the batch.
Principle:
The test of the uniformity of the content of single dose preparations is based on the assay of individual
contents of active substance(s) of a number of dosage units to determine whether the individual contents
are within the limits set with reference to the average content of the sample.
Method:
 Using a suitable analytical method, determine the individual contents of active substance(s) of 10
33

dosage units taken at random.

 Now apply the criteria of Test A, Test B or Test C as specified in the monograph for any specified
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dosage form.
  Use Test A for liquid dosage form given below:
Test A:
 For tablets, powders for parenteral use, ophthalmic inserts, suspension for injection.
 The preparation complies with the test if each individual content is between 85-115% of the
Average content.
 The preparation fails to comply with the test if more than 1 individual content is outside 85-115%
of the average content or if 1 individual content is outside 75-125% of the average content.
 If 1 individual content is outside the limit of 85-115% but within the limit of 75-125%, determine
the individual content of another 20 dosage units taken at random.
 The preparation complies with the test if more than 1 individual contents of the 30 units is outside
85-115% of the average content and none is outside the limit of 75-125% of the average content.

3.2. GENERAL ORAL LIQUID DOSAGE FORM QC TESTS

1. For Syrups
2. For Elixir
3. For Emulsion
4. For Suspension

1. FOR SYRUPS:
i. Refractive index:
Refractrometer is used to determine the refractive index of syrup, in which quality of material is
check.
Limits: 1.4608—1.4630
ii. Optical rotation:
Polarimeter is used to check the optical rotation of sugar, which in term tells about the inversion of
sugar.
Limits: syrup should have less than 56° and more than 50° optical rotation.

2. FOR ELIXIR
Alcohol Content Determination
As elixir contain alcohol 15 to 50%.
I. Distillation Method:
Used for determination of alcohol, unless otherwise specified in the individual monograph, it is
suitable for examining mostly for
 Fluid extracts
 Tinctures
Procedure:
a) For liquids presumed to contain 30% of alcohol or less:
 By using pipette transfer liquid dosage form in distilling apparatus equal to 25ml.
 Note temperature and add equal volume of water
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 Pour into distillation apparatus and start the process

 Collect the volume of distillate about 2ml less than volume of original test liquid
 Maintain the temperature as it was at the start of process
 Add some quantity of water to make original volume of test liquid
 Resulted distillate may be clear or not if not then clear it by adding the talc or CaCO3
 Now determine the specific gravity at 25 oC
 So we can calculate the %age v/v of alcohol.
b) For liquids presumed to contain 50% of alcohol or less:
 By using pipette transfer liquid dosage form in distilling apparatus equal to 25ml and note
the temperature
 Add double volume of water as compared to sample
 Collect a volume of distillate 2ml less than, twice the volume of original test liquid
34

 Maintain temperature of distillate as of original temperature

 Add some amount of water to double exactly the volume as taken at start
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 Mix and note specific gravity

  Now determine the %age of alcohol v/v.
 That must be ½ of original liquid examined
c) For liquids presumed to contain more than 50% of alcohol:
Adjust the specimen under examination by diluting it with water up to 25% ad then performed
process as above.
II. Gas Liquid Chromatography Method:
USP Reference Standards
1. USP Alcohol Determination—Acetonitrile RS
2. USP Alcohol Determination—Alcohol RS.
Method IIa
Apparatus
Gas chromatography specification
1. Flame-ionization detector
2. 4-mm × 1.8-m glass column packed with 100- to 120-mesh chromatographic column packing
support S3
3. Nitrogen or helium as the carrier.
Temperature specification
1. Prior to use, condition the column overnight at 235oC with a slow flow of carrier gas.
2. The column temperature is maintained at 120o
3. The injection port and detector temperatures are maintained at 210o.
4. Adjust the carrier flow and temperature so that acetonitrile, the internal standard, elutes in 5 to 10
minutes.
Solutions:
Test Stock Preparation: Dilute the specimen under examination stepwise with water to obtain a
solution containing approximately 2% (v/v) of alcohol.
Test Preparation: Pipet 5 mL each of the Test Stock Preparation and the USP Alcohol Determination—
Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as
the internal standard solution] into a 50-mL volumetric flask, dilute with water to volume, and mix.
Standard Preparation: Pipet 5 mL each of the USP Alcohol Determination—Alcohol RS and the USP
Alcohol Determination—Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of
suitable quality may be used as the internal standard solution] into a 50-mL volumetric flask, dilute with
water to volume, and mix.
Procedure
 Inject about 5 µL each of the Test Preparation and the Standard Preparation, in duplicate, into the
gas chromatograph, record the chromatograms, and determine the peak response ratios.
 Calculate the percentage of alcohol (v/v) in the specimen under test according to the formula:
CD(RU / RS)
o C is the labeled concentration of USP Alcohol Determination—Alcohol RS
o D is the dilution factor (the ratio of the volume of the Test Stock Preparation to the volume of
the specimen taken);
o RU and RS are the peak response ratios obtained from the Test Preparation and the
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Standard Preparation, respectively.

System Suitability Test
 In a suitable chromatogram, the resolution factor, R, is not less than 2;
 The tailing factor of the alcohol peak is not greater than 2.0
 Six replicate injections of the Standard Preparation show a relative standard deviation of not
more than 2.0% in the ratio of the peak of alcohol to the peak of the internal standard.

Method IIb
Apparatus:
Gas chromatography specification
1. Split injection port with a split ratio of 5:1
2. A flame-ionization detector
35

3. 0.53-mm × 30-m capillary column coated with a 3.0-µm film of phase G43.
4. Helium is used as the carrier gas at a linear velocity of 34.0 cm per second.
Temperature specification
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1. The chromatograph is programmed to maintain the column temperature at 50o for 5 minutes
 2. Then to increase the temperature at a rate of 10o per minute to 200o, and maintain at this temperature
for 4 minutes.
3. The injection port temperature is maintained at 210o
4. The detector temperature at 280o.
Solutions:
Test Stock Preparation: Dilute the specimen under examination stepwise with water to obtain a
solution containing approximately 2% (v/v) of alcohol.
Test Preparation: Pipet 5 mL each of the Test Stock Preparation and the USP Alcohol Determination—
Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as
the internal standard solution] into a 25-mL volumetric flask, dilute with water to volume, and mix.
Standard Preparation: Pipet 5 mL each of the USP Alcohol Determination—Alcohol RS and the USP
Alcohol Determination—Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of
suitable quality may be used as the internal standard solution] into a 25-mL volumetric flask, dilute with
water to volume, and mix.
Procedure:
 Inject about 0.2 to 0.5 µL each of the Test Preparation and the Standard Preparation, in duplicate,
into the gas chromatograph, record the chromatograms, and determine the peak response ratios.
 Calculate the percentage of alcohol (v/v) in the specimen under test according to the formula:
CD(RU / RS)
o C is the labeled concentration of USP Alcohol Determination—Alcohol RS
o D is the dilution factor (the ratio of the volume of the Test Stock Preparation to the volume of
the specimen taken);
o RU and RS are the peak response ratios obtained from the Test Preparation and the Standard
Preparation, respectively.
System Suitability Test:
 In a suitable chromatogram, the resolution factor, R, between alcohol and the internal standard is
not less than 4
 The tailing factor of the alcohol peak is not greater than 2.0
 Six replicate injections of the Standard Preparation show a relative standard deviation of not
more than 4.0% in the ratio of the peak of alcohol to the peak of the internal standard.Used only
when specified in individual monograph.

3. FOR EMULSION
i. Type of Emulsion:
Type of emulsion is determined whether it w/o or o/w as follow.
a) Physical examination test:
Emulsion is applied on skin and if is easily removable then it is o/w emulsion and if it is not easily
removable then it is w/o emulsion.
b) Dilution test:
Suitable diluents like water are added and stability is checked if it is stable then it is o/w type of
emulsion if emulsion break down then it is w/o emulsion.
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c) Conductivity test:
This test is based on conduction of electric current in emulsion for which a pair of electrodes
is applied in emulsion and outer circuit is completed. If electricity passed then it is o/w type
of emulsion and if not pass then it is w/o type of emulsion.
d) Dye test:
Dye is added to emulsion and it may be water soluble (methylene blue) will distribute
throughout in o/w emulsion or may be Oil soluble (scarlet red) will distribute throughout in
w/o emulsion.
e) CoCl2 test:
Simply dip the filter paper into solution of CoCl2, it will turn blue upon drying and dip it into
emulsion for test. If paper turns pink emulsion is w/o and if it remains unchanged then it is
o/w type of emulsion.
36

f) Fluorescence test:
As oils are exposed to UV rays, they become fluorescent so when o/w emulsion exposed to UV-
light emulsion globules become fluorescent and when w/o type of emulsion exposed to UV light
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the continuous phase will be fluorescent.

 ii. Dispersibility/pourability:
It is used to check the spreadability & pourability of emulsion.
Process:
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.
iii. Physical stability of emulsion:
It is checked by visual inspection of emulsion in which coalescence of internal phase, and creaming,
elegance of emulsion, odour, colour and other physical properties are checked.
iv. Water content:
Water content of emulsion is determined by titration method called Karl Fischer titration
It is a classic titration method that uses volumetric titration to determine trace amounts of water in a
sample.
v. Globule size determination:
Use for assessment of shelf life of emulsion in which we consider about the
a. Uniform size of globule
b. Uniform distribution of globule
Globule size analysis is carried out by
a. Microscopic measurement:
It gives an average value dependent on the no of globules of each size
b. Coulter counter:
It measures the globule volume and check the uniformity of distribution.

4. FOR SUSPENSION
i. Particle size determination of suspension:
 It predicts the shelf life of material.
 In which the some quantity of suspension is mixed with the equal quantity of glycerol and further
dilution is carried out by mixture of glycerol and water.
 Now mount the sample material on slide and examine under microscope, measure the diameter of
particles above the maximum size permitted in individual monograph.
 %age is calculated from observation on at least 1000 particles other diluents may be paraffin.
ii. Sedimentation volume of suspensions:
Physical stability of suspension depends upon
a. Size of particle
b. Suspending agent (↑ Suspending agent, ↑ Viscosity, ↑ Sedimentation volume, ↑ Stability)
Following two parameter determine the pharmaceutical acceptability of suspensions,
a. Sedimentation volume
b. Degree of flocculation
Sedimentation volume F can be defined as “The ratio of final or ultimate volume of sediment Vu to the
original volume of suspension Vo before settling”.
𝐕𝐮
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F = 𝐕𝐨
 Sedimentation volume can have values ranging from less than 1 to greater than 1.
 When volume of sediment in a flocculated suspension equals the original volume of
suspension then F=1.
 Such a product is said to be in “flocculation equilibrium” and shows no clear supernatant on
standing it is pharmaceutically acceptable.
iii. 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
37

cycling time of 16 min.

 Maintain temperature at 23-33 oC, alter temperature from this range after every 16 minutes then
crystal growth observed
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 Crystal growth depends upon the particles concentration, the bulk particles solubility, the slope of
solubility curve, the temperature fluctuation range and frequency of fluctuation.

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38
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 39
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 QUALITY CONTROL OF STERILE PRODUCTS

4.1 STERILE PRODUCTS (BP)

Sterile products are the dosage form of therapeutic agents that are free from viable micro-organisms.
These sterile products include the followings:
1. Parenterals
2. Ophthalmic
3. Irrigating preparations
Of these parenteral products are unique among the dosage forms of the drugs because they are injected through
skin or mucous membranes into the internal body compartments.
Ophthalmics (BP):
These are the sterile liquids, semisolids or solid preparations intended for administration upon the Eyeball and/
or to the conjunctiva or for insertion in the conjunctival sac.
Categories of ophthalmics:
1. Eye drops
2. Eye lotions
3. Powders for eye drops
4. Powders for eye lotions
5. Semisolid eye preparations
6. Ophthalmic inserts
Irrigating Preparations:
These solutions are applied topically to bathe open wounds and body cavities. They are sterile solutions for
single use only.
 Water for irrigation is sterilized distilled water that is free of pyrogens.
 The water is packed in containers and is intended for use on one occasion only.
 The containers are sealed and sterilized by moist heat.
Examples:
1. 0.9% w/v sodium chloride solution
2. Sterile water for irrigation.

4.2 PARENTERAL PREPARATIONS (BP)

Parenteral preparations are sterile preparations intended for administration by injection, infusion or
implantation into the human or animal body.
Parenteral preparations are supplied in glass, plastic containers and prefilled syringes with closure are
made up of plastic or elastomers.

4.3 QUALITY CONTROL TESTS

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Six types of test are used to check the quality if sterile products which are as follow:
1. Pyrogen Test
2. Clarity Test
3. Sterility Test
4. Leaker’s Test
5. Safety Test
6. Content Uniformity Test

4.3.1 Pyrogen Test

Pyrogens are fever producing substances released from either bacteria or viruses.
Pyro means ‘pyrexia’
Gen means ‘producing’
40

Two types are there:

1. Invivo Pyrogen Test (Rabbit test)
2. Invitro Pyrogen Test (LAL test)
Page
1. Invivo Pyrogen Test (Rabbit test) (EP)
Principle:
The test consists of measuring the rise in body temperature evoked in rabbits by the intravenous injection of
a sterile solution of the substance to be examined.
Selection of animals:
Selection criteria
 Use healthy, adult rabbits of either sex weighing not less than 1.5 kg
 Fed a complete and balanced diet not containing antibiotics
 Not showing loss of body mass during the week preceding the test.
Rejection criteria
A rabbit is not be used in a pyrogen test:
a) If it has been used in a negative pyrogen test in the preceding 3 days, or
b) If it has been used in the preceding 3 weeks in a pyrogen test in which the substance under examination
failed to pass the test.
Animal’s quarters:
 Keep the rabbits individually in a quiet area with a uniform appropriate temperature.
 Withhold food from the rabbits overnight and until the test is completed; withhold water during the test.
 Carry out the test in a quiet room where there is no risk of disturbance exciting the animals and in which
the room temperature is within 3 °C of that of the rabbits' living quarters, or in which the rabbits have
been kept for at least 18 h before the test.
Materials
 Glassware, syringes and needles.
 Thoroughly wash all glassware, syringes and needles with water for injections and heat in a hot-air oven
at 250 °C for 30 min or at 200 °C for 1 h.
Retaining boxes:
 The retaining boxes for rabbits whose temperature is being measured by an electrical device are made
in such a way that the animals are retained only by loosely fitting neck-stocks; the rest of the body
remains relatively free so that the rabbits may sit in a normal position.
 They are not restrained by straps or other similar methods which may harm the animal.
 The animals are put into the boxes not less than 1 h before the first record of the temperature and remain
in them throughout the test.
Thermometers:
 Use a thermometer or electrical device which indicates the temperature with a precision of 0.1 °C and
insert into the rectum of the rabbit to a depth of about 5 cm.
 The depth of insertion is constant for any one rabbit in any one test.
 When an electrical device is used it may be left in position throughout the test.
Preliminary test:
 After selection of the animals, one to three days before testing the product to be examined, treat those
animals that have not been used during the previous 2 weeks by intravenous injection of 10 ml per
kilogram of body mass of a pyrogen-free 9 g/l solution of sodium chloride R warmed to about 38.5
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°C.
 Record the temperatures of the animals, beginning at least 90 min before injection and continuing
for 3 h after the injection of the solution.
 Any animal showing a temperature variation greater than 0.6 °C is not used in the main test.
Main test
Carry out the test using a group of three rabbits.
Preparation and injection of the product
 Warm the liquid to be examined to approximately 38.5 °C before the injection.
 The product to be examined may be dissolved in, or diluted with, a pyrogen-free 9 g/l solution of
sodium chloride R or another prescribed liquid.
 Inject the solution slowly into the marginal vein of the ear of each rabbit over a period not
exceeding 4 min, unless otherwise prescribed in the monograph.
 The amount of the product to be injected varies according to the product to be examined and is
41

prescribed in the monograph.

 The volume injected is not less than 0.5 ml per kilogram and not more than 10 ml per kilogram of body
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mass.
 Determination of the initial and maximum temperatures
 The "initial temperature" of each rabbit is the mean of two temperature readings recorded for that rabbit
at an interval of 30 min in the 40 min immediately preceding the injection of the product to be examined.
 The "maximum temperature" of each rabbit is the highest temperature recorded for that rabbit in the 3
h after the injection.
 Record the temperature of each rabbit at intervals of not more than 30 min, beginning at least 90 min
before the injection of the product to be examined and continuing 3 h after the injection.
 The difference between the maximum temperature and the initial temperature of each rabbit is taken to
be its response.
 When this difference is negative, the result is counted as a zero response.
 Rabbits showing a temperature variation greater than 0.2 °C between two successive readings in the
determination of the initial temperature are withdrawn from the test.
 In any one test, only rabbits having initial temperatures which do not differ from one another by more
than 1 °C are used.
 All rabbits having an initial temperature higher than 39.8 °C or less than 38.0 °C are withdrawn from
the test.
Interpretation of results
 Having carried out the test first on a group of three rabbits, repeat if necessary on further groups of three
rabbits to a total of four groups, depending on the results obtained.
 If the summed response of the first group does not exceed the figure given in the second column of the
Table 2.6.8.-1, the substance passes the test.
 If the summed response exceeds the figure given in the second column of the table but does not exceed
the figure given in the third column of the table, repeat the test as indicated above.
 If the summed response exceeds the figure given in the third column of the table, the product fails the
test.
 Rabbits used in a test for pyrogens where the mean rise in the rabbits' temperature has exceeded 1.2 °C
are permanently excluded.
No of rabbits Product passes Product fails
If summed response does not If summed response exceed
exceed
3 1.15°C 2.65°C
6 2.80°C 4.30°C
9 4.45°C 5.95°C
12 6.60°C 6.60°C

2. Invitro Pyrogen Test (LAL test) (EP)

This test is used to check bacterial endotoxin.
Principle:
Extract protenious in nature
Nature:
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Containing enzyme and proteins

Source:
Limulus polyphemus (horse shoe crab)
Take heart of mature crab and get blood ameobocyte cells by lysis so called Limulus Amebocyte
Lysate test.
Prepration:
Protein + lipopolysaccharides Clot or gel formed
Specific conditions:
Temperature: 36 - 38 °C during incubation or centrifugation
pH: 5-7
Instrument & Equipment: Depyrogenated (wash with water for injection or heat in hot air oven at
250 °C for 30 minutes.
Techniques:
42

i. Gel clot technique:

Apparatus:
Page

Incubator 36-38 oC, Hot air oven 250 oC, Pipette.

 Precaution:
Avoid agitation otherwise gel not formed
Types:
a. Qualitative:
Take Sample and LAL reagent in test tube and incubate for 1 hour.
Gel or clot formed
Rotate 180o or invert it.
If clot break then test is negative otherwise positive.
b. Quantitative:
Make dilutions of sample other procedure is same as in qualitative.
Check lamda at different wavelengths.
ii. Photometric technique:
a. Turbidimetric technique
This technique is a photometric test to measure the increase in turbidity. Based on the test
principle employed, this technique may be classified as being either the end- point-turbidimetric
test or the kinetic-turbidimetric test.
 The end-point-turbidimetric test is based on the quantitative relationship between the
endotoxin concentration and the turbidity (absorbance or transmission) of the reaction
mixture at the end of an incubation period.
 The kinetic-turbidimetric test is a method to measure either the time (onset time) needed for
the reaction mixture to reach a predetermined absorbance or transmission, or the rate of
turbidity development.
The test is carried out at the incubation temperature recommended by the lysate manufacturer
(usually 37 ± 1 °C).
b. Chromogenic technique
This technique is used to measure the chromophore released from a suitable chromogenic peptide
by the reaction of endotoxins with the lysate. Depending on the test principle employed, this
technique may be classified as being either the end- point-chromogenic test or the kinetic-
chromogenic test.
 The end-point-chromogenic test is based on the quantitative relationship between the
endotoxin concentration and the quantity of chromophore released at the end of an
incubation period.
 The kinetic-chromogenic test measures either the time (onset time) needed for the reaction
mixture to reach a predetermined absorbance, or the rate of colour development.
The test is carried out at the incubation temperature recommended by the lysate manufacturer
(usually 37 ± 1 °C).
4.3.2 Clarity Test (USP)
Particulate matter in injections and parenteral infusions consists of mobile undissolved particles, other than gas
bubbles, unintentionally present in the solutions.
There are two types of clarity test depend upon the type of particles present:
1. Subvisible particles
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2. Visible particles
1. Subvisible particles
For the determination of particulate matter, two procedures are as follows:
i. Method 1 (Light Obscuration Particle Count Test)
ii. Method 2 (Microscopic Particle Count Test)
When Method 1 is not applicable, e.g., in the case of preparations having reduced clarity or increased
viscosity, the test should be carried out according to Method 2. Emulsions, colloids, and liposomal
preparations are examples.
Similarly, products that produce air or gas bubbles when drawn into the sensor may also require
microscopic particle count testing.
i. Method 1 (Light Obscuration Particle Count Test)
Principle:
43

Based on the principle of light blockage that allows for an automatic determination of the size of
particles and the number of particles according to size.
Page
 Apparatus:
An electronic liquid borne particle counting system that uses a light obstruction sensor with a suitable
feeding device is used.
Precautions:
 The test is carried out in a laminar flow cabinet (Class 100 area).
 Very carefully wash the glassware and filtration equipment used, except for the membrane filters,
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.
Procedure:
 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 allowing to stand for 2 minutes 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 are
combined in a cleaned container to obtain a volume of not less than 25 mL
 Remove four portions of sample, not less than 5 mL each, and count the number of particles equal
to or greater than 10 µm and 25 µm.
 Disregard the result obtained for the first portion, and calculate the mean number of particles for
the preparation to be examined.
Acceptance Criteria:
Test-1.A (more than 100 mL) (25/ml = 10µm, 3/ml = 25 µm)
The preparation complies with the test if the average number of particles present in the units tested
does not exceed 25 per mL equal to or greater than 10 µm and does not exceed 3 per mL equal to or
greater than 25 µm.
Test-1.B (less than 100 mL) (6000/container = 10 µm, 600/container = 25 µm)
The preparation complies with the test if the average number of particles present in the units tested
does not exceed 6000 per container equal to or greater than 10 µm and does not exceed 600 per
container equal to or greater than 25 µm.
Disadvantage:
Not used for viscous materials.
ii. Method 2 (Microscopic Particle Count Test)
Use a suitable binocular microscope, a filter assembly for retaining particulate matter, and a membrane
filter for examination.
Binocular Microscope
a. Ocular micrometer (circular diameter graticule)
The large circle divided by crosshairs into quadrants is designated the graticule field of view
(GFOV).
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Transparent and black circles having 10-µm and 25-µm diameters at 100× are provided as
comparison scales for particle sizing.
A relative error of the linear scale of the graticule within ±2% is acceptable.
44
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 b. Stage micrometer
Ocular micrometer is calibrated using a stage micrometer that is certified by either a domestic or
international standard institution. The large circle is designated the graticule field of view (GFOV).
c. Mechanical stage
It hold the entire filteration area of the membrane filter
d. Illuminators
Two illuminators are required. One is an episcopic brightfield illuminator internal to the
microscope, the other is an external, focusable auxiliary illuminator that can be adjusted to give
reflected oblique illumination at an angle of 10 to 20.
Filter Assembly
The filter assembly for retaining particulate matter consists of a filter holder made of glass or other
suitable material, and is equipped with a vacuum source and a suitable membrane filter.
The membrane filter is of suitable size, black or dark gray in color, nongridded or gridded, and 1.0
µm or finer in nominal pore size.
Procedure:
 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 allowing to stand for 2 minutes 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 are
combined in a cleaned container to obtain a volume of not less than 25 mL
 Wet the inside of the filter holder fitted with the membrane filter with several mL of particle-free
water. Filter all the solution.
 Place the membrane filter in a Petri dish, and allow the membrane filter to air-dry.
 After the membrane filter has been dried, place the Petri dish on the stage of the microscope, scan
the entire membrane filter under the reflected light from the illuminating device, and count the
number of particles that are equal to or greater than 10 µm and the number of particles that are
equal to or greater than 25 µm.
 Alternatively, partial membrane filter count and determination of the total filter count by
calculation is allowed. Calculate the mean number of particles for the preparation to be examined.
Acceptance Criteria
Test-2.A (more than 100 mL) (12/ml = 10µm, 2/ml = 25 µm)
The preparation complies with the test if the average number of particles present in the units tested
does not exceed 12 per mL equal to or greater than 10 µm and does not exceed 2 per mL equal to or
greater than 25 µm.
Test-1.B (less than 100 mL) (3000/container = 10 µm, 300/container = 25 µm)
The preparation complies with the test if the average number of particles present in the units tested
does not exceed 3000 per container equal to or greater than 10 µm and does not exceed 300 per
container equal to or greater than 25 µm.
P. QUALITY CONTROL

2. Visible Test (visual assessment of parenterals)

Principle:
Visual assessment of parenterals
Apparatus:
1. Viewing station
2. Black panel (vertical position)
3. White panel (vertical position to the black panel)
4. Adjustable lamp holder (fitted with shaded white light source)
The intensity of illumination at the viewing point is maintained between 2000 lux and 3750 lux.
45
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 Procedure:
 Remove label from the bottle.
 Wash the bottle from out side and dry it.
 Invert, shake and swirl (rounding) to remove the gas bubbles not so hard otherwise bubbles are produced
and observe for about 5 sec in front of white panel.
 Repeat the procedure in front of the black panel.
 Record the presence of any particles.
Acceptance Criteria:
If particles are seen than reject otherwise accept.
4.2.3 Sterility Test (EP)
The test for sterility is carried out under aseptic conditions. In order to achieve such conditions, the test
environment has to be adapted to the way in which the sterility test is performed.
The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are
to be revealed in the test.
The working conditions in which the tests are performed are monitored regularly by appropriate sampling of
the working area and by carrying out appropriate controls.
Two methods are used for sterilization of parenterals:
1. Membrane Filteration
2. Direct Inoculation
1. Membrane Filteration
Apparatus:
 A filter assembly with membrane filter of pore size 0.45µm ± 0.02µm.
 Membrane filter should be pre-sterilized with steam under pressure or by radition method.
Examples:
Cellulose nitrate filter Cellulose acetate filter Specially adapted filter
Used for aqueous, oily and Used for strongly Used for antibiotics
weakly alcoholic solutions alcoholic solutions
Procedure:
i. Dilution:
a. Aqueous Solutions or soluble solids:
 The product is dissolved in small quantity of a suitable, sterile diluent such as a 1 g/l neutral
solution of meat or casein peptone pH 7.1 ± 0.2 on to the membrane in the apparatus and filter.
 The diluent may contain suitable neutralising substances and/or appropriate inactivating
substances for example in the case of antibiotics.
b. Oils and oily solutions:
 Oils and oily solutions of sufficiently low viscosity may be filtered without dilution through a
dry membrane.
 Viscous oils may be diluted as necessary with a suitable sterile diluent such as isopropyl
myristate.
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c. Ointments and creams:

 Ointments in a fatty base and emulsions of the water-in-oil type may be diluted to 1 per cent
in isopropyl myristate, by heating, if necessary, to not more than 40 °C.
 In exceptional cases it may be necessary to heat to not more than 44 °C.
ii. Filteration:
 Filter the solution immediately after dilution.
 If solution is in smaller quanity than pass it in a single membrane and cut this membrane into
two parts for both bacterial and fungal media separately.
 If solution is in larger quantity than use two filter papers.
iii. Incubation:
 Incubate with fungal medium at 20-25 oC.
 Incubate with bacterial medium at 30-35 oC.
46

 Incubate the media for 2 weeks and finally after 2 weeks check the growth.
Acceptance Criteria
Page

If growth occur than reject it otherwise accept it.

2. Direct Inoculation:
 Transfer the quantity of the preparation to be examined directly into the culture medium so that the
volume of the product is not more than 10 per cent of the volume of the medium, unless otherwise
prescribed.
 If the product to be examined has antimicrobial activity, carry out the test after neutralising this with a
suitable neutralising substance or by dilution in a sufficient quantity of culture medium.
 When it is necessary to use a large volume of the product it may be preferable to use a concentrated
culture medium prepared in such a way that it takes account of the subsequent dilution.
 Where appropriate, the concentrated medium may be added directly to the product in its container.
4.3.4 Leakers Test (Remington)
A leak is a way for fluid to escape a container or fluid-containing system, through which the contents of
container can escape or outside matter can enter the container.
 Ampoules are leakers.
 Leaking through tiny holes or cracks.
 To check the leakage from ampoules only.
Types:
Sealing is of two types:
1. Tip sealing (heating until tip seal)
They are made by melting sufficient glass at the tip of the ampoule neck to form a bead of the glass and
close the opening.
2. Pull sealing (pull the red hot ampoule to seal)
They are made by heating the neck of a rotating ampoule below the tip, then pulling the tip away
to form a small twisted capillary just prior to being melted closed.
Purpose:
 To check the incompletely sealed ampoules so that they may be discarded.
 Can cause the interchange between the content of ampoules and environment.
 Microorganisms and other contaminants can enter the ampoules and deteriorate it or content release outside
and spoil the packaging.
Procedure:
 This test is performed by producing negative pressure within an incompletely sealed ampoule while the
ampoule is submerged entirely in a deeply dye solution.
 Most often, approximately 1% methylene blue solution is employed.
 After carefully rinsing the dye solution from the outside, colour from the dye will be visible within a leaker.
 Leakers of course, are discarded.
Disadvantages:
 Not for vials and bottles.
 Capillaries of about 15 µm may or may not be detected.
P. QUALITY CONTROL

4.3.5 Content Uniformity Test (EP)

This test is used to confirm that every unit should contains same amount of drug or active ingredients with
little variation in the batch.
Principle:
The test of the uniformity of the content of single dose preparations is based on the assay of individual
contents of active substance(s) of a number of dosage units to determine whether the individual contents
are within the limits set with reference to the average content of the sample.
Method:
 Using a suitable analytical method, determine the individual contents of active substance(s) of 10
dosage units taken at random.
 Now apply the criteria of Test A, Test B or Test C as specified in the monograph for any specified
dosage form.
47

 Use Test A for liquid dosage form given below:

Test A:
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 For tablets, powders for parenteral use, ophthalmic inserts, suspension for injection.
 The preparation complies with the test if each individual content is between 85-115% of the Average
content.
 The preparation fails to comply with the test if more than 1 individual content is outside 85-115% of the
average content or if 1 individual content is outside 75-125% of the average content.
 If 1 individual content is outside the limit of 85-115% but within the limit of 75-125%, determine the
individual content of another 20 dosage units taken at random.
 The preparation complies with the test if more than 1 individual contents of the 30 units is outside 85-115%
of the average content and none is outside the limit of 75-125% of the average content.

4.3.6 Safety Test (Remington)

It is entirely possible for a parenteral product to pass the routine Sterility test, Pyrogen test, and Chemical
analysis, and still cause unfavourable reactions when injected.
So, a safety test in animal is essential to provide the additional assurance that the product does not have
unexpected toxic properties.

P. QUALITY CONTROL

48
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 49
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P. QUALITY CONTROL
 QUALITY CONTROL OF SUPPOSITORIES

5.1 DEFINITION (BP)

Parenteral preparations are sterile preparations intended for administration by injection, infusion or
implantation into the human or animal body.

5.2 QUALITY CONTROL TESTS

A. Physical Testing:
1. Visual Examination
2. Weight Variation Test
3. Melting Range Test
4. Breaking Test
5. Disintegration Test (Liquefaction Test or Softening Test)
B. Chemical Testing:
1. Content Uniformity Test
2. Dissolution Test
5.2.1 Physical Testing
1. Visual Examination:
Visual evaluation of suppositories is necessary and important to check for the absence of fissuring,
pitting, fat blooming, exudation, sedimentation and migration of active ingredient.
Purpose:
Purpose of this test is to check uniformity between batches and physical quality assessment.
i. Shape & Size
Supossitories must be of proper shape and size because change in shape effect the suppositories.
Change in shape can effect the formulation
Check the shape of the suppositories to see wheter it is consistent or intact or not
ii. Colour
Intensity, nature and homogenesity of colour should be checked and verified.
iii. Odour
Must be of desired odour because change in odour indicate the degradation process.
iv. Stability
Check the stability there should be no crack and bubbles in the suppositories.
2. Weight Variation Test (EP):
 Weigh 20 suppositories individually and calculate average weight.
 Now calculate the percentage deviation by following formula.

𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 − 𝑰𝒏𝒅𝒊𝒗𝒊𝒅𝒖𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕

P. QUALITY CONTROL

𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝒅𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 = × 𝟏𝟎𝟎

𝐀𝐯𝐞𝐫𝐚𝐠𝐞 𝐰𝐞𝐢𝐠𝐡𝐭
Acceptance criteria:
Not more than 2 of the individual masses deviate from the average mass by more than 5% and no
suppository should deviate from average weight by more than 10%. All weight must be range in
10%.
3. Melting Range Test
This test is also called macro melting range test. This test is a measure of time it takes for entire
suppository to melt when immersed in a constant temperature water bath, i.e. 37 oC.
Purpose:
This test is performed to check either suppository is melt within body cavity at optimum time or not.
Apparatus:
USP-Tablet Disintegration Apparatus is used to find melting range.
50

Procedure:
Set the apparatus and maintain the temperature at 37 oC.
Page

Immerse the suppository in glass tube and immerse the tube in water bath and run the apparatus.
Time for the entire suppository to melt or dispersed or disintegrated in surrounding water is measured.
 Disintegration:
Suppository is considered to be disintegrated when:
 It is completely dissolved or disintegrated.
 It is dispersed into its components or parts.
 It become soft in shape with the formation of core which is not resistant to pressure of glass rod.
Acceptance criteria:
Fat base: 30 minutes
Water base: 60 minutes.
4. Breaking Test
Purpose:
This test applied to suppositories and passeries based on fatty excipients. It is not suited to suppositories
and passeries based on hydrophilic excipients such as gelatin-glycerol mixture.
This test is design to check:
 Hardness
 Fragility
 Brittlness
Apparatus:
 A thermostattted chamber closed in front by a glass window and containing a device that is to hold
the suppository or passery.
 Two opposite jaws, the upper jaw descending vertically toward the lower jaw. The crushing
surfaces of the jaw are flat perpendicular, to the direction of movement and larger than zone of
contact with the suppository or passery.
 A plastic sample holder is fixed in the centre of the jaw (half a holder in each jaw).
 The upper jaw (top pressure block) is connected to suspension to which can be added each of which
weight 200g. The initial mass of the device is 600g. Crushing of the sample is carried out by
successive adding 200g disc to the initial mass of 600g.
Method:
 Check that apparatus is vertical. Heat the thermostattted chamber to 25 oC.
 The dosage form to be tested has been maintained for at least 24 hours at the required measuring
temperature. Place the suppository or passery vertically between the jaws in the sample holder with
the point upwards.
 The top pressure block of the suspension loading rod is carefully positioned and the test chamber is
closed with its glass window; for each determination position the suppository or passery in the same
manner with respect to force applied.
 Wait for 1min and add the first 200g disc, again wait for 1min and add another disc. Repeat the
operation until the suppository or passery collapses.
 The mass required to crush the suppository or pessary is calculated by the sum of the masses
weighing on the suppository or pessary when it collapses (including the initial mass of the device)
assessed as following
o If the suppository or passery collapses within 20 seconds of placing the last disc do not take its
P. QUALITY CONTROL

mass into account.

o If the suppository or passery collapse between 20 seconds and 40 seconds of placing the
last disc, use only half of this mass in the calculations i.e. 100g.
o If the suppository or passery remains uncrushed for more than 40 sec after the last disc is
placed, use all the mass in the calculation.
 Carry out each measurement on 10 suppositories or 10 passeries, making sure that no residue
remains before each determination.
5. Disintegration Test (Liquefaction Test or Sogtening Test)
Suppository is considered to be disintegrated when:
 It is completely dissolved or disintegrated.
 It is dispersed into its components or parts.
 It become soft in shape with the formation of core which is not resistant to pressure of glass rod.
Methods:
51

There are two methods are used for disintegration:

1. Method-I
Page

2. Method-II
1. Method-I (water & fat base) (EP)
This method is used for both water and fat base suppositories.
Apparatus:
The apparatus consists of 2 main parts:
i. Cylinder/vessel/beaker of glass or suitable transparent plastic,
ii. Metal device with two perforated stainless metal discs which contains 39 holes or perforations.

Assembly:
 Metal device is inserted into cylinder and attach to the rim of the cylinder with 3 spring clips.
 Each disc containing 39 holes 4 mm in diameter held 30mm a part evenly spaced in a concentric
pattern.
Test criteria:
The test is carried out using three such apparatuses each containing a single sample.
Procedure:
 Each apparatus is placed in a beaker with a capacity of at least 4 L filled with water maintained
at 36-37 °C, unless otherwise specified in monograph.
 The beaker is fitted with a slow stirrer and a device that will hold the cylinders vertically not less
than 90 mm below the surface of the water and allow them to be inverted without emerging from
the water.
 Place one suppository on the lower disc of a device.
 Invert the apparatus every 10 min. and note the time of disintegration.
Acceptance criteria:
Fat base: 30 minutes
Water base: 60 minutes.
2. Method-II (fat base)
This test is used for the disintegration of fat base or lipophilic suppositories.
Priniciple:
This test is intended to determine the time (under defined conditions) which elapses until a
suppository, maintained in a water, soften to extent so that no longer offers resistance when a
P. QUALITY CONTROL

defined weight is applied.

Apparatus
Two types of apparatus are used:
i. Apparatus-A
ii. Apparatus-B
i. Apparatus-A:
It consists of two parts:
a) Glass tube
b) Glas rod
a) Glass tube:
 It is tube of glass having internal diameter 15.5mm.
 Flat bottom.
 Length is 140mm.
52

 Tube is closed with removeable plastic cover having opening of 5.2 mm for rod.
Page
 b) Glas rod:
 Upper part is made of plastic or metal having diameter 5.0mm with a weight disc. Upper
part containing sliding mark ring when the rod is introduce into glass tube it touches the
bottom, sliding mark ring is adjusted to concide with upper level of the plastic cover.
 Lower part made up of plastic and wider having diameter12mm.
 Metal needle is fixed at flat lower side with 2mm length and 1mm diameter.

Procedure:
 Place glass tube having 10ml of water in water bath maintained at 36.5±0.5 oC.
 Fix the glass tube vertically and immersed at a depth of at least 7cm below the surface but
without touching the bottom of water bath.
 Introduce a suppository tip downward followed by the rod with a free gliding plastic cover
into the glass tube until the metal needle touches the flat end of the suppository.
 Put the cover on the tube and start the time measurement.
 Note the time which passes until the rod sinks downward to the bottom of glass tube and
sliding mark reach the upper level of the plastic cover.
ii. Apparatus-B:
It consists of three parts:
a) A water bath
b) An inner tube which is inserted in water bath and fixed with stopper. The inner tube is
closed by a stopper at the bottom.
c) Thermometer: The apparatus is also fixed with a thermometer to note the temperature.
d) Two insets are available
i. Glass rod (C1): It is in the form of a tube sealed at both ends, carrying a ring at its
P. QUALITY CONTROL

lower end weighted with lead shot, which has weight of 30±0.4g.
ii. A penetration inset (C2): it consists of a rod (7.5±0.1g) in a tube which has an
enlargement for the suppository both made of stainless steel.
Method:
 Pour 5ml of water at 36.5±0.5ᵒC into the inner tube (A).
 Introduce a suppository with the tip downward.
 Place the inset at the suppository.
 Note the time which elapses between this moment and the moment when the lower rim
end of the glass rod (C1) or penetration inset rod (C2) reaches the narrow part of the
inner glass tube. Melting or dissolution is then considered as complete.
Acceptance and rejection criteria:
Fat base: 30 minutes
Water base: 60 minutes.
53

[Difference between apparatus A and apparatus B is that apparatus B also has a penetration inset].
Page
5.2.2. Chemical Tests:
1. Content Uniformity (EP):
This test is used to confirm that every unit should contains same amount of drug or active ingredients with
little variation in the batch.
Principle:
The test of the uniformity of the content of single dose preparations is based on the assay of individual
contents of active substance(s) of a number of dosage units to determine whether the individual contents
are within the limits set with reference to the average content of the sample.
Method:
 Using a suitable analytical method, determine the individual contents of active substance(s) of 10
dosage units taken at random.
 Now apply the criteria of Test A, Test B or Test C as specified in the monograph for any specified
dosage form.
 Use Test B for Suppositories given below:
Test B
 Take 10 tablets randomly and determine the individual content of active substance. The preparation
compiles with the test if not more than 1 individual content is outside the limit of ±15% of the
average content and none is the outside the limit of ±25% of the average content.
 The preparation fail to comply with the test if more than 3 individual contents are outside the limit of
±15% of the average content or if one or more individual contents are outside the limit of 75% of the
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average content.
 If 2 or more individual contents are outside the limits of ±15% but within the limit of ±25%.
Then determine the individual contents of 20 dosage units taken at random.
 The preparation compiles with the test if not more than 3 individual contents of 30 units are
outside the limit of the average content.
2. Dissolution Test:
The test determines the amount of dosage form that gets dissolve in body fluid per unit time or it is
the measure of rate of drug release from the suppositories.
Type of apparatus:
Two types of apparatus are used
i. Suppository dialysis cell
ii. Stationary basket
i. Suppository dialysis cell:
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It is also called flow through cell apparatus. Lipophilic suppositories are tested in it.
ii. Stationary basket:
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This apparatus is used for hydrophilic suppositories.

This apparatus consists of a basket and rotating paddles.
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 BIOLOGICAL ASSAY

6.1. INTRODUCTION
Assay
Determination of activity, potency or strength of substances either on absolute basis or in comparison with that
of standard.
Bioassay (USP)
Measurement of the effectiveness of a compound by its effect on animals or cells in comparison with a standard
preparation
Principle of Bioassay
The basic principle of bioassay is to compare the test substance with Standard Preparation of the same and to
find out how much test substance is required to produce the same biological effect as produced by the standard.
Standard preparation:
The standards are internationally accepted samples of drugs maintained and recommended by the Expert
Committee of Biological Standardization of W.H.O. They represent the fixed units of activity (definite weight
of preparation) for drugs.
Purposes
 Measurement of pharmacological activity of substances
 Investigation of function of endogenous mediators.
 Determination of side effect relating to toxicity.
 Measurement of concentration of known substances.
 Specificity of certain enzymes to certain substrates.
 Assessing the amount of pollutants being released by a particular source, such as waste water or urban run-
off
Why we need Bioassays
 When chemical identity not elucidated.
 When chemical assay not adequate, although chemical identity present e.g. Insulin
 When drug composed of complex mixture of substances e.g. digitalis
 When sufficient purification not possible e.g. Vitamin D from irradiated oils.
 When chemical assay not a valid indication of biological activity e.g. active & inactive isomers.
Difference b/w Chemical and Biological assay

Chemical Assay Biological Assay

Less time consuming More time consuming
Economical Expensive
More precise & accurate Less precise & accurate
Measure the specific compound in Measures the actual biological
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sample activity of sample

Less chance of error More chance of error
Less reliable More reliable
Types of bioassays
1. Quantal
2. Graded
1. Quantal (direct end-point)
All or none response like in case of toxicity studies, the animal receiving a dose of drug either die or
not.
Examples:
i. Digitalis induced cardiac arrest in guinea pigs
ii. Hypoglycaemic convulsions in mice with insulin.
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iii. Digitalis induced head drop in rabbits.

Limitation:
This method is not precise due to following reasons:
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i. Comparison of threshold response

 ii. Comparison of median effective dose (ED50) nad median lethal dose (LD50)
2. Graded
Proportionate increase in the observed response following an increase in concentration or dose (graded
effect).
Examples:
i. Study of blood pressure in case of adrenaline.
ii. Study of intestinal motility in case of acetylcholine.
Merits:
The choice of this procedure depends on:
i. The precision of the assay required.
ii. The quantity of the sample substance available.
iii. The availability of experimental animal (not died as in Quantal method)
Methods of Bioassays
1. Matching Bioassay
2. Interpolation Bioassay
3. Bracketing Method
4. Multiple Point Bioassay
1. Matching Bioassays
 First response of the test is taken and then match with response of the standard.
 Done till a closed matching is observed.
 Corresponding concentration is then calculated by using following formula:
𝑑𝑜𝑠𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
𝐶𝑜𝑛𝑐. 𝑜𝑓 𝑢𝑛𝑘𝑛𝑜𝑤𝑛 = × 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
𝑑𝑜𝑠𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒

2. Interpolation Bioassay
 Determine the amount of preparation of unknown potency required to produce a definite effect on
suitable animal.
 Concentration response curve of standard is established.
 Then 2-3 responses of test is recorded.
 Effect of sample is compared with standard and potency is calculated.
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3. Bracketed Method
 When sample is too small Bracketed method is used
 Response of the test is bracketed between standard responses.
 Then compare the test with standard response and calculate the potency
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4. Multi point bioassay
Take multiple doses of standard and match with test product.

6.2. BIOASSAY OF DIGITALIS

Introduction:
 Digitalis is Cardio-active glycoside.
 Acts directly on myocardium and increase force of contraction
 Used in CHF
Principle:
Potency of test is compared with that of standard preparation by determining the action on cardiac muscles.
Standard Preparation & Units
 It consists of a mixture of dried and powdered digitalis leaves.
 Supplied in ampoules having 2.5g digitalis.
1 unit = 76 mg of standard
0.01316 unit = 1 mg of standard
Preparation of Extract
 Transfer the known quantity of powder in container approx. 50ml capacity.
 Add 10ml of alcohol (80%) for each gram of powder.
 Close the container and shake continuously:
o For 24 hours at 20-30 ℃
o For 48 hours at 10-20 ℃
 Centrifuge and filter it
 Store in dry and tightly closed bottle at 5 to -5 ℃
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Procedure:
1. Bioassay by Guinea Pig
 Take 12 guinea pig, each weighing between 200-600g.
 Weight of the heaviest and lightest animal should not differ > 100g.
 Distribute the pigs at random into two groups.
 Mean body weights of the two groups should not differ by >10%.
 Extract of both test and standard are diluted with normal saline (1g in 80ml).
 The jugular vein is traced out by removing the adhering tissues and cannulated by means of
venous cannula.
 A pin is inserted in the heart such that it gets inserted in the apex of heart. In this way we can
observe the heart beats by up and down movements of the pin.
 Extract is injected at slow uniform rate into jugular vein of anesthetized animal between duration
of 20-40 min.
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 Injection is continued until heart is arrested.

 Volume of extract administered is taken as lethal dose.
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2. Bioassay by Pigeons
 Take 12 pigeons and divided into 2 groups.
 Weight of largest pigeon should not exceed twice the weight of smallest pigeon.
 Mean body weights of the two groups should not differ by >30%.
 Extract of both test and standard are diluted with normal saline (1g in 80ml).
 Extract is injected at slow uniform rate into alar vein of slightly anesthetized and immobilized pigeon.
 Dose 1ml/kg is administered within few seconds and repeated at 5 min interval until heart is arrested.
 In pigeons, stoppage of heart is associated with a characteristic vomiting response called ‘Emesis’ (the
milk from the crop sac of pigeon is ejected out). This may take as the end point response of the
digitalis.
 Lethal dose per kg is equal to number of doses received.
Acceptance Criteria:
Acceptable limits for the sample is 85-125% of estimated potency.

6.3. BIOASSAY OF ANTIBIOTICS (EP)

Antibiotics
An antibiotic is a medicinal preparation containing a significant quantity of a chemical substance that is
produced naturally by a microorganism or artificially by synthesis that has the capacity to inhibit or destroy the
microorganisms.
Principle
The potency of an antibiotic is estimated by comparing the inhibition of growth of sensitive micro-organisms
produced by known concentrations of the antibiotic to be examined and a reference substance.
Acceptance Criteria:
Acceptable limits for the sample is 95-105% of estimated potency.
Methods:
There are two methods for Antibiotics Assay
I. Diffusion Method
II. Turbidimetric Method
I. Diffusion Method
Principle:
Potency of antibiotic is determined by measuring the diameter of zones of microbial growth inhibition that
occur due to the diffusion of antibiotic sample into semisolid medium.
Procedure
A. Preparation of Petri dish
 Liquefy a suitable medium (Agar medium)
 Inoculate it with suitable micro-organisms sensitive to antibiotic examined.
 Pour this inoculated medium into petri dish.
 Layer of the medium will be of uniform thickness i.e. 2-5mm.
 Store the dishes so that no appreciable growth or death of the micro-organisms occurs.
B. Preparation of reference & sample solutions
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 Using the solvent and the buffer solution indicated in Table, prepare solutions of the reference
substance and of the antibiotic to be examined.
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 Types of diffusion method
1. Cavity/well method
2. Disc method
1. Cavity/well method
 Holes of 5-8 mm in diameter are bored in medium with sterile borer.
 The solutions are placed in the holes by micropipette sufficient to fill the hole (1-10µg).
 Place petri dish for 1-4 hrs at RT or at 4℃.
 Incubate at a suitable temperature for about 18 hrs.
 Measure the diameters of the areas of the circular inhibition zones with accuracy.
 In order to assess the validity of the assay, use not fewer than 3 doses of the reference substance and
3 doses of the antibiotic to be examined
2. Disc method
 Use sterile absorbent paper discs of suitable quality.
 Impregnate the disks with reference or test solutions.
 Place the disc on the surface of medium.
 Place petri dish for 1-4 hrs at RT or at 4℃.
 Incubate at a suitable temperature for about 18 hrs.
 Measure the diameters of the areas of the circular inhibition zones with accuracy.
II. Turbidimetric 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.
Procedure
 Prepare a suitable medium e.g. Agar broth.
 Inoculate the medium (9ml) with a suspension of chosen micro-organism (1ml) sensitive to the
antibiotic to be examined.
 Using the solvent and the buffer solution indicated in Table, prepare solutions of the reference substance
and of the antibiotic to be examined.
 Distribute an equal volume of each solution into two identical test tubes.
 Add equal volume of inoculated medium to each tube.
 Prepare at the same time 2 control tubes without antibiotic, both containing the inoculated medium and
to one of which is added immediately 0.5 mL of formaldehyde. These tubes are used to set the optical
apparatus used to measure the growth.
 Incubate all test tubes for 3-4 hrs at a suitable temperature i.e. 37 ℃ in water bath.
 After incubation, stop the growth of micro-organisms by adding 0.5ml of formaldehyde.
 Determine the amount of growth by measuring transmittance with SP.
 Compare the growth of micro-organisms in test with reference.
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 Simply check the clarity with formaldehyde control test tube for 100% inhibition.
Acceptance Criteria:
Clarity of solution
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6.4. BIOASSAY OF INSULIN (USP)
Principle:
The most prominent manifestation of insulin activity, an abrupt decrease in blood glucose, was the basis for
biological assay from the time of its first clinical use.
RABBIT BLOOD SUGAR METHOD—QUANTITATIVE
USP Reference Standards
 USP Dextrose RS
 USP Insulin RS
 USP Insulin (Beef) RS
 USP Insulin Human RS
 USP Insulin (Pork) RS
Diluent
 Prepare an aqueous solution containing:
o 0.1% to 0.25% (w/v) of either cresol or phenol
o 1.4% to 1.8% (w/v) of glycerine
 Add sufficient hydrochloric acid to produce a pH between 2.5 and 3.5
Standard Stock Solution
 Dissolve either a suitable quantity of accurately weighed USP insulin RS in Diluent to make a Standard
Stock Solution containing 40 USP Insulin Units per mL and having a pH between 2.5 and 3.5, unless
otherwise directed in the individual monograph.
 Store in a cold place, protected from freezing, and use within 6 months.
Standard Solutions
Dilute portions of the Standard Stock Solution with Diluent to make two solutions:
1. Standard Solution 1
First containing 1.0 USP Insulin Unit per mL
2. Standard Solution 2
Second containing 2.0 USP Insulin Units per mL
Assay Stock Solution
 Dissolve either a suitable quantity of accurately weighed Sample preparation in Diluent to make Assay
Stock Solution containing 40 USP Insulin Units per mL and having a pH between 2.5 and 3.5, unless
otherwise directed in the individual monograph (same preparation as in Standard Stock Preparation).
 Store in a cold place, protected from freezing, and use within 6 months.
Assay Solutions
Dilute portions of the Assay Stock Solution with Diluent to make two solutions:
1. Assay Solution 1
First containing 1.0 USP Insulin Unit per mL
2. Assay Solution 2
Second containing 2.0 USP Insulin Units per mL
Doses of the Solutions to be Injected
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Select on the basis of trial or experience the dose of the dilutions to be injected, the volume of which
usually will be between 0.30 mL and 0.50 mL. For each animal the volume of the Standard Solution is the
same as that of the Assay Solution.
Preparation of Animal
 Select suitable, healthy rabbits each weighing not less than 1.8 kg.
 Keep the rabbits in the laboratory for not less than 1 week before use in the assay, maintaining them
on an adequate uniform diet, with water available at all times.
Procedure
 Divide the rabbits into four equal groups of preferably not less than six rabbits each.
 On the preceding day, approximately 20 hours before the assay, provide each rabbit with an amount
of food that will be consumed within 6 hours.
 Follow the same feeding schedule before each test day.
 During the assay, withhold all food until after the final blood specimen is taken.
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 Handle the rabbits with care in order to avoid undue excitement, and inject subcutaneously the doses
indicated in the following design (as in Table), the second injection being made on the day after the first
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injection, or not more than 1 week later.

 The time between the first and second injection is the same for all rabbits.
 Group First Injection Second Injection
1 Standard Solution 2 Assay Solution 1
2 Standard Solution 1 Assay Solution 2
3 Assay Solution 2 Standard Solution 1
4 Assay Solution 1 Standard Solution 2
Blood Samples
At 1 hour ± 5 minutes and 2½ hours ± 5 minutes after the time of injection, obtain from each rabbit a suitable
blood specimen from a marginal ear vein. Blood can also be collected effectively from the central auricular
artery.

Dextrose Determination
Determine the dextrose content of the blood specimens by a suitable procedure that is adapted to automated
analysis. The following procedure may be used.
1. Anticoagulant Solution:
Dissolve 1 g of edetate sodium and 200 mg of sodium fluoride in 1 L of water, and mix.
2. Dextrose Standard Preparations
Transfer known concentrations of USP Dextrose RS to suitable vessels, and dilute quantitatively and
stepwise with Anticoagulant Solution (1:9) to obtain a range of Dextrose Standard Preparations containing
between 20 and 100 mg per 100 mL, having known concentrations similar to the concentrations in the
rabbit blood samples.
3. Test Preparations
Pipet into separate, suitable vessels 0.1 mL of each Blood Sample and 0.9 mL of Anticoagulant Solution.
4. Procedure
 Subject the Test Preparations to dialysis across a semipermeable membrane for a sufficient time so
that the dextrose passes through the membrane into a saline TS solution containing glucose oxidase,
horseradish peroxidase, 3-methyl-2-benzothiazolinone hydrazone hydrochloride TS, and N,N-
dimethylaniline.
 The absorbances of the Test Preparations are determined at 600 nm in a recording colorimeter.
 The absorbances of the Dextrose Standard Preparations are similarly determined at the start and the
end of each run.
Calculation
 Determine the 95% confidence interval for the log-relative potency using Fieller's Theorem.
OR
 Percentage calculation by Percentage yield method

6.5. BIOASSAY OF VITAMIN-D (USP)

Vitamin-D
 Vitamin D is fat soluble vitamin.
 It maintain the adequate plasma level of calcium in the body.
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 Deficiency results in rickets in children and osteomalacia in adults

Principle
The activity of a preparation of vitamin D is determined by comparing its antirachitic activity with that of
standard preparation by a suitable method
USP Reference Standard:
USP Cholecalciferol RS
Subject:
Very young rats (litters)
Procedure
1. Preliminary Period
2. Depletion Period
3. Rachitogenic Diet
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4. Assigning Rats to Groups for Assay Period

5. Administration of Standard and Sample Doses
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6. Assay Period
7. Line Test
 8. Observations and Acceptability
1. Preliminary Period
 30 days period and extends from birth to the first day of the depletion period
 During the preliminary period, use a dietary regimen that provides for normal development but is limited
in its content of vitamin D.
 At the end of the preliminary period, reject any rat that weighs less than 44 g or more than 60 g, or that
shows evidence of injury, disease, or anatomical abnormality.
2. Depletion Period
 Depletion period, which extends from the end of the preliminary period to the first day of the assay
period.
 Provide each rat with the Rachitogenic Diet and water
3. Rachitogenic Diet
 The Rachitogenic Diet consists of a uniform mixture of the following ingredients in the proportions
shown in the accompanying table.
Ingredient Parts by weight
Whole yellow corn, ground 76
Wheat gluten, ground 20
Calcium carbonate 3
Sodium chloride 1
4. Assigning Rats to Groups for Assay Period
 Consider a litter suitable for the assay period when individual rats in the litter show evidence of rickets
such as enlarged joints and a distinctive wobbly, rachitic gait, provided that the depletion period is not
less than 19 or more than 25 days.
 The presence of rickets may be established also from the width of the rachitic metaphysis upon X-ray
examination or by applying the Line Test (described below) to a leg bone of one member of each litter.
 Record the weight of each rat, and assign it to a group
 Litters are divided into 4 groups each containing 7 or more litters.
5. Administration of Standard and Sample Doses
 The litters in two reference groups receive x and nx doses respectively.
 The litters in two sample group receive the dose in same ratio.
 Give the dose at once or in 8 divided doses.
 Ratio of larger to smaller dose not less than 1.5 or more than 2.5.
 Before feeding, the Reference Standard and/or sample may be diluted with cottonseed oil, provided that
not more than 0.2 mL is fed on any one day.
 Store the oil solutions in well-closed bottles, protected from light, at a temperature not exceeding 10°,
and use within 5 weeks.
6. Assay Period
 Assay period last for 7-10 days.
 Throughout the assay period, maintain as uniform environmental conditions as possible for all rats, and
exclude exposure to antirachitic radiations.
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 At the end of a fixed period of 7 to 10 days, weigh and kill each rat.
 Dissect out one or more leg bones for examination by the Line Test.
7. Line Test
 Remove the proximal end of a tibia or the distal end of a radius, and clean adhering tissue from it,
in any one assay using the same bone from all animals.
 Rinse in purified water, immerse immediately in silver nitrate solution (1 in 50) for 1 minute, and
rinse again in purified water.
 Expose the cut surface of bone, in water, to daylight or another source of actinic light until the
calcified areas develop a clearly defined stain without marked discoloration of the uncalcified
areas.
 The staining procedure may be modified to differentiate more clearly between calcified and
uncalcified areas.
63

8. Observations And Acceptability

 Calcified and uncalcified areas are observed and relative potency of standard and sample is calculated.
 Score the degree of calcification of the rachitic metaphysis in each rat, according to a scale that allows
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the average response to be plotted as a straight line against the logarithm of the dose.
 64
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 SUSTAINED ACTION PRODUCTS

7.1. INTRODUCTION
Modified Release Drug Products:
Sustained release, sustained action, modified release, extended release and time release dosage forms are the
terms used to describe drug delivery system that are designed to achieve prolonged therapeutic effects over
extended period of time by continuous release of medications. Modified-release drug products are designed for
different routes of administration (e.g. oral drug products, transdermal drug delivery system, parenteral drug
delivery, implants).
The goal to develop this new and novel drug delivery system is
 To increase patient compliance
 To reduce frequency of administration
 To maintain plasma drug concentration over extended period of time
Modified Release Dosage Form:
A modified-release dosage form is a formulation in which the drug –release characteristics of time course and/or
location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage
forms such as solutions, ointments, or promptly dissolving dosage forms.
History:
The history of controlled release or sustain release technology is divided into three time periods:
1. From 1950-1970 was the period of sustained drug release product.
2. From 1970-1990 was the period in the determination of needs of the control drug delivery.
3. Post 1990 modern era of controlled release technology.

7.2. TYPES OF MODIFIED-RELEASE ORAL DRUG PRODUCTS:

1. Extended-release drug products:
A dosage form that allows at least a twofold reduction in dosage frequency as compared to that drug
presented as an immediate release (conventional) dosage form. Examples Of extended release dosage forms
include sustained-release and long acting drug products.
2. Delayed- release drug products:
A dosage form that releases discrete portion or portions of drug at a time other than promptly after
administration. An initial portion may be released promptly after administration.
Example: enteric coated dosage form (e.g. enteric coated aspirin)
3. Targeted-release drug products:
A dosage form that releases drug at or near the intended physiologic site of action. Targeted- release dosage
forms may have either immediate or extended release characteristics.
CONTROLLED RELEASE DRUG SUSTAINED RELEASE DRUG
PRODUCT PRODUCT
Pharmaceutical dosage form which is Pharmaceutical dosage form that
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designed to deliver the drug at a release the drug slowly thereby

predetermined rate for a specified period achieving therapeutic level which is
of time. prolonged but not maintained constant.
Controlled release is perfectly zero-order Sustained release products usually
release that is the drug release over time follow first order release that is drug
irrespective of concentration. release dependent to concentration.

7.3. DRUG RELEASE RATE CONSIDERATIONS:

 Ideally, an extended release system should deliver drug to the desired site at a rate according to the
needs of the body (i.e a self- regulated system).
 Although some researchers are investigating possible approaches to develop such an ideal dosage
form.
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 This is still a difficult goal to achieve, especially for oral drug administration.
 Zero-order release has been idealized as the ultimate goal for ER delivery system. This means that drug
Page

release must be independent of the amount of drug in the dosage form and should remain constant over
time.
 But the majority of ER systems follow first-order kinetics, where the drug release is dependent on the
amount remaining in the dosage form at any given time.

Figure 1: Plasma level time curve showing zero-order kinetics by controlled release, slow first order by
sustained release and first order kinetics by conventional release dosage form.

7.4. MERITS AND DEMERITS OF MODIFIED RELEASE DRUG PRODUCTS

Merits
1. Improved patient compliance
Improved patient convenience and compliance due to less frequent drug administration.
2. Reduce fluctuation of plasma drug conc.
Reduction in fluctuation of steady state drug concentration and therefore better control of disease
condition.
3. Increased safety margin
Increased safety margin of high potency drug due to better control of plasma level.
4. Efficiency in treatment
Maximum utilization of drug enabling reduction in total amount of dose administration.
5. Reduce health care cost
Reduction in health care cost through improved therapy, shorter treatment period.
6. Lesser time for dispensing
Less frequency of dosing and reduction in personnel time to dispense, administer, monitor the patients.
Demerits
1. Possibility of Dose Dumping
Possibility of dose dumping due to food, physiologic or formulated variable or chewing of oral
formulation by the patient and thus increase risk of toxicity.
2. Stability problems
The complexity of formulation causes stability problem.
3. less flexibility in dose adjustment
The physician has less flexibility in adjusting dosage regimens.
4. Retrieval of drug
Retrieval of drug is difficult in case of toxicity, and hypersensitivity.
5. Economic factor
Economic factors must also be accessed, since most costly processes and equipments are involved in
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manufacturing of many modified release drug products.

7.5. QUALITY CONTROL TEST FOR SUSTAINED ACTION DRUG

A. In-vitro Evaluation
B. In-vivo Evaluation
C. Stability Test
7.5.1. IN-VITRO EVALUATION
Dissolution Test
Purpose:
This test is provided to determine compliance with the dissolution requirements for solid dosage form
administered orally.
Apparatus:
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Apparatus 1(Basket- apparatus)

Apparatus 2(Paddle- apparatus)
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Apparatus 3(Reciprocating -apparatus)

Apparatus 4(flow through cell)
The Basket apparatus and paddle apparatus are most commonly used for dissolution testing.
Apparatus Suitability:
The determination of suitability of the apparatus to perform dissolution testing must include conformance to
the dimensions and tolerances of the apparatus. In addition, critical test parameters that have to be monitored
periodically during use include volume and temperature of the dissolution medium, rotation speed (Apparatus
1 and 2), dip rate (Apparatus 3), and flow rate of medium (Apparatus 4).
Recommended Dissolution Media:
pH Dissolution media
pH1.0 Hal
pH 1.2 NaCl, Hal
pH 1.5 NaCl, Hal
pH 4.5 Phosphate or acetate buffer
pH 5.5 and 5.8 Phosphate or acetate buffer
pH 6.8 Phosphate buffer
pH 7.2 and 7.5 Phosphate buffer

1. Prolonged-Release Solid Dosage Forms

Procedure:
 Place the stated volume of the dissolution medium (+1%) in the vessel of the specified apparatus.
 Assemble the apparatus, equilibrate the dissolution medium to 37+0.50C.
 Place 1 dosage unit in the apparatus and operate the apparatus at specified rate (e.g. Apparatus 1 with
100 rpm and Apparatus 2 with 50 rpm).
 Within specified time interval, withdraw a specimen from the dissolution medium.
 If multiple sampling times are specified, replace the aliquots withdraw for analysis with equal
volumes of fresh dissolution medium.
 Perform the analysis using suitable assay method.
 Repeat the test with additional dosage units.
Acceptance criteria:
Level Number Acceptance Criteria
Tested
L1 6 No individual value lies outside each of the stated ranges and no
individual value is less than the stated amount at the final test
time.
L2 6 The average value of the 12 units (L1 + L2) lies within each of
the stated ranges and is not less than the stated amount at the
final test time; none is more than 10% of labeled content
outside each of the stated ranges; and none is more than 10% of
labeled content below the stated amount at the final test time..
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L3 12 The average value of the 24 units (L1 + L2 + L3) lies within

each of the stated ranges, and is not less than the stated amount
at the final test time; not more than 2 of the 24 units are more
than 10% of labeled content outside each of the stated ranges;
not more than 2 of the 24 units are more than 10% of labeled
content below the stated amount at the final test time; and none
of the units is more than 20% of labeled content outside each of
the stated ranges or more than 20% of labeled content below the
stated amount at the final test time.
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2. Delayed-Release Solid Dosage Forms
APPARATUS 1 AND 2
Procedure:
Use method A and B.
i. Method A
Method A is also called sequential method. It is divided into two stages.
Acid stage
 Place 750 ml of 0.1 M hydrochloric acid in the vessel, and assemble the apparatus.
 Allow the medium to equilibrate to a temprature of 37±0.5 oC.
 Place 1 dosage unit in the apparatus, cover the vessel and operate the apparatus at the specified rate.
 After 2 hours of operation in 0.1M hydrochloric acid, withdraw an aliquot of the fluid and proceed
immediately.
 Perform an analysis of the aliquot using a suitable assay method.
Buffer stage
 With the apparatus operating at specified speed, add to the fluid in the vessel 250 ml of 0.20 M
solution of trisodium phosphate dodecahydrate R that has been equilibrated to 37±0.5 oC.
 Adjust, if necessary, with 2 M sodium hydroxide to a pH of 6.8±0.05.
 Continue to operate for 45 min, or for the specified time.
 At the end of the time period, withdraw an aliquot of the fluid and perform the analysis using a
suitable assay method.
ii. Method B (Parallel Method)
Acid stage
 Place 1000 ml of 0.1 M hydrochloric acid in the vessel and assemble the apparatus.
 Allow the medium to equilibrate to a temprature of 37±0.5 oC.
 Place 1 dosage unit in the apparatus, cover the vessel, and operate the apparatus at the specified rate.
 After 2 hours of operation in 0.1M hydrochloric acid, withdraw an aliquot of the fluid, and perform
an analysis using a suitable assay method.
Buffer stage
 For this stage of the procedure use the buffer that has previously been equilibrated to a temprature
of 37±0.5 oC.
 Drain the acid from the vessel and add 1000 ml of pH phosphate buffer, prepared by mixing 3
volumes of 0.1 M hydrochloric acid with 1 volume 0.20 M solution of trisodium phosphate
dodecahydrate R and adjusting, if necessary, with 2 M hydrochloric acid or 2 M sodium hydroxide
to a pH of 6.8± 0.05.
 This may also be accomplished by removing from the vessel containing the acid and replacing it
with another vessel, containing the buffer and transferring the dosage unit to the vessel containing
the buffer.
 Continue to operate the apparatus for 45 min, or for the specified time.
 Withdraw an aliquot of the fluid and perform the analysis using a suitable assay method.
APPARATUS 3
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Procedure
Procedure is same as , method B , under apparatus 1 and 2 , using one row of vessels for the acid
stage media and the following row of vessels for the buffer stage media and using the volume of
medium specified(usually 300 ml).
Time:
Proceed as directed for delayed-release dosage forms under apparatus 1 and 2.
APPARATUS 4
Procedure
Proceed as described for delayed-release dosage forms under apparatus 1 and 2, using the specified
media.
Time:
Proceed as described for delayed-release dosage forms under apparatus 1 and 2.
Acceptance Criteria
69

 Dissolution is done in two stages to simulate with GI environment.

 The purpose of the acid stage is to show the intactness of dosage form and of the buffer stage is to
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evidence the drug release in specific site.

  At least two time points should be included in the specification on in-vitro dissolution of gastro-resistant
products.
 An early time point to exclude release in the acidic medium (less than 10 % dissolved after 2 hr) and
second to ensure that the majority of the active substance has been released in a neutral medium (after
45 min).
i. Acid Stage
Testing is continued upto 3 levels untill the results conform at an earlier level.

Level Number Tested Acceptance Criteria

A1 6 No individual value exceeds 10 percent dissolved.

A2 6 The average value of 12 units (A1+A2) is not more than 10
percent dissolved , and no individual unit is greater than 25
percent dissolved.
A3 12 The average value of the 24 units (A1+A2+A3) is not more
than 10 percent dissolved , and no individual unit is greater
than 25 percent dissolved.

ii. Buffer Stage

The quantity, Q is the percentage of the labeled contents. The value of Q is 75 per cent dissolved
unless otherwise specified.

Level Number Acceptance Criteria

Tested

B1 6 No unit is less than Q+5 percent.

B2 6 The average value of the 12 units (B1+B2) is equal to or greater
than Q, and no unit is less than Q-15 percent.
B3 12 The average value of the 24 units (B1+B2+B3) is equal to or
greater than Q, not more than 2 units are less than Q-15 percent,
and no unit is less than Q-25 percent.

7.5.2. IN-VIVO EVALUATION OF MODIFIED RELEASE DOSAGE FORMS

In-vivo studies are performed within the body of living subjects.
Objective
There are following objectives of in vivo studies
1. To determine that drug product meets the controlled release claim made for that product.
2. To determine occurrence of dose dumping.
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3. To determine safety and efficacy.

4. To determine that steady state performance of modified release dosage form is equal to immediate
release dosage form.
Study designs
Two study designs are used for evaluation of modified release dosage forms.
1. Single dose cross-over study design
2. Multiple-dose, steady-state study design
Parameters
 Area under curve
 Cmax
 Tmax
 Fluctuation in plasma drug concentration
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Studies
The type of studies generally conducted can be categorized as follows.
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Case A
Applies to the original modified release dosage form of an active drug entity already marketed in immediate-
release form and for which extensive pharmacodynamic and pharmacokinetic data exist.
1. Single Dose Crossover Study
Three treatments are involved in single-dose crossover study.
i. Modified release dosage form administered under fasting conditions.
ii. Immediate release dosage form under fasting conditions.
iii. Modified release dosage form with high fat meal.
 If there is a significant difference in bioavailability, it is necessary to determine how food affects the
modified release dosage forms.
 If there is no significant difference in the rate and extent of bioavailability (AUC, Cmax, and Tmax)
as a function of meal, then additional food effect studies are not necessary.
Objective
 To determine if there is a need for labeling instructions to patient when taking drug with meal.
 To provide the information of the pattern of absorption of modified release dosage forms.
2. Multiple- Dose, Steady-State Studies
 Multiple dose, steady state study is conducted with modified release dosage forms using immediate
release dosage form as a control.
 At least three trough plasma drug concentrations (Cmin) should be determined to make sure that steady
state conditions have been achieved.
 The modified release dosage form should produce an AUC that is equivalent to immediate release
dosage form, and there should be no fluctuations in plasma drug concentration.
 Data can be misleading when obtained from subjects having individual variations.
 Subject selection should be randomized or from a target population. Regardless of whether a drug
exhibits linear or non-linear pharmacokinetics, the basis of characterization should be the equivalence
of the AUC and of the relative degree of fluctuations in plasma concentrations of immediate and
modified release dosage forms.
Case B
Case B is applicable to non-oral modified release dosage forms.
Case C
Case C is applicable to generic equivalents of an already marketed approved modified release dosage form.
A generic equivalent should be equivalent to standard modified release dosage form in its rate and extent of
bioavailability (i.e., AUC, Cmax, Cmin, and degree of fluctuations ) in crossover and steady state studies

7.5.3. STABILITY OF FINISHED DOSAGE FORMS

Objectives
 Quality
 Shelf life
 Recommended storage conditions
 Stability
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Stability:
The term stability is defined as ability of drug substance or drug product to retain its physical, Stability
of sustain release dosage form during storage chemical and microbiological properties within specified
limits throughout its shelf- life.
Stability Protocols
 The stability parameters of drug dosage form can be influenced by environmental condition of
storage (temperature, light, air and humidity) as well as packaged component.
 The detailed plan is applied to generate and analyze acceptable data that is used to detect the
changes with time in:
o Chemical
o Physical
o Microbiological properties of drug substance in support of the shelf- life.
Stability Test
71

Adequate stability data of the drug and its dosage forms is essential to ensure the strength, safety, identity,
quality and purity and in vitro in vivo release rates that they claim to have at the time of use.
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 A controlled release product should release a pre determined amount of drug at specified time intervals,
which should not change on storage.
 Any considerable deviation from the appropriate release would render the controlled release product
useless.
 The stability programmes of a controlled release product include storage at both nominal and accelerated
conditions such as temperature and humidity to ensure that the product will withstand these conditions.
 There is a difference between a product being kept at room temperature in plains area for a month and for
that is kept in hilly areas at room temp.

General Storage Condition

Study Storage condition Minimum time period covered

by date at submission
Long term 30±2 oC/65%R 12 months
Accelerated 40±2 oC/75%R 6 months

Recommended Description of Labeled Storage Condition

Storage condition Storage statement

Room temperature Store below 30 0r 35 c
Refrigerator Store in refrigerator b/w 2-8'C
Freezer Store in freezer b/w -5 to -20'C

Real Time Stability Studies

It is the study carried out by manufacturer on production batches according to predetermined schedule in order
to confirm the projected shelf life.
 Studies are conducted at 30 ± 2 oC for 12 months
 Sampling should be done at 3, 6, 9, 12, 18, 24, 36 months.
 Samples are withdrawn from three different batches.

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 73
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 ALCOHOL DETERMINATION
“The measurement of percentage of alcohol in different dosage forms.”
Properties of Alcohol:
Alcohol means ‘ethanol’, used in preparations because of its important properties:
 Used as solvent
 As co-solvent along with water
 Used because of its preservative properties.
 Non toxic in nature.

8.1.METHODS TO DETERMINE ALCOHOL

There are two methods for determination of alcohol:
1. Distillation method.
2. Gas Liquid Chromatography.
8.1.1. METHOD I (DISTILLATION METHOD)
 Method I is to be used for determination of alcohol unless otherwise specified in individual monograph.
 It is suitable for examining fluid extracts, tinctures, provided the capacity of the distilling flask is
sufficient (commonly 2 to 4 times the volume of the liquid to be heated) and the rate of distillation is
such that clear distillates are produced.
Procedure:
Two types of procedures are there:
1. Normal Procedure
2. Special Procedure
1. Normal Procedure:
i. For liquids containing 30% alcohol or less
 By using pipette transfer liquid dosage form in distilling apparatus equal to 25ml.
 Note temperature and add equal volume of water
 Pour into distillation apparatus and start the process
 Collect the volume of distillate about 2ml less than volume of original test liquid
 Maintain the temperature as it was at the start of process
 Add some quantity of water to make original volume of test liquid
 Resulted distillate may be clear or not if not then clear it by adding the talc or CaCO3
 Now determine the specific gravity at 25 oC
 So we can calculate the %age v/v of alcohol.
 The distillate is clear or not more than slightly cloudy, and does not contain more than traces of
volatile substances other than alcohol and water. If distillate is not clear than add talc or calcium
carbonate, and filtered, after which the temperature of the filtrate is adjusted and the alcohol
content determined from the specific gravity.
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 Take precautions to minimize the loss of alcohol by evaporation during all manipulations.
ii. For liquids containing more than 30% of alcohol
 Take 25ml of the liquid in which the alcohol is to be determined
 Add twice volume of water in the sample liquid.
 Subject this liquid to distillate.
 Collect a volume of distillate about 2ml less than the twice volume of sample liquid taken
(about 48ml) and bring the temperature at which original liquid was measured.
 Add sufficient water to measure exactly the double volume of sample liquid taken i.e.50ml
 Determine the specific gravity of liquid.
 The proportion of ethanol, by volume, in this distillate, as ascertained from its specific
gravity, equals one half that in the liquid examined.
2. Special Procedure
74

i. For liquids containing 50% alcohol or less

 Take 25ml of the sample to be examined in separating funnel.
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 Mix an equal volume of water in it.

 Saturate this mixture with sodium chloride.
  Add 25ml of solvent hexane, shake the mixture to extract the interfering volatile ingredients.
 Draw off the lower layer into second separating funnel.
 Repeat the extraction twice with two further 25ml portions of solvent hexane.
 Extract the combined solvent hexane solutions with three 10ml portions of a saturated solution
of sodium chloride.
 Separate aqueous layer i.e. NaCl solution.
 Combine the saline solution and distill in usual manner, collecting a volume of distillate having
a simple ratio to the volume of the original sample. (via same procedure as for less than 30%
alcohol)
 Collect the distillate and determine %age of alcohol & specific gravity.
ii. For liquids containing more than 50% alcohol
 Adjust the specimen under examination by diluting it with water up to 25% ad then performed
process as above.
 In preparing collodion or flexible collodion, use water in place of saturated NaCl solution as
directed above.
 The remaining procedure is same.
 If volatile oils are presents in small proportion only, and a cloudy distillate is obtained , the
solvent hexane treatment not having been employed. The distillate may be clarified by shaking
it with about one-fifth its volume of solvent hexane, or by filtering it through a thin layer of
talc.
Special Consideration
a) Volatile Acids & Bases:
If preparations contain volatile bases make it slightly acidic with diluted sulfuric acidic before distilling.
If volatile acids are present make the preparation slightly alkaline with sodium hydroxideTS.(4% NaOH)
b) Glycerin:
If sample contain glycerin add sufficient water so that the residue, after distillation, contains not less than
50% of water.
c) Iodine:
If the sample solution contain free iodine than treat sample with powdered zinc before distillation.
OR
Decolorize with just sufficient sodium thiosulfate solution (1in10), followed by a few drops of sodium
hydroxideTS.
d) Other Volatile Substances:
Spirits, elixirs , tinctures, and similar preparations that contain appreciable proportions of volatile
materials other than alcohol and water, such as volatile oils , chloroform , ether , camphor , etc., require
special treatment, as follows:
Problems during Distillation of Alcohol
Following problems occurred during distillation of alcohol:
1. Rothing / Foam Formation:
 The foam formation in the distillation flask is frothing due to presence of surface active agents.
 It may cause problem in accurate measuring.
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Remedy:
 Make it strongly acidic by adding sulfuric acid, phosphoric acid or tannic acid.
 Treat with slight excess of CaCl₂ solution.
 Addition of small amount of paraffin oil or silicone oil before starting distillation.
2. Bumping:
 Bubble formation in distillation flask is called bumping.
 It may exert pressure on the walls of vessel which may break the vessel.
Remedy:
It can be prevented by adding porous chips of insoluble material e.g., silicon carbide, glass chips,
beads.
3. Cloudy / Milky Distillation:
Cause problem in measuring.
75

Remedy
Clarification is done by adding talc or calcium carbonate, chalk and then filter.
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4. Azeotrope Formation:
 Complex formation between two agents which may change the nature of individual components is
called Azeotrope.
 Azeotropes are also called constant boiling mixtures.
 They are binary mixtures having the same composition in liquid and vapor phase and boil at a
constant temperature. In such a case it is not possible to separate the components by fractional
distillation.
Example:
Water+ acetonitrile (Azeotrope formation)
5. Emulsification:
It can be problematic for the process of distillation.
Remedy:
 Distillate is saturated with brine and light petroleum.
 Excessive temperature can break emulsion.

8.1.2. METHOD II (GAS LIQUID CHROMATOGRAPHY):

USP Reference Standards
1. USP Alcohol Determination—Acetonitrile RS
2. USP Alcohol Determination—Alcohol RS.
Method IIa
Apparatus
Gas chromatography specification
1. Flame-ionization detector
2. 4-mm × 1.8-m glass column packed with 100- to 120-mesh chromatographic column packing support S3
3. Nitrogen or helium as the carrier.
Temperature specification
1. Prior to use, condition the column overnight at 235oC with a slow flow of carrier gas.
2. The column temperature is maintained at 120o
3. The injection port and detector temperatures are maintained at 210o.
4. Adjust the carrier flow and temperature so that acetonitrile, the internal standard, elutes in 5 to 10 minutes.
Solutions:
Test Stock Preparation:
Dilute the specimen under examination stepwise with water to obtain a solution containing approximately 2%
(v/v) of alcohol.
Test Preparation:
Pipet 5 mL each of the Test Stock Preparation and the USP Alcohol Determination—Acetonitrile RS
[Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as the internal standard
solution] into a 50-mL volumetric flask, dilute with water to volume, and mix.
Standard Preparation:
Pipet 5 mL each of the USP Alcohol Determination—Alcohol RS and the USP Alcohol Determination—
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Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as

the internal standard solution] into a 50-mL volumetric flask, dilute with water to volume, and mix.
Procedure
 Inject about 5 µL each of the Test Preparation and the Standard Preparation, in duplicate, into the gas
chromatograph, record the chromatograms, and determine the peak response ratios.
 Calculate the percentage of alcohol (v/v) in the specimen under test according to the formula:
CD(RU / RS)
o C is the labeled concentration of USP Alcohol Determination—Alcohol RS
o D is the dilution factor (the ratio of the volume of the Test Stock Preparation to the volume of
the specimen taken);
o RU and RS are the peak response ratios obtained from the Test Preparation and the Standard
Preparation, respectively.
System Suitability Test
76

 In a suitable chromatogram, the resolution factor, R, is not less than 2;

 The tailing factor of the alcohol peak is not greater than 2.0
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 Six replicate injections of the Standard Preparation show a relative standard deviation of not more than
2.0% in the ratio of the peak of alcohol to the peak of the internal standard.
 Method IIb
Apparatus:
Gas chromatography specification
1. Split injection port with a split ratio of 5:1
2. A flame-ionization detector
3. 0.53-mm × 30-m capillary column coated with a 3.0-µm film of phase G43.
4. Helium is used as the carrier gas at a linear velocity of 34.0 cm per second.
Temperature specification
1. The chromatograph is programmed to maintain the column temperature at 50o for 5 minutes
2. Then to increase the temperature at a rate of 10o per minute to 200o, and maintain at this temperature for 4
minutes.
3. The injection port temperature is maintained at 210o
4. The detector temperature at 280o.
Solutions:
Test Stock Preparation:
Dilute the specimen under examination stepwise with water to obtain a solution containing approximately 2%
(v/v) of alcohol.
Test Preparation:
Pipet 5 mL each of the Test Stock Preparation and the USP Alcohol Determination—Acetonitrile RS
[Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as the internal standard
solution] into a 25-mL volumetric flask, dilute with water to volume, and mix.
Standard Preparation:
Pipet 5 mL each of the USP Alcohol Determination—Alcohol RS and the USP Alcohol Determination—
Acetonitrile RS [Alternatively, a 2% aqueous solution of acetonitrile of suitable quality may be used as the
internal standard solution] into a 25-mL volumetric flask, dilute with water to volume, and mix.
Procedure:
 Inject about 0.2 to 0.5 µL each of the Test Preparation and the Standard Preparation, in duplicate, into the
gas chromatograph, record the chromatograms, and determine the peak response ratios.
 Calculate the percentage of alcohol (v/v) in the specimen under test according to the formula:
CD(RU / RS)
o C is the labeled concentration of USP Alcohol Determination—Alcohol RS
o D is the dilution factor (the ratio of the volume of the Test Stock Preparation to the volume of the
specimen taken);
o RU and RS are the peak response ratios obtained from the Test Preparation and the Standard Preparation,
respectively.
System Suitability Test:
 In a suitable chromatogram, the resolution factor, R, between alcohol and the internal standard is not less
than 4
 The tailing factor of the alcohol peak is not greater than 2.0
 Six replicate injections of the Standard Preparation show a relative standard deviation of not more
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than 4.0% in the ratio of the peak of alcohol to the peak of the internal standard.Used only when
specified in individual monograph.
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 79
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 MISCELLANEOUS DETERMINATION & TESTS

9.1. DETERMINATION OF WEIGHT/mL

 The weight per milliliter is determined by dividing the weight in air, expressed in g, of the quantity of
liquid that fills a pycnometer at the specified temperature by the capacity, expressed in ml, of the
pycnometer at the same temperature.
 The capacity of the pycnometer is ascertained from the weight in air, expressed in g, of the quantity of
water required to fill the pycnometer at that temperature.
 The weight of a litre of water at specified temperatures when weighed against brass weights in air of density
0.0012 g per ml is given in the following table.
 Ordinary deviations in the density of air from the above value, here taken as the mean, do not affect the
result of a determination in the significant figures prescribed for Pharmacopoeial substances.
Temperature °C Weight of a litre of water
20 997.18
25 996.02
30 994.62
9.2. WATER CONTENT DETERMINATION (BP)
There are following methods:
A. Method 1 (Titrimetric)
B. Method 2 (Azeotropic)
C. Method 3 (Gravimetric)
9.2.1. METHOD 1 (TITRIMETRIC)
1. Karl Fischer
2. Residual
3. Coulometric
1. Karl Fischer Titration:
 Classical method of titration
 Invented in 1935
Karl Fischer Reagent:
A solution of iodine and sulphur dioxide in a mixture of pyridine and methanol.
Principle:
The titrimetric determination of water is based upon the quantitative reaction of water with an anhydrous
solution of sulphur dioxide and in the presence of a buffer that reacts with hydrogen ions.
2H₂O+ SO₂+I₂→2HI+H₂SO₄
Characteristics of the Karl Fischer Titration Method:
a) Moisture content can be determined accurately
b) Measurements can be taken over short periods of time
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c) Measurements can be taken using small samples

d) The method can be used with liquid, solid and gaseous sample
e) The method is suitable for unstable substances that can alter when heated
Working:
 Water and iodine are consumed in a 1:1 ratio in the above reaction.
 Once all of the water present is consumed, the presence of excess iodine is detected Volta
metrically by the titrator’s indicator electrode.
 That signals the end point of the titration. The amount of the water present in the sample is
calculated based on the concentration of iodine in the Karl Fischer titrating reagent (i.e., titer) &
amount of Karl Fischer reagent consumed in the titration.
Apparatus:
 A closed system consisting one or two automatic burets
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 A tightly covered titration vessel fitted with electrodes

 A magnetic stirrer
 A suitable desiccant
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 Standardization:
 To the titration vessel, add methanol R, dried if necessary, or the solvent recommended by the supplier
of the titrant.
 Where applicable for the apparatus used, eliminate residual water from the measurement cell or carry
out a pre-titration.
 Introduce a suitable amount of water in an appropriate form and carry out the titration, stirring for the
necessary time.
 The water equivalent is not less than 80% of that indicated by the supplier.
 Standardize the titrant before the first use and at suitable intervals thereafter.
Procedure:
 Unless otherwise specified, transfer 35 to 40 ml of methanol or other suitable solvent to the titration
vessel, and titrate with the Reagent to the electrometric or visual endpoint to consume any moisture that
may be present.
 Quickly add the test preparation, mix, and again titrate with the reagent to the electrometric or visual
endpoint.
 Calculate the water content of the specimen, in mg, taken by the formula (SF)

o S is the volume, in ml, of the reagent consumed in the second titration

o F is the water equivalence of the reagent.
Visual Detection:
In case of colorless solution that is titrated directly, the end point may be observed visually as a change in
color from canary yellow to amber.
Electrical Detection:
When alkyl sulphate moiety is developed then ammeter show deflection. When ammeter show persistent
deflection for 30 sec then it is assumed that end point is reached.
2. Residual Titration
Principle:
 In the residual titration, excess reagent is added to the test specimen, sufficient time is allowed for the
reaction to reach completion and the unconsumed reagent is titrated with a standard solution of water
in a solvent such as methanol.
 The residual titration procedure avoids the difficulties that may be encountered in the direct titration
of substances from which the bound water is released slowly.
Apparatus, Reagents and test preparation:
Use same as above
Procedure:
 Were the individual monograph specifies that the water content is to be determined by method 1b,
transfer 35-40 ml of methanol or other suitable solvent to the titration vessel, and titrate with the reagent
to the electrometric or visual endpoint.
 Quickly add the test preparation, mix , and add an accurately measured excess of the reagent.
 Allow sufficient time for the reaction to reach completion, and titrate the unconsumed reagent with
standardized water solution to the electrometric or visual endpoint.
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 Calculate the water content of the specimen, in mg, taken by the formula:
F(X’-XR)
o F is the water equivalence factor for the reagent
o X’ is the volume, in ml, of the reagent added after introduction of the specimen
o X is the volume, in ml, of the standardized water solution required to neutralized the
unconsumed reagent
o R is the ratio, determined by the standardization of the water solution for residual titration
(ml of the reagent/ ml of water solution)
3. Coulometric Titration
Principle:
 The coulometric titration of water is based upon the quantitative reaction of water with sulphur
dioxide and iodine in an anhydrous medium in the presence of a base with sufficient buffering
81

capacity.
 In contrast to the volumetric method, iodine is produced electrochemically in the reaction cell by
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oxidation of iodide.
 The iodine produced at the anode reacts immediately with water and the sulphur dioxide contained in
the reaction cell.
 The amount of water in the substance is directly proportional to the quantity of electricity up until the
titration endpoint.
 When all of the water in the cell has been consumed, the endpoint is reached and thus an excess of
iodine appears. 1mole of iodine corresponds to 1mole of water, a quantity of the electricity of 10.71C
corresponds to 1mg of water.
 Moisture is eliminated from the system by pre-electrolysis. Individual determinations can be carried out
successively in the same reagent solution, under the following conditions:
o Each component of the test mixture is compatible with the other components
o No other reactions take place
o The volume and the water capacity of the electrolyte reagent are sufficient
Apparatus:
 Absolutely tight system fitted with the necessary electrodes and a magnetic stirrer
 The reaction cell consists of a large anode compartment and a smaller cathode compartment separated
by diaphragm.

Test Preparation:
 Where the specimen is a soluble solid, dissolve an appropriate quantity, accurately weighed, in
anhydrous methanol or other suitable solvents.
 Liquids may be used as such or as accurately prepared solutions in appropriate anhydrous solvents.
 Where specimen is an insoluble solid the water may be extracted using a suitable anhydrous solvent
from which an appropriate quantity, accurately weighed, may be injected into anolyte solution.
 Alternatively an evaporation technique may be used in which water is released & evaporated by heating
the specimen in a tube in a stream of dry inert gas, this gas being then passed into the cell.
Procedure:
 Using a dry syringe, quickly inject the test preparation accurately measured and estimated to contain
0.5 to 5 mg of water, or as recommended by the instrument manufacture into the anolyte, mix, and
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perform the coulometric titration to the electrometric end point.

 Read the water content of the test preparation directly from instrument’s display, and calculate the
% that is present in the substance.
 Perform a blank determination, and make any necessary corrections.
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9.2.2. METHOD 2 (AZEOTROPIC-TOULENE DISTILLATION):
Apparatus:
Use a 500 ml glass flask A connected by means of a trap B to a
reflux condenser C by ground glass joints.

Procedure:
 Place the accurately weighed quantity of the substance in the flash to the nearest centigram.
 Place about 200ml of toluene in the flask.
 Connect the apparatus.
 Fill the receiving tube E with toluene poured through top of condenser.
 Heat the flask gently for 15 minutes until the toluene begins to boil.
 Distill out the water at the rate 2 drops per second.
 Continue distillation for 5 minutes.
 Remove the heat. Allow the flask to cool to room temp
 Scrub any adhered water droplets to the walls of the tube, with the brush.
 When the water and toluene have separated completely, read volume of water and calculate the %age that
was present in the substance.

9.2.3. METHOD 3 (GRAVIMETRIC)

Procedure for Chemicals
Proceed as directed in the individual monograph preparing the chemicals as directed under Loss On Drying.
Procedure for Biologics
Proceed as directed in the individual monograph.

9.3. LOSS ON DRYING

Definition:
The loss on drying test is designed to measure the amount of water and volatile matters in a sample when
sample is dried under specified condition.
Procedure USP:
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 Mix and weigh the substance.

 Unless otherwise directed in the monograph.
 Reduce size about 2mm if in crystal form.
 Take a covered petri-dish dry it for about 30 minutes under same conditions.
 Weigh it without cover.
 Put the sample in it.
 Weigh content with petri-dish.
 Spread the sample not more than 10mm in depth.
 Place it in oven under conditions given in monograph.
 Upon opening the oven promptly cover the dish, allow to come in room temperature in dessicator.
 If sample melt at specified temperature then kept sample for one to two hours at temperature 5-10℃
below its melting point.
83

 In case of capsules and tablets use content of not less than 4 capsules.
 If drying in vacuum over a dessicator is directed suitable vacuum drying apparatus should use.

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If drying in a capillary –stoppered bottle in vacuum is directed, use bottle with stopper having
225±25𝑢𝑚 diameter and maintain pressure of 5mm or less of mercury.
Formula:
LOD is calculated by:-
𝑳𝒐𝒔𝒔 𝒊𝒏 𝒘𝒆𝒊𝒈𝒉𝒕
𝑳𝒐𝒔𝒔 𝒐𝒏 𝒅𝒓𝒚𝒊𝒏𝒈 = × 𝟏𝟎𝟎
𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒘𝒆𝒕 𝒔𝒂𝒎𝒑𝒍𝒆

9.4. IDENTIFICATION TESTS

Definition:
It is performed to measure;
 Severity and
 Potential of health risk due to drug problems
Purpose:
The purpose of identification test is to check drug:
 Bio-analysis e.g., mechanism of action
 Bioavailability
 Interaction of one drug with other
Methods:
These are perfomed both;
A. In vivo
B. In vitro
9.4.1. IN VIVO IDENTIFICATION TESTS:
In vivo identification tests discussed are for:
1. Atropine
2. Insulin Injection
3. Ophthalmic ointments and Ophthalmic solutions
1. Test for Atropine:
General characteristics of atropine are:
 Belladona Alkaloid
 Affinity for muscarinic receptors
 Cause mydriasis in eye by blocking cholinergic activity
Procedure for test:
 First take sufficient number of tablets or injection which contain 0.6 to 1 mg Atropine sulphate
 Dissolve it in 10 ml water
 Take 1 ml from this and instill in rabbits eye and observe
Result:
If dilation occurs within 2 hrs atopine is confirmed
2. Test For Insulin Injection:
Subject: Rabbit
Number of subjects: 6
Weight range: 1.8 – 2.2 kg
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Requirement: These rabbits are placed under observation and food is not given for 18-24 hrs before
experiment
Procedure:
 Inject subcutaneously, a quantity of insulin injection, which should cause convulsion in atleast 3
of the animals
 Immediately after convulsions, inject 5ml of 50% dextrose solution intravenously… convulsions
should be stopped in atleast 4 of the animals and they should remain alive for atleast 3 days after
Specification:
This is specified in the monograph
3. Test For Ophthalmic Ointment And Ophthalmic Solution (Isophlurophate):
Requirement:
Subject: Rabbit
84

Number of subjects: 3
Procedure:
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 Take 100mg of isoflurophate ointment or 0.1ml of solution in the right eye of each of three rabbits
 Observe the constriction of pupil
 RESULT:
If atleast 20mm decrease occurs then it is confirmed
9.4.2. IDENTIFICATION TESTS IN VITRO:
For some drugs in vitro tests are performed and compared with standard
 The IR absorption spectra for chlorthiazide dispersed in mineral oil should show maxima equal to the
chlorthiazide refrence standard.
 When transmitted light is passed through Quabin dissolved in sulphuric acid produces dark red color and
produce greenish fluorescence in reflected light..
 Blue fluorescence is exhibited in case of solution of quinine gluconate in dilute sulphuric acid, on
addition of few drops of HCl fluorescence disappears.

9.5. TOXICITY TESTS

Toxicology:
It is the study of the adverse effects of the physical, chemical or biologic agents on living organisms and the
ecosystem.
Tests for Toxicity
1. Single Dose Acute Toxicity Test
Toxicity produced by a pharmaceutical when it is administered in one or more doses during a period not
exceeding 24 hrs.
Purpose & Procedure:
Test is performed to identify doses causing no and maximum toxic effects.
For acute toxicity tests two routes are used:
i. The route intended for human administration &
ii. Intravenous administration if feasible
Observation:
Animals are kept under observation for 14 days after administration of pharmaceutical and following
observations are noted and recorded
 Mortalities
 Time of onset
 Duration
 Reversibility of toxicity
If primary data supports single dose safety/kinetic studies in humans, the toxicity studies should be
designed to access dose response relationship and pharmacokinetics.
2. Repeated Dose Toxicity
Purpose:
 Development of safe medicinal products that need repeated administration to patients.
 To characterize toxicological profile of test compound following repeated administration.
 To identify potential target organs of toxicity & potential reversibility of toxic effects.
General Recommendations Concerning The Experimental Animal:
 Species: it should be chosen based on similarity to humans with regard to pharmacokinetic
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profile and biotransformation.

 Sexes: Equal no. of male and female animals should be taken.
 Size: It should be sufficient to allow meaningful scientific interpretation of data generated.
 No. of Species: It should be carried out in two species of mammals, one of which, must be non-
rodent
 Animal Husbandry: A high standard of animal husbandry is required & Environmental
conditions should be controlled.
General Recommendations Concerning Dose and Administration:
 Duration of Administration: it depends on the duration of proposed therapeutic use in
humans.
 Route of Administration: same route should be chosen as intended for humans. Other routes
are selected if justified on the basis of pharmacology.
85

 Frequency of Administration: it should be taken on the basis of intended clinical dosing regimen
and toxicological/pharmacokinetic/pharmacodynamics profile of test compound.
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 Dose levels: Treatment should include:

o A low dose: sufficient to produce a pharmacodynamic effect or the desired therapeutic effect.
 o A high dose: selected to enable identification of toxicity for target organ.
o Intermediate dose: geometric mean between high and low dose.
Observation:
 Pre-treatment and control values; historical control data should be available for morphological,
biochemical and physiologic variables studied, both for rodents and non-rodents. For non-rodents
pre-treatment values should be obtained from animal used in the study.
 Monitoring during the study; followings are monitored: Food intake, General behavior, Body
weight, Heamatological parameters, Clinical chemistry, Urinalysis and ophthalmology.
Data Analysis, Presentation Of Results And Conclusions:
 Study report should reflect all the raw data and information gathered during the study.
 Group summary values should be presented.
 Finally, a conclusion based on study results should be drawn.
Methods for Toxicity Testing:
1. Clinical investigation; chemicals are administered to human subjects with careful observation and lab
measurements.
2. Epidemiological studies; observation of humans that have been exposed to xenobiotics in normal course
of their life and occupation.
3. Reports of drug adverse reactions; Reports are submitted to FDA after drug has been approved.
Phases of Clinical Investigation:
Phase 1
 Drug is tested in a small group of 20-80 patients
 In it we determine drug’s:
o Pharmacokinetics
o Metabolism &
o Mechanism of action
Phase 2
 Determine the short term effects of drugs &
 Evaluate effectiveness of treatment
Phase 3
 It is conducted with several thousand patients to gather information about effectiveness and safety.
 It is done prior to human clinical investigations
Advantages:
 Any type of toxicity test can be evaluated
 Mechanism of toxicity test can be evaluated
Why Required:
Toxicity tests are required for different
 Drugs
 Toxoids &
 Containers
Example:
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Diphtheria toxoid
Procedure:
For toxicity test of diphtheria toxoid
 Take four healthy pigs of weight 300-400g
 Inject 2ml diphtheria toxoid subcutaneously
Result
No symptoms of diphtheria toxoid should appear within 30 days
86
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9.6. EVALUATION OF OINTMENT
Ointment:
 The word ointment derived from Latin word “UNGUENT” means anoint with oil.
 Anoint with oil means rub with oil, or covered with oil.
Ointments are semisolids intended for external application to the skin or mucous membranes
OR
Ointments are semisolids intended for external application to the skin or mucous membranes that usually
contain less than 20% water and volatiles and more than 50% hydrocarbons, waxes, or polyols as the vehicle
and provide emollient, protectants, or lubricants properties.
 Ointments are semisolid preparations intended for topical application. They are used to provide
protective and emollient effects on the skin or carry medicaments for treating certain topical ailments.
 Any greasy or oily semi solid preparation, usually medicated, that can be applied externally to the skin in
order to heal, soothe or protect it.

Types of Ointment bases:

There are five (5) classes or types of ointment bases which are differentiated on the basis of their physical
composition.
These are:
1. Oleaginous bases/hydrocarbon bases
2. Absorption bases
3. Water in oil emulsion ointment bases
4. Oil in water emulsion ointment bases
5. Water soluble or water miscible bases

Oleaginous Absorption Water/Oil Oil/Water Water-

Ointment Bases Ointment Bases Emulsion Emulsion miscible
Ointment Bases Ointment Bases Ointment
Bases
Composition oleaginous oleaginous base + oleaginous base + oleaginous base + Polyethylene
compound w/o surfactant water (< 45% w/w) water (> 45% Glycols
s + w/o surfactant w/w) + o/w (PEGs)
(HLB <8) surfactant
(HLB >9)
Water anhydrous anhydrous Hydrous hydrous anhydrous,
Content hydrous
Affinity for hydrophob hydrophilic Hydrophilic hydrophilic hydrophilic
Water ic
Spreadability difficult difficult moderate to easy easy moderate to
easy
Washability Non Non washable non- or poorly washable washable
washable washable
Stability oils poor; oils poor; unstable, especially unstable, stable
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hydrocarbo hydrocarbons alkali soaps and especially alkali

ns better better natural colloids soaps and natural
colloids; non
ionics better
Drug solids or solids, oils, and solids, oils, and solid and aqueous solid and
Incorporatio oils (oil aqueous solutions aqueous solutions solutions (small aqueous
n Potential solubles (small amounts) (small amounts) amounts) solutions
only)
Drug Release poor poor, but > fair to good fair to good good
Potential* oleaginous
Occlusiveness yes yes Sometimes no no
Uses protectants protectants, emollients, emollients, drug vehicles
, emollients (+/-), cleansing creams, vehicles for solid,
87

emollients vehicles for vehicles for solid, liquid, or non-

(+/-), aqueous solutions, liquid, or non- hydrolyzable
vehicles solids, and non- hydrolyzable drugs drugs
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for hydrolyzable
drugs
 hydrolyzab
le drugs
Examples White Hydrophilic Cold Cream type, Hydrophilic PEG
Petrolatum Petrolatum, Hydrous Lanolin, Ointment, Ointment,
, White Anhydrous Rose Water Dermabase™, Polybase™
Ointment Lanolin, Ointment, Velvachol®,
Aquabase™, Hydrocream™, Unibase®
Aquaphor®, Eucerin®, Nivea®
Polysorb®

Quality Control Tests:

1. Appearance and colour USP
2. Weight variation tests BP or minimum fill
3. Particle size determination BP
4. Microbial content test USP
5. Metal contents in ophthalmic ointments BP
6. Sterility tests
7. Potency or content uniformity test BP
8. Viscosity
9. Leakage tests
10.Homogenicity
1. Appearance and colour:
Procedure:
Transfer the ointment to a suitable test tube and examine the sample in front of light source.
Acceptance rejection criteria:
Translucent sample is accepted.
Color:
Colour should be colourless to yellow.
2. Weight variation tests or minimum fill:
 Test is applied only to those containers that contain not more than 150 g or mL of preparation.
 Select 10 filled containers, remove label and weight them individually
 Remove the contents and weigh the empty containers individually.
 Take difference of full and empty container for getting contents.
 Take average of 10 tablets and its weight should not be less than labelled amount.
Acceptance/rejection criteria:
In 2 stages, in S1 select 6 units if not meet criteria then on S2 select 20 more units and total units is 30.
Sample size S1= for 10 units S2=for 30
For ≤60g Net wt of contents of any single unit Net wt of contents of not more than 3
should not be less than 90% of the units should be less than 90% of labelled
labelled amount. amount.
For 60-150g Net wt of contents of any single unit Net wt of contents of not more than 1
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should not less than 95% of the unit should be less than 95% of labelled
labelled amount. amount.

3. Particle size determination:

 Dilute a specific quantity of sample with equal volume of glycerol or liquid paraffin or specified
in monograph.
 Mount the diluted sample on slide and examine random fields microscopically using microscope
providing adequate resolution for observation of small particles.
 Count the number of particles with diameter above or below than that specified in monograph.
 Compare the percentage with official limits.
Acceptance rejection criteria:
88

Diameter of particles ≥10µm ≥ 25µm ≥ 50µm

No. Of particles Not more than 12/ml Not more than 5/ml Not more than 2/ml
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4. Microbial content test:
 Except ophthalmic preparations, tropical applications are not required to be sterile.
 They must meet acceptable standards for microbial content.
 Microbial limits are stated for certain articles in USP.
Example:
 Betamethasone valerate ointment, must clear the test for absence of Staphyloccus aureous and
Pseudomonas aeruginosa.
 These microbes effect the skin and the ointment also used for the skin condition is already
compromised.
5. Metal particle test:
 This test is required only for ophthalmic ointments. It is performed using 10 ointment tubes.
 The content from each tube is completely removed onto a clean 60 - mm - diameter petri dish which
possesses a fl at bottom
 The lid is closed and the product is heated at 85 ° C for 2 h. Once the product is melted and distributed
uniformly, it is cooled to room temperature. The lid is removed after solidification.
 The bottom surface is then viewed through an optical microscope at 30 magnification. The viewing
surface is illuminated using an external light source positioned at 45° on the top.
 The entire bottom surface of the ointment is examined, and the number of particles are 50µm or above
counted using a calibrated eyepiece micrometer.
Acceptance rejection criteria:
 The number of such particles in 10 tubes should not exceed 50, with not more than 8 particles in any
individual tube.
 If these limits are not met, the test is repeated with an additional 20 tubes.
 In this case, the total number of particles in 30 tubes should not exceed 150, and not more than 3 tubes
are allowed to contain more than 8 particles.
6. Sterility test:
 Applied to the products that required to be sterile such as ophthalmic preparations.
 Ophthalmic semisolids should be free from anaerobic and aerobic bacteria and fungi.
Methods:
i. Direct inoculation method
ii. Membrane filtration method
i. Membrane filtration method:
 In the membrane filtration method, a solution of test product (1%) is prepared in isopropyl
myristate and allowed to penetrate through cellulose nitrate filter with pore size less than0.45µm.
 If necessary, gradual suction or pressure is applied to aid filtration.
 The membrane is then washed three times with 100 - mL quantities of sterile diluting and rinsing
fluid and transferred aseptically into fluid thioglycolate (FTG) and soybean – casein digest
(SBCD) medium.
 The membrane is finally incubated for 14 days.
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 Growth on FTG medium indicates the presence of anaerobic and aerobic bacteria, and
SBCD medium indicates fungi and aerobic bacteria.
 Absence of any growth in both these media establishes the sterility of the product.
ii. Direct inoculation method:
 In the direct - inoculation technique, 1 part of the product is diluted with 10 parts of sterile
diluting and rinsing fluid with the help of an emulsifying agent.
 Incubated in FTG and SBCD media for 14 days.
Criteria:
In both techniques, the number of test articles is based on the batch size of the product. If the batch
size is less than 200 the containers, either 5% of the containers or 2 containers (whichever is
greater) are used. If the batch size is more than 200, 10 containers are used for sterility testing.
Acceptance rejection criteria:
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 If no evidence of microbial growth, sample is sterile.

 If evidence of microbial growth, sample is non sterile.
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7. Potency/content uniformity test:
In this test the different analytical techniques are used.
 Assay the 10 units individually, if test failed 20 more containers are tested.
 Drug assay is performed by using different analytical techniques e.g. titrimetric assay.
 Conduct the assay on the amount of the material that drains from the individual container.
 Adjust the degree of the dilution so that the concentration of the active ingredient in the final solution
is of same order as that obtained in the assay procedure.
Acceptance rejection criteria:
For 10 doses unit acceptance criteria:
 Not more than 1 individual content, is outside the limit of 85%-115% of average labelled amount.
 Not a single unit should be outside the limit of 75%-125% of average labelled amount.
Rejection criteria:
 More than 3 individual content is outside the limit of 85%-115% of average labelled amount or
 One or more is outside the limit of 75%-125% of average labelled amount.
If this step reject then take 20 more units and tested individually.
For 30 unit acceptance criteria:
 Not more than 3 units are outside the 85%-115% of average labelled amount
 No one is outside the 75%-125% of average content.
8. Viscosity:
 Viscosity is a property of liquids that is closely related to resistance to flow.
 Force required to moves one plane surface continuously past another under specified conditions.
 Basic unit is poise=100 centipoises.
 Viscosity of the water as a reference material and all viscosities compare with this standard.
 Specifying of temperature is more important because viscosity decreases as temperature raised.
 Viscosity take at 20 degree centigrade, and for water is 1.
Measurement of viscosity:
Many capillary tube viscometers are used.
i. Ostwald viscometers
ii. Ubbelohde viscometers

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(i) (ii)

9. Leakage test:
 This test is mandatory for ophthalmic ointments, which evaluates the intactness of the ointment
tube and its seal.
 Ten sealed containers are selected, and their exterior surfaces are cleaned.
 They are horizontally placed over absorbent blotting paper and maintained at 60± 3 ° C for 8 h.
90

Acceptance rejection criteria:

 The test passes if leakage is not observed from any tube.
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 If leakage is observed, the test is repeated with an additional 20 tubes.

 The test passes if not more than 1 tube shows leakage out of 30 tubes.
10. Homogenicity:
Content of ointment is placed on glass slab and spread in form of thin layer. And then observe under
light source.
Acceptance criteria:
Ointment is homogenous if there are no granules present.

9.7. ASH CONTENT DETERMINATION:

Definition:
Ash content is measure of total amount of minerals present in a product.
Mineral Content:
It is the measure of total amount of specific inorganic compounds present in a product e.g Ca, Na, Cl, Mg, Cu,
Mn, Zn etx
Determination Methods BP:
There are two methods:
1. Acid insoluble ash value
2. Determination of sulphated ash
1. Acid insoluble ash value:
Method 1:
 Ash content is boiled with 25ml Hcl (1:2:5) for 5 min in water bath,covering the dish with watch
glass.
 It is then filtered through ashless filter paper No.40.
 The residue is washed with water until free of acid.
 It is then ignited at 600˚C for 20min.
 It is then cooled and weighed.
Method 2:
 Ash insoluble in Hydrochloric acids the residue obtained after extracting the Sulfated or Total ash
with Hydrochloric acid, calculated with reference to 100g of drug.
 To the crucible containing the residue from the determination of Sulfated or Total ash, add 15ml of
water and 10ml of Hydrochloric acid, cover with a watch glass, boil the mixture gently for 10min and
allow to cool.
 Filter through an ashless filter.
 Wash the residue with hot water until the filtrate is neutral, dry, ignite to dull redness, allow to cool it
in a dessicator and weigh.
 Reheat until the difference between 2 consecutive weighing is not more than 1mg.
2. Sulphated –Ash value:
Method 1:
 Heat platinum dish to redness for 10min.
 Allow to cool in a dessicator and weigh.
 Unless otherwise specified in the monograph, place 1g of substance in dish moisten with Sulphuric
acid.
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 Ignite gently.
 Again moisten with Sulphuric acid, again ignite at 800˚c.
 Cool, weigh, ignite for 15min and repeat this procedure until two successive weighing do not
differ by more than 0.5mg.
Method 2:
 Ignite suitable crucible at 600± 50℃ for 30min.
 Cool in a dessicator.
 Place sample in crucible n weigh.
 Moisten with sulphuric acid usually (1ml).
 Heat gently at low temperature.
 Cool, again moisten, heat until white fumes no longer evolved.
 Then at 600±50℃ until residue completely incinerated.
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 Cool in a dessicator, weigh and calculate %age of residue.

 If amount of residue exceed the prescribed limit, repeat the procedure.
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9.8. ALKALINITY OF GLASS
“It is measure of ability of solution to neutralize acids to equivalence point of carbonates & bicarbonates”.
 Alkalinity is basically/mainly due to carbonates due to its common presence in atmosphere.
 It may also be due to borates, hydroxides, silicates & sulfides, etc.
 Unit of alkalinity is mEq/L.
 Commercially, ppm is used.
How Alkalinity of Glass Occurs?
 Glass contains sodium & potassium oxides which are hygroscopic & absorbs the moisture from air. This
causes the oxides to get converted to sodium & potassium carbonates. These are also hygroscopic.
 In sea water, Na & K carbonates in unstable glass may leach out leaving fragile, cracked, flaked glass
with frosty appearance.
Treatment of Unstable Glass:
They can be treated in different ways but the one is as follows:
 Wash the glass in running tap water.
 Soak in distilled water.
 Dry in 2 baths of alcohol (This prevents disintegration, breakdown of glass & improves appearance).
 To impede (delay) disintegration, apply organic lacquer.
 Store in environment of humidity not higher than 40%.
Types of Glass:

Type General Description Type of Test

I Highly resistant, Borosilicate glass Powdered glass test
II Treated Soda-lime glass Water attack test
III Soda-lime glass Powdered glass test
IV or O General purpose soda-lime Powdered glass test

These are classified by Pharmacopoeial preparations according to the resistance to chemical attack. Types I, II
& III are for parenteral use While types O is for non-parenteral use.
Type of Glass Test
1. Crushed Glass Test:
 This test is official in USP.
 Container may be crushed & sieved to produce definite particles.
 The control of particle size & weight of powder ensures constant surface area exposed to solution.
 All of glass is examined & extraction is enhanced by rough surface of particles.
 This test can be used to determine nature of glass.
2. Whole Container Test:
 This test is official in European, British & International Pharmacopoeias.
 In USP, used only for Soda-lime glass.
 Containers are filled with test solution & exposed to the test conditions.
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 Glass wares may pass this test more easily because surface layer is smooth & Creative.
3. Chemical Resistance of Test:
Equipments used in this test should be of high quality & area should be from fumes & dust.
Apparatus:
 Auto clave (Temp. 121 ± 0.5)
 Mortar & pestle
 8 inch sieves (20, 40)
Reagents:
 Special distilled water
 Methyl red solution
4. Powdered Glass Test:
 This test is official in USP & IP.
 In this test, alkalinity as well as other glass constituents are measured.
92

Procedure:
 Rinse with water (6 or more containers)
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 Dry in air
  Crush (25mm in size)
 Divide in 3 portions & place 1 in the mortar.
 Empty mortar in sieve no. 20 & repeat the same procedure for the remaining two portions.
 Then pass through the sieve no. 40 & empty in the mechanical shaker for 5 minutes.
 Spread sample on glazed paper & pass the magnet over the material to eradicate the iron particles of
sieves if present.
 Then transfer it in conical flask & wash.
 Dry for 20 minutes at 140 degrees.
 Use test solution within 48 hours.
5. Water Attack Test:
 This test is used for determining alkali leaching out from surface of container.
 Mainly based on the alkali released from glass under influence of medium used.
 The amount of acid to neutralize released alkali from surface is estimated.
 Methyl red is used to determine end point.

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 94
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 95
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 ALKALOIDAL DRUG ASSAY

Alkaloids
Alkaloids are the organic compounds normally with basic chemical properties and usually containing at least
one nitrogen atom in a heterocyclic ring, occurring chiefly in many vascular plants and some fungi; having
marked physiological effects on humans or animals. Many alkaloids, such as nicotine, quinine, cocaine, and
morphine, are known for their poisonous or medicinal attributes.
Assay
The determination of the activity, potency, strength, etc. of a substance, either on an absolute basis or in
comparison with that of a standard preparation.
OR
Qualitative or quantitative analysis of a substance, especially of a drug, to determine its components.
OR
Determination of the amount of a particular constituent of a mixture, or of the potency of a drug.

10.1. ALKALOIDAL DRUG ASSAY

The assay of the drugs containing alkaloids is called “Alkaloidal Drug Assay”.
Examples
Alkaloidal Drug Brand
Morphine sulphate MS CONTIN® KADIAN®
Epinephrine ADRENALIN®
Hyoscyamine HYOSPAZ®
Atropine ATROPISOL®
Papaverine PARA-TIME® SR
Codeine DIHISTINE®
Reserpine SERPASIL®
Theophylline ASMALIX®
Caffeine Present in PANADOL EXTRA®

Introduction
 Alkaloids are slightly or very slightly soluble in water.
 Alkaloids are soluble in certain organic solvents immiscible with water e.g. chloroform.
 Salts of alkaloids are soluble in water but almost insoluble in organic solvents.
 The process of assay is carried out by treating the drug with a solvent immiscible with water in the presence
of excess of alkali that liberates the alkaloid.

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The free alkaloid is dissolved by the immiscible solvent from which it is removed by means of excess
of dilute acid.
 The acid solution is then extracted with an immiscible solvent in the presence of alkali.
 The immiscible solvent is then evaporated to obtain the alkaloid which is either weighed or
determined volumetrically.
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Scheme
Alkaloid Salt + Alkali


Alkaloid base (free) in immiscible solvent (Chloroform)

+
Dilute
 Acid


Acid alkaloidal salt in aqueous solution

+
Alkali

Alkaloid base (free) in immiscible solvent (Chloroform)

Chloroform is evaporated giving free alkaloid

+
Excess dilute acid of known normality

Alkaloid salt; the excess acid back titrated against standard alkali.

Preparation of Drug for Assay

 Grind the drug to be extracted to a powder of fineness grade.
 Care should be taken to avoid the loss of water.
Weighing for Assay
a. Weighing of bulky crude drugs
In weighing bulky crude drugs for the assay, accuracy to within 10 mg for quantities of 5 gm and over is
sufficient.
b. Portions of soft extracts or ointments
These may be weighed on a tarred piece of wax paper and transferred to the vessel containing the solvent
for extraction.
Extraction of drugs
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The alkaloidal content of alkaloid-bearing drugs is usually extracted by one of the following methods.
1. Maceration
2. Percolation
3. Continuous Extraction
1. Maceration
Introduction
 The term maceration comes from the Latin ‘macerare’ which means ‘to soak’.
 It is a process in which the properly crushed drug is permitted to soak in the menstrum until the
cellular structure is softened and penetrated by the menstrum.
 By this method, nearly all the soluble contents are dissolved in the menstrum.
Menstrum: A solvent, especially one used in extracting compounds from plant and animal
tissues and preparing drugs.
 Maceration is different from water based infusions and decoctions in following respects:
97

o The menstrum is usually alcohol.

o The herb remains in the menstruum for a longer period of time.
Page

o The process is conducted at ordinary temperature.

 Procedure
Method 1
 The drug to be extracted is placed in a wide-mouth container containing certain menstruum.
 The vessel is closed tightly to prevent the loss of menstruum.
 The contents are shaken/agitated repeated (preferably on daily basis) for a period of 2 to 14 days.
 The agitation allows the repeated flow of fresh menstrum over the entire surface area of soaked
comminuted drug.
 After the specific time, the liquid is drained from the mark. The mark is then pressed to retrieve more
of the menstruum.
 The expressed liquid is mixed with the strained liquid and the mixture is left to stand until it is clear,
after which it is filtered.
Method 2
 Place the drug in a porous cloth bag that is tied and suspended in the upper portion of menstruum.
(Just like a teabag).
 As the soluble contents dissolve in the menstruum, they tend to settle to the bottom because of an
increase in the specific gravity of the liquid.
 The extractive is separated from the marc by expressing the bag of drug and washing it with additional
menstruum.
 The menstruum is then filtered.
Specifications for Maceration
 Maceration is usually conducted at a temperature between 15 to 20 ° C.
 Duration is from minimum 2 days to maximum 14 days.
 Wide mouth vessel is used.
 Mouth of vessel is stoppered tightly.
 Contents are agitated repeatedly.
Examples:
 Benzoin
 Aloe
 Tolu
2. Percolation
Introduction
The term percolation is derived from the Latin ‘per’ meaning ‘through’ and ‘colare’ meaning ‘to strain’.
So
“It is a process in which a comminuted drug is extracted of its soluble constituents by a slow passage of a
suitable solvent through a column of the drug”.
According to USP
“Percolation consists in subjecting a comminuted substance or a mixture of substances contained in a vessel
called a percolator, to the solvent action of a liquid termed as menstruum in such a manner that the liquid
shall extract the soluble constituents and pass from the percolator”.
Procedure
 The comminuted drug is packed in a special extraction apparatus termed as percolator by packing
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the outlet with some porous material e.g. cotton.

 Saturate the drug with specified solvent and allow standing for 5 minutes.
 Add some ammonia sufficient to make the mixture distinctly alkaline and mix thoroughly with
drug.
 From this percolator, the suitable solvent(menstruum) is passed through slowly which dissolves
the soluble constituents of the comminuted drug.
 Allow the drug to macerate for about 10 to 12 hours.
 That menstruum is now called “Percolate” and is collected from the bottom of percolator until the
drug is completely exhausted of its alkaloidal content.
 The flow of the menstruum is generally downwards due to gravity.
 In some specialized and more sophisticated percolation apparatus, additional pressure is exerted
98

on the column with positive air pressure at the inlet and suction at the outlet.
Determination of the completeness of extraction of alkaloid
 Take about 4 ml of the last percolate.
Page

 Evaporate it to dryness.
  Dissolve the residue in 0.5 ml of 0.5 N acid.
 Add a drop of mercuric iodide(Valser’s Reagent)
 A slight turbidity is produced.
Percolators
 Percolators employed on large scale industrial preparations are generally made up of stainless steel or
glass lined metal vessels and vary greatly in size & operation.
 For example percolators used to extract from leaves may be 6 to 8 feet in diameter and 12 to 18 feet
high.
 Percolators used on small scale are usually made up of glass.
Shapes
 Cylindrical
 Roundish
 Conical
 Funnel shaped

3. Continuous Extraction
Procedure
 Moisten the drug with a specified solvent.
 Allow to stand for 5 mins.
 Make the mixture alkaline using ammonia and mix
thoroughly.
 Allow the drug to macerate for 6-12 hrs.
 Then pack the drug in the thimble and cover it with a
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pledge of cotton.
 Take the thimble and insert it into a suitable extractor
e.g. Soxhlet extractor.
 Add sufficient quantity of solvent and extract the drug.
99
Page
10.2. PURIFICATION OF ALKALOIDS
 The alkaloidal solution obtained by any of the extraction methods is usually contaminated with other
extractives which interfere with the quantitative determination of alkaloids.
 For effective purification, remove the alkaloids from the immiscible solvent by shaking out with an acid.
 Then make the acidic solution alkaline with an alkali hydroxide and extract with an immiscible solvent.
 The volume & strength of the acid vary case to case. However total volume should be as small as possible.
 Shake the combined acid extracts with one or more 10 ml portions of the appropriate immiscible solvent
until the acid solution is clear. Then wash the immiscible solvent extracts with one or more 5 ml portion of
water acidified with HCl or H2SO4 and add these washings to the acid solution.
 Then make the acid solution alkaline with ammonia and extract it with some immiscible solvent. Repeat
the operation as long as any alkaloid is extracted by the immiscible solvent. The completeness of the
extraction can be tested by mercuric iodide TS.
 In all assays, continues the extraction until 0.5 ml of the last acid washing shows a very slight turbidity on
the addition of a drop of mercuric iodide.

10.3. DETERMINATION OF ALKALOIDS

1. Evaporate the solution of the purified alkaloids in the immiscible solvent to dryness on a steam bath or
with a current of air.
2. Soften the alkaloidal residue by addition of about 1 ml of the neutralized alcohol or ether.
3. Add an accurately measured volume of standard acid.
4. Warm the mixture gently.
5. Dissolve the alkaloidal residue in chloroform and add standard acid of higher normality.
6. Remove the chloroform completely by evaporation.
7. The add water (q.s) to make the volume of mixture at least 25 ml.
8. Titrate the excess of the acid with standard alkali.
9. Dry the alkaloidal residue at 105 °C to a constant weight.

10.4. ESTIMATION OF ALKALOIDS

Following tests are used to detect the presence of alkaloids:
1. Mayer’s Test:
It gives white or yellow ppt except with alkaloids of purine group.
2. Dragendroff’s Test
Orange colour ppt formed.
3. Wagner’s Test
Brown or reddish brown ppt.
4. Hagger’s Test
Characteristics crystalline ppt.
5. Tannic Acid Test
Freshly prepared tannic acid solution gives ppt which is insoluble in dilute acid.
6. Melting Range Test
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Every alkaloid has specific melting range like Atropine has 114-118 oC.

10.5. MACERATION & PERCOLATION

Maceration Percolation
Involves just soaking Involve just rinsing
Material to be extracted remain in Solvent moves through the material to
solvent be extracted
Slow process Faster process
May take weeks Can be completed within day
May be better when using fresh Percolation extracts more when using
100

plants dried plants

Requires pressing the plant material It does not require pressing
after soaking
Page
 101 Page
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 GENERAL KNOWLEDGE OF APPENDICES

PHARMACOPOEIA
The term Pharmacopoeia is derived from Greek words “pharmakon” means “Drug” and “poien” means
“Make”.
It is Official collection of approved pharmaceutical standards.
Definition:
The books containing the standards for drugs and other related substances are known as Pharmacopoeias and
Formularies. Collectively these books are known as Drug Compendia.
 The pharmacopoeias contain a list of drugs and other related substances regarding their source,
descriptions, tests, formulas for preparing the same, action and uses, doses, storage conditions etc.
 These books are prepared under the authority of the government of the respective countries.
Classification of Drug Compendia:
There are 2 types of drug compendia
1. Official compendia
2. Non official compendia
7. Official Compendia:
Official compendia are the compilations of drugs and other related substances which are recognized as
legal standards of purity, quality and strength by a government of respective countries.
These include:
i. British pharmacopoeia
ii. British pharmaceutical codex
iii. Indian pharmacopoeia
iv. United states pharmacopoeia
v. National formulary
vi. International pharmacopoeia
8. Non Official Compendia :
The books other than official drug compendia which are used as secondary reference sources for drugs
and other related substances.
These include
i. Merck index
ii. Remington’s pharmaceutical sciences
iii. Extra pharmacopoeia ( Martindale )
Significance of Pharmacopoeia:
 Provides requirements on the quality of medicinal products, of the substances used to manufacture them.
 Provides quality control methods.
 Important component of drug safety system
 Support the availability of safe, effective, good quality pharmaceutical care for all.
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 Ensure public health.

11.1 BRITISH PHARMACOPOEIA

The British Pharmacopoeia (BP) is an annual published collection of quality standards for UK medicinal
substances. It is used by individuals and organizations involved in pharmaceutical research, development,
manufacture and testing.
History of BP:
It was first published in 1864 and prepared by Pharmacopoeia Commission. Since 1948 the new edition
of BP is published at interval of five years. After 2008 the new edition is published every year.
The new edition of British pharmacopoeia which is released in august of 1 year becomes official on 1st
102

January of next year.

Volumes of BP:
Volume I Monographs of medicinal & pharmaceutical substances (A-I)
Volume II Monographs of medicinal & pharmaceutical substances (J-Z)
Page

Volume III General & specific monographs of formulated preparations

 Blood related products
Immunological products
Radiopharmaceuticals
Surgical materials
Herbal drugs and herbal drug preparations, herbal medicinal-products
Materials used in homeopathic preparations
Volume IV Infrared Reference Spectra, Appendices, Supplementry chapters and Index
Volume V British Pharmacopoeia Veterinary
Volume VI British approved names (BAN), CD ROM version
General Notice:
Genral notices has three parts:
1. Part I
2. Part II
3. Part III
1. Part I:
It consist of information about the monographs of European Pharmacopoeia which are reproduce in this
edition of British Pharmacopoia.
They are included for the convinence of users of British Pharmacopoia.
Monographs of the European Pharmacopoeia are distinguished by a chaplet of stars against the title and
by reference to the European Pharmacopoeia monograph number included immediately below the title in
italics.
The beginning and end of text from the Europea Pharmacopoeia are denoted by means of horizontal lines
with the symbol 'Ph Eur' ranged left and right, respectively.
2. Part II:
It consists of explanation of different terms like expression of standards, temperature, weights &
measures, atomic weight, constant weight, water bath, reagents etc
3. Part III:
It consists information about additions, ommision, technical changes, changes in title, reference
substances included in current BP edition.
Appendices:
The word appendix is derived from Latin word “appendere” meaning to hang upon.
Definition:
The section at the end of a book that gives additional information on the topic explored in the contents of
texts”.
 In BP there are twenty five appendices represented by roman numerals as I, II.............XXV.
 Each appendix is further subdivided alphabetically as IA, IB, IC etc.
 The detail of each appendix are as follows:
Appendix I:
It consistsof information about the reagents, solutions and reference materials.
Appendix II:
It has information about the different types of spectrophotometry as:
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Infrared spectrophotometry, NMR spectrophotometry, Ultraviolet and visible spectrophotometry etc

Appendix III:
It includes different types of chromatographic separation techniques like:TLC,gas chromatography,size
exclusion chromatography etc.
Appendix IV:
It contains information about clarity and color of solutions.
Appendix V:
It consists of information of determination of physical parameters like boiling point,melting
point.distillation,freezing point etc
Appendix VI:
It consist of qualitative reactions tests.
103

Appendix VII:
It consists of information of:
 Nesseler cylinders
 Tubes for comparative tests
Page

 Limit test for different elements

Appendix VIII:
It have different types of titration methods and methods of determinition of ethanol, methanol, nitrogen etc
Appendix IX:
It has methods of determination of sulphated ash, SO2, water, loss on drying, water and oxygen in
medicinal gases etc.
Appendix X:
It has values of different functional group like acetyl value, iodine value, acid value etc.
Appendix XI:
It consist information about total solids, ash, stomata, swelling index etc Appendix XII:
It has three subparts as
A. Disintegration test
B. Dissolution test
C. Consistency of formulated praparationsl products
Appendix XIII:
It has test of particulate contamination for visible and subvisible particles
Appendix XIV:
It consists of biological assays & tests, immunologic products etc
Appendix XV:
It consist of information about production and testing of vaccines etc
Appendix XVI:
It consist of test of sterility and microbilogical examination of non-sterile products
Appendix XVII:
It consists of particle size of powders, sieves and filters sizes, flowability, frability and resistance to crushing
of tablets etc
Appndix XVIII:
It contains methods of sterilisation.
Appendix XIX:
It consist of information about different type of containers & closures.
Appendix XX:
It consist of materials used in the manufacturing of containers.
Appendix XXI:
It consist of:
A. Abbreviated titles
B. Approved synonyms
C. Codes for eye drops in single dose containers
Appendix XXII:
It has methods of viral safety.
Appendix XXIII:
It consist of International system of units.
Apendix XXIV:
It has tables of abbreviations.
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Appendix XXV:
It contains names, symbols and atomic weight of elements.
Supplementry Chapters:
 Supplementry chapters contain no standards, tests, or assays nor any other mandatory specifications
with respect to any pharmacopoeial article.
 They comprises explanatory and other ancillary texts and are provided for the assistance and
information of users of pharmacopoeia.
 There are seven supplementary chapters represented by roman numerals as I-VII. Each chapter
is subdivided by alphabetically.
Index:
Index is an alphabetically arranged list of items (such as names or terms) given at the end of a printed text
104

with page number on which the item can be found.

Page
11.2. BRITISH PHARMACEUTICAL CODEX
British Pharmaceutical Codex is a reference book which is used to provide drugs related information to
medical practitioners and dispensing pharmacist.
History:
In 1903 the Council of Pharmaceutical Society of Great Britain decided to produce a reference book that provide
authoritative guidance to those who are engaged in prescribing and dispensing of medicines throughout British
Empire. As a result the first edition of BPC was published in 1907 by the ROYAL PHARMACEUTICAL
SOCITY OF GREAT BRITAIN.
How BPC Differ From BP?
 BPC contains many more drugs and preparations, which have been obsolete by BP but are retained in
codex.
 BPC provides standards for drugs, surgical dressings and pharmaceutical preparations which are not
present in BP.
 BPC provides information on the action, uses of drug, undesirable effects and precautions.
 BPC contains formulae, methods of preparations and storage conditions of preparation which are not
included in B P.
British Pharmacopoeia
In 1972, the MEDICINE COMMISSION recommended that there should be only one compendium of standards
for all medicines used in the United Kingdom and that is BRITISH PHARMACOPOEIA therefore the provision
of standards in BPC was discontinued.
The Pharmaceutical Codex 11th Edition:
In 1979, a major reconstruction of BPC was undertaken and as a result a new edition was published under the
title THE PHARMACEUTICAL CODEX in this compendium all the drug information was arranged in
encyclopaedic style.
The Pharmaceutical Codex 12th Edition:
In 1994, the 12th edition was published by Royal Pharmaceutical Society of Great Britain and retitled as: THE
PHARMACEUTICAL CODEX THE PRINCIPLES AND PRACTICE OF PHARMACEUTICS.
This CODEX provides a reference source on those aspects of pharmaceutical science and technology that are
applied in development and provision of therapeutically active dosage form.
Contents:
1. Part I
2. Part II
1. PART 1:
Preparation and presentation of drugs as medicine
SECTION 1 Dosage Form.
SECTION 2 Product Desige, Development and presentation.
SECTION 3 Preparation and Supply of Medicine.
SECTION 4 Pharmaceutical Microbiology, Sterile Processing and Contamination Control.
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SECTION 5 Electrolyte Replacement, Nutrient Fluids and Dialysis Solutions.

SECTION 6 Nomenclature and Miscellaneous Data.
2. PART II:
Monographs on 154 Drug Substances.

11.3. UNITED STATES PHARMACOPIEA –NATIONAL FOLMULARY

Introduction:
 The United States Pharmacopeia and The National Formulary (USP–NF) is a book of public
pharmacopeial standards.
 It contains standards for (chemical and biological drug substances, dosage forms, and compounded
preparations), excipients, medical devices, and dietary supplements.
105

History:
United States Pharmacopiea:
The United States Pharmacopoeia was originally published in 1820 under the authority of the United States
Pharmacopoeial Convention.
Page

National Formulary:
National formulary was published in 1888 under the guidance of American Pharmaceutical Association.
Combination of USP & NF:
 In 1974 the NF was purchased by the United States Pharmacopoeial Convention.
 There is difference of 5 editions between USP and NF e.g. If USP 30 then NF 25.
 From that time it is published as a single compendia.
 From 2000 it is released annually.
 The Edition is released on November 1 of each year, and becomes Official on May 1 of next year.
 USP–NF is a combination of two compendia, the United States Pharmacopeia (USP) and the National
Formulary (NF).
 Monographs for drug substances, dosage forms, and compounded preparations are featured in the USP.
 Monographs for dietary supplements and ingredients appear in a separate section of the USP. Excipient
monographs are in the NF.
Official Recognition:
 The U.S. Federal Food, Drug, and Cosmetics Act designates the USP–NF as official compendia for drugs
marketed in the United States.
 A drug product in the U.S. market must conform to the standards in USP–NF to avoid possible charges of
adulteration and misbranding.
 USP standards are used in more than 140 countries around the world including Pakistan.
Standard Establishment:
 USP creates and continuously revises USP–NF standards through a unique public-private collaborative
process, which involves pharmaceutical scientists in industry, academia, and government as well as other
interested parties from anywhere in the world.
 The standards generally originate from sponsors who provide draft standards and supporting data to either
create new or revise existing monographs and general chapters.
 USP's scientific staff and volunteer experts review this input, conduct laboratory tests (if necessary), and
forward the new or revised monograph or general chapter to Pharmacopeial Forum (PF) for public review
and comment (as described in below figure)

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106
Page
Contents Of USP-NF :
The major components of USP-NF are
1. General Notices
2. General Chapters
3. Official Monographs
1. General Notices :
 The General Notices provide definitions for terms used in the monographs, as well as information that
is necessary to interpret the monograph requirements.
 USP is proposing to revise the General Notices for the USP and NF. These are given at the start of
each volume.
2. General Chapters :
 Tests and procedures referred to in multiple monographs are described in detail in the USP–NF
general chapters.
3. Monographs :
 A monograph includes the name of the ingredient or preparation; the definition; packaging, storage,
and labeling requirements; and the specification.
 The specification consists of a series of tests, procedures for the tests, and acceptance criteria.
 These tests and procedures require the use of official USP Reference Standards.
 Medicinal ingredients and products will have the stipulated strength, quality, and purity if they
conform to the requirement the monograph and relevant general chapters.
VOLUMES OF USP-NF :
USP-NF has 3 volumes:
1. Volume 1
2. Volume2
3. Volume 3
4. First Supplement, USP-NF.
1. Volume 1
Mission Statement and Preface (v)
People (xi)
It include details about people who contributed in the peparation of Pharmacopeia
Preambles (xxiv)
It include articles of incorporation and importance of USP by law.
Admissions (xli)
It include Articles Admitted to USP, revisions appearing in USP, Changes in Official Titles.
Commentary (xliv)
It includes public and official comments about USP.
Notices
It include USP General Notices and Requirements. It includes:
 “Official” and “Official Articles”
 Atomic Weights and Chemical Formulas
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 Abbreviation
 Significant figures and Tolerances
 Pharmacopial Forum
 Supplements
 Reagent standard
 Refrence standards
 Unit of potency
 Ingredients of processing
 Test and Assays
 Dietry suplements
107

General chapters
It contain general tests and Assays.
 General requirements for tests and assays
 Apparatus for tests
Page

 Microbial tests
  Biological tests and Assays
 Chemical tests and Assays
 Physical tests and Assays
 General information
 Dietary Suplements
Reagents, indicators, solutions
It contains:
 Reagents
 Indicators and Indicator test Papers
 Solutions
Reference Tables
 Containers f or dispensing
 Describtion and relative solubility of USP& NF
 Articles
 Atomic weights
 Alcholometric table
 Intrinsic viscosity table
 Thermometric Equivalent
Dietary Supplements
 Official monographs
NF 25
Admissions
Articles Admitted to NF 25
Excipients
USP and NF excipients listed by category
Notices
NF General Notices and Requirements
Monographs
Official monographs for NF 25
Index
Combined USP index to USP 30 and NF25
2. Volume 2
Guide to General Chapters
Same as that of volume 1
Notices
Same as that of Volume 1
USP 30:
Official Monographs (A-L)
Index
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Combined index to USP30 and NF25

3. Volume 3
Guide to General Chapters
Same as that of volume 1
Notices
Same as that of volume 1
USP 30:
Official Monographs (M-Z)
Index
Combined index to USP 30 and NF 25
4. First Supplement, USP-NF.
108

People
Admissions
Notices
Page

General Chapters
Reagents
Reference tables
Dietary Supplements (Official monographs)
Excipients
Monographs for NF 25 (New monographs admitted to NF 25)
Monograph for USP 30 (New monographs Admitted to USP 30)
Index

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109
Page
 110 Page
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 111 Page
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 STATISTICAL QUALITY CONTROL

Statistic:
The collection, organization and interpretation of data.
12.1. STATISTICAL QUALITY CONTROL
Statistical quality control is the term use to describe the set of statistical tools used by quality professionals to
evaluate organization quality.
Statistical quality control can be divided into three broad categories.
1. Descriptive statistics
2. Statistical process control
3. Acceptance sampling
1. Descriptive statistics:
Statistics used to describe quality characteristic and relationships, included are statistics such as the mean,
Standard deviation, the range, and a measure of the distribution of data.
a) Mean (Average)
 The arithmetic average, or the mean, is a statistic that measures of central tendency of a data set.
 To compute the mean, simple sum all the observations and divide by the total number of
observations.
 The equation for computing the mean is
∑ 𝑥𝑖
𝑥̅ =
𝑛
Where
𝑥̅ = the mean
xi = observation i, 1, 2, 3,……., n
n = number of observations.
b) Range
It is the difference between the largest and smallest observant ions in data set.
c) Standard Deviation
 It is a statistic that measures the amount of data dispersion around the mean.
 It is a measure of average spread around the mean.
 The equation for computing the standard deviation is

Where
S = standard deviation of a sample
X = the mean
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Xk = observation i, I 1, 2, 3,……., n
n = number of observations in the sample.
112

d) Distribution Of Data
Page

A descriptive statistic used to measure quality characteristics is the shape of the distribution of the
observed data.
 i. Symmetric distribution
When a distribution is symmetric, there is the same number of observations blew or above the
mean. This is what we commonly find when only normal variation is present in the data.
ii. Skewness distribution
When a disproportionate number of observations are either above or below the mean, the data has
a skewed distribution.
𝑆𝑘𝑒𝑤𝑛𝑒𝑠𝑠 = (𝑚𝑒𝑎𝑛 – 𝑚𝑒𝑑𝑖𝑎𝑛) / 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛

Positive skewness
If the data is skewed toward right than it’s called positive skewness. A long right tail in graph.
Mean > Median / Mode = Positive Skeweness
Negative skewness
If the data is skewed toward left than it’s called negative skewness. A long left tail in graph.
Mean < Median / Mode = Negative Skeweness

iii. Kurtosis:
Kurtosis provides the visual estimation of variance in a sample. It is a measure whether the data is
peak or flat related to the normal distribution.
Leptokurtic
Kurtosis value greater than two is leptokurtic. It’s sharper than the normal distribution; values are
concentrated around the mean and little variance.
Platykurtic
Kurtosis for the negative number more than -1 is called platykurtic distribution. It’s flatter than the
normal distribution; values are spread out wider from the mean and greater variance.
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2. Statistical process control (SPC)

Involves inspecting a random sample of the output from a process and deciding whether the process is
producing products with characteristics that fall within a predetermined range. SPC answers the
113

question of whether the process is functioning properly or not.

3. Acceptance sampling
Is the process of randomly inspecting a sample of goods and deciding whether to accept the entire lot based
on the results. Acceptance sampling determines whether a batch of goods should be accepted or rejected.
Page
12.2. CONTROL CHARTS
A control chart (also called a process chart, shewart or quality control chart) is a graph that shows whether a
sample of data falls within the common or normal range of variation.
The most common used tool for monitoring the process is a control chart.
The standard deviation decreases as the process become more capable.
12.2.1. COMPONENTS OF CONTROL CHART:
There are five components of control chart:
1. X-Axis
The x-axis represents sample, sequence or time.
2. Y-Axis
The Y-axis represents the quality characteristic that is being monitored i.e. weight of tablets, volume of
injection fills.
3. Control Line
The central line of the control chart is the mean, or average, of the quality characteristic that is being
measured.
4. Control Limit
i. Upper control limit (UCL)
Upper control limit is the maximum acceptable variation from the mean for a process that is in a state
of control.
UCL=mean+3*sigma /n (1/2)
ii. Lower control limit (LCL)
Lower control limit is the minimum acceptable variation from the mean for a process that is in a state
of control.
LCL=mean-3*sigma /n (1/2)
5. Data Points
 The upper and lower control limits on a control chart are usually set at ±3 standard deviation from the
mean. If we assume that the data exhibit a normal distribution, these control limits will capture
99.74min of the normal variation.
 Control limit can be set at ±2 standard deviation from the mean. In the case, control limits would
capture 95.44% of the values.
 Control limit can be set at ±1 standard deviation from the mean. In the case, control limits would
capture 68% of the values.

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12.2.2. IMPORTANCE OF QUALITY CONTROL CHARTS
1. Control charts are a proven technique for improving productivity.
2. Control charts are effective in defect prevention.
3. Determine what process adjustments need to be made.
4. Determine if process is “in” or “out” of control.
5. Provide the information about the process capability.
6. Control charts are used as aid in controlling and analyzing physically, chemically, analytical and biological
parameters of production.
i. Weight variation of tablets.
ii. Thickness of tablets.
iii. Volume of filled liquid in a container.
iv. The number of defective or %age of defective in parenteral products.
v. The number or fraction of defective in the sample of packages.
vi. Useful to highlight inter or intra batch variation

12.2.3. TYPES OF CONTROL CHARTS

Control charts can be divided into two groups:
1. Charts for variables
2. Charts for attributes
1. Control Charts For Variables
Control charts for variables monitor characteristics that can be monitored and have a continuous scale , such
as height, weight , volume, or width .
Control charts for variables are of following types:
i. X chart
ii. R chart
iii. S chart
iv. S2 chart
i. Mean (X Bar) Charts
 A mean control chart is often referred to as an x-bar chart. It is used to monitor changes in the mean
value of a process.
 In X – bar chart the sample means are plotted in order to control the mean value of a variable. (e.g.,
size of piston rings, strength of materials)
 Centre line and control limits are calculated as
𝑥̅ 1 + 𝑥̅ 2 + 𝑥̅ 3 + ⋯ + 𝑥̅ 𝑘
𝐶𝐿 = 𝑥̿ =
𝑘
𝑈𝐶𝐿 = 𝑥 + 𝑧 𝜎𝑥
𝐿𝐶𝐿 = 𝑥 − 𝑧𝜎𝑥
Where
k= no of sample mean
z= standard normal variation (2 for 95.44% confidence, 3 for 99.74% confidence)
σx= standard deviation of the distribution of sample means, computed as
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𝜎𝑥 = 𝜎/√𝑛
σ= population (process) standard deviation.
n= number of observations.
 Another way to construct the control limits when σ is not known is to use the sample range as an
estimate of the variability of the process.
 In this case control limit would be constructed as follow:
UCL= X+A2R
LCL= X-A2R
Where
X = average of sample mean
R = average range of samples
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A2 = factor for x bar chart

ii. Range (R) Chart:
R chart are another type of control chart for variables. Range chart monitor the dispersion or variability
of the process, where as x bar charts measure shift in the central tendency of the process.
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In this chart, the sample range are plotted in order to control the variability of a variable.
 The method for same as the developing and using R charts is that for x bar charts. The central line of the
control chart is the average range, and the upper and lower control limits are computer as follows,
CL = R
UCL = D4R
LCL = D3R
Where
R = average range
D4 and D3 = factor for R chart
iii. S Chart
In this chart, the sample standard deviations are plotted in order to control the variability of a variable.
iv. S2 Chart
In this chart, the sample variances are plotted in order to control the variation of a variable.
2. Control Charts For Attributes
 The control chart for attributes is used to monitor characteristics that have discrete values and can be
counted rather than measured. Often they can be evaluated with a simple yes or no decision.
 For quality characteristic that can discrete and involve yes/no or good/bad decision. e.g. colour, taste,
or smell of product.
 Control charts for attributes are of following types:
i. P chart
ii. C chart
iii. U chart
iv. Np chart
i. P- CHARTS
 Control chart that monitors the proportion of defects in a sample is called P chart. P chart are
appropriate when both number of defectives measured and the size of the total sample can be
counted.
 In this chart, we plot the percent of defectives (per batch, per day, per machine, etc.) as in the U
chart. However, the control limits in this chart are not based on the distribution of rare events but
rather on the binomial distribution (of proportions).
 Therefore, this chart is most applicable to situations where the occurrence of defectives is not rare
(e.g., we expect the percent of defectives to be more than 5% of the total number of units produced).
 The computation of the central line as well as upper and lower control limits is similar to the other
types of control charts.
 The central line is computed as the average proportion defective in the population.
 To construct the upper and lower control limits for a P chart, we use the following formulas,
CL = p

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UCL = P+zσP
LCL = P+zσP
Where
z = standard normal variable
p = the sample proportion defective
σP = standard deviation of the average proportion defective calculated as
σP = √p(1-p)/n
ii. C-CHARTS
 A control chart used to monitor the number of defects per unit.
 C chart used for discrete defects when there can be more than one defect per unit.
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 Unit may be time, space or distance. Examples are the number of returned meals in a
restaurant, the number of trucks that exceed their weight limit in a month.
 C chart count the actual number of defects. However, we cannot compute the proportion of
complaints. Control limits are calculated as
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CL = c
 UCL = c + z√c
LCL = c - z√c
Where
c = average number of defects
z = standard normal variable
iii. U chart
 In this chart we plot the rate of defectives, that is, the number of defectives divided by the number
of units inspected (the n; e.g., feet of pipe, number of batches).
 Unlike the C chart, this chart does not require a constant number of units, and it can be used, for
example, when the batches (samples) are of different sizes.
iv. Np chart
 In this chart, we plot the number of defectives (per batch, per day, per machine) as in the C chart.
 However, the control limits in this chart are not based on the distribution of rare events, but rather
on the binomial distribution.
 Therefore, this chart should be used if the occurrence of defectives is not rare (e.g., they occur in
more than 5% of the units inspected).
 For example, we may use this chart to control the number of units produced with minor flaws

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12.2.4. OUT OF CONTROL PROCESS

Zone A, B, C. Customarily, to define the runs tests, the area above and below the chart center line is
divided into three "zones."
By default, Zone A is defined as the area between 2 and 3 times sigma above and below the center
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line; Zone B is defined as the area between 1 and 2 times sigma, and Zone C is defined as the area between
the center line and 1 times sigma.
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 Out of control when the situation in which a plot of data falls outside present control limits.

 A plot of data reveals that one or more sample fall outside the control limits.
 9 points in zone C or beyond (on one side of central line)
 2 out of 3 points in a row in zone A or beyond
 4 out of 5 point in a row in zone B or beyond
 15 point in a row in zone C (above or below the central line)
 8 point in a row in zone B, A, or beyond, on either side of the center line (without points in zone C)
 6 point in a row steadily increasing or decreasing.
 14 point in a row alternating up and down.

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Chart Process observation Process Process Size of shift
observations observations to detect
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relationships type
and R chart Quality characteristic Independent Variables Large (≥ 1.5σ)
measurement within one
subgroup
and s chart Quality characteristic Independent Variables Large (≥ 1.5σ)
measurement within one
subgroup
p-chart Fraction nonconforming Independent Attributes† Large (≥ 1.5σ)
within one subgroup
np-chart Number nonconforming Independent Attributes† Large (≥ 1.5σ)
within one subgroup
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c-chart Number of Independent Attributes† Large (≥ 1.5σ)

nonconformances within
one subgroup
u-chart Nonconformances per Independent Attributes† Large (≥ 1.5σ)
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unit within one subgroup

12.2.5. COMPARISON B/W CONTROL CHARTS OF VARIABLES AND ATTRIBUTES
VARIABLES CONTROL CHARTS ATTRIBUTES CONTROL CHARTS
Monitor the quality characteristic that can be Monitor the quality characteristic that can be
measured counted rather than measured
Based on the continuous values Based on the discrete values
Uneconomical Economical
Less easily understandable More easily understandable
More sensitive or efficient Less sensitive or efficient

12.3. PROCESS CAPABILITY

 The ability of the production process to meet or exceed preset specifications, this is called the process
capability.
 Product specifications often called tolerances are present ranges of acceptable quality characteristics.
 The process capability involves evaluation process variability relative to preset product specification in
order to determine whether the process is capable of producing an acceptable product.
Measuring Process Capability
 Simple setting up control charts to monitor whether a process is in control does not guarantee process
capability.
 To produce an acceptable product, the process much be capable and in control before production begins.
 Process capability is measured by the process capability index, Cp, which is computed as the specification
width of the process variability:
𝒔𝒑𝒆𝒄𝒊𝒇𝒊𝒄𝒂𝒕𝒊𝒐𝒏 𝒘𝒊𝒅𝒕𝒉 𝑼𝑺𝑳 − 𝑳𝑺𝑳
𝑪𝒑 = =
𝒑𝒓𝒐𝒄𝒆𝒔𝒔 𝒘𝒊𝒅𝒕𝒉 𝟔𝝈
Where
Specification width is the difference between the upper specification limit (USL) and the lower
specification limit (LSL) of the process.
Process width is computed as 6 standard deviations (6σ) of the process being monitored.
 Another measure for the process capability is used more frequently:
𝑈𝑆𝐿 − µ 𝐿𝑆𝐿 − µ
𝐶𝑝𝑘 = 𝑚𝑖𝑛 ( , )
3𝜎 3𝜎
Where
µ = the mean of the process
σ = the standers deviation of the process
 To use this measure, the process capability of each half of the normal distribution is computed and the
minimum of the two is used.
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Interpretation Of Cp Value
There are three possible ranges of values for Cp that also helps us interpret its value:
Cp=1: A value of Cp equal to 1 means that the process variability just meets specifications, as in figure (a). We
could then say the process is minimally capable.
Cp <1: A value of Cp below 1 means that the process variability is outside the range of specification as in figure
(b). This means that the process is not capable of producing within the specification and the process much be
improved.
Cp > 1: A value of Cp above 1means that the process variability is tighter than specification and the process
exceed minimal capability, as in figure (c)

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 Factor for Computing Central Line and Three Sigma Limits
Observation A A2 D1 D2 D3 D4 A3 B3 B4 d2 c4
in Sample
(n)
2 2.121 1.880 0 3.686 0 3.267 2.659 0 3.267 1.128 0.7979
3 1.732 1.023 0 4.358 0 2.574 1.954 0 2.568 1.693 0.8862
4 1.500 0.729 0 4.698 0 2.282 1.628 0 2.266 2.059 0.9213
5 1.342 0.577 0 4.918 0 2.114 1.427 0 2.089 2.326 0.9400
6 1.225 0.483 0 5.078 0 2.004 1.287 0.030 1.970 2.534 0.9515
7 1.134 0.419 0.204 5.204 0.076 1.924 1.182 0.118 1.882 2.704 0.9594
8 1.061 0.373 0.388 5.306 0.136 1.864 1.099 0.185 1.815 2.847 0.9650
9 1.000 0.337 0.547 5.393 0.184 1.816 1.032 0.239 1.761 2.970 0.9693
10 0.949 0.308 0.687 5.469 0.223 1.777 0.975 0.284 1.716 3.078 0.9727
11 0.905 0.285 0.811 5.535 0.256 1.744 0.927 0.321 1.679 3.173 0.9754
12 0.866 0.266 0.922 5.594 0.283 1.717 0.886 0.354 1.646 3.258 0.9776
13 0.832 0.249 1.025 5.647 0.307 1.693 0.850 0.382 1.618 3.336 0.9794
14 0.802 0.235 1.118 5.696 0.328 1.672 0.817 0.406 1.594 3.407 0.9810
15 0.775 0.223 1.203 5.741 0.347 1.653 0.789 0.428 1.572 3.472 0.9823
16 0.750 0.212 1.282 5.782 0.363 1.637 0.763 0.448 1.552 3.532 0.9835
17 0.728 0.203 1.356 5.820 0.378 1.622 0.739 0.466 1.534 3.588 0.9845
18 0.071 0.194 1.424 5.856 0.391 1.608 0.718 0.482 1.518 3.640 0.9854
19 0.688 0.187 1.487 5.891 0.403 1.597 0.698 0.497 1.503 3.689 0.9862
20 0.671 0.180 1.549 5.921 0.415 1.585 0.680 0.510 1.490 3.735 0.9869
21 0.655 0.173 1.605 5.951 0.425 1.575 0.663 0.523 1.477 3.778 0.9876
22 0.640 0.167 1.659 5.979 0.434 1.566 0.647 0.534 1.466 3.819 0.9882
23 0.626 0.162 1.710 6.006 0.443 1.557 0.633 0.545 1.455 3.858 0.9887
24 0.612 0.157 1.759 6.031 0.451 1.548 0.619 0.555 1.445 3.895 0.9892
25 0.600 0.153 1.806 6.056 0.459 1.541 0.606 0.565 1.435 3.931 0.9896

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