Introduction to Pharmaceutical Analysis I

By: Yehualashet T. (BSc, MSc. Of pharmaceutical Analysis & Quality Assurance)

Pharmaceutical Chemistry Course and Research Unit

1
 Introduction to Pharmaceutical analysis

 It is a science which deals with identification, characterization

and quantification a given sample of pharmaceutical interest

and biological fluids .

 Or it is an applied science that ensures the safety, efficacy and

stability of pharmaceutical products by using different methods.

 It is also a technique that is used in elucidation of drug

entities from natural products.

2
 Cont.…
 A substance to be identified or quantified is called the analyte.
 The samples in pharmaceutical analysis are typically
Pharmaceutical raw materials,
Finished pharmaceutical products, or
Biological samples like human blood or urine containing
one or more drug substances.

3
 Why do we analyze
drug?
 It is one way (and often the only way) to judge
quality.
Any hazards associated with the drug can be
ascertained and the necessary correction made before
marketing.

4
 pharmaceutical analysis can answer questions with regard to:

 The Identity and purity of a pure drug substance meet

specification or not?
 Percentage amount of the stated API content in a
formulation?
 Does this formulation contain solely the active ingredient or
are additional impurities present?
 stability of a drug in the formulation
 Identity and purity of excipients meet specification?
 What are the physical constants like Pka value (s),
solubilities, etc of a drug substance under development?
 At what rate is the drug released from its formulation so that
it can be absorbed by the body
5
 Introduction to pharmaceutical
Scope (Where )?
 Pharmaceutical industry
 Raw material control, In-process control, Dosage form control
 Government drug control laboratory (regulatory agencies)
 Manufacture lab. Procedure control, Products control
 Pharmaceutical research labs
 Advanced research: Development of analytical method, and Process development, Dosage form
evaluation, Stability studies,
 Basic research: Separation, Identification , Quantization and Molecular
characterization

6
 Cont.….
 Therefore pharmaceutical analysis is one of applied chemistry that used
to quantitvely or qualitatively determine a given sample or
pharmaceutical interest.
 The following are questions answered by Pharmaceutical Analysis. State
whether they are qualitative analysis or quantitative analysis. Please try
to justify your answers

7
 Cont.….
• Is the identity of the drug in the formulated product correct?
• What is the percentage of the stated content of a drug present in a
formulation?
• Does this formulation contain solely the active ingredient or
are additional impurities present?
• What is the stability of a drug in the formulation and hence the shelf–
life of the product?
• At what rate is the drug released from its formulation so that it can be
absorbed by the body.

8
 Cont.….
 What to test? The drug product
 Why to test? Ensures the safety, efficacy and
quality of pharmaceutical products.
 How to test? compendial and non
compendial
methods presented in regulatory documents.
 Analytical method used to achieve Qualitative and
quantitative analysis:
 Classical method
 Physico-chemical method
 Micro-biological method
9
 1.1. Introduction to quality, quality control and
Quality Assurance

 What is pharmaceutical product?

It is also known as medicines or drugs which are special

preparations used to enhance personal health.
 What is the term quality?
 It is the totality of features and characteristics of a product
or service that bear on its ability to satisfy given needs.

10
 Cont.….
Pharmaceutical products have a significant role in
improving the quality of life.
by treating and preventing the disease condition.

Hence, the medication must be promoted as safe,

therapeutically active formulations with consistent
and predictable results.

11
 Cont...
In the UHC providing high product quality for the community
is a sustainable requirement in the health system.

However the possibility of spreading poor quality drug

is increase in alarming rate specially in middle east Africa.

This is may be due to the globalization of

different pharmaceutical industry.

12
 Cont...
 To get a better final outcomes, every pharmaceutical
products should be ; Safe,Effective and having a standard quality.
 This is because poor qualities of pharmaceutical product
may leads to
 Therapeutic flirty which may lead Prolonged illness or death
 Unwanted toxic reaction
 Wastage of financial resources & diminish program credibility
 Less effective & less desirable to improve the entire health care system.

13
 Cont.….

 The official like USP and other

pharmacopeia’s BP,
pharmacopeia’s set a for pharmaceutical
standard
quality products.
 By specifying the acceptance limits of API present in a
given pharmaceutical product.

14
 Cont...
 Hence, formulation having API above or below
the specified level is called poor quality.
 As result, the two main types of poor-
quality medicines according to WHO classification.
 Those are substandard and falsified product

15
 Cont...

 Any pharmaceutical formulation might be substandard.

 if it contains too much or too little API compared to the
formulation standards.
 Substandard manufacturing of legitimate drugs is due to a lack
of proper quality management procedures during manufacturing.

16
 Cont...
 It may occurred by accidental manufacturing defects or
deterioration due to inadequate storage/handling
 Whereas falsified drugs are a type of poor-quality medicines
containing hazardous quality or wrong ingredients.
 It is disguised intentionally or deliberately false
manufacturing as genuine medications for the matter of profit.

17
 Cont.….
 Their quality is unpredictable as they may contain wrong amount of
active ingredients, wrong ingredients or no active or
ingredients
with fake
packaging.
 In all cases falsified (counterfeit) medicines are manufactured
in
clandestine with no possibility of control.
laboratories

18
 Cont...
 Know a day all this issue is a critical and underreported issue in health
system, mainly in developing countries.

 Thus, it causes avoidable morbidity and mortality.

 It also causes a lack of consumer confidence in drugs and healthcare systems.

19
 Determinants of Medicine Quality
 Identity: Active ingredient

 Purity: Not contaminated with harmful

potentially substances
 Potency: Usually 90–110% of the labeled amount

 Uniformity: Consistency of color, shape, size and….

 Bioavailability/ bioequivalence: Interchangeable products?

 Stability: Ensuring medicine activity for stated 20

 Quality control
 It is the part of GMP concerned with sampling, specification, testing and

organization, documentation and release of procedure to ensure the quality of

the drug

– a planned coherent system of activities designed to provide quality product

 The 4 Main responsibilities of quality control in pharmaceutical industry

include :Efficacy, Safety, Quality & Compliance

21
 Cont.….

⚫Quality control can be expressed as internal and external

– Internal QC refers to all measures taken to achieve precision and

accuracy in the laboratory

– External QC compares and measures the performance of

different laboratory

22
 Cont.…..

 Internal Quality control: IQC

 Nature: Concurrent

 Performed by: lab staff

 Objective: Reliable results on a daily basis

 External quality assessment: EQ C

 Nature: Retrospective to evaluate IQC

 Performed by: Independent agency

 Objective: Ensure inter-laboratory comparability

23
Cont...

24
 Good Manufacturing Practice (GMP)

– The part of QA that products are consistently produced & controlled

to the quality standard appropriate to their intended use.
– The basic concept of GMP is – Do as you have written (i.e. SOPs)
and write what you have done
– quality can’t be tested into a batch of product but must be built into
each one during all stages of the manufacturing process

25
 Advantages of GMP
a. Prevent errors that can’t be eliminated through
quality control of finished products
b. Grantees credibility Via avoiding poor quality medicines
C. Ensure all units of a medicine are of the same quality (with in
specified parameters)

d. GMP helps boost pharmaceutical export

opportunities Governments can do so by
o making GMP mandatory for all pharmaceutical production, &
o training their inspectors in GMP requirements
26
 Basic Requirements for GMP
1. Appropriate resources to produce a quality product
Qualified and trained personnel
Suitable equipment
Correct materials, containers and
labels Approved procedures and
instructions Suitable storage and
transport

27
 Cont…

2. Validated critical steps of production process

Validation is the process of checking and
documenting variability
Validation carries out due to variability in quality of
materials & performance of equipments.

Validation is required if there is a change in site or any part of the

process (material/equipment)

28
 Cont…
3. Clearly written manufacturing procedure
Batch manufacturing and testing instructions, and every
department’s SOPs

4. Proper storage and distribution of the products

Stability testing for determining the storage condition and
shelf life
Minimize the risk to product quality

29
 Cont…

5. Complete document of manufacturing process

appropriate investigations are carried out if
quality problem come up
6. A recall system, in case quality problems are detected after
release of products
Recall can be done in d/t way & with d/t
degrees of severity depending up on the reason for
recall

30
 Quality Assurance (QA)
 The sum of all activities and responsibilities intended to ensure the products
meet all the applicable quality specifications in the final dosage form.

– A planned system of activities designed to ensure effective QC

 is used for the review and approval of all procedures related to production &
maintenance
 review of associated records & auditing

 performing trend analysis

31
 The Difference between QA and QC
QA QC

 Quality assurance is all about  In quality control, you find defects and
prevention of defects correct them while making the

 The goal of QA is to develop product.

a process so that defects do  Quality control is all about the

detection.
not arise when you are
 Quality control identifies the defects after
producing the product
the product is produced but is not yet
released or is still in the production
phase 32
 Head of Production: Responsibilities
1. Approval & implementation of production instructions, in-process QC & ensure
strict implementation
2. Ensures that production records are evaluated and signed by designated person
3. Checks maintenance of production department, premises and equipment
4. Ensures process validation and equipment calibration
5. Ensures initial and continuous training of production personnel

Head of Quality Control: Responsibilities

6. Approval/rejection of materials in accordance with specifications
7. Ensures carrying out of necessary testing
8. Approval of quality control procedures, e.g. sampling & testing; specifications
9. Checks maintenance of quality department, premises & equipment
10. Ensures validation of analytical procedures/methods
11. Ensures initial & continuous training of QC personnel

33
 The Quality Assurance Head: Responsibilities

1.Ensuring compliance with regulatory requirements

& international standards
2. Approval of the release of finished product for sale
3. Establishment & implementation of quality system
4. Review of all
 QC testing results,
 Production documents,
 Results of in-process control, &
 Overall compliance to the specification for the finished product prior to
release

34
 Cont.….

 Medicines are exposed to strict quality control. Why?

₰ Poor quality medicines lead to therapeutic failure or drug
resistance
₰ Toxicity, in many cases, it can lead to death.
₰ Poor quality medicines lead to loss of customer’s confidence in
the brand
₰ Poor quality medicines lead to enormous economic losses (e.g.
costs incurred in solving post sale problems)

35
 Cont.…
Objectives of quality control
• Make sure that proper sampling and analytical test are done on:
- Starting materials
- Packaging materials
- Intermediate and bulk products
- Finished products.
• Make sure that products are made which demonstrate that all the
required sampling, inspecting and testing procedures were actually
carried out.
• Make sure that the finished products contain active ingredients
complying with the qualitative and quantitative composition, are of the
required quantity, and enclosed within their proper container and
correctly labeled.
36
 Cont.….

• Make sure that no batch of product is released to sale of supply prior to

certification by qualified person.
• Make sure that sufficient reference sample of starting materials and
products are retained to permit future examination of the product if
necessary.

37
 1.1.1. QC procedures in pharmaceutical industries

 The main areas of QC procedures include the various steps to ascertain

that within reasonable limits the drug should have the following
characteristics:
I. Genuine quality, good nature and purity

Identification test: should be performed to ensure

the pharmaceutical product to be genuine

38
 Cont.…
 This is normally achieved by comparison of a property of the
sample (e.g., Ir spectrum, chromatographic behavior, chemical
reactivity, etc) to that of a reference standard.
Purity: the drug should be physically and chemically pure.

 Purity is the state of a chemical compound when no impurity can

be detected

39
 Cont.…
 The purity of pharmaceuticals can be ascertained through purity test.
 The tests for purity involve tests for the presence of impurity and fix
the limits of tolerance for these impurities.
 Some tests which may be run to ascertain the purity of substances
are:

a. physical comparison (colour, odour and taste tests.)

b. Determination of Physico -chemical constants

c. Determinations using analytical techniques
d. Miscellaneous characteristics
40
a) physical comparison with pure
standard
 They are used when other tests of purity are not available.

 It is the simplest method of checking purity a particular compound by

comparing pure sample.
 It has limitation but can
it show significantly visualized
like impurity
colored impurities and dirt.
 Smell tests will also reveal the differences between the
chemical
undergoing tests and the actual and pure state of a particular compound.
 It is used only when the sample is non-toxic through touch, taste, and
smell
41
b) Determination of Physico -chemical constants

 Pure chemicals has defined boiling and melting point

at
specific temperatures, and that is documented.
 However, if the melting or boiling point of a particular
chemical is changed from the normal, there is a great sense
that the chemical is not pure.

42
c. Determinations using analytical techniques

 Identification of drug purity can be accomplished by

utilizing simple to more sophisticated analytical methods.

 These techniques are very sensitive to control impurities in

very minute concentrations.
d. Miscellaneous characteristics: A large number of characteristics
still need to be monitored to ascertain the purity and authenticity of
drugs.
 Loss on drying
Sulfated ash

43
 Sulfated ash: used to test synthetic organic compounds
Determined by ignition with conc. sulphuric acid- metals thus
remain as sulphides that are stable to heat
E.g. Ascorbic acid — sulphated ash should not be more than
0.1% determined on 1 gm
 Loss on drying: to the net

refers pharmaceutical weight of a being

substances temperature. dried at a

 E.g. Aspirin: for 1g sample when dried (under reduced pressure
specified
over silica gel) for 5 hrs. It should not loss more than 0.5% of its weight.

44
2. A drug should contain the same amount of active ingredients as
stated on the label

This is achieved through:

 assay of active ingredients/finished product

 dosage form uniformity - content uniformity

- weight variation

The standards of pharmaceutical dosage forms should fulfill the

criteria set by various official compendia

E.g. Chloramphenicol: 98-102% (BP)

Aspirin: 99.5-101% (BP)

45
3. A drug should retain quality in terms of shelf-life and stability

Shelf-life: is the period at which the drug remains to its 90%

Stability is defined as the time from the date of manufacturing of

the formulation until its chemical or biological activity is not less

than a pre-determined level of the labeled potency and its physical

characteristics have not changed appreciably

There are factors which can contribute to instability of a

pharmaceutical product

46
 Cont.….
a. Incompatibility:
• Instability of a pharmaceutical product due to undesired reaction
between two or more components of the product
 The undesired reaction may lead to:
 Physical incompatibility
 Chemical incompatibility
 Therapeutic incompatibility

47
 Cont.….
b. Oxidation/reductionreaction, hydrolysis, …
Influenced by temperature, radiation, catalysis,
moisture, etc.
c. Photochemical reactions
Light can cause degradation thus colored glass
containers are used
“Hence these all factors should be controlled in the
production process, raw material as well as upon storage
of the finished product as they do have influence on the
shelf-life as well”

48
 Cont.….

4. A drug should not contain undesirable impurities

Impurities: are defined as undesired or extraneous

materials in a given substance

In pharmaceuticals, impurities can be defined as

unwanted chemicals

49
 Impurity
 Impurities in pharmaceuticals are the unwanted chemicals that:
– remain with the active pharmaceutical ingredients (APIs), develop
during formulation upon aging of both API and formulated APIs to
medicines.
– Can arise during the synthesis, purification, and storage of a
new drug substance and/ or drug
product. affect safety and
do not have
– efficacy any therapeutic value
and ultimately of theand may
product.
quality

50
 Source of impurity

They are two major sources of

impurity
1. API Synthesis related impurity
2. Formulation related impurity

51
1. API Synthesis related impurity
 Impurities in pharmaceutical compound or New
Chemical Entities(NCE) arise mainly during synthetic process from
raw material,
solvent , intermediate and by-products.
 Hence, impurities during synthesis process and it can be
Categorized as:
 Organic impurity
 Inorganic impurity
 Residual solvents

52
 Organic impurities

 Mainly arise during synthetic process and /or storage of drug

substance
 Impurity classified as:
 Starting material
 Intermediate
 By-product
 Degradation product

53
 Inorganic impurity
 Inorganic impurities may also be derived from the manufacturing
processes used for bulk drugs.
I. Reagents, ligands, and catalysts: chances of having these impurities are
rare: however, in some processes, these could create problem unless the
manufacturers take proper care during production.

II. Heavy metals: the main sources of heavy metals are the water
used in the processes and the reactors. These impurities of heavy
metals can easily be avoided using demineralized water and glass-
lined reactors.

54
 Residual solvent
 Are potentially undesirable substance lead to modify the property of the
compound or hazardous to human health.
 It is very difficult to remove these solvents completely by the work-up process
 Affect the physico-chemical property of the bulk drug substance
like
crystalinity , dissolution properties, odor and color in finished
change
product.
 Solvent used in the manufacturing of drug substance is categorized in to four
types, according to ICH guideline.

55
 Class I solvent
 Not used in the manufacturing of drug substance, excipients
and formulations
 Due to unacceptable toxicity or their deleterious effects
 If use of these solvents is unavoidable, then their usage
should be restricted.

56
 Class II solvents
 Usage should be limited in pharmaceutical products
because of their inherent toxicity.

57
 Class III solvents

 Are less toxic and possess lower risk to human health than
class I and II solvents.
 It has permitted daily exposures of 50 mg or less

 The use of these solvents in pharmaceuticals does not have

any serious health hazard.
 Example: ethanol, pentene, methyl ether, isopropyl acetate,
heptane, 1-pentanol, 2-propanol etc.

58
 Class IV solvents

 Have toxicological data is not available.

 Example of such solvents are: 1, 1-diethoxy propane,

1,1- dimethoxy propane, isooctane, trichloro acetic acid etc.

59
 2. Formulation related impurity
A. Dosage form related impurity
 liquid dosage forms are very much to both
susceptible

degradation and microbiological contaminations.

 Solutions and suspensions are inherently prone to
degradation

due to hydrolysis or solvolysis.

 In this regard, water content, pH of the solution/suspension,

compatibility of anions and cations, mutual interactions of

ingredients and the primary container are also some critical

factors. 60
 b. Method related impurity:
 may be caused by improper manufacturing processes which don’t
follow the optimized conditions like pressure, temperature during
processing.
 For example: Indolinone derivative and sodium hydroxide may be
formed during the production of dosage form of
parenteral diclofenac sodium.
 terminally sterilized by autoclave method (i.e., 123±2°C)

 that enforced to intramolecular cyclic reaction

61
 C. Environmental related impurity

 Temperature: vitamin, vaccine, antibiotics…

 Light: vitamins, diphenhydramine …
 Humidity: aspirin , ranitidine…
D. Formulation related impurities
 Impurities Formed Due To Functional Group Reaction
Degradation

62
 Cont.…
 Hydrolysis: Hydrolysis is a common phenomenon ester
and
for amide type of drugs, especially in liquid dosage forms.
 benzylpenicillin, barbitol, chloramphenicol,
chlordiazepoxide, lincomycin and oxazepam.
 Oxidation: The oxidative decomposition of pharmaceutical
compounds mediated either by free radicals or by molecular
oxygen.
 hydrocortisone, methotrexate, adinazolam,
 hydroxyl group directly bonded to an aromatic ring (eg,
phenol derivatives such as catecholamines and morphine),
 conjugated dienes (eg, vitamin A and unsaturated free fatty acids),
 heterocyclic aromatic rings

63
 Cont.…
 Photolysis: Fluoroquinolones antibiotics are found to
be susceptible to photolytic cleavage.
 In ciprofloxacin eye drops preparation (0.3%),
sunlight induces photo cleavage reaction.
 producing ethylenediamine analog of ciprofloxacin.
 Impurity profiling – helps in detection, identification and
quantification of various types impurities as well as residual
solvents in bulk drugs and in pharmaceutical formulations.

64
 Identified An impurity for which a structural
characterization
Impurity:– has been achieved
 Specified An impurity that is individually listed and
limited with a specific acceptance criterion in specification.
Impurity:
 Unidentified Impurity: An impurity for which a structural
characterization has not been achieved and that is defined solely by
qualitative analytical properties (e.g. chromatographic retention time)

 Unspecified Impurity: An impurity that is limited by a general

acceptance criterion, but not individually listed with own specific
acceptance criterion in the specification

 Qualification: Process of acquiring and evaluating data

that
establishes the biological safety of an individual impurity or a given
impurity profile at the level(s) specified

65
 Quiz 1
1. What is term drug shelf-life and stability?

2. What are the factors which can contribute to instability

of a pharmaceutical product and discuss it?

3. What characteristics makes a given drug have a

standard quality?

4. Why we do impurity profiling in pharmaceutical dosage

form?

66
1.2.The compendia

67
 1.2. Drug compendia

 It is a collection of concise but detailed information about a

drug.
 Pharmacopeias and formularies are
pharmaceuticals and medical devices containing
books
specifications and procedures of tests.
of
 They are collectively referred as the drug compendia.
standards

for

68
 Cont.….

 The main objective of the compendia:

🞄 To control the quality of medicine as pre

standard

🞄 To ensure the public health

🞄 To support the availability of effective, good
safe,
quality pharmaceutical care for all

69
 Cont.….
 Drug compendia are sub classified as official and non-official

1. Official compendia
 It is a collection of list of drugs and devices which have been
recognized as legal standards of purity, quality and strength that is
accepted by some recognized authority.
 The books contains the standards of drugs and related substances are
known as pharmacopeias and formularies, collectively this books
known as drug compendia.

70
 Cont.….
 Pharmacopeia- is a book containing a list of medicinal
substances (drugs) with descriptions, specifications, tests, and
acceptance criteria, specified by some recognized authority.
 Such books of standards include the British pharmacopeia
(BP), European pharmacopeia (EP), International
pharmacopeia (IP), Japanese pharmacopeia (JP), United States
pharmacopeia (USP), etc.

71
72
 The compendia (cont…)

 Some specification requirements for pure drugs (API):

– Description: Crystalline, amorphous, etc.

– Solubility

– Identification tests

– Physical constants

– Limit tests for classically well known toxicants

– Assay

– Limit of degradation products

73
 The compendia (cont…)

 Specification requirements of dosage

forms:

 Identification

 Assay ( with reference to label claim)

 Disintegration

 Dissolution

 Content uniformity tests

 Packaging and storage

74
 cont.…
2. Non official compendia
 These are secondary reference sources that are not subject to
categories of official compendia. They include monographs and
text books.
 Monograph- is a detailed written description about principal
feature of the substance and the ways that the features can be
determined.
 A monograph is written document about a single product and
usually written by a single author.
 Text books: They concentrate on principle rather than on details of the
last
minute (very latest) development in the field.
 Martindale (Extra pharmacopeia)- reading assignment
75
The law and
pharmaceutical
 Law – is the body of principles that govern conduct
and observance of which can be enforced in courts.
 Assuring continuous availability of the required pharmaceuticals, of
proven quality, safety and efficacy requires pharmaceutical policy which
does have legal instrument such as pharmaceutical legislation and
pharmaceutical regulation.

76
 Food and drug Administration
(FDA)
 The largest of the world’s drug regulatory agencies.
 FDA is responsible for the approval
– Foods are safe, wholesome and sanitary
– Human and veterinary drugs, biological products and medical
devices are safe and effective
– Cosmetics are safe
– Electronic products that emit radiation are safe.

77
 FDA….
 Currently, the FDA is divided into five major Centers, each
with its own origins and history:
 The Center for Drug Evaluation and Research (CDER)

 The Center for Biologics Evaluation and Research (CBER)

 The Center for Devices and Radiological Health (CDRH)

 The Center for Food Safety and Applied Nutrition (CFSAN)

 The Center for Veterinary Medicine (CVM)

78
 Does the FDA test
drugs?
 FDA does not develop, manufacture or test drugs.
 Drug manufacturers submit full reports of a drug's studies so
that the Center can evaluate its data.
 The studies answer the question: "Does this drug work for the
proposed use?"
 By analyzing the data, CDER reviewers assess the benefit-to-
risk relationship and determine if the drug will be approved.
 Once FDA approves a drug, does this mean that the product is
perfectly safe?

79
 Local drug regulation (Ethiopian Food and Drug Authority),

 EFDA MISSION: To protect and promote public health by

ensuring the safety, effectiveness, quality and proper use of
regulated products through licensing, inspection, registration,
laboratory testing, post-marketing surveillance, community
participation, and provision of up-to-date regulatory
information.

What it does? Visit

–www.EFDA.org
80
Scope of Control

 Products Controlled by FMHACA/

EFDA
 Human drugs

 Radio-pharmaceuticals

 Traditional medicines

 Pesticides

 Medical supplies and instruments

 Sanitary items

 Cosmetics

 Raw and packaging materials

 Food and food products

81
 Institutions Controlled by FMHACA/EFDA
 Manufacturers

 Importers and/wholesalers

 Exporters

 Retail-outlets

 Drug quality control Labs

 Scientific offices,

 Commission agents

 Health institutions

82
Thank you

83
1.3 Analytical Errors and validation of analytical procedures

84
 Analytical Errors

 Error refers to the difference in the numerical values between a measured value and
the true value.
 It has become universally accepted in methods of comparison that the percentage
composition of a ‘standard sample’ provided and certified by the National Institute of
Standards and Technology (NIST) or the British Pharmacopoea Chemical Reference
Substance (BPCRS) or the European Pharmacopoea Chemical Reference Substance
(EPCRS) must be regarded and treated as absolutely correct, pure and authentic while
evaluating a new analytical method.
 Consequently, the differences thus obtained between the standard values and those
by the new analytical methods are then treated as ‘errors’ in the latest procedure.

85
 Cont.….
 Or errors can be generated due to human mistakes, improper
adjustment of instruments, error in instruments, etc. removal of
these errors are necessary before preceding the analysis of the
survey data.
 There are three types of errors which may occur in the course of analysis:

1. Gross error

2. Random (or indeterminate) error and

3. Systematic (or determinate) error

86
 1. Gross error

 Gross errors are easily recognized since they involve a major

breakdown in the analytical process such as
samples being spilt, wrong dilutions being prepared or
instruments breaking down or being used in the wrong way
 If a gross error occurs the results are rejected and the analysis is
repeated from the beginning
 Often the product of human errors & usually occur only
occasionally.
 may cause a result to be either high or low lead to outliers,
Outliers results that appear to differ markedly from all other data in a
set of replicate measurements
87
 2. Determinate (systematic) errors

 These are errors that possess a definite value together with a

reasonable assignable cause.

 Mostly the cause for this type of error can be identified
and

corrected but not be easy.

 Errors affecting the accuracy of an analysis are called determinate &

characterized by a systematic deviation from the true value; that is,

all the individual measurements are either too large or too small.

88
 Cont.…
• A positive determinate error results in a central value that is
larger than the true value, and a negative determinate error
leads to a central value that is smaller than the true value.
• Both positive and negative determinate errors may affect the
result of an analysis, with their cumulative effect leading
to a net positive or negative determinate error.

• 4 types

89
 I. Personal Errors

 Result from the carelessness, inattention, or

personal
limitations of the experimenter.
 Many measurements require personal judgment leads personal
error or an error due to biases introduced by the analyst
 Most personal errors can be minimized by care and
self- discipline

90
 Cont.…
 Analytical work is always subject to a variety of
personal errors, which can include
– the ability to see a change in the color of an indicator used to signal
the end point of a titration;
– biases, such as consistently overestimating or underestimating the
value on an instrument’s readout scale;
– failing to calibrate glassware and instrumentation; and
– misinterpreting procedural directions.

91
 II. Sampling error

 An error introduced during the process of collecting a sample for

analysis.
– We introduce determinate sampling errors when our sampling strategy
fails to provide a representative sample.
III. Method error: an error due to limitations in the analytical
method used to analyze a sample.
IV.Measurement error: an error due to limitations in the equipment
and instruments used to make measurements.
– Determinate measurement errors can be minimized by
calibration.

92
 Other types of errors
a) Instrumental Errors
 Are caused by the imperfections in measuring devices , faulty
and uncaliberated instruments such as : pH meters, single pan
electric balances, UV spectrophotometers,
 In this case errors will decrease by periodic calibration of
equipment

93
 (b) Reagent Errors

 due to individual reagents. For instance: impurities inherently

present in reagents; unwanted introduction of ‘foreign
substances’ caused by the action of reagents on either
porcelain or glass apparatus.

94
 C). Constant Errors

 They are observed to be rather independent of the

magnitude of the measured amount; and turn out to be
relatively less significant as the magnitude enhances.
– Example : Assuming a constant equivalence point
error of 0.10 ml is introduced in a series of titrations,
hence for a specific titration needing only 10.0 ml of
titrant shall represent a relative error of 1% and only
0.2% for a corresponding 50 ml of titrant consumed

95
 3. Random (or indeterminate) error

 It is indeterminate errors cannot be pin-pointed to any specific well-

defined reasons.
 Random (or indeterminate) errors affect the precision
of measurement and are non-traceable
 These errors are mostly random in nature and ultimately give rise to
high as well as low results with equal probability.
 They can neither be corrected nor eliminated, and therefore, form
the ‘ultimate limitation’ on the specific measurements.

96
 cont.…

 They can not be eliminated or corrected

 They can only be minimized by performing repeated
measurements & giving particular attention during
analytical procedure

97
 Generally, systematic errors can be minimized

 Calibration of instruments, Apparatus and

applying
necessary corrections
 Blank Determination

 Cross-checking Results by Different Methods of Analysis

 Analysis of standard samples

98
 Minimizing systematic errors
 Systematic errors may be reduced substantially and significantly
by adopting one of the following procedures firmly, such as:
(i) Calibration of Instruments, Apparatus and Applying
Necessary Corrections

 Most of the instruments, commonly used in an analytical laboratory.

For example: UV-Spectrophotometer, IR-Spectrophotometer, single—pan

electric balance, pH-meter and the like must be calibrated duly, before use so
as to eliminate any possible errors.

99
 In the same manner all apparatus, namely : pipettes, burettes, volumetric flasks,

thermometers, weights etc., must be calibrated duly, and the

necessary

corrections incorporated to the original measurements.

 In some specific instances where an error just cannot be avoided it may be

convenient to enforce an appropriate correction for the effect that it ultimately

causes ;

 For instance : the inherent impurity present in a weighed

precipitate can be estimated first and then deducted duly from
its weight.

100
(ii) Blank Determination :

 In order to ascertain the effect of the impurities present in the

reagents employed and reaction vessels used ; a blank
determination is an absolute necessity.
 It may be accomplished by performing a separate parallel
estimation, without using the sample at all, and under identical
experimental parameters as employed in the actual analysis of the
given sample.

101
iii) Using standard substance

a. External standard

b. Internal standard

c.Standard Addition

Reading assignment

102
External standard

It essentially comprises of performing an altogether separate

estimation under almost identical experimental parameters with a

quantity of a std substance that consists of similar weight of the

component as is present in the unknown sample.

Thus, the conc of the component present in the unknown sample:

Rsample
Csample  Cstd
Rstd
Internal standard

•An internal standard is a known amount of a compound,

different from analyte, that is added to the unknown but very

similar in physicochemical properties.

Employed if there is variation in experimental parameters.

The effect of such variations can be eliminated by use of

internal std.

An equal amount of an internal std, a component that is not

present in the sample, is added to both the sample and std

solutions.

Signal from analyte is compared with signal from the internal

standard to find out how much analyte is present.

10
4
Internal
standards
…

10
5
Standard Addition
 This is especially useful when there is a problem with
interference from the sample matrix, since it cancels out
these effects.

 Here, a small known quantity of the component under

estimation is added to the sample, which is subsequently
subjected to analysis for the total amount of component
present.

 The actual difference in the quantity of components present

in samples with or without the added component ultimately
 Validation of Analytical Procedures

 Method validation is the process of proving that an analytical

method is acceptable for its intended purpose.
 The object of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose determined
by means of well-documented experimental studies.

107
 General recommendation in method validation

 The principle of the test procedure should be described briefly.

 Procedure should be sufficiently detailed to make repletion by experts or

other

analysts & the following description should be provided:

 Parameters evaluated or tested

 Reagents preparation techniques

 Calculation technique of assessed parameters

 Equipment and parameters

 Reference standard used

 Precaution to be taken 108

 Method validation#3
Method validation parameters
Specificity

Range Linearity

Sensitivity

109
 Accuracy
 The accuracy of an analytical method is the extent to which test results
generated by the method and the true value agree.
 Accuracy can also be described as the closeness of agreement between
the true or accepted reference value, and the value found
– comparison of the results of the proposed analytical procedure with
those of a second well-characterized procedure, the accuracy of which
is stated and/or defined;
– accuracy may be inferred once precision, linearity and specificity
have been established.

110
 Cont…
 Accuracy is usually reported as percent recovery by assay, using the

proposed analytical procedure, of known amount of analyte added to

the sample.

 Typical accuracy of the recovery of the drug substance in

the

mixture is expected to be about 98 to 102%.

 Values of accuracy of the recovery data beyond this range need to be

investigated.
111
 Precision

 Precision is the degree of agreement among a series of measurements of the

same quantity; it is a measure of the repeatability/reproducibility of results

rather than their correctness

 It does not imply anything with respect to their relation to the ‘true value’
 Precision is usually expressed as the standard deviation (s) or the relative
standard deviation (% RSD) of the mean (m) of a series of
measurements

 According to the ICH document, precision may be considered at three

levels:
 Repeatability, intermediate precision and reproducibility
112
 Repeatability
 is a measure of the precision under the same operating conditions over a
short interval of time.
 It is sometimes referred to as intraassay precision

Intermediate Precision.
 Intermediate precision is defined as the variation within the same
laboratory
(intra-laboratory precision).

 The extent to which intermediate precision needs to be established depends

on the circumstances under which the procedure is intended to be used.

 Typical parameters that are investigated include day-to-day variation, analyst

variation, and equipment variation

 The intermediate precision is also called ruggedness.

113
 Reproducibility.

 Reproducibility measures the precision between laboratories

 This parameter should be considered in the standardization of an

analytical procedure (e.g., inclusion of procedures in
pharmacopoeias and method transfer between different
laboratories).

 To validate this characteristic, similar studies need to be performed

at other laboratories using the same homogeneous sample lot and
the same experimental design.
114
 Accuracy without
Accuracy & Precision
Precision

Precision without No Precision &

Accuracy No Accuracy 115
 Range

• The range of a method is related to its sensitivity.

• The interval b/n the upper & lower conc (amounts) of the analyte in the sample

(including these concs) for which it has been shown that the analytical

procedure has a suitable level of precision, accuracy & linearity.

• The following minimum specified ranges should be considered:
– For the assay of a drug substance or a finished (drug) product: normally from 80 to 120
percent of the test concentration;
– For content uniformity, covering a minimum of 70 to 130 percent of the test concentration,
unless a wider more appropriate range, based on the nature of the dosage form (e.g.,
metered dose inhalers), is justified;
– For dissolution testing: +/-20 % over the specified range

116
 Detection Limit
(LOD)
• The detection limit of an individual analytical procedure is the

lowest amount of analyte in a sample which can be detected

but not necessarily quantitated as an exact value.

• ICH defines LOD as the lowest amount of analyte in a sample

which can be detected but not necessarily quantitated as an

exact value.

117
 Quantitation Limit (LOQ)
#1
 ICH defines LOQ of an individual analytical procedure as the
lowest amount of analyte in a sample which can be
quantitatively determined with suitable precision and accuracy.
 The quantitation limit is a parameter of quantitative assays for
low levels of compounds in sample matrices, and is used
particularly for the determination of impurities and/or
degradation products.

118
 Robustnes
s
 The robustness of an analytical procedure is a measure of its
capacity to remain unaffected by small, but deliberate
variations in method parameters and provides an indication
of its reliability during normal usage.
– stability of analytical solutions;
– extraction time.
– influence of variations of pH in a mobile phase
– influence of variations in mobile phase composition
– different columns (different lots and/or suppliers)
– Temperature
– flow rate.

119
Summar
y

120