Introduction to Pharmaceutical

Analysis
Definitions
Pharmaceutical analysis:
 is a science which deals with identification and
quantification of drugs in raw materials, dosage
forms and biological fluids.
Or
 An applied science that insures 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 of drug entities from natural products.
Why analyzing drugs?
– 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
Generally, pharmaceutical analysis procedures may be
used to answer:

1. Is the identity of the drug in the formulated product correct?

2. What is the percentage of the stated content of a drug present

in a formulation?

3. Does this formulation contain solely the active ingredient or

are additional impurities present?

4. What is the stability of a drug in the formulation and hence

the shelf–life of the product?

5. At what rate is the drug released from its formulation so that

it can be absorbed by the body.
6. Do the identity and purity of a pure drug substance to be used in
the preparation of a formulation meet specification?

7. Do the identity and purity of excipients to be used in the

preparation of a formulation meet specification?

8. What are the concentrations of specified impurities in the pure

drug substance?

9. What is the concentration of the drug in a sample of tissue or

biological fluid?

10. What are the Pka value (s), partition coefficients, solubilities,
and stability of a drug substance under development?
Drug Analysis

– Estimates the purity and quality of drugs and fine

chemicals

– It also involves in the analysis of medicinal agents and

their metabolites in human or animal body
Scope of pharmaceutical analysis:
• Pharmaceutical industry
– Raw material control
– In-process control
– Dosage form control

• Government drug control laboratory

(regulatory agencies) …EFDA
– Manufacture lab. Procedure control
– Products control
• Pharmaceutical research labs
– Advanced research
• Development of analytical method
• Process development
– Dosage form evaluation
– Stability studies
– Basic research
• Separation
• Identification
• Quantization
• Medicinal characterization
 Introduction to quality control and
quality assurance
What is Quality?
ISO definition:
 "The totality of features and characteristics of a product or

service that bear on its ability to satisfy stated or implied needs".

 When projected on analytical work, quality can be defined
as "delivery of reliable information within an agreed span of
time under agreed conditions, at agreed costs, and with
necessary aftercare".
Quality Assurance
"the assembly of all planned and systematic actions necessary to
provide adequate confidence that a product, process, or service will
satisfy given quality requirements."
Quality Control:

 A major part of the quality assurance "the operational

techniques and activities that are used to satisfy quality
requirements.

 Quality of drugs should meet the standards related to safety,

potency, and efficacy.

 evaluated by various quality control methods.

A QC program for drug industry involves with
– batch-to-batch uniformity of a product
 Desired characteristics of identity, purity, potency,
uniformity, safety, efficacy and stability
QC also includes
Packing and storing,
Labeling,
Expiry date and
The distribution in a manner that retains potency
QC is concerned with
– Sampling
– Specification and testing
– Documentation and release procedures

Aim of QC
 To evaluate whether the sample of drug complies with the

appropriate specifications, based on various tests .

Types of tests for QC
– Chemical methods
– Physico-chemical methods
– Microbiological methods
– Biological methods
Objectives of Quality Control:
Make sure that
– Proper sampling and analytical test are done

– Products are made which demonstrate that all the required

sampling, inspecting and testing procedures were actually carried
out.

– The finished products contain active ingredients and enclosed with

in their proper container and correctly labeled.

– No batch of product is released to sale of supply prior to

certification by qualified person.

– Sufficient reference sample of starting materials and products are

retained to permit future examination of the product if necessary.
Sources of Impurities in Drug Products
• Raw materials used
• Method of manufacturer adopted – e.g. reagent, solvent
• Due to the instability of the product…degradation
• From atmospheric contaminates
 Official Methods of Standardization
 Quality of pharmaceuticals is based on pharmacopoeia
specifications.
 Official methods of standardization i.e. monographs of
pharmaceutical chemicals and formulated products are
– descriptive
– informative
– Contain limits of purity
– standards of the product and
– storage conditions
Monograph:
• A written description of the principal features of
the substance and the ways that these features can
be determined
Pharmacopoeia:
• Monographs of pharmaceutical substances
when collected together.
National pharmacopoeia:
• Legally-binding definition of the drugs and
preparations whose monographs are contained
there in.
E.g. UK – BP
USA – USP
Reasons for having a standard:
• To protect the user (patient, pharmacist,
manufacturer, government office, health
authority) against drugs of poor quality.
• To guarantee /ensure that the analytical methods
used are;
– standard,
– keep a breast of recent developments both in
analytical techniques and in quality considerations of
the drug
Contents of Official monographs
1. Description
2. Minimum standard of purity
3. Identification tests
4. Limit tests to exclude excessive impurities
5. Physical constants
6. Storage conditions and packaging
7. Labeling
8. Dosage
9. Therapeutic category
10.Quantitative assays
Titles:
Main titles
• Subsidiary titles or synonyms
– Molecular formula and
– Molecular weights
– Chemical names have also been provided (IUPAC)

Identification:
• Tests for identity are provided only as an aid to identification.

• Identifications tests are usually based on qualitative tests for basic

and acidic radicals for inorganic chemicals.

• Organic substances are identified by the various characteristic

reactions of one or more of the functional groups present in the
molecule
Dosage:
• Doses mentioned in monographs are intended for
general guidance
Minimum standards of purity, Assay Tolerance:
• Upper limit is not stated, the upper limit is not more
than the equivalent of 100.5%.
• The range is inclusive of the two limits and values
outside the range is not acceptable
• Allow for
– Analytical errors
– Variations in manufacture and compounding, and
– Deterioration to an extent considered insignificant under
practical conditions
Description:
• Is relatively general in nature
• It is provided to indicate the properties of the chemicals
• The properties are not in themselves standards or tests
for purity though they may help in the preliminary
evaluation of chemical.

Solubility:
• It provided primarily as information
• However, where quantitative solubility test is given under
standards the drug should comply with all these
requirements.
E.g. Very soluble
Freely soluble
Packaging, storage and labeling:
The container should
– not interact physically or chemically with the chemical
– be sight sensitive,
– well-closed, etc…

Storage conditions do not form a part of the standards

• Cold: any TO not exceeding 8O and usually between 2O
and 8OC.
• Cool: Any TO between 8O and 15OC.
• 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.

• In general labeling of drugs and pharmaceuticals

is governed by Dugs and Cosmetic Act (EFDA).

• Additional information which must be stated on

label is mentioned in the monograph.
Limit Tests:
• Limit tests are important to determine the
permissive limits of tolerance

• Usually involve simple comparison of

opalescence, color, or turbidity with that of
standard sample prescribed in pharmacopoeia.

• In these tests, concentration of impurity is

expressed as PPM or as percentage.
Physical Constants:
• Are characteristic properties useful for both
identification and maintenance of standards of purity

– included in monograph for the standardization of

pharmaceuticals.

These are:-
 melting point,
 boiling point,
 refractive index,
 optical rotation,
 light absorption,
 solubility etc..
Quantitative Assays:
• Assay methods
– specific for the chemical
– stability determination.
• The procedures of quantitative analytical chemistry are
applied to the analysis of materials used in pharmaceuticals.

• In analytical chemistry
– Qualitative (what a substance is composed of ) and
– Quantitative (exactly how much)
• In qualitative analysis
– the presence or absence of one or more components
• Quantitative analysis,
– how much of the pure component is present
• There are various methods of quantitative analysis.
 Chemical methods
Volumetric

Gravimetric

Gasometric

 Physico-chemical methods (instrumental)

 Microbiological methods

 Biological methods
Steps in Atypical Quantitative Analysis
1. Formulating the questions to be answered through
chemical measurement

2. Selecting analytical procedures

– Search the chemical literature
– Developing original procedure

3. Sampling
– Bulk sample
4. Sample preparation
1. Homogenous laboratory sample

2. Converting lab. sample to the one suitable for analysis –

dissolving, making concentration prior to analysis

3. Extraction– to remove mask species or interferences

5. Analysis
– Clear written report with limitations

6. Reporting and interpretation

– Conclusions should be consistent with the data
Methods of Quantitative Analysis
1. Chemical methods
a. Volumetric methods
– Preferred to gravimetric processes especially because of
speed and convenience.

– Assay is based on the measurement of volume of solution of

known strength that is required to react completely with the
substance to be analyzed.

– On the basis of this measurement of volume, quantity of pure

component is estimated.
• Criterion for selection of reagent is that
– The reagent must react rapidly
– Reaction must be complete
– There should be some method to detect the completion of reaction

• This method is classified in to different types

depending up on the type of reactions involved in the
titration process.
They are:
– Neutralization titrations
– Non-aqueous titrations
– Precipitation titrations
– Oxidation reduction titrations
– Complexometric titrations
b. Gravimetric methods
– Is a quantitative analysis by weight
– It is a process of isolating and weighting the compound of
known composition i.e. purest form.

– The separation of compound is affected by number of ways

like:
Precipitation,
Volatilization,
Electro analytical etc.
– Although gravimetric analysis is time-consuming, the
constituent may be examined in the presence of impurities
and correction can be applied if necessary
C. Gasometric methods:
 Involve measurement of the volume of gases
• They measure the:
– Volume of gas librated in the given chemical reaction under the
conditions that are described in the process

– Decrease in the volume of gas when a suitable agent is placed to

absorb one of the gases present and reduced to standard
conditions of temperature and pressure

E.g. Cyclopropane, CO2, nitrous oxide, octylnitrate, nitrogen,

amylnitrate, ethylene and helium etc.

• The measurement of gas is generally made in gas burettes or

by nitro meters.
2. Instrumental methods
• They are based on the relation between the content and
corresponding physicochemical and physical properties
of the chemical system being analyzed.

• The changes in the properties of the system are generally

detected through the measurement of
– Current,
– Potential,
– Electrical conductivity,
– Optical density,
– Refractive index etc. with suitable and sensitive instruments
E.g. Electrical potential – Potentiometery
Mass to charge ratio – Mass spectrometry
• In addition to above methods, the chromatographic methods
are available

• A procedure by which active ingredient, excipients and purities

are separated by passage of mixture through affixed porous bed
(stationary phase) possessing varying but reversible affinity for
individual components.

• Is a separation technique or a device by which a mixture of

substances is separated into its various components

• These methods are also used for identification as well as

quality control of various pharmaceuticals.

E.g. column, paper, thin layer, gas, ion exchange, HPLC, etc.
3. Microbiological methods
• The inhibition of microbial growth under the standard conditions
may be utilized for knowing therapeutic efficacy of antibiotics.

• Very important for resolving doubts regarding possible loss of

potency of antibiotics and their preparations.

• Is based up on a comparison of the inhibition of growth of bacteria

by measured concentration of antibiotics to be examined with that
produced by
 Known concentration of standard preparation of the antibiotic
having a known activity

• Two general methods are usually employed,

– The cylinder plate (or cup plate) method and
– The turbidimetric (tube assay) method.
–
4. Biological Methods
• Are prescribed when the potency of a drug or its
preparation can not be adequately determined
by chemical or physical means,
• But it may be possible to observe the biological
effect of the drug on some type of living matter.
E.g. a stimulus applied to a subject
 Analytical errors and their control
 A quantitative analysis is not great deal of use unless there is some
estimation of how prone to error the analytical procedure is

 Analytical result may lead to acceptance or rejection of a product

on the basis of faulty analysis.
 Repeated measurements of the same sample to determine the
degree of agreement b/n them
Three types of errors
 Gross errors- easily recognized b/c involve major breakdown in
the analytical process
Sources - Wrong dilutions being prepared
- Instruments breaking down or being used in wrong way
 The result is rejected and the analysis is repeated
Random and systematic errors
Example

Calculate the mean of measurements of each student by using the

following formula:
• Student 1 found a mean which is very close to the
true value
– Precise and accurate
– The analysis procedure is done carefully
• Student 2
– Closely clustered results but a mean which is less than
the true value
– Precise but not completely accurate
– No random errors but systematic errors
• Random errors lead to the production of scattered
measurements and systematic errors lead to a
mean which is not close to the true value
Factors giving rise to inaccuracy and imprecision in an
assay
 Validation of Analytical Methods

• Validation of analytical procedures is the process of

determining the suitability of a given methodology for

providing useful analytical data.

• Validation is the formal and systematic proof that a method

compiles with the requirements for testing a product when

observing a defined procedures.

44
• Method validation is the process of demonstrating that
analytical procedures are suitable for their intended use and
that they support the identity, strength, quality, purity and
potency of the drug substances and drug products
Primarily concerned with:
Identification of the sources of potential errors
Quantification of the potential errors in the method

45
Examples of Methods That Require Validation Documentation

• Chromatographic Methods - HPLC, GC, TLC, GC/MS, etc.

Pharmaceutical Analysis
Bioanalytical Analysis - In support of PK/PD/Clinical Studies.
• Spectrophotometric Methods – UV/VIS, IR, NIR, AA, NMR
• Capillary Electrophoresis - Zone, Isoelectric Focusing
• Particle Size Analysis Methods - Laser, Microscopic, Sieving, SEC,
etc
• Automated Analytical Methods - Robots, Automated Analysis

46
Considerations Prior to Method Validation

Suitability of Instrument
• Status of Qualification and Calibration
Suitability of Materials
• Status of Reference Standards, Reagents, Placebo Lots
Suitability of Analyst
• Status of Training and Qualification Records
Suitability of Documentation
• Written analytical procedure and proper approved
protocol with pre-established acceptance criteria

47
 Validation Steps
 Define the application, purpose and scope of the method
Analyte? Concentration? Sample matrices?
 Develop an analytical method
 Develop a validation protocol
 Qualification of instrument
 Qualify/train operator
 Qualification of material
 Perform pre-validation experiments
 Adjust method parameters and/or acceptance criteria if necessary
 Perform full validation experiments.
 Develop SOP for executing the method in routine analysis.
 Document validation experiments and results in the validation
report 48
 Purpose of Method Validation

 Identification of Sources and Quantitation of Potential errors

 Determination if Method is Acceptable for Intended Use

 Establish a Proof that a Method Can be Used for Decision Making

 To prove that the method satisfy official method requirements

49
What is not Analytical Method Validation?

• Calibration

 The Process of Performing Tests on Individual System

Components to Ensure Proper function

For example: HPLC detector calibration

– Wavelength : Accuracy/ Linear Range/ Noise Level/ Drift

50
• System Suitability
 Test to verify the proper functioning of the operating system,

i.e., the electronics, the equipment, the specimens and the

analytical operations.

– Minimum Resolution of 3.0 between the analyte peak and internal

standard peaks

– Relative Standard Deviation of replicate standard injections of not

more than 2.0%

51
 Method Life Cycle

Validation

Development Optimization

52
 Published Validation Guidelines
• 1978 Current Good Manufacturing Practices (cGMPs)
• 1987 FDA Validation Guideline
• 1989 Supplement 9 to USP XXI
• 1994 CDER Reviewer Guidance: Validation of Chromatographic Method
• 1995 ICH Validation Definitions:
Q2A, Text on Validation of Analytical procedures
• 1997 ICH Validation Methodology:
Q2B, Validation of Analytical Procedures: Methodology
• 1999 Supplement 10 to USP 23 <1225>: Validation of Compendial Methods
• 1999 CDER “Bioanalytical Method Validation for Human Studies”
• 2000 CDER Draft “Analytical Procedures and Method Validation”

53
 ICH/USP Validation Requirements &
Parameters
USP ICH
 Specificity
 Specificity
 Linearity
 Linearity and Range
 Range
 Accuracy
 Accuracy
 Precision
 Precision
 Limit of Detection
 Repeatability
 Limit of Quantitation
 Intermediate Precision
 Ruggedness
 Reproducibility
 Robustness
 Limit of Detection
 Limit of Quantitation
54
 USP Data Elements Required For Assay Validation

Assay Category 2
Analytical
Assay Cate- Assay Cate-
Performance
gory 1 gory 3
Parameter Quantitative Limit Tests

Accuracy Yes Yes * *

Precision Yes Yes No Yes
Specificity Yes Yes Yes *
LOD No No Yes *
LOQ No Yes No *
Linearity Yes Yes No *
Range Yes Yes * *
Ruggedness Yes Yes Yes Yes
* May be required, depending on the nature of the specific test.
55
 USP Categories
 Category 1: Quantitation of major components or active
ingredients
 Category 2: Determination of impurities or degradation
products
 Category 3: Determination of performance characteristics

56
ICH Validation Characteristics vs. Type of Analytical
Procedure
Impurity testing
Type of Analytical
Identification Assay
Procedure Quantitative Limit Tests

Accuracy No Yes No Yes

Precision
Repeatabil-
No Yes No Yes
ity
Interm.
No Yes No Yes
Prec.
Specificity Yes Yes Yes Yes
LOD No No Yes No
LOQ No Yes No No
Linearity No Yes No Yes
Range No Yes No Yes
57
 Specificity/Selectivity
• Ability of an analytical method to measure the analyte
free from any interference due to other components.

• Selectivity describes the ability of an analytical

method to differentiate various substances in a
sample

58
 Specificity: Impurities Assay
• Chromatographic Methods
– Demonstrate Resolution
• Impurities/Degradants Available
– Spike with impurities/degradants
– Show resolution and a lack of interference
• Impurities/Degradants Not Available
– Stress Samples
– For assay, Stressed and Unstressed Samples should be compared.
– For impurity test, impurity profiles should be compared.

59
 Forced Degradation Studies

• Temperature (50-60℃)
• Humidity (70-80%)
• Acid Hydrolysis (0.1 N HCl)
• Base Hydrolysis (0.1 N NaOH)
• Oxidation (3-30%)
• Light (UV/Vis/Fl)

Intent is to create 10 to 30 % Degradation

60
 Linearity

 Ability of an assay to elicit a

direct and proportional response
to changes in analyte
concentration.

61
 Linearity Should be Evaluated;

• By Visual Inspection of plot of signals vs. analyte

concentration
• By Appropriate statistical methods
– Linear Regression (y = mx + b)
– Correlation Coefficient, y-intercept (b), slope (m)
• Acceptance criteria: Linear regression r2 > 0.95
 Requires a minimum of 5 concentration levels

62
 Accuracy

• Closeness of the test

results obtained by the
method to the true value.

63
 Accuracy…
• Should be established across specified range of analytical
procedure.
• Should be assessed using a minimum of 3 concentration
levels, each in triplicate (total of 9 determinations)
• Should be reported as:
– Percent recovery of known amount added or
– The difference between the mean assay result and the
accepted value (RSD)

64
 Precision
• The closeness of agreement (degree of

scatter) between a series of

measurements obtained from multiple

samplings of the same homogeneous

sample.

65
Precision… Considered at 3 Levels

• Repeatability

• Intermediate Precision

• Reproducibility

66
 Repeatability

• Express the precision • Should be assessed using

under the same operating minimum of 9
conditions over a short determinations
interval of time. • (3 concentrations/ 3
• Also referred to as Intra- replicates) or
assay precision • Minimum of 6
determinations at the
100% level.
67
 Intermediate Precision

Express within-laboratory Depends on the circumstances

variations. under which the procedure is

Expressed in terms of standard intended to be used.

deviation, relative standard Studies should include varying

deviation (coefficient of days, analysts, equipment, etc.

variation) and confidence

interval.

68
Repeatability & Intermediate Precision

Day 1 Day 2
100.6 99.5
100.8 99.9
100.1 98.9
100.3 99.2
100.5 99.7
100.4 99.6

Mean = 100.5 Mean = 99.5

RSD = 0.24% RSD = 0.35%
Grand
Mean = 100.0
RSD = 0.59%
69
 Reproducibility

• Ability to reproduce data Lab 1 Lab 2 Lab 3

within the predefined precision
Day Day Day Day Day Day
1 2 1 2 1 2
• Determination: SD, RSD and
Man Ma Ma Man Ma Man
confidence interval 1 n2 n1 2 n1 2

– Repeatability test at two

3 3 3 3 3 3
Pre Pre Pre Prep Pre Pre
different labs. p p p p p

70
 Detection Limit (LOD)/ Quantitation Limit (LOQ)

LOD LOQ

Lowest amount of analyte in a Lowest amount of analyte in a

sample that can be detected sample that can be quantified

but not necessarily with suitable accuracy and

quantitated. precision.

Estimated by Signal to Noise Estimated by Signal to Noise

Ratio of 3:1. Ratio of 10:1

71
LOD and LOQ are Estimated Based on:

1. Visual Evaluations
- Used for non-instrumental methods
2. Signal-to Noise-Ratio
- 3:1 for Detection Limit
- 10:1 for Quantitation Limit
3. Standard Deviation of the Response and the Slope

72
 LOD and LOQ Estimated by

3.3s 10s
DL = QL =
S S

• S = slope of calibration curve

• s = standard deviation of blank readings or
standard deviation of regression line

Validated by assaying samples at DL or QL

73
 Ruggedness
 Degree of reproducibility of test results under a variety of
conditions
• Different Laboratories
• Different Analysts
• Different Instruments
• Different Reagents
• Different Days…..etc.
 Expressed as %RSD

74
 Robustness
• Capacity to remain unaffected by small but deliberate variations in
method parameters
• Determination: Comparison results under differing conditions with
precision under normal conditions
• Examples of typical variations in LC

– 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
75
 Re-validation
• When
– Method parameters have been changed
– The scope of the method has been changed
– Synthetic methods have been changed
– Impurity profile has been changed
• What
– Preferably everything
– Exceptions should be scientifically justified

76
 Basic Calculations in Pharmaceutical Analysis

Percent concentration (parts per hundred)

• Percent composition of a solution can be expressed as:
massofsolute
weightpercent ( w / w)  x100
massofsolution
volumeofso lute
volumepercent (v / v)  x100
volumeofso lution
weightofso lute
weight / volumepercent ( w / v)  x100
volumeofso lution

• 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 employed to indicate the

composition of dilute aqueous solutions of solid
reagents
Example, 5% aqueous silver nitrate often refers to a
solution prepared by dissolving 5g of silver nitrate in
sufficient water to give 100ml of solution.
Molar Concentration
– Is the number of moles of a 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 moles solute = no m mole solute

L solution no ml solution
Example:
1. 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.(ans. 0.0143M)
Parts Per Million & Parts Per Billion

• For very dilute solutions, parts per million (PPM) is convenient way to express
concentration:
massofthes olute

Cppm massofthes olution
X 106 ppm

Where Cppm is the concentration in parts per million.

• 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
Reading Assignment
– Normality
– Dilutions
– Density and specific gravity (definitions and worked examples)
Physical and chemical properties of drug molecules
Calculations of pH value

Dissociation of water
The pH of a solution is defined as
-log [H+]
Where [H+] is the concentration of hydrogen ions in solution
In pure water the concentration of hydrogen ions is governed by the equilibrium:

Where Ka is the dissociation constant for the equilibrium, Kw in case of water

dissociation and is determined by the following expression:
Since the concentration of water does not change appreciably due
to dissociation, ----don’t have an effect on the equilibrium

Strong Acids and Bases

If an acid is introduced into an aqueous soln the [H+] increases.
A strong acid is completely ionized in water and the [H+] is equal to
its molarity
Eg. 0.1M HCl contains 0.1M [H+] and has a pH of 1

For strong base like 0.1M NaOH, [OH-] = 0.1M and [H+]= and
its pH is 13
 Weak acids and bases
• Not completely ionized in solution
• Are in equilibrium with the undissociated acid or base
Ka for a weak acid is given by:

For instance for a 0.1M of acetic acid(Ka= )

Acid and Base Strength
• Strong acids are
completely dissociated in
water
– Their conjugate bases are
quite weak.

• Weak acids only

dissociate partially in
water.
Acid and Base Strength

• Substances with
negligible acidity do not
dissociate in water.
– Their conjugate bases are
exceedingly strong.
 Other “p” Scales
• The “p” in pH tells us to take the negative log
of the quantity (in this case, hydrogen ions).

Some similar examples are

pOH = −log [OH−]
pKw = −log Kw
 Watch This!
Because
[H3O+] [OH−] = Kw = 1.0  10−14,
we know that

−log [H3O+] + −log [OH−] = −log Kw = 14.00

or, in other words,

pH + pOH = pKw = 14.00
How Do We Measure pH?
 For less accurate
measurements, one can
use
– Litmus paper
• “Red” paper turns blue
above ~pH = 8
• “Blue” paper turns red
below ~pH = 5
– An indicator
 How Do We Measure pH?
 For more accurate
measurements, one uses
a pH meter, which
measures the voltage in
the solution.

90
 Dissociation Constants
The greater the value of Ka, the stronger the acid

91
 Calculating Ka from the pH
 The pH of a 0.10 M solution of formic acid, HCOOH, at 25°C

is 2.38. Calculate Ka for formic acid at this temperature.

 We know that [H3O+] [COO−]
Ka =
[HCOOH]

 To calculate Ka, we need the equilibrium concentrations of

all three things.
 We can find [H3O+], which is the same as [HCOO−], from
the pH.
 pH = −log [H3O+]
2.38 = −log [H3O+]
−2.38 = log [H3O+]

10−2.38 = 10log [H3O+] = [H3O+]

4.2  10−3 = [H3O+] = [HCOO−]
[4.2  10−3] [4.2  10−3]
Ka =
[0.10]

= 1.8  10−4
 Calculating Percent Ionization
• Percent Ionization = [H3O+] eq  100
[HA]initial
• In this example
[H3O+]eq = 4.2  10−3 M
[HCOOH]initial = 0.10 M
Percent Ionization =  100 = 4.2%
 Calculating pH from Ka
Calculate the pH of a 0.30 M solution of acetic
acid, HC2H3O2, at 25°C.

HC2H3O2(aq) + H2O(l) H3O+(aq) + C2H3O2−(aq)

Ka for acetic acid at 25°C is 1.8  10−5.

 Calculating pH from Ka

The equilibrium constant expression is

[H3O+] [C2H3O2−]
Ka =
[HC2H3O2]
 Calculating pH from Ka
We next set up a table…
[C2H3O2], M [H3O+], M [C2H3O2−], M

Initially 0.30 0 0

Change −x +x +x

At Equilibrium 0.30 − x  0.30 x x

We are assuming that x will be very small compared to 0.30 and

can, therefore, be ignored.
 Calculating pH from Ka
Now,
(x) 2
1.8  10−5 =
(0.30)
(1.8  10−5) (0.30) = x2
5.4  10−6 = x2
2.3  10−3 = x

pH = −log [H3O+]
pH = −log (2.3  10−3)
pH = 2.64
 Weak Bases

Bases react with water to produce hydroxide ion.

 Weak Bases

The equilibrium constant expression for this

reaction is

[HB] [OH−]
Kb =
[B−]

where Kb is the base-dissociation constant.

 Weak Bases

Kb can be used to find [OH−] and, through it, pH.

 pH of Basic Solutions

What is the pH of a 0.15 M solution of NH3?

NH3(aq) + H2O(l) NH4+(aq) + OH−(aq)

[NH4+] [OH−]
Kb = = 1.8  10−5
[NH3]
 pH of Basic Solutions

Tabulate the data.

[NH3], M [NH4+], M [OH−], M

Initially 0.15 0 0
At Equilibrium 0.15 - x  x x
0.15
pH of Basic Solutions

(x) 2
1.8  10−5 =
(0.15)
(1.8  10−5) (0.15) = x2
2.7  10−6 = x2
1.6  10−3 = x2
 pH of Basic Solutions

Therefore,
[OH−] = 1.6  10−3 M
pOH = −log (1.6  10−3)
pOH = 2.80
pH = 14.00 − 2.80
pH = 11.20
 Ka and Kb

Ka and Kb are related in this way:

Ka  Kb = Kw
Therefore, if you know one of them, you can
calculate the other.
 Factors Affecting Acid Strength

• The more polar the H-X bond and/or the weaker the H-
X bond, the more acidic the compound.
• Acidity increases from left to right across a row and
from top to bottom down a group.
Factors Affecting Acid Strength

In oxyacids, in which
an OH is bonded to
another atom, Y, the
more electronegative
Y is, the more acidic
the acid.
Factors Affecting Acid Strength

For a series of oxyacids, acidity increases with the

number of oxygens.
Factors Affecting Acid Strength

Resonance in the conjugate bases of carboxylic

acids stabilizes the base and makes the conjugate
acid more acidic.
 The Henderson-Hasselbalch Equation

Take the equilibrium ionization of a weak acid:

HA(aq) + H2O(aq) = H3O+(aq) + A-(aq) [H3O+] [A-]
Ka =
[HA]
Solving for the hydronium ion concentration gives:

[H3O+] = Ka x [HA]
[A-]
Taking the negative logarithm of both sides:

-log[H3O +] = -log Ka - log ( )

[HA]
[A ]
-
pH = -log Ka - log ( )
[HA]
[A-]
Generalizing for any conjugate acid-base pair :

pH = -log Ka + log
( [base]
[acid] ) Henderson-Hasselbalch
equation
 [A-]
pH = pKa + log
[HA]

a useful concept:

when [H-A] = [ A ] pKa = pH + log (1)

pKa = pH

Biological fluids are often buffered (constant pH) and it is useful to know
the predominant species present at a given pH.
 BUFFERS

Mixture of an acid and its conjugate base.

Buffer solution  resists change in pH when acids

or bases are added or when dilution occurs.
Mix:
A moles of weak acid + B moles of conjugate base
Find:
• moles of acid remains close to A, and
• moles of base remains close to B
 Very little reaction
HA  H+ + A- Le Chatelier’s principle
 Why does a buffer resist change in pH when small
amounts of strong acid or bases is added? ?

The acid or base is consumed

by A- or HA respectively

Buffer capacity, :

 Measure of how well solution resists change in

pH when strong acid/base is added.

dCb  dCa
 
dpH dpH

Larger   more resistance to pH change

 How a Buffer Works

Consider adding H3O+ or OH- to water and also to a buffer

For 0.01 mol H3O+ to 1 L water:

[H3O+] = 0.01 mol/1.0 L = 0.01 M pH = -log([H3O+]) = 2.0

So, change in pH from pure water: dpH = 7.00 – 2.00 = 5.0

For the H2CO3- / HCO3- system:

pH of buffer = 7.38

Addition of 0.01 mol H3O+ changes pH to 7.46

So change in pH from buffer: dpH = 7.46 – 7.38 = 0.08 !!!

 Consider a buffer made from acetic acid and sodium acetate:

CH3COOH(aq) + H2O(l) CH3COO-(aq) + H3O+(aq)

[CH3COO-] [H3O+]
Ka = or
[CH3COOH]

[CH3COOH]
[H3O ] = Ka x
+

[CH3COO-]
 Let’s consider a buffer made by placing 0.25 mol of acetic acid
and 0.25 mol of sodium acetate per liter of solution.

 What is the pH of the buffer?

 And what will be the pH of 100.00 mL of the buffer before and
after 1.00 mL of concentrated HCl (12.0 M) is added to the
buffer?
 What will be the pH of 300.00 mL of pure water if the same acid is
added?

[CH3COOH] (0.25)
[H3O ] = Ka x
+
= 1.8 x 10 x
-5
= 1.8 x 10-5
[CH3COO-] (0.25)

pH = -log[H3O+] = -log(1.8 x 10-5) = pH = 4.74 Before acid added!

What is pH if added to pure water?

1.00 mL conc. HCl 1.00 mL x 12.0 mol/L = 0.012 mol H3O+

Added to 300.00 mL of water :

0.012 mol H3O+

= 0.0399 M H3O+ pH = -log(0.0399 M)
301.00 mL soln.
pH = 1.40 Without buffer!
After acid is added to buffer:
Conc. (M) CH3COOH(aq) + H2O(aq) CH3COO- + H3O+
Initial 0.250 ---- 0.250 0
Change +0.012 ---- -0.012 0.012
Equilibrium 0.262 ---- 0.238 0.012
Solving for the quantity ionized:
Conc. (M) CH3COOH(aq) + H2O(aq) CH3COO- + H3O+
Initial 0.262 ---- 0.238 0
Change -x ---- +x +x
Equilibrium 0.262 - x ---- 0.238 + x x
Assuming: 0.262 - x = 0.262 & 0.238 + x = 0.238
[CH3COOH]
[H3O ] = Ka x
+
=1.8 x 10-5 x (0.262) = 1.982 x 10-5
[CH3COO-] (0.238)

pH = -log(1.982 x 10-5) = 5.000 - 0.297 = 4.70 After the acid is added!

Suppose we add 1.0 mL of a concentrated base instead of an acid. Add
1.0 mL of 12.0 M NaOH to pure water and our buffer, and let’s see what
the impact is: 1.00 mL x 12.0 mol OH-/1000mL = 0.012 mol OH-
This will reduce the quantity of acid present and force the equilibrium
to produce more hydronium ion to replace that neutralized by the
addition of the base!
Conc. (M) CH3COOH(aq) + H2O(aq) CH3COO- + H3O+
Initial 0.250 ---- 0.250 0
Change - 0.012 ---- +0.012 +0.012
Equilibrium 0.238 ---- 0.262 +0.012
Assuming: Again, using x as the quantity of acid dissociated we get:
our normal assumptions: 0.262 + x = 0.262 & 0.238 - x = 0.238
0.238
[H3O+] = 1.8 x 10-5 x = 1.635 x 10-5
0.262
pH = -log(1.635 x 10-5) = 5.000 - 0.214 = 4.79 After base is added!
By adding the 1.00mL base to 300.00 mL of pure water we would get a
hydroxide ion concentration of:
0.012 mol OH -
[OH-] = = 3.99 x 10-5 M OH-
301.00 mL
The hydrogen ion concentration is:
Kw 1 x 10-14
[H3O ] =
+
= = 2.506 x 10-10
[OH-] 3.99 x 10-5 M
This calculates out to give a pH of:
pH = -log(2.5 6 x 10-10) = 10.000 - 0.408 = 9.59 With 1.0 mL of the
base in pure water!
In summary:
Buffer alone pH = 4.74
Buffer plus 1.0 mL base pH = 4.79 Base alone, pH = 9.59
Buffer plus 1.0 mL acid pH = 4.70 Acid alone, pH = 1.40
Problem:
Calculate the pH of a solution containing 0.200 M NH3
and 0.300 M NH4Cl given that the acid dissociation
constant for NH4+ is 5.7x10-10.

NH3 + H2O  NH4+ + OH- pKa = 9.244

Ka
base acid

[B] pKa applies

pH pK a  log
[BH ] to this acid

(0.200)
pH = 9.244 + log
(0.300)
pH = 9.07
Drug stability and stability studies
 Stability of drugs
• Stability: is the capacity of a drug product to remain within
specifications established to ensure its identity, strength
quality and purity.

• Instability may cause

- Undesired change in performance, i.e.
dissolution/bioavailability
- Substantial changes in physical appearance of the dosage form
- Causing product failures
 Factors affecting Stability
1- Environmental factors
- Temperature - Light
- Oxygen - Moisture
- Carbon dioxide

2- Drugs or excipients in the dosage form

- Particle size of drug
- pH of the vehicle

3- Microbial contamination
4- Trace metal Contamination
5- Leaching from containers
 Types of stability :

۞ Physical

۞ Chemical

۞ Microbiological
 Physical stability
Physical stability implies that:
- The formulation is totally unchanged throughout its shelf
life and has not suffered any changes by way of
appearance, organoleptic properties, hardness,
brittleness, particle size etc.

- It is significant as it affects:
Pharmaceutical elegance
Drug content uniformity
Drug release rate
 Physical stability (Cont.)

Formulatio Likely physical Effects

n instability problems

Oral 1- Loss of flavour Change in

solutions 2- Change in taste smell or
3- Presence of off feel or
flavours due to taste
interaction with plastic
bottle
4- Loss of dye
5- Precipitation
6- discoloration
 Physical stability (Cont.)
Formulatio Likely physical Effects
n instability problems
Parenteral 1. Discoloration due to photo Change in
chemical reaction or appearan
solutions oxidation ce and in
2. Presence of precipitate bio-
due to interaction with availabilit
container or stopper y

3. Presence of “whiskers”
4. Clouds due to:
(i) Chemical changes
(ii) The original preparation
of a supersaturated
solution
 Physical stability (Cont.)

Formulatio Likely physical Effects

n instability
problems
Suspensions 1- settling 1-Loss of
2- caking drug content
3- crystal growth uniformity in
different
doses from
the bottle

2- loss of
elegance.
 Physical stability (Cont.)

Formulatio Likely physical Effects

n instability
problems
Emulsions 1- Creaming 1- Loss of
2- coalescence drug content
uniformity in
different
doses from
the bottle

2- loss of
elegance
 Physical stability (Cont.)
Formulatio Likely physical Effects
n instability
problems
Semisolids 1. Changes in: 1-Loss of drug
(Ointments a) Particle size content
and b) Consistency uniformity
suppositorie
s) 2- loss of
2. Caking or
coalescence elegance

3. Bleeding 3-change in
drug release
rate.
 Physical stability (Cont.)

Formulatio Likely physical Effects

n instability
problems
Tablets Change in: Change in
a) Disintegration drug
time release
b) Dissolution profile
c) Hardness
d) Appearance (soft
and ugly or
become very
hard)
 Physical stability (Cont.)

Formulatio Likely physical Effects

n instability
problems
Capsules Change in: Change in
a) Appearance drug
b) Dissolution release
c) Strength
 Chemical stability:

Chemical stability implies:

The lack of any decomposition in the chemical moiety that
is incorporated in the formulation as the drug,
preservatives or any other excipients.

This decomposition may influence the physical and

chemical stability of the drug
 Mechanisms Of Degradation
1- Hydrolysis:Hydrolysis means “splitting by water’’

Some Functional Groups Subject to Hydrolysis

Drug type Examples

Esters Aspirin, alkaloids
Dexmethasne sodium phosphate
Nitroglycerin
Lactones Pilocarpine
Spironolactone
Amides Chloramphenicol

Lactams Penicillins
Cephalosporins
 Some Functional Groups Subject to Hydrolysis

Drug type Examples

Imides Glutethimide

Malonic ureas Barbiturates

 Mechanisms Of Degradation

2- Oxidation

Oxidation of inorganic and organic compounds is explained

by a loss of electrons and the loss of a molecule of
hydrogen.
Some Functional Groups Subject to Autoxidation

Functional Examples
group
Catechols Catecholamines (dopamine)
Ethers Diethylether

Thiols Dimercaprol (BAL)

Thioethers Chlorpromazine
Carboxylic acids Fatty acids
 Mechanisms Of Degradation

3- Photolysis
It means: decomposition by light

e.g. Sodium nitroprusside is administered by intravenous

infusion for the management of acute hypertension.

If the solution is protected from light, it is stable for at least 1

year; if exposed to normal room light, it has a shelf life of
only 4 hours.
 Mechanisms Of Degradation

Photolysis is prevented by:

1- suitable packing in amber coloured bottles
2- cardboard outers
3- aluminium foil over wraps
 Factors Affecting Rates Of Degradation

1- pH
The acidity or the alkalinity of a solution has a profound
influence on the decomposition of drug compound.
- Aspirin buffered solution is maximum stable at a pH of
2.4, above a pH of 10 the decomposition rate rapidly
increases.

pH can also influence the rate of oxidation.

- The system is less readily oxidized when the pH is low.
Factors Affecting Rates Of Degradation

2- Complexation
Complex formation reduces the rate of hydrolysis and
oxidation.

e.g. caffeine complexes with local anesthetics, such as

benzocaine, procaine and tetracaine to cause a reduction
in their rate of hydrolytic degradation.
Factors Affecting Rates Of Degradation

3- Surfactants
Nonionic, cationic and anionic surfactants when added to
solutions containing drugs form micelle and the drug
particles become trapped in the micelle.

The hydrolytic groups such as OH cannot penetrate this

micelle cover and reach the drug particles, hence hydrolysis
rate is decreased.
Factors Affecting Rates Of Degradation
4- Presence of heavy metals
Heavy metals, such as copper, iron, cobalt and nickel
increase the rate of formation of free radicals and
enhance oxidative decomposition.

5- Light and humidity

Light, especially ultraviolet light enhances photolysis and
humidity enhances hydrolytic decomposition.
Stabilization of drugs against hydrolysis, oxidation
and photolysis

1- Temperature
All the drug products are stored at suitable temperatures
to avoid thermal acceleration of decomposition.
Three varieties of temperatures are suggested for storage of
drug products. Room temperature, cool storage and cold
storage.

2- Light
Light sensitive materials are stored in ambered colour
bottles.
Stabilization of drugs against hydrolysis, oxidation
and photolysis

3- Humidity
Packing materials are chosen (usually glass and plastic) to
prevent exposure of drug products to high humid
condition.

4- Oxygen
Proper packing keeping the oxygen content of the solution
less and leaving very little head space in the bottle above
the drug products are methods to fight against oxidation.
 Stabilization of drugs against hydrolysis,
oxidation and photolysis

5- Chelating Agents
Chelating agents form complexes with heavy metal ions
and prevent them from catalyzing oxidative
decomposition.
e.g. ethylenediamine tetracetic acid (EDTA) derivatives and
salts, citric acid and tartaric acid.

6- Solvents
By the addition of a suitable solvent hydrolysis rate may be
decreased.
 Microbiological stability
Microbiological stability implies that:

The formulation has not suffered from any

microbiological attack and is meeting the
standards with respect to lack of
contamination/sterility.
Microbiological stability

Sources of Microbial Contamination:

Water gram-negative groups: Pseudomonas,

Xanthamonas, Flavobacterium

Air Mould spores: Penicillium, Aspergillus

Bacterial spores: Bacillus spp. Yeasts

Raw Micrococci
materials
Starches Coliforms
Pigments Salmonella
151
To prevent contamination to the formulation during storage

(1) suitably designing the containers

(2) usually using single dose containers

(3) sticking to proper storage conditions

(4) adding an antimicrobial substance as

preservative.
Preservatives used in pharmaceutical
preparations:

Preparation Preservative Concentratio

n
% w.v
Injections Phenol 0.5
Cresol 0.3
Chlorocresol 0.1
Eye drops Chlorhexidine 0.01
acetate 0.01
Benzalkonium
chloride
Mixtures Benzoic acid 0.1
Methyl paraben 0.1
Alcohol 12-20
 Packaging And Stability :

• The immediate container and closure are particularly

important in affecting product stability.
 Packaging and Stability:

• Glass
- Glass is resistant to chemical and physical change and is
the most commonly used material.
Limitations Overcome
1. Its alkaline surface use of Borosilicate glass

2. Ions may precipitate the use of buffers

insoluble crystals from the
glass
3- Permits the transmission Amber coloured glass
of light which may
accelerate decomposition.
155
 Packaging and Stability :

Plastics
The problems with plastic are:
1. Migration of the drug through the plastic into the
environment…leakage
2. Transfer of environmental moisture, oxygen, and other
elements into the pharmaceutical product.
3. Leaching of container ingredients into the drug.
4. Adsorption of the active drug or excipients by the plastic.
 Packaging and Stability :

• Metals
- Various alloys and aluminium tubes may be utilized as
containers for emulsions, ointments, creams and pastes.

- Limitation: They may cause corrosion and precipitation in

the drug product.

- Overcome: Coating the tubes with polymers may reduce

these tendencies.
 Packaging and Stability:

• Rubber
- Rubber also has the problems of extraction of drug
ingredients and leaching of container ingredients.

- The pretreatment of rubber vial stoppers and closures

with water and steam reduces potential leaching.
Stability Testing
 Forced degradation tests
• To identify potential degradants (degradation pathways)
of the API and assess if they can be formed during
manufacture or storage of the product (intrinsic stability of
the API).

• To validate the stability indicating power of the analytical

procedures.

• To identify stability-affecting factors such as ambient

temperature, humidity and light and to select packing
materials, which protect the product against such effects.
 Requirements for predictive stress conditions

Recommendations:
• Should lead to the degradation of the main
compound, but not more than 5-15%.

• Should lead to a good predictability of degradation

pathways (i.e., a low probability of "drastic" or
"false" degradation)

• Should be conducted for no longer than three

months.
 Regulatory or formal stability testing

Relative Minimum time

Storage temperature humidity period covered by
(°C) (%) data at submission
(months)
Accelerated: 40±2 75±5 6

Intermediate: 30±2 65±5 12

Long term: 25±2 60±5 12 +

 Potential instability issues
• Loss/increase in concentration of API
• Formation of (toxic) degradation products
• Modification of any attribute of functional relevance
• Alteration of dissolution time/profile or bioavailability
• Decline of microbiological status
• Loss of package integrity
• Reduction of label quality
• Loss of pharmaceutical elegance and patient
acceptability
163/61
 Stability-indicating quality parameters

Stability studies should include testing of those

attributes of the product that are susceptible to change
during storage and are likely to influence quality, safety
and/or efficacy.
For instance, in case of tablets:

♦ appearance ♦ hardness
♦ friability ♦ moisture content
♦ dissolution time ♦ degradants
♦ assay ♦ microbial purity
 Main points
• Stability studies should be planned on the basis of
pharmaceutical R&D and regulatory requirements.

• Forced degradation studies reveal the intrinsic chemical

properties of the API, while formal stability studies establish the
retest date.

• The shelf life (expiry date) of FPPs is derived from formal stability
studies.

• Variability and time trends of stability data must be evaluated by

the manufacturer in order to propose a retest date or expiry date.
 Measurement of optical rotation
• Biological discrimination
 Optical Activity

 A substance is optically active if it rotates

the plane of polarized light.
 In order for a substance to exhibit optical activity, it must

be chiral and one enantiomer must be present in excess

of the other.
 Light

optical activity is usually measured using light having a

wavelength of 589 nm
this is the wavelength of the yellow light from a sodium lamp and
is called the D line of sodium
 Polarized light

ordinary
(nonpolarized)
light consists of
many beams
vibrating in
different planes

plane-polarized
light consists of
only those beams
that vibrate in the
same plane
Polarization of light

Nicol prism
Rotation of plane-polarized light
Rotation of plane-polarized light

a
 Polarimetry
• Use monochromatic light, usually sodium D
• Movable polarizing filter to measure angle
• Enantiomers rotate light in opposite directions, but same number of
degrees.

• Clockwise = dextrorotatory = d or (+)

• Counterclockwise = levorotatory = l or (-)
 Specific rotation

observed rotation (a) depends on the number

of molecules encountered and is proportional to:

path length (l), and concentration (c)

therefore, define specific rotation [a] as:

[] =  (observed)
c l
c is concentration in g/mL
l is length of path in decimeters.
 Calculate []

• A 1.00-g sample is dissolved in 20.0 mL

ethanol. 5.00 mL of this solution is placed in
a 20.0-cm polarimeter tube at 25C. The
observed rotation is 1.25 counterclockwise.
 Racemic Products

If optically inactive reagents combine to form

a chiral molecule, a racemic mixture of
enantiomers is formed.

=>
a sample that is optically inactive can be either
an achiral substance or a racemic mixture
 Optical Purity
• Also called enantiomeric excess (ee).
• Amount of pure enantiomer in excess of the
racemic mixture.
• If o.p. = 50%, then the observed rotation will
be only 50% of the rotation of the pure
enantiomer.
• Mixture composition would be 75-25.
=>
 Calculate % Composition

The specific rotation of (S)-2-iodobutane is

+15.90. Determine the % composition of a
mixture of (R)- and (S)-2-iodobutane if the
specific rotation of the mixture is -3.18.
06/25/2025 181
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