UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA

COLLEGE OF PHARMACY

ENRICHMENT ACTIVITY NO.2

Name : Unlayao, Diana Mae
FINISHED PRODUCT QUALITY Date :______________________
CONTROL TESTS FOR PARENTERALS Rating :______________________

I. Objective
To identify the different finished product quality control tests conducted for parenteral.

II. Materials
Search engines like google, yahoo
Reference books like USP, BP, PP

III. Procedure
1. Research for at least 15 quality control tests conducted for finished product parenteral.

QUALITY CONTROL TESTS FOR PARENTERALS

Bacterial endotoxin test

LAL (Limulus Amebocyte Lysate) test is used to characterize the bacterial endotoxin that may be present.
The USP reference standard contains 10,000 USP endotoxins per vial. The LAL reagent is used for gel-clot
formation. The test is performed using stated amounts of volumes of products, standard, positive
control, negative control of endotoxin. The tubes are incubated at 37±1ºC FOR 60 ±2 minutes. When the
tubes are inverted at 180ºC angle, formation of firm gel confirms positive reaction. While formation of a
viscous gel that doesn't maintain its integrity or absence of a firm gel confirms negative reaction. The
test is invalid if the standard endotoxin or positive product control doesn't show end point within ± 1.
Two fold dilution from label claim sensitivity of LAL reagent or if the negative control shows gel-clot end
point

The following methods can be used to monitor the endotoxin concentration:

Method A - Gel- clot limit test method

Method B -Semi quantitative gel clot method
Method C - Kinetic turbidimetric method
Method D - Kinetic chromogenic method
Method E - End point chromogenic method

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 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

Gel clot technique

 A solid gel is formed in the presence of endotoxins

 This technique requires positive and negative controls

 Positive controls - a known concentration of endotoxin added to the lysate

solution

 Negative controls - water, free from endotoxins, added to the lysate solution

Turbidimetric technique
 This test is based on the measurement of opacity change due to the formation of insoluble
coagulin

 Opacity is directly proportional to the endotoxin concentration

 This technique is used for water systems and simple pharmaceutical products

Chromogenic technique
This is based on the measurement of color change which is caused by the release of the chromogenic
chemical

p-nitroanilide

The quantity of the p-nitroanilide produced is directly proportional to the endotoxin concentration

Uniformity of content
Procedures
 30 sterile units are selected from each batch

 The weight of 10 individual sterile unit is noted and the content is removed from them and
empty individual sterile unit is weighed accurately again

 Then, the net weight is calculated by subtracting empty sterile unit weight from gross weight

 The dose uniformity is met if the amount of active ingredient is within the range of 85-115.0% of
label claim

 Relative standard deviation is equal to or less than 6.0%

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 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

 If one unit is outside the range of 85-115.0%, and none of the sterile unit is outside the range of
75-125.0% or if the relative standard deviation of the resultant is greater than 6.0%, or if both
condition prevail, an additional 20 sterile unit should be tested.

 The sterile units meet the requirements if not more than one unit is outside the range of 85-
115%, no unit is outside the range of 75-125.0% and the calculated relative standard deviation is
7.8%

Sham test (Preliminary test)

 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 to 3 days before testing the substance by injecting IV 10ml per
kg pyrogen free saline solution warmed to about 38.5 degree

 Record the temperature of the animals beginning at least 90 min before injection and
continuing for 3 hours after injection

 Any animal showing a temperature variation of 0.6 degree or more must not be used in main
test.

Pyrogen Test
The test involves measurement of the rise in body temperature of rabbits following the IV injection of a
sterile solution into ear vein of rabbit

Procedure:

 Dose not exceeding 10ml per kg injected intravenously within a period of not more than 10min

 Test animals: Use healthy, adult rabbits of either sex, preferably of the same variety.

 Recording of temperature: Clinical thermometer

Rabbit test
This test basically involves the injection sample solution which is to be tested into a Rabbits which are
use as test animals through ear vein. The Temperature sensing probe (Clinical Thermometer,
Thermosistor or similar probe) into a rectum cavity of Rabbit at the depth of 7.5 cm the test solution
must be warmed at 37 degrees prior to injection. Then Rectal temperature is recorded at 1,2,3 hr
subsequent to injection. This test is performed in separate area designed solely for this purpose under
environmental conditions similar to animal house should be free from disturbances that likely to excite
them. Initially this test is performed on 3 Rabbits but if required results are not obtained this test is
repeated on 5 additional Rabbits with same sample solution administer to initial 3 rabbits. Prior to 1hr of
injecting sample solutions the control temperatures of rabbits are determined. Use only those rabbits
whose control temperature is no vary by more than 1 degree Celsius.

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 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

*Interpretation:- The solution is judged to be non pyrogenic if no single rabbit show rise in temperature
of 0.5 degree Celsius but if this condition is not met then the test if repeated on 5 additional rabbits with
same preparation administer to initial first 3 rabbits the solution is judged to be non pyrogenic if NMT 3
of 8 rabbits show individual temperature rise of 0.5 degree

Particulate matter in injections

The preparations intended for parenteral use should be free from particulate matter and should be
clear when inspected visually. Two methods are described by USP according to the filled volume of the
product to be tested. For large volume parenterals (LVP's), a filtration followed by microscopical
examination procedure is used. For small volume parenterals (SVP's) a light obscuration based sensor
containing electronic liquidborne particle counter system is used. The USP standards are met if the LVP's
under test contain NMT 50 particles per ml of 10μ m, and NMT 5 particles per ml of 25μm in an effective
linear dimensional fashion. The USP standards are met if the SVP's under test contain NMT 10,000
particles per container of 10 μm, and NMT 1000 particles per container of 25μm in an effective spherical
diameter.

Table No: 3: Limits for particle number as per IP, BP, EP, JP.

Volume of solution Particle size ≥ 10 μm Particle size ≥25 μm

Small volume injections (< 100 3000 per container 300 per container
ml)

Large volume injections (> 100 12 per ml 2 per ml

ml)

Sterility test
Growth promotion medium and incubation conditions are selected based on the test microorganism.
The sterility test is done using direct transfer and membrane filtration techniques. Membrane filtration
technique is suitable for liquids, soluble powders with bacteriostatic or fungi static properties, oils,
creams and ointments. Sterility test by direct transfer is performed by aseptic transfer of specified
volume from test container to culture medium and incubated for 14 days and visual observation of
medium is done on 3rd, 4th, 5th, 7th, 8th and 14th day. A membrane filter with porosity of 0.45μm with
diameter of 47mm with flow rate of 55-75 ml of water per minute at a pressure of 70 cm of mercury
should be used. The test meets the requirements when no growth is observed and if growth is observed
then the test is repeated in the second stage and generally second stage is repeated with double the
number of specimens tested in first stage when the test was found to be conducted under faulty or
inadequate aseptic techniques.

PHARMACEUTICAL ANALYSIS 2
 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

Direct Transfer method

It is an traditional sterility test method which involves a direct inoculation of required volume of a
sample in two tests tube containing a culture medium that is FTM, SCDM. This method is simple in
theory but difficult in  practice  when  the  demand  for  repetition  in  opening  container,  sampling
Transferring, and mixing increases causes potential fatigue to the operator and detoriation in operator
technique.  So chances of Accidental contamination  is there.

Membrane Filtration method

It is official in U.S.P. 1970. It is more popular and widely used method over direct transfer method. 
Successful Employment Requires a more skill and knowledge than Direct transfer method. This method
basically involves filtration of Sample through membrane filters of porosity 0.22 micron and Diameter
47mm with hydrophobic characteristics. The filtration is assisted under Vaccum, After filtration
completion the membrane is cut into 2 halves and one halve is placed in two test tubes containing FTM,
SCDM medium. 

*Interpretation: - If no visible evidence of microbial growth in culture medium in test tube then it is
interpreted that the sample representing lot is without intrinsic contamination. If visible microbial
growth is seen or if the test is judged to be invalid because of inadequate environmental conditions the
sterility test is repeated such  interpretation  must  be  made  by  those  personnel  who  have  adequate
knowledge  of  aseptic  processing,  industrial  sterilization  methods,  and environmental control
procedures used in test facility. 

Clarity of Solution
Clarity is performed to ensure that parenteral product is free from foreign particles Constitute the
injection as directed on the label.

a) The solid dissolves completely, leaving no visible residue as undissolved matter.

b) The constituted injection is not significantly less clear than an equal volume of diluents for water for
injections contained in a similar container and examined in the same manner.

Test for volume of liquid

This test applies to liquid supplied in single dose, only part of the content is used.

PHARMACEUTICAL ANALYSIS 2
 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

Procedure

 Empty the contents of one container & determine the volume of contents

 Emulsions & suspensions shake the container before the determination

 The volume is not less than the amount stated on the label

Uniformity of weight
Procedure

 Remove the label & wash the container & dry

 Weight the container along with content

 Empty the container completely

 Rinse with water & ethanol, dry at 100 degree Celsius to a constant weight

 Cool & weigh

 Net weight should be calculated

 CLARITY TESTING (DETECTION OF PARTICULATE MATTER)

Particulate matter can be detected in parenteral product by two methods, including visual inspection
and electronic particulate counting.

Visual method

 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 back ground. 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 injectable. Visual
inspection gives the qualitative estimation of the particulate matter.

 Acceptance Standards is that each container checked must not contain any visible particulate
matter.

Automated techniques

 The automatic systems are also called as the electron particles counter. the electronic particles
counter evaluates the particles in injectables automatically.

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 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

 However, this method requires destruction of the ampoule/container for the particle
examination.

 Electronic particles counting may be based on any one of the following principles: a) change in
electrical resistance, b) light blockages principle and c) light scattering. Some of the automated
systems for visual particle inspection include Autoskan, Eisai Ampoule inspection machine,
Schering PDS/A-V system, etc. Autoskan System.

 The Autoskan system is based on light scattering principle whereby the particle in the path of a
light source causes the scattering of light. The scattered light is measured and provides the
corresponding information regarding the presence of particulate in the sample. This is a non-
destructive test.

Leakage test
Leakage test is employed to test the package integrity. Package integrity reflects its ability to keep the
product in and to keep potential contamination out.

It is because leakage occurs when a discontinuity exists in the wall of a package that can allow the
passage of gas under pressure or concentration differential existing across the wall. leak test methods.

VISUAL INSPECTION

 Visual inspection is the easiest leak test method to perform. But this method is least sensitive..

 The method is used for the evaluation of large volume parenteral. 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. However, the method is insensitive, operator
dependent, and qualitative.

 Sometimes, the method is used in combination with pressure and /or temperature cycling to
accelerate leakage to improve sensitivity.

BUBBLE TEST

The test package is submerged in liquids. A differential pressure is applied on the container. The
container is observed for bubbles.

 Sometimes, surfactant added liquid is used for immersion of test package. Any leakage is
evident after the application of differential pressure as the generation of foaming in immersion
liquid. The method is simple and inexpensive.
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 UNIVERSITY OF PERPETUAL HELP SYSTEM DALTA
COLLEGE OF PHARMACY

 The location of the leaks can be observed in this method. 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. 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. Positive leak test result. Air bubbles

https://www.pharmatutor.org/articles/review-quality-control-of-parenteral-products

https://e-
currentscience.com/storage/app/archive/pdf/czcx1ekbW0o8rymgyPd6fLIYUeI8snpaCBX35jDa.pdf

https://www.slideshare.net/bharathpharmacist/ipqc-for-parenterals-39635925

PHARMACEUTICAL ANALYSIS 2