REFERENCE

STANDARDS
AND DRUG PURITY

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 • REFERENCE STANDARDS
• For any development/validation processes we should consider
first obtaining a sufficient quantity of a good qualified reference
standard material and appropriate samples to support
development and validation processes.

• Material selected for use as a standard should be of high purity

and stability

• Requirements for reference standards differ, depending on

objectives and stage in drug development cycle.
•
• * Reference Standard according to European
• Pharmacopoeia (EPCRS): A substance or mixture
• of substances intended for use as stated in a
• monograph or general chapter of European
• Pharmacopoeia. EPCRS are considered as
• primary standards. 2
Reference standard according to FDA guidelines:
A particular lot or batch of drug substance specifically prepared, either by
independent synthesis or by additional purification of production material
and shown by an extensive set of analytical tests to be authentic material
of the highest purity reasonably attainable. It is usually used for
structural elucidation, and is the benchmark for working standards.

Reference Standard according to ICH guidelines:

- A reference standard or reference material is a substance prepared for
use as a standard in an assay, identification or purity test.
- It has a quality appropriate to its intended use.
- For new drug substances reference standards intended for use in assays,
purity should be measured by a quantitative procedure.
Note that, The impurities should be adequately identified
and/or controlled and

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• Reference Standards are characterized according to
the intended use as follows:
• (A) Primary Standards:
• Materials which are accepted without reference to other
standards.
• Their identity must be provided and their purity must be high
and stated (>99.0%)
• Characterization of primary standards generally involves
elucidation of chemical structure by tools such as; IR, UV, H-
NMR, C-NMR, MS, ….. etc.
• Purity determination using HPLC, TLC, GC, DSC, residue of
• ignition, water content …..etc.
• Assay: Titration, DSC, Chromatography.
• (N.B.) Analytical procedures (purity and assay) used
• for characterization must be validated.

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• (B) Pharmacopeial Standards
• Commonly used for certain tests and assays to achieve accuracy
and precision of analytical results required in the compendial
monographs.
• It may be used only for purpose for which it is intended.
• Note: Pharmacopeial standards are basically regarded as
primary standards.
• All the pharmacopeial standards are reference standards.

• (C) Working Standards

• Materials are designed for daily use in analysis
• such as routine quality control.
• They are characterized (standardized) by
• comparison with Primary or Pharmacopoeial
• standards.
• Their purity corresponds to a "typical batch".

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• (D) Impurity Standards:
• They are mainly required for development and validation of
analytical procedures (Specificity, LOD & LOQ)
• For routine controls, the impurity standards are not generally
needed.

• (E) Related compound standards:

• These standards are typically characterized (standardized) , but
not to the same extent as primary or reference standard of the
active drug substance.

(F) Other standards:

Reagents and chemicals that are commercially
available at high purity and have been
characterized by trusted methods can be used
as standard materials.

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 Need and Use
Analytical procedures that may require a chemical reference
substance are:
1- IR spectrophotometry for identification or quantitative purposes.
2- UV-Vis absorption spectrophotometry (Quantitative).
3- Methods based on chromatographic separation for identification
or quantitative purposes.
4- Quantitative methods based on other separation techniques e.g.
Capillary electrophoresis etc.
5- Quantitative methods, often titrimetric but sometimes
gravimetric, that are based on non stoichiometric relationships.
6- Assay methods based on measurement of optical rotation.
7- Methods that might require a chemical reference substance
consisting of a fixed ratio of known components (such as
cis/trans isomers and spiked samples)

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 Reference substances required for:
• identification (of substance for pharmaceutical use)

• system suitability (to ensure adequate performance of the

applied technique)

• purity (identification and estimation of specified impurities in a

substance for pharmaceutical use manufactured by a particular
route of synthesis)

• assay (of content of the substance for pharmaceutical use or its

products).

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• Dealing with the reference standards
• 1. Storage:
•Standard material should be protected from light, heat, and
moisture & housed in a controlled environment.

•Typical storage conditions would be a 2-80C stability room,

protected from light and from humidity.

•Reference stock solutions and working solutions should also be l

with the required storage conditions and expiration dates.

• - Method procedure should specify; the storage

• conditions and expiry dates.
• - Containers should be sized to minimize headspace.
•

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2.Handling within storage time:
- Reference material must be handled in such a way that
preserve integrity of the sample.

- Standard materials that at stored at sub-ambient temp. must

be equilibrated to ambient before opening to prevent water
condensation.

- If primary standard materials are used frequently, it can

be subdivided into several smaller containers to limit
contamination and changes in assay.

- Certain standards require oven drying prior to use.

- Drying conditions should be adjusted to minimize
any potential decomposition.

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 3.Documentation
• Used to support its use as a standard, establish its assigned assay
and define re-test date (or expiry date). Documentation includes:
• (a) Reference Standard Qualification Report
1- A detailed qualification report is written for primary reference
standards of active compounds. Less comprehensive documents
is required for other standards (e.g. impurity standards).
2- Description of the assay method used in characterization of the
standard.
3- Report results of characterization supported with data (UV, NMR
spectra, HPLC fig., etc.)
4- Verification of data to establish purity and assay
of standard
5- Appendix detailing additional requalification and
stability data as they are obtained.

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 (b) Certificate of Analysis
• All standards should have a certificate of analysis generated
either from a qualified supplier or through analysis of in-house
data.
• The certificate should contain the following information.
1- Standard name 2- Lot number and supplier
3- Effective data and recertification date
4- Purity 5- Assay results

Chemical Purity and Quality Control

Purity Is freedom from foreign substances.
Purity Is a relative term.
Absolute purity
Is unobtainable, and hard to be achieved.
Reasons include The expenses, purification
limitations and stability properties.

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• Impure substance;
• Is a substance of interest mixed with an extraneous or inferior
substances. Thus a simple definition for an impurity, is that, any
material can affect the purity of the material of interest.

Sources of Impurities: includes;

1.Synthesis-related impurities:
Originate mainly during synthetic process from raw materials
reaction vessels, reagents, catalysts and solvents, intermediates
and by-products.

2.Formulation-related impurities:
Originate mainly from inert ingredients used to
formulate a drug substance, degradation or other
reactions of drug during formulation process.

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 General terminology
• There are a number of terms that commonly used to describe
impurities:
Intermediates;
• Compounds produced during synthesis of desired material
Penultimate intermediate;
• The last compound in the synthesis chain before production of
the final desired compound.
By-products;
• The unplanned compounds that produced in a reaction. It may or
may not theorize all of them.
 Transformation products;
• Very similar to by-products, except that more
information is known about the transformation
reaction products.

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o Interaction products;
• Compounds produced through interactions that could occur
between various involved chemicals intentionally or
unintentionally.
o Degradation products;
• Compounds produced due to decomposition of the material of
interest or active ingredient.
o Related products;
• Products that can have similar chemical structure as the active
ingredient and potentially similar biological activity.
o Chiral products;
• Related products that having the same chemical
structure of active ingredient but different spatial
orientation, leading to different optical rotation.
The undesired optical isomer is considered a chiral
impurity.

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 Compendial (Pharmacopeial) terminology
• USP and BP describe impurities with many terms as:
oForeign substances, Compounds that introduced by
contamination or adulteration and not by consequences of
synthesis or preparation.
oToxic impurities, They have significant undesirable biological
activity and require individual identification & quantification by
specific tests.
oConcomitant compounds, Bulk pharmaceutical chemicals may
contain concomitant compounds, e.g. geometric & optical isomers
of antibiotics that are present in mixtures.
o Ordinary impurities, Species of impurities in bulk
pharmaceutical chemicals that having no significant undesirable
biological activity in amounts present.
o Signal impurities, They include some process-related
impurities or degradation products that provide key information
about process.
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o Organic volatile impurities; This term relates to residual
solvents that may be found in the drug substance.
o Inorganic impurities; these impurities include common salts
(NaCl, NaHCO3 etc..) or heavy metals (Pb, As, Hg … etc.)
Limits for impurities in bulk drug substances must consider a
number of factors from which:
1)Toxicology of a drug containing typical levels of impurities and
toxicology of impurities relative to a drug substance.
2)The route of administration, e.g., oral, topical or parenteral.
3) The daily dose, (frequency and amount administered).
4)The target population (age and disease state).
5)The pharmacology of an impurity.
6)The source of a drug substance, e.g., synthetic, natural product, or
produced by biotechnology.
7) Duration of therapy, i.e., administration over a long period
(chronic conditions) or over a short period (of acute conditions).
8) Capability of a manufacturer to produce high-quality material with
a reasonable cost to consumers

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Thus chemicals can be classified depending on their
purity to different grades including:
• 1) Industrial chemicals
• These are known as technical or commercial grade.
• This is the major part of chemistry production, which contains
amount of impurities.
• 2) Pure chemicals
• These are reagent-grade chemicals, which have been purified as
possible as could be.
• They are used mainly for synthesis of drugs and drug design.
• 3) Analytical chemicals
• They are one of the most pure chemicals .
• Used for preparation of standard solutions,
• reference materials and for analytical research
• work.e.g. Spectroscopic-grade and HPLC-grade
• chemicals
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4) Pharmaceutical grade
They are very pure chemicals, which may contain traces of
impurities but these impurities must be:

oNot harmful
oNot cause any undesirable effects and
oNot contraindicated or interfere with the intended use of
the pharmaceutical product.

These chemicals should be confirmed with one of pharmacopoeial

standards and they should have labels bear the abbreviation of the
pharmacopoeia to which they confirmed.

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STABILITY OF DRUGS AND
DRUG PRODUCTS

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 Pharmaceutical product stability
STABILITY is defined as the extent to which a product retains the
same properties and characteristics that it possessed at the time
of its manufacturing within specified limits, and throughout its
period of storage and use.
• Factors affecting product stability;
• Stability of the active ingredient(s).
• Potential interaction between active and inactive ingredients
• Manufacturing process
• Dosage form
• Container-liner-closure system and environmental conditions
encountered during shipment.
• Handling, storage conditions and length of time between
manufacturing and usage.

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• Pharmaceutical product stability separated into chemical and
physical stability of formulations.
• There is no absolute separation between the two classes.
• Physical factors such as heat, light and moisture may initiate or
accelerate chemical reactions.
• While every time a measurement is made on a chemical property,
physical dimensions are included in the study
• Physical stability of pharmaceutical formulations:
• Importance:
(a) Pharmaceutical product must appear fresh, elegant and professional so
long as it remains on the shelf. Any change such as color fading can cause
patient to lose confidence in the product.
(b) Since some products are dispensed in multiple-dose containers,
uniformity of dose content of active ingredient over time must be assured.
(c) Active ingredient must be available to the patient throughout the
expected shelf life of preparation.
(Note that) Physical changes in pharmaceutical products mainly
depend upon the type of dosage form itself

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 Examples of such physical changes;
Formulation Physical instability problems Effects
1- Oral solutions 1- Loss of flavor 2- Change in Change in smell or feel
taste 3- Discoloration or taste
4- Presence of odd flavors due to
interaction with plastic bottles
5- Loss of dye 6- Precipitation
2- Suspensions 1- settling 2- caking 1-Loss of drug content
3- crystal growth uniformity in different
doses 2- loss of
elegance.
3- Tablets Change in: a) Disintegration time Change in drug release
b) Dissolution profile c) Hardness
d) Appearance
4- Capsules Change in: a) Appearance Change in drug release
b) Dissolution c) Strength
Formulation physical instability problems Effects

5- Parenteral 1. Discoloration due to photochemical Change in appearance

solutions reactions or oxidation & in bioavailability
2. Presence of ppt due to interaction
with container or stopper
3. Presence of “whiskers”
4. Clouds due to: (i) Chemical changes
(ii) Supersaturated solutions.
6- Emulsions 1- Creaming 1- Loss of drug content
2- coalescence uniformity in different
doses from the bottle
2- loss of elegance
7- Semisolids 1- Changes in: a) Particle size 1-Loss of drug content
(Ointments b) Consistency uniformity
&suppositorie 2. Caking or coalescence 2- loss of elegance
s) 3-change in drug
3. Bleeding
5/5/2020 release rate. 24
Pharmaceutical Analysis & Quality Control (1) Dr.Abdel-Maaboud Ismail
• Chemical stability of drug products;
• Incompatibility:
• Undesirable reactions between two or more components are
said to result in a:
• (a) Physical incompatibility; visibly recognizable change e.g., a
gross precipitate or color change.
• (b) Chemical incompatibility changes in chemical properties
accompanied with visible or non visible changes.
• (c) Therapeutic incompatibility; includes increase of a
therapeutic effect, destruction of effectiveness and occurrence
of toxic manifestations.

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(A) Physical Incompatibility
Interaction between two or more substances which lead to change
in color, odor, taste, viscosity and morphology.
Ex (1): Benzalkonium chloride & Sodium lauryl sulfate:
Benzalkonium chloride is positively charged while sodium lauryl
sulfate has a negative charge. By mixing a precipitate is formed.
Ex (2) Ephedrine sulfate, Menthol &Liquid paraffin: Ephedrine
sulfate is a water soluble salt but insoluble in organic solvents, oil
and paraffin.
(B) Therapeutic incompatibility
Modification of therapeutic effect of one drug by prior concomitant
administration of another. (It is also called drug-drug interactions).
1. Pharmacokinetics:
Involve effect of a drug on absorption, distribution
, metabolism and/or excretion of another one.
2. Pharmacodynamics:
are related to the pharmacological activity of the
interacting drugs e.g; synergism, antagonism
effects and effect on the receptor sites ….. etc.
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Examples of Pharmacokinetic interactions:
Ex(1) Tetracycline interacts with iron preparations or calcium salts in
milk to form un-absorpable complexes.
Ex (2) Antacids (containing aluminium and magnesium hydroxides)
decrease absorption of ciprofloxacin by about 85% due to chelation.
Examples of Pharmacodynamic interactions
Ex (1); β-adreno-receptor antagonists diminish the effectiveness
of β-receptor agonists, such as salbutamol.
Ex (2); Many diuretics lower plasma potassium concentration, and
thereby enhance some actions of digoxin and may lead to
glycoside toxicity.
Ex (3); Non-steroidal anti-inflammatory drugs
(NSAIDs), such as ibuprofen or indomethacin,
inhibit biosynthesis of prostaglandins.

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(C) Chemical Incompatibility
Reaction between two or more substances which lead to change in
chemical properties of pharmaceutical dosage form.
Factors that can induce chemical incompatibility:
1. Presence of oxygen & oxygenated compounds (e.g. peroxides)
2. Light and other types of electromagnetic radiations .
3. Elevated temperature accelerates chemical interactions.
4. pH: each drug has its ideal pH for stability. Any change in pH
can affect drug stability and may accelerate certain interactions
5. Type of dosage form, excipients & additives or vehicles used.
6. Presence of pre-oxidants as some metal ions and peroxides
7. Presence or absence of water & other types of solvents used.
8. Presence of certain impurities (amount & type).

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1- Light & Photochemical Decomposition:
Generally, light can cause photo-chemical reactions. UV radiation is
the main cause of many degradation reactions. The intensity and
wavelength of the light and the size, shape, composition and color
of container may affect the velocity of such photo-reactions
R1
R2 O Radical induced
O
O oxidation
R3 OH
R1 R1
h
R2 H R2 C
R3 R3 R1 R1 R1 Radical formation
R2 C R2 R2 and polymerization
R3 R3 R3

Example:Photodegradation of Indomethacin:
CH2COOH R
MeO MeO
h, Hg lamp
CH3 CH3
N MeOH N
COC6H4Cl COC6H4Cl
R = Me, CHO
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2- Ultrasonic energy and ionizing radiation:
• Ultrasonic energy consists of vibrations with frequency greater
than 20,000 Hz, It may cause formation of free radicals and alter
drug molecules.
• Example;
• Changes in prednisone acetate and de-oxycorticosterone acetate
suspensions in an ultrasonic field have been observed.
• From other hand, ionizing radiations, e.g. gamma rays and X-
rays, which used for the sterilization of certain pharmaceutical
products may cause a chemical degradation such as UV radiations
3- Presence of ions & its ionic Strength:
In general, some rate constants such as the rate of
hydrolysis is inversely proportional to the ionic
strength with oppositely-charged ions (e.g., drug is
a cation and excipient is an anion) and directly
proportional to the ionic strength with ions of
similar charges.

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4- pH Effect:
Degradation of many drugs in solution accelerates or decelerates as
the pH is changed over a specific range of pH values.
Improper pH values may cause a clinically significant loss of drug
activity resulting from hydrolysis and/or oxidation reactions.

A drug solution or suspension, may be stable for weeks, or even

years in its original formulation, but when mixed with another
liquid that changes itsbpH, it degrades in minutes or days.
The most affected drugs are those containing weakly acidic and/or
weakly basic groups. (Ex. Atropine: optimal pH=3.1-4.5 and
penicillines, optimal pH around pH=6)

5- Inter-ionic compatibility:
Compatibility or solubility of oppositely charged
ions depends mainly on the number of charges per
ion and the size of ions.
In general, polyvalent ions of opposite charge are
more likely to be incompatible.
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6- Temperature:
In general, rate of a chemical reaction increases exponentially
for each 10° increase in temperature.
This relationship has been observed for nearly all drug hydrolysis
and some drug oxidation reactions.
Suitable storage condition is a way of limiting such decompositions.
Examples:
- Use of darkened glass - Displacement of air with nitrogen
- Use of sealed containers. - Temperature controlling
- Including of antioxidant e.g., ascorbic acid.

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• Types of chemical changes that can occur:
1. Combination
2. Hydrolysis
3. Oxidation
4. Polymerization
5. Isomerization
6. Decarboxylation
7. Absorption of Co2
8. Formation of insoluble complexes
9. Dehydration