Yanet-Liyana College of Health Science

Department of Pharmacy
 Course Title: Pharmaceutical Analysis I
 Course EtCTS: 7
 Program: Regular Students’

By: Rashed E. ([Link], MSc in PQARA)

E-mail:- rashafro60@[Link]

11/03/2025 1
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 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 of drug

entities from natural products.
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

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

– 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
[Link] 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
1.3. Analytical Errors and Validation of
Analytical procedures

39
 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

Example NIST- National Institute of Standards and Technology, PCRS- British

Pharmacopoea Chemical Reference Substance, EPCRS -European Pharmacopoea Chemical
Reference Substance
40
• 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

41
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.

42
 Determinate (systematic) errors…
(a) Personal Errors : due to ‘personal equation’ of an analyst and have no bearing
whatsoever either on the prescribed procedure or methodology involved.

(b) Instrumental Errors : due to faulty and uncalibrated instruments, such as : pH

meters, single pan electric balances, UV spectrophotometers,

(c) Reagent Errors : due to individual reagents

For instance: impurities inherently present in reagents ; unwanted

introduction of ‘foreign substances’ caused by the action of reagents on either

porcelain or glass apparatus.

43
(d) Constant Errors :
– 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

44
 Errors in pharmaceutical…

 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:

45
Indeterminate (Random) Errors:
 It cannot be pin-pointed to any specific well-defined reasons.
 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.
 Mostly random in nature and ultimately give rise to high as well as low results with equal
probability.
 Neither be corrected nor eliminated, and therefore, form the ‘ultimate limitation’ on the
specific measurements.

46
Minimizing Systematic Errors
Systematic errors may be reduced substantially and significantly by adopting
one of the following procedures rigidly.

(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.

47
 In the same manner all apparatus, namely: pipettes, burettes, volumetric

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

corrections incorporated to the original measurements.

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

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

causes ;

 For instance: the inherent impurity present in a weighed precipitate can be estimated first

and then deducted duly from its weight.

48
(ii) Blank Determination:
 In order to ascertain the effect of the impurities present in the reagents

employed and reaction vessels used; a blank determination is an absolute

necessity.

 It may be accomplished by performing a separate parallel estimation, without

using the sample at all, and under identical experimental parameters as

employed in the actual analysis of the given sample.

49
iii) Using Standard Substance

– External standard

– Internal standard

– Standard addition

50
Factors giving rise to inaccuracy and imprecision in an assay

Pharm. Analysis I 51
 Validation of Analytical Methods
 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.

52
• 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

53
 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

54
 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

55
 Common Parameters That Should Be
Verified In Method Validation
ICH/USP Validation Requirements & Parameters
USP ICH
 Specificity  Specificity
 Linearity and Range  Linearity
 Accuracy  Range
 Precision  Accuracy
 Limit of Detection  Precision
 Limit of Quantitation  Repeatability
 Ruggedness  Intermediate Precision
 Robustness  Reproducibility
 Limit of Detection
 Limit of Quantitation

56
 USP Data Elements Required For Assay Validation
Assay Category 2
Analytical
Assay Cate- Assay Cat-
Performance
gory 1 Quantitative Limit Tests egory 3
Parameter

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.
57
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
Repeatability No Yes No Yes
Interm. Prec. No Yes No Yes
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
58
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

59
 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.

60
 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

61
Linearity

 Ability of an assay to elicit a direct and

proportional response to changes in
analyte concentration.

62
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

63
 Accuracy

• Closeness of the test results

obtained by the method to the
true value.

64
 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)

65
 Precision
• The closeness of agreement (degree of scatter)

between a series of measurements obtained from

multiple samplings of the same homogeneous

sample.

66
Precision… Considered at 3 Levels

 Repeatability

 Intermediate Precision

 Reproducibility

67
 Repeatability

• Express the precision under the • Should be assessed using

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

68
Intermediate Precision
Express within-laboratory Depends on the circumstances under

variations. which the procedure is intended to be

Expressed in terms of standard used.

deviation, relative standard Studies should include varying days,

deviation (coefficient of variation) analysts, equipment, etc.

and confidence interval.

69
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%
70
 Reproducibility

• Ability to reproduce data within the Lab 1 Lab 2 Lab 3

predefined precision
Day Day Day Day Day Day
1 2 1 2 1 2
• Determination: SD, RSD and
Man Man Man Man Man Man
confidence interval 1 2 1 2 1 2

– Repeatability test at two different

3 3 3 3 3 3
Prep Prep Prep Prep Prep Prep
labs.

71
 Detection Limit (LOD)/ Quantitation Limit (LOQ)

LOD LOQ

Lowest amount of analyte in a Lowest amount of analyte in a sample that

sample that can be detected but not can be quantified with suitable

necessarily quantitated. accuracy and precision.

Estimated by Signal to Noise Ratio of Estimated by Signal to Noise Ratio of

3:1. 10:1

72
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

73
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

74
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

75
 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
76
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

77
1.4. Basic calculations in
pharmaceutical analysis
Pharm. Analysis I 78
Basic calculations in pharmaceutical analysis………

 There are various units used to express amounts and concentrations

in pharmaceutical analysis and these units includes:
• Percentages:
 Percentage volume/volume (% V/V)
 Percentage weight/volume (% W/V)
 Percentage weight/weight (% W/W)
• Parts per million (ppm) and parts per billion (ppb)
• Molarity (M)
• Normality (N)

Pharm. Analysis I 79
 Percentage Volume/Volume (%V/V)
 Volume percent is commonly used to specify the concentration of a
solution prepared by diluting a pure liquid with another liquid.
 Most often encountered in relation to the composition of mobile phases
used in HPLC
 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.
volume of subs tan ce
Volume percent  x(100)
volume of total solution or total sample
Pharm. Analysis I 80
 Percentage Weight In Volume (%W/V)
 Concentration expressed in terms of mass of substance versus the total volume of
the sample.
 It used to express the content of active ingredient in liquid formulations such as
injections, infusions and eye drops
 For 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
 The density of the solvent in this case is irrelevant; thus, a 1g/100ml solution of
a drug is 1%w/v whether it is dissolved in ethanol or water
mass of subs tan ce
weight  volume percent  x(100)
volume of total solution or total sample
81
 Percentage Weight/Weight (%W/W)
 Concentration expressed in terms of mass of substance versus the total mass of
the sample.
 A common measure used to express the concentration of active ingredient in a
formulation such as a cream or to express the content of a minor impurity in a
drug substance
 Thus, a cream containing 10 mg (0.01 g) of drug per gram is a:

(0.01/1)x100% = 1% w/w formulation.

mass of subs tan ce

Weight percent  x ( 100 )
mass of total solution or total sample

Pharm. Analysis I 82
 Percentage weight/weight (%w/w)…
 Equally, if a drug contains 0.5 mg (0.0005 g) per gram of an impurity the
impurity is at a concentration of (0.0005/1) x100% = 0.05% w/w.
 It is generally accepted that, for a drug, all impurities above 0.05% w/w
should be characterized, i.e. their structures should be known and their
toxicities should be assessed.
 Also in determining impurities in a drug, 1 g of the drug might be dissolved in
100 ml of solvent.
 If an analysis was carried out and the drug solution was found to contain 3
mg/100 ml of an impurity, then the %w/w referring back to the original
weight of drug substance would be: (0.003/1)100%=0.3% w/w: 83
 Exercise

1. 0.1521 g of a corticosteroid drug was dissolved in 100 ml

of methanol/water (1:1 v/v).The sample was analyzed by
HPLC and was found to contain a known impurity in a
concentration of 0.354 mg/100 ml. What is the %w/w of
the impurity in the drug?

ANS---0.2327% w/w

Pharm. Analysis I 84
 Parts Per Million/Billion (ppm/ppb) Calculations
 Parts per million (ppm) on a w/w basis is 1 mg/kg or 1 µgm/gm.
 It is a common measure used for impurities in drug substances, particularly
heavy metals and solvents.
 1 ppm is also 0.0001%w/w.
 For very dilute solutions, parts per million (PPM) is convenient way to express
concentration:

W/Wppm  Mass of the solute X 106

Mass of the solution

•The units of mass in the numerator & denominator must agree.

•For even more (extremely)dilute solutions, we use parts per billion.

W/W ppb = Mass of solute

Mass of solution X 109
 ppm/ppb
Example
The potassium content of an intravenous infusion containing sodium
chloride was determined. The infusion was found to contain
0.9092%w/v NaCl. The undiluted infusion was measured for potas-
sium content in comparison with a KCl standard. The potassium con-
tent of the undiluted infusion was found to be 0.141 mg/100 ml.
Calculate the potassium content in the sodium chloride in ppm:
NaCl, 0.9092%w/v = 0.9092 g/100 ml
Potassium content = 0.141 mg/100 ml
Relative to the sodium chloride, there are 0.141mg/0.9092 g = 141/0.9092
ppm= 155 ppm = 155000 ppb
86
 Exercise
1. 125.1 mg of streptomycin sulphate are dissolved in 10ml of water.
A GC headspace analysis is carried out in order to determine the
methanol content of the drug. A peak for methanol is produced
which has 73.2% of the area of a peak for a methanol standard
containing 0.532 mg/100 ml of methanol in water analyzed under
exactly the same conditions. What is the methanol content of the
streptomycin sulphate in ppm and %w/w?
Answer: 311.3 ppm, 0.03113%w/w
Pharm. Analysis I 87
2. If 14 L of commercial ethyl alcohol are found to contain 0.010ml of butanol, what is
the concentration of butanol, in ppm?
1000ml=1L

1000ml = Xml
1L 14L

 X=14L * 1000ml = 14,000ml

1L
0.01ml = Y ml
14,000ml 1,000,000
 Y=0.71ml ppm
3. A sample of solution for injection is found to contain 1.4ppm of lead chloride. How
much solution will contain 50 µg of lead chloride?
Ans. Y=35.7 ml ppm
 Molarity(M)
The number of moles of solute per liter of solution.
M = mol/L = given mass(g)/[Mwt x L]
Example:
 Find the concentration in Molarity (M) of 12.00g of benzene (C6H6) dissolved up
to a total volume of 250.00 ml in hexane.

Pharm. Analysis I 89
 Normality (N)
 The number of equivalents of solute per liter of solution independent of species.

N = EW/L = FW/nL = nM
 Where: EW-equivalent weight , Fw= formula weight, n= no. of equivalents
 Equivalent: The moles of a species that can donate one reaction unit.
 The number of equivalents, n, is based on a reaction unit
 Equivalent weight: The mass of a compound containing one equivalent
 Formula weight: The mass of a compound containing one mole

Type of rxn Rxn unit (n)

1. Acid-base rxn No. of H+ donated(acid) / accepted(base)
2. Precipitation rxn Charge of cation / anion
3. Redox rxn No. of e- s donated(RA)/ accepted(OA)
4. Complexation rxn No. of e- s donated(ligand)/ accepted (metal)

90
 Normality examples
• For precipitation rxn
Pb2+(aq) + 2I–(aq) PbI2(s)
n = 2 for Pb2+ and n = 1 for I–
• For the reaction between sulfuric acid and ammonia
H2SO4(aq) + 2NH3(aq) 2NH4+(aq) + SO42–(aq)
n = 2 for H2SO4 and n = 1 for NH3.
• For a complexation reaction:
Ag+(aq) + 2NH3(aq) Ag(NH3)2 +(aq)
n for Ag+ is 2 and that for NH3 is 1
• For an oxidation–reduction rxn
2Fe3+(aq) + Sn2+(aq) Sn4+(aq) + 2Fe2+(aq)
n = 1 for Fe3+ and n = 2 for Sn2+
• NB. Determining the number of equivalents for a chemical species requires an
understanding of how it reacts.
Pharm. Analysis I 91
NORMALITY: EXAMPLE

Pharm. Analysis I 92
 Summary:
Common Units For Reporting Concentration

Pharm. Analysis I 93
 Dilutions
 In order for an extract from a formulation or a solution of a pure drug substance to be

measured it must be diluted so that it falls within the working range of the in-
strument used to make the measurement.
 Thus an understanding of dilution factors is fundamental to calculations based
on analytical data.
C1V1 = C2V2 Dilution Factor
C2= C1[V1/V2]

Pharm. Analysis I 94
 Dilution …
Example
An infusion stated to contain 0.95% w/v NaCl was diluted so that its Na con-
tent could be determined by flame photometry. The following dilutions were
carried out:
I. 10 ml of the sample was diluted to 250 ml with water.

II. 10 ml of the diluted sample was diluted to 200 ml with water.

 The diluted sample was found to contain 0.74 mg/100 ml of Na.

Atomic weights: Na=23, Cl=35.5

Calculate: The % w/v of NaCl present in the infusion.

Dilution factor = 10 to 250 (× 25), 10 to 200 (× 20). Total dilution =25x20=500.

Therefore, in the original injection, conc. of Na=0.74x500 mg/100 ml=370 mg/100 ml.
Conc. of NaCl in the injection=370x(58.5/23)=941 mg/100 ml=0.941 g/100 ml= 0.941%w/v

Pharm. Analysis I 95
 Dilution (exercise)…
A 2 ml volume of eyedrops containing the local anaesthetic proxymetacaine. HCl is diluted to 100
ml and then 5 ml of the dilution is diluted to 200 ml. The diluted sample was measured by UV
spectrophotometry and was found to contain 0.512 mg/100 ml of the drug.

Calculate the %w/v of the drug in the original eye drops.

Dilution factors: 2/100 (50), 5/ 200 (40). Total dilution 40x50=2000.
Original concentration = 2000x0.512=1024 mg/100 ml=1.024 g/100 ml= 1.024% w/v.

Also; 2 ml volume of eyedrops contain 20.48mg/2ml of the drug

Pharm. Analysis I 96
 Delusion (exercise)…
• Eg3. A 5 ml sample of an injection of a steroid phosphate was diluted to 100 ml.

Then 10 ml of the diluted injection was diluted to 100 ml and this dilution was

further diluted 10 to100 ml. From measurement by UV the diluted sample was

found to contain 0.249 mg/100 ml of the steroid. What was the original

concentration of the injection in %w/v and in mg/ml?

Answers: 0.498% w/v, 4.98 mg/ml

Pharm. Analysis I 97
 Quiz one
1. 0.5321 g of α-adrenergic blocking drug is dissolved in 50 ml of methanol/0.1%
w/v acetic acid (20:80 v/v) and the sample is then diluted by taking 10 ml of
the solution and diluting it to 50 ml. The diluted sample was analyzed by
HPLC and was found to contain 0.153 mg/100 ml of an impurity. What is the
%w/w of the impurity in the drug?
2. An infusion which was stated to contain 0.5000% w/v KCl was diluted so that
its K content could be determined by flame photometry. The following
dilutions were carried out:
(i) 10 ml of the sample was diluted to 200 ml with water.
(ii) 10 ml of the diluted sample was diluted to 200 ml with water.
The sample was found to contain 0.6670 mg/100 ml of K.
(Atomic weights: K = 39.1 Cl = 35).
Calculate:
(i) The % w/v of KCl present in the infusion
(ii) The % of stated content of [Link]. Analysis I 98
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)
2. For example, Find the number of gram equivalents (mole)
present in 0.5 g of HCl, & Normality if it is found in 500 ml
of solution. (ans. No of equivalent mole = 0.0137 mol)
3. How would you prepare 500mL of 0.200 M NaOH (aq) from
a stock solution of 1.5 M NaOH? Ans. V1=0.067L

Pharm. Analysis I 99
1.5. Physical And Chemical Properties Of

Drug Molecules

Pharm. Analysis I 100

 Chemical analysis is an important part of the quality assessment of

drugs.
 A deeper understanding of the analytical method requires

knowledge about both the analytical technique and the chemical

properties of the analytes.
 Therefore a basic knowledge of a number of physicochemical

properties of molecules is needed to be able to understand and

further develop analytical chemical methods.
101
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

Pharm. Analysis I 103

 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.

111
 Dissociation Constants
The greater the value of Ka, the stronger the acid

112
 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 above 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  0.15 x x
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] (0.262)
[H3O ] = Ka x
+
= 1.8 x 10 x
-5 = 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:
K w 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.)

Formulation Likely physical Effects

instability problems

Oral solutions 1- Loss of flavour Change in

2- Change in taste smell or
3- Presence of off flavours feel or taste
due to interaction with
plastic bottle
4- Loss of dye
5- Precipitation
6- discoloration
 Physical stability (Cont.)
Formulation Likely physical instability Effects
problems
Parenteral 1. Discoloration due to photo Change in
chemical reaction or oxidation appearance
solutions and in bio-
2. Presence of precipitate due to
interaction with container or availability
stopper
3. Presence of “whiskers”
4. Clouds due to:
(i) Chemical changes
(ii) The original preparation of a
supersaturated solution
 Physical stability (Cont.)

Formulation Likely physical Effects

instability problems

Suspensions 1- settling 1-Loss of drug

2- caking content
3- crystal growth uniformity in
different doses
from the bottle

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

Formulation Likely physical Effects

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

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

3. Bleeding 3-change in drug

release rate.
 Physical stability (Cont.)

Formulation Likely physical Effects

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

Formulation Likely physical Effects

instability problems
Capsules Change in: Change in
a) Appearance drug release
b) Dissolution
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 group Examples

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 materials Micrococci

Starches Coliforms
Pigments Salmonella

171
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 Concentration

% 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 insoluble the use of buffers

crystals from the glass

3- Permits the transmission of Amber coloured glass

light which may accelerate
decomposition.
175
 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 period

Storage temperature humidity covered by data at
(°C) (%) 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

183/61
 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.
Pharm. Analysis I 201
Pharm. Analysis I 202