In-Process Quality Control Tests for Parenterals

1. IN-PROCESS AND
FINISHED PRODUCT
QUALITY CONTROL
TESTS FOR
PARENTERALS
Prapti Pattanayak
Pharmaceutical Analysis
M.Pharm 1st Year
1

2. CONTENTS
1. Introduction
2. Definition
3. Types Of Parenterals
4. Routes Of Administration
5. Significance Of IPQC Tests
6. IPQC Tests According To IP, BP And USP
2

3. INTRODUCTION
• Derived from Greek words “Para” means
“outside” and “enteron” means “intestine”.
• Route of administration- mostly
intravenous, intramuscular, subcutaneous,
intradermal .
• Sterile solution or suspension of drug in
aqueous or oily vehicle.
• Directly administered directly under the
skin, veins, muscles or more specialized
tissues like spinal cord.
• Drug delivery doesn’t utilize alimentary
canal for entering into the tissues.
3
Fig 1: Injections, vial,
ampoule

4. DEFINITION
• According to IP
Preparations that are sterile and intended for administration by
injection, infusion or implantation.
• According to USP
Preparations that includes injections and implanted products
that are injected through the skin or other external boundary
tissue or implanted within the body to allow direct
administration.
• According to BP
Sterile products that are intended for administration by
injection, infusion or implantation into the body.
4

5. TYPES OF PARENTERALS
1. Small Volume Parenterals (SVPs)
 They are given as multiple dose.
 Volume- <100ml
 Types: Ampoules, vials, dry powders etc.
a. Ampoules- These are sealed glass container with an
elongated neck that must be broken off.
A 5 micron filter needle should be used when drawing the
contents of an ampoule into a syringe since glass particles
may have fallen inside the ampoule when the top was
snapped off.
b. Vials- It is made of glass or plastic and sealed with a rubber
stopper.
Drugs and other additives are packaged as liquid or
lyophilized powders.
It is designed for both single and multiple dose use.
Multiple dose vials contain a preservative to inhibit bacterial
contamination.
5
Fig 2: Ampoules
Fig 3: Vial

6. 6
c. Dry Powders- These are lyophilized or freeze dried
powders that must be reconstituted with some
suitable solvent to make a liquid formulation before
being withdrawn from the vial.
Solvents may be WFI, BWFI, NaCl injection etc.
2. Large Volume Parenterals (LVPs)
They are designed to provide fluid (water), calories
(dextrose solution) and electrolytes ( saline solution).
Volume- > 100ml
 4 Types:
a. Hyperalimentation solution/ Total Parenteral
Nutrition- administration of large amount of
nutrients to patients who are unable to take food
orally.
• Use- administration of life saving drugs to the
comatose patients
Fig 4: LVPs

7. 7
b. Cardioplegic Solution- it is used for heart surgery to prevent
injury to myocardium during reperfusion.
• Maintains diastolic arrest.
c. Peritoneal Dialysis Solution- It is infused continuously into
the abdominal cavity, bathing peritoneum and are then
continuously withdrawn.
Use- To minimize reperfusion injury resulting from tissue
edema.
Formulations- Glucose, Antibiotics as prophylactic .
d. Irrigating Solution- It is used to irrigate, flush and aid in
cleansing body cavities and wounds.
Formulation- Normal saline
Use- For the treatment of serious wounds infused into blood
stream.

8. ROUTE OF ADMINISTRATION
• Angle- 15ﾟ
• Dose- 0.1ml
• Ex. BCG vaccineINTRADERMAL
• Angle- 25ﾟ
• Dose- >1litre
• Ex. DNS (Dextrose Normal Saline)
INTRAVENOUS
• Angle- 45ﾟ
• Dose- 1-1.3ml
• Ex. Insulin
SUBCUTANEOUS
• Angle- 90ﾟ
• Dose- 2-5ml
• Ex. Atropine, Haloperidol
INTRAMUSCULAR
8

9. SIGNIFICANCE OF IPQC(In Process
Quality Control)
• We can remove error from every stage in production and
maintain the quality of the final product with the compendial
standards as specified in the pharmacopoeia’s.
• We can determine the number and types of microorganisms
present in the product by sterility testing.
• We can ensure the exact quantity of the solution mentioned
on the label claim.
• We can prevent the contamination of the drug caused due to
poor package integrity.
• Identification of any crack or leakage present on the container
which may hamper the package integrity.
9

10. IPQC TESTS
FOR
PARENTERALS
CONTENT
UNIFORMITY
EXTRACTABLE
VOLUME
PYROGEN
TEST
STERILITY
TEST
LEAKAGE
TEST
BACTERIAL
ENDOTOXIN
TEST
CLARITY TEST
10Fig 5: IPQC tests for Parenterals

11. CONTENT UNIFORMITY
• ACCORDING TO IP(Method)
Weight of 10 individual
sterile units are noted
Content is removed from them and
empty individual sterile unit is
weighed accurately.
Net weight is calculated by
substracting empty sterile unit
weight from gross weight.
11

12. Acceptance Criteria (IP)
DESCRIPTION RANGES INFERENCE
If all individual values are
within the range of
85-115% of avg. values and RSD
≤6%
Passes
If more than 1 individual
values
OR
If any one individual value
>85-115% of the avg. value
OR
>75-125% of the avg. value
Fails
If one individual value is >85-115% but within the limits 75-
125% of the avg. value
RSD >6%
Repeat the test using
additional 20 containers
If in 30 containers, NMT 1
individual value
OR
None of the units
Not exceeds 85-115% of avg. value
OR
Exceeds 75-125% of avg. value
RSD NMT 7.8%
Passes
12
Table no. 1

13. According to BP
• Individual contents of the 10 dosage units are taken randomly.
TEST DESCRIPTION RANGES INFERENCE
TEST A
(tab, powders for
parenterals
administration,
opthalmic inserts,
suspensions for
injection)
1.If each individual
contents is
2.If 1 or more than 1
individual content is
3.If 1 individual
content
4. If in 30 units, NMT 1
individual
b/w 85-115% of avg.
contents
>75-125% of the avg.
value
Exceeds 85-115% but
within 75-125% limits
Exceeds 85-115% of
the avg. value and
none exceeds 75-125%
avg. value
Passes
Fails
Another 20 dosage
forms taken randomly
Passes
TEST B
(cap, powders other
than for parenterals
administration,
granules,
suppositories,
pessaries)
1.If NMT 1 individual
content
Exceeds 85-115% and
none exceeds 75-125%
avg. contents
Passes
13
Table no. 2

14. Continue..
2. If more than 3
individual contents
OR
If 1 or more individual
content
3. If 2 or 3 individual
contents
4. If in 30 individual
units, NMT 3
individual contents
Exceeds 85-115%avg.
content
OR
Exceeds 75-125% avg.
content
Exceeds 85-115% avg.
value but within 75-
125%
Exceeds 85-115% and
none exceeds 75-125%
of avg. contents
Fails
Randomly 20 dosages
forms are added
Passes
TEST C
(Transdermal patches)
1. If avg. content of
10 dosages units
is
2. If the individual
content of each
dosage unit is
b/w 90-110% of the
content
b/w 75-125% of avg.
content
Passes
14

15. According to USP
• 10 dosage units are taken randomly.
DESCRIPTION RANGES INFERENCE
If 10 individual units Within 85-115% avg.
content and RSD <6%
Passes
If more than 1 individual
value
OR
If any one individual value
Exceeds 85-115% avg.
content
OR
Exceeds 75-125% avg.
content
Fails
Fails
If one individual Exceeds 85-115% but with
75-125% avg. content
Repeat test with another
20 units randomly
If in 30 containers, NMT 1
individual value
Exceeds 85-115% and none
exceeds 75-125% avg.
content value
Passes
15
Table no.3

16. LEAKAGE TEST
16
 To test package integrity.
 Leakage test is employed to detect incompletely
sealed ampoule and vials so that they may be
discarded.
WHAT IS THE NEED ?
• Presence of capillary pores or tiny cracks can cause
microbes or other contaminants to enter in the
ampoules.
• Change in temperature during storage can cause
expansion and contraction of the ampoule and
thereby causing interchange of its contents if any
opening exists.
• Leakage test is done by visual inspection, bubble
test and dye bath test .
A. METHOD ( DYE BATH TEST)
TEST CONTAINER
IMMERSED IN A
DYE BATH UNDER
VACUUM AND
PRESSURE
REMOVED THE
CONTAINER
FROM DYE BATH
AND WASHED
CONTAINER IS
INSPECTED FOR
THE PRESENCE OF
DYE EITHER
VISUALLY OR BY
UV
SPECTROSCOPY
DYES USED : Mainly methylene
blue is used. Other than that
green, yellowish-green color are
also used.
OPTIMIZATION OF DYE TEST:
• Addition of surfactant or a low
viscosity fluid in the dye.
• These will increase the
capillary migration of the
pores.
ADVANTAGES:
• Inexpensive
• No special equipment
required for identification
• Only for Qualitative analysis
DISADVANTAGES:
• Destructive and slow test
• Not for Quantitative analysis

17. 17
B. VISUAL INSPECTION
• It is the easiest and least sensitive leakage test
method .
• Used for 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.
• It is a very simple , inexpensive and operator
dependent method.
C. BUBBLE TEST
• The test package is submerged in liquids.
• A differential pressure is applied on the container.
• The container is observed for bubbles.
• For optimization of leakage test, surfactant(ex.
polysorbate 80) added to the liquid along with dark
background and high intensity lighting.
• Differential positive pressure of 3 psi inside the vial is
generated and observation of any leakage using
magnifying glass within a maximum test time of 15
minutes.
• It can detect leaks as small as 10 mbar-L/sec.
• It is also insensitive, operator dependent and
qualitative test.
Fig 6: Visual Inspection

18. 18
STERILITY TEST
 Sterile means free from microorganisms like
bacteria, fungi, yeast etc.
PRINCIPLE: If the bacteria and fungi are placed in a
medium which provides nutrition to the organisms
and kept at favorable temperature, the organisms
will grow and their presence can be indicated by
turbidity in the clear medium.
WHAT IS THE NEED?
• To detect the presence of viable organisms in
the parenterals.
• Presence of microbes may contaminate the
sterile products.
 For sterility testing, aseptic conditions should
be maintained.
MAJOR FACTORS AFFECTING STERILITY TESTING
• The environment in which test is conducted
• The quality of culture
• Test method
• Test procedure
• Sample size
Fig 7: Sterility testing

19. 19
CULTURE MEDIA USED:
There are basically 2 types of culture
media:
a. Fluid thioglycollate medium
b. Soyabean casein digest medium
1. FLUID THIOGLYCOLATE MEDIUM (FTM)
 FTM is an excellent medium for the
detection of microbial contamination.
 It can provide both aerobic and
anaerobic environment.
COMPOSITIONS OF FTM:
a. L- cysteine- it supply carbon and
nitrogenous compounds.
b. Dextrose- acts as an energy source.
c. Yeast extract- act as growth enhancer.
d. Sodium chloride- it maintains osmotic
equilibrium.
e. Sodium thioglycollate- acts as
reducing agent which maintains a low
oxygen tension by removing
molecular oxygen from the
environment i.e., it creates anaerobic
conditions when it reduces molecular
oxygen to water.
f. Resazurin- it is a redox indication that turns
pink when oxidation increased and colorless
when reduction occurred.
g. Agar- addition of small amount of agar in
medium initiate the growth of small inocula and
anaerobes.
h. Purified water
i. Adjusted to pH 7.1± 0.2
 Incubation of the media – 14 days at 30-
35℃
Components Quantity
L-Cysteine 0.5g
Sodium chloride 2.5g
Dextrose monohydrate 5.5g
Yeast extract 5.0g
Pancreatic digest of casein 15.0g
Sodium thioglycollate/thioglycollic
acid
0.5g/0.3g
Resazurin sodium solution 1.0ml
Distilled water q.s to 1000ml
pH of medium after sterilization 7.1± 0.2
Table no. 4

20. 20
B. SOYABEAN CASEIN DIGEST MEDIUM
 It is used for culture of both fungi and aerobic bacteria.
COMPOSITIONS:
a.Trypticase soya broth- it is used to grow aerobic bacteria
b.Trypticase peptone- it is an animal origin pancreatic digest of casein which provides
nutrition.
c. Sodium chloride- it maintains osmotic equilibrium
d. Dipotassium phosphate- it provides buffering capacity
e. Dextrose- provides energy
f. Purified water
g. Adjusted to pH 7.3± 0.2
 Incubation period- 14 days at 20- 25℃
Components Quantity
Pancreatic digest of casein 17.0g
Papaic digest of soyabean meal 3.0g
Sodium chloride 5.0g
Dipotassium hydrogen phosphate 2.5g
Dextrose monohydrate 2.5g
Distilled water q.s. to 1000ml
pH of medium after sterilization 7.3± 0.2
Table no. 5

21. 21
STERILITY TESTING METHODS:
There are 2 types of sterility testing methods:
a. Membrane filtration method
b. Direct inoculation method
1. METHOD A (MEMBRANE FILTRATION METHOD)
Membrane filtration is preferred for:
a. An oil
b. An ointment that can be put into solution
c. A non bacteriostatic solid not readily soluble in the culture medium
d. A soluble powder or a liquid that possesses bacteriostatic or fungistatic properties
 It is also used for the positive and negative controls.
Medium Test microorganism Incubation
Temp.
(℃)
Duration Type of
microorganism
Fluid thioglycollate Clostridium sporogenes
Staphylococcus aureus
Pseudomonas aeruginosa
30-35
30-35
30-35
3days
3days
3days
Anaerobic
Aerobic
Aerobic
Soyabean casein digest Asperigillus niger
Candida albicans
Bacillus subtilis
20-25
20-25
20-25
5days
5days
3days
Aerobic
Aerobic
Aerobic
Table no. 6: Strains of microorganisms used in the test
Fig 8:
Membrane
filtration unit

22. 22
TYPES OF FILTERS:
 Cellulose nitrate filters are used for aqueous, oily and weakly alcoholic solutions .
 Cellulose acetate filters are used for strongly alcoholic solutions.
Diluting Fluids (IP, BP):
Fluid A: Dissolve 1 g of bacteriological peptone in water to make 1liter, filter/centrifuge to
clarify, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20 minutes.
Fluid B: If the test sample contains lecithin or oil, use fluid A to each liter, add1 ml of
polysorbate 80, adjust to pH 7.1 ± 0.2, dispense into flasks and sterilize at 121℃ for 20
minutes.
Quantity of parenteral sample Minimum quantity to be used for each culture
medium
Less than 1ml Total contents of the container
1 ml or more but less than 40 ml Half the contents of the container
40 ml or more but less than 100 ml 20 ml
100 ml or more 10% of the contents of container but not less than
20 ml
Antibiotic liquids 1ml
Table no.7: Minimum quantity of sample to be used for each culture medium

23. 23
APPARATUS:
• Suitable unit consists of a closed reservoir and a receptacle between which a
supported membrane of appropriate porosity is placed.
• Membrane should has a nominal pore size < 0.45µm and diameter is 50mm(approx.)
METHOD OF TEST:
• For aqueous preparations, minimum quantity of sample (as mentioned in table no.7)
is mixed with diluting fluid A.
• Prepare each filter membrane by aseptically transferring a small quantity of fluid A
onto the membrane(to moisten it) and filter it.
• For each medium to be used, transfer aseptically into 2 separate membrane filter
funnels.
• Alternatively, transfer aseptically the combined quantities of the preparations under
examination in 2 media onto one membrane.
• Draw the liquid rapidly through the filter with the aid of vacuum.
• If the solution under examination has antimicrobial properties, wash the membrane
by filtering through it not less than 3 successive quantities, each of 100ml/filter.
• After filtration, aseptically remove the membrane from the holder and transfer the
whole membrane or cut it into 2 equal parts.
• Transfer one half to each of the 2 suitable media.
• Incubate the media not less than 14 days. Observe the containers and if the test
specimen is positive before 14 days of incubation, further incubation is not
required.

24. 24
• For liquids immiscible with aqueous vehicles, and suspensions, again repeat the
method for aqueous preparations but add a sufficient quantity of Fluid A to the
pooled sample to achieve rapid filtration.
• Sterilize enzyme preparations such as penicillinase or cellulose may be added to
Fluid A to dissolve insoluble substances.
• If the substance being examined contains lecithin, use fluid B for diluting.
2. METHOD B (DIRECT INOCULATION METHOD)
Quantities of sample to be used: The quantity of the substance or preparation under
examination to be used for inoculation in the culture media varies according to the
quantity in each container which is mentioned in Table no.7.
METHOD OF TEST:
• For aqueous solutions and suspensions, remove the liquid from the test containers
with a sterile pipette or needle or syringe.
• Transfer the quantity of the preparation under examination directly into the culture
medium so that the volume of preparation under examination is NMT 10% of the
volume of medium.
• If the preparation under examination has antimicrobial activity, carry out the test
after neutralizing this with a suitable neutralizing substance or by dilution in a
sufficient quantity of culture medium.
• Incubate the inoculated media for not less than 14 days . Observe the containers and
if the test specimen is positive before 14 days of incubation, further incubation is not
required.

25. 25
Table no.8 : Minimum number of items to be tested
No. of items in a batch Minimum no. of items to be tested
Not more than 100 containers 10%/ 4 items whichever is greater
More than 100 but not more than 500
containers
10 items
For more than 500 2%/ 20 items whichever is less
For large volume parenterals 2%/ 10 items whichever is less
Advantages of membrane filtration method over direct inoculation method are:
1. Entire contents can be tested which will increase the detection ability of the
microorganisms.
2. Organisms present in an oleaginous product can be separated from the product during
filtration .
3. It minimize the occurance of false results.

26. 26
PYROGEN TEST
 Pyrogen = fever producing organisms
 The test involves measurement of the rise in body temperature
of rabbits following the intravenous injection of a sterile
solution of the substance under examination.
 It is designed for products that can be tolerated by the test
rabbit in a dose not exceeding 10ml/kg injected through i.v
route within a period of NMT 10 minutes.
Test Animals:
• Healthy, adult rabbits of either sex, preferably of the same
variety.
• Weight NLT 1.5kg
• Fed on a complete balanced diet
• Rabbits should be kept under uniform temperature(± 2℃), with
uniform humidity and free from disturbances.
Note: 1.Don’t use animals for pyrogen tests more frequently
than once every 48 hours.
2.After a pyrogen test, if the rabbit’s temperature has risen 0.6ﾟ
or more, then atleast 2 weeks must be allowed to elapse before
the animal is used again.
Fig 9: Pyrogen
testing on rabbit

27. 27
Materials:
 All glasswares, syringes and needles must be washed with water for injection and
heated in hot air oven at 250ﾟfor 30 minutes or 200ﾟfor 1 hour.
 Treat all the diluents and solutions for washing and rinsing of devices in a manner
that will they become sterile.
 The animal must put in the box 1 hour before the test and remain in them
throughout the test.
 The room temperature should be within 3ﾟ of that of rabbits living quarters.
 Withhold food from the animals overnight and until the test is completed.
 withhold water during the test.
Recording of Temperature:
• Thermometer or thermistor have been caliberated to assure an accuracy of 0.1℃.
• Insert the thermometer into the rectum of the test rabbit to a depth of about 5 cm
(according to USP- 7.5cm).
• If any electrical device is used, it should be inserted in the rectum of the rabbit 90
minutes before the injection of the solution being examined and left in position
throughout the test.
• After a period of time, record the rabbit’s body temperature.

28. 28
 SHAM TEST /PRELIMINARY TEST/ RABBIT TEST(IN-VIVO)
• If animals are used for the first time in a pyrogen test or have not been used during the
2 previous weeks, condition them 1-3 days before testing the substance under
examination by injecting intravenously into them 10ml/kg of body weight of a pyrogen-
free saline solution warmed to about 38.5ﾟ.
• Record the temperatures of the animals, beginning at least 90 minutes before injection
and continuing for 1,2,3 hours after injection of the solution being examined.
• Any animal showing a temperature variation of 0.6ﾟ or more must not be used in the
main test.
 MAIN TEST:
Carry out the test using a group of 3 rabbits.
Preparation of the sample: Dissolve the substance with pyrogen-free saline solution.
Warm the liquid under examination to approximately 38.5°C before injection.
Method of test:
• Record the temperature of each animal 90 minutes before the injection and continue
for 3 hours after the injection for every 30 minutes.
• Record the "initial temperature" of each rabbit which is the mean of 2 temperatures
recorded after 30 minutes.
• Rabbits showing a temperature variation greater than 0.2°C between two successive
readings in the determination of "initial temperature" should not be used for the test.
Do not use any rabbit having a temperature higher than 39.8°C and lower than 38°C.

29. 29
• Inject the solution slowly into the marginal vein of the ear of each rabbit over a period
not exceeding 4 minutes.
• The volume of injection is not less than 0.5 ml per kg and not more than 10 ml per kg of
body weight.
• The difference between the "initial temperature" and the "maximum temperature"
which is the highest temperature recorded for a rabbit is taken as its response. When
this difference is negative, the result is counted as a zero response.
Table no.9: Interpretation of results
Pharmacopoeia No. of rabbits in a
group
Passes if the temp. is
not more than
Fails if temp. is more
than
IP 3
8
1.4
3.7
Each rabbit temp. raise
should not be more
than 0.6℃
BP 3
6
9
12
1.15
2.80
4.45
6.6
2.65
4.30
5.95
6.6
USP 3
8
-
3.3
Each rabbit temp. raise
should not be more
than 0.6℃

30. 30
BACTERIAL ENDOTOXIN TEST(USP)
 It measures the concentration of bacterial endotoxins that may be present in the
sample or in the articles to which the test is applied using a lysate derived from
amoebocytes of horseshoe crab, Limulus polyphemus.
 Other species of horseshoe crab namely Tachypleus gigas, Trachypleus tridentatus.
 LAL(Limulus Amoebocyte Lysate) test(IN-VITRO)
 Amoebocyte- crab blood cell from which active component is derived.
 Lysate – component obtained by separating amoebocyte from plasma and then lysing
them.
What is endotoxin?
• Produced by gram negative bacteria
• Outer covering – lipopolysaccharide
• Thermostable and water soluble
PRINCIPLE: When endotoxin solution is injected
to horseshoe crab, the chemical component
lysate is secreted by amoebocyte which reacts
with the endotoxin and inactivate it.
Inactivation of endotoxin by lysate results in
formation of gel or turbidity or precipitation. Fig 10: Horseshoe crab

31. 31
METHODS OF ENDOTOXIN TEST:
1. METHOD A: GEL- CLOT LIMIT TEST METHOD
2. METHOD B: SEMI-QUANTITATIVE GEL-CLOT METHOD
3. METHOD C: KINETIC TURBIDIMETRIC METHOD
4. METHOD D: KINETIC CHROMOGENIC METHOD
5. METHOD E: END-POINT CHROMOGENIC METHOD
Fig 11: LAL reagent reaction cascade mechanism
MECHANISM:
 The cascade reaction starts by
the presence of a bacterial
endotoxin which reacts with
lysate, and initially activates
factor C i.e., serine protease
precursor followed by activation
of factor B i.e., serine protease
precursor.
 Activated Factor B further
activates proclotting enzyme into
clotting enzyme which
hydrolyzes coagulogen into
coagulin, forming an insoluble
gel.
 When (1 → 3)-β-D-glucan (a
fungal cell wall component),
reacts with lysate, activates
factor G.
 Activated factor G activates
proclotting enzyme and futher
forms a gel-clot.

32. 32
METHOD A: GEL-CLOT LIMIT TEST METHOD (Qualitative method)
• Prepare the solutions and dilutions with water BET. If necessary, adjust the pH of the
solution to 6.0 to 8.0 using sterile 0.1M HCl BET, 0.1M NaOH BET of suitable buffer
prepared with water BET.
• Prepare the sample solution . Use two positive controls, one having the concentration of
2λ and other is spiked to get the concentration of 2λ.(where λ=0.125 EU/ml)
• Add an appropriate volume of negative control (NC), standard CSE solutions in water
BET, test solution and positive control(PPC). At regular intervals add an equal volume of
the appropriately constituted lysate. Mix it and place it in an incubator.
• Incubation should be done at 37º±1º undisturbed for 60±2 minutes.
• Remove and examine the receptacles carefully.
• A positive reaction is recorded in dilution 2λ when firm gel is formed that retains the
integrity when inverted through 180º in one smooth motion.
• If no firm gel is formed then it is a negative reaction.
Fig 12: After tilting 180ﾟ, if clot retains then
test positive and if not then negative
CALCULATION:
Geometric mean end-point
concentration= Antilog(Ʃe/f)
Where,
Ʃe = sum of the log end-point
concentrations of the series of dilutions
used
f= number of replicate test-tubes

33. 33
METHOD B: SEMI-QUANTITATIVE GEL-CLOT METHOD
Preparation of test solutions: Prepare test solutions at concentration of MVD, 0.5
MVD,0.25MVD (Maximum Valid Dilution).
[MVD= Endotoxin limit(EU/mg) X concentration of the test solution(mg/ml)/λ]
Method: Same as Method A
Calculation and interpretation of results
• To calculate the endotoxin concentration in the product, determine for the series of
test solutions the lowest concentration or the highest dilution giving a positive (+)
reaction.
• Multiply this dilution with to obtain the endotoxin concentration of the product.
• For example, if MVD is equal to 8 and the positive reaction was obtained at 0.25
MVD and λ was equal to 0.125EU/ml, then endotoxin concentration in the test will
be, 8x 0.25x 0.125=0.25 EU/ml.
• If none of the dilutions of the series gives a positive reaction, the endotoxin
concentration will be less than the value obtained by multiplying the lowest diltution
factor with λ.
• If all the dilutions of the series give a positive reaction, the endotoxin concentration
will be more than the value obtained by multiplying the highest dilution factor with
λ.

34. 34
METHOD C: KINETIC TURBIDIMETRIC METHOD
• Quantitative method
• Using CSE( Control Standard Endotoxin), prepare solutions of not less than 3 endotoxin
concentrations to get a standard curve.
• It is a photometric assay measuring the increase in turbidity caused by reaction of the
endotoxin with the lysate.
• It is a method measuring either the time needed to reach a predetermined absorbance
of the reaction mixture or the rate of turbidity development.
METHOD D: KINETIC CHROMOGENIC METHOD
• Quantitative method
• It is a photometric assay measuring the colour developed by the chromophore released
from a chromogenic substrate by the reaction of the endotoxin with the lysate.
• It is a method measuring either the time needed to reach a predetermined absorbance
of the reaction mixture or the rate of colour development.
Preparation of test solutions:
Solution A: Solution of the product under examination at the initial dilution (test solution)
Solution B: Test solution spiked with CSE at a concentration at or near the middle of the
standard curve (PPC)
Solution C: Standard solutions of CSE in water BET covering the linear part of the standard
curve
Solution D: water BET (NC)

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Method:
Add solution D, followed by solutions C, A, B. Add lysate and carry out the assay .
Interpretation of results:
The assay is valid only if
1) The standard curve is linear for the range of CSE concentrations used
2) The coefficient of correlation r, is not greater than 0.980;
3) The mean % recovery of the added endotoxin in the positive product control is between
50% and 150%.
METHOD E: END POINT CHROMOGENIC METHOD
Add solution D, followed by solutions C, A, B. The chromogenic substrate and lysate are
added to the solution and incubated for the recommended time. Stop the reaction and
measure the absorbance at the specified wavelength in accordance with the instructions
of the lysate manufacture.
Interpretation of results:
The assay is valid only if:
1) The standard curve is linear for the range of CSE concentrations used
2) The coefficient of correlation r, is not less than 0.980;
3) The mean % recovery of the added endotoxin in the positive product control is between
50% and 150%.

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CLARITY TEST
• 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.
• Used to check the presence of particulate matter.
• The preparations intended for parenteral use should be free from particulate matter
and should be clear when inspected visually.
• Particulate contamination of injections and infusions consists of extraneous, mobile
undissolved particles, other than gas bubbles, unintentionally present in the
solutions.
• According to USP Pharmacopoeia, for sub-visible particles mainly two methods are
employed:
1. Microscopic particle count method
2. Light obscuration particle count method
• Visible particles are inspected visually by using clarity test apparatus.

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For visible particles:
Apparatus: viewing station comprising:
-a matt black panel of appropriate size held in a vertical position
-a non-glare white panel of appropriate size held in a vertical
position next to the black panel
-an adjustable lamp holder fitted with a suitable shaded, white-
light source and with a suitable light diffuser(a viewing
illuminator containing two 13W fluorescent tubes, each 525mm
in length)
For clear glass ampoules, the intensity of illumination is
maintained between 2000lux to 3750 lux and higher for colored
glass and plastic container.
Method:
• Remove the labels on the container, wash and dry the
outside.
• Gently swirl or invert the container ensuring that air bubbles
are not introduced and observe for 5 seconds in front of the
white panel.
• Repeat in front of black panel.
• Record the presence of any particles.
• Repeat the procedure for further 19 containers.
ACCEPTANCE CRITERIA:
For liquid preparations, if 1 or more particles are found in more
than 1 container, then test fails.
Fig 13: clarity testing
apparatus

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For sub-visible particles: Two methods are specified, one involving the counting of particles
viewed under the microscope and the other based on the count of particles causing light
obscuration. Both methods are applied on small samples.
Method 1:
MICROSCOPIC PARTICLE COUNT TEST:
This method is suitable for revealing the presence of particles the longest axis or effective
linear dimension of which is 10m or more.
 Mainly used for large volume parenterals.
Apparatus: Binocular microscope, filter assembly, membrane filter for retention of
particles.
Method:
• Invert the container of the preparation 20 times in order to mix the contents.
• For large volume parenterals, single units should be tested.
• For small volume parenterals less than 25 ml in volume, the contents of 10 or more units
should be combined in a clean container where the volume of liquid in a container is
very small, the test solution may be prepared by mixing the contents of a suitable
number of containers and diluting to 25 ml with particle free water.
• Small-volume parenterals having a volume of 25ml or more may be tested individually.
• Fit the membrane filter on to the membrane filter holder. Transfer the pooled solution
or of a single unit, and allowed to stand for a minute and then apply vacuum and filter.
• Direct the jet of particle-free water in such a manner as to wash the walls of the funnel.
• Remove the membrane filter and allowed to dry in air with a cover on the petri dish.
After drying, place the petri dish on the stage of microscope and scan under reflected
light.

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Sample Particle size(µm)
equal to or greater
than
Maximum number
of particles
Containers with
nominal content of
more than 100ml(LVP)
10
25
Average in the units
tested
12per ml
2 per ml
Containers with
nominal content of
100ml
10
25
3000 per container
300 per container
Less than 100ml(SVP) 10
25
3000 per container
300 per container
Table no.10: Limits of microscopic particle count test
METHOD 2:
LIGHT OBSCURATION PARTICLE COUNT TEST
 Not suitable for reduced clarity or increased viscosity such as emulsion, colloids,
liposomal preparations.
 Mainly used for small volume parenterals.
Apparatus:
A suitable particle counter based on the principle of light blockage and capable of
automatic counting and sizing of particles.
Fig 14: microscopic particle
count test apparatus

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METHOD:
• Invert the container of the examination 20 times in order to mix the contents.
• Wash the outer surface of the container with a jet of particle-free water and remove
the closure carefully, without any contamination.
• Eliminate the gas bubbles by sonicating.
• For LVP, single units should be tested.
• For SVP less than 25 ml, the contents of 1o or more units should be combined in a
clean container.
• SVP having volume more than 25ml may be tested individually.
• Count the number of particles equal to or greater than 10µm and 25µm. Calculate
the average number of particles.
Fig 15: Diagram of light
obscuration particle count test

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Sample Particle size(µm)
equal to or greater
than
Maximum number of
particles
Containers with nominal
content of more than
100ml(LVP)
10
25
Average in the units
tested
25per ml
3 per ml
Containers with nominal
content of 100ml
10
25
6000 per container
600 per container
Less than 100ml(SVP) 10
25
6000 per container
600 per container
Table no. 11: Limits for light obscuration particle count test

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EXTRACTABLE VOLUME(USP)
• Deliverable volume
• To ensure that deliver the volume of dosage form that is mentioned on the label
claim.
Single dose containers :-
METHOD 1:
(Where the nominal volume does not exceed 5ml)
• Use 6 containers, 5 for the tests and 1 for rinsing the syringe used.
• Using a syringe with appropriate capacity, rinse the syringe and withdraw as much
as possible the contents of one of the containers reserved for the test and
transfer, without emptying the needle, to a dry graduated cylinder of such
capacity that the total combined volume to be measured occupies not less than
40% of the nominal volume of the cylinder.
• Repeat the procedure until the contents of the 5 containers have been transferred
and measure the volume. The average content of the 5 containers is not less than
the nominal volume and not more than 115% of the nominal volume.
• Alternatively the volume of contents in milliliter can be calculated as mass in
grams divided by the density.

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Volume of the solution Number of containers to be
used for the test
≥10ml 1
3-10ml 3
≤10ml 5
Table no. 12: limits for extractable volume
METHOD 2:
(Where the nominal volume is more than 5ml)
• Transfer the contents of not less than 3 containers separately to dry
graduated cylinders such that the volume to be measured occupies not less
than 40% of the nominal volume of the cylinder and measure the volume
transferred.
• The contents of each container are not less than the nominal volume and
not more than 110% of the nominal volume.

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