PHARMACEUTICAL ANALYSIS MODULE 3

UNIFORMITY OF DOSAGE FORM

DRUG PRODUCT TESTING

TEST FOR TABLETS 1. Weight variation test

➢ Dissolution tests 2. Content uniformity test
➢ Disintegration test
➢ Friability testing WEIGHT VARIATION
➢ Tablet hardness ● Weight variation is used to ensure that each
➢ Diameter and thickness
➢ Weight variation test tablet contains the proper amount of drug
● It involves the determination of individual
● Drug product testing is a process of measuring weights of the samples tested and computation
and analyzing the properties of drug products for the average weight
before, during, and after the manufacturing
processes. ● the test was carried out by weighing 20 tablets
● This is a means of ensuring a product’s individually using an analytical balance, then
performance, safety, quality and compliance calculating the average weight, and comparing
with established standards.
the individual tablet weight to the average.

COMPENDIAL REQUIREMENT FOR SOLID

DOSAGE FORMS FORMULA WEIGHT VARIATION TEST

➢ The weight of the tablet is the quantity of the

● Testing of pharmaceutical products involves granulation which contains the labeled
chemical, physical, and some microbiological amount of the therapeutic ingredients.
evaluation or tests. This would vary depending ➢ Twenty tablets are weighed individually and
on the dosage form the average weight is calculated
● According to WHO, the term quality control
𝐴𝑐𝑡𝑢𝑎𝑙 𝑤𝑡 − 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑡
refers to the sum of all procedures undertaken %𝑤𝑡 𝑣𝑎𝑟𝑖𝑎𝑡𝑖𝑜𝑛 = 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑡
𝑥 100
to ensure the identity and purity of a particular
pharmaceutical product.
● Quality control involves testing of units and
determining if they are within the specifications Average Weight (mg) % Weight Variation
for the final product.
● The purpose of the testing is to determine any < 150 ± 10
need for corrective actions in the 150 to 324 ± 7.5
manufacturing process to uncover defects from
the decision whether to allow or deny product > 324 ±5
release.
● Good quality control improves the overall Criteria:
quality of the drug and helps companies meet
● NMT 2 tablets should differ by more than the
consumer demands for better products
% weight variation.
● Quality control tests for tablets include weight
● No tablet differs by more than double the %
variation, content uniformity, disintegration,
weight variation
dissolution, friability, hardness, and thickness
↳ Second criteria if more than 2 fail the test
and double the weight variation (ex. ±5 → ±10)

ACCEPT IF BOTH CRITERIA IS SATISFIED!

 PHARMACEUTICAL ANALYSIS MODULE 3

EXAMPLE

CONTENT UNIFORMITY TEST

LCL = Average weight X __%
● It is intended to establish within limits that a
product has a uniform amount of an active
ingredient in a batch. It is done to determine
uniformity in dosage units for samples whose
active ingredient is less than 50 mg.
● A sample of 30 tablets is selected and ten are
individually assayed
● NLT 85% and NMT 115%

CRITERIA:

STAGES CONDITION SPECIFICATION REMARKS

None of the
samples
1 10 samples 85% - 115%
exceed the
specifications

Repeat the
If more than 1
test with
exceeds the 85% - 115%
specification additional 20
samples

Repeat the
test with
1 sample 75% - 125%
additional 20
samples

None of the
samples
Additional 20
2
samples
85% - 115% exceed out
the
specification
 PHARMACEUTICAL ANALYSIS MODULE 3

● If more than 1 exceeds 85% to 115% or 1 prescribed time when placed in a liquid
exceeds 75% to 125%, repeat the test with medium under experimental conditions.
additional 20 samples.
● Complete disintegration is a state in which any
● With the 20 samples, the requirements are met
if none of the tablets falls out within the limits residue of the unit except fragments of
of 85% to 115% insoluble coatings and capsule shells
remaining on the screen of the test apparatus
or adhering to the lower surface of the disk, if
EXAMPLE used, is a soft mass having no palpably firm
core.
● An in-vitro test that measures the amount of
time required for a given percentage of the
drug substance (in a tablet form) to go into
solution under specified set of conditions
● A process by which solid of only fair
characteristics enters into solution. It provides
steps towards the evaluation of physiological
availability of drug substances and serves as
an approximation of the drug’s absorption.

PARTS OF DISINTEGRATION APPARATUS

● 1000-mL low form beaker

- For immersion of fluids
↪ Contain 800 mL water or dissolution
medium
● A thermostatic arrangement for heating
fluid
- 35 - 39 Celcius
● Basket rack assembly
- A device for raising and lowering the basket
- Moves vertically along its axis
↪ 29 - 32 per cycle minute

DISINTEGRATION
● Involves mechanical break up of compressed SAMPLE PROBLEM OF DISINTEGRATION
tablet into smaller granules upon ingestion
● This test is conducted to determine whether
tablets or capsules disintegrate within the
 PHARMACEUTICAL ANALYSIS MODULE 3

USES:

1.Quality Control

◦ Examining batch to batch homogeneity

◦ Examining batch to batch conformity

◦ Examining stability

2.Research and Development

◦ Examining drug release behavior of pre-formulations

◦ In- vitro stimulation of the gastrointestinal passage

3.IVIVC (In vitro-in vivo correlation)

SAMPLE PROBLEM

TABLETS/CAPSULES DELIVER DRUG

THROUGH A SERIES OF PHYSICAL AND
PHASE CHANGES AS DESCRIBE

● Tablet or Capsule
● ↪Disintegration → Aggregates
● Aggregates
● ↪De-aggregation → Particles
● Particles
● ↪ Dissolution → Drug in solution

USP GUIDELINES ON DISSOLUTION TESTING

● Preferred medium is distilled water

● Experiment conducted should be at 37 C
● Usual volume is 500 mL to 1000 mL

Other dissolution medium used:

● Buffered aqueous solution (pH 4 - 8)

● Diluted acid ( 0.001 N to 0,1 N HCl)
 PHARMACEUTICAL ANALYSIS MODULE 3
HARDNESS, THICKNESS, DIAMETER AND
FRIABILITY TESTS
● Hardness Test
○ Also known as “crushing strength”
○ Hardness refers to the resistance of the tablet
to chipping, abrasion or breakage under
conditions of storage, transportation and
handling before usage.
● Hardness determinations are made throughout the
tablet runs to determine the need for pressure
adjustment on the tableting machine
➔ If the tablet is too hard, it may not disintegrate
in the required period of time or meet the
dissolution specification
➔ If the tablet is too soft, it will not withstand the
handling during subsequent processing such
as coating or packaging and shipping
operations
RULE OF THUMB
Most primitive and practical test for the hardness of
tablets.
● A commonly used rule of thumb describes a
tablet to be proper hardness if it is firm enough
to break with a sharp snap when it is held
between the second and third fingers and
using the thumb as the fulcrum, yet, doesn’t
break when it falls on the floor
STOKES-MONSANTO
● An instrument that measures the force
required to break the tablet when the force
generated by a coil spring is applied
diametrically to the tablet
● The force is measured in kilograms and when
used in production, hardness of 4kg is
considered to be min. For a satisfactory tablet
STRONG-COBB HARDNESS TESTER
● Measures the diametrically applied force
required to break the tablet
● The force is produced by a manually operated
air pump. As the pressure has increased a
plunger is forced against the tablet and placed
on an anvil.
● The final breaking point is indicated on a dial
calibrated into 30 arbitrary units.
PFIZER HARDNESS TESTER
● Operates on the same mechanical principle as
ordinary pliers. The force required to break the
tablet is recorded on a dial and may be
expressed as either kilograms or pounds of
force.
SCHLEUNIGER HARDNESS TESTER
● Known as the heberlein and electrically
operated test equipment and most widely used
apparatus to measure tablet hardness.
 PHARMACEUTICAL ANALYSIS MODULE 3

TABLET THICKNESS

● Tablet thickness refers to the depth or extent

from one surface to another surface.
● Criteria: Average thickness ± 5% deviation.
● Determined with a caliper or thickness gauge
which measures the thickness in mm.

FORMULA

FINAL READING = Main scale reading + Circular

scale reading x least count (LC)

LC = 0.01 mm.#

Example:

Sample problem

ACTIVITY
 PHARMACEUTICAL ANALYSIS MODULE 3
DIAMETER TEST
● Tablet diameter refers to the straight line from one
side to the other side of a circle that passes
through the center point
● Criteria: Average diameter ± 5% deviation.
FRIABILITY
● It is a tablet property related to hardness. It is
the ability to withstand abrasion in packaging,
handling, and shipping.
● Friable is the tendency of a tablet to crumble,
chip, or break
FRIABILITOR
DRUM
- Made of transparent synthetic polymer with
polished internal surface and subject to
static build-up - 285 mm (i.d)
- Rotates at a speed of 25 rpm
- Each turn the tablet fall about 130 mm onto
the drum
FRIABILITY TEST
● Tablets weighing < 650 mg - 20 tablets
● Tablets weighing > 650 mg - 10 tablets
● Tablets are to be rotated 100 times
● Loose dust are removed by air pressure or soft
brush using no. 10 sieve
● % weight loss is calculated
Requirement:
● The weight loss should not be more than
1%.
● The test should be repeated twice if did not
meet the specification
COMPENDIAL REQUIREMENT FOR
PARENTERALS AND SEMISOLIDS
● Quality control is a fundamental segment that
refers to a process of striving to produce a
product by a series of measures requiring an
organized effort by an entire pharmaceutical
company to eliminate or prevent error at any
stage of production.
● Quality control deals with testing, sampling,
specification, documentation, release
procedure which ensure that all tests are
actually carried out prior to release of material
for sale or use. Until its quality meet the
specification in accordance with the
international standard.
● In parenteral dosage forms, its quality control
have 4 basic area these are: Sterility, Freedom
form Pyrogens, Freedom from particulate
matter and leakers.
● The achievement of sterile, non pyrogenic and
particulate free parenteral product provides a
quality product for the patients and consumers.
QUALITY CONTROL TESTS FOR
PARENTERALS
STERILITY TEST
● Sterility test is a microbiological test suitable for
revealing the presence of viable forms of
bacteria, fungi and yeasts in or on
pharmaceutical articles
● Used as a validity tool in any aseptic processing
 PHARMACEUTICAL ANALYSIS MODULE 3
(i.e. Sterilization process) of a parenteral
product
● Aqueous parenterals for IV INJECTION requires
the most critical evaluation for sterility
● Test Organism and Culture Media
○ Soybean Casein Digest Medium for
Streptococcus species
○ Fluid Thioglycollate Medium for Clostridium
species
● METHODS OF STERILITY TEST
○ Direct Inoculation Method
➔ involves the direct transfer of the
pharmacopeial article to test media
➔ Method of choice for medical devices,
non-filterable sterile products, parenteral
emulsions
➔ Turbidity/cloudiness in the test samples is a
indication of microbial contamination
○ Membrane Filtration Method
➔ method of choice for sterility testing; it uses
a suitable membrane filter consisting of an
assembly that facilitates handling of articles
and that allows the processed membrane to
be removed aseptically for inoculation of
appropriate media
➔ Applicable for aqueous/filterable solutions
➔ It involves the passage of a solution through
a sterile membrane filter. Any contaminating
microorganisms will be retained on the
surface of the filter.
➔ The membrane filter is cut in half. Then
separately cultured and incubated. The
filters are examined for microbial growth.
SPECIFICATIONS OF MEMBRANE
➔ Nominal porosity of 0.45 um
➔ Diameter of 47 mm
➔ Flow rate of 55 to 75 mL/minute at a
pressure of 70 cm of Hg
○ Biological Indicators - the most preferred
method which makes use of the different
methods of sterilization
■ Moist heat and Ethylene oxide -
Bacillus stearothermophilus;
■ Dry heat - Bacillus subtili
■ Ethylene oxide - Bacillus
stearothermophilus
USP REQUIREMENT FOR PARENTERAL
STERILITY TESTING
● Absolutely NO GROWTH on the surface of
the membrane filter for the entire incubation
period.
● Absolutely NO GROWTH / NO TURBIDITY
in the culture tubes for the entire incubation
period.
● No Retest is required unless an evidence
had been established that the aseptic/sterile
technique has been violated.
LEAKER’S TEST
● Done using negative pressure within
incompletely sealed ampules while
submerging them in a dye solution
Leaker Test for Ophthalmic Ointments
○ By Classical Blotting Paper Method – the
sealed and filled ophthalmic ointment
collapsible tubes are placed in an absorbent
paper, heated in a 60C (+/- 3) oven for 8
hours
SAFETY TEST
● A test done using white mice or guinea pigs.
● It is used for determining the safety of the
plastic devices used in parenteral products.
PARTICULATE MATTER TEST
● Equipment
○ Electronic Particulate Counter
○ Membrane Filtration Technique
CLARITY TEST
● A test done to prevent the distribution and
use of parenteral products that contain any
 PHARMACEUTICAL ANALYSIS MODULE 3

particulate matter. SIGNS AND SYMPTOMS OF PYROGEN

CONTAMINATION
MINIMUM FILL TEST FOR SEMISOLIDS

- Febrile reaction in human beings

● Done for the assessment of content - Chills
uniformity in semisolid products like creams, - Pains in back an legs
ointments, lotions, aerosols, jellies and - Malaise
pastes.
SOURCES OF PYROGEN
PYROGEN TEST

- Water
● Bacterial Endotoxin [LAL] Test - Equipment
○ “Limulus Amoebocyte Lysate test” - Solute
○ A test for estimating the concentration of
bacterial endotoxins that maybe present
in a sample
● Pyrogen Test
○ Also known as “rabbit test”
PYROGEN TEST
○ It is designed to limit to an unacceptable
level the risks of febrile reactions in the RABBIT TEST
patient to the administration or injection
of a product ● In vivo pyrogen testing
○ It involves measuring the rise in ● Use healthy mature rabbits
temperature of rabbits following ● Qualitative Fever Response in Rabbits
administration of a test solution (QFRiR)
PYROGEN ● Rabbits are used because of its similarity
with the human body's physiological
- Any substance that produces fever response to a parenteral product
- Products of the metabolism of contaminated with pyrogens.
microorganisms or portions of the protein ● Uses healthy rabbits, weighing at least 1.5
coat of bacteria. kg BW, temperature interval is NMT 1C,
● Don not use rabbit have a temperature
exceeding to 39.8C
● Use rabbits whose temperature do not vary
(between) by more than one degrees
MOST POTENT PYROGENIC SUBSTANCES ARE centigrade from each rabbit
PRODUCED BY ● Unless otherwise specified in the individual
monograph inject to an ear vein of three
● Gram-negative bacteria (endotoxin
rabbits 10mL of the test solution per kg of
secretion)
body weight, completing each injection
● Gram-positive bacteria
within 10 minutes after the start of
● Fungi
administration.
PROPERTIES ● 3 rabbits are used (for each drug tested)
● Positive control - Brewer's Yeast
● Negative control - NSS
- Lipid in nature ● Test drug - not exceeding 10 mL/ kg BW
- Phosphorus attached to a polysaccharide or Dose
a protein or both ● Sham Test - conducted as preparatory
stage to the test proper. Designed to
 PHARMACEUTICAL ANALYSIS MODULE 3
establish a normal baseline of the body
temperature of the test rabbits.
PROCEDURE
● Get the baseline temperature of the rabbits
● inject into an ear vein 10mL/kg of the test
solution, in portions, completing each
injection within 10 minutes after start of
administration
● Record the temperature at 1, 2 , and 3 hours
subsequent to the injection
TEST INTERPRETATION
● If no rabbit shows an indiviual rise in
temperature of 0.6 Celcius or move above
its respective control temperature and if the
sum of three (3) individual maximum
temperature rises does not exceed 1.4
Celcius, the product meets the requirements
for the absence of pyrogens
● If any rabbit shows an individual rise of 0.6
Celcius or more, or if the sum of the three
individual temperature rises exceeds 1.4
Celcius, continue the test using other
rabbits.
● If not more than three of the eight rabbits
show individual rises in temperature of 0.6
Celcius or more and if the sum of the eight
individual maximum temperature rises does
not exceed a 3.7 Celcius, the material under
examination meets the requirements for the
absence of pyrogens.
ACCEPT IF
● No rabbit shows an individual rise in
temperature that is 0.5C
● Sum of the three individual temperature
rises should not exceed 1.4C
● Otherwise, repeat test using an additional 5
rabbits
INTERPRETATION AFTER REPEAT TEST
REJECT IF
● More than 3 of the 8 rabbits showed a
temperature rise of 0.5C
● Sum of the readings of temperature rise for
the 8 rabbits is more than 3.3C
PYROGEN TEST
LAL TEST (LIMULUS AMOEBOCYTE LYSATE )
● IN VITRO TEST
● Also known as bacterial endotoxin testing
(BET)
● Aqueous extract of blood cell (amoebocytes)
from the blue-blooded horseshoe crab
Limulus polyphemus
● Use for endotoxin detection
● Developed in the 1960s by Drs. Bang and
levin
● Based on the gel-clot formation of a
pyrogen-contaminated product, in the
presence of lysate(obtained from the
horseshoe crab)
● Detects only pyrogens from gram-negative
bacteria.
● Simpler, more rapid method than QFRiR
● Quantitative ( measures EU/ mL)
● Faster
- More economical
- More sensitive than rabbit pyrogen
test
3 principles
1. Gel - Clot formation (clot formation)
2. Chromogenic (color development)
3. Kinetic (turbidimetric)
METHODS OF ENDOTOXIN TESTING
Since 1940s - Rabbit pyrogen test
 PHARMACEUTICAL ANALYSIS MODULE 3

SInce 1970s - LAL test ○ Carr’s Index of Compressibility

TYPE OF LAL TEST
● GEL-CLOT
● TURBIDIMETRIC
● CHROMOGENIC
PROCEDURE OF LAL TEST
● To use the commercial product, a laboratory
analyst reconstitutes the vial of freeze-dried
LAL with endotoxin-free water
● An equal amount of reconstituted LAL,
usually 0.1 mL, is then added to the sample
of solution in a small, glass, endotoxin-free
test tube
● The mixture is then incubated at 37 Celcius
for one hour
● At the end of this time, the mixture is
examined for gel formation by gently
inverting the tube
○ Hausner’s Ratio
○ Porosity
○ Moisture Content
● Microbial Assays
○ Microbial assays are biological assays
performed with microorganisms such as
bacteria and fungi
○ In a typical microbial assay, each
evaluation is performed with a culture of
microorganisms and the measurements
represent the average response of an
extremely large population of the test
organism

QUALITY CONTROL TESTS FOR SOLUTIONS,

SUSPENSIONS, EMULSIONS, GRANULES AND
MICROBIOLOGICAL ASSAYS

● QC Tests for Solutions

○ Appearance Test
○ Stability Test
○ Physical and Chemical Tests
● QC Tests for Suspensions
○ Sedimentation Volume Test
○ Redispersability Test
○ Particle Size Measurement
○ Rheologic Property
○ Temperature and Gravitational Stress
○ Zeta Potential Determination
● QC Tests for Emulsions
○ Dye Solubility Test
○ UV Fluorescence
○ Cobalt chloride test
○ Direction of Creaming
● QC Tests for Granules
○ Particle Size Determination
○ Angle of Repose
○ Bulk Density
○ Tapped Density