Raw Material Quality Control (RMQC) B.

TEST FOR PURITY (POTENCY)

 ADULTERATION 1. Monograph (Assay)
Debasement of article  Chemical
 Sophistication - Titration
"true adulteration". Addition of inferior material
 Substitution  Instrumental
Entirely different from the one requested - HPLC, GC
 Admixture
Add through accident, ignorance, carelessness  Biological
 Spoilage
Quality destroyed by microorganism C. LIMIT TEST
 Deterioration 1. Gross impurities (insoluble matter, dirt)
Quality impaired due to environment, insects, 2. Biological impurities (microorganisms, cell
etc debris, degradation products)
3. Chemical impurities (trace metals)
Chioride - AgNO, → Agcl
A. ID TEST Sulphates
1. Chemical - BaCl, → BasO,
: Indication: change in color, precipitation, * Arsenic
evolution of gas - Diethylthiocarbamate(vis-spec 535 45mm)
2. Physical + Na and Ca
Specific gravity - Flame spectroscopy
- wt of sample/wt of std (water) (Pycnometer + tron
and Westphai balance) - Ammonium thiocyanate → blood red complex
Alcohols at 15.56°C, Water at 25°C * Heavy metals
- H.S black
Solubility
- Dissolution of compound at a suitable solvent Except:
 Zinc (white)
Refractive index  Cadmium (yellow)
- Bending of light (Abbe refractometer)  Tin (orange)
Ratio of velocity of light in air and velocity of  Mn (pink)
light in a sample

Optical activity
- Angle of optical rotation of a plane polarized
light (Polarimeter)

Boiling point
- Vapor pressure is equal to atmospheric
pressure

Melting point

Instrumental

 IR spec
- Vibration, compared to standard
 UV-vis spec
- Excitation - absorbed and transmitted light
 HPLC
- Retention factor
 NMR
- Amount of magnetic signal/field resonance
COMPENDIAL REQUIREMENTS FOR SOLID AND
SEMI-SOLID DOSAGE FORMS (cont.)
a. LC – means label claim
b. nmt – means not more than
c. pyrogen – a substance, typically produced by
a bacterium, which produces fever when
introduced or released into the blood
d. assay – subjecting the drug product to test to
determine and assess its quality and purity
Acceptance Criteria:
1. Total number of metal particles (>50 μm) is
NMT 50.
2. NMT 1 tube contains > 8 metal particles.
k. Microbial Content
Ophthalmic – sterile
Topical – P. aeruginosa, S. aureus
Urethral, Rectal, Vaginal – molds and yeasts

II. CAPSULES
a. Identification
b. Assay
c. Disintegration test
d. Dissolution Test
e. Uniformity of Dosage Units
f. Weight Variation
g. Content Uniformity
For Hard Gelatin Capsules - remove content and
weigh A Brookfield viscometer.
For Soft Gelatin Capsules - wash capsule with COMPENDIAL REQUIREMENTS FOR LIQUID
solvent DOSAGE FORMS AND PARENTERALS
Acceptance Limit:
1. 9 of 10 units is within 85% - 115% of LC LIQUID DOSAGE FORMS
2. no unit below 75% of LC a. Identification
b. Assay
III. SEMISOLID DOSAGE FORMS c. pH
a. Identification d. Viscosity
b. Assay e. Deliverable Volume - ensures correctness of
c. pH volume
d. Consistency - apparatus: penetrometer TIME: Multiple Unit – 30 minutes
e. Viscosity - apparatus: Brookfield Viscometer Single Unit – 5 seconds
f. Spreadability
g. Spatula Feel - grittiness 1. SUSPENSION
h. Melting point i. Sedimentation Volume
i. Minimum Fill - ensures correctness of net - ideal sedimentation volume = 1
weight of contents of filled containers Formula: SV = setteled volume / total volume of
compared with labeled amount suspension
- limitation: nmt 150g or 150mL ii. Ease of Redispersibility
- sample size: initial (10 containers); retest (+20 - 100% redispersion with minimum agitation
containers) - No sediment should remain at the base after
shaking
Acceptance Criteria: iii. Particle Size Distribution
1. average net content is NLT the labeled - Optical Microscopy
amount. - Sedimentation Rate
2. If the labeled amount is <60g or 60mL, the iv. Rheological Property
%LC of any single container is NLT 90%. - Pour out readily and evenly from container
3. If the labeled amount is >60g or 60mL but - Pseudoplastic/shear thinning
<150g or 150 mL, the %LC of any single v. Zeta Potential Determination
container is NLT 95%. - repulsive forces among particles
- increase zeta potential = slower settling
j. Metal Particles in Ophthalmic Ointments -
microscopic examination of heat-melted
2. EMULSION
ointments for presence of metal particles
i. Dilution Test
- SAMPLE SIZE: 10 tubes
- This method is based on the principle that
emulsion is always miscible with the external
phase. If water is added to water in oil w/o type
emulsion, it will not be mixed. On the other
hand, oil mixes well. Same is the case with o/w
type emulsion. If oil is added to oil in water o/w
type emulsion, it will not be mixed. On the
other hand, water mixes well.
Dilution test of emulsion.
ii. Dye Solubility Test
- It is based on the solubility of any dye in the
external phase. Amaranth green, a water
soluble dye gives color to oil in water o/w type
emulsion, but not to water in oil w/o type
emulsion which will only give color to sudan
red.
iii. Fluorescent Test
- Based on the fluorescence of
oils under ultraviolet light, the emulsion is
examined under the light in the microscope. If
the whole fluid is fluorescent, it is water in oil
w/o but in case of oil in water o/w spotty
fluorescence will appear.
v. Conductivity test
- Based on the electrical conductivity of aqueous
solutions, the electric current is supplied and
electrodes are placed in the emulsion. If the
current is passed, it is oil in water o/w and if is
not passed, it is water in oil w/o.
v. Direction of Creaming
- Creaming is the sedimentation of the
dispersed phase which is either upwards or
downwards, upon which this test is based. The
density of oil is mostly less than water. Thus if
creaming is at the upper side, it is oil in water
o/w and if it is downwards, it is water in oil w/o
type of emulsion.
STERILE DOSAGE FORMS
 These are the products which are
manufactured using sterilization or aseptic
processing conditions.
 There are two types of sterile dosage
forms: Parenteral preparation and
Ophthalmic formulations
1. Leaker's Test
- uses autoclave or vacuum chamber (negative
pressure)
- Dye: 1% Methylene blue
- 100% inspection
2. Clarity Test
- visual inspection for particulate matter
- done by swirling the solution and visually
inspect against light and dark background, using
a clarity testing lamp
- 100% inspection
3. Sterility Test
The tests for sterility are intended for
detecting the presence of viable microorganism
in pharmaceutical preparation that is designed
to be sterile. The test is based on the principle
that if micro-organism are placed in a medium
that provide optimum condition of nutrition,
moisture, PH, aeration, temperature, they can
grow and their presence will be indicated by the
presence of turbidity in clear medium. Test for
sterility may be carried out by one of the
following two methods:
a. Membrane Filtration
Use membrane filters having a nominal
pore size not greater than 0.4μm whose
effectiveness to retain microorganisms has
been established. The filtration apparatus and
membrane are sterilized by appropriate
means. The apparatus is designed so that the
solution to be examined can be introduced
and filtered under aseptic conditions: it
permits the aseptic removal of the membrane
for transfer to the medium, or it is suitable for
carrying out the incubation after adding the
medium to the apparatus itself. After filtration
the preparation membrane is cut into two
halves. One halve is transferred in to 100ml of
culture medium meant for the growth of the
bacteria and incubated at 30 to 35°C for not
 less than 7 days. The another halve is rabbit is NMT 0.5oC.
transferred to 100 ml of culture medium • If 1 rabbit has a temperature rise of >
meant for fungi and incubated at 20 - 25 oC 0.5oC → retest.
for not less than 7 days. RETEST:
• NMT 3 rabbits have a temperature rise of
> 0.5oC.
• Total temperature rise for all rabbits is ≤
3.3oC.

5. Bacterial Endotoxin Test

- also a test for pyrogen
- more rapid, simpler and greater sensitivity
- uses LAL Reagent (Limulus Ameobacyte Lysate)
from Limulus polyphemus (Horseshoe Crab)

METHODS:
a. Gel Clot Technique
b. Photometric Technique
A membrane filtration apparatus Turbidimetric Method = produce turbid
Chromogenic Method = produce clot
b. Direct Inoculation
In this method an aliquot quantity of PACKAGING MATERIALS
the material being tested is drawn aseptically 1. Glass
from the container and transferred to a vessel  Mainly made up of:
containing a measured quantity of a suitable a. Sand – pure silica
culture medium. The culture is incubated at b. Soda-ash – sodium carbonate
appropriate temperature for not less than 14 c. Limestone – calcium carbonate
days. The culture medium is observed at d. Cullet – broken glass that is mixed with the
periodic intervals during the incubation period batch & acts as a fusion agent for the entire
and at the end to detect presence of any mixture.
microbial growth.

NUTRIENT MEDIUM
a. Fluid Thioglycolate Medium - anaerobic
bacteria (Clostridium sp.), P. aeruginosa, and S.
aureus

b. Soybean-Casein Digest Medium - aerobic

bacteria (B. subtilis) and fungi (C. albicans)

BIOLOGICAL INDICATORS
a. Moist heat (autoclave)  B.
stearothermophilus
b. Dry heat (oven)  B. subtilis
c. Ethylene oxide  B.
stearothermophilus
d. Radiation  Both + B. pumilus

4. Pyrogen Test
- qualitative biological test based on
fever response of rabbits
Sample Size: 3 rabbits (initial)
+5 rabbits (retest)
Acceptance Criteria:
a. Leaching Property
• Initial temperature rise for any single
b. Light Transmission Test
- done for colored glass
c. Arsenic Release Test
- for Types I and III
2. Plastic

Polymers used for the production of plastic.

a. Biological Reactivity Test In Vivo

- determines the classification of plastic
materials

a. Biological Reactivity Test In Vitro

- determines the classification of plastic
materials
i. Agar diffusion test
ii. Direct contact test
iii. Elution test

BIOLOGICAL ASSAY

Biological essay (or “bioassay”), in its classical
form, is concerned with the measurement of
the concentration of an active ingredient in a
test preparation, by estimation of its potency
relative to that of a standard preparation.
Assays, usually by animal experimentation, form
an essential tool of biological standardization.
Standard designs and methods of analysis are
described here, including stochastic
approximation, radioimmunoassays,
semiparametric and nonparametric methods,
and bioequivalence studies. In a broader sense,
Bioassay refers to experiments, with biological
units, to detect possible dose–response (or
toxicity‐adverse effect) relationships (Sen,
2014).
The purpose of bioassay is to ascertain
the potency of a drug and hence it serves as the
quantitative part of any screening procedure
(Research). Other purpose of bioassay is to
standardize the preparation so that each
contains the uniform specified pharmacological
activity. In this way, it serves as a pointer in the
Commercial Production of drugs when chemical
assays are not available or do not suffice. From
the clinical point of view, bioassay may help in
the diagnosis of various conditions, e.g.
gonadotrophins for pregnancy (Goyal, 2008).
a. cardiac glycoside - are medicines for treating
heart failure and certain irregular heartbeats
b. bioassay – means biological assay
c. HPLC – means High Pressure/Performance
Liquid Chromatography
d. hypoglycemic – low blood sugar
e. oxytocic – a substance that stimulates
contraction of uterine smooth muscle or
hastens childbirth
f. galactokinetic – a substance that can increase
the production of human milk
Definition:
 includes the quantitative assay of drugs by
biological methods as well as the application of
qualitative biological tests
 generally less precise, more time consuming,
and more expensive to conduct
 measures the actual biological activity of a
given sample, which may represent the sum of
the interaction of a number of chemical and
physico-chemical factors
It is used when:
1. If the chemical identity of the active principal
has not been elucidated fully.
2. If no adequate chemical assay has been
devised for the active principle, although its
chemical structure has been established
(insulin).
3. If the drug is composed of a complex mixture
of substances of varying structure and activity
(digitalis).
4. If purification of the crude drug is not
possible or practical (Vit. D).
5. If the chemical assay is not a valid indication
of biological activity (due to lack of
differentiation between active and inactive
isomers).
1. Digitaloid Drugs
 from Digitalis purpurea
 a cardiac glycoside
 uses anesthesized pigeon – death due to
cardiac arrest
2. Insulin
 a hormone synthesized and secreted by the
beta cells of the pancreatic islets
 used to control blood-glucose levels
 uses hyperglycemic rabbit or HPLC
3. Glucagon
 a polypeptide hormone
 increase blood-glucose concentrations
 treatment for acute hypoglycemic reactions
(insulin-induced)
 in Glucagon Hydrochloride
 uses anesthetized cats (16-hour fasted) and
both femoral veins are exposed
 The glucagon samples are injected into one
femoral vein and blood samples are taken from
the vein on the opposite sides.
4. Parathyroid
 hormone
 responsible for maintaining extracellular
calcium ions at a constant concentration in the
body
 uses dogs
 safely administered by calcium and/or Vit. D
5. Posterior Pituitary, Oxytocin and Vasopressin
 oxytocic and galactokinetic activities
(Oxytcin)
 antidiuretic activities (Vasopressin)
A. Oxytocin – uses anesthesized chicken
- currently, isolated rat uterus
- RS: Oxytocin Nasal Solution (lactational
stimulant)
- used to induce labor, control postpartum
uterine bleeding and treat incomplete abortion  current: spectrophotometric assay
 treatment of heparin overdosage
B. Vasopressin – antidiuretic hormone
- potent vasopressor 10. Vitamin D
- uses male rat (elevation of blood pressure)  uses young rats (not older than 55 days) that
- available as Vasopressin Tannate have developed rickets (Vitamin D deficient)
 stimulate calcification
6. Corticotropin  leg bone is dissected out and assayed for
 or adrenocorticotropic hormone (ACTH) amount of recalcification
 a polypeptide hormone
 stimulate the release of corticosteroids from
the adrenal cortex
 uses rats (pituitary gland removed) injected
SC 16-48 hours after
7. Chorionic Gonadtropin
 a gonad-stimulating principle prepared from
the urine of pregnant women
 treatment for infertility in women
 may promote spermatogenesis
 uses young female rats (increase in the
weight of uterus excised two days after the last
of three daily SC injections)
 estrogenic activity test (vaginal smears)

Flowchart of corticotropin biological assay.

8. Heparin
 an anticoagulant that prolongs the clotting
time of blood
 citrated sheep plasma (previous)
 measures Anti-Factor IIa potency and Anti-
Factor Xa activity (current)

Flowchart of heparin bioassay.

9. Protamine Sulfate STABILITY OF PHARMACEUTICAL PRODUCTS

 simple proteins rich in arginine
 previous: citrated sheep plasma (neutralize The stability studies of pharmaceutical products
heparin activity)
are one of the very important parameter for
development of new drugs as well as new
formulations. The shelf-life prediction is a major
role for the pharmaceutical product
development of all the dosage forms and also it
is utilized to determine the particular storage
conditions and to suggest label instructions.
Stability studies of pharmaceutical products
ensuring the maintenance of product quality,
safety and efficacy throughout the shelf life are
considered as prerequisite for the acceptance
and approval of any pharmaceutical products.
These studies are required to be conducted in a
planned way following the guidelines issued by
ICH, WHO or other agencies (Sneha et. al.,
2008).
a. prototype – the first or preliminary sample of
a drug product manufactured
b. nlt – means not less than
c. API – means active pharmaceutical ingredient
Stability
 capability of a particular formulation, in a
specific container/closure system, to remain
within its physical, chemical, microbiological,
therapeutic and toxicological specifications at a
defined storage condition
I. Preformulation Stage – drug substance
determined
- stability of related salts
- compatibility with various solvents, buffered
solutions and excipients
II. Preclinical studies – preparation of a “first in
human” formulation
- short-term
III. Phase II clinical studies – proof of concept
phase
- prototype development
- longer-term
IV. Phase III clinical studies – formulation of true
representative
- final market image
EXPIRATION DATE
– expresses the stability of a commercial
pharmaceutical product
- depends on the shelf life
- the time in which a drug product in a specific
packaging configuration will remain stable when
stored under recommended conditions
- denotes the last day of the month
- shall appear on the immediate container and
the outer retail package
Shelf life
– the duration of time during which a
preparation will remain physically, chemically
and biologically stable
- time from the date of manufacture of the
formulation until its chemical or biological
activity is nlt 90% of the labelled potency
Factors that accelerate instability:
a. temperature
b. light
c. moisture
d. gravity
e. agitation
f. inversion
g. method of manufacture
Type of Stability:
1. Chemical – Each AI retains its chemical
integrity and labeled potency, within the
specified limits.
2. Physical – The original physical properties,
including appearance, palatability, uniformity,
dissolution, and suspendability are retained.
Physical stability is of importance to formulators
for three primary reasons:
a. Appearance – look fresh, elegant and
professional
b. Uniformity
c. Availability
3. Microbiological – Sterility or resistance to
microbial growth is retained according to the
specified requirements.
4. Therapeutic – The therapeutic effect remains
unchanged.
5. Toxicological – No significant increase in
toxicity occurs.
REGULATORY REQUIREMENTS
A. GMP
1. There shall be a written testing program
designed to assess the stability characteristics of
drug products.
2. Results shall be used to determine
appropriate storage conditions and expiration
dating.
3. “After marketing, the stability of the
medicinal product should be monitored
according to a continuous appropriate program
that will permit the detection of any stability
issue associated with the formulation in the
marketed package”, as stated by the European
Union GMP.
B. Compendia
1. Freezer - -25ºC to -10ºC
 2. Cold – not exceeding 8ºC
3. Controlled Cold Temperature
4. Cool – 8 to 15ºC
5. Room Temperature – temp. in working
area
6. Controlled Room Temperature – 20 to
25ºC
7. Warm – 30 to 40ºC
8. Excessive Heat – above 40ºC
9. Protect from Freezing
10. Dry Place – does not exceed 490%
average RH @ controlled room temp.
FDA and ICH
ICH - International Conference on
Harmonisation of Technical Requirements for
Registration of Pharmaceuticals for Human Use
ICH Harmonized Guidelines (related to stability):
1. Q1A (R2) – provides the general requirements
for Stability Testing of New Drug Substances and
Products
2. Q1B – for assessing the intrinsic
photostability characeristics
3. Q1C – new products dosage forms that use AI
already approved in another DF
4. Q1D – allows for bracketing and matrixing
designs
5. Q1E – assess the shelf life of the product in a
registration application
6. Q1F – for testing conditions to register drugs
marketed in Climatic Zones III and IV
(withdrawn)
STABILITY PROTOCOLS
The stability of finished pharmaceutical
products depends, on the one hand, on
environmental factors such as ambient
temperature, humidity and light, and, on the
other, on product-related factors, e.g. the
chemical and physical properties of the active
substance and of pharmaceutical excipients, the
dosage form and its composition, the
manufacturing process, the nature of the
container-closure system and the properties of
the packaging materials.
Conditions Minimum
Time Period
at
Submission
Long-term Testing 25ºC +/- 12 months
2ºC
60% +/-
5% RH
Accelerated Testing 40ºC 6 months
+/- 2ºC
75%
+/- 6% RH
Alternate Testing 30ºC +/- 12 months
2ºC
65% +/-
5% RH
Purpose:
Based on ICH Q1A(R2), “the purpose of stability
testing is to provide evidence on how the
quality of a drug substance or drug product
varies with time under the influence of a variety
of environmental factors such as temperature,
humidity and light”.
OVERAGES
 “as the voluntary introduction of a specific
excess during the manufacture of
pharmaceutical forms of medicaments that are
unstable by nature and difficult to stabilize, in
order to maintain during their period of use an
active content within the limits compatible with
therapeutic requirements”, General Assembly
of the International Pharmaceutical Federation
(FIP) on Sept. 1964
Overages are justifiable when:
1. The labile (unstable) active ingredients
cannot possibly be standardized.
2. The overage allows an even equilibrium of
the content of the active ingredient within the
acceptable limits.
3. The overage would not present a possibility
of a therapeutic overdosage if the preparation
were used during the early part of the product’s
shelf life.
4. The clinical studies show that overage is safe
therapeutically.
5. The lower limit proposed for the decrease in
strength applies only at the end of the period of
validity of unstable preparations.
Maximum amount of overages:
antibiotics – 15%
dry dosage forms – 15%
fluids – 20%
ointments, supp., creams, aerosols and foams –
25%

Two types:
1. manufacturing overage – to compensate loss
during manufacturing
2. stability overage – excess added to a
preparation to extend its shelf life