MANJUL PRATAP SINGH & ANITA SINGH

KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA,

GORAKHPUR

09628373561
K.I.P.M. GIDA, GORAKHPUR, U.P.
DEFINATION:
Parenteral refers injectable route of administration.
It derived from Greek words Para (Outside) and enteron (Intestine). So it
is a route of administration other than the oral route. This route of
administration bypasses the alimentary canal

Pyrogens, fever-producing substances are primarily lipid polysaccharide
product of metabolism of microorganism; they may be soluble,
insoluble, or colloid.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Parenteral Dose Forms
Parenteral preparations must be sterile
free of microorganisms
To ensure sterility, parenterals are prepared using
aseptic techniques
special clothing (gowns, masks, hair net, gloves)
laminar flow hoods placed in special rooms
K.I.P.M. GIDA, GORAKHPUR, U.P.
Advantages of the Parenteral Route
The IV route is the fastest method for delivering systemic drugs
preferred administration in an emergency situation
It can provide fluids, electrolytes, and nutrition
patients who cannot take food or have serious problems with the
GI tract
It provides higher concentration of drug to bloodstream or tissues
advantageous in serious bacterial infection
IV infusion provides a continuous amount of needed medication
without fluctuation in blood levels of other routes
infusion rate can be adjusted
to provide more or less medication as the situation dictates
Drug action can be prolonged by modifying the formulation.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Disadvantages of the Parenteral Route
Traumatic injury from the insertion of needle
Potential for introducing:
Toxic agents
Microbes
Pyrogens
Impossible to retrieve if adverse reaction occurs
injected directly into the body
Correct syringe, needle, and technique must be used
Rotation of injection sites with long-term use
prevents scarring and other skin changes
can influence drug absorption

K.I.P.M. GIDA, GORAKHPUR, U.P.
Routes of Administration of parenteral products
Various types of route of administration of parenteral products are:
Intradermal injection
Subcutaneous (Hypodermis) injection
Intramuscular injection
Intravenous injection
Intra-arterial injection
Intracardiac injection
Intrathecal injection
Intracisternal injection
Peridural injection
Intra- articular injection
Intracerebral injection
K.I.P.M. GIDA, GORAKHPUR, U.P.
Subcutaneous Injections
Given at a 45-degree angle
25- or 26-gauge needle, 3/8 to 5/8
inch length
No more then 1.5 ml should be injected
into the site
to avoid pressure on sensory nerves
causing pain and discomfort
Administer medications below the skin into the subcutaneous fat
outside of the upper arm
top of the thigh
lower portion of each side of the abdomen
not into grossly adipose, hardened, inflamed, or swollen tissue
Often have a longer onset of action and a longer duration of action
compared with IM or IV injection
K.I.P.M. GIDA, GORAKHPUR, U.P.
Intramuscular Injections
Care must be taken with deep IM injections to avoid hitting a vein,
artery, or nerve
In adults, IM injections are given into upper, outer portion of the
gluteus maximus
large muscle on either side of the buttocks
For children and some adults, IM injections are given into the deltoid
muscles of the shoulders
Typical needle is 22- 25 gauge - to 1-inch needle
IM injections are administered at a 90
0
angle
volume limited to less than 3 ml
K.I.P.M. GIDA, GORAKHPUR, U.P.
Intravenous Injections or Infusions
Fast-acting route because the drug goes directly into the
bloodstream
often used in the emergency department and in critical care
areas
Commonly used
for fluid and electrolyte replacement
to provide necessary nutrition to the patient who is critically ill
Intravenous (IV) injections are
administered at a 15- to 20-degree
angle
K.I.P.M. GIDA, GORAKHPUR, U.P.
Intra-arterial injection



Intracardiac injection




Intrathecal injection
These are given into the subachonoid space the
surround the spinal cord. This route is used for
spinal anesthesia.
These are given into the heart muscle or ventricle
at the time of emergency only.
The inaction are given directly in to the artery
K.I.P.M. GIDA, GORAKHPUR, U.P.
Intracisternal injection
These are given in b/w first & second cervical nerve.
Used for CSF for diagnostic purpose.
Peridural injection
These are given in b/w the dura matter & inner aspect
of vertebra.
Used for given spinal anesthesia.

Intra- articular injection
These are given in into the articulating ends of bones in
a joint.
Used for lubricating the joints.
Intracerebral injection
These are given into the cerebrum.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Official types of injections
Injection: Liquid preparation there are drug substance or
drug solution thereof e.g. insulin injection USP.

For injection: Dry solid that upon addition of suitable vehicles yield
solutions confirming in all respect to the requirements to the
injection. e.g. Cefuroxime injection USP.

Injectable emulsions: Liquid preparation of drug substance
dispersed in a suitable emulsion medium. e.g. Propofol USP.

Injectable suspension: Liquid preparation of solid suspended in a
suitable medium. e.g. Methyl Prednisolone Acetate Suspension USP.

For injectable suspension: Dry solid that upon addition of
suitable vehicle yields preparation confirming in all respect
to the requirements for Injectable suspension.
e.g. Imipenem and Cilastatin injectable suspension USP.

K.I.P.M. GIDA, GORAKHPUR, U.P.
General requirements of parenteral preparations
Stability
Sterility
Free from Pyrogens
Free from foreign particles
Isotonicity
Specific gravity
Chemical purity
K.I.P.M. GIDA, GORAKHPUR, U.P.
Formulation of parenteral products
In the preparation of parenteral products, the following substances
are added to make a stable preparation:
The active drug
Vehicles
Aqueous vehicle (e.g. water for injection, water for injection free from CO
2
)
Non-aqueous vehicle (e.g. Ethyl alcohol, propylene glycol, almond oil)
Adjuvants
Solubilizing agents (e.g. Tweens & polysorbates)
Stabilizers & antioxidants (e.g. thiourea, ascorbic acid, tocopherol)
Buffering agents (e.g. citric acid, sodium citrate)
Antibacterial agents (e.g. benzyl alcohol, metacresol, phenol)
Chelating agents (e.g. EDTA)
Suspending, emulsifying & wetting agents (e.g. MC, CMC)
Tonicity factor (e.g. sodium chloride, dextrose)
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PREFORMULATION FACTORS
It is study about physical & chemical properties of drug substance
prior formulation is called as preformulation.
They are
pH /pka
Solubility
Thermal/heat effect
Dissociation constant
Compatabilty studies- FTIR / DSC
Oxidation & reduction
particle size
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pH and pKa SOLUBILITY PROFILE
pKa Determination: The Henderson Hasseslebach equation
provides an estimate of the ionized and un ionized durg concentration
at a particular pH.
For acidic drugs,
pH = pKa + log (ionized drug) / un-ionized drug)
For basic drugs,
pH = pKa + log (unionized drug / ionized drug )
Buffers, temperature, ionic strength and cosolvent can affect the pKa
value.
Potentiometric titration offers maximum sensitivity for
compounds with pKa values in the range of 3-10.
K.I.P.M. GIDA, GORAKHPUR, U.P.
SOLUBILIZATION
Solubilization is increased by co solvent addition.
E.g.- propylene glycol solubilize drug molecules by
disrupting the hydrophobic interactions of water.
More non polar the solute

greater is the solubilisation
achieved by co solvent addition
K.I.P.M. GIDA, GORAKHPUR, U.P.
THERMAL/HEAT EFFECTS
Drugs which are unstable to heat requires refrigerative storage or
lyophilisation (these products must be used within short periods)
If it is endothermic ---> H is +
ve

increase in temp ---> increase in drug solubility
If it is exothermic ---> H is
ve

increase in temp ---> decrease in drug solubility
For determining H
ln S= - H /RT + C
S=molar solubility at temperature, T=temperature in Kelvin,
R= gas constant
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Particle size
FINE PARTICLE CHARACTERISATION Very imp. Property and
here smallest particle should be tested to facilitate homogeneous
sample preparation.
Coulter Counter Technique To check particle size and particle volume
BET (BRUNAUER, EMMET, TELLER) NITROGEN
ADSORPTION APPARATUS Measurement of surface area
SEM( SCANNING ELECTRON MICROSCOPY) to check surface
morphology

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Oxidation & reduction
Adding oxygen to form an oxide (oxidation) or 2H
2
+ O
2
-> 2H
2
O
Removing oxygen (reduction).
Due to oxidation ,
- shelf life reduces
- changes in color
- Potency of drug becomes less
So antioxidants are used to prevent oxidation e.g sodium bisulphate,
sodium metabisulphate
K.I.P.M. GIDA, GORAKHPUR, U.P.
Processing of parenteral preparation
Following steps are involved in the processing of parenteral
preparation:
1) Cleaning of containers, closures & equipments
2) Collection of materials
3) Preparation of parenteral products
4) Filtration
5) Filling the preparation in final container
6) Sealing the container
7) Sterilization
8) Evaluation of the parenteral preparation
9) Labeling & packaging
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1. Cleaning of containers, closures & equipments: Thoroughly cleaned
with detergents with tap water distilled water finally
rinsed with water for injection.
Rubber closures are washed with 0.5% sod. pyrophosphate in water.

2. Collection of materials: All raw material of preparation should be
collect from warehouse after accurate weighed.
Water for injection should be Pyrogens free.

3. Preparation of parenteral products: The parenteral preparation
must be prepared in aseptic conditions.
The ingredients are accurately weighed separately and dissolved in
vehicle as per method of preparation to be followed.

4. Filtration: The parenteral preparation must be filtered by
bacteria proof filter such as, filter candle, membrane filter.
K.I.P.M. GIDA, GORAKHPUR, U.P.
5. Filling the preparation in final container: The filling operation is
carried out under strict aseptic precautions.
6. Sealing the container: Sealing should be done immediate after filling
in aseptic environment.
7. Sterilization: For thermostable substances the parenteral products
are sterilized by autoclaving method at different temp. & pressure.
10 lb pressure (115.5
0
C, or 240
0
F) for 30 minutes
15 lb pressure (121.5
0
C, or 250
0
F ) for 20 minutes
20 lb pressure (126.5
0
C, or 260
0
F) for 15 minutes
Heat sensitive or moisture sensitive material are sterilized by
exposure to ethylene oxide or propylene oxide gas .
8. Evaluation of the parenteral preparation: The following tests are
performed in order to maintain quality control:
1. Sterility test 2. Clarity test 3. Leakage test
4. Pyrogen test 5. Assay
9. Labeling & packaging
K.I.P.M. GIDA, GORAKHPUR, U.P.
Evaluation of Parenteral products
Sterility testing
Particulate matter monitoring
Faculty seal packaging or leakage test
Pyrogen testing
LAL test
Assay or drug content uniformity
K.I.P.M. GIDA, GORAKHPUR, U.P.
Sterility testing
DEFINITION: Sterility Testing: It is a procedure carried out to detect and
conform absence of any viable form of microbes in or on pharmacopeia
preparation or product.
PRINCIPLE : Sterility testing only shows that organisms capable of growing
in selected conditions are absent from the fraction of batch that has been
tested. If the microorganism are present in the product can be indicated by
a turbidity in the clear medium.
OBJECTIVE OF STERILITY TESTING:
For validation of sterilization process.
To check presence of microorganisms in preparation which are sterile.
To prevent issue of contaminated product in market.
K.I.P.M. GIDA, GORAKHPUR, U.P.
STEPS INVOLVED IN STERILITY TE TESTING
1) Sampling
2) Selection of the quantity of the product to be used
3) Method of sterility testing
i ) METHOD 1 Membrane filtration method
ii) METHOD 2 Direct inoculation method
4) Observation and interpretation Must be carried out under
aseptic condition.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Sampling
The sample must be representative of the whole of the bulk material
& a lot of final containers.
MAINLY FOLLOWED BY TWO RULES:
A fixed percentage of the final container are selected.
A fixed number of container are taken independent of the lot
or batch size.
According to Indian Pharmacopoeia following guidelines for
determining the minimum number of items are:
K.I.P.M. GIDA, GORAKHPUR, U.P.
Selection of the quantity of the product to be used
Selection of the quantity of the product to be used for sterility testing
depends mainly on the volume or weight in the container.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method of sterility testing
Membrane filtration method (METHOD 1):
Membrane filtration Appropriate for : (advantage)
Filterable aqueous preparations
Alcoholic preparations
Oily preparations
Preparations miscible with or soluble in aqueous or oily (solvents
with no antimicrobial effect)
All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood
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Membrane filter 0.45 porosity
Filter the test solution
After filtration remove the filter
Cut the filter in to two halves
First halves (For Bacteria) Second halves (For Fungi)
Transfer in 100 ml culture media
(Fluid Thioglycollate medium)
Incubate at 30-35
0
C for not less then 7 days

Transfer in 100 ml culture media
(Soyabeans-Casein Digest medium)
Incubate at 20-25
0
C for not less then 7 days

Observe the growth in the media

Observe the growth in the media

K.I.P.M. GIDA, GORAKHPUR, U.P.
Suitable for samples with small
volumes
volume of the product is not more
than 10% of the volume of the
medium
suitable method for aqueous
solutions, oily liquids, ointments an
creams
Direct inoculation of the culture
medium suitable quantity of the
preparation to be examined is
transferred directly into the
appropriate culture medium &
incubate for not less than 14 days.
Direct inoculation method (METHOD 2):
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Observation and results
Culture media is examined during and after at the end of incubation.
The following observations are possible:
1) No evidence of growth Pass the test for sterility.
2) There is evidence of growth Re-testing is performed same no.
of sample, volume & media as in original test No evidence of
growth Pass the test for sterility.
3) There is evidence of growth isolate & identify the organism.
Re-testing is performed with twice no. of sample if:
No evidence of growth Pass the test for sterility.
There is evidence of growth Fail the test for sterility


K.I.P.M. GIDA, GORAKHPUR, U.P.
Particulate matter monitoring
Particulate matter is defined as unwanted mobile insoluble matter
other than gas bubble present in the product.
If the particle size of foreign matter is larger than the size of R.B.C.. It
can block the blood vessel.
The permit limits of particulate matter as per I.P. are follows:




Source of particulate matter:
1. Intrinsic contamination: The material which are originally
present in the parenteral solution e.g. Barium ions leach in
parenteral & react with sulphur ions in the product to form
barium sulphate crystals.
2. Extrinsic contamination: The material which comes from the
environment e.g. Shedding of material from cloth, body, &
cotton, paper, rubber, tissue etc.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Methods for monitoring particulate matter contamination:
1) Visual method
2) Coulter counter method
3) Filtration method
4) Light blockage method
Identification of particulate matter:
1) Microscopy
2) X-ray powder diffraction
3) Mass spectroscopy
4) Polarized light spectroscopy
5) Scanning electron microscopy (SEM)
K.I.P.M. GIDA, GORAKHPUR, U.P.
Faculty seal packaging / leaking testing
The sealed ampoules are subjected to small cracks which occur due
to rapid temperature changes or due to mechanical shocks.









Vials & bottles are not suitable for this test because the sealing
material used is not rigid.
Filled & sealed ampoules
Dipped in 1% Methylene blue solution
Under negative pressure in vacuum chamber
Vacuum released colored solution enter into the ampoule
Defective sealing
K.I.P.M. GIDA, GORAKHPUR, U.P.
Pyrogen Testing
Pyrogen = Pyro (Greek = Fire) + gen (Greek = beginning).
Fever producing, metabolic by-products of microbial growth and
death.
Bacterial pyrogens are called Endotoxins. Gram negative bacteria
produce more potent endotoxins than gram + bacteria and fungi.
Endotoxins are heat stable lipopolysaccharides (LPS) present in
bacterial cell walls, not present in cell-free bacterial filtrates
Stable to at least 175
o
C; steam sterilization ineffective
Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)
so endotoxins can easily pass through 0.22m filters
Sources: Water (main), raw materials, equipment, process
environment, people, and protein expression systems if using gram
negative bacteria.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Biological properties of endotoxin
Pyrogen elevate the circulatory levels of inflammatory cytokines
which may be followed by fever, blood coagulation, hypotension
Low doses of Pyrogen: asymptomatic inflammation reaction
Moderate doses: fever & changes in plasma composition
High doses: cardiovascular dysfunction, vasodilation,
vasoconstriction, endothelium dysfunction, multiple organ failure
& finally death.
Sources of pyrogen
1) Equipment
2) Containers (Glass , plastic , metal)
3) Solvent
4) Solute
K.I.P.M. GIDA, GORAKHPUR, U.P.
Elimination of pyrogens
Dry heat sterilization : For glass wares, metal equipments,
powders, waxes, oils, heat stable drugs
650
o
C temp - 1 min
250
o
C temp - 30 min
180
o
C temp - 240 min
Ultra filtration
Reverse osmosis : RO membrane is composed of cellulose acetate
phthalate/ polyamide
Distillation
Adsorption method

K.I.P.M. GIDA, GORAKHPUR, U.P.
Principal:
Rabbits are used to perform this test because their body temp
increases when pyrogen are introduced into their bodies by
parenteral route
3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are
selected
Do not use any rabbit
having a temp higher than 39.8
o
C
Showing temp variation >0.2
o
C between two successive reading
in the determination of initial temp
Sham test is performed within 7 days of actual test
Animal showing temp increase over 0.6
o
C should be removed from
pyrogen testing
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method :
Dissolve the subs being examined in, or dilute it with a pyrogen free
saline solution
Warm the liquid being examined to approx. 38.5
o
C temp before
injection
The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body
weight
Withhold water during test
Clinical thermometer is inserted into the rectum of rabbit to record
body temp
2 normal reading of rectal temp are should be taken prior to the test
injection at an interval of half an hr & its mean is calculated- initial
temp
The solution under test is injected through an ear vein
Record the temp of each rabbit in an interval of 30 min for 3 hrs
The difference between initial temp & maximum temp is recorded-
taken as response
K.I.P.M. GIDA, GORAKHPUR, U.P.
Interpretation of results
K.I.P.M. GIDA, GORAKHPUR, U.P.
Bacterial endotoxin (LAL) test )
To detect or quantify endotoxins of gram-ve bacterial origin
Reagent: amoebocyte lysate from horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus).
The name of the test is also Limulus amebocyte lysate (LAL) test

Mechanism of LAL Test:

The test is based on the primitive blood-clotting mechanism of
the horseshoe crab
enzymes located with the crab's amebocyte blood cells endotoxin


Initiation of an enzymatic coagulation cascade

proteinaceous gel
K.I.P.M. GIDA, GORAKHPUR, U.P.
Test performance (short)
Avoid endotoxin contamination
Before the test:
interfering factors should not be present
equipment should be depyrogenated the sensitivity of the lysate
should be known
Test:
equal Volume of LAL reagent and test solution (usually 0.1 ml of
each) are mixed in a depyrogenated test-tube
Incubation at 37C, 1 hour
remove the tube - invert at (180) observe the result
pass-fail test
K.I.P.M. GIDA, GORAKHPUR, U.P.
LAL test
Three different techniques:
1. The gel-clot technique - gel formation
2. The turbidimetric technique - the development of Turbidity after
cleavage of an endogenous substrate
3. The chromogenic technique - the development of color after
cleavage of a synthetic peptide- chromogen complex
Advantages of LAL test
Fast - 60 minutes vs. 180 minutes
Greater Sensitivity ,Less Variability
Much Less False, Positives ,Much Less Expensive
Alternative to Animal Model, cheaper,
particularly useful for:
Radiopharmaceuticals and cytotoxic agents
Blood products
Water for injection
K.I.P.M. GIDA, GORAKHPUR, U.P.
Production facilities of parenterals
The production area where the parenteral preparation are
manufactured can be divided into five sections:
Clean-up area
Preparation area
Aseptic area
Quarantine area
Finishing & packaging area
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Clean-up area:
It is not aseptic area.
All the parenteral products must be free from foreign particles &
microorganism.
Clean-up area should be withstand moisture, dust & detergent.
This area should be kept clean so that contaminants may not be carried out
into aseptic area.
Preparation area:
In this area the ingredients of the parenteral preparation are mixed &
preparation is made for filling operation.
It is not essentially aseptic area but strict precautions are required to prevent
any contamination from outside.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Aseptic area:
The parenteral preparations are filtered, filled into final container
& sealed should be in aseptic area.
The entry of personnel into aseptic area should be limited &
through an air lock.
Ceiling, wall & floor of that area should be sealed & painted.
The air in the aseptic area should be free from fibers, dust and
microorganism.
The High efficiency particulate air filters (HEPA) is used for air.
UV lamps are fitted in order to maintain sterility.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Quarantine area:
After filling, sealing & sterilization the parenteral product are held
up in quarantine area.
Randomly samples were kept foe evaluation.
The batch or product pass the evaluation tests are transfer in to
finishing or packaging area.
Finishing & packaging area:
Parenteral products are properly labelled and packed.
Properly packing is essential to provide protection against
physical damage.
The labelled container should be packed in cardboard or plastic
container.
Ampoules should be packed in partitioned boxes


K.I.P.M. GIDA, GORAKHPUR, U.P.
Lyophilization or freeze drying
Lyophilization or freeze drying is a process in which water is removed
from a product after it is frozen and placed under a vacuum, allowing
the ice to change directly from solid to vapor without passing through
a liquid phase.
The process consists of three separate, unique, and interdependent
processes;
Freezing,
Primary drying (sublimation), and
Secondary drying (desorption).

K.I.P.M. GIDA, GORAKHPUR, U.P.
The advantages of Lyophilization include:
Ease of processing a liquid, which simplifies aseptic handling
Enhanced stability of a dry powder
Removal of water without excessive heating of the product
Enhanced product stability in a dry state
Rapid and easy dissolution of reconstituted product
Disadvantages of Lyophilization include:
Increased handling and processing time
Need for sterile diluent upon reconstitution
Cost and complexity of equipment
K.I.P.M. GIDA, GORAKHPUR, U.P.
The Lyophilization process generally includes the following steps:
Dissolving the drug and excipients in a suitable solvent, generally
water for injection (WFI).
Sterilizing the bulk solution by passing it through a 0.22 micron
bacteria-retentive filter.
Filling into individual sterile containers and partially stoppering
the containers under aseptic conditions.
Transporting the partially stoppered containers to the lyophilizer
and loading into the chamber under aseptic conditions.
Freezing the solution by placing the partially stoppered containers
on cooled shelves in a freeze-drying chamber or pre-freezing in
another chamber.
Applying a vacuum to the chamber and heating the shelves in
order to evaporate the water from the frozen state.
Complete stoppering of the vials usually by hydraulic or screw rod
stoppering mechanisms installed in the lyophilizers.

K.I.P.M. GIDA, GORAKHPUR, U.P.
There are many new parenteral products, including anti-infectives,
biotechnology derived products, and in-vitro diagnostics which are
manufactured as lyophilized products.
Additionally, inspections have disclosed potency, sterility and stability
problems associated with the manufacture and control of lyophilized
products.

K.I.P.M. GIDA, GORAKHPUR, U.P.
K.I.P.M. GIDA, GORAKHPUR, U.P.

HIGH PERFORMANCE THIN
LAYER CHROMATOGRAPHY


CHROMATOGRAPHY



Chromatography is a physical process of
separation in which the components to be
separated are distributed between 2 immiscible
phases a stationary phase which has a large
surface area and mobile phase which is in constant
motion through the stationary phase.


Introduction of HPTLC


HPTLC is the improved method of TLC which utilizes the
conventional technique of TLC in more optimized way.
HPTLC takes place in highspeed capillary flow range of
the mobile phase.
There are three main steps HPTLC procedure, they are

1] Sample to analyzed to chromatogram layer, volume precision
and exact position are achieved by use of suitable instrument.
2] Solvent (mobile phase) migrates the planned distance in layer
(stationary phase) by capillary action. In this process sample
separated into its components.
3] Separation tracks are scanned in densitometer with light
beams in visible or uv region




Steps Involving in HPTLC
Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
57
Sample preparation
Normal phase chromatography: non polar solvent
Reversed phase chromatography: polar solvent

Selection of chromatography layer
Depends on nature of material to be separated
Commonly used(silica gel, alumina)
58
Pre-washing
It is purification step
Mainly methanol is used
Essential for quantitative evaluation
Stability testing

59
Pre-conditioning
Also called Chamber Saturation
Low polarity mob. Phase:- no need
High polar mob. Phase:- desirable
For reverse phase saturate chamber with polar
solvent
Sample application
1-5 l
Linomat IV Applicator
60
Linomat lV applicator
61
Fig. Hptlc instrumentation
Selection of HPTLC plates
Previously hand made plates is used in TLC for both
qualitative and quantitative work. Certain drawbacks with
that is nonuniform layer, formation of thick layer, paved for
advent of precoated plates.

Nowadays precoated plates are available in different format
and thickness by various manufactures. Precaoted plates can
be used for both qualitative and quantitative work in
HPTLC, they are

GLASS PLATES
POLY ESTER/POLYETHYLYNE
ALUMINIUM PLATES





Glass Plates:

Offers superior flat and smooth surface.
- fragile
- high weight
- higher production cost


Polyester/polyethylene plates:
Thickness of plate is 0.2mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packing material is required.
- Development of plate cannt be above temperature
120
0
c loses its shape.





Aluminium plates:
- Thickness of plate is 0.1mm.
- It can be produced in roll forms.
- Unbreakable.
- Less packaging material is required.


SORBENTS USED IN HPTLC PLATES:

sorbents which are used in convential TLC are
also used in HPTLC with or without modification.
- silica gel 65F
- highly purified silicagel 60
- aluminium oxide
- cellulose microcrystalline
- silica gel
- reversed stationary phase


Layer thickness
The layer thickness in HPTLC is around 100-
200cm,in conventional it is 250mm.

Layer prewashing:
- Ascending method
- Dipping method
- Continuous method

ACTIVATION OF PRECOATED PLATES

The plates are activated by placing in an oven
at 110120
0
C for 30 min, this step will removes
water that has been physically absorbed on surface
at solvent layer.

Freshly opened box of HPTLC plates usually
does not require activation.

Activation at higher temp and for longer time is
avoided which leads to very active layer and there
is risk of sample being decomposed.


Solvents used in HPTLC

- Methanol (commonly used)
- Chloroform:methanol:ammonia(90:10:1)
- Chloroform:methanol(1:1)
- Methylene chloride:methanol(1:1)
- Ammonia(1%)solution

Application of sample and standard

Usual concentration range is 0.1-1g / l,above
this causes poor separation.

Linomat IV (automatic applicator) - nitrogen gas
sprays sample and standard from syringe on TLC
plates as bands.

Band wise application - better separation - high
response to densitometer.

Chromatographic development and drying

After development, remove the plate and
mobile phase is removed from the plate - to avoid
contamination of lab atmosphere.

Dry in vacuum desiccator - avoid hair drier
because essential oil components may evaporate.

Detection and visualization


Detection under UV light is first choice -
non destructive.

Spots of fluorescent compounds can be
seen at 254 nm (short wave length) or at 366 nm
(long wave length).

Spots of non fluorescent compounds can be
seen - fluorescent stationary phase is used - silica
gel GF.


Non UV absorbing compounds like
ethambutol, dicylomine etc - dipping the plates in
0.1% iodine solution.

When individual component does not
respond to UV - derivatisation required for
detection .


HPTLC
100m
High due to smaller particle size
generated
3 - 5 cm
Shorter migration distance and the
analysis time is greatly reduced
Wide choice of stationary phases
like silica gel for normal phase and
C8 , C18 for reversed phase modes
New type that require less amount
of mobile phase
Auto sampler
Use of UV/ Visible/ Fluorescence
scanner scans the entire
chromatogram qualitatively and
quantitatively and the scanner is
an advanced type of densitometer
TLC
250m
Less

10 - 15 cm

Slower

Silica gel , Alumina &
Kiesulguhr

More amount

Manual spotting


Not possible


APPLICATIONS

Pharmaceutical Researches
Biomedical Analysis
Clinical Analysis
Environmental Analysis
Food Industry
Therapeutic drug monitoring to determine concentration of drug
and its metabolite in blood, urine etc
Analysis of environmental pollutions levels
Quantitative determination of prostaglandins and thromboxanes
in plasma
Determination of mercury in water
Analysis of nitrosoamines in food and body fluids
Determination of sorbic acid in wine
Characterization of hazards in industrial waste

REFERENCES
1. Principles of instrumental analysis skoog, Holler,
Nieman
2. Instrumental methods of analysis Willard
,Merrit, Dean
3. Pharmaceutical analysis Munson
4. Sharma J.friedB.Handbook of TLC
5. Kasture A.V A text book of Pharmaceutical Analysis
(Instrumental methods), 14
th
edition, page no.28-30
6. Elke Hahn Deinstrop Applied TLC, 2
nd
edition
7. Site www.Google.com (Google images)


THANK YOU
Sterility testing
DEFINITION: Sterility Testing: It is a procedure carried out to detect and
conform absence of any viable form of microbes in or on pharmacopeia
preparation or product.
PRINCIPLE : Sterility testing only shows that organisms capable of growing
in selected conditions are absent from the fraction of batch that has been
tested. If the microorganism are present in the product can be indicated by
a turbidity in the clear medium.
OBJECTIVE OF STERILITY TESTING:
For validation of sterilization process.
To check presence of microorganisms in preparation which are sterile.
To prevent issue of contaminated product in market.
K.I.P.M. GIDA, GORAKHPUR, U.P.
STEPS INVOLVED IN STERILITY TE TESTING
1) Sampling
2) Selection of the quantity of the product to be used
3) Method of sterility testing
i ) METHOD 1 Membrane filtration method
ii) METHOD 2 Direct inoculation method
4) Observation and interpretation Must be carried out under
aseptic condition.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Sampling
The sample must be representative of the whole of the bulk material
& a lot of final containers.
MAINLY FOLLOWED BY TWO RULES:
A fixed percentage of the final container are selected.
A fixed number of container are taken independent of the lot
or batch size.
According to Indian Pharmacopoeia following guidelines for
determining the minimum number of items are:
K.I.P.M. GIDA, GORAKHPUR, U.P.
Selection of the quantity of the product to be used
Selection of the quantity of the product to be used for sterility testing
depends mainly on the volume or weight in the container.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method of sterility testing
Membrane filtration method (METHOD 1):
Membrane filtration Appropriate for : (advantage)
Filterable aqueous preparations
Alcoholic preparations
Oily preparations
Preparations miscible with or soluble in aqueous or oily (solvents
with no antimicrobial effect)
All steps of this procedure are performed aseptically in a Class 100
Laminar Flow Hood
K.I.P.M. GIDA, GORAKHPUR, U.P.
Membrane filter 0.45 porosity
Filter the test solution
After filtration remove the filter
Cut the filter in to two halves
First halves (For Bacteria) Second halves (For Fungi)
Transfer in 100 ml culture media
(Fluid Thioglycollate medium)
Incubate at 30-35
0
C for not less then 7 days

Transfer in 100 ml culture media
(Soyabeans-Casein Digest medium)
Incubate at 20-25
0
C for not less then 7 days

Observe the growth in the media

Observe the growth in the media

K.I.P.M. GIDA, GORAKHPUR, U.P.
Suitable for samples with small
volumes
volume of the product is not more
than 10% of the volume of the
medium
suitable method for aqueous
solutions, oily liquids, ointments an
creams
Direct inoculation of the culture
medium suitable quantity of the
preparation to be examined is
transferred directly into the
appropriate culture medium &
incubate for not less than 14 days.
Direct inoculation method (METHOD 2):
K.I.P.M. GIDA, GORAKHPUR, U.P.
Observation and results
Culture media is examined during and after at the end of incubation.
The following observations are possible:
1) No evidence of growth Pass the test for sterility.
2) There is evidence of growth Re-testing is performed same no.
of sample, volume & media as in original test No evidence of
growth Pass the test for sterility.
3) There is evidence of growth isolate & identify the organism.
Re-testing is performed with twice no. of sample if:
No evidence of growth Pass the test for sterility.
There is evidence of growth Fail the test for sterility


K.I.P.M. GIDA, GORAKHPUR, U.P.
Particulate matter monitoring
Particulate matter is defined as unwanted mobile insoluble matter
other than gas bubble present in the product.
If the particle size of foreign matter is larger than the size of R.B.C.. It
can block the blood vessel.
The permit limits of particulate matter as per I.P. are follows:




Source of particulate matter:
1. Intrinsic contamination: The material which are originally
present in the parenteral solution e.g. Barium ions leach in
parenteral & react with sulphur ions in the product to form
barium sulphate crystals.
2. Extrinsic contamination: The material which comes from the
environment e.g. Shedding of material from cloth, body, &
cotton, paper, rubber, tissue etc.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Methods for monitoring particulate matter contamination:
1) Visual method
2) Coulter counter method
3) Filtration method
4) Light blockage method
Identification of particulate matter:
1) Microscopy
2) X-ray powder diffraction
3) Mass spectroscopy
4) Polarized light spectroscopy
5) Scanning electron microscopy (SEM)
K.I.P.M. GIDA, GORAKHPUR, U.P.
Faculty seal packaging / leaking testing
The sealed ampoules are subjected to small cracks which occur due
to rapid temperature changes or due to mechanical shocks.









Vials & bottles are not suitable for this test because the sealing
material used is not rigid.
Filled & sealed ampoules
Dipped in 1% Methylene blue solution
Under negative pressure in vacuum chamber
Vacuum released colored solution enter into the ampoule
Defective sealing
K.I.P.M. GIDA, GORAKHPUR, U.P.
Pyrogen Testing
Pyrogen = Pyro (Greek = Fire) + gen (Greek = beginning).
Fever producing, metabolic by-products of microbial growth and
death.
Bacterial pyrogens are called Endotoxins. Gram negative bacteria
produce more potent endotoxins than gram + bacteria and fungi.
Endotoxins are heat stable lipopolysaccharides (LPS) present in
bacterial cell walls, not present in cell-free bacterial filtrates
Stable to at least 175
o
C; steam sterilization ineffective
Water soluble; monomer unit of LPS can be 10,000 Daltons (1.8 nm)
so endotoxins can easily pass through 0.22m filters
Sources: Water (main), raw materials, equipment, process
environment, people, and protein expression systems if using gram
negative bacteria.
K.I.P.M. GIDA, GORAKHPUR, U.P.
Biological properties of endotoxin
Pyrogen elevate the circulatory levels of inflammatory cytokines
which may be followed by fever, blood coagulation, hypotension
Low doses of Pyrogen: asymptomatic inflammation reaction
Moderate doses: fever & changes in plasma composition
High doses: cardiovascular dysfunction, vasodilation,
vasoconstriction, endothelium dysfunction, multiple organ failure
& finally death.
Sources of pyrogen
1) Equipment
2) Containers (Glass , plastic , metal)
3) Solvent
4) Solute
K.I.P.M. GIDA, GORAKHPUR, U.P.
Elimination of pyrogens
Dry heat sterilization : For glass wares, metal equipments,
powders, waxes, oils, heat stable drugs
650
o
C temp - 1 min
250
o
C temp - 30 min
180
o
C temp - 240 min
Ultra filtration
Reverse osmosis : RO membrane is composed of cellulose acetate
phthalate/ polyamide
Distillation
Adsorption method

K.I.P.M. GIDA, GORAKHPUR, U.P.
Principal:
Rabbits are used to perform this test because their body temp
increases when pyrogen are introduced into their bodies by
parenteral route
3 healthy adult rabbits of either sex, each weighing NLT 1.5 kg are
selected
Do not use any rabbit
having a temp higher than 39.8
o
C
Showing temp variation >0.2
o
C between two successive reading
in the determination of initial temp
Sham test is performed within 7 days of actual test
Animal showing temp increase over 0.6
o
C should be removed from
pyrogen testing
K.I.P.M. GIDA, GORAKHPUR, U.P.
Method :
Dissolve the subs being examined in, or dilute it with a pyrogen free
saline solution
Warm the liquid being examined to approx. 38.5
o
C temp before
injection
The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of body
weight
Withhold water during test
Clinical thermometer is inserted into the rectum of rabbit to record
body temp
2 normal reading of rectal temp are should be taken prior to the test
injection at an interval of half an hr & its mean is calculated- initial
temp
The solution under test is injected through an ear vein
Record the temp of each rabbit in an interval of 30 min for 3 hrs
The difference between initial temp & maximum temp is recorded-
taken as response
K.I.P.M. GIDA, GORAKHPUR, U.P.
Interpretation of results
K.I.P.M. GIDA, GORAKHPUR, U.P.
Bacterial endotoxin (LAL) test )
To detect or quantify endotoxins of gram-ve bacterial origin
Reagent: amoebocyte lysate from horseshoe crab (Limulus
polyphemus or Tachypleus tridentatus).
The name of the test is also Limulus amebocyte lysate (LAL) test

Mechanism of LAL Test:

The test is based on the primitive blood-clotting mechanism of
the horseshoe crab
enzymes located with the crab's amebocyte blood cells endotoxin


Initiation of an enzymatic coagulation cascade

proteinaceous gel
K.I.P.M. GIDA, GORAKHPUR, U.P.
Test performance (short)
Avoid endotoxin contamination
Before the test:
interfering factors should not be present
equipment should be depyrogenated the sensitivity of the lysate
should be known
Test:
equal Volume of LAL reagent and test solution (usually 0.1 ml of
each) are mixed in a depyrogenated test-tube
Incubation at 37C, 1 hour
remove the tube - invert at (180) observe the result
pass-fail test
K.I.P.M. GIDA, GORAKHPUR, U.P.
LAL test
Three different techniques:
1. The gel-clot technique - gel formation
2. The turbidimetric technique - the development of Turbidity after
cleavage of an endogenous substrate
3. The chromogenic technique - the development of color after
cleavage of a synthetic peptide- chromogen complex
Advantages of LAL test
Fast - 60 minutes vs. 180 minutes
Greater Sensitivity ,Less Variability
Much Less False, Positives ,Much Less Expensive
Alternative to Animal Model, cheaper,
particularly useful for:
Radiopharmaceuticals and cytotoxic agents
Blood products
Water for injection
K.I.P.M. GIDA, GORAKHPUR, U.P.
Production facilities of parenterals
The production area where the parenteral preparation are
manufactured can be divided into five sections:
Clean-up area
Preparation area
Aseptic area
Quarantine area
Finishing & packaging area
K.I.P.M. GIDA, GORAKHPUR, U.P.
Clean-up area:
It is not aseptic area.
All the parenteral products must be free from foreign particles &
microorganism.
Clean-up area should be withstand moisture, dust & detergent.
This area should be kept clean so that contaminants may not be carried out
into aseptic area.
Preparation area:
In this area the ingredients of the parenteral preparation are mixed &
preparation is made for filling operation.
It is not essentially aseptic area but strict precautions are required to prevent
any contamination from outside.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Aseptic area:
The parenteral preparations are filtered, filled into final container
& sealed should be in aseptic area.
The entry of personnel into aseptic area should be limited &
through an air lock.
Ceiling, wall & floor of that area should be sealed & painted.
The air in the aseptic area should be free from fibers, dust and
microorganism.
The High efficiency particulate air filters (HEPA) is used for air.
UV lamps are fitted in order to maintain sterility.

K.I.P.M. GIDA, GORAKHPUR, U.P.
Quarantine area:
After filling, sealing & sterilization the parenteral product are held
up in quarantine area.
Randomly samples were kept foe evaluation.
The batch or product pass the evaluation tests are transfer in to
finishing or packaging area.
Finishing & packaging area:
Parenteral products are properly labelled and packed.
Properly packing is essential to provide protection against
physical damage.
The labelled container should be packed in cardboard or plastic
container.
Ampoules should be packed in partitioned boxes


K.I.P.M. GIDA, GORAKHPUR, U.P.
Lyophilization or freeze drying
Lyophilization or freeze drying is a process in which water is removed
from a product after it is frozen and placed under a vacuum, allowing
the ice to change directly from solid to vapor without passing through
a liquid phase.
The process consists of three separate, unique, and interdependent
processes;
Freezing,
Primary drying (sublimation), and
Secondary drying (desorption).

K.I.P.M. GIDA, GORAKHPUR, U.P.
The advantages of Lyophilization include:
Ease of processing a liquid, which simplifies aseptic handling
Enhanced stability of a dry powder
Removal of water without excessive heating of the product
Enhanced product stability in a dry state
Rapid and easy dissolution of reconstituted product
Disadvantages of Lyophilization include:
Increased handling and processing time
Need for sterile diluent upon reconstitution
Cost and complexity of equipment
K.I.P.M. GIDA, GORAKHPUR, U.P.
The Lyophilization process generally includes the following steps:
Dissolving the drug and excipients in a suitable solvent, generally
water for injection (WFI).
Sterilizing the bulk solution by passing it through a 0.22 micron
bacteria-retentive filter.
Filling into individual sterile containers and partially stoppering
the containers under aseptic conditions.
Transporting the partially stoppered containers to the lyophilizer
and loading into the chamber under aseptic conditions.
Freezing the solution by placing the partially stoppered containers
on cooled shelves in a freeze-drying chamber or pre-freezing in
another chamber.
Applying a vacuum to the chamber and heating the shelves in
order to evaporate the water from the frozen state.
Complete stoppering of the vials usually by hydraulic or screw rod
stoppering mechanisms installed in the lyophilizers.

K.I.P.M. GIDA, GORAKHPUR, U.P.
There are many new parenteral products, including anti-infectives,
biotechnology derived products, and in-vitro diagnostics which are
manufactured as lyophilized products.
Additionally, inspections have disclosed potency, sterility and stability
problems associated with the manufacture and control of lyophilized
products.

K.I.P.M. GIDA, GORAKHPUR, U.P.
K.I.P.M. GIDA, GORAKHPUR, U.P.