QUALITY CONTROL OF PARENTERALS,STERILE PRODUCT

By: Dr.Sadia Urooj

(Pharm.D),(M.Phil. Pharmaceutics)

Assistant Professor (L.M.D.C)

QUALITY CONTROL OF

STERILE PRODUCTS

STERILE PRODUCTS

Def:

Sterile products are the dosage form of

therapeutic agents that are free from viable

microorganisms.

These sterile products include the following:

1. Parenterals

2. Ophthalmic

3. Irrigating preparations

Of these parenteral products are unique among

the dosage forms of the drugs because they are

injected through skin or mucous membranes into

the internal body compartment.

1.PARENTERALS Term derived from Greek words “Para” outside &

“Enteron” intestine.

Parenterals are sterile solution/suspension of drug in

aqueous or oily vehicle.

Parenteral drugs are administered directly into the

veins, muscles or under the skin, or more specialized

tissues such as the spinal cord.

Term parenteral used for any drug/fluid whose

delivery does not utilize the alimentary canal for

entering into the body tissues.

Parenteral preparations are supplied in glass ,

plastic container and prefilled syringes with

closures are made up of plastic or elastomer.

Categories of parenteral preparations:

Injections or infusions

Concentrates for injections or infusions

Powders for injection or infusions

Gels for injections

Implants

Unique Characteristics of Parenterals

Sterile

Particulate-free

Pyrogen free

Stable for intended use

pH – not vary significantly

Osmotic pressure similar to blood

2. OPHTHALMICS These are the sterile liquids, semisolids or solid

preparations intended for administration upon the eyeball and/or conjunctiva in the conjunctival sac.

Categories of ophthalmics:

Eye drops

Eye lotions

Powder for eye drops

Powders for eye lotions

Semisolids eye preparations

Ophthalmic inserts

3. IRRIGATING

PREPARATIONS

These solutions are applied topically to bath open

wounds and body cavities.

These are sterile solutions for single use only.

Examples of irrigating solutions are

0.9% w/v sodium chloride solution.

Sterile water for irrigation

Most irrigation fluids are now available in rigid

plastic bottles.

The containers are sealed and sterilized by moist

heat.

History Of Parenteral Therapy

1657: First recorded injection in animals

– Sir Christopher Wren

1855: First subcutaneous injections of drugs

using hypodermic needles

– Dr. Alexander Wood

1920s: Proof of microbial growth resulting in

infections

– Dr. Florence Seibert

1926: inclusion in the National Formulary

1944: Discovery of ethylene oxide(used for

sterilization)

1946: Organization of Parenteral drug

Association

1961: Development of laminar air flow

concept

1965: Development of Total Parenteral

nutrition(TPN)

Routes of Parenteral

Administration

Intradermal (I.D.)

Subcutaneous (S.C)

Intramuscular (I.M.)

Intravenous (I.V.)

Intra-arterial

Intracardiac

Intra-articular (joint)

Intrasynovial (joint fluid area)

Intraspinal, Intrathecal (spinal fluid)

Advantages Of Parenteral

Administration

Fastest method of drug delivery

Viable alternative

Use for Uncooperative patients

Nauseous patients

Unconscious patients

Less patient control

For the patient who can have nothing by mouth

Prolonged action

Correcting serious fluids and electrolyte imbalance

TPN

Disadvantages Of Parenteral

Administration

Trained personnel

Pain

Difficult to reverse an administered drug’s effects

Manufacturing and Packaging requirements

Cost

Needle sticks Injury

Characteristics of parentral

Preparations

Intravenous (IV) preparations are either:

solutions (in which ingredients are dissolved)

suspensions (in which ingredients are suspended)

Most parenteral preparations are made of

ingredients in a sterile water medium.

Some parenteral preparations may be

oleaginous (oily).

Parenteral IV preparations must have

chemical properties that will not:

damage vessels or blood cells

alter the chemical properties of the blood serum

With blood, IVs must be: iso-osmoticIsotonic

Human blood plasma has a pH of 7.4slightly alkalineparenteral IV solutions should have a pH that is

neutral (near 7)

Characteristics of parenteral preparations that areimportant to adjust:

Tonicity,

Osmolality,

pH are characteristics of parenteral preparations.

It is important that they be adjusted to be as close aspossible to the values for human blood, to preventdamage to blood cells and organs.

Parenteral Added Substances

Antibacterial Agents

Antioxidants

Buffers

Tonicity Contributors

Surfactants (Emulsifying, Solubilizing, Wetting

Agents)

Suspending or Viscosity Increasing Agents

Local anesthetics

Inert Gases

Vehicles For Injection Aqueous vehicles:

– Frequently, isotonic (to blood) to which drug may be added at

time of use.

Water-miscible vehicles:

– portion of the vehicle in the formulation

– used primarily to effect solubility of drugs and/or reduce

hydrolysis

– ethyl alcohol; polyethylene glycol (liquid) and propylene glycol

Nonaqueous vehicles:

– Fixed oils (vegetable origin, and rancid resistance) used in hormone preparations

– Peanut oil , corn oil, cotton seed oil, sesame oil,

Types of Water for Injection

Water for Injection USP

Sterile Water for Injection USP (SWFI)

Bacteriostatic Water for Injection USP

Sterile Water for Irrigation USP

Water for Injection USP: Is water obtained by distillation or by reverse osmosis.

It conforms to the standards of purified water (It can not contain more than 10ppm of total solid and should have a pH between 5 and 7).

Is also free of pyrogens.

Is used as solvent for preparation of parentral solutions.

Sterile Water for Injection USP (SWFI):

Is water for injection that is sterilized and packaged in single dose container of type 1 and 2 glass.

These containers do not exceed a capacity of 1 L.

Bacteriostatic Water for Injection USP: Is sterile water for injection that contains one or more

suitable antimicrobial agents.

It also packaged in single or multiple dose container of type 1 and 2 glass.

These containers do not exceed the capacity of 30ml.

Sterile Water for Irrigation USP: Is water for injection that is sterilized and suitably

packaged.

It contains no antimicrobial agents or other added substances.

Classification Small Volume Parenterals (25-50 ml)

Requires little or no manipulation

Extended stability

Little wastage

Do not offer flexibility in quantity/concentration

Primary uses of SVP

Therapeutic injections

Ophthalmic products

Diagnostic agents including

o Diagnostic radiopharmaceuticals

o Allergenic extracts

Large Volume Parenterals (>100ml):

Flexible but requires manipulation

Used for maintenance or replacement therapy

Free of Preservatives

Volume must not exceed 1L (except irrigation sols)

Clinical Utilization of LVP

Basic nutrition

Restoration of electrolyte balance

Fluid replacement

Blood and blood products drug carriers

QUALITY CONTROL

TESTING

General areas of Quality

control

Three General Areas are:

1. Incoming Stock:

Routine work testing

2. Manufacturing:

Include numerable tests, reading and

observations through out the manufacturing

process

3. Finished Products:

Sterility test, Pyrogen test, Clarity test, Leaker

test,

In process Quality control

tests

Conductivity measurement

Volume filled

Temperature for heat sterilized product

Environmental control tests

Visual inspection

TESTS FOR PARENTERALS

Finished product Quality control tests:

There are mainly five Quality control test for

the parenterals are performed.

1) LEAKER TEST

2) CLARITY TEST

3) PYROGEN TEST

4) STERILITY TEST

5) CONTENT UNIFORMITY TEST

1) LEAKER TEST Leakage occurs when a discontinuity exists in the

wall of a package that can allow the passage of gas under the action of a pressure or concentration differential existing across the wall.

Presence of capillary pores or tiny cracks can cause microbes or other dangerous contaminants to enter the ampoules or package or may lead to the leakage of contents to outside. This may cause contamination of the sterile contents and also spoilage of appearance of the package.

Changes in temperature during storage can cause expansion and contraction of the ampoule or package and thereby causing interchange of its contents if an opening exists.

Leaker test is employed to detectincompletely sealed ampoule sothat they may be discarded.

To test the package integrity.

Package integrity reflects its abilityto keep the product in and to keeppotential contamination out.

Leaker tests are 4 types

a) visual inspection

b) bubble test

c) dye test

d) vacuum ionization test

a) Visual inspection

Visual inspection is the easiest leaker test method

to perform.

The method is used for the evaluation of large

volume parenterals.

To increase the sensitivity of the method the

visual inspection of the sample container may be

coupled with the application of vacuum to make

leakage more readily observable.

This method is simple and inexpensive.

Disadvantage: less sensitive

Sensitivity is increased by applying

pressure/vacuum.

b)Bubble test The test package is submerged in liquids.

A differential pressure is applied on thecontainer.

The container is observed for bubbles.

Sometimes, surfactant added liquid is usedfor immersion of test package.

Any leakage is evident after the application ofdifferential pressure as the generation offoaming in immersion liquid.

The method is simple and inexpensive.

The location of the leaks can be observed inthis method.

Generation of a differential positive pressure of 3

psi inside the vial and observation of any

leakage using magnifying glass within a

maximum test time of 15 minutes.

However, it is relatively insensitive and the

findings are operator dependent and are

qualitative.

The optimized conditions can be achieved

using a surfactant immersion fluid along with

the dark background and High intensity

lighting.

C)Dye test

The test container is immersed in a dye bath.

Vacuum and pressure is applied for

sometime.

The container is removed from the dye bath

and washed.

The container is then inspected for the

presence of dye either visually or by means of

UV spectroscopy.

The dye used is usually 0.5% to 1%

methylene blue.

The dye test can be optimized by use of a

surfactant and or a low viscosity fluid in the

The dye test is widely accepted in industry

and is approved in drug use.

The test is inexpensive and is requires no

special equipment required for visual dye

detection.

However, the test is qualitative, destructive

and slow.

The test is used for ampoules.

D)Vacuum ionization test

Vacuum ionization test is useful for testing

leakage in the vials or bottled sealed

under vacuum.

This test is used for testing of the lyophilized

products.

High voltage, high frequency field is applied

to vials which to cause residual gas, if present

to glow.

Glow intensity is the function of headspace

vacuum level.

The blue glow is the indicative of vacuum

while the purple glow indicative of no vacuum.

The sensitivity of the method is not

documented.

This test is rapid and is non destructive test.

However, the proteins present in the test

sample may be decomposed.

This method is used for the lyophilized vials

of biopharmaceuticals.

2) CLARITY TEST(PARTICLE CONTAINMENT TEST)

Definition:

Clarity is a relative term, its mean a clear solution

having a high polish conveys to the observer that

the product is of exceptional quality and purity.

Clarity test is carried out to check the

particulate matter in the sample.

It is practically impossible that every unit of lot

is perfectly free from visible particulate matter

,that is, from particles that are 30 to 40

micrometer and large in size.

PRINCIPLE:

This test is performed to check the particulate

contamination of injections and infusions

consists of extraneous, mobile and

undissolved particles, other than gas bubbles,

unintentionally present in the solution.

USP limits for large volume infusionParticle size Particle limit

10 um (or) larger/ml 50

25 um (or) larger/ml 5

TYPES OF TEST:

Particulate matter can be detected in

parenteral product by two methods,

1. Test for visible particles

2. Test for sub visible particles

1) Test for Visible particles

Visual inspection by naked eye:

The test is intended to provide a simple procedure for the visual assessment of the quality of parenteral solutions as regards visible particles.

In visual inspection, each injectable is inspected visually against white and black backgrounds.

The white background aids in detection of dark colored particles.

The light or reflective particles will appear against the black background.

Some visual-enhancing aids can increase the

efficiency.

A magnifying lens at 2.5 × magnification set at

the eye level facilitates the inspection.

Microscopic examination enhances detection

of particulate matter in injectables.

Visual inspection gives the qualitative

estimation of the particulate matter.

Acceptance Standards is that each

container checked must not contain any

visible particulate matter.

2. Test for Sub visible particle

This test is performed to check particulate

contamination of injections and infusions consists

of extraneous, mobile un-dissolved particles,

other than gas bubbles, unintentionally present in

the solutions.

This is further divided into two methods:

1. Method 1 ((Light Obscuration Particle Count

Test)

2. Method 2 (Microscopic Particle Count Test)

When examining injections and infusions for sub-visible particles, Method 1 is preferably applied.

But in case of preparations having reduced clarity or increased viscosity, the test is carried out according to Method 2. e. g. Emulsions, colloids, and liposomal preparations.

METHOD 1 (Light obscuration particle count test):

Principle:

This test is based on the principle of light blockage which allows an automatic determination of the size of particles and the number of particles according to size.

Apparatus:

An electronic particle counting system that uses a

light obstruction sensor with a suitable feeding

device is used.

General precautions:

The test is carried out under conditions limiting

particulate contamination, preferably in a laminar-

flow cabinet.

Very carefully wash the glassware and filtration

equipment used, except for the membrane filters,

with a warm detergent solution and rinse with

abundant amounts of water to remove all traces

of detergent.

Immediately before use, rinse the equipment from

top to bottom, outside and then inside, with

particle-free water R.

Take care not to introduce air bubbles into the

preparation to be examined, especially when

fractions of the preparation are being transferred

to the container in which the determination is to

be carried out.

PROCEDURE:

Mix the contents of the sample by slowly inverting

the container 20 times successively.

Clean the outer surfaces of the container opening using a jet of particle-free water R, avoiding any contamination of the contents.

Eliminate gas bubbles by appropriate measures such as allowing to stand for 2 min or sonicating.

For large-volume parenterals, single units are tested.

For small-volume parenterals less than 25mL in volume, the contents of 10 or more units are combined in a cleaned container to obtain a volume of not less than 25 ml.

Small-volume parenterals having a volume of 25 mL or more may be tested individually.

For large-volume parenterals or for small-volume

parenterals having a volume of 25 mL or more,

fewer than 10 units may be tested, based on an

appropriate sampling plan.

Remove 4 portions, each of not less than 5 mL,

and count the number of particles equal to or

greater than 10 μm and 25 μm.

Calculate the mean number of particles for the

preparation to be examined.

Evaluation:

Test 1.A – Solutions for infusion or solutions

for injection supplied in containers with a

nominal content of more than 100 mL:

The preparation complies with the test if the

average number of particles present in the units

tested does not exceed 25 per millilitre equal to or

greater than 10 μm and does not exceed 3 per

millilitre equal to or greater than 25 μm.

Test 1.B – Solutions for infusion or solutions for injection supplied in containers with a nominal content of less than 100 mL

The preparation complies with the test if the average number of particles present in the units tested does not exceed 6000 per container equal to or greater than 10 μm and does not exceed 600 per container equal to or greater than 25 μm.

For preparations supplied in containers with a nominal volume of 100 mL, apply the criteria of test 1.B.

If the average number of particles exceeds the limits, test the preparation by the microscopic particle count test.

METHOD 2 (Microscopic Particle Count Test):

Use a suitable binocular microscope, filter

assembly for retaining particulate contamination

and membrane filter for examination.

The microscope is equipped with an ocular

micrometer calibrated with an objective

micrometer,

a mechanical stage and, 2 suitable

illuminators.

The ocular micrometer is a circular diameter

graticule and consists of a large circle divided by

crosshairs into quadrants, transparent and black

reference circles 10 μm and 25 μm in diameter at

100 magnifications, and a linear scale graduated

in 10 μm increments.

The large circle is designated the graticule field of

view (GFOV).

Mechanical stage capable of holding and

traversing the entire filtration area of the

membrane filter.

Illuminators:

2 illuminators are required.

an episcopic brightfield illuminator internal to the

microscope,

the other is an external, focusable auxiliary

illuminator.

Filter assembly:

The filter assembly for retaining particulate

contamination consists of a filter holder made of

glass or other suitable material, and is equipped

with a vacuum source and a suitable membrane

filter.

Membrane filter:

The membrane filter is of suitable size, black or

dark grey in colour, non-gridded or gridded, and

1.0 μm or finer in nominal pore size.

Precautions:

The test is carried out in laminar-flow cabinet.

Very carefully wash the glassware and filter

assembly used, except for the membrane

filter, with a warm detergent solution and rinse

with abundant amounts of water to remove all

traces of detergent.

Air bubbles should not be present.

PROCEDURE:

Mix the contents of the samples by slowly inverting the container 20 times successively.

Clean the outer surfaces of the container opening using a jet of particle-free water R and remove the closure, avoiding any contamination of the contents.

Eliminate gas bubbles by appropriate measures such as allowing to stand for 2 min or sonicating.

For large-volume parenterals, single units are tested.

For small-volume parenterals less than 25 mL in volume, the contents of 10 or more units are combined in a cleaned container;

Small-volume parenterals having a volume of 25

mL or more may be tested individually.

Powders for parenteral administration are

constituted with particle-free water R.

Wet the inside of the filter holder fitted with the

membrane filter with particle-free water R and

apply vacuum.

Entire volume is filtered.

Maintain the vacuum until the surface of the

membrane filter is free from liquid.

Place the filter in a Petri dish and allow the filter

to air-dry.

After the filter has been dried, place the Petri dish

on the stage of the microscope.

Scan the entire membrane filter under the

reflected light from the illuminating device,

And count the number of particles that are equal

to or greater than 10 μm and the number of

particles that are equal to or greater than 25 μm

under the microscope.

Then Calculate the mean number of particles for

the preparation to be examined.

Evaluation:

Test 2.A – Solutions for infusion or

solutions for injection supplied in

containers with a nominal content of more

than 100 mL


average number of particles present in the

units tested does not exceed 12 per millilitre

equal to or greater than 10 μm and does not

exceed 2 per millilitre equal to or greater than

25 μm.

Test 2.B – Solutions for infusion or solutions

for injection supplied in containers with a

nominal content of less than 100 mL:


average number of particles present in the units

tested does not exceed 3000 per container equal

to or greater than 10 μm and does not exceed

300 per container equal to or greater than 25 μm.

For preparations supplied in containers with a

nominal volume of 100 mL, apply the criteria of

test 2.B.

3. PYROGENS TESTPYROGENS:

Pyrogens are fever producing substances.

Pyro means ‘pyrexia’, Gen means ‘producing’.

Pyrogens are the by-products of microorganisms mainly of bacteria, molds and viruses.

During the processing these pyrogens may come from water, active constituent or the excipient or from the equipments.

Chemically these pyrogens are lipid substances associated with carrier usually polysaccharides or may be proteins.

Parenteral solutions are officially tested for

the presence of pyrogens by a biological test

in which “FEVER” response of rabbits is used

as criteria.

Depyrogenation:

Depyrogenation is the removal of pyrogen.

This is achieved by the following methods.

Inactivation - Application of very high dry heat

(250 °C) for not less than 30 minutes is the

desired method for rendering material

pyrogen free.

Types of Pyrogen test

For Detection and quantification of Pyrogens:

Basically there are 2 tests performed to detect the

presence of pyrogens in sterile parenteral products

they are

1) In Vivo pyrogen test (Rabbit Test)

2) In Vitro pyrogen test(Limulus Amebocyte

Lysate Test)

1) In Vivo Pyrogen test (Rabbit Test)

PRINCIPLE:

This test consists of measuring the rise in

body temperature evoked in rabbits by the

injection of a sterile solution of the substance

being examined.

SELECTION & PROTOCOL

SELECTION OF ANIMALS:

Use healthy adult rabbits of any sex weighing not less

than 1.5kg.

Feed them a well balanced diet not containing any

antibiotics.

Not showing loss of body mass during one week

preceding the test.

A rabbit should not be used in the pyrogen test if:

It has been used in a negative pyrogen test in the

preceding three days or

It has been used in the preceding three weeks in a

pyrogen test in which the substance under

examination fails to pass the test.

MATERIALS NEEDED:

(a) ANIMALS' QUARTERS:

Keep the rabbits individually in a quiet area

with a uniform appropriate temperature.

Carry out the test in a quiet room where there

is no risk of disturbance exciting the animals

in which the room temperature is within 3 °C

of that of the rabbits' living quarters, or

In which the rabbits have been kept for at

least 18 h before the test.

(b) THERMOMETERS:

The thermometer or electrical device which indicates the temperature with a precision of 0.1 °C is used.

and insert into the rectum of the rabbit to a depth of about 5 cm. (B.P specification) or 7.2cm (USP specification). The depth of insertion is constant for any rabbit in every group.

When an electrical device is used, it should be inserted in the rectum of the rabbit 90 minutes before injection of the solution to be examined and left in position throughout the test.

(c) GLASSWARE, SYRINGES AND NEEDLES:

All the glassware, syringes and needles must be

thoroughly washed with water and heated in a hot air

oven at 250°C for 30 minutes or at 200°C for an hour.

(d) RETAINING BOXES:

The retaining boxes for rabbit in which the

temperature is being measured by an electrical device

should be made in such a way that the animals are

retained only by loosely fitting neck stocks, the rest of

the body remains relatively free, so the rabbit may sit

in a normal position.

The animals must be put in box not less than one

hour before the test and remain there throughout the

test.

PRELIMINARY TEST (SHAM TEST):

One to three days before testing the product, inject

pyrogen free isotonic NaCl solution (10ml/kg body

weight warmed at 38.5°C intravenously) into animal,

which has not been used during the two previous

weeks.

Record the temperature of animal, beginning at least

90 minutes before injection and continuing for 3 hours

after injection of solution.

Any animal showing a temperature difference greater

than 0.6°C must not be used in the main test.

MAIN TEST:

Carry out the test using a group of three

rabbits.

PREPARATION AND INJECTION OF

SAMPLE

Warm the liquids to be examined to

approximately 38.5°C before injection. The

sample liquid to be injected may be diluted

with a pyrogen free isotonic NaCl solution.

Inject the solution slowly into the marginal

vein of the ear of each rabbit over a period of

four minutes, unless otherwise mentioned in

the monograph.

The volume of the injection should be not less

DETERMINATION OF INITIAL AND MAXIMUM TEMPERATURE

The initial temperature of each rabbit is the mean of two temperature readings, recorded for that rabbit at an interval of 30 minutes in the 40min immediately preceding the injection.

While the maximum temperature is the highest temperature recorded for that rabbit three hours after the injection of the preparation being tested.

Record the temperature of each animal at an interval of not more than 30 minutes, beginning at least 90 minutes before the injection of the product to be examined and continuing 3 h after the injection.

The difference between the initial temperature and the maximum temperature of each rabbit is taken to be its response.

When this difference is negative, the result is counted as zero response.

REJECT THE RABBIT IF:

Rabbits showing a temperature variation greater than 0.2 °C between two successive readings in the determination of the initial temperature are withdrawn from the test.

All rabbits having an initial temperature higher than 39.8 °C or less than 38.0 °C are withdrawn from the test.

INTERPRETATION OF RESULTS

Having carried out the test on a group of three rabbits,

repeat if necessary on further groups of three rabbits

to a total of four groups.

If the summed response of the first group does not

exceed the figure given in the second column of the

Table, the substance passes the test.

If the summed response exceeds the figure given in

the second column of the table but does not exceed

the figure given in the third column of the table, repeat

the test as indicated above.

If the summed response exceeds the figure given in

the third column of the table, the product fails the test.

Depending on the results obtained tabulate the results in the following manner.

NUMBER

OF

RABBITS

MATERIAL PASSED IF SUM OF RESPONSE DOES NOT EXCEED.

MATERIAL FAILED IF

SUM OF RESPONSE EXCEEDS.

3 RABBITS 1.15C 2.65C




Sometimes the difference of initial and the

final temperature is negative. If the difference

is negative, the result of the rabbit test is

counted as zero response and the sample is

considered apyrogenic.

Rabbits used in a test for pyrogens where the

mean rise in the rabbits' temperature has

exceeded 1.2 °C are permanently excluded.

Advantages of Rabbit Test

The human and rabbits are equally

responsive to threshold levels of the pyrogens

2) In Vitro Pyrogen test

(Limulus Amebocyte Lysate, LAL Test)

The limulus amebocyte lysate test is also called as

in-vitro pyrogen test (USP XXI Specified new test).

Officially it is termed as bacterial endotoxin test (BET)

used to detect or quantify endotoxins from gram

negative bacteria.

The test principle is based on the clotting of lysate of

amebocyte (an enzyme obtained from the horse shoe

crab) in the presence of pyrogens.

The extract from the blood cells of horse shoe crab,

Limulus Polyphemus contains an enzyme system

called "Limulus- Amebocyte Lysate" (LAL) which

reacts with pyrogens so that an assay mixture

increases in viscosity and opacity until an opaque gel

is formed.

Amebocyte + Pyrogen ~ Opaque gel

The reaction accomplishes within 15-60 minutes, depending on concentration of pyrogens after mixing. The concentrated pyrogens make the gel more turbid and thick.

REQUIREMENTS:

Limulus-Ambocyte Lysate is prepared by bleeding healthy mature specimens by heart puncture.

The amebocytes are carefully concentrated, washed and lysed by osmotic effects.

Prior to perform the LAL test, lysate assay is carried out with purified endotoxins and are accepted if it detects 0.001ug/ml or less concentration of the purified endotoxins.

The glassware, such as glass test tubes (10 x 75mm) used in the test must be thoroughly cleaned, dry and heat sterilized.

A buffer solution of potassium phosphate 2mEq/ml is used to adjust the pH of test sample at 7.

The alcoholic content in sample is to be removed as it causes precipitation of lysate. If the sample contains proteins, it produces gel thus the proteins must be diluted to appropriate concentration before the test.

Similarly other interfering substances present in sample must also be removed before the test.

PROCEDURE:

The pH of test sample if specified is adjusted.

The test solution and standardized LAL are

separately mixed in equal parts (0.05-0.2ml).

The mixture is incubated immediately at 36-

38°C for 1 hour in assay tube.

The assay tube must be remained

undisturbed completely because agitation

may irreversibly destroy the gel leading to a

false negative result.

The test tube is observed after the specified

time and is examined for the formation of

opaque gel.

Formation of gel represents a positive test

endpoint reaction.

The test is performed using a commercial LAL

test kit.

This kit contains a lyophilized LAL, and E. coli

endotoxin and pure water as standards and

these later two are used to check the

sensitivity of the test.

ADVANTAGE OF LAL TEST

1. It is in-vitro and does not require animal handling, thus is more convenient.

2. It is 10 times more sensitive than that of the in-vivo rabbit test.

3. It is economical.

4. It consume less time, i.e., 1 vs 3 hours required by rabbits test.

5. It requires less laboratory facilities and minimum equipments.

6. It requires less test volume( as little as 0.1ml of test solution).

7. It is more accurate.

4) Uniformity of contents

The test for uniformity of content of single-dose

preparations is based on the assay of the

individual contents of active substance(s) of a

number of single-dose units to determine whether

the individual contents are within limits set with

reference to the average content of the sample.

The test is not required for multivitamin and trace-

element preparations and in other justified and

authorised circumstances.

METHOD:

Using a suitable analytical method, determine the individual contents of active substance(s) of 10 dosage units taken at random.

TEST A:

Criteria for Tablets, powders for parenteral administration, ophthalmic inserts, suspensions for injection:

The preparation complies with the test if each individual content is between 85 per cent and 115 percent of the average content.

The preparation fails to comply with the test if more than one

individual content is outside these limits or if one individual content is outside the limits of 75 per cent to 125 per cent of the average content.

If one individual content is outside the limits of 85

per cent to 115 percent but within the limits of 75

per cent to 125 percent, determine the individual

contents of another 20 dosage units taken at

random.

The preparation complies with the test if not more

than one of the individual contents of the 30 units

is outside 85 per cent to 115 percent of the

average content and none is outside the limits of

75 percent to 125 per cent of the average

content.

5) TEST FOR STERILITY

The test is applied to substances, preprations or

articles which, according to pharmacopiea, are

required to be sterile.

However, a satisfactory result only indicates that

no contaminating microorganisms has been

found in the sample examined in the conditions of

the test.

PRECAUTIONS:

The test for sterility is carried out under aseptic

conditions.

The working conditions in which the tests are

performed are regularly monitored by appropriate

sampling of working area.

Culture media & incubation

temperature

The following culture media have been found to

be suitable for test of sterility.

Fluid thioglcollate medium:

is primarily intended for culture of anaerobic

bacteria.

Can also detect aerobic bacteria.

Soya bean casein digest medium:

Is suitable for culture of both fungi and aerobic

bacteria.

FLUID THIOGLCOLLATE MEDIUM:

Composition:

L. Cystine 0.5g

Sodium chloride 0.75g

Glucose monohydrate 5.5g

Yeast extract 5g

Pancreatic digest of casein 15g

Sodium thioglycollate 0.5g

Resazurin sodium soln. 1ml, freshly prepared

Water R 1000ml

pH after sterilisation 7.1 ± 0.2

Prepare the medium and sterilise using a

validation process.

If medium is stored, store a temperature between

2˚ C to 25˚ C in a sterile, airtight container.

Do not use medium for a longer storage period.

Fluid thioglcollate medium is incubated at 30 – 35

˚ C.

SOYA BEAN CASEIN DIGEST MEDIUM:

Composition:

Pancreatic digest of casein 17.0 g

Papaic digest of casein 3g

Sodium chloride 5.0 g

Dipotassium hydrogen phosphate 2.5g

Glucose monohydrate 2.5g

Water R 1000ml

pH after sterilisation 7.3 ± 0.2

Prepare and sterilise the medium.

If medium is stored, store a temperature between

2˚ C to 25˚ C in a sterile, well closed container.

Do not use medium for a longer storage period.

Soya bean casein digest medium is incubated at

20 – 25 ˚ C.

Sterility of medium:

The media used comply with the following tests,

carried out before or in parallel with the test on

the product to be examined.

Incubate portions of media for 14 days.

No growth of microorganisms occur.

Growth promotion test of aerobes,

anaerobes and fungi

Perform to check the suitability of medium for sterility test.

Test each batch of medium.

Inoculate portions of thioglycolate medium with a small no. (not more than 100CFU) of following microorganisms;

Clostridium sporogenes

Pseudomonas aeruginnosa

Staphylococcus aureus

Use separate portion of media for each specie of microorganisms.

Inoculate portions of soya bean casein digest medium with a small no. (not more than 100CFU) of following microorganisms;

Aspergillus brasiliensis

Bacillus subtilis

Candida albicans

Use separate portion of media for each specie of microorganisms.

Incubate for not more than 3 days in the case of bacteria.

Not more than 5 days in the case of fungi.

RESULT: The media are suitable if a clearly visible growth of microorganisms occur.

BACTERIOSTASIS &

FUNGISTASIS In addition to the foregoing medium tests, prior to

conducting a sterility test on a product, determine its level of bacteriostatic and fungistatic activity by the following procedure:

To each of several vessels containing the specified quantity (15, 40, 80ml) of appropriate test medium, add the specified quantity of product.

Inoculate these vessels of product-medium mixtures and the control vessels of medium with dilute culture of bacteria and fungi that are sensitive to the product being tested, including spores of aerobic and anaerobic bacilli.

Incubate all vessels at an appropriate temp. for not less than 7 days.

If growth of the test organisms is comparable in

control vessels and in product-medium mixture

vessels, the product is not bacteriostatic or

fungistatic.

If the product is fungstatic or bacteriostatic,

Either use a suitable sterile inactivating agent or

Diluting the product with sufficient quantity of

culture medium.

Test procedure for sterility of the

product

The test may be carried out using the technique

of membrane filtration or by direct inoculation of

the culture media with the product to be

examined.

OPENING CONTAINERS:

Clean the exterior surface of ampules and

closures of vials and bottles with antimicrobial

agents and make access to the contents in a

suitable manner.

SAMPLING:

For each unit, use not less than the volume of

product and medium specified in BP.

1. Direct inoculation of culture medium:

Transfer the specified quantity of the preparation

to be examined directly in to culture medium so

that the volume of the product is not more than

10% of the volume of the medium.

If the product has antimicrobial activity, carry out

test after neutralizing this with a suitable

neutralizing substance or by diluting in a sufficient

quantity of culture medium.

When it is necessary to use a large volume of the

product it may be preferable to use a

concentrated medium.

2. Membrane Filtration:

The technique of membrane filtration is used whenever the nature of the product permits, that is,

For filterable aqueous preparation

For alcoholic or oily preparations

For preparations miscible with or soluble in aqueous or oily solvents provided these solvents do not have antimicrobial activity.

Use membrane filters having a nominal pore size of not greater than 0.45um with effectiveness to retain microorganisms.

For example:

Cellulose nitrate filters are used for aqueous, oily and

weakly alcoholic solutions.

Cellulose acetate filters are used for strongly

alcoholic solutions.

Special filters may be needed for certain products

.e.g. Antibiotics.

The diameter of membrane is about 50mm.

The filtration apparatus and membrane are sterilized

under aseptic conditions.

If appropriate, transfer a small quantity of a suitable,

sterile diluent such as a 1 g/L neutral soln. Of meat or

casein peptone on to membrane in the apparatus and

filter.

Transfer the contents of the container to be tested to the membrane.

Filter immediately.

If the product has antimicrobial activity, wash the membrane not less than 3 times by filtering through it the volume of chosen sterile diluent.

Transfer the whole membrane to the culture medium or cut it aseptically into equal parts and transfer one half to each of suitable media.

Alternately transfer the medium on to the membrane in the apparatus.

Incubate the medium for not less than 14 days.

OBSERVATIONS &

INTERPRETATION OF RESULTS

At intervals during the incubation and at its

conclusion, examine media for microbial growth.

If the material being tested rendered the medium

turbid, so that the presence or absence of

microbial growth can not be readily determined by

visual inspection.

In this case, 14 days after the beginning of

incubation transfer portion of the medium not less

than 1ml to fresh vessels of the same medium

and then incubate the original and transfer

vessels not less than 4 days.

If no evidence of microbial growth is found, the

product to be examined complies with the test for

sterility.

If evidence of microbial growth is found, the

product to be examined does not complies with

the test for sterility.

When using the technique of membrane filtration ,

use whenever possible, the whole contents of

container but not less than specified quantity.

When the volume or the quantity of in a single

container is insufficient to carryout test, the

contents of 2 or more containers are used to

inoculate different media.

Health & Medicine

QUALITY CONTROL OF PARENTERALS,STERILE PRODUCT