pharmaceutical analysis I

Phar3121
By Teshale E.
(B. Pharm, MSc)

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1. Introduction to pharmaceutical
analysis

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 1.1 Definitions
Pharmaceutical analysis:
 It is a science which deals with identification, characterization and
quantification of drugs in raw materials, dosage forms and biological
fluids or

 It is an applied science that ensures the safety, efficacy and stability

of pharmaceutical products by using physical, chemical, biological,
pharmacological and biopharmaceutical methods; and

 It is also a technique that is used in elucidation(make clear,explain) of

drug entities from natural products.
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 Generally, pharmaceutical analysis procedures may be used to answer any of the
questions below:
– Do the identity and purity of a pure drug substance meet specification?

– 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?

– Do the identity and purity of excipients meet specification?

– What are the physical constants like Pka value (s), solubilities, stability 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.

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 Scope (areas) of pharmaceutical (drug) analysis:

 Pharmaceutical industry

 Raw material control

 In-process control

 Dosage form control

 Government drug control laboratory (regulatory agencies)

» Manufacture lab. Procedure control

» Products control

 Analysis means a detailed examination or study

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 Pharmaceutical research labs

 Advanced research

 Development of analytical method

 Process development

• Stability studies

 Basic research

 Separation, Identification, Quantification, Molecular characterization

 Laboratories requiring pharmaceutical analysis:

 Government regulatory agencies

 Manufacture of raw materials for drugs

 University and other non-commercial research centers

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 1.2. Introduction to quality control and Quality
Assurance
• Quality:
o A measure of a product’s or service’s ability to satisfy the customer’s stated or
implied needs.

o In the pharmaceutical industry represents both function and process.

“Fitness for purpose”

• Quality Control (QC):

 The operational techniques and activities that are used to Check or test the
specifications are met OR

 Steps taken during the generation of a product or service to ensure that it meets
requirements and that the product or service is reproducible.

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 Quality assurance (QA): The sum total of the organized
arrangements made with the objective of ensuring that all materials are of
the quality required for their intended use and that quality systems are

maintained.

 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

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• Quality control is also concerned with

 sampling, specification and testing with the organization documentation

and release procedures which insure that the necessary and relevant tests

are actually carried out.

 no products released for sale or supply until their quality has been judged to

be satisfactory.

 Good manufacturing practice (GMP):

 GMP is that part of QA which ensures products are consistently produced

and controlled to the quality standards appropriate to their intended use

and as required by the marketing authorization or product specification.

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 Comparison of QA and QC
Quality assurance Quality control
• Process oriented • product oriented,
• Defect prevention • Defect identification
• Proactive measure • Reactive measure
• Responsibility of all staff • Responsibility of team
• Create the deliverables • Verify deliverables
• Defines standards and • Confirms standards are
methodologies followed
• Low Level Activity • High Level Activity

pharmaceutical QA II 10
 The tests of quality control may belong to the following types:
 Physico-chemical methods

 Microbiological methods

 Biological methods

 It is better to use Total Quality Control (TQC or QA) rather than QC.

 By TQC it means to include all those aspects which start with the

procurement(obtain) of raw materials to the finished product available at the medical

store until it gets consumed by the patient.

Input Black box Output Patient

 In robust modern quality systems:

“Quality should be built into the product, and testing alone cannot be relied on
to ensure product quality”
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• There is a common theme to all quality systems whether used for
pharmaceutical production or not.

– The quality system must be described in written documents approved by

management (policies, quality manual, etc).

– The quality system must be regularly reviewed.(a formal examination or

reconsidration)

– All operations that can effect quality must be described in written and approved

procedures (standard operating procedures, SOPs).

– Materials must be appropriately approved prior to use.

– Output (product) must be appropriately inspected prior to release.

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– Equipment used must be fit for purpose (qualified/validated, calibrated and

maintained).

– Personnel must be trained in the quality system and in operations they

perform.

– Written records must be kept to demonstrate quality procedures have been

followed.

– There must be regular internal quality audits to ensure quality is maintained.

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Objectives of Quality Control

• Make sure that proper sampling and analytical test are done.

• Make sure that the finished products contain active ingredients complying with the

qualitative and quantitative composition and enclosed with in their proper container and

correctly labeled.

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

• In conclusion, all quality systems are to do with people, materials, equipment, records

and procedures.

– Generally speaking Objectives of Quality Control:-to render good of quality

products and/or service to the customer.

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 1.2.1.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:

1. Genuine(truly what it said to be) quality, good nature and

purity

 Identification test: should be performed to ensure the pharmaceutical

product to be genuine

o Usually an IR spectrum of a reference or authentic(genuine) compound is

utilized to be compared with.

 Purity: the drug should be physically and chemically pure(not mixed).

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

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 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 of the tests which may be run to ascertain the purity of substances are:

I. colour, odour and taste tests

 They are used when other tests of purity are not available,

 They have limited value, but determine whether a substance is reasonably pure or not

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II. Determination of physico -chemical constants

 This ensures whether the substances are reasonably free from other substances,

although they fail to indicate the nature of impurities present.

 Solubility of substances in different solvents, determination of mpt. and bpt., optical

rotation and refractive index have reliable values which can reveal the purity of the

substances.

 mpt. and bpt. are usually expressed in ranges to define purity

E.g. –Metronidazole, MP: 159-162°C

 Specific optical rotation-

E.g. Ergometrine:+50 to +56o( 1% w/v solution in water)

Phenylepherine: -46 to -47.5o (2% w/v solution in water)

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III. Determinations using analytical techniques

 Identification of drug purity can be accomplished by utilizing simple to more

sophisticated analytical methods. It includes:

 Titrimetric techniques - Acidimetry and alkalinimetry, Non-aqueous titrations

- Karl- Fischer titrations, Gravimetric titrations

 Separation techniques - Chromatographic methods (TLC, GC, HPLC, etc.)

- Hyphenated techniques (GC-MS, HPLC-MS. etc.)

 Spectroscopic techniques - UV-Visible, NMR, IR, MS. etc.

o These techniques are very sensitive to control impurities in very minute conc

o They are most of the time utilized to assay active ingredients or finished product and

determine potency.

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IV. Miscellaneous(consisting of many d/t kinds) characteristics

 A large number of characteristics still need to be monitored to ascertain the purity

and authenticity of drugs. These include:

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%

Loss on drying:

 refers to the net weight of a pharmaceutical substances being dried at a specified T

E.g. Aspirin: for 1g sample when dried (under reduced pressure over silica gel) for 5hrs.

It should not loss more than 0.5% of its weight.

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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 chemicals and dosage forms should fulfill

the criteria set by various official compendia. These include:

 highest attainable standards for chemical purity; there may be

differences in methods of manufacture and changing pattern of stability.

E.g. Chloramphenicol: 98-102%

Acetylsalicylic acid: 99.5-100.5%

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

 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.

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

 Stability indicating assay is the assessment of a pharmaceutical dosage form

(excipients and active ingredients) under stressful conditions

 There are factors which can contribute to instability of a pharmaceutical product

a. Incompatibility:

a. Instability of a pharmaceutical product due to undesired reaction between two or

more components of the product.

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 The undesired reaction may lead to:

 Physical instability: physical changes (colour, odour, gross precipitation)

 Chemical instability: identified by chemical analysis.

 Therapeutic incompatibility: leads to excessive potentiation or decrease effectiveness.

b. Oxidation/reduction reaction, 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”
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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 that

remain with the API or developed during formulation or upon aging of the active

ingredient (dosage form) that arose in either toxicity or decrease or increase

potency.

 The presence of impurities even in small amount may influence efficacy and

safety of pharmaceutical products.

 There are two types of impurities:

 Impurities associated with the API

 Impurities created during formulation and with aging or that are related to the

formulated forms.
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 According to ICH guidelines, impurities associated with APIs are

classified into the following categories:

A. Organic impurities (process and drug-related):

 May arise during the manufacturing process and/or storage of the drug subs.

 They may be identified or unidentified, volatile or non-volatile, and include

the following:

i. Starting materials or intermediates: the most common impurities found in

every API.

Eg. In paracetamol bulk, there is a limit test for p-aminophenol, which could be a

starting material for some one manufacturer or be an intermediate for another.

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 ii. By-products: In the case of paracetamol bulk, diacetylated paracetamol is the by

product
.

Fig. Production of Paracetamol from intermediate, p-Aminophenol.

iii. Degradation products: degradation products resulting from storage or formulation to

different dosage forms or aging of API are common impurities in the medicines. Eg. The degradation

of penicillins and cephalosporins

 The presence of a ß-lactam ring as well as that of an α-amino group in the C6/C7 side chain plays

a critical role in their degradation

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iv. Reagents,ligands,and catalysts: These chemicals are less commonly found in

APIs.

“In general, an individual API may contain all of the above-mentioned types of

organic impurities at levels varying from negligible to significant”

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B. Inorganic impurities: Inorganic impurities may also derive from the

manufacturing processes used for bulk drugs (API).

 They are normally known and identified and include the following:

 Reagents, ligands, and catalysts - The chances of having these impurities

are rare

 Heavy metals - The main sources of heavy metals are the water used in the

processes and the reactors (if stainless steel reactors are used), where

acidification or acid hydrolysis takes place.

 can be easily avoided using demineralized water and glass-lined reactors.

 Other materials (eg, filter aids, charcoal):

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C. Solvent residues: are organic volatile chemicals used during the

manufacturing process or generated during the production.

 It is very difficult to remove these solvents completely by the work-up process;

however, efforts should be taken to the extent possible to meet the safety

data.

 Depending on the possible risk to human health, residual solvents are divided

into 3 classes.

Class I solvents: Solvents to be avoided, known to cause unacceptable toxicities,

strongly suspected human carcinogens

 such as benzene (2 ppm limit) and CCl4 (4 ppm limit) have to be avoided.

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Class II solvents: Solvents to be limited, known non-genotoxic, animal

carcinogens or are possible causative agents of irreversible toxicity

 such as methylene chloride (600 ppm), methanol (3000 ppm), pyridine (200 ppm),

toluene (890 ppm), N,N-dimethylformamide (880 ppm), and acetonitrile (410 ppm).

Class III solvents: have permitted daily exposures of 50 mg or less.

 acetic acid, acetone, isopropyl alcohol, butanol, ethanol, and ethyl acetate

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Impurities related to formulation

 Apart from bulk drug-related impurities, the formulated form of API may contain

impurities that form in various ways. Impurity formed during formulation

a) method related

 A known impurity, 1-(2,6-diclorophenyl)indolin-2-one is formed in the production of a

parenteral dosage form of diclofenac sodium if it is terminally sterilized by autoclave.

Fig. Formation of impurity on autoclaving of Diclofenac sodium

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b) environmental related

 environmental factors that can reduce stability include the following :

Exposures to adverse temperatures-

 Eg. Vitamins as drug substances are very heat-sensitive and degradation frequently
leads to loss of potency in vitamin products, especially in liquid formulations.

Light-especially UV light –

 Eg. Ergometrine as well as methyl ergometrine injection is unstable under tropical

conditions such as light and heat.

Humidity-

 For hygroscopic products, humidity is considered detrimental to both bulk powder and

formulated solid dosage forms. Aspirin and ranitidine are classical examples.

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c) dosage form factors related

 In general, liquid dosage forms are very much susceptible to both degradation

and microbiological contamination.

 In this regard,

 water content,

 pH of the solution/suspension,

 compatibility of anions and cations,

 mutual interactions of ingredients, and the primary container are

critical factors.

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Formation of impurities on aging(irreversible biological change)

a) Mutual interaction amongst ingredients

 Most vitamins are very labile and on aging they cause a problem of

instability in different dosage forms, especially in liquid dosage forms.

 Degradation of vitamins such as folic acid, pantothenic acid,

cyanocobalamin, and thiamine do not give toxic impurities;

 however, potency of active ingredients drops below pharmacopoeias

specifications.

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b) Functional group-related typical degradation

Hydrolysis -Examples included the following: Aspirin, benzocaine, cefotaxime, cocaine

and echothiophate. especially in liquid dosage forms.

Oxidative degradation –

 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, nitroso and nitrite derivatives, and aldehydes

(eg, flavorings) are all susceptible to oxidative degradation.

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Photolytic cleavage - Pharmaceutical products are exposed to light while being

manufactured as a solid or solution, packaged, held in pharmacy shops or

hospitals pending use, or held by the consumer pending use

Eg. In ciprofloxacin eye drops preparation (0.3%), sunlight induce photocleavage

Decarboxylation- Some dissolved carboxylic acids lose carbon dioxide from the

carboxyl group when heated.

Eg. >> p-aminosalicylic acid

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1.3.The compendia

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 1.3. Drug compendia(collection of
facts on a subject)
 It is a collection of concise but detailed information about a drug.

 Pharmacopeias and formularies are books of standards for pharmaceuticals

and medical devices containing specifications and procedures of tests.

 They are collectively referred as the drug compendia.

 Published by the authority of a government or a medical or pharmaceutical

society. 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 safe, effective, good quality

pharmaceutical care for all

 Drug compendia are sub classified as official and non-official

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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.

Pharmacopeia

 It is a book containing a list of medicinal substances, their dosage forms or

devices with descriptions of test methods, specifications for purity and strength

accepted by some recognized authority.

 The recognized authority which issue the books of standards in most

countries is the government

 In some countries e.g. USA both the national pharmacopoeia and national

formularies are published by a private non profit making organizations

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39
 The compendia (cont…)

 Components and specification requirements of pharmaceutical dosages:

Excipients: therapeutically inactive but have indirect effect on like the

rate of release (dissolution and disintegration) of the medicament.

 They should be safe( non toxic), not having any interference with

the therapeutic activity and analytical procedure

Active components: therapeutically active form of a drug.

 They should have the highest attainable standard in terms of

chemical purity and biological response

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 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

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 The compendia (cont…)

Specification requirements of dosage forms:

– Identification

– Assay ( with reference to label claim)

– Disintegration

– Dissolution

– Content uniformity tests

– Packaging and storage

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 The compendia (cont…)

The following are some of the representatives:

 United States Pharmacopeia and National formulary (USP and NF)

 British Pharmacopoeia ( BP)

 International Pharmacopoeia (PhI)

 European Pharmacopoeia (Ph. Eur.)

 Indian Pharmacopeia (IP), etc

 Pharmacopeias or formularies are generally national in origin and scope

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 The compendia (cont…)

2. Non official compendia

 These are secondary reference sources that are not subject to categories of

official compendia. They include treatises, monographs and text books.

 Treatise: it is a comprehensive, exhaustive, systematic or critical approach to a

broad topic or whole field of knowledge.

 It is generally written for specialist in a given field.

 Monograph: is a work of writing upon a single subject, usually by a single author.

 It is by definition a single document that forms a complete text in itself

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 The compendia (cont…)

 Text books: They concentrate on principle rather than on details of the

last minute (very latest) development in the field.

 Martindale (Extrapharmacopeia)- is based on published information

 it is not a book of standard hence the inclusion of a substance or

preparation is neither to be considered as a recommendation for

use nor confer any status on substance or preparation.

 Its main objectives are to provide practicing pharmacists and

physicians with unbiased evaluated information on drugs and

medicaments (proprietary preparations) used through out the world

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 The compendia (cont…)
Components of pharmacopoeia
a) General notices:
 General notices provide the overall guiding principles for using the monographs and general

chapters. It mainly clarifies information provided in the pharmacopoeia to the reader.

EXAMPLES

 Tolerances: e.g. Aspirin:-should have not less than 99.5% and not more than 100.5% of

the label claim. Reason for tolerances: -Assay errors

- Basic compounding or manufacturing errors

- Detection of chemical substances

 Tests and assays:

 About: approximately (fairly correct or accurate; near to the actual value).

 Exactly: implies an error of 0.1% in the case of weighing or 0.05ml in a 50.0ml burette

 Corresponds: similar or equivalent in character or quantity.

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 The compendia (cont…)

 water bath' means a bath of boiling water, unless water at some other

temperature is indicated in the text

 Drying to constant weight: means that drying shall be continued until two

consecutive weighing do not differ by more than 0.5mg per gram and weighing

should be 1hr apart.

 Blank determination: conducted in exactly same manner as in the assay or test

except that there is no sample.

 Indicators: implies usually approximately 0.2ml or 3 drops of indicators

solution

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 The compendia (cont…)

 Packaging storage and labeling:

 The container should not interact physically or chemically with the chemical

placed in it

 The container should be

– Light sensitive container: which does not transmit more than 18% of UV

radiant energy falling up on it.

– Well-closed container: protects contents from extraneous solids and from

loss of drug under ambient conditions.

– Tightly closed container: protects contents from contamination by

extraneous liquids solids or vapors or from loss of drug due to evaporation.

– Tamper –Evident Packing, airtight container, Single–dose container etc

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 The compendia (cont…)

• Storage conditions

 Cold: any TO not exceeding 8OC and usually between 2O and 8OC.

 Cool: Any TO between 8O and 25OC.

 Room Temperature: TO prevailing in working area

 Warm: Any TO between 20O and 40OC.

 Excessive heat: Any To above 40OC.

• Where no specific conditions are indicated it is to be understood that

storage conditions includes protection from moisture, freezing and

excessive heat.

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 The compendia (cont…)

 Other provisions applied to general chapters and monographs

 Quantities

 Apparatus and procedures

 Drying and ignition to constant mass

 Reagents

 Solvents

 Expression of content

 Temperature

 Abbreviation and symbols

 Units of the international system (SI) used in the pharmacopoeia and

equivalence with other units

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 The compendia (cont…)

b) Monograph:

 A detailed and documented article on a particular subject.

 a statement that specifies

 the kinds and amounts of ingredients a drug or class of drugs may

contain,

 the directions for the drug's use,

 the conditions in which it may be used, and the contraindications to its

use.

 It also defines qualitative and quantitative characteristics with the

test procedures and their acceptance limits.

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 The compendia (cont…)

 Official monograph generally is both descriptive and informative: includes the

following

1. Title : generic name, molecular structure, formula and weights are given,

Chemical names have also been provided

2. Therapeutic category

3. a description of its physical characteristics

4. a statement of the minimum standard of purity as determined by the

assay

5. Identification tests for use in verifying the identity of the product

6. limit tests to exclude excessive contamination and /or decomposition

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 The compendia (cont…)

7. Assay method: official quantitative procedures for determination of the

active ingredient, solvents and other constituents require to asses

compliance with the standard

8. physical constants and tests which supplement the standard

9. other information on:

 packaging and storage conditions

 labeling and other regulatory requirements

 Dosage

 cautionary notices on cytotoxic and other such dangerous

materials

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 The compendia (cont…)

General tests

 Contain information basic test procedures which are needed as general

requirements to be tested for the generalized dosage form (tablet, injection,

capsules, suppositories, etc.). It includes

 Apparatus for tests and assay

 Physical tests and determinations : - Disintegration, Dissolution, Color, Loss

on drying, Refractive index, Optical rotation

 Chemical tests and assays : - Identification tests, Limit tests and assays

- Nitrogen determination, Water determination

- Vitamins assay

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 The compendia (cont…)

 Microbial tests

 Antimicrobial preservative effectiveness tests

 Microbial limit tests

 Biological tests and assays

o Antibiotic assay

o Bacterial endotoxin tests

o Bacterial reactivity tests

LIMIT TESTS

 The substances that are used in pharmaceutical field should be almost pure

so that they can be used safely.

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 The compendia (cont…)
 It is almost impossible to get an absolutely pure material as impurities get

incorporated in to them either during manufacture, purification or storage

 The total impurities should be in certain minimal range, i.e. there should be

acceptance criteria for specified impurities.

 In general, specification limit not more than 0.1% for any unspecified

impurity should be included.

 Limit tests are designed to identify and control small quantities of impurity

which are likely to be present in the substance

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 The compendia (cont…)

 Sources of Impurities in Drug Products

1. Raw materials used in the manufacture

2. Method or the process used by the manufacturer

 Reagents, Solvents, Reaction vessels, Catalysts used in the process,

3. Due to the instability of the product

4. From atmospheric contaminates:- dust(aluminum oxide, silica, sulfur, soot,

etc), sulfur dioxide, hydrogen sulfides, arsenic

5. Manufacturing hazards

-microbial contamination, packing errors,

6. Storage conditions- Temperature effects, Physical changes

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 The Law and pharmaceutical analysis
Law – is the body of principles that govern conduct and observance of which can be

enforced in courts.

 It can distinguish what is permissible from what is not.

FDA, Food and Drug Administration, FDA overview

 FDA is an agency of the United States Department of Health and Human Services and is

responsible for:

 Regulating the safety and effectiveness of:

 drug and food (humans and animal), vaccines, ,

 medical devices (human and animal) and radiation emitting devices (including non-

medical devices)

 Regulating biologics, and blood products.

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 The Law and pharmaceutical…….
Authorization and mandate

 The FDA derives its authority and jurisdiction from various Congressional acts.

 The main source of the FDA's authority is the Federal Food, Drug, and

Cosmetic Act.

 The main purpose of the FDA is to protect citizens from products that are

inherently unsafe

 Regulations may take several forms, including but not limited to outright ban,

controlled distribution, and controlled marketing

 Additionally, the FDA sets the standards under which individuals may be

licensed to prescribe and dispense drugs or other medical devices.

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 The Law and pharmaceutical…….
The Organization Components
• 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)

CDER operations

 regulates human pharmaceuticals

 approves new drugs

 determine whether new drugs are unsafe or present risks not

disclosed in the product's labeling 63
 The Law and pharmaceutical…….

The drug approval process

 The FDA is charged with the task of approving or rejecting drugs that

pharmaceutical companies want to market.

 The FDA ensures that newly approved drugs have passed vigorous testing,

which includes:

 animal testing
 clinical trials of healthy individuals, and

 clinical trials of individuals suffering from the disease the drug is

meant to treat.

 The FDA also verifies safety, quality, efficacy, along with drug interactions,

and how various drugs may work depending on age and sex.

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 The Law and pharmaceutical…….

Ethiopian Food and Medicine Administration (EFDA).

 FDA of Ethiopia is a federal agency established by Proclamation No. 661/2018 .

Mission

 To promote and protect public health by ensuring the safety, efficacy, and

quality as well as the proper use of the drugs

 FDA of Ethiopia has four major Departments:

1. Food, Drug Evaluation and Registration Department (DERD)

2. Planning, Drug Information Establishment and Distribution Department (PDIEDD)

3. Drug Control and Abuse Prevention Department (DCAPD)

4. Drug Quality Control and Toxicology Laboratory Department (DQCTLD)

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Scope of Control

 Products Controlled by EFDA

 Human drugs
 Radio-pharmaceuticals
 Traditional medicines
 Medical supplies and instruments
 Sanitary items
 Cosmetics
 Raw and packaging materials
 Food and food products

66
 Institutions Controlled by EFDA
 Manufacturers

 Importers and/wholesalers

 Exporters

 Retail-outlets

 Drug quality control Labs

 Scientific offices,

 Commission agents

67
1.4. Analytical Errors and Validation of
Analytical procedures

68
 Errors in pharmaceutical analysis

• The terminology ‘error’ invariably 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 NIST, BPCRS or 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

69
 Errors in pharmaceutical…

• Analytical errors may be broadly categorized into two heads, namely :

(i) Determinate (systematic) Errors, and

(ii) Indeterminate (random) Errors.

• It is pertinent to mention here that it becomes rather difficult at times to

place a particular ‘error’ into one of the above mentioned categories

i) Determinate (systematic) errors

• These are errors that possess a definite value together with a reasonable

assignable cause;

• however, in principle these avoidable errors may be measured and accounted

for conveniently.
70
 Errors in pharmaceutical…
 The most important errors belonging to this particular class are:

(a) Personal Errors : They are exclusively caused due to ‘personal equation’ of an analyst

and have no bearing whatsoever either on the prescribed procedure or methodology

involved.

(b) Instrumental Errors : These are invariably(never changed) caused due to faulty and

uncalibrated instruments(measuring device), such as : pH meters, single pan electric

balances, UV spectrophotometers,

(c) Reagent Errors : The errors that are solely introduced by virtue of the individual

reagents(substance used to produce a chemical rxn), for instance : impurities

inherently present in reagents ; unwanted introduction of ‘foreign substances’ caused

by the action of reagents on either porcelain or glass apparatus.

71
 Errors in pharmaceutical…

(d) 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

72
 Errors in pharmaceutical…

(f) Errors due to Methodology : Both improper (incorrect) sampling and

incompleteness of a reaction often lead to serious errors.

• A few typical examples invariably encountered in titrimetric and gravimetric

analysis are cited below :

73
 Errors in pharmaceutical…

Indeterminate (random) errors

• As the name suggests, indeterminate errors cannot be pin-pointed to any

specific well-defined reasons.

• They are usually manifested due to the minute variations which take place

inadvertently in several successive measurements performed by the same

analyst, using utmost care, under almost identical experimental parameters.

• 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.

74
Random errors Systematic errors
Affect precision – repeatability or Produce bias – an overall deviation of a
reproducibility result from the true value
Cause replicate results to fall on either side Cause all results to be affected in one sense
of an accepted true value only, all too high or all too low
Can be estimated using replicate Cannot be detected simply by using
measurements replicate measurements
Can be minimised by good technique but Can be corrected, e.g. by using
not eliminated standard methods and materials

Caused by both humans and equipment Caused by both humans and equipment

pharmaceutical QA II 75
pharmaceutical QA II 76
 Errors in pharmaceutical…

Minimizing systematic errors

• Systematic errors may be reduced substantially and significantly by

adopting one of the following procedures rigidly, such as :

(i) Calibration of Instruments, Apparatus and Applying Necessary

Corrections

• Most of the instruments, commonly used in an analytical laboratory, such as

: 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.

77
 Errors in pharmaceutical…

(ii) Performing a parallel control determination

• It essentially comprises of performing an altogether separate estimation

under almost identical experimental parameters with a quantity of a

standard substance that consists of exactly the same weight of the

component as is present in the unknown sample.

• Thus, the weight of the component present in the unknown sample may be

calculated with the help of the following expression :

where, X = Weight of the component present in the Unknown Sample.

78
 Errors in pharmaceutical…

(iii) 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 parmeters as

employed in the actual analysis of the given sample.

79
 Errors in pharmaceutical…

(iv) Method of Standard Addition

• 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 gives the recovery of the quantum

added component.

• Note : The method of ‘standard addition’ is particularly useful to

physicochemical techniques of analysis, for instance : spectrophotometry,

turbidimetry.
80
1.5 Validation of Analytical
procedures

81
 Validation of Analytical procedures

• Validation is the conformation of a newly developed analytical methods that

is going to be used in QC to produce analytical results as designed and

expected.

• 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.

• Accuracy and reliability of the analytical results is crucial for ensuring

quality, safety and efficacy of pharmaceuticals. For this reason, regulatory

requirements have been published for many years.

82
 Validation of Analytical……

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 analyst and 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

83
 Validation of Analytical……
• The common parameters(a limit defining the scope of a process or activity)
that should be verified in method validation are:

Linearity

• The ICH defines the linearity of an analytical procedure as the ability (within a
given range) to obtain test results of variable data which are directly proportional
to the concentration (amount of analyte) in the sample.

• The equation of a straight line takes the form: y = ax + b, wher b intercept of y-

axis, a slope of the line.

• At least five concentration levels should be used. Under normal circumstances,

linearity is achieved when the coefficient of determination (r2) is ≥0.997

84
 Validation of Analytical……

Range

• The range of a method is related to its sensitivity, although there are

methods such as immunoassay w/c are capable of measuring very small

amount of materials, but are not very sensitive in that they measure over

a restrict range of low conc.

• Thus, some typed of detection have very wide dynamic range and other

may only function over a restriction range before linearity is lost. E.g. A

UV detector has a dynamic range of about 1x103 and for a particular cpd

it might measure conc b/n 0.1-100μg/ml.

85
 Validation of Analytical……

Accuracy

• The ICH defines the accuracy of an analytical procedure as the closeness of

agreement between the values that are accepted either as conventional true

values or an accepted reference value and the value found.

• 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.

86
 Validation of Analytical……

Precision

• The precision of an analytical procedure expresses the closeness of agreement

(degree of scatter) between a series of measurements obtained from multiple

samples of the same homogeneous sample under prescribed conditions.

• it does not imply anything with respect to their relation to the ‘true value’

• Precision is usually investigated at three levels: repeatability, intermediate

precision, and reproducibility

• Example : A sample of pure Peppermint Oil is known to contain 31.10 ± 0.03 per cent of

Menthone. The results obtained by two Analysts-1 and 2 are as stated below :

87
 Validation of Analytical……

Re

The arithmetic mean stands at 31.12%

The arithmetic mean is 31.44%

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

88
 Validation of Analytical……

Intermediate Precision.

• Intermediate precision is defined as the variation within the same laboratory.

• 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

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).

89
 Validation of Analytical……

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

other laboratories using the same homogeneous sample lot and the same

experimental design.

 the most common approach is the direct method transfer from the

originating laboratory to the receiving laboratory.

90
 Validation of Analytical……
Selectivity

• The selectivity of a method is a measure of how capable it is of measuring the

analyte alone in the presence of other copds cotained in the sample.

• The most selected analytical methods involves a chromatography separation.

• Detection methods can be ranked according to their selectivity

• A simple comparison is b/n fluorescence and UV spectrophotometery; there are

many more cpds w/c exhibit UV absorption than fluorescence, thus fluorescence

spectrophotometry is more selective methods.

• B/c selective methods are based on more complex principles than non selective

methods they may be less robust, e.g. fluorescence spectrophotometry is more

affected by changes in the analytical method than UV.

91
 Validation of Analytical……

Robustness

• Refers to how resistance the precision and accuracy of an assay is to small variation in the

method. E.g. changes of instrumentation, slight variation in extraction procedure etc.

• Robust assays may not be capable of the highest precision or specificity but they are

regarded as fit for the purpose for w/c they are designed.

Sensitivity

• It indicates how responsive the method is to a small change in the conc of the analyte.

• It can be viewed as the slope on a response curve and may be a function of the method it

self or of the way in w/c the instrument in has been calibrated

92
1.6. Basic calculations in
pharmaceutical analysis

93
 1.6. Basic calculations……

Molarity
 The molar concentration (Cx) of the solution of the chemical species X is the number of
moles of that species that is contained in one liter of the solution (not one liter of the
solvent).
 The unit of molar concentration is molarity, M, which has the dimensions of mol L-1.
Cx= no mole solute = no m mole solute
no L solution no ml solution
 One liter of one molar solution will consist of one mole of solute plus enough solvent to
make a final volume of one liter.
Example:
 Calculate the molar concentration of ethanol in an aqueous solution that contains 2.30g of
C2H5OH (46.07 g/mol) in 3.50L of solution.
 Describe the preparation of 2.00L of 0.108M Bacl2 from BaCl2.2 H2O (FW= 244.3g /mol )

94
 1.6. Basic calculations……
Normality

• Normality (N) is defined as the number of equivalents weight of solute

dissolved in one liter of solution.
N = no of equivalent weight no of equivalent weight = weight of solute

Volume of so/n in Liter equivalent weight

• An equivalent weight is defined as the ratio of a chemical species’ formula

weight (FW) to the number of its equivalents

• The number of equivalents, n, is based on a reaction unit, which is that

part of a chemical species involved in a reaction

• Normality makes use of the chemical equivalent, which is the amount of

one chemical species reacting stiochiometrically with another chemical

species. 95
 1.6. Basic calculations……

• Note that this definition makes an equivalent, and thus normality, a

function of the chemical reaction in which the species participates.

• Although a solution of H2SO4 has a fixed molarity, its normality

depends on how it reacts.

• In an acid–base reaction, the reaction unit is the number of H+ ions

donated by an acid or accepted by a base. For the reaction between

sulfuric acid and ammonia

n = 2 for H2SO4 and n = 1 for NH3

96
 1.6. Basic calculations……

• In a precipitation reaction, for example, the reaction unit is the charge of

the cation or anion involved in the reaction; thus for the reaction

n = 2 for Pb2+ and n = 1 for I–.

• For a complexation reaction, the reaction unit is the number of electron

pairs that can be accepted by the metal or donated by the ligand. In the

reaction between Ag+ and NH3

n for Ag+ is 2 and that for NH3 is 1

97
 1.6. Basic calculations……

• Finally, in an oxidation–reduction reaction the reaction unit is the number of

electrons released by the reducing agent or accepted by the oxidizing

agent; thus, for the reaction

n = 1 for Fe3+ and n = 2 for Sn2+

Example:

 The equivalent weight of H2 SO4 is (FW=98 gm/mol)

 If there is a one liter solution that contains 78.32 grams H2 SO4 , the

number of equivalents is

98
 1.6. Basic calculations……

Weight, Volume, and Weight-to-Volume Ratios

• Weight percent (% w/w), volume percent (% v/v) and weight-to-volume percent

(% w/v) express concentration as units of solute per 100 units of sample

• Percent composition of a solution can be expressed as:

• Weight percent (w/w) = mass of solute X 100%

mass of soln

• Volume percent (v/v) = volume of solute X 100%

volume of solution

• Weight /volume percent (w/v) = mass of solute g X 100%

• volume soln ml

99
 1.6. Basic calculations……

• Weight percent is frequently employed to express the concentration of commercial

aqueous reagents. E.g. 37% hydrochloric solution – this means the reagent contains

37g of HCl per 100g of solution.

• Volume percent is commonly used to specify the concentration of a solution prepared

by diluting a pure liquid with another liquid.

E.g. 5% aqueous solution of methanol – usually means a solution prepared by diluting

5.0ml of pure methanol to give 100ml of solution with enough water.

• Weight /volume percent is after employed to indicate the composition of dilute

aqueous solutions of solid reagents.

E.g., 5% aqueous silver nitrate often refers to a solution prepared by dissolving 5g of

silver nitrate in sufficient water to give 100ml of solution

100
 1.6. Basic calculations……
Parts per million & parts per billion
 For very dilute solutions, parts per million (PPM) is convenient way to express
concentration:
Cppm = Mass of solute X 106 ppm
Mass of solution
 The units of mass in the numerator & denominator must agree.
 For even more dilute solutions we use parts per billion.
Cppb = Mass of solute X109 ppb
Mass of solution
 If we approximate the density of an aqueous solution as 1.00 g/mL, then solution
concentrations can be expressed in parts per million or parts per billion using the following
relationships.

 Example
 What is the molarity of K+ in aqueous solution that contains 63.3 ppm of K3 Fe
(CN)6 (329.2 g/mol)?
101
Dilution and Concentration of Liquids

102
1.7. Physical and chemical
properties of drug molecules

103
 1.7. Physical and chemical……
• The physical properties of a drug molecules along with simple chemical derivatization
and degradation reaction play an important part in the development of analytical
methods.

• Drug molecules can be complex, containing multiple functional group that in combination
produce the overall properties of the drug

Calculation of pH value of aqueous solution of strong and weak acids and

bases
• The pH of a solution is defined as – log [H+], where [H+] is the conc of hydrogen ion in
so/n

• In pure water the conc. of hydrogen ion governed by the equilibrium:

• Ka is the dissociation constant for the equilibrium, is known as Kw in the case of the
dissociation of water

104
 1.7. Physical and chemical……
• Since the conc. of water does not change appreciably as a result of ionization its
conc. Can be regarded as not having an effect on the equiblirium and it can be
omitted from the equation and this mean that in pure water:

• If an acid is introduced into an aqueous so/n the [H+] increase

• Strong acid is completely ionized in water and [H+] is equal to its Molarity e.g. 0.1M
HCl contains 0.1M H+ and has a pH of log [0.1]=1

• For a so/n of a strong base such as 0.1NaOH, [OH-]=1M and

[H+] [OH-]=1x 10-14, therefore [H+]=1x10-13 and pH=13

• Weak acids are not completely ionized in aqueous so/n and are in equilibrium with
the undissociated acid, as is the case for water, w/c is a very weak acid.

105
 1.7. Physical and chemical……
• The dissociation constant Ka is given by the expression below:

• For instance in a 0.1M so/n of acetic acid (Ka=1.75 x 10-5) the equilibrium can be written
as follows:

• The pH can be calculated as follows:

• Since the dissociation of the acetic acid does not greatly change the conc. of the
unionized acid the above expression can be approximated to:

• In comparison the pH of 0.1M HCl is 1

106
1.7. Physical and chemical……

107
 1.7. Physical and chemical……

Acidic and basic strength and pKa

• The pKa value of a cpd is defined as :pKa= - log Ka

• If pKa is used as a measure of acidic or basic strength, for an acid the smaller the pKa
vallue the strongest the acid. For a base the largest the pKa value the stronger the base

• For an acid the forward reaction used

• In the case of a base it is the protonated form of the base that act as a proton donor

Buffer solution
• A solution containing a weak acid/ base and its conjugate base/acid that is resistant to a

change in pH when a strong acid or strong base is added.

• Adding as little as 0.1 mL of concentrated HCl to a liter of H2O shifts the pH from 7.0 to
3.0. The same addition of HCl to a liter solution that is 0.1 M in both a weak acid and its
conjugate weak base, however, results in only a negligible change in pH

108
 1.7. Physical and chemical……
 A mixture of acetic acid and sodium acetate is one example of an acid/base buffer

 The equilibrium position of the buffer is governed by the reaction

 The relationship between the pH of an acid–base buffer and the relative amounts of
CH3COOH and CH3COO– is derived by taking the negative log of both sides of the
above equation and solving for the pH

 Buffering occurs because of the logarithmic relationship between pH and the ratio of
the weak base and weak acid concentrations.

 For example, if the equilibrium concentrations of CH3COOH and CH3COO– are equal,
the pH of the buffer is 4.76.

109
 1.7. Physical and chemical……

• A more useful relationship relates the buffer’s pH to the initial concentrations of weak
acid and weak base.

• A general buffer equation can be derived by considering the following reactions for a
weak acid, HA, and the salt of its conjugate weak base, NaA.

• After several rearrangement, it provide us a general formula known as henderson-

Hasselbalch equation

• Where CNaA conc of salt, CHA conc of the acid

• Hasselbalch equation provides a simple way to calculate the pH of a buffer and to
determine the change in pH upon adding a strong acid or strong base.

• For a base, henderson-Hasselbalch equation is written as

110
 1.7. Physical and chemical……
• Using hasselbach equation it is possible to determine degree of ionization of a
drug at a given pH. E.g. degree of ionization of acetic acid at pH of 4.76

acetic acid ionized 50% at pH of 4.76

111
 1.7. Physical and chemical……

Stability of drugs
 Many drugs are quite stable but functional groups such as esters and lactam rings
w/c occur in some drugs are susceptible to hydrolysis and functional groups such as
catechols and phenols are quite readily oxidized.
 The most common type of degradation w/c occur and formulated drugs obey zero or
first order kinetics

Zero order degradation

 In zero order kinetics the rate of degradation is independent of the conc of the
reactant.
 Thus, if the rate constant for the zero order degradation of a subs is 0.01mole/h
then after 10hr 0.1 mole of the subs will have degraded
 This type of degradation is typically of hydrolysis of drugs in suspension or tablets
where the drugs is initially in the solid state and gradually dissolve at the same rate
as the drug in so/n id degraded

112
 1.7. Physical and chemical……
First order degradation
• This type of degradation would be typical of hydrolysis of a drug in so/n

• In first order kinetics the rate constant k has units h-1 or s-1 and the rate of the
reaction for a drug is governed by the expression

• From this expression by integration and rearrangement the following expression arises:

• The half life of the drug (the time taken for 50% of a sample drug to degrade, i.e. where
x is a/2) is thus given by the following expression

113
The End

114