QUALITY CONTROL I:

DRUG TESTING AND

ASSAY

PEARLYN GRACE O. BANGAAN, RPh

QUALITATIVE-QUANTITATIVE
CHEMISTRY
• AKA:
–Analytical Chemistry
–Wet Chemistry Methods
 USES
1.) Manufacturing Pharmacy
-Purity – Raw Materials
-% Label Claim – Finished Products

2.) Body
- Chemical Constituents – for diagnosis
 USES
3.) Analyze medicinal agents and their
metabolites/ KINETICS

-active ingredients – used in monitoring

patients

-metabolites – active, inactive/water

soluble, reactive/toxic
DIVISIONS OF ANALYTICAL
CHEMISTRY

1. Qualitative
2. Quantitative
 1. Qualitative
• Test for the NATURE OF THE
CONSTITUENTS of a given material

• COMPOSITION determination
– Elements
– Species
– Compounds
 1. Qualitative

• Ascertainment of IMPURITIES
present in a given sample

• Answers the question:

What is present in a
given sample?
 2. Quantitative

• Determines the PROPORTION

of a certain component in a sample

• Answers the question:

How much is present
in the sample?
 Classification of Quantitative
Method of Analysis

• Based on the extent of determination

• Based on the sample size
• Based on materials used
• Based on the amount of analyte present
• Based on the type of final measurement
 Based on the extent of determination

• PROXIMATE – total amount of class or

groupof active plant principles in a given
sample (ex. % alkaloid in Belladona leaf)

• ULTIMATE – one or more, but not all the

constituents are determined (ex. % atropine,
% hyoscyamine in Belladona leaf)
 Based on the sample size

Types Sample size

Ultra-microanalysis <1 mg

Microanalysis 1 mg-10 mg

Semi-microanalysis 10 mg -100 mg

Macroanalysis: 100 mg or more

 Based on materials used

• Chemical
– Chemical reagents
• Physical
– Instruments and special apparatus
• Biological
– Living organisms, microorganisms, animals
Based on the amount of analyte present

• MACRO/MAJOR – analyte is present in

high concentrations

• TRACE ANALYSIS – analyte is present in

low concentrations
3. Based on nature of method

• Classical
• Instrumentation
• Miscellaneous/Specific
Method
 A. CLASSICAL
• AKA: general/ chemical
• Titrimetric:
– VOLUME of standard reagent solution
reacting with the analyte
• Gravimetric:
– WEIGHT of pure analyte or compound
of known stoichiometry
 B. INSTRUMENTATION
• Based on a specific PHYSICAL or
CHEMICAL PROPERTIES of the
analyte
• More accurate analysis
• Examples:
– Spectrophotometry, polarimetry,
chromatography
C. MISCELLANEOUS/ SPECIFIC METHODS

• Involves some drugs, and plant

products
• Examples:
–Water content
–Ash content
–Acid value
QUALITY CONTROL I:
DRUG TESTING AND
ASSAY
 QUALITY
• The totality of features and
characteristics of a product or
service that bears on its ability to
satisfy stated or implied needs
 Quality in drugs
• Sum of all factors which
contribute directly or indirectly
to the safety, effectiveness and
reliability of the product.
 Quality in drugs
• Free from impurities
• Is physically and chemically stable
• Contains the amount of AI as
stated on the label
• Provides optimal release of AI
when the product is administered
Quality assurance

• Sum totalof activities to ensure that

products are consistently of quality
• Monitors the integrity of the
production process

• Process oriented
 QA Departmental Functions
• Ensures quality policies adopted are
followed
• Contact with regulatory agency
• Identification and preparation of the policies
and Standard Operating Procedures
• Quality monitoring or audit function
 Quality control
• A tool that gives the assurance that
a product conforms to standards
and specifications through a system
of inspection, analysis, and action.
• Monitors the finished product
Quality Control Departmental Functions

• Sampling and analytical testing

• In- Process Testing
• Environmental monitoring
• Tests on finished dosage form
• Monitoring product quality
Sampling and analytical testing

• Analytical testing of raw materials

• Packaging materials
• Labeling
• Selection and qualification of
vendors
 IN-PROCESS Testing

• Give in-process alert or action

levels
• Provides early warnings of
conditions that can lead to out-of-
control situations
 Environmental conditions
monitoring
• Different levels of control are
established depending on the
intended use of the dosage form
– Air and water systems
– Microbial matter
– Levels of particulates
 Final Product Testing

• Analytical testing of products

before release
 MAIN AREAS OF
QUALITY CONTROL
 Main areas of quality control
• Raw Material Quality Control
• In-Process Quality Control
• Finished Product Quality Control
 Raw materials QC
• For Parenterals
– Identification Test: Active Ingredient
– Solvent: Pyrogen Test

• Glass:
-Powdered Glass Test: Leaching Potential
-Water Attack Test: Intact surface

• Rubber: Durometer
 In-Process QC
• Volume Fill
• Detection of Particles
 Final Product QC

• Done on final products on its

final container
–Stability Test
–Leaker’s Test
–Sterility Test
 Documentation
“If it wasn’t documented, it wasn’t done”
• Raw material and final product
specifications
• Validated test methods
• Equipment
 Certificate of analysis
• Results of all tests conducted
• Show compliance or non-
compliance
 MONOGRAPH
• a publication that specifies for a drug
the kinds and amounts of ingredients it may
contain, the conditions and limitations for
which it may be offered, directions for use,
warnings, and other information that its
labeling must contain
• It may contain important information
concerning interactions with other drugs.
 MONOGRAPH
• All the TESTS to be conducted on
a product
• Appropriate REFERENCES
containing
– Details of procedure
– Expected result
 Components of a Monograph
• Molecular Formula
• Added Substances, Excipients, and
Ingredients
• Description and Solubility
• Identification Test
• Assay
• Example of a monograph
-Calcium Citrate
-Arginine
-Ascorbyl Palmitate
 Assignment
• Group yourselves into 4.
• List 3 examples of monograph
from the USP-NF
• Present your work tomorrow.
 STEPS IN A TYPICAL
QUANTITATIVE ANALYSIS
1. SELECTING A METHOD OF ANALYSIS

• Based on the extent of determination

• Based on the sample size
• Based on materials used
• Based on the amount of analyte present
• Based on the type of final measurement
 2. SAMPLING
• Get representative amount from the whole
sample
 3. PREPARING THE LABORATORY
SAMPLE

• Washing the sample to remove

unnecessary dirt
 4. DEFINING REPLICATE SAMPLE

• Portions of a material of
approximately same size carried thru
same analytical procedure
 5. DISSOLVING THE SAMPLE
• Dissolving the sample in the appropriate
solvent (HCl, Sulfuric acid, Hypochloric acid)
 6. ELIMINATING INTERFERENCES

• Species that causes an error by

enhancing/attenuating the quantity being
measured
• SEPARATION/ MASKING
7. TREATING THE SAMPLE AND
MEASURING THE ANALYTE
8. CALCULATING THE AMOUNT
AND EVALUATING THE RESULTS
CENTRAL VALUES
 MEAN/AVERAGE
• Most reliable measure of central tend
 MEDIAN
• It is a point of measure that divide
the distribution of arranged test
scores from highest to lowest or vice
versa in half
• It is the most stable measure of
central tendency
 MODE
• Score that occurs most frequently
• The mode for a set of test scores need not be
unique
• It is possible to have two or more modes
EXAMPLE
 EXAMPLE
8, 19, 22, 86, 43, 19, 25, 98, 76, 19, 7

a. Mean? -38.36
b. Median?-22
c. Mode?-19
 EXAMPLE
89, 87, 93, 91, 90, 87, 89, 99, 89, 75, 91

a. Mean?- 89.09
b. Median?- 89
c. Mode?- 89
 EXAMPLE
13, 9, 18, 89, 65, 17, 23, 23, 9, 11, 9, 17

a. Mean?-25.25
b. Median?-17
c. Mode?-9
 ACCURACY
• Indicates the closeness of the measurement to
its true or accepted value
• Expressed as degree of error (absolute error
or relative error)
• ABSOLUTE ERROR

Absolute error= Theoretical value-

experimental value

• RELATIVE ERROR

Relative error= TV-EV

TV
 PRECISION
• Describes the reproducibility of
measurements or the closeness of results that
have been obtained in exactly the same way
• Described using standard deviation, variance
and coefficient of variation
 TYPES OF ERRORS IN EXPERIMENTAL
DATA

No analysis is free of error or

“uncertainty”

• Systematic/ Determinate Error

• Random/ Indeterminate Error
• Gross Error
 SYSTEMATIC ERROR

• AKA: Determinate Error

• The error is reproducible and can
be discovered and corrected.
 TYPES OF SYSTEMATIC ERRORS

1. INSTRUMENT ERRORS
2. METHOD ERRORS
3. PERSONAL ERRORS
 INSTRUMENT ERRORS
• Failure to calibrate, degradation of parts
in the instrument, power fluctuations,
variation in temperature, etc.

• Can be corrected by calibration or

proper instrumentation maintenance
 METHOD ERRORS
• Errors due to no ideal physical or chemical
behavior

Example: completeness and speed of reaction,

interfering side reactions, sampling problems

• Can be corrected with proper method

development
 PERSONAL ERROR
• Result from carelessness, inattention or
personal limitations of the analyst and result
from prejudice and color acuity problems

• Can be minimized or eliminated with proper

training and experience.
 RANDOM ERROR

• AKA: Indeterminate Error

• no identifiable cause
• Always present, cannot be
eliminated
• The ultimate limitation on the
determination of a quantity
 RANDOM ERROR
Example:
• Reading a scale on an instrument caused by
the finite thickness of the lines on the scale
• Electrical noise
 GROSS ERROR
• are caused by experimenter carelessness or
equipment failure
• Occurs occasionally, are often large and may
cause a result to be either too high or too low;
leads to outliers
• These "outliers" are so far above or below the
true value that they are usually discarded
when assessing data
 BASIC
PRINCIPLES OF
ANALYSIS
Basic Principles of Analysis
• pH
• Buffer
pH (Power of Hydrogen)
• pH is the number of gram equivalent
of H+ per Liter of solution

*In solution, pH means the range of

acidity and basicity
 Theories of pH
• ARRHENIUS THEORY
• BRONSTED-LOWRY
THEORY
• LEWIS THEORY
 ARRHENIUS THEORY

• Acids releases H+

• Bases releases OH -
BRONSTED-LOWRY THEORY

• Acid donated proton/ p+

• Base accept proton/ p+

 LEWIS THEORY
• A~A

• Acid Accept electron/ e-

• Base donate electron/ e-

 PROTOLYSIS
• Transfer of a proton/ p+ from one
molecule to another

*monoprotic acid- gives up one proton/ p+

per molecule

*monoprotic base- accept one proton/ p+

per molecule
 PROTOLYSIS

• If an acid gives up greater number

of proton/ p+, then it has greater
strength acid

• If a base accepts greater number

of protons/ p+, then it has greater
strength base
 AUTOPROTOLYSIS
• Transfer of one molecule to one identical
molecule

Example:

H2O + H2O H3O+ + OH-

hydronium ion hydroxide ion
 AMPHOTERIC
• Property where a substance can
act either as acid or base

Example:

H2O- water
 pH equation
• SORENSEN’S EQUATION

pH= -log (H+) pOH= -log (OH-)

(acid) (base)

pH= log 1/H+ pH= log 1/OH-

(acid) (base)
 EXAMPLE
• Calculate the pH of a
solution containing
0.000273 g ion per
Liter of H .
+
 EXAMPLE
• What is the pH of the
solution, if the
concentration of H is
+

1x10 -10?
 EXAMPLE
• What is the pH of the
solution, if the
concentration of H is
+

1.8x10 -5?
 EXAMPLE
• What is the pH of a
solution which has a
OH ion concentration
-

of 3.0x10 mol/L?
-6
pKw (dissociation constant
of water)

pKw= pH + pOH

pKw= 14
 EXAMPLE
• What is the pH of
NaOH with a
concentration of
3.0x10 M?
-2
 EXAMPLE
• A KOH solution has a
concentration of 0.05M,
what will be the pH of the
resulting solution if water is
added?
 EXAMPLE
• Calculate theH + ion
concentration in a
solution with a pH of
5.84.
 BUFFER
• a solution that resists changes
in pH when acid or alkali is
added to it
• Buffers typically involve a weak
acid or alkali together with one
of its salts
 BUFFER PAIRS
• weak acid + salt of weak acid
Example:
acetic acid + Na acetate
(HAC) (NAC)

• Weak base + salt of weak base

Example:
Ammonia + Ammonium chloride
 BUFFER EQUATION
• HENDERSON-HASSELBALCH
EQUATION

For weak acid:

pH= pKa + log (salt/acid)

For weak base:

pH= pKa + log (conjugate base/ base)
 BUFFER CAPACITY
• Ability of a buffer solution to resist
changes in pH upon addition of acid and
alkali

VAN SLYKE
- measure of buffer capacity
- was responsible for a quantitative
expression
- The higher the buffer capacity, the
lower change in pH
 EXAMPLE
• Calculate the pH of the buffer solution
containing:

Acid= 3.7x10-2
Salt= 4.8x10-2
pKa= 9.26
 EXAMPLE
• Calculate the pH of a buffer
composed of 0.1 M acetic acid
(CH3COOH) and 0.6 M acetate
(CH3COO-) knowing that the
acid dissociation constant Ka is
1.8x10-5
 EXAMPLE
• Calculate the pKa and pH of the buffer
solution containing:

acid concentration: 1.8x10-5

acid= 0.1 M
Salt= 0.1 M
 EXAMPLE
• Calculate the concentration of the
acid and its salt in the buffer
solution with a pH of 4.76 and a
dissociation constant of 4.5.
Other Methods of
Analysis
 Ash content
determination

Importance Methods
Inorganic salts Total Ash
(naturally-occurring or as Acid-Insoluble ash
adulterants)
Sulfated ash
 Total Ash
• Residue remaining after incineration
 Acid insoluble ash
• Part of the total ash which is insoluble in
diluted HCl
• Represents the silica content of the sample

• % AIA= acid insoluble ash x100

• wt of the sample
 Sulfated ash
• Ash treated with H2SO4
Approximate Temperature
Equivalent

Very dull red heat 500C- 550C

Dull red heat 550C-700C
Bright red heat 800C-1000C
Yellow Heat 1000C-1200C
White Heat 1200C-1600C
 Water content
determination

• Method I
• Method II
• Method III
 Method I
• Titrimetric (Karl Fischer)
• Reagent is freshly prepared (80% efficient)
• Pyridine, methanol, SO2 and iodine
• Primary standard: Na tartrate

%H2O= V of KFR in mL X EF x 100

weight of sample in mg

EF= water equivalence factor

Karl Fischer reagents
• Anhydrous methanol
– prevents pyridine- sulfur complex
• Iodine
– Reacts with water to form hydroiodic acid
• Sulfuric acid
– reacts with water to form sulfur trioxide
• Pyridine
– Prevent the reversal of reaction
 Problem 1
Calculate the water content of streptomycin
powder using 3.50 g sample. The water
equivalence factor is 4.6 and the volume
consumed was 9.2 mL. The water content was:
%H2O= V of KFR in mL X EF x 100
weight of sample in mg
% H2O= 9.2 mL x 4.6 x 100
3500mg
%%H20= 1.21%
 Method II
•Azeotropic Distillation
•Xylene Method
•Toluene Moisture Apparatus
•For samples containing 2-4 mL moisture
•Also used in Alcohol content determination
%water= mL reading x 100
weight of sample
 Method III: Gravimetric

• For vegetable drugs

Assay of fatty substances,
waxes, balsams and resins
 Acid Value
Acid number or Acidity index
No of mg of KOH necessary to neutralize the free
acids in 1 gram of a substance
Acid value= N x V x 56.11
wt in g
 Ester value
• AKA: Ester number
• No. of mg of KOH required to saponify the
esters contained in 1 g of oil, fat, wax, resin or
balsam
 Saponification value
• AKA:
• Saponification number
• Koettsdorfer number
– No of mg of KOH necessary to neutralize the free
fatty acids and saponify the esters in 1 g of a
substance
SV=AV + EV
 Iodine value
• AKA: Iodine number
• Numbe of mg of Iodine absorbed by 100 g of
oil, fat, wax or other substances
• Measure degree of UNSATURATION
• Method I: Hanus Method (use of
Iodobromide)
• Method II: Wij’s method
• Method III: Hubl’s method< unofficial>
 Iodine value
IV= (N x V)blank- (N x V) actual x 0.1269/meq x 100
wt in g

Type IV Examples
Drying oils >120 Linseed oil
Fish oil
Cod liver oil
Non-Drying oils <100 Olive oil
Almond oil
Semi-drying oils 100-120 Cottonseed oil
Sesame seed oil